Null and missense mutations of ERI1 cause a recessive phenotypic dichotomy in humans.

ERI1 is a 3'-to-5' exoribonuclease involved in RNA metabolic pathways including 5.8S rRNA processing and turnover of histone mRNAs. Its biological and medical significance remain unclear. Here, we uncover a phenotypic dichotomy associated with bi-allelic ERI1 variants by reporting eight affected individuals from seven unrelated families. A severe spondyloepimetaphyseal dysplasia (SEMD) was identified in five affected individuals with missense variants but not in those with bi-allelic null variants, who showed mild intellectual disability and digital anomalies. The ERI1 missense variants cause a loss of the exoribonuclease activity, leading to defective trimming of the 5.8S rRNA 3' end and a decreased degradation of replication-dependent histone mRNAs. Affected-individual-derived induced pluripotent stem cells (iPSCs) showed impaired in vitro chondrogenesis with downregulation of genes regulating skeletal patterning. Our study establishes an entity previously unreported in OMIM and provides a model showing a more severe effect of missense alleles than null alleles within recessive genotypes, suggesting a key role of ERI1-mediated RNA metabolism in human skeletal patterning and chondrogenesis.

Parkin deficiency promotes liver cancer metastasis by TMEFF1 transcription activation via TGF-ß/Smad2/3 pathway.

Parkin (PARK2) deficiency is frequently observed in various cancers and potentially promotes tumor progression. Here, we showed that Parkin expression is downregulated in liver cancer tissues, which correlates with poor patient survival. Parkin deficiency in liver cancer cells promotes migration and metastasis as well as changes in EMT and metastasis markers. A negative correlation exists between TMEFF1 and Parkin expression in liver cancer cells and tumor tissues. Parkin deficiency leads to upregulation of TMEFF1 which promotes migration and metastasis. TMEFF1 transcription is activated by Parkin-induced endogenous TGF-ß production and subsequent phosphorylation of Smad2/3 and its binding to TMEFF1 promotor. TGF-ß inhibitor and TMEFF1 knockdown can reverse shParkin-induced cell migration and changes of EMT markers. Parkin interacts with and promotes the ubiquitin-dependent degradation of HIF-1α/HIF-1ß and p53, which accounts for the suppression of TGF-ß production. Our data have revealed that Parkin deficiency in cancer leads to the activation of the TGF-ß/Smad2/3 pathway, resulting in the expression of TMEFF1 which promotes cell migration, EMT, and metastasis in liver cancer cells.

Ras inhibitor farnesylthiosalicylic acid conjugated with IR783 dye exhibits improved tumor-targeting and altered anti-breast cancer mechanisms in mice.

Ras has long been viewed as a promising target for cancer therapy. Farnesylthiosalicylic acid (FTS), as the only Ras inhibitor has ever entered phase II clinical trials, has yielded disappointing results due to its strong hydrophobicity, poor tumor-targeting capacity, and low therapeutic efficiency. Thus, enhancing hydrophilicity and tumor-targeting capacity of FTS for improving its therapeutic efficacy is of great significance. In this study we conjugated FTS with a cancer-targeting small molecule dye IR783 and characterized the anticancer properties of the conjugate FTS-IR783. We showed that IR783 conjugation greatly improved the hydrophilicity, tumor-targeting and therapeutic potential of FTS. After a single oral administration in Balb/c mice, the relative bioavailability of FTS-IR783 was increased by 90.7% compared with FTS. We demonstrated that organic anion transporting polypeptide (OATP) and endocytosis synergistically drove the uptake of the FTS-IR783 conjugate in breast cancer MDA-MB-231 cells, resulting in superior tumor-targeting ability of the conjugate both in vitro and in vivo. We further revealed that FTS-IR783 conjugate could bind with and directly activate AMPK rather than affecting Ras, and subsequently regulate the TSC2/mTOR signaling pathway, thus achieving 2-10-fold increased anti-cancer therapeutic efficacy against 6 human breast cancer cell lines compared to FTS both in vivo and in vitro. Overall, our data highlights a promising approach for the modification of the anti-tumor drug FTS using IR783 and makes it possible to return FTS back to the clinic with a better efficacy.

