Muscle Type Research Articles

Determination of qPCR reference genes suitable for normalizing gene expression in a novel model of Duchenne muscular dystrophy, the D2-mdx mouse

Duchenne muscular dystrophy (DMD) is a X-linked neuromuscular disorder arising from mutations in the dystrophin gene, leading to a progressive muscle wasting and disability. Currently there is no universal therapy, and there is thus a strong interest in preclinical studies for finding novel treatments. The most widely used and characterized mouse model for DMD is the C57BL/10ScSn-Dmdmdx/J (BL10-mdx), but this model exhibits mild pathology and does not replicate key features of human disease. The D2.B10-Dmdmdx/J (D2-mdx) mouse is a more recent model which seems to better mimics the complex human DMD phenotype. However, the D2-mdx mouse remains less extensively characterised than its BL10-mdx counterpart. Quantitative PCR analysis of gene expression is an important tool to monitor disease progression and evaluate therapeutic efficacy, but measurements must be normalised to stably expressed reference genes, which should ideally be determined and validated empirically. We examined gene expression in the gastrocnemius (GC), diaphragm (DIA) and heart in the D2-mdx mouse, the BL10-mdx mouse, and appropriate strain-matched wild-type controls (D2-wt and BL10-wt), from 4 to 52 weeks of age, using a large panel of candidate references (ACTB, AP3D1, CSNK2A2, GAPDH, HPRT1, PAK1IP1, RPL13A, SDHA, and in the heart, also HTATSF1 and HMBS). Data was analyzed using GeNorm, Bestkeeper, deltaCt and Normfinder algorithms to identify stable references under multiple possible scenarios. We show that CSNK2A2, AP3D1 and ACTB represent strong universal reference genes in both GC and DIA, regardless of age, muscle type, strain and genotype, while HTATSF1 and SDHA are optimal for the heart. GAPDH, HPRT1 and RPL13A were conversely revealed to be poor references, showing tissue-, age- or disease-specific changes in expression. Our results illustrate the importance of determining appropriate reference genes for specific comparative scenarios, but also reconfirm that universal panels can nevertheless be identified for normalising gene expression studies in even complex pathological states.

Platelet-activating Factor (PAF) in Urinary Bladder Smooth Muscle (UBSM): Its Novel Role as a Potential Inducer of Detrusor Overactivity and the Mechanisms Underlying Its Effects in Enhancing UBSM Mechanical Activities

Platelet-activating factor (PAF), a phospholipid mediator, was discovered in 1972 as an inducer of platelet aggregation. Subsequent studies have revealed that PAF has a variety of biological functions, such as its role as a potent proinflammatory mediator. Additionally, PAF regulates the contractile functions of various types of smooth muscle (SM), such as the (1) endothelium-dependent relaxation of vascular SM; (2) contraction and epithelium-dependent relaxation of airway SM; (3) contraction of gastrointestinal SM; and (4) contraction of uterine SM, which occurs more strongly in pregnant females. PAF is produced in platelets, monocytes, neutrophils, and macrophages, which are cells related to thrombus formation and inflammation/immune responses. Furthermore, PAF is produced in various other cells throughout the body. Interestingly, recent studies have focused on the urinary bladder (UB) as a PAF-producing organ since the accumulation of this phospholipid is enhanced in patients with bladder cancer and interstitial cystitis/bladder pain syndrome, especially those who smoke. Therefore, in UB tissue, PAF may play a substantial role as an inducer or enhancer of cancers and inflammatory diseases. However, the effects of PAF on the immediate mechanical activities of UBSM have not been investigated to date. In this regard, we recently discovered that PAF strongly enhances mechanical activities (muscle tone and spontaneous contractile activity) in UBSM tissues isolated from guinea pigs and mice. In this review article, we present our data on these PAF effects together with the possible underlying mechanisms. We also discuss the potential pathophysiological roles of this phospholipid in UB diseases and disorders.

