Clinical significance of reopening anterior chamber angle for severe acute angle -closure glaucoma / 国际眼科杂志(Guoji Yanke Zazhi)

·AIM: To explore whether the drainage angle could be reopened by surgery in patients with severe acute angle-closure glaucoma at " the greatest degree " of angle closure, and to study the treatment methods, such as double-paracentesis, phacoemulsification combined with goniosychialysis, and the effectiveness. ·METHODS: Retrospective observational case series. From November 2008, to November 2015, there were 33 patients with severe acute angle-closure glaucoma and 360° angle closure. Drug treatment showed no effect on them, so initial double-paracentesis ( anterior chamber paracentesis combined with vitreous paracentesis ) was applied. Then, either phacoemulsification combined with goniosychialysis or trabeculectomy surgery was performed after 7-14d, which was chosen based on the result of gonioscope during the surgery. The intraocular pressure, angle changes, and complications were observed. The follow-up period was 6mo to 3a. ·RESULTS: Of 33 participants enrolled, 32 had normal intraocular pressure after " double-paracentesis" ( 2 had normal intraocular pressure after laser peripheral iridotomy ). The mean intraocular pressure was significantly reduced from 53. 4 ± 10. 7mmHg to 16. 9 ± 13. 2mmHg ( t= 9. 21, P<0. 001 ) by applying " double-paracentesis", and 1 still had higher intraocular pressure. The mean intraocular pressure ( 16. 7 ± 4. 8mmHg ) was 0. 2mmHg lower after phacoemulsification than after" double- paracentesis " while there was no significant difference (t=0. 38,P>0. 05). One patient had abnormal intraocular pressure until 30d after phacoemulsification. Every participant had 360° angle closed before " double-paracentesis", 32 patients had opened angle ( mean 131. 8°± 111. 3°) after " double-paracentesis " and mean (228. 6°± 108. 3°) during phacoemulsification, and mean (234. 6°± 107. 2°) at 3mo after phacoemulsification. There was a significant difference between the post -paracentesis and intraoperative values ( t = 4. 52, P <0. 001 ). There was no difference between the intraoperative and postoperative values ( t = 0. 46, P>0. 05). No patients had serious adverse events. · CONCLUSION: For the " maximum degree " angle closure of severe acute angle-closure glaucoma, "double-paracentesis" combined with phacoemulsification can be chosen to open the angle gradually, and reduce intraocular pressure in vast majority of patients.

Shenfu Yangrong Decoction Combined with Routine Western Therapy for Treatment of Viral Myocarditis of Ⅰ– Ⅲ Grades in Children / 中国中医药信息杂志

Objective To observe the efficacy of Shenfu Yangrong Decoction combined with routine Western therapy in the treatment of viral myocarditis (VMC) of Ⅰ– Ⅲ grades in children. Methods Totally 100 cases of VMC children were selected and divided into observation group and control group according to random number table method, with 50 cases in both groups. The control group was treated with routine Western therapy, while the observation group was combined with Shenfu Yangrong Decoction based on the control group, one dosage a day, twice a day, orally. The TCM syndrome scores, the serum levels of interleukin (IL)-17, IL-27 and nuclear factor - κB (NF-κB), the levevs of troponin Ⅰ (cTnⅠ) and cardiac free fatty acid binding protein (H-FABP) before and after treatment in both groups were compared, and the total incidence of adverse reactions during treatment was monitored. Results The total effective rate was 90% (45/50) in the observation group and 74% (37/50) in the control group. The observation group was significantly higher than the control group (χ2=4.336, P=0.037). Compared with before treatment, the scores of the main symptoms, the scores of the secondary symptoms, and the total scores of the two groups decreased significantly after treatment (P＜0.01). Comparing the two groups after treatment, the scores of main symptoms, scores of secondary symptoms and total scores of the observation group were significantly lower than those of the control group (P＜0.05, P＜0.01). Compared with before treatment, serum IL-17, NF-κB, cTnⅠ, H-FABP levels were significantly reduced (P＜0.01), while serum IL-27 levels were significantly increased (P＜0.01) in both group. After treatment, the levels of serum IL-17, NF-κB, cTnⅠ, and H-FABP in the observation group were significantly lower than those in the control group (P＜0.01), and serum IL-27 level in the observation group was significantly higher than in the control group (P＜0.01). The adverse reaction rate was 12% (6/50) in the observation group and 8% (4/50) in the control group, without statistical significance between the two groups (χ2=0.368, P=0.544). Conclusion Shenfu Yangrong Decoction combined with routine Western therapy for the treatment of viral myocarditis children of Ⅰ– Ⅲ grades can effectively reduce the symptoms of patients, inhibit inflammation, reduce myocardial injury, with high safety.

