Fnr (EtrA) acts as a fine-tuning regulator of anaerobic metabolism in Shewanella oneidensis MR-1

BackgroundEtrA in Shewanella oneidensis MR-1, a model organism for study of adaptation to varied redox niches, shares 73.6% and 50.8% amino acid sequence identity with the oxygen-sensing regulators Fnr in E. coli and Anr in Pseudomonas aeruginosa, respectively; however, its regulatory role of anaerobic metabolism in Shewanella spp. is complex and not well understood.ResultsThe expression of the nap genes, nrfA, cymA and hcp was significantly reduced in etrA deletion mutant EtrA7-1; however, limited anaerobic growth and nitrate reduction occurred, suggesting that multiple regulators control nitrate reduction in this strain. Dimethyl sulfoxide (DMSO) and fumarate reductase gene expression was down-regulated at least 2-fold in the mutant, which, showed lower or no reduction of these electron acceptors when compared to the wild type, suggesting both respiratory pathways are under EtrA control. Transcript analysis further suggested a role of EtrA in prophage activation and down-regulation of genes implicated in aerobic metabolism.ConclusionIn contrast to previous studies that attributed a minor regulatory role to EtrA in Shewanella spp., this study demonstrates that EtrA acts as a global transcriptional regulator and, in conjunction with other regulators, fine-tunes the expression of genes involved in anaerobic metabolism in S. oneidensis strain MR-1. Transcriptomic and sequence analyses of the genes differentially expressed showed that those mostly affected by the mutation belonged to the "Energy metabolism" category, while stress-related genes were indirectly regulated in the mutant possibly as a result of a secondary perturbation (e.g. oxidative stress, starvation). We also conclude based on sequence, physiological and expression analyses that this regulator is more appropriately termed Fnr and recommend this descriptor be used in future publications

Fnr (EtrA) acts as a fine-tuning regulator of anaerobic metabolism in \u3cem\u3eShewanella oneidensis\u3c/em\u3e MR-1

Background EtrA in Shewanella oneidensis MR-1, a model organism for study of adaptation to varied redox niches, shares 73.6% and 50.8% amino acid sequence identity with the oxygen-sensing regulators Fnr in E. coli and Anr in Pseudomonas aeruginosa, respectively; however, its regulatory role of anaerobic metabolism in Shewanella spp. is complex and not well understood. Results The expression of the nap genes, nrfA, cymA and hcp was significantly reduced in etrA deletion mutant EtrA7-1; however, limited anaerobic growth and nitrate reduction occurred, suggesting that multiple regulators control nitrate reduction in this strain. Dimethyl sulfoxide (DMSO) and fumarate reductase gene expression was down-regulated at least 2-fold in the mutant, which, showed lower or no reduction of these electron acceptors when compared to the wild type, suggesting both respiratory pathways are under EtrA control. Transcript analysis further suggested a role of EtrA in prophage activation and down-regulation of genes implicated in aerobic metabolism. Conclusion In contrast to previous studies that attributed a minor regulatory role to EtrA in Shewanella spp., this study demonstrates that EtrA acts as a global transcriptional regulator and, in conjunction with other regulators, fine-tunes the expression of genes involved in anaerobic metabolism in S. oneidensis strain MR-1. Transcriptomic and sequence analyses of the genes differentially expressed showed that those mostly affected by the mutation belonged to the Energy metabolism category, while stress-related genes were indirectly regulated in the mutant possibly as a result of a secondary perturbation (e.g. oxidative stress, starvation). We also conclude based on sequence, physiological and expression analyses that this regulator is more appropriately termed Fnr and recommend this descriptor be used in future publications

Stochastic electrotransport selectively enhances the transport of highly electromobile molecules

Nondestructive chemical processing of porous samples such as fixed biological tissues typically relies on molecular diffusion. Diffusion into a porous structure is a slow process that significantly delays completion of chemical processing. Here, we present a novel electrokinetic method termed stochastic electrotransport for rapid nondestructive processing of porous samples. This method uses a rotational electric field to selectively disperse highly electromobile molecules throughout a porous sample without displacing the low-electromobility molecules that constitute the sample. Using computational models, we show that stochastic electrotransport can rapidly disperse electromobile molecules in a porous medium. We apply this method to completely clear mouse organs within 1–3 days and to stain them with nuclear dyes, proteins, and antibodies within 1 day. Our results demonstrate the potential of stochastic electrotransport to process large and dense tissue samples that were previously infeasible in time when relying on diffusion.Simons Foundation. Postdoctoral FellowshipLife Sciences Research FoundationBurroughs Wellcome Fund (Career Awards at the Scientific Interface)Searle Scholars ProgramMichael J. Fox Foundation for Parkinson's ResearchUnited States. Defense Advanced Research Projects AgencyJPB FoundationNational Institutes of Health (U.S.)National Institutes of Health (U.S.) (Grant 1-U01-NS090473-01

