Functional integration of a semi-synthetic azido-queuosine derivative into translation and a tRNA modification circuitAbstract
Substitution of the queuine nucleobase precursor preQ1 by an azide-containing derivative (azido-propyl-preQ1) led to incorporation of this clickable chemical entity into tRNA via transglycosylation in vitro as well as in vivo in Escherichia coli, Schizosaccharomyces pombe and human cells. The resulting semi-synthetic RNA modification, here termed Q-L1, was present in tRNAs on actively translating ribosomes, indicating functional integration into aminoacylation and recruitment to the ribosome. The azide moiety of Q-L1 facilitates analytics via click conjugation of a fluorescent dye, or of biotin for affinity purification. Combining the latter with RNAseq showed that TGT maintained its native tRNA substrate specificity in S. pombe cells. The semi-synthetic tRNA modification Q-L1 was also functional in tRNA maturation, in effectively replacing the natural queuosine in its stimulation of further modification of tRNAAsp with 5-methylcytosine at position 38 by the tRNA methyltransferase Dnmt2 in S. pombe. This is the first demonstrated in vivo integration of a synthetic moiety into an RNA modification circuit, where one RNA modification stimulates another. In summary, the scarcity of queuosinylation sites in cellular RNA, makes our synthetic q/Q system a ‘minimally invasive’ system for placement of a non-natural, clickable nucleobase within the total cellular RNA.
Imp interacts with Lin28 to regulate adult stem cell proliferation in the Drosophila intestineAbstract
Stem cells are essential for the development and long-term maintenance of tissues and organisms. Preserving tissue homeostasis requires exquisite control of all aspects of stem cell function: cell potency, proliferation, fate decision and differentiation. RNA binding proteins (RBPs) are essential components of the regulatory network that control gene expression in stem cells to maintain self-renewal and long-term homeostasis in adult tissues. While the function of many RBPs may have been characterized in various stem cell populations, how these interact and are organized in genetic networks remains largely elusive. In this report, we show that the conserved RNA binding protein IGF2 mRNA binding protein (Imp) is expressed in intestinal stem cells (ISCs) and progenitors in the adult Drosophila midgut. We demonstrate that Imp is required cell autonomously to maintain stem cell proliferative activity under normal epithelial turnover and in response to tissue damage. Mechanistically, we show that Imp cooperates and directly interacts with Lin28, another highly conserved RBP, to regulate ISC proliferation. We found that both proteins bind to and control the InR mRNA, a critical regulator of ISC self-renewal. Altogether, our data suggests that Imp and Lin28 are part of a larger gene regulatory network controlling gene expression in ISCs and required to maintain epithelial homeostasis.
Mitochondrial complex I dysfunction alters the balance of soluble and membrane-bound TNF during chronic experimental colitisAbstract
Inflammatory bowel disease (IBD) is a complex, chronic, relapsing and heterogeneous disease induced by environmental, genomic, microbial and immunological factors. MCJ is a mitochondrial protein that regulates the metabolic status of macrophages and their response to translocated bacteria. Previously, an acute murine model of DSS-induced colitis showed increased disease severity due to MCJ deficiency. Unexpectedly, we now show that MCJ-deficient mice have augmented tumor necrosis factor α converting enzyme (TACE) activity in the context of chronic inflammation. This adaptative change likely affects the balance between soluble and transmembrane TNF and supports the association of the soluble form and a milder phenotype. Interestingly, the general shifts in microbial composition previously observed during acute inflammation were absent in the chronic model of inflammation in MCJ-deficient mice. However, the lack of the mitochondrial protein resulted in increased alpha diversity and the reduction in critical microbial members associated with inflammation, such as Ruminococcus gnavus, which could be associated with TACE activity. These results provide evidence of the dynamic metabolic adaptation of the colon tissue to chronic inflammatory changes mediated by the control of mitochondrial function.
Tissue remodeling by an opportunistic pathogen triggers allergic inflammationAbstract
Different effector arms of the immune system are optimized to protect from different classes of pathogens. In some cases, pathogens manipulate the host immune system to promote the wrong type of effector response—a phenomenon known as immune deviation. Typically, immune deviation helps pathogens to avoid destructive immune responses. Here, we report on a type of immune deviation whereby an opportunistic pathogen, Pseudomonas aeruginosa (P. aeruginosa), induces the type 2 immune response resulting in mucin production that is used as an energy source by the pathogen. Specifically, P. aeruginosa-secreted toxin, LasB, processed and activated epithelial amphiregulin to induce type 2 inflammation and mucin production. This “niche remodeling” by P. aeruginosa promoted colonization and, as a by-product, allergic sensitization. Our study thus reveals a type of bacterial immune deviation by increasing nutrient supply. It also uncovers a mechanism of allergic sensitization by a bacterial virulence factor.
Cross-tissue and generation predictability of relative Wolbachia densities in the mosquito Aedes aegyptiAbstract
The insect endosymbiotic bacterium Wolbachia is being deployed in field populations of the mosquito Aedes aegypti for biological control. This microbe prevents the replication of human disease-causing viruses inside the vector, including dengue, Zika and chikungunya. Relative Wolbachia densities may in part predict the strength of this ‘viral blocking’ effect. Additionally, Wolbachia densities may affect the strength of the reproductive manipulations it induces, including cytoplasmic incompatibility (CI), maternal inheritance rates or induced fitness effects in the insect host. High rates of CI and maternal inheritance and low rates of fitness effects are also key to the successful spreading of Wolbachia through vector populations and its successful use in biocontrol. The factors that control Wolbachia densities are not completely understood.
We used quantitative PCR-based methods to estimate relative density of the Wolbachia wAlbB strain in both the somatic and reproductive tissues of adult male and female mosquitoes, as well as in eggs. Using correlation analyses, we assessed whether densities in one tissue predict those in others within the same individual, but also across generations.
We found little relationship among the relative Wolbachia densities of different tissues in the same host. The results also show that there was very little relationship between Wolbachia densities in parents and those in offspring, both in the same and different tissues. The one exception was with ovary–egg relationships, where there was a strong positive association. Relative Wolbachia densities in reproductive tissues were always greater than those in the somatic tissues. Additionally, the densities were consistent in females over their lifetime regardless of tissue, whereas they were generally higher and more variable in males, particularly in the testes.
Our results indicate that either stochastic processes or local tissue-based physiologies are more likely factors dictating Wolbachia densities in Ae. aegypti individuals, rather than shared embryonic environments or heritable genetic effects of the mosquito genome. These findings have implications for understanding how relative Wolbachia densities may evolve and/or be maintained over the long term in Ae. aegypti.
A versatile transposon-based technology to generate loss- and gain-of-function phenotypes in the mouse liverAbstract
Understanding the contribution of gene function in distinct organ systems to the pathogenesis of human diseases in biomedical research requires modifying gene expression through the generation of gain- and loss-of-function phenotypes in model organisms, for instance, the mouse. However, methods to modify both germline and somatic genomes have important limitations that prevent easy, strong, and stable expression of transgenes. For instance, while the liver is remarkably easy to target, nucleic acids introduced to modify the genome of hepatocytes are rapidly lost, or the transgene expression they mediate becomes inhibited due to the action of effector pathways for the elimination of exogenous DNA. Novel methods are required to overcome these challenges, and here we develop a somatic gene delivery technology enabling long-lasting high-level transgene expression in the entire hepatocyte population of mice.
We exploit the fumarylacetoacetate hydrolase (Fah) gene correction-induced regeneration in Fah-deficient livers, to demonstrate that such approach stabilizes luciferase expression more than 5000-fold above the level detected in WT animals, following plasmid DNA introduction complemented by transposon-mediated chromosomal gene transfer. Building on this advancement, we created a versatile technology platform for performing gene function analysis in vivo in the mouse liver. Our technology allows the tag-free expression of proteins of interest and silencing of any arbitrary gene in the mouse genome. This was achieved by applying the HADHA/B endogenous bidirectional promoter capable of driving well-balanced bidirectional expression and by optimizing in vivo intronic artificial microRNA-based gene silencing. We demonstrated the particular usefulness of the technology in cancer research by creating a p53-silenced and hRas G12V-overexpressing tumor model.
We developed a versatile technology platform for in vivo somatic genome editing in the mouse liver, which meets multiple requirements for long-lasting high-level transgene expression. We believe that this technology will contribute to the development of a more accurate new generation of tools for gene function analysis in mice.