Expression of neuraminidase gene of influenza virus H1N1 in baculovirus-expression system / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To construct the recombinant baculovirus with NA gene of Influenza H1N1 virus.</p><p><b>METHODS</b>Full-length NA gene of Influenza virus H1N1 (A/PR/8/34) was amplified by PCR and inserted into pFastBacdual vector to construct the recombinant baculovirus transfer vector pFBD-NA. Recombinant shuttle vectors rBacmid-NA was then obtained after transforming DH10B competent cells containing bacmid plasmids. After transfecting into sf9 cells, recombinant baculovirus rBac-NA was obtained. The rBac-NA genome was extracted and identified by PCR. NA protein expressed by recombinant baculovirus-infected sf9 cells was determined by IFA, Western Bolt and ELISA.</p><p><b>RESULTS</b>PCR results proved that recombinant shuttle vectors rBacmid-NA was successfully constructed. NA protein was detected by IFA and showed strong specific green fluorescence on the surface of infected cells. NA protein was recognized by two polyclonal antibodies specific for NA in Western Blot. ELISA showed specific reaction of recombinant NA protein with mouse polyclonal antibody against influenza virus (PR8), indicating high antigenicity.</p><p><b>CONCLUSION</b>Recombinant baculovirus rBac-NA that expresse NA protein of influenza virus was successfully constructed. This work provides a basis for further study on NA protein function and novel influenza vaccine development.</p>

Catestatin could ameliorate proliferating changes of target organs in spontaneously hypertensive rats / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Catestatin, a chromogranin A-derived peptide, is a potent antagonist of nicotine-evoked catecholamine release. We know that catecholamine plays an important role in cardiovascular remodeling induced by hypertension, therefore we hypothesized that catestatin would affect target-organ structure during hypertension.</p><p><b>METHODS</b>Twelve spontaneously hypertensive rats (SHRs) were randomized to SHR control group and catestatin group, the normal control group was comprised of six healthy Wistar-Kyoto rats of the same age. Tail-cuff blood pressure and pulse rate were obtained at weeks 1, 4 and 8. At the end of the eight-week period, the heart, abdominal aorta and left kidney were excised and weighed, VG staining was done and the intima-media thickness of vessels and the collagen volume fraction were assessed by an image acquisition and analysis system. The proliferating cell nuclear antigen (PCNA) was observed by immunohistochemistry, and real time reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the mRNA levels of proliferative genes including cyclin A, ki67 and PCNA in the abdominal aorta.</p><p><b>RESULTS</b>All the parameters in SHR observed in the present study increased significantly compared to Wistar Kyoto rats (P < 0.01). With intervention with catestatin, the systolic blood pressure decreased slightly but it was not significantly different from the SHR control, the cardiac mass index and left ventricular mass index both decreased significant ly, the collagen volume fraction decreased by nearly 30% in the heart, by 25% in vessels and by 10% in the kidney, and the intima-media thickness and expression of proliferative genes, including cyclin A, ki67 and PCNA, in the abdominal aorta also decreased significant ly.</p><p><b>CONCLUSIONS</b>The present study indicated that catestatin could ameliorate proliferating changes of heart, kidney and vessels during hypertension, especially to the deposition of interstitial collagen. Blood pressure was not the main factor to mediate this effect, which suggested that catestatin could become a novel protective factor for hypertensive target organs.</p>

Establishment of mammalian cell lines for constitutive expression of influenza virus matrix protein 2 / 病毒学报

