Using exome sequencing and a variant prioritization strategy that focuses on loss-of-function variants, we identified biallelic, loss-of-function CEP57 mutations as a cause of constitutional mosaic aneuploidies. CEP57 is a centrosomal protein and is involved in nucleating and stabilizing microtubules. Our findings indicate that these and/or additional functions of CEP57 are crucial for maintaining correct chromosomal number during cell division.

The khmer package is a freely available software library for working efficiently with fixed length DNA words, or k-mers. khmer provides implementations of a probabilistic k-mer counting data structure, a compressible De Bruijn graph representation, De Bruijn graph partitioning, and digital normalization. khmer is implemented in C++ and Python, and is freely available under the BSD license at  https://github.com/dib-lab/khmer/.

To study the social determinants and cardiovascular risk factors for multimorbidity and the acquisition sequence of multimorbidity.

Opioid prescribing has more than doubled in the UK between 1998 and 2016. Potential adverse health implications include dependency, falls and increased health expenditure.

Crystallographic and solution studies have shown that IgE molecules are acutely bent in their Fc region. Crystal structures reveal the Cɛ2 domain pair folded back onto the Cɛ3-Cɛ4 domains, but is the molecule exclusively bent or can the Cɛ2 domains adopt extended conformations and even 'flip' from one side of the molecule to the other? We report the crystal structure of IgE-Fc captured in a fully extended, symmetrical conformation and show by molecular dynamics, calorimetry, stopped-flow kinetic, surface plasmon resonance (SPR) and Förster resonance energy transfer (FRET) analyses that the antibody can indeed adopt such extended conformations in solution. This diversity of conformational states available to IgE-Fc offers a new perspective on IgE function in allergen recognition, as part of the B-cell receptor and as a therapeutic target in allergic disease.

A growing body of evidence suggests that ATP-gated P2X3 receptors (P2X3Rs) are implicated in chronic pain. We address the possibility that stable, synthetic analogs of diadenosine tetraphosphate (Ap4A) might induce antinociceptive effects by inhibiting P2X3Rs in peripheral sensory neurons.

Tropical forests are fundamental ecosystems, essential for providing terrestrial primary productivity, global nutrient cycling, and biodiversity. Despite their importance, tropical forests are currently threatened by deforestation and associated activities. Moreover, tropical regions are now mostly represented by secondary forest regrowth, with half of the remaining tropical forests as secondary forest. Soil invertebrates are an important component to the functioning and biodiversity of these soil ecosystems. However, it remains unclear how these past land-use activities and subsequent secondary forest developments have altered the soil invertebrate communities and any potential ecological consequences associated with this. DNA metabarcoding offers an effective approach to rapidly monitor soil invertebrate communities under different land-use practices and within secondary forests. In this study, we used DNA metabarcoding to detect community-based patterns of soil invertebrate composition across a primary forest, a 23-year-old secondary forest, and a 33-year-old secondary forest and the associated soil environmental drivers of the soil invertebrate community structure in the Maquenque National Wildlife Refuge of Costa Rica (MNWR). We also used a species contribution analysis (SIMPER) to determine which soil invertebrate groups may be an indication of these soils reaching a pre-disturbed state such as a primary forest. We found that the soil invertebrate community composition at class, order, family, and ESV level were mostly significantly different across that habitats. We also found that the primary forest had a greater richness of soil invertebrates compared to the 23-year-old and 33-year-old secondary forest. Moreover, a redundancy analysis indicated that soil moisture influenced soil invertebrate community structure and explained up to 22% of the total variation observed in the community composition across the habitats; whereas soil invertebrate richness was structured by soil microbial biomass carbon (C) (Cmic) and explained up to 52% of the invertebrate richness across the primary and secondary forests. Lastly, the SIMPER analysis revealed that Naididae, Entomobryidae, and Elateridae could be important indicators of soil and forest recuperation in the MNWR. This study adds to the increasing evidence that soil invertebrates are intimately linked with the soil microbial biomass carbon (Cmic) and that even after 33 years of natural regrowth of a forest, these land use activities can still have persisting effects on the overall composition and richness of the soil invertebrate communities.

