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Disruption of persistent, stress-associated memories is relevant for treating posttraumatic stress disorder (PTSD) and related syndromes, which develop in a subset of individuals following a traumatic event. We previously developed a stress-enhanced fear learning (SEFL) paradigm in inbred mice that produces PTSD-like characteristics in a subset of mice, including persistently enhanced memory and heightened cFos in the basolateral amygdala complex (BLC) with retrieval of the remote (30-day-old) stress memory. Here, the contribution of BLC microRNAs (miRNAs) to stress-enhanced memory was investigated because of the molecular complexity they achieve through their ability to regulate multiple targets simultaneously. We performed small-RNA sequencing (smRNA-Seq) and quantitative proteomics on BLC tissue collected from mice 1 month after SEFL and identified persistently changed microRNAs, including mir-135b-5p, and proteins associated with PTSD-like heightened fear expression. Viral-mediated overexpression of mir-135b-5p in the BLC of stress-resilient animals enhanced remote fear memory expression and promoted spontaneous renewal 14 days after extinction. Conversely, inhibition of BLC mir-135b-5p in stress-susceptible animals had the opposite effect, promoting a resilient-like phenotype. mir-135b-5p is highly conserved across mammals and was detected in post mortem human amygdala, as well as human serum samples. The mir-135b passenger strand, mir-135b-3p, was significantly elevated in serum from PTSD military veterans, relative to combat-exposed control subjects. Thus, miR-135b-5p may be an important therapeutic target for dampening persistent, stress-enhanced memory and its passenger strand a potential biomarker for responsivity to a mir-135-based therapeutic.

Arsenic (As) contamination in groundwater is responsible for numerous adverse health outcomes among millions of people. Epigenetic alterations are among the most widely studied mechanisms of As toxicity. To understand how As exposure alters gene expression through epigenetic modifications, a systematic genome-wide study was designed to address the impact of multiple important single nucleotide polymorphisms (SNPs) related to As exposure on the methylome of drinking water As-exposed rural subjects from Pakistan. Urinary As levels were used to stratify subjects into low, medium and high exposure groups. Genome-wide DNA methylation was investigated using MeDIP in combination with NimbleGen 2.1 M Deluxe Promotor arrays. Transcriptome levels were measured using Agilent 8 × 60 K expression arrays. Genotyping of selected SNPs (As3MT, DNMT1a, ERCC2, EGFR and MTHFR) was measured and an integrated genetic risk factor for each respondent was calculated by assigning a specific value to the measured genotypes based on known risk allele numbers. To select a representative model related to As exposure we compared 9 linear mixed models comprising of model 1 (including the genetic risk factor), model 2 (without the genetic risk factor) and models with individual SNPs incorporated into the methylome data. Pathway analysis was performed using ConsensusPathDB. Model 1 comprising the integrated genetic risk factor disclosed biochemical pathways including muscle contraction, cardio-vascular diseases, ATR signaling, GPCR signaling, methionine metabolism and chromatin modification in association with hypo- and hyper-methylated gene targets. A unique pathway (direct P53 effector) was found associated with the individual DNMT1a polymorphism due to hyper-methylation of CSE1L and TRRAP. Most importantly, we provide here the first evidence of As-associated DNA methylation in relation with gene expression of ATR, ATF7IP, TPM3, UBE2J2. We report the first evidence that integrating SNPs data with methylome data generates a more representative epigenome profile and discloses a better insight in disease risks of As-exposed individuals.

Four complementary approaches were used to investigate acetaminophen overdose as a risk factor for Parkinson's disease (PD). Circulating microRNAs (miRNAs) serum profiles from acetaminophen-overdosed patients were compared with patients with terminal PD, revealing four shared miRNAs. Similarities were found among molecular structures of dopamine (DA), acetaminophen, and two known PD inducers indicating affinity for dopaminergic transport. Potential interactions between acetaminophen and the human DA transporter were confirmed by molecular docking modeling and binding free energy calculations. Thus, it is plausible that acetaminophen is taken up by the dopaminergic transport system into the substantia nigra (SN). A ChEMBL query identified proteins that are similarly targeted by DA and acetaminophen. Here, we highlight CYP3A4, present in the SN, a predominant metabolizer of acetaminophen into its toxic metabolite N-acetyl-p-benzoquinone imine and shown to be regulated in PD. Overall, based on our results, we hypothesize that overdosing of acetaminophen is a potential risk factor for parkinsonism.

