Triaging patients in the outbreak of COVID-2019 / 中国感染控制杂志

In the outbreak of COVID-19,triage procedures based on epidemiology were implemented in a local hospital in Changsha to control the transmission of SARS-CoV-2 and avoid healthcare-associated infection.This re-trospective study analyzed the data collected during the triage period and found that COVID-19 patients were en-riched 7 folds into the Section A designated for patients with obvious epidemiological history.On the other side,nearly triple amounts of visits were received at the Section B for patients without obvious epidemiological history.8 COVID-19 cases were spotted out of 247 suspected patients.More than 50％of the suspected patients were submi-tted to multiple rounds of nucleic acid analysis for SARS-CoV-2 infection.Of the 239 patients who were diagnosed as negative of the virus infection,188 were successfully revisited and none was reported as COVID-19 case.Of the 8 COVID-19 patients,3 were confirmed only after multiple rounds of nucleic acid analysis.Besides comorbidities,delayed sharing of epidemiological history added complexity to the diagnosis in practice.The triaging experience and strategy will be helpful for the control of infectious diseases in the future.

Establishment of System Suitability Standard for N-glycan Profile Analysis of Monoclonal Antibodies / 中国药学杂志

OBJECTIVE: To establish the system suitability standard for N-glycan profile analysis of monoclonal antibodies. METHODS: LC-MS was used to characterize the N-linked glycoform of the system suitability standard, and the stability was evaluated. The acceptance criteria of system suitability was set according to the method property and the data from method validation. RESULTS: The data of accelerated and long-term stability test indicated that the system suitability standard was stable under storage condition. The N-glycoform of this standard was representative, which covered the main glycoform of monoclonal antibodies. The acceptance criteria of system suitability set for the three analytical METHODS: were as below: the detected chromatogram should be visually similar to representative chromatogram; the resolution between G1F(1, 6) and G1F(1, 3), and G0F percentage should meet the specific requirements; the RSD of G0F retention time should be ≤4%. CONCLUSION: The standard substance for system suitability test and acceptance criteria for three N-glycan profile analytical METHODS are established.

Interlaboratory Validation of HILIC-UPLC Method for N-glycan Profile Analysis of Monoclonal Antibodies / 中国药学杂志

OBJECTIVE: To validate the HILIC-UPLC method for N-glycan profile analysis of therapeutic antibodies. METHODS: The interlaboratory method validation was performed according to ICH_Q2_R1 guideline and general principles of China Pharmacopoeia (2015 edition) 9101. The validation items included specificity, linearity, accuracy, precision, quantitation limit, range and robustness. RESULTS: The method showed good specificity, accuracy, precision and robustness, and showed a good linearity at a protein range from 100 to 400 μg. The r2 of linear regression equation was above 0.98, and the recoveries were between 86% and 117%. Both the RSDs of peak area percentage and retention time were below 10%, which indicated good precision. The lower quantitation limit of the method was 0.040%, and the range was from 0.040% to 78.751%, which means that single peak at this range could be quantified accurately. Furthermore, robustness evaluation under a series of conditions showed that this method was robust, where the RSD of peak area percentage was below 5% and RSD of retention time was below 1%. CONCLUSION: Interlaboratory validation of HILIC-UPLC provides a methodological verification basis for the improvement of Chinese Pharmacopoeia standards.

Influence of influenza A and B virus infections on human aortic smooth muscle cells and the expressions of cytokines / 医学研究生学报

Objective　Vascular smooth muscle cells are the main cells in atherosclerosis. Reports are rarely seen on influenza virus infection on human aortic smooth muscle cells (HASMC) and its influence on the expressions of the related cytokines. This study was to investigate the impact of influenza A virus (IAV) and influenza B virus (IBV) infection on HASMCs and the expressions of cytokines. Methods　HASMCs were stimulated with IAV or IBV or not stimulated with virus (the control). The nucleoprotein of the influenza virus in the cells was detected by immunofluorescence assay, the proliferation of the cells determined with CCK8, and the level of influenza virus RNA in the supernatant measured by qPCR. The collected supernatant was used to infect Madin-Darby canine kidney (MDCK) cells and detect the influenza virus nucleoprotein. The expressions of the cytokines of the influenza virus after 24 hours of infection were determined by qPCR.　Results　At 3 and 4 days after infected with influenza virus, the proliferation of the HASMCs was significantly inhibited in the IAV and IBV groups as compared with the control (P<0.05). The expression of virus RNA in the supernatant of the IBV group at 3 days was 5.75 times as high as that at 2 days (P<0.05), dropped at 4 days but still higher than that at 2 days (P<0.01). Compared with the normal culture medium, the medium with virus growth fluid significantly elevated the RNA level of IAV (0.842±0.148 vs 15.182±1.932, P< 0.01) and IBV (0.962±0.033 vs 4.029±0.681, P<0.01). After infection, the expression of MCP-1 was remarkably up-regulated in the IAV and IBV groups (4.364±0.193 and 3.348±0.507) in comparison with that in the control group (1.001±0.001) (P<0.05), and so were the expressions of IL-6 and TNF-α (P<0.05).　Conclusion　Both IAV and IBV can infect HASMCs and increase the expressions of the cytokines MCP-1, IL-6 , and TNF-α.

