The risk of posttraumatic stress disorder (PTSD) following trauma is heritable, but robust common variants have yet to be identified. In a multi-ethnic cohort including over 30,000 PTSD cases and 170,000 controls we conduct a genome-wide association study of PTSD. We demonstrate SNP-based heritability estimates of 5–20%, varying by sex. Three genome-wide significant loci are identified, 2 in European and 1 in African-ancestry analyses. Analyses stratified by sex implicate 3 additional loci in men. Along with other novel genes and non-coding RNAs, a Parkinson’s disease gene involved in dopamine regulation, PARK2, is associated with PTSD. Finally, we demonstrate that polygenic risk for PTSD is significantly predictive of re-experiencing symptoms in the Million Veteran Program dataset, although specific loci did not replicate. These results demonstrate the role of genetic variation in the biology of risk for PTSD and highlight the necessity of conducting sex-stratified analyses and expanding GWAS beyond European ancestry populations.

Epstein-Barr virus (EBV), an oncogenic human herpesvirus, induces cell proliferation after infection of resting B lymphocytes, its reservoir in vivo. The viral latent proteins are necessary for permanent B cell growth, but it is unknown whether they are sufficient. EBV was recently found to encode microRNAs (miRNAs) that are expressed in infected B cells and in some EBV-associated lymphomas. EBV miRNAs are grouped into two clusters located either adjacent to the BHRF1 gene or in introns contained within the viral BART transcripts. To understand the role of the BHRF1 miRNA cluster, we have constructed a virus mutant that lacks all its three members (Δ123) and a revertant virus. Here we show that the B cell transforming capacity of the Δ123 EBV mutant is reduced by more than 20-fold, relative to wild type or revertant viruses. B cells exposed to the knock-out virus displayed slower growth, and exhibited a two-fold reduction in the percentage of cells entering the cell cycle S phase. Furthermore, they displayed higher latent gene expression levels and latent protein production than their wild type counterparts. Therefore, the BHRF1 miRNAs accelerate B cell expansion at lower latent gene expression levels. Thus, this miRNA cluster simultaneously enhances expansion of the virus reservoir and reduces the viral antigenic load, two features that have the potential to facilitate persistence of the virus in the infected host. Thus, the EBV BHRF1 miRNAs may represent new therapeutic targets for the treatment of some EBV-associated lymphomas.

Infection of resting primary human B cells by Epstein-Barr virus (EBV) results in their transformation into indefinitely proliferating lymphoblastoid cell lines (LCLs). Epstein-Barr virus (EBV) is a human gammaherpesvirus that is able to establish a long-term, latent infection in human B cells for the life of the host (40). EBV infection is associated with a range of human cancers, including Burkitt's lymphoma (BL) and Hodgkin's disease, as well as several AIDS-associated cancers, of which the most prevalent is diffuse large B-cell lymphoma (DLBCL) (7). It has been proposed that EBV initially infects naïve B cells and then induces these to undergo a period of rapid proliferation leading eventually to differentiation into a pool of latently EBV-infected cells that resemble memory B cells (45). These two successive phases are referred to as EBV latency III and EBV latency I. During latency III, EBV expresses a set of nine proteins-the EBV nuclear antigen (EBNA) proteins EBNA-1, -2, -3A, -3B, -3C, and -LP and the latent membrane proteins (LMPs) LMP-1, -2A, and -2B-that are thought to mimic the ordered activation process that a naïve B cell would normally undergo after antigenic stimulation (22, 45). Eventually, these cells cycle back into a differentiation state that resembles that of a memory B cell, at which point EBV protein expression becomes restricted to EBNA-1.Previously published models for EBV latency proposed that the reprogramming of B cells after EBV infection largely reflected the action of viral proteins (22). These were thought to modify the pattern of cellular gene expression either directly, by acting as transcription factors, or indirectly, by mimicking the signals normally generated at the cell surface during antigenic stimulation. However, recent data have also revealed that EBV encodes no fewer than 25 distinct microRNAs (miRNAs) that have the potential to also play a critical role in the establishment and/or maintenance of EBV latency (5,19,36,56). Moreover, it is now clear that EBV infection greatly perturbs not only the pattern of cellular mRNA expression in infected B cells but also the pattern of cellular miRNA expression. In particular, EBV infection of resting primary B cells has been shown to strongly activate the expression of miR-155 and a small number of other cellular miRNAs (6,15,21,32).miRNAs are a class of ϳ22-nucleotide (nt)-long RNAs that function as negative posttranscriptional regulators of gene expression by binding to complementary sites located on target mRNAs (2). Particularly critical for efficient downregulation of mRNA expression is the miRNA seed region, nucleotides 2 to 8 from the 5Ј end of the miRNA, which generally must base pair to a fully complementary mRNA sequence. The human genome encodes Ͼ700 distinct miRNAs, and miRNAs are also encoded by a range of different herpesviruses, including EBV and the related gammaherpesvirus Kaposi's sarcoma-associated herpesvirus (KSHV) (47). While most viral miRNAs lack sequence homology to known cellular miRNAs, there is an ex...

