Development and Implementation of a High Throughput Screen for the Human Sperm-Specific Isoform of Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDHS)

Glycolytic isozymes that are restricted to the male germline are potential targets for the development of reversible, non-hormonal male contraceptives. GAPDHS, the sperm-specific isoform of glyceraldehyde-3-phosphate dehydrogenase, is an essential enzyme for glycolysis making it an attractive target for rational drug design. Toward this goal, we have optimized and validated a high-throughput spectrophotometric assay for GAPDHS in 384-well format. The assay was stable over time and tolerant to DMSO. Whole plate validation experiments yielded Z’ values >0.8 indicating a robust assay for HTS. Two compounds were identified and confirmed from a test screen of the Prestwick collection. This assay was used to screen a diverse chemical library and identified fourteen small molecules that modulated the activity of recombinant purified GAPDHS with confirmed IC50 values ranging from 1.8 to 42 µM. These compounds may provide useful scaffolds as molecular tools to probe the role of GAPDHS in sperm motility and long term to develop potent and selective GAPDHS inhibitors leading to novel contraceptive agents

Recombinant human sperm-specific glyceraldehyde-3-phosphate dehydrogenase (GAPDHS) is expressed at high yield as an active homotetramer in baculovirus-infected insect cells

The sperm-specific glyceraldehyde-3-phosphate dehydrogenase (GAPDHS) isoform is a promising contraceptive target because it is specific to male germ cells, essential for sperm motility and male fertility, and well suited to pharmacological inhibition. However, GAPDHS is difficult to isolate from native sources and recombinant expression frequently results in high production of insoluble enzyme. We chose to use the Bac-to-Bac baculovirus-insect cell system to express a His-tagged form of human GAPDHS (Hu his-GAPDHS) lacking the proline-rich N-terminal sequence. This recombinant Hu his-GAPDHS was successfully produced in Spodoptera frugiperda 9 (Sf9) cells by infection with recombinant virus as a soluble, enzymatically active form in high yield, >35mg/L culture. Biochemical characterization of the purified enzyme by mass spectrometry and size exclusion chromatography confirmed the presence of the tetrameric form. Further characterization by peptide ion matching mass spectrometry and Edman sequencing showed that unlike the mixed tetramer forms produced in bacterial expression systems, human his-GAPDHS expressed in baculovirus-infected insect cells is homotetrameric. The ability to express and purify active human GAPDHS as homotetramers in high amounts will greatly aid in drug discovery efforts targeting this enzyme for discovery of novel contraceptives and three compounds were identified as inhibitors of Hu his-GAPDHS from a pilot screen of 1120 FDA-approved compounds

Long wavelength-sensing cones of zebrafish retina exhibit multiple layers of transcriptional heterogeneity

IntroductionUnderstanding how photoreceptor genes are regulated is important for investigating retinal development and disease. While much is known about gene regulation in cones, the mechanism by which tandemly-replicated opsins, such as human long wavelength-sensitive and middle wavelength-sensitive opsins, are differentially regulated remains elusive. In this study, we aimed to further our understanding of transcriptional heterogeneity in cones that express tandemly-replicated opsins and the regulation of such differential expression using zebrafish, which express the tandemly-replicated opsins lws1 and lws2.MethodsWe performed bulk and single cell RNA-Seq of LWS1 and LWS2 cones, evaluated expression patterns of selected genes of interest using multiplex fluorescence in situ hybridization, and used exogenous thyroid hormone (TH) treatments to test selected genes for potential control by thyroid hormone: a potent, endogenous regulator of lws1 and lws2 expression.ResultsOur studies indicate that additional transcriptional differences beyond opsin expression exist between LWS1 and LWS2 cones. Bulk RNA-Seq results showed 95 transcripts enriched in LWS1 cones and 186 transcripts enriched in LWS2 cones (FC > 2, FDR < 0.05). In situ hybridization results also reveal underlying heterogeneity within the lws1- and lws2-expressing populations. This heterogeneity is evident in cones of mature zebrafish, and further heterogeneity is revealed in transcriptional responses to TH treatments.DiscussionWe found some evidence of coordinate regulation of lws opsins and other genes by exogenous TH in LWS1 vs. LWS2 cones, as well as evidence of gene regulation not mediated by TH. The transcriptional differences between LWS1 and LWS2 cones are likely controlled by multiple signals, including TH

