Society for endocrinology guideline for understanding, diagnosing and treating female hypogonadism.

Female hypogonadism (FH) is a relatively common endocrine disorder in women of premenopausal age, but there are significant uncertainties and wide variation in its management. Most current guidelines are monospecialty and only address premature ovarian insufficiency (POI); some allude to management in very brief and general terms, and most rely upon the extrapolation of evidence from the studies relating to physiological estrogen deficiency in postmenopausal women. The Society for Endocrinology commissioned new guidance to provide all care providers with a multidisciplinary perspective on managing patients with all forms of FH. It has been compiled using expertise from Endocrinology, Primary Care, Gynaecology and Reproductive Health practices, with contributions from expert patients and a patient support group, to help clinicians best manage FH resulting from both POI and hypothalamo-pituitary disorders, whether organic or functional.

PADLOC: a web server for the identification of antiviral defence systems in microbial genomes.

Most bacteria and archaea possess multiple antiviral defence systems that protect against infection by phages, archaeal viruses and mobile genetic elements. Our understanding of the diversity of defence systems has increased greatly in the last few years, and many more systems likely await discovery. To identify defence-related genes, we recently developed the Prokaryotic Antiviral Defence LOCator (PADLOC) bioinformatics tool. To increase the accessibility of PADLOC, we describe here the PADLOC web server (freely available at https://padloc.otago.ac.nz), allowing users to analyse whole genomes, metagenomic contigs, plasmids, phages and archaeal viruses. The web server includes a more than 5-fold increase in defence system types detected (since the first release) and expanded functionality enabling detection of CRISPR arrays and retron ncRNAs. Here, we provide user information such as input options, description of the multiple outputs, limitations and considerations for interpretation of the results, and guidance for subsequent analyses. The PADLOC web server also houses a precomputed database of the defence systems in > 230,000 RefSeq genomes. These data reveal two taxa, Campylobacterota and Spriochaetota, with unusual defence system diversity and abundance. Overall, the PADLOC web server provides a convenient and accessible resource for the detection of antiviral defence systems.

Transcription factors GAF and HSF act at distinct regulatory steps to modulate stress-induced gene activation.

The coordinated regulation of gene expression at the transcriptional level is fundamental to development and homeostasis. Inducible systems are invaluable when studying transcription because the regulatory process can be triggered instantaneously, allowing the tracking of ordered mechanistic events. Here, we use precision run-on sequencing (PRO-seq) to examine the genome-wide heat shock (HS) response in Drosophila and the function of two key transcription factors on the immediate transcription activation or repression of all genes regulated by HS. We identify the primary HS response genes and the rate-limiting steps in the transcription cycle that GAGA-associated factor (GAF) and HS factor (HSF) regulate. We demonstrate that GAF acts upstream of promoter-proximally paused RNA polymerase II (Pol II) formation (likely at the step of chromatin opening) and that GAF-facilitated Pol II pausing is critical for HS activation. In contrast, HSF is dispensable for establishing or maintaining Pol II pausing but is critical for the release of paused Pol II into the gene body at a subset of highly activated genes. Additionally, HSF has no detectable role in the rapid HS repression of thousands of genes.

Identification and classification of antiviral defence systems in bacteria and archaea with PADLOC reveals new system types.

To provide protection against viral infection and limit the uptake of mobile genetic elements, bacteria and archaea have evolved many diverse defence systems. The discovery and application of CRISPR-Cas adaptive immune systems has spurred recent interest in the identification and classification of new types of defence systems. Many new defence systems have recently been reported but there is a lack of accessible tools available to identify homologs of these systems in different genomes. Here, we report the Prokaryotic Antiviral Defence LOCator (PADLOC), a flexible and scalable open-source tool for defence system identification. With PADLOC, defence system genes are identified using HMM-based homologue searches, followed by validation of system completeness using gene presence/absence and synteny criteria specified by customisable system classifications. We show that PADLOC identifies defence systems with high accuracy and sensitivity. Our modular approach to organising the HMMs and system classifications allows additional defence systems to be easily integrated into the PADLOC database. To demonstrate application of PADLOC to biological questions, we used PADLOC to identify six new subtypes of known defence systems and a putative novel defence system comprised of a helicase, methylase and ATPase. PADLOC is available as a standalone package (https://github.com/padlocbio/padloc) and as a webserver (https://padloc.otago.ac.nz).

An AR-ERG transcriptional signature defined by long-range chromatin interactomes in prostate cancer cells.