Spatial learning and memory deficits induced by prenatal glucocorticoid exposure depend on hippocampal CRHR1 and CXCL5 signaling in rats.

BACKGROUND: Prenatal synthetic glucocorticoid (sGC) exposure increases the susceptibility to cognitive and affective disorders in postnatal life. We previously demonstrated that prenatal sGC exposure results in an increase in corticotropin-releasing hormone (CRH) receptor type 1 (CRHR1) expression in the hippocampus of rats, and CRHR1 is involved in synapse formation via regulation of C-X-C chemokine ligand 5 (CXCL5) in hippocampus. We sought to investigate that the roles of CRHR1 and CXCL5 in learning and memory impairment caused by prenatal sGC exposure. METHODS: Pregnant rats were administered with saline or dexamethasone (DEX) from gestational day (GD) 14 to GD21. DEX offspring at 2-day old were treated with saline and CRHR1 antagonists (antalarmin and CP154526) for 7 days. Some DEX offspring received intra-hippocampal injection of AAV9 carrying CXCL5 gene. Spatial learning and memory was assessed by Morris water maze test. Immunofluorescence analysis was applied to show synapsin I and PSD95 signals in hippocampus. Synapsin I and PSD95 protein level and CXCL5 concentration were determined by western blotting and ELISA, respectively. Organotypic hippocampal slice cultures were used to investigate the effect of DEX on CXCL5 production in vitro. RESULTS: Both male and female DEX offspring displayed impairment of spatial learning and memory in adulthood. Synapsin I and PSD95 signals and CXCL5 levels were decreased in DEX offspring. DEX offspring with antalarmin and CP154526 treatment showed improved spatial learning and memory. Antalarmin and CP154526 treatment increased synapsin I and PSD95 signals and CXCL5 concentration in hippocampus. Bilaterally hippocampal injection of AAV9 carrying CXCL5 gene improved the spatial learning and memory and increased CXCL5 concentration and synapsin I and PSD95 levels in hippocampus. DEX dose-dependently suppressed CXCL5 production in cultured hippocammpal slices, which was prevented by antalarmin treatment. CONCLUSION: CRHR1 and CXCL5 signaling in the hippocampus are involved in spatial learning and memory deficits caused by prenatal DEX exposure. CRHR1 activation contributes to decreased CXCL5 production in hippocampus induced by prenatal DEX treatment. Our study provides a molecular basis of prenatal GC exposure programming spatial learning and memory.

Gender- and age-related differences in distinct phenotypes of hypertrophic cardiomyopathy-associated mutation MYBPC3-E334K.

The mutation MYBPC3-E334K is a culprit mutation of hypertrophic cardiomyopathy (HCM). The pathogenicity of MYBPC3-E334K is conflicting in ClinVar because of the limited segregation data and the relatively high frequency in gnomAD (0.03% overall, with 0.3% in East Asians and 0.8% in Japanese). The main aim is to clarify the clinical importance and phenotype-genotype correlations in subjects with or without MYBPC3-E334K alone. The prevalence of MYBPC3-E334K was sequenced in 1017 HCM unrelated probands. The clinical features, morphology phenotypes, and electrical phenotypes were further analyzed according to the phenotype and genotype status in families with single-mutation MYBPC3-E334K. Nine of 1017 (0.88%) unrelated HCM probands were detected harboring MYBPC3-E334K, and three of them harbored a second variant in sarcomere protein gene. Family study and co-segregation analyses indicated that patients with single-mutation MYBPC3-E334K showed autosomal dominant mode of inheritance with incomplete penetrance. The overall disease penetrance was 52.6%, and the disease penetrance was higher in males than in females (100% in men vs 25% in women, p = 0.003). The mean age at diagnosis of males was approximately 25 years younger than females (36.57 ± 18.65 vs 62.33 ± 12.10, p = 0.062). The variant MYBPC3-E334K was classified as a likely pathogenic variant, and a second sarcomere variant did not reveal obvious cumulative effects. The patients harboring single-mutation MYBPC3-E334K had incomplete penetrance, and males demonstrated higher penetrance and early onset HCM than females. A second sarcomere variant did not reveal obvious cumulative effects.

