Effects and mechanisms of ultrasound-assisted emulsification treatment on the curcumin delivery and digestive properties of myofibrillar protein-carboxymethyl cellulose complex emulsion gel.

Different emulsion gel systems are widely applied to deliver functional ingredients. The effects and mechanisms of ultrasound-assisted emulsification (UAE) treatment and carboxymethyl cellulose (CMC) modifying the curcumin delivery properties and in vitro digestibility of the myofibrillar protein (MP)-soybean oil emulsion gels were investigated. The rheological properties, droplet size, protein and CMC distribution, ultrastructure, surface hydrophobicity, sulfhydryl groups, and zeta potential of emulsion gels were also measured. Results indicate that UAE treatment and CMC addition both improved curcumin encapsulation and protection efficiency in MP emulsion gel, especially for the UAE combined with CMC (UAE-CMC) treatment which encapsulation efficiency, protection efficiency, the release rate, and bioaccessibility of curcumin increased from 86.75 % to 97.67 %, 44.85 % to 68.85 %, 18.44 % to 41.78 %, and 28.68 % to 44.93 % respectively. The protein digestibility during the gastric stage was decreased after the CMC addition and UAE treatment, and the protein digestibility during the intestinal stage was reduced after the CMC addition. The fatty acid release rate was increased after CMC addition and UAE treatment. Apparent viscosity, storage modulus, and loss modulus were decreased after CMC addition while increased after UAE and UAE-CMC treatment especially the storage modulus increased from 0.26 Pa to 41 Pa after UAE-CMC treatment. The oil size was decreased, the protein and CMC concentration around the oil was increased, and a denser and uniform emulsion gel network structure was formed after UAE treatment. The surface hydrophobicity, free SH groups, and absolute zeta potential were increased after UAE treatment. The UAE-CMC treatment could strengthen the MP emulsion gel structure and decrease the oil size to increase the curcumin delivery properties, and hydrophobic and electrostatic interaction might be essential forces to maintain the emulsion gel.

Thawed drip and its membrane-separated components: Role in retarding myofibrillar protein gel deterioration during freezing-thawing cycles.

Myofibrillar proteins are crucial for gel formation in processed meat products such as sausages and meat patties. Freeze-thaw cycles can alter protein properties, impacting gel stability and product quality. This study aims to investigate the potential of thawed drip and its membrane-separated components as potential antifreeze agents to retard denaturation, oxidation and gel deterioration of myofibrillar proteins during freezing-thawing cycles of pork patties. The thawed drip and its membrane-separated components of > 10 kDa and < 10 kDa, along with deionized water, were added to minced pork at 10 % mass fraction and subjected to increasing freeze-thaw cycles. Results showed that the addition of thawed drip and its membrane separation components inhibited denaturation and structural changes of myofibrillar proteins, evidenced by reduced surface hydrophobicity and carbonyl content, increased free sulfhydryl groups, protein solubility and α-helix, as compared to the deionized water group. Correspondingly, improved gel properties including water-holding capacity, textural parameters and denser network structure were observed with the addition of thawed drip and its membrane separation components. Denaturation and oxidation of myofibrillar proteins were positively correlated with gel deterioration during freezing-thawing cycles. We here propose a role of thawed drip and its membrane separation components as cryoprotectants against myofibrillar protein gel deterioration during freeze-thawing cycles.

Insight into the evolution of textural properties and juiciness of ready-to-eat chicken breasts upon different thermal sterilization: From the perspective of protein degradation.

Texture deterioration of meat products upon high-temperature sterilization is a pressing issue in the meat industry. This study evaluated the effect of different thermal sterilization temperatures on the textural and juiciness of ready-to-eat (RTE) chicken breast. In this study, by dynamically monitoring the texture and juiciness of chicken meat products during the process of thermal sterilization, it has been observed that excessively high sterilization temperatures (above 100°C) significantly diminish the shear force, springiness and water-holding capacity of the products. Furthermore, from the perspective of myofibrillar protein degradation, molecular mechanisms have been elucidated, unveiling that the thermal sterilization treatment at 121°C/10 min triggers the degradation of myosin heavy chains and F-actin, disrupting the lattice arrangement of myofilaments, compromising the integrity of sarcomeres, and resulting in an increase of approximately 40.66% in the myofibrillar fragmentation index, thus diminishing the quality characteristics of the products. This study unravels the underlying mechanisms governing the dynamic changes in quality of chicken meat products during the process of thermal sterilization, thereby providing theoretical guidance for the development of high-quality chicken products.

