A deep learning-based approach for fully automated segmentation and quantitative analysis of muscle fibers in pig skeletal muscle.

Muscle fiber properties exert a significant influence on pork quality, with cross-sectional area (CSA) being a crucial parameter closely associated with various meat quality indicators, such as shear force. Effectively identifying and segmenting muscle fibers in a robust manner constitutes a vital initial step in determining CSA. This step is highly intricate and time-consuming, necessitating an accurate and automated analytical approach. One limitation of existing methods is their tendency to perform well on high signal-to-noise ratio images of intact, healthy muscle fibers but their lack of validation on more complex image datasets featuring significant morphological changes, such as the presence of ice crystals. In this study, we undertake the fully automatic segmentation of muscle fiber microscopic images stained with myosin adenosine triphosphate (mATPase) activity using a deep learning architecture known as SOLOv2. Our objective is to efficiently derive accurate measurements of muscle fiber size and distribution. Tests conducted on actual images demonstrate that our method adeptly handles the intricate task of muscle fiber segmentation, yielding quantitative results amenable to statistical analysis and displaying reliability comparable to manual analysis.

Comparative evaluation of different modalities for measuring in vivo carnosine levels.

Carnosine is an endogenous di-peptide (ß-alanine -L- histidine) involved in maintaining tissue homeostasis. It is most abundant in skeletal muscle where its concentration has been determined in biopsy samples using tandem mass spectrometry (MS-MS). Carnosine levels can also be assessed in intact leg muscles by proton magnetic resonance spectroscopy (1H-MRS) or in blood and urine samples using mass spectrometry. Nevertheless, it remains uncertain how carnosine levels from these distinct compartments are correlated with each other when measured in the same individual. Furthermore, it is unclear which measurement modality might be most suitable for large-scale clinical studies. Hence, in 31 healthy volunteers, we assessed carnosine levels in skeletal muscle, via 1H-MRS, and in erythrocytes and urine by MS-MS. While muscle carnosine levels were higher in males (C2 peak, p = 0.010; C4 peak, p = 0.018), there was no sex-associated difference in urinary (p = 0.433) or erythrocyte (p = 0.858) levels. In a linear regression model adjusted for age, sex, race, and diet, there was a positive association between erythrocyte and urinary carnosine. However, no association was observed between 1H-MRS and erythrocytes or urinary measures. In the relationship between muscle versus urinary and erythrocyte measures, females had a positive association, while males did not show any association. We also found that 1H-MRS measures were highly sensitive to location of measurement. Thus, it is uncertain whether 1H-MRS can accurately and reliably predict endogenous carnosine levels. In contrast, urinary and erythrocyte carnosine measures may be stable and in greater synchrony, and given financial and logistical concerns, may be a feasible alternative for large-scale clinical studies.

Dietary iron or inulin supplementation alters iron status, growth performance, intramuscular fat and meat quality in finisher pigs.

Forty LW × L pigs (20 boars and 20 gilts) (51.1 ± 0.41 kg) were allocated to a 2 × 2 × 2 factorial design with the respective factors being supplemental organic iron (Fe, 0 and 500 mg/kg), inulin (In, 0 and 50 g/kg) and sex (boars and gilts). After 5 weeks the animals were transported to an abattoir before slaughter and collection of samples. Serum iron was increased by supplemental Fe (28.4 v. 30.9 µmol/L, P = 0.05), although there was an interaction (P = 0.03) such that pigs fed diets with In had lower serum Fe concentrations than those without In (26.8 v. 32.3 µmol/L). Boars had lower (P < 0.01) haemoglobin (116 vs 125), haematocrit (36.7 v. 39.7%) and erythrocyte (6.6 v. 7.1 × 106/mL) concentrations than gilts. Dietary In increased liveweight gain (795 v. 869 g/d, P < 0.02) and carcass weight (62.9 v. 65.2 kg, P < 0.02). Dietary Fe or In supplementation did not improve muscle Longissimus thoracis et lumborum (LTL) total Fe concentration (P > 0.05). Muscle non-heme Fe concentration was higher in Fe-supplemented pigs (P < 0.04) and gilts (P < 0.05) than their counterparts. Muscle heme Fe concentration was greater (3.04 vs 2.51, P < 0.05) in boars than in gilts. The LTL marbling score was greater (P < 0.01) for In-supplemented pigs, and the response was more notable when Fe and In were fed together. These data show that dietary supplementation of Fe increased serum Fe and muscle non-heme Fe concentrations. Supplementation of In at 5% in the diet of finisher pigs improved liveweight gain and the marbling score of pork.

