OBJECTIVE To analyze the types and control measures of major microorganisms in pharmaceutical water systems, so as to provide guidance for effective control of pharmaceutical water systems. METHODS The main microbial species, abundance and harmfulness of drinking water, purified water and water for injection were reviewed, and the control measures on microorganisms in pharmaceutical water were discussed. RESULTS There were differences in the main microbial types in pharmaceutical water. Burkholderia cepacia complex and Ralstonia pickettii were conditioned pathogens in pharmaceutical water, thus causing certain biological safety hazards. CONCLUSION Pharmaceutical companies can strengthen the control of microorganisms in the water system by establishing microbial databases and common microbial strain banks at all levels. Trend analysis should to be conducted based on alert limits and action limits, so as to strengthen the control of microorganisms in the water system.

OBJECTIVE To establish a rapid and accurate PCR method for detecting 24 strains of Burkholderia cepacia complex(Bcc) by comparing three detection methods of loop-mediated isothermal amplification(LAMP), SYTO 9 dye method based on polymerase chain reaction(PCR) and TaqMan probe real-time fluorescent quantitative PCR method( TaqMan probe method). METHODS According to the molecular biological information of 24 strains of Bcc in the NCBI database, multiple candidate sequence fragments unique to Bcc were screened out, and specific primer and probe that could simultaneously detect 24 strains of Bcc were designed. At the same time, the detection methods of LAMP, SYTO 9 dye method based on PCR and Taqman probe were explored, and the optimal annealing temperature was optimized and screened. The 39 experimental strains were used to verify the Bcc detection method. RESULTS LAMP method could not effectively detect Bcc, SYTO 9 dye method and TaqMan probe method could effectively detect more than 20 strains of Bcc, while TaqMan probe method had higher amplification effect, better detection sensitivity, repeatability and stability, which could meet the requirements of this study. CONCLUSION In this study, a TaqMan probe method for rapid detection of Bcc was established. Compared with LAMP method and SYTO 9 dye method, this method has the advantages of fast, simple and high sensitivity, and provides technical support for the rapid detectionof Bcc.

Pharmaceutical water is the most widely used raw material and solvent in the process of pharmaceutical products, which is widely used in the cleaning and disinfecting process of appliances, equipment, facilities and systems, and is involved in the whole process of pharmaceutical products. Pharmaceutical water is one of the main sources of microbial contamination of pharmaceutical products, so the microbial safety of pharmaceutical water is crucial for pharmaceutical quality. This paper mainly introduces the characterization of the pharmaceutical water system, and conducts biofilm analysis. Due to the characteristics of oligotrophic and slow-growing of microorganisms in the pharmaceutical water system, this paper also further discusses the microbiological testing and culture methods of pharmaceutical water, so as to provide reference for the establishment of a scientific and reasonable microbiological testing system, as well as provide an important theoretical basis and new ideas for further research on hard-to-culture microorganisms.

AIM:To explore the association between single nucleotide polymorphism in exon 33 (E33SNP) of thyroglobulin gene and Graves ’ disease ( GD) relapse after antithyroid drug ( ATD) withdrawal .METHODS:The healthy controls (232 cases) and GD patients with discontinued treatment (243 cases) were selected.According to the time of re-lapse, the GD patients were divided into A, B and C subgroups.The A group contained 77 cases of relapse within 1 year, B group contained 86 cases of relapse 1~2 years after treatment and C group contained 80 cases without recurrence within 2 years.The genotypes of E33SNP were identified by RT-PCR.The genotype ratio of thyroglobulin between control group and observation group was comparatively analyzed , and the levels of thyroid-stimulating hormone ( TSH) , free triiodothyro-nine (FT3), free thyroxine (FT4) and thyrotropin receptor antibody (TRAb), ophthalmopathy and goiter size in A , B and C subgroups in different genotype GD patients were investigated .Moreover , cumulative efficiency for patients with different genotypes in the observation group after ATD treatment within 2 years were analyzed .RESULTS:The genotype of E33SNP between observation group and control group had no significant difference , but a significant difference between A , B and C subgroups was observed (P<0.05).The levels of TSH, FT3 and FT4, and goiter size of the patients with different geno-types had no significant difference , while the TRAb levels and ophthalmopathy presented a significant difference ( P <0.05).In addition, the cumulative efficiency within 2 years for GD patients with E33SNP T/T, E33SNP T/C and E33SNP C/C genotypes was 61.8%, 42.6% and 21.3%, respectively, all with significant differences (P<0.05).CONCLU-SION:The GD patients with E33SNP C/C genotype have significantly higher TRAb level and ophthalmopathy rate than those in the patients with E33SNP C/T and E33SNP C/C genotypes, and are more likely to relapse after ATD treatment . The GD patients with E33SNP T/T genotype show a lower recurrence rate .Therefore, combination treatment or other treat-ment modalities may be more reasonable for the GD patients with E 33SNP C/C genotype.