Purpose Identify differentially expressed microRNAs (miRNAs) in aqueous humor (AH) and blood of primary open-angle glaucoma (POAG) patients by using small RNA sequencing. These may provide insight into POAG pathophysiology or serve as diagnostic biomarker. Methods AH and plasma of nine POAG patients and 10 cataract control patients were small RNA sequenced on Illumina NovaSeq 6000. Identification of gene transcripts targeted by differentially expressed miRNAs was done with miRWalk and MirPath. These targets were used for pathway analysis and Gene Ontology enrichment. Diagnostic potential was evaluated by receiver operating characteristics analysis. Results We identified 715 miRNAs in plasma and 62 miRNAs in AH. Plasma miRNA profile did not differ between POAG and control. In contrast, in AH, seven miRNAs were differentially expressed. Hsa-miR-30a-3p, hsa-miR-143-3p, hsa-miR-211-5p, and hsa-miR-221-3p were upregulated, whereas hsa-miR-92a-3p, hsa-miR-451a, and hsa-miR-486-5p were downregulated in POAG. Compared to previous studies, hsa-mir-143-3p, hsa-miR-211-5p, and hsa-miR-221-3p were reported previously, strengthening their involvement in POAG whereas hsa-miR-30a-3p, hsa-miR-92a-3p, and hsa-miR-486-5p are implicated in POAG for the first time. Identified gene transcripts were involved in several pathways, some implicated in glaucoma before (e.g., TGF-β and neurotrophin signaling), whereas others are new (e.g., prolactin and apelin signaling). In respect to diagnostics, AH concentration of hsa-mir-143-3p had an area under the curve (AUC) of 0.889. Combined with hsa-miR-221-3p, AUC improved to 0.96. Conclusions Small RNA sequencing identified seven differentially expressed miRNAs in AH of POAG patients. The differentially expressed miRNAs may be useful as POAG biomarkers or could become targets for new therapeutic strategies.

Alzheimer's disease (AD) is a neurodegenerative disease which is manifested by a progressive and irreversible decline of cognition, memory loss, a shortened attention span, and changes in personality. Aging and genetic pre-dispositions, particularly the presence of a specific form of apolipoprotein E (APOE), are main risk factors of sporadic AD; however, a large body of evidence has shown that multiple environmental factors, including exposure to toxic metals, increase the risk for late onset AD. Lead (Pb) and cadmium (Cd) are ubiquitous toxic metals with a wide range of applications resulting in global distribution in the environment and exposure of all living organisms on earth. In addition to being classified as carcinogenic (Cd) and possibly carcinogenic (Pb) to humans by the International Agency for Research on Cancer, both compounds disrupt metal homeostasis and can cause toxic responses at the cellular and organismal levels. Pb toxicity targets the central nervous system and evidence for that has emerged also for Cd. Recent epidemiological studies show that both metals possibly are etiological factors of multiple neurodegenerative diseases, including Alzheimer's disease (AD). To further explore the association between metal exposure and AD risk we applied whole transcriptome gene expression analysis in peripheral blood leukocytes (PBLs) from 632 subjects of the general population, taken from the EnviroGenomarkers project. We used linear mixed effect models to associate metal exposure to gene expression after adjustment for gender, age, BMI, smoking, and alcohol consumption. For Pb exposure only few associations were identified, including a downregulation of the human eukaryotic translation initiation factor 5 (eIF5). In contrast, Cd exposure, particularly in males, revealed a much stronger transcriptomic response, featuring multiple pathways related to pathomolecular mechanisms of AD, such as endocytosis, neutrophil degranulation, and Interleukin-7 signaling. A gender stratified analysis revealed that the Cd responses were male-specific and included a downregulation of the APOE gene in men. This exploratory study revealed novel hypothetical findings which might contribute to the understanding of the neurotoxic effects of chronic Pb and Cd exposure and possibly improve our knowledge on the molecular mechanisms linking metal exposure to AD risk.

