OBJECTIVE To investigate the metabolic changes in MEG-01 megakaryocytes after treatment with linezolid （LZD） from metabolomic perspective and its impact on the the proliferation of cells and generation of platelet precursors. METHODS MEG-01 cells were seeded in proliferation medium and divided into blank control group （untreated）， solvent control group （4‰DMSO）， and 100， 200， 400， 800 μg/mL LZD groups. The proliferation status of cells in each group was observed under the microscope； cell proliferation and viability were assessed. Cells were also seeded in differentiation medium and divided into blank control group （untreated）， solvent control group （4‰DMSO）， and 400 μg/mL LZD group； after 14 days of culture， platelet precursor generation was observed under the microscope； immunofluorescence staining was performed to count the proportion of cells producing pseudopodia， the relative length of pseudopodia was measured， and the expression levels of CD41 and CD42b mRNA were assessed. Cells from the solvent control group and the 400 μg/mL LZD group， cultured in differentiation medium for 14 days， were extracted and subjected to non-targeted metabolomics and targeted energy metabolomics analysis using liquid chromatography-tandem mass spectrometry. The relative content of pyruvate in cells， after being cultured for 14 days with the addition of pyruvate （10 mmol/L） or LZD （400 μg/mL）+pyruvate （10 mmol/L）， was measured and observed， as well as its effects on cell proliferation and platelet precursor generation. RESULTS 400 μg/mL LZD could significantly inhibit the proliferation of MEG-01 cells and the generation of platelet precursors （P＜0.01）. Non-targeted metabolomic analysis of MEG-01 cells after 400 μg/mL LZD treatment revealed significant changes in energy metabolism-related pathways such as mTOR signaling pathway， alanine， aspartate and glutamate metabolism， and central carbon metabolism in cancer. Targeted energy metabolomic analysis further showed that the relative contents of adenosine triphosphate， guanosine triphosphate， and pyruvate in MEG-01 cells were significantly reduced （P＜0.01）， while the relative contents of lactate were significantly increased （P＜0.01）. Compared with the LZD group， the relative content of pyruvate， cell count， the proportion of cells producing pseudopodia and the relative length of pseudopodia were significantly increased in the LZD+pyruvate group （P＜0.01）. CONCLUSIONS LZD may reduce pyruvate production by inhibiting mitochondrial energy metabolism， thereby suppressing megakaryocyte proliferation and platelet precursor production， ultimately leading to the occurrence of thrombocytopenia.

