Development of highly efficient electrocompetent cells for electroporation of Geobacillus thermoglucosidasius NCIMB 11955 / 生物工程学报

Geobacillus thermoglucosidasius is a kind of Gram-positive facultative anaerobic bacteria. The fast growth rate under high temperature and less susceptibility to microbial contamination enable G. thermoglucosidasius to be a desirable producer of biofuels and high-value-added chemicals for the next-generation industrial biotechnology. However, compared with the classical model strain Escherichia coli, the applications of G. thermoglucosidasius are hampered by its low transformation efficiency. This study aimed at obtaining competent cells with high transformation efficiency through inactivating restriction enzymes, adding cell membrane inhibitors and cell wall weakening agents. The results showed that the electro-transformation efficiency achieved 1.2×104 CFU/(μg DNA) by knocking out four genes encoding restriction enzymes. Adding a certain amount of tween 80, dl-threonine and glycine further increased the competent efficiency about 22.5, 44, and 334 times, respectively. The electro-transformation efficiency was enhanced to 4.6×106 CFU/(μg DNA) under the optimized conditions, laying a foundation for genetic manipulation and metabolic engineering of G. thermoglucosidasius.

Integrating PCR-free amplification and synergistic sensing for ultrasensitive and rapid CRISPR/Cas12a-based SARS-CoV-2 antigen detection

Antigen detection provides particularly valuable information for medical diagnoses; however, the current detection methods are less sensitive and accurate than nucleic acid analysis. The combination of CRISPR/Cas12a and aptamers provides a new detection paradigm, but sensitive sensing and stable amplification in antigen detection remain challenging. Here, we present a PCR-free multiple trigger dsDNA tandem-based signal amplification strategy and a de novo designed dual aptamer synergistic sensing strategy. Integration of these two strategies endowed the CRISPR/Cas12a and aptamer-based method with ultra-sensitive, fast, and stable antigen detection. In a demonstration of this method, the limit of detection was at the single virus level (0.17 fM, approximately two copies/L) in SARS-CoV-2 antigen nucleocapsid protein analysis of saliva or serum samples. The entire procedure required only 20 minutes. Given our systems simplicity and modular setup, we believe that it could be adapted reasonably easily for general applications in CRISPR/Cas12a-aptamer-based detection.

Protein-Degrading Enzymes in Osteoarthritis.

OBJECTIVE: TGFß1 plays an important role in the metabolism of articular cartilage and bone; however, the pathological mechanism and targets of TGFß1 in cartilage degradation and uncoupling of subchondral bone remodeling remain unclear. Therefore, in this study, we investigated the relationship between TGFß1 and major protein-degrading enzymes, and evaluated the role of high levels of active TGFß1 in the thickening of subchondral bone and calcification of articular cartilage. MATERIALS AND METHODS: The expression of TGFß1 and protein-degrading enzymes in clinical samples of articular cartilage and subchondral bone obtained from the knee joint of patients with osteoarthritis was detected by immunohistochemistry. The expression levels of TGFß1, MMP-3, MMP-13 and IL-1ß in cartilage and subchondral bone tissues were detected by absolute real-time quantitative RT-PCR. The expression of TGFß1, nestin and osterix in subchondral bone was detected by Western blot analysis and immunohistochemistry. The degree of subchondral bone thickening was determined by micro-computed tomography (CT) imaging. RESULTS: Expression of TGFß1 and cartilage-degrading enzymes was higher in the cartilage-disrupted group than that in the intact group. Furthermore, expression of TGFß1, nestin and osterix was significantly higher in the OA group than that in the control group. Micro-CT imaging showed that in the OA group, the subchondral bone plate is thickened and the density is increased. The trabecular bone structure is thick plate-like structure, the thickness of the trabecular bone is increased and the gap is small. CONCLUSIONS: The data suggest that highly active TGFß1 activates the expression of cartilage-degrading enzymes. Abnormally activated TGFß1 may induce formation of the subchondral bone and expansion of the calcified cartilage area, eventually leading to degradation of the cartilage tissue.

