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  • Sample Preparation
    https://academic.oup.com/hmg/advance-article-abstract/doi/10.1093/hmg/ddy292/5068613?redirectedFrom=fulltext
    Feiran Zhang, Human Molecular Genetics - 2018
    Abstract
    N6-methyladenosine (m6A) is the most prevalent internal modification of mammalian messenger RNAs (mRNAs) and long non-coding RNAs. The biological functions of this reversible RNA modification can be interpreted by cytoplasmic and nuclear “m6A reader” proteins to fine-tune gene expression, such as mRNA degradation and translation initiation. Here we profiled transcriptome-wide m6A sites in adult mouse cerebral cortex, underscoring that m6A is a widespread epitranscriptomic modification in brain. Interestingly, the mRNA targets of fragile X mental retardation protein (FMRP), a selective RNA-binding protein, are enriched for m6A marks. Loss of functional FMRP leads to Fragile X syndrome (FXS), the most common inherited form of intellectual disability. Transcriptome-wide gene expression profiling identified 2,035 genes differentially expressed in the absence of FMRP in cortex, and 92.5% of 174 downregulated FMRP targets are marked by m6A. Biochemical analyses indicate that FMRP binds to the m6A sites of its mRNA targets and interacts with m6A reader YTHDF2 in an RNA-independent manner. FMRP maintains the stability of its mRNA targets while YTHDF2 promotes the degradation of these mRNAs. These data together suggest that FMRP regulates the stability of its m6A-marked mRNA targets through YTHDF2, which could potentially contribute to the molecular pathogenesis of FXS.
    Transcriptome of Xenopus andrei, an octoploid frog, during embryonic development
    Mark E. Pownall, Data in Brief - 2018
    Abstract
    Although polyploidy occurs throughout the fish and amphibian lineages, the Xenopus genus exhibits a high incidence of polyploidy, with 25 out of the 26 known species being polyploid. However, transcriptomic information is currently available for only one of these species, the tetraploid Xenopus laevis. Xenopus andrei, an octoploid species within the Xenopus genus, offers an opportunity for assessing a novel polyploid transcriptome during vertebrate development. RNA-Seq data was generated at nine different developmental stages ranging from unfertilized eggs through swimming tadpole stages and raw FASTQ files were deposited in the NCBI SRA database (accession number SRP134281). Additionally, RNA-seq data from all nine stages were pooled to create a de novo assembly of the transcriptome using Trinity and has been deposited in the NCBI GEO database (accession number GSE111639). To our knowledge, this represents the first published assembly of an octoploid vertebrate transcriptome. In total, 849Mb were assembled, which led to the identification of 1,650,048 transcripts in the assembly with a contig N50 of 630 bases. This RNA-Seq and transcriptome data will be valuable for comparing polyploid transcriptomes across Xenopus species, as well as understanding evolutionary implications of whole-genome duplication and polyploidy in vertebrates.
    Maternal obesity during lactation may protect offspring from high fat diet-induced metabolic dysfunction
    Jenifer Monks, Nutrition & Diabetes - 2018
    Abstract
    Background/Objectives The current obesity epidemic has spurred exploration of the developmental origin of adult heath and disease. A mother’s dietary choices and health can affect both the early wellbeing and lifelong disease-risk of the offspring. Subjects/Methods To determine if changes in the mother’s diet and adiposity have long-term effects on the baby’s metabolism, independently from a prenatal insult, we utilized a mouse model of diet-induced-obesity and cross-fostering. All pups were born to lean dams fed a low fat diet but were fostered onto lean or obese dams fed a high fat diet. This study design allowed us to discern the effects of a poor diet from those of mother’s adiposity and metabolism. The weaned offspring were placed on a high fat diet to test their metabolic function. Results In this feeding challenge, all male (but not female) offspring developed metabolic dysfunction. We saw increased weight gain in the pups nursed on an obesity-resistant dam fed a high fat diet, and increased pathogenesis including liver steatosis and adipose tissue inflammation, when compared to pups nursed on either obesity-prone dams on a high fat diet or lean dams on a low fat diet. Conclusion Exposure to maternal over-nutrition, through the milk, is sufficient to shape offspring health outcomes in a sex- and organ-specific manner, and milk from a mother who is obesity-prone may partially protect the offspring from the insult of a poor diet.
    The BPA-substitute bisphenol S alters the transcription of genes related to endocrine, stress response and biotransformation pathways in the aquatic midge Chironomus riparius (Diptera, Chironomidae)
    Óscar Herrero, PLOS ONE - 2018
    Abstract
    Bisphenol S (BPS) is an industrial alternative to the endocrine disruptor bisphenol A (BPA), and can be found in many products labeled “BPA-free”. Its use has grown in recent years, and presently it is considered a ubiquitous emerging pollutant. To date there is a lack of information on the effects of BPS on invertebrates, although they represent more than 95% of known species in the animal kingdom and are crucial for the structure and proper function of ecosystems. In this study, real-time RT-PCR was used to determine the early detrimental effects of BPS on the transcriptional rate of genes in the model species Chironomus riparius, specifically those related to the ecdysone pathway (EcR, ERR, E74, Vtg, cyp18a1) crucial for insect development and metamorphosis, stress and biotransformation mechanisms (hsp70, hsp40, cyp4g, GPx, GSTd3) that regulate adaptive responses and determine survival, and ribosome biogenesis (its2, rpL4, rpL13) which is essential for protein synthesis and homeostasis. While 24-hour exposure to 0.5, 5, 50, and 500 μg/L BPS had no effect on larval survival, almost all the studied genes were upregulated following a non-monotonic dose-response curve. Genes with the greatest increases in transcriptional activity (fold change relative to control) were EcR (3.8), ERR (2), E74 (2.4), cyp18a1 (2.5), hsp70 (1.7), hsp40 (2.5), cyp4g (6.4), GPx (1.8), and GST (2.1), while others including Vtg, GAPDH, and selected ribosomal genes remained stable. We also measured the transcriptional activity of these genes 24 hours after BPS withdrawal and a general downregulation compared to controls was observed, though not significant in most cases. Our findings showed that BPS exposure altered the transcriptional profile of these genes, which may have consequences for the hormone system and several metabolic pathways. Although further research is needed to elucidate its mode of action, these results raise new concerns about the safety of BPA alternatives.
    Compartmentalized partnered replication for the directed evolution of genetic parts and circuits
    Zhanar Abil, Nature Protocols - 2017
    Abstract

    This protocol describes the procedures for compartmentalized partnered replication (CPR), an emulsion-based directed evolution method for the generation of proteins, genetic elements, and genetic circuits with improved or altered function.

    Quantitative Bromodeoxyuridine Immunoprecipitation Analyzed by High-Throughput Sequencing (qBrdU-Seq or QBU)
    Joanna E. Haye-Bertolozzi, Genome Instability - 2017
    Abstract
    Incorporation into DNA of nucleoside analogs like 5-bromo-2′-deoxyuridine (BrdU) is a powerful tool for in vivo studies of DNA synthesis during replication and repair. Immunoprecipitation of BrdU-labeled DNA analyzed by DNA sequencing (BrdU-IP-seq) allows for genome-wide, sequence-specific tracking of replication origin and replication fork dynamics under different conditions, such as DNA damage and replication stress, and in mutant strains. We have recently developed a quantitative method for BrdU-IP-seq (qBrdU-seq) involving DNA barcoding to enable quantitative analysis of multiple experimental samples subjected to BrdU-IP-seq. After initial barcoding of multiple, individually BrdU-labeled genomic DNA samples, a pooling strategy is used for all subsequent steps including immunoprecipitation, amplification, and sequencing, which eliminates sample-to-sample variability in these steps. Parallel processing of an aliquot of the pooled input sample provides a direct control for the normalization of the data and yields results that allow quantitative comparisons of the experimental samples. Though developed for the analysis of S. cerevisiae, this method should be directly adaptable to other model systems.
    Parasitic nematodes of the genus Syphacia Seurat, 1916 infecting Muridae in the British Isles, and the peculiar case of Syphacia frederici
    Alex Stewart, Parasitology - 2017
    Abstract
    SUMMARY Syphacia stroma (von Linstow, 1884) Morgan, 1932 and Syphacia frederici Roman, 1945 are oxyurid nematodes that parasitize two murid rodents, Apodemus sylvaticus and Apodemus flavicollis, on the European mainland. Only S. stroma has been recorded previously in Apodemus spp. from the British Isles. Despite the paucity of earlier reports, we identified S. frederici in four disparate British sites, two in Nottinghamshire, one each in Berkshire and Anglesey, Wales. Identification was based on their site in the host (caecum and not small intestine), on key morphological criteria that differentiate this species from S. stroma (in particular the tail of female worms) and by sequencing two genetic loci (cytochrome C oxidase 1 gene and a section of ribosomal DNA). Sequences derived from both genetic loci of putative British S. frederici isolates formed a tight clade with sequences from continental worms known to be S. frederici, clearly distinguishing these isolates from S. stroma which formed a tight clade of its own, distinct from clades representative of Syphacia obvelata from Mus and S. muris from Rattus. The data in this paper therefore constitute the first record of S. frederici from British wood mice, and confirm the status of this species as distinct from both S. obvelata and S. stroma.
  • Reverse Transcription
    First-Strand cDNA Synthesis
    Alzheimer’s disease neuropathology may not predict functional impairment in HIV: a report of two individuals
    Susan Morgello, Journal of NeuroVirology - 2018
    Abstract
    With aging of HIV populations, there is concern that Alzheimer’s disease (AD) may become prevalent and difficult to distinguish from HIV-associated neurocognitive disorders. To date, there are no reports documenting histologically verified Alzheimer’s neuropathology in individuals with HIV and dementia. Herein, we report two antiretroviral-treated, virally suppressed, HIV-infected individuals autopsied by the Manhattan HIV Brain Bank. Subject A presented to study at 52 years, already dependent in instrumental activities of daily living (ADLs), with severe cognitive impairment inclusive of learning and memory dysfunction. Her history was significant for educational disability and head trauma. She had rapid cognitive decline and, by death at age 59 years, was bed-bound, incontinent, and non-communicative. At autopsy, she exhibited severe AD neuropathologic change (NIA-AA score A3B3C3) and age-related tau astrogliopathy (ARTAG). She was homozygous for APOE ε3/ε3. No HIV DNA was detected in frontal lobe by nested polymerase chain reaction. Subject B was a community dwelling 81-year-old woman who experienced sudden death by pulmonary embolus. Prior to death, she was fully functional, living independently, and managing all ADLs. At autopsy, she displayed moderate amyloid and severe tau AD neuropathologic changes (A2B3C2), ARTAG, and cerebral congophilic angiopathy. She was an APOE ε3/ε4 heterozygote, and HIV DNA, but not RNA, was detected in frontal lobe, despite 20 years of therapy-induced viral suppression. We conclude that in the setting of HIV, AD neuropathology may occur with or without symptomatic cognitive dysfunction; as with seronegative individuals, there are likely to be complex factors in the generation of clinically relevant impairments.
    EZH1 is an antipsychotic-sensitive epigenetic modulator of social and motivational behavior that is dysregulated in schizophrenia
    Andrea L. Johnstone, Neurobiology of Disease - 2018
    Abstract
    Background With the capacity to modulate gene networks in an environmentally-sensitive manner, the role of epigenetic systems in mental disorders has come under intense investigation. Dysregulation of epigenetic effectors, including microRNAs and histone-modifying enzymes, may better explain the role of environmental risk factors and the observed heritability rate that cannot be fully attributed to known genetic risk alleles. Here, we aimed to identify novel epigenetic targets of the schizophrenia-associated microRNA 132 (miR-132). Methods Histone modifications were quantified by immunodetection in response to viral-mediated overexpression of miR-132 while a luminescent reporter system was used to validate targets of miR-132 in vitro. Genome-wide profiling, quantitative PCR and NanoSting were used to quantify gene expression in post-mortem human brains, neuronal cultures and prefrontal cortex (PFC) of mice chronically exposed to antipsychotics. Following viral-mediated depletion of Enhancer of Zeste 1 (EZH1) in the murine PFC, behaviors including sociability and motivation were assessed using a 3-chambered apparatus and forced-swim test, respectively. Results Overexpression of miR-132 decreased global histone 3 lysine 27 tri-methylation (H3K27me3), a repressive epigenetic mark. Moreover, the polycomb-associated H3K27 methyltransferase, EZH1, is regulated by miR-132 and upregulated in the PFC of schizophrenics. Unlike its homolog EZH2, expression of EZH1 in the murine PFC decreased following chronic exposure to antipsychotics. Viral-mediated depletion of EZH1 in the mouse PFC attenuated sociability, enhanced motivational behaviors, and affected gene expression pathways related to neurotransmission and behavioral phenotypes. Conclusions EZH1 is dysregulated in schizophrenia, sensitive to antipsychotic medications, and a brain-enriched miR-132 target that controls neurobehavioral phenotypes.
    A piRNA utilizes HILI and HIWI2 mediated pathway to down-regulate ferritin heavy chain 1 mRNA in human somatic cells
    Sumirtha Balaratnam, Nucleic Acids Research - 2018
    Abstract
    The piwi interacting RNAs (piRNAs) are small non-coding RNAs that specifically bind to the PIWI proteins, a functional requirement. The piRNAs regulate germline development, transposons control, and gene expression. However, piRNA-mediated post-transcriptional gene regulation in human somatic cells is not well understood. We discovered a human piRNA (piR-FTH1) which has a complementary sequence in the ferritin heavy chain 1 (Fth1) mRNA. We demonstrated that expression of piR-FTH1 and Fth1 are inversely correlated in the tested tumor cell lines. We found that piR-FTH1 negatively regulates the Fth1 expression at post-transcriptional level in triple negative breast cancer (TNBC) cells. Additionally, we confirmed that transfected piR-FTH1 knocks down the Fth1 mRNA via the HIWI2 and HILI mediated mechanism. piR-FTH1 mediated Fth1 repression also increased doxorubicin sensitivity by a remarkable 20-fold in TNBC cells. Since the current piRNA-mediated knockdowns of target mRNA are mostly reported in germ line cells, piRNA-mediated post-transcriptional gene regulation in somatic cells is rather unique in its application and mechanistically uses an alternative pathway to siRNA and miRNA. This work begins to lay the groundwork with a broader impact on treatment of various diseases that are linked to elevated levels of specific mRNAs which have a piRNA target.
    Resident brain neural precursor cells develop age-dependent loss of therapeutic functions in Alzheimer's mice
    Nina Fainstein, Neurobiology of Aging - 2018
    Abstract
    There is vast knowledge on pathogenic mechanisms in Alzheimer's disease, but very little on means by which the brain protects itself from disease. A major candidate in providing neuroprotection is the resident brain neural stem/precursor cell (NPC) pool. Transplanted NPCs possess powerful immune-modulatory and trophic properties in vivo and in vitro, underscoring the question whether resident brain NPCs have any role in regulating disease pathology in Alzheimer's disease, and particularly whether they fail to protect the brain from degeneration. To evaluate brain NPC function in relation to disease pathology, we first characterized the pathological properties of 5xFAD transgenic mouse model of Alzheimer's disease at different ages. We found that age seven months is a critical time point of heavy amyloid deposition and gliosis but prior to neurodegeneration and a normal basal rate of NPC turnover in the subventricular zone (SVZ) of 5xFAD mice as compared to wild type mice. Analysis of NPC functional properties showed that despite preserved rate of turnover, there was substantial SVZ NPC dysfunction as indicated by both ex vivo and in vivo assays. Freshly isolated NPCs from seven months old 5xFAD mice exhibited reduced expansion rate, and diminished immune-modulatory and trophic properties. Moreover, there was slowed recovery of SVZ NPCs after Cytosine-arabinoside insult and markedly reduced migratory response following a Lysolecithine-induced lesion in the Corpus-Callosum in vivo. Importantly, these functions were fully preserved in two-months old 5xFAD mice, a time-point prior to Alzheimer's-specific pathological changes. There was reduced expression of key genes involved in NPC proliferative and migratory response in NPCs derived from seven months old 5xFAD mice. The dysfunctional properties and down-regulation of gene expression were reversible in NPCs derived from seven-months old 5xFAD mice following in-vitro expansion and were reproduced in wild type NPC by addition of amyloid beta peptide. Thus, there is age-dependent acquired NPC dysfunction, with loss of immune-modulatory and neurotrophic properties, which is induced by the pathological Alzheimer's brain environment at a critical time point prior to neurodegeneration. We suggest that failure of resident NPC to provide tissue support may be involved in promoting neurodegeneration.
    Gja1 expression is regulated by cooperation between SOX8/SOX9 and cJUN transcription factors in TM4 and 15P-1 Sertoli cell lines
    Firas Ghouili, Molecular Reproduction and Development - 2018
    Abstract
    Within the seminiferous tubules of the testis, Gja1-encoded connexin43 plays a critical role in intercellular communication between Sertoli cells. These cells nurture, protect and stimulate the developing germ cells and spermatids. SOX transcription factors are known to play an important role in male fertility and sex determination; however, their physiological function and the identity of their target genes in postnatal Sertoli cells remain to be defined. Members of the AP-1 family have been shown to regulate Gja1 expression in myometrial and testicular cells and to physically interact with SOX members, suggesting that these transcription factors may regulate its expression within the testis. Hence, we performed co-transfections of expression plasmids encoding SOX4, SOX8, SOX9 and cJUN with different mouse Gja1 promoter/luciferase reporter constructs within TM4 and 15P-1 Sertoli cells. We showed that a functional cooperation between cJUN and SOX8 or SOX9 regulates Gja1 expression and may involve DNA regulatory elements located between -132 and -26 bp. Such synergy relies on the recruitment of cJUN to the -47 bp AP-1 DNA regulatory element of the mouse Gja1 promoter. Hence, SOX and AP-1 members cooperate to regulate Gja1 within testicular Sertoli cells. This article is protected by copyright. All rights reserved.
    CSDC2, a cold shock domain RNA-binding protein in decidualization
    Griselda Vallejo, Journal of Cellular Physiology - 2018
    Abstract
    RNA-binding proteins (RBPs) have been described for cancer cell progression and differentiation, although there is still much to learn about their mechanisms. Here, using in vivo decidualization as a model, we describe the role of RBP cold shock domain containing C2 (CSDC2) in the endometrium. Csdc2 messenger RNA expression was differentially regulated depending on time and areas of decidua development, with the most variation in antimesometrium (AM) and, to a lesser degree, in the junctional zone (JZ). Immunohistochemistry of CSDC2 showed a preferentially cytoplasmic localization at AM and JZ, and nuclear localization in underneath myometrium and mesometrium (M). Cytoplasmic localization coincided with differentiated, DESMIN-marked areas, while nuclear localization coincides with proliferative zones. Uterine suppression of CSDC2 through intrauterine-injected-specific small interfering RNA (siRNA) led to abnormal decidualization in early pregnancy, with more extended antimesometrial area and with poor M development if compared with control siRNA-injected animals. These results suggest that CSDC2 could be a regulator during decidua development.
    Induction of brain Nrf2-HO-1 pathway and antinociception after different physical training paradigms in mice
    Abdulkarim Tutakhail, Life Sciences - 2018
    Abstract
    Aim Activation of the Nrf2-antioxidant response element signaling pathway is a major mechanism in the cellular defense against oxidative or electrophilic stress through conjugative reactions and by enhancing cellular antioxidant capacity. Although exercise training up-regulates antioxidant defenses system, while information regarding the intensity levels of physical exercise that acts on the cellular protection systems is limited. Main methods The present study evaluated the effects of different durations and intensities of physical exercise on the hippocampus, cortex and hypothalamus Nrf2 and HO-1 gene expression and related protein content and the nociception thresholds in adult C57Bl male mice. Exercise training consisted of daily running on a 10-lane rodent motor-driven treadmill for either 3 or 7 weeks at three different intensities. Pain responses were evaluated after exercise and in untrained mice by Von Frey hair test and cold plate test. Key findings This study confirmed that only vigorous and longer duration aerobic exercise increased Nrf2 protein level in the hippocampus and HO-1 protein level in the cortex and reduced pain perception. Mechanical and thermal hypoalgesia were only observed in exercise groups after 7 weeks of physical training. Significance The overall findings in this study confirm that only the long duration intensive forced exercise reduced inflammatory pain by induction of Nrf2/HO-1 antioxidant signaling pathway.
    Deletion of Axin1 in condylar chondrocytes leads to osteoarthritis-like phenotype in temporomandibular joint via activation of β-catenin and FGF signaling
    Yachuan Zhou, Journal of Cellular Physiology - 2018
    Abstract
    Osteoarthritis (OA) in the temporomandibular joint (TMJ) is a degenerative disease in the adult, which is characterized by the pathological degeneration of condylar cartilage. Axin1 plays a critical role in the regulation of cartilage development and homeostasis. To determine the role of Axin1 in TMJ tissue at the adult stage, we generated Axin1Agc1ER mice, in which Axin1 was deleted in aggrecan-expressing chondrocytes at 2 months of age. Histology, histomorphometry, and immunostaining analyses were performed using TMJ tissues harvested from 4- and 6-month-old mice after tamoxifen administration. Total RNA isolated from TMJ cartilage of 6-month-old mice was used for gene expression analysis. Progressive OA-like degeneration was observed in condylar cartilage in Axin1 knockout (KO) mice with loss of surface continuity and the formation of vertical fissures. In addition, reduced alcian blue staining in condylar cartilage was also found in Axin1 KO mice. Immunostaining and reverse transcription quantitative polymerase chain reaction (qRT-PCR) assays revealed disturbed homeostasis in condylar cartilage with increased expressions of MMP13 and Adamts5 and decreased lubricin expression in Axin1-deficient chondrocytes. Less proliferative cells with increased hypertrophic and apoptotic activities were presented in the condylar cartilage of Axin1Agc1ER KO mice. As a scaffolding protein, the deletion of Axin1 stimulated not only the β-catenin but also the fibroblast growth factor (FGF) signaling via extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) activation. The qRT-PCR results showed an increased expression of Fgfr1 in Axin1 KO cartilage. Overall, the deletion of Axin1 in condylar chondrocytes altered the β-catenin and FGF/ERK1/2 signaling pathways, thus cooperatively contribute to the cartilage degeneration.
    Cultivation of Purified Primary Purkinje Cells from Rat Cerebella
    Jonas Tjaden, Cellular and Molecular Neurobiology - 2018
    Abstract
    Primary neurons are difficult to cultivate because they are often part of a complex tissue, and synaptically connected to numerous other cell types. These circumstances often prevent us from unveiling molecular and metabolic mechanisms of distinct cells, as functional signals or assays cannot clearly be correlated with them due to interfering signals from other parts of the culture. We therefore present an up-to-date method for obtaining a highly purified neuronal culture of Purkinje cells. In the past, Purkinje cells were successfully isolated from young mouse cerebella, but this protocol was never adapted to other mammals. We therefore provide an updated and adjusted protocol for Purkinje cell isolation from rat instead of mouse cerebella. To purify Purkinje cells, we obtained perinatal rat cerebella, dissociated them and performed a Percoll gradient centrifugation to segregate the smaller and larger cell fractions. In a second step, we performed an immunopanning procedure to enrich only Purkinje cells from the large cell fraction. Based on former protocols, we used a different antibody for the immunopanning procedure and adjusted several aspects from the initial protocol to improve the yield and vitality of Purkinje cells. We provide RT-qPCR-based purity data obtained with this protocol and show the behaviour and the growth of these purified Purkinje cells. We provide a highly reproducible purification protocol for Purkinje cell cultures of high purity that allows functional analysis and downstream assays on living rat Purkinje cells and further morphological growth analysis in future.
    Serotonin uptake is required for Rac1 activation in Kras-induced acinar-to-ductal metaplasia in the pancreas
    Enrica Saponara, The Journal of Pathology - 2018
    Abstract
    Pancreatic ductal adenocarcinoma (PDAC), the primary cause of pancreatic cancer mortality, is poorly responsive to currently available interventions. Identifying new targets that drive PDAC formation and progression is critical to develop alternative therapeutic strategies to treat this lethal malignancy. Using genetic and pharmacologic approaches, we investigated in vivo and in vitro whether uptake of the monoamine serotonin is required for PDAC development. We demonstrated that pancreatic acinar cells have the ability to readily take up serotonin in a transport-mediated manner. Serotonin uptake promoted the activation of the small GTPase Ras-Related C3 Botulinum Toxin Substrate 1 (Rac1), which is required for trans-differentiation of acinar cells into acinar-to-ductal metaplasia (ADM), a key determinant in PDAC development. Consistent with the central role played by Rac1 in ADM formation, inhibition of the serotonin transporter Sert (Slc6a4) with fluoxetine reduced ADM formation both in vitro and in vivo in a cell autonomous manner. In addition, fluoxetine treatment profoundly compromised the stromal reaction and affected proliferation and lipid metabolism of malignant PDAC cells. We propose that Sert is a promising therapeutic target to counteract the early event of acinar-to-ductal metaplasia with the potential to stall initiation and progression of pancreatic carcinogenesis. This article is protected by copyright. All rights reserved.
    Assessment of Sep1virus interaction with stationary cultures by transcriptional and flow cytometry studies
    Luís D. R. Melo, FEMS Microbiology Ecology - 2018
    Abstract
    The majority of phage infection studies are performed in bacteria that are growing exponentially, although in nature, phages usually interact also with non-replicating cells. These stationary-phase cells differ from exponential cells morphologically, physiologically and metabolically. The interaction of a Sep1virus with Staphylococcus epidermidis stationary and exponential phase cells was explored. Phage SEP1 efficiently infected both cell culture states, without the addition of any fresh nutrients to stationary cultures. Phage-host interactions, analysed by flow cytometry, showed stationary-phase cells response to phage immediately after SEP1 addition. Quantitative PCR experiments corroborate that phage genes are expressed within 5 min of contact with stationary phase cells. The increase of host RNA polymerase transcripts in stationary cells, suggests that SEP1 infection leads to upregulation of host machinery fundamental for completion of its lytic life cycle. SEP1 infection and replication process highlights its potential clinical interest targeting stationary phase cells.
    Loss of IGF1R in human astrocytes alters complex I activity and support for neurons
    Laura E. Ratcliffe, Neuroscience - 2018
    Abstract
    The insulin/insulin-like growth factor 1 (IGF1) signalling pathways are implicated in longevity and in progression of Alzheimer’s disease. Previously, we showed that insulin-like growth factor 1 receptor (IGF1R) and downstream signalling transcripts are reduced in astrocytes in human brain with progression of Alzheimer’s neuropathology and developed a model of IGF1 signalling impairment in human astrocytes using an IGF1R-specific monoclonal antibody, MAB391. Here, we have established a novel human astrocyte-neuron co-culture system to determine whether loss of astrocytic IGF1R affects their support for neurons. Astrocyte-neuron co-cultures were developed using human primary astrocytes and differentiated Lund Human Mesencephalic Cells (LUHMES). Neurite outgrowth assays, performed to measure astrocytic support for neurons, showed astrocytes provided contact-mediated support for neurite outgrowth. Loss of IGF1R did not affect neurite outgrowth under control conditions but when challenged with hydrogen peroxide IGF1R-impaired astrocytes were less able to protect LUHMES. To determine how loss of IGF1R affects neuronal support MAB391-treated astrocytes were FACS sorted from GFP-LUHMES and their transcriptomic profile was investigated using microarrays. Changes in transcripts involved in astrocyte energy metabolism were identified, particularly NDUFA2 and NDUFB6, which are related to complex I assembly. Loss of complex I activity in MAB391-treated astrocytes validated these findings. In conclusion, reduced IGF1 signalling in astrocytes impairs their support for neurons under conditions of stress and this is associated with defects in the mitochondrial respiratory chain in astrocytes.
    Differential gene expression to an LPS challenge in relation to exogenous corticosterone in the invasive cane toad (Rhinella marina)
    Steven Gardner, Developmental & Comparative Immunology - 2018
    Abstract
    The cane toad (Rhinella marina) is an invasive amphibian in several parts of the world. Much of the research performed on assessing the dispersal potential of invasive species has focused immunity. Invaders are predicted to rely less on pro-inflammatory immunity, allowing them to allocate energy to dispersal. Elevated stress may play a role in regulation of immune responses used by invasive species. RNA sequencing of spleen tissue from cane toads subjected to an acute LPS challenge revealed genes coding for cytokines involved in typical innate responses such as phagocytic cell recruitment, extravasation, inflammation, and lymphocyte differentiation were significantly upregulated, while toads receiving transdermal application of corticosterone in addition to an LPS injection showed downregulation of genes involved with cell mediated immunity. These results indicate hormonal changes associated with acute stress may alter investment into mounting cell-mediated or humoral responses while allowing for prolonged phagocytic innate responses in this invasive species.
    NLRP3 inflammasome mediates oxidative stress-induced pancreatic islet dysfunction.
    Marina Sokolova, American Journal of Physiology-Endocrinology and Metabolism - 2018
    Abstract
    Inflammasomes are multi-protein inflammatory platforms that induce caspase-1 activation and subsequently interleukin (IL)-1β and IL-18 processing. The NLRP3 inflammasome is activated by different forms of oxidative stress, and based on the central role of IL-1β in the destruction of pancreatic islets, it could be related to the development of diabetes. We therefore investigated responses in wild-type C57Bl/6 (WT), NLRP3-/- and ASC-/- mice after exposing islets to short-term hypoxia or alloxan induced islet damage. NLRP3 deficient islets compared to WT islets had preserved function ex vivo and were protected against hypoxia-induced cell death. Further, NLRP3 and ASC deficient mice were protected against oxidative stress-induced diabetes caused by repetitive low dose alloxan administration, and this was associated with reduced β-cell death and reduced macrophage infiltration. This suggests that the beneficial effect of NLRP3 inflammasome deficiency on oxidative stress-mediated β-cell damage could involve reduced macrophage infiltration and activation. To support the role of macrophage activation in alloxan-induced diabetes, we injected WT mice with liposomal clodronate which causes macrophage depletion prior to induction of a diabetic phenotype by alloxan treatment resulting in improved glucose homeostasis in WT mice. We show here that the NLRP3 inflammasome acts as a mediator of hypoxia and oxidative stress in insulin producing cells, suggesting that inhibition of the NLRP3 inflammasome could have beneficial effects on β-cell preservation.
    https://www.nature.com/articles/s41401-018-0068-9
    Xiao Zhu, Acta Pharmacologica Sinica - 2018
    Abstract
    Perivascular adipose tissue (PVAT), a special type of adipose tissue, closely surrounds vascular adventitia and produces numerous bioactive substances to maintain vascular homeostasis. PVAT dysfunction has a crucial role in regulating vascular remodeling, but the exact mechanisms remain unclear. In this study, we investigated whether and how obesity-induced PVAT dysfunction affected adventitia remodeling in early vascular injury stages. Mini pigs were fed a high sugar and fat diet for 6 months to induce metabolic syndrome and obesity. In the mini pigs, left carotid vascular injury was then generated using balloon dilation. Compared with normal mini pigs, obese mini pigs displayed significantly enhanced vascular injury-induced adventitial responses, evidenced by adventitia fibroblast (AF) proliferation and differentiation, and adventitia fibrosis, as well as exacerbated PVAT dysfunction characterized by increased accumulation of resident macrophages, particularly the M1 pro-inflammatory phenotype, increased expression of leptin and decreased expression of adiponectin, and production of pro-inflammatory cytokines interleukin (IL)-1β and IL-18. Primary AFs cultured in PVAT-conditioned medium from obese mini pigs also showed significantly increased proliferation and differentiation. We further revealed that activated nod-like receptor protein 3 (NLRP3) inflammasome and its downstream products, i.e., IL-1 family members such as IL-1β and IL-18 were upregulated in the PVAT of obese mini pigs; PVAT dysfunction was also demonstrated in preadipocytes treated with palmitic acid. Finally, we showed that pretreatment with IL-1 receptor (IL-1R) antagonist or IL-1R knockdown blocked AF proliferation and differentiation in AFs cultured in PVAT-conditioned medium. These results demonstrate that obesity-induced PVAT dysfunction aggravates adventitial remodeling after early vascular injury with elevated AF proliferation and differentiation via activating the NLRP3/IL-1 signaling pathway.
    MED31 involved in regulating self-renewal and adipogenesis of human mesenchymal stem cells
    Erik P. Beadle, Molecular Biology Reports - 2018
    Abstract
    Regulation of gene expression is critical for the maintenance of cell state and homeostasis. Aberrant regulation of genes can lead to unwanted cell proliferation or misdirected differentiation. Here we investigate the role of MED31, a highly conserved subunit of the Mediator complex, to determine the role this subunit plays in the maintenance of human mesenchymal stem cell (hMSC) state. Using siRNA-mediated knockdown of MED31 we demonstrate a decrease in self-renewal based on cell assays and monitoring of gene expression. In addition, in the absence of MED31, hMSCs also displayed a reduction in adipogenesis as evidenced by diminished lipid vesicle formation and expression of specific adipogenic markers. These data present evidence for a significant role for MED31 in maintaining adult stem cell homeostasis, thereby introducing potential novel targets for future investigation and use in better understanding stem cell behavior and adipogenesis.
    Viral RNA load and histological changes in tissues following experimental infection with an arterivirus of possums (wobbly possum disease virus)
    Julia Giles, Virology - 2018
    Abstract
    Tissues from Australian brushtail possums (Trichosurus vulpecula) that had been experimentally infected with wobbly possum disease (WPD) virus (WPDV) were examined to elucidate pathogenesis of WPDV infection. Mononuclear inflammatory cell infiltrates were present in livers, kidneys, salivary glands and brains of WPD-affected possums. Specific staining was detected by immunohistochemistry within macrophages in the livers and kidneys, and undefined cell types in the brains. The highest viral RNA load was found in macrophage-rich tissues. The detection of viral RNA in the salivary gland, serum, kidney, bladder and urine is compatible with transmission via close physical contact during encounters such as fighting or grooming, or by contact with an environment that has been contaminated with saliva or urine. Levels of viral RNA remained high in all tissues tested throughout the study, suggesting that on-going virus replication and evasion of the immune responses may be important in the pathogenesis of disease.
    MPZL2 is a novel gene associated with autosomal recessive nonsyndromic moderate hearing loss
    Guney Bademci, Human Genetics - 2018
    Abstract
    While recent studies have revealed a substantial portion of the genes underlying human hearing loss, the extensive genetic landscape has not been completely explored. Here, we report a loss-of-function variant (c.72delA) in MPZL2 in three unrelated multiplex families from Turkey and Iran with autosomal recessive nonsyndromic hearing loss. The variant co-segregates with moderate sensorineural hearing loss in all three families. We show a shared haplotype flanking the variant in our families implicating a single founder. While rare in other populations, the allele frequency of the variant is ~ 0.004 in Ashkenazi Jews, suggesting that it may be an important cause of moderate hearing loss in that population. We show that Mpzl2 is expressed in mouse inner ear, and the protein localizes in the auditory inner and outer hair cells, with an asymmetric subcellular localization. We thus present MPZL2 as a novel gene associated with sensorineural hearing loss.
    The search for proteins involved in the formation of crustacean cuticular structures
    Shai Abehsera, Hydrobiologia - 2018
    Abstract
    Crustacean cuticular structures are key features formed during a molt cycle. These structures are complex biomaterials comprising chitin and different mineral forms in distinct scaffold organizations. The formation of these complex biomaterials is controlled by the organic extracellular matrix including structural proteins. Since cuticular structures are formed de novo during each molt cycle, the spatial and temporal expression patterns of structural proteins are tightly linked to molt cycle events. As a model scenario, we demonstrate the molt-related pattern of expression of the gene encoding GAP65, a core structural protein involved in the formation of the cuticular structures of Cherax quadricarinatus. Based on this typical pattern of expression and using a binary-patterning approach, which is a specialized tool for the study of molt-related proteins, we revealed and characterized additional candidate proteins involved in the formation of crustacean cuticular structures. We propose that our approach be applied as a framework in the search for proteins involved in the formation of the crustacean cuticle. To stimulate research on this important aspect of structural biology, we put forward a schematic representation of the extracellular matrix and its proteins in three cuticular structures of C. quadricarinatus, the gastroliths, the mandibles, and the mineralized cuticle.
    Lactobacillus casei BL23 modulates the innate immune response in Staphylococcus aureus-stimulated bovine mammary epithelial cells
    R.f.s. Souza, Beneficial Microbes - 2018
    Abstract
    Probiotics have been adopted to treat and prevent various diseases in humans and animals. They were notably shown to be a promising alternative to prevent mastitis in dairy cattle. This inflammation of the mammary gland is generally of infectious origin and generates extensive economic losses worldwide. In a previous study, we found that Lactobacillus casei BL23 was able to inhibit the internalisation of Staphylococcus aureus, one of the major pathogens involved in mastitis, into bovine mammary epithelial cells (bMEC). In this study, we further explored the capacity of this strain to modulate the innate immune response of bovine mammary epithelial cells during S. aureus infection. L. casei BL23 was able to decrease the expression of several pro-inflammatory cytokines, including interleukins 6, 8, 1α and 1β and tumour necrosis factor alpha, in S. aureus-stimulated bMEC, 8 h post-infection. On the other hand, L. casei did not impair the induction of defensins, such as lingual antimicrobial peptide and defensin β1 in the presence of S. aureus, and even slightly increased the induction of tracheal antimicrobial peptide during S. aureus infection. Finally, this strain did not alter the expression of the pattern recognition receptor nucleotide-binding oligomerisation domain proteins (NOD2). This study demonstrates that L. casei BL23 displayed anti-inflammatory properties on S. aureus-stimulated bMEC. These results open the way to further characterisation of the BL23 probiotic potential in a bovine mammary gland context and to a better understanding of how all these beneficial properties combine in vivo to combat mastitis pathogens.
    The von Hippel Lindau tumour suppressor gene is a novel target of E2F4-mediated transcriptional repression in preeclampsia
    Sruthi Alahari, Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease - 2018
    Abstract
    The von Hippel Lindau tumour suppressor (VHL) protein is essential for proper placental development and is downregulated in preeclampsia (PE), a devastating disorder of pregnancy typified by chronic hypoxia. To date, knowledge on VHL genetic and epigenetic regulation is restricted to inactivating mutations and loss-of-heterozygosity in renal cell carcinomas. Herein, we sought to examine whether VHL DNA is subject to differential methylation, and if so, whether it is altered in early-onset PE (E-PE). Sodium bisulfite modification and methylation-specific PCR analysis revealed that VHL is subject to extensive methylation in a CpG-rich region within its promoter in the human placenta. Notably, we detected significant differences in methylation in E-PE placentae relative to normotensive age-matched controls at key transcription factor binding sites, including that of the transcriptional repressor E2F4. Treatment of JEG3 cells with 5-Aza-2′-deoxycytidine, revealed that loss of DNA methylation promoted VHL transcription by attenuating VHL association with E2F4. RNAi knockdown of E2F4 in vitro confirmed its function on VHL repression. Exposure of JEG3 cells to transforming growth factor beta (TGFβ) downregulated VHL mRNA. In line with elevated levels of TGFβ3 in E-PE, chromatin immunoprecipitation assays revealed that E2F4-VHL association was enhanced upon TGFβ3 treatment, indicative of VHL transcriptional inhibition. In line with decreased VHL expression in E-PE, augmented E2F4-VHL association was also observed in E-PE placental tissue relative to controls. In conclusion, we demonstrate for the first time that hypomethylation of VHL DNA at a key transcription factor binding site has significant consequences for its transcriptional repression in early-onset preeclampsia.
    Microphysiological flux balance platform unravels the dynamics of drug induced steatosis
    Avner Ehrlich, Lab on a Chip - 2018
    Abstract
    Drug development is currently hampered by the inability of animal experiments to accurately predict human response. While emerging organ on chip technology offers to reduce risk using microfluidic models of human tissues, the technology still mostly relies on end-point assays and biomarker measurements to assess tissue damage resulting in limited mechanistic information and difficulties to detect adverse effects occurring below the threshold of cellular damage. Here we present a sensor-integrated liver on chip array in which oxygen is monitored using two-frequency phase modulation of tissue-embedded microprobes, while glucose, lactate and temperature are measured in real time using microfluidic electrochemical sensors. Our microphysiological platform permits the calculation of dynamic changes in metabolic fluxes around central carbon metabolism, producing a unique metabolic fingerprint of the liver's response to stimuli. Using our platform, we studied the dynamics of human liver response to the epilepsy drug Valproate (Depakine™) and the antiretroviral medication Stavudine (Zerit™). Using E6/E7LOW hepatocytes, we show TC50 of 2.5 and 0.8 mM, respectively, coupled with a significant induction of steatosis in 2D and 3D cultures. Time to onset analysis showed slow progressive damage starting only 15–20 hours post-exposure. However, flux analysis showed a rapid disruption of metabolic homeostasis occurring below the threshold of cellular damage. While Valproate exposure led to a sustained 15% increase in lipogenesis followed by mitochondrial stress, Stavudine exposure showed only a transient increase in lipogenesis suggesting disruption of β-oxidation. Our data demonstrates the importance of tracking metabolic stress as a predictor of clinical outcome.
    A STAT4 variant increases liver fibrosis risk in Caucasian patients with chronic hepatitis B
    R. El Sharkawy, Alimentary Pharmacology & Therapeutics - 2018
    Abstract
    Background Host genetic modifiers of the natural history of chronic hepatitis B (CHB) remain poorly understood. Recently, a genome-wide association study (GWAS)-identified polymorphism in the STAT4 gene that contributes to the risk for hepatocellular carcinoma (HCC) was shown to be associated with the full spectrum of hepatitis B virus (HBV) outcomes in Asian patients. However, the functional mechanisms for this effect are unknown and the role of the variant in modulating HBV disease in Caucasians has not been investigated. Aims To determine whether STAT4 genetic variation is associated with liver injury in Caucasian patients with CHB and to investigate potential mechanisms mediating this effect. Methods STAT4 rs7574865 was genotyped in 1085 subjects (830 with CHB and 255 healthy controls). STAT4 expression in liver, PBMCs and NK cells, STAT4 phosphorylation and secretion of interferon-gamma (IFN-γ) according to STAT4 genetic variation was examined. Results STAT4 rs7574865 genotype was independently associated with hepatic inflammation (OR: 1.42, 95% CI: 1.07-2.06, P = 0.02) and advanced fibrosis (OR: 1.83, 95% CI: 1.19-2.83, P = 0.006). The minor allele frequency of rs7574865 was significantly lower than that in healthy controls. rs7574865 GG risk carriers expressed lower levels of STAT4 in liver, PBMCs and in NK cells, while NK cells from patients with the risk genotype had impaired STAT4 phosphorylation following stimulation with IL-12/IL-18 and a reduction in secretion of IFN-γ. Conclusion Genetic susceptibility to HBV persistence, hepatic inflammation and fibrosis in Caucasians associates with STAT4 rs7574865 variant. Downstream effects on NK cell function through STAT4 phosphorylation-dependent IFN-γ production likely contribute to these effects.
    Striatal Rgs4 regulates feeding and susceptibility to diet-induced obesity
    Michael Michaelides, Molecular Psychiatry - 2018
    Abstract
    Consumption of high fat, high sugar (western) diets is a major contributor to the current high levels of obesity. Here, we used a multidisciplinary approach to gain insight into the molecular mechanisms underlying susceptibility to diet-induced obesity (DIO). Using positron emission tomography (PET), we identified the dorsal striatum as the brain area most altered in DIO-susceptible rats and molecular studies within this region highlighted regulator of G-protein signaling 4 (Rgs4) within laser-capture micro-dissected striatonigral (SN) and striatopallidal (SP) medium spiny neurons (MSNs) as playing a key role. Rgs4 is a GTPase accelerating enzyme implicated in plasticity mechanisms of SP MSNs, which are known to regulate feeding and disturbances of which are associated with obesity. Compared to DIO-resistant rats, DIO-susceptible rats exhibited increased striatal Rgs4 with mRNA expression levels enriched in SP MSNs. siRNA-mediated knockdown of striatal Rgs4 in DIO-susceptible rats decreased food intake to levels comparable to DIO-resistant animals. Finally, we demonstrated that the human Rgs4 gene locus is associated with increased body weight and obesity susceptibility phenotypes, and that overweight humans exhibit increased striatal Rgs4 protein. Our findings highlight a novel role for involvement of Rgs4 in SP MSNs in feeding and DIO-susceptibility.
    Prenatal stress disrupts social behavior, cortical neurobiology and commensal microbes in adult male offspring
    Tamar L. Gur, Behavioural Brain Research - 2018
    Abstract
    In utero and early neonatal exposure to maternal stress is linked with psychiatric disorders, and the underlying mechanisms are currently being elucidated. We used a prenatal stressor in pregnant mice to examine novel relationships between prenatal stress exposure, changes in the gut microbiome, and social behavior. Here, we show that males exposed to prenatal stress had a significant reduction in social behavior in adulthood, with increased corticosterone release following social interaction. Male offspring exposed to prenatal stress also had neuroinflammation, decreased oxytocin receptor, and decreased serotonin metabolism in their cortex in adulthood, which are linked to decreased social behavior. Finally, we found a significant difference in commensal microbes, including decreases in Bacteroides and Parabacteroides, in adult male offspring exposed to prenatal stress when compared to non-stressed controls. Our findings indicate that gestation is a critical window where maternal stress contributes to the development of aberrant social behaviors and alterations in cortical neurobiology, and that prenatal stress is sufficient to disrupt the male gut-brain axis into adulthood.
    Development of Escherichia coli based gene expression profiling of sewage sludge leachates
    Manish Goswami, Journal of Applied Microbiology - 2018
    Abstract
    Aims The impact of municipal waste on pathogenic microorganisms released into the environment is a public health concern. The present study aims to evaluate the effects of sewage sludge and antibiotic contaminants on stress response, virulence and antibiotic resistance in a pathogenic Escherichia coli. Methods and Results The effects of sewage sludge leachates on uropathogenic E. coli CFT073 were determined by monitoring the expression of 45 genes associated with antibiotic/metal resistance, stress response and virulence using RT-qPCR. The E. coli gene expression was validated using sub-inhibitory concentrations of tetracycline and ciprofloxacin. E. coli exposed to sewage sludge or sewage sludge-fly ash leachates altered the expression of 5 antibiotic and metal resistance, 3 stress response and 2 virulence associated genes. When antibiotics were combined with sludge or sludge-fly ash the antibiotic-associated gene expression was altered. Conclusions E. coli treated with two sludge leachates had distinct gene expression patterns that were altered when the sludge leachates were combined with tetracycline, although to a lesser extent with ciprofloxacin. Significance and Impact of Study The E. coli multigene expression analysis is a potential new tool for assessing the effects of pollutants on pathogenic microbes in environmental waters for improved risk assessment. This article is protected by copyright. All rights reserved.
    High MUC2 Mucin Biosynthesis in Goblet Cells Impedes Restitution and Wound Healing by Elevating Endoplasmic Reticulum Stress and Altered Production of Growth Factors
    Adelaide Tawiah, The American Journal of Pathology - 2018
    Abstract
    Intestinal epithelial cell wound healing involves cell migration, proliferation, and differentiation. Although numerous studies have analyzed the migration of absorptive epithelial cells during wound healing, it remains unclear how goblet cells restitute and how MUC2 mucin production affects this process. In this study, we examined the role of high MUC2 production in goblet cell migration during wound healing and demonstrated that during high MUC2 output, goblet cells migrated slower because of impaired production of wound healing factors and endoplasmic reticulum (ER) stress. Two goblet cell lines, HT29-H and HT29-L, that produced high and low MUC2 mucin, respectively, were used. HT29-L healed wounds faster than HT29-H cells by producing significantly higher amounts of fibroblast growth factor (FGF) 1, FGF2, vascular endothelial growth factor-C, and matrix metallopeptidase 1. Predictably, treatment of HT29-H cells with recombinant FGF2 significantly enhanced migration and wound healing. High MUC2 biosynthesis in HT29-H cells induced ER stress and delayed migration that was abrogated by inhibiting ER stress with tauroursodeoxycholic acid and IL-22. FGF2- and IL-22–induced wound repair was dependent on STAT1 and STAT3 signaling. During wound healing after dextran sulfate sodium–induced colitis, restitution of Math1M1GFP+ goblet cells occurred earlier in the proximal colon, followed by the mid and then distal colon, where ulceration was severe. We conclude that high MUC2 output during colitis impairs goblet cell migration and wound healing by reducing production of growth factors critical in wound repair.
    PTCH1 isoform 1b is the major transcript in the development of basal cell nevus syndrome
    Robbert-Jan C. A. M. Gielen, Journal of Human Genetics - 2018
    Abstract
    Basal cell nevus syndrome (BCNS) is an autosomal dominant disorder most commonly caused by a germline mutation in the PTCH1 gene. PTCH1 is known to have different isoforms with different functional properties and expression patterns among tissues. We detected a novel, pathogenic de novo mutation in PTCH1 isoform 1b (c.114delG) in a BCNS patient. Furthermore, we elucidated the specific expression pattern of PTCH1 isoforms in normal skin, BCC and peripheral blood by studying expression of different PTCH1 isoforms. Human skin showed expression of isoforms 1b and 1d, while peripheral blood additionally showed 1a and 1e expression. BCCs showed expression of all isoforms. Here we report a patient with a novel, isoform 1b specific mutation in PTCH1 and thereby distinguish PTCH1 isoform 1b as the major transcript in the development of BCNS.
    Roflumilast promotes memory recovery and attenuates white matter injury in aged rats subjected to chronic cerebral hypoperfusion
    Amanda Santiago, Neuropharmacology - 2018
    Abstract
    Chronic cerebral hypoperfusion (CCH) has been associated with aging-related vascular dementia, including Alzheimer's disease. It can be induced by the four-vessel occlusion/internal carotid artery (4VO/ICA) model in aged rats, resulting in persistent memory deficits, white matter injury, and significant neuronal loss in the hippocampus and cerebral cortex. The phosphodiesterase type 4 inhibitor (PDE4-I) roflumilast has been reported to have pro-cognitive effects in several behavioral paradigms. The present study evaluated the effects of repeated roflumilast treatment in aged rats that were subjected to CCH. After surgery, roflumilast (0.003 and 0.01 mg/kg) was administered intraperitoneally once per day for 29 days. Memory performance was assessed in the aversive radial maze (AvRM) 7, 14, and 21 days after CCH. The effects of roflumilast on hippocampal neurodegeneration and white matter injury were investigated using Nissl and Kluver-Barrera staining, respectively. Western blot and RT-qPCR were used to explore microglial polarization using M1 (Iba-1 and iNOS) and M2 (Arginase-1) markers. Chronic cerebral hypoperfusion caused persistent memory deficits, hippocampal neurodegeneration, and vacuolization and fiber disarrangement in white matter. Repeated roflumilast treatment restored CCH-induced cognitive impairments in aged rats but in the absence of the rescue of hippocampal neurons. Attenuation of white matter injury was detected in the optic tract in aged CCH rats that were treated with roflumilast. In vitro, roflumilast increased Arg-1 gene expression in myelin-laden primary microglia. The present data suggest that roflumilast might be useful for the treatment of cognitive sequelae associated with CCH.
    The origins of global invasions of the German wasp (Vespula germanica) and its infection with four honey bee viruses
    Evan C. Brenton-Rule, Biological Invasions - 2018
    Abstract
    A successful control or eradication programme using biological control or genetically-mediated methods requires knowledge of the origin and the extent of wasp genetic diversity. Mitochondrial DNA variation in the native and invaded range of the social wasp Vespula germanica was used to examine intra-specific genetic variation and invasive source populations. We also examined wasps for the presence of four viruses found in honey bees: Acute bee paralysis virus, Deformed wing virus, Israeli acute paralysis virus and Kashmir bee virus. German wasps showed reduced genetic diversity in the invaded range compared to that of their native range. Populations in the introduced range are likely to have arrived from different source populations. All four viral honey bee pathogens were found in V. germanica, although they varied in their distribution and strain. Multiple introductions of German wasps have occurred for most invaded regions, though some populations are genetically homogenous. The differing locations of origin will guide researchers searching for biocontrol agents and the reduced genetic diversity may make these wasps a potentially viable target for control via gene drives.
    Novel key roles for Structural maintenance of chromosome flexible domain containing 1 (Smchd1) during preimplantation mouse development
    Uros Midic, Molecular Reproduction and Development - 2018
    Abstract
    Structural maintenance of chromosome flexible domain containing 1 (Smchd1) is a chromatin regulatory gene for which mutations are associated with facioscapulohumeral muscular dystrophy and arhinia. The contribution of oocyte- and zygote-expressed SMCHD1 to early development was examined in mice (Mus musculus) using an siRNA knockdown approach. Smchd1 knockdown compromised long-term embryo viability, with reduced embryo nuclear volumes at the morula stage, reduced blastocyst cell number, formation and hatching, and reduced viability to term. RNAseq analysis of Smchd1 knockdown morulae revealed aberrant increases in expression of a small number of trophectoderm-related genes and reduced expression of cell proliferation genes, including S-phase kinase-associated protein 2 (Skp2). Smchd1 expression was elevated in embryos deficient for Caudal type homeobox transcription factor 2 (Cdx2, a key regulator of trophectoderm specification), indicating that Smchd1 is normally repressed by CDX2. These results indicate that Smchd1 plays an important role in the preimplantation embryo, regulating early gene expression and contributing to long-term embryo viability. These results extend the known functions of SMCHD1 to the preimplantation period and highlight important function for maternally expressed Smchd1 mRNA and protein. This article is protected by copyright. All rights reserved.
    Elevation of the TP53 isoform Δ133p53β in glioblastomas: an alternative to mutant p53 in promoting tumour development
    Marina Kazantseva, The Journal of Pathology - 2018
    Abstract
    As tumour protein 53 (p53) isoforms have tumour promoting, migration and inflammatory properties, this study investigated whether p53 isoforms contributed to glioblastoma progression. The expression levels of full-length TP53α (TAp53α) and six TP53 isoforms were quantitated by RT-qPCR in 89 glioblastomas and correlated with TP53 mutation status, tumour-associated macrophage content and various immune cell markers. Elevated levels of Δ133p53β mRNA characterised glioblastomas with increased CD163-positive macrophages and wild-type TP53. In situ based analyses found Δ133p53β expression localised to malignant cells in areas with increased hypoxia, and in cells with the monocyte chemoattractant protein C-C motif chemokine ligand 2 (CCL2) expressed. Tumours with increased Δ133p53β had increased numbers of cell positive for macrophage colony stimulating factor 1 receptor (CSF1R) and programmed death ligand 1 (PDL1). In addition, cells expressing a murine ‘mimic’ of Δ133p53 (Δ122p53) were resistant to temozolomide treatment and oxidative stress. Our findings suggest elevated Δ133p53β is an alternative pathway to TP53 mutation in glioblastoma that aids tumour progression by promoting an immunosuppressive and chemoresistant environment. Adding Δ133p53β to a TP53 signature along with TP53 mutation status will better predict treatment resistance in glioblastoma. This article is protected by copyright. All rights reserved.
    Isolation and profiling of plasma microRNAs: Biomarkers for asthma and allergic rhinitis
    Ronaldo P. Panganiban, Methods - 2018
    Abstract
    Chronic inflammatory diseases can be particularly challenging to diagnose and characterize, as inflammatory changes in tissue may not be present in blood. There is a crucial need to develop non-invasive biomarkers that would be useful in diagnosing disease and selecting medical therapies. For example, there are no blood tests to diagnose asthma, a common inflammatory lung disease. MicroRNA (miRNA) expression profiling in blood is emerging as a potentially sensitive and useful biomarker of many diseases. In particular, we have characterized a cost-effective PCR-based array technology to measure and profile circulating miRNAs in the plasma of patients with allergic rhinitis and asthma. Here, we describe the methods to isolate, quantify, and analyze miRNAs in the plasma of human subjects as well as ways to determine their diagnostic utility.
    Fabrication of Plant Virus-Based Thin Films to Modulate the Osteogenic Differentiation of Mesenchymal Stem Cells
    Kamolrat Metavarayuth, Virus-Derived Nanoparticles for Advanced Technologies - 2018
    Abstract
    Stem cells can interact and respond to the extracellular nanoscale environment. Viral nanoparticles have been utilized as building blocks to control cell growth and differentiation. By integrating stem cell research and virus nanoparticle chemistry together, a systematic analysis of the effects of nanotopography on stem cell differentiation can be accomplished. The fabrication of thin films of the viral nanoparticles is particularly valuable for such studies. Here, we describe two methods to fabricate plant virus-based thin films and procedures to study the osteogenic differentiation of mesenchymal stem cells on plant virus-based substrates. The method makes use of wild-type tobacco mosaic virus (wt-TMV), RGD-modified TMV (TMV-RGD), turnip yellow mosaic virus (TYMV), cowpea mosaic virus (CPMV), turnip vein clearing virus (TVCV), and potato virus X (PVX) for development of bone tissue engineering biomaterials.
    Antioxidant treatment ameliorates phenotypic features of SMC1A-mutated Cornelia de Lange syndrome in vitro and in vivo
    Dubravka Cukrov, Human Molecular Genetics - 2018
    Abstract
    Cornelia de Lange syndrome (CdLS) is a rare disease characterized by cognitive impairment, multisystemic alterations and premature aging. Furthermore, CdLS cells display gene expression dysregulation and genomic instability. Here, we demonstrated that treatment with antioxidant drugs, such as ascorbic acid and riboceine, reduced the level of genomic instability and extended the in vitro lifespan of CdLS cell lines. We also found that antioxidant treatment partially rescued the phenotype of a zebrafish model of CdLS. Gene expression profiling showed that antioxidant drugs caused dysregulation of gene transcription; notably, a number of genes coding for the zinc finger (ZNF)-containing Krueppel-associated box (KRAB) protein domain (KRAB-ZNF) were found to be downregulated. Taken together, these data suggest that antioxidant drugs have the potential to ameliorate the developmental phenotype of CdLS.
    Gemcitabine resistance mediated by ribonucleotide reductase M2 in lung squamous cell carcinoma is reversed by GW8510 through autophagy induction
    Ping Chen, Clinical Science - 2018
    Abstract
    Although chemotherapeutic regimen containing gemcitabine is the first-line therapy for advanced lung squamous cell carcinoma (LSCC), gemcitabine resistance remains an important clinical problem. Some studies suggest that overexpressions of ribonucleotide reductase subunit M2 (RRM2) may be involved in gemcitabine resistance. We used a novel RRM2 inhibitor, GW8510, as a gemcitabine sensitization agent to investigate the therapeutic utility in reversing gemcitabine resistance in LSCC. Results showed that the expressions of RRM2 were increased in gemcitabine intrinsic resistant LSCC cells upon gemcitabine treatment. GW8510 not only suppressed LSCC cell survival, but also sensitized gemcitabine-resistant cells to gemcitabine through autophagy induction mediated by RRM2 downregulation along with decreases of deoxyribonucleotide triphosphate (dNTP) levels. The combination of GW8510 and gemcitabine produced a synergistic effect on killing LSCC cells. The synergism of the two agents was impeded by addition of autophagy inhibitors chloroquine or bafilomycin A1, or knockdown of the autophagy gene BECN1. Moreover, GW8510-caused LSCC cell sensitization to gemcitabine through autophagy induction was parallel with impairment of DNA double strand break repair and marked increase of cell apoptosis, revealing a crosstalk between autophagy and DNA damage repair, and an interplay between autophagy and apoptosis. Finally, gemcitabine sensitization mediated by autophagy induction through GW8510-caused RRM2 downregulation was demonstrated in vivo gemcitabine-resistant LSCC tumor xenograft, further indicating that the sensitization is dependent on autophagy activation. In conclusions, GW8510 can reverses gemcitabine resistance in LSCC cells through RRM2 downregulation-mediated autophagy induction, and GW850 may be a promising therapeutic agent against LSCC as it combined with gemcitabine.
    Comparative transcriptome analysis of the swimbladder reveals expression signatures in response to low oxygen stress in channel catfish, Ictalurus punctatus.
    Yujia Yang, Physiological Genomics - 2018
    Abstract
    Channel catfish is the leading aquaculture species in the US, and one of the reasons for its application in aquaculture is its relatively high tolerance against hypoxia. However, hypoxia can still cause huge economic losses to the catfish industry. Studies on hypoxia tolerance, therefore, are important for aquaculture. Fish swimbladder has been considered as an accessory respiration organ surrounded by a dense capillary countercurrent exchange system. In this regard, we conducted RNA-Seq analysis with swimbladder samples of catfish under hypoxic and normal conditions to determine if swimbladder was responsive to low oxygen treatment, and to reveal genes, their expression patterns and pathways involved in hypoxia responses in catfish. A total of 155 differentially expressed genes (DEGs) were identified from swimbladder of adult catfish, whereas a total of 2,127 DEGs were identified from swimbladder of fingerling catfish, under hypoxic condition as compared to untreated controls. Subsequent pathway analysis revealed that many DEGs under hypoxia were involved in HIF signaling pathway (nos2, eno2, camk2d2, prkcb, cdkn1a, eno1, and tfrc), MAPK signaling pathway (voltage-dependent calcium channel subunit genes), PI3K/Akt/mTOR signaling pathway (itga6, g6pc, and cdkn1a), Ras signaling pathway (efna3 and ksr2), and signaling by VEGF (fn1, wasf3, and hspb1) in catfish swimbladder. This study provided insights into regulation of gene expression and their involved gene pathways in catfish swimbladder in response to low oxygen stresses.
    Heparanase is expressed in adult human osteoarthritic cartilage and drives catabolic responses in primary chondrocytes
    G. Gibor, Osteoarthritis and Cartilage - 2018
    Abstract
    Summary Objectives The chondrocytes' pericellular matrix acts as a mechanosensor by sequestering growth factors that are bound to heparan sulfate (HS) proteoglycans. Heparanase is the sole mammalian enzyme with HS degrading endoglycosidase activity. Here, we aimed to ascertain whether heparanase plays a role in modulating the anabolic or catabolic responses of human articular chondrocytes. Methods Primary chondrocytes were incubated with pro-heparanase and catabolic and anabolic gene expression was analyzed by quantitative polymerase chain reaction (PCR). MMP13 enzymatic activity in the culture medium was measured with a specific fluorescent assay. Extracellular regulated kinase (ERK) phosphorylation was evaluated by Western blot. Human osteoarthritis (OA) cartilage was assessed for heparanase expression by reverse-transcriptase PCR, by Western blot and by a heparanase enzymatic activity assay. Results Cultured chondrocytes rapidly associated with and activated pro-heparanase. Heparanase induced the catabolic genes MMP13 and ADAMTS4 and the secretion of active MMP13, and down-regulated the anabolic genes ACAN and COL2A1. PG545, a HS-mimetic, inhibited the effects of heparanase. Heparanase expression and enzymatic activity were demonstrated in adult human osteoarthritic cartilage. Heparanase induced ERK phosphorylation in cultured chondrocytes and this could be inhibited by PG545, by fibroblast growth factor 2 (FGF2) neutralizing antibodies and by a FGF-receptor inhibitor. Conclusions Heparanase is active in osteoarthritic cartilage and induces catabolic responses in primary human chondrocytes. This response is due, at least in part, to the release of soluble growth factors such as FGF2.
    Functional characterization of oxazolone-induced colitis and survival improvement by vagus nerve stimulation
    Elisa Meroni, PLOS ONE - 2018
    Abstract
    Background Oxazolone-induced colitis has been frequently used in literature as a model of IBD, but insights into the underlying immune response and pathological features are surprisingly still very limited. Vagus nerve stimulation (VNS) has proven to be effective in innate and Th1/Th17 predominant inflammatory models, including pre-clinical models of colitis, however to what extent VNS is also effective in ameliorating Th2-driven colitis remains to be studied. In the present study, we therefore further characterized the immune response in oxazolone-induced colitis and investigated the potential therapeutic effect of VNS. Methods Colitis was induced in Balb/c mice by cutaneous sensitization with 3% oxazolone followed by intracolonic administration of 1% oxazolone 7 days later. To evaluate the effect of VNS on the development of Th2-driven colitis, VNS and sham-treated mice were challenged with 1% oxazolone. Results Intracolonic oxazolone administration resulted in a severe destruction of the colonic mucosa and a rapid drop in body temperature leading to a 65% mortality rate at day 5. Severe infiltration of neutrophils and monocytes was detected 6h after oxazolone administration which was associated with a Th2-type inflammatory response. VNS significantly improved survival rate which correlated with decreased levels of HMGB1 and reduced colonic (il6 and cxcl1 mRNA) and serum cytokine levels (IL-6, TNFα and CXCL1) compared to sham treated mice. Conclusions Oxazolone-induced colitis rather represents a model of sepsis and, at best, may resemble a severe type of ulcerative colitis, associated with early and severe mucosal damage and a high mortality rate. VNS reduces colonic inflammation and improves survival in this model, supporting the anti-inflammatory properties of VNS, even in an aggressive model as oxazolone-induced colitis.
    Prebiotic effects of white button mushroom (Agaricus bisporus) feeding on succinate and intestinal gluconeogenesis in C57BL/6 mice
    Yuan Tian, Journal of Functional Foods - 2018
    Abstract
    The mechanisms by which white button (WB) mushrooms (Agaricus bisporus) may influence health are unclear. WB feeding (1%) resulted in changes in the composition of microbiota in conventional (CV) mice to expand a population of Prevotella that produce propionate and succinate. Microbial propionate and succinate production induced expression of genes important for intestinal gluconeogenesis (IGN) via the gut-brain neural circuit. Reduced hepatic glucose production was a metabolic benefit of IGN that was found in WB fed CV mice. In the absence of microbiota or in mice with disruptions in the ability to sense microbiota there was no WB mediated effect. WB-fed lean mice had a small but significant improvement in glucose sensitivity. WB feeding resulted in shifts in the microbiota that induced IGN and improved glucose homeostasis.
    Characterization of carfilzomib-resistant non-small cell lung cancer cell lines
    Neale T. Hanke, Journal of Cancer Research and Clinical Oncology - 2018
    Abstract
    PurposeWe previously showed that carfilzomib (CFZ) has potent anti-proliferative and cytotoxic activity in a broad range of lung cancer cell lines. Here we investigate possible mechanisms of CFZ acquired resistance in lung cancer cell lines.MethodsCFZ-resistant non-small cell lung cancer (NSCLC) cell lines were developed by exposing A549 and H520 cells to stepwise increasing concentrations of CFZ. Resistance to CFZ and cross-resistance to bortezomib and other chemotherapy drugs was measured using the MTT assay. Cytotoxicity to CFZ was determined using a CytoTox assay. Western blot was used to measure apoptosis, autophagy, and drug efflux transporter-related proteins. Quantitative targeted whole transcriptome sequencing and quantitative RT-PCR was used to measure gene expression. Flow cytometry was used to analyze intracellular accumulation of doxorubicin.ResultsThe CFZ IC50 value of the resistant cells increased versus parental lines (2.5-fold for A549, 122-fold for H520). Resistant lines showed reduced expression of apoptosis and autophagy markers and reduced death versus parental lines following CFZ treatment. Both resistant lines exhibited higher P-glycoprotein (Pgp) gene (TempO-Seq® analysis, increased 1.2-fold in A549, > 9000-fold in H520) and protein expression levels versus parental lines. TempO-Seq® analysis indicated other drug resistance pathways were upregulated. The resistant cell lines demonstrated less accumulation of intracellular doxorubicin, and were cross-resistant to other Pgp client drugs: bortezomib, doxorubicin, and paclitaxel, but not cisplatin.ConclusionsUpregulation of Pgp appears to be an important, but not the only, mechanism of CFZ resistance in NSCLC cell lines.
    Regulated intratumoral expression of IL-12 using a RheoSwitch Therapeutic System ® (RTS ® ) gene switch as gene therapy for the treatment of glioma
    John A. Barrett, Cancer Gene Therapy - 2018
    Abstract
    The purpose of this study was to determine if localized delivery of IL-12 encoded by a replication-incompetent adenoviral vector engineered to express IL-12 via a RheoSwitch Therapeutic System® (RTS®) gene switch (Ad-RTS-IL-12) administered intratumorally which is inducibly controlled by the oral activator veledimex is an effective approach for glioma therapy. Mice bearing 5–10-day-old intracranial GL-261 gliomas were intratumorally administered Ad-RTS-mIL-12 in which IL-12 protein expression is tightly controlled by the activator ligand, veledimex. Local tumor viral vector levels concomitant with veledimex levels, IL-12-mRNA expression, local and systemic cytokine expression, tumor and systemic flow cytometry and overall survival were studied. Ad-RTS-mIL-12+veledimex elicited a dose-related increase in tumor IL-12 mRNA and IL-12 protein and discontinuation of veledimex resulted in a return to baseline levels. These changes correlated with local immune and antitumor responses. Veledimex crossed the blood–brain barrier in both orthotopic GL-261 mice and cynomolgus monkeys. We have demonstrated that this therapy induced localized controlled production of IL-12 which correlates with an increase in tumor-infiltrating lymphocytes (TILs) leading to the desired biologic response of tumor growth inhibition and regression. At day 85 (study termination), 65% of the animals that received veledimex at 10 or 30 mg/m2/day were alive and tumor free. In contrast, the median survival for the other groups were: vehicle 23 days, bevacizumab 20 days, temozolomide 33 days and anti-PD-1 37 days. These findings suggest that the controlled intratumoral production of IL-12 induces local immune cell infiltration and improved survival in glioma, thereby demonstrating that this novel regulated immunotherapeutic approach may be an effective form of therapy for glioma.
    Effect of green light on nitrate reduction and edible quality of hydroponically grown lettuce (Lactuca sativa L.) under short-term continuous light from red and blue light-emitting diodes
    Zhonghua Bian, Environmental and Experimental Botany - 2018
    Abstract
    Most leafy vegetables can accumulate large amounts of nitrate, which are often associated with harmful effects on human health. Nitrate assimilation in plants is determined by various growth conditions, especially light conditions including light intensity, light duration and light spectral composition. Red and blue light are the most important since both drive photosynthesis. Increasingly, recent evidence demonstrates a role for green light in the regulation of plant growth and development by regulating the expression of some specific genes. However, the effect of green light on nitrate assimilation has been underestimated. In this study, lettuce (Lactuca sativa L. cv. Butterhead) was treated with continuous light (CL) for 48 h by combined red and blue light-emitting diodes (LEDs) supplemented with or without green LED in an environment-controlled growth chamber. The results showed that nitrate reductase (NR) and nitrite reductase (NiR) related-gene expression and nitrate assimilation enzyme activities were affected by light spectral composition and light duration of CL. Adding green light to red and blue light promoted NR and NiR expressions at 24 h, subsequently, it reduced expression of these genes during CL. Compared with red and blue LEDs, green light supplementation significantly increased NR, NiR, glutamate synthase (GOGAT) and glutamine synthetase (GS) activities. Green-light supplementation under red and blue light was more efficient in promoting nutritional values by maintaining high net photosynthetic rates (Pn) and maximal photochemical efficiency (Fv/Fm).
    Myristate-induced endoplasmic reticulum stress requires ceramide synthases 5/6 and generation of C14-ceramide in intestinal epithelial cells
    Songhwa Choi, The FASEB Journal - 2018
    Abstract
    Saturated fatty acids (SFAs) have been shown to induce endoplasmic reticulum (ER) stress and chronic inflammatory responses, as well as alter sphingolipid metabolism. Disruptions in ER stress and sphingolipid metabolism have also been implicated in intestinal inflammation. Therefore, to elucidate the roles of SFAs in ER stress and inflammation in intestinal epithelial cells, we examined myristate (C14:0) and palmitate (C16:0). Myristate, but not palmitate, induced ER stress signaling, including activation of inositol-requiring enzyme 1 (IRE1) and X-box binding protein 1 (XBP1) signaling. Myristate significantly increased C14-ceramide levels, whereas palmitate increased several long-chain ceramides. To define the role of ceramide synthases (CerSs) in myristate-induced ER stress, we used the pharmacologic inhibitor, fumonisin B1 (FB1), and small interfering RNA (siRNA) for CerS5 and 6, the primary isoforms that are involved in C14-ceramide generation. FB1 and siRNA for CerS5 or 6 suppressed myristate-induced C14-ceramide generation and XBP1 splicing (XBP1s). Moreover, increased XBP1s induced the downstream expression of IL-6 in a CerS5/6-dependent manner. In addition, a myristate-enriched milk fat–based diet, but not a lard-based diet, increased C14-ceramide, XBP1s, and IL-6 expression in vivo. Taken together, our data suggest that myristate modulates ER stress and cytokine production in the intestinal epithelium via CerS5/6 and C14-ceramide generation.—Choi, S., Snider, J. M., Olakkengil, N., Lambert, J. M., Anderson, A. K., Ross-Evans, J. S., Cowart, L. A., Snider, A. J. Myristate-induced endoplasmic reticulum stress requires ceramide synthases 5/6 and generation of C14-ceramide in intestinal epithelial cells.
    Poly (ethylene glycol) hydrogel elasticity influences human mesenchymal stem cell behavior
    Anna K. Whitehead, Regenerative Biomaterials - 2018
    Abstract
    Abstract. Coordinated investigations into the interactions between biologically mimicking (biomimetic) material constructs and stem cells advance the potential
    The role of zinc in calprotectin expression in human myeloid cells
    Simone Lienau, Journal of Trace Elements in Medicine and Biology - 2018
    Abstract
    Elevated levels of calprotectin and other inflammatory mediators have been observed in inflammatory diseases paralleling serum hypozincemia. While a role of zinc in the regulation of tumor necrosis factor α, interleukin (IL)-1β and IL-6 expression has been established, the direct interrelation of zinc and calprotectin (S100A8/S100A9 heterodimer) expression is so far missing. In the present study, we analyzed mRNA and protein levels of S100A8 and S100A9 in monocytic Mono Mac (MM)1 and early myeloid THP-1 and U937 cells to elucidate the effect of zinc deficiency on their expression. We could depict that zinc deficiency alone enhances mRNA and protein expression of calprotectin in myeloid cells, independently from maturity stage. Moreover, pre-existing zinc deficiency augmented lipopolysaccharide (LPS)-induced calprotectin expression in CD14+ MM1, but not in CD14− U937 or CD14− THP-1 cells. Zinc deficiency and LPS seem therefore to activate different intracellular pathways. Our findings suggest that zinc does not only regulate the activity of calprotectin but also its expression by human myeloid cells.
    Traditional kefir reduces weight gain and improves plasma and liver lipid profiles more successfully than a commercial equivalent in a mouse model of obesity
    Benjamin C. T. Bourrie, Journal of Functional Foods - 2018
    Abstract
    Kefir, a fermented milk beverage, has shown promise in alleviating obesity and associated metabolic dysfunction. However, microbial characteristics are variable among traditional kefirs, and commercial kefirs drastically differ from traditional kefir. This study investigated the ability of four traditional and one commercial kefir to control weight gain, plasma cholesterol, and liver triglycerides in a high fat diet-induced obesity mouse model. Two traditional kefirs decreased weight gain and plasma cholesterol levels. Conversely, commercial kefir had no beneficial effect. Additionally, one of the four traditional kefirs lowered liver triglycerides, which corresponded with decreases in the expression of fatty acid synthase, a gene involved in liver lipogenesis. Together with evidence of gut microbiome modulation, this study shows that traditional kefir has the potential for improving metabolic dysfunction associated with obesity. Notably, differences in kefir microbial populations may influence the ability of traditional kefir to positively impact host metabolic health.
    Long Noncoding RNAs AC009014.3 and Newly Discovered XPLAID Differentiate Aggressive and Indolent Prostate Cancers
    Anthony J. Cesnik, Translational Oncology - 2018
    Abstract
    The molecular mechanisms underlying aggressive versus indolent disease are not fully understood. Recent research has implicated a class …
    Salmon louse rhabdoviruses: Impact on louse development and transcription of selected Atlantic salmon immune genes
    Aina-Cathrine Øvergård, Developmental & Comparative Immunology - 2018
    Abstract
    Recently, it has been shown that the salmon louse (Lepeophtheirus salmonis) is commonly infected by one or two vertically transmitted Lepeophtheirus salmonis rhabdoviruses (LsRVs). As shown in the present study, the viruses have limited effect on louse survival, developmental rate and fecundity. Since the LsRVs were confirmed to be present in the louse salivary glands, the salmon cutaneous immune response towards LsRV positive and negative lice was analyzed. In general, L. salmonis increased the expression of IL1β, IL8 and IL4/13A at the attachment site, in addition to the non-specific cytotoxic cell receptor protein 1 (NCCRP-1). Interestingly, LsRV free lice induced a higher skin expression of IL1β, IL8, and NCCRP-1 than the LsRV infected lice. The inflammatory response is important for louse clearance, and the present results suggest that the LsRVs can be beneficial for the louse by dampening inflammation. Further research is, however; needed to ascertain whether this is a direct modulatory effect of secreted virions, or if virus replication is altering the level of louse salivary gland proteins.
    Salmon louse rhabdoviruses: Impact on louse development and transcription of selected Atlantic salmon immune genes
    Aina-Cathrine Øvergård, Developmental & Comparative Immunology - 2018
    Abstract
    Recently, it has been shown that the salmon louse (Lepeophtheirus salmonis) is commonly infected by one or two vertically transmitted Lepeophtheirus salmonis rhabdoviruses (LsRVs). As shown in the present study, the viruses have limited effect on louse survival, developmental rate and fecundity. Since the LsRVs were confirmed to be present in the louse salivary glands, the salmon cutaneous immune response towards LsRV positive and negative lice was analyzed. In general, L. salmonis increased the expression of IL1β, IL8 and IL4/13A at the attachment site, in addition to the non-specific cytotoxic cell receptor protein 1 (NCCRP-1). Interestingly, LsRV free lice induced a higher skin expression of IL1β, IL8, and NCCRP-1 than the LsRV infected lice. The inflammatory response is important for louse clearance, and the present results suggest that the LsRVs can be beneficial for the louse by dampening inflammation. Further research is, however; needed to ascertain whether this is a direct modulatory effect of secreted virions, or if virus replication is altering the level of louse salivary gland proteins.
    Chronic Lymphocytic Leukemia–Derived IL-10 Suppresses Antitumor Immunity
    Sara S. Alhakeem, The Journal of Immunology - 2018
    Abstract
    Chronic lymphocytic leukemia (CLL) patients progressively develop an immunosuppressive state. CLL patients have more plasma IL-10, an anti-inflammatory cytokine, than healthy controls. In vitro human CLL cells produce IL-10 in response to BCR cross-linking. We used the transgenic Eμ–T cell leukemia oncogene-1 (TCL1) mouse CLL model to study the role of IL-10 in CLL associated immunosuppression. Eμ-TCL mice spontaneously develop CLL because of a B cell–specific expression of the oncogene, TCL1. Eμ-TCL1 mouse CLL cells constitutively produce IL-10, which is further enhanced by BCR cross-linking, CLL-derived IL-10 did not directly affect survival of murine or human CLL cells in vitro. We tested the hypothesis that the CLL-derived IL-10 has a critical role in CLL disease in part by suppressing the host immune response to the CLL cells. In IL-10R−/− mice, wherein the host immune cells are unresponsive to IL-10–mediated suppressive effects, there was a significant reduction in CLL cell growth compared with wild type mice. IL-10 reduced the generation of effector CD4 and CD8 T cells. We also found that activation of BCR signaling regulated the production of IL-10 by both murine and human CLL cells. We identified the transcription factor, Sp1, as a novel regulator of IL-10 production by CLL cells and that it is regulated by BCR signaling via the Syk/MAPK pathway. Our results suggest that incorporation of IL-10 blocking agents may enhance current therapeutic regimens for CLL by potentiating host antitumor immune response.