Objective:To investigate the effects of Anhydroicaritin (AHI) , an isopentenylated flavo-noid compound, on proliferation, migration and apoptosis of human hepatocarcinoma cell line MHCC-97H.Methods:Human hepatocarcinoma cell line MHCC-97H and human normal liver cell line L02 were cultured in vitro. MHCC-97H cells were treated with 0, 20, 40, 80, 120, 160, 200 μg/ml of AHI respectively and L02 cells were treated with 0, 25, 50, 100, 150, 200, 400, 500 μg/ml of AHI respectively. CCK-8 and clone formation assay were used to detect cell proliferation. Scratch test was used to explore cell migration ability. Hoechst33342 assay and flow cytometer were used to detect cell apoptosis. The expressions of apoptosis-related proteins were detected by Western blotting. Results:The cell viabilities of MHCC-97H cells treated with 0, 20, 40, 80, 120, 160, 200 μg/ml of AHI for 24 h were (100.00±0.00) %, (97.41±2.10) %, (96.58±3.23) %, (87.72±4.85) %, (78.33±3.76) %, (56.97±2.61) % and (15.25±2.51) % respectively, and there was a statistically significant difference ( F=429.20, P<0.001) . There were statistically significant differences between 0 μg/ml and 80, 120, 160, 200 μg/ml of AHI treatment (all P<0.001) . The cell viabilities of L02 cells treated with 0, 25, 50, 100, 150, 200, 400, 500 μg/ml of AHI for 24 h were (100.00±0.00) %, (96.82±3.79) %, (95.36±3.43) %, (90.79±5.75) %, (77.67±5.66) %, (63.98±5.22) %, (34.22±4.01) % and (33.84±4.41) % respectively, and there was a statistically significant difference ( F=233.20, P<0.001) . There were statistically significant differences between 0 μg/ml and 100, 150, 200, 400, 500 μg/ml of AHI treatment (all P<0.05) . The 24 h half maximal inhibitory concentration (IC 50) value of AHI treated L02 cells was (300.20±17.10) μg/ml, which was significantly higher than that of MHCC-97H cells [ (158.60±5.50) μg/ml], and there was a statistically significant difference ( t=13.65, P<0.001) . The cell clone numbers of MHCC-97H cells treated with 0, 120, 160 and 200 μg/ml of AHI for 24 h were 1 993.00±46.29, 1 355.00±54.84, 998.33±21.03 and 218.33±35.95 respectively, and there was a statistically significant difference ( F=954.80, P<0.001) . There were statistically significant differences between 0 μg/ml and 120, 160, 200 μg/ml of AHI treatment (all P<0.001) . The healing rates of MHCC-97H cells treated with 0, 120, 160 and 200 μg/ml of AHI for 24 h were (51.68±1.93) %, (16.04±0.73) %, (8.88±0.31) % and (-6.94±0.46) % respectively, and there was a statistically significant difference ( F=1 616.00, P<0.001) . There were statistically significant differences between 0 μg/ml and 120, 160, 200 μg/ml of AHI treatment (all P<0.001) . Hoechst33342 experiment showed that MHCC-97H cells treated with 0 μg/ml AHI showed uniform dark blue with a complete nuclear state under inverted microscope. Compared with 0 μg/ml AHI treated cells, cells in the 120, 160, 200 μg/ml AHI treatment groups wrinkled and broken, and nuclei were also morphologically abnormal, with some nuclei stained bright blue, and the situation became more obvious with increasing dose. The apoptosis rates of MHCC-97H cells treated with 0, 120, 160 and 200 μg/ml AHI for 24 h were (10.51±0.56) %, (42.23±0.87) %, (61.92±0.52) % and (72.05±0.74) % respectively, and there was a statistically significant difference ( F=4 677.00, P<0.001) . There were statistically significant differences between 0 μg/ml and 120, 160, 200 μg/ml of AHI treatment (all P<0.001) . There were statistically significant differences among the different expression levels of Bax, Cleaved Caspase-3/Caspase-3, Cleaved Caspase-9/Caspase-9, and Bcl-2 proteins in MHCC-97H cells of 0, 120, 160, and 200 μg/ml of AHI treatment ( F=30.43, P<0.001; F=212.80, P<0.001; F=475.30, P<0.001; F=10.75, P=0.004) . The Bax protein expression of 160 and 200 μg/ml was significantly increased than that of 0 μg/ml AHI (both P<0.001) . The Cleaved Caspase-3/Caspase-3, Cleaved Caspase-9/Caspase-9 protein expressions of 120, 160 and 200 μg/ml were significantly increased than those of 0 μg/ml AHI (all P<0.001) . The Bcl-2 protein expression of 120, 160, 200 μg/ml was significantly decreased compared with that of 0 μg/ml AHI (all P<0.05) . Conclusion:AHI can inhibit the proliferation and migration of hepatocellular carcinoma cell line MHCC-97H, and promote its apoptosis.

