[Effects of PUMA knockout on the apoptosis of H9C2 cardiomyocytes induced by high glucose].

OBJECTIVE: To investigate the effects of p53 upregulated modulator of apoptosis (PUMA) on the apoptosis of H9C2 cardiomyocytes induced by high glucose and its mechanisms. METHODS: H9C2 cardiomyocytes were treated with 5.5mmol/L (control group) or 35 mmol/L glucose (HG group) for 6 h, 12 h, 24 h or 48 h respectively to induce apoptosis, each group sets 5 multiple wells. Apoptosis was tested by TUNEL assay. PUMA mRNA was measured by RT-PCR and protein expression was measured by Western blot assay. The mitochondrial membrane potential was detected by JC-1 method. The expressions of cleaved caspase-3 and cytochrome C (Cyt C) protein in mitochondria and cytoplasm were determined by Western blot assay. H9C2 cardiomyocytes were randomly divided into four groups, control group (5.5 mmol/L), HG (35 mmol/L) group, HG+si-scramble group(si-scramble treatment for 24 h, then 35 mmol/L high glucose treatment for 24 h) and HG-si-PUMA group (si-PUMA treatment for 24 h, then 35mmol/L high glucose treatment for 24 h). Si-PUMA was transfected into cardiomyocytes and the effects of PUMA on high glucose-induced apoptosis were studied. RESULTS: Compared with the control group, high glucose increased cardiomyocyte apoptosis and enhanced PUMA mRNA and protein expressions significantly (P＜0.05 or P＜0.01). Cell injury and increased PUMA expression were time-dependent and there was no significant difference between the high glucose 24 h group and the high glucose 48h group. The following experiment used high glucose 24 h as the stimulation time. The cardiomyocytes transfected with si-PUMA to inhibit PUMA expression had decreased apoptotic rate and cleaved caspase-3, increased mitochondria membrane potential and decreased Cyt C release (P＜0.05 or P＜0.01). There were no significant differences between the HG+si-scramble group and the high glucose group (P＞0.05). CONCLUSION: PUMA mediates high glucose-induced cardiomyocyte apoptosis suggesting PUMA may be an important target gene of diabetic cardiomyopathy.

[Differences of Energy Spectrum CT Findings between Small Cell Lung Cancer with Mediastinal Lymph Node Metastasis and Mediastinal Sarcoidosis].

Objective To compare the differences of energy spectrum CT between small cell lung cancer(SCLC)with mediastinal lymph node metastasis and mediastinal sarcoidosis.Methods Twenty-five SCLC patients with mediastinal lymph node metastasis(SCLC group)and 26 patients with mediastinal sarcoidosis(sarcoidosis group)confirmed by bronchoscopy and biopsy in Tangshan People's Hospital from January 2018 to June 2019 were selected as the research objects.The CT value,iodine concentration,water concentration and energy spectrum curve slope under different single energy levels were compared between SCLC group and sarcoidosis group.Results The single-energy CT values of 40-80 keV segments in the arterial phase of the SCLC group were significantly higher than those in the sarcoidosis group(all P <0.05).The single-energy CT values of 90-140 keV segments were not significantly different from those in the sarcoidosis group(all P >0.05).The single-energy CT values of 40-90 keV segments in venous phase of the SCLC group were significantly higher than those of the sarcoidosis group(all P <0.05),and the single-energy CT values of 100-140 keV segments were not significantly different from those of the sarcoidosis group(all P >0.05).The concentrations of iodine in the arterial phase and venous phase of the SCLC group were(11.56±4.06)µg/cm 3 and(13.39±0.87)µg/cm 3,respectively,which were significantly higher than those [(4.43±3.85)µg/cm 3,t=11.564,P=0.026;(7.23±2.71)µg/cm 3,t=13.653,P=0.021] in the sarcoidosis group.The concentrations of water in the arterial and venous phases of the SCLC group were(1040.67±5.62)mg/cm 3 and(1035.23±8.57)mg/cm 3,respectively,which showed no statistically significant difference compared with those [(1028.87±6.94)mg/cm 3,t=3.155,P=1.861;(1021.53±4.68)mg/cm 3,t=3.265,P=1.687] in the sarcoidosis group.The slopes of energy spectrum curve at 40-70 keV,70-100 keV and 100-140 keV in venous phase of the SCLC group were significantly higher than those of the sarcoidosis group(all P <0.05),whereas they showed no significant difference between the two groups in arterial phase(all P >0.05).Conclusion The differences between SCLC with mediastinal lymph node metastasis and mediastinal sarcoidosis can be shown on the single-energy CT values of 40-80 keV in arterial phase and 40-90 keV in venous phase,iodine concentrations in arterial phase and venous phase,and the slope of energy spectrum curve in venous phase.

