Distinctive genes and signaling pathways associated with type 2 diabetes-related periodontitis: Preliminary study.

The biological mechanisms underlying the pathogenesis of type 2 diabetes (T2DM)-related periodontitis remain unclear. This cross-sectional study evaluated the distinctive transcriptomic changes between tissues with periodontal health and with periodontitis in patients with T2DM. In this cross-sectional study, whole transcriptome sequencing was performed on gingival biopsies from non-periodontitis and periodontitis tissues from non-diabetic and diabetic patients. A differentially expressed gene (DEG) analysis and Ingenuity Pathway Analysis (IPA) assessed the genes and signaling pathways associated with T2DM-related periodontitis. Immunohistochemistry was performed to validate selected DEGs possibly involved in T2DM-related periodontitis. Four hundred and twenty and one thousand five hundred and sixty-three DEGs (fold change ≥ 2) were uniquely identified in the diseased tissues of non-diabetic and diabetic patients, respectively. The IPA predicted the activation of Phagosome Formation, Cardiac ß-adrenergic, tRNA Splicing, and PI3K/AKT pathways. The IPA also predicted the inhibition of Cholesterol Biosynthesis, Adrenomedullin, and Inositol Phosphate Compounds pathways in T2DM-related periodontitis. Validation of DEGs confirmed changes in protein expression of PTPN2, PTPN13, DHCR24, PIK3R2, CALCRL, IL1RN, IL-6R and ITGA4 in diseased tissues in diabetic subjects. Thus, these preliminary findings indicate that there are specific genes and functional pathways that may be involved in the pathogenesis of T2DM-related periodontitis.

Investigating miR-9 as a mediator in laryngeal cancer health disparities.

Background: For several decades, Black patients have carried a higher burden of laryngeal cancer among all races. Even when accounting for sociodemographics, a disparity remains. Differentially expressed microRNAs have been linked to racially disparate clinical outcomes in breast and prostate cancers, yet an association in laryngeal cancer has not been addressed. In this study, we present our computational analysis of differentially expressed miRNAs in Black compared with White laryngeal cancer and further validate microRNA-9-5p (miR-9-5p) as a potential mediator of cancer phenotype and chemoresistance. Methods: Bioinformatic analysis of 111 (92 Whites, 19 Black) laryngeal squamous cell carcinoma (LSCC) specimens from the TCGA revealed miRNAs were significantly differentially expressed in Black compared with White LSCC. We focused on miR-9-5 p which had a significant 4-fold lower expression in Black compared with White LSCC (p<0.05). After transient transfection with either miR-9 mimic or inhibitor in cell lines derived from Black (UM-SCC-12) or White LSCC patients (UM-SCC-10A), cellular migration and cell proliferation was assessed. Alterations in cisplatin sensitivity was evaluated in transient transfected cells via IC50 analysis. qPCR was performed on transfected cells to evaluate miR-9 targets and chemoresistance predictors, ABCC1 and MAP1B. Results: Northern blot analysis revealed mature miR-9-5p was inherently lower in cell line UM-SCC-12 compared with UM-SCC-10A. UM -SCC-12 had baseline increase in cellular migration (p < 0.01), proliferation (p < 0.0001) and chemosensitivity (p < 0.01) compared to UM-SCC-10A. Increasing miR-9 in UM-SCC-12 cells resulted in decreased cellular migration (p < 0.05), decreased proliferation (p < 0.0001) and increased sensitivity to cisplatin (p < 0.001). Reducing miR-9 in UM-SCC-10A cells resulted in increased cellular migration (p < 0.05), increased proliferation (p < 0.05) and decreased sensitivity to cisplatin (p < 0.01). A significant inverse relationship in ABCC1 and MAP1B gene expression was observed when miR-9 levels were transiently elevated or reduced in either UM-SCC-12 or UM-SCC-10A cell lines, respectively, suggesting modulation by miR-9. Conclusion: Collectively, these studies introduce differential miRNA expression in LSCC cancer health disparities and propose a role for low miR-9-5p as a mediator in LSCC tumorigenesis and chemoresistance.

