SARS-CoV-2 population dynamics in immunocompetent individuals in a closed transmission chain shows genomic diversity over the course of infection.

BACKGROUND: SARS-CoV-2 remains rapidly evolving, and many biologically important genomic substitutions/indels have characterised novel SARS-CoV-2 lineages, which have emerged during successive global waves of the pandemic. Worldwide genomic sequencing has been able to monitor these waves, track transmission clusters, and examine viral evolution in real time to help inform healthcare policy. One school of thought is that an apparent greater than average divergence in an emerging lineage from contemporary variants may require persistent infection, for example in an immunocompromised host. Due to the nature of the COVID-19 pandemic and sampling, there were few studies that examined the evolutionary trajectory of SARS-CoV-2 in healthy individuals. METHODS: We investigated viral evolutionary trends and participant symptomatology within a cluster of 16 SARS-CoV-2 infected, immunocompetent individuals with no co-morbidities in a closed transmission chain. Longitudinal nasopharyngeal swab sampling allowed characterisation of SARS-CoV-2 intra-host variation over time at both the dominant and minor genomic variant levels through Nimagen-Illumina sequencing. RESULTS: A change in viral lineage assignment was observed in individual infections; however, there was only one indel and no evidence of recombination over the period of an acute infection. Minor and dominant genomic modifications varied between participants, with some minor genomic modifications increasing in abundance to become the dominant viral sequence during infection. CONCLUSIONS: Data from this cohort of SARS-CoV-2-infected participants demonstrated that long-term persistent infection in an immunocompromised host was not necessarily a prerequisite for generating a greater than average frequency of amino acid substitutions. Amino acid substitutions at both the dominant and minor genomic sequence level were observed in immunocompetent individuals during infection showing that viral lineage changes can occur generating viral diversity.

Modulation of a critical period for motor development in Drosophila by BK potassium channels.

Critical periods are windows of heightened plasticity occurring during neurodevelopment. Alterations in neural activity during these periods can cause long-lasting changes in the structure, connectivity, and intrinsic excitability of neurons, which may contribute to the pathology of neurodevelopmental disorders. However, endogenous regulators of critical periods remain poorly defined. Here, we study this issue using a fruit fly (Drosophila) model of an early-onset movement disorder caused by BK potassium channel gain of function (BK GOF). Deploying a genetic method to place robust expression of GOF BK channels under spatiotemporal control, we show that adult-stage neuronal expression of GOF BK channels minimally disrupts fly movement. In contrast, limiting neuronal expression of GOF BK channels to a short window during late neurodevelopment profoundly impairs locomotion and limb kinematics in resulting adult flies. During this critical period, BK GOF perturbs synaptic localization of the active zone protein Bruchpilot and reduces excitatory neurotransmission. Conversely, enhancing neural activity specifically during development rescues motor defects in BK GOF flies. Collectively, our results reveal a critical developmental period for limb control in Drosophila that is influenced by BK channels and suggest that BK GOF causes movement disorders by disrupting activity-dependent aspects of synaptic development.

Applications of Nanopore sequencing in precision cancer medicine.

Oxford Nanopore Technologies sequencing, also referred to as Nanopore sequencing, stands at the forefront of a revolution in clinical genetics, offering the potential for rapid, long read, and real-time DNA and RNA sequencing. This technology is currently making sequencing more accessible and affordable. In this comprehensive review, we explore its potential regarding precision cancer diagnostics and treatment. We encompass a critical analysis of clinical cases where Nanopore sequencing was successfully applied to identify point mutations, splice variants, gene fusions, epigenetic modifications, non-coding RNAs, and other pivotal biomarkers that defined subsequent treatment strategies. Additionally, we address the challenges of clinical applications of Nanopore sequencing and discuss the current efforts to overcome them.

Phage anti-CRISPR control by an RNA- and DNA-binding helix-turn-helix protein.

