Protein tyrosine phosphatase 69D is a substrate of protein O-mannosyltransferases 1-2 that is required for the wiring of sensory axons in Drosophila.

Mutations in protein O-mannosyltransferases (POMTs) result in severe brain defects and congenital muscular dystrophies characterized by abnormal glycosylation of α-dystroglycan (α-Dg). However, neurological phenotypes of POMT mutants are not well understood, and the functional substrates of POMTs other than α-Dg remain unknown. Using a Drosophila model, here we reveal that Dg alone cannot account for the phenotypes of POMT mutants, and identify Protein tyrosine phosphatase 69D (PTP69D) as a gene interacting with POMTs in producing the abdomen rotation phenotype. Using RNAi-mediated knockdown, mutant alleles, and a dominant-negative form of PTP69D, we reveal that PTP69D is required for the wiring of larval sensory axons. We also found that PTP69D and POMT genes interact in this process, and that their interactions lead to complex synergistic or antagonistic effects on axon wiring phenotypes, depending on the mode of genetic manipulation. Using glycoproteomic approaches, we further characterized the glycosylation of the PTP69D transgenic construct expressed in genetic strains with different levels of POMT activity. We found that the PTP69D construct carries many O-linked mannose modifications when expressed in Drosophila with wild-type or ectopically upregulated expression of POMTs. These modifications were absent in POMT mutants, suggesting that PTP69D is a substrate of POMT-mediated O-mannosylation. Taken together, our results indicate that PTP69D is a novel functional substrate of POMTs that is required for axon connectivity. This mechanism of POMT-mediated regulation of receptor-type protein tyrosine phosphatase functions could potentially be conserved in mammals and may shed new light on the etiology of neurological defects in muscular dystrophies.

Identification of a novel base J binding protein complex involved in RNA polymerase II transcription termination in trypanosomes.

Base J, ß-D-glucosyl-hydroxymethyluracil, is a modification of thymine DNA base involved in RNA Polymerase (Pol) II transcription termination in kinetoplastid protozoa. Little is understood regarding how specific thymine residues are targeted for J-modification or the mechanism of J regulated transcription termination. To identify proteins involved in J-synthesis, we expressed a tagged version of the J-glucosyltransferase (JGT) in Leishmania tarentolae, and identified four co-purified proteins by mass spectrometry: protein phosphatase (PP1), a homolog of Wdr82, a potential PP1 regulatory protein (PNUTS) and a protein containing a J-DNA binding domain (named JBP3). Gel shift studies indicate JBP3 is a J-DNA binding protein. Reciprocal tagging, co-IP and sucrose gradient analyses indicate PP1, JGT, JBP3, Wdr82 and PNUTS form a multimeric complex in kinetoplastids, similar to the mammalian PTW/PP1 complex involved in transcription termination via PP1 mediated dephosphorylation of Pol II. Using RNAi and analysis of Pol II termination by RNA-seq and RT-PCR, we demonstrate that ablation of PNUTS, JBP3 and Wdr82 lead to defects in Pol II termination at the 3'-end of polycistronic gene arrays in Trypanosoma brucei. Mutants also contain increased antisense RNA levels upstream of transcription start sites, suggesting an additional role of the complex in regulating termination of bi-directional transcription. In addition, PNUTS loss causes derepression of silent Variant Surface Glycoprotein genes involved in host immune evasion. Our results suggest a novel mechanistic link between base J and Pol II polycistronic transcription termination in kinetoplastids.

Identification and characterization of the Streptococcus pneumoniae type 3 capsule-specific glycoside hydrolase of Paenibacillus species 32352.

