Effects of repeated alcohol abstinence on within-subject prefrontal cortical gene expression in rhesus macaques.

Male rhesus monkeys (n = 24) had a biopsy of prefrontal cortical area 46 prior to chronic ethanol self-administration (n = 17) or caloric control (n = 7). Fourteen months of daily self-administration (water vs. 4% alcohol, 22 h access/day termed "open-access") was followed by two cycles of prolonged abstinence (5 weeks) each followed by 3 months of open-access alcohol and a final abstinence followed by necropsy. At necropsy, a biopsy of Area 46, contralateral to the original biopsy, was obtained. Gene expression data (RNA-Seq) were collected comparing biopsy/necropsy samples. Monkeys were categorized by drinking status during the final post-abstinent drinking phase as light (LD), binge (BD), heavy (HD) and very heavy (VHD drinkers). Comparing pre-ethanol to post-abstinent biopsies, four animals that converted from HD to VHD status had significant ontology enrichments in downregulated genes (necropsy minus biopsy n = 286) that included immune response (FDR < 9 × 10-7) and plasma membrane changes (FDR < 1 × 10-7). Genes in the immune response category included IL16 and 18, CCR1, B2M, TLR3, 6 and 7, SP2 and CX3CR1. Upregulated genes (N = 388) were particularly enriched in genes associated with the negative regulation of MAP kinase activity (FDR < 3 × 10-5), including DUSP 1, 4, 5, 6 and 18, SPRY 2, 3, and 4, SPRED2, BMP4 and RGS2. Overall, these data illustrate the power of the NHP model and the within-subject design of genomic changes due to alcohol and suggest new targets for treating severe escalated drinking following repeated alcohol abstinence attempts.

Mayaro virus pathogenesis and immunity in rhesus macaques.

Mayaro virus (MAYV) is a mosquito-transmitted alphavirus that causes debilitating and persistent arthritogenic disease. While MAYV was previously reported to infect non-human primates (NHP), characterization of MAYV pathogenesis is currently lacking. Therefore, in this study we characterized MAYV infection and immunity in rhesus macaques. To inform the selection of a viral strain for NHP experiments, we evaluated five MAYV strains in C57BL/6 mice and showed that MAYV strain BeAr505411 induced robust tissue dissemination and disease. Three male rhesus macaques were subcutaneously challenged with 105 plaque-forming units of this strain into the arms. Peak plasma viremia occurred at 2 days post-infection (dpi). NHPs were taken to necropsy at 10 dpi to assess viral dissemination, which included the muscles and joints, lymphoid tissues, major organs, male reproductive tissues, as well as peripheral and central nervous system tissues. Histological examination demonstrated that MAYV infection was associated with appendicular joint and muscle inflammation as well as presence of perivascular inflammation in a wide variety of tissues. One animal developed a maculopapular rash and two NHP had viral RNA detected in upper torso skin samples, which was associated with the presence of perivascular and perifollicular lymphocytic aggregation. Analysis of longitudinal peripheral blood samples indicated a robust innate and adaptive immune activation, including the presence of anti-MAYV neutralizing antibodies with activity against related Una virus and chikungunya virus. Inflammatory cytokines and monocyte activation also peaked coincident with viremia, which was well supported by our transcriptomic analysis highlighting enrichment of interferon signaling and other antiviral processes at 2 days post MAYV infection. The rhesus macaque model of MAYV infection recapitulates many of the aspects of human infection and is poised to facilitate the evaluation of novel therapies and vaccines targeting this re-emerging virus.

Optic Nerve Head Gene Transcription Sequelae to a Single Elevated IOP Exposure Provides Insights Into Known Responses to Chronically Elevated IOP.