LC-MS analysis of sintilimab as an anti-PD-1 therapeutic mab for its improved hinge stability study / 药学学报

Monoclonal antibodies (mAbs) have been widely used as therapeutic drugs for treating diseases such as cancers and auto-immune diseases. When using an IgG4 isotype, one of the challenges is the instability of its hinge which is prone to Fab-arm exchange (FAE). The hinge sequence of a wild type IgG4 is -CPSC-, however, a single point mutation S228P from -CPSC- to -CPPC- can effectively diminish FAE, thereby improving hinge stability of the IgG4 molecule. Sintilimab is the fully human anti-PD-1 monoclonal antibody designed and developed for immuno-oncology, in which serine 228 in the hinge was engineered to proline to mitigate FAE. In this study, LC-MS is used to study hinge stability of sintilimab in both in vitro (PBS and human serum) and in vivo (SCID mouse) studies. The studies demonstrate that LC-MS is a fast and simple way to monitor for the occurrence of FAE in vitro and in vivo, and FAE can be eliminated by antibody engineering with a single point mutation.

Establishment of Capillary Zone Electrophoresis Method for Charge Heterogeneity Analysis of IgG2 Monoclonal Antibody / 中国药学杂志

OBJECTIVE: To establish a capillary zone electrophoresis(CZE) -based method for charge heterogeneity analysis of IgG2 monoclonal antibody. METHODS: The concentrations of TETA and EACA, pH of separation buffer and temperature of capillary were optimized, to obtain a method which can efficiently separate the charge variants of IgG2 mabs. The reproducibility and repeatability of the optimized CZE method were validated and a battery of mabs were evaluated. RESULTS: The separation effect was significantly influenced by TETA/EACA concentration, pH and separation temperature. Through a series of optimization, a CZE method which showed good resolution and precision(RSD of area percentage was below 3.0% was established and the method parameters were as below: 400 mmol•L-1 EACA/0.05% TETA, pH 6.2, separation under 30 kV voltage at 35℃. This method was also suitable for IgG1 and IgG4 mabscharge heterogeneity analysis. Based on the specific electrophorogram and migration time, it could also be used as an identification method for monoclonal antibodies. CONCLUSION: The optimized CZE method can efficiently separate the charge variants of IgG2 mabs and shows good precision and specificity, which can be used to analyze the charge heterogeneity and identification of monoclonal antibodies.

Analysis of N-linked Glycan Profile of Therapeutic Antibodies by UPLC-Qda MS / 中国药学杂志

OBJECTIVE: To analyze the N-glycan profile of therapeutic antibodies by UPLC-Qda MS. METHODS: The Nlinked glycan in Fc region was released by PNGase F digestion and labeled by RapiFluor-MS, and labeled glycans were analyzed by UPLC-Qda MS. RESULTS: The UPLC-Qda MS method showed good accuracy in N-glycan quantitaion and qualification, and the Δm/z value of individual N-glycan was in a range of ± 0.5. Three similar biotherapeutic products (SBP) showed a nearly same N-glycan profile with the reference biotherapeutic products (RBP) and the total percentage of fucosylated glycans was comparable, only one fraction of N-glycans had some difference. The major N-glycans of antibodies expressed by CHO cells, NS0 cells and SP2/0 cells were G0F-N, G0F, Man5, G1Fa, G1Fb and G2F, accounting for above 80% of total glycans. Eleven glycoforms were detected in CHO cell expressed antibodies, 22 and 26 glycoforms were detected in NS0 cell and SP2/0 cell expressed antibodies respectively. The N-glycans of NS0 cell and SP2/0 cell expressed antibodies contained more sialylated and galactosylated complex glycoforms, which was related to the antibody half-life in vivo and immunogenicity. CONCLUSION: The HILIC UPLC-Qda MS, as a fast and accurate analytical method, can be used in the quality control of N-glycan profile of antibody.

Correlation Study Between Percutaneous Coronary Intervention Complicated Depression and Serum Levels of Brain-derived Neurotrophic Factor, Ghrelin in Coronary Artery Disease Patients / 中国循环杂志

Objective: To explore the relationship between the incidences of percutaneous coronary intervention (PCI) complicated depression and serum levels of brain-derived neurotrophic factor (BDNF), ghrelin in coronary artery disease (CAD) patients. Methods: A total of 90 CAD patients after PCI were enrolled. According to Hamilton depression (HAMD) scale, the patients were divided into 2 groups: Depression group, n=40 and Non-depression group, n=50. Serum levels of BDNF and Ghrelin were examined by ELISA and compared between 2 groups. Results: Compared with Non-depression group, Depression group had reduced serum levels of BDNF and Ghrelin, both P<0.05. As increased severity of depression, BDNF and Ghrelin were decreased accordingly; serum levels of BDNF and Ghrelin were negatively related to HAMD score (r=-0.711, P<0.05 and r=-0.711, P<0.05). Conclusion: Serum levels of BDNF and Ghrelin have an early warning effect on depression in CAD patients after PCI, it may reflect the severity of depression at certain degree in relevant patients.

