Sleep Deprivation Promotes Endothelial Inflammation and Atherogenesis by Reducing Exosomal miR-182-5p.

BACKGROUND: Insufficient or disrupted sleep increases the risk of cardiovascular disease, including atherosclerosis. However, we know little about the molecular mechanisms by which sleep modulates atherogenesis. This study aimed to explore the potential role of circulating exosomes in endothelial inflammation and atherogenesis under sleep deprivation status and the molecular mechanisms involved. METHODS: Circulating exosomes were isolated from the plasma of volunteers with or without sleep deprivation and mice subjected to 12-week sleep deprivation or control littermates. miRNA array was performed to determine changes in miRNA expression in circulating exosomes. RESULTS: Although the total circulating exosome levels did not change significantly, the isolated plasma exosomes from sleep-deprived mice or human were a potent inducer of endothelial inflammation and atherogenesis. Through profiling and functional analysis of the global microRNA in the exosomes, we found miR-182-5p is a key exosomal cargo that mediates the proinflammatory effects of exosomes by upregulation of MYD88 (myeloid differentiation factor 88) and activation of NF-ĸB (nuclear factor kappa-B)/NLRP3 pathway in endothelial cells. Moreover, sleep deprivation or the reduction of melatonin directly decreased the synthesis of miR-182-5p and led to the accumulation of reactive oxygen species in small intestinal epithelium. CONCLUSIONS: The findings illustrate an important role for circulating exosomes in distant communications, suggesting a new mechanism underlying the link between sleep disorder and cardiovascular disease.

Tra2ß exerts tumor-promoting effects via GSK3/ß-catenin signaling in oral squamous cell carcinoma.

OBJECTIVES: The splicing factor transformer-2 homolog beta (Tra2ß) plays a pivotal role in various cancers. Nonetheless, its role in oral squamous cell carcinoma (OSCC) has not been comprehensively explored. This study sought to discern the influence of Tra2ß on OSCC and its underlying mechanisms. MATERIALS AND METHODS: We assessed Tra2ß expression in OSCC utilizing immunohistochemistry, qRT-PCR, and western blotting techniques. siRNA transfection was used to silence Tra2ß. Whole transcriptome RNA sequencing (RNA-seq) analysis was carried out to reveal the alternative splicing (AS) events. KEGG pathway analysis enriched the related pathways. Colony formation, transwell, wound healing, and Annexin V-FITC/PI were employed to appraise the consequences of Tra2ß silencing on OSCC. RESULTS: Tra2ß was highly expressed in both OSCC tissues and cell lines. Knockdown of Tra2ß-regulated AS events with skipped exon (SE) accounts for the highest proportion. Meanwhile, downregulation of Tra2ß reduced cell proliferation, migration, and invasion, however increasing cell apoptosis. Moreover, Wnt signaling pathway involved in the function of Tra2ß knockdown which was demonstrated directly by a discernible reduction in the expression of GSK3/ß-catenin signaling axis. CONCLUSIONS: These findings suggest that knockdown of Tra2ß may exert anti-tumor effects through the GSK3/ß-catenin signaling pathway in OSCC.

MicroRNA-191 regulates oral squamous cell carcinoma cells growth by targeting PLCD1 via the Wnt/ß-catenin signaling pathway.