Patient: Female, 44-year-old Final Diagnosis: Acute liver failure Symptoms: APAP overdose Medication: Acetaminophen Clinical Procedure: — Specialty: Molecular Biology Objective: Unusual clinical course Background: Acetaminophen overdose is the most common cause of acute liver failure. Nevertheless, new biomarker approaches enabling early prediction of the outcome of the acetaminophen overdose are needed. Recently, using next-generation sequencing analysis of serum from human study participants we uncovered injury-specific signatures of circulating microRNAs (miRNAs) that represented underlying molecular mechanisms of toxicity. This case study is first to show the application of miRNA profiling to assess prognosis of acetaminophen poisoning. Case Report: The patient was admitted to the hospital following supra therapeutic acetaminophen ingestion. The patient showed elevated levels of biomarkers of hepatocellular injury alanine aminotransferase, aspartate transaminase, and glutamate dehydrogenase. Even though treatment with N-acetyl cysteine was initiated 24 hours post-ingestion, levels of alanine-aminotransferase and aspartate transaminase peaked at about 40 hours post ingestion of acetaminophen. We analyzed global circulating miRNA levels from 24 consecutive serum samples from this study participant covering the period from admission to time of death. Conclusions: The resulting global miRNA profiles were compared with profiles from study participants with non-lethal acetaminophen poisoning and healthy controls. At the admission, the miRNA profiles of both lethal and non-lethal acetaminophen poisoning showed induction of cellular stress and oxidative damage. Later, the miRNA profiles of the lethal poisoning featured fibrosis and coagulation pathways while profiles of non-lethal cases resembled those of healthy study participants. Although additional confirmatory studies are needed, our case study is first to indicate that global miRNA profiles to be used as liquid biopsies have potential to facilitate the assessment of acetaminophen poisoning.

Circulating microRNAs (cimiRNAs) are a class of non-encoding RNAs found in bodily fluids such as blood, cerebrospinal fluid (CSF) and tears. CimiRNAs have been implicated as promising biomarkers for central nervous system (CNS) disorders because they are actively secreted as messengers and are profoundly involved in fine-tuning of developmental and differentiation processes. Furthermore, they are attractive biomarkers because they are extremely stable, tissue enriched and can be determined in a quantitative manner. This review aims to provide a comprehensive assessment on the current progress regarding the potential value of cimiRNAs as CNS biomarkers. Within this framework five CNS disorders are explored which share a common pathological hallmark namely cognitive impairment. The CNS disorders include Major depression disorder (MDD), Bipolar disorder (BD), Schizophrenia (SZ), Alzheimer's disease (AD) and Parkinson disease (PD). The similarities and differences between altered cimiRNAs in the different disorders are described. The miR-29 family, miR-34a-5p and miR-132-3p are discussed as common dysregulated cimiRNAs found in the CNS disorders. Furthermore, it is shown that the type of bodily fluid used for measuring cimiRNAs is important as inconsistencies in cimiRNAs expression directions are found when comparing CSF, blood cell-free and blood cell-bound samples.

Diesel vehicle emissions are the major source of genotoxic compounds in ambient air from urban areas. These pollutants are linked to risks of cardiovascular diseases, lung cancer, respiratory infections and adverse neurological effects. Biological events associated with exposure to some air pollutants are widely unknown but applying omics techniques may help to identify the molecular processes that link exposure to disease risk. Most data on health risks are related to long-term exposure, so the aim of this study is to investigate the impact of short-term exposure (two hours) to air pollutants on the blood transcriptome and microRNA expression levels.We analyzed transcriptomics and microRNA expression using microarray technology on blood samples from volunteers participating in studies in London, the Oxford Street cohort, and, in Barcelona, the TAPAS cohort. Personal exposure levels measurements of particulate matter (PM10 PM2.5), ultrafine particles (UFPC), nitrogen oxides (NO2, NO and NOx), black carbon (BC) and carbon oxides (CO and CO2) were registered for each volunteer. Associations between air pollutant levels and gene/microRNA expression were evaluated using multivariate normal models (MVN).MVN-models identified compound-specific expression of blood cell genes and microRNAs associated with air pollution despite the low exposure levels, the short exposure periods and the relatively small sized cohorts. Hsa-miR-197-3p, hsa-miR-29a-3p, hsa-miR-15a-5p, hsa-miR-16-5p and hsa-miR-92a-3p are found significantly expressed in association with exposures. These microRNAs target also relevant transcripts, indicating their potential relevance in the research of omics-biomarkers responding to air pollution. Furthermore, these microRNAs are also known to be associated with diseases previously linked to air pollution exposure including several cancers such lung cancer and Alzheimer's disease. In conclusion, we identified in this study promising compound-specific mRNA and microRNA biomarkers after two hours of exposure to low levels of air pollutants during two hours that suggest increased cancer risks. (C) 2018 Published by Elsevier Ltd.