Double-headed binding of myosin II to F-actin shows the effect of strain on head structure.

Force production in muscle is achieved through the interaction of myosin and actin. Strong binding states in active muscle are associated with Mg·ADP bound to the active site; release of Mg·ADP allows rebinding of ATP and dissociation from actin. Thus, Mg·ADP binding is positioned for adaptation as a force sensor. Mechanical loads on the lever arm can affect the ability of myosin to release Mg·ADP but exactly how this is done is poorly defined. Here we use F-actin decorated with double-headed smooth muscle myosin fragments in the presence of Mg·ADP to visualize the effect of internally supplied tension on the paired lever arms using cryoEM. The interaction of the paired heads with two adjacent actin subunits is predicted to place one lever arm under positive and the other under negative strain. The converter domain is believed to be the most flexible domain within myosin head. Our results, instead, point to the segment of heavy chain between the essential and regulatory light chains as the location of the largest structural change. Moreover, our results suggest no large changes in the myosin coiled coil tail as the locus of strain relief when both heads bind F-actin. The method would be adaptable to double-headed members of the myosin family. We anticipate that the study of actin-myosin interaction using double-headed fragments enables visualization of domains that are typically noisy in decoration with single-headed fragments.

Effects of linseed oil supplementation duration on fatty acid profile and fatty acid metabolism-related genes in the muscles of Chinese crested white ducks.

Meat rich in polyunsaturated fatty acids is considered beneficial to health. Supplementing the diet with linseed oil promotes the deposition of polyunsaturated fatty acids (PUFAs) in poultry, a conclusion that has been confirmed multiple times in chicken meat. However, fewer studies have focused on the effects of dietary fatty acids on duck meat. Therefore, this study aims to evaluate the effects of the feeding time of a linseed oil diet on duck meat performance and gene expression, including meat quality performance, plasma biochemical indicators, fatty acid profile, and gene expression. For this study, we selected 168 Chinese crested ducks at 28 days old and divided them into three groups, with 56 birds in each group. The linseed oil content in the different treatment groups was as follows: the control group (0% flaxseed oil), the 14d group (2% linseed oil), and the 28d group (2% linseed oil). Ducks in the two experimental groups were fed a linseed oil diet for 28 and 14 days at 28 and 42 days of age, respectively. The results showed that linseed oil had no negative effect on duck performance (slaughter rate, breast muscle weight, and leg muscle weight) or meat quality performance (pH, meat color, drip loss, and shear force) (P > 0.05). The addition of linseed oil in the diet increased plasma total cholesterol and high-density lipoprotein cholesterol levels (P < 0.05), while decreasing triglyceride content (P < 0.05). Furthermore, the supplementation of linseed oil for four weeks affected the composition of muscle fatty acids. Specifically, levels of α-linolenic acid, eicosapentaenoic acid, and docosahexaenoic acid were increased (P < 0.05), while eicosatetraenoic acid content was negatively correlated with flaxseed oil intake (P < 0.05). qRT-PCR analysis further revealed that the expression of FATP1, FABP5, and ELOVL5 genes in the breast muscle, as well as FABP3 and FADS2 genes in the thigh muscle, increased after four weeks of linseed oil supplementation (P < 0.05). However, after two weeks of feeding, CPT1A gene expression inhibited fatty acid deposition, suggesting an increase in fatty acid oxidation (P < 0.05). Overall, the four-week feeding time may be a key factor in promoting the deposition of n-3 PUFAs in duck meat. However, the limitation of this study is that it remains unknown whether longer supplementation time will continue to affect the deposition of n-3 PUFAs. Further experiments are needed to explain how prolonged feeding of linseed oil will affect the meat quality traits and fatty acid profile of duck meat.