BACKGROUND: Studies have shown that microRNA-191 (miR-191) is involved in the development and progression of a variety of tumors. However, the function and mechanism of miR-191 in oral squamous cell carcinoma (OSCC) have not been clarified. METHODS: The expression level of miR-191 in tumor tissues of patients with primary OSCC and OSCC cell lines were detected using real-time quantitative polymerase chain reaction (RT-qPCR) and western blot. OSCC cells were treated with miR-191 enhancers and inhibitors to investigate the effects of elevated or decreased miR-191 expression on OSCC cells proliferation, migration, cell cycle, and tumorigenesis. The target gene of miR-191 in OSCC cells were analyzed by dual-Luciferase assay, and the downstream signaling pathway of the target genes was detected using western blot assay. RESULTS: The expression of miR-191 was significantly upregulated in OSCC tissues and cell lines. Upregulation of miR-191 promoted proliferation, migration, invasion, and cell cycle progression of OSCC cells, as well as tumor growth in nude mice. Meanwhile, reduced expression of miR-191 inhibited these processes. Phospholipase C delta1 (PLCD1) expression was significantly downregulated, and negatively correlated with the expression of miR-191 in OSCC tissues. Dual-Luciferase assays showed that miR-191-5p could bind to PLCD1 mRNA and regulate PLCD1 protein expression. Western blot assay showed that the miR-191 regulated the expression of ß-catenin and its downstream gene through targeting PLCD1. CONCLUSION: MicroRNA-191 regulates oral squamous cell carcinoma cells growth by targeting PLCD1 via the Wnt/ß-catenin signaling pathway. Thus, miR-191 may serve as a potential target for the treatment of OSCC.

Screening and identification of miR-181a-5p in oral squamous cell carcinoma and functional verification in vivo and in vitro.

BACKGROUND: Oral squamous cell carcinoma (OSCC) is a common malignant tumor associated with poor prognosis. MicroRNAs (miRNAs) play crucial regulatory roles in the cancer development. However, the role of miRNAs in OSCC development and progression is not well understood. METHODS: We sought to establish a dynamic Chinese hamster OSCC animal model, construct miRNA differential expression profiles of its occurrence and development, predict its targets, and perform functional analysis and validation in vitro. RESULTS: Using expression and functional analyses, the key candidate miRNA (miR-181a-5p) was selected for further functional research, and the expression of miR-181a-5p in OSCC tissues and cell lines was detected. Subsequently, transfection technology and a nude mouse tumorigenic model were used to explore potential molecular mechanisms. miR-181a-5p was significantly downregulated in human OSCC specimens and cell lines, and decreased miR-181a-5p expression was observed in multiple stages of the Chinese hamster OSCC animal model. Moreover, upregulated miR-181a-5p significantly inhibited OSCC cell proliferation, colony formation, invasion, and migration; blocked the cell cycle; and promoted apoptosis. BCL2 was identified as a target of miR-181a-5p. BCL2 may interact with apoptosis- (BAX), invasion- and migration- (TIMP1, MMP2, and MMP9), and cell cycle-related genes (KI67, E2F1, CYCLIND1, and CDK6) to further regulate biological behavior. Tumor xenograft analysis indicated that tumor growth was significantly inhibited in the high miR-181a-5p expression group. CONCLUSION: Our findings indicate that miR-181a-5p can be used as a potential biomarker and provide a novel animal model for mechanistic research on oral cancer.

A randomized, placebo-controlled clinical trial of hydrogen/oxygen inhalation for non-alcoholic fatty liver disease.

Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease worldwide with increasing incidence consistent with obesity, type 2 diabetes and cardiovascular diseases. No approved medication was currently available for NAFLD treatment. Molecular hydrogen (H2 ), an anti-oxidative, anti-inflammatory biomedical agent is proved to exhibit therapeutic and preventive effect in various diseases. The purpose of this study was to investigate the effect of hydrogen/oxygen inhalation on NAFLD subjects and explore the mechanism from the perspective of hepatocyte autophagy. We conducted a randomized, placebo-controlled clinical trial of 13-week hydrogen/oxygen inhalation (China Clinical Trial Registry [#ChiCTR-IIR-16009114]) including 43 subjects. We found that inhalation of hydrogen/oxygen improved serum lipid and liver enzymes. Significantly improved liver fat content detected by ultrasound and CT scans after hydrogen/oxygen inhalation was observed in moderate-severe cases. We also performed an animal experiment based on methionine and choline-deficient (MCD) diet-induced mice model to investigate effect of hydrogen on mouse NASH. Hydrogen/oxygen inhalation improved systemic inflammation and liver histology. Promoted autophagy was observed in mice inhaled hydrogen/oxygen and treatment with chloroquine blocked the beneficial effect of hydrogen. Moreover, molecular hydrogen inhibited lipid accumulation in AML-12 cells. Autophagy induced by palmitic acid (PA) incubation was further promoted by 20% hydrogen incubation. Addition of 3-methyladenine (3-MA) partially blocked the inhibitory effect of hydrogen on intracellular lipid accumulation. Collectively, hydrogen/oxygen inhalation alleviated NAFLD in moderate-severe patients. This protective effect of hydrogen was possibly by activating hepatic autophagy.

