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Mitochondrial DNA (mtDNA) encodes essential subunits of the oxidative phosphorylation system, but is also a major damage-associated molecular pattern (DAMP) that engages innate immune sensors when released into the cytoplasm, outside of cells or into circulation. As a DAMP, mtDNA not only contributes to anti-viral resistance, but also causes pathogenic inflammation in many disease contexts. Cells experiencing mtDNA stress caused by depletion of the mtDNA-packaging protein, transcription factor A, mitochondrial (TFAM) or during herpes simplex virus-1 infection exhibit elongated mitochondria, enlargement of nucleoids (mtDNA–protein complexes) and activation of cGAS–STING innate immune signalling via mtDNA released into the cytoplasm. However, the relationship among aberrant mitochondria and nucleoid dynamics, mtDNA release and cGAS–STING activation remains unclear. Here we show that, under a variety of mtDNA replication stress conditions and during herpes simplex virus-1 infection, enlarged nucleoids that remain bound to TFAM exit mitochondria. Enlarged nucleoids arise from mtDNA experiencing replication stress, which causes nucleoid clustering via a block in mitochondrial fission at a stage when endoplasmic reticulum actin polymerization would normally commence, defining a fission checkpoint that ensures mtDNA has completed replication and is competent for segregation into daughter mitochondria. Chronic engagement of this checkpoint results in enlarged nucleoids trafficking into early and then late endosomes for disposal. Endosomal rupture during transit through this endosomal pathway ultimately causes mtDNA-mediated cGAS–STING activation. Thus, we propose that replication-incompetent nucleoids are selectively eliminated by an adaptive mitochondria–endosomal quality control pathway that is prone to innate immune system activation, which might represent a therapeutic target to prevent mtDNA-mediated inflammation during viral infection and other pathogenic states. Newman et al. show that, upon mitochondrial DNA (mtDNA) replication stress, enlarged nucleoids are trafficked to endosomes. Endosomal rupture releases mtDNA into the cytoplasm, triggering cGAS–STING activation and innate immune signalling.

A suite of river discharge, tree-ring, sedimentary and climate data shows that the Mississippi’s flood magnitude has risen by about twenty per cent over the past half-century, largely owing to engineering works. Engineering magnifies Mississippi's rising rivers Instrumental records of river discharge do not go far enough back in time to place recent flood activity in a longer-term context, making it difficult to understand how climate variability and human activity might have affected flooding. Now, Samuel Munoz and colleagues reconstruct the past flood frequency of the Mississippi River from a compilation of river-discharge, tree-ring, sedimentary and climate data. The results show that the magnitude of the 100-year flood has gone up by about 20 per cent over the past 500 years. Climate cycles account for most of the variability in flooding on multidecadal timescales, but engineering works account for about three-quarters of the long-term increase. Over the past century, many of the world’s major rivers have been modified for the purposes of flood mitigation, power generation and commercial navigation 1 . Engineering modifications to the Mississippi River system have altered the river’s sediment levels and channel morphology 2 , but the influence of these modifications on flood hazard is debated 3 , 4 , 5 . Detecting and attributing changes in river discharge is challenging because instrumental streamflow records are often too short to evaluate the range of natural hydrological variability before the establishment of flood mitigation infrastructure. Here we show that multi-decadal trends of flood hazard on the lower Mississippi River are strongly modulated by dynamical modes of climate variability, particularly the El Niño–Southern Oscillation and the Atlantic Multidecadal Oscillation, but that the artificial channelization (confinement to a straightened channel) has greatly amplified flood magnitudes over the past century. Our results, based on a multi-proxy reconstruction of flood frequency and magnitude spanning the past 500 years, reveal that the magnitude of the 100-year flood (a flood with a 1 per cent chance of being exceeded in any year) has increased by 20 per cent over those five centuries, with about 75 per cent of this increase attributed to river engineering. We conclude that the interaction of human alterations to the Mississippi River system with dynamical modes of climate variability has elevated the current flood hazard to levels that are unprecedented within the past five centuries.