Purpose: To clarify the optic nerve head (ONH) gene expression responses associated with a single, axon-damaging exposure to elevated IOP in relation to the composite cellular events previously identified in models of chronically elevated IOP. Methods: Anesthetized rats were exposed unilaterally to an 8-hour pulse-train controlled elevation of IOP (PT-CEI) at 60 mm Hg, while others received normotensive CEI at 20 mm Hg. ONH RNA was harvested at 0 hours and 1, 2, 3, 7, and 10 days after either CEI and from naïve animals. RNA sequencing was performed to analyze ONH gene expression. DAVID Bioinformatics tools were used to identify significant functional annotation clusters. Gene function was compared between PT-CEI and two models of chronic ocular hypertension from the literature. Results: The number of significantly changed genes peaked immediately (n = 1354) after PT-CEI (0 hours). This was followed by a lull (<4 genes per time point) at 1 and 2 days after PT-CEI. Gene activity increased again at 3 days (136 genes) and persisted at 7 (78 genes) and 10 (339 genes) days. Significant gene functional categories included an immediate upregulation of Defense Response at 0 hours, followed by upregulation in Cell Cycle, a reduction in Axonal-related genes at 3 to 10 days, and upregulation of Immune Response-related genes at 10 days following PT-CEI. The most commonly upregulated gene expression across our PT-CEI study and two chronic models of ocular hypertension were cell cycle related. Conclusions: The PT-CEI model places in sequence ONH gene expression responses previously reported in models with chronically elevated IOP and may provide insights into their role in optic nerve damage.

Transcriptional profiling of mucus production and modification in rhesus macaque endocervical cells under hormonal regulation.

Objective: Endocervical mucus production is a key regulator of fertility throughout the menstrual cycle. With cycle-dependent variability in mucus quality and quantity, cervical mucus can either facilitate or block sperm ascension into the upper female reproductive tract. This study seeks to identify genes involved in the hormonal regulation of mucus production, modification, and regulation through profiling the transcriptome of endocervical cells from the non-human primate, the Rhesus Macaque (Macaca mulatta). Design: Experimental. Setting: Translational science laboratory. Intervention: We treated differentiated primary endocervical cultures with estradiol (E2) and progesterone (P4) to mimic peri-ovulatory and luteal-phase hormonal changes. Using RNA-sequencing, we identified differential expression of gene pathways and mucus producing and modifying genes in cells treated with E2 compared to hormone-free conditions and E2 compared to E2-primed cells treated with P4. Main Outcome Measures: We pursued differential gene expression analysis on RNA-sequenced cells. Sequence validation was done using qPCR. Results: Our study identified 158 genes that show significant differential expression in E2-only conditions compared to hormone-free control, and 250 genes that show significant differential expression in P4-treated conditions compared to E2-only conditions. From this list, we found hormone-induced changes in transcriptional profiles for genes across several classes of mucus production, including ion channels and enzymes involved in post-translational mucin modification that have not previously been described as hormonally regulated. Conclusion: Our study is the first to use an in vitro culture system to create an epithelial-cell specific transcriptome of the endocervix. As a result, our study identifies new genes and pathways that are altered by sex-steroids in cervical mucus production.

The combined impact of testosterone and Western-style diet on endometriosis severity and progression in rhesus macaques†.

Polycystic ovary syndrome (PCOS) is associated with irregular menstrual cycles, hyperandrogenemia, and obesity. It is currently accepted that women with PCOS are also at risk for endometriosis, but the effect of androgen and obesity on endometriosis has been underexplored. The goal of this study was to determine how testosterone (T) and an obesogenic diet impact the progression of endometriosis in a nonhuman primate (NHP) model. Female rhesus macaques were treated with T (serum levels approximately 1.35 ng/ml), Western-style diet (WSD; 36% of calories from fat compared to 16% in standard monkey chow) or the combination (T + WSD) at the time of menarche as part of a longitudinal study for ~7 years. Severity of endometriosis was determined based on American Society for Reproductive Medicine (ASRM) revised criteria, and staged 1-4. Stages 1 and 2 were associated with extent of abdominal adhesions, while stages 3 and 4 were associated with presence of chocolate cysts. The combined treatment of T + WSD resulted in earlier onset of endometriosis and more severe types associated with large chocolate cysts compared to all other treatments. There was a strong correlation between glucose clearance, homeostatic model assessment for insulin resistance (HOMA-IR), and total percentage of body fat with presence of cysts, indicating possible indirect contribution of hyperandrogenemia via metabolic dysfunction. An RNA-seq analysis of omental adipose tissue revealed significant impacts on a number of inflammatory signaling pathways. The interactions between obesity, hyperandrogenemia, and abdominal inflammation deserve additional investigation in NHP model species.

