In-Cell Biochemistry Using NMR Spectroscopy

Biochemistry and structural biology are undergoing a dramatic revolution. Until now, mostly in vitro techniques have been used to study subtle and complex biological processes under conditions usually remote from those existing in the cell. We developed a novel in-cell methodology to post-translationally modify interactor proteins and identify the amino acids that comprise the interaction surface of a target protein when bound to the post-translationally modified interactors. Modifying the interactor proteins causes structural changes that manifest themselves on the interacting surface of the target protein and these changes are monitored using in-cell NMR. We show how Ubiquitin interacts with phosphorylated and non-phosphorylated components of the receptor tyrosine kinase (RTK) endocytic sorting machinery: STAM2 (Signal-transducing adaptor molecule), Hrs (Hepatocyte growth factor regulated substrate) and the STAM2-Hrs heterodimer. Ubiquitin binding mediates the processivity of a large network of interactions required for proper functioning of the RTK sorting machinery. The results are consistent with a weakening of the network of interactions when the interactor proteins are phosphorylated. The methodology can be applied to any stable target molecule and may be extended to include other post-translational modifications such as ubiquitination or sumoylation, thus providing a long-awaited leap to high resolution in cell biochemistry

Non Linear Programming (NLP) Formulation for Quantitative Modeling of Protein Signal Transduction Pathways

Modeling of signal transduction pathways plays a major role in understanding cells' function and predicting cellular response. Mathematical formalisms based on a logic formalism are relatively simple but can describe how signals propagate from one protein to the next and have led to the construction of models that simulate the cells response to environmental or other perturbations. Constrained fuzzy logic was recently introduced to train models to cell specific data to result in quantitative pathway models of the specific cellular behavior. There are two major issues in this pathway optimization: i) excessive CPU time requirements and ii) loosely constrained optimization problem due to lack of data with respect to large signaling pathways. Herein, we address both issues: the former by reformulating the pathway optimization as a regular nonlinear optimization problem; and the latter by enhanced algorithms to pre/post-process the signaling network to remove parts that cannot be identified given the experimental conditions. As a case study, we tackle the construction of cell type specific pathways in normal and transformed hepatocytes using medium and large-scale functional phosphoproteomic datasets. The proposed Non Linear Programming (NLP) formulation allows for fast optimization of signaling topologies by combining the versatile nature of logic modeling with state of the art optimization algorithms.National Institutes of Health (U.S.) (Grant P50-GM068762)National Institutes of Health (U.S.) (Grant R24-DK090963)United States. Army Research Office (Grant W911NF-09-0001)German Research Foundation (Grant GSC 111

Air pollution from household solid fuel combustion in India: an overview of exposure and health related information to inform health research priorities

Environmental and occupational risk factors contribute to nearly 40% of the national burden of disease in India, with air pollution in the indoor and outdoor environment ranking amongst leading risk factors. It is now recognized that the health burden from air pollution exposures that primarily occur in the rural indoors, from pollutants released during the incomplete combustion of solid fuels in households, may rival or even exceed the burden attributable to urban outdoor exposures. Few environmental epidemiological efforts have been devoted to this setting, however. We provide an overview of important available information on exposures and health effects related to household solid fuel use in India, with a view to inform health research priorities for household air pollution and facilitate being able to address air pollution within an integrated rural–urban framework in the future

Th17 Cells and IL-17 in Protective Immunity to Vaginal Candidiasis

Background: Th17 cells play a major role in coordinating the host defence in oropharyngeal candidiasis. In this study we investigated the involvement of the Th17 response in an animal model of vulvovaginal candidiasis (VVC). Methods: To monitor the course of infection we exploited a new in vivo imaging technique. Results: i) The progression of VVC leads to a strong influx of neutrophils in the vagina soon after the challenge which persisted despite the resolution of infection; ii) IL-17, produced by vaginal cells, particularly CD4 T cells, was detected in the vaginal wash during the infection, reaching a maximum 14 days after the challenge; iii) The amount and kinetics of IL-23 in vaginal fluids were comparable to those in vaginal cells; iv) The inhibition of Th17 differentiation led to significant inhibition of IL-17 production with consequent exacerbation of infection; v) An increased production of bdefensin 2 was manifested in cells of infected mice. This production was strongly reduced when Th17 differentiation was inhibited and was increased by rIL-17 treatment. Conclusions: These results imply that IL-17 and Th17, along with innate antimicrobial factors, have a role in the immune response to vaginal candidiasis

Effects of anthropogenic activities on the heavy metal levels in the clams and sediments in a tropical river

The present study aimed to assess the effects of anthropogenic activities on the heavy metal levels in the Langat River by transplantation of Corbicula javanica. In addition, potential ecological risk indexes (PERI) of heavy metals in the surface sediments of the river were also investigated. The correlation analysis revealed that eight metals (As, Co, Cr, Fe, Mn, Ni, Pb and Zn) in total soft tissue (TST) while five metals (As, Cd, Cr, Fe and Mn) in shell have positively and significantly correlation with respective metal concentration in sediment, indicating the clams is a good biomonitor of the metal levels. Based on clustering patterns, the discharge of dam impoundment, agricultural activities and urban domestic waste were identified as three major contributors of the metals in Pangsun, Semenyih and Dusun Tua, and Kajang, respectively. Various geochemical indexes for a single metal pollutant (geoaccumulation index (I geo), enrichment factors (EF), contamination factor (C f) and ecological risk (Er)) all agreed that Cd, Co, Cr, Cu, Fe, Mn, Ni and Zn are not likely to cause adverse effect to the river ecosystem, but As and Pb could pose a potential ecological risk to the river ecosystem. All indexes (degree of contamination (C d), combined pollution index (CPI) and PERI) showed that overall metal concentrations in the tropical river are still within safe limit. River metal pollution was investigated. Anthropogenic activities were contributors of the metal pollution. Geochemical indexes showed that metals are within the safe limit

