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UNLABELLED: We sought to evaluate the efficacy of WEE1 inhibitor adavosertib in patients with solid tumor malignancies (cohort A) and clear cell renal cell carcinoma (ccRCC; cohort B). NCT03284385 was a parallel cohort, Simon two-stage, phase II study of adavosertib (300 mg QDAY by mouth on days 1-5 and 8-12 of each 21-day cycle) in patients with solid tumor malignancies harboring a pathogenic SETD2 mutation. The primary endpoint was the objective response rate. Correlative assays evaluated the loss of H3K36me3 by IHC, a downstream consequence of SETD2 loss, in archival tumor tissue. Eighteen patients were enrolled (9/cohort). The median age was 60 years (range 45-74). The median duration of treatment was 1.28 months (range 0-24+). No objective responses were observed in either cohort; accrual was halted following stage 1. Minor tumor regressions were observed in 4/18 (22%) evaluable patients. Stable disease (SD) was the best overall response in 10/18 (56%) patients, including three patients with SD > 4 months. One patient with ccRCC remains on treatment for >24 months. The most common adverse events of any grade were nausea (59%), anemia (41%), diarrhea (41%), and neutropenia (41%). Nine patients (50%) experienced a Grade ≥3 adverse event. Of eight evaluable archival tissue samples, six (75%) had a loss of H3K36me3 by IHC. Adavosertib failed to exhibit objective responses in SETD2-altered ccRCC and other solid tumor malignancies although prolonged SD was observed in a subset of patients. Combination approaches may yield greater depth of tumor response. SIGNIFICANCE: WEE1 inhibition with adavosertib monotherapy demonstrated limited clinical activity in patients with SETD2-altered solid tumors despite compelling preclinical data indicating a synthetic lethal effect, which did not translate into robust tumor regression. Loss of the H3K36me3 trimethylation mark caused by SETD2-deficiency was confirmed in the majority of evaluable tumors. A subset of patients derived clinical benefit as manifested by minor tumor regressions and prolonged SD.

The de novo design of small molecule-binding proteins has seen exciting recent progress; however, high-affinity binding and tunable specificity typically require laborious screening and optimization after computational design. We developed a computational procedure to design a protein that recognizes a common pharmacophore in a series of poly(ADP-ribose) polymerase-1 inhibitors. One of three designed proteins bound different inhibitors with affinities ranging from

ObjectiveWith the shift toward utilization of sentinel lymph node biopsy (SLNB) in oral cavity cancer, improved techniques for intraoperative sentinel node identification are needed. This study investigates the feasibility of fluorescently labeled tilmanoscept in SLNB in an oral cancer rabbit model.MethodsAn animal study was designed using 21 healthy male New Zealand rabbits. Gallium-68-labeled tilmanocept labeled with IRDye800CW was injected submucosally into the buccal mucosa (n = 6) or lateral tongue (n = 7) followed by PET imaging. One hour after injection, SLNB was performed using fluorescence imaging followed by a bilateral neck dissection and sampling of non-nodal surrounding tissue. All tissues were measured for radioactivity and fluorescence. In addition, eight rabbits were injected with delayed SLNB performed 48 h after injection.ResultsBuccal injections all had ipsilateral SLN drainage and tongue injections exhibited 18.2% contralateral drainage. An average of 1.9 ± 1.0 SLN (range 1-5) were identified. In addition, an average of 16.9 ± 3.3 non-sentinel lymph nodes were removed per animal. SLNs had an average of 0.69 ± 0.60 percent-of-injected dose (%ID) compared with non-sentinel nodes with 0.012 ± 0.025 %ID and surrounding tissue with 0.0067 ± 0.015 %ID. There was 98.0% agreement between sentinel lymph nodes identified using fluorescence compared to radioactivity with Cohen's kappa coefficient of 0.879. In 48-h delayed SLNB, results were consistent with 97.8% agreement with radioactivity and Cohen's Kappa coefficient of 0.884. Fluorescence identified additional lymph nodes that were not identified by radioactivity, and with one false negative.ConclusionFluorescent-labeled Tc-99 m-tilmanocept represents a highly accurate adjunct to enhance SLNB for oral cavity cancer.Level of evidenceN/A Laryngoscope, 134:1299-1307, 2024.

Mucopolysaccharidosis type VI (MPS VI) is an autosomal recessive lysosomal storage disorder characterized by deficient activity of arylsulfatase B enzyme (ASB) resulting in cellular accumulation of dermatan sulfate (DS) and chondroitin sulfate (CS) that leads to cell injury. Urinary glycosaminoglycans (GAG) are often used as a biomarker in MPS diseases for diagnosis and to monitor treatment efficacy. This study evaluated leukocyte GAGs (leukoGAG) and skin GAGs as alternate biomarkers representing intracellular GAG changes in patients with MPS VI and treated with enzyme replacement therapy (ERT). In addition, we evaluated corneal opacification measurements (COM) and carotid intima media thickness (CIMT) as indicators of GAG accumulation and tissue injury. The study was performed in a serial two-step design in a single center. A quantitative method to measure leukoGAG levels in leukocytes was developed in Study 1 to compare the GAG levels between MPS VI patients and a control group and to assess correlations between leukoGAG and urineGAG. Study 2 validated the leukoGAG measurement, assessed the effect of ERT infusion on leukoGAG and ASB activity in leukocytes, identified correlations between leukoGAG and other biomarkers, and assessed differences in GAG accumulation between MPS VI patients and control subjects. In Study 1, leukoCS and leukoDS levels were significantly higher in the MPS VI group than the control group (leukoCS: 37.9 ± 10.2 and 2.9 ± 1.5 μg/μg protein, respectively, p = 0.005; leukoDS: 0.26 ± 0.2 and 0.0 ± 0.0 μg/μg protein, respectively, p = 0.028) with positive correlations between leukoCS and urine CS and leukoDS and urineDS. In Study 2, leukoCS (32.0 ± 11.8 vs 6.9 ± 3.1 μg/mg protein, p = 0.005) and leukoDS (0.4 ± 0.1 and 0.2 ± 0.1 μg/mg protein, p = 0.020) were significantly higher compared with control subjects. Thus, these results highlight the potential of leukoGAG as a new biomarker representing intracellular GAG accumulation in MPS VI patients and may be valuable for patient management.