Effects of high-intensity gymnastics on skeletal muscle proteins / Efeitos da ginástica de alta intensidade sobre as proteínas musculares esqueléticas / Efectos de la gimnasia de alta intensidad en las proteínas musculares esqueléticas

ABSTRACT Introduction: Although the current method of muscle stretching in gymnastics teaching in colleges and universities can reduce sports fatigue, it has been shown to have little effect on the well-being of athletes because it requires a long recovery time from psychological fatigue. Progressive muscle relaxation training is a method that uses the basic principle of sympathetic nerve activity to reduce the impact of negative emotions psychologically and relieve fatigue physiologically, requiring a further study of its impact on muscle protein. Objective: Explore the effect of high-intensity gymnastics on skeletal muscle protein and study the progressive muscle relaxation training method post-workout adjustment. Methods: After three weeks of training, excluding the standard deviations in the experimental data caused by the athletes' irregular movements, the athletes' blood lactate content and heart rate were counted and recorded. The collected data were analyzed using Excel software to integrate and compare the data using the T-test method. Results: After exercise training, the skeletal muscle function indices of the subjects increased to different degrees. From the point of view of heart rate recovery efficiency, the rate of heart rate decline of progressive relaxation training was higher than that of the two groups, and the degree of fluctuation was lower than that of the two groups, indicating that the level of recovery in heart rate of progressive relaxation training was better. Conclusion: The action of the high-intensity gymnastics team has a good effect on improving the athletes' skeletal muscle and skeletal muscle proteins. Post-exercise conditioning training plays an important role in athletes' physical recovery. Level of evidence II; Therapeutic studies - investigation of treatment outcomes.

RESUMO Introdução: Embora o método de alongamento muscular atual no ensino de ginástica em faculdades e universidades consiga reduzir a fadiga esportiva, tem se mostrado pouco eficaz no bem-estar dos atletas por exigir grande tempo de recuperação da fadiga psicológica. O treinamento progressivo de relaxamento muscular é um método que usa o princípio básico da atividade nervosa simpática para reduzir o impacto das emoções negativas psicologicamente e aliviar a fadiga fisiologicamente, necessitando de mais estudos do seu impacto sobre a proteína muscular. Objetivo: Explorar o efeito da ginástica de alta intensidade sobre as proteínas musculares esqueléticas e estudar o método de treinamento progressivo de relaxamento muscular no ajuste pós-treino. Métodos: Após 3 semanas de treinamento, excluídos os desvios-padrão nos dados experimentais causados pelos movimentos irregulares dos atletas, foram contabilizados e registrados os conteúdos de lactato sanguíneo e frequência cardíaca dos atletas. Analisou-se os dados coletados, com o software Excel, para integrar e comparar os dados pelo método de teste-T. Resultados: Após o treinamento do exercício, os índices de função muscular esquelética dos sujeitos aumentaram em diferentes graus. Do ponto de vista da eficiência da recuperação da frequência cardíaca, a taxa de declínio da frequência cardíaca do treinamento de relaxamento progressivo foi maior do que a dos dois grupos, o grau de flutuação foi menor do que o dos dois grupos, indicando que o nível de recuperação na frequência cardíaca do treinamento de relaxamento progressivo foi melhor. Conclusão: A ação da equipe de ginástica de alta intensidade tem um bom efeito na melhoria do músculo esquelético e das proteínas musculares esqueléticas dos atletas. O treinamento de condicionamento pós-exercício desempenha um papel importante na recuperação física dos atletas. Nível de evidência II; Estudos terapêuticos - investigação dos resultados do tratamento.

RESUMEN Introducción: Aunque el método actual de elongación muscular en la enseñanza de la gimnasia en colegios y universidades consigue reducir la fatiga deportiva, se ha demostrado que tiene poco efecto en el bienestar de los atletas porque requiere un largo tiempo de recuperación de la fatiga psicológica. El entrenamiento de la relajación muscular progresiva es un método que utiliza el principio básico de la actividad nerviosa simpática para reducir el impacto de las emociones negativas desde el punto de vista psicológico y aliviar la fatiga desde el punto de vista fisiológico, lo que requiere un estudio más profundo de su impacto en la proteína muscular. Objetivo: Explorar el efecto de la gimnasia de alta intensidad sobre la proteína del músculo esquelético y estudiar el método de entrenamiento de relajación muscular progresiva en el ajuste posterior al entrenamiento. Métodos: Después de 3 semanas de entrenamiento, excluyendo las desviaciones estándar en los datos experimentales causadas por los movimientos irregulares de los atletas, se contó y registró el contenido de lactato en sangre y la frecuencia cardíaca de los atletas. Los datos recogidos se analizaron, con el programa informático Excel, para integrar y comparar los datos mediante el método de la prueba T. Resultados: Tras el entrenamiento con ejercicios, los índices de función del músculo esquelético de los sujetos aumentaron en diferentes grados. Desde el punto de vista de la eficacia de la recuperación de la frecuencia cardíaca, el índice de disminución de la frecuencia cardíaca del entrenamiento de relajación progresiva fue mayor que el de los dos grupos, el grado de fluctuación fue menor que el de los dos grupos, lo que indica que el nivel de recuperación de la frecuencia cardíaca del entrenamiento de relajación progresiva fue mejor. Conclusión: La acción del equipo de gimnasia de alta intensidad tiene un buen efecto en la mejora del músculo esquelético y de las proteínas del músculo esquelético de los atletas. El entrenamiento de acondicionamiento posterior al ejercicio desempeña un papel importante en la recuperación física de los deportistas. Nivel de evidencia II; Estudios terapéuticos - investigación de los resultados del tratamiento.