Oxidative Stress and X-ray Exposure Levels-Dependent Survival and Metabolic Changes in Murine HSPCsAbstract
Haematopoietic bone marrow cells are amongst the most sensitive to ionizing radiation (IR), initially resulting in cell death or genotoxicity that may later lead to leukaemia development, most frequently Acute Myeloid Leukaemia (AML). The target cells for radiation-induced Acute Myeloid Leukaemia (rAML) are believed to lie in the haematopoietic stem and progenitor cell (HSPC) compartment. Using the inbred strain CBA/Ca as a murine model of rAML, progress has been made in understanding the underlying mechanisms, characterisation of target cell population and responses to IR. Complex regulatory systems maintain haematopoietic homeostasis which may act to modulate the risk of rAML. However, little is currently known about the role of metabolic factors and diet in these regulatory systems and modification of the risk of AML development. This study characterises cellular proliferative and clonogenic potential as well as metabolic changes within murine HSPCs under oxidative stress and X-ray exposure. Ambient oxygen (normoxia; 20.8% O2) levels were found to increase irradiated HSPC-stress, stimulating proliferative activity compared to low oxygen (3% O2) levels. IR exposure has a negative influence on the proliferative capability of HSPCs in a dose-dependent manner (0–2 Gy) and this is more pronounced under a normoxic state. One Gy x-irradiated HSPCs cultured under normoxic conditions displayed a significant increase in oxygen consumption compared to those cultured under low O2 conditions and to unirradiated HSPCs. Furthermore, mitochondrial analyses revealed a significant increase in mitochondrial DNA (mtDNA) content, mitochondrial mass and membrane potential in a dose-dependent manner under normoxic conditions. Our results demonstrate that both IR and normoxia act as stressors for HSPCs, leading to significant metabolic deregulation and mitochondrial dysfunctionality which may affect long term risks such as leukaemia.
Evolution of a novel cell type in Dictyostelia required gene duplication of a cudA-like transcription factorAbstract
The evolution of novel cell types has been proposed to result from duplication of gene regulatory networks, but proven examples are rare. In addition to stalk cells and spores that make up the fruiting bodies of three major groups of Dictyostelia, those in group 4 additionally evolved basal disc and cup cells that respectively anchor the stalk to the substratum and the spore mass to the stalk. We noted a putative group-4-specific duplication of a cudA-like transcription factor (TF) in a comparative analysis of group-representative genomes. Using increased taxon sampling, we here confirmed that this TF, cdl1, duplicated into cdl1a and cdl1b in the common ancestor to group 4. cdl1a, but not cdl1b, showed signatures of positive selection, indicative of functional innovation. Deletion of cdl1a in Dictyostelium discoideum resulted in fruiting bodies with sagging spore heads that lacked the supporting cup cells and expression of cup-specific genes. Deletion of cdl1b resulted in thinner fruiting body stalks, while a cdl1b−cdl1a− double knockout showed more severe stalk defects, suggesting an ancestral role of cdl1 in stalk formation. This was confirmed in a closely related non-group 4 species, Polysphondylium violaceum, where cdl1 knockout caused defective stalk formation. These data indicate that the group-specific duplication of cdl1 and subsequent diversification of cdl1a played a pivotal role in the evolution of a novel somatic cell type in group 4 Dictyostelia.
Effect of Periodized Resistance Training on Skeletal Muscle During Androgen Deprivation Therapy for Prostate Cancer: A Pilot Randomized TrialAbstract
Purpose: Prostate cancer survivors (PCS) receive androgen deprivation therapy (ADT) as treatment for recurrent cancer, yet ADT is associated with loss of skeletal muscle and physical function. Resistance training can counter both muscle and physical function loss; however, an understanding of the molecular responses of skeletal muscle to resistance training during ADT is still undefined. This sub-analysis of the original randomized, controlled pilot trial investigated effects of 12 weeks of periodized resistance training on mRNA expression of the anabolic genes IGF-1, myogenin, PGC-1α4 and the catabolic genes myostatin and MuRF-1 in skeletal muscle of PCS on ADT. Secondary aims investigated if changes in lean mass and physical function correlated with changes in mRNA expression. Methods: PCS on ADT (n=17) were randomized to 12 weeks of supervised resistance training (EXE, n=9) or home-based stretching (STRETCH, n=8) 3 days per week. Outcomes were assessed at baseline and post-intervention. Muscle biopsies were analyzed by RT-PCR for mRNA expression. Body composition was assessed through dual-energy X-ray absorptiometry, and physical function through muscular strength, timed up and go, stair climb, and 400m walk. Results: MuRF-1 mRNA expression was significantly greater in EXE compared to STRETCH post-intervention (P=.005). Change in MuRF-1 mRNA expression significantly correlated with improvements in strength and physical function (P < .05), while change in IGF-1 expression correlated with change in lean mass (P =.015). Conclusion: Twelve weeks of resistance training increased mRNA expression of MuRF-1 in skeletal muscle of PCS on ADT. Elevations in resting mRNA expression of IGF-1, myogenin and PGC-1α4, and reduction in mRNA expression of myostatin that are typically expected following resistance training were not observed.
ENTRY AND EARLY INFECTION OF NON-SEGMENTED NEGATIVE SENSE RNA VIRUSESAbstract
Paramyxoviruses, pneumoviruses, and other non-segmented negative
sense (NNS) RNA viruses have historically been of public health concern.
Although their genomes are typically small (up to 19kbs) they are able to inflict
large-scale detrimental pathologies on host cells. Human metapneumovirus
(HMPV) is a widespread pathogen and is a NNS RNA virus. HMPV results
respiratory tract infections and is particularly dangerous for preterm infants, the
elderly, and immunocompromised individuals. Other viruses within the NNS
RNA virus order include the deadly Ebola, Hendra, and Nipah viruses (EBOV,
HeV, and NiV), as well as the re-emerging measles virus (MeV). Despite their
public impact, there are currently very limited available FDA-approved
therapeutics and antivirals against NNS RNA viruses.
During the infectious cycle, viral surface glycoproteins play critical roles
in establishing infection. For most NNS RNA viruses, the attachment protein is
important for the tethering of a viral membrane to host cells, while the fusion
protein is responsible for the membrane merger of the virus and host. The fusion
protein of paramyxo-and pneumovirus proteins are class I proteins that are
folded into trimers, must be proteolytically cleaved to be functional, and are held
in a metastable prefusion conformation until the signal for fusion occurs. Upon
being signaled, the fusion protein undergoes dramatic essentially irreversible
conformational changes for membrane mixing. Because of its important role in
starting infection, F has garnered interest as a potentially powerful target against
infection. For paramyxoviruses, the ectodomain regions of F have been wellstudied; however, the hydrophobic nature of the transmembrane domain (TMD)
of the protein has resulted in difficulties in crystallization. To address this,
several biochemical assays have been utilized to address the function of the
TMDs of paramyxo-and pneumovirus fusion proteins. Although initially thought
to be solely a membrane anchor, the transmembrane domains of several
viruses have been shown to be important for the functionality of fusion proteins.
For some paramyxoviruses, replacement of the proteinaceous TMD resulted in
the premature triggering. Further studies showed that the TMDs of
paramyxoviruses and several other viral F proteins exist in isolation as trimers,
and these trimeric associations in turn drive trimeric associations of the full
protein. Studies of the HeV F TMD in isolation identified a leucine/isoleucine
(L/I) zipper as an important motif for TMD-TMD trimerization. Mutations to this
L/I zipper motif in the context of the full protein resulted in reduced surface
expression, and a loss of functionality. The L/I zipper was found to be present
in 140 paramyxo- and pneumovirus fusion protein TMDs. This work examines
whether the importance of the wh iimporether L/Izipper in the context of another paramyxvovirus. Weused the model system, PIV5 F to dissect the role of the TMD L/I zipper
fusogenic activity. We found that the (L/I) zipper plays important roles in in expression and
functionality of the PIV5 F protein, but not surface expression of the protein.
Following membrane merging, a series of events occur that facilitate the
release of viral contents into the host cell. The NNS RNA carried by the virus
into the cell is used as a template for viral replication and transcription; two
important steps in generation of viral progeny. In the life cycle of NNS viruses,
viral proteins assume multi-functional roles to optimize their replication and
spread. One of the key players during the course of infection is the matrix
protein (M). The matrix protein has been identified as a master regulator of viral
infection with most studies focusing on its roles in late-stage infection, during
assembly and budding of viral progeny. The matrix proteins of many enveloped
viruses have been shown to associate in high order oligomers to form a gridlike array underneath the plasma membrane, where they can induce
membrane curvature to allow for the budding of viral particles. Not surprisingly,
the absence of M in some NNS RNA viruses results in a significant viral titer
decrease. Interestingly, some recent studies show that the matrix protein has
other critical roles in viral infection such as immune modulation and host cell
translation antagonism. One of these newly uncovered roles for viral matrix
proteins involves the regulation of viral RNA synthesis. Studies with EBOV and
MeV demonstrate that the matrix protein is involved in early infection events,
as inhibits viral replication. To study the roles of the HMPV M protein in early
infection, we performed a spatiotemporal analysis of M in HMPV-infected cells.
We noted the presence of HMPV M within the nucleus during early infection.
