Mechanism of programmed cell death in follicular atresia / 生理学报

Apoptosis and autophagy of follicular granulosa cells play an important regulatory role in the process of ovarian follicular atresia in animals. Recent studies have shown that ferroptosis and pyroptosis are also involved in the process of ovarian follicular atresia. Ferroptosis is a form of cell death caused by iron-dependent lipid peroxidation and reactive oxygen species (ROS) accumulation. Studies have confirmed that autophagy- and apoptosis-mediated follicular atresia also have typical characteristics of ferroptosis. Pyroptosis is a pro-inflammatory cell death dependent on Gasdermin protein, which can regulate ovarian reproductive performance by regulating follicular granulosa cells. This article reviews the roles and mechanisms of several types of programmed cell death independently or interactively regulating follicular atresia, in order to expand the theoretical research on follicular atresia mechanism and provide the theoretical reference for the mechanism of programmed cell death-induced follicular atresia.

Safety and efficacy of the early administration of levosimendan in patients with acute non-ST-segment elevation myocardial infarction and elevated NT-proBNP levels: An Early Management Strategy of Acute Heart Failure (EMS-AHF) / 中华内科杂志

Objectives: To investigated the safety and efficacy of treating patients with acute non-ST-segment elevation myocardial infarction (NSTEMI) and elevated levels of N-terminal pro-hormone B-type natriuretic peptide (NT-proBNP) with levosimendan within 24 hours of first medical contact (FMC). Methods: This multicenter, open-label, block-randomized controlled trial (NCT03189901) investigated the safety and efficacy of levosimendan as an early management strategy of acute heart failure (EMS-AHF) for patients with NSTEMI and high NT-proBNP levels. This study included 255 patients with NSTEMI and elevated NT-proBNP levels, including 142 males and 113 females with a median age of 65 (58-70) years, and were admitted in the emergency or outpatient departments at 14 medical centers in China between October 2017 and October 2021. The patients were randomly divided into a levosimendan group (n=129) and a control group (n=126). The primary outcome measure was NT-proBNP levels on day 3 of treatment and changes in the NT-proBNP levels from baseline on day 5 after randomization. The secondary outcome measures included the proportion of patients with more than 30% reduction in NT-proBNP levels from baseline, major adverse cardiovascular events (MACE) during hospitalization and at 6 months after hospitalization, safety during the treatment, and health economics indices. The measurement data parameters between groups were compared using the t-test or the non-parametric test. The count data parameters were compared between groups using the χ² test. Results: On day 3, the NT-proBNP levels in the levosimendan group were lower than the control group but were statistically insignificant [866 (455, 1 960) vs. 1 118 (459, 2 417) ng/L, Z=-1.25,P=0.21]. However, on day 5, changes in the NT-proBNP levels from baseline in the levosimendan group were significantly higher than the control group [67.6% (33.8%,82.5%)vs.54.8% (7.3%,77.9%), Z=-2.14, P=0.03]. There were no significant differences in the proportion of patients with more than 30% reduction in the NT-proBNP levels on day 5 between the levosimendan and the control groups [77.5% (100/129) vs. 69.0% (87/126), χ²=2.34, P=0.13]. Furthermore, incidences of MACE did not show any significant differences between the two groups during hospitalization [4.7% (6/129) vs. 7.1% (9/126), χ²=0.72, P=0.40] and at 6 months [14.7% (19/129) vs. 12.7% (16/126), χ²=0.22, P=0.64]. Four cardiac deaths were reported in the control group during hospitalization [0 (0/129) vs. 3.2% (4/126), P=0.06]. However, 6-month survival rates were comparable between the two groups (log-rank test, P=0.18). Moreover, adverse events or serious adverse events such as shock, ventricular fibrillation, and ventricular tachycardia were not reported in both the groups during levosimendan treatment (days 0-1). The total cost of hospitalization [34 591.00(15 527.46,59 324.80) vs. 37 144.65(16 066.90,63 919.00)yuan, Z=-0.26, P=0.80] and the total length of hospitalization [9 (8, 12) vs. 10 (7, 13) days, Z=0.72, P=0.72] were lower for patients in the levosimendan group compared to those in the control group, but did not show statistically significant differences. Conclusions: Early administration of levosimendan reduced NT-proBNP levels in NSTEMI patients with elevated NT-proBNP and did not increase the total cost and length of hospitalization, but did not significantly improve MACE during hospitalization or at 6 months.

