Echocardiographic Strain Abnormalities Precede Left Ventricular Hypertrophy Development in Hypertrophic Cardiomyopathy Mutation Carriers.

Hypertrophic cardiomyopathy (HCM) is a genetic disease characterized by unexplained left ventricular hypertrophy (LVH), diastolic dysfunction, and increased sudden-death risk. Early detection of the phenotypic expression of the disease in genetic carriers without LVH (Gen+/Phen-) is crucial for emerging therapies. This clinical study aims to identify echocardiographic predictors of phenotypic development in Gen+/Phen-. Sixteen Gen+/Phen- (one subject with troponin T, six with myosin heavy chain-7, and nine with myosin-binding protein C3 mutations), represented the study population. At first and last visit we performed comprehensive 2D speckle-tracking strain echocardiography. During a follow-up of 8 ± 5 years, five carriers developed LVH (LVH+). At baseline, these patients were older than those who did not develop LVH (LVH-) (30 ± 8 vs. 15 ± 8 years, p = 0.005). LVH+ had reduced peak global strain rate during the isovolumic relaxation period (SRIVR) (0.28 ± 0.05 vs. 0.40 ± 0.11 1/s, p = 0.048) and lower global longitudinal strain (GLS) (-19.8 ± 0.4 vs. -22.3 ± 1.1%; p < 0.0001) than LVH- at baseline. SRIVR and GLS were not correlated with age (overall, p > 0.08). This is the first HCM study investigating subjects before they manifest clinically significant or relevant disease burden or symptomatology, comparing at baseline HCM Gen+/Phen- subjects who will develop LVH with those who will not. Furthermore, we identified highly sensitive, easily obtainable, age- and load-independent echocardiographic predictors of phenotype development in HCM gene carriers who may undergo early preventive treatment.

The Aryl Hydrocarbon Receptor Regulates Invasiveness and Motility in Acute Myeloid Leukemia Cells through Expressional Regulation of Non-Muscle Myosin Heavy Chain IIA.

Acute myeloid leukemia (AML) is the most prevalent type of hematopoietic malignancy. Despite recent therapeutic advancements, the high relapse rate associated with extramedullary involvement remains a challenging issue. Moreover, therapeutic targets that regulate the extramedullary infiltration of AML cells are still not fully elucidated. The Aryl Hydrocarbon Receptor (AHR) is known to influence the progression and migration of solid tumors; however, its role in AML is largely unknown. This study explored the roles of AHR in the invasion and migration of AML cells. We found that suppressed expression of AHR target genes correlated with an elevated relapse rate in AML. Treatment with an AHR agonist on patient-derived AML cells significantly decreased genes associated with leukocyte trans-endothelial migration, cell adhesion, and regulation of the actin cytoskeleton. These results were further confirmed in THP-1 and U937 AML cell lines using AHR agonists (TCDD and FICZ) and inhibitors (SR1 and CH-223191). Treatment with AHR agonists significantly reduced Matrigel invasion, while inhibitors enhanced it, regardless of the Matrigel's stiffness. AHR agonists significantly reduced the migration rate and chemokinesis of both cell lines, but AHR inhibitors enhanced them. Finally, we found that the activity of AHR and the expression of NMIIA are negatively correlated. These findings suggest that AHR activity regulates the invasiveness and motility of AML cells, making AHR a potential therapeutic target for preventing extramedullary infiltration in AML.

Identification of Variants of Uncertain Significance in the Genes Associated with Thoracic Aortic Disease in Russian Patients with Nonsyndromic Sporadic Subtypes of the Disorder.