Driving response analysis of twisted and coiled polymer actuators by considering the viscoelastic behavior of their precursor fibers

Twisted and coiled polymer actuators (TCPA) represent a type of artificial muscle predominantly composed of viscoelastic polymers in their precursor fibers. An integral form of the viscoelastic constitutive model has been developed to predict the mechanical deformation of the precursor fibers. The model successfully accounts for the first-cycle effect and the creep deformation near the glass transition temperature of the precursor fibers. Combined with the multilayer model by Gao and Wang (2024 Smart Mater. Struct. 33 045031), which predicts the effective mechanical and thermal properties of TCPA, the proposed viscoelastic constitutive model accurately predicts the thermo-mechanical response of TCPA under the heating and cooling cycle and applied loads. It is noted that the driving strain of TCPA is sensitive to the heating and cooling cycles. When the cycle duration is short, the proposed viscoelastic model can be simplified to a linear elastic model. The numerical implementation of the viscoelastic constitutive model is detailed, with validation conducted on two polymer materials, polypropylene, and polyamide 66. The proposed constitutive model can effectively predict the driving response of TCPA under complex loading conditions.

Mitochondrial-targeted plastoquinone therapy ameliorates early onset muscle weakness that precedes ovarian cancer cachexia in mice.

Cancer cachexia, and the related loss of muscle and strength, worsens quality of life and lowers overall survival. Recently, a novel 'pre-atrophy' muscle weakness was identified during early-stage cancer. While mitochondrial stress responses are associated with early-stage pre-atrophy weakness, a causal relationship has not been established. Using a robust mouse model of metastatic epithelial ovarian cancer (EOC)-induced cachexia, we found the well-established mitochondrial-targeted plastoquinone SkQ1 partially prevents pre-atrophy weakness in the diaphragm. Furthermore, SkQ1 improved force production during atrophy without preventing atrophy itself in the tibialis anterior and diaphragm. EOC reduced flexor digitorum brevis (FDB) force production and myoplasmic free calcium ([Ca 2+ ] i ) during contraction in single muscle fibers, both of which were prevented by SkQ1. Remarkably, changes in mitochondrial reactive oxygen species and pyruvate metabolism were heterogeneous across time and between muscle types which highlights a considerable complexity in the relationships between mitochondria and muscle remodeling throughout EOC. These discoveries identify that muscle weakness can occur independent of atrophy throughout EOC in a manner that is linked to improved calcium handling. The findings also demonstrate that mitochondrial-targeted therapies exert a robust effect in preserving muscle force during the early pre-atrophy period and in late-stage EOC once cachexia has become severe.

On the rate-limiting dynamics of force development in muscle.

Skeletal muscles produce forces relatively slowly compared with the action potentials that excite them. The dynamics of force production are governed by multiple processes, such as calcium activation, cycling of cross-bridges between myofilaments, and contraction against elastic tissues and the body. These processes have been included piecemeal in some muscle models, but not integrated to reveal which are the most rate limiting. We therefore examined their integrative contributions to force development in two conventional types of muscle models: Hill-type and cross-bridge. We found that no combination of these processes can self-consistently reproduce classic data such as twitch and tetanus. Rather, additional dynamics are needed following calcium activation and facilitating cross-bridge cycling, such as for cooperative myofilament interaction and reconfiguration. We provisionally lump such processes into a simple first-order model of 'force facilitation dynamics' that integrate into a cross-bridge-type muscle model. The proposed model self-consistently reproduces force development for a range of excitations including twitch and tetanus and electromyography-to-force curves. The model's step response reveals relatively small timing contributions of calcium activation (3%), cross-bridge cycling (3%) and contraction (27%) to overall force development of human quadriceps, with the remainder (67%) explained by force facilitation. The same set of model parameters predicts the change in force magnitude (gain) and timing (phase delay) as a function of excitatory firing rate, or as a function of cyclic contraction frequency. Although experiments are necessary to reveal the dynamics of muscle, integrative models are useful for identifying the main rate-limiting processes.

Bag of Feature-Based Ensemble Subspace KNN Classifier in Muscle Ultrasound Diagnosis of Diabetic Peripheral Neuropathy