Fine-mapping and replication of EWAS loci harboring putative epigenetic alterations associated with AD neuropathology in a large collection of human brain tissue samples.

INTRODUCTION: Our previous epigenome-wide association study (EWAS) of Alzheimer's disease (AD) in human brain identified 71 CpGs associated with AD pathology. However, due to low coverage of the Illumina platform, many important CpGs might have been missed. METHODS: In a large collection of human brain tissue samples (N = 864), we fine-mapped previous EWAS loci by targeted bisulfite sequencing and examined their associations with AD neuropathology. DNA methylation was also linked to gene expression of the same brain cortex. RESULTS: Our targeted sequencing captured 130 CpGs (∼1.2 kb), 93 of which are novel. Of the 130 CpGs, 57 sites (only 17 included in previous EWAS) and 12 gene regions (e.g., ANK1, BIN1, RHBDF2, SPG7, PODXL) were significantly associated with amyloid load. DNA methylation in some regions was associated with expression of nearby genes. DISCUSSION: Targeted methylation sequencing can validate previous EWAS loci and discover novel CpGs associated with AD pathology.

Transcriptional profiling of two muscadine grape cultivars "Carlos" and "Noble" to reveal new genes, gene regulatory networks, and pathways that involved in grape berry ripening.

In commercial fruit production, synchronized ripening and stable shelf life are important properties. The loosely clustered or non-bunching muscadine grape has unrealized potential as a disease-resistant cash crop, but requires repeated hand harvesting due to its unsynchronized or long or heterogeneous maturation period. Genomic research can be used to identify the developmental and environmental factors that control fruit ripening and postharvest quality. This study coupled the morphological, biochemical, and genetic variations between "Carlos" and "Noble" muscadine grape cultivars with RNA-sequencing analysis during berry maturation. The levels of antioxidants, anthocyanins, and titratable acids varied between the two cultivars during the ripening process. We also identified new genes, pathways, and regulatory networks that modulated berry ripening in muscadine grape. These findings may help develop a large-scale database of the genetic factors of muscadine grape ripening and postharvest profiles and allow the discovery of the factors underlying the ripeness heterogeneity at harvest. These genetic resources may allow us to combine applied and basic research methods in breeding to improve table and wine grape ripening uniformity, quality, stress tolerance, and postharvest handling and storage.

Development of Human Adrenocortical Adenoma (HAA1) Cell Line from Zona Reticularis.

The human adrenal cortex is composed of distinct zones that are the main source of steroid hormone production. The mechanism of adrenocortical cell differentiation into several functionally organized populations with distinctive identities remains poorly understood. Human adrenal disease has been difficult to study, in part due to the absence of cultured cell lines that faithfully represent adrenal cell precursors in the early stages of transformation. Here, Human Adrenocortical Adenoma (HAA1) cell line derived from a patient's macronodular adrenocortical hyperplasia and was treated with histone deacetylase inhibitors (HDACis) and gene expression was examined. We describe a patient-derived HAA1 cell line derived from the zona reticularis, the innermost zone of the adrenal cortex. The HAA1 cell line is unique in its ability to exit a latent state and respond with steroidogenic gene expression upon treatment with histone deacetylase inhibitors. The gene expression pattern of differentiated HAA1 cells partially recreates the roster of genes in the adrenal layer that they have been derived from. Gene ontology analysis of whole genome RNA-seq corroborated increased expression of steroidogenic genes upon HDAC inhibition. Surprisingly, HDACi treatment induced broad activation of the Tumor Necrosis Factor (TNF) alpha pathway. This novel cell line we developed will hopefully be instrumental in understanding the molecular and biochemical mechanisms controlling adrenocortical differentiation and steroidogenesis.

Sequencing of Argonaute-bound microRNA/mRNA hybrids reveals regulation of the unfolded protein response by microRNA-320a.

