Molecular cloning and expression of bovine nucleoplasmin 2 (NPM2): a maternal effect gene regulated by miR-181a

Background Nucleoplasmin 2 (NPM2) is an oocyte-specific nuclear protein essential for nuclear and nucleolar organization and early embryonic development. The aims of this study were to clone the bovine NPM2 gene, determine its temporal expression during oocyte development and early embryogenesis, and evaluate the potential role of miRNA-181a in regulation of its expression. Methods A 329 bp cDNA fragment was amplified from bovine fetal ovary using primers designed based on the conserved regions of the human and mouse NPM2 cDNA sequences. RACE experiments were performed to obtain the 5\u27 and 3\u27 ends of the bovine NPM2 cDNA. Real time PCR and Western blot analysis were used to examine the expression of bovine NPM2 in oocytes and early embryos. Co-expression of bovine NPM2 and miRNA-181a in Hela cells was performed to determine if expression of bovine NPM2 is regulated by miRNA-181a. Results The bovine NPM2 cDNA is 851 bp in length encoding a protein of 200 amino acids. The protein contains the conserved bipartite nuclear localization sequence and shows 53% and 62% identity with mouse and human NPM2, respectively. Expression of bovine NPM2 mRNA is restricted to ovaries. NPM2 mRNA is abundant in GV and MII stage oocytes, decreases in early cleavage stage embryos, and barely detectable in morula and blastocyst stage embryos. Similarly, expression of NPM2 protein is high in oocytes and early embryos but extremely low in blastocysts. The abundance of NPM2 mRNA is significantly lower in oocytes isolated from persistent versus growing dominant follicles (P \u3c 0.05). A miR-181a binding site in the 3\u27UTR of the NPM2transcript was identified. Transfection experiments showed that bovine NPM2 protein expression is reduced in Hela cells expressing miR-181a compared to control cells without miR-181a, indicating that translation of NPM2 is repressed by miR-181a. Conclusions Our data suggest that expression of bovine NPM2 is temporally regulated during early embryogenesis and miR-181a may play a role in its regulation

High-throughput sequencing of the paired human immunoglobulin heavy and light chain repertoire.

Each B-cell receptor consists of a pair of heavy and light chains. High-throughput sequencing can identify large numbers of heavy- and light-chain variable regions (VH and VL) in a given B-cell repertoire, but information about endogenous pairing of heavy and light chains is lost after bulk lysis of B-cell populations. Here we describe a way to retain this pairing information. In our approach, single B cells (>5 × 104 capacity per experiment) are deposited in a high-density microwell plate (125 pl/well) and lysed in situ. mRNA is then captured on magnetic beads, reverse transcribed and amplified by emulsion VH:VL linkage PCR. The linked transcripts are analyzed by Illumina high-throughput sequencing. We validated the fidelity of VH:VL pairs identified by this approach and used the method to sequence the repertoire of three human cell subsets-peripheral blood IgG+ B cells, peripheral plasmablasts isolated after tetanus toxoid immunization and memory B cells isolated after seasonal influenza vaccinatio

Neurosurgeons' experiences of conducting and disseminating clinical research in low-income and middle-income countries: a reflexive thematic analysis.

OBJECTIVES: Low-income and-middle-income countries (LMICs) are increasing investment in research and development, yet there remains a paucity of neurotrauma research published by those in LMICs. The aim of this study was to understand neurosurgeons' experiences of, aspirations for, and ability to conduct and disseminate clinical research in LMICs. DESIGN: This was a two-stage inductive qualitative study situated within the naturalistic paradigm. This study committed to an interpretivist way of knowing (epistemology), and considered reality subjective and multiple (ontology). Data collection used online methods and included a web-based survey tool for demographic data, an asynchronous online focus group and follow-up semistructured interviews. Data were analysed using Braun and Clarke's Reflexive Thematic Analysis supported by NVivo V.12. SETTING: LMICs. PARTICIPANTS: In April-July 2020, 26 neurosurgeons from 11 LMICs participated in this study (n=24 in the focus groups, n=20 in follow-up interviews). RESULTS: The analysis gave rise to five themes: The local landscape; creating capacity; reach and impact; collaborative inquiry; growth and sustainability. Each theme contained an inhibitor and stimulus to neurosurgeons conducting and disseminating clinical research, interpreted as 'the neurosurgical research potential in LMICs'. Mentorship, education, infrastructure, impact and engagement were identified as specific accelerators. Whereas lack of generalisability, absence of dissemination and dissemination without peer review may desensitise the impact of research conducted by neurosurgeons. CONCLUSION: The geographical, political and population complexities make research endeavour challenging for neurosurgeons in LMICs. Yet in spite of, and because of, these complexities LMICs provide rich opportunities to advance global neurosurgery. More studies are required to evaluate the specific effects of accelerators of research conducted by neurosurgeons and to understand the effects of desensitisers on high-quality, high-impact clinical research

Genome sequence of an Australian kangaroo, Macropus eugenii, provides insight into the evolution of mammalian reproduction and development.