Molecular contribution to embryonic aneuploidy and karyotypic complexity in initial cleavage divisions of mammalian development.

Embryonic aneuploidy is highly complex, often leading to developmental arrest, implantation failure or spontaneous miscarriage in both natural and assisted reproduction. Despite our knowledge of mitotic mis-segregation in somatic cells, the molecular pathways regulating chromosome fidelity during the error-prone cleavage-stage of mammalian embryogenesis remain largely undefined. Using bovine embryos and live-cell fluorescent imaging, we observed frequent micro-/multi-nucleation of mis-segregated chromosomes in initial mitotic divisions that underwent unilateral inheritance, re-fused with the primary nucleus or formed a chromatin bridge with neighboring cells. A correlation between a lack of syngamy, multipolar divisions and asymmetric genome partitioning was also revealed, and single-cell DNA-seq showed propagation of primarily non-reciprocal mitotic errors. Depletion of the mitotic checkpoint protein BUB1B (also known as BUBR1) resulted in similarly abnormal nuclear structures and cell divisions, as well as chaotic aneuploidy and dysregulation of the kinase-substrate network that mediates mitotic progression, all before zygotic genome activation. This demonstrates that embryonic micronuclei sustain multiple fates, provides an explanation for blastomeres with uniparental origins, and substantiates defective checkpoints and likely other maternally derived factors as major contributors to the karyotypic complexity afflicting mammalian preimplantation development.

Effects of early daily alcohol exposure on placental function and fetal growth in a rhesus macaque model.

BACKGROUND: Prenatal alcohol exposure is the most common cause of birth defects and intellectual disabilities and can increase the risk of stillbirth and negatively impact fetal growth. OBJECTIVE: To determine the effect of early prenatal alcohol exposure on nonhuman primate placental function and fetal growth. We hypothesized that early chronic prenatal alcohol would alter placental perfusion and oxygen availability that adversely affects fetal growth. STUDY DESIGN: Rhesus macaques self-administered 1.5 g/kg/d of ethanol (n=12) or isocaloric maltose-dextrin (n=12) daily before conception through the first 60 days of gestation (term is approximately 168 days). All animals were serially imaged with Doppler ultrasound to measure fetal biometry, uterine artery volume blood flow, and placental volume blood flow. Following Doppler ultrasound, all animals underwent both blood oxygenation level-dependent magnetic resonance imaging to characterize placental blood oxygenation and dynamic contrast-enhanced magnetic resonance imaging to quantify maternal placental perfusion. Animals were delivered by cesarean delivery for placental collection and fetal necropsy at gestational days 85 (n=8), 110 (n=8), or 135 (n=8). Histologic and RNA-sequencing analyses were performed on collected placental tissue. RESULTS: Placental volume blood flow was decreased at all gestational time points in ethanol-exposed vs control animals, but most significantly at gestational day 110 by Doppler ultrasound (P<.05). A significant decrease in total volumetric blood flow occurred in ethanol-exposed vs control animals on dynamic contrast-enhanced magnetic resonance imaging at both gestation days 110 and 135 (P<.05); moreover, a global reduction in T2∗, high blood deoxyhemoglobin concentration, occurred throughout gestation (P<.05). Similarly, evidence of placental ischemic injury was notable by histologic analysis, which revealed a significant increase in microscopic infarctions in ethanol-exposed, not control, animals, largely present at middle to late gestation. Fetal biometry and weight were decreased in ethanol-exposed vs control animals, but the decrease was not significant. Analysis with RNA sequencing suggested the involvement of the inflammatory and extracellular matrix response pathways. CONCLUSION: Early chronic prenatal alcohol exposure significantly diminished placental perfusion at mid to late gestation and also significantly decreased the oxygen supply to the fetal vasculature throughout pregnancy, these findings were associated with the presence of microscopic placental infarctions in the nonhuman primate. Although placental adaptations may compensate for early environmental perturbations to fetal growth, placental blood flow and oxygenation were reduced, consistent with the evidence of placental ischemic injury.