MicroRNAs (miRNA) are short non-coding RNAs widely implicated in gene regulation. Most metazoan miRNAs utilize the RNase III enzymes Drosha and Dicer for biogenesis. One notable exception is the RNA polymerase II transcription start sites (TSS) miRNAs whose biogenesis does not require Drosha. The functional importance of the TSS-miRNA biogenesis is uncertain. To better understand the function of TSS-miRNAs, we applied a modified Crosslinking, Ligation, and Sequencing of Hybrids on Argonaute (AGO-qCLASH) to identify the targets for TSS-miRNAs in HCT116 colorectal cancer cells with or without DROSHA knockout. We observed that miR-320a hybrids dominate in TSS-miRNA hybrids identified by AGO-qCLASH. Targets for miR-320a are enriched for the eIF2 signaling pathway, a downstream component of the unfolded protein response. Consistently, in miR-320a mimic- and antagomir- transfected cells, differentially expressed gene products are associated with eIF2 signaling. Within the AGO-qCLASH data, we identified the endoplasmic reticulum (ER) chaperone calnexin as a direct miR-320a down-regulated target, thus connecting miR-320a to the unfolded protein response. During ER stress, but not amino acid deprivation, miR-320a up-regulates ATF4, a critical transcription factor for resolving ER stress. In summary, our study investigates the targetome of the TSS-miRNAs in colorectal cancer cells and establishes miR-320a as a regulator of unfolded protein response.

Biological features between miRNAs and their targets are unveiled from deep learning models.

MicroRNAs (miRNAs) are ~ 22 nucleotide ubiquitous gene regulators. They modulate a broad range of essential cellular processes linked to human health and diseases. Consequently, identifying miRNA targets and understanding how they function are critical for treating miRNA associated diseases. In our earlier work, a hybrid deep learning-based approach (miTAR) was developed for predicting miRNA targets. It performs substantially better than the existing methods. The approach integrates two major types of deep learning algorithms: convolutional neural networks (CNNs) and recurrent neural networks (RNNs). However, the features in miRNA:target interactions learned by miTAR have not been investigated. In the current study, we demonstrated that miTAR captures known features, including the involvement of seed region and the free energy, as well as multiple novel features, in the miRNA:target interactions. Interestingly, the CNN and RNN layers of the model perform differently at capturing the free energy feature: the units in RNN layer is more unique at capturing the feature but collectively the CNN layer is more efficient at capturing the feature. Although deep learning models are commonly thought "black-boxes", our discoveries support that the biological features in miRNA:target can be unveiled from deep learning models, which will be beneficial to the understanding of the mechanisms in miRNA:target interactions.

miTAR: a hybrid deep learning-based approach for predicting miRNA targets.

BACKGROUND: microRNAs (miRNAs) have been shown to play essential roles in a wide range of biological processes. Many computational methods have been developed to identify targets of miRNAs. However, the majority of these methods depend on pre-defined features that require considerable efforts and resources to compute and often prove suboptimal at predicting miRNA targets. RESULTS: We developed a novel hybrid deep learning-based (DL-based) approach that is capable of predicting miRNA targets at a higher accuracy. This approach integrates convolutional neural networks (CNNs) that excel in learning spatial features and recurrent neural networks (RNNs) that discern sequential features. Therefore, our approach has the advantages of learning both the intrinsic spatial and sequential features of miRNA:target. The inputs for our approach are raw sequences of miRNAs and genes that can be obtained effortlessly. We applied our approach on two human datasets from recently miRNA target prediction studies and trained two models. We demonstrated that the two models consistently outperform the previous methods according to evaluation metrics on test datasets. Comparing our approach with currently available alternatives on independent datasets shows that our approach delivers substantial improvements in performance. We also show with multiple evidences that our approach is more robust than other methods on small datasets. Our study is the first study to perform comparisons across multiple existing DL-based approaches on miRNA target prediction. Furthermore, we examined the contribution of a Max pooling layer in between the CNN and RNN and demonstrated that it improves the performance of all our models. Finally, a unified model was developed that is robust on fitting different input datasets. CONCLUSIONS: We present a new DL-based approach for predicting miRNA targets and demonstrate that our approach outperforms the current alternatives. We supplied an easy-to-use tool, miTAR, at https://github.com/tjgu/miTAR . Furthermore, our analysis results support that Max Pooling generally benefits the hybrid models and potentially prevents overfitting for hybrid models.