The aberrant activities of transcription factors such as the androgen receptor (AR) underpin prostate cancer development. While the AR cis-regulation has been extensively studied in prostate cancer, information pertaining to the spatial architecture of the AR transcriptional circuitry remains limited. In this paper, we propose a novel framework to profile long-range chromatin interactions associated with AR and its collaborative transcription factor, erythroblast transformation-specific related gene (ERG), using chromatin interaction analysis by paired-end tag (ChIA-PET). We identified ERG-associated long-range chromatin interactions as a cooperative component in the AR-associated chromatin interactome, acting in concert to achieve coordinated regulation of a subset of AR target genes. Through multifaceted functional data analysis, we found that AR-ERG interaction hub regions are characterized by distinct functional signatures, including bidirectional transcription and cotranscription factor binding. In addition, cancer-associated long noncoding RNAs were found to be connected near protein-coding genes through AR-ERG looping. Finally, we found strong enrichment of prostate cancer genome-wide association study (GWAS) single nucleotide polymorphisms (SNPs) at AR-ERG co-binding sites participating in chromatin interactions and gene regulation, suggesting GWAS target genes identified from chromatin looping data provide more biologically relevant findings than using the nearest gene approach. Taken together, our results revealed an AR-ERG-centric higher-order chromatin structure that drives coordinated gene expression in prostate cancer progression and the identification of potential target genes for therapeutic intervention.

Lurking in the water: testing eDNA metabarcoding as a tool for ecosystem-wide parasite detection.

In the light of global biodiversity change and emerging disease, there is an urgent need to establish efficient monitoring programmes of parasites in aquatic ecosystems. However, parasite identification is time-consuming, requires a high degree of taxonomic expertize and in general requires lethal sampling. The use of environmental DNA methodology to identify parasites has the potential to circumvent these limitations. This study evaluates the use of eDNA metabarcoding to detect the presence of all species of nematode and platyhelminth parasites in two New Zealand lakes. We developed two novel metabarcoding primer pairs targeting a region of cytochrome oxidase I gene (COI) specific to platyhelminths and nematodes. We successfully detected parasite DNA in both lakes. Platyhelminth DNA yield was in general greater than nematode DNA yield. This most likely results from the larger biomass of the former quantified using traditional methods, or the presence of free-swimming life stages in the life cycle of many platyhelminths. By using eDNA, we did not detect all expected parasite families revealed through traditional methods, likely due to a lack of sequencing data available from public databases such as GenBank. As such, genetic resources need to include full reference sequences if parasitology is to truly harness eDNA to characterize and monitor parasite biodiversity in natural systems.

The people vs science: can passively crowdsourced internet data shed light on host-parasite interactions?

Every internet search query made out of curiosity by anyone who observed something in nature, as well as every photo uploaded to the internet, constitutes a data point of potential use to scientists. Researchers have now begun to exploit the vast online data accumulated through passive crowdsourcing for studies in ecology and epidemiology. Here, we demonstrate the usefulness of iParasitology, i.e. the use of internet data for tests of parasitological hypotheses, using hairworms (phylum Nematomorpha) as examples. These large worms are easily noticeable by people in general, and thus likely to generate interest on the internet. First, we show that internet search queries (collated with Google Trends) and photos uploaded to the internet (specifically, to the iNaturalist platform) point to parts of North America with many sightings of hairworms by the public, but few to no records in the scientific literature. Second, we demonstrate that internet searches predict seasonal peaks in hairworm abundance that accurately match scientific data. Finally, photos uploaded to the internet by non-scientists can provide reliable data on the host taxa that hairworms most frequently parasitize, and also identify hosts that appear to have been neglected by scientific studies. Our findings suggest that for any parasite group likely to be noticeable by non-scientists, information accumulating through internet search activity, photo uploads, social media or any other format available online, represents a valuable source of data that can complement traditional scientific data sources in parasitology.

Extensive polymerase pausing during Drosophila axis patterning enables high-level and pliable transcription.

Cascades of zygotic gene expression pattern the anterior-posterior (AP) and dorsal-ventral (DV) axes of the early Drosophila embryo. Here, we used the global run-on sequencing assay (GRO-seq) to map the genome-wide RNA polymerase distribution during early Drosophila embryogenesis, thus providing insights into how genes are regulated. We identify widespread promoter-proximal pausing yet show that the presence of paused polymerase does not necessarily equate to direct regulation through pause release to productive elongation. Our data reveal that a subset of early Zelda-activated genes is regulated at the level of polymerase recruitment, whereas other Zelda target and axis patterning genes are predominantly regulated through pause release. In contrast to other signaling pathways, we found that bone morphogenetic protein (BMP) target genes are collectively more highly paused than BMP pathway components and show that BMP target gene expression requires the pause-inducing negative elongation factor (NELF) complex. Our data also suggest that polymerase pausing allows plasticity in gene activation throughout embryogenesis, as transiently repressed and transcriptionally silenced genes maintain and lose promoter polymerases, respectively. Finally, we provide evidence that the major effect of pausing is on the levels, rather than timing, of transcription. These data are discussed in terms of the efficiency of transcriptional activation required across cell populations during developmental time constraints.