In all organisms, regulation of gene expression must be adjusted to meet cellular requirements and frequently involves helix-turn-helix (HTH) domain proteins1. For instance, in the arms race between bacteria and bacteriophages, rapid expression of phage anti-CRISPR (acr) genes upon infection enables evasion from CRISPR-Cas defence; transcription is then repressed by an HTH-domain-containing anti-CRISPR-associated (Aca) protein, probably to reduce fitness costs from excessive expression2-5. However, how a single HTH regulator adjusts anti-CRISPR production to cope with increasing phage genome copies and accumulating acr mRNA is unknown. Here we show that the HTH domain of the regulator Aca2, in addition to repressing Acr synthesis transcriptionally through DNA binding, inhibits translation of mRNAs by binding conserved RNA stem-loops and blocking ribosome access. The cryo-electron microscopy structure of the approximately 40 kDa Aca2-RNA complex demonstrates how the versatile HTH domain specifically discriminates RNA from DNA binding sites. These combined regulatory modes are widespread in the Aca2 family and facilitate CRISPR-Cas inhibition in the face of rapid phage DNA replication without toxic acr overexpression. Given the ubiquity of HTH-domain-containing proteins, it is anticipated that many more of them elicit regulatory control by dual DNA and RNA binding.

Comprehensive meta-analysis of surgical procedure for congenital diaphragmatic hernia: thoracoscopic versus open repair.

PURPOSE: Previous studies have shown a higher recurrence rate and longer operative times for thoracoscopic repair (TR) of congenital diaphragmatic hernia (CDH) compared to open repair (OR). An updated meta-analysis was conducted to re-evaluate the surgical outcomes of TR. METHODS: A comprehensive literature search comparing TR and OR in neonates was performed in accordance with the PRISMA statement (PROSPERO: CRD42020166588). RESULTS: Fourteen studies were selected for quantitative analysis, including a total of 709 patients (TR: 308 cases, OR: 401 cases). The recurrence rate was higher [Odds ratio: 4.03, 95% CI (2.21, 7.36), p < 0.001] and operative times (minutes) were longer [Mean Difference (MD): 43.96, 95% CI (24.70, 63.22), p < 0.001] for TR compared to OR. A significant reduction in the occurrence of postoperative bowel obstruction was observed in TR (5.0%) compared to OR (14.8%) [Odds ratio: 0.42, 95% CI (0.20, 0.89), p = 0.02]. CONCLUSIONS: TR remains associated with higher recurrence rates and longer operative times. However, the reduced risk of postoperative bowel obstruction suggests potential long-term benefits. This study emphasizes the importance of meticulous patient selection for TR to mitigate detrimental effects on patients with severe disease.

Risk of Delayed Percutaneous Coronary Intervention for STEMI in the Southeast United States.

Background: Emergent reperfusion by percutaneous coronary intervention (PCI) within 90 minutes of first medical contact (FMC) is indicated in patients with ST-segment elevation myocardial infarction (STEMI). However, long transport times in rural areas in the Southeast US make meeting this goal difficult. The objective of this study was to determine the number of Southeast US residents with prolonged transport times to the nearest 24/7 primary PCI (PPCI) center. Methods: A cross-sectional study of residents in the Southeastern US was conducted based on geographical and 2022 5-Year American Community Survey data. The geographic information system (GIS) ArcGIS Pro was used to estimate Emergency Medical Services (EMS) transport times for Southeast US residents to the nearest PPCI center. All 24/7 PPCI centers in North Carolina, South Carolina, Georgia, Florida, Mississippi, Alabama, and Tennessee were included in the analysis, as well as nearby PPCI centers in surrounding states. To identify those at risk of delayed FMC-to-device time, the primary outcome was defined as a >30-minute transport time, beyond which most patients would not have PCI within 90 minutes. A secondary outcome was defined as transport >60 minutes, the point at which FMC-to-device time would be >120 minutes most of the time. These cutoffs are based on national median EMS scene times and door-to-device times. Results: Within the Southeast US, we identified 62,880,528 residents and 350 PPCI centers. Nearly 11 million people living in the Southeast US reside greater than 30 minutes from a PPCI center (17.3%, 10,866,710, +/- 58,143), with 2% (1,271,522 +/- 51,858) living greater than 60 minutes from a PPCI hospital. However, most patients reside in short transport zones; 82.7% (52,013,818 +/- 98,741). Within the Southeast region, 8.4% (52/616) of counties have more than 50% of their population in a long transport zone and 42.3% (22/52) of those have more than 90% of their population in long transport areas. Conclusions: Nearly 11 million people in the Southeast US do not have access to timely PCI for STEMI care. This disparity may contribute to increased morbidity and mortality.

