Whether microbiome characteristics of induced sputum or oral samples demonstrate unique relationships to features of atopy or mild asthma in adults is unknown.
We sought to determine sputum and oral ...microbiota relationships to clinical or immunologic features in mild atopic asthma and the impact on the microbiota of inhaled corticosteroid (ICS) treatment administered to ICS-naive subjects with asthma.
Bacterial microbiota profiles were analyzed in induced sputum and oral wash samples from 32 subjects with mild atopic asthma before and after inhaled fluticasone treatment, 18 atopic subjects without asthma, and 16 nonatopic healthy subjects in a multicenter study (NCT01537133). Associations with clinical and immunologic features were examined, including markers of atopy, type 2 inflammation, immune cell populations, and cytokines.
Sputum bacterial burden inversely associated with bronchial expression of type 2 (T2)-related genes. Differences in specific sputum microbiota also associated with T2-low asthma phenotype, a subgroup of whom displayed elevations in lung inflammatory mediators and reduced sputum bacterial diversity. Differences in specific oral microbiota were more reflective of atopic status. After ICS treatment of patients with asthma, the compositional structure of sputum microbiota showed greater deviation from baseline in ICS nonresponders than in ICS responders.
Novel associations of sputum and oral microbiota to immunologic features were observed in this cohort of subjects with or without ICS-naive mild asthma. These findings confirm and extend our previous report of reduced bronchial bacterial burden and compositional complexity in subjects with T2-high asthma, with additional identification of a T2-low subgroup with a distinct microbiota-immunologic relationship.
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Mapping protein–protein interactions is an invaluable tool for understanding protein function. Here, we report the first large‐scale study of protein–protein interactions in human cells using a mass ...spectrometry‐based approach. The study maps protein interactions for 338 bait proteins that were selected based on known or suspected disease and functional associations. Large‐scale immunoprecipitation of Flag‐tagged versions of these proteins followed by LC‐ESI‐MS/MS analysis resulted in the identification of 24 540 potential protein interactions. False positives and redundant hits were filtered out using empirical criteria and a calculated interaction confidence score, producing a data set of 6463 interactions between 2235 distinct proteins. This data set was further cross‐validated using previously published and predicted human protein interactions. In‐depth mining of the data set shows that it represents a valuable source of novel protein–protein interactions with relevance to human diseases. In addition, via our preliminary analysis, we report many novel protein interactions and pathway associations.
Synopsis
Understanding the roles and consequences of protein–protein interactions is a fundamental goal in cellular biology and a prerequisite for the development of molecular systems biology. The endeavor of cataloging protein interactions is primarily hindered by the throughput and reproducibility of existing technologies. Different techniques for mapping protein interactions are available, such as the two‐hybrid approach (Chien et al, 1991) and the LUMIER approach (Barrios‐Rodiles et al, 2005) and assay whether two proteins interact in a pair‐wise fashion. We have developed a high‐throughput platform combining immunoprecipitation and high‐throughput mass spectrometry (IP‐HTMS) to rapidly identify potentially novel protein interactions for a bait protein of interest. We (Ho et al, 2002) and others (Gavin et al, 2002) previously used this approach to map protein–protein interactions in yeast, creating invaluable data sets for yeast biology and extrapolation into mammalian biology.
Mapping protein interactions in human cells has its own set of challenges owing to the number of potentially expressed genes, the number of different cell types, and the numbers of internal and external factors that impact the cellular system. Although a complete mapping of the human interactome is still beyond current capabilities, more focused studies are possible. Here we report the first large‐scale application of IP‐HTMS to the mapping of protein–protein interactions in human cells using 338 human bait proteins of significant biomedical interest. The complete data set is available from the Intact database (http://www.ebi.ac.uk/intact/site/) (accession EBI‐1059370) or as a table of bait–prey pairs with associated confidence values (Supplementary Table II).
There has been much focus and discussion over the last few years on the quality and reproducibility of interactions in high‐throughput protein–protein interaction datasets (e.g. von Mering et al, 2002). A guiding principle in our study has therefore been to implement stringent quality controls. The final data set includes protein interactions for 338 human bait proteins (Supplementary Table I). For over half of these baits, two or more replicate immunoprecipitation experiments were performed, requiring a total of 1034 individual immunoprecipitation experiments with associated SDS–PAGE. These experiments yielded over 16 000 gel bands for which over 400 000 MS/MS spectra were assigned peptide sequences. Approximately 1/5 of our immunoprecipitation experiments were control (no‐bait) experiments allowing us to build a comprehensive list of spurious and ubiquitously binding proteins that could then be filtered out of the interaction network. Another 1/5 of the experiments were directed towards a study of the reproducibility of prey protein identification using our platform. These 202 immunoprecipitation experiments, derived from 18 baits, were used to train a statistical model that associates interaction reproducibility with various observed experimental parameters, such as the number of peptides identified for the given prey protein. This model was used to assign confidence values (taking a value between 0 and 1) to each of the 6486 interactions in the data set.
