Short CDRL1 in intermediate VRC01-like mAbs is not sufficient to overcome key glycan barriers on HIV-1 Env.

VRC01-class broadly neutralizing antibodies (bnAbs) have been isolated from people with HIV-1, but they have not yet been elicited by vaccination. They are extensively somatically mutated and sometimes accumulate CDRL1 deletions. Such indels may allow VRC01-class antibodies to accommodate the glycans expressed on a conserved N276 N-linked glycosylation site in loop D of the gp120 subunit. These glycans constitute a major obstacle in the development of VRC01-class antibodies, as unmutated antibody forms are unable to accommodate them. Although immunizations of knock-in mice expressing human VRC01-class B-cell receptors (BCRs) with specifically designed Env-derived immunogens lead to the accumulation of somatic mutations in VRC01-class BCRs, CDRL1 deletions are rarely observed, and the elicited antibodies display narrow neutralizing activities. The lack of broad neutralizing potential could be due to the absence of deletions, the lack of appropriate somatic mutations, or both. To address this point, we modified our previously determined prime-boost immunization with a germline-targeting immunogen nanoparticle (426c.Mod.Core), followed by a heterologous core nanoparticle (HxB2.WT.Core), by adding a final boost with a cocktail of various stabilized soluble Env trimers. We isolated VRC01-like antibodies with extensive somatic mutations and, in one case, a seven-amino acid CDRL1 deletion. We generated chimeric antibodies that combine the vaccine-elicited somatic mutations with CDRL1 deletions present in human mature VRC01 bnAbs. We observed that CDRL1 indels did not improve the neutralizing antibody activities. Our study indicates that CDRL1 length by itself is not sufficient for the broadly neutralizing phenotype of this class of antibodies. IMPORTANCE: HIV-1 broadly neutralizing antibodies will be a key component of an effective HIV-1 vaccine, as they prevent viral acquisition. Over the past decade, numerous broadly neutralizing antibodies (bnAbs) have been isolated from people with HIV. Despite an in-depth knowledge of their structures, epitopes, ontogenies, and, in a few rare cases, their maturation pathways during infection, bnAbs have, so far, not been elicited by vaccination. This necessitates the identification of key obstacles that prevent their elicitation by immunization and overcoming them. Here we examined whether CDRL1 shortening is a prerequisite for the broadly neutralizing potential of VRC01-class bnAbs, which bind within the CD4 receptor binding site of Env. Our findings indicate that CDRL1 shortening by itself is important but not sufficient for the acquisition of neutralization breadth, and suggest that particular combinations of amino acid mutations, not elicited so far by vaccination, are most likely required for the development of such a feature.

Survey of Current Simulation Based Training in the US Military Health System.

INTRODUCTION: Simulation-based medical training has been shown to be effective and is widely used in civilian hospitals; however, it is unclear how widely and how effectively simulation is utilized in the U.S. Military Health System (MHS). The current operational state of medical simulation in the MHS is unknown, and there remains a need for a system-wide assessment of whether and how the advances in simulation-based medical training are employed to meet the evolving needs of the present-day warfighter. Understanding the types of skills and methods used within simulation programs across the enterprise is important data for leaders as they plan for the future in terms of curriculum development and the investment of resources. The aim of the present study is to survey MHS simulation programs in order to determine the prevalence of skills taught, the types of learners served, and the most common methodologies employed in this worldwide health care system. MATERIALS AND METHODS: A cross-sectional survey of simulation activities was distributed to the medical directors of all 93 simulation programs in the MHS. The survey was developed by the authors based on lists of critical wartime skills published by the medical departments of the US Army, Navy, and Air Force. Respondents were asked to indicate the types of learners trained at their program, which of the 82 unique skills included in the survey are trained at their site, and for each skill the modalities of simulation used, i.e., mannequin, standardized patients, part task trainers, augmented/virtual reality tools, or cadaver/live tissue. RESULTS: Complete survey responses were obtained from 75 of the 93 (80%) MHS medical simulation training programs. Across all skills included in the survey, those most commonly taught belonged predominantly to the categories of medic skills and nursing skills. Across all sites, the most common category of learner was the medic/corpsman (95% of sites), followed by nurses (87%), physicians (83%), non-medical combat lifesavers (59%), and others (28%) that included on-base first responders, law enforcement, fire fighters, and civilians. The skills training offered by programs included most commonly the tasks associated with medics/corpsmen (97%) followed by nursing (81%), advanced provider (77%), and General Medical Officer (GMO) skills (47%). CONCLUSION: The survey demonstrated that the most common skills taught were all related to point of injury combat casualty care and addressed the most common causes of death on the battlefield. The availability of training in medic skills, nursing skills, and advanced provider skills were similar in small, medium, and large programs. However, medium and small programs were less likely to deliver training for advanced providers and GMOs compared to larger programs. Overall, this study found that simulation-based medical training in the MHS is focused on medic and nursing skills, and that large programs are more likely to offer training for advanced providers and GMOs. Potential gaps in the availability of existing training are identified as over 50% of skills included in the nursing, advanced provider, and GMO skill categories are not covered by at least 80% of sites serving those learners.