Introduction: Accurate diagnosis of traumatic arthrotomy of the knee (TAK) is critical for patients presenting to the emergency department (ED) to ensure timely treatment. Current diagnostic modalities including plain radiography, computed tomography (CT), and the saline load test (SLT) have advantages and disadvantages. Point-of-care-ultrasonography (POCUS) offers a possible timely, low-cost, and efficient alternative method of diagnosing TAK. In this case series we present three cases where POCUS was used to diagnose TAK in the ED.Case Series: Three patients in their early 20s presented to the ED complaining of knee trauma with wounds in proximity to the joint. Mechanisms of injury included a gunshot wound in one case and blunt trauma (motor vehicle collision and bicycle crash) in two cases. In all three cases TAK was suggested on POCUS examinations by the presence of intra-articular hyperechoic foci consistent with air artifact. All three cases had TAK confirmed by orthopedic evaluation.Discussion: Ultrasound may have utility in the evaluation of patients presenting with knee trauma where TAK is a concern. The SLT is generally considered the gold standard test for diagnosis of TAK, but it is invasive and has a wide range of diagnostic accuracy. Intra-articular air has been found to be a sensitive marker for TAK in CT studies. Thus, additional investigations into the diagnostic accuracy of POCUS for this finding should be undertaken.

Abstract The harmful effects of mining waste on human and ecosystem health make reclamation of former mine sites an environmental management priority. However, field-based monitoring of reclamation requires significant investments of time, labor, and money. Remote sensing offers a less expensive alternative to field-based monitoring, but work is still needed to determine which metrics can be reliably estimated using remote sensing techniques. This study uses remote sensing to examine over 20 years of reclamation efforts at Tar Creek Superfund Site and to assess revegetation after site restoration. Using the Normalized Difference Vegetation Index (NDVI), we quantify key factors affecting vegetation recovery and stability after reclamation across 123 surface mining waste cleanup locations within Tar Creek. Leveraging long-term imagery from Landsat and high-resolution PlanetScope imagery, we combine time series analysis of vegetation regrowth and landcover change detection for a comprehensive picture of recovery at each site. Across all reclamation sites, the average recovery duration was 3.5 years, and average recovery rate was 0.1 NDVI year−1. After vegetative growth had plateaued, reclaimed sites had an average NDVI of 0.70. All reclamation areas were converted from majority barren landcover to vegetated landcover classes after reclamation, and vegetative stability at reclamation sites was high (70% of reclaimed area saw continuous vegetative cover from 2017 to 2023). These results demonstrate a strong potential for multi-method remote sensing techniques in tracking and explaining vegetation recovery after reclamation and represent a cost-effective approach for real-time monitoring of reclamation progress and outcomes.

Exponential population increases are threatening food security, particularly in mountainous areas. One potential solution is dual-use intercropped agroforestry systems such as olive (Olea europaea)–maize (Zea mays), which may mitigate risk by providing multiple market sources (oil and grain) for smallholder producers. Several studies have conducted integrated agroforestry land suitability analyses; however, few studies have used machine learning (ML) algorithms to evaluate multiple variables (i.e., soil physicochemical properties and climatic and topographic data) for the selection of suitable rainfed sites in mountainous terrain systems. The goal of this study is therefore to identify suitable land classes for an integrated olive–maize agroforestry system based on the Food and Agriculture Organization (FAO) land suitability assessment framework for 1757 km2 in Khyber Pakhtunkhwa province, Pakistan. Information on soil physical and chemical properties was obtained from 701 soil samples, along with climatic and topographic data. After determination of land suitability classes for an integrated olive–maize-crop agroforestry system, the region was then mapped through ML algorithms using random forest (RF) and support vector machine (SVM), as well as using traditional techniques of weighted overlay (WOL). Land suitability classes predicted by ML techniques varied greatly. For example, the S1 area (highly suitable) classified through RF was 9%↑ than that of SVM, and 8%↓ than that through WOL. The area of S2 (moderately suitable) classified through RF was 18%↑ than that of SWM and was 17%↓ than the area classified through WOL; similarly, the S3 (marginally suitable) class area via RF was 27%↓ than that of SVM, and 45%↓ than the area classified through WOL. Conversely, the area of N2 (permanently not suitable class) classified through RF and SVM was 6%↑ than the area classified through WOL. Model performance was assessed through overall accuracy and Kappa Index and indicated that RF performed better than SVM and WOL. Crop suitability limitations of the study area included high elevation, slope, pH, and large gravel content. Results can be used for sustainable intensification in mountainous rainfed regions by expanding intercrop agroforestry systems in developing nations to close yield gaps.

Abstract The sequencing platform and workflow strongly influence microbial community analyses through potential errors at each step. Effective diagnostics and experimental controls are needed to validate data and improve reproducibility. This cross-laboratory study evaluates sources of variability and error at three main steps of a standardized amplicon sequencing workflow (DNA extraction, polymerase chain reaction [PCR], and sequencing) using Oxford Nanopore MinION to analyze agricultural soils and a simple mock community. Variability in sequence results occurs at each step in the workflow with PCR errors and differences in library size greatly influencing diversity estimates. Common bioinformatic diagnostics and the mock community are ineffective at detecting PCR abnormalities. This work outlines several diagnostic checks and techniques to account for sequencing depth and ensure accuracy and reproducibility in soil community analyses. These diagnostics and the inclusion of a reference soil can help ensure data validity and facilitate the comparison of multiple sequencing runs within and between laboratories.

