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ABSTRACT: The objective was to evaluate the performance of exploratory models containing routinely available on-farm data, behavior data, and the combination of both to predict metritis self-cure (SC) and treatment failure (TF). Holstein cows (n = 1,061) were fitted with a collar-mounted automated-health monitoring device (AHMD) from −21 ± 3 to 60 ± 3 d relative to calving to monitor rumination time and activity. Cows were examined for diagnosis of metritis at 4 ± 1, 7 ± 1, and 9 ± 1 d in milk (DIM). Cows diagnosed with metritis (n = 132), characterized by watery, fetid, reddish/brownish vaginal discharge (VD), were randomly allocated to 1 of 2 treatments: control (CON; n = 62), no treatment at the time of metritis diagnosis (d 0); or ceftiofur (CEF; n = 70), subcutaneous injection of 6.6 mg/kg of ceftiofur crystalline-free acid on d 0 and 3 relative to diagnosis. Cure was determined 12 d after diagnosis and was considered when VD became mucoid and not fetid. Cows in CON were used to determine SC, and cows in CEF were used to determine TF. Univariable analyses were performed using farm-collected data (parity, calving season, calving-related disorders, body condition score, rectal temperature, and DIM at metritis diagnosis) and behavior data (i.e., daily averages of rumination time, activity generated by AHMD, and derived variables) to assess their association with metritis SC or TF. Variables with P-values ≤0.20 were included in the multivariable logistic regression exploratory models. To predict SC, the area under the curve (AUC) for the exploratory model containing only data routinely available on-farm was 0.75. The final exploratory model to predict SC combining routinely available on-farm data and behavior data increased the AUC to 0.87, with sensitivity (Se) of 89% and specificity (Sp) of 77%. To predict TF, the AUC for the exploratory model containing only data routinely available on-farm was 0.90. The final exploratory model combining routinely available on-farm data and behavior data increased the AUC to 0.93, with Se of 93% and Sp of 87%. Cross-validation analysis revealed that generalizability of the exploratory models was poor, which indicates that the findings are applicable to the conditions of the present exploratory study. In summary, the addition of behavior data contributed to increasing the prediction of SC and TF. Developing and validating accurate prediction models for SC could lead to a reduction in antimicrobial use, whereas accurate prediction of cows that would have TF may allow for better management decisions.

Abstract The spatial and temporal variability of gravity waves (GWs) potential energy (Ep) over South America (SA) was examined by analyzing temperature profiles obtained through the utilization of Sounding of the Atmosphere using Broadband Emission Radiometry (SABER) from January 2002 to December 2021. We used the empirical orthogonal function (EOF) analysis to decompose GWs parameters and to analyze the GW variations over SA. We considered the first three eigenmodes (EOF1, EOF2, and EOF3) and their principal components (PC1, PC2, and PC3) of the EOF decomposition, which accounts for ∼80–90% of the total GWs variation over SA. Further, we analyzed the coupled variation of Ep and zonal mean wind (U) to verify their inter-dependencies using the singular value decomposition (SVD). The spatial variation showed that different localized mechanisms generate GWs at different sectors of the continent. The EOF1 of Ep comprised more than 50%, the EOF2 ∼20–25%, and the third ∼10–15% of the total GWs variation. The positive variation of GWs energy in the EOF1 is localized in the tropical region from the lower stratosphere to the lower mesosphere and southward below 1.5° S in the upper mesosphere. The spectral analysis of GWs energy showed biannual, annual, semiannual, and 11-year variations at different eigenvectors. Relative Ep (REp) showed an asymmetric hemispheric response to solar flux over South America. The REp response to QBO showed a modulating effect below 70 km and a positive response above 70 km. There is a good positive correlation between the temporal component of EOF2 of Ep and the quasi-biennial oscillation (QBO) at 30 mb and 50 mb in the PC2 temporal variation. Graphical Abstract

Abstract Hypotension is one of the main characteristics of the systemic inflammation, basically caused by endothelial dysfunction. Studies have shown that the amino acid L‐kynurenine (KYN) causes vasodilation in mammals, leading to hypotensive shock. In hypotensive shock, when activated by the KYN, the voltage‐gated potassium channel encoded by the family KCNQ (Kv7) gene can cause vasodilation. Fructose‐1,6‐bisphosphate (FBP) it is being considered in studies an anti‐inflammatory, antioxidant, immunomodulator, and a modulator of some ion channels (Ca2+, Na+, and K+). We analyzed the effects of KYN and FBP on mean blood pressure (MBP), systolic and diastolic (DBP) blood pressure, and heart rate variability (HRV) in Wistar rats. Results demonstrated that the administration of KYN significant decreased MBP, DBP, and increased HRV. Importantly, the FBP treatment reversed the KYN effects on MBP, DBP, and HRV. Molecular Docking Simulations suggested that KYN and FBP present a very close estimated free energy of binding and the same position into structure of KCNQ4. Our results did demonstrate that FBP blunted the decrease in BP, provoked by KYN. Results raise new hypotheses for future and studies in the treatment of hypotension resulting from inflammation.

Resumo Modelagem dos processos de negócio possui um papel fundamental na representação do fluxo de atividades realizadas por uma organização. Este fluxo é registrado de forma clara, a partir de informações e conhecimento fornecidos pelos envolvidos no processo. Como resultado, os processos modelados podem ser compartilhados, possibilitando a institucionalização desse conhecimento. Para tornar tomadas de decisão mais eficientes e fidedignas, este trabalho tem como objetivo propor um método de conversão capaz de extrair informações de modelos de processos de negócio, descritos com a notação Business Process Model and Notation, registrando-as em grafos de conhecimento semânticos, com uso da ontologia Business Process Model and Notation 1.1 Ontology. Portanto, propõe-se a construção de uma base de conhecimento formalmente estruturada a partir dos modelos, a fim de viabilizar consultas precisas e extração de indicadores. Para tal, a metodologia utilizada compreende uma revisão da literatura, seguida da elaboração do método de conversão proposto e da validação do mesmo por meio de um estudo empírico. Analisando os resultados, pode-se afirmar que o método obteve sucesso na conversão do modelo do processo na base de conhecimento subjacente, e esta, por sua vez, permitiu a extração automática de indicadores para auxiliar às tomadas de decisão por parte dos gestores envolvidos.

Abstract Using COSMIC-2 and METOP radio occultation measurements during the years 2020 and 2021, the study presents the first direct and independent relationship between the potential energy (Ep) in the stratosphere, precipitable water vapour (PWV), tropopause heights (TPH), and cold-point heights (CPH) over South America. The South American continent comprises the tropical region, the Andes Mountain range, and mid-latitude climates. The seasonal mean of the potential energy (Ep), the PWV, and the tropopause parameters height (TPH and CPH) were obtained to investigate the relationship between the stratospheric gravity wave (SGW) Ep and the tropospheric parameters (PWV, TPH, and CPH). Around the Andes Mountains to the east, there is significantly less water vapour (PWV 8 kJ kg −1). A good correlation of variability was found between the PWV and the lower SGW Ep in summer over the tropical region (± 20 ◦ ). Generally, good and strong correlations were observed in the summer and spring, with negative/no correlations in the winter in 2020 and 2021. Also, good and strong correlations between SGW, PWV, and TPH were observed in the summer at $${20}^{o}N - {10}^{o}N$$ 20 o N - 10 o N in 2020 and 2021. Our result demonstrated the possibility that convective activity was a major driver of the tropical gravity waves over South America. In the subtropical (30 $$^\circ$$ ∘ –40 $$^\circ$$ ∘ ) region, especially in the winter, the tropospheric parameters make little or no contribution to gravity wave activity in the region. The CPH generally showed a no/negative with SGW over the South American tropics. The SGW activities in the tropical region showed an impact on the structure of the tropopause parameters, which could be a result of the convective activity in this region. Graphical Abstract

The main objective was to characterize behavioral changes in metritic primiparous cows treated with chitosan microparticles (CM) or ceftiofur (CEF). A secondary objective was to compare behavioral patterns of metritic cows with nonmetritic (NMET) cows. Nulliparous Holstein cows (n = 311) were fitted with a neck-mounted automated health-monitoring device (AHMD) from −21 to 60 d relative to calving. Cows diagnosed with metritis (d 0), characterized by watery, fetid, red-brownish uterine discharge within 21 d in milk were assigned randomly to CM (n = 45), intrauterine infusion of 24 g of CM dissolved in 40 mL of sterile distilled water on d 0, 2, and 4; CEF (n = 47), subcutaneous injection of 6.6 mg/kg ceftiofur crystalline-free acid on d 0 and 3; and control (CON; n = 39), no treatment. For comparison, NMET cows (n = 180) were matched with metritic cows according to age at calving and calving date. Postdiagnosis, there was an effect of treatment and an interaction between treatment and time on rumination and activity. The interaction showed that CM had lesser rumination than CEF from d 1 to 11, d 18, and d 20; CM had lesser rumination than CON from d 2 to 8; and CEF was not different from CON. The interaction showed that CM had lesser activity than CON on d 2, from d 6 to 11, and d 13 to 14; CM was not different from CEF; and CEF had lesser activity than CON on d 8, 9, 13, and 14. Prediagnosis, cows in CM, CEF, and CON had lesser rumination and activity than cows in NMET. Postdiagnosis, cows in CM, CEF, and CON had lesser rumination than NMET from d 0 to 2 and had lesser activity than NMET from d 0 to 5. In summary, CM decreased rumination and activity compared with CON, which indicates a negative systemic effect of CM. This may be associated with exacerbated inflammation in the uterus. Additionally, metritic cows had decreased rumination and activity prediagnosis, which may allow for the use of AHMD for metritis diagnosis.