Circulating microRNAs (miRNAs) have emerged as candidate biomarkers of various diseases and conditions including malignancy and pregnancy. This approach requires sensitive and accurate quantitation of miRNA concentrations in body fluids. Herein we report that enzyme-based miRNA quantitation, which is currently the mainstream approach for identifying differences in miRNA abundance among samples, is skewed by endogenous serum factors that co-purify with miRNAs and anticoagulant agents used during collection. Of importance, different miRNAs were affected to varying extent among patient samples. By developing measures to overcome these interfering activities, we increased the accuracy, and improved the sensitivity of miRNA detection up to 30-fold. Overall, the present study outlines key factors that prevent accurate miRNA quantitation in body fluids and provides approaches that enable faithful quantitation of miRNA abundance in body fluids.

Adverse posttraumatic neuropsychiatric sequelae (APNS) are common among civilian trauma survivors and military veterans. These APNS, as traditionally classified, include posttraumatic stress, post-concussion syndrome, depression, and regional or widespread pain. Traditional classifications have come to hamper scientific progress because they artificially fragment APNS into siloed, syndromic diagnoses unmoored to discrete components of brain functioning and studied in isolation. These limitations in classification and ontology slow the discovery of pathophysiologic mechanisms, biobehavioral markers, risk prediction tools, and preventive/ treatment interventions. Progress in overcoming these limitations has been challenging, because such progress would require studies that both evaluate a broad spectrum of posttraumatic sequelae (to overcome fragmentation) and also perform in-depth biobehavioral evaluation (to index sequelae to domains of brain function). This article summarizes the methods of the Advancing Understanding of RecOvery afteR traumA (AURORA) Study. AURORA conducts a large scale (n = 5,000 target sample) in-depth assessment of APNS development using a state-of-the-art battery of self-report, neurocognitive, physiologic, digital phenotyping, psychophysical, neuroimaging, and genomic assessments, beginning in the early aftermath of trauma and continuing for one year. The goals of AURORA are to achieve improved phenotypes, prediction tools, and understanding of molecular mechanisms to inform the future development and testing of preventive and treatment interventions.

