Gene Transfer of Skeletal Muscle-Type Myosin Light Chain Kinase via Adeno-Associated Virus 6 Improves Muscle Functions in an Amyotrophic Lateral Sclerosis Mouse Model.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that shows progressive muscle weakness. A few treatments exist including symptomatic therapies, which can prolong survival or reduce a symptom; however, no fundamental therapies have been found. As a therapeutic strategy, enhancing muscle force is important for patients' quality of life. In this study, we focused on skeletal muscle-specific myosin regulatory light chain kinase (skMLCK), which potentially enhances muscle contraction, as overexpression of skMLCK was thought to improve muscle function. The adeno-associated virus serotype 6 encoding skMLCK (AAV6/skMLCK) and eGFP (control) was produced and injected intramuscularly into the lower limbs of SOD1G37R mice, which are a familial ALS model. AAV6/skMLCK showed the successful expression of skMLCK in the muscle tissues. Although the control did not affect the muscle force in both of the WT and SOD1G37R mice, AAV6/skMLCK enhanced the twitch force of SOD1G37R mice and the tetanic force of WT and SOD1G37R mice. These results indicate that overexpression of skMLCK can enhance the tetanic force of healthy muscle as well as rescue weakened muscle function. In conclusion, the gene transfer of skMLCK has the potential to be a new therapy for ALS as well as for other neuromuscular diseases.

The CR9 element is a novel mechanical load-responsive enhancer that regulates natriuretic peptide genes expression.

Enhancers regulate gene expressions in a tissue- and pathology-specific manner by altering its activities. Plasma levels of atrial and brain natriuretic peptides, encoded by the Nppa and Nppb, respectively, and synthesized predominantly in cardiomyocytes, vary depending on the severity of heart failure. We previously identified the noncoding conserved region 9 (CR9) element as a putative Nppb enhancer at 22-kb upstream from the Nppb gene. However, its regulatory mechanism remains unknown. Here, we therefore investigated the mechanism of CR9 activation in cardiomyocytes using different kinds of drugs that induce either cardiac hypertrophy or cardiac failure accompanied by natriuretic peptides upregulation. Chronic treatment of mice with either catecholamines or doxorubicin increased CR9 activity during the progression of cardiac hypertrophy to failure, which is accompanied by proportional increases in Nppb expression. Conversely, for cultured cardiomyocytes, doxorubicin decreased CR9 activity and Nppb expression, while catecholamines increased both. However, exposing cultured cardiomyocytes to mechanical loads, such as mechanical stretch or hydrostatic pressure, upregulate CR9 activity and Nppb expression even in the presence of doxorubicin. Furthermore, the enhancement of CR9 activity and Nppa and Nppb expressions by either catecholamines or mechanical loads can be blunted by suppressing mechanosensing and mechanotransduction pathways, such as muscle LIM protein (MLP) or myosin tension. Finally, the CR9 element showed a more robust and cell-specific response to mechanical loads than the -520-bp BNP promoter. We concluded that the CR9 element is a novel enhancer that responds to mechanical loads by upregulating natriuretic peptides expression in cardiomyocytes.

Detection of pancreatic tumor cell nuclei via a hyperspectral analysis of pathological slides based on stain spectra.

Hyperspectral imaging (HSI) provides more detailed information than red-green-blue (RGB) imaging, and therefore has potential applications in computer-aided pathological diagnosis. This study aimed to develop a pattern recognition method based on HSI, called hyperspectral analysis of pathological slides based on stain spectrum (HAPSS), to detect cancers in hematoxylin and eosin-stained pathological slides of pancreatic tumors. The samples, comprising hyperspectral cubes of 420-750 nm, were harvested for HSI and tissue microarray (TMA) analysis. As a result of conducting HAPSS experiments with a support vector machine (SVM) classifier, we obtained maximal accuracy of 94%, a 14% improvement over the widely used RGB images. Thus, HAPSS is a suitable method to automatically detect tumors in pathological slides of the pancreas.

Tissue amino acid profiles are characteristic of tumor type, malignant phenotype, and tumor progression in pancreatic tumors.

Tissue amino acid profiles depend on the cell types and extracellular components that constitute the tissue, and their functions and activities. We aimed to characterize the tissue amino acid profiles in several types of pancreatic tumors and lesions. We examined tissue amino acid profiles in 311 patients with pancreatic tumors or lesions. We used newly developed LC-MS/MS methods to obtain the profiles, which were compared with clinicopathological data. Each tumor or lesion presented a characteristic tissue amino acid profile. Certain amino acids were markedly altered during the multistep pancreatic carcinogenesis and pancreatic ductal adenocarcinoma (PDAC) progression. A tissue amino acid index (TAAI) was developed based on the amino acids that were notably changed during both carcinogenesis and cancer progression. Univariate and multivariate survival analyses revealed that PDAC patients with a high TAAI exhibited a significantly shorter survival rate, and these findings were validated using a second cohort. We suggest that tissue amino acid profiles are characteristic for normal tissue type, tumor histological type, and pathological lesion, and are representative of the cancer grade or progression stage in multistep carcinogenesis and of malignant characteristics. The TAAI could serve as an independent prognosticator for patients with PDAC.

Prognostic impact of peak mitral inflow velocity in asymptomatic degenerative mitral regurgitation.

OBJECTIVE: Appropriate timing of mitral valve surgery in asymptomatic mitral regurgitation (MR) remains controversial. Peak mitral inflow velocity (peak E wave velocity) has been reported as a simple and easy predictor of quantitative MR severity; however, its prognostic significance in asymptomatic MR remains unclear. Therefore, we sought to investigate the prognostic impact of peak E wave velocity in asymptomatic MR. METHODS: Among 529 consecutive patients with degenerative MR of grade 3+ (moderate to severe) or 4+ (severe), 188 asymptomatic patients in sinus rhythm without left ventricular (LV) dysfunction (end-systolic dimension ≥40 mm or ejection fraction <60%) or pulmonary hypertension were studied. Cardiovascular events were defined as a composite endpoint of cardiovascular death or events that indicated mitral surgery including congestive heart failure, atrial fibrillation, LV dysfunction or pulmonary hypertension. RESULTS: Average peak E wave velocity was 1.05±0.26 m/s, and was significantly higher in grade 4+ than grade 3+ (1.20±0.28 vs 0.98±0.21 m/s, p<0.001). Peak E wave velocity was associated with quantitative MR severity, as well as clinical characteristics of advanced MR (higher brain natriuretic peptide, larger LV and left atrium, higher tricuspid regurgitation pressure gradient and dilated inferior vena cava). During a median follow-up of 4.3 years, 66 (35%) patients developed cardiovascular events. Multivariate Cox proportional hazards analysis showed that peak E wave velocity was an independent predictor of cardiovascular events (adjusted HR 1.245 (95% CI 1.126 to 1.378) per 0.1 m/s, p<0.001). CONCLUSIONS: Peak E wave velocity was an independent predictor of cardiovascular events in asymptomatic degenerative MR with preserved LV function.