Muscle ultrasound quantification is a valuable complementary diagnostic tool for diabetic peripheral neuropathy (DPN), enhancing physicians’ diagnostic capabilities. Quantitative assessment is generally regarded as more reliable and sensitive than visual evaluation, which often necessitates specialized expertise. This work develops a computer-aided diagnostic (CAD) system based on muscle ultrasound that integrates the bag of features (BOF) and an ensemble subspace k-nearest neighbor (KNN) algorithm for DPN detection. The BOF creates a histogram of visual word occurrences to represent the muscle ultrasound images and trains an ensemble classifier through cross-validation, determining optimal parameters to improve classification accuracy for the ensemble diagnosis system. The dataset includes ultrasound images of six muscles from 53 subjects, consisting of 27 control and 26 patient cases. An empirical analysis was conducted for each binary classifier based on muscle type to select the best vocabulary tree properties or K values for BOF. The result indicates that ensemble subspace KNN classification, based on the bag of features, achieved an accuracy of 97.23%. CAD systems can effectively diagnose muscle pathology, thereby addressing limitations and identifying issues in individuals with diabetes. This research underscores muscle ultrasound as a promising diagnostic tool to aid physicians in making accurate diagnoses, streamlining workflow, and uncovering muscle-related complications in DPN patients.

Skeletal muscle fibre type-dependent effects of atorvastatin on the PI3K/Akt/mTOR signalling pathway and atrophy-related genes in rats

BackgroundOne of the probable causes of statin myotoxicity is an imbalance between protein synthesis and degradation. These processes are regulated by the PI3K/Akt/mTOR pathway and the ubiquitin‒proteasome system (UPS). The aim of this study was to assess whether the effects of atorvastatin on PI3K/Akt/mTOR pathway downstream proteins, the FoxO3a transcription factor and the UPS genes, i.e., MuRF-1 and MAFbx, depend on muscle fibre type.Methods and resultsAtorvastatin (50 mg/kg) was administered to Wistar rats. The levels of selected PI3K/Akt/mTOR pathway proteins were assayed via Western blotting, whereas MuRF-1, MAFbx and FoxO3a mRNA levels were measured using reverse transcription quantitative polymerase chain reaction (RT‒qPCR). Gomöri trichrome staining was performed to assess skeletal muscle pathology. A decrease in the P-Akt/Akt ratio was observed in the gastrocnemius muscle (MG), whereas an increase in the P-Akt/Akt ratio was observed in the soleus muscle (SOL). FoxO3a gene expression increased in the SOL and extensor digitorum longus (EDL) muscles. MuRF-1 gene expression increased in the MG, and MAFbx expression increased in the EDL. No histopathological changes were observed in any of the tested muscles.ConclusionsIn the absence of overt muscle damage, atorvastatin decreased the P-Akt/Akt ratio in the MG, indicating an increase in inactive Akt. Consistent with the decrease in Akt activation, rpS6 phosphorylation decreased. In SOL, atorvastatin increased the P-Akt/Akt ratio, indicating Akt activation. P-FoxO3a and the P-FoxO3a/FoxO3a ratio increased, suggesting that FoxO3a inactivation occurred. Moreover, in the SOL, atorvastatin did not affect the expression of atrophy-related genes. These findings indicate that atorvastatin has no adverse effect on the Akt pathway in the SOL. Our results showed that the effects of atorvastatin on the Akt signalling pathway and atrophy-related gene expression depend on muscle type.

Outcomes of ADM-Assisted Implant-based Breast Reconstructions According to Division Types of the Pectoralis Major Muscle.

In implant-based breast reconstruction (IBR), several surgical techniques are based on the implant insertion planes. Ideal location for breast implants has become a popular topic in IBRs because each technique has advantages and disadvantages. Herein, we report the outcomes of three different division types of the pectoralis muscle and suggest an algorithm for selecting the division types. Charts of patients who underwent IBRs between October 2017 and December 2020 were retrospectively reviewed. The patients were classified into three groups (prepectoral, high subpectoral, and subpectoral) according to the division types of the pectoralis muscle. Aesthetic outcomes were assessed in terms of animation deformity, rippling deformity, and visibility of the upper implant border. Total 104 cases were included. 25, 38, and 41 were in the prepectoral, high-subpectoral, subpectoral IBR groups, respectively. Animation deformities frequently appeared in the subpectoral group. Nipple displacement was most prominent in the subpectoral group. Skin rippling at rest was mostly detected in the prepectoral group, whereas ripping during contraction was commonly observed in the subpectoral group. Visibility of upper border was more prominent in the prepectoral group than the other groups. We propose an algorithm for selecting the division types of the pectoralis muscle. If the pectoralis muscle is thick, prepectoral plane is most preferred to avoid the animation deformity. In cases with thin pectoralis muscle, thickness of the mastectomy flap is considered next. If it is thicker than 1cm, prepectoral plane is better than the subpectoral plane; otherwise, high-subpectoral plane is preferable for better aesthetic outcomes. This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266.

