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For over a decade, a large research community has relied on a flawed reference assembly of the genome ofRattus norvegicusknown as Rnor_6.0. The seventh assembly of the rat reference genome4mRatBN7.2, based on the inbred Brown Norway rat, corrects numerous misplaced segments, reduces base-level errors by approximately 9-fold, and increases contiguity by 290-fold, despite some remaining regions of potential misassembly. Gene annotations are now more complete, significantly improving the mapping precision of genomic, transcriptomic, and proteomics data sets. SimpleLiftOverfrom Rnor_6.0 to mRatBN7.2 misses ∼12% of variants. To facilitate the transition to mRatBN7.2, we performed a joint analysis of 163 whole genomes representing 120 strains/substrains. We defined 20.0 million sequence variations, of which 18.7 thousand are predicted to potentially impact the function of 6,677 genes. Phylogenetic analysis confirmed historical records and prior results and refined the ancestral relationship of these strains. Sixteen million polymorphisms segregate in the widely studied heterogeneous stock rat population, and 11313 million variants segregate collectively in the HXB/BXH and FXLE/LEXF strain families. Some inbred strains differ by only 132 M variants, and closely related substrains segregate by even fewer variants. We generated a new rat genetic map based on data from 1,893 heterogeneous stock rats and annotated transcription start sites and alternative polyadenylation sites.

Many aspects of innate immune responses to SARS viruses remain unclear. Of particular interest is the role of emerging neutralizing antibodies against the receptor-binding domain (RBD) of SARS-CoV-2 in complement activation and opsonization. To overcome challenges with purified virions, here we introduce "pseudovirus-like" nanoparticles with ∼70 copies of functional recombinant RBD to map complement responses. Nanoparticles fix complement in an RBD-dependent manner in sera of all vaccinated, convalescent, and naı̈ve donors, but vaccinated and convalescent donors with the highest levels of anti-RBD antibodies show significantly higher IgG binding and higher deposition of the third complement protein (C3). The opsonization

Background and aim Metabolic syndrome (MetS) is a complex disease of physiological imbalances interrelated to abnormal metabolic conditions, such as abdominal obesity, type II diabetes, dyslipidemia and hypertension. In the present pilot study, we investigated the nutraceutical bitter melon (Momordica charantia L) -intake induced transcriptome and metabolome changes and the converging metabolic signaling networks underpinning its inhibitory effects against MetS-associated risk factors. Experimental procedure Metabolic effects of lyophilized bitter melon juice (BMJ) extract (oral gavage 200 mg/kg/body weight-daily for 40 days) intake were evaluated in diet-induced obese C57BL/6J male mice [fed-high fat diet (HFD), 60 kcal% fat]. Changes in a) serum levels of biochemical parameters, b) gene expression in the hepatic transcriptome (microarray analysis using Affymetrix Mouse Exon 1.0 ST arrays), and c) metabolite abundance levels in lipid-phase plasma [liquid chromatography mass spectrometry (LC-MS)-based metabolomics] after BMJ intervention were assessed. Results and conclusion BMJ-mediated changes showed a positive trend towards enhanced glucose homeostasis, vitamin D metabolism and suppression of glycerophospholipid metabolism. In the liver, nuclear peroxisome proliferator-activated receptor (PPAR) and circadian rhythm signaling, as well as bile acid biosynthesis and glycogen metabolism targets were modulated by BMJ (p

Introduction: Relatively little is known about the molecular pathways influenced by cannabis use in humans. We used a multi-omics approach to examine protein, metabolomic, and lipid markers in plasma differentiating between cannabis users and nonusers to understand markers associated with cannabis use. Methods: Eight discordant twin pairs and four concordant twin pairs for cannabis use completed a blood draw, urine and plasma toxicology testing, and provided information about their past 30-day cannabis use and other substance use patterns. The 24 twins were all non-Hispanic whites. Sixty-six percent were female. Median age was 30 years. Fifteen participants reported that they had used cannabis in the last 30 days, including eight participants that used every day or almost every day (29–30 of 30 days). Of these 15 participants, plasma 11-nor-9-carboxy-Δ(9)-tetrahydrocannabinol (THC-COOH) and total tetrahydrocannabinol (THC) concentrations were detectable in 12 participants. Among the eight “heavy users” the amount of total THC (sum of THC and its metabolites) and plasma THC-COOH concentrations varied widely, with ranges of 13.1–1713 ng/mL and 2.7–284 ng/mL, respectively. A validated liquid chromatography–tandem mass spectrometry (LC-MS/MS) assay measured plasma THC-COOH, THC, and other cannabinoids and metabolites. Plasma THC-COOH was used as the primary measure. Expression levels of 1305 proteins were measured using SOMAScan assay, and 34 lipid mediators and 314 metabolites were measured with LC-MS/MS. Analyses examined associations between markers and THC-COOH levels with and without taking genetic relatedness into account. Results: Thirteen proteins, three metabolites, and two lipids were identified as associated with THC-COOH levels. Myc proto-oncogene was identified as associated with THC-COOH levels in both molecular insight and potential marker analyses. Five pathways (interleukin-6 production, T lymphocyte regulation, apoptosis, kinase signaling pathways, and nuclear factor kappa-light-chain-enhancer of activated B cells) were linked with molecules identified in these analyses. Conclusions: THC-COOH levels are associated with immune system-related pathways. This study presents a feasible approach to identify additional molecular markers associated with THC-COOH levels.

The complement system plays a key role in opsonization and immune clearance of engineered nanoparticles. Understanding the efficiency, inter-subject, and inter-strain differences of complement opsonization in preclinical species can help with translational nanomedicine development and improve our ability to model complement response in humans. Dextran-coated superparamagnetic iron oxide (SPIO) nanoparticles and a wide range of non-magnetic iron oxide nanoparticle formulations are widely used in magnetic resonance imaging and as clinically approved iron supplements. Previously we found that opsonization of SPIO nanoworms (NW) with the third complement protein (C3) proceeds mostly via the alternative pathway in humans, and via the lectin pathway in mice. Here, we studied the pathway and efficiency of opsonization of 106 nm SPIO NW with C3 in different preclinical species and commonly used laboratory strains. In sera of healthy human donors (n = 6), C3 opsonization proceeded exclusively through the alternative pathway. On the other hand, the C3 opsonization in dogs (6 breeds), rats (4 strains) and mice (5 strains) sera was either partially or completely dependent on the complement Ca2+-sensitive pathways (lectin and/or classical). Specifically, C3 opsonization in sera of Long Evans rat strain, and mouse strains widely used in nanomedicine research (BALB/c, C57BL/6 J, and A/J) was only through the Ca2+-dependent pathways. Dogs and humans had the highest between-subject variability in C3 opsonization levels, while rat and mouse sera showed the lowest between-strain variability. Furthermore, using a panel of SPIO nanoparticles of different sizes and dextran coatings, we found that the level of C3 opsonization (C3 molecules per milligram Fe) in human sera was lower than in animal sera. At the same time, there was a strong predictive value of complement opsonization in dog and rat sera; nanoparticles with higher C3 deposition in animals showed higher deposition in humans, and vice versa. Notably, the opsonization decreased with decreasing size in all sera. The studies highlight the importance of the consideration of species and strains for predicting human complement responses (opsonization) towards nanomedicines.