Nonsyndromic sporadic thoracic aortic aneurysm (nssTAA) is characterized by diverse genetic variants that may vary in different populations. Our aim was to identify clinically relevant variants in genes implicated in hereditary aneurysms in Russian patients with nssTAA. Forty-one patients with nssTAA without dissection were analyzed. Using massive parallel sequencing, we searched for variants in exons of 53 known disease-causing genes. Patients were found to have no (likely) pathogenic variants in the genes of hereditary TAA. Six variants of uncertain significance (VUSs) were identified in four (9.8%) patients. Three VUSs [FBN1 c.7841C>T (p.Ala2614Val), COL3A1 c.2498A>T (p.Lys833Ile), and MYH11 c.4993C>T (p.Arg1665Cys)] are located in genes with "definitive" disease association (ClinGen). The remaining variants are in "potentially diagnostic" genes or genes with experimental evidence of disease association [NOTCH1 c.964G>A (p.Val322Met), COL4A5 c.953C>G (p.Pro318Arg), and PLOD3 c.833G>A (p.Gly278Asp)]. Russian patients with nssTAA without dissection examined in this study have ≥1 VUSs in six known genes of hereditary TAA (FBN1, COL3A1, MYH11, NOTCH1, COL4A5, or PLOD3). Experimental studies expanded genetic testing, and clinical examination of patients and first/second-degree relatives may shift VUSs to the pathogenic (benign) category or to a new class of rare "predisposing" low-penetrance variants causing the pathology if combined with other risk factors.

Motor domain phosphorylation increases nucleotide exchange and turns MYO6 into a faster and stronger motor.

Myosin motors perform many fundamental functions in eukaryotic cells by providing force generation, transport or tethering capacity. Motor activity control within the cell involves on/off switches, however, few examples are known of how myosins regulate speed or processivity and fine-tune their activity to a specific cellular task. Here, we describe a phosphorylation event for myosins of class VI (MYO6) in the motor domain, which accelerates its ATPase activity leading to a 4-fold increase in motor speed determined by actin-gliding assays, single molecule mechanics and stopped flow kinetics. We demonstrate that the serine/threonine kinase DYRK2 phosphorylates MYO6 at S267 in vitro. Single-molecule optical-tweezers studies at low load reveal that S267-phosphorylation results in faster nucleotide-exchange kinetics without change in the working stroke of the motor. The selective increase in stiffness of the acto-MYO6 complex when proceeding load-dependently into the nucleotide-free rigor state demonstrates that S267-phosphorylation turns MYO6 into a stronger motor. Finally, molecular dynamic simulations of the nucleotide-free motor reveal an alternative interaction network within insert-1 upon phosphorylation, suggesting a molecular mechanism, which regulates insert-1 positioning, turning the S267-phosphorylated MYO6 into a faster motor.

Fast and slow myofiber nuclei, satellite cells, and size distribution with lifelong endurance exercise in men and women.

We previously observed lifelong endurance exercise (LLE) influenced quadriceps whole-muscle and myofiber size in a fiber-type and sex-specific manner. The current follow-up exploratory investigation examined myofiber size regulators and myofiber size distribution in vastus lateralis biopsies from these same LLE men (n = 21, 74 ± 1 years) and women (n = 7, 72 ± 2 years) as well as old, healthy nonexercisers (OH; men: n = 10, 75 ± 1 years; women: n = 10, 75 ± 1 years) and young exercisers (YE; men: n = 10, 25 ± 1 years; women: n = 10, 25 ± 1 years). LLE exercised ~5 days/week, ~7 h/week for the previous 52 ± 1 years. Slow (myosin heavy chain (MHC) I) and fast (MHC IIa) myofiber nuclei/fiber, myonuclear domain, satellite cells/fiber, and satellite cell density were not influenced (p > 0.05) by LLE in men and women. The aging groups had ~50%-60% higher proportion of large (>7000 µm2) and small (<3000 µm2) myofibers (OH; men: 44%, women: 48%, LLE; men: 42%, women: 42%, YE; men: 27%, women: 29%). LLE men had triple the proportion of large slow fibers (LLE: 21%, YE: 7%, OH: 7%), while LLE women had more small slow fibers (LLE: 15%, YE: 8%, OH: 9%). LLE reduced by ~50% the proportion of small fast (MHC II containing) fibers in the aging men (OH: 14%, LLE: 7%) and women (OH: 35%, LLE: 18%). These data, coupled with previous findings, suggest that myonuclei and satellite cell content are uninfluenced by lifelong endurance exercise in men ~60-90 years, and this now also extends to septuagenarian lifelong endurance exercise women. Additionally, lifelong endurance exercise appears to influence the relative abundance of small and large myofibers (fast and slow) differently between men and women.

MYO5B gene mutations may promote the occurrence of very early onset inflammatory bowel disease: a case report.