Precipitation is the most important driver of the hydrological cycle, but it is challenging to estimate it over large scales from satellites and models. Here, we assessed the performance of six global and quasi-global high-resolution precipitation datasets (European Centre for Medium-Range Weather Forecasts (ECMWF) Reanalysis version 5 (ERA5), Climate Hazards group Infrared Precipitation with Stations version 2.0 (CHIRPS), Multi-Source Weighted-Ensemble Precipitation version 2.80 (MSWEP), TerraClimate (TERRA), Climate Prediction Centre Unified version 1.0 (CPCU), and Precipitation Estimation from Remotely Sensed Information using Artificial Neural Networks-Cloud Classification System-Climate Data Record (PERSIANN-CCS-CDR, hereafter PERCCDR) for hydrological modelling globally and quasi-globally. We forced the WBMsed global hydrological model with the precipitation datasets to simulate river discharge from 1983 to 2019 and evaluated the predicted discharge against 1825 hydrological stations worldwide, using a range of statistical methods. The results show large differences in the accuracy of discharge predictions when using different precipitation input datasets. Based on evaluation at annual, monthly, and daily timescales, MSWEP followed by ERA5 demonstrated a higher correlation (CC) and Kling–Gupta efficiency (KGE) than other datasets for more than 50 % of the stations, whilst ERA5 was the second-highest-performing dataset, and it showed the highest error and bias for about 20 % of the stations. PERCCDR is the least-well-performing dataset, with a bias of up to 99 % and a normalised root mean square error of up to 247 %. PERCCDR only show a higher KGE and CC than the other products for less than 10 % of the stations. Even though MSWEP provided the highest performance overall, our analysis reveals high spatial variability, meaning that it is important to consider other datasets in areas where MSWEP showed a lower performance. The results of this study provide guidance on the selection of precipitation datasets for modelling river discharge for a basin, region, or climatic zone as there is no single best precipitation dataset globally. Finally, the large discrepancy in the performance of the datasets in different parts of the world highlights the need to improve global precipitation data products.

Abstract Remote sensing tools, along with Global Navigation Satellite System cattle collars and digital soil maps, may help elucidate spatiotemporal relationships among soils, terrain, forages, and animals. However, standard computational procedures preclude systems‐level evaluations across this continuum due to data inoperability and processing limitations. Deep learning, a subset of neural network, may elucidate efficiency of livestock production and linkages within the livestock‐grazing environment. Consequently, we applied deep learning to environmental remote sensing data to (1) develop predictive models for yield and forage nutrition based on vegetation indices and (2) at a pixel‐level (per 55 m2), identify how grazing is linked to soil properties, forage growth and nutrition, and terrain attributes in silvopasture and pasture‐only systems. Remotely sensed data rapidly and non‐destructively estimated herbage mass and nutritive value for enhanced net and primary productivity management in livestock and grazing systems. Cattle grazed big bluestem (Andropogon gerardii ‘Vitman’) with 182% greater frequency than orchardgrass (Dactylis glomerata L.) in the pasture‐only system. Real‐time estimates of vegetative bands may assist in predicting grazing pressure for more efficient pasture resource management. Cattle grazing followed distinct soil‐landscape patterns, namely reduced cattle grazing preference occurred in areas of water accumulation, which highlights linkages among terrain, soil‐water movement, soil properties, forage nutrition, and animal grazing response spatially and temporally. Results from this study could be scaled up to improve grazing management among the largest land‐use category in the United States, that is, grasslands, which would allow for sustainable intensification of forage‐based livestock production to meet growing demands for environmentally responsible protein.

Background The long-term outcomes of COVID-19 hospitalisation in individuals with pre-existing airway diseases are unknown. Methods Adult participants hospitalised for confirmed or clinically suspected COVID-19 and discharged between 5 March 2020 and 31 March 2021 were recruited to the Post-hospitalisation COVID-19 (PHOSP-COVID) study. Participants attended research visits at 5 months and 1 year post discharge. Clinical characteristics, perceived recovery, burden of symptoms and health-related quality of life (HRQoL) of individuals with pre-existing airway disease (i.e., asthma, COPD or bronchiectasis) were compared to the non-airways group. Results A total of 615 out of 2697 (22.8%) participants had a history of pre-existing airway diseases (72.0% diagnosed with asthma, 22.9% COPD and 5.1% bronchiectasis). At 1 year, the airways group participants were less likely to feel fully recovered (20.4% versus 33.2%, p

Background Pre-existing cardiovascular disease (CVD) or cardiovascular risk factors have been associated with an increased risk of complications following hospitalisation with COVID-19, but their impact on the rate of recovery following discharge is not known.Objectives To determine whether the rate of patient-perceived recovery following hospitalisation with COVID-19 was affected by the presence of CVD or cardiovascular risk factors.Methods In a multicentre prospective cohort study, patients were recruited following discharge from the hospital with COVID-19 undertaking two comprehensive assessments at 5 months and 12 months. Patients were stratified by the presence of either CVD or cardiovascular risk factors prior to hospitalisation with COVID-19 and compared with controls with neither. Full recovery was determined by the response to a patient-perceived evaluation of full recovery from COVID-19 in the context of physical, physiological and cognitive determinants of health.Results From a total population of 2545 patients (38.8% women), 472 (18.5%) and 1355 (53.2%) had CVD or cardiovascular risk factors, respectively. Compared with controls (n=718), patients with CVD and cardiovascular risk factors were older and more likely to have had severe COVID-19. Full recovery was significantly lower at 12 months in patients with CVD (adjusted OR (aOR) 0.62, 95% CI 0.43 to 0.89) and cardiovascular risk factors (aOR 0.66, 95% CI 0.50 to 0.86).Conclusion Patients with CVD or cardiovascular risk factors had a delayed recovery at 12 months following hospitalisation with COVID-19. Targeted interventions to reduce the impact of COVID-19 in patients with cardiovascular disease remain an unmet need.Trail registration number ISRCTN10980107.

