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Abstract Wastewater treatment plants (WWTPs) are hotspots for pathogens, and can facilitate horizontal gene transfer, potentially releasing harmful genetic material and antimicrobial resistance genes into the environment. Little information exists on the composition and behavior of microbes in WWTPs, especially in developing countries. This study used environmental DNA (eDNA) techniques to examine the microbiome load of wastewater from WWTPs. The DNA was isolated from wastewater samples collected from the treatment trains of three WWTPs in Kimberley, South Africa, and the microbial diversity and composition was compared through 16 S rRNA gene sequencing. The microbes detected were of the Kingdom Bacteria, and of these, 48.27% were successfully identified to genus level. The majority of reads from the combined bacterial data fall within the class Gammaproteobacteria, which is known to adversely impact ecological and human health. Arcobacteraceae constituted 19% of the bacterial reads, which is expected as this family is widespread in aquatic environments. Interestingly, the most abundant bacterial group was Bacteroides, which contain a variety of antibiotic-resistant members. Overall, various antibiotic-resistant taxa were detected in the wastewater, indicating a concerning level of antibiotic resistance within the bacterial community. Therefore, eDNA analysis can be a valuable tool in monitoring and assessing the bacterial microbiome in wastewater, thus providing important information for the optimization and improvement of wastewater treatment systems and mitigate public health risks.

Abstract Neck pain associated with helmet‐wear is an occupational health problem often observed in helicopter pilots and aircrew. Whether aircrew helmet wearing is associated with physiological and biomechanical differences between sexes is currently unknown. This study investigated neuromuscular activation patterns during different helmet‐wearing conditions. The helmet load was manipulated through a novel Helmet Balancing System (HBS) in healthy, non‐pilot male and female participants (n = 10 each, age 19–45 years) in two phases. Phase A assessed the acute effects of helmet‐wear on neck muscles activation during head movements. Phase B examined changes in muscle activity and cervical disc height after wearing a helmet for 45 min. In Phase A, muscle activity was similar between sexes in many movements, but it was higher in female participants when wearing a helmet than in males. The HBS reduced muscle activity in both sexes. In Phase B, female participants exhibited a greater level of muscular fatigue, and male participants’ cervical disc height was significantly decreased [5.7 (1.4) vs. 4.4 (1.5) mm, P

Abstract Background A prediction model can be a useful tool to quantify the risk of a patient developing dementia in the next years and take risk-factor-targeted intervention. Numerous dementia prediction models have been developed, but few have been externally validated, likely limiting their clinical uptake. In our previous work, we had limited success in externally validating some of these existing models due to inadequate reporting. As a result, we are compelled to develop and externally validate novel models to predict dementia in the general population across a network of observational databases. We assess regularization methods to obtain parsimonious models that are of lower complexity and easier to implement. Methods Logistic regression models were developed across a network of five observational databases with electronic health records (EHRs) and claims data to predict 5-year dementia risk in persons aged 55–84. The regularization methods L1 and Broken Adaptive Ridge (BAR) as well as three candidate predictor sets to optimize prediction performance were assessed. The predictor sets include a baseline set using only age and sex, a full set including all available candidate predictors, and a phenotype set which includes a limited number of clinically relevant predictors. Results BAR can be used for variable selection, outperforming L1 when a parsimonious model is desired. Adding candidate predictors for disease diagnosis and drug exposure generally improves the performance of baseline models using only age and sex. While a model trained on German EHR data saw an increase in AUROC from 0.74 to 0.83 with additional predictors, a model trained on US EHR data showed only minimal improvement from 0.79 to 0.81 AUROC. Nevertheless, the latter model developed using BAR regularization on the clinically relevant predictor set was ultimately chosen as best performing model as it demonstrated more consistent external validation performance and improved calibration. Conclusions We developed and externally validated patient-level models to predict dementia. Our results show that although dementia prediction is highly driven by demographic age, adding predictors based on condition diagnoses and drug exposures further improves prediction performance. BAR regularization outperforms L1 regularization to yield the most parsimonious yet still well-performing prediction model for dementia.

Abstract Low skeletal muscle index/density (SMI/SMD) is prevalent in cancer, adversely prognostic and associated with tumour stage and the systemic inflammatory response (SIR). Age and SMI/SMD has not been widely studied. The present study analyses the association between age and SMI/SMD after adjustment for other clinicopathological factors. Patients undergoing resectional surgery for TNM Stage I-III disease within the West of Scotland between 2011 and 2014 were identified. A single CT slice was obtained from each patients staging CT scan. SMI and SMD were stratified normal/abnormal. The SIR was stratified using Systemic Inflammatory Grade (SIG). When stratified by age (

Abstract Objective Mid‐life cardiovascular risk factors are associated with later cognitive decline. Whether repetitive head injury among professional athletes impacts cardiovascular risk is unknown. We investigated associations between concussion burden and postcareer hypertension, high cholesterol, and diabetes among former professional American‐style football (ASF) players. Methods In a cross‐sectional study of 4080 professional ASF players conducted between January 2015 and March 2022, we used an mulitsymptom concussion symptom score (CSS) and the number of loss‐of‐consciousness (LOC) episodes as a single severe symptom to quantify football‐related concussion exposure. Primary outcomes were hypertension, dyslipidemia, and diabetes, defined by current or recommended prescription medication use. Results The prevalence of hypertension, high cholesterol, and diabetes among former players (52 ± 14 years of age) was 37%, 34%, and 9%. Concussion burden was significantly associated with hypertension (lowest vs. highest CSS quartile, odds ratio (OR) = 1.99; 95%CI: 1.33–2.98; p

Abstract The use of patient‐reported outcomes (PROMs) of quality of life (QOL) is common in cachexia trials. Patients' self‐report on health, functioning, wellbeing, and perceptions of care, represent important measures of efficacy. This review describes the frequency, variety, and reporting of QOL endpoints used in cancer cachexia clinical trials. Electronic literature searches were performed in Medline, Embase, and Cochrane (1990–2023). Seven thousand four hundred thirty‐five papers were retained for evaluation. Eligibility criteria included QOL as a study endpoint using validated measures, controlled design, adults (>18 years), ≥40 participants randomized, and intervention exceeding 2 weeks. The Covidence software was used for review procedures and data extractions. Four independent authors screened all records for consensus. Papers were screened by titles and abstracts, prior to full‐text reading. PRISMA guidance for systematic reviews was followed. The protocol was prospectively registered via PROSPERO (CRD42022276710). Fifty papers focused on QOL. Twenty‐four (48%) were double‐blind randomized controlled trials. Sample sizes varied considerably (n = 42 to 469). Thirty‐nine trials (78%) included multiple cancer types. Twenty‐seven trials (54%) featured multimodal interventions with various drugs and dietary supplements, 11 (22%) used nutritional interventions alone and 12 (24%) used a single pharmacological intervention only. The median duration of the interventions was 12 weeks (4–96). The most frequent QOL measure was the EORTC QLQ‐C30 (60%), followed by different FACIT questionnaires (34%). QOL was a primary, secondary, or exploratory endpoint in 15, 31 and 4 trials respectively, being the single primary in six. Statistically significant results on one or more QOL items favouring the intervention group were found in 18 trials. Eleven of these used a complete multidimensional measure. Adjustments for multiple testing when using multicomponent QOL measures were not reported. Nine trials (18%) defined a statistically or clinically significant difference for QOL, five with QOL as a primary outcome, and four with QOL as a secondary outcome. Correlation statistics with other study outcomes were rarely performed. PROMs including QOL are important endpoints in cachexia trials. We recommend using well‐validated QOL measures, including cachexia‐specific items such as weight history, appetite loss, and nutritional intake. Appropriate statistical methods with definitions of clinical significance, adjustment for multiple testing and few co‐primary endpoints are encouraged, as is an understanding of how interventions may relate to changes in QOL endpoints. A strategic and scientific‐based approach to PROM research in cachexia trials is warranted, to improve the research base in this field and avoid the use of QOL as supplementary measures.

