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qPCR Instrumentation
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  • Next Generation Sequencing (NGS)
    Mixed transmission modes and dynamic genome evolution in an obligate animal-bacterialsymbiosis
    Russell SL, ISME Journal - 2017
    Reliable transmission of symbionts between host generations facilitates the evolution of beneficial and pathogenic associations. Although transmission mode is typically characterized as either vertical or horizontal, the prevalence of intermediate transmission modes, and their impact on symbiont genome evolution, are understudied. Here, we use population genomics to explore mixed transmission modes of chemosynthetic bacterial symbionts in the bivalve Solemya velum. Despite strong evidence for symbiont inheritance through host oocytes, whole-genome analyses revealed signatures of frequent horizontal transmission, including discordant mitochondrial-symbiont genealogies, widespread recombination and a dynamic symbiont genome structure consistent with evolutionary patterns of horizontally transmitted associations. Population-level analyses thus provide a tractable means of ascertaining the fidelity of vertical versus horizontal transmission. Our data support the strong influence horizontal transmission can have on symbiont genome evolution, and shed light on the dynamic evolutionary pressures shaping symbiotic bacterial genomes.
    Association of the gut microbiota mobilome with hospital location and birth weight in preterm infants
    Ravi A, Pediatric Research - 2017
    BackgroundThe preterm infant gut microbiota is vulnerable to different biotic and abiotic factors. Although the development of this microbiota has been extensively studied, the mobilome-i.e. the mobile genetic elements (MGEs) in the gut microbiota-has not been considered. Therefore, the aim of this study was to investigate the association of the mobilome with birth weight and hospital location in the preterm infant gut microbiota.MethodsThe data set consists of fecal samples from 62 preterm infants with and without necrotizing enterocolitis (NEC) from three different hospitals. We analyzed the gut microbiome by using 16S rRNA amplicon sequencing, shot-gun metagenome sequencing, and quantitative PCR. Predictive models and other data analyses were performed using MATLAB and QIIME.ResultSThe microbiota composition was significantly different between NEC-positive and NEC-negative infants and significantly different between hospitals. An operational taxanomic unit (OTU) showed strong positive and negative correlation with NEC and birth weight, respectively, whereas none showed significance for mode of delivery. Metagenome analyses revealed high levels of conjugative plasmids with MGEs and virulence genes. Results from quantitative PCR showed that the plasmid signature genes were significantly different between hospitals and in NEC-positive infants.ConclusionOur results point toward an association of the mobilome with hospital location in preterm infants.
    Abundance of antibiotics, antibiotic resistance genes and bacterial community composition in wastewater effluents from different Romanian hospitals
    Szekeres E, Environmental Pollution - 2017
    Antimicrobial resistance represents a growing and significant public health threat, which requires a global response to develop effective strategies and mitigate the emergence and spread of this phenomenon in clinical and environmental settings. We investigated, therefore, the occurrence and abundance of several antibiotics and antibiotic resistance genes (ARGs), as well as bacterial community composition in wastewater effluents from different hospitals located in the Cluj County, Romania. Antibiotic concentrations ranged between 3.67 and 53.05 μg L-1, and the most abundant antibiotic classes were β-lactams, glycopeptides, and trimethoprim. Among the ARGs detected, 14 genes confer resistance to β-lactams, aminoglycosides, chloramphenicol, macrolide-lincosamide-streptogramin B (MLSB) antibiotics, sulfonamides, and tetracyclines. Genes encoding quaternary ammonium resistance and a transposon-related element were also detected. The sulI and qacEΔ1 genes, which confer resistance to sulfonamides and quaternary ammonium, had the highest relative abundance with values ranging from 5.33 × 10-2 to 1.94 × 10-1 and 1.94 × 10-2 to 4.89 × 10-2 copies/16 rRNA gene copies, respectively. The dominant phyla detected in the hospital wastewater samples were Proteobacteria, Bacteroidetes, Firmicutes, and Actinobacteria. Among selected hospitals, one of them applied an activated sludge and chlorine disinfection process before releasing the effluent to the municipal collector. This conventional wastewater treatment showed moderate removal efficiency of the studied pollutants, with a 55-81% decrease in antibiotic concentrations, 1-3 order of magnitude lower relative abundance of ARGs, but with a slight increase of some potentially pathogenic bacteria. Given this, hospital wastewaters (raw or treated) may contribute to the spread of these emerging pollutants in the receiving environments. To the best of our knowledge, this study quantified for the first time the abundance of antibiotics and ARGs in wastewater effluents from different Romanian hospitals.