Toward a global reference database of COI barcodes for marine zooplankton

© The Author(s), 2021. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Bucklin, A., Peijnenburg, K. T. C. A., Kosobokova, K. N., O'Brien, T. D., Blanco-Bercial, L., Cornils, A., Falkenhaug, T., Hopcroft, R. R., Hosia, A., Laakmann, S., Li, C., Martell, L., Questel, J. M., Wall-Palmer, D., Wang, M., Wiebe, P. H., & Weydmann-Zwolicka, A. Toward a global reference database of COI barcodes for marine zooplankton. Marine Biology, 168(6), (2021): 78, https://doi.org/10.1007/s00227-021-03887-y.Characterization of species diversity of zooplankton is key to understanding, assessing, and predicting the function and future of pelagic ecosystems throughout the global ocean. The marine zooplankton assemblage, including only metazoans, is highly diverse and taxonomically complex, with an estimated ~28,000 species of 41 major taxonomic groups. This review provides a comprehensive summary of DNA sequences for the barcode region of mitochondrial cytochrome oxidase I (COI) for identified specimens. The foundation of this summary is the MetaZooGene Barcode Atlas and Database (MZGdb), a new open-access data and metadata portal that is linked to NCBI GenBank and BOLD data repositories. The MZGdb provides enhanced quality control and tools for assembling COI reference sequence databases that are specific to selected taxonomic groups and/or ocean regions, with associated metadata (e.g., collection georeferencing, verification of species identification, molecular protocols), and tools for statistical analysis, mapping, and visualization. To date, over 150,000 COI sequences for ~ 5600 described species of marine metazoan plankton (including holo- and meroplankton) are available via the MZGdb portal. This review uses the MZGdb as a resource for summaries of COI barcode data and metadata for important taxonomic groups of marine zooplankton and selected regions, including the North Atlantic, Arctic, North Pacific, and Southern Oceans. The MZGdb is designed to provide a foundation for analysis of species diversity of marine zooplankton based on DNA barcoding and metabarcoding for assessment of marine ecosystems and rapid detection of the impacts of climate change.Funding sources for authors of the review paper are described here: Scientific Committee on Oceanic Research (SCOR), and a grant to SCOR from the U.S. National Science Foundation (OCE-1840868). Netherlands Organization for Scientific Research (NWO) Vidi Grant/Award Number: 016.161.351 to K.T.C.A.P. European Union Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement No. 746186 (POSEIDoN) to D.W.P. The work of K.N.K. was performed in the framework of the state assignment of IO RAS (Theme No. 0128-2021-0007) and partially supported by Russian Foundation for Basic Research grants No. 18-05-60158 and No. 19-04-00955. The work of A.W.Z. was supported by a grant from HIDEA—Hidden diversity of the Arctic Ocean (No. 2017/27/B/NZ8/01056) from the National Science Centre, Poland, and a Fulbright Senior Award. The Norwegian Taxonomy Initiative of the Norwegian Biodiversity Information Centre provided funding for A.H. and L.M. (Project Nos. 70184233/HYPNO and 70184240/NORHYDRO), and for T.F. (Project Nos. 70184233/COPCLAD and 70184241/HYPCOP). The work of R.R.H. and J.M.Q. was supported by Census of Marine Life and NOAA Ocean Exploration and Research (NA05OAR4601079 and NA15OAR0110209). The work of S.L. was conducted at the Helmholtz Institute for Functional Marine Biodiversity at the University of Oldenburg (HIFMB). HIFMB is a collaboration between the Alfred-Wegener-Institute, Helmholtz-Center for Polar and Marine Research, and the Carl-von-Ossietzky University Oldenburg, initially funded by the Ministry for Science and Culture of Lower Saxony and the Volkswagen Foundation through the Niedersächsisches Vorab’ grant program (Grant No. ZN3285)

The Founder Strains of the Collaborative Cross Express a Complex Combination of Advantageous and Deleterious Traits for Male Reproduction

Surveys of inbred strains of mice are standard approaches to determine the heritability and range of phenotypic variation for biomedical traits. In addition, they may lead to the identification of novel phenotypes and models of human disease. Surprisingly, male reproductive phenotypes are among the least-represented traits in the Mouse Phenome Database. Here we report the results of a broad survey of the eight founder inbred strains of both the Collaborative Cross (CC) and the Diversity Outbred populations, two new mouse resources that are being used as platforms for systems genetics and sources of mouse models of human diseases. Our survey includes representatives of the three main subspecies of the house mice and a mix of classical and wild-derived inbred strains. In addition to standard staples of male reproductive phenotyping such as reproductive organ weights, sperm counts, and sperm morphology, our survey includes sperm motility and the first detailed survey of testis histology. As expected for such a broad survey, heritability varies widely among traits. We conclude that although all eight inbred strains are fertile, most display a mix of advantageous and deleterious male reproductive traits. The CAST/EiJ strain is an outlier, with an unusual combination of deleterious male reproductive traits including low sperm counts, high levels of morphologically abnormal sperm, and poor motility. In contrast, sperm from the PWK/PhJ and WSB/EiJ strains had the greatest percentages of normal morphology and vigorous motility. Finally, we report an abnormal testis phenotype that is highly heritable and restricted to the WSB/EiJ strain. This phenotype is characterized by the presence of a large, but variable, number of vacuoles in at least 10% of the seminiferous tubules. The onset of the phenotype between 2 and 3 wk of age is temporally correlated with the formation of the blood-testis barrier. We speculate that this phenotype may play a role in high rates of extinction in the CC project and in the phenotypes associated with speciation in genetic crosses that use the WSB/EiJ strain as representative of the Mus muculus domesticus subspecies

Safety and Immunogenicity of Neonatal Pneumococcal Conjugate Vaccination in Papua New Guinean Children: A Randomised Controlled Trial