As the interaction confidence score is calculated solely from IP‐HTMS experimental parameters, an initial focus was to confirm that the confidence score was an accurate means of ranking the interactions for further study. We observed, for example, that known interactions in the data set have, on average, significantly higher interaction confidence scores. For example, the set of baits corresponding to core and regulatory components of the proteasome enabled reconstruction of a proteasome interaction network (Figure 6C), comprising many known proteasome components and enriched for high‐scoring interactions.
We also integrated the IP‐HTMS data set with several other genomic‐scale data including other protein–protein interaction data sets, gene co‐expression data, and annotations from the gene ontology project. In the latter case, we analyzed the frequency of co‐occurrence of both bait and prey protein in the same biological process or cellular component category (Figure 3). We find that there is significant enrichment of bait–prey pairs sharing the same annotation category, indicating a strong tendency for bait proteins to bind prey proteins with related functions. Integration with gene co‐expression data showed that interaction data sets, this one included, are enriched for gene pairs that are co‐expressed. This enabled identification of tightly clustered sets of protein interactors that are also co‐expressed at the mRNA level. For example, the LYAR bait protein (Ly1 antibody reactive clone) is a nucleolar protein of unknown function (Su et al, 1993). This bait identified a set of nucleolar‐localized prey proteins that are also very tightly co‐expressed (Figure 5). These results along with the other protein–protein interaction data sources provided a powerful means of cross‐validating the human IP‐HTMS data set and associated methodology.
Our focus in this paper has been to prepare a quality‐controlled, large‐scale human protein interaction data set that will add significantly to our knowledge of the human protein interactome. Given the focus on baits of significant biomedical interest (through functional or disease associations), we anticipate that this data set alongside other sources of human protein–protein interactions will be an important starting point for functional characterization of disease‐related interactions and complexes. The IP‐HTMS platform utilized here shows great promise as an effective means of protein interaction discovery and we anticipate that future applications will include broadening to a larger set of disease associated proteins, to other cell lines and coupling with drug treatments.
We present a dataset of 6486 interactions between 2371 distinct proteins from a large‐scale application of immunoprecipitation and high‐throughput mass‐spectrometry (IP‐HTMS) on 338 human bait proteins expressed in human cells.
The dataset is cross‐validated using previously published and predicted human protein interactions. In depth mining of the dataset shows that it represents a valuable source of novel protein‐protein interactions with relevance to human diseases. In addition, our analysis reveals many novel protein interactions and pathway associations.
Protein interactions in the dataset are accompanied by a confidence score which is derived by combining several experimental and protein identification analysis metrics.
Successful treatment of chronic graft-versus-host disease (GvHD) often requires long-term systemic therapy (ST). Durable discontinuation of ST reflects the resolution of active chronic GvHD. We ...evaluated the factors associated with durable ST discontinuation, defined as cessation of all ST for ≥12 months, using data from two prospectively followed cohorts from the Chronic GvHD Consortium (n=684). Transplant sources were peripheral blood (89%), bone marrow (6.6%), and cord blood (4.4%) from HLA matched related (37.6%), HLA matched unrelated (45%), and other donor types (18%). Half of the patients received non-myeloablative conditioning. The median time from transplantation to chronic GvHD diagnosis was 7.7 months (range, 1.0-141.3) and the median time from chronic GvHD onset to enrollment into the cohorts was 0.9 months (range, 0.0-12.0). The cumulative incidence estimate of durable ST discontinuation was 32% (95% confidence interval: 28%-37%) at 10 years after enrollment into the cohort. Among patients who discontinued ST, the median time from chronic GvHD diagnosis to durable ST discontinuation was 3.6 years (range, 1.2-10.5). In multivariate analysis, patients who received myeloablative conditioning, had chronic GvHD manifested as moderate/severe lower gastrointestinal involvement, and had a higher (worse) Lee symptom overall score were less likely to attain durable ST discontinuation. In contrast, mild lower gastrointestinal involvement and cord blood (vs. peripheral blood) as the graft source were associated with a greater likelihood of ST discontinuation. Although a minority of patients can discontinue ST permanently, most patients require prolonged ST. Viewing chronic GvHD in this way has implications for management approaches.