Programmable genome integration of large, diverse DNA cargo without DNA repair of exposed DNA double-strand breaks remains an unsolved challenge in genome editing. We present programmable addition via site-specific targeting elements (PASTE), which uses a CRISPR–Cas9 nickase fused to both a reverse transcriptase and serine integrase for targeted genomic recruitment and integration of desired payloads. We demonstrate integration of sequences as large as ~36 kilobases at multiple genomic loci across three human cell lines, primary T cells and non-dividing primary human hepatocytes. To augment PASTE, we discovered 25,614 serine integrases and cognate attachment sites from metagenomes and engineered orthologs with higher activity and shorter recognition sequences for efficient programmable integration. PASTE has editing efficiencies similar to or exceeding those of homology-directed repair and non-homologous end joining-based methods, with activity in non-dividing cells and in vivo with fewer detectable off-target events. PASTE expands the capabilities of genome editing by allowing large, multiplexed gene insertion without reliance on DNA repair pathways. Large sequences are integrated site specifically into the human genome without double-strand DNA cleavage.

Background Increasing evidence highlights that accumulating sitting time in prolonged bouts is detrimental to cardiometabolic health. Objectives This systematic review aimed to compare the effects of fractionating prolonged sitting with frequent short bouts of standing and light-intensity walking on cardiometabolic health markers and conduct a meta-analysis for differences in systolic blood pressure (SBP), postprandial glucose and insulin. Methods Experimental randomised crossover trials with at least three intervention arms that assessed interrupting sitting with frequent short bouts of standing and light-intensity walking over a single day compared to a prolonged sitting condition were retrieved. These studies measured at minimum one marker of cardiometabolic health in adults > 18 years. An electronic search was completed on the 2nd of August 2021, searching PubMed and Web of Science Core Collection, Scopus, Embase, Cochrane Library and APA PsycINFO. Risk of bias was assessed using a modified Downs and Black checklist. A meta-analysis was conducted using calculated Cohen’s d quantifying the magnitude of difference between experimental conditions. Results Seven studies met the inclusion criteria for the systematic review. All seven studies were included within the meta-analysis for postprandial glucose, four studies were pooled for postprandial insulin and three for SBP. Biomarkers of cardiometabolic health were discussed qualitatively if fewer than three studies measured and reported the variable. A meta-analysis of seven acute, 1-day randomised crossover trials that sampled mixed-sex adults (aged > 18 years) who were predominately overweight or participants with obesity found that standing as an interruption to prolonged sitting significantly reduced postprandial glucose (∆ = − 0.31, 95% CI − 0.60, − 0.03; z  = − 2.15, p  < 0.04) but had no significant effect on insulin or SBP. Light-intensity walking was shown to significantly attenuate postprandial glucose (∆ = − 0.72, 95% CI − 1.03, − 0.41; z  = − 4.57, p  < 0.001) and insulin (∆ = − 0.83, 95% CI − 1.18, − 0.48; z  = − 4.66, p  < 0.001) compared to continued sitting. When comparing light-intensity walking breaks compared to standing breaks a significant reduction in glucose (∆ = − 0.30, 95% CI − 0.52, − 0.08; z  = -2.64, p  < 0.009) and insulin (∆ = − 0.54, 95% CI − 0.75, − 0.33; z  = -4.98, p  < 0.001) was observed. Both standing and light-intensity walking showed no effect on SBP. Conclusions Frequent short interruptions of standing significantly attenuated postprandial glucose compared to prolonged sitting; however, light-intensity walking was found to represent a superior physical activity break. The feasibility and longitudinal implications of breaking sedentary behaviour with light-intensity walking should be investigated in a free-living setting. Registration Not available.

Peter Donnelly and colleagues report fine mapping of 14 susceptibility loci in 8,000 cases and controls for type 2 diabetes, coronary artery disease and Graves' disease. They apply a new Bayesian method for analysis of fine-mapping data sets, using this to define sets of SNPs likely to contain causal disease-associated variants. To further investigate susceptibility loci identified by genome-wide association studies, we genotyped 5,500 SNPs across 14 associated regions in 8,000 samples from a control group and 3 diseases: type 2 diabetes (T2D), coronary artery disease (CAD) and Graves' disease. We defined, using Bayes theorem, credible sets of SNPs that were 95% likely, based on posterior probability, to contain the causal disease-associated SNPs. In 3 of the 14 regions, TCF7L2 (T2D), CTLA4 (Graves' disease) and CDKN2A - CDKN2B (T2D), much of the posterior probability rested on a single SNP, and, in 4 other regions ( CDKN2A - CDKN2B (CAD) and CDKAL1 , FTO and HHEX (T2D)), the 95% sets were small, thereby excluding most SNPs as potentially causal. Very few SNPs in our credible sets had annotated functions, illustrating the limitations in understanding the mechanisms underlying susceptibility to common diseases. Our results also show the value of more detailed mapping to target sequences for functional studies.

