Characterisation of the Putative Effector Interaction Site of the Regulatory HbpR Protein from Pseudomonas azelaica by Site-Directed Mutagenesis

Bacterial transcription activators of the XylR/DmpR subfamily exert their expression control via σ54-dependent RNA polymerase upon stimulation by a chemical effector, typically an aromatic compound. Where the chemical effector interacts with the transcription regulator protein to achieve activation is still largely unknown. Here we focus on the HbpR protein from Pseudomonas azelaica, which is a member of the XylR/DmpR subfamily and responds to biaromatic effectors such as 2-hydroxybiphenyl. We use protein structure modeling to predict folding of the effector recognition domain of HbpR and molecular docking to identify the region where 2-hydroxybiphenyl may interact with HbpR. A large number of site-directed HbpR mutants of residues in- and outside the predicted interaction area was created and their potential to induce reporter gene expression in Escherichia coli from the cognate PC promoter upon activation with 2-hydroxybiphenyl was studied. Mutant proteins were purified to study their conformation. Critical residues for effector stimulation indeed grouped near the predicted area, some of which are conserved among XylR/DmpR subfamily members in spite of displaying different effector specificities. This suggests that they are important for the process of effector activation, but not necessarily for effector specificity recognition

An analysis of first-time enquirers to the CancerBACUP information service: variations with cancer site, demographic status and geographical location

A retrospective comparison of cancer incidence data and, where relevant, population data with 16 955first-time users (patients, relatives and friends) of a national cancer information service (CancerBACUP) during the period April1995 to March 1996 is presented. The number of events observed was compared with the number of events expected, were the nationalrates of cancer incidence and population demographics apply. Standardized incidence ratios (SIRs) (observed – expectedratios) were used to indicate any differences. Statistically significant differences (P< 0.001) in the observed andexpected sex, age and primary site distribution of patients enquired about were found. Statistically significant differences(P< 0.001) were also identified for the age, employment status, socioeconomic class and geographical location offirst-time enquirers (patients, relatives and friends). Enquiries about brain, testis and breast cancers and non-Hodgkin'slymphoma (NHL) were substantially higher than expected; enquiries about bladder, lung, stomach and colorectal cancers were muchlower than expected. As the service is provided via a freephone number, it is available to all, and users might be expected to berandomly distributed across the variables listed. The underlying reasons for the differences identified need to be investigated,and the role of information in the care of cancer patients should be formally evaluated. © 1999 Cancer Research Campaig

Utilisation of an operative difficulty grading scale for laparoscopic cholecystectomy

Background A reliable system for grading operative difficulty of laparoscopic cholecystectomy would standardise description of findings and reporting of outcomes. The aim of this study was to validate a difficulty grading system (Nassar scale), testing its applicability and consistency in two large prospective datasets. Methods Patient and disease-related variables and 30-day outcomes were identified in two prospective cholecystectomy databases: the multi-centre prospective cohort of 8820 patients from the recent CholeS Study and the single-surgeon series containing 4089 patients. Operative data and patient outcomes were correlated with Nassar operative difficultly scale, using Kendall’s tau for dichotomous variables, or Jonckheere–Terpstra tests for continuous variables. A ROC curve analysis was performed, to quantify the predictive accuracy of the scale for each outcome, with continuous outcomes dichotomised, prior to analysis. Results A higher operative difficulty grade was consistently associated with worse outcomes for the patients in both the reference and CholeS cohorts. The median length of stay increased from 0 to 4 days, and the 30-day complication rate from 7.6 to 24.4% as the difficulty grade increased from 1 to 4/5 (both p < 0.001). In the CholeS cohort, a higher difficulty grade was found to be most strongly associated with conversion to open and 30-day mortality (AUROC = 0.903, 0.822, respectively). On multivariable analysis, the Nassar operative difficultly scale was found to be a significant independent predictor of operative duration, conversion to open surgery, 30-day complications and 30-day reintervention (all p < 0.001). Conclusion We have shown that an operative difficulty scale can standardise the description of operative findings by multiple grades of surgeons to facilitate audit, training assessment and research. It provides a tool for reporting operative findings, disease severity and technical difficulty and can be utilised in future research to reliably compare outcomes according to case mix and intra-operative difficulty

Overview and statistical analysis of boundary layer clouds and precipitation over the western North Atlantic Ocean