Hirudin as a novel anticoagulant and antithromotic agent, is a kind of protein that is obtained from the salivary gland secretion of one kind blood-sucking leech.It increases the difficulty extraction and purification of hirudin that composition of no anticoagulant activity of peseudo-hirudin in the body of leech and a large number of complex material from the salivary gland secretion of one kind blood-sucking leech.This article focuses on reviewing the structure of hirudin, genetic makeup, extraction and purification of hirudin according to the related research.Analysis of the pros and cons of various extraction and purification methods.Looking forward to its developing tendency, so as to provide reference for extraction and purification of Hirudin.

OBJECTIVE To investigate the characteristics and laws of changes in electrically evoked auditory brainstem responses(EABR),electrically evoked stapedius reflex(ESRT) after cochlear implantation in children with inner ear malformation.METHODS 88 cases of Australian 24 multi-channel cochlear implants were divided into normal group and inner ear malformation group,The EABR and ESRT thresholds were measured at different periods within one year after operation.Behavior auditory responses,T-level and C-level were measured 1 year postoperatively to analyze their characteristics and changes.RESULTS The EABR and ESRT thresholds in the patients with inner ear malformation were significantly higher than those in the normal cochlear group(P＜0.05).The changing trend of EABR and ESRT were the same in the 2 groups,the overall trend is that low frequency value is low,high frequency value is higher.EABR,ESRT threshold increased gradually after 1 year,EABR was significantly correlated with T-level,and ESRT was significantly correlated with C-level.CONCLUSION The changes of EABR and ESRT thresholds in the cochlear implantation of the inner ear malformation group were similar to those of the normal cochlear group,the thresholds can be used to guide mapping for the inner ear malformation of the cochlear implant.

Objective To analyze the feasibility of body surface electrodes instead of multipair esophageal electrodes for the evaluation of neural respiratory drive in patients with COPD. Methods Diaphragm electromyo-gram(EMG)from body surface electrodes and multipair esophageal electrodes,was recorded in 29 patients with stable COPD recruited from outpatient clinic. Changes of neural respiratory drive of two kinds of electrodes during resting and maximal isocapnic ventilation (MIV) were observed before and after inhalation of bronchodilators. Results Ventilation significantly improved ,RMS-sur and RMS-eso significantly decreased after the inhalation of bronchodilators during resting and MIV. RMS-sur and RMS-eso were significantly correlated(r=0.660,P<0.01). Conclusion EMG from the surface electrodes may be a useful and noninvasive technique to evaluate neural respi-ratory drive in patients with COPD.

Objective To explore the efficacy of noninvasive positive pressure ventilation technology com-bined with respiratory stimulant in the treatment of pulmonary encephalopathy in patients with COPD.Methods For-ty -eight COPD patients with early pulmonary encephalopathy were included.They were randomly divided into the treatment group and control group.The treatment group was treated by not only regular treatment and noninvasive posi-tive pressure ventilation technology,but also respiratory stimulant.The control group was only treated by regular treat-ment and noninvasive positive pressure ventilation technology.The indexes were recorded,including tracheal intubation rate,clinical recovery rate,mortality,Glasgow coma score,arterial blood gas analysis and adverse reactions.Results Compared with the control group,the arterial blood gas analysis of treatment group on the 2h,24h and 72h were statis-tically significant different(P <0.05).The GCS score on the 24h and 72h were also statistically significant different [24h,(13.4 ±1.6)vs.(11.8 ±2.1),P <0.05;72h,(14.6 ±1.5)vs.(12.4 ±2.4),P <0.05].The treatment group had lower tracheal intubation rate(12% vs.56.5%,P =0.001),shorter consciousness recovery time[(3.0 ± 0.5)h vs.(5.6 ±0.7)h,P =0.023],shorter hospitalization time in ICU[(12.6 ±1.5)d vs.(19.2 ±1.8)d,P =0.004]and better clinical recovery(22 /25 vs.10 /23,P =0.001)than the control group.But the mortality rate had no significant difference between the two groups(1 /25 vs.4 /23,P =0.129).Conclusion NIPPV combined with respiratory stimulant is an effective,safe and simple method for treatment of COPD complicated with pulmonary encephalopathy.It can significantly improve the clinical symptoms and the index of arterial blood gas analysis,and it can shorten consciousness recovery time and decrease tracheal intubation rate.But close observation must be carried out,and the invasive mechanical ventilation should be promptly executed in case of emergency or exacerbation.