BACKGROUND: The objective was to assess the relationship between single nucleotide polymorphisms in mycophenolate and cytomegalovirus antiviral drug pharmacokinetic and pharmacodynamic genes and drug-induced leukopenia in adult heart transplant recipients. METHODS: This retrospective analysis included N=148 patients receiving mycophenolate and a cytomegalovirus antiviral drug. In total, 81 single nucleotide polymorphisms in 21 pharmacokinetic and 23 pharmacodynamic genes were selected for investigation. The primary and secondary outcomes were mycophenolate and/or cytomegalovirus antiviral drug-induced leukopenia, defined as a white blood cell count C (p.I27L) was associated with drug-induced leukopenia (unadjusted p=0.002; false discovery rate

Down syndrome (DS) is the most common genetic cause of intellectual disability. Mechanistically, oxidative stress and mitochondrial dysfunction are reported to be etiological factors for many of the DS-related comorbidities and have previously been reported in a number of in vitro and in vivo models of DS. The purpose of this study was to test for the presence of mitochondrial dysfunction in fibroblast cells obtained via skin biopsy from individuals with DS, and to assess the impact of trisomy 21 on central carbon metabolism. Using extracellular flux assays in matched dermal fibroblasts from euploid and DS individuals, we found that basal mitochondrial dysfunction is quite mild. Stressing the cells with a cocktail of mitochondrial stressors revealed a significant mitochondrial deficit in DS cells compared to euploid controls. Evaluation of extracellular acidification rate did not reveal a baseline abnormality in glycolysis; however, metabolomic assessments utilizing isotopically labeled glucose and glutamine revealed altered central carbon metabolism in DS cells. Specifically, we observed greater glucose dependency, uptake and flux into the oxidative phase of the pentose phosphate pathway in DS fibroblasts. Furthermore, using induced pluripotent stem cells (iPSC) we found that mitochondrial function in DS iPSCs was similar to the previously published studies employing fetal cells. Together, these data indicate that aberrant central carbon metabolism is a candidate mechanism for stress-related mitochondrial dysfunction in DS.

Tenofovir diphosphate (TFV-DP) concentrations measured with dried blood spots (DBS) can be used to classify adherence to emtricitabine/tenofovir disoproxil fumarate (F/TDF) for HIV pre-exposure prophylaxis (PrEP). A TFV-DP of 700 fmol/punch was previously associated with high PrEP efficacy, and was estimated to represent ≥4 doses/week on average. However, interindividual variability in TFV-DP concentrations may lead to adherence misclassification and decrease the precision of adherence–efficacy relationships. The purpose of this analysis was to evaluate sources of TFV-DP variability to improve the precision of TFV-DP for adherence assessments by incorporating individual characteristics. Data and samples from a 36-week study of TFV-DP in DBS, collected biweekly, among 48 HIV-negative volunteers (25 Females/26 Caucasian/10 African American/14 Hispanic) receiving F/TDF at 33%, 67%, and 100% of daily dosing under directly observed therapy were used for analysis. The simplest pharmacokinetic model to describe TFV-DP accumulation with acceptable performance was a one-compartment constant input model. Covariates, including laboratory values and demographics were ranked in importance of their association with post hoc pharmacokinetic (PK) parameters using random forest analyses. Weight and platelet count were included in the final model and simulations were conducted to generate benchmarks for 110 kg) doses/week, amounting to a much lower rate of misspecification (17% vs. 30%) with this individualized model versus previous interpretations. Incorporating body weight and platelet count improved the precision of TFV-DP concentrations for adherence assessments. Previous benchmarks were conservative, indicating that the pharmacological forgiveness of F/TDF may be higher than currently recognized and supports continued investigation of intermittent PrEP dosing regimens. Clinical Trial Registration number, NCT02022657.

We developed a framework for identifying trait-associated genes in rats and facilitating the transfer of polygenic evidence across species by expanding the transcriptome-wide association (TWAS) approach to rats. Our analysis successfully trained transcript predictors for over 8000 genes in each of the five brain regions of rats, revealing several shared properties of gene regulation with humans. Moreover, mirroring trends observed in humans, our findings showed that sparse predictors using variants in cis are more effective than polygenic predictors and that gene expression prediction in rats is highly correlated across brain regions. Importantly, our analysis also identified a significant overlap between genes associated with rat and human body length and BMI, indicating rat models may be useful for studying the genetic basis of complex traits in humans. RatXcan represents a valuable tool for uncovering shared biological mechanisms of complex traits across species, with potential applications in a wide range of research fields.

The R7 and R8 photoreceptor cells of the Drosophila compound eye mediate color vision. Throughout the majority of the eye, these cells occur in two principal types of ommatidia. Approximately 35% of ommatidia are of the pale type and express Rh3 in R7 cells and Rh5 in R8 cells. The remaining 65% are of the yellow type and express Rh4 in R7 cells and Rh6 in R8 cells. The specification of an R8 cell in a pale or yellow ommatidium depends on the fate of the adjacent R7 cell. However, pale and yellow R7 cells are specified by a stochastic process that requires the genes spineless, tango and klumpfuss. To identify additional genes involved in this process we performed genetic screens using a collection of 480 P{EP} transposon insertion strains. We identified genes in gain of function and loss of function screens that significantly altered the percentage of Rh3 expressing R7 cells (Rh3%) from wild-type. 36 strains resulted in altered Rh3% in the gain of function screen where the P{EP} insertion strains were crossed to a sevEP-GAL4 driver line. 53 strains resulted in altered Rh3% in the heterozygous loss of function screen. 4 strains showed effects that differed between the two screens, suggesting that the effect found in the gain of function screen was either larger than, or potentially masked by, the P{EP} insertion alone. Analyses of homozygotes validated many of the candidates identified. These results suggest that R7 cell fate specification is sensitive to perturbations in mRNA transcription, splicing and localization, growth inhibition, post-translational protein modification, cleavage and secretion, hedgehog signaling, ubiquitin protease activity, GTPase activation, actin and cytoskeletal regulation, and Ser/Thr kinase activity, among other diverse signaling and cell biological processes.

Complement is one of the critical branches of innate immunity that determines the recognition of engineered nanoparticles by immune cells. Antibody-targeted iron oxide nanoparticles are a popular platform for magnetic separations, in vitro diagnostics, and molecular imaging. We used 60 nm cross-linked iron oxide nanoworms (CLIO NWs) modified with antibodies against Her2/neu and EpCAM, which are common markers of blood-borne cancer cells, to understand the role of complement in the selectivity of targeting of tumor cells in whole blood. CLIO NWs showed highly efficient targeting and magnetic isolation of tumor cells spiked in lepirudin-anticoagulated blood, but specificity was low due to high uptake by neutrophils, monocytes, and lymphocytes. Complement C3 opsonization in plasma was predominantly via the alternative pathway regardless of the presence of antibody, PEG, or fluorescent tag, but was higher for antibody-conjugated CLIO NWs. Addition of various soluble inhibitors of complement convertase (compstatin, soluble CD35, and soluble CD55) to whole human blood blocked up to 99% of the uptake of targeted CLIO NWs by leukocytes, which resulted in a more selective magnetic isolation of tumor cells. Using well-characterized nanomaterials, we demonstrate here that complement therapeutics can be used to improve targeting selectivity.

