Objective To investigate the molecular epidemiology and mechanism of carbapenem resistance of Escherichia coli collected from intensive care units(ICUs)of general surgery.Methods Agardilution were carried out to confirmed the drug-susceptibility,pulsed-field gel electrophoresis(PFGE)were performed to analyze the molecular epidcmiology of carbapenem-resistance isolates.Specific PCR,DNA sequencing,conjugation experiments,plasmids extraction,plasmid transformation assays and SDS-PAGE of outer membrane proteins(OMPs)were carried to confirm genotype of carbapenemase and its transmission mechanism.Results PFGE showed the isolates belonged to 10 clonotype,and all the clinical isolates were resistant to β-lactams including imipenem and meropenem,but uncertain to aminoglycosides,specific PCR and DNA sequencing revealed that all isolates encoded carbapenem-hydrolyzing enzyme gene,KPC-2.Plasmid DNA extraction and plasmid transformation assays from some isolates comfirmed that KPC-2 encoded on a 56 kb plasmid.SDS-PAGE analysis confirmed that there are alterations in OMPs of Escherichia coli.Conclusion Escherichia coli isolates with carbapenem resistance are collected from our hospital,production of KPC-2 carbapenemase mainly contributed to reduced susceptibility of carbapenem in Escherichia coli,the alterations in OMPs may as a cofactor in high-level drug-resistance in Escherichia coli.

OBJECTIVETo observe the effect of tensile stress on human heel skin fibroblast proliferation in vitro, providing a theoretical basis for preventing the wound edge skin necrosis and nonunion after calcaneal fracture surgery.

METHODSFibroblast cells were taken from lateral heel skin of a 40 year-old-man, then cultured and subcultured in vitro. After that, they were divided into three groups: 0 hours group, 6 hours group and 24 hours group and were tested by tensile stress testing. The levels of TGF-β1 and IL-6 in nutrient fluid were measured. Transmission electron microscope and light microscope was applied for observe mitochondria and nucleus.

RESULTSUnder 10% of the tensile stress, mitochondria decreased, the levels of TGF-β1 and IL-6 in nutrient fluid were decreased and cell proliferation was inhibited gradually with time increasing.

CONCLUSIONThe human lateral heel skin in a long-time tensile stress state is an important cause of wound edge skin necrosis and nonunion after calcaneus fracture surgery.

Adult ; Cell Proliferation ; Cells, Cultured ; Fibroblasts ; chemistry ; cytology ; Heel ; physiology ; Humans ; In Vitro Techniques ; Interleukin-6 ; metabolism ; Male ; Skin ; chemistry ; cytology ; metabolism ; Tensile Strength ; Transforming Growth Factor beta1 ; metabolism

Objective To report the clinical outcome and prognostic factors for locally recurrent nasopharyngeal carcinoma(NPC)treated with intensity modulated radiotherapy(IMRT).Methods From January 2001 to August 2004,the data of 132 such NPC patients were analyzed retrospectively;104 male and 28 female with a median of 44.5 years(range 21-73 years).Ninety-eight patients(74.2%)were confirmed by biopsy as having NPC:9 with WHO TypeⅡand 89 WHO TypeⅢ.The other 34 patients were only diagnosed by MRI scan because of the extension/invasion was in the base of skull and/or cavernous sinus.Median interval time were 24 months(range 6-184 months).According to the 1992 Chinese Fuzhou Staging System:stageⅠ3.8 %,Ⅱ10.6 %,Ⅲ22.0% andⅣa 63.6%;T1 5.3%,T2 10.6%,T3 22.7% and T4 55.3%.Twenty-two patients had recurrence in the neck lymph nodes.IMRT was given with the sequential tomotherapy system(NOMOS Peacock systems)of 6 MV X-rays.Prescription dose was 60-70 Gy in GTV,with the fractional dose of 1.94-2.8 Gy.Sixty patients were also supplemented with two to six courses of cisplatin-based chemotherapy.Results The median volume of GTV was 39.5 cm~3(range 0.8-158.9 cm~3).The D95,V95,mean dose and fractionation dose of GTV was 66.9 Gy,98.3%,69.8 Gy and 2.32 Gy,respectively.The median follow-up time was 12 months(range,2-47 months).The 1-,2-and 3-year local progression-free rate was 96.4%,88.4% and 85.3%,respectively.The overall 1-,2-and 3-year survival rate was 6.5.9%,49.6% and 41.6%,respectively.Eleven patients developed distant metastases.Forty-seven patients were observed to devdop mucosa necrosis and/or massive hemorrhage in the nasopharynx.On univariate and multivariate analysis,fractional dose and vohane of GTV were significant prognostic factors for overall survival(P=0.016,0.009).Conclusions The local control and survival rate can be improved for patients with locally recurrent nasopharygeal carcinoma after treatment of intensity modulated radiotherapy.The fractional dose and volume of GTV are independent prognostic factors for the overall survival. The main death reasons are mucosa necrosis and/or massive hemorrhage in the nasopharynx.

