Hydrogen Sulfide Delivery to Enhance Bone Tissue Engineering Cell Survival.

Though crucial for natural bone healing, local calcium ion (Ca2+) and phosphate ion (Pi) concentrations can exceed the cytotoxic limit leading to mitochondrial overload, oxidative stress, and cell death. For bone tissue engineering applications, H2S can be employed as a cytoprotective molecule to enhance mesenchymal stem cell (MSC) tolerance to cytotoxic Ca2+/Pi concentrations. Varied concentrations of sodium hydrogen sulfide (NaSH), a fast-releasing H2S donor, were applied to assess the influence of H2S on MSC proliferation. The results suggested a toxicity limit of 4 mM for NaSH and that 1 mM of NaSH could improve cell proliferation and differentiation in the presence of cytotoxic levels of Ca2+ (32 mM) and/or Pi (16 mM). To controllably deliver H2S over time, a novel donor molecule (thioglutamic acid-GluSH) was synthesized and evaluated for its H2S release profile. Excitingly, GluSH successfully maintained cytoprotective level of H2S over 7 days. Furthermore, MSCs exposed to cytotoxic Ca2+/Pi concentrations in the presence of GluSH were able to thrive and differentiate into osteoblasts. These findings suggest that the incorporation of a sustained H2S donor such as GluSH into CaP-based bone graft substitutes can facilitate considerable cytoprotection, making it an attractive option for complex bone regenerative engineering applications.

Motor unit firing rates increase in prepubescent youth following linear periodization resistance exercise training.

PURPOSE: The purpose was to examine the effects of 8-weeks (3 days/week) of linear periodization resistance exercise training (RET) on neuromuscular function in prepubescent youth. METHODS: Twenty-five healthy prepubescent youth (11 males, 14 females, age = 9.1 ± 0.8 years) completed the RET (n = 17) or served as controls (CON, n = 8). Isometric maximal voluntary contractions (MVCs) and trapezoidal submaximal contractions at 35 and 60% MVC of the right leg extensors were performed with surface electromyography (EMG) recorded from the leg extensors [vastus lateralis (VL), rectus femoris, and vastus medialis] and flexors (biceps femoris and semitendinosus). EMG amplitude of the leg extensors and flexors were calculated during the MVCs. Motor unit (MU) action potential trains were decomposed from the surface EMG of the VL for the 35 and 60% MVCs. MU firing rates and action potential amplitudes were regressed against recruitment threshold with the y-intercepts and slopes calculated for each contraction. Total leg extensor muscle cross-sectional area (CSA) was collected using ultrasound images. ANOVA models were used to examine potential differences. RESULTS: Isometric strength increased post-RET (P = 0.006) with no changes in leg extensor and flexor EMG amplitude. Furthermore, there were no changes in total CSA or the MU action potential amplitude vs. recruitment threshold relationships. However, there were increases in the firing rates of the higher-threshold MUs post-RET as indicated with greater y-intercepts (P = 0.003) from the 60% MVC and less negative slope (P = 0.004) of the firing rates vs. recruitment threshold relationships at 35% MVC. CONCLUSIONS: MU adaptations contribute to strength increases following RET in prepubescent youth.

Multimodal exercise training to reduce frailty in people with multiple sclerosis: study protocol for a pilot randomized controlled trial.

BACKGROUND: Frailty, a syndrome characterized by decreased reserve and resistance to stressors across multiple physiologic systems, is highly prevalent in people living with multiple sclerosis (pwMS), independent of age or disability level. Frailty in MS is strongly associated with adverse clinical outcomes, such as falls, and may aggravate MS-related symptoms. Consequently, there is a pressing necessity to explore and evaluate strategies to reduce frailty levels in pwMS. The purpose of this pilot randomized controlled trial (RCT) will be to examine the feasibility and preliminary efficacy of a multimodal exercise training program to reduce frailty in pwMS. METHODS: A total of 24 participants will be randomly assigned to 6 weeks of multimodal exercise or to a waitlist control group with a 1:1 allocation. PwMS aged 40-65 years and living with frailty will be eligible. The multimodal exercise program will consist of cognitive-motor rehabilitation (i.e., virtual reality treadmill training) combined with progressive, evidence-based resistance training. At baseline and post-intervention, participants will complete the Evaluative Frailty Index for Physical Activity (EFIP), measures of fall risk, and quality of life. Frailty-related biomarkers will also be assessed. In addition, the feasibility of the multimodal exercise program will be systematically and multidimensionally evaluated. DISCUSSION: To date, no RCT has yet been conducted to evaluate whether targeted exercise interventions can minimize frailty in MS. The current study will provide novel data on the feasibility and preliminary efficacy of multimodal exercise training as a strategy for counteracting frailty in pwMS. TRIAL REGISTRATION: ClinicalTrials.gov, NCT06042244 (registered in September 2023).

