Comparison of a Newly Developed HPV Genotyping Assay (Mojin HPV Kit) with Cobas 4800 HPV Test for Detection of High-Risk HPV in Specimens Collected in SurePath Solution.

BACKGROUND: Human papillomavirus (HPV) detection based on cervical cytology specimens is useful for cervical cancer screening. The aim of this study was to compare Mojin HPV kit (a newly developed HPV genotyping assay) with the Cobas 4800 HPV test in detecting high-risk (HR) HPV. METHODS: A total of 347 cervical exfoliated cell specimens were tested using the Mojin HPV kit and Cobas 4800 HPV test. When the results from the two tests were inconsistent, gene sequencing was performed for correction. RESULTS: For HR-HPV, the results of the two assays agreed by 96.3% [Kappa = 0.911; 95% confidence interval (CI): 0.863 - 0.958)]. The positive and negative coincidence rates between the two tests were 96.0% (95% CI: 92.7% - 98.0%) and 97.0% (95% CI: 91.5% - 99.4%), respectively. Of the 13 samples with discordant results, 3 samples were false positive and 10 samples were true negative for Mojin HPV test, according to the identification by sequencing. For HPV16 genotyping, the total coincidence rate between the 2 tests was 100% (Kappa = 1.000), and 99.7% (Kappa = 0.973; 95% CI: 0.905 - 1.000) for HPV18. CONCLUSIONS: Mojin HPV kit may be as effective as Cobas 4800 HPV assay in detecting the total HR-HPV, especially HPV16 or HPV18.

Chinese Xibe population genetic composition according to linkage groups of X-chromosomal STRs: population genetic variability and interpopulation comparisons.

BACKGROUND: The Xibe population is one of China's officially recognised populations and is now distributed separately from west to east in the northern part of China. X-chromosomal short tandem repeats have a special inheritance pattern, and could be used as complements in forensic application, especially for complex or deficiency cases. SUBJECTS AND METHODS: This study obtained the allelic and haplotypic frequencies of 19 X-STR loci in the Xibe population from Xinjiang Uygur Autonomous Region, China, and studied the genetic differentiations between the Xibe and other populations. RESULTS: The combined power of discrimination in females and males and mean exclusion chances in deficiency cases, normal trios and duo cases was at least 0.999 999 994. In the haplotypic study, the Xibe population showed a more similar pattern of haplotype distribution with Asian populations than populations from other continents, while allelic study also indicated a closer relationship between the Xibe and Asian populations. CONCLUSIONS: The 19 X-STR loci would be useful in forensic application in the studied population. The Xibe population showed a closer genetic relationship with Asian populations in the study, and more population data would be necessary for more detailed genetic relationship studies.

[Pathogens in expressed prostatic secretion and their correlation with serum prostate specific antigen: analysis of 320 cases].

OBJECTIVE: To investigate the pathogenic infection and its drug resistance in expressed prostatic secretion (EPS) and its correlation with serum PSA, and provide some evidence for the systematic and normalized diagnosis and treatment of prostatitis. METHODS: Three EPS swabs were collected from each of the 320 prostatis patients following measurement of the serum PSA level, 1 for bacterial culture and identification, 1 for detection of Mycoplasma and drug sensitivity, and the other for examination of Chlamydia trachomatis antigen by colloidal gold immunoblot. RESULTS: Totally 244 strains were isolated from the 320 EPS samples, including 188 bacterial strains (dominated by Staphylococcus and sensitive to vancomycin or linezolid) and 44 Mycoplasma and Chlamydia strains (mainly Ureaplasma urealyticum and susceptible to josamycin or doxycycline). The serum PSA level was significantly higher in the pathogen-positive than in the pathogen-negative group ([6.98 +/- 0.56] microg/L vs [2.32 +/- 0.12] microg/L, P < 0.05). CONCLUSION: Prostatitis may lead to the elevation of the serum PSA level and the pathogens involved vary in their resistance to different antibacterial spectrums. Therefore, appropriate and individualized antibiotic therapy should be selected according to etiological diagnosis and the results of drug sensitivity test.