One night of sleep deprivation induces release of small extracellular vesicles into circulation and promotes platelet activation by small EVs.

Extracellular vesicles (EVs) are emerging as key players in intercellular communication. Few studies have focused on EV levels in subjects with sleep disorders. Here, we aimed to explore the role of acute sleep deprivation on the quantity and functionality of circulating EVs, and their tissue distribution. EVs were isolated by ultracentrifugation from the plasma of volunteers and animals undergoing one night of sleep deprivation. Arterio-venous shunt, FeCl3 thrombus test and thrombin-induced platelet aggregation assay were conducted to evaluate the in vivo and in vitro bioactivity of small EVs. Western blotting was performed to measure the expression of EV proteins. The fate and distribution of circulating small EVs were determined by intravital imaging. We found that one night of sleep deprivation triggers release of small EVs into the circulation in both healthy individuals and animals. Injection of sleep deprivation-liberated small EVs into animals increased thrombus formation and weight in thrombosis models. Also, sleep deprivation-liberated small EVs promoted platelet aggregation induced by thrombin. Mechanistically, sleep deprivation increased the levels of HMGB1 protein in small EVs, which play important roles in platelet activation. Furthermore, we found sleep deprivation-liberated small EVs are more readily localize in the liver. These data suggested that one night of sleep deprivation is a stress for small EV release, and small EVs released here may increase the risk of thrombosis. Further, small EVs may be implicated in long distance signalling during sleep deprivation-mediated adaptation processes.

Construction of Fzd6Q152E mice through CRISPR/Cas9 technology and their reproduction and identification.

BACKGROUND: The CRISPR/Cas9 system is widely used for genome editing in human, rat and mouse cells. In this study, we established Fzd6 mutant mice using CRISPR/Cas9 technology, and obtained Fzd6 homozygous mutant (Fzd6Q152E) mice through breeding. Fzd6 plays a role in depression, but there are few related reports. We used this model to investigate the mechanism of Fzd6 involved in depression, and build a solid foundation for subsequent in-depth studies. METHODS AND RESULTS: The target of Fzd6 mutation was obtained by CRISPR/Cas9 technology and hippocampal tissue was collected for Nissl staining and histological analysis. Blood was collected for enzyme linked immunosorbent assay (ELISA); The gene expression of Fzd6 and the related genes expression in wnt pathway was quantified by quantitative real-time PCR (qRT-PCR), and then expression of Fzd6 and proteins in the Wnt pathway were identified by western blotting. ELISA results showed that the expression levels of brain derived neurotrophic factor (BDNF), 5-hydroxytryptamine (5-HT), and Noradrenaline (NE) in serum were significantly decreased in Fzd6Q152E mice, whereas the mRNA expression of Lrp5, Lrp6, and Dkk2 is increased. The western blotting revealed that the expression of Fzd6 and Lrp6 is decreased, although the expression of Dkk2 and Gsk-3ß increased. CONCLUSION: Our study successfully established homozygous Fzd6 mutant mice model. The relationship between Fzd6-Wnt and depression was preliminarily clarified, which provides an ideal animal model for subsequent research on diseases induced by the Fzd6 mutation.

Evaluation of basal hormone levels and androgen receptor gene mutations in individuals with recurrent abortion.