Our knockdown studies of HMPV M indicate that HMPV M is a positive
regulator of viral replication and transcription, as in its absence, the rates of
mRNA and viral genomic RNA synthesis are dramatically reduced. Additionally,
within the NNS RNA virus order, HMPV M is the only matrix protein found to
bind calcium. We created alanine mutants to the calcium coordinating residues
of HMPV M and found that these residues were important in properly folding
the protein. Together, these findings contribute to our understanding of the
mechanisms of NNS RNA viral infection
Polycomb-group recruitment to a Drosophila target gene is the default state that is inhibited by a transcriptional activatorAbstract
Polycomb-group (PcG) proteins are epigenetic regulators that maintain the transcriptional repression of target
genes following their initial repression by transcription factors. PcG target genes are repressed in some cells, but
active in others. Therefore, a mechanism must exist by which PcG proteins distinguish between the repressed and
active states and only assemble repressive chromatin environments at target genes that are repressed. Here, we
present experimental evidence that the repressed state of a Drosophila PcG target gene, giant (gt), is not identified by the presence of a repressor. Rather, de novo establishment of PcG-mediated silencing at gt is the default
state that is prevented by the presence of an activator or coactivator, which may inhibit the catalytic activity of
Polycomb-repressive complex 2 (PRC2).
Mutations that increase expression of the EmrAB-TolC efflux pump confer increased resistance to nitroxoline in Escherichia coliAbstract
To determine the mechanism of resistance to the antibiotic nitroxoline in Escherichia coli.
Spontaneous nitroxoline-resistant mutants were selected at different concentrations of nitroxoline. WGS and strain reconstruction were used to define the genetic basis for the resistance. The mechanistic basis of resistance was determined by quantitative PCR (qPCR) and by overexpression of target genes. Fitness costs of the resistance mutations and cross-resistance to other antibiotics were also determined.
Mutations in the transcriptional repressor emrR conferred low-level resistance to nitroxoline [nitroxoline MIC (MICNOX)=16 mg/L] by increasing the expression of the emrA and emrB genes of the EmrAB-TolC efflux pump. These resistant mutants showed no fitness reduction and displayed cross-resistance to nalidixic acid. Second-step mutants with higher-level resistance (MICNOX=32–64 mg/L) had mutations in the emrR gene, together with either a 50 kb amplification, a mutation in the gene marA, or an IS upstream of the lon gene. The latter mutations resulted in higher-level nitroxoline resistance due to increased expression of the tolC gene, which was confirmed by overexpressing tolC from an inducible plasmid in a low-level resistance mutant. Furthermore, the emrR mutations conferred a small increase in resistance to nitrofurantoin only when combined with an nfsAB double-knockout mutation. However, nitrofurantoin-resistant nfsAB mutants showed no cross-resistance to nitroxoline.
Mutations in different genes causing increased expression of the EmrAB-TolC pump lead to an increased resistance to nitroxoline. The structurally similar antibiotics nitroxoline and nitrofurantoin appear to have different modes of action and resistance mechanisms.
Sphingomyelin-Based Nanosystems (SNs) for the Development of Anticancer miRNA TherapeuticsAbstract
Gene replacement therapy with oncosuppressor microRNAs (miRNAs) is a promising alternative to interfere with cancer progression. However, miRNAs are highly inefficient in a biological environment, hampering a successful translation to clinics. Nanotechnology can tackle this drawback by providing delivery systems able to efficiently deliver them to cancer cells. Thus, the objective of this work was to develop biocompatible nanosystems based on sphingomyelin (SM) for the intracellular delivery of miRNAs to colorectal cancer cells. We pursued two different approaches to select the most appropriate composition for miRNA delivery. On the one hand, we prepared sphingomyelin-based nanosystems (SNs) that incorporate the cationic lipid stearylamine (ST) to support the association of miRNA by the establishment of electrostatic interactions (SNs–ST). On the other hand, the cationic surfactant (DOTAP) was used to preform lipidic complexes with miRNA (Lpx), which were further encapsulated into SNs (SNs-Lpx). Restitution of miRNA145 levels after transfection with SNs-Lpx was related to the strongest anticancer effect in terms of tumor proliferation, colony forming, and migration capacity assays. Altogether, our results suggest that SNs have the potential for miRNA delivery to develop innovative anticancer therapies.
THROUGH THE LOOKING GLASS: Real-Time Imaging in Brachypodium Roots and Osmotic Stress AnalysisAbstract
To elucidate dynamic developmental processes in plants, live tissues and organs must be visualised frequently and for extended periods. The development of roots is studied at a cellular resolution not only to comprehend the basic processes fundamental to maintenance and pattern formation but also study stress tolerance adaptation in plants. Despite technological advancements, maintaining continuous access to samples and simultaneously preserving their morphological structures and physiological conditions without causing damage presents hindrances in the measurement, visualisation and analyses of growing organs including plant roots. We propose a preliminary system which integrates the optical real-time visualisation through light microscopy with a liquid culture which enables us to image at the tissue and cellular level horizontally growing Brachypodium roots every few minutes and up to 24 h. We describe a simple setup which can be used to track the growth of the root as it grows including the root tip growth and osmotic stress dynamics. We demonstrate the system’s capability to scale down the PEG-mediated osmotic stress analysis and collected data on gene expression under osmotic stress.
α-Lipoic Acid Reduces Iron-induced Toxicity and Oxidative Stress in a Model of Iron OverloadAbstract
Iron toxicity is associated with organ injury and has been reported in various clinical
conditions, such as hemochromatosis, thalassemia major, and myelodysplastic syndromes. Therefore, iron chelation therapy represents a pivotal therapy for these patients during their lifetime. The aim of the present study was to assess the iron chelating properties of α-lipoic acid (ALA) and how such an effect impacts on iron overload mediated toxicity. Human mesenchymal stem cells (HS-5) and animals (zebrafish, n = 10 for each group) were treated for 24 h with ferric ammonium citrate (FAC, 120 µg/mL) in the presence or absence of ALA (20 µg/mL). Oxidative stress was evaluated by reduced glutathione content, reactive oxygen species formation, mitochondrial dysfunction, and gene expression of heme oxygenase-1b and mitochondrial superoxide dismutase; organ injury, iron accumulation, and autophagy were measured by microscopical, cytofluorimetric analyses, and inductively coupled plasma-optical mission Spectrometer (ICP-OES). Our results showed that FAC results in a significant increase of tissue iron accumulation, oxidative stress, and autophagy and such detrimental effects were reversed by ALA treatment. In conclusion, ALA possesses excellent iron chelating properties that may be exploited in a clinical setting for organ preservation, as well as exhibiting a good safety profile and low cost for the national health system.
Single-Cell Heterogeneity Analysis and CRISPR Screen Identify Key β-Cell-Specific Disease GenesAbstract
Identification of human disease signature genes typically requires samples from many donors to achieve statistical significance. Here, we show that single-cell heterogeneity analysis may overcome this hurdle by significantly improving the test sensitivity. We analyzed the transcriptome of 39,905 single islets cells from 9 donors and observed distinct β cell heterogeneity trajectories associated with obesity or type 2 diabetes (T2D). We therefore developed RePACT, a sensitive single-cell analysis algorithm to identify both common and specific signature genes for obesity and T2D. We mapped both β-cell-specific genes and disease signature genes to the insulin regulatory network identified from a genome-wide CRISPR screen. Our integrative analysis discovered the previously unrecognized roles of the cohesin loading complex and the NuA4/Tip60 histone acetyltransferase complex in regulating insulin transcription and release. Our study demonstrated the power of combining single-cell heterogeneity analysis and functional genomics to dissect the etiology of complex diseases.
Rhodnius prolixus: Identification of missing components of the IMD immune signaling pathway and functional characterization of its role in eliminating bacteriaAbstract
The innate immune system in insects is regulated by specific signalling pathways. Most immune related pathways were identified and characterized in holometabolous insects such as Drosophila melanogaster, and it was assumed they would be highly conserved in all insects. The hemimetabolous insect, Rhodnius prolixus, has served as a model to study basic insect physiology, but also is a major vector of the human parasite, Trypanosoma cruzi, that causes 10,000 deaths annually. The publication of the R. prolixus genome revealed that one of the main immune pathways, the Immune-deficiency pathway (IMD), was incomplete and probably non-functional, an observation shared with other hemimetabolous insects including the pea aphid (Acyrthosiphon pisum) and the bedbug (Cimex lectularius). It was proposed that the IMD pathway is inactive in R. prolixus as an adaptation to prevent eliminating beneficial symbiont gut bacteria. We used bioinformatic analyses based on reciprocal BLAST and HMM-profile searches to find orthologs for most of the “missing” elements of the IMD pathway and provide data that these are regulated in response to infection with Gram-negative bacteria. We used RNAi strategies to demonstrate the role of the IMD pathway in regulating the expression of specific antimicrobial peptides (AMPs) in the fat body of R. prolixus. The data indicate that the IMD pathway is present and active in R. prolixus, which opens up new avenues of research on R. prolixus-T. cruzi interactions.
