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Objective:To determine the expression of matrix metalloproteinase 13 (MMP13) in patients with psoriasis, and to evaluate the effect of tazarotene and narrow-band ultraviolet B (NB-UVB) on the expression of MMP13 in mice with psoriasis-like dermatitis.Methods:Lesional skin tissues and normal skin tissues were collected from 18 patients with psoriasis vulgaris and 10 healthy controls respectively, who were enrolled from General Hospital of Tianjin Medical University between May 2019 and August 2019, and serum samples were collected from all the subjects. A total of 25 specific pathogen-free (SPF) male BALB/c mice were randomly divided into control group, imiquimod group, imiquimod+NB-UVB group, imiquimod+tazarotene group and imiquimod+tazarotene+NB-UVB group. The control group received topical vaseline cream on the back once every morning; imiquimod group and imiquimod+NB-UVB group received imiquimod cream on the back once every morning; imiquimod+tazarotene group and imiquimod+tazarotene+NB-UVB group received imiquimod cream on the back once every morning, and tazarotene cream on the back once at night; imiquimod+NB-UVB group and imiquimod+tazarotene+NB-UVB group received NB-UVB irradiation on the back every other day at noon, with the dose being 300 mJ/cm 2 in the first session and increasing by 50 mJ/cm 2 in every session. The modeling lasted 7 days. After successful modeling, blood samples were obtained from the eyeballs of the mice, and skin tissues were resected from the back of the mice after being sacrificed by cervical dislocation on day 8. Changes in the epidermal thickness and pathological manifestations were observed by hematoxylin and eosin (HE) staining, protein expression of MMP13 in skin tissues was determined by immunohistochemical study, and the serum level of MMP13 was detected by enzyme-linked immunosorbent assay. Comparisons between 2 groups were performed by using two-independent-sample t test, comparisons among several groups by using one-way analysis of variance, multiple comparisons by using least significant difference- t test, and comparisons of enumeration data by using chi-square test. Results:The skin lesions of the patients with psoriasis were strongly positive for MMP13, and the MMP13 expression levels in the epidermis and serum (84.11±17.16, 13.29±3.95 μg/L, respectively) were significantly higher in the patients with psoriasis than in the healthy controls (11.98±4.08, 7.46±1.58 μg/L, respectively, both P< 0.01) . Compared with the control group (1.26±0.04 μm, 25.40±2.34, 185.76±7.22 μg/L, respectively) , a significant increase was observed in the epidermis thickness (7.93±0.59 μm, P< 0.01) , as well as MMP13 levels in the epidermis and serum in the imiquimod group (147.14±5.53, 215.98±15.17 μg/L, respectively, both P< 0.01) . Compared with the imiquimod group, the imiquimod+tazarotene group, imiquimod+NB-UVB group, and imiquimod+tazarotene+NB-UVB group all showed significantly decreased epidermal thickness (3.56±0.37 μm, 3.83±0.39 μm, 2.14±0.34 μm, respectively, all P< 0.05) , MMP13 levels in the epidermis (120.42±3.23, 91.08±0.46, 71.12±7.11, respectively, all P< 0.05) and serum (197.39±3.92 μg/L, 196.13±11.76 μg/L, 183.21±14.99 μg/L, respectively, all P< 0.05) . Conclusions:MMP13 protein expression markedly increased in the skin lesions and sera of patients with psoriasis, and decreased in skin lesions and sera of mice with psoriasis-like dermatitis after the treatment with tazarotene and NB-UVB. MMP13 may be involved in the development of psoriasis, and tazarotene and NB-UVB may inhibit the development of psoriasis by reducing the expression of MMP13.
