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Regulation of gene expression is integral to the development and survival of all organisms. Transcription begins with the assembly of a pre-initiation complex at the gene promoter 1 , followed by initiation of RNA synthesis and the transition to productive elongation 2 , 3 , 4 . In many cases, recruitment of RNA polymerase II (Pol II) to a promoter is necessary and sufficient for activation of genes. However, there are a few notable exceptions to this paradigm, including heat shock genes and several proto-oncogenes, whose expression is attenuated by regulated stalling of polymerase elongation within the promoter-proximal region 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 . To determine the importance of polymerase stalling for transcription regulation, we carried out a genome-wide search for Drosophila melanogaster genes with Pol II stalled within the promoter-proximal region. Our data show that stalling is widespread, occurring at hundreds of genes that respond to stimuli and developmental signals. This finding indicates a role for regulation of polymerase elongation in the transcriptional responses to dynamic environmental and developmental cues.

The kinetics and magnitude of cytokine gene expression are tightly regulated to elicit a balanced response to pathogens and result from integrated changes in transcription and mRNA stability. Yet, how a single microbial stimulus induces peak transcription of some genes (TNFα) within minutes whereas others (IP-10) require hours remains unclear. Here, we dissect activation of several lipopolysaccharide (LPS)-inducible genes in macrophages, an essential cell type mediating inflammatory response in mammals. We show that a key difference between the genes is the step of the transcription cycle at which they are regulated. Specifically, at TNFα, RNA Polymerase II initiates transcription in resting macrophages, but stalls near the promoter until LPS triggers rapid and transient release of the negative elongation factor (NELF) complex and productive elongation. In contrast, no NELF or polymerase is detectible near the IP-10 promoter before induction, and LPS-dependent polymerase recruitment is rate limiting for transcription. We further demonstrate that this strategy is shared by other immune mediators and is independent of the inducer and signaling pathway responsible for gene activation. Finally, as a striking example of evolutionary conservation, the Drosophila homolog of the TNFα gene, eiger, displayed all of the hallmarks of NELF-dependent polymerase stalling. We propose that polymerase stalling ensures the coordinated, timely activation the inflammatory gene expression program from Drosophila to mammals.

Antisense transcription is a prevalent feature at mammalian promoters. Previous studies have primarily focused on antisense transcription initiating upstream of genes. Here, we characterize promoter-proximal antisense transcription downstream of gene transcription starts sites in human breast cancer cells, investigating the genomic context of downstream antisense transcription. We find extensive correlations between antisense transcription and features associated with the chromatin environment at gene promoters. Antisense transcription downstream of promoters is widespread, with antisense transcription initiation observed within 2 kb of 28% of gene transcription start sites. Antisense transcription initiates between nucleosomes regularly positioned downstream of these promoters. The nucleosomes between gene and downstream antisense transcription start sites carry histone modifications associated with active promoters, such as H3K4me3 and H3K27ac. This region is bound by chromatin remodeling and histone modifying complexes including SWI/SNF subunits and HDACs, suggesting that antisense transcription or resulting RNA transcripts contribute to the creation and maintenance of a promoter-associated chromatin environment. Downstream antisense transcription overlays additional regulatory features, such as transcription factor binding, DNA accessibility, and the downstream edge of promoter-associated CpG islands. These features suggest an important role for antisense transcription in the regulation of gene expression and the maintenance of a promoter-associated chromatin environment.

Organogenesis requires the complex interactions of multiple cell lineages that coordinate their expansion, differentiation, and maturation over time. Here, we profile the cell types within the epithelial and mesenchymal compartments of the murine pancreas across developmental time using a combination of single-cell RNA sequencing, immunofluorescence, in situ hybridization, and genetic lineage tracing. We identify previously underappreciated cellular heterogeneity of the developing mesenchyme and reconstruct potential lineage relationships among the pancreatic mesothelium and mesenchymal cell types. Within the epithelium, we find a previously undescribed endocrine progenitor population, as well as an analogous population in both human fetal tissue and human embryonic stem cells differentiating toward a pancreatic beta cell fate. Further, we identify candidate transcriptional regulators along the differentiation trajectory of this population toward the alpha or beta cell lineages. This work establishes a roadmap of pancreatic development and demonstrates the broad utility of this approach for understanding lineage dynamics in developing organs. Coordinated proliferation and differentiation of diverse cell populations drive pancreatic epithelial and mesenchymal development. Here, the authors profile cell type dynamics in the developing mouse pancreas using single-cell RNA sequencing, identifying mesenchymal subtypes and undescribed endocrine progenitors.

The western clawed frog Xenopus tropicalis is an important model for vertebrate development that combines experimental advantages of the African clawed frog Xenopus laevis with more tractable genetics. Here we present a draft genome sequence assembly of X. tropicalis. This genome encodes more than 20,000 protein-coding genes, including orthologs of at least 1700 human disease genes. Over 1 million expressed sequence tags validated the annotation. More than one-third of the genome consists of transposable elements, with unusually prevalent DNA transposons. Like that of other tetrapods, the genome of X. tropicalis contains gene deserts enriched for conserved noncoding elements. The genome exhibits substantial shared synteny with human and chicken over major parts of large chromosomes, broken by lineage-specific chromosome fusions and fissions, mainly in the mammalian lineage.