Adverse events related to physical restraint use in intensive care units: A review of the literature.

Physical restraints are widely used and accepted as protective measures during treatment in intensive care unit (ICU). This review of the literature summarizes the adverse events and outcomes associated with physical restraint use, and the risk factors associated with their use during treatment in the ICU. The PubMed, Scopus, and Google Scholar databases were screened using predefined search terms to identify studies pertaining to adverse events and/or outcomes associated with physical restraint use, and the factors associated with their use in adult patients admitted to the ICU. A total of 24 articles (including 6126 patients) that were published between 2006 and 2022 were identified. The described adverse events associated with physical restraint use included skin injuries, subsequent delirium, neurofunctional impairment, and a higher rate of post-traumatic stress disorder. Subsequent delirium was the most frequent adverse event to be reported. No alternative measures to physical restraints were discussed, and only one study reported a standardized protocol for their use. Although physical restraint use has been reported to be associated with adverse events (including neurofunctional impairment) in the literature, the available evidence is limited. Although causality cannot be confirmed, a definite association appears to exist. Our findings suggest that it is essential to improve awareness regarding their adverse impact and optimize approaches for their detection, management, and prevention using protocols or checklists.

The free vastus lateralis-And conjoined vastus lateralis anterolateral thigh/tensor fascia lata flap for oncological chest wall reconstruction.

INTRODUCTION: A reconstructive option for extensive chest wall reconstruction is the free myocutaneous vastus lateralis muscle (VL) flap which can be performed in isolation or in conjunction with a fasciocutaneus anterolateral thigh (cVLALT) and/or myofasciocutaneous tensor fascia lata flap (cVLTFL). We aimed to directly compare the outcomes of these reconstructive options. METHODS: Patients who underwent oncological chest wall reconstruction with a free VL, cVLALT, or cVLTFL flap between February 2010 and 2022 were included in this retrospective study. Patient demographics, surgical characteristics, as well as medical and reconstructive outcomes, were evaluated. The operative outcomes between myocutaneous VL, cVLALT, and cVLTFL flap reconstructions were compared. RESULTS: A total of 41 patients underwent chest wall reconstruction with a free myocutaneous VL (n = 25; 61%), cVLALT (n = 14; 34%), or cVLTFL Three acute flap thromboses occurred in the entire cohort (3/41, 7%), with one myocutaneous VL flap failing because of recurrent venous thrombosis during the salvage procedure. Total flap necrosis was seen in two cases (5%; VL flap: n = 1; cVLALT flap: n = 1), and partial flap necrosis in one VL flap (1/25, 4%) and in the distal ALT portion of three cVLALT flaps (3/14, 21%). No significant difference was seen between isolated VL and conjoined VL flaps regarding the partial (p = .28) or total flap necrosis rate (p = .9). CONCLUSION: The free (conjoined) VL flap provides reliable outcomes for obliterating dead space achieving durable reconstruction of complex chest wall defects.

Complex vaccination strategies prevent the emergence of vaccine resistance.

Vaccination is the most effective tool to control infectious diseases. However, the evolution of vaccine resistance, exemplified by vaccine-resistance in SARS-CoV-2, remains a concern. Here, we model complex vaccination strategies against a pathogen with multiple epitopes - molecules targeted by the vaccine. We found that a vaccine targeting one epitope was ineffective in preventing vaccine escape. Vaccine resistance in highly infectious pathogens was prevented by the full-epitope vaccine, that is, one targeting all available epitopes, but only when the rate of pathogen evolution was low. Strikingly, a bet-hedging strategy of random administration of vaccines targeting different epitopes was the most effective in preventing vaccine resistance in pathogens with low rate of infection and high rate of evolution. Thus, complex vaccination strategies, when biologically feasible, may be preferable to the currently used single-vaccine approaches for long-term control of disease outbreaks, especially when applied to livestock with near 100% vaccination rates.

Exploring the role of E. faecalis enterococcal polysaccharide antigen (EPA) and lipoproteins in evasion of phagocytosis.