Objective To explore the accuracy of Vitek 2 compact advanced expert system (AES) in indicating and analyze the carbapenemases-resisting Enterobacteriaceae phenotypes,and further investigate the methods to make up the AES.Methods 28 Enterobacteriaceae strains with Imipenem-Nonsusceptible by Vitek 2 compact,but AES suggested all production of carbapenemases were isolated.And imipenem susceptibility was determined by the disk diffusion method.Modified Hodge test (MHT) and the metallo-β-1actamase was detected by the double disk synergy method.Resistance genes were detected by the PCR amplification.Results ESBLs gene was amplified from all 28 selected strains,16 of which was detected KPC gene,and no strain of metallo-β-1actamases-producing bacteria.With carbapenemase gene detection as the gold standard,the accuracy of AES was 57.1％.Disc diffusion method detection accuracy rate of imipenem was 100％,and for 100％ of MHT accuracy.PCR amplification,MHT and the disk diffusion displayed the same result in detecting carbapenemases,but different with AES (x2 =10.08,P＜0.05).Conclusion The indications of the presence of carbapenemases using AES was not completely correct with a certain false-positive,and it is necessary to take other methods,such as disk diffusion or MHT methods,and improve the reliability of medicine-sensitivity tests.

OBJECTIVE:To optimize the extraction technology for organic acids in Crataegi fructus. METHODS:Based on sin-gle factor test,using liquid-solid ratio,extraction time and ethanol volume fraction as independent variables,extraction yield of or-ganic acids as dependent variable,central composite design-response surface method was used to optimize the extraction technology of organic acids in Crataegi fructus. RESULTS:The optimal extraction technology was as follow as liquid-solid ratio of 18.5:1, adding 75% ethanol,reflux extraction twice,2.0 h each time. Average extraction yield of organic acids in verification test was 5.22%(RSD=2.70%,n=3),with 1.75% relative error of the predicted value(5.13%). CONCLUSIONS:Optimized extraction technology for organic acids in Crataegi fructus is simple,with good reproducibility and predictability.

Objective Laboratory tests of yeast, with their disadvantages of long identification, complicated operation, and low positive rate, cannot meet the needsof rapid diagnosis and timely treatment.This studyinvestigated the feasibility ofapplying matrix-assisted laser desorption ionization-time of flight mass spectrometry ( MALDI-TOF-MS) in rapid identification and clinical isolationof yeast strains. Methods A total of 120 clinical non-repetitive isolates were collected from clinical culture samples and identified by MALDI-TOF-MS and VITEK 2-Compact/API, respectively.The discordant results were resolved by ITS gene sequencing. Results Of the 120 yeast isolates analyzed, 117 (97.5%) were correctly identified at the species level by MALDI-TOF-MS, 1(0.8%) misidenti-fied, and 2 ( 1.7%) unidentified.ITS gene sequencing of the 3 isolates showed the coincidence rate to be 0( 0/3) for MALDI-TOF-MS and Vitek 2-Compact/API. Conclusion MALDI-TOF-MScan be used as a rapid, accurate, and inexpensive tool for the identification of clinical yeast strains.

This study was purposed to investigate the expression of RPL36A (ribosomal protein 36a) in the newly diagnosed acute myeloid leukemia (AML) cells and its mechanism at the molecular level. The RPL36A mRNA expression in the newly diagnosed AML cells, U937 cells and normal MNCs was determined by RT-PCR. Small interfering RNA (siRNA) targeting to RPL36A was transfected into U937 cells by Lipofectamine 2000 system. Proliferation, cell cycle, apoptosis of U937 were observed through MTT assay, flow cytometry, acridine orange/ethidium bromide (AO/EB) double staining, TUNEL and Annexin V/FITC respectively. RPL36A mRNA and protein expression levels were detected by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot analysis respectively. The results showed that RPL36A expression in the newly diagnosed AML cells and U937 cells was significantly upregulated. The average OD value of U937 cells transfected with RPL36A siRNA was significantly lower as compared with 3 control groups. The cell percentage in G2-and S-phase increased, which indicated the inhibition effect of RPL36A siRNA on cell proliferation. Remarkable cell apoptosis in U937 cells treated with RPL36A siRNA was observed by AO/EB, TUNEL analysis and Annexin V/FITC assay; RPL36A mRNA and protein expression level of U937 cells treated with siRNA were significantly declined in a time-dependent manner (r=0.9813 and 0.9537). It is concluded that the RPL36A expression in the AML cells is significantly enhanced and the RPL36A gene may be involved in regulation of cell cycle and cell apoptosis of AML, which promotes proliferation of AML cells and inhibits apoptosis of cells.
Adolescent ; Adult ; Aged ; Apoptosis ; drug effects ; Cell Proliferation ; drug effects ; Female ; Humans ; Leukemia, Myeloid, Acute ; pathology ; Male ; Middle Aged ; RNA Interference ; RNA, Small Interfering ; genetics ; pharmacology ; Ribosomal Proteins ; genetics ; pharmacology ; U937 Cells ; Young Adult