Chloropicrin (CP), a warfare agent now majorly used as a soil pesticide, is a strong irritating and lacrimating compound with devastating toxic effects. To elucidate the mechanism of its ocular toxicity, toxic effects of CP (0-100 μM) were studied in primary human corneal epithelial (HCE) cells. CP exposure resulted in reduced HCE cell viability and increased apoptotic cell death with an up-regulation of cleaved caspase-3 and poly ADP ribose polymerase indicating their contribution in CP-induced apoptotic cell death. Following CP exposure, cells exhibited increased expression of heme oxygenase-1, and phosphorylation of H2A.X and p53 as well as 4-hydroxynonenal adduct formation, suggesting oxidative stress, DNA damage and lipid peroxidation. CP also caused increases in mitogen activated protein kinase-c-Jun N-terminal kinase and inflammatory mediator cyclooxygenase-2. Proteomic analysis revealed an increase in the carbonylation of 179 proteins and enrichment of pathways (including proteasome pathway and catabolic process) in HCE cells following CP exposure. CP-induced oxidative stress and lipid peroxidation can enhance protein carbonylation, prompting alterations in corneal epithelial proteins as well as perturbing signaling pathways resulting in toxic effects. Pathways and major processes identified following CP exposure could be lead-hit targets for further biochemical and molecular characterization as well as therapeutic intervention.

Simple Summary Mitotane is the only approved drug for the treatment of advanced adrenocortical carcinoma and for postoperative adjuvant therapy. It is known that mitotane destroys the adrenal cortex impairing steroidogenesis, although its exact molecular mechanism is still unclear. However, confounding factors affecting in vitro experiments could reduce the relevance of the studies. In this review, we explore in vitro studies on mitotane effects, highlighting how different experimental conditions might contribute to the controversial findings. On this basis, it may be necessary to re-evaluate the experiments taking into account their potential confounding factors such as cell strains, culture serum, lipoprotein concentration, and culture passages, which could hide important molecular results. As a consequence, the identification of novel pharmacological molecular pathways might be used in the future to implement personalized therapy, maximizing the benefit of mitotane treatment while minimizing its toxicity. Abstract Mitotane is the only approved drug for the treatment of advanced adrenocortical carcinoma and is increasingly used for postoperative adjuvant therapy. Mitotane action involves the deregulation of cytochromes P450 enzymes, depolarization of mitochondrial membranes, and accumulation of free cholesterol, leading to cell death. Although it is known that mitotane destroys the adrenal cortex and impairs steroidogenesis, its exact mechanism of action is still unclear. The most used cell models are H295-derived cell strains and SW13 cell lines. The diverging results obtained in presumably identical cell lines highlight the need for a stable in vitro model and/or a standard methodology to perform experiments on H295 strains. The presence of several enzymatic targets responsive to mitotane in mitochondria and mitochondria-associated membranes causes progressive alteration in mitochondrial structure when cells were exposed to mitotane. Confounding factors of culture affecting in vitro experiments could reduce the significance of any molecular mechanism identified in vitro. To ensure experimental reproducibility, particular care should be taken in the choice of culture conditions: aspects such as cell strains, culture serum, lipoproteins concentration, and culture passages should be carefully considered and explicated in the presentation of results. We aimed to review in vitro studies on mitotane effects, highlighting how different experimental conditions might contribute to the controversial findings. If the concerns pointed out in this review will be overcome, the new insights into mitotane mechanism of action observed in-vitro could allow the identification of novel pharmacological molecular pathways to be used to implement personalized therapy.

BackgroundCardiac hypertrophy can be considered a maladaptive response which in many cases results in heart failure and sudden death. Genetic predisposition to cardiac hypertrophy is ill-defined, but current research has given credence to a role of non-protein-coding RNAs in the development of cardiac hypertrophy.ResultsWe used microarrays, RNA-Seq and quantitative genetics with the spontaneously hypertensive, SHR (SHR/OlaIpcv) rat strain, the normotensive Brown Norway (BN-Lx/Cub) rat strain, and a recombinant inbred (RI) panel of rats (HXB/BXH) derived from these strains to examine the areas of the genome associated with cardiac hypertrophy. We identified circular (circ) RNAs coded within these areas. Of the 122 differentially expressed circRNAs in left ventricle of SHR and BN-Lx rats, three were transcribed from areas corresponding to the QTLs we identified for cardiac hypertrophy using the HXB/BXH rat panel. We then identified microRNAs which are “sponged” by the circ RNAs and the mRNAs which are destabilized by the microRNAs. 265 miRNAs could be identified as targets for the three circRNAs. We focused on the four miRNAs that were also differentially expressed between SHR and BN-Lx rats. One of the miRNAs (Mir-210-5p) was a target of the differentially expressed circ H2afy and circ H2afy was located within the QTL on Chr 17 (genome wide p-value = 0.011). Mir-210-5p has a binding site on the 3’ UTR of DR6, which is differentially expressed and in turn controls the expression level of NFκB. NFκB is an important component of the oxidative stress response leading to hypertrophy.ConclusionsOur work identified a novel circRNA that may be the mediator of a cascade of events that influence a cardiac hypertrophy phenotype. The circRNA and miRNA which we identified may become useful as markers of the risk for cardiac hypertrophy.

Metabolic syndrome (MetS) is a complex disease of physiological imbalances interrelated to abnormal metabolic conditions, such as abdominal obesity, type II diabetes, dyslipidemia and hypertension. In the present pilot study, we investigated the nutraceutical bitter melon (Metabolic effects of lyophilized bitter melon juice (BMJ) extract (oral gavage 200 mg/kg/body weight-daily for 40 days) intake were evaluated in diet-induced obese C57BL/6J male mice [fed-high fat diet (HFD), 60 kcal% fat]. Changes inBMJ-mediated changes showed a positive trend towards enhanced glucose homeostasis, vitamin D metabolism and suppression of glycerophospholipid metabolism. In the liver, nuclear peroxisome proliferator-activated receptor (PPAR) and circadian rhythm signaling, as well as bile acid biosynthesis and glycogen metabolism targets were modulated by BMJ (p 0.05). Thus, our in-depth transcriptomics and metabolomics analysis suggests that BMJ-intake lowers susceptibility to the onset of high-fat diet associated MetS risk factors partly through modulation of PPAR signaling and its downstream targets in circadian rhythm processes to prevent excessive lipogenesis, maintain glucose homeostasis and modify immune responses signaling.

Objective Many patients with adrenocortical carcinoma (ACC) suffer from tumor recurrence despite radical surgery. Evidence on the post-operative use of mitotane is controversial and no predictors of response are available. We aimed to assess whether adjuvant mitotane treatment may prolong survival in patients with non-metastatic ACC following complete resection and whether ACC patients at high risk of recurrence may benefit from treatment. Design and methods We retrospectively reviewed data from 152 non-metastatic ACC patients followed at the San Luigi Gonzaga Hospital: 100 patients were treated with adjuvant mitotane and 52 patients were left untreated following surgery. We assessed a number of potential predictive factors of recurrence and death. Mitotane effect was explored stratifying patients by staging (stage I–II vs stage III), hormone secretion (yes vs no) and Ki67 index. Results The non-treated group had a higher risk of recurrence (HR: 2.79, 95%CI: 1.58–4.91; P P = 0.005) and in patients with stage III ACC (P = 0.02). Conclusions Adjuvant mitotane may prolong recurrence-free survival in radically resected ACC patients with acceptable toxicity and may also prolong overall survival in a subgroup of ACC patients at high risk of recurrence.

