Spindle assembly checkpoint genes reveal distinct as well as overlapping expression that implicates MDF-2/Mad2 in postembryonic seam cell proliferation in Caenorhabditis elegans

Background: The spindle assembly checkpoint (SAC) delays anaphase onset by inhibiting the activity of theanaphase promoting complex/cyclosome (APC/C) until all of the kinetochores have properly attached to thespindle. The importance of SAC genes for genome stability is well established; however, the roles these genes play,during postembryonic development of a multicellular organism, remain largely unexplored.Results: We have used GFP fusions of 5’ upstream intergenic regulatory sequences to assay spatiotemporalexpression patterns of eight conserved genes implicated in the spindle assembly checkpoint function inCaenorhabditis elegans. We have shown that regulatory sequences for all of the SAC genes drive ubiquitous GFPexpression during early embryonic development. However, postembryonic spatial analysis revealed distinct, tissuespecificexpression of SAC genes with striking co-expression in seam cells, as well as in the gut. Additionally, weshow that the absence of MDF-2/Mad2 (one of the checkpoint genes) leads to aberrant number and alignment ofseam cell nuclei, defects mainly attributed to abnormal postembryonic cell proliferation. Furthermore, we showthat these defects are completely rescued by fzy-1(h1983)/CDC20, suggesting that regulation of the APC/CCDC20 bythe SAC component MDF-2 is important for proper postembryonic cell proliferation.Conclusion: Our results indicate that SAC genes display different tissue-specific expression patterns duringpostembryonic development in C. elegans with significant co-expression in hypodermal seam cells and gut cells,suggesting that these genes have distinct as well as overlapping roles in postembryonic development that may ormay not be related to their established roles in mitosis. Furthermore, we provide evidence, by monitoring seamcell lineage, that one of the checkpoint genes is required for proper postembryonic cell proliferation. Importantly,our research provides the first evidence that postembryonic cell division is more sensitive to SAC loss, in particularMDF-2 loss, than embryonic cell division

The PRECISE (PREgnancy Care Integrating translational Science, Everywhere) database: open-access data collection in maternal and newborn health

In less-resourced settings, adverse pregnancy outcome rates are unacceptably high. To effect improvement, we need accurate epidemiological data about rates of death and morbidity, as well as social determinants of health and processes of care, and from each country (or region) to contextualise strategies. The PRECISE database is a unique core infrastructure of a generic, unified data collection platform. It is built on previous work in data harmonisation, outcome and data field standardisation, open-access software (District Health Information System 2 and the Baobab Laboratory Information Management System), and clinical research networks. The database contains globally-recommended indicators included in Health Management Information System recording and reporting forms. It comprises key outcomes (maternal and perinatal death), life-saving interventions (Human Immunodeficiency Virus testing, blood pressure measurement, iron therapy, uterotonic use after delivery, postpartum maternal assessment within 48 h of birth, and newborn resuscitation, immediate skin-to-skin contact, and immediate drying), and an additional 17 core administrative variables for the mother and babies. In addition, the database has a suite of additional modules for ‘deep phenotyping’ based on established tools. These include social determinants of health (including socioeconomic status, nutrition and the environment), maternal comorbidities, mental health, violence against women and health systems. The database has the potential to enable future high-quality epidemiological research integrated with clinical care and discovery bioscience

The ability and safety of community-based health workers to safely initiate lifesaving therapies for pre-eclampsia in Ogun State, Nigeria: An analysis of 260 community treatments with MgSO4 and/or methyldopa

Objectives: To evaluate community-based health workers\u27 ability to identify cases of hypertension in pregnancy, safely deliver methyldopa and magnesium sulphate and make referrals when appropriate.Study design: This was part of Nigeria Community-Level Interventions for Pre-eclampsia (CLIP) cluster randomized controlled trial (NCT01911494). Community-based Health Workers (CHW) recruited pregnant women from five Local Government Areas (clusters) and used mobile health aid for clinical assessment of pre-eclampsia.Main outcome measures: The primary outcome was the number of adverse events that occurred after the administration of magnesium sulphate and/or methyldopa to pregnant women by CHWs.Findings: Of 8790 women receiving mobile health-guided care, community-based health workers in Nigeria provided 309 women with hypertension (4.2% of delivered women), and safely administered 142 doses of intramuscular magnesium sulphate. Community Heath Extension Workers (CHEWs) and nurses gave fifty-two and sixty-seven doses of intramuscular magnesium sulphate respectively, twenty-three doses were given by other health care workers (midwives, community health officers, health assistants). The high rate of administration by nurses can be explained by turf protection as well as their seniority within the health system. Also, CHEWs and nurses gave 124 doses of oral methyldopa and 126 urgent referrals were completed. There were no complications related to administration of treatment or referral.Interpretation: These findings demonstrate the ability of community-based health workers to safely administer methyldopa and intramuscular magnesium sulphate. The use of task-sharing, therefore, could drastically reduce the three delays (triage, transport and treatment) associated with high maternal mortality and morbidity in rural communities in low- and middle-income countries

Allelic Ratios and the Mutational Landscape Reveal Biologically Significant Heterozygous SNVs