The polycomb group mutant esc leads to augmented levels of paused Pol II in the Drosophila embryo.

Many developmental control genes contain paused RNA polymerase II (Pol II) and are thereby "poised" for rapid and synchronous activation in the early Drosophila embryo. Evidence is presented that Polycomb group (PcG) repressors can influence paused Pol II. ChIP-Seq and GRO-Seq assays were used to determine the genome-wide distributions of Pol II, H3K27me3, and H3K4me3 in extra sex combs (esc) mutant embryos. ESC is a key component of the Polycomb repressive complex 2 (PRC2), which mediates H3K27me3 modification. Enhanced Pol II occupancy is observed for thousands of genes in esc mutant embryos, including genes not directly regulated by PRC2. Thus, it would appear that silent genes lacking promoter-associated paused Pol II in wild-type embryos are converted into "poised" genes with paused Pol II in esc mutants. We suggest that this conversion of silent genes into poised genes might render differentiated cell types susceptible to switches in identity in PcG mutants.

Regulating RNA polymerase pausing and transcription elongation in embryonic stem cells.

Transitions between pluripotent stem cells and differentiated cells are executed by key transcription regulators. Comparative measurements of RNA polymerase distribution over the genome's primary transcription units in different cell states can identify the genes and steps in the transcription cycle that are regulated during such transitions. To identify the complete transcriptional profiles of RNA polymerases with high sensitivity and resolution, as well as the critical regulated steps upon which regulatory factors act, we used genome-wide nuclear run-on (GRO-seq) to map the density and orientation of transcriptionally engaged RNA polymerases in mouse embryonic stem cells (ESCs) and mouse embryonic fibroblasts (MEFs). In both cell types, progression of a promoter-proximal, paused RNA polymerase II (Pol II) into productive elongation is a rate-limiting step in transcription of ∼40% of mRNA-encoding genes. Importantly, quantitative comparisons between cell types reveal that transcription is controlled frequently at paused Pol II's entry into elongation. Furthermore, "bivalent" ESC genes (exhibiting both active and repressive histone modifications) bound by Polycomb group complexes PRC1 (Polycomb-repressive complex 1) and PRC2 show dramatically reduced levels of paused Pol II at promoters relative to an average gene. In contrast, bivalent promoters bound by only PRC2 allow Pol II pausing, but it is confined to extremely 5' proximal regions. Altogether, these findings identify rate-limiting targets for transcription regulation during cell differentiation.

Identification of active transcriptional regulatory elements from GRO-seq data.

Modifications to the global run-on and sequencing (GRO-seq) protocol that enrich for 5'-capped RNAs can be used to reveal active transcriptional regulatory elements (TREs) with high accuracy. Here, we introduce discriminative regulatory-element detection from GRO-seq (dREG), a sensitive machine learning method that uses support vector regression to identify active TREs from GRO-seq data without requiring cap-based enrichment (https://github.com/Danko-Lab/dREG/). This approach allows TREs to be assayed together with gene expression levels and other transcriptional features in a single experiment. Predicted TREs are more enriched for several marks of transcriptional activation­including expression quantitative trait loci, disease-associated polymorphisms, acetylated histone 3 lysine 27 (H3K27ac) and transcription factor binding­than those identified by alternative functional assays. Using dREG, we surveyed TREs in eight human cell types and provide new insights into global patterns of TRE function.

X chromosome dosage compensation via enhanced transcriptional elongation in Drosophila.

The evolution of sex chromosomes has resulted in numerous species in which females inherit two X chromosomes but males have a single X, thus requiring dosage compensation. MSL (Male-specific lethal) complex increases transcription on the single X chromosome of Drosophila males to equalize expression of X-linked genes between the sexes. The biochemical mechanisms used for dosage compensation must function over a wide dynamic range of transcription levels and differential expression patterns. It has been proposed that the MSL complex regulates transcriptional elongation to control dosage compensation, a model subsequently supported by mapping of the MSL complex and MSL-dependent histone 4 lysine 16 acetylation to the bodies of X-linked genes in males, with a bias towards 3' ends. However, experimental analysis of MSL function at the mechanistic level has been challenging owing to the small magnitude of the chromosome-wide effect and the lack of an in vitro system for biochemical analysis. Here we use global run-on sequencing (GRO-seq) to examine the specific effect of the MSL complex on RNA Polymerase II (RNAP II) on a genome-wide level. Results indicate that the MSL complex enhances transcription by facilitating the progression of RNAP II across the bodies of active X-linked genes. Improving transcriptional output downstream of typical gene-specific controls may explain how dosage compensation can be imposed on the diverse set of genes along an entire chromosome.