A recurrent miscarriage is at least two consecutive miscarriages in the first trimester of pregnancy. Due to the dependence of pregnancy on endocrine changes in the menstrual cycle, its disorders can also affect the outcome of pregnancy. In addition to hormonal disorders, genetic changes are essential factors in recurrent miscarriage. The development and maturation of ovulation depend on the molecular signaling pathways that respond to androgens. Hundreds of mutations leading to resistance to androgen receptor (AR) gene function have been recorded, including the 5'UTR polymorphic region. Therefore, considering the role of androgen receptors and hormonal changes in recurrent miscarriage, this study was performed to investigate the relationship between hormonal changes and AR gene mutations in patients with recurrent miscarriage. In this regard, a case-control study was performed on 150 patients with miscarriage referred to the infertility center. Hysterosalpingography, parental karyotype, vaginal ultrasound, antiphospholipid antibody measurement, anticardiolipin antibody, history and physical examination were performed to evaluate the possible causes of recurrent miscarriage. Hormone levels of LH, FSH, TSH, and Prolactin were measured and compared in two groups with known and unknown causes. Blood samples were also taken from patients, and after DNA extraction, the PCR method was used to determine AR gene mutations. The mean age was 30.2 ± 7.1 years, the mean number of abortions was 2.6 ± 1.2, and the mean duration of marriage was 6.1 ± 2.1 years. The mean of hormones in the two groups with known and unknown causes was compared, that TSH was significantly lower in the group with unknown cause (P = 0.031) and prolactin was higher in recurrent miscarriage patients with polycystic ovaries (P = 0.048). Regarding genetic evaluation, in the 5'UTR region of the androgen receptor gene, deletion of T nucleotide was observed in the +25 position, but no significant difference was found between the two groups. Generally, the findings of this study showed that thyroid dysfunction and hyperprolactinemia should be considered as an endocrine disorder in people with recurrent miscarriage, and genetic evaluation showed that the AR gene mutation was not associated with recurrent miscarriage.

miR-450b promotes cell migration and invasion by inhibiting SERPINB2 in oral squamous cell carcinoma.

OBJECTIVE: microRNA-450b (miR-450b) plays an important role in cancer progression; however, its function in oral squamous cell carcinoma (OSCC) remains largely unknown. This study aimed to investigate the action mechanisms of miR-450b in OSCC. MATERIALS AND METHODS: OSCC animal model was established via continuous induction with single-drug 7, 12-dimethylbenzo[a]anthracene (DMBA). Animal tissue samples were pathologically typed using haematoxylin-eosin (HE) staining. The Cancer Genome Atlas (TCGA) database was used to predict miR-450b and SERPINB2 expression in head and neck squamous cell carcinoma (HNSCC). qRT-PCR and Western blotting were used to detect gene and protein expression in OSCC tissue and cells, respectively. OSCC cell proliferation, growth, migration and invasion were detected using CCK-8, colony formation, transwell migration and matrigel invasion assays, respectively. Bioinformatic tools were used to predict miR-450b target genes. Dual-luciferase reporter assay was used to verify targeting between miR-450b and SERPINB2. Finally, small interfering RNA (siRNA) was used to reduce SERPINB2 expression to detect its effect on tumourigenesis. RESULTS: Four stages of OSCC carcinogenesis (normal oral epithelium, simple epithelial hyperplasia, dysplasia and OSCC) were identified. miR-450b was found to be overexpressed in OSCC animal samples, HNSCC samples and human OSCC cells. Upregulation of miR-450b significantly promoted OSCC cell proliferation, colony formation, migration and invasion, while its downregulation had the opposite effect. SERPINB2 was found to be a miR-450b target gene, and its expression was negatively correlated with miR-450b expression. Altering SERPINB2 expression effectively inhibited OSCC cell invasion, metastasis and epithelial-mesenchymal transition (EMT). CONCLUSIONS: miR-450b plays a key role in OSCC tumourigenesis by regulating OSCC cell migration, invasion and EMT via SERPINB2.

Mitochondrial DNA Diversity of Mesocricetus auratus and Other Cricetinae Species among Cricetidae Family.