Rhizobium sphaerophysae sp. nov., a novel species isolated from root nodules of Sphaerophysa salsula in China.

Four gram-negative, aerobic, motile, non-spore, forming rods with a wide pH and temperature range for growth (pH 7.0-11.0, optimum pH 8.0; 20-45°C, optimum 28°C) strains were isolated from root nodules of Sphaerophysa salsula and characterized by means of a polyphasic approach. Phylogenetic analysis based on 16S rRNA gene sequences revealed that the four strains formed a new lineage related to the genus Rhizobium and the sequence similarities between the isolate and the most related type strain Rhizobium giardinii was 96.5%. These strains also formed a distinctive group from the reference strains for defined Rhizobium species based on housekeeping gene sequences (atpD and recA), BOX-PCR fingerprinting, phenotypic features and symbiotic properties. The representative strain CCNWGS0238(T) has DNA-DNA relatedness of less than 33.4% with the most closely related species R. giardinii. It is therefore proposed as a new species, Rhizobium sphaerophysae sp. nov., with isolate CCNWGS0238(T) (=ACCC17498(T) = HAMBI3074(T)) as the type strain.

[Clinical observation of transforaminal endoscopy combined with interspinous fusion in the treatment of lumbar spinal stenosis with instability in the elderly].

OBJECTIVE: To observe the clinical effect of transforaminal endoscopy combined with interspinous fusion in the treatment of lumbar spinal stenosis with instability in the elderly. METHODS: From February 2018 to February 2019, 82 elderly patients with lumbar spinal stenosis and instability were divided into control group and observation group. In the control group, there were 23 males and 18 females;the age was (68.9±4.1) years;the course of disease was (14.1±5.7) months;there were 5 cases of single segment lesions and 36 cases of multi segment lesions;simple bacfuse interspinous fusion was used. In the observation group, there were 22 males and 19 females;the age was (69.1±4.0) years;the course of diseasewas (14.4±5.5) months;there were 6 cases of single segment lesions and 35 cases of multi segment lesions;they were treated with transforaminal endoscopic surgery combined with Bacfuse interspinous fusion. The clinical efficacy, visual analogue scale (VAS), Japanese Orthopaedic Association scores (JOA), Oswestry disability index (ODI), Lehmann lumbar function score, posterior disc height and intervertebral foramen height, complication rate and recurrence rate of the two groups were compared. RESULTS: The clinical efficacy of the observation group was better than that of the control group;the VAS score of the observation group was lower than that of the control group, the JOA score was higher than that of the control group, and the ODI index at 3 months after operation and at the last follow-up was lower than that of the control group, the Lehmann lumbar function score was higher than that of the control group;the posterior edge height of intervertebral disc and intervertebral foramen height were higher than those of the controlgroup;the incidence of complications and recurrence rate (4.9% and 0.0%) of the observation group were lower than those of the control group (19.5%, 9.8%), the difference was statistically significant (P<0.05). CONCLUSION: The clinical effect of transforaminal endoscopy combined with interspinous process fusion in the treatment of lumbar spinal stenosis with instability in the elderly is ideal. It can reduce postoperative pain, improve lumbar function, improve the height of posterior edge of intervertebral disc and intervertebral foramen, and reduce the incidence and recurrence rate. It is worthy of clinical promotion.

[Preliminary study of the dual release baicalin and rhBMP-2 system to improve periodontal tissue regeneration in minipigs].

