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The oomycete Aphanomyces invadans causes epizootic ulcerative syndrome (EUS), a World Organization for Animal Health (WOAH)-listed disease that has seriously impacted a wide range of fish worldwide. Currently, only three conventional polymerase chain reaction (PCR) assays are recommended for the detection of A. invadans. The robust quantitative PCR (qPCR) assay has recently become more important due to its highly accurate nature and the applicability of qPCR-based environmental DNA (eDNA) detection in the monitoring of pathogens in aquatic environments. Therefore, in this study, we developed a novel TaqMan probe-based qPCR method to sensitively and quantitatively detect A. invadans. The assay limit of detection was determined using 10-fold serial dilutions of linearized A. invadans plasmid. Assay sensitivity was assessed in the presence of interfering substances and compared to three WOAH-listed primers using the mycelia and zoospores of A. invadans with and without fish muscle tissue. The assay specificity was also theoretically and experimentally assessed against other oomycetes, fish muscle tissue, and water samples. The assay's repeatability and reproducibility were determined. In this study, the limit of detection of the developed assay was 7.24 copies of A. invadans genomic DNA per reaction (95% confidence interval (CI): 2.75 to 19.05 copies/reaction). The assay showed the same sensitivity in the presence of other substances. Compared to the WOAH-recommended PCR assays, this assay had 10-times higher sensitivity for all tested samples. There were no cross-reactions with other closely related oomycetes, fish muscle, or water samples, indicating that the assay was highly specific for A. invadans. The repeatability and reproducibility tests showed little variation, ranging from 0.1–0.9% and 0.04–1.1%, respectively, indicating the high consistency, repeatability, and reliability of the developed assay. This highly rapid, sensitive, specific, and consistent EUS qPCR assay would be of importance in transboundary disease management and the monitoring of pathogens in aquatic environments. [ABSTRACT FROM AUTHOR]

Purpose: Research on whether wearable device interventions can effectively prevent metabolic syndrome remains insufficient. This study aimed to evaluate the effect of feedback on clinical indicators in patients with metabolic syndrome on activities measured using wearable devices, such as smartphone apps. Methods: Patients with metabolic syndrome were recruited and prescribed to live for 12 weeks using a wrist-wearable device (B.BAND, B Life Inc., Korea). A block randomization method was used to distribute participants between the intervention (n=35) and control groups (n=32). In the intervention group, an experienced study coordinator provided feedback on physical activity to individuals through telephonic counseling every other week. Results: The mean number of steps in the control group was 8892.86 (4473.53), and those in the intervention group was 10,129.31 (4224.11). After 12 weeks, metabolic syndrome was resolved. Notably, there were statistically significant differences in the metabolic composition among the participants who completed the intervention. The mean number of metabolic disorder components per person remained at 3 in the control group, and decreased from 4 to 3 in the intervention group. Additionally, waist circumference, systolic and diastolic blood pressure, and triglyceride levels were significantly reduced, while HDL-cholesterol levels were significantly increased in the intervention group. Conclusion: Overall, 12 weeks of telephonic counseling intervention using wearable device-based physical activity confirmation improved the damaged metabolic components of patients with metabolic syndrome. Telephonic intervention can help increase physical activity and reduce waist circumference, which is a typical clinical indicator of metabolic syndrome. [ABSTRACT FROM AUTHOR]

Metabolic abnormalities, such as preexisting diabetes or hyperglycemia or hypoglycemia during hospitalization aggravated the severity of COVID-19. We evaluated whether diabetes history, hyperglycemia before and during extracorporeal membrane oxygenation (ECMO) support, and hypoglycemia were risk factors for mortality in patients with COVID-19. This study included data on 195 patients with COVID-19, who were aged ≥19 years and were treated with ECMO. The proportion of patients with diabetes history among nonsurvivors was higher than that among survivors. Univariate Cox regression analysis showed that in-hospital mortality after ECMO support was associated with diabetes history, renal replacement therapy (RRT), and body mass index (BMI) < 18.5 kg/m2. Glucose at admission >200 mg/dL and glucose levels before ventilator >200 mg/dL were not associated with in-hospital mortality. However, glucose levels before ECMO >200 mg/dL and minimal glucose levels during hospitalization <70 mg/dL were associated with in-hospital mortality. Multivariable Cox regression analysis showed that glucose >200 mg/dL before ECMO and minimal glucose <70 mg/dL during hospitalization remained risk factors for in-hospital mortality after adjustment for age, BMI, and RRT. In conclusion, glucose >200 mg/dL before ECMO and minimal glucose level <70 mg/dL during hospitalization were risk factors for in-hospital mortality among COVID-19 patients who underwent ECMO. [ABSTRACT FROM AUTHOR]

