OBJECTIVETo 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.

METHODSThe 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.

RESULTSNinety-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.

CONCLUSIONSPF2D 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.

Antigens, Bacterial ; analysis ; Mass Spectrometry ; Membrane Proteins ; analysis ; Porphyromonas gingivalis ; immunology ; Reproducibility of Results ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ; Vaccines

OBJECTIVETo investigate therapeutic effects of minimally invasive arthroscopic internal fixation with plates and screws in treating tibial plateau fractures.

METHODSA retrospective study from December 2006 to June 2010 was done on 69 patients with tibial plateau fractures. According to Schatzker classification, 5 patients were type I, 5 patients were type II, 25 patients were type III, 20 patients were type IV, 9 patients were type V and 5 patients were type VI. Thirty-six patients were treated with arthroscopy-assisted reduction and internal fixation, including 21 males and 15 females, ranging in age from 17 to 59 years (averaged, 34.2 years); another 33 patients were treated with small incision and fixed with plates and screws,including 19 males and 14 females, ranging in age from 18 to 62 years (averaged, 35.4 years). The operation time, blood loss during operation,drainage volume of blood, healing time, weight-bearing time and function of effected knee were compared between the two groups.

RESULTSAll the patients were followed up,and the duration ranged from 6 to 12 months (averaged, 10.3 months). All the patients had no complications such as infection, articular collapse, re-fracture and joint stiffness. There were no significant differences in weight-bearing time, complications and Rasmussen scores between two groups (P>0.05); but there were significant differences in the operative time, blood loss, drainage volume of blood, hospital stay time, the healing time between two groups (P<0.05). The results showed that arthroscopy-assisted technique was better than minimally invasive internal fixation in operation duration, blood loss during operation and the healing time.

CONCLUSIONDifferent types of fracture of tibial plateau should be treated with different surgical treatments. Arthroscopic technique for reduction of fractures, which has less influence on bony union and minimally invasive, and can provide a good clinical outcome.

Adolescent ; Adult ; Arthroscopy ; methods ; Case-Control Studies ; Female ; Fracture Fixation, Internal ; methods ; Humans ; Male ; Middle Aged ; Minimally Invasive Surgical Procedures ; methods ; Tibial Fractures ; surgery ; Young Adult

OBJECTIVETo clone the glyceraldehydes 3-phosphate dehydrogenase (GAPDH) gene of Porphyromonas gingivalis (P. gingivalis) and to induce its fusion expression in E. coli.

METHODSGAPDH 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.

RESULTSDNA 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.

CONCLUSIONThe 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.

Cells, Cultured ; Cloning, Molecular ; Cloning, Organism ; Escherichia coli ; Genetic Vectors ; Glyceraldehyde ; Oxidoreductases ; Phosphates ; Polymerase Chain Reaction ; Porphyromonas gingivalis

OBJECTIVETo 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.

METHODSFour 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.

RESULTSFour 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.

CONCLUSIONSSELDI-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.

Antigens, Bacterial ; analysis ; Membrane Proteins ; analysis ; Porphyromonas gingivalis ; chemistry ; immunology ; Proteins ; Proteome ; Reproducibility of Results ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

OBJECTIVETo 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.

METHODSTo 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.

RESULTSCloned 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.

CONCLUSIONThe 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.

Cloning, Molecular ; Escherichia coli ; Porphyromonas gingivalis ; Recombinant Fusion Proteins ; Recombinant Proteins

OBJECTIVETo clone the catalytic domain gene sequence of RgpAcd of Porphyromonas gingivalis (P. gingivalis) and to induce its fusion expression in E. coli.

METHODSThe desired DNA fragment RgpAcd was obtained by PCR and was separately sequenced and identified by inserting into inter-vector pMD18-T vector. The correctly fragment was linked with and cloned into a prokaryotic expression vector pET-15b. The recombinant expression plasmid which had been confirmed by enzymes digestion was transformed to E. coli competent cells BL21 (DE3) and expression of fusion protein was induced by IPTG.

RESULTSA 1 476 bp specific fragment was obtained and DNA sequencing showed that the fragment was consistent with those of the published. After induction with IPTG, a fusion protein of 5 x 10(4) was visualized on SDS-PAGE gel.

CONCLUSIONThe protein of RgpAcd will be obtained for further study and its protein was correctly expressed in E. coli BL21 cells.

Cloning, Molecular ; Cloning, Organism ; Escherichia coli ; Genetic Vectors ; Polymerase Chain Reaction ; Porphyromonas gingivalis ; Recombinant Fusion Proteins ; Recombinant Proteins

Objective:To study the clinical characteristics and risk factors of nephrocalcinosis in preterm infants.Methods:From March 2021 to August 2021, all preterm infants admitted to NICU of our hospital were retrospectively analyzed. The infants were assigned into nephrocalcinosis group and non-nephrocalcinosis group according to urinary tract ultrasound. Clinical data including gestational age, birth weight(BW), nutritional support strategy and complications were reviewed.Results:A total of 40 preterm infants (<34 weeks) were enrolled. 9 cases were in the nephrocalcinosis group and 31 cases in the non-nephrocalcinosis group. The nephrocalcinosis group had lower BW[(1 167±214) g vs.(1 586±215) g], higher calcium [6.9 (5.1, 8.7) g vs.3.3 (2.1, 6.8) g] and vitamin D intake [3.2(2.5, 4.2)×10 4U vs.1.7(1.1, 3.2)×10 4U] during hospitalization. No significant differences existed between the two groups on the following items:blood calcium and phosphate, 25-hydroxyvitamin D, feeding strategy, time to reach full enteral feeding(TFF), furosemide dosage and respiratory support duration ( P>0.05). In the nephrocalcinosis group, the median age of diagnosing nephrocalcinosis was 40.0(30.0, 52.5)d after birth. 5 cases showed bilateral nephrocalcinosis. 5 cases in the nephrocalcinosis group received renal tubule function examination,4 cases had increased urine β2 microglobulin and 2 cases had increased urine α1 microglobulin. 7 cases had elevated urine calcium in the nephrocalcinosis group. Follow-up showed that nephrocalcinosis disappeared 3-9 months after birth. Conclusions:BW, total calcium and vitamin D intake are risk factors for nephrocalcinosis in preterm infants. Increased urine β2 microglobulin and calcium levels are common co-morbidities in preterm infants with nephrocalcinosis.