In this work, a model of the degradation of the Mg–Zn–Mn–Ca alloy system in SBF (Simulated Body Fluid) is proposed by applying the isoconversion method. Alloy ingots with nominal compositions of Mg–2Zn–0.2Mn–0.2Ca (wt.%) designed A.2 alloy and Mg–4Zn–0.2Mn–0.2Ca (wt.%), A.4 alloy, was induction melted at 680 °C. In a eudiometer, the alloys were immersed for 600 min in Kokubo solution at temperatures of 23.5 °C, 36.5 °C, 49.5 °C, and the evolution of hydrogen was monitored. In the isoconversion method, the Arrhenius' equations and the kinetic mechanism models of chemical and diffusion control were used to construct the model. The degradation rate in A.2 alloy varied from 6.66 mg of Mg/cm2 to 9.40 mg of Mg/cm2, and in A.4 alloy, from 5.48 mg to 6.70 mg of Mg/cm2 with a test temperature variation of 23.5–49.5 °C. The control mechanism observed was the mixed one, being predominantly of chemical control. The activation energy found for the isoconversion model that used the kinetic mechanism model of chemical control was 3.24 kJ/mol for A.2 alloy and 3.45 kJ/mol for A.4 alloy, the pre-exponential factor of 4.34 × 10−3 mm−1 for A.2 alloy and 4.21 × 10−3 mm−1 for A.4 alloy. It is concluded that the isoconversion model that best represents the degradation of A.2 and A.4 alloys in Kokubo solution is the model of the kinetics mechanism of chemical control associated with the Arrhenius equation, which presents good correlations results comparing the experimental results with the proposed model.

Abstract Demand for iron ore worldwide has been steadily increasing, thus implying an increase in the volume of mining resulting in the extraction of ores with higher levels of silicon, phosphorus and sulfur impurities. An extensively used alternative is to perform a pre-treatment of the particles in order to reach the acceptable phosphorus limit in iron ore before its use in steel production. This study aims at the evaluation of the heating process and decrease of the phosphorus content of the particles using microwave energy in iron ore samples percolated by sulfuric acid followed by the leaching technique as a feasible route for the remotion of the phosphorus content in iron ore particles. Using the scanning electron microscopy (SEM) and statistical analysis techniques, it was possible to observe the characteristics of the iron ore particles after treatment with microwave energy. The results indicated that the developed process is feasible. The results of the kinetic simulation contribute to a better understanding of the phenomena of heating using microwave energy and also to the development of new cleaner technologies aiming to improve the efficiency of the acid baking - leaching technique for the reduction of the phosphorus content in iron ore particles.

Unlike the available mineral resources, the steelmaking processes have demanded raw material with lower phosphorus content to decrease the costs, energy use, and the residue generated within the steel plant. One alternative is to develop pretreatment of the iron ore concentrates producing raw materials with lower phosphorus. Depending on the mineral structure, a heat treatment combined with leaching could be an efficient way to achieve concentrates with low phosphorus (less than 0.01%), suitable for steelmaking processes. A fast and efficient way of applying energy to iron ore particles is the use of microwave to heat the particles. Thus, we propose a treatment using microwave heating while mixing with a dilute aqueous solution of sulfuric acid, followed by quenching during leaching with water, as a feasible route for the phosphorus removal from iron ore particles. We performed a design of experiment (DOE) to investigate the optimal conditions of heating and leaching, which maximize the rate of phosphorus removal. The structure of the iron ore particles after their treatment with microwave energy was observed by scanning electron microscopy (SEM). Thus, we analyze the optimal conditions for heating and leaching, the structure of the iron ore particles and which mechanism and rate equations are controlling the phosphorus removal. The results indicated that the average heating time is 10 min, the size of the crack of the order of 20µm and a leaching time of 8 min are adequate to achieve less than 0.01% of phosphorus. We demonstrated that under the most favorable combination of conditions for heating followed by leaching proposed in this study, the removal of the phosphorus content in the iron ore samples could reach 100%.

Welding is a widely used process that requires continuous developments to meet new application demands of mechanical projects under severe conditions. The homogeneity of metallurgical and mechanical properties in welded joints is the key factor for any welding process. The applications of external magnetic fields, mechanical vibration, and ultrasound are the fundamental steps to achieve success in improving these properties. The present work aimed at determining suitable processing conditions to achieve the desired balance between metallurgical and mechanical properties of 304L steel in TIG (Tungsten Inert Gas) welding under the application of an external magnetic field. The microstructural characteristics of the weld bead were analyzed by optical microscopy (OM) and scanning electron microscopy (SEM). In order to evaluate the mechanical properties of the welded specimen, its Vickers microhardness map and Charpy impact energy at −20 °C were obtained. In addition, corrosion tests were carried out in the saline medium to compare the corrosion resistance of the joint with that of the base metal and that without the magnetic field. It was found that the external magnetic field decreased the percentage of delta ferrite, improved the filling of the weld pool with the weld metal, and decreased the primary and secondary dendritic spacings. The Vickers microhardness value under the magnetic field was found to be lower than that without the magnetic field, and the Charpy test showed no significant variation in energy absorption. Moreover, the welded joint produced under the external magnetic field manifested less resistance to corrosion.

A chuva é um dos fatores meteorológicos que apresenta grande variação espacial e temporal. Diversos trabalhos ao ar livre são afetados de forma direta e indireta pela sua ausência ou excesso de precipitação. Este trabalho tem como objetivo caracterizar a distribuição sazonal e espacial da precipitação mensal e anual no Estado de Santa Catarina. Foram utilizados os dados de precipitação diária do período de 1970 a 2012 de 92 estações pluviométricas distribuídas no estado de Santa Catarina. Para estimar a precipitação provável em período mensal foi usada a distribuição gama e para a precipitação anual foram avaliadas as distribuições normal e gama. A aderência das séries de precipitação às distribuições de probabilidade foi avaliada pelo teste de Kolmogorov-Smirnov, ao nível de significância de 5%. Para representar a variação espacial e sazonal da precipitação foram gerados mapas das variáveis analisadas usando o software ArcGIS. Foram determinados os mapas de precipitação e anual com probabilidades de 25%, 50% e 75 %, em que se pode observar a variação sazonal e também espacial da precipitação no estado de Santa Catarina. Com base nos dados, observou-se que a distribuição Gama se mostrou adequada para estimar as probabilidades de chuva mensal. Para as séries de precipitação anual, embora ambas distribuições foram consideradas adequadas verificou-se que em 75% das estações o ajuste obtido com a distribuição gama foi melhor que a distribuição normal. A precipitação mensal e anual apresenta grande variação espacial e sazonal no estado de Santa Catarina. Nos meses de outubro a março as maiores precipitações mensais ocorrem na região do litoral norte e menor no litoral sul. Nos meses de abril a setembro observa-se maiores precipitações na região oeste do estado

Dihydrolipoamide dehydrogenase (DLD) deficiency is a recessive mitochondrial disease caused by variants in DLD, the E3 subunit of mitochondrial α-keto (or 2-oxo) acid dehydrogenase complexes. DLD disease symptoms are multisystemic, variably manifesting as Leigh syndrome, neurodevelopmental disability, seizures, cardiomyopathy, liver disease, fatigue, and lactic acidemia. While most DLD disease symptoms are attributed to dysfunction of the pyruvate dehydrogenase complex, the effects of other α-keto acid dehydrogenase deficiencies remain unclear. Current therapies for DLD deficiency are ineffective, with no vertebrate animal model available for preclinical study. We created a viable Danio rerio (zebrafish) KO model of DLD deficiency, dldhcri3. Detailed phenotypic characterization revealed shortened larval survival, uninflated swim bladder, hepatomegaly and fatty liver, and reduced swim activity. These animals displayed increased pyruvate and lactate levels, with severe disruption of branched-chain amino acid catabolism manifest as increased valine, leucine, isoleucine, α-ketoisovalerate, and α-ketoglutarate levels. Evaluation of mitochondrial ultrastructure revealed gross enlargement, severe cristae disruption, and reduction in matrix electron density in liver, intestines, and muscle. Therapeutic modeling of candidate therapies demonstrated that probucol or thiamine improved larval swim activity. Overall, this vertebrate model demonstrated characteristic phenotypic and metabolic alterations of DLD disease, offering a robust platform to screen and characterize candidate therapies.