BACKGROUND: We present the genome sequence of the tammar wallaby, Macropus eugenii, which is a member of the kangaroo family and the first representative of the iconic hopping mammals that symbolize Australia to be sequenced. The tammar has many unusual biological characteristics, including the longest period of embryonic diapause of any mammal, extremely synchronized seasonal breeding and prolonged and sophisticated lactation within a well-defined pouch. Like other marsupials, it gives birth to highly altricial young, and has a small number of very large chromosomes, making it a valuable model for genomics, reproduction and development. RESULTS: The genome has been sequenced to 2 × coverage using Sanger sequencing, enhanced with additional next generation sequencing and the integration of extensive physical and linkage maps to build the genome assembly. We also sequenced the tammar transcriptome across many tissues and developmental time points. Our analyses of these data shed light on mammalian reproduction, development and genome evolution: there is innovation in reproductive and lactational genes, rapid evolution of germ cell genes, and incomplete, locus-specific X inactivation. We also observe novel retrotransposons and a highly rearranged major histocompatibility complex, with many class I genes located outside the complex. Novel microRNAs in the tammar HOX clusters uncover new potential mammalian HOX regulatory elements. CONCLUSIONS: Analyses of these resources enhance our understanding of marsupial gene evolution, identify marsupial-specific conserved non-coding elements and critical genes across a range of biological systems, including reproduction, development and immunity, and provide new insight into marsupial and mammalian biology and genome evolution

GOALS-JWST: Small neutral grains and enhanced 3.3 micron PAH emission in the Seyfert galaxy NGC 7469

We present James Webb Space Telescope (JWST) Near Infrared Spectrograph (NIRSpec) integral-field spectroscopy of the nearby luminous infrared galaxy, NGC 7469. We take advantage of the high spatial/spectral resolution and wavelength coverage of JWST /NIRSpec to study the 3.3 um neutral polycyclic aromatic hydrocarbon (PAH) grain emission on ~60 pc scales. We find a clear change in the average grain properties between the star-forming ring and the central AGN. Regions in the vicinity of the AGN, with [NeIII]/[NeII]>0.25, tend to have larger grain sizes and lower aliphatic-to-aromatic (3.4/3.3) ratios indicating that smaller grains are preferentially removed by photo-destruction in the vicinity of the AGN. We find an overall suppression of the total PAH emission relative to the ionized gas in the central 1 kpc region of the AGN in NGC 7469 compared to what has been observed with Spitzer on 3 kpc scales. However, the fractional 3.3 um to total PAH power is enhanced in the starburst ring, possibly due to a variety of physical effects on sub-kpc scales, including recurrent fluorescence of small grains or multiple photon absorption by large grains. Finally, the IFU data show that while the 3.3 um PAH-derived star formation rate (SFR) in the ring is 8% higher than that inferred from the [NeII] and [NeIII] emission lines, the integrated SFR derived from the 3.3 um feature would be underestimated by a factor of two due to the deficit of PAHs around the AGN, as might occur if a composite system like NGC 7469 were to be observed at high-redshift.Comment: 14 pages, 5 figures, 2 tables, Submitted to ApJ

Mismatch repair deficiency predicts response of solid tumors to PD-1 blockade.

The genomes of cancers deficient in mismatch repair contain exceptionally high numbers of somatic mutations. In a proof-of-concept study, we previously showed that colorectal cancers with mismatch repair deficiency were sensitive to immune checkpoint blockade with antibodies to programmed death receptor-1 (PD-1). We have now expanded this study to evaluate the efficacy of PD-1 blockade in patients with advanced mismatch repair-deficient cancers across 12 different tumor types. Objective radiographic responses were observed in 53% of patients, and complete responses were achieved in 21% of patients. Responses were durable, with median progression-free survival and overall survival still not reached. Functional analysis in a responding patient demonstrated rapid in vivo expansion of neoantigen-specific T cell clones that were reactive to mutant neopeptides found in the tumor. These data support the hypothesis that the large proportion of mutant neoantigens in mismatch repair-deficient cancers make them sensitive to immune checkpoint blockade, regardless of the cancers\u27 tissue of origin

A connectome and analysis of the adult Drosophila central brain.