OBJECTIVE: Proton-pump inhibitors (PPIs) have been associated with adverse renal outcomes in older adults; however, there are little data regarding the magnitude of the change in renal function in this population. The objective of this study was to quantify the change in kidney function associated with chronic PPI therapy at two years in older adults using estimated glomerular filtration rate (eGFR). DESIGN: The study was a retrospective, pre/post, observational cohort. SETTING/PATIENTS/INTERVENTIONS/MAIN OUTCOME MEASURE(S): The study included University of Colorado Health primary care patients 60 to 89 years of age who were newly initiated on a PPI between August 1, 2012, and March 1, 2015, and remained on therapy for at least two years. The primary outcome was the change in kidney function, measured by eGFR, two years after starting PPI therapy. Secondary outcomes included change in kidney function and incidence of reduction in eGFR to < 60 mL/min/1.73 m2 two years post-index date between patients with and without diabetes mellitus. RESULTS: Of 877 electronic health records reviewed, 100 patients met inclusion criteria. The mean change in eGFR was -6.15 mL/min/1.73 m2 (standard error of the mean = 1.03) at two years compared with baseline (95% confidence interval -8.20 to -4.10; P < 0.0001). There were no differences in the secondary outcomes based on concomitant diabetes mellitus. CONCLUSIONS: Chronic PPI use was associated with a significant reduction in eGFR in ambulatory older adults at two years, beyond that expected based on increased age alone. Prescribers should be aware of the potential adverse renal effects of chronic PPI use.

Low molecular weight polycyclic aromatic hydrocarbons (LMW PAHs

Mitochondrial dysfunction is one of many key factors in the etiology of alcoholic liver disease (ALD). Lysine acetylation is known to regulate numerous mitochondrial metabolic pathways, and recent reports demonstrate that alcohol-induced protein acylation negatively impacts these processes. To identify regulatory mechanisms attributed to alcohol-induced protein post-translational modifications, we employed a model of alcohol consumption within the context of wild type (WT), sirtuin 3 knockout (SIRT3 KO), and sirtuin 5 knockout (SIRT5 KO) mice to manipulate hepatic mitochondrial protein acylation. Mitochondrial fractions were examined by label-free quantitative HPLC-MS/MS to reveal competition between lysine acetylation and succinylation. A class of proteins defined as "differential acyl switching proteins" demonstrate select sensitivity to alcohol-induced protein acylation. A number of these proteins reveal saturated lysine-site occupancy, suggesting a significant level of differential stoichiometry in the setting of ethanol consumption. We hypothesize that ethanol downregulates numerous mitochondrial metabolic pathways through differential acyl switching proteins. Data are available via ProteomeXchange with identifier PXD012089.

Genes often work together to perform complex biological processes, and “networks” provide a versatile framework for representing the interactions between multiple genes. Differential network analysis (DiNA) quantifies how this network structure differs between two or more groups/phenotypes (e.g., disease subjects and healthy controls), with the goal of determining whether differences in network structure can help explain differences between phenotypes. In this paper, we focus on gene co-expression networks, although in principle, the methods studied can be used for DiNA for other types of features (e.g., metabolome, epigenome, microbiome, proteome, etc.). Three common applications of DiNA involve (1) testing whether the connections to a single gene differ between groups, (2) testing whether the connection between a pair of genes differs between groups, or (3) testing whether the connections within a “module” (a subset of 3 or more genes) differs between groups. This article focuses on the latter, as there is a lack of studies comparing statistical methods for identifying differentially co-expressed modules (DCMs). Through extensive simulations, we compare several previously proposed test statistics and a new p-norm difference test (PND). We demonstrate that the true positive rate of the proposed PND test is competitive with and often higher than the other methods, while controlling the false positive rate. The R package discoMod (differentially co-expressed modules) implements the proposed method and provides a full pipeline for identifying DCMs: clustering tools to derive gene modules, tests to identify DCMs, and methods for visualizing the results.

Background: The goal of the study was to assess the relationship between single nucleotide variants (SNVs) in calcineurin inhibitor (CNI) pharmacokinetic and pharmacodynamic genes and renal dysfunction in adult heart transplant (HTx) recipients.Methods: This retrospective analysis includedN= 192 patients receiving a CNI at 1-year post-HTx. Using a candidate gene approach, 93 SNVs in eight pharmacokinetic and 35 pharmacodynamic genes were chosen for investigation. The primary outcome was renal dysfunction 1-year after HTx, defined as an estimated glomerular filtration rate (eGFR) 2.Results:Renal dysfunction was present in 28.6% of patients 1-year after HTx. Two SNVs [transforming growth factor beta 1 (TGFB1) rs4803455 C > A and phospholipase C beta 1 (PLCB1) rs170549 G > A] were significantly associated with renal dysfunction after accounting for a false discovery rate (FDR) of 20%. In a multiple-SNV adjusted model, variant A allele carriers ofTGFB1rs4803455 had lower odds of renal dysfunction compared to C/C homozygotes [odds ratio (OR) 0.28, 95% CI 0.12–0.62;p= 0.002], whereasPLCB1rs170549 variant A allele carriers had higher odds of the primary outcome vs. patients with the G/G genotype (OR 2.66, 95% CI 1.21–5.84,p= 0.015).Conclusion: Our data suggest that genetic variation inTGFB1andPLCB1may contribute to the occurrence of renal dysfunction in HTx recipients receiving CNIs. Pharmacogenetic markers, such asTGFB1rs4803455 andPLCB1rs170549, could help identify patients at increased risk of CNI-associated renal dysfunction following HTx, potentially allowing clinicians to provide more precise and personalized care to this population.

The National Institute on Drug Abuse and Joint Institute for Biological Sciences at the Oak Ridge National Laboratory hosted a meeting attended by a diverse group of scientists with expertise in substance use disorders (SUDs), computational biology, and FAIR (Findability, Accessibility, Interoperability, and Reusability) data sharing. The meeting's objective was to discuss and evaluate better strategies to integrate genetic, epigenetic, and 'omics data across human and model organisms to achieve deeper mechanistic insight into SUDs. Specific topics were to (a) evaluate the current state of substance use genetics and genomics research and fundamental gaps, (b) identify opportunities and challenges of integration and sharing across species and data types, (c) identify current tools and resources for integration of genetic, epigenetic, and phenotypic data, (d) discuss steps and impediment related to data integration, and (e) outline future steps to support more effective collaboration—particularly between animal model research communities and human genetics and clinical research teams. This review summarizes key facets of this catalytic discussion with a focus on new opportunities and gaps in resources and knowledge on SUDs., This report discusses gaps in knowledge and possibilities for the next phase of functional discovery for addiction.

Annotation of polyadenylation sites from short-read RNA sequencing alone is a challenging computational task. Other algorithms rooted in DNA sequence predict potential polyadenylation sites; however, in vivo expression of a particular site varies based on a myriad of conditions. Here, we introduce aptardi (alternative polyadenylation transcriptome analysis from RNA-Seq data and DNA sequence information), which leverages both DNA sequence and RNA sequencing in a machine learning paradigm to predict expressed polyadenylation sites. Specifically, as input aptardi takes DNA nucleotide sequence, genome-aligned RNA-Seq data, and an initial transcriptome. The program evaluates these initial transcripts to identify expressed polyadenylation sites in the biological sample and refines transcript 3′-ends accordingly. The average precision of the aptardi model is twice that of a standard transcriptome assembler. In particular, the recall of the aptardi model (the proportion of true polyadenylation sites detected by the algorithm) is improved by over three-fold. Also, the model—trained using the Human Brain Reference RNA commercial standard—performs well when applied to RNA-sequencing samples from different tissues and different mammalian species. Finally, aptardi’s input is simple to compile and its output is easily amenable to downstream analyses such as quantitation and differential expression., Short read RNA sequencing and DNA sequence contain useful information for profiling polyadenylation sites, but each also possesses inherent limitations when examined independently. Aptardi combines these data and significantly improves annotation of polyadenylation sites in the expressed transcriptome.