Building, Breaking, and Repairing Neuromuscular Synapses.

A coordinated and complex interplay of signals between motor neurons, skeletal muscle cells, and Schwann cells controls the formation and maintenance of neuromuscular synapses. Deficits in the signaling pathway for building synapses, caused by mutations in critical genes or autoantibodies against key proteins, are responsible for several neuromuscular diseases, which cause muscle weakness and fatigue. Here, we describe the role that four key genes, Agrin, Lrp4, MuSK, and Dok7, play in this signaling pathway, how an understanding of their mechanisms of action has led to an understanding of several neuromuscular diseases, and how this knowledge has contributed to emerging therapies for treating neuromuscular diseases.

Structural domain in the Titin N2B-us region binds to FHL2 in a force-activation dependent manner.

Titin N2B unique sequence (N2B-us) is a 572 amino acid sequence that acts as an elastic spring to regulate muscle passive elasticity. It is thought to lack stable tertiary structures and is a force-bearing region that is regulated by mechanical stretching. In this study, the conformation of N2B-us and its interaction with four-and-a-half LIM domain protein 2 (FHL2) are investigated using AlphaFold2 predictions and single-molecule experimental validation. Surprisingly, a stable alpha/beta structural domain is predicted and confirmed in N2B-us that can be mechanically unfolded at forces of a few piconewtons. Additionally, more than twenty FHL2 LIM domain binding sites are predicted to spread throughout N2B-us. Single-molecule manipulation experiments reveals the force-dependent binding of FHL2 to the N2B-us structural domain. These findings provide insights into the mechano-sensing functions of N2B-us and its interactions with FHL2.

Deciphering the Therapeutic Role of Lactate in Combating Disuse-Induced Muscle Atrophy: An NMR-Based Metabolomic Study in Mice.

Disuse muscle atrophy (DMA) is a significant healthcare challenge characterized by progressive loss of muscle mass and function resulting from prolonged inactivity. The development of effective strategies for muscle recovery is essential. In this study, we established a DMA mouse model through hindlimb suspension to evaluate the therapeutic potential of lactate in alleviating the detrimental effects on the gastrocnemius muscle. Using NMR-based metabolomic analysis, we investigated the metabolic changes in DMA-injured gastrocnemius muscles compared to controls and evaluated the beneficial effects of lactate treatment. Our results show that lactate significantly reduced muscle mass loss and improved muscle function by downregulating Murf1 expression, decreasing protein ubiquitination and hydrolysis, and increasing myosin heavy chain levels. Crucially, lactate corrected perturbations in four key metabolic pathways in the DMA gastrocnemius: the biosynthesis of phenylalanine, tyrosine, and tryptophan; phenylalanine metabolism; histidine metabolism; and arginine and proline metabolism. In addition to phenylalanine-related pathways, lactate also plays a role in regulating branched-chain amino acid metabolism and energy metabolism. Notably, lactate treatment normalized the levels of eight essential metabolites in DMA mice, underscoring its potential as a therapeutic agent against the consequences of prolonged inactivity and muscle wasting. This study not only advances our understanding of the therapeutic benefits of lactate but also provides a foundation for novel treatment approaches aimed at metabolic restoration and muscle recovery in conditions of muscle wasting.

The Influence of Stress and Binge-Patterned Alcohol Drinking on Mouse Skeletal Muscle Protein Synthesis and Degradation Pathways.

