The rsmS (ybaM) mutation causes bypass suppression of the RsmAB post-transcriptional virulence regulation system in enterobacterial phytopathogens.

Plant cell wall degrading enzymes (PCWDEs) are the primary virulence determinants of soft rotting bacteria such as the potato pathogen, Pectobacterium atrosepticum. The regulation of secondary metabolite (Rsm) system controls production of PCWDEs in response to changing nutrient conditions. This work identified a new suppressor of an rsmB mutation - ECA1172 or rsmS (rsmB suppressor). Mutants defective in rsmB (encoding a small regulatory RNA), show reduced elaboration of the quorum sensing molecule (N-3-oxohexanoyl-homoserine lactone; OHHL) and PCWDEs. However, OHHL and PCWDE production were partially restored in an rsmB, rsmS double mutant. Single rsmS mutants, overproduced PCWDEs and OHHL relative to wild type P. atrosepticum and exhibited hypervirulence in potato. RsmS overproduction also resulted in increased PCWDEs and OHHL. Homology searches revealed rsmS conservation across pathogens such as Escherichia coli (ybaM), Dickeya solani, Klebsiella pneumoniae and Shigella flexneri. An rsmS mutant of Pectobacterium carotovorum ATCC39048 showed bypass of rsmB-dependent repression of PCWDEs and OHHL production. P. carotovorum ATCC39048 produces the β-lactam antibiotic, 1-carbapen-2-em-3-carboxylic acid (a carbapenem). Production of the antibiotic was repressed in an rsmB mutant but partially restored in an rsmB, rsmS double mutant. This work highlights the importance of RsmS, as a conserved pleiotropic regulator of virulence and antibiotic biosynthesis.James Hutton Institut

The impact of physical inactivity on glucose homeostasis when diet is adjusted to maintain energy balance in healthy, young males

Background & aims: It is unclear if dietary adjustments to maintain energy balance during reduced physical activity can offset inactivity-induced reductions in insulin sensitivity and glucose disposal to produce normal daily glucose concentrations and meal responses. Therefore, the aim of the present study was to examine the impact of long-term physical inactivity (60 days of bed rest) on daily glycemia when in energy balance.Methods: Interstitial glucose concentrations were measured using Continuous Glucose Monitoring Systems (CGMS) for 5 days before and towards the end of bed rest in 20 healthy, young males (Age: 34 ± 8 years; BMI: 23.5 ± 1.8 kg/m2). Energy intake was reduced during bed rest to match energy expenditure, but the types of foods and timing of meals was maintained. Fasting venous glucose and insulin concentrations were determined, as well as the change in whole-body glucose disposal using a hyperinsulinemic-euglycemic clamp (HIEC).Results: Following long-term bed rest, fasting plasma insulin concentration increased 40% (p = 0.004) and glucose disposal during the HIEC decreased 24% (p < 0.001). Interstitial daily glucose total area under the curve (tAUC) from pre-to post-bed rest increased on average by 6% (p = 0.041), despite a 20 and 25% reduction in total caloric and carbohydrate intake, respectively. The nocturnal period (00:00–06:00) showed the greatest change to glycemia with glucose tAUC for this period increasing by 9% (p = 0.005). CGMS measures of daily glycemic variability (SD, J-Index, M-value and MAG) were not changed during bed rest.Conclusions: Reduced physical activity (bed rest) increases glycemia even when daily energy intake is reduced to maintain energy balance. However, the disturbance to daily glucose homeostasis was much more modest than the reduced capacity to dispose of glucose, and glycemic variability was not negatively affected by bed rest, likely due to positive mitigating effects from the contemporaneous reduction in dietary energy and carbohydrate intake.Clinical trials record: NCT03594799 (registered July 20, 2018) (https://clinicaltrials.gov/ct2/show/NCT03594799)