There is compelling evidence that sex and gender have crucial roles in excessive alcohol (ethanol) consumption. Here we review some of the data from the perspective of brain transcriptional differences between males and females, focusing on rodent animal models. A key emerging transcriptional feature is the role of neuroimmune processes. Microglia are the resident neuroimmune cells in the brain, and exhibit substantial functional differences between males and females. Selective breeding for binge ethanol consumption, as well as the impacts of chronic ethanol consumption and withdrawal from chronic ethanol exposure, all demonstrate sex-dependent neuroimmune signatures. A focus is on resolving sex-dependent differences in transcriptional responses to ethanol at the neurocircuitry level. Sex-dependent transcriptional differences are found in the extended amygdala and the nucleus accumbens. Telescoping of ethanol consumption is found in some, but not all, studies to be more prevalent in females. Recent transcriptional studies suggest that some sex differences may be due to female-dependent remodeling of the primary cilium. An interesting theme appears to be developing: at least from the animal model perspective, even when males and females are phenotypically similar, they differ significantly at the level of the transcriptome.

LncRNAs are important regulators of quantitative and qualitative features of the transcriptome. We have used QTL and other statistical analyses to identify a gene coexpression module associated with alcohol consumption. The “hub gene” of this module, Lrap (Long non‐coding RNA for alcohol preference), was an unannotated transcript resembling a lncRNA. We used partial correlation analyses to establish that Lrap is a major contributor to the integrity of the coexpression module. Using CRISPR/Cas9 technology, we disrupted an exon of Lrap in Wistar rats. Measures of alcohol consumption in wild type, heterozygous and knockout rats showed that disruption of Lrap produced increases in alcohol consumption/alcohol preference. The disruption of Lrap also produced changes in expression of over 700 other transcripts. Furthermore, it became apparent that Lrap may have a function in alternative splicing of the affected transcripts. The GO category of “Response to Ethanol” emerged as one of the top candidates in an enrichment analysis of the differentially expressed transcripts. We validate the role of Lrap as a mediator of alcohol consumption by rats, and also implicate Lrap as a modifier of the expression and splicing of a large number of brain transcripts. A defined subset of these transcripts significantly impacts alcohol consumption by rats (and possibly humans). Our work shows the pleiotropic nature of non‐coding elements of the genome, the power of network analysis in identifying the critical elements influencing phenotypes, and the fact that not all changes produced by genetic editing are critical for the concomitant changes in phenotype., Lrap, a novel lncRNA, is a mediator of alcohol consumption and a modifier of brain RNA expression and splicing. Categories from GO and KEGG pathways that are enriched within the group of differentially expressed transcripts in brains of the Lrap +/+ versus Lrap −/− rats (A). B and C depict heat maps illustrating the direction and magnitude of difference in expression of the genes included in the category “response to ethanol” (C) and “tight junction” between Lrap +/+, Lrap +/− and Lrap −/− rats. Relations between these genes and differential alcohol consumption of genetically, Lrap modified rats are discussed in the manuscript.

Liquid chromatography-tandem mass spectrometry is an increasingly powerful tool for studying proteins in the context of disease. As technological advances in instrumentation and data analysis have enabled deeper profiling of proteomes and peptidomes, the need for a rigorous, standardized approach to validate individual peptide-spectrum matches (PSMs) has emerged. To address this need, we developed a novel and broadly applicable workflow: PSM validation with internal standards (P-VIS). In this approach, the fragmentation spectrum and chromatographic retention time of a peptide within a biological sample are compared with those of a synthetic version of the putative peptide sequence match. Similarity measurements obtained for a panel of internal standard peptides are then used to calculate a prediction interval for valid matches. If the observed degree of similarity between the biological and the synthetic peptide falls within this prediction interval, then the match is considered valid. P-VIS enables systematic and objective assessment of the validity of individual PSMs, providing a measurable degree of confidence when identifying peptides by mass spectrometry.

Magnesium is an essential trace metal and a necessary factor for multiple biochemical functions in humans. Its role in biology is fundamental in over 600 enzymatic reactions implicated in protein synthesis, mitochondrial functions, neuromuscular activity, bone formation, and immune system competence. Magnesium status is relevant in fetal development during gestation and in the newborn growth during the perinatal period. Moreover, magnesium is able to influence fetal programming and disease presentation in childhood or adulthood. The aim of this review is to focus on this metal homeostasis, analyzing its normal values, the causes of hypomagnesemia, the interaction with drugs and other conditions, and the diseases associated with magnesium value alteration during pregnancy, in order to study its role in fetal programming of adult diseases. The data here reported clearly indicated the existence of a connection between magnesium status and human pathology starting from intrauterine life and extending into childhood and adulthood.

Background We assessed the relationship between circadian blood pressure (BP) patterns and clinical outcomes in a contemporary cohort of adult heart transplant recipients. Methods This retrospective, cross-sectional study included adult heart transplant recipients at least 6 months post-transplant. Ambulatory BP measurements were recorded over 24 hours. Nondippers were defined as a decline in average nighttime BP ≤ 10% compared with daytime. Primary outcomes were the presence of end organ damage, that is, microalbuminuria, chronic kidney disease, and/or left ventricular hypertrophy. Secondary outcomes were measures of diastolic dysfunction (ie, mitral valve deceleration time, e/e', E/A, and isovolumetric relaxation time), microalbumin/creatinine ratio, eGFR, interventricular septal thickness, and left ventricular posterior wall thickness. Results Of 30 patients, 53.3% (n = 16) were systolic nondippers and 40% (n = 12) were diastolic nondippers. Diastolic nondippers had three times higher urine microalbumin/creatinine ratios than diastolic dippers (P = .03). Systolic nondippers had 16.3% lower mitral valve deceleration time (P = .05) than systolic dippers, while diastolic nondippers had 20.4% higher e/e' (P = .05) than diastolic dippers. There were no significant relationships between BP dipping status and any of the primary outcomes. Conclusions These data suggest that systolic and diastolic nondipping BP patterns are associated with subclinical kidney damage and diastolic dysfunction in heart transplant recipients.

BACKGROUND: Glutathione-S-transferase A4-4 (GSTA4) is a key enzyme for removal of toxic lipid peroxidation products such as 4-hydroxynonenal (4-HNE). In the current study, we examined the potential role of GSTA4 on protein carbonylation and progression of alcoholic liver disease (ALD) by examining the development of liver injury in male wild type SV/J mice (WT) and SV/J mice lacking functional GSTA4 (GSTA4(−/−) mice). METHODS: Adult male WT and GSTA4(−/−) mice were fed chow (N = 10-12) or high fat Lieber-DeCarli liquid diets containing up to 28% calories as ethanol (EtOH) (N = 18-20) for 116 days. At the end of the study, half of the ethanol-fed mice were acutely challenged with an ethanol binge (3 g/kg given intragastrically) 12 hours before sacrifice. Carbonylation of liver proteins was assessed by immunohistochemical staining for 4-HNE adduction and by comprehensive liquid chromatography tandem mass spectrometry, LC-MS/MS, of purified carbonylated proteins. RESULTS: Chronic EtOH intake significantly increased hepatic 4-HNE adduction and protein carbonylation, including carbonylation of ribosomal proteins. EtOH intake also resulted in steatosis and increased serum ALTs. Hepatic infiltration with B-cells, T-cells, and neutrophils and mRNA expression of pro-inflammatory cytokines TNFα and IFNγ was modest in WT mice. However, an ethanol binge increased hepatic necrosis, hepatic cell proliferation and expression of TNFα mRNA (P