Simple, Scalable Proteomic Imaging for High-Dimensional Profiling of Intact Systems

Combined measurement of diverse molecular and anatomical traits that span multiple levels remains a major challenge in biology. Here, we introduce a simple method that enables proteomic imaging for scalable, integrated, high-dimensional phenotyping of both animal tissues and human clinical samples. This method, termed SWITCH, uniformly secures tissue architecture, native biomolecules, and antigenicity across an entire system by synchronizing the tissue preservation reaction. The heat- and chemical-resistant nature of the resulting framework permits multiple rounds (>20) of relabeling. We have performed 22 rounds of labeling of a single tissue with precise co-registration of multiple datasets. Furthermore, SWITCH synchronizes labeling reactions to improve probe penetration depth and uniformity of staining. With SWITCH, we performed combinatorial protein expression profiling of the human cortex and also interrogated the geometric structure of the fiber pathways in mouse brains. Such integrated high-dimensional information may accelerate our understanding of biological systems at multiple levels.Simons Foundation. Postdoctoral FellowshipLife Sciences Research FoundationBurroughs Wellcome Fund (Career Award at the Scientific Interface)Searle Scholars ProgramMichael J. Fox Foundation for Parkinson's ResearchUnited States. Defense Advanced Research Projects AgencyNational Institutes of Health (U.S.) (1-U01-NS090473-01

Comparative systems biology across an evolutionary gradient within the Shewanella genus

Author Posting. © The Authors, 2009. This is the author's version of the work. It is posted here by permission of National Academy of Sciences for personal use, not for redistribution. The definitive version was published in Proceedings of the National Academy of Sciences 106 (2009): 15909-15914, doi:10.1073/pnas.0902000106.To what extent genotypic differences translate to phenotypic variation remains a poorly understood issue of paramount importance for several cornerstone concepts of microbiology including the species definition. Here, we take advantage of the completed genomic sequences, expressed proteomic profiles, and physiological studies of ten closely related Shewanella strains and species to provide quantitative insights into this issue. Our analyses revealed that, despite extensive horizontal gene transfer within these genomes, the genotypic and phenotypic similarities among the organisms were generally predictable from their evolutionary relatedness. The power of the predictions depended on the degree of ecological specialization of the organisms evaluated. Using the gradient of evolutionary relatedness formed by these genomes, we were able to partly isolate the effect of ecology from that of evolutionary divergence and rank the different cellular functions in terms of their rates of evolution. Our ranking also revealed that whole-cell protein expression differences among these organisms when grown under identical conditions were relatively larger than differences at the genome level, suggesting that similarity in gene regulation and expression should constitute another important parameter for (new) species description. Collectively, our results provide important new information towards beginning a systems-level understanding of bacterial species and genera.The authors have been supported by the DOE through the Shewanella Federation consortium and the Proteomics Application project. The MSU work relevant to speciation was also supported by NSF (DEB 0516252)

Communications Biophysics

Contains research objectives and reports on eight research projects split into three sections.National Institutes of Health (Grant 2 PO1 NS13126)National Institutes of Health (Grant 5 RO1 NS18682)National Institutes of Health (Grant 5 RO1 NS20322)National Institutes of Health (Grant 1 RO1 NS 20269)National Institutes of Health (Grant 5 T32 NS 07047)Symbion, Inc.National Institutes of Health (Grant 5 R01 NS10916)National Institutes of Health (Grant 1 RO NS 16917)National Science Foundation (Grant BNS83-19874)National Science Foundation (Grant BNS83-19887)National Institutes of Health (Grant 5 RO1 NS12846)National Institutes of Health (Grant 1 RO1 NS21322-01)National Institutes of Health (Grant 5 T32-NS07099-07)National Institutes of Health (Grant 1 RO1 NS14092-06)National Science Foundation (Grant BNS77-21751)National Institutes of Health (Grant 5 RO1 NS11080

Effect of Hydrocortisone on Mortality and Organ Support in Patients With Severe COVID-19: The REMAP-CAP COVID-19 Corticosteroid Domain Randomized Clinical Trial.

