Performance of the Xpert® MTB/RIF assay in the rapid diagnosis of tracheobronchial tuberculosis using bronchial washing fluid.

OBJECTIVE: To assess the diagnostic value of the Xpert® MTB/RIF (GeneXpert) assay for tracheobronchial tuberculosis (TBTB) using bronchial washing fluid (BWF). METHODS: This retrospective study enrolled patients suspected of having TBTB and patients with non-TB pulmonary disease as controls. BWF were used to undertake acid-fast bacillus (AFB) smears, the GeneXpert assay and the LÓ§wenstein-Jensen (LJ) culture method. The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) were compared among BWF AFB smears, BWF GeneXpert and the BWF LJ culture method. RESULTS: A total of 130 patients with TBTB and 102 patients with non-TB pulmonary disease were enrolled in the study. Sputum AFB smears were positive in 62 of 130 patients (47.7%) with TBTB. Using the clinical diagnosis of TBTB as the gold standard, the sensitivity, specificity, PPV and NPV of the three methods using BWF were as follows: 93.1%, 99.0%, 99.2% and 91.8% for BWF GeneXpert; 73.1%, 100.0%, 100.0% and 74.5% for BWF LJ cultures; 53.8%, 99.0%, 98.6% and 62.7% for BWF AFB smears. The diagnostic yield of BWF GeneXpert was significantly higher compared with BWF cultures for type III and IV TBTB. CONCLUSION: The Xpert® MTB/RIF assay using BWF exhibited higher sensitivity than bacteriological diagnostic methods and was particularly useful for the early diagnosis of smear-negative TBTB.

The effect of nano-TiO2 photocatalysis on the antioxidant activities of Cu, Zn-SOD at physiological pH.

Security issues of nanoparticles on biological toxicity and potential environmental risk have attracted more and more attention with the rapid development and wide applications of nanotechnology. In this work, we explored the effect and probable mechanism of nano-TiO2 on antioxidant activity of copper, zinc superoxide dismutase (Cu, Zn-SOD) under natural light and mixed light at physiological pH. Nano-TiO2 was prepared by sol-hydrothermal method, and then characterized by X-ray Diffraction (XRD) and Transmission electron micrographs (TEM). The Cu, Zn-SOD was purified by sephadex G75 chromatography and qualitatively analyzed by sodium dodecyl sulfate polypropylene amide gel electrophoresis (SDS-PAGE). The effect and mechanism were elucidated base on Fourier Transform Infrared Spectrometer (FT-IR), Circular Dichroism (CD), zeta potential, and electron spin resonance (ESR) methods. Accompanying the results of FT-IR, CD and zeta potential, it could be concluded that nano-TiO2 had no effect on the antioxidant activity of Cu, Zn-SOD by comparing the relative activity under natural light at physiological pH. But the relative activity of Cu, Zn-SOD significantly decreased along with the increase of nano-TiO2 concentration under the mixed light. The results of ESR showed the cause of this phenomenon was the Cu(II) in the active site of Cu, Zn-SOD was reduced to Cu(I) by H2O2 and decreased the content of active Cu, Zn-SOD. The reduction can be inhibited by catalase. Excess O2·- produced by nano-TiO2 photocatalysis under mixed light accumulated a mass of H2O2 through disproportionation reaction in this experimental condition. The results show that nano-TiO2 cannot affect the antioxidant activity of Cu, Zn-SOD in daily life. The study on the effect of nano-TiO2 on Cu, Zn-SOD will provide a valid theory support for biological safety and the toxicological effect mechanism of nanomaterials on enzyme.

Ultra-performance liquid chromatography tandem mass spectrometry combined with automated MetaboLynx analysis approach to screen the bioactive components and their metabolites in Wen-Xin-Formula.

Wen-Xin-Formula (WXF), a famous traditional prescription, has been widely used to treat myocardial ischemia syndrome for thousands of years. However, the constituents absorbed into blood after oral administration of WXF remain unknown. Here, an integrative ultra performance liquid chromatography coupled with electrospray ionization/quadrupole-time-of-flight mass spectrometry (UPLC-ESI-Q-TOF-MS) combined with the MetaboLynx approach was established to investigate the absorbed constituents in rats after oral administration of WXF. A hyphenated electrospray ionization and quadrupole-time-of-flight analyzer was used for the determination of accurate mass of the molecule and fragment ions. With this rapid and automated analysis method, a total of 32 peaks were tentatively characterized in vivo based on MS and MS/MS data and comparison with available databasess, 26 of which were parent components and six metabolites. These components mainly were ginsenosides, paeoniflorin, galloyl glucose, berberis alkaloids, phenolic, phenolic glycosides and unsaturated fatty acids, glucuronide products of original berberis alkaloids. The present study demonstrates that integrative UPLC-ESI-Q-TOF-MS technique and MetaboLynx data processing method were successfully applied for the rapid discovery of potentially bioactive components and metabolites from WXF, and proved that the established method could help to explore the effective substances for further research into WXF.