The breast cancer resistance protein (BCRP/ABCG2) is a maternally-facing efflux transporter that regulates the placental disposition of chemicals. Transcription factors and gene variants are important regulatory factors that influence transporter expression. In this study, we sought to identify the genetic and transcriptional mechanisms underlying the interindividual expression of BCRP mRNA and protein across 137 term placentas from uncomplicated pregnancies. Placental expression of BCRP and regulatory transcription factor mRNAs was measured using multiplex-branched DNA analysis. BCRP expression and ABCG2 genotypes were determined using Western blot and Fluidigm Biomark genetic analysis, respectively. Placentas were obtained from a racially and ethnically diverse population, including Caucasian (33%), African American (14%), Asian (14%), Hispanic (15%), and mixed (16%) backgrounds, as well as unknown origins (7%). Between placentas, BCRP mRNA and protein varied up to 47-fold and 14-fold, respectively. In particular, BCRP mRNA correlated significantly with known transcription factor mRNAs, including nuclear factor erythroid 2-related factor 2 and aryl hydrocarbon receptor. Somewhat surprisingly, single-nucleotide polymorphisms (SNPs) in the ABCG2 noncoding regions were not associated with variation in placental BCRP mRNA or protein. Instead, the coding region polymorphism (C421A/Q141K) corresponded with 40%–50% lower BCRP protein in 421C/A and 421A/A placentas compared with wild types (421C/C). Although BCRP protein and mRNA expression weakly correlated (r = 0.25, P = 0.040), this relationship was absent in individuals expressing the C421A variant allele. Study results contribute to our understanding of the interindividual regulation of BCRP expression in term placentas and may help to identify infants at risk for increased fetal exposure to chemicals due to low expression of this efflux protein.

Brown adipose tissue (BAT) has been suggested to play an important role in lipid and glucose metabolism in rodents and possibly also in humans. In the current study, we used genetic and correlation analyses in the BXH/HXB recombinant inbred (RI) strains, derived from Brown Norway (BN) and spontaneously hypertensive rats (SHR), to identify genetic determinants of BAT function. Linkage analyses revealed a quantitative trait locus (QTL) associated with interscapular BAT mass on chromosome 4 and two closely linked QTLs associated with glucose oxidation and glucose incorporation into BAT lipids on chromosome 2. Using weighted gene coexpression network analysis (WGCNA) we identified 1,147 gene coexpression modules in the BAT from BXH/HXB rats and mapped their module eigengene QTLs. Through an unsupervised analysis, we identified modules related to BAT relative mass and function. The Coral4.1 coexpression module is associated with BAT relative mass (includes Cd36 highly connected gene), and the Darkseagreen coexpression module is associated with glucose incorporation into BAT lipids (includes Hiat1, Fmo5, and Sort1 highly connected transcripts). Because multiple statistical criteria were used to identify candidate modules, significance thresholds for individual tests were not adjusted for multiple comparisons across modules. In summary, a systems genetic analysis using genomic and quantitative transcriptomic and physiological information has produced confirmation of several known genetic factors and significant insight into novel genetic components functioning in BAT and possibly contributing to traits characteristic of the metabolic syndrome.

In the liver, a contributing factor in the pathogenesis of non-alcoholic fatty liver disease is oxidative stress leading to the accumulation of highly reactive electrophilic α/β unsaturated aldehydes. The objective of this study was to determine if significant differences were evident when evaluating carbonylation in human end-stage fatty nonalcoholic steatohepatitis (fNASH) compared to end-stage nonfatty NASH (nfNASH).Using hepatic tissue obtained from healthy humans and patients diagnosed with end stage nfNASH or fNASH, overall carbonylation was assessed by immunohistochemistry (IHC) and LC-MS/MS followed by bioinformatics.Picrosirius red staining revealed extensive fibrosis in both fNASH and nfNASH which corresponded with increased reactive aldehyde staining. Although significantly elevated when compared to normal hepatic tissue, no significant differences in overall carbonylation and fibrosis were evident when comparing fNASH with nfNASH. Examining proteins that are critical for anti-oxidant defense revealed elevated expression of thioredoxin, thioredoxin interacting protein, glutathione S-transferase p1 and mitochondrial superoxide dismutase in human NASH. As important, using immunohistochemistry, significant colocalization of the aforementioned proteins occurred in cytokeratin 7 positive cells indicating that they are part of the ductular reaction. Expression of catalase and Hsp70 decreased in both groups when compared to normal human liver. Mass spectrometric analysis revealed a total of 778 carbonylated proteins. Of these, 194 were common to all groups, 124 unique to tissue prepared from healthy individuals, 357 proteins exclusive to NASH, 124 proteins distinct to samples from patients with fNASH and 178 unique to nfNASH. Using functional enrichment analysis of hepatic carbonylated proteins revealed a propensity for increased carbonylation of proteins regulating cholesterol and Huntington's disease related pathways occurred in nfNASH. Examining fNASH, increased carbonylation was evident in proteins regulating Rho cytoskeletal pathways, nicotinic acetylcholine receptor signaling and chemokine/cytokine inflammatory pathways. Using LC-MS/MS analysis and trypsin digests, sites of carbonylation were identified on peptides isolated from vimentin, endoplasmin and serum albumin in nfNASH and fNASH respectively.These results indicate that cellular factors regulating mechanisms of protein carbonylation may be different depending on pathological diagnosis of NASH. Furthermore these studies are the first to use LC-MS/MS analysis of carbonylated proteins in human NAFLD and explore possible mechanistic links with end stage cirrhosis due to fatty liver disease and the generation of reactive aldehydes.

Ethyl alcohol is a toxin that, when consumed at high levels, produces organ damage and death. One way to prevent or ameliorate this damage in humans is to reduce the exposure of organs to alcohol by reducing alcohol ingestion. Both the propensity to consume large volumes of alcohol and the susceptibility of human organs to alcohol-induced damage exhibit a strong genetic influence. We have developed an integrative genetic/genomic approach to identify transcriptional networks that predispose complex traits, including propensity for alcohol consumption and propensity for alcohol-induced organ damage. In our approach, the phenotype is assessed in a panel of recombinant inbred (RI) rat strains, and quantitative trait locus (QTL) analysis is performed. Transcriptome data from tissues/organs of naïve RI rat strains are used to identify transcriptional networks using Weighted Gene Coexpression Network Analysis (WGCNA). Correlation of the first principal component of transcriptional coexpression modules with the phenotype across the rat strains, and overlap of QTLs for the phenotype and the QTLs for the coexpression modules (module eigengene QTL) provide the criteria for identification of the functionally related groups of genes that contribute to the phenotype (candidate modules). While we previously identified a brain transcriptional module whose QTL overlapped with a QTL for levels of alcohol consumption in HXB/BXH RI rat strains and 12 selected rat lines, this module did not account for all of the genetic variation in alcohol consumption. Our search for QTL overlap and correlation of coexpression modules with phenotype can, however, be applied to any organ in which the transcriptome has been measured, and this represents a holistic approach in the search for genetic contributors to complex traits. Previous work has implicated liver/brain interactions, particularly involving inflammatory/immune processes, as influencing alcohol consumption levels. We have now analyzed the liver transcriptome of the HXB/BXH RI rat panel in relation to the behavioral trait of alcohol consumption. We used RNA-Seq and microarray data to construct liver transcriptional networks, and identified a liver candidate transcriptional coexpression module that explained 24% of the genetic variance in voluntary alcohol consumption. The transcripts in this module focus attention on liver secretory products that influence inflammatory and immune signaling pathways. We propose that these liver secretory products can interact with brain mechanisms that affect alcohol consumption, and targeting these pathways provides a potential approach to reducing high levels of alcohol intake and also protecting the integrity of the liver and other organs.