    Microbial diversity of consumption milk during processing and storage
    Porcellato D, International Journal of Food Microbiology - 2018
    Bovine milk contains a complex microbial community that affects the quality and safety of the product. Detailed knowledge of this microbiota is, therefore, of importance for the dairy industry. In this study, the bacterial composition of consumption milk was assessed during different stages in the production line and throughout the storage in cartons by using culturing techniques and 16S rRNA marker gene sequencing. Monthly samples from two dairies were analyzed to capture the seasonal variations in the milk microbiota. Although there was a core microbiota present in milk samples from both dairies, the composition of the bacterial communities were significantly influenced by sampling month, processing stage and storage temperature. Overall, a higher abundance of operational taxonomic units (OTUs) within the order Bacillales was detected in samples of raw and pasteurized milk from the spring and summer months, while Pseudomonadales and Lactobacillales OTUs were predominant in the winter months. OTUs belonging to the order Lactobacillales, Pseudomonadales, Clostridiales and Bacillales were significantly more abundant in milk samples taken immediately after pasteurization compared to raw milk samples. During storage of milk in cartons at 4°C, the bacterial composition remained stable throughout the product shelf life, while storage at 8°C significantly increased the abundance of OTUs belonging to the genus Bacillus and the plate count levels of presumptive Bacillus cereus. The knowledge obtained in this work will be useful to the dairy industry during their quality assurance work and risk assessment practices.
    Application of a novel amplicon-based sequencing approach reveals the diversity of the Bacillus cereus group in stored raw and pasteurized milk
    Davide Porcellato, Food Microbiology - 2018
    Members of the Bacillus cereus sensu lato (B. cereus group) are spore-forming organisms commonly associated with spoilage of milk and dairy products. Previous studies have shown, by using 16S marker gene sequencing, that the genus Bacillus is part of the core microbiota of raw bovine milk and that some members of this genus are able to grow during sub-optimal storage (8 °C) of pasteurized consumption milk. Here, the composition of this genus in pasteurized consumption milk samples, collected from two dairies, over a one-year period and stored at 4 or 8 °C up to the end of shelf life is uncovered. Our results show that the B. cereus group is the dominant Bacillus group in stored consumption milk. By applying a new marker gene sequencing approach, several dominating phylogenetic clusters were identified within the B. cereus group populations from the milk samples. There was a higher phylogenetic diversity among bacteria from milk stored at 8 °C compared to milk stored at 4 °C. Sampling period and the dairy the samples were collected from, also significantly influenced the diversity, which shows that the B. cereus group population in consumption milk is heterogeneous and subjected to temporal and spatial changes. The new approach applied in this study will facilitate the identification of isolates within the B. cereus group, of which some are potential spoilage bacteria and pathogenic contaminants of milk and dairy products.