Isoptericola halalbus sp. nov., a halotolerant actinobacterium isolated from saline lake sediment.

A novel Gram-stain-positive bacterium, designated CFH 91151T, was isolated from sediment collected from a saline lake in Yuncheng, Shanxi Province, PR China. Cells of strain CFH 91151T were rod-or v-shaped, aerobic, non-motile, non-spore-forming and halotolerant. Results of 16S rRNA gene sequence analysis revealed that strain CFH 91151T was closely related to Isoptericola variabilis MX5T and Isoptericola nanjingensis H17T (98.7 and 98.4% sequence similarity, respectively). The strain grew at 4-45 °C, pH 5.0-9.0 and with 0-14.0 % (w/v) NaCl. Cells were positive for catalase, nitrate was not used and H2S was not produced. Major cellular fatty acids were anteiso-C15 : 0 (62.76 %), anteiso-C17 : 0 (12.09 %) and iso-C15 : 0 (9.46 %). The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol, two unidentified phospholipids and three unidentified glycolipids. The menaquinone was MK-9 (H4). The genome size was 4.10 Mbp with a G+C content of 72.4 mol%. The average amino acid identity (ANI) and in silico DNA-DNA hybridization (DDH) values between CFH 91151T and the other species of the genus Isoptericola were found to be low (ANIm <87.19 %, ANIb <84.38 % and DDH <29.30 %). Based on physiological properties, chemotaxonomic characteristics and low ANI and DDH results, strain CFH 91151T is considered to represent a novel species, for which the name Isoptericola halalbus sp. nov. is proposed. The type strain is CFH 91151T (=DSM 105976T=KCTC 49061T).

Paracoccus luteus sp. nov., isolated from the intestine of grass carp.

A novel Gram-negative bacterium, designated CFH 10530T, was isolated from the intestine of grass carp. The sample was collected from the aquaculture training base at the College of Fisheries, Henan Normal University, Xinxiang, PR China. Cells of strain CFH 10530T were coccoid, ovoid or short-rod-shaped, aerobic, non-spore-forming and non-motile. 16S rRNA gene sequence analysis demonstrated that strain CFH 10530T was closely related to Paracoccus endophyticus SYSUP0003T (97.7 % sequence similarity), Paracoccus halophilus HN-182T (96.5 %) and Paracoccus panacisoli DCY94T (96.1 %). The strain grew optimally at 25-28 °C, at pH 7.0 and with 0-2 % (w/v) NaCl. Cells were positive for catalase and oxidase, nitrate was reduced and H2S was not produced. The isoprenoid quinone was Q-10. Major cellular fatty acids were summed feature 8, C18 : 0 and C18 : 03-OH. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine, one unidentified aminolipid and five unidentified polar lipids. The genome size was 3 331 229 bp with a G+C content of 69.6 mol%. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between CFH 10530T and the other species of the genus Paracoccus were found to be below the recommended levels for species delineation (ANIm <85, ANIb <80 and dDDH <24 %). Based on its physiological properties, chemotaxonomic characteristics and low ANI and dDDH results, strain CFH 10530T is considered to represent a novel species for which the name Paracoccus luteus sp. nov., is proposed. The type strain is CFH 10530T (=KCTC 62919T=CGMCC 1.16597T).

Azospirillum thermophilum sp. nov., isolated from a hot spring.