Development and Validation of a Western Blot Method to Quantify Mini-Dystrophin in Human Skeletal Muscle Biopsies.

Duchenne muscular dystrophy (DMD) is a degenerative muscular disease affecting roughly one in 5000 males at birth. The disease is often caused by inherited X-linked recessive pathogenic variants in the dystrophin gene, but may also arise from de novo mutations. Disease-causing variants include nonsense, out of frame deletions or duplications that result in loss of dystrophin protein expression. There is currently no cure for DMD and the few treatment options available aim at slowing muscle degradation. New advances in gene therapy and understanding of dystrophin (DYS) expression in other muscular dystrophies have opened new opportunities for treatment. Therefore, reliable methods are needed to monitor dystrophin expression and assess the efficacy of new therapies for muscular dystrophies such as DMD and Becker muscular dystrophy (BMD). Here, we describe the validation of a novel Western blot (WB) method for the quantitation of mini-dystrophin protein in human skeletal muscle tissues that is easy to adopt in most laboratory settings. This WB method was assessed through precision, accuracy, selectivity, dilution linearity, stability, and repeatability. Based on mini-DYS standard performance, the assay has a dynamic range of 0.5-15 ng protein (per 5 µg total protein per lane), precision of 3.3 to 25.5%, and accuracy of - 7.5 to 3.3%. Our stability assessment showed that the protein is stable after 4 F/T cycles, up to 2 h at RT and after 7 months at - 70°C. Furthermore, our WB method was compared to the results from our recently published LC-MS method. Workflow for our quantitative WB method to determine mini-dystrophin levels in muscle tissues (created in Biorender.com). Step 1 involves protein extraction from skeletal muscle tissue lysates from control, DMD, or BMD biospecimen. Step 2 measures total protein concentrations. Step 3 involves running gel electrophoresis with wild-type dystrophin (wt-DYS) from muscle tissue extracts alongside mini-dystrophin STD curve and mini-DYS and protein normalization with housekeeping GAPDH.

Study on nucleotide, myofibrillar protein biochemical properties and microstructure of freeze-dried scallop striated muscle during storage and rehydration.

The biochemical properties and microstructural changes of freeze-dried Japanese scallop (Patinopecten yessoensis) striated muscle during room temperature storage and rehydration were investigated. The results showed that the content of ATP in freeze-dried scallop muscle remained stable with no significant difference (p > 0.05). However, ATP was rapidly decomposed and AMP accumulated within 1.5 min of rehydration, and HxR and Hx were gradually produced from AMP decomposition with the extension of rehydration time. Besides, the results of chymotryptic digestion patterns demonstrated that the rod of myosin was unstable after dehydration, reflecting lower salt solubility than that of frozen-thawed scallop. In contrast, the myosin subfragment-1 (S-1) was stable, as indicated by the constant of Ca2+-ATPase activity of freeze-dried scallops throughout the storage and rehydration (p > 0.05). Furthermore, the microstructural analysis revealed that the Z line of the freeze-dried scallop was broken after dehydration process. This study might be useful for developing high-quality dehydrated scallops in the future.

Dietary Plukenetia conophora seed alters muscle cholesterol, antioxidant enzymes, and fatty acids in growing rabbits.