TCO, a Putative Transcriptional Regulator in Arabidopsis, Is a Target of the Protein Kinase CK2Abstract
As multicellular organisms grow, spatial and temporal patterns of gene expression are strictly regulated to ensure that developmental programs are invoked at appropriate stages. In this work, we describe a putative transcriptional regulator in Arabidopsis, TACO LEAF (TCO), whose overexpression results in the ectopic activation of reproductive genes during vegetative growth. Isolated as an activation-tagged allele, tco-1D displays gene misexpression and phenotypic abnormalities, such as curled leaves and early flowering, characteristic of chromatin regulatory mutants. A role for TCO in this mode of transcriptional regulation is further supported by the subnuclear accumulation patterns of TCO protein and genetic interactions between tco-1D and chromatin modifier mutants. The endogenous expression pattern of TCO and gene misregulation in tco loss-of-function mutants indicate that this factor is involved in seed development. We also demonstrate that specific serine residues of TCO protein are targeted by the ubiquitous kinase CK2. Collectively, these results identify TCO as a novel regulator of gene expression whose activity is likely influenced by phosphorylation, as is the case with many chromatin regulators.
Octopus maya white body show sex-specific transcriptomic profiles during the reproductive phase, with high differentiation in signaling pathwaysAbstract
White bodies (WB), multilobulated soft tissue that wraps the optic tracts and optic lobes, have been considered the hematopoietic organ of the cephalopods. Its glandular appearance and its lobular morphology suggest that different parts of the WB may perform different functions, but a detailed functional analysis of the octopus WB is lacking. The aim of this study is to describe the transcriptomic profile of WB to better understand its functions, with emphasis on the difference between sexes during reproductive events. Then, validation via qPCR was performed using different tissues to find out tissue-specific transcripts. High differentiation in signaling pathways was observed in the comparison of female and male transcriptomic profiles. For instance, the expression of genes involved in the androgen receptorsignaling pathway were detected only in males, whereas estrogen receptor showed higher expression in females. Highly expressed genes in males enriched oxidation-reduction and apoptotic processes, which are related to the immune response. On the other hand, expression of genes involved in replicative senescence and the response to cortisol were only detected in females. Moreover, the transcripts with higher expression in females enriched a wide variety of signaling pathways mediated by molecules like neuropeptides, integrins, MAPKs and receptors like TNF and Toll-like. In addition, these putative neuropeptide transcripts, showed higher expression in females’ WB and were not detected in other analyzed tissues. These results suggest that the differentiation in signaling pathways in white bodies of O. maya influences the physiological dimorphism between females and males during the reproductive phase.
Specific sequences of infectious challenge lead to secondary hemophagocytic lymphohistiocytosis-like disease in miceAbstract
Secondary hemophagocytic lymphohistiocytosis (sHLH) is a highly mortal complication associated with sepsis. In adults, it is often seen in the setting of infections, especially viral infections, but the mechanisms that underlie pathogenesis are unknown. sHLH is characterized by a hyperinflammatory state and the presence hemophagocytosis. We found that sequential challenging of mice with a nonlethal dose of viral toll-like receptor (TLR) agonist followed by a nonlethal dose of TLR4 agonist, but not other permutations, produced a highly lethal state that recapitulates many aspects of human HLH. We found that this hyperinflammatory response could be recapitulated in vitro in bone marrow-derived macrophages. RNA sequencing analyses revealed dramatic up-regulation of the red-pulp macrophage lineage-defining transcription factor SpiC and its associated transcriptional program, which was also present in bone marrow macrophages sorted from patients with sHLH. Transcriptional profiling also revealed a unique metabolic transcriptional profile in these macrophages, and immunometabolic phenotyping revealed impaired mitochondrial function and oxidative metabolism and a reliance on glycolytic metabolism. Subsequently, we show that therapeutic administration of the glycolysis inhibitor 2-deoxyglucose was sufficient to rescue animals from HLH. Together, these data identify a potential mechanism for the pathogenesis of sHLH and a potentially useful therapeutic strategy for its treatment.
Impact of malaria and hepatitis B co-infection on clinical and cytokine profiles among pregnant womenAbstract
The overlap of malaria and chronic hepatitis B (CHB) is common in endemic regions, however, it is not known if this co-infection could adversely influence clinical and immunological responses. This study investigated these interactions in pregnant women reporting to antenatal clinics in Ghana.
Clinical parameters (hemoglobin, liver function biomarker, peripheral malaria parasitemia, and hepatitis B viremia) and cytokine profiles were assayed and compared across four categories of pregnant women: un-infected, mono-infected with Plasmodium falciparum (Malaria group), mono-infected with chronic hepatitis B virus (CHB group) and co-infected (Malaria+CHB group).
Women with Malaria+CHB maintained appreciably normal hemoglobin levels (mean±SEM = 10.3±0.3 g/dL). That notwithstanding, Liver function test showed significantly elevated levels of alanine aminotransferase, aspartate aminotransferase and total bilirubin [P<0.001 for all comparisons]. Similarly, the Malaria+CHB group had significantly elevated pro-inflammatory cytokines, including tumour necrosis factor alpha (TNF-α), interleukin (IL)-1β, and IL-6 [P<0.05 for all comparisons]. In women with Malaria+CHB, correlation analysis showed significant negative association of the pro-inflammatory cytokines responses with malaria parasitemia [IL-1β (P<0.001; r = -0.645), IL-6 (P = 0.046; r = -0.394) and IL-12 (P = 0.011; r = -0.49)]. On the other hand, the pro-inflammatory cytokine levels positively correlated with HBV viremia [TNF-α (P = 0.004; r = 0.549), IL-1β (P<0.001; r = 0.920), IL-6 (P<0.001; r = 0.777), IFN-γ (P = 0.002; r = 0.579), IL-2 (P = 0.008; r = 0.512) and IL-12 (P<0.001; r = 0.655)]. Also, for women in the Malaria+CHB group, parasitemia was observed to diminish HBV viremia [P = 0.003, r = -0.489].
Put together the findings suggests that Malaria+CHB could exacerbate inflammatory cytokine responses and increase susceptibility to liver injury among pregnant women in endemic settings.
Selection for novel metabolic capabilities in Salmonella entericaAbstract
Bacteria are known to display extensive metabolic diversity and many studies have shown that they can use an extensive repertoire of small molecules as carbon‐ and energy sources. However, it is less clear to what extent a bacterium can expand its existing metabolic capabilities by acquiring mutations that, for example, rewire its metabolic pathways. To investigate this capability and potential for evolution of novel phenotypes, we sampled large populations of mutagenized Salmonella enterica to select very rare mutants that can grow on minimal media containing 124 low molecular weight compounds as sole carbon sources. We found mutants growing on 18 of these novel carbon sources, and identified the causal mutations that allowed growth for four of them. Mutations that relieve physiological constraints or increase expression of existing pathways were found to be important contributors to the novel phenotypes. For the remaining 14 novel phenotypes, whole genome sequencing of independent mutants and genetic analysis suggested that these novel metabolic phenotypes result from a combination of multiple mutations. This work, by virtue of identifying the genetic and mechanistic basis for new metabolic capabilities, sheds light on the properties of adaptive landscapes underlying the evolution of novel phenotypes.
Bacterial diet and weak cadmium stress affect the survivability of Caenorhabditis elegans and its resistance to severe stressAbstract
Stress may have negative or positive effects in dependence of its intensity (hormesis). We studied this phenomenon in Caenorhabditis elegans by applying weak or severe abiotic (cadmium, CdCl2) and/or biotic stress (different bacterial diets) during cultivation/breeding of the worms and determining their developmental speed or survival and performing transcriptome profiling and RT-qPCR analyses to explore the genetic basis of the detected phenotypic differences. To specify weak or severe stress, developmental speed was measured at different cadmium concentrations, and survival assays were carried out on different bacterial species as feed for the worms. These studies showed that 0.1 μmol/L or 10 mmol/L of CdCl2 were weak or severe abiotic stressors, and that E. coli HT115 or Chitinophaga arvensicola feeding can be considered as weak or severe biotic stress. Extensive phenotypic studies on wild type (WT) and different signaling mutants (e.g., kgb-1Δ and pmk-1Δ) and genetic studies on WT revealed, inter alia, the following results. WT worms bred on E. coli OP50, which is a known cause of high lipid levels in the worms, showed high resistance to severe abiotic stress and elevated gene expression for protein biosynthesis. WT worms bred under weak biotic stress (E. coli HT115 feeding which causes lower lipid levels) showed an elevated resistance to severe biotic stress, elevated gene expression for the innate immune response and signaling but reduced gene expression for protein biosynthesis. WT worms bred under weak biotic and abiotic stress (E. coli HT115 feeding plus 0.1 μmol/L of CdCl2) showed high resistance to severe biotic stress, elevated expression of DAF-16 target genes (e.g., genes for small heat shock proteins) but further reduced gene expression for protein biosynthesis. WT worms bred under weak biotic but higher abiotic stress (E. coli HT115 feeding plus 10 μmol/L of CdCl2) showed re-intensified gene expression for the innate immune response, signaling, and protein biosynthesis, which, however, did not caused a higher resistance to severe biotic stress. E. coli OP50 feeding as well as weak abiotic and biotic stress during incubations also improved the age-specific survival probability of adult WT worms. Thus, this study showed that a bacterial diet resulting in higher levels of energy resources in the worms (E. coli OP50 feeding) or weak abiotic and biotic stress promote the resistance to severe abiotic or biotic stress and the age-specific survival probability of WT.