The mammalian Ste20-like kinase (MST) pathway or Hippo pathway plays essential roles in cell proliferation, apoptosis, organ size control, and development. Runx2 is a key transcription factor in osteoblast differentiation. The objective of this study was to investigate whether the MST pathway could modulate Runx2 and osteoblast differentiation. First, we found that Runx2 interacted with MST2 and SAV1 via the WW domain of SAV1 and amino acid 292–445 of Runx2 containing a PY motif. Results of OSE luciferase reporter assay revealed that co-expression of MST2 and SAV1 inhibited the transcriptional activity of Runx2 whereas siRNA-mediated down-regulation of Mst1 and Mst2 increased its activity. MST2 and SAV1 significantly reduced mRNA levels of osteoblast differentiation marker genes such as alkaline phosphatase and osteocalcin in differentiating C2C12 cells. MST2 and SAV1 also hampered osteoblast differentiation of C2C12 cells induced by Runx2 as shown by alkaline phosphatase activity assay and Alizarin Red staining. Mass spectrometric analysis of immunoprecipitated Runx2 protein from HEK293 cells overexpressing MST2 and SAV1 revealed two novel phosphorylation sites at Ser-339 and Ser-370 residues of mouse Runx2 protein. Mutation of both serine residues to alanine interfered with the inhibitory effect of MST2 and SAV1 on the transcriptional activity of Runx2 and osteoblast differentiation induced by Runx2. Our results suggest that the MST kinase pathway can directly regulate osteoblast differentiation by modulating Runx2 activity through phosphorylation. • MST pathway is a regulator of Runx2 activity. • MST2 and SAV1 interacts with Runx2 • MST2 and SAV1 inhibits the transcriptional activity of Runx2, the expression of osteoblast differentiation marker genes, and osteoblast differentiation of C2C12 cells. • Two novel MST2 phosphorylation residues in the Runx2 protein were identified and shown to be critical for the regulation of Runx2 activity by MST2 during osteoblast differentiation. [ABSTRACT FROM AUTHOR]

Background: Oxidative stress induced by chronic hyperglycemia is recognized as a significant mechanistic contributor to the development of diabetic kidney disease (DKD). Nonphagocytic nicotinamide adenine dinucleotide phosphate oxidase 4 (Nox4) is a major source of reactive oxygen species (ROS) in many cell types and in the kidney tissue of diabetic animals. We designed this study to explore the therapeutic potential of chloroquine (CQ) and amodiaquine (AQ) for inhibiting mitochondrial Nox4 and diabetic tubular injury., Methods: Human renal proximal tubular epithelial cells (hRPTCs) were cultured in high-glucose media (30 mM D-glucose), and diabetes was induced with streptozotocin (STZ, 50 mg/kg i.p. for 5 days) in male C57BL/6J mice. CQ and AQ were administered to the mice via intraperitoneal injection for 14 weeks., Results: CQ and AQ inhibited mitochondrial Nox4 and increased mitochondrial mass in hRPTCs under high-glucose conditions. Reduced mitochondrial ROS production after treatment with the drugs resulted in decreased endoplasmic reticulum (ER) stress, suppressed inflammatory protein expression and reduced cell apoptosis in hRPTCs under high-glucose conditions. Notably, CQ and AQ treatment diminished Nox4 activation and ER stress in the kidneys of STZ-induced diabetic mice. In addition, we observed attenuated inflammatory protein expression and albuminuria in STZ-induced diabetic mice after CQ and AQ treatment., Conclusion: We substantiated the protective actions of CQ and AQ in diabetic tubulopathy associated with reduced mitochondrial Nox4 activation and ER stress alleviation. Further studies exploring the roles of mitochondrial Nox4 in the pathogenesis of DKD could suggest new therapeutic targets for patients with DKD., Competing Interests: The authors have no potential conflicts of interest to disclose., (© 2020 The Korean Academy of Medical Sciences.)