A novel Gram-staining negative, aerobic, motile by flagellum, rod-shaped bacterium, designated CFH 70021T was isolated from a hot spring soil sample collected from Tengchong, Yunnan province, PR China. Growth of CFH 70021T occurred at 15-50 °C (optimum 50 °C), pH 5.0-7.0 (optimum pH 7.0) and with 0-3.0 % (w/v) NaCl (optimum 0 %, w/v). The genome of CFH 70021T consisted of four complete circular chromosomes and five plasmids, the genomic DNA G+C content was 69.3 mol%. Comparison of the 16S rRNA gene sequences indicated that CFH 70021T represented a member of the genus Azospirillum and showed close relationship with the type strains of Azospirillum agricola CC-HIH038T (97.8 %), Azospirillum rugosum IMMIB AFH-6T (97.6 %), Azospirillum doebereinerae GSF71T (97.6 %), Azospirillum thiophilum DSM 21654T (97.4 %) and Azospirillum picis IMMIB TAR-3T (97.2 %). The polar lipids of CFH 70021T contained diphosphatidylglycerol, phosphatidylmehtylethanolamine, phosphatidylglycerol, phosphatidylcholine, two aminolipids and an unidentified phospholipid. The predominant cellular fatty acids (>10 %) included C19:0cyclo ω8c (11.4 %), C16 : 0 (27.6 %) and summed feature 8 (C18:1ω7c/C18:1ω6c, 40.9 %). The major isoprenoid quinone was Q-10. On the basis of the low ANIb result (<78 %) and different phenotypic and chemotaxonomic characters, we conclude that strain CFH 70021T represents a novel member of the genus Azospirillum, for which the name Azospirillum thermophilum sp. nov. is proposed. The type strain is CFH 70021T (=KCTC 62259T= CCTCC AB2018121T).

CRH/CRHR1 mediates prenatal synthetic glucocorticoid programming of depression-like behavior across 2 generations.

Pregnant women at risk of preterm labor usually receive synthetic glucocorticoids (sGCs) to promote fetal lung development. Emerging evidence indicates that antenatal sGC increases the risk of affective disorders in offspring. Data from animal studies show that such disorders can be transmitted to the second generation. However, the molecular mechanisms underlying the intergenerational effects of prenatal sGC remain largely unknown. Here we show that prenatal dexamethasone (Dex) administration in late pregnancy induced depression-like behavior in first-generation (F1) offspring, which could be transmitted to second-generation (F2) offspring with maternal dependence. Moreover, corticotropin-releasing hormone (CRH) and CRH receptor type 1 (CRHR1) expression in the hippocampus was increased in F1 Dex offspring and F2 offspring from F1 Dex female rats. Administration of a CRHR1 antagonist to newborn F1 Dex offspring alleviated depression-like behavior in these rats at adult. Furthermore, we demonstrated that increased CRHR1 expression in F1 and F2 offspring was associated with hypomethylation of CpG islands in Crhr1 promoter. Our results revealed that prenatal sGC exposure could program Crh and Crhr1 gene expression in hippocampus across 2 generations, thereby leading to depression-like behavior. Our study indicates that prenatal sGC can cause epigenetic instability, which increases the risk of disease development in the offspring's later life.-Xu, Y.-J., Sheng, H., Wu, T.-W., Bao, Q.-Y., Zheng, Y., Zhang, Y.-M., Gong, Y.-X., Lu, J.-Q., You, Z.-D., Xia, Y., Ni, X. CRH/CRHR1 mediates prenatal synthetic glucocorticoid programming of depression-like behavior across 2 generations.

Design, synthesis and structure-activity relationship of diaryl-ureas with novel isoxazol[3,4-b]pyridine-3-amino-structure as multi-target inhibitors against receptor tyrosine kinase.

Inspired by that the multi-target inhibitors against receptor tyrosine kinases (RTKs) have significantly improved the effect of clinical treatment for cancer, and based on the chemical structure of Linifanib (ABT-869, Abbott), two series of diaryl-ureas with novel isoxazol[3,4-b]pyridine-3-amino-structure were designed and synthesized as multi-target inhibitors against RTKs. The preliminary biological evaluation showed that several compounds exhibited comparable potency with Linifanib. Compound S21 was identified as the most potent inhibitor against Fms-like tyrosine kinase 3 (FLT-3), kinase insert domain containing receptor (KDR) and platelet-derived growth factor receptor ß (PDGFR-ß) with its IC50 values were 4 nM, 3 nM and 8 nM respectively, it also showed potent inhibitory activities against several cancer cells.

Investigation of myocardial dysfunction using three-dimensional speckle tracking echocardiography in a genetic positive hypertrophic cardiomyopathy Chinese family.

