Proteins circulating in the blood are critical for age-related disease processes; however, the serum proteome has remained largely unexplored. To this end, 4137 proteins covering most predicted ...extracellular proteins were measured in the serum of 5457 Icelanders over 65 years of age. Pairwise correlation between proteins as they varied across individuals revealed 27 different network modules of serum proteins, many of which were associated with cardiovascular and metabolic disease states, as well as overall survival. The protein modules were controlled by cis- and trans-acting genetic variants, which in many cases were also associated with complex disease. This revealed co-regulated groups of circulating proteins that incorporated regulatory control between tissues and demonstrated close relationships to past, current, and future disease states.
Although it has long been recognized that the enteric community of bacteria that inhabit the human distal intestinal track broadly impacts human health, the biochemical details that underlie these ...effects remain largely undefined. Here, we report a broad MS-based metabolomics study that demonstrates a surprisingly large effect of the gut "microbiome" on mammalian blood metabolites. Plasma extracts from germ-free mice were compared with samples from conventional (conv) animals by using various MS-based methods. Hundreds of features were detected in only 1 sample set, with the majority of these being unique to the conv animals, whereas almost equal to10% of all features observed in both sample sets showed significant changes in their relative signal intensity. Amino acid metabolites were particularly affected. For example, the bacterial-mediated production of bioactive indole-containing metabolites derived from tryptophan such as indoxyl sulfate and the antioxidant indole-3-propionic acid (IPA) was impacted. Production of IPA was shown to be completely dependent on the presence of gut microflora and could be established by colonization with the bacterium Clostridium sporogenes. Multiple organic acids containing phenyl groups were also greatly increased in the presence of gut microbes. A broad, drug-like phase II metabolic response of the host to metabolites generated by the microbiome was observed, suggesting that the gut microflora has a direct impact on the drug metabolism capacity of the host. Together, these results suggest a significant interplay between bacterial and mammalian metabolism.
Significant innovations in molecular biology methods have vastly improved the speed and efficiency of traditional restriction site and ligase-based cloning strategies. "Enzyme-free" methods eliminate ...the need to incorporate constrained sequences or modify Polymerase Chain Reaction (PCR)-generated DNA fragment ends. The Polymerase Incomplete Primer Extension (PIPE) method further condenses cloning and mutagenesis to a very simple two-step protocol with complete design flexibility not possible using related strategies. With this protocol, all major cloning operations are achieved by transforming competent cells with PCR products immediately following amplification. Normal PCRs generate mixtures of incomplete extension products. Using simple primer design rules and PCR, short, overlapping sequences are introduced at the ends of these incomplete extension mixtures which allow complementary strands to anneal and produce hybrid vector/insert combinations. These hybrids are directly transformed into recipient cells without any post-PCR enzymatic manipulations. We have found this method to be very easy and fast as compared to other available methods while retaining high efficiencies. Using this approach, we have cloned thousands of genes in parallel using a minimum of effort. The method is robust and amenable to automation as only a few, simple processing steps are needed.
Translocation of bacteria and their products across the intestinal barrier is common in patients with liver disease, and there is evidence that experimental liver fibrosis depends on bacterial ...translocation. The purpose of our study was to investigate liver fibrosis in conventional and germ‐free (GF) C57BL/6 mice. Chronic liver injury was induced by administration of thioacetamide (TAA) in the drinking water for 21 wk or by repeated intraperitoneal injections of carbon tetrachloride (CCl4). Increased liver fibrosis was observed in GF mice compared with conventional mice. Hepatocytes showed more toxin‐induced oxidative stress and cell death. This was accompanied by increased activation of hepatic stellate cells, but hepatic mediators of inflammation were not significantly different. Similarly, a genetic model using Myd88/Trif‐deficient mice, which lack downstream innate immunity signaling, had more severe fibrosis than wild‐type mice. Isolated Myd88/Trif‐deficient hepatocytes were more susceptible to toxin‐induced cell death in culture. In conclusion, the commensal microbiota prevents fibrosis upon chronic liver injury in mice. This is the first study describing a beneficial role of the commensal microbiota in maintaining liver homeostasis and preventing liver fibrosis.—Mazagova, M., Wang, L., Anfora, A. T., Wissmueller, M., Lesley, S. A., Miyamoto, Y., Eckmann, L., Dhungana, S., Pathmasiri, W., Sumner, S., Westwater, C., Brenner, D. A., Schnabl, B., Commensal microbiota is hepatoprotective and prevents liver fibrosis in mice. FASEB J. 29, 1043–1055 (2015). www.fasebj.org
The COVID-19 global incidence rate among young adults (age 19–40) drastically increased since summer 2020, and young adults were often portrayed by popular media as the "main spreader" of the ...pandemic. However, young adults faced unique challenges during the pandemic due to working in high-risk, low-paying essential service occupations, as well as having higher levels of financial insecurity and mental burden. This qualitative study aims to examine the attitudes and perceptions of health orders of young adults to better inform public health messaging to reach this demographic and increase compliance to public health orders. A total of 50 young adults residing in British Columbia, Canada, were recruited to participate in focus group in groups of four to six. Focus group discussions were conducted via teleconferencing. Thematic analysis revealed four major themes: 1) risks of contracting the disease, 2) the perceived impact of COVID-19, 3) responsibility of institutions, 4) and effective public health messaging. Contrary to existing literature, our findings suggest young adults feel highly responsible for protecting themselves and others. They face a higher risk of depression and anxiety compared to other age groups, especially when they take on multiple social roles such as caregivers and parents. Our findings suggest young adults face confusion due to inconsistent messaging and are not reached due to the ineffectiveness of existing strategies. We recommend using evidence-based strategies proven to promote behaviour change to address the barriers identified by young adults through tailoring public health messages, specifically by using positive messaging, messaging that considers the context of the intended audiences, and utilizing digital platforms to facilitate two-way communication.