Changes of aqueous cytomegalovirus DNA in patients with cytomegalovirus retinitis after allogeneic bone marrow hematopoietic stem cell transplantation / 中华眼底病杂志

Objective:To observe aqueous cytomegalovirus (CMV) DNA load in patients with cytomegalovirus retinitis (CMVR) after allogeneic hematopoietic stem cell transplantation (Allo-HSCT), and to explore influencing factors for transient elevation of CMV-DNA load during the treatment.Methods:A retrospective study. From January 2016 to July 2020, 28 eyes of 19 patients with CMVR after Allo-HSCT diagnosed in the Department of Ophthalmology of Peking University People's Hospital were included in the study. Among them, there were 8 males with 12 eyes, 11 females with 16 eyes; the mean age was 28 years; 10 patients were unilateral and 9 patients were bilateral. During the course of treatment and follow-up, the blood CMV-DNA remained negative. All patients were treated with intravitreal injection of 60 mg/ml ganciclovir 0.05 ml (containing ganciclovir 3 mg), twice a week for two weeks in induction phase and weekly injection in maintenance phase. Aqueous humor sample was collected during injection of ganciclovir (IVG) and CMV-DNA load was determined by real-time quantitative polymerase chain reaction. Intravitreal treatment was terminated if aqueous CMV-DNA load turned negative after the fourth or later intravitreal injection. The patients were followed up every 2 weeks for at least 6 months. Serum CMV-DNA was negative in all patients during treatment and follow-up. All the eyes were divided into continuous decline group and non-continuous decline group depending on whether there was transient elevation of aqueous CMV-DNA load, and data between two groups were compared. Pearson linear regression analysis was used to analyze the correlation between aqueous CMV-DNA load and injection times or treatment duration.Results:At the end of treatment, the median number of IVG in the affected eye was 7 (4, 9). The results of correlation analysis showed that the aqueous humor CMV-DNA load of the affected eye was related to the number of treatments [ R2=0.385, P<0.000 1, B=-0.237 log 10 copies/(ml·time)], and the duration of treatment [ R2=0.394, P <0.000 1, B=-0.301 log 10 copies/(ml·week)] were negatively correlated. Among the 28 eyes, 13 eyes (46.4%, 13/28) in the continuous decline group and 15 eyes (53.6%, 15/28) in the non-sustained decline group. Baseline visual acuity ( t=-1.223), intraocular pressure ( t=1.538), aqueous humor CMV-DNA load ( t=-0.109), retinitis lesion area ( Z=-0.308) in the continuous decline group and the non-continuous decline group), the number of quadrants involved ( Z=-0.024) and whether the macula was involved ( Z=-1.826), combined with anterior segment inflammation ( Z =-0.499), combined with high intraocular pressure ( Z=-1.342), terminal visual acuity ( t =-0.845), intraocular pressure ( t=-0.068), total IVG times ( Z=0.907), age ( Z=-0.832), gender composition ( Z=-1.074), etc. The difference was not statistically significant ( P>0.05). Conclusion:The CMV-DNA load in aqueous humor decreases by about 50% every week during the treatment of CMVR eyes after Allo-HSCT; the transient increase in the CMV-DNA load in the aqueous humor during treatment does not affect the treatment process and clinical prognosis.

Relationship between Expression of Runt-related Transcription Factor 3 and Enhancer of zeste Homolog 2 Proteins and Sensitivity to Neoadjuvant Chemotherapy in Locally Advanced Rectal Cancer / 中国医学科学院学报

Objective To investigate the expression and correlation of Runt-related transcription factor 3(RUNX3)and enhancer of zeste homolog 2(EZH2)in rectal cancer,and to reveal the relationship between the expression of RUNX3 and EZH2 and the sensitivity of XELOX regimen to neoadjuvant chemotherapy in locally advanced rectal cancer patients. Methods The carcinoma and paracancerous tissues of 31 patients with rectal adenocarcinoma and no preoperative antitumor therapy were selected as cancer group and paracancer group,respectively.The relative mRNA levels of RUNX3 and EZH2 in the two groups were measured by real-time quantitative reverse transcription-polymerase chain reaction,and the protein levels were determined by immunohistochemical assay.The expression of RUNX3 and EZH2 was compared between cancer tissue and paracancerous tissue.The pre-treatment wax blocks of 26 patients with locally advanced rectal cancer who received 3 cycles of XELOX regimen as neoadjuvant chemotherapy before surgery were selected as the pre-neoadjuvant therapy group,and the postoperative pathological wax blocks were selected as the post-neoadjuvant treatment group.Tumor regression grade(TRG)was determined to evaluate the efficacy of neoadjuvant therapy.Immunohistochemical assay was used to detect the protein levels of RUNX3 and EZH2 in the two groups,and then the relationship between the expression patterns of the two proteins and the efficacy of neoadjuvant chemotherapy was analyzed. Results Compared with paracancerous tissue,the cancer tissue showed down-regulated mRNA level and reduced positive protein expression rate of RUNX3,while up-regulated mRNA level(

