Uremia Impedes Skeletal Myocyte Myomixer Expression and Fusogenic Activity: Implication for Uremic Sarcopenia.

In patients with chronic kidney disease (CKD), skeletal muscle mass and function are known to occasionally decline. However, the muscle regeneration and differentiation process in uremia has not been extensively studied. In mice with CKD induced by adenine-containing diet, the tibialis anterior muscle injured using a barium chloride injection method recovered poorly as compared to control mice. In the cultured murine skeletal myocytes, stimulation with indoxyl sulfate (IS), a representative uremic toxin, morphologically jeopardized the differentiation, which was counteracted by L-ascorbic acid (L-AsA) treatment. Transcriptome analysis of cultured myocytes identified a set of genes whose expression was down-regulated by IS stimulation but up-regulated by L-AsA treatment. Gene silencing of myomixer, one of the genes in the set, impaired myocyte fusion during differentiation. By contrast, lentiviral overexpression of myomixer compensated for a hypomorphic phenotype caused by IS treatment. The split-luciferase technique demonstrated that IS stimulation negatively affected early myofusion activity that was rescued by L-AsA treatment. Lastly, in mice with CKD compared with control mice, myomixer expression in the muscle tissue in addition to the muscle weight after the injury was reduced, both of which were restored with L-AsA treatment. Collectively, data showed that the uremic milieu impairs the expression of myomixer and impedes the myofusion process. Considering frequent musculoskeletal injuries in uremic patients, defective myocyte fusion followed by delayed muscle damage recovery could underlie their muscle loss and weakness.

Dual-specificity phosphatases 13 and 27 as key switches in muscle stem cell transition from proliferation to differentiation.

Muscle regeneration depends on muscle stem cell (MuSC) activity. Myogenic regulatory factors, including myoblast determination protein 1 (MyoD), regulate the fate transition of MuSCs. However, the direct target of MYOD in the process is not completely clear. Using previously established MyoD knock-in (MyoD-KI) mice, we revealed that MyoD targets dual-specificity phosphatase (Dusp) 13 and Dusp27. In Dusp13:Dusp27 double knock-out mice, the ability for muscle regeneration after injury was reduced. Moreover, single-cell RNA sequencing of MyoD-high expressing MuSCs from MyoD-KI mice revealed that Dusp13 and Dusp27 are expressed only in specific populations within MyoD-high MuSCs, which also express Myogenin. Overexpressing Dusp13 in MuSCs causes premature muscle differentiation. Thus, we propose a model where DUSP13 and DUSP27 contribute to the fate transition of MuSCs from proliferation to differentiation during myogenesis.

Metasurface Biosensors Enabling Single-Molecule Sensing of Cell-Free DNA.

In this study, we have revealed that highly fluorescence (FL)-enhancing all-dielectric metasurface biosensors are capable of detecting single-target DNA, which is cell-free DNA (cfDNA) specific to the human practice effect. The ultimately high-precision detection was achieved in a scheme combining the metasurface biosensors with a short-time nucleic acid amplification technique, that is, a reduced-cycle polymerase chain reaction (PCR). In this combined scheme, we obtained a series of FL signals at a single-molecule concentration, reflecting the Poisson distribution, and moreover elucidated that the FL signals exhibit the single-molecule cfDNA detection with more than 84% statistical confidence in an automated FL detection system and with 99.9% statistical confidence in confocal FL microscopy. As a result, we have found a simple and practical test to discriminate the target of 1 copy/test from zero using metasurface biosensors, which has not been realized by other elaborate techniques such as digital PCR.

Female athlete triad affects rat intestinal morphology and sucrase-isomaltase expression.

