The Proteomics of N-terminal Methionine Cleavage

Methionine aminopeptidase (MAP) is a ubiquitous, essential enzyme involved in protein N-terminal methionine excision. According to the generally accepted cleavage rules for MAP, this enzyme cleaves all proteins with small side chains on the residue in the second position (P1′), but many exceptions are known. The substrate specificity of Escherichia coli MAP1 was studied in vitro with a large (\u3e120) coherent array of peptides mimicking the natural substrates and kinetically analyzed in detail. Peptides with Val or Thr at P1′ were much less efficiently cleaved than those with Ala, Cys, Gly, Pro, or Ser in this position. Certain residues at P2′, P3′, and P4′ strongly slowed the reaction, and some proteins with Val and Thr at P1′ could not undergo Met cleavage. These in vitro data were fully consistent with data for 862 E. coli proteins with known N-terminal sequences in vivo. The specificity sites were found to be identical to those for the other type of MAPs, MAP2s, and a dedicated prediction tool for Met cleavage is now available. Taking into account the rules of MAP cleavage and leader peptide removal, the N termini of all proteins were predicted from the annotated genome and compared with data obtained in vivo. This analysis showed that proteins displaying N-Met cleavage are overrepresented in vivo. We conclude that protein secretion involving leader peptide cleavage is more frequent than generally thought

A comparative study of three methods for detecting association of quantitative traits in samples of related subjects

We used Genetic Analysis Workshop 16 Problem 3 Framingham Heart Study simulated data set to compare methods for association analysis of quantitative traits in related individuals. More specifically, we investigated type I error and relative power of three approaches: the measured genotype, the quantitative transmission-disequilibrium test (QTDT), and the quantitative trait linkage-disequilibrium (QTLD) tests. We studied high-density lipoprotein and triglyceride (TG) lipid variables, as measured at Visit 1. Knowing the answers, we selected three true major genes for high-density lipoprotein and/or TG. Empirical distributions of the three association models were derived from the first 100 replicates. In these data, all three models were similar in error rates. Across the three association models, the power was the lowest for the functional SNP with smallest size effects (i.e., α2), and for the less heritable trait (i.e., TG). Our results showed that measured genotype outperformed the two orthogonal-based association models (QTLD, QTDT), even after accounting for population stratification. QTDT had the lowest power rates. This is consistent with the amount of marker and trait data used by each association model. While the effective sample sizes varied little across our tested variants, we observed some large power drops and marked differences in performances of the models. We found that the performances contrasted the most for the tightly linked, but not associated, functional variants

Comparative study of statistical methods for detecting association with rare variants in exome-resequencing data

Genome-wide association studies for complex traits are based on the common disease/common variant (CDCV) and common disease/rare variant (CDRV) assumptions. Under the CDCV hypothesis, classical genome-wide association studies using single-marker tests are powerful in detecting common susceptibility variants, but under the CDRV hypothesis they are not as powerful. Several methods have been recently proposed to detect association with multiple rare variants collectively in a functional unit such as a gene. In this paper, we compare the relative performance of several of these methods on the Genetic Analysis Workshop 17 data. We evaluate these methods using the unrelated individual and family data sets. Association was tested using 200 replicates for the quantitative trait Q1. Although in these data the power to detect association is often low, our results show that collapsing methods are promising tools. However, we faced the challenge of assessing the proper type I error to validate our power comparisons. We observed that the type I error rate was not well controlled; however, we did not find a general trend specific to each method. Each method can be conservative or nonconservative depending on the studied gene. Our results also suggest that collapsing and the single-locus association approaches may not be affected to the same extent by population stratification. This deserves further investigation

Multi-scale high-throughput phenotyping of apple architectural and functional traits in orchard reveals genotypic variability under contrasted watering regimes

