Requirement for DNA Ligase IV during Embryonic Neuronal Development

The embryonic ventricular and subventricular zones (VZ/SVZ) contain the neuronal stem and progenitor cells and undergo rapid proliferation. The intermediate zone (IZ) contains nonreplicating, differentiated cells. The VZ/SVZ is hypersensitive to radiation-induced apoptosis. Ablation of DNA non-homologous end-joining (NHEJ) proteins, XRCC4 or DNA ligase IV (LigIV), confers ataxia telangiectasia mutated (ATM)-dependent apoptosis predominantly in the IZ. We examine the mechanistic basis underlying these distinct sensitivities using a viable LigIV (Lig4(Y288C)) mouse, which permits an examination of the DNA damage responses in the embryonic and adult brain. Via combined analysis of DNA breakage, apoptosis, and cell-cycle checkpoint control in tissues, we show that apoptosis in the VZ/SVZ and IZ is activated by low numbers of DNA double-strand breaks (DSBs). Unexpectedly, high sensitivity in the VZ/SVZ arises from sensitive activation of ATM-dependent apoptosis plus an ATM-independent process. In contrast, the IZ appears to be hypersensitive to persistent DSBs. NHEJ functions efficiently in both compartments. The VZ/SVZ and IZ regions incur high endogenous DNA breakage, which correlates with VZ proliferation. We demonstrate a functional G(2)/M checkpoint in VZ/SVZ cells and show that it is not activated by low numbers of DSBs, allowing damaged VZ/SVZ cells to transit into the IZ. We propose a novel model in which microcephaly in LIG4 syndrome arises from sensitive apoptotic induction from persisting DSBs in the IZ, which arise from high endogenous breakage in the VZ/SVZ and transit of damaged cells to the IZ. The VZ/SVZ, in contrast, is highly sensitive to acute radiation-induced DSB formation

Clinical observation of diminished bone quality and quantity through longitudinal HR-pQCT-derived remodeling and mechanoregulation.

High resolution peripheral quantitative computed tomography (HR-pQCT) provides methods for quantifying volumetric bone mineral density and microarchitecture necessary for early diagnosis of bone disease. When combined with a longitudinal imaging protocol and finite element analysis, HR-pQCT can be used to assess bone formation and resorption (i.e., remodeling) and the relationship between this remodeling and mechanical loading (i.e., mechanoregulation) at the tissue level. Herein, 25 patients with a contralateral distal radius fracture were imaged with HR-pQCT at baseline and 9-12 months follow-up: 16 patients were prescribed vitamin D3 with/without calcium supplement based on a blood biomarker measures of bone metabolism and dual-energy X-ray absorptiometry image-based measures of normative bone quantity which indicated diminishing (n = 9) or poor (n = 7) bone quantity and 9 were not. To evaluate the sensitivity of this imaging protocol to microstructural changes, HR-pQCT images were registered for quantification of bone remodeling and image-based micro-finite element analysis was then used to predict local bone strains and derive rules for mechanoregulation. Remodeling volume fractions were predicted by both average values of trabecular and cortical thickness and bone mineral density (R2 > 0.8), whereas mechanoregulation was affected by dominance of the arm and group classification (p < 0.05). Overall, longitudinal, extended HR-pQCT analysis enabled the identification of changes in bone quantity and quality too subtle for traditional measures

Precision of bone mechanoregulation assessment in humans using longitudinal high-resolution peripheral quantitative computed tomography in vivo.

Local mechanical stimuli in the bone microenvironment are essential for the homeostasis and adaptation of the skeleton, with evidence suggesting that disruption of the mechanically-driven bone remodelling process may lead to bone loss. Longitudinal clinical studies have shown the combined use of high-resolution peripheral quantitative computed tomography (HR-pQCT) and micro-finite element analysis can be used to measure load-driven bone remodelling in vivo; however, quantitative markers of bone mechanoregulation and the precision of these analyses methods have not been validated in human subjects. Therefore, this study utilised participants from two cohorts. A same-day cohort (n = 33) was used to develop a filtering strategy to minimise false detections of bone remodelling sites caused by noise and motion artefacts present in HR-pQCT scans. A longitudinal cohort (n = 19) was used to develop bone imaging markers of trabecular bone mechanoregulation and characterise the precision for detecting longitudinal changes in subjects. Specifically, we described local load-driven formation and resorption sites independently using patient-specific odds ratios (OR) and 99 % confidence intervals. Conditional probability curves were computed to link the mechanical environment to the remodelling events detected on the bone surface. To quantify overall mechanoregulation, we calculated a correct classification rate measuring the fraction of remodelling events correctly identified by the mechanical signal. Precision was calculated as root-mean-squared averages of the coefficient of variation (RMS-SD) of repeated measurements using scan-rescan pairs at baseline combined with a one-year follow-up scan. We found no significant mean difference (p < 0.01) between scan-rescan conditional probabilities. RMS-SD was 10.5 % for resorption odds, 6.3 % for formation odds, and 1.3 % for correct classification rates. Bone was most likely to be formed in high-strain and resorbed in low-strain regions for all participants, indicating a consistent, regulated response to mechanical stimuli. For each percent increase in strain, the likelihood of bone resorption decreased by 2.0 ± 0.2 %, and the likelihood of bone formation increased by 1.9 ± 0.2 %, totalling 38.3 ± 1.1 % of strain-driven remodelling events across the entire trabecular compartment. This work provides novel robust bone mechanoregulation markers and their precision for designing future clinical studies