Assessing the Ability of a Large Language Model to Score Free-Text Medical Student Clinical Notes: Quantitative Study.

Background: Teaching medical students the skills required to acquire, interpret, apply, and communicate clinical information is an integral part of medical education. A crucial aspect of this process involves providing students with feedback regarding the quality of their free-text clinical notes. Objective: The goal of this study was to assess the ability of ChatGPT 3.5, a large language model, to score medical students' free-text history and physical notes. Methods: This is a single-institution, retrospective study. Standardized patients learned a prespecified clinical case and, acting as the patient, interacted with medical students. Each student wrote a free-text history and physical note of their interaction. The students' notes were scored independently by the standardized patients and ChatGPT using a prespecified scoring rubric that consisted of 85 case elements. The measure of accuracy was percent correct. Results: The study population consisted of 168 first-year medical students. There was a total of 14,280 scores. The ChatGPT incorrect scoring rate was 1.0%, and the standardized patient incorrect scoring rate was 7.2%. The ChatGPT error rate was 86%, lower than the standardized patient error rate. The ChatGPT mean incorrect scoring rate of 12 (SD 11) was significantly lower than the standardized patient mean incorrect scoring rate of 85 (SD 74; P=.002). Conclusions: ChatGPT demonstrated a significantly lower error rate compared to standardized patients. This is the first study to assess the ability of a generative pretrained transformer (GPT) program to score medical students' standardized patient-based free-text clinical notes. It is expected that, in the near future, large language models will provide real-time feedback to practicing physicians regarding their free-text notes. GPT artificial intelligence programs represent an important advance in medical education and medical practice.

A smart mask for exhaled breath condensate harvesting and analysis.

Recent respiratory outbreaks have garnered substantial attention, yet most respiratory monitoring remains confined to physical signals. Exhaled breath condensate (EBC) harbors rich molecular information that could unveil diverse insights into an individual's health. Unfortunately, challenges related to sample collection and the lack of on-site analytical tools impede the widespread adoption of EBC analysis. Here, we introduce EBCare, a mask-based device for real-time in situ monitoring of EBC biomarkers. Using a tandem cooling strategy, automated microfluidics, highly selective electrochemical biosensors, and a wireless reading circuit, EBCare enables continuous multimodal monitoring of EBC analytes across real-life indoor and outdoor activities. We validated EBCare's usability in assessing metabolic conditions and respiratory airway inflammation in healthy participants, patients with chronic obstructive pulmonary disease or asthma, and patients after COVID-19 infection.

A Novel Method for Determining Ventilatory and Gas Exchange Dynamics During Exercise: The "Chirp" Waveform.