Progesterone decreases gut permeability through upregulating occludin expression in primary human gut tissues and Caco-2 cellsAbstract
Progesterone plays a protective role in preventing inflammation and preterm delivery during pregnancy. However, the mechanism involved is unknown. Microbial product translocation from a permeable mucosa is demonstrated as a driver of inflammation. To study the mechanism of the protective role of progesterone during pregnancy, we investigated the effect of physiologic concentrations of progesterone on tight junction protein occludin expression and human gut permeability in vitro and systemic microbial translocation in pregnant women in vivo. Plasma bacterial lipopolysaccharide (LPS), a representative marker of in vivo systemic microbial translocation was measured. We found that plasma LPS levels were significantly decreased during 24 to 28 weeks of gestation compared to 8 to 12 weeks of gestation. Moreover, plasma LPS levels were negatively correlated with plasma progesterone levels but positively correlated with plasma tumor necrosis factor-alpha (TNF-α) levels at 8 to 12 weeks of gestation but not at 24 to 28 weeks of gestation. Progesterone treatment increased intestinal trans-epithelial electrical resistance (TEER) in primary human colon tissues and Caco-2 cells in vitro through upregulating tight junction protein occludin expression. Furthermore, progesterone exhibited an inhibitory effect on nuclear factor kappa B (NF-κB) activation following LPS stimulation in Caco-2 cells. These results reveal a novel mechanism that progesterone may play an important role in decreasing mucosal permeability, systemic microbial translocation, and inflammation during pregnancy.
Chronic liver injury drives non‐traditional intrahepatic fibrin(ogen) crosslinking via tissue transglutaminaseAbstract
Intravascular fibrin clots and extravascular fibrin deposits are often implicated in the progression of liver fibrosis. However, evidence supporting a pathological role of fibrin in hepatic fibrosis is indirect and based largely on studies using anticoagulant drugs that inhibit activation of the coagulation protease thrombin, which has other downstream targets that promote fibrosis. Therefore, the goal of this study was to determine the precise role of fibrin deposits in experimental hepatic fibrosis.
Liver fibrosis was induced in mice expressing mutant fibrinogen insensitive to thrombin‐mediated proteolysis (i.e. locked in the monomeric form), termed FibAEK mice, and factor XIII A2 subunit‐deficient (FXIII−/−) mice. Female wild‐type mice, FXIII−/− mice and homozygous FibAEK mice were challenged with carbon tetrachloride (CCl4) twice weekly for 4 weeks or 6 weeks (1 mL kg−1, intraperitoneal).
Hepatic injury and fibrosis induced by CCl4 challenge were unaffected by FXIII deficiency or inhibition of thrombin‐catalyzed fibrin polymer formation (in FibAEK mice). Surprisingly, hepatic deposition of crosslinked fibrin(ogen) was not reduced in CCl4‐challenged FXIII−/− mice or FibAEK mice as compared with wild‐type mice. Rather, deposition of crosslinked hepatic fibrin(ogen) following CCl4 challenge was dramatically reduced in tissue transglutaminase‐2 (TGM2)‐deficient (TGM2−/−) mice. However, the reduction in crosslinked fibrin(ogen) in TGM2−/− mice did not affect CCl4‐induced liver fibrosis.
These results indicate that neither traditional fibrin clots, formed by the thrombin–activated FXIII pathway nor atypical TGM2‐crosslinked fibrin(ogen) contribute to experimental CCl4‐induced liver fibrosis. Collectively, the results indicate that liver fibrosis occurs independently of intrahepatic fibrin(ogen) deposition.
MiRNA-27a sensitizes breast cancer cells to treatment with Selective Estrogen Receptor ModulatorsAbstract
MicroRNA-27a (miR-27a) is a small non-coding RNA, shown to play a role in multiple cancers, including the regulation of ERα expression in breast cancer. Most ERα positive tumors are treated with Selective Estrogen Receptor Modulators (SERMs) and thus the role of miR-27a expression in response to SERM treatment is of interest.
Tamoxifen resistant cells were generated by molecular evolution with six cycles of tamoxifen treatment. MCF7 and T47D luminal A breast cancer cell lines were either treated with miR-27a mimics, or ER-signaling was modulated ectopically. The changes were analyzed with RT-qPCR, western blotting and transcriptional activity ERE-reporter assays. Moreover, the response to SERM treatments (tamoxifen, endoxifen and toremifen) was investigated by cell viability and apoptosis measurements. An in silico analysis of survival data from the METABRIC study was performed in order to assess the prognostic value of miR-27a for response to SERM treatment.
Tamoxifen-resistant cells showed decreased expression of ERα and miR-27a. The overexpression of miR-27a increased the levels of ERα, while modulation of ERα decreased miR-27a expression. High miR-27a expression increased the sensitivity of MCF7 and T47D cells to SERM treatments and re-sensitized the cells to tamoxifen. Patient survival of luminal A breast cancer patients that underwent endocrine therapies was better in groups with high miR-27a expression.
MiR-27a sensitizes luminal A breast cancer cells to SERM treatments based on a positive feedback loop with ERα. An increased overall-survival of ER-positive breast cancer patients that underwent endocrine treatments and displayed high miR-27a levels was found.
Presence of Circulating miR-145, miR-155, and miR-382 in Exosomes Isolated from Serum of Breast Cancer Patients and Healthy DonorsAbstract
miR-145, miR-155, and miR-382 have been proposed as noninvasive biomarkers to distinguish breast cancer patients from healthy individuals. However, it is unknown if these three miRNAs are secreted by exosomes. Thus, we hypothesized that miR-145, miR-155, and miR-382 in breast cancer patients are present in exosomes. We isolated exosomes from serum of breast cancer patients and healthy donors, then we characterized them according to their shape, size, and exosome markers by scanning electron microscopy, atomic force microscopy, nanoparticle tracking analysis (NTA), and Western blot and determined the exosome concentration in all samples by NTA. Later, exosomal small RNA extraction was done to determine the expression levels of miR-145, miR-155, and miR-382 by qRT-PCR. We observed a round shape of exosomes with a mean size of 119.84 nm in breast cancer patients and 115.4 nm in healthy donors. All exosomes present the proteins CD63, Alix, Tsg, CD9, and CD81 commonly used as markers. Moreover, we found a significantly high concentration of exosomes in breast cancer patients with stages I, III, and IV compared to healthy donors. We detected miR-145, miR-155, and miR-382 in the exosomes isolated from serum of breast cancer patients and healthy donors. Our results show that the exosomes isolated from the serum of breast cancer patients and healthy donors contains miR-145, miR-155, and miR-382 but not in a selective manner in breast cancer patients. Moreover, our data support the association between exosome concentration and the presence of breast cancer, opening the possibility to study how miRNAs packaged into exosomes play a role in BC progression.
Quantitative PCR Measurement of miR-371a-3p and miR-372-p Is Influenced by HemolysisAbstract
Cell-free microRNAs have been reported as biomarkers for several diseases. For testicular germ cell tumors (GCT), circulating microRNAs 371a-3p and 372-3p in serum and plasma have been proposed as biomarkers for diagnostic and disease monitoring purposes. The most widely used method for quantification of specific microRNAs in serum and plasma is reverse transcriptase real-time quantitative PCR (RT-qPCR) by the comparative Ct-method. In this method one or several reference genes or reference microRNAs are needed in order to normalize and calculate the relative microRNA levels across samples. One of the pitfalls in analysis of microRNAs from serum and plasma is the release of microRNAs from blood cells during hemolysis. This is an important issue because varying degrees of hemolysis are not uncommon in routine blood sampling. Thus, hemolysis must be taken into consideration when working with circulating microRNAs from blood. miR-93-5p, miR-30b-5p, and miR-20a-5p have been reported as reference microRNA in analysis of the miR-371a-373 cluster. We here show how these three microRNAs are influenced by hemolysis. We also propose a new reference microRNA, miR-191-5p, which is relatively stable in serum samples with mild hemolysis. In addition, we show how hemolysis can have effect on the reported microRNA levels in patient samples when these reference microRNAs are used in samples with varying levels of hemolysis.