To establish a mammalian cell line for stable expression of the matrix protein 2 (M2) of influenza virus type A. M2 gene was amplified by PCR from the influenza virus strain A/PR/8/34. The PCR product was cloned into eukaryotic expression vector pcDNA5/FRT. After identification with restriction enzyme digestion, the plasmid was co-transfected with plasmid pOG44 which expressed Flp in Flp-In-CHO cells. The target gene was integrated into chromosome of CHO cells by homologous recombination in vivo. Recombinant CHO-M2 cell lines were selected for hygromycin B resistance. A total of 15 recombinant cell strains with high expression of M2 protein were screened by hygromycin, and the expression of M2 protein was determined by IFA and Western blot. After subculturing for 10 passages, the presence of M2 gene in the CHO-M2 cells was confirmed by PCR, and the expression of M2 protein were proved by IFA and Western blot. We successfully constructed a mammalian cell line which stably expressed M2 protein of influenza virus type A. The cell line will be useful for studies on function of M2 protein and provide tools for novel influenza virus vaccine development.

Effect of aliskiren on arterial stiffness, compared with ramipril in patients with mild to moderate essential hypertension / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Aliskiren is a novel blood pressure-lowering agent acting as an oral direct renin inhibitor. The aim of this study was to assess the effect of aliskiren on arterial stiffness, compared with that of ramipril in mild to moderate essential hypertensive patients.</p><p><b>METHODS</b>Following a two week placebo run-in period, patients with a mean sitting diastolic blood pressure (ms-DBP) ≥ 95 and < 110 mmHg (1 mmHg = 0.133 kPa), and a mean sitting systolic blood pressure (ms-SBP) < 180 mmHg were randomly allocated to treatment with aliskiren (150 mg/d, n = 20) or ramipril (5 mg/d, n = 20) for eight weeks. Blood pressure, plasma renin activity, and the brachial-ankle pulse wave velocity (ba-PWV) were measured before and after eight weeks of treatment.</p><p><b>RESULTS</b>Eight weeks of treatment significantly decreased systolic blood pressure and diastolic blood pressure in both the aliskiren group and ramipril group. The hypotensive effect did not differ between the two groups. Plasma renin activity decreased after aliskiren treatment and increased after ramipril treatment. There was no significant difference in baseline ba-PWV between the aliskiren and ramipril groups (P = 0.892). The ba-PWV was significantly reduced in both the aliskiren group (1535 (1405 - 1666) vs. 1464 (1360 - 1506) cm/s) (P < 0.01) and the ramipril group (1544 (1433 - 1673) vs. 1447 (1327 - 1549) cm/s) (P < 0.01). No statistically significant difference was found in the decline of ba-PWV between the two groups (P = 0.766).</p><p><b>CONCLUSIONS</b>The current study revealed that aliskiren (150 mg/d) could ameliorate arterial stiffness and its effect was similar to ramipril (5 mg/d) in mild to moderate hypertensive patients, indicating that in addition to lowering blood pressure, aliskiren had beneficial effect on vascular protection.</p>

Construction of recombinant baculovirus co-expressing M1 and HA of influenza A virus / 病毒学报

The M1 and HA genes of H1N1 influenza virus were amplified and then cloned into the pFastBac dual donor plasmid. The recombinant pFastBac Dual-M1-HA was identified by restriction enzyme digestion. After the pFastBacdual-M1-HA was transformed into the baculovirus shuttle plasmid (bacmid) in DH10Bac competent cells, the colonies were identified by antibiotics and blue-white selection. The rBac-mid-M1-HA was verified by PCR and transfected into S f9 cells to produce recombinant baculovirus (rBac-M1-HA). Gene insertion of rBac-M1-HA was verified and the expression of M1 and HA genes was analyzed by IFA and Western-blot, demonstrating M1 and HA were co-expressed successfully. This study provides the foundation for researching the formation mechanism of influenza VLP and developing new influenza vaccines.

Subcloning of M1 gene fragment of H5N1 influenza virus and its expression in Escherichia coli / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To generate the Escherichia col vector expressing human H5N1 influenza virus M1 protein. To provide useful tools for detection of human H5N1 influenza virus and study on biological function of M1 protein.</p><p><b>METHODS</b>M1 gene fragment was amplified by PCR using the influenza virus gene segment 7 as template, and was subcloned into pQE80-L vector. The recombinant plasmid pQE80-L/M1 was transformed into Escherichia coil BL21 (DE3) strain. The expression of M1 was induced by isopropy-beta3-D-thiogalactopyranoside. We purified the recombinant M1 protein with polyhistidine tag with Ni2+ affinity chromatography. Mouse were immunized with the purified M1 protein for preparing antibodies against M1.</p><p><b>RESULTS</b>The recombinant Ml protein was recognized by antiserum against H5N1 subtype influenza virus, elicit specific antibody in immunized animals.</p><p><b>CONCLUSION</b>These confirmed that we successfully constructed the Escherichia coli vector expressing human H5N1 influenza virus M1 protein.</p>

