The biotechnology of glycolipid fermented b y a bacillus coagulans was studied and the fermentation pro cess in 10L bioreactor was conducted.Suitable medium contained 6% bean oil as ca rbon source,3 5g/L NaNO 3 as nitrogen source,0 75g/L yeast extract and some i no rganic salts.The fermentation temperature of 30℃,initial pH of 8 5,strring rev olution of 150～240r/min and fermentation period of 96h proved to be optimal.The yield of glycolipid in 10L bioreactor was 7 073g/L.

OBJECTIVETo investigate the role of Bcl-2 in the cytoprotective effect of thyroid hormone against hippocampal cell apoptosis in rats with chronic cerebral ischemia.

METHODSFifty adult male SD rats were randomized into sham-operated group, 2-vessel occlusion (2VO) group and triiodothyronine (T3) treatment group. At 7 and 14 days after the operation, the tissue structure of the CA1 region was observed with Nissl staining, and TUNEL staining was used to determine the apoptosis index (AI) in the dentate gyrus; Western blotting was performed to detect the expression level of Bcl-2 in the hippocampus.

RESULTSIn the 2VO group, the CA1 region of the hippocampus showed obvious structural damages with reduced number of neurons, and these changes were significantly improved in T3 treatment group. At 7 days after the operation, no significant difference was found in AI between the sham-operated group (17.714∓2.553), 2VO group (20.868∓2.090) and T3 group (20.365∓1.055) (P=0.060); the expression level of Bcl-2 was higher in T3 group than in 2VO group. On day 14, AI was 66.532∓3.249 in 2VO group, significantly higher than that in T3 treatment group (56.153∓4.556, P=0.001); Bcl-2 expression was the highest in T3 group and the lowest in 2VO group.

CONCLUSIONThyroid hormone can reduce cell apoptosis in the hippocampus of rats with chronic cerebral ischemia possibly by up-regulating the expression of Bcl-2.

Animals ; Apoptosis ; drug effects ; Brain Ischemia ; pathology ; CA1 Region, Hippocampal ; cytology ; pathology ; Male ; Neurons ; cytology ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Rats ; Rats, Sprague-Dawley ; Triiodothyronine ; pharmacology

As a therapeutic agent in thrombosis the fibrinolytic enzymes are of interest and the search for a new enzyme continues. A novel fibrinolytic enzyme was produced from Rhizopus chinensis 120, which was screened from the starter for brewing rice wine in the South of China, by solid fermentation, and purified through ammonium sulfate precipitation, hydrophobic interaction, ionic exchange and gel filtration chromatographies. The purified enzyme hydrolyzed fibrin, it cleaved the alpha-, beta- and gamma-chains of fibrinogen simultaneously, and it also activated plasminogen to plasmin. The enzyme hydrolyzed N-Succinyl-Ala-Ala- Pro-Phe-pNA, and Km was 0.23 mmol/L and Kcat 16.36 s(-1). The optimal temperature of the enzyme for hydrolying fibrin was 45 degrees C, and the optimal pH range of 6.8 - 8.8. The isoelectric point of the enzyme estimated by isoelectric focusing electrophoresis was 8.5 +/- 0.1. The enzyme was a glycoprotein. EDTA, PCMB, PMSF inhibited the activety of the enzyme, and SBTI, Lys, TPCK, Aprotinine had none obvious inhibition, which suggested that the activity centre of the enzyme had hydrosulfuryl, metal and serine. The first 12 amino acids of the N-termimal sequence of the enzyme were NH2-Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly, and had none homology with that of other fibrinolytic enzyme from other microbes. The novel fibrinolytic enzyme from Rhizopus chinensis 12# has potential to become a therapeutic agent in thrombosis.
Enzyme Stability ; Fermentation ; Fibrinolysin ; metabolism ; Fibrinolysis ; Fibrinolytic Agents ; chemistry ; Humans ; Plasminogen ; metabolism ; Rhizopus ; enzymology

OBJECTIVEInflammatory reaction and injury in immature lungs are associated with activation of nuclear factor-kappa B (NF-kappaB) to trigger proinflammatory cytokine release, but the mechanism thereof is not fully understood. The present study was conducted to understand possible relationship between expression of NF-kappaB and its inhibitor and severity and outcome of neonates with hyaline membrane disease (HMD).