In this first worldwide synthesis of in situ and satellite‐derived lake data, we find that lake summer surface water temperatures rose rapidly (global mean = 0.34°C decade−1) between 1985 and 2009. ...Our analyses show that surface water warming rates are dependent on combinations of climate and local characteristics, rather than just lake location, leading to the counterintuitive result that regional consistency in lake warming is the exception, rather than the rule. The most rapidly warming lakes are widely geographically distributed, and their warming is associated with interactions among different climatic factors—from seasonally ice‐covered lakes in areas where temperature and solar radiation are increasing while cloud cover is diminishing (0.72°C decade−1) to ice‐free lakes experiencing increases in air temperature and solar radiation (0.53°C decade−1). The pervasive and rapid warming observed here signals the urgent need to incorporate climate impacts into vulnerability assessments and adaptation efforts for lakes.
Key Points
Lake surface waters are warming rapidly but are spatially heterogeneous
Ice‐covered lakes are typically warming at rates greater than air temperatures
Both geomorphic and climate factors influence lake warming rates
The African continent is regarded as the cradle of modern humans and African genomes contain more genetic variation than those from any other continent, yet only a fraction of the genetic diversity ...among African individuals has been surveyed
. Here we performed whole-genome sequencing analyses of 426 individuals-comprising 50 ethnolinguistic groups, including previously unsampled populations-to explore the breadth of genomic diversity across Africa. We uncovered more than 3 million previously undescribed variants, most of which were found among individuals from newly sampled ethnolinguistic groups, as well as 62 previously unreported loci that are under strong selection, which were predominantly found in genes that are involved in viral immunity, DNA repair and metabolism. We observed complex patterns of ancestral admixture and putative-damaging and novel variation, both within and between populations, alongside evidence that Zambia was a likely intermediate site along the routes of expansion of Bantu-speaking populations. Pathogenic variants in genes that are currently characterized as medically relevant were uncommon-but in other genes, variants denoted as 'likely pathogenic' in the ClinVar database were commonly observed. Collectively, these findings refine our current understanding of continental migration, identify gene flow and the response to human disease as strong drivers of genome-level population variation, and underscore the scientific imperative for a broader characterization of the genomic diversity of African individuals to understand human ancestry and improve health.
Although protein hydroxylation is a relatively poorly characterized posttranslational modification, it has received significant recent attention following seminal work uncovering its role in oxygen ...sensing and hypoxia biology. Although the fundamental importance of protein hydroxylases in biology is becoming clear, the biochemical targets and cellular functions often remain enigmatic. JMJD5 is a "JmjC-only" protein hydroxylase that is essential for murine embryonic development and viability. However, no germline variants in JmjC-only hydroxylases, including JMJD5, have yet been described that are associated with any human pathology. Here we demonstrate that biallelic germline JMJD5 pathogenic variants are deleterious to JMJD5 mRNA splicing, protein stability, and hydroxylase activity, resulting in a human developmental disorder characterized by severe failure to thrive, intellectual disability, and facial dysmorphism. We show that the underlying cellular phenotype is associated with increased DNA replication stress and that this is critically dependent on the protein hydroxylase activity of JMJD5. This work contributes to our growing understanding of the role and importance of protein hydroxylases in human development and disease.
BACKGROUND:Medication errors continue to be a significant source of patient harm in the operating room with few concrete countermeasures. The organization and identification of medication syringes ...may have an impact on the commission of medication errors in anesthesia, so a team of physicians and designers at the University of Washington created the Anesthesia Medication Template (AMT) to define a formal way of organizing the anesthesia workspace. The purpose of this study is to assess the ability of the AMT to reduce perioperative medication errors by anesthesia providers.
METHODS:This study evaluated the AMT in 2 phases(1) 41 anesthesia providers administered medications in 2 prospective, randomized operating room simulations with or without the AMT, while medication errors were directly observed; and (2) around 200 providers prospectively self-reported medication errors from all anesthetizing locations during a 2-year period at Seattle Children’s Hospital, an academic, pediatric medical center.
RESULTS:In simulated emergencies, the odds of medication dosing errors using the AMT were 0.21 times the odds of medication dosing errors without AMT (95% confidence interval CI, 0.07, 0.66), controlling for scenario, session, training level, and years at training level. During the year after implementation of the AMT, the mean monthly error rate for all reported medication errors that reached patients decreased from 1.24 (95% CI, 0.85–1.79) to 0.65 (95% CI, 0.39–1.09) errors per 1000 anesthetics. The mean monthly error rate of reported swap, preparation, miscalculation, and timing errors decreased from 0.97 (95% CI, 0.64–1.48) to 0.35 (95% CI, 0.17–0.70) errors per 1000 anesthetics. Medication errors that resulted in patient harm did not change after implementation of the AMT.