Paused Pol II captures enhancer activity and acts as a potent insulator.

Enhancers act over many kilobase pairs to activate target promoters, but their activity is constrained by insulator elements that prevent indiscriminate activation of nearby genes. In the July 1, 2009, issue of Genes & Development, Chopra and colleagues (pp. 1505-1509) report that promoters containing a stalled Pol II are activated by enhancers, but these promoters also serve as insulators that block enhancers from reaching more distal genes. This new class of insulators provide critical clues to regulatory mechanisms.

Double-balloon enteroscopy in Crohn's disease: findings and impact on management in a multicenter retrospective study.

BACKGROUND: Double-balloon enteroscopy (DBE) is effective in visualizing the small bowel to perform biopsy sampling and interventions. Few studies have evaluated the utility of DBE in patients with known or suspected Crohn's disease (CD). OBJECTIVE: To evaluate the use of DBE in the diagnosis and impact on patient management in known and suspected CD and to compare capsule endoscopy (CE) with DBE findings. DESIGN: Retrospective study from August 2004 to August 2009 of DBE procedures. SETTING: Five academic, tertiary U.S. centers. PATIENTS: Patients with known or suspected CD. MAIN OUTCOME MEASURES: Diagnostic yield, impact on patient management, and comparison of DBE to CE findings in patients with known and suspected CD. RESULTS: We analyzed 98 DBE procedures performed in 81 patients (38 with known CD and 43 with suspected CD). For patients with CD, common indications were abdominal pain and bleeding/anemia. The diagnostic yield was 87% (33/38 patients). The impact on subsequent management decisions was 82% (31/38). Common indications for DBE in patients with suspected CD were abnormal CE or other imaging. The diagnostic yield was 79% (34/43 patients). The impact on subsequent management decisions was 77% (33/43). In 17% of patients (14/81), DBE failed to reach the target lesion. There was 1 perforation, 3 strictures dilated, and 1 of 2 retained capsules recovered. When CE was followed by DBE, 46% of lesions were confirmed on DBE. LIMITATIONS: Retrospective analysis, imperfect criterion standard. CONCLUSIONS: DBE is an effective technique for assessment of the small bowel in known and suspected CD and affects management. Failure to reach target areas with DBE is not uncommon, and perforations can occur. There is poor correlation between CE and DBE.

Single administration of a psychedelic [(R)-DOI] influences coping strategies to an escapable social stress.

Psychedelic compounds have potentially rapid, long-lasting anxiolytic, antidepressive and anti-inflammatory effects. We investigated whether the psychedelic compound (R)-2,5-dimethoxy-4-iodoamphetamine [(R)-DOI], a selective 5-HT2A receptor partial agonist, decreases stress-related behavior in male mice exposed to repeated social aggression. Additionally, we explored the likelihood that these behavioral changes are related to anti-inflammatory properties of [(R)-DOI]. Animals were subjected to the Stress Alternatives Model (SAM), an escapable social stress paradigm in which animals develop reactive coping strategies - remaining in the SAM arena (Stay) with a social aggressor, or dynamically initiated stress coping strategies that involve utilizing the escape holes (Escape) to avoid aggression. Mice expressing these behavioral phenotypes display behaviors like those in other social aggression models that separate animals into stress-vulnerable (as for Stay) or stress-resilient (as for Escape) groups, which have been shown to have distinct inflammatory responses to social stress. These results show that Stay animals have heightened cytokine gene expression, and both Stay and Escape mice exhibit plasma and neural concentrations of the inflammatory cytokine tumor necrosis factor-α (TNFα) compared to unstressed control mice. Additionally, these results suggest that a single administration of (R)-DOI to Stay animals in low doses, can increase stress coping strategies such as increasing attention to the escape route, promoting escape behavior, and reducing freezing during socially aggressive interaction in the SAM. Lower single doses of (R)-DOI, in addition to shifting behavior to suggest anxiolytic effects, also concomitantly reduce plasma and limbic brain levels of the inflammatory cytokine TNFα.