The issue of heterozygosity continues to be a challenge in the analysis of genome sequences. In this article, we describe the use of allele ratios to distinguish biologically significant single-nucleotide variants from background noise. An application of this approach is the identification of lethal mutations in Caenorhabditis elegans essential genes, which must be maintained by the presence of a wild-type allele on a balancer. The h448 allele of let-504 is rescued by the duplication balancer sDp2. We readily identified the extent of the duplication when the percentage of read support for the lesion was between 70 and 80%. Examination of the EMS-induced changes throughout the genome revealed that these mutations exist in contiguous blocks. During early embryonic division in self-fertilizing C. elegans, alkylated guanines pair with thymines. As a result, EMS-induced changes become fixed as either G→A or C→T changes along the length of the chromosome. Thus, examination of the distribution of EMS-induced changes revealed the mutational and recombinational history of the chromosome, even generations later. We identified the mutational change responsible for the h448 mutation and sequenced PCR products for an additional four alleles, correlating let-504 with the DNA-coding region for an ortholog of a NFκB-activating protein, NKAP. Our results confirm that whole-genome sequencing is an efficient and inexpensive way of identifying nucleotide alterations responsible for lethal phenotypes and can be applied on a large scale to identify the molecular basis of essential genes

High-Throughput In Vivo Analysis of Gene Expression in Caenorhabditis elegans

Using DNA sequences 5′ to open reading frames, we have constructed green fluorescent protein (GFP) fusions and generated spatial and temporal tissue expression profiles for 1,886 specific genes in the nematode Caenorhabditis elegans. This effort encompasses about 10% of all genes identified in this organism. GFP-expressing wild-type animals were analyzed at each stage of development from embryo to adult. We have identified 5′ DNA regions regulating expression at all developmental stages and in 38 different cell and tissue types in this organism. Among the regulatory regions identified are sequences that regulate expression in all cells, in specific tissues, in combinations of tissues, and in single cells. Most of the genes we have examined in C. elegans have human orthologs. All the images and expression pattern data generated by this project are available at WormAtlas (http://gfpweb.aecom.yu.edu/index) and through WormBase (http://www.wormbase.org)

SARS-CoV-2 seroprevalence in pregnant women in Kilifi, Kenya from March 2020 to March 2022

BackgroundSeroprevalence studies are an alternative approach to estimating the extent of transmission of SARS-CoV-2 and the evolution of the pandemic in different geographical settings. We aimed to determine the SARS-CoV-2 seroprevalence from March 2020 to March 2022 in a rural and urban setting in Kilifi County, Kenya.MethodsWe obtained representative random samples of stored serum from a pregnancy cohort study for the period March 2020 to March 2022 and tested for antibodies against the spike protein using a qualitative SARS-CoV-2 ELISA kit (Wantai, total antibodies). All positive samples were retested for anti-SARS-CoV-2 anti-nucleocapsid antibodies (Euroimmun, ELISA kits, NCP, qualitative, IgG) and anti-spike protein antibodies (Euroimmun, ELISA kits, QuantiVac; quantitative, IgG).ResultsA total of 2,495 (of 4,703 available) samples were tested. There was an overall trend of increasing seropositivity from a low of 0% [95% CI 0–0.06] in March 2020 to a high of 89.4% [95% CI 83.36–93.82] in Feb 2022. Of the Wantai test-positive samples, 59.7% [95% CI 57.06–62.34] tested positive by the Euroimmun anti-SARS-CoV-2 NCP test and 37.4% [95% CI 34.83–40.04] tested positive by the Euroimmun anti-SARS-CoV-2 QuantiVac test. No differences were observed between the urban and rural hospital but villages adjacent to the major highway traversing the study area had a higher seroprevalence.ConclusionAnti-SARS-CoV-2 seroprevalence rose rapidly, with most of the population exposed to SARS-CoV-2 within 23 months of the first cases. The high cumulative seroprevalence suggests greater population exposure to SARS-CoV-2 than that reported from surveillance data

Characterization of the octamer, a cis-regulatory element that modulates excretory cell gene-expression in Caenorhabditis elegans

Background: We have previously demonstrated that the POU transcription factor CEH-6 is required for driving aqp-8 expression in the C. elegans excretory (canal) cell, an osmotic regulatory organ that is functionally analogous to the kidney. This transcriptional regulation occurs through a CEH-6 binding to a cis-regulatory element called the octamer (ATTTGCAT), which is located in the aqp-8 promoter. Results Here, we further characterize octamer driven transcription in C. elegans. First, we analyzed the positional requirements of the octamer. To do so, we assayed the effects on excretory cell expression by placing the octamer within the well-characterized promoter of vit-2. Second, using phylogenetic footprinting between three Caenorhabditis species, we identified a set of 165 genes that contain conserved upstream octamers in their promoters. Third, we used promoter::GFP fusions to examine the expression patterns of 107 of the 165 genes. This analysis demonstrated that conservation of octamers in promoters increases the likelihood that the gene is expressed in the excretory cell. Furthermore, we found that the sequences flanking the octamers may have functional importance. Finally, we altered the octamer using site-directed mutagenesis. Thus, we demonstrated that some nucleotide substitutions within the octamer do not affect the expression pattern of nearby genes, but change their overall expression was changed. Therefore, we have expanded the core octamer to include flanking regions and variants of the motif. Conclusions Taken together, we have demonstrated that octamer-containing regions are associated with excretory cell expression of several genes that have putative roles in osmoregulation. Moreover, our analysis of the octamer sequence and its sequence variants could aid in the identification of additional genes that are expressed in the excretory cell and that may also be regulated by CEH-6.Medical Genetics, Department ofMedicine, Faculty ofMolecular Medicine and Therapeutics, Centre forReviewedFacult

An antibiotic selection marker for nematode transgenesis

We have developed a nematode transformation vector carrying the bacterial neomycin resistance gene (NeoR) and shown that it could confer resistance to G-418 on both wild-type Caenorhabditis elegans and C. briggsae. This selection system allows hands-off maintenance and enrichment of transgenic worms carrying non-integrated transgenes on selective plates. We also show that this marker can be used for Mos1-mediated single-copy insertion in wild-type genetic backgrounds (MosSCI-biotic)