Multi-omics analysis of chemical composition variation among different muscle types in Hu lamb.

Consumers' preferences for lamb meat vary greatly depending on the specific cut. Variations in the chemical composition across different muscle types play a crucial role in determining meat quality, particularly with regard to flavor. Therefore, it is essential to study the variations in chemical composition among different muscle types in lamb, as well as the mechanisms behind their formation, aiming to understand the flavor variation across the muscle types. Flank muscles showed significantly higher intramuscular fat content and muscle fiber diameter compared to triceps brachii and biceps femoris (BF), at the same time as displaying a significantly lower percentage of type I muscle fibers. Forty-three differentially abundant volatile compounds (DAVC) were identified across five muscles, with the majority of DAVCs being more abundant in the BF. In total, 161 differentially abundant lipids were identified across five muscles, with triglycerides (TG), phosphatidylcholines (PC), phosphatidyl ethanolamines (PE) and phosphatidylmethanol (PMeOH) showing a strong correlation with DAVCs. A lipid-gene regulatory network was established, encompassing 664 lipids and 11 107 genes, leading to the identification of pathways and genes that regulate the metabolism of PEs, PMeOH, PCs and TGs. The present study showed the significant variation in flavor compounds among the five edible muscles, as well as the potential reasons for their formation. The results potentially provide a theoretical foundation for improving the meat quality of lamb. © 2024 Society of Chemical Industry. Published by John Wiley & Sons Ltd.

Changes in Skeletal Muscle Atrophy over Time in a Rat Model of Adenine-Induced Chronic Kidney Disease

This study evaluated changes over time in skeletal muscle atrophy, expressions of skeletal muscle anabolic and catabolic genes, and mitochondrial activity by skeletal muscle type in an adenine-induced chronic kidney disease (CKD) model. A CKD model was successfully established by feeding male Wistar rats a 0.75% adenine diet for 4 weeks starting at 8 weeks of age. Control and CKD groups were sacrificed at 12 and 20 weeks of age. The back muscles were analyzed histologically, and succinate dehydrogenase (SDH) staining was performed to evaluate mitochondrial activity. Gene expressions of myogenic determination gene number 1 and myogenin as indicators of muscle anabolism, atrogin-1 and muscle RING-finger protein-1 (MuRF1) as indicators of muscle catabolism, and peroxisome proliferator-activated receptor-γ coactivator-1-α as a marker of mitochondrial biogenesis were assessed. Type I and type II muscle cross-sectional areas (CSAs) were decreased at 12 weeks, but type I muscle CSA was recovered at 20 weeks. SDH staining was lower in CKD than in control rats at 12 weeks, but no significant difference was observed at 20 weeks. Increased expressions of myogenin, atrogin-1, and MuRF-1 were observed only at 12 weeks, but no differences were observed at 20 weeks. The adenine-induced CKD rat model appears to show changes in muscle atrophy over time.