OBJECTIVETo study the genotyping of Bacillus anthracis based on multiple-locus variable-number tandem repeats(VNTR) in the B. anthracis genome.

METHODSWe selected 13 VNTR loci (which cited from published articles) to study 88 strains of B. anthracis isolated from China. The methods used were: (1) Selecting the primers which were at both ends of the tandem repeat locus; (2) Amplifying the sequence of the locus by PCR; (3)cDetecting the PCR products by agarose gel and polyacrylamide electrophoresis; (4)Analyzing the PCR products and computing the molecular weight by analysis software of gel images;(5) Double-checking with sequencing results; (6)Reckoning the repeat numbers and study the VNTRs loci characters.

RESULTS(1) We used multiple-locus variable-number tandem repeat analysis (MLVA) to characterize 88 B. anthracis isolates from diverse geographic locations which were divided into 45 MLVA genotypes and 3 groups through cluster analysis. The genotypes was relative to restricted geographical region. It seemed clear that the multiple isolates from the same anthrax outbreak frequently having identical genotypes. (2)Results from VNTR analysis showed that A16R vaccine strain isolated from China was having the nature of representativeness in the country.

CONCLUSIONAnalysis showed that the VNTR patterns was an appropriate study method for B. anthracis genetic diversity from different geographical areas and different time. Isolates from the same anthrax outbreak had identical

Anthrax ; epidemiology ; genetics ; Bacillus anthracis ; genetics ; isolation & purification ; China ; epidemiology ; Genetic Variation ; Genotype ; Geography ; Polymerase Chain Reaction ; Sequence Analysis, DNA ; Tandem Repeat Sequences

OBJECTIVETo analyze the genetic characterization of enterovirus type71 (EV71) associated with hand foot and mouth disease (HFMD) epidemics in Jilin province, during 2009-2010.

METHODSRandomly selected 31 representative EV71 strains from the cases of 8 prefectures to amplify and sequences of VP1 genes of EV71 strains, and analyzed with Bioedit and Mega4.0 program.

RESULTSAll representative 31 EV71 strains belong to C4a subgenotype, the homology of nucleotide in VP1 region among the 31 EV71 strains were 94. 5%-100. 0%, and were clustered into 5 transmission chains respectively. 25 strains out of 31 strains were associated with a predominant transmission chain, and circulating in 8 prefectures, while other 6 strains clustered into 4 lineages.

CONCLUSIONMultiple transmission chains of EV71 C4a subgenotype were co-circulating in Jilin province during 2009-2010, and a predominant transmission chain was circulating in 8 prefectures, associated with HFMD outbreaks of Jilin province.

China ; epidemiology ; Disease Outbreaks ; Enterovirus D, Human ; classification ; genetics ; isolation & purification ; Feces ; virology ; Hand, Foot and Mouth Disease ; epidemiology ; transmission ; virology ; Humans ; Molecular Sequence Data ; Phylogeny

OBJECTIVES: Safe and effective anticoagulation is essential for hemodialysis patients who are at high risk of bleeding. The purpose of this trial is to evaluate the effectiveness and safety of two-stage regional citrate anticoagulation (RCA) combined with sequential anticoagulation and standard calcium-containing dialysate in intermittent hemodialysis (IHD) treatment. METHODS: Patients at high risk of bleeding who underwent IHD from September 2019 to May 2021 were prospectively enrolled in 13 blood purification centers of nephrology departments, and were randomly divided into RCA group and saline flushing group. In the RCA group, 0.04 g/mL sodium citrate was infused from the start of the dialysis line during blood draining and at the venous expansion chamber. The sodium citrate was stopped after 3 h of dialysis, which was changed to sequential dialysis without anticoagulant. The hazard ratios for coagulation were according to baseline. RESULTS: A total of 159 patients and 208 sessions were enrolled, including RCA group (80 patients, 110 sessions) and saline flushing group (79 patients, 98 sessions). The incidence of severe coagulation events of extracorporeal circulation in the RCA group was significantly lower than that in the saline flushing group (3.64% vs. 20.41%, P<0.001). The survival time of the filter pipeline in the RCA group was significantly longer than that in the saline flushing group ((238.34±9.33) min vs. (221.73±34.10) min, P<0.001). The urea clearance index (Kt/V) in the RCA group was similar to that in the saline flushing group with no statistically significant difference (1.12±0.34 vs. 1.08±0.34, P=0.41). CONCLUSIONS Compared with saline flushing, the two-stage RCA combined with a sequential anticoagulation strategy significantly reduced extracorporeal circulation clotting events and prolonged the dialysis time without serious adverse events.
Humans ; Citric Acid/adverse effects* ; Prospective Studies ; Sodium Citrate ; Hemorrhage/chemically induced* ; Citrates/adverse effects* ; Anticoagulants/adverse effects* ; Renal Dialysis/adverse effects*