Quantitating exercise ventilatory and gas exchange dynamics affords insights into physiological control processes and cardiopulmonary dysfunction. We designed a novel waveform, the chirp waveform, to efficiently extract moderate intensity exercise response dynamics. In the chirp waveform, work rate fluctuates sinusoidally with constant amplitude as sinusoidal period decreases progressively from approximately 8.5 to 1.4 minutes over 30 minutes of cycle ergometry. We hypothesized that response dynamics of pulmonary ventilation (V̇E) and gas exchange (V̇O2 and V̇CO2) extracted from chirp waveform are similar to those obtained from step-wise transitions. Thirty-one participants (14 young-healthy, 7 older-healthy, 10 COPD patients) exercised on three occasions. Participants first performed ramp-incremental exercise for gas exchange threshold (GET) determination. In randomized order, the next two visits involved either chirp or step-wise waveforms. Work rate amplitude (20W to ∼95% GET work rate) and exercise duration (30 min) were the same for both waveforms. A first-order linear transfer function with system gain (G) and time constant (τ) characterized response dynamics. Agreement between model parameters extracted from chirp and step-wise waveforms was established using Bland-Altman analysis and Rothery's Concordance Coefficient (RCC). V̇E, V̇O2, and V̇CO2 Gs showed no systematic bias (p>0.178) and moderate-to-good agreement (RCC>0.772, p<0.01) between waveforms. Similarly, no systematic bias (p=0.815) and good agreement (RCC=0.837, p<0.001) was found for τV̇O2. Despite moderate agreement for τV̇CO2 (RCC=0.794, p<0.001) and τV̇E (RCC=0.722, p=0.083), chirp τ was less (-6.9(11.7)s and -12.2(22.5)s, respectively). We conclude that the chirp waveform is a promising method for measuring exercise response dynamics and investigating physiological control mechanisms.

Deletion of miPEP in adipocytes protects against obesity and insulin resistance by boosting muscle metabolism.

Mitochondria facilitate thousands of biochemical reactions, covering a broad spectrum of anabolic and catabolic processes. Here we demonstrate that the adipocyte mitochondrial proteome is markedly altered across multiple models of insulin resistance and reveal a consistent decrease in the level of the mitochondrial processing peptidase miPEP. OBJECTIVE: To determine the role of miPEP in insulin resistance. METHODS: To experimentally test this observation, we generated adipocyte-specific miPEP knockout mice to interrogate its role in the aetiology of insulin resistance. RESULTS: We observed a strong phenotype characterised by enhanced insulin sensitivity and reduced adiposity, despite normal food intake and physical activity. Strikingly, these phenotypes vanished when mice were housed at thermoneutrality, suggesting that metabolic protection conferred by miPEP deletion hinges upon a thermoregulatory process. Tissue specific analysis of miPEP deficient mice revealed an increment in muscle metabolism, and upregulation of the protein FBP2 that is involved in ATP hydrolysis in the gluconeogenic pathway. CONCLUSION: These findings suggest that miPEP deletion initiates a compensatory increase in skeletal muscle metabolism acting as a protective mechanism against diet-induced obesity and insulin resistance.

Reverse Genetics of Murine Rotavirus: A Comparative Analysis of the Wild-Type and Cell-Culture-Adapted Murine Rotavirus VP4 in Replication and Virulence in Neonatal Mice.

Small-animal models and reverse genetics systems are powerful tools for investigating the molecular mechanisms underlying viral replication, virulence, and interaction with the host immune response in vivo. Rotavirus (RV) causes acute gastroenteritis in many young animals and infants worldwide. Murine RV replicates efficiently in the intestines of inoculated suckling pups, causing diarrhea, and spreads efficiently to uninoculated littermates. Because RVs derived from human and other non-mouse animal species do not replicate efficiently in mice, murine RVs are uniquely useful in probing the viral and host determinants of efficient replication and pathogenesis in a species-matched mouse model. Previously, we established an optimized reverse genetics protocol for RV and successfully generated a murine-like RV rD6/2-2g strain that replicates well in both cultured cell lines and in the intestines of inoculated pups. However, rD6/2-2g possesses three out of eleven gene segments derived from simian RV strains, and these three heterologous segments may attenuate viral pathogenicity in vivo. Here, we rescued the first recombinant RV with all 11 gene segments of murine RV origin. Using this virus as a genetic background, we generated a panel of recombinant murine RVs with either N-terminal VP8* or C-terminal VP5* regions chimerized between a cell-culture-adapted murine ETD strain and a non-tissue-culture-adapted murine EW strain and compared the diarrhea rate and fecal RV shedding in pups. The recombinant viruses with VP5* domains derived from the murine EW strain showed slightly more fecal shedding than those with VP5* domains from the ETD strain. The newly characterized full-genome murine RV will be a useful tool for dissecting virus-host interactions and for studying the mechanism of pathogenesis in neonatal mice.

Viroporin activity from rotavirus nonstructural protein 4 induces intercellular calcium waves that contribute to pathogenesis.

