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Improved RNA virus understanding is critical to studying animal and plant health, and environmental processes. However, the continuous and rapid RNA virus evolution makes their identification and characterization challenging. While recent sequence-based advances have led to extensive RNA virus discovery, there is growing variation in how RNA viruses are identified, analyzed, characterized, and reported. To this end, an RdRp Summit was organized and a hybrid meeting took place in Valencia, Spain in May 2023 to convene leading experts with emphasis on early career researchers (ECRs) across diverse scientific communities. Here we synthesize key insights and recommendations and offer these as a first effort to establish a consensus framework for advancing RNA virus discovery. First, we need interoperability through standardized methodologies, data-sharing protocols, metadata provision and interdisciplinary collaborations and offer specific examples as starting points. Second, as an emergent field, we recognize the need to incorporate cutting-edge technologies and knowledge early and often to improve omic-based viral detection and annotation as novel capabilities reveal new biology. Third, we underscore the significance of ECRs in fostering international partnerships to promote inclusivity and equity in virus discovery efforts. The proposed consensus framework serves as a roadmap for the scientific community to collectively contribute to the tremendous challenge of unveiling the RNA virosphere.

Classical metabolomic and new metabolic network methods were used to study the developmental features of autism spectrum disorder (ASD) in newborns (n = 205) and 5-year-old children (n = 53). Eighty percent of the metabolic impact in ASD was caused by 14 shared biochemical pathways that led to decreased anti-inflammatory and antioxidant defenses, and to increased physiologic stress molecules like lactate, glycerol, cholesterol, and ceramides. CIRCOS plots and a new metabolic network parameter, V° net, revealed differences in both the kind and degree of network connectivity. Of 50 biochemical pathways and 450 polar and lipid metabolites examined, the developmental regulation of the purine network was most changed. Purine network hub analysis revealed a 17-fold reversal in typically developing children. This purine network reversal did not occur in ASD. These results revealed previously unknown metabolic phenotypes, identified new developmental states of the metabolic correlation network, and underscored the role of mitochondrial functional changes, purine metabolism, and purinergic signaling in autism spectrum disorder.

Escalating vector disease burdens pose significant global health risks, as such innovative tools for targeting mosquitoes are critical. CRISPR-Cas technologies have played a crucial role in developing powerful tools for genome manipulation in various eukaryotic organisms. Although considerable efforts have focused on utilizing class II type II CRISPR-Cas9 systems for DNA targeting, these modalities are unable to target RNA molecules, limiting their utility against RNA viruses. Recently, the Cas13 family has emerged as an efficient tool for RNA targeting; however, the application of this technique in mosquitoes, particularly Aedes aegypti, has yet to be fully realized. In this study, we engineered an antiviral strategy termed REAPER (vRNA Expression Activates Poisonous Effector Ribonuclease) that leverages the programmable RNA-targeting capabilities of CRISPR-Cas13 and its potent collateral activity. REAPER remains concealed within the mosquito until an infectious blood meal is uptaken. Upon target viral RNA infection, REAPER activates, triggering programmed destruction of its target arbovirus such as chikungunya. Consequently, Cas13-mediated RNA targeting significantly reduces viral replication and viral prevalence of infection, and its promiscuous collateral activity can even kill infected mosquitoes within a few days. This innovative REAPER technology adds to an arsenal of effective molecular genetic tools to combat mosquito virus transmission.

Abstract The spread of carbapenemase-producing gram-negative pathogens, especially those producing metallo-β-lactamases (MBLs), has become a major health concern. MBLs are molecularly the most diverse carbapenemases, produced by a wide spectrum of gram-negative organisms, including the Enterobacterales, Pseudomonas spp., Acinetobacter baumannii, and Stenotrophomonas maltophilia, and can hydrolyze most β-lactams using metal ion cofactors in their active sites. Over the years, the prevalence of MBL-carrying isolates has increased globally, particularly in Asia. MBL infections are associated with adverse clinical outcomes including longer length of hospital stay, ICU admission, and increased mortality across the globe. The optimal treatment for MBL infections not only depends on the pathogen but also on the underlying resistance mechanisms. Currently, there are only few drugs or drug combinations that can efficiently offset MBL-mediated resistance, which makes the treatment of MBL infections challenging. The rising concern of MBLs along with the limited treatment options has led to the need and development of drugs that are specifically targeted towards MBLs. This review discusses the prevalence of MBLs, their clinical impact, and the current treatment options for MBL infections and their limitations. Furthermore, this review will discuss agents currently in the pipeline for treatment of MBL infections.