Congenital heart defects (CHDs) have a neonatal incidence of 0.8-1% (refs. 1,2). Despite abundant examples of monogenic CHD in humans and mice, CHD has a low absolute sibling recurrence risk (∼2.7%), suggesting a considerable role for de novo mutations (DNMs) and/or incomplete penetrance. De novo protein-truncating variants (PTVs) have been shown to be enriched among the 10% of 'syndromic' patients with extra-cardiac manifestations. We exome sequenced 1,891 probands, including both syndromic CHD (S-CHD, n = 610) and nonsyndromic CHD (NS-CHD, n = 1,281). In S-CHD, we confirmed a significant enrichment of de novo PTVs but not inherited PTVs in known CHD-associated genes, consistent with recent findings. Conversely, in NS-CHD we observed significant enrichment of PTVs inherited from unaffected parents in CHD-associated genes. We identified three genome-wide significant S-CHD disorders caused by DNMs in CHD4, CDK13 and PRKD1. Our study finds evidence for distinct genetic architectures underlying the low sibling recurrence risk in S-CHD and NS-CHD.

Rationale: Image-guided, triggerable, drug delivery systems allow for precisely placed and highly localised anti-cancer treatment. They contain labels for spatial mapping and tissue uptake tracking, providing key location and timing information for the application of an external stimulus to trigger drug release. High Intensity Focused Ultrasound (HIFU or FUS) is a non-invasive approach for treating small tissue volumes and is particularly effective at inducing drug release from thermosensitive nanocarriers. Here, we present a novel MR-imageable thermosensitive liposome (iTSL) for drug delivery to triple-negative breast cancers (TNBC). Methods: A macrocyclic gadolinium-based Magnetic Resonance Imaging (MRI) contrast agent was covalently linked to a lipid. This was incorporated at 30 mol% into the lipid bilayer of a thermosensitive liposome that was also encapsulating doxorubicin. The resulting iTSL-DOX formulation was assessed for physical and chemical properties, storage stability, leakage of gadolinium or doxorubicin, and thermal- or FUS-induced drug release. Its effect on MRI relaxation time was tested in phantoms. Mice with tumours were used for studies to assess both tumour distribution and contrast enhancement over time. A lipid-conjugated near-infrared fluorescence (NIRF) probe was also included in the liposome to facilitate the real time monitoring of iTSL distribution and drug release in tumours by NIRF bioimaging. TNBC (MDA-MB-231) tumour-bearing mice were then used to demonstrate the efficacy at retarding tumour growth and increasing survival. Results: iTSL-DOX provided rapid FUS-induced drug release that was dependent on the acoustic power applied. It was otherwise found to be stable, with minimum leakage of drug and gadolinium into buffers or under challenging conditions. In contrast to the usually suggested longer FUS treatment we identified that brief (~3 min) FUS significantly enhanced iTSL-DOX uptake to a targeted tumour and triggered near-total release of encapsulated doxorubicin, causing significant growth inhibition in the TNBC mouse model. A distinct reduction in the tumours' average T1 relaxation times was attributed to the iTSL accumulation. Conclusions: We demonstrate that tracking iTSL in tumours using MRI assists the application of FUS for precise drug release and therapy.

Hypertension, especially if poorly controlled, is a key determinant of chronic kidney disease (CKD) development and progression to end stage renal disease (ESRD).

Traditional languages are a key element of Indigenous peoples' identity, cultural expression, autonomy, spiritual and intellectual sovereignty, and wellbeing. While the links between Indigenous language loss and poor mental health have been demonstrated in several settings, little research has sought to identify the potential psychological benefits that may derive from language reclamation. The revival of the Barngarla language on the Eyre Peninsula, South Australia, offers a unique opportunity to examine whether improvements in mental health and social and emotional wellbeing can occur during and following the language reclamation process. This paper presents findings from 16 semi-structured interviews conducted with Barngarla community members describing their own experienced or observed mental health and wellbeing impacts of language reclamation activities. Aligning with a social and emotional wellbeing framework from an Aboriginal and Torres Strait Islander perspective, key themes included connection to spirituality and ancestors; connection to Country; connection to culture; connection to community; connection to family and kinship; connection to mind and emotions; and impacts upon identity and cultural pride at an individual level. These themes will form the foundation of assessment of the impacts of language reclamation in future stages of the project.

