Objective To clone and express the polymorphic membrane protein I(PmpI)gene of Chlamydia trachomatis(Ct), and to assess the immunogenicity and biological characteristics of PmpI. Methods A bioinformatic software was used to analyze the sequence of the PmpI gene of Ct, and to predict B cell epitopes in PmpI. With Ct serovar D DNA as the template, PCR was performed to amplify the N?terminal region(from position 90 to 1464)of the PmpI gene, which was cloned into a prokaryotic expression vector pET28a to express the recombinant protein PmpI. A Ni?ion affinity chromatography column was used to purify the recombinant protein, which was used to immunize New Zealand rabbits for preparation of polyclonal antibodies. Western blot analysis was conducted to evaluate the immunogenicity of this protein. Results A comprehensive analysis was carried out on the secondary structure, flexible regions, hydrophilicity plot, antigenic index and surface probability plot of the protein, which suggested that PmpI had 8 dominant B?cell epitopes. The product of PCR targeting the PmpI gene of Ct serovar D showed a total length of 1 375 bp. The recombinant prokaryotic expression vector pET28a?PmpI was successfully constructed. A recombi?nant protein with a relative molecular mass of approximately 50 000 was successfully expressed after isopropylβ?d?1?thiogalactopyranoside (IPTG) induction, and purified by affinity chromatography. Polyclonal antibodies against the recombinant protein were successfully prepared. Conclusion The N?PmpI protein of Ct serovar D is cloned and expressed successfully, laying a foundation for further studies on its biological functions.

Two sample pretreatment methods of pesticide residues in Panax notoginseng of Chinese traditional medicine were developed. For Method I, the residues were extracted from homogenized tissue with n-hexane-dichloromethane (6:4) by means of ultrasonication, the crude extract was purified by an Envi-carb/NH2 solid-phase extraction (SPE) column. For Method II, matrix solid-phase dispersion (MSPD) technique was used for extracting and cleaning up. The eluates were concentrated by rotary evaporation, and then were redissolved in dichloromethane prior to GC-MS determination. The determination was performed in selected ion monitoring (SIM) mode with the external calibration for quantitative analysis. Under the optimal conditions, the results indicated that the methods are easier and faster, the recoveries of method I for the spiked standards at concentration of 0.01, 0.5, and 2.0 mg x kg(-1) were 81.90%-102.10% with the relative standard deviations (RSDs) of 3.60%-7.10%. The recoveries of method II were 96.26%-104.20% with the RSDs of 3.52%-7.94%. The detection limits (S/N) for residues of pesticides were in the range of 0.48-1.34 ng x g(-1). The results indicated that these multiresidue analysis methods can meet the requirements for determination of residue pesticides and can be appropriate for trace analysis of residue pesticides in Panax notoginseng.

New strategies to improve vaccine efficacy against human immunodeficiency virus type 1 (HIV-1) are still required. DNA vaccines, exhibiting potential advantages over conventional vaccines for their simplicity and versatility, can induce specific humoral and cellular immune responses. We developed a recombinant pVAX1 DNA vector carrying p24 gene of HIV-1. The results showed that pVAX1 mediated gene possessed the ability of effective expression in both transfected 293T cells and BALB/c mice. And pVAX1-p24 DNA prime and boost immunization can induce significant P24-specific humoral immune responses and cellular immune responses in BALB/c mice. Furthermore, immunization with pVAX1-p24 DNA prime and protein boost induced 7.3 to 8.0-fold greater p24-specific humoral responses than pVAX1-p24 DNA prime and boost, while the cellular immune responses induced by combined immunization was lower. The results suggested that pVAX1-p24 DNA and P24 protein vaccine is a promising HIV-1 vaccine, and the selections of the immunization strategies are important for the immunization results.
AIDS Vaccines ; genetics ; immunology ; Animals ; DNA ; genetics ; immunology ; HEK293 Cells ; HIV Core Protein p24 ; genetics ; immunology ; Humans ; Immunization ; Mice ; Mice, Inbred BALB C ; Vaccines, DNA ; genetics ; immunology