9291 Hypothyroidism impairs skeletal muscle regeneration after injury

Abstract Disclosure: P. Aguiari: None. V. Villani: None. K.Y. Liu: None. G.A. Brent: None. L. Perin: None. A. Milanesi: None. Thyroid hormone (TH) signaling plays an essential role in muscle development and function, in the maintenance of muscle mass, and in regeneration after injury. Disruption of TH signaling results in an abnormal skeletal muscle phenotype, impaired regeneration, and sarcopenia with aging, reflecting the myopathic changes described in hypothyroid patients. Muscle stem cells (MuSCs) are the mediators of post-natal skeletal muscle plasticity. Normally quiescent, in response to injury they enter the cell cycle (myoblasts) and proliferate. Myoblasts then exit the cell cycle and differentiate into myocytes that will fuse with existing myofibers to restore tissue architecture and function. Via TH receptor alpha, TH regulates all the stages of the regeneration program and regulates the expression of multiple myogenic genes. Despite all this evidence, to date there are no studies investigating MuSCs behavior in response to injury during hypothyroidism. We characterized injury-induced regeneration of the tibialis anterior muscle (TAM) in euthyroid and hypothyroid mice, fed a low iodine + 0.15% propylthiouracil diet. To investigate the cell cycle dynamics of MuSCs, we generated Pax7-Fucci mice carrying the fluorescent ubiquitination-based cell-cycle indicator (Fucci) system under the Pax7 promoter. Muscle injury was induced via cardiotoxin injection in the TAM of 3-month-old Pax7-Fucci mice. Hypothyroid mice present signs of impaired regeneration compared to euthyroid mice. From histological analysis, at different time points, we observed smaller fiber diameters, myofibers with central nuclei, and a significantly reduced number of fast glycolytic type IIb fibers, the prevalent muscle fiber type in the TA muscle. ScRNAseq analysis shows that at a late phase of regeneration (14 days after injury) hypothyroid MuSCs and myoblasts are still in the activation state and overexpress genes related to DNA replication and cell proliferation, while genes related to myogenic differentiation and cell cycle exit are downregulated. Differentiated myocytes in the hypothyroid fibers present an immature phenotype and are characterized by overexpression of embryonic/temporary and slow myosin genes and downregulation of mature fast myosins. Cell cycle analysis of the Fucci signal through Flow Cytometry confirmed a higher number of activated hypothyroid MuSCs at 4, 7, and 28 days after injury. These cells accumulate in the S phase and are unable to exit the cell cycle and differentiate towards myocytes. These data demonstrate that hypothyroidism impairs muscle tissue regeneration halting MuSCs progression through the cell cycle, myoblast cell cycle exit, and fiber maturation. Investigating muscle stem cell behavior and response to injury during hypothyroidism is crucial to understanding the mechanism behind thyroid hormone-induced myopathies and identifying possible therapeutical targets. Presentation: 6/2/2024

8670 Hypothyroidism impairs skeletal muscle regeneration after injury

Abstract Disclosure: P. Aguiari: None. V. Villani: None. K.Y. Liu: None. G.A. Brent: None. L. Perin: None. A. Milanesi: None. Thyroid hormone (TH) signaling plays an essential role in muscle development and function, in the maintenance of muscle mass, and in regeneration after injury. Disruption of TH signaling results in an abnormal skeletal muscle phenotype, impaired regeneration, and sarcopenia with aging, reflecting the myopathic changes described in hypothyroid patients. Muscle stem cells (MuSCs) are the mediators of post-natal skeletal muscle plasticity. Normally quiescent, in response to injury they enter the cell cycle (myoblasts) and proliferate. Myoblasts then exit the cell cycle and differentiate into myocytes that will fuse with existing myofibers to restore tissue architecture and function. Via TH receptor alpha, TH regulates all the stages of the regeneration program and regulates the expression of multiple myogenic genes. Despite all this evidence, to date there are no studies investigating MuSCs behavior in response to injury during hypothyroidism. We characterized injury-induced regeneration of the tibialis anterior muscle (TAM) in euthyroid and hypothyroid mice, fed a low iodine + 0.15% propylthiouracil diet. To investigate the cell cycle dynamics of MuSCs, we generated Pax7-Fucci mice carrying the fluorescent ubiquitination-based cell-cycle indicator (Fucci) system under the Pax7 promoter. Muscle injury was induced via cardiotoxin injection in the TAM of 3-month-old Pax7-Fucci mice. Hypothyroid mice present signs of impaired regeneration compared to euthyroid mice. From histological analysis, at different time points, we observed smaller fiber diameters, myofibers with central nuclei, and a significantly reduced number of fast glycolytic type IIb fibers, the prevalent muscle fiber type in the TA muscle. ScRNAseq analysis shows that at a late phase of regeneration (14 days after injury) hypothyroid MuSCs and myoblasts are still in the activation state and overexpress genes related to DNA replication and cell proliferation, while genes related to myogenic differentiation and cell cycle exit are downregulated. Differentiated myocytes in the hypothyroid fibers present an immature phenotype and are characterized by overexpression of embryonic/temporary and slow myosin genes and downregulation of mature fast myosins. Cell cycle analysis of the Fucci signal through Flow Cytometry confirmed a higher number of activated hypothyroid MuSCs at 4, 7, and 28 days after injury. These cells accumulate in the S phase and are unable to exit the cell cycle and differentiate towards myocytes. These data demonstrate that hypothyroidism impairs muscle tissue regeneration halting MuSCs progression through the cell cycle, myoblast cell cycle exit, and fiber maturation. Investigating muscle stem cell behavior and response to injury during hypothyroidism is crucial to understanding the mechanism behind thyroid hormone-induced myopathies and identifying possible therapeutical targets. Presentation: 6/2/2024

Dynamic and static nasolabial muscle anatomy of unilateral cleft lip adult patients based on magnetic resonance imaging data.

