Molecular technologies are sensitive, fast, and cheap in the study on microbial ecology. However, these methods do not provide information about morphology, number, and spatial distribution of the microorganisms. In contrast, Fluorescence in situ hybridization (FISH) combines the precision of molecular biology with the visual information from microscope, and permits visualization and identification of individual miciobial cells within their natural habitat. FISH not only allows the detection of slow growing microorganisms, but also of yet-to-be cultured. It is useful for many applications in diagnosis and assessment of the population structure of complex microbial communities, and is a powerful tool for molecular ecology studies in microbiology, In this paper, major techniques and progresses of FTSH were described. Its application in microbial ecology, as well as problems , pitfalls, and perspectives of FISH are discussed.

Designed the degenerate primers of alcohol dehydeogenase and L-lactate dehydrogenase to aug- ment Ethanoligenens harbinense B49 genomic DNA, and obtained about 780 bp and 610 bp PCR product respectively. Augmented flank sequences of the two PCR fragments with the Cassette PCR method. Similar- ity alignment showed that the products of the cloned DNA were very high similar to those of alcohol dehy- drogenase genes and L-lactate dehydrogenase genes respectively. One of the two sequences was 1902 bp long, and the ORF of adh was 1101 bp long and encoded 366 amino acids. Its putative molecular weight was about 39.71 kD, its calculational isoionic point was pH 5.93. The maximal identity and positive was 51% and 73% with Clostridium thermocellum ATCC 27405 adh. The other one was 2490 bp long, and the ORF of adh was 951 bp long and encoded 316 amino acids. Its putative molecular weight was 34.23 kD, its calcula-tional isoionic point was pH 6.09. The maximal identity and positive was 55% and 74% with Bacillus megaterium L-ldh. Successfully cloning these two genes would not only enrich the gene resources of L-lactate dehydrogenase and alcohol dehydrogenase genes, but also give the scientific warrant for the meta- bolic engineering research and the construction of the gene-engineering bacteria.

A anaerobic hydrogen-producing strain HR-1 was isolated from compost. Phylogenetic analysis based on 16S rRNA sequence similarity indicates that strain HR-1 is the closest relative to Clostridium ace- tobutylicum ATCC 824, with the similarity of 96%. Biological characteristics and phylogenetic analysis of 16S rRNA gene indicate that HR-1 is a new species named Clostridium sp. HR-1. Cells are Gram-positive, mobile rod-shaped. Spores and flagellums were no observed. Temperature range for growth is 10?C to 45?C (optimum temperature 37?C~39?C), and range pH for growth is 4.0 to 10.0 (optimum pH 7.5~8.0). H2, CO2, acetate, butyrate and a little ethanol are the end products of PYG fermentation. Strain HR-1 has the ability to use organic nitrogen and inorganic nitrogen sources for growth and hydrogen production, and yeast extract is the optimum nitrogen source for hydrogen production. Strain HR-1 produces hydrogen from xylose (3 g/L) at 37?C and initial pH 6.5, the hydrogen yields and maximal hydrogen production rate are 1.84 mol-H2/mol-xylose and 10.52 mmol-H2/h?g-cdw, respectively. Strain HR-1 is able to utilize glucose, galactose, fructose, mannose and cellobiose for hydrogen production and the hydrogen yields from glucose is 2.36 mol-H2/mol-glucose.

Based on the clinical skill competition of college students of the advanced medical colleges and universities nationwide,we aimed at the analysis on the weaknesses of medical emergency clinical practical teaching and emphasis on the theoretical education and neglect the practical education,and humanistic care,etc.In the clinical practice teaching of emergency,we combined the clinical skill training with physician occupation quality training,pay attention to the practice of advanced simulation exercises,gradual transition,clinical thinking,training medical students hands-on,team cooperation ability and humane accomplishment,to improve their ability of analyzing and solving problems and eventually optimize medical emergency clinical practical teaching,formulate the clinical practice standards as well as promote the reform and innovation of clinical teaching.

Objective To investigate the mutations ACTN4 and SYNPO genes promoter in sporadic primary focal segmental glomerulosclerosis (FSGS) and to analyze the role of mutations in FSGS. Methods The study consisted of 82 Chinese primary FSGS, including 39 females and 43 males, ranged from 12 to 76 years old. Seventy volunteers were selected as healthy control group. Genomie DNA was extracted from peripheral blood cells of FSGS patients and hair of patients' parents by polymerase chain reaction (PCR) and direct sequencing to analyze ACTN4 and SYNPO gene promoter mutations. Mutations were matched with GenBank and TRANSFAC software database (www.ncbi.nlm.nih.gov; www.genometix.de; www.gene-regulation, corn). Dual luciferase assay system was used to analyze the promoter region mutations, based on PGL3-Basie vector, pRL-SV40 and PCI2 cell line. Hair DNA of novel mutation patients' parents was sequenced. Expression of alpha-actinin-4 and synaptopodin in patients' kidney tissue was examined by immunofluorescence. Results Three patients with 1-34C>T, 1-590delA and (1-1044delT)+ (I-797T >C) +(1-769A >G) heterozygous mutations were found in ACTN4 gene promoter respectively, and two patients with 1-24G>A and 1-851C>T heterozygous mutations in SYNPO gene promoter respectively. The same mutations were not found in the control group of 70 healthy people. Except one patient accepting her parents' 1-1044delT and 1-797T>C mutated chromosome respectively, no same mutations were found in patients' parents. Protein expression of alpha-actinin-4 and synaptopodin was reduced in mutated patients' kidneys. Except 1-1044delT group, luciferase activity in mutated groups decreased. (1-1044delT)+(1-797T>C)+(1-769A>G) mutation was associated with poor outcome and patient with these mutations progressed to end-stage renal failure. Conclusion Mutations of ACTN4 and SYNPO gene promoters affect gene transcription and protein translation, which may contribute to the onset of sporadic primary FSGS.