Abstract Background The incidence of Pulmonary Hypertension (PH) and Pulmonary Arterial Hypertension (PAH) is believed to be on the rise and is associated with poor outcomes. Methods We extracted age-standardized mortality rates (ASMRs) for decedents ≥ 18 years of age from the World Health Organization Mortality Database, using International Classification of Diseases 10th edition codes for PH and PAH, covering the period from 2001 to 2019. The UK and European Union countries with at least 1,000,000 inhabitants and at least 75% of available data points over the study period were included. Results Between 2001 and 2019, in countries with available data, the median ASMR for PH increased by + 1.19 per 1,000,000 (+ 22.51%) in females and + 0.36 per 1,000,000 (+ 6.06%) in males. Out of 19 countries, 13 demonstrate an increase in female PH ASMR, and 12 reported an increase in male PH ASMR. In contrast, median PAH ASMR decreased by -0.29 per 1,000,000 (-28.74%) in females and remained relatively unchanged in males, with a minor increase of + 0.01 per 1,000,000 (+ 1.07%). Notably, there was significant inter-country heterogeneity, with countries such as Hungary, Romania, and Poland displaying results incongruous with the rest of Europe. Conclusions While publicly available mortality statistics for PH may be unreliable, these data suggest an overall increase in mortality across Europe from 2001 to 2019. However, mortality from PAH has shown a decrease in females and a modest increase in males. This underscores the urgent need for robust and high-quality mortality reporting, including international registries, for both PH and PAH.

A lesser tuberosity osteotomy (LTO) is commonly performed during total shoulder arthroplasty to access the glenohumeral joint. Healing of the LTO is critical to optimizing the outcome of the procedure and is enhanced by a repair that provides stability and compression across the osteotomy site. The purpose of this article is to describe a technique that uses a tensionable suture construct to repair the LTO during anatomic total shoulder arthroplasty using a stemless humeral component. The technique involves passing a row of high-tensile sutures through bone tunnels lateral to the osteotomy site (transosseous sutures) and another row of sutures through the humeral implant (implant sutures). One limb of each bone tunnel suture is then tied to its corresponding limb of implant suture and the remaining free strands of the tied sutures are manually tensioned and tied to each other. This technique is an efficient and reproducible method for creating compression and stability across the osteotomy site that facilitates bony healing.

Abstract We aimed to characterize the cognitive profile of post-acute COVID-19 syndrome (PACS) patients with cognitive complaints, exploring the influence of biological and psychological factors. Participants with confirmed SARS-CoV-2 infection and cognitive complaints ≥ 8 weeks post-acute phase were included. A comprehensive neuropsychological battery (NPS) and health questionnaires were administered at inclusion and at 1, 3 and 6 months. Blood samples were collected at each visit, MRI scan at baseline and at 6 months, and, optionally, cerebrospinal fluid. Cognitive features were analyzed in relation to clinical, neuroimaging, and biochemical markers at inclusion and follow-up. Forty-nine participants, with a mean time from symptom onset of 10.4 months, showed attention-executive function (69%) and verbal memory (39%) impairment. Apathy (64%), moderate-severe anxiety (57%), and severe fatigue (35%) were prevalent. Visual memory (8%) correlated with total gray matter (GM) and subcortical GM volume. Neuronal damage and inflammation markers were within normal limits. Over time, cognitive test scores, depression, apathy, anxiety scores, MRI indexes, and fluid biomarkers remained stable, although fewer participants (50% vs. 75.5%; p = 0.012) exhibited abnormal cognitive evaluations at follow-up. Altered attention/executive and verbal memory, common in PACS, persisted in most subjects without association with structural abnormalities, elevated cytokines, or neuronal damage markers.