Objective:To investigate the effect of Ginkgo leaf extract and dipyridamole injection on serum tumor necrosis factor-α, procalcitonin, lipopolysaccharide and 28-day mortality in patients with sepsis.Methods:Ninety patients with sepsis who received treatment in Ningbo Yinzhou People's Hospital from February 2018 to February 2019 were included in this study. They were randomly assigned to receive routine treatment (control group, n = 45) or Ginkgo leaf extract and dipyridamole injection treatment combined with routine treatment (treatment group, n = 45). Clinical efficacy, preoperative and postoperative Acute Physiology and Chronic Health Evaluation II score, Sequential Organ Failure Assessment score, serum tumor necrosis factor-α, procalcitonin, bacterial lipopolysaccharide levels as well as 28-day mortality were compared between the control and treatment groups. Results:There was significant difference in total effective rate between the treatment and control groups [91.11% (41/45) vs. 75.56% (34/45), χ 2 = 6.690, P = 0.039]. After treatment, Acute Physiology and Chronic Health Evaluation II score and Sequential Organ Failure Assessment score in the treatment group were (15.93 ± 1.01) points and (6.25 ± 1.83) points, respectively, which were significantly lower than those in the control group [(18.62 ± 1.75) points, (8.54 ± 2.19) points, t = 8.931, 5.383, all P < 0.001]. After treatment, serum tumor necrosis factor-α, procalcitonin and lipopolysaccharide levels in the treatment group were (43.62 ± 3.39) ng/L, (1.46 ± 0.79) μg/L, (23.62 ± 7.19) ng/L, respectively, which were significantly lower than those in the control group [(56.28 ± 4.54) ng/L, (2.12 ± 1.03) ng/L, (33.75 ± 8.67) ng/L, t = 14.989, 3.411, 6.033, P < 0.001, P = 0.001, P < 0.001]. The 28-day mortality in the treatment group was significantly lower than that in the control group [6.67%(3/45) vs. 22.22%(10/45), χ 2 = 8.160, P = 0.017]. Conclusion:Based on routine treatment, Ginkgo leaf extract and dipyridamole injection can improve the clinical symptoms of patients with sepsis, reduce inflammatory reaction and decrease mortality.

OBJECTIVE：To provide reference for clinical treatment of Acinetobacter baumannii infection and rational use of antibiotics. METHODS ：By retrospective analysis ，64 500 strains of bacteria were isolated from the inpatients of our hospital during Jan. 2015 to Dec. 2018. WHONET 5.6 software was used to analyze the detection rate ，specimen type ，departments of A. baumannii. The resistance of A. baumannii to 18 commonly used antibiotics in 4 years was analyzed by RxC table χ 2 test. RESULTS：A total of 2 072，2 040，2 017 and 2 143 strains of A. baumannii were isolated during 2015-2018，accounting for 12.85％，13.38％，13.60％，11.71％ of positive specimens. The main specimen types of 8 272 strains of A. baumannii were sputum（4 368 strains，52.81％），pus（1 106 strains，13.37％），ascites（804 strains，9.72％）. The main departments were burn department（1 605 strains，19.40％），hepatobiliary department （1 200 strains，14.51％），brain surgery department （977 strains， 11.81％）. The drug resistance rate to 18 kinds of antibiotics showed a wave-like decreasing trend （P＜0.001）. In 2018，drug resistance rate to ampicillin and aztreonam was more than 80％，and that to ampicillin/sulbactam ，ceftazidime，levofloxacin， Compound sulfamethoxazole ，gentamicin，amikacin，tobramycin and tegacyclin was less than 50％ ，among which the drug resistance rate to amikacin and tegacyclin were 14.7％ and 0，respectively. CONCLUSIONS ：There is no significant change in the number of isolates and detection rate of A. baumannii in our hospital between 2015 and 2018. The bacteria mainly cause respiratory tract infection. Amikacin or tegacyclin are recommended for treatment.