Bacillus circulans Jordan 32352 was isolated from decaying organic matter in the New Jersey soil in the early 1930s. This soil-dwelling bacterium produced an enzyme capable of degrading the type 3 capsular polysaccharide (Pn3P) of Streptococcus pneumoniae (Spn). Early reports of this enzyme, Pn3Pase, demonstrated its inducibility by, and specificity for Pn3P. We set out to identify and clone this enzyme for its recombinant expression and characterization. We first sequenced the genome of this bacterial species, and reclassified the Pn3Pase producing bacterium as Paenibacillus species 32352. We identified the putative protein of Pn3Pase through mass spectrometry-based proteomics and cloned the gene for recombinant expression. We then characterized the oligosaccharide products generated upon the enzymatic depolymerization of Pn3P. Sequence analysis suggests that this glycoside hydrolase belongs to a new carbohydrate-active enzyme GH family. To our knowledge, this is the only enzyme to demonstrate Pn3P depolymerization activity.

EUROCarbDB(CCRC): a EUROCarbDB node for storing glycomics standard data.

MOTIVATION: In the field of glycomics research, several different techniques are used for structure elucidation. Although multiple techniques are often used to increase confidence in structure assignments, most glycomics databases allow storing of only a single type of experimental data. In addition, the methods used to prepare a sample for analysis is seldom recorded making it harder to reproduce the analytical data and results. RESULTS: We have extended the freely available EUROCarbDB framework to allow the submission of experimental data and the reporting of several orthogonal experimental datasets. The features aim to increase the understandability and reproducibility of the reported data. AVAILABILITY AND IMPLEMENTATION: The installation with the glycan standards is available at http://glycomics.ccrc.uga.edu/eurocarb/. The source code of the project is available at https://code.google.com/p/ucdb/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Using the HSE stress indicator tool in a military context.

An assessment of the Health and Safety Executive's (HSE) stress indicator tool was conducted to determine whether it was suitable for use with Ministry of Defence (MoD) personnel. A total of 1031 respondents from the Army, Navy, Air Force and MoD civilians completed a questionnaire containing the HSE tool and supplementary measures of work-life balance, engagement, deployment, leave taken and hours of work. Six measures of adverse reaction to the stressors were also reported: perceptions of job stress, job stress and health, psychological strain, fatigue after work, work ability and quality of working life. The stressor scales, particularly the 'demands' and 'relationships' scales, were associated with adverse outcomes as was the work-life balance scale. The HSE tool had some validity when used with MoD personnel, but its content was too narrow. The content validity of the tool can be improved for use in a military context with the addition of a 'work-life balance' scale'. PRACTITIONER SUMMARY: The HSE stress tool was tested with a mixed sample of MoD employees. The 'Demands and "Relationships" scales were associated with adverse outcomes. An additional Work­Life Balance' scale improved the content validity, demonstrating the importance of assessing the psychometric qualities of scales when used within particular contexts to ensure validity.

Site-specific glycan microheterogeneity of inter-alpha-trypsin inhibitor heavy chain H4.

Inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4) is a 120 kDa acute-phase glycoprotein produced primarily in the liver, secreted into the blood, and identified in serum. ITIH4 is involved in liver development and stabilization of the extracellular matrix (ECM), and its expression is altered in liver disease. In this study, we aimed to characterize glycosylation of recombinant and serum-derived ITIH4 using analytical mass spectrometry. Recombinant ITIH4 was analyzed to optimize glycopeptide analyses, followed by serum-derived ITIH4. First, we confirmed that the four ITIH4 N-X-S/T sequons (N81, N207, N517, and N577) were glycosylated by treating ITIH4 tryptic/GluC glycopeptides with PNGaseF in the presence of (18)O water. Next, we performed glycosidase-assisted LC-MS/MS analysis of ITIH4 trypsin-GluC glycopeptides enriched via hydrophilic interaction liquid chromatography to characterize ITIH4 N-glycoforms. While microheterogeneity of N-glycoforms differed between ITIH4 protein expressed in HEK293 cells and protein isolated from serum, occupancy of N-glycosylation sites did not differ. A fifth N-glycosylation site was discovered at N274 with the rare nonconsensus NVV motif. Site N274 contained high-mannose N-linked glycans in both serum and recombinant ITIH4. We also identified isoform-specific ITIH4 O-glycoforms and documented that utilization of O-glycosylation sites on ITIH4 differed between the cell line and serum.