BACKGROUND: With recent advances in gene sequencing technology, more than 60 genetic mutations associated with very early onset inflammatory bowel disease (VEO-IBD) have been reported. Most of the genes are associated with immune deficiencies. The Myosin 5B (MYO5B) gene is primarily involved in cell motility and material transport which is associated with congenital intractable diarrhea and cholestasis. No studies have examined the relationship between the MYO5B gene and VEO-IBD. We report a case of a child with a mutation in the MYO5B gene who was diagnosed with VEO-IBD, then we investigated the association between the MYO5B gene and VEO-IBD. CASE PRESENTATION: A 7-month-old baby girl with a chief complaint of "blood in the stool for more than 4 months and vaginal pus and blood discharge for 3 weeks" was diagnosed with VEO-IBD, and her symptoms improved after treatment with mesalazine. The whole-exome sequencing was performed with peripheral blood. Immunohistochemistry was performed on the terminal ileal tissue. Western blotting, quantitative polymerase chain reaction (Q-PCR) and immunofluorescence were performed with cultured organoid tissue from the terminal ileum. Whole-exome sequencing identified heterozygous missense of MYO5B variant of unknown significance (p. [I769N]; [T1546M]). Immunohistochemistry revealed a significant decrease in the expression of MYO5B protein in the terminal ileum of the child with MYO5B mutation; Q-PCR revealed a decrease in the mRNA levels of occludin and ZO-1 and both the mRNA levels and protein levels of MYO5B was downregulated in the patient. Immunofluorescence images showed that MYO5B gene mutation disrupted the apical delivery of transporters SGLT1, NHE3 and AQP7. CONCLUSIONS: MYO5B gene mutation leading to the downregulation of MYO5B protein may promote the occurrence of VEO-IBD by decreasing mRNA and protein levels of intestinal tight junction genes and dislocating the apical transporters.

The downregulation of genes encoding muscle proteins have a potential role in the development of scrotal hernia in pigs.

BACKGROUND: Testicular descent is a physiological process regulated by many factors. Eventually, disturbances in the embryological/fetal development path facilitate the occurrence of scrotal hernia, a congenital malformation characterized by the presence of intestinal portions within the scrotal sac due to the abnormal expansion of the inguinal ring. In pigs, some genes have been related to this anomaly, but the genetic mechanisms involved remain unclear. This study aimed to investigate the expression profile of a set of genes potentially involved with the manifestation of scrotal hernia in the inguinal ring tissue. METHODS AND RESULTS: Tissue samples from the inguinal ring/canal of normal and scrotal hernia-affected male pigs with approximately 30 days of age were used. Relative expression analysis was performed using qPCR to confirm the expression profile of 17 candidate genes previously identified in an RNA-Seq study. Among them, the Myosin heavy chain 1 (MYH1), Desmin (DES), and Troponin 1 (TNNI1) genes were differentially expressed between groups and had reduced levels of expression in the affected animals. These genes encode proteins involved in the formation of muscle tissue, which seems to be important for increasing the resistance of the inguinal ring to the abdominal pressure, which is essential to avoid the occurrence of scrotal hernia. CONCLUSIONS: The downregulation of muscular candidate genes in the inguinal tissue clarifies the genetic mechanisms involved with this anomaly in its primary site, providing useful information for developing strategies to control this malformation in pigs and other mammals.

Inhibition of skeletal muscle differentiation by calciprotein particles in human primary myoblasts.

Sarcopenia is a common complication of chronic kidney disease (CKD) and has a detrimental effect on prognosis. Previous studies have explored the role of secondary calciprotein particles (CPP2) in determining the progression of complications and poor outcomes in patients with CKD. However, no study has demonstrated that CPP2 impairs skeletal myogenesis. Our study revealed that CPP2 exposure inhibits skeletal myogenesis by suppressing myotube formation and expression of skeletal muscle-specific myosin heavy chain and actin in human primary myoblasts. Moreover, CPP2 exposure altered the expression patterns of lineage-determinative transcription factors responsible for regulating myotube differentiation marker genes. This study first demonstrated that CPP2 interferes with myoblast differentiation and myotube formation in vitro.

Heart proteomic profiling discovers MYH6 and COX5B as biomarkers for sudden unexplained death.