Unique anatomical and physiological features have made hamster species desirable research models. Comparative genomics and phylogenetic analysis of the hamster family members to clarify their evolution and genetic relationship, can provide a genetic basis for the comprehension of the variable research results obtained using different hamster models. The Syrian golden hamster (Mesocricetus auratus) is the most widely used species. In this study, we sequenced the complete mitochondrial genome (mitogenome) of M. auratus, compared it with the mitogenome of other Cricetinae subfamily species, and defined its phylogenetic position in the Cricetidae family. Our results show that the mitogenome organization, gene arrangement, base composition, and genetic analysis of the protein coding genes (PCGs) of M. auratus are similar to those observed in previous reports on Cricetinae species. Nonetheless, our analysis clarifies some striking differences of M. auratus relative to other subfamily members, namely distinct codon usage frequency of TAT (Tyr), AAT (Asn), and GAA (Glu) and the presence of the conserved sequence block 3 (CSB-3) in the control region of M. auratus mitogenome and other hamsters (not found in Arvicolinae). These results suggest the particularity of amino acid codon usage bias of M. auratus and special regulatory signals for the heavy strand replication in Cricetinae. Additionally, Bayesian inference/maximum likelihood (BI/ML) tree shows that Cricetinae and Arvicolinae are sister taxa sharing a common ancestor, and Neotominae split prior to the split between Cricetinae and Arvicolinae. Our results support taxonomy revisions in Cricetulus kamensis and Cricetulus migratorius, and further revision is needed within the other two subfamilies. Among the hamster research models, Cricetulus griseus is the species with highest sequence similarity and closer genetic relationship with M. auratus. Our results show mitochondrial DNA diversity of M. auratus and other Cricetinae species and provide genetic basis for judgement of different hamster models, promoting the development and usage of hamsters with regional characteristics.

Animal model and bioinformatics analyses suggest the TIMP1/MMP9 axis as a potential biomarker in oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) is a common malignant tumor of the head and neck. However, the molecular mechanism underlying its development and progression is yet unclear. Genes that are differentially expressed, that is, differentially expressed genes (DEGs), between normal and diseased tissues are believed to be involved in disease development and progression. To identify the DEGs in OSCC and explore their role in occurrence and progression, we established a Chinese hamster OSCC model, determined the DEG, screened the identified DEGs, and performed Gene Ontology (GO) and KEGG enrichment analyses. A protein-protein interaction (PPI) network was generated to screen potential candidate genes. We then analyzed the expression, tumor stage and prognosis of candidate genes using the Gene Expression Profiling Interactive Analysis (GEPIA) database. Finally, we verified the candidate DEGs by quantitative real-time PCR and Gene Expression Omnibus analysis. The results showed 194 significantly DEGs, 140 enriched GO terms, and 8 KEGG pathways, which suggested that OSCC was closely related to the immune system, cell migration, and extracellular matrix. GEPIA and PPI network analysis revealed that SPP1, TNC, and ACTA1 were significantly related to tumor staging; SPP1, tissue inhibitors of matrix metallopeptidases (MMPs) 1 (TIMP1), and ACTA1 were closely related to prognosis. The scores for the top five highest degree genes were close, and the TIMP1/MMP9 axis appeared to be at the center of the PPI network, indicating that expression changes in the TIMP1/MMP9 axis and related genes may be involved in tumor invasion and metastasis. These findings provide novel insights into the mechanism of oral cancer.

Demonstration of critical role of GRIN3A in nicotine dependence through both genetic association and molecular functional studies.