Qualification of cardiac troponins for nonclinical use: a regulatory perspective.

The US Food and Drug Administration (FDA) Biomarker Qualification Review Team presents its perspective on the recent qualification of cardiac troponins for use in nonclinical safety assessment studies. The goal of this manuscript is to provide greater transparency into the qualification process and factors that were considered in reaching a regulatory decision. This manuscript includes an overview of the data that were submitted and a discussion of the strengths and shortcomings of these data supporting the qualification decision. The cardiac troponin submission is the first literature-based biomarker application to be reviewed by the FDA and insights gained from this experience may aid future submissions and help streamline the characterization and qualification of future biomarkers.

Bacillus subtilis RapA phosphatase domain interaction with its substrate, phosphorylated Spo0F, and its inhibitor, the PhrA peptide.

Rap proteins in Bacillus subtilis regulate the phosphorylation level or the DNA-binding activity of response regulators such as Spo0F, involved in sporulation initiation, or ComA, regulating competence development. Rap proteins can be inhibited by specific peptides generated by the export-import processing pathway of the Phr proteins. Rap proteins have a modular organization comprising an amino-terminal alpha-helical domain connected to a domain formed by six tetratricopeptide repeats (TPR). In this study, the molecular basis for the specificity of the RapA phosphatase for its substrate, phosphorylated Spo0F (Spo0F∼P), and its inhibitor pentapeptide, PhrA, was analyzed in part by generating chimeric proteins with RapC, which targets the DNA-binding domain of ComA, rather than Spo0F∼P, and is inhibited by the PhrC pentapeptide. In vivo analysis of sporulation efficiency or competence-induced gene expression, as well as in vitro biochemical assays, allowed the identification of the amino-terminal 60 amino acids as sufficient to determine Rap specificity for its substrate and the central TPR3 to TPR5 (TPR3-5) repeats as providing binding specificity toward the Phr peptide inhibitor. The results allowed the prediction and testing of key residues in RapA that are essential for PhrA binding and specificity, thus demonstrating how the widespread structural fold of the TPR is highly versatile, using a common interaction mechanism for a variety of functions in eukaryotic and prokaryotic organisms.

Early Prophylactic Gastrectomy for the Management of Gastric Adenomatous Proximal Polyposis Syndrome (GAPPS).

Background: Gastric adenomatous proximal polyposis syndrome (GAPPS) is a recently described, rare, autosomal dominant condition characterized by the extensive involvement of the proximal stomach with hundreds of heterogeneous fundic gland polyps with antral and duodenal sparing. GAPPS is caused by a point mutation of the APC gene promoter 1B and is associated with a risk of malignant transformation, distant metastasis, and death. There are no surveillance, screening, or treatment guidelines for managing GAPPS. The few reported cases have been variably managed with endoscopic surveillance or prophylactic gastrectomy. However, there is no consensus on the optimal management approach. Summary: In this case series, we review the relevant literature on GAPPS and present two siblings who underwent early prophylactic total gastrectomies with good outcomes. Conclusion: Due to the poor correlation between the endoscopic findings on sampled polyps and the risk of harboring invasive gastric cancer, patients with GAPPS should be strongly considered for early prophylactic total gastrectomies in the absence of prohibitive comorbidities.

Design considerations for piezoelectrically powered electrical stimulation: The balance between power generation and fatigue resistance.

Quality and timing of bone healing from orthopedic surgeries, especially lumbar spinal fusion procedures, is problematic for many patients. To address this issue, clinicians often use electrical stimulation to improve surgery success rates and decrease healing time in patients with increased risk of pseudarthrosis, including smokers and diabetics. Current invasive electrical stimulation devices require an implantable battery and a second surgery for removal. Piezoelectric composites within an interbody implant generate sufficient power under physiologic loads to deliver pulsed electrical stimulation without a battery and have demonstrated promising preclinical bone growth and fusion success. The objective of the current study was to assess the power generation and fatigue resistance of three commercially manufactured piezocomposite configurations in a modified implant design to demonstrate efficacy as a robust biomaterial within osteogenic implants. The three configurations were electromechanically assessed under physiological lumbar loading conditions, and all configurations produced sufficient power to promote bone healing. Additionally, electrical and mechanical fatigue performance was assessed under high load, low cycle conditions. All configurations demonstrated runout with no gross mechanical failure and two configurations demonstrated electrical fatigue resistance. Future piezoelectric implant design decisions should be based on power generation needs to stimulate bone growth, as mechanical fatigue efficacy was proven for all piezocomposite configurations tested.