    Cross-talk between androgen and Wnt signaling potentially contributes to age-related skeletal muscle atrophy in rats
    Petey W. Mumford, Journal of Applied Physiology - 2018
    Abstract
    We sought to determine whether age-related gastrocnemius muscle mass loss was associated with parallel decrements in androgen receptor (AR) or select Wnt signaling markers. To test this hypothesis, serum free and total testosterone (TEST) as well as gastrocnemius AR and Wnt signaling markers were analyzed in male Fischer 344 rats that were 3/6/12/18 and 24 months (mo) old (n=9 per group). Free and total TEST were greatest in 6 mo rats, and AR protein and Wnt5 protein levels linearly declined with aging. There were associations between Wnt5 protein levels and relative gastrocnemius mass (r=0.395, p=0.007) as well as AR and Wnt5 protein levels (r=0.670, p<0.001). We next tested the hypothesis that Wnt5 affects muscle fiber size by treating C2C12-derived myotubes lower (75 ng/mL) and higher (150 ng/mL) concentrations of recombinant Wnt5a protein. Both treatments increased myotube size (p<0.05) suggesting this ligand may affect muscle fiber size in vivo. We next tested if Wnt5a protein levels were androgen-modulated by examining 10 mo old male Fischer 344 rats (n=10-11 per group) that were orchiectomized and treated with testosterone-enanthate (TEST-E), trenbolone enanthate (TREN), a non-aromatizable synthetic testosterone analogue, or a vehicle (ORX only) for 4 weeks. Interestingly, TEST-E and TREN treatments increased Wnt5a protein in the androgen-sensitive levator ani/bulbocavernosus (LABC) muscle compared ORX only (p<0.05). To summarize, aromatizable and non-aromatizable androgens increase Wnt5a protein expression in skeletal muscle, age-related decrements in muscle AR may contribute Wnt5a protein decrements, and our in vitro data imply this mechanism may contribute to age-related muscle loss.
    Distinct roles for REV-ERBα and REV-ERBβ in oxidative capacity and mitochondrial biogenesis in skeletal muscle
    Ariadna Amador, PLOS ONE - 2018
    Abstract
    The nuclear receptors REV-ERBα and REV-ERBβ have been demonstrated to be core members of the circadian clock and participate in the regulation of a diverse set of metabolic functions. Due to their overlapping tissue expression patterns and gene expression profiles, REV-ERBβ is thought to be redundant to REV-ERBα. Recent work has highlighted REV-ERBα’s role in the regulation of skeletal muscle oxidative capacity and mitochondrial biogenesis. Considering the similarity between the REV-ERBs and the hypothesized overlap in function, we sought to determine whether REV-ERBβ-deficiency presented with a similar skeletal muscle phenotype as REV-ERBα-deficiency. Ectopic overexpression in C2C12 cells demonstrated that REV-ERBβ drives mitochondrial biogenesis and the expression of genes involved in fatty acid oxidation. Intriguingly, knock down of REV-ERBβ in C2C12 cultures also resulted in mitochondrial biogenesis and increased expression of genes involved in fatty acid β-oxidation. To determine whether these effects occurred in vivo, we examined REV-ERBβ-deficient mice and observed a similar increase in expression of genes involved in mitochondrial biogenesis and fatty acid β-oxidation. Consistent with these results, REV-ERBβ-deficient mice exhibited an altered metabolic phenotype compared to wild-type littermate controls when measured by indirect calorimetry. This likely compensated for the increased food consumption that occurred, possibly aiding in the maintenance of their weight over time. Since feeding behaviors are a direct circadian output, this study suggests that REV-ERBβ may have more subtle effects on circadian behaviors than originally identified. Furthermore, these data implicate REV-ERBβ in the control of skeletal muscle metabolism and energy expenditure and suggest that development of REV-ERBα versus REV-ERBβ selective ligands may have therapeutic utility in the treatment of metabolic syndrome.
    Cutting Edge: The Histone Methyltransferase G9a Is Required for Silencing of Helper T Lineage–Associated Genes in Proliferating CD8 T Cells
    Daniel J. Verbaro, The Journal of Immunology - 2018
    Abstract
    Helper versus cytotoxic T lineage decision in the thymus has been studied as a model for silencing of alternative lineage genes. Although the transcription factor RUNX3 is required for the initiation of Cd4 silencing in developing CD8 T cells, it is unknown how silencing of Cd4 and other helper T lineage genes is maintained. We show that the histone methyltransferase G9a is necessary for silencing helper T lineage genes in proliferating mouse CD8 T cells. Despite normal initial Cd4 downregulation, G9a-deficient CD8 T cells derepress Cd4 and other helper lineage genes during repeated division in lymphopenia or in response to tumor Ag. However, G9a was dispensable for continued silencing of those genes in CD8 T cells that respond to infection by Listeria monocytogenes. These results demonstrate that G9a facilitates maintenance of cellular identity of CD8 T cells during cell division, which is further reinforced by inflammatory signals.
    Obesity in mares promotes uterine inflammation and alters embryo lipid fingerprints and homeostasis
    Dawn R. Sessions-Bresnahan, Biology of Reproduction - 2018
    Abstract
    Abstract. Maternal body composition can be an important determinant for development of obesity and metabolic syndrome in adult offspring. Obesity-related outco
    Estrogens and selective estrogen receptor modulators differentially antagonize Runx2 in ST2 mesenchymal progenitor cells
    Yonatan Amzaleg, The Journal of Steroid Biochemistry and Molecular Biology - 2018
    Abstract
    Estrogens attenuate bone turnover by inhibiting both osteoclasts and osteoblasts, in part through antagonizing Runx2. Apparently conflicting, stimulatory effects in osteoblast lineage cells, however, sway the balance between bone resorption and bone formation in favor of the latter. Consistent with this dualism, 17ß-estradiol (E2) both stimulates and inhibits Runx2 in a locus-specific manner, and here we provide evidence for such locus-specific regulation of Runx2 by E2 in vivo. We also demonstrate dual, negative and positive, regulation of Runx2-driven alkaline phosphatase (ALP) activity by increasing E2 concentrations in ST2 osteoblast progenitor cells. We further compared the effects of E2 to those of the Selective Estrogen Receptor Modulators (SERMs) raloxifene (ral) and lasofoxifene (las) and the phytoestrogen puerarin. We found that E2 at the physiological concentrations of 0.1-1 nM, as well as ral and las, but not puerarin, antagonize Runx2-driven ALP activity. At ≥10 nM, E2 and puerarin, but not ral or las, stimulate ALP relative to the activity measured at 0.1-1 nM. Contrasting the difference between E2 and SERMs in ST2 cells, they all shared a similar dose-response profile when inhibiting pre-osteoclast proliferation. That ral and las poorly mimic the locus- and concentration-dependent effects of E2 in mesenchymal progenitor cells may help explain their limited clinical efficacy.
    Gastrointestinal microbiota and mucosal immune gene expression in neonatal pigs reared in a cross-fostering model
    Nidia Maradiaga, Microbial Pathogenesis - 2018
    Abstract
    Cross fostering is employed to equalize the number of piglet between litters ensuring colostrum intake for their survival and growth. However, little is known about the impact of cross fostering on the intestinal microbiota and mucosal immune gene expression of the neonatal pig. The objective of this study was to determine the influence of maternal microbial communities on the gastrointestinal (GI) microbiota and mucosal immune gene expression in young pigs reared in a cross-fostering model. Piglets were given high quality colostrum from birth dam or foster dam upon birth. Twenty-four piglets were randomly assigned at birth to 1 of 3 treatments according to colostrum source and postcolostral milk feeding during, as follow: treatment 1 (n = 8), received colostrum and post-colostral milk feeding from their own dam; treatment 2 (n = 8), received colostrum from foster dam and returned to their own dam for post-colostral milk feeding; and treatment 3 (n = 8), received colostrum and post-colostral milk feeding from foster dam. Genomic DNA was extracted, and the V1-V3 hypervariable region of the bacterial 16S rRNA gene was amplified and sequenced using the Illumina MiSeq platform. Quantitative real-time PCR analysis was also performed to quantify the expression of toll-like receptors (TLR) 2, TLR 4, TLR 10, tumor necrosis factor alpha (TNFα), interferon gamma (IFNγ), and interleukin (IL) 4 and IL 10. Data analysis revealed that microbial communities were varied according to the GI biogeographical location, with colon being the most diverse section. Bacterial communities in both maternal colostrum and vaginal samples were significantly associated with those present in the fecal samples of piglets. Cross-fostering did not affect bacterial communities present in the piglet GI tract. However, the mRNA expression of TLR and inflammatory cytokines changed (P < 0.05) with biogeographical location in the GI tract. Higher mRNA expression of TLR and inflammatory cytokines was observed in ileum and ileum associated lymph tissues. This study suggests an impact of colostrum and maternal microbial communities on the microbiota development and mucosal immune gene expression in the newly born piglet. This study revealed novel information about the distribution and expression patterns of TLR and inflammatory cytokines in the GI tract of the young pig. Future studies are needed to determine the role and clinical importance of the mucosal microbiota and mucosal gene expression in health, productivity, and susceptibility to the development of GI disease, in piglets.
    Cyanidin Curtails Renal Cell Carcinoma Tumorigenesis
    Xiaobing Liu, Cellular Physiology and Biochemistry - 2018
    Abstract
    Cyanidin is an anthocyanin found in many foods. Although its variable antioxidant levels are well-documented, little is known about its effects on renal cell carcinoma (RCC) tumorigenesis. This study, therefore, investigated the effects of cyanidin on the proliferation, migration, and invasion of renal cell carcinoma lines and demonstrated, for the first time, significant inhibitory effects of cyanidin on RCC tumorigenesis. Methods: RCC cells were treated with different doses of cyanidin and the effects were tested by Cell Counting Kit-8 reagent, clone formation assay, transwell assay, and flow cytometry. Moreover, the cyanidin-mediated mechanism that curtailed tumorigenesis was analyzed by RNA sequencing (RNA-seq). Sequencing data from The Cancer Genome Atlas (TCGA) were used to compare the expression of both early growth response protein 1 (EGR1) and selenoprotein W (SEPW1) in RCC and tumor-free adjacent normal tissue samples. Real-time PCR (RT-PCR) and/or western blot were used to assess the expression of E-cadherin, cleaved-caspase3, Bcl2, p62, and ATG4. Results: We found significantly greater induction of cell-cycle arrest, apoptosis, and suppression of RCC cell invasion and migration at concentrations of 25 µM and 100 µM than at a concentration of 50 µM. It was also discovered, first through RNA-seq then confirmed by RT-PCR, that cyanidin (100 µM) inhibited RCC carcinogenesis through EGR1 and SEPW1. TCGA data indicated that the expression level of EGR1 was lower and that of SEPW1 was higher in RCC tumor tissue than in normal tissues. Moreover, western blot and/or RT-PCR indicated that cleaved-caspase3 was enhanced and E-cadherin was inhibited by cyanidin treatment. Furthermore, western blot and RT-PCR also showed regulation of p62 and ATG4, which are associated with autophagy. Cyanidin in vivo significantly inhibited the growth of xenografts in nude mice. Conclusions: The results of this study showed the therapeutic potential of cyanidin for the treatment of RCC and the prevention of recurrence and metastasis.
    Low-dose irradiated mesenchymal stromal cells break tumor defensive properties in vivo
    Francesca Romana Stefani, International Journal of Cancer - 2018
    Abstract
    Solid tumors, including gliomas, still represent a challenge to clinicians and first line treatments often fail, calling for new paradigms in cancer therapy. Novel strategies to overcome tumor resistance are mainly represented by multi-targeted approaches, and cell vector-based therapy is one of the most promising treatment modalities under development. Here, we show that mouse bone marrow-derived mesenchymal stromal cells (MSCs), when primed with low-dose irradiation (irMSCs), undergo changes in their immunogenic and angiogenic capacity and acquire anti-tumoral properties in a mouse model of glioblastoma (GBM). Following grafting in GL261 glioblastoma, irMSCs migrate extensively and selectively within the tumor and infiltrate predominantly the perivascular niche, leading to rejection of established tumors and cure in 29% of animals. The therapeutic radiation dose window is narrow, with effects seen between 2 and 15 Gy, peaking at 5 Gy. A single low-dose radiation decreases MSCs inherent immune suppressive properties in vitro as well as shapes their immune regulatory ability in vivo. Intra-tumorally grafted irMSCs stimulate the immune system and decrease immune suppression. Additionally, irMSCs enhance peri-tumoral reactive astrocytosis and display anti-angiogenic properties. Hence, the present study provides strong evidence for a therapeutic potential of low-dose irMSCs in cancer as well as giving new insight into MSC biology and applications. This article is protected by copyright. All rights reserved.
    Targeted Gene Knock Out Using Nuclease-Assisted Vector Integration: Hemi- and Homozygous Deletion of JAG1
    Michael Gapinske, Synthetic Biology - 2018
    Abstract
    Gene editing technologies are revolutionizing fields such as biomedicine and biotechnology by providing a simple means to manipulate the genetic makeup of essentially any organism. Gene editing tools function by introducing double-stranded breaks at targeted sites within the genome, which the host cells repair preferentially by Non-Homologous End Joining. While the technologies to introduce double-stranded breaks have been extensively optimized, this progress has not been matched by the development of methods to integrate heterologous DNA at the target sites or techniques to detect and isolate cells that harbor the desired modification. We present here a technique for rapid introduction of vectors at target sites in the genome that enables efficient isolation of successfully edited cells.
    Triggering the activation of Activin A type II receptor in human adipose stem cells towards tenogenic commitment using mechanomagnetic stimulation
    A. I. Gonçalves, Nanomedicine: Nanotechnology, Biology and Medicine - 2018
    Abstract
    Stem cell therapies hold potential to stimulate tendon regeneration and homeostasis, which is maintained in response to the native mechanical environment. Activins are members of the mechano-responsive TGF-β superfamily that participates in the regulation of several downstream biological processes. Mechanosensitive membrane receptors such as activin can be activated in different types of stem cells via magnetic nanoparticles (MNPs) through remote magnetic actuation resulting in cell differentiation. In this work, we target the Activin receptor type IIA (ActRIIA) in human adipose stem cells (hASCs), using anti-ActRIIA functionalized MNPs, externally activated through a oscillating magnetic bioreactor. Upon activation, the phosphorylation of Smad2/3 is induced allowing translocation of the complex to the nucleus, regulating tenogenic transcriptional responses. Our study demonstrates the potential remote activation of MNPs tagged hASCs to trigger the Activin receptor leading to tenogenic differentiation. These results may provide insights toward tendon regeneration therapies.
    Insecticide toxicity associated with detoxification enzymes and genes related to transcription of cuticular melanization among color morphs of Asian citrus psyllid
    Xue Dong Chen, Insect Science - 2018
    Abstract
    The Asian citrus psyllid (Diaphorina citri Kuwayama) is known to exhibit abdominal color polymorphisms. In the current study, susceptibility to four insecticides was compared among orange/yellow, blue/green and gray/brown color morphs of field collected D. citri. The LD50 values and 95% fiducial limits were quantified for each insecticide and color morph combination and ranged between 0.10 ng/μL (0.06–0.10) and 6.16 ng/μL (3.30–12.50). Second, we measured the detoxification enzyme activity levels of orange/yellow, blue/green and gray/brown color morphs for cytochrome P450, glutathione S-transferase, and general esterase. The mean P450 activity (equivalent units) was significantly lower in gray/brown (0.152 ± 0.006) and blue/green morphs (0.149 ± 0.005) than in the orange/yellow morphs (0.179 ± 0.008). GST activity (μmol/min/mg protein) was significantly lower in the orange/yellow morph (299.70 ±1.24) than gray/brown (350.86 ± 1.19) and blue/green (412.25 ± 1.37) morphs. The mean EST activity (μmol/min/mg protein) was significantly higher in blue/green (416.72 ± 5.12) and gray/brown morphs (362.19 ± 4.69) than in the orange/yellow morphs (282.56 ± 2.93). Additionally, we analyzed the relative expression of assortment genes involved in cuticular melanization and basal immunity. The transcripts of Dopa Decarboxylase and Tyrosine Hydroxylase were expressed higher in blue/green and gray/brown than orange/yellow morphs. The transcription results paralleled the susceptibility of D. citri to organophosphate, neonicotinoid and pyrethroid insecticides. GST and EST activity may also be correlated with low levels of insecticide susceptibility. Cuticular melanization could be a factor for the development of resistance to insecticides among different color morphs.
    Nrf2 deletion from adipocytes, but not hepatocytes, potentiates systemic metabolic dysfunction after long-term high-fat diet-induced obesity in mice
    Dionysios V Chartoumpekis, American Journal of Physiology-Endocrinology and Metabolism - 2018
    Abstract
    Nrf2 is a canonical regulator of cytoprotective gene expression but evidence of its crosstalk with other pathways, including metabolic ones, is ever increasing. Pharmacologic or systemic genetic activation of the Nrf2 pathway partially protects from obesity in mice and ameliorates fasting hyperglycemia in mice and humans. However, systemic Nrf2 deletion also protected from diet-induced obesity and insulin resistance in mice. To further investigate the effect of the disruption of Nrf2 on obesity in a tissue-specific manner, we focused on adipocytes and hepatocytes with targeted deletion of Nrf2. To this end, mice with cell-specific deletion of Nrf2 in adipocytes (ANKO) or hepatocytes (HeNKO) were fed high-fat diet (HFD) for 6 months and showed similar increases in body weight and body fat content. ANKO mice showed a partially deteriorated glucose tolerance, higher fasting glucose levels and higher levels of cholesterol, and non-esterified fatty acids compared to their control counterparts. The HeNKO mice though, had lower insulin levels and trended towards improved insulin sensitivity without having any difference in liver triglyceride accumulation. This study compared for the first time two conditional Nrf2 knock-out models in adipocytes and in hepatocytes during HFD-induced obesity. None of these models could completely recapitulate the unexpected protection against obesity observed in the whole body Nrf2 knock-out mice but this study points out the differential roles that Nrf2 may play, beyond cytoprotection, in different target tissues and rather suggests systemic activation of the Nrf2 pathway as an effective means of prevention and treatment of obesity and type 2 diabetes.
    Study of the beneficial effects of green light on lettuce grown under short-term continuous red and blue light-emitting diodes
    Zhonghua Bian, Physiologia Plantarum - 2018
    Abstract
    Red and blue light are the most important light spectra for driving photosynthesis to produce adequate crop yield. It is also believed that green light may contribute to adaptations to growth. However, the effects of green light, which can trigger specific and necessary responses of plant growth, have been underestimated in the past. In this study, lettuce (Lactuca sativa L.) was exposed to different continuous light (CL) conditions for 48 h by a combination of red and blue light-emitting diodes (LEDs) supplemented with or without green LEDs, in an environmental-controlled growth chamber. Green light supplementation enhanced photosynthetic capacity by increasing net photosynthetic rates (Pn), maximal photochemical efficiency (Fv/Fm), electron transport for carbon fixation (JPSII) and chlorophyll content in plants under the CL treatment. Green light decreased malondialdehyde and H2O2 accumulation by increasing the activities of superoxide dismutase (SOD; EC 1.15.1.1) and ascorbate peroxidase (APX; EC 1.11.1.11) after 24 h of CL. Supplemental green light significantly increased the expression of photosynthetic genes LHCb and PsbA from 6 to 12 h, and these gene expression were maintained at higher levels than those under other light conditions between 12 and 24 h. However, a notable down-regulation of both LHCb and PsbA was observed during 24 to 48 h. These results indicate that the effects of green light on lettuce plant growth, via enhancing activity of particular components of antioxidantive enzyme system and promoting of LHCb and PsbA expression to maintain higher photosynthetic capacity, alleviated a number of the negative effects caused by CL.
    Morphine-potentiated cognitive deficits correlate to suppressed hippocampal iNOS RNA expression and an absent type 1 interferon response in LP-BM5 murine AIDS
    Virginia D. McLane, Journal of Neuroimmunology - 2018
    Abstract
    Opioid use accelerates neurocognitive impairment in HIV/AIDS patients. We assessed the effect of chronic morphine treatment and LP-BM5/murine AIDS (MAIDS) infection on cognition, cytokine production, and type 1 interferon (IFN) expression in the murine CNS. Morphine treatment decreased expression of pro-inflammatory factors (CCL5, iNOS) and reduced cognitive performance in LP-BM5-infected mice, correlating to increased hippocampal viral load and a blunted type 1 IFN response. In the striatum, morphine reduced viral load while increasing IFN-α RNA expression. Our results suggest that differentially regulated type 1 IFN responses may contribute to distinct regional outcomes in the hippocampus and striatum in LP-BM5/MAIDS.
    Liver damage, proliferation, and progenitor cell markers in experimental necrotizing enterocolitis
    Hiromu Miyake, Journal of Pediatric Surgery - 2018
    Abstract
    Background Necrotizing enterocolitis (NEC) is a disease known to cause injury to multiple organs including the liver. Liver regeneration is essential for the recovery after NEC-induced liver injury. Our aim was to investigate hepatic proliferation and progenitor cell marker expression in experimental NEC. Methods Following ethical approval (#32238), NEC was induced in mice by hypoxia, gavage feeding of hyperosmolar formula, and lipopolysaccharide. Breastfed pups were used as control. We analyzed serum ALT level, liver inflammatory cytokines, liver proliferation markers, and progenitor cell marker expression. Comparison was made between NEC and controls. Results Serum ALT level was higher in NEC (p<0.05). The mRNA expression of inflammatory cytokines in the liver was also higher in NEC (IL6: p<0.05, TNF-α: p<0.01). Conversely, mRNA expression of proliferation markers in the liver was lower in NEC (Ki67; p<0.01, PCNA: p<0.01). LGR5 expression was also significantly decreased in NEC as demonstrated by mRNA (p<0.05) and protein (p<0.01) levels. Conclusions Inflammatory injury was present in the liver during experimental NEC. Proliferation and LGR5 expression were impaired in the NEC liver. Modulation of progenitor cell expressing LGR5 may result in stimulation of liver regeneration in NEC-induced liver injury and improved clinical outcome. Level of evidence Level IV.
    Improved apple latent spherical virus-induced gene silencing in multiple soybean genotypes through direct inoculation of agro-infiltrated Nicotiana benthamiana extract
    C. R. Gedling, Plant Methods - 2018
    Abstract
    Virus induced gene silencing (VIGS) is a powerful genomics tool for interrogating the function of plant genes. Unfortunately, VIGS vectors often produce disease symptoms that interfere with the silencing phenotypes of target genes, or are frequently ineffective in certain plant genotypes or tissue types. This is especially true in crop plants like soybean [Glycine max (L.) Merr]. To address these shortcomings, we modified the inoculation procedure of a VIGS vector based on Apple latent spherical virus (ALSV). The efficacy of this new procedure was assessed in 19 soybean genotypes using a soybean Phytoene desaturase (GmPDS1) gene as the VIGS target. Silencing of GmPDS1 was easily scored as photo-bleached leaves and/or stems.
    Is 8% O2 more normoxic than 21% O2 for long-term in vitro cultures of human primary term cytotrophoblasts?
    C. L. Depoix, MHR: Basic science of reproductive medicine - 2018
    Abstract
    STUDY QUESTIONIs 8% O2 a better percentage of atmospheric oxygen for long-term cultures of human primary term cytotrophoblasts than the conventional 21% O2 traditionally used in cell culture?SUMMARY ANSWERHuman primary term cytotrophoblasts are able to differentiate into syncytiotrophoblasts under both atmospheric oxygen levels.WHAT IS KNOWN ALREADYCell culture is traditionally done under 21% O2, which is equal to a pO2 of ~160 mm Hg. Based on the pO2 measured after instauration of the blood circulation within the placenta, it has been proposed that cytotrophoblasts culture should be under 8% O2, which is equivalent to 60 mm Hg, and that this percentage should be considered as the physiological normoxia for cytotrophoblasts.STUDY, DESIGN, SIZE, DURATIONCytotrophoblasts were isolated and purified from human term placentas (n > 4). Cells were cultured under 21% O2 and 8% O2 for 12 days. Several cellular parameters were assessed on Days 2, 4, 8 and 12.PARTICIPANTS/MATERIALS, SETTING, METHODSPlacentas were obtained after vaginal or elective cesarean delivery from uncomplicated pregnancies at term (n ≥ 4). Cell viability was measured by a luminescent assay based on quantitation of the ATP content of living cells. Cell fusion was assessed by quantification of syncytin and e-cadherin mRNA expression by real-time PCR and determination of the fusion index by immunofluorescent microscopy. Trophoblast differentiation was assessed by measuring the expression levels of hCGβ, inhibin α subunit (InhA) and placental growth factor (PlGF) by real-time PCR and ELISA. Finally, the effect of the two oxygen levels on apoptosis and cellular oxidative stress was also investigated by quantifying caspase 3/7 activation, superoxide dismutase 1 (SOD-1) mRNA expression and H2O2 generation.MAIN RESULTS AND THE ROLE OF CHANCEThere was no difference between 21% O2 and 8% O2 on cell viability. Cell fusion seemed to be enhanced during the first 4 days when the cells were cultured under 21% O2 compared to 8% O2. The expression level of hCGβ was equivalent in both oxygen conditions, indicating that there was no difference in trophoblast differentiation. Interestingly, InhA expression was higher under 8% O2, while PlGF expression was inhibited compared to 21% O2. This latter result indicates that 8% O2 may be more hypoxic than normoxic for in vitro culture of primary term cytotrophoblast. This is further corroborated by the fact that 21% O2 did not significantly increase caspase 3/7 activities and the oxidative stress (SOD-1 mRNA expression and H2O2 generation) in our cell cultures.LARGE SCALE DATANot applicable.LIMITATIONS, REASONS FOR CAUTIONThe in vitro culture of cytotrophoblasts is artificial and does not reflect the in vivo situation. The cell population is nearly 100% pure, cultured as a monolayer, and the cells bath in a chemically defined culture medium deprived of any oxygen carrier. The oxygen molecules available to the cells are passively dissolved in the medium. The gas dissolution properties of the medium and the cellular consumption rate of oxygen may allow the cells to sustain a wide range of oxygen percentages from 8% to 21%.WIDER IMPLICATIONS OF THE FINDINGSIt is possible to culture human primary term cytotrophoblasts for at least 12 days. The O2 percentage of the air does not negatively affect in vitro cytotrophoblast differentiation. For in vitro culture of cytotrophoblasts, it is not necessary to lower the percentage of atmospheric oxygen to 8%.STUDY FUNDING/COMPETING INTEREST(S)This work was fully supported by ‘Fetus for Life’ charity. The authors state that there is no conflict of interest to declare regarding the publication of this paper.
    Redundant regulation of localization and protein stability of DmPar3
    Lars Kullmann, Cellular and Molecular Life Sciences - 2018
    Abstract
    Apical–basal polarity is an important characteristic of epithelia and Drosophila neural stem cells. The conserved Par complex, which consists of the atypical protein kinase C and the scaffold proteins Baz and Par6, is a key player in the establishment of apical–basal cell polarity. Membrane recruitment of Baz has been reported to be accomplished by several mechanisms, which might function in redundancy, to ensure the correct localization of the complex. However, none of the described interactions was sufficient to displace the protein from the apical junctions. Here, we dissected the role of the oligomerization domain and the lipid-binding motif of Baz in vivo in the Drosophila embryo. We found that these domains function in redundancy to ensure the apical junctional localization of Baz: inactivation of only one domain is not sufficient to disrupt the function of Baz during apical–basal polarization of epithelial cells and neural stem cells. In contrast, mutation of both domains results in a strongly impaired protein stability and a phenotype characterized by embryonic lethality and an impaired apical–basal polarity in the embryonic epithelium and neural stem cells, resembling a baz-loss of function allele. Strikingly, the binding of Baz to the transmembrane proteins E-Cadherin, Echinoid, and Starry Night was not affected in this mutant protein. Our findings reveal a redundant function of the oligomerization and the lipid-binding domain, which is required for protein stability, correct subcellular localization, and apical–basal cell polarization.
    Short-term effect of FSH on gene expression in bovine granulosa cells in vitro
    Anne-Laure Nivet, Reproduction, Fertility and Development - 2018
    Abstract
    In reproduction, FSH is one of the most important hormones, especially in females, because it controls the number of follicles and the rate of follicular growth. Although several studies have examined the follicular response at the transcriptome level, it is difficult to obtain a clear and complete picture of the genes responding to an increase in FSH in an in vivo context because follicles undergo rapid morphological and physical changes during their growth. To help define the transcriptome downstream response to FSH, an in vitro model was used in the present study to observe the short-term (4 h) cellular response. Gene expression analysis highlighted a set of novel transcripts that had not been reported previously as being part of the FSH response. Moreover, the results of the present study indicate that the epithelial to mesenchymal transition pathway is inhibited by short-term FSH stimuli, maintaining follicles in a growth phase and preventing differentiation. Modulating gene expression in vitro has physiological limitations, but it can help assess the potential downstream response and begin the mapping of the granulosa cell transcriptome in relation to FSH. This information is a key feature to help discriminate between the effects of FSH and LH, or to elucidate the overlapping of insulin-like growth factor 1 and FSH in the granulosa mitogenic response.
    Effect of metformin on estrogen and progesterone receptor-positive (MCF-7) and triple-negative (MDA-MB-231) breast cancer cells
    Inês Amaral, Biomedicine & Pharmacotherapy - 2018
    Abstract
    This work aimed to investigate the effect of metformin on cellular glucose uptake and metabolism by breast cancer cells, as a mechanism contributing to its anticancer properties. Estrogen and progesterone receptor-positive (MCF-7) and triple-negative (MDA-MB-231) breast cancer cell lines were used as in vitro models of breast cancer. Short-term (26 min) exposure of MCF-7 and MDA-MB-231 cells to metformin inhibited uptake of 3H-deoxy-D-glucose (3H-DG). In contrast, long-term (24 h) exposure to metformin (5 μM–1 mM) concentration-dependently increased 3H-DG uptake in both cell lines. This effect was associated with an increase in lactate production but was not associated with changes in GLUT1 mRNA expression. Long-term exposure of MCF-7 and MDA-MB-231 cells to metformin (5 μM–1 mM) concentration-dependently reduced cell viability and culture mass and slightly increased cell proliferation rates. Combination of metformin (1 mM) with the facilitative glucose transporter (GLUT) inhibitor kaempferol (30 μM) did not change the effect of metformin on culture growth. In conclusion, short-term exposure to metformin reduces cellular glucose uptake, probably by direct inhibition of GLUT1. However, after long-term exposure to metformin, cellular uptake of glucose is significantly increased, not associated to changes in GLUT1 transcription rates. We suggest that, in the long-term, metformin induces a compensatory increase in glucose uptake in response to cellular energy depletion resulting from its inhibitory effect on mitochondrial oxidative phosphorylation machinery. Metformin-induced dependence of breast cancer cells on glycolytic pathway, associated with an anticarcinogenic effect of the drug, provides a biochemical basis for the design of new therapeutic strategies.
    Hydroxychavicol, a key ingredient of Piper betle induces bacterial cell death by DNA damage and inhibition of cell division
    Deepti Singh, Free Radical Biology and Medicine - 2018
    Abstract
    Antibiotic resistance is a global problem and there is an urgent need to augment the arsenal against pathogenic bacteria. The emergence of different drug resistant bacteria is threatening human lives to be pushed towards the pre-antibiotic era. Botanical sources remain a vital source of diverse organic molecules that possess antibacterial property as well as augment existing antibacterial molecules. Piper betle, a climber, is widely used in south and south-east Asia whose leaves and nuts are consumed regularly. Hydroxychavicol (HC) isolated from Piper betle has been reported to possess antibacterial activity. It is currently not clear how the antibacterial activity of HC is manifested. In this investigation we show HC generates superoxide in E. coli cells. Antioxidants protected E. coli against HC induced cell death while gshA mutant was more sensitive to HC than wild type. DNA damage repair deficient mutants are hypersensitive to HC and HC induces the expression of DNA damage repair genes that repair oxidative DNA damage. HC treated E. coli cells are inhibited from growth and undergo DNA condensation. In vitro HC binds to DNA and cleaves it in presence of copper. Our data strongly indicates HC mediates bacterial cell death by ROS generation and DNA damage. Damage to iron sulfur proteins in the cells contribute to amplification of oxidative stress initiated by HC. Further HC is active against a number of Gram negative bacteria isolated from patients with a wide range of clinical symptoms and varied antibiotic resistance profiles.
    Growth characteristics of Chlamydia trachomatis in human intestinal epithelial Caco-2 cells
    Ildikó Lantos, Pathogens and Disease - 2018
    Abstract
    Chlamydia trachomatis is an obligate intracellular bacterium causing infections of the eyes, urogenital and respiratory tracts. Asymptomatic, repeat and chronic infections with C. trachomatis are common in the urogenital tract potentially causing severe reproductive pathology. Animal models of infection and epidemiological studies suggested the gastrointestinal tract as a reservoir of chlamydiae and as a source of repeat urogenital infections. Thus, we investigated the growth characteristics of C. trachomatis in human intestinal epithelial Caco-2 cells and the infection-induced defensin production. Immunofluorescence staining and transmission electron microscopy showed the presence of chlamydial inclusions in the cells. Chlamydial DNA and viable C. trachomatis were recovered from Caco-2 cells in similar quantity compared to that detected in the usual in vitro host cell of this bacterium. The kinetics of expression of selected C. trachomatis genes in Caco-2 cells indicated prolonged replication with persisting high expression level of late genes and of heat shock protein gene groEL. Replication of C. trachomatis induced moderate level of β-defensin-2 production by Caco-2 cells, which might contribute to avoidance of immune recognition in the intestine. According to our results, Caco-2 cells support C. trachomatis replication, suggesting that the gastrointestinal tract is a site of residence for these bacteria.
    Macrophage-Derived Protein S Facilitates Apoptotic Polymorphonuclear Cell Clearance by Resolution Phase Macrophages and Supports Their Reprogramming
    Delphine Lumbroso, Frontiers in Immunology - 2018
    Abstract
    The complete resolution of inflammation requires the uptake of apoptotic polymorphonuclear cells (PMN) by local macrophages (efferocytosis) and the consequent reprogramming of the engulfing phagocytes to reparative and pro-resolving phenotypes. The tyrosine kinase receptors TYRO3, AXL, and MERTK (collectively named TAM) are fundamental mediators in regulating inflammatory responses and efferocytosis. Protein S (PROS1) is a ligand for all TAM receptors that mediates various aspects of their activity. However, the involvement of PROS1 in the resolution of inflammation is incompletely understood. Here, we report the upregulation of Pros1 in macrophages during the resolution of inflammation. Selective knockout of Pros1 in the myeloid lineage significantly downregulated macrophage pro-resolving properties. Hence, Pros1-deficient macrophages engulfed fewer apoptotic PMN remnants in vivo, and exogenous PROS1 rescued impaired efferocytosis ex vivo. Moreover, Pros1-deficient peritoneal macrophages secreted higher levels of the pro-inflammatory mediators TNFα and CCL3, while they secreted lower levels of the reparative/anti-inflammatory IL-10 following exposure to lipopolysaccharide in comparison to their WT counterparts. Moreover, Pros1-deficient macrophages expressed less of the anti-inflammatory/pro-resolving enzymes arginase-1 and 12/15-lipoxygenase and produced less of the specialized pro-resolving mediator resolvin D1. Altogether, our results suggest that macrophage-derived PROS1 is an important effector molecule in regulating the efferocytosis, maturation, and reprogramming of resolution phase macrophages, and imply that PROS1 could provide a new therapeutic target for inflammatory and fibrotic disorders.
    RANKL/RANK Pathway and its Inhibitor RANK-Fc in Uterine Leiomyoma Growth
    Deborah E. Ikhena, The Journal of Clinical Endocrinology & Metabolism - 2018
    Abstract
    ContextUterine leiomyomas are the most common type of gynecologic tumor in women.ObjectiveTo determine the role of the cytokine RANKL, its receptor RANK, and the RANKL/RANK pathway inhibitor RANK-Fc in leiomyoma growth.DesignmRNA or protein levels of RANKL, RANK and proliferation markers cyclin D1 and Ki67 were assessed in various leiomyoma tissues and cell populations. Human xenograft experiments were performed to determine the effects of RANK-Fc on leiomyoma growth in vivo.SettingResearch laboratoryPatientsTwenty-four regularly cycling premenopausal women (age 28-49 years) who were not on hormone therapy.InterventionsnoneMain Outcome MeasureTumor growth in a murine xenograft model following targeting of the RANKL/RANK pathway with RANK-Fc.ResultsRANKL mRNA levels in leiomyoma were significantly higher than those in myometrial tissues. The highest RANK levels were found in the leiomyoma stem cell (LSC) population, which is deficient in progesterone receptor (PR). Conversely, the highest RANKL levels were found in the PR-rich leiomyoma intermediate cell (LIC) population. R5020, a PR agonist, specifically increased RANKL expression in LICs. RANK-Fc blocked RANKL-induced expression of the proliferative gene cyclin D1. Treatment with RANK-Fc also significantly decreased tumor growth in vivo and diminished the expression of proliferation marker Ki67 in tumors (p<0.01, n=4).ConclusionsTreatment with the RANKL/RANK pathway inhibitor RANK-Fc significantly decreased human leiomyoma cell proliferation and tumor growth. This suggests that the RANKL/RANK pathway could serve as a potential target for the prevention and treatment of uterine leiomyoma.
    Inflammation arising from obesity reduces taste bud abundance and inhibits renewal
    Andrew Kaufman, PLOS Biology - 2018
    Abstract
    Despite evidence that the ability to taste is weakened by obesity and can be rescued with weight loss intervention, few studies have investigated the molecular effects of obesity on the taste system. Taste bud cells undergo continual turnover even in adulthood, exhibiting an average life span of only a few weeks, tightly controlled by a balance of proliferation and cell death. Recent data reveal that an acute inflammation event can alter this balance. We demonstrate that chronic low-grade inflammation brought on by obesity reduces the number of taste buds in gustatory tissues of mice—and is likely the cause of taste dysfunction seen in obese populations—by upsetting this balance of renewal and cell death.
    Surfactant protein A suppresses preterm delivery induced by live Escherichia coli in mice
    Varkha Agrawal, Biology of Reproduction - 2018
    Abstract
    Preterm birth accounts for the majority of neonatal morbidity and mortality in the developed world. A significant proportion of cases of spontaneous preterm labor are attributable to infections within gestational tissues. Surfactant protein A (SP-A), a collectin produced in the fetal lung and other tissues, has been shown previously in mice to suppress preterm delivery due to intrauterine (IU) instillation of sterile proinflammatory substances. Here we report a powerful antilabor effect for SP-A after IU infection with live Escherichia coli. SP-A abolished preterm birth (rate reduced from 100% to 0%) when it was administered into the uterus simultaneously with bacterial infection, reducing it by 75% when administered intravenously at the same time as IU bacterial inoculation, and by 48% when administered intravenously 4 h after IU bacterial infection. This effect on preterm delivery was accompanied by a parallel benefit on fetal survival in utero. SP-A had no effect on bacterial growth but reversed several major consequences of infection, including increased production of inflammatory mediators and a shift in macrophage polarization to the M1 phenotype. These findings suggest that exogenous SP-A has potential use to counteract infection-induced labor by reversing its proinflammatory consequences.
    MCP-1 is overexpressed in triple-negative breast cancers and drives cancer invasiveness and metastasis
    Pranabananda Dutta, Breast Cancer Research and Treatment - 2018
    Abstract
    BackgroundTriple-negative breast cancer (TNBC) is the most aggressive type of breast cancer that lacks ER/PR and HER2 receptors. Hence, there is urgency in developing new or novel therapeutic strategies for treatment of TNBC. Our study shows that the Monocyte Chemoattractant Protein-1 (MCP-1) is a marker associated with TNBC and may play a key role in TNBC disease progression.Experimental designELISA method was used to measure secreted MCP-1, and mRNA levels were determined by Real-time PCR in numerous cancer cell lines, representing various breast cancer subtypes. Cellular invasiveness was determined by Boyden chamber assay.ResultsOur data show that MCP-1 is upregulated in TNBC cell lines both transcriptionally as well as in secreted protein levels compared to ER-positive luminal cell line, MCF-7. Breast cancer patients, with Basal or Claudin-low subtypes, also showed high expression of MCP-1. MCP-1 treatment induced cell invasion in various breast cancer cell types, without affecting cell proliferation. Small molecule antagonists against Chemokine Receptor 2 (CCR2), cognate receptor for MCP-1 as well as the MAP kinase pathway inhibitor U0126 negatively affected MCP-1 induced MCF-7 cell invasion. This suggests that MCP-1-CCR2 axis may regulate invasiveness via the MAP Kinase pathway. Knocking down MCP-1 decreased cell invasion in TNBC cell line BT-549, along with downregulation of key epithelial to mesenchymal transition markers, N-cadherin and Vimentin.ConclusionOur study suggests that MCP-1 mediated pathways could be potential therapeutic targets for the treatment of TNBC, and could reduce cancer health disparities.
    Cryosurvival of rabbit embryos obtained after superovulation with corifollitropin alfa with or without LH
    José Salvador Vicente, Animal Reproduction Science - 2018
    Abstract
    The efficiency of an embryo bank depends on provision of optimal conditions for recovery, cryopreservation and transfer to a breed or strain. In this sense, increasing the number of embryos available using superovulation should improve the cryobank efficiency. However, vagueness of response to conventional protocols to control or increase ovarian response and the quality of oocytes and embryos and their cryotolerance remain a challenge. The aim of our study was to evaluate the effect of corifollitropin alpha (CTP) and a recombinant human FSH (rhFSH), alone or supplemented with rhLH, on embryo cryosurvival by in vitro development and OCT4 and NANOG mRNA abundance at blastocyst stage and offspring rate. In vitro development of vitrified embryos was not significantly affected by superstimulation with or without rhLH supplementation, resulting in similar development rates to those of the control groups (fresh and vitrified embryos from non-superstimulated donor does). Blastocysts developed from vitrified embryos showed higher levels of OCT4 transcript abundance than fresh control, while NANOG transcript abundance was only higher in the blastocysts developed from vitrified embryos after superstimulation treatment in comparison with control groups. The implantation and offspring rates at birth were negatively affected by supplementation with rhLH. Both rhFSH or CTP vitrified embryo groups showed an implantation rate similar to those of the control groups, but an offspring rate lower than control. In conclusion, embryos produced using corifollitropin alpha did not compromise the cryosurvival of vitrified embryos in the rabbit. In addition, this study points out the negative effect of rhLH supplementation in terms of offspring rate on embryo vitrification.
    Involvement of organic acids and amino acids in ameliorating Ni(II) toxicity induced cell cycle dysregulation in Caulobacter crescentus: a metabolomics analysis
    Abhishek Jain, Applied Microbiology and Biotechnology - 2018
    Abstract
    Nickel (Ni(II)) toxicity is addressed by many different bacteria, but bacterial responses to nickel stress are still unclear. Therefore, we studied the effect of Ni(II) toxicity on cell proliferation of α-proteobacterium Caulobacter crescentus. Next, we showed the mechanism that allows C. crescentus to survive in Ni(II) stress condition. Our results revealed that the growth of C. crescentus is severely affected when the bacterium was exposed to different Ni(II) concentrations, 0.003 mM slightly affected the growth, 0.008 mM reduced the growth by 50%, and growth was completely inhibited at 0.015 mM. It was further shown that Ni(II) toxicity induced mislocalization of major regulatory proteins such as MipZ, FtsZ, ParB, and MreB, resulting in dysregulation of the cell cycle. GC-MS metabolomics analysis of Ni(II) stressed C. crescentus showed an increased level of nine important metabolites including TCA cycle intermediates and amino acids. This indicates that changes in central carbon metabolism and nitrogen metabolism are linked with the disruption of cell division process. Addition of malic acid, citric acid, alanine, proline, and glutamine to 0.015 mM Ni(II)-treated C. crescentus restored its growth. Thus, the present work shows a protective effect of these organic acids and amino acids on Ni(II) toxicity. Metabolic stimulation through the PutA/GlnA pathway, accelerated degradation of CtrA, and Ni-chelation by organic acids or amino acids are some of the possible mechanisms suggested to be involved in enhancing C. crescentus’s tolerance. Our results shed light on the mechanism of increased Ni(II) tolerance in C. crescentus which may be useful in bioremediation strategies and synthetic biology applications such as the development of whole cell biosensor.
    27-Hydroxycholesterol increases α-synuclein protein levels through proteasomal inhibition in human dopaminergic neurons
    Jared Schommer, BMC Neuroscience - 2018
    Abstract
    Accumulation of the α-synuclein (α-syn) protein is a hallmark of a group of brain disorders collectively known as synucleinopathies. The mechanisms responsible for α-syn accumulation are not well understood. Several studies suggest a link between synucleinopathies and the cholesterol metabolite 27-hydroxycholesterol (27-OHC). 27-OHC is the major cholesterol metabolite in the blood that crosses the blood brain barrier, and its levels can increase following hypercholesterolemia, aging, and oxidative stress, which are all factors for increased synucleinopathy risk. In this study, we determined the extent to which 27-OHC regulates α-syn levels in human dopaminergic neurons, the cell type in which α-syn accumulates in PD, a major synucleinopathy disorder.
    Transcription and microRNA Profiling of Cultured Human Tympanic Membrane Epidermal Keratinocytes
    Peder Aabel, Journal of the Association for Research in Otolaryngology - 2018
    Abstract
    The human tympanic membrane (TM) has a thin outer epidermal layer which plays an important role in TM homeostasis and ear health. The specialised cells of the TM epidermis have a different physiology compared to normal skin epidermal keratinocytes, displaying a dynamic and constitutive migration that maintains a clear TM surface and assists in regeneration. Here, we characterise and compare molecular phenotypes in keratinocyte cultures from TM and normal skin. TM keratinocytes were isolated by enzymatic digestion and cultured in vitro. We compared global mRNA and microRNA expression of the cultured cells with that of human epidermal keratinocyte cultures. Genes with either relatively higher or lower expression were analysed further using the biostatistical tools g:Profiler and Ingenuity Pathway Analysis. Approximately 500 genes were found differentially expressed. Gene ontology enrichment and Ingenuity analyses identified cellular migration and closely related biological processes to be the most significant functions of the genes highly expressed in the TM keratinocytes. The genes of low expression showed a marked difference in homeobox (HOX) genes of clusters A and C, giving the TM keratinocytes a strikingly low HOX gene expression profile. An in vitro scratch wound assay showed a more individualised cell movement in cells from the tympanic membrane than normal epidermal keratinocytes. We identified 10 microRNAs with differential expression, several of which can also be linked to regulation of cell migration and expression of HOX genes. Our data provides clues to understanding the specific physiological properties of TM keratinocytes, including candidate genes for constitutive migration, and may thus help focus further research.
    Regulation of dual specificity phosphatases in breast cancer during initial treatment with Herceptin: a Boolean model analysis
    Petronela Buiga, BMC Systems Biology - 2018
    Abstract
    25% of breast cancer patients suffer from aggressive HER2-positive tumours that are characterised by overexpression of the HER2 protein or by its increased tyrosine kinase activity. Herceptin is a major drug used to treat HER2 positive breast cancer. Understanding the molecular events that occur when breast cancer cells are exposed to Herceptin is therefore of significant importance. Dual specificity phosphatases (DUSPs) are central regulators of cell signalling that function downstream of HER2, but their role in the cellular response to Herceptin is mostly unknown. This study aims to model the initial effects of Herceptin exposure on DUSPs in HER2-positive breast cancer cells using Boolean modelling.
    Genomic and transcriptomic alterations in Leishmania donovani lines experimentally resistant to antileishmanial drugs
    Alberto Rastrojo, International Journal for Parasitology: Drugs and Drug Resistance - 2018
    Abstract
    Leishmaniasis is a serious medical issue in many countries around the World, but it remains largely neglected in terms of research investment for developing new control and treatment measures. No vaccines exist for human use, and the chemotherapeutic agents currently used are scanty. Furthermore, for some drugs, resistance and treatment failure are increasing to alarming levels. The aim of this work was to identify genomic and trancriptomic alterations associated with experimental resistance against the common drugs used against VL: trivalent antimony (SbIII, S line), amphotericin B (AmB, A line), miltefosine (MIL, M line) and paromomycin (PMM, P line). A total of 1006 differentially expressed transcripts were identified in the S line, 379 in the A line, 146 in the M line, and 129 in the P line. Also, changes in ploidy of chromosomes and amplification/deletion of particular regions were observed in the resistant lines regarding the parental one. A series of genes were identified as possible drivers of the resistance phenotype and were validated in both promastigotes and amastigotes from Leishmania donovani, Leishmania infantum and Leishmania major species. Remarkably, a deletion of the gene LinJ.36.2510 (coding for 24-sterol methyltransferase, SMT) was found to be associated with AmB-resistance in the A line. In the P line, a dramatic overexpression of the transcripts LinJ.27.T1940 and LinJ.27.T1950 that results from a massive amplification of the collinear genes was suggested as one of the mechanisms of PMM resistance. This conclusion was reinforced after transfection experiments in which significant PMM-resistance was generated in WT parasites over-expressing either gene LinJ.27.1940 (coding for a D-lactate dehydrogenase-like protein, D-LDH) or gene LinJ.27.1950 (coding for an aminotransferase of branched-chain amino acids, BCAT). This work allowed to identify new drivers, like SMT, the deletion of which being associated with resistance to AmB, and the tandem D-LDH-BCAT, the amplification of which being related to PMM resistance.
    Follicle capacitation: A meta-analysis to investigate the transcriptome dynamics following FSH decline in bovine granulosa cells.
    David A. Landry, Biology of Reproduction - 2018
    Abstract
    In recent years, exciting progress was made to improve the embryo outcome after ovarian stimulation in domestic animals. The practice of follicle-stimulating hormone (FSH) withdrawal, which is defined as the period of time between the last injection of FSH and oocyte retrieval, resulted in embryo yields significantly superior. Since then, specific changes in the transcriptome of granulosa cells were associated with the increase and also the decline in oocyte developmental competence following the FSH decline. In this study, we integrated large data sets from a public depository using a meta-analysis in order to elucidate the molecular changes occurring in granulosa cells following FSH decline in association with oocyte developmental competence. The meta-analysis revealed that the gene expression patterns observed during this period resulted from the downregulation of proliferative signals, and the upregulation of differentiation signals and early apoptotic signals. Additionally, FSH decline induced cellular hypoxia and triggered the expression of pro-inflammatory molecules which resulted in early atresia and mimicked the luteinizing hormone (LH) surge signaling to ovulation. To characterize this unique differentiation period, we suggest using the term “follicle capacitation” to refer to the functional changes occurring within the follicle in order to prepare the molecular machinery for the LH surge and ovulation following FSH decline. During this period, the follicle confers the oocyte with developmental competence to become a viable embryo. However, if this period is not rapidly followed by a LH surge, apoptosis signals are increased to generate follicular atresia and decrease oocyte quality.
    The integrated analysis of transcriptome and proteome for exploring the biodegradation mechanism of 2, 4, 6-trinitrotoluene by Citrobacter sp
    Hung-Yu Liao, Journal of Hazardous Materials - 2018
    Abstract
    Citrobacter sp. has been shown to degrade 2,4,6-trinitrotoluene (TNT). However, the mechanism of its TNT biodegradation is poorly understood. An integrated proteome and transcriptome analysis was performed for investigating the differential genes and differential proteins in bacterial growth at the onset of experiments and after 12 h treatment with TNT. With the RNA sequencing, we found a total of 3792 transcripts and 569 differentially expressed genes (≥2 fold, P < 0.05) by. Genes for amino acid transport, cellular metabolism and stress-shock proteins were up-regulated, while carbohydrate transport and metabolism were down-regulated. A total of 42 protein spots (≥1.5 fold, P < 0.05) showed differential expression on two-dimensional gel electrophoresis and these proteins were identified by mass spectrometry. The most prominent proteins up-regulated were involved in energy production and conversion, amino acid transport and metabolism, posttranslational modification, protein turnover and chaperones. Proteins involved in carbohydrate transport and metabolism were down-regulated. Most notably, we observed that nemA encoding N-ethylmaleimide reductase was the most up-regulated gene involved in TNT degradation, and further proved that it can transform TNT to 4-amino-2,6-dinitrotoluene (4-ADNT) and 2-amino-4,6-dinitrotoluene (2-ADNT). This study highlights the molecular mechanisms of Citrobacter sp. for TNT removal.
    Genetic defects in mtDNA-encoded protein translation cause pediatric, mitochondrial cardiomyopathy with early-onset brain disease
    Rick Kamps, European Journal of Human Genetics - 2018
    Abstract
    This study aims to identify gene defects in pediatric cardiomyopathy and early-onset brain disease with oxidative phosphorylation (OXPHOS) deficiencies. We applied whole-exome sequencing in three patients with pediatric cardiomyopathy and early-onset brain disease with OXPHOS deficiencies. The brain pathology was studied by MRI analysis. In consanguineous patient 1, we identified a homozygous intronic variant (c.850-3A > G) in the QRSL1 gene, which was predicted to cause abnormal splicing. The variant segregated with the disease and affected the protein function, which was confirmed by complementation studies, restoring OXPHOS function only with wild-type QRSL1. Patient 2 was compound heterozygous for two novel affected and disease-causing variants (c.[253G > A];[938G > A]) in the MTO1 gene. In patient 3, we detected one unknown affected and disease-causing variants (c.2872C > T) and one known disease-causing variant (c.1774C > T) in the AARS2 gene. The c.1774C > T variant was present in the paternal copy of the AARS2 gene, the c.2872C > T in the maternal copy. All genes were involved in translation of mtDNA-encoded proteins. Defects in mtDNA-encoded protein translation lead to severe pediatric cardiomyopathy and brain disease with OXPHOS abnormalities. This suggests that the heart and brain are particularly sensitive to defects in mitochondrial protein synthesis during late embryonic or early postnatal development, probably due to the massive mitochondrial biogenesis occurring at that stage. If both the heart and brain are involved, the prognosis is poor with a likely fatal outcome at young age.
    Targeting the nonmevalonate pathway in Burkholderia cenocepacia increases susceptibility to certain β-lactam antibiotics
    Andrea Sass, Antimicrobial Agents and Chemotherapy - 2018
    Abstract
    The non-mevalonate pathway is the sole pathway for isoprenoid biosynthesis in Burkholderia cenocepacia and possibly a novel target for the development of antibacterial chemotherapy. The goal of the present study was to evaluate the essentiality of dxr, the second gene of the non-mevalonate pathway, in B. cenocepacia and to determine whether interfering with the non-mevalonate increases susceptibility towards antibiotics. To this end, a rhamnose-inducible conditional dxr knock-down mutant of B. cenocepacia K56-2 (B. cenocepacia K56-2dxr) was constructed, by using a plasmid which enables the delivery of a rhamnose-inducible promotor in the chromosome. Expression of dxr is essential for bacterial growth; this growth defect could be complemented by expressing dxr in trans under control of a constitutive promotor, but not by providing 2-C-methyl-D-erythritol-4-phosphate, the reaction product of DXR. B. cenocepacia K56-2dxr showed markedly increased susceptibility to the β-lactam antibiotics aztreonam, ceftazidime and cefotaxime, while susceptibility to other antibiotics was not (or much less) affected, and also this increased susceptibility could be complemented by in trans expression of dxr. A similar increased susceptibility was observed when antibiotics were combined with FR900098, a known DXR inhibitor. Our data confirm that the non-mevalonate pathway is essential in B. cenocepacia and suggest that combining potent DXR inhibitors with selected β-lactam antibiotics is a useful strategy to combat B. cenocepacia infections.
    Germ cell depletion in zebrafish leads to incomplete masculinization of the brain
    Ajay Pradhan, General and Comparative Endocrinology - 2018
    Abstract
    Zebrafish sex differentiation is under the control of multiple genes, but also relies on germ cell number for gonadal development. Morpholino and chemical mediated germ cell depletion leads to sterile male development in zebrafish. In this study we produced sterile males, using a dead end gene morpholino, to determine gonadal-brain interactions. Germ cell depletion following dnd inhibition downregulated the germ cell markers, vasa and ziwi, and later the larvae developed as sterile males. Despite lacking proper testis, the gonadal 11-ketotestosterone (11-KT) and estradiol (E2) levels of sterile males were similar to wild type males. Qualitative analysis of sexual behavior of sterile males demonstrated that they behaved like wild type males. Furthermore, we observed that brain 11-KT and E2 levels in sterile males remained the same as in the wild type males. In female brain, 11-KT was lower in comparison to wild type males and sterile males, while E2 was higher when compared to wild type males. qRT-PCR analysis revealed that the liver transcript profile of sterile adult males was similar to wild type males while the brain transcript profile was similar to wild type females. The results demonstrate that proper testis development may not be a prerequisite for male brain development in zebrafish but that it may be needed to fully masculinize the brain.
    Characterization of Caco-2 cells stably expressing the protein-based zinc probe eCalwy-5 as a model system for investigating intestinal zinc transport
    Maria Maares, Journal of Trace Elements in Medicine and Biology - 2018
    Abstract
    Intestinal zinc resorption, in particular its regulation and mechanisms, are not yet fully understood. Suitable intestinal cell models are needed to investigate zinc uptake kinetics and the role of labile zinc in enterocytes in vitro. Therefore, a Caco-2 cell clone was produced, stably expressing the genetically encoded zinc biosensor eCalwy-5. The aim of the present study was to reassure the presence of characteristic enterocyte-specific properties in the Caco-2-eCalwy clone. Comparison of Caco-2-WT and Caco-2-eCalwy cells revealed only slight differences regarding subcellular localization of the tight junction protein occludin and alkaline phosphatase activity, which did not affect basic integrity of the intestinal barrier or the characteristic brush border membrane morphology. Furthermore, introduction of the additional zinc-binding protein in Caco-2 cells did not alter mRNA expression of the major intestinal zinc transporters (zip4, zip5, znt-1 and znt-5), but increased metallothionein 1a-expression and cellular resistance to higher zinc concentrations. Moreover, this study examines the effect of sensor expression level on its saturation with zinc. Fluorescence cell imaging indicated considerable intercellular heterogeneity in biosensor-expression. However, FRET-measurements confirmed that these differences in expression levels have no effect on fractional zinc-saturation of the probe.
    REV-ERBβ is required to maintain normal wakefulness and the wake-inducing effect of dual REV-ERB agonist SR9009
    Ariadna Amador, Biochemical Pharmacology - 2018
    Abstract
    Circadian signaling regulates and synchronizes physiological and behavioral processes, such as feeding, metabolism, and sleep cycles. The endogenous molecular machinery that regulates circadian activities is located in the suprachiasmatic nucleus of the hypothalamus. The REV-ERBs are transcription factors that play key roles in the regulation of the circadian clock and metabolism. Using pharmacological methods, we recently demonstrated the involvement of the REV-ERBs in sleep architecture. Another group reported a delayed response to sleep deprivation and altered sleep cycles in REV-ERBα null mice, indicating a role of REV-ERBα in sleep. Given that REV-ERBβ is structurally and functionally similar to REV-ERBα, we investigated the role of REV-ERBβ in sleep and wakefulness by assessing electroencephalographic recordings in REV-ERBβ deficient mice and the mechanism underlying effects of loss of REV-ERBβ on sleep. Our data suggest that REV-ERBβ is involved in the maintenance of wakefulness during the activity period. In addition, REV-ERBβ-deficient mice administered with dual REV-ERB agonist SR9009, failed to show drug-induced wake increase. Finally, the expression of a number of genes known to mediate sleep and wakefulness were altered in REV-ERBβ null mice.
    Oncogenic RAC1 and NRAS drive resistance to endoplasmic reticulum stress through MEK/ERK signalling
    Michael D. Bright, Cellular Signalling - 2018
    Abstract
    Cancer cells are able to survive under conditions that cause endoplasmic reticulum stress (ER-stress), and can adapt to this stress by upregulating cell-survival signalling pathways and down-regulating apoptotic pathways. The cellular response to ER-stress is controlled by the unfolded protein response (UPR). Small Rho family GTPases are linked to many cell responses including cell growth and apoptosis. In this study, we investigate the function of small GTPases in cell survival under ER-stress. Using siRNA screening we identify that RAC1 promotes cell survival under ER-stress in cells with an oncogenic N92I RAC1 mutation. We uncover a novel connection between the UPR and N92I RAC1, whereby RAC1 attenuates phosphorylation of EIF2S1 under ER-stress and drives over-expression of ATF4 in basal conditions. Interestingly, the UPR connection does not drive resistance to ER-stress, as knockdown of ATF4 did not affect this. We further investigate cancer-associated kinase signalling pathways and show that RAC1 knockdown reduces the activity of AKT and ERK, and using a panel of clinically important kinase inhibitors, we uncover a role for MEK/ERK, but not AKT, in cell viability under ER-stress. A known major activator of ERK phosphorylation in cancer is oncogenic NRAS and we show that knockdown of NRAS in cells, which bear a Q61 NRAS mutation, sensitises to ER-stress. These findings highlight a novel mechanism for resistance to ER-stress through oncogenic activation of MEK/ERK signalling by small GTPases.
    3D-Printed Gelatin Scaffolds of Differing Pore Geometry Modulate Hepatocyte Function and Gene Expression
    Phillip L. Lewis, Acta Biomaterialia - 2018
    Abstract
    Three dimensional (3D) printing is highly amenable to the fabrication of tissue-engineered organs of a repetitive microstructure such as the liver. The creation of uniform and geometrically repetitive tissue scaffolds can also allow for the control over cellular aggregation and nutrient diffusion. However, the effect of differing geometries, while controlling for pore size, has yet to be investigated in the context of hepatocyte function. In this study, we show the ability to precisely control pore geometry of 3D-printed gelatin scaffolds. An undifferentiated hepatocyte cell line (HUH7) demonstrated high viability and proliferation when seeded on 3D-printed scaffolds of two different geometries. However, hepatocyte specific functions (albumin secretion, CYP activity, and bile transport) increases in more interconnected 3D-printed gelatin cultures compared to a less interconnected geometry and to 2D controls. Additionally, we also illustrate the disparity between gene expression and protein function in simple 2D culture modes, and that recreation of a physiologically mimetic 3D environment is necessary to induce both expression and function of cultured hepatocytes. Statement of Significance Three dimensional (3D) printing provides tissue engineers the ability spatially pattern cells and materials in precise geometries, however the biological effects of scaffold geometry on soft tissues such as the liver have not been rigorously investigated. In this manuscript, we describe a method to 3D print gelatin into well-defined repetitive geometries that show clear differences in biological effects on seeded hepatocytes. We show that a relatively simple and widely used biomaterial, such as gelatin, can significantly modulate biological processes when fabricated into specific 3D geometries. Furthermore, this study expands upon past research into hepatocyte aggregation by demonstrating how it can be manipulated to enhance protein function, and how function and expression may not precisely correlate in 2D models.
    Atlantic salmon adapted to seawater for 9 weeks develop a robust immune response to salmonid alphavirus upon bath challenge
    L. J. Moore, Fish & Shellfish Immunology - 2018
    Abstract
    Pancreas disease (PD) caused by salmonid alphavirus (SAV) is the most serious viral disease in Norwegian aquaculture. Study of the immune response to SAV will aid preventative measures including vaccine development. The innate immune response was studied in Atlantic salmon infected by either bath immersion (BI) or by intra-muscular (i.m.) injection (IM) with SAV subtype 3, two and nine weeks after seawater transfer (Phases A and B respectively). Phase A results have been previously published (Moore et al., 2017) and Phase B results are presented here together with a comparison of results achieved in Phase A. There was a rapid accumulation of infected fish in the IM-B (IM Phase B) group and all fish sampled were SAV RNA positive by 7 dpi (days post infection). In contrast, only a few SAV RNA positive (infected) fish were identified at 14, 21 and 28 dpi in the BI-B (BI Phase B) group. Differences in the transcription of several immune genes were apparent when compared between the infected fish in the IM-B and BI-B groups. Transcription of the analysed genes peaked at 7 dpi in the IM-B group and at 14 dpi in the BI-B group. However, this latter finding was difficult to interpret due to the low prevalence of SAV positive fish in this group. Additionally, fish positive for SAV RNA in the BI-B group showed higher transcription of IL-1β, IFNγ and CXCL11_L1, all genes associated with the inflammatory response, compared to the IM-B group. Histopathological changes in the heart were restricted to the IM-B group, while (immune) cell filtration into the pancreas was observed in both groups. Compared to the Phase A fish that were exposed to SAV3 two weeks after seawater transfer, the Phase B fish in the current paper, showed a higher and more sustained innate immune gene transcription in response to the SAV3 infection. In addition, the basal transcription of several innate immune genes in non-infected control fish in Phase B (CT-B) was also significantly different when compared to Phase A control fish (CT-A).
    Aldehyde Dehydrogenase Activity in Adipose Tissue: Isolation and Gene Expression Profile of Distinct Sub-population of Mesenchymal Stromal Cells
    Mehdi Najar, Stem Cell Reviews and Reports - 2018
    Abstract
    Thanks to their relative abundance and easier collection, adipose tissue (AT) is considered an alternative source for the isolation of mesenchymal stromal cells (MSCs). MSCs have great therapeutic values and are thus under investigations for several clinical indications such as regenerative medicine and immunomodulation. In this work, we aimed to identify, isolate and characterize AT-MSCs based on their aldehyde dehydrogenase (ALDH) activity known to be a classical feature of stem cells. FACS technology allowed to isolate two different populations of AT-MSCs according to their ALDH activity (referred as ALDH+ and ALDH−). Depending on their ALDH activity, the transcriptome analysis of both cell populations demonstrated a differential pattern of genes related to the main properties of MSCs (proliferation, response to hypoxia, angiogenesis, phenotype, stemness, multilineage, hematopoiesis, immunomodulation). Based on these profiling, both AT-MSC populations could differ in terms of biological responses and functionalities. Collectively, the use of ALDH for isolating and identifying sub-populations of MSCs with specific gene profile may represent an alternative method to provide solutions for targeted therapeutic applications.
    The effects of platelet lysate patches on the activity of tendon-derived cells
    Raquel Costa-Almeida, Acta Biomaterialia - 2018
    Abstract
    Platelet-derived biomaterials are widely explored as cost-effective sources of therapeutic factors, holding a strong potential for endogenous regenerative medicine. Particularly for tendon repair, treatment approaches that shift the injury environment are explored to accelerate tendon regeneration. Herein, genipin-crosslinked platelet lysate (PL) patches are proposed for the delivery of human-derived therapeutic factors in patch augmentation strategies aiming at tendon repair. Developed PL patches exhibited a controlled release profile of PL proteins, including bFGF and PDGF-BB. Additionally, PL patches exhibited an antibacterial effect by preventing the adhesion, proliferation and biofilm formation by S. aureus, a common pathogen in orthopaedic surgical site infections. Furthermore, these patches supported the activity of human tendon-derived cells (hTDCs). Cells were able to proliferate over time and an up-regulation of tenogenic genes (SCX, COL1A1 and TNC) was observed, suggesting that PL patches may modify the behavior of hTDCs. Accordingly, hTDCs deposited tendon-related extracellular matrix proteins, namely collagen type I and tenascin C. In summary, PL patches can act as a reservoir of biomolecules derived from PL and support the activity of native tendon cells, being proposed as bioinstructive patches for tendon regeneration. Statement of significance Platelet-derived biomaterials hold great interest for the delivery of therapeutic factors for applications in endogenous regenerative medicine. In the particular case of tendon repair, patch augmentation strategies aiming at shifting the injury environment are explored to improve tendon regeneration. In this study, PL patches were developed with remarkable features, including the controlled release of growth factors and antibacterial efficacy. Remarkably, PL patches supported the activity of native tendon cells by up-regulating tenogenic genes and enabling the deposition of ECM proteins. This patch holds great potential towards simultaneously reducing post-implantation surgical site infections and promoting tendon regeneration for prospective in vivo applications.
    Cancer Stem Cell Phenotypes in ER+ Breast Cancer Models are Promoted by PELP1/AIB1 Complexes
    Thu H. Truong, Molecular Cancer Research - 2018
    Abstract
    Abstract: Proline, glutamic acid, and leucine rich protein 1 (PELP1) is overexpressed in approximately 80% of invasive breast tumors. PELP1 dynamically shuttles between the nucleus and cytoplasm, but is primarily nuclear in normal breast tissue. However, altered localization of PELP1 to the cytoplasm is an oncogenic event that promotes breast cancer initiation and progression. Herein, interacting partners unique to cytoplasmic PELP1 and the mechanisms by which these interactions promote oncogenic PELP1 signaling were sought. AIB1 (amplified in breast cancer 1; also known as SRC-3 or NCOA3) was identified as a novel binding partner of cytoplasmic PELP1 in both estrogen receptor-positive (ER+) and ER-negative cell lines. Cytoplasmic PELP1 expression elevated basal phosphorylation levels (i.e. activation) of AIB1 at Thr24, enhanced ALDH+ tumorsphere formation, and upregulated specific target genes independently of hormone stimulation. Direct manipulation of AIB1 levels using shRNA abrogated cytoplasmic PELP1-induced tumorsphere formation and down-regulated cytoplasmic PELP1-specific target genes. SI-2, an AIB1 inhibitor, limited the PELP1/AIB1 interaction and decreased cytoplasmic PELP1-induced tumorsphere formation. Similar results were observed in a murine-derived MMTV-AIB1 tumor cell line. Furthermore, in vivo syngeneic tumor studies revealed that PELP1 knockdown resulted in increased survival of tumor-bearing mice as compared to mice injected with control cells. Implications: These data demonstrate that cytoplasmic PELP1/AIB1 containing complexes function to promote advanced cancer phenotypes, including outgrowth of stem-like cells, associated with estrogen-independent breast cancer progression.
    The splicing factor transformer2 (tra2) functions in the Drosophila fat body to regulate lipid storage
    Cezary Mikoluk, Biochemical and Biophysical Research Communications - 2018
    Abstract
    Excess nutrients are stored as triglycerides mainly in the adipose tissue of an animal and these triglycerides are located in structures called lipid droplets. Previous genome-wide RNAi screens in Drosophila cells identified splicing factors as playing a role in lipid droplet formation. Our lab has recently identified the SR protein, 9G8, as an important factor in fat storage as decreasing its levels results in augmented triglyceride storage in the fat body. Previous in vitro studies have implicated 9G8 in the regulation of splicing of the sex determination gene doublesex (dsx) by binding to transformer (tra) and transformer2 (tra2); however, any function of these sex determination proteins in regulating metabolism is unknown. In this study, we have uncovered a role of tra2 to regulate fat storage in vivo. Inducing tra2dsRNA in the adult fat body resulted in an increase in triglyceride levels but had no effect on glycogen storage. Consistent with the triglyceride phenotype, tra2 knockdown flies lived longer under starvation conditions. In addition, this increase in triglycerides is due to more fat storage per cell and not an increase in the number of fat cells. Interestingly, the splicing of CPT1, an enzyme involved in the breakdown of lipids, was altered in flies with decreased tra2. The less-catalytically active isoform of CPT1 accumulated in tra2dsRNA flies suggesting a decrease in lipid breakdown, which is consistent with the increased triglyceride levels observed in these flies. Together, these results suggest a link between mRNA splicing, sex determination and lipid metabolism and may provide insight into the mechanisms underlying tissue-specific splicing and nutrient storage.
    Characterization of three salmon louse (Lepeophtheirus salmonis) genes with fibronectin II domains expressed by tegumental type 1 glands
    Ewa Harasimczuk, Molecular and Biochemical Parasitology - 2018
    Abstract
    The salmon louse, Lepeophtheirus salmonis (Copepoda: Caligidae), is currently the most significant pathogen affecting the salmon farming industry in the Northern Hemisphere. Exocrine glands of blood-feeding parasites are believed to be important for the host-parasite interaction, but also in the production of substances for integument lubrication and antifouling. In L. salmonis; however, we have limited knowledge about the exocrine glands. The aim of this study was therefore to examine three genes containing fibronectin type II (FNII) domains expressed in L. salmonis tegumental type 1 (teg 1) glands, namely LsFNII1, 2 and 3. LsFNII1, 2 and 3 contains four, three, and two FNII domains respectively. Sequence alignment of LsFNII domains showed conservation of amino acids that may indicate a possible involvement of LsFNII domains in collagen binding. Ontogenetic analysis of LsFNII1, 2 and 3 revealed highest expression in pre-adult and adult lice. Localization of LsFNII1, 2 and 3 transcripts showed expression in teg 1 glands only, which are the most abundant exocrine gland type in L. salmonis. LsFNII1, 2 and 3 were successfully knocked-down by RNAi, however, alteration in gland morphology was not detected between the knock-down and control groups. Overall, this study gives first insight into FNII domain containing proteins in L. salmonis.
    Adrenergic and glucocorticoid receptor antagonists reduce ozone-induced lung injury and inflammation
    Andres R. Henriquez, Toxicology and Applied Pharmacology - 2018
    Abstract
    Recent studies showed that the circulating stress hormones, epinephrine and corticosterone/cortisol, are involved in mediating ozone-induced pulmonary effects through the activation of the sympathetic-adrenal-medullary (SAM) and hypothalamus-pituitary-adrenal (HPA) axes. Hence, we examined the role of adrenergic and glucocorticoid receptor inhibition in ozone-induced pulmonary injury and inflammation. Male 12-week old Wistar-Kyoto rats were pretreated daily for 7 days with propranolol (PROP; a non-selective β adrenergic receptor [AR] antagonist, 10 mg/kg, i.p.), mifepristone (MIFE; a glucocorticoid receptor [GR] antagonist, 30 mg/kg, s.c.), both drugs (PROP + MIFE), or respective vehicles, and then exposed to air or ozone (0.8 ppm), 4 h/d for 1 or 2 consecutive days while continuing drug treatment. Ozone exposure alone led to increased peak expiratory flow rates and enhanced pause (Penh); with greater increases by day 2. Receptors blockade minimally affected ventilation in either air- or ozone-exposed rats. Ozone exposure alone was also associated with marked increases in pulmonary vascular leakage, macrophage activation, neutrophilic inflammation and lymphopenia. Notably, PROP, MIFE and PROP + MIFE pretreatments significantly reduced ozone-induced pulmonary vascular leakage; whereas PROP or PROP + MIFE reduced neutrophilic inflammation. PROP also reduced ozone-induced increases in bronchoalveolar lavage fluid (BALF) IL-6 and TNF-α proteins and/or lung Il6 and Tnfα mRNA. MIFE and PROP + MIFE pretreatments reduced ozone-induced increases in BALF N-acetyl glucosaminidase activity, and lymphopenia. We conclude that stress hormones released after ozone exposure modulate pulmonary injury and inflammatory effects through AR and GR in a receptor-specific manner. Individuals with pulmonary diseases receiving AR and GR-related therapy might experience changed sensitivity to air pollution.
    Myrcia sylvatica essential oil mitigates molecular, biochemical and physiological alterations in Rhamdia quelen under different stress events associated to transport
    Etiane M. H. Saccol, Research in Veterinary Science - 2018
    Abstract
    The effects of pre-transport handling and addition of essential oil of Myrcia sylvatica (EOMS) during transport on stress pathways activation in Rhamdia quelen were investigated. Fish (n = 400, 25.2 ± 2.9 g) were captured in production ponds and transferred to 100-L tank (density 100 g L− 1). After 24 h, 10 fish were sampled (before transport group). The remaining fish were placed in plastic bags (n = 30 or 32 fish per bag, density 150 g L− 1) containing 5 L of water (control), ethanol (315 μL L− 1, vehicle) or EOMS (25 or 35 μL L− 1), in triplicate, transported for 6 h and sampled (n = 10 animals per group). Indicators of stress and metabolism, as well as mRNA expression of brain hormones were evaluated. Previously, full-length cDNAs, encoding specific corticotropin-releasing hormone (crh) and proopiomelanocortins (pomca and pomcb), were cloned from whole brain of R. quelen. Crh expression increased after 24 h of capture and handling, whereas cortisol and glucose plasmatics enhanced their values in the control group. Transport with EOMS reduced plasma cortisol and lactate levels, while ethanol and EOMS groups increased Na+/K+-ATPase gill activity compared to control. Gene expression of crh, pomcb, prolactin and somatolactin mRNAs were lower after transport with EOMS compared to control. EOMS was able to mitigate the stress pathways activation caused by transport, maintaining a balance in body homeostasis. Thus, EOMS is recommended as sedative in procedures as transport and the pre-transport handling requires greater attention and use of tranquilizers.
    Effect of heifer age on the granulosa cell transcriptome after ovarian stimulation
    David A. Landry, Reproduction, Fertility and Development - 2018
    Abstract
    Genomic selection is accelerating genetic gain in dairy cattle. Decreasing generation time by using younger gamete donors would further accelerate breed improvement programs. Although ovarian stimulation of peripubertal animals is possible and embryos produced in vitro from the resulting oocytes are viable, developmental competence is lower than when sexually mature cows are used. The aim of the present study was to shed light on how oocyte developmental competence is acquired as a heifer ages. Ten peripubertal Bos taurus Holstein heifers underwent ovarian stimulation cycles at the ages of 8, 11 (mean 10.8) and 14 (mean 13.7) months. Collected oocytes were fertilised in vitro with spermatozoa from the same adult male. Each heifer served as its own control. The transcriptomes of granulosa cells recovered with the oocytes were analysed using microarrays. Differential expression of certain genes was measured using polymerase chain reaction. Principal component analysis of microarray data revealed that the younger the animal, the more distinctive the gene expression pattern. Using ingenuity pathway analysis (IPA) and NetworkAnalyst (www.networkanalyst.ca), the main biological functions affected in younger donors were identified. The results suggest that cell differentiation, inflammation and apoptosis signalling are less apparent in peripubertal donors. Such physiological traits have been associated with a lower basal concentration of LH.
    Expression Pattern of Individual IFNA Subtypes in Chronic HIV Infection
    Yanpeng Li, Journal of Interferon & Cytokine Research - 2018
    Abstract
    Interferon-α (IFN-α) plays an important role in HIV pathogenesis. IFN-α consists of 13 individual IFN-α subtypes, which exhibit individual antiviral and immunomodulatory activities in HIV infection. Here, we determined the expression profiles of all IFN-α subtypes in treated and treatment-naive HIV+ patients and their impact on the induction of distinct HIV restriction factors. We collected blood samples of chronic HIV+ patients, which underwent antiretroviral therapy or were treatment-naive, and determined the individual expression levels of different IFN-α subtypes and HIV restriction factors. HIV infection transiently enhanced the expression of IFNA mRNA. The IFN-α response was dominated by the most abundantly expressed subtypes IFNA4, A5, A7, and A14 in all individuals. HIV infection affected the expression pattern of the IFN-α response, in particular for IFNA2 and IFNA16, which were elevated by chronic HIV infection. Elevated expression of HIV restriction factors was observed in chronically HIV-infected patients, which partly decreased during successful antiretroviral treatment. In vitro stimulation of peripheral blood mononuclear cells revealed that IFN-α6, -α14, and -α21 were most effective in inducing the expression of HIV restriction factors. These results indicate that HIV infection induces a specific expression pattern of IFN-α subtypes, which in turn induce the expression of various HIV restriction factors.