Abstract Regulatory agencies require evidence that endpoints correlate with clinical benefit before they can be used to approve drugs. Biomarkers are often considered surrogate endpoints. In cancer cachexia trials, the measurement of biomarkers features frequently. The aim of this systematic review was to assess the frequency and diversity of biomarker endpoints in cancer cachexia trials. A comprehensive electronic literature search of MEDLINE, Embase and Cochrane (1990–2023) was completed. Eligible trials met the following criteria: adults (≥18 years), prospective design, more than 40 participants, use of a cachexia intervention for more than 14 days and use of a biomarker(s) as an endpoint. Biomarkers were defined as any objective measure that was assayed from a body fluid, including scoring systems based on these assays. Routine haematology and biochemistry to monitor intervention toxicity were not considered. Data extraction was performed using Covidence, and reporting followed PRISMA guidance (PROSPERO: CRD42022276710). A total of 5975 studies were assessed, of which 52 trials (total participants = 6522) included biomarkers as endpoints. Most studies (n = 29, 55.7%) included a variety of cancer types. Pharmacological interventions (n = 27, 51.9%) were most evaluated, followed by nutritional interventions (n = 20, 38.4%). Ninety‐nine different biomarkers were used across the trials, and of these, 96 were assayed from blood. Albumin (n = 29, 55.8%) was assessed most often, followed by C‐reactive protein (n = 22, 42.3%), interleukin‐6 (n = 16, 30.8%) and tumour necrosis factor‐α (n = 14, 26.9%), the latter being the only biomarker that was used to guide sample size calculations. Biomarkers were explicitly listed as a primary outcome in six trials. In total, 12 biomarkers (12.1% of 99) were used in six trials or more. Insulin‐like growth factor binding protein 3 (IGFBP‐3) and insulin‐like growth factor 1 (IGF‐1) levels both increased significantly in all three trials in which they were both used. This corresponded with a primary outcome, lean body mass, and was related to the pharmacological mechanism. Biomarkers were predominately used as exploratory rather than primary endpoints. The most commonly used biomarker, albumin, was limited by its lack of responsiveness to nutritional intervention. For a biomarker to be responsive to change, it must be related to the mechanism of action of the intervention and/or the underlying cachexia process that is modified by the intervention, as seen with IGFBP‐3, IGF‐1 and anamorelin. To reach regulatory approval as an endpoint, the relationship between the biomarker and clinical benefit must be clarified.

Abstract Significant variation exists in the outcomes used in cancer cachexia trials, including measures of body composition, which are often selected as primary or secondary endpoints. To date, there has been no review of the most commonly selected measures or their potential sensitivity to detect changes resulting from the interventions being examined. The aim of this systematic review is to assess the frequency and diversity of body composition measures that have been used in cancer cachexia trials. MEDLINE, Embase and Cochrane Library databases were systematically searched between January 1990 and June 2021. Eligible trials examined adults (≥18 years) who had received an intervention aiming to treat or attenuate the effects of cancer cachexia for >14 days. Trials were also of a prospective controlled design and included body weight or at least one anthropometric, bioelectrical or radiological endpoint pertaining to body composition, irrespective of the modality of intervention (e.g., pharmacological, nutritional, physical exercise and behavioural) or comparator. Trials with a sample size of

Summary: Background: Contemporary surgical practices for traumatic brain injury (TBI) remain unclear. We describe the clinical profile of an 18-centre US TBI cohort with cranial surgery. Methods: The prospective, observational Transforming Research and Clinical Knowledge in Traumatic Brain Injury Study (2014–2018; ClinicalTrials.gov #NCT02119182) enrolled subjects who presented to trauma centre and received head computed tomography within 24-h (h) post-TBI. We performed a secondary data analysis in subjects aged ≥17-years with hospitalisation. Clinical characteristics, surgery type/timing, hospital and six-month outcomes were reported. Findings: Of 2032 subjects (age: mean = 41.4-years, range = 17–89-years; male = 71% female = 29%), 260 underwent cranial surgery, comprising 65% decompressive craniectomy, 23% craniotomy, 12% other surgery. Subjects with surgery (vs. without surgery) presented with worse neurological injury (median Glasgow Coma Scale = 6 vs. 15; midline shift ≥5 mm: 48% vs. 2%; cisternal effacement: 61% vs. 4%; p

ABSTRACT The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus continues to cause severe disease and deaths in many parts of the world, despite massive vaccination efforts. Antiviral drugs to curb an ongoing infection remain a priority. The virus-encoded 3C-like main protease (MPro; nsp5) is seen as a promising target. Here, with a positive selection genetic system engineered in Saccharomyces cerevisiae using cleavage and release of MazF toxin as an indicator, we screened in a robotized setup small molecule libraries comprising ~2,500 compounds for MPro inhibitors. We detected eight compounds as effective against MPro expressed in yeast, five of which are characterized proteasome inhibitors. Molecular docking indicates that most of these bind covalently to the MPro catalytically active cysteine. Compounds were confirmed as MPro inhibitors in an in vitro enzymatic assay. Among those were three previously only predicted in silico; the boron-containing proteasome inhibitors bortezomib, delanzomib, and ixazomib. Importantly, we establish reaction conditions in vitro preserving the MPro-inhibitory activity of the boron-containing drugs. These differ from the standard conditions, which may explain why boron compounds have gone undetected in screens based on enzymatic in vitro assays. Our screening system is robust and can find inhibitors of a specific protease that are biostable, able to penetrate a cell membrane, and are not generally toxic. As a cellular assay, it can detect inhibitors that fail in a screen based on an in vitro enzymatic assay using standardized conditions, and now give support for boron compounds as MPro inhibitors. This method can also be adapted for other viral proteases.IMPORTANCEThe coronavirus disease 2019 (COVID-19) pandemic triggered the realization that we need flexible approaches to find treatments for emerging viral threats. We implemented a genetically engineered platform in yeast to detect inhibitors of the virus’s main protease (MPro), a promising target to curb severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections. Screening molecule libraries, we identified candidate inhibitors and verified them in a biochemical assay. Moreover, the system detected boron-containing molecules as MPro inhibitors. Those were previously predicted computationally but never shown effective in a biochemical assay. Here, we demonstrate that they require a non-standard reaction buffer to function as MPro inhibitors. Hence, our cell-based method detects protease inhibitors missed by other approaches and provides support for the boron-containing molecules. We have thus demonstrated that our platform can screen large numbers of chemicals to find potential inhibitors of a viral protease. Importantly, the platform can be modified to detect protease targets from other emerging viruses.

Background: Proximal pole scaphoid fractures pose a complex surgical challenge. Fractures proximal to the vessels entering the dorso-radial surface can result in avascular necrosis (AVN) due to ischemia of the proximal pole. Management of these types of fractures can be difficult due to the delayed presentation of patients. Surgical management is limited when the proximal pole is deemed unreconstructible. Viable options for young, non-arthritic patients include vascularised or non-vascularised bone grafting. Additionally, an adaptive proximal scaphoid implant with a pyrocarbon implant has also been shown to provide adequate outcomes. This case highlights a patient who had a failed pyrocarbon implant and then went on to have a successful non-vascularised rib autograft procedure. Case report: A 22-year-old male athlete presented with a proximal pole scaphoid fracture. He suffered a hyper-extension injury to his wrist 3 years previous to his presentation. The patient subsequently developed AVN and underwent proximal pole pyrocarbon replacement. This replacement failed due to excessive early mobilisation. The following revision procedure involved incorporating an osteochondral rib autograft. 4 months later the patient was pain-free and returned to previous high-level activity. At 1 year follow-up, he had a Patient Rated Wrist Evaluation score of 6.5/100 with full range of motion. Conclusion: Osteochondral rib graft is a viable option for the management of failed proximal scaphoid replacement failure and provides good functional outcomes in a young active patient.

Abstract In this contribution, we investigate two sparassodonts from the Sarmiento Formation (Colhuehuapian Age; Early Miocene) recovered at the Patagonian locality of Sacanana, Chubut Province, Argentina. The first specimen (MACN-Pv CH1911), identified as Sipalocyon externus, is an almost complete cranium with upper dentition. The second specimen (MACN-Pv CH40), referred to Borhyaena macrodonta, consists of a fragmentary rostral portion of the face with associated m3–m4. The cranium of S. externus was studied through µCT-scanning to investigate its internal anatomy and infer paleoecological aspects of olfaction and hearing. In general, the endocranial anatomy and encephalization quotient are like those of other metatherians. We report the presence of an accessory transverse diploic sinus that has not previously been described in marsupials. Elements of the nasal cavity (e.g., turbinals, ossified nasal septum, cribriform plate) of S. externus exhibit features that are widespread among marsupials. Sense of olfaction, as evaluated from the cribriform plate and the 3D models of the olfactory bulbs, appears to have grossly resembled that of the domestic cat, a well-researched therian proxy. The dimensions of the tympanic membrane, as estimated from the preserved ectotympanic, suggest that the optimal hearing frequency of S. externus would have been similar to that of extant carnivorous marsupials. In sum, our study suggests that S. externus was a typical small-bodied (2–3 kg) hypercarnivorous metatherian that primarily relied on vision to hunt its prey, complemented by other sensory capabilities (olfaction and hearing), in a similar manner to small felids.