Effects of low-load resistance training with blood flow restriction on muscle fiber myofibrillar and extracellular area.

Blood flow restriction applied during low-load resistance training (LL-BFR) induces a similar increase in the cross-sectional area of muscle fibers (fCSA) compared to traditional high-load resistance training (HL-RT). However, it is unclear whether LL-BFR leads to differential changes in myofibrillar spacing in muscle fibers and/or extracellular area compared to HL-RT. Therefore, this study aimed to investigate whether the hypertrophy of type I and II fibers induced by LL-BFR or HL-RT is accompanied by differential changes in myofibrillar and non-myofibrillar areas. In addition, we examined if extracellular spacing was differentially affected between these two training protocols. Twenty recreationally active participants were assigned to LL-BFR or HL-RT groups and underwent a 6-week training program. Muscle biopsies were taken before and after the training period. The fCSA of type I and II fibers, the area occupied by myofibrillar and non-myofibrillar components, and extracellular spacing were analyzed using immunohistochemistry techniques. Despite the significant increase in type II and mean (type I + II) fCSA (p < 0.05), there were no significant changes in the proportionality of the myofibrillar and non-myofibrillar areas [∼86% and ∼14%, respectively (p > 0.05)], indicating that initial adaptations to LL-BFR are primarily characterized by conventional hypertrophy rather than disproportionate non-myofibrillar expansion. Additionally, extracellular spacing was not significantly altered between protocols. In summary, our study reveals that LL-BFR, like HL-RT, induces skeletal muscle hypertrophy with proportional changes in the areas occupied by myofibrillar, non-myofibrillar, and extracellular components.

Identification of temporal shifts of oral bacteria in bone regeneration following mandibular bone defect injury and therapeutic surgery in a porcine model.

BACKGROUND: Considered the second largest and most diverse microbiome after the gut, the human oral ecosystem is complex with diverse and niche-specific microorganisms. Although evidence is growing for the importance of oral microbiome in supporting a healthy immune system and preventing local and systemic infections, the influence of craniomaxillofacial (CMF) trauma and routine reconstructive surgical treatments on community structure and function of oral resident microbes remains unknown. CMF injuries affect a large number of people, needing extensive rehabilitation with lasting morbidity and loss of human productivity. Treatment efficacy can be complicated by the overgrowth of opportunistic commensals or multidrug-resistant pathogens in the oral ecosystem due to weakened host immune function and reduced colonization resistance in a dysbiotic oral microbiome. AIMS: To understand the dynamics of microbiota's community structure during CMF injury and subsequent treatments, we induced supra-alveolar mandibular defect in Hanford miniature swine (n = 3) and compared therapeutic approaches of immediate mandibullar reconstructive (IMR) versus delayed mandibullar reconstructive (DMR) surgeries. METHODS: Using bacterial 16S ribosomal RNA gene marker sequencing, the composition and abundance of the bacterial community of the uninjured maxilla (control) and the injured left mandibula (lingual and buccal) treated by DMR were surveyed up to 70-day post-wounding. For the injured right mandibula receiving IMR treatment, the microbial composition and abundance were surveyed up to 14-day post-wounding. Moreover, we measured sera level of biochemical markers (e.g., osteocalcin) associated with bone regeneration and healing. Computed tomography was used to measure and compare mandibular bone characteristics such as trabecular thickness between sites receiving DMR and IMR therapeutic approaches until day 140, the end of study period. RESULTS: Independent of IMR versus DMR therapy, we observed similar dysbiosis and shifts of the mucosal bacteria residents after CMF injury and/or following treatment. There was an enrichment of Fusobacterium, Porphyromonadaceae, and Bacteroidales accompanied by a decline in Pasteurellaceae, Moraxella, and Neisseria relative abundance in days allotted for healing. We also observed a decline in species richness and abundance driven by reduction in temporal instability and inter-animal heterogeneity on days 0 and 56, with day 0 corresponding to injury in DMR group and day 56 corresponding to delayed treatment for DMR or injury and immediate treatment for the IMR group. Analysis of bone healing features showed comparable bone-healing profiles for IMR vs. DMR therapeutic approach.