The domestication history of the avocado (Persea americana) remains unclear. We created a reference genome from the Gwen varietal, which is closely related to the economically dominant Hass varietal. Our genome assembly had an N50 of 3.37 megabases, a BUSCO score of 91%, and was scaffolded with a genetic map, producing 12 pseudo-chromosomes with 49,450 genes. We used the Gwen genome as a reference to investigate population genomics, based on a sample of 34 resequenced accessions that represented the 3 botanical groups of P. americana. Our analyses were consistent with 3 separate domestication events; we estimated that the Mexican group diverged from the Lowland (formerly known as "West Indian") and Guatemalan groups >1 million years ago. We also identified putative targets of selective sweeps in domestication events; within the Guatemalan group, putative candidate genes were enriched for fruit development and ripening. We also investigated divergence between heterodichogamous flowering types, providing preliminary evidence for potential candidate genes involved in pollination and floral development.

Medical graduates are expected to apply scientific principles and explain the processes underlying common and important diseases. Evidence shows that integrated medical curricula, which deliver biomedical science within the context of clinical cases, facilitate student learning in preparation for practice. However, research has also shown that the student's perception of their knowledge can be lower in integrated compared to traditional courses. Thus the development of teaching methods to support both integrated learning and build student confidence in clinical reasoning is a priority. In this study, we describe the use of an audience response system to support active learning in large classes. Sessions, delivered by medical faculty from both academic and clinical backgrounds, were designed to build on the knowledge of the respiratory system in both health and disease through the interpretation of clinical cases. Results showed that student engagement was high throughout the session and students strongly agreed that the application of knowledge to real-life cases was a better way to understand clinical reasoning. Qualitative free text comments revealed that students liked the link between theory and practice and the active, integrated method of learning. In summary, this study describes a relatively simple but highly effective way of delivering integrated medical science teaching, in this case respiratory medicine, to improve student confidence in clinical reasoning. This educational approach was applied within the early years of the curriculum in preparation for teaching within a hospital setting, but the format could be applied across many different settings.

Abstract Intrauterine growth restriction (IUGR) occurs both in humans and domestic species. It has a particularly high incidence in pigs, and is a leading cause of neonatal morbidity and mortality as well as impaired postnatal growth. A key feature of IUGR is impaired muscle development, resulting in decreased meat quality. Understanding the developmental origins of IUGR, particularly at the molecular level, is important for developing effective strategies to mitigate its economic impact on the pig industry and animal welfare. The aim of this study was to characterise transcriptional profiles in the muscle of growth restricted pig foetuses at different gestational days (GD; gestational length ~ 115 days), focusing on selected genes (related to development, tissue injury and metabolism) that were previously identified as dysregulated in muscle of GD90 fetuses. Muscle samples were collected from the lightest foetus (L) and the sex-matched foetus with weight closest to the litter average (AW) from each of 22 Landrace x Large White litters corresponding to GD45 (n = 6), GD60 (n = 8) or GD90 (n = 8), followed by analyses, using RT-PCR and protein immunohistochemistry, of selected gene targets. Expression of the developmental genes, MYOD, RET and ACTN3 were markedly lower, whereas MSTN expression was higher, in the muscle of L relative to AW littermates beginning on GD45. Levels of all tissue injury-associated transcripts analysed (F5, PLG, KNG1, SELL, CCL16) were increased in L muscle on GD60 and, most prominently, on GD90. Among genes involved in metabolic regulation, KLB was expressed at higher levels in L than AW littermates beginning on GD60, whereas both IGFBP1 and AHSG were higher in L littermates on GD90 but only in males. Furthermore, the expression of genes specifically involved in lipid, hexose sugar or iron metabolism increased or, in the case of UCP3, decreased in L littermates on GD60 (UCP3, APOB, ALDOB) or GD90 (PNPLA3, TF), albeit in the case of ALDOB this only involved females. In conclusion, marked dysregulation of genes with critical roles in development in L foetuses can be observed from GD45, whereas for a majority of transcripts associated with tissue injury and metabolism differences between L and AW foetuses were apparent by GD60 or only at GD90, thus identifying different developmental windows for different types of adaptive responses to IUGR in the muscle of porcine foetuses.

Introduction: Accurate diagnosis of traumatic arthrotomy of the knee (TAK) is critical for patients presenting to the emergency department (ED) to ensure timely treatment. Current diagnostic modalities including plain radiography, computed tomography (CT), and the saline load test (SLT) have advantages and disadvantages. Point-of-care-ultrasonography (POCUS) offers a possible timely, low-cost, and efficient alternative method of diagnosing TAK. In this case series we present three cases where POCUS was used to diagnose TAK in the ED. Case Series: Three patients in their early 20s presented to the ED complaining of knee trauma with wounds in proximity to the joint. Mechanisms of injury included a gunshot wound in one case and blunt trauma (motor vehicle collision and bicycle crash) in two cases. In all three cases TAK was suggested on POCUS examinations by the presence of intra-articular hyperechoic foci consistent with air artifact. All three cases had TAK confirmed by orthopedic evaluation. Discussion: Ultrasound may have utility in the evaluation of patients presenting with knee trauma where TAK is a concern. The SLT is generally considered the gold standard test for diagnosis of TAK, but it is invasive and has a wide range of diagnostic accuracy. Intra-articular air has been found to be a sensitive marker for TAK in CT studies. Thus, additional investigations into the diagnostic accuracy of POCUS for this finding should be undertaken.