Adverse experiences (e.g., acute stress) and alcohol misuse can both impair skeletal muscle homeostasis, resulting in reduced protein synthesis and greater protein breakdown. Exposure to acute stress is a significant risk factor for engaging in alcohol misuse. However, little is known about how these factors together might further affect skeletal muscle health. To that end, this study investigated the effects of acute stress exposure followed by a period of binge-patterned alcohol drinking on signaling factors along mouse skeletal muscle protein synthesis (MPS) and degradation (MPD) pathways. Young adult male C57BL/6J mice participated in the Drinking in the Dark paradigm, where they received 2-4 h of access to 20% ethanol (alcohol group) or water (control group) for four days to establish baseline drinking levels. Three days later, half of the mice in each group were either exposed to a single episode of uncontrollable tail shocks (acute stress) or remained undisturbed in their home cages (no stress). Three days after stress exposure, mice received 4 h of access to 20% ethanol (alcohol) to model binge-patterned alcohol drinking or water for ten consecutive days. Immediately following the final episode of alcohol access, mouse gastrocnemius muscle was extracted to measure changes in relative protein levels along the Akt-mTOR MPS, as well as the ubiquitin-proteasome pathway (UPP) and autophagy MPD pathways via Western blotting. A single exposure to acute stress impaired Akt singling and reduced rates of MPS, independent of alcohol access. This observation was concurrent with a potent increase in heat shock protein seventy expression in the muscle of stressed mice. Alcohol drinking did not exacerbate stress-induced alterations in the MPS and MPD signaling pathways. Instead, changes in the MPS and MPD signaling factors due to alcohol access were primarily observed in non-stressed mice. Taken together, these data suggest that exposure to a stressor of sufficient intensity may cause prolonged disruptions to signaling factors that impact skeletal muscle health and function beyond what could be further induced by periods of alcohol misuse.

Salt reduction in myofibrillar protein gel via inhomogeneous distribution of sodium-containing encapsulated fish oil coacervate: Mucopenetration ability of sodium carboxymethyl cellulose.

The potential application of fish oil microcapsules as salt reduction strategies in low-salt myofibrillar protein (MP) gel was investigated by employing soy protein isolates/carboxymethyl cellulose sodium (SPI-CMC) coacervates enriched with 25 mM sodium chloride and exploring their rheological characteristics, taste perception, and microstructure. The results revealed that the SPI-CMC coacervate phase exhibited the highest sodium content under 25 mM sodium level, albeit with uneven distribution. Notably, the hydrophilic and adhesive properties of CMC to sodium facilitated the in vitro release of sodium during oral digestion, as evidenced by the excellent wettability and mucopenetration ability of CMC. Remarkably, the fish oil microcapsules incorporating SPI-CMC as the wall material, prepared at pH 3.5 with a core-to-wall ratio of 1:1, demonstrated the highest encapsulation efficiency, which was supported by the strong hydrogen bonding. Interestingly, the presence of SPI-CMC coacervates and fish oil microcapsules enhanced the interaction between MPs and strengthened the low-salt MP gel network. Coupled with electronic tongue analysis, the incorporation of fish oil microcapsules slightly exacerbated the non-uniformity of sodium distribution. This ultimately contributed to an enhanced perception of saltiness, richness, and aftertaste in low-salt protein gels. Overall, the incorporation of fish oil microcapsules emerged as an effective salt reduction strategy in low-salt MP gel.

Correlation analysis of phosphorylation of myofibrillar protein and muscle quality of tilapia during storage in ice.

In this study, the correlation between protein phosphorylation and deterioration in the quality of tilapia during storage in ice was examined by assessing changes in texture, water-holding capacity (WHC), and biochemical characteristics of myofibrillar protein throughout 7 days of storage. The hardness significantly decreased from 471.50 to 252.17 g, whereas cooking and drip losses significantly increased from 26.5% to 32.6% and 2.9% to 9.1%, respectively (P < 0.05). Myofibril fragmentation increased, while myofibrillar protein sulfhydryl content and Ca2+-ATPase activity decreased from 119.33 to 89.29 µmol/g prot and 0.85 to 0.46 µmolPi/mg prot/h, respectively (P < 0.05). Correlation analysis revealed that the myofibrillar protein phosphorylation level was positively correlated with hardness and Ca2+-ATPase activity but negatively correlated with WHC. Myofibrillar protein phosphorylation affects muscle contraction by influencing the dissociation of actomyosin, thereby regulating hardness and WHC. This study provides novel insights for the establishment of quality control strategies for tilapia storage based on protein phosphorylation.

Myofibrillar protein lipoxidation in fish induced by linoleic acid and 4-hydroxy-2-nonenal: Insights from LC-MS/MS analysis.