Due to their fast evolution and large natural variability in macro- and microphysical properties, the accurate representation of boundary layer clouds in current climate models remains a challenge. One of the regions with large intermodel spread in the Coupled Model Intercomparison Project Phase 6 ensemble is the western North Atlantic Ocean. Here, statistically representative in situ measurements can help to develop and constrain the parameterization of clouds in global models. To this end, we performed comprehensive measurements of boundary layer clouds, aerosol, trace gases, and radiation in the western North Atlantic Ocean during the NASA Aerosol Cloud meTeorology Interactions oVer the western ATlantic Experiment (ACTIVATE) mission. In total, 174 research flights with 574 flight hours for cloud and precipitation measurements were performed with the HU-25 Falcon during three winter (February–March 2020, January–April 2021, and November 2021–March 2022) and three summer seasons (August–September 2020, May–June 2021, and May–June 2022). Here we present a statistical evaluation of 16 140 individual cloud events probed by the fast cloud droplet probe and the two-dimensional stereo cloud probe during 155 research flights in a representative and repetitive flight strategy allowing for robust statistical data analyses. We show that the vertical profiles of distributions of the liquid water content and the cloud droplet effective diameter (ED) increase with altitude in the marine boundary layer. Due to higher updraft speeds, higher cloud droplet number concentrations (Nliquid) were measured in winter compared to summer despite lower cloud condensation nucleus abundance. Flight cloud cover derived from statistical analysis of in situ data is reduced in summer and shows large variability. This seasonal contrast in cloud coverage is consistent with a dominance of a synoptic pattern in winter that favors conditions for the formation of stratiform clouds at the western edge of cyclones (post-cyclonic). In contrast, a dominant summer anticyclone is concomitant with the occurrence of shallow cumulus clouds and lower cloud coverage. The evaluation of boundary layer clouds and precipitation in the Nliquid ED phase space sheds light on liquid, mixed-phase, and ice cloud properties and helps to categorize the cloud data. Ice and liquid precipitation, often masked in cloud statistics by a high abundance of liquid clouds, is often observed throughout the cloud. The ACTIVATE in situ cloud measurements provide a wealth of cloud information useful for assessing airborne and satellite remote-sensing products, for global climate and weather model evaluations, and for dedicated process studies that address precipitation and aerosol–cloud interactions.</p

Genome analysis and physiological comparison of Alicycliphilus denitrificans strains BC and K601T

The genomes of the Betaproteobacteria Alicycliphilus denitrificans strains BC and K601T have been sequenced to get insight into the physiology of the two strains. Strain BC degrades benzene with chlorate as electron acceptor. The cyclohexanol-degrading denitrifying strain K601T is not able to use chlorate as electron acceptor, while strain BC cannot degrade cyclohexanol. The 16S rRNA sequences of strains BC and K601T are identical and the fatty acid methyl ester patterns of the strains are similar. Basic Local Alignment Search Tool (BLAST) analysis of predicted open reading frames of both strains showed most hits with Acidovorax sp. JS42, a bacterium that degrades nitro-aromatics. The genomes include strain-specific plasmids (pAlide201 in strain K601T and pAlide01 and pAlide02 in strain BC). Key genes of chlorate reduction in strain BC were located on a 120 kb megaplasmid (pAlide01), which was absent in strain K601T. Genes involved in cyclohexanol degradation were only found in strain K601T. Benzene and toluene are degraded via oxygenase-mediated pathways in both strains. Genes involved in the meta-cleavage pathway of catechol are present in the genomes of both strains. Strain BC also contains all genes of the ortho-cleavage pathway. The large number of mono- and dioxygenase genes in the genomes suggests that the two strains have a broader substrate range than known thus far.This research was supported by the Technology Foundation, the Applied Science Division (STW) of the Netherlands Organization for Scientific Research (NWO), project number 08053, the graduate school WIMEK (Wageningen Institute for Environment and Climate Research, which is part of SENSE Research School for Socio-Economic and Natural Sciences of the Environment, www.wimek-new.wur.nl and www.sense.nl), SKB (Dutch Centre for Soil Quality Management and Knowledge Transfer, www.skbodem.nl) and the Consolider project CSD-2007-00055. The research was incorporated in the TRIAS (TRIpartite Approaches 469 toward Soil systems processes) program (http://www.nwo.nl/en/research-and-results/programmes/alw/trias-tripartite-approach-to-soil-system-processes/index. html). Flávia Talarico Saia was supported by a FAPESP (the State of São Paulo Research Foundation) scholarship (2006-01997/5). The work conducted by the DOE JGI is supported by the Office of Science of the United States Department of Energy under contract number DE-AC02-05CH11231. Alfons Stams acknowledges support by an ERC (European Research Counsil) advanced grant (project 323009). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