COL6A3 promotes cellular malignancy of osteosarcoma by activating the PI3K/AKT pathway

SUMMARY OBJECTIVE In this study, we aimed to investigate the role of COL6A3 on cell motility and the PI3K/AKT signaling pathway in osteosarcoma. METHODS The relative expression of COL6A3 was achieved from a GEO dataset in osteosarcoma tissue. siRNA technology was applied to decrease the COL6A3 expression in cells, and cell counting kit-8 (CCK-8) assay and colony formation analysis were used to examine the cell proliferation potential. Knockdown COL6A3 made the proliferation and colony formation abilities worse than the COL6A3 without interference. Likewise, in contrast to the si-con group, cell invasion and migration were inhibited in the si-COL6A3 group. Moreover, the western blot results suggested that the PI3K/AKT signaling pathway was manipulated by measuring the protein expression of the PI3K/AKT pathway-related markers, due to the COL6A3 inhibition. CONCLUSION COL6A3 plays a crucial role in modulating various aspects of the progression of osteosarcoma, which would provide a potentially effective treatment for osteosarcoma.

RESUMO OBJETIVO Neste estudo, investigamos a função do COL6A3 na mobilidade celular e na via PI3K/AKT em osteossarcomas. METODOLOGIA A expressão relativa do COL6A3 foi obtida a partir de dados GEO em tecidos de osteossarcoma. O RNA de interferência (siRNA) foi utilizado para reduzir a expressão do COL6A3 nas células, e o teste de contagem de células kit-8 (CCK-8) e a análise de formação de colônias foram realizados para examinar o potencial de proliferação celular. Além disso, o Transwell comprovou os efeitos do si-COL6A3 na invasão celular e migração em células de osteossarcoma. Para medir os níveis de expressão das proteínas e mRNAs, utilizamos transcriptase reversa quantitativa (qRT-PCR) e western blot. RESULTADOS O COL6A3 foi regulado nos tecidos e células do osteossarcoma quando comparado com o controle normal. A redução de COL6A3 reduziu a proliferação e a capacidades de formação de colônias em relação ao COL6A3 sem interferência. Do Mesmo modo, ao contrário do observado no grupo si-con, a invasão e migração celular foram inibidas no grupo si-COL6A3. Além disso, o resultado do western blot sugere que a via PI3K/AKT foi manipulada, medindo a expressão proteica dos marcadores relacionados à PI3K/AKT, devido à inibição do COL6A3. CONCLUSÃO O COL6A3 desempenha um papel crucial na modulação de vários aspectos da progressão do osteossarcoma, o que pode representar um possível tratamento eficaz para a doença.

Elimination of a disulfide bond in the light chain of coagulation factor VIII improves secretion of a BDD-FVIII variant with an engineered inter-chain disulfide / 药学学报

The coagulation VIII factor (FVIII) contains eight pairs of disulfide bonds, which are involved in maintaining its structure and function. It has been demonstrated that the disulfide bond between Cys1899/Cys1903 of the A3 domain in the light chain impedes secretion. In our previous work, an engineered inter-chain disulfide in the B domain-deleted FVIII (BDD-FVIII) promoted heterodimer assembly and secretion of separately expressed heavy and light chains. In this study, we constructed two BDD-FVIII variants, one of which contains an engineered inter-chain disulfide bond (F8C) between Met662 > Cys and Asp1828 > Cys mutations and another contains an endogenous A3 domain with a disrupted disulfide bond from F8C (F8CG) by replacement of Cys1899 and Cys1903 with Gly in F8C. We explored their function and secretion. By transducing F8C and F8CG into HEK293 and COS-7 cells, the formation of disulfide bonds and the secretion and coagulation activity of the two variants in the culture media and their binding affinity for von Willebrand factor (vWF) could be observed. The results show that variants F8C and F8CG are mainly the disulfide bonded heavy and light chain dimer, while the wild type BDD-FVIII (F8) is dominated by the easily dissociated heavy and light chain dimer. The secretion and activity of F8C was significantly higher than that of F8, while the secretion and activity of F8CG was significantly higher than that of F8C. The vWF binding of the two variants is similar to F8. This indicates that the BDD-FVIII variant F8CG may be attractive molecule for protein replacement and as a transgene in gene-therapy strategies. These findings are encouraging for future studies targeting disulfide bond elimination for further enhancement of FVIII secretion.