N -linked glycoproteins function in numerous biological processes, modulating enzyme activities as well as protein folding, stability, oligomerization, and trafficking. While N -glycosylation of mitochondrial proteins has been detected by untargeted MS-analyses, the physiological existence and roles of mitochondrial protein N -linked glycosylation remain under debate. Here, we report that MRS2, a mitochondrial inner membrane protein that functions as the high flux magnesium transporter, is N -glycosylated to various extents depending on cellular bioenergetic status. Both N -glycosylated and unglycosylated isoforms were consistently detected in mitochondria isolated from mouse liver, rat and mouse liver fibroblast cells (BRL 3A and AFT024, respectively) as well as human skin fibroblast cells. Immunoblotting of MRS2 showed it was bound to, and required stringent elution conditions to remove from, lectin affinity columns with covalently bound concanavalin A or Lens culinaris agglutinin. Following peptide: N -glycosidase F (PNGase F) digestion of the stringently eluted proteins, the higher M r MRS2 bands gel-shifted to lower M r and loss of lectin affinity was seen. BRL 3A cells treated with two different N -linked glycosylation inhibitors, tunicamycin or 6-diazo-5-oxo-l-norleucine, resulted in decreased intensity or loss of the higher M r MRS2 isoform. To investigate the possible functional role of MRS2 N - glycosylation, we measured rapid Mg 2+ influx capacity in intact mitochondria isolated from BRL 3A cells in control media or following treatment with tunicamycin or 6-diazo-5-oxo-l-norleucine. Interestingly, rapid Mg 2+ influx capacity increased in mitochondria isolated from BRL 3A cells treated with either N -glycosylation inhibitor. Forcing reliance on mitochondrial respiration by treatment with either galactose media or the glycolytic inhibitor 2-deoxyglucose or by minimizing glucose concentration similarly reduced the N -glycosylated isoform of MRS2, with a correlated concomitant increase in rapid Mg 2+ influx capacity. Conversely, inhibiting mitochondrial energy production in BRL 3A cells with either rotenone or oligomycin resulted in an increased fraction of N -glycosylated MRS2, with decreased rapid Mg 2+ influx capacity. Collectively, these data provide strong evidence that MRS2 N -glycosylation is directly involved in the regulation of mitochondrial matrix Mg 2+ , dynamically communicating relative cellular nutrient status and bioenergetic capacity by serving as a physiologic brake on the influx of mitochondrial matrix Mg 2+ under conditions of glucose excess or mitochondrial bioenergetic impairment., Competing Interests: M L., D.I., C.R., P.K., C.B., N.D.M., R.X., C.S., E.N.O. and V.E.A., have no relevant financial disclosures. M.J.F. is an inventor on US Patent No. PCT/US 17/256,406 entitled, “Compositions and Methods for Treatment of Mitochondrial Respiratory Chain Dysfunction and Other Mitochondrial Disorders,” filed in the Name of The Children’s Hospital of Philadelphia on 12/28/20. M.J.F is co-founder and chief scientific advisor of Rarefy Therapeutics, a scientific advisory board member with equity interest in RiboNova, Inc., and scientific board member as paid consultant with Khondrion and with Larimar Therapeutics. M.J.F. has previously been or is currently engaged as a paid consultant with Abliva [formerly Neurovive], Astellas [formerly Mitobridge] Pharma Inc., Casma Therapeutics, Cyclerion Therapeutics, Epirium Bio, HealthCap VIII Advisor AB, Imel Therapeutics, Minovia Therapeutics, Reneo Therapeutics, Stealth BioTherapeutics, Taysha Therapeutics, Zogenix, Inc. and/or as a sponsored research collaborator with AADI Therapeutics, Astellas [formerly Mitobridge] Pharma Inc., Cyclerion Therapeutics, Epirium Bio [formerly Cardero Therapeutics], Imel Therapeutics, Merck, Minovia Therapeutics Inc., Mission Therapeutics, NeuroVive, Raptor Therapeutics, REATA Inc., Reneo Therapeutics, RiboNova Inc., Standigm Therapeutics, and Stealth BioTherapeutics. M.J.F has received royalties from Elsevier and a speaker’s honorarium from PlatformQ and Agios Pharma.

Background: Underutilization of therapies to reduce ischemic risk in peripheral artery disease (PAD) persists., Objectives: The purpose was to conduct an implementation trial of lipid management in vascular disease., Methods: The OPTIMIZE PAD-1 (Implementation of Vascular Care Team to Improve Medical Management of PAD Patients) trial randomized patients with peripheral artery disease with low-density lipoprotein cholesterol (LDL-C) ≥70 mg/dL to management via a vascular care team including a clinical pharmacist and an algorithm of intensive lipid management to achieve goal LDL-C in 1 step vs usual care plus provider education. Medications were obtained using commercial insurance. The primary endpoint was percent change in LDL-C at 12 months., Results: Of 166 enrolled patients, 74.2% did not have an LDL-C level at goal. Among 114 randomized patients (mean age 66 years, 36.0% women, and 15.8% Black), 50.9% received high-intensity statin, and 7.9% received ezetimibe at baseline. The mean 12-month LDL-C change was -49.1% (95% CI: -58.7% to -39.5%) with vascular care team management and -5.4% (95% CI: -15.3% to 4.6%) with usual care; the between-group least-squares mean difference was -43.7% (95% CI: -57.6% to -29.9%; P < 0.0001). Mean LDL-C was reduced in vascular care team patients from 100.6 mg/dL at baseline to 54.8 and 50.1 mg/dL by week 4 and month 12, respectively. At 12 months, vascular care team patients were >3 times as likely to achieve LDL-C <70 mg/dL and 8 times as likely to achieve LDL-C <55 mg/dL (P < 0.0001) than usual care., Conclusions: OPTIMIZE PAD-1 showed that an interprofessional, algorithm-based program can achieve rapid LDL-C lowering in vascular patients using available insurance and therapies, and LDL-C targets can be met in most patients if enabled by optimized systems of care., Competing Interests: Funding Support and Author Disclosures This was an investigator-initiated study funded through a research grant from Amgen to CPC Clinical Research and a Quality Initiative grant awarded by the American College of Cardiology Accreditation Services Foundation (to Dr Hess). The funders had no role in the design, data collection, data analysis, and reporting of this study. Ms Anderson, Drs Hess, Nehler, Szarek, Cannon, Hsia, and Bonaca all receive salary support from CPC, a nonprofit academic research organization affiliated with the University of Colorado, that receives or has received research grant/consulting funding between July 2021 and July 2023 from the following organizations: Agios Pharmaceuticals, Inc, Alexion Pharma Godo Kaisha, Amgen Inc, Anthos Therapeutics, Inc, ARCA biopharma, Inc, AstraZeneca Pharma India, AstraZeneca Pharmaceuticals LP, AstraZeneca UK Ltd, AstraZeneca, Produtos Farmaceuticos, Lda, Atentiv, LLC, Bayer, Bayer (Proprietary) Limited, Bayer Aktiengesellschaft, Bayer Pharma AG, Beth Israel Deaconess Medical Center, Better Therapeutics, Bionest Partners Inc, Boston Clinical Research Institute, LLC, Bristol Myers Squibb, CellResearch Corporation Pte Ltd, Cleerly, Inc, Colorado Dept of Public Health and Environment, Cook Regentec LLC, CSL Behring LLC, Eidos Therapeutics, Inc, EPG Communication Holdings Ltd, Esperion Therapeutics, Inc, Faraday Pharmaceuticals, Inc, HeartFlow Inc, Hummingbird Bioscience PTE Ltd, Insmed, Ionis Pharmaceuticals, IQVIA Inc, Janssen Pharmaceuticals, Inc, Janssen Research and Development, LLC, Janssen Scientific Affairs LLC, Lexicon Pharmaceuticals, Inc, LSG Corporation, MedImmune Limited, Medpace, Inc, Medscape, Merck Sharp and Dohme Corp, Northwell Health, Novartis Pharmaceuticals Corporation, Novo Nordisk, Osiris Therapeutics, Inc, Pfizer, PPD Development, LP, Prothena Biosciences Limited, Regeneron, Regents of the University of Colorado (aka UCD), Sanifit Therapeutics SA, Sanofi, Silence Therapeutics PLC, Stanford University, Stealth BioTherapeutics Inc, The Brigham and Women's Hospital, Inc, Thrombosis Research Institute, UCD iC42 Lab, University of Colorado Denver, University of Pittsburgh, VarmX, and WraSer, LLC. Dr Hess has received a research grant from the American College of Cardiology Accreditation Services Foundation. Dr Szarek has received consulting fees from Amarin, CiVi, NewAmsterdam Pharma, and Silence Therapeutics PLC. Dr Cannon from 2021-2023 has received research grants from Amgen, Better Therapeutics, Boehringer Ingelheim, Daiichi-Sankyo, Merck, Novo Nordisk, and Pfizer; has received consulting fees from Amryt/Chiesi, Alnylam, Amarin, Amgen, Applied Therapeutics, Ascendia, Biogen, Boehringer Ingelheim, Bristol Myers Squibb, CSL Behring, Eli Lilly, Janssen, Lexicon, Merck, Milestone, Pfizer, Rhoshan, and Sanofi; and is a member of the Data and Safety Monitoring Boards for Applied Therapeutics, Areteia, Novo Nordisk, and the Veterans Administration. Dr Hsia owns AstraZeneca stock. Dr Saseen is a member of the Amgen Lipid Monitoring Committee for the VESALIUS and OCEAN(a) outcome trials. Dr Bonaca has received support from the AHA SFRN under award numbers 18SFRN3390085 (BWH-DH SFRN Center) and 18SFRN33960262 (BWH-DH Clinical Project); and has stock in Medtronic and Pfizer. All other authors have reported that they have no relationships relevant to the contents of this paper to disclose., (Copyright © 2024 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.)

