Early‐life stress can result in life‐long effects that impact adult health and disease risk, but little is known about how such programming is established and maintained. Here, we show that such epigenetic memories can be initiated in the Drosophila embryo before the major wave of zygotic transcription, and higher‐order chromatin structures are established. An early short heat shock results in elevated levels of maternal miRNA and reduced levels of a subgroup of zygotic genes in stage 5 embryos. Using a Dicer‐1 mutant, we show that the stress‐induced decrease in one of these genes, the insulator‐binding factor Elba1, is dependent on functional miRNA biogenesis. Reduction in Elba1 correlates with the upregulation of early developmental genes and promotes a sustained weakening of heterochromatin in the adult fly as indicated by an increased expression of the PEV w m4h reporter. We propose that maternal miRNAs, retained in response to an early embryonic heat shock, shape the subsequent de novo heterochromatin establishment that occurs during early development via direct or indirect regulation of some of the earliest expressed genes, including Elba1.

Aims: Increasing concentrations of dietary sugar results in a linear accumulation of triglycerides in male Drosophila, while inducing a U-shaped obesity response in their offspring. Here, using a combination of proteomics and small RNA (sRNA) sequencing, we aimed at understanding the molecular underpinning in sperm for such plasticity. Results: Proteomic analysis of seminal vesicles revealed that increasing concentrations of dietary sugar resulted in a bell-shaped induction of proteins involved in metabolic/redox regulation. Using stains and in vivo redox reporter flies, this pattern could be explained by changes in sperm production of reactive oxygen species (ROS), more exactly mitochondria-derived H2O2. By quenching ROS with the antioxidant N-acetyl cysteine and performing sRNA-seq on sperm, we found that sperm miRNA is increased in response to ROS. Moreover, we found sperm mitosRNA to be increased in high-sugar diet conditions (independent of ROS). Reanalyzing our previously published data revealed a similar global upregulation of human sperm mitosRNA in response to a high-sugar diet, suggesting evolutionary conserved mechanisms. Innovation: This work highlights a fast response to dietary sugar in mitochondria-produced H2O2 in Drosophila sperm and identifies redox-sensitive miRNA downstream of this event. Conclusions: Our data support a model where changes in the sperm mitochondria in response to dietary sugar are the primary event, and changes in redox homoeostasis are secondary to mitochondrial ROS production. These data provide multiple candidates for paternal intergenerational metabolic responses as well as potential biomarkers for human male fertility. Antioxid. Redox Signal. 38, 1167-1183.

Summary A wide spectrum of exogenous factors, including diet, environmental pollutants, stress, and seasonal changes have major impact on sperm quality and function. The molecular basis, however, that explains this susceptibility remains largely unknown. Using a combination of proteomics and small RNA (sRNA) sequencing, we show that Drosophila sperm display rapid molecular changes in response to dietary sugar, both in terms of metabolic/redox proteins and sRNA content, particularly miRNA and mitochondria derived sRNA (mt-sRNA). Thus, results from two independent omics point at the dynamics of mitochondria as the central aspect in rapid metabolic adjustments in sperm. Using specific stains and in vivo redox reporter flies, we show that diet indeed rapidly alters the production of mitochondrial derived reactive oxygen species (ROS). Quenching ROS via supplementation of N acetyl cysteine reduces diet-upregulated miRNA, but not mitochondrial-sRNA. Together, these results open new territories in our search for the mechanistic understanding of sperm health and disease. Highlights Diet rapidly changes the proteomic and sRNA profiles in sperm Diet sensitive sperm proteins are found in human infertility studies Sperm mitochondrial ROS levels are modulated by diet dme-miR-10 regulation is secondary to diet-induced ROS Diet, but not diet-induced ROS, alters the expression of mitochondrial small RNA, especially tsRNA