METHODSSerial samples of bronchoalveolar lavage fluid (BALF) were obtained during mechanical ventilation from 31 preterm infants with HMD. These infants were divided into two groups: survivors group [n = 22, birth weight (1500 +/- 320) g and gestational age (31.2 +/- 1.8) weeks] and nonsurvivors group [birth weight (1340 +/- 280) g, gestational age (30.8 +/- 2.1) weeks]. Nineteen preterm infants [birth weight (1470 +/- 280) g, gestational age (30.6 +/- 1.9) weeks] without respiratory disorders were enrolled as control subjects. Alveolar macrophages (AM) were isolated by differential adherence. AM was cultured and treated with lipopolysaccharide (LPS) for 1 hr. Then, nuclear extracts of AM were analyzed by electrophoretic mobility shift assay (EMSA) for NF-kappaB expression. NF-kappaB inhibitor (IkappaB-alpha protein) in cytoplasmic extracts was detected by using Western blotting and IL-1beta and IL-8 in BALF by enzyme-linked immunosorbent assay (ELISA).

RESULTSNF-kappaB complexes were observed by EMSA, they were characterized by competition with cold oligonucleotide and p65-specific antibodies. The addition of an excess of cold oligonucleotide, corresponding to the NF-kappaB binding site, turned off the signal of the band, showing that the band was specific. An excess of an irrelevant oligonucleotide (corresponding to the SP-1) did not show any effect. The addition of an anti-p65 antibody caused the supershift of the two upper bands. After EMSA, the NF-kappaB complexes were quantified by using a ImageQuant software. NF-kappaB expression in AM at 24 hrs was higher in all the patients with HMD as compared with control subjects (survives/control, 34.1 vs 11.4 RDU, P < 0.01; nonsurvivors/control, 55.2 vs 11.4 RDU, P < 0.01). The NF-kappaB expression in AM at 72 hrs was higher than that in control subjects but not for nonsurvivors (survivors/control, 47.8 vs 25.6 RDU, P < 0.01; nonsurvivors/control, 21.8 vs 25.6, P > 0.05). The NF-kappaB expression in AM from nonsurvivors was depressed at 72 hrs as compared to 24 hrs (21.8 vs 55.2, P < 0.01), whereas the NF-kappaB expression in AM from survivors was still higher at 72 hrs than that at 24 hrs (47.8 vs 34.1, t = 4.43, P < 0.01).

CONCLUSIONAltered NF-kappaB activation in AM of BALF of neonates with HMD was observed, and it may be mediated by decreased IkappaB synthesis, increased IkappaB degradation, or both. In HMD nonsurvivors NF-kappaB translocation was hampered upon LPS activation.

Birth Weight ; Blotting, Western ; Bronchoalveolar Lavage Fluid ; cytology ; Cell Culture Techniques ; Cell Nucleus ; drug effects ; metabolism ; Cytoplasm ; drug effects ; metabolism ; Electrophoretic Mobility Shift Assay ; Enzyme-Linked Immunosorbent Assay ; Female ; Gestational Age ; Humans ; Hyaline Membrane Disease ; immunology ; therapy ; I-kappa B Proteins ; immunology ; Infant, Newborn ; Infant, Premature ; immunology ; Interleukin-1beta ; immunology ; Interleukin-8 ; immunology ; Lipopolysaccharides ; pharmacology ; Macrophages, Alveolar ; drug effects ; immunology ; Male ; NF-KappaB Inhibitor alpha ; NF-kappa B ; immunology ; Respiration, Artificial ; Severity of Illness Index ; Time Factors

Cardiac Catheterization ; Heart Arrest ; therapy ; Humans ; Male ; Middle Aged ; Pulmonary Embolism ; complications ; therapy ; Thrombolytic Therapy ; methods

OBJECTIVETo study the effect of RNA interference (RNAi)-mediated aggrecanase-1 gene silencing on extracellular matrix metabolism of cultured rat costochondral chondrocytes.

METHODSRat costochondral chondrocyte monolayers were obtained by microdissection and digestion. The growth and morphological changes of the chondrocytes were observed after RNAi of aggrecanase-1 gene. The mRNA expression of aggrecanase-1 was detected by RT-PCR method, and aggrecan content was determined by Western blotting.

RESULTSThe specific inhibition of aggrecanase-1 by RNAi produced no adverse effect on the morphology and growth of the chondrocytes. The mRNA of aggrecanase-1 decreased and aggrecan content increased significantly after transfection of the chondrocytes.