Relationship between the gene polymorphisms of transforming growth factor beta 1 and interleukin 1 beta and primary knee osteoarthritis in Xinjiang Han and Uygur populations / 中国组织工程研究

BACKGROUND:Transforming growth factor β1 (TGF-β1) and interleukin 1β (IL-1β) have been reported to play an important role in the occurrence and development of primary osteoarthritis. OBJECTIVE:To investigate the association between gene polymorphisms of TGF-β1 and IL-1β and primary knee osteoarthritis. METHODS: Han and Uyghur elderly populations in Xinjiang Uygur Autonomous Region, China were surveyed on the prevalence of osteoarthritis, followed by allotted to osteoarthritis and health groups according the symptoms and radiography. Genotyping TGF-β1-509C/T and -1348C/T and IL-1β-511C/T was performed to analyze the relationship between the gene polymorphisms of TGF-β1 and IL-1β and osteoarthritis. RESULTS AND CONCLUSION:Alleles T and C with genotypes CC, CT and TT were detected in both two groups. In the Uygur population, the genotype frequency of TGF-β1-509C/T and IL-1β-511C/T showed significant difference between osteoarthritis and health groups (P < 0.05). In the Han population, the genotype frequency of TGF-β1-1348C/T showed significant difference between two groups (P < 0.05). These results suggest that the Uygur individuals carrying TT genotype of TGF-β1-509C/T and IL-1β-511C/T, and Han people carrying the TT genotype of TGF-β -1348T are more susceptible to osteoarthritis.

Investigation on influence factors of pathogenic bacterial detection results in children lower respiratory tract infections / 国际检验医学杂志

Objective To investigate the distribution characteristics of pathogenic bacteria among different seasons and ages in children lower respiratory tract infection.Methods To retrospectively analyze the results of bacterial culture in pediatric outpatients and inpatients with lower respiratory tract infections in our hospital from September 2014 to August 2015.The pathogenic bacterial detection situation of lower respiratory tract infection among different age groups and different were statistically analyzed.Results A total of 2 809 pathogenic bacterial strains were isolated from 4 629 lower respiratory tract specimens,and detection rate was 60.7%.The pathogenic bacteria distribution was different among different age groups.Haemophilus influenzae (33.3%) ranked the first place in the lower respiratory infection among children aged from 19 d to 7 year old.The detection rates of Haemophilus parainfluenzae,Moraxella catarrhalis and staphylococcus aureus had obvious seasonality,while Streptococcus pneumoniae had no obvious seasonality.Conclusion Pathogenic bacteria causing lower respiratory tract infections in children are different from those in adults,which are affected by age and seasonal change.

Construction and identification of FRET-based MMP3 biosensor / 吉林大学学报(医学版)

Objective:To study the construction of matrix metalloproteinase 3 (MMP3)biosensor vector,and to illuminate the activated process of MMP3 in the living cells.Methods:The ECFP-MMP3-YPet biosensor vector anchored on cellular surface was constructed and identified.The MMP3 biosensor was transfected into the 293T cells.The transfection efficiency was observed 24 h after transfection.The flurorescence resonance energy transfer (FRET )-based MMP3 biosensor was observed by inversion fluorescence microscope. Results:The MMP3 biosensor vector was successfully constructed.The length of MMP3-YPet identified by double enzyme digestion and PCR was about 780 bp.The transfection efficiency of MMP3 biosensor was about 40%,and which was evenly presented in cytoplasm of 293T cells.And the FRET ratio of MMP3 biosensor was decreased after stimulation with uPA on the 293T cells. The FRET ratio reached its minimum about 30 min later. Conclusion:The MMP3 biosensor can sensitively and reliably monitor the MMP3 activation in living cells.

Comparison of effect between tranexamic acid versus fibrin sealant after joint arthroplasty:a Meta-analysis / 重庆医学