[Different Energy Spectrum CT Findings between Anterior Mediastinal Lymphoma and Thymic Carcinoma].

Objective To investigate the differences in energy spectrum CT findings between anterior mediastinal lymphoma and thymic carcinoma. Methods Twenty-two cases of anterior mediastinal lymphoma and 28 cases of thymic carcinoma confirmed by biopsy in Tangshan People's Hospital were selected.The CT values and changes of iodine content and water content in lesion sites were measured by energy spectrum analysis software.The differences between anterior mediastinal lymphoma and thymic carcinoma were compared. Results The single-energy CT value of 40-80 keV in thymus carcinoma was higher than that in anterior mediastinal lymphoma(P=0.001,P=0.037,P=0.042,P=0.034,P=0.002;P=0.016,P=0.013,P=0.018,P=0.024,P=0.012).The difference in the single-energy CT value of 90-110 keV between anterior mediastinal lymphoma and thymic carcinoma showed no statistical significance(all P>0.05).The concentrations of water in the arterial and venous stages of thymic carcinoma were significantly lower than those in the anterior mediastinal lymphoma(P=0.030,P=0.037),whereas the iodine concentrations were significantly higher(P=0.026,P=0.000). Conclusion Anterior mediastinal lymphoma and thymic carcinoma have remarkably different 40-80 keV single energy CT value and iodine concentration in arterial and venous phases,which may be helpful for the differential diagnosis of these two malignancies.

Microbiomes of China's Space Station During Assembly, Integration, and Test Operations.

Sufficient evidence indicates that orbiting space stations contain diverse microbial populations, which may threaten astronaut health and equipment reliability. Understanding the composition of microbial communities in space stations will facilitate further development of targeted biological safety prevention and maintenance practices. Therefore, this study systematically investigated the microbial community of China's Space Station (CSS). Air and surface samples from 46 sites on the CSS and Assembly Integration and Test (AIT) center were collected, from which 40 bacteria strains were isolated and identified. Most isolates were cold- and desiccation-resistant and adapted to oligotrophic conditions. Bacillus was the dominant bacterial genus detected by both cultivation-based and Illumina MiSeq amplicon sequencing methods. Microbial contamination on the CSS was correlated with encapsulation staff activities. Analysis by spread plate and qPCR revealed that the CSS surface contained 2.24 × 103-5.47 × 103 CFU/100 cm2 culturable bacteria and 9.32 × 105-5.64 × 106 16S rRNA gene copies/100cm2; BacLight™ analysis revealed that the viable/total bacterial cell ratio was 1.98-13.28%. This is the first study to provide important systematic insights into the microbiome of the CSS during assembly that describes the pre-launch microbial diversity of the space station. Our findings revealed the following. (1) Bacillus strains and staff activities should be considered major concerns for future biological safety. (2) Autotrophic and multi-resistant microbial communities were widespread in the AIT environment. Although harsh cleaning methods reduced the number of microorganisms, stress-resistant strains were not completely removed. (3) Sampling, storage and analytical methods for the space station were thoroughly optimized, and are expected to be applicable to low-biomass environments in general. Microbiology-related future works will follow up to comprehensively understand the changing characteristics of microbial communities in CSS.

[Analysis of transicriptomes and exploring flavonoid biosynthetic pathway genes in Lithocarpus polystachyus].

The sweet taste and health effect of Lithocarpus polystachyus are mainly related flavonoid. To obtain Lithocarpus transcriptome database and flavonoid biosynthesis-related genes, the RNA-Seq techology (Illumina HiSeq 4000) was used to sequence its transcriptome. Six Gb database was assembled after assembly steps, and 41 043 of L. polystachyus unigenes were obtained. With blasting them with 7 data banks, all unigenes were involved in 51 GO-terms and 237 metabolic pathways. And furthermore 28 genes of the flavonoid biosynthesis-related were found. After using the MicroSatallite, 18 161 SSR were obtained, the single-nucleotide-repeated was the richest at 7 346. These data represent abundant messages about transcripts and provide valuable genome data sources in molecular biology of L. polystachyus.

Sphingosine kinase 1/sphingosine 1-phosphate signalling pathway as a potential therapeutic target of pulmonary hypertension.