Acute gastroenteritis remains the second leading cause of death among children under the age of 5 worldwide. While enteric viruses are the most common etiology, the drivers of their virulence remain incompletely understood. We recently found that cells infected with rotavirus, the most prevalent enteric virus in infants and young children, initiate hundreds of intercellular calcium waves that enhance both fluid secretion and viral spread. Understanding how rotavirus triggers intercellular calcium waves may allow us to design safer, more effective vaccines and therapeutics, but we still lack a mechanistic understanding of this process. In this study, we used existing virulent and attenuated rotavirus strains, as well as reverse engineered recombinants, to investigate the role of rotavirus nonstructural protein 4 (NSP4) in intercellular calcium wave induction using in vitro , organoid, and in vivo model systems. We found that the capacity to induce purinergic intercellular calcium waves (ICWs) segregated with NSP4 in both simian and murine-like rotavirus backgrounds, and NSP4 expression alone was sufficient to induce ICWs. NSP4's ability to function as a viroporin, which conducts calcium out of the endoplasmic reticulum, was necessary for ICW induction. Furthermore, viroporin activity and the resulting ICWs drove transcriptional changes indicative of innate immune activation, which were lost upon attenuation of viroporin function. Multiple aspects of RV disease severity in vivo correlated with the generation of ICWs, identifying a critical link between viroporin function, intercellular calcium waves, and enteric viral virulence.

Copper(II) coordination to the intrinsically disordered region of SARS-CoV-2 Nsp1.

The intrinsically disordered C-terminal peptide region of severe acute respiratory syndrome coronavirus 2 nonstructural protein-1 (Nsp1-CT) inhibits host protein synthesis by blocking messenger RNA (mRNA) access to the 40S ribosome entrance tunnel. Aqueous copper(II) ions bind to the disordered peptide with micromolar affinity, creating a possible strategy to restore protein synthesis during host infection. Electron paramagnetic resonance (EPR) and tryptophan fluorescence measurements on a 10-residue model of the disordered protein region (Nsp1-CT10), combined with advanced quantum mechanics calculations, suggest that the peptide binds to copper(II) as a multidentate ligand. Two optimized computational models of the copper(II)-peptide complexes were derived: One corresponding to pH 6.5 and the other describing the complex at pH 7.5 to 8.5. Simulated EPR spectra based on the calculated model structures are in good agreement with experimental spectra.

Differentially co-expressed myofibre transcripts associated with abnormal myofibre proportion in chronic obstructive pulmonary disease.

BACKGROUND: Skeletal muscle dysfunction is a common extrapulmonary manifestation of chronic obstructive pulmonary disease (COPD). Alterations in skeletal muscle myosin heavy chain expression, with reduced type I and increased type II myosin heavy chain expression, are associated with COPD severity when studied in largely male cohorts. The objectives of this study were (1) to define an abnormal myofibre proportion phenotype in both males and females with COPD and (2) to identify transcripts and transcriptional networks associated with abnormal myofibre proportion in COPD. METHODS: Forty-six participants with COPD were assessed for body composition, strength, endurance and pulmonary function. Skeletal muscle biopsies from the vastus lateralis were assayed for fibre-type distribution and cross-sectional area via immunofluorescence microscopy and RNA-sequenced to generate transcriptome-wide gene expression data. Sex-stratified k-means clustering of type I and IIx/IIax fibre proportions was used to define abnormal myofibre proportion in participants with COPD and contrasted with previously defined criteria. Single transcripts and weighted co-expression network analysis modules were tested for correlation with the abnormal myofibre proportion phenotype. RESULTS: Abnormal myofibre proportion was defined in males with COPD (n = 29) as <18% type I and/or >22% type IIx/IIax fibres and in females with COPD (n = 17) as <36% type I and/or >12% type IIx/IIax fibres. Half of the participants with COPD were classified as having an abnormal myofibre proportion. Participants with COPD and an abnormal myofibre proportion had lower median handgrip strength (26.1 vs. 34.0 kg, P = 0.022), 6-min walk distance (300 vs. 353 m, P = 0.039) and forced expiratory volume in 1 s-to-forced vital capacity ratio (0.42 vs. 0.48, P = 0.041) compared with participants with COPD and normal myofibre proportions. Twenty-nine transcripts were associated with abnormal myofibre proportions in participants with COPD, with the upregulated NEB, TPM1 and TPM2 genes having the largest fold differences. Co-expression network analysis revealed that two transcript modules were significantly positively associated with the presence of abnormal myofibre proportions. One of these co-expression modules contained genes classically associated with muscle atrophy, as well as transcripts associated with both type I and type II myofibres, and was enriched for genetic loci associated with bone mineral density. CONCLUSIONS: Our findings indicate that there are significant transcriptional alterations associated with abnormal myofibre proportions in participants with COPD. Transcripts canonically associated with both type I and type IIa fibres were enriched in a co-expression network associated with abnormal myofibre proportion, suggesting altered transcriptional regulation across multiple fibre types.