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Objective:To investigate the application value of "four doors of the liver" approach in the laparoscopic anatomical hepatectomy.Methods:The retrospective and descriptive study was conducted. The clinicopathological data of 52 patients with liver cancer who were admitted to West China Hospital of Sichuan University from September 2018 to September 2019 were collected.Patients underwent laparoscopic anatomical hepatectomy by opening "four doors of the liver" approach. There were 36 males and 16 females, aged (53±16)years, with a range from 35 to 78 years. Observation indicators: (1) surgical situations; (2) postoperative situations; (3) follow-up and survival. Follow-up using outpatient examination or telephone interview was conducted to detect the physical situations, liver function and recurrence of liver cancer in patients up to March 2020. Measurement data with normal distribution were represented as Mean± SD, and measurment data with skewed distribution were represented as M (range). Count data were expressed as absolute numbers or percentages. Results:(1) Surgical situations: all the 52 patients underwent laparoscopic anatomical hepatectomy successfully, without perioperative death. Eight and 8 patients underwent laparoscopic left hemihepatectomy and right hemihepatectomy by opening "the first door of the liver" respectively, the operation time of which was (151±31)minutes and (190±43)minutes, the volume of blood loss was (151±20)mL and (361±51)mL. Eight and 8 patients underwent laparoscopic left inner hepatic lobotomy and left outer hepatic lobotomy by opening "the second door of the liver" respectively, the operation time of which was (171±41)minutes and (90±26)minutes, the volume of blood loss was (221±31)mL and (111±21)mL. Eight and 8 patients underwent laparoscopic right posterior hepatic lobotomy and right anterior hepatic lobotomy by opening "the third door of the liver" respectively, the operation time of which was (172±29)minutes and (220±40)minutes, the volume of blood loss was volume of (351±41)mL and (451±47)mL. Four patients underwent laparoscopic hepatic caudate lobotomy by opening "the fourth door of the liver" , the operation time of which was (246±36)minutes, the volume of blood loss was (261±31)mL. None of the 52 patients had blood transfusion. (2) Postoperative situations: all the 52 patients recovered well after surgery, with no complications such as bleeding, biliary fistula, infection or liver failure. The duration of postoperative hospital stay was (7±4)days. (3) Follow-up and survival: all the 52 patients were followed up for 6-17 months, with a median follow-up time of 10 months. At 6 months after operation, all the 52 patients achieved of Eastern Cooperative Oncology Group performance status grade 1, Child-Pugh A of liver function, without tumor recurrence or metastasis. The overall survival rate was 100%(52/52).Conclusion:It is safe and feasible to perform laparoscopic anatomical hepatectomy by the "four doors of the liver" approach.
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Objective@#To establish a New Zealand rabbit animal model of laryngopharyngeal reflux disease (LPRD) using esophageal balloon together with metal internal stent dilation and to investigate the changes of mucosa.@*Methods@#20 New Zealand rabbits were randomly divided into experimental group and control group, with 10 in each group. Balloon dilatation and metal internal stent dilation were carried out in experimental group to reproduce the animal model of LPRD.The middle of balloon was placed at the lower esophageal sphincter (LES) while the stent was placed at the upper esophageal sphincter (UES). The guide wire was placed in the control group, but the balloon was not expanded and the stent was not placed. The general condition, pH value of hypopharynx, laryngeal histopathology and changes of pepsin content of New Zealand rabbits were observed regularly. The difference between experimental group and control group was compared.@*Results@#The 24-hour Dx-pH monitoring results showed that the number of reflux episodes(20.0[9.5, 35.0], 13.0[6.5, 22.0]), and the percent time below pH 5.5 (1.36%[0.60%, 4.57%], 1.36%[0.43%, 2.77%]) in the experimental group at the 2nd and 4th week were significantly different from those in the control group (0[0,3.0], 1.0[0.5, 3.8]; 0[0, 0.01%], 0[0, 0], respectively, all P<0.01), suggesting that the experimental group New Zealand rabbits developed LPRD. Compared with the control group under microscope, lymphocytes infiltration and submucosal gland hyperplasia increased in the mucosa of the throat of the experimental group. The results of pepsin immunohistochemical staining between the two groups were statistically significant (P=0.014).@*Conclusion@#The use of balloon dilatation of the LES combined with metal stent dilatation of the UES can successfully establish a laryngopharyngeal reflux model, and lesions in the throat tissue can be observed.
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From bacteria to mammalian cells, protein-DNA interactions are indispensable for all living organisms. In order to perform cell growth, differentiation, division, and regulation of cell function when stimulated by the external environment, DNA-encoded genetic information must be accurately transcribed, which is extremely important for the growth, development, and evolution of organisms. Manipulating the interaction of protein and DNA through biotechnology can modulate the expression of certain virulence genes, so as to contributing to the treatment of diseases. With the deepening of scientific research, more and more scientific technologies are applied in the field of protein and DNA interaction research. In this paper, the principles, advantages, disadvantages and applications of commonly used techniques in the study of protein-DNA interactions were reviewed, which will help researchers evaluate and select appropriate methods in their researches.