    RNA‐seq: Applications and Best Practices
    Michele Araújo Pereira, Intech - 2017
    RNA‐sequencing (RNA‐seq) is the state‐of‐the‐art technique for transcriptome analysis that takes advantage of high‐throughput next‐generation sequencing. Although being a powerful approach, RNA‐seq imposes major challenges throughout its steps with numerous caveats. There are currently many experimental options available, and a complete comprehension of each step is critical to make right decisions and avoid getting into inconclusive results. A complete workflow consists of: (1) experimental design; (2) sample and library preparation; (3) sequencing; and (4) data analysis. RNA‐seq enables a wide range of applications such as the discovery of novel genes, gene/transcript quantification, and differential expression and functional analysis. This chapter will encompass the main aspects from sample preparation to downstream data analysis. It will be discussed how to obtain high‐quality samples, replicates amount, library preparation, sequencing platforms and coverage, focusing on best recommended practices based on specialized literature. Basic techniques and well‐known algorithms are presented and discussed, guiding both beginners and experienced users in the implementation of reliable experiments.
    A Method to Assess Bacteriocin Effects on the Gut Microbiota of Mice
    Chrstine Bäuerl, JoVE (Journal of Visualized Experiments) - 2017
    Very intriguing questions arise with our advancing knowledge on gut microbiota composition and the relationship with health, particularly relating to the factors that contribute to maintaining the population balance. However, there are limited available methodologies to evaluate these factors. Bacteriocins are antimicrobial peptides produced by many bacteria that may confer a competitive advantage for food acquisition and/or niche establishment. Many probiotic lactic acid bacteria (LAB) strains have great potential to promote human and animal health by preventing the growth of pathogens. They can also be used for immuno-modulation, as they produce bacteriocins. However, the antagonistic activity of bacteriocins is normally determined by laboratory bioassays under well-defined but over-simplified conditions compared to the complex gut environment in humans and animals, where bacteria face multifactorial influences from the host and hundreds of microbial species sharing the same niche. This work describes a complete and efficient procedure to assess the effect of a variety of bacteriocins with different target specificities in a murine system. Changes in the microbiota composition during the bacteriocin treatment are monitored using compositional 16S rDNA sequencing. Our approach uses both the bacteriocin producers and their isogenic non-bacteriocin-producing mutants, the latter giving the ability to distinguish bacteriocin-related from non-bacteriocin-related modifications of the microbiota. The fecal DNA extraction and 16S rDNA sequencing methods are consistent and, together with the bioinformatics, constitute a powerful procedure to find faint changes in the bacterial profiles and to establish correlations, in terms of cholesterol and triglyceride concentration, between bacterial populations and health markers. Our protocol is generic and can thus be used to study other compounds or nutrients with the potential to alter the host microbiota composition, either when studying toxicity or beneficial effects.
    Circadian rhythm disruption impairs tissue homeostasis and exacerbates chronic inflammation in the intestine
    René Pagel, The FASEB Journal - 2017
    Endogenous circadian clocks regulate 24 h rhythms of physiology and behavior. Circadian rhythm disruption (CRD) is suggested as a risk factor for inflammatory bowel disease. However, the underlying molecular mechanisms remain unknown. Intestinal biopsies from Per1/2 mutant and wild-type (WT) mice were investigated by electron microscopy, immunohistochemistry, and bromodeoxyuridine pulse–chase experiments. TNF-α was injected intraperitoneally, with or without necrostatin-1, into Per1/2 mice or rhythmic and externally desynchronized WT mice to study intestinal epithelial cell death. Experimental chronic colitis was induced by oral administration of dextran sodium sulfate. In vitro, caspase activity was assayed in Per1/2-specific small interfering RNA–transfected cells. Wee1 was overexpressed to study antiapoptosis and the cell cycle. Genetic ablation of circadian clock function or environmental CRD in mice increased susceptibility to severe intestinal inflammation and epithelial dysregulation, accompanied by excessive necroptotic cell death and a reduced number of secretory epithelial cells. Receptor-interacting serine/threonine-protein kinase (RIP)-3-mediated intestinal necroptosis was linked to increased mitotic cell cycle arrest via Per1/2-controlled Wee1, resulting in increased antiapoptosis via cellular inhibitor of apoptosis-2. Together, our data suggest that circadian rhythm stability is pivotal for the maintenance of mucosal barrier function. CRD increases intestinal necroptosis, thus rendering the gut epithelium more susceptible to inflammatory processes.—Pagel, R., Bär, F., Schröder, T., Sünderhauf, A., Künstner, A., Ibrahim, S. M., Autenrieth, S. E., Kalies, K., König, P., Tsang, A. H., Bettenworth, D., Divanovic, S., Lehnert, H., Fellermann, K., Oster, H., Derer, S., Sina, C. Circadian rhythm disruption impairs tissue homeostasis and exacerbates chronic inflammation in the intestine.