Construction of vectors expressing M2 and NA genes of H5N1 influenza virus / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To construct vectors expressing M2 and NA genes of H5N1 influenza virus.</p><p><b>METHODS</b>Based on the human H5N1 avian influenza virus (A/Anhui/1/2005) isolated in china, M2 and NA genes were amplified by PCR. M2 or NA gene was subcloned into pStar vector to construct recombinant pStar-M2/, pStar-/M2, pStar-NA/and pStar-NA/. Furthermore, both of the M2 and NA genes were subcloned into pStar to construct two genes co-expressing recombinant pStar-M2/NA and pStar-NA/M2. Expression of the genes were detected by IFA after transfection of 293 cells with the recombinant plasmids.</p><p><b>RESULTS</b>Recombinant plasmids were constructed and identified by restriction endonuclease digestion. Expression of the genes cloned into the recombinant plasmids was confirmed by IFA.</p><p><b>CONCLUSION</b>Recombinant plasmids expressing M2 and/or NA genes of H5N1 influenza virus were constructed, which provided basis for development of influenza DNA vaccine.</p>

Construction, expression and immunogenicity analysis of a fusion protein containing M2e of influenza A virus fused to a modified Pseudomonas aeruginosa exotoxin A / 病毒学报

M2 protein of type A influenza virus is a good candidate for universal influenza vaccine, exotoxin A of Pseudomonas aeruginosa may facilitate the immunogenicity of M2 protein. We constructed and expressed a prokaryotic expression plasmid containing a chimeric gene of M2 extracellular coding region and a partial PEA gene, and observed the immunoprotection in BALB/c mice vaccinated with the fusion protein. The fusion protein (ntPE-M2e) was generated by inserting the coding sequence of the M2e in place of Ib loop in PEA. This fusion protein was used to immunize BALB/c mice by subcutaneously injection with incomplete Freund's adjuvant and boost at weeks 3 and 7. The immunized mice were challenged with influenza virus strain A/PR/34/8. The fusion protein (ntPE-M2e) immunization protected mice against lethal viral challenge. ELISA and ELISPOT results demonstrated that the fusion protein could induce a strong systemic immune response against synthetic M2e peptide, and virus replication in the lungs of mice was inhibited in comparison with the control. This study provides foundation for developing broad-spectrum vaccines against type A influenza viruses.

Epidemiologic and clinical characteristics of 59 persons suspecting of being infected by the HIV virus despite having repeated negative laboratory findings / 中华流行病学杂志

Objective To describe the epidemiologic characteristics and clinical manifestations of 59 persons recruited via an intemet chat group who complained of AIDS-like symptoms, so as to formulate effective intervention strategies and measures. Methods Case was defined as onset of any three of the following self-reported AIDS-like symptoms in a member of relevant "intemet chat groups": persistent low grade fever, rash, swollen lymph node, fatigue, diarrhea, weight loss and low CD4+T count. We administered an internet-based questionnaire, and invited 59 of the 88 casepersons for voluntary physical examination and laboratory testing. Results The 59 case-persons came from 22 provinces; 54 (91.5 %)were men; the median age was 34 (range: 22-53)years; 84.7% of them had high-risk sexual behaviors before the onset of self-reported symptoms. The median time interval from exposure to onset was 15 d (range: 1-365 d). Blood specimens for all the 59 case-persons were tested negative for HIV and syphilis antibodies. There was also no evidence of Xenotropic murine leukemia virus-related virus infection. One case-person was tested positive for hepatitis C virus antibody. The average CD4'T lymphocyte count was 707/μl. Of the 59 case-persons,57 (96.6%) sought medical care from multiple providers; 40 were diagnosed to have no physical disorders. Conclusion None of the 59 case-persons had any evidence of infection with HIV or any other infectious agents that could explain their self-reported symptoms.