Background: Canonical α/β T-cell receptors (TCRs) bind to human leukocyte antigen (HLA) displaying antigenic peptides to elicit T cell-mediated cytotoxicity. TCR-engineered T-cell immunotherapies targeting cancer-specific peptide-HLA complexes (pHLA) are generating exciting clinical responses, but owing to HLA restriction they are only able to target a subset of antigen-positive patients. More recently, evidence has been published indicating that naturally occurring α/β TCRs can target cell surface proteins other than pHLA, which would address the challenges of HLA restriction. In this proof-of-concept study, we sought to identify and engineer so-called HLA-independent TCRs (HiTs) against the tumor-associated antigen mesothelin., Methods: Using phage display, we identified a HiT that bound well to mesothelin, which when expressed in primary T cells, caused activation and cytotoxicity. We subsequently engineered this HiT to modulate the T-cell response to varying levels of mesothelin on the cell surface., Results: The isolated HiT shows cytotoxic activity and demonstrates killing of both mesothelin-expressing cell lines and patient-derived xenograft models. Additionally, we demonstrated that HiT-transduced T cells do not require CD4 or CD8 co-receptors and, unlike a TCR fusion construct, are not inhibited by soluble mesothelin. Finally, we showed that HiT-transduced T cells are highly efficacious in vivo, completely eradicating xenografted human solid tumors., Conclusion: HiTs can be isolated from fully human TCR-displaying phage libraries against cell surface-expressed antigens. HiTs are able to fully activate primary T cells both in vivo and in vitro. HiTs may enable the efficacy seen with pHLA-targeting TCRs in solid tumors to be translated to cell surface antigens., Competing Interests: This study was funded by Adaptimmune. Adaptimmune contributed to study design, data analysis, decision to publish, and preparation of the manuscript. Writing and editorial support was funded by Adaptimmune. All authors are employees of Adaptimmune or were at the time of the study. Authors may hold stock in Adaptimmune. This does not alter our adherence to PLOS ONE policies on sharing data and materials, (Copyright: © 2024 Hiscox et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

SURF1 deficiency (OMIM # 220110) causes Leigh syndrome (LS, OMIM # 256000), a mitochondrial disorder typified by stress-induced metabolic strokes, neurodevelopmental regression and progressive multisystem dysfunction. Here, we describe two novel surf1-/- zebrafish knockout models generated by CRISPR/Cas9 technology. While gross larval morphology, fertility, and survival into adulthood appeared unaffected, surf1-/- mutants manifested adult-onset ocular anomalies and decreased swimming activity, as well as classical biochemical hallmarks of human SURF1 disease, including reduced complex IV expression and enzymatic activity and increased tissue lactate. surf1-/- larvae also demonstrated oxidative stress and stressor hypersensitivity to the complex IV inhibitor, azide, which exacerbated their complex IV deficiency, reduced supercomplex formation, and induced acute neurodegeneration typical of LS including brain death, impaired neuromuscular responses, reduced swimming activity, and absent heartrate. Remarkably, prophylactic treatment of surf1-/- larvae with either cysteamine bitartrate or N-acetylcysteine, but not other antioxidants, significantly improved animal resiliency to stressor-induced brain death, swimming and neuromuscular dysfunction, and loss of heartbeat. Mechanistic analyses demonstrated cysteamine bitartrate pretreatment did not improve complex IV deficiency, ATP deficiency, or increased tissue lactate but did reduce oxidative stress and restore glutathione balance in surf1-/- animals. Overall, two novel surf1-/- zebrafish models recapitulate the gross neurodegenerative and biochemical hallmarks of LS, including azide stressor hypersensitivity that was associated with glutathione deficiency and ameliorated by cysteamine bitartrate or N-acetylcysteine therapy., (© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Adoptive cell therapy with T cells expressing affinity-enhanced T-cell receptors (TCRs) is a promising treatment for solid tumors. Efforts are ongoing to further engineer these T cells to increase the depth and durability of clinical responses and broaden efficacy toward additional indications. In the present study, we investigated one such approach: T cells were transduced with a lentiviral vector to coexpress an affinity-enhanced HLA class I-restricted TCR directed against MAGE-A4 alongside a CD8α coreceptor. We hypothesized that this approach would enhance CD4 + T-cell helper and effector functions, possibly leading to a more potent antitumor response. Activation of transduced CD4 + T cells was measured by detecting CD40 ligand expression on the surface and cytokine and chemokine secretion from CD4 + T cells and dendritic cells cultured with melanoma-associated antigen A4 + tumor cells. In addition, T-cell cytotoxic activity against 3-dimensional tumor spheroids was measured. Our data demonstrated that CD4 + T cells coexpressing the TCR and CD8α coreceptor displayed enhanced responses, including CD40 ligand expression, interferon-gamma secretion, and cytotoxic activity, along with improved dendritic cell activation. Therefore, our study supports the addition of the CD8α coreceptor to HLA class I-restricted TCR-engineered T cells to enhance CD4 + T-cell functions, which may potentially improve the depth and durability of antitumor responses in patients., (Copyright © 2023 The Author(s). Published by Wolters Kluwer Health, Inc.)

Dihydrolipoamide dehydrogenase (DLD) deficiency is a recessive mitochondrial disorder caused by depletion of DLD from α-ketoacid dehydrogenase complexes. Caenorhabditis elegans animal models of DLD deficiency generated by graded feeding of dld-1(RNAi) revealed that full or partial reduction of DLD-1 expression recapitulated increased pyruvate levels typical of pyruvate dehydrogenase complex deficiency and significantly altered animal survival and health, with reductions in brood size, adult length, and neuromuscular function. DLD-1 deficiency dramatically increased mitochondrial unfolded protein stress response induction and adaptive mitochondrial proliferation. While ATP levels were reduced, respiratory chain enzyme activities and in vivo mitochondrial membrane potential were not significantly altered. DLD-1 depletion directly correlated with the induction of mitochondrial stress and impairment of worm growth and neuromuscular function. The safety and efficacy of dichloroacetate, thiamine, riboflavin, 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR), l-carnitine, and lipoic acid supplemental therapies empirically used for human DLD disease were objectively evaluated by life span and mitochondrial stress response studies. Only dichloroacetate and thiamine showed individual and synergistic therapeutic benefits. Collectively, these C. elegans dld-1(RNAi) animal model studies demonstrate the translational relevance of preclinical modeling of disease mechanisms and therapeutic candidates. Results suggest that clinical trials are warranted to evaluate the safety and efficacy of dichloroacetate and thiamine in human DLD disease.

Importance: Hospitalized patients with COVID-19 are at risk for venous and arterial thromboembolism and death. Optimal thromboprophylaxis dosing in high-risk patients is unknown., Objective: To evaluate the effects of therapeutic-dose low-molecular-weight heparin (LMWH) vs institutional standard prophylactic or intermediate-dose heparins for thromboprophylaxis in high-risk hospitalized patients with COVID-19., Design, Setting, and Participants: The HEP-COVID multicenter randomized clinical trial recruited hospitalized adult patients with COVID-19 with D-dimer levels more than 4 times the upper limit of normal or sepsis-induced coagulopathy score of 4 or greater from May 8, 2020, through May 14, 2021, at 12 academic centers in the US., Interventions: Patients were randomized to institutional standard prophylactic or intermediate-dose LMWH or unfractionated heparin vs therapeutic-dose enoxaparin, 1 mg/kg subcutaneous, twice daily if creatinine clearance was 30 mL/min/1.73 m2 or greater (0.5 mg/kg twice daily if creatinine clearance was 15-29 mL/min/1.73 m2) throughout hospitalization. Patients were stratified at the time of randomization based on intensive care unit (ICU) or non-ICU status., Main Outcomes and Measures: The primary efficacy outcome was venous thromboembolism (VTE), arterial thromboembolism (ATE), or death from any cause, and the principal safety outcome was major bleeding at 30 ± 2 days. Data were collected and adjudicated locally by blinded investigators via imaging, laboratory, and health record data., Results: Of 257 patients randomized, 253 were included in the analysis (mean [SD] age, 66.7 [14.0] years; men, 136 [53.8%]; women, 117 [46.2%]); 249 patients (98.4%) met inclusion criteria based on D-dimer elevation and 83 patients (32.8%) were stratified as ICU-level care. There were 124 patients (49%) in the standard-dose vs 129 patients (51%) in the therapeutic-dose group. The primary efficacy outcome was met in 52 of 124 patients (41.9%) (28.2% VTE, 3.2% ATE, 25.0% death) with standard-dose heparins vs 37 of 129 patients (28.7%) (11.7% VTE, 3.2% ATE, 19.4% death) with therapeutic-dose LMWH (relative risk [RR], 0.68; 95% CI, 0.49-0.96; P = .03), including a reduction in thromboembolism (29.0% vs 10.9%; RR, 0.37; 95% CI, 0.21-0.66; P < .001). The incidence of major bleeding was 1.6% with standard-dose vs 4.7% with therapeutic-dose heparins (RR, 2.88; 95% CI, 0.59-14.02; P = .17). The primary efficacy outcome was reduced in non-ICU patients (36.1% vs 16.7%; RR, 0.46; 95% CI, 0.27-0.81; P = .004) but not ICU patients (55.3% vs 51.1%; RR, 0.92; 95% CI, 0.62-1.39; P = .71)., Conclusions and Relevance: In this randomized clinical trial, therapeutic-dose LMWH reduced major thromboembolism and death compared with institutional standard heparin thromboprophylaxis among inpatients with COVID-19 with very elevated D-dimer levels. The treatment effect was not seen in ICU patients., Trial Registration: ClinicalTrials.gov Identifier: NCT04401293.