Sudden unexplained death (SUD) is not uncommon in forensic pathology. Yet, diagnosis of SUD remains challenging due to lack of specific biomarkers. This study aimed to screen differentially expressed proteins (DEPs) and validate their usefulness as diagnostic biomarkers for SUD cases. We designed a three-phase investigation, where in the discovery phase, formalin-fixed paraffin-embedded (FFPE) heart specimens were screened through label-free proteomic analysis of cases dying from SUD, mechanical injury and carbon monoxide (CO) intoxication. A total of 26 proteins were identified to be DEPs for the SUD cases after rigorous criterion. Bioinformatics and Adaboost-recursive feature elimination (RFE) analysis further revealed that three of the 26 proteins (MYH6, COX5B and TNNT2) were potential discriminative biomarkers. In the training phase, MYH6 and COX5B were verified to be true DEPs in cardiac tissues from 29 independent SUD cases as compared with a serial of control cases (n = 42). Receiver operating characteristic (ROC) analysis illustrated that combination of MYH6 and COX5B achieved optimal diagnostic sensitivity (89.7 %) and specificity (84.4 %), with area under the curve (AUC) being 0.91. A diagnostic software based on the logistic regression formula derived from the training phase was then constructed. In the validation phase, the diagnostic software was applied to eight authentic SUD cases, seven (87.5 %) of which were accurately recognized. Our study provides a valid strategy towards practical diagnosis of SUD by integrating cardiac MYH6 and COX5B as dual diagnostic biomarkers.

Circ_0000395 promotes cell growth, metastasis and oxaliplatin resistance by regulating miR-153-5p/MYO6 in colorectal cancer.

BACKGROUND: Circular RNAs (circRNAs) are involved in the regulation of colorectal cancer (CRC) progression and chemoresistence. Here, we attempted to reveal the function and mechanism of circ_0000395 in CRC chemoresistence. METHODS: The expression levels of circ_0000395, microRNA (miR)-153-5p, and myosin VI (MYO6) were determined by quantitative real-time PCR. Cell growth, metastasis and oxaliplatin resistance were evaluated via EdU assay, colony formation assay, flow cytometry, transwell assay, and cell counting kit 8 assay. Xenograft tumor model was adopted to evaluate the role of circ_0000395 on CRC tumor growth and oxaliplatin sensitivity. Protein expression of drug-resistance markers and MYO6 was analyzed by western blot. The target relationship between miR-153-5p and circ_0000395 or MYO6 was validated via dual-luciferase reporter assay and RIP assay. RESULTS: Circ_0000395 expression was enhanced in CRC tissues and cells. Silencing of circ_0000395 repressed CRC cell proliferation, migration and invasion, while promoted apoptosis and oxaliplatin sensitivity. Besides, circ_0000395 knockdown also reduced CRC tumor growth and enhanced the sensitivity of tumor to oxaliplatin. Additionally, circ_0000395 acted as a sponge for miR-153-5p, and miR-153-5p targeted MYO6. Functional experiments suggested that miR-153-5p inhibitor or MYO6 overexpression could reverse the suppressive effect of circ_0000395 knockdown on CRC cell growth, metastasis and oxaliplatin resistance. CONCLUSION: Circ_0000395 promoted CRC cell growth, metastasis and oxaliplatin resistance via the miR-153-5p/MYO6 axis, which might provide new insights into the treatment of CRC.