Molecular characterization and phylogenetic analysis of porcine epidemic diarrhea virus (PEDV) field strains in south China.

A total of 127 porcine samples were collected from 48 farms in six provinces in south China. The positive rate of porcine epidemic diarrhea virus (PEDV) was 43.0 % (55/127), and the co-infection rate of PEDV and transmissible gastroenteritis virus (TGEV) was 12.0 % (15/127). The partial S gene and complete M gene were amplified from PEDV-positive strains by RT-PCR, cloned, sequenced and compared with each other, as well as with the reference strains in GenBank. Sequence homology results of the partial S gene and complete M gene showed that all south China field PEDV strains had nucleotide (deduced amino acid) sequence identities of 86.7-98.7 % (83.2-99.3 %) and 96.1-100 % (95.0-100%), respectively, with the foreign reference strains reported in GenBank. Phylogenetic analysis of the partial S gene showed that all the south China PEDV strains and two Thailand strains (08UB01 and 08RB07) belong to the same group and differ genetically from European strains and early domestic strains. Phylogenetic analysis of the complete M gene showed that all south China PEDV strains have a close relationship with most of the strains in Korea and Thailand, but differ genetically from the vaccine strain (CV777).

Molecular cloning, sequencing, and expression of the outer membrane protein P2 gene of Haemophilus parasuis.

Haemophilus parasuis is the etiological agent of Glässer's disease characterized by fibrinous polyserositis, polyarthritis, and meningitis in young pigs. But it is difficult to develop universal serological diagnostic tools and effective vaccines against this disease because of the serovar diversity of the isolates. In this study, enterobacterial repetitive intergenic consensus-polymerase chain reaction, were performed to investigate the gene profile of 111 isolates of H. parasuis from China. And a specific common gene of H. parasuis was cloned and identified as the outer-membrane protein (OMP) P2 gene. Sequencing results of OMP P2 genes of 22 isolates showed that they had high homology and could be divided into 2 genetic types. Moreover, the OMPP2 protein was expressed in Escherichia coli expressing system. And the purified recombinant protein provided partial protection against H. parasuis infection in mice. It suggested the OMP P2 was an immunogenic protein and had great potential to serve as a vaccine and diagnostic antigen.

[Molecular characterization of a porcine reproductive and respiratory syndrome virus isolate NT0801 from China].

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the important pathogens causing serious economic losses to swine industry worldwide. PRRSV is genetically and pathologically heterogenous. PRRSV NT0801 strain was isolated in a pig farm with clinical signs and had high pathogenesis in piglets. But its NSP2 gene did not have 30 amino acids deletion as highly pathogenic JXA1 strain. To elucidate the genetic characteristics of PRRSV NT0801 strain, the full-length genome of NT0801 isolate was sequenced and analyzed. The results showed that the genome of PRRSV NT0801 was 15439bp in length, including 29nt Poly(A) tail. Compared with the highly pathogenic JXA1 strain, it had the nucleotide sequence identity of 96.7%, amino acid sequence homology of 97.2% and 98.5% in GP3 and GP5, respectively. Phylogenetic analysis indicated that NT0801 isolate was located between the traditional strain and the highly pathogenic strain. But no obvious recombination signal was observed, compared with other PRRSV isolates with different virulence. The alignment of amino acid sequence of NT0801 with other PRRSV isolates demonstrated that three out of nine sites, being consistent with the highly pathogenic strain, were different from those in highly pathogenic while same as those in traditional strains and JXA1 vaccine strain. And one out of 9 sites was same as that of JXA1 vaccine strain exclusively, two out of 9 sites were different from all the strains. These results indicated that PRRSV NT0801 strain is closely related to highly pathogenic PRRSV, although there has no 30 amino acids deletions in NSP2 region. The epidemic PRRSV strains variation results from the gene mutation. It should be useful for studying on the virulence genes located in different ORFs of PRRSV in the future.