The NLRP3 inflammasome assembles in response to a variety of pathogenic and sterile danger signals, resulting in the production of interleukin-1β and interleukin-18. NLRP3 is a key component of the innate immune system and has been implicated as a driver of a number of acute and chronic diseases. We report the 2.8 Å crystal structure of the NLRP3 NACHT domain in complex with an inhibitor. The structure defines a binding pocket formed by the four subdomains of the NACHT domain, and shows the inhibitor acts as an intramolecular glue, which locks the protein in an inactive conformation. It provides further molecular insight into our understanding of NLRP3 activation, helps to detail the residues involved in subdomain coordination within the NLRP3 NACHT domain, and gives molecular insights into how gain-of-function mutations de-stabilize the inactive conformation of NLRP3. Finally, it suggests stabilizing the auto-inhibited form of the NACHT domain is an effective way to inhibit NLRP3, and will aid the structure-based development of NLRP3 inhibitors for a range of inflammatory diseases.

Delineating the genetic causes of developmental disorders is an area of active investigation. Mosaic structural abnormalities, defined as copy number or loss of heterozygosity events that are large and present in only a subset of cells, have been detected in 0.2-1.0% of children ascertained for clinical genetic testing. However, the frequency among healthy children in the community is not well characterized, which, if known, could inform better interpretation of the pathogenic burden of this mutational category in children with developmental disorders. In a case-control analysis, we compared the rate of large-scale mosaicism between 1303 children with developmental disorders and 5094 children lacking developmental disorders, using an analytical pipeline we developed, and identified a substantial enrichment in cases (odds ratio = 39.4, P-value 1.073e - 6). A meta-analysis that included frequency estimates among an additional 7000 children with congenital diseases yielded an even stronger statistical enrichment (P-value 1.784e - 11). In addition, to maximize the detection of low-clonality events in probands, we applied a trio-based mosaic detection algorithm, which detected two additional events in probands, including an individual with genome-wide suspected chimerism. In total, we detected 12 structural mosaic abnormalities among 1303 children (0.9%). Given the burden of mosaicism detected in cases, we suspected that many of the events detected in probands were pathogenic. Scrutiny of the genotypic-phenotypic relationship of each detected variant assessed that the majority of events are very likely pathogenic. This work quantifies the burden of structural mosaicism as a cause of developmental disorders.

Oxyntomodulin (OXM) is a glucagon-like peptide 1 (GLP-1) receptor (GLP1R)/glucagon receptor (GCGR) dual agonist peptide that reduces body weight in obese subjects through increased energy expenditure and decreased energy intake. The metabolic effects of OXM have been attributed primarily to GLP1R agonism. We examined whether a long acting GLP1R/GCGR dual agonist peptide exerts metabolic effects in diet-induced obese mice that are distinct from those obtained with a GLP1R-selective agonist.

Parathyroid hormone receptor 1 (PTH1R) belongs to the secretin class of G protein coupled receptors (GPCRs) and natively binds parathyroid hormone (PTH) and parathyroid hormone related peptide (PTHrP). Ligand binding to PTH1R involves binding to the large extracellular domain (ECD) and the orthosteric pocket, inducing conformational changes in the transmembrane domain and receptor activation. PTH1R regulates bone metabolism, signaling mainly through Gs and Gq/11 G-proteins. Here, we used phage display to generate PTH1R ECD-specific antibodies with the aim of modulating receptor functionality. We identified ECD-scFvhFc, which exhibited high affinity binding to both the isolated ECD and to the full-length receptor in styrene-maleic acid (SMA) lipid particles. Epitope mapping using hydrogen-deuterium exchange mass spectrometry (HDX-MS) indicates that the α1 helix of the ECD is ECD-scFvhFc's epitope which may partially overlap with the known PTH (1-34) binding site. However, PTH (1-34)-mediated Gs activation is Undisturbed by ECD-scFvhFc binding. In contrast, ECD-scFvhFc potently inhibits β-arrestin-2 recruitment after PTH (1-34)-driven receptor activation and thus represents the first monoclonal antibody to selectively inhibit distinct PTH1R signaling pathways. Given the complexity of PTH1R signaling and the emerging importance of biased GPCR activation in drug development, ECD-scFvhFc could be a valuable tool to study PTH1R signaling bias.

Protein:protein interactions are fundamental in living organism homeostasis. Here we introduce VHH6, a junctional epitope antibody capable of specifically recognizing a neo-epitope when two proteins interact, albeit transiently, to form a complex. Orthogonal biophysical techniques have been used to prove the "junctional epitope" nature of VHH6, a camelid single domain antibody recognizing the IL-6-gp80 complex but not the individual components alone. X-ray crystallography, HDX-MS and SPR analysis confirmed that the CDR regions of VHH6 interact simultaneously with IL-6 and gp80, locking the two proteins together. At the cellular level, VHH6 was able to alter the response of endothelial cells to exogenous IL-6, promoting a sustained STAT3 phosphorylation signal, an accumulation of IL-6 in vesicles and an overall pro-inflammatory phenotype supported further by transcriptomic analysis. Junctional epitope antibodies, like VHH6, not only offer new opportunities in screening and structure-aided drug discovery, but could also be exploited as therapeutics to modulate complex protein:protein interactions.