This study aims to obtain a three-dimensional reconstruction model based on magnetic resonance imaging (MRI) data of patients with different degrees of unilateral cleft lip and analyze the anatomy and changes in multiple groups of nasolabial muscles under dynamic and static conditions. One normal person and four adult patients with unilateral cleft lip were included, and MRI was performed under static (upper and lower lips closed naturally) and dynamic (pout and grin) conditions. 3D Slicer software was used to reconstruct the model and draw the anatomic morphology of nasolabial muscles. The distance between the junction (where the muscle merges into the orbicularis oris) of the levator muscle, zygomaticminor muscle, and zygomatic major muscle to the median sagittal plane, the starting point to the junction point, the dynamic and static junction points, and the angle between the connection of dynamic and static junctions and the horizontal plane were measured under three kinds of movements, and the ratio was calculated. In all patients, under dynamic and static conditions, the distance from the muscle junction to the median sagittal plane, their ratios of the cleft side to the non-cleft side were all greater than 1. While the ratio of the distance from the starting point of the muscle to the junction point is less than 1. At static conditions, the two ratios of the same muscle increased gradiently with the severity of the cleft, and the ratio of the zygomatic minor muscle was prominent in the same patient. The ratio of the cleft side to the non-cleft side was greater than 1, and the value for comparison was the angle of the line from the static to the dynamic junction and the horizontal plane. The symmetry of the insertion site of the orbicularis oris and the linear distance of both sides of the muscle are related to muscle and cleft types. The angle of muscle contraction on the cleft side is greater than that on the non-cleft side.

Targeted Bmal1 restoration in muscle prolongs lifespan with systemic health effects in aging model.

Disruption of the circadian clock in skeletal muscle worsens local and systemic health, leading to decreased muscle strength, metabolic dysfunction, and aging-like phenotypes. Whole-body knockout mice that lack Bmal1, a key component of the molecular clock, display premature aging. Here, by using adeno-associated viruses, we rescued Bmal1 expression specifically in the skeletal muscle fibers of Bmal1-KO mice and found that this engaged the circadian clock and clock output gene expression contributing to extended lifespan. Time course phenotypic analyses found that muscle strength, mobility, and glucose tolerance were improved with no effects on muscle mass, fiber size or type. A multi-omics approach at two ages further determined that restored muscle Bmal1 improved glucose handling pathways while concomitantly reducing lipid and protein metabolic pathways. The improved glucose tolerance and metabolic flexibility resulted in the systemic reduction of inflammatory signatures across peripheral tissues including liver, lung, and white adipose fat. Together, these findings highlight the critical role of muscle Bmal1 and downstream target genes for skeletal muscle homeostasis with considerable implications for systemic health.

Upstream and downstream pathways of diacylglycerol kinase : Novel phosphatidylinositol turnover-independent signal transduction pathways

Diacylglycerol kinase (DGK) phosphorylates diacylglycerol (DG) to produce phosphatidic acid (PA). Mammalian DGK comprise ten isozymes (α–κ) that regulate a wide variety of physiological and pathological events. Recently, we revealed that DGK isozymes use saturated fatty acid (SFA)/monosaturated fatty acid (MUFA)-containing and docosahexaenoic acid (22:6)-containing DG species, but not phosphatidylinositol (PI) turnover-derived 18:0/20:4-DG. For example, DGKδ, which is involved in the pathogenesis of type 2 diabetes, preferentially uses SFA/MUFA-containing DG species, such as 16:0/16:0- and 16:0/18:1-DG species, in high glucose-stimulated skeletal muscle cells. Moreover, DGKδ, which destabilizes the serotonin transporter (SERT) and regulates the serotonergic system in the brain, primarily generates 18:0/22:6-PA. Furthermore, 16:0/16:0-PA is produced by DGKζ in Neuro-2a cells during neuronal differentiation. We searched for SFA/MUFA-PA- and 18:0/22:6-PA-selective binding proteins (candidate downstream targets of DGKδ) and found that SFA/MUFA-PA binds to and activates the creatine kinase muscle type, an energy-metabolizing enzyme, and that 18:0/22:6-PA interacts with and activates Praja-1, an E3 ubiquitin ligase acting on SERT, and synaptojanin-1, a key player in the synaptic vesicle cycle. Next, we searched for SFA/MUFA-DG-generating enzymes upstream of DGKδ. We found that sphingomyelin synthase (SMS)1, SMS2, and SMS-related protein (SMSr) commonly act as phosphatidylcholine (PC)-phospholipase C (PLC) and phosphatidylethanolamine (PE)-PLC, generating SFA/MUFA-DG species, in addition to SMS and ceramide phosphoethanolamine synthase. Moreover, the orphan phosphatase PHOSPHO1 showed PC- and PE-PLC activities that produced SFA/MUFA-DG. Although PC- and PE-PLC activities were first described 70–35 years ago, their proteins and genes were not identified for a long time. We found that DGKδ interacts with SMSr and PHOSPHO1, and that DGKζ binds to SMS1 and SMSr. Taken together, these results strongly suggest that there are previously unrecognized signal transduction pathways that include DGK isozymes and generate and utilize SFA/MUFA-DG/PA or 18:0/22:6-DG/PA but not PI-turnover-derived 18:0/20:4-DG/PA.