Postgraduate medical education (PGME) is an essential part of medical education and increasingly shifts into focus of educational stakeholders. Structured postgraduate medical training programs are required in the U.S. through the “American Council for Graduate Medical Education” (ACGME) guidelines with their six core competencies as common program requirements. The basis for this development was provided in Germany with the implementation of the “Standard Framework for Postgraduate Medical Training” (Musterweiterbildungsordnung) issued by the German Medical Association (Bundesärztekammer). However, implementation has been gradual and program development is often conducted in a time consuming, lengthy and top-down approach without that trainee experiences or needs are being assessed or evaluated for their impact on successful medical training.We demonstrate how application of agile working can enable rapid and efficient creation and implementation of a novel postgraduate training program. The postgraduate training program ped.tracks aims to achieve a high-quality, structured and reliable postgraduate training. Moreover, it provides the opportunity to select a priority on scientific or clinical education. The entire process from the first draft to full release of the program was completed within 8 months through agile working. Our team worked using agile working techniques, creating a trainee- customized and -centred program. We anticipate that the quantity of structured postgraduate training programs will significantly increase in Germany and Europe to improve training quality and employee satisfaction. Therefore, the use of agile methods for the creation and implementation of structured training programs represents a useful approach to support program directors rapidly and effectively in this effort.

Abstract Classical metabolomic and new metabolic network methods were used to study the developmental features of autism spectrum disorder (ASD) in newborns (n = 205) and 5-year-old children (n = 53). Eighty percent of the metabolic impact in ASD was caused by 14 shared biochemical pathways that led to decreased anti-inflammatory and antioxidant defenses, and to increased physiologic stress molecules like lactate, glycerol, cholesterol, and ceramides. CIRCOS plots and a new metabolic network parameter, $$\dot{{{\boldsymbol{V}}}}\!$$ V ° net , revealed differences in both the kind and degree of network connectivity. Of 50 biochemical pathways and 450 polar and lipid metabolites examined, the developmental regulation of the purine network was most changed. Purine network hub analysis revealed a 17-fold reversal in typically developing children. This purine network reversal did not occur in ASD. These results revealed previously unknown metabolic phenotypes, identified new developmental states of the metabolic correlation network, and underscored the role of mitochondrial functional changes, purine metabolism, and purinergic signaling in autism spectrum disorder.

Abstract Background Cutaneous squamous cell carcinoma (CSCC) has a propensity for perineural spread (PNS) which is associated with poorer treatment outcomes. Immunotherapy is the new standard of care treatment for advanced CSCC resulting in durable responses. PNS is not captured by traditional response assessment criteria used in clinical trials, e.g. RECIST 1.1, and there is limited literature documenting radiological PNS responses to immunotherapy. In this study we assess PNS responses to immunotherapy using a modified grading system. Methods This is an Australian single-center retrospective review of patients with advanced CSCC who were treated with immunotherapy between April 2018 and February 2022 who had evidence of PNS on pre-treatment magnetic-resonance imaging (MRI). The primary outcome was blinded overall radiological response in PNS using graded radiological criteria, post-commencement of immunotherapy. Three defined timepoints ( 10 months) were reviewed. Secondary outcomes included a correlation between RECIST 1.1 and PNS assessments and the assessment of PNS on fluorodeoxyglucose (FDG)-positron emission tomography (PET)/computed tomography (CT). Results Twenty CSCC patients treated with immunotherapy were identified. Median age was 75.7 years and 75% (n = 15) were male. All patients had locoregionally advanced disease and no distant metastases. Median follow-up was 18.5 months (range: 2–59). 70% (n = 14) demonstrated a PNS response by 5 months. Three patients experienced pseudoprogression. One patient had PNS progression by the end of study follow up. RECIST 1.1 and PNS responses were largely concordant at > 10 months (Cohen’s Kappa 0.62). 5/14 cases had features suspicious for PNS on FDG-PET/CT. Conclusions PNS response to immunotherapy can be documented on MRI using graded radiological criteria. High response rates were seen in PNS with the use of immunotherapy in this cohort and these responses were largely concordant with RECIST 1.1 assessments. FDG-PET/CT demonstrated limited sensitivity in the detection of PNS.

We previously reported that nonobese diabetic (NOD) congenic mice (NOD.c3c4 mice) developed an autoimmune biliary disease (ABD) with similarities to human primary biliary cholangitis (PBC), including anti-mitochondrial antibodies and organ-specific biliary lymphocytic infiltrates. We narrowed the possible contributory regions in a novel NOD.Abd3 congenic mouse to a B10 congenic region on chromosome 1 ("Abd3") and a mutated Pkhd1 gene (Pkhd1del36-67) upstream from Abd3, and we showed via backcrossing studies that the NOD genetic background was necessary for disease. Here, we show that NOD.Abd3 mice develop anti-PDC-E2 autoantibodies at high levels, and that placing the chromosome 1 interval onto a scid background eliminates disease, demonstrating the critical role of the adaptive immune system in pathogenesis. While the NOD genetic background is essential for disease, it was still unclear which of the two regions in the Abd3 locus were necessary and sufficient for disease. Here, using a classic recombinant breeding approach, we prove that the mutated Pkhd1del36-67 alone, on the NOD background, causes ABD. Further characterization of the mutant sequence demonstrated that the Pkhd1 gene is disrupted by an ETnII-beta retrotransposon inserted in intron 35 in an anti-sense orientation. Homozygous Pkhd1 mutations significantly affect viability, with the offspring skewed away from a Mendelian distribution towards NOD Pkhd1 homozygous or heterozygous genotypes. Cell-specific abnormalities, on a susceptible genetic background, can therefore induce an organ-specific autoimmunity directed to the affected cells. Future work will aim to characterize how mutant Pkhd1 can cause such an autoimmune response.