On resolving ambiguities in microbial community analysis of partial nitritation anammox reactorsAbstract
PCR-based methods have caused a surge for integration of eco-physiological approaches into research on partial nitritation anammox (PNA). However, a lack of rigorous standards for molecular analyses resulted in widespread data misinterpretation and consequently lack of consensus. Data consistency and accuracy strongly depend on the primer selection and data interpretation. An in-silico evaluation of 16S rRNA gene eubacterial primers used in PNA studies from the last ten years unraveled the difficulty of comparing ecological data from different studies due to a variation in the coverage of these primers. Our 16S amplicon sequencing approach, which includes parallel sequencing of six 16S rRNA hypervariable regions, showed that there is no perfect hypervariable region for PNA microbial communities. Using qPCR analysis, we emphasize the significance of primer choice for quantification and caution with data interpretation. We also provide a framework for PCR based analyses that will improve and assist to objectively interpret and compare such results.
Metformin induces the AP-1 transcription factor network in normal dermal fibroblastsAbstract
Metformin is a widely-used treatment for type 2 diabetes and is reported to extend health and lifespan as a caloric restriction (CR) mimetic. Although the benefits of metformin are well documented, the impact of this compound on the function and organization of the genome in normal tissues is unclear. To explore this impact, primary human fibroblasts were treated in culture with metformin resulting in a significant decrease in cell proliferation without evidence of cell death. Furthermore, metformin induced repositioning of chromosomes 10 and 18 within the nuclear volume indicating altered genome organization. Transcriptome analyses from RNA sequencing datasets revealed that alteration in growth profiles and chromosome positioning occurred concomitantly with changes in gene expression profiles. We further identified that different concentrations of metformin induced different transcript profiles; however, significant enrichment in the activator protein 1 (AP-1) transcription factor network was common between the different treatments. Comparative analyses revealed that metformin induced divergent changes in the transcriptome than that of rapamycin, another proposed mimetic of CR. Promoter analysis and chromatin immunoprecipitation assays of genes that changed expression in response to metformin revealed enrichment of the transcriptional regulator forkhead box O3a (FOXO3a) in normal human fibroblasts, but not of the predicted serum response factor (SRF). Therefore, we have demonstrated that metformin has significant impacts on genome organization and function in normal human fibroblasts, different from those of rapamycin, with FOXO3a likely playing a role in this response.
Cytoprotective effects of Avenathramide C against oxidative and inflammatory stress in normal human dermal fibroblastsAbstract
Natural polyphenols are promising anti-aging compounds not only for their antioxidant activity, but also their ability to activate specific cellular pathways mediating the aging process. Avenanthramide C (Avn C), found exclusively in oats, is a natural antioxidant associated with free radical scavenging; however, it is how this compound elicits other protective effects. We investigated the intracellular antioxidant activity of Avn C and other cytoprotective potential in normal human skin fibroblasts exposed to extracellular stress. Avn C reduced H2O2-induced oxidative stress by reducing intracellular free radical levels and antioxidant gene transcripts. Avn C also resulted in decreased levels of gene transcripts encoding pro-inflammatory cytokines in response to H2O2 or tumor necrosis factor-α (TNF-α). This reduction in cytokine gene transcription occurred concomitantly with reduced phosphorylated nuclear factor-κB (NF-κB) p65, and decreased NF-κB DNA binding. Avn C further induced heme oxygense-1 (HO-1) expression through increased Nrf2 DNA binding activity, demonstrating a second mechanism by which Avn C attenuates cellular stress. Collectively, our findings indicate that Avn C protects normal human skin fibroblasts against oxidative stress and inflammatory response through NF-κB inhibition and Nrf2/HO-1 activation
Three Types of Functional Regulatory T Cells Control T Cell Responses at the Human Maternal-Fetal InterfaceAbstract
During pregnancy, maternal regulatory T cells (Tregs) are important in establishing immune tolerance to invading fetal extravillous trophoblasts (EVTs). CD25HIFOXP3+ Tregs are found at high levels in decidual tissues and have been shown to suppress fetus-specific and nonspecific responses. However, limited data are available on additional decidual Treg types and the mechanisms by which they are induced. This study investigated three distinct decidual CD4+ Treg types in healthy pregnancies with a regulatory phenotype and the ability to suppress T cell responses: CD25HIFOXP3+, PD1HIIL-10+, and TIGIT+FOXP3dim. Moreover, co-culture of HLA-G+ EVTs or decidual macrophages with blood CD4+ T cells directly increased the proportions of CD25HIFOXP3+ Tregs compared to T cells cultured alone. EVTs also increased PD1HI Tregs that could be inhibited by HLA-C and CD3 antibodies, suggesting an antigen-specific induction. The presence of distinct Treg types may allow for the modulation of a variety of inflammatory responses in the placenta.
Surmounting Cytarabine-resistance in acute myeloblastic leukemia cells and specimens with a synergistic combination of hydroxyurea and azidothymidineAbstract
Acute myeloid leukemia (AML) patients display dismal prognosis due to high prevalence of refractory and relapsed disease resulting from chemoresistance. Treatment protocols, primarily based on the anchor drug Cytarabine, remained chiefly unchanged in the past 50 years with no standardized salvage regimens. Herein we aimed at exploring potential pre-clinical treatment strategies to surmount Cytarabine resistance in human AML cells. We established Cytarabine-resistant sublines derived from human leukemia K562 and Kasumi cells, and characterized the expression of Cytarabine-related genes using real-time PCR and Western blot analyses to uncover the mechanisms underlying their Cytarabine resistance. This was followed by growth inhibition assays and isobologram analyses testing the sublines’ sensitivity to the clinically approved drugs hydroxyurea (HU) and azidothymidine (AZT), compared to their parental cells. All Cytarabine-resistant sublines lost deoxycytidine kinase (dCK) expression, rendering them refractory to Cytarabine. Loss of dCK function involved dCK gene deletions and/or a novel frameshift mutation leading to dCK transcript degradation via nonsense-mediated decay. Cytarabine-resistant sublines displayed hypersensitivity to HU and AZT compared to parental cells; HU and AZT combinations exhibited a marked synergistic growth inhibition effect on leukemic cells, which was intensified upon acquisition of Cytarabine-resistance. In contrast, HU and AZT combination showed an antagonistic effect in non-malignant cells. Finally, HU and AZT synergism was demonstrated on peripheral blood specimens from AML patients. These findings identify a promising HU and AZT combination for the possible future treatment of relapsed and refractory AML, while sparing normal tissues from untoward toxicity.
CYLD Regulates Centriolar Satellites Proteostasis by Counteracting the E3 Ligase MIB1Abstract
The tumor suppressor CYLD is a deubiquitinatingenzyme that removes non-degradative ubiquitin link-ages bound to a variety of signal transduction adap-tors. CYLD participates in the formation of primarycilia, a microtubule-based structure that protrudesfrom the cell body to act as a ‘‘sensing antenna.’’Yet, how exactly CYLD regulates ciliogenesis is notfully understood. Here, we conducted an unbiasedproteomic screen of CYLD binding partners andidentified components of the centriolar satellites.These small granular structures, tethered to the scaf-fold protein pericentriolar matrix protein 1 (PCM1),gravitate toward the centrosome and orchestrateciliogenesis. CYLD knockdown promotes PCM1degradation and the subsequent dismantling of thecentriolar satellites. We found that CYLD marshalsthe centriolar satellites by deubiquitinating andpreventing the E3 ligase Mindbomb 1 (MIB1) frommarking PCM1 for proteasomal degradation. Theseresults link CYLD to the regulation of centriolar satel-lites proteostasis and provide insight into howreversible ubiquitination finely tunes ciliogenesis.
Disruption of Intestinal Homeostasis and Intestinal Microbiota During Experimental Autoimmune UveitisAbstract
Purpose: We determine the changes in intestinal microbiota and/or disruptions in intestinal homeostasis during uveitis.
Methods: Experimental autoimmune uveitis (EAU) was induced in B10.RIII mice with coadministration of interphotoreceptor retinoid-binding protein peptide (IRBP) and killed mycobacterial antigen (MTB) as an adjuvant. Using 16S rRNA gene sequencing, we looked at intestinal microbial differences during the course of uveitis, as well as intestinal morphologic changes, changes in intestinal permeability by FITC-dextran leakage, antimicrobial peptide expression in the gastrointstinal tract, and T lymphocyte prevalence before and at peak intraocular inflammation.