Enzymatic basis for N-glycan sialylation: structure of rat α2,6-sialyltransferase (ST6GAL1) reveals conserved and unique features for glycan sialylation.

Glycan structures on glycoproteins and glycolipids play critical roles in biological recognition, targeting, and modulation of functions in animal systems. Many classes of glycan structures are capped with terminal sialic acid residues, which contribute to biological functions by either forming or masking glycan recognition sites on the cell surface or secreted glycoconjugates. Sialylated glycans are synthesized in mammals by a single conserved family of sialyltransferases that have diverse linkage and acceptor specificities. We examined the enzymatic basis for glycan sialylation in animal systems by determining the crystal structures of rat ST6GAL1, an enzyme that creates terminal α2,6-sialic acid linkages on complex-type N-glycans, at 2.4 Å resolution. Crystals were obtained from enzyme preparations generated in mammalian cells. The resulting structure revealed an overall protein fold broadly resembling the previously determined structure of pig ST3GAL1, including a CMP-sialic acid-binding site assembled from conserved sialylmotif sequence elements. Significant differences in structure and disulfide bonding patterns were found outside the sialylmotif sequences, including differences in residues predicted to interact with the glycan acceptor. Computational substrate docking and molecular dynamics simulations were performed to predict and evaluate the CMP-sialic acid donor and glycan acceptor interactions, and the results were compared with kinetic analysis of active site mutants. Comparisons of the structure with pig ST3GAL1 and a bacterial sialyltransferase revealed a similar positioning of donor, acceptor, and catalytic residues that provide a common structural framework for catalysis by the mammalian and bacterial sialyltransferases.

ß-N-Acetylglucosamine (O-GlcNAc) is a novel regulator of mitosis-specific phosphorylations on histone H3.

O-Linked ß-N-acetylglucosamine, or O-GlcNAc, is a dynamic post-translational modification that cycles on and off serine and threonine residues of nucleocytoplasmic proteins. The O-GlcNAc modification shares a complex relationship with phosphorylation, as both modifications are capable of mutually inhibiting the occupation of each other on the same or nearby amino acid residue. In addition to diabetes, cancer, and neurodegenerative diseases, O-GlcNAc appears to play a significant role in cell growth and cell cycle progression, although the precise mechanisms are still not well understood. A recent study also found that all four core nucleosomal histones (H2A, H2B, H3, and H4) are modified with O-GlcNAc, although no specific sites on H3 were reported. Here, we describe that histone H3, a protein highly phosphorylated during mitosis, is modified with O-GlcNAc. Several biochemical assays were used to validate that H3 is modified with O-GlcNAc. Mass spectrometry analysis identified threonine 32 as a novel O-GlcNAc site. O-GlcNAc was detected at higher levels on H3 during interphase than mitosis, which inversely correlated with phosphorylation. Furthermore, increased O-GlcNAcylation was observed to reduce mitosis-specific phosphorylation at serine 10, serine 28, and threonine 32. Finally, inhibiting OGA, the enzyme responsible for removing O-GlcNAc, hindered the transition from G2 to M phase of the cell cycle, displaying a phenotype similar to preventing mitosis-specific phosphorylation on H3. Taken together, these data indicate that O-GlcNAcylation regulates mitosis-specific phosphorylations on H3, providing a mechanistic switch that orchestrates the G2-M transition of the cell cycle.

Occupational stress and employee turnover.

Questionnaire data captured in January-March 2007 were examined in relation to turnover in males and females during the next five years. In general, most of the workplace stressors (such as role conflict or peer support) were not antecedents of turnover in any group. Junior personnel with psychological strain in 2007 had an increased risk of turnover in the next five years. Low commitment to the service in 2007 increased the odds of turnover in male and female juniors and in female officers. Female juniors with less effective skills for coping with stress and who exercised less frequently on a weekly basis were more likely to leave. An incidental finding was that the odds of turnover were three times greater in female officers with children than in female officers with no children. Stress management interventions focusing on effective coping and sports and exercise participation which are targeted appropriately may improve retention.