Morphological parameters of corneal endothelium in patients with rhegmatogenous retinal detachment complicated with choroidal detachment / 国际眼科杂志(Guoji Yanke Zazhi)

@#AIM: To explore the changes of morphological parameters of corneal endothelial cell in patients with choroidal detachment following rhegmatogenous retinal detachment(RDD-CHD). <p>METHODS: Seventy patients(70 eyes)with RDD-CHD were collected consecutively. In patients with RDD-CHD, thirty-eight cases(38 eyes)not with high myopia were enrolled in group A; 32 cases(32 eyes)associated with high myopia were enrolled in group B. Thirty-six normal controls(36 eyes)were enrolled in group C. We measured the related morphological parameters of corneal endothelial cells in all subjects using corneal specular microscope. The parameters of corneal endothelial cells included minimum size of cell area(S_min), maximum size of cell area(S_max), average size of cell area, standard deviation of cell area(SD), coefficient of variability cell area(CV), cell density of corneal endothelial cells(CD)and hexagonality(HG). <p>RESULTS: There were statistically differences in the CD(<i>P</i><0.001)and hexagonality(<i>P</i><0.001)between the patients with RDD-CHD and normal subjects. There were statistically differences in the CD between groups A and B(<i>P</i><0.05), between groups A and C(<i>P</i><0.05), between groups B and C(<i>P</i><0.001). SD correlated with axis length(<i>r</i>_s=-0.426, <i>P</i><0.01); CV correlated with axis length(<i>r</i>_s=0.494, <i>P</i><0.01), CD correlated with intraocular pressure(<i>r</i>_s=-0.025, <i>P</i><0.05), CD correlated with axis length(<i>r</i>_s=0.368, <i>P</i><0.05), HG correlated with course(<i>r</i>_s=0.552, <i>P</i><0.05). In patients with RDD-CHD, SD correlated with axis length(<i>r</i>_s=0.236, <i>P</i><0.05); CV correlated with axis length(<i>r</i>_s=0.159, <i>P</i><0.05), HG correlated with course(<i>r</i>_s=0.142, <i>P</i><0.05), S_max correlated with intraocular pressure(<i>r</i>_s=-0.314, <i>P</i><0.01).<p>CONCLUSION: The valus of HG and CD of corneal endothelial cells in patients with RDD-CHD were both lower than that of the normal subjects. Axis length, course and intraocular pressure might influence the morphological parameters of corneal endothelium in RDD-CHD patients.

Characteristics, Prognostic Factors, and Survival of Patients with NK/T-Cell Lymphoma of Non-upper Aerodigestive Tract: A 17-Year Single-Center Experience / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: The extranodal natural killer (NK)/T-cell lymphoma (NKTCL) of non-upper aerodigestive tract (NUAT) was found to have clinical heterogeneity compared with NKTCL of the upper aerodigestive tract (UAT) in small scale studies. We conducted this study in a much larger cohort to analyze the clinical characteristics, prognostic factors, treatment modality, and clinical outcomes of patients with NUAT-NKTCL. MATERIALS AND METHODS: From January 2001 to December 2017, a total of 757 NKTCL patients were identified and included in this study, including 92 NUAT-NKTCL patients (12.2%) and 665 UAT-NKTCLpatients (87.8%). RESULTS: NUAT-NKTCL patients had relatively poorer performance status, more unfavorable prognostic factors, and more advanced stage, compared with UAT-NKTCL patients. The 5-year overall survival (OS) was 34.7% for NUAT-NKTCL, which was significantly worse than UAT-NKTCL (64.2%, p<0.001). The median OS duration was 30.9 months for NUAT-NKTCL. Multivariate analysis showed that presence with B symptoms and elevated serum lactate dehydrogenase independently predicted worse OS. International prognostic index score and prognostic index of natural killer lymphoma score still had prognostic values in NUAT-NKTCL, while the Ann Arbor system could not accurately predict the OS. CONCLUSION: NUAT-NKTCL is a distinctive subtype of NKTCL in many aspects. Patients with NUAT-NKTCL have relatively poorer performance status, more unfavorable prognostic factors, more advanced stage, and poorer prognosis.