Results: We demonstrate that increased intestinal permeability and antimicrobial peptide expression in the intestinal tract coincide in timing with increased effector T cells in the mesenteric lymph nodes, during the early stages of uveitis, before peak inflammation. Morphologic changes in the intestine were most prominent during this phase, but also occurred with adjuvant MTB alone, whereas increased intestinal permeability was found only in IRBP-immunized mice that develop uveitis. We also demonstrate that the intestinal microbiota were altered during the course of uveitis, and that some of these changes are specific to uveitic animals, whereas others are influenced by adjuvant MTB alone. Intestinal permeability peaked at 2 weeks, coincident with an increase in intestinal bacterial strain differences, peak lipocalin production, and peak uveitis.
Conclusions: An intestinal dysbiosis accompanies a disruption in intestinal homeostasis in autoimmune uveitis, although adjuvant MTB alone promotes intestinal disruption as well. This may indicate a novel axis for future therapeutic targeting experimentally or clinically.
Tart Cherry Prevents Bone Loss through Inhibition of RANKL in TNF-Overexpressing MiceAbstract
Current drugs for the treatment of rheumatoid arthritis-associated bone loss come with concerns about their continued use. Thus, it is necessary to identify natural products with similar effects, but with fewer or no side effects. We determined whether tart cherry (TC) could be used as a supplement to prevent inflammation-mediated bone loss in tumor necrosis factor (TNF)-overexpressing transgenic (TG) mice. TG mice were assigned to a 0%, 5%, or 10% TC diet, with a group receiving infliximab as a positive control. Age-matched wild-type (WT) littermates fed a 0% TC diet were used as a normal control. Mice were monitored by measurement of body weight. Bone health was evaluated via serum biomarkers, microcomputed tomography (µCT), molecular assessments, and mechanical testing. TC prevented TNF-mediated weight loss, while it did not suppress elevated levels of interleukin (IL)-1β and IL-6. TC also protected bone structure from inflammation-induced bone loss with a reduced ratio of receptor activator of nuclear factor kappa-B ligand (RANKL)/osteoprotegerin (OPG) to a degree comparable to infliximab. Furthermore, unlike with infliximab, TC exhibited a moderate improvement in TNF-mediated decline in bone stiffness. Thus, TC could be used as a prophylactic regimen against future fragility fractures in the context of highly chronic inflammation.
Insulin-like Growth Factor (IGF)-1 treatment stabilizes the microvascularcytoskeleton under ischemic conditionsAbstract
Our previous studies showed that Insulin-like Growth Factor (IGF)-1 reduced blood brain barrier permeabilityand decreased infarct volume caused by middle cerebral artery occlusion (MCAo) in middle aged female rats.Similarly, cultures of primary brain microvessel endothelial cells from middle-aged female rats and exposed tostroke-like conditions (oxygen glucose deprivation; OGD) confirmed that IGF-1 reduced dye transfer across thiscell monolayer. Surprisingly, IGF-1 did not attenuate endothelial cell death caused by OGD. To reconcile thesefindings, the present study tested the hypothesis that, at the earliest phase of ischemia, IGF-1 promotes barrierfunction by increasing anchorage and stabilizing cell geometry of surviving endothelial cells. Cultures of humanbrain microvessel endothelial cells were subject to oxygen-glucose deprivation (OGD) in the presence of IGF-1,IGF-1 + JB-1 (IGFR inhibitor) or vehicle. OGD disrupted the cell monolayer and reduced cell-cell interactions,which was preserved in IGF-1-treated cultures and reversed by concurrent treatment with JB-1. IGF-1-mediatedpreservation of the endothelial monolayer was reversed with LY294002 treatment, but not by Rapamycin, in-dicating that IGF-1 s actions on cell-cell contacts are likely mediated via the PI3K pathway. In vivo, microvesselmorphology was evaluated in middle-aged female rats that were subjected to ischemia by MCAo, and treated ICVwith IGFeI, IGF-1 + JB-1, or artificial CSF (aCSF; vehicle) after reperfusion. Compared to vehicle controls, IGF-1treated animals displayed larger microvessel diameters in the peri-infarct area and increased staining density forvinculin, an anchorage protein. Both these measures were reversed by concurrent IGF-1 + JB-1 treatment.Moreover these effects were restricted to 24 h after ischemia-reperfusion and no treatment effects were seen at5d post stroke. Collectively, these data suggest that in the earliest hours during ischemia, IGF-1 promotes re-ceptor-mediated anchorage of endothelial cells, and its actions may be accurately characterized as vasculo-protective.
High diagnostic yield and novel variants in very late-onset spasticityAbstract
Hereditary spastic paraplegias (HSPs) are a diverse group of genetic conditions with variable severity
and onset age. From a neurogenetic clinic, we identified 14 patients with very late-onset HSP, with
symptoms starting after the age of 35. In this cohort, sequencing of known genetic causes was performed using clinically available HSP sequencing panels. We identified 4 patients with mutations in
SPG7 and 3 patients with SPAST mutations, representing 50% of the cohort and indicating a very high
diagnostic yield. In the SPG7 group, we identified novel variants in two patients. We have also identified two novel mutations in the SPAST group. We present sequencing data from cDNA and RT-qPCR to
support the pathogenicity of these variants, and provide observations regarding the poor genotypephenotype correlation in these conditions that should be the subject of future study
Three-Dimensional Printed Polylactic Acid Scaffolds Promote Bonelike Matrix Deposition in VitroAbstract
Large bone defects represent a significant challenge for
clinicians and surgeons. Tissue engineering for bone regeneration represents
an innovative solution for this dilemma and may yield attractive alternate bone
substitutes. Three-dimensional (3D) printing with inexpensive desktop printers
shows promise in generating high-resolution structures mimicking native
tissues using biocompatible, biodegradable, and cost-effective thermoplastics,
which are already FDA-approved for food use, drug delivery, and many medical
devices. Microporous 3D-printed polylactic acid scaffolds, with different pore
sizes (500, 750, and 1000 μm), were designed and manufactured using an
inexpensive desktop 3D printer, and the mechanical properties were assessed.
The scaffolds were compared for cell growth, activity, and bone-like tissue
formation using primary human osteoblasts. Osteoblasts showed high
proliferation, metabolic activity, and osteogenic matrix protein production, in
which 750 μm pore-size scaffolds showed superiority. Further experimentation
using human mesenchymal stem cells on 750 μm pore scaffolds showed their ability in supporting osteogenic differentiation.
These findings suggest that even in the absence of any surface modifications, low-cost 750 μm pore-size 3D-printed scaffolds
may be suitable as a bone substitute for repair of large bone defects.
Biallelic CCM3 mutations cause a clonogenic survival advantage and endothelial cell stiffeningAbstract
CCM3, originally described as PDCD10, regulates blood‐brain barrier integrity and vascular maturation in vivo. CCM3 loss‐of‐function variants predispose to cerebral cavernous malformations (CCM). Using CRISPR/Cas9 genome editing, we here present a model which mimics complete CCM3 inactivation in cavernous endothelial cells (ECs) of heterozygous mutation carriers. Notably, we established a viral‐ and plasmid‐free crRNA:tracrRNA:Cas9 ribonucleoprotein approach to introduce homozygous or compound heterozygous loss‐of‐function CCM3 variants into human ECs and studied the molecular and functional effects of long‐term CCM3 inactivation. Induction of apoptosis, sprouting, migration, network and spheroid formation were significantly impaired upon prolonged CCM3 deficiency. Real‐time deformability cytometry demonstrated that loss of CCM3 induces profound changes in cell morphology and mechanics: CCM3‐deficient ECs have an increased cell area and elastic modulus. Small RNA profiling disclosed that CCM3 modulates the expression of miRNAs that are associated with endothelial ageing. In conclusion, the use of CRISPR/Cas9 genome editing provides new insight into the consequences of long‐term CCM3 inactivation in human ECs and supports the hypothesis that clonal expansion of CCM3‐deficient dysfunctional ECs contributes to CCM formation.
Leishmania-derived trimannose modulates inflammatory response to significantly reduce Leishmania (L.) major-induced lesionsAbstract
Leishmania lipophosphoglycan (LPG) is a key virulence factor, initiating inflammation resulting in cutaneous lesions. LPG is capped by various oligosaccharides. How these glycans are recognized and how they alter the course of Leishmania infection is poorly understood. Previous studies synthesized α-1,2-trimannose cap sugars on latex beads demonstrated that C57BL/6 mice co-inoculated with L. major and trimannose-coated beads produced significantly higher levels of IL-12 p40 and other pro-inflammatory, type 1 cytokines compared L. major infection alone within the first 48 h of infection. However, as L. major infection typically progress over weeks to months, the role of trimannose in altering disease progression over the course of infection was unknown. Wild-type mice were inoculated with either trimannose or carrier (uncoated) beads, infected with L. major alone, co-inoculated with carrier beads and L. major, or co-inoculated with trimannose beads and L. major. Trimannose treatment of L. major-infected mice decreased parasite load and significantly decreased lesion size at 14 days post infection (pi) compared to non-treated, infected mice. Infected, trimannose-treated mice had decreased IL-12p40 and IL-10 secretion and increased IFN-γ at 14 days pi. Mice lacking the ability to detect trimannose, mannose-receptor deleted mice (MR-/-), when treated with trimannose beads and infected with L. major did not have decreased lesion size. Leishmania-derived trimannose represents a novel immunomodulator that provides early type 1-skewed cytokine production to control parasite load and alter the course of cutaneous leishmaniasis.