    Characterization of a novel variant in siblings with Asparagine Synthetase Deficiency
    Stephanie J. Sacharow, Molecular Genetics and Metabolism - 2018
    Abstract
    Asparagine Synthetase Deficiency (ASD) is a recently described inborn error of metabolism caused by bi-allelic pathogenic variants in the asparagine synthetase (ASNS) gene. ASD typically presents congenitally with microcephaly and severe, often medically refractory, epilepsy. Development is generally severely affected at birth. Tone is abnormal with axial hypotonia and progressive appendicular spasticity. Hyperekplexia has been reported. Neuroimaging typically demonstrates gyral simplification, abnormal myelination, and progressive cerebral atrophy. The present report describes two siblings from consanguineous parents with a homozygous Arg49Gln variant associated with a milder form of ASD that is characterized by later onset of symptoms. Both siblings had a period of normal development before onset of seizures, and development regression. Primary fibroblast studies of the siblings and their parents document that homozygosity for Arg49Gln blocks cell growth in the absence of extracellular asparagine. Functional studies with these cells suggest no impact of the Arg49Gln variant on basal ASNS mRNA or protein levels, nor on regulation of the gene itself. Molecular modelling of the ASNS protein structure indicates that the Arg49Gln variant lies near the substrate binding site for glutamine. Collectively, the results suggest that the Arg49Gln variant affects the enzymatic function of ASNS. The clinical, cellular, and molecular observations from these siblings expand the known phenotypic spectrum of ASD.
    Neonatal Systemic Inflammation Induces Inflammatory Reactions and Brain Apoptosis in a Pathogen-Specific Manner
    Mari Falck, Neonatology - 2018
    Abstract
    After neonatal asphyxia, therapeutic hypothermia (HT) is the only proven treatment option. Although established as a neuroprotective therapy, benefit
    Hippocampal GABAA antagonism reverses the novel object recognition deficit in sub-chronic phencyclidine-treated rats
    Nichole M. Neugebauer, Behavioural Brain Research - 2018
    Abstract
    Background Abnormalities in prefrontal cortical and hippocampal GABAergic function are postulated to be major causes of the cognitive impairment associated with schizophrenia (CIAS). There are conflicting views on whether diminished or enhanced GABAergic activity contributes to the deficit in short-term novel object recognition (NOR) in the sub-chronic phencyclidine (scPCP) rodent model of CIAS. This study assessed the role of GABAA signaling in the medial prefrontal cortex (mPFC) and ventral hippocampus (vHPC) in NOR in saline (scSAL)- and scPCP-treated rats. Methods The effects of local administration of a GABAA agonist (muscimol) into the vHPC or mPFC and an antagonist (bicuculline) or a GABAA/benzodiazepine partial agonist (bretazenil) into the vHPC on NOR in scSAL and scPCP-treated rats were determined. Results In scSAL-treated rats, injection of muscimol into the vHPC, but not mPFC, induced a deficit in NOR. The scPCP-induced NOR deficit was significantly reversed by intra-vHPC bicuculline, while intra-vHPC bretazenil produced a non-significant trend for reversal (p = 0.06). scPCP treatment increased mRNA expression of GABAA γ2 in PFC and GABAA α5 and GABAA β1 in the HPC. However, GABA concentration in the PFC or HPC was not altered. Conclusions These findings indicate that the scPCP-induced NOR deficit can be rescued by reducing GABAA receptor stimulation in vHPC, indicating that increased vHPC GABAA inhibition may contribute to the scPCP-induced NOR deficit in rats. These results also indicate that excessive GABAA receptor signalling in the vHPC has a deleterious effect on NOR in normal rats.
    Anti-nociceptive role of CXCL1 in a murine model of peripheral nerve injury-induced neuropathic pain
    Ling Cao, Neuroscience - 2018
    Abstract
    Both spinal cord infiltrating CD4+ T lymphocytes and microglial CD40 contribute to the maintenance of neuropathic pain-like behaviors induced by spinal nerve L5 transection (L5Tx), a murine model of neuropathic pain. Here, we sought to investigate the involvement of multiple chemokines in microglial CD40-mediated and CD4+ T lymphocytes-mediated L5Tx-induced sensory hypersensitivity. Spinal cord chemokine expression in CD4 knockout (KO), CD40 KO, and wild type (WT) BALB/c mice was determined at the protein level via multiplex assays and at the RNA level via quantitative real-time PCR. In WT mice, L5Tx induced significant increases in CCL2, CCL3, and CCL5 expression (protein and RNA) up to day 21 post-L5Tx, while CD4 KO mice displayed blunted, predominantly non-significant, responses in these chemokines at protein levels post-L5Tx. L5Tx also induced increased expression of these chemokines in CD40 KO mice; however, the overall protein levels of these chemokines were significantly lower than that in WT mice. Further, L5Tx induced a significant increase in CXCL1 at the protein level and in CXCR2 at RNA level only in CD40 KO mice. Intrathecal administration of CXCL1 in WT mice significantly reduced L5Tx-induced mechanical hypersensitivity. CD40 KO mice also displayed higher levels of Ly6G (neutrophil marker) RNA expression in the lumbar spinal cord post-L5Tx. Altogether, our data suggest that CD4+ T lymphocytes and microglial CD40 mediate their pro-nociceptive effects in part by promoting selected chemokine responses, and more importantly, CXCL1 can play an anti-nociceptive role in peripheral nerve injury induced neuropathic pain, which is possibly mediated by infiltrating neutrophils.
    Expression and clinical role of long non-coding RNA in high-grade serous carcinoma
    Natalie Filippov-Levy, Gynecologic Oncology - 2018
    Abstract
    Objective To profile long non-coding RNA (lncRNA) expression at the various anatomic sites of high-grades serous carcinoma (HGSC) and in effusion-derived exosomes. Methods LncRNA profiling was performed on 60 HGSC specimens, including 10 ovarian tumors, 10 solid metastases and 10 malignant effusions, as well as exosomes from 30 effusion supernatants. Anatomic site-related expression of ESRG, Link-A, GAS5, MEG3, GATS, PVT1 H19, Linc-RoR, HOTAIR and MALAT1 was validated by quantitative PCR and assessed for clinical relevance in a series of 77 HGSC effusions, 40 ovarian carcinomas, 21 solid metastases and 42 supernatant exosomes. Results Significantly different (p < 0.05) expression of 241, 406 and 3634 lncRNAs was found in comparative analysis of the ovarian tumors to solid metastases, effusions and exosomes, respectively. Cut-off at two-fold change in lncRNA expression identified 54 lncRNAs present at the 3 anatomic sites and in exosomes. Validation analysis showed significantly different expression of 5 of 10 lncRNAs in the 4 specimen groups (ESRG, Link-A, MEG3, GATS and PVT1, all p < 0.001). Higher ESRG levels in HGSC effusions were associated with longer overall survival in the entire effusion cohort (p = 0.023) and in patients with pre-chemotherapy effusions tapped at diagnosis (p = 0.048). Higher Link-A levels were associated with better overall (p = 0.015) and progression-free (p = 0.023) survival for patients with post-chemotherapy effusions. Link-A was an independent prognostic marker in Cox multivariate analysis in the latter group (p = 0.045). Conclusions We present the first evidence of differential LncRNA expression as function of anatomic site in HGSC. LncRNA levels in HGSC effusions are candidate prognostic markers.
    Trichloroethylene perturbs HNF4a expression and activity in the developing chick heart
    Alondra P. Harris, Toxicology Letters - 2018
    Abstract
    Exposure to trichloroethylene (TCE) is linked to formation of congenital heart defects in humans and animals. Prior interactome analysis identified the transcription factor, Hepatocyte Nuclear Factor 4 alpha (HNF4a), as a potential target of TCE exposure. As a role for HNF4a is unknown in the heart, we examined developing avian hearts for HNF4a expression and for sensitivity to TCE and the HNF4a agonist, Benfluorex. In vitro analysis using a HNF4a reporter construct showed both TCE and HFN4a to be antagonists of HNF4a-mediated transcription at the concentrations tested. HNF4a mRNA is expressed transiently in the embryonic heart during valve formation and cardiac development. Embryos were examined for altered gene expression in the presence of TCE or Benfluorex. TCE altered expression of selected mRNAs including HNF4a, TRAF6 and CYP2C45. There was a transition between inhibition and induction of marker gene expression in embryos as TCE concentration increased. Benfluorex was largely inhibitory to selected markers. Echocardiography of exposed embryos showed reduced cardiac function with both TCE and Benfluorex. Cardiac contraction was reduced by 29% and 23%, respectively at 10 ppb. The effects of TCE and Benfluorex on autocrine regulation of HNF4a, selected markers and cardiac function argue for a functional interaction of TCE and HNF4a. Further, the dose-sensitive shift between inhibition and induction of marker expression may explain the nonmonotonic-like dose response observed with TCE exposure in the heart.
    CXCR4/CXCR7/CXCL12 axis promotes an invasive phenotype in medullary thyroid carcinoma
    Thomas A. Werner, British Journal of Cancer - 2017
    Abstract
    CXCR4/CXCR7/CXCL12 axis promotes an invasive phenotype in medullary thyroid carcinoma
    Glycogen Synthase Kinase-3 Modulates Cbl-b and Constrains T Cell Activation
    Charles W. Tran, The Journal of Immunology - 2017
    Abstract
    The decision between T cell activation and tolerance is governed by the spatial and temporal integration of diverse molecular signals and events occurring downstream of TCR and costimulatory or coinhibitory receptor engagement. The PI3K–protein kinase B (PKB; also known as Akt) signaling pathway is a central axis in mediating proximal signaling events of TCR and CD28 engagement in T cells. Perturbation of the PI3K–PKB pathway, or the loss of negative regulators of T cell activation, such as the E3 ubiquitin ligase Cbl-b, have been reported to lead to increased susceptibility to autoimmunity. In this study, we further examined the molecular pathway linking PKB and Cbl-b in murine models. Our data show that the protein kinase GSK-3, one of the first targets identified for PKB, catalyzes two previously unreported phosphorylation events at Ser476 and Ser480 of Cbl-b. GSK-3 inactivation by PKB abrogates phosphorylation of Cbl-b at these two sites and results in reduced Cbl-b protein levels. We further show that constitutive activation of PKB in vivo results in a loss of tolerance that is mediated through the downregulation of Cbl-b. Altogether, these data indicate that the PI3K–PKB–GSK-3 pathway is a novel regulatory axis that is important for controlling the decision between T cell activation and tolerance via Cbl-b.
    Effect of different salinities on gene expression and activity of digestive enzymes in the thick-lipped grey mullet (Chelon labrosus)
    I. M. Pujante, Fish Physiology and Biochemistry - 2017
    Abstract
    The effects of different environmental salinities (0, 12, 40, and 55 ppt) on pepsinogen 2 (pga2), trypsinogen 2 (try2), chymotrypsinogen (ctr), and pancreatic alpha-amylase (amy2a) gene expression, and on the total activities of their corresponding enzymes, were assessed in Chelon labrosus juveniles, after their corresponding full-complementary DNA sequences were cloned. Furthermore, the quantitative effect of different salinities on the hydrolysis of feed protein by fish digestive enzymes was evaluated using an in vitro system. Relative pga2 expression levels were significantly higher in animals maintained at 12 ppt, while a significantly higher gene expression level for ctr and try2 was observed at 40 ppt. amy2a gene expression showed its maximum level at 40 ppt and the lowest at 55 ppt. A significant reduction in the activity of amylase with the increase in salinity was observed, whereas the maximum activity for alkaline proteases was observed in individuals maintained at 40 ppt. A negative effect of high salinity on the action of proteases was confirmed by the in vitro assay, indicating a decreased efficiency in the digestive function in C. labrosus when maintained at high environmental salinities. Nevertheless, individuals can live under different environmental salinities, even though gene expression is different and the enzymatic activities are not maintained at the highest studied salinity. Therefore, compensatory mechanisms should be in place. Results are discussed on the light of the importance as a new species for aquaculture.
    Comparative transcriptome analysis reveals conserved branching morphogenesis related genes involved in chamber formation of catfish swimbladder
    Qiang Fu, Physiological Genomics - 2017
    Abstract
    The swimbladder is an internal gas-filled organ in teleosts. Its major function is to regulate buoyancy. Swimbladder exhibits great variations in size, shape, number of compartments or chambers among teleosts. However, the genomic control of swimbladder variations is unknown. Channel catfish (Ictalurus punctatus), blue catfish (Ictalurus furcatus), and their F1 hybrids of female channel catfish x male blue catfish (C×B hybrid catfish) provide a good model to investigate the swimbladder morphology, because channel catfish possess a single-chambered swimbladder whereas blue catfish possess a bi-chambered swimbladder; and C×B hybrid catfish possess a bi-chambered swimbladder but with significantly reduced posterior chamber. Here we determined the transcriptional profiles of swimbladder from channel catfish, blue catfish, and C×B hybrid catfish. We examined their transcriptomes at both the fingerling and adult stages. Through comparative transcriptome analysis, approximately 4,000 differentially expressed genes (DEGs) were identified. Among these DEGs, members of the Wnt signaling pathway (wnt1, wnt2, nfatc1, rac2), Hedgehog signaling pathway (shh), and growth factors (fgf10, igf-1) were identified. As these genes were known to be important for branching morphogenesis of mammalian lung and of mammary glands, their association with budding of posterior chamber primordium and progressive development of bi-chambered swimbladder in fish suggested that these branching morphogenesis related genes and their functions in branching are evolutionarily conserved across a broad spectrum of species.
    Phenotypic and functional characterization of porcine bone marrow monocyte subsets
    Fernández-Caballero, Developmental & Comparative Immunology - 2017
    Abstract
    Monocytes comprise several subsets with distinct phenotypes and functional capacities. Based on CD163 expression, two major monocyte subsets can be discriminated in the porcine bone marrow. The CD163+ cells expressed higher levels of SLA-DR, Siglec-1, CD11R1 and CD16 when compared to CD163- monocytes, whereas no remarkable differences were observed in the expression of other markers analyzed. Gene expression analysis showed differential expression of several chemokine receptor and TLR genes. Both subsets phagocytosed microspheres with similar efficiency. However, CD163- cells tended to produce higher levels of ROS in response to PMA, whereas CD163+ cells were more efficient in endocytosing and processing antigens (DQ-OVA). CD163- monocytes produced higher levels of TNF-α and IL-10 than CD163+ cells when stimulated with LPS or Imiquimod. Both subsets produced similar amounts of IL-8 in response to LPS; however, CD163+ cells produced more IL-8 after Imiquimod stimulation. Whether these subsets represent different developmental stages, and how are they related remain to be investigated.
    Transcriptomic difference in bovine blastocysts following vitrification and slow freezing at morula stage
    Alisha Gupta, PLOS ONE - 2017
    Abstract
    Cryopreservation is known for its marked deleterious effects on embryonic health. Bovine compact morulae were vitrified or slow-frozen, and post-warm morulae were cultured to the expanded blastocyst stage. Blastocysts developed from vitrified and slow-frozen morulae were subjected to microarray analysis and compared with blastocysts developed from unfrozen control morulae for differential gene expression. Morula to blastocyst conversion rate was higher (P < 0.05) in control (72%) and vitrified (77%) than in slow-frozen (34%) morulae. Total 20 genes were upregulated and 44 genes were downregulated in blastocysts developed from vitrified morulae (fold change ≥ ± 2, P < 0.05) in comparison with blastocysts developed from control morulae. In blastocysts developed from slow-frozen morulae, 102 genes were upregulated and 63 genes were downregulated (fold change ≥ ± 1.5, P < 0.05). Blastocysts developed from vitrified morulae exhibited significant changes in gene expression mainly involving embryo implantation (PTGS2, CALB1), lipid peroxidation and reactive oxygen species generation (HSD3B1, AKR1B1, APOA1) and cell differentiation (KRT19, CLDN23). However, blastocysts developed from slow-frozen morulae showed changes in the expression of genes related to cell signaling (SPP1), cell structure and differentiation (DCLK2, JAM2 and VIM), and lipid metabolism (PLA2R1 and SMPD3). In silico comparison between blastocysts developed form vitrified and slow-frozen morulae revealed similar changes in gene expression as between blastocysts developed from vitrified and control morulae. In conclusion, blastocysts developed form vitrified morulae demonstrated better post-warming survival than blastocysts developed from slow-frozen morulae but their gene expression related to lipid metabolism, steroidogenesis, cell differentiation and placentation changed significantly (≥ 2 fold). Slow freezing method killed more morulae than vitrification but those which survived up to blastocyst stage did not express ≥ 2 fold change in their gene expression as compared with blastocysts from control morulae.
    Carbohydrates digestion and metabolism in the spiny lobster (Panulirus argus): biochemical indication for limited carbohydrate utilization
    Leandro Rodríguez-Viera, PeerJ - 2017
    Abstract
    As other spiny lobsters, Panulirus argus is supposed to use preferentially proteins and lipids in energy metabolism, while carbohydrates are well digested but poorly utilized. The aim of this study was to evaluate the effect of dietary carbohydrate level on digestion and metabolism in the spiny lobster P. argus. We used complementary methodologies such as post-feeding flux of nutrients and metabolites, as well as measurements of α-amylase expression and activity in the digestive tract. Lobsters readily digested and absorbed carbohydrates with a time-course that is dependent on their content in diet. Lobster showed higher levels of free glucose and stored glycogen in different tissues as the inclusion of wheat flour increased. Modifications in intermediary metabolism revealed a decrease in amino acids catabolism coupled with a higher use of free glucose as carbohydrates rise up to 20%. However, this effect seems to be limited by the metabolic capacity of lobsters to use more than 20% of carbohydrates in diets. Lobsters were not able to tightly regulate α-amylase expression according to dietary carbohydrate level but exhibited a marked difference in secretion of this enzyme into the gut. Results are discussed to highlight the limitations to increasing carbohydrate utilization by lobsters. Further growout trials are needed to link the presented metabolic profiles with phenotypic outcomes.
    Increased Chalcone Synthase (CHS) expression is associated with dicamba resistance in Kochia scoparia
    Dean J. Pettinga, Pest Management Science - 2017
    Abstract
    BACKGROUND Resistance to the synthetic auxin herbicide dicamba is increasingly problematic in Kochia scoparia. The resistance mechanism in an inbred dicamba-resistant K. scoparia line (9425R) was investigated using physiological and transcriptomics (RNA-Seq) approaches. RESULTS No differences were found in dicamba absorption or metabolism between 9425R and a dicamba-susceptible line, but 9425R was found to have significantly reduced dicamba translocation. Known auxin-responsive genes ACC synthase (ACS) and indole-3-acetic acid amino synthetase (GH3) were transcriptionally induced following dicamba treatment in dicamba-susceptible K. scoparia but not in 9425R. Chalcone synthase (CHS), the gene regulating synthesis of the flavonols quertecin and kaemperfol, was found to have two-fold higher transcription in 9425R both without and 12 h after dicamba treatment. Increased CHS transcription co-segregated with dicamba resistance in a forward genetics screen using an F2 population. CONCLUSION Prior work has shown that the flavonols quertecin and kaemperfol compete with auxin for intercellular movement and vascular loading via ATP-binding cassette subfamily B (ABCB) membrane transporters. The results of this study support a model in which constitutively increased CHS expression in the meristem produces more flavonols that would compete with dicamba for intercellular transport by ABCB transporters, resulting in reduced dicamba translocation.
    High-fat diet exposure, regardless of induction of obesity, is associated with altered expression of genes critical to normal ovulatory function
    Natalie M. Hohos, Molecular and Cellular Endocrinology - 2017
    Abstract
    We evaluated the impact of high-fat diet (HFD) on ovarian gene expression. Female 5-week-old C57BL/6J mice were fed a 60% HFD or standard chow for 10 weeks. HFD-fed mice were then separated into obese (HF-Ob) and lean (HF-Ln) based on body weight. HFD exposure led to impairment of the estrous cycle, changes in hormones affecting reproduction, and decreased primordial follicles regardless of the development of obesity. RNA-sequencing of whole ovaries identified multiple genes with altered expression after HFD, with 25 genes displaying decreased expression in both HF-Ln and HF-Ob mice compared to the chow-fed controls (q < 0.05). Several of these 25 genes are involved in normal ovarian functions, including ovulation (Edn2, Tnfaip6, Errfi1, Prkg2, and Nfil3), luteinization (Edn2), and luteolysis (Nr4a1). Taken together, elevated dietary fat intake, regardless of obesity, is associated with impaired estrous cycle, depletion of the ovarian reserve, and altered expression of genes critical to normal ovulatory function.
    CmpX Affects Virulence in Pseudomonas aeruginosa Through the Gac/Rsm Signaling Pathway and by Modulating c-di-GMP Levels
    Anjali Y. Bhagirath, The Journal of Membrane Biology - 2017
    Abstract
    Pseudomonas aeruginosa is an ubiquitous organism which is able to infect and colonize many types of hosts including humans. Colonization of P. aeruginosa in chronic infections leads to the formation of biofilms, which are difficult to eradicate. P. aeruginosa is capable of regulating its virulence factors in response to external environment triggers and its signaling mechanism involves two-component regulatory systems and small molecules such as bis-(3′–5′)-cyclic dimeric guanosine monophosphate. PA1611-RetS-GacS/A-RsmA/Y/Z is a key regulatory pathway in P. aeruginosa that controls several virulence factors and biofilm formation. We have previously identified a conserved cytoplasmic membrane protein cmpX (PA1775), as a regulator for PA1611 expression. In this study, we demonstrate that cmpX regulates virulence, and controls biofilm formation in P. aeruginosa as well as provide evidence showing that cmpX affects Gac/Rsm pathway, possibly by modulating intra-cellular c-di-GMP levels. A cmpX knockout showed significantly decreased promoter activity of exoS (PA1362) and increased activity of small RNA, RsmY. As compared to the wild-type PAO1, cmpX mutant had elevated intracellular c-di-GMP level as measured indirectly by cdrA (PA4625) activity, as well as increased expression of wspR (PA3702), a c-di-GMP synthase. The transcription of the major outer membrane porin gene oprF (PA1777), and sigma factor sigX (PA1776) was also significantly decreased in the cmpX mutant. Biolog phenotype microarray experiments further indicated that the cmpX knockout mutant had increased sensitivity to membrane detergents and antibiotics such as lauryl sulfobetaine, tobramycin, and vancomycin. These results point to a significant role of cmpX in P. aeruginosa virulence and colonization.
    Unraveling vasotocinergic, isotocinergic and stress pathways after food deprivation and high stocking density in the gilthead sea bream
    Arleta Krystyna Skrzynska, Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology - 2017
    Abstract
    The influence of chronic stress, induced by food deprivation (FD) and/or high stocking density (HSD), was assessed on stress, vasotocinergic and isotocinergic pathways of the gilthead sea bream (Sparus aurata). Fish were randomly assigned to one of the following treatments: (1) fed at low stocking density (LSD-F; 5kg·m−3); (2) fed at high stocking density (HSD-F, 40kg·m−3); (3) food-deprived at LSD (LSD-FD); and (4) food-deprived at HSD (HSD-FD). After 21days, samples from plasma, liver, hypothalamus, pituitary and head-kidney were collected. Both stressors (FD and HSD) induced a chronic stress situation, as indicated by the elevated cortisol levels, the enhancement in corticotrophin releasing hormone (crh) expression and the down-regulation in corticotrophin releasing hormone binding protein (crhbp) expression. Changes in plasma and liver metabolites confirmed a metabolic adjustment to cope with energy demand imposed by stressors. Changes in avt and it gene expression, as well as in their specific receptors (avtrv1a, avtrv2 and itr) at central (hypothalamus and pituitary) and peripheral (liver and head-kidney) levels, showed that vasotocinergic and isotocinergic pathways are involved in physiological changes induced by FD or HSD, suggesting that different stressors are handled through different stress pathways in S. aurata.
    Concomitant external pneumatic compression treatment with consecutive days of high intensity interval training reduces markers of proteolysis
    Cody T. Haun, European Journal of Applied Physiology - 2017
    Abstract
    PurposeTo compare the effects of external pneumatic compression (EPC) and sham when used concurrently with high intensity interval training (HIIT) on performance-related outcomes and recovery-related molecular measures.MethodsEighteen recreationally endurance-trained male participants (age: 21.6 ± 2.4 years, BMI: 25.7 ± 0.5 kg/m2, VO2peak: 51.3 ± 0.9 mL/kg/min) were randomized to balanced sham and EPC treatment groups. Three consecutive days of HIIT followed by EPC/sham treatment (Days 2–4) and 3 consecutive days of recovery (Days 5–7) with EPC/sham only on Days 5–6 were employed. Venipuncture, flexibility and pressure-to-pain threshold (PPT) measurements were made throughout. Vastus lateralis muscle was biopsied at PRE (i.e., Day 1), 1-h post-EPC/sham treatment on Day 2 (POST1), and 24-h post-EPC/sham treatment on Day 7 (POST2). 6-km run time trial performance was tested at PRE and POST2.ResultsNo group × time interaction was observed for flexibility, PPT, or serum measures of creatine kinase (CK), hsCRP, and 8-isoprostane. However, there was a main effect of time for serum CK (p = 0.005). Change from PRE in 6-km run times at POST2 were not significantly different between groups. Significant between-groups differences existed for change from PRE in atrogin-1 mRNA (p = 0.018) at the POST1 time point (EPC: − 19.7 ± 8.1%, sham: + 7.7 ± 5.9%) and atrogin-1 protein concentration (p = 0.013) at the POST2 time point (EPC: − 31.8 ± 7.5%, sham: + 96.0 ± 34.7%). In addition, change from PRE in poly-Ub proteins was significantly different between groups at both the POST1 (EPC: − 26.0 ± 10.3%, sham: + 34.8 ± 28.5%; p = 0.046) and POST2 (EPC: − 33.7 ± 17.2%, sham: + 21.4 ± 14.9%; p = 0.037) time points.ConclusionsEPC when used concurrently with HIIT and in subsequent recovery days reduces skeletal muscle markers of proteolysis.
    Shear Stress Upregulates Regeneration-Related Immediate Early Genes in Liver Progenitors in 3D ECM-like Microenvironments
    Kenichiro Nishii, Journal of Cellular Physiology - 2017
    Abstract
    The role of fluid stresses in activating the hepatic stem/progenitor cell regenerative response is not well understood. This study hypothesized that immediate early genes (IEGs) with known links to liver regeneration will be upregulated in liver progenitor cells (LPCs) exposed to in vitro shear stresses on the order of those produced from elevated interstitial flow after partial hepatectomy. The objectives were: (1) to develop a shear flow chamber for application of fluid stress to LPCs in 3D culture; and (2) to determine the effects of fluid stress on IEG expression in LPCs. Two hours of shear stress exposure at ∼4 dyn/cm2 was applied to LPCs embedded individually or as 3D spheroids within a hyaluronic acid/collagen I hydrogel. Results were compared against static controls. Quantitative reverse transcriptase polymerase chain reaction was used to evaluate the effect of experimental treatments on gene expression. Twenty-nine genes were analyzed, including IEGs and other genes linked to liver regeneration. Four IEGs (CFOS, IP10, MKP1, ALB) and three other regeneration-related genes (WNT, VEGF, EpCAM) were significantly upregulated in LPCs in response to fluid mechanical stress. LPCs maintained an early to intermediate stage of differentiation in spheroid culture in the absence of the hydrogel, and addition of the gel initiated cholangiocyte differentiation programs which were abrogated by the onset of flow. Collectively the flow-upregulated genes fit the pattern of an LPC-mediated proliferative/regenerative response. These results suggest that fluid stresses are potentially important regulators of the LPC-mediated regeneration response in liver. This article is protected by copyright. All rights reserved
    Chronic BMY7378 treatment alters behavioral circadian rhythms
    Jhenkruthi Vijaya Shankara, European Journal of Neuroscience - 2017
    Abstract
    The mammalian circadian clock is synchronized to the day:night cycle by light. Serotonin modulates the circadian effects of light, with agonists inhibiting response to light and antagonists enhancing responses to light. A special class of serotonergic compounds, the mixed 5-HT1A agonist/antagonists, potentiate light-induced phase advances by up to 400% when administered acutely. In this study, we examine the effects of one of these mixed 5-HT1A agonist/antagonists, BMY7378, when administered chronically. Thirty adult male hamsters were administered either vehicle or BMY7378 via surgically implanted osmotic minipumps over a period of 28 days. In a light:dark cycle, chronic BMY7378 advanced the phase angle of entrainment, prolonged the duration of the active phase, and attenuated the amplitude of the wheel running rhythm during the early night. In constant darkness, chronic treatment with BMY7378 significantly attenuated light-induced phase advances, but had no significant effect on light-induced phase delays. Non-photic phase shifts to daytime administration of a 5-HT1A/7 agonist were also attenuated by chronic BMY7378 treatment. qRT-PCR analysis revealed that chronic BMY7378 treatment upregulated mRNA for 5-HT1A and 5-HT1B receptors in the hypothalamus, and downregulated mRNA for 5-HT1A and monoamine oxidase-A in the brainstem. These results highlight adaptive changes of serotonin receptors in the brain to chronic treatment with BMY7378, and link such up- and down-regulation to changes in important circadian parameters. Such long-term changes to the circadian system should be considered when patients are treated chronically with drugs that alter serotonergic function. This article is protected by copyright. All rights reserved.
    Competitive elimination and virulence property alteration of Campylobacter jejuni by genetically engineered Lactobacillus casei
    Zajeba Tabashsum, Food Control - 2017
    Abstract
    Probiotics, prebiotics, or a combination of these two referred to as synbiotics, have emerged as a promising natural and alternative approach to make the sustainable animal farming. Previously, we reported that in the presence of prebiotic like components such as peanut flour, Lactobacillus produced more metabolites and inhibited several enteric pathogens. In this study, we tested a genetically modified lactic acid-producing bacterial strain Lactobacillus casei (LC), that produced large amounts of bioactive compounds including conjugated linoleic acid (CLA), in inhibiting enteric bacterial pathogens and improving host immune systems. The genetically engineered LC strain, LC+mcra (overexpressed mcra gene in LC) effectively eliminated Campylobacter jejuni (CJ) in co-culture condition without any stimulation with prebiotic like components. LC+mcra alone inhibited the growth of CJ completely by 48 h (P < 0.05) similarly the combine effect of LC with prebiotic like component, peanut flour. Cell free culture supernatants (CFCSs) of LC+mcra was also effective in growth reduction of CJ most efficiently (p < 0.05), followed by CFCSs of LC with peanut flour (p < 0.05). In co-culture conditions, LC with peanut flour, LC+mcra and their CFCSs reduced the adherence and invasion ability of CJ to both HD-11 and HeLa cells. Physicochemical properties and gene expressions related to CJ virulence were also altered by CFCSs treatments significantly. These findings suggest, LC+mcra can be an alternative in controlling CJ infection along with other beneficial attributes of LC.
    Exercise training attenuates experimental autoimmune encephalomyelitis by peripheral immunomodulation rather than direct neuroprotection
    Ofira Einstein, Experimental Neurology - 2017
    Abstract
    Conflicting results exist on the effects of exercise training (ET) on Experimental Autoimmune Encephalomyelitis (EAE), nor is it known how exercise impacts on disease progression. We examined whether ET ameliorates the development of EAE by modulating the systemic immune system or exerting direct neuroprotective effects on the CNS. Healthy mice were subjected to 6weeks of motorized treadmill running. The Proteolipid protein (PLP)-induced transfer EAE model in mice was utilized. To assess effects of ET on systemic autoimmunity, lymph-node (LN)-T cells from trained- vs. sedentary donor mice were transferred to naïve recipients. To assess direct neuroprotective effects of ET, PLP-reactive LN-T cells were transferred into recipient mice that were trained prior to EAE transfer or to sedentary mice. EAE severity was assessed in vivo and the characteristics of encephalitogenic LN-T cells derived from PLP-immunized mice were evaluated in vitro. LN-T cells obtained from trained mice induced an attenuated clinical and pathological EAE in recipient mice vs. cells derived from sedentary animals. Training inhibited the activation, proliferation and cytokine gene expression of PLP-reactive T cells in response to CNS-derived autoantigen, but strongly enhanced their proliferation in response to Concanavalin A, a non-specific stimulus. However, there was no difference in EAE severity when autoreactive encephalitogenic T cells were transferred to trained vs. sedentary recipient mice. ET inhibits immune system responses to an auto-antigen to attenuate EAE, rather than generally suppressing the immune system, but does not induce a direct neuro-protective effect against EAE.
    Influence of stress factors on intestinal epithelial injury and regeneration
    Carol Lee, Pediatric Surgery International - 2017
    Abstract
    PurposeLgr5+ intestinal epithelial stem cells (ISCs) crucial for intestinal epithelial regeneration are impaired during necrotizing enterocolitis. This study aims to investigate the influence of different stressors on intestinal epithelial injury and regeneration in vitro.MethodsIntestinal epithelial cells (IEC-18) were exposed to stressors such as lipopolysaccharide, hydrogen peroxide, and serum. Cell viability was assessed using MTT assay at 18 and 24 h. IL-6 and Lgr5 gene expressions were measured using qPCR.ResultsIEC-18 cell viability decreased 18 h following administration of lipopolysaccharide, hydrogen peroxide, and low serum concentration. However, after 24 h, the decrease in cell viability was observed only in higher, but not in lower concentrations of lipopolysaccharide and hydrogen peroxide. IL-6 expression increased in all groups compared to control. Lgr5 expression was up-regulated in cells exposed to a single stressor, but down-regulated when multiple stressors were administered.ConclusionLipopolysaccharide, hydrogen peroxide, or low serum induced IEC-18 injury. The upregulation of Lgr5 expression after exposure to a single stressor suggests that minor injury to IEC-18 induces Lgr5+ ISCs to stimulate repair. Conversely, when IEC-18 cells were exposed to multiple stressors, Lgr5 expression was reduced. We speculate that this finding is similar to what happens in NEC when multiple stressors cause impairment of intestinal epithelium regeneration.
    Pegylated interferon beta in the treatment of the Theiler's murine encephalomyelitis virus mouse model of multiple sclerosis
    Francesca Gilli, Journal of Neuroimmunology - 2017
    Abstract
    We evaluated the effects of pegylated-interferonβ-1a (pegIFNβ) therapy on intrathecal antibody responses, disability progression, and viral load in the CNS in mice infected with the Theiler's virus (TMEV), an animal model of progressive disability in Multiple Sclerosis (MS). The lack of a direct antiviral activity in the CNS, the absence of any effect upon the intrathecal immune response, and the failure to treat disease progression, indicate that the immunomodulatory effects of pegIFNβ-1a likely occur in the systemic circulation rather than within the CNS. These results may be relevant to the relative lack of effect of IFNβ in progressive MS relative to relapsing MS.
    In roots of Arabidopsis thaliana, the damage-associated molecular pattern AtPep1 is a stronger elicitor of immune signalling than flg22 or the chitin heptamer
    Lorenzo Poncini, PLOS ONE - 2017
    Abstract
    Plants interpret their immediate environment through perception of small molecules. Microbe-associated molecular patterns (MAMPs) such as flagellin and chitin are likely to be more abundant in the rhizosphere than plant-derived damage-associated molecular patterns (DAMPs). We investigated how the Arabidopsis thaliana root interprets MAMPs and DAMPs as danger signals. We monitored root development during exposure to increasing concentrations of the MAMPs flg22 and the chitin heptamer as well as of the DAMP AtPep1. The tissue-specific expression of defence-related genes in roots was analysed using a toolkit of promoter::YFPN lines reporting jasmonic acid (JA)-, salicylic acid (SA)-, ethylene (ET)- and reactive oxygen species (ROS)- dependent signalling. Finally, marker responses were analysed during invasion by the root pathogen Fusarium oxysporum. The DAMP AtPep1 triggered a stronger activation of the defence markers compared to flg22 and the chitin heptamer. In contrast to the tested MAMPs, AtPep1 induced SA- and JA-signalling markers in the root and caused a severe inhibition of root growth. Fungal attack resulted in a strong activation of defence genes in tissues close to the invading fungal hyphae. The results collectively suggest that AtPep1 presents a stronger danger signal to the Arabidopsis root than the MAMPs flg22 and chitin heptamer.
    Sex-specific phenotypes and metabolism-related gene expression in juvenile sticklebacks
    Alberto Velando, Behavioral Ecology - 2017
    Abstract
    To fully understand the evolution of sexual dimorphism, it is necessary to study how genetic and developmental systems function to generate sex-specific phenotype as well as sex-specific selection. Males and females show different patterns of energy storage and mitochondrial metabolism from early stages of life, and this may underlie sex-specific developmental pathway to shape both juvenile and adult phenotype. Here, we examined sex-specific relationships between juvenile morphology and behavior, and transcriptional profiles of 4 candidate genes related to mitochondrial function in the 3-spined stickleback. This study provides, for the first time to our knowledge, evidence for sex differences in melanin pigmentation and antipredator behavior as well as the expression of mitochondria-related genes in juvenile sticklebacks. Males were paler and bolder, and overexpressed genes involved in mitochondrial respiration and antioxidant enzymes compared to females. Relationships between phenotypic traits and gene expression were also sex-specific. In general, females showed stronger positive correlations between body size or pigmentation and the expression of genes involved in mitochondrial biogenesis and activity. In both sexes, more fearful individuals overexpressed those genes. Our results suggest that mitochondrial function may either facilitate or constrain sex-specific responses to selection on dimorphic phenotype, possibly generating intralocus sexual conflict on the transcriptional regulation of mito-nuclear genes during ontogeny. This study highlights that mitochondrial regulation plays an important role in the process of phenotypic differentiation between the 2 sexes from early stages of life before apparent sexual dimorphism appears.
    More than just antibodies: Protective mechanisms of a mucosal vaccine against fish pathogen Flavobacterium columnare
    Dongdong Zhang, Fish & Shellfish Immunology - 2017
    Abstract
    A recently developed attenuated vaccine for Flavobacterium columnare has been demonstrated to provide superior protection for channel catfish, Ictalurus punctatus, against genetically diverse columnaris isolates. We were interested in examining the mechanisms of this protection by comparing transcriptional responses to F. columnare challenge in vaccinated and unvaccinated juvenile catfish. Accordingly, 58 day old fingerling catfish (28 days post-vaccination or unvaccinated control) were challenged with a highly virulent F. columnare isolate (BGSF-27) and gill tissues collected pre-challenge (0 h), and 1 h and 2 h post infection, time points previously demonstrated to be critical in early host-pathogen interactions. Following RNA-sequencing and transcriptome assembly, differential expression (DE) analysis within and between treatments revealed several patterns and pathways potentially underlying improved survival of vaccinated fish. Most striking was a pattern of dramatically higher basal expression of an array of neuropeptides (e.g. somatostatin), hormones, complement factors, and proteases at 0 h in vaccinated fish. Previous studies indicate these are likely the preformed mediators of neuroendocrine cells and/or eosinophilic granular (mast-like) cells within the fish gill. Following challenge, these elements fell to almost undetectable levels (>100-fold downregulated) by 1 h in vaccinated fish, suggesting their rapid release and/or cessation of synthesis following degranulation. Concomitantly, levels of pro-inflammatory cytokines (IL-1b, IL-8, IL-17) were induced in unvaccinated fish. In contrast, in vaccinated catfish, we observed widespread induction of genes needed for collagen deposition and tissue remodeling. Taken together, our results indicate an important component of vaccine protection in fish mucosal tissues may be the sensitization, proliferation and arming of resident secretory cells in the period between primary and secondary challenge.
    Di(2-ethylhexyl) phthalate and diethyl phthalate disrupt lipid metabolism, reduce fecundity and shortens lifespan of Caenorhabditis elegans
    Ajay Pradhan, Chemosphere - 2017
    Abstract
    The widespread use of phthalates is of major concern as they have adverse effects on many different physiological functions, including reproduction, metabolism and cell differentiation. The aim of this study was to compare the toxicity of the widely-used di (2-ethydlhexyl) phthalate (DEHP) with its substitute, diethyl phthalate (DEP). We analyzed the toxicity of these two phthalates using Caenorhabditis elegans as a model system. Gene expression analysis following exposure during the L1 to young adult stage showed that DEHP and DEP alter the expression of genes involved in lipid metabolism and stress response. Genes associated with lipid metabolism, including fasn-1, pod-2, fat-5, acs-6 and sbp-1, and vitellogenin were upregulated. Among the stress response genes, ced-1 wah-1, daf-21 and gst-4 were upregulated, while ctl-1, cdf-2 and the heat shock proteins (hsp-16.1, hsp-16.48 and sip-1) were downregulated. Lipid staining revealed that DEHP significantly increased lipid content following 1 μM exposure, however, DEP required 10 μM exposure to elicit an effect. Both DEHP and DEP reduced the fecundity at 1 μM concentration. Lifespan analysis indicated that DEHP and DEP reduced the average lifespan from 14 days in unexposed worms to 13 and 12 days, respectively. Expression of lifespan associated genes showed a correlation to shortened lifespan in the exposed groups. As reported previously, our data also indicates that the banned DEHP is toxic to C. elegans, however its substitute DEP has not been previously tested in this model organism and our data revealed that DEP is equally potent as DEHP in regulating C. elegans physiological functions.
    Impaired fertility and motor function in a zebrafish model for classic galactosemia
    Jo M. Vanoevelen, Journal of Inherited Metabolic Disease - 2017
    Abstract
    Classic galactosemia is a genetic disorder of galactose metabolism, caused by severe deficiency of galactose-1-phosphate uridylyltransferase (GALT) enzyme activity due to mutations of the GALT gene. Its pathogenesis is still not fully elucidated, and a therapy that prevents chronic impairments is lacking. In order to move research forward, there is a high need for a novel animal model, which allows organ studies throughout development and high-throughput screening of pharmacologic compounds. Here, we describe the generation of a galt knockout zebrafish model and present its phenotypical characterization. Using a TALEN approach, a galt knockout line was successfully created. Accordingly, biochemical assays confirm essentially undetectable galt enzyme activity in homozygotes. Analogous to humans, galt knockout fish accumulate galactose-1-phosphate upon exposure to exogenous galactose. Furthermore, without prior exposure to exogenous galactose, they exhibit reduced motor activity and impaired fertility (lower egg quantity per mating, higher number of unsuccessful crossings), resembling the human phenotype(s) of neurological sequelae and subfertility. In conclusion, our galt knockout zebrafish model for classic galactosemia mimics the human phenotype(s) at biochemical and clinical levels. Future studies in our model will contribute to improved understanding and management of this disorder.
    Tissue-engineered magnetic cell sheet patches for advanced strategies in tendon regeneration
    Ana I. Gonçalves, Acta Biomaterialia - 2017
    Abstract
    Tendons are powerful 3D biomechanically structures combining a few cells in an intrincated and highly hierarchical niche environment. When tendon homeostasis is compromised, restoration of functionality upon injury is limited and requires alternatives to current augmentation or replacement strategies. Cell sheet technologies are a powerful tool for the fabrication of living extracellular-rich patches towards regeneration of tenotopic defects. Thus, we originally propose the development of magnetically responsive tenogenic patches through magnetic cell sheet (magCSs) technology that enable the remote control upon implantation of the tendon-mimicking constructs. A Tenomodulin positive (TNMD+) subpopulation of cells sorted from a crude population of human adipose stem cells (hASCs) previously identified as being prone to tenogenesis was selected for the magCSs patch construction. We investigated the stability, the cellular co-location of the iron oxide nanoparticles (MNPs), as well as the morphology and mechanical properties of the developed magCSs. Moreover, the expression of tendon markers and collagenous tendon-like matrix were further assessed under the actuation of an external magnetic field. Overall, this study confirms the potential to bioengineer tendon patches using a magnetic cell sheet construction with magnetic responsiveness, good mechanoelastic properties and a tenogenic prone stem cell population envisioning cell-based functional therapies towards tendon regeneration. The concept of magnetic force-based tissue engineering may assist the development of innovative solutions to treat tendon (or other tissues) disorders upon remote control of biological processes as cell migration or differentiation. Herein, we originally fabricated magnetic responsive cell sheets (magCSs) with a Tenomodulin positive subpopulation of adipose tissue derived stem cells identified to commit to the tenogenic lineage. To the best of authors knowledge, this is the first time a tendon oriented strategy resorting on magCSsis reported. Moreover, the promising role of tenogenic living constructs fabricated as magnetically responsive ECM-rich patches is highlighted, envisioning the stimulation of endogenous regenerative mechanisms. Altogether, these findings contribute to future stem cell studies and their translation toward tendon therapies.
    Treatment of Theiler’s virus-induced demyelinating disease with teriflunomide
    Francesca Gilli, Journal of NeuroVirology - 2017
    Abstract
    Teriflunomide is an oral therapy approved for the treatment of relapsing remitting multiple sclerosis (MS), showing both anti-inflammatory and antiviral properties. Currently, it is uncertain whether one or both of these properties may explain teriflunomide’s beneficial effect in MS. Thus, to learn more about its mechanisms of action, we evaluated the effect of teriflunomide in the Theiler’s encephalomyelitis virus-induced demyelinating disease (TMEV-IDD) model, which is both a viral infection and an excellent model of the progressive disability of MS. We assessed the effects of the treatment on central nervous system (CNS) viral load, intrathecal immune response, and progressive neurological disability in mice intracranially infected with TMEV. In the TMEV-IDD model, we showed that teriflunomide has both anti-inflammatory and antiviral properties, but there seemed to be no impact on disability progression and intrathecal antibody production. Notably, benefits in TMEV-IDD were mostly mediated by effects on various cytokines produced in the CNS. Perhaps the most interesting result of the study has been teriflunomide’s antiviral activity in the CNS, indicating it may have a role as an antiviral prophylactic and therapeutic compound for CNS viral infections.
    Mechanisms of pathogen virulence and host susceptibility in virulent Aeromonas hydrophila infections of channel catfish (Ictalurus punctatus)
    Eric Peatman, Aquaculture - 2017
    Abstract
    An emerging pathotype of Aeromonas hydrophila (vAh) has been responsible for widespread farm losses in the US catfish industry over the last decade. While our genetic and biochemical understanding of vAh has been greatly enhanced in this time frame, our ability to reliably induce the disease in the laboratory has remained limited. Taking cues from observed farm conditions associated with outbreaks, here we perturbed iron scavenging dynamics and catfish feeding status. Addition of a xenosiderophore, deferoxamine mesylate (DFO), to vAh cultures prior to immersion challenge significantly increased virulence in several vAh isolates but not in a non-epidemic strain. DFO addition did not impact vAh growth dynamics or perturb iron-sensitive gene pathways, but did significantly enhance hemolysis of catfish blood. Furthermore, hours between last feeding and immersion challenge (postprandial status), was observed to be a critical determinant of catfish susceptibility. Fish with a full gastrointestinal tract had significantly lower survival than those in a fasted state, and this effect was cumulative with that of DFO-enhanced vAh virulence. Taken together, our results not only provide a more robust challenge model, they offer actionable insights into pond level host-pathogen-environmental interactions potentially underlying vAh pathogenesis.
    Biopsy-derived intestinal epithelial cell cultures for pathway based stratification of patients with inflammatory bowel disease
    Wiebe Vanhove, Journal of Crohn's and Colitis - 2017
    Abstract
    BackgroundEndoplasmic reticulum stress was shown to be pivotal in the pathogenesis of inflammatory bowel disease. Despite progress in IBD drug development, not more than one third of patients achieve steroid-free remission and mucosal healing with current therapies. Furthermore, patient stratification tools for therapy selection are lacking. We aimed to identify and quantify epithelial ER stress in a patient-specific manner in an attempt towards personalized therapy.MethodsA biopsy-derived intestinal epithelial cell culture system was developed and characterized. ER stress was induced by thapsigargin and quantified with a BiP ELISA on cell lysates from 35 patients with known genotypes who were grouped based on the number of IBD-associated ER stress and autophagy risk alleles.ResultsThe epithelial character of the cells was confirmed by E-cadherin, ZO-1 and MUC2 staining and CK-18,CK-20 and LGR5 gene expression. Patients with 3 risk alleles had higher median epithelial BiP-induction (vs. untreated) levels compared to patients with 1 or 2 risk alleles (p=0.026 and 0.043, respectively). When autophagy risk alleles were included and patients were stratified in genetic risk quartiles, patients in Q2, Q3 and Q4 had significantly higher ER stress (BiP) when compared to Q1 (p=0.034, 0.040 and 0.034, respectively).ConclusionWe developed and validated an ex vivo intestinal epithelial cell culture system and showed that patients with more ER stress and autophagy risk alleles have augmented epithelial ER stress responses. We thus presented a personalized approach whereby patient-specific defects can be identified which in turn could help in selecting tailored therapies.
    Fetal sex alters maternal anti-Mullerian hormone during pregnancy in cattle
    Anja Stojsin-Carter, Animal Reproduction Science - 2017
    Abstract
    Anti-Mullerian hormone (AMH) is expressed by both male and female fetuses during mammalian development, with males expressing AMH earlier and at significantly higher concentration. The aim of the current study was to explore the potential impact of pregnancy and fetal sex on maternal AMH and to determine if plasma (Pl) AMH or placenta intercotyledonary membrane and cotyledonary AMH receptor 2 (AMHR2) mRNA expression differ in pregnant cows carrying male vs. female fetuses. AMH levels in blood were measured using a bovine optimized ELISA kit. Cows pregnant with a male fetus were observed to have a significantly greater difference in Pl AMH between day 35 and 135 of gestation. Average fetal AMH level between 54 and 220 days of gestation was also observed to be significantly higher in male vs. female fetuses. Intercotyledonary membranes and cotyledons were found to express AMHR2 between days 38 and 80 of gestation at similar levels in both fetal sexes. These findings support the hypothesis that fetal sex alters maternal Pl AMH during pregnancy in cattle.
    Data supporting the functional role of Eleven-nineteen Lysine-rich Leukemia 3 (ELL3) in B cell lymphoma cell line cells
    Lou-Ella M. M. Alexander, Data in Brief - 2017
    Abstract
    The data presented here are related to the research article entitled “Selective expression of the transcription elongation factor ELL3 in B cells prior to ELL2 drives proliferation and survival” (Alexander et al., 2017) [1]. The cited research article characterizes Eleven-nineteen Lysine-rich Leukemia 3 (ELL3) expression in the B cell compartment and functional dependence in B lymphoma cell lines. This data report describes the mRNA expression pattern in a panel of cell lines representing the B cell compartment, supplementing the protein expression data presented in the associated research report. In addition, a reanalysis is presented of publicly available mRNA expression data from primary murine B cells to reveal dynamic regulation of the ELL family members post LPS stimulation (Barwick et al., 2016) [2]. The effect of ELL3 depletion on cell morphology, latent Epstein Barr Virus (EBV) lytic replication and differentiation markers in a Burkitt's lymphoma (BL) cell line cells are presented.
    Susceptibility of Human Cumulus Cells to Bisphenol A In Vitro
    Abdallah Mansur, Reproductive Toxicology - 2017
    Abstract
    Bisphenol A (BPA) is detectable in follicular fluid. However, the effect of BPA exposure on human cumulus cells (CC) that surround the oocyte and are crucial for oocyte competence has been largely unexplored. We exposed primary cultures of CC to increasing concentrations of BPA [0,0.002, 0.02 and 20 μg/mL] and tested the effects of BPA on the expression of genes associated with apoptosis using quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR); we also assessed the effect of BPA on apoptosis by staining with anti-caspase 3. Exposure to 20 μg/mL BPA led to significantly decreased expression of CDC20, BUB1 B and HAS2 (p < 0.03), increased expression of TRIB3 and LUM (p ≤ 0.005), and increased frequency of cells positive for anti-CASP3 (p = 0.03), compared to control. Our results imply that BPA may lead to ovarian toxicity by increasing CC apoptosis and provide an important molecular mechanism for the effect of BPA on human CC in vitro.
    The role of Tec kinase signaling pathways in the development of Mallory Denk Bodies in balloon cells in alcoholic hepatitis
    N. Afifiyan, Experimental and Molecular Pathology - 2017
    Abstract
    Several research strategies have been used to study the pathogenesis of alcoholic hepatitis (AH). These strategies have shown that various signaling pathways are the target of alcohol in liver cells. However, few have provided specific mechanisms associated with Mallory-Denk Bodies (MDBs) formed in Balloon cells in AH. The formation of MDBs in these hepatocytes is an indication that the mechanisms of protein quality control have failed. The MDB is the result of aggregation and accumulation of proteins in the cytoplasm of balloon degenerated liver cells. To understand the mechanisms that failed to degrade and remove proteins in the hepatocyte from patients suffering from alcoholic hepatitis, we investigated the pathways that showed significant up regulation in the AH liver biopsies compared to normal control livers (Liu et al., 2015). Analysis of genomic profiles of AH liver biopsies and control livers by RNA-seq revealed different pathways that were up regulated significantly. In this study, the focus was on Tec kinase signaling pathways and the genes that significantly interrupt this pathway. Quantitative PCR and immunofluorescence staining results, indicated that several genes and proteins are significantly over expressed in the livers of AH patients that affect the Tec kinase signaling to PI3K which leads to activation of Akt and its downstream effectors.
    Didox (3,4-dihydroxybenzohydroxamic acid) suppresses IgE-mediated mast cell activation through attenuation of NFκB and AP-1 transcription
    Jamie Josephine Avila McLeod, Cellular Immunology - 2017
    Abstract
    Mast cell activation via the high-affinity IgE receptor (FcεRI) elicits production of inflammatory mediators central to allergic disease. As a synthetic antioxidant and a potent ribonucleotide reductase (RNR) inhibitor, Didox (3,4-dihyroxybenzohydroxamic acid) has been tested in clinical trials for cancer and is an attractive therapeutic for inflammatory disease. We found that Didox treatment of mouse bone marrow-derived mast cells (BMMC) reduced IgE-stimulated degranulation and cytokine production, including IL-6, IL-13, TNF and MIP-1a (CCL3). These effects were consistent using BMMC of different genetic backgrounds and peritoneal mast cells. While the RNR inhibitor hydroxyurea had little or no effect on IgE-mediated function, high concentrations of the antioxidant N-acetylcysteine mimicked Didox-mediated suppression. Furthermore, Didox increased expression of the antioxidant genes superoxide dismutase and catalase, and suppressed DCFH-DA fluorescence, indicating reduced reactive oxygen species production. Didox effects were not due to changes in FcεRI expression or cell viability, suggesting it inhibits signaling required for inflammatory cytokine production. In support of this, we found that Didox reduced FcεRI-mediated AP-1 and NFκB transcriptional activity. Finally, Didox suppressed mast cell-dependent, IgE-mediated passive systemic anaphylaxis in vivo. These data demonstrate the potential use for Didox as a means of antagonizing mast cell responses in allergic disease.
    A phenotype combining hidradenitis suppurativa with Dowling-Degos disease caused by a founder mutation in PSENEN
    M. Pavlovsky, British Journal of Dermatology - 2017
    Abstract
    Dowling-Degos disease, featuring reticulate pigmentation, and familial hidradenitis suppurativa share many clinical features including autosomal dominant inheritance, flexural location and follicular defects. The co-existence of the two disorders was recently found to result from mutations in PSENEN, encoding protein presenilin enhancer gamma-secretase subunit. Here we report 4 additional families of Jewish Ashkenazi origin who presented with clinical features characteristic of both disorders. All patients were found to carry the same, heterozygous mutation in PSENEN (c.168T>G, p.Y56X). Haplotype analysis revealed that the mutation originated from a common ancestor. Dowling-Degos disease- as well as hidradenitis suppurativa-associated genes have been shown to encode important regulators of Notch signaling. Accordingly, using a reporter assay, we demonstrated decreased Notch activity in patient's keratinocytes. The present data confirm the genetic basis of the combined Dowling-Degos disease-hidradenitis suppurativa phenotype and suggest that Notch signaling may play a central role in the pathogenesis of this rare condition. This article is protected by copyright. All rights reserved.
    Design of Boron Nitride/Gelatin Electrospun Nanofibers for Bone Tissue Engineering
    Sakthivel Nagarajan, ACS Applied Materials & Interfaces - 2017
    Abstract
    Gelatin is a biodegradable biopolymer obtained by collagen denaturation, which shows poor mechanical properties. Hence, improving its mechanical properties is very essential towards the fabrication of efficient nontoxic material for biomedical applications. For this aim, various methods are employed using external fillers such as ceramics or bioglass. In this report, we introduce boron nitride (BN) reinforced gelatin as a new class of two dimensional biocompatible nanomaterials. The effect of the nanofiller on the mechanical behavior is analyzed. BN is efficiently exfoliated using the biopolymer gelatin as shown through Fourier transform infrared (FTIR) spectroscopy and X-ray diffraction (XRD). The exfoliated BN reinforces gelatin electrospun fibers, which results in an increase in the Young’s modulus. The ESM are stable after the glutaraldehyde cross-linking and the fibrous morphology is preserved. The cross-linked gelatin/BN ESM is highly bioactive in forming bone like hydroxyapatite as shown by scanning electron microscopy. Due to their enhanced mineralization ability, the cross-linked ESM have been tested on human bone cells (HOS osteosarcoma cell line). The cell attachment, proliferation and biocompatibility results show that the ESM are nontoxic and biodegradable. The analysis of osteoblast gene expression and the measurement of alkaline phosphatase activity confirm that these materials are suitable for bone tissue engineering.
    Wnt7a induces a unique phenotype of monocyte-derived macrophages with lower phagocytic capacity and differential expression of pro-and anti-inflammatory cytokines
    Jennillee Wallace, Immunology - 2017
    Abstract
    The variation of macrophage functions suggests the involvement of multiple signaling pathways in fine tuning their differentiation. Macrophages that originate from monocytes in the blood migrate to tissue in response to homeostatic or “danger” signals and undergo substantial morphological and functional modifications to meet the needs of the dominant signals in the microenvironment. Wnts are secreted glycoproteins that play a significant role in organ and cell differentiation, yet their impact on monocyte differentiation is not clear. In this study, we assessed the role of Wnt1 and Wnt7a on the differentiation of monocytes and the subsequent phenotype and function of monocyte-derived macrophages (MDMs). We show that Wnt7a decreased the expression of CD14, CD11b, CD163 and CD206 whereas Wnt1 had no effect. Wnt7a effect on CD11b was also observed in the brain and spleen of Wnt7a -/- adult brain mouse tissue and in embryonic Wnt7a -/- tissue. Wnt7a reduced the phagocytic capacity of M-MDM and M1-like MDMs, decreased IL-10 and IL-12 secretion and increased IL-6 secretion. Collectively, these findings demonstrate that Wnt7a generates an MDM phenotype with both pro-inflammatory and alternative MDM cytokine profiles and reduced phagocytic capacity. As such, Wnt7a can have a significant impact on macrophage responses in health and disease. This article is protected by copyright. All rights reserved.
    An amphipathic trans -acting phosphorothioate RNA element delivers an uncharged phosphorodiamidate morpholino sequence in mdx mouse myotubes
    H.V. Jain, RSC Advances - 2017
    Abstract
    An efficient method for the delivery of uncharged polyA-tailed phosphorodiamidate morpholino sequences (PMO) in mammalian cells consists of employing a synthetic 8-mer amphipathic trans-acting poly-2′-O-methyluridylic thiophosphate triester element (2′-OMeUtaPS) as a transfection reagent. Unlike the dTtaPS DNA-based element, this RNA element is potent at delivering polyA-tailed PMO sequences to HeLa pLuc 705 cells or to myotube muscle cells. However, much like dTtaPS, the 2′-OMeUtaPS-mediated internalization of PMO sequences occurs through an energy-dependent mechanism; macropinocytosis appears to be the predominant endocytic pathway used for cellular uptake. The transfected PMO sequences induce alternate splicing of either the pre-mRNA encoding luciferase in HeLa pLuc 705 cells or the excision of exon 23 from the pre-mRNA encoding dystrophin in myotube muscle cells of the mdx mouse model of muscular dystrophy with an efficiency comparable to that of commercial cationic lipid reagents but without detrimental cytotoxicity.
    Trichostatin A Sensitizes Hepatocellular Carcinoma Cells to Enhanced NK Cell-mediated Killing by Regulating Immune-related Genes
    Sangsu Shin, Cancer Genomics - Proteomics - 2017
    Abstract
    Background/Aim: Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related death worldwide. The ability of HCC to avoid immune detection is considered one of the main factors making it difficult to cure. Abnormal histone deacetylation is thought to be one of the mechanisms for HCC immune escape, making histone deacetylases (HDACs) attractive targets for HCC treatment. Here, we investigated the effect of trichostatin A (TSA), a highly potent HDAC inhibitor, on HCC (HepG2) gene expression and function. Materials and Methods: A genome wide-transcriptional microarray was used to identify genes regulated by TSA in HepG2 cells. Gene Ontology was used to identify pathways regulated by TSA, and these changes were confirmed by qPCR. The effect of TSA on natural killer (NK) cell-mediated killing of HCC cell lines were analyzed by both flow cytometry and LDH cytotoxicity assay. A study was also conducted in a Balb/c nude mice xenograft model to assess the anti-tumor activity of TSA. Results: TSA regulated the transcription of numerous innate immunity & tumor antigen recognition-associated genes, such as ULBP1 and RAET1G, in HCC cells. In vivo, TSA reduced tumor cell growth in an NK cell-dependent manner. In vitro, TSA treatment of HepG2 cells rendered them more susceptible to NK cell-mediated killing while increasing the expression of NKGD2 ligands, including ULBP1/2/3 and MICA/B. TSA also induced direct killing of HCC cells by stimulating apoptosis. Conclusion: TSA likely increases killing of HCC cells indirectly by increasing NK cell-directed killing and directly by increasing apoptosis.
    Helicobacter pylori infection perturbs iron homeostasis in gastric epithelial cells
    Sebastian E. Flores, PLOS ONE - 2017
    Abstract
    The iron deficiency anaemia that often accompanies infection with Helicobacter pylori may reflect increased uptake of iron into gastric epithelial cells. Here we show an infection-associated increase in total intracellular iron levels was associated with the redistribution of the transferrin receptor from the cell cytosol to the cell surface, and with increased levels of ferritin, an intracellular iron storage protein that corresponded with a significant increase in lysosomal stores of labile iron. In contrast, the pool of cytosolic labile iron was significantly decreased in infected cells. These changes in intracellular iron distribution were associated with the uptake and trafficking of H. pylori through the cells, and enhanced in strains capable of expressing the cagA virulence gene. We speculate that degradation of lysosomal ferritin may facilitate H. pylori pathogenesis, in addition to contributing to bacterial persistence in the human stomach.
    Selective expression of the transcription elongation factor ELL3 in B cells prior to ELL2 drives proliferation and survival
    Lou-Ella M. M. Alexander, Molecular Immunology - 2017
    Abstract
    B cell activation is dependent on a large increase in transcriptional output followed by focused expression on secreted immunoglobulin as the cell transitions to an antibody producing plasma cell. The rapid transcriptional induction is facilitated by the release of poised RNA pol II into productive elongation through assembly of the super elongation complex (SEC). We report that a SEC component, the Eleven -nineteen Lysine-rich leukemia (ELL) family member 3 (ELL3) is dynamically up-regulated in mature and activated human B cells followed by suppression as B cells transition to plasma cells in part mediated by the transcription repressor PRDM1. Burkitt’s lymphoma and a sub-set of Diffuse Large B cell lymphoma cell lines abundantly express ELL3. Depletion of ELL3 in the germinal center derived lymphomas results in severe disruption of DNA replication and cell division along with increased DNA damage and cell death. This restricted utilization and survival dependence reveal a key step in B cell activation and indicate a potential therapeutic target against B cell lymphoma’s with a germinal center origin.
    In silico analysis and effects of environmental salinity in the expression and activity of digestive α-amylase and trypsins from the euryhaline crab Neohelice granulata
    Antonela Asaro, Canadian Journal of Zoology - 2017
    Abstract
    Studies on molecular characteristics and modulation of expression of α-amylase and trypsin in the hepatopancreas of intertidal euryhaline crabs are lacking. In this work, we cloned and studied by in silico approaches the characteristics of cDNA sequences for α-amylase and two trypsins isoforms, as well as the effect of environmental salinity on gene expression and protein activities in hepatopancreas of Neohelice granulata (Dana,1852), as a good invertebrate model species. The cDNA sequence of -amylase is 1,637 bp long, encoding 459 amino acid residues. Trypsin 1 and 2 are 689 bp and 1,174 bp long, encoding 204 and 151 amino acid residues, respectively. Multiple sequence alignment of deduced protein sequences revealed the presence of conserved motifs found in other invertebrates. In crabs acclimated at 37 psu (hypo-regulation),α-amylase mRNA level and total pancreatic amylase activity were higher than at 10 psu (hyper-regulation) and 35 psu (osmoconformation). Trypsin 1 mRNA levels increased at 37 psu wh...
    Artemisinin permeability via Caco-2 cells increases after simulated digestion of Artemisia annua leaves
    Matthew R. Desrosiers, Journal of Ethnopharmacology - 2017
    Abstract
    Ethnopharmacological relevance Artemisia annua has been used for > 2000 yrs to treat fever and is more recently known for producing the important antimalarial drug, artemisinin. Aim of the study Artemisinin combination therapies (ACTs) are effective for treating malaria, but are often unavailable to those in need. Dried leaves of A. annua (DLA) have recently been studied as a cost effective alternative to traditional ACTs. DLA was shown to dramatically increase oral bioavailability compared to pure artemisinin, so more investigation into the mechanisms causing this increased bioavailability is needed. Materials and methods In this study, we used a simulated digestion system coupled with Caco-2 cell permeability assays to investigate the intestinal permeability of DLA compared to pure artemisinin. We also determined the effects of different phytochemicals (7 flavonoids, 3 monoterpenes, 2 phenolic acids, scopoletin and inulin) and the cytochrome P450 isoform CYP3A4 on artemisinin intestinal permeability. Results Artemisinin permeability, when delivered as digested DLA, significantly increased by 37% (Papp = 8.03 × 10−5 cm s−1) compared to pure artemisinin (Papp = 5.03 × 10−5 cm s−1). However, none of the phytochemicals tested or CYP3A4 had any significant effect on the intestinal permeability of artemisinin. We also showed that essential oil derived from A. annua negatively affected the intestinal permeability of artemisinin, but only after simulated digestion. Finally, we showed that A. annua essential oil reduced the transepithelial electrical resistance of Caco-2 monolayers, but only in the presence of bile. Although also reduced by essential oils, artemisinin Papp subsequently recovered in the presence of plant matrix. Conclusions These results shed light on the mechanisms by which DLA enhances the oral bioavailability of artemisinin.
    Chronic stress targets adult neurogenesis preferentially in the suprapyramidal blade of the rat dorsal dentate gyrus
    Nuno D. Alves, Brain Structure and Function - 2017
    Abstract
    The continuous generation of new neurons and glial cells in the adult hippocampal dentate gyrus (DG) represents an important form of adult neuroplasticity, involved in normal brain function and behavior but also associated with the etiopathogenesis and treatment of psychiatric disorders. Despite the large number of studies addressing cell genesis along the septotemporal axis, data on the anatomical gradients of cytogenesis along the DG transverse axis is scarce, especially after exposure to stress. As such, in this study we characterized both basal proliferation and survival of adult-born neural cells along the transverse axis of the rat dorsal DG, and after stress exposure. In basal conditions, both proliferating cells and newborn neurons and glial cells were preferentially located at the subgranular zone and suprapyramidal blade. Exposure to chronic stress induced an overall decrease in the generation of adult-born neural cells and, more specifically, produced a regional-specific decrease in the survival of adult-born neurons at the suprapyramidal blade. No particular region-specific alterations were observed on surviving adult-born glial cells. This work reveals, for the first time, a distinct survival profile of adult-born neural cells, neurons and glial cells, among the transverse axis of the DG, in both basal and stress conditions. Our results unveil that adult-born neurons are preferentially located in the suprapyramidal blade and suggest a regional-specific impact of chronic stress in this blade with potential repercussions for its functional significance.
    Assessment of Response of Kidney Tumors to Rapamycin and Atorvastatin in Tsc1+/− Mice
    Ming Hong Shen, Translational Oncology - 2017
    Abstract
    Atorvastatin is widely used to lower blood cholesterol and to reduce risk of cardiovascular disease–associated complications. Epidemiological investigations and preclinical studies suggest that statins such as atorvastatin have antitumor activity for various types of cancer. Tuberous sclerosis (TSC) is a tumor syndrome caused by TSC1 or TSC2 mutations that lead to aberrant activation of mTOR and tumor formation in multiple organs. Previous studies have demonstrated that atorvastatin selectively suppressed growth and proliferation of mouse Tsc2 null embryonic fibroblasts through inhibition of mTOR. However, atorvastatin alone did not reduce tumor burden in the liver and kidneys of Tsc2+/− mice as assessed by histological analysis, and no combination therapy of rapamycin and atorvastatin has been tried. In this study, we used T2-weighted magnetic resonance imaging to track changes in tumor number and size in the kidneys of a Tsc1+/− mouse model and to assess the efficacy of rapamycin and atorvastatin alone and as a combination therapy. We found that rapamycin alone or rapamycin combined with atorvastatin significantly reduced tumor burden, while atorvastatin alone did not. Combined therapy with rapamycin and atorvastatin appeared to be more effective for treating renal tumors than rapamycin alone, but the difference was not statistically significant. We conclude that combined therapy with rapamycin and atorvastatin is unlikely to provide additional benefit over rapamycin as a single agent in the treatment of Tsc-associated renal tumors.
    AR-V7 in Peripheral Whole Blood of Patients with Castration-resistant Prostate Cancer: Association with Treatment-specific Outcome Under Abiraterone and Enzalutamide
    Anna Katharina Seitz, European Urology - 2017
    Abstract
    Background It has been demonstrated that androgen receptor splice variant 7 (AR-V7) expression in circulating tumor cells (CTCs) predicts poor treatment response in metastatic castration-resistant prostate cancer (mCRPC) patients treated with abiraterone or enzalutamide. Objective To develop a practical and robust liquid profiling approach for direct quantification of AR-V7 in peripheral whole blood without the need for CTC capture and to determine its potential for predicting treatment response in mCRPC patients. Design, setting, and participants Whole blood samples from a prospective biorepository of 85 mCRPC patients before treatment initiation with abiraterone (n = 56) or enzalutamide (n = 29) were analyzed via droplet digital polymerase chain reaction. Outcome measurements and statistical analysis The association of AR-V7 status with prostate-specific antigen (PSA) response defined by PSA decline ≥50% and with PSA–progression-free survival (PSA-PFS), clinical PFS, and overall survival (OS) was assessed. Results and limitations High AR-V7 expression levels in whole blood were detectable in 18% (15/85) of patients. No patient with high AR-V7 expression achieved a PSA response, and AR-V7 status was an independent predictor of PSA response in multivariable logistic regression analysis (p = 0.03). High AR-V7 expression was associated with shorter PSA-PFS (median 2.4 vs 3.7 mo; p < 0.001), shorter clinical PFS (median 2.7 vs 5.5 mo; p < 0.001), and shorter OS (median 4.0 vs. 13.9 mo; p < 0.001). On multivariable Cox regression analysis, high AR-V7 expression remained an independent predictor of shorter PSA-PFS (hazard ratio [HR] 7.0, 95% confidence interval [CI] 2.3–20.7; p < 0.001), shorter clinical PFS (HR 2.3, 95% CI 1.1–4.9; p = 0.02), and shorter OS (HR 3.0, 95% CI 1.4–6.3; p = 0.005). Conclusions Testing of AR-V7 mRNA levels in whole blood is a simple and promising approach to predict poor treatment outcome in mCRPC patients receiving abiraterone or enzalutamide. Patient summary We established a method for determining AR-V7 status in whole blood. This test predicted treatment resistance in patients with metastatic castration-resistant prostate cancer undergoing treatment with abiraterone or enzalutamide. Prospective validation is needed before application to clinical practice.
    Variation in the response of tomato (Solanum lycopersicum) breeding lines to the effects of benzo (1,2,3) thiadiazole-7-carbothioic acid S-methyl ester (BTH) on systemic acquired resistance and seed germination
    Paul H. Goodwin, Journal of Phytopathology - 2017
    Abstract
    Genetic variation may play a major role in how plants respond to activators of systemic acquired resistance. To examine this, the defence activator benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester (BTH) was applied to seed of different breeding lines of tomato (Solanum lycopersicum) with diverse pedigrees, and the levels of induced resistance against Pseudomonas syringae pv. tomato, changes in defence gene expression and detrimental effects on seed germination and seedling emergence were measured. Two breeding lines, 7007 and 7024, were selected as non-responsive and responsive to BTH. The SAR-associated genes, SlPR1a and SlPR3b, were induced earlier or more strongly over the control prior to inoculation for line 7024 but not for line 7007. This was not observed for the ISR-related genes, SlPin2 and SlPR2b. BTH inhibition of seed germination and seedling emergence was more delayed in line 7024 than 7007. However, applying BTH as a seed or soil drip reduced the delay. Thus, greater levels of BTH response have both positive (i.e., induced resistance and expression of SAR-related gene expression) and negative (i.e., inhibition of seed germination and seedling emergence) effects and can differ significantly between genotypes. Thus, recommendations for use of induced resistance activators should include plant genotype recommendations and consider possible negative impacts of greater responsiveness.
    Fibroblast Growth Factor 15 Deficiency Increases Susceptibility but does not Improves Repair to Acetaminophen-induced Liver Injury in Mice
    Mingxing Huang, Digestive and Liver Disease - 2017
    Abstract
    The leading cause of acute liver failure (ALF) is hepatotoxicity from acetaminophen (APAP) overdose. However, limited options are available to treat this ALF so stimulating liver regeneration maybe a potential treatment. Our previous study has shown that fibroblast growth factor 15 (FGF15) plays a crucial role in liver regeneration, but the roles of FGF15 in liver injury and repair following APAP-overdose are unknown. In this study, treatment of FGF15 knockout (KO) male mice with APAP at 200, 250, or 300 mg/kg significantly increased the degree of liver injury compared to wild type (WT) mice. To determine the effects of FGF15 deficiency on liver repair following APAP overdose, a similar degree of liver injury was first obtained 24 hrs after treatment of WT and Fgf15 KO mice with APAP at different dosage. Fgf15 KO mice did not differ from WT mice in liver repair following similar degree of liver injury. In conclusion, we showed that FGF15 deficiency renders mice more susceptible to APAP-induced liver injury but did not seem to affect liver repair or regeneration. This study suggests that in contrast to the critical role that FGF15 plays in promoting liver regeneration following partial hepatectomy, this intestine factor is less involved in liver repair after APAP-induced liver injury.
    Validation of a Targeted RNA Sequencing Assay for Kinase Fusion Detection in Solid Tumors
    Julie W.Reeser, The Journal of Molecular Diagnostics - 2017
    Abstract
    Kinase gene fusions are important drivers of oncogenic transformation and can be inhibited with targeted therapies. Clinical grade diagnostics using RNA sequencing to detect gene rearrangements in solid tumors are limited, and the few that are available require prior knowledge of fusion break points. To address this, we have analytically validated a targeted RNA sequencing assay (OSU-SpARKFuse) for fusion detection that interrogates complete transcripts from 93 kinase and transcription factor genes. From a total of 74 positive and 36 negative control samples, OSU-SpARKFuse had 93.3% sensitivity and 100% specificity for fusion detection. Assessment of repeatability and reproducibility revealed 96.3% and 94.4% concordance between intrarun and interrun technical replicates, respectively. Application of this assay on prospective patient samples uncovered OLFM4 as a novel RET fusion partner in a small-bowel cancer and led to the discovery of a KLK2-FGFR2 fusion in a patient with prostate cancer who subsequently underwent treatment with a pan–fibroblast growth factor receptor inhibitor. Beyond fusion detection, OSU-SpARKFuse has built-in capabilities for discovery research, including gene expression analysis, detection of single-nucleotide variants, and identification of alternative splicing events.
    Chronic traumatic stress impairs memory in mice: Potential roles of acetylcholine, neuroinflammation and corticotropin releasing factor expression in the hippocampus
    Ami Bhakta, Behavioural Brain Research - 2017
    Abstract
    Chronic stress in humans can result in multiple adverse psychiatric and neurobiological outcomes, including memory deficits. These adverse outcomes can be more severe if each episode of stress is very traumatic. When compared to acute or short term stress relatively little is known about the effects of chronic traumatic stress on memory and molecular changes in hippocampus, a brain area involved in memory processing. Here we studied the effects of chronic traumatic stress in mice by exposing them to adult Long Evan rats for 28 consecutive days and subsequently analyzing behavioral outcomes and the changes in the hippocampus. Results show that stressed mice developed memory deficits when assayed with radial arm maze tasks. However, chronic traumatic stress did not induce anxiety, locomotor hyperactivity or anhedonia. In the hippocampus of stressed mice interleukin-1β protein expression was increased along with decreased corticotropin releasing hormone (CRH) gene expression. Furthermore, there was a reduction in acetylcholine levels in the hippocampus of stressed mice. There were no changes in brain derived neurotrophic factor (BDNF) or nerve growth factor (NGF) levels in the hippocampus of stressed mice. Gene expression of immediate early genes (Zif268, Arc, C-Fos) as well as glucocorticoid and mineralocorticoid receptors were also not affected by chronic stress. These data demonstrate that chronic traumatic stress followed by a recovery period might lead to development of resilience resulting in the development of selected, most vulnerable behavioral alterations and molecular changes in the hippocampus.
    An inducible form of Nrf2 confers enhanced protection against acute oxidative stresses in RPE cells
    Khiem T. Vu, Experimental Eye Research - 2017
    Abstract
    Increasing evidence suggests that overt oxidative stress within the retina plays an important role in the progression of age-related retinal decline, and in particular, in the disease age-related macular degeneration (AMD). Nuclear factor erythroid 2-like 2 (Nrf2) is a master transcription factor that upregulates numerous of antioxidant/detoxification genes. Nrf2−/− mice develop progressive retinal degeneration that includes the formation of drusen-like deposits, lipofuscin, and sub-retinal pigment epithelium (RPE) deposition of inflammatory proteins. Furthermore, strategies that promote Nrf2 activation have shown promise for the treatment of cone/rod dystrophies and other forms of retinal degeneration. Herein we explored whether utilizing a small molecule-inducible version of Nrf2 confers additional protection against oxidative stresses when compared to a constitutively expressed version of Nrf2. Stable populations of human ARPE-19 cells were generated that express either constitutive FLAG-tagged (FT) Nrf2 (FT cNrf2) or doxycycline (dox)-inducible FT Nrf2 (FT iNrf2) at low levels (∼4.5 fold vs. endogenous). Expression of either FT cNRF2 or FT iNrf2 upregulated canonical antioxidant genes (e.g., NQO1, GCLC). Both FT cNrf2 and FT iNrf2 ARPE-19 cells were protected from cigarette smoke extract-induced nitric oxide generation to similar extents. However, only FT iNrf2 cells demonstrated enhanced resistance to doxorubicin and cumene hydroperoxide-mediated increases in mitochondrial superoxide and lipid peroxidation, respectively, and did so in a dox-dependent manner. These results suggest that therapeutic approaches which conditionally control Nrf2 activity may provide additional protection against acute oxidative stresses when compared to constitutively expressed Nrf2 strategies.