MYADM binds human parechovirus 1 and is essential for viral entry.

Human parechoviruses (PeV-A) are increasingly being recognized as a cause of infection in neonates and young infants, leading to a spectrum of clinical manifestations ranging from mild gastrointestinal and respiratory illnesses to severe sepsis and meningitis. However, the host factors required for parechovirus entry and infection remain poorly characterized. Here, using genome-wide CRISPR/Cas9 loss-of-function screens, we identify myeloid-associated differentiation marker (MYADM) as a host factor essential for the entry of several human parechovirus genotypes including PeV-A1, PeV-A2 and PeV-A3. Genetic knockout of MYADM confers resistance to PeV-A infection in cell lines and in human gastrointestinal epithelial organoids. Using immunoprecipitation, we show that MYADM binds to PeV-A1 particles via its fourth extracellular loop, and we identify critical amino acid residues within the loop that mediate binding and infection. The demonstrated interaction between MYADM and PeV-A1, and its importance specifically for viral entry, suggest that MYADM is a virus receptor. Knockout of MYADM does not reduce PeV-A1 attachment to cells pointing to a role at the post-attachment stage. Our study suggests that MYADM is a multi-genotype receptor for human parechoviruses with potential as an antiviral target to combat disease associated with emerging parechoviruses.

The Dynamics of Locomotor Neuromuscular Fatigue during Ramp-Incremental Cycling to Intolerance.

INTRODUCTION: Traditional neuromuscular fatigue assessments are not task-specific and are unable to characterize neuromuscular performance decline during dynamic whole-body exercise. This study used interleaved maximal isokinetic cycling efforts to characterize the dynamics of the decline in neuromuscular performance during ramp-incremental (RI) cycle ergometry exercise to intolerance. METHODS: Eleven young healthy participants (10 male/1 female) performed two RI cycle ergometry exercise tests to intolerance: [1] RI-exercise with peak isokinetic power (Piso) at 80 rev·min-1 measured at baseline and immediately at intolerance from a maximal ~6 s effort; [2] RI-exercise where additional Piso measurements were interleaved every 90 s to characterize the decline in neuromuscular performance during the RI-test. Muscle excitation was measured using EMG during all Piso assessments, and pulmonary gas exchange was measured throughout. RESULTS: Baseline Piso was 832 ± 140 W and RI-exercise reduced Piso to 349 ± 96 W at intolerance (p = 0.001), which was not different from flywheel power at intolerance (303 ± 96 W; p = 0.292). There was no reduction in Piso between baseline cycling and gas exchange threshold (GET; baseline Piso vs. mean Piso below GET: 828 ± 146 vs. 815 ± 149 W; p = 1.00). Piso fell progressively above GET until intolerance (Piso every 90 s above GET: 759 ± 139; 684 ± 141; 535 ± 144; 374 ± 117 W; each p < 0.05 vs. baseline and mean Piso below GET). Peak muscle excitation (EMG) was also reduced only above GET (73 ± 14 % of baseline, at intolerance; p < 0.05). However, the reduction in peak Piso preceded the reduction in peak muscle excitation. CONCLUSIONS: The dynamics of the decline in neuromuscular performance (reduction in Piso and EMG) during RI-exercise are consistent with known intensity-dependent metabolic and traditional pre-post neuromuscular fatigue responses to discrete bouts of constant-power exercise.

Thematic evolution of smart learning environments, insights and directions from a 20-year research milestones: A bibliometric analysis.