A comparative evaluation of crowding stress on muscle HSP90 and myostatin expression in salmonidsAbstract
Stress is a major factor that contributes to poor production and animal welfare concerns in aquaculture. As such, a thorough understanding of mechanisms involved in the stress response is imperative to developing strategies to mitigate the negative side effects of stressors, including the impact of high stocking densities on growth. The purpose of this study was to determine how the muscle growth inhibitor, myostatin, and the stress-responsive gene HSP90 are regulated in response to crowding stress in rainbow trout (Oncorhynchus mykiss), cutthroat trout (Oncorhynchus clarki), brook trout (Salvelinus fontinalis), and Atlantic salmon (Salmo salar). All species exhibited higher cortisol and glucose levels following the handling stress, indicating physiological response to the treatment. Additionally, all species, except rainbow trout, exhibited higher HSP90 levels in muscle after a 48h crowding stress. Crowding stress resulted in a decrease of myostatin-1a in brook trout white muscle but not red muscle, while, myostatin-1a and -2a levels increased in white muscle and myostatin-1b levels increased in red muscle in Atlantic salmon. In rainbow trout, no significant changes were detected in either muscle type, but myostatin-1a was upregulated in both white and red skeletal muscle in the closely related cutthroat trout. The variation in response to crowding suggests a complex and species-specific interaction between stress and the muscle gene regulation in these salmonids. Only Atlantic salmon and cutthroat trout exhibited increased muscle myostatin transcription, and also exhibited the largest increase in circulating glucose in response to crowding. These results suggest that species-specific farming practices should be carefully examined in order to optimize low stress culture conditions.
T regulatory cell induced Foxp3 binds the IL2, IFNγ, and TNFα promoters in virus-specific CD8+ T cells from feline immunodeficiency virus infected cats.Abstract
Polyfunctional CD8+ T cells play a critical role in controlling viremia during AIDS lentiviral infections. However, for most HIV infected individuals, virus-specific CD8+ T cells exhibit loss of polyfunctionality including loss of IL2, TNFα, and IFNγ. Using the feline immunodeficiency virus (FIV) model for AIDS lentiviral persistence, our laboratory has demonstrated that FIV-activated Treg cells target CD8+ T cells, leading to a reduction in IL2 and IFNγ production. Further, we have demonstrated that Treg cells induce expression of the repressive transcription factor, Foxp3 in CD8+ T cells. Based upon these findings, we asked if Treg-induced Foxp3 could bind to the IL2, TNFα, and IFNγ promoter regions in virus-specific CD8+ T cells. Following coculture with autologous Treg cells, we demonstrated decreased mRNA levels of IL2 and IFNγ at weeks 4 and 8 post-infection and decreased TNFα at week 4 post infection in virus-specific CD8+ T cells. We also clearly demonstrated Treg cell induced Foxp3 expression in virus-specific CD8+ T cells at weeks 1, 4, and 8 post-infection. Finally, we documented Foxp3 binding to the IL2, TNFα and IFNγ promoters at 8 weeks and 6 months post-infection in virus-specific CD8+ T cells following Treg cell coculture. In summary, the results here clearly demonstrate that Foxp3 inhibits IL2, TNFα and IFNγ transcription by binding to their promoter regions in lentivirus-specific CD8+ T cells. We believe this is the first description of this process during the course of AIDS lentiviral infection.
H3K27 Methylation Dynamics during CD4 T Cell Activation: Regulation of JAK/STAT and IL12RB2 Expression by JMJD3Abstract
The changes to the epigenetic landscape in response to Ag during CD4 T cell activation have not been well characterized. Although CD4 T cell subsets have been mapped globally for numerous epigenetic marks, little has been done to study their dynamics early after activation. We have studied changes to promoter H3K27me3 during activation of human naive and memory CD4 T cells. Our results show that these changes occur relatively early (1 d) after activation of naive and memory cells and that demethylation is the predominant change to H3K27me3 at this time point, reinforcing high expression of target genes. Additionally, inhibition of the H3K27 demethylase JMJD3 in naive CD4 T cells demonstrates how critically important molecules required for T cell differentiation, such as JAK2 and IL12RB2, are regulated by H3K27me3. Our results show that H3K27me3 is a dynamic and important epigenetic modification during CD4 T cell activation and that JMJD3-driven H3K27 demethylation is critical for CD4 T cell function.
Dietary broccoli impacts microbial community structure and attenuates chemically induced colitis in mice in an Ah receptor dependent mannerAbstract
Consumption of broccoli mediates numerous chemo-protective benefits through the intake of phytochemicals, some of which modulate aryl hydrocarbon receptor (AHR) activity. Whether AHR activation is a critical aspect of the therapeutic potential of dietary broccoli is not known. Here we administered isocaloric diets, with or without supplementation of whole broccoli (15% w/w), to congenic mice expressing the high-affinity Ahrb/b or low-affinity Ahrd/d alleles, for 24 days and examined the effects on AHR activity, intestinal microbial community structure, inflammatory status, and response to chemically induced colitis. Cecal microbial community structure and metabolic potential were segregated according to host dietary and AHR status. Dietary broccoli associated with heightened intestinal AHR activity, decreased microbial abundance of the family Erysipelotrichaceae, and attenuation of colitis. In summary, broccoli consumption elicited an enhanced response in ligand-sensitive Ahrb/b mice, demonstrating that in part the beneficial aspects of dietary broccoli upon intestinal health are associated with heightened AHR activity.
Reptin regulates insulin-stimulated Akt phosphorylation in hepatocellular carcinoma via the regulation of SHP-1/PTPN6Abstract
Hepatocellular carcinoma (HCC) is the main primary cancer of the liver. Many studies have shown that insulin resistance is a risk factor for HCC. We previously discovered the overexpression and oncogenic role of the Reptin/RUVBL2 ATPase in HCC. Here, we found that Reptin silencing enhanced insulin sensitivity in 2 HCC cell lines, as shown by a large potentiation of insulin-induced AKT phosphorylation on Ser473 and Thr308, and of downstream signalling. Reptin silencing did not affect the tyrosine phosphorylation of the insulin receptor nor of IRS1, but it enhanced the tyrosine phosphorylation of the p85 subunit of PI3K. The expression of the SHP-1/PTPN6 phosphatase, which dephosphorylates p85, was reduced after Reptin depletion. Forced expression of SHP-1 restored a normal AKT phosphorylation after insulin treatment in cells where Reptin was silenced, demonstrating that the downregulation of SHP1 is mechanistically linked to increased Akt phosphorylation. In conclusion, we have uncovered a new function for Reptin in regulating insulin signalling in HCC cells via the regulation of SHP-1 expression. We suggest that the regulation of insulin sensitivity by Reptin contributes to its oncogenic action in the liver.
Long-term correction of diabetic hyperglycemia through glucose-responsive hepatic insulin production using lentivirusAbstract
Type 1 diabetes mellitus (T1DM) is caused by the autoimmune destruction of the insulin-producing β cells of the pancreas. Insulin gene therapy is a promising strategy capable of overcoming the limitations of current treatments, but to become a viable option, it must provide long-term, glucose-responsive control of insulin production. We have previously achieved glucose-responsivity by incorporating glucose-inducible response elements (GIREs) upstream of a liver-specific insulin expression cassette (3xGIRE.ALB.Ins1-2xfur). In this study, 3xGIRE.ALB.Ins1-2xfur was delivered into streptozotocin-induced diabetic rats using lentivirus, resulting in remission of diabetic hyperglycemia for at least 482 days while restoring rate of weight gain in a dose-dependent fashion. Insulin immunostaining showed abundant insulin production in the liver, and qPCR showed 13-20 lentiviral integrations per cell in the liver of rats treated with high dose lentivirus. Negligible integration was found in the pancreas, kidney, spleen and muscle of LV-treated rats, confirming liver specificity. In vitro, LV.3xGIRE.ALB.Ins1-2xfur produced a 4.5-fold increase in insulin production in high glucose conditions, and in vivo, a 1.7-fold increase in insulin levels was found during an intraperitoneal glucose tolerance test. Unfortunately, limitations in large-scale lentivirus production and use of a tissue-specific promoter prevented treatment of more than one rat per batch of lentivirus. Thus, two of the LV-treated diabetic rats were undertreated, while another two rats were over treated, becoming hypoglycemic in the fed state. Nonetheless, we have established the framework for a long-term, glucose-responsive treatment for T1DM from which further improvements can be made.