The oxidation of fish lipids and proteins is interconnected. The LOX (lipoxygenase)-catalyzed LA (linoleic acid) oxidation system on MPs (myofibrillar proteins) was established in vitro, to investigate the impact of lipoxidation on the physicochemical properties of fish MPs. By detecting HNE (4-hydroxy-2-nonenal) concentration during LA oxidation, the HNE treatment system was established to investigate the role of HNE in this process. In addition, the site specificity of modification on MPs was detected utilizing LC-MS/MS. Both treatments could induce sidechain modification, increase particle size, and cause loss of nutritional value through the reduction in amino acid content of MPs. The HNE group is more likely to alter the MPs' surface hydrophobicity compared to the LA group. By increasing the exposure of modification sites in MPs, the HNE group has more types and number of modifications compared to the LA group. LA group mainly induced the modification of single oxygen addition on MPs instead, which accounted for over 50 % of all modifications. The LA group induced a more pronounced reduction in the solubility of MPs as compared to the HNE group. In conclusion, HNE binding had a high susceptibility to Lys on MPs. Protein aggregation, peptide chain fragmentation, and decreased solubility occurred in the LA group mainly induced by peroxide generated during lipid oxidation or the unreacted LA instead of HNE. This study fills in the mechanism of lipoxidation on protein oxidation in fish and sheds light on the HNE modification sites of MPs, paving the way for the development of oxidation control technology.

Inhibitive effect of trehalose and sodium pyrophosphate on oxidation and structural changes of myofibrillar proteins in silver carp surimi during frozen storage.

This work investigated the cryoprotective effect of trehalose (TH) and sodium pyrophosphate (SPP) alone and in combination on myofibrillar protein (MP) oxidation and structural changes in silver carp surimi during 90 days of frozen storage (-20 °C). TH combined with SPP was significantly more effective than single TH or SPP in preventing MP oxidation (P < 0.05), showing a higher SH content (6.05 nmol/mg protein), and a lower carbonyl (4.24 nmol/mg protein) and dityrosine content (1280 A.U.). SDS-PAGE results indicated that TH combined with SPP did not differ significantly from TH and SPP in inhibiting protein degradation but was more effective in inhibiting protein crosslinking. Moreover, all cryoprotectants could stabilise the secondary and tertiary structures and inhibit unfolded and aggregation of MP, with the combination of TH and SPP being the best. It's worth noting that TH combined with SPP had a synergistic effect on inhibiting the decrease in α-helix content and gel-forming ability, and the increase in surface hydrophobicity. Overall, TH combined with SPP could significantly inhibited MP oxidation and structural changes in surimi during frozen storage and improve the gel-forming ability, which was significantly better than single TH or SPP.

Proximal tubular FHL2, a novel downstream target of hypoxia inducible factor 1, is a protector against ischemic acute kidney injury.

Four-and-a-half LIM domains protein 2 (FHL2) is an adaptor protein that may interact with hypoxia inducible factor 1α (HIF-1α) or ß-catenin, two pivotal protective signaling in acute kidney injury (AKI). However, little is known about the regulation and function of FHL2 during AKI. We found that FHL2 was induced in renal tubular cells in patients with acute tubular necrosis and mice model of ischemia-reperfusion injury (IRI). In cultured renal proximal tubular cells (PTCs), hypoxia induced FHL2 expression and promoted the binding of HIF-1 to FHL2 promoter. Compared with control littermates, mice with PTC-specific deletion of FHL2 gene displayed worse renal function, more severe morphologic lesion, more tubular cell death and less cell proliferation, accompanying by downregulation of AQP1 and Na, K-ATPase after IRI. Consistently, loss of FHL2 in PTCs restricted activation of HIF-1 and ß-catenin signaling simultaneously, leading to attenuation of glycolysis, upregulation of apoptosis-related proteins and downregulation of proliferation-related proteins during IRI. In vitro, knockdown of FHL2 suppressed hypoxia-induced activation of HIF-1α and ß-catenin signaling pathways. Overexpression of FHL2 induced physical interactions between FHL2 and HIF-1α, ß-catenin, GSK-3ß or p300, and the combination of these interactions favored the stabilization and nuclear translocation of HIF-1α and ß-catenin, enhancing their mediated gene transcription. Collectively, these findings identify FHL2 as a direct downstream target gene of HIF-1 signaling and demonstrate that FHL2 could play a critical role in protecting against ischemic AKI by promoting the activation of HIF-1 and ß-catenin signaling through the interactions with its multiple protein partners.