Psychometric properties of the Cognitive Failures Questionnaire.

The Cognitive Failures Questionnaire (CFQ) is used in ergonomics research to measure behavioural problems associated with attentiveness and memory in everyday life. CFQ scores have been related to constructs such as accident proneness and outcomes such as human error and psychological strain. The two-year test-retest reliability of the CFQ is reported together with the findings of factor analyses of CFQ data from 535 respondents. Evidence for the predictive and criterion validity and internal reliability of the CFQ is provided. Psychological strain was measured concurrently with CFQ on both testing occasions, two years apart. The test-retest reliability of the summated CFQ score was found to be 0.71, while for the General Health Questionnaire (GHQ-12) strain measure it was 0.32.The relative variance stability was five times greater for the CFQ than the GHQ, indicating that scores on these questionnaires are not covariates. The use of the CFQ as a measure of cognitive control capacity is also discussed. PRACTITIONER SUMMARY: Ergonomists have long been interested in human error and the role of high work demands due to poor equipment design and excessive workload. The CFQ measures attentiveness in daily life and is shown to have excellent psychometric properties that make it suitable for use in both laboratory and field studies as a trait measure of attentiveness in daily life.

MaHPIC malaria systems biology data from Plasmodium cynomolgi sporozoite longitudinal infections in macaques.

Plasmodium cynomolgi causes zoonotic malarial infections in Southeast Asia and this parasite species is important as a model for Plasmodium vivax and Plasmodium ovale. Each of these species produces hypnozoites in the liver, which can cause relapsing infections in the blood. Here we present methods and data generated from iterative longitudinal systems biology infection experiments designed and performed by the Malaria Host-Pathogen Interaction Center (MaHPIC) to delve deeper into the biology, pathogenesis, and immune responses of P. cynomolgi in the Macaca mulatta host. Infections were initiated by sporozoite inoculation. Blood and bone marrow samples were collected at defined timepoints for biological and computational experiments and integrative analyses revolving around primary illness, relapse illness, and subsequent disease and immune response patterns. Parasitological, clinical, haematological, immune response, and -omic datasets (transcriptomics, proteomics, metabolomics, and lipidomics) including metadata and computational results have been deposited in public repositories. The scope and depth of these datasets are unprecedented in studies of malaria, and they are projected to be a F.A.I.R., reliable data resource for decades.

Cognitive task demands, self-control demands and the mental well-being of office workers.

The cognitive task demands of office workers and the self-control demands of their work roles were measured in a sample of 196 employees in two different office layouts using a self-report questionnaire, which was circulated electronically. Multiple linear regression analysis revealed that both factors were associated with mental well-being, but not with physical well-being, while controlling for exposure to psychosocial stressors. The interaction between cognitive task demands and self-control demands had the strongest association with mental well-being, suggesting that the deleterious effect of one was greater when the other was present. An exploratory analysis revealed that the association was stronger for employees working in a large open-plan office than for those working in smaller offices with more privacy. Frustration of work goals was the cognitive task demand having the strongest negative impact on mental well-being. Methodological limitations and scale psychometrics (particularly the use of the NASA Task Load Index) are discussed. STATEMENT OF RELEVANCE: Modern office work has high mental demands and low physical demands and there is a need to design offices to prevent adverse psychological reactions. It is shown that cognitive task demands interact with self-control demands to degrade mental well-being. The association was stronger in an open-plan office.

Cumulative psychological strain and future strain in Naval personnel: is executive function the elephant in the room?