    Microbial Composition and Diversity Patterns in Deep Hyperthermal Aquifers from the Western Plain of Romania
    Cecilia M. Chiriac, Microbial Ecology - 2017
    A limited number of studies have investigated the biodiversity in deep continental hyperthermal aquifers and its influencing factors. Here, we present the first description of microbial communities inhabiting the Pannonian and Triassic hyperthermal aquifers from the Western Plain of Romania, the first one being considered a deposit of “fossilized waters,” while the latter is embedded in the hydrological cycle due to natural refilling. The 11 investigated drillings have an open interval between 952 and 3432 m below the surface, with collected water temperatures ranging between 47 and 104 °C, these being the first microbial communities characterized in deep continental water deposits with outflow temperatures exceeding 80 °C. The abundances of bacterial 16S rRNA genes varied from approximately 105–106 mL−1 in the Pannonian to about 102–104 mL−1 in the Triassic aquifer. A 16S rRNA gene metabarcoding analysis revealed distinct microbial communities in the two water deposits, especially in the rare taxa composition. The Pannonian aquifer was dominated by the bacterial genera Hydrogenophilus and Thermodesulfobacterium, together with archaeal methanogens from the Methanosaeta and Methanothermobacter groups. Firmicutes was prevalent in the Triassic deposit with a large number of OTUs affiliated to Thermoanaerobacteriaceae, Thermacetogenium, and Desulfotomaculum. Species richness, evenness, and phylogenetic diversity increased alongside with the abundance of mesophiles, their presence in the Triassic aquifer being most probably caused by the refilling with large quantities of meteoric water in the Carpathian Mountains. Altogether, our results show that the particular physico-cheminal characteristics of each aquifer, together with the water refilling possibilities, seem to determine the microbial community structure.
    The Potential of Class II Bacteriocins to Modify Gut Microbiota to Improve Host Health
    Özgün C. O. Umu, PLOS ONE - 2016
    Production of bacteriocins is a potential probiotic feature of many lactic acid bacteria (LAB) as it can help prevent the growth of pathogens in gut environments. However, knowledge on bacteriocin producers in situ and their function in the gut of healthy animals is still limited. In this study, we investigated five bacteriocin-producing strains of LAB and their isogenic non-producing mutants for probiotic values. The LAB bacteriocins, sakacin A (SakA), pediocin PA-1 (PedPA-1), enterocins P, Q and L50 (enterocins), plantaricins EF and JK (plantaricins) and garvicin ML (GarML), are all class II bacteriocins, but they differ greatly from each other in terms of inhibition spectrum and physicochemical properties. The strains were supplemented to mice through drinking water and changes on the gut microbiota composition were interpreted using 16S rRNA gene analysis. In general, we observed that overall structure of the gut microbiota remained largely unaffected by the treatments. However, at lower taxonomic levels, some transient but advantageous changes were observed. Some potentially problematic bacteria were inhibited (e.g., Staphylococcus by enterocins, Enterococcaceae by GarML, and Clostridium by plantaricins) and the proportion of LAB was increased in the presence of SakA-, plantaricins- and GarML-producing bacteria. Moreover, the treatment with GarML-producing bacteria co-occurred with decreased triglyceride levels in the host mice. Taken together, our results indicate that several of these bacteriocin producers have potential probiotic properties at diverse levels as they promote favorable changes in the host without major disturbance in gut microbiota, which is important for normal gut functioning.