The neural circuits responsible for animal behavior remain largely unknown. We summarize new methods and present the circuitry of a large fraction of the brain of the fruit fly Drosophila melanogaster. Improved methods include new procedures to prepare, image, align, segment, find synapses in, and proofread such large data sets. We define cell types, refine computational compartments, and provide an exhaustive atlas of cell examples and types, many of them novel. We provide detailed circuits consisting of neurons and their chemical synapses for most of the central brain. We make the data public and simplify access, reducing the effort needed to answer circuit questions, and provide procedures linking the neurons defined by our analysis with genetic reagents. Biologically, we examine distributions of connection strengths, neural motifs on different scales, electrical consequences of compartmentalization, and evidence that maximizing packing density is an important criterion in the evolution of the fly's brain

BRITICE Glacial Map, version 2: a map and GIS database of glacial landforms of the last British-Irish Ice Sheet

During the last glaciation, most of the British Isles and the surrounding continental shelf were covered by the British–Irish Ice Sheet (BIIS). An earlier compilation from the existing literature (BRITICE version 1) assembled the relevant glacial geomorphological evidence into a freely available GIS geodatabase and map (Clark et al. 2004: Boreas 33, 359). New high-resolution digital elevation models, of the land and seabed, have become available casting the glacial landform record of the British Isles in a new light and highlighting the shortcomings of the V.1 BRITICE compilation. Here we present a wholesale revision of the evidence, onshore and offshore, to produce BRITICE version 2, which now also includes Ireland. All published geomorphological evidence pertinent to the behaviour of the ice sheet is included, up to the census date of December 2015. The revised GIS database contains over 170 000 geospatially referenced and attributed elements – an eightfold increase in information from the previous version. The compiled data include: drumlins, ribbed moraine, crag-and-tails, mega-scale glacial lineations, glacially streamlined bedrock (grooves, roches moutonnées, whalebacks), glacial erratics, eskers, meltwater channels (subglacial, lateral, proglacial and tunnel valleys), moraines, trimlines, cirques, trough-mouth fans and evidence defining ice-dammed lakes. The increased volume of features necessitates different map/database products with varying levels of data generalization, namely: (i) an unfiltered GIS database containing all mapping; (ii) a filtered GIS database, resolving data conflicts and with edits to improve geo-locational accuracy (available as GIS data and PDF maps); and (iii) a cartographically generalized map to provide an overview of the distribution and types of features at the ice-sheet scale that can be printed at A0 paper size at a 1:1 250 000 scale. All GIS data, the maps (as PDFs) and a bibliography of all published sources are available for download from: https://www.sheffield.ac.uk/geography/staff/clark_chris/britice

Molecular Ecology and Natural History of Simian Foamy Virus Infection in Wild-Living Chimpanzees

Identifying microbial pathogens with zoonotic potential in wild-living primates can be important to human health, as evidenced by human immunodeficiency viruses types 1 and 2 (HIV-1 and HIV-2) and Ebola virus. Simian foamy viruses (SFVs) are ancient retroviruses that infect Old and New World monkeys and apes. Although not known to cause disease, these viruses are of public health interest because they have the potential to infect humans and thus provide a more general indication of zoonotic exposure risks. Surprisingly, no information exists concerning the prevalence, geographic distribution, and genetic diversity of SFVs in wild-living monkeys and apes. Here, we report the first comprehensive survey of SFVcpz infection in free-ranging chimpanzees (Pan troglodytes) using newly developed, fecal-based assays. Chimpanzee fecal samples (n = 724) were collected at 25 field sites throughout equatorial Africa and tested for SFVcpz-specific antibodies (n = 706) or viral nucleic acids (n = 392). SFVcpz infection was documented at all field sites, with prevalence rates ranging from 44% to 100%. In two habituated communities, adult chimpanzees had significantly higher SFVcpz infection rates than infants and juveniles, indicating predominantly horizontal rather than vertical transmission routes. Some chimpanzees were co-infected with simian immunodeficiency virus (SIVcpz); however, there was no evidence that SFVcpz and SIVcpz were epidemiologically linked. SFVcpz nucleic acids were recovered from 177 fecal samples, all of which contained SFVcpz RNA and not DNA. Phylogenetic analysis of partial gag (616 bp), pol-RT (717 bp), and pol-IN (425 bp) sequences identified a diverse group of viruses, which could be subdivided into four distinct SFVcpz lineages according to their chimpanzee subspecies of origin. Within these lineages, there was evidence of frequent superinfection and viral recombination. One chimpanzee was infected by a foamy virus from a Cercopithecus monkey species, indicating cross-species transmission of SFVs in the wild. These data indicate that SFVcpz (i) is widely distributed among all chimpanzee subspecies; (ii) is shed in fecal samples as viral RNA; (iii) is transmitted predominantly by horizontal routes; (iv) is prone to superinfection and recombination; (v) has co-evolved with its natural host; and (vi) represents a sensitive marker of population structure that may be useful for chimpanzee taxonomy and conservation strategies