Coronavirus disease-2019 (COVID-19) has been associated with significant risk of venous thromboembolism (VTE), arterial thromboembolism (ATE), and mortality particularly among hospitalized patients with critical illness and elevated D-dimer (Dd) levels. Conflicting data have yet to elucidate optimal thromboprophylaxis dosing. HEP-COVID (NCT04401293) is a phase 3, multicenter, pragmatic, prospective, randomized, pseudo-blinded, active control trial to evaluate efficacy and safety of therapeutic-dose low-molecular-weight heparin (LMWH) versus prophylactic-/intermediate-dose LMWH or unfractionated heparin (UFH) for prevention of a primary efficacy composite outcome of VTE, ATE, and all-cause mortality 30 ± 2 days post-enrollment. Eligible patients have COVID-19 diagnosis by nasal swab or serologic testing, requirement for supplemental oxygen per investigator judgment, and Dd >4 × upper limit of normal (ULN) or sepsis-induced coagulopathy score ≥4. Subjects are randomized to enoxaparin 1 mg/kg subcutaneous (SQ)/two times a day (BID) (creatinine clearance [CrCl] ≥ 30 mL/min) or 0.5 mg/kg (CrCl 15-30 mL/min) versus local institutional prophylactic regimens including (1) UFH up to 22,500 IU (international unit) daily (divided BID or three times a day), (2) enoxaparin 30 and 40 mg SQ QD (once daily) or BID, or (3) dalteparin 2,500 IU or 5,000 IU QD. The principal safety outcome is major bleeding. Events are adjudicated locally. Based on expected 40% relative risk reduction with treatment-dose compared with prophylactic-dose prophylaxis, 308 subjects will be enrolled (assuming 20% drop-out) to achieve 80% power. Distinguishing design features include an enriched population for the composite endpoint anchored on Dd >4 × ULN, stratification by intensive care unit (ICU) versus non-ICU, and the ability to capture asymptomatic proximal deep venous thrombosis via screening ultrasonography prior to discharge., Competing Interests: M.G.: research grant from Janssen; M.C.: speakers bureau Sanofi; A.T.: educational grant: Janssen, research grant: Janssen, Bristol Meyers Squibb, Idorsia, consultant: Recovery Force, J.M.K.: consultant for Bayer Healthcare, Jansen, the ATLAS Group, AMAG pharmaceuticals, Agile Therapeutics for advisory and steering committee activities. Data and safety monitoring committees for Cystic Fibrosis Foundation Therapeutics, Novo Nordisk, Pfizer, Genentech, and BioMarin pharmaceuticals. M.B.: research grant support to CPC Clinical Research from Amgen, AstraZeneca, Bayer, Janssen, Merck, NovoNordisk; J.L.H.: consultancies with the ATLAS Group, Bayer Healthcare, Boehringer Ingelheim, and Population Health Research Institute; J.I.W.: consultant for Anthos, ATLAS Group, Bayer, Boehringer Ingelheim, Bristol Myers Squibb, Daiichi Sankyo, Ionis, Janssen, Johnson and Johnson, Merck, Pfizer, PhaseBio, and Servier, institutional research grants from Bayer and Boehringer Ingelheim; A.C.S.: Janssen, Boehringer Ingelheim, Bayer, BMS, Portola, ATLAS Group: consultancy; Janssen, Boehringer Ingelheim: research funding. D.G., W.D., J.W., S.K., G.S., J.M.L., A.M., E.G., P.A.L., K.C., M.L., C.P.S., H.R., K.O., W.R.H., R.A.D., V.E.A.: no conflict of interest., (Thieme. All rights reserved.)

Introduction: Few data exist to understand the recovery phase of pit viper envenomation. A recently published placebo-controlled clinical trial affords this opportunity. The purpose of this study is to examine the time course of recovery from copperhead snake (Agkistrodon contortrix) envenomation patients managed with and without the use of antivenom, stratified by age, sex, anatomic site of envenomation, initial severity of envenomation, and geographic region., Methods: This is a post-hoc subgroup analysis of data from a multi-center double-blinded clinical trial of Fab antivenom (FabAV) vs. placebo. Outcomes were the Patient-Specific Functional Scale (PSFS) score at 3, 7, 10, and 14 days after envenomation. Least-squares mean PSFS score curves were calculated for each subgroup, and repeated measures ANOVA was used to estimate between-group comparisons., Results: Seventy-two subjects were included, of whom 44 received FabAV. Males demonstrated better overall recovery than females (model predicted PSFS score 6.18 vs 4.99; difference 1.19; 95% CI 0.12 to 2.25; p = 0.029). No sex difference was found in response to FabAV. Overall recovery and effect of FabAV were similar in adult vs adolescent patients, patients with upper vs lower extremity envenomation, and patients with initially mild vs moderate envenomation signs. Analysis by geographic location was not successful due to ANOVA mode instability., Conclusions: Male victims of copperhead snake envenomation demonstrate slightly better recovery than females, but response to Fab antivenom overall is similar across all subgroups studied.

Objectives: Although more than 1.8 million people survive snakebite envenomation each year, their recovery is understudied. Obtaining long-term follow-up is challenging in both high- and low-resource settings. The Patient-Specific Functional Scale (PSFS) is an easily administered, well-accepted patient-reported outcome that is validated for assessing limb recovery from snakebite envenomation. We studied whether the PSFS is valid and reliable when administered by telephone., Methods: This is a secondary analysis of data from a randomized clinical trial. We analyzed the results of PSFS collected in-person on days 3, 7, 14, 21, and 28 and by telephone on days 10, 17, and 24. We assessed the following scale psychometric properties: (a) content validity (ceiling and floor effects), (b) internal structure and consistency (Cronbach's alpha), and (c) temporal and external validity using Intraclass Correlation Coefficient (ICC). Temporal stability was assessed using Spearman's correlation coefficient and agreement between adjacent in-person and telephonic assessments with Cohen's kappa. Bland Altman analysis was used to assess differential bias in low and high score results., Results: Data from 74 patients were available for analysis. Floor effects were seen in the early post-injury time points (median: 3 (IQR: 0, 5) at 3 days post-enrollment) and ceiling effects in the late time points (median: 9 (IQR: 8, 10). Internal consistency was good to excellent with both in-person (Cronbach α: 0.91 (95%CI 0.88, 0.95)) and telephone administration (0.81 (0.73, 0.89). Temporal stability was also good (ICC: 0.83 (0.72, 0.89) in-person, 0.80 (0.68, 0.88) telephone). A strong linear correlation was found between in-person and telephone administration (Spearman's ρ: 0.83 (CI: 0.78, 0.84), consistency was assessed as excellent (Cohen's κ 0.81 (CI: 0.78, 0.84), and Bland Altman analysis showed no systematic bias., Conclusions: Telephone administration of the PSFS provides valid, reliable, and consistent data for the assessment of recovery from snakebite envenomation., Competing Interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: Dr. Rose reports speakers bureau fees from BTG International Inc outside the submitted work. Dr. Greene reports receiving consulting fees and honoraria from BTGInternational Inc outside the submitted work. Drs. Gerardo, Charlton, Mullins, Lavonas and Kleinschmidt and Ms. Anderson report receiving prior study-related grants from BTG International Inc.

