[Changes of resistant phenotype and CRISPR/Cas system of four Shigella strains passaged for 90 times without antibiotics].

Objective: To explore the stability of resistant phenotypes and changes of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) gene system on four Shigella strains in the absence of antibiotics. Methods: Four clinical isolated Shigella strains that resistant to different antibiotics were consecutive passaged for 90 times without antibiotics. Agar dilution method was used to determine the minimum inhibitory concentration of Shigella strains. After sequence analysis with PCR, CRISPR Finder and Clustal X 2.1 were applied to identify the changes of CRISPR loci in the Shigella strains. Results: After the consecutive transfer of 90 generations, sensitivity to certain antibiotics of four Shigella strains with different drug resistant spectrums increased. Mel-sf1998024/zz resistance to ampicillin, cephalexin, cefotaxime, chloramphenicol decreased, mel-s2014026/sx resistance to norfloxacin, trimethoprim decreased, mel-sf2004004/sx drug resistance to ampicillin, cefuroxime, cefotaxime, chloramphenicol, trimethoprim decreased and mel-sf2013004/bj resistance to chloramphenicol decreased. The spacer of which matched gene codes Cas and its upstream repeat in 3'end of CRISPR3 got lost in mel-sf1998024/zz and mel-sf2013004/bj. Conclusions:Shigella strains could reduce or lose their resistance to some antibiotics after consecutive transfers, without the interference of antibiotics. CRISPR3 locus had dynamic spacers in Shigella strains while CRISPR3 locus and cas genes might have been co-evolved.

Effect of nucleoside analogues in the treatment of hepatitis B cirrhosis and its effect on Th17 cell.

OBJECTIVE: We conducted this study to analyze the effects of nucleoside analogues in the treatment of hepatitis B cirrhosis and its effect on Th17 cells. PATIENTS AND METHODS: 120 patients were randomly divided into lamivudine combined with adefovir dipivoxil group (combined group) and entecavir group. There were 59 cases in the combined group and 59 cases in entecavir group. The combined group was administered lamivudine 100 mg/d + adefovir dipivoxil 10 mg/d and entecavir group was administered entecavir 0.5 mg/d. The treatment was continued until there was virus negativity and it maintains for at least 3 months. RESULTS: The treatment effects were compared. We compared the average rate of viral clearance time and virus clearance of two groups of patients; the difference was not statistically significant (p>0.05). The relapse rate after a negative test of entecavir group was lower than that of the combined group (p<0.05). Before and after treatment, the levels of TBIL, ALT and ALB in the two groups were compared; the differences were not statistically significant (p>0.05). The Th17 cell proportion and the level of IL-17 after treatment of the entecavir group were lower than those before treatment. The combined group exhibited no change, and the entecavir group was lower than combined group; the differences were statistically significant (p<0.05). CONCLUSIONS: Therefore, the effects of the combination of lamivudine and adefovir dipivoxil is the same as single entecavir treatment of hepatitis B cirrhosis suppression of viral replication. It does not increase liver injury and the antiviral effects of entecavir may be related to inhibition of the expression of Th17 cells and effector molecules IL-17.

[A surveillance study on CRISPR/Cas molecular biomarker in Escherichia coli].

OBJECTIVE: A new method related to molecular biomarker with CRISPR/Cas (clustered regularly interspaced short palindromic repeats-cas) in Escherichia (E.) coli was developed and used for surveillance programs. METHODS: CRISPR/Cas sequence that containing 135 strains with complete sequence and 203 strains with whole genome shotgun sequence of E. coli in GenBank by BLAST and 361 strains of E. coli (including 38 strains of E. coli O157∶H7) in laboratory were identified by PCR and analyzed with the CRISPR Finder. Spacers were compared with DANMAN and the phylogenetic trees of cas gene were constructed under Clustal Ⅹ and Mega 5.1. RESULTS: With new perspective, a descriptive method was developed targeting on the position of CRISPR/cas in E. coli. The CRISPR1 was detected in 77.04%, 100.00% and 75.62% and the CRISPR2 was detected in 74.81%, 100.00% and 92.24% and the CRISPR3 and CRISPR4 were detected in 11.85%, 0 and 1.39% for 135 strains with complete sequence, 203 strains with whole genome shotgun sequence and 361 strains in the laboratory, respectively. One strain downloaded in GenBank with whole genome sequencing and 2 strains in the our laboratory were identified that containing four CRISPR locus. The other E. coli strain was with insertion sequence in downstream of the non-cas CRISPR1. The unique CRISPR was found in 8 strains of O55∶H7, in 180 strains of O157∶H7, in 8 strains of O157∶HNM, in 40 strains of O104∶H4, in 4 strains of O145∶H28, in all the 699 E. coli strains. The phylogenetic tree could be divided into two groups-cas with type I-E or type I-F. CONCLUSIONS: CRISPR/Cas might be used as a valuable molecular biomarker in epidemiological surveillance studies to identify the high virulent strains or new strains of E. coli. Phage night be related to the missing or obtaining of spacers.