The aftermath of traumatization lives on in the neural and epigenetic traces creating a momentum of affliction in the psychological and social realm. Can psychotherapy reorganise these memories through changes in DNA methylation signatures? Using a randomised controlled parallel group design, we examined methylome-wide changes in saliva samples of 84 female former child soldiers from Eastern DR Congo before and six months after Narrative Exposure Therapy. Treatment predicted differentially methylated positions (DMPs) related to ALCAM , RIPOR2 , AFAP1 and MOCOS . In addition, treatment associations overlapped at gene level with baseline clinical and social outcomes. Treatment related DMPs are involved in memory formation—the key agent in trauma focused treatments—and enriched for molecular pathways commonly affected by trauma related disorders. Results were partially replicated in an independent sample of 53 female former child soldiers from Northern Uganda. Our results suggest a molecular impact of psychological treatment in women with war-related childhood trauma. Trial registration : Addressing Heightened Levels of Aggression in Traumatized Offenders With Psychotherapeutic Means (ClinicalTrials.gov Identifier: NCT02992561, 14/12/2016).

Summary Preconception parental environment can reproducibly program offspring phenotype without altering the DNA sequence, yet the mechanisms underpinning this ‘epigenetic inheritance’ remains elusive. Here, we demonstrate the existence of an intact piRNA-pathway in mature Drosophila sperm and show that pathway modulation alters offspring gene transcription in a sequence-specific manner. We map a dynamic small RNA content in developing sperm and find that the mature sperm carry a highly distinct small RNA cargo. By biochemical pulldown, we identify a small RNA subset bound directly to piwi protein. And, we show that piRNA-pathway controlled sperm small RNAs are linked to target gene repression in offspring. Critically, we find that full piRNA-pathway dosage is necessary for the intergenerational metabolic and transcriptional reprogramming events triggered by high paternal dietary sugar. These data provide a direct link between regulation of endogenous mature sperm small RNAs and transcriptional programming of complementary sequences in offspring. Thus, we identify a novel mediator of paternal intergenerational epigenetic inheritance.

Small RNA sequencing (sRNA-seq) has become important for studying regulatory mechanisms in many cellular processes. Data analysis remains challenging, mainly because each class of sRNA—such as miRNA, piRNA, tRNA- and rRNA-derived fragments (tRFs/rRFs)—needs special considerations. Analysis therefore involves complex workflows across multiple programming languages, which can produce research bottlenecks and transparency issues. To make analysis of sRNA more accessible and transparent we present seqpac: a tool for advanced group-based analysis of sRNA completely integrated in R. This opens advanced sRNA analysis for Windows users—from adaptor trimming to visualization. Seqpac provides a framework of functions for analyzing a PAC object, which contains 3 standardized tables: sample phenotypic information (P), sequence annotations (A), and a counts table with unique sequences across the experiment (C). By applying a sequence-based counting strategy that maintains the integrity of the fastq sequence, seqpac increases flexibility and transparency compared to other workflows. It also contains an innovative targeting system allowing sequence counts to be summarized and visualized across sample groups and sequence classifications. Reanalyzing published data, we show that seqpac’s fastq trimming performs equal to standard software outside R and demonstrate how sequence-based counting detects previously unreported bias. Applying seqpac to new experimental data, we discovered a novel rRF that was down-regulated by RNA pol I inhibition (anticancer treatment), and up-regulated in previously published data from tumor positive patients. Seqpac is available on github (<https://github.com/Danis102/seqpac>), runs on multiple platforms (Windows/Linux/Mac), and is provided with a step-by-step vignette on how to analyze sRNA-seq data. ### Competing Interest Statement The authors have declared no competing interest.