    A Novel Egr-1-Agrin Pathway and Potential Implications for Regulation of Synaptic Physiology and Homeostasis at the Neuromuscular Junction
    Ryen MacDonald, Frontiers in Aging Neuroscience - 2017
    Abstract
    Synaptic transmission requires intricate coordination of the components involved in processing of incoming signals, formation and stabilization of synaptic machinery, neurotransmission, and in all related signaling pathways. Changes to any of these components cause synaptic imbalance and disruption of neuronal circuitry. Extensive studies at the neuromuscular junction (NMJ) have greatly aided in the current understanding of synapses and served to elucidate the underlying physiology as well as associated adaptive and homeostatic processes. The heparansulfate proteoglycan agrin is a vital component of the NMJ, mediating synaptic formation and maintenance in both brain and muscle, but very little is known about direct control of its expression. Here, we investigated the relationship between agrin and transcription factor early growth response-1 (Egr-1), as Egr-1 regulates the expression of many genes involved in synaptic homeostasis and plasticity. Using chromatin immunoprecipitation, cell culture with cell lines derived from brain and muscle, and animal models, we show that Egr-1 binds to the AGRN gene locus and suppresses its expression. When compared with wild type (WT), mice deficient in Egr-1 (Egr-1-/-) display a marked increase in AGRN mRNA and agrin full-length and cleavage fragment protein levels, including the 22 kDa, C-terminal fragment in brain and muscle tissue homogenate. Because agrin is a crucial component of the NMJ, we explored possible physiological implications of the Egr-1-agrin relationship. In the diaphragm, Egr-1-/- mice display increased NMJ motor endplate density, individual area, and area of innervation. In addition to increased density, soleus NMJs also display an increase in fragmented and faint endplates in Egr-1-/- versus WT mice. Moreover, the soleus NMJ electrophysiology of Egr-1-/- mice revealed increased quantal content and motor testing showed decreased movement and limb muscle strength compared with WT. This study provides evidence for the potential involvement of a novel Egr-1-agrin pathway in synaptic homeostatic and compensatory mechanisms at the NMJ. Synaptic homeostasis is greatly affected by the process of aging. These and other data suggest that changes in Egr-1 expression may directly or indirectly promote age-related pathologies.
    Isobutanol production in Synechocystis PCC 6803 using heterologous and endogenous alcohol dehydrogenases
    Rui Miao, Metabolic Engineering Communications - 2017
    Abstract
    Isobutanol is a flammable compound that can be used as a biofuel due to its high energy density and suitable physical and chemical properties. In this study, we examined the capacity of engineered strains of Synechocystis PCC 6803 containing the α-ketoisovalerate decarboxylase from Lactococcus lactis and different heterologous and endogenous alcohol dehydrogenases (ADH) for isobutanol production. A strain expressing an introduced kivd without any additional copy of ADH produced 3 mg L−1 OD750−1 isobutanol in 6 days. After the cultures were supplemented with external addition of isobutyraldehyde, the substrate for ADH, 60.8 mg L−1 isobutanol was produced after 24 h when OD750 was 0.8. The in vivo activities of four different ADHs, two heterologous and two putative endogenous in Synechocystis, were examined and the Synechocystis endogenous ADH encoded by slr1192 showed the highest efficiency for isobutanol production. Furthermore, the strain overexpressing the isobutanol pathway on a self-replicating vector with the strong Ptrc promoter showed significantly higher gene expression and isobutanol production compared to the corresponding strains expressing the same operon introduced on the genome. Hence, this study demonstrates that Synechocystis endogenous AHDs have a high capacity for isobutanol production, and identifies kivd encoded α-ketoisovalerate decarboxylase as one of the likely bottlenecks for further isobutanol production.
    Choroid plexus-cerebrospinal fluid route for monocyte-derived macrophages after stroke
    Ruimin Ge, Journal of Neuroinflammation - 2017
    Abstract
    Choroid plexus (CP) supports the entry of monocyte-derived macrophages (MDMs) to the central nervous system in animal models of traumatic brain injury, spinal cord injury, and Alzheimer’s disease. Whether the CP is involved in the recruitment of MDMs to the injured brain after ischemic stroke is unknown.
    Short-day photoperiods affect expression of genes related to dormancy and freezing tolerance in Norway spruce seedlings
    Elisabeth Wallin, Annals of Forest Science - 2017
    Abstract
    Key MessageGene expression analysis showed that prolonged short day (SD) treatment deepened dormancy and stimulated development of freezing tolerance ofPicea abiesseedlings. Prolonged SD treatment also caused later appearance of visible buds in autumn, reduced risks for reflushing, and promoted earlier spring bud break. ContextShort day (SD) treatment of seedlings is a common practice in boreal forest tree nurseries to regulate shoot growth and prepare the seedlings for autumn planting or frozen storage. AimsThe aim of this study was to examine responses of Norway spruce (Picea abies (L.) Karst.) to a range of SD treatments of different length and evaluate gene expression related to dormancy induction and development of freezing tolerance. MethodsThe seedlings were SD treated for 11 h a day during 7, 14, 21, or 28 days. Molecular tests were performed, and the expression profiles of dormancy and freezing tolerance-related genes were analyzed as well as determination of shoot growth, bud set, bud size, reflushing, dry matter content, and timing of spring bud break. ResultsThe 7-day SD treatment was as effective as longer SD treatments in terminating apical shoot growth. However, short (7 days) SD treatment resulted in later activation of dormancy-related genes and of genes related to freezing tolerance compared to the longer treatments which had an impact on seedling phenology. ConclusionGene expression analysis indicated an effective stimulus of dormancy-related genes when the SD treatment is prolonged for at least 1–2 weeks after shoot elongation has terminated and that seedlings thereafter are exposed to ambient outdoor climate conditions.
    RNAi screen identifies essential regulators of human brain metastasis-initiating cells
    Mohini Singh, Acta Neuropathologica - 2017
    Abstract
    Brain metastases (BM) are the most common brain tumor in adults and are a leading cause of cancer mortality. Metastatic lesions contain subclones derived from their primary lesion, yet their functional characterization is limited by a paucity of preclinical models accurately recapitulating the metastatic cascade, emphasizing the need for a novel approach to BM and their treatment. We identified a unique subset of stem-like cells from primary human patient brain metastases, termed brain metastasis-initiating cells (BMICs). We now establish a BMIC patient-derived xenotransplantation (PDXT) model as an investigative tool to comprehensively interrogate human BM. Using both in vitro and in vivo RNA interference screens of these BMIC models, we identified SPOCK1 and TWIST2 as essential BMIC regulators. SPOCK1 in particular is a novel regulator of BMIC self-renewal, modulating tumor initiation and metastasis from the lung to the brain. A prospective cohort of primary lung cancer specimens showed that SPOCK1 was overexpressed only in patients who ultimately developed BM. Protein–protein interaction network mapping between SPOCK1 and TWIST2 identified novel pathway interactors with significant prognostic value in lung cancer patients. Of these genes, INHBA, a TGF-β ligand found mutated in lung adenocarcinoma, showed reduced expression in BMICs with knockdown of SPOCK1. In conclusion, we have developed a useful preclinical model of BM, which has served to identify novel putative BMIC regulators, presenting potential therapeutic targets that block the metastatic process, and transform a uniformly fatal systemic disease into a locally controlled and eminently more treatable one.
    miR-130b directly targets Arhgap1 to drive activation of a metastatic CDC42-PAK1-AP1 positive feedback loop in Ewing sarcoma
    Laura Satterfield, International Journal of Cancer - 2017
    Abstract
    Ewing Sarcoma (ES) is a highly aggressive bone tumor with peak incidence in the adolescent population. It has a high propensity to metastasize, which is associated with dismal survival rates of approximately 25%. To further understand mechanisms of metastasis we investigated microRNA regulatory networks in ES. Our studies focused on miR-130b due to our analysis that enhanced expression of this microRNA has clinical relevance in multiple sarcomas, including ES. Our studies provide insights into a novel positive feedback network involving the direct regulation of miR-130b and activation of downstream signaling events contributing towards sarcoma metastasis. Specifically, we demonstrated miR-130b induces proliferation, invasion, and migration in vitro and increased metastatic potential in vivo. Using microarray analysis of ES cells with differential miR-130b expression we identified alterations in downstream signaling cascades including activation of the CDC42 pathway. We identified Arhgap1, which is a negative regulator of CDC42, as a novel, direct target of miR-130b. In turn, downstream activation of PAK1 activated the JNK and AP-1 cascades and downstream transcriptional targets including IL-8, MMP1 and CCND1. Furthermore, chromatin immunoprecipitation of endogenous AP-1 in ES cells demonstrated direct binding to an upstream consensus binding site within the miR-130b promoter. Finally, small molecule inhibition of PAK1 blocked miR-130b activation of JNK and downstream AP-1 target genes, including primary miR-130b transcripts, and mir-130b oncogenic properties, thus identifying PAK1 as a novel therapeutic target for ES. Taken together, our findings identify and characterize a novel, targetable miR-130b regulatory network that promotes ES metastasis. This article is protected by copyright. All rights reserved.
    Hepatitis C virus NS5A region mutation in chronic hepatitis C genotype 1 patients who are non-responders to two or more treatments and its relationship with response to a new treatment
    Paloma Muñoz de Rueda, World Journal of Gastroenterology - 2017
    Abstract
    AIM To determine the number of mutations in the NS5A region of the hepatitis C virus (HCV) and its relationship to the response to antiviral therapy in patients with chronic hepatitis C genotype 1 who are non-responders to two or more treatments. METHODS Sequences within HCV NS5A [PKR binding domain (PKRBD) and the interferon-sensitivity-determining region (ISDR)] were analysed via direct sequencing in a selected cohort of 72 patients, with a total of 201 treatments [interferon-alpha (IFN-α), n = 49; IFN-α + ribavirin (RBV), n = 75; pegylated (peg) IFN-α + RBV, n = 47; first-generation direct-acting antivirals (DAAs), n = 13; and second-generation DAAs, n = 17]. Of these, 48/201 achieved a sustained virological response (SVR) and 153/201 achieved no virological response (NVR). RESULTS For both regions, treatments resulting in SVR were associated with more baseline mutations than were treatments resulting in NVR (SVR vs NVR; PKRBD: 5.82 ± 3 vs 4.86 ± 2 mutations, P = 0.045; ISDR: 2.65 ± 2 vs 1.51 ± 1.7 mutations, P = 0.005). A decrease or no change in the number of mutations over time between treatments in the PKRBD or ISDR, as shown by sequencing, was associated with patients who usually failed to respond to treatment (PKRBD, P = 0.02; ISDR, P = 0.001). Moreover, patients showing a post-treatment baseline viral load > 600000 IU/mL and increased ISDR mutations with respect to the previous treatment were 9.21 times more likely to achieve SVR (P = 0.001). CONCLUSION The obtained results show that among patients who have shown no response to two or more antiviral treatments, the likelihood of achieving SVR increases with the genetic variability in the ISDR region (≥ 2 mutations or number of substitutions from the HCV-J and HCV-1 prototype), especially when the viral load is greater than 600000 IU/mL.
    The roles of NF-κB and ROS in regulation of pro-inflammatory mediators of inflammation induction in LPS-stimulated zebrafish embryos
    Eun-Yi Ko, Fish & Shellfish Immunology - 2017
    Abstract
    In this study, the roles of reactive oxygen species (ROS) and NF-κB on inflammation induction in lipopolysaccharide (LPS)-stimulated zebrafish embryos were evaluated using N-acetyl-l-cysteine (NAC) and pyrrolidine dithiocarbamate (PDTC), specific inhibitors of ROS and NF-κB, respectively. LPS-stimulated zebrafish embryos showed increasing production of NO and ROS and expression of iNOS and COX-2 protein, compared to a control group without LPS. However, NAC significantly inhibited production of NO and ROS and markedly suppressed expression of iNOS and COX-2 protein in LPS-stimulated zebrafish embryos. The mRNA expressions of NF-κB such as p65NF-κB and IκB-A were significantly increased after LPS stimulation, whereas PDTC attenuated mRNA expression of NF-κB. IκB was suppressed by PDTC, but not significantly. PDTC also inhibited production of NO and reduced expression of iNOS and COX-2 protein in LPS-stimulated zebrafish embryos. Taken together, these results indicated that LPS increases pro-inflammatory mediators in zebrafish embryos through ROS and NF-κB regulation.
    Alternative strategy for visceral leishmaniosis control: HisAK70-Salmonella Choleraesuis-pulsed Dendritic Cells
    Gustavo Domínguez-Bernal, Comparative Immunology, Microbiology and Infectious Diseases - 2017
    Abstract
    Here, we describe a novel approach that exploits an attenuated mutant of Salmonella enterica serovar Choleraesuis as carrier to deliver a plasmid encoding protein HisAK70. Subsequently, dendritic cells (DCs) were pulsed with this vaccine vector. The aim of this study was to evaluate the effectiveness of the prepared HisAK70-S. Choleraesuis-pulsed DCs (HisAK70-SAL DCs) against visceral leishmaniosis (VL). In our ex vivo model of infection, the prepared formulations could decrease parasite growth by up to 80% by augmenting the production of IL-12p40 and by reducing arginase activity (ARG). Also, BALB/c mice when immunised with this formulation showed significant reduction in parasite burden in both spleen (20% of reduction) and liver (75% of reduction). The balance of the immune ratios IFN-γ/IL-10, TNF-α/IL-10, and IgG2a/IgG1 reflected the acquisition of an improved resistant phenotype in HisAK70-SAL DCs vaccinated mice compared to control mice. Our results suggest that HisAK70-SAL DCs could be a promising alternative approach for vaccine delivery that has the potential to fight Leishmania infantum (L. infantum) infection.
    Next Generation Sequencing to characterise the breaking of bud dormancy using a natural biostimulant in kiwifruit (Actinidia deliciosa)
    Frank A. Hoeberichts, Scientia Horticulturae - 2017
    Abstract
    Dormancy occurs in many woody perennials and fruit trees, and is characterised mainly by the temporal absence of growth. This strategy has an ecological role in ensuring plant survival under non-permissive growing conditions. In kiwifruit (Actinidia deliciosa), buds must be exposed to a certain amount of chilling to complete dormancy and achieve optimal bud break and flowering in spring. However, in warmer geographical areas bud break can be limited by insufficient winter chilling, leading to less and uneven flowering and lower productivity. Several bud break-enhancing compounds have been shown to be effective in kiwifruit, overcoming the negative effects of poor winter chilling. However, their mode of action is still largely unknown. In this work, using a molecular approach based on Next Generation Sequencing, we characterised dormancy and its breaking after the application of the biostimulant Erger®. Samples were collected at different time-points and in distinct geographical areas, namely Italy and New Zealand. Our results indicated that the effect of Erger® depends on the moment of application. Furthermore, molecular mechanisms that characterise a biostimulant treatment for bud break, irrespective of the region and seasonality, were identified. This characterisation opens new perspectives to improve bud break-enhancing compound formulations and identify their best moment of application.
    Biofilm formation and antibiotic resistance in Klebsiella pneumoniae urinary strains
    Claudia Vuotto, Journal of Applied Microbiology - 2017
    Abstract
    Aims Multi-drug resistant Klebsiella pneumoniae has become a relevant healthcare-associated pathogen. Capsule, type 1 and 3 fimbriae (mrkA gene), type 2 quorum-sensing system (luxS), synthesis of D-galactan I (wbbM), LPS transport (wzm) and poly-beta-1,6-N-acetyl-D-glucosamine (pgaA) seem involved in K. pneumoniae biofilm. Non-enzymatic antibiotic resistance is related to non-expression or mutation of porins (OmpK35 and OmpK36), and efflux pump (acrB) over-expression. The aim of this study was to analyse some virulence factors of K. pneumoniae isolates, and to evaluate possible correlations between their antibiotic resistance profile and ability to form biofilm. Methods and Results Quantitative biofilm production assay, congo red agar test and string test were performed on 120 isolates clustered in 56 extensively drug resistant (XDR, 40 MDR and 24 susceptible (S)). Nine representative strains were analyzed by real-time RT-PCR for the expression of antibiotic resistance (OmpK35, OmpK36, acrB) and biofilm production genes (mrkA, luxS,, pga, wbbM, wzm) during planktonic and sessile growth. XDR isolates showed a higher ability to form biofilm (91.07%) and to produce polysaccharides (78.57%) when compared to MDR and S strains. In biofilm-growing XDR strains, 7 out of 8 genes were upregulated, with the only exception of OmpK36. Conclusions XDR strains exhibited phenotypic and genotypic features supporting a significant growth as biofilm. Significance and Impact of Study this study produces new findings that highlight a positive correlation between antibiotic resistance profile and biofilm-forming ability in XDR K. pneumoniae strains. These new evidences might contribute to the progress in selection of therapeutic treatments of infections caused by Klebsiella pneumoniae resistant also to the “last line of defense” antibiotics, i.e. carbapenems. This article is protected by copyright. All rights reserved.
    The receptor tyrosine kinase AXL promotes migration and invasion in colorectal cancer
    Diana J. Uribe, - 2017
    Abstract
    The receptor tyrosine kinases (RTKs) TYRO3, AXL and MERTK (TAM) have well-described oncogenic functions in a number of cancers. Notwithstanding, TAM RTKs are also potent and indispensable inhibitors of inflammation. The combined deletion of Axl and Mertk in mice enhances chronic inflammation and autoimmunity, including increased inflammation in the gut and colitis-associated cancer. On the other hand, deletion of Tyro3 increases the risk of allergic responses. Therefore, the indiscriminate inhibition of these TAM RTKs could result in undesirable immunological diseases. Here we show that AXL, but not MERTK or TYRO3 expression is enhanced in late stage colorectal cancer (CRC) and AXL expression associates with a cell migration gene signature. Silencing AXL or the inhibition of AXL kinase activity significantly inhibits tumor cell migration and invasion. These results indicate that the selective inhibition of AXL alone might confer sufficient therapeutic benefit in CRC, while preserving at least some of the beneficial, anti-inflammatory effects of MERTK and TYRO3 RTKs.
    Cytokine profiling in the prefrontal cortex of Parkinson's Disease and Multiple System Atrophy patients
    Rasmus Rydbirk, Neurobiology of Disease - 2017
    Abstract
    Parkinson's Disease (PD) and Multiple System Atrophy (MSA) are neurodegenerative diseases characterized neuropathologically by alpha-synuclein accumulation in brain cells. This accumulation is hypothesized to contribute to constitutive neuroinflammation, and to participate in the neurodegeneration. Cytokines, which are the main inflammatory signalling molecules, have been identified in blood and cerebrospinal fluid of PD patients, but studies investigating the human brain levels are scarce. It is documented that neurotrophins, necessary for survival of brain cells and known to interact with cytokines, are altered in the basal ganglia of PD patients. In regards to MSA, no major study has investigated brain cytokine or neurotrophin protein expression. Here, we measured protein levels of 18 cytokines (IL-2, 4–8, 10, 12, 13, 17, G-CSF, GM-CSF, IFN-γ, MCP-1, MIP-1α and 1β, TNF-α) and 5 neurotrophins (BDNF, GDNF, bFGF, PDGF-BB, VEGF) in the dorsomedial prefrontal cortex in brains of MSA and PD patients and control subjects. We found altered expression of IL-2, IL-13, and G-CSF, but no differences in neurotrophin levels. Further, in MSA patients we identified increased mRNA levels of GSK3β that is involved in neuroinflammatory pathways. Lastly, we identified increased expression of the neurodegenerative marker S100B, but not CRP, in PD and MSA patients, indicating local rather than systemic inflammation. Supporting this, in both diseases we observed increased MHC class II+ and CD45+ positive cells, and low numbers of infiltrating CD3+ cells. In conclusion, we identified neuroinflammatory responses in PD and MSA which seems more widespread in the brain than neurotrophic changes.
    Effect of dietary polyphenols on fructose uptake by human intestinal epithelial (Caco-2) cells
    Nelson Andrade, Journal of Functional Foods - 2017
    Abstract
    Intake of high-fructose products is associated with metabolic syndrome development. We investigated if dietary polyphenols can interfere with fructose absorption by the human intestinal epithelial Caco-2 cell line. Acutely, several polyphenols induced a significant decrease (15–20%) in 14C-fructose uptake. Chronically, 14C-fructose uptake was also affected by polyphenols; quercetin, chrysin and apigenin (100 µM) were the most effective (±25% decrease). These compounds (100 µM) appear to interfere with both GLUT2 and GLUT5 activity; accordingly, they caused a very marked decrease in the mRNA expression levels of GLUT2 (≅90%) and GLUT5 (≅75%). The effect of these compounds on 14C-fructose uptake does not involve interference with PKC, PKA, PI3K and p38 MAPK intracellular signaling pathways. Quercetin, apigenin and chrysin might decrease the intestinal absorption of fructose. Further studies are needed to test for their beneficial effects on metabolic syndrome.
    Mdm2 selectively suppresses DNA damage arising from inhibition of topoisomerase II independent of p53
    Senturk J.C., Oncogene - 2017
    Abstract
    Mdm2 is often overexpressed in tumors that retain wild-type TP53 but may affect therapeutic response independently of p53. Herein is shown that tumor cells with MDM2 amplification are selectively resistant to treatment with topoisomerase II poisons but not other DNA damaging agents. Tumor cells that overexpress Mdm2 have reduced DNA double-strand breaks in response to doxorubicin or etoposide. This latter result is not due to altered drug uptake. The selective attenuation of DNA damage in response to these agents is dependent on both Mdm2 levels and an intact ubiquitin ligase function. These findings reveal a novel, p53-independent activity of Mdm2 and have important implications for the choice of chemotherapeutic agents in the treatment of Mdm2-overexpressing tumors.
    The Absence of Laminin Alpha 4 in Male Mice Results in Enhanced Energy Expenditure and Increased Beige Subcutaneous Adipose Tissue
    Marcella K. Vaicik, Endocrinology - 2017
    Abstract
    Laminin alpha 4 (LAMA4) is located in the extracellular basement membrane that surrounds each individual adipocyte. Here we show that LAMA4 null (Lama4-/-) mice exhibit significantly higher energy expenditure relative to wild type mice at room temperature and when exposed to a cold challenge, despite similar levels of food intake and locomotor activity. The Lama4-/- mice are resistant to age- and diet-induced obesity. Expression of uncoupling protein 1 (UCP1) is higher in subcutaneous white adipose tissue (sWAT) of Lama4-/- mice relative to wild type animals on either a chow diet or a high-fat diet. In constrast, UCP1 expression was not increased in brown adipose tissue (BAT). Lama4-/- mice exhibit significantly improved insulin sensitivity when compared to WT mice, suggesting improved metabolic function. Overall, these data provide critical evidence for a role of the basement membrane in energy expenditure, weight gain, and systemic insulin sensitivity.
    Didox (3,4-dihydroxybenzohydroxamic acid) suppresses IL-33-induced cytokine production in primary mouse mast cells
    Heather L. Caslin, Cellular Immunology - 2017
    Abstract
    While IgE is considered the primary mediator of mast cell activation, IL-33 contributes substantially in asthma, allergic rhinitis, and atopic dermatitis. To develop effective treatments for allergic disease, it is important to understand the role of therapeutic agents on IL-33 activation. We examined the effect of Didox (3,4-dihydroxybenzohydroxamic acid), an antioxidant and ribonucleotide reductase (RNR) inhibitor, on IL-33-mediated mast cell activation. Didox suppressed IL-6, IL-13, TNF, and MIP-1α (CCL3) production in bone marrow derived mast cells following IL-33 activation. This suppression was observed in different genetic backgrounds and extended to peritoneal mast cells. The antioxidant N-acetylcysteine mimicked the suppression of Didox, albeit at a much higher dose, while the RNR inhibitor hydroxyurea had no effect. Didox substantially suppressed IL-33-mediated NFκB and AP-1 transcriptional activities. These results suggest that Didox attenuates IL-33-induced mast cell activation and should be further studied as a potential therapeutic agent for inflammatory diseases involving IL-33.
    Cellular zinc homeostasis modulates polarization of THP-1-derived macrophages
    Laura Dierichs, European Journal of Nutrition - 2017
    Abstract
    PurposePolarization of macrophages by environmental stimuli leads to the characteristic of different phenotypes that exhibit distinct functions, ranging in a continuous spectrum from pro-inflammatory M1 up to immunoregulatory and wound-healing M2 macrophages. Diseases like cancer, allergic asthma or diabetes are associated with an M1/M2 imbalance. Owing to the importance of the essential trace element zinc for the immune system and its involvement in signal transduction as a second messenger, we investigated the impact of zinc on M1 and M2 polarization of macrophages in vitro.MethodsA polarization model with human THP-1 cells was established and validated with previously described markers using quantitative real-time PCR, Western blot and flow cytometry. Intracellular free Zn2+ was determined with FluoZin-3-AM.ResultsWhereas pSTAT1 and HLA-DR or pSTAT6 and Dectin-1 distinguish between M1 and M2 macrophages, respectively, CD86 and CD206 failed. Depending on the used markers, both zinc supplementation in physiological dose (50 µM) and zinc deficiency promote M1 polarization of THP-1-derived macrophages. Furthermore, zinc supplementation strongly inhibits M2 polarization.ConclusionFor the first time, we show a modulating effect of zinc for the polarization of human macrophages. The strong inhibitory effect of zinc supplementation on M2 polarization indicates a relevance regarding M2-dominated diseases like allergic asthma or cancer. All in all, zinc achieves a great potential for modulating macrophage polarization.
    Maternal Antiviral Immunoglobulin Accumulates in Neural Tissue of Neonates To Prevent HSV Neurological Disease
    Yike Jiang, mBio - 2017
    Abstract
    While antibody responses to neurovirulent pathogens are critical for clearance, the extent to which antibodies access the nervous system to ameliorate infection is poorly understood. In this study on herpes simplex virus 1 (HSV-1), we demonstrate that HSV-specific antibodies are present during HSV-1 latency in the nervous systems of both mice and humans. We show that antibody-secreting cells entered the trigeminal ganglion (TG), a key site of HSV infection, and persisted long after the establishment of latent infection. We also demonstrate the ability of passively administered IgG to enter the TG independently of infection, showing that the naive TG is accessible to antibodies. The translational implication of this finding is that human fetal neural tissue could contain HSV-specific maternally derived antibodies. Exploring this possibility, we observed HSV-specific IgG in HSV DNA-negative human fetal TG, suggesting passive transfer of maternal immunity into the prenatal nervous system. To further investigate the role of maternal antibodies in the neonatal nervous system, we established a murine model to demonstrate that maternal IgG can access and persist in neonatal TG. This maternal antibody not only prevented disseminated infection but also completely protected the neonate from neurological disease and death following HSV challenge. Maternal antibodies therefore have a potent protective role in the neonatal nervous system against HSV infection. These findings strongly support the concept that prevention of prenatal and neonatal neurotropic infections can be achieved through maternal immunization. IMPORTANCE Herpes simplex virus 1 is a common infection of the nervous system that causes devastating neonatal disease. Using mouse and human tissue, we discovered that antiviral antibodies accumulate in neural tissue after HSV-1 infection in adults. Similarly, these antibodies pass to the offspring during pregnancy. We found that antiviral maternal antibodies can readily access neural tissue of the fetus and neonate. These maternal antibodies then protect neonatal mice against HSV-1 neurological infection and death. These results underscore the previously unappreciated role of maternal antibodies in protecting fetal and newborn nervous systems against infection. These data suggest that maternal immunization would be efficacious at preventing fetal/neonatal neurological infections.
    Polyphenols and tri-terpenoids from Olea europaea L. in alleviation of enteric pathogen infections through limiting bacterial virulence and attenuating inflammation
    Mengfei Peng, Journal of Functional Foods - 2017
    Abstract
    Olea europaea L. was known associated with health benefits, however the role of single bioactive component remains to be elucidated. This study was to investigate the operational feasibility of maslinic/oleanolic acid, hydroxytyrosol, and oleuropein from olive in control and alleviation of enteric pathogen infections. Our results indicated that the growth of Lactobacillus was stimulated by maslinic/oleanolic acid up to 4% (w/v) in dose-dependent manner. Hydroxytyrosol (>0.05%) and oleuropein (>0.5%) significantly reduced Salmonella Typhimurium/EHEC within 12 h, altered their physicochemical properties, influenced host cell-pathogen interactions, and especially 0.6–6.0 folds down-regulated the virulence gene expressions. Further molecular analysis revealed that these olive compounds up-regulated anti-inflammatory cytokine IL-10 gene by 3.0–8.0 folds and down-regulated multiple inflammation related genes at various levels. These results suggest that polyphenols in olive may serve as potential preservatives in post-harvest food processing while tri-terpenoids may assist in modulation of gut microflora and reduction of enteric pathogen colonization.
    Bone marrow mesenchymal stem cell-derived CD63+ exosomes transport Wnt3a exteriorly and enhance dermal fibroblast proliferation, migration and angiogenesis in vitro
    Jeffrey D. McBride, Stem Cells and Development - 2017
    Abstract
    Wnts are secreted glycoproteins that regulate stem cell self-renewal, differentiation, and cell-to-cell communication during embryonic development and in adult tissues. Bone marrow mesenchymal stem cells (BM-MSCs) have been shown to stimulate dermis repair and regeneration; however, it is unclear how BM-MSCs may modulate downstream Wnt signaling. While recent reports implicate that Wnt ligands and Wnt messenger RNAs (such as Wnt4) exist within the interior compartment of exosomes, it has been debated whether or not Wnts exist on the exterior surface of exosomes to travel in the extracellular space. To help answer this question, we utilized flow cytometry of magnetic beads coated with anti-CD63 antibodies, and found for the first time, that Wnt3a protein is detectable exteriorly on CD63+ exosomes derived from BM-MSCs over-secreting Wnt3a into serum-free conditioned media (Wnt3a CM). Our data suggests that CD63+ exosomes significantly help transport exterior Wnt3a signal to recipient cells to promote fibroblast and endothelial functions. During purification of exosomes, we unexpectedly found that use of ultracentrifugation alone significantly decreased the ability to detect exteriorly bound Wnt3a on CD63+ exosomes, however, polyethylene glycol-mediated exosome-enrichment prior to exosome-purification (with ultracentrifugation into a sucrose cushion) resulted in exosomes more likely to retain exterior Wnt3a detectability and downstream Wnt/beta-catenin activity. Our findings indicate the important role that purification methods may have on stem cell-derived Wnt-exosome activity in downstream assays. The ability for BM-MSC Wnt3a CM and exosomes to stimulate dermal fibroblast proliferation and migration, as well as endothelial angiogenesis in vitro, was significantly decreased after CD63+-exosome depletion or knockdown of Wnt coreceptor LRP6 in recipient cells, suggesting both are required for optimal Wnt-exosome activity in our system. Thus, BM-MSC-derived CD63+ exosomes are a significant carrier of exterior Wnt3a within high Wnt environments, resulting in downstream fibroblast proliferation, migration and angiogenesis in vitro.
    Dual loss of succinate dehydrogenase (SDH) and complex I activity is necessary to recapitulate the metabolic phenotype of SDH mutant tumors
    Doriane Lorendeau, ResearchGate - 2016
    Abstract
    Mutations in succinate dehydrogenase (SDH) are associated with tumor development and neurodegenerative diseases. Only in tumors, loss of SDH activity is accompanied with the loss of complex I...
    ROS mediates interferon gamma induced phosphorylation of Src, through the Raf/ERK pathway, in MCF-7 human breast cancer cell line
    Kazem Zibara, Journal of Cell Communication and Signaling - 2016
    Abstract
    Interferon gamma (IFN-ɣ) is a pleiotropic cytokine which plays dual contrasting roles in cancer. Although IFN-ɣ has been clinically used to treat various malignancies, it was recently shown to have protumorigenic activities. Reactive oxygen species (ROS) are overproduced in cancer cells, mainly due to NADPH oxidase activity, which results into several changes in signaling pathways. In this study, we examined IFN-ɣ effect on the phosphorylation levels of key signaling proteins, through ROS production, in the human breast cancer cell line MCF-7. After treatment by IFN-ɣ, results showed a significant increase in the phosphorylation of STAT1, Src, raf, AKT, ERK1/2 and p38 signaling molecules, in a time specific manner. Src and Raf were found to be involved in early stages of IFN-ɣ signaling since their phosphorylation increased very rapidly. Selective inhibition of Src-family kinases resulted in an immediate significant decrease in the phosphorylation status of Raf and ERK1/2, but not p38 and AKT. On the other hand, IFN-ɣ resulted in ROS generation, through H2O2 production, whereas pre-treatment with the ROS inhibitor NAC caused ROS inhibition and a significant decrease in the phosphorylation levels of AKT, ERK1/2, p38 and STAT1. Moreover, pretreatment with a selective NOX1 inhibitor resulted in a significant decrease of AKT phosphorylation. Finally, no direct relationship was found between ROS production and calcium mobilization. In summary, IFN-ɣ signaling in MCF-7 cell line is ROS-dependent and follows the Src/Raf/ERK pathway whereas its signaling through the AKT pathway is highly dependent on NOX1.
    Insights into a novel nuclear function for Fascin in the regulation of the amino-acid transporter SLC3A2
    Amine Saad, Scientific Reports - 2016
    Abstract
    Fascin 1 (FSCN1) is a cytoskeleton-associated protein recognized to function primarily in the regulation of cytoskeleton structure and formation of plasma membrane protrusions.
    MicroRNA-126 overexpression rescues diabetes-induced impairment in efferocytosis of apoptotic cardiomyocytes
    Sahana Suresh Babu, Scientific Reports - 2016
    Abstract
    Efferocytosis, a process of clearance of apoptotic cells by phagocytes, is essential for successful resolution of inflammation and maintenance of tissue homeostasis.
    A Ketogenic Diet in Rodents Elicits Improved Mitochondrial Adaptations in Response to Resistance Exercise Training Compared to an Isocaloric Western Diet
    Hayden W. Hyatt, Frontiers in Physiology - 2016
    Abstract
    Purpose: Ketogenic diets (KD) can facilitate weight loss, but their effects on skeletal muscle remain equivocal. In this experiment we investigated the effects of two diets on skeletal muscle mitochondrial coupling, mitochondrial complex activity, markers of oxidative stress, and gene expression in sedentary and resistance exercised rats. Methods: Male Sprague-Dawley rats (9-10 weeks of age, 300-325 g) were fed isocaloric amounts of either a KD (17 g/day, 5.2 kcal/g, 20.2% protein, 10.3% CHO, 69.5% fat, n=16) or a Western diet (WD) (20 g/day, 4.5 kcal/g, 15.2% protein, 42.7% CHO, 42.0% fat, n=16) for 6 weeks. During these six weeks animals were either sedentary (SED, n=8 per diet group) or voluntarily exercised using resistance-loaded running wheels (EXE, n=8 per diet group). Gastrocnemius was excised and used for mitochondrial isolation and biochemical analyses. RESULTS: In the presence of a complex II substrate, the respiratory control ratio (RCR) of isolated gastrocnemius mitochondria was higher (p<0.05) in animals fed the KD compared to animals fed the WD. Complex I and IV enzyme activity was higher (p<0.05) in EXE animals regardless of diet. SOD2 protein levels and GLUT4 and PGC1α mRNA expression were higher (p<0.05) in EXE animals regardless of diet. CONCLUSION: Our data indicate that skeletal muscle mitochondrial coupling of complex II substrates is more efficient in chronically resistance trained rodents fed a KD. These findings may provide merit for further investigation, perhaps on humans.
    Disinhibition of somatostatin-positive GABAergic interneurons results in an anxiolytic and antidepressant-like brain state
    T. Fuchs, Molecular Psychiatry - 2016
    Abstract
    Major depressive disorder (MDD) is associated with reduced concentrations of γ-aminobutyric acid (GABA) that are normalized by antidepressant therapies. Moreover, depressive-like phenotypes of GABAA receptor mutant mice can be reversed by treatment with conventional antidepressants drugs, as well as by subanesthetic doses of ketamine. Thus GABAergic deficits may causally contribute to depressive disorders, while antidepressant therapies may enhance GABAergic synaptic transmission. Here we tested the hypothesis that sustained enhancement of GABAergic transmission alone is sufficient to elicit antidepressant-like behavior, using disinhibition of GABAergic interneurons. We focused on somatostatin-positive (SST+) GABAergic interneurons because of evidence that their function is compromised in MDD. To disinhibit SST+ interneurons, we inactivated the γ2 subunit gene of GABAA receptors selectively in these neurons (SSTCre:γ2f/f mice). Loss of inhibitory synaptic input resulted in increased excitability of SST+ interneurons. In turn, pyramidal cell targets of SST+ neurons showed an increased frequency of spontaneous inhibitory postsynaptic currents. The behavior of SSTCre:γ2f/f mice mimicked the effects of anxiolytic and antidepressant drugs in a number of behavioral tests, without affecting performance in a spatial learning- and memory-dependent task. Finally, brain extracts of SSTCre:γ2f/f mice showed decreased phosphorylation of the eukaryotic elongation factor eEF2, reminiscent of the effects of ketamine. Importantly, these effects occurred without altered activity of the mammalian target of rapamycin pathway nor did they involve altered expression of SST. However, they were associated with reduced Ca2+/calmodulin-dependent auto-phosphorylation of eEF2 kinase, which controls the activity of eEF2 as its single target. Thus enhancing GABAergic inhibitory synaptic inputs from SST+ interneurons to pyramidal cells and corresponding chronic reductions in the synaptic excitation:inhibition ratio represents a novel strategy for antidepressant therapies that reproduces behavioral and biochemical end points of rapidly acting antidepressants.
    A miR-200c/141-BMI1 autoregulatory loop regulates oncogenic activity of BMI1 in cancer cells.
    Manjari Dimri, George Washington University research paper - 2016
    Abstract
    MicroRNAs (miRNAs) are known to function as oncomiRs or tumor suppressors and are important noncoding RNA regulators of oncogenesis. The miR-200c/141 locus on chromosome 12 encodes miR-200c and miR-141, two members of the miR-200 family, which have been shown to function as tumor suppressive miRNAs by targeting multiple oncogenic factors such as polycomb group protein BMI1. Here, we show that BMI1 reciprocally functions as a transcriptional repressor of the miR-200c/141 cluster and that BMI1 inhibitors upregulate expression of miR-200c and miR-141. Our data suggest that BMI1 binds to the miR-200c/141 promoter and regulates it through transcription factor binding motifs E-box 2 and Z-box 1 to repress expression of miR-200c/141 cluster. We also show that PTC-209, a small molecule inhibitor of BMI1 gene expression induces cellular senescence and transcriptionally upregulates expression of miR-200c/141 cluster in breast cancer cells. Furthermore, inhibition of expression of miR-200c or miR-141 overcomes tumor suppressive effects of PTC-209 including induction of cellular senescence and downregulation of breast cancer stem cell phenotype. Therefore, our studies suggest a reciprocal regulation between BMI1 and miR-200c/141 cluster, and that BMI1 inhibitory drugs can further amplify their inhibitory effects on BMI1 via multiple mechanisms including posttranscriptional regulation by upregulating BMI1 targeting miRNAs
    EGF-Induced VEGF Exerts a PI3K-Dependent Positive Feedback on ERK and AKT through VEGFR2 in Hematological In Vitro Models
    Lilian Saryeddine, PLOS ONE - 2016
    Abstract
    EGFR and VEGFR pathways play major roles in solid tumor growth and progression, however, little is known about these pathways in haematological tumors. This study investigated the crosstalk between EGFR and VEGFR2 signaling in two hematological in vitro models: THP1, a human monocytic leukemia, and Raji, a Burkitt’s lymphoma, cell lines. Results showed that both cell lines express EGFR and VEGFR2 and responded to EGF stimulation by activating EGFR, triggering VEGF production and phosphorylating ERK, AKT, and p38 very early, with a peak of expression at 10–20min. Blocking EGFR using Tyrphostin resulted in inhibiting EGFR induced activation of ERK, AKT, and p38. In addition, EGF stimulation caused a significant and immediate increase, within 1min, in pVEGFR2 in both cell lines, which peaked at ~5–10 min after treatment. Selective inhibition of VEGFR2 by DMH4, anti-VEGFR2 antibody or siRNA diminished EGF-induced pAKT and pERK, indicating a positive feedback exerted by EGFR-induced VEGF. Similarly, the specific PI3K inhibitor LY294002, suppressed AKT and ERK phosphorylation showing that VEGF feedback is PI3K-dependent. On the other hand, phosphorylation of p38, initiated by EGFR and independent of VEGF feedback, was diminished using PLC inhibitor U73122. Moreover, measurement of intracellular [Ca2+] and ROS following VEGFR2 inhibition and EGF treatment proved that VEGFR2 is not implicated in EGF-induced Ca2+ release whereas it boosts EGF-induced ROS production. Furthermore, a significant decrease in pAKT, pERK and p-p38 was shown following the addition of the ROS inhibitor NAC. These results contribute to the understanding of the crosstalk between EGFR and VEGFR in haematological malignancies and their possible combined blockade in therapy.
    Relationship between FGF21 and UCP1 levels under time-restricted feeding and high-fat diet
    Nava Chapnik, Journal of Nutritional Biochemistry - 2016
    Abstract
    FGF21 (fibroblast growth factor 21) exhibits a circadian oscillation and its induction is critical during fasting. When secreted by liver and skeletal muscle, FGF21 enhances thermogenic activity in brown adipose tissue (BAT) by utilizing uncoupling protein 1 (UCP1) to dissipate energy as heat. Recently, it has been reported that UCP1 is not required for FGF21-mediated reduction in body weight or improvements in glucose homeostasis. As the relationship between FGF21 and UCP1 induction in tissues other than BAT is less clear, we tested the effect of restricted feeding (RF) and high dietary fat on FGF21 circadian expression and its correlation with UCP1 expression in liver and white adipose tissue (WAT).
    TSAP6 is a novel candidate marker of poor survival in metastatic high-grade serous carcinoma
    Esther Channah Broner, Human Pathology - 2016
    Abstract
    The objective of this study was to analyze the expression and clinical role of molecules involved in exosome synthesis and secretion in high-grade serous carcinoma (HGSC), with focus on malignant effusions. The mRNA expression levels of ARF6, nSMase2, TSAP6, Rab27a and Rab27b by qRT-PCR were analyzed in 103 HGSC effusions and 65 solid specimens (35 ovarian, 30 abdominal metastases). Protein expression of ARF6, nSMase2, TSAP6 and Rab27a by Western blotting was analyzed in 150 specimens (94 effusions, 29 ovarian carcinomas, 27 solid metastases). Secreted ARF6, nSMase2 and Rab27a protein levels in exosomes were analyzed in supernatants from 75 effusions. Expression levels were analyzed for association with anatomic site and clinical parameters, including survival. nSMase2 and TSAP6 mRNA was overexpressed in effusions compared to solid lesions (P < .001 and P = .003, respectively), whereas ARF6, nSMase2, TSAP6 and Rab27a protein was overexpressed in solid specimens (ovarian and peritoneal) compared to effusions (P < .001 for all). Secreted ARF6, nSMase2 and Rab27a levels were found in all effusion supernatants. In univariate survival analysis, higher TSAP6 protein levels in effusions were associated with shorter overall survival (P = .01), a finding which was reproduced in analysis of specimens from patients with pre-chemotherapy effusions tapped at diagnosis (P = .026). Higher levels of exosomal Rab27a protein were significantly related to longer overall survival (P = .025). Molecules which are part of the exosome secretion machinery are differentially expressed in HGSC effusions and solid lesions and are present in effusion supernatant-derived exosomes. TSAP6 and Rab27a may be novel prognostic markers in metastatic HGSC.
    Impact of oral and waterborne administration of rhamnolipids on the susceptibility of channel catfish (Ictalurus punctatus) to Flavobacterium columnare infection
    Dongdong Zhang, Fish & Shellfish Immunology - 2016
    Abstract
    Flavobacterium columnare is the causative agent of columnaris disease and causes tremendous morbidity and mortality of farmed fish globally. Previously, we identified a potential lectin-mediator (a rhamnose-binding lectin; RBL1a) of F. columnare adhesion and showed higher RBL1a expression in susceptible channel catfish under basal conditions and following infection. Exposure of challenged fish to the carbohydrate ligand L-rhamnose just prior to a challenge substantially decreased columnaris mortality and pathogen adherence via the down-regulation of RBL1a. While highly effective in protecting fish from columnaris, L-rhamnose is prohibitively expensive, underscoring the need for alternative cost-effective sources of rhamnose for disease control. One such alternative may be microbially produced glycolipid compounds termed rhamnolipids (RLs), which feature abundant L-rhamnose moieties and are readily available from commercial sources. In the present study, we examined whether commercially available RLs (administered either by immersion or via feed) would function similarly to L-rhamnose in affording host protection against F. columnare. A four-week feeding trial with basal and RL top-coated diets (basal diet + RLs) was conducted in channel catfish fingerlings. Surprisingly, columnaris challenges revealed significantly lower survival following the 10 d challenge period in RL diet fed fish when compared with the basal treatment group (p < 0.001). In fish fed RLs, we observed a rapid and large-scale upregulation of RBL1a immediately after challenge combined with a suppression of mucin and lysozyme transcripts. Similarly, fish that were briefly pre-exposed to RLs by immersion and then challenged exhibited lower survival as compared to unexposed fish during a 4 d trial. In conclusion, RLs do not represent an alternative to rhamnose as an experimental treatment for protecting catfish from columnaris mortality. Further research is needed to find other affordable and efficacious alternative sources of L-rhamnose.
    Naturil - EFFECT OF FEED RESTRICTION IN A RABBIT LINE SELECTED FOR GROWTH RATE ON REPRODUCTIVE PE....PDF
    Carmen Naturil Alfonso, Thesis UNIVERSITAT POLITÈCNICA DE VALÈNCIA - 2016
    Abstract
    The general aim of this thesis was to reproductively characterize females from a rabbit line selected for growth rate (line R) and to evaluate the effect of different nutritional strategies (ad libitum and restricted before reproduction), in order to improve the reproductive performance of these females.In chapter 1, the influence of maternal and embryonic genotype on prenatal survival and fetal growth over gestation was evaluated and contrasted with a maternal line. Prenatal survival, fetal weight and fetal placenta weight were affected by both embryonic and maternal genotype. Firstly, embryonic genotype was a main factor at Days 14 and 24 and maternal genotype contributed at Day 30 of prenatal survival. Secondly, differences in fetal weight were only manifested at Day 14, the embryonic/maternal genotype from the maternal line being the heaviest (0.29±0.01 g vs. 0.19±0.01 g, for line R genotypes). However, while for fetal placenta weight both genotypes showed an effect at Day 24, for maternal placenta weight they were relevant at Day 30. Nevertheless, no differences were detected either at transcriptomic level in fetal placenta or in progesterone and IGF I plasma levels in these females. From this chapter it may be concluded that in rabbit females from paternal lines both embryonic and maternal genotypes are key factors in the reproductive performance of these females.The aim of chapter 2 was to explore the causes of ovulation failures in these rabbit females. Results showed that non ovulated females presented lower LH plasma concentration as well as higher body weight and leptin and BOHB II plasma levels than ovulated females. Thus, ovulation failures in females from line R could be attributed to decreased LH plasma concentrations in these females which may be related with their higher body weight and leptin levels. In the light of the previous results, the following three chapters were focused on improvement of the reproductive performance of these females through a different nutritional strategy: a feed to appetite diet of these females after the rearing period and prior to insemination. The initial hypothesis was that the females are submitted to a restricted nutritional regimen which is not enough to cope with their needs during reproduction, causing long term disturbances of energy balance which leads to the subsequent reproductive problems. Chapter 3 aimed to determine if a feed-to-appetite nutritional strategy would affect the hypothalamus-hypophysis axis and the quality of the produced oocytes, by transcriptomic analysis. While no differences were found in the microarray analysis of the hypothalamus hypophysis, small differences were detected in the transcript expression analysis in oocytes of a group of genes selected. MSY2 was found to be downregulated in oocytes from restricted females. As a key regulator of maternal RNA transcription and translation, changes in this essential gene could explain some of the reproductive problems of these females with high growth potential. Whether the differences found at oocyte level were inherent at embryonic level and so involved in the drop of fertility found in these females was studied in Chapter 4. Although no significant differences were revealed in ovulation, embryo recovery, and implantation rate, higher fetal and gestational losses were found in restricted females, as well as lower fetal growth. Thus, we concluded that the nutritional strategy employed may have an impact on the III oocyte (Chapter 3), but we also demonstrated that these changes were inherited by the embryo, and result in disturbances in gestational losses and fetal growth.For this reason, the final chapter of this thesis was conducted to determine whether these effects on reproductive and metabolic elements were also evident in females following the common semi-intensive farm production system. Although the results obtained showed small variances in NEFAs and BOHB plasma levels, and also in body weight,no differences were detected in global reproductive performance in terms of fertility, prolificacy and productivity. The results obtained established that although differences are found at oocyte level and inherited by embryo and fetus,no improvements are reached with the proposed nutritional strategy in terms of reproductive performance when females selected for growth rate lead a normal semi intensive production system
    Understanding Tamoxifen Resistance In Breast cancer
    Raie Taye Bekele, Department of Biochemistry University of Alberta Thesis - 2016
    Abstract
    Tamoxifen is the accepted therapy for patients with estrogen receptor α (ERα)positive breast cancer. However, clinical resistance to tamoxifen, as demonstrated by recurrence or progression on therapy, is frequent and precedes death from metastases. To improve breast cancer treatment it is vital to understand the mechanisms that result in tamoxifen resistance. The study presented in this thesis shows that concentration of tamoxifen and its metabolites, which accumulate in tumors of patients, killed breast cancer cells by inducing oxidative stress. Breast cancer cells responded to tamoxifen induced oxidation by increasing Nrf2 expression and subsequent activation of the anti-oxidant response element (ARE). This increased the transcription of anti-oxidant genes and multidrug resistance transporters. As a result, breast cancer cells are able to destroy or export toxic oxidation products leading to increased survival from tamoxifen-induced oxidative damage. These responses in cancer cells also occur in breast tumors of tamoxifen treated mice. Additionally, high levels of expression of Nrf2 and its downstream targets in breast tumors of patients at the time of diagnosis were prognostic of poor survival after tamoxifen therapy. The oxidative stress induced by tamoxifen also activated phospholipase D (PLD) and led to the up regulation of the RALBP1 (Ral-binding protein 1). Tamoxifen resistant cells also had a significant increase in both basal and stimulated PLD activity along with increased PLD1 and RALBP1 levels. The activity of PLD provides survival signals to cancer cells, whereas RALBP1 iii exports chemotherapeutic drugs. Thus both RALBP1 and PLD in concert can lead to development of an aggressive and metastatic breast cancers and also contribute to chemo-resistance. In our study, cancerous breast tissues from patients have a significantly higher expression of RALBP1 compared to normal breast tissue. Furthermore, cytotoxic chemotherapy combination offered no significant advantage in patient cohorts with high RALBP1 expression as compared to those patients receiving mono or non-cytotoxic chemotherapies. Moreover, patients with high expression of PLD1 also had poor prognostic outcomes to different treatments. Thus, overcoming adaptive responses to tamoxifen induced oxidative stress could improve the survival of breast cancer patients.
    The DPYSL2 gene connects mTOR and schizophrenia
    X Pham, Translational Psychiatry - 2016
    Abstract
    We previously reported a schizophrenia-associated polymorphic CT di-nucleotide repeat (DNR) at the 5′-untranslated repeat (UTR) of DPYSL2, which responds to mammalian target of Rapamycin (mTOR) signaling with allelic differences in reporter assays. Now using microarray analysis, we show that the DNR alleles interact differentially with specific proteins, including the mTOR-related protein HuD/ELAVL4. We confirm the differential binding to HuD and other known mTOR effectors by electrophoretic mobility shift assays. We edit HEK293 cells by CRISPR/Cas9 to carry the schizophrenia risk variant (13DNR) and observe a significant reduction of the corresponding CRMP2 isoform. These edited cells confirm the response to mTOR inhibitors and show a twofold shortening of the cellular projections. Transcriptome analysis of these modified cells by RNA-seq shows changes in 12.7% of expressed transcripts at a false discovery rate of 0.05. These transcripts are enriched in immunity-related genes, overlap significantly with those modified by the schizophrenia-associated gene, ZNF804A, and have a reverse expression signature from that seen with antipsychotic drugs. Our results support the functional importance of the DPYSL2 DNR and a role for mTOR signaling in schizophrenia.
    AAV-mediated gene delivery attenuates neuroinflammation in feline Sandhoff disease
    Allison M.Bradbury, Neuroscience - 2016
    Abstract
    Sandhoff disease (SD) is a lysosomal storage disorder characterized by the absence of hydrolytic enzyme β-N-acetylhexosaminidase (Hex), which results in storage of GM2 ganglioside in neurons and unremitting neurodegeneration. Neuron loss initially affects fine motor skills, but rapidly progresses to loss of all body faculties, a vegetative state, and death by five years of age in humans. A well-established feline model of SD allows characterization of the disease in a large animal model and provides a means to test the safety and efficacy of therapeutic interventions before initiating clinical trials. In this study, we demonstrate a robust central nervous system (CNS) inflammatory response in feline SD, primarily marked by expansion and activation of the microglial cell population. Quantification of major histocompatibility complex II (MHC-II) labeling revealed significant up-regulation throughout the CNS with areas rich in white matter most severely affected. Expression of the leukocyte chemokine macrophage inflammatory protein-1 alpha (MIP-1α) was also up-regulated in the brain. SD cats were treated with intracranial delivery of adeno-associated viral (AAV) vectors expressing feline Hex, with a study endpoint 16 weeks post treatment. AAV-mediated gene delivery repressed the expansion and activation of microglia and normalized MHC-II and MIP-1α levels. These data reiterate the profound inflammatory response in SD and show that neuroinflammation is abrogated after AAV-mediated restoration of enzymatic activity.
    CD133+ brain tumor-initiating cells are dependent on STAT3 signaling to drive medulloblastoma recurrence
    N Garg, Oncogene - 2016
    Abstract
    Medulloblastoma (MB), the most common malignant paediatric brain tumor, is currently treated using a combination of surgery, craniospinal radiotherapy and chemotherapy. Owing to MB stem cells (MBSCs), a subset of MB patients remains untreatable despite standard therapy. CD133 is used to identify MBSCs although its functional role in tumorigenesis has yet to be determined. In this work, we showed enrichment of CD133 in Group 3 MB is associated with increased rate of metastasis and poor clinical outcome. The signal transducers and activators of transcription-3 (STAT3) pathway are selectively activated in CD133+ MBSCs and promote tumorigenesis through regulation of c-MYC, a key genetic driver of Group 3 MB. We screened compound libraries for STAT3 inhibitors and treatment with the selected STAT3 inhibitors resulted in tumor size reduction in vivo. We propose that inhibition of STAT3 signaling in MBSCs may represent a potential therapeutic strategy to treat patients with recurrent MB.
    Intestinal epithelial injury induced by maternal separation is protected by hydrogen sulfide
    Bo Li, Journal of Pediatric Surgery - 2016
    Abstract
    Oxidative stress has been implicated in the pathogenesis of various neonatal diseases involving the intestine. Hydrogen sulfide (H2S) has been shown to protect against oxidative stress. We hypothesized that administration of sodium hydrosulfide (NaHS), an H2S donor, to neonatal mice can decrease the intestinal epithelial injury associated with maternal separation (MS).
    Osmolality of enteral formula and severity of experimental necrotizing enterocolitis
    Hiromu Miyake, Pediatric Surgery International - 2016
    Abstract
    PurposeAdministration of hyperosmolar formula is regarded as a risk factor for the development of necrotizing enterocolitis (NEC). However, there are limited number of reports about the relationship between formula osmolality and NEC. The aim of this study is to evaluate the effects of formula concentration in an experimental model of NEC.MethodsWe studied experimental NEC in C57BL/6 mice. NEC was induced by giving hypoxia, gavage administration of lipopolysaccharide and gavage formula feeding from postnatal day 5–9. We used two types of formula: (1) hyperosmolar formula (HF): 15 g Similac + 75 ml Esbilac (849 mOsm/kg); (2) diluted formula (DF): dilute hyperosmolar formula with an equal amount of water (325 mOsm/kg). Controls were fed by the mother. On postnatal day 9, the ileum was harvested and evaluated for severity of mucosal injury (hematoxylin/eosin staining) and inflammation (PCR for IL6 and TNFα mRNA expression).ResultsThe incidence of NEC was same in both HF and DF (80%). The intestinal inflammatory response was similar between HF and DF (IL6: p = 0.26, TNFα: p = 0.69).ConclusionsThis study indicates the osmolality of enteral formula does not affect incidence of experimental NEC. This experimental study provides new insights into the relationship between formula feeding and NEC.
    Muscle injury and impaired function, and insulin resistance in Chromogranin A knockout mice
    Kechun Tang, Journal of Endocrinology - 2016
    Abstract
    Chromogranin A (CgA) is widely expressed in endocrine and neuroendocrine tissues as well as in the central nervous system. We observed CgA expression (mRNA and protein) in the gastrocnemius (GAS) muscle and found that performance of CgA-deficient Chga-KO mice in treadmill exercise was impaired. Supplementation with CgA in Chga-KO mice restored exercise ability suggesting a novel role for endogenous CgA in skeletal muscle function. Chga-KO mice display (i) lack of exercise-induced stimulation of pAKT, pTBC1D1 and phospho-p38 kinase signaling, (ii) loss of GAS muscle mass, (iii) extensive formation of tubular aggregates (TA), (iv) disorganized cristae architecture in mitochondria, (v) increased expression of the inflammatory cytokines Tnfα, Il6 and Ifnɣ, and fibrosis. The impaired maximum running speed and endurance in the treadmill exercise in Chga-KO mice correlated with decreased glucose uptake and glycolysis, defects in glucose oxidation and decreased mitochondrial cytochrome C oxidase activity. The lack of adaptation to endurance training correlated with the lack of stimulation of p38MAPK that is known to mediate the response to tissue damage. Since CgA sorts proteins to the regulated secretory pathway, we speculate that lack of CgA could cause misfolding of membrane proteins inducing aggregation of sarcoplasmic reticulum (SR) membranes and formation of tubular aggregates that is observed in Chga-KO mice. In conclusion, CgA deficiency renders the muscle energy deficient, impairs performance in treadmill exercise and prevents regeneration after exercise-induced tissue damage.
    A robust vitronectin-derived peptide for the scalable long-term expansion and neuronal differentiation of human pluripotent stem cell (hPSC)-derived neural progenitor cells (hNPCs)
    Divya Varun, Acta Biomaterialia - 2016
    Abstract
    Despite therapeutic advances, neurodegenerative diseases and disorders remain some of the leading causes of mortality and morbidity in the United States. Therefore, cell-based therapies to replace lost or damaged neurons and supporting cells of the central nervous system (CNS) are of great therapeutic interest. To that end, human pluripotent stem cell (hPSC) derived neural progenitor cells (hNPCs) and their neuronal derivatives could provide the cellular ‘raw material’ needed for regenerative medicine therapies for a variety of CNS disorders. In addition, hNPCs derived from patient-specific hPSCs could be used to elucidate the underlying mechanisms of neurodegenerative diseases and identify potential drug candidates. However, the scientific and clinical application of hNPCs requires the development of robust, defined, and scalable substrates for their long-term expansion and neuronal differentiation. In this study, we rationally designed a vitronectin-derived peptide (VDP) that served as an adhesive growth substrate for the long-term expansion of several hNPC lines. Moreover, VDP-coated surfaces allowed for the directed neuronal differentiation of hNPC at levels similar to cells differentiated on traditional extracellular matrix protein-based substrates. Overall, the ability of VDP to support the long-term expansion and directed neuronal differentiation of hNPCs will significantly advance the future translational application of these cells in treating injuries, disorders, and diseases of the CNS.
    Whey protein-derived exosomes increase protein synthesis and hypertrophy in C2C12 myotubes
    C. Brooks Mobley, Journal of Dairy Science - 2016
    Abstract
    We sought to examine potential amino acid independent mechanisms whereby hydrolyzed whey protein (WP) affects muscle protein synthesis (MPS) and anabolism in vitro. Specifically, we tested (1) whether 3-h and 6-h treatments of WP, essential amino acids, or l-leucine (Leu) affected MPS, and whether 6-h treatments with low-, medium-, or high doses of WP versus Leu affected MPS; (2) whether knockdown of the primary Leu transporter affected WP- and Leu-mediated changes in MPS, mammalian target of rapamycin (mTOR) signaling responses, or both, following 6-h treatments; (3) whether exosomes isolated from WP (WP-EXO) affected MPS, mTOR signaling responses, or both, compared with untreated (control) myotubes, following 6-h, 12-h, and 24-h treatments, and whether they affected myotube diameter following 24-h and 48-h treatments.
    Therapeutic reversal of food allergen sensitivity by mature retinoic acid–differentiated dendritic cell induction of LAG3+CD49b−Foxp3− regulatory T cells
    Wojciech Dawicki, Journal of Allergy and Clinical Immunology - 2016
    Abstract
    Anaphylaxis is a life-threatening condition for which we have limited therapeutic options. Although specific immunotherapy for food allergies is becoming more effective, it is still laborious and carries substantial risk of adverse events. On the other hand, regulatory dendritic cell (DC) therapy is effective in mouse models of allergic disease and has been shown to work with TH2 cells from atopic asthmatic patients.
    Replication-Independent Histone Variant H3.3 Controls Animal Lifespan through the Regulation of Pro-longevity Transcriptional Programs
    Antonia Piazzesi, Cell Reports - 2016
    Abstract
    Summary Chromatin structure orchestrates the accessibility to the genetic material. Replication-independent histone variants control transcriptional plasticity in postmitotic cells. The life-long accumulation of these histones has been described, yet the implications on organismal aging remain elusive. Here, we study the importance of the histone variant H3.3 in Caenorhabditis elegans longevity pathways. We show that H3.3-deficient nematodes have negligible lifespan differences compared to wild-type animals. However, H3.3 is essential for the lifespan extension of C. elegans mutants in which pronounced transcriptional changes control longevity programs. Notably, H3.3 loss critically affects the expression of a very large number of genes in long-lived nematodes, resulting in transcriptional profiles similar to wild-type animals. We conclude that H3.3 positively contributes to diverse lifespan-extending signaling pathways, with potential implications on age-related processes in multicellular organisms.
    http://www.sciencedirect.com/science/article/pii/S1550413116304995
    Yaarit Adamovich, Cell Metabolism - 2016
    Abstract
    Summary The mammalian circadian system consists of a master clock in the brain that synchronizes subsidiary oscillators in peripheral tissues. The master clock maintains phase coherence in peripheral cells through systemic cues such as feeding-fasting and temperature cycles. Here, we examined the role of oxygen as a resetting cue for circadian clocks. We continuously measured oxygen levels in living animals and detected daily rhythms in tissue oxygenation. Oxygen cycles, within the physiological range, were sufficient to synchronize cellular clocks in a HIF1α-dependent manner. Furthermore, several clock genes responded to changes in oxygen levels through HIF1α. Finally, we found that a moderate reduction in oxygen levels for a short period accelerates the adaptation of wild-type but not of HIF1α-deficient mice to the new time in a jet lag protocol. We conclude that oxygen, via HIF1α activation, is a resetting cue for circadian clocks and propose oxygen modulation as therapy for jet lag.
    Protein S Regulates Neural Stem Cell Quiescence and Neurogenesis
    Katya Zelentsova, STEM CELLS - 2016
    Abstract
    Neurons are continuously produced in brains of adult mammalian organisms throughout life—a process tightly regulated to ensure a balanced homeostasis. In the adult brain, quiescent Neural Stem Cells (NSCs) residing in distinct niches engage in proliferation, to self-renew and to give rise to differentiated neurons and astrocytes. The mechanisms governing the intricate regulation of NSC quiescence and neuronal differentiation are not completely understood. Here, we report the expression of Protein S (PROS1) in adult NSCs, and show that genetic ablation of Pros1 in neural progenitors increased hippocampal NSC proliferation by 47%. We show that PROS1 regulates the balance of NSC quiescence and proliferation, also affecting daughter cell fate. We identified the PROS1-dependent downregulation of Notch1 signaling to correlate with NSC exit from quiescence. Notch1 and Hes5 mRNA levels were rescued by reintroducing Pros1 into NCS or by supplementation with purified PROS1, suggesting the regulation of Notch pathway by PROS1. Although Pros1-ablated NSCs show multilineage differentiation, we observed a 36% decrease in neurogenesis, coupled with a similar increase in astrogenesis, suggesting PROS1 is instructive for neurogenesis, and plays a role in fate determination, also seen in aged mice. Rescue experiments indicate PROS1 is secreted by NSCs and functions by a NSC-endogenous mechanism. Our study identifies a duple role for PROS1 in stem-cell quiescence and as a pro-neurogenic factor, and highlights a unique segregation of increased stem cell proliferation from enhanced neuronal differentiation, providing important insight into the regulation and control of NSC quiescence and differentiation. Stem Cells 2016
    Development of TRACER: tissue roll for analysis of cellular environment and response
    Darren Rodenhizer, Biofabrication - 2016
    Abstract
    The tumour microenvironment is heterogeneous and consists of multiple cell types, variable extracellular matrix (ECM) composition, and contains cell-defined gradients of small molecules, oxygen, nutrients and waste. Emerging in vitro cell culture systems that attempt to replicate these features often fail to incorporate design strategies to facilitate efficient data collection and stratification. The tissue roll for analysis of cellular environment and response (TRACER) is a novel strategy to assemble layered, three-dimensional tumours with cell-defined, graded heterogeneous microenvironments that also facilitates cellular separation and stratification of data from different cell populations from specific microenvironments. Here we describe the materials selection and development of TRACER. We find that cellulose fibre scaffolding is an ideal support to generate tissue constructs having homogenous cell seeding and consistent properties. We explore ECM remodeling and long-term cell growth in the scaffold, and characterize the tumour microenvironment in assembled TRACERs using multiple established analysis methods. Finally, we confirm that TRACERs replicate small molecule gradients of glucose and lactate, and explore cell phenotype associated with these gradients using confocal microscopy, flow cytometry, and quantitative PCR analysis. We envision this technology will provide a platform to create complex, yet controlled tumour microenvironments that can be easily disassembled for snapshot analysis of cell phenotype and response to therapy in relation to microenvironment properties.
    HOXB4 Gene Expression Is Regulated by CDX2 in Intestinal Epithelial Cells
    Steffen Jørgensen, PLOS ONE - 2016
    Abstract
    The mammalian Caudal-related homeobox transcription factor 2 (CDX2) plays a key role in the homeobox regulatory network and is essential in regulating the expression of several homeobox (HOX) genes during embryonic development, particularly in the gut. Genome-wide CDX2 chromatin immunoprecipitation analysis and expression data from Caco2 cells also suggests a role for CDX2 in the regulation of HOXB4 gene expression in the intestinal epithelium. Thus, the aim of this study was to investigate whether HOXB4 gene expression is regulated by CDX2 in the intestinal epithelium. We demonstrated binding of CDX2 to four different CDX2 binding sites in an enhancer region located upstream of the HOXB4 transcription start site. Mutations in the CDX2 binding sites reduced HOXB4 gene activity, and knock down of endogenous CDX2 expression by shRNA reduced HOXB4 gene expression. This is the first report demonstrating the CDX2 regulation of HOXB4 gene expression in the developed intestinal epithelium, indicating a possible role for HOXB4 in intestinal homeostasis.
    Acquired CDK6 amplification promotes breast cancer resistance to CDK4/6 inhibitors and loss of ER signaling and dependence
    C Yang, Oncogene - 2016
    Abstract
    Dysregulated activation of the CDK4/6 kinases is a hallmark of most mammary-derived carcinomas. ATP-competitive inhibitors against this complex have been recently advanced in the clinic and have shown significant activity, particularly against tumors driven by the estrogen receptor (ER). However, resistance to these compounds has begun to emerge often months to years after their initiation. We investigated potential mechanisms of resistance using cell line models that are highly sensitive to this class of drugs. After prolonged exposure to the selective and potent CDK4/6 inhibitor LY2835219, clones emerged and several were found to harbor amplification of the CDK6 kinase. Amplification of CDK6 resulted in a marked increase in CDK6 expression and reduced response of the CDK4/6 target, phospho-Rb (pRb), to CDK4/6 inhibitors. Knockdown of CDK6 restored drug sensitivity, while enforced overexpression of CDK6 was sufficient to mediate drug resistance. Not only did CDK6 overexpression mediate resistance to CDK4/6 inhibitors but it also led to reduced expression of the ER and progesterone receptor (PR), and diminished responsiveness to ER antagonism. The reduced ER/PR expression after CDK4/6 inhibitor resistance was additionally observed in tumor biopsy specimens from patients treated with these drugs. Alternative mechanisms of resistance to CDK4/6 inhibitors such as loss of pRb and cyclin E1 overexpression also exhibited decreased hormone responsiveness, suggesting that the clinical paradigm of sequential endocrine-based therapy may be ineffective in some settings of acquired CDK4/6 resistance.
    A novel FRET-based screen in high-throughput format to identify inhibitors of malarial and human glucose transporters
    Thomas E. Kraft, Antimicrobial Agents and Chemotherapy - 2016
    Abstract
    The glucose transporter PfHT is essential to the survival of the malaria parasite Plasmodium falciparum and has been shown to be a druggable target with high potential for pharmacological intervention. Identification of compounds against novel drug targets is crucial to combating resistance against current therapeutics. Here, we describe the development of a cell-based assay system readily adaptable to high-throughput screening that directly measures compound effects on PfHT-mediated glucose transport. Intracellular glucose concentrations are detected using a genetically encoded fluorescence resonance energy transfer (FRET)-based glucose-sensor. This allows assessment of the ability of small molecules to inhibit glucose uptake with high accuracy (Z'-factor of >0.8), thereby eliminating the need for radiolabeled substrates. Furthermore, we have adapted this assay to counter screen PfHT hits against the human orthologues GLUT1, 2, 3 and 4. We report the identification of several hits after screening the Medicines for Malaria Venture (MMV) Malaria Box, a library of 400 compounds known to inhibit erythrocytic development of P. falciparum. Hit compounds were characterized by determining the half-maximal inhibitory concentration (IC50) for the uptake of radiolabeled glucose into isolated P. falciparum parasites. One of our hits, compound MMV009085, shows high potency and ortholog selectivity, thereby successfully validating our assay for anti-malarial screening.
    An RNAi-Based Control of Fusarium graminearum Infections Through Spraying of Long dsRNAs Involves a Plant Passage and Is Controlled by the Fungal Silencing Machinery
    Aline Koch, PLOS Pathog - 2016
    Abstract
    Author Summary RNA interference has emerged as a powerful genetic tool for scientific research. The demonstration that agricultural pests, such as insects and nematodes, are killed by exogenously supplied RNA targeting their essential genes has raised the possibility that plant predation can be controlled by lethal RNA signals. We show that spraying barley with a 791 nt long dsRNA ( CYP3 -dsRNA) targeting the three fungal ergosterol biosynthesis genes ( CYP51A , CYP51B , CYP51C ), whose respective proteins also are known as azole fungicide targets, efficiently inhibited the necrotrophic fungus Fusarium graminearum in directly sprayed and systemic leaf tissue. Strong inhibition of fungal growth required an operational fungal RNA interference mechanism as demonstrated by the fact that a Fusarium DICER-LIKE-1 mutant was insensitive to CYP3 -dsRNA in systemic, non-sprayed leaf areas. Our findings will help in the efficient design of RNAi-based plant disease control. We provide essential information on a fundamentally new plant protection strategy, thereby opening novel avenues for improving crop yields in an environmentally friendly and sustainable manner.
    Farnesoid X Receptor Signaling Shapes the Gut Microbiota and Controls Hepatic Lipid Metabolism
    Limin Zhang, mSystems - 2016
    Abstract
    The gut microbiota modulates obesity and associated metabolic phenotypes in part through intestinal farnesoid X receptor (FXR) signaling. Glycine-β-muricholic acid (Gly-MCA), an intestinal FXR antagonist, has been reported to prevent or reverse high-fat diet (HFD)-induced and genetic obesity, insulin resistance, and fatty liver; however, the mechanism by which these phenotypes are improved is not fully understood. The current study investigated the influence of FXR activity on the gut microbiota community structure and function and its impact on hepatic lipid metabolism. Predictions about the metabolic contribution of the gut microbiota to the host were made using 16S rRNA-based PICRUSt (phylogenetic investigation of communities by reconstruction of unobserved states), then validated using 1H nuclear magnetic resonance-based metabolomics, and results were summarized by using genome-scale metabolic models. Oral Gly-MCA administration altered the gut microbial community structure, notably reducing the ratio of Firmicutes to Bacteroidetes and its PICRUSt-predicted metabolic function, including reduced production of short-chain fatty acids (substrates for hepatic gluconeogenesis and de novo lipogenesis) in the ceca of HFD-fed mice. Metabolic improvement was intestinal FXR dependent, as revealed by the lack of changes in HFD-fed intestine-specific Fxr-null (FxrΔIE) mice treated with Gly-MCA. Integrative analyses based on genome-scale metabolic models demonstrated an important link between Lactobacillus and Clostridia bile salt hydrolase activity and bacterial fermentation. Hepatic metabolite levels after Gly-MCA treatment correlated with altered levels of gut bacterial species. In conclusion, modulation of the gut microbiota by inhibition of intestinal FXR signaling alters host liver lipid metabolism and improves obesity-related metabolic dysfunction. IMPORTANCE The farnesoid X receptor (FXR) plays an important role in mediating the dialog between the host and gut microbiota, particularly through modulation of enterohepatic circulation of bile acids. Mounting evidence suggests that genetic ablation of Fxr in the gut or gut-restricted chemical antagonism of the FXR promotes beneficial health effects, including the prevention of nonalcoholic fatty liver disease in rodent models. However, questions remain unanswered, including whether modulation of FXR activity plays a role in shaping the gut microbiota community structure and function and what metabolic pathways of the gut microbiota contribute in an FXR-dependent manner to the host phenotype. In this report, new insights are gained into the metabolic contribution of the gut microbiota to the metabolic phenotypes, including establishing a link between FXR antagonism, bacterial bile salt hydrolase activity, and fermentation. Multiple approaches, including unique mouse models as well as metabolomics and genome-scale metabolic models, were employed to confirm these results.
    Lack of effects of ooplasm transfer on early development of interspecies somatic cell nuclear transfer bison embryos
    L. Antonio González-Grajales, BMC Developmental Biology - 2016
    Abstract
    BackgroundSuccessful development of iSCNT (interspecies somatic cell nuclear transfer) embryos depends on complex interactions between ooplasmic and nuclear components, which can be compromised by genetic divergence. Transfer of ooplasm matching the genetic background of the somatic cell in iSCNT embryos is a valuable tool to study the degree of incompatibilities between nuclear and ooplasmic components. This study investigated the effects of ooplasm transfer (OT) on cattle (Bos taurus) and plains bison (Bison bison bison) embryos produced by iSCNT and supplemented with or without ooplasm from cattle or plains bison oocytes.ResultsEmbryos in all groups were analysed for developmental competence that included cleavage rates, ATP content, and expression of nuclear- and mitochondrial- encoded genes at 8–16 cell stage. Interestingly, no significant differences were observed in embryo development, ATP content, and expression of nuclear respiratory factor 2 (NRF2), mitochondrial transcription factor A (TFAM) and mitochondrial subunit 2 of cytochrome c oxidase (mt-COX2) among groups. Thus, although OT did not result in any detrimental effects on the reconstructed embryos due to invasive manipulation, significant benefits of OT were not observed up to the 8–16 cell stage.ConclusionsThis study showed that a viable technique for OT + SCNT is possible, however, further understanding of the effects of OT on blastocyst development is necessary.
    Conjugates of HA2 with octaarginine-grafted HPMA copolymer offer effective siRNA delivery and gene silencing in cancer cells
    Moran Golan, Science Direct - 2016
    Abstract
    The key for successful gene silencing is to design a safe and efficient siRNA delivery system for the transfer of therapeutic nucleic acids into the target cells. Here, we describe the design of hydrophilic N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer displaying multiple copies of octaarginine (R8) and its use in promoting the effective delivery of small interfering RNA (siRNA) molecules intracellularly. Fluorescein-5-isothiocyanate (FITC)-labeled HPMA copolymer-bound R8 (P-R8-FITC) was synthesized with increasing R8 molar ratios (4–9.5 mol-%) to define the optimal R8 content that allowed the polymer to serve both as a siRNA-binding domain and as an intracellular transduction moiety mediating improved cellular delivery. A subunit of the influenza virus hemagglutinin (HA2), known for its ability to disrupt endosomal membranes, was further conjugated to P-R8-FITC copolymer to promote endosomal escape. Of the different P-(R8)-FITC conjugates considered, only that polymer containing the highest mol-% of R8 (P-(R8)9.5-FITC) was able to encapsulate siRNA molecules into nano-sized polyion complexes (PICs) presenting positive surface charge, low in vitro cytotoxicity, and high serum stability. P-(R8)9.5-FITC/cy5-siRNA complexes can efficiently deliver siRNA molecules into cells, while naked siRNA or siRNA encapsulated within polymers with lower R8 mol-% were unable to transfect the same cells. Conjugation of HA2 fusogenic peptide to P-(R8)-FITC significantly decreased the oncogenic RAC1 mRNA levels in cancer cells. This indicates that P-(R8)-(HA2)-FITC can deliver siRNA into target cells, and that the siRNA can reach the perinuclear region where it interacts with the RNA-induced silencing complex.
    An endoplasmic reticulum stress-regulated lncRNA hosting a microRNA megacluster induces early features of diabetic nephropathy
    Mitsuo Kato, Nature Communications - 2016
    Abstract
    Nephropathy is a common and hard-to-treat consequence of diabetes. Here Kato et al. show that a megacluster of microRNAs regulates early development of diabetic nephropathy in mice, and that inhibition of the cluster's host long non-coding RNA transcript attenuates disease symptoms, suggesting a new therapy for diabetic nephropathy.
    microRNA-122 target sites in the hepatitis C virus RNA NS5B coding region and 3′ untranslated region: function in replication and influence of RNA secondary structure
    Gesche K.Gerresheim, Cellular and Molecular Life Sciences - 2016
    Abstract
    We have analyzed the binding of the liver-specific microRNA-122 (miR-122) to three conserved target sites of hepatitis C virus (HCV) RNA, two in the non-structural protein 5B (NS5B) coding region and one in the 3′ untranslated region (3′UTR). miR-122 binding efficiency strongly depends on target site accessibility under conditions when the range of flanking sequences available for the formation of local RNA secondary structures changes. Our results indicate that the particular sequence feature that contributes most to the correlation between target site accessibility and binding strength varies between different target sites. This suggests that the dynamics of miRNA/Ago2 binding not only depends on the target site itself but also on flanking sequence context to a considerable extent, in particular in a small viral genome in which strong selection constraints act on coding sequence and overlapping cis-signals and model the accessibility of cis-signals. In full-length genomes, single and combination mutations in the miR-122 target sites reveal that site 5B.2 is positively involved in regulating overall genome replication efficiency, whereas mutation of site 5B.3 showed a weaker effect. Mutation of the 3′UTR site and double or triple mutants showed no significant overall effect on genome replication, whereas in a translation reporter RNA, the 3′UTR target site inhibits translation directed by the HCV 5′UTR. Thus, the miR-122 target sites in the 3′-region of the HCV genome are involved in a complex interplay in regulating different steps of the HCV replication cycle.
    Acute Loss of Cited2 Impairs Nanog Expression and Decreases Self-Renewal of Mouse Embryonic Stem Cells
    Kranc, KR, The University of Edinburgh - 2016
    Abstract
    Identifying novel players of the pluripotency gene regulatory network centered on Oct4, Sox2, and Nanog as well as delineating the interactions within the complex network is key to understanding self-renewal and early cell fate commitment of embryonic stem cells (ESC). While over-expression of the transcriptional regulator Cited2 sustains ESC pluripotency, its role in ESC functions remains unclear. Here, we show that Cited2 is important for proliferation, survival, and self-renewal of mouse ESC. We position Cited2 within the pluripotency gene regulatory network by defining Nanog, Tbx3, and Klf4 as its direct targets. We also demonstrate that the defects caused by Cited2 depletion are, at least in part, rescued by Nanog constitutive expression. Finally, we demonstrate that Cited2is required for and enhances reprogramming of mouse embryonic fibroblasts to induced pluripotent stem cells.