Exploring the immune landscape of disulfidptosis in ulcerative colitis and the role of modified gegen qinlian decoction in mediating disulfidptosis to alleviate colitis in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Ulcerative colitis (UC), a recurrent inflammatory bowel disease, continues to challenge effective pharmacologic management. Disulfidptosis, a recently identified form of cell death, appears implicated in the progression of various diseases. Scientific studies have demonstrated that Modified Gegen Qinlian decoction (MGQD) alleviates UC symptoms. However, the underlying mechanisms remain inadequately elucidated. AIM OF THE STUDY: This study investigated the role of disulfidptosis in UC and explored the potential of MGQD to ameliorate UC by mediating disulfidptosis. METHODS: Microarray data were utilized to identify disulfidptosis-related genes stably expressed in UC, and integrated genomic analyses were conducted to elucidate the landscape of disulfidptosis in UC. Subsequently, C57BL/6J mice were administered 3% dextran sodium sulfate (DSS) to induce experimental colitis and treated with MGQD. Quantitative real-time polymerase chain reaction and immunohistochemical analysis of colonic tissues from colitis mice were performed to validate the microarray data findings. Finally, molecular docking was employed to explore the binding interactions between MGQD components and disulfidptosis biomarkers. RESULTS: Myosin heavy chain 10 (MYH10) and filamin A (FLNA) were identified as stably expressed in UC, demonstrating high diagnostic value for the disease. Correlation analysis indicated that disulfidptosis-related genes are associated with elevated levels of immune cells in UC. Single gene set enrichment analysis further clarified that these genes might be involved in the pathological processes of UC via immune-related pathways. Subsequent animal experiments revealed that MYH10 and FLNA were significantly upregulated in mice with colitis, a condition reversed by MGQD treatment. Molecular docking results showed that MYH10 and FLNA serve as stable binding targets for the primary components of MGQD. CONCLUSIONS: The study identified a connection between the disulfidptosis-related landscape and immune infiltration in UC, suggesting that MGQD may modulate disulfidptosis by inhibiting MYH10 and FLNA, thereby alleviating UC.

Does sex matter in the cheetah? Insights into the skeletal muscle of the fastest land animal.

The cheetah is considered the fastest land animal, but studies on their skeletal muscle properties are scarce. Vastus lateralis biopsies, obtained from male and female cheetahs as well as humans, were analysed and compared for fibre type and size, and metabolism. Overall, cheetah muscle had predominantly type IIX fibres, which was confirmed by the myosin heavy chain isoform content (mean±s.d. type I: 17±8%, type IIA: 21±6%, type IIX: 62±12%), whereas human muscle contained predominantly type I and IIA fibres (type I: 49±14%, type IIA: 43±8%, type IIX: 7±7%). Cheetahs had smaller fibres than humans, with larger fibres in the males compared with their female counterparts. Citrate synthase (16±6 versus 28±7 µmol min-1 g-1 protein, P<0.05) and 3-hydroxyacyl co-enzyme A dehydrogenase (30±11 versus 47±15 µmol min-1 g-1 protein, P<0.05) activities were lower in cheetahs than in humans, whereas lactate dehydrogenase activity was 6 times higher in cheetahs (2159±827 versus 382±161 µmol min-1 g-1 protein, P<0.001). The activities of creatine kinase (4765±1828 versus 6485±1298, P<0.05 µmol min-1 g-1 protein) and phosphorylase (111±29 versus 216±92 µmol min-1 g-1 protein) were higher in humans, irrespective of the higher type IIX fibres in cheetahs. Superoxide dismutase and catalase, markers of antioxidant capacity, were higher in humans, but overall antioxidant capacity was higher in cheetahs. To conclude, fibre type, fibre size and metabolism differ between cheetahs and humans, with limited differences between the sexes.

Rab10-CAV1 mediated intraluminal vesicle transport to migrasomes.

Migrasomes, vesicular organelles generated on the retraction fibers of migrating cells, play a crucial role in migracytosis, mediating intercellular communication. The cargoes determine the functional specificity of migrasomes. Migrasomes harbor numerous intraluminal vesicles, a pivotal component of their cargoes. The mechanism underlying the transportation of these intraluminal vesicles to the migrasomes remains enigmatic. In this study, we identified that Rab10 and Caveolin-1 (CAV1) mark the intraluminal vesicles in migrasomes. Transport of Rab10-CAV1 vesicles to migrasomes required the motor protein Myosin Va and adaptor proteins RILPL2. Notably, the phosphorylation of Rab10 by the kinase LRRK2 regulated this process. Moreover, CSF-1 can be transported to migrasomes through this mechanism, subsequently fostering monocyte-macrophage differentiation in skin wound healing, which served as a proof of the physiological importance of this transporting mechanism.

Targeted Next-Generation Sequencing Analysis Reveals a Novel Genetic Variant in MYO6 Gene in an Indian Family with Postlingual Nonsyndromic Hearing Loss.