Synaptic effects of IL-1ß and CRF in the central amygdala after protracted alcohol abstinence in male rhesus macaques.

A major barrier to remission from an alcohol use disorder (AUD) is the continued risk of relapse during abstinence. Assessing the neuroadaptations after chronic alcohol and repeated abstinence is important to identify mechanisms that may contribute to relapse. In this study, we used a rhesus macaque model of long-term alcohol use and repeated abstinence, providing a platform to extend mechanistic findings from rodents to primates. The central amygdala (CeA) displays elevated GABA release following chronic alcohol in rodents and in abstinent male macaques, highlighting this neuroadaptation as a conserved mechanism that may underlie excessive alcohol consumption. Here, we determined circulating interleukin-1ß (IL-1ß) levels, CeA transcriptomic changes, and the effects of IL-1ß and corticotropin releasing factor (CRF) signaling on CeA GABA transmission in male controls and abstinent drinkers. While no significant differences in peripheral IL-1ß or the CeA transcriptome were observed, pathway analysis identified several canonical immune-related pathways. We addressed this potential dysregulation of CeA immune signaling in abstient drinkers with an electrophysiological approach. We found that IL-1ß decreased CeA GABA release in controls while abstinent drinkers were less sensitive to IL-1ß's effects, suggesting adaptations in the neuromodulatory role of IL-1ß. In contrast, CRF enhanced CeA GABA release similarly in controls and abstinent drinkers, consistent with rodent studies. Notably, CeA CRF expression was inversely correlated with intoxication, suggesting that CRF levels during abstinence may predict future intoxication. Together, our findings highlight conserved and divergent actions of chronic alcohol on neuroimmune and stress signaling on CeA GABA transmission across rodents and macaques.

Identifying RNA Biomarkers and Molecular Pathways Involved in Multiple Subtypes of Uveitis.

PURPOSE: Uveitis is a heterogeneous collection of diseases. We tested the hypothesis that despite the diversity of uveitides, there could be common mechanisms shared by multiple subtypes, and that evidence of these common mechanisms may be detected as gene expression profiles in whole blood. DESIGN: Cohort study. METHODS: Ninety subjects with uveitis including axial spondyloarthritis (n = 17), sarcoidosis (n = 13), inflammatory bowel disease (n = 12), tubulointerstitial nephritis with uveitis (n = 10), or idiopathic uveitis (n = 38) as well as 18 healthy controls were enrolled, predominantly at Oregon Health & Science University. RNA-Seq data generated from peripheral, whole blood identified 19,859 unique transcripts. We analyzed gene expression pathways via Kyoto Encyclopedia of Genes and Genomes and Gene Ontology (GO). We validated our list of upregulated genes by comparison to a previously published study on peripheral blood gene expression among 50 subjects with diverse forms of uveitis. RESULTS: Both the Kyoto Encyclopedia of Genes and Genomes and GO analysis identified multiple shared pathways or GO terms with a P value of <.0001. Almost all pathways related to the immune response and/or response to an infection. A total of 119 individual transcripts were upregulated by at least 1.5-fold and false discovery rate <.05, and 61 were downregulated by similar criteria. Comparing mRNA from our study with a false discovery rate <.05 and the prior report, we identified 10 common gene transcripts: ICAM1, IL15RA, IL15, IRF1, IL10RB, GSK3A, TYK2, MEF2A, MEF2B, and MEF2D. CONCLUSIONS: Many forms of uveitis share overlapping mechanisms. These data support the concept that a single therapeutic approach could benefit diverse forms of this disease.

Blocking the IL-1 receptor reduces cardiac transplant ischemia and reperfusion injury and mitigates CMV-accelerated chronic rejection.

