OBJECTIVE: To explore the expression of the RNA-binding protein tristetraprolin in lung adenocarcinoma cells and its molecular mechanism for inhibiting autophagy. METHODS: Quantitative real-time PCR and Western blotting were performed to detect the expression of autophagy-related genes (including Beclin1, LC3-Ⅱ/LC3-Ⅰ and SQSTM1/p62) in cultured lung adenocarcinoma cells at 24, 48 and 72 h after transient transfection with a tristetraprolin-overexpressing plasmid and the empty plasmid. The effects of transfection with the tristetraprolin-overexpressing plasmid and empty plasmids in the presence or absence of tumor necrosis factor- (TNF-) on the expressions of nuclear factor-κB (NF-κB) p65, c-rel, and p50 were examined in lung adenocarcinoma cells using immunofluorescence assay and Western blotting. The cells were also transfected with the IκBα-mut plasmid and the tristetraprolin-overexpressing plasmid, either alone or in combination, and the changes in the expressions of tristetraprolin and autophagy-related genes were detected using RT-qPCR and Western blotting. RESULTS: The expressions of tristetraprolin were significantly reduced at both the mRNA and protein levels in lung adenocarcinoma cells ( < 0.001). Overexpression of tristetraprolin in the cells significantly lowered the expressions of autophagy-related genes Beclin1 and the ratio of LC3-Ⅱ/LC3-Ⅰ at the mRNA and protein levels ( < 0.001), obviously lowered the expressions of NF-κB p65 and c-rel, and almost totally blocked the nuclear translocation of NF-κB p65 and c-rel ( < 0.05); the expression of p50, however, did not undergo significant changes in response to tristetraprolin overexpression ( > 0.05). The inhibitory effect of tristetraprolin overexpression on autophagy was abrogated by transfection of the cells with IκBα-mut plasmid, which blocked the NF-κB signaling pathway. Co-transfection of the cells with IκBα-mut also attenuated the inhibitory effect of tristetraprolin overexpression on Beclin1 and the LC3-Ⅱ/LC3-Ⅰ ratio at both the mRNA and protein levels ( < 0.05). CONCLUSIONS The expression of tristetraprolin is low in lung adenocarcinoma cells. Tristetraprolin overexpression causes inhibition of autophagy in lung adenocarcinoma cells possibly by blocking NF-κB p65 and c-rel nuclear translocation.
Autophagy ; Cell Line ; Humans ; Lung Neoplasms ; NF-kappa B ; Signal Transduction ; Tristetraprolin

Objective To explore the relationship between hemoglobin (Hb) level during the first trimester of pregnancy and gestational diabetes mellitus (GDM). Methods A total of 1 276 participants, who underwent scheduled prenatal examination and normal singleton delivery at the Fifth People′s Hospital of Shanghai and Hospital of Intergrated Chinese and Western Medicine in Minhang District, from January 2016 to May 2018 were included. There were 99 cases of GDM (GDM group) and 1 177 cases of normal (control group) pregnant women.Based on the serum Hb level during the first trimester of pregnancy, participants were divided into three groups, 236 cases of low Hb level group (Hb<110 g/L), 868 cases of normal Hb level group (110 g/L≤Hb<130 g/L), and 172 cases of high Hb level group (Hb≥130 g/L). Maternal clinical data were collected, including Hb level during the first trimester of pregnancy, three-point blood glucose (BG) of oral glucose tolerance test (OGTT) and fasting insulin during the second trimester of pregnancy. Homeostasis model assessment of insulin resistance index (HOMA-IR) and homeostasis model assessment of pancreatic β cell function index (HOMA-β) were used to evaluate insulin resistance and pancreatic β cell function. Results (1) Hb level during the first trimester of pregnancy in GDM group was significantly higher than that in control group [(123±10),(119±11) g/L, P<0.05]. There were no significant difference in gravidity, parity, index of liver and renal function (all P>0.05). (2) Pre-pregnancy body mass index (BMI), 1-hour BG and 2-hour BG of OGTT were significantly increased in the high Hb level group during the first trimester of pregnancy, which were (23±4) kg/m2, (7.3±2.0) mmol/L, and (6.5±1.4) mmol/L (P<0.05), respectively. The pre-pregnancy BMI, 1-hour BG and 2-hour BG of the normal or low Hb level group were (22±3) kg/m2, (6.7±1.6) mmol/L, (6.1± 1.2) mmol/L; (22±3) kg/m2, (6.5±1.5) mmol/L, (5.9±1.1) mmol/L, respectively. There were no statistically significant difference in levels of fasting blood glucose, fasting insulin, HOMA-IR and HOMA-β within 3 groups (all P>0.05). (3) In the high Hb level group, prevalence of pregnancy overweight or obesity and GDM were the highest, which were 37.2%(64/172) and 15.1%(26/172), respectively; the differences were statistically significant (all P<0.05). (4) The serum Hb level in the first trimester was positively related with pre-pregnancy BMI (r=0.130, P<0.05), 1-hour BG (r=0.129, P<0.05), 2-hour BG (r=0.134, P<0.05), fasting insulin (r=0.096, P<0.05), and HOMA-IR (r=0.101, P<0.05).Logistic regression indicated that Hb≥130 g/L during the first trimester of pregnancy was an independent risk factor for GDM ( OR=2.799, 95% CI :1.186-6.604; P<0.05). Conclusion The high level of Hb (Hb≥130 g/L) during the first trimester of pregnancy is associated with GDM.