Objective To establish an aging model of rat bone marrow stromal cells (BMSCs) in vitro and in vivo, in order to study the senescence biology of aging BMSCs .Methods The control cell group ( in vitro):isolating, puri-fying and culturing BMSCs from healthy male SD rats .collecting the third generation ( P3) of BMSCs for analysis . The aging model group (in vitro):the P3 BMSCs were incubated with D-Galactose (D-Gal, 30 g/L) for 48 hours. The aging rat model group ( in vivo): the rats were given 120 mg D-Gal by the way of daily neck subcutaneous injection for 42 consecutive days .The control rat group ( in vivo):the rats were administrated with the same volume of saline for the same times .On the second day after the aging model was established , the BMSCs were collecting and culturing for study.1)The proliferative potency was detected by cell counting Kit-8(CCK-8);the distribution of cell cycle and apoptosis was detected by flow cytometry (FCM);2)the ratio of aging BMSCs was examined by the senescence-associated β-Galactosidase(SA-β-Gal) staining;3)malonaldehyde(MDA) content and total super-oxide dismutase(SOD) was examined activity by enzymatic assay; the level of reactive oxygen species (ROS) by DCFH-DA fluorescent staining was counted with FCM;4 ) the expression level of senescence-related signaling was proteins of P16 , P21 , P53 , CDK2 and cyclin D by Western blot .Results Compared with the matched control group, the BMSCs of aging model group displayed a significant decrease in proliferation; the BMSCs were held in G1 phase arrest as the proportion of the cells in G 1 phase increased , while that decreased in S phase ( P<0.05 );and the positive ratio of SA-β-Gal stained BMSCs also significantly increased ( P <0.05 ); BMSCs in the aging model group showed an increasing level of ROS and MDA , meanwhile a decline in total SOD activity was decreased (P<0.05);P16,P21 and P53 protein expression in aging BMSCs was obviously enhanced (P<0.05), at the same time the expression of CDK2 and cyclin D was also decreased ( P<0.05 ) .Conclusions D-Gal can be used to develope an aging model of BMSCs .It acts through up-regulation of expressions of aging-related proteins and in-hibition of oxidative stress injury and chronic inflammation .

Objective To study effect of piracetam combine with hyperbaric oxygen in treatment of acute carbon monoxide poisoning patients with electrocardiogram and its effects on Lactate clearance.Methods 60 patients of acute carbon monoxide poisoning who received therapy from February 2011 to February 2016 in our hospital were selected as research objects.All accord with the diagnostic criteria of acute carbon monoxide poisoning.According to draw method,those patients were divided into the experimental group(n=30)and the control group(n=30).The two groups were given a large number of sustained oxygen,intracranial pressure,protect brain cells,promote blood circulation and improve microcirculation and other basic symptomatic treatment.The control group on the basis,was treated with hyperbaric oxygen,one times a day,a total of ten times.while the experimental group was treated with piracetam combine with hyperbaric oxygen,hyperbaric oxygen method with the control group,intravenous drip of Piracetam and Sodium Chloride Injection,each 100ml,two times a day,a total of treatment for ten days.Then abnormal ECG,creatine kinase isoenzymes(CK-MB),troponin(cTnl),lactate clearance,incidence of delayed encephalopathy,mortality,therapeutic effect of two groups were compared.Results ECG abnormal rate there was no difference between the two groups before treatment,after treatment,the abnormal rate of the experimental group was significantly lower than the control group [6.66(2/30)vs.33.33％(10/30)](P<0.05); CK-MB、cTnl、6h and 24h after treatment,Lactate clearance rate was significantly higher than control group[(15.80±2.03)％vs.(10.26±2.01)％,(20.75±3.12)％vs.(13.07±2.56)％](P<0.05);DEACMP rate and mortality was significantly lower than the control group[6.66％(2/30)vs.33.33％(10/30),3.33％(1/30)vs.30.00％(9/30)](P<0.05); The total effective rate was significantly higher than the control group[95.56％(28/30)vs.75.56％(22/30)](P<0.05).Conclusion Piracetam combine with hyperbaric oxygen is well for acute carbon monoxide poisoning,which can improve the clearance rate of lactic acid,improve hypoxia and myocardial injury,and reduce the abnormal ecg.