A method for simultaneous determination of PCDDs, dl-PCBs, BFRs and PBDD/Fs in flue gas from stationary source was developed. The sample was extracted by Soxhlet apparatus with toluene, and followed by purification through sulfuric acid partition and multi-layer silica gel column separation. The target compounds were then all separated by passing through the active carbon-dispersed silica gel column and reversal eluting. Gas chromatography coupled with a thermostable capillary column ( short length, thin stationary phase film) was operated at pulse injection mode. High resolution mass spectrometry set at low-electron-energy ionization was used for quantification. The high- and low-brominated compounds were determined simultaneously. The detection limits of this method were 0. 081-1. 2 pg for PCDD/Fs, 0. 10-0. 32 pg for dl-PCBs, 0. 14-12 pg for PBDEs, 0. 26-16 pg for new BFRs, 0. 44-3. 6 pg for tetra- to hepta-BDD/Fs and 8. 2-12 pg for OBDD/F. Recoveries ( RSDs) in spiked flue gas samples were 88%-115%(2. 9%-6. 1%) for PCDD/Fs, 84%-118% (3. 2%-10%) for dl-PCBs, 71%-135% (2. 1%-18%) for PBDEs, 71%-114% (2. 9%-7. 4%) for new BFRs, 83%-127% (5. 2%-10%) for tetra-to hepta-BDD/Fs and 52%-149% ( 23%-24%) for OBDD/F. All quality control data fell within the acceptable range specified in analysis standards for flue gas.

Single-Strand Conformation Polymorphism(SSCP) is an effect method for investigating environment microbial genetic polymorphism, with its characterization of rapidness, simplicity, and sensitivity. However, many factors can influence the results of SSCP in the analysis of complex environment samples, and its optimization is highly needed. In this paper, optimal PCR-SSCP conditions were discussed based on PAGE concentration, formamide deionized in denaturing loading buffer, electrophoresis time and temperature. The resluts showed that the optimal conditions were as follows: 16S rDNA V1~V3 was selected as the targeted gene, the ratio of acrylamide to N, N-dimethylacrylamide in 12% polyacrylamide gel electrophoresis(PAGE)gel was 49:1, the ratio of formamide deionized in denaturing loading buffer was 1:3, running the SSCP gel at 300 V for 18 h (under 4 ℃). Aside from this, the validations using samples from a simultaneous desulfurification and denitrification bioreactor were conducted under this optimal conditions.

OBJECTIVETo establish the median of serum markers for second trimester screening in Qingdao region and to assess the influence of median correction on the performance of screening.

METHODSMaternal serum alpha-fetoproteins (AFP), human chorionic gonadotrophin, free beta subunit (β -HCG) and unconjugated oestriol (uE3) were assayed for prenatal screening of 18 188 singleton pregnancies at 15-20(+ 6) weeks gestation from January 2009 to July 2010. The median of serum markers was calculated based on above results and applied for risk estimation in screening for fetal aneuploidy from August 2010 to March 2011. The screening performance, specified in terms of detection rates (DRs), false positive rates (FPRs) and odds of being affected given a positive result (OAPR) were compared between the two groups. The risks of 45 affected pregnancies detected during the study were estimated with both Caucasian and corrected medians.

RESULTSThe average level of AFP in local pregnancies was similar to that of the Caucasian population, whilst β -HCG and uE3 were respectively 11% and 33% higher than those of Caucasians. The multiple of median (MoM) value was between 0.94 and 1.02 for the dataset based on the corrected median. At a cut-off of l in 270, FPR has decreased from 5.2% to 4.9%, and DR of Down syndrome has increased from 60% to 69.2%, and OAPR has increased from 1:79 to 1:59 when evaluating risk based on the corrected median. For the 45 affected pregnancies, three Down syndrome pregnancies could be missed because their risk estimates were lower than the cut-off level based on Caucasian median.

CONCLUSIONIt is useful to establish and apply population and laboratory-specific medians in order to improve the performance of prenatal screening and diagnosis.