A substantial obstacle to the success of adoptive T cell-based cancer immunotherapy is the sub-optimal affinity of T-cell receptors (TCRs) for most tumor antigens. Genetically engineered TCRs that have enhanced affinity for specific tumor peptide-MHC complexes may overcome this barrier. However, this enhancement risks increasing weak TCR cross-reactivity to other antigens expressed by normal tissues, potentially leading to clinical toxicities. To reduce the risk of such adverse clinical outcomes, we have developed an extensive preclinical testing strategy, involving potency testing using 2D and 3D human cell cultures and primary tumor material, and safety testing using human primary cell and cell-line cross-reactivity screening and molecular analysis to predict peptides recognized by the affinity-enhanced TCR. Here, we describe this strategy using a developmental T-cell therapy, ADP-A2M4, which recognizes the HLA-A2-restricted MAGE-A4 peptide GVYDGREHTV. ADP-A2M4 demonstrated potent anti-tumor activity in the absence of major off-target cross-reactivity against a range of human primary cells and cell lines. Identification and characterization of peptides recognized by the affinity-enhanced TCR also revealed no cross-reactivity. These studies demonstrated that this TCR is highly potent and without major safety concerns, and as a result, this TCR is now being investigated in two clinical trials (NCT03132922, NCT04044768)., (© 2019 The Author(s). Published with license by Taylor & Francis Group, LLC.)

Study Objective: The antivenom currently available for treatment of systemic black widow envenomation (latrodectism) is composed of equine whole immunoglobin. Although considered effective, it has been associated with anaphylaxis and 2 reported fatalities. We test the efficacy and safety of new equine antivenom composed of purified F(ab') 2 antibody fragments., Methods: A randomized, double-blind, placebo-controlled trial was conducted at 16 sites across the United States. Subjects aged 10 years or older with moderate to severe pain because of black widow spider envenomation received F(ab') 2 antivenom or placebo. The primary outcome measure was treatment failure, which was defined as failure to achieve and maintain clinically significant reduction in pain for 48 hours posttreatment. Secondary measures of pain intensity differences and summed pain intensity difference were computed. Adverse events were recorded., Results: Sixty patients were treated (29 antivenom and 31 placebo). The mean age was 39 years and 68% were male. There were 15 treatment failures in the antivenom group and 24 in the placebo group (P=.019). Differences in pain intensity difference between groups were lower at each postbaseline point, and the mean summed pain intensity difference was greater for the antivenom group (difference 2,133; 95% confidence interval 177 to 4,090). No deaths or serious drug-related adverse events were detected., Conclusion: The F(ab') 2 antivenom met the predefined primary outcome of reduced treatment failures. Secondary outcomes of pain intensity difference and summed pain intensity difference also supported efficacy. The rate of symptom improvement in the placebo group was higher than expected, which may be related to enrollment criteria or placebo effect., (Copyright © 2019 American College of Emergency Physicians. Published by Elsevier Inc. All rights reserved.)

Introduction: Copperhead envenomation causes local tissue destruction, leading people to seek treatment for the pain and swelling. First-line treatment for the pain is opioid medications. There is rising concern that an initial opioid prescription from the emergency department (ED) can lead to long-term addiction. This analysis sought to determine whether use of Fab antivenom (FabAV) for copperhead envenomation affected opioid use., Methods: We performed a secondary analysis using data from a randomized clinical trial designed to determine the effect of FabAV on limb injury recovery following mild to moderate copperhead envenomation. Opioid use was a defined secondary outcome in the parent trial. Patients were contacted after discharge, and data were obtained regarding medications used for pain and the patients' functional status. This analysis describes the proportion of patients in each treatment group reporting opioid use at each time point. It also assesses the interaction between functional status and use of opioids., Results: We enrolled 74 patients in the parent trial (45 received FabAV, 29 placebo), of whom 72 were included in this secondary analysis. Thirty-five reported use of any opioids after hospital discharge. A smaller proportion of patients treated with FabAV reported opioid use: 40.9% vs 60.7% of those in the placebo group. The proportion of patients using opioids remained smaller in the FabAV group at each follow-up time point. Controlling for confounders and interactions between variables, the model estimated that the odds ratio of using opioids after hospital discharge among those who received placebo was 5.67 times that of those who received FabAV. Patients who reported higher baseline pain, those with moderate as opposed to mild envenomation, and females were more likely to report opioid use at follow-up. Patients with ongoing limitations to functional status had an increased probability of opioid use, with a stronger association over time. Opioid use corresponded with the trial's predefined criteria for full recovery, with only two patients reporting opioid use in the 24 hours prior to achieving full limb recovery and no patients in either group reporting opioid use after full limb recovery., Conclusion: In this study population, the proportion of patients using opioids for pain related to envenomation was smaller in the FabAV treatment group at all follow-up time points., Competing Interests: Conflicts of Interest: By the WestJEM article submission agreement, all authors are required to disclose all affiliations, funding sources and financial or management relationships that could be perceived as potential sources of bias. The original trial was funded by BTG International Inc. Funding for the statistical analysis for this secondary analysis was also provided by BTG International Inc. Drs Gerardo and Lavonas report receiving grants from BTG during the original trial and outside of the submitted work. Dr. Kleinschmidt and Ms. Anderson report receiving study-related funds from BTG.

Patients with hepatocellular carcinoma (HCC) have a poor prognosis and limited therapeutic options. Alpha-fetoprotein (AFP) is often expressed at high levels in HCC and is an established clinical biomarker of the disease. Expression of AFP in nonmalignant liver can occur, particularly in a subset of progenitor cells and during chronic inflammation, at levels typically lower than in HCC. This cancer-specific overexpression indicates that AFP may be a promising target for immunotherapy. We verified expression of AFP in normal and diseased tissue and generated an affinity-optimized T-cell receptor (TCR) with specificity to AFP/HLA-A*02 + tumors. Expression of AFP was investigated using database searches, by qPCR, and by immunohistochemistry (IHC) analysis of a panel of human tissue samples, including normal, diseased, and malignant liver. Using in vitro mutagenesis and screening, we generated a TCR that recognizes the HLA-A*02-restricted AFP 158-166 peptide, FMNKFIYEI, with an optimum balance of potency and specificity. These properties were confirmed by an extension of the alanine scan (X-scan) and testing TCR-transduced T cells against normal and tumor cells covering a variety of tissues, cell types, and human leukocyte antigen (HLA) alleles. Conclusion: We have used a combination of physicochemical, in silico, and cell biology methods for optimizing a TCR for improved affinity and function, with properties that are expected to allow TCR-transduced T cells to differentiate between antigen levels on nonmalignant and cancer cells. T cells transduced with this TCR constitute the basis for a trial of HCC adoptive T-cell immunotherapy., (© 2018 The Authors. Hepatology published by Wiley Periodicals, Inc., on behalf of American Association for the Study of Liver Diseases.)

Objective: Valid, reliable, and clinically relevant outcome measures are necessary in clinical studies of snake envenomation. The aim of this study was to evaluate the psychometric (validity and reliability) and clinimetric (minimal clinically important difference [MCID]) properties of the Patient-Specific Functional Scale (PSFS) in snakebite envenomation., Methods: We performed a secondary analysis of two existing snakebite trials that measured clinical outcomes using the PSFS as well as other quality of life and functional assessments. Data were collected at 3, 7, 10, and 17 days. Reliability was determined using Cronbach's alpha for internal consistency and the intraclass correlation coefficient (ICC) for temporal stability at 10 and 17 days. Validity was assessed using concurrent validity correlating with the other assessments. The MCID was evaluated using the following criteria: (1) the distribution of stable patients according to both standard error of measurement (SEM) and responsiveness techniques, and (2) anchor-based methods to compare between individuals and to detect discriminant ability of a positive change with a receiver operator characteristic (ROC) curve and optimal cutoff point., Results: A total of 86 patients were evaluated in this study. The average PSFS scores were 5.37 (SD 3.23), 7.95 (SD 2.22), and 9.12 (SD 1.37) at 3, 7, and 10 days, respectively. Negligible floor effect was observed (maximum of 8% at 3 days); however, a ceiling effect was observed at 17 days (25%). The PSFS showed good reliability with an internal consistency of 0.91 (Cronbach's alpha) (95% CI 0.88, 0.95) and a temporal stability of 0.83 (ICC) (95% CI 0.72, 0.89). The PSFS showed a strong positive correlation with quality of life and functional assessments. The MCID was approximately 1.0 for all methods., Conclusions: With an MCID of approximately 1 point, the PSFS is a valid and reliable tool to assess quality of life and functionality in patients with snake envenomation., Competing Interests: Dr. Rose reports speakers bureau fees from BTG International Inc outside the submitted work. Dr. Greene reports receiving consulting fees and honoraria from BTGInternational Inc outside the submitted work. Drs. Gerardo, Charlton, Mullins, Lavonas and Kleinschmidt and Ms. Anderson report receiving prior study-related grants from BTG International Inc. The parent studies from which data were derived, were funded by BTG International Inc. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Background: No previous research has studied whether early snake antivenom administration leads to better clinical outcomes than late antivenom administration in North American pit viper envenomation., Methods: A secondary analysis of data from a clinical trial of Fab antivenom (FabAV) versus placebo for copperhead snake envenomation was conducted. Patients treated before the median time to FabAV administration were classified as receiving early treatment and those treated after the median time were defined as the late treatment group. A Cox proportional hazards model was used to compare time to full recovery on the Patient-Specific Functional Scale (PSFS) instrument between groups. Secondary analyses compared estimated mean PSFS scores using a generalized linear model and the estimated proportion of patients with full recovery at each time point using logistic regression. To evaluate for confounding, the main analysis was repeated using data from placebo-treated subjects., Results: Forty-five subjects were treated with FabAV at a median of 5.47 h after envenomation. Patients in the early treatment group had a significantly shorter time to full recovery than those treated late (median time: 17 versus 28 days, p = .025). Model-estimated PSFS scores were numerically higher at each time point in the early group. No difference was found between patients treated early versus late with placebo., Conclusions: In this secondary analysis of trial data, recovery of limb function was faster when Fab antivenom was administered soon after envenomation, as opposed to late administration.