PURPOSE: To explore the feasibility of dual release baicalin and rhBMP-2 system for the treatment of periodontal diseases in minipigs. METHODS: Four miniature swines were selected to establish the model of periodontitis and randomly divided them into four groups: Dual release group, hydrogel with baicalin-GMS and rhBMP-2; Single Huang group, hydrogel with baicalin-GMS; BMP group, hydrogel with rhBMP-2; Negative control group, blank hydrogel. All parameters including clinical indicators of periodontitis, the level of IL-1 and TNF-α in the gingival crevicular fluid (GCF) were measured before and 6 weeks after modeling, and 4, 8 weeks after treatment. The data was analyzed using SPSS13.0 software package. The periodontitis model animals were sacrificed at the end of 8 weeks after treatment. The histological changes of periodontal tissues were observed under microscope with HE staining. RESULTS: (1)In all groups, there were significant differences of clinical indicators and levels of IL-1 and TNF-α in the GCF between periodontitis was established and 4, 8 weeks after treatment (P<0.05). Dual release group was significantly superior than that of other three groups (P<0.05). Moreover, all the parameters improved continuously. X-ray showed that bone mineral density and height in the remaining three groups increased compared with the control group, with highest in dual release group. (2)The results of histological observation showed that the inflammation reaction of periodontal tissues in negative control group was more serious than that in other groups 8 weeks after treatment. There was no obvious repair reaction in the control group, but different amount of new bone was seen in other three groups. Among them, dual release group had most obvious repair reaction. CONCLUSIONS: The dual-slow-release chitosan thermosensitive hydrogel system is employable to be used effectively in the regeneration of periodontal defects, which laid a foundation for further clinical use.

[Recombinant hFOXA2 and hPDX1 lentivirus induced dental pulp stem cells from deciduous teeth reprogramming for insulin-producing cells].

PURPOSE: The purpose of this study was to culture and identify dental pulp stem cells(DPSCs) from deciduous teeth in vitro and construct the recombinant hFOXA2 and hPDX1 lentivirus vectors and transfect the DPSCs to induce insulin-producing cells (IPCs). METHODS: DPSCs were separated and cultured by enzyme digest method, and purified by limited dilution method. Flow cytometry was used to determine the surface marker expression of the DPSCs, and the ability of multiple differentiations was determined by specific staining. hFOXA2 and hPDX1 genes were amplified by PCR, and the recombinant hFOXA2 and hPDX1 lentivirus vectors were reconstructed and transfected into 293T cells by lipofectamine2000 for virus packaging. The viral infection efficiency and titer were determined through fluorescence cell count. The recombinant virus was used to infect the DPSCs cells via multiplicity of infection (MOI) and induce the DPSCs reprogramming for IPCs. Immunofluorescence staining was used to measure the expression of proinsulin, FOXA2 and PDX1. ELISA method was used to detect the insulin secretion. The data was analyzed Using SPSS13.0 software package. RESULTS: DPSCs were isolated and cultured successfully. Cell surface highly expressed STRO-1 (98.01%), CDl46 (98.51%), CD34 (99.54%) and CD45 (24.08%). The multi-lineage differentiation capacity into osteoblasts, chondrocytes, and adipose was achieved. The recombinant hFOXA2 and hPDX1 lentivirus vectors were successfully constructed. Double enzyme digestion and sequencing appraisal showed that the sequence was fully consistent with GenBank retrieval. Virus packing efficiency was (96.15±0.17) % and (95.49±0.21) % respectively, and the infection titer was about 1.80±108 GTU/mL. The best MOI of the virus was 20. After inducing the cells to express proinsulin, FOXA2 and PDX1, insulin secretion volume was about 1.92 µmol/L. Compared with the uninduced group and control group, insulin secretion increased significantly (P<0.01). CONCLUSIONS: The recombinant transcription factor virus can activate cell reprogramming mechanism, form insulin-producing cells, and can be used for gene therapy of diabetes seed cells. Supported by Science and Technology Research Program of Shaanxi Province (2009K17-06) and Science and Technology Innovation as a Whole Plan Resources Leading Industry Key Technology (Chain) Project of Shaanxi Province (2011KTCL03-24).