Genomic and Targeted Approaches Unveil the Cell Membrane as a Major Target of the Antifungal Cytotoxin Amantelide A.

Amantelide A, a polyhydroxylated macrolide isolated from a marine cyanobacterium, displays broad-spectrum activity against mammalian cells, bacterial pathogens, and marine fungi. We conducted comprehensive mechanistic studies to identify the molecular targets and pathways affected by amantelide A. Our investigations relied on chemical structure similarities with compounds of known mechanisms, yeast knockout mutants, yeast chemogenomic profiling, and direct biochemical and biophysical methods. We established that amantelide A exerts its antifungal action by binding to ergosterol-containing membranes followed by pore formation and cell death, a mechanism partially shared with polyene antifungals. Binding assays demonstrated that amantelide A also binds to membranes containing epicholesterol or mammalian cholesterol, thus suggesting that the cytotoxicity to mammalian cells might be due to its affinity to cholesterol-containing membranes. However, membrane interactions were not completely dependent on sterols. Yeast chemogenomic profiling suggested additional direct or indirect effects on actin. Accordingly, we performed actin polymerization assays, which suggested that amantelide A also promotes actin polymerization in cell-free systems. However, the C-33 acetoxy derivative amantelide B showed a similar effect on actin dynamics in vitro but no significant activity against yeast. Overall, these studies suggest that the membrane effects are the most functionally relevant for amantelide A mechanism of action.

Transfer of oral bacteria to the fetus during late gestation.

The fetus develops in a privileged environment, as the placenta serves as both a gateway for nutrients and a barrier for pathogen transfer to the fetus. Regardless, recent evidence suggests the presence of bacterial DNA in both placenta and fetus, and we have reported that DNA and protein from small numbers of bacteria gain access to the fetus from the maternal bloodstream. Other routes of environmental bacterial transfer from the mother to fetus remain unknown, as well as the physiological relevance of their presence. In these experiments, we examine multiple routes by which bacterial cellular components can enter the fetus and the fetal response to influx of bacterial DNA and protein. We inoculated maternal sheep with genetically-labeled S. aureus (Staphylococcus aureus) using three routes: intravenously, orally, and intra-vaginally. The inoculum did not produce sepsis or fever in the ewes, therefore mimicking incidental exposure to bacteria during pregnancy. 3-5 days post inoculation, we assessed the presence of bacterial components in the fetal tissues and analyzed fetal brain tissue to identify any alterations in gene expression. Our results demonstrate that components of bacteria that were introduced into the maternal mouth were detected in the fetal brain and that they stimulated changes in gene expression. We conclude that an oral route of transmission is relevant for transfer of bacterial cellular components to the fetus.

Systematic identification of A-to-I editing associated regulators from multiple human cancers.

A-to-I editing is the most common editing type in humans that is catalyzed by ADAR family members (ADARs), ADAR1 and ADAR2. Although millions of A-to-I editing sites have recently been discovered, the regulation mechanisms of the RNA editing process are still not clear. Herein, we developed a two-step logistic regression model to identify genes that are potentially involved in the RNA editing process in four human cancers. In the first step, we tested the association of each editing site with known enzymes. To validate the logistic regression model, we collected 10 genes with 168 editing sites from multiple published studies and obtained a nearly 100% validation rate. ADAR1 was identified as the enzyme associated with the majority of the A-to-I editing sites. Thus, ADAR1 was taken as a control gene in the second step to identify genes that have a stronger regulation effect on editing sites than ADAR1. Using our advanced method, we successfully found a set of genes that were significantly positively or negatively associated (PA or NA) with specific sets of RNA editing sites. 51 of these genes had been reported in at least one previous study. We highlighted two genes: 1), SRSF5, supported by three previous studies, and 2) MIR22HG, supported by one previous study and two of our cancer datasets. The PA and NA genes were cancer-specific but shared common pathways. Interestingly, the PA genes from kidney cancer were enriched for survival-associated genes while the NA genes were not, indicating that the PA genes may play more important roles in kidney cancer progression.