Adult ; Biomarkers ; blood ; Estriol ; blood ; Female ; Humans ; Lindane ; blood ; Pregnancy ; Pregnancy Trimester, Second ; Prenatal Diagnosis ; methods ; alpha-Fetoproteins ; analysis

BACKGROUNDPolymorphonuclear neutrophil (PMN), one of the most important inflammatory cells, functions throughout the initiation, progression and resolution of inflammation. This study aimed at investigating the relationship between PMN apoptosis and the lung injury after chest impact trauma.

METHODSPMNs were purified from rabbits subjected to the chest impact trauma and their apoptosis, necrosis, survival and respiratory burst were detected by flow cytometry. Meanwhile, lactate dehydrogenase and (LDH) [Ca2+]i were measured.

RESULTSThe delayed apoptosis of PMNs in bronchoalveolar lavage fluid was observed from 2 hours to 12 hours after trauma, and viable cells increased. Respiratory burst of PMNs in bronchoalveolar lavage fluid was increased significantly from 2 hours with the peak at 8 hours. Meanwhile, lactate dehydrogenase in bronchoalveolar lavage fluid was higher than that in control (P < 0.05) from 4 hours to 24 hours, and intracellular free Ca2+ in PMN was increased temporarily.

CONCLUSIONSRetention of PMN in tissues and the abnormality in apoptotic pathway inevitably generate persistent activation of PMN and excessive release of toxic substances, resulting in tissue injury. The temporary increase of intracellular free Ca2+ may be responsible for the delayed apoptosis of PMN.

Animals ; Apoptosis ; physiology ; Lung Injury ; Neutrophils ; physiology ; Rabbits ; Respiratory Burst ; physiology ; Thoracic Injuries ; complications

OBJECTIVETo explore the role and mechanism of the Fas/Fas ligand (FasL) system and its downstream signaling pathway related to the progression of alcoholic steatohepatitis and liver fibrosis.

METHODSEighteen C57BL/6J mice were randomly divided into three groups: controls; alcoholic steatohepatitis model, given four-weeks of a 4% ethanol-containing Lieber-DeCarli liquid diet; alcoholic steatohepatitis and liver fibrosis model, given the four-week alcohol diet followed by twice weekly intraperitoneal injections of carbon tetrachloride (5% olive oil solution; 2 mL/kg dose) during the fifth to eighth weeks. Mice in the model groups were sacrificed at the end of week 4 and 8, respectively, along with control mice for comparative analyses. Liver tissue sections were evaluated for hepatocellular apoptosis by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay. The mRNA expression of Fas, FasL, cysteine aspartate-specific proteases 3 (caspase 3), and cytochrome P450 2E1 (CYP 2E1) in liver tissues was detected by reverse transcription (RT)-PCR, visualized by ethidium bromide staining, and normalized to the gray-value of GAPDH expression. The protein expression of Fas and caspase 3 were detected by western blotting (b-actin normalized), and of FasL and CYP 2E1 by immunohistochemistry staining. Intergroup differences and statistical significance were evaluated by single factor analysis of variance and the least squares difference-t test or the Kruskal-Wallis H test and the Mann-Whitney U test.

RESULTSThe number of apoptotic cells in the liver sections was significantly higher in both model groups with alcoholic steatohepatitis (vs. controls) and the amount in the alcoholic steatohepatitis plus liver fibrosis model was significantly higher than that in the model with only alcoholic steatohepatitis. In addition, activation of Fas, FasL and its downstream signaling pathway showed an increasing trend with extent of liver injury. The hepatic mRNA (by RT-PCR) and protein (by western blotting) normalized expression levels in the controls, alcoholic steatohepatitis models, and alcoholic steatohepatitis plus liver fibrosis models were, respectively: Fas mRNA: 0.50+/-0.05, 0.61+/-0.10, 0.76+/-0.03 (H=12.137, P less than 0.05), protein: 0.52+/-0.14, 0.86+/-0.10, 0.99+/-0.09 (F=12.758, P less than 0.01); FasL mRNA: 0.31+/-0.03, 0.53+/-0.02, 1.02+/-0.04 (F=153.260, P less than 0.01); caspase 3 mRNA: 0.86+/-0.11, 0.85+/-0.05, 1.33+/-0.16 (F=8.740, P less than 0.01), protein: 0.40+/-0.03, 0.69+/-0.06, 1.02+/-0.10 (F=90.785, P less than 0.01); CYP 2E1 mRNA: 0.72+/-0.14, 1.00+/-0.15, 1.30+/-0.20 (H=4.713, P less than 0.01). The changes in hepatic FasL and CYP 2E1 expression detected by immunohistochemistry were consistent with the mRNA expression.

CONCLUSIONActivation of Fas/FasL and its downstream signaling pathway, which induces hepatocellular apoptosis, contributes to the development of alcoholic steatohepatitis and liver fibrosis.

Animals ; Apoptosis ; Cytochrome P-450 CYP2E1 ; metabolism ; Fas Ligand Protein ; metabolism ; Fatty Liver, Alcoholic ; metabolism ; pathology ; Liver Cirrhosis ; metabolism ; pathology ; Male ; Mice ; Mice, Inbred C57BL ; Signal Transduction ; fas Receptor ; metabolism