Radiotherapy is a treatment for cancer with undesired by-effects. In order to develop a new radiation protective agent that could reduce the by-effects, we tried to express and purify human cryptochrome 1 (hCRY1). The coding sequence of hCRY1 was inserted into prokaryotic expression plasmid pET28a(+), and this protein was purified from Escherichia coli BL21(DE3) after IPTG induction, ultrasonication, inclusion body dissolution, gradient dialysis, nickel column purification and ultrafiltration. The yield of hCRY1 in 1 L E. coli culture (LB medium) was about 10-15 mg. The radiation protective efficiency of hCRY1 was monitored by detecting X-ray-induced H2A.X foci in HaCaT cells. The results of immunofluorescence show that hCRY1 significantly reduces X-ray stimulated DNA damage response. The apoptosis of HaCaT cell was also detected, and the repression of H2A.X foci formation was not due to hCRY1's cytotoxity. All these data suggest a potential application of recombinant hCRY1 as a protective agent for radiotherapy.
Cryptochromes ; biosynthesis ; Escherichia coli ; Humans ; Plasmids ; Radiation-Protective Agents ; Recombinant Proteins ; biosynthesis

RNA silencing is a conserved eukaryotic pathway involved in the suppression of gene expression via sequence-specific interactions that are mediated by 21-23 nt RNA molecules. During infection, RNAi can act as an innate immune system to defend against viruses. As a counter-defensive strategy, silencing suppressors are encoded by viruses to inhibit various stages of the silencing process. These suppressors are diverse in sequence and structure and act via different mechanisms. In this review, we discuss whether RNAi is a defensive strategy in mammalian host cells and whether silencing suppressors can be encoded by mammalian viruses. We also review the modes of action proposed for some silencing suppressors.
Animals ; Gene Expression Regulation, Viral ; Gene Silencing ; Host-Pathogen Interactions ; Humans ; Mammals ; virology ; MicroRNAs ; genetics ; metabolism ; Plant Viruses ; physiology ; Plants ; virology ; RNA, Small Interfering ; genetics ; metabolism ; Repressor Proteins ; genetics ; metabolism ; Viral Proteins ; genetics ; metabolism ; Viruses ; growth & development