Author Correction: Integrating multi-platform genomic datasets for kidney renal clear cell carcinoma subtyping using stacked denoising autoencoders.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Integrating multi-platform genomic datasets for kidney renal clear cell carcinoma subtyping using stacked denoising autoencoders.

Clear cell renal cell carcinoma (ccRCC) is highly heterogeneous and is the most lethal cancer of all urologic cancers. We developed an unsupervised deep learning method, stacked denoising autoencoders (SdA), by integrating multi-platform genomic data for subtyping ccRCC with the goal of assisting diagnosis, personalized treatments and prognosis. We successfully found two subtypes of ccRCC using five genomics datasets for Kidney Renal Clear Cell Carcinoma (KIRC) from The Cancer Genome Atlas (TCGA). Correlation analysis between the last reconstructed input and the original input data showed that all the five types of genomic data positively contribute to the identification of the subtypes. The first subtype of patients had significantly lower survival probability, higher grade on neoplasm histology and higher stage on pathology than the other subtype of patients. Furthermore, we identified a set of genes, proteins and miRNAs that were differential expressed (DE) between the two subtypes. The function annotation of the DE genes from pathway analysis matches the clinical features. Importantly, we applied the model learned from KIRC as a pre-trained model to two independent datasets from TCGA, Lung Adenocarcinoma (LUAD) dataset and Low Grade Glioma (LGG), and the model stratified the LUAD and LGG patients into clinical associated subtypes. The successful application of our method to independent groups of patients supports that the SdA method and the model learned from KIRC are effective on subtyping cancer patients and most likely can be used on other similar tasks. We supplied the source code and the models to assist similar studies at https://github.com/tjgu/cancer_subtyping.

Clinical Relevance of Noncoding Adenosine-to-Inosine RNA Editing in Multiple Human Cancers.

PURPOSE: RNA editing is a post-transcriptional process that alters the nucleotide sequences of certain transcripts, in vertebrate most often converting adenosines to inosines. Multiple studies have recently implicated RNA editing in cancer development; however, most studies have focused on recoding RNA editing events. The function and clinical relevance of noncoding RNA (ncRNA) editing events in cancers have not been systematically examined. PATIENTS AND METHODS: We improved our previously published pipeline to identify ncRNA editing sites from four human cancers: liver hepatocellular carcinoma, lung adenocarcinoma, kidney renal clear-cell carcinoma, and thyroid carcinoma. We then developed multiple advanced statistical models to identify significantly differential edited (DE) sites between tumor and normal samples and clinical relevance ncRNA editing sites, as well as to investigate the association between gene expression, ncRNA editing, and microRNAs. Finally, we validated computational results with experiments. RESULTS: We identified 3,788 ncRNA editing sites of high confidence from the four cancers. We found thousands of DE sites which had distinct profiles across the four cancers. In kidney cancer, which had the largest uncensored survival data among the four cancers, 80 DE sites were significantly associated with patient survival. We identified 3' untranslated region (UTR) RNA editing sites that can affect gene expression, either independent of or by working with microRNAs. We validated that the 3'UTR RNA editing sites in CWF19L1 and F11R genes resulted in increased protein levels and that alterations of the expression of the two genes affected the proliferation of human embryonic kidney cells. CONCLUSION: On the basis of our computational and experimental results, we hypothesize that 3'UTR editing sites may affect their host gene expression, thereby affecting cell proliferation.

Sex-specific airway hyperreactivity and sex-specific transcriptome remodeling in neonatal piglets challenged with intra-airway acid.