Enterococcus faecalis is an opportunistic pathogen frequently causing nosocomial infections. The virulence of this organism is underpinned by its capacity to evade phagocytosis, allowing dissemination in the host. Immune evasion requires a surface polysaccharide produced by all enterococci, known as the enterococcal polysaccharide antigen (EPA). EPA consists of a cell wall-anchored rhamnose backbone substituted by strain-specific polysaccharides called 'decorations', essential for the biological activity of this polymer. However, the structural determinants required for innate immune evasion remain unknown, partly due to a lack of suitable validated assays. Here, we describe a quantitative, in vitro assay to investigate how EPA decorations alter phagocytosis. Using the E. faecalis model strain OG1RF, we demonstrate that a mutant with a deletion of the locus encoding EPA decorations can be used as a platform strain to express heterologous decorations, thereby providing an experimental system to investigate the inhibition of phagocytosis by strain-specific decorations. We show that the aggregation of cells lacking decorations is increasing phagocytosis and that this process does not involve the recognition of lipoproteins by macrophages. Collectively, our work provides novel insights into innate immune evasion by enterococci and paves the way for further studies to explore the structure/function relationship of EPA decorations.

Achieving realistic ozonation conditions with synthetic water matrices comprising low-molecular-weight scavenger compounds.

Ozonation is used worldwide for drinking water disinfection and increasingly also for micropollutant abatement from wastewater. Identification of transformation products formed during the ozonation of micropollutants is challenging due to several factors including (i) the reactions of both oxidants, ozone and hydroxyl radicals with the micropollutants, as well as with intermediate transformation products, (ii) effects of the water matrix on the ozone and hydroxyl radical chemistry and (iii) the generation of oxidation by-products. In this study, a simple approach to achieve realistic ozonation conditions in the absence of dissolved organic matter has been developed. It is based on composing synthetic water matrices with low-molecular-weight scavenger compounds (phenol, methanol, acetate, and carbonate) that mimic the chemical interactions of ozone and hydroxyl radicals with real water matrices. Synthetic waters composed of only four low-molecular-weight compounds successfully replicated two lake waters and two secondary wastewater effluents, matching instantaneous ozone demand, ozone and hydroxyl radical exposures in the initial phase, as well as the ozone evolution in the second phase of the ozonation process. The synthetic water matrices also reproduced the effects of temperature and pH changes observed in real waters. The abatement of two micropollutants, bezafibrate and atrazine, and the formation of the corresponding transformation products during ozonation were in agreement for synthetic and real waters. Furthermore, the kinetics and extent of bromate formation during ozonation in synthetic water were comparable to real lake water and wastewater. This supports the robustness of the proposed approach because bromate formation is very sensitive to the interplay of ozone and hydroxyl radicals. Furthermore, with the novel reaction system, a significant effect of hydroxyl radicals scavenging by carbonate on bromate formation was demonstrated. Overall, the herein-developed approach based on synthetic water matrices allows to perform realistic ozonation studies including both oxidants, ozone and hydroxyl radicals, without the constraints of using dissolved organic matter.

Community consensus for Heparan sulfate as a biomarker to support accelerated approval in Neuronopathic Mucopolysaccharidoses.

Mucopolysaccharidoses (MPS) disorders are a group of ultra-rare, inherited, lysosomal storage diseases caused by enzyme deficiencies that result in accumulation of glycosaminoglycans (GAGs) in cells throughout the body including the brain, typically leading to early death. Current treatments do not address the progressive cognitive impairment observed in patients with neuronopathic MPS disease. The rarity and clinical heterogeneity of these disorders as well as pre-existing brain disease in clinically diagnosed patients make the development of new therapeutics utilizing a traditional regulatory framework extremely challenging. Children with neuronopathic MPS disorders will likely sustain irreversible brain damage if randomized to a placebo or standard-of-care treatment arm that does not address brain disease. The United States Food and Drug Administration (FDA) recognized these challenges, and, in 2020, issued final guidance for industry on slowly progressive, low-prevalence, rare diseases with substrate deposition that result from single enzyme defects, outlining a path for generating evidence of effectiveness to support accelerated approval based on reduction of substrate accumulation [1]. Neuronopathic MPS disorders, which are characterized by the accumulation of the GAG heparan sulfate (HS) in the brain, fit the intended disease characteristics for which this guidance was written, but to date, this guidance has not yet been applied to any therapeutic candidate for MPS. In February 2024, the Reagan-Udall Foundation for the FDA convened a public workshop for representatives from the FDA, patient advocacy groups, clinical and basic science research, and industry to explore a case study of using cerebrospinal fluid (CSF) HS as a relevant biomarker to support accelerated approval of new therapeutics for neuronopathic MPS disorders. This review provides a summary of the MPS presentations at the workshop and perspective on the path forward for neuronopathic MPS disorders.