Psychological strain was measured in 592 Naval personnel at four phases using the General Health Questionnaire (GHQ-12). The GHQ-12 Likert score was summed to generate a measure of cumulative strain, which was best predicted by the Cognitive Failures Questionnaire (CFQ) score, effort-reward imbalance and a measure of organisational commitment. Point strain at phase IV was best predicted by CFQ score, whereas need for recovery was best predicted by over-commitment to work role. Susceptibility to strain, both cumulatively over 2 years and 12 months in the future appears to depend on a cognitive factor measured by the CFQ. This factor was more strongly associated with strain than with fatigue. Psychosocial stress exposure at work played a lesser role in predicting cumulative and future strain, possibly because these stressors were of an acute nature. The findings are discussed with reference to recent research on attention and anxiety and some of the practical implications are considered. STATEMENT OF RELEVANCE: There is renewed interest in intrinsic variables that predict adverse reactions to occupational stress. The paper presents evidence that self-reported cognitive failure is a vulnerability factor for future episodes of psychological strain. The paper strengthens the conclusions of a previous article and adds empirical support to recent research on the links between anxiety and attention.

Effects of chronic exposure to low levels of IR on Medaka (Oryzias latipes): a proteomic and bioinformatic approach.

PURPOSE: Chronic exposure to ionizing radiation (IR) at low doses (<100 mGy) has been insufficiently studied to understand fully the risk to health. Relatively little knowledge exists regarding how species and healthy tissues respond at the protein level to chronic exposure to low doses of IR, and mass spectrometric-based profiling of protein expression is a powerful tool for studying changes in protein abundance. MATERIALS AND METHODS: SDS gel electrophoresis, LC-MS/MS mass spectrometry-based approaches and bioinformatic data analytics were used to detect proteomic changes following chronic exposure to moderate/low doses of radiation in adults and normally developed Medaka fish (Oryzias latipes). RESULTS: Significant variations in the abundance of proteins involved in thyroid hormone signaling and lipid metabolism were detected, which could be related to the gonadal regression phenotype observed after 21.04 mGy and 204.3 mGy/day exposure. The global proteomic change was towards overexpression of proteins in muscle and skin, while the opposite effect was observed in internal organs. CONCLUSION: The present study provides information on the impacts of biologically relevant low doses of IR, which will be useful in future research for the identification of potential biomarkers of IR exposure and allow for a better assessment of radiation biosafety regulations.

Job strain related to cognitive failure in naval personnel.

The Naval Service Stress Study (2007-2012) is investigating job strain, its characteristics, causes and distribution in the Service. Data from phases I, II and III of the study (January 2007, June 2007 and January 2008) were analysed to determine the relationship between General Health questionnaire scores and a score on the Cognitive Failures Questionnaire (CFQ) completed at phase III. Of 791 personnel who completed questionnaires at all phases, 43.6% had no job strain at any phase, whereas 9.9% had strain on all three occasions ('chronic strain'). 27% had strain at one of the three phases and 19% had strain at two of the three phases. The particular phase at which job strain was experienced was not related to CFQ score at phase III, whereas the total strain experienced over the period was related. High strain over the year was the strongest predictor of high CFQ score. A 'strain dose' variable, which combined both the amount of strain exposure and the timing of the exposure, explained little additional variance in CFQ score. The findings might be interpreted to indicate that a high CFQ score is a vulnerability factor for adverse reactions to work stress. The hypothesis that recent job strain elevates CFQ score was not supported. STATEMENT OF RELEVANCE: Current models of occupational stress focus on psychosocial factors and much of the advice about stress management in organisations is centred on the identification and control of psychosocial risk factors. The present paper provides evidence that cognitive factors are also important and suggests that support for those with poor executive function should be part of stress management in complex environments.

Occupational stress in submariners: the impact of isolated and confined work on psychological well-being.