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Objective To explore the therapeutic effect of sphincteric fistula ligation in the treatment of complex anal fistula. Methods From August 2016 to April 2017,66 patients with complicated anal fistula in the People's Hospital of Liulin County were selected as in the study. They were divided into two groups according to the principle of average and complete randomization, with 33 cases in each group. The control group was treated by hanging line. The treatment group was treated with ligation of sphincter fistula. The clinical effect,operation condition, postoperative pain and adverse events were observed in the two groups. Results The total effective rate of the observation group was 93. 94％ ,which was higher than 75. 76％ of the control group (χ2 = 4. 24,P < 0. 05). In the observation group,the wound healing time,the length of hospital stay and the postoperative pain score were better than those in the control group[(15. 69 ± 1. 28)d vs. (19. 47 ± 1. 93)d,(8. 93 ± 0. 71)d vs. (12. 61 ± 1. 08)d,(2. 29 ± 0. 56)points vs. (4. 29 ± 0. 74)points,t = 9. 38,16. 36,12. 38,all P < 0. 05]. The incidence rate of adverse events in the observation group was lower than that in the control group,but the difference was not statistically significant (9. 09％ vs. 21. 21％ ,χ2 = 1. 89,P > 0. 05),and the recurrence rate of the observation group was significantly lower than that of the control group(3. 03％ vs. 21. 21％ ,χ2 = 5. 12,P < 0. 05). Conclusion Sphincteric fistula ligation in the treatment of complex anal fistula can effectively control the disease,promote wound healing,and has a good prognosis.
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Objective To investigate the correlation between serum vitamin D level in human body and Chlamydia trachomatis (Ct) genitourinary tract infection. Methods This study enrolled 174 outpa-tients (male: 95, female: 79, 20-49 years) infected with Ct and 380 healthy subjects (male: 211, female:169, 20-49 years) in Tianjin from November 1, 2016 to March 15, 2017. Blood samples were collected from all subjects after fasting overnight and the time points for sample collection in the Ct infection group were before and after a course of antibiotic treatment. Serum 25-hydroxyvitamin D [25-(OH)D] levels were measured with enzyme-linked immunosorbent assay (ELISA). PCR assay was used to assess the recovery in those patients one month after a course of treatment. Two case-control studies were respectively conducted, in which 161 patients and 161 healthy subjects as well as 41 uncured patients and 41 cured patients were randomly selected and matched for gender and age. Statistical analysis was performed using SPSS19. 0. Re-sults The two case-control studies showed that vitamin D deficiency was a risk factor for both Ct genital tract infection and poor efficacy, of which the adjusted ORs were 2. 281 (95％ CI: 1. 438, 3. 619) and 7. 266 (95％ CI: 2. 551, 21. 036). Among all subjects aged 20-39, male patients had lower 25-(OH)D level in serum than healthy men [(40. 10±17. 93) nmol/ L vs (53. 72±18. 00) nmol/ L, P< 0. 01] and fe-male patients also had lower 25-(OH)D level in serum than healthy women [(35. 71±19. 99) nmol/ L vs (45. 42±16. 08) nmol/ L, P<0. 01]. The levels of 25-(OH)D in uncured male and female patients were re-spectively lower than those in cured male and female patients [(30. 50±14. 53) nmol/ L vs (41. 32±17. 24) nmol/ L; (29. 47±16. 66) nmol/ L vs (41. 37±21. 03) nmol/ L; both P<0. 05]. Conclusion Vitamin D deficiency related to Ct infection in human genitourinary tract and poor prognosis. Lower serum vitamin D levels might increase the risk of Ct genitourinary tract infection and reduce the efficacy of treatment.
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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.