MCT4 knockdown by tumor microenvironment-responsive nanoparticles remodels the cytokine profile and eradicates aggressive breast cancer cells.

Breast cancer is a wide-spread threat to the women's health. The drawbacks of conventional treatments necessitate the development of alternative strategies, where gene therapy has regained hope in achieving an efficient eradication of aggressive tumors. Monocarboxylate transporter 4 (MCT4) plays pivotal roles in the growth and survival of various tumors, which offers a promising target for treatment. In the present study, pH-responsive lipid nanoparticles (LNPs) based on the ionizable lipid,1,2-dioleoyl-3-dimethylammonium propane (DODAP), were designed for the delivery of siRNA targeting MCT4 gene to the breast cancer cells. Following multiple steps of characterization and optimization, the anticancer activities of the LNPs were assessed against an aggressive breast cancer cell line, 4T1, in comparison with a normal cell line, LX-2. The selection of the helper phospholipid to be incorporated into the LNPs had a dramatic impact on their gene delivery performance. The optimized LNPs enabled a powerful MCT4 silencing by ∼90 % at low siRNA concentrations, with a subsequent ∼80 % cytotoxicity to 4T1 cells. Meanwhile, the LNPs demonstrated a 5-fold higher affinity to the breast cancer cells versus the normal cells, in which they had a minimum effect. Moreover, the MCT4 knockdown by the treatment remodeled the cytokine profile in 4T1 cells, as evidenced by 90 % and ∼64 % reduction in the levels of TNF-α and IL-6; respectively. The findings of this study are promising for potential clinical applications. Furthermore, the simple and scalable delivery vector developed herein can serve as a breast cancer-targeting platform for the delivery of other RNA therapeutics.

Aspartate ß-Hydroxylase Is Upregulated in Head and Neck Squamous Cell Carcinoma and Regulates Invasiveness in Cancer Cell Models.

Aspartate ß-hydroxylase (ASPH) is a protein associated with malignancy in a wide range of tumors. We hypothesize that inhibition of ASPH activity could have anti-tumor properties in patients with head and neck cancer. In this study, we screened tumor tissues of 155 head and neck squamous cell carcinoma (HNSCC) patients for the expression of ASPH using immunohistochemistry. We used an ASPH inhibitor, MO-I-1151, known to inhibit the catalytic activity of ASPH in the endoplasmic reticulum, to show its inhibitory effect on the migration of SCC35 head and neck cancer cells in cell monolayers and in matrix-embedded spheroid co-cultures with primary cancer-associated fibroblast (CAF) CAF 61137 of head and neck origin. We also studied a combined effect of MO-I-1151 and HfFucCS, an inhibitor of invasion-blocking heparan 6-O-endosulfatase activity. We found ASPH was upregulated in HNSCC tumors compared to the adjacent normal tissues. ASPH was uniformly high in expression, irrespective of tumor stage. High expression of ASPH in tumors led us to consider it as a therapeutic target in cell line models. ASPH inhibitor MO-I-1151 had significant effects on reducing migration and invasion of head and neck cancer cells, both in monolayers and matrix-embedded spheroids. The combination of the two enzyme inhibitors showed an additive effect on restricting invasion in the HNSCC cell monolayers and in the CAF-containing co-culture spheroids. We identify ASPH as an abundant protein in HNSCC tumors. Targeting ASPH with inhibitor MO-I-1151 effectively reduces CAF-mediated cellular invasion in cancer cell models. We propose that the additive effect of MO-I-1151 with HfFucCS, an inhibitor of heparan 6-O-endosulfatases, on HNSCC cells could improve interventions and needs to be further explored.

Oleocanthal Protects C2C12 Myotubes against the Pro-Catabolic and Anti-Myogenic Action of Stimuli Able to Induce Muscle Wasting In Vivo.