Despite previous reports on the genotypic variation of architectural and functional traits in fruit trees, phenotyping large populations in the field remains challenging. In this study, we used high-throughput phenotyping methods on an apple tree core-collection (1000 individuals) grown under contrasted watering regimes. First, architectural phenotyping was achieved using T-LiDAR scans for estimating convex and alpha hull volumes and the silhouette to total leaf area ratio (STAR). Second, a semi-empirical index (IPL) was computed from chlorophyll fluorescence measurements, as a proxy for leaf photosynthesis. Last, thermal infrared and multispectral airborne imaging was used for computing canopy temperature variations, water deficit, and vegetation indices. All traits estimated by these methods were compared to low-throughput in planta measurements. Vegetation indices and alpha hull volumes were significantly correlated with tree leaf area and trunk cross sectional area, while IPL values showed strong correlations with photosynthesis measurements collected on an independent leaf dataset. By contrast, correlations between stomatal conductance and canopy temperature estimated from airborne images were lower, emphasizing discrepancies across measurement scales. High heritability values were obtained for almost all the traits except leaf photosynthesis, likely due to large intra-tree variation. Genotypic means were used in a clustering procedure that defined six classes of architectural and functional combinations. Differences between groups showed several combinations between architectural and functional traits, suggesting independent genetic controls. This study demonstrates the feasibility and relevance of combining multi-scale high-throughput methods and paves the way to explore the genetic bases of architectural and functional variations in woody crops in field conditions

Nucleoprotein Nanostructures Combined with Adjuvants Adapted to the Neonatal Immune Context: A Candidate Mucosal RSV Vaccine

BACKGROUND: The human respiratory syncytial virus (hRSV) is the leading cause of severe bronchiolitis in infants worldwide. The most severe RSV diseases occur between 2 and 6 months-of-age, so pediatric vaccination will have to be started within the first weeks after birth, when the immune system is prone to Th2 responses that may turn deleterious upon exposure to the virus. So far, the high risk to prime for immunopathological responses in infants has hampered the development of vaccine. In the present study we investigated the safety and efficacy of ring-nanostructures formed by the recombinant nucleoprotein N of hRSV (N(SRS)) as a mucosal vaccine candidate against RSV in BALB/c neonates, which are highly sensitive to immunopathological Th2 imprinting. METHODOLOGY AND PRINCIPAL FINDINGS: A single intranasal administration of N(SRS) with detoxified E. coli enterotoxin LT(R192G) to 5-7 day old neonates provided a significant reduction of the viral load after an RSV challenge at five weeks of age. However, neonatal vaccination also generated an enhanced lung infiltration by neutrophils and eosinophils following the RSV challenge. Analysis of antibody subclasses and cytokines produced after an RSV challenge or a boost administration of the vaccine suggested that neonatal vaccination induced a Th2 biased local immune memory. This Th2 bias and the eosinophilic reaction could be prevented by adding CpG to the vaccine formulation, which, however did not prevent pulmonary inflammation and neutrophil infiltration upon viral challenge. CONCLUSIONS/SIGNIFICANCE: In conclusion, protective vaccination against RSV can be achieved in neonates but requires an appropriate combination of adjuvants to prevent harmful Th2 imprinting

Analysis of the human monocyte-derived macrophage transcriptome and response to lipopolysaccharide provides new insights into genetic aetiology of inflammatory bowel disease

The FANTOM5 consortium utilised cap analysis of gene expression (CAGE) to provide an unprecedented insight into transcriptional regulation in human cells and tissues. In the current study, we have used CAGE-based transcriptional profiling on an extended dense time course of the response of human monocyte-derived macrophages grown in macrophage colony-stimulating factor (CSF1) to bacterial lipopolysaccharide (LPS). We propose that this system provides a model for the differentiation and adaptation of monocytes entering the intestinal lamina propria. The response to LPS is shown to be a cascade of successive waves of transient gene expression extending over at least 48 hours, with hundreds of positive and negative regulatory loops. Promoter analysis using motif activity response analysis (MARA) identified some of the transcription factors likely to be responsible for the temporal profile of transcriptional activation. Each LPS-inducible locus was associated with multiple inducible enhancers, and in each case, transient eRNA transcription at multiple sites detected by CAGE preceded the appearance of promoter-associated transcripts. LPS-inducible long non-coding RNAs were commonly associated with clusters of inducible enhancers. We used these data to re-examine the hundreds of loci associated with susceptibility to inflammatory bowel disease (IBD) in genome-wide association studies. Loci associated with IBD were strongly and specifically (relative to rheumatoid arthritis and unrelated traits) enriched for promoters that were regulated in monocyte differentiation or activation. Amongst previously-identified IBD susceptibility loci, the vast majority contained at least one promoter that was regulated in CSF1-dependent monocyte-macrophage transitions and/or in response to LPS. On this basis, we concluded that IBD loci are strongly-enriched for monocyte-specific genes, and identified at least 134 additional candidate genes associated with IBD susceptibility from reanalysis of published GWA studies. We propose that dysregulation of monocyte adaptation to the environment of the gastrointestinal mucosa is the key process leading to inflammatory bowel disease