CONCLUSIONInhibition of aggrecanase-1 decreases aggrecan degradation in cultured rat chondrocytes. RNAi technique can be a useful means for studying extracellular matrix metabolism in the cartilage.

ADAM Proteins ; genetics ; metabolism ; ADAMTS4 Protein ; Aggrecans ; metabolism ; Animals ; Cells, Cultured ; Chondrocytes ; cytology ; metabolism ; Extracellular Matrix ; metabolism ; Female ; Procollagen N-Endopeptidase ; genetics ; metabolism ; RNA Interference ; RNA, Messenger ; genetics ; Rats ; Rats, Sprague-Dawley ; Transfection

Objective This study aimed to comprehensively analyze and compare the clinicopathological features and prognosis of Chinese patients with human epidermal growth factor receptor 2(HER2)-low early breast cancer(BC)and HER2-IHC0 BC. Methods Patients diagnosed with HER2-negative BC(N=999)at our institution between January 2011 and December 2015 formed our study population.Clinicopathological characteristics,association between estrogen receptor(ER)expression and HER2-low,and evolution of HER2 immunohistochemical(IHC)score were assessed.Kaplan-Meier method and log-rank test were used to compare the long-term survival outcomes(5-year follow-up)between the HER2-IHC0 and HER2-low groups. Results HER2-low BC group tended to demonstrate high expression of ER and more progesterone receptor(PgR)positivity than HER2-IHC0 BC group(P<0.001).The rate of HER2-low status increased with increasing ER expression levels(Mantel-Haenszel χ2 test,P<0.001,Pearson's R=0.159,P<0.001).Survival analysis revealed a significantly longer overall survival(OS)in HER2-low BC group than in HER2-IHC0 group(P=0.007)in the whole cohort and the hormone receptor(HR)-negative group.There were no significant differences between the two groups in terms of disease-free survival(DFS).The discordance rate of HER2 IHC scores between primary and metastatic sites was 36.84%. Conclusion HER2-low BC may not be regarded as a unique BC group in this population-based study due to similar clinicopathological features and prognostic roles.

ObjectiveUsing Chromium-51 release assay, lactate dehydrogenase release assayand other methods to detect the cytotoxicity of CD19 CAR-T cells is cumbersome, with low repeatability and poor stability. This study aims to establish a label-free and real-time method for detectingspecific cytotoxicity of CD19 CAR-T cells.MethodsIn order to establish target cell models for cytotoxic assay of CD19 CAR-T cells by using Real Time Cellular Analysis (RTCA) system,the adherent human breast cancer cells were infected with lentiviral vectors encoding CD19. CD19 expression on the transduced cells was detected by flow cytometry. The cellsexpressing CD19 stably werethen sorted by fluorescence activated cell sorting (FACS).With such cells as target cells, CD19 CAR-T cells and BCMA CAR-T cells as effector cells, RTCAsystem was used to evaluate the cytotoxicity of CAR-T cells against target cells.ResultsMDA-MB-231 and SKBR3cells with stable expression CD19were obtained in this study.The results of flow cytometry showed that positive expression rate of CD19 in MDA-MB-231/CD19 cells and SKBR3/CD19 monoclonal cells were 99.03% and 98.91%,respectively.RTCA results showed that with MDA-MB-231 and MDA-MB-231/CD19 cells as target cells,CD19 CAR-T cells showed significant cytotoxicity to MDA-MB-231/CD19 cellsat the effector-target ratio of 5∶1, 1∶1 and 1∶5,but not to MDA-MB-231 cells. With SKBR3 and SKBR3/CD19 cells as target cells, CD19 CAR-T cells showed significant cytotoxicity to SKBR3/CD19 cellsat the effector-target ratio of 5∶1and 1∶1. When the effector-target ratio was 1∶5, there was no obvious cytotoxicity.The data of MDA-MB-231/CD19 or SKBR3/CD19 as target cells and CD19 CAR-T as effector cells were analyzed separately, showing that when the number of target cells was the same, the cytotoxicity detected by RTCA increased as the number of CD19 CAR-T cells increased.The cytotoxic assays of CD19 CAR-T cells showed specificity and dose-response relationship of CD19 CAR-T cytotoxicity against the target cells.ConclusionThis study established a method for evaluating cytotoxicity of CD19 CAR-T cells that is real-time, label-free, simple and convenient.