Protocol for a single-arm, pilot trial of creatine monohydrate supplementation in patients with Alzheimer's disease.

BACKGROUND: Impaired brain bioenergetics is a pathological hallmark of Alzheimer's disease (AD) and is a compelling target for AD treatment. Patients with AD exhibit dysfunction in the brain creatine (Cr) system, which is integral in maintaining bioenergetic flux. Recent studies in AD mouse models suggest Cr supplementation improves brain mitochondrial function and may be protective of AD peptide pathology and cognition. AIMS: The Creatine to Augment Bioenergetics in Alzheimer's disease (CABA) study is designed to primarily assess the feasibility of supplementation with 20 g/day of creatine monohydrate (CrM) in patients with cognitive impairment due to AD. Secondary aims are designed to generate preliminary data investigating changes in brain Cr levels, cognition, peripheral and brain mitochondrial function, and muscle strength and size. METHODS: CABA is an 8-week, single-arm pilot study that will recruit 20 patients with cognitive impairment due to AD. Participants attend five in-person study visits: two visits at baseline to conduct screening and baseline assessments, a 4-week visit, and two 8-week visits. Outcomes assessment includes recruitment, retention, and compliance, cognitive testing, magnetic resonance spectroscopy of brain metabolites, platelet and lymphocyte mitochondrial function, and muscle strength and morphology at baseline and 8 weeks. DISCUSSION: CABA is the first study to investigate CrM as a potential treatment in patients with AD. The pilot data generated by this study are pertinent to inform the design of future large-scale efficacy trials. TRIAL REGISTRATION: ClinicalTrials.gov, NCT05383833 , registered on 20 May 2022.

Creatine as a Therapeutic Target in Alzheimer's Disease.

Alzheimer's disease (AD) is the most prevalent neurodegenerative disease, affecting approximately 6.5 million older adults in the United States. Development of AD treatment has primarily centered on developing pharmaceuticals that target amyloid-ß (Aß) plaques in the brain, a hallmark pathological biomarker that precedes symptomatic AD. Though recent clinical trials of novel drugs that target Aß have demonstrated promising preliminary data, these pharmaceuticals have a poor history of developing into AD treatments, leading to hypotheses that other therapeutic targets may be more suitable for AD prevention and treatment. Impaired brain energy metabolism is another pathological hallmark that precedes the onset of AD that may provide a target for intervention. The brain creatine (Cr) system plays a crucial role in maintaining bioenergetic flux and is disrupted in AD. Recent studies using AD mouse models have shown that supplementing with Cr improves brain bioenergetics, as well as AD biomarkers and cognition. Despite these promising findings, no human trials have investigated the potential benefits of Cr supplementation in AD. This narrative review discusses the link between Cr and AD and the potential for Cr supplementation as a treatment for AD.

Long-Read High-Throughput Sequencing (HTS) Revealed That the Sf-Rhabdovirus X+ Genome Contains a 3.7 kb Internal Duplication.

We previously reported a novel rhabdovirus produced from the Spodoptera frugiperda Sf9 cell line, designated as Sf-rhabdovirus X+ since it contained a unique accessory gene X. The Sf-rhabdovirus X+ genome sequence was generated using Sanger sequencing and short-read high-throughput sequencing (HTS). In this study, we have used long-read HTS technologies, PacBio's single-molecule real-time sequencing and Oxford's Nanopore RNA direct sequencing, to analyze the parent Sf9 cell line transcriptome and the virus RNA produced from an X+ cell clone, respectively. A unique 3.7 kb duplication was identified in the L gene between nucleotide position 8523 and 8524, preceded by a GA dinucleotide insertion. This duplication contained a partial G gene, the complete X gene, and a partial L gene, which extended from nucleotide positions 4767-8523 in the X+ virus. Thus, the X+ genome length is 17,361 nucleotides, and we have re-designated the virus as Sf-rhabdovirus X+3.7. The 3.7 kb duplication was found in all Sf9 cell clones producing the X+ variant virus. Furthermore, the Sf-rhabdovirus X+3.7 genome was stable at passage 30, which was the highest passage tested. These results highlight the importance of combining short-read and long-read technologies for accurately sequencing virus genomes using HTS.

A comparison of techniques for verifying the accuracy of precision decomposition-derived relationships between motor unit firing rates and recruitment thresholds from surface EMG signals.