A benzene-degrading nitrate-reducing microbial consortium displays aerobic and anaerobic benzene degradation pathways

All sequence data from this study were deposited at the European Bioinformatics Institute under the accession numbers ERS1670018 to ERS1670023. Further, all assigned genes, taxonomy, function, sequences of contigs, genes and proteins can be found in Table S3.In this study, we report transcription of genes involved in aerobic and anaerobic benzene degradation pathways in a benzene-degrading denitrifying continuous culture. Transcripts associated with the family Peptococcaceae dominated all samples (2136% relative abundance) indicating their key role in the community. We found a highly transcribed gene cluster encoding a presumed anaerobic benzene carboxylase (AbcA and AbcD) and a benzoate-coenzyme A ligase (BzlA). Predicted gene products showed >96% amino acid identity and similar gene order to the corresponding benzene degradation gene cluster described previously, providing further evidence for anaerobic benzene activation via carboxylation. For subsequent benzoyl-CoA dearomatization, bam-like genes analogous to the ones found in other strict anaerobes were transcribed, whereas gene transcripts involved in downstream benzoyl-CoA degradation were mostly analogous to the ones described in facultative anaerobes. The concurrent transcription of genes encoding enzymes involved in oxygenase-mediated aerobic benzene degradation suggested oxygen presence in the culture, possibly formed via a recently identified nitric oxide dismutase (Nod). Although we were unable to detect transcription of Nod-encoding genes, addition of nitrite and formate to the continuous culture showed indication for oxygen production. Such an oxygen production would enable aerobic microbes to thrive in oxygen-depleted and nitrate-containing subsurface environments contaminated with hydrocarbons.This study was supported by a grant of BE-Basic-FES funds from the Dutch Ministry of Economic Affairs. The research of A.J.M. Stams is supported by an ERC grant (project 323009) and the gravitation grant “Microbes for Health and Environment” (project 024.002.002) of the Netherlands Ministry of Education, Culture and Science. F. Hugenholtz was supported by the same gravitation grant (project 024.002.002). B. Hornung is supported by Wageningen University and the Wageningen Institute for Environment and Climate Research (WIMEK) through the IP/OP program Systems Biology (project KB-17-003.02-023).info:eu-repo/semantics/publishedVersio

Comparative analyses imply that the enigmatic sigma factor 54 is a central controller of the bacterial exterior

Contains fulltext : 95738.pdf (publisher's version ) (Open Access)BACKGROUND: Sigma-54 is a central regulator in many pathogenic bacteria and has been linked to a multitude of cellular processes like nitrogen assimilation and important functional traits such as motility, virulence, and biofilm formation. Until now it has remained obscure whether these phenomena and the control by Sigma-54 share an underlying theme. RESULTS: We have uncovered the commonality by performing a range of comparative genome analyses. A) The presence of Sigma-54 and its associated activators was determined for all sequenced prokaryotes. We observed a phylum-dependent distribution that is suggestive of an evolutionary relationship between Sigma-54 and lipopolysaccharide and flagellar biosynthesis. B) All Sigma-54 activators were identified and annotated. The relation with phosphotransfer-mediated signaling (TCS and PTS) and the transport and assimilation of carboxylates and nitrogen containing metabolites was substantiated. C) The function annotations, that were represented within the genomic context of all genes encoding Sigma-54, its activators and its promoters, were analyzed for intra-phylum representation and inter-phylum conservation. Promoters were localized using a straightforward scoring strategy that was formulated to identify similar motifs. We found clear highly-represented and conserved genetic associations with genes that concern the transport and biosynthesis of the metabolic intermediates of exopolysaccharides, flagella, lipids, lipopolysaccharides, lipoproteins and peptidoglycan. CONCLUSION: Our analyses directly implicate Sigma-54 as a central player in the control over the processes that involve the physical interaction of an organism with its environment like in the colonization of a host (virulence) or the formation of biofilm