Ischemia-reperfusion injury (IRI) is an important risk factor for accelerated cardiac allograft rejection and graft dysfunction . Utilizing a rat heart isogeneic transplant model, we identified inflammatory pathways involved in IRI in order to identify therapeutic targets involved in disease. Pathway analyses identified several relevant targets, including cytokine signaling by the IL-1 receptor (IL-1R) pathway and inflammasome activation. To investigate the role of IL-1R signaling pathways during IRI, we treated syngeneic cardiac transplant recipients at 1-hour posttransplant with Anakinra, a US Food and Drug Administration (FDA)-approved IL-1R antagonist; or parthenolide, a caspase-1 and nuclear factor kappa-light-chain-enhancer of activated B cells inhibitor that blocks IL-1ß maturation. Both Anakinra and parthenolide significantly reduced graft inflammation and cellular recruitment in the treated recipients relative to nontreated controls. Anakinra treatment administered at 1-hour posttransplant to recipients of cardiac allografts from CMV-infected donors significantly increased the time to rejection and reduced viral loads at rejection. Our results indicate that reducing IRI by blocking IL-1Rsignaling pathways with Anakinra or inflammasome activity with parthenolide provides a promising approach for extending survival of cardiac allografts from CMV-infected donors.

Revising the Diagnosis of Idiopathic Uveitis by Peripheral Blood Transcriptomics.

PURPOSE: To test the hypothesis that idiopathic uveitis can be categorized into subtypes based on gene expression from blood. DESIGN: Case control study. METHODS: We applied RNA-Seq to peripheral blood from patients with uveitis associated with 1 of 4 systemic diseases, including axial spondyloarthritis (n = 17), sarcoidosis (n = 13), inflammatory bowel disease (n = 12), tubulo-interstitial nephritis with uveitis (n = 10), or idiopathic uveitis (n = 38) as well as 18 healthy control subjects evaluated predominantly at Oregon Health and Science University. A high-dimensional negative binomial regression model implemented in the edgeR R package compared each disease group with the control subjects. The 20 most distinctive genes for each diagnosis were extracted. Of 80 genes, there were 75 unique genes. A classification algorithm was developed by fitting a gradient boosting tree with 5-fold cross-validation. Messenger RNA from subjects with idiopathic uveitis were analyzed to see if any fit clinically and by gene expression pattern with one of the diagnosable entities. RESULTS: For uveitis associated with a diagnosable systemic disease, gene expression profiling achieved an overall accuracy of 85% (balanced average of sensitivity plus specificity, P < .001). Although most patients with idiopathic uveitis presumably have none of these 4 associated systemic diseases, gene expression profiles helped to reclassify 11 of 38 subjects. CONCLUSIONS: Peripheral blood gene expression profiling is a potential adjunct in accurate differential diagnosis of the cause of uveitis. Validation of these results and characterization of the gene expression profile from additional discrete diagnoses could enhance the value of these observations.

RNA-Seq of human whole blood: Evaluation of globin RNA depletion on Ribo-Zero library method.

Peripheral blood is a highly accessible biofluid providing a rich source of information about human physiology and health status. However, for studies of the blood transcriptome with RNA sequencing (RNA-Seq) techniques, high levels of hemoglobin mRNAs (hgbRNA) present in blood can occupy valuable sequencing space, impacting detection and quantification of non-hgbRNAs. In this study, we evaluated two methods for preparing ribosomal RNA (rRNA)-depleted sequencing libraries for RNA-Seq of whole blood, one of which is also designed to deplete hgbRNAs. Two experiments were performed: one evaluating library performance across 6 human blood samples and the other examining library reproducibility and performance in a two-subject subset. We find that addition of hgbRNA depletion to the rRNA-depletion protocol for library preparation from blood RNA effectively reduces highly abundant hgbRNA reads; however, it does not result in a statistically significant increase in differentially expressed genes in our patient-control study. Bioinformatic removal of globin gene counts in non-hgbRNA depleted libraries provides improvement in overall performance of these libraries. We conclude that use of a standard ribosomal RNA depletion method for library preparation coupled with bioinformatic removal of globin gene counts is sufficient for reproducible and sensitive measurement of both coding and noncoding RNAs in the blood transcriptome.