Approaches for validating motor unit firing times following surface electromyographic (EMG) signal decomposition with the precision decomposition III (PDIII) algorithm have not been agreed upon. Two approaches have been common: (1) "reconstruct-and-test" and (2) spike-triggered averaging (STA). We sought to compare motor unit results following the application of these approaches. Surface EMG signals were recorded from the vastus lateralis of 13 young males performing trapezoidal, isometric knee extensions at 50% and 80% of maximum voluntary contraction (MVC) force. The PDIII algorithm was used to quantify motor unit firing rates. Motor units were excluded using eight combinations of the reconstruct-and-test approach with accuracy thresholds of 0, 90, 91, and 92% with and without STA. The mean firing rate versus recruitment threshold relationship was minimally affected by STA. At 80% MVC, slopes acquired at the 0% accuracy threshold were significantly greater (i.e., less negative) than when 91% (p = .010) and 92% (p = .030) accuracy thresholds were applied. The application of STA has minimal influence on surface EMG signal decomposition results. Stringent reconstruct-and-test accuracy thresholds influence motor unit-derived relationships at high forces, perhaps explained through the increased presence of large motor unit action potentials. Investigators using the PDIII algorithm can expect negligible changes in motor unit-derived linear regression relationships with the application of secondary validation procedures.

Sex-related differences in motor unit behavior are influenced by myosin heavy chain during high- but not moderate-intensity contractions.

AIMS: Motor unit recruitment and firing rate patterns of the vastus lateralis (VL) have not been compared between sexes during moderate- and high-intensity contraction intensities. Additionally, the influence of fiber composition on potential sex-related differences remains unquantified. METHODS: Eleven males and 11 females performed 40% and 70% maximal voluntary contractions (MVCs). Surface electromyographic (EMG) signals recorded from the VL were decomposed. Recruitment thresholds (RTs), MU action potential amplitudes (MUAPAMP ), initial firing rates (IFRs), mean firing rates (MFRs), and normalized EMG amplitude (N-EMGRMS ) at steady torque were analyzed. Y-intercepts and slopes were calculated for MUAPAMP , IFR, and MFR versus RT relationships. Type I myosin heavy chain isoform (MHC) was determined with muscle biopsies. RESULTS: There were no sex-related differences in MU characteristics at 40% MVC. At 70% MVC, males exhibited greater slopes (p = 0.002) for the MUAPAMP , whereas females displayed greater slopes (p = 0.001-0.007) for the IFR and MFR versus RT relationships. N-EMGRMS at 70% MVC was greater for females (p < 0.001). Type I %MHC was greater for females (p = 0.006), and was correlated (p = 0.018-0.031) with the slopes for the MUAPAMP , IFR, and MFR versus RT relationships at 70% MVC (r = -0.599-0.585). CONCLUSION: Both sexes exhibited an inverse relationship between MU firing rates and recruitment thresholds. However, the sex-related differences in MU recruitment and firing rate patterns and N-EMGRMS at 70% MVC were likely due to greater type I% MHC and smaller twitch forces of the higher threshold MUs for the females. Evidence is provided that muscle fiber composition may explain divergent MU behavior between sexes.

Mineral composition of serially slaughtered Holstein steers supplemented with zilpaterol hydrochloride.