Gene typing of Legionella pneumophila serogroup 1 in Sichuan Province, China / 中国人兽共患病学报

The aim of the research is to investigate the genetic characteristics of Legionella pneumophila serogroup1 (LP1)in Sichuan Province.The sequence-based typing (SBT) and multiple-locus VNTR analysis (MLVA) were used to describe the genetic polymorphism of 42 strains which were isolated from 1989-2016 in Sichuan Province,China.According to the reference,PCR was used to detect the 8-VNTR loci and 7 housekeeping genes respectively.The VNTR results were determined by using capillary electrophoresis,and the SBT results were sequenced and compared with the database of EWGLI.Results showed that totally 42 stains were divided into 8 MLVA types with the advantage types were M08 (47.6 ％) and M07 (23.8 ％).Twelve ST types were obtained with 3 main clonal complex and 2 singleton,including 2 novel ST types,among those,ST1 was the predominant type,accounting for 52.3 ％,following by ST630 (14.2 ％).In conclusion,our results demonstrated MLVA and SBT were both applied to the research for molecular epidemiological investigation of LP1 and showed the high genetic polymorphism and the regional specificity.The results also suggest that the isolates are a potential threat to the public,effective control and prevention strategies are urgently needed.

Gene typing of Legionella pneumophila serogroup 1 in Sichuan Province, China / 中国人兽共患病学报

The aim of the research is to investigate the genetic characteristics of Legionella pneumophila serogroup1 (LP1)in Sichuan Province.The sequence-based typing (SBT) and multiple-locus VNTR analysis (MLVA) were used to describe the genetic polymorphism of 42 strains which were isolated from 1989-2016 in Sichuan Province,China.According to the reference,PCR was used to detect the 8-VNTR loci and 7 housekeeping genes respectively.The VNTR results were determined by using capillary electrophoresis,and the SBT results were sequenced and compared with the database of EWGLI.Results showed that totally 42 stains were divided into 8 MLVA types with the advantage types were M08 (47.6 ％) and M07 (23.8 ％).Twelve ST types were obtained with 3 main clonal complex and 2 singleton,including 2 novel ST types,among those,ST1 was the predominant type,accounting for 52.3 ％,following by ST630 (14.2 ％).In conclusion,our results demonstrated MLVA and SBT were both applied to the research for molecular epidemiological investigation of LP1 and showed the high genetic polymorphism and the regional specificity.The results also suggest that the isolates are a potential threat to the public,effective control and prevention strategies are urgently needed.

Differences in the changes of blood clotting state after initial trauma fractures and twice trauma fractures / 中国骨伤

<p><b>OBJECTIVE</b>To compare changes of blood clotting state after initial trauma fractures and twice trauma fractures, investigate the effect of fracture history to the state of the blood clotting after secondary trauma fractures.</p><p><b>METHODS</b>Thirty New Zealand rabbits were selected, aged 5-6 months, males and females unlimited, weighted 2.4-2.6 kg, non-pregnant. Postoperative model of initial trauma fractures was established, and then postoperative model after twice trauma fractures was built. Prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT), fiber fibrinogen (FIB) and D-dimer (DD) were detected by venous blood at 1 day and 5 days after surgery. Changes of indicators were compared between after twice fractures at the same time.</p><p><b>RESULTS</b>Comparing with 1 day after the secondary trauma fracture and initial trauma fracture surgery, PT, APTT values showed no significant difference (P > 0.05), TT, FIB, DD values were increased, and the difference was statistically significant (P < 0.05); Comparing with 5 days after the secondary trauma fracture and initial trauma fracture surgery, PT values showed no significant difference (P < 0 .05), APTT, TT, FIB, DD values were increased, and the difference was statistically significant (P< 0.05).</p><p><b>CONCLUSION</b>Blood after the secondary trauma fractures is in hypercoagulable state after fracture surgery.</p>

Aqueous extracts of Ocimum grasstimum inhibits lipopolysaccharide-induced interleukin-6 and interleukin-8 expression in airway epithelial cell BEAS-2B / 中国结合医学杂志

<p><b>OBJECTIVE</b>To investigate the antiinflammatory activities of aqueous extract of Occimum gratissmium (OGE) with emphasis on expression of proinflammatory cytokines in Lipopolysaccharide (LPS)-stimulated epithelial cell BEAS-2B.</p><p><b>METHODS</b>Effects of OGE on cell viability were determined by MTT assay. mRNA expression were analyzed by and reverse transcription polymerase chain reaction (RT-PCR) and quantitative real-time PCR. Activation of kinase cascades was investigated by immunoblot. Intracellular reactive oxygen species (ROS) was analyzed by flow cytometry.</p><p><b>RESULTS</b>OGE (<200 μg/mL) treatment or pretreatment and following LPS exposure slightly affected viability of BEAS-2B cells. Increase of interleukin (IL)-6 and IL-8 and the elevated level of intracellular ROS in LPS-stimulated BEAS-2B cells were diminished by OGE pretreatment in a dose-dependent manner. OGE suppressed inflammatory response-associated mitogen-activated protein kinases (MAPKs) and Akt activation. Additionally, OGE pretreatment increased level of cellular inhibitor of κBα (IκBα) and inhibited nuclear translocation of nuclear factor kappa B (NF-κB).</p><p><b>CONCLUSION</b>These findings indicate that significant suppression of IL-6 and IL-8 expressions in LPS-stimulated BEAS-2B cells by OGE may be attributed to inhibiting activation of MAPKs and Akt and consequently suppressing nuclear translocation of NF-κB.</p>