Pulmonary hypertension is characterized by extensive vascular remodelling, leading to increased pulmonary vascular resistance and eventual death due to right heart failure. The pathogenesis of pulmonary hypertension involves vascular endothelial dysfunction and disordered vascular smooth muscle cell (VSMC) proliferation and migration, but the exact processes remain unknown. Sphingosine 1-phosphate (S1P) is a bioactive lysophospholipid involved in a wide spectrum of biological processes. S1P has been shown to regulate VSMC proliferation and migration and vascular tension via a family of five S1P G-protein-coupled receptors (S1P1-SIP5). S1P has been shown to have both a vasoconstrictive and vasodilating effect. The S1P receptors S1P1 and S1P3 promote, while S1P2 inhibits VSMC proliferation and migration in vitro in response to S1P. Moreover, it has been reported recently that sphingosine kinase 1 and S1P2 inhibitors might be useful therapeutic agents in the treatment of empirical pulmonary hypertension. The sphingosine kinase 1/S1P signalling pathways may play a role in the pathogenesis of pulmonary hypertension. Modulation of this pathway may offer novel therapeutic strategies.

Prognostic factors influencing clinical outcomes of malignant glioblastoma multiforme: clinical, immunophenotypic, and fluorescence in situ hybridization findings for 1p19q in 816 chinese cases.

Malignant glioblastoma multiforme (GBM) is the most malignant brain tumor and despite recent advances in diagnostics and treatment prognosis remains poor. In this retrospective study, we assessed the clinical and radiological parameters, as well as fluorescence in situ hybridization (FISH) of 1p19q deletion, in a series of cases. A total of 816 patients with GBM who received surgery and radiation between January 2010 and May 2014 were included in this study. Kaplan-Meier survival analysis and Cox regression analysis were used to find the factors independently influencing patient progression free survival (PFS) and overall survival (OS). Age at diagnosis, preoperative Karnofsky Performance Scale (KPS) score, KPS score change at 2 weeks after operation, neurological deficit symptoms, tumor resection extent, maximal tumor diameter, involvement of eloquent cortex or deep structure, involvement of brain lobe, Ki-67 and MMP9 expression level and adjuvant chemotherapy were statistically significant factors (p<0.05) for both PFS and OS in the univariate analysis. Cox proportional hazards modeling revealed that age ≤50 years, preoperative KPS score ≥80, KPS score change after operation ≥0, involvement of single frontal lobe, deep structure involvement, low Ki-67 and MMP9 expression and adjuvant chemotherapy were independent favorable factors (p<0.05) for patient clinical outcomes.

Radixin knockdown by RNA interference suppresses human glioblastoma cell growth in vitro and in vivo.

Radixin, a member of the ERM (ezrin-radixin-moesin) family, plays important roles in cell motility, invasion and tumor progression. It is expressed in a variety of normal and neoplastic cells, including many types of epithelial and lymphoid examples. However, its function in glioblastomas remains elusive. Thus, in this study, radixin gene expression was first examined in the glioblastoma cells, then suppressed with a lentivirus-mediated short-hairpin RNA (shRNA) method.We found that there were high levels of radixin expression in glioblastoma U251cells. Radixin shRNA caused down-regulation of radixin gene expression and when radixin-silenced cells were implanted into nude mice, tumor growth was significantly inhibited as compared to blank control cells or non- sense shRNA cells. In addition, microvessel density in the tumors was significantly reduced. Thrombospondin-1 (TSP-1) and E-cadherin were up-regulated in radixin- suppressed glioblastoma U251 cells. In contrast, MMP9 was down-regulated. Taken together, our findings suggest that radixin is involved in GBM cell migration and invasion, and implicate TSP-1, E-cadherin and MMP9 as metastasis-inducing factors.

[Construction, expression and purification of trichosanthin mutant gene TCS(FYY163-165CSA);].

AIM: To construct and express a trichosanthin (TCS) gene mutant and purify the expressed product in E.coli. METHODS: The potential antigenic determinant was predicted on TCS molecule by computer modeling and induced for site-directed mutation. The gene mutant TCS(FYY163-165CSA); was amplified by PCR using the genomic DNA of Trichosanthes kirilowii as a template and cloned into expression vector pRSET-A, then transfected into E.coli BL21 (DE3) for expression by inducing with IPTG. The expressed product was identified by Western blotting and purified by Ni-NTA affinity column chromatography. RESULTS: The soluble target protein was successfully expressed in E.coli. Homogenous TCS mutant protein was obtained after purification of expressed product. CONCLUSION: The site-directed mutagenesis, expression and purification of TCS provide a new approach for reconstructing TCS.

[Construction and expression of trichosanthin mutant gene TCS(RL28-29CG) and purification of expressed product].