Abstract Changes in myonuclear architecture and positioning are associated with exercise adaptations and ageing. However, data on the positioning and number of myonuclei following exercise are inconsistent. Additionally, whether myonuclear domains (MNDs; i.e., the theoretical volume of cytoplasm within which a myonucleus is responsible for transcribing DNA) and myonuclear positioning are altered with age remains unclear. The aim of this investigation was to investigate relationships between age and activity status and myonuclear domains and positioning. Vastus lateralis muscle biopsies from younger endurance‐trained (YT) and older endurance‐trained (OT) individuals were compared with age‐matched untrained counterparts (YU and OU; OU samples were acquired during surgical operation). Serial, optical z‐slices were acquired throughout isolated muscle fibres and analysed to give three‐dimensional coordinates for myonuclei and muscle fibre dimensions. The mean cross‐sectional area (CSA) of muscle fibres from OU individuals was 33%–53% smaller compared with the other groups. The number of nuclei relative to fibre CSA was 90% greater in OU compared with YU muscle fibres. Additionally, scaling of MND volume with fibre size was altered in older untrained individuals. The myonuclear arrangement, in contrast, was similar across groups. Fibre CSA and most myonuclear parameters were significantly associated with age in untrained individuals, but not in trained individuals. These data indicate that regular endurance exercise throughout the lifespan might better preserve the size of muscle fibres in older age and maintain the relationship between fibre size and MND volumes. Inactivity, however, might result in reduced muscle fibre size and altered myonuclear parameters.

Traumatic brain injury (TBI) is independently associated with hypertension and ischemic stroke. The goal of this study was to determine the interplay between TBI and incident hypertension in the occurrence of post-TBI stroke. This prospective study used a hospital-based registry to identify patients without pre-existing comorbidities. TBI patients (n?=?3664) were frequency matched on age, sex, and race to non-TBI patients (n?=?1848). Follow-up started 6 months post-TBI or study entry and extended up to 10 years. To examine hypertension's role in post-TBI stroke, we used logistic regression models to calculate the effect estimates for stroke in four exposure categories that included TBI or hypertension in isolation and in combination. Second, we calculated the conditional direct effect (CDE) of TBI in models that considered hypertension as intermediary. Third, we examined whether TBI effect was modified by antihypertensive medication use. The 10-year cumulative incidence of stroke was higher in the TBI group (4.7%) than the non-TBI group (1.3%; p?0.001). TBI patients who developed hypertension had the highest risk of stroke (odds ratio [OR]?=?4.83, 95% confidence interval [CI]?=?2.53?9.23, p?

Abstract There is no consensus on the optimal endpoint(s) in cancer cachexia trials. Endpoint variation is an obstacle when comparing interventions and their clinical value. The aim of this systematic review was to summarize and evaluate endpoints used to assess appetite and dietary intake in cancer cachexia clinical trials. A search for studies published from 1 January 1990 until 2 June 2021 was conducted using MEDLINE, Embase and Cochrane Central Register of Controlled Trials. Eligible studies examined cancer cachexia treatment versus a comparator in adults with assessments of appetite and/or dietary intake as study endpoints, a sample size ≥40 and an intervention lasting ≥14 days. Reporting was in line with PRISMA guidance, and a protocol was published in PROSPERO (2022 CRD42022276710). This review is part of a series of systematic reviews examining cachexia endpoints. Of the 5975 articles identified, 116 were eligible for the wider review series and 80 specifically examined endpoints of appetite (65 studies) and/or dietary intake (21 studies). Six trials assessed both appetite and dietary intake. Appetite was the primary outcome in 15 trials and dietary intake in 7 trials. Median sample size was 101 patients (range 40–628). Forty‐nine studies included multiple primary tumour sites, while 31 studies involved single primary tumour sites (15 gastrointestinal, 7 lung, 7 head and neck and 2 female reproductive organs). The most frequently reported appetite endpoints were visual analogue scale (VAS) and numerical rating scale (NRS) (40%). The appetite item from the European Organisation for Research and Treatment of Cancer Quality of Life Questionnaire (EORTC QLQ) C30/C15 PAL (38%) and the appetite question from North Central Cancer Treatment Group anorexia questionnaire (17%) were also frequently applied. Of the studies that assessed dietary intake, 13 (62%) used food records (prospective registrations) and 10 (48%) used retrospective methods (24‐h recall or dietary history). For VAS/NRS, a mean change of 1.3 corresponded to Hedge's g of 0.5 and can be considered a moderate change. For food records, a mean change of 231 kcal/day or 11 g of protein/day corresponded to a moderate change. Choice of endpoint in cachexia trials will depend on factors pertinent to the trial to be conducted. Nevertheless, from trials assessed and available literature, NRS or EORTC QLQ C30/C15 PAL seems suitable for appetite assessments. Appetite and dietary intake endpoints are rarely used as primary outcomes in cancer cachexia. Dietary intake assessments were used mainly to monitor compliance and are not validated in cachexia populations. Given the importance to cachexia studies, dietary intake endpoints must be validated before they are used as endpoints in clinical trials.

Summary: Background: Apomorphine is a dopaminergic candidate therapy to improve recovery in patients with prolonged disorders of consciousness (PDoC). Behavioural improvements were previously described in non-controlled case series, but its efficacy and neural mechanisms remain largely unknown. This open-label controlled study using multimodal outcome measures investigates the action of apomorphine in severely brain-injured patients. Methods: Thirteen PDoC patients received 30-day subcutaneous apomorphine treatment (n = 6) or standard care (control group, n = 7) in a neurological rehabilitation centre between February 2018 and January 2021. The apomorphine group was monitored 30 days before treatment initiation, during treatment and one year after treatment. Primary outcome measure was defined as changes in behavioural diagnosis using the Coma Recovery Scale—Revised (CRS-R). CRS-R index, recovery of new conscious behaviours, DoC-feeling scores, high-density electroencephalography, and fluorodeoxyglucose positron emission tomography were employed as secondary outcome measures. The control group was monitored with repeated CRS-R only. Registration: EudraCT 2018-003144-23; Clinicaltrials.gov NCT03623828. Findings: Groups (apomorphine vs. control: odds ratio 8.9, 95% CI 3.3–17.8) and study phase (treatment vs. baseline, apomorphine group only: odds ratio 3.9, 95% CI 1.5–10.1) significantly influenced positive changes in behavioural diagnosis. At one-year post-injury, 4/6 patients in the apomorphine group and 1/7 patients in the control group had improved their diagnosis. Similarly, CRS-R index was significantly influenced by study phase (treatment vs. baseline). All items on the DoC-feeling score were rated higher after treatment than before by both family and medical staff. Patients in the apomorphine group recovered more conscious behaviours than control patients. Alpha-band whole-brain connectivity and participation coefficient, as well as alpha-band parieto-temporal connectivity and frontal participation coefficient were higher after treatment than at baseline. Whole-brain metabolism increased by a relative mean of 13.8% after treatment compared to baseline, with a significant effect of timing (pre-vs. post-treatment scans) on regional SUV. Interpretation: Long-lasting consciousness improvements were observed in patients treated with apomorphine, compared to controls and compared to baseline. Changes in brain connectivity and metabolism were observed after treatment, providing insights into possible neurophysiological mechanisms and target areas. This open-label study confirmed the feasibility and safety of apomorphine treatment, which may represent a key therapeutic option for PDoC. Funding: University and University Hospital of Liege, Belgian National Funds for Scientific Research, Fund Generet of the King Baudouin Foundation, AstraZeneca Foundation, Leon Fredericq Foundation and NeuroHealing Pharmaceuticals Inc.