Objective To compare the effectiveness and safety of tranexamic acid and fibrin sealant in joint arthroplasty . Methods The literatures on the application of tranexamic acid and fibrin sealant in joint arthroplasty were retrieved from PubMed , Embase ,Cochrane Library ,CNKI and other internet databases .Two reviewers independently screened the literatures according to the inclusion and exclusion standard .The RevMan 5 .2 software was adopted to conduct the statistical analysis on the extracted da‐ta .Results Four randomized controlled trials(RCTs) and 2 retrospective experiments were included ,involving 449 patients .The meta analysis results showed that tranexamic acid and fibrin sealant had no statistically significant difference in total blood loss[MD= -192 .24 ,95% CI(-496 .16 ,111 .69) ,P=0 .22] and hemoglobin loss amount[MD= -0 .49 ,95% CI(-1 .19 ,0 .20) ,P=0 .16] , had statistically significant difference in the blood transfusion rate[OR=0 .30 ,95% CI(0 .18 ,0 .52) ,P<0 .01] ,and had no statisti‐cally significant difference in the postoperative complications[OR=1 .20 ,95% CI(0 .36 ,3 .99) ,P=0 .76] .Conclusion Tranexamic acid is more effective than fibrin sealant of controlling postoperative transfusion rate in joint arthroplasty .

Regression analysis of factors affecting efficacy of arthroscopic treatment for knee osteoarthritis / 局解手术学杂志

Objective To investigate the factors that affecting the efficacy of arthroscopic debridement in the treatment of knee osteoar-thritis. Methods The clinical data of patients with knee osteoarthritis treated with arthroscopic debridement in our hospital from January 2009 to December 2013 was retrospectively analyzed,and the treatment effect was evaluated by Lysholm score. The factors that may affect the efficacy were selected to make a single factor analysis,and then made a multiple logistic regression analysis with the factors which were of sta-tistical significance through single factor analysis. Results Single factor analysis showed that age,course of disease,body mass index,psycho-logical factor,preoperative VAS score,preoperative Kellgren-Lawrence grade,preoperative Lysholm score and postoperative rehabilitation exer-cise were the factors that affected the efficacy of knee arthroscopy in the treatment of knee osteoarthritis,and the difference between the two groups was statistically significant. Further multivariate analysis showed that high body mass index,high preoperative VAS pain score,low Ly-sholm score,high psychological expectations and high preoperative Kellgren-Lawrence grading were expected to be the independent risk fac-tors of knee arthroscopy in the treatment of knee osteoarthritis. Strict compliance with the postoperative rehabilitation exercise was the protect factor. Conclusion High body mass index,high preoperative VAS pain score,low preoperative Lysholm score,high preoperative Kellgren-Lawrence grading,and high psychological expectations were expected to reduce the effect of surgery,while strict compliance with the postoper-ative rehabilitation exercise can improve the curative effect.

Characteristics of pathogens and risk factors of surgical site infection in orthopedic patients in five consecutive years / 中国感染控制杂志

Objective To investigate the incidence of surgical site infection (SSI)in patients undergoing orthopedic surgery,analyze the risk factors,and provide basis for the prevention and control of SSI.Methods All hospitalized orthopedic patients undergoing surgery in a hospital from January 2010 to December 2014 were retrospectively sur-veyed,questionnaires were designed,patients’medical records were reviewed,incidence of SSI was analyzed,risk factors for SSI were analyzed with univariate and logistic regression methods.Results A total of 14 300 orthopedic patients undergoing surgery were investigated,576 (4.03%)patients had SSI,predominantly were superficial inci-sion infection (n=429,74.48%),615 strains of pathogenic bacteria were isolated from 576 patients,mainly were Staphylococcus aureus (n=137,22.28%),Escherichia coli (n=84,13.66%),and Enterobacter cloacae (n=73, 11 .87%).The incidence of SSI decreased year by year in patients undergoing orthopedics surgery(χ2 =24.706,P <0.001);the incidence of SSI in patients with amputation was the highest (22.67%),followed by patients with de-bridement (7.16%);multivariate logistic regression analysis indicated that long duration of operation,long length of hospital stay,underlying diseases,use of implants,contaminated incision,more intraoperative blood loss,irra-tional perioperative use of antimicrobial agents,and without using negative pressure drainage were independent risk factors for SSI in patients undergoing orthopedic surgery.Conclusion The incidence of SSI is high in orthopedic pa-tients undergoing surgery,effective preventive measures should be actively taken according to the related risk factors of SSI,so as to reduce the occurrence of SSI.