CONCLUSIONS:Standardizing medication organization with the AMT is an intuitive, low-cost strategy with the potential to improve patient safety through reducing medication errors by anesthesia providers.
Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an ion channelopathy characterized by ventricular arrhythmia during exertion or stress. Mutations in RYR2-coded Ryanodine Receptor-2 ...(RyR2) and CASQ2-coded Calsequestrin-2 (CASQ2) genes underlie CPVT1 and CPVT2, respectively. However, prognostic markers are scarce. We sought to better characterize the phenotypic and genotypic spectrum of CPVT, and utilize molecular modelling to help account for clinical phenotypes.
This is a Pediatric and Congenital Electrophysiology Society multicentre, retrospective cohort study of CPVT patients diagnosed at <19 years of age and their first-degree relatives. Genetic testing was undertaken in 194 of 236 subjects (82%) during 3.5 (1.4-5.3) years of follow-up. The majority (60%) had RyR2-associated CPVT1. Variant locations were predicted based on a 3D structural model of RyR2. Specific residues appear to have key structural importance, supported by an association between cardiac arrest and mutations in the intersubunit interface of the N-terminus, and the S4-S5 linker and helices S5 and S6 of the RyR2 C-terminus. In approximately one quarter of symptomatic patients, cardiac events were precipitated by only normal wakeful activities.
This large, multicentre study identifies contemporary challenges related to the diagnosis and prognostication of CPVT patients. Structural modelling of RyR2 can improve our understanding severe CPVT phenotypes. Wakeful rest, rather than exertion, often precipitated life-threatening cardiac events.
Atmospheric carbon dioxide enrichment (eCO
) can enhance plant carbon uptake and growth
, thereby providing an important negative feedback to climate change by slowing the rate of increase of the ...atmospheric CO
concentration
. Although evidence gathered from young aggrading forests has generally indicated a strong CO
fertilization effect on biomass growth
, it is unclear whether mature forests respond to eCO
in a similar way. In mature trees and forest stands
, photosynthetic uptake has been found to increase under eCO
without any apparent accompanying growth response, leaving the fate of additional carbon fixed under eCO
unclear
. Here using data from the first ecosystem-scale Free-Air CO
Enrichment (FACE) experiment in a mature forest, we constructed a comprehensive ecosystem carbon budget to track the fate of carbon as the forest responded to four years of eCO
exposure. We show that, although the eCO
treatment of +150 parts per million (+38 per cent) above ambient levels induced a 12 per cent (+247 grams of carbon per square metre per year) increase in carbon uptake through gross primary production, this additional carbon uptake did not lead to increased carbon sequestration at the ecosystem level. Instead, the majority of the extra carbon was emitted back into the atmosphere via several respiratory fluxes, with increased soil respiration alone accounting for half of the total uptake surplus. Our results call into question the predominant thinking that the capacity of forests to act as carbon sinks will be generally enhanced under eCO
, and challenge the efficacy of climate mitigation strategies that rely on ubiquitous CO
fertilization as a driver of increased carbon sinks in global forests.
Glucocorticoids potently inhibit expression of many inflammatory mediators, and have been widely used to treat both acute and chronic inflammatory diseases for more than seventy years. However, they ...can have several unwanted effects, amongst which immunosuppression is one of the most common. Here we used microarrays and proteomic approaches to characterise the effect of dexamethasone (a synthetic glucocorticoid) on the responses of primary mouse macrophages to a potent pro-inflammatory agonist, lipopolysaccharide (LPS). Gene ontology analysis revealed that dexamethasone strongly impaired the lipopolysaccharide-induced antimicrobial response, which is thought to be driven by an autocrine feedback loop involving the type I interferon IFNβ. Indeed, dexamethasone strongly and dose-dependently inhibited the expression of IFNβ by LPS-activated macrophages. Unbiased proteomic data also revealed an inhibitory effect of dexamethasone on the IFNβ-dependent program of gene expression, with strong down-regulation of several interferon-induced antimicrobial factors. Surprisingly, dexamethasone also inhibited the expression of several antimicrobial genes in response to direct stimulation of macrophages with IFNβ. We tested a number of hypotheses based on previous publications, but found that no single mechanism could account for more than a small fraction of the broad suppressive impact of dexamethasone on macrophage type I interferon signaling, underlining the complexity of this pathway. Preliminary experiments indicated that dexamethasone exerted similar inhibitory effects on primary human monocyte-derived or alveolar macrophages.
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