Nicotine dependence (ND) is a chronic disease with catastrophic effects on individual and public health. The glutamate receptor subunit gene, ionotropic N-methyl-d-aspartate 3A (GRIN3A), encodes a crucial subunit of N-methyl-d-aspartate receptors (NMDARs), which play an essential role in synaptic plasticity in the brain. Although various variants of GRIN3A have been associated with ND in European-American and African-American samples, no study has been reported for the association between GRIN3A and ND in Chinese Han population. We performed an association study of 16 single nucleotide polymorphisms (SNPs) in GRIN3A with ND in 2616 Chinese individuals. SNP-based association analysis indicated that SNP rs1323423 was significantly associated with the Fagerström Test for Nicotine Dependence (FTND) score after correction for multiple testing (P = 0.0026). Haplotype-based association analysis revealed that Block 3, formed by rs1323423-rs10989591, was significantly associated with the FTND score after correction for multiple testing (global P = 0.0183). Furthermore, luciferase reporter assay demonstrated that the DNA region containing rs1323423 was an enhancer element, the activity of which was significantly impacted by rs1323423 genotype. Considering that rs1323423 is located in a potential enhancer region, we performed GRIN3A editing in HEK293T cells with CRISPR/Cas9 and found that the DNA region around rs1323423 has a regulatory function and the expression of GRIN3A affects the expression of other NMDA subunits. Moreover, we demonstrated that nicotine at a concentration of 100 µM decreased expression of GRIN3A in SH-SY5Y and HEK293T cells at the RNA and protein level, respectively. This study provides novel evidence for the involvement of GRIN3A in ND.

Lipopolysaccharide-Binding Protein and Bactericidal/Permeability-Increasing Protein in Lipid Metabolism and Cardiovascular Diseases.

Lipopolysaccharide-binding protein (LBP) and bactericidal/permeability-increasing protein (BPI) are the main members of BPI-like family based on the similar protein structure and conserved gene homology. Both LBP and BPI participate in lipid metabolism and thereby involve in pathogenesis of certain cardiovascular diseases. This chapter describes four aspects: (1) the loci of BPI and LBP in genome, (2) the characteristics of the cDNAs and expression patterns of LBP and BPI, (3) the structures and functions of LBP and BPI, and (4) the LBP and BPI in lipid metabolism and cardiovascular research.

ITRAQ-based proteomics reveals the potential mechanism of fluoride-induced myocardial contraction function damage.

Fluorosis is a worldwide public health problem, and its adverse effects on the heart have been confirmed by many studies. Abnormal myocardial contractions are often associated with impairment of cardiac function as a cause or consequence. We designed two-part experiments to search for biomarkers and clarify the underlying molecular mechanism of fluoride on myocardial contraction. First, we used Pressure-volume Loop analysis to evaluate changes in myocardial function indexes with multiple fluoride exposure levels in mice (0, 30, 70, and 150 mg/L) exposed for 4 weeks. The results showed that fluoride exposure affects the heart pump function and reduces cardiac contractility. Then, we established a rat model of fluoride exposure (0, 30, 60, and 90 mg/L) for 6 months to carry out proteomic analysis of fluoride-induced myocardial contractile injury. Hematoxylin-eosin (H&E) staining was used to determine the severity of myocardial injury, and myocardial tissue samples were submitted for isobaric tags for relative and absolute quantitation (ITRAQ) analysis. A total of 1607 proteins were successfully identified with 294 differentially expressed proteins (DEPs) in fluoride treated groups. According to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, 12 DEPs were confirmed to be involved in pathways related to myocardial contraction. Furthermore, we constructed a protein-protein interaction (PPI) network for these 12 core DEPs to illustrate the role and location of each DEP in the myocardial contraction pathway. The results of this study are helpful for identify a potential mechanism and biomarkers of fluoride-induced myocardial contraction function damage, moreover, which can provide a new insight into the heart toxicity of fluoride in animals at the proteomics level.

Influence of cecotrophy on fat metabolism mediated by caecal microorganisms in New Zealand white rabbits.