Calf-fed Holstein steers (n = 115; 449 ± 20 kg) were utilized in a serial harvest experiment. A baseline group of five steers was harvested after 226 d on feed (DOF), which was designated day 0. The remaining cattle were assigned randomly to 11 harvest groups, with slaughter every 28 d. Cattle were either not (CON) or were fed zilpaterol hydrochloride for 20 d followed by a 3 d withdrawal (ZH). There were five steers per treatment in each slaughter group ranging from days 28 to 308. Whole carcasses were divided into lean, bone, internal cavity, hide, and fat trim components. Apparent mineral retention (Ca, P, Mg, K, and S) within the body was calculated as the difference between mineral concentration at slaughter and day 0. Mineral concentration at day 0 was determined from body composition of steers harvested at day 0 multiplied by individual live body weight (BW) at day 0. All data were analyzed as a 2 × 11 factorial arrangement with individual animal as the experimental unit. Orthogonal contrasts were used to analyze linear and quadratic contrasts over time (11 slaughter dates). There were no differences in concentration of Ca, P, and Mg in bone tissue as feeding duration increased (P ≥ 0.89); concentration of K, Mg, and S in lean tissue did fluctuate across DOF (P < 0.01). Averaged across treatment and DOF, 99% of Ca, 92% of P, 78% of Mg, and 23% of S present in the body were in bone tissue; 67% of K and 49% of S were in lean tissue. Expressed as gram per day, apparent retention of all minerals decreased linearly across DOF (P < 0.01). Expressed relative to empty body weight (EBW) gain, apparent Ca, P, and K retention decreased linearly as BW increased (P < 0.01) whereas Mg and S increased linearly (P < 0.01). Apparent retention of Ca was greater for CON cattle (greater bone fraction) and apparent retention of K was greater for ZH cattle (greater muscle fraction) when expressed relative to EBW gain (P ≤ 0.02), demonstrating the increase in lean gain by ZH cattle. There were no differences in apparent retention of Ca, P, Mg, K, or S due to treatment (P ≥ 0.14) or time (P ≥ 0.11) when expressed relative to protein gain. Apparent retention averaged 14.4 g Ca, 7.5 g P, 0.45 g Mg, 1.3 g K, and 1.0 g S/100 g protein gain. Expressing apparent mineral retention on a protein gain basis minimized effects of rate and type of gain, allowing for better comparison across treatments and time. Feeding zilpaterol hydrochloride did not affect apparent mineral retention when expressed relative to protein gain.

Mineral requirements for feedlot cattle are largely based on measured mineral concentration in the body at harvest. Fairly extensive research has been done quantifying Ca and P in the body of cattle, but data on Mg, K, and S are sparse. Serial harvest experiments are expensive and labor intensive and therefore not conducted frequently. A group of 115 Holstein steers was fed a finishing diet with serial harvest every 28 d. Two treatments were evaluated, control and cattle fed zilpaterol hydrochloride to increase lean tissue growth. Every 28 d, five steers from each treatment group were harvested with the whole carcass divided into lean, bone, internal cavity, hide, and fat trim components. Apparent mineral retention was calculated as the difference between mineral composition at day 0 (baseline harvest group) and each 28 d harvest group. Averaged across treatment and days on feed, 99% of Ca, 92% of P, 78% of Mg, and 23% of S present in the body were measured in bone tissue; 67% of K and 49% of S were in lean tissue. Apparent retention averaged 14.4 g Ca, 7.5 g P, 0.45 g Mg, 1.3 g K, and 1.0 g S/100 g protein gain.

High-Intensity Cycling Training Necessitates Increased Neuromuscular Demand of the Vastus Lateralis During a Fatiguing Contraction.

Purpose: To examine the effects of a 5-week continuous cycling training intervention on electromyographic amplitude (EMGRMS)- and mechanomyographic amplitude (MMGRMS)-torque relationships of the vastus lateralis (VL) during a prolonged contraction. Methods: Twenty-four sedentary, young adults performed maximal voluntary contractions (MVCs) and a prolonged isometric trapezoidal contraction at the same absolute 40% MVC for the knee extensors before (PRE) and after training (POSTABS). Individual b- (slopes) and a-terms (y-intercepts) were calculated from the log-transformed electromyographic amplitude (EMGRMS)- and mechanomyographic amplitude (MMGRMS)-torque relationships during the increasing and decreasing segments of the trapezoid. EMGRMS and MMGRMS was normalized for the 45-s steady torque segment. Results: At PRE, b-terms for the EMGRMS-torque relationships during the linearly decreasing segment were greater than the increasing segment (p < .001), and decreased from PRE to POSTABS (p = .027). a-terms were greater during the linearly increasing than decreasing segment at PRE, while the a-terms for the linearly decreasing segment increased from PRE to POSTABS (p = .027). For the MMGRMS-torque relationships, b-terms during the linearly decreasing segment decreased from PRE to POSTABS (p = .013), while a-terms increased from PRE to POSTABS when collapsed across segments (p = .022). Steady torque EMGRMS increased for POSTABS (p < .001). Conclusion: Although cycling training increased aerobic endurance, incorporating resistance training may benefit athletes/individuals as the alterations in neuromuscular parameters post-training suggest a greater neural cost (EMGRMS) and mechanical output (MMGRMS) to complete the same pre-training fatiguing contraction.

Live and carcass production traits for progeny of purebred sires in comparison with the clone of a USDA prime yield grade one carcass.