[Genetic polymorphisms of five STR gene loci GATA30F04, GATA23B10, D18S847, GATA83B04 and GATA167A05 alleles in Chinese Han population of Chengdu area].

OBJECTIVE: To get the preliminary genotype and allele frequency distributions of GATA30F04, GATA23B10, D18S847, GATA83B04 and GATA167A05 loci in Chinese Han population in Chengdu area, and to validate more short tandem repeat (STR) systems for forensic application. METHODS: The polymerase chain reaction (PCR) was used to this study. Five STRs (GATA30F04, GATA23B10, D18S847, GATA83B04 and GATA167A05) were amplified from DNA samples, which were extracted with Chelex-100 method from EDTA-blood of 100 unrelated individuals. The PCR products were analyzed by PAG vertical electrophoresis. RESULTS: No deviations from Hardy-Weinberg equilibrium were observed. The expected heterozygosities observed were 0.694, 0.918, 0.836, 0.889 and 0.880 for GATA30F04, GATA23B10, D18S847, GATA83B04 and GATA167A05 respectively. The discriminating powers were 0.697, 0.901, 0.875, 0.900 and 0.891. CONCLUSION: The last four loci in this study are useful markers for genetics purposes in individual identification and paternity test.

Ascorbic acid alleviates pancreatic damage induced by dibutyltin dichloride (DBTC) in rats.

PURPOSE: Because previous studies have reported depleted antioxidant capacity in patients with chronic pancreatitis (CP), prevention of free radical production has gained importance in antifibrotic treatment strategies for CP. The aim of this study was to investigate the effects of ascorbic acid on oxidative capacity and pancreatic damage in experimental CP. MATERIALS AND METHODS: CP was induced in male Sprague-Dawley rats by infusion of dibutyltin dichloride (DBTC) into the tail vein. Ascorbic acid was given intraperitoneally at a daily dose of 10 mg/kg body weight. The treatment groups were as follows: group 1, DBTC plus intraperitoneal physiologic saline; group 2, DBTC plus intraperitoneal ascorbic acid; group 3, solvent plus intraperitoneal physiologic saline; group 4, no operation plus intraperitoneal physiologic saline. Each group contained 15 animals. Treatment was started after CP was established. After 4 weeks of treatment, serum hyaluronic acid and laminin levels were determined by radioimmunoassay, pancreatic tissue oxidative stress was analyzed, and the degree of pancreatic damage was determined. RESULTS: Ascorbic acid treatment markedly increased superoxide dismutase (SOD) activity and decreased malondialdehyde (MDA) concentrations in pancreatic tissue (p < 0.01 for both). Significant serum hyaluronic acid and laminin reductions were observed in group 2 as compared with group 1 (p < 0.05). However, the serum hyaluronic acid and laminin levels remained elevated when compared with those of groups 3 and 4 (p < 0.05). Histopathologic scores were also lower in animals with CP that underwent ascorbic acid-treatment (p < 0.05). CONCLUSION: Ascorbic acid treatment alleviated the degree of oxidative stress and pancreatic damage in rat CP. Antioxidant treatment might be considered a potential option to improve the pathologic process in CP.

[Genetic polymorphisms at five STR loci in Chengdu Han population].

OBJECTIVE: To understand the allele structure and genetic polymorphisms at five STR loci in Chengdu Han population, to obtain a preliminary database and to analyze the value in forensic medicine. METHODS: EDTA-blood specimens were collected from unrelated individuals. The DNA of samples was extracted with Chelex-100 method. And then the PCR amplification, PAGE horizontal electrophoresis and gel silver staining were performed for analyzing the polymorphisms of five STR loci in Chengdu Han population. RESULTS: In the five STR loci,10, 7, 5, 7 and 7 alleles were found respectively. The observed heterozygosity of 83%, 80%, 64%, 66% and 77% and discrimination power of 95.44%, 91.0%, 84.3%, 86.1% and 86.8% were identified for the five STR loci respectively. All loci obeyed Hardy-Weinberg equilibrium. CONCLUSION: The five STR loci display the high discrimination power. The obtained data are desirable for forensic analysis and benefiting to the research of the population genetics.

[Polymorphism of five short tandem repeat systems in Chinese Han population in Chengdu].