This study aimed to identify work-related and personal factors associated with occupational stress in submariners. Work and well-being questionnaires were distributed to 219 male submariners (mean age 34 years), as part of a larger cohort study involving a stratified sample of 4951 Royal Navy (RN) personnel. The stress rate in submariners was 40%; significantly higher than the stress rate in the general RN, although once demographic factors were controlled for in a matched control sample, this difference was no longer significant. A summary model accounted for 49% of the variance in submariner stress, with key differences emerging between the occupational factors associated with stress in submariners and in the general RN. The longitudinal nature of this study permits stress in submariners to be monitored over 5 years, which will provide valuable insights into the chronicity of stress in this specialised occupational group. STATEMENT OF RELEVANCE: This paper contributes to the current literature on the negative impact of working in isolated conditions. It is demonstrated that occupational stress in submarines can be partially explained using current theories of stress in the workplace. However, the constraints of a restricted environment introduce additional factors which can also be associated with occupational stress.

Virus-Receptor Interactions of Glycosylated SARS-CoV-2 Spike and Human ACE2 Receptor.

The SARS-CoV-2 betacoronavirus uses its highly glycosylated trimeric Spike protein to bind to the cell surface receptor angiotensin converting enzyme 2 (ACE2) glycoprotein and facilitate host cell entry. We utilized glycomics-informed glycoproteomics to characterize site-specific microheterogeneity of glycosylation for a recombinant trimer Spike mimetic immunogen and for a soluble version of human ACE2. We combined this information with bioinformatics analyses of natural variants and with existing 3D structures of both glycoproteins to generate molecular dynamics simulations of each glycoprotein both alone and interacting with one another. Our results highlight roles for glycans in sterically masking polypeptide epitopes and directly modulating Spike-ACE2 interactions. Furthermore, our results illustrate the impact of viral evolution and divergence on Spike glycosylation, as well as the influence of natural variants on ACE2 receptor glycosylation. Taken together, these data can facilitate immunogen design to achieve antibody neutralization and inform therapeutic strategies to inhibit viral infection.

Virus-Receptor Interactions of Glycosylated SARS-CoV-2 Spike and Human ACE2 Receptor.

The current COVID-19 pandemic is caused by the SARS-CoV-2 betacoronavirus, which utilizes its highly glycosylated trimeric Spike protein to bind to the cell surface receptor ACE2 glycoprotein and facilitate host cell entry. We utilized glycomics-informed glycoproteomics to characterize site-specific microheterogeneity of glycosylation for a recombinant trimer Spike mimetic immunogen and for a soluble version of human ACE2. We combined this information with bioinformatic analyses of natural variants and with existing 3D-structures of both glycoproteins to generate molecular dynamics simulations of each glycoprotein alone and interacting with one another. Our results highlight roles for glycans in sterically masking polypeptide epitopes and directly modulating Spike-ACE2 interactions. Furthermore, our results illustrate the impact of viral evolution and divergence on Spike glycosylation, as well as the influence of natural variants on ACE2 receptor glycosylation that, taken together, can facilitate immunogen design to achieve antibody neutralization and inform therapeutic strategies to inhibit viral infection.

Smoking, BMI and psychological strain and fitness in the Naval Service.

BACKGROUND: Data from the Naval Service (NS) cohort study of psychological strain were extracted and analysed to investigate the relationship between self-reported health and lifestyle factors and medical fitness. Identification of factors associated with medical downgrading is of obvious value in shaping future health and safety policy and in understanding the relative contributions of physical and psychosocial factors to adverse occupational health outcomes. AIMS: To identify variables associated with a lack of fitness to serve. METHOD: Extraction and analysis of data from the Phase I of the study, with a binary outcome of fitness as the dependent variable, controlling for psychosocial and other confounders. RESULTS: Stepwise logistic regression analysis found statistically significant effects due to smoking, body mass index (BMI), General Health Questionnaire (GHQ)-12 and work-family conflict. The model accounted for 5.6% of the variance in medical grading, 3% of which was due to smoking. CONCLUSIONS: With psychosocial factors and GHQ-12 scores accounted for, personnel who were not fully fit for NS were found to be more likely to be smokers and to have a high BMI (> or =25).