Female athletes follow a strict diet and perform rigorous exercise to boost their performance, which induces health issues called the female athlete triad (FAT), defined as the combination of disordered eating, amenorrhoea and low bone mineral density. It is known to have a significant effect on bones. However, its effects on the small intestine, which is responsible for nutrient uptake into the body, remain unclear. In this study, we created an animal model of FAT to examine its effects on digestive and absorptive molecules in the small intestine. Thirty 5-week-old female Sprague-Dawley (sd) rats with an initial body weight of about 147 g were divided into control (Con, n = 7), exercise (Ex, n = 7), food restriction (FR, n = 8) and exercise plus food restriction (FAT, n = 8) groups. The rats were subjected to 4 weeks of wheel running (Ex, FAT) and 50-40 % food restriction (FR, FAT) to examine the effects on bone and typical digestive enzymes and transporters in the jejunum. Two-way ANOVA and the Kruskal-Wallis test were used for statistical analysis of normal and non-normal data, respectively. Four weeks of exercise and food restriction decreased bone weight (vs. other group P < 0·01) and bone breaking power (vs. other group P < 0·01). Villus height decreased in the jejunum (vs. other group P < 0·01), but the expression of typical macronutrients digestive enzyme and absorptive molecules remained unchanged. In contrast, sucrase-isomaltase gene (v. Ex P = 0·02) and protein expression were increased (vs. other group P < 0·05). The study findings show that FAT affects sucrase-isomaltase without histone methylation changes.

Single-cell dissection, hdWGCNA and deep learning reveal the role of oxidatively stressed plasma cells in ulcerative colitis.

Ulcerative colitis (UC) develops as a result of complex interactions between various cell types in the mucosal microenvironment. In this study, we aim to elucidate the pathogenesis of ulcerative colitis at the single-cell level and unveil its clinical significance. Using single-cell RNA sequencing and high-dimensional weighted gene co-expression network analysis, we identify a subpopulation of plasma cells (PCs) with significantly increased infiltration in UC colonic mucosa, characterized by pronounced oxidative stress. Combining 10 machine learning approaches, we find that the PC oxidative stress genes accurately distinguish diseased mucosa from normal mucosa (independent external testing AUC=0.991, sensitivity=0.986, specificity=0.909). Using MCPcounter and non-negative matrix factorization, we identify the association between PC oxidative stress genes and immune cell infiltration as well as patient heterogeneity. Spatial transcriptome data is used to verify the infiltration of oxidatively stressed PCs in colitis. Finally, we develop a gene-immune convolutional neural network deep learning model to diagnose UC mucosa in different cohorts (independent external testing AUC=0.984, sensitivity=95.9%, specificity=100%). Our work sheds light on the key pathogenic cell subpopulations in UC and is essential for the development of future clinical disease diagnostic tools.

Glycoprotein NMB promotes tumor formation and malignant progression of laryngeal squamous cell carcinoma.

Laryngeal squamous cell carcinoma (LSCC), although one of the most common head and neck cancers, has a static or slightly decreased survival rate because of difficulties in early diagnosis, lack of effective molecular targeting therapy, and severe dysfunction after radical surgical treatments. Therefore, a novel therapeutic target is crucial to increase treatment efficacy and survival rates in these patients. Glycoprotein NMB (GPNMB), whose role in LSCC remains elusive, is a type 1 transmembrane protein involved in malignant progression of various cancers, and its high expression is thought to be a poor prognostic factor. In this study, we showed that GPNMB expression levels in LSCC samples are significantly higher than those in normal tissues, and GPNMB expression is observed mostly in growth-arrested cancer cells. Furthermore, knockdown of GPNMB reduces monolayer cellular proliferation, cellular migration, and tumorigenic growth, while GPNMB protein displays an inverse relationship with Ki-67 levels. Therefore, we conclude that GPNMB may be an attractive target for future LSCC therapy.

Acute cold stress induces transient MuRF1 upregulation in the skeletal muscle of zebrafish.

Cryotherapy is one of the most common treatments for trauma or fatigue in the field of sports medicine. However, the molecular biological effects of acute cold exposure on skeletal muscle remain unclear. Therefore, we used zebrafish, which have recently been utilized as an animal model for skeletal muscle, to comprehensively investigate and selectively clarify the time-course changes induced by cryotherapy. Zebrafish were exposed intermittently to cold stimulation three times for 15 min each. Thereafter, skeletal muscle samples were collected after 15 min and 1, 2, 4, and 6 h. mRNA sequencing revealed the involvement of trim63a, fbxo32, fbxo30a, and klhl38b in "protein ubiquitination" from the top 10 most upregulated genes. Subsequently, we examined the time-course changes of the four genes by quantitative PCR, and their expression peaked 2 h after cryotherapy and returned to baseline after 6 h. Moreover, the proteins encoded by trim63a and fbxo32 (muscle-specific RING finger protein 1 [MuRF1] and muscle atrophy F-box, respectively), which are known to be major genes encoding E3 ubiquitin ligases, were examined by western blotting, and MuRF1 expression displayed similar temporal changes as trim63a expression. These findings suggest that acute cold exposure transiently upregulates E3 ubiquitin ligases, especially MuRF1; thus, cryotherapy may contribute to the treatment of trauma or fatigue by promoting protein processing.