The influence of dietary inclusion of Plukenetia conophora seed (PCS) on growth, carcass, muscle antioxidant enzymes, fatty acids, meat quality, and sensory attributes of Longissimus thoracis et lumburum muscle in rabbits was examined. Seventy-two, 28 d old male New Zealand rabbits (750 ± 20 g) were randomly allotted to diets containing either no PCS (PCS-0), 2.5% PCS (PCS-2.5) or 5% PCS (PCS-5) for eight weeks, and euthanized. PCS-5 rabbits had higher (P < 0.05) body and carcass weights than the PCS-0 rabbits. Dietary PCS improved feed efficiency in rabbits. Muscle antioxidant enzymes activities and total phenols were higher while muscle cholesterol was lower (P < 0.05) in supplemented meat than the PCS-0 meat. The concentration of C22:6n-3, C20:5n-3 and C18:3n-3 was higher (P < 0.05) in the supplemented meat than the PCS-0 meat. Sensory attributes, carbonyl, and TBARS values and physicochemical properties of meat did not differ among diets. Supplementation of PCS-5 enhances muscle n-3 fatty acids without impairing the sensory properties, and oxidative stability of rabbit meat.

Effect of energy metabolism and proteolysis on the toughness of intermediate ultimate pH beef.

The objective of this study was to identify the tenderness and energy metabolism attributes in normal ultimate pH (pHu, 5.4-5.8, NpHu), intermediate pHu (5.8-6.2, IpHu) and high pHu (> 6.2, HpHu) Longissimus lumborum from beef. During 21 days of aging, the IpHu group exhibited a higher Warner-Bratzler shear force, lower activity of µ-calpain, and a higher content of heat shock protein 27 and greater protein oxidation. Metabolomics analysis revealed that 24 differential metabolites were detected during the first 24 h postmortem between pH groups, and these were mainly the products of glycolysis, the tricarboxylic acid (TCA) cycle and oxidative phosphorylation. The relatively higher adenosine triphosphate content in the IpHu group could delay tenderization by being used as key substrate for protein phosphorylation. In addition, the relationship between guanosine triphosphate, diphosphate and monophosphate, and beef tenderness was worthy to be verified in the future study.

Preparation, Identification, Molecular Docking Study and Protective Function on HUVECs of Novel ACE Inhibitory Peptides from Protein Hydrolysate of Skipjack Tuna Muscle.

To prepare bioactive peptides with high angiotensin-I-converting enzyme (ACE)-inhibitory (ACEi) activity, Alcalase was selected from five kinds of protease for hydrolyzing Skipjack tuna (Katsuwonus pelamis) muscle, and its best hydrolysis conditions were optimized using single factor and response surface experiments. Then, the high ACEi protein hydrolysate (TMPH) of skipjack tuna muscle was prepared using Alcalase under the optimum conditions of enzyme dose 2.3%, enzymolysis temperature 56.2 °C, and pH 9.4, and its ACEi activity reached 72.71% at 1.0 mg/mL. Subsequently, six novel ACEi peptides were prepared from TMPH using ultrafiltration and chromatography methods and were identified as Ser-Pro (SP), Val-Asp-Arg-Tyr-Phe (VDRYF), Val-His-Gly-Val-Val (VHGVV), Tyr-Glu (YE), Phe-Glu-Met (FEM), and Phe-Trp-Arg-Val (FWRV), with molecular weights of 202.3, 698.9, 509.7, 310.4, 425.6, and 606.8 Da, respectively. SP and VDRYF displayed noticeable ACEi activity, with IC50 values of 0.06 ± 0.01 and 0.28 ± 0.03 mg/mL, respectively. Molecular docking analysis illustrated that the high ACEi activity of SP and VDRYF was attributed to effective interaction with the active sites/pockets of ACE by hydrogen bonding, electrostatic force, and hydrophobic interaction. Furthermore, SP and VDRYF could significantly up-regulate nitric oxide (NO) production and down-regulate endothelin-1 (ET-1) secretion in HUVECs after 24 h treatment, but also abolish the negative effect of 0.5 µM norepinephrine (NE) on the generation of NO and ET-1. Therefore, ACEi peptides derived from skipjack tuna (K. pelamis) muscle, especially SP and VDRYF, are beneficial components for functional food against hypertension and cardiovascular diseases.