BackgroundManagement of adults with atrial fibrillation (AF) or atrial flutter in the emergency department (ED) includes rate reduction, cardioversion, and stroke prevention. Different approaches to these components of care may lead to variation in frequency of hospitalization and stroke prevention actions, with significant implications for patient experience, cost of care, and risk of complications. Standardization using evidence-based recommendations could reduce variation in management, preventable hospitalizations, and stroke risk.MethodsWe describe the rationale for our ED-based AF treatment recommendations. We also describe the development of an electronic clinical decision support system (CDSS) to deliver these recommendations to emergency physicians at the point of care. We implemented the CDSS at three pilot sites to assess feasibility and solicit user feedback. We will evaluate the impact of the CDSS on hospitalization and stroke prevention actions using a stepped-wedge cluster randomized pragmatic clinical trial across 13 community EDs in Northern California.DiscussionWe hypothesize that the CDSS intervention will reduce hospitalization of adults with isolated AF or atrial flutter presenting to the ED and increase anticoagulation prescription in eligible patients at the time of ED discharge and within 30 days. If our hypotheses are confirmed, the treatment protocol and CDSS could be recommended to other EDs to improve management of adults with AF or atrial flutter.Trial registrationClinicalTrials.gov NCT05009225 .  Registered on 17 August 2021.

Cysteine, the rate-controlling amino acid in cellular glutathione synthesis is imported as cystine, by the cystine/glutamate antiporter, xCT, and subsequently reduced to cysteine. As glutathione redox is important in muscle regeneration in aging, we hypothesized that xCT exerts upstream control over skeletal muscle glutathione redox, metabolism and regeneration. Bioinformatic analyses of publicly available datasets revealed that expression levels of xCT and GSH-related genes are inversely correlated with myogenic differentiation genes. Muscle satellite cells (MuSCs) isolated from Slc7a11sut/sut mice, which harbour a mutation in the Slc7a11 gene encoding xCT, required media supplementation with 2-mercaptoethanol to support cell proliferation but not myotube differentiation, despite persistently lower GSH. Slc7a11sut/sut primary myotubes were larger compared to WT myotubes, and also exhibited higher glucose uptake and cellular oxidative capacities. Immunostaining of myogenic markers (Pax7, MyoD, and myogenin) in cardiotoxin-damaged tibialis anterior muscle fibres revealed greater MuSC activation and commitment to differentiation in Slc7a11sut/sut muscle compared to WT mice, culminating in larger myofiber cross-sectional areas at 21 days post-injury. Slc7a11sut/sut mice subjected to a 5-week exercise training protocol demonstrated enhanced insulin tolerance compared to WT mice, but blunted muscle mitochondrial biogenesis and respiration in response to exercise training. Our results demonstrate that the absence of xCT inhibits cell proliferation but promotes myotube differentiation by regulating cellular metabolism and glutathione redox. Altogether, these results support the notion that myogenesis is a redox-regulated process and may help inform novel therapeutic approaches for muscle wasting and dysfunction in aging and disease.

Abstract Pulmonary arterial hypertension (PAH) is characterised by pulmonary vascular remodelling causing premature death from right heart failure. Established DNA variants influence PAH risk, but susceptibility from epigenetic changes is unknown. We addressed this through epigenome-wide association study (EWAS), testing 865,848 CpG sites for association with PAH in 429 individuals with PAH and 1226 controls. Three loci, at Cathepsin Z (CTSZ, cg04917472), Conserved oligomeric Golgi complex 6 (COG6, cg27396197), and Zinc Finger Protein 678 (ZNF678, cg03144189), reached epigenome-wide significance (p