Acinetobacter baumannii Regulates Its Stress Responses via the BfmRS Two-Component Regulatory System

Acinetobacter baumannii is a common nosocomial pathogen that utilizes numerous mechanisms to aid its survival in both the environment and the host. Coordination of such mechanisms requires an intricate regulatory network. We report here that A. baumannii can directly regulate several stress-related pathways via the two-component regulatory system BfmRS. Similar to previous studies, results from transcriptomic analysis showed that mutation of the BfmR response regulator causes dysregulation of genes required for the oxidative stress response, the osmotic stress response, the misfolded protein/heat shock response, Csu pilus/fimbria production, and capsular polysaccharide biosynthesis. We also found that the BfmRS system is involved in controlling siderophore biosynthesis and transport, and type IV pili production. We provide evidence that BfmR binds to various stress-related promoter regions and show that BfmR alone can directly activate transcription of some stress-related genes. Additionally, we show that the BfmS sensor kinase acts as a BfmR phosphatase to negatively regulate BfmR activity. This work highlights the importance of the BfmRS system in promoting survival of A. baumannii. IMPORTANCE Acinetobacter baumannii is a nosocomial pathogen that has extremely high rates of multidrug resistance. This organism’s ability to endure stressful conditions is a key part of its ability to spread in the hospital environment and cause infections. Unlike other members of the gammaproteobacteria, A. baumannii does not encode a homolog of the RpoS sigma factor to coordinate its stress response. Here, we demonstrate that the BfmRS two-component system directly controls the expression of multiple stress resistance genes. Our findings suggest that BfmRS is central to a unique scheme of general stress response regulation by A. baumannii

Genetic perturbation of PU.1 binding and chromatin looping at neutrophil enhancers associates with autoimmune disease.

Neutrophils play fundamental roles in innate immune response, shape adaptive immunity, and are a potentially causal cell type underpinning genetic associations with immune system traits and diseases. Here, we profile the binding of myeloid master regulator PU.1 in primary neutrophils across nearly a hundred volunteers. We show that variants associated with differential PU.1 binding underlie genetically-driven differences in cell count and susceptibility to autoimmune and inflammatory diseases. We integrate these results with other multi-individual genomic readouts, revealing coordinated effects of PU.1 binding variants on the local chromatin state, enhancer-promoter contacts and downstream gene expression, and providing a functional interpretation for 27 genes underlying immune traits. Collectively, these results demonstrate the functional role of PU.1 and its target enhancers in neutrophil transcriptional control and immune disease susceptibility

Germline mutations in the transcription factor IKZF5 cause thrombocytopenia.

To identify novel causes of hereditary thrombocytopenia, we performed a genetic association analysis of whole-genome sequencing data from 13 037 individuals enrolled in the National Institute for Health Research (NIHR) BioResource, including 233 cases with isolated thrombocytopenia. We found an association between rare variants in the transcription factor-encoding gene IKZF5 and thrombocytopenia. We report 5 causal missense variants in or near IKZF5 zinc fingers, of which 2 occurred de novo and 3 co-segregated in 3 pedigrees. A canonical DNA-zinc finger binding model predicts that 3 of the variants alter DNA recognition. Expression studies showed that chromatin binding was disrupted in mutant compared with wild-type IKZF5, and electron microscopy revealed a reduced quantity of α granules in normally sized platelets. Proplatelet formation was reduced in megakaryocytes from 7 cases relative to 6 controls. Comparison of RNA-sequencing data from platelets, monocytes, neutrophils, and CD4+ T cells from 3 cases and 14 healthy controls showed 1194 differentially expressed genes in platelets but only 4 differentially expressed genes in each of the other blood cell types. In conclusion, IKZF5 is a novel transcriptional regulator of megakaryopoiesis and the eighth transcription factor associated with dominant thrombocytopenia in humans

Transcriptional characterization of human megakaryocyte polyploidization and lineage commitment

Funder: National Institute for Health Research; Id: http://dx.doi.org/10.13039/501100014338Funder: NHS Blood and Transplant; Id: http://dx.doi.org/10.13039/100009033Funder: Bristol‐Myers Squibb; Id: http://dx.doi.org/10.13039/100002491Funder: European Commission; Id: http://dx.doi.org/10.13039/100013273Abstract: Background: Megakaryocytes (MKs) originate from cells immuno‐phenotypically indistinguishable from hematopoietic stem cells (HSCs), bypassing intermediate progenitors. They mature within the adult bone marrow and release platelets into the circulation. Until now, there have been no transcriptional studies of primary human bone marrow MKs. Objectives: To characterize MKs and HSCs from human bone marrow using single‐cell RNA sequencing, to investigate MK lineage commitment, maturation steps, and thrombopoiesis. Results: We show that MKs at different levels of polyploidization exhibit distinct transcriptional states. Although high levels of platelet‐specific gene expression occur in the lower ploidy classes, as polyploidization increases, gene expression is redirected toward translation and posttranslational processing transcriptional programs, in preparation for thrombopoiesis. Our findings are in keeping with studies of MK ultrastructure and supersede evidence generated using in vitro cultured MKs. Additionally, by analyzing transcriptional signatures of a single HSC, we identify two MK‐biased HSC subpopulations exhibiting unique differentiation kinetics. We show that human bone marrow MKs originate from these HSC subpopulations, supporting the notion that they display priming for MK differentiation. Finally, to investigate transcriptional changes in MKs associated with stress thrombopoiesis, we analyzed bone marrow MKs from individuals with recent myocardial infarction and found a specific gene expression signature. Our data support the modulation of MK differentiation in this thrombotic state. Conclusions: Here, we use single‐cell sequencing for the first time to characterize the human bone marrow MK transcriptome at different levels of polyploidization and investigate their differentiation from the HSC