Acute airway acidification is a potent stimulus of sensory nerves and occurs commonly with gastroesophageal reflux disease, cystic fibrosis, and asthma. In infants and adults, airway acidification can acutely precipitate asthma-like symptoms, and treatment-resistant asthma can be associated with gastroesophageal reflux disease. Airway protective behaviors, such as mucus secretion and airway smooth muscle contraction, are often exaggerated in asthma. These behaviors are manifested through activation of neural circuits. In some populations, the neural response to acid might be particularly important. For example, the immune response in infants is relatively immature compared with adults. Infants also have a high frequency of gastroesophageal reflux. Thus, in the current study, we compared the transcriptomes of an airway-nervous system circuit (e.g., tracheal epithelia, nodose ganglia, and brain stem) in neonatal piglets challenged with intra-airway acid. We hypothesized that the identification of parallel changes in the transcriptomes of two neutrally connected tissues might reveal the circuit response, and, hence, molecules important for the manifestation of asthma-like features. Intra-airway acid induced airway hyperreactivity and airway obstruction in male piglets. In contrast, female piglets displayed airway obstruction without airway hyperreactivity. Pairwise comparisons revealed parallel changes in genes directly implicated in airway hyperreactivity ( scn10a) in male acid-challenged piglets, whereas acid-challenged females exhibited parallel changes in genes associated with mild asthma ( stat 1 and isg15). These findings reveal sex-specific responses to acute airway acidification and highlight distinct molecules within a neural circuit that might be critical for the manifestation of asthma-like symptoms in pediatric populations.

Dicer cleaves 5'-extended microRNA precursors originating from RNA polymerase II transcription start sites.

MicroRNAs (miRNAs) are approximately 22 nucleotide (nt) long and play important roles in post-transcriptional regulation in both plants and animals. In animals, precursor (pre-) miRNAs are ∼70 nt hairpins produced by Drosha cleavage of long primary (pri-) miRNAs in the nucleus. Exportin-5 (XPO5) transports pre-miRNAs into the cytoplasm for Dicer processing. Alternatively, pre-miRNAs containing a 5' 7-methylguanine (m7G-) cap can be generated independently of Drosha and XPO5. Here we identify a class of m7G-capped pre-miRNAs with 5' extensions up to 39 nt long. The 5'-extended pre-miRNAs are transported by Exportin-1 (XPO1). Unexpectedly, a long 5' extension does not block Dicer processing. Rather, Dicer directly cleaves 5'-extended pre-miRNAs by recognizing its 3' end to produce mature 3p miRNA and extended 5p miRNA both in vivo and in vitro. The recognition of 5'-extended pre-miRNAs by the Dicer Platform-PAZ-Connector (PPC) domain can be traced back to ancestral animal Dicers, suggesting that this previously unrecognized Dicer reaction mode is evolutionarily conserved. Our work reveals additional genetic sources for small regulatory RNAs and substantiates Dicer's essential role in RNAi-based gene regulation.

Remodeling of the Streptococcus mutans proteome in response to LrgAB and external stresses.

The Streptococcus mutans Cid/Lrg system represents an ideal model to study how this organism withstands various stressors encountered in the oral cavity. Mutation of lrgAB renders S. mutans more sensitive to oxidative, heat, and vancomycin stresses. Here, we have performed a comprehensive proteomics experiment using label-free quantitative mass spectrometry to compare the proteome changes of wild type UA159 and lrgAB mutant strains in response to these same stresses. Importantly, many of identified proteins showed either a strikingly large fold-change, or were completely suppressed or newly induced in response to a particular stress condition. Notable stress proteome changes occurred in a variety of functional categories, including amino acid biosynthesis, energy metabolism, protein synthesis, transport/binding, and transcriptional/response regulators. In the non-stressed growth condition, mutation of lrgAB significantly altered the abundance of 76 proteins (a fold change >1.4, or <0.6, p-value <0.05) and several of these matched the stress proteome of the wild type strain. Interestingly, the statistical correlation between the proteome changes and corresponding RNA-seq transcriptomic studies was relatively low (rho(ρ) <0.16), suggesting that adaptation to a new environment may require radical proteome turnover or metabolic remodeling. Collectively, this study reinforces the importance of LrgAB to the S. mutans stress response.

GSR is not essential for the maintenance of antioxidant defenses in mouse cochlea: Possible role of the thioredoxin system as a functional backup for GSR.