OBJECTIVE: To obtain population genetic data of loci D11S4951, D11S4957, GATA193H05, D2S2951, and D6S2421 in Han population in Chengdu area and to validate the value of their forensic application. METHODS: Blood samples were collected in EDTA tubes from unrelated individuals. DNAs were extracted with Chelex-100 and were analyzed by PCR and horizontal PAGE followed by silver staining. RESULTS: Alleles 7, 10, 8, 6 and 8 were found in 5 STR loci, respectively. No deviations from Hardy-Weinberg balance were observed. The heterozygosities observed were 0.743, 0.772, 0.833, 0.650 and 0.800, respectively. The chances of exclusion were 0.497, 0.549, 0.662, 0.356 and 0.599, and the discrimination powers were 0.863, 0.912, 0.947, 0.829 and 0.931. CONCLUSION: All of the five loci studied may be useful markers for individual identification and paternity testing.

[Primary culture and morphologic observation of eutopic and ectopic endometrial cells from patients with endometriosis].

OBJECTIVE: To explore the method of primary culture for endometriotic cells and to find out the differences in morphological manifestations among endometriotic cells and eutopic endometrial cells sampled from patients with endometriosis and endometriosis-free women. METHODS: Endometriotic and eutopic endometrial cells were cultured by modified method of primary culture. The endometriotic cell types were observed and differentiated under optical and electron microscopes. RESULTS: The success rates for culture of eutopic endometrial cells from endometriosis-free women and patients with endometriosis were 91.67% and 93.75% respectively. The success rate for culture of endometriotic cells was 75.00%. The size of endometriotic glandular cells was similar to those of eutopic endometrial glandular cells from endometriosis-free women and patients with endometriosis. The chromatin was manifold and the nucleus was augmented in the endometriotic glandular cells. The endometriotic stromal cells were smaller than the eutopic endometrial stromal cells from endometriosis-free women and patients with endometriosis. Many tiny villi and protuberances on plasma membrane could be seen in the endometriotic stromal cells. CONCLUSION: The success rate for culture of endometriotic cells can be elevated through improving the method of primary culture. The ultrastructures of endometriotic glandular and stromal cells are obviously different from those of eutopic endometrial glandular and stromal cells from endometriosis-free women and patients with endometriosis.

[A method for DNA template preparation using a single embryo].

We established a simple method for the preparation of DNA template from a single oocyte or early embryo by KOH/DTT-Triton X disintegration. The PCR amplification efficiency of DNA template prepared by this method was compared with that prepared by TE-proteinase K. Single oocyte, 2-cell embryo, 8-cell embryo, morula or blastocyst were separately treated by KOH/DTT-Triton X, then the DNA template was directly used to amplify mitochondrial DNA segment by PCR. The overall PCR success rate of the 3 pairs of primers was 100% (70/70), while the overall PCR success rate of single oocyte treated by TE-proteinase K was 92.9% (65/70). Difference between the two results was significant (P<0.05), and the PCR false positive rates in both groups were 0. The designed KOH/DTT-Triton X disintegrate method was efficient to the preparation of DNA template of a single early embryo. It needed only one cycle of PCR amplification to get clear aimed DNA stripe and the efficiency was high enough to meet the need of early embryonic genetic material detection.

Upregulation of phosphodiesterase-4 in the lung of allergic rats.

Inhibitors of phosphodiesterase-4 (PDE4) are efficacious for allergic asthma in animal models and have shown some efficacy in human asthma. Regulation of PDE4 in allergy and asthma has been widely investigated in blood leukocytes, with discrepant results. This study investigated PDE4 regulation in the lung in a rat model of allergic asthma. Ovalbumin sensitization and challenge significantly increased pulmonary resistance and lung interleukin (IL)-4 production. The increases in pulmonary resistance and IL-4 production were both suppressed by the PDE4-selective inhibitor rolipram or the corticosteroid drug dexamethasone. Furthermore, cAMP-PDE enzyme activity in the lung was also significantly increased by the sensitization and challenge. mRNA analysis confirmed that PDE4 gene expression was increased in the lung of the allergic rats. A highly significant correlation was observed between the increases in PDE activity and IL-4 production. Our data suggest, for the first time, that PDE4 may be upregulated in the lung and play a role in the pathogenesis of allergic asthma.

Spinal dorsal horn calcium channel alpha2delta-1 subunit upregulation contributes to peripheral nerve injury-induced tactile allodynia.