Transcriptional diversity during lineage commitment of human blood progenitors.

Blood cells derive from hematopoietic stem cells through stepwise fating events. To characterize gene expression programs driving lineage choice, we sequenced RNA from eight primary human hematopoietic progenitor populations representing the major myeloid commitment stages and the main lymphoid stage. We identified extensive cell type-specific expression changes: 6711 genes and 10,724 transcripts, enriched in non-protein-coding elements at early stages of differentiation. In addition, we found 7881 novel splice junctions and 2301 differentially used alternative splicing events, enriched in genes involved in regulatory processes. We demonstrated experimentally cell-specific isoform usage, identifying nuclear factor I/B (NFIB) as a regulator of megakaryocyte maturation-the platelet precursor. Our data highlight the complexity of fating events in closely related progenitor populations, the understanding of which is essential for the advancement of transplantation and regenerative medicine.The work described in this article was primarily supported by the European Commission Seventh Framework Program through the BLUEPRINT grant with code HEALTH-F5-2011-282510 (D.H., F.B., G.C., J.H.A.M., K.D., L.C., M.F., S.C., S.F., and S.P.G.). Research in the Ouwehand laboratory is further supported by program grants from the National Institute for Health Research (NIHR, www.nihr.ac.uk; to A.A., M.K., P.P., S.B.G.J., S.N., and W.H.O.) and the British Heart Foundation under nos. RP-PG-0310-1002 and RG/09/12/28096 (www.bhf.org.uk; to A.R. and W.J.A.). K.F. and M.K. were supported by Marie Curie funding from the NETSIM FP7 program funded by the European Commission. The laboratory receives funding from the NHS Blood and Transplant for facilities. The Cambridge BioResource (www.cambridgebioresource.org.uk), the Cell Phenotyping Hub, and the Cambridge Translational GenOmics laboratory (www.catgo.org.uk) are supported by an NIHR grant to the Cambridge NIHR Biomedical Research Centre (BRC). The BRIDGE-Bleeding and Platelet Disorders Consortium is supported by the NIHR BioResource—Rare Diseases (http://bioresource.nihr.ac.uk/; to E.T., N.F., and Whole Exome Sequencing effort). Research in the Soranzo laboratory (L.V., N.S., and S. Watt) is further supported by the Wellcome Trust (Grant Codes WT098051 and WT091310) and the EU FP7 EPIGENESYS initiative (Grant Code 257082). Research in the Cvejic laboratory (A. Cvejic and C.L.) is funded by the Cancer Research UK under grant no. C45041/A14953. S.J.S. is funded by NIHR. M.E.F. is supported by a British Heart Foundation Clinical Research Training Fellowship, no. FS/12/27/29405. E.B.-M. is supported by a Wellcome Trust grant, no. 084183/Z/07/Z. Research in the Laffan laboratory is supported by Imperial College BRC. F.A.C., C.L., and S. Westbury are supported by Medical Research Council Clinical Training Fellowships, and T.B. by a British Society of Haematology/NHS Blood and Transplant grant. R.J.R. is a Principal Research Fellow of the Wellcome Trust, grant no. 082961/Z/07/Z. Research in the Flicek laboratory is also supported by the Wellcome Trust (grant no. 095908) and EMBL. Research in the Bertone laboratory is supported by EMBL. K.F. and C.v.G. are supported by FWO-Vlaanderen through grant G.0B17.13N. P.F. is a compensated member of the Omicia Inc. Scientific Advisory Board. This study made use of data generated by the UK10K Consortium, derived from samples from the Cohorts arm of the project.This is the author’s version of the work. It is posted here by permission of the AAAS for personal use, not for redistribution. The definitive version was published in Science on 26/9/14 in volume 345, number 6204, DOI: 10.1126/science.1251033. This version will be under embargo until the 26th of March 2015

Lineage-Specific Genome Architecture Links Enhancers and Non-coding Disease Variants to Target Gene Promoters

This is the final version of the article. It first appeared from Elsevier (Cell Press) via https://doi.org/10.1016/j.cell.2016.09.03

Improved annotation of the insect vector of citrus greening disease: Biocuration by a diverse genomics community