    Dongmei Cheng, Journal of Biological Chemistry - 2016
    Abstract
    Hepatic apolipoprotein A-IV (apoA-IV) expression is correlated with hepatic triglyceride (TG) content in mouse models of chronic hepatosteatosis and steatosis-induced hepatic apoA-IV gene expression is regulated by nuclear transcription factor cAMP responsive element-binding protein H (CREBH) processing. To define what aspects of TG homeostasis regulates hepatic CREBH processing and apoA-IV gene expression, several mouse models of attenuated VLDL particle assembly were subjected to acute hepatosteatosis induced by an overnight fast or short-term ketogenic diet feeding. Compared with chow-fed C57BL/6 mice, fasted or ketogenic diet-fed mice displayed increased hepatic TG content, which was highly correlated (r2=0.95) with apoA-IV gene expression, and secretion of larger, TG-enriched VLDL, despite a lower rate of TG secretion and similar or reduced rate of apoB100 secretion. When VLDL particle assembly and secretion was inhibited by hepatic shRNA-induced apoB silencing or genetic or pharmacologic reduction in microsomal triglyceride transfer protein (MTP) activity, hepatic TG content increased dramatically; however, CREBH processing and apoA-IV gene expression was attenuated compared to controls. Adenovirus-mediated reconstitution of MTP expression proportionately restored CREBH processing and apoA-IV expression in liver-specific MTP knockout mice. These results reveal that hepatic TG content per se, does not regulate CREBH processing. Instead TG mobilization into the ER for nascent VLDL particle assembly activates CREBH processing and enhances apoA-IV gene expression in the setting of acute steatosis. We conclude that VLDL assembly and CREBH activation play key roles in the response to hepatic steatosis by upregulating apoA-IV and promoting assembly and secretion of larger, more TG-enriched VLDL particles.
    Low concentrations of bromodichloromethane induce a toxicogenomic response in porcine embryos in vitro
    Florence Pagé-Larivièrea,, Reproductive Toxicology - 2016
    Abstract
    Bromodichloromethane (BDCM) is one of the trihalomethanes present in chlorinated water. Humans are thus daily exposed. Previous contradictory results failed to clearly establish the adverse effects of low concentrations of BDCM. By using the porcine preimplantation embryo as a sensitive model, we showed that exposure to low concentrations of BDCM (10 and 100 ppb) during the first week of embryo development induced adverse effect on the blastocyst rate and alteration of the estradiol pathway. Our results also suggest that blastocysts exposed to BDCM present transcriptomic and epigenomic adaptive modifications compatible with the cardiac anomalies observed by previous studies of newborns exposed to BDCM during gestation. Thus, phenotypic observations and toxicogenomic adaptations of embryo to low concentration of BDCM provide insights for BDCM risk assessment. Indeed, our results support the use of sensitive toxicogenomic models using environmentally relevant concentrations to which humans are exposed in order to conduct the risk assessment.
    Delayed glial clearance of degenerating axons in aged Drosophila is due to reduced PI3K/Draper activity
    Maria D. Purice, Nature Communications - 2016
    Abstract
    Advanced age is the greatest risk factor for neurodegenerative disorders, but the mechanisms that render the senescent brain vulnerable to disease are unclear. Glial immune responses provide neuroprotection in a variety of contexts. Thus, we explored how glial responses to neurodegeneration are altered with age. Here we show that glia–axon phagocytic interactions change dramatically in the aged Drosophila brain. Aged glia clear degenerating axons slowly due to low phosphoinositide-3-kinase (PI3K) signalling and, subsequently, reduced expression of the conserved phagocytic receptor Draper/MEGF10. Importantly, boosting PI3K/Draper activity in aged glia significantly reverses slow phagocytic responses. Moreover, several hours post axotomy, early hallmarks of Wallerian degeneration (WD) are delayed in aged flies. We propose that slow clearance of degenerating axons is mechanistically twofold, resulting from deferred initiation of axonal WD and reduced PI3K/Draper-dependent glial phagocytic function. Interventions that boost glial engulfment activity, however, can substantially reverse delayed clearance of damaged neuronal debris.
    Diverse fates of uracilated HIV-1 DNA during infection of myeloid lineage cells
    Erik C. Hansen, eLife - 2016
    Abstract
    Human immunodeficiency virus type 1 (HIV-1) infects and kills immune cells known as CD4+ T cells, leading to the disease AIDS. Current drug treatments enable HIV-1 infected patients to live relatively long and healthy lives. However, no cure for HIV-1 exists because the virus lives indefinitely in a resting state within the genetic material – or genome – of the infected cell, where it is not susceptible to drug treatments. Most HIV-1 research focuses on T cells, but another type of immune cell – the macrophage – may also harbor resting HIV-1 in its genome. Compared to other cells, macrophages are unusual because they produce large amounts of a molecule called deoxyuridine triphosphate (dUTP). Most cells, including T cells, keep dUTP levels very low because it closely resembles molecules that are used to make DNA and so it can be accidentally incorporated into the cell’s DNA. When this happens, the cell removes the dUTP from the DNA using enzymes in a process called uracil base excision repair (UBER). To hide inside the cell’s genome, HIV-1 needs to produce a DNA copy of its own genome, but it was not known what happens when HIV-1 tries to do this within a macrophage that contains high levels of dUTP and UBER enzymes. Here, Hansen et al. reveal that about 90% of macrophages have exceptionally high levels of dUTP and are poorly infected by HIV-1. The high levels of dUTP result in the virus incorporating dUTP into its DNA, which is then attacked and fragmented by UBER enzymes. However, about one in a hundred viral DNA molecules do manage to successfully integrate into the genome of the macrophage. This viral DNA later gives rise to new virus particles through an error-prone process that, by introducing new mutations into the virus genome, may help HIV-1 to evolve and persist. Further experiments examined cells that give rise to macrophages from infected patients who had been on anti-HIV drug therapy for several years. Hansen et al. found that there was lots of dUTP in the DNA sequences of HIV-1 viruses found in these “precursor” cells. These precursor cells only live for several days before being eliminated, so the presence of viruses containing dUTP suggests these cells were infected recently. A future challenge will be to identify new anti-HIV drugs that specifically target macrophages and to understand the role of error-prone production of new viral genomes.
    A Single Injection of Hypertrophied Androgenic Gland Cells Produces All-Female Aquaculture
    Tom Levy, Marine Biotechnology - 2016
    Abstract
    Monosex culture, common in animal husbandry, enables gender-specific management. Here, production of all-female prawns (Macrobrachium rosenbergii) was achieved by a novel biotechnology comprising three steps: (a) A single injection of suspended hypertrophied androgenic gland cells caused fully functional sex reversal of females into “neo-males” bearing the WZ genotype; (b) crossing neo-males with normal females (WZ) yielded genomically validated WW females; and (c) WW females crossed with normal males (ZZ) yielded all-female progeny. This is the first sustainable biotechnology for large-scale all-female crustacean aquaculture. The approach is particularly suited to species in which females are superior to males and offers seedstock protection, thereby ensuring a quality seed supply. Our technology will thus revolutionize not only the structure of the crustacean aquaculture industry but can also be applied to other sectors. Finally, the production of viable and reproducible females lacking the Z chromosome questions its role, with respect to sexuality.
    A Single Injection of Hypertrophied Androgenic Gland Cells Produces All-Female Aquaculture
    Tom Levy, Marine Biotechnology - 2016
    Abstract
    Monosex culture, common in animal husbandry, enables gender-specific management. Here, production of all-female prawns (Macrobrachium rosenbergii) was achieved by a novel biotechnology comprising three steps: (a) A single injection of suspended hypertrophied androgenic gland cells caused fully functional sex reversal of females into “neo-males” bearing the WZ genotype; (b) crossing neo-males with normal females (WZ) yielded genomically validated WW females; and (c) WW females crossed with normal males (ZZ) yielded all-female progeny. This is the first sustainable biotechnology for large-scale all-female crustacean aquaculture. The approach is particularly suited to species in which females are superior to males and offers seedstock protection, thereby ensuring a quality seed supply. Our technology will thus revolutionize not only the structure of the crustacean aquaculture industry but can also be applied to other sectors. Finally, the production of viable and reproducible females lacking the Z chromosome questions its role, with respect to sexuality.
    Sex-related factors influence expression of mood-related genes in the basolateral amygdala differentially depending on age and stress exposure | Biology of Sex Differences | Full Text
    Rachel Puralewski, BioMed Central - 2016
    Abstract
    Women are twice as likely to be diagnosed with major depressive disorder (MDD) compared to men, but the molecular mechanisms underlying this sex difference are unclear. Previous studies in the human postmortem brain suggest dysfunction in basolateral amygdala (BLA) inhibitory gamma-aminobutyric acid (GABA) signaling and brain-derived neurotrophic factor (BDNF) function, specifically in females with MDD.
    Direct reprogramming of urine-derived cells with inducible MyoD for modeling human muscle disease
    Ellis Y. Kim, Skeletal Muscle - 2016
    Abstract
    Cellular models of muscle disease are taking on increasing importance with the large number of genes and mutations implicated in causing myopathies and the concomitant need to test personalized therapies. Developing cell models relies on having an easily obtained source of cells, and if the cells are not derived from muscle itself, a robust reprogramming process is needed. Fibroblasts are a human cell source that works well for the generation of induced pluripotent stem cells, which can then be differentiated into cardiomyocyte lineages, and with less efficiency, skeletal muscle-like lineages. Alternatively, direct reprogramming with the transcription factor MyoD has been used to generate myotubes from cultured human fibroblasts. Although useful, fibroblasts require a skin biopsy to obtain and this can limit their access, especially from pediatric populations.
    The Kaposi's-sarcoma-associated herpesvirus orf35 gene product is required for efficient lytic virus reactivation
    Shir Bergson, Virology - 2016
    Abstract
    Kaposi's sarcoma-associated herpesvirus (KSHV) is implicated in the etiology of several human malignancies. KSHV open reading frame (orf) 35 encodes a conserved gammaherpesvirus protein with an, as yet, unknown function. Employing the bacterial artificial chromosome (BAC) system, we generated a recombinant viral clone that fails to express ORF35 (BAC16-ORF35-stop) but preserves intact adjacent and overlapping reading frames. Using this construct, we studied the role of this previously uncharacterized gene product during lytic reactivation of KSHV. Upon lytic reactivation, the ORF35-stop recombinant virus displayed significantly reduced lytic viral gene expression, viral DNA replication, and progeny virus production as compared to control wild-type virus. Exogenous expression of ORF35-Flag reversed the effects of ORF35 deficiency. These results demonstrate that ORF35 is important for efficient lytic virus reactivation.
    Progesterone induces progesterone receptor gene (PGR) expression via rapid activation of protein kinase pathways required for cooperative estrogen receptor alpha (ER) and progesterone receptor (PR) genomic action at ER/PR target genes
    Caroline H. Diep, Science Direct - 2016
    Abstract
    Progesterone Receptors (PRs) are critical effectors of estrogen receptor (ER) signaling required for mammary gland development and reproductive proficiency. In breast and reproductive tract malignancies, PR expression is a clinical prognostic marker of ER action. While estrogens primarily regulate PR expression, other factors likely contribute to a dynamic range of receptor expression across diverse tissues. In this study, we identified estrogen-independent but progestin (R5020)-dependent regulation of ER target genes including PGR in ER+/PR+ cancer cell lines. R5020 (10 nM-10 μM range) induced dose-dependent PR mRNA and protein expression in the absence of estrogen but required both PR-B and ERα. Antagonists of either PR (RU486, onapristone) or ERα (ICI 182,780) attenuated R5020 induction of TFF1, CTSD, and PGR. Chromatin immunoprecipitation (ChIP) assays performed on ER+/PR+ cells demonstrated that both ERα and PR were recruited to the same ERE/Sp1 site-containing region of the PGR proximal promoter in response to high dose progestin (10 μM). Recruitment of ERα and PR to chromatin and subsequent PR mRNA induction were dependent upon rapid activation of MAPK/ERK and AKT; inhibition of these kinase pathways via U0126 or LY294002 blocked these events. Overall, we have identified a novel mechanism of ERα activation initiated by rapid PR-dependent kinase pathway activation and associated with phosphorylation of ERα Ser118 for estrogen-independent but progestin-dependent ER/PR cross talk. These studies may provide insight into mechanisms of persistent ER-target gene expression during periods of hormone (i.e. estrogen) ablation and suggest caution following prolonged treatment with aromatase or CYP17 inhibitors (i.e. contexts when progesterone levels may be abnormally elevated).
    Denervation drives mitochondrial dysfunction in skeletal muscle of octogenarians
    Sally Spendiff, The Journal of Physiology - 2016
    Abstract
    Experimental denervation modulates mitochondrial function, where changes in both reactive oxygen species (ROS) and sensitivity to permeability transition are implicated in the resultant muscle atrophy. Notably, although denervation occurs sporadically in ageing muscle, its impact on ageing muscle mitochondria is unknown. As this information has important therapeutic implications concerning targeting the mitochondrion in ageing muscle, we examined mitochondrial function in skeletal muscle from four groups of humans, comprising two active (mean age 23.7 (SD 2.7) yr & 71.2 (4.9) yr) and two inactive groups (64.8 (3.1) yr & 82.5 (4.8) yr), and compared this to a murine model of sporadic denervation. We tested the hypothesis that while some alterations of mitochondrial function in aged muscle are attributable to a primary organelle defect, in advanced age, mitochondrial dysfunction would be impacted by persistent denervation. Both ageing in humans, and sporadic denervation in mice, increased mitochondrial sensitivity to permeability transition (Humans P = 0.004; Mice P = 0.01). To ascertain the contribution of sporadic denervation to mitochondrial function we pharmacologically inhibited the denervation-induced ROS response. This reduced ROS emission by 60% (P = 0.02) in sporadically denervated mouse muscle, similar to what was seen in humans older than 75 years (-66%, P = 0.02) but not those younger than 75 years. We conclude that an increased sensitivity to permeability transition is a primary mitochondrial defect in ageing muscle. However, at more advanced age when muscle atrophy becomes more clinically severe, mitochondrial function changes are markedly impacted by persistent sporadic denervation, making the mitochondrion a less viable therapeutic target. This article is protected by copyright. All rights reserved
    PLOS ONE: Ezrin Is Associated with Disease Progression in Ovarian Carcinoma
    Vered Horwitz, PLOS ONE - 2016
    Abstract
    Objective Ezrin and p130Cas are structural proteins with an important role in signaling pathways and have been shown to promote cancer dissemination. We previously reported on overexpression of both ezrin and p130Cas in breast carcinoma effusions compared to primary carcinomas. Since ovarian and breast carcinomas share the ability to disseminate by forming malignant effusions, we sought to study the role of these molecules in ovarian carcinoma (OC). Methods OC cell lines were cultured in two different 3-dimensional conditions, on alginate scaffolds and as spheroids, which served as models for solid tumor and malignant effusions, respectively. shRNA was used to reduce protein expression in the cells. The malignant potential was evaluated by chemo-invasion assay, branching capacity on Matrigel and rate of proliferation. Subsequently, clinical specimens of high-grade serous carcinoma effusions, ovarian tumors and solid metastases were analyzed for ezrin and p130Cas expression. Results Higher ezrin expression was found in cells composing the spheroids compared to their counterparts cultured on alginate scaffold and in clinical samples of malignant effusions compared to solid tumors. In addition, reduced Ezrin expression impaired the invasion ability and the branching capacity of OC cells to a greater extent than reduced p130Cas expression. However, ezrin and p130Cas expression in effusions was unrelated to clinical outcome. Conclusions The 3-dimensional cell cultures were found to mimic the different tumor sites and be applicable as a model. The in vitro results concur with the clinical specimen analysis, suggesting that in OC, the role of ezrin in disease progression is more pronounced than that of p130Cas.
    β-Catenin signaling positively regulates glutamate uptake and metabolism in astrocytes
    Victoria Lutgen, Journal of Neuroinflammation - 2016
    Abstract
    Neurological disorders have been linked to abnormal excitatory neurotransmission. Perturbations in glutamate cycling can have profound impacts on normal activity, lead to excitotoxicity and neuroinflammation, and induce and/or exacerbate impairments in these diseases. Astrocytes play a key role in excitatory signaling as they both clear glutamate from the synaptic cleft and house enzymes responsible for glutamate conversion to glutamine. However, mechanisms responsible for the regulation of glutamate cycling, including the main astrocytic glutamate transporter excitatory amino acid transporter 2 (EAAT2 or GLT-1 in rodents) and glutamine synthetase (GS) which catalyzes the ATP-dependent reaction of glutamate and ammonia into glutamine, remain largely undefined.
    Failed reinnervation in aging skeletal muscle
    Sudhakar Aare, Skeletal Muscle - 2016
    Abstract
    Skeletal muscle displays a marked accumulation of denervated myofibers at advanced age, which coincides with an acceleration of muscle atrophy.
    High fat diet drives obesity regardless the composition of gut microbiota in mice
    Sylvie Rabot, Scientific Reports - 2016
    Abstract
    The gut microbiota is involved in many aspects of host physiology but its role in body weight and glucose metabolism remains unclear. Here we studied the compositional changes of gut microbiota in diet-induced obesity mice that were conventionally raised or received microbiota transplantation. In conventional mice, the diversity of the faecal microbiota was weakly associated with 1st week weight gain but transferring the microbiota of mice with contrasting weight gain to germfree mice did not change obesity development or feed efficiency of recipients regardless whether the microbiota was taken before or after 10 weeks high fat (HF) feeding. Interestingly, HF-induced glucose intolerance was influenced by microbiota inoculation and improved glucose tolerance was associated with a low Firmicutes to Bacteroidetes ratio. Transplantation of Bacteroidetes rich microbiota compared to a control microbiota ameliorated glucose intolerance caused by HF feeding. Altogether, our results demonstrate that gut microbiota is involved in the regulation of glucose metabolism and the abundance of Bacteroidetes significantly modulates HF-induced glucose intolerance but has limited impact on obesity in mice. Our results suggest that gut microbiota is a part of complex aetiology of insulin resistance syndrome, individual microbiota composition may cause phenotypic variation associated with HF feeding in mice.
    Estrogen receptor expression and gene promoter methylation in non-small cell lung cancer - a short report
    Xavier Tekpli, Cellular Oncology - 2016
    Abstract
    PurposeIn the past, anomalous estrogen receptor (ER) regulation has been associated with various lung pathologies, but so far its involvement in lung cancer initiation and/or progression has remained unclear. Here, we aimed at assessing in vivo and in vitro ER expression and its possible epigenetic regulation in non-small cell lung cancer (NSCLC) samples and their corresponding normal tissues and cells.MethodsERα and ERβ gene expression levels were assessed using real time quantitative PCR (RT-qPCR), whereas ERα and ERβ gene promoter methylation levels were assessed using DNA bisulfite conversion followed by pyrosequencing. We included NSCLC (n = 87) and adjacent histologically normal lung tissue samples from lung cancer patients (n = 184), primary normal bronchial epithelial-derived cell cultures (n = 11), immortalized bronchial epithelial-derived cell lines (n = 3) and NSCLC derived cell lines (n = 9).ResultsUsing RT-qPCR we found significantly lower ERα and ERβ expression levels in the NSCLC tissue samples compared to their normal adjacent tissue samples. These lower ER expression levels were confirmed in vitro using primary normal bronchial epithelial-derived cell cultures, immortalized bronchial epithelial-derived cell lines and NSCLC-derived cell lines. By using this latter panel of cells, we found that ER gene promoter hypermethylation was associated with decreased ER expression. In addition we found that in tumor and normal lung tissues, smoking was associated with decreased ER expression and that normal lung tissues with a low ERβ expression level exhibited increased smoking-related DNA adducts.ConclusionsTaken together, our results indicate that decreased ER expression mediated by DNA methylation may play a role in NSCLC development.
    Ehrlichia chaffeensis TRP32 is a Nucleomodulin that Directly Regulates Expression of Host Genes Governing Differentiation and Proliferation
    Tierra R. Farris, Infection and Immunity - 2016
    Abstract
    Ehrlichia chaffeensis is an obligately intracellular bacterium that reprograms the mononuclear phagocyte through diverse effector-host interactions to modulate numerous host cell processes, including transcription. In a previous study, we reported that E. chaffeensis TRP32, a type 1 secreted effector, interacts with multiple host nucleus-associated proteins and also auto-activates reporter gene expression in yeast. In this study, we demonstrate that TRP32 is a nucleomodulin that binds host DNA and alters host gene transcription. TRP32 enters the host cell nucleus via a noncanonical translocation mechanism that involves phosphorylation of Y179 located in a C-terminal tri-tyrosine motif. Both genistein and mutation of Y179 inhibited TRP32 nuclear entry. An electromobility shift assay (EMSA) demonstrated TRP32 host DNA binding via its tandem repeat domain. TRP32 DNA binding and motif preference were further confirmed by supershift assays, as well as competition and mutant probe analyses. Using ChIP-Seq, we determined that TRP32 binds a G-rich motif primarily within ±500 bp of the gene transcription start site. An ontology analysis identified genes involved in processes such as immune cell differentiation, chromatin remodeling, and RNA transcription and processing, as primary TRP32 targets. TRP32 bound genes (n=1223) were distributed on all chromosomes and included several global regulators of proliferation and inflammation such as FOS and JUN, AKT3 and NRAS, and non-coding RNA genes, miRNA 21 and miRNA 142. TRP32 target genes were differentially regulated during infection, the majority of which were repressed, and direct repression/activation of these genes by TRP32 was confirmed in vitro with a cellular luciferase reporter assay.
    Dependence-induced increase of alcohol self-administration and compulsive drinking mediated by the histone methyltransferase PRDM2
    E. Barbier, Molecular Psychiatry - 2016
    Abstract
    Epigenetic processes have been implicated in the pathophysiology of alcohol dependence, but the specific molecular mechanisms mediating dependence-induced neuroadaptations remain largely unknown. Here, we found that a history of alcohol dependence persistently decreased the expression of Prdm2, a histone methyltransferase that monomethylates histone 3 at the lysine 9 residue (H3K9me1), in the rat dorsomedial prefrontal cortex (dmPFC). Downregulation of Prdm2 was associated with decreased H3K9me1, supporting that changes in Prdm2 mRNA levels affected its activity. Chromatin immunoprecipitation followed by massively parallel DNA sequencing showed that genes involved in synaptic communication are epigenetically regulated by H3K9me1 in dependent rats. In non-dependent rats, viral-vector-mediated knockdown of Prdm2 in the dmPFC resulted in expression changes similar to those observed following a history of alcohol dependence. Prdm2 knockdown resulted in increased alcohol self-administration, increased aversion-resistant alcohol intake and enhanced stress-induced relapse to alcohol seeking, a phenocopy of postdependent rats. Collectively, these results identify a novel epigenetic mechanism that contributes to the development of alcohol-seeking behavior following a history of dependence.
    Estrogen receptor expression and gene promoter methylation in non-small cell lung cancer - a short report
    Xavier Tekpli, Cellular Oncology - 2016
    Abstract
    PurposeIn the past, anomalous estrogen receptor (ER) regulation has been associated with various lung pathologies, but so far its involvement in lung cancer initiation and/or progression has remained unclear. Here, we aimed at assessing in vivo and in vitro ER expression and its possible epigenetic regulation in non-small cell lung cancer (NSCLC) samples and their corresponding normal tissues and cells.MethodsERα and ERβ gene expression levels were assessed using real time quantitative PCR (RT-qPCR), whereas ERα and ERβ gene promoter methylation levels were assessed using DNA bisulfite conversion followed by pyrosequencing. We included NSCLC (n = 87) and adjacent histologically normal lung tissue samples from lung cancer patients (n = 184), primary normal bronchial epithelial-derived cell cultures (n = 11), immortalized bronchial epithelial-derived cell lines (n = 3) and NSCLC derived cell lines (n = 9).ResultsUsing RT-qPCR we found significantly lower ERα and ERβ expression levels in the NSCLC tissue samples compared to their normal adjacent tissue samples. These lower ER expression levels were confirmed in vitro using primary normal bronchial epithelial-derived cell cultures, immortalized bronchial epithelial-derived cell lines and NSCLC-derived cell lines. By using this latter panel of cells, we found that ER gene promoter hypermethylation was associated with decreased ER expression. In addition we found that in tumor and normal lung tissues, smoking was associated with decreased ER expression and that normal lung tissues with a low ERβ expression level exhibited increased smoking-related DNA adducts.ConclusionsTaken together, our results indicate that decreased ER expression mediated by DNA methylation may play a role in NSCLC development.
    Investigating the physiological role of HDAC1 and HDAC2 in embryonic stem cells
    Shereen Jamal Jamaladdin, University of Leicester Thesis - 2016
    Abstract
    Histone deacetylases 1 and 2 (HDAC1/2) are highly similar proteins (83% identical) that form the core catalytic components of corepressor complexes that modulate gene expression. Germline deletion of Hdac1 in mice results in early embryonic lethality and conditional deletion of Hdac1 but not Hdac2 causes precocious differentiation in ES cells. Therefore to further investigate the role of HDAC1/2 during the early embryogenesis, we have generated a compound conditional knockout ES cell line Hdac1ko; Hdac2Het in which HDAC1/2 activity is reduced but not entirely lost. Hdac1ko; Hdac2He cells have a significant reduction in total deacetylase activity and disruption of core pressor complex integrity. The prolifration capicity of Hdac1ko; Hdac2He cells is not inhibited, however, upon differentiation they were predisposed to toward the cardiomyocyte lineage. In most cell types, deletion of both Hdac1 and Hdac2 is required to produce a henotype, suggesting their activity is redundant. To circumvent this functional edundancy, we generated a double conditional knockout (DKO) cells in which both dac1 and Hdac2 can be inactivated simultaneously. Loss of HDAC1/2 results in a 0% reduction in total HDAC activity and a loss of cell viability, which is associated ith increased abnormal mitotic spindle, chromatin bridges and miconuclei, suggesting that HDAC1/2 are necessary for accurate chromosome segregation. Transcriptome analysis reveals 1,708 differentially expressed genes in DKO cells including a reduction in the expression of the ES cells core pluripotent factors. HDAC1/2 activity can be regulated in vitro through the binding of inositol tetraphosphate (IP4). By rescuing the viability of DKO cells using wt and mutant forms of HDAC1, we demonstrated that mutations that abolish IP4 binding reduce the activity of HDAC1/2 in vivo. We have also shown that treatment of DKO ES cells with RA results in reduces induction of HOX genes, suggesting a positive role of HDAC1/2 in gene activation as well as gene repression.
    Osteopontin has a protective role in prostate tumor development in mice
    Keiko Danzaki, European Journal of Immunology - 2016
    Abstract
    Osteopontin (OPN) is a protein, generally considered to play a pro-tumorigenic role, whereas several reports have demonstrated the anti-tumorigenic function of OPN during tumor development. These opposing anti- and pro-tumorigenic functions are not fully understood. Here, we report that host-derived OPN plays an anti-tumorigenic role in the transgenic adenocarcinoma of the mouse prostate (TRAMP) model and a TRAMP tumor transplant model. Tumor suppression mediated by OPN in Rag2−/− mice suggests that OPN is dispensable in the adaptive immune response. We found that host-derived OPN enhanced infiltration of natural killer (NK) cells into TRAMP tumors. The requirement of OPN in NK cell migration towards TRAMP cells was confirmed by an ex vivo cell migration assay. In contrast to TRAMP cells, in vivo B16 tumor development was not inhibited by OPN, and B16 tumors did not show OPN-mediated cell recruitment. It is possible that low levels of chemokine expression by B16 cells do not allow OPN to enhance immune cell recruitment. In addition to demonstrating the anti-tumorigenic role of OPN in TRAMP tumor development, this study also suggests that the contribution of OPN to tumor development depends on the type of tumor as well as the source and isoform of OPN. This article is protected by copyright. All rights reserved
    Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids
    Purvi C. Trivedi, Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids - 2016
    Abstract
    Impaired cardiac metabolism in the obese and diabetic heart leads to glucolipotoxicity and ensuing cardiomyopathy. Glucolipotoxicity causes cardiomyocyte injury by increasing energy insufficiency, impairing proteasomal-mediated protein degradation and inducing apoptosis. Proteasome-evading proteins are degraded by autophagy in the lysosome, whose metabolism and function is regulated by master regulator transcription factor EB (TFEB). Limited studies have examined the impact of glucolipotoxicity on intra-lysosomal signaling proteins and their regulators. By utilizing a mouse model of diet-induced obesity, type-1 diabetes (Akita) and ex-vivo model of glucolipotoxicity (H9C2 cells and NRCM, neonatal rat cardiomyocyte), we examined whether glucolipotoxicity negatively targets TFEB and lysosomal proteins to dysregulate autophagy and cause cardiac injury. Despite differential effects of obesity and diabetes on LC3B-II, expression of proteins facilitating autophagosomal clearance such as TFEB, LAMP-2 A, Hsc70, Hsp90 were decreased in the obese and diabetic heart. In-vivo data was recapitulated in H9C2 and NRCM cells, which exhibited impaired autophagic flux and reduced TFEB content when exposed to a glucolipotoxic milieu. Notably, overloading myocytes with a saturated fatty acid (palmitate) but not an unsaturated fatty acid (oleate) depleted cellular TFEB and supressed autophagy, suggesting a fatty acid specific regulation of TFEB and autophagy in the cardiomyocyte. The effect of glucolipotoxicity to reduce TFEB content was also confirmed in heart tissue from patients with Class-I obesity. Therefore during glucolipotoxicity, suppression of lysosomal autophagy was associated with reduced lysosomal content, decreased cathepsin-B activity and diminished cellular TFEB content likely rendering myocytes susceptible to cardiac injury.
    Isolation of TGF-β-neutralizing single-domain antibodies of predetermined epitope specificity using next-generation DNA sequencing
    Kevin A. Henry, Protein Engineering Design and Selection - 2016
    Abstract
    The epitope specificity of therapeutic antibodies is often critical to their efficacy and mode of action. Here, we report the isolation of single-domain antibodies (sdAbs) against a pre-specified epitope of TGF-β3: namely, the site of interaction between the cytokine and its cell-surface type II receptor. By panning a phage-displayed immune llama VhH library against TGF-β3 using competitive elution with soluble dimeric type II receptor ectodomain in tandem with next-generation DNA sequencing, we identified several sdAbs that competed with the receptor for TGF-β3 binding and neutralized TGF-β3 in in vitro cellular assays. In contrast, all other sdAbs identified using conventional panning approaches (i.e., without regard to epitope specificity) did not target the site of receptor:cytokine interaction. We expect this strategy to be generally applicable for identifying epitope-specific sdAbs when binding reagents directed against the epitope of interest are available. The sdAbs identified here are of potential interest as cancer immunotherapeutics.
    Cell type specific modulation of innate immune signalling by vitamin D in human mononuclear phagocytes
    Rhiannon Kundu, Immunology - 2016
    Abstract
    Vitamin D is widely reported to inhibit innate immune signalling and dendritic cell (DC) maturation as a potential immunoregulatory mechanism. It is not known whether vitamin D has global or gene-specific effects on transcriptional responses downstream of innate immune stimulation, or whether vitamin D inhibition of innate immune signalling is common to different cells. We confirmed vitamin D inhibition of NFκB and p38 mitogen activated protein kinase (MAPK) signalling in monocyte derived DC (MDDC) stimulated with lipopolysaccharide (LPS). This was associated with global but modest attenuation of LPS-induced transcriptional changes at genome-wide level. Surprisingly, vitamin D did not inhibit innate immune NFκB activation in monocyte derived macrophages. Consistent with our findings in MDDC, ex vivo vitamin D treatment of primary peripheral blood myeloid DC also lead to significant inhibition of LPS inducible NFκB activation. Unexpectedly, in the same samples, vitamin D enhanced activation of both NFκB and MAPK signalling in primary peripheral blood monocytes. In a cross sectional clinical cohort, we found no relationship between peripheral blood vitamin D levels and LPS inducible activation of NFκB and MAPK pathways in monocytes of myeloid DC. Remarkably however, in vivo supplementation of vitamin D deficient people in this clinical cohort also enhanced LPS inducible MAPK signalling in peripheral blood monocytes. Therefore, we report that vitamin D differentially modulates the molecular response to innate immune stimulation in monocytes, macrophages and dendritic cells. These results are of importance in the design of studies on vitamin D supplementation in infectious and immunological diseases. This article is protected by copyright. All rights reserved.
    Inhibition of adhesion, migration and of α5β1 integrin in the HCT-116 colorectal cancer cells treated with the ruthenium drug NAMI-A
    Chiara Pelillo, Journal of Inorganic Biochemistry - 2016
    Abstract
    NAMI-A, imidazolium trans-imidazoledimethylsulfoxidetetrachlororuthenate, is a ruthenium-based drug characterised by the selective activity against tumour metastases. Previously we have shown the influence of the hepatic microenvironment to direct the arrest of the metastatic cells of colorectal cancer. Here we used the experimental model of HCT-116 colorectal cancer cells in vitro to explore whether the interference with α5β1 integrin may mechanistically explain the anti-metastatic effect of NAMI-A. NAMI-A inhibits two important steps of the tumour metastatic progression of colorectal cancer, i.e. the adhesion and migration of the tumour cells on the extracellular matrix proteins. The fibronectin receptor α5β1 integrin is likely involved in the anti-adhesive effects of NAMI-A on the HCT-116 colorectal cancer cells during their interaction with the extracellular matrix. Mechanistically, NAMI-A decreases the α5β1 integrin expression, and reduces FAK (Focal Adhesion Kinase) auto-phosphorylation on Tyr397, an important signalling event, involved in α5β1 integrin activation. These effects were validated by siRNA-induced knock down of the α5 integrin subunit and/or by the use of specific blocking mAbs against the active site of the integrin. Our results demonstrate the relevance of α5β1 integrin for colorectal cancer. We also show that the anti-metastatic effect of NAMI-A depends on the modulation of this integrin. Thus, our data on NAMI-A support the new concept that metal-based drugs can inhibit tumour metastases through targeting of integrins and of other proteins which mediate tumour progression-related cell functions such as adhesion and migration.
    Effect of Roughness on in Situ Biomineralized CaP-Collagen Coating on the Osteogenesis of Mesenchymal Stem Cells
    Xingjie Zan, Langmuir - 2016
    Abstract
    Because of its outstanding osteo-conductive property, a calcium phosphate (CaP) coating has been used as an implant coating for bone tissue engineering. Nevertheless, the issues, such as harsh fabrication conditions, long-term stability and biocompatibility, and the requirement for expensive instruments, still exist in current coating techniques. To address these issues, the CaP coatings doped with collagen (CaP-Col) were in situ generated on polyelectrolyte multilayers (PEMs) by incubating PEMs in a mixture of the collagen, phosphate, and calcium ions. The resulting coatings have controllable physical properties (chemical composition, crystallinity, and roughness) and good stability before and after incubation with cell culture medium. We also found that both the cellular viability and osteogenesis of mesenchymal stem cells (MSCs) were closely related to the roughness of PEMs/CaP-Col, one of the easily ignored physical factors in current coating designs but very critical. The existed roughness window (between 18 ± 1.2 and 187 ± 7.3 nm) suitable for MSC proliferation on PEMs/CaP-Col coating and the optimal roughness (∼98 ± 3.5 nm) for MSC osteogenesis further demonstrated that the roughness was a critical factor for bone formation. Therefore, we envision that our exploration of the effects of surface roughness on MSC behaviors would provide better guidance for the future design of material coating and eventual medical success.
    The Piwi P athway in Bovine Gametes and Preimplantation Embryo s
    Stewart James Russell, The University of Guelph A Thesis - 2016
    Abstract
    Epigenetic reprogramming is required during both embryo and gamete development to restore the potential for cellular pluripotency. Changes in DNA methylation during reprogramming can allow the expression of integrated endogenous transposable elements (TEs) that may lead to genomic instability should widespread reintegration occur. Small RNA mediated mechanisms have therefore evolved to constrain TE expression during reprogramming. PIWI proteins are a subfamily of the Argonaute clade of proteins which have functions in small RNA (sRNA) mediated TE and gene silencing. PIWI proteins direct silencing through RNA-mediated interactions with complementary nucleic acid targets. TE expression has been documented in early bovine embryos, although mechanisms regulating their expression are not widely studied. This period is further complicated by extensive targeted decay of maternally-derived mRNAs which may utilize related mechanisms of gene silencing. Based on these observations, it was hypothesized that the PIWI pathway is present in bovine gametes and embryos, and regulates endogenous TEs and mRNAs during reprogramming. PIWIL1 expression was characterized in bovine gonads, oocytes, and embryos. The testis-specific expression of full-length PIWIL1 showed localization to spermatocytes and spermatids, and association with piRNAs of 29-32 nucleotides in length. Additionally, two shorter, 3ʹtruncated PIWIL1 transcript isoforms were found to be expressed in testis, oocytes and embryos, and showed dynamic regulation throughout embryogenesis. Full-length PIWIL2 and PIWIL3 transcripts were also quantified throughout embryo genesis. To address potential roles for the PIWI pathway, sRNAs from oocytes and zygotes were profiled through next-generation sequencing and compared to those from testis, ovary and sperm. An analysis “pipeline” was developed to identify putative piRNAs from the sequencing libraries. The piRNA-“like” RNAs (pilRNAs) were characterized based on their size and canonical biogenesis features and were abundant in oocytes and embryos. When mapped to bovine repeats, up to one third of embryonic pilRNAs targeted TE classes including LINEs, SINEs, and ERVs. Through comparisons with published embryonic mRNA transcriptomes this analysis also revealed the potential for maternally-deposited, zygotic pilRNAs to direct mRNA degradation. These data provide one of the first characterizations of the PIWI pathway outside of classical models, and provide evidence for novel functions in embryo TE and gene regulation.
    Characterization of cAMP-phosphodiesterase activity in bovine seminal plasma
    A. Bergeron, Andrology - 2016
    Abstract
    The second messenger cyclic adenosine monophosphate (cAMP) has a central role in sperm physiology. Extracellular cAMP can be sequentially degraded into 5′AMP and adenosine by ecto-phosphodiesterases (ecto-PDE) and ecto-nucleotidases, a phenomenon called extracellular cAMP-adenosine pathway. As cAMP-adenosine pathway is involved in sperm capacitation, we hypothesize that extracellular PDEs are functionally present in seminal plasma. Exclusively measuring cAMP-PDE activity, total activity in bovine seminal plasma was 10.1 ± 1.5 fmoles/min/μg. Using different family-specific PDE inhibitors, we showed that in seminal plasma, the major cAMP-PDE activity was papaverine sensitive (47.5%). These data support the presence of PDE10 in bovine seminal plasma and was further confirmed by western blot. In epididymal fluid, total cAMP-PDE activity was 48.2 ± 14.8 fmoles/min/μg and we showed that the major cAMP-PDE activity was 3-isobutyl-methylxanthine insensitive and thus ascribed to PDE8 family. PDE10A mRNAs were found in the testis, epididymis, and seminal vesicles. cAMP-PDE activity is present in bovine seminal plasma and epididymal fluid. The results suggest a role for ecto-PDEs present in those fluids in the signaling pathways involved in sperm functions.
    Role of the orexin (hypocretin) system in contextual fear conditioning in rats
    Huiying Wang, Behavioural Brain Research - 2016
    Abstract
    Orexin (hypocretin) neurons located in the posterior hypothalamus send projections to multiple areas of the brain involved in arousal and experimental evidence indicates that these neurons play a role in the physiological and behavioral responses to stress. This study was done to determine if the orexin system was involved in mediating the fear associated with shock context (5 × 2 s of 1.5 mA). First, real-time RT-PCR was used to examine changes in the mRNA levels for prepro-orexin (ppOX), the orexin-1 receptor (OX1R) and the orexin-2 receptor (OX2R) at two weeks post-shock. We found that the mRNA levels for ppOX and OX1R were increased in the posterior hypothalamus of shocked rats. In contrast, no significant difference was found in the midline thalamus or the locus coeruleus/parabrachial region. Second, the study examined if systemic injections of antagonists for orexin receptors attenuated the freezing related to contextual fear. The OX1R antagonist SB334867 (20 or 30 mg/kg; i.p.) decreased freezing while the same doses of the OX2R antagonist TCSOX229 had no effect. The dual orexin antagonist TCS1102 (20 mg/kg; i.p.) also decreased the freezing to the shock context. The results of the present study show upregulation of orexin activity and of the OX1R in the hypothalamus following exposure of rats to footshocks and highlight a specific role of OX1R in contextual fear.
    The antiandrogen flutamide is a novel aryl hydrocarbon receptor ligand that disrupts bile acid homeostasis in mice through induction of Abcc4
    Xiaoxia Gao, Biochemical Pharmacology - 2016
    Abstract
    Flutamide (FLU), an oral, nonsteroidal antiandrogen drug used in the treatment of prostate cancer, is associated with idiosyncratic hepatotoxicity that sometimes causes severe liver damage, including cholestasis, jaundice, and liver necrosis. To understand the mechanism of toxicity, a combination of aryl hydrocarbon receptor (Ahr)-deficient (Ahr−/−) mice, primary hepatocytes, luciferase reporter gene assays, in silico ligand docking and ultra-performance chromatography-quadrupole time-of-flight mass spectrometry-based metabolomics was used. A significant increase of liver weights, and liver and serum bile acid levels was observed after FLU treatment, indicating hepatomegaly and disrupted bile acid homeostasis. Expression of the AhR gene battery was markedly increased in livers of wild-type mice Ahr+/+ treated with FLU, while no change was noted in Ahr−/− mice. Messenger RNAs encoded by AhR target genes were induced in primary mouse hepatocytes cultured with FLU, which confirmed the in vivo results. Ligand-docking analysis further predicted that FLU is an AhR agonist ligand which was confirmed by luciferase reporter gene assays. Multivariate data analysis showed that bile acids were responsible for the separation of vehicle- and FLU-treated Ahr+/+ mice, while there was no separation in Ahr−/− mice. Expression of mRNA encoding the bile acid transporter ABCC4 was increased and farnesoid X receptor signaling was inhibited in the livers of Ahr+/+ mice, but not in Ahr−/− mice treated with FLU, in agreement with the observed downstream metabolic alterations. These findings provide new insights into the mechanism of liver injury caused by FLU treatment involving activation of AhR and the alterations of bile acid homeostasis, which could guide clinical application.
    Disruption of the carA gene in Pseudomonas syringae results in reduced fitness and alters motility
    Bronwyn G. Butcher, BMC Microbiology - 2016
    Abstract
    Pseudomonas syringae infects diverse plant species and is widely used in the study of effector function and the molecular basis of disease. Although the relationship between bacterial metabolism, nutrient acquisition and virulence has attracted increasing attention in bacterial pathology, there is limited knowledge regarding these studies in Pseudomonas syringae. The aim of this study was to investigate the function of the carA gene and the small RNA P32, and characterize the regulation of these transcripts.
    Inhibition of IL-13-induced periostin in airway epithelium attenuates cellular protein expression of MUC5AC
    Isao Suzaki, Respirology - 2016
    Abstract
    Background and objective Serum periostin is increased in asthma and serves as a surrogate marker for IL-13 activity in the lung. Serum levels of periostin are the most robust biomarker predicting a favourable response to the anti-IL-13 drug, lebrikizumab. We investigated the mechanisms of IL-13 stimulation of periostin, the polarized secretion of periostin and whether periostin would have a direct effect on mucin secretion by airway cells. Methods Normal human bronchial epithelial (NHBE) cells were cultured at air–liquid interface (ALI) in the presence of IL-13, and we evaluated the effect of the specific inhibitors, leflunomide (Janus kinase (JAK)/signal transducer and activator of transcription factor 6 (STAT6) inhibitor) or PD98059 (MEK/extracellular regulated protein kinase (ERK) inhibitor), on periostin production. We examined MUC5AC secretion from NHBE cells exposed to recombinant human (rh) periostin or IL-13 in the presence and absence of OC-20, a periostin-neutralizing antibody. Results IL-13 induced periostin protein which was predominantly secreted towards the basal surface of the cells. Periostin production was much greater from goblet cells than ciliated cells (P < 0.001). Periostin production after exposure to IL-13 was attenuated by both leflunomide (P < 0.001) and PD98059 (P < 0.001). The addition of exogenous periostin modestly increased MUC5AC secretion (P < 0.01), but did not visibly change cell morphology. IL-13-induced MUC5AC secretion was attenuated by OC-20 (P < 0.01). Conclusion Periostin production in differentiated airway cells is mediated by JAK/STAT6 and MEK/ERK pathways. Periostin secretion is much greater from immunologically active goblet cells. IL-13-driven mucin production is partially inhibited by OC-20.
    Reinforcement of poly-l-lactic acid electrospun membranes with strontium borosilicate bioactive glasses for bone tissue engineering
    João S. Fernandes, Acta Biomaterialia - 2016
    Abstract
    Herein, for the first time, we combined poly-l-lactic acid (PLLA) with a strontium borosilicate bioactive glass (BBG-Sr) using electrospinning to fabricate a composite bioactive PLLA membrane loaded with 10% (w/w) of BBG-Sr glass particles (PLLA-BBG-Sr). The composites were characterised by scanning electron microscopy (SEM) and microcomputer tomography (μ-CT), and the results showed that we successfully fabricated smooth and uniform fibres (1–3 μm in width) with a homogeneous distribution of BBG-Sr microparticles (<45 μm). Degradation studies (in phosphate buffered saline) demonstrated that the incorporation of BBG-Sr glass particles into the PLLA membranes increased their degradability and water uptake with a continuous release of cations. The addition of BBG-Sr glass particles enhanced the membrane’s mechanical properties (69% higher Young modulus and 36% higher tensile strength). Furthermore, cellular in vitro evaluation using bone marrow-derived mesenchymal stem cells (BM-MSCs) demonstrated that PLLA-BBG-Sr membranes promoted the osteogenic differentiation of the cells as demonstrated by increased alkaline phosphatase activity and up-regulated osteogenic gene expression (Alpl, Sp7 and Bglap) in relation to PLLA alone. These results strongly suggest that the composite PLLA membranes reinforced with the BBG-Sr glass particles have potential as an effective biomaterial capable of promoting bone regeneration. Statement of Significance PLLA membranes were reinforced with 10% (w/w) of strontium-bioactive borosilicate glass microparticles, and their capacity to induce the osteogenic differentiation of bone marrow mesenchymal stem cells (BM-MSCs) was evaluated. These membranes presented an increased: degradability, water uptake, Young modulus and tensile strength. We also demonstrated that these membranes are non-cytotoxic and promote the attachment of BM-MSCs. The addition of the glass microparticles into the PLLA membranes promoted the increase of ALP activity (under osteogenic conditions), as well as the BM-MSCs osteogenic differentiation as shown by the upregulation of Alpl, Sp7 and Bglap gene expression. Overall, we demonstrated that the reinforcement of PLLA with glass microparticles results in a biomaterial with the appropriate properties for the regeneration of bone tissue.
    Characterization of the peripheral thyroid system of gilthead seabream acclimated to different ambient salinities
    I. Ruiz-Jarabo, Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology - 2016
    Abstract
    Thyroid hormones are involved in many developmental and physiological processes, including osmoregulation. The regulation of the thyroid system by environmental salinity in the euryhaline gilthead seabream (Sparus aurata) is still poorly characterized. To this end seabreams were exposed to four different environmental salinities (5, 15, 40 and 55 ppt) for 14 days, and plasma free thyroid hormones (fT3, fT4), outer ring deiodination and Na+/K+-ATPase activities in gills and kidney, as well as other osmoregulatory and metabolic parameters were measured. Low salinity conditions (5 ppt) elicited a significant increase in fT3 (29%) and fT4 (184%) plasma concentrations compared to control animals (acclimated to 40 ppt, natural salinity conditions in the Bay of Cádiz, Spain), while the amount of pituitary thyroid stimulating hormone subunit β (tshb) transcript abundance remained unchanged. In addition, plasma fT4 levels were positively correlated to renal and branchial deiodinase type 2 (dio2) mRNA expression. Gill and kidney T4-outer ring deiodination activities correlated positively with dio2 mRNA expression and the highest values were observed in fish acclimated to low salinities (5 and 15 ppt). The high salinity (55 ppt) exposure caused a significant increase in tshb expression (65%), but deiodinase gene expression (dio1 and dio2) and activity did not change and were similar to controls (40 ppt). In conclusion, acclimation to different salinities led to changes in the peripheral regulation of thyroid hormone metabolism in seabream. Therefore, thyroid hormones are involved in the regulation of ion transport and osmoregulatory physiology in this species. The conclusions derived from this study may also allow aquaculturists to modulate thyroid metabolism in seabream by adjusting culture salinity.
    Testosterone inhibits expression of lipogenic genes in visceral fat by an estrogen-dependent mechanism
    A. Maleah Holland, Journal of Applied Physiology - 2016
    Abstract
    The influence of the aromatase enzyme on the chronic fat-sparing effects of testosterone requires further elucidation. Our purpose was to determine whether chronic anastrozole (AN; aromatase inhibitor) treatment alters testosterone-mediated lipolytic/lipogenic gene expression in visceral fat. 10 month old F344 rats (n=6/group) received SHAM surgery, orchiectomy(ORX), ORX+testosterone-enanthate(TEST; 7.0mg/week), or ORX+TEST+AN(0.5mg/day), with drug treatment beginning 14 days post-surgery. At day 42, ORX animals exhibited nearly undetectable serum testosterone and 29% higher retroperitoneal fat mass versus SHAM (p<0.001). Testosterone treatment produced a ~380-415% higher serum testosterone versus SHAM (p<0.001) and completely prevented ORX-induced visceral fat gain (p<0.001). Retroperitoneal fat was 21% and 16% lower in ORX+TEST versus SHAM (p<0.001) and ORX+TEST+AN (p=0.007), while serum estradiol was 62% (p=0.024) and 87% (p=0.010) higher, respectively. ORX stimulated lipogenic-related gene expression in visceral fat, demonstrated by ~84-154% higher SREBP-1 (p=0.023), FASN (p=0.01), and LPL mRNA (p<0.001) versus SHAM, effects that were completely prevented in ORX+TEST animals (p<0.01 versus ORX for all). FASN (p=0.061, trend) and LPL mRNA (p=0.043) were lower in ORX+TEST+AN versus ORX and not different than SHAM, but remained higher than ORX+TEST (p<0.05). In contrast, the ORX-induced elevation in SREBP-1 mRNA was not prevented by TEST+AN, with SREBP-1 expression remaining ~117-171% higher than SHAM and ORX+TEST (p<0.01). Across groups, visceral fat mass and lipogenic-related gene expressions were negatively associated with serum testosterone, but not estradiol. Aromatase inhibition constrains testosterone-induced visceral fat loss and the down-regulation of key lipogenic genes at the mRNA level, indicating that estradiol influences the visceral fat-sparing effects of testosterone.
    Molecular and Kinetic Characterization of The Fermentative Behavior of Saccharomyces cerevisiae strains Isolated from North Patagonia
    Adriana Beatriz Simes, Scientific Research Publishing - 2016
    Abstract
    At present, Argentina does not count with a production of indigenous yeast strains with suitable technological and oenological features to be used in the regional winery industry. Isolation and molecular characterization of these microorganisms and its fermentation attributes would be relevant to the sustainable development of the activity in the country and to recognize and preserve the biodiversity of the region. Eight strains isolated from grapes and musts from the North Patagonian region and genetically identified as Saccharomyces cerevisiae, were Studied for their fermentation behavior, emphasizing in hexose transport through the plasma membrane, which is the limiting step of the process. Thus, Sugar consumption profiles were analyzed in different media at laboratory scale, to be subsequently applied to the fermentation of natural musts. Three Of the eight initial strains were selected, named ÑNM10, ÑIF 8 and ÑMN16 According to their fermentation profiles. The Expression of hexose transporters during fermentations revealed interesting differences in the response of each strain to sugar consumption, where transporters HXT 2 and HXT 5 showed Significant changes in expression in Patagonian strains, which are normally associated to endurance to culture stress conditions. The Results obtained by combining the characteristics studied, at molecular and physiological level, are extremely encouraging. Native Strain ÑMN16, showed a high potential for application in local winemaking. Assays Carried out on a pilot scale will determine the feasibility of applying this strain with promising technological features at industrial scale
    N-methylation of a bactericidal compound as a resistance mechanism in Mycobacterium tuberculosis
    Kenan C Murphy, UMass - 2016
    Abstract
    The rising incidence of antimicrobial resistance (AMR) makes it im- perative to understand the underlying mechanisms. Mycobacterium tuberculosis (Mtb) is the single leading cause of death from a bacterial pathogen and estimated to be the leading cause of death from AMR. A pyrido-benzimidazole, 14, was rep orted to have potent bactericidal activity against Mtb. Here, we isolated multiple Mtb clones resistant to 14. Each had mutations in the put ative DNA-binding and dimeriza- tion domains of rv2887 , a gene encoding a transcriptional repressor of the MarR family. The mutations in Rv2887 led to markedly increased expression of rv0560c. We characterized Rv0560c as an S -adenosyl- L - methionine-dependent methyltransferase that N -methylates 14, abolishing its mycobactericidal activity. An Mtb strain lacking rv0560c became resistant to 14 by mutating decaprenylphosphoryl- β - D -ribose 2-oxidase (DprE1), an essential enzyme in arabinogalactan synthesis; 14 proved to be a nanomolar inhibitor of DprE1, and methylation of 14 by Rv0560c abrogated this activity. Thus, 14 joins a growing list of DprE1 inhibitors that are potently mycobactericidal. Bacterial methylation of an antibacterial agent, 14, catalyzed by Rv0560c of Mtb, is a previously unreported mechanism of AMR.
    Transcriptomic evaluation of the American oyster, Crassostrea virginica, deployed during the Deepwater Horizon oil spill: Evidence of an active hydrocarbon response pathway
    Matthew J. Jenny, Marine Environmental Research - 2016
    Abstract
    Estuarine organisms were impacted by the Deepwater Horizon oil spill which released ∼5 million barrels of crude oil into the Gulf of Mexico in the spring and summer of 2010. Crassostrea virginica, the American oyster, is a keystone species in these coastal estuaries and is routinely used for environmental monitoring purposes. However, very little is known about their cellular and molecular responses to hydrocarbon exposure. In response to the spill, a monitoring program was initiated by deploying hatchery-reared oysters at three sites along the Alabama and Mississippi coast (Grand Bay, MS, Fort Morgan, AL, and Orange Beach, AL). Oysters were deployed for 2-month periods at five different time points from May 2010 to May 2011. Gill and digestive gland tissues were harvested for gene expression analysis and determination of aliphatic and polycyclic aromatic hydrocarbon (PAH) concentrations. To facilitate identification of stress response genes that may be involved in the hydrocarbon response, a nearly complete transcriptome was assembled using Roche 454 and Illumina high-throughput sequencing from RNA samples obtained from the gill and digestive gland tissues of deployed oysters. This effort resulted in the assembly and annotation of 27,227 transcripts comprised of a large assortment of stress response genes, including members of the aryl hydrocarbon receptor (AHR) pathway, Phase I and II biotransformation enzymes, antioxidant enzymes and xenobiotic transporters. From this assembly several potential biomarkers of hydrocarbon exposure were chosen for expression profiling, including the AHR, two cytochrome P450 1A genes (CYP1A-like 1 and CYP1A-like 2), Cu/Zn superoxide dismutase (CuZnSOD), glutathione S-transferase theta (GST theta) and multidrug resistance protein 3 (MRP3). Higher expression levels of GST theta and MRP3 were observed in gill tissues from all three sites during the summer to early fall 2010 deployments. Linear regression analysis indicated a statistically significant relationship between total PAH levels in digestive gland tissue samples with CYP1A-like 2, CuZnSOD, GST theta and MRP3 induction. These observations provide evidence of a potentially conserved AHR pathway in invertebrates and yield new insight into the development of novel biomarkers for use in environmental monitoring activities.
    IL-6 blockade reprograms the lung tumor microenvironment to limit the development and progression of K-ras mutant lung cancer
    Mauricio S. Caetano, Cancer Research - 2016
    Abstract
    Activating mutations of K-ras are the most common oncogenic alterations found in lung cancer. Unfortunately, attempts to target K-ras mutant lung tumors have thus far failed, clearly indicating the need for new approaches in patients with this molecular profile. We have previously shown NF-κB activation, release of IL-6, and activation of its responsive transcription factor STAT3 in K-ras mutant lung tumors, which was further amplified by the tumor enhancing effect of chronic obstructive pulmonary disease (COPD)-type airway inflammation. These findings suggest an essential role for this inflammatory pathway in K-ras mutant lung tumorigenesis and its enhancement by COPD. Therefore, here we blocked IL-6 using a monoclonal anti-IL-6 antibody in a K-ras mutant mouse model of lung cancer in the absence or presence of COPD-type airway inflammation. IL-6 blockade significantly inhibited lung cancer promotion, tumor cell intrinsic STAT3 activation, tumor cell proliferation, and angiogenesis markers. Moreover, IL-6 inhibition reduced expression of pro-tumor type 2 molecules (Arginase 1, Fizz 1, Mgl, and IDO), number of M2 type macrophages and G-MDSCs, and pro-tumor T-regulatory/T helper 17 cell responses. This was accompanied by increased expression of anti-tumor type 1 molecule (Nos2), and anti-tumor T helper 1/CD8 T cell responses. Our study demonstrates that IL-6 blockade not only has direct intrinsic inhibitory effect on tumor cells, but also re-educates the lung microenvironment toward an anti-tumor phenotype by altering the relative proportion between pro-tumor and anti-tumor immune cells. This information introduces IL-6 as a potential druggable target for prevention and treatment of K-ras mutant lung tumors.
    The two Dps proteins, NpDps2 and NpDps5, are involved in light-induced oxidative stress tolerance in the N2-fixing cyanobacterium Nostoc punctiforme
    Vamsi K. Moparthi, Biochimica et Biophysica Acta (BBA) - Bioenergetics - 2016
    Abstract
    Cyanobacteria are photosynthetic prokaryotes that are considered biotechnologically prominent organisms for production of high-value compounds. Cyanobacteria are subject to high-light intensities, which is a challenge that needs to be addressed in design of efficient bio-engineered photosynthetic organisms. Dps proteins are members of the ferritin superfamily and are omnipresent in prokaryotes. They play a major role in oxidative stress protection and iron homeostasis. The filamentous, heterocyst-forming Nostoc punctiforme, has five Dps proteins. In this study we elucidated the role of these Dps proteins in acclimation to high light intensity, the gene loci organization and the transcriptional regulation of all five dps genes in N. punctiforme was revealed, and dps-deletion mutant strains were used in physiological characterization. Two mutants defective in Dps2 and Dps5 activity displayed a reduced fitness under increased illumination, as well as a differential Photosystem (PS) stoichiometry, with an elevated Photosystem II to Photosystem I ratio in the dps5 deletion strain. This work establishes a Dps-mediated link between light tolerance, H2O2 detoxification, and iron homeostasis, and provides further evidence on the non-redundant role of multiple Dps proteins in this multicellular cyanobacterium.
    Uropathogenic Escherichia coli Releases Extracellular Vesicles That Are Associated with RNA
    Cherie Blenkiron, PLOS ONE - 2016
    Abstract
    Background Bacterium-to-host signalling during infection is a complex process involving proteins, lipids and other diffusible signals that manipulate host cell biology for pathogen survival. Bacteria also release membrane vesicles (MV) that can carry a cargo of effector molecules directly into host cells. Supported by recent publications, we hypothesised that these MVs also associate with RNA, which may be directly involved in the modulation of the host response to infection. Methods and Results Using the uropathogenic Escherichia coli (UPEC) strain 536, we have isolated MVs and found they carry a range of RNA species. Density gradient centrifugation further fractionated and characterised the MV preparation and confirmed that the isolated RNA was associated with the highest particle and protein containing fractions. Using a new approach, RNA-sequencing of libraries derived from three different ‘size’ RNA populations (<50nt, 50-200nt and 200nt+) isolated from MVs has enabled us to now report the first example of a complete bacterial MV-RNA profile. These data show that MVs carry rRNA, tRNAs, other small RNAs as well as full-length protein coding mRNAs. Confocal microscopy visualised the delivery of lipid labelled MVs into cultured bladder epithelial cells and showed their RNA cargo labelled with 5-EU (5-ethynyl uridine), was transported into the host cell cytoplasm and nucleus. MV RNA uptake by the cells was confirmed by droplet digital RT-PCR of csrC . It was estimated that 1% of MV RNA cargo is delivered into cultured cells. Conclusions These data add to the growing evidence of pathogenic bacterial MV being associated a wide range of RNAs. It further raises the plausibility for MV-RNA-mediated cross-kingdom communication whereby they influence host cell function during the infection process.
    Development of an Injectable Calcium Phosphate/Hyaluronic Acid Microparticles System for Platelet Lysate Sustained Delivery Aiming Bone Regeneration
    Pedo Babo, Macromolecular Bioscience - 2016
    Abstract
    Despite the biocompatibility and osteoinductive properties of calcium phosphate (CaP) cements their low biodegradability hampers full bone regeneration. Herein the incorporation of CaP cement with hyaluronic acid (HAc) microparticles loaded with platelet lysate (PL) to improve the degradability and biological performance of the cements is proposed. Cement formulations incorporating increasing weight ratios of either empty HAc microparticles or microparticles loaded with PL (10 and 20 wt%) are developed as well as cements directly incorporating PL. The direct incorporation of PL improves the mechanical properties of the plain cement, reaching values similar to native bone. Morphological analysis shows homogeneous particle distribution and high interconnectivity between the HAc microparticles. The cements incorporating PL (with or without the HAc microparticles) present a sustained release of PL proteins for up to 8 d. The sustained release of PL modulates the expression of osteogenic markers in seeded human adipose tissue derived stem cells, thus suggesting the stimulatory role of this hybrid system toward osteogenic commitment and bone regeneration applications.
    CKIP-1 silencing promotes the new bone formation in the rat mandibular distraction osteogenesis
    Zi-chao Zhou, Oral Surgery, Oral Medicine, Oral Pathology and Oral Radiology - 2016
    Abstract
    Objective This study investigated the effects and possible molecular mechanism of CKIP-1 silencing on bone regeneration during rat mandibular distraction osteogenesis (DO).Study Design CKIP-1 silencing by chitosan/si-CKIP-1 was employed and analyzed both in the rat mandibular DO models in vivo and in cultured rat mandible BMSCs in vitro. Results Gross observation, MicroCT analysis, and H&E staining revealed that the new bone formation in the distraction gap of the chitosan/si-CKIP-1-treated group was better compared with the chitosan/si-NC- or PBS-treated groups both in quantity and quality. Proliferation assay, flow cytometry, and alizarin red staining indicated that CKIP-1 silencing significantly inhibited apoptosis, but promoted the osteogenic differentiation of cultured BMSCs. Additionally, CKIP-1 silencing significantly promoted the expression of Wnt3a, β-catenin, and OCN both in the new bone formation of DO models in vivo and in the osteogenic differentiation process of BMSCs in vitro. ConclusionsThe promotion of bone formation after CKIP-1 silencing in the rat mandibular distraction osteogenesis appears to be mediated through the Wnt3a/β-catenin signaling pathway.
    A multi-step model for facilitated unwinding of the yeast U4/U6 RNA duplex
    Margaret L. Rodgers, Oxford Journals - 2016
    Abstract
    The small nuclear RNA (snRNA) components of the spliceosome undergo many conformational rearrangements during its assembly, catalytic activation and disassembly. The U4 and U6 snRNAs are incorporated into the spliceosome as a base-paired complex within the U4/U6.U5 small nuclear ribonucleoprotein (tri-snRNP). U4 and U6 are then unwound in order for U6 to pair with U2 to form the spliceosome's active site. After splicing, U2/U6 is unwound and U6 annealed to U4 to reassemble the tri-snRNP. U6 rearrangements are crucial for spliceosome formation but are poorly understood. We have used single-molecule Förster resonance energy transfer and unwinding assays to identify interactions that promote U4/U6 unwinding and have studied their impact in yeast. We find that U4/U6 is efficiently unwound using DNA oligonucleotides by coupling unwinding of U4/U6 stem II with strand invasion of stem I. Unwinding is stimulated by the U6 telestem, which transiently forms in the intact U4/U6 RNA complex. Stabilization of the telestem in vivo results in accumulation of U4/U6 di-snRNP and impairs yeast growth. Our data reveal conserved mechanisms for U4/U6 unwinding and indicate telestem dynamics are critical for tri-snRNP assembly and stability.
    PLOS Genetics: Exposure to the BPA-Substitute Bisphenol S Causes Unique Alterations of Germline Function
    Yichang Chen, PLOS ONE - 2016
    Abstract
    Concerns about the safety of Bisphenol A, a chemical found in plastics, receipts, food packaging and more, have led to its replacement with substitutes now found in a multitude of consumer products. However, several popular BPA-free alternatives, such as Bisphenol S, share a high degree of structural similarity with BPA, suggesting that these substitutes may disrupt similar developmental and reproductive pathways. We compared the effects of BPA and BPS on germline and reproductive functions using the genetic model system Caenorhabditis elegans. We found that, similarly to BPA, BPS caused severe reproductive defects including germline apoptosis and embryonic lethality. However, meiotic recombination, targeted gene expression, whole transcriptome and ontology analyses as well as ToxCast data mining all indicate that these effects are partly achieved via mechanisms distinct from BPAs. These findings therefore raise new concerns about the safety of BPA alternatives and the risk associated with human exposure to mixtures.
    PLOS ONE: Nutritional Supplement of Hatchery Eggshell Membrane Improves Poultry Performance and Provides Resistance against Endotoxin Stress
    S.K Makkar, PLOS ONE - 2016
    Abstract
    Eggshells are significant part of hatchery waste which consist of calcium carbonate crust, membranes, and proteins and peptides of embryonic origins along with other entrapped contaminants including microbes. We hypothesized that using this product as a nutritional additive in poultry diet may confer better immunity to the chickens in the paradigm of mammalian milk that enhances immunity. Therefore, we investigated the effect of hatchery eggshell membranes (HESM) as a short term feed supplement on growth performance and immunity of chickens under bacterial lipopolysaccharide (LPS) challenged condition. Three studies were conducted to find the effect of HESM supplement on post hatch chickens. In the first study, the chickens were fed either a control diet or diets containing 0.5% whey protein or HESM as supplement and evaluated at 5 weeks of age using growth, hematology, clinical chemistry, plasma immunoglobulins, and corticosterone as variables. The second and third studies were done to compare the effects of LPS on control and HESM fed birds at 5 weeks of age following at 4 and 24 h of treatment where the HESM was also sterilized with ethanol to deplete bacterial factors. HESM supplement caused weight gain in 2 experiments and decreased blood corticosterone concentrations. While LPS caused a significant loss in body weight at 24 h following its administration, the HESM supplemented birds showed significantly less body weight loss compared with the control fed birds. The WBC, heterophil/lymphocyte ratio, and the levels of IgG were low in chickens fed diets with HESM supplement compared with control diet group. LPS challenge increased the expression of pro-inflammatory cytokine gene IL-6 but the HESM fed birds showed its effect curtailed, also, which also, favored the up-regulation of anti-inflammatory genes compared with control diet fed chickens. Post hatch supplementation of HESM appears to improve performance, modulate immunity, and increase resistance of chickens to endotoxin.
    The C/ebp-Atf response element (CARE) location reveals two distinct Atf4-dependent, elongation-mediated mechanisms for transcriptional induction of aminoacyl-tRNA synthetase genes in response to amino acid limitation
    Jixiu Shan,, Oxford Journals - 2016
    Abstract
    The response to amino acid (AA) limitation of the entire aminoacyl-tRNA synthetase (ARS) gene family revealed that 16/20 of the genes encoding cytoplasmic-localized enzymes are transcriptionally induced by activating transcription factor 4 (Atf4) via C/ebp-Atf-Response-Element (CARE) enhancers. In contrast, only 4/19 of the genes encoding mitochondrial-localized ARSs were weakly induced. Most of the activated genes have a functional CARE near the transcription start site (TSS), but for others the CARE is downstream. Regardless of the location of CARE enhancer, for all ARS genes there was constitutive association of RNA polymerase II (Pol II) and the general transcription machinery near the TSS. However, for those genes with a downstream CARE, Atf4, C/ebp-homology protein (Chop), Pol II and TATA-binding protein exhibited enhanced recruitment to the CARE during AA limitation. Increased Atf4 binding regulated the association of elongation factors at both the promoter and the enhancer regions, and inhibition of cyclin-dependent kinase 9 (CDK9), that regulates these elongation factors, blocked induction of the AA-responsive ARS genes. Protein pull-down assays indicated that Atf4 directly interacts with CDK9 and its associated protein cyclin T1. The results demonstrate that AA availability modulates the ARS gene family through modulation of transcription elongation.
    Hypoxia upregulates Malat1 expression through a CaMKK/AMPK/HIF-1α axis
    Sandrine Sallï-Lefort, International Journal of Oncology - 2016
    Abstract
    Increased expression levels of the long non-coding RNA metastasis-associated lung adenocarcinoma transcript 1 (Malat1) have been associated with enhanced proliferation and metastasis of several cancer cell types. Hypoxia, a hallmark characteristic of solid tumors, has been linked to an increase in the activity of the ATP-generating AMPK protein. Since Malat1 was recently shown to be upregulated during hypoxia, the objective of this study was to determine the contribution of AMPK in the mechanistic pathways regulating Malat1 expression in low oxygen conditions. Compared to those cultured in 21% O2 conditions, HeLa cells incubated in 1.5% O2 expressed more Malat1 transcripts. This observation was mimicked in HEK293T cells using a synthetic reporter construct containing 5.6 kb of the human Malat1 promoter, suggesting that hypoxia directly impacted Malat1 gene transcription. Interestingly, pharmacological stimulation of AMPK increased Malat1 promoter transactivation in 21% O2 conditions, whereas inhibition of either AMPK or its upstream activator CaMKK completely abolished the augmentation of Malat1 under hypoxia. Pharmacological modulation of LKB1, another major regulator of AMPK, had no impact on Malat1 promoter transactivation, suggesting that calcium inputs are important in the control of Malat1 expression by AMPK. Overexpression of hypoxia-inducible factor-1α (HIF-1α) increased Malat1 expression in 21% O2 conditions, whereas pharmacological inhibition of HIF-1α blocked the impact of hypoxia on the Malat1 promoter. Taken together, these findings strongly suggest that Malat1 expression is regulated in hypoxic conditions by a CaMKK/AMPK/HIF-1α axis. More research is needed in physiological settings to test the clinical relevance of this pathway.
    Sam68/KHDRBS1 is critical for colon tumorigenesis by regulating genotoxic stress-induced NF-κB activation
    Kai Fu, eLife - 2016
    Abstract
    Cells use signaling pathways to detect and respond to harmful conditions by switching on genes that keep the cell healthy. One important pathway is the nuclear factor kappa B (NF-κB) signaling pathway, which is activated by many stimuli. These stimuli may come from infections from outside the cell or may originate inside the cell, as seen for DNA damage caused by irradiation, chemicals or rapid DNA replication in cancer cells. Most of a cell’s DNA is located in the cell nucleus. However, NF-κB proteins are normally located outside the nucleus, in the cell’s cytoplasm. Damage to DNA triggers a signal from the nucleus to the cytoplasm. This signal activates the NF-κB proteins, which move into the nucleus and turn on genes that help the cell to recover from the damage. These genes include those that prevent the cell from self-destructing. In one step of the NF-κB activation process, chain-like molecules called polymers are made from a compound called poly(ADP-ribose), or PAR for short. However, few other details are known about how the damaged DNA in the nucleus signals to the cytoplasm. A protein called Sam68, which is found in the cell nucleus, has been linked to DNA damage signaling. Fu, Sun et al. now present evidence that suggests that if mouse cells lack Sam68, they do not produce PAR polymers in response to DNA damage. In addition, these cells could not trigger the PAR-dependent signaling cascade that is essential for activating NF-κB and for turning on the protective genes. Consequently, cells that lacked Sam68 were extremely sensitive to agents that cause DNA damage, such as chemicals and irradiation. The NF-κB pathway is regulated incorrectly in some cancers, but is also activated by DNA damage caused by cancer treatments. Therefore, Fu, Sun et al. also explored the role of Sam68 in cancer. Reducing the levels of Sam68 made human colon cancer cells more likely to self-destruct when they were exposed to DNA-damaging agents. Furthermore, removing Sam68 from mice that spontaneously grow colon cancer caused their tumors to develop more slowly than mice that retained Sam68 in their cells. Overall, the findings presented by Fu, Sun et al. suggest that Sam68 regulates the signal from the nucleus to the cytoplasm that activates NF-κB proteins in response to DNA damage. Sam68 also appears to be important for helping colon cancer cells grow and survive. Future challenges will be to understand how Sam68 regulates the production of the PAR polymer in this response and to explore whether Sam68 can be targeted for treating cancer.
    USE OF PLASMA-TREATED LIQUIDS TO TREAT HERPES KERATITIS
    Jane Azizkhan-clifford, United States Patent - 2016
    Abstract
    The present invention is directed toward the use of non-thermal plasma-treated liquids as treatment options for herpes keratitis.
    CD84 mediates CLL-microenvironment interactions
    A. Marom, Oncogene - 2016
    Abstract
    Chronic lymphocytic leukemia (CLL) is a malignant disease of small mature lymphocytes. Signals from the CLL microenvironment promote progression of the disease and induce drug resistance. This phenomenon is largely dependent on direct contact between the malignant B cells and stromal cells. CD84 belongs to the signaling lymphocyte activation molecule family of immunoreceptors, which self-associates, forming an orthogonal homophilic dimer. We therefore hypothesized that CD84 may bridge between CLL cells and their microenvironment, promoting cell survival. Our in vitro results show that CD84 expressed on CLL cells interact with CD84 expressed on cells in their microenvironment, inducing cell survival in both sides. Blocking CD84 in vitro and in vivo disrupt the interaction of CLL cells with their microenvironment, resulting in induced cell death. Thus, our findings suggest novel therapeutic strategies based on the blockade of this CD84-dependent survival pathway.
    Adipocyte nuclei captured from VAT and SAT | BMC Obesity | Full Text
    Suresh Ambati, BioMed Central - 2016
    Abstract
    Obesity-related comorbidities are thought to result from the reprogramming of the epigenome in numerous tissues and cell types, and in particular, mature adipocytes within visceral and subcutaneous adipose tissue, VAT and SAT. The cell-type specific chromatin remodeling of mature adipocytes within VAT and SAT is poorly understood, in part, because of the difficulties of isolating and manipulating large fragile mature adipocyte cells from adipose tissues.
    Interleukin (IL)‐1 in rat parturition: IL‐1 receptors 1 and 2 and accessory proteins abundance in pregnant rat uterus at term – regulation by progesterone
    Tomohito Ishiguro, Physiological Reports - 2016
    Abstract
    The role of interleukin‐1 (IL‐1), a pro‐inflammatory cytokine, in parturition is typically noted by changes in its concentrations. Studying the expression of its receptor family, IL‐1 receptor (IL‐1R) 1, IL‐1R2, IL‐1R accessory protein (IL‐1RAcP), and its predominantly brain isoform, IL‐1RAcPb, during late gestation in the uterus in the Long‐Evans rat is another. We assessed changes in their mRNA and protein relative abundance in the uterus and compared IL‐1RAcP and IL‐1RAcPb mRNA abundance in uterus, cervix, ovaries, placenta, and whole blood of Long‐Evans rats during late gestation or in RU486 and progesterone‐treated dams using quantitative real‐time PCR and western immunoblotting. IL‐1R1, IL‐1RAcP, and IL‐1RAcPb mRNA abundance significantly increased in the uterus at delivery whereas IL‐1R2 mRNA abundance significantly decreased. IL‐1R1 protein increased at term and IL‐1R2 protein decreased at term compared to nonpregnant uteri. IL1‐RAcPb mRNA abundance was less than IL‐1RAcP, but in the lower uterine segment it was the highest of all tissues examined. RU486 stimulated preterm delivery and an increase in IL‐1R1 mRNA abundance whereas progesterone administration extended pregnancy and suppressed the increase in IL‐1R1. These data suggest that changes in uterine sensitivity to IL‐1 occur during late gestation and suggest another level of regulation for the control of delivery. The roles for IL‐1RAcP and IL‐1RAcPb need to be determined, but may relate to different intracellular signaling pathways.
    Netrin-1 Augments Chemokinesis in CD4+ T Cells In Vitro and Elicits a Proinflammatory Response In Vivo
    Leo Boneschansker, The Journal of Immunology - 2016
    Abstract
    Netrin-1 is a neuronal guidance cue that regulates cellular activation, migration, and cytoskeleton rearrangement in multiple cell types. It is a chemotropic protein that is expressed within tissues and elicits both attractive and repulsive migratory responses. Netrin-1 has recently been found to modulate the immune response via the inhibition of neutrophil and macrophage migration. However, the ability of Netrin-1 to interact with lymphocytes and its in-depth effects on leukocyte migration are poorly understood. In this study, we profiled the mRNA and protein expression of known Netrin-1 receptors on human CD4+ T cells. Neogenin, uncoordinated-5 (UNC5)A, and UNC5B were expressed at low levels in unstimulated cells, but they increased following mitogen-dependent activation. By immunofluorescence, we observed a cytoplasmic staining pattern of neogenin and UNC5A/B that also increased following activation. Using a novel microfluidic assay, we found that Netrin-1 stimulated bidirectional migration and enhanced the size of migratory subpopulations of mitogen-activated CD4+ T cells, but it had no demonstrable effects on the migration of purified CD4+CD25+CD127dim T regulatory cells. Furthermore, using a short hairpin RNA knockdown approach, we observed that the promigratory effects of Netrin-1 on T effectors is dependent on its interactions with neogenin. In the humanized SCID mouse, local injection of Netrin-1 into skin enhanced inflammation and the number of neogenin-expressing CD3+ T cell infiltrates. Neogenin was also observed on CD3+ T cell infiltrates within human cardiac allograft biopsies with evidence of rejection. Collectively, our findings demonstrate that Netrin-1/neogenin interactions augment CD4+ T cell chemokinesis and promote cellular infiltration in association with acute inflammation in vivo.
    Plasmalogen Deficit: A New and Testable Hypothesis for the Etiology of Alzheimer’s Disease - wood_2011.pdf
    Tara Smith, Phenomenome Discoveries, - 2011
    Abstract
    Alzheimer’s disease (AD) is a complex cognitive disorder for which the single greatest risk factor is age. The pathophysiological basis for AD is still a matter of debate with no current hypothesis explaining all of the complex pathological changes observed. These include neurofibrillary tangles, amyloid plaques, neuroinflammation, hypomyelination, neuronal shrinkage (eg. N- basalis cholinergic neurons, resulting in a dramatic cholinergic deficit), ocular pathology, microvascular pathology and liver peroxisomal dysfunction. The hypothesis that we are presenting, namely peroxisomal dysfunction resulting in decreased supply of critical ethanolamine plasmalogens to the brain, eye and vascular endothelium, is the first hypothesis that can potentially explai n all of these complex pathologies in AD. The value of this hypothesis is that it is imminently testable via resupply of critical ether lipid precursors of plasmalogens. PPI-1011 is such a drug candidate and will be presented after a review of the basis of this hypothesis.