MicroRNAs, a class of regulatory small non-coding RNAs, are emerging as promising biomarkers for different health outcomes. Due to their tissue specificity, stability in extracellular space and high conservation between preclinical test species, applications of novel miRNA-based biomarkers for drug safety testing regarding hepatotoxicity and cardiotoxicity are investigated. Furthermore, miRNA expression is altered by environmental exposure such as cigarette smoke or polychlorinated biphenyls. As a consequence, miRNAs potentially influence tumor suppressor genes and oncogenes and may influence carcinogenesis. This has raised the interest in the use of miRNA profiles for health risk assessment. This review summarizes the recent developments in miRNA research with focus on biomarkers for drug safety testing and biomarkers for health outcomes related to environmental exposures.

Background B-cell chronic lymphocytic leukemia (CLL) is a common type of adult leukemia. It often follows an indolent course and is preceded by monoclonal B-cell lymphocytosis, an asymptomatic condition, however it is not known what causes subjects with this condition to progress to CLL. Hence the discovery of prediagnostic markers has the potential to improve the identification of subjects likely to develop CLL and may also provide insights into the pathogenesis of the disease of potential clinical relevance. Results We employed peripheral blood buffy coats of 347 apparently healthy subjects, of whom 28 were diagnosed with CLL 2.0–15.7 years after enrollment, to derive for the first time genome-wide DNA methylation, as well as gene and miRNA expression, profiles associated with the risk of future disease. After adjustment for white blood cell composition, we identified 722 differentially methylated CpG sites and 15 differentially expressed genes (Bonferroni-corrected p

MicroRNAs (miRNAs) released into the peripheral circulation upon cellular injury have shown a promise as a new class of tissue-specific biomarkers. We were first to demonstrate that next-generation sequencing analysis of serum from human subjects with acetaminophen-induced liver injury revealed a specific signature of circulating miRNAs. We consequently hypothesized that different types of hepatic liver impairments might feature distinct signatures of circulating miRNAs and that this approach might be useful as minimally invasive diagnostic "liquid biopsies" enabling the interrogation of underlying molecular mechanisms of injury in distant tissues. Therefore we examined serum circulating miRNAs in a total of 72 serum samples from a group of 53 subjects that included patients with accidental acetaminophen overdose, hepatitis B infection, liver cirrhosis and type 2 diabetes as well as gender-and age-matched healthy subjects with no evidence of liver disease. The miRNA signatures were identified using next-generation sequencing that provided analysis for the whole miRNome, including miRNA isoforms. Compared to the healthy subjects, a total of 179 miRNAs showed altered serum levels across the diseased subjects. Although many subjects have elevated alanine aminotransferase suggesting liver impairments, we identified distinct miRNA signatures for different impairments with minimum overlap. Furthermore, the bioinformatics analysis of miRNA signatures revealed relevant molecular pathways associated with the mechanisms of toxicity and or pathogenesis of disease. Interestingly, the high proportion of miRNA isoforms present in the respective signatures indicated a new level of complexity in cellular response to stress or disease. Our study demonstrates for the first time that signatures of circulating miRNAs show specificity for liver injury phenotypes and, once validated, might become useful for diagnosis of organ pathologies as "liquid biopsies".

The development of biomarkers based on high-throughput techniques, cutting-edge biostatistics and bioinformatics tools is revolutionizing molecular cancer epidemiology. Once validated, such biomarkers will open new and promising perspectives for human health risk assessment and identification of at-risk populations. The application of OMICS to environmental health research is currently resulting in the production of large data sets on gene expression, transcription factors, proteins, metabolites, adducts and epigenetic regulation of the genome in relation to dietary and environmental exposures. The assessment of whole genome transcriptomic and epigenetic profiles (where microRNA analysis has raised special attention in recent years) are regarded as potentially powerful approaches to reduce uncertainties in health risk assessments and to improve strategies for disease prevention, which is a shared aim of the new European Union (EU) research programme, Horizon 2020 ( http://ec.europa.eu/programmes/horizon2020/ ). However, to guarantee appropriate application of OMICS, some challenges need to be addressed in the coming years regarding study design, technicalities in methodology and data analysis.