Abstract Introduction The present study sought to examine the relationships between systemic inflammatory composite ratios/cumulative scores, magnitude of systemic inflammatory response (SIR) and survival in good performance status patients (ECOG‐PS 0/1) with advanced NSCLC receiving palliative radiotherapy. Methods Systemic inflammatory composite ratios/cumulative scores included the neutrophil‐lymphocyte ratio (NLR), platelet‐lymphocyte ratio (PLR), lymphocyte‐monocyte ratio (LMR), C‐reactive protein, (CRP)‐albumin ratio (CAR), neutrophil‐ lymphocyte score (NLS), platelet‐lymphocyte score (PLS), lymphocyte‐monocyte score (LMS), neutrophil‐platelet score (NPS), modified Glasgow prognostic score (mGPS). The magnitude of SIR was determined by serum CRP concentration, with a median CRP concentration of >10 m mg/L considered to be systemically inflamed. Relationships between systemic inflammatory composite ratios/ cumulative scores and clinicopathological characteristics were examined using chi‐square analysis. Relationships between overall survival (OS) and systemic inflammatory composite ratios/ cumulative scores were examined using cox regression analysis. Results 479 patients were included. 48% (n = 231) of patients were male and 70% (n = 338) were ≥65 years of age. 29% (n = 140) patients were ECOG‐PS 0 and 71% (n = 339) were ECOG‐PS 1. 98% (n = 469) of patients died during follow‐up. The median survival was 5 months (2–11). A similar prevalence of systemic inflammation was noted across the various ratios/scores (NLR >3 68%; LMR 150 70%; CAR >0.20 83%; NLS ≥1 66%; LMS ≥1 71%; NPS≥1 50%; PLS≥1 60% and mGPS≥1 75%). Despite not considered to be systemically inflamed, an NLR 10 mg/L. When adjusted for ECOG‐PS, CAR>0.40 (p

Abstract The interest in recirculating aquaculture systems (RAS) is growing due to their benefits such as increased productivity, better control over animal care, reduced environmental effects, and less water consumption. However, in some regions of the world, traditional aquaculture methods remain prevalent, and selective breeding has often been designed for performance within these systems. Therefore, it is important to evaluate how current fish populations fare in RAS to guide future breeding choices. In a commercial setting, we explore the genetic structure of growth characteristics, measure genotype–environment interactions (GxE) in salmon smolts, and examine genetic markers related to growth in freshwater lochs and RAS. Young salmon were raised together until they reached the parr stage, after which they were divided equally between freshwater net‐pens and RAS. After an 8‐week period, we sampled fish from each environment and genotyped them. Our findings revealed that fish reared in RAS were generally smaller in weight and length but exhibited a higher condition factor and uniformity. We found a notably smaller component of unexplained variance in the RAS, leading to higher heritability estimates. We observed a low GxE effect for length and condition factor, but significant re‐ranking for whole‐body weight, as well as noticeable differences in trait associations across environments. Specifically, a segment of chromosome 22 was found to be linked with the condition factor in the RAS population only. Results suggests that if the use of RAS continues to expand, the efficiency of existing commercial populations may not reach its full potential unless breeding programs specific to RAS are implemented.

Objectives An estimated 14–23% of patients with traumatic brain injury (TBI) incur multiple lifetime TBIs. The relationship between prior TBI and outcomes in patients with moderate to severe TBI (msTBI) is not well delineated. We examined the associations between prior TBI, in-hospital mortality, and outcomes up to 12 months after injury in a prospective US msTBI cohort.Methods Data from hospitalized subjects with Glasgow Coma Scale score of 3–12 were extracted from the Transforming Research and Clinical Knowledge in Traumatic Brain Injury Study (enrollment period: 2014–2019). Prior TBI with amnesia or alteration of consciousness was assessed using the Ohio State University TBI Identification Method. Competing risk regressions adjusting for age, sex, psychiatric history, cranial injury and extracranial injury severity examined the associations between prior TBI and in-hospital mortality, with hospital discharged alive as the competing risk. Adjusted HRs (aHR (95% CI)) were reported. Multivariable logistic regressions assessed the associations between prior TBI, mortality, and unfavorable outcome (Glasgow Outcome Scale-Extended score 1–3 (vs. 4–8)) at 3, 6, and 12 months after injury.Results Of 405 acute msTBI subjects, 21.5% had prior TBI, which was associated with male sex (87.4% vs. 77.0%, p=0.037) and psychiatric history (34.5% vs. 20.7%, p=0.010). In-hospital mortality was 10.1% (prior TBI: 17.2%, no prior TBI: 8.2%, p=0.025). Competing risk regressions indicated that prior TBI was associated with likelihood of in-hospital mortality (aHR=2.06 (1.01–4.22)), but not with hospital discharged alive. Prior TBI was not associated with mortality or unfavorable outcomes at 3, 6, and 12 months.Conclusions After acute msTBI, prior TBI history is independently associated with in-hospital mortality but not with mortality or unfavorable outcomes within 12 months after injury. This selective association underscores the importance of collecting standardized prior TBI history data early after acute hospitalization to inform risk stratification. Prospective validation studies are needed.Level of evidence IV.Trial registration number NCT02119182.

Habitat fragmentation can negatively impact wildlife populations by simplification of ecological interactions, but little is known about how these impacts extend to host-associated symbiotic communities. The symbiotic communities of amphibians play important roles in anti-pathogen defences, particularly against the amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd). In this study, we analyse the role of macroparasitic helminth communities in concert with microbial communities in defending the host against Bd infection within the context of forest fragmentation. We found that skin microbial and helminth communities are disrupted at fragmented habitats, while gut microbiomes appear more resilient to environmental change. We also detected potential protective roles of helminth diversity and anti-pathogen microbial function in limiting Bd infection. Microbial network analysis revealed strong patterns of structure in both skin and gut communities, with helminths playing central roles in these networks. We reveal consistent roles of microbial and helminth diversity in driving host–pathogen interactions and the potential implications of fragmentation on host fitness.

Introduction: The prognostic value of CT-derived liver volume in terms of cancer outcomes is not clear. The aim of the present study was to examine the relationship between liver area on a single axial CT-slice and the total liver volume in patients with colonic cancer. Furthermore, we examine the relationship between liver volume, determined using this novel method, clinicopathological variables and survival. Methods: Consecutive patients who underwent potentially curative surgery for colonic cancer were identified from a prospectively maintained database. Maximal liver area on axial CT-slice (cm2) and total volume (cm3), were obtained by the manual segmentation of pre-operative CT-images in a PACS viewer. The maximal liver area was normalized for body height2 to create the liver index (LI) and values, categorized into tertiles. The primary outcome of interest was overall survival (OS). Relationships between LI and clinico-pathological variables were examined using chi-square analysis and binary logistic regression. The relationship between LI and OS was examined using cox proportional hazard regression. Results: A total of 359 patients were included. A total of 51% (n = 182) of patients were male and 73% (n = 261) were aged 65 years or older. 81% (n = 305) of patients were alive 3-years post-operatively. The median maximal liver area on the axial CT slice was 178.7 (163.7–198.4) cm2. The median total liver volume was 1509.13 (857.8–3337.1) cm3. Maximal liver area strongly correlated with total liver volume (R2 = 0.749). The median LI was 66.8 (62.0–71.6) cm2/m2. On multivariate analysis, age (p < 0.001), sex (p < 0.05), BMI (p < 0.001) and T2DM (p < 0.05) remained significantly associated with LI. On univariate analysis, neither LI (continuous) or LI (tertiles) were significantly associated with OS (p = 0.582 and p = 0.290, respectively). Conclusions: The simple, reliable method proposed in this study for quantifying liver volume using CT-imaging was found to have an excellent correlation between observers and provided results consistent with the contemporary literature. This method may facilitate the further examination of liver volume in future cancer studies.

Abstract Bulk bismuth has a complex Landau spectrum. The small effective masses and the large g-factors are anisotropic. The chemical potential drifts at high magnetic fields. Moreover, twin boundaries further complexify the interpretation of the data by producing extra anomalies in the extreme quantum limit. Here, we present a study of angle dependence of magnetoresistance up to 65 T in bismuth complemented with Nernst, ultrasound, and magneto-optic data. All observed anomalies can be explained in a single-particle picture of a sample consisting of two twinned crystals tilted by 108° and with two adjacent crystals keeping their own chemical potentials despite a shift between chemical potentials as large as 68 meV at 65 T. This implies an energy barrier between adjacent twinned crystals reminiscent of a metal- semiconductor Schottky barrier or a p-n junction. We argue that this barrier is built by accumulating charge carriers of opposite signs across a twin boundary.