Cecotrophy is a special behaviour of rabbits. Eating soft faeces can improve feed efficiency and maintain gut flora in rabbits. In our previous study, we found that fasting from soft faeces significantly reduced growth rate and total cholesterol (TC) in New Zealand white rabbits (NZW rabbits), thereby resulting in lower values for body weight and fat deposition in the soft faeces fasting group than in the control group. However, it has not been demonstrated whether cecotrophy by NZW rabbits can regulate lipid metabolism by changing the diversity of caecal microorganisms. In this study, thirty-six 28-day-old weaned NZW female rabbits were randomly divided into two groups (the soft faeces fasting group and the control group) and fed to 90 days. Rabbits in the experimental group were treated with an Elizabeth circle to prevent them from eating their soft faeces. Then, the caecal contents of three rabbits from the soft faeces fasting group and three rabbits from the control group were collected for metagenomic sequencing. We found that the abundance of Bacteroides increased, while Ruminococcus decreased, compared with the control group after fasting from soft faeces. Relative abundance was depressed for genes related to metabolic pathways such as ascorbate and aldarate metabolism, riboflavin metabolism and bile secretion. Moreover, there was a general correlation between variation in microbial diversity and fat deposition. Bacteroides affects body weight and TC by participating in the riboflavin metabolism pathway. By investigating the effect of cecotrophy on caecal microorganisms of rabbits, we identified the key microorganisms that regulate the rapid growth performance of NZW rabbits, which may provide useful reference for the future research and development of microecological preparations for NZW rabbits.

Enhancing the CRISPR/Cas9 system based on multiple GmU6 promoters in soybean.

Small guide RNA (sgRNA) is an important component of the CRISPR/Cas9 system. The gene editing efficiency of the CRISPR/Cas9 system could be enhanced by using highly active U6 promoters to drive the expression of sgRNA. Therefore, we constructed various expression vectors based on the 11 GmU6 promoters predicted and cloned in the whole soybean genome. The expression of truncated GUS driven by 11 GmU6 promoters was tested in hairy roots and by Arabidopsis thaliana transformation. The results indicated that higher transcriptional levels were driven by 5 GmU6 promoters (GmU6-4, GmU6-7, GmU6-8, GmU6-10 and GmU6-11) in both soybean hairy roots and Arabidopsis thaliana. In addition, three genes, Glyma03g36470, Glyma14g04180 and Glyma06g136900, were selected as targets to detect the transcriptional levels of multiple GmU6 promoters. Mutations in these three genes were detected in soybean hairy roots after Agrobacterium rhizogenes infection, indicating efficient target gene editing, including nucleotide insertion, deletion, and substitution. Mutation efficiencies differed among the 11 GmU6 promoters, ranging from 2.8% to 20.6%, and markedly higher efficiencies were obtained with all three genes using the GmU6-8 (20.3%) and GmU6-10 (20.6%) promoters. These two GmU6 promoters also showed higher ability to drive truncated GUS transcription in both soybean hairy roots and transformed Arabidopsis thaliana. These results will help to construct an efficient CRISPR-Cas9 gene editing system and promote the application of the CRISPR-Cas9 genome editing system in soybean molecular breeding.

Molecular hydrogen: current knowledge on mechanism in alleviating free radical damage and diseases.

Ever since molecular hydrogen was first reported as a hydroxyl radical scavenger in 2007, the beneficial effect of hydrogen was documented in more than 170 disease models and human diseases including ischemia/reperfusion injury, metabolic syndrome, inflammation, and cancer. All these pathological damages are concomitant with overproduction of reactive oxygen species (ROS) where molecular hydrogen has been widely demonstrated as a selective antioxidant. Although it is difficult to construe the molecular mechanism of hydrogen's biomedical effect, an increasing number of studies have been helping us draw the picture clearer with days passing by. In this review, we summarized the current knowledge on systemic and cellular modulation by hydrogen treatment. We discussed the antioxidative, anti-inflammatory, and anti-apoptosis effects of hydrogen, as well as its protection on mitochondria and the endoplasmic reticulum, regulation of intracellular signaling pathways, and balancing of the immune cell subtypes. We hope that this review will provide organized information that prompts further investigation for in-depth studies of hydrogen effect.

CpG oligodeoxynucleotide preconditioning improves cardiac function after myocardial infarction via modulation of energy metabolism and angiogenesis.