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Objective To explore the clinical characteristics and prognostic factors of patients with hepatolithiasis associated with intrahepatic cholangiocarcinoma. Methods The clinical data of 39 patients with hepatolithiasis associated with intrahepatic cholangiocarcinoma in the First Affiliated Hospital of Chongqing Medical University from Jan 2006 to Jun 2013 was retrospectively analyzed. Results The main clinical manifestations of hepatolithiasis associated with intrahepatic cholangiocarcinoma included recurrent fever, abdominal pain, jaundice and hepatic percussion pain. Among the 39 cases, the patients older than 60 years accounted for 69.2 % (27/39), and the duration of hepatolithiasis more than 10 years accounted for 76.9 % (30/39). Remarkable differences were found in serum CA19-9 and surgery methods (both P< 0.05). Conclusions Recurrent fever, abdominal pain, jaundice and hepatic percussion pain are the main clinical manifestations for the patients who are diagnosed with hepatolithiasis associated with intrahepatic cholangiocarcinoma. Advanced age and longer duration of the disease may be the high risk factors. Serum CA19-9 levels and surgical methods are the important prognostic factors.
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Objective To investigate the inhibitory effect of IN5 from chlamydiaphage phiCPG1 capsid protein Vp1 on Chlamydia trachomatis (Ct).Methods PCR was used to amplify IN5 gene from Vp1 DNA of phiCPG1, then the recombinant plasmid pET28a/IN5 was constructed.After transformation, the fusion protein IN5 was induced,identified and purified.Ct was incubated with the purified IN5 protein or Vp1 protein.After 48 h of incubation, the inclusion bodies were counted with iodine staining and indirect immunofluorescence.One-way ANOVA was used to compare the difference of inclusion bodies among groups.If the difference among the groups was statistically significant, the Bonferroni method was used to compare any two mean values.Finally, the inhibitory rate of IN5 protein and Vp1 protein to Ct was calculated.Results IN5 protein from chlamydiaphage phiCPG1 capsid protein Vp1 was successfully obtained.At the same concentration of 53μg/mL,the inhibitory rates of Ct growth in IN5 and in Vp1 groups were 52.42% and 78.04%, respectively.Conclusion IN5 protein has inhibitory effect on the growth of Ct,but the inhibitory rate is lower than that of Vp1, which provides a preliminary clue for searching the dominant region of Vp1 protein inhibiting the growth of Ct.
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Objective@#To evaluate the therapeutic efficacy of penciclovir combined with foscarnet sodium in the treatment of herpes zoster.@*Methods@#The clinical datas of 135 herpes zoster patients from the ward of Department of Dermatology, Tianjin Medical University General Hospital were collected. Among them 64 patients received penciclovir and foscarnet sodium, and the remaining 71 patients only received penciclovir alone.Their general information, the time for vesicle stopped emerging, rash began to scab, pain to relief obviously, the adverse reaction and if they got the postherpetic neuralgia were recorded and included into statistical analysis.@*Results@#The general information showed no significant differences between the 2 groups(all P>0.05). The time for vesicle stopped emerging, rash began to scab, pain to relief obviously in combination group was shorter than the penciclovir group (all P<0.001). The number of patients who developed postherpetic neuralgia of combination group was fewer than that of penciclovir group(P=0.013). There was no statistical significance between the 2 groups the adverse reaction(P=0.928).@*Conclusions@#The penciclovir and foscarnet sodium combination therapy showed rapid therapeutic effects on herpes zoster patients, the incidence of postherpetic neuralgia was low, and there was no more side effects than penciclovir alone therapy. The combined therapy may be a reliable way to treat herpes zoster.
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We investigated the effects of γ-interferon and exogenous indole on the growth of domestic dominant standard strains and clinical straìns of Chlamydia trachomatis E-UW-5/Cx,and compared with the dominant strains of D-UW-5/Cx abroad.We used DMEM-10,DMEM-10 containing 5 ng/mL recombinant human interferon gamma (referred to as DMEM-10+IFN) and DMEM-10 containing 5 ng/mL recombinant human interferon gamma and 50 μM exogenous indole (referred to as DMEM-10+IFN+IND) to culture C.trachomatis,and then we fixed it with methanol to count inclusions after 48 hours,observing the influence of r-interferon and exogenous indole on the growth of C.trachomatis standard strains(E,D) and clinical strains.Results showed that the count of Chlamydia inclusion bodies in DMEM-10+IFN group was significantly lower than others (P＜0.05);no significant difference was found (P＞0.05) between the count of DMEM-10 group between DMEM-10+IFN+IND group.There were no significant difference between the E and D standard or clinical strains (P＞0.05).Under the effect of IFN-γ,the growth of domestic dominant strain E-UW-5/Cx C.trachomatis was significantly inhibited.After adding exogenous indole,C.trachomatis can escape the scavenging activity of IFN-γγto restore the infection vitality.