Chemical investigation of an undescribed Australian fungus, Aspergillus nanangensis , led to the identification of the nanangenines – a family of seven new and three previously reported drimane sesquiterpenoids. The structures of the nanangenines were elucidated by detailed spectroscopic analysis supported by single crystal X-ray diffraction studies. The compounds were assayed for in vitro activity against bacteria, fungi, mammalian cells and plants. Bioinformatics analysis, including comparative analysis with other acyl drimenol-producing Aspergilli, led to the identification of a putative nanangenine biosynthetic gene cluster that corresponds to the proposed biosynthetic pathway for nanangenines.

Appendectomy for presumed appendicitis is the most common surgical emergency during pregnancy. Delayed diagnosis and treatment of appendicitis carries risk for the fetus and mother. We sought to evaluate the accuracy of MRI in pregnant patients with suspected appendicitis. All pregnant patients with suspected appendicitis between January 2014 and April 2016 were included. MRI reports were categorized into positive, negative, and inconclusive groups. Diagnosis of appendicitis was based on pathology report. Fifty-two patients were included in the study. The MRI was positive in two, negative in 29, and inconclusive in 21 patients. Twelve patients had surgery, 11 of which had positive appendicitis on pathology. Both positive MRI patients had appendicitis. In the negative MRI group, 3 of 29 (10%) had appendicitis. In the inconclusive MRI group, 6 of 21 (29%) had appendicitis. A positive MRI result was very specific with a 100 per cent positive predictive value; however, the sensitivity was as low as 18 per cent (diagnosed only 2 of 11 cases). Although a positive MRI finding was reliable in making a decision to operate, a negative or inconclusive MRI was not. In patients with a high clinical suspicion of appendicitis, surgery should still be considered even without definitive positive MRI findings.

Novel and rare fungal species continue to serve as an invaluable source of new natural products. In this study, we conducted a comprehensive metabologenomic assessment of the endemic Australian fungus Aspergillus luteorubrus MST-FP2246. Genome sequencing revealed that A. luteorubrus harbours a rich biosynthetic potential, including 42 biosynthetic gene clusters (BGCs) involved in secondary metabolite biosynthesis. Extensive chemical profiling of A. luteorubrus led to the identification of ten metabolites, including one novel compound, luteolactone A (1), and nine previously reported metabolites, dimethoxyphthalide (2), marilone B (3), (+)-dihydrocanadensolide (4), ascosteroside C (5), ascosteroside D (6), viridicatumtoxin A (7), aszonalenin (8), 6-hydroxyaszonalenin (9), and the recently described polyketide glycoside, luteodienoside A (10). By integrating prior knowledge with BGC analysis, eight of these metabolites were mapped to their corresponding BGCs, while the remaining BGCs represent an opportunity for novel metabolite discovery. Together, these findings underscore the rich biosynthetic capacity of A. luteorubrus and establish it as a promising target for future genome mining and novel secondary metabolites discovery.

Background. An estimated 1 million children die each year before their fifth birthday from diarrhea. Previous population-based surveys of pediatric diarrheal diseases have identified the protozoan parasite Entamoeba histolytica, the etiological agent of amebiasis, as one of the causes of moderate-to-severe diarrhea in sub-Saharan Africa and South Asia. Methods. We prospectively studied the natural history of E. histolytica colonization and diarrhea among infants in an urban slum of Dhaka, Bangladesh. Results. Approximately 80% of children were infected with E. histolytica by the age of 2 years. Fecal anti-galactose/N-acetylgalactosamine lectin immunoglobulin A was associated with protection from reinfection, while a high parasite burden and expansion of the Prevotella copri level was associated with diarrhea. Conclusions. E. histolytica infection was prevalent in this population, with most infections asymptomatic and diarrhea associated with both the amount of parasite and the composition of the microbiota.

ObjectiveThe primary objective is to develop an automated method for detecting patients that are ready for discharge from intensive care.DesignWe used two datasets of routinely collected patient data to test and improve on a set of previously proposed discharge criteria.SettingBristol Royal Infirmary general intensive care unit (GICU).PatientsTwo cohorts derived from historical datasets: 1870 intensive care patients from GICU in Bristol, and 7592 from Medical Information Mart for Intensive Care (MIMIC)-III.ResultsIn both cohorts few successfully discharged patients met all of the discharge criteria. Both a random forest and a logistic classifier, trained using multiple-source cross-validation, demonstrated improved performance over the original criteria and generalised well between the cohorts. The classifiers showed good agreement on which features were most predictive of readiness-for-discharge, and these were generally consistent with clinical experience. By weighting the discharge criteria according to feature importance from the logistic model we showed improved performance over the original criteria, while retaining good interpretability.ConclusionsOur findings indicate the feasibility of the proposed approach to ready-for-discharge classification, which could complement other risk models of specific adverse outcomes in a future decision support system. Avenues for improvement to produce a clinically useful tool are identified.