Glutathione reductase (GSR), a key member of the glutathione antioxidant defense system, converts oxidized glutathione (GSSG) to reduced glutathione (GSH) and maintains the intracellular glutathione redox state to protect the cells from oxidative damage. Previous reports have shown that Gsr deficiency results in defects in host defense against bacterial infection, while diquat induces renal injury in Gsr hypomorphic mice. In flies, overexpression of GSR extended lifespan under hyperoxia. In the current study, we investigated the roles of GSR in cochlear antioxidant defense using Gsr homozygous knockout mice that were backcrossed onto the CBA/CaJ mouse strain, a normal-hearing strain that does not carry a specific Cdh23 mutation that causes progressive hair cell degeneration and early onset of hearing loss. Gsr-/- mice displayed a significant decrease in GSR activity and GSH/GSSG ratios in the cytosol of the inner ears. However, Gsr deficiency did not affect ABR (auditory brainstem response) hearing thresholds, wave I amplitudes or wave I latencies in young mice. No histological abnormalities were observed in the cochlea of Gsr-/- mice. Furthermore, there were no differences in the activities of cytosolic glutathione-related enzymes, including glutathione peroxidase and glutamate-cysteine ligase, or the levels of oxidative damage markers in the inner ears between WT and Gsr-/- mice. In contrast, Gsr deficiency resulted in increased activities of cytosolic thioredoxin and thioredoxin reductase in the inner ears. Therefore, under normal physiological conditions, GSR is not essential for the maintenance of antioxidant defenses in mouse cochlea. Given that the thioredoxin system is known to reduce GSSG to GSH in multiple species, our findings suggest that the thioredoxin system can support GSSG reduction in the mouse peripheral auditory system.

Modification of the Streptococcus mutans transcriptome by LrgAB and environmental stressors.

The Streptococcus mutans Cid/Lrg system is central to the physiology of this cariogenic organism, affecting oxidative stress resistance, biofilm formation and competence. Previous transcriptome analyses of lytS (responsible for the regulation of lrgAB expression) and cidB mutants have revealed pleiotropic effects on carbohydrate metabolism and stress resistance genes. In this study, it was found that an lrgAB mutant, previously shown to have diminished aerobic and oxidative stress growth, was also much more growth impaired in the presence of heat and vancomycin stresses, relative to wild-type, lrgA and lrgB mutants. To obtain a more holistic picture of LrgAB and its involvement in stress resistance, RNA sequencing and bioinformatics analyses were used to assess the transcriptional response of wild-type and isogenic lrgAB mutants under anaerobic (control) and stress-inducing culture conditions (aerobic, heat and vancomycin). Hierarchical clustering and principal components analyses of all differentially expressed genes revealed that the most distinct gene expression profiles between S. mutans UA159 and lrgAB mutant occurred during aerobic and high-temperature growth. Similar to previous studies of a cidB mutant, lrgAB stress transcriptomes were characterized by a variety of gene expression changes related to genomic islands, CRISPR-C as systems, ABC transporters, competence, bacteriocins, glucosyltransferases, protein translation, tricarboxylic acid cycle, carbohydrate metabolism/storage and transport. Notably, expression of lrgAB was upregulated in the wild-type strain under all three stress conditions. Collectively, these results demonstrate that mutation of lrgAB alters the transcriptional response to stress, and further support the idea that the Cid/Lrg system acts to promote cell homeostasis in the face of environmental stress.

Genetic Architectures of Quantitative Variation in RNA Editing Pathways.

RNA editing refers to post-transcriptional processes that alter the base sequence of RNA. Recently, hundreds of new RNA editing targets have been reported. However, the mechanisms that determine the specificity and degree of editing are not well understood. We examined quantitative variation of site-specific editing in a genetically diverse multiparent population, Diversity Outbred mice, and mapped polymorphic loci that alter editing ratios globally for C-to-U editing and at specific sites for A-to-I editing. An allelic series in the C-to-U editing enzyme Apobec1 influences the editing efficiency of Apob and 58 additional C-to-U editing targets. We identified 49 A-to-I editing sites with polymorphisms in the edited transcript that alter editing efficiency. In contrast to the shared genetic control of C-to-U editing, most of the variable A-to-I editing sites were determined by local nucleotide polymorphisms in proximity to the editing site in the RNA secondary structure. Our results indicate that RNA editing is a quantitative trait subject to genetic variation and that evolutionary constraints have given rise to distinct genetic architectures in the two canonical types of RNA editing.