SUMMARY Early-life stress can generate persistent life-long effects that impact adult health and disease risk, but little is known of how such programming is established and maintained. Previous use of the Drosophila strain w m4h show that an early embryonic heat shock result in stable epigenetic alteration in the adult fly. To investigate the potential role of small non-coding RNA (sncRNA) in the initiation of such long-term epigenetic effects, we here generated a fine timeline of sncRNA expression during the first 5 stages of Drosophila embryogenesis in this strain. Building on this, we show that (1) miRNA is increased following early embryonic heat shock, and (2) the increased miRNA is coming from two separate sources, maternal and zygotic. By performing long RNA sequencing on the same single embryo, we found that a subgroup of miRNA with maternal origin, had a strong negative correlation with a group of early zygotic transcripts. Critically, we found evidence that one such early zygotic transcript, the insulator binding factor Elba1, is a Su(var) for w m4h . The findings provide insights of the dynamics and stress-sensitivity of sncRNA during the first embryonic stages in Drosophila and suggest an interplay between miRNA, Elba1 and long-term epigenetic alteration. HIGHLIGHTS We provide a high-resolution timeline for sncRNA for Drosophila stage 1-5 embryos Heat shock before midblastula transition (MBT) results in a massive upregulation of miRNA at cellularization Heat shock-induced miRNAs negatively associate with downregulation of a specific subset of pre-MBT genes Elba1 is a position-effect-variegation (PEV) modifier for w m4h Heat shock-induces an “leaky” expression of genes that overlap with Elba 1-3 binding sites

Small RNA (sRNA) sequencing has been critical for our understanding of the transcriptional regulation in most biological processes. Nonetheless, the varying biochemical properties of sRNA, such as 59 nucleotide modifications, make many sRNA subspecies incompatible with common protocols for sRNA sequencing. Here we describe 5XP-seq that outlines a novel strategy on how to overcome this limitation. By specifically tagging 59-phosphorylated sRNA (59P sRNA), that enriches miRNA and piRNA, we successfully discriminate these sRNA from all other sRNA species. Our results underscore the importance of more holistic approaches, in which 59 insensitive library strategies are combined with 59 sensitive tagging. Further advances using such strategies will overcome the increasing bias in reports of easily caught sRNA and ensure assessment of the previously overlooked transcriptome.

ABSTRACT Small RNA (sRNA) sequencing has been critical for our understanding of many cellular processes, including gene regulation. Nonetheless, the varying biochemical properties of sRNA, such as 5’ nucleotide modifications, makes many sRNA subspecies incompatible with common protocols for sRNA sequencing. Here we describe 5XP-seq that outlines a novel strategy that solves this problem. By tagging 5’P sRNA during library preparation, 5XP-seq combines an open approach that includes all types of 5’-terminal modifications (5’X), with a selective approach for 5-phosphorylated sRNA (5’P). We show that 5XP-seq not only enriches phosphorylated miRNA and piRNA but successfully discriminates these sRNA from all other sRNA species. We further demonstrate the importance of this strategy by successful inter-species validation of sRNAs that would have otherwise failed, including human to insect translation of several tRNA (tRFs) and rRNA (rRFs) fragments. By combining 5’ insensitive library strategies with 5’ sensitive tagging, we have solved an intrinsic bias in modern sRNA sequencing that will help us reveal the true complexity and the evolutionary significance of the sRNA world.

The world has recently experienced a decline in male reproductive (e.g. sperm counts and motility) and metabolic (e.g. obesity and diabetes) health. Accumulated evidence from animal models also shows that the metabolic health of the father may influence the metabolic health in his offspring. Vectors for such paternal intergenerational metabolic responses (IGMRs) involve small noncoding RNAs (sncRNAs) that often increase in spermatozoa during the last days of maturation in the epididymis. We and others have shown that the metabolic state - depending on factors such as diet, obesity and physical exercise - may affect sperm quality and sperm sncRNA. Together, this suggests that there are overlapping aetiologies between the male metabolic syndrome, male factor infertility and intergenerational responses. In this review, we present a theoretical framework for an overlap of these aetiologies by exploring the advances in our understanding of the roles of sncRNA in spermatogenesis and offspring development. A special focus will lie on novel findings about tRNA-derived small RNA (tsRNA), rRNA-derived small RNA (rsRNA) and small mitochondrial RNA (mitoRNA), and their emerging roles in intergenerational metabolic and reproductive health.