We examined the effects of empagliflozin, a selective inhibitor of Na + -glucose cotransporter 2, on mitochondrial quality control and autophagy in renal tubular cells in a diabetic environment in vivo and in vitro. Human renal proximal tubular cells (hRPTCs) were incubated under high-glucose conditions. Diabetes was induced with streptozotocin in male C57BL/6J mice. Improvements in mitochondrial biogenesis and balanced fusion-fission protein expression were noted in hRPTCs after treatment with empagliflozin in high-glucose media. Empagliflozin also increased autophagic activities in renal tubular cells in the high-glucose environment, which was accompanied with mammalian target of rapamycin inhibition. Moreover, reduced mitochondrial reactive oxygen species production and decreased apoptotic and fibrotic protein expression were observed in hRPTCs after treatment with empagliflozin, even in the hyperglycemic circumstance. Importantly, empagliflozin restored AMP-activated protein kinase-α phosphorylation and normalized levels of AMP-to-ATP ratios in hRPTCs subjected to a high-glucose environment, which suggests the way that empagliflozin is involved in mitochondrial quality control. Empagliflozin effectively suppressed Na + -glucose cotransporter 2 expression and ameliorated renal morphological changes in the kidneys of streptozotocin-induced diabetic mice. Electron microscopy analysis showed that mitochondrial fragmentation was decreased and 8-hydroxy-2'-deoxyguanosine content was low in renal tubular cells of empagliflozin treatment groups compared with those of the diabetic control group. We suggest one mechanism related to the renoprotective actions of empagliflozin, which reverse mitochondrial dynamics and autophagy.

Background: The concept of adaptive evolution implies underlying genetic mutations conferring a selective advantage to an organism under particular environmental conditions. Thus, a flow cytometry-based strategy was used to study the adaptive evolution in Chlamydomonas reinhardtii wild-type strain CC124 and starchless mutant sta6-1 cells, with respect to lipid metabolism under nitrogen-(N) depleted and -replete conditions., Results: The successive sorting and regeneration of the top 25,000 high-lipid content cells of CC124 and sta6-1, combined with nitrogen starvation, led to the generation of a new population with an improved lipid content when compared to the original populations (approximately 175% and 50% lipid increase in sta6-1 and CC124, respectively). During the adaptive evolution period, the major fatty acid components observed in cells were C16:0, C16:1, C18:0, and C18:1-3, and elemental analysis revealed that cellular carbon to nitrogen ratio increased at the end of adaptive evolution period In order to gain an insight into highly stimulated intracellular lipid accumulation in CC124 and sta6-1 resulting from the adaptive evolution, proteomics analyses of newly generated artificial high-lipid content populations were performed. Functional classifications showed the heightened regulation of the major chlorophyll enzymes, and the enzymes involved in carbon fixation and uptake, including chlorophyll-ab-binding proteins and Rubisco activase. The key control protein (periplasmic L-amino acid oxidase (LAO1)) of carbon-nitrogen integration was specifically overexpressed. Glutathione-S-transferases and esterase, the enzymes involved in lipid-metabolism and lipid-body associated proteins, were also induced during adaptive evolution., Conclusions: Adaptive evolution results demonstrate the potential role of photosynthesis in terms of carbon partitioning, flux, and fixation and carbon-nitrogen metabolism during lipid accumulation in microalgae. This strategy can be used as a new tool to develop C. reinhardtii strains and other microalgal strains with desired phenotypes such as high lipid accumulation.