Carcass characteristics and meat evaluation of cattle finished in temperate pasture and supplemented with natural additive containing clove, cashew oil, castor oils, and a microencapsulated blend of eugenol, thymol, and vanillin.

BACKGROUND: Forty crossbred steers were supplemented with different doses (from 0 control to 6000 mg/animal/day) of natural additive blend containing clove essential oil, cashew oil, castor oil, and a microencapsulated blend of eugenol, thymol, and vanillin for 80 days. Carcass characteristics, drip loss, and antioxidant activity were evaluated 24 h post mortem on longissimus thoracis, and the effects of aging (until 14 days) were evaluated for water losses (thawing/aging and cooking), texture, color, and lipid oxidation. RESULTS: The use of the natural additive blend did not modify (P > 0.05) carcass characteristics but did, however, modify body composition (P < 0.05). Drip losses were unaffected by the treatments tested (P > 0.05). There was an observed quadratic effect (P < 0.05) on losses from thawing/aging on the first day of storage. Regarding the effects of natural additives on cooking losses, there was a quadratic effect (P < 0.05) among the treatments on day 7 of aging. Differences between days of aging were only observed with control treatment. Shear force was similar among treatments on days 1 and 7 of aging. On day 14 a linear effect (P < 0.05) was observed. Also, a linear effect (P < 0.05) appeared on meat lightness, meat from the control group being clearer on day 1. No changes were observed in redness among treatments or days of storage (P > 0.05). Yellowness was not modified by the treatments (P > 0.05)but only by the days of storage in control and the lowest dosage used. CONCLUSION: The blend of natural additives has potential use in pasture feeding and could improve meat quality. However, doses should be adjusted. © 2021 Society of Chemical Industry.

Biochemical and textural changes in beef from bulls and steers of different crossbreeds shortly after slaughter and during ageing.

The aim of this study was to investigate the course of glycogenolysis, ATP breakdown and fragmentation of myofibrillar proteins in the semitendinosus muscle of a progeny of Limousin×Holstein-Friesian (LMx) and Charolaise×Holstein-Friesian (CHx) (bulls and steers) and to describe the changes in the above parameters over time and its relationship with beef texture. The hypothesis that beef from bulls and steers of different crossbreeds required the same ageing time to achieve satisfactory tenderness was also tested. Cattle crossbreeding did not affect the amount of muscle glycogen, and castration did not differentiate it until 3 h post-mortem. The interaction between crossbreeding and castration was found, and the highest shear force values were observed in CHx bulls, whereas the lowest was in CHx steers. Beef obtained from CHx was found to be more predestined to short ageing, and LMx required longer ageing to achieve good tenderness. The R-values more strongly influenced subsequent beef texture than pH values.

Effects of yeast hydrolysate on growth performance, serum parameters, carcass traits, meat quality and antioxidant status of broiler chickens.

BACKGROUND: Yeast hydrolysate (YH) has multiple salutary biological activities. Nevertheless, the application of YH in broiler production is limited. This study was conducted to evaluate the protective effects of YH derived from Saccharomyces cerevisiae by exploring growth performance, serum parameters, organs relative weight, carcass traits, meat quality and antioxidant status of broilers. RESULTS: Supplementing YH linearly and quadratically improved (P < 0.05) body weight gain and gain-to-feed ratio compared to that in the control group. Triglycerides, low-density lipoprotein cholesterol and total cholesterol in serum, the decline in pH and cooking loss of breast muscle, and malonaldehyde concentration in serum and liver were decreased linearly and/or quadratically by YH (P < 0.05), whereas high-density lipoprotein cholesterol in serum, superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities in serum, GSH-Px activity in liver, glutathione content in serum and liver, eviscerated yield rate and chest muscle yield, and the relative weight of spleen and liver were linearly and/or quadratically increased (P < 0.05). Moreover, YH enhanced the mRNA levels of nuclear factor erythroid 2-related factor 2, heme oxygennase-1 (HO-1), GSH-Px1 and SOD1 (linear and/or quadratic, P < 0.05). CONCLUSION: Dietary YH beneficially affected growth performance, serum parameters, organ relative weight, carcass traits, meat quality and antioxidant status in broilers, indicating its potential application as a promising feed additive in broiler production. © 2021 Society of Chemical Industry.

Structural and functional interactions between the Ca2+-, ATP-, and caffeine-binding sites of skeletal muscle ryanodine receptor (RyR1).