Molecular analysis of lymphoid tissue from rhesus macaque rhadinovirus-infected monkeys identifies alterations in host genes associated with oncogenesis.

Rhesus macaque (RM) rhadinovirus (RRV) is a simian gamma-2 herpesvirus closely related to human Kaposi's sarcoma-associated herpesvirus (KSHV). RRV is associated with the development of diseases in simian immunodeficiency virus (SIV) co-infected RM that resemble KSHV-associated pathologies observed in HIV-infected humans, including B cell lymphoproliferative disorders (LPD) and lymphoma. Importantly, how de novo KSHV infection affects the expression of host genes in humans, and how these alterations in gene expression affect viral replication, latency, and disease is unknown. The utility of the RRV/RM infection model provides a novel approach to address these questions in vivo, and utilizing the RRV bacterial artificial chromosome (BAC) system, the effects of specific viral genes on host gene expression patterns can also be explored. To gain insight into the effects of RRV infection on global host gene expression patterns in vivo, and to simultaneously assess the contributions of the immune inhibitory viral CD200 (vCD200) molecule to host gene regulation, RNA-seq was performed on pre- and post-infection lymph node (LN) biopsy samples from RM infected with either BAC-derived WT (n = 4) or vCD200 mutant RRV (n = 4). A variety of genes were identified as being altered in LN tissue samples due to RRV infection, including cancer-associated genes activation-induced cytidine deaminase (AICDA), glypican-1 (GPC1), CX3C chemokine receptor 1 (CX3CR1), and Ras dexamethasone-induced 1 (RasD1). Further analyses also indicate that GPC1 may be associated with lymphomagenesis. Finally, comparison of infection groups identified the differential expression of host gene thioredoxin interacting protein (TXNIP), suggesting a possible mechanism by which vCD200 negatively affects RRV viral loads in vivo.

Mild hyperandrogenemia in presence/absence of a high-fat, Western-style diet alters secretory phase endometrial transcriptome in nonhuman primates.

OBJECTIVE: To identify novel transcriptomic changes to eutopic endometrium by exposure to chronic mild hypernadrogenemia (testosterone [T]) with/without exposure to an obesogenic Western-style diet (WSD). DESIGN: Two-by-two factorial arrangement of treatments. SETTING: National primate research center. ANIMALS: Rhesus macaque females were chronically exposed to T and/or consumed a WSD from menarche through adulthood. After 4.5 years of treatment, Tru-Cut endometrial biopsies were obtained at the midsecretory phase (n = 6-4/group), and paired-end sequencing of RNA was performed. Several females in the T, WSD, and T+WSD cohorts developed endometriosis within 6 months of biopsy; a separate analysis was performed contrasting diagnosis of endometriosis stage 0-2 versus stages 3 and 4 (American Society for Reproductive Medicine revised criteria). INTERVENTIONS: Chronic exposure to mild elevation of T (~five-fold elevation) and/or WSD from menarche until adulthood. MAIN OUTCOME MEASURES: Limma voom empirical Bayes pipeline was performed to detect differentially expressed RNAs (DEs) significantly impacted by treatments and endometriosis severity. Differentially expressed RNAs were then interrogated by Ingenuity Pathway Analyses and Protein Analysis through Evolutionary Relationships. RESULTS: Total DEs included C versus T, 469; C versus WSD, 525; C versus T+WSD, 549; and T versus T+WSD, 1,505. The majority of DEs mapped to the ontology pathways: heterotrimeric G-protein signaling pathways Gi alpha and Gs alpha (C vs. T), WNT signaling (C vs. WSD and T vs. T+WSD), and Huntington disease (C vs. T+WSD). A total of 2,171 DEs from eutopic endometrium were altered by the presence of stage 3 and 4 endometriosis lesions. CONCLUSIONS: The present global transcriptomic analyses demonstrate that the greatest magnitude of changes occurred in contrasts of C and T versus T+WSD, adding to the evidence that these two insults have a synergistic effect on female physiology. These data also support the concept that prior alterations to the function of eutopic endometrium increase the risk for endometriosis.