Objective: To identify the epidemiological characteristics of the common diarrhea-related virusesof children under 5 years of age in Chengdu area, and provide the objective evidences for prevention and control of diarrhea. Methods: Fecal specimens collected from children with acute gastroenteritis between March 2006 and June 2015 were sent to Center for Disease Control and Prevention(CDC) of Sichuan province for detection of viral RNA. Clinical data were also documented. Enzyme-linked immunosorbent assay(ELISA) and/or Reverse transcriptase-polymerase chain reaction(RT-PCR) were used to detect and classify rotavirus, human calicivirus, adenovirus and astrovirus. Results: A total of 2 331 fecal specimens from children (1 446 male and 885 female) under 5 were collected. 1 351 were identified as having viral gastroenteritis with the overall positive rate of 58.0%. Children at the age from 7 to 12 months were the susceptible population. Rotavirus was detected in 659 specimens (28.3%) with epidemic time from November to December. Human calicivirus was detected in 542 specimens (23.3%) and September was its epidemic time. Norovirus GII was the main strain of the virus, but no outbreak was observed in our study. Prevalence of rotavirus declined after 2007, while the detection rate of calicivirus was increasing, which led it to be one of the primary pathogens related to viral gastroenteritis in children under 5. Astrovirus was detected in only 35 patients (1.5%) mainly identified from January to March. Adenovirus was detected in 118 patients (5.1%) mainly from May to August with limited epidemic in 2011. Most patients had acute progress(91.2%), none have chronic progress. Mild dehydration was the most common symptom among all the children, followed by moderate dehydration, while none of the patient had severe symptom. Digestive symptoms are usually accompanied by extra-intestinal symptoms in both virus infection. However, extra-intestinal symptoms had higher incidences in rotavirus infection than in calicivirus infection, while the patients with these symptoms recovered during the follow-up period. Conclusions Virus infection is the common cause of acute gastroenteritis in children under 5. Rotavirus and human calicivirus were the leading pathogens in Chengdu area.

Objective To identify the epidemiological characteristics of the common diarrhearelated virusesof children under 5 years of age in Chengdu area,and provide the objective evidences for prevention and control of diarrhea.Methods Fecal specimens collected from children with acute gastroenteritis between March 2006 and June 2015 were sent to Center for Disease Control and Prevention (CDC) of Sichuan province for detection of viral RNA.Clinical data were also documented.Enzyme-linked immunosorbent assay(ELISA) and/or Reverse transcriptase-polymerase chain reaction(RT-PCR) were used to detect and classify rotavirus,human calicivirus,adenovirus and astrovirus.Results A total of 2 331 fecal specimens from children (1 446 male and 885 female) under 5 were collected.1 351 were identified as having viral gastroenteritis with the overall positive rate of 58.0％.Children at the age from 7 to 12 months were the susceptible population.Rotavirus was detected in 659 specimens (28.3％) with epidemic time from November to December.Human calicivirus was detected in 542 specimens (23.3％) and September was its epidemic time.Norovirus GII was the main strain of the virus,but no outbreak was observed in our study.Prevalence of rotavirus declined after 2007,while the detection rate of calicivirus was increasing,which led it to be one of the primary pathogens related to viral gastroenteritis in children under 5.Astrovirus was detected in only 35 patients (1.5％) mainly identified from January to March.Adenovirus was detected in 118 patients (5.1％) mainly from May to August with limited epidemic in 2011.Most patients had acute progress(91.2％),none have chronic progress.Mild dehydration was the most common symptom among all the children,followed by moderate dehydration,while none of the patient had severe symptom.Digestive symptoms are usually accompanied by extra-intestinal symptoms in both virus infection.However,extraintestinal symptoms had higher incidences in rotavirus infection than in calicivirus infection,while the patients with these symptoms recovered during the follow-up period.Conclusions Virus infection is the common cause of acute gastroenteritis in children under 5.Rotavirus and human calicivirus were the leading pathogens in Chengdu area.

OBJECTIVETo study the effects of inhibitory peptide of Staphylococcus epidermidis (SE) biofilm (briefly referred to as inhibitory peptide) on adhesion and biofilm formation of SE at early stage.