Lysine demethylase 2B regulates angiogenesis via Jumonji C dependent suppression of angiogenic transcription factors.

Vascular endothelial growth factor (VEGF) signaling plays a central role in vascular development and maintenance of vascular homeostasis. In endothelial cells (ECs), VEGF activates the gene expression of angiogenic transcription factors (TFs), followed by induction of downstream angiogenic responsive genes. Recent findings support that histone modification dynamics contribute to the transcriptional control of genes that are important for EC functions. Lysine demethylase 2B (KDM2B) demethylates histone H3K4me3 and H3K36me2/3 and mediates the monoubiquitination of histone H2AK119. KDM2B functions as a transcriptional repressor in somatic cell reprogramming and tumor development. However, the role of KDM2B in VEGF signaling remains to be elucidated. Here, we show that KDM2B knockdown enhances VEGF-induced angiogenesis in cultured human ECs via increased migration and proliferation. In contrast, ectopic expression of KDM2B inhibits angiogenesis. The function of KDM2B may depend on its catalytic Jumonji C domain. Genome-wide analysis further reveals that KDM2B selectively controls the transcription of VEGF-induced angiogenic TFs that are associated with increased H3K4me3/H3K36me3 and decreased H2AK119ub. These findings suggest an essential role of KDM2B in VEGF signaling in ECs. As dysregulation of VEGF signaling in ECs is involved in various diseases, including cancer, KDM2B may be a potential therapeutic target in VEGF-mediated vasculopathic diseases.

High protein diet-induced metabolic changes are transcriptionally regulated via KLF15-dependent and independent pathways.

High protein diet (HPD) is an affordable and positive approach in prevention and treatment of many diseases. It is believed that transcriptional regulation is responsible for adaptation after HPD feeding and Kruppel-like factor 15 (KLF15), a zinc finger transcription factor that has been proved to perform transcriptional regulation over amino acid, lipid and glucose metabolism, is known to be involved at least in part in this HPD response. To gain more insight into molecular mechanisms by which HPD controls expressions of genes involved in amino acid metabolism in the liver, we performed RNA-seq analysis of mice fed HPD for a short period (3 days). Compared to a low protein diet, HPD feeding significantly increased hepatic expressions of enzymes involved in the breakdown of all the 20 amino acids. Moreover, using KLF15 knockout mice and in vivo Ad-luc analytical system, we were able to identify Cth (cystathionine gamma-lyase) as a new target gene of KLF15 transcription as well as Ast (aspartate aminotransferase) as an example of KLF15-independent gene despite its remarkable responsiveness to HPD. These findings provide us with a clue to elucidate the entire transcriptional regulatory mechanisms of amino acid metabolic pathways.

CtBP2 confers protection against oxidative stress through interactions with NRF1 and NRF2.

While molecular oxygen is essential for aerobic organisms, its utilization is inseparably connected with generation of oxidative insults. To cope with the detrimental aspects, cells evolved antioxidative defense systems, and insufficient management of the oxidative insults underlies the pathogenesis of a wide range of diseases. A battery of genes for this antioxidative defense are regulated by the transcription factors nuclear factor-erythroid 2-like 1 and 2 (NRF1 and NRF2). While the regulatory steps for the activation of NRFs have been investigated with particular emphasis on nuclear translocation and proteosomal degradation, unknown redundancy may exist considering the indispensable nature of these defense systems. Here we unraveled that C-terminal binding protein 2 (CtBP2), a transcriptional cofactor with redox-sensing capability, is an obligate partner of NRFs. CtBP2 forms transcriptional complexes with NRF1 and NRF2 that is required to promote the expression of antioxidant genes in response to oxidative insults. Our findings illustrate a basis for understanding the transcriptional regulation of antioxidative defense systems that may be exploited therapeutically.