Background: Hereditary nonsyndromic hearing loss (NSHL) is an extremely heterogeneous disorder, both genetically and clinically. Myosin VI (MYO6) pathogenic variations have been reported to cause both prelingual and postlingual forms of NSHL. Postlingual autosomal dominant cases are often overlooked for genetic etiology in clinical setups. In this study, we used next-generation sequencing (NGS)-based targeted deafness gene panel assay to identify the cause of postlingual hearing loss in an Indian family. Methods: The proband and his father from a multigenerational Indian family affected by postlingual hearing loss were examined via targeted capture of 129 deafness genes, after excluding gap junction protein beta 2 (GJB2) pathogenic variants by Sanger sequencing. NGS data analysis and co-segregation of the candidate variants in the family were carried out. The variant effect was predicted by in silico tools and interpreted following American College of Medical Genetics and Genomics-Association for Molecular Pathology guidelines. Results: A novel heterozygous transversion c.3225T>G, p.(Tyr1075*) in MYO6 gene was identified as the disease-causing variant in this family. This stop-gained variant is predicted to form a truncated myosin VI protein, which is devoid of crucial cargo-binding domain. PCR-RFLP screening in 200 NSHL cases and 200 normal-hearing controls showed the absence of this variant indicating its de novo nature in the population. Furthermore, we reviewed MYO6 variants reported from various populations to date. Conclusions: To the best of our knowledge, this is the first family with MYO6-associated hearing loss from an Indian population. The study also highlights the importance of deafness gene panels in molecular diagnosis of GJB2-negative pedigrees, contributing to genetic counseling in the affected families.

Prognostic prediction of patients having classical papillary thyroid carcinoma with a 4 mRNA-based risk model.

The dysregulation of protein-coding genes involved in various biological functions is closely associated with the progression of thyroid cancer. This study aimed to investigate the effects of dysregulated gene expressions on the prognosis of classical papillary thyroid carcinoma (cPTC). Using expression profiling datasets from the Cancer Genome Atlas (TCGA) database, we performed differential expression analysis to identify differentially expressed genes (DEGs). Cox regression and Kaplan-Meier analysis were used to identify DEGs, which were used to construct a risk model to predict the prognosis of cPTC patients. Functional enrichment analysis unveiled the potential significance of co-expressed protein-encoding genes in tumors. We identified 4 DEGs (SALL3, PPBP, MYH1, and SYNDIG1), which were used to construct a risk model to predict the prognosis of cPTC patients. These 4 genes were independent of clinical parameters and could be functional in cPTC carcinogenesis. Furthermore, PPBP exhibited a strong correlation with poorer overall survival (OS) in the advanced stage of the disease. This study suggests that the 4-gene signature could be an independent prognostic biomarker to improve prognosis prediction in cPTC patients older than 46.

OLFM4 promotes the progression of intestinal metaplasia through activation of the MYH9/GSK3ß/ß-catenin pathway.

BACKGROUND: Intestinal metaplasia (IM) is classified into complete intestinal metaplasia (CIM) and incomplete intestinal metaplasia (IIM). Patients diagnosed with IIM face an elevated susceptibility to the development of gastric cancer, underscoring the critical need for early screening measures. In addition to the complexities associated with diagnosis, the exact mechanisms driving the progression of gastric cancer in IIM patients remain poorly understood. OLFM4 is overexpressed in several types of tumors, including colorectal, gastric, pancreatic, and ovarian cancers, and its expression has been associated with tumor progression. METHODS: In this study, we used pathological sections from two clinical centers, biopsies of IM tissues, precancerous lesions of gastric cancer (PLGC) cell models, animal models, and organoids to explore the role of OLFM4 in IIM. RESULTS: Our results show that OLFM4 expression is highly increased in IIM, with superior diagnostic accuracy of IIM when compared to CDX2 and MUC2. OLFM4, along with MYH9, was overexpressed in IM organoids and PLGC animal models. Furthermore, OLFM4, in combination with Myosin heavy chain 9 (MYH9), accelerated the ubiquitination of GSK3ß and resulted in increased ß-catenin levels through the Wnt signaling pathway, promoting the proliferation and invasion abilities of PLGC cells. CONCLUSIONS: OLFM4 represents a novel biomarker for IIM and could be utilized as an important auxiliary means to delimit the key population for early gastric cancer screening. Finally, our study identifies cell signaling pathways involved in the progression of IM.