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Objective To obtain the full length (FL ) and C‐terminal fragment of polymorphic membrane protein I (PmpI) of Chlamydia trachomatis serovar D ,and to study the immunogenicity of these proteins .Methods The target genes of PmpI‐FL and PmpI‐C were amplified by polymerase chain reaction (PCR) and inserted into the prokaryotic plasmid vector pGEX‐6P‐1 .The recombinant plasmids pGEX‐6P‐1/PmpI‐FL and pGEX‐6P‐1/PmpI‐C were separately transformed into Escherichia .coli ( E . coli) DH5αand were identified by enzyme digestion ,sequencing and PCR .After the identification ,the recombinant plasmids were separately transformed into E .coli BL21 and induced to express the proteins . The expected proteins were identified by Coomassie brilliant blue staining and Western blot ,then purified by glutathione S‐transferase (GST) MagBeads .The purified proteins were then injected into BALB/c mice to prepare the polyclonal antibodies against PmpI‐FL or PmpI‐C .Enzyme‐linked immune sorbent assay (ELISA) was used for the quantitative detection of the specific antibody .Results The lengths of cloned target genes PmpI‐FL and PmpI‐C were 2 659 bp and 1 195 bp ,respectively ,and the sequences were consistent with those of Chlamydia trachomatis serovar D in GenBank .The molecular masses of target proteins were 122 000 and 69 000 ,respectively ,which were confirmed by Coomassie brilliant blue staining and Western blot and then purified .The titers of the antibodies (anti‐PmpI‐FL and anti‐PmpI‐C) in sera of immunized mice detected by ELISA were 1∶12 800 and 1∶6 400 ,respectively .Conclusion The PmpI‐FL‐GST and PmpI‐C‐GST fusion proteins with high immunogenicity are successfully expressed and purified , which lays the foundation for further study .
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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.
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Objective To evaluate inhibitory effects of the Chlamydiaphage phiCPG1 capsid protein Vp1 on Chlamydia psittaci strain guinea pig inclusion conjunctivitis (GPIC) and Chlamydia trachomatis (Ct) serovar E,and to provide new ideas for the treatment of Ct infection.Methods The Chlamydiaphage phiCPG1 capsid protein Vp1 was expressed in Escherichia coli BL21 transfected with the recombinant plasmid Vp1-pET30a (+),identified by Western blot analysis and purified by using dialysis bags.Bicinchonininc acid (BCA) assay was performed to determine the concentration of Vp1 protein.GPIC and Ct serovar E strains were both classified into 4 groups to be firstly incubated with Vp1 protein (Vp1 group),Tris-glycine solution (Tris group),S protein (S group) or Dulbecco's Modified Eagle Medium (DMEM,DMEM group) at room temperature for 3 hours,then were used to infect Hela cells followed by 72-hour (GPIC) or 48-hour (Ct serovar E) culture with the presence of Vp 1 protein (Vp 1 group),Tris-glycine solution (Tris group),S protein (S group) or DMEM (DMEM group).Subsequently,immunofluorescence staining was conducted to observe and count chlamydial inclusions.Results The number of GPIC inclusions was significantly different between the 4 groups after 72-hour culture (F=476.632,P＜ 0.05),and lower in the Vp1 group (5.0 ± 1.5) than in the Tris group (24 ± 1.2,P＜ 0.05),S group (25 ± 1.7,P＜ 0.05) and DMEM group (25 ± 1.5,P＜ 0.05),but insignificantly different between the latter 3 groups (P ＞ 0.05).Compared with the DMEM group,the Vp1 group showed a significant decrease of 80.2％ ± 3.99％ and 77.2％ ± 1.79％ in the number of GPIC and Ct serovar E inclusions respectively,with no significant difference in the inhibitory effect of Vp1 on GPIC versus Ct serovar E (t =2.057,P ＞ 0.05).Conclusion The phiCPG1 capsid protein Vp1 can obviously inhibit GPIC and Ct serovar E infections to a similar degree.