Abstract Background Second‐line immunotherapy is currently recognized to help only a subset of patients with advanced forms of non‐small cell lung cancer (NSCLC). The current study analyzes the connection between prior treatment host/tumor characteristics and survival in advanced NSCLC patients receiving nivolumab as a second‐line therapy. Methods A retrospective cohort analysis was carried out on individuals with advanced NSCLC receiving second‐line Nivolumab with palliative intent between February 2016 and May 2019 across three health boards in NHS Greater Glasgow and Clyde, Lanarkshire, Ayrshire, and Arran in Scotland to examine the association between systemic inflammation, body composition, and survival were determined using computed tomography (CT). Results The current study investigates the connection between prior treatment host/tumor characteristics and survival in advanced NSCLC patients receiving nivolumab as a second‐line therapy. The majority were 65 years of age or older (51%), female (53%), had adenocarcinoma (53%), and had good performance status (ECOG 0/1) (86%). Most patients had high SFI (70%) or VFA (54%). The median overall survival after starting Nivolumab was 15 months. ECOG‐PS and hypoalbuminemia were significant predictors of 12‐month survival in patients with advanced NSCLC following Nivolumab treatment, according to Cox regression (p‐value = 0.047 and 0.014, respectively). Conclusion In patients with advanced NSCLC receiving Nivolumab as a second‐line therapy, ECOG‐PS and hypoalbuminemia were strongly associated with survival. Systemic inflammation and hypoalbuminemia measurements may enhance the ECOG‐PS stratification of expected outcomes.

Abstract The amphibian skin microbiome is an important component of anti-pathogen defense, but the impact of environmental change on the link between microbiome composition and host stress remains unclear. In this study, we used radiotelemetry and host translocation to track microbiome composition and function, pathogen infection, and host stress over time across natural movement paths for the forest-associated treefrog, Boana faber. We found a negative correlation between cortisol levels and putative microbiome function for frogs translocated to forest fragments, indicating strong integration of host stress response and anti-pathogen potential of the microbiome. Additionally, we observed a capacity for resilience (resistance to structural change and functional loss) in the amphibian skin microbiome, with maintenance of putative pathogen-inhibitory function despite major temporal shifts in microbiome composition. Although microbiome community composition did not return to baseline during the study period, the rate of microbiome change indicated that forest fragmentation had more pronounced effects on microbiome composition than translocation alone. Our findings reveal associations between stress hormones and host microbiome defenses, with implications for resilience of amphibians and their associated microbes facing accelerated tropical deforestation.

Background/Objectives: Diagnosing concussions is problematic, in part due to the invisible nature of concussion symptoms, in addition to personal and interpersonal factors that influence symptom reporting. As a result, observable signs of concussion can ensure concussions are identified and appropriately treated. Here, we define a potential novel sign, the spontaneous headshake after a kinematic event (SHAAKE) and evaluate its utility in the diagnosis of concussion. Methods: A cross-sectional survey study of 347 athletes (age 27, IQR: 25–29; 47.6% female; highest level of play: college—46.1%, high school—41.2%) identified whether SHAAKE occurred, the reasons underlying SHAAKEs, and its utility for self-reported concussion. Sensitivity and positive predictive value were calculated across all sports and these parameters, as well as estimates for specificity and negative predictive value leveraging published helmet sensor data, were calculated for football players. Results: The median number of times participants reported SHAAKE was 5 (IQR: 3–10), with 4 (IQR: 2–7) associated with a self-reported concussion. Overall, 84.9% of participants reported concussion symptoms as the most common reason for their SHAAKEs. Across all sports, SHAAKE had a sensitivity of 49.6% and positive predictive value 72.4% for diagnosing concussion. In football players, SHAAKE had a sensitivity of 52.3%, estimated specificity of 99.9%, positive predictive value of 91.9%, and estimated negative predictive value of 99.5% for diagnosing self-reported concussion. Conclusions: These results demonstrate that nearly three-quarters of athletes reported a SHAAKE associated with a self-reported concussion, which supports the potential for SHAAKE to be used as a concussion screening tool.

Insects are one of the most species-rich groups on Earth. They comprise much of animal diversity and play vital roles in ecosystems, including pollination, pest control, and decomposition. However, only a fraction of this diversity has been formally described. South Africa is recognised as one of the most biologically diverse countries globally, with an estimated 44 000 insect species. Many crops rely on insect pollinators, including canola, apples, oranges, and sunflowers. A shortage of wild pollinators currently threatens crop yields, yet our knowledge of insect diversity within South Africa is sparse. There are few taxonomic specialists relative to South Africa’s biodiversity, and the methods used for insect identification can be time-consuming and expensive. DNA barcoding provides an important research tool to accelerate insect biodiversity research. In this review, we queried the public DNA barcoding BOLD (Barcode of Life Data System) database for records of “Insecta” within South Africa, and 416 211 published records assigned to 28 239 unique BINs (Barcode Index Numbers) were returned. We identified five taxonomic orders with more BINs than known species in southern Africa (Hymenoptera, Diptera, Thysanoptera, Plecoptera, and Strepsiptera). Most of the barcoded records were derived from Malaise trap sampling in Gauteng, Mpumalanga and Limpopo, while the rest of South Africa remains poorly sampled. We suggest that there is a need for a comprehensive national sampling effort alongside increased investment in taxonomic expertise to generate critical baseline data on insect biodiversity before species are lost to extinction. Significance: Insects provide critical ecosystem services, but our knowledge of insect biodiversity is limited. DNA barcoding can help fill biodiversity knowledge gaps; however, within South Africa, sampling has been uneven. Well-sampled provinces include Gauteng, Mpumalanga and Limpopo, while the Eastern Cape, Free State, KwaZulu-Natal, Northern Cape, North-West and Western Cape remain under-sampled. We argue for a broad national Malaise trapping effort to generate crucial baseline data on insect biodiversity. Without urgent investment in taxonomic expertise and biomonitoring, we risk losing much of our biodiversity before it is even described.

The high rates of protein synthesis and processing render multiple myeloma (MM) cells vulnerable to perturbations in protein homeostasis. The induction of proteotoxic stress by targeting protein degradation with proteasome inhibitors (PIs) has revolutionized the treatment of MM. However, resistance to PIs is inevitable and represents an ongoing clinical challenge. Our first-in-human study of the selective inhibitor of RNA polymerase I transcription of ribosomal RNA genes, CX-5461, has demonstrated a potential signal for anti-tumor activity in three of six heavily pre-treated MM patients. Here, we show that CX-5461 has potent anti-myeloma activity in PI-resistant MM preclinical models in vitro and in vivo. In addition to inhibiting ribosome biogenesis, CX-5461 causes topoisomerase II trapping and replication-dependent DNA damage, leading to G2/M cell-cycle arrest and apoptotic cell death. Combining CX-5461 with PI does not further enhance the anti-myeloma activity of CX-5461 in vivo. In contrast, CX-5461 shows synergistic interaction with the histone deacetylase inhibitor panobinostat in both the Vk∗MYC and the 5T33-KaLwRij mouse models of MM by targeting ribosome biogenesis and protein synthesis through distinct mechanisms. Our findings thus provide strong evidence to facilitate the clinical development of targeting the ribosome to treat relapsed and refractory MM.

Pacific oysters (Crassostrea or Magallana gigas) are one of the most economically important aquaculture species globally. Over the past two decades, ostreid herpesvirus (OsHV-1) has become a major pathogen of cultured Pacific oysters, resulting in widespread mortality with a global distribution. Experimental use of OsHV-1 is challenging for many reasons, including both complexity of host–pathogen dynamics and a lack of functioning model systems. The goal of this study was to improve the tools available for working with OsHV-1 in both whole animals and in tissue explants established from oysters maintained in controlled laboratory conditions. Tissue explants were taken from oysters originating from two different sources that have different levels of mortality in experimental OsHV-1 infections and were exposed to OsHV-1. A whole-animal infection experiment was run concurrently as a comparison. Quantitative PCR and electron microscopy were used to confirm that the explants were capable of replicating OsHV-1. Furthermore, the quantitative PCR results suggest that the source of the oysters was significant in determining the outcome of infection in the explants, supporting the validity of the explant model for OsHV-1 infection. This tissue explant approach for studying OsHV-1 allows for the control of confounding factors in the disease outcome that is not possible in whole-animal experiments, providing a new tool for the study of OsHV-1 in Pacific oysters.