Mortality and pulmonary complications in patients undergoing surgery with perioperative SARS-CoV-2 infection: an international cohort study

Background: The impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on postoperative recovery needs to be understood to inform clinical decision making during and after the COVID-19 pandemic. This study reports 30-day mortality and pulmonary complication rates in patients with perioperative SARS-CoV-2 infection. Methods: This international, multicentre, cohort study at 235 hospitals in 24 countries included all patients undergoing surgery who had SARS-CoV-2 infection confirmed within 7 days before or 30 days after surgery. The primary outcome measure was 30-day postoperative mortality and was assessed in all enrolled patients. The main secondary outcome measure was pulmonary complications, defined as pneumonia, acute respiratory distress syndrome, or unexpected postoperative ventilation. Findings: This analysis includes 1128 patients who had surgery between Jan 1 and March 31, 2020, of whom 835 (74·0%) had emergency surgery and 280 (24·8%) had elective surgery. SARS-CoV-2 infection was confirmed preoperatively in 294 (26·1%) patients. 30-day mortality was 23·8% (268 of 1128). Pulmonary complications occurred in 577 (51·2%) of 1128 patients; 30-day mortality in these patients was 38·0% (219 of 577), accounting for 81·7% (219 of 268) of all deaths. In adjusted analyses, 30-day mortality was associated with male sex (odds ratio 1·75 [95% CI 1·28–2·40], p\textless0·0001), age 70 years or older versus younger than 70 years (2·30 [1·65–3·22], p\textless0·0001), American Society of Anesthesiologists grades 3–5 versus grades 1–2 (2·35 [1·57–3·53], p\textless0·0001), malignant versus benign or obstetric diagnosis (1·55 [1·01–2·39], p=0·046), emergency versus elective surgery (1·67 [1·06–2·63], p=0·026), and major versus minor surgery (1·52 [1·01–2·31], p=0·047). Interpretation: Postoperative pulmonary complications occur in half of patients with perioperative SARS-CoV-2 infection and are associated with high mortality. Thresholds for surgery during the COVID-19 pandemic should be higher than during normal practice, particularly in men aged 70 years and older. Consideration should be given for postponing non-urgent procedures and promoting non-operative treatment to delay or avoid the need for surgery. Funding: National Institute for Health Research (NIHR), Association of Coloproctology of Great Britain and Ireland, Bowel and Cancer Research, Bowel Disease Research Foundation, Association of Upper Gastrointestinal Surgeons, British Association of Surgical Oncology, British Gynaecological Cancer Society, European Society of Coloproctology, NIHR Academy, Sarcoma UK, Vascular Society for Great Britain and Ireland, and Yorkshire Cancer Research

Soil Microbial Community Changes in Wooded Mountain Pastures due to Simulated Effects of Cattle Grazing

The effect of cattle activity on pastures can be subdivided into three categories of disturbances: herbage removal, dunging and trampling. The objective of this study was to assess separately or in combination the effect of these factors on the potential activities of soil microbial communities and to compare these effects with those of soil properties and plant composition or biomass. Controlled treatments simulating the three factors were applied in a fenced area including a light gradient (sunny and shady situation): (i) repeated mowing; (ii) trampling; (iii) fertilizing with a liquid mixture of dung and urine. In the third year of the experiment, community level physiological profiles (CLPP) (Biolog Ecoplates¿) were measured for each plots. Furthermore soil chemical properties (pH, total organic carbon, total nitrogen and total phosphorus), plant species composition and plant biomass were also assessed. Despite differences in plant communities and soil properties, the metabolic potential of the microbial community in the sunny and in the shady situations were similar. Effects of treatments on microbial communities were more pronounced in the sunny than in the shady situation. In both cases, repeated mowing was the first factor retained for explaining functional variations. In contrast, fertilizing was not a significant factor. The vegetation explained a high proportion of variation of the microbial community descriptors in the sunny situation, while no significant variation appeared under shady condition. The three components of cattle activities influenced differently the soil microbial communities and this depended on the light conditions within the wooded pasture. Cattle activities may also change spatially at a fine scale and short-term and induce changes in the microbial community structure. Thus, the shifting mosaic that has been described for the vegetation of pastures may also apply for below-ground microbial communities