Powdery mildew (PM, caused by Podosphaera fusca) and downy mildew (DM, caused by Pseudoperonospora cubensis) are important diseases of cucumber (Cucumis sativus). Breeding for resistance has been undertaken since the 1940s, but underlying resistance genes have not been functionally analysed yet. The published genome sequence of cucumber catalyses the search for such genes. Genetic studies have indicated that resistances to PM and DM in cucumber are often inherited recessively, which indicates the presence of susceptibility genes (S-genes). Therefore we analyzed the cucumber genome for homologs of functionally proven S-genes known from other plant species. We identified 13 MLO-like genes in cucumber, three of which cluster in Clade V, the clade that contains all known MLO-like susceptibility genes to powdery mildews in other dicots. The expression of one of these three genes, CsaMLO1, located on chromosome 1, was upregulated after PM inoculation. It co-localizes with a QTL for PM resistance previously identified. Also homologs of the susceptibility genes PMR4 and PMR5 are located at this QTL. The second MLO-like gene from Clade V (CsaMLO8) resides in a recessively inherited major QTL for PM resistance at the bottom of chromosome 5, together with a PMR6-like gene. Two major QTL for DM recessive resistance at the top of chromosome 5 co-localize with CsaDMR6-2, which is homologous to the DMR6 susceptibility gene in Arabidopsis. This study has identified several candidate genes for susceptibility to PM and DM in cucumber that may explain QTL for recessively inherited resistance, reported earlier.

Polychlorinated biphenyls (PCBs) are a class of widespread environmental pollutants, commonly found in human blood, that have been suggested to be linked to the occurrence of sporadic Parkinson's disease (PD). It has been reported that some non-coplanar PCBs accumulate in the brains of female PD patients. To improve our understanding of the association between PCB exposure and PD risk we have applied whole transcriptome gene expression analysis in blood cells from 594 PCB-exposed subjects (369 female, 225 male).Interestingly, we observe that in females, blood levels of non-coplanar PCBs appear to be associated with expression levels of PD-specific genes. However, no such association was detected in males. Among the 131 PD-specific genes affected, 39 have been shown to display similar changes in expression levels in the substantia nigra of deceased PD patients. Especially among the down-regulated genes, transcripts of genes involved in neurotransmitter vesicle-related functions were predominant. (C) 2019 Elsevier Ltd. All rights reserved.

In the original version of this article, the author ‘Christiaan H. Vinkers’ was incorrectly associated with ‘Brain Center Rudolf Magnus, Department of Psychiatry, University Medical Center Utrecht, Utrecht, The Netherlands’. This authors affiliations have now been corrected to: ‘Department of Psychiatry, Amsterdam UMC (location VUmc), Amsterdam, The Netherlands’ and ‘Department of Anatomy and Neurosciences, Amsterdam UMC (location VUmc), Amsterdam, The Netherlands’. This has been corrected in both the PDF and HTML versions of the article.

Persistent organic pollutants (POPs) are synthetic chemical substances that accumulate in our environment. POPs such as polychlorinated biphenyls (PCBs), hexachlorobenzene (HCB) and dichlorodiphenyltrichloroethane (DDT) have been classified as carcinogenic to humans and animals. Due to their resistance to biodegradation humans are still exposed to these compounds worldwide. We aim to evaluate the miRNA and transcriptomic response of a human population exposed to POPs. The miRNA and transcriptomic response was measured in blood of healthy subjects by microarray technology and associated with the serum concentrations of six PCB congeners, DDE (a common DDT metabolite), and HCB. A total of 93 miRNA levels appeared significantly associated with the POP-exposure (FDR MYC, CCND1, BCL2 and VEGFA. We have shown that exposure to POPs is associated with human miRNA and transcriptomic responses. The identified miRNAs and target genes are related to various types of cancer and involved in relevant signaling pathways like wnt and p53. Therefore, these miRNAs may have great potential to contribute to biomarker-based environmental health risk assessment.