Study on proficiency level of upper limb function and grip force characteristics of subjective satisfaction according to proficiency level in artificial muscle type myoelectric splints

Study on proficiency level of upper limb function and grip force characteristics of subjective satisfaction according to proficiency level in artificial muscle type myoelectric splints

Fast, variable stiffness-induced braided coiled artificial muscles

Biomimetic actuation technologies with high muscle strokes, cycle rates, and work capacities are necessary for robotic systems. We present a muscle type that operates based on changes in muscle stiffness caused by volume expansion. This muscle is created by coiling a mechanically strong braid, in which an elastomer hollow tube is adhesively attached inside. We show that the muscle reversibly contracts by 47.3% when driven by an oscillating input air pressure of 120 kilopascals at 10 Hz. It generates a maximum power density of 3.0 W/g and demonstrates a mechanical contractile efficiency of 74%. The muscle's low-pressure operation allowed for portable, thermal pneumatical actuation. Moreover, the muscle demonstrated bipolar actuation, wherein internal pressure leads to muscle length expansion if the initial muscle length is compressed and contraction if the muscle is not compressed. Modeling indicates that muscle expansion significantly alters its stiffness, which causes muscle actuation. We demonstrate the utility of BCMs for fast running and climbing robots.

Unraveling the Complexities of Beef Marination: Effect of Marinating Time, Marination Treatments, and Breed.

The present study focused on evaluating the effects of beef marination on quality traits and consumer acceptability. In this context, m. longissimus thoracis et lumborum and m. semimembranosus samples (n = 192) were obtained from Aberdeen Angus, Hereford, Charolais, and Limousine bulls and were marinated with milk (pasteurized, 100%), garlic and olive oil (2.35 g/500 mL), and lemon (citrus) juice (31% orange juice, 31% lemon juice, 38% distilled water) for 12, 24, and 72 h. Marinade components were selected based on traditional culinary practices and their scientifically proven effects on meat quality. Beef samples on day 0 and non-marinated samples were used as control groups. Beef color, water holding capacity, pH, cooking loss, and Warner-Bratzler shear force were measured three times for each sample. A taste panel assessment was performed to determine the sensory characteristics. Statistical analysis was performed using general linear model (GLM) procedures followed by Tukey's post-hoc comparison. Results revealed that marination time, as well as its two- and three-way interactions, significantly influenced beef quality parameters. These results indicate that the cattle breed is an important factor in evaluating the effectiveness of beef marination applications. The olive oil-garlic marinade was the most preferred by the panel across both types of muscle, as indicated by sensory evaluation results. The findings will not only enrich the scientific literature but also have practical implications for the beef industry.