Cloning is a technology by which an animal's tissue can be salvaged and replicated. Carcasses that grade USDA prime-yield grade 1 (P1) represent a rare and antagonistic outcome and are a goal for terminal sire selection in the United States. A terminal sire progeny test generated offspring for a crossbred bull (14% Zebu, 86% Angus; ALPHA), born in 2012 via somatic cell nuclear transfer (SCNT) from a carcass that graded P1. ALPHA progeny (steers and heifers) were compared against progeny of three purebred (Angus; Charolais; Simmental) reference sires. Live production traits included weaning weight, morbidity, mortality, and days on feed; carcass traits included abscessed liver frequency and lung lesion frequency, individual quality and yield grade (YG) parameters, and carcass value. Observed carcass traits for progeny from the Angus, Charolais, and Simmental sires were reflective of the carcass outcomes expected for each sire's respective breed. Calves sired by the Angus were the earliest maturing indicated by the youngest chronological age at harvest (P ≤ 0.02) concomitant with the most backfat (P < 0.01), and the greatest marbling scores (P < 0.01). Calves sired by the Charolais had the heaviest carcass weight (P = 0.04), greatest cutability as assessed by USDA calculated YG (P < 0.01) and were the heaviest muscled based on "longissimus" muscle area (P < 0.01). ALPHA-sired calves were the most similar in carcass outcomes to calves sired by the Simmental, combining advantageous quality and yield parameters to produce an intermediate for carcass quality and yield. The economic value of moderate carcass outcomes is reflected in the carcass value per century weight, in which ALPHA-sired steers tended (P = 0.07) to be of the greatest value compared to other sire groups. ALPHA progeny performed comparably to high-performing reference sires for terminal sire production traits and the P1 genetics in which ALPHA was cloned have economical and biological value in modern U.S. beef production.

Myofiber hypertrophy adaptations following 6 weeks of low-load resistance training with blood flow restriction in untrained males and females.

The effects of low-load resistance training with blood flow restriction (BFR) on hypertrophy of type I/II myofibers remains unclear, especially in females. The purpose of the present study is to examine changes in type I/II myofiber cross-sectional area (fCSA) and muscle CSA (mCSA) of the vastus lateralis (VL) from before (Pre) to after (Post) 6 wk of high-load resistance training (HL; n = 15, 8 females) and low-load resistance training with BFR (n = 16, 8 females). Mixed-effects models were used to analyze fCSA with group (HL, BFR), sex (M, F), fiber type (I, II), and time (Pre, Post) included as factors. mCSA increased from pre- to posttraining (P < 0.001, d = 0.91) and was greater in males compared with females (P < 0.001, d = 2.26). Type II fCSA increased pre- to post-HL (P < 0.05, d = 0.46) and was greater in males compared with females (P < 0.05, d = 0.78). There were no significant increases in fCSA pre- to post-BFR for either fiber type or sex. Cohen's d, however, revealed moderate effect sizes in type I and II fCSA for males (d = 0.59 and 0.67), although this did not hold true for females (d = 0.29 and 0.34). Conversely, the increase in type II fCSA was greater for females than for males after HL. In conclusion, low-load resistance training with BFR may not promote myofiber hypertrophy to the level of HL resistance training, and similar responses were generally observed for males and females. In contrast, comparable effect sizes for mCSA and 1-repetition maximum (1RM) between groups suggest that BFR could play a role in a resistance training program.NEW & NOTEWORTHY This is the first study, to our knowledge, to examine myofiber hypertrophy from low-load resistance training with blood flow restriction (BFR) in females. Although this type of training did not result in myofiber hypertrophy, there were comparable increases in muscle cross-sectional area compared with high-load resistance training. These findings possibly highlight that males and females respond in a similar manner to high-load resistance training and low-load resistance training with BFR.

Circulating tumor DNA analysis of the phase III VOYAGER trial: KIT mutational landscape and outcomes in patients with advanced gastrointestinal stromal tumor treated with avapritinib or regorafenib.