    The ER membrane-anchored ubiquitin ligase Hrd1 is a positive regulator of T-cell immunity
    Yuanming Xu, Nature Communications - 2016
    Abstract
    Identification of positive regulators of T-cell immunity induced during autoimmune diseases is critical for developing novel therapies. The endoplasmic reticulum resident ubiquitin ligase Hrd1 has recently emerged as a critical regulator of dendritic cell antigen presentation, but its role in T-cell immunity is unknown. Here we show that genetic deletion of Hrd1 in mice inhibits T-cell proliferation, production of IL-2, and differentiation of Th1 and Th17 cells, and consequently protects mice from experimental autoimmune encephalomyelitis. Hrd1 facilitates T-cell proliferation by the destruction of cyclin-dependent kinase inhibitor p27kip1, and deletion of p27kip1 in Hrd1-null T-cells rescues proliferative capacity but not the production of cytokines, including IL-2, IFN-γ and IL-17. T-cell expression of Hrd1 is higher in patients with multiple sclerosis than in healthy individuals, and knockdown of Hrd1 in human CD4+ T cells inhibits activation and differentiation to Th1 and Th17 cells. Our study identifies Hrd1 as a previously unappreciated positive regulator of T cells and implies that Hrd1 is a potential therapeutic target for autoimmune diseases.
    Responses of bovine early embryos to S-adenosyl methionine supplementation in culture
    Habib A Shojaei Saadi, Epigenomics - 2016
    Abstract
    Aim: There is a growing concern about the potential adverse effects of high dose folic acid (FA) supplementation before and during pregnancy. FA metabolism generates S-adenosyl methionine (SAM) which is an important cofactor of epigenetic programming. We sought to assess the impact of a large dose of SAM on early embryo development. Materials & methods: In vitro cultured bovine embryos were treated with SAM from the eight-cell stage to the blastocyst stage. In addition to the phenotype, the genome-wide epigenetic and transcription profiles were analyzed. Results: Treatment significantly improved embryo hatching and caused a shift in sex ratio in favor of males. SAM caused genome-wide hypermethylation mainly in exonic regions and in CpG islands. Although differentially expressed genes were associated with response to nutrients and developmental processes, no correspondence was found with the differentially methylated regions, suggesting that cellular responses to SAM treatment during early embryo development may not require DNA methylation-driven changes. Conclusion: Since bovine embryos were not indifferent to SAM, effects of large-dose FA supplements on early embryonic development in humans cannot be ruled out.
    Rare novel variants in the ZIC3 gene cause X-linked heterotaxy
    Aimee D. C. Paulussen, European Journal of Human Genetics - 2016
    Abstract
    Variants in the ZIC3 gene are rare, but have demonstrated their profound clinical significance in X-linked heterotaxy, affecting in particular male patients with abnormal arrangement of thoracic and visceral organs. Several reports have shown relevance of ZIC3 gene variants in both familial and sporadic cases and with a predominance of mutations detected in zinc-finger domains. No studies so far have assessed the functional consequences of ZIC3 variants in an in vivo model organism. A study population of 348 patients collected over more than 10 years with a large variety of congenital heart disease including heterotaxy was screened for variants in the ZIC3 gene. Functional effects of three variants were assessed both in vitro and in vivo in the zebrafish. We identified six novel pathogenic variants (1,7%), all in either male patients with heterotaxy (n=5) or a female patient with multiple male deaths due to heterotaxy in the family (n=1). All variants were located within the zinc-finger domains or leading to a truncation before these domains. Truncating variants showed abnormal trafficking of mutated ZIC3 proteins, whereas the missense variant showed normal trafficking. Overexpression of wild-type and mutated ZIC protein in zebrafish showed full non-functionality of the two frame-shift variants and partial activity of the missense variant compared with wild-type, further underscoring the pathogenic character of these variants. Concluding, we greatly expanded the number of causative variants in ZIC3 and delineated the functional effects of three variants using in vitro and in vivo model systems.
    HLA peptides derived from tumor antigens induced by inhibition of DNA methylation for development of drug-facilitated immunotherapy
    Bracha Shraibman, Molecular & Cellular Proteomics - 2016
    Abstract
    Treatment of cancer cells with anti-cancer drugs often fails to achieve complete remission. Yet, such drug treatments may induce alteration in the tumor's gene expression patterns, including those of Cancer/Testis Antigens (CTA). The degradation products of such antigens can be presented as HLA peptides on the surface of the tumor cells and be developed into anti-cancer immunotherapeutics. For example, the DNA methyl transferase inhibitor, 5-aza-2-deoxycytidine (Decitabine) has limited anti-tumor efficacy, yet it induces the expression of many genes, including CTAs that are normally silenced in the healthy adult tissues. In this study, the presentation of many new HLA peptides derived from CTAs and induced by Decitabine was demonstrated in three human Glioblastoma cell lines. Such presentation of CTA-derived HLA peptides can be exploited for development of new treatment modalities, combining drug treatment with anti-CTA targeted immunotherapy. The Decitabine-induced HLA peptidomes include many CTAs that are not normally detected in healthy tissues or in cancer cells, unless treated with the drug. In addition, the study included large-scale analyses of the simultaneous effects of Decitabine on the transcriptomes, proteomes and HLA peptidomes of the human Glioblastoma cells. It demonstrates the poor correlations between these three levels of gene expression, both in their total levels and in their response to the drug. The proteomics and HLA peptidomics data are available via ProteomeXchange with identifier PXD003790 and the transcriptomics data are available via GEO with identifier GSE80137.
    Investigating essential gene function in Mycobacterium tuberculosis using an efficient CRISPR interference system
    Atul K. Singh, Nucleic Acids Research - 2016
    Abstract
    Despite many methodological advances that have facilitated investigation of Mycobacterium tuberculosis pathogenesis, analysis of essential gene function in this slow-growing pathogen remains difficult. Here, we describe an optimized CRISPR-based method to inhibit expression of essential genes based on the inducible expression of an enzymatically inactive Cas9 protein together with gene-specific guide RNAs (CRISPR interference). Using this system to target several essential genes of M. tuberculosis, we achieved marked inhibition of gene expression resulting in growth inhibition, changes in susceptibility to small molecule inhibitors and disruption of normal cell morphology. Analysis of expression of genes containing sequences similar to those targeted by individual guide RNAs did not reveal significant off-target effects. Advantages of this approach include the ability to compare inhibited gene expression to native levels of expression, lack of the need to alter the M. tuberculosis chromosome, the potential to titrate the extent of transcription inhibition, and the ability to avoid off-target effects. Based on the consistent inhibition of transcription and the simple cloning strategy described in this work, CRISPR interference provides an efficient approach to investigate essential gene function that may be particularly useful in characterizing genes of unknown function and potential targets for novel small molecule inhibitors.
    An investigation of BMP-7 mediated alterations to BMP signalling components in human tenocyte-like cells : Scientific Reports
    Franka Klatte-Schulz, Scientific Reports - 2016
    Abstract
    The incidence of tendon re-tears post-surgery is an ever present complication. It is suggested that the application of biological factors, such as bone morphogenetic protein 7 (BMP-7), can reduce complication rates by promoting tenogenic characteristics in in vitro studies. However, there remains a dearth of information in regards to the mechanisms of BMP-7 signalling in tenocytes. Using primary human tenocyte-like cells (hTLCs) from the supraspinatus tendon the BMP-7 signalling pathway was investigated: induction of the BMP associated Smad pathway and non-Smad pathways (AKT, p38, ERK1/2 and JNK); alterations in gene expression of BMP-7 associated receptors, Smad pathway components, Smad target gene (ID1) and tenogenic marker scleraxis. BMP-7 increases the expression of specific BMP associated receptors, BMPR-Ib and BMPR-II, and Smad8. Additionally, BMP-7 activates significantly Smad1/5/8 and slightly p38 pathways as indicated by an increase in phosphorylation and proven by inhibition experiments, where p-ERK1/2 and p-JNK pathways remain mainly unresponsive. Furthermore, BMP-7 increases the expression of the Smad target gene ID1, and the tendon specific transcription factor scleraxis. The study shows that tenocyte-like cells undergo primarily Smad8 and p38 signalling after BMP-7 stimulation. The up-regulation of tendon related marker genes and matrix proteins such as Smad8/9, scleraxis and collagen I might lead to positive effects of BMP-7 treatment for rotator cuff repair, without significant induction of osteogenic and chondrogenic markers.
    Metformin elicits antitumor effects and downregulates the histone methyltransferase multiple myeloma SET domain (MMSET) in prostate cancer cells
    Nicole M. A. White-Al Habeeb, The Prostate - 2016
    Abstract
    BACKGROUND This study explored the biological effects of metformin on prostate cancer (PCa) cells and determined molecular pathways and epigenetic regulators implicated in its mechanism of action. METHODS We performed mRNA expression profiling in 22Rv1 cells following 2.5 mM and 5 mM metformin treatment. Genes significantly modified by metformin treatment were ranked based on altered expression, involvement with cancer-related processes, and reported dysregulation in PCa. The effects of the top ranked gene, MMSET, on the proliferative and invasive capabilities of PCa cells were investigated via siRNA knockdown alone and also combined with metformin treatment. RESULTS Metformin treatment decreased cell growth of PCa cell line 22Rv1 and stalled cells at the G1/S checkpoint in a time- and dose-dependent manner, resulting in increased cells in G1 (P < 0.05) and decreased cells in S (P < 0.05) phase. Metformin activated the AMPK/mTOR signaling pathway as shown by increased p-AMPK and decreased p-p70S6K. mRNA expression profiling following metformin treatment identified significant changes in 136 chromatin-modifying genes. The top ranked gene, multiple myeloma SET domain (MMSET) showed increased expression in PCa cell lines (22Rv1 and DU145) when compared to the benign prostate epithelium-derived cell-line RWPE-1, and its expression was decreased upon metformin treatment. siRNA-mediated knockdown of MMSET showed decreased cellular migration and invasion in DU-145 cells. MMSET knockdown in combination with metformin treatment resulted in further reduction in the capacity of PCa cells to migrate and invade. CONCLUSIONS These data suggest MMSET may play a role in the inhibitory effect of metformin on PCa and could serve as a potential novel therapeutic target for PCa. Prostate © 2016 Wiley Periodicals, Inc.
    Gluten-Free Diet Only during Pregnancy Efficiently Prevents Diabetes in NOD Mouse Offspring
    Julie C. Antvorskov,, Journal of Diabetes Research - 2016
    Abstract
    Gluten exposure is an important factor in the development of type 1 diabetes (T1D) [1]. Both in nonobese diabetic (NOD) mice and in Biobreeding- (BB-) rats a gluten-free (GF) [2, 3] or hydrolyzed [4] diet lowers the incidence of the diseasemarkedly. In humans, a GF, low carbohydrate diet was recently reported to induce remission in a newly diagnosed T1D patient [5], and improved insulin secretion has been observed after 6 months of GF diet [6]. The appearance of beta cell autoimmunity may be related to the age at introduction of cereals into the infant diet [7, 8]. Further, a high dietary content of monosaccharides increases the risk of developing T1D [9, 10]. The mechanism by which gliadin influences disease development is not known. It is likely that gut immunity is involved since we [11] and others [12, 13] have reported that gluten intake confers a proinflammatory cytokine profile in multiple regulatory T cell populations, including 𝛾𝛿 T cells, inmucosal lymphoid tissues. It is likely that these changes are facilitated by or even dependent on gluten peptides being able to transverse the epithelium,whichwe recently demonstrated in NOD and BALB/c mice (unpublished) and which was previously reported in T1D patients [14]. It is also possible that a direct effect of gluten peptides on the beta cells can influence disease development. We have shown that a 33-mer gliadin peptide can directly close the K channel and induce insulin secretion [15]. Although it is not clear at the present time whether gliadin interacts directly with the K channel or the signal is relayed through other receptors, such as TLR4, this cellular activation might increase the risk of disease development through beta cell stress [16]. Previous experiments with GF diet in animal models of autoimmune diabetes [2, 3, 17] were all carried out in animals exposed to GF diet both during pregnancy and during the neonatal period. However, the potential of gluten to affect the diabetogenic process seems to be dependent on the time of gluten introduction, both in animals [12, 13] and in humans [7, 8]. It is therefore not known if there is a critical period, during which the effect of a GF diet is most efficient, or if both time slots are equally important. This was investigated in this study, which showed that GF diet, exclusively during pregnancy, far exceeds the efficiency of other treatment periods.
    Transcriptome meta-analysis of three follicular compartments and its correlation with ovarian follicle maturity and oocyte developmental competence in cows
    Daulat Raheem Khan, Physiological Genomics - 2016
    Abstract
    Oocyte developmental competence in super-stimulated cows is dependent in part on the duration of the FSH coasting. FSH coasting refers to super-stimulation with FSH (two days of endogenous FSH following follicle ablation and three days of FSH injections) followed by no FSH for a specific duration. The optimal duration varies among individuals. FSH coasting appears to modulate the transcriptome of different follicular compartments, which cooperate as a single functional unit. However, the integrative effects of FSH coasting on different follicular compartments remain ambiguous. Meta-analysis of three independent transcriptome studies each focused on a single cell type (granulosa, cumulus and oocyte) during FSH coasting allowed the identification of 12 gene clusters with similar time-course expression patterns in all three compartments. Network analysis identified HNF4A (involved in metabolic functions) and ELAVL1 (an RNA-binding protein) as hub genes regulated respectively upwards and downwards in the clusters enriched at the optimal coasting time, and APP (involved in mitochondrial functions) and COPS5 (a member of the COP9 signalosome) as hub genes regulated respectively upwards and downwards in the clusters enriched progressively throughout the coasting period. We confirmed the effects on HNF4A downstream targets (TTR, PPL) and other hub genes (ELAVL1, APP, MYC and PGR) in 30 cows using RT-qPCR. The correlation of hub gene expression levels with FSH coasting indicated that a combination of these genes could predict oocyte competence with 83% sensitivity, suggesting that they are potential biomarkers of follicle differentiation. These findings could be used to optimize FSH coasting on an individual basis.
    MITF depletion elevates expression levels of ERBB3 receptor and its cognate ligand NRG1-beta in melanoma (PDF Download Available)
    t Tine Norman Alver, Oncotarget - 2016
    Abstract
    The phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) pathway is frequently hyper-activated upon vemurafenib treatment of melanoma. We have here investigated the relationship between SRY-box 10 (SOX10), forkhead box 3 (FOXD3) and microphthalmia-associated transcription factor (MITF) in the regulation of the receptor tyrosine-protein kinase ERBB3, and its cognate ligand neuregulin 1-beta (NRG1-beta). We found that both NRG1-beta and ERBB3 mRNA levels were elevated as a consequence of MITF depletion, induced by either vemurafenib or MITF small interfering RNA (siRNA) treatment. Elevation of ERBB3 receptor expression after MITF depletion caused increased activation of the PI3K pathway in the presence of NRG1-beta ligand. Together, our results suggest that MITF may play a role in the development of acquired drug resistance through hyper-activation of the PI3K pathway.
    Influence of energy level and nicotinic acid supplementation on apoptosis of blood leukocytes of periparturient dairy cows
    S. Bühlera, Science Direct - 2016
    Abstract
    The periparturient period of dairy cows is accompanied by an immunosuppression that leaves the animal more susceptible to infections and metabolic disorders. Non-esterified fatty acids (NEFA) and beta-hydroxybutyrate (BHB) which peak shortly after parturition due to lipolysis are known to impair immune cell functions. Niacin with its well-known anti-lipolytic effect may have the ability to ameliorate this situation. Additionally, niacin shows also anti-inflammatory effects that may be beneficial to the immune status of the cow. To address this 29 multiparous and 18 primiparous German Holstein cows were subjected to four different feeding groups. They were fed either a ration with a high concentrate proportion of 60% (HC), or a low concentrate proportion of 30% (LC). After parturition both concentrate levels were reduced to 30% and increased again to 50% either within 16 days (LC-group) or within 24 days (HC-group). Half of the animals received either 24 g per day of nicotinic acid from 42 days prepartum until 24 days postpartum (LC-NA, HC-NA) or no supplement (LC-CON, HC-CON). Apoptosis in polymorphonuclear leukocytes (PMN) and peripheral blood mononuclear cells (PBMC) was examined with an Annexin V and propidium iodide (PI) based fluorescence flow cytometry assay and distinguished into early apoptotic (Annexin V positive and PI negative) and late apoptotic (Annexin V and PI positive) cells. Additionally, the pro-apoptotic gene BAX, the effector caspase CASP3, and the anti-apoptotic genes BCL2 and BCL-xL, as well as the NFκB subunit RELA were quantified by real-time PCR in blood leukocytes. All variables showed time dependencies that were mainly related to parturition (p < 0.01). Early apoptotic PBMC were significantly affected by concentrate level showing higher numbers of apoptotic cells in the HC groups (p = 0.029). PBMC were characterized by a more pronounced apoptosis than PMN and seemed to be more susceptible to the changes that occur around parturition. The genes BAX and CASP3 were positively correlated (0.631) and their peak preceded the apoptotic peak around parturition in the blood leukocytes. The LC animals showed a decrease in BCL2 expression before parturition, whereas the HC animals showed a continuous increase in BCL2 mRNA abundance (p = 0.059). RELA correlated stronger with the pro-apoptotic genes (0.715 and 0.650 with BAX and CASP3 respectively) and its expression was higher in primiparous than in multiparous cows (p = 0.011). Nicotinic acid supplementation did show some influence in increasing numbers of early apoptotic PMN and late apoptotic PBMC between 42 and 100 DIM.
    THERAPEUTIC AGENTS FOR MODULATING THYMIC FUNCTION AND/OR GROWTH AND/OR TREATING VARIOUS DISORDERS
    Adam Giangreco, United States Patent - 2016
    Abstract
    The present disclosure relates to a therapeutic agent for use in a method for modulating the function and/or growth of a thymus in a subject, wherein the therapeutic agent comprises an HER2 or HER1 pathway antagonist or agonist, and/or a CCR/CCL5 antagonist the method involving administering the therapeutic agent to the subject. Also disclosed herein is a therapeutic agent for use in a method for treating a disorder in a subject, the disorder selected from systemic autoimmunity, peripheral autoimmunity and Systemic Lupus Erythematosus,
    Ehrlichia chaffeensis TRP120 Activates Canonical Notch Signaling To Downregulate TLR2/4 Expression and Promote Intracellular Survival
    Taslima T. Lina, mBio - 2016
    Abstract
    Ehrlichia chaffeensis preferentially targets mononuclear phagocytes and survives through a strategy of subverting innate immune defenses, but the mechanisms are unknown. We have shown E. chaffeensis type 1 secreted tandem repeat protein (TRP) effectors are involved in diverse molecular pathogen-host interactions, such as the TRP120 interaction with the Notch receptor-cleaving metalloprotease ADAM17. In the present study, we demonstrate E. chaffeensis, via the TRP120 effector, activates the canonical Notch signaling pathway to promote intracellular survival. We found that nuclear translocation of the transcriptionally active Notch intracellular domain (NICD) occurs in response to E. chaffeensis or recombinant TRP120, resulting in upregulation of Notch signaling pathway components and target genes notch1, adam17, hes, and hey. Significant differences in canonical Notch signaling gene expression levels (>40%) were observed during early and late stages of infection, indicating activation of the Notch pathway. We linked Notch pathway activation specifically to the TRP120 effector, which directly interacts with the Notch metalloprotease ADAM17. Using pharmacological inhibitors and small interfering RNAs (siRNAs) against γ-secretase enzyme, Notch transcription factor complex, Notch1, and ADAM17, we demonstrated that Notch signaling is required for ehrlichial survival. We studied the downstream effects and found that E. chaffeensis TRP120-mediated activation of the Notch pathway causes inhibition of the extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (MAPK) pathways required for PU.1 and subsequent Toll-like receptor 2/4 (TLR2/4) expression. This investigation reveals a novel mechanism whereby E. chaffeensis exploits the Notch pathway to evade the host innate immune response for intracellular survival. IMPORTANCE E. chaffeensis is an obligately intracellular bacterium and the etiologic agent of human monocytotropic ehrlichiosis (HME), an emerging life-threatening tick-borne zoonosis. Mechanisms by which E. chaffeensis establishes intracellular infection and avoids innate host defenses are not understood, but functionally relevant host-pathogen interactions with type 1 secreted TRP effectors are essential for the ehrlichial cellular reprogramming strategy. This study provides further insight into the molecular strategies used by obligately intracellular pathogens such as E. chaffeensis, which have small genomes and a limited number of effector proteins and exploit evolutionarily conserved host cell programs such as Notch signaling to promote infection and intracellular survival.
    PTBP1 modulation of MCL1 expression regulates cellular apoptosis induced by antitubulin chemotherapeutics
    J. Cui, Cell Death & Differentiation - 2016
    Abstract
    Myeloid cell leukemia sequence 1 (MCL1), an anti-apoptotic BCL2 family protein, is a key regulator of intrinsic apoptosis. Normal cells require strict control over MCL1 expression with aberrant MCL1 expression linked to the emergence of various diseases and chemoresistance. Previous studies have detailed how MCL1 expression is regulated by multiple mechanisms both transcriptionally and translationally. However, characterization of the post-transcriptional regulators of MCL1 mRNA is limited. Polypyrimidine tract binding protein 1 (PTBP1) is a known regulator of post-transcriptional gene expression that can control mRNA splicing, translation, stability and localization. Here we demonstrate that PTBP1 binds to MCL1 mRNA and that knockdown of PTBP1 upregulates MCL1 expression in cancer cells by stabilizing MCL1 mRNA and increasing MCL1 mRNA accumulation in cytoplasm. Further, we show that depletion of PTBP1 protects cancer cells from antitubulin agent-induced apoptosis in a MCL1-dependent manner. Taken together, our findings suggest that PTBP1 is a novel regulator of MCL1 mRNA by which it controls apoptotic response to antitubulin chemotherapeutics.
    Misregulation of Alternative Splicing in a Mouse Model of Rett Syndrome
    Ronghui Li, PLOS Genet - 2016
    Abstract
    Author Summary Rett syndrome (RTT) is a debilitating neurodevelopmental disorder with no cure or effective treatment. To fully understand the disease mechanism and develop therapies, it is necessary to study all aspects of the molecular function of methyl-CpG binding protein 2 (MeCP2), mutations in which have been identified as the genetic cause of RTT. Over the years, MeCP2 has been shown to maintain DNA methylation, regulate transcription and chromatin structure, control microRNA processing, and modulate RNA splicing. Among these known functions, the role of MeCP2 in modulating RNA splicing is less well understood. We took several unbiased approaches to investigate the how MeCP2 may regulate splicing, what splicing changes are caused by the loss of MeCP2, and what functional consequences are caused by altered splicing. We discovered that MeCP2 interacts with splicing factors to regulated the splicing of glutamate receptor genes, which mediate the vast majority of excitatory synaptic transmission in the brain; and linked the altered splicing of glutamate receptor genes to specific synaptic changes in a RTT mouse model. Our findings not only advance the understanding of RTT disease mechanism, but also reveal a potential drug target for future development of therapies.
    Paternal age and diet: The contributions of a father’s experience to susceptibility for post-concussion symptomology
    Harleen Hehar, Neuroscience - 2016
    Abstract
    In an attempt to improve current understanding of risk factors that influence individual susceptibility to poor outcomes following mild traumatic brain injury (mTBI) or concussion, this project investigated whether modifications to paternal experiences (Advanced Age (AA) or High-Fat Diet (HFD)) affected offspring susceptibility to behavioral symptomology and changes in gene expression following pediatric concussion in a rodent model. The study demonstrated that paternal treatment prior to conception altered behavioral outcomes and molecular characterization of offspring. Offspring of AA fathers demonstrated abnormal behavioral performance when compared to offspring of control fathers. Similarly, paternal HFD altered pathophysiological outcomes for offspring, contributing to the heterogeneity in post-concussion syndrome. Additionally, this study provided insight into the mechanisms that mediate non-genetic paternal inheritance. Paternal treatment and the mTBI significantly influenced expression of a majority of the genes under examination in the prefrontal cortex, hippocampus, and nucleus accumbens, with changes being dependent upon sex and the brain region examined. These epigenetic changes may have contributed to the differences in offspring susceptibility to concussion.
    Intraarticular slow-release triamcinolone acetate reduces allodynia in an experimental mouse knee osteoarthritis model
    Jeffrey S. Kroina, Redearch Paper - 2016
    Abstract
    Intraarticular steroid injection has been the mainstay of short-term treatment of knee osteoarthritis (OA) pain. However, the duration of therapeutic effect from a single injection is not as long as desired. In this study we use a viscous formulation of triamcinolone acetate (TCA) in hyaluronic acid to prolong the anti-allodynia effect of that steroid. OA was induced in mice by a partial medial meniscectomy. Over time the animals' developed a mechanical allodynia in the injected leg. Mice were then given a single intraarticular injection of TCA in a short-acting DMSO formulation, or a standard commercial suspension, or the drug formulated in 5% hyaluronic acid for slow-release. Control injections in OA mice were PBS or 5% hyaluronic acid vehicle. Mechanical allodynia was then monitored over the therapeutic period. Organotypic spinal cord slices and DRG culture were performed to assess whether TCA attenuates expressions of pain mediators induced by interleukin 1β. TCA 40 μg in a fast-releasing DMSO formulation produced relief from mechanical allodynia for a few days compared to PBS control injections (P = 0.007). Similarly, the commercial suspension of TCA 40 μg also produced relief from mechanical allodynia for a few days compared to PBS control injections (P = 0.001). However, TCA 100 μg in 5% hyaluronic acid produced relief from mechanical allodynia for at least 28 days compared to PBS control or 5% hyaluronic acid vehicle injections (P = 0.0005). Furthermore, TCA significantly suppressed expression of pain mediators induced by interleukin 1β in spinal cord and DRG organotypic culture. Intraarticular TCA in a sustained release formulation of viscous 5% hyaluronic acid will produce a long-term attenuation of mechanical allodynia in the OA knees of mice.
    Trypanosomatid parasites rescue heme from endocytosed hemoglobin through lysosomal HRG transporters
    María Cabello-Donayre, Molecular Microbiology - 2016
    Abstract
    Pathogenic trypanosomatid parasites are auxotrophic for heme and they must scavenge it from their human host. Trypanosoma brucei (responsible for sleeping sickness) and Leishmania (leishmaniasis) can fulfill heme requirement by receptor-mediated endocytosis of host hemoglobin. However, the mechanism used to transfer hemoglobin-derived heme from the lysosome to the cytosol remains unknown. Here we provide strong evidence that HRG transporters mediate this essential step. In bloodstream T. brucei, TbHRG localizes to the endolysosomal compartment where endocytosed hemoglobin is known to be trafficked. TbHRG overexpression increases cytosolic heme levels whereas its downregulation is lethal for the parasites unless they express the Leishmania orthologue LmHR1. LmHR1, known to be an essential plasma membrane protein responsible for the uptake of free heme in Leishmania, is also present in its acidic compartments which colocalize with endocytosed hemoglobin. Moreover, LmHR1 levels modulated by its overexpression or the abrogation of an LmHR1 allele correlate with the mitochondrial bioavailability of heme from lysosomal hemoglobin. In addition, using heme auxotrophic yeasts we show that TbHRG and LmHR1 transport hemoglobin-derived heme from the digestive vacuole to the cytosol. Collectively, these results show that trypanosomatid parasites rescue heme from endocytosed hemoglobin through endolysosomal HRG transporters, which could constitute novel drug targets.
    The Effects of Fortetropin Supplementation on Body Composition, Strength, and Power in Humans and Mechanism of Action in a Rodent Model
    Matthew H. Sharp, Journal of the American College of Nutrition - 2016
    Abstract
    Objective: The purpose of this study was to investigate the effects of Fortetropin on skeletal muscle growth and strength in resistance-trained individuals and to investigate the anabolic and catabolic signaling effects using human and rodent models.Methods: In the rodent model, male Wistar rats (250 g) were gavage fed with either 1.2 ml of tap water control (CTL) or 0.26 g Fortetropin for 8 days. Then rats participated in a unilateral plantarflexion exercise bout. Nonexercised and exercised limbs were harvested at 180 minutes following and analyzed for gene and protein expression relative to mammalian target of rapamycin (mTOR) and ubiquitin signaling. For the human model, 45 (of whom 37 completed the study), resistance-trained college-aged males were divided equally into 3 groups receiving a placebo macronutrient matched control, 6.6 or 19.8 g of Fortetropin supplementation during 12 weeks of resistance training. Lean mass, muscle thickness, and lower and upper body strength were measured before and after 12 weeks of training.Results: The human study results indicated a Group × Time effect (p ≤ 0.05) for lean mass in which the 6.6 g (+1.7 kg) and 19.8 g (+1.68 kg) but not placebo (+0.6 kg) groups increased lean mass. Similarly, there was a Group × Time effect for muscle thickness (p ≤ 0.05), which increased in the experimental groups only. All groups increased equally in bench press and leg press strength. In the rodent model, a main effect for exercise (p ≤ 0.05) in which the control plus exercise but not Fortetropin plus exercise increased both ubiquitin monomer protein expression and polyubiquitination. mTOR signaling was elevated to a greater extent in the Fortetropin exercising conditions as indicated by greater phosphorylation status of 4EBP1, rp6, and p70S6K for both exercising conditions.Conclusions: Fortetropin supplementation increases lean body mass (LBM) and decreases markers of protein breakdown while simultaneously increasing mTOR signaling.
    Flagellin Induces β-Defensin 2 in Human Colonic Ex vivo Infection with Enterohemorrhagic Escherichia coli
    Steven B. Lewis, Frontiers in Cellular and Infection Microbiology - 2016
    Abstract
    Enterohemorrhagic E.coli (EHEC) is an important foodborne pathogen in the developed world and can cause life-threatening disease particularly in children. EHEC persists in the human gut by adhering intimately to colonic epithelium and forming characteristic attaching/effacing lesions. In this study, we investigated the innate immune response to EHEC infection with particular focus on antimicrobial peptide and protein expression by colonic epithelium. Using a novel human colonic biopsy model and polarized T84 colon carcinoma cells, we found that EHEC infection induced expression of human β-defensin 2 (hBD2), whereas hBD1, hBD3, LL-37, and lysozyme remained unchanged. Infection with specific EHEC deletion mutants demonstrated that this was dependent on flagellin, and apical exposure to purified flagellin was sufficient to stimulate hBD2 and also interleukin (IL)-8 expression ex vivo and in vitro. Flagellin-mediated hBD2 induction was significantly reduced by inhibitors of NF-κB, MAP kinase p38 and JNK but not ERK1/2. Interestingly, IL-8 secretion by polarized T84 cells was vectorial depending on the side of stimulation, and apical exposure to EHEC or flagellin resulted in apical IL-8 release. Our results demonstrate that EHEC only induces a modest immune response in human colonic epithelium characterized by flagellin-dependent induction of hBD2 and low levels of IL-8.
    DNA methylation in lung fibroblasts and its role in pulmonary fibrosis
    Ian Matthew Garner, UCL thesis - 2015
    Abstract
    Altered methylation and subsequent changes in gene expression have been implicated in several fibroses including lung however, the full extent and role of altered DNA methylation in fibrotic lung fibroblasts is unknown. Emerging evidence also suggests gender specific methylation differences are common in disease and could elucidate why diseases characterised by pulmonary fibrosis including idiopathic pulmonary fibrosis (IPF) and systemic sclerosis (SSc) have a sex biased prevalence.Using a Genome wide array based approach, this thesis investigates differentially methylated and expressed genes in fibrotic compared to control lung fibroblasts, gender specific methylation and expression differences and the effects of modulating DNA methylation using a DNA methyltransferase (DNMT)inhibitor, 5 Aza 2’deoxycytidine(5Aza). Data show primary human IPF and SSc lung fibroblasts have multiple genes with altered DNA methylation and expression compared to control lung fibroblasts. Multiple biological processes were enriched in these genes, many of which are relevant to fibrosis including, transcriptional regulation, extracellular matrix (ECM) organisation, Wnt signalling and apoptosis. Using siRNA knockdown and collagen gel contraction assays, novel genes including Tenascin XB (TNXB), which encodes the ECM glycoprotein TenasicnX (TNX), were identified as having potential functional significance in the pathogenesis of pulmonary fibrosis. Furthermore, multiple genes including TNXB had altered methylation and expression in IPF compared to SSc lung fibroblasts and may distinguish IPF from other diseases associated with pulmonary fibrosis. Multiple genes were identified with gender specific differences in methylation and expression in lung fibroblasts.Interestingly, multiple genes with altered methylation in IPF males compared to control males were not the same genes with altered methylation in IPF females compared to control females, which may in part explain why IPF predominates in males. The final chapter of my thesis shows 5 Aza treatment alters the methylation and expression of multiple genes in primary human lung fibroblasts. Strong correlation between changes in methylation and changes in expression were identified suggesting DNA methylation can directly regulate the expression of multiple genes in lung fibroblasts
    CHARACTERIZATION OF PRE-CANCER BIOMARKER FOR PROGNOSTIC SCREEN - Worcester Polytechnic Institute
    Sarah Hernandez, United States Patent - 2016
    Abstract
    The invention features compositions and methods for a pre-cancer prognostic screen
    AlgU controls expression of virulence genes in Pseudomonas syringae pv. tomato DC3000
    Erik Markel, Journal of Bacteriology - 2016
    Abstract
    Plant pathogenic bacteria are able to integrate information about their environment and adjust gene expression to provide adaptive functions. AlgU, an ECF sigma factor encoded by Pseudomonas syringae, controls expression of genes for alginate biosynthesis and genes involved with resisting osmotic and oxidative stress. AlgU is active while these bacteria are associated with plants where its presence supports bacterial growth and disease symptoms. We found that AlgU is an important virulence factor for P. syringae pv. tomato DC3000 but that alginate production is dispensable for disease in host plants. This implies that AlgU regulates additional genes that facilitate bacterial pathogenesis. We used RNA-seq to characterize the AlgU regulon and ChIP-seq to identify AlgU-regulated promoters associated with genes directly controlled by this sigma factor. We found that in addition to genes involved with alginate and osmotic and oxidative stress responses, AlgU regulates genes with known virulence functions including components of the Hrp type III secretion system, virulence effectors and the hrpL and hrpRS transcription regulators. These data suggest that P. syringae pv. tomato DC3000 has adapted to use signals that activate AlgU to induce expression of important virulence functions that facilitate survival and disease in plants. Importance Plant immune systems produce antimicrobial and bacteriostatic conditions in response to bacterial infection. Plant pathogenic bacteria are adapted to suppress and/or tolerate these conditions, however, the mechanisms controlling these bacterial systems are largely uncharacterized. The work presented here provides a mechanistic explanation for how P. syringae pv. tomato DC3000 coordinates expression of multiple genetic systems, including those dedicated to pathogenicity, in response to environmental conditions. This work demonstrates the scope of AlgU regulation in P. syringae pv. tomato DC3000, and characterizes the promoter sequence regulated by AlgU in these bacteria.
    The serine protease, dipeptidyl peptidase IV as a myokine: dietary protein and exercise mimetics as a stimulus for transcription and release
    Leslie E. Neidert, Physiological Reports - 2016
    Abstract
    Dipeptidyl‐peptidase IV (DPP‐IV) is an enzyme with numerous roles within the body, mostly related to regulating energy metabolism. DPP‐IV is also a myokine, but the stimulus for its release is poorly understood. We investigated the transcription and release of DPP‐IV from skeletal muscle in a three‐part study using C2C12 myotube cultures, an acute rat exercise and postexercise feeding model, and human feeding or human exercise models. When myotubes were presented with leucine only, hydrolyzed whey protein, or chemicals that cause exercise‐related signaling to occur in cell culture, all caused an increase in the mRNA expression of DPP‐IV (1.63 to 18.56 fold change, P < 0.05), but only whey protein caused a significant increase in DPP‐IV activity in the cell culture media. When rats were fed whey protein concentrate immediately following stimulated muscle contractions, DPP‐IV mRNA in both the exercised and nonexercised gastrocnemius muscles significantly increased 2.5‐ to 3.7‐fold (P < 0.05) 3–6 h following the exercise/feeding bout; of note exercise alone or postexercise leucine‐only feeding had no significant effect. In humans, plasma and serum DPP‐IV activities were not altered by the ingestion of whey protein up to 1 h post consumption, after a 10 min bout of vigorous running, or during the completion of three repeated lower body resistance exercise bouts. Our cell culture and rodent data suggest that whey protein increases DPP‐IV mRNA expression and secretion from muscle cells. However, our human data suggest that DPP‐IV is not elevated in the bloodstream following acute whey protein ingestion or exercise.
    A critical postnatal period of heightened vulnerability to lipopolysaccharide
    Kyle S. Rourke, Respiratory Physiology & Neurobiology - 2016
    Abstract
    Evidence of respiratory abnormalities and vulnerability to infection during a critical period of development have been implicated in Sudden Infant Death Syndrome (SIDS). Here we investigated whether the acute hypoxic ventilatory response (HVR) exhibits a heightened vulnerability to the endotoxin lipopolysaccharide (LPS) during a critical period of development. The acute HVR was measured 2 h after an i.p. injection of saline or LPS (0.1 mg/kg) at various postnatal (P) ages (P5, P10, or P20 days). LPS attenuated the early (1–2 min) and late (4–6 min) phase of the acute HVR in P10 but not P5 or P20 rats. The P10 age group exhibited the largest increase in brainstem TNFα and iNOS mRNA expression following LPS. LPS also caused a higher mortality rate in P10 rats (48%) compared to P5 (12%) and P20 (0%) age groups. After stratifying LPS treated P10 rats into survivors vs non-survivors, only the latter exhibited an attenuated HVR (specifically the early phase). Thus, the heightened vulnerability to endotoxin exposure during this critical period of development is characterized by a depression of the ventilatory response to acute hypoxia in association with an increased incidence of mortality. These data share similarities with some of the circumstances surrounding a SIDS scenario, including evidence of infection, increased brainstem cytokine expression, a disturbance in respiratory control, and a peak incidence of mortality during a critical period of development.
    GLUT3 is present in Clone 9 liver cells and translocates to the plasma membrane in response to insulin
    Danielle M. Defries, Biochemical and Biophysical Research Communications - 2016
    Abstract
    Clone 9 cells have been reported to express only the GLUT1 facilitative glucose transporter; however, previous studies have not examined Clone 9 cells for GLUT3 content. The current study sought to profile the presence of glucose transporters in Clone 9 cells, H4IIE hepatoma cells, and L6 myoblasts and myotubes. While the other cell types contained the expected complement of transporters, Clone 9 cells had GLUT3 which was previously not reported. Interestingly, both GLUT3 mRNA and protein were detected in Clone 9 cells, but only mRNA for GLUT1 was detected. Glucose transport in Clone 9 cells was insulin-sensitive in a concentration-dependent manner, concomitant with the presence of GLUT3 in the plasma membrane after insulin treatment. Although basal glucose uptake was unaffected, insulin-stimulated glucose uptake was abolished with siRNA-mediated GLUT3 knockdown. These results contradict previous reports that Clone 9 cells exclusively express GLUT1 and suggest GLUT3 is a key insulin-sensitive glucose transporter required for insulin-stimulated glucose uptake by Clone 9 cells.
    Forkhead box O member FOXO1 regulates the majority of follicle-stimulating hormone responsive genes in ovarian granulosa cells
    Maria K. Herndon, Molecular and Cellular Endocrinology - 2016
    Abstract
    FSH promotes maturation of ovarian follicles. One pathway activated by FSH in granulosa cells (GCs) is phosphatidylinositol-3 kinase/AKT. The AKT target FOXO1 is reported to function primarily as a repressor of FSH genes, including Ccnd2 and Inha. Based on its broad functions in other tissues, we hypothesized that FOXO1 may regulate many more GC genes. We transduced GCs with empty adenovirus or constitutively active FOXO1 followed by treatment with FSH for 24 h, and conducted RNA deep sequencing. Results show that FSH regulates 3772 genes ≥2.0-fold; 60% of these genes are activated or repressed by FOXO1. Pathway Studio Analysis revealed enrichment of genes repressed by FOXO1 in metabolism, signaling, transport, development, and activated by FOXO1 in signaling, cytoskeletal functions, and apoptosis. Gene regulation was verified by q-PCR (eight genes) and ChIP analysis (two genes). We conclude that FOXO1 regulates the majority of FSH target genes in GCs.
    CHARACTERIZATION OF DEFB103 Gene Structure, Gene Expression and its haplotype Association with three Common Diseases in Beef and Holstein Cattle
    Ali Mirabzadeh-Ardakani, M.Sc., University of Saskatchewan Thesis - 2016
    Abstract
    The DEF B103 gene is a member of the β defensin gene family, and it has not been well studied in cattle. In this study DEFB103 gene expression, gene structure and its haplotypes association with three diseases were studied. DEFB103 gene expression was profiled for 27 tissues in nine two week old calves and the transcript was most abundant in tissues with stratified squamous epithelium. An age dependent decrease (P <0.05) in DEFB103 gene expression was observed in buccal epithelium when comparing healthy two week old and 10 to 12 Month Old calves. A bovine herpes virus 1 respiratory infection did, however, significantly (P < 0.05) up regulate DEFB103 Gene expression in the buccal epithelium of 6 to 8 Month old calves. Immunohistochemistry was used to identify cells expressing DEFB103 protein within tissues with stratified squamous epitheliums to confirm the DEFB103 gene expression results. DEFB103 protein was most abundant in basal epithelial cells and was present in these cells before birth. A close association was observed between the dendritic cells and epithelial cells expressing DEFB103 in both the fetus and newborn calf, and this is consistent with the β defensin regulatory effect on dendritic cell responses.
    Melanopsin Regulates Both Sleep-Promoting and Arousal-Promoting Responses to Light
    Violetta Pilorz, PLOS Biol - 2016
    Abstract
    Light can produce either sleep or arousal in mice. This study reveals that these opposing effects depend upon the wavelength of light and appear to involve separate pathways, both modulated by the photopigment melanopsin.
    Using Fluorescence Imaging to Track Drug Delivery and Guide Treatment Planning In Vivo
    Qiaoya Lin, In Vivo Fluorescence Imaging - 2016
    Abstract
    Imaging has become an indispensable tool in both clinical medicine and preclinical sciences. It enables doctors to locate sites of cancer/disease, track drug delivery, and guide operative planning, thus enhancing the treatment efficacy. Recently, we developed a multimodal theranostic lipid nanoparticles, named HPPS(NIR)-chol-siRNA with its built-in near-infrared (NIR) fluorescent probe core as a useful surrogate for tracking small interfering RNA (siRNA) delivery. By using the image co-registration of computed tomography (CT) and fluorescence molecular tomography (FMT), we achieved noninvasive assessment and treatment planning of siRNA delivery into the orthotopic tumor, thus enabling efficacious RNA interference (RNAi) therapy. In this chapter, we introduce this method to illustrate the use of CT-FMT co-registration for tracking drug delivery and guiding treatment planning in vivo.
    c-Jun is Required for NF-κB-dependent, LPS-stimulated Fos-related Antigen-1 Transcription in Alveolar Macrophages
    Rakesh K Mishra, American Journal of Respiratory Cell and Molecular Biology - 2016
    Abstract
    Previously, we have reported that Fos-related antigen-1 (Fra-1) transcription factor promotes LPS-induced acute lung injury and mortality, and LPS-induced Fra-1 expression in the lung occurs predominantly in alveolar macrophages. NF-κB and c-Jun transcription factors play key roles in modulating inflammatory and immune responses induced by infectious and non-infectious insults. Here, we report that NF-κB and c-Jun co-regulate Fra-1 induction by LPS in alveolar macrophages, and this regulation occurs through both NF-κB and extracellular signal-regulated protein kinase (ERK) signaling pathways. Transient transfections with Fra-1 promoter-reporter constructs and inhibitor studies revealed that the transcriptional activation of Fra-1 by LPS in alveolar macrophages mediated by NF-κB and ERK1/2 signaling. Importantly, chromatin immunoprecipitation assays revealed the recruitment of c-Jun and NF-κB to the endogenous Fra-1 promoter following LPS stimulation. We found that inhibition of ERK1/2 signaling reduces LPS-stimulated c-Jun and NF-κB recruitment to the promoter. Likewise, NF-κB inhibitor blocked LPS-induced NF-κB and c-Jun binding to the promoter. ERK1/2 inhibition had no effect on c-Jun activation, but suppressed LPS-stimulated NF-κB phosphorylation. Finally, functional assays show reduced levels of LPS-stimulated NF-κB regulated pro-inflammatory IL-1β and MIP1-α expression and increased anti-inflammatory IL-10 expression in lung alveolar macrophages of Fra-1-null mice in vivo. Thus, our studies define that NF-κB and c-Jun co-regulate LPS-induced Fra-1 transcription, and that Fra-1 selectively modulates LPS-stimulated inflammatory cytokine expression in lung alveolar macrophages during inflammatory lung injury.
    Osteopontin regulates proliferation, apoptosis, and migration of murine claudin-low mammary tumor cells
    S. Saleh, BMC Cancer - 2016
    Abstract
    Osteopontin is a secreted phosphoglycoprotein that is expressed by a number of normal cells as well as a variety of tumor cells. With respect to breast cancer, osteopontin has been implicated in regulating tumor cell proliferation and migration/metastasis and may serve as a prognostic indicator. However it remains unclear whether osteopontin has the same impact in all breast cancer subtypes and in particular, osteopontin’s effects in claudin-low breast cancer are poorly understood.
    http://www.sciencedirect.com/science/article/pii/S004484861630312X
    Nir Shpak, Aquaculture - 2016
    Abstract
    The recent introduction into aquaculture of RNA interference (RNAi) for producing the preferred all-male monosex cultures, which give improved yields, has raised awareness of the need to investigate the consequences of such novel biotechnological manipulations. Here, we present meta-analysis style study on data from observations of three consecutive cultured all-male (ZZ) generations of the giant freshwater prawn Macrobrachium rosenbergii (De Man). Each consecutive generation comprised the progeny of RNAi-manipulated sex-reversed males. The manipulation was achieved through the administration of dsRNA encoding the insulin-like androgenic hormone into males (which transformed them into ‘neofemales,’ ZZ) of the previous all-male generation. Each generation was cultured in a separate earthen pond for a short (~ 4–5 months) growout period. At harvest of each of the generations, the typical M. rosenbergii population structure comprising three male morphotypes was obtained. An anatomical examination of the male reproductive system of a representative specimen of the third all-male generation showed normal reproductive outputs, even though the prawns had been grown without the presence of females (WZ) for three generations. At the molecular level, expression of vital male-specific genes in the third generation of all-male M. rosenbergii culture was demonstrated. Thus, the present study showing the lack of any overtly apparent long-term consequences of the RNAi-based biotechnology provides support for the responsible use of temporal RNAi in aquaculture. Finally, the absence of the W chromosome for three generations raises questions with regard to its role and content with respect to crustacean sexual determination and differentiation.
    HIF1A and EPAS1 mRNA and protein expression during in vitro culture of human primary term cytotrophoblasts and effect of oxygen tension on their expression
    Christophe Louis Depoix, Reproductive Biology - 2016
    Abstract
    During the first trimester of pregnancy, placenta formation probably occurs in a low-oxygen environment necessary to protect cytotrophoblasts from oxidative stress and to allow proper gene regulation. Transcription factors involved in gene regulation under low oxygen tension are the hypoxia-inducible factors, mainly HIF1A, EPAS1 and their dimerization partner HIF1B. Little is known about their expression during in vitro culture of cytotrophoblasts under chronic hypoxia. We assessed HIF1A and EPAS1 expression in a 4-day in vitro culture of primary term cytotrophoblasts under 21% O2 and 2.5% O2. Copy numbers and relative mRNA expression were assessed by real-time quantitative polymerase chain reaction. Protein levels were quantified by immunoblot and densitometric analysis. In undifferentiated cytotrophoblasts, EPAS1 transcripts were four times more abundant than HIF1A transcripts (2.14e7 and 5e6 copies/μg total RNA, respectively). During cell culture, HIF1A mRNA expression increased after 24 h and then decreased to stay stable. The expression was even lower when cells were grown under 2.5% O2. EPAS1 mRNA expression increased during cytotrophoblast differentiation. The expression was higher when cells were under 21% O2 than when they were under 2.5% O2. Interestingly, HIF1A, but not EPAS1, was detected in the nuclei of undifferentiated cytotrophoblasts, and in the nuclei of cytotrophoblasts that grew under 21% O2. During cytotrophoblast differentiation, no variation in HIF1A protein levels was detected. To the contrary, EPAS1 protein level increased during differentiation, and oxygen tension had no effect on EPAS1 protein level. In conclusion, HIF1A and EPAS1 expression was not inhibited by chronic hypoxia during in vitro cytotrophoblast differentiation.
    Decreased antimony uptake and overexpression of genes of thiol metabolism are associated with drug resistance in a canine isolate of Leishmania infantum
    Verónica Gómez Pérez, International Journal for Parasitology: Drugs and Drug Resistance - 2016
    Abstract
    Visceral leishmaniasis (VL) caused by the protozoan parasite Leishmania infantum, is one of the most important zoonotic diseases affecting dogs and humans in the Mediterranean area. The presence of infected dogs as the main reservoir host of L. infantum is regarded as the most significant risk for potential human infection. We have studied the susceptibility profile to antimony and other anti-leishmania drugs (amphotericin B, miltefosine, paromomycin) in Leishmania infantum isolates extracted from a dog before and after two therapeutic interventions with meglumine antimoniate (subcutaneous Glucantime®, 100 mg/kg/day for 28 days). After the therapeutic intervention, these parasites were significantly less susceptible to antimony than pretreatment isolate, presenting a resistance index of 6-fold to SbIII for promastigotes and >3-fold to SbIII and 3-fold to SbV for intracellular amastigotes. The susceptibility profile of this resistant L. infantum line is related to a decreased antimony uptake due to lower aquaglyceroporin-1 expression levels. Additionally, other mechanisms including an increase in thiols and overexpression of enzymes involved in thiol metabolism, such as ornithine decarboxylase, trypanothione reductase, mitochondrial tryparedoxin and mitochondrial tryparedoxin peroxidase, could contribute to the resistance as antimony detoxification mechanisms. A major contribution of this study in a canine L. infantum isolate is to find an antimony-resistant mechanism similar to that previously described in other human clinical isolates.
    KAT8 Regulates Androgen Signaling in Prostate Cancer Cells
    Ji-Young Kim, Original Research - 2016
    Abstract
    Androgen receptor (AR) plays pivotal roles in prostate cancer. Upon androgen stimulation, AR recruits the Protein kinase N1 (PKN1), which phosphorylates histone H3 at threonine 11, with subsequent recruitment of tryptophan, aspartic acid (WD) repeat-containing protein 5 (WDR5) and the su(var)3–9, enhancer of zeste, trithorax/mixed-lineage leukemia (SET1/MLL) histone methyltransferase complex to promote AR target gene activation and prostate cancer cell growth. However, the underlying mechanisms of target gene activation and cell growth subsequent to WDR5 recruitment are not well understood. Here, we demonstrate an epigenetic cross talk between histone modifications and AR target gene regulation. We discovered that K(lysine) acetyltransferase 8 (KAT8), a member of the MOZ, YBF2/SAS2, and TIP 60 protein 1 (MYST) family of histone acetyltransferases that catalyzes histone H4 lysine 16 acetylation, colocalized with WDR5 at AR target genes, resulting in hormone-dependent gene activation in prostate cancer cells. PKN1 or WDR5 knockdown severely inhibited KAT8 association with AR target genes and histone H4 lysine 16 acetylation upon androgen treatment. Knockdown of KAT8 significantly decreased AR target gene expression and prostate cancer cell proliferation. Collectively, these data describe a transhistone modification pathway involving PKN1/histone H3 threonine 11 phosphorylation followed by WDR5/MLL histone methyltransferase and KAT8/histone acetyltransferase recruitment to effect androgen-dependent gene activation and prostate cancer cell proliferation.(Molecular Endocrinology30:925–936, 2016)
    High alpha-methylacyl-CoA racemase (AMACR) is associated with ERG expression and with adverse clinical outcome in patients with localized prostate cancer
    Adrian Box, Tumor Biology - 2016
    Abstract
    Alpha-methylacyl-CoA racemase (AMACR) is a well-characterized marker extensively utilized in prostate cancer (PCA) diagnosis. However, the prognostic value of AMACR expression and its relation to TMPRSS2-ERG gene rearrangement as one of the most common molecular alterations in PCA is not fully explored. AMACR expression was investigated in a cohort of 218 men with localized PCA treated by radical prostatectomy and correlated with ERG and various clinical and pathological parameters. In vitro studies assessed AMACR changes to ERG knockdown and other related genes. In addition, bioinformatics validated the significance of AMACR/ERG expression and assessed relevant genetic signatures in relation to AMACR/ERG expression. AMACR expression was significantly associated with disease progression and with ERG (p ∼0). Seventeen percent of cancer foci showed negative/weak AMACR expression while being ERG positive. High AMACR expression was significantly associated with positive surgical margins (p = 0.01), specifically in tumors with lower Gleason score <7, with ∼95 % exhibiting positive surgical margin (p = 0.008). High AMACR showed marginal association with PSA biochemical recurrence (BCR) (p = 0.06) which was slightly more pronounced in ERG-positive tumors (p = 0.04). This was validated in other public cohorts. However, in this cohort, the association with BCR was not statistically significant in multivariate analysis (p = 0.09). Using in vitro cellular models, AMACR messenger RNA (mRNA) expression, but not protein levels, showed an association with ERG expression. We report for the first time a significant association between AMACR and ERG with prognostic implication. Patients with high AMACR/ERG-positive PCA may be at higher risk for disease progression, and additional studies in larger cohorts are needed to confirm the above findings. Functional studies investigating the molecular pathways connecting AMACR and ERG may provide an additional insight into PCA progression pathways.
    METHODS OF PREPARING ANTI-HUMAN PAPILLOMAVIRUS ANTIGEN T CELLS
    Christian S. Hinrichs, United States Patent - 2016
    Abstract
    Disclosed are methods of preparing an isolated population of human papillomavirus (HPV)-specific T cells comprise dividing an HPV-positive tumor sample into multiple fragments; separately culturing the multiple fragments; obtaining T cells from the cultured multiple fragments; testing the T cells for specific autologous HPV-positive tumor recognition; selecting the T cells that exhibit specific autologous HPV-positive tumor recognition; and expanding the number of selected T cells to produce a population of HPV-specific T cells for adoptive cell therapy. Related methods of treating or preventing cancer using the T cells are also disclosed.
    Impact of Storage Temperature on the Expression of Cell Survival Genes in Cultured ARPE-19 Cells
    Lara Pasovic, Current Eye Research - 2016
    Abstract
    Purpose: The development of a suitable storage method for retinal pigment epithelium (RPE) is necessary in the establishment of future RPE replacement therapy, and storage temperature has proven to be pivotal for cell survival. ARPE-19, a widely used model for RPE, has been shown to yield the greatest number of viable cells when stored at 16°C compared to other storage temperatures. In this study, we analyze the gene expression profile of cultured ARPE-19 cells after seven days of storage at different temperatures in an effort to predict the gene-level consequences of storage of RPE transplants.Materials and Methods: ARPE-19 cells were cultured until confluence and then stored in minimum essential medium at 4°C, 16°C, and 37°C for seven days. The total RNA was isolated and the gene expression profile was determined using DNA microarrays. The Results were validated using qPCR.Results: Principal component and hierarchical clustering analyses show that the gene expression profiles of cell cultures stored at different temperatures cluster into separate groups. Cultures stored at 4°C cluster closest to the control cultures that were not stored and display the least change in gene expression after storage (157 differentially expressed genes). Cultures stored at 16°C and 37°C display a much larger change in differential gene expression (1787 and 1357 differentially expressed genes, respectively). At 16°C, the expression of several genes with proposed tumor suppressor functions was markedly increased. Changes in regulation of several known signaling pathways and of oxidative stress markers were discovered at both 16°C and 37°C, and activation of the angiogenesis marker vascular endothelial growth factor (VEGF) was discovered at 37°C. There was no evidence of the activation of inflammatory processes in stored cell cultures.Conclusion: ARPE-19 cultures stored at 16°C show the greatest propensity to modulate their gene expression profile in a manner that supports cell survival during storage.
    Hypoxia and Reactive Oxygen Species Homeostasis in Mesenchymal Progenitor Cells Define a Molecular Mechanism for Fracture Nonunion
    Emma Muinos-López, STEM CELLS - 2016
    Abstract
    Fracture nonunion is a major complication of bone fracture regeneration and repair. The molecular mechanisms that result in fracture nonunion appearance are not fully determined. We hypothesized that fracture nonunion results from the failure of hypoxia and hematoma, the primary signals in response to bone injury, to trigger Bmp2 expression by mesenchymal progenitor cells (MSCs). Using a model of nonstabilized fracture healing in transgenic 5′Bmp2BAC mice we determined that Bmp2 expression appears in close association with hypoxic tissue and hematoma during the early phases of fracture healing. In addition, BMP2 expression is induced when human periosteum explants are exposed to hypoxia ex vivo. Transient interference of hypoxia signaling in vivo with PX-12, a thioredoxin inhibitor, results in reduced Bmp2 expression, impaired fracture callus formation and atrophic-like nonunion by a HIF-1α independent mechanism. In isolated human periosteum-derived MSCs, BMP2 expression could be induced with the addition of platelets concentrate lysate but not with hypoxia treatment, confirming HIF-1α-independent BMP2 expression. Interestingly, in isolated human periosteum-derived mesenchymal progenitor cells, inhibition of BMP2 expression by PX-12 is accomplished only under hypoxic conditions seemingly through dis-regulation of reactive oxygen species (ROS) levels. In conclusion, we provide evidence of a molecular mechanism of hypoxia-dependent BMP2 expression in MSCs where interference with ROS homeostasis specifies fracture nonunion-like appearance in vivo through inhibition of Bmp2 expression. Stem Cells 2016.
    Zinc supplementation induces CD4+CD25+Foxp3+ antigen-specific regulatory T cells and suppresses IFN-γ production by upregulation of Foxp3 and KLF-10 and downregulation of IRF-1
    Martina Maywald, European Journal of Nutrition - 2016
    Abstract
    PurposeThe essential trace element zinc plays a fundamental role in immune function and regulation since its deficiency is associated with autoimmunity, allergies, and transplant rejection. Thus, we investigated the influence of zinc supplementation on the Th1-driven alloreaction in mixed lymphocyte cultures (MLC), on generation of antigen-specific T cells, and analyzed underlying molecular mechanisms.MethodsCell proliferation and pro-inflammatory cytokine production were monitored by [3H]-thymidine proliferation assay and ELISA, respectively. Analysis of surface and intracellular T cell marker was performed by flow cytometry. Western blotting and mRNA analysis were used for Foxp3, KLF-10, and IRF-1 expression.ResultsZinc supplementation on antigen-specific T cells in physiological doses (50 µM) provokes a significant amelioration of cell proliferation and pro-inflammatory cytokine production after reactivation compared to untreated controls. Zinc administration on MLC results in an increased induction and stabilization of CD4+CD25+Foxp3+ and CD4+CD25+CTLA-4+ T cells (p < 0.05). The effect is based on zinc-induced upregulation of Foxp3 and KLF-10 and downregulation of IRF-1. However, in resting lymphocytes zinc increases IRF-1.ConclusionIn summary, zinc is capable of ameliorating the allogeneic immune reaction by enhancement of antigen-specific iTreg cells due to modulation of essential molecular targets: Foxp3, KLF-10, and IRF-1. Thus, zinc can be seen as an auspicious tool for inducing tolerance in adverse immune reactions.
    Characterization of catecholamine receptors and transporters in murine macrophages - viewcontent.cgi
    Elizabeth Gon, University of Richmon Student Research - 2016
    Abstract
    Macrophages are a critical part of the immune response. When circulating monocytes move into tissues they differentiate into macrophages to mount the first line of defense against pathogens. Macrophages are primarily phagocytic cells, physically engulfing pathogens and cell debris at the site of an infection (Abbas et al. 2009). They play a key role in the innate immune system, initiating and maintaining the inflammatory response and acting as antigen presenting cells to active adaptive immunity (Abbas et al. 2009). Macrophages are activated in two different ways: the binding of a pro-inflammatory cytokine or by the recognition of a pathogen-associated molecular pattern (PAMP) by a receptor on the macrophage's surface (Abbas et al. 2009, Chi et al. 2003). Lipopolysaccharide (LPS) is a component of gram-negative bacterial cell walls and is a common activating PAMP that triggers NF-KB mediated pro-inflammatory effects (Abbas et al. 2009). When an immune response is mounted, macrophages act by phagocytosing pathogens and by releasing nitric oxide (NO) and other reactive intermediates as well as by secreting pro-inflammatory cytokines (Abbas et al. 2009). Cytokine release enhances the activation of phagocytes and promotes the recruitment of other immune cells to the site of infection (Abbas et al. 2009). Macrophages are able to control the spread of the pathogen through these non-specific destructive effects as well as by recruiting other immune cells to initiate a targeted immune response.
    Detection of Murine TRPA1 Transcripts in Keratinocytes
    Kelvin Y Kwan, Journal of Skin and Stem Cell - 2016
    Abstract
    Background: Transient receptor potential ankyrin 1 (TRPA1) is expressed by nociceptive neurons of the dorsal root ganglia (DRGs) and trigeminal ganglia, but its expression in other cell types is largely unexplored. TRPA1 contributes to chemical, mechanical and cold nociception and expression of TRPA1 in keratinocytes may extend the detection of chemical and physical stimuli from the nerve endings to the surface of the skin. Results: Comparison of TRPA1 genes from multiple inbred mouse lines revealed a large number of genetic variations in the TRPA1 locus. These genetic variations may affect annealing of primers to detect TRPA1 transcript when using nucleic acid-based methods. To consistently detect TRPA1 in genetically diverse mouse strains and in different tissue types, an optimized qPCR protocol for TRPA1 detection was created. Conclusions: TRPA1 transcripts are consistently detected in murine keratinocytes.
    Testosterone and trenbolone enanthate increase mature myostatin protein expression despite increasing skeletal muscle hypertrophy and satellite cell number in rodent muscle
    V.J Dalbo, Andrologia - 2016
    Abstract
    The androgen-induced alterations in adult rodent skeletal muscle fibre cross-sectional area (fCSA), satellite cell content and myostatin (Mstn) were examined in 10-month-old Fisher 344 rats (n = 41) assigned to Sham surgery, orchiectomy (ORX), ORX + testosterone (TEST; 7.0 mg week−1) or ORX + trenbolone (TREN; 1.0 mg week−1). After 29 days, animals were euthanised and the levator ani/bulbocavernosus (LABC) muscle complex was harvested for analyses. LABC muscle fCSA was 102% and 94% higher in ORX + TEST and ORX + TREN compared to ORX (p < .001). ORX + TEST and ORX + TREN increased satellite cell numbers by 181% and 178% compared to ORX, respectively (p < .01), with no differences between conditions for myonuclear number per muscle fibre (p = .948). Mstn protein was increased 159% and 169% in the ORX + TEST and ORX + TREN compared to ORX (p < .01). pan-SMAD2/3 protein was ~30–50% greater in ORX compared to SHAM (p = .006), ORX + TEST (p = .037) and ORX + TREN (p = .043), although there were no between-treatment effects regarding phosphorylated SMAD2/3. Mstn, ActrIIb and Mighty mRNAs were lower in ORX, ORX + TEST and ORX + TREN compared to SHAM (p < .05). Testosterone and trenbolone administration increased muscle fCSA and satellite cell number without increasing myonuclei number, and increased Mstn protein levels. Several genes and signalling proteins related to myostatin signalling were differentially regulated by ORX or androgen therapy.
    Axonal localization and mitochondrial association of precursor microRNA 338
    Jose Norberto S.Vargas, Cellular and Molecular Life Sciences - 2016
    Abstract
    MicroRNAs (miRNAs) selectively localize to subcompartments of the neuron, such as dendrites, axons, and presynaptic terminals, where they regulate the local protein synthesis of their putative target genes. In addition to mature miRNAs, precursor miRNAs (pre-miRNAs) have also been shown to localize to somatodendritic and axonal compartments. miRNA-338 (miR-338) regulates the local expression of several nuclear-encoded mitochondrial mRNAs within axons of sympathetic neurons. Previous work has shown that precursor miR-338 (pre-miR-338) introduced into the axon can locally be processed into mature miR-338, where it can regulate local ATP synthesis. However, the mechanisms underlying the localization of pre-miRNAs to the axonal compartment remain unknown. In this study, we investigated the axonal localization of pre-miR-338. Using proteomic and biochemical approaches, we provide evidence for the localization of pre-miR-338 to distal neuronal compartments and identify several constituents of the pre-miR-338 ribonucleoprotein complex. Furthermore, we found that pre-miR-338 is associated with the mitochondria in axons of superior cervical ganglion (SCG) neurons. The maintenance of mitochondrial function within axons requires the precise spatiotemporal synthesis of nuclear-encoded mRNAs, some of which are regulated by miR-338. Therefore, the association of pre-miR-338 with axonal mitochondria could serve as a reservoir of mature, biologically active miRNAs, which could coordinate the intra-axonal expression of multiple nuclear-encoded mitochondrial mRNAs.
    Autonomous osteogenic differentiation of hASCs encapsulated in methacrylated gellan-gum hydrogels
    Mariana B. Oliveira,, Science Direct - 2016
    Abstract
    Methacrylated gellan-gum (GG-MA) alone and combined with collagen type I (Coll) is suggested here for the first time as a cell-laden injectable biomaterial for bone regeneration. On-chip high-throughput studies allowed rapidly assessing the suitability of 15 biomaterials/media combinations for the osteodifferentiation of human adipose stem cells (hASCs). Hydrogels composed solely of GG-MA (GG100:0Coll) led hASCs from three different donors into the osteogenic lineage after 21 days of cell culture, in the absence of any osteogenic or osteoconductive factors. Hydrogels containing more than 30% of Coll promoted increased cellular proliferation and led hASCs into osteogenic differentiation under basal conditions. Studies using isolated individual hydrogels – excluding eventual on-chip crosstalk – and standard biochemical assays corroborated such findings. The formation of focal adhesions of hASCs on GG100:0Coll hydrogels was verified. We hypothesize that the hydrogels osteogenic effect could be guided by mechanotransduction phenomena. Indeed, the hydrogels showed elastic modulus in ranges previously reported as osteoinductive and the inhibition of the actin-myosin contractility pathway impaired hASCs’ osteodifferentiation. GG-MA hydrogels also did not promote hASCs’ adipogenesis while used in basal conditions. Overall, GG-MA showed promising properties as an innovative and off-the shelf self-inducing osteogenic injectable biomaterial.
    Acrolein enhances epigenetic modifications, FasL expression and hepatocyte toxicity induced by anti-HIV drug Zidovudine
    Smita S. Ghare, Toxicology in Vitro - 2016
    Abstract
    Zidovudine (AZT) remains the mainstay of antiretroviral therapy against HIV in resource-poor countries; however, its use is frequently associated with hepatotoxicity. Not all HIV patients on AZT develop hepatotoxicity, and the determining factors are unclear. Alcohol consumption and cigarette smoking are known risk factors for HIV hepatotoxicity, and both are significant sources of acrolein, a highly reactive and toxic aldehyde. This study examines the potential hepatotoxic interactions between acrolein and AZT. Our data demonstrate that acrolein markedly enhanced AZT-induced transcriptionally permissive histone modifications (H3K9Ac and H3K9Me3) allowing the recruitment of transcription factor NF-kB and RNA polymerase II at the FasL gene promoter, resulting in FasL upregulation and apoptosis in hepatocytes. Notably, the acrolein scavenger, hydralazine prevented these promoter-associated epigenetic changes and inhibited FasL upregulation and apoptosis induced by the combination of AZT and acrolein, as well as AZT alone. Our data strongly suggest that acrolein enhancement of promoter histone modifications and FasL upregulation are major pathogenic mechanisms driving AZT-induced hepatotoxicity. Moreover, these data also indicate the therapeutic potential of hydralazine in mitigating AZT hepatotoxicity.
    Differential effect of fructose on fat metabolism and clock gene expression in hepatocytes vs. myotubes
    Nava Chapnik, The International Journal of Biochemistry & Cell Biology - 2016
    Abstract
    In the liver, fructose bypasses the main rate-limiting step of glycolysis at the level of phosphofructokinase, allowing it to act as an unregulated substrate for de novo lipogenesis. It has been reported that consumption of large amounts of fructose increases de novo lipogenesis in the liver. However, the effect of fructose on ectopic deposition of muscle fat has been under dispute. Our aim was to study the effect of fructose on levels of genes and proteins involved in fatty acid oxidation and synthesis in hepatocytes vs. muscle cells. In addition, as fat accumulation leads to disruption of daily rhythms, we tested the effect of fructose treatment on clock gene expression. AML-12 hepatocytes and C2C12 myotubes were treated with fructose or glucose for 2 consecutive 24-h cycles and harvested every 6 h. In contrast to glucose, fructose disrupted clock gene rhythms in hepatocytes, but in myotubes, it led to more robust rhythms. Fructose led to low levels of phosphorylated AMP-activated protein kinase (pAMPK) and high levels of LIPIN1 in hepatocytes compared with glucose. In contrast, fructose led to high pAMPK and low LIPIN1 and microsomal triacylglycerol transfer protein (MTTP) levels in myotubes compared with glucose. Analysis of fat content revealed that fructose led to less fat accumulation in myotubes compared to hepatocytes. In summary, fructose shifts metabolism towards fatty acid synthesis and clock disruption in hepatocytes, but not in myotubes.
    Effect of ovarian stimulation treatments on the embryo quality in rabbit
    José Isaí Cedano Castro, Original Paper - 2016
    Abstract
    Superovulation in animals is used to produce a maximum number of transferable embryos per donor, in order to support both genetic improvement programs and ex situ conservation or to optimize other biotechnologies. Over time, the use of this biotechnology has shown variable outcomes as a consequence of several factors, such as the origin of exogenous hormone, its administration mode, the donor and the environment. Nowadays the use of gonadotropins such as FSH, LH, hCG and eCG has enabled us to achieve the superovulation in different species successfully. However,the posology and the effect of both gonadotropins when used simultaneously (e.g. FSH and LH) it is not yet clearly defined. The superovulation in rabbit does and in other species, has had a great advance in recent years, changing the eCG use by the recombinant gonadotropins and jumping the morphological classification to the molecular analysis of embryos. The objective of this study was to evaluate the effect of long acting FSH CTP and rhFSH alone or supplemented with rhLH on ovarian stimulation in rabbit does and determine the impact of its stimulation on in vitro and in vivo development of fresh and cryopreserved embryos.
    Experimental Infection of Calves by Two Genetically-Distinct Strains of Rift Valley Fever Virus
    William C. Wilson, Viruses - 2016
    Abstract
    Recent outbreaks of Rift Valley fever in ruminant livestock, characterized by mass abortion and high mortality rates in neonates, have raised international interest in improving vaccine control strategies. Previously, we developed a reliable challenge model for sheep that improves the evaluation of existing and novel vaccines in sheep. This sheep model demonstrated differences in the pathogenesis of Rift Valley fever virus (RVFV) infection between two genetically-distinct wild-type strains of the virus, Saudi Arabia 2001 (SA01) and Kenya 2006 (Ken06). Here, we evaluated the pathogenicity of these two RVFV strains in mixed breed beef calves. There was a transient increase in rectal temperatures with both virus strains, but this clinical sign was less consistent than previously reported with sheep. Three of the five Ken06-infected animals had an early-onset viremia, one day post-infection (dpi), with viremia lasting at least three days. The same number of SA01-infected animals developed viremia at 2 dpi, but it only persisted through 3 dpi in one animal. The average virus titer for the SA01-infected calves was 1.6 logs less than for the Ken06-infected calves. Calves, inoculated with either strain, seroconverted by 5 dpi and showed time-dependent increases in their virus-neutralizing antibody titers. Consistent with the results obtained in the previous sheep study, elevated liver enzyme levels, more severe liver pathology and higher virus titers occurred with the Ken06 strain as compared to the SA01 strain. These results demonstrate the establishment of a virulent challenge model for vaccine evaluation in calves.
    Epithelial-mesenchymal transition and FOXA genes during tobacco smoke carcinogen induced transformation of human bronchial epithelial cells
    Audun Bersaas, Toxicology in Vitro - 2016
    Abstract
    Lung cancer is largely an environmentally caused disease with poor prognosis. An in vitro transformation model of human bronchial epithelial cells (HBEC) was used to study long-term effects of tobacco smoke carcinogens on epithelial-mesenchymal transition (EMT) and the forkhead box transcription factors FOXA1 and FOXA2. CDK4 and hTERT immortalized HBEC2 and HBEC12 cell lines were exposed weekly to either cigarette smoke condensate (CSC), benzo[a]pyrene, or methylnitrosourea. Transformed cell lines were established from soft-agar colonies after 12 weeks of exposure. HBEC12 was transformed by all exposures while HBEC2 was only transformed by CSC. Untransformed HBEC2 showed little invasive capacity, whereas transformed cell lines completely closed the gap in a matrigel scratch wound assay. CDH1 was down-regulated in all of the transformed cell lines. In contrast, CDH2 was up-regulated in both HBEC2 and one of the HBEC12 transformed cell lines. Furthermore, transformed cells showed activation of EMT markers including SNAI1, ZEB1, VIM, and MMP2. All transformed cell lines had significant down-regulation of FOXA1 and FOXA2, indicating a possible role in cell transformation and EMT. ChIP analysis showed increased binding of Histone-H3 and macroH2A in FOXA1 and FOXA2 in the transformed HBEC2 cell lines, indicating a compact chromatin. In conclusion, long-term carcinogen exposure lead to down-regulation of FOXA1 and FOXA2 concomitantly with the occurrence of EMT and in vitro transformation in HBEC cells.
    Molecules Associated with Fatty Acid Biosynthetic Pathways and Uses Thereof - Synthetic Genomics, Inc.
    Gerardo V.Toledo, US Patent - 2016
    Abstract
    The present disclosure relates in part to recombinant microorganisms that include non-native genes encoding PUFA-PKS polypeptides, and to methods of making and using such microorganisms for producing at least one PUFA. In particular, the disclosure further relates to methods and related materials useful for the production of at least one PUFA by heterologous expression of the nucleic acid sequences disclosed herein encoding PUFA-PKS polypeptides.
    Effects of Arachidonic Acid Supplementation on Acute Anabolic Signaling and Chronic Functional Performance and Body Composition Adaptations
    Eduardo O. De Souza, PLOS ONE - 2016
    Abstract
    Background The primary purpose of this investigation was to examine the effects of arachidonic acid (ARA) supplementation on functional performance and body composition in trained males. In addition, we performed a secondary study looking at molecular responses of ARA supplementation following an acute exercise bout in rodents. Methods Thirty strength-trained males (age: 20.4 ± 2.1 yrs) were randomly divided into two groups: ARA or placebo (i.e. CTL). Then, both groups underwent an 8-week, 3-day per week, non-periodized training protocol. Quadriceps muscle thickness, whole-body composition scan (DEXA), muscle strength, and power were assessed at baseline and post-test. In the rodent model, male Wistar rats (~250 g, ~8 weeks old) were pre-fed with either ARA or water (CTL) for 8 days and were fed the final dose of ARA prior to being acutely strength trained via electrical stimulation on unilateral plantar flexions. A mixed muscle sample was removed from the exercised and non-exercised leg 3 hours post-exercise. Results Lean body mass (2.9%, p<0.0005), upper-body strength (8.7%, p<0.0001), and peak power (12.7%, p<0.0001) increased only in the ARA group. For the animal trial, GSK-β (Ser9) phosphorylation (p<0.001) independent of exercise and AMPK phosphorylation after exercise (p-AMPK less in ARA, p = 0.041) were different in ARA-fed versus CTL rats. Conclusions Our findings suggest that ARA supplementation can positively augment strength-training induced adaptations in resistance-trained males. However, chronic studies at the molecular level are required to further elucidate how ARA combined with strength training affect muscle adaptation.
    ERK5 signalling rescues intestinal epithelial turnover and tumour cell proliferation upon ERK1/2 abrogation
    Petrus R. de Jong, Nature Communications - 2016
    Abstract
    The ERK1/2 MAPK signalling module integrates extracellular cues that induce proliferation and differentiation of epithelial lineages, and is an established oncogenic driver, particularly in the intestine. However, the interrelation of the ERK1/2 module relative to other signalling pathways in intestinal epithelial cells and colorectal cancer (CRC) is unclear. Here we show that loss of Erk1/2 in intestinal epithelial cells results in defects in nutrient absorption, epithelial cell migration and secretory cell differentiation. However, intestinal epithelial cell proliferation is not impeded, implying compensatory mechanisms. Genetic deletion of Erk1/2 or pharmacological targeting of MEK1/2 results in supraphysiological activity of the ERK5 pathway. Furthermore, targeting both pathways causes a more effective suppression of cell proliferation in murine intestinal organoids and human CRC lines. These results suggest that ERK5 provides a common bypass route in intestinal epithelial cells, which rescues cell proliferation upon abrogation of ERK1/2 signalling, with therapeutic implications in CRC.
    Functional reprogramming of human prostate cancer to promote local attraction of effector CD8+ T cells
    Ravikumar Muthuswamy, The Prostate - 2016
    Abstract
    BACKGROUND Local infiltration of CD8+ T cells (CTLs) in tumor lesions predicts overall clinical outcomes and the clinical benefit of cancer patients from immune checkpoint blockade. In the current study, we evaluated local production of different classes of chemokines in prostate cancer lesions, and the feasibility of their modulation to promote selective entry of CTLs into prostate tumors. METHODS Chemokine expression in prostate cancer lesion was analyzed by TaqMan-based quantitative PCR, confocal fluorescence microscopy and ELISA. For ex vivo chemokine modulation analysis, prostate tumor explants from patients undergoing primary prostate cancer resections were cultured for 24 hr, in the absence or presence of the combination of poly-I:C, IFNα, and celecoxib (PAC). The numbers of cells producing defined chemokines in the tissues were analyzed by confocal microscopy. Chemotaxis of effector CD8+ T cells towards the untreated and PAC-treated tumor explant supernatants were evaluated in a standard in vitro migration assays, using 24 well trans-well plates. The number of effector cells that migrated was enumerated by flow cytometry. Pearson (r) correlation was used for analyzing correlations between chemokines and immune filtrate, while paired two tailed students t-test was used for comparison between treatment groups. RESULTS Prostate tumors showed uniformly low levels of CTL/NK/Th1-recruiting chemokines (CCL5, CXCL9, CXCL10) but expressed high levels of chemokines implicated in the attraction of myeloid derived suppressor cells (MDSC) and regulatory T cells (Treg): CCL2, CCL22, and CXCL12. Strong positive correlations were observed between CXCL9 and CXCL10 and local CD8 expression. Tumor expression levels of CCL2, CCL22, and CXCL12 were correlated with intratumoral expression of MDSC/Treg markers: FOXP3, CD33, and NCF2. Treatment with PAC suppressed intratumoral production of the Treg-attractant CCL22 and Treg/MDSC-attractant, CXCL12, while increasing the production of the CTL attractant, CXCL10. These changes in local chemokine production were accompanied by the reduced ability of the ex vivo-treated tumors to attract CD4+ FOXP3+ Treg cells, and strongly enhanced attraction of the CD8+Granzyme B+ CTLs. CONCLUSIONS Our data demonstrate that the chemokine environment in prostate cancer can be reprogrammed to selectively enhance the attraction of type-1 effector immune cells and reduce local attraction of MDSCs and Tregs. Prostate 76:1095–1105, 2016. © 2016 Wiley Periodicals, Inc.
