For engineering an efficient butanol-producing Escherichia coli strain, many efforts have been paid on the known genes or pathways based on current knowledge. However, many genes in the genome could also contribute to butanol production in an unexpected way. In this work, we used Tn5 transposon to construct a mutant library including 1 196 strains in a previously engineered butanol-producing E. coli strain. To screen the strains with improved titer of butanol production, we developed a high-throughput method for pyruvate detection based on dinitrophenylhydrazine reaction using 96-well microplate reader, because pyruvate is the precursor of butanol and its concentration is inversely correlated with butanol in the fermentation broth. Using this method, we successfully screened three mutants with increased butanol titer. The insertion sites of Tn5 transposon was in the ORFs of pykA, tdk, and cadC by inverse PCR and sequencing. These found genes would be efficient targets for further strain improvement. And the genome scanning strategy described here will be helpful for other microbial cell factory construction.
Butanols ; chemistry ; DNA Transposable Elements ; Escherichia coli ; metabolism ; Fermentation ; Gene Library ; Hydrazines ; Industrial Microbiology ; Mutagenesis ; Open Reading Frames ; Organisms, Genetically Modified ; Polymerase Chain Reaction ; Pyruvic Acid ; chemistry

Objective To analyze the clinical characteristics of systemic lupus erythematosus (SLE) patients with septic arthritis.Methods Twelve SLE patients with septic arthritis admitted to Peking Union Medical College Hospital from January 1990 to December 2012 were retrospectively analyzed.Results The median duration from onset of SLE to septic arthritis was 8.5 months (ranged from 1 to 120 months).All patients had received higher doses of steroid therapy (equal to prednisolone 1 mg ·kg-1 ·d-1) and immunosuppressant,and 5 of them had received methylprednisolone pulse therapy.The infectious manifestations included joint pain (all cases),swelling (5 cases),reduced joint function (8 cases) and fever (10 cases).All patients were oligo-arthritis.The hip and knee joints were most commonly involved.Three patients were infected by Salmonella,4 patients were infected by S.aureus,and 3 patients were infected by tuber culosis.Conclusion When SLE patients with long term steroid and immunosuppressant therapy developedacute joint pain and swelling,septic arthritis should be considered.Synovial fluid Gram stain,culture,blood culture and arthroscopy should be performed promptly.Appropriate antibiotics should be administered,and early treatment can improve the outcomes.

OBJECTIVE To investigate the degree of risk of viral prevalence of HBV,HCV and HIV through blood transfusion in Beijing Hospital in China,and to assess the need of a national Haemovigilance System. METHODS Retrospectively,7883 blood bank specimens (collected from 2004 to 2007) were re-examined using 8 indicators (including 5-item Hepatitis B,anti-HCV,anti-HIV and Syphilis) for the prevalence of most common viral infection. RESULTS From the blood bank specimens,the prevalence of HBsAg was 0.88% (69),the anti-HBc positive blood,only the anti-HBc was found in 2.65% of the specimens,while both the anti-HBc and the anti-HBe were found in 2.09% of the specimens. The prevalence of anti-HCV was 0.09% (7). CONCLUSIONS We need to establish the national Haemovigilance System to strengthen the monitoring of the above HbsAg,HBcAb and HCV indicators to prevent the transfusion-transmitted infection. Only in this way can the public confidence in blood safety be improved.

The template-independent teminal transferase activity of Taq DNA polymerase results in an overhanging dA at the 3′end of its PCR products. The pGEMX vector constructed in this experiment forms a single overhanging dT at its 3′end as the result of cleavage with Xcm I restriction enzyme. This vector is very efficient for direct cloning of PCR product obtained by using Taq DNA polymerase.Recombinant colonies can be selected by Blue/white screening. Moreover,insertion fragment can be easily released from the vector simply with either BamH I or Hind III digestion.

OBJECTIVE: To investigate the influences of staphylococcus aureus in planktonic and biofilm forms on the expression of lysozyme, SLPI and gp340 in the human sinonasal explant model. METHOD: Mucosa samples from ethmoid sinus were collected from ten patients of cerebrospinal fluid leak and were cultured with and without S. aureus biofilms and planktonic cells. After the infection, the explant model was confirmed by CLSM, and the secretion of lysozyme, SLPI and gp340 was detected by enzyme-linked immunosorbent assay (ELISA) at 8, 16, and 24 h after S. aureus challenge. Expressions of lysozyme, SLPI and gp340 in mRNA and protein levels after 24 h S. aureus challenge were detected using RT-PCR, immunohistochemistry and Western bolt assay respectively. RESULT: The secretion of lysozyme, SLPI and gp340 in the explant model was observed with a trend to increase in a time-dependent manner. At 8 and 16 h after S. aureus challenge, the secretion of lysozyme, SLPI and gp340 in biofilms group was significantly higher than these in planktonic cells group and control group (P<0. 05). S. aureus biofilms enhanced the mRNA expressions of lysozyme, SLPI and gp340 significantly compared with planktonic cells and controls, and the mRNA expressions in the explant model challenged by planktonic cells were significantly higher than controls (P < 0.05). Although the Western bolt assay showed no differences between the lysozyme expression in the planktonic cells group and control group (P > 0.05), the biofilms enhanced the expressions of lysozyme, SLPI and gp340 significantly compared with planktonic cells and controls (P < 0.05). CONCLUSION S. aureus biofilm induced the expressions of lysozyme, SLPI and gp340 to a higher level than planktonic cells, indicating that S. aureus biofilm was an influencing factor on the innate immune system.
Biofilms ; Enzyme-Linked Immunosorbent Assay ; Ethmoid Sinus ; metabolism ; microbiology ; Humans ; Immunity, Innate ; Muramidase ; metabolism ; RNA, Messenger ; metabolism ; Receptors, Cell Surface ; metabolism ; Secretory Leukocyte Peptidase Inhibitor ; metabolism ; Staphylococcal Infections ; metabolism ; Tissue Culture Techniques