Unmethylated CpG oligodeoxynucleotide (CpG-ODN), a Toll-like receptor 9 (TLR9) ligand, has been shown to protect against myocardial ischemia/reperfusion injury. However, the potential effects of CpG-ODN on myocardial infarction (MI) induced by persistent ischemia remains unclear. Here, we investigated whether and how CpG-ODN preconditioning protects against MI in mice. C57BL/6 mice were treated with CpG-ODN by i.p. injection 2 hr prior to MI induction, and cardiac function, and histology were analyzed 2 weeks after MI. Both 1826-CpG and KSK-CpG preconditioning significantly improved the left ventricular (LV) ejection fraction (LVEF) and LV fractional shortening (LVFS) when compared with non-CpG controls. Histological analysis further confirmed the cardioprotection of CpG-ODN preconditioning. In vitro studies further demonstrated that CpG-ODN preconditioning increases cardiomyocyte survival under hypoxic/ischemic conditions by enhancing stress tolerance through TLR9-mediated inhibition of the SERCA2/ATP and activation of AMPK pathways. Moreover, CpG-ODN preconditioning significantly increased angiogenesis in the infarcted myocardium compared with non-CpG. However, persistent TLR9 activation mediated by lentiviral infection failed to improve cardiac function after MI. Although CpG-ODN preconditioning increased angiogenesis in vitro, both the persistent stimulation of CpG-ODN and stable overexpression of TLR9 suppressed the tube formation of cardiac microvascular endothelial cells. CpG-ODN preconditioning significantly protects cardiac function against MI by suppressing the energy metabolism of cardiomyocytes and promoting angiogenesis. Our data also indicate that CpG-ODN preconditioning may be useful in MI therapy.

Species-habitat associations in an old-growth temperate forest in northeastern China.

BACKGROUND: Species coexistence mechanisms and maintenance of biodiversity have long been considered important components of community ecology research. As one of the important mechanisms, species coexistence theory based on niche differentiation has received attention in past years. Thus, topography, through the formation of habitat heterogeneity, affects species distributions and coexistence. A 30-ha dynamic plot of mixed broadleaved-Korean pine (Pinus koraiensis) forest is located in the Heilongjiang Fenglin National Nature Reserve. We examined species-habitat associations using the torus-translation method. We aim to understand the habitat associations of different species, life forms (shrubs, trees), and shade tolerance (light-demanding, midtolerant, shade-tolerant) across life stages (sapling, juvenile and mature), providing further evidence for the role of niche theory in temperate forests. RESULTS: Of the 33 species we tested, 28 species (84.8%) were at least significantly associated with one habitat type. Positive associations were more frequent in the valley and slope (shady and sunny) and less frequent on the ridge. Thirty-four significant positive associations with the five habitats were detected at three life stages (11, 11 and 12 at the sapling stage, juvenile stage, and mature stage, respectively). The trees were positively associated with the valley, and the shrubs were positively associated with sunny and ridge. The majority of species' habitat preferences shifted among different life stages; the exceptions were Corylus mandshurica, Maackia amurensis, Quercus mongolica, Picea jezoensis and Acer ukurunduense, which had consistent associations with the same habitat at all stages. The midtolerant trees and midtolerant shrubs were positively correlated with sunny across the three life stages. CONCLUSIONS: Most species show habitat preferences in the plot. These results indicate that niche theory plays an important role in species coexistence. Most species have no consistent association with habitat at different life stages.

[Research advances on preventive and therapeutic effects of hydrogen on cardiovascular and cerebrovascular diseases and underlying mechanisms].

For a long time, hydrogen (H2) has been considered as a physiological inert gas. However, recent studies have demonstrated that molecular H2 exerts significant therapeutic effects on various disease models due to its antioxidative, anti-inflammatory and anti-apoptotic capabilities, which have also been well confirmed in many clinical trials. Cardiovascular and cerebrovascular diseases (CCVDs) are the leading cause of death in the world, constituting a serious threat to human life and public health. In this paper, we reviewed the latest research progress of the biomedical effects of H2 in CCVDs and its possible molecular mechanisms, in the hope of providing new clues for the treatment of some CCVDs.