BACKGROUND: We previously reported four heterozygous missense mutations of MYH7, KCNQ1, MYLK2, and TMEM70 in a single three-generation Chinese family with dual Long QT and hypertrophic cardiomyopathy phenotypes for the first time. However, the clinical course among the family members was various, and the potential myocardial dysfunction has not been investigated. OBJECTIVES: The objective of this study was to investigate the echocardiographic and electrocardiographic characteristics in a genetic positive Chinese family with hypertrophic cardiomyopathy and further to explore the association between myocardial dysfunction and electric activity, and the identified mutations. METHODS: A comprehensive echocardiogram - standard two-dimensional Doppler echocardiography and three-dimensional speckle tracking echocardiography - and electrocardiogram were obtained for members in this family. RESULTS: As previously reported, four missense mutations - MYH7-H1717Q, KCNQ1-R190W, MYLK2-K324E, and TMEM70-I147T - were identified in this family. The MYH7-H1717Q mutation carriers had significantly increased left ventricular mass indices, elevated E/e' ratio, deteriorated global longitudinal stain, but enhanced global circumferential and radial strain compared with those in non-mutation patients (all p<0.05). The KCNQ1-R190W carriers showed significantly prolonged QTc intervals, and the MYLK2-K324E mutation carriers showed inverted T-waves (both p<0.05). However, the TMEM70-I147T mutation carriers had similar echocardiography and electrocardiographic data as non-mutation patients. CONCLUSIONS: Three of the identified four mutations had potential pathogenic effects in this family: MYH7-H1717Q was associated with increased left ventricular thickness, elevated left ventricular filling pressure, and altered myocardial deformation; KCNQ1-R190W and MYLK2-K324E mutations were correlated with electrocardiographic abnormalities reflected in long QT phenotype and inverted T-waves, respectively.

The Cumulative Effects of the MYH7-V878A and CACNA1C-A1594V Mutations in a Chinese Family with Hypertrophic Cardiomyopathy.

AIMS: We investigated the pathogenesis of MYH7-V878A and CACNA1C-A1594V mutations in a Chinese family with hypertrophic cardiomyopathy. METHODS: Clinical, electrocardiographic (ECG), echocardiographic, and cardiac magnetic resonance (CMR) examinations of members of a Chinese family were followed by exon and boarding intron analyses of 96 genes in the proband using second-generation sequencing. We confirmed the mutations by bidirectional Sanger sequencing in the members and in 300 healthy controls. RESULTS: We detected MYH7-V878A and CACNA1C-A1594V mutations in this family. The members with both mutations showed inverted T-waves and ST-segment depression in ECG recordings, severe left ventricular (LV) hypertrophy in echocardiography, and myocardial fibrosis in CMR; subject II-11 did not show late gadolinium enhancement. Among those with only the MYH7-V878A mutation, subject III-7 showed abnormal ECG recordings, asymmetric septal hypertrophy, and myocardial fibrosis, and subjects II-13 and III-15 showed some abnormal repolarization, borderline LV wall thickness, and normal CMR findings. Those with only the CACNA1C-A1594V mutation showed nearly normal readings in all examinations. The members with both mutations displayed more severe LV hypertrophy and elevated LV filling pressure than those with 1 or no mutation (p < 0.05). CONCLUSION: Our results suggest that the pathogenesis of MYH7-V878A and CACNA1C-A1594V mutations may have a cumulative effect.

Improvement of soybean transformation via Agrobacterium tumefaciens methods involving α-aminooxyacetic acid and sonication treatments enlightened by gene expression profile analysis.