    Transition from infant- to adult-like gut microbiota
    E. Avershina, Environmental Microbiology - 2016
    Transition from an infant to an adult associated gut microbiota with age through establishment of strict anaerobic bacteria remains one of the key unresolved questions in gut microbial ecology. Here a comprehensive comparative analysis of stool microbiota in a large cohort of mothers and their children sampled longitudinally up until 2 years of age using sequencing analysis tool was presented that allows realistic microbial diversity estimates. In this work, evidence for the switch from children to adult associated microbial profile between 1 and 2 years of age was provided, suggestively driven by Bifidobacterium breve. An Operational Taxonomic Unit (OTU) belonging to B. breve was highly prevalent in the population throughout the first year of life, and was negatively associated with detection of a range of adult-like OTUs. Although an adult profile was not fully established by 2 years of age, it was demonstrated that with regards to the most prevalent OTUs, their prevalence in the child population by then already resembled that of the adult population. Taken together, it was proposed that late-colonizing OTUs were recruited at a later stage and were not acquired at birth with the recruitment being controlled by gatekeeping OTUs until the age of 1 year.
    Microbial biogeography of a university campus
    Ashley A. Ross, Microbiome - 2015
    Microorganisms are distributed on surfaces within homes, workplaces, and schools, with the potential to impact human health and disease. University campuses represent a unique opportunity to explore the distribution of microorganisms within built environments because of high human population densities, throughput, and variable building usage. For example, the main campus of the University of Waterloo spans four square kilometres, hosts over 40,000 individuals daily, and is comprised of a variety of buildings, including lecture halls, gyms, restaurants, residences, and a daycare.
    Role of two single nucleotide polymorphisms in secreted frizzled re-lated protein 1 and bladder cancer risk
    Anja Rogler, IJCEP Int J Clin Exp Pathol - 2013
    In this study- we determined the genotype distribution of two single nucleotide polymorphisms (SNPs) in secreted frizzled related protein 1 (SFRP1)- rs3242 and rs921142- in a Caucasian bladder cancer case-control study. Allelic variants of the SNPs were determined using restriction fragment length polymorphism (RFLP) analysis and partly verified by sequencing analysis. Overall- DNA from 188 consecutive and 215 early-onset bladder cancer patients (?45 years) as well as from 332 controls was investigated. Potential microRNA binding sites were determined for rs3242- and microRNA expression was analysed in cell lines and tumour specimens. We observed a remarkable distribution difference in rs3242 between bladder cancer patients and healthy controls (p=0.05). Additionally- we found a significant difference in genotype distribution (p=0.032)- resulting from the difference of early-onset patients and the control group (p=0.007). The risk allele T showed increased frequency in the early-onset patient group (p=0.002). Genotype-dependent differences of microRNA binding capacity were predicted in SFRP1 mRNA for two microRNAs. Hsa-miR-3646 showed strong expression in cell lines and tumour tissue- whereas hsa-miR-603 exhibited weak expression. The rs921142 SNP showed no significant association with bladder cancer risk. This is the first study to describe an association of the SFRP1 SNP rs3242 and bladder cancer risk as well as the influence of rs3242 on genotype-dependent microRNA capacity on SFRP1 mRNA. The onset of bladder seems to be associated with the increased occurrence of the T-allele in rs3242. (IJCEP1308034).