Background: Approximately 826,000 children, mostly young infants, die annually from invasive pneumococcal disease. A 6-10-14-week schedule of pneumococcal conjugate vaccine (PCV) is efficacious but neonatal PCV may provide earlier protection and better coverage. We conducted an open randomized controlled trial in Papua New Guinea to compare safety, immunogenicity and priming for memory of 7-valent PCV (PCV7) given in a 0-1-2-month (neonatal) schedule with that of the routine 1-2-3-month (infant) schedule. Methods: We randomized 318 infants at birth to receive PCV7 in the neonatal or infant schedule or no PCV7. All infants received 23-valent pneumococcal polysaccharide vaccine (PPV) at age 9 months. Serotype-specific serum IgG for PCV7 (VT) serotypes and non-VT serotypes 2, 5 and 7F were measured at birth and 2, 3, 4, 9, 10 and 18 months of age. Primary outcomes were geometric mean concentrations (GMCs) and proportions with concentration ≥0.35 µg/ml of VT serotype-specific pneumococcal IgG at age 2 months and one month post-PPV.Results: We enrolled 101, 105 and 106 infants, respectively, into neonatal, infant and control groups. Despite high background levels of maternally derived antibody, both PCV7 groups had higher GMCs than controls at age 2 months for serotypes 4 (p<0.001) and 9V (p<0.05) and at age 3 months for all VTs except 6B. GMCs for serotypes 4, 9V, 18C and 19F were significantly higher (p<0.001) at age 2 months in the neonatal (one month post-dose2 PCV7) than in the infant group (one month post-dose1 PCV7). PPV induced significantly higher VT antibody responses in PCV7-primed than unprimed infants, with neonatal and infant groups equivalent. High VT and non-VT antibody concentrations generally persisted to age 18 months. Conclusions: PCV7 is well-tolerated and immunogenic in PNG neonates and young infants and induces immunologic memory to PPV booster at age 9 months with antibody levels maintained to age 18 months

Assessment of habitat and survey criteria for the great crested newt (Triturus cristatus) in Scotland: a case study on a translocated population

The great crested newt Triturus cristatus has declined across its range due to habitat loss, motivating research into biotic and abiotic species determinants. However, research has focused on populations in England and mainland Europe. We examined habitat and survey criteria for great crested newts in Scotland, with focus on a large, translocated population. Adult counts throughout the breeding season were obtained annually using torchlight surveys, and Habitat Suitability Index (HSI) assessed at created ponds (N = 24) in 2006 (immediately post-translocation) and 2015 (9 years post-translocation). In 2006, ‘best case’ HSI scores were calculated to predict habitat suitability should great crested newts have unrestricted access to terrestrial habitat. Abiotic criteria included in and omitted from current great crested newt survey guidelines were assessed using data recorded in 2015. Some ponds had improved HSI scores in 2015, but overall failure to meet predicted scores suggests management is needed to improve habitat suitability. Great crested newt activity was positively associated with moon visibility and phase, air temperature, and pH, but negatively correlated with water clarity. Importantly, our results indicate there are abiotic determinants specific to Scottish great crested newts. Principally, survey temperature thresholds should be lowered to enable accurate census of Scottish populations

Rehabilitation of memory following brain injury (ReMemBrIn): study protocol for a randomised controlled trial

Background Impairments of memory are commonly reported by people with traumatic brain injuries (TBI). Such deficits are persistent, debilitating, and can severely impact quality of life. Currently, many do not routinely receive follow-up appointments for residual memory problems following discharge. Methods/Design This is a multi-centre, randomised controlled trial investigating the clinical and cost-effectiveness of a group-based memory rehabilitation programme. Three hundred and twelve people with a traumatic brain injury will be randomised from four centres. Participants will be eligible if they had a traumatic brain injury more than 3 months prior to recruitment, have memory problems, are 18 to 69 years of age, are able to travel to one of our centres and attend group sessions, and are able to give informed consent. Participants will be randomised in clusters of 4 to 6 to the group rehabilitation intervention or to usual care. Intervention groups will receive 10 weekly sessions of a manualised memory rehabilitation programme, which has been developed in previous pilot studies. The intervention will include restitution strategies to retrain impaired memory functions and compensation strategies to enable participants to cope with their memory problems. All participants will receive a follow-up postal questionnaire and an assessment by a research assistant at 6 and 12 months post-randomisation. The primary outcome is the Everyday Memory Questionnaire at 6 months. Secondary outcomes include the Rivermead Behavioural Memory Test-3, General Health Questionnaire-30, health related quality of life, cost-effectiveness analysis determined by the EQ-5D and a service use questionnaire, individual goal attainment, European Brain Injury Questionnaire (patient and relative versions), and the Everyday Memory Questionnaire-relative version. The primary analysis will be based on intention to treat. A mixed-model regression analysis of the Everyday Memory Questionnaire at 6 months will be used to estimate the effect of the group memory rehabilitation programme. Discussion The study will hopefully provide robust evidence regarding the clinical and cost-effectiveness of a group-based memory rehabilitation intervention for civilians and military personnel following TBI. We discuss our decision-making regarding choice of outcome measures and control group, and the unique challenges to recruiting people with memory problems to trials