Assessing the cost of global biodiversity and conservation knowledge

Knowledge products comprise assessments of authoritative information supported by stan-dards, governance, quality control, data, tools, and capacity building mechanisms. Considerable resources are dedicated to developing and maintaining knowledge productsfor biodiversity conservation, and they are widely used to inform policy and advise decisionmakers and practitioners. However, the financial cost of delivering this information is largelyundocumented. We evaluated the costs and funding sources for developing and maintain-ing four global biodiversity and conservation knowledge products: The IUCN Red List ofThreatened Species, the IUCN Red List of Ecosystems, Protected Planet, and the WorldDatabase of Key Biodiversity Areas. These are secondary data sets, built on primary datacollected by extensive networks of expert contributors worldwide. We estimate that US$160million (range: US$116–204 million), plus 293 person-years of volunteer time (range: 278–308 person-years) valued at US$14 million (range US$12–16 million), were invested inthese four knowledge products between 1979 and 2013. More than half of this financingwas provided through philanthropy, and nearly three-quarters was spent on personnelcosts. The estimated annual cost of maintaining data and platforms for three of these knowl-edge products (excluding the IUCN Red List of Ecosystems for which annual costs were notpossible to estimate for 2013) is US$6.5 million in total (range: US$6.2–6.7 million). We esti-mated that an additional US$114 million will be needed to reach pre-defined baselines ofdata coverage for all the four knowledge products, and that once achieved, annual mainte-nance costs will be approximately US$12 million. These costs are much lower than those tomaintain many other, similarly important, global knowledge products. Ensuring that biodi-versity and conservation knowledge products are sufficiently up to date, comprehensiveand accurate is fundamental to inform decision-making for biodiversity conservation andsustainable development. Thus, the development and implementation of plans for sustain-able long-term financing for them is critical

Non‐canonical autophagy functions of ATG16L1 in epithelial cells limit lethal infection by influenza A virus

Influenza A virus (IAV) and SARS-CoV-2 (COVID-19) cause pandemic infections where cytokine storm syndrome and lung inflammation lead to high mortality. Given the high social and economic cost of respiratory viruses, there is an urgent need to understand how the airways defend against virus infection. Here we use mice lacking the WD and linker domains of ATG16L1 to demonstrate that ATG16L1-dependent targeting of LC3 to single-membrane, non-autophagosome compartments - referred to as non-canonical autophagy - protects mice from lethal IAV infection. Mice with systemic loss of non-canonical autophagy are exquisitely sensitive to low-pathogenicity IAV where extensive viral replication throughout the lungs, coupled with cytokine amplification mediated by plasmacytoid dendritic cells, leads to fulminant pneumonia, lung inflammation and high mortality. IAV was controlled within epithelial barriers where non-canonical autophagy reduced IAV fusion with endosomes and activation of interferon signalling. Conditional mouse models and ex vivo analysis showed that protection against IAV infection of lung was independent of phagocytes and other leucocytes. This establishes non-canonical autophagy in airway epithelial cells as a novel innate defence that restricts IAV infection and lethal inflammation at respiratory surfaces

Spiroindolines Identify the Vesicular Acetylcholine Transporter as a Novel Target for Insecticide Action

The efficacy of all major insecticide classes continues to be eroded by the development of resistance mediated, in part, by selection of alleles encoding insecticide insensitive target proteins. The discovery of new insecticide classes acting at novel protein binding sites is therefore important for the continued protection of the food supply from insect predators, and of human and animal health from insect borne disease. Here we describe a novel class of insecticides (Spiroindolines) encompassing molecules that combine excellent activity against major agricultural pest species with low mammalian toxicity. We confidently assign the vesicular acetylcholine transporter as the molecular target of Spiroindolines through the combination of molecular genetics in model organisms with a pharmacological approach in insect tissues. The vesicular acetylcholine transporter can now be added to the list of validated insecticide targets in the acetylcholine signalling pathway and we anticipate that this will lead to the discovery of novel molecules useful in sustaining agriculture. In addition to their potential as insecticides and nematocides, Spiroindolines represent the only other class of chemical ligands for the vesicular acetylcholine transporter since those based on the discovery of vesamicol over 40 years ago, and as such, have potential to provide more selective tools for PET imaging in the diagnosis of neurodegenerative disease. They also provide novel biochemical tools for studies of the function of this protein family