Abstract Background Flavobacterium columnare is the pathogen agent of columnaris disease, a major emerging disease that affects rainbow trout aquaculture. Selective breeding using genomic selection has potential to achieve cumulative improvement of the host resistance. However, genomic selection is expensive partly because of the cost of genotyping large numbers of animals using high-density single nucleotide polymorphism (SNP) arrays. The objective of this study was to assess the efficiency of genomic selection for resistance to F. columnare using in silico low-density (LD) panels combined with imputation. After a natural outbreak of columnaris disease, 2874 challenged fish and 469 fish from the parental generation (n = 81 parents) were genotyped with 27,907 SNPs. The efficiency of genomic prediction using LD panels was assessed for 10 panels of different densities, which were created in silico using two sampling methods, random and equally spaced. All LD panels were also imputed to the full 28K HD panel using the parental generation as the reference population, and genomic predictions were re-evaluated. The potential of prioritizing SNPs that are associated with resistance to F. columnare was also tested for the six lower-density panels. Results The accuracies of both imputation and genomic predictions were similar with random and equally-spaced sampling of SNPs. Using LD panels of at least 3000 SNPs or lower-density panels (as low as 300 SNPs) combined with imputation resulted in accuracies that were comparable to those of the 28K HD panel and were 11% higher than the pedigree-based predictions. Conclusions Compared to using the commercial HD panel, LD panels combined with imputation may provide a more affordable approach to genomic prediction of breeding values, which supports a more widespread adoption of genomic selection in aquaculture breeding programmes.

Abstract Chronic traumatic encephalopathy (CTE) is a neurodegenerative tauopathy associated with repetitive head impacts (RHI), but the components of RHI exposure underlying this relationship are unclear. We create a position exposure matrix (PEM), composed of American football helmet sensor data, summarized from literature review by player position and level of play. Using this PEM, we estimate measures of lifetime RHI exposure for a separate cohort of 631 football playing brain donors. Separate models examine the relationship between CTE pathology and players’ concussion count, athletic positions, years of football, and PEM-derived measures, including estimated cumulative head impacts, linear accelerations, and rotational accelerations. Only duration of play and PEM-derived measures are significantly associated with CTE pathology. Models incorporating cumulative linear or rotational acceleration have better model fit and are better predictors of CTE pathology than duration of play or cumulative head impacts alone. These findings implicate cumulative head impact intensity in CTE pathogenesis.

Abstract Nile tilapia (Oreochromis niloticus) is among the most farmed finfish worldwide, distributed across different environmental conditions. Its wide distribution has mainly been facilitated by several breeding programs and widespread dissemination of genetically improved strains. In the first Nile tilapia study exploiting a whole‐genome pooled sequencing (Poolseq) approach, we identified the genetic structure and signatures of selection in diverse, farmed Nile tilapia populations, with a particular focus on the GIFT strain, developed in the 1980s, and currently managed by WorldFish (GIFTw). We also investigated important farmed strains from The Philippines and Africa. Using both SNP array data and Poolseq SNPs, we characterized the population structure of these samples. We observed the greatest separation between the Asian and African populations and greater admixture in the Asian populations than in the African ones. We also established that the SNP array data were able to successfully resolve relationships between these diverse Nile tilapia populations. The Poolseq data identified genomic regions with high levels of differentiation (FST) between GIFTw and the other populations. Gene ontology terms associated with mesoderm development were significantly enriched in the genes located in these regions. A region on chromosome Oni06 was genetically differentiated in pairwise comparisons between GIFTw and all other populations. This region contains genes associated with muscle‐related traits and overlaps with a previously published QTL for fillet yield, suggesting that these traits may have been direct targets for selection on GIFT. A nearby region was also identified using XP‐EHH to detect genomic differentiation using the SNP array data. Genomic regions with high or extended homozygosity within each population were also identified. This study provides putative genomic landmarks associated with the recent domestication process in several Nile tilapia populations, which could help to inform their genetic management and improvement.

Abstract Batrachochytrium salamandrivorans (Bsal) is a fungal pathogen of amphibians that is emerging in Europe and could be introduced to North America through international trade or other pathways. To evaluate the risk of Bsal invasion to amphibian biodiversity, we performed dose-response experiments on 35 North American species from 10 families, including larvae from five species. We discovered that Bsal caused infection in 74% and mortality in 35% of species tested. Both salamanders and frogs became infected and developed Bsal chytridiomycosis. Based on our host susceptibility results, environmental suitability conditions for Bsal, and geographic ranges of salamanders in the United States, predicted biodiversity loss is expected to be greatest in the Appalachian Region and along the West Coast. Indices of infection and disease susceptibility suggest that North American amphibian species span a spectrum of vulnerability to Bsal chytridiomycosis and most amphibian communities will include an assemblage of resistant, carrier, and amplification species. Predicted salamander losses could exceed 80 species in the United States and 140 species in North America.

Modern societies rely heavily on centralized industrial processes to generate a multitude of products ranging from electrical energy to synthetic chemical building blocks to construction materials. To date, these processes have relied extensively on energy produced from fossil fuels, which has led to dramatically increased quantities of greenhouse gases (including carbon dioxide) being released into the atmosphere; the effects of the ensuing change to our climate are easily observed in day-to-day life. Some of the reactions catalyzed by these industrial processes can be catalyzed in nature by metal-containing enzymes (metalloenzymes) that have evolved over the course of up to 3.8 billion years to do so under mild physiological conditions using Earth-abundant metals. While such metalloenzymes could in principle facilitate the implementation of carbon-neutral processes around the globe, either in “bio-inspired” catalyst design or even by direct exploitation, many remaining questions surrounding their mechanisms often preclude both options. Here, our recent efforts in understanding and applying metalloenzymes that catalyze reactions such as dinitrogen reduction to ammonia or proton reduction to molecular hydrogen are discussed. In closing, an opinion on the question: “Can these types of enzymes really be used in new biotechnologies?” is offered.

BackgroundLow skeletal muscle mass and density, as assessed by CT-body composition (CT-BC), are recognised to have prognostic value in non-cancer and cancer patients. The aim of the present study was to compare CT-BC parameters between non-cancer (abdominal aortic aneurysm, AAA) and cancer (colorectal cancer, CRC) patients.MethodsTwo retrospective multicentre cohorts were compared. Thresholds of visceral fat area (VFA, Doyle), skeletal fat index (SFI, Ebadi), skeletal muscle index (SMI, Martin), and skeletal muscle density (SMD, Martin) were applied to these cohorts and compared. The systemic inflammatory response (SIR) was measured by the systemic inflammatory grade (SIG).Results1695 patients were included; 759 patients with AAA and 936 patients with CRC. Low SMD (33% vs. 66%, p 2 (OR 1.37, 95% CI 1.08-1.73, p = 0.01), and elevated SIG (OR 1.19, 95% CI 1.03-1.37, p = 0.02) were independently associated with increased odds of low SMD.ConclusionsIncreasing age and systemic inflammation appear to be important determinants of loss of skeletal muscle mass and quality irrespective of disease.

Abstract Blue mussels from the genus Mytilus are an abundant component of the benthic community, found in the high latitude habitats. These foundation species are relevant to the aquaculture industry, with over 2 million tonnes produced globally each year. Mussels withstand a wide range of environmental conditions and species from the Mytilus edulis complex readily hybridize in regions where their distributions overlap. Significant effort has been made to investigate the consequences of environmental stress on mussel physiology, reproductive isolation, and local adaptation. Yet our understanding on the genomic mechanisms underlying such processes remains limited. In this study, we developed a multi species medium‐density 60 K SNP‐array including four species of the Mytilus genus. SNPs included in the platform were called from 138 mussels from 23 globally distributed mussel populations, sequenced using a whole‐genome low coverage approach. The array contains polymorphic SNPs which capture the genetic diversity present in mussel populations thriving across a gradient of environmental conditions (~59 K SNPs) and a set of published and validated SNPs informative for species identification and for diagnosis of transmissible cancer (610 SNPs). The array will allow the consistent genotyping of individuals, facilitating the investigation of ecological and evolutionary processes in these taxa. The applications of this array extend to shellfish aquaculture, contributing to the optimization of this industry via genomic selection of blue mussels, parentage assignment, inbreeding assessment and traceability. Further applications such as genome wide association studies (GWAS) for key production traits and those related to environmental resilience are especially relevant to safeguard aquaculture production under climate change.