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Objective To evaluate the susceptibility of Chlamydia trachomatis clinical isolates to rifampin, and assess the relationship between rpoB mutations and antibiotic resistance in them.Methods A microculture method was used to determine the minimal inhibitory concentration (MIC) of rifampin in 52 Chlamydia trachomatis clinical isolates.The rpoB gene was amplified from all the clinical isolates and a standard strain of Chlamydia trachomatis followed by single-strand conformation polymorphism (SSCP)analysis.Sequencing of PCR products was carried out for two clinical isolates.Results No rifampin-resistant strain was found among these clinical isolates.The MIC of rifampin varied from 0.004 to 0.030 mg/L Neither SSCP analysis nor sequencing showed rpoB mutations.Conclusions No rpoB mutations were found in Chlamydia trachomatis isolates from patients unresponsive to rifampin.The unresponsiveness to rifampin may be attributed to multiple factors.
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Objective To compare the infectivity of several transformed Chlamydia trachomatis (Ct) mouse pneumonitis (Mopn) strains to host cells, and to identify cell invasion-related virulence genes in Chlamydial plasmids. Methods Several Ct strains, including wild-type Ct Mopn strain(WT strain), plasmid-free Ct strain(CMUT3 strain), Ct Mopn strain transformed with the shuttle vector carrying pGFP and the complete C. muridarum (CM) plasmid (pGFP::CM strain)and Ct Mopn strains transformed with shuttle vectors carrying pGFP and mutant CM plasmids with in-frame deletions of Pgp3, 4, 5 or 7 (pGFP::CM△Pgp3, 4, 5, 7 strains), were cultured, amplified and collected. After the concentrations of Ct were determined, each of these strains was divided into four groups to be inoculated at a same amount(1.5 × 104 inclusion forming units(IFU)) followed by four different treatments respectively: centrifugalization +DEAE group treated with centrifugalization followed by ion-exchange chromatography on diethylaminoethyl(DEAE)-cellulose columns, centrifugalization group treated with centrifugalization only, DEAE group treated with chromatography on DEAE-cellulose columns only, control group receiving no treatment. After additional culture for 20 - 24 hours, indirect immunofluorescence assay was performed to count the number of chlamydial inclusions. At 20, 40 and 60 hours after infection, the growth rate and area of chlamydial plaques were assessed after three continuous passages. Lugol′s iodine staining was conducted to observe glycogen synthesis in bacterial inclusions. Results The inclusion number in the centrifugalization + DEAE group, centrifugalization group, DEAE group and control group was 10.20 ± 1.30, 6.80 ± 0.44, 3.00 ± 1.22 and 0.80 ± 0.45 respectively for the pGFP::CM△Pgp4 strain, 6.40 ± 0.89, 3.80 ± 0.83, 1.60 ± 0.89 and 0.60 ± 0.54 respectively for the CMUT3 strain. Under same experiment conditions, the pGFP::CM△Pgp4 strain and CMUT3 strain showed similar infectivity, and formed less inclusions compared with the other Ct strains (all P < 0.01). The number of inclusions formed by the same Ct strains were significantly different among the 4 groups(F = 845.310, P <0.01), and were highest in the centrifugalization + DEAE group for all the strains. The pGFP::CM△Pgp4 strain showed significantly lower growth rate and area of plaques with an abnormality in glycogen accumulation compared with the other strains at 20, 40 and 60 hours after infection. Conclusion The plasmid-encoding gene Pgp4 may be a cell invasion-associated virulence gene in chlamydial plasmids.
