The Transcription Factor Nfatc2 Regulates ß-Cell Proliferation and Genes Associated with Type 2 Diabetes in Mouse and Human Islets.

Human genome-wide association studies (GWAS) have shown that genetic variation at >130 gene loci is associated with type 2 diabetes (T2D). We asked if the expression of the candidate T2D-associated genes within these loci is regulated by a common locus in pancreatic islets. Using an obese F2 mouse intercross segregating for T2D, we show that the expression of ~40% of the T2D-associated genes is linked to a broad region on mouse chromosome (Chr) 2. As all but 9 of these genes are not physically located on Chr 2, linkage to Chr 2 suggests a genomic factor(s) located on Chr 2 regulates their expression in trans. The transcription factor Nfatc2 is physically located on Chr 2 and its expression demonstrates cis linkage; i.e., its expression maps to itself. When conditioned on the expression of Nfatc2, linkage for the T2D-associated genes was greatly diminished, supporting Nfatc2 as a driver of their expression. Plasma insulin also showed linkage to the same broad region on Chr 2. Overexpression of a constitutively active (ca) form of Nfatc2 induced ß-cell proliferation in mouse and human islets, and transcriptionally regulated more than half of the T2D-associated genes. Overexpression of either ca-Nfatc2 or ca-Nfatc1 in mouse islets enhanced insulin secretion, whereas only ca-Nfatc2 was able to promote ß-cell proliferation, suggesting distinct molecular pathways mediating insulin secretion vs. ß-cell proliferation are regulated by NFAT. Our results suggest that many of the T2D-associated genes are downstream transcriptional targets of NFAT, and may act coordinately in a pathway through which NFAT regulates ß-cell proliferation in both mouse and human islets.

Modeling fibrosis using fibroblasts isolated from scarred rat vocal folds.

Following injury, pathologically activated vocal fold fibroblasts (VFFs) can engage in disordered extracellular matrix (ECM) remodeling, leading to VF fibrosis and impaired voice function. Given the importance of scar VFFs to phenotypically appropriate in vitro modeling of VF fibrosis, we pursued detailed characterization of scar VFFs obtained from surgically injured rat VF mucosae, compared with those obtained from experimentally naïve, age-matched tissue. Scar VFFs initially exhibited a myofibroblast phenotype characterized by increased proliferation, increased Col1a1 transcription and collagen, type I synthesis, increased Acta2 transcription and α-smooth muscle actin synthesis, and enhanced contractile function. These features were most distinct at passage 1 (P1); we observed a coalescence of the scar and naïve VFF phenotypes at later passages. An empirical Bayes statistical analysis of the P1 cell transcriptome identified 421 genes that were differentially expressed by scar, compared with naïve, VFFs. These genes were primarily associated with the wound response, ECM regulation, and cell proliferation. Follow-up comparison of P1 scar VFFs and their in vivo tissue source showed substantial transcriptomic differences. Finally, P1 scar VFFs responded to treatment with hepatocyte growth factor and transforming growth factor-ß3, two biologics with reported therapeutic value. Despite the practical limitations inherent to working with early passage cells, this experimental model is easily implemented in any suitably equipped laboratory and has the potential to improve the applicability of preclinical VF fibrosis research.

R/EBcoexpress: an empirical Bayesian framework for discovering differential co-expression.

UNLABELLED: R/EBcoexpress implements the approach of Dawson and Kendziorski using R, a freely available, open source statistical programming language. The approach identifies differential co-expression (DC) by examining the correlations among gene pairs using an empirical Bayesian approach, producing a false discovery rate controlled list of DC pairs. This interrogation of DC gene pairs complements but is distinct from differential expression analyses, under the general goal of understanding differential regulation across biological conditions. AVAILABILITY AND IMPLEMENTATION: R/EBcoexpress is freely available and hosted on Bioconductor; a source file and vignette may be found at http://www.bioconductor.org/packages/release/bioc/html/EBcoexpress.html

Mechanotransduction of vocal fold fibroblasts and mesenchymal stromal cells in the context of the vocal fold mechanome.