Management of patients with advanced prostate cancer—metastatic and/or castration-resistant prostate cancer: report of the Advanced Prostate Cancer Consensus Conference (APCCC) 2022

Background: Innovations in imaging and molecular characterisation together with novel treatment options have improved outcomes in advanced prostate cancer. However, we still lack high-level evidence in many areas relevant to making management decisions in daily clinical practise. The 2022 Advanced Prostate Cancer Consensus Conference (APCCC 2022) addressed some questions in these areas to supplement guidelines that mostly are based on level 1 evidence. Objective: To present the voting results of the APCCC 2022. Design, setting, and participants: The experts voted on controversial questions where high- level evidence is mostly lacking: locally advanced prostate cancer; biochemical recurrence after local treatment; metastatic hormone-sensitive, non-metastatic, and metastatic castration- resistant prostate cancer; oligometastatic prostate cancer; and managing side effects of hormonal therapy. A panel of 105 international prostate cancer experts voted on the consensus questions. Outcome measurements and statistical analysis: The panel voted on 198 pre-defined questions, which were developed by 117 voting and non-voting panel members prior to the conference following a modified Delphi process. A total of 116 questions on metastatic and/or castration- resistant prostate cancer are discussed in this manuscript. In 2022, the voting was done by a web-based survey because of COVID-19 restrictions. Results and limitations: The voting reflects the expert opinion of these panellists and did not incorporate a standard literature review or formal meta-analysis. The answer options for the consensus questions received varying degrees of support from panellists, as reflected in this article and the detailed voting results are reported in the supplementary material. We report here on topics in metastatic, hormone-sensitive prostate cancer (mHSPC), non-metastatic, castration-resistant prostate cancer (nmCRPC), metastatic castration-resistant prostate cancer (mCRPC), and oligometastatic and oligoprogressive prostate cancer. Conclusions: These voting results in four specific areas from a panel of experts in advanced prostate cancer can help clinicians and patients navigate controversial areas of management for which high-level evidence is scant or conflicting and can help research funders and policy makers identify information gaps and consider what areas to explore further. However, diagnostic and treatment decisions always have to be individualised based on patient characteristics, including the extent and location of disease, prior treatment(s), co-morbidities, patient preferences, and treatment recommendations and should also incorporate current and emerging clinical evidence and logistic and economic factors. Enrolment in clinical trials is strongly encouraged. Importantly, APCCC 2022 once again identified important gaps where there is non-consensus and that merit evaluation in specifically designed trials. Patient summary: The Advanced Prostate Cancer Consensus Conference (APCCC) provides a forum to discuss and debate current diagnostic and treatment options for patients with advanced prostate cancer. The conference aims to share the knowledge of international experts in prostate cancer with healthcare providers worldwide. At each APCCC, an expert panel votes on pre-defined questions that target the most clinically relevant areas of advanced prostate cancer treatment for which there are gaps in knowledge. The results of the voting provide a practical guide to help clinicians discuss therapeutic options with patients and their relatives as part of shared and multidisciplinary decision-making. This report focuses on the advanced setting, covering metastatic hormone-sensitive prostate cancer and both non-metastatic and metastatic castration-resistant prostate cancer. Twitter summary: Report of the results of APCCC 2022 for the following topics: mHSPC, nmCRPC, mCRPC, and oligometastatic prostate cancer. Take-home message: At APCCC 2022, clinically important questions in the management of advanced prostate cancer management were identified and discussed, and experts voted on pre-defined consensus questions. The report of the results for metastatic and/or castration- resistant prostate cancer is summarised here