Abstract Pseudomonas aeruginosa is a major nosocomial pathogen that causes severe disease including sepsis. Carbapenem-resistant P. aeruginosa is recognised by the World Health Organisation as a priority 1 pathogen, with urgent need for new therapeutics. As such, there is renewed interest in using bacteriophages as a therapeutic. However, the dynamics of treating pan-resistant P. aeruginosa with phage in vivo are poorly understood. Using a pan-resistant P. aeruginosa in vivo infection model, phage therapy displays strong therapeutic potential, clearing infection from the blood, kidneys, and spleen. Remaining bacteria in the lungs and liver displays phage resistance due to limiting phage adsorption. Yet, resistance to phage results in re-sensitisation to a wide range of antibiotics. In this work, we use phage steering in vivo, pre-exposing a pan resistant P. aeruginosa infection with a phage cocktail to re-sensitise bacteria to antibiotics, clearing the infection from all organs.

Abstract Background Pre-Hospital Emergency Anaesthesia (PHEA) has undergone significant developments since its inception. However, optimal drug dosing remains a challenge for both medical and trauma patients. Many prehospital teams have adopted a drug regimen of 3 mcg/kg fentanyl, 2 mg/kg ketamine and 1 mg/kg rocuronium (‘3:2:1’). At Essex and Herts Air Ambulance Trust (EHAAT) a new standard dosing regimen was introduced in August 2021: 1 mcg/kg fentanyl, 2 mg/kg ketamine and 2 mg/kg rocuronium (up to a maximum dose of 150 mg) (‘1:2:2’). The aim of this study was to evaluate the cardiorespiratory consequences of a new attenuated fentanyl and augmented rocuronium dosing regimen. Methods A retrospective study was conducted at EHAAT as a service evaluation. Anonymized records were reviewed from an electronic database to compare the original (‘3:2:1’) drug dosing regimen (December 2019-July 2021) and the new (‘1:2:2’) dosing regimen (September 2021-May 2023). The primary outcome was the incidence of absolute hypotension within ten minutes of induction. Secondary outcomes included immediate hypertension, immediate hypoxia and first pass success (FPS) rates. Results Following exclusions (n = 121), 720 PHEA cases were analysed (360 new vs. 360 original, no statistically significant difference in demographics). There was no difference in the rate of absolute hypotension (24.4% ‘1:2:2’ v 23.8% ‘3:2:1’, p = 0.93). In trauma patients, there was an increased first pass success (FPS) rate with the new regimen (95.1% v 86.5%, p = 0.01) and a reduced incidence of immediate hypoxia (7.9% v 14.8%, p = 0.05). There was no increase in immediate hypertensive episodes (22.7% vs. 24.2%, p = 0.73). No safety concerns were identified. Conclusion An attenuated fentanyl and augmented rocuronium dosing regimen showed no difference in absolute hypotensive episodes in a mixed cohort of medical and trauma patients. In trauma patients, the new regimen was associated with an increased FPS rate and reduced episodes of immediate hypoxia. Further research is required to understand the impact of such drug dosing in the most critically ill and injured subpopulation.

Hyperspectral measurements can help with rapid decision-making and collecting data across multiple locations. However, there are multiple data processing methods (Savisky-Golay [SG], first derivative [FD], and normalization) and analyses (partial least squares regression [PLS], weighted k-nearest neighbor [KKNN], support vector machine [SVM], and random forest [RF]) that can be used to determine the best relationship between physical measurements and hyperspectral data. In the current study, FD was the best method for data processing and SVM was the best model for predicting average cotton (Gossypium spp. Malvaceae) height and nodes. However, the combination of FD and RF were best at predicting cotton leaf area index, canopy cover, and chlorophyll content across the growing season. Additionally, results from models developed by both SVM and RF were closely related to pseudo-CHIME satellite wavebands, where in-situ hyperspectral data were matched to the spectral resolutions of a future hyperspectral satellite. The information and results presented will aid producers and other members of the cotton industry to make rapid and meaningful decisions that could result in greater yield and sustainable intensification.

The nonstructural protein 3 (NSP3) of the severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) contains a conserved macrodomain enzyme (Mac1) that is critical for pathogenesis and lethality. While small-molecule inhibitors of Mac1 have great therapeutic potential, at the outset of the COVID-19 pandemic, there were no well-validated inhibitors for this protein nor, indeed, the macrodomain enzyme family, making this target a pharmacological orphan. Here, we report the structure-based discovery and development of several different chemical scaffolds exhibiting low- to sub-micromolar affinity for Mac1 through iterations of computer-aided design, structural characterization by ultra-high-resolution protein crystallography, and binding evaluation. Potent scaffolds were designed with in silico fragment linkage and by ultra-large library docking of over 450 million molecules. Both techniques leverage the computational exploration of tangible chemical space and are applicable to other pharmacological orphans. Overall, 160 ligands in 119 different scaffolds were discovered, and 153 Mac1-ligand complex crystal structures were determined, typically to 1 Å resolution or better. Our analyses discovered selective and cell-permeable molecules, unexpected ligand-mediated conformational changes within the active site, and key inhibitor motifs that will template future drug development against Mac1.

Despite unprecedented efforts, our therapeutic arsenal against SARS-CoV-2 remains limited. The conserved macrodomain 1 (Mac1) in NSP3 is an enzyme exhibiting ADP-ribosylhydrolase activity and a possible drug target. To determine the role of Mac1 catalytic activity in viral replication, we generated recombinant viruses and replicons encoding a catalytically inactive NSP3 Mac1 domain by mutating a critical asparagine in the active site. While substitution to alanine (N40A) reduced catalytic activity by ~10-fold, mutations to aspartic acid (N40D) reduced activity by ~100-fold relative to wild-type. Importantly, the N40A mutation rendered Mac1 unstable in vitro and lowered expression levels in bacterial and mammalian cells. When incorporated into SARS-CoV-2 molecular clones, the N40D mutant only modestly affected viral fitness in immortalized cell lines, but reduced viral replication in human airway organoids by 10-fold. In mice, the N40D mutant replicated at >1000-fold lower levels compared to the wild-type virus while inducing a robust interferon response; all animals infected with the mutant virus survived infection. Our data validate the critical role of SARS-CoV-2 NSP3 Mac1 catalytic activity in viral replication and as a promising therapeutic target to develop antivirals.