[Study on hepatitis C virus genotypes in Yantai district, Shandong province].

OBJECTIVE: To understand the hepatitis C virus (HCV) genotype distribution in Yantai district of Shandong province, and to explore whether the HCV genotypes was relevant to the injure of liver through the index of liver function. METHODS: Using specific PCR primers to amplify the HCV RNA 5' UTR/NS5B,then PCR products were sequenced by genetic analyzer. The genotypes were identified by alignment to the GenBank reference sequences and construction the phylogenetic tree of 5' UTR. RESULTS: Among 9 unpaid blood donors we detected two kinds of genotypes of 1b and 3a, respectively, 8 cases (88.9%) and 1 case (11.1%). Among 33 cases of hepatitis C patients we detected the 1b, 2a and 6a the three kinds of genotypes, respectively, 22 (66.7%), 10 (30.3%) and 1 (3.03%) cases. Subtype 1b is the advantage of popular genotype in HCV carriers from Yantai district, and the distribution of 1b was no significant difference in the different population (chi2 = 0.796, P = 0.373); The difference of indicator of liver damage in the different genotypes of subjects were significant (P < 0.05), the mean of ALT, AST of 2a-subtype carriers was significantly higher than the 1b-subtype population. CONCLUSIONS: The genetic diversity of HCV in Shandong Yantai district was presented. The main genotypes were 1b-subtype, and 3a and 6a-subtype was detected firstly. The genotype of HCV were relevant to the liver damage indicators, 2a-subtype hepatitis C virus infection in the liver cells may play an important role in the disease process.

[Cloning of the glyceraldehydes 3-phosphate dehydrogenase gene of porphyromonas gingivalis and its expression in E. coli].

OBJECTIVE: To clone the glyceraldehydes 3-phosphate dehydrogenase (GAPDH) gene of Porphyromonas gingivalis (P. gingivalis) and to induce its fusion expression in E. coli. METHODS: GAPDH was obtained by PCR and was inserted into cloning vector pMD-18-T to construct clone recon. Double enzymes digest the recon pMD18-T-GAPDH and the prokaryotic expression vector pET-32a and then connect to get the expressing recon pET-32a-GAPDH. The recombinant expression plasmid which had been confirmed by enzymes digestion was transformed to E. coli competent cells BL21 and induced the expression of GAPDH with isopropyl beta-D-1-thiogalactopyranoside (IPTG) of different density. RESULTS: DNA sequencing showed that the fragment was 99.802% the same to the sequence published in NCBI. Under the best density, IPTG could be highly expressed. CONCLUSION: The GAPDH had been successfully cloned and expressed in E. coli which gets ready for the following experiment to study the immunity of GAPDH and the homologues antibody preparation.

[Construction and identification of BIRC5 shRNA lentiviral expression vector].

AIM: Design and synthesis complementary DNA sequences of 3 pairs of short hairpin structure and a pair of negative control sequence by RNA interference technique and construct and identify a lentiviral interference vector with human BIRC5 gene as target gene. METHODS: The designed and synthesised Single-Stranded primer were annealed to Double-Stranded oligo sequences and subcloned into linear pMAGic lentiviral plasmid vector digested by enzyme Age I and EcoR I. Screening positive clone after transformed into DH5α competent cells and identified by PCR amplification and DNA sequencing. RESULTS: 335 bp straps of positive clone and 298 bp straps of negative clone form PCR amplification production have been obtained after gel electrophoresis, the designed and synthesised sequences have been contained in these clone straps confirmed by the result of DNA sequencing. CONCLUSION: Four pairs of BIRC5 shRNA recombinant lentiviral expression vector were constructed successfully, which laid the foundation for researching the inhibition of BIRC5 siRNA target against tumor cells proliferation, induction apoptosis and gene therapy.

[Cloning and expression of peptidylarginine deiminase of Porphyromonas gingivalis].