AIM: To construct and express a trichosanthin(TCS)gene mutant and purify the expressed product. METHODS: Predict the potential antigenic determinant on TCS molecule by computer modeling and induce site-directed mutation. Amplify gene mutant TCS(RL28-29CG); by PCR using the genomic DNA of Trichosanthes kirilowii as a template and insert into expression vector pRSET-A, then transform to E.coli BL21(DE3)for expression under induction of IPTG. Purify the expressed product by Ni-NTA afinity column chromatography. RESULTS: The target protein in a soluble form was successfully expressed in E.coli. Homogenous TCS mutant protein was obtained after purification of expressed product. CONCLUSION: TCS mutant gene TCS(RL28-29CG); is succ-essfully constructed and expressed.

Analysis of the immune response to Hantaan virus nucleocapsid protein C-terminal-specific CD8(+) T cells in patients with hemorrhagic fever with renal syndrome.

Hantaan virus (HTNV), the prototype member of the Hantavirus genus in the family Bunyaviridae, causes hemorrhagic fever with renal syndrome (HFRS), which is characterized by capillary leakage, hemorrhage, and renal injury, and is an important public health problem in China. Some kinds of immune cells, particularly CD8(+) T cells, are involved in the pathogenesis of Hantavirus infection. The nucleocapsid protein (NP) of the Hantavirus is the most conserved structural protein and the most abundant viral protein produced during infection. It is one of the important target antigens that induce the CD8(+) T-cell response. In this study, we examined the CD8(+) T-cell response to HTNV NP C-terminal polypeptides. We synthesized 23 overlapping C-terminal polypeptides and detected the antigen-specific CD8(+) T cell response in 15 patients with HFRS. The results demonstrated that there were NP-specific T-cell responses in bulk cultures of peripheral blood mononuclear cells (PBMCs) from 9 of 15 patients. The peptide 51 (aa 301-315: SPSSIWVFAGAPDRC), peptide 60 (aa 355-369: LRKKSSFYQSYLRRT), and peptide 70 (aa 415-429: DVKVKEISNQEPLKL) induced strong CD8(+) T-cell responses. Among them, peptide 70 induced CTL responses in donors 7, 9, and 11, and the strongest responses were seen in donor 11. Depletion of CD8(+) T cells from PBMCs completely abrogated the peptide-specific T-cell response, while depletion of CD4(+) T cells did not diminish the number of IFN-gamma spot-forming cells. These data suggest that infection with HTNV results in CTL responses to immunodominant regions on the NP.

Boty-II, a novel LTR retrotransposon in Botrytis cinerea B05.10 revealed by genomic sequence

Botrytis cinerea is a necrotrophic pathogen causing pre- and post-harvest diseases in at least 235 plant species. It manifests extraordinary genotype and phenotype variation. One of the causes of this variation is transposable elements. Two transposable elements have been discovered in this fungus, the retrotransposon (Boty), and the transposon (Flipper). In this work, two complete (Boty-II-76 and Boty-II-103) and two partial (Boty-II-95 and Boty-II-141) long terminal repeat (LTR) retrotransposons were identified by an in silico genomic sequence analysis. Boty-II-76 and Boty-II-103 contain 6439 bp nucleotides with a pair of LTRs at both ends, and an internal deduced pol gene encoding a polyprotein with reverse transcriptase and DDE integrase domains. They are flanked by 5 bp direct repeats (ACCAT, CTTTC). In Boty-II-141, two LTRs at both ends, and a partial internal pol gene encoding a protein with a DDE integrase domain were identified. In Boty-II-95, a right LTR and a partial internal pol gene encoding a protein with no conserved domains were identified. Boty-II uses a self-priming mechanism to initiate synthesis of reverse transcripts. The sequence of the presumed primer binding site for first-strand reverse transcription is 5’-TTGTACCAT-3’. The polypurine-rich sequence for plus-strand DNA synthesis is 5’-GCCTTGAGCGGGGGGTAC-3’. Fourteen Boty-II LTRs that contain 125-158 bp nucleotides and share 69.1 ~ 100 percent identities with the short inverted terminal repeats of 5 bp (TGTCA…TGACA) were discovered. Analysis of structural features and phylogeny revealed that Boty-II is a novel LTR retrotransposon. It could potentially be used as a novel molecular marker for the investigation of genetic variation in B. cinerea.