Breast cancer prognosis predicted by nuclear receptor-coregulator networks

Although molecular signatures based on transcript expression in breast cancer samples have provided new insights into breast cancer classification and prognosis, there are acknowledged limitations in current signatures. To provide rational, pathway-based signatures of disrupted physiology in cancer tissues that may be relevant to prognosis, this study has directly quantitated changed gene expression, between normal breast and cancer tissue, as a basis for signature development. The nuclear receptor (NR) family of transcription factors, and their coregulators, are fundamental regulators of every aspect of metazoan life, and were rigorously quantified in normal breast tissues and ERα positive and ERα negative breast cancers. Coregulator expression was highly correlated with that of selected NR in normal breast, particularly from postmenopausal women. These associations were markedly decreased in breast cancer, and the expression of the majority of coregulators was down-regulated in cancer tissues compared with normal. While in cancer the loss of NR-coregulator associations observed in normal breast was common, a small number of NR (Rev-ERBβ, GR, NOR1, LRH-1 and PGR) acquired new associations with coregulators in cancer tissues. Elevated expression of these NR in cancers was associated with poorer outcome in large clinical cohorts, as well as suggesting the activation of ERα -related, but ERα-independent, pathways in ERα negative cancers. In addition, the combined expression of small numbers of NR and coregulators in breast cancer was identified as a signature predicting outcome in ERα negative breast cancer patients, not linked to proliferation and with predictive power superior to existing signatures containing many more genes. These findings highlight the power of predictive signatures derived from the quantitative determination of altered gene expression between normal breast and breast cancers. Taken together, the findings of this study identify networks of NR-coregulator associations active in normal breast but disrupted in breast cancer, and moreover provide evidence that signatures based on NR networks disrupted in cancer can provide important prognostic information in breast cancer patients

Gene expression profiling of the astrocyte transcriptome in multiple sclerosis normal appearing white matter reveals a neuroprotective role

Multiple sclerosis (MS) is a chronic, inflammatory, demyelinating disease of the central nervous system (CNS). White matter lesions in MS are surrounded by areas of non-demyelinated normal appearing white matter (NAWM) with complex pathology, including blood brain barrier dysfunction, axonal damage and glial activation. Astrocytes, the most abundant cell type within the CNS, may respond and/or contribute to lesion pathogenesis. We aimed to characterise the transcriptomic profile of astrocytes in NAWM to determine whether specific glial changes exist in the NAWM which contribute to lesion development or prevent disease progression. Astrocytes were isolated from control and NAWM by laser capture microdissection (LCM), using glial fibrillary acidic protein (GFAP) as a marker, and the astrocyte transcriptome determined using microarray analysis. 452 genes were significantly differentially expressed (208 up-regulated and 244 down-regulated, FC ≥ 1.5 and p-value ≤ 0.05). Within the NAWM, astrocytes were associated with significant upregulation of genes involved in the control of iron homeostasis (including metallothionein-1 and -2, ferritin light chain and transferrin), oxidative stress responses, the immune response and neurotrophic support. These findings suggest a neuroprotective role of astrocytes in the NAWM in M

Immune System Dysregulation During Spaceflight: Potential Countermeasures for Deep Space Exploration Missions

Recent studies have established that dysregulation of the human immune system and the reactivation of latent herpesviruses persists for the duration of a 6-month orbital spaceflight. It appears certain aspects of adaptive immunity are dysregulated during flight, yet some aspects of innate immunity are heightened. Interaction between adaptive and innate immunity also seems to be altered. Some crews experience persistent hypersensitivity reactions during flight. This phenomenon may, in synergy with extended duration and galactic radiation exposure, increase specific crew clinical risks during deep space exploration missions. The clinical challenge is based upon both the frequency of these phenomena in multiple crewmembers during low earth orbit missions and the inability to predict which specific individual crewmembers will experience these changes. Thus, a general countermeasure approach that offers the broadest possible coverage is needed. The vehicles, architecture, and mission profiles to enable such voyages are now under development. These include deployment and use of a cis-Lunar station (mid 2020s) with possible Moon surface operations, to be followed by multiple Mars flyby missions, and eventual human Mars surface exploration. Current ISS studies will continue to characterize physiological dysregulation associated with prolonged orbital spaceflight. However, sufficient information exists to begin consideration of both the need for, and nature of, specific immune countermeasures to ensure astronaut health. This article will review relevant in-place operational countermeasures onboard ISS and discuss a myriad of potential immune countermeasures for exploration missions. Discussion points include nutritional supplementation and functional foods, exercise and immunity, pharmacological options, the relationship between bone and immune countermeasures, and vaccination to mitigate herpes (and possibly other) virus risks. As the immune system has sentinel connectivity within every other physiological system, translational effects must be considered for all potential immune countermeasures. Finally, we shall discuss immune countermeasures in the context of their individualized implementation or precision medicine, based on crewmember specific immunological biases

The RNA polymerase I transcription inhibitor CX-5461 cooperates with topoisomerase 1 inhibition by enhancing the DNA damage response in homologous recombination-proficient high-grade serious ovarian cancer