    Nolan Gokey, Molecular and Cellular Biology - 2012
    Schwann cell differentiation and subsequent myelination of the peripheral nervous system require the action of several transcription factors- including Sox10- which is vital at multiple stages of development. The transition from immature to myelinating Schwann cell is also regulated posttranscriptionally and depends upon Dicer-mediated processing of microRNAs (miRNAs). Although specific miRNA targets have begun to be identified- the mechanisms establishing the dynamic regulation of miRNA expression have not been elucidated. We performed expression profiling studies and identified 225 miRNAs differentially expressed during peripheral myelination. A subset of 9 miRNAs is positively regulated by Sox10- including miR-338 which has been implicated in oligodendrocyte maturation. In vivo chromatin immunoprecipitation (ChIP) of sciatic nerve cells revealed a Sox10 binding site upstream of an alternate promoter within the Aatk gene- which hosts miR-338. Sox10 occupied this site in spinal cord ChIP experiments- suggesting a similar regulatory mechanism in oligodendrocytes. Cancer profiling studies have identified clusters of miRNAs that regulate proliferation- termed "oncomirs." In Schwann cells- the expression of many of these proproliferative miRNAs was reduced in the absence of Sox10. Finally- Schwann cells with reduced Sox10 and oncomir expression have an increase in the CDK inhibitor p21 and a concomitant reduction in cell proliferation.
    Amanda Favreau, American Journal of Hematology - 2012
    Chen-Hua Chuang, Nucleic Acids Research - 2012
    The MiniChromosome Maintenance 2-7 (MCM2-7) complex provides essential replicative helicase function. Insufficient MCMs impair the cell cycle and cause genomic instability (GIN)- leading to cancer and developmental defects in mice. Remarkably- depletion or mutation of one Mcm can decrease all Mcm levels. Here- we use mice and cells bearing a GIN-causing hypomophic allele of Mcm4 (Chaos3)- in conjunction with disruption alleles of other Mcms- to reveal two new mechanisms that regulate MCM protein levels and pre-RC formation. First- the Mcm4(Chaos3) allele- which disrupts MCM4:MCM6 interaction- triggers a Dicer1 and Drosha-dependent ≈ 40\% reduction in Mcm2-7 mRNAs. The decreases in Mcm mRNAs coincide with up-regulation of the miR-34 family of microRNAs- which is known to be Trp53-regulated and target Mcms. Second- MCM3 acts as a negative regulator of the MCM2-7 helicase in vivo by complexing with MCM5 in a manner dependent upon a nuclear-export signal-like domain- blocking the recruitment of MCMs onto chromatin. Therefore- the stoichiometry of MCM components and their localization is controlled post-transcriptionally at both the mRNA and protein levels. Alterations to these pathways cause significant defects in cell growth reflected by disease phenotypes in mice.
    Regulation of Inflammatory Phenotype in Macrophages by a Diabetes-Induced Long Non-coding RNA
    Marpadga Reddy, Diabetes - 2014
    The mechanisms by which macrophages mediate the enhanced inflammation associated with diabetes complications are not completely understood. We used RNA-seq to profile the transcriptome of bone marrow macrophages isolated from diabetic db/db mice and identified 1648 differentially expressed genes compared to control db/+ mice. Data analyses revealed that diabetes promoted a pro-inflammatory- pro-fibrotic and dysfunctional alternatively activated macrophage phenotype possibly via transcription factors involved in macrophage function. Notably- diabetes altered levels of several long non-coding RNAs (lncRNAs). Because- the role of lncRNAs in diabetic complications is unknown- we further characterized the function of lncRNA E330013P06 which was up-regulated in macrophages from db/db and diet-induced insulin resistant type-2 diabetic mice- but not from type-1 diabetic mice. It was also up-regulated in monocytes from type-2 diabetes patients. E330013P06 was also increased along with inflammatory genes- in mouse macrophages treated with high glucose and palmitic acid. E330013P06 overexpression in macrophages induced inflammatory genes- enhanced responses to inflammatory signals- and increased foam cell formation. In contrast- siRNA-mediated E330013P06 gene silencing inhibited inflammatory genes induced by the diabetic stimuli. These results define the diabetic macrophage transcriptome- and novel functional roles for lncRNAs in macrophages that could lead to lncRNA based therapies for inflammatory diabetic complications.