Inhibitory Effect of Melanoma Differentiation Associated Gene-7/Interleukin-24 on Invasion In Vitro of Human Melanoma Cancer Cells

The acquisition of metastasis potential is a critical point for malignant tumors. Melanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24) is a potential tumor suppress gene and frequently down-regulated in malignant tumors. It has been implicated that overexpression of MDA-7 led to proliferation inhibition in many types of human tumor. Invasion is an important process which is potential to promote tumor metastasis. However, the role and potential molecular mechanism of mda-7/IL-24 to inhibit the invasion of human melanoma cancer is not fully clear. In this report, we identified a solid role for mda-7/IL-24 in invasion inhibition of human melanoma cancer LiBr cells, including decreasing of adhesion and invasion in vitro, blocking cell cycle, down-regulating the expression of ICAM-1, MMP-2/9, CDK1, the phosphorylation of ERK and Akt, NF-kappaB and AP-1 transcription activity. Meanwhile, there was an increased expression of PTEN in mda-7/IL-24 over-expression LiBr cells. Our results demonstrated that mda-7/IL-24 is a potential invasion suppress gene, which inhibits the invasion of LiBr cells by the down-regulation of ICAM-1, MMP-2/9, PTEN, and CDK1 expression. The molecular pathways involved were the MAPK/ERK, PI3K-Akt, NF-kappaB, and AP-1. These findings suggest that mda-7/IL-24 may be used as a possible therapeutic strategy for human melanoma cancer.

Myocardial injury and inflammatory response after interventional therapy in children with congenital heart disease / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To study myocardial injury and inflammatory response within 7 days after interventional therapy in children with congenital heart disease (CHD).</p><p><b>METHODS</b>A total of 77 children with CHD, including 12 cases of ventricular septal defect (VSD), 14 cases of atrial septal defect (ASD), 14 cases of pulmonary stenosis (PS) and 37 cases of patent ductus arteriosus (PDA), were enrolled. The levels of myocardial enzyme (AST, CK and CKMB), cardiac troponin I (cTnI) and CRP in serum were measured before operation, immediately after operation, and 6 hrs, 24 hrs, 72 hrs and 7 days after operation.</p><p><b>RESULTS</b>Serum AST levels in the VSD group were significantly higher than the other CHD groups immediately after operation, and 6 hrs and 24 hrs after operation (P<0.05). There were significant differences in serum CK and CKMB levels among the four CHD groups immediately and 6 hrs after operation (P<0.05), and the highest serum CK and CKMB levels were found in the VSD group. Serum CRP levels in the PDA group were significantly higher than the other CHD groups 72 hrs and 7 days after operation (P<0.05). Compared with before operation, serum AST levels increased significantly in all four CHD groups 6 and 24 hrs after operation groups (P<0.05). Serum CK and CKMB levels increased significantly in the VSD group immediately and 6 hrs after operation (P<0.05). Serum cTnI levels increased significantly in the VSD, PDA and PS groups immediately and 6 hrs after operation (P<0.05). The PDA group showed increased CRP levels 24 hrs, 72 hrs and 7 days after operation (P<0.05).</p><p><b>CONCLUSIONS</b>Minor myocardial injury can be noted within 7 days after interventional therapy in children with CHD and mainly occurs between immediately and 24 hrs after operation. The injury is more significant in VSD cases. The interventional therapy does not cause significant inflammation.</p>

Leucine zippers improves protein splicing-mediated coagulation factor VIII gene delivery by dual-vector system / 药学学报