KEY MESSAGE: Antagonists and sonication treatment relieved the structural barriers of Agrobacterium entering into cells; hindered signal perception and transmission; alleviated defense responses and increased cell susceptibility to Agrobacterium infection. Soybean gene expression analysis was performed to elucidate the general response of soybean plant to Agrobacterium at an early stage of infection. Agrobacterium infection stimulated the PAMPs-triggered immunity (BRI1, BAK1, BZR1, FLS2 and EFR) and effector-triggered immunity (RPM1, RPS2, RPS5, RIN4, and PBS1); up-regulated the transcript factors (WRKY25, WRKY29, MEKK1P, MKK4/5P and MYC2) in MAPK pathway; strengthened the biosynthesis of flavonoid and isoflavonoid in the second metabolism; finally led to a fierce defense response of soybean to Agrobacterium infection and thereby lower transformation efficiency. To overcome it, antagonist α-aminooxyacetic acid (AOA) and sonication treatment along with Agrobacterium infection were applied. This novel method dramatically decreased the expression of genes coding for F3'H, HCT, ß-glucosidase and IF7GT, etc., which are important for isoflavone biosynthesis or the interconversion of aglycones and glycon; genes coding for peroxidase, FLS2, PBS1 and transcription factor MYC2, etc., which are important components in plant-pathogen interaction; and genes coding for GPAT and α-L-fucosidase, which are important in polyesters formation in cell membrane and the degradation of fucose-containing glycoproteins and glycolipids on the external surface of cell membrane, respectively. This analysis implied that AOA and sonication treatment not only relieved the structural membrane barriers of Agrobacterium entering into cells, but also hindered the perception of 'invasion' signal on cell membrane and intercellular signal transmission, thus effectively alleviated the defense responses and increased the cell susceptibility to Agrobacterium infection. All these factors benefit the transformation process; other measures should also be further explored to improve soybean transformation.

The wheat NHX antiporter gene TaNHX2 confers salt tolerance in transgenic alfalfa by increasing the retention capacity of intracellular potassium.

Previous studies have shown that TaNHX2 transgenic alfalfa (Medicago sativa L.) accumulated more K(+) and less Na(+) in leaves than did the wild-type plants. To investigate whether the increased K(+) accumulation in transgenic plants is attributed to TaNHX2 gene expression and whether the compartmentalization of Na(+) into vacuoles or the intracellular compartmentalization of potassium is the critical mechanism for TaNHX2-dependent salt tolerance in transgenic alfalfa, aerated hydroponic culture was performed under three different stress conditions: control condition (0.1 mM Na(+) and 6 mM K(+) inside culture solution), K(+)-sufficient salt stress (100 mM NaCl and 6 mM K(+)) and K(+)-insufficient salt stress (100 mM NaCl and 0.1 mM K(+)). The transgenic alfalfa plants had lower K(+) efflux through specific K(+) channels and higher K(+) absorption through high-affinity K(+) transporters than did the wild-type plants. Therefore, the transgenic plants had greater K(+) contents and [K(+)]/[Na(+)] ratios in leaf tissue and cell sap. The intracellular compartmentalization of potassium is critical for TaNHX2-induced salt tolerance in transgenic alfalfa.

Studies on [5,6]-Fused Bicyclic Scaffolds Derivatives as Potent Dual B-RafV600E/KDR Inhibitors Using Docking and 3D-QSAR Approaches.

Research and development of multi-target inhibitors has attracted increasing attention as anticancer therapeutics. B-RafV600E synergistically works with vascular endothelial growth factor receptor 2 (KDR) to promote the occurrence and progression of cancers, and the development of dual-target drugs simultaneously against these two kinds of kinase may offer a better treatment advantage. In this paper, docking and three-dimensional quantitative structure activity relationship (3D-QSAR) studies were performed on a series of dual B-Raf/KDR inhibitors with a novel hinge-binding group, [5,6]-fused bicyclic scaffold. Docking studies revealed optimal binding conformations of these compounds interacting with both B-Raf and KDR. Based on these conformations, comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) 3D-QSAR models were constructed, and the best CoMFA (q²=0.542, r²=0.989 for B-Raf; q²=0.768, r²=0.991 for KDR) and CoMSIA models (q²=0.519, r²=0.992 for B-Raf; q²=0.849, r²=0.993 for KDR) were generated. Further external validations confirmed their predictability, yielding satisfactory correlation coefficients (r²pred=0.764 (CoMFA), r²pred=0.841 (CoMSIA) for B-Raf, r²pred=0.912 (CoMFA), r²pred=0.846 (CoMSIA) for KDR, respectively). Through graphical analysis and comparison on docking results and 3D-QSAR contour maps, key amino acids that affect the ligand-receptor interactions were identified and structural features influencing the activities were discussed. New potent derivatives were designed, and subjected to preliminary pharmacological evaluation. The study may offer useful references for the modification and development of novel dual B-Raf/KDR inhibitors.