The utility of blood-based omic profiles for linking environmental exposures to their potential health effects was evaluated in 649 individuals, drawn from the general population, in relation to tobacco smoking, an exposure with well-characterised health effects. Using disease connectivity analysis, we found that the combination of smoking-modified, genome-wide gene (including miRNA) expression and DNA methylation profiles predicts with remarkable reliability most diseases and conditions independently known to be causally associated with smoking (indicative estimates of sensitivity and positive predictive value 94% and 84%, respectively). Bioinformatics analysis reveals the importance of a small number of smoking-modified, master-regulatory genes and suggest a central role for altered ubiquitination. The smoking-induced gene expression profiles overlap significantly with profiles present in blood cells of patients with lung cancer or coronary heart disease, diseases strongly associated with tobacco smoking. These results provide proof-of-principle support to the suggestion that omic profiling in peripheral blood has the potential of identifying early, disease-related perturbations caused by toxic exposures and may be a useful tool in hazard and risk assessment.

Drug-induced liver injury (DILI) is a leading cause of acute liver failure and the major reason for withdrawal of drugs from the market. Preclinical evaluation of drug candidates has failed to detect about 40% of potentially hepatotoxic compounds in humans. At the onset of liver injury in humans, currently used biomarkers have difficulty differentiating severe DILI from mild, and/or predict the outcome of injury for individual subjects. Therefore, new biomarker approaches for predicting and diagnosing DILI in humans are urgently needed. Recently, circulating microRNAs (miRNAs) such as miR-122 and miR-192 have emerged as promising biomarkers of liver injury in preclinical species and in DILI patients. In this study, we focused on examining global circulating miRNA profiles in serum samples from subjects with liver injury caused by accidental acetaminophen (APAP) overdose. Upon applying next generation high-throughput sequencing of small RNA libraries, we identified 36 miRNAs, including 3 novel miRNA-like small nuclear RNAs, which were enriched in the serum of APAP overdosed subjects. The set comprised miRNAs that are functionally associated with liver-specific biological processes and relevant to APAP toxic mechanisms. Although more patients need to be investigated, our study suggests that profiles of circulating miRNAs in human serum might provide additional biomarker candidates and possibly mechanistic information relevant to liver injury.

Background Plants are sessile organisms that deal with their -sometimes adverse- environment in well-regulated ways. Chromatin remodeling involving SWI/SNF2-type ATPases is thought to be an important epigenetic mechanism for the regulation of gene expression in different developmental programs and for integrating these programs with the response to environmental signals. In this study, we report on the role of chromatin remodeling in Arabidopsis with respect to the variability of growth and gene expression in relationship to environmental conditions. Results Already modest (2-fold) over-expression of the AtCHR23 ATPase gene in Arabidopsis results in overall reduced growth compared to the wild-type. Detailed analyses show that in the root, the reduction of growth is due to reduced cell elongation. The reduced-growth phenotype requires sufficient light and is magnified by applying deliberate abiotic (salt, osmotic) stress. In contrast, the knockout mutation of AtCHR23 does not lead to such visible phenotypic effects. In addition, we show that over-expression of AtCHR23 increases the variability of growth in populations of genetically identical plants. These data indicate that accurate and controlled expression of AtCHR23 contributes to the stability or robustness of growth. Detailed RNAseq analyses demonstrate that upon AtCHR23 over-expression also the variation of gene expression is increased in a subset of genes that associate with environmental stress. The larger variation of gene expression is confirmed in individual plants with the help of independent qRT-PCR analysis. Conclusions Over-expression of AtCHR23 gives Arabidopsis a phenotype that is markedly different from the growth arrest phenotype observed upon over-expression of AtCHR12, the paralog of AtCHR23, in response to abiotic stress. This demonstrates functional sub-specialization of highly similar ATPases in Arabidopsis. Over-expression of AtCHR23 increases the variability of growth among genetically identical individuals in a way that is consistent with increased variability of expression of a distinct subset of genes that associate with environmental stress. We propose that ATCHR23-mediated chromatin remodeling is a potential component of a buffer system in plants that protects against environmentally-induced phenotypic and transcriptional variation.