    Physico-chemical properties and biological effects of diesel and biomass particles
    Eleonora Longhin, Environmental Pollution - 2016
    Abstract
    Diesel combustion and solid biomass burning are the major sources of ultrafine particles (UFP) in urbanized areas. Cardiovascular and pulmonary diseases, including lung cancer, are possible outcomes of combustion particles exposure, but differences in particles properties seem to influence their biological effects. Here the physico-chemical properties and biological effects of diesel and biomass particles, produced under controlled laboratory conditions, have been characterized. Diesel UFP were sampled from a Euro 4 light duty vehicle without DPF fuelled by commercial diesel and run over a chassis dyno. Biomass UFP were collected from a modern automatic 25 kW boiler propelled by prime quality spruce pellet. Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) images of both diesel and biomass samples showed aggregates of soot particles, but in biomass samples ash particles were also present. Chemical characterization showed that metals and PAHs total content was higher in diesel samples compared to biomass ones. Human bronchial epithelial (HBEC3) cells were exposed to particles for up to 2 weeks. Changes in the expression of genes involved in xenobiotic metabolism were observed after exposure to both UFP already after 24 h. However, only diesel particles modulated the expression of genes involved in inflammation, oxidative stress and epithelial-to-mesenchymal transition (EMT), increased the release of inflammatory mediators and caused phenotypical alterations, mostly after two weeks of exposure. These results show that diesel UFP affected cellular processes involved in lung and cardiovascular diseases and cancer. Biomass particles exerted low biological activity compared to diesel UFP. This evidence emphasizes that the study of different emission sources contribution to ambient PM toxicity may have a fundamental role in the development of more effective strategies for air quality improvement.
    FGFR2IIIb-MAPK Activity Is Required for Epithelial Cell Fate Decision in the Lower Müllerian Duct - me.2016-1027
    Jumpei Terakawa, Original Research - 2016
    Abstract
    Cell fate of lower Müllerian duct epithelium (MDE), to become uterine or vaginal epithelium, is determined by the absence or presence of Np63 expression, respectively. Previously, we showed that SMAD4 and runt-related transcription factor 1 (RUNX1) were independently required for MDE to express Np63. Here, we report that vaginal mesenchyme directs vaginal epithelial cell fate in MDE through paracrine activation of fibroblast growth factor (FGF) receptor-MAPK pathway. In the developing reproductive tract, FGF7 and FGF10 were enriched in vaginal mesenchyme, whereas FGF receptor 2IIIb was expressed in epithelia of both the uterus and vagina. When Fgfr2 was inactivated, vaginal MDE underwent uterine cell fate, and this differentiation defect was corrected by activation of MEK-ERK pathway. In vitro, FGF10 in combination with bone morpho genetic protein 4 and activin A (ActA) was sufficient to induce Np63 in MDE, and ActA was essential for induction of RUNX1 through SMAD-independent pathways. Accordingly, inhibition of type 1 receptors for activin in neonatal mice induced uterine differentiation in vaginal epithelium by down-regulating RUNX1, whereas conditional deletion of Smad2 and Smad3 had no effect on vaginal epithelial differentiation. In conclusion, vaginal epithelial cell fate in MDE is induced by FGF7/10-MAPK, bone morphogenetic protein 4-SMAD, and ActA-RUNX1 pathway activities, and the disruption in any one of these pathways results in conversion from vaginal to uterine epithelial cell fate.(Molecular Endocrinology30: 783–795, 2016
    Effect of Aronia melanocarpa (Black Chokeberry) supplementation on the development of obesity in mice fed a high-fat diet
    Jamie Baum, Journal of Berry Research - 2016
    Abstract
    BACKGROUND: Products derived from black chokeberry are claimed to be beneficial in treating chronic diseases, such as obesity and diabetes. OBJECTIVE: The objective of this study was to determine if supplementation with Aronia melanocarpa (black chokeberry) juice concentrate (AJC) has anti-obesity properties in mice fed obesogenic diets. METHODS: Male C57BL/6J mice (n = 10/dietary treatment) were placed on either a low-fat, high-sucrose (LFHS; 5% fat), LFHS+AJC (1.44 g AJC/kg diet), high-fat (HF; 30% fat), or HF+AJC for 12-weeks. RESULTS: Final body weight was lower in LFHS+AJC compared to LFHS, HF and HF+AJC (∼14, 20% and ∼16%, respectively; P < 0.05). Mice receiving LFHS and LFHS+AJC had significantly higher (P = 0.001) energy intake than HF and HF+AJC. LFHS-fed mice had less (–30%) epididymal fat (p < 0.05) than HF-fed mice, however mice on the LFHS+AJC had less epididymal fat per gram body weight than LFHS controls. There was no effect of diet or AJC on adipose tissue gene expression. There was no difference in plasma insulin, glucose or triglycerides between groups, however there was a positive effect of AJC on adiponectin (P = 0.059). There was also a significant effect of diet (LFHS versus HF) on HOMA-IR (P = 0.004) and HOMA-BCF (P = 0.002). CONCLUSIONS: The results from this study demonstrate that AJC supplementation has the potential to prevent weight gain and markers of obesity. Further research is needed to determine mechanisms of action.
    LANTHIONINE SYNTHETASE C-LIKE 2-BASED THERAPEUTICS - Biotherapeutics, Inc.
    Josep Bassaganya-riera,, US Patent - 2016
    Abstract
    Provided are compounds that target the lanthionine synthetase C-like protein 2 pathway. The compounds can be used to treat a number of conditions, including infectious disease, autoimmune disease, diabetes, and a chronic inflammatory disease.
    Functional and Structural Mimicry of Cellular Protein Kinase A Anchoring Proteins by a Viral Oncoprotein
    Cason King, PLOS Pathog - 2016
    Abstract
    Author Summary Studies of human adenovirus (HAdV), a small DNA tumor virus, illustrate the profound impact of viral proteins on multiple host functions. The multifunctional E1A proteins of HAdV are particularly adept at targeting key cellular regulators. Mechanistically, E1A alters or inhibits the normal function of the cellular proteins that it targets, and also establishes new connections in the cellular protein interaction network. Through these interactions, E1A creates a cellular milieu more conducive for replication. Here we show that HAdV E1A mimics cellular A-kinase anchoring proteins (AKAPs) in both appearance and function. We found that the protein kinase A (PKA) regulatory subunits are conserved targets of most HAdV E1A species. Structural modeling and a docking analysis predict a remarkable similarity between the binding of E1A and cellular AKAPs to PKA, which was confirmed experimentally. In addition, we observed E1A-mediated relocalization of PKA subunits and competition between E1A and cellular AKAPs during infection that contribute to HAdV gene expression and overall viral replication. Together, our studies identify E1A as the first known viral AKAP, and reveal a unique example of viral subversion of the PKA pathway via structural mimicry.
    DNA cytosine hydroxymethylation levels are distinct among non-overlapping classes of peripheral blood leukocytes
    Natalie M.Hohos, Journal of Immunological Methods - 2016
    Abstract
    Background Peripheral blood leukocytes are the most commonly used surrogates to study epigenome-induced risk and epigenomic response to disease-related stress. We considered the hypothesis that the various classes of peripheral leukocytes differentially regulate the synthesis of 5-methylcytosine (5mCG) and its removal via Ten-Eleven Translocation (TET) dioxygenase catalyzed hydroxymethylation to 5-hydroxymethylcytosine (5hmCG), reflecting their responsiveness to environment. Although it is known that reductions in TET1 and/or TET2 activity lead to the over-proliferation of various leukocyte precursors in bone marrow and in development of chronic myelomonocytic leukemia and myeloproliferative neoplasms, the role of 5mCG hydroxymethylation in peripheral blood is less well studied. Results We developed simplified protocols to rapidly and reiteratively isolate non-overlapping leukocyte populations from a single small sample of fresh or frozen whole blood. Among peripheral leukocyte types we found extreme variation in the levels of transcripts encoding proteins involved in cytosine methylation (DNMT1, 3A, 3B), the turnover of 5mC by demethylation (TET1, 2, 3), and DNA repair (GADD45A, B, G) and in the global and gene-region-specific levels of DNA 5hmCG (CD4 + T cells ≫ CD14 + monocytes > CD16 + neutrophils > CD19 + B cells > CD56 + NK cells > Siglec8 + eosinophils > CD8 + T cells). Conclusions Our data taken together suggest a potential hierarchy of responsiveness among classes of leukocytes with CD4 +, CD8 + T cells and CD14 + monocytes being the most distinctly poised for a rapid methylome response to physiological stress and disease.
    INTRINSIC AND EXTRINSIC REGULATION OF PINEAL MELATONIN RHYTHMS - viewcontent.cgi
    Ye Li, University of Kentucky - 2016
    Abstract
    . Circadian rhythm is a biological rhythm with period of about 24 hours. Circadian rhythm is universal in phyla from bacteria to mammals and exist in different level from gene expression to behavior. Circadian system consists of three components: 1) a self-sustained oscillator; 2) an input pathway which can alter the phase of the oscillator; and 3) an output such as gene expression, enzyme activity, hormone production, heart rate, body temperature or loco motor activities. The way the oscillator regulates its outputs is complicated, in that on one hand usually the oscillator is not the only factor affecting the outputs, and on the other, the oscillator itself is incorporated in intricate pathways. Chicken pineal cell culture is a well-established in vitro model to study circadian rhythm. It contains a self-sustained oscillator which can be phase-shifted by light as input and rhythmically releases melatonin as an output. Here I have characterized the role of norepinephrine (NE), the sympathetic regulatory input of pineal gland, and the microenvironment of pineal cells in melatonin rhythmicity of cultured chicken pineal cells. Chapter 1 of this dissertation provides a review of circadian rhythm with a focus on melatonin regulation in pineal gland. Chapter 2 describes the methods to build up a fraction collector which offers high time resolution of sampling for a super fusion system. Chapter 3 is a technical report of a melatonin enzyme-linked immunosorbent assay suitable for high throughput measurement of melatonin. Chapter 4 presents data demonstrating that daily administration of NE recovers damped melatonin rhythm in constant darkness. In addition, NE does not change the expression of clock genes but the recovery effect of NE depends on the internal clock. Furthermore, the data indicates that NE administration stimulates the gene expression of phosphodiesterase 4D (PDE4D) and adenylate cyclase 1 (AC1) in a time order, potentially corresponding to the trough and peak of recovered melatonin rhythm. Chapter 5 presents data showing that the amplitude of melatonin rhythm in cultured pineal cells is affected by microenvironments of the cell culture and connexin plays a role in this effect. Finally, in Chapter 6I discuss how the results of each chapter demonstrate multiple regulatory mechanism of the melatonin rhythm of chicken pineal cells. Furthermore, I discuss the implications of this work in the field of developmental biology and how the current data will shape future investigations. My dissertation incorporates engineering, immunocytochemistry, chicken genetics, and biochemical analyses, and will help in better understanding the regulation mechanism of output in a circadian system.
    Activation of PI3K signaling prevents aminoglycoside-induced hair cell death in the murine cochlea
    Azadeh Jadali, Biology Open - 2016
    Abstract
    Loss of sensory hair cells of the inner ear due to aminoglycoside exposure is a major cause of hearing loss. Using an immortalized multipotent otic progenitor (iMOP) cell line, specific signaling pathways that promote otic cell survival were identified. Of the signaling pathways identified, the PI3K pathway emerged as a strong candidate for promoting hair cell survival. In aging animals, components for active PI3K signaling are present but decrease in hair cells. In this study, we determined whether activated PI3K signaling in hair cells promotes survival. To activate PI3K signaling in hair cells, we used a small molecule inhibitor of PTEN or genetically ablated PTEN using a conditional knockout animal. Hair cell survival was challenged by addition of gentamicin to cochlear cultures. Hair cells with activated PI3K signaling were more resistant to aminoglycoside-induced hair cell death. These results indicate that increased PI3K signaling in hair cells promote survival and the PI3K signaling pathway is a target for preventing aminoglycoside-induced hearing loss.
    Transcriptome analysis of bovine oocytes from distinct follicle sizes: Insights from correlation network analysis
    Rémi Labrecque, Molecular Reproduction and Development - 2016
    Abstract
    Follicle size is recognized as a predictor of the potential for the enclosed oocyte to yield an embryo following in vitro maturation and in vitro fertilization. Oocytes from larger follicles are more likely to reach the blastocyst stage than those from smaller follicles. A growing oocyte accumulates all the transcripts needed to ensure development until the maternal embryonic transition, and this accumulation must be completed before the period of transcriptional arrest. Accordingly, the transcriptomes of bovine germinal-vesicle-stage oocytes collected from follicles of increasing sizes (<3, 3–5, >5–8, and >8 mm) were evaluated, using the EmbryoGENE bovine transcriptomic platform (custom Agilent 4 × 44 K), to better understand transcriptional modulation in the oocyte as the follicle becomes larger. Microarray analyses revealed very few differences between oocytes from small follicles (<3 vs. 3–5 mm), whereas an important number of differences were detected at the mRNA level between oocytes from larger follicles. Weighted gene correlation network analysis allowed for the identification of several hub genes involved in crucial functions such as transcriptional regulation (TAF2), chromatin remodeling (PPP1CB), energy production (SLC25A31), as well as transport of key molecules within the cell (NAGPA, CYHR1, and SLC3A12). The results presented here thus reinforce the hypothesis that developmental competence acquisition cannot be seen as a simple one-step process, especially in regards to the modulation of mRNA. Mol. Reprod. Dev. 83: 558–569, 2016. © 2016 Wiley Periodicals, Inc.
    Non-canonical role of matrix metalloprotease (MMP) in activation and migration of hepatic stellate cells (HSCs)
    Mirza S. Baiga,, Science Direct - 2016
    Abstract
    Aims Matrix metalloproteinases (MMPs) that degrade extracellular matrix (ECM) and help to resolve the excess matrix are considered to be under-expressed in fibrosis. MMPs are generally anti-fibrotic, however others can have pro-fibrotic functions. Therefore, the aim of this study was to find out the mechanism of pro-fibrotic function of MMPs in hepatic stellate cells' (HSC's) activation and migration. Main methods Human MMP Antibody Array from Abcam was used to profile MMPs in macrophages. Gelatin or casein zymography was performed using 10% SDS-polyacrylamide gels (SDS-PAGE) containing gelatin (1 mg/ml) or Casein (1 mg/ml) as substrate. HSCs migration assay was performed using Boyden chamber as described previously (Guo et al., 2007, McGarrigle et al., 2006, Shan et al., 2006 and Yang and Huang, 2005). Real-time PCR with SYBR green was performed using iTaq™ universal SYBR® Green supermix (BIO-RAD) and a 7500 Real-Time PCR System (Applied Biosystems). Collagen, type I, alpha 1 (COL1A1), alpha smooth muscle actin (α-SMA) expression was determined by immunoblot analysis. Key findings We first profiled the expression of all MMPs in primary murine bone marrow-derived macrophages (BMDMs) and differentiated THP-1 cells and found that MMP-8, -10, & -13, were significantly overexpressed after 12 h of lipopolysaccharide (LPS) treatment. Based on this pattern of expression, we speculated that macrophage MMP-8,-10, &-13 might play a non-canonical role in HSCs activation. Further, we found that exogenous active MMP-8 (Collagenase-2) treated HSC shows markedly increased migration and COL1A1 expression as compared to MMP-10 and MMP-13 treated HSCs. Thus, macrophage MMP-8 (Collagenase-2) expression in macrophages emerges as an important moderator of HSC cell migration and invasion.
    The bright side of plasmonic gold nanoparticles; activation of Nrf2, the cellular protective pathway
    Alona Goldstein, Nanoscale - 2016
    Abstract
    Plasmonic gold nanoparticles (AuNPs) are widely investigated for cancer therapy, due to their ability to strongly absorb light and convert it to heat and thus selectively destroy tumor cells. In this study we shed light on a new aspect of AuNPs and their plasmonic excitation, wherein they can provide anti-oxidant and anti-inflammatory protection by stimulating the cellular protective Nrf2 pathway. Our study was carried out on cells of the immune system, macrophages, and on skin cells, keratinocytes. A different response to AuNPs was noted in the two types of cells, explained by their distinct uptake profiles. In keratinocytes, the exposure to AuNPs, even at low concentrations, was sufficient to activate the Nrf2 pathway, without any irradiation, due to the presence of free AuNPs inside the cytosol. In contrast, in macrophages, the plasmonic excitation of the AuNPs by a low, non-lethal irradiation dose was required for their release from the constraining vesicles. The mechanism by which AuNPs activate the Nrf2 pathway was studied. Direct and indirect activation were suggested, based on the inherent ability of the AuNPs to react with thiol groups and to generate reactive oxygen species, in particular, under plasmonic excitation. The ability of AuNPs to directly activate the Nrf2 pathway renders them good candidates for treatment of disorders in which the up-regulation of Nrf2 is beneficial, specifically for topical treatment of inflammatory skin diseases.
    Transcriptome Analysis of Piperlongumine-Treated Human Pancreatic Cancer Cells Reveals Involvement of Oxidative Stress and Endoplasmic Reticulum Stress Pathways
    Harsharan Dhillon, Journal of Medicinal Food - 2016
    Abstract
    Piperlongumine (PL), an alkaloid obtained from long peppers, displays antitumorigenic properties for a variety of human cell- and animal-based models. The aim of this study was to identify the underlying molecular mechanisms for PL anticancer effects on human pancreatic cancer cells. RNA sequencing (RNA-seq) was used to identify the effects of PL on the transcriptome of MIA PaCa-2 human pancreatic cancer cells. PL treatment of pancreatic cancer cells resulted in differential expression of 683 mRNA transcripts with known protein functions, 351 of which were upregulated and 332 of which were downregulated compared to control-treated cells. Transcripts associated with oxidative stress, endoplasmic reticulum (ER) stress, and unfolded protein response pathways were significantly overexpressed with PL treatment. Reverse transcription-quantitative polymerase chain reaction and western blotting were used to validate the RNA-seq results, which included upregulation of HO-1, IRE1α, cytochrome c, and ASNS. The results provide key insight into the mechanisms by which PL alters cancer cell physiology and identify that activation of oxidative stress and ER stress pathways is a critical avenue for PL anticancer effects.
    TMEM184b Promotes Axon Degeneration and Neuromuscular Junction Maintenance
    Martha R. C. Bhattacharya, The Journal of Neuroscience - 2016
    Abstract
    Complex nervous systems achieve proper connectivity during development and must maintain these connections throughout life. The processes of axon and synaptic maintenance and axon degeneration after injury are jointly controlled by a number of proteins within neurons, including ubiquitin ligases and mitogen activated protein kinases. However, our understanding of these molecular cascades is incomplete. Here we describe the phenotype resulting from mutation of TMEM184b, a protein identified in a screen for axon degeneration mediators. TMEM184b is highly expressed in the mouse nervous system and is found in recycling endosomes in neuronal cell bodies and axons. Disruption of TMEM184b expression results in prolonged maintenance of peripheral axons following nerve injury, demonstrating a role for TMEM184b in axon degeneration. In contrast to this protective phenotype in axons, uninjured mutant mice have anatomical and functional impairments in the peripheral nervous system. Loss of TMEM184b causes swellings at neuromuscular junctions that become more numerous with age, demonstrating that TMEM184b is critical for the maintenance of synaptic architecture. These swellings contain abnormal multivesicular structures similar to those seen in patients with neurodegenerative disorders. Mutant animals also show abnormal sensory terminal morphology. TMEM184b mutant animals are deficient on the inverted screen test, illustrating a role for TMEM184b in sensory-motor function. Overall, we have identified an important function for TMEM184b in peripheral nerve terminal structure, function, and the axon degeneration pathway. SIGNIFICANCE STATEMENT Our work has identified both neuroprotective and neurodegenerative roles for a previously undescribed protein, TMEM184b. TMEM184b mutation causes delayed axon degeneration following peripheral nerve injury, indicating that it participates in the degeneration process. Simultaneously, TMEM184b mutation causes progressive structural abnormalities at neuromuscular synapses and swellings within sensory terminals, and animals with this mutation display profound weakness. Thus, TMEM184b is necessary for normal peripheral nerve terminal morphology and maintenance. Loss of TMEM184b results in accumulation of autophagosomal structures in vivo, fitting with emerging studies that have linked autophagy disruption and neurological disease. Our work recognizes TMEM184b as a new player in the maintenance of the nervous system.
    Suppressor of hairy-wing, modifier of mdg4 and centrosomal protein of 190 gene orthologues of the gypsy insulator complex in the malaria mosquito, Anopheles stephensi
    R Carballar-Lejarazú, Insect Molecular Biology - 2016
    Abstract
    DNA insulators organize independent gene regulatory domains and can regulate interactions amongst promoter and enhancer elements. They have the potential to be important in genome enhancing and editing technologies because they can mitigate chromosomal position effects on transgenes. The orthologous genes of the Anopheles stephensi putative gypsy-like insulator protein complex were identified and expression characteristics studied. These genes encode polypeptides with all the expected protein domains (Cysteine 2 Histidine 2 (C2H2) zinc fingers and/or a bric-a-brac/poxvirus and zinc finger). The mosquito gypsy transcripts are expressed constitutively and are upregulated in ovaries of blood-fed females. We have uncovered significant experimental evidence that the gypsy insulator protein complex is widespread in vector mosquitoes.
    Effects of carbon nanotubes on intercellular communication and involvement of IL-1 genes
    Yke Jildouw Arnoldussen, Journal of Cell Communication and Signaling - 2016
    Abstract
    An increasing amount of products containing engineered nanoparticles is emerging. Among these particles are carbon nanotubes (CNTs) which are of interest for a wide range of industrial and biomedical applications. There have been raised concerns over the effects of CNTs on human health. Some types of CNTs are classified as group 2B carcinogens by the International Agency for Research on Cancer. CNTs may also induce pulmonary inflammatory and fibrotic effects. By utilizing CNTs of different lengths, we investigated the role of the proinflammatory cytokine, interleukin-1 (IL-1) on gap junctional intercellular communication (GJIC) by using IL-1 wild-type (IL1-WT) and IL-1 knock-out (IL1-KO) cells. GJIC decreased equally in both cell types after CNT exposure. Immunofluorescence staining showed Gja1 and Gjb2 in gap junctions and hemichannels for both cell types. Gjb1 and Gjb2 expression was low in IL1-KO cells, which was confirmed by protein analysis. Gja1 was upregulated with both CNTs, whereas Gjb1 was down-regulated by CNT-2 in IL1-WT cells. Connexin mRNA expression was regulated differently by the CNTs. CNT-1 affected Gja1 and Gjb2, whereas CNT-2 had an effect on Gjb1. CNTs negatively affect GJIC through gap junctions independently of the length of CNT and IL-1 status. Furthermore, connexin gene expression was affected by IL-1 at transcriptional and translational levels. As both CNTs used in this study are cytotoxic to the cells and reduce cell survival, we suggest that CNT-induced reduction in GJIC may be important for inhibiting transfer of cell survival signals between cells.
    PROMOTION OF WOUND HEALING - Northwestern University
    Amy Paller, US Patent - 2016
    Abstract
    SUMMARY OF THE INVENTION In some embodiments, the present invention provides methods of promoting wound healing comprising depleting gangliosides in a subject. In some embodiments, a ganglioside depletion agent (e.g., glucosylceramide synthase inhibitor) is administered to a subject to promote wound healing. In some embodiments, the present invention provides methods of promoting wound healing comprising administering one or more glucosylceramide synthase inhibitors to a subject with one or more cutaneous wounds. In some embodiments, the subject is diabetic (e.g., Type I diabetes, Type II diabetes, gestational diabetes, etc.). In some embodiments, one or more ganglioside precursors (e.g., GM3) are targeted to promote wound healing. In some embodiments, conversion of ganglioside precursors (e.g., GM3) into gangliosides is inhibited to promote wound healing. In some embodiments, the subject is not diabetic. In some embodiments, one or more cutaneous wounds comprise one or more of incisions, lacerations, abrasions, puncture wounds, and closed wounds (e.g., diabetic ulcers, such as a foot ulcer). In some embodiments, a glucosylceramide synthase inhibitor is selected from PDMP, D-threo-EtDO-P4, ((1R, 2R)-nonanoic acid[2-(2′,3′-dihydro-benzo[1,4]dioxin-6′-yl)-2-hydroxy-1-pyrrolidin-1-ylmethyl-ethyl]-amide-L-tartaric acid salt, AMP-DNM and analogues, homologues, and functional equivalents thereof. In some embodiments, a glucosylceramide synthase inhibitor is administered systemically. In some embodiments, a glucosylceramide synthase inhibitor is administered locally. In some embodiments, a glucosylceramide synthase inhibitor is administered topically. In some embodiments, administering a glucosylceramide synthase inhibitor accelerates the rate of wound repair (e.g., the wound heals twice as fast as without said inhibitor). In some embodiments, administering a glucosylceramide synthase inhibitor reduces the chance of said wound becoming infected. In some embodiments, a ganglioside depletion agent is administered systemically. In some embodiments, a ganglioside depletion agent is administered locally. In some embodiments, a ganglioside depletion agent is administered topically. In some embodiments, administering a ganglioside depletion agent accelerates the rate of wound repair (e.g., the wound heals at least 1.5×, 2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, or 10× as fast as without said inhibitor). In some embodiments, administering a ganglioside depletion agent reduces the chance of said wound becoming infected. In some embodiments, a composition that inhibits conversion of ganglioside precursors (e.g., GM3) into gangliosides is administered systemically. In some embodiments, a composition that inhibits conversion of ganglioside precursors (e.g., GM3) into gangliosides is administered locally. In some embodiments, a composition that inhibits conversion of ganglioside precursors (e.g., GM3) into gangliosides is administered topically. In some embodiments, administering a composition that inhibits conversion of ganglioside precursors (e.g., GM3) into gangliosides accelerates the rate of wound repair (e.g., the wound heals twice as fast as without said inhibitor). In some embodiments, administering a composition that inhibits conversion of ganglioside precursors (e.g., GM3) into gangliosides reduces the chance of said wound becoming infected. In some embodiments, the present invention provides a composition for wound care comprising one or more ganglioside depletion agents (e.g., a glucosylceramide synthase inhibitors) and an application element. In some embodiments, the present invention provides a composition for wound care comprising one or more glucosylceramide synthase inhibitors and an application element. In some embodiments, a glucosylceramide synthase inhibitor is selected from PDMP, D-threo-EtDO-P4, ((1R, 2R)-nonanoic acid[2-(2′,3′-dihydro-benzo[1,4]dioxin-6′-yl)-2-hydroxy-1-pyrrolidin-1-ylmethyl-ethyl]-amide-L-tartaric acid salt, AMP-DNM and analogues, homologues, and functional equivalents thereof. In some embodiments, the application element is configured for topical application to a wound. In some embodiments, the application element comprises a liquid, cream, paste, salve, balm, or semi-solid. In some embodiments, the application element comprises a patch, wrap, or bandage. In some embodiments, a composition further comprises one or more additional wound care agents. In some embodiments, wound care agents are selected from antiseptic, antibiotic, local anesthetic, anti-inflammatory, pain reliever, etc.
    Compositions Useful for Treating Herpes Simplex Labialis and/or Herpes Esophagitis, and Methods Using Same - DREXEL UNIVERSITY
    Clifford, Jane E., US Patent - 2016
    Abstract
    1. A composition comprising an anti-herpetic agent and at least one inhibitor selected from the group consisting of an ATM inhibitor, a Chk2 inhibitor, and a salt, solvate or N-oxide thereof, wherein the composition treats or prevents a HSV-1 infection in a subject in need thereof, wherein the HSV-1 infection comprises at least one selected from the group consisting of herpes simplex labialis and herpes esophagitis. 2. The composition of claim 1, wherein the ATM inhibitor and the Chk2 inhibitor are independently selected from the group consisting of a nucleic acid, siRNA, antisense nucleic acid, ribozyme, peptide, small molecule, antagonist, aptamer, peptidomimetic, and any combinations thereof. 3. The composition of claim 2, wherein the ATM inhibitor is at least one small molecule selected from the group consisting of caffeine, wortmannin, chloroquine, CP-466722, KU-55933, KU-59403, KU-60019, and a salt, N-oxide or solvate thereof. 4. The composition of claim 2, wherein the Chk2 inhibitor is at least one small molecule selected from the group consisting of Chk2 inhibitor II, SC-203885, NSC-109555, and a salt, N-oxide or solvate thereof. 5. The composition of claim 1, wherein the anti-herpetic agent is at least one selected from the group consisting of acyclovir, famciclovir, penciclovir, valacyclovir, acyclovir, trifluridine, penciclovir and valacyclovir. 6. A method of treating or preventing a HSV-1 infection in a subject in need thereof, the method comprising administering to the subject an effective amount of an anti-herpetic agent and an effective amount of at least one inhibitor selected from the group consisting of an ATM inhibitor and a Chk2 inhibitor, whereby the HSV-1 infection is treated or prevented in the subject, wherein the HSV-1 infection comprises at least one selected from the group consisting of herpes simplex labialis and herpes esophagitis. 7. The method of claim 6, wherein the ATM inhibitor and the Chk2 inhibitor are independently selected from the group consisting of a nucleic acid, siRNA, antisense nucleic acid, ribozyme, peptide, small molecule, antagonist, aptamer, peptidomimetic and any combinations thereof. 8. The method of claim 7, wherein the ATM inhibitor is at least one small molecule selected from the group consisting of caffeine, wortmannin, chloroquine, CP-466722, KU-55933, KU-59403, KU-60019, and a salt, N-oxide or solvate thereof. 9. The method of claim 7, wherein the Chk2 inhibitor is at least one small molecule selected from the group consisting of Chk2 inhibitor II, SC-203885, NSC-109555, and a salt, N-oxide or solvate thereof. 10. The method of claim 6, wherein the anti-herpetic agent is selected from the group consisting of acyclovir, famciclovir, penciclovir, valacyclovir, acyclovir, trifluridine, penciclovir and valacyclovir. 11. The method of claim 6, wherein the at least one inhibitor and the anti-herpetic agent are co-administered to the subject. 12. The method of claim 6, wherein administration of the inhibitor to the subject reduces the amount of the anti-herpetic agent required to be administered to the subject to obtain the same therapeutic benefit obtained when the effective dose of the anti-herpetic agent in the absence of the inhibitor is administered to the subject. 13. The method of claim 6, wherein the subject experiences less frequent or less severe side effects of the anti-herpetic agent, as compared to when the effective dose of the anti-herpetic agent in the absence of the inhibitor is administered to the subject. 14. The method of claim 6, wherein development of resistance to the anti-herpetic agent is prevented or minimized in the subject, as compared to when the effective dose of the anti-herpetic agent in the absence of the inhibitor is administered to the subject. 15. A method of treating or preventing a HSV-1 infection in a subject in need thereof, wherein the infection is caused by a drug-resistant HSV-1 strain, the method comprising administering to the subject an effective amount of at least one inhibitor selected from the group consisting of an ATM inhibitor and a Chk2 inhibitor, wherein the subject is optionally further administered an effective amount of an anti-herpetic agent, wherein administration of the at least one inhibitor treats or prevents the HSV-1 infection in the subject, wherein the HSV-1 infection comprises at least one selected from the group consisting of herpes simplex labialis and herpes esophagitis. 16. The method of claim 15, wherein the ATM inhibitor and the Chk2 inhibitor are independently selected from the group consisting of a nucleic acid, siRNA, antisense nucleic acid, ribozyme, peptide, small molecule, antagonist, aptamer, peptidomimetic and any combinations thereof. 17. The method of claim 16, wherein the ATM inhibitor is at least one small molecule selected from the group consisting of caffeine, wortmannin, chloroquine, CP-466722, KU-55933, KU-59403, KU-60019, and a salt, N-oxide or solvate thereof. 18. The method of claim 16, wherein the Chk2 inhibitor is at least one small molecule selected from the group consisting of Chk2 inhibitor II, SC-203885, NSC-109555, and a salt, N-oxide or solvate thereof. 19. The method of claim 15, wherein the anti-herpetic agent is at least one selected from the group consisting of acyclovir, famciclovir, penciclovir, valacyclovir, acyclovir, trifluridine, penciclovir and valacyclovir. 20. The method of claim 15, wherein the drug-resistant HSV-1 strain has a TK mutation. 21. The method of claim 15, wherein the strain is resistant to at least one selected from the group consisting of acyclovir, famciclovir, penciclovir, valacyclovir, acyclovir, trifluridine, penciclovir and valacyclovir. 22. A kit comprising at least one inhibitor selected from the group consisting of an ATM inhibitor and a Chk2 inhibitor, the kit further comprising an applicator; and an instructional material for the use of the kit, wherein the instruction material comprises instructions for treating, ameliorating or preventing a HSV-1 infection in a subject in need thereof, wherein the HSV-1 infection comprises at least one selected from the group consisting of herpes simplex labialis and herpes esophagitis. 23. The kit of claim 22, wherein the kit further comprises an anti-herpetic agent.
    IGSF10 mutations dysregulate gonadotropin-releasing hormone neuronal migration resulting in delayed puberty
    Sasha R Howard, EMBO Molecular Medicine - 2016
    Abstract
    Early or late pubertal onset affects up to 5% of adolescents and is associated with adverse health and psychosocial outcomes. Self-limited delayed puberty (DP) segregates predominantly in an autosomal dominant pattern, but the underlying genetic background is unknown. Using exome and candidate gene sequencing, we have identified rare mutations in IGSF10 in 6 unrelated families, which resulted in intracellular retention with failure in the secretion of mutant proteins. IGSF10 mRNA was strongly expressed in embryonic nasal mesenchyme, during gonadotropin-releasing hormone (GnRH) neuronal migration to the hypothalamus. IGSF10 knockdown caused a reduced migration of immature GnRH neurons in vitro, and perturbed migration and extension of GnRH neurons in a gnrh3:EGFP zebrafish model. Additionally, loss-of-function mutations in IGSF10 were identified in hypothalamic amenorrhea patients. Our evidence strongly suggests that mutations in IGSF10 cause DP in humans, and points to a common genetic basis for conditions of functional hypogonadotropic hypogonadism (HH). While dysregulation of GnRH neuronal migration is known to cause permanent HH, this is the first time that this has been demonstrated as a causal mechanism in DP.
    Monocyte-Derived Macrophages Contribute to Spontaneous Long-Term Functional Recovery after Stroke in Mice
    Somsak Wattananit, The Journal of Neuroscience - 2016
    Abstract
    Stroke is a leading cause of disability and currently lacks effective therapy enabling long-term functional recovery. Ischemic brain injury causes local inflammation, which involves both activated resident microglia and infiltrating immune cells, including monocytes. Monocyte-derived macrophages (MDMs) exhibit a high degree of functional plasticity. Here, we determined the role of MDMs in long-term spontaneous functional recovery after middle cerebral artery occlusion in mice. Analyses by flow cytometry and immunocytochemistry revealed that monocytes home to the stroke-injured hemisphere., and that infiltration peaks 3 d after stroke. At day 7, half of the infiltrating MDMs exhibited a bias toward a proinflammatory phenotype and the other half toward an anti-inflammatory phenotype, but during the subsequent 2 weeks, MDMs with an anti-inflammatory phenotype dominated. Blocking monocyte recruitment using the anti-CCR2 antibody MC-21 during the first week after stroke abolished long-term behavioral recovery, as determined in corridor and staircase tests, and drastically decreased tissue expression of anti-inflammatory genes, including TGFβ, CD163, and Ym1. Our results show that spontaneously recruited monocytes to the injured brain early after the insult contribute to long-term functional recovery after stroke. SIGNIFICANCE STATEMENT For decades, any involvement of circulating immune cells in CNS repair was completely denied. Only over the past few years has involvement of monocyte-derived macrophages (MDMs) in CNS repair received appreciation. We show here, for the first time, that MDMs recruited to the injured brain early after ischemic stroke contribute to long-term spontaneous functional recovery through inflammation-resolving activity. Our data raise the possibility that inadequate recruitment of MDMs to the brain after stroke underlies the incomplete functional recovery seen in patients and that boosting homing of MDMs with an anti-inflammatory bias to the injured brain tissue may be a new therapeutic approach to promote long-term improvement after stroke.
    Enhanced Muscular Dystrophy from Loss of Dysferlin Is Accompanied by Impaired Annexin A6 Translocation after Sarcolemmal Disruption
    Alexis Demonbreun, The American Journal of Pathology - 2016
    Abstract
    Dysferlin is a membrane-associated protein implicated in membrane resealing; loss of dysferlin leads to muscular dystrophy. We examined the same loss-of-function Dysf mutation in two different mouse strains, 129T2/SvEmsJ (Dysf129) and C57BL/6J (DysfB6). Although there are many genetic differences between these two strains, we focused on polymorphisms in Anxa6 because these variants were previously associated with modifying a pathologically distinct form of muscular dystrophy and increased the production of a truncated annexin A6 protein. Dysferlin deficiency in the C57BL/6J background was associated with increased Evan's Blue dye uptake into muscle and increased serum creatine kinase compared to the 129T2/SvEmsJ background. In the C57BL/6J background, dysferlin loss was associated with enhanced pathologic severity, characterized by decreased mean fiber cross-sectional area, increased internalized nuclei, and increased fibrosis, compared to that in Dysf129 mice. Macrophage infiltrate was also increased in DysfB6 muscle. High-resolution imaging of live myofibers demonstrated that fibers from DysfB6 mice displayed reduced translocation of full-length annexin A6 to the site of laser-induced sarcolemmal disruption compared to Dysf129 myofibers, and impaired translocation of annexin A6 associated with impaired resealing of the sarcolemma. These results provide one mechanism by which the C57BL/6J background intensifies dysferlinopathy, giving rise to a more severe form of muscular dystrophy in the DysfB6 mouse model through increased membrane leak and inflammation.
    LNCRHOXF1: a long noncoding RNA from the X-chromosome that suppresses viral response genes during development of the early human placenta
    Ian Penkala, Molecular and Cellular Biology - 2016
    Abstract
    Long noncoding RNAs (lncRNAs) can regulate gene expression in a cell-specific fashion during development. Here we identify a novel lncRNA from the X-chromosome that we named lncRHOXF1 and which is abundantly expressed in trophectoderm and primitive endoderm cells of human blastocyst-stage embryos. LncRHOXF1 is a spliced and polyadenylated lncRNA about 1 kb in length, found in both the nuclear and cytoplasmic compartments of in vitro differentiated human trophectoderm progenitor cells. Gain of function experiments in human embryonic stem cells, which normally lack lncRHOXF1 RNA, revealed that lncRHOXF1 reduced proliferation and favored cellular differentiation. LncRHOXF1 knockdown using siRNAs in human trophectoderm progenitors increased expression of viral response genes, including type I interferon. Sendai virus infection of human trophectoderm progenitor cells increased lncRHOXF1 RNA levels and siRNA-mediated disruption of lncRHOXF1 during infection reduced the expression of viral response genes leading to higher virus replication. Thus, lncRHOXF1 RNA is the first example of a lncRNA that regulates the host response to viral infections in human placental progenitor cells, and we propose that it functions as a repressor of the viral response during early human development.
    Bcl-2 Regulates Reactive Oxygen Species Signaling and a Redox-Sensitive Mitochondrial Proton Leak in Mouse Pancreatic β-Cells: Endocrinology: Vol 157, No 6
    Michal Aharoni-Simon, Endocrine Society - 2016
    Abstract
    In pancreatic β-cells, controlling the levels of reactive oxygen species (ROS) is critical to counter oxidative stress, dysfunction and death under nutrient excess. Moreover, the fine-tuning of ROS and redox balance is important in the regulation of normal β-cell physiology. We recently demonstrated that Bcl-2 and Bcl-xL, in addition to promoting survival, suppress β-cell glucose metabolism and insulin secretion. Here, we tested the hypothesis that the nonapoptotic roles of endogenous Bcl-2 extend to the regulation of β-cell ROS and redox balance. We exposed mouse islet cells and MIN6 cells to the Bcl-2/Bcl-xL antagonist Compound 6 and the Bcl-2-specific antagonist ABT-199 and evaluated ROS levels, Ca2+ responses, respiratory control, superoxide dismutase activity and cell death. Both acute glucose stimulation and the inhibition of endogenous Bcl-2 progressively increased peroxides and stimulated superoxide dismutase activity in mouse islets. Importantly, conditional β-cell knockout of Bcl-2 amplified glucose-induced formation of peroxides. Bcl-2 antagonism also induced a mitochondrial proton leak that was prevented by the antioxidant N-acetyl-L-cysteine and, therefore, secondary to redox changes. We further established that the proton leak was independent of uncoupling protein 2 but partly mediated by the mitochondrial permeability transition pore. Acutely, inhibitor-induced peroxides promoted Ca2+ influx, whereas under prolonged Bcl inhibition, the elevated ROS was required for induction of β-cell apoptosis. In conclusion, our data reveal that endogenous Bcl-2 modulates moment-to-moment ROS signaling and suppresses a redox-regulated mitochondrial proton leak in β-cells. These noncanonical roles of Bcl-2 may be important for β-cell function and survival under conditions of high metabolic demand. - See more at: http://press.endocrine.org/doi/abs/10.1210/en.2015-1964?journalCode=endo&#sthash.PLRoyXBy.dpuf
    Hyper-Variability in Circulating Insulin, High Fat Feeding Outcomes, and Effects of Reducing Ins2 Dosage in Male Ins1 -Null Mice in a Specific Pathogen-Free Facility
    Nicole M. Templeman, PLOS ONE - 2016
    Abstract
    Insulin is an essential hormone with key roles in energy homeostasis and body composition. Mice and rats, unlike other mammals, have two insulin genes: the rodent-specific Ins1 gene and the ancestral Ins2 gene. The relationships between insulin gene dosage and obesity has previously been explored in male and female Ins2 -/- mice with full or reduced Ins1 dosage, as well as in female Ins1 -/- mice with full or partial Ins2 dosage. We report herein unexpected hyper-variability in Ins1 -null male mice, with respect to their circulating insulin levels and to the physiological effects of modulating Ins2 gene dosage. Two large cohorts of Ins1 -/- : Ins2 +/- mice and their Ins1 -/- : Ins2 +/+ littermates were fed chow diet or high fat diet (HFD) from weaning, and housed in specific pathogen-free conditions. Cohort A and cohort B were studied one year apart. Contrary to female mice from the same litters, inactivating one Ins2 allele on the complete Ins1 -null background did not consistently cause a reduction of circulating insulin in male mice, on either diet. In cohort A, all HFD-fed males showed an equivalent degree of insulin hypersecretion and weight gain, regardless of Ins2 dosage. In cohort B the effects of HFD appeared generally diminished, and cohort B Ins1 -/- : Ins2 +/- males showed decreased insulin levels and body mass compared to Ins1 -/- : Ins2 +/+ littermates, on both diets. Although experimental conditions were consistent between cohorts, we found that HFD-fed Ins1 -/- : Ins2 +/- mice with lower insulin levels had increased corticosterone. Collectively, these observations highlight the phenotypic characteristics that change in association with differences in circulating insulin and Ins2 gene dosage, particularly in male mice.
    Lumpfish (Cyclopterus lumpus L.) develop amoebic gill disease (AGD) after experimental challenge with Paramoeba perurans and can transfer amoebae to Atlantic salmon (Salmo salar L.)
    Gyri T. Haugland, Aquaculture - 2016
    Abstract
    Cleaner fish such as lumpfish (Cyclopterus lumpus L.) and ballan wrasse (Labrus bergylta A.) are increasingly used to delouse farmed Atlantic salmon (Salmo salar L.). In 2014, > 20 million cleaner fish were placed into net-pens with farmed salmon in Norway. Amoebic gill disease (AGD), caused by the opportunistic, parasitic amoeba Paramoeba perurans, is emerging in salmon farming in Northern Europe. The amoeba displays low host specificity as it has been isolated from a range of fish species in addition to salmonids, such as wrasse and lumpfish cohabitating with farmed salmon. It is, however, not known to which degree lumpfish respond to P. perurans challenge, to which extent lumpfish may develop AGD, and if they can function as a vector for the spread of P. perurans to salmon. The present study shows that lumpfish can be infected with P. perurans under experimental conditions and develop AGD. However, lumpfish are more resistant and the development of pathology is slower compared to salmon. It is also shown that lumpfish can act as carriers and transmit parasitic amoebae to Atlantic salmon. Importantly, it is demonstrated that the gill lesion score system extensively used for evaluating AGD in Atlantic salmon is less suitable for lumpfish infected with P. perurans as the disease develops more slowly in lumpfish and because lumpfish may be non-symptomatic carriers. Statement of relevance 1) The last few years, lumpfish have been used as cleaner fish in order to delouse farmed Atlantic salmon. However, nothing is yet known about its susceptibility to the parasitic amoeba P. perurans. 2) Currently, nothing is known about amoebic gill disease (AGD) development in lumpfish and the possibility that amoeba may be transferred from lumpfish to salmon. 3) There is currently little information about the pathology (macroscopic and histological changes) in lumpfish. 4) We have found that the commonly used gill scoring system for salmon is not applicable for lumpfish, and we recommend that the lumpfish is screened, not scored before transfer to net-pens with salmon.
    Glucocorticoids Hijack Runx2 to Stimulate Wif1 for Suppression of Osteoblast Growth and Differentiation
    Eri Morimoto, Journal of Cellular Physiology - 2016
    Abstract
    Inhibition of Runx2 is one of many mechanisms that suppress bone formation in glucocorticoid (GC)-induced osteoporosis (GIO). We profiled mRNA expression in ST2/Rx2dox cells after treatment with doxycycline (dox; to induce Runx2) and/or the synthetic GC dexamethasone (dex). As expected, dex typically antagonized Runx2-driven transcription. Select genes, however, were synergistic stimulated and this was confirmed by RT-qPCR. Among the genes synergistically stimulated by GCs and Runx2 was Wnt inhibitory Factor 1 (Wif1), and Wif1 protein was readily detectable in medium conditioned by cultures co-treated with dox and dex, but neither alone. Cooperation between Runx2 and GCs in stimulating Wif1 was also observed in primary preosteoblast cultures. GCs strongly inhibited dox-driven alkaline phosphatase (ALP) activity in control ST2/Rx2dox cells, but not in cells in which Wif1 was silenced. Unlike its anti-mitogenic activity in committed osteoblasts, induction of Runx2 transiently increased the percentage of cells in S-phase and accelerated proliferation in the ST2 mesenchymal pluripotent cell culture model. Furthermore, like the inhibition of Runx2-driven ALP activity, dex antagonized the transient mitogenic effect of Runx2 in ST2/Rx2dox cultures, and this inhibition eased upon Wif1 silencing. Plausibly, homeostatic feedback loops that rely on Runx2 activation to compensate for bone loss in GIO are thwarted, exacerbating disease progression through stimulation of Wif1. J. Cell. Physiol. 9999: 1–9, 2016. © 2016 Wiley Periodicals, Inc.
    Epigenetic silencing of miR-124 prevents spermine oxidase regulation: implications for Helicobacter pylori-induced gastric cancer
    T. Murray-Stewart, Oncogene - 2016
    Abstract
    Chronic inflammation contributes to the development of various forms of cancer. The polyamine catabolic enzyme spermine oxidase (SMOX) is induced in chronic inflammatory conditions, including Helicobacter pylori-associated gastritis, where its production of hydrogen peroxide contributes to DNA damage and subsequent tumorigenesis. MicroRNA expression levels are also altered in inflammatory conditions; specifically, the tumor suppressor miR-124 becomes silenced by DNA methylation. We sought to determine if this repression of miR-124 is associated with elevated SMOX activity and concluded that miR-124 is indeed a negative regulator of SMOX. In gastric adenocarcinoma cells harboring highly methylated and silenced mir-124 gene loci, 5-azacytidine treatment allowed miR-124 re-expression and decreased SMOX expression. Overexpression of an exogenous miR-124-3p mimic repressed SMOX mRNA and protein expression as well as H2O2 production by >50% within 24 h. Reporter assays indicated that direct interaction of miR-124 with the 3′-untranslated region of SMOX mRNA contributes to this negative regulation. Importantly, overexpression of miR-124 before infection with H. pylori prevented the induction of SMOX believed to contribute to inflammation-associated tumorigenesis. Compelling human in vivo data from H. pylori-positive gastritis tissues indicated that the mir-124 gene loci are more heavily methylated in a Colombian population characterized by elevated SMOX expression and a high risk for gastric cancer. Furthermore, the degree of mir-124 methylation significantly correlated with SMOX expression throughout the population. These results indicate a protective role for miR-124 through the inhibition of SMOX-mediated DNA damage in the etiology of H. pylori-associated gastric cancer.
    Maleah dissertation Final (3-29-16).pdf
    Angelia Maleah Holland, A dissertation submitted to the Graduate Faculty of Auburn University - 2016
    Abstract
    We investigated the effects of a low carbohydrate ketogenic diet ( KD),versus other diets, on adipose tissue, liver and serum biomarkers in exercise trained versus sedentary rodents . METHODS: Male Sprague -Dawley rats (~9 -10 weeks of age) remained sedentary (SED) or exercised daily with resistance loaded running wheels (EX) over 6 weeks. Rats were provided isocaloric amounts of KD (20.2% protein, 10.3% carbohydrate, 69.5% fat), Western diet (WD; 15.2% protein, 42.7 carbohydrate, 42.0% fat), or Standard Chow(SC; 24.0% protein, 58.0% carbohydrate, 18.0% fat); n=8 10 in each diet group for SED and EX rats.Upon euthanasia, body and select adipose tissue masses were recorded and preserved for analyses, and liver and serum were also removed and preserved for analyses. RESULTS: Body mass and feed efficiency was greater in WD and SC fed vs. KD fed rats (p<0.001). Diet (WD>KD=SC, p<0.05)and activity (SED>EX, p<0.05) effects existed for raw and relative (body mass adjusted) omental adipose tissue (OMAT)masses. OMAT adipocyte diameters were lowest in KD fed rats p<0.01). Activity effects (EX>SED, p<0.05 to p<0.001)existed for OMAT acetyl Co A carboxylase (ACC),phospho/pan p65 (Ser536), phosphopan-AMPK (Thr172), phosphor /pan-HSL (Ser563),and uncoupling protein 1 (UCP1) protein expression patterns. Raw inguinal/subcutaneous (SQ)masses were greater in WD vs. KD fed rats (p<0.01), and diet (WD>KD, p<0.05) and activity (SED>EX, p<0.01) effects existed for raw subscapular brown adipose tissue (BAT)masses. Diet effects existed for liver iii triglycerides (WD>KD=SC, p<0.001), and diet (WD=SC>KD, p<0.05) and activity effects (SED>EX, p=0.01) existed for liver phosphopan p65.Diet (WD=SC>KD, p<0.001) and activity (SED>EX, p<0.001) effects existed for serum insulin,and diet (KD>SC, p<0.01) and activity (SED>EX, p<0.01) effects existed for serum β hydroxybutyrate (BHB). In all rats, serum insulin was positively associated with body mass (r=0.54, p<0.001), feed efficiency (r=0.57, p<0.001), relative OMAT mass (r=0.57, p<0.001), and relative SQ mass (r=0.31, p<0.05). CONCLUSIONS: While KD fed rats weighed less and had lower adipose tissue masses compared to WD and/or SC-fed rats, exercise did not enhance body or fat mass maintenance and/or further alter adipose tissue, liver or serum biomarkers in KD-fed rats. Notwithstanding, the association data suggest lower insulin with KD and/or exercise may facilitate attenuation of adipose tissue mass accretion in rodents
    Storage Temperature Alters the Expression of Differentiation-Related Genes in Cultured Oral Keratinocytes
    Tor Paaske Utheim, PLOS ONE - 2016
    Abstract
    Purpose Storage of cultured human oral keratinocytes (HOK) allows for transportation of cultured transplants to eye clinics worldwide. In a previous study, one-week storage of cultured HOK was found to be superior with regard to viability and morphology at 12°C compared to 4°C and 37°C. To understand more of how storage temperature affects cell phenotype, gene expression of HOK before and after storage at 4°C, 12°C, and 37°C was assessed. Materials and Methods Cultured HOK were stored in HEPES- and sodium bicarbonate-buffered Minimum Essential Medium at 4°C, 12°C, and 37°C for one week. Total RNA was isolated and the gene expression profile was determined using DNA microarrays and analyzed with Partek Genomics Suite software and Ingenuity Pathway Analysis. Differentially expressed genes (fold change > 1.5 and P < 0.05) were identified by one-way ANOVA. Key genes were validated using qPCR. Results Gene expression of cultures stored at 4°C and 12°C clustered close to the unstored control cultures. Cultures stored at 37°C displayed substantial change in gene expression compared to the other groups. In comparison with 12°C, 2,981 genes were differentially expressed at 37°C. In contrast, only 67 genes were differentially expressed between the unstored control and the cells stored at 12°C. The 12°C and 37°C culture groups differed most significantly with regard to the expression of differentiation markers. The Hedgehog signaling pathway was significantly downregulated at 37°C compared to 12°C. Conclusion HOK cultures stored at 37°C showed considerably larger changes in gene expression compared to unstored cells than cultured HOK stored at 4°C and 12°C. The changes observed at 37°C consisted of differentiation of the cells towards a squamous epithelium-specific phenotype. Storing cultured ocular surface transplants at 37°C is therefore not recommended. This is particularly interesting as 37°C is the standard incubation temperature used for cell culture.
    Dissociated sterol-based liver X receptor agonists as therapeutics for chronic inflammatory diseases
    Shan Yu, The FASEB Journal - 2016
    Abstract
    Liver X receptor (LXR), a nuclear hormone receptor, is an essential regulator of immune responses. Activation of LXR-mediated transcription by synthetic agonists, such as T0901317 and GW3965, attenuates progression of inflammatory disease in animal models. However, the adverse effects of these conventional LXR agonists in elevating liver lipids have impeded exploitation of this intriguing mechanism for chronic therapy. Here, we explore the ability of a series of sterol-based LXR agonists to alleviate inflammatory conditions in mice without hepatotoxicity. We show that oral treatment with sterol-based LXR agonists in mice significantly reduces dextran sulfate sodium colitis-induced body weight loss, which is accompanied by reduced expression of inflammatory markers in the large intestine. The anti-inflammatory property of these agonists is recapitulated in vitro in mouse lamina propria mononuclear cells, human colonic epithelial cells, and human peripheral blood mononuclear cells. In addition, treatment with LXR agonists dramatically suppresses inflammatory cytokine expression in a model of traumatic brain injury. Importantly, in both disease models, the sterol-based agonists do not affect the liver, and the conventional agonist T0901317 results in significant liver lipid accumulation and injury. Overall, these results provide evidence for the development of sterol-based LXR agonists as novel therapeutics for chronic inflammatory diseases.—Yu, S., Li, S., Henke, A., Muse, E. D., Cheng, B., Welzel, G., Chatterjee, A. K., Wang, D., Roland, J., Glass, C. K., Tremblay, M. Dissociated sterol-based liver X receptor agonists as therapeutics for chronic inflammatory diseases.
    GPR18 undergoes a high degree of constitutive trafficking but is unresponsive to N-Arachidonoyl Glycine
    David B. Finlay, PeerJ - 2016
    Abstract
    he orphan receptor GPR18 has become a research target following the discovery of a putative endogenous agonist, N-arachidonoyl glycine (NAGly). Chemical similarity between NAGly and the endocannabinoid anandamide suggested the hypothesis that GPR18 is a third cannabinoid receptor. GPR18-mediated cellular signalling through inhibition of cyclic adenosine monophosphate (cAMP) and phosphorylation of extracellular signal-regulated kinase (ERK), in addition to physiological consequences such as regulation of cellular migration and proliferation/apoptosis have been described in response to both NAGly and anandamide. However, discordant findings have also been reported. Here we sought to describe the functional consequences of GPR18 activation in heterologously-expressing HEK cells. GPR18 expression was predominantly intracellular in stably transfected cell lines, but moderate cell surface expression could be achieved in transiently transfected cells which also had higher overall expression. Assays were employed to characterise the ability of NAGly or anandamide to inhibit cAMP or induce ERK phosphorylation through GPR18, or induce receptor trafficking. Positive control experiments, which utilised cells expressing hCB1 receptors (hCB1R), were performed to validate assay design and performance. While these functional pathways in GPR18-expressing cells were not modified on treatment with a panel of putative GPR18 ligands, a constitutive phenotype was discovered for this receptor. Our data reveal that GPR18 undergoes rapid constitutive receptor membrane trafficking—several-fold faster than hCB1R, a highly constitutively active receptor. To enhance the likelihood of detecting agonist-mediated receptor signalling responses, we increased GPR18 protein expression (by tagging with a preprolactin signal sequence) and generated a putative constitutively inactive receptor by mutating the hGPR18 gene at amino acid site 108 (alanine to asparagine). This A108N mutant did cause an increase in surface receptor expression (which may argue for reduced constitutive activity), but no ligand-mediated effects were detected. Two glioblastoma multiforme cell lines (which endogenously express GPR18) were assayed for NAGly-induced pERK phosphorylation, with negative results. Despite a lack of ligand-mediated responses in all assays, the constitutive trafficking of GPR18 remains an interesting facet of receptor function and will have consequences for understanding the role of GPR18 in physiology.
    Retinoic acid receptor signaling preserves tendon stem cell characteristics and prevents spontaneous differentiation in vitro | Stem Cell Research & Therapy | Full Text
    Stuart Webb, Stem Cell Research & Therapy - 2016
    Abstract
    Previous studies have reported that adult mesenchymal stem cells (MSCs) tend to gradually lose their stem cell characteristics in vitro when placed outside their niche environment. They subsequently undergo spontaneous differentiation towards mesenchymal lineages after only a few passages. We observed a similar phenomenon with adult tendon stem cells (TSCs) where expression of key tendon genes such as Scleraxis (Scx), are being repressed with time in culture. We hypothesized that an environment able to restore or maintain Scleraxis expression could be of therapeutic interest for in vitro use and tendon cell-based therapies.
    Phenotypical and biochemical characterisation of resistance for parasitic weed (Orobanche foetida Poir.) in radiation-mutagenised mutants of chickpea
    Ines Brahmi, Pest Management Science - 2016
    Abstract
    BACKGROUND Some radiation-mutagenised chickpea mutants potentially resistant to the broomrape, Orobanche foetida Poir., were selected through field trials. The objectives of this work were to confirm resistance under artificial infestation, in pots and mini-rhizotron systems, and to determine the developmental stages of broomrape affected by resistance and the relevant resistance mechanisms induced by radiation mutagenesis. RESULTS Among 30 mutants tested for resistance to O. foetida, five shared strong resistance in both pot experiments and mini-rhizotron systems. Resistance was not complete, but the few individuals that escaped resistance displayed high disorders of shoot development. Results demonstrated a 2–3-fold decrease in stimulatory activity of root exudates towards broomrape seed germination in resistant mutants in comparison with non-irradiated control plants and susceptible mutants. Resistance was associated with an induction of broomrape necrosis early during infection. When infested, most of the resistant mutants shared enhanced levels of soluble phenolic contents, phenylalanine ammonia lyase activity, guaiacol peroxidase activity and polyphenol oxidase activity, in addition to glutathione and notably ascorbate peroxidase gene expression in roots. CONCLUSION Results confirmed enhanced resistance in chickpea radiation-mutagenised mutants, and demonstrated that resistance is based on alteration of root exudation, presumed cell-wall reinforcement and change in root oxidative status in response to infection. © 2016 Society of Chemical Industry
    A Closed Chondromimetic Environment within Magnetic-Responsive Liquified Capsules Encapsulating Stem Cells and Collagen II/TGF-β3 Microparticles
    Clara R. Correia, Advanced Healthcare Materials - 2016
    Abstract
    TGF-β3 is enzymatically immobilized by transglutaminase-2 action to poly(l-lactic acid) microparticles coated with collagen II. Microparticles are then encapsulated with stem cells inside liquified spherical compartments enfolded with a permselective shell through layer-by-layer adsorption. Magnetic nanoparticles are electrostatically bound to the multilayered shell, conferring magnetic-response ability. The goal of this study is to engineer a closed environment inside which encapsulated stem cells would undergo a self-regulated chondrogenesis. To test this hypothesis, capsules are cultured in chondrogenic differentiation medium without TGF-β3. Their biological outcome is compared with capsules encapsulating microparticles without TGF-β3 immobilization and cultured in normal chondrogenic differentiation medium containing soluble TGF-β3. Glycosaminoglycans quantification demosntrates that similar chondrogenesis levels are achieved. Moreover, collagen fibrils resembling the native extracellular matrix of cartilage can be observed. Importantly, the genetic evaluation of characteristic cartilage markers confirms the successful chondrogenesis, while hypertrophic markers are downregulated. In summary, the engineered capsules are able to provide a suitable and stable chondrogenesis environment for stem cells without the need of TGF-β3 supplementation. This kind of self-regulated capsules with softness, robustness, and magnetic responsive characteristics is expected to provide injectability and in situ fixation, which is of great advantage for minimal invasive strategies to regenerate cartilage.
    Gluten-free diet increases beta-cell volume and improves glucose tolerance in an animal model of type 2 diabetes
    Karsten Buschard, Diabetes/Metabolism Research and Reviews - 2016
    Abstract
    Background Gluten-free (GF) diet alleviates type 1 diabetes in animal models and possibly in humans. We recently showed that fatty acid-induced insulin secretion is enhanced by enzymatically digested gluten (gliadin) stimulation in INS-1E insulinoma cells. We therefore hypothesized that GF diet would induce beta-cell rest and ameliorate type 2 diabetes. Methods C57BL/6JBomTac (B6) mice were fed a high-fat (HF), gluten-free high-fat (GF–HF), standard (STD) or gluten-free (GF) diet for 42 weeks. Results Short-term (6–24 weeks) GF–HF versus HF feeding impaired glucose tolerance and increased fasting glucose. Long-term (36–42 weeks) GF–HF versus HF feeding improved glucose tolerance and decreased fasting leptin. Mice fed a GF–HF versus HF diet for 42 weeks showed higher volumes of beta cells, islets and pancreas. The beta-cell volume correlated with the islet- and pancreas volume as well as body weight. GF–HF versus HF diet did not influence toll-like receptor 4 (Tlr4), interleukin 1 (IL-1), interleukin 6 (IL-6) or tumour necrosis factor-alpha (TNF-alpha) mRNA expression in intestine. STD versus GF feeding did not affect any parameter studied. Conclusions Long-term feeding with GF–HF versus HF increases beta-cell volume and improves glucose tolerance in B6 mice. The mechanism may include beta-cell rest, but is unlikely to include TLR4 and proinflammatory cytokines in the intestine. Beta-cell volume correlates with pancreas volume and body weight, indicating that insulin secretion capacity controls pancreas volume. Thus, long-term GF diets may be beneficial for obese type 2 diabetes patients and trials should be performed. Copyright © 2016 John Wiley & Sons, Ltd.
    Impact of KRAS, BRAF, PIK3CA, TP53 status and intraindividual mutation heterogeneity on outcome after liver resection for colorectal cancer metastases
    Inger Marie Løes, International Journal of Cancer - 2016
    Abstract
    We determined prognostic impact of KRAS, BRAF, PIK3CA and TP53 mutation status and mutation heterogeneity among 164 colorectal cancer (CRC) patients undergoing liver resections for metastatic disease. Mutation status was determined by Sanger sequencing of a total of 422 metastatic deposits. In univariate analysis, KRAS (33.5%), BRAF (6.1%) and PIK3CA (13.4%) mutations each predicted reduced median time to relapse (TTR) (7 vs. 22, 3 vs. 16 and 4 vs. 17 months; p < 0.001, 0.002 and 0.023, respectively). KRAS and BRAF mutations also predicted a reduced median disease-specific survival (DSS) (29 vs. 51 and 16 vs. 49 months; p <0.001 and 0.008, respectively). No effect of TP53 (60.4%) mutation status was observed. Postoperative, but not preoperative chemotherapy improved both TTR and DSS (p < 0.001 for both) with no interaction with gene mutation status. Among 94 patients harboring two or more metastatic deposits, 13 revealed mutation heterogeneity across metastatic deposits for at least one gene. Mutation heterogeneity predicted reduced median DSS compared to homogeneous mutations (18 vs. 37 months; p = 0.011 for all genes; 16 vs. 26 months; p < 0.001 analyzing BRAF or KRAS mutations separately). In multivariate analyses, KRAS or BRAF mutations consistently predicted poor TRR and DSS. Mutation heterogeneity robustly predicted DSS but not TTR, while postoperative chemotherapy improved both TTR and DSS. Our findings indicate that BRAF and KRAS mutations as well as mutation heterogeneity predict poor outcome in CRC patients subsequent to liver resections and might help guide treatment decisions.
    MicroRNA Expression during Bovine Oocyte Maturation and Fertilization
    Graham C. Gilchrist, International Journal of Molecular Sciences - 2016
    Abstract
    Successful fertilization and subsequent embryo development rely on complex molecular processes starting with the development of oocyte competence through maturation. MicroRNAs (miRNAs) are small non-coding RNA molecules that function as gene regulators in many biological systems, including the oocyte and embryo. In order to further explore the roles of miRNAs in oocyte maturation, we employed small RNA sequencing as a screening tool to identify and characterize miRNA populations present in pools of bovine germinal vesicle (GV) oocytes, metaphase II (MII) oocytes, and presumptive zygotes (PZ). Each stage contained a defined miRNA population, some of which showed stable expression while others showed progressive changes between stages that were subsequently confirmed by quantitative reverse transcription polymerase chain reaction (RT-PCR). Bta-miR-155, bta-miR-222, bta-miR-21, bta-let-7d, bta-let-7i, and bta-miR-190a were among the statistically significant differentially expressed miRNAs (p < 0.05). To determine whether changes in specific primary miRNA (pri-miRNA) transcripts were responsible for the observed miRNA changes, we evaluated pri-miR-155, -222 and let-7d expression. Pri-miR-155 and -222 were not detected in GV oocytes but pri-miR-155 was present in MII oocytes, indicating transcription during maturation. In contrast, levels of pri-let-7d decreased during maturation, suggesting that the observed increase in let-7d expression was likely due to processing of the primary transcript. This study demonstrates that both dynamic and stable populations of miRNAs are present in bovine oocytes and zygotes and extend previous studies supporting the importance of the small RNA landscape in the maturing bovine oocyte and early embryo.
    Glucagon-like peptide-1 and vitamin D: anti-inflammatory response in diabetic kidney disease in db/db mice and in cultured endothelial cells
    Yael Einbinder, Diabetes/Metabolism Research and Reviews - 2016
    Abstract
    Background Glucagon-like peptide-1 (GLP-1) is a gut incretin hormone that stimulates insulin secretion and may affect the inflammatory pathways involved in diabetes mellitus. Calcitriol, an active form of vitamin D, plays an important role in renal, endothelial and cardiovascular protection. We evaluated the anti-inflammatory and histologic effects of a GLP-1 analogue (liraglutide) and of calcitriol in a db/db mouse diabetes model and in endothelial cells exposed to a diabetes-like environment. Methods Diabetic db/db mice were treated with liraglutide and calcitriol for 14 weeks, after which the kidneys were perfused and removed for mRNA and protein analysis and histology. Endothelial cells were stimulated with advanced glycation end products (AGEs), glucose, liraglutide and calcitriol. Total RNA and protein were extracted and analysed for the expression of selected inflammatory markers. Results Typical histological changes, glomerular enlargement and mesangial expansion were seen in db/db mice compared with control mice. Glomerular hypertrophy was ameliorated with liraglutide, compared with db/db controls. Liraglutide up-regulated endothelial nitric oxide synthase protein expression compared with the db/db control group and down-regulated p65 protein expression. Calcitriol did not further improve the beneficial effect observed on protein expression. In endothelial cells, liraglutide treatment exhibited a dose-dependent ability to prevent an inflammatory response in the selected markers: thioredoxin-interacting protein, p65, IL6 and IL8. In most gene and protein expressions, addition of calcitriol did not enhance the effect of liraglutide. Conclusions The GLP-1 analogue liraglutide prevented the inflammatory response observed in endothelial cells exposed to a diabetes-like environment and in db/db mice at the level of protein expression and significantly ameliorated the glomerular hypertrophy seen in the diabetic control group. Copyright © 2016 John Wiley & Sons, Ltd.
    Acidified nitrite inhibits proliferation of Listeria monocytogenes — Transcriptional analysis of a preservation method
    Stefanie Müller-Herbst, International Journal of Food Microbiology - 2016
    Abstract
    Sodium nitrite (NaNO2) is added as a preservative during raw meat processing such as raw sausage production to inhibit growth of pathogenic bacteria. In the present study it was shown in challenge assays that the addition of sodium nitrite indeed inhibited growth and survival of Listeria monocytogenes in short-ripened spreadable raw sausages. Furthermore, in vitro growth analyses were performed, which took into account combinations of various parameters of the raw sausage ripening process like temperature, oxygen availability, pH, NaCl concentration, and absence or presence of NaNO2. Data based on 300 growth conditions revealed that the inhibitory effect of nitrite was most prominent in combination with acidification, a combination that is also achieved during short-ripened spreadable raw sausage production. At pH 6.0 and below, L. monocytogenes was unable to replicate in the presence of 200 mg/l NaNO2. During the adaptation of L. monocytogenes to acidified nitrite stress (pH 6.0, 200 mg/l NaNO2) in comparison to acid exposure only (pH 6.0, 0 mg/l NaNO2), a massive transcriptional adaptation was observed using microarray analyses. In total, 202 genes were up-regulated and 204 genes were down-regulated. In accordance with growth inhibition, a down-regulation of genes encoding for proteins which are involved in central cellular processes, like cell wall/membrane/envelope biogenesis, translation and ribosomal structure and biogenesis, transcription, and replication, recombination and repair, was observed. Among the up-regulated genes the most prominent group belonged to poorly characterized genes. A considerable fraction of the up-regulated genes has been shown previously to be up-regulated intracellularly in macrophages, after exposure to acid shock or to be part of the SigB regulon. These data indicate that the adaptation to acidified nitrite partly overlaps with the adaptation to stress conditions being present during host colonization.
    Daily rhythms of digestive enzyme activity and gene expression in gilthead seabream (Sparus aurata) during ontogeny
    José Antonio Mata-Sotresa, Elsevier - 2016
    Abstract
    In order to identify daily changes in digestive physiology in developing gilthead seabream larvae, the enzyme activity (trypsin, lipases and α-amylase) and gene expression (trypsinogen-try, chymotrypsinogen-ctrb, bile salt-activated lipase-cel1b, phospholipase A2-pla2 and α-amylase-amy2a) were measured during a 24 h cycle in larvae reared under a 12 h light/12 h dark photoperiod. Larvae were sampled at 10, 18, 30 and 60 days post-hatch. In each sampling day, larvae were sampled every 3 h during a complete 24 h cycle. The enzyme activity and gene expression exhibited a marked dependent behavior to the light/darkness cycle in all tested ages. The patterns of activity and expression of all tested enzymes were compared to the feeding pattern found in the same larvae, which showed a rhythmic feeding pattern with a strong light synchronization. In the four tested ages, the activities of trypsin, and to a lesser extent lipases and amylase, were related to feeding activity. Molecular expression of the pancreatic enzymes tended to increase during the night, probably as an anticipation of the forthcoming ingestion of food that will take place during the next light period. It follows that the enzymatic activities are being regulated at translational and/or post-translational level. The potential variability of enzyme secretion along the whole day is an important factor to take into account in future studies. A particularly striking consequence of the present results is the reliability of studies based in only one daily sample taken at the same hour of the day, as those focused to assess ontogeny of digestive enzymes.
    Transforming Growth Factor-β Is an Upstream Regulator of Mammalian Target of Rapamycin Complex 2–Dependent Bladder Cancer Cell Migration and Invasion
    Sounak Gupta, The American Journal of Pathology - 2016
    Abstract
    Our prior work identified the mammalian target of rapamycin complex 2 (mTORC2) as a key regulator of bladder cancer cell migration and invasion, although upstream growth factor mediators of this pathway in bladder cancer have not been well delineated. We tested whether transforming growth factor (TGF)-β, which can function as a promotility factor in bladder cancer cells, could regulate mTORC2-dependent bladder cancer cell motility and invasion. In human bladder cancers, the highest levels of phosphorylated SMAD2, a TGF-β signaling intermediate, were present in high-grade invasive bladder cancers and associated with more frequent recurrence and decreased disease-specific survival. Increased expression of TGF-β isoforms, receptors, and signaling components was detected in invasive high-grade bladder cancer cells that expressed Vimentin and lacked E-cadherin. Application of TGF-β induced phosphorylation of the Ser473 residue of AKT, a selective target of mTORC2, in a SMAD2- and SMAD4-independent manner and increased bladder cancer cell migration in a modified scratch wound assay and invasion through Matrigel. Inhibition of TGF-β receptor I using SB431542 ablated TGF-β–induced migration and invasion. A similar effect was seen when Rictor, a key mTORC2 component, was selectively silenced. Our results suggest that TGF-β can induce bladder cancer cell invasion via mTORC2 signaling, which may be applicable in most bladder cancers.
    Fluid flow and Smad2 affects the response of vascular endothelial cells
    Linda Selene Tamez, UNIVERSITY OF CALGARY Thesis - 2016
    Abstract
    Human aortic endothelial cells (HAECs) have been observed to respond to fluid flow and shear stress by activating different signaling molecules both in vitro and in vivo. An important example of these flow activated molecules is Smad2. Smad2 is a signaling molecule and transcription factor that has shown to be indispensable for the maintenance of vascular integrity. The aim of this study was to understand the effect of shear stress and Smad2 knockdown on endothelial gene expression.HAEC were transfected with Smad2 siRNA, and exposed to steady laminar shear stress (10 dyne/cm 2). Our results showed that Smad2 siRNA and shear stress significantly up regulated genes involved in atherosclerosis, heart dysfunction, and angiogenes is. Furthermore, Smad2 siRNA had a negative impact on athero-protective genes under static conditions. This is the first reported Smad2 siRNA gene expression profile of endothelial cells. Our findings suggest that Smad2 may a have a protective role against cardiovascular diseases.
    Expression Atlas of the Deubiquitinating Enzymes in the Adult Mouse Retina, Their Evolutionary Diversification and Phenotypic Roles - 657534.pdf
    Mariona Esquerdo, PLOS One - 2016
    Abstract
    Ubiquitination is a relevant cell regulatory mechanism to determine protein fate and function. Most data has focused on the role of ubiquitin as a tag molecule to target substrates to proteasome degradation, and on its impact in the control of cell cycle, protein homeostasis and cancer. Only recently, systematic assays have pointed to the relevance of the ubiquitin pathway in the development and differentiation of tissues and organs, and its implication in hereditary diseases. Moreover, although the activity and composition of ubiquitin ligases has been largely addressed, the role of the deubiquitinating enzymes (DUBs) in specific tissues, such as the retina, remains mainly unknown. In this work, we undertook a systematic analysis of the transcriptional levels of DUB genes in the adult mouse retina by RT-qPCR and analyzed the expression pattern by in situhybridization and fluorescent immunohistochemistry, thus providing a unique spatial reference map of retinal DUB expression. We also performed a systematic phylogenetic analysis to understand the origin and the presence/absence of DUB genes in the genomes of diverse animal taxa that represent most of the known animal diversity. The expression landscape obtained supports the potential sub-functionalization of paralogs in those families that expanded in vertebrates. Overall, our results constitute a reference framework for further characterization of the DUB roles in the retina and suggest new candidates for inherited retinal disorders.
    PLOS ONE: Pharmacological and Genetic Modulation of REV-ERB Activity and Expression Affects Orexigenic Gene Expression
    Ariadna Amador, Plos One - 2016
    Abstract
    The nuclear receptors REV-ERBα and REV-ERBβ are transcription factors that play pivotal roles in the regulation of the circadian rhythm and various metabolic processes. The circadian rhythm is an endogenous mechanism, which generates entrainable biological changes that follow a 24-hour period. It regulates a number of physiological processes, including sleep/wakeful cycles and feeding behaviors. We recently demonstrated that REV-ERB-specific small molecules affect sleep and anxiety. The orexinergic system also plays a significant role in mammalian physiology and behavior, including the regulation of sleep and food intake. Importantly, orexin genes are expressed in a circadian manner. Given these overlaps in function and circadian expression, we wanted to determine whether the REV-ERBs might regulate orexin. We found that acute in vivo modulation of REV-ERB activity, with the REV-ERB-specific synthetic ligand SR9009, affects the circadian expression of orexinergic genes in mice. Long term dosing with SR9009 also suppresses orexinergic gene expression in mice. Finally, REV-ERBβ-deficient mice present with increased orexinergic transcripts. These data suggest that the REV-ERBs may be involved in the repression of orexinergic gene expression.
    A simple, accurate and universal method for quantification of PCR
    Nicky Boulter, BMC Biotechnology - 2016
    Abstract
    Research into gene expression enables scientists to decipher the complex regulatory networks that control fundamental biological processes. Quantitative real-time PCR (qPCR) is a powerful and ubiquitous method for interrogation of gene expression. Accurate quantification is essential for correct interpretation of qPCR data. However, conventional relative and absolute quantification methodologies often give erroneous results or are laborious to perform.
    Molecular endocrine changes of Gh/Igf1 axis in gilthead sea bream (Sparus aurata L.) exposed to different environmental salinities during larvae to post-larvae stages
    Manuel Yúfera, Fish Physiology and Biochemistry - 2016
    Abstract
    The influence of acclimation of the euryhaline gilthead sea bream (Sparus aurata) larvae/post-larvae to brackish water on growth, energetic contents, and mRNA levels of selected hormones and growth-regulating hypothalamic neurohormones was assessed. Specimens from 49 days post-hatching were acclimated during 28 days to two different environmental salinities: 38 and 20 psu (as brackish water). Both groups were then transferred to 38 psu and acclimated for an additional week. Early juveniles were sampled after 28 days of acclimation to both salinities and one week after transfer to 38 psu. Pituitary adenylate cyclase-activating peptide (adcyap1; pacap), somatostatin-I (sst1), growth hormone (gh1), insulin-like growth factor-I (igf1), and prolactin (prl) mRNA expression were all studied by QPCR. Post-larvae acclimated to 20 psu showed better growth performance and body energetic content than post-larvae maintained at 38 psu. prl, adcyap1, and igf1 mRNA expression levels increased in 20-psu-acclimated post-larvae but decreased upon transfer to 38 psu. GH1 expression did not show significant changes under both experimental conditions. Our results suggested an enhanced general performance for post-larvae in brackish water, supported by the actions of adcyap1, igf1, and prl.
    Requirement for Microglia for the Maintenance of Synaptic Function and Integrity in the Mature Retina
    Xu Wang, The Journal of Neuroscience - 2016
    Abstract
    Microglia, the principal resident immune cell of the CNS, exert significant influence on neurons during development and in pathological situations. However, if and how microglia contribute to normal neuronal function in the mature uninjured CNS is not well understood. We used the model of the adult mouse retina, a part of the CNS amenable to structural and functional analysis, to investigate the constitutive role of microglia by depleting microglia from the retina in a sustained manner using genetic methods. We discovered that microglia are not acutely required for the maintenance of adult retinal architecture, the survival of retinal neurons, or the laminar organization of their dendritic and axonal compartments. However, sustained microglial depletion results in the degeneration of photoreceptor synapses in the outer plexiform layer, leading to a progressive functional deterioration in retinal light responses. Our results demonstrate that microglia are constitutively required for the maintenance of synaptic structure in the adult retina and for synaptic transmission underlying normal visual function. Our findings on constitutive microglial function are relevant in understanding microglial contributions to pathology and in the consideration of therapeutic interventions that reduce or perturb constitutive microglial function. SIGNIFICANCE STATEMENT Microglia, the principal resident immune cell population in the CNS, has been implicated in diseases in the brain and retina. However, how they contribute to the everyday function of the CNS is unclear. Using the model of the adult mouse retina, we examined the constitutive role of microglia by depleting microglia from the retina. We found that in the absence of microglia, retinal neurons did not undergo overt cell death or become structurally disorganized in their processes. However, connections between neurons called synapses begin to break down, leading to a decreased ability of the retina to transmit light responses. Our results indicate that retinal microglia contribute constitutively to the maintenance of synapses underlying healthy vision.