Objective: To compare the incidence of hypersensitivity reactions following copperhead envenomation treated with Fab antivenom (FabAV) or placebo., Methods: Patients with copperhead snakebites received treatment and follow-up in a prospective, randomized, double-blind, placebo-controlled trial of FabAV or placebo. The treatment allocation ratio was 2:1 (FabAV:placebo). All of the included patients received at least one dose of study treatment. We reviewed all treatment-emergent adverse events (AEs) using a previously published scale to classify likely hypersensitivity reactions as mild, moderate, or severe., Results: We enrolled 74 patients at 13 sites. Forty-five patients received FabAV, and 29 patients received placebo. Five FabAV patients and 4 placebo patients had moderate envenomations; the rest were mild. Twenty-five FabAV patients and 8 placebo patients had at least 1 AE. Mild skin reactions occurred in 11 (24%) FabAV patients (pruritis, urticaria, rash, ecchymosis, erythema) and 1 (3%) placebo patient (pruritis). Moderate gastrointestinal AEs occurred in 7 (16%) FabAV patients (nausea, vomiting, constipation, diarrhea, oral paresthesia) and in 2 (7%) placebo patients (nausea). Respiratory AEs occurred in 3 (7%) FabAV patients (dyspnea, pulmonary embolism, nasal congestion, sneezing) and no placebo patients. Hypotension occurred in 1 patient in each group., Conclusions: In a randomized controlled trial of FabAV for copperhead bites, the incidence of hypersensitivity reactions was low. Most reactions were mild skin reactions.

Context: Serum paracetamol-protein adducts (PPAs) are a novel potential biomarker of paracetamol exposure. The relationship between serum PPA concentrations and reported paracetamol use in ambulatory adults has not been previously described., Materials and Methods: This was a cross-sectional study of ambulatory adults. A detailed medication history was obtained from all subjects and subjects were stratified by reported paracetamol use in the 2 weeks prior to enrolment. Serum PPAs were measured in all subjects and correlated with reported dose, time of last ingestion and demographics., Results: We enrolled 230 in the paracetamol exposure arm and 74 in the no exposure arm. 98/230 (42.6%)of subjects who reported paracetamol exposure had PPA detected and 68/74 (91.9%) of subjects who denied paracetamol exposure had no PPA detected. PPA concentrations were positively correlated with total paracetamol dose and with more recent ingestion., Discussion: Detection of serum PPA generally reflects paracetamol exposure histories in ambulatory adults. Concentrations are well correlated with reported dose and time from last dose., Conclusions: Serum PPA can be detected with reported therapeutic use of paracetamol but may not be detected in all patients who report taking paracetamol.

We provide recommendations for stocking of antidotes used in emergency departments (EDs). An expert panel representing diverse perspectives (clinical pharmacology, medical toxicology, critical care medicine, hematology/oncology, hospital pharmacy, emergency medicine, emergency medical services, pediatric emergency medicine, pediatric critical care medicine, poison centers, hospital administration, and public health) was formed to create recommendations for antidote stocking. Using a standardized summary of the medical literature, the primary reviewer for each antidote proposed guidelines for antidote stocking to the full panel. The panel used a formal iterative process to reach their recommendation for both the quantity of antidote that should be stocked and the acceptable timeframe for its delivery. The panel recommended consideration of 45 antidotes; 44 were recommended for stocking, of which 23 should be immediately available. In most hospitals, this timeframe requires that the antidote be stocked in a location that allows immediate availability. Another 14 antidotes were recommended for availability within 1 hour of the decision to administer, allowing the antidote to be stocked in the hospital pharmacy if the hospital has a mechanism for prompt delivery of antidotes. The panel recommended that each hospital perform a formal antidote hazard vulnerability assessment to determine its specific need for antidote stocking. Antidote administration is an important part of emergency care. These expert recommendations provide a tool for hospitals that offer emergency care to provide appropriate care of poisoned patients., (Copyright © 2017 American College of Emergency Physicians. Published by Elsevier Inc. All rights reserved.)

Study Objective: Copperhead snake (Agkistrodon contortrix) envenomation causes limb injury resulting in pain and disability. It is not known whether antivenom administration improves limb function. We determine whether administration of antivenom improves recovery from limb injury in patients envenomated by copperhead snakes., Methods: From August 2013 through November 2015, we performed a multicenter, randomized, double-blind, placebo-controlled, clinical trial to evaluate the effect of ovine Crotalidae polyvalent immune Fab (ovine) (CroFab; FabAV) antivenom therapy on recovery of limb function in patients with copperhead snake envenomation at 14 days postenvenomation. The study setting was 18 emergency departments in regions of the United States where copperhead snakes are endemic. Consecutive patients aged 12 years or older with mild- to moderate-severity envenomation received either FabAV or placebo. The primary outcome was limb function 14 days after envenomation, measured by the Patient-Specific Functional Scale. Additional outcomes included the Patient-Specific Functional Scale at other points; the Disorders of the Arm, Shoulder, and Hand, Lower Extremity Functional Scale, and Patient's Global Impression of Change instruments; grip strength; walking speed; quality of life (Patient-Reported Outcomes Measurement Information System Physical Fucntion-10); pain; and analgesic use., Results: Seventy-four patients received study drug (45 FabAV, 29 placebo). Mean age was 43 years (range 12 to 86 years). Fifty-three percent were men, 62% had lower extremity envenomation, and 88% had mild initial severity. The primary outcome, the least square mean Patient-Specific Functional Scale score at 14 days postenvenomation, was 8.6 for FabAV-treated subjects and 7.4 for placebo recipients (difference 1.2; 95% confidence interval 0.1 to 2.3; P=.04). Additional outcome assessments generally favored FabAV. More FabAV-treated subjects experienced treatment-emergent adverse events (56% versus 28%), but few were serious (1 in each group)., Conclusion: Treatment with FabAV reduces limb disability measured by the Patient-Specific Functional Scale 14 days after copperhead envenomation., (Copyright © 2017 American College of Emergency Physicians. Published by Elsevier Inc. All rights reserved.)

The breaking of RNA strands by 2'-O-transphosphorylation is a ubiquitous reaction in biology, and enzymes that catalyze this reaction play key roles in RNA metabolism. The mechanisms of 2'-O-transphosphorylation in solution are relatively well studied, but complex and can involve different transition states depending on how the reaction is catalyzed. Because of this complexity and the lack of experimental information on transition-state structure, pinning down the chemical details of enzyme-catalyzed RNA strand cleavage has been difficult. Kinetic isotope effects (KIEs) provide information about changes in bonding as a reaction proceeds from ground state to transition state, and therefore they provide a powerful tool for revealing mechanistic detail. Application of kinetic isotope analyses to RNA 2'-O-transphosphorylation faces three fundamental challenges: synthesis of RNA substrate isotopomers with 18 O substitutions at the 2'-O, 5'-O and nonbridging phosphoryl oxygens; determination of the 18 O/ 16 O ratios in the residual unreacted substrate or product RNAs; and analyzing these data to allow calculation of the KIEs for use in evaluating different mechanistic scenarios. In this chapter, we outline methods for surmounting these challenges for solution RNA 2'-O-transphosphorylation reactions, and we describe their initial application to understand nonenzymatic solution reactions and reactions catalyzed by the enzyme ribonuclease A., (© 2017 Elsevier Inc. All rights reserved.)