ADP-ribosylation, a modification of proteins, nucleic acids, and metabolites, confers broad functions, including roles in stress responses elicited, for example, by DNA damage and viral infection and is involved in intra- and extracellular signaling, chromatin and transcriptional regulation, protein biosynthesis, and cell death. ADP-ribosylation is catalyzed by ADP-ribosyltransferases (ARTs), which transfer ADP-ribose from NAD+ onto substrates. The modification, which occurs as mono- or poly-ADP-ribosylation, is reversible due to the action of different ADP-ribosylhydrolases. Importantly, inhibitors of ARTs are approved or are being developed for clinical use. Moreover, ADP-ribosylhydrolases are being assessed as therapeutic targets, foremost as antiviral drugs and for oncological indications. Due to the development of novel reagents and major technological advances that allow the study of ADP-ribosylation in unprecedented detail, an increasing number of cellular processes and pathways are being identified that are regulated by ADP-ribosylation. In addition, characterization of biochemical and structural aspects of the ARTs and their catalytic activities have expanded our understanding of this protein family. This increased knowledge requires that a common nomenclature be used to describe the relevant enzymes. Therefore, in this viewpoint, we propose an updated and broadly supported nomenclature for mammalian ARTs that will facilitate future discussions when addressing the biochemistry and biology of ADP-ribosylation. This is combined with a brief description of the main functions of mammalian ARTs to illustrate the increasing diversity of mono- and poly-ADP-ribose mediated cellular processes.

Alterations in the ATM gene are among the most common somatic and hereditary cancer mutations, and ATM-deficient tumors are hypersensitive to DNA-damaging agents. A synthetic lethal combination of DNA-damaging agents and DNA repair inhibitors could have widespread utility in ATM-deficient cancers. However, overlapping normal tissue toxicities from these drug classes have precluded their clinical translation. We investigated PLX038, a releasable polyethylene glycol-conjugate of the topoisomerase I inhibitor SN-38, in ATM wild-type and null isogenic xenografts and in a BRCA1-deficient xenograft. PLX038 monotherapy and combination with PARP inhibition potently inhibited the growth of both BRCA1- and ATM-deficient tumors. A patient with an ATM-mutated breast cancer treated with PLX038 and the PARP inhibitor rucaparib achieved rapid, symptomatic, and radiographic complete response lasting 12 months. Single-agent PLX038 or PLX038 in combination with DNA damage response inhibitors are novel therapeutic paradigms for patients with ATM-loss cancers.

Over one million cases of gastric cancer are diagnosed each year globally, and the metastatic disease continues to have a poor prognosis. A significant proportion of gastric tumors have defects in the DNA damage response pathway, creating therapeutic opportunities through synthetic lethal approaches. Several small-molecule inhibitors of ATR, a key regulator of the DNA damage response, are now in clinical development as targeted agents for gastric cancer. Here, we performed a large-scale CRISPR interference screen to discover genetic determinants of response and resistance to ATR inhibitors (ATRi) in gastric cancer cells. Among the top hits identified as mediators of ATRi response were UPF2 and other components of the nonsense-mediated decay (NMD) pathway. Loss of UPF2 caused ATRi resistance across multiple gastric cancer cell lines. Global proteomic, phosphoproteomic, and transcriptional profiling experiments revealed that cell-cycle progression and DNA damage responses were altered in UPF2-mutant cells. Further studies demonstrated that UPF2-depleted cells failed to accumulate in G1 following treatment with ATRi. UPF2 loss also reduced transcription-replication collisions, which has previously been associated with ATRi response, thereby suggesting a possible mechanism of resistance. Our results uncover a novel role for NMD factors in modulating response to ATRi in gastric cancer, highlighting a previously unknown mechanism of resistance that may inform the clinical use of these drugs.SignificanceLoss of NMD proteins promotes resistance to ATR inhibitors in gastric cancer cells, which may provide a combination of therapeutic targets and biomarkers to improve the clinical utility of these drugs.

Analysis of DNA methylation is a valuable tool to understand disease progression and is increasingly being used to create diagnostic and prognostic clinical biomarkers. While conversion of cytosine to 5-methylcytosine (5mC) commonly results in transcriptional repression, further conversion to 5-hydroxymethylcytosine (5hmC) is associated with transcriptional activation. Here we perform the first study integrating whole-genome 5hmC with DNA, 5mC, and transcriptome sequencing in clinical samples of benign, localized, and advanced prostate cancer. 5hmC is shown to mark activation of cancer drivers and downstream targets. Furthermore, 5hmC sequencing revealed profoundly altered cell states throughout the disease course, characterized by increased proliferation, oncogenic signaling, dedifferentiation, and lineage plasticity to neuroendocrine and gastrointestinal lineages. Finally, 5hmC sequencing of cell-free DNA from patients with metastatic disease proved useful as a prognostic biomarker able to identify an aggressive subtype of prostate cancer using the genes TOP2A and EZH2, previously only detectable by transcriptomic analysis of solid tumor biopsies. Overall, these findings reveal that 5hmC marks epigenomic activation in prostate cancer and identify hallmarks of prostate cancer progression with potential as biomarkers of aggressive disease.SignificanceIn prostate cancer, 5-hydroxymethylcytosine delineates oncogene activation and stage-specific cell states and can be analyzed in liquid biopsies to detect cancer phenotypes. See related article by Wu and Attard, p. 3880.