    Expression Atlas of the Deubiquitinating Enzymes in the Adult Mouse Retina, Their Evolutionary Diversification and Phenotypic Roles
    Mariona Esquerdo, PLOS ONE - 2016
    Abstract
    Ubiquitination is a relevant cell regulatory mechanism to determine protein fate and function. Most data has focused on the role of ubiquitin as a tag molecule to target substrates to proteasome degradation, and on its impact in the control of cell cycle, protein homeostasis and cancer. Only recently, systematic assays have pointed to the relevance of the ubiquitin pathway in the development and differentiation of tissues and organs, and its implication in hereditary diseases. Moreover, although the activity and composition of ubiquitin ligases has been largely addressed, the role of the deubiquitinating enzymes (DUBs) in specific tissues, such as the retina, remains mainly unknown. In this work, we undertook a systematic analysis of the transcriptional levels of DUB genes in the adult mouse retina by RT-qPCR and analyzed the expression pattern by in situ hybridization and fluorescent immunohistochemistry, thus providing a unique spatial reference map of retinal DUB expression. We also performed a systematic phylogenetic analysis to understand the origin and the presence/absence of DUB genes in the genomes of diverse animal taxa that represent most of the known animal diversity. The expression landscape obtained supports the potential subfunctionalization of paralogs in those families that expanded in vertebrates. Overall, our results constitute a reference framework for further characterization of the DUB roles in the retina and suggest new candidates for inherited retinal disorders.
    Inhibition of adhesion, migration and of α5β1 integrin in the HCT-116 colorectal cancer cells treated with the ruthenium drug NAMI-A
    Chiara Pelillo, Journal of Inorganic Biochemistry - 2016
    Abstract
    NAMI-A, imidazolium trans-imidazoledimethylsulfoxidetetrachlororuthenate, is a ruthenium-based drug characterised by the selective activity against tumour metastases. Previously we have shown the influence of the hepatic microenvironment to direct the arrest of the metastatic cells of colorectal cancer. Here we used the experimental model of HCT-116 colorectal cancer cells in vitro to explore whether the interference with α5β1 integrin may mechanistically explain the anti-metastatic effect of NAMI-A. NAMI-A inhibits two important steps of the tumour metastatic progression of colorectal cancer, i.e. the adhesion and migration of the tumour cells on the extracellular matrix proteins. The fibronectin receptor α5β1 integrin is likely involved in the anti-adhesive effects of NAMI-A on the HCT-116 colorectal cancer cells during their interaction with the extracellular matrix. Mechanistically, NAMI-A decreases the α5β1 integrin expression, and reduces FAK (Focal Adhesion Kinase) auto-phosphorylation on Tyr397, an important signalling event, involved in α5β1 integrin activation. These effects were validated by siRNA-induced knock down of the α5 integrin subunit and/or by the use of specific blocking mAbs against the active site of the integrin. Our results demonstrate the relevance of α5β1 integrin for colorectal cancer. We also show that the anti-metastatic effect of NAMI-A depends on the modulation of this integrin. Thus, our data on NAMI-A support the new concept that metal-based drugs can inhibit tumour metastases through targeting of integrins and of other proteins which mediate tumour progression-related cell functions such as adhesion and migration.
    Nicotinic Acetylcholine Receptors Modulate Bone Marrow-Derived Pro-Inflammatory Monocyte Production and Survival
    Stéphanie St-Pierre, PLOS ONE - 2016
    Abstract
    It is increasingly clear that nicotinic acetylcholine receptors (nAChRs) are involved in immune regulation, and that their activation can protect against inflammatory diseases. Previous data have shown that nicotine diminishes the numbers of peripheral monocytes and macrophages, especially those of the pro-inflammatory phenotype. The goal of the present study was to determine if nicotine modulates the production of bone marrow -derived monocytes/macrophages. In this study, we first found that murine bone marrow cells express multiple nAChR subunits, and that the α7 and α9 nAChRs most predominant subtypes found in immune cells and their precursors. Using primary cultures of murine bone marrow cells, we then determined the effect of nicotine on monocyte colony-stimulating factor and interferon gamma (IFNγ)-induced monocyte production. We found that nicotine lowered the overall number of monocytes, and more specifically, inhibited the IFNγ-induced increase in pro-inflammatory monocytes by reducing cell proliferation and viability. These data suggested that nicotine diminishes the ratio of pro-inflammatory versus anti-inflammatory monocyte produced in the bone marrow. We thus confirmed this hypothesis by measuring cytokine expression, where we found that nicotine inhibited the production of the pro-inflammatory cytokines TNFα, IL-1β and IL-12, while stimulating the secretion of IL-10, an anti-inflammatory cytokine. Finally, nicotine also reduced the number of pro-inflammatory monocytes in the bone marrow of LPS-challenged mice. Overall, our data demonstrate that both α7 and α9 nAChRs are involved in the regulation of pro-inflammatory M1 monocyte numbers.
    Amplified in Breast Cancer Regulates Transcription and Translation in Breast Cancer Cells
    Aleksandra M. Ochnik, Neoplasia - 2016
    Abstract
    Background Control of mRNA translation is fundamentally altered in cancer. Insulin-like growth factor-I (IGF-I) signaling regulates key translation mediators to modulate protein synthesis (e.g. eIF4E, 4E-BP1, mTOR, and S6K1). Importantly the Amplified in Breast Cancer (AIB1) oncogene regulates transcription and is also a downstream mediator of IGF-I signaling. Materials and Methods To determine if AIB1 also affects mRNA translation, we conducted gain and loss of AIB1 function experiments in estrogen receptor alpha (ERα)+ (MCF-7L) and ERα- (MDA-MB-231, MDA-MB-435 and LCC6) breast cancer cells. Results AIB1 positively regulated IGF-I-induced mRNA translation in both ERα+ and ERα- cells. Formation of the eIF4E-4E-BP1 translational complex was altered in the AIB1 ERα+ and ERα- knockdown cells, leading to a reduction in the eIF4E/4E-BP1 and eIF4G/4E-BP1 ratios. In basal and IGF-I stimulated MCF-7 and LCC6 cells, knockdown of AIB1 decreased the integrity of the cap-binding complex, reduced global IGF-I stimulated polyribosomal mRNA recruitment with a concomitant decrease in ten of the thirteen genes tested in polysome-bound mRNAs mapping to proliferation, cell cycle, survival, transcription, translation and ribosome biogenesis ontologies. Specifically, knockdown of AIB1 decreased ribosome-bound mRNA and steady-state protein levels of the transcription factors ERα and E2F1 in addition to reduced ribosome-bound mRNA of the ribosome biogenesis factor BYSL in a cell-line specific manner to regulate mRNA translation. Conclusion The oncogenic transcription factor AIB1 has a novel role in the regulation of polyribosome recruitment and formation of the translational complex. Combinatorial therapies targeting IGF signaling and mRNA translation in AIB1 expressing breast cancers may have clinical benefit and warrants further investigation.
    Seasonal variation in nifH abundance and expression of cyanobacterial communities associated with boreal feather mosses
    Denis Warshan, The ISME Journal - 2016
    Abstract
    Dinitrogen (N2)-fixation by cyanobacteria living in symbiosis with pleurocarpous feather mosses (for example, Pleurozium schreberi and Hylocomium splendens) represents the main pathway of biological N input into N-depleted boreal forests. Little is known about the role of the cyanobacterial community in contributing to the observed temporal variability of N2-fixation. Using specific nifH primers targeting four major cyanobacterial clusters and quantitative PCR, we investigated how community composition, abundance and nifH expression varied by moss species and over the growing seasons. We evaluated N2-fixation rates across nine forest sites in June and September and explored the abundance and nifH expression of individual cyanobacterial clusters when N2-fixation is highest. Our results showed temporal and host-dependent variations of cyanobacterial community composition, nifH gene abundance and expression. N2-fixation was higher in September than June for both moss species, explained by higher nifH gene expression of individual clusters rather than higher nifH gene abundance or differences in cyanobacterial community composition. In most cases, ‘Stigonema cluster’ made up less than 29% of the total cyanobacterial community, but accounted for the majority of nifH gene expression (82–94% of total nifH expression), irrespective of sampling date or moss species. Stepwise multiple regressions showed temporal variations in N2-fixation being greatly explained by variations in nifH expression of the ‘Stigonema cluster’. These results suggest that Stigonema is potentially the most influential N2-fixer in symbiosis with boreal forest feather mosses.
    USE OF CD36 TO IDENTIFY CANCER SUBJECTS FOR TREATMENT - CHILDREN'S MEDICAL CENTER CORPORATION
    Watnick, Randolph S, US Patent - 2016
    Abstract
    A method for evaluating a subject's responsiveness to treatment with a Psap peptide, the method comprising: determining a level of CD36 in a sample obtained from a subject having cancer, wherein an elevated level of CD36 in the sample compared to a control level indicates that the subject is responsive to or likely to be responsive to treatment with a Psap peptide. 2. The method of claim 1, wherein the level of CD36 in the sample is determined by performing an assay. 3. The method of claim 1, wherein the method further comprises: identifying the subject with an elevated level of CD36 in the sample compared to the control level as responsive to or likely to be responsive to treatment with a Psap peptide. 4. The method of claim 3, wherein the method further comprises: administering to the subject identified as responsive to or likely to be responsive to treatment with a Psap peptide an effective amount of a Psap peptide to treat the cancer. 5. A method for treating a subject with cancer, the method comprising: administering to a subject with cancer characterized by an elevated level of CD36 in a sample compared to a control level an effective amount of a Psap peptide to treat the cancer. 6. A method for treating a subject with cancer, the method comprising: (a) selecting a subject with cancer on the basis that the subject is known to have an elevated level of CD36 in a sample compared to a control level; and (b) administering an effective amount of a Psap peptide to the subject because the subject has an elevated level of CD36 in the sample compared to the control level. 7. The method of claim 1, wherein the control level is a level of CD36 from a non-cancerous cell or tissue obtained from the subject having the cancer. 8. The method of claim 1, wherein the control level is a level of CD36 in a cell or tissue obtained from a healthy subject or a population of healthy subjects. 9. The method of claim 1, wherein the control level is a predetermined level. 10. The method of claim 1, wherein the level of CD36 is a CD36 protein level. 11. The method of claim 1, wherein the cancer is prostate cancer, breast cancer, ovarian cancer, lung cancer, leukemia, pancreatic cancer, glioblastoma multiforme, astrocytoma, or melanoma. 12. The method of claim 1, wherein the Psap peptide comprises the amino acid sequence CDWLPK (SEQ ID NO: 1), DWLPK (SEQ ID NO: 2), or DWLP (SEQ ID NO: 3), or an amino acid substitution variant thereof, wherein the amino acid substitution is: a) Tyrosine (Y) for Tryptophan (W); b) an amino acid substitution for Leucine (L) selected from Valine (V), Alanine (A) or Glycine (G), or a non-canonical amino acid of similar size, or a derivative thereof; c) Arginine (R) for Lysine (K); d) a D-isomer of Aspartic Acid (D) for an L-isomer of Aspartic Acid (D) and/or a D-isomer of Leucine (L) for a L-isomer of Leucine (L); e) a D-isomer of Tryptophan (W) for an L-isomer of Tryptophan (W) and/or a D-isomer of Proline (P) for an L-isomer of Proline (P); or combinations thereof. 13. The method of claim 12, wherein the Psap peptide is 50 amino acids or fewer in length. 14. The method of claim 13, wherein the Psap peptide is 30 amino acids or fewer in length. 15. The method of claim 14, wherein the Psap peptide is 15 amino acids or fewer in length. 16. The method of claim 15, wherein the Psap peptide is 6 amino acids or fewer in length. 17. The method of claim 12, wherein the Psap peptide is a cyclic peptide. 18. The method of claim 12, wherein the non-canonical amino acid of similar size is methylvaline, methylleucine, or sarcosine. 19. 19-33. (canceled) 34. The method of claim 5, wherein the control level is a level of CD36 from a non-cancerous cell or tissue obtained from the subject having the cancer. 35. The method of claim 6, wherein the control level is a level of CD36 from a non-cancerous cell or tissue obtained from the subject having the cancer.
    Disruption of the sigS gene attenuates the local innate immune response to Staphylococcus aureus in a mouse mastitis model
    Vincent Peton, Veterinary Microbiology - 2016
    Abstract
    Staphylococcus aureus (S. aureus) is a major pathogen involved in ruminant mastitis and present worldwide. Clinical signs of S. aureus mastitis vary considerably and are largely dependent on strain-specific factors. A comparison of two S. aureus strains that reproducibly induced either severe (O11) or mild (O46) mastitis in ewes revealed that the transcriptional regulator sigS was mutated in O46 (Le Maréchal et al., 2011. PLoS One. 6 (11) e27354. doi:10.1371/journal.pone.0027354). In the present paper, we analysed the sigS sequence in 18 other S. aureus strains isolated from goat or ewe mastitis and found a 4-bp deletion similar to that of the O46 sigS gene in three strains associated with subclinical ewe mastitis. This sigS gene was disrupted in strain O11 (O11ΔsigS), so our aim was to investigate its involvement in the severity of infections in the context of mastitis. The wild type (wt) and mutant strains were then characterized in vitro to determine the involvement of sigS in the response S. aureus under various stress conditions, and assess its influence on the cytotoxicity of the pathogen, its invasive capacity and biofilm formation. The strains were compared in vivo in an experimental mouse mastitis model in which clinical signs and cytokine production were evaluated at 24 h post-infection. While no significant differences in the effect on bacterial growth between O11 and O11ΔsigS were observed either in vitro or in vivo, a significantly weaker in vivo production of interleukin (IL)-1α, IL-1β, and Tumor Necrosis Factor (TNF)-α was measured in the mammary glands infected with the mutant strain, suggesting that infection with O11ΔsigS induced an attenuated local innate immune response. These results suggest an impact of sigS disruption on S. aureus pathogenesis in a ruminant mastitis context. This disruption is probably involved in, and may partly explain, the milder symptoms previously observed in S. aureus O46-induced mastitis in ewes.
    microRNA 338-3p exhibits tumor suppressor role and its down-regulation is associated with adverse clinical outcome in prostate cancer patients
    Ashraf Bakkar, Molecular Biology Reports - 2016
    Abstract
    MicroRNAs (miRNAs) are small non-coding RNAs that function in transcriptional and post-transcriptional regulation of gene expression. Several miRNAs have been implicated in regulating prostate cancer (PCa) progression. Deregulations of miRNA regulatory networks have been reported in ERG positive PCa, which accounts for ~50 % of PCa and have been suggested to affect tumor aggressiveness. The function of miR338-3p, its prognostic significance, and its association with ERG positive PCa has not been fully investigated. Using microarray expression profiling, we identified miRNA338-3p as among the top deregulated miRNAs associated with ERG status in PCa. We investigated miR338-3p function using in vitro and in vivo experimental models and its expression was assessed and validated in clinical samples and a public cohort of localized and metastatic prostate cancer. miR338-3p was significantly down-regulated with disease progression from benign prostate tissue to primary and metastatic lesions. In localized disease, patients with lower miR338-3p expression levels showed increased association to biochemical recurrence and several adverse pathological parameters compared to patients with higher miRNA338-3p tissue expression levels. Using in vitro PCa cell models, overexpression of miR338-3p resulted in a decrease in cell invasion and expression of chemokine signalling genes CXCL12, CXCR4, and CXCR7. In vivo, orthotropic implantation of PC3 cells stably expressing miR338-3p was associated with a significant decrease in tumor weights compared to control cells. miR338-3p has anti-proliferative and anti-invasive properties. It affects CXCR4 axis, and its down-regulation is associated with adverse clinical outcomes in PCa patients.
    microRNA 338-3p exhibits tumor suppressor role and its down-regulation is associated with adverse clinical outcome in prostate cancer patients
    Mohammed Alshalalfa, Molecular Biology Reports - 2016
    Abstract
    MicroRNAs (miRNAs) are small non-coding RNAs that function in transcriptional and post-transcriptional regulation of gene expression. Several miRNAs have been implicated in regulating prostate cancer (PCa) progression. Deregulations of miRNA regulatory networks have been reported in ERG positive PCa, which accounts for ~50 % of PCa and have been suggested to affect tumor aggressiveness. The function of miR338-3p, its prognostic significance, and its association with ERG positive PCa has not been fully investigated. Using microarray expression profiling, we identified miRNA338-3p as among the top deregulated miRNAs associated with ERG status in PCa. We investigated miR338-3p function using in vitro and in vivo experimental models and its expression was assessed and validated in clinical samples and a public cohort of localized and metastatic prostate cancer. miR338-3p was significantly down-regulated with disease progression from benign prostate tissue to primary and metastatic lesions. In localized disease, patients with lower miR338-3p expression levels showed increased association to biochemical recurrence and several adverse pathological parameters compared to patients with higher miRNA338-3p tissue expression levels. Using in vitro PCa cell models, overexpression of miR338-3p resulted in a decrease in cell invasion and expression of chemokine signalling genes CXCL12, CXCR4, and CXCR7. In vivo, orthotropic implantation of PC3 cells stably expressing miR338-3p was associated with a significant decrease in tumor weights compared to control cells. miR338-3p has anti-proliferative and anti-invasive properties. It affects CXCR4 axis, and its down-regulation is associated with adverse clinical outcomes in PCa patients.
    Inhibition of O-GlcNAc transferase activity reprograms prostate
    Harri M. Itkonen, Oncotarget - 2016
    Abstract
    Metabolic networks are highly connected and complex, but a single enzyme, O-GlcNAc transferase (OGT) can sense the availability of metabolites and also modify target proteins. We show that inhibition of OGT activity inhibits the proliferation of prostate cancer cells, leads to sustained loss of c-MYC and suppresses the expression of CDK1, elevated expression of which predicts prostate cancer recurrence (p=0.00179). Metabolic profiling revealed decreased glucose consumption and lactate production after OGT inhibition. This decreased glycolytic activity specifically sensitized prostate cancer cells, but not cells representing normal prostate epithelium, to inhibitors of oxidative phosphorylation (rotenone and metformin). Intra-cellular alanine was depleted upon OGT inhibitor treatment. OGT inhibitor increased the expression and activity of alanine aminotransferase (GPT2), an enzyme that can be targeted with a clinically approved drug, cycloserine. Simultaneous inhibition of OGT and GPT2 inhibited cell viability and growth rate, and additionally activated a cell death response. These combinatorial effects were predominantly seen in prostate cancer cells, but not in a cell-line derived from normal prostate epithelium. Combinatorial treatments were confirmed with two inhibitors against both OGT and GPT2. Taken together, here we report the reprogramming of energy metabolism upon inhibition of OGT activity, and identify synergistically lethal combinations that are prostate cancer cell specific.
    Laser capture microdissection of intestinal tissue from sea bass larvae using an optimized RNA integrity assay and validated reference genes : Scientific Reports
    M. Schaeck, Scientific Reports - 2016
    Abstract
    The increasing demand for a sustainable larviculture has promoted research regarding environmental parameters, diseases and nutrition, intersecting at the mucosal surface of the gastrointestinal tract of fish larvae. The combination of laser capture microdissection (LCM) and gene expression experiments allows cell specific expression profiling. This study aimed at optimizing an LCM protocol for intestinal tissue of sea bass larvae. Furthermore, a 3′/5′ integrity assay was developed for LCM samples of fish tissue, comprising low RNA concentrations. Furthermore, reliable reference genes for performing qPCR in larval sea bass gene expression studies were identified, as data normalization is critical in gene expression experiments using RT-qPCR. We demonstrate that a careful optimization of the LCM procedure allows recovery of high quality mRNA from defined cell populations in complex intestinal tissues. According to the geNorm and Normfinder algorithms, ef1a, rpl13a, rps18 and faua were the most stable genes to be implemented as reference genes for an appropriate normalization of intestinal tissue from sea bass across a range of experimental settings. The methodology developed here, offers a rapid and valuable approach to characterize cells/tissues in the intestinal tissue of fish larvae and their changes following pathogen exposure, nutritional/environmental changes, probiotic supplementation or a combination thereof.
    A Neurobiological Pathway That Mediates Stress-Induced Drug Use - viewcontent.cgi
    Sarit Aviel-Ronen, Scientific Report - 2016
    Abstract
    Aims: Chordomas and chondrosarcomas are malignant mesenchymal tumours with overlapping morphological and immunohistochemical (IHC) characteristics. Our aim was to evaluate the IHC expression of α-methylacyl-CoA racemase (AMACR/P504S), β-catenin and E-cadherin in chordomas relative to chondrosarcomas and assess the utility of these markers for differential diagnosis. Methods: Archival sections of 18 chordomas, 19 chondrosarcomas and 10 mature cartilage samples were immunostained and scored for AMACR, β-catenin and E-cadherin and the relative differential capacity of each marker was calculated. In addition, AMACR mRNA level was assessed in 5 chordomas by RT-PCR and evaluated by comparative CT method. Results: AMACR and β-catenin stained 88.9% and 94.1% of the chordomas respectively, 21.1% and 10.5% of the chondrosarcomas correspondingly and none of the mature cartilage samples. E-cadherin stained positively 82.4% of the chordomas, 36.8% of the chondrosarcomas and 42.9% of the mature cartilage cases. Both AMACR and β-catenin showed statistically significant difference between chordomas and chondrosarcomas (p < 0.001 for both), unlike E-cadherin. AMACR was detected at the mRNA level. Conclusions: AMACR is expressed in most of the chordomas but only in a minority of chondrosarcomas. AMACR may serve as IHC marker of chordoma with differentiating ability comparable to that of β-catenin.
    Onyango1362015BJMMR23764.pdf
    B.M Habiyambere, British Journal of Medicine & Medical Research - 2016
    Abstract
    Objectives: The aim for this study was to investigate the effects of chronic restraint stress on the expression of mucins that comprises the integral component of the gastrointestinal barrier. Design and Methods: A completely randomized design was used for the study. Sixteen ICR male mice were randomly divided into treated and control animals. Restraint stress was applied to the treated mice for 21 days. Body weight and feed consumed were regularly recorde d. After 21 days mice were euthanized and blood and gastrointestinal tissues were collected. Serum corticosterone levels were determined. Intestinal sections were fixed, paraffin embedded, sectioned, stained with alcian blue periodic acid Schiff stain to visualize goblet cells. Expression of mucosa mucins was determined using qPCR and western blotting.Results: As expected stressed mice showed higher corticosterone levels than the control mice (5082±1975 vs 1566±1236 pg/ml, P= 0.002). At the end of the study the body weight was lower in the stressed group (32±2 vs 36.0±3 g) even though the cumulative food consumed in both groups was not different. Stressed mice had lower goblet cell count in the duodenum, jejunum, ileum and colon (8±2, 7±1, 9±1, and 25±8 vs14±1, 14±1, 11±2 and 40±9, respectively), and shorter villi in the Original Research Article Habiyambere and Onyango ; BJMMR, 13(6): 1-14, 2016; Article no.BJMMR.237642 duodenum and jejunum (316±82 and 256±73 vs 440±73 and 357±35 μm, respectively). Expressions of mRNA for Muc1 and Muc4 in the colon of stressed animals were upregulated (1.5 and 2.2 fold increase, and P values of 0.05 and 0.01, respectively). This mRNA upregulation was accompanied by increased expression of Muc1 and 4 proteins in the colon.Conclusion: These findings suggest that chronic stress affects the intestinal barrier by reducing the number of the mucin-producing goblet cells, shortening the villi and upregulating Muc1 and Muc4 in the colon.
    Mesenchymal stromal cells from the foreskin: Tissue isolation, cell characterization and immunobiological properties
    Mehdi Najar, Cytotherapy - 2016
    Abstract
    Background aims Because of their self-renewal capacity, multilineage potential and immunomodulatory properties, MSCs are an attractive tool for cell-based immunotherapy strategies. Foreskin, considered as a biological waste material, has been shown to be a reservoir of therapeutic cells. Methods MSCs were isolated from different foreskin samples, maintained under in vitro culture and defined according to the International Society for Cellular Therapy (ISCT) criteria. We subsequently determined their main cell characteristics as well as their immunobiological properties. The following parameters were determined: (i) morphology and phenotype, (ii) proliferative and clonogenic potentials, (iii) tri-lineage differentiation ability, (iv) immunological profile, (v) immunomodulatory properties and (vi) protein and messenger RNA expression/secretion profile of immunoregulatory cytokines/factors as well as the pattern of toll-like receptors (TLRs). By using a pro-inflammatory cytokine cocktail, we also evaluated the influence of an inflammatory environment on their biology. Results With a typical fibroblast-like morphology and an ISCT-compliant phenotype, foreskin-MSCs (FSK-MSCs) were highly proliferative and had a great clonogenic potential. They displayed multilineage capacities and interesting immunomodulatory properties. Of importance, FSK-MSCs were not immunogenetic and were further able to inhibit T-cell proliferation. We showed that several immunoregulatory cytokines and factors might be potentially involved in FSK-MSC immunomodulation with particular attention to hepatocyte growth factor and interleukin-11. Moreover, FSK-MSCs expressed several TLRs and were sensitive to the inflammatory environment by properly adjusting their profile and fate. Conclusions Foreskin represents a new alternative source for MSCs that is compliant with ISCT criteria. Their unique immunobiological properties allow consideration of FSK-MSCs as a valuable tolerogenic product for cell-based immunotherapy.
    Characterization of aromatic aminotransferases from Ephedra sinica Stapf
    Korey Kilpatrick, Amino Acids - 2016
    Abstract
    Ephedra sinica Stapf (Ephedraceae) is a broom-like shrub cultivated in arid regions of China, Korea and Japan. This plant accumulates large amounts of the ephedrine alkaloids in its aerial tissues. These analogs of amphetamine mimic the actions of adrenaline and stimulate the sympathetic nervous system. While much is known about their pharmacological properties, the mechanisms by which they are synthesized remain largely unknown. A functional genomics platform was established to investigate their biosynthesis. Candidate enzymes were obtained from an expressed sequence tag collection based on similarity to characterized enzymes with similar functions. Two aromatic aminotransferases, EsAroAT1 and EsAroAT2, were characterized. The results of quantitative reverse transcription-polymerase chain reaction indicated that both genes are expressed in young stem tissue, where ephedrine alkaloids are synthesized, and in mature stem tissue. Nickel affinity-purified recombinant EsAroAT1 exhibited higher catalytic activity and was more homogeneous than EsAroAT2 as determined by size-exclusion chromatography. EsAroAT1 was highly active as a tyrosine aminotransferase with α-ketoglutarate followed by α-ketomethylthiobutyrate and very low activity with phenylpyruvate. In the reverse direction, catalytic efficiency was similar for the formation of all three aromatic amino acids using l-glutamate. Neither enzyme accepted putative intermediates in the ephedrine alkaloid biosynthetic pathway, S-phenylacetylcarbinol or 1-phenylpropane-1,2-dione, as substrates.
    GAPDH Binding to TNF-α mRNA Contributes to Posttranscriptional Repression in Monocytes: A Novel Mechanism of Communication between Inflammation and Metabolism
    Patrick Millet, The Journal of Immunology - 2016
    Abstract
    Expression of the inflammatory cytokine TNF is tightly controlled. During endotoxin tolerance, transcription of TNF mRNA is repressed, although not entirely eliminated. Production of TNF cytokine, however, is further controlled by posttranscriptional regulation. In this study, we detail a mechanism of posttranscriptional repression of TNF mRNA by GAPDH binding to the TNF 3′ untranslated region. Using RNA immunoprecipitation, we demonstrate that GAPDH–TNF mRNA binding increases when THP-1 monocytes are in a low glycolysis state, and that this binding can be reversed by knocking down GAPDH expression or by increasing glycolysis. We show that reducing glycolysis decreases TNF mRNA association with polysomes. We demonstrate that GAPDH–TNF mRNA binding results in posttranscriptional repression of TNF and that the TNF mRNA 3′ untranslated region is sufficient for repression. Finally, after exploring this model in THP-1 cells, we demonstrate this mechanism affects TNF expression in primary human monocytes and macrophages. We conclude that GAPDH–TNF mRNA binding regulates expression of TNF based on cellular metabolic state. We think this mechanism has potentially significant implications for treatment of various immunometabolic conditions, including immune paralysis during septic shock.
    Unraveling the Tissue-Specific Gene Signatures of Gilthead Sea Bream (Sparus aurata L.) after Hyper- and Hypo-Osmotic Challenges
    Juan Antonio Martos-Sitcha, PLoS ONE - 2016
    Abstract
    A custom microarray was used for the transcriptomic profiling of liver, gills and hypothalamus in response to hypo- (38‰ → 5‰) or hyper- (38‰ → 55‰) osmotic challenges (7 days after salinity transfer) in gilthead sea bream (Sparus aurata) juveniles. The total number of differentially expressed genes was 777. Among them, 341 and 310 were differentially expressed in liver after hypo- and hyper-osmotic challenges, respectively. The magnitude of changes was lower in gills and hypothalamus with around 131 and 160 responsive genes in at least one osmotic stress condition, respectively. Regardless of tissue, a number of genes were equally regulated in either hypo- and hyper-osmotic challenges: 127 out of 524 in liver, 11 out of 131 in gills and 19 out of 160 in hypothalamus. In liver and gills, functional analysis of differentially expressed genes recognized two major clusters of overlapping canonical pathways that were mostly related to “Energy Metabolism” and “Oxidative Stress”. The later cluster was represented in all the analyzed tissues, including the hypothalamus, where differentially expressed genes related to “Cell and tissue architecture” were also over-represented. Overall the response for “Energy Metabolism” was the up-regulation, whereas for oxidative stress-related genes the type of response was highly dependent of tissue. These results support common and different osmoregulatory responses in the three analyzed tissues, helping to load new allostatic conditions or even to return to basal levels after hypo- or hyper-osmotic challenges according to the different physiological role of each tissue.
    Estrogen receptor β regulates endometriotic cell survival through serum and glucocorticoid–regulated kinase activation
    Diana Monsivais, Fertility and Sterility - 2016
    Abstract
    Objective To determine the expression and biological roles of serum and glucocorticoid–regulated kinase (SGK1) in tissues and cells from patients with endometriosis and from healthy control subjects. Design Case-control. Setting University research setting. Patient(s) Premenopausal women. Intervention(s) Endometriotic tissues were obtained from women with ovarian endometriosis, and normal endometrial tissues were obtained from women undergoing hysterectomy for benign conditions. Main Outcome Measure(s) Expression levels of SGK1, the role of SGK1 in endometriosis pathology, and regulation of SGK1 by estrogen receptor (ER) β. Result(s) Transcript and protein levels of SGK1 were significantly higher in endometriotic tissues and cells compared with normal endometrium. SGK1 mRNA and protein levels were stimulated by E2, by the ERβ-selective agonist diarylpropionitrile, and by prostaglandin E2. SGK1 was transcriptionally regulated by ERβ based on small interfering RNA knockdown and chromatin immunoprecipitation of ERβ followed by quantitative polymerase chain reaction. SGK1 knockdown led to increased cleavage of poly(ADP-ribose) polymerase, and SGK1 activation was correlated with the phosphorylation of FOXO3a, a proapoptotic factor. Conclusion(s) ERβ leads to SGK1 overexpression in endometriosis, which contributes to the survival of endometriotic lesions through inhibition of apoptosis.
    Methods and Compounds for the Inhibition of Cellular Proliferation
    Gerhard Wagner, US Patent - 2015
    Abstract
    Methods for inhibiting translation, treating a cellular proliferative disorder, and inhibiting proliferation of cells using the compounds disclosed herein are provided.
    Effect of genetic background and postinfectious stress on visceral sensitivity in Citrobacter rodentium-infected mice
    S. U. Mondelaers, Neurogastroenterology & Motility - 2016
    Abstract
    Background Infectious gastroenteritis is a major risk factor to develop postinfectious irritable bowel syndrome (PI-IBS). It remains unknown why only a subgroup of infected individuals develops PI-IBS. We hypothesize that immunogenetic predisposition is an important risk factor. Hence, we studied the effect of Citrobacter rodentium infection on visceral sensitivity in Th1-predominant C57BL/6 and Th2-predominant Balb/c mice. Methods Eight-week-old mice were gavaged with C. rodentium, followed by 1 h of water avoidance stress (WAS) at 5 weeks PI. At 10, 14 days, and 5 weeks PI, samples were assessed for histology and inflammatory gene expression by RT-qPCR. Visceral sensitivity was evaluated by visceromotor response recordings (VMR) to colorectal distension. Key Results Citrobacter rodentium evoked a comparable colonic inflammatory response at 14 days PI characterized by increased crypt length and upregulation of Th1/Th17 cytokine mRNA levels (puncorrected < 0.05) in both C57BL/6 and Balb/c mice. At 5 weeks PI, inflammatory gene mRNA levels returned to baseline in both strains. The VMR was maximal at 14 days PI in C57BL/6 (150 ± 47%; p = 0.02) and Balb/c mice (243 ± 52%; p = 0.03). At 3 weeks PI, the VMR remained increased in Balb/c (176 ± 23%; p = 0.02), but returned to baseline in C57BL/6 mice. At 5 weeks PI, WAS could not re-introduce visceral hypersensitivity (VHS). Conclusions & Inferences Citrobacter rodentium infection induces transient VHS in C57BL/6 and Balb/c mice, which persisted 1 week longer in Balb/c mice. Although other strain-related differences may contribute, a Th2 background may represent a risk factor for prolonged PI-VHS. As PI-VHS is transient, other factors are crucial for persistent VHS development as observed in PI-IBS.
    Targeting NF-κB in glioblastoma: A therapeutic approach
    Dinorah Friedmann-Morvinski, Science Advances - 2016
    Abstract
    Glioblastoma multiforme (GBM) is the most common and lethal form of intracranial tumor. We have established a lentivirus-induced mouse model of malignant gliomas, which faithfully captures the pathophysiology and molecular signature of mesenchymal human GBM. RNA-Seq analysis of these tumors revealed high nuclear factor κB (NF-κB) activation showing enrichment of known NF-κB target genes. Inhibition of NF-κB by either depletion of IκB kinase 2 (IKK2), expression of a IκBαM super repressor, or using a NEMO (NF-κB essential modifier)–binding domain (NBD) peptide in tumor-derived cell lines attenuated tumor proliferation and prolonged mouse survival. Timp1, one of the NF-κB target genes significantly up-regulated in GBM, was identified to play a role in tumor proliferation and growth. Inhibition of NF-κB activity or silencing of Timp1 resulted in slower tumor growth in both mouse and human GBM models. Our results suggest that inhibition of NF-κB activity or targeting of inducible NF-κB genes is an attractive therapeutic approach for GBM. Inhibition of the transcription factor NF-κB or its target genes should be considered for the treatment of patients with glioblastoma multiforme. Inhibition of the transcription factor NF-κB or its target genes should be considered for the treatment of patients with glioblastoma multiforme.
    The auxin response factor MONOPTEROS controls meristem function and organogenesis in both the shoot and root through the direct regulation of PIN genes
    Naden Krogan, New Phytologist - 2016
    Abstract
    * The regulatory effect auxin has on its own transport is critical in numerous self-organizing plant patterning processes. However, our understanding of the molecular mechanisms linking auxin signal transduction and auxin transport is still fragmentary, and important regulatory genes remain to be identified. * To track a key link between auxin signaling and auxin transport in development, we established an Arabidopsis thaliana genetic background in which fundamental patterning processes in both shoot and root were essentially abolished and the expression of PIN FORMED (PIN) auxin efflux facilitators was dramatically reduced. * In this background, we demonstrate that activating a steroid-inducible variant of the auxin response factor (ARF) MONOPTEROS (MP) is sufficient to restore patterning and PIN gene expression. Further, we show that MP binds to distinct promoter elements of multiple genetically defined PIN genes. * Our work identifies a direct regulatory link between central, well-characterized genes involved in auxin signal transduction and auxin transport. The steroid-inducible MP system directly demonstrates the importance of this molecular link in multiple patterning events in embryos, shoots and roots, and provides novel options for interrogating the properties of self-regulated auxin-based patterning in planta.
    Soybean Meal and Soy Protein Concentrate in Early Diet Elicit Different Nutritional Programming Effects on Juvenile Zebrafish
    Erick Perera, Zebrafish - 2015
    Abstract
    There is now strong evidence that early nutrition plays an important role in shaping later physiology. We assessed here whether soy protein concentrate (SPC) or soybean meal (SBM) in early diet would modify zebrafish responses to these products in later life. We fed zebrafish larvae with SPC-, SBM-, or a control-diet for the first 3 days of feeding and then grew all larvae on the control diet up to juveniles. Finally, we assessed the expression in juveniles of genes involved in inflammation/immunity, the breakdown of extracellular matrix, luminal digestion, and intestinal nutrient absorption/trafficking. First feeding SBM had wider, stronger, and more persistent effects on gene expression with respect to SPC. Juveniles fed with SPC at first feeding were more prone to inflammation after refeeding with SPC than fish that never experienced SPC before. Conversely, zebrafish that faced SBM at first feeding were later less responsive to refeeding with SBM through inflammation and had higher expression of markers of peptide absorption and fatty acid transport. Results indicate that some features of inflammation/remodeling, presumably at the intestine, and nutrient absorption/transport in fish can be programmed by early nutrition. These findings sustain the rationale of using zebrafish for depicting molecular mechanisms involved in nutritional programming.
    A genome-wide association study identifies four novel susceptibility loci underlying inguinal hernia
    Eric Jorgenson, Nature Communications - 2016
    Abstract
    Inguinal hernia repair is one of the most commonly performed operations in the world, yet little is known about the genetic mechanisms that predispose individuals to develop inguinal hernias. We perform a genome-wide association analysis of surgically confirmed inguinal hernias in 72,805 subjects (5,295 cases and 67,510 controls) and confirm top associations in an independent cohort of 92,444 subjects with self-reported hernia repair surgeries (9,701 cases and 82,743 controls). We identify four novel inguinal hernia susceptibility loci in the regions of EFEMP1, WT1, EBF2 and ADAMTS6. Moreover, we observe expression of all four genes in mouse connective tissue and network analyses show an important role for two of these genes (EFEMP1 and WT1) in connective tissue maintenance/homoeostasis. Our findings provide insight into the aetiology of hernia development and highlight genetic pathways for studies of hernia development and its treatment.
    Caspase-3 cleaved p65 fragment dampens NF-κB-mediated anti-apoptotic transcription by interfering with the p65/RPS3 interaction - 56420a8508aec448fa621bec.pdf
    Eric M. Wier, Federation of European Biochemical Societies. - 2015
    Abstract
    Caspase-3-mediated p65 cleavage is believed to suppress nuclear factor-kappa B (NF- j B)-mediated anti-apoptotic transactivation in cells undergoing apoptosis. However, only a small percentage of p65 is cleaved during apoptosis, not in proportion to the dramatic reduction in NF- j B transactiva- tion. Here we show that the p65 1-97 fragment generated by Caspase-3 cleavage interferes with ribo- somal protein S3 (RPS3), an NF- j B ‘‘specifier ” subunit, and selectively retards the nuclear translocation of RPS3, thus dampening the RPS3/NF- j B-dependent anti-apoptotic gene expression. Our findings reveal a novel cell fate determination mechanism to ensure cells undergo programed cell death through interfering with RPS3/NF- j B-conferred anti-apoptotic transcription by the frag- ment from partial p65 cleavage by activated Caspase-3.
    In-Vitro Adhesion and Invasion Properties of Salmonella Typhimurium Competing with Bacteriophage in Epithelial Cells and Chicken Macrophages
    H.Y Lee, Revista Brasileira de Ciência Avícola - 2015
    Abstract
    This study was designed to assess the role of bacteriophage P22 in the adhesion, invasion, intracellular survival of, and cellular immune response to Salmonella Typhimurium in intestinal epithelial INT-407 and chicken macrophage-like HD11 cells. The ability of S. Typhimurium to adhere, invade, and survive to INT-407 and HD11cells was evaluated under Salmonella infection alone (control), phage treatment followed by Salmonella infection (PS), Salmonella infection followed by phage treatment (SP), and a combination treatment with Salmonella and phage (S+P). The number of S. Typhimurium associated on INT-407 cells was reduced from 4.2 to 2.7 log cfu/cm2 by phage treatment (SP). The number of intracellular S. Typhimurium within INT-407 cells was significantly reduced to below the detection limit (0.7 log cfu/cm2) compared with the control (3.4 log cfu/cm2). S. Typhimurium remained inside HD11 cells at 49% and 17% levels in the absence and presence of phages, respectively, at 24 h post-infection (hpi). The expression levels of IFN-g, IL-10, IL-1b, IL-6, IL-8, iNOS, and IL-12 increased in HD11 cells regardless the absence and presence of phages, while those of IL-16, TLR2-1, TLR3, and TLR7 were decreased at 0 and 24 hpi. This study sheds new light on our understanding of the role of phages in Salmonella adhesion, invasion, survival, and cellular immune responses.Key words: Adhesion; Bacteriophage; Invasion; Salmonella; Toll-like receptor
    Insulin Receptor Substrate 1: The Hub Linking Follicle-stimulating Hormone to Phosphatidylinositol-3 Kinase Activation
    Nathan Law, Journal of Biological Chemistry - 2015
    Abstract
    The ubiquitous phosphatidylinositol-3 kinase (PI3K) signaling pathway regulates many cellular functions. However, the mechanism by which G protein-coupled receptors (GPCRs) signal to activate PI3K is poorly understood. We have used ovarian granulosa cells (GCs) as a model to investigate this pathway, based on evidence that the GPCR agonist follicle-stimulating hormone (FSH) promotes the protein kinase A (PKA)- dependent phosphorylation of insulin receptor substrate 1 (IRS1) on tyrosine residues that activate PI3K. We report that in the absence of FSH, GCs secrete a subthreshold concentration of insulin-like growth factor-1 (IGF-1) that primes the IGF-1 receptor (IGF-1R) but fails to promote tyrosine phosphorylation of IRS1. FSH via PKA acts to sensitize IRS1 to the tyrosine kinase activity of the IGF-1R by activating protein phosphatase 1 (PP1) to promote dephosphorylation of inhibitory Ser/Thr residues on IRS1, including Ser789. Knockdown of PP1β blocks the ability of FSH to activate PI3K in the presence of endogenous IGF-1. Activation of PI3K thus requires both PKA-mediated relief of IRS1 inhibition and IGF-1R-dependent tyrosine phosphorylation of IRS1. Treatment with FSH and increasing concentrations of exogenous IGF-1 triggers synergistic IRS1 tyrosine phosphorylation at PI3K-activating residues that persists downstream through AKT and forkhead box O1 (FOXO1) to drive synergistic expression of genes that underlies follicle maturation. Based on the ability of GPCR agonists to synergize with IGFs to enhance gene expression in other cell types, PP1 activation to relieve IRS1 inhibition may be a more general mechanism by which GPCRs act with the IGF-1R to activate PI3K/AKT.
    20-HETE Activates the Transcription of Angiotensin Converting Enzyme (ACE) via NF-kB Translocation and Promoter Binding
    Victor Garcia, Journal of Pharmacology and Experimental Therapeutics - 2015
    Abstract
    Increased vascular 20-hydroxyeicosatetraenoic acid (20-HETE), a cytochrome P450 arachidonic acid metabolite, promotes vascular dysfunction, injury and hypertension that is dependent, in part, on the renin angiotensin system (RAS). We have shown that in human microvascular endothelial cells (HMVECs), 20-HETE increases angiotensin converting enzyme (ACE) mRNA, protein, and ACE activity via an EGFR-tyrosine kinase-MAPK-IKKβ-mediated signaling pathway. Here we show that similar to EGF, 20-HETE (10nM) activates EGFR by stimulating tyrosine phosphorylation; however, unlike 20-HETE, EGF does not induce ACE expression, and pretreatment with a neutralizing antibody against EGF does not prevent the 20-HETE-mediated ACE induction. Inhibition of NF-kB activation prevented the 4.58-fold (±0.78; p<0.05) 20-HETE-mediated induction of ACE. 20-HETE increased NF-kB binding activity in nuclear extracts and the activity of both the somatic and germinal ACE promoters by 4.37-fold (±0.18; p<0.05) and 2.53-fold (± 0.24; p<0.05), respectively. 20-HETE-stimulated ACE promoter activity was abrogated by the 20-HETE antagonist 20-HEDE and by inhibitors of EGFR, MAPK, IKKβ and NF-kB activation. Sequence analysis demonstrated the presence of two and one putative NF-kB binding sites on the human somatic and germinal ACE promoters, respectively. ChIP assay indicated that 20-HETE stimulates the translocation and subsequent binding of NF-kB to each of the putative binding sites (S1: 3.43±0.3, S2: 3.72±0.68, S3: 3.20±0.18-fold enrichment vs. vehicle; p<0.05). This is the first study to identify NF-kB as a transcriptional factor for ACE and to implicate a distinct EGFR/MAPK/IKK/NF-kB signaling cascade underlying 20-HETE-mediated transcriptional activation of ACE mRNA and stimulation of ACE activity.
    Visualizing estrogen receptor-α-expressing neurons using a new ERα-ZsGreen reporter mouse line
    Kenji Saito, Metabolism - 2016
    Abstract
    Background A variety of biological functions of estrogens, including regulation of energy metabolism, are mediated by neurons expressing estrogen receptor-α (ERα) in the brain. However, complex intracellular processes in these ERα-expressing neurons are difficult to unravel, due to the lack of strategy to visualize ERα-expressing neurons, especially in unfixed brain tissues. Results and conclusions Here we generated a novel ERα-ZsGreen reporter mouse line in which expression of a green fluorescent reporter protein, ZsGreen, is driven by a 241 kb ERα gene promoter. We validated that ZsGreen is highly colocalized with endogenous ERα in the brain. Native ZsGreen signals were visualized in unfixed brain tissue, and were used to assist single cell collection and electrophysiological recordings. Finally, we demonstrated that this ERα-ZsGreen mouse allele can be used in combination with other genetic reporter alleles to allow experiments in highly selective neural populations.
    Discrete Responses to Limitation for Iron and Manganese in Agrobacterium tumefaciens: Influence on Attachment and Biofilm Formation
    Jason E. Heindl, Journal of Bacteriology - 2015
    Abstract
    Transition metals such as iron and manganese are crucial trace nutrients for the growth of most bacteria, functioning as catalytic cofactors for many essential enzymes. Dedicated uptake and regulatory systems have evolved to ensure their acquisition for growth, while preventing toxicity.Transcriptomic analysis of the iron- and manganese-responsive regulons of Agrobacterium tumefaciens revealed that there are discrete regulatory networks that respond to changes in iron and manganese levels. Complementing earlier studies, the iron-responsive gene network is quite large, and includes many aspects of iron-dependent metabolism and the iron-sparing response. In contrast, the manganese-responsive network is restricted to a limited number of genes, many of which can be linked to transport and utilization of the transition metal. Several of the target genes predicted to drive manganese uptake are required for growth under manganese-limited conditions, and an A. tumefaciens mutant with a manganese transport deficiency is attenuated for plant virulence. Iron and manganese limitation independently inhibit biofilm formation by A. tumefaciens, and several candidate genes that could impact biofilm formation were identified in each regulon. The biofilm inhibitory effects of iron and manganese do not rely on recognized metal-responsive transcriptional regulators, suggesting alternate mechanisms influencing biofilm formation. However, under low manganese the dcpA operon is upregulated, a system that controls levels of the cyclic-diguanosine monophosphate second messenger. Mutation of this regulatory pathway dampens the effect of manganese limitation. IMPORTANCE Responses to changes in transition metal levels, such as those of manganese and iron, are important for normal metabolism and growth in bacteria. Our study uses global gene expression profiling to understand the response of the plant pathogen Agrobacterium tumefaciens to changes of transition metal availability. Among the properties that are affected by both iron and manganese levels are those required for normal surface attachment and biofilm formation, but the requirement for each of these transition metals is mechanistically independent from the other.
    Nerve Growth Factor is Regulated by Toll-Like Receptor 2 in Human Intervertebral Discs
    Krock Emerson, Journal of Biological Chemistry - 2015
    Abstract
    Nerve growth factor (NGF) contributes to the development of chronic pain associated with degenerative connective tissue pathologies, such as intervertebral disc degeneration and osteoarthritis. However, surprisingly little is known about the regulation of NGF in these conditions. Toll-like receptors (TLR) are pattern recognition receptors classically associated with innate immunity, but more recently were found to be activated by endogenous alarmins such as fragmented extra-cellular matrix proteins found in degenerating discs or cartilage. In this study we investigated if TLR activation regulates NGF and which signaling mechanisms control this response in intervertebral discs. TLR2 agonists, TLR4 agonists, or IL-1β (control) treatment increased NGF, BDNF and IL-1β gene expression in human disc cells isolated from healthy, pain-free organ donors. However, only TLR2 activation or IL-1β treatment increased NGF protein secretion. TLR2 activation increased p38, ERK1/2 and p65 activity and increased p65 translocation to the cell nucleus. JNK activity was not affected by TLR2 activation. Inhibition of NF-κB, and to a lesser extent p38, but not ERK1/2 activity blocked TLR2-driven NGF upregulation at both the transcript and protein levels. These results provide a novel mechanism of NGF regulation in the intervertebral disc and potentially other pathogenic connective tissues. TLR2 and NF-κB signaling are known to increase cytokines and proteases, which accelerate matrix degradation. Therefore, TLR2 or NF-κB inhibition may both attenuate chronic pain and slow the degenerative progress in vivo.
    Protein Kinase C Epsilon Promotes Cerebral Ischemic Tolerance Via Modulation of Mitochondrial Sirt5
    Kahlilia C. Morris-Blanco, Scientific Reports - 2016
    Abstract
    not listed
    Antisense RNA-Mediated Epigenetic Regulation of Brain-Derived Neurotrophic Factor. - viewcontent.cgi
    Roya Pedram Fatemi, University of Miami Scholarly Repository DISSERTATION - 2015
    Abstract
    Long noncoding RNAs (lncRNAs) regulate chromatin remodeling through their interactions with epigenetic enzymes during development and disease. The inhibition of the natural antisense transcript of Brain-derived neurotrophic factor (BDNF-AS), results in BDNF promoter de-repression and transcriptional upregulation, both in vitro and in vivo. Recently, we showed that BDNF-AS interacts with the histone methyltransferase enhancer of zeste homolog 2 (EZH2) to suppress BDNF mRNA and protein expression. BDNF is an important neurotrophin that is required for neural development and maintenance of the nervous system. Dysregulation of BDNF occurs in a number of neurological disorders, including: Alzheimer’s Disease, Parkinson’s Disease, Rett syndrome, and amyotrophic lateral sclerosis. Previous attempts to upregulate BDNF by administering the recombinant form in various parts of the central nervous system have failed, mostly due to the challenge of delivering BDNF to the correct cells and neural networks. Our approach to upregulating BDNF by modulating its interaction with an epigenetic enzyme is a highly specific target with potential therapeutic value. To achieve this, we developed a novel pharmacological assay to characterize the interaction between long noncoding RNAs and their epigenetic targets using Amplified Luminescent Proximity Homogeneous Assay (AlphaScreen) technology. With this assay, we are able to quantify lncRNA-protein interactions rapidly for the purpose of high throughput screening, enabling drug discovery efforts for this novel class of drug targets. In this work, we present our assay development and screening findings, including the identification of potential small molecule modulators of lncRNA-protein interactions. Furthermore, we describe the application of this lncRNA-protein interaction assay to detect RNA requirements for EZH2 recruitment, a much debated and important question that lingers in the field. From our work, it is evident that BDNF-AS has several regions of RNA that are required for EZH2 recruitment, potentially due to the importance of this transcript in regulating BDNF. This work describes exploratory drug discovery for a novel class of drug targets as well as applications to understand the basic biochemistry
    Transcriptome profiling of bovine inner cell mass and trophectoderm derived from in vivo generated blastocysts
    S. M. Hosseini, BMC Developmental Biology - 2015
    Abstract
    This study describes the generation and analysis of the transcriptional profile of bovine inner cell mass (ICM) and trophectoderm (TE), obtained from in vivo developed embryos by using a bovine-embryo specific array (EmbryoGENE) containing 37,238 probes.
    Macrophage and adipocyte IGF1 maintain adipose tissue homeostasis during metabolic stresses
    Hye Rim Chang, Obesity - 2016
    Abstract
    Objective Insulin-like growth factor-1 (IGF1) regulates differentiation and growth of tissues and reduces stress and injury. IGF1 also in a tissue-specific manner modulates the differentiation and lipid storage capacity of adipocytes in vitro, but its roles in adipose tissue development and response to stress are not known. Methods To study IGF1 in vivo, the cellular sources of adipose tissue Igf1 expression were identified and mice were generated with targeted deletion in adipocytes and macrophages. The effects of adipocyte and macrophage deficiency of IGF1 on adipose tissue development and the response to chronic (high-fat feeding) and acute (cold challenge) stress were studied. Results The expression of Igf1 by adipose tissue was derived from multiple cell types including adipocytes and macrophages. In lean animals, adipocytes were the primary source of IGF1, but in obesity expression by adipocytes was reduced and by macrophages increased, so as to maintain overall adipose tissue Igf1 expression. Genetic deletion studies revealed that adipocyte-derived IGF1 regulated perigonadal but not subcutaneous adipose tissue mass during high-fat feeding and the development of obesity. Conversely, macrophage-derived IGF1 acutely modulated perigonadal adipose tissue mass during thermogenic challenges. Conclusions Local IGF1 is not required in lean adipose tissue development but is required to maintain homeostasis during both chronic and acute metabolic stresses.
    Conserved and unique features of the homeologous maize Aux/IAA proteins ROOTLESS WITH UNDETECTABLE MERISTEM 1 and RUM1-like 1
    Yanxiang Zhang, Journal of Experimental Botany - 2015
    Abstract
    The maize (Zea mays L.) Aux/IAA protein RUM1 (ROOTLESS WITH UNDETECTABLE MERISTEM 1) is a key regulator of lateral and seminal root formation. An ancient maize genome duplication resulted in the emergence of its homeolog rum1-like1 (rul1), which displays 92% amino acid sequence identity with RUM1. Both, RUL1 and RUM1 exhibit the canonical four domain structure of Aux/IAA proteins. Moreover, both are localized to the nucleus, are instable and have similar short half-lives of ~23min. Moreover, RUL1 and RUM1 can be stabilized by specific mutations in the five amino acid degron sequence of domain II. In addition, proteins encoded by both genes interact in vivo with auxin response factors (ARFs) such as ZmARF25 and ZmARF34 in protoplasts. Although it was demonstrated that RUL1 and RUM1 can homo and heterodimerize in vivo, rul1 expression is independent of rum1. Moreover, on average rul1 expression is ~84-fold higher than rum1 in the 12 tested tissues and developmental stages, although the relative expression levels in different root tissues are very similar. While RUM1 and RUL1 display conserved biochemical properties, yeast-two-hybrid in combination with BiFC experiments identified a RUM1-associated protein 1 (RAP1) that specifically interacts with RUM1 but not with RUL1. This suggests that RUM1 and RUL1 are at least in part interwoven into different molecular networks.
    FMR6 may play a role in the pathogenesis of fragile X-associated premature ovarian insufficiency
    Shai E. Elizur, Gynecological Endocrinology - 2015
    Abstract
    The aim of this study was to evaluate whether long noncoding RNA accumulation play a role in the pathophysiology of fragile X-associated premature ovarian insufficiency (FXPOI). The study population consisted of 22 consecutive fragile X mental retardation 1 (FMR1) premutation carriers (CGGn 55–199 repeats) undergoing in vitro fertilization and pre-implantation genetic diagnosis (IVF–PGD) treatment. The control group consists of 11 patients, with <55 CGG repeats, undergoing IVF–ICSI for male factor infertility, matched by age, treated in the same period. After oocyte retrieval, granulosa cells from follicular fluid were washed and stored at −80 °C. RNA was transcribed to generate cDNA and the RNA levels were measured using RT–PCR. Transcripts levels in granulosa cells of long noncoding RNA’s FMR4 and FMR6 were measured. In FMR1 premutation carriers there was a significant nonlinear association between the number of CGG repeats and the levels of FMR6 (p = 0.03), but not FMR4. The highest level of FMR6 was seen in women with mid-size CGG repeats (80–120). In addition, a significant negative linear correlation was observed between the number of oocytes retrieved and the RNA levels in granulosa cells of FMR6 (r = −0.53, p = 0.01) but not FMR4. Our study supports previous findings suggesting RNA toxic gain-of-function as one of the possible pathophysiologic mechanisms underlying FXPOI.
    An Immunofluorescence-assisted Microfluidic Single Cell Quantitative Reverse Transcription Polymerase Chain Reaction Analysis of Tumour Cells Separated from Blood
    Kazunori Hoshino, Journal of Circulating Biomarkers - 2015
    Abstract
    Circulating tumour cells (CTCs) are important indicators of metastatic cancer and may provide critical information for individualized treatment. As CTCs are usually very rare, the techniques to obtain information from very small numbers of cells are crucial. Here, we propose a method to perform a single cell quantitative reverse transcription polymerase chain reaction (qPCR) analysis of rare tumour cells. We utilized a microfluidic immunomagnetic assay to separate cancer cells from blood. A combination of detailed immunofluorescence and laser microdissection enabled the precise selection of individual cells. Cancer cells that were spiked into blood were successfully separated and picked up for a single cell PCR analysis. The breast cancer cell lines MCF7, SKBR3 and MDAMB231 were tested with 10 different genes. The result of the single cell analysis matched the results from a few thousand cells. Some markers (e.g., ER, HER2) that are commonly used for cancer identification showed relatively large deviations in expression levels. However, others (e.g., GRB7) showed deviations that are small enough to supplement single cell disease profiling.
    Detection of emergent strains of West Nile virus with a blood screening assay
    Helen M. Faddy, Transfusion - 2015
    Abstract
    BACKGROUND West Nile virus (WNV) is a threat to transfusion safety. WNV Kunjin strain (WNVKUN) is endemic across parts of Australia; however, human infection is believed to be infrequent and is often associated with relatively minor symptoms. A virulent strain, closely related to WNVKUN (termed WNVNSW2011) was recently identified as the etiologic agent of encephalitis in Australian horses. The aim of this project was to investigate whether a commercially available WNV blood screening assay can detect different strains of WNVKUN, including the virulent WNVNSW2011, in human blood donor samples. STUDY DESIGN AND METHODS Plasma samples were spiked with four different strains of WNVKUN, as well as a prototype WNV strain, at high, medium, and low viral loads. Spiking was confirmed with real-time reverse transcription–polymerase chain reaction (RT-PCR), before testing with the Procleix WNV transcription-mediated amplification (TMA) blood screening assay (Grifols). RESULTS All WNV strains used were detectable by RT-PCR after being spiked into plasma. Additionally, all viral spiked samples were reactive by WNV TMA. CONCLUSION We experimentally demonstrate that a commercially available WNV blood screening assay can detect different strains of WNVKUN. Given that WNV can be transfusion transmissible, it is essential to confirm that emergent strains are detectable by existing blood screening methods.
    Blood-Borne Activity-Dependent Neuroprotective Protein (ADNP) is Correlated with Premorbid Intelligence, Clinical Stage, and Alzheimer’s Disease Biomarkers
    Malishkevich, Anna, Journal of Alzheimer's Disease - 2015
    Abstract
    Biomarkers for Alzheimer’s disease (AD) are vital for disease detection in the clinical setting. Discovered in our laboratory, activity-dependent neuroprotective protein (ADNP) is essential for brain formation and linked to cognitive functions. Here, we revealed that blood borne expression of ADNP and its paralog ADNP2 is correlated with premorbid intelligence, AD pathology, and clinical stage. Age adjustment showed significant associations between: 1) higher premorbid intelligence and greater serum ADNP, and 2) greater cortical amyloid and lower ADNP and ADNP2 mRNAs. Significant increases in ADNP mRNA levels were observed in patients ranging from mild cognitive impairment (MCI) to AD dementia. ADNP2 transcripts showed high correlation with ADNP transcripts, especially in AD dementia lymphocytes. ADNP plasma/serum and lymphocyte mRNA levels discriminated well between cognitively normal elderly, MCI, and AD dementia participants. Measuring ADNP blood-borne levels could bring us a step closer to effectively screening and tracking AD.
    High-content screening identifies a role for Na+ channels in insulin production
    Marta Szabat, Royal Society Open Science - 2015
    Abstract
    Insulin production is the central feature of functionally mature and differentiated pancreatic β-cells. Reduced insulin transcription and dedifferentiation have been implicated in type 2 diabetes, making drugs that could reverse these processes potentially useful. We have previously established ratiometric live-cell imaging tools to identify factors that increase insulin promoter activity and promote β-cell differentiation. Here, we present a single vector imaging tool with eGFP and mRFP, driven by the Pdx1 and Ins1 promoters, respectively, targeted to the nucleus to enhance identification of individual cells in a high-throughput manner. Using this new approach, we screened 1120 off-patent drugs for factors that regulate Ins1 and Pdx1 promoter activity in MIN6 β-cells. We identified a number of compounds that positively modulate Ins1 promoter activity, including several drugs known to modulate ion channels. Carbamazepine was selected for extended follow-up, as our previous screen also identified this use-dependent sodium channel inhibitor as a positive modulator of β-cell survival. Indeed, carbamazepine increased Ins1 and Ins2 mRNA in primary mouse islets at lower doses than were required to protect β-cells. We validated the role of sodium channels in insulin production by examining Nav1.7 (Scn9a) knockout mice and remarkably islets from these animals had dramatically elevated insulin content relative to wild-type controls. Collectively, our experiments provide a starting point for additional studies aimed to identify drugs and molecular pathways that control insulin production and β-cell differentiation status. In particular, our unbiased screen identified a novel role for a β-cell sodium channel gene in insulin production.
    Electroporation Enhanced Effect of Dystrophin Splice Switching PNA Oligomers in Normal and Dystrophic Muscle
    Camilla Brolin, Molecular Therapy — Nucleic Acids - 2015
    Abstract
    Peptide nucleic acid (PNA) is a synthetic DNA mimic that has shown potential for discovery of novel splice switching antisense drugs. However, in vivo cellular delivery has been a limiting factor for development, and only few successful studies have been reported. As a possible modality for improvement of in vivo cellular availability, we have investigated the effect of electrotransfer upon intramuscular (i.m.) PNA administration in vivo. Antisense PNA targeting exon 23 of the murine dystrophin gene was administered by i.m. injection to the tibialis anterior (TA) muscle of normal NMRI and dystrophic mdx mice with or without electroporation. At low, single PNA doses (1.5, 3, or 10 µg/TA), electroporation augmented the antisense exon skipping induced by an unmodified PNA by twofold to fourfold in healthy mouse muscle with optimized electric parameters, measured after 7 days. The PNA splice switching was detected at the RNA level up to 4 weeks after a single-dose treatment. In dystrophic muscles of the MDX mouse, electroporation increased the number of dystrophin-positive fibers about 2.5-fold at 2 weeks after a single PNA administration compared to injection only. In conclusion, we find that electroporation can enhance PNA antisense effects in muscle tissue.
    Activation of Autophagy by Unfolded Proteins during Endoplasmic Reticulum Stress
    Xiaochen Yang, The Plant Journal - 2015
    Abstract
    Endoplasmic reticulum stress is defined as the accumulation of unfolded proteins in the endoplasmic reticulum and is caused by conditions such as heat or endoplasmic reticulum stress agents, including tunicamycin (TM) and dithiothreitol (DTT). Autophagy, a major pathway for degradation of macromolecules in the vacuole, is activated by these stress agents in an INOSITOL-REQUIRING ENZYME 1b (IRE1b)-dependent manner and delivers endoplasmic reticulum fragments to the vacuole for degradation. In this study, we examined the mechanism for activation of autophagy during endoplasmic reticulum stress in Arabidopsis thaliana. The chemical chaperones sodium 4-phenylbutyrate and tauroursodeoxycholic acid were found to reduce tunicamycin- or dithiothreitol-induced autophagy, but not autophagy caused by unrelated stresses. Similarly, overexpression of BINDING IMMUNOGLOBULIN PROTEIN (BIP), encoding a heat shock protein 70 (HSP70) molecular chaperone, reduced autophagy. Autophagy activated by heat stress was also found to be partially dependent on IRE1b and to be inhibited by sodium 4-phenylbutyrate, suggesting that heat-induced autophagy is due to accumulation of unfolded proteins in the endoplasmic reticulum. Expression in Arabidopsis of the misfolded protein mimics zeolin or a mutated form of carboxypeptidase Y (CPY*) also induced autophagy in an IRE1b-dependent manner. Moreover, zeolin and CPY* partially co-localized with the autophagic body marker green fluorescent protein-ATG8e, indicating delivery to the vacuole by autophagy. We conclude that accumulation of unfolded proteins in the endoplasmic reticulum is a trigger for autophagy under conditions that cause endoplasmic reticulum stress. This article is protected by copyright. All rights reserved.
    Depletion of elongation initiation factor 4E binding proteins by CRISPR/Cas9 enhances the antiviral response in porcine cells
    Lisbeth Ramírez-Carvajala, Antiviral Research - 2015
    Abstract
    Type I interferons (IFNs) are key mediators of the innate antiviral response in mammalian cells. Elongation initiation factor 4E binding proteins (4E-BPs) are translational controllers of interferon regulatory factor 7 (IRF-7), the “master regulator” of IFN transcription. Previous studies have suggested that mouse cells depleted of 4E-BPs are more sensitive to IFNβ treatment and had lower viral loads as compared to wild type (WT) cells. However, such approach has not been tested as an antiviral strategy in livestock species. In this study, we tested the antiviral activity of porcine cells depleted of 4E-BP1 by a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9) genome engineering system. We found that 4E-BP1 knockout (KO) porcine cells had increased expression of IFNα and β, IFN stimulated genes, and significant reduction in vesicular stomatitis virus titer as compare to WT cells. No phenotypical changes associated with CRISPR/Cas9 manipulation were observed in 4E-BP1 KO cells. This work highlights the use of the CRISPR/Cas9 system to enhance the antiviral response in porcine cells.
    Zinc enhances the number of regulatory T cells in allergen-stimulated cells from atopic subjects
    Eva Rosenkranz, European Journal of Nutrition - 2015
    Abstract
    Purpose The trace element zinc is essential for immune function and its regulation. Since zinc deficiency and allergic hyperresponsive reactions are often accompanied, the influence of zinc on allergen-induced cell growth, CD4+ regulatory T (Treg) cell numbers and cytokine expression during allergic immune reactions was investigated. Methods Peripheral blood mononuclear cells (PBMCs) from non-atopic and atopic subjects were treated with timothy grass allergen pre-incubated with or without zinc. Proliferation was determined by analyzing the incorporation of 3H-thymidine. Intracellular zinc and Foxp3 levels and cell surface antigens were measured by FACS, cytokine expression by ELISA and real-time PCR. Results Incubation with 50 μM zinc sulfate (Zn50) enhances cytosolic zinc concentrations in CD3+ T cells. The data also reveal that the combination of Zn50 plus allergen significantly reduces PBMC proliferation of atopic subjects. Additionally, Zn50 plus allergen enhances Th1 cytokine responses shown by increased interferon (IFN)-γ/interleukin (IL)-10 ratios as well as enhanced tumor necrosis factor-α release. In response to allergen, zinc increases Treg cells and upregulates the mRNA expression of cytotoxic T-lymphocyte antigen-4 in atopic subjects. Interestingly, Zn50 alone leads to an increase of CD4+CD25high(hi)+ cells in atopic and non-atopic subjects. Conclusions Zinc may regulate unwanted hyperresponsive immune reactions by suppressing proliferation through a significant shift from IL-10 to the Th1 cytokine IFN-γ, and enhanced regulatory T cell numbers. Therefore, zinc supplementation may be a promising tool for the therapy of allergies, without negatively affecting the immune system.
    Active FOXO1 is a Key Determinant of Isoform-Specific Progesterone Receptor Transactivation and Senescence Programming
    Caroline H. Diep, Molecular Cancer Research - 2015
    Abstract
    Progesterone promotes differentiation coupled to proliferation and pro-survival in the breast, but inhibits estrogen-driven growth in the reproductive tract and ovaries. Herein, it is demonstrated, using progesterone receptor (PR) isoform-specific ovarian cancer model systems, that PR-A and PR-B promote distinct gene expression profiles that differ from PR-driven genes in breast cancer cells. In ovarian cancer models, PR-A primarily regulates genes independently of progestin, while PR-B is the dominant ligand-dependent isoform. Notably, FOXO1 and the PR/FOXO1 target-gene p21 (CDKN1A) are repressed by PR-A, but induced by PR-B. In the presence of progestin, PR-B, but not PR-A, robustly induced cellular senescence via FOXO1-dependent induction of p21 and p15 (CDKN2B). Chromatin immunoprecipitation (ChIP) assays performed on PR-isoform specific cells demonstrated that while each isoform is recruited to the same PRE-containing region of the p21 promoter in response to progestin, only PR-B elicits active chromatin marks. Overexpression of constitutively active FOXO1 in PR-A-expressing cells conferred robust ligand-dependent upregulation of the PR-B target genes GZMA, IGFBP1, and p21, and induced cellular senescence. In the presence of endogenous active FOXO1, PR-A was phosphorylated on Ser294 and transactivated PR-B at PR-B target genes; these events were blocked by the FOXO1 inhibitor (AS1842856). PR isoform-specific regulation of the FOXO1/p21 axis recapitulated in human primary ovarian tumor explants treated with progestin; loss of progestin sensitivity correlated with high AKT activity. Implications: This study indicates FOXO1 as a critical component for progesterone signaling to promote cellular senescence and reveals a novel mechanism for transcription factor control of hormone sensitivity.
    Migration of CD8+ T Cells into the Central Nervous System Gives Rise to Highly Potent Anti-HIV CD4dimCD8bright T Cells in a Wnt Signaling–Dependent Manner
    Maureen H. Richards, The Journal of Immunology - 2015
    Abstract
    The role of CD8+ T cells in HIV control in the brain and the consequences of such control are unclear. Approximately 3% of peripheral CD8+ T cells dimly express CD4 on their surface. This population is known as CD4dimCD8bright T cells. We evaluated the role of CD4dimCD8bright and CD8 single positive T cells in HIV-infected brain using NOD/SCID/IL-2rcγ−/− mice reconstituted with human PBMCs (NSG-huPBMC). All three T cell populations (CD4 single positive, CD8 single positive, and CD4dimCD8bright) were found in NSG-huPBMC mouse brain within 2 wk of infection. Wnts secreted from astrocytes induced CD4dimCD8bright T cells by 2-fold in vitro. Injection of highly purified CD8 single positive T cells into mouse brain induced CD4dimCD8bright T cells by 10-fold, which were proliferative and exhibited a terminally differentiated effector memory phenotype. Brain CD4dimCD8bright T cells from HIV-infected mice exhibited anti-HIV–specific responses, as demonstrated by induction of CD107ab post exposure to HIV peptide–loaded targets. Further, higher frequency of CD4dimCD8bright T cells (R = −0.62; p ≤ 0.001), but not CD8 single positive T cells (R = −0.24; p ≤ 0.27), negatively correlated with HIV gag mRNA transcripts in HIV-infected NSG-huPBMC brain. Together, these studies indicate that single positive CD8+ T cells entering the CNS during HIV infection can give rise to CD4dimCD8bright T cells, likely through a Wnt signaling–dependent manner, and that these cells are associated with potent anti-HIV control in the CNS. Thus, CD4dimCD8bright T cells are capable of HIV control in the CNS and may offer protection against HIV-associated neurocognitive disorders.
    Anti-inflammatory Effects of Poly-l-lysine in Intestinal Mucosal System Mediated by Calcium-Sensing Receptor Activation
    Yoshinori Mine, Journal of Agricultural and Food Chemistry - 2015
    Abstract
    Calcium-sensing receptor (CaSR) is involved in maintaining cellular homeostasis and promoting recovery of damaged intestinal epithelial cells (IECs). Poly-l-lysine (PL) is a basic polypeptide identified for its role in the activation of CaSR through allosteric binding. The primary goal of the current study was to identify the modulatory effect of PL on intestinal inflammation and to determine whether these effects were mediated by CaSR activation. We used human intestinal epithelial cell lines, Caco-2 and HT-29, to assess PL anti-inflammatory activities in vitro. We found that PL reduced the IL-8 secretion from tumor necrosis factor (TNF)-α-treated human intestinal epithelial cell lines. On the other hand, the gene expression of pro-inflammatory cytokines TNF-α, IL-6, and IL-1? was inhibited by PL supplementation. We subsequently evaluated the anti-inflammatory activity of PL in vivo using a DSS-induced mouse colitis model. PL supplementation was shown to prevent dextran sulfate sodium salt (DSS)-induced loss of weight, colitic symptoms, and shortening of colon length but maintained colonic morphology. The pro-inflammatory cytokine expression in the mouse colon, including TNF-α, IL-6, INF-?, IL-17, and IL-1?, was significantly up-regulated by DSS treatment, but was inhibited upon PL administration. As shown by the results from both in vitro and in vivo studies, the reduction of inflammatory cytokine production caused by PL was reversed by NPS-2143 pretreatment. In the present study, we provide evidence that PL exerts anti-inflammatory effects on the gut system, which is primarily mediated by allosteric ligand activation of CaSR.
    Epithelial-to-mesenchymal transition is not required for lung metastasis but contributes to chemoresistance : Nature : Nature Publishing Group
    Kari R. Fischer, Nature - 2015
    Abstract
    The role of epithelial-to-mesenchymal transition (EMT) in metastasis is a longstanding source of debate, largely owing to an inability to monitor transient and reversible EMT phenotypes in vivo. Here we establish an EMT lineage-tracing system to monitor this process in mice, using a mesenchymal-specific Cre-mediated fluorescent marker switch system in spontaneous breast-to-lung metastasis models. We show that within a predominantly epithelial primary tumour, a small proportion of tumour cells undergo EMT. Notably, lung metastases mainly consist of non-EMT tumour cells that maintain their epithelial phenotype. Inhibiting EMT by overexpressing the microRNA miR-200 does not affect lung metastasis development. However, EMT cells significantly contribute to recurrent lung metastasis formation after chemotherapy. These cells survived cyclophosphamide treatment owing to reduced proliferation, apoptotic tolerance and increased expression of chemoresistance-related genes. Overexpression of miR-200 abrogated this resistance. This study suggests the potential of an EMT-targeting strategy, in conjunction with conventional chemotherapies, for breast cancer treatment.
    CDX2 downregulation is associated with poor differentiation and MMR deficiency in colon cancer
    J. Olsen, Experimental and Molecular Pathology - 2015
    Abstract
    Background Homeobox genes are often deregulated in cancer and can have both oncogenic and tumor-suppressing potential. The Caudal-related homeobox transcription factor 2 (CDX2) is an intestine-specific transcription factor. CDX2 has been implicated in differentiation, proliferation, cell adhesion, and migration. In this study, we investigated CDX2 mRNA and protein expression in relation to the clinicopathological characteristics of colon cancer, including mismatch repair status and recurrence risk. Methods Tumor samples were obtained from colon cancer patients. Biopsies from tumor tissue and normal adjacent tissue were fixed in liquid nitrogen for RNA extraction or in formalin and paraffin embedded (FFPE) for immunohistochemical staining. CDX2 mRNA expression was evaluated by RT-qPCR. FFPE sections were stained for MLH1, MSH2, MSH6, PMS2, and CDX2. Results A total of 191 patient samples were included in the study and analyzed by immunohistochemistry. Of these samples, 97 were further evaluated by RT-qPCR. There was no significant difference in CDX2 mRNA expression between tumor and normal tissues. CDX2 mRNA expression was significantly lower in right-sided tumors (p < 0.05), poorly differentiated tumors (p < 0.05), and MMR-deficient tumors (p < 0.05). Similarly, CDX2 protein expression was more often low or absent in right-sided tumors (p < 0.01), poorly differentiated tumors (p < 0.001), and MMR-deficient tumors (p < 0.001). Low CDX2 protein or mRNA expression was not associated with recurrence risk. Conclusion We found that CDX2 downregulation is associated with MMR deficiency, right-sided tumors, and poor differentiation at both the mRNA and protein level. Whether CDX2 plays an active role in tumor progression in MSI/MMR-deficient tumors remains to be elucidated.
    Induction of lytic cycle sensitizes Epstein-Barr virus infected B cells to NK cell killing that is counteracted by virus-mediated NK cell evasion mechanisms in late lytic cycle
    Luke R. Williams, Journal of Virology - 2015
    Abstract
    Epstein-Barr Virus (EBV) persists for the lifetime of the infected host despite eliciting strong immune responses. This persistence requires a fine balance between the host immune system and EBV immune evasion. Accumulating evidence suggests an important role for natural killer (NK) cells in this balance. NK cells can kill EBV infected cells undergoing lytic replication in-vitro and studies in both humans, and mice with reconstituted human immune systems have shown NK cells can limit EBV replication and prevent infectious mononucleosis. We now show that NK cells, via NKG2D and DNAM-1 interactions, recognize and kill EBV infected cells undergoing lytic replication, and that expression of a single EBV lytic gene, BZLF1, is sufficient to trigger sensitization to NK cell killing. We also present evidence suggesting the possibility of the existence of an as yet unidentified DNAM-1 ligand which may be particularly important for killing lytically infected normal B cells. Furthermore, whilst cells entering lytic cycle become sensitized to NK cell killing, we observed that cells in late lytic cycle are highly resistant. We identified expression of the vBcl-2 protein, BHRF1, as one effective mechanism by which EBV mediates this protection. Thus, contrary to the view expressed in some reports, EBV has evolved the ability to evade NK cell responses. IMPORTANCE This report extends our understanding of the interaction between EBV and host innate responses. It provides the first evidence that the susceptibility to NK cell lysis of EBV infected B cells undergoing lytic replication is dependent upon the phase of lytic cycle. Induction of lytic cycle is associated with acquired sensitization to NK cell killing, while progress through late lytic cycle is associated with acquired resistance to killing. We provide mechanistic explanations for this novel observation, implicating important roles for the BZLF1 immediate-early transactivator, the BHRF1 vBcl-2 homologue, and a novel ligand for the DNAM-1 NK cell receptor.
    Chronic exposure to low doses of lipopolysaccharide and high‐fat feeding increases body mass without affecting glucose tolerance in female rats
    Anete Dudele, Physiological Reports - 2015
    Abstract
    Obesity‐related inflammation may have a causal role in the development of diabetes and insulin resistance, and studies using animal models of chronic experimental endotoxemia have shown the link. However, many studies use only males, and much less is known about the role of obesity‐related inflammation in females. Therefore, we addressed how experimentally induced chronic inflammation affects body mass, energy intake, and glucose metabolism in female rats. Adult female Sprague Dawley rats were instrumented with slow release pellets that delivered a constant daily dose of 53 or 207 μg of lipopolysaccharide (LPS) per rat for 60 days. Control rats were instrumented with vehicle pellets. Due to inflammatory nature of high‐fat diet (HFD) half of the rats received HFD (60% of calories from lard), while the other half remained on control diet to detect possible interactions between two modes of induced inflammation. Our results showed that chronic LPS administration increased female rat body mass and calorie intake in a dose‐dependent manner, and that HFD further exacerbated these effects. Despite these effects, no effects of LPS and HFD were evident on female rat glucose metabolism. Only LPS elevated expression of inflammatory markers in the hypothalamus. To conclude, female rats respond to experimentally induced chronic inflammation by increasing body mass, but do not develop glucose intolerance in the given period of time.