PURPOSE: To clone the peptidylarginine deiminase(PAD) gene of Porphyromonas gingivalis(P.gingivalis) and to be expressed in E. coli under the best conditions. METHODS: With P.gingivalis strains ATCC33277 genomic DNA as a template, PCR was applied to obtain gene PAD which was then inserted into linear cloning vector PMD-18-T to construct clone recon. Recombinant PMD18-T-PAD was cloned and analyzed with PCR and restriction endonuclease,and PET-28a expression vector was digested by Xhol and Ncol,their products were linked to construct expression plasmid PET-28a-PAD under certain connection system. The recombinant expression plasmid PET-28a-PAD which had been confirmed correctly was transformed to E. coli competent cells BL21 and induce the expression of PAD with IPTG of different density and time. With His Tag monoclonal antibody as the first antibody, the expressed fusion protein was characterized by Western blot. RESULTS: DNA sequencing showed that the fragment was same as the sequence published in NCBI. Under the condition of 37 degrees centigrade, 0.5mmol/L IPTG, 250r/min shaking for 6 hours, the PAD could be highly expressed. CONCLUSIONS: The PAD is successfully cloned and expressed in E. coli which can be further uesd to study the immunity of PAD and the homologues antibody preparation.

[Experimental study on the functional regulation of naringin in human periodontal ligament cells].

PURPOSE: To detect the effect of naringin on human periodontal ligament cells' (hPDLCs) proliferation,bone formation and OPG mRNA expression. METHODS: hPDLCs were primarily cultured and identified in vitro through enzyme digestion combined tissue culture method. With 3-(4, 5-Dimethylthiazol-2- yl)-2, 5-diphenyltetrazolium bromide (MTT) method, enzyme linked immunosorbent assay and RT-PCR, the hPDLCs' proliferation, alkaline phosphatase (ALP) activity, expression of collagen protein-I and OPG mRNA were observed after treatment with different concentrations (100,10,1.0,0.1,0.01mg/L) of naringin at different times. SPSS16.0 software package was used for statistical analysis. RESULTS: Primary cultured hPDLCs had good shape; Significant promotion of proliferation,ALP activity and collagen protein-I expression of hPDLCs with naringin was found at the dose of 1.0 mg/L; and 1.0mg/L naringin regulated the expression of OPG mRNA in time-dependent manner. CONCLUSION: Naringin might significantly promote the proliferation of hPDLCs and conversion into the osteoblast.

[A preliminary study on screening for Porphyromonas gingivalis outer membrane protein antigen with two-dimensional liquid phase fractionation and matrix-assisted laser desorption ionization time-of-flight/time-of-flight mass spectrometry].

OBJECTIVE: To screen a variety of Porphyromonas gingivalis (Pg) common outer membrane proteins with two-dimensional liquid phase fractionation (PF2D) and matrix-assisted laser desorption ionization time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF-MS) and provide candidate target antigen for the design of vaccines with cross protection against a variety of Pg. METHODS: The outer membrane proteins of Pg301, PgATCC33277 and PgW83 were extracted through ultracentrifugation, and then they were separated by ProteomeLab PF2D protein fractionation system. After separation, the outer membrane proteins were obtained through comparison, and the primary structure of the proteins was identified by MALDI-TOF/TOF-MS and database. RESULTS: Ninety-nine protein samples out of 3 strains of Pg were obtained after the high performance chromato focusing (HPCF) separation process. B7 fractions of 3 strains of Pg were separated by the reversed-phase high performance liquid chromatography (RP-HPCL) separation process. After comparison of peak and retention time of chromatogram, the 8 common protein peaks of 3 strains of Pg were confirmed. The protein samples were identified by MALDI-TOF/TOF-MS, and one of them was known protein arg-gingipain A. CONCLUSIONS: PF2D protein fractionation system is of good reproducibility and high resolution. A combination of PF2D and MALDI-TOF/TOF-MS can be used to identify the common outer membrane proteins of Pg.