Adaptations to Submarine Hydrothermal Environments Exemplified by the Genome of Nautilia profundicola

Submarine hydrothermal vents are model systems for the Archaean Earth environment, and some sites maintain conditions that may have favored the formation and evolution of cellular life. Vents are typified by rapid fluctuations in temperature and redox potential that impose a strong selective pressure on resident microbial communities. Nautilia profundicola strain Am-H is a moderately thermophilic, deeply-branching Epsilonproteobacterium found free-living at hydrothermal vents and is a member of the microbial mass on the dorsal surface of vent polychaete, Alvinella pompejana. Analysis of the 1.7-Mbp genome of N. profundicola uncovered adaptations to the vent environment—some unique and some shared with other Epsilonproteobacterial genomes. The major findings included: (1) a diverse suite of hydrogenases coupled to a relatively simple electron transport chain, (2) numerous stress response systems, (3) a novel predicted nitrate assimilation pathway with hydroxylamine as a key intermediate, and (4) a gene (rgy) encoding the hallmark protein for hyperthermophilic growth, reverse gyrase. Additional experiments indicated that expression of rgy in strain Am-H was induced over 100-fold with a 20°C increase above the optimal growth temperature of this bacterium and that closely related rgy genes are present and expressed in bacterial communities residing in geographically distinct thermophilic environments. N. profundicola, therefore, is a model Epsilonproteobacterium that contains all the genes necessary for life in the extreme conditions widely believed to reflect those in the Archaean biosphere—anaerobic, sulfur, H2- and CO2-rich, with fluctuating redox potentials and temperatures. In addition, reverse gyrase appears to be an important and common adaptation for mesophiles and moderate thermophiles that inhabit ecological niches characterized by rapid and frequent temperature fluctuations and, as such, can no longer be considered a unique feature of hyperthermophiles

G-protein α-subunit (GPA1) regulates stress, nitrate and phosphate response, flavonoid biosynthesis, fruit/seed development and substantially shares GCR1 regulation in A. <i>thaliana</i>

Heterotrimeric G-proteins are implicated in several plant processes, but the mechanisms of signal-response coupling and the roles of G-protein coupled receptors in general and GCR1 in particular, remain poorly understood. We isolated a knock-out mutant of the Arabidopsis G-protein α subunit (gpa1-5) and analysed its transcriptome to understand the genomewide role of GPA1 and compared it with that of our similar analysis of a GCR1 mutant (Chakraborty et al. 2015, PLoS ONE 10(2):e0117819). We found 394 GPA1-regulated genes spanning 79 biological processes, including biotic and abiotic stresses, development, flavonoid biosynthesis, transcription factors, transporters and nitrate/phosphate responses. Many of them are either unknown or unclaimed explicitly in other published gpa1 mutant transcriptome analyses. A comparison of all known GPA1-regulated genes (including the above 394) with 350 GCR1-regulated genes revealed 114 common genes. This can be best explained by GCR1–GPA1 coupling, or by convergence of their independent signaling pathways. Though the common genes in our GPA1 and GCR1 mutant datasets constitute only 26 % of the GPA1-regulated and 30 % of the GCR1-responsive genes, they belong to nearly half of all the processes affected in both the mutants. Thus, GCR1 and GPA1 regulate not only some common genes, but also different genes belonging to the same processes to achieve similar outcomes. Overall, we validate some known and report many hitherto unknown roles of GPA1 in plants, including agronomically important ones such as biotic stress and nutrient response, and also provide compelling genetic evidence to revisit the role of GCR1 in G-protein signalling

Intraperitoneal drain placement and outcomes after elective colorectal surgery: international matched, prospective, cohort study

Despite current guidelines, intraperitoneal drain placement after elective colorectal surgery remains widespread. Drains were not associated with earlier detection of intraperitoneal collections, but were associated with prolonged hospital stay and increased risk of surgical-site infections.Background Many surgeons routinely place intraperitoneal drains after elective colorectal surgery. However, enhanced recovery after surgery guidelines recommend against their routine use owing to a lack of clear clinical benefit. This study aimed to describe international variation in intraperitoneal drain placement and the safety of this practice. Methods COMPASS (COMPlicAted intra-abdominal collectionS after colorectal Surgery) was a prospective, international, cohort study which enrolled consecutive adults undergoing elective colorectal surgery (February to March 2020). The primary outcome was the rate of intraperitoneal drain placement. Secondary outcomes included: rate and time to diagnosis of postoperative intraperitoneal collections; rate of surgical site infections (SSIs); time to discharge; and 30-day major postoperative complications (Clavien-Dindo grade at least III). After propensity score matching, multivariable logistic regression and Cox proportional hazards regression were used to estimate the independent association of the secondary outcomes with drain placement. Results Overall, 1805 patients from 22 countries were included (798 women, 44.2 per cent; median age 67.0 years). The drain insertion rate was 51.9 per cent (937 patients). After matching, drains were not associated with reduced rates (odds ratio (OR) 1.33, 95 per cent c.i. 0.79 to 2.23; P = 0.287) or earlier detection (hazard ratio (HR) 0.87, 0.33 to 2.31; P = 0.780) of collections. Although not associated with worse major postoperative complications (OR 1.09, 0.68 to 1.75; P = 0.709), drains were associated with delayed hospital discharge (HR 0.58, 0.52 to 0.66; P &lt; 0.001) and an increased risk of SSIs (OR 2.47, 1.50 to 4.05; P &lt; 0.001). Conclusion Intraperitoneal drain placement after elective colorectal surgery is not associated with earlier detection of postoperative collections, but prolongs hospital stay and increases SSI risk