Background: Intrinsic and acquired drug resistance represent fundamental barriers to the cure of high-grade serous ovarian carcinoma (HGSC), the most common histological subtype accounting for the majority of ovarian cancer deaths. Defects in homologous recombination (HR) DNA repair are key determinants of sensitivity to chemotherapy and poly-ADP ribose polymerase inhibitors. Restoration of HR is a common mechanism of acquired resistance that results in patient mortality, highlighting the need to identify new therapies targeting HR-proficient disease. We have shown promise for CX-5461, a cancer therapeutic in early phase clinical trials, in treating HR-deficient HGSC. Methods: Herein, we screen the whole protein-coding genome to identify potential targets whose depletion cooperates with CX-5461 in HR-proficient HGSC. Results: We demonstrate robust proliferation inhibition in cells depleted of DNA topoisomerase 1 (TOP1). Combining the clinically used TOP1 inhibitor topotecan with CX-5461 potentiates a G2/M cell cycle checkpoint arrest in multiple HR-proficient HGSC cell lines. The combination enhances a nucleolar DNA damage response and global replication stress without increasing DNA strand breakage, significantly reducing clonogenic survival and tumour growth in vivo. Conclusions: Our findings highlight the possibility of exploiting TOP1 inhibition to be combined with CX-5461 as a non-genotoxic approach in targeting HR-proficient HGSC.The China Scholarship Council University of Melbourne Ph.D. Scholarship supported S.Y. A National Health and Medical Research Council (NHMRC) Grant and NHMRC Senior Research Fellowship to R.B.P. supported this work. The Victorian Centre for Functional Genomics (K.J.S.) is funded by the Australian Cancer Research Foundation (ACRF), the Australian Phenomics Network (APN) through funding from the Australian Government’s National Collaborative Research Infrastructure Strategy (NCRIS) programme, the Peter MacCallum Cancer Centre Foundation and the University of Melbourne Research Collaborative Infrastructure Programm

Original research: longitudinal evaluation of cognitively demanding daily function using performance-based functional assessment highlights heterogeneous trajectories in cognitive and functional abilities in people with Parkinson’s disease

BackgroundLongitudinal assessment of functional abilities in Parkinson’s disease (PD) is needed to determine the efficacy of cognitive interventions in providing meaningful improvements in daily life. Additionally, subtle changes in instrumental activities of daily living may precede a clinical diagnosis of dementia and could aid earlier detection of and intervention for cognitive decline.ObjectiveThe primary goal was to validate the longitudinal application of the University of California San Diego Performance-Based Skills Assessment (UPSA). An exploratory secondary goal was to determine whether UPSA may identify individuals at higher risk of cognitive decline in PD.MethodsSeventy participants with PD completed the UPSA with at least one follow-up visit. Linear mixed effects modeling was used to identify associations between baseline UPSA score and cognitive composite score (CCS) over time. Descriptive analysis of four heterogeneous cognitive and functional trajectory groups and individual case examples was performed.ResultsBaseline UPSA score predicted CCS at each timepoint for functionally impaired and unimpaired groups (p < 0.01) but did not predict the rate change in CCS over time (p = 0.83). Participants displayed heterogenous trajectories in both UPSA and CCS during the follow-up period. Most participants maintained both cognitive and functional performance (n = 54), though some displayed cognitive and functional decline (n = 4), cognitive decline with functional maintenance (n = 4), and functional decline with cognitive maintenance (n = 8).ConclusionThe UPSA is a valid measure of cognitive functional abilities over time in PD. Given the heterogeneity of functional and cognitive trajectories, this performance-based assessment did not predict cognitive decline with this relatively short follow-up. Further work is needed to understand longitudinal functional assessments in PD-associated cognitive impairment

The structure of the bacterial oxidoreductase enzyme DsbA in complex with a peptide reveals a basis for substrate specificity in the catalytic cycle of DsbA enzymes

Oxidative protein folding in Gram-negative bacteria results in the formation of disulfide bonds between pairs of cysteine residues. This is a multistep process in which the dithiol-disulfide oxidoreductase enzyme, DsbA, plays a central role. The structure of DsbA comprises an all helical domain of unknown function and a thioredoxin domain, where active site cysteines shuttle between an oxidized, substrate-bound, reduced form and a DsbB-bound form, where DsbB is a membrane protein that reoxidizes DsbA. Most DsbA enzymes interact with a wide variety of reduced substrates and show little specificity. However, a number of DsbA enzymes have now been identified that have narrow substrate repertoires and appear to interact specifically with a smaller number of substrates. The transient nature of the DsbA-substrate complex has hampered our understanding of the factors that govern the interaction of DsbA enzymes with their substrates. Here we report the crystal structure of a complex between Escherichia coli DsbA and a peptide with a sequence derived from a substrate. The binding site identified in the DsbA-peptide complex was distinct from that observed for DsbB in the DsbA-DsbB complex. The structure revealed details of the DsbA-peptide interaction and suggested a mechanism by which DsbA can simultaneously show broad specificity for substrates yet exhibit specificity for DsbB. This mode of binding was supported by solution nuclear magnetic resonance data as well as functional data, which demonstrated that the substrate specificity of DsbA could be modified via changes at the binding interface identified in the structure of the comple

Pooled sequencing of 531 genes in inflammatory bowel disease identifies an associated rare variant in BTNL2 and implicates other immune related genes.