Alluvial plains are vexing landscapes for soil mapping and spatial soil property predictions. Alluvial sediments often exhibit unpredictable spatial variability from both fluvial and anthropogenic disturbance. The determination of optimal number of soil sampling points for capturing soil variability remains a persistent issue for mapping and monitoring soil conditions. Here, soil organic matter (SOM) and cation exchange capacity (CEC) were estimated in an alluvial plain from a dense array of 2145 soil samples over 250 ha. The primary goals were to i) use pixel-based statistics from time-series Sentinel-2 satellite reflectance data to estimate SOM and CEC; ii) evaluate the use of images with vegetation cover versus bare soil images; and, iii) investigate the optimal number of sampling points to map alluvial soils using different sampling strategies. The optimal sample density was 1 sample per 2.5 ha based on the overlapping of prediction distributions (OV > 0.9). Conditioned Latin Hypercube Sampling (cLHS) was the most efficient sampling strategy. Random grid sampling provided the least consistent results. The use of cLHS coupled with the mean and standard deviation bands was the optimal sampling strategy. Pixel-based statistics from readily available satellite imagery captured persistent soil-reflectance relationships that enabled the use of row crops as proxies to predict soil properties. The combined use of pixel-based statistics as inputs to cLHS can reduce the chances of oversampling and associated costs. Pixel-based statistics provided persistent spatial information that can enhance precision agriculture management, carbon mapping, and is likely applicable to other soil properties and remotely sensed products. Use of pixel-based statistics from Sentinel-2 and similar global-coverage imagery could provide readily-calculable inputs for improved soil mapping and more optimal site sample selections in other agricultural row crop areas, globally.

Background: Individuals with substance use disorder (SUD) and recovery support services often face significant social stigma, especially in rural areas. One method of addressing stigma is through education and personal recovery stories. It is unclear if such messages will work similarly across rural and non-rural areas. Methods: We conduct an exploratory analysis of data from a national randomized controlled trial (N = 2,721) to determine if there are differences in the effectiveness of messages at reducing stigma across rurality. Specifically, we test four interventions to reduce stigma: education about the effectiveness of recovery housing and three versions of a personal recovery story that varied social distance and delivery medium (identified written story, anonymous written story, and video). Results: We find that messages may not have the same effect across rurality, with non-rural participants in the identified and anonymous written recovery story groups having lower stigma scores and only rural participants exposed to the anonymous written story having lower stigma scores compared to their counterparts in the control group. Further, non-rural participants exposed to both written story treatments had higher positive feelings towards those in recovery compared to the control group, but only rural participants in the anonymous written story group had higher positive feelings compared to the control group. Conclusion: Our results suggest that messages may have different effects on stigma across rurality and that rural participants’ beliefs may be particularly hard to change. Future research should examine what types of stigma reduction interventions are most effective in rural areas.

The efficacy of adoptive T cell therapies for cancer treatment can be limited by suppressive signals from both extrinsic factors and intrinsic inhibitory checkpoints1,2. Targeted gene editing has the potential to overcome these limitations and enhance T cell therapeutic function3-10. Here we performed multiple genome-wide CRISPR knock-out screens under different immunosuppressive conditions to identify genes that can be targeted to prevent T cell dysfunction. These screens converged on RASA2, a RAS GTPase-activating protein (RasGAP) that we identify as a signalling checkpoint in human T cells, which is downregulated upon acute T cell receptor stimulation and can increase gradually with chronic antigen exposure. RASA2 ablation enhanced MAPK signalling and chimeric antigen receptor (CAR) T cell cytolytic activity in response to target antigen. Repeated tumour antigen stimulations in vitro revealed that RASA2-deficient T cells show increased activation, cytokine production and metabolic activity compared with control cells, and show a marked advantage in persistent cancer cell killing. RASA2-knockout CAR T cells had a competitive fitness advantage over control cells in the bone marrow in a mouse model of leukaemia. Ablation of RASA2 in multiple preclinical models of T cell receptor and CAR T cell therapies prolonged survival in mice xenografted with either liquid or solid tumours. Together, our findings highlight RASA2 as a promising target to enhance both persistence and effector function in T cell therapies for cancer treatment.

Soil nutrient content varies spatially across agricultural fields in hard-to-predict ways, particularly in floodplains with complex fluvial depositional history. Satellite reflectance data from the Sentinel-2 (S2) mission provides spatially continuous land reflectance data that can aid model development when used with point observations of nutrients. Reflectance from vegetation is assumed to obstruct land reflectance of bare soil, such that researchers have masked vegetation in models. We developed a routine for masking vegetation within Google Earth Engine (GEE) using Random Forest classification for iterative application to libraries of S2-images. Using gradient boosting, we then developed soil nutrient models for surface soils at a 250-ha agricultural site using S2 images. Soils were sampled at 2145 point locations to a 23-cm depth and analyzed for Ca, K, Mg, P, pH, S, and Zn. Results showed that masking vegetation improved model performance for models from subsets of the data (80% of samples used for model development, 20% validation), but full data sets did not require masking to achieve accuracy. Models of Ca, K, Mg, and S were successful (validation R2 > 0.60 to 0.96), but models for pH, P, and Zn failed. Bare soil composite images from S2 data are helpful in predicting soil fertility in low-relief floodplains.