Photoactivated Chromophore for Keratitis-Corneal Cross-linking (PACK-CXL)-A Scoping Review Based on Preclinical Studies.

Purpose: Photoactivated chromophore for keratitis-corneal cross-linking (PACK-CXL) stabilizes the corneal stroma and eliminates microorganisms. Numerous PACK-CXL protocols, using different energy sources and chromophores, have been applied in preclinical studies, including live animal studies, with various experimental designs and endpoints. So far, a systematic mapping of the applied protocols and consistency across studies seems lacking but is essential to guide future research. Methods: The scoping review protocol was in line with the JBI Manual for Evidence Synthesis. Electronic databases were searched (Embase, MEDLINE, Scopus, Web of Science) to identify eligible records, followed by a two-step selection process (title and abstract screening, full text screening) for record inclusion. We extracted information on (1) different PACK-CXL protocol characteristics; (2) infectious pathogens tested; (3) study designs and experimental settings; and (4) endpoints used to determine antimicrobial and tissue stabilizing effects. The information was charted in frequency maps. Results: The searches yielded 3654 unique records, 233 of which met the inclusion criteria. With 103 heterogeneous endpoints, the researchers investigated a wide range of PACK-CXL protocols. The tested microorganisms reflected pathogens commonly associated with infectious keratitis. Bacterial solutions and infectious keratitis rabbit models were the most widely used models to study the antimicrobial effects of PACK-CXL. Conclusions: If preclinical PACK-CXL studies are to guide future translational research, further cross-disciplinary efforts are needed to establish, promote, and facilitate acceptance of common endpoints relevant to PACK-CXL. Translational Relevance: Systematic mapping of PACK-CXL protocols in preclinical studies guides future translational research.

Exploring the Cost-Effectiveness of Newborn Screening for Metachromatic Leukodystrophy (MLD) in the UK.

Metachromatic leukodystrophy (MLD) is a fatal inherited lysosomal storage disease that can be detected through newborn bloodspot screening. The feasibility of the screening assay and the clinical rationale for screening for MLD have been previously demonstrated, so the aim of this study is to determine whether the addition of screening for MLD to the routine newborn screening program in the UK is a cost-effective use of National Health Service (NHS) resources. A health economic analysis from the perspective of the NHS and Personal Social Services was developed based on a decision-tree framework for each MLD subtype using long-term outcomes derived from a previously presented partitioned survival and Markov economic model. Modelling inputs for parameters related to epidemiology, test characteristics, screening and treatment costs were based on data from three major UK specialist MLD hospitals, structured expert opinion and published literature. Lifetime costs and quality-adjusted life years (QALYs) were discounted at 1.5% to account for time preference. Uncertainty associated with the parameter inputs was explored using sensitivity analyses. This health economic analysis demonstrates that newborn screening for MLD is a cost-effective use of NHS resources using a willingness-to-pay threshold appropriate to the severity of the disease; and supports the inclusion of MLD into the routine newborn screening programme in the UK.

Hypoeutectic Liquid Metal Printing of 2D Indium Gallium Oxide Transistors.