The design of cell-based therapies for vocal fold tissue engineering requires an understanding of how cells adapt to the dynamic mechanical forces found in the larynx. Our objective was to compare mechanotransductive processes in therapeutic cell candidates (mesenchymal stromal cells from adipose tissue and bone marrow, AT-MSC and BM-MSC) to native cells (vocal fold fibroblasts-VFF) in the context of vibratory strain. A bioreactor was used to expose VFF, AT-MSC, and BM-MSC to axial tensile strain and vibration at human physiological levels. Microarray, an empirical Bayes statistical approach, and geneset enrichment analysis were used to identify significant mechanotransductive pathways associated with the three cell types and three mechanical conditions. Two databases (Gene Ontology, Kyoto Encyclopedia of Genes and Genomes) were used for enrichment analyses. VFF shared more mechanotransductive pathways with BM-MSC than with AT-MSC. Gene expression that appeared to distinguish the vibratory strain condition from polystyrene condition for these two cells types related to integrin activation, focal adhesions, and lamellipodia activity, suggesting that vibratory strain may be associated with cytoarchitectural rearrangement, cell reorientation, and extracellular matrix remodeling. In response to vibration and tensile stress, BM-MSC better mimicked VFF mechanotransduction than AT-MSC, providing support for the consideration of BM-MSC as a cell therapy for vocal fold tissue engineering. Future research is needed to better understand the sorts of physical adaptations that are afforded to vocal fold tissue as a result of focal adhesions, integrins, and lamellipodia, and how these adaptations could be exploited for tissue engineering.

Reversal of Vocal Fold Mucosal Fibrosis Using siRNA against the Collagen-Specific Chaperone Serpinh1.

Vocal fold (VF) mucosal fibrosis results in substantial voice impairment and is recalcitrant to current treatments. To reverse this chronic disorder, anti-fibrotic therapies should target the molecular pathology of aberrant collagen accumulation in the extracellular matrix. We investigated the therapeutic potential of siRNA against Serpinh1, a collagen-specific chaperone that enables cotranslational folding and assembly of procollagens in the endoplasmic reticulum. We implemented a previously validated siRNA construct, conducted transfection experiments using in vitro and in vivo rat models, and measured knockdown efficiency, dose responses, delivery strategies, and therapeutic outcomes. Liposome-mediated delivery of Serpinh1-siRNA downregulated collagen production in naive and scar VF fibroblasts as well as naive VF mucosa; moreover, sustained Serpinh1 knockdown in fibrotic VF mucosa reversed scar-associated collagen accumulation within 4 weeks. Analysis of therapeutic effects at the transcriptome level showed evidence of cell cycle upregulation, catabolism, matrix disassembly, and morphogenesis. These findings indicate that Serpinh1-siRNA holds potential as a molecular therapy for chronic VF mucosal fibrosis.

Correction: Identification of a serum-induced transcriptional signature associated with metastatic cervical cancer.

[This corrects the article DOI: 10.1371/journal.pone.0181242.].

Statistical Methods for Latent Class Quantitative Trait Loci Mapping.

Identifying the genetic basis of complex traits is an important problem with the potential to impact a broad range of biological endeavors. A number of effective statistical methods are available for quantitative trait loci (QTL) mapping that allow for the efficient identification of multiple, potentially interacting, loci under a variety of experimental conditions. Although proven useful in hundreds of studies, the majority of these methods assumes a single model common to each subject, which may reduce power and accuracy when genetically distinct subclasses exist. To address this, we have developed an approach to enable latent class QTL mapping. The approach combines latent class regression with stepwise variable selection and traditional QTL mapping to estimate the number of subclasses in a population, and to identify the genetic model that best describes each subclass. Simulations demonstrate good performance of the method when latent classes are present as well as when they are not, with accurate estimation of QTL. Application of the method to case studies of obesity and diabetes in mouse gives insight into the genetic basis of related complex traits.

Identification of a serum-induced transcriptional signature associated with metastatic cervical cancer.