Objective To certify that Chlamydia can spread from the genital tract to the gastrointestinal tract for long-lasting colonization.Methods Totally,120 female C57BL/6J mice aged 5-6 weeks were divided into 4 experimental groups to be inoculated with purified Chlamydia muridarum (C.muridarum) elementary bodies in the vagina (n =35),gastric area (n =30),anus and rectum (n =30),retro-orbital venous plexus (n =5) respectively.Moreover,corresponding negative groups inoculated with sucrose phosphate glutamate buffer (n =5) were set up for each experimental group.On days 3,7,and every 7 days,vaginal and rectal discharges were collected with swabs from the mice,and the number of live C muridarum orgnisms in exfoliated cells infected with C muridarum in the swabs was determined.Indirect immunofluorescence assay and quantitative PCR (qPCR) were performed to determine the number of live chlamydial organisms and the copy number of chlamydial genomes in the mouse genital tract (vagina,uterus,oviduct and ovary),gastrointestinal tract (stomach,small intestine,cecum,colon,rectum)and parenteral tissues (heart,liver,spleen,lung,kidney) on days 7,14,28,56 and 105 after the inoculation.The number of live chlamydial organisms and copy number of chlamydial genomes were transformed logarithmically with a base of 10.The degree of hydrosalpinx and inflammation in the genital tract,and histopathological changes of the gastrointestinal tract were observed.The infectivity and virulence of C.muridarum in the genital tract and gastrointestinal tract were evaluated in the intragastric inoculation group and intra-anal and intrarectal inoculation group on days 28 and 56 after the inoculation.Blood samples were obtained from the mouse caudal vein in the retro-orbital venous plexus inoculation group on days 3,5,7,10 and 14 after the inoculation,the number of live chlamydial organisms and the copy number of chlamydial genomes in the blood samples were determined,and chlamydial infectivity in the genital tract and gastrointestinal tract was evaluated on day 56.Results On day 7 after the inoculation in the vagina,both C.muridarum live organisms and genomes were detected in the genital tract,gastrointestinal tract and parenteral tissues of all the mice.The largest common logarithm of the number of C.muridarum inclusion forming units (IFU) was observed in the vagina (6.26 ± 0.56),with the common logarithm of the copy number of chlamydial genomes in the vagina being 7.30 ± 0.23,and the common logarithms of the number of Chlamydia IFU and genomic copy were 2.60 ± 1.95 and 4.87 ± 0.09 respectively in the rectum.On day 28,no live Chlamydia was detected in the heart,lung or other parenteral tissues,while live Chlamydia could be found in the genital tract and gastrointestinal tract.The common logarithms of the number of Chlamydia IFU and genomic copy were 3.47 ± 1.06 and 5.80 ± 1.49 respectively in the vagina,and 4.00 ±0.35 and 5.14 ± 0.81 respectively in the rectum.On day 56,live Chlamydia could only be detected in the gastrointestinal tract.On day 105,live Chlamydia and its genomes could be still detected in the gastrointestinal tract,and the common logarithms of the number of Chlamydia IFU and genomic copy could be up to 2.60 ± 0.65 and 4.29 ± 0.57 respectively in the rectum.On days 28 and 56 after the inoculation,both live Chlamydia and its genomes could be detected in the gastrointestinal tract of all the mice in the intragastric inoculation group and intra-anal and intrarectal inoculation group.Chlamydia could survive in the blood for about 14 days in the retro-orbital venous plexus inoculation group,and live Chlamydia was detected in anal-rectal swabs in all the mice on day 14.On day 56 after the intravaginal inoculation with C.muridarum,severe hydrosalpinx,chronic inflammation and oviduct dilation occurred in the genital tract of 5 mice,but there was no obvious infiltration of inflammatory cells in the gastrointestinal tract,and inflammatory pathological changes were not observed in the gastrointestinal tract of mice after inoculation with Chlamydia through other routes either.Conclusion The infection with Chlamydia in the genital tract can lead to systemic dissemination,and Chlamydia can be spread to the gastrointestinal tract,and colonize and survive in the gastrointestinal tract for a long time.

Objective To construct active phages against Chlamydia trachomatis,and to evaluate its effect on Chlamydia trachomatis.Methods The M13 phage was recombined with the IN5 sequences encoding the capsid protein VP1 of chlamydiophage phiCPG1,and then the recombinant M13-IN5 phage was obtained.PCR amplification,enzyme digestion and sequencing were performed to verify whether the target fragment was inserted into the phage successfully.The viability of the phage was evaluated by plaque formation assay.Cell counting kit-8 (CCK8) assay was conducted to evaluate the effect of M13 phage and recombinant M13-IN5 phage at the titer of 1011 plaque-forming units (PFU)/ml on the proliferation of Hela cells,and Hela cells uninfected with chlamydia served as the blank control group.Western blot analysis was performed to determine the expression of the IN5 loop protein in the recombinant M13-IN5 phage,M13 phage and Escherichia coli ER2738 at exponential growth phase.Cultured standard Chlamydia trachomatis serovar E strain was treated with M13 phage and recombinant M13-IN5 phage at the titer of 1011 PFU/ml separately,and chlamydia control group without the treatment with phages was set up.After 36-hour infection,confocal microscopy was performed to detect the location of the M13 phage and the recombinant M13-IN5 phage.Moreover,iodine staining was conducted to count inclusion bodies at 36,48,60 and 72 hours separately after infection.Statistical analysis was carried out by a two-sample t-test for comparisons between two groups,one-way analysis of variance (ANOVA) for intergroup comparison,and Bonferroni test for multiple comparisons.Results The bioactive recombinant M13 phage containing the IN5 loop gene was constructed successfully,and Western blot analysis confirmed that the recombinant phage expressed IN5 loop/p Ⅲ fusion protein with a high titer of 3.05 × 1011 PFU/ml.As CCK8 assay showed,there was no significant difference in proliferation of Hela cells among the blank control group,M 13 phage group and recombinant M13-IN5 phage group (A450 values:3.63 ± 0.01,3.55 ± 0.02,3.70 ± 0.01,respectively,F =12.0,P ＞ 0.05).Confocal microscopy showed overlap between the phage fluorescence and chlamydial inclusion body fluorescence.The M13-IN5 phage group and M13 phage group both showed significantly decreased number of inclusion bodies compared with the control group (both P ＜ 0.05) at 36 and 72 hours after chlamydial infection,and the number of inclusion bodies was significantly lower in the M 13-IN5 phage group than in the M13 phage group (P ＞ 0.05).After 48,and 60 hours of chlamydial infection,the number of inclusion bodies did not differ among the M13 phage group,M13-IN5 phage group and control group (both P ＞ 0.05).Conclusions The recombinant M13-IN5 phage was bioactive and could successfully express the IN5 loop protein.In the in vitro experiments,the recombinant phage could enter into chlamydia inclusion bodies,and markedly inhibited the infection of Chlamydia trachomatis.