Abstract High vagal nerve activity, reliability measured by HRV, is considered protective in cancer, reducing oxidative stress, inflammation and opposing sympathetic nerve activity. The present monocentric study examines the relationship between HRV, TNM stage, co-morbidity, systemic inflammation and survival in patients who underwent potentially curative resections for colorectal cancer (CRC). Time-domain HRV measures, Standard Deviation of NN-intervals (SDNN) and Root Mean Square of Successive Differences (RMSSD), were examined as categorical (median) and continuous variables. Systemic inflammation was determined using systemic inflammatory grade (SIG) and co-morbidity using ASA. The primary end point was overall survival (OS) and was analysed using Cox regression. There were 439 patients included in the study and the median follow-up was 78 months. Forty-nine percent (n = 217) and 48% (n = 213) of patients were categorised as having low SDNN (

Abstract Saccharomyces cerevisiae is a very well studied organism, yet ∼20% of its proteins remain poorly characterized. Moreover, recent studies seem to indicate that the pace of functional discovery is slow. Previous work has implied that the most probable path forward is via not only automation but fully autonomous systems in which active learning is applied to guide high-throughput experimentation. Development of tools and methods for these types of systems is of paramount importance. In this study we use constrained dynamical flux balance analysis (dFBA) to select ten regulatory deletant strains that are likely to have previously unexplored connections to the diauxic shift. We then analyzed these deletant strains using untargeted metabolomics, generating profiles which were then subsequently investigated to better understand the consequences of the gene deletions in the metabolic reconfiguration of the diauxic shift. We show that metabolic profiles can be utilised to not only gaining insight into cellular transformations such as the diauxic shift, but also on regulatory roles and biological consequences of regulatory gene deletion. We also conclude that untargeted metabolomics is a useful tool for guidance in high-throughput model improvement, and is a fast, sensitive and informative approach appropriate for future large-scale functional analyses of genes. Moreover, it is well-suited for automated approaches due to relative simplicity of processing and the potential to make massively high-throughput.

Abstract Background Infectious Salmon Anaemia Virus (ISAV) is an Orthomixovirus that represents a large problem for salmonid aquaculture worldwide. Current prevention and treatment methods are only partially effective. Genetic selection and genome engineering have the potential to develop ISAV resistant salmon stocks. Both strategies can benefit from an improved understanding of the genomic regulation of ISAV pathogenesis. Here, we used single-cell RNA sequencing of an Atlantic salmon cell line to provide the first high dimensional insight into the transcriptional landscape that underpins host-virus interaction during early ISAV infection. Results Salmon head kidney (SHK-1) cells were single-cell RNA sequenced at 24, 48 and 96 h post-ISAV challenge. At 24 h post infection, cells showed expression signatures consistent with viral entry, with genes such as PI3K, FAK or JNK being upregulated relative to uninfected cells. At 48 and 96 h, infected cells showed a clear anti-viral response, characterised by the expression of IFNA2 or IRF2. Uninfected bystander cells at 48 and 96 h also showed clear transcriptional differences, potentially suggesting paracrine signalling from infected cells. These bystander cells expressed pathways such as mRNA sensing, RNA degradation, ubiquitination or proteasome; and up-regulation of mitochondrial ribosome genes also seemed to play a role in the host response to the infection. Correlation between viral and host genes revealed novel genes potentially key for this fish-virus interaction. Conclusions This study has increased our understanding of the cellular response of Atlantic salmon during ISAV infection and revealed host-virus interactions at the cellular level. Our results highlight various potential key genes in this host-virus interaction, which can be manipulated in future functional studies to increase the resistance of Atlantic salmon to ISAV.

The unique orientation of the orbits in the extinct hypercarnivorous sabertooth Thylacosmilus atrox shows that it was able to achieve binocular depth perception even though its cranium evolved for the growth and accommodation of its enlarged canines

The freshwater crayfish, Procambarus clarkii is an excellent aquatic animal model that is highly adaptable and tolerant. P. clarkii is widely used as a toxicity model to study various pharmaceutical exposure. This animal model has complex behavioral traits and is considered sensitive to environmental changes, making it an excellent candidate to study psychoactive drugs based on a behavioral approach. However, up to now, most behavioral studies on crayfish use manual observation and scoring that require panelists. In this study, we aim to develop an automation pipeline to analyze crayfish behavior automatically. We use a deep-learning approach to label body parts in multiple crayfish, and based on the trajectory results, the intra- or inter-individual crayfish were calculated. Reliable and fast results of several behavior endpoints in multiple crayfish were retrieved. We then validated the detection performance of numerous crayfish in specific gender groups (male-male and female-female). Based on the result, the male crayfish displayed significantly higher aggression than females. We also tested the antidepressant exposure on this animal model to evaluate the psychoactive effects of this drug. As male crayfish display more distinct agonistic behavior than females, we exposed them to sertraline (SRT) 1 ppb for 7 and 14 days. It was revealed that sertraline was able to alter several behavioral endpoints in crayfish. Significant increases in extend claw ratio, total distance moved, average speed, and rapid movement were displayed in sertraline-exposed crayfish but decreased interaction time and longest interaction time. In addition, SRT 14 days exposure could atler the aggressiveness and bold behavior In the present method, DeepLabCut (DLC) has been utilized to analyze the locomotion behavior of multiple crayfish. This established method provides rapid and accurate ecotoxicity measurements using freshwater crayfish, which beneficient and applicable for environmental research.

Summary: The mechanism by which morphogenetic signals engage the regulatory networks responsible for early embryonic tissue patterning is incompletely understood. Here, we developed a minimal gene regulatory network (GRN) model of human pluripotent stem cell (hPSC) lineage commitment and embedded it into “cellular” agents that respond to a dynamic morphogenetic signaling microenvironment. Simulations demonstrated that GRN wiring had significant non-intuitive effects on tissue pattern order, composition, and dynamics. Experimental perturbation of GRN connectivities supported model predictions and demonstrated the role of OCT4 as a master regulator of peri-gastrulation fates. Our so-called GARMEN strategy provides a multiscale computational platform to understand how single-cell-based regulatory interactions scale to tissue domains. This foundation provides new opportunities to simulate the impact of network motifs on normal and aberrant tissue development.

Abstract Background While the current literature suggests an association with frailty and clinical outcomes in patients undergoing surgery for colorectal cancer (CRC), the basis of this relationship is unclear. Aim Examine the relationship between frailty, malnutrition, body composition, systemic inflammation and short-term clinical outcomes in patients undergoing surgery for colorectal cancer. Methods Consecutive patients who underwent potentially curative resection for colorectal cancer, between April 2008 and April 2018, were identified from a prospectively maintained database. Frailty was defined using the modified five-item frailty index (mFI-5). Body composition measures included CT-derived skeletal muscle index (SMI) and density (SMD). Systemic inflammatory status was determined using Systemic Inflammatory Grade (SIG). Outcomes of interest were the incidence of post-operative complications and thirty-day mortality. Associations between categorical variables were examined using χ2 test and binary logistics regression analysis. Results 1002 patients met the inclusion criteria. 28% (n = 221) scored 2 or more on the mFI-5. 39% (n = 388) of patients had a post-operative complication (Clavien-Dindo I-IV) and 1% (n = 11) died within thirty days of surgery. On univariate analysis, mFI-5 frailty score, was significantly associated with advanced age (p

Abstract Background Infectious Salmon Anaemia virus (ISAV) is an orthomyxovirus responsible for large losses in Atlantic salmon (Salmo salar) aquaculture. Current available treatments and vaccines are not fully effective, and therefore selective breeding to produce ISAV-resistant strains of Atlantic salmon is a high priority for the industry. Genomic selection and potentially genome editing can be applied to enhance the disease resistance of aquaculture stocks, and both approaches can benefit from increased knowledge on the genomic mechanisms of resistance to ISAV. To improve our understanding of the mechanisms underlying resistance to ISAV in Atlantic salmon we performed a transcriptomic study in ISAV-infected salmon with contrasting levels of resistance to this virus. Results Three different tissues (gills, head kidney and spleen) were collected on 12 resistant and 12 susceptible fish at three timepoints (pre-challenge, 7 and 14 days post challenge) and RNA sequenced. The transcriptomes of infected and non-infected fish and of resistant and susceptible fish were compared at each timepoint. The results show that the responses to ISAV are organ-specific; an important response to the infection was observed in the head kidney, with up-regulation of immune processes such as interferon and NLR pathways, while in gills and spleen the response was more moderate. In addition to immune related genes, our results suggest that other processes such as ubiquitination and ribosomal processing are important during early infection with ISAV. Moreover, the comparison between resistant and susceptible fish has also highlighted some interesting genes related to ubiquitination, intracellular transport and the inflammasome. Conclusions Atlantic salmon infection by ISAV revealed an organ-specific response, implying differential function during the infection. An immune response was observed in the head kidney in these early timepoints, while gills and spleen showed modest responses in comparison. Comparison between resistance and susceptible samples have highlighted genes of interest for further studies, for instance those related to ubiquitination or the inflammasome.