Objective: To explore the relationship between hemoglobin (Hb) level during the first trimester of pregnancy and gestational diabetes mellitus (GDM). Methods: A total of 1 276 participants, who underwent scheduled prenatal examination and normal singleton delivery at the Fifth People's Hospital of Shanghai and Hospital of Intergrated Chinese and Western Medicine in Minhang District, from January 2016 to May 2018 were included. There were 99 cases of GDM (GDM group) and 1 177 cases of normal (control group) pregnant women.Based on the serum Hb level during the first trimester of pregnancy, participants were divided into three groups, 236 cases of low Hb level group (Hb<110 g/L), 868 cases of normal Hb level group (110 g/L≤Hb<130 g/L), and 172 cases of high Hb level group (Hb≥130 g/L). Maternal clinical data were collected, including Hb level during the first trimester of pregnancy, three-point blood glucose (BG) of oral glucose tolerance test (OGTT) and fasting insulin during the second trimester of pregnancy. Homeostasis model assessment of insulin resistance index (HOMA-IR) and homeostasis model assessment of pancreatic β cell function index (HOMA-β) were used to evaluate insulin resistance and pancreatic β cell function. Results: (1) Hb level during the first trimester of pregnancy in GDM group was significantly higher than that in control group [(123±10),(119±11) g/L, P<0.05]. There were no significant difference in gravidity, parity, index of liver and renal function (all P>0.05). (2) Pre-pregnancy body mass index (BMI), 1-hour BG and 2-hour BG of OGTT were significantly increased in the high Hb level group during the first trimester of pregnancy, which were (23±4) kg/m², (7.3±2.0) mmol/L, and (6.5±1.4) mmol/L (P<0.05), respectively. The pre-pregnancy BMI, 1-hour BG and 2-hour BG of the normal or low Hb level group were (22±3) kg/m², (6.7±1.6) mmol/L, (6.1±1.2) mmol/L; (22±3) kg/m², (6.5±1.5) mmol/L, (5.9±1.1) mmol/L, respectively. There were no statistically significant difference in levels of fasting blood glucose, fasting insulin, HOMA-IR and HOMA-β within 3 groups (all P>0.05). (3) In the high Hb level group, prevalence of pregnancy overweight or obesity and GDM were the highest, which were 37.2%(64/172) and 15.1%(26/172), respectively; the differences were statistically significant (all P<0.05). (4) The serum Hb level in the first trimester was positively related with pre-pregnancy BMI (r=0.130, P<0.05), 1-hour BG (r=0.129, P<0.05), 2-hour BG (r=0.134, P<0.05), fasting insulin (r=0.096, P<0.05), and HOMA-IR (r=0.101, P<0.05).Logistic regression indicated that Hb≥130 g/L during the first trimester of pregnancy was an independent risk factor for GDM (OR=2.799, 95%CI: 1.186-6.604; P<0.05). Conclusion The high level of Hb (Hb≥130 g/L) during the first trimester of pregnancy is associated with GDM.

Objective To establish an intestinal organoid-based assay to investigate the radiation mitigation effect of epiregulin in vitro. Methods Intestinal crypts were released from tissue incubated with EDTA. Intestinal crypts seeded in 3D matrigel were irradiated at 24 h after plating. The radiation mitigation effect of epiregulin was evaluated by measuring the survival rate, size and budding numbers of the organoid after irradiation, and the basic FGF was used as a positive control of epiregulin. Results Radiation-induced lethality and dose-dependent survival curve of the intestinal organoid were consistent with in vivo data. Treatment with epiregulin (400 ng/ml) at 24 h post-radiation significantly increased survival rate of 8 Gy X-ray irradiated intestinal organoid in comparison with non-treated group [(12.56 ± 1.02)％vs. (4.73 ± 0.38)％, t=12.43,P<0.05]. Conclusions Epiregulin has radiation mitigation effect on intestinal organoid and could serve as a potential medical countermeasure to mitigate gastrointestinal toxicity.