PSLBI-11 Relevance of mitochondrial-mediated apoptotic processes in color and oxidative stabilities of five different beef muscles

Abstract Fresh meat color stands as one of the most critical quality indicators influencing consumer perception of meat freshness and purchase decision. Variation in color and color stability among different muscle types exists due to differences in chemical compositions, including lipids and metabolites, and other redox stability-related biochemical properties. Understanding biochemical mechanisms governing color stability in different muscles will offer insights for the industry to develop practical strategies aimed at reducing food waste attributed to discoloration. The objective of the current study was to identify metabolites and lipid compounds potentially linked to oxidative stabilities and relevant biochemical properties across different bovine muscles. Five different muscles [longissimus lumborum (LL), gluteus medius (GM), biceps femoris (BF), semitendinosus (ST), and infraspinatus (IF)] were collected from 16 beef carcasses (USDA Choice) at 2-d postmortem. Multiple cuts were made from each muscle for meat color measurements including instrumental and sensory color evaluations during display, and biochemical attributes, including total reducing activity (TRA), mitochondria-swelling, mitochondrial-lipid oxidation, cytochrome c redox ability (CytC). For the lipidome and metabolome analyses, beef samples were extracted and profiled using non-targeted UPLC-MS analyses. Data analysis was conducted with Metaboanalyst 6.0. The correlation analysis was conduct with Pearson’s correlation and considered the significance level at P < 0.05. In brief, LL and ST maintained greater color stability, redox stability, and lipid/protein oxidative stabilities compared with BF and GM, with IF showing the least (P < 0.05). Also, IF had the greatest levels of mitochondrial-swelling, mitochondrial-lipid oxidation, and oxidized CytC compared with LL (P < 0.05). The ANOVA results found 1,410 significant features in the metabolome analysis and 531 in the lipidome analysis of beef samples. Utilizing partial least square-discriminant analysis (PLS-DA), scatter plots grouped the five muscle types into three distinct clusters: LL and ST, BF and GM, and IF. Pro-apoptotic factors such as phosphatidylserine and lysophosphatidylserine were greater in IF compared with LL and ST (P < 0.05). From the metabolome results, anti-apoptotic factors like creatine and glutathione were increased in LL and ST compared with IF, whereas pro-apoptotic spermidine and adenosine monophosphate (AMP) were greatest in IF (P < 0.05). CIE a* showed a positive correlation with phospholipids and mitochondria-swelling (P < 0.05). Chroma exhibited a positive correlation with TRA, creatine, cyclic AMP, and GMP (P < 0.05). The results suggest that variations in meat color stability among the five beef muscles were likely attributed to differences in the abundance of lipids and metabolites associated with redox and oxidative stabilities, as well as mitochondrial-mediated apoptotic pathways. Specifically, the varying abundance of pro-/anti-apoptotic factors across muscles indicates potential mechanistic biochemical linkages between the apoptosis and meat color. Further research is warranted to investigate the effects of extended aging periods on changes in apoptotic features and color stability of beef muscles.

Development of a scintillation proximity assay for [3H]epibatidine binding sites of Tetronarce californica muscle-type nicotinic acetylcholine receptor

The therapy of intoxication with distinct organophosphorus (OP) compounds is still limited today. Especially chemical warfare agents like tabun and soman as well as novichok intoxications are difficult to address using established oxime therapeutics. These neurotoxins inhibit acetylcholinesterase (AChE), a pivotal enzyme in the synaptic cleft. The following accumulation of acetylcholine in the synaptic cleft leads to a dysfunctional, desensitized state of nicotinic acetylcholine receptors (nAChR). Without adequate treatment, the resulting cholinergic crisis leads to death by respiratory arrest. Consequently, the research approach for new therapeutic options needs to be expanded. A promising option would be substances interacting directly with nAChRs. Therefore, screening methods for new drug candidates are needed, with affinity assays playing an important role. In the present work, a saturation and competition scintillation proximity assay (SPA) for binding studies at [3H]epibatidine binding sites, conventionally classified as orthosteric binding sites of the muscle type nAChR was developed. This method offers several advantages over other assay technologies because no separation as well as washing steps are required to remove unbound ligands. Assay precision and solvent tolerance were validated according to the guidelines for validation of bioanalytical methods of the Food and Drug Administration (FDA) and European Medicines Agency (EMA). The newly developed binding assay was successfully implemented on an automated pipetting platform and is suitable for high-throughput-screening of receptor-ligand interactions at the nAChR. Furthermore, it allows to investigate/quantify competition of highly toxic agents such as nerve agents or structurally similar pesticides at the orthosteric binding site. Related to further pharmacological results, the affinity to [3H]epibatidine binding sites can provide additional information on whether potential drug candidates would be suitable for treatment of nerve agent poisoning.