Linking Metabolic Capacity and Molecular Biology of Methylocystis sp. Strain SC2 by a Newly Developed Proteomics Workflow

Microbial methane oxidation is one of the fundamental processes in global methane cycle. Methane-oxidizing bacteria, or methanotrophs, are the major biological sink for the methane produced from anthropogenic and natural sources. Our model organism, Methylocystis sp. strain SC2, is one of the best-studied representatives of alphaproteobacterial (type IIa) methanotrophs. Proteobacterial methanotrophs possess a unique cell architecture characterized by intracytoplasmic membranes (ICMs). The cellular amount of the ICMs is increasing with methanotrophic activity. The presence of ICMs makes molecular biology approaches, but in particular global proteomics, highly challenging. In this study, we therefore aimed to develop an efficient proteomics workflow for strain SC2 and to apply this state-of-the-art tool for investigation of the strain SC2 response to environmental factors. To successfully develop the proteomics workflow, we particularly focused on an efficient solubilization and digestion of the integral membrane proteins of strain SC2 for further downstream analysis. We introduced the so-called crude-MS proteomics workflow, upon assessing and optimizing all the major steps in the proteomics workflow, including cell lysis, protein solubilization, and protein digestion. Our new SC2 proteomics workflow greatly increased not only the protein quantification accuracy (mean coefficient of variation 3.2 %) but also the proteome coverage to 62%, with up to 10-fold increase in the detection intensity of membrane-associated proteins. Previous studies have shown that the LysC/trypsin tandem digestion resulted in higher coverage of fully cleaved tryptic peptides than a trypsin-only digestion. Therefore, the development of our optimized proteomics workflow involved the application of the LysC/trypsin tandem digestion in detergent environment to increase the SC2 proteome coverage. Prior to publication of our crude-MS approach, all systematic assessments of LysC/trypsin proteolysis were conducted in chaotropic environments, like urea. As a spin-off, we therefore initiated a follow-up study to compare the efficiency of the LysC/trypsin tandem digestion in detergent environments (e.g., SDC, SLS) relative to chaotropic environments. The study revealed that the LysC/trypsin tandem digestion could be efficiently carried out not only in chaotropic environments but also in MS-compatible detergent environments. In fact, the LysC/trypsin tandem digestion in both environments resulted in a higher coverage of fully cleaved peptides than the trypsin-only digestion. After successful development of the crude-MS proteomics workflow, we used this high-throughput method to assess the molecular response of strain SC2 to the availability of hydrogen as a potentially alternative energy source. Starting point of this research was the knowledge that strain SC2 and other Methylocystis spp. possess the genetic potential to produce various hydrogenases. In fact, the addition of 2% hydrogen to the headspace atmosphere led, under limiting concentrations of methane and oxygen, to the complete hydrogen consumption by strain SC2. Concurrently, the SC2 biomass yield was significantly increased, while the methane consumption rate was significantly decreased. Global proteome analyses revealed that the addition of hydrogen induced an increase in the production of Group 1d and Group 2b [NiFe]-hydrogenases, and hydrogenase accessory proteins. Notably, the upregulation of the Group 1d, 2b [NiFe]-hydrogenases was concomitantly linked to a reconstruction of the energy metabolism in strain SC2. In another project, genome-scale metabolic modeling and growth experiments were applied to show that strain SC2 has the capacity to utilize acetate through the glyoxylate assimilation pathway. In addition, the study revealed that in type II methanotrophs, energy demand for methane oxidation is covered by complex I of the electron transport chain. In summary, our research demonstrates how to experimentally link the metabolic potential of Methylocystis sp. strain SC2 with the underlying proteome complexity. Thus, the newly developed highly reproducible SC2 proteomics workflow represents a high-throughput method that makes it possible to achieve in future research an understanding of the molecular adaptation mechanisms of strain SC2 to environmental change