Opsonization (coating) of nanoparticles with complement C3 component is an important mechanism that triggers immune clearance and downstream anaphylactic and proinflammatory responses. The variability of complement C3 binding to nanoparticles in the general population has not been studied. We examined complement C3 binding to dextran superparamagnetic iron oxide nanoparticles (superparamagnetic iron oxide nanoworms, SPIO NWs, 58 and 110 nm) and clinically approved nanoparticles (carboxymethyl dextran iron oxide ferumoxytol (Feraheme, 28 nm), highly PEGylated liposomal doxorubicin (LipoDox, 88 nm), and minimally PEGylated liposomal irinotecan (Onivyde, 120 nm)) in sera from healthy human individuals. SPIO NWs had the highest variation in C3 binding (n = 47) between subjects, with a 15-30 fold range in levels of C3. LipoDox (n = 12) and Feraheme (n = 18) had the lowest levels of variation between subjects (an approximately 1.5-fold range), whereas Onivyde (n = 18) had intermediate between-subject variation (2-fold range). There was no statistical difference between males and females and no correlation with age. There was a significant correlation in complement response between small and large SPIO NWs, which are similar structurally and chemically, but the correlations between SPIO NWs and other types of nanoparticles, and between LipoDox and Onivyde, were not significant. The calculated average number of C3 molecules bound per nanoparticle correlated with the hydrodynamic diameter but was decreased in LipoDox, likely due to the PEG coating. The conclusions of this study are (1) all nanoparticles show variability of C3 opsonization in the general population; (2) an individual's response toward one nanoparticle cannot be reliably predicted based on another nanoparticle; and (3) the average number of C3 molecules per nanoparticle depends on size and surface coating. These results provide new strategies to improve nanomedicine safety.

Objective Mitotane, a drug used to treat adrenocortical cancer (ACC), inhibits multiple enzymatic steps of adrenocortical steroid biosynthesis, potentially causing adrenal insufficiency. Recent studies in vitro have also documented a direct inhibitory effect of mitotane at the pituitary level. The present study was aimed to assess the hypothalamic–pituitary–adrenal axis in patients with ACC receiving mitotane. Design and methods We prospectively enrolled 16 patients on adjuvant treatment with mitotane after radical surgical resection of ACC, who underwent standard hormone evaluation and h-CRH stimulation. A group of 10 patients with primary adrenal insufficiency (PAI) served as controls for the CRH test. Results We demonstrated a close correlation between cortisol-binding globulin (CBG) and plasma mitotane levels, and a non-significant trend between mitotane dose and either serum or salivary cortisol in ACC patients. We did not find any correlation between the dose of cortisone acetate and either ACTH or cortisol levels. ACTH levels were significantly higher in patients with PAI than that in patients with ACC, both in baseline conditions (88.99 (11.04–275.00) vs 24.53 (6.16–121.88) pmol/L, P = 0.031) and following CRH (158.40 (34.32–275.00) vs 67.43 (8.8–179.52) pmol/L P = 0.016). Conclusions The observation of lower ACTH levels in patients with ACC than that in patients with PAI, both in basal conditions and after CRH stimulation, suggests that mitotane may play an inhibitory effect on ACTH secretion at the pituitary levels. In conclusion, the present study shows that mitotane affects the HPA axis at multiple levels and no single biomarker may be used for the assessment of adrenal insufficiency.

Rats remain a major model for studying disease mechanisms and discovery, validation, and testing of new compounds to improve human health. The rat’s value continues to grow as indicated by the more than 1.4 million publications (second to human) at PubMed documenting important discoveries using this model. Advanced sequencing technologies, genome modification techniques, and the development of embryonic stem cell protocols ensure the rat remains an important mammalian model for disease studies. The 2004 release of the reference genome has been followed by the production of complete genomes for more than two dozen individual strains utilizing NextGen sequencing technologies; their analyses have identified over 80 million variants. This explosion in genomic data has been accompanied by the ability to selectively edit the rat genome, leading to hundreds of new strains through multiple technologies. A number of resources have been developed to provide investigators with access to precision rat models, comprehensive datasets, and sophisticated software tools necessary for their research. Those profiled here include the Rat Genome Database, PhenoGen, Gene Editing Rat Resource Center, Rat Resource and Research Center, and the National BioResource Project for the Rat in Japan.

Seizures that occur during early development are associated with adverse neurodevelopmental outcomes. Causation and mechanisms are currently under investigation. Induction of an early life seizure by kainic acid (KA) in immature rats on post-natal day (P) 7 results in behavioral changes in the adult rat that reflect social and intellectual deficits without overt cellular damage. Our previous work also demonstrated increased expression of CA1 hippocampal long-term potentiation (LTP) and reduced desensitization of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type ionotropic glutamate receptors (AMPA-R) one week following a kainic acid induced seizure (KA-ELS). Here we used RNA sequencing (RNAseq) of mRNA from dorsal hippocampal CA1 to probe changes in mRNA levels one week following KA-ELS as a means to investigate the mechanisms for these functional changes. Ingenuity pathway analysis (IPA) confirmed our previous results by predicting an up-regulation of the synaptic LTP pathway. Differential gene expression results revealed significant differences in 7 gene isoforms. Additional assessments included AMPA-R splice variants and adenosine deaminase acting on RNA 2 (ADAR2) editing sites as a means to determine the mechanism for reduced AMPA-R desensitization. Splice variant analysis demonstrated that KA-ELS result in a small, but significant decrease in the "flop" isoform of Gria3, and editing site analysis revealed significant changes in the editing of a kainate receptor subunit, Grik2, and a serotonin receptor, Htr2c. While these specific changes may not account for altered AMPA-R desensitization, the differences indicate that KA-ELS alters gene expression in the hippocampal CA1 one week after the insult.

Genetic mutations in Duchenne muscular dystrophy (DMD) gene affecting the expression of dystrophin protein lead to a number of muscle disorders collectively called dystrophinopathies. In addition to muscle dystrophin, mutations in brain-specific dystrophin isoforms, in particular those that are expressed in the brain cortex and Purkinje neurons, result in cognitive impairment associated with DMD. These isoforms carry minor variations in the flanking region of the N-terminal actin-binding domain (ABD1) of dystrophin, which is composed of two calponin-homology (CH) domains in tandem. Determining the effect of these sequence variations is critical for understanding the mechanisms that govern varied symptoms of the disease. We studied the impact of differences in the N-terminal flanking region on the structure and function of dystrophin tandem CH domain isoforms. The amino acid changes did not affect the global structure of the protein but drastically affected the thermodynamic stability, with the muscle isoform more stable than the brain and Purkinje isoforms. Actin binding investigated with actin from different sources (skeletal muscle, smooth muscle, cardiac muscle, and platelets) revealed that the muscle isoform binds to filamentous actin (F-actin) with a lower affinity compared to the brain and Purkinje isoforms, and a similar trend was observed with actin from different sources. In addition, all isoforms showed a higher affinity to smooth muscle actin in comparison to actin from other sources. In conclusion, tandem CH domain isoforms might be using minor sequence variations in the N-terminal flanking regions to modulate their thermodynamic stability and actin-binding function, thus leading to specificity in dystrophin-actin interactions in various tissues.