Protective effect of melatonin on bone marrow mesenchymal stem cells against hydrogen peroxide-induced apoptosis in vitro.

Bone marrow mesenchymal stem cells (MSCs) transplantation has shown great promises for treating various central nervous system (CNS) diseases. However, poor viability of transplanted MSCs in injured CNS has limited the therapeutic efficiency. Oxidative stress is one of major mechanisms underlying the pathogenesis of CNS diseases and has a negative impact on the survival of transplanted MSCs. Melatonin has recently been reported to have the antioxidant and anti-apoptotic properties in serial of cells. This study was designed to investigate the protective effect and potential mechanisms of melatonin against hydrogen peroxide (H2O2)-induced apoptosis of MSCs. MSCs were pretreated with melatonin (1, 10, and 100 nM, respectively) for 30 min, followed by exposure to 400 µM H2O2 and melatonin together for 12 h. The present study reports that melatonin pretreatment significantly attenuated H2O2-induced MSC apoptosis in a dose-dependent manner. Consistently, melatonin effectively suppressed the generation of intracellular ROS, expression ratio of Bax/Bcl-2, activation of caspase-3 and expression of phospho-P38MAPK in H2O2-induced MSCs. Luzindole, a nonselective melatonin receptor antagonist, significantly counteracted melatonin's promotion effect on cell survival, indicating that melatonin exerts its protective effect on MSCs, at least in part, through the activation of melatonin receptors. The findings suggest that melatonin may be an effectively protective agent against oxidative stress-induced MSC apoptosis.

Activation of cannabinoid type 2 receptor by JWH133 protects heart against ischemia/reperfusion-induced apoptosis.

BACKGROUND: Cannabinoid type 2 (CB2) receptor agonists can protect myocardium against ischemia/reperfusion (I/R) injury although the underlying mechanism remains unclear. Here we report the antiapoptotic effect of CB2 receptor agonist, JWH133, during myocardial ischemia/reperfusion injury and potential underlying mechanisms. METHODS: Ischemia was performed by blocking left coronary artery of rat for 30 min. After ischemia for 30 min, the rat heart was reperfused for 120 min by loosing the ligation of blocking left coronary artery. JWH133 (20 mg/kg), a CB2 receptor selective agonist, or vehicles were injected intravenously 5 minutes before ischemia. Infarct size of myocardium was assessed by histological stain, myocardial apoptosis index (AI) was determined by TUNEL, and mitochondrial membrane potential (∆Ψm) was measured by flow cytometry. Western blots were performed to measure the cytochrome c release, cleaved caspase 3, cleaved caspase 9 and PI3K/Akt kinase phosphorylation. RESULTS: JWH133 significantly reduced the infarct size and AI of myocardium suffering I/R compared to vehicle-treated group. Further mechanistic study revealed that activation of CB2 receptor by JWH133 inhibited the loss of ΔΨm, reduction of the cleaved caspases-3 and -9, release of mitochondrial cytochrome c to the cytosol, and increase of phosphorylated Akt. These JWH133-mediated effects could be totally abrogated by PI3K inhibitor wortmanin or CB2 receptor antagonist AM630. CONCLUSION: Our results demonstrate that activation of CB2 receptor by JWH133 prevent apoptosis during ischemia/reperfusion through inhibition of the intrinsic mitochondria-mediated apoptotic pathway and involvement of the PI3K/Akt signal pathway.

[Significance of high sensitivity C-reactive protein level for predicting risk of nonalcoholic fatty liver in type 2 diabetes mellitus patients].