ObjectiveTo estimate the incidence of avoidable significant harm in primary care in England; describe and classify the associated patient safety incidents and generate suggestions to mitigate risks of ameliorable factors contributing to the incidents.DesignRetrospective case note review. Patients with significant health problems were identified and clinical judgements were made on avoidability and severity of harm. Factors contributing to avoidable harm were identified and recorded.SettingPrimary care.ParticipantsThirteen general practitioners (GPs) undertook a retrospective case note review of a sample of 14 407 primary care patients registered with 12 randomly selected general practices from three regions in England (total list size: 92 255 patients).Main outcome measuresThe incidence of significant harm considered at least ‘probably avoidable’ and the nature of the safety incidents.ResultsThe rate of significant harm considered at least probably avoidable was 35.6 (95% CI 23.3 to 48.0) per 100 000 patient-years (57.9, 95% CI 42.2 to 73.7, per 100 000 based on a sensitivity analysis). Overall, 74 cases of avoidable harm were detected, involving 72 patients. Three types of incident accounted for more than 90% of the problems: problems with diagnosis accounted for 45/74 (60.8%) primary incidents, followed by medication-related problems (n=19, 25.7%) and delayed referrals (n=8, 10.8%). In 59 (79.7%) cases, the significant harm could have been identified sooner (n=48) or prevented (n=11) if the GP had taken actions aligned with evidence-based guidelines.ConclusionThere is likely to be a substantial burden of avoidable significant harm attributable to primary care in England with diagnostic error accounting for most harms. Based on the contributory factors we found, improvements could be made through more effective implementation of existing information technology, enhanced team coordination and communication, and greater personal and informational continuity of care.

Abstract Rationale “Target trial emulation” has been proposed as an observational method to answer comparative effectiveness questions, but it has rarely been attempted concurrently with a randomized clinical trial (RCT). Objectives We tested the hypothesis that blinded analysts applying target trial emulation to existing observational data could predict the results of an RCT. Methods PreVent (Preventing Hypoxemia with Manual Ventilation during Endotracheal Intubation) was a multicenter RCT examining the effects of positive-pressure ventilation during tracheal intubation on oxygen saturation and severe hypoxemia. Analysts unaware of PreVent’s results used patient-level data from three previous trials evaluating airway management interventions to emulate PreVent’s eligibility criteria, randomization procedure, and statistical analysis. After PreVent’s release, results of this blinded observational analysis were compared with those of the RCT. Difference-in-differences estimates for comparison of treatment effects between the observational analysis and the PreVent trial are reported on the absolute scale. Results Using observational data, we were able to emulate PreVent’s randomization procedure to produce balanced groups for comparison. The lowest oxygen saturation during intubation was higher in the positive-pressure ventilation group than the no positive-pressure ventilation group in the observational analysis (n = 360; mean difference = 1.8%; 95% confidence interval [CI] = −1.0 to 4.6) and in the PreVent trial (n = 401; mean difference = 3.9%; 95% CI = 1.4 to 6.4), though the observational analysis could not exclude no difference. Difference-in-differences estimates comparing treatment effects showed reasonable agreement for lowest oxygen saturation between the observational analysis and the PreVent trial (mean difference = −2.1%; 95% CI = −5.9 to 1.7). Positive-pressure ventilation resulted in lower rates of severe hypoxemia in both the observational analysis (risk ratio = 0.60; 95% CI = 0.38 to 0.93) and in the PreVent trial (risk ratio = 0.48; 95% CI = 0.30 to 0.77). The absolute reduction in the incidence of severe hypoxemia with positive-pressure ventilation was similar in the observational analysis (9.4%) and the PreVent trial (12.0%), though the difference between these estimates had wide CIs (mean difference = 2.5%; 95% CI = −8.0 to 13.6%). Conclusions Applying target trial emulation methods to existing observational data for the evaluation of a novel intervention produced results similar to those of a randomized trial. These findings support the use of target trial emulation for comparative effectiveness research.