In our recent study by exploring an intein-based dual-vector to deliver a B-domain-deleted FVIII (BDD-FVIII) gene, it showed that covalently ligated intact BDD-FVIII molecules with a specific coagulant activity could be produced from expressed heavy and light chains by protein trans-splicing. Here, we assessed the hypothesis that the efficiency of trans-splicing may be increased by adding to the intein sequences a pair of leucine zippers that are known to bring about specific and strong protein binding. The intein-fused heavy and light chain genes were co-transferred into cultured COS-7 cells using a dual-vector system. After transient expression, the intracellular BDD-FVIII splicing was observed and the spliced BDD-FVIII and bioactivity secreted to culture media were quantitatively analyzed. An enhanced splicing of BDD-FVIII with decreased protein precursors from gene co-transfected cells was observed by Western blotting. The amount of spliced BDD-FVIII and bioactivity secreted to the culture media were 106 +/- 12 ng x mL(-1) and 0.89 +/- 0.11 U x mL(-1) analyzed by ELISA and Coatest method respectively, which was greater than leucine zipper free intein-fused heavy and light chain genes co-transfected cells (72 +/- 10 ng x mL(-1) and 0.62 +/- 0.07 U x mL(-1)). The activity of cellular mechanism-independent protein splicing was also improved, as showed by the increasing of spliced BDD-FVIII and bioactivity in culture media from combined cells separately transfected with heavy and light chain genes which was 36 +/- 11 ng x mL(-1) and 0.28 +/- 0.09 U x mL(-1). It demonstrated that the leucine zippers could be used to increase the efficiency of protein trans-splicing to improve the efficacy of a dual-vector mediated BDD-FVIII gene delivery by strengthening the interaction between the two intein-pieces fused to heavy and light chains. It provided evidence for further study in animal model using a dual-adeno-associated virus vector to deliver FVIII gene in vivo.

Construction of recombinant lentiviral vectors containing Rheb gene and its mutant Rheb'D60K gene and their expression in human liver cancer cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To construct recombinant lentiviral vectors carrying Rheb gene and its mutant Rheb'D60K gene, and examine their expression in human liver cancer cells.</p><p><b>METHODS</b>Rheb gene was amplified by PCR to construct the recombinant plasmid LV31-Rheb-WT and LV31-Rheb-D60K. HEK-293 FT cells were contransfected with the recombinant lentiviral vector together with a lentiviral package plasmid to produce the lentiviral particles. The expression of PS6 protein was detected in the lentivirus-infected MCF-7 cells. The apoptosis of SK-HEP-1 cells transfected with LV31-Rheb-WT or LV31-Rheb-D60K was observed.</p><p><b>RESULTS</b>The recombinant LV31-Rheb-WT and LV31-Rheb-D60K vectors were confirmed by PCR and DNA sequencing. Western blotting showed that PS6 protein expression was increased in LV31-Rheb-WT-transfected cells while decreased in LV31-Rheb-D60K-transfected cells. LV31-Rheb-D60K-transfected SK-HEP-1 cells showed more obvious apoptosis after starvation than LV31-Rheb-WT-transfected cells.</p><p><b>CONCLUSION</b>Lentiviral vectors carrying Rheb gene and its mutant has been successfully constructed, which can be useful in further investigation of the role of Rheb gene in cancer cells.</p>

Trans-splicing of Cys mutated coagulation factor VIII / 药学学报

To investigate the improving effect of inter-chain disulfide formation on protein trans-splicing, we introduce a Cys point mutation at Tyr(664) in heavy chain and at Thr(1826) in light chain of B-domain-deleted FVIII (BDD-FVIII). By co-transfection of COS-7 cell with the two Cys mutated chain genes, the intracellular protein splicing, inter-chain disulfide formation, secreted BDD-FVIII and bioactivity in culture supernatant were observed. The data showed that a strengthened spliced BDD-FVIII with an inter-chain disulfide detected by Western blotting and an elevated secretion of spliced BDD-FVIII (128 +/- 24 ng mL(-1)) compared to control (89 +/- 15 ng mL(-1)), assayed by a sandwich ELISA. A Coatest was performed to assay the secretion of bioactivity in culture supernatant and shown a much higher value (0.94 +/- 0.08 u mL(-1)) compared to that of control (0.62 +/- 0.15 u mL(-1)). It suggests that inter-chain disulfide formation could improve protein trans-splicing based dual-vector delivery of BDD-FVIII gene providing experimental evidence for ongoing in vivo study.