OBJECTIVE: To investigate the significance of high sensitivity C-reactive protein (hsCRP) levels in serum for detecting type 2 diabetes mellitus (T2DM) patients at risk of developing nonalcoholic fatty liver (NAFLD). METHODS: Individuals with T2DM (n = 9489) were recruited from the Kailuan Company between 2006 and 2007 for the first phase of this community-based prospective cohort study. For the second phase of the study, the original cohort was recruited for follow-up (at two years from each subject's original enrollment date (baseline)). The total followed-up subjects (n = 2802; 2344 males, 458 females, 22-88 years old) were categorized into quartiles according to baseline measurements of serum hsCRP levels (less than or equal to 0.30, > 0.30-0.60, > 0.60-1.92 and > 1.92 mg/L) and used to determine the relationship between change in incidence rates of NAFLD and predictive value of baseline serum hsCRP levels by logistic regression analysis. RESULTS: Twenty-nine percent (n = 813) of the followed-up subjects developed NAFLD. The incidence (%) of NAFLD at the two-year follow-up had increased in conjunction with the level of serum hsCRP detected at baseline (quartile 1: 22.5%, 2: 27.3%, 3: 32.1%, and 4: 34.3%; all, P less than 0.01). It was found that the subjects in the highest quartile had an increased risk of NAFLD (odds ratio (OR) = 1.80, 95% confidence interval (CI): 1.42-2.28, P less than 0.01), as compared with those in the lowest quartile. Moreover, when the regression model was adjusted for baseline factors of age, sex, triglycerides, total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, fasting serum glucose, and body mass index, the risk of NAFLD remained significantly higher for the highest quartile (vs. the lowest quartile; OR = 1.49, 95% CI: 1.16-1.91, P less than 0.01). CONCLUSION: Serum hsCRP levels may be predictive of development of NAFLD in individuals with type 2 diabetes mellitus. The risk of NAFLD increases in parallel with increasing levels of serum hsCRP.

[Study on antitumor effect of wutousan].

OBJECTIVE: To screen the active extracts from three traditional Chinese medicines. METHODS: Five traditional Chinese medicines including Unprocessed Aconiti Radix, Aconiti Radix Cocta, Phellodendri Chinensis Cortex were extracted with 70% alcohol or water respectively. The cell proliferation effect of these extracts was tested with MTT assay in SMMC7721, MCF7, A549, Hela and SGC7901. RESULTS: The 70% alcohol extract of Unprocessed Aconiti Radix, Aconiti Radix Cocta, Phellodendri Chinensis Cortex displayed remarkable inhibitory effect in the A549 and Hela in a dose-dependent manner. CONCLUSION: The alcohol extract of Unprocessed Aconiti Radix, Aconiti Radix Cocta, Phellodendri Chinensis Cortex and the water extraction of Phellodendri Chinensis Cortex present obviously inhibitory effect in both A549 and Hela. The combination of Aconiti Radix with Phellodendri Chinensis Cortex has synergistic inhibitory effect on Hela.

[Site-directed mutagenesis and protein expression of SCN5A gene associated with congenital long QT syndrome].

OBJECTIVE: To construct the sodium channel gene SCN5A-delQKP1507-1509 mutation associated with congenital long QT syndrome, and its eukaryotic expression vector, and to examine the expression of mutation protein in human embryonic kidney (HEK) 293 cells. METHODS: Eukaryotic expression vector PEGFP-delQKP-hH1 for SCN5A-delQKP1507-1509 mutation was constructed by rapid site-directed mutagenesis. HEK293 cells were transfected with the wild or mutant vector using lipofectamine, and then subjected to confocal microscopy. The transfected cells were immunostained to visualize intracellular expression of the mutant molecules. RESULTS: Direct sequence and electrophoresis analysis revealed 9 basic group absences at position 1507-1509. The delQKP1507-1509 mutation eukaryotic expression vector was expressed in HEK293 cells. Immunostaining of transfected cells showed the expression of both wild type and mutant molecules on the plasma membrane and there was no difference in the amount of protein, which suggested that the mutant delQKP1507-1509 did not impair normal protein expression in HEK293 cells. CONCLUSIONS: Successful construction of mutant SCN5AdelQKP1507-1509 eukaryotic expression vector and expression of SCN5A protein in HEK293 cells provides a basis for further study on the functional effects of congenital long QT syndrome as a cause of SCN5A mutation.