METHODSBy using peptide synthesizer, the inhibitory peptide was synthesized with purity of 96.8% and relative molecular mass of 874.4. (1) Solution of SE ATCC 35984 (the same below) was cultivated with inhibitory peptide in the final concentrations of 1-256 µg/mL, and the M-H broth without bacteria solution was used as blank control. The MIC of the inhibitory peptide against SE was determined (n=3). (2) Solution of SE was cultivated with trypticase soy broth (TSB) culture solution containing inhibitory peptide in the final concentrations of 16, 32, 64, 128, and 256 µg/mL (set as inhibitory peptide groups in corresponding concentration), and solution of SE being cultivated with TSB culture medium was used as negative control group. Growth of SE was observed every one hour from immediately after cultivation (denoted as absorbance value), and the growth curve of SE during the 24 hours of cultivation was drawn, with 3 samples in each group at each time point. (3) Solution of SE was cultivated with TSB culture solution containing inhibitory peptide in the final concentrations of 16, 32, 64, 128, and 256 µg/mL (set as inhibitory peptide groups in corresponding concentration), and solution of SE being cultivated with TSB culture medium was used as negative control group. Adhesive property of SE was observed after cultivation for 4 hours (denoted as absorbance value, n=10); biofilm formation of SE was observed after cultivation for 20 hours (denoted as absorbance value, n=10). (4) Solution of SE was cultivated with TSB culture solution containing inhibitory peptide in the final concentration of 128 µg/mL (set as 128 µg/mL inhibitory peptide group), and solution of SE being cultivated with TSB culture medium was used as negative control group. Adhesive property of SE and its biofilm formation were observed with confocal laser scanning microscope (CLSM), and the sample numbers were both 3. Data were processed with one-way analysis of variance, LSD test, and Dunnett T3 test.

RESULTS(1) The MIC of inhibitory peptide against SE exceeded 256 µg/mL. (2) There was no significant difference in the growth curve of SE between inhibitory peptide groups in different concentrations and negative control group. (3) After 4 hours of cultivation, the absorbance values of adhesive property of SE in 256, 128, 64, and 32 µg/mL inhibitory peptide groups were respectively 0.20 ± 0.04, 0.27 ± 0.03, 0.35 ± 0.04, and 0.40 ± 0.04, which were significantly lower than the absorbance value in negative control group (0.53 ± 0.10, P<0.05 or P<0.01); the absorbance value of adhesive property of SE in 16 µg/mL inhibitory peptide group was 0.47 ± 0.09, which was close to the absorbance value in negative control group (P>0.05). After 20 hours of cultivation, the absorbance values of biofilm formation of SE in 256, 128, and 64 µg/mL inhibitory peptide groups were respectively 0.49 ± 0.10, 0.68 ± 0.06, and 0.93 ± 0.13, which were significantly less than the absorbance value in negative control group (1.21 ± 0.18, P<0.05 or P<0.01); the absorbance values of biofilm formation in 32 and 16 µg/mL inhibitory peptide groups were respectively 1.18 ± 0.22 and 1.15 ± 0.26, which were close to the absorbance value in negative control group (with P values above 0.05). (4) CLSM showed that more adhering bacteria and compact structure of biofilm were observed in negative control group, but less adhering bacteria and loose structure of biofilm were observed in 128 µg/mL inhibitory peptide group.

CONCLUSIONSThe inhibitory peptide can inhibit adhesion and biofilm formation of SE at early stage, but its structure still needs to be further modified.

Bacterial Adhesion ; Biofilms ; growth & development ; Humans ; Microscopy, Confocal ; Peptides ; Staphylococcus epidermidis ; genetics ; metabolism ; physiology

Objective To investigate the genetic mutation of the norA gene and its promotor from the wild-type drug-resistance Staphylococeus aureus(S.aureus)strains. Methods A total of 10 antibiotic-resistant S.aureus strains were isolated and screened from the burn wound for the sequencing and analysis of the nora gene and its promoter. Results There isolated 87 S.aureus strains from the burn wound flora,which were completely sensitive to vacomycin,highly sensitive to Quinupristin and Nitrofurantoin,but highly resistant to the other antibiotics,even up to91.7% of MRSA.There found the same point mutation(G→A) located at 1 349 sites of the norA gene coding region in all the S.aureus strains,saying that the amino acid was changed from Gly(glycin)to Asp(agpartic acid) in 291 sites.The resetpine reverse test showed that the MICs value of three antibiotics was lowered at various degrees in all 10strains.Conclusion NorA gene mutation is one of the mechanisms for antibiotic-resistance of S.aureus.