Expression of stromal cell-derived factor-1 in mouse retina with oxygen-induced retinopathy / 中华实验眼科杂志

Background The development of retinopathy of prematurity(ROP) is associated with many regulatory cytokines related to neovascularization;however,the retinal expression and regulated mechanism of stromal cell-derived factor-1 (SDF-1) in mouse model of oxygen-induced retinopathy (OIR) remain uncertain.Objective This study was to investigate the expression of SDF-1 in retina of mouse model of OIR.Methods Forty 7-day-old C57BL/6J mice were divided into OIR group and control group.In OIR group,20 mice were exposed to 75% oxygen for 5 days and then to room air for 5 days.In control group,20 mice were raised in room air.The expression of SDF-1 in retina of mice was studied by immunochemistry and quantified by real time reverse transcriptase polymerase chain reaction (RT-PCR).Results The positive immunohistochemical staining for SDF-1 was found mainly locating at the ganglion cell layer in 12-day-old mice of OIR group;the stronger positive immunohistochemical staining for SDF-1 was noted mainly locating at the ganglion cell layer,vascular endothelial cells of inner retina,neovascular endothelial cells in 17-day-old mice of OIR group;the delicate positive immunohistochemical staining for SDF-1 was both found mainly locating at the inner retina and being around the retinal vascular in 12-day-old mice of control group and 17-day-old mice of control group.The expression of SDF-1 mRNA in 17-day-old mice of OIR group was higher than that of 12-day-old mice of OIR group (t=8.072,P＜0.05)and 17-day-old mice of control group(t=10.026,P＜0.05),respectively.The expression of SDF-1 mRNA in 12-day-old mice of OIR group was lower than that of 12-day-old mice of control group (t=4.336,P＜0.05).Conclusion SDF-1 might improve the onset of retinal neovascularization of OIR.

Enhancing effect of deoxynivalenol-mediated GRP78 down-regulation on heavy chain secretion and bioactivity of two-chain FVIII gene co-transfected cells / 药学学报

Although two chain transfering separately could be used to overcome the volume limitation of adeno-associated virus vectors (AAV) in coagulation factor VIII (FVIII) gene delivery, it leads to chain imbalance for inefficient heavy chain secretion. In this study we aimed to improve the efficacy of two chain strategy in FVIII gene delivery through the degradation of glucose-regulated protein 78 (GRP78) known as a protein chaperone in endoplasmic reticulum (ER) by deoxynivalenol (DON) to decrease GRP78-bound FVIII heavy chain. By treating the two-chain gene transduced 293 cells with DON, the heavy chain (HC) secretion and FVIII bioactivity were observed. Data showed that 293 cells after three hours post-treatment with DON at a concentration of 500 ng mL(-1) resulted in obvious decrease the level of GRP78 but no effect on the cell proliferation. The HC secreted from DON-treated cells transfected with HC gene alone was 59 +/- 11 ng mL(-1), higher than that secreted by control cells (15 +/- 4 ng mL(-1)), and the HC secretion was further increasing to 146 +/- 34 ng mL(-1) in light chain (LC) gene co-transfected cells with an activity measured up to 0.66 +/- 0.15 U mL(-1), also greater than control cells (76 +/- 17 ng mL(-1) and 0.35 +/- 0.09 U mL(-1)). Taken together, these data suggest that DON-mediated GRP78 down-regulation could improve the efficacy of two-chain FVIII gene transfering by facilitating HC secretion, providing an experimental basis for in vivo dual-AAV application in FVIII gene delivery.

Post-translational ligation and function of dual-vector transferred split CFTR gene / 药学学报

The mutation of cystic fibrosis transmembrane conductance regulator (CFTR) gene leads to an autosomal recessive genetic disorder cystic fibrosis (CF). The gene therapy for CF using adeno-associated virus (AAV) vectors delivering CFTR gene is restricted by the contents limitation of AAV vectors. In this study the split CFTR genes severed at its regulatory domain were delivered by a dual-vector system with an intein-mediated protein trans-splicing as a technique to investigate the post-translational ligation of CFTR half proteins and its function as a chloride ion channel. A pair of eukaryotic expression vectors was constructed by breaking the human CFTR cDNA before Ser712 codon and fusing with Ssp DnaB intein coding sequences. After co-transfection into baby hamster kidney (BHK) cells followed by transient expression, patch clamps were carried out to record the chloride current of whole-cell and the activity of a single channel, and the ligation of two halves of CFTR was observed by Western blotting. The results showed that the intein-fused half genes co-transfected cells displayed a high whole cell chloride current and activity of a single channel indicating the functional recovery of chloride channel, and an intact CFTR protein band was figured out by CFTR-specific antibodies indicating that intein can efficiently ligate the separately expressed half CFTR proteins. The data demonstrated that protein splicing strategy could be used as a strategy in delivering CFTR gene by two vectors, encouraging our ongoing research program on dual AAV vector system based gene transfer in gene therapy for cystic fibrosis.