RNAi has considerable potential as a cancer therapeutic approach, but effective and efficient delivery of short interfering RNA (siRNA) to tumors remains a major hurdle. It remains a challenge to prepare a functional siRNA complex, target enough dose to the tumor, and stimulate its internalization into tumor cells and its release to the cytoplasm. Here, we show how these key barriers to siRNA delivery can be overcome with a complex─comprising siRNA, cationic lipids, and pH-responsive peptides─that is suited to tumor uptake enhancement via focused ultrasound (FUS). The complex provides effective nucleic acid encapsulation, nuclease protection, and endosomal escape such that gene silencing in cells is substantially more effective than that obtained with either equivalent lipoplexes or commercial reagents. In mice bearing MDA-MB-231 breast cancer xenografts, both lipid and ternary, lipid:peptide:siRNA complexes, prepared with near-infrared fluorescently labeled siRNA, accumulate in tumors following FUS treatments. Therefore, combining a well-designed lipid:peptide:siRNA complex with FUS tumor treatments is a promising route to achieve robust in vivo gene delivery.

Low back pain (LBP) is commonly treated with opioid analgesics despite evidence that these medicines provide minimal or no benefit for LBP and have an established profile of harms. International guidelines discourage or urge caution with the use of opioids for back pain; however, doctors and patients lack practical strategies to help them implement the guidelines. This trial will evaluate a multifaceted intervention to support general practitioners (GPs) and their patients with LBP implement the recommendations in the latest opioid prescribing guidelines.

Recent research on genomic profiling of pancreatic ductal adenocarcinoma (PDAC) has identified many potentially actionable alterations. However, the feasibility of using genomic profiling to guide routine clinical decision making for PDAC patients remains unclear. We retrospectively reviewed PDAC patients between October 2013 and December 2017, who underwent treatment at the Johns Hopkins Hospital and had clinical tumor next-generation sequencing (NGS) through commercial resources. Ninety-two patients with 93 tumors tested were included. Forty-eight (52%) patients had potentially curative surgeries. The median time from the tissue available to the NGS testing ordered was 229 days (interquartile range 62-415). A total of three (3%) patients had matched targeted therapies based on genomic profiling results. Genomic profiling guided personalized treatment for PDAC patients is feasible, but the percentage of patients who receive targeted therapy is low. The main challenges are ordering NGS testing early in the clinical course of the disease and the limited evidence of using a targeted approach in these patients. A real-time department level genomic testing ordering system in combination with an evidence-based flagging system for potentially actionable alterations could help address these shortcomings.

KPTN-related disorder is an autosomal recessive disorder associated with germline variants in KPTN (previously known as kaptin), a component of the mTOR regulatory complex KICSTOR. To gain further insights into the pathogenesis of KPTN-related disorder, we analysed mouse knockout and human stem cell KPTN loss-of-function models. Kptn -/- mice display many of the key KPTN-related disorder phenotypes, including brain overgrowth, behavioural abnormalities, and cognitive deficits. By assessment of affected individuals, we have identified widespread cognitive deficits (n = 6) and postnatal onset of brain overgrowth (n = 19). By analysing head size data from their parents (n = 24), we have identified a previously unrecognized KPTN dosage-sensitivity, resulting in increased head circumference in heterozygous carriers of pathogenic KPTN variants. Molecular and structural analysis of Kptn-/- mice revealed pathological changes, including differences in brain size, shape and cell numbers primarily due to abnormal postnatal brain development. Both the mouse and differentiated induced pluripotent stem cell models of the disorder display transcriptional and biochemical evidence for altered mTOR pathway signalling, supporting the role of KPTN in regulating mTORC1. By treatment in our KPTN mouse model, we found that the increased mTOR signalling downstream of KPTN is rapamycin sensitive, highlighting possible therapeutic avenues with currently available mTOR inhibitors. These findings place KPTN-related disorder in the broader group of mTORC1-related disorders affecting brain structure, cognitive function and network integrity.