Advanced malignant pleural or peritoneal effusion in patients treated with recombinant adenovirus p53 injection plus cisplatin.

The aim of this study was to evaluate the efficacy of recombinant adenovirus p53 agent (rAd-p53) injection combined with cisplatin (CDDP) for the treatment of malignant pleural or peritoneal effusion. After puncture drainage, patients in the treatment group (n = 27) received intracavitary administration of rAd-p53 (2 x 10(12) virus particles) once a week for 4 weeks. At 48 h after each rAd-p53 injection, patients were given intracavitary administration of cisplatin 60 mg/m(2). This administration procedure continued once a week for 4 weeks. The control group (n = 21) received the same intracavitary therapy as the treatment group but without rAd-p53 therapy. Efficacy was evaluated by clinical observations, computed tomography, tumour markers, Karnofsky score and short-term follow-up. The total effective rates for the treatment group (63.0%) were significantly higher than for the control group (42.9%), suggesting that the treatment group benefited over the control group. In conclusion, rAd-p53 therapy is a safe and effective treatment for advanced malignant pleural or peritoneal effusion.

Enhanced visualization of weak colloidal iron signals with Bodian's protein silver for demonstration of perineuronal nets of proteoglycans in the central nervous system.

The present study aimed for a clear visualization of faintly deposited colloidal iron in tissue sections for light microscopy. Paraffin blocks containing paraformaldehyde-fixed brain tissue from healthy adult mice were cut into sections 10-15 microm thick. After deparaffinization, the sections were stained with fine cationic iron colloid at a pH value of 1.0-1.5, and treated with a mixture of potassium ferrocyanide and hydrochloride for Prussian blue reaction. Some sections were further treated with Bodian's protein silver after the Prussian blue reaction. This sensitized development of Prussian blue reaction with Bodian's protein silver more clearly visualized the faintly deposited cationic colloidal irons than the demonstration by Prussian blue reaction alone, and allowed an enhanced visualization of the perineuronal nets of sulfated proteoglycans in the brain. Thus, such fine perineuronal sulfated proteoglycans as those in the CA3 field of the hippocampus, which are weakly stained with cationic iron colloid and usually overlooked by a demonstration with only a Prussian blue reaction, could be clearly visualized with striking contrast by the sensitized development with Bodian's protein silver after the Prussian blue reaction. Preliminary hyaluronidase digestion erased Bodian's protein silver development of perineuronal sulfated proteoglycans. Though some axonal fibers were also additionally stained with Bodian's protein silver itself, this sensitized development is useful to enhance such weak colloidal iron signals as are hardly detectable by only Prussian blue reaction.

Perineuronal sulfated proteoglycans and cell surface glycoproteins in adult and newborn mouse brains, with special reference to their postnatal developments.

Sections of the retrosplenial cortex from adult and newborn mouse brains were observed with a light microscope. The retrosplenial cortex of the adult animals contained many neurons (10% of the total), including some dark neurons, with perineuronal sulfated proteoglycans detectable with cationic iron colloid and aldehyde fuchsin. The retrosplenial cortex of the adult animals also contained many neurons (10% of the total) with cell surface glycoproteins reactive to lectin Vicia villosa, soybean or Wisteria floribunda agglutinin. Double staining showed that the majority (75%) of the neurons labeled with lectins were stained with cationic iron colloid, and that some (25%) of them were not stained with this colloid. Double staining also showed that some (25%) of the neurons stained with cationic iron colloid were not labeled with lectins. These findings indicate that the perineuronal sulfated proteoglycans are, at least partly, independent from the cell surface glycoproteins. Observations of the sections from the newborn animals revealed that the perineuronal sulfated proteoglycans were produced by the associated satellite astrocytes 3-4 weeks after birth, and that the cell surface glycoproteins were produced by the associated nerve cells at earlier stages, or 2-3 weeks after birth. Dark neurons began to appear 3-4 weeks after birth. These dark neurons or their Golgi complexes were also reactive to lectins, suggesting the production of cell surface glycoproteins.