Oleocanthal (OC) is a monophenol of extra-virgin olive oil (EVOO) endowed with antibiotic, cardioprotective and anticancer effects, among others, mainly in view of its antioxidant and anti-inflammatory properties. OC has been largely investigated in terms of its anticancer activity, in Alzheimer disease and in collagen-induced arthritis; however, the possibility that it can also affect muscle biology has been totally overlooked so far. This study is the first to describe that OC modulates alterations induced in C2C12 myotubes by stimuli known to induce muscle wasting in vivo, namely TNF-α, or in the medium conditioned by the C26 cachexia-inducing tumor (CM-C26). C2C12 myotubes were exposed to CM-C26 or TNF-α in the presence or absence of OC for 24 and 48 h and analyzed by immunofluorescence and Western blotting. In combination with TNF-α or CM-C26, OC was revealed to be able to restore both the myotube's original size and morphology and normal levels of both atrogin-1 and MuRF1. OC seems unable to impinge on the autophagic-lysosomal proteolytic system or protein synthesis. Modulations towards normal levels of the expression of molecules involved in myogenesis, such as Pax7, myogenin and MyHC, were also observed in the myotube cultures exposed to OC and TNF-α or CM-C26. In conclusion, the data presented here show that OC exerts a protective action in C2C12 myotubes exposed to TNF-α or CM-C26, with mechanisms likely involving the downregulation of ubiquitin-proteasome-dependent proteolysis and the partial relief of myogenic differentiation impairment.

A retrospective study on the clinical and molecular outcomes of calpainopathy in a Turkish patient cohort.

Background and aim: Calpainopathy, also known as limb-girdle muscular dystrophy recessive type 1, is a progressive muscle disorder that impacts the muscles around the hips and shoulders. The disease is caused by defects in the CAPN3 gene and can be inherited in both recessive and dominant forms. In this retrospective study, we aimed to evaluate the clinical and molecular results of our patients with calpainopathy and to examine the CAPN3 variants in Turkish and global populations. Materials and methods: Molecular analyses were performed using the next-generation sequencing (NGS) method. CAPN3 variants were identified through the examination of various databases. Results: In this retrospective study, the cohort consisted of seven patients exhibiting the CAPN3 (NM_000070.3) mutation and a phenotype compatible with calpainopathy at a single center in Türkiye. All patients displayed high CK levels and muscle weakness. We report a novel missense c.2437G>A variant that causes the autosomal dominant form of calpainopathy. Interestingly, the muscle biopsy report for the patient with the novel mutation indicated sarcoglycan deficiency. Molecular findings for the remaining individuals in the cohort included a compound heterozygous variant (frameshift and missense), one homozygous nonsense, one homozygous intronic deletion, and three homozygous missense variants. The most common variant in the Turkish population was c.550del. In both populations, pathogenic variants were most frequently located in exon 21, according to exon length. Variants were stochastically distributed based on consequences in CAPN3 domains. Conclusion: Therefore, the NGS method proves highly effective in diagnosing rare diseases characterized by clinical heterogeneity. Assessing variants based on ethnicity holds significance in the development of precise therapies.

Increased ANKRD1 Levels in Early Senescence Mediated by RBMS1-Elicited ANKRD1 mRNA Stabilization.

Cellular senescence is a dynamic biological process triggered by sublethal cell damage and driven by specific changes in gene expression programs. We recently identified ANKRD1 (ankyrin repeat domain 1) as a protein strongly elevated after triggering senescence in fibroblasts. Here, we set out to investigate the mechanisms driving the elevated production of ANKRD1 in the early stages of senescence. Our results indicated that the rise in ANKRD1 levels after triggering senescence using etoposide (Eto) was the result of moderate increases in transcription and translation, and robust mRNA stabilization. Antisense oligomer (ASO) pulldown followed by mass spectrometry revealed a specific interaction of the RNA-binding protein RBMS1 with ANKRD1 mRNA that was confirmed by ribonucleoprotein immunoprecipitation analysis. RBMS1 abundance decreased in the nucleus and increased in the cytoplasm during Eto-induced senescence; in agreement with the hypothesis that RBMS1 may participate in post-transcriptional stabilization of ANKRD1 mRNA, silencing RBMS1 reduced, while overexpressing RBMS1 enhanced ANKRD1 mRNA half-life after Eto treatment. A segment proximal to the ANKRD1 coding region was identified as binding RBMS1 and conferring RBMS1-dependent increased expression of a heterologous reporter. We propose that RBMS1 increases expression of ANKRD1 during the early stages of senescence by stabilizing ANKRD1 mRNA.