Two LTR retrotransposon elements within the abscisic acid gene cluster in Botrytis cinerea B05.10, but not in SAS56

The plant hormone abscisic acid has huge economic potential and can be applied in agriculture and forestry for it is considered to be involved in plant resistance to stresses such as cold, heat, salinity, drought, pathogens and wounding. Now overproducing strains of Botrytis cinerea are used for biotechnological production of abscisic acid. An LTR retrotransposon, Boty-aba, and a solo LTR were identified by in silico genomic sequence analysis, and both were detected within the abscisic acid gene cluster in B. cinerea B05.10, but not in B. cinerea SAS56. Boty-aba contains a pair of LTRs and two internal genes. The LTRs and the first gene have features characteristic of Ty3/gypsy LTR retrotransposons. The second gene is a novel gene, named brtn, which encodes for a protein (named BRTN) without putative conserved domains. The impressive divergence in structure of the abscisic acid gene clusters putatively gives new clues to investigate the divergence in the abscisic acid production yields of different B. cinerea strains.

Hantaan virus induces toll-like receptor 4 expression, leading to enhanced production of beta interferon, interleukin-6 and tumor necrosis factor-alpha.

Hantaan virus (HTNV) infects endothelial cells and is associated with increased vascular permeability during hemorrhagic fever with renal syndrome (HFRS). The pattern of increased vascular permeability is mediated by immune response. Therefore, it is necessary to characterize the mechanism of HTNV involvement in the host's innate immune. In this study, the expression of five toll-like receptors (TLRs) was analyzed in Endothelial vein cells (EVC-304) following HTNV infection in vitro. TLR4 showed an altered expression after HTNV infection. HTNV infection significantly increased IFN-beta, IL-6 and TNF-alpha secretion from EVC-304 cells, particularly after lipopolysaccharide stimulation. The increased IFN-beta, IL-6 and TNF-alpha production was mediated by TLR4 induction, since the introduction of the small interfering RNA against TLR4 specifically inhibited the HTNV-induced cytokine production. In conclusion, HTNV infection directly induces TLR4 expression and thereby enhanced production of IFN-beta, IL-6 and TNF-alpha, which may contribute to the host's innate immune response.

Cloning and characterization of farnesyl pyphosphate synthase gene from the ABA-producing fungi Botrytis cinerea.

Farnesyl pyphosphate synthase (FPPS) catalyzes the systhesis of farnesyl pyphosphate and appears to be a promising regulation site of Abscisic acid (ABA) biosynthesis pathway in fungi. Here we reported the isolation and characterization of Botrytis cinerea (FPPS) gene. The cloned FPPS gene carries an open reading frame of 1044-bp encoding a deduced protein of 347 amino acids with a molecular weight of 39.83 kDa, and the coding region is interrupted with a 63-bp intron. Comparison analysis showed that the deduced amino acids sequence share high similarity with other known FPPS gene. Southern blot revealed a single copy of FPPS gene in the genomic DNA. The result of transcription analysis indicated that the cloned FPPS gene expressed constitutively and was not induced in ABA accumulation phase.

[Photochemical degradation of petroleum on soil surfaces under simulated visible light].

The factors influencing photochemical degradation of petroleum on soil surfaces under simulated visible light, such as the initial oil concentrations, soil types and pH, were investigated. The concentrations of petroleum in photolytic processes were analyzed by UV-Vis. GC-FID and FTIR were used for the characterization of petroleum hydrocarbons. Experimental results indicated that photochemical degradation of petroleum on soil surfaces followed pseudo first-order kinetics with the rate constant of 0.0026-0.0172 h(-1) and half-life of 40-267 h. GC-FID analysis showed that, after 50 h of irradiation, the content of saturated hydrocarbons with longer chain decreased, while the proportion of shorter chain ones increased slightly; in addition, most chromatogram peaks of aromatics weakened or disappeared. After 60 h of photolysis, the appearance of carbonyl compounds in FTIR spectra of the extract from soil demonstrated that petroleum was gradually oxidized in the photolytic process.

[The interaction of the intracellular domain of beta-amyloid precursor protein with JKTBP2].

APP (beta-amyloid precursor protein) plays an important role in the formation of Alzheimer's Disease (AD), and its proteolytic product, the intracellular domain AID is believed to be involved in this process by promoting cell apoptosis. To further understand the function of AID in the pathology of AD, we utilized AID as a bait to identify AID interacting proteins in a yeast two-hybrid system. One of the positive clones encodes the fragment corresponding to amino acids 90-204 of heterogeneous nuclear ribonucleoprotein D-like JKTBP2. The interaction between JKTBP2(90)-204 and AID was further confirmed by co-immunoprecipitation in the mammalian 293T cells. These results indicate that JKTBP2 may have an important function in AD formation.