Stress during pregnancy widely associates with epigenetic changes and psychiatric problems during childhood. Animal studies, however, show that under specific postnatal conditions prenatal stress may have other, less detrimental consequences for the offspring. Here we studied mental health and epigenome-wide DNA methylation in saliva following intimate partner violence (IPV) during pregnancy in São Gonçalo, a Brazilian city with high levels of violence. Not surprisingly, mothers exposed to pregnancy IPV expressed elevated depression, PTSD and anxiety symptoms. Children had similar psychiatric problems when they experienced maternal IPV after being born. More surprisingly, when maternal IPV occurred both during (prenatal) and after pregnancy these problems were absent. Following prenatal IPV, genomic sites in genes encoding the glucocorticoid receptor (NR3C1) and its repressor FKBP51 (FKBP5) were among the most differentially methylated and indicated an enhanced ability to terminate hormonal stress responses in prenatally stressed children. These children also showed more DNA methylation in heterochromatin-like regions, which previously has been associated with stress/disease resilience. A similar relationship was seen in prenatally stressed middle-eastern refugees of the same age as the São Gonçalo children but exposed to postnatal war-related violence. While our study is limited in location and sample size, it provides novel insights on how prenatal stress may epigenetically shape resilience in humans, possibly through interactions with the postnatal environment. This translates animal findings and emphasizes the importance to account for population differences when studying how early life gene-environment interactions affects mental health.

The global rise in obesity and steady decline in sperm quality are two alarming trends that have emerged during recent decades. In parallel, evidence from model organisms shows that paternal diet can affect offspring metabolic health in a process involving sperm tRNA-derived small RNA (tsRNA). Here, we report that human sperm are acutely sensitive to nutrient flux, both in terms of sperm motility and changes in sperm tsRNA. Over the course of a 2-week diet intervention, in which we first introduced a healthy diet followed by a diet rich in sugar, sperm motility increased and stabilized at high levels. Small RNA-seq on repeatedly sampled sperm from the same individuals revealed that tsRNAs were up-regulated by eating a high-sugar diet for just 1 week. Unsupervised clustering identified two independent pathways for the biogenesis of these tsRNAs: one involving a novel class of fragments with specific cleavage in the T-loop of mature nuclear tRNAs and the other exclusively involving mitochondrial tsRNAs. Mitochondrial involvement was further supported by a similar up-regulation of mitochondrial rRNA-derived small RNA (rsRNA). Notably, the changes in sugar-sensitive tsRNA were positively associated with simultaneous changes in sperm motility and negatively associated with obesity in an independent clinical cohort. This rapid response to a dietary intervention on tsRNA in human sperm is attuned with the paternal intergenerational metabolic responses found in model organisms. More importantly, our findings suggest shared diet-sensitive mechanisms between sperm motility and the biogenesis of tsRNA, which provide novel insights about the interplay between nutrition and male reproductive health.

Post-traumatic stress disorder (PTSD) is a common, debilitating condition with limited treatment options. Extinction of fear memories through prolonged exposure therapy, the primary evidence-based behavioral treatment for PTSD, has only partial efficacy. In mice, pharmacological inhibition of fatty acid amide hydrolase (FAAH) produces elevated levels of anandamide (AEA) and promotes fear extinction, suggesting that FAAH inhibitors may aid fear extinction-based treatments. A human FAAH 385C->A substitution encodes an FAAH enzyme with reduced catabolic efficacy. Individuals homozygous for the FAAH 385A allele may therefore offer a genetic model to evaluate the impact of elevations in AEA signaling in humans, helping to inform whether FAAH inhibitors have the potential to facilitate fear extinction therapy for PTSD. To overcome the challenge posed by low frequency of the AA genotype (appr. 5%), we prospectively genotyped 423 individuals to examine the balanced groups of CC, AC, and AA individuals (n = 25/group). Consistent with its loss-of-function nature, the A allele was dose dependently associated with elevated basal AEA levels, facilitated fear extinction, and enhanced the extinction recall. Moreover, the A-allele homozygotes were protected against stress-induced decreases in AEA and negative emotional consequences of stress. In a humanized mouse model, AA homozygous mice were similarly protected against stress-induced decreases in AEA, both in the periphery, and also in the amygdala and prefrontal cortex, brain structures critically involved in fear extinction and regulation of stress responses. Collectively, these data suggest that AEA signaling can temper aspects of the stress response and that FAAH inhibition may aid the treatment for stress-related psychiatric disorders, such as PTSD.