Objective: To evaluate the in vitro antibiotic effects of colistin combined with meropenem, levofloxacin or fosfomycin against colistin-heteroresistant Acinetobacter baumannii (A.baumannii). Methods: A total of 576 A. baumannii clinical isolates were collected from the First Affiliated Hospital of Wenzhou Medical University from 2014 to 2015. Minimal inhibitory concentrations (MICs) of colistin against A. baumannii were detected by broth dilution method. Colistin-heteroresistant A. baumannii isolates were screened using population analysis profiles (PAPs). MICs of colistin combined with meropenem, levofloxacin or fosfomycin, and the four drugs used alone against colistin-heteroresistant A. baumannii were detected by checkerboard method and broth dilution method. Fractional inhibitory concentration index (FICI) was calculated to evaluate antibiotic effects. Results: None of the 576 A. baumannii isolates was resistant to colistin as indicated by the broth dilution method. Nine colistin-heteroresistant A. baumannii isolates were identified using PAPs. Compared with the MICs of colistin used alone, the MICs of colistin used in combination with meropenem, levofloxacin or fosfomycin against colistin-heteroresistant isolates were all decreased. Colistin-meropenem combination showed synergistic (55.6%), additive (33.3%) and indifferent effects (11.1%), but no antagonistic effect. Colistin-levofloxacin combination showed synergistic (55.6%), additive (22.2%) and indifferent effects (22.2%), but no antagonistic effect. Colistin-fosfomycin combination showed synergistic (77.8%) and additive (22.2%) effects, but no indifferent or antagonistic effect. Conclusion In vitro use of colistin in combination with meropenem, levofloxacin or fosfomycin has synergistic and additive antibacterial effects against colistin-heteroresistant A. baumannii. Combinations of colistin-meropenem and colistin-levofloxacin have fewer indifferent effects and no antagonistic effect.

In the presence of silver ion, Mn2+ could be electro-oxidized to potassium hypermanganate in phosphoric acid solution, which could effectively react with pyrocatechol in acid solution and luminol in sodium hydroxide solution to produce chemiluminescence. On the basis of this, a novel indirect approach for the detection of Mn2+ was established. The effect of silver ions on the electrochemical oxidation of Mn2+was studied. when 1. 5 ×10-5 mol/L Ag+ and 0. 01 mol/L phosphoric acid solution were used in the process of electrochemical oxidation, the CL intensity could be up to the maximum value after the above solution was electrolyzed for 2 min. The relation of CL intensity and Mn2+concentration in the solutions at different pH and the selectivity were also investigated. when the pyrocatechol was used as luminescent reagent in the acidic medium, the CL intensity was linearly to the Mn2+concentration in the range of 1. 82×10-7-7. 27×10-5 mol/L with excellent selectivity. Common ions had little interferences in the determination of Mn2+. The method was successfully applied to the determination of Mn2+ in surface water and drinking water with satisfactory results.

Objective To investigate the isolation rate, distribution and trend of nontuberculous mycobacteria (NTM) in Wenzhou during 2014 to 2016.Methods Sputum or alveolar lavage specimens of patients with suspected pulmonary tuberculosis were collected for mycobacteria culture from January 2014 to December 2016.Mycobacterium culture positive strains were further identified by gene chip, 16S rRNA and hsp65 gene sequencing.SPSS 19.0 software was used to analyze the data.Results After excluding repetitive strains, 3 295 mycobacteria strains (MTB) were isolated from respiratory specimens, included 3 032 mycobacterium tuberculosis complex strains, 238 NTM strains, 20 Gordon genera strains, 3 Nocardia genera strains and 2 Tsukamurella genera strains.The proportion of NTM among confirmed mycobacteria was 8.5％ (86/1 006), 6.7％ (72/1 079) and 6.8％ (80/1 185) in 2014, 2015 and 2016, respectively (x2 =2.459,P ＞ 0.05).The overall prevalence of NTM was 7.3 ％ (238/3 270).There were 15 species of NTM, and the most common NTM strain was Mycobacterium intracellulare (52.5％,125/238), followed by Mycobacterium abscessus (22.7％, 54/238) and Mycobacterium avium (10.1％, 24/238), other species were only accounted for 14.7％ (35/238).The ranking of Mycobacterium avium went up rapidly from the fifth in 2014 to the second in 2016 (x2 =18.259, P ＜ 0.01), while proportion of Mycobacterium abscess, dropped from 34.9％ (30/86) in 2014 to 17.5％ (14/80) in 2016 (x2 =7.335, P＜0.01).Among patients from whom the NTM strains were isolated, 56.7％ (135/238) were male and most of them were aged 45 years or above (79.8％, 190/238).Conclusions In the past three years, the trend of NTM isolation rate in Wenzhou is steady.The most prevalent NTM species is Mycobacterium intracellulare, followed by Mycobacterium abscessus and Mycobacterium avium.Mycobacterium avium shows a continuously upward trend, while the separation of Mycobacterium abscessus shows a downward trend.