Ryanodine receptor type 1 (RyR1) releases Ca2+ ions from the sarcoplasmic reticulum of skeletal muscle cells to initiate muscle contraction. Multiple endogenous and exogenous effectors regulate RyR1, such as ATP, Ca2+, caffeine (Caf), and ryanodine. Cryo-EM identified binding sites for the three coactivators Ca2+, ATP, and Caf. However, the mechanism of coregulation and synergy between these activators remains to be determined. Here, we used [3H]ryanodine ligand-binding assays and molecular dynamics simulations to test the hypothesis that both the ATP- and Caf-binding sites communicate with the Ca2+-binding site to sensitize RyR1 to Ca2+. We report that either phosphomethylphosphonic acid adenylate ester (AMPPCP), a nonhydrolyzable ATP analog, or Caf can activate RyR1 in the absence or the presence of Ca2+. However, enhanced RyR1 activation occurred in the presence of Ca2+, AMPPCP, and Caf. In the absence of Ca2+, Na+ inhibited [3H]ryanodine binding without impairing RyR1 activation by AMPPCP and Caf. Computational analysis suggested that Ca2+-, ATP-, and Caf-binding sites modulate RyR1 protein stability through interactions with the carboxyterminal domain and other domains in the activation core. In the presence of ATP and Caf but the absence of Ca2+, Na+ is predicted to inhibit RyR1 by interacting with the Ca2+-binding site. Our data suggested that ATP and Caf binding affected the conformation of the Ca2+-binding site, and conversely, Ca2+ binding affected the conformation of the ATP- and Caf-binding sites. We conclude that Ca2+, ATP, and Caf regulate RyR1 through a network of allosteric interactions involving the Ca2+-, ATP-, and Caf-binding sites.

The impact of different Hormonal Growth Promotants (HGP) on desmin degradation and collagen content of various muscles from pasture and feedlot finished steer carcasses.

The impacts of several hormonal growth promotants (HGP) on Warner-Bratzler Shear Force (WBSF), desmin degradation ratio (DDR) and collagen content (COLL) were assessed. Treatments within feedlot and pasture finished steer carcasses (n = 60, n = 40, respectively) were control (CON-100-F and CON-400-P), oestradiol HGPs (OES-100-F and OES-400-P) and trenbolone acetate/oestradiol HGPs (TBA+OES-100-F only). The longissimus lumborum (LL), gluteus medius (GM), infraspinatus (IS), semitendinosus (ST,) and the LL and biceps femoris (BF) were collected from feedlot and pasture finished steers, respectively. All muscles were aged between 3 and 35 days. The LL from TBA+OES-100-F carcasses had increased WBSF and decreased DDR, which varied in magnitude with ageing (P < 0.05). The GM from OES-100-F steers also had lower DDR (P < 0.05). The feedlot HGP treatments had no impact on the WBSF of the IS, ST or GM and no impact on COLL in the LL. The OES-400-P had no impact on WBSF, DDRor COLL for both muscles (P > 0.05).

Zinc-containing Mohs' paste affects blood flow and angiogenesis suppression.

PURPOSE: Mohs' paste, which is composed of zinc chloride and zinc oxide starch, is used for hemostasis of superficial malignancy in the clinical setting. We investigated the concentration of intramuscular zinc in mice after Mohs' paste application and evaluated its relationship with angiogenesis from the perspective of blood flow levels within 24 h. METHODS: Male C57BL/6JJmsSlc mice were administered single dose of Mohs' paste at 25%, 50%, and 75% after unilateral hind limb surgery, and glycerin, a viscosity modifier, was administered to the control group (0%). Hind limb blood flow levels were measured with a laser Doppler perfusion imaging system (n = 6). The amounts of intramuscular zinc and vascular endothelial growth factor-A (VEGF-A) expression were analyzed using inductively coupled plasma mass spectrometry (ICP-MS) and western blotting, respectively (n = 5 or 3). RESULTS: Blood flow levels were significantly decreased in the 50% group after 8 h, and significantly decreased in the 25% and 50% groups after 24 h. Intramuscular zinc was significantly increased in the 50% and 75% groups after 8 h. Western blotting showed that VEGF-A levels were significantly increased in the 25% and 50% groups after 8 h. Based on analytical experiments and biological investigation, we predicated the pharmacological effect of Mohs' paste and found over 50% of it is critical in the blood flow and angiogenesis suppression after more than 8 h of its application. CONCLUSIONS: The results suggest that the mechanism of blood flow suppression is independent of VEGF-A levels and might suppress future angiogenesis. Our findings support that of previous studies, in which Mohs' paste was expected to induce hemostasis and suppress angiogenesis. It is an excellent ointment that facilitates hemostasis by suppressing blood flow regardless of angiogenesis, and may be apt for situations where hemostasis is required in the clinical setting.