BACKGROUND: The current treatment paradigm of imatinib-resistant metastatic gastrointestinal stromal tumor (GIST) does not incorporate KIT/PDGFRA genotypes in therapeutic drug sequencing, except for PDGFRA exon 18-mutant GIST that is indicated for avapritinib treatment. Here, circulating tumor DNA (ctDNA) sequencing was used to analyze plasma samples prospectively collected in the phase III VOYAGER trial to understand how the KIT/PDGFRA mutational landscape contributes to tyrosine kinase inhibitor (TKI) resistance and to determine its clinical validity and utility. PATIENTS AND METHODS: VOYAGER (N = 476) compared avapritinib with regorafenib in patients with KIT/PDGFRA-mutant GIST previously treated with imatinib and one or two additional TKIs (NCT03465722). KIT/PDGFRA ctDNA mutation profiling of plasma samples at baseline and end of treatment was assessed with 74-gene Guardant360® CDx. Molecular subgroups were determined and correlated with outcomes. RESULTS: A total of 386/476 patients with KIT/PDGFRA-mutant tumors underwent baseline (pre-trial treatment) ctDNA analysis; 196 received avapritinib and 190 received regorafenib. KIT and PDGFRA mutations were detected in 75.1% and 5.4%, respectively. KIT resistance mutations were found in the activation loop (A-loop; 80.4%) and ATP-binding pocket (ATP-BP; 40.8%); 23.4% had both. An average of 2.6 KIT mutations were detected per patient; 17.2% showed 4-14 different KIT resistance mutations. Of all pathogenic KIT variants, 28.0% were novel, including alterations in exons/codons previously unreported. PDGFRA mutations showed similar patterns. ctDNA-detected KIT ATP-BP mutations negatively prognosticated avapritinib activity, with a median progression-free survival (mPFS) of 1.9 versus 5.6 months for regorafenib. mPFS for regorafenib did not vary regardless of the presence or absence of ATP-BP/A-loop mutants and was greater than mPFS with avapritinib in this population. Secondary KIT ATP-BP pocket mutation variants, particularly V654A, were enriched upon disease progression with avapritinib. CONCLUSIONS: ctDNA sequencing efficiently detects KIT/PDGFRA mutations and prognosticates outcomes in patients with TKI-resistant GIST treated with avapritinib. ctDNA analysis can be used to monitor disease progression and provide more personalized treatment.

Chronic training status affects muscle excitation of the vastus lateralis during repeated contractions.

This study examined electromyographic amplitude (EMGRMS)-force relationships during repeated submaximal knee extensor muscle actions among chronic aerobically-(AT), resistance-trained (RT), and sedentary (SED) individuals. Fifteen adults (5/group) attempted 20 isometric trapezoidal muscle actions at 50% of maximal strength. Surface electromyography (EMG) was recorded from vastus lateralis (VL) during the muscle actions. For the first and last successfully completed contractions, linear regression models were fit to the log-transformed EMGRMS-force relationships during the linearly increasing and decreasing segments, and the b terms (slope) and a terms (antilog of y-intercept) were calculated. EMGRMS was averaged during steady force. Only the AT completed all 20 muscle actions. During the first contraction, the b terms for RT (1.301 â€‹± â€‹0.197) were greater than AT (0.910 â€‹± â€‹0.123; p â€‹= â€‹0.008) and SED (0.912 â€‹± â€‹0.162; p â€‹= â€‹0.008) during the linearly increasing segment, and in comparison to the linearly decreasing segment (1.018 â€‹± â€‹0.139; p â€‹= â€‹0.014), respectively. For the last contraction, the b terms for RT were greater than AT during the linearly increasing (RT â€‹= â€‹1.373 â€‹± â€‹0.353; AT â€‹= â€‹0.883 â€‹± â€‹0.129; p â€‹= â€‹0.018) and decreasing (RT â€‹= â€‹1.526 â€‹± â€‹0.328; AT â€‹= â€‹0.970 â€‹± â€‹0.223; p â€‹= â€‹0.010) segments. In addition, the b terms for SED increased from the linearly increasing (0.968 â€‹± â€‹0.144) to decreasing segment (1.268 â€‹± â€‹0.126; p â€‹= â€‹0.015). There were no training, segment, or contraction differences for the a terms. EMGRMS during steady force increased from the first- ([64.08 â€‹± â€‹51.68] â€‹µV) to last-contraction ([86.73 â€‹± â€‹49.55] â€‹µV; p â€‹= â€‹0.001) collapsed across training statuses. The b terms differentiated the rate of change for EMGRMS with increments in force among training groups, indicating greater muscle excitation to the motoneuron pool was necessary for the RT than AT during the linearly increasing and decreasing segments of a repetitive task.

Enantiomeric purity analysis of synthetic peptide therapeutics by direct chiral high-performance liquid chromatography-electrospray ionization tandem mass spectrometry.