Proteomics studies often explore phenotypic differences between whole organs and systems. Within the heart, more subtle variation exists. To date, differences in the underlying proteome are only described between whole cardiac chambers. This study, using the bovine heart as a model, investigates inter-regional differences and assesses the feasibility of measuring detailed, cross-tissue variance in the cardiac proteome. Using a bovine heart, we created a two-dimensional section through a plane going through two chambers. This plane was further sectioned into 4 × 4 mm cubes and analysed using label-free proteomics. We identified three distinct proteomes. When mapped to the extracted sections, the proteomes corresponded largely to the outer wall of the right ventricle and secondly to the outer wall of the left ventricle, right atrial appendage, tricuspid and mitral valves, modulator band, and parts of the left atrium. The third separate proteome corresponded to the inner walls of the left and right ventricles, septum, and left atrial appendage. Differential protein abundancies indicated differences in energy metabolism between regions. Data analyses of the mitochondrial proteins revealed a variable pattern of abundances of complexes I–V between the proteomes, indicating differences in the bioenergetics of the different cardiac sub-proteomes. Mapping of disease-associated proteins interestingly showed desmoglein-2, for which defects in this protein are known to cause Arrhythmogenic Right Ventricular Dysplasia/Cardiomyopathy, which was present predominantly in the outer wall of the left ventricle. This study highlights that organs can have variable proteomes that do not necessarily correspond to anatomical features.

Inhibitors directed toward PARP1 and PARP2 are approved agents for the treatment of BRCA1 and BRCA2-related cancers. Other members of the PARP family have also been implicated in cancer and are being assessed as therapeutic targets in cancer and other diseases. Recently, an inhibitor of PARP7 (RBN-2397) has reached early-stage human clinical trials. Here, we performed a genome-wide CRISPR screen for genes that modify the response of cells to RBN-2397. We identify the polycyclic aromatic hydrocarbon receptor AHR and multiple components of the cohesin complex as determinants of resistance to this agent. Activators and inhibitors of AHR modulate the cellular response to PARP7 inhibition, suggesting potential combination therapy approaches.

T cell exhaustion limits antitumor immunity, but the molecular determinants of this process remain poorly understood. Using a chronic stimulation assay, we performed genome-wide CRISPR-Cas9 screens to systematically discover regulators of T cell exhaustion, which identified an enrichment of epigenetic factors. In vivo CRISPR screens in murine and human tumor models demonstrated that perturbation of the INO80 and BAF chromatin remodeling complexes improved T cell persistence in tumors. In vivo Perturb-seq revealed distinct transcriptional roles of each complex and that depletion of canonical BAF complex members, including Arid1a, resulted in the maintenance of an effector program and downregulation of exhaustion-related genes in tumor-infiltrating T cells. Finally, Arid1a depletion limited the acquisition of exhaustion-associated chromatin accessibility and led to improved antitumor immunity. In summary, we provide an atlas of the genetic regulators of T cell exhaustion and demonstrate that modulation of epigenetic state can improve T cell responses in cancer immunotherapy.

The nonstructural protein 3 (NSP3) macrodomain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (Mac1) removes adenosine diphosphate (ADP) ribosylation posttranslational modifications, playing a key role in the immune evasion capabilities of the virus responsible for the coronavirus disease 2019 pandemic. Here, we determined neutron and x-ray crystal structures of the SARS-CoV-2 NSP3 macrodomain using multiple crystal forms, temperatures, and pHs, across the apo and ADP-ribose-bound states. We characterize extensive solvation in the Mac1 active site and visualize how water networks reorganize upon binding of ADP-ribose and non-native ligands, inspiring strategies for displacing waters to increase the potency of Mac1 inhibitors. Determining the precise orientations of active site water molecules and the protonation states of key catalytic site residues by neutron crystallography suggests a catalytic mechanism for coronavirus macrodomains distinct from the substrate-assisted mechanism proposed for human MacroD2. These data provoke a reevaluation of macrodomain catalytic mechanisms and will guide the optimization of Mac1 inhibitors.

Background & aims: Exploring Being Autistic is an autistic-led group-based programme providing psychoeducation and peer support to newly identified/diagnosed autistic adults. In 2020, due to social distancing measures implemented following the coronavirus pandemic, Exploring Being Autistic was adapted for online delivery. Here, we aimed to replicate previous research into the in-person delivery of Exploring Being Autistic, to determine whether similar results were obtained when the programme was delivered online. Further, we aimed to identify the unique opportunities and challenges that online delivery afforded. Methods: We used a community-based participatory research (CBPR) approach, whereby the autistic developer and facilitator of Exploring Being Autistic worked collaboratively with a team of academic researchers throughout the research process. Together, we evaluated two iterations of the online Exploring Being Autistic programme, involving 16 attendees. Attendees completed questionnaires before, during and after the programme. Attendees were also invited to participate in two post-programme (group or individual) interviews: one following the completion of the programme (time one) and another 6--8 months later (time two). Attendees were included in the research if they completed at least one questionnaire or interview. Data were analysed qualitatively, using reflexive thematic analysis. Results: Experiences of participating in the programme tended to be positive. Participants appreciated the autistic-led nature of the programme, found unity in the diversity of the group, and developed a positive and practical outlook as a result of the programme. Further analyses of our data revealed mixed views regarding the online delivery of the programme. Opportunities of online delivery were noted, such as this mode of participation reducing cognitive load, enabling the programme to be accessible to more participants, and fostering meaningful social connections among participants. However, technology and practical issues were felt to cause barriers, and some human aspects of participation were felt to be "lost in translation" (e.g., in breakout groups). Conclusions: The online delivery of the Exploring Being Autistic programme yielded similar results to previous, in-person evaluations of the programme. While we identified positive aspects of online delivery, this mode did not entirely suit everyone's needs. Implications: From the current findings, we can make several recommendations to develop online support for autistic people. First, flexibility is key. To make support accessible and inclusive to a broad range of autistic people, the option for attendees to engage in-person, online or in hybrid formats should be considered. Second, if delivering support online, the use of breakout rooms should be carefully considered. While participants appreciated the opportunity to meet different people, some participants found the unpredictability and lack of scaffolding associated with breakout rooms challenging. To mitigate these challenges, groups could be pre-determined and shared with the attendees in advance (although consideration should be given to how the groups "fit" together, and whether groupings should be changed at set intervals). Gentle warnings should also be given to those in breakout rooms, to alert them of the need to re-join the main group. Finally, support with technological aspects relating to engagement should be prioritised.