[Analysis of outer membrane proteins in various strains of Porphyromonas gingivalis by surface enhanced laser desorption/ionization time-of-flight mass spectrometry].

OBJECTIVE: To analyze the common outer membrane proteins (OMP) from Porphyromonas gingivalis (Pg) by surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) and to provide antigens for the subsequent experiments in screening vaccine for periodontitis therapy. METHODS: Four strains of Pg were cultured under anaerobic conditions. The common OMP was extracted through ultracentrifugation and SELDI-TOF-MS was employed to detect the expressions of proteomes by chip H(50). The data was analyzed by Biomarker Wizard. RESULTS: Four kinds of strains of OMP fingerprint spectrum were obtained. Seventy-one proteins of PgATCC33277, 74 proteins of PgW83, 76 proteins of PgW301 and 72 proteins of Pg381 were captured by chip H(50). Thirteen common proteins were identified according to fingerprint spectrum. There was only 1 of the 13 common proteins identified in NCBI protein bank. CONCLUSIONS: SELDI-TOF-MS has good reproducibility and high sensibility and can be used to identify the common OMP of Pg. The 13 proteins have a potential value in the screening vaccine candidate antigen sites for periodontitis.

[Construction of prokaryotic expression vector of FimA gene from Porphyromonas gingivalis, fusion expression and purification in E. coli BL21(DE3)pLyS].

OBJECTIVE: To clone the FimA gene of fimbriae from Porphyromonas gingivalis (P. gingivalis) and to construct prokaryotic expression vector which was induced in E.coli BL21(DE3)pLyS in the form of fusion protein expression and to identify, purify the product of its expression. METHODS: To clone the FimA gene of fimbriae from P. gingivalis and to construct prokaryotic expression vector pET15b-FimA vector which was transformed into the competent cells of BL21(DE3)pLyS. The expression of fusion protein was induced by isopropyl beta-D-1-thiogalactopyranoside (IPTG). With anti-6xHis Tag monoclonal antibody as the first antibody, the expressed fusion protein was characterized by Western blot and purified by Co(2+)-NTA affinity chromatography. RESULTS: Cloned FimA gene sequences and inserted into expression vector of the FimA sequences were related to the sequence in GenBank database showed 100% homology. IPTG induced and then identified by Western blot showed a fragment of 4.1 x 10(4) has been expressed. Co(2+)-NTA affinity chromatography column was used to obtain high concentrations of FimA purified protein. CONCLUSION: The recombinant prokaryotic expression vector of pET15b-FimA was constructed and was expressed and purified successfully in E. coli BL21 (DE3)pLyS. This study laid the experimental foundation to further prepare for monoclonal antibodies of fimbriae of P. gingivalis and to develop the subunit protein vaccine of prevention of periodontitis.

[Isolation, culture and purification of olfactory ensheathing cells from human fetal olfactory mucosa].

OBJECTIVE: To explore the method for obtaining olfactory ensheathing cells from human fetal olfactory mucosa by cell culture for selective adhesion in the presence of neurotrophin-3 (NT3) and low-concentration serum. METHODS: The olfactory ensheathing cells were cultured alternatively in DMEM/F12 culture medium containing 10% fetal bovine serum (FBS) and the medium containing NT3 and 2.5% FBS every 72 h. The cells were observed for morphological changes and identified using immunocytochemistry with P75NTR and GFAP, and the cell purity was estimated. RESULTS: The olfactory ensheathing cells from human fetal olfactory mucosa were positive for P75(NTR) and GFAP, and in in vitro culture, the cells exhibited dipolar or tripolar appearance with long thin neurites. On the 9th day of cell culture, the purity of the olfactory ensheathing cells reached about 83%. CONCLUSION: The olfactory ensheathing cells can be obtained by in vitro culture for selective adhesion in the presence of NT3 and low-concentration serum.