Cost-Efficient Expression of Human Cardiac Myosin Heavy Chain in C2C12 Cells with a Non-Viral Transfection Reagent.

Production of functional myosin heavy chain (MHC) of striated muscle myosin II for studies of isolated proteins requires mature muscle (e.g., C2C12) cells for expression. This is important both for fundamental studies of molecular mechanisms and for investigations of deleterious diseases like cardiomyopathies due to mutations in the MHC gene (MYH7). Generally, an adenovirus vector is used for transfection, but recently we demonstrated transfection by a non-viral polymer reagent, JetPrime. Due to the rather high costs of JetPrime and for the sustainability of the virus-free expression method, access to more than one transfection reagent is important. Here, we therefore evaluate such a candidate substance, GenJet. Using the human cardiac ß-myosin heavy chain (ß-MHC) as a model system, we found effective transfection of C2C12 cells showing a transfection efficiency nearly as good as with the JetPrime reagent. This was achieved following a protocol developed for JetPrime because a manufacturer-recommended application protocol for GenJet to transfect cells in suspension did not perform well. We demonstrate, using in vitro motility assays and single-molecule ATP turnover assays, that the protein expressed and purified from cells transfected with the GenJet reagent is functional. The purification yields reached were slightly lower than in JetPrime-based purifications, but they were achieved at a significantly lower cost. Our results demonstrate the sustainability of the virus-free method by showing that more than one polymer-based transfection reagent can generate useful amounts of active MHC. Particularly, we suggest that GenJet, due to its current ~4-fold lower cost, is useful for applications requiring larger amounts of a given MHC variant.

RGS19 activates the MYH9/ß-catenin/c-Myc positive feedback loop in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most common fatal cancers worldwide, and the identification of novel treatment targets and prognostic biomarkers is urgently needed because of its unsatisfactory prognosis. Regulator of G-protein signaling 19 (RGS19) is a multifunctional protein that regulates the progression of various cancers. However, the specific function of RGS19 in HCC remains unclear. The expression of RGS19 was determined in clinical HCC samples. Functional and molecular biology experiments involving RGS19 were performed to explore the potential mechanisms of RGS19 in HCC. The results showed that the expression of RGS19 is upregulated in HCC tissues and is significantly associated with poor prognosis in HCC patients. RGS19 promotes the proliferation and metastasis of HCC cells in vitro and in vivo. Mechanistically, RGS19, via its RGS domain, stabilizes the MYH9 protein by directly inhibiting the interaction of MYH9 with STUB1, which has been identified as an E3 ligase of MYH9. Moreover, RGS19 activates ß-catenin/c-Myc signaling via MYH9, and RGS19 is also a transcriptional target gene of c-Myc. A positive feedback loop formed by RGS19, MYH9, and the ß-catenin/c-Myc axis was found in HCC. In conclusion, our research revealed that competition between RGS19 and STUB1 is a critical mechanism of MYH9 regulation and that the RGS19/MYH9/ß-catenin/c-Myc feedback loop may represent a promising strategy for HCC therapy.

Muscle-Protective Effect of Carnosine against Dexamethasone-Induced Muscle Atrophy in C2C12 Myotube.

This study investigated the protective effect of carnosine and its components (L-histidine and ß-alanine [HA]) against dexamethasone (Dex)-induced muscle atrophy in C2C12 myotubes. Myotubes were treated with Dex (10 µM) to induce muscle atrophy manifested by decreased myotube diameter, low myosin heavy chain content, and increased expression of muscle atrophy-associated ubiquitin ligases (Atrogin-1, MuRF-1, and Cbl-b). Carnosine (20 mM) treatment significantly improved the myotube diameter and MyHC protein expression level in Dex-treated C2C12 myotubes. It also downregulated the expression of Atrogin-1, MuRF-1, and Cbl-b and suppressed the expression of forkhead box O3 (FoxO3a) mediated by Dex. Furthermore, reactive oxygen species production was increased by Dex but was ameliorated by carnosine treatment. However, HA (20 mM), the component of carnosine, treatment was found ineffective in preventing Dex-induced protein damage. Therefore, based on above results it can be suggested that carnosine could be a potential therapeutic agent to prevent Dex-induced muscle atrophy compared to its components HA.