One Week of CDAHFD Induces Steatohepatitis and Mitochondrial Dysfunction with Oxidative Stress in Liver.

The prevalence of nonalcoholic fatty liver disease (NAFLD) has been rapidly increasing worldwide. A choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD) has been used to create a mouse model of nonalcoholic steatohepatitis (NASH). There are some reports on the effects on mice of being fed a CDAHFD for long periods of 1 to 3 months. However, the effect of this diet over a short period is unknown. Therefore, we examined the effect of 1-week CDAHFD feeding on the mouse liver. Feeding a CDAHFD diet for only 1-week induced lipid droplet deposition in the liver with increasing activity of liver-derived enzymes in the plasma. On the other hand, it did not induce fibrosis or cirrhosis. Additionally, it was demonstrated that CDAHFD significantly impaired mitochondrial respiration with severe oxidative stress to the liver, which is associated with a decreasing mitochondrial DNA copy number and complex proteins. In the gene expression analysis of the liver, inflammatory and oxidative stress markers were significantly increased by CDAHFD. These results demonstrated that 1 week of feeding CDAHFD to mice induces steatohepatitis with mitochondrial dysfunction and severe oxidative stress, without fibrosis, which can partially mimic the early stage of NASH in humans.

Plasma free metanephrine and normethanephrine levels correlated to plasma catecholamine after acute running in amateur runner.

BACKGROUND: Catecholamine is a typical index of exercise intensity, but it is difficult to detect. Plasma metanephrine (MN) and normethanephrine (NMN) levels are more stable than those of catecholamines. This study aimed to investigate plasma MN and NMN levels during acute exercise running in amateur runners. METHODS: Samples were collected from eight healthy male participants. They were either sedentary or running at low or high intensity for 30 min. Blood samples were collected under these conditions. Measurements taken included plasma adrenaline, noradrenaline, MN, and NMN. RESULTS: Plasma adrenaline levels increased after high-intensity exercise compared with sedentary subjects. Plasma noradrenaline, MN, and NMN levels increased after both low- and high-intensity exercise compared with sedentary subjects. In addition, these levels were also significantly higher at high intensity than at low intensity. Plasma adrenaline and noradrenaline levels were positively correlated with plasma free MN and NMN levels after acute running, respectively. CONCLUSION: This study revealed that plasma MN and NMN levels transiently increased depending on exercise intensity in amateur runners. In addition, plasma NMN levels are better markers than plasma MN levels because of their stronger correlation with plasma catecholamine levels.

Influence of Intermittent Cold Stimulations on CREB and Its Targeting Genes in Muscle: Investigations into Molecular Mechanisms of Local Cryotherapy.

Local cryotherapy is widely used as a treatment for sports-related skeletal muscle injuries. The molecular mechanisms are unknown. To clarify these mechanisms, we applied one to three 15-min cold stimulations at 4 °C to various cell lines (in vitro), the tibialis anterior (TA) muscle (ex vivo), and mouse limbs (in vivo). In the in vitro assay, cyclic AMP (cAMP) response element binding protein 1 (CREB1) was markedly phosphorylated (p-CREB1), and the CREB-binding protein (CBP) was recruited to p-CREB-1 in response to two or three cold stimulations. In a reporter assay with the cAMP-responsive element, the signals significantly increased after two to three cold stimulations at 4 °C. In the ex vivo study, CREB-targeting genes were significantly upregulated following two or three cold stimulations. The in vivo experiment disclosed that cold stimulation of a mouse limb for 9 days significantly increased mitochondrial DNA copy number and upregulated genes involved in mitochondrial biogenesis. The results suggest that local cryotherapy increases CREB transcription and upregulates CREB-targeting genes, in a manner dependent on cold stimulation frequency and duration. This information will inform further investigations into local cryotherapy as a treatment for sports-related skeletal muscle trauma.