Study Objective: Medication histories are collected to measure drug exposure in epidemiologic studies, to identify adverse drug events and in clinical practice. The objective of this study was to compare the accuracy of a structured medication history obtained by using the Medication History Assessment Tool (MedHAT) with a medication diary in which subjects recorded real-time medication use., Design: Prospective observational study., Setting: Outpatient research center., Subjects: Sixty-seven adult healthy volunteers., Interventions: Subjects were provided diaries to record the product name, dose quantity, and time that they used a prescription or nonprescription medication, supplement, or vitamin. After a minimum of 30 continuous days of diary use, a final interview was conducted, and medication history data were collected by using the MedHAT., Measurements and Main Results: We compared the medications reported during the interview with the medications recorded in the diary for each day to determine the accuracy of the verbal history. The primary outcome was the proportion of matches for each day for each subject. Overall accuracy for medication use for the day preceding the interview was 90%, and accuracy during the 30 days preceding the interview was 76%. Recall was higher for subjects taking prescription medications, those who had more recent medication use, older subjects, and subjects taking a higher proportion of prescription medications., Conclusion: The MedHAT system provided relatively high accuracy for immediate past use and for prescription medications and may offer better accuracy than other methods. Medication histories are often inaccurate, however, and may represent an important source of misclassification in epidemiologic studies. Thus medication histories alone should be used cautiously when measuring associations between drug exposure and health outcomes., (© 2016 Pharmacotherapy Publications, Inc.)

Solvent D2O and (18)O kinetic isotope effects on RNA 2'-O-transphosphorylation catalyzed by Zn(2+) demonstrate an altered transition state relative to specific base catalysis. A recent model from DFT calculations involving inner sphere coordination to the non-bridging and leaving group oxygens is consistent with the data.

Background: IV acetaminophen at 4 g per day is considered safe, producing no hepatic failure in more than 1400 cases. Oxidation of acetaminophen forms a reactive intermediate that binds to cellular proteins resulting in acetaminophen-protein adducts (APAP-CYS). Serum concentrations of APAP-CYS have been found to correlate with acetaminophen-induced hepatotoxicity. We report a case of hepatotoxicity associated with therapeutic doses of IV acetaminophen, with elevated serum APAP-CYS., Case Details: The patient was a 92-year-old, 68 kg woman without known hepatic disease or ethanol abuse. On hospital day 3 she underwent laparoscopic reduction of internal hernias under general anesthesia. Surgery was uncomplicated and postoperatively she was treated with subcutaneous heparin and IV acetaminophen, 1 g every 6 h for almost 4 days (total dose = 13 g). At the start of therapy, transaminases were normal. On hospital day 5, she was noted to have marked transaminase elevations (AST: 4698 IU/L; ALT: 3914 IU/L) with increases in INR (1.68), ammonia (60 mcg/dL), and total bilirubin (1.8 mg/dL). Serum acetaminophen concentration was 15.3 mcg/mL 26 h after her last dose. Acetaminophen was discontinued and IV acetylcysteine was given and continued at the second maintenance dose rate for a second 16-hour infusion, at which time transaminases, INR, ammonia and total bilirubin were all improving. The patient was discharged 2 days later. Serum APAP-CYS concentrations in serum samples obtained during her hospitalization were elevated (peak = 4.81 μM on hospital day 5; expected range for therapeutic dosing <1.1 μM)., Case Discussion: We have identified a case of acute liver injury associated with therapeutic dosing of IV acetaminophen. The serum APAP-CYS concentrations are consistent with that seen in cases of hepatotoxicity following repeated supratherapeutic acetaminophen ingestion. Several factors that likely contributed to her susceptibility included advanced age, post-operative status, a likely catabolic state and multiple acetaminophen doses over several days. These uncommon circumstances limit the generalizability of risk. We believe the findings are most consistent with acetaminophen-induced liver injury., Conclusion: This case illustrates a potential hazard of IV acetaminophen and demonstrates the potential utility of APAP-CYS adducts in evaluating causality in acute liver injury.

The specificity of enzymes for their respective substrates has been a focal point of enzyme kinetics since the initial characterization of metabolic chemistry. Various processes to quantify an enzyme's specificity using kinetics have been utilized over the decades. Fersht's definition of the ratio kcat/Km for two different substrates as the "specificity constant" (ref [7]), based on the premise that the important specificity existed when the substrates were competing in the same reaction, has become a consensus standard for enzymes obeying Michaelis-Menten kinetics. The expansion of the theory for the determination of the relative specificity constants for a very large number of competing substrates, e.g. those present in a combinatorial library, in a single reaction mixture has been developed in this contribution. The ratio of kcat/Km for isotopologs has also become a standard in mechanistic enzymology where kinetic isotope effects have been measured by the development of internal competition experiments with extreme precision. This contribution extends the theory of kinetic isotope effects to internal competition between three isotopologs present at non-tracer concentrations in the same reaction mix. This article is part of a special issue titled: Enzyme Transition States from Theory and Experiment., (Published by Elsevier B.V.)

We developed an isotopic technique to assess mitochondrial acetyl-CoA turnover (≈citric acid flux) in perfused rat hearts. Hearts are perfused with buffer containing tracer [(13)C2,(2)H3]acetate, which forms M5 + M4 + M3 acetyl-CoA. The buffer may also contain one or two labeled substrates, which generate M2 acetyl-CoA (e.g. [(13)C6]glucose or [1,2-(13)C2]palmitate) or/and M1 acetyl-CoA (e.g. [1-(13)C]octanoate). The total acetyl-CoA turnover and the contributions of fuels to acetyl-CoA are calculated from the uptake of the acetate tracer and the mass isotopomer distribution of acetyl-CoA. The method was applied to measurements of acetyl-CoA turnover under different conditions (glucose ± palmitate ± insulin ± dichloroacetate). The data revealed (i) substrate cycling between glycogen and glucose-6-P and between glucose-6-P and triose phosphates, (ii) the release of small excess acetyl groups as acetylcarnitine and ketone bodies, and (iii) the channeling of mitochondrial acetyl-CoA from pyruvate dehydrogenase to carnitine acetyltransferase. Because of this channeling, the labeling of acetylcarnitine and ketone bodies released by the heart are not proxies of the labeling of mitochondrial acetyl-CoA., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Nucleic-acid-binding proteins are generally viewed as either specific or nonspecific, depending on characteristics of their binding sites in DNA or RNA. Most studies have focused on specific proteins, which identify cognate sites by binding with highest affinities to regions with defined signatures in sequence, structure or both. Proteins that bind to sites devoid of defined sequence or structure signatures are considered nonspecific. Substrate binding by these proteins is poorly understood, and it is not known to what extent seemingly nonspecific proteins discriminate between different binding sites, aside from those sequestered by nucleic acid structures. Here we systematically examine substrate binding by the apparently nonspecific RNA-binding protein C5, and find clear discrimination between different binding site variants. C5 is the protein subunit of the transfer RNA processing ribonucleoprotein enzyme RNase P from Escherichia coli. The protein binds 5' leaders of precursor tRNAs at a site without sequence or structure signatures. We measure functional binding of C5 to all possible sequence variants in its substrate binding site, using a high-throughput sequencing kinetics approach (HITS-KIN) that simultaneously follows processing of thousands of RNA species. C5 binds different substrate variants with affinities varying by orders of magnitude. The distribution of functional affinities of C5 for all substrate variants resembles affinity distributions of highly specific nucleic acid binding proteins. Unlike these specific proteins, C5 does not bind its physiological RNA targets with the highest affinity, but with affinities near the median of the distribution, a region that is not associated with a sequence signature. We delineate defined rules governing substrate recognition by C5, which reveal specificity that is hidden in cellular substrates for RNase P. Our findings suggest that apparently nonspecific and specific RNA-binding modes may not differ fundamentally, but represent distinct parts of common affinity distributions.

Improving patient outcome by personalized therapy involves a thorough understanding of an agent's mechanism of action. β-Lapachone (clinical forms, Arq501/Arq761) has been developed to exploit dramatic cancer-specific elevations in the phase II detoxifying enzyme NAD(P)H:quinone oxidoreductase (NQO1). NQO1 is dramatically elevated in solid cancers, including primary and metastatic [e.g., triple-negative (ER-, PR-, Her2/Neu-)] breast cancers. To define cellular factors that influence the efficacy of β-lapachone using knowledge of its mechanism of action, we confirmed that NQO1 was required for lethality and mediated a futile redox cycle where ∼120 moles of superoxide were formed per mole of β-lapachone in 2 minutes. β-Lapachone induced reactive oxygen species (ROS), stimulated DNA single-strand break-dependent poly(ADP-ribose) polymerase-1 (PARP1) hyperactivation, caused dramatic loss of essential nucleotides (NAD(+)/ATP), and elicited programmed necrosis in breast cancer cells. Although PARP1 hyperactivation and NQO1 expression were major determinants of β-lapachone-induced lethality, alterations in catalase expression, including treatment with exogenous enzyme, caused marked cytoprotection. Thus, catalase is an important resistance factor and highlights H2O2 as an obligate ROS for cell death from this agent. Exogenous superoxide dismutase enhanced catalase-induced cytoprotection. β-Lapachone-induced cell death included apoptosis-inducing factor (AIF) translocation from mitochondria to nuclei, TUNEL+ staining, atypical PARP1 cleavage, and glyceraldehyde 3-phosphate dehydrogenase S-nitrosylation, which were abrogated by catalase. We predict that the ratio of NQO1:catalase activities in breast cancer versus associated normal tissue are likely to be the major determinants affecting the therapeutic window of β-lapachone and other NQO1 bioactivatable drugs., (©2013 AACR.)