BACKGROUND: Acellular vascular matrix as vascular scaffold has following advantages: acellular vascular matrix possesses complicated three-dimensional structure of natural blood vessels. Growth factor and structural domain on the surface of acellular matrix helps for cell adhesion and infiltration.OBJECTIVE: To prepare acellular vascular matrix material and to evaluate its biocompatibility in vivo and in vitro.METHODS: Trypsin and Triton X-100 were used to gradually dispose pig carotid artery and to prepare acellular vascular matrix. The biocompstibility of the material was evaluated by implantation in muscle, acute toxicity experiment and cytotoxicity test in vitro.RESULTS AND CONCLUSION: The acallular vascular matrix material possessed good chemical stability and did not release harmful factors that produced destruction and dissolution in erythrocytes, without acute hemolytic reaction or toxic effects on cell growth. The acellular vascular matrix material showed lots of inflammatory cell infiltration in eady stage of implantation, and no significant inflammatory cell infiltration in late stage of observation. Fibroblasts were visible in the acellular matrix. In addition, the acellular matrix material did not exhibit toxic effects on surrounding tissues, showing wound stage I healing. Simultaneously,histological sections demonstrated that there were good compatibility of scaffold material and surrounding tissues, without rejection. These indicated that acellular matrix material presented good biocompatibility in animals.

Based on the subject classification of biomedical engineering in military hospital,the development modes of the discipline are discussed in this paper.

Objective To retrospectively analyze our experience of embolization of ruptured intracranial aneurysms during the period of cerebral vasospasm (CVS). Methods Thirty-seven patients with ruptured intracranial aneurysms were embolized with electrolytic detachable coils during the period of CVS (days 4 to 14). Group A included the 14 patients with angiographic CVS and group B included 23 patients without angiographic CVS. All except 2 patients were transferred to our department during the CVS period. Glasgow outcome score (GOS) was evaluated 3 months after the treatment. Results Twelve patients in group A successfully received the aneurysm embolization and treatment of the CVS with intraarterial papaverine injection and balloon angioplasty. GOS in 3 months was good recovery in 7 patients, moderate disability in 2, severe disability in 1, and dead in 2, respectively. Embolization failed in 2 patients because the microcatheters could't pass the spasmodic parent arteries. All the aneurysms in group B were successfully embolized. GOS were good recovery in 18 patients, moderate disability in 2, severe disability in 2, and dead in 1, respectively. There was no intraprocedural aneurysmal rupture but with 2 thromboembolic events. No rebleeding occurred during the mean 11 months follow-up. Conclusions The so-called “period of CVS” isn′t always associated with CVS in angiograpy. Embolization of ruptured intracranial aneurysms during the period of pure CVS doesn′t carry an increased risk. Both the aneurysms and CVS can be treated during the single procedure. It can reduce the rebleeding rate in hospital and improve the prognosis of the patients with CVS.

AIM: To explore the effect of berbamine(BER) on apoptosis in K562 cells and its possible molecular mechanisms. METHODS: The apoptosis rate was measured by flow cytometry while electron microscopy and DNA electrophoresis were used to evaluate the characteristic changes of apoptosis, RT-PCR and Western blot were used to examine the expression levels of apoptosis related gene bcr/abl and BCR/ABL protein. RESULTS: By FCM, the apoptosis rate of K562 cells treated with 8.0 mg/L BER for 24 h and 72 h increased from (29.20?3.82)% to (61.77?4.35)% (P

BackgroundThe morphological examination of macular disease is very important information for the early diagnosis.Spectral domain optical coherence tomography (SD-OCT) is a gold criteria in the diagnosis of macular diseases,but its clinical application is limited in the eyes with refractive medium opacity.The resolution ratio of conventional B-scan sonography is unsatisfactory.20 MHz B-scan sonography may be a new approach to maculopathy.ObjectiveThis study was to evaluate the clinical value of 20 MHz B-scan ultrasound by comparing morphological changes of different maculopathies observed by 20 MHz ultrasound and spectral-domain OCT.MethodsA prospective study was designed.51 eyes of 51 patients with macular diseases who were determined by Tianjin Medical University Eye Center were enrolled in this study.Spectral-domain OCT was used to image the macular area,and then the 20 MHz B-scan ultrasound was used to examine the same eye to assess the value of 20 MHz B-scan ultrasound diagnosis.The macular diseases were typed according to the OCT manifestation.The characteristics of 20 MHz ultrasound for the same maculopathy were analyzed.All the patients subscribe the informed consent before the study.ResultsIn 10 eyes with macular hole determined by spectral-domain OCT,shallow dome-shaped lesion with central hollow was exhibited on the ultrasound image.The macular edema,retinal neuroepithelium layer detachment and retinoschisis were found in 16 eyes,6 eyes and 4 eyes by spectral-domain OCT,but the 20 M Hz B-Scan ultrasound presented with the focal belt-shaped elevation with non-echo zone below.In contrast,in 15 eyes of retinal pigment epithelium detachment in OCT,spectral-domain OCT showed stronger and thicker belt-shaped echo.Conclusions20 MHz B-scan ultrasound has certain value in the diagnosis and differentiation for several common maculopathies.Although it is not accurate as OCT in recognizing diseases,it could play an important role in refractive media opacity eye.