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Objective To add an open reading frame in the shuttle vector of pGFP ∷ CM for transfection of exogenous genes into Chlamydia muridarum.Methods The sequence of plasmid pGFP ∷ CM and new open reading frame (including promoter of pgp4,mCherry gene of red fluorescence protein and transcription termination sequence of Chlamydia trachomatis CT579) were amplified by polymerase chain reaction (PCR),and the products were transfected into Stellar competent cells.The recombinant plasmids were identified by PCR,enzyme digestion and sequencing.Then the recombinant plasmid was transfected into plasmid-free strain CMUT3,and the GFP-and mCherry-positive inclusions were observed under the fluorescence microscope.After the ampicillin selection and plaque purification,the purified CMUT3-pGFP-mCherry-CM was identified by indirect immunofluorecesent stain using anti-pgp3 and anti-glgA antibodies.Results The correct recombinant plasmid after sequencing identification,enzyme digestion and PCR amplification was successfully transfected into CMUT3,and the GFP-and mCherry-positive inclusions were observed.The transfected strain CMUT3-pGFP-mCherry-CM was purified after ampicillin selection and plaque purification.The expression of pgp3 and glgA protein in CMUT3-pGFP-mCherry-CM was similar to that in CMUT3-pGFP ∷ CM.Conclusion An open reading frame is successfully added in the plasmid pGFP ∷ CM,and the new plasmid can be transfected into CMUT3 and express exogenous protein,which can be used for further study on the function of single chlamydial protein.
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Retmperitoneal ganglioneuroma is a rare neurogenic benign tumor.The prognosis of patients was good when the tumor was completely resected,while the surgical procedure is complicated.In March of 2013,a male patient with complex retroperitoneal ganglioneuroma was treated at the First Affiliated Hospital of Chongqing Medical University.A hypoechoic solid lesion (size,6.5 cm ×4.5 cm) adjacent to the head of the pancreas was detected by color Doppler ultra-sonography 9 months ago,and no any other clinical symptoms were detected.Perioperative abdominal computed tomography and the surgery confirmed that the tumor (size,8.5 cm × 7.5 cm × 4.5 cm) was located beneath the pancreas,encompassing thc ccliac artery,hepatic artery,splenic artery and superior mesenteric artery,surrounding the head and uncinate process of the pancreas,making it impossible to be separated.The tumor was closely connected with the portal vein,superior mesenteric vein,splenic vein and left renal vein.The tumor was separated from the major blood vessels,the body and tail of the pancreas,while the tumor could not be resected from the pancreatic head,and thus tumor resection and pancreaticoduodenectomy were performed.The surgery was extremely diffcult,but the complete removal of tumor was successfully achieved without excision of the major blood vessels and the patient recovered well.
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Objective To investigate the relationship of point mutations in ERG11,TAC1,MRR1 and UPC2 genes to fluconazole resistance in Candida albicans.Methods This study included 11 fluconazole-resistant Candida albicans strains.DNA was extracted from these Candida isolates by urea pyrolysis method.PCR was carried out to amplify the full-length EGR11 gene,as well as the corresponding fragments of TAC1,MRR1 and UPC2 genes followed by DNA sequencing.Results Eight mutations were detected in the ERG11 gene,including D116E,Y132H,Y132F,K143Q,T229A,E266D,G448E and G464S.Among them,K143Q was a novel mutation and had not been reported.A G648D mutation was detected in the UpC2 gene.No mutations were detected in the TAC1 or MRR1 gene.Conclusions There is a definite association between fluconazole resistance and ERG11 mutations,while further research is warranted to clarify the relationship between the mutations of TAC1,MRR1,UPC2 genes and resistance to fluconazole.
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Objective To investigate the feasibility of C.trachomatis culture in HaCaT human keratinocytes.Methods According to the procedure for C.trachomatis culture in McCoy cells,clinical swab specimens and standard strains of C.trachomatis serotype E were inoculated into HaCaT cells.Iodine staining,a fluorescent monoclonal antibody test and PCR amplification of the endogenous plasmid of C.trachomatis were performed to detect the growth of C.trachomatis in HaCaT cells.Five passages of subculture were carried out for the standard strain of C.trachomatis serotype E in HaCaT cells,and inclusion bodies were counted after each passage.One-factor analysis of variance was conducted by using the software SPSS17 to determine if C.trachomatis was propagated in HaCaT cells.Results Iodine staining showed typical inclusion bodies of C.trachomatis in the cytoplasm of HaCaT cells.Yellow fluorescence-labeled granules were observed in the HaCaT cells under a microscope.Endogenous plasmids of C.rachomatis were successfully amplified by PCR from the infected HaCaT cells.The number of inclusions in HaCaT cells gradually increased at passage 1 through 5.Conclusions C.trachomatis is successfully cultivated in HaCaT cells in vitro,and the standard strain of C.trachomatis serotype E can propagate in HaCaT cells.