This work was mainly studied the effects of the four alkaloids from Coptidis Rhizoma on the mouse peritoneal macrophages in vitro and preliminarily discussed the regulating mechanisms. The effect of alkaloids from Coptidis Rhizoma on the vitality of macrophages was measured by the MTT assay. The effect of alkaloids on the phagocytosis of macrophages was determined by neutral red trial and respiratory burst activity was tested by NBT. The expressions of respiratory-burst-associated genes influenced by alkaloids were detected by qRT-PCR. The conformation change of membrane protein in macrophages by the impact of alkaloids was studied by fluorospectro-photometer. Results showed that the four alkaloids from Coptidis Rhizoma could increase the phagocytosis of macrophages in different level and berberine had the best effect. Berberine, coptisine and palmatine had up-regulation effects on respiratory burst activity of mouse peritoneal macrophages stimulated by PMA and regulatory activity on the mRNA expression of PKC, p40phox or p47phox, whereas the epiberberine had no significant influence on respiratory burst. Moreover, alkaloids from Coptidis Rhizoma could change the conformation of membrane protein and the berberine showed the strongest activity. The results suggested that the four alkaloids from Coptidis Rhizoma might activate macrophages through changing the conformation of membrane protein of macrophages and then enhanced the phagocytosis and respiratory burst activity of macrophages. Furthermore, the regulatory mechanism of alkaloids on the respiratory burst activity of macrophages may be also related to the expression level of PKC, p40phox and p47phox.
Alkaloids ; pharmacology ; Animals ; Cells, Cultured ; Coptis ; chemistry ; Drugs, Chinese Herbal ; pharmacology ; Female ; Gene Expression ; drug effects ; Macrophages, Peritoneal ; drug effects ; Mice ; Phosphoproteins ; genetics ; metabolism ; Protein Kinase C ; genetics ; metabolism ; Rhizome ; chemistry

The growing status of Anoectochilus roxburghii seedling was observed and the survival rate of seedlings, height, stem diameter and plant fresh weight under the conditions of different transplanting substrate compositions, planting density, shading rate were measured. The results showed that the effects of different transplanting substrates, planting densities, shading rates and nutrient solutions on the growing status of A. roxburghii plantlets varied greatly. A. roxburghii plantlets demonstrated a high survival rate and better growing status under the Following conditions: the ratio of peat and river sand as 2: 1, the planting density as 3 cm x 3 cm, the shading rate as 70%, and the nutrient solution as 1/4MS. The findings of the study provide a solid technical solution for the artificial cultivation of A. roxburghii plantlets.
Breeding ; methods ; Culture Media ; chemistry ; pharmacology ; Orchidaceae ; drug effects ; growth & development ; Seedlings ; drug effects ; growth & development ; Survival Analysis

OBJECTIVETo analyze the constituent proteins in donkey hide, the key ingredient for Ejiao, an important traditional Chinese medicine for the blood-related conditions, in hope to eventually decipher the biochemical mechanism behind Ejiao's prominent medicinal efficacy.

METHODTwo methods were employed to extract proteins in donkey skin. One used TriPure isolation reagent to extract the total proteins in donkey skin. Another used 1% sodium dodecyl sulfate (SDS) to heat the sample at 100 degrees C overnight. And then sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and capillary HPLC were used to analyze the component of proteins.

RESULTThere are not only collagen alpha1 (I) and collagen alpha2 (I), but also serum albumin in donkey skin. The content is over 25% in total proteins with the method of TriPure isolation reagent. The content of donkey serum albumin is up to 20% with the method of 1% SDS heating. And two bands, molecular weight are nearly 200 kDa,were found on 7.5% SDS-PAGE. Extracted these proteins to analyze with capillary HPLC, they were found to be the complex products of collagen and serum albumin of donkey.

CONCLUSIONDonkey serum albumin is a main protein component in the hide, which is a clue to expose is the effect of Ejiao on blood.

Animals ; Chromatography, High Pressure Liquid ; Collagen Type I ; analysis ; chemistry ; metabolism ; Collagen Type II ; analysis ; chemistry ; metabolism ; Drug Interactions ; Electrophoresis, Polyacrylamide Gel ; Equidae ; Molecular Weight ; Protein Binding ; Serum Albumin ; analysis ; chemistry ; metabolism ; Skin ; chemistry