Mesenchymal stromal cells (MSCs) are multipotent progenitor cells that are of considerable clinical potential in transplantation and anti-inflammatory therapies due to their capacity for tissue repair and immunomodulation. However, MSCs rapidly differentiate once in culture, making their large-scale expansion for use in immunomodulatory therapies challenging. Although the differentiation mechanisms of MSCs have been extensively investigated using materials, little is known about how materials can influence paracrine activities of MSCs. Here, we show that nanotopography can control the immunomodulatory capacity of MSCs through decreased intracellular tension and increasing oxidative glycolysis. We use nanotopography to identify bioactive metabolites that modulate intracellular tension, growth and immunomodulatory phenotype of MSCs in standard culture and during larger scale cell manufacture. Our findings demonstrate an effective route to support large-scale expansion of functional MSCs for therapeutic purposes. Mesenchymal Stromal Cells are hard to expand whilst retaining immunomodulatory properties due to spontaneous differentiation and ageing in culture. Here, the authors describe a mechanotransductive link between metabolism and functional activity and identify bioactive metabolites to expand functional MSCs at cell therapy scale.

River ecosystems in semi-arid environments provide an array of resources that concentrate biodiversity, but also attract human settlement and support economic development. In the southwestern United States, land-use change, drought, and anthropogenic disturbance are compounding factors which have led to departures from historical conditions of river ecosystems, consequently affecting wildlife habitat, including important wintering areas for migratory birds. The Rio Grande (River) in central New Mexico is the lifeblood of the Middle Rio Grande Valley (MRGV), maintaining large urban and agricultural centers and riparian and wetland resources, which disproportionately support a diversity of wildlife. The MRGV has been identified as the most important wintering area for the Rocky Mountain Population of greater sandhill cranes (Antigone canadensis tabida). Presently, however, changes in the hydrogeomorphology of the Rio Grande and landscape modification by humans have reshaped the MRGV and winter habitat for sandhill cranes. To evaluate these impacts, we investigated how land-use practices, anthropogenic disturbance, and river morphology influenced patterns of diurnal and roosting habitat selection by sandhill cranes. During the diurnal period, sandhill cranes relied heavily on managed public lands selecting agriculture crops, such as corn fields, and wetlands for foraging and loafing while avoiding areas with increasing densities of human structures. Sandhill cranes selected areas for roosting in the Rio Grande characterized by shallower water interspersed with sandbars, wide channel width, low bank vegetation, and farther away from disturbances associated with bridges. Our results establish and identify the central processes driving patterns of diel habitat selection by wintering sandhill cranes. Land use and riverine trends have likely gradually reduced winter habitat to managed public lands and limited reaches of the Rio Grande, underscoring the importance of natural resources agencies in supporting migratory birds and challenges involved when managing for wildlife in highly pressured semi-arid environments.

Managers typically estimate wildlife abundance using surveys within a timeframe that favors increased detectability; however, the ability to account for probabilities of inclusion, detection, and/or presence within a given sampling area is often limited. Cranes provide a good opportunity to research count accuracy because they are large, conspicuous, and often congregate during part of the year, typically on staging areas (i.e., fall and spring) or on wintering grounds. The objectives of this paper are twofold: (1) to evaluate how environmental factors influence crane movement in and out of crane survey areas to identify the best window of availability for annual survey counts; and (2) to evaluate environmental factors that influence overall crane survey counts from year to year. For Objective 1, a generalized linear mixed model was selected to model the probability of crane presence within survey areas using GPS transmitter data. A binary response variable representing crane movement within and outside formal survey areas was used as the dependent variable to investigate environmental covariates that influence movement into survey areas. Probability of crane presence was explained by seven covariates plus a quadratic term for Julian day number. Interactions between Palmer drought severity index (PDSI) and normalized difference vegetation index supported higher probability of crane presence in survey areas during times of drought. Probability of crane presence increased throughout the entire study period (May–October), suggesting that formal surveys in September could be augmented or replaced by surveys in October. For Objective 2, a negative binomial model with linear parameterization was selected to model crane counts using census data compiled from 1995 to 2019. Covariates were acquired at the watershed scale using Hydrologic Unit Code 6 boundaries. Of the 17 covariates investigated, we found that 18‐month precipitation (PPTgss), PDSI, and minimum temperature (Tempmin) explained most of the variability in crane census counts. High PPTgss (antecedent moisture), low PDSI (drought conditions), and low Tempmin (cold extremes) result in higher annual crane counts. The ability to link ecological processes to wildlife movement and population abundance both locally and at landscape scales has long‐ranging implications on resource projections, conservation, and the ability to deploy adaptive management.