Smart learning environments (SLEs) have been developed to create an effective learning environment gradually and sustainably by applying technology. Given the growing dependence on technology daily, SLE will inevitably be incorporated into the teaching and learning process. Without transforming technology-enhanced learning environments into SLE, they are restricted to adding sophistication and lack pedagogical benefits, leading to wasteful educational investments. SLE research has grown over time, particularly during the COVID-19 pandemic in 2020-2021, which fundamentally altered the "landscape" of technology use in education. This study aims to discover how the stages of SLE transform from time to time by applying two bibliometric analysis approaches: publication performance analysis and science mapping. The dataset was created by extracting bibliometric data from Scopus, including 427 articles, 162 publication sources (journals and proceeding), and 1080 authors from 2002 to 2022. Three kinds of SLE research subjects were identified by keyword synthesis: SLE features, technological innovation, and adaptive learning systems. Adaptive learning and personalized learning are consistently used interchangeably to demonstrate the significance of supporting the diversity of student and teacher conditions. Learning analytics, essential to employing big data technology for educational data mining, is a new theme being considered increasingly in the future to achieve adaptive and personalized learning. The 20-year SLE research milestone, broken down into five stages with various focuses on goals and served as the foundation for creating a maturity model of SLE.

The genetic and dietary landscape of the muscle insulin signalling network.

Metabolic disease is caused by a combination of genetic and environmental factors, yet few studies have examined how these factors influence signal transduction, a key mediator of metabolism. Using mass spectrometry-based phosphoproteomics, we quantified 23,126 phosphosites in skeletal muscle of five genetically distinct mouse strains in two dietary environments, with and without acute in vivo insulin stimulation. Almost half of the insulin-regulated phosphoproteome was modified by genetic background on an ordinary diet, and high-fat high-sugar feeding affected insulin signalling in a strain-dependent manner. Our data revealed coregulated subnetworks within the insulin signalling pathway, expanding our understanding of the pathway's organisation. Furthermore, associating diverse signalling responses with insulin-stimulated glucose uptake uncovered regulators of muscle insulin responsiveness, including the regulatory phosphosite S469 on Pfkfb2, a key activator of glycolysis. Finally, we confirmed the role of glycolysis in modulating insulin action in insulin resistance. Our results underscore the significance of genetics in shaping global signalling responses and their adaptability to environmental changes, emphasising the utility of studying biological diversity with phosphoproteomics to discover key regulatory mechanisms of complex traits.

When we eat, the pancreas releases a hormone called insulin, which helps our tissues absorb glucose. Insulin works by triggering a cascade of events in cells, which include adding chemical tags called phosphate groups at thousands of specific locations on proteins. This tag causes the changes needed to move glucose from the blood into cells and also regulates many other essential functions in the cell. If this process stops working and the body becomes resistant to the effects of insulin, it can lead to type 2 diabetes. This can result from a complex combination of genetic and lifestyle factors, which are difficult to study systematically in people. An alternative approach to understand these influences is to study mice, which are commonly used to investigate metabolic diseases and have contributed to our understanding of the mechanisms of type 2 diabetes. Using carefully bred mice allows precise control of their genetics and environment, revealing the independent and joint effects of these factors. Monitoring differences in the phosphate groups on proteins, van Gerwen et al. studied five distinct inbred mouse strains fed either an ordinary diet or one that was high in fat and sugar. Nearly half of the biochemical events triggered by insulin were altered by genetics on the ordinary diet. High-fat, high-sugar feeding also reshaped the pattern of phosphate tags depending on the mouse strain. By examining these cellular responses, van Gerwen et al. identified proteins that may regulate the insulin response in muscle cells. Increasing the activity of one of these enzymes reversed insulin resistance in skeletal muscle cells grown in the laboratory. This research underscores the importance of genetics in controlling insulin responses and shaping the impact of environmental challenges. It establishes a new opportunity in personalised medicine, which seeks to understand how an individual's genetics combine with their lifestyle to shape health. Furthermore, it identifies potential new targets for treating insulin resistance, paving the way for future research to develop more effective diabetes treatments.

A Viral Protein 4-Based Trivalent Nanoparticle Vaccine Elicited High and Broad Immune Responses and Protective Immunity against the Predominant Rotaviruses.