Stress during pregnancy may impact subsequent generations, which is demonstrated by an increased susceptibility to childhood and adulthood health problems in the children and grandchildren. Although the importance of the prenatal environment is well reported with regards to future physical and emotional outcomes, little is known about the molecular mechanisms that mediate the long-term consequences of early stress across generations. Recent studies have identified DNA methylation as a possible mediator of the impact of prenatal stress in the offspring. Whether psychosocial stress during pregnancy also affects DNA methylation of the grandchildren is still not known. In the present study we examined the multigenerational hypothesis, that is, grandmaternal exposure to psychosocial stress during pregnancy affecting DNA methylation of the grandchildren. We determined the genome-wide DNA methylation profile in 121 children (65 females and 56 males) and tested for associations with exposure to grandmaternal interpersonal violence during pregnancy. We observed methylation variations of five CpG sites significantly (FDR<0.05) associated with the grandmother’s report of exposure to violence while pregnant with the mothers of the children. The results revealed differential methylation of genes previously shown to be involved in circulatory system processes (FDR<0.05). This study provides support for DNA methylation as a biological mechanism involved in the transmission of stress across generations and motivates further investigations to examine prenatal-dependent DNA methylation as a potential biomarker for health problems.

This search, a type not previously performed at ATLAS, uses a comparison of the production cross sections for e+μ− and e−μ+ pairs to constrain physics processes beyond the Standard Model. It uses 139fb−1 of proton–proton collision data recorded at s=13 TeV at the LHC. Targeting sources of new physics which prefer final states containing e+μ− to e−μ+, the search contains two broad signal regions which are used to provide model-independent constraints on the ratio of cross sections at the 2% level. The search also has two special selections targeting supersymmetric models and leptoquark signatures. Observations using one of these selections are able to exclude, at 95% confidence level, singly produced smuons with masses up to 640 GeV in a model in which the only other light sparticle is a neutralino when the R-parity-violating coupling λ231′ is close to unity. Observations using the other selection exclude scalar leptoquarks with masses below 1880 GeV when g1Reu=g1Rμc=1, at 95% confidence level. The limit on the coupling reduces to g1Reu=g1Rμc=0.46 for a mass of 1420 GeV.

Childhood malnutrition is a risk factor for mental disorders, such as major depression and anxiety. Evidence shows that similar early life adversities induce sex-dependent epigenetic reprogramming. However, little is known about how genes are specifically affected by early malnutrition and the implications for males and females respectively. One relevant target is neuropeptide Y (NPY), which regulates both stress and food-intake. We studied maternal low protein diet (LPD) during pregnancy/lactation in mice. Male, but not female, offspring of LPD mothers consistently displayed anxiety-and depression-like behaviors under acute stress. Transcriptome-wide analysis of the effects of acute stress in the amygdala, revealed a list of transcription factors affected by either sex or perinatal LPD. Among these immediate early genes (IEG), members of the Early growth response family (Egr1/2/4) were consistently upregulated by perinatal LPD in both sexes. EGR1 also bound the NPY receptor Y1 gene (Npy1r), which co-occurred with sex-specific effects of perinatal LPD on both Npy1r DNA-methylation and gene transcription. Our proposed pathway connecting early malnutrition, sex-independent regulatory changes in Egr1, and sex-specific epigenetic reprogramming of its effector gene, Npy1r, represents the first molecular evidence of how early life risk factors may generate sex-specific epigenetic effects relevant for mental disorders.