The modified bacterial two-hybrid system / 生物工程学报

Bacterial two-hybrid system is a newly developed method for studying protein-protein interactions. However, in our studies of the interaction of regulatory proteins in Streptomyces, it was found that the bacterial two-hybrid system is not sensitive enough by the blue-and-white selection on X-gal plate. To overcome this drawback, the reason of false positive clone was firstly determined, which was the disturbance of other direct or indirect regulation on lacZ promoter. Then the disturbance was diluted by introducing multicopy lacZ promoter, which drive another reporter gene gfp. By such design, the sensitivity of the modified bacterial two-hybrid system was significantly inproved and the two different reporters also help to decrease the rate of the false positive clones. Further the evaluation of the modifiedd bacterial two-hybrid system indicated that the sensitivity was significantly improved.

Analysis of locking plate in the treatment of proximal humerus fractures / 重庆医学

Objective To evaluate the clinical and radiological results of locking plate for treatment of proximal humerus frac‐tures and the efficacy of anatomical healing of tuberosities .Methods A total of 57 patients with proximal humerus fractures were treated with locking‐plate from July 2008 to March 2012 .A standardized radiological evaluation was conducted .Patients were divid‐ed into two groups :group A (anatomical healing of tuberosities) with 31 cases and group B (without anatomical healing of tuberosi‐ties) with 26 cases .Clinical assessment was performed using the Neer rating scale .Results Considering the entire sample ,the mean Neer score was 87 .96 ± 5 .06 points ,the excellent rate was 94 .74℅ .Comparing these parameters in the two groups ,group A was significant higher in the Neer scores and the range of movement than that of group B (P0 .05) .Conclusion the locing plate for treatment of complex proximal humerus fractures has a high subjective satisfaction rate .A good functional result depends on anatomical reestablishment of proximal humerus anatomy , particularly the healing of the greater tuberosity .

Construction and identification of urokinase-type plasminogen activator biosensor plasmid / 吉林大学学报(医学版)

Objective To construct the eukaryotic expression vector urokinase-type plasminogen activator (uPA) biosensor which was the composition of the fusion protein enhanced cyan fluorescent protein-uPA (substrate)-yellow fluorescent protein variant (ECFP-uPA substrate-linker-YPet).Methods By the template Src-biosensor, the YPet primers were designed by Primer Premier 5.0 software,and the restriction enzyme sites,uPA substrate gene sequence and linker were added in its 5′ end. With the intermediate vector pDMTM-18T, an eukaryotic expression vector which contained a fusion protein of ECFP-uPA substrate-linker-YPet was constructed by genetic engineering.Then the uPA biosensor was transfected into 293T cells.The transfection efficiency and expression of fusion proteins were observed after 24 h.Fluorescence resonance energy transfer (FRET)was observed by the inversion fluorescence microscope and measured by the MetaFlour FRET 4.6 software. Results The uPA biosensor vector was confirmed by the fragment of PCR and double restriction enzyme digestion.The transfection efficiency was nearly 40%.The immunofluorescence detection results displayed that uPA biosensor fusion protein expressed in the 293T cells membrane and the FRET of uPA biosensor in the living 293T cells was observed after incubation with the recombinant human uPA (rhuPA).Conclusion uPA biosensor is successfully constructed and it could be used as a molecular probe to study the temporal and spatial variation of uPA in living cells.

The epidemiological characteristics of Salmonella in children with infectious diarrhea in Zhuhai City from 2009 to 2014 / 国际检验医学杂志

Objective To investigate epidemiological characteristics of Salmonella infection in children with infectious diarrhea in Zhuhai City from 2009 to 2014 .Methods Isolation ,culturing ,biochemical identification and serotyping of Salmonella were carried out in strains isolated from stool specimens of 4 395 children with infectious diarrhea in inpatient and outpatient settings in Zhuhai Municipal Maternal and Child Healthcare Hospital from 2009 to 2014 ,and data were statistically analysed .Results A total of 546 strains of Salmonella was isolated(the overall isolation rate was 12 .42% ) and 30 serotypes were identified .Among children with Salmonella infection ,those under 3 years old accounted for 93 .22% .There was no statistically significant difference of isolation rate between male and female children with infectious diarrhea(P<0 .05) .June and July were peak period of Salmonella infection ,and the isolation rate was 16 .40% and 16 .09% respectively .Isolates of Salmonella typhimurium ,Salmonella enteritidis and Salmonella stanley account for 50 .92% ,13 .55% and 10 .26% respectively ,which were dominant serotypes .Conclusion Salmonella is the main pathogenic bacteria in children with infectious diarrhea in Zhuhai area ,and Salmonella typhimurium is the main serotype .The infec‐tion of Salmonella might be correlated with children′s age and climate change .