    Shawn Rice, The Journal of Biological Chemistry - 2013
    In lung cancers- TTF-1 displays seemingly paradoxical activities. Although TTF-1 is amplified in primary human lung cancers- it inhibits primary lung tumors from metastasizing in a mouse model system. It was reported that the oncogenic proepithelial mesenchymal transition (EMT) high mobility group AT-hook 2 gene (HMGA2) mediates the antimetastatic function of TTF-1. To gain mechanistic insight into the metastasis-critical signaling axis of TTF-1 to HMGA2- we used both reverse and forward strategies and discovered that microRNA-33a (miR-33a) is under direct positive regulation of TTF-1. By chromatin immunoprecipitation- we determined that TTF-1 binds to the promoter of SREBF2- the host gene of miR-33a. The 3'-untranslated region (UTR) of HMGA2 contains three predicted binding sites of miR-33a. We showed that the first two highly conserved sites are conducive to HMGA2 repression by miR-33a- establishing HMGA2 as a genuine target of miR-33a. Functional studies revealed that enforced expression of miR-33a inhibits the motility of lung cancer cells- and this inhibition can be rescued by overexpression of the form of HMGA2 without the 3'-UTR- suggesting that TTF-1 keeps the prometastasis gene HMGA2 in check via up-regulating miR-33a. This study reports the first miRNAs directly regulated by TTF-1 and clarifies how TTF-1 controls HMGA2 expression. Moreover- the documented importance of SREBF2 and miR-33a in regulating cholesterol metabolism suggests that TTF-1 may be a modulator of cholesterol homeostasis in the lung. Future studies will be dedicated to understanding how miRNAs influence the oncogenic activity of TTF-1 and the role of TTF-1 in cholesterol metabolism.
    FOG2 Protein Down-regulation by Transforming Growth Factor-Beta1-induced MicroRNA-200b/c Leads to Akt Kinase Activation and Glomerular Mesangial Hypertrophy Related to Diabetic Nephropathy
    Jung Park, Journal of Biological Chemistry - 2013
    Glomerular hypertrophy is a hallmark of diabetic nephropathy. Akt kinase activated by transforming growth factor-Beta1 (TGF-Beta) plays an important role in glomerular mesangial hypertrophy. However- the mechanisms of Akt activation by TGF-Beta are not fully understood. Recently- miR-200 and its target FOG2 were reported to regulate the activity of phosphatidylinositol 3-kinase (the upstream activator of Akt) in insulin signaling. Here- we show that TGF-Beta activates Akt in glomerular mesangial cells by inducing miR-200b and miR-200c- both of which target FOG2- an inhibitor of phosphatidylinositol 3-kinase activation. FOG2 expression was reduced in the glomeruli of diabetic mice as well as TGF-Beta-treated mouse mesangial cells (MMC). FOG2 knockdown by siRNAs in MMC activated Akt and increased the protein content/cell ratio suggesting hypertrophy. A significant increase of miR-200b/c levels was detected in diabetic mouse glomeruli and TGF-Beta-treated MMC. Transfection of MMC with miR-200b/c mimics significantly decreased the expression of FOG2. Conversely- miR-200b/c inhibitors attenuated TGF-Beta-induced decrease in FOG2 expression. Furthermore- miR-200b/c mimics increased the protein content/cell ratio- whereas miR-200b/c inhibitors abrogated the TGF-Beta-induced increase in protein content/cell. In addition- down-regulation of FOG2 by miR-200b/c could activate not only Akt but also ERK- which was also through PI3K activation. These data suggest a new mechanism for TGF-Beta-induced Akt activation through FOG2 down-regulation by miR-200b/c- which can lead to glomerular mesangial hypertrophy in the progression of diabetic nephropathy.