A common genetic variant of the chromogranin A-derived peptide catestatin is associated with atherogenesis and hypertension in a Japanese population.

Chromogranin A (CHGA) is a major protein in the secretory granules of chromaffin cells. CHGA also gives rise to cardiovascular/metabolism regulatory peptides, such as catestatin (CST) and pancreastatin (PST). While CST is a potent inhibitor of catecholamine secretion, PST is a potent physiological inhibitor of glucose-induced insulin secretion. Recently, several SNPs were identified in the CST and PST domains of CHGA locus in different populations. Among the discovered SNPs, CST variant allele Ser-364 was associated with blood pressure alteration and PST variant allele Ser-297 was associated with significantly higher plasma glucose level. In this study, we examined whether these CST and PST variant alleles exist and influence cardiovascular and metabolic phenotypes in Japanese population. Our study comprised of 343 Japanese subjects aged 45-85 years (143 men and 200 women, mean age 66 ± 8 years). We determined the genotypes of CST and PST by PCR-direct sequencing method and carried out genotype-phenotype association analysis. In 343 participants, the minor allele frequency of CST variant Ser-364 was 6.10%. On the other hand, we did not detect the PST variant Ser-297 in this entire study population. The presence of Ser-364 allele was associated with increased in baPWV (an index of systemic arterial stiffness) that suggests an initiation and/or progression atherogenesis and hypertension. The Ser-364 allele was also associated with elevated systolic blood pressure and pulse pressure, consistent with increased baPWV. In conclusion, the CST Ser-364 allele may increase the risk for cardiovascular diseases in Japanese population.

Detection Method for Gene Doping in a Mouse Model Expressing Human Erythropoietin from Adeno-Associated Virus Vector-9.

With the rapid development of gene therapy technology in recent years, its abuse as a method of sports doping in athletics has become a concern. However, there is still room for improvement in gene-doping testing methods, and a robust animal model needs to be developed. Therefore, the purposes of this study were to establish a model of gene doping using recombinant adeno-associated virus vector-9, including the human erythropoietin gene (rAAV9-hEPO), and to establish a relevant testing method. First, it was attempted to establish the model using rAAV9-hEPO on mice. The results showed a significant increase in erythrocyte volume accompanied by an increase in spleen weight, confirming the validity of the model. Next, we attempted to detect proof of gene doping by targeting DNA and RNA. Direct proof of gene doping was detected using a TaqMan-qPCR assay with certain primers/probes. In addition, some indirect proof was identified in RNAs through the combination of a TB Green qPCR assay with RNA sequencing. Taken together, these results could provide the foundation for an effective test for gene doping in human athletes in the future.

Hypoxia and lactate influence VOC production in A549 lung cancer cells.

Introduction: Cancer cells emit characteristic volatile organic compounds (VOCs), which are potentially generated from ROS-based lipid peroxidation of polyunsaturated fatty acids. The metabolism of such VOCs and their regulation remain to be fully investigated. In fact, the enzymes involved in the synthesis of these VOCs have not been described yet. Methods: In this study, we firstly conducted in vitro enzyme assays and demonstrated that recombinant alcohol dehydrogenase (ADH) converted Trans 2-hexenal into Trans 2-hexenol. The latter has previously been reported as a cancer VOC. To study VOC metabolism, 14 different culture conditions were compared in view of Trans 2-hexenol production. Results and discussion: The data indicate that hypoxia and the addition of lactate positively influenced Trans 2-hexenol production in A549 cancer cells. The RNAseq data suggested certain gene expressions in the VOC pathway and in lactate signaling, parallel to VOC production. This implies that hypoxia and lactate signaling with a VOC production can be characteristic for cancer in vitro.

Systematic single-cell dissecting reveals heterogeneous oncofetal reprogramming in the tumor microenvironment of gastric cancer.