Application of polyetheretherketone cages and titanium cages combined with pedicle screw fixation operation in lumbar degenerative disease / 局解手术学杂志

Objective To explore the clinical efficacy of polyetheretherketone( PEEK) cages and titanium cages combined with pedicle screw fixation operation in treatment of lumbar degenerative disease,and provide reference for clinic. Methods The data of 50 patients with lumbar degenerative disease in our hospital from March 2011 to March 2013 were retrospectively analyzed. All patients were treated with pos-terior decompression,bone graft and transpedicular screw internal fixation,according to the different cages,they were divided into PEEK group and titanium group. The PEEK group with 25 patients used polyetheretherketone cages,and the titanium group with 25 patients used titanium cages. The JOA scores of patient before surgery,1 month,6 months and 1 year after surgery between two groups were recorded and compared. The ROM of fusion levels and adjacent segment of patients before and after operation between two groups were compared. Results There was no statistical significance of difference in JOA score,Oswestry score and fusion rate between two groups(P>0. 05). There were statistical significance of differences intervertebral disc height change between 2 groups(P<0. 05). The ROM of fusion levels and adjacent segment of patients in PEEK group was better than those in titanium group(P<0. 05). Conclusion The polyetheretherketone cages combined with pedicle screw fixation operation have a good effect in the treatment of patients with lumbar degenerative disease,especially in matter of lumbar spinal motion and intervertebral height lost.

Progress in transcriptional studies / 生物工程学报

Gene expression exhibits temporal and spatial patterns to response environmental changes and growth cycle. Gene expression is under strict control at different levels among which control at transcription level is the predominant mode, especially in prokaryotes. In this review, we summarized the new developments of methods used in transcriptional studies, including modifications and improvements of the classic methods, such as gel-shift assay, DNA foot printing, and in vivo reporter system. In addition, we introduced examples to apply new methods, such as surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) to characterize protein-DNA, ligand-protein, and ligand-protein-DNA interactions. The collection of these methods and their application could guide and accelerate relevant studies.

Randomized,controlled clinical trial comparison of sodium hyaluronate versus celecoxib in knee osteoarthritis patients / 实用医学杂志

Objective To compare the effects of sodium hyaluronate (SH) and celecoxib (CO) administration on the treatment of knee osteoarthritis (OA) and to investigate their influences on levels of uPA and MMP-3 in synovial fluid. Methods One hundred and thirty-six knee osteoarthritis (OA) patients from January 2010 to October 2011 were randomly enrolled into two groups: the SH group and the CO group. In the SH group, patients were injected with 2 mL sodium hyaluronate intra articulation once a week for 5 weeks. In the CO group , patients were given oral administration of celecoxib daily at a dosage of 200 mg for 5 weeks. Before and at 1 ,6 months after treatment, Lequesne′s index and VAS-pain were detected to assess the clinical results of these two drugs. The levels of uPA and MMP-3 in synovial fluid were measured by using ELISA assay. Results All patients were followed up for 6 months to 12 months. The Lequesne′s index and VAS-pain score were lowered at 1 and 6 moths after treatment in both the SH group and the CO group(P0.05). Dramatic reduction of the levels of uPA and MMP-3 in synovial fluid were observed in the SH group after treatment(P0.05). Conclusion The redueced levels of uPA and MMP-3 in synovial fluid after treatment of sodium hyaluronate may contribute to its longer-lasting effect than that of celecoxib. Therefore , the combination of sodium hyaluronate with celecoxib may lead to better therapeutic effect on OA patients.

The expression and significance of MMP3 in synovium of knee joint of different stage in osteoarthritis patients / 实用医学杂志

Objective To investigate the expression and significance of MMP-3 in knee joint synovium of different stage in osteoarthritis (OA) patients. Methods MMP-3 protein were detected by immunohistochemical method in knee synovial tissues from 90 OA patients (the OA group) and OA group was divided into 3 subgroups according to Kellgren and LawrenceX-ray diagnosing standards: the gradeⅠ(n = 30), gradeⅡ (n = 30) and grade Ⅲ subgroups (n = 30). 30 patients were enrolled as control group. Immunohistochemistry was used to detect the expression of MMP-3 protein in the tissue. Results The expression level of MMP-3 protein in OA group was significantly higher that in the normal synovium (P < 0.05). There existed significant difference in the expression of MMP-3 protein between the grade Ⅲ subgroup and the gradeⅠ, or Ⅱsubgroups (P < 0.05). The expression of MMP-3 protein related positively to the severity of OA (r = 0.912, P < 0.05). Conclusion The expression of MMP-3 protein related closely to the pathogenic mechanism of OA. It may serve as an important indicator of early diagnosis and the activity of OA.