Study on preparation and identification of monoclonal antibodies immunized with H5N1 influenza virus M1 protein / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To prepare monoclonal antibodies specific for M1 protein of H5N1 subtype human influenza virus, this work may provide new tool in rapid diagnosis and study of type A influenza virus.</p><p><b>METHODS</b>BALB/c mice were immunized with purified recombinant H5N1 (A/Anhui/1/2005)/M1 protein expressed in E. coli. Spleen cells of the immunized mice were fused with sp2/0 cells to produce hybridoma cell lines. ELISA was performed to identify the monoclonal antibody against M1 protein of H5N1. Immunofluorescence assay (IFA) were applied to identify the specificity of these antibodies.</p><p><b>RESULTS</b>Three hybridoma cell lines steadily secreting anti-H5N1/M1 McAb were obtained, and their cross reactivity was confirmed by cross-reaction test and IFA.</p><p><b>CONCLUSION</b>Monoclonal antibodies immunized with H5N1 subtype influenza virus M1 protein are cross-reactive, which can be used to detect different subtype of influenze virus type A.</p>

Construction of recombinant adenovirus co-expressing M1 and HA genes of influenza virus type A / 病毒学报

Based on the human H5N1 influenza virus strain A/Anhui/1/2005, recombinant adenovirus co-expressing M1 and HA genes of H5N1 influenza virus was constructed using an internal ribosome entry site (IRES) sequence to link the two genes. The M1 and HA genes of H5N1 influenza virus were amplified by PCR and subcloned into pStar vector separately. Then the M1-IRES-HA fragment was amplified and subcloned into pShuttle-CMV vector, the shuttle plasmid was then linearized and transformed into BJ5183 bacteria which contained backbone vector pAd-Easy. The recombinant vector pAd-Easy was packaged in 293 cells to get recombinant adenovirus Ad-M1/HA. CPE was observed after 293 cells were transfected by Ad-M1/HA. The co-expression of M1 and HA genes was confirmed by Western-blot and IFA (immunofluorescence assay). The IRES containing recombinant adenovirus allowed functional co-expression of M1 and HA genes and provided the foundation for developing new influenza vaccines with adenoviral vector.

Construction and immunogenicity study of DNA vaccine expressing human H5N1 influenza virus hemagglutinin / 病毒学报

Based on the first isolated human H5N1 influenza virus strain A/Anhui/1/2005 in China, HA and HA1 genes were amplified and cloned into the eukaryotic expression vector pStar. The recombinant plasmids pStar-HA and pStar-HA1 were transfected into COS7 cells. Western blot and IFA showed that the two recombinant DNA plasmids were successfully expressed in eukaryotic cells. BALB/c mice were immunized with the plasmids DNA by intramuscular injection. Anti-HA specific antibody in peripheral blood of immunized mice was tested by ELISA. The results showed that the recombinant plasmids successfully induced the anti-HA humoral immune response, and there was no significant difference between HA and HA1 as immunogen. This work provides basis for future development of novel avian flu vaccine.

Molecular cloning and expression of extremely thermostable and acid-stable amylase gene in Pichia pastoris / 生物工程学报

The gene encoding a extremely thermostable and acid-stable alpha-Amylase was amplified by PCR using hyperthermophilic archaebacterium pyrococcus furiosus genomic DNA as template. Then the gene was cloned into the vector of pPIC9K. The recombinant vector pPIC9K-amy was then transformed into E. coli DH5alpha strain. Sequencing test showed that the a-amylase gene cloned consisted of 1305 base pairs and the mature protein encoded by the gene consisted of 435 amino acids. The recombinant vector was transformed into chromosome of methylotrophic yeast Pichia pastoris GS115 strain. Regulated by the alpha-Factor, promoter of AOX1 gene and termination signal of yeast genomic, the recombinant a-Amylase was expressed and excreted out of the cells. The expression of the recombinant alpha-amylase was strictly induced by methanol. As induction time increased, the activity of amylase per milliliter medium went up accordingly. After 7 days induction, the activity of the amylase reached the max. The recombinant alpha-amylase exhibited maximal activity at 90 to approximately 100 degrees C and at pHranging from 4.5 to 5.0. The enzyme is so thermostable that after disposed at 100 degrees C for 5 hours over 60% of activity was retained.