Objective:This study aims to observe the effect of baicalein on the clonal formation of triple negative breast cancer MDA-MB-231 and MDA-MB-468 cells， and to explore the mediation role of Yes- related protein （YAP） in it. Method:MDA-MB-231 and MDA-MB-468 cells were treated with baicalein. Thiazole blue （MTT） colorimetric method was used to detect cell proliferation ability. Plate cloning experiments was used to detect the colony forming ability. Immunofluorescence method was used to detect the nuclear distribution of YAP， and Western blot test was used to detect the protein expression levels of YAP large tumor suppressor factor 1 （LATS1）， YAP， phosphorylated Yes- related protein（p-YAP） and phosphorylated YAP large tumor suppressor factor 1 （p-LATS1）. Result:Compared with the blank group， baicalein （40， 80， 160 μmol·L^-1） significantly inhibited the proliferation ability of MDA-MB-468 and MDA-MB-231 cells （P<0.05， P<0.01）， and the inhibitory effect was dose-dependent. The half inhibit concentration（IC₅₀） of baicalein against MDA-MB-468 and MDA-MB-231 cells were （80.3±7.2），（70.4±6.5） μmol·L^-1， respectively. Compared with blank group， baicalein （5， 10， 20 μmol·L^-1） had no significant effect on the proliferation of MDA-MB-468 and MDA-MB-231 cells， and the difference was not statistically significant. Compared with the blank group， baicalein （5， 10， 20 μmol·L^-1） significantly dose-dependently reduced the cell colony formation rates of MDA-MB-468 and MDA-MB-231 cells （P<0.05， P<0.01）， and baicalein （10， 20 μmol·L^-1） significantly inhibited the nuclear expression of YAP in MDA-MB-468 and MDA-MB-231 cells in a dose-dependent manner（P<0.01）. Also， baicalin （5， 10， 20 μmol·L^-1） significantly up-regulated p-YAP and p-LATS1 protein expressions in MDA-MB-468 cells in a dose-dependent manner （P<0.05， P<0.01）. Baicalein （10， 20 μmol·L^-1） significantly up-regulated p-YAP and p-LATS1 protein expressions in MDA-MB-231 cells in a dose-dependent manner （P<0.01）. Conclusion:Baicalein can inhibit colony formation of triple negative breast cancer MDA-MB-468 and MDA-MB-231 cells by mediating the reduction of YAP entry into the nucleus.

OBJECTIVE: To investigate the current status of antibiotic use for very and extremely low birth weight (VLBW/ELBW) infants in neonatal intensive care units (NICUs) of Hunan Province. METHODS: The use of antibiotics was investigated in multiple level 3 NICUs of Hunan Province for VLBW and ELBW infants born between January, 2017 and December, 2017. RESULTS: The clinical data of 1 442 VLBW/ELBW infants were collected from 24 NICUs in 2017. The median antibiotic use duration was 17 days (range: 0-86 days), accounting for 53.0% of the total length of hospital stay. The highest duration of antibiotic use was up to 91.4% of the total length of hospital stay, with the lowest at 14.6%. In 16 out of 24 NICUs, the antibiotic use duration was accounted for more than 50.0% of the hospitalization days. There were 113 cases with positive bacterial culture grown in blood or cerebrospinal fluid, making the positive rate of overall bacterial culture as 7.84%. The positive rate of bacterial culture in different NICUs was significantly different from 0% to 14.9%. The common isolated bacterial pathogens Klebsiella pneumoniae was 29 cases (25.7%); Escherichia coli 12 cases (10.6%); Staphylococcus aureus 3 cases (2.7%). The most commonly used antibiotics were third-generation of cephalosporins, accounting for 41.00% of the total antibiotics, followed by penicillins, accounting for 32.10%, and followed by carbapenems, accounting for 13.15%. The proportion of antibiotic use time was negatively correlated with birth weight Z-score and the change in weight Z-score between birth and hospital discharge (r=-0.095, -0.151 respectively, P<0.01), positively correlated with death/withdrawal of care (r=0.196, P<0.01). CONCLUSIONS Antibiotics used for VLBW/ELBW infants in NICUs of Hunan Province are obviously prolonged in many NICUs. The proportion of routine use of third-generation of cephalosporins and carbapenems antibiotics is high among the NICUs.
Anti-Bacterial Agents ; Birth Weight ; Humans ; Infant ; Infant, Extremely Low Birth Weight ; Infant, Newborn ; Intensive Care Units, Neonatal ; Surveys and Questionnaires