The WD and linker domains of ATG16L1 required for non-canonical autophagy limit lethal influenza A virus infection at epithelial surfaces

Phagocytosis and autophagy represent two evolutionarily ancient pathways that provide an important defense against infection by delivering pathogens to lysosomes for degradation. Phagocytosis and autophagy are linked by non-canonical autophagy pathways that conjugate LC3 to endo-lysosome compartments to facilitate phagosome maturation and lysosome fusion. A role for non-canonical autophagy in host defence is implied from cellular studies in vitro, but critically, these studies have rarely been extended to infection of model organisms with intact epithelial barriers and complex immune systems. To address this, we developed a mouse model with specific loss of non-canonical autophagy by removing the WD and linker domain of ATG16L1 required for recruitment of LC3 to endo-lysosome compartments. The mice retain the coiled-coiled domain of ATG16L1 required for conventional autophagy and maintain tissue and immunological homeostasis. Mice with systemic loss of non-canonical autophagy are exquisitely sensitive to low-pathogenicity murine-adapted influenza A virus leading to extensive viral replication throughout the lungs, cytokine dysregulation, fulminant pneumonia and lung inflammation leading to high mortality associated with virulent strains. Conditional mouse models and ex vivo analysis showed that protection against IAV infection of lung required non-canonical autophagy within epithelial barriers but was independent of phagocytes and other leukocytes. This establishes non-canonical autophagy pathways in epithelial cells as a novel innate defence mechanism that can restrict IAV infection at mucosal surfaces

Role of Intraspecies Recombination in the Spread of Pathogenicity Islands within the Escherichia coli Species

Horizontal gene transfer is a key step in the evolution of bacterial pathogens. Besides phages and plasmids, pathogenicity islands (PAIs) are subjected to horizontal transfer. The transfer mechanisms of PAIs within a certain bacterial species or between different species are still not well understood. This study is focused on the High-Pathogenicity Island (HPI), which is a PAI widely spread among extraintestinal pathogenic Escherichia coli and serves as a model for horizontal transfer of PAIs in general. We applied a phylogenetic approach using multilocus sequence typing on HPI-positive and -negative natural E. coli isolates representative of the species diversity to infer the mechanism of horizontal HPI transfer within the E. coli species. In each strain, the partial nucleotide sequences of 6 HPI–encoded genes and 6 housekeeping genes of the genomic backbone, as well as DNA fragments immediately upstream and downstream of the HPI were compared. This revealed that the HPI is not solely vertically transmitted, but that recombination of large DNA fragments beyond the HPI plays a major role in the spread of the HPI within E. coli species. In support of the results of the phylogenetic analyses, we experimentally demonstrated that HPI can be transferred between different E. coli strains by F-plasmid mediated mobilization. Sequencing of the chromosomal DNA regions immediately upstream and downstream of the HPI in the recipient strain indicated that the HPI was transferred and integrated together with HPI–flanking DNA regions of the donor strain. The results of this study demonstrate for the first time that conjugative transfer and homologous DNA recombination play a major role in horizontal transfer of a pathogenicity island within the species E. coli

Performance evaluation of a non-invasive one-step multiplex RT-qPCR assay for detection of SARS-CoV-2 direct from saliva

Polymerase chain reaction (PCR) has proven to be the gold-standard for SARS-CoV-2 detection in clinical settings. The most common approaches rely on nasopharyngeal specimens obtained from swabs, followed by RNA extraction, reverse transcription and quantitative PCR. Although swab-based PCR is sensitive, swabbing is invasive and unpleasant to administer, reducing patient compliance for regular testing and resulting in an increased risk of improper sampling. To overcome these obstacles, we developed a non-invasive one-step RT-qPCR assay performed directly on saliva specimens. The University of Nottingham Asymptomatic Testing Service protocol simplifies sample collection and bypasses the need for RNA extraction, or additives, thus helping to encourage more regular testing and reducing processing time and costs. We have evaluated the assay against the performance criteria specified by the UK regulatory bodies and attained accreditation (BS EN ISO/IEC 17,025:2017) for SARS-CoV-2 diagnostic testing by the United Kingdom Accreditation Service. We observed a sensitivity of 1 viral copy per microlitre of saliva, and demonstrated a concordance of > 99.4% between our results and those of other accredited testing facilities. We concluded that saliva is a stable medium that allows for a highly precise, repeatable, and robust testing method