The Asian citrus psyllid (Diaphorina citri Kuwayama) is the insect vector of the bacterium Candidatus Liberibacter asiaticus (CLas), the pathogen associated with citrus Huanglongbing (HLB, citrus greening). HLB threatens citrus production worldwide. Suppression or reduction of the insect vector using chemical insecticides has been the primary method to inhibit the spread of citrus greening disease. Accurate structural and functional annotation of the Asian citrus psyllid genome, as well as a clear understanding of the interactions between the insect and CLas, are required for development of new molecular-based HLB control methods. A draft assembly of the D. citri genome has been generated and annotated with automated pipelines. However, knowledge transfer from well-curated reference genomes such as that of Drosophila melanogaster to newly sequenced ones is challenging due to the complexity and diversity of insect genomes. To identify and improve gene models as potential targets for pest control, we manually curated several gene families with a focus on genes that have key functional roles in D. citri biology and CLas interactions. This community effort produced 530 manually curated gene models across developmental, physiological, RNAi regulatory and immunity-related pathways. As previously shown in the pea aphid, RNAi machinery genes putatively involved in the microRNA pathway have been specifically duplicated. A comprehensive transcriptome enabled us to identify a number of gene families that are either missing or misassembled in the draft genome. In order to develop biocuration as a training experience, we included undergraduate and graduate students from multiple institutions, as well as experienced annotators from the insect genomics research community. The resulting gene set (OGS v1.0) combines both automatically predicted and manually curated gene models.Peer reviewedBiochemistry and Molecular BiologyEntomology and Plant Patholog

PANC Study (Pancreatitis: A National Cohort Study): national cohort study examining the first 30 days from presentation of acute pancreatitis in the UK

Abstract Background Acute pancreatitis is a common, yet complex, emergency surgical presentation. Multiple guidelines exist and management can vary significantly. The aim of this first UK, multicentre, prospective cohort study was to assess the variation in management of acute pancreatitis to guide resource planning and optimize treatment. Methods All patients aged greater than or equal to 18 years presenting with acute pancreatitis, as per the Atlanta criteria, from March to April 2021 were eligible for inclusion and followed up for 30 days. Anonymized data were uploaded to a secure electronic database in line with local governance approvals. Results A total of 113 hospitals contributed data on 2580 patients, with an equal sex distribution and a mean age of 57 years. The aetiology was gallstones in 50.6 per cent, with idiopathic the next most common (22.4 per cent). In addition to the 7.6 per cent with a diagnosis of chronic pancreatitis, 20.1 per cent of patients had a previous episode of acute pancreatitis. One in 20 patients were classed as having severe pancreatitis, as per the Atlanta criteria. The overall mortality rate was 2.3 per cent at 30 days, but rose to one in three in the severe group. Predictors of death included male sex, increased age, and frailty; previous acute pancreatitis and gallstones as aetiologies were protective. Smoking status and body mass index did not affect death. Conclusion Most patients presenting with acute pancreatitis have a mild, self-limiting disease. Rates of patients with idiopathic pancreatitis are high. Recurrent attacks of pancreatitis are common, but are likely to have reduced risk of death on subsequent admissions. </jats:sec

Alarm calls of a cooperative bird are referential and elicit context-specific antipredator behavior

Although functionally referential signals have been extensively studied, largely in mammals (e.g., nonhuman primates, see Cheney and Seyfarth (1988); mongooses, see Manser et al. (2002); and other ground-dwelling species, see Blumstein and Armitage (1997), other social taxa such as birds would similarly benefit from the use of referential signals. We therefore investigated alarm calling in the cooperative noisy miner (Manorina melanocephala), a species that has been anecdotally recorded producing aerial alarms to flying predators and empirically recorded generating terrestrial alarms to ground-based threats. For these signals to be truly referential however, they must meet 3 criteria. First, calls must be structurally distinct, a requirement that these 2 call types meet. Second, calls must be stimulus-specific and reliably associated with a given stimulus. We tested this on free-living birds by exposing them to a simulated aerial predator that was either in flight or subsequently perched and thus presented one of the first studies on functionally referential alarm systems where both aerial and terrestrial alarm calls have been tested. Miners only produced aerial alarms while the stimulus was in flight, switching to terrestrial alarms once it landed. Third, referential signals must elicit different escape responses that are "appropriate" to the associated threat. Under field conditions, aerial alarm playback alone provoked an almost instantaneous response of fleeing to vegetation cover, whereas terrestrial alarm playback elicited significantly slower responses by receivers and an increase in scanning behavior. During laboratory experiments, aerial alarms stimulated birds to spend more time looking upwards, whereas terrestrial alarm calls stimulated individuals to scan perpendicularly, as expected if these stimuli provided information on likely predator location. Although other avian taxa have been shown to use referential alarm signals, this system provides novel evidence of referential calls based on the behavior rather than the type of predator, providing a highly adaptive means of communicating risk to other members of the social group in this cooperative species