Abstract During development, cell state transitions are coordinated through changes in the identity of molecular regulators in a cell type‐ and dose‐specific manner. The ability to rationally engineer such transitions in human pluripotent stem cells (hPSC) will enable numerous applications in regenerative medicine. Herein, we report the generation of synthetic gene circuits that can detect a desired cell state using AND‐like logic integration of endogenous miRNAs (classifiers) and, upon detection, produce fine‐tuned levels of output proteins using an miRNA‐mediated output fine‐tuning technology (miSFITs). Specifically, we created an “hPSC ON” circuit using a model‐guided miRNA selection and circuit optimization approach. The circuit demonstrates robust PSC‐specific detection and graded output protein production. Next, we used an empirical approach to create an “hPSC‐Off” circuit. This circuit was applied to regulate the secretion of endogenous BMP4 in a state‐specific and fine‐tuned manner to control the composition of differentiating hPSCs. Our work provides a platform for customized cell state‐specific control of desired physiological factors in hPSC, laying the foundation for programming cell compositions in hPSC‐derived tissues and beyond.

Abstract The European flat oyster (Ostrea edulis L.) is a bivalve naturally distributed across Europe, which was an integral part of human diets for centuries, until anthropogenic activities and disease outbreaks severely reduced wild populations. Despite a growing interest in genetic applications to support population management and aquaculture, a reference genome for this species is lacking to date. Here, we report a chromosome‐level assembly and annotation for the European Flat oyster genome, generated using Oxford Nanopore, Illumina, Dovetail OmniC™ proximity ligation and RNA sequencing. A contig assembly (N50: 2.38 Mb) was scaffolded into the expected karyotype of 10 pseudochromosomes. The final assembly is 935.13 Mb, with a scaffold‐N50 of 95.56 Mb, with a predicted repeat landscape dominated by unclassified elements specific to O. edulis. The assembly was verified for accuracy and completeness using multiple approaches, including a novel linkage map built with ddRAD‐Seq technology, comprising 4016 SNPs from four full‐sib families (eight parents and 163 F1 offspring). Annotation of the genome integrating multitissue transcriptome data, comparative protein evidence and ab‐initio gene prediction identified 35,699 protein‐coding genes. Chromosome‐level synteny was demonstrated against multiple high‐quality bivalve genome assemblies, including an O. edulis genome generated independently for a French O. edulis individual. Comparative genomics was used to characterize gene family expansions during Ostrea evolution that potentially facilitated adaptation. This new reference genome for European flat oyster will enable high‐resolution genomics in support of conservation and aquaculture initiatives, and improves our understanding of bivalve genome evolution.

Abstract This volume of Evolutionary Applications sees the publication of two genomes for the European native flat oyster Ostrea edulis, a species of significant evolutionary, ecological and commercial value. Each is a highly contiguous chromosome‐level assembly from individuals of different genetic backgrounds, which have been benchmarked against one another. This situation has resulted from the serendipitous discovery that two independent research groups were both deep into the process of building, annotating and investigating separately produced assemblies. Due to constraints with funder requirements and the need to recognize early career researchers for their work, alongside the technical challenge of integrating assemblies from two very different genomes, there was limited capacity to merge the sequences into one publication at the stage of discovery. This issue is likely to become very common over the next few years until the technologies for working with multiple genomes at once, for example, graph genomes, become commonplace in nonmodel species. Consequently, both of our teams have decided to collaborate rather than compete, recognizing the benefit to copublishing two separate genome resources for the research community, each with distinct scientific investigations, and working collaboratively to benchmark the assemblies.

Abstract European flat oyster (Ostrea edulis) is an ecologically and economically important marine bivalve, that has been severely affected by the intracellular parasite Bonamia ostreae. In this study, a flat oyster SNP array (~14,000 SNPs) was used to validate previously reported outlier loci for divergent selection associated with B. ostreae exposure in the Northeast Atlantic Area. A total of 134 wild and hatchery individuals from the North Sea, collected in naïve (NV) and long‐term affected (LTA) areas, were analysed. Genetic diversity and differentiation were related to the sampling origin (wild vs. hatchery) when using neutral markers, and to bonamiosis status (NV vs. LTA) when using outlier loci for divergent selection. Two genetic clusters appeared intermingled in all sampling locations when using outlier loci, and their frequency was associated with their bonamiosis status. When both clusters were compared, outlier data sets showed high genetic divergence (FST > 0.25) unlike neutral loci (FST not ≠ 0). Moreover, the cluster associated with LTA samples showed much higher genetic diversity and significant heterozygote excess with outlier loci, but not with neutral data. Most outliers mapped on chromosome 8 (OE‐C8) of the flat oyster genome, supporting a main genomic region underlying resilience to bonamiosis. Furthermore, differentially expressed genes previously reported between NV and LTA strains showed higher mapping density on OE‐C8. A range of relevant immune functions were specifically enriched among genes annotated on OE‐C8, providing hypotheses for resilience mechanisms to an intracellular parasite. The results suggest that marker‐assisted selection could be applied to breed resilient strains of O. edulis to bonamiosis, if lower parasite load and/or higher viability of the LTA genetic cluster following B. ostreae infection is demonstrated.

Background The tumor microenvironment (TME) contributes to cancer progression and treatment response to therapy, including in renal cell carcinoma (RCC). Prior profiling studies, including single-cell transcriptomics, often involve limited sample sizes and lack spatial orientation. The TME of RCC brain metastases, a major cause of morbidity, also remains largely uncharacterized.Methods We performed digital spatial profiling on the NanoString GeoMx platform using 52 validated immuno-oncology markers on RCC tissue microarrays representing progressive stages of RCC, including brain metastases. We profiled 76 primary tumors, 27 adjacent histologically normal kidney samples, and 86 metastases, including 24 brain metastases.Results We observed lower immune checkpoint (TIM-3 and CTLA-4), cytolytic (GZMA and GZMB), and T cell activation (CD25) protein expression in metastases compared with primary tumors in two separate cohorts. We also identified changes in macrophages in metastases, with brain metastases-susceptible patients showing less M1-like, inflammatory macrophage markers (HLA-DR and CD127) in metastatic samples. A comparison of brain metastases to extracranial metastases revealed higher expression of the anti-apoptotic, BCL-2-family protein BCL-XL and lower expression of the innate immune activator STING in brain metastases. Lower TIM-3 and CD40 in the TME of brain metastases appear to be associated with longer survival, a finding that requires further validation.Conclusions Compared with primary tumors, RCC metastases, including brain metastases, express lower levels of numerous markers of immune activation and current or investigational therapeutic targets. Our findings may have important implications for designing future biomarker and treatment studies and may aid in development of brain metastases-specific therapies.

Immune checkpoint inhibitors (ICIs) are increasingly being used to manage multiple tumor types. Unfortunately, immune-related adverse events affect up to 60% of recipients, often leading to treatment discontinuation in settings where few alternative cancer therapies may be available. Checkpoint inhibitor induced colitis (ICI-colitis) is a common toxicity for which the underlying mechanisms are poorly defined. To better understand the changing colon-specific and peripheral immune environments over the course of progression and treatment of colitis, we collected blood and colon tissue from a patient with Merkel cell carcinoma who developed colitis on treatment with pembrolizumab. We performed single-cell RNA sequencing and T-cell receptor sequencing on samples collected before, during and after pembrolizumab and after various interventions to mitigate toxicity. We report T-cells populations defined by cytotoxicity, memory, and proliferation markers at various stages of colitis. We show preferential depletion of CD8+ T cells with biologic therapy and nominate both circulating and colon-resident T-cell subsets as potential drivers of inflammation and response to immune suppression. Our findings highlight the need for further exploration of the colon immune environment and rationalize future studies evaluating biologics for ICI-colitis, including in the context of ICI re-challenge.