Lipid Dynamics due to Muscle Atrophy Induced by Immobilization.

Muscle atrophy refers to skeletal muscle loss and dysfunction that affects glucose and lipid metabolism. Moreover, muscle atrophy is manifested in cancer, diabetes, and obesity. In this study, we focused on lipid metabolism during muscle atrophy. We observed that the gastrocnemius muscle was associated with significant atrophy with 8 days of immobilization of hind limb joints and that muscle atrophy occurred regardless of the muscle fiber type. Further, we performed lipid analyses using thin layer chromatography, liquid chromatography-mass spectrometry, and mass spectrometry imaging. Total amounts of triacylglycerol, phosphatidylserine, and sphingomyelin were found to be increased in the immobilized muscle. Additionally, we found that specific molecular species of phosphatidylserine, phosphatidylcholine, and sphingomyelin were increased by immobilization. Furthermore, the expression of adipose triglyceride lipase and the activity of cyclooxygenase-2 were significantly reduced by atrophy. From these results, it was revealed that lipid accumulation and metabolic changes in specific fatty acids occur during disuse muscle atrophy. The present study holds implications in validating preventive treatment strategies for muscle atrophy.

Depletion of clomiphene residues in eggs and muscle after oral administration to laying hens.

The selective oestrogen receptor modulator (SERM) clomiphene is therapeutically used to induce ovulation. While prohibited as a doping agent in sports, it is frequently detected in sports drug testing urine samples. Few reports exist on clomiphene's (illicit) use in the farming industry to increase the egg production rate of laying hens, which creates a risk that eggs as well as edible tissue of these hens contain residues of clomiphene. To investigate the potential transfer of clomiphene into eggs and muscle tissue, laying hens were orally administered with clomiphene citrate at 10 mg/day for 28 days. To determine clomiphene residues in eggs, chicken breast and chicken thigh, the target analyte was extracted from homogenised material with acetonitrile and subjected to ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) analysis. The test method reached a limit of quantification (LOQ) of 1 µg/kg and was characterised concerning specificity, precision, trueness and linearity. Analyses were performed on whole egg, egg white and yolk separately, and chicken muscle from breast and thigh. Clomiphene was detectable in eggs two days after the beginning of the drug administration period. The drug concentrations increased to 10-20 µg per egg within one week, and after withdrawal of clomiphene, residues decreased after 4 days, but traces of clomiphene were still detectable until the end of the study (14 days after the last administration). In the chicken's muscle tissue, clomiphene levels up to 150 µg/kg (thigh) and 36 µg/kg (breast) were found. Six days after the last dose, tissue clomiphene concentrations fell below the LOQ. Overall, these results underline the concerns that clomiphene may be transferred into animal-derived food and future research will therefore need to focus on assessing and minimising the risk of unintentional adverse analytical findings in doping controls.

Myosin-based regulation of twitch and tetanic contractions in mammalian skeletal muscle.

Time-resolved X-ray diffraction of isolated fast-twitch muscles of mice was used to show how structural changes in the myosin-containing thick filaments contribute to the regulation of muscle contraction, extending the previous focus on regulation by the actin-containing thin filaments. This study shows that muscle activation involves the following sequence of structural changes: thin filament activation, disruption of the helical array of myosin motors characteristic of resting muscle, release of myosin motor domains from the folded conformation on the filament backbone, and actin attachment. Physiological force generation in the 'twitch' response of skeletal muscle to single action potential stimulation is limited by incomplete activation of the thick filament and the rapid inactivation of both filaments. Muscle relaxation after repetitive stimulation is accompanied by a complete recovery of the folded motor conformation on the filament backbone but by incomplete reformation of the helical array, revealing a structural basis for post-tetanic potentiation in isolated muscles.