The R7 and R8 photoreceptor cells of theDrosophilacompound eye mediate color vision. Throughout the majority of the eye, these cells occur in two principal types of ommatidia. Approximately 35% of ommatidia are of the pale type and express Rh3 in R7 cells and Rh5 in R8 cells. The remaining 65% are of the yellow type and express Rh4 in R7 cells and Rh6 in R8 cells. The specification of an R8 cell in a pale or yellow ommatidium depends on the fate of the adjacent R7 cell. However, pale and yellow R7 cells are specified by a stochastic process that requires the genesspineless,tangoandklumpfuss. To identify additional genes involved in this process we performed a genetic screen using a collection of 480P{EP}transposon insertion strains. We identified genes that when inactivated and/or ectopically expressed in R7 cells resulted in a significantly altered percentage of Rh3 expressing R7 cells (Rh3%) from wild-type. 53 strains resulted in altered Rh3% in the heterozygous inactivation arm of the screen. 36 strains resulted in altered Rh3% in the ectopic expression arm of the screen, where the P{EP} insertion strains were crossed to asevEP-GAL4driver line. 4 strains showed differential effects between the two screens. Analyses of these results suggest that R7 cell fate specification is sensitive to perturbations in transcription, growth inhibition, glycoprotein ligand binding, WNT signaling, ubiquitin protease activity and Ser/Thr kinase activity, among other diverse signaling and cell biological processes.

Background Chronic alcohol consumption is a significant cause of liver disease worldwide. Several biochemical mechanisms have been linked to the initiation and progression of alcoholic liver disease (ALD) such as oxidative stress, inflammation, and metabolic dysregulation, including the disruption of NAD+/NADH. Indeed, an ethanol-mediated reduction in hepatic NAD+ levels is thought to be one factor underlying ethanol-induced steatosis, oxidative stress, steatohepatitis, insulin resistance, and inhibition of gluconeogenesis. Therefore, we applied a NAD+ boosting supplement to investigate alterations in the pathogenesis of early-stage ALD. Methods To examine the impact of NAD+ therapy on the early stages of ALD, we utilized nicotinamide mononucleotide (NMN) at 500 mg/kg intraperitoneal injection every other day, for the duration of a Lieber-DeCarli 6-week chronic ethanol model in mice. Numerous strategies were employed to characterize the effect of NMN therapy, including the integration of RNA-seq, immunoblotting, and metabolomics analysis. Results Our findings reveal that NMN therapy increased hepatic NAD+ levels, prevented an ethanol-induced increase in plasma ALT and AST, and changed the expression of 25% of the genes that were modulated by ethanol metabolism. These genes were associated with a number of pathways including the MAPK pathway. Interestingly, our analysis revealed that NMN treatment normalized Erk1/2 signaling and prevented an induction of Atf3 overexpression. Conclusions These findings reveal previously unreported mechanisms by which NMN supplementation alters hepatic gene expression and protein pathways to impact ethanol hepatotoxicity in an early-stage murine model of ALD. Overall, our data suggest further research is needed to fully characterize treatment paradigms and biochemical implications of NAD+-based interventions.

Introduction Smoking is a leading cause of death, and genetic variation contributes to smoking behaviors. Identifying genes and sets of genes that contribute to risk for addiction is necessary to prioritize targets for functional characterization and for personalized medicine. Methods We performed a gene set–based association and heritable enrichment study of two addiction-related gene sets, those on the Smokescreen Genotyping Array and the nicotinic acetylcholine receptors, using the largest available GWAS summary statistics. We assessed smoking initiation, cigarettes per day, smoking cessation, and age of smoking initiation. Results Individual genes within each gene set were significantly associated with smoking behaviors. Both sets of genes were significantly associated with cigarettes per day, smoking initiation, and smoking cessation. Age of initiation was only associated with the Smokescreen gene set. Although both sets of genes were enriched for trait heritability, each accounts for only a small proportion of the single nucleotide polymorphism-based heritability (2%–12%). Conclusions These two gene sets are associated with smoking behaviors, but collectively account for a limited amount of the genetic and phenotypic variation of these complex traits, consistent with high polygenicity. Implications We evaluated evidence for the association and heritable contribution of expert-curated and bioinformatically identified sets of genes related to smoking. Although they impact smoking behaviors, these specifically targeted genes do not account for much of the heritability in smoking and will be of limited use for predictive purposes. Advanced genome-wide approaches and integration of other ‘omics data will be needed to fully account for the genetic variation in smoking phenotypes.

Background micro RNA (miRNA) are important regulators of gene expression and may influence phenotypes and disease traits. The connection between genetics and miRNA expression can be determined through expression quantitative loci (eQTL) analysis, which has been extensively used in a variety of tissues, and in both human and model organisms. miRNA play an important role in brain-related diseases, but eQTL studies of miRNA in brain tissue are limited. We aim to catalog miRNA eQTL in brain tissue using miRNA expression measured on a recombinant inbred mouse panel. Because samples were collected without any intervention or treatment (naïve), the panel allows characterization of genetic influences on miRNAs’ expression levels. We used brain RNA expression levels of 881 miRNA and 1416 genomic locations to identify miRNA eQTL. To address multiple testing, we employed permutation p-values and subsequent zero permutation p-value correction. We also investigated the underlying biology of miRNA regulation using additional analyses, including hotspot analysis to search for regions controlling multiple miRNAs, and Bayesian network analysis to identify scenarios where a miRNA mediates the association between genotype and mRNA expression. We used addiction related phenotypes to illustrate the utility of our results. Results Thirty-eight miRNA eQTL were identified after appropriate multiple testing corrections. Ten of these miRNAs had target genes enriched for brain-related pathways and mapped to four miRNA eQTL hotspots. Bayesian network analysis revealed four biological networks relating genetic variation, miRNA expression and gene expression. Conclusions Our extensive evaluation of miRNA eQTL provides valuable insight into the role of miRNA regulation in brain tissue. Our miRNA eQTL analysis and extended statistical exploration identifies miRNA candidates in brain for future study.

One of the most fruitful resources for systems genetic studies of nonhuman mammals is a panel of inbred strains that exhibits significant genetic diversity between strains but genetic stability (isogenicity) within strains. These characteristics allow for fine mapping of complex phenotypes (QTLs) and provide statistical power to identify loci which contribute nominally to the phenotype. This type of resource also allows the planning and performance of investigations using the same genetic backgrounds over several generations of the test animals. Often, rats are preferred over mice for physiologic and behavioral studies because of their larger size and more distinguishable anatomy (particularly for their central nervous system). The Hybrid Rat Diversity Panel (HRDP) is a panel of inbred rat strains, which combines two recombinant inbred panels (the HXB/BXH, 30 strains; the LEXF/FXLE, 34 strains and 35 more strains of inbred rats which were selected for genetic diversity, based on their fully sequenced genomes and/or thorough genotyping). The genetic diversity and statistical power of this panel for mapping studies rivals or surpasses currently available panels in mouse. The genetic stability of this panel makes it particularly suitable for collection of high-throughput omics data as relevant technology becomes available for engaging in truly integrative systems biology. The PhenoGen website ( http://phenogen.org ) is the repository for the initial transcriptome data, making the raw data, the processed data, and the analysis results, e.g., organ-specific protein coding and noncoding transcripts, isoform analysis, expression quantitative trait loci, and co-expression networks, available to the research public. The data sets and tools being developed will complement current efforts to analyze the human transcriptome and its genetic controls (the Genotype-Tissue Expression Project (GTEx)) and allow for dissection of genetic networks that predispose to particular phenotypes and gene-by-environment interactions that are difficult or even impossible to study in humans. The HRDP is an essential population for exploring truly integrative systems genetics.