Oncofetal reprogramming of the tumor microenvironment is clinically relevant. This study used the non-negative matrix factorial (NMF) algorithm for single-cell RNA sequencing data of gastric cancer (GC) based on embryonic stem genes. Pseudotime analysis, cell-cell interaction analysis, and SCENIC analysis revealed that cancer-associated fibroblasts (CAFs), tumor-associated endothelial cells (TECs), and tumor-associated macrophages (TAMs) have different oncofetal reprogramming that affects cell function, enhances intercellular communication, and activates multiple transcription factors in these cells. Furthermore, based on the signatures of the newly defined oncofetal cell subtypes and expression profiles of large cohorts in GC patients, we determined that GJA1 + TEC-C2, IFITM1 + CAF-C3, PODXL + TEC-C1, SFRP2 + CAF-C2, and SRSF7 + CAF-C1 are crucial prognostic factors for GC patients and predictors of immune checkpoint blockade in GC. Cell subtypes were validated by immunohistochemical methods. Our novel, profound, and systematic analysis of the oncofetal reprogramming of GC may facilitate the development of improved drugs for treating GC.

Generation of a MyoD knock-in reporter mouse line to study muscle stem cell dynamics and heterogeneity.

Myoblast determination protein 1 (MyoD) dynamics define the activation status of muscle stem cells (MuSCs), aiding in muscle tissue regeneration after injury. However, the lack of experimental platforms to monitor MyoD dynamics in vitro and in vivo has hampered the investigation of fate determination and heterogeneity of MuSCs. Herein, we report a MyoD knock-in (MyoD-KI) reporter mouse expressing tdTomato at the endogenous MyoD locus. Expression of tdTomato in MyoD-KI mice recapitulated the endogenous MyoD expression dynamics in vitro and during the early phase of regeneration in vivo. Additionally, we showed that tdTomato fluorescence intensity defines MuSC activation status without immunostaining. Based on these features, we developed a high-throughput screening system to assess the effects of drugs on the behavior of MuSCs in vitro. Thus, MyoD-KI mice are an invaluable resource for studying the dynamics of MuSCs, including their fate decisions and heterogeneity, and for drug screening in stem cell therapy.

Identification of antigen-presentation related B cells as a key player in Crohn's disease using single-cell dissecting, hdWGCNA, and deep learning.

Crohn's disease (CD) arises from intricate intercellular interactions within the intestinal lamina propria. Our objective was to use single-cell RNA sequencing to investigate CD pathogenesis and explore its clinical significance. We identified a distinct subset of B cells, highly infiltrated in the CD lamina propria, that expressed genes related to antigen presentation. Using high-dimensional weighted gene co-expression network analysis and nine machine learning techniques, we demonstrated that the antigen-presenting CD-specific B cell signature effectively differentiated diseased mucosa from normal mucosa (Independent external testing AUC = 0.963). Additionally, using MCPcounter and non-negative matrix factorization, we established a relationship between the antigen-presenting CD-specific B cell signature and immune cell infiltration and patient heterogeneity. Finally, we developed a gene-immune convolutional neural network deep learning model that accurately diagnosed CD mucosa in diverse cohorts (Independent external testing AUC = 0.963). Our research has revealed a population of B cells with a potential promoting role in CD pathogenesis and represents a fundamental step in the development of future clinical diagnostic tools for the disease.

Establishing a Sequencing Method for the Whole Mitochondrial DNA of Domestic Dogs.

In human beings, whole mitochondrial DNA (mtDNA) sequencing has been widely used in many research fields, including medicine, forensics, and genetics. With respect to the domestic dog (Canis lupus familiaris), which is commonly recognized as being an additional member of the traditional human family structure, research studies on mtDNA should be developed to expand and improve our collective knowledge of dog medicine and welfare as it seems that there is still room for further development in these areas. Moreover, a simple and robust method for sequencing whole mtDNA that can be applied to various dog breeds has not yet been described in the literature. In the present study, we aim to establish such a method for the whole mtDNA sequencing of the domestic dog. In the experiments we conducted, oral mucosa DNA samples obtained from six Japanese domestic dogs were used as a template. We designed four primer pairs that could amplify approximately 5 kbp from each region of the mtDNA and validated several PCR conditions. Subsequently, the PCR amplicons were pooled and subjected to library preparation. The sequencing of the libraries was performed using next-generation sequencing (NGS), followed by bioinformatics analysis. Our results demonstrate that the proposed method can be used to perform highly accurate resequencing. We believe that this method may be useful for future research conducted to better understand dog medicine and welfare.