2D native surface oxides formed on low melting temperature metals such as indium and gallium offer unique opportunities for fabricating high-performance flexible electronics and optoelectronics based on a new class of liquid metal printing (LMP). An inherent property of these Cabrera-Mott 2D oxides is their suboxide nature (e.g., In2O3-x), which leads high mobility LMP semiconductors to exhibit high electron concentrations (ne > 1019 cm-3) limiting electrostatic control. Binary alloying of the molten precursor can produce doped, ternary metal oxides such as In-X-O with enhanced electronic performance and greater bias-stress stability, though this approach demands a deeper understanding of the native oxides of alloys. This work presents an approach for hypoeutectic rapid LMP of crystalline InGaOx (IGO) at ultralow process temperatures (180 °C) beyond the state of the art to fabricate transistors with 10X steeper subthreshold slope and high mobility (≈18 cm2 Vs-1). Detailed characterization of IGO crystallinity, composition, and morphology, as well as measurements of its electronic density of states (DOS), show the impact of Ga-doping and reveal the limits of doping induced amorphization from hypoeutectic precursors. The ultralow process temperatures and compatibility with high-k Al2O3 dielectrics shown here indicate potential for 2D IGO to drive low-power flexible transparent electronics.

Pilot Implementation of Guiando Buenas Decisiones, an Evidence-Based Parenting Program for Spanish-Speaking Families, in Pediatric Primary Care in a Large, U.S. Health System: A Qualitative Interview Study.

Adolescent substance use is a significant public health problem in the United States and Hispanic youth engage in substance use services at lower rates than other groups. For this under-served group, prevention services delivered in non-stigmatized, non-specialty care settings may increase access to the services. We describe findings from a feasibility pilot of the implementation of a virtual version of Guiando Buenas Decisiones (GBD), a universal, group-based substance use prevention program for parents. It was conducted with Spanish-speaking families and delivered, virtually, in pediatric primary care in a large healthcare system in the U.S. Through qualitative interviews with pediatricians (n =7) and parents (n = 26), we explored potential barriers and facilitators of GBD enrollment and engagement. Parents and pediatricians alike noted the dearth of universal prevention programming in Spanish and that GBD could help address the need for linguistically appropriate programming. Parents liked the curriculum content, materials and videos; they felt the focus on strengthening family bonds, setting clear expectations and guidelines, the use of family meetings, and the positive tools provided for navigating family conflict were well-aligned with their cultural and family values. Feedback from parents was helpful for informing more personalized and attentive approaches to program outreach and recruitment methods, and for adaptation of recruitment fliers and letters. In this pediatric primary care context serving an underserved population, we found virtual GBD feasible to implement, acceptable and appealing to parents, and judged by pediatricians as a promising, much-needed addition to their prevention armamentarium.

Chromatin plasticity predetermines neuronal eligibility for memory trace formation.

Memories are encoded by sparse populations of neurons but how such sparsity arises remains largely unknown. We found that a neuron's eligibility to be recruited into the memory trace depends on its epigenetic state prior to encoding. Principal neurons in the mouse lateral amygdala display intrinsic chromatin plasticity, which when experimentally elevated favors neuronal allocation into the encoding ensemble. Such chromatin plasticity occurred at genomic regions underlying synaptic plasticity and was accompanied by increased neuronal excitability in single neurons in real time. Lastly, optogenetic silencing of the epigenetically altered neurons prevented memory expression, revealing a cell-autonomous relationship between chromatin plasticity and memory trace formation. These results identify the epigenetic state of a neuron as a key factor enabling information encoding.

Influential individuals can promote prosocial practices in heterogeneous societies: a mathematical and agent-based model.

In this paper, we examine how different governance types impact prosocial behaviors in a heterogenous society. We construct a general theoretical framework to examine a game-theoretic model to assess the ease of achieving a cooperative outcome. We then build a dynamic agent-based model to examine three distinct governance types in a heterogenous population: monitoring one's neighbors, despotic leadership, and influencing one's neighbors to adapt strategies that lead to better fitness. In our research, we find that while despotic leadership may lead towards high prosociality and high returns it does not exceed the effects of a local individual who can exert positive influence in the community. This may suggest that greater individual gains can be had by cooperating and that global hierarchical leadership may not be essential as long as influential individuals exert their influence for public good and not for public ill.

Geobiology: Machine learning puts bioturbation on the map.

Bioturbation, the mixing of sediment through the actions of organisms, is a crucial ecosystem engineering process that controls biogeochemical cycles and helps structure marine ecosystems. Machine learning is helping to develop global maps of the intensity and depth of bioturbation.