OBJECTIVE: Tumor cells that escape local tissue control can convert inflammatory cells from tumor suppressors to tumor promoters. Moreover, soluble immune-modulating factors secreted from the tumor environment can be difficult to identify in patient serum due to their low abundance. We used an alternative strategy to infer a metastatic signature induced by sera of cervical cancer patients. METHODS: Sera from patients with local and metastatic cervical cancer were used to induce a disease-specific transcriptional signature in cultured, healthy peripheral blood mononuclear cells (PBMCs). An empirical Bayesian method, EBarrays, was used to identify differentially expressed (DE) genes with a target false discovery rate of <5%. Ingenuity Pathway Analysis (IPA) software was used to detect the top molecular and cellular functions associated with the DE genes. IPA and in silco analysis was used to pinpoint candidate upstream regulators, including cancer-related microRNAs (miRNAs). RESULTS: We identified enriched pathways in the metastatic cervical group related to immune surveillance functions, such as downregulation of engulfment, accumulation, and phagocytosis of hematopoietic cells. The predicted top upstream genes were IL-10 and immunoglobulins. In silco analysis identified miRNAs predicted to drive the transcriptional signature. Two of the 4 miRNAs (miR-23a-3p and miR-944) were validated in a cohort of women with local and metastatic cervical cancer. CONCLUSIONS: This study supports the use of a cell-based assay that uses PBMC "reporters" to predict biologically relevant factors in patient serum. Further, disease-specific transcriptional signatures induced by patient sera have the potential to differentiate patients with local versus metastatic disease.

Biomechanical Screening of Cell Therapies for Vocal Fold Scar.

Candidate cell sources for vocal fold scar treatment include mesenchymal stromal cells from bone marrow (BM-MSC) and adipose tissue (AT-MSC). Mechanosensitivity of MSC can alter highly relevant aspects of their behavior, yet virtually nothing is known about how MSC might respond to the dynamic mechanical environment of the larynx. Our objective was to evaluate MSC as a potential cell source for vocal fold tissue engineering in a mechanically relevant context. A vibratory strain bioreactor and cDNA microarray were used to evaluate the similarity of AT-MSC and BM-MSC to the native cell source, vocal fold fibroblasts (VFF). Posterior probabilities for each of the microarray transcripts fitting into specific expression patterns were calculated, and the data were analyzed for Gene Ontology (GO) enrichment. Significant wound healing and cell differentiation GO terms are reported. In addition, proliferation and apoptosis were evaluated with immunohistochemistry. Results revealed that VFF shared more GO terms related to epithelial development, extracellular matrix (ECM) remodeling, growth factor activity, and immune response with BM-MSC than with AT-MSC. Similarity in glycosaminoglycan and proteoglycan activity dominated the ECM analysis. Analysis of GO terms relating to MSC differentiation toward osteogenic, adipogenic, and chondrogenic lineages revealed that BM-MSC expressed fewer osteogenesis GO terms in the vibrated and scaffold-only conditions compared to polystyrene. We did not evaluate if vibrated BM-MSC recover osteogenic expression markers when returned to polystyrene culture. Immunostaining for Ki67 and cleaved caspase 3 did not vary with cell type or mechanical condition. We conclude that VFF may have a more similar wound healing capacity to BM-MSC than to AT-MSC in response to short-term vibratory strain. Furthermore, BM-MSC appear to lose osteogenic potential in the vibrated and scaffold-only conditions compared to polystyrene, potentially attenuating the risk of osteogenesis for in vivo applications.

Extending information retrieval methods to personalized genomic-based studies of disease.

Genomic-based studies of disease now involve diverse types of data collected on large groups of patients. A major challenge facing statistical scientists is how best to combine the data, extract important features, and comprehensively characterize the ways in which they affect an individual's disease course and likelihood of response to treatment. We have developed a survival-supervised latent Dirichlet allocation (survLDA) modeling framework to address these challenges. Latent Dirichlet allocation (LDA) models have proven extremely effective at identifying themes common across large collections of text, but applications to genomics have been limited. Our framework extends LDA to the genome by considering each patient as a "document" with "text" detailing his/her clinical events and genomic state. We then further extend the framework to allow for supervision by a time-to-event response. The model enables the efficient identification of collections of clinical and genomic features that co-occur within patient subgroups, and then characterizes each patient by those features. An application of survLDA to The Cancer Genome Atlas ovarian project identifies informative patient subgroups showing differential response to treatment, and validation in an independent cohort demonstrates the potential for patient-specific inference.