Peripheral nerve injury induces upregulation of the calcium channel alpha2delta-1 structural subunit in dorsal root ganglia (DRG) and dorsal spinal cord of spinal nerve-ligated rats with neuropathic pain, suggesting a role of the calcium channel alpha2delta-1 subunit in central sensitization. To investigate whether spinal dorsal horn alpha2delta-1 subunit upregulation derives from increased DRG alpha2delta-1 subunit and plays a causal role in neuropathic pain development, we examined spinal dorsal hornalpha2delta-1 subunit expression with or without dorsal rhizotomy in spinal nerve-ligated rats and its correlation with tactile allodynia, a neuropathic pain state defined as reduced thresholds to non-noxious tactile stimulation. We also examined the effects of intrathecal alpha2delta-1 antisense oligonucleotides on alpha2delta-1 subunit expression and neuropathic allodynia in the nerve-ligated rats. Our data indicated that spinal nerve injury resulted in time-dependentalpha2delta-1 subunit upregulation in the spinal dorsal horn that correlated temporally with neuropathic allodynia development and maintenance. Dorsal rhizotomy diminished basal level expression and blocked injury-induced expression of the spinal dorsal hornalpha2delta-1 subunit and reversed injury-induced tactile allodynia. In addition, intrathecal alpha2delta-1 antisense oligonucleotides blocked injury-induced dorsal horn alpha2delta-1 subunit upregulation and diminished tactile allodynia. These findings indicate that alpha2delta-1 subunit basal expression occurs presynaptically and postsynaptically in spinal dorsal horn. Nerve injury induces mainly presynaptic alpha2delta-1 subunit expression that derives from increased alpha2delta-1 subunit in injured DRG neurons. Thus, changes in presynaptic alpha2delta-1 subunit expression contribute to injury-induced spinal neuroplasticity and central sensitization that underlies neuropathic pain development and maintenance.

Coupling gene chip analyses and rat genetic variances in identifying potential target genes that may contribute to neuropathic allodynia development.

Genetic factors and nerve injury-induced changes of gene expression in sensory neurons are potential contributors to tactile allodynia, a neuropathic pain state manifested as hypersensitivity to innocuous mechanical stimulation. To uncover genes relevant to neuropathic allodynia, we analyzed gene expression profiles in dorsal root ganglia (DRG) of spinal nerve-ligated Harlan and Holtzman Sprague Dawley rats, strains with different susceptibilities to neuropathic allodynia. Using Affymetrix gene chips, we identified genes showing differential basal-level expression in these strains without injury-induced regulation. Of more than 8000 genes analyzed, less than 180 genes in each strain were regulated after injury, and 19-22% of that was regulated in a strain-specific manner. Importantly, we identified functionally related genes that were co-regulated post injury in one or both strains. In situ hybridization and real-time PCR analyses of a subset of identified genes confirmed the patterns of the microarray data, and the former also demonstrated that injury-induced changes occurred, not only in neurons, but also in non-neuronal cells. Together, our studies provide a global view of injury plasticity in DRG of these rat stains and support a plasticity-based mechanism mediating variations in allodynia susceptibility, thus providing a source for further characterization of neuropathic pain-relevant genes and potential pathways.

[Comparison between intra-operative fine needle aspiration cytology and rapid histopathologic examination of a pancreatic mass].

OBJECTIVE: To evaluate the value in diagnosis by fine needle aspiration cytology and rapid histopathologic examination during operation of a pancreatic mass. METHODS: Both fine needle aspiration cytology and rapid histopathologic examination were performed during the operation of 56 patients with pancreatic mass. RESULTS: The accuracy, sensitivity and specificity of intraoperative cytologic examination were 94.6%, 95.3% and 92.3%, as compared with 92.9%, 90.7% and 100% in rapid paraffin section intraoperative examination. CONCLUSION: Fine needle aspiration is rapid, practical and safe. It is able to improve the diagnosis, pathologic grade and histological origin of pancreatic tumor when combined with rapid histopathologic examination.

[Cyclic nucleotides phosphodiesterase activity in a rat lung model of asthma]

OBJECTIVE: To assess the change of cGMP-PDE and cAMP-PDE activity in a rat lung model of asthma. METHODS: cGMP-PDE and cAMP-PDE activity were determined by HPLC. RESULTS: Both cAMP-PDE activity and cGMP-PDE activity in the lung tissue of antigen-challenged rats were higher than that from the normal rats (P <0.05). CONCLUSION: In the rat lung model of asthma, cyclic nucleotides phosphodiesterase activity was elevated. This may be significant in the pathogenesis of asthma.