Ranpirnase (onconase, ONC) is a new drug, with weak RNase activity and strong cytotoxicity to various tumor cells in vitro and in vivo. This study is to obtain recombination onconase (rONC) with high bioactivity. Based on the codon preference of Pichia pastoris, we designed and synthesized the gene according to cDNA sequences of ONC and the α mating factor's prepeptide. We screened positive clones after transforming the recombination plasmids into P. pastoris X-33, GSS115 and SMD1168. We screened the best combination of seven different vectors and host strains. Moreover, we optimized culture condition in shake flasks and 10 L bioreactor, and purified rONC from the supernatant after inducing it with 0.25% methanol by aqueous two-phase extraction coupling G50 molecular exclusion method. The highest rONC production was 13 mg/L in pPICZα-A/X-33/ONC combination under the condition of pH 5.5 and 23 degrees C in shake flasks for 7 d; and that the highest rONC production was 180 mg/L when the induction is performed in the lower basic salt medium with pH 5.5 in the 10 L bioreactor for 7 d. The yield of rONC is more than 90% at a purity of above 95%. rONC can kill various tumor cells in vitro. The expression and purification of rONC would be useful for further investigation of this new drug.
Antineoplastic Agents ; metabolism ; Bioreactors ; Cell Line, Tumor ; Codon ; DNA, Complementary ; Genetic Vectors ; Humans ; Pichia ; metabolism ; Recombinant Proteins ; biosynthesis ; Ribonucleases ; biosynthesis

OBJECTIVETo investigate the molecular?mechanisms by which triptolide induces apoptosis of human acute T lymphocytic leukemia Jurkat cells.

METHODSMTT assay was employed to detect the proliferation inhibition of Jurkat cells by triptolide, and the IC50 was calculated by OriginPro8. Flow cytometry was used to analyze apoptosis of Jurkat cells. Np9 mRNA levels were detected by RT-PCR and analyzed quantitatively by Kodak 1D 3.6 software. Correlation between the inhibition of Np9 transcription and the cell apoptosis was analyzed by SPSS 19.0.Western blotting was employed to determine Np9 downstream signaling molecules c-myc, β-catenin, ERK, AKT and Notch1 protein level in Jurkat cells after exposure to different concentrations of triptolide for 48 h.

RESULTSTriptolide treatment resulted in dose-dependent inhibition of Jurkat cells proliferation and its IC50 was 12.7 nmol/L. Triptolide induced apoptosis of Jurkat cells in dose- dependent manner. Furthermore, triptolide inhibited Np9 mRNA transcription level in Jurakt cells in a dose-dependent manner. There was a correlation between the triptolide-mediated the apoptosis and the inhibition of Np9 transcription of Jurkat cells (R(2)=0.907). Western blotting results displayed that triptolide inhibited transcription levels of Np9 mRNA with a concomitant decrease of its downstream signaling molecules c-myc, β-catenin, ERK, AKT and Notch1 at protein levels.

CONCLUSIONInhibition of HERV-K Np9 mRNA and its downstream signaling molecules c-myc, β-catenin, ERK, Akt and Notch1 protein might be one of important molecular?mechanisms by which triptolide induces apoptosis of human acute T lymphocytic leukemia Jurkat cells.

Apoptosis ; drug effects ; Diterpenes ; pharmacology ; Endogenous Retroviruses ; genetics ; Epoxy Compounds ; pharmacology ; Flow Cytometry ; Gene Products, env ; genetics ; Humans ; Jurkat Cells ; drug effects ; Phenanthrenes ; pharmacology ; Precursor T-Cell Lymphoblastic Leukemia-Lymphoma ; pathology ; Transcription, Genetic