The determination of chiral purity is critical to the evaluation of the quality of peptide pharmaceutical products. For synthetic peptides, the undesirable d-isomers can be introduced as impurities in amino acid starting materials and can also be formed during peptide synthesis and in some cases during product shelf life. A chiral high-performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) method is described that facilitates rapid and accurate determination of amino acid chiral purity of a peptide. The peptide is hydrolyzed in deuterated acid to facilitate correction for any racemization occurring during this step of sample preparation, and the amino acids are subsequently separated by chiral chromatography interfaced with ESI-MS/MS for quantitation. The amino acid samples are analyzed directly following hydrolysis using high-low chromatography and extraction of selected ion response, providing efficiency and simplicity by avoiding the derivatization steps and multiple external standards required by traditional methodologies. GMP method validation feasibility is described for all nineteen chiral proteogenic amino acids. The practical application of the chiral HPLC-ESI-MS/MS method was demonstrated through the recovery of d-amino acid substitutions at each residue of an octapeptide across the 0.1-1.0 % range of interest. The method was applied to the analysis of four model peptides, each consisting of 8-14 amino acid residues, and the results were comparable to those provided by traditional testing methods.

A noninvasive test for estimating myosin heavy chain of the vastus lateralis in females with mechanomyography.

This study examined relationships between percent myosin heavy chain (%MHC) expression and mechanomyographic amplitude (MMGRMS). Fifteen females (age ± SD=21.3 ± 5.3 yrs) completed isometric trapezoidal contractions at 30% and 70% maximal voluntary contraction (MVC). MMG was recorded from the vastus lateralis (VL). Participants gave a muscle biopsy of the VL post-testing. MMGRMS-torque relationships during the linearly varying segments were log-transformed and linear regressions were applied to calculate b terms (slopes). For the steady torque segment, MMGRMS was averaged. Correlations were performed for type I%MHC with the MMG variables. Multiple regression was utilized to examine prediction equations for type I%MHC. Type I%MHC was significantly correlated with the b terms during the increasing segment of the 70% MVC (p = 0.003; r = -0.718), and MMGRMS during steady torque at 30% (p = 0.008; r = -0.652) and 70% MVC (p = 0.040; r = -0.535). Type I%MHC reduced the linearity of the MMGRMS-torque relationship during the high-intensity linearly increasing segment, and MMGRMS at a low- and high-intensity steady torque. A combination of MMG variables estimated type I%MHC expression with 81.2% accuracy. MMG recorded during a low- and high-intensity isometric trapezoidal contraction may offer a simple, noninvasive test for estimating type I%MHC expression of the VL in sedentary females.

Acute muscle swelling effects of a knee rehabilitation exercise performed with and without blood flow restriction.

This study assessed the acute effect of adding blood flow restriction (BFR) to quad sets on muscle-cross sectional area (mCSA), muscle thickness (MT), echo intensity (EI), and subcutaneous fat-normalized EI (EINORM) of the superficial quadriceps muscles. Twelve males and 12 females (mean±SD; age (yrs): 21.4±2.9; stature (m): 1.76±0.1; body mass (kg): 77.7±2.9) performed 70 repetitions (one set of 30, three sets of 15 repetitions) of bodyweight quad sets separately on each leg, with or without BFR (CON) applied. Rating of perceived exertion was recorded following each set. Panoramic ultrasound images of the vastus lateralis (VL), vastus medialis (VM), and rectus femoris (RF) were captured prior to (PRE), immediately after (IMM-POST), 30- (30-POST), and 60-minutes after (60-POST) after exercise. Sex x condition x time repeated measures ANOVAs assessed differences at p<0.05 for each muscle and dependent variable separately. Although males had larger VM and VL mCSA and VL MT (p<0.05), there were no acute changes from PRE to IMM-POST (p>0.05). There was a 3-way interaction in VL mCSA (p = 0.025) which indicated BFR was greater than CON at IMM-POST by 7.6% (p = 0.019) for males only. Females had greater EI in the VM and VL than males (p<0.05), yet males had greater EINORM for each muscle (p>0.05) and EINORM did not change over time or treatment (p>0.05). The lack of changes in MT, EI, and EINORM indicate that unloaded quad sets do not provide a stimulus to promote fluid shifts or acute changes in muscle size with the exception of IMM-POST in the VL for males. Future research should attempt to elucidate the acute muscular responses of BFR application for lightly loaded rehabilitation exercises in the clinical populations for which they are prescribed.