Application of MATLAB in -Omics and Systems Biology

Biological data analysis has dramatically changed since the introduction of high-throughput -omics technologies, such as microarrays and next-generation sequencing. The key advantage of obtaining thousands of measurements from a single sample soon became a bottleneck limiting transformation of generated data into knowledge. It has become apparent that traditional statistical approaches are not suited to solve problems in the new reality of “big biological data.” From the other side, traditional computing languages such as C/C++ and Java, are not flexible enough to allow for quick development and testing of new algorithms, while MATLAB provides a powerful computing environment and a variety of sophisticated toolboxes for performing complex bioinformatics calculations

Genome scale metabolic modeling reveals the metabolic potential of three Type II methanotrophs of the genus Methylocystis

Producción CientíficaGenome Scale Metabolic Models (GSMMs) of the recently sequenced Methylocystis hirsuta and two other methanotrophs from the genus Methylocystis have been reconstructed. These organisms are Type II methanotrophs with the ability of accumulating Polyhydroxyalkanoates under nutrient limiting conditions. For the first time, GSMMs have been reconstructed for Type II methanotrophs. These models, combined with experimental biomass and PHB yields of Methylocystis hirsuta, allowed elucidating the methane oxidation mechanism by the enzyme pMMO (particulate methane monooxygenase) in these organisms. In contrast to Type I methanotrophs, which use the “direct coupling mechanism”, Type II methanotrophs appear to use the so called “redox arm mechanism”. The utilization of the “redox arm mechanism”, which involves the coupling between methane oxidation and complex I of the respiratory chain, was confirmed by inhibition of complex I with catechol. Utilization of the “redox arm” mechanism leads to lower biomass yields on methane compared to Type I methanotrophs. However, the ability of Type II methanotrophs to redirect high metabolic carbon fluxes towards acetoacetyl-CoA under nitrogen limiting conditions makes these organisms promising platforms for metabolic engineering.Marie Curie grant H2020-MSCA-IF-2016 CH4BioVal (GA nº 750126).Junta de Castilla y León (Ref. Project VA281P18)Ministerio de Ciencia e Innovación (Proyect CLU 2017-09, CTM2015-70442-R

Unmet need for family planning, contraceptive failure, and unintended pregnancy among HIV-infected and HIV-uninfected women in Zimbabwe.

BackgroundPrevention of unintended pregnancies among women living with HIV infection is a strategy recommended by the World Health Organization for prevention of mother-to-child transmission of HIV (PMTCT). We assessed pregnancy intentions and contraceptive use among HIV-positive and HIV-negative women with a recent pregnancy in Zimbabwe.MethodsWe analyzed baseline data from the evaluation of Zimbabwe's Accelerated National PMTCT Program. Eligible women were randomly sampled from the catchment areas of 157 health facilities offering PMTCT services in five provinces. Eligible women were ≥16 years old and mothers of infants (alive or deceased) born 9 to 18 months prior to the interview. Participants were interviewed about their HIV status, intendedness of the birth, and contraceptive use.ResultsOf 8,797 women, the mean age was 26.7 years, 92.8% were married or had a regular sexual partner, and they had an average of 2.7 lifetime births. Overall, 3,090 (35.1%) reported that their births were unintended; of these women, 1,477 (47.8%) and 1,613 (52.2%) were and were not using a contraceptive method prior to learning that they were pregnant, respectively. Twelve percent of women reported that they were HIV-positive at the time of the survey; women who reported that they were HIV-infected were significantly more likely to report that their pregnancy was unintended compared to women who reported that they were HIV-uninfected (44.9% vs. 33.8%, p<0.01). After adjustment for covariates, among women with unintended births, there was no association between self-reported HIV status and lack of contraception use prior to pregnancy.ConclusionsUnmet need for family planning and contraceptive failure contribute to unintended pregnancies among women in Zimbabwe. Both HIV-infected and HIV-uninfected women reported unintended pregnancies despite intending to avoid or delay pregnancy, highlighting the need for effective contraceptive methods that align with pregnancy intentions