The current live rotavirus (RV) vaccines show reduced effectiveness in developing countries, calling for vaccine strategies with improved efficacy and safety. We generated pseudovirus nanoparticles (PVNPs) that display multiple ectodomains of RV viral protein 4 (VP4), named S-VP4e, as a nonreplicating RV vaccine candidate. The RV spike protein VP4s that bind host receptors and facilitate viral entry are excellent targets for vaccination. In this study, we developed scalable methods to produce three S-VP4e PVNPs, each displaying the VP4e antigens from one of the three predominant P[8], P[4], and P[6] human RVs (HRVs). These PVNPs were recognized by selected neutralizing VP4-specific monoclonal antibodies, bound glycan receptors, attached to permissive HT-29 cells, and underwent cleavage by trypsin between VP8* and VP5*. 3D PVNP models were constructed to understand their structural features. A trivalent PVNP vaccine containing the three S-VP4e PVNPs elicited high and well-balanced VP4e-specific antibody titers in mice directed against the three predominant HRV P types. The resulting antisera neutralized the three HRV prototypes at high titers; greater than 4-fold higher than the neutralizing responses induced by a trivalent vaccine consisting of the S60-VP8* PVNPs. Finally, the trivalent S-VP4e PVNP vaccine provided 90-100% protection against diarrhea caused by HRV challenge. Our data supports the trivalent S-VP4e PVNPs as a promising nonreplicating HRV vaccine candidate for parenteral delivery to circumvent the suboptimal immunization issues of all present live HRV vaccines. The established PVNP-permissive cell and PVNP-glycan binding assays will be instrumental for further investigating HRV-host cell interactions and neutralizing effects of VP4-specific antibodies and antivirals.

Dual Tracer Test to Measure Tissue-Specific Insulin Action in Individual Mice Identifies In Vivo Insulin Resistance Without Fasting Hyperinsulinemia.

The ability of metabolically active tissues to increase glucose uptake in response to insulin is critical to whole-body glucose homeostasis. This report describes the Dual Tracer Test, a robust method involving sequential retro-orbital injection of [14C]2-deoxyglucose ([14C]2DG) alone, followed 40 min later by injection of [3H]2DG with a maximal dose of insulin to quantify both basal and insulin-stimulated 2DG uptake in the same mouse. The collection of both basal and insulin-stimulated measures from a single animal is imperative for generating high-quality data since differences in insulin action may be misinterpreted mechanistically if basal glucose uptake is not accounted for. The approach was validated in a classic diet-induced model of insulin resistance and a novel transgenic mouse with reduced GLUT4 expression that, despite ubiquitous peripheral insulin resistance, did not exhibit fasting hyperinsulinemia. This suggests that reduced insulin-stimulated glucose disposal is not a primary contributor to chronic hyperinsulinemia. The Dual Tracer Test offers a technically simple assay that enables the study of insulin action in many tissues simultaneously. By administering two tracers and accounting for both basal and insulin-stimulated glucose transport, this assay halves the required sample size for studies in inbred mice and demonstrates increased statistical power to detect insulin resistance, relative to other established approaches, using a single tracer. The Dual Tracer Test is a valuable addition to the metabolic phenotyping toolbox.

Lack of effect of an in-line filter on cardiopulmonary exercise testing variables in healthy subjects.

PURPOSE: Pathogen transmission during cardio-pulmonary exercise testing (CPET) is caused by carrier aerosols generated during respiration. METHODS: Ten healthy volunteers (age range: 34 ± 15; 4 females) were recruited to see if the physiological reactions to ramp-incremental CPET on a cycle ergometer were affected using an in-line filter placed between the mouthpiece and the flow sensor. The tests were in random order with or without an in-line bacterial/viral spirometer filter. The work rate aligned, time interpolated 10 s bin data were compared throughout the exercise period. RESULTS: From rest to peak exercise, filter use increased only minute ventilation ([Formula: see text]E) (Δ[Formula: see text]E = 1.56 ± 0.70 L/min, P < 0.001) and tidal volume (VT) (ΔVT = 0.10 ± 0.11 L, P = 0.014). Over the entire test, the slope of the residuals for [Formula: see text]CO2 was positive (0.035 ± 0.041 (ΔL/L), P = 0.027). During a ramp-incremental CPET in healthy subjects, an in-line filter increased [Formula: see text]E and VT but not metabolic rate. CONCLUSION: In conclusion, using an in-line filter is feasible, does not affect appreciably the physiological variables, and may mitigate risk of aerosol dispersion during CPET.