Objective To observe BQ123 on lung tissue injury following ischemia-reperfusion of hind limbs in rats.Methods A total of 30 male SD rats were randomly divided into control,I-R,and BQ123 groups,with 10 rats in each group. After 4- hour of ischemia and 4-hour of reperfusion to the hind limbs, ET-1 was measured by radioimmunoassay, levels of myeloperoxidase (MPO) and malondialehyde (MDA) were examined by biochemical method,and the content of P-selectin was examined by ELISA. Immunohistochemical method was used to detect the expressions of Fas,Bcl-2 and Caspase-3 in lung tissues. Pulmonary apoptosis was examined by means of TdT-mediated dUTP nick end labeling ( TUNEL) . Results Compared with the control group,the levels of ET-1,MPO,MDA and P-selectin in lung tissues were all increased significantly in I-R group ( P<0. 01). The expressions of Fas,Bcl-2 and Caspase-3 were 0. 294±0. 003,0. 108±0. 005,and 0. 174±0. 003,significantly up-regulated in the I-R group. The apoptosis rate [(18. 83±2. 86)%] was significantly increased in the I-R group (P<0. 01). Compared with I-R group,the tissue contents of ET-1,MPO,MDA and P-selectin were significantly lowered in BQ123 group (P<0. 01). The expression levels of Fas (0. 115±0. 007) and Caspase-3 (0. 159±0. 006) were decreased,but the expression of Bcl-2 was increased (0. 128±0. 005). The apoptosis rate in BQ123 group was significantly lower [(10. 67±2. 16)%,P<0. 01].Conclusion BQ123 may have protective effects on lung tissue after limb ischemia-reperfusion in rats by means of improving neutrophil aggregation and reducing the expression of proteins related to cell apoptosis.

OBJECTIVETo construct a prokaryotic expression system of groEL gene of Leptospira interrogans serogroup Icterohaemorrhagia serovar Lai strain Lai, and to determine the immunoprotective effect of recombinant GroEL protein (rGroEL) in LVG hamsters.

METHODSThe groEL gene was amplified by high fidelity PCR and the amplification products were then sequenced. A prokaryotic expression system of groEL gene was constructed using routine genetic engineering technique. SDS-PAGE plus Bio-Rad Gel Image Analyzer was applied to examine the expression and dissolubility of rGroEL protein while Ni-NTA affinity chromatography was used to extract the expressed rGroEL. The immunoprotective rate in rGroEL-immunized LVG hamsters was determined after challenge with L.interrogans strain Lai. The cross agglutination titers of sera from immunized hamsters with different L.interrogans serogroups were detected using MAT.

RESULTSThe nucleotide and amino acid sequences of the cloned groEL gene were the same as those reported in GenBank. The constructed prokaryotic expression system of groEL gene expressed soluble rGroEL. The immunoprotective rates of 100 and 200 μg rGroEL in LVG hamsters were 50.0 % and 75.0%, respectively. The sera from the rGroEL-immunized LVG hamsters agglutinated all the L.interrogans serogroups tested with different levels.

CONCLUSIONThe GroEL protein is a genus-specific immunoprotective antigen of L.interrogans and can be used to develop an universal genetically engineering vaccine of Leptospira.

Agglutination Tests ; Animals ; Antigens, Bacterial ; immunology ; Chaperonin 60 ; genetics ; immunology ; Cricetinae ; Gene Expression ; Leptospira interrogans ; genetics ; immunology ; Recombinant Proteins ; genetics ; immunology