Epstein-Barr virus (EBV) transforms B lymphocytes through the expression of the latent viral proteins EBNA and latent membrane protein (LMP).Recently, it has become apparent that microRNAs (miRNAs) also contribute to EBV's oncogenic properties; recombinant EBVs that lack the BHRF1 miRNA cluster display a reduced ability to transform B lymphocytes in vitro. Furthermore, infected cells evince a marked upregulation of the EBNA genes. Using recombinant viruses that lack only one member of the cluster, we now show that all three BHRF1 miRNAs contribute to B-cell transformation. Recombinants that lacked miR-BHRF1-2 or miR-BHRF1-3 displayed enhanced EBNA expression initiated at the Cp and Wp promoters. Interestingly, we find that the deletion of miR-BHRF1-2 reduced the expression level of miR-BHRF1-3 and possibly that of miR-BHRF1-1, demonstrating that the expression of one miRNA can potentiate the expression of other miRNAs located in the same cluster. Therefore, the phenotypic traits of the miR-BHRF1-2 null mutant could result partly from reduced miR-BHRF1-1 and miR-BHRF1-3 expression levels. Nevertheless, using an miR-BHRF1-1 and miR-BHRF1-3 double mutant, we could directly assess and confirm the contribution of miR-BHRF1-2 to B-cell transformation. Furthermore, we found that the potentiating effect of miR-BHRF1-2 on miR-BHRF1-3 synthesis can be reproduced with simple expression plasmids, provided that both miRNAs are processed from the same transcript. Therefore, this enhancing effect does not result from an idiosyncrasy of the EBV genome but rather reflects a general property of these miRNAs. This study highlights the advantages of arranging the BHRF1 miRNAs in clusters: it allows the synchronous and synergistic expression of genetic elements that cooperate to transform their target cells.

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Bacterial spore heat resistance is primarily dependent upon dehydration of the spore cytoplasm, a state that is maintained by the spore peptidoglycan wall, the spore cortex. A peptidoglycan structural modification found uniquely in spores is the formation of muramic ␦-lactam. Production of muramic ␦-lactam in Bacillus subtilis requires removal of a peptide side chain from the N-acetylmuramic acid residue by a cwlD-encoded muramoyl-L-Alanine amidase. Expression of cwlD takes place in both the mother cell and forespore compartments of sporulating cells, though expression is expected to be required only in the mother cell, from which cortex synthesis derives. Expression of cwlD in the forespore is in a bicistronic message with the upstream gene ybaK. We show that ybaK plays no apparent role in spore peptidoglycan synthesis and that expression of cwlD in the forespore plays no significant role in spore peptidoglycan formation. Peptide cleavage by CwlD is apparently followed by deacetylation of muramic acid and lactam ring formation. The product of pdaA (yfjS), which encodes a putative deacetylase, has recently been shown to also be required for muramic ␦-lactam formation. Expression of CwlD in Escherichia coli results in muramoyl L-Alanine amidase activity but no muramic ␦-lactam formation. Expression of PdaA alone in E. coli had no effect on E. coli peptidoglycan structure, whereas expression of CwlD and PdaA together resulted in the formation of muramic ␦-lactam. CwlD and PdaA are necessary and sufficient for muramic ␦-lactam production, and no other B. subtilis gene product is required. PdaA probably carries out both deacetylation and lactam ring formation and requires the product of CwlD activity as a substrate.Bacterial endospores can maintain a dormant, highly resistant state for long periods and then, under favorable conditions, rapidly germinate to produce vegetative cells. Spore dormancy and heat resistance are dependent on the relative dehydration of the spore core (6, 24, 28). Spore core dehydration requires the integrity of a thick spore peptidoglycan wall, and peptidoglycan hydrolysis is required for rehydration and resumption of spore core metabolism during spore germination (27,31).The spore peptidoglycan is composed of two contiguous layers that are synthesized between the two membranes surrounding the developing forespore. The inner layer, the germ cell wall, makes up only 10 to 15% of the total spore peptidoglycan (23) and is apparently synthesized by proteins expressed on the surface of the inner forespore membrane (22, 35). The germ cell wall has a structure resembling the peptidoglycan of the vegetative cell wall (23), is maintained during spore germination to serve as the initial wall of the outgrowing spore (5), and appears to function as a template for proper synthesis of the outer spore peptidoglycan layers, the cortex (22). The cortex makes up Ͼ80% of the spore peptidoglycan (23), is synthesized by proteins present on the surface of the outer forespore membrane (10, 35), is rapidly degraded...