The Significance of Person-Centered Care for Satisfaction With Care and Well-Being Among Informal Caregivers of Persons With Severe Intellectual Disability

Person-centered care (PCC) delivery and co-creation of care (establishing productive patient-professional interaction) are expected to lead to better patient outcomes. Given the prominent role of informal caregivers in care delivery processes to persons with intellectual disabilities (PWID), they are expected to benefit from person-centered care (PCC) and co-creation of care as well. This study aims to identify the relationship between PCC, co-creation of care and outcomes among informal caregivers of PWID. A cross-sectional survey was conducted in 2015 among informal caregivers of PWID (45.8% parents, 44.1% siblings, 10.1% other family member). All PWID were living in residential homes of a long-term care organization in the Eastern part of the Netherlands. For every PWID, the most important informal caregiver was invited to participate. Nine hundred and forty-one invitations were sent out and 289 of them responded (31% response rate). Mean age of informal caregivers was 61.80 years old. About half of the respondents were female and 23% were single. Most of the respondents were providing informal care for more than 10 years and 29% provided informal care for 8 hours per week or more. Correlation analyses indicated that PCC and co-creation of care were positively related to informal caregivers’ satisfaction with care and their own well-being. Regression analyses showed that PCC is associated with satisfaction with care and well-being while controlling for background characteristics. Relational co-creation was also positively associated with satisfaction with care and well-being. This study provided the first empirical evidence that PCC and cocreation of care matter for satisfaction with care and the well-being of informal caregivers of PWID

THE NECESSITY OF INTRODUCTION THE DRUG INSURANCE SYSTEM IN ARMENIA

The increase in the cost of the medicinal component of the treatment, the spread of chronic diseases, and the maintenance of socio-economic inequality in access to health services require the provision of adequate access to medicines. These issues create prerequisites for the improvement of the state health policy and, first, the drug supply system, which is an integral part of the treatment process. The financing of healthcare in Armenia is mainly formed from budget allocations and out of pocket expenditures of the population. Reducing the financial burden on the state and ensuring the rational use of drugs contributes to improving the health of the population. The implementation of a drug insurance scheme, which partially or fully cover the cost of drugs in RA, is one of the solutions for resolving the issue of access to medicines. This article studies the problems of financing healthcare system in Armenia and highlights the need of introduction a drug insurance system in Armeni

Functional characterization of the complement receptor type 1 and its circulating ligands in patients with schizophrenia

<p>Abstract</p> <p>Background</p> <p>Whereas the complement system alterations contribute to schizophrenia, complement receptors and regulators are little studied. We investigated complement receptor type 1 (CR1) expression on blood cells, the levels of circulating immune complexes (CIC) containing ligands of CR1, C1q complement protein and fragments of C3 complement protein (C1q-CIC, C3d-CIC), and CR1 C5507G functional polymorphism in schizophrenia patients and controls.</p> <p>Results</p> <p>We found an increased C1q-CIC level and CR1 expression on blood cells, elevated number of CR1 positive erythrocytes and reduced number of CR1 positive lymphocytes and monocytes in patients compared to controls. No difference in the levels of C3d-CIC between groups was observed. Higher CR1 expression on erythrocytes in CC genotype versus CG+GG for both groups was detected, whereas no difference was observed for other cell populations. Our results indicated that schizophrenia is associated with the increased CR1 expression and C1q-CIC level.</p> <p>Conclusions</p> <p>Our study for the first time indicated that schizophrenia is associated with the increased CR1 expression and C1q-CIC level. Further studies in other ethnic groups are needed to replicate these findings.</p