Importance: Evidence regarding corticosteroid use for severe coronavirus disease 2019 (COVID-19) is limited. Objective: To determine whether hydrocortisone improves outcome for patients with severe COVID-19. Design, Setting, and Participants: An ongoing adaptive platform trial testing multiple interventions within multiple therapeutic domains, for example, antiviral agents, corticosteroids, or immunoglobulin. Between March 9 and June 17, 2020, 614 adult patients with suspected or confirmed COVID-19 were enrolled and randomized within at least 1 domain following admission to an intensive care unit (ICU) for respiratory or cardiovascular organ support at 121 sites in 8 countries. Of these, 403 were randomized to open-label interventions within the corticosteroid domain. The domain was halted after results from another trial were released. Follow-up ended August 12, 2020. Interventions: The corticosteroid domain randomized participants to a fixed 7-day course of intravenous hydrocortisone (50 mg or 100 mg every 6 hours) (n = 143), a shock-dependent course (50 mg every 6 hours when shock was clinically evident) (n = 152), or no hydrocortisone (n = 108). Main Outcomes and Measures: The primary end point was organ support-free days (days alive and free of ICU-based respiratory or cardiovascular support) within 21 days, where patients who died were assigned -1 day. The primary analysis was a bayesian cumulative logistic model that included all patients enrolled with severe COVID-19, adjusting for age, sex, site, region, time, assignment to interventions within other domains, and domain and intervention eligibility. Superiority was defined as the posterior probability of an odds ratio greater than 1 (threshold for trial conclusion of superiority >99%). Results: After excluding 19 participants who withdrew consent, there were 384 patients (mean age, 60 years; 29% female) randomized to the fixed-dose (n = 137), shock-dependent (n = 146), and no (n = 101) hydrocortisone groups; 379 (99%) completed the study and were included in the analysis. The mean age for the 3 groups ranged between 59.5 and 60.4 years; most patients were male (range, 70.6%-71.5%); mean body mass index ranged between 29.7 and 30.9; and patients receiving mechanical ventilation ranged between 50.0% and 63.5%. For the fixed-dose, shock-dependent, and no hydrocortisone groups, respectively, the median organ support-free days were 0 (IQR, -1 to 15), 0 (IQR, -1 to 13), and 0 (-1 to 11) days (composed of 30%, 26%, and 33% mortality rates and 11.5, 9.5, and 6 median organ support-free days among survivors). The median adjusted odds ratio and bayesian probability of superiority were 1.43 (95% credible interval, 0.91-2.27) and 93% for fixed-dose hydrocortisone, respectively, and were 1.22 (95% credible interval, 0.76-1.94) and 80% for shock-dependent hydrocortisone compared with no hydrocortisone. Serious adverse events were reported in 4 (3%), 5 (3%), and 1 (1%) patients in the fixed-dose, shock-dependent, and no hydrocortisone groups, respectively. Conclusions and Relevance: Among patients with severe COVID-19, treatment with a 7-day fixed-dose course of hydrocortisone or shock-dependent dosing of hydrocortisone, compared with no hydrocortisone, resulted in 93% and 80% probabilities of superiority with regard to the odds of improvement in organ support-free days within 21 days. However, the trial was stopped early and no treatment strategy met prespecified criteria for statistical superiority, precluding definitive conclusions. Trial Registration: ClinicalTrials.gov Identifier: NCT02735707

Pseudomonas aeruginosa-specific IgG1 and IgG2 subclasses in enhancement of pulmonary clearance following passive immunisation in the rat

Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen, which causes serious debilitating infections in patients with compromised lung function. The mechanism by which P. aeruginosa is cleared from the lung is not fully defined, although our previous studies have established a role for cellular immunity in protection against P. aeruginosa infections. This study aimed to evaluate the role of P. aeruginosa-specific IgG in protection against P. aeruginosa in a rat model of acute pulmonary infection. Immunoaffinity chromatography was used to purify total rat IgG from rat immune serum (rats immunised with P. aeruginosa) and non-immune serum. Untreated recipient rats were injected intravenously with different concentrations of pure IgG prepared from serum of unimmunised rats (non-immune IgG) or from rats immunised intestinally with killed P. aeruginosa (immune IgG) and infected intratracheally with P. aeruginosa 18 h later. The protective capability of the purified IgG against P. aeruginosa was assessed by measurement of reduction in P. aeruginosa infection in the lung 4 h after instillation of bacteria. Enhanced bacterial clearance induced by IgG was determined to be dose-dependent with a 1 mg dose failing to enhance clearance, whereas 5 mg of immune IgG enhanced clearance from the airways and the lung tissue. Measurement of the IgG1, IgG2a and IgG2b isotypes in serum and the lung lavage following transfer of P. aeruginosa-specific IgG found that all three were present. These results demonstrate that anti-P. aeruginosa IgG can enhance bacterial clearance from the airways in an acute infection and identify an important role for IgG in acute respiratory infections caused by P. aeruginosa