Transcriptome analysis during photostimulated recrudescence reveals distinct patterns of gene regulation in Siberian hamster ovaries†.

In Siberian hamsters, exposure to short days (SDs, 8 h light:16 h dark) reduces reproductive function centrally by decreasing gonadotropin secretion, whereas subsequent transfer of photoinhibited hamsters to stimulatory long days (LDs, 16 L:8 D) promotes follicle stimulating hormone (FSH) release inducing ovarian recrudescence. Although differences between SD and LD ovaries have been investigated, a systematic investigation of the ovarian transcriptome across photoperiod groups to identify potentially novel factors that contribute to photostimulated restoration of ovarian function had not been conducted. Hamsters were assigned to one of four photoperiod groups: LD to maintain ovarian cyclicity, SD to induce ovarian regression, or post transfer (PT), where females housed in SD for 14-weeks were transferred to LD for 2-days or 1-week to reflect photostimulated ovaries prior to (PTd2) and following (PTw1) the return of systemic FSH. Ovarian RNA was extracted to create RNA-sequencing libraries and short-read sequencing Illumina assays that mapped and quantified the ovarian transcriptomes (n = 4/group). Ovarian and uterine masses, plasma FSH, and numbers of antral follicles and corpora lutea decreased in SD as compared to LD ovaries (P < 0.05). When reads were aligned to the mouse genome, 18 548 genes were sufficiently quantified. Most of the differentially expressed genes noted between functional LD ovaries and regressed SD ovaries; however, five main expression patterns were identified across photoperiod groups. These results, generally corroborated by select protein immunostaining, provide a map of photoregulated ovary function and identify novel genes that may contribute to the photostimulated resumption of ovarian activity.

RNA-seq from archival FFPE breast cancer samples: molecular pathway fidelity and novel discovery.

BACKGROUND: Formalin-fixed, paraffin-embedded (FFPE) tissues for RNA-seq have advantages over fresh frozen tissue including abundance and availability, connection to rich clinical data, and association with patient outcomes. However, FFPE-derived RNA is highly degraded and chemically modified, which impacts its utility as a faithful source for biological inquiry. METHODS: True archival FFPE breast cancer cases (n = 58), stored at room temperature for 2-23 years, were utilized to identify key steps in tissue selection, RNA isolation, and library choice. Gene expression fidelity was evaluated by comparing FFPE data to public data obtained from fresh tissues, and by employing single-gene, gene set and transcription network-based regulon analyses. RESULTS: We report a single 10 µm section of breast tissue yields sufficient RNA for RNA-seq, and a relationship between RNA quality and block age that was not linear. We find single-gene analysis is limiting with FFPE tissues, while targeted gene set approaches effectively distinguish ER+ from ER- breast cancers. Novel utilization of regulon analysis identified the transcription factor KDM4B to associate with ER+ disease, with KDM4B regulon activity and gene expression having prognostic significance in an independent cohort of ER+ cases. CONCLUSION: Our results, which outline a robust FFPE-RNA-seq pipeline for broad use, support utilizing FFPE tissues to address key questions in the breast cancer field, including the delineation between indolent and life-threatening disease, biological stratification and molecular mechanisms of treatment resistance.

Synergistic Effects of Hyperandrogenemia and Obesogenic Western-style Diet on Transcription and DNA Methylation in Visceral Adipose Tissue of Nonhuman Primates.