Antibiotic resistance of the Haemophilus inf luenzae strains isolated from respiratory tract in children / 中国感染与化疗杂志

Objective To investigate the antibiotic resistance of Haemophilus influenzae isolates collected from the children with respiratory tract infection for rational use of antibiotics in clinical practice .Methods The H .influenzae strains were isolated from children and subjected to antimicrobial susceptibility testing by Kirby-Bauer method .Nitrocefin disc test was used to detect the production of beta-lactamases .WHONET 5 .6 software was used to analyze the susceptibility data .Results A totalof1256strainsof H.influenzaewereisolated.About37.8% ,65.5% and16.5% ofthe1256strainsof H.influenzae were resistant to ampicillin ,trimethoprim-sulfamethoxazole ,and ampicillin-sulbactam ,respectively .Less than 10 .0% of these strains were resistant to any other antibiotics tested .Beta-lactamase was produced in 33 .5% of the 1 256 strains of H . influenzae .Conclusions The H . influenzae strains in this study are mainly resistant ampicillin and trimethoprim-sulfamethoxazole .About 80 .0% of these H . influenzae strains were still susceptible to cefaclor ,ampicillin-sulbactam , cefixime ,ceftazidime ,azithromycin ,ciprofloxacin ,meropenem and rifampin .The primary mechanism of ampicillin resistance in Haemophilus is production of beta-lactamases .

Analysis of Salmonella infection in 6 417 cases of infectious diarrhea / 国际检验医学杂志

Objective To understand the status quo of Salmonella infection in the patients with infectious diarrhea in Zhongshan city .Methods The fecal samples collected from 6 417 outpatients or inpatients with infection diarrhea in our hospital from January 2010 to December 2013 were performed the Salmonella culture ,isolation and identification and at the same time the serological clas-sification and the pulsed-field gel electrophoresis (PFGE) were performed .Results Among 6 417 samples ,460 strains of Salmonel-la were detected with the isolation rate of 7 .17% .Salmonella infection was dominated by children infection .The Salmonella infec-tion rate in children aged less than 1 years was 52 .4% (241 cases) ,followed by children aged 1- < 3 years ,which was 36 .7% (169 cases) .In the serological classification ,the most of detected Salmonella was salmonella typhimurium 46 .1% ,accounting for 46 .1% , Stanley salmonella accounting for 13 .5% and Salmonella enteritidis accounting for 8 .7% .The PFGE homology analysis showed that in the XbaI enzyme digestion spectral band ,26 PFGE band types had 100% homology .Conclusion Salmonella is one of the main pathogenic bacteria causing diarrhea and serious impact on health ,which is worthy of clinicians to pay attention to .

Expression and significance of matrix metalloproteinase 14 in synovial tissue and syrovial fluid of osteoarthritis / 重庆医学

Objective To evaluate the expression and clinical significance of matrix metalloproteinase 14(MMP-14) in osteoar-thritis(OA) patients in synovium and synovial fluid of knee joint .Methods Semi-quantitative RT-PCR were used to detect the ex-pression of MMP-14 mRNA in 32 case of OA (experimental group)and 26 non-OA patients(control group)synovium ;protein level expression of MMP-14 in synovial fluid were analyzed by ELISA .Results The expression of MMP-14 mRNA in experimental group synovium of knee joint was significantly increased compared with control group (P<0 .05);but the protein level of MMP-14 was low in experimental group synovial fluid compared with control group (P<0 .05) .Conclusion The expression of MMP-14 is increased in OA patients synovium and maybe promote the progress of OA directly ;the protein level of MMP-14 is decreased in OA synovial fluid ,indicated the function of MMP-14 in OA synoviocyte indirectly .