The contribution of rare coding sequence variants to genetic susceptibility in complex disorders is an important but unresolved question. Most studies thus far have investigated a limited number of genes from regions which contain common disease associated variants. Here we investigate this in inflammatory bowel disease by sequencing the exons and proximal promoters of 531 genes selected from both genome-wide association studies and pathway analysis in pooled DNA panels from 474 cases of Crohn's disease and 480 controls. 80 variants with evidence of association in the sequencing experiment or with potential functional significance were selected for follow up genotyping in 6,507 IBD cases and 3,064 population controls. The top 5 disease associated variants were genotyped in an extension panel of 3,662 IBD cases and 3,639 controls, and tested for association in a combined analysis of 10,147 IBD cases and 7,008 controls. A rare coding variant p.G454C in the BTNL2 gene within the major histocompatibility complex was significantly associated with increased risk for IBD (p = 9.65x10-10, OR = 2.3[95% CI = 1.75-3.04]), but was independent of the known common associated CD and UC variants at this locus. Rare (T) or decreased risk (IL12B p.V298F, and NICN p.H191R) of IBD. These results provide additional insights into the involvement of the inhibition of T cell activation in the development of both sub-phenotypes of inflammatory bowel disease. We suggest that although rare coding variants may make a modest overall contribution to complex disease susceptibility, they can inform our understanding of the molecular pathways that contribute to pathogenesis

A comprehensive assessment of somatic mutation detection in cancer using whole-genome sequencing.

As whole-genome sequencing for cancer genome analysis becomes a clinical tool, a full understanding of the variables affecting sequencing analysis output is required. Here using tumour-normal sample pairs from two different types of cancer, chronic lymphocytic leukaemia and medulloblastoma, we conduct a benchmarking exercise within the context of the International Cancer Genome Consortium. We compare sequencing methods, analysis pipelines and validation methods. We show that using PCR-free methods and increasing sequencing depth to ∼ 100 × shows benefits, as long as the tumour:control coverage ratio remains balanced. We observe widely varying mutation call rates and low concordance among analysis pipelines, reflecting the artefact-prone nature of the raw data and lack of standards for dealing with the artefacts. However, we show that, using the benchmark mutation set we have created, many issues are in fact easy to remedy and have an immediate positive impact on mutation detection accuracy.We thank the DKFZ Genomics and Proteomics Core Facility and the OICR Genome Technologies Platform for provision of sequencing services. Financial support was provided by the consortium projects READNA under grant agreement FP7 Health-F4-2008-201418, ESGI under grant agreement 262055, GEUVADIS under grant agreement 261123 of the European Commission Framework Programme 7, ICGC-CLL through the Spanish Ministry of Science and Innovation (MICINN), the Instituto de Salud Carlos III (ISCIII) and the Generalitat de Catalunya. Additional financial support was provided by the PedBrain Tumor Project contributing to the International Cancer Genome Consortium, funded by German Cancer Aid (109252) and by the German Federal Ministry of Education and Research (BMBF, grants #01KU1201A, MedSys #0315416C and NGFNplus #01GS0883; the Ontario Institute for Cancer Research to PCB and JDM through funding provided by the Government of Ontario, Ministry of Research and Innovation; Genome Canada; the Canada Foundation for Innovation and Prostate Cancer Canada with funding from the Movember Foundation (PCB). PCB was also supported by a Terry Fox Research Institute New Investigator Award, a CIHR New Investigator Award and a Genome Canada Large-Scale Applied Project Contract. The Synergie Lyon Cancer platform has received support from the French National Institute of Cancer (INCa) and from the ABS4NGS ANR project (ANR-11-BINF-0001-06). The ICGC RIKEN study was supported partially by RIKEN President’s Fund 2011, and the supercomputing resource for the RIKEN study was provided by the Human Genome Center, University of Tokyo. MDE, LB, AGL and CLA were supported by Cancer Research UK, the University of Cambridge and Hutchison-Whampoa Limited. SD is supported by the Torres Quevedo subprogram (MI CINN) under grant agreement PTQ-12-05391. EH is supported by the Research Council of Norway under grant agreements 221580 and 218241 and by the Norwegian Cancer Society under grant agreement 71220-PR-2006-0433. Very special thanks go to Jennifer Jennings for administrating the activity of the ICGC Verification Working Group and Anna Borrell for administrative support.This is the final version of the article. It first appeared from Nature Publishing Group via http://dx.doi.org/10.1038/ncomms1000