Association of C1QB gene polymorphism with schizophrenia in Armenian population

<p>Abstract</p> <p>Background</p> <p>Schizophrenia is a complex, multifactorial psychiatric disorder. Our previous findings indicated that altered functional activity of the complement system, a major mediator of the immune response, is implicated in the pathogenesis of schizophrenia. In order to explore whether these alterations are genetically determined or not, in the present study we evaluated the possible association of complement C1Q component gene variants with susceptibility to schizophrenia in Armenian population, focusing on four frequent single nucleotide polymorphisms (SNPs) of <it>C1QA </it>and <it>C1QB </it>genes.</p> <p>Methods</p> <p>In the present study four SNPs of the complement C1Q component genes (<it>C1QA</it>: rs292001, <it>C1QB </it>rs291982, rs631090, rs913243) were investigated in schizophrenia-affected and healthy subjects. Unrelated Caucasian individuals of Armenian nationality, 225 schizophrenic patients and the same number of age- and sex-matched healthy subjects, were genotyped. Genotyping was performed using polymerase chain reaction with sequence-specific primers (PCR-SSP) and quantitative real-time (qRT) PCR methods.</p> <p>Results</p> <p>While there was no association between <it>C1QA </it>rs292001, <it>C1QB </it>rs913243 and rs631090 genetic variants and schizophrenia, the <it>C1QB </it>rs291982*G minor allele was significantly overrepresented in schizophrenic patients (G allele frequency 58%) when compared to healthy subjects (46%, OR = 1.64, <it>p</it>_corr= 0.0008). Importantly, the susceptibility for schizophrenia was particularly associated with <it>C1QB </it>rs291982 GG genotype (OR = 2.5, <it>p</it>_corrected= 9.6E-5).</p> <p>Conclusions</p> <p>The results obtained suggest that <it>C1QB </it>gene may be considered as a relevant candidate gene for susceptibility to schizophrenia, and its rs291982*G minor allele might represent a risk factor for schizophrenia at least in Armenian population. Replication in other centers/populations is necessary to verify this conclusion.</p

Monitoring and evaluating the impact of national school-based deworming in Kenya: study design and baseline results.

BACKGROUND: An increasing number of countries in Africa and elsewhere are developing national plans for the control of neglected tropical diseases. A key component of such plans is school-based deworming (SBD) for the control of soil-transmitted helminths (STHs) and schistosomiasis. Monitoring and evaluation (M&E) of national programmes is essential to ensure they are achieving their stated aims and to evaluate when to reduce the frequency of treatment or when to halt it altogether. The article describes the M&E design of the Kenya national SBD programme and presents results from the baseline survey conducted in early 2012. METHODS: The M&E design involves a stratified series of pre- and post-intervention, repeat cross-sectional surveys in a representative sample of 200 schools (over 20,000 children) across Kenya. Schools were sampled based on previous knowledge of STH endemicity and were proportional to population size. Stool (and where relevant urine) samples were obtained for microscopic examination and in a subset of schools; finger-prick blood samples were collected to estimate haemoglobin concentration. Descriptive and spatial analyses were conducted. The evaluation measured both prevalence and intensity of infection. RESULTS: Overall, 32.4% of children were infected with at least one STH species, with Ascaris lumbricoides as the most common species detected. The overall prevalence of Schistosoma mansoni was 2.1%, while in the Coast Province the prevalence of S. haematobium was 14.8%. There was marked geographical variation in the prevalence of species infection at school, district and province levels. The prevalence of hookworm infection was highest in Western Province (25.1%), while A. lumbricoides and T. trichiura prevalence was highest in the Rift Valley (27.1% and 11.9%). The lowest prevalence was observed in the Rift Valley for hookworm (3.5%), in the Coast for A. lumbricoides (1.0%), and in Nyanza for T. trichiura (3.6%). The prevalence of S. mansoni was most common in Western Province (4.1%). CONCLUSIONS: The current findings are consistent with the known spatial ecology of STH and schistosome infections and provide an important empirical basis on which to evaluate the impact of regular mass treatment through the school system in Kenya