Myofibrillar protein hydrolysis under hydroxyl radical oxidative stress: Structural changes and their impacts on binding to selected aldehydes.

In this study, a hydroxyl radical oxidation system was established to simulate the oxidation process in fermented meat products. This system was employed to examine the structural changes in myofibrillar proteins (MPs) resulting from tryptic hydrolysis after a hydroxyl radical oxidative regime. The effect of these changes on the ability of MPs to bind selected aldehydes (3-methyl butanal, pentanal, hexanal, and heptanal) was also investigated. Moderate oxidation (H2O2 ≤ 1.0 mM) unfolded the structure of MPs, facilitating trypsin-mediated hydrolysis and increasing their binding capacity for the four selected aldehydes. However, excessive oxidation (H2O2 ≥ 2.5 mM) led to cross-linking and aggregation of MPs, inhibiting trypsin-mediated hydrolysis. The oxidised MPs had the best binding capacity for heptanal. The interaction of the oxidised trypsin-hydrolysed MPs with heptanal was driven by hydrophobic interactions. The binding of heptanal affected the structure of the oxidised trypsin-hydrolysed MPs and reduced their α-helix content.

The Influence of a Genetic Variant in CCDC78 on LMNA-Associated Skeletal Muscle Disease.

Mutations in the LMNA gene-encoding A-type lamins can cause Limb-Girdle muscular dystrophy Type 1B (LGMD1B). This disease presents with weakness and wasting of the proximal skeletal muscles and has a variable age of onset and disease severity. This variability has been attributed to genetic background differences among individuals; however, such variants have not been well characterized. To identify such variants, we investigated a multigeneration family in which affected individuals are diagnosed with LGMD1B. The primary genetic cause of LGMD1B in this family is a dominant mutation that activates a cryptic splice site, leading to a five-nucleotide deletion in the mature mRNA. This results in a frame shift and a premature stop in translation. Skeletal muscle biopsies from the family members showed dystrophic features of variable severity, with the muscle fibers of some family members possessing cores, regions of sarcomeric disruption, and a paucity of mitochondria, not commonly associated with LGMD1B. Using whole genome sequencing (WGS), we identified 21 DNA sequence variants that segregate with the family members possessing more profound dystrophic features and muscle cores. These include a relatively common variant in coiled-coil domain containing protein 78 (CCDC78). This variant was given priority because another mutation in CCDC78 causes autosomal dominant centronuclear myopathy-4, which causes cores in addition to centrally positioned nuclei. Therefore, we analyzed muscle biopsies from family members and discovered that those with both the LMNA mutation and the CCDC78 variant contain muscle cores that accumulated both CCDC78 and RyR1. Muscle cores containing mislocalized CCDC78 and RyR1 were absent in the less profoundly affected family members possessing only the LMNA mutation. Taken together, our findings suggest that a relatively common variant in CCDC78 can impart profound muscle pathology in combination with a LMNA mutation and accounts for variability in skeletal muscle disease phenotypes.

Macrophage MCT4 inhibition activates reparative genes and protects from atherosclerosis by histone H3 lysine 18 lactylation.

Macrophage activation is a hallmark of atherosclerosis, accompanied by a switch in core metabolism from oxidative phosphorylation to glycolysis. The crosstalk between metabolic rewiring and histone modifications in macrophages is worthy of further investigation. Here, we find that lactate efflux-associated monocarboxylate transporter 4 (MCT4)-mediated histone lactylation is closely related to atherosclerosis. Histone H3 lysine 18 lactylation dependent on MCT4 deficiency activated the transcription of anti-inflammatory genes and tricarboxylic acid cycle genes, resulting in the initiation of local repair and homeostasis. Strikingly, histone lactylation is characteristically involved in the stage-specific local repair process during M1 to M2 transformation, whereas histone methylation and acetylation are not. Gene manipulation and protein hydrolysis-targeted chimerism technology are used to confirm that MCT4 deficiency favors ameliorating atherosclerosis. Therefore, our study shows that macrophage MCT4 deficiency, which links metabolic rewiring and histone modifications, plays a key role in training macrophages to become repair and homeostasis phenotypes.