    Pierre Lau, EMBO molecular medicine - 2013
    An overview of miRNAs altered in Alzheimer's disease (AD) was established by profiling the hippocampus of a cohort of 41 late-onset AD (LOAD) patients and 23 controls- showing deregulation of 35 miRNAs. Profiling of miRNAs in the prefrontal cortex of a second independent cohort of 49 patients grouped by Braak stages revealed 41 deregulated miRNAs. We focused on miR-132-3p which is strongly altered in both brain areas. Downregulation of this miRNA occurs already at Braak stages III and IV- before loss of neuron-specific miRNAs. Next-generation sequencing confirmed a strong decrease of miR-132-3p and of three family-related miRNAs encoded by the same miRNA cluster on chromosome 17. Deregulation of miR-132-3p in AD brain appears to occur mainly in neurons displaying Tau hyper-phosphorylation. We provide evidence that miR-132-3p may contribute to disease progression through aberrant regulation of mRNA targets in the Tau network. The transcription factor (TF) FOXO1a appears to be a key target of miR-132-3p in this pathway.
    Trichostatin A alters the expression of cell cycle controlling genes and miRNAs in donor cells and subsequently improves the yield and quality of cloned bovine embryos in-vitro
    M. Saini, Theriogenology - 2014
    Trichostatin A (TSA)- a histone deacetylase inhibitor- has been used to improve nuclear reprogramming in SCNT embryos. However- the molecular mechanism of TSA for the improvement of the pre- and post-implantation embryonic development is unknown. In the present study- we investigated mechanism of cell cycle arrest caused by TSA and also determined embryo quality and gene expression in cloned bovine embryos produced from TSA treated donor cells compared to embryos produced by in vitro fertilization or parthenogenetic activation. We observed that- 50 nM TSA treated cells were synchronized at G0/G1 stage with concomitant decrease in the proportion of these cells in the S stage of the cell cycle- which was also supported by significant changes in cell morphology and decreased proliferation (P{\textless}0.05). Measurement of relative expression using real-time PCR of a some cell cycle related genes and miRNAs in treated donor cells showed- decreased expression of HDAC1- DNMT1- P53- CYC E1 and CDK4 and increased expression of DNMT3a- CDKN1A- CDK2- CDK3- miR-15a- miR-16 and miR-34a (P{\textless}0.05). No change in relative expression of miR-449a was noticed. TSA treatment of donor cells signi�cantly improved both cleavage and blastocyst rate (P{\textless}0.05) compared to the control embryos- also apoptotic index in treated cloned blastocysts were significantly decreased compared to the non-treated blastocysts (P{\textless}0.05)- and was at the level of IVF counterpart. Relative expression of HDAC1 and DNMT3a was significantly lower in treated cloned and parthenogenetic embryos than that of non-treated and IVF counterpart- while in case P53- expression level between treated and IVF embryos was similar- which as significantly lower than non-treated cloned and parthenogenetic embryos. In conclusion- our data suggested that TSA improves yield and quality of cloned bovine embryos by modulating the expression of G0/G1 cell cycle stage related miRNA in donor cells- which support that TSA might be great cell cycle synchronizer apart from potent epigenetic modulator in cloning research in future.
    Evaluation of quantitative miRNA expression platforms in the microRNA quality control (miRQC) study
    Pieter Mestdagh, Nature Methods - 2014
    MicroRNAs are important negative regulators of protein-coding gene expression and have been studied intensively over the past years. Several measurement platforms have been developed to determine relative miRNA abundance in biological samples using different technologies such as small RNA sequencing- reverse transcription-quantitative PCR (RT-qPCR) and (microarray) hybridization. In this study- we systematically compared 12 commercially available platforms for analysis of microRNA expression. We measured an identical set of 20 standardized positive and negative control samples- including human universal reference RNA- human brain RNA and titrations thereof- human serum samples and synthetic spikes from microRNA family members with varying homology. We developed robust quality metrics to objectively assess platform performance in terms of reproducibility- sensitivity- accuracy- specificity and concordance of differential expression. The results indicate that each method has its strengths and weaknesses- which help to guide informed selection of a quantitative microRNA gene expression platform for particular study goals.
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