Here, we took base construction of neurosurgery as example to discuss and analyze according to requirements and evaluation indexes of base construction in Xinqiao Hospital, and put forward the specific objectives, measures and implementations of base construction. Foremost, we summarized experiences and overcame shortcomings through interpreting and implementing scheme of our base construction, which would help to improve the construction of standardized residency training base in China.

Objective:To investigate the clinicopathological features and prognosis of medullary thyroid carcinoma (MTC) co-existent with Hashimoto's thyroiditis (HT).Methods:Seven cases of MTC co-existent with HT were collected from Fujian Cancer Hospital between Jan 2007 and May 2021. Its clinicopathological features were analyzed.Results:All patients were adult female with a median age of 49 years, and 6 of them were unilateral MTC. The tumor diameter ranged from 0.4 to 7.0 cm. The thyroid tissue surrounding the tumor showed HT. The follow-up time was 16 to 152 months, with a median time of 61 months, 5 patients (tumor diameter <1 cm) were biochemically cured, 1 patient (tumor diameter >1 cm) was anatomically cured. The another patient died due to tumor progression 30 months after surgery, the tumor cells in which were highly atypia and more mitoses (5~8/10HPF) necrosis and vascular invasion were also observed.Conclusion:MTC with HT are mostly sporadic cases, and most of them have a fair prognosis.

BACKGROUND: There are few multi-city studies on the association between temperature and mortality in basin climates. This study was based on the Sichuan Basin in southwest China to assess the association of basin temperature with non-accidental mortality in the population and with the temperature-related mortality burden. METHODS: Daily mortality data, meteorological and air pollution data were collected for four cities in the Sichuan Basin of southwest China. We used a two-stage time-series analysis to quantify the association between temperature and non-accidental mortality in each city, and a multivariate meta-analysis was performed to obtain the overall cumulative risk. The attributable fractions (AFs) were calculated to access the mortality burden attributable to non-optimal temperature. Additionally, we performed a stratified analyses by gender, age group, education level, and marital status. RESULTS: A total of 751,930 non-accidental deaths were collected in our study. Overall, 10.16% of non-accidental deaths could be attributed to non-optimal temperatures. A majority of temperature-related non-accidental deaths were caused by low temperature, accounting for 9.10% (95% eCI: 5.50%, 12.19%), and heat effects accounted for only 1.06% (95% eCI: 0.76%, 1.33%). The mortality burden attributable to non-optimal temperatures was higher among those under 65 years old, females, those with a low education level, and those with an alternative marriage status. CONCLUSIONS Our study suggested that a significant association between non-optimal temperature and non-accidental mortality. Those under 65 years old, females, and those with a low educational level or alternative marriage status had the highest attributable burden.
Female ; Humans ; China/epidemiology* ; Cities ; Cold Temperature ; Hot Temperature ; Mortality ; Temperature ; Time Factors ; Middle Aged ; Male