Polycystic ovary syndrome (PCOS) is a major reproductive disorder that is responsible for 80% of anovulatory infertility and that is associated with hyperandrogenemia, increased risk of obesity, and white adipose tissue (WAT) dysfunction. We have previously demonstrated that the combination of chronic testosterone (T) treatment and an obesogenic Western-style diet (WSD) exerts synergistic functional effects on WAT, leading to increased lipid accumulation in visceral adipocytes by an unknown mechanism. In this study, we examined the whole-genome transcriptional response in visceral WAT to T and WSD, alone and in combination. We observed a synergistic effect of T and WSD on gene expression, resulting in upregulation of lipid storage genes concomitant with adipocyte hypertrophy. Because DNA methylation is known to be associated with body fat distribution and the etiology of PCOS, we conducted whole-genome DNA methylation analysis of visceral WAT. While only a fraction of differentially expressed genes also exhibited differential DNA methylation, in silico analysis showed that differentially methylated regions were enriched in transcription factor binding motifs, suggesting a potential gene regulatory role for these regions. In summary, this study demonstrates that hyperandrogenemia alone does not induce global transcriptional and epigenetic response in young female macaques unless combined with an obesogenic diet.

Sex differences in sympathetic gene expression and cardiac neurochemistry in Wistar Kyoto rats.

The stellate ganglia are the predominant source of sympathetic innervation to the heart. Remodeling of sympathetic nerves projecting to the heart has been observed in several cardiovascular diseases, and sympathetic dysfunction contributes to cardiac pathology. Wistar Kyoto rats are a common model for the study of cardiovascular diseases, but we lack a profile of the baseline transcriptomic and neurochemical characteristics of their cardiac sympathetic neurons. Most studies of cardiovascular disease have used male animals only, but in the future both male and female animals will be used for these types of studies; therefore, we sought to characterize the transcriptome of male and female stellate ganglia and to correlate that with catecholamine and acetylcholine content in the heart. We have generated a dataset of baseline RNA expression in male and female Wistar Kyoto rat stellate ganglia using RNA-seq, and have measured neurotransmitter levels in heart and stellate ganglia using HPLC and mass spectrometry. We identified numerous gene expression differences between male and female stellates, including genes encoding important developmental factors, receptors and neuropeptides. Female hearts had significantly higher neurotransmitter content than male hearts; however, no significant differences were detected in expression of the genes encoding neurotransmitter synthetic enzymes. Similarly, no statistically significant differences were identified between the sexes in cardiac tyrosine hydroxylase levels.

Single-cell sequencing of primate preimplantation embryos reveals chromosome elimination via cellular fragmentation and blastomere exclusion.

Aneuploidy that arises during meiosis and/or mitosis is a major contributor to early embryo loss. We previously showed that human preimplantation embryos encapsulate missegregated chromosomes into micronuclei while undergoing cellular fragmentation and that fragments can contain chromosomal material, but the source of this DNA was unknown. Here, we leveraged the use of a nonhuman primate model and single-cell DNA-sequencing (scDNA-seq) to examine the chromosomal content of 471 individual samples comprising 254 blastomeres, 42 polar bodies, and 175 cellular fragments from a large number (N = 50) of disassembled rhesus cleavage-stage embryos. Our analysis revealed that the aneuploidy and micronucleation frequency is conserved between humans and macaques, and that fragments encapsulate whole and/or partial chromosomes lost from blastomeres. Single-cell/fragment genotyping showed that these chromosome-containing cellular fragments (CCFs) can be maternally or paternally derived and display double-stranded DNA breaks. DNA breakage was further indicated by reciprocal subchromosomal losses/gains between blastomeres and large segmental errors primarily detected at the terminal ends of chromosomes. By combining time-lapse imaging with scDNA-seq, we determined that multipolar divisions at the zygote or two-cell stage were associated with CCFs and generated a random mixture of chromosomally normal and abnormal blastomeres with uniparental or biparental origins. Despite frequent chromosome missegregation at the cleavage-stage, we show that CCFs and nondividing aneuploid blastomeres showing extensive DNA damage are prevented from incorporation into blastocysts. These findings suggest that embryos respond to chromosomal errors by encapsulation into micronuclei, elimination via cellular fragmentation, and selection against highly aneuploid blastomeres to overcome chromosome instability during preimplantation development.