Perineuronal sulfated proteoglycans and cell surface glycoproteins in the visual cortex of adult and newborn cats.

Sections of the visual cortex of newborn (1-4 weeks after birth) and adult cats were stained with cationic iron colloid, aldehyde fuchsin or lectins (lectin Vicia villosa, soybean and Wisteria floribunda agglutinins). Many neurons in the adult cat visual cortex contained perineuronal sulfated proteoglycans detectable with cationic iron colloid and aldehyde fuchsin, or cell surface glycoproteins reactive to lectins. Double staining indicated that some of the lectin-labeled neurons were not stained with cationic iron colloid, and also that some of the cationic iron colloid-stained neurons were not labeled with lectins. The perineuronal sulfated proteoglycans and cell surface glycoproteins developed 3 weeks after birth. In the newborn cats 1-2 weeks after birth, no neurons were reactive to cationic iron colloid, aldehyde fuchsin or lectins. In the newborn cats 3-4 weeks after birth, it was clearly observed that the cytoplasm of the glial cells closely associated with the neurons containing the perineuronal sulfated proteoglycans showed an intense reaction to cationic iron colloid and aldehyde fuchsin, and that the Golgi complexes of the neurons with cell surface glycoproteins were intensely labeled with lectins. These findings suggest that the perineuronal sulfated proteoglycans are derived from the associated glial cells, and that the cell surface glycoproteins are produced by the associated nerve cells.

An alpha 5 beta 1-like integrin receptor mediates the binding of less pathogenic Candida species to fibronectin.

The present study was undertaken to investigate whether less pathogenic Candida species (C. tropicalis, C. stellatoidea, C. krusei and C. glabrata) express a fibronectin receptor (FNr) antigenically related to alpha 5 beta 1 integrin, which mediates their binding to fibronectin (FN). By flow cytometric analysis, a monoclonal antibody (MAb) directed against human alpha 5 integrin subunit (clone SAM-1) and two different antisera to FNr positively stained C. tropicalis, C. stellatoidea and C. glabrata, with the greatest expression observed for C. tropicalis. No or only marginal immunoreactivity was found on C. krusei. C. tropicalis, C. stellatoidea, C. glabrata, but not C. krusei yeasts specifically adhered to FN; higher levels of adhesion were found for C. tropicalis and C. stellatoidea with respect to C. glabrata. Less pathogenic Candida spp. bound to the Arg-Gly-Asp (RGD) containing 120-kDa fragment of FN and adhesion to intact FN was markedly inhibited by Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP), but not by Gly-Arg-Gly-Glu-Ser-Pro (GRGESP) peptides. In addition, anti-alpha 5 SAM-1 MAb and both anti-FNr antisera strongly blocked binding of less pathogenic Candida spp. to FN. Overall, these results indicate that less pathogenic Candida spp., including C. tropicalis, C. stellatoidea and C. glabrata, express a receptor antigenically related to alpha 5 beta 1 integrin which mediates their adhesion to FN.

[Studies on the changes in infection rates of whipworm and ascaris in population after continuous chemotherapy].

The inhabitants with positive eggs of whipworm and ascaris in a village of Jiangdou County were examined and treated with different antihelmintic drugs twice a year. The uncured population were treated by drug administration once again. On an average the efficiency of drug treatment (cure rate x treatment rate) was 56.7% in whipworm and 64.5% in ascaris. The program had been carried out for five years. The positive rate of eggs of whipworm and ascaris in the population decreased from 64.7% and 52.1% to 2.7% and 4.1%, respectively, the mean increase in infection rates among population at 5 or 7 months after treatment was 2.68 (0.86-5.74)% and 6.94(1.72-17.27)%, respectively.