Pili are proteinaceous polymers of linked pilins that protrude from the cell surface of many bacteria and often mediate adherence and virulence. We investigated a set of 20 Bacteroidia pilins from ...the human microbiome whose structures and mechanism of assembly were unknown. Crystal structures and biochemical data revealed a diverse protein superfamily with a common Greek-key β sandwich fold with two transthyretin-like repeats that polymerize into a pilus through a strand-exchange mechanism. The assembly mechanism of the central, structural pilins involves proteinase-assisted removal of their N-terminal β strand, creating an extended hydrophobic groove that binds the C-terminal donor strands of the incoming pilin. Accessory pilins at the tip and base have unique structural features specific to their location, allowing initiation or termination of the assembly. The Bacteroidia pilus, therefore, has a biogenesis mechanism that is distinct from other known pili and likely represents a different type of bacterial pilus.
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•A new pilin superfamily uncovered by structural exploration of the gut microbiome•A distinct mechanism of pilus biogenesis in Bacteroidia of the human microbiome•Mechanistic insights into proteinase-mediated pilin polymerization•Key features of the tip, stalk, and anchor subunits of the pilus assembly
Structural exploration of the gut microbiome uncovers a new pilin superfamily that is likely critical for microbe-host or microbe-microbe interactions.
ALK (anaplastic lymphoma kinase) is an RTK (receptor tyrosine kinase) of the IRK (insulin receptor kinase) superfamily, which share an YXXXYY autophosphorylation motif within their A-loops ...(activation loops). A common activation and regulatory mechanism is believed to exist for members of this superfamily typified by IRK and IGF1RK (insulin-like growth factor receptor kinase-1). Chromosomal translocations involving ALK were first identified in anaplastic large-cell lymphoma, a subtype of non-Hodgkin's lymphoma, where aberrant fusion of the ALK kinase domain with the NPM (nucleophosmin) dimerization domain results in autophosphosphorylation and ligand-independent activation. Activating mutations within the full-length ALK kinase domain, most commonly R1275Q and F1174L, which play a major role in neuroblastoma, were recently identified. To provide a structural framework for understanding these mutations and to guide structure-assisted drug discovery efforts, the X-ray crystal structure of the unphosphorylated ALK catalytic domain was determined in the apo, ADP- and staurosporine-bound forms. The structures reveal a partially inactive protein kinase conformation distinct from, and lacking, many of the negative regulatory features observed in inactive IGF1RK/IRK structures in their unphosphorylated forms. The A-loop adopts an inhibitory pose where a short proximal A-loop helix (alphaAL) packs against the alphaC helix and a novel N-terminal beta-turn motif, whereas the distal portion obstructs part of the predicted peptide-binding region. The structure helps explain the reported unique peptide substrate specificity and the importance of phosphorylation of the first A-loop Tyr1278 for kinase activity and NPM-ALK transforming potential. A single amino acid difference in the ALK substrate peptide binding P-1 site (where the P-site is the phosphoacceptor site) was identified that, in conjunction with A-loop sequence variation including the RAS (Arg-Ala-Ser)-motif, rationalizes the difference in the A-loop tyrosine autophosphorylation preference between ALK and IGF1RK/IRK. Enzymatic analysis of recombinant R1275Q and F1174L ALK mutant catalytic domains confirms the enhanced activity and transforming potential of these mutants. The transforming ability of the full-length ALK mutants in soft agar colony growth assays corroborates these findings. The availability of a three-dimensional structure for ALK will facilitate future structure-function and rational drug design efforts targeting this receptor tyrosine kinase.
Early detection of immunotherapy-induced tumor response is of major benefit for patients but can be complicated by therapy-induced pseudoprogression. A consensus guideline-iRECIST- was developed as a ...modification of Response Evaluation Criteria in Solid Tumours (RECIST version 1.1). Here we describe which next steps are required to test its validity and how novel approaches for response criteria might be developed and included.
Abstract
Marine heatwaves cause widespread environmental, biological, and socio-economic impacts, placing them at the forefront of 21st-century management challenges. However, heatwaves vary in ...intensity and evolution, and a paucity of information on how this variability impacts marine species limits our ability to proactively manage for these extreme events. Here, we model the effects of four recent heatwaves (2014, 2015, 2019, 2020) in the Northeastern Pacific on the distributions of 14 top predator species of ecological, cultural, and commercial importance. Predicted responses were highly variable across species and heatwaves, ranging from near total loss of habitat to a two-fold increase. Heatwaves rapidly altered political bio-geographies, with up to 10% of predicted habitat across all species shifting jurisdictions during individual heatwaves. The variability in predicted responses across species and heatwaves portends the need for novel management solutions that can rapidly respond to extreme climate events. As proof-of-concept, we developed an operational dynamic ocean management tool that predicts predator distributions and responses to extreme conditions in near real-time.
XtalPred is a web server for prediction of protein crystallizability. The prediction is made by comparing several features of the protein with distributions of these features in TargetDB and ...combining the results into an overall probability of crystallization. XtalPred provides: (1) a detailed comparison of the protein's features to the corresponding distribution from TargetDB; (2) a summary of protein features and predictions that indicate problems that are likely to be encountered during protein crystallization; (3) prediction of ligands; and (4) (optional) lists of close homologs from complete microbial genomes that are more likely to crystallize. Availability: The XtalPred web server is freely available for academic users on http://ffas.burnham.org/XtalPred Contact: adam@burnham.org
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