It has been shown that maternal stress and malnutrition, or experience of other adverse events, during the perinatal period may alter susceptibility in the adult offspring in a time-of-exposure dependent manner. The mechanism underlying this may be epigenetic in nature. Here, we summarize some recent findings on the effects on gene-regulation following maternal malnutrition, focusing on epigenetic regulation of peptidergic activity. Numerous neuropeptides within the central nervous system are crucial components in regulation of homeostatic energy-balance, as well as affective health (i.e. health events related to affective disorders, psychiatric disorders also referred to as mood disorders). It is becoming evident that expression, and function, of these neuropeptides can be regulated via epigenetic mechanisms during fetal development, thereby contributing to the development of the adult phenotype and, possibly, modulating disease susceptibility. Here, we focus on two such neuropeptides, neuropeptide Y (NPY) and corticotropin-releasing hormone (CRH), both involved in regulation of endocrine function, energy homeostasis, as well as affective health. While a number of published studies indicate the involvement of epigenetic mechanisms in CRH-dependent regulation of the offspring adult phenotype, NPY has been much less studied in this context and needs further work.

Epigenetic processes have been implicated in the pathophysiology of alcohol dependence, but the specific molecular mechanisms mediating dependence-induced neuroadaptations remain largely unknown. Here, we found that a history of alcohol dependence persistently decreased the expression of Prdm2, a histone methyltransferase that monomethylates histone 3 at the lysine 9 residue (H3K9me1), in the rat dorsomedial prefrontal cortex (dmPFC). Downregulation of Prdm2 was associated with decreased H3K9me1, supporting that changes in Prdm2 mRNA levels affected its activity. Chromatin immunoprecipitation followed by massively parallel DNA sequencing showed that genes involved in synaptic communication are epigenetically regulated by H3K9me1 in dependent rats. In non-dependent rats, viral-vector-mediated knockdown of Prdm2 in the dmPFC resulted in expression changes similar to those observed following a history of alcohol dependence. Prdm2 knockdown resulted in increased alcohol self-administration, increased aversion-resistant alcohol intake and enhanced stress-induced relapse to alcohol seeking, a phenocopy of postdependent rats. Collectively, these results identify a novel epigenetic mechanism that contributes to the development of alcohol-seeking behavior following a history of dependence.

Transposons are playing an important role in the evolution of eukaryotic genomes. These endogenous virus-like elements often amplify within their host genomes in a species specific manner. Today we have limited understanding when and how these amplification events happens. What we do know is that cells have evolved multiple line of defenses to keep these potentially invasive elements under control, often involving epigenetic mechanisms such as DNA-methylation and histone modifications. Emerging evidence shows a strong link between transposon activity and human aging and diseases, as well as a role for transposons in normal brain development. Controlling transposon activity may therefore uphold the fine balance between health and disease. In this article we investigate this balance, and sets it in relation to allostatic load, which conceptualize the link between stress and the "wear and tear" of the organism that leads to aging and disease. We hypothesize that stress-induced retrotransposon reactivation in humans may be used to estimate allostatic load, and may be a possible mechanism in which transposons amplify within species genomes.

Early stress can have long-lasting phenotypic effects. Previous research shows that male and female chickens differ in many behavioural aspects, and respond differently to chronic stress. The present experiment aimed to broadly characterize long-term sex differences in responses to brief events of stress experienced during the first weeks of life. Chicks from a commercial egg-laying hybrid were exposed to stress by inducing periods of social isolation during their first three weeks of life, followed by a broad behavioural, physiological and genomic characterization throughout life. Early stressed males, but not females, where more anxious in an open field-test, stayed shorter in tonic immobility and tended to have delayed sexual maturity, as shown by a tendency for lower levels of testosterone compared to controls. While early stressed females did not differ from non-stressed in fear and sexual maturation, they were more socially dominant than controls. The differential gene expression profile in hypothalamus was significantly correlated from 28 to 213 days of age in males, but not in females. In conclusion, early stress had a more pronounced long-term effect on male than on female chickens, as evidenced by behavioral, endocrine and genomic responses. This may either be attributed to inherent sex differences due to evolutionary causes, or possibly to different stress related selection pressures on the two sexes during commercial chicken breeding.