The effects of ramipril in individuals at risk for Alzheimer's disease: results of a pilot clinical trial.

Research shows that certain antihypertensives taken during midlife confer Alzheimer's disease (AD) related benefits in later life. We conducted a clinical trial to evaluate the extent to which the angiotensin converting enzyme inhibitor (ACE-I), ramipril, affects AD biomarkers including cerebrospinal fluid (CSF) amyloid-ß (Aß) levels and ACE activity, arterial function, and cognition in participants with a parental history of AD. This four month randomized, double-blind, placebo-controlled, pilot clinical trial evaluated the effects of ramipril, a blood-brain-barrier crossing ACE-I, in cognitively healthy individuals with mild, or Stage I hypertension. Fourteen participants were stratified by gender and apolipoprotein E ε4 (APOE ε4) status and randomized to receive 5 mg of ramipril or matching placebo daily. Participants were assessed at baseline and month 4 on measures of CSF Aß(1-42) and ACE activity, arterial function, and cognition. Participants were middle-aged (mean 54 y) and highly educated (mean 15.4 y), and included 50% men and 50% APOE ε4 carriers. While results did not show a treatment effect on CSF Aß(1-42) (p = 0.836), data revealed that ramipril can inhibit CSF ACE activity (p = 0.009) and improve blood pressure, however, there were no differences between groups in arterial function or cognition. In this study, ramipril therapy inhibited CSF ACE activity and improved blood pressure, but did not influence CSF Aß1-42. While larger trials are needed to confirm our CSF Aß results, it is possible that prior research reporting benefits of ACE-I during midlife may be attributed to alternative mechanisms including improvements in cerebral blood flow or the prevention of angiotensin II-mediated inhibition of acetylcholine.

Protective effect of Radix Astragali injection on immune organs of rats with obstructive jaundice and its mechanism.

AIM: To observe the protective effect of Radix Astragali injection on immune organs (lymph nodes, spleen and thymus) of rats with obstructive jaundice (OJ) and its mechanism. METHODS: SD rats were randomly divided into sham-operation group, model control group and Radix Astragali treatment group. On days 7, 14, 21 and 28 after operation, mortality rate of rats, pathological changes in immune organs, expression levels of Bax and nuclear factor (NF)-kappaB p65 proteins, apoptosis indexes and serum tumor necrosis factor (TNF)-alpha level in spleen and thymus were observed, respectively. RESULTS: Compared to model control group, the number of dead OJ rats in Radix Astragali treatment group decreased (P > 0.05). The TNF-alpha level (27.62 +/- 12.61 vs 29.55 +/- 18.02, 24.61 +/- 9.09 vs 31.52 +/- 10.95) on days 7 and 21, the pathological severity score for spleen [0.0 (0.0) vs 0.0 (2.0) on days 7 and 14 and for lymph nodes [0.0 (1.0) vs 1.0 (2.0), 1.0 (0.0) vs 2.0 (1.0)] on days 21 and 28, the product staining intensity and positive rate of Bax protein in spleen [0.0 (0.0) vs 1.0 (2.0), 0.0 (1.0) vs 2.0 (1.5) and thymus [0.0 (0.0) vs 1.0 (2.0), 0.0 (1.0) vs 2.0 (1.5)] on days 14 and 28, the apoptotic indexes [0.0 (0.0) vs 0.0 (0.01)] in spleen and thymus [0.0 (0.0) vs 0.0 (0.01) on days 14 and 21 were significantly lower in Radix Astragali treatment group than in model control group (P < 0.05). CONCLUSION: Radix Astragali has protective effects on immune organs of OJ rats by relieving the pathological changes in immune organs, reducing TNF-alpha level and inhibiting Bax expression and apoptosis in spleen and thymus.