Transpedicular screw fixation for thoracolumbar fracture based on finite element analysis / 中国组织工程研究

BACKGROUND: Thoracolumbar fracture is commonly seen in spinal injuries, which causes loss of stability of the spine, as well as spinal cord and nerve compression, even deformity and paralysis. The diagnosis and treatment of thoracolumbar fracture remain controversial.OBJECTIVE: To summarize the mechanism of thoracolumbar fracture based on finite element method, its classification and transpedicular screw fixation.METHODS: The first author retrieved CNKI and PubMed databases for the relevant literature published between January 2000 and December 2016. The keywords were "finite element method, thoracolumbar spine fracture,transpedicular screw fixation" in Chinese and English, respectively.RESULTS AND CONCLUSION: (1) The finite element analysis method can simulate the mechanism of thoracolumbar fracture and provides a reference for the studies on the occurrence, development and treatment of thoracolumbar fracture. (2) The classification of thoracolumbar fracture is beneficial for planning a rational treatment strategy and evaluating prognosis. (3) Compared with the traditional screw fixation, the transpedicular screw fixation holds advantages in biomechanical stability and postoperative correction effect. (4) There are various classifications for thoracolumbar fracture; differences in severity and cartilage injury are difficult to simulate completely. (5) The finite element analysis method shows certain application limitations due to long learning curve and modeling time as well as complicated calculations.

Evaluation of Basal Serum Adrenocorticotropic Hormone and Cortisol Levels and Their Relationship with Nonalcoholic Fatty Liver Disease in Male Patients with Idiopathic Hypogonadotropic Hypogonadism / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Prolonged gonadal hormone deficiency in patients with idiopathic hypogonadotropic hypogonadism (IHH) may produce adverse effects on the endocrine homeostasis and metabolism. This study aimed to compare basal serum adrenocorticotropic hormone (ACTH) and cortisol levels between male IHH patients and healthy controls. Moreover, this study compared the basal hypothalamic-pituitary-adrenal (HPA) axis in patients with and without nonalcoholic fatty liver disease (NAFLD), and also evaluated the relationship between basal HPA axis and NAFLD in male IHH patients.</p><p><b>METHODS</b>This was a retrospective case-control study involving 75 Chinese male IHH patients (mean age 21.4 ± 3.8 years, range 17-30 years) and 135 healthy controls after matching for gender and age. All subjects underwent physical examination and blood testing for serum testosterone, luteinizing hormone, follicle-stimulating hormone, ACTH, and cortisol and biochemical tests.</p><p><b>RESULTS</b>Higher basal serum ACTH levels (8.25 ± 3.78 pmol/L vs. 6.97 ± 2.81 pmol/L) and lower cortisol levels (366.70 ± 142.48 nmol/L vs. 452.82 ± 141.53 nmol/L) were observed in male IHH patients than healthy subjects (all p<0.05). IHH patients also showed higher metabolism parameters and higher prevalence rate of NAFLD (34.9% vs. 4.4%) than the controls (all P < 0.05). Basal serum ACTH (9.91 ± 4.98 pmol/L vs. 7.60 ± 2.96 pmol/L) and dehydroepiandrosterone sulfate (2123.7 ± 925.8 μg/L vs. 1417.1 ± 498.4 μg/L) levels were significantly higher in IHH patients with NAFLD than those without NAFLD (all P < 0.05). We also found that basal serum ACTH levels were positively correlated with NAFLD (r = 0.289,p<0.05) and triglyceride levels (r = 0.268, P< 0.05) in male IHH patients. Furthermore, NAFLD was independently associated with ACTH levels in male IHH patients by multiple linear regression analysis.</p><p><b>CONCLUSIONS</b>The male IHH patients showed higher basal serum ACTH levels and lower cortisol levels than matched healthy controls. NAFLD was an independent associated factor for ACTH levels in male IHH patients. These preliminary findings provided evidence of the relationship between basal serum ACTH and NAFLD in male IHH patients.</p>

Development of quality control methods of recombinant anti-EBOV antibodies / 中国药学杂志

OBJECTIVE: To establish quality control methods for recombinant anti-EBOV mAbs which are comprised by three anti-EBOV glycoprotein mAbs. METHODS: The binding-activity of recombinant humanized anti-EBOV mAbs to EBOV glycoprotein was evaluated by ELISA. Peptide map by LC-MS was used in the identification tests. Purity was analyzed by CE-SDS and SEC-HPLC. iCIEF was performed to measure the PI value and the charge heterogeneity. 2AB was labeled on the released glycan, and was analyzed by NP-HPLC and mass spectrometry. The concentration of polysorbate 20 was tested by HPLC. RESULTS: The EC50s of recombinant anti-EBOV mAbs were (12.53±1.62), (11.10±0.62) and (6.09±0.35) ng·mL-1, respectively. The theoretical sequence coverage rates of three mAbs were all above 95%. The main peak area percentages shown by non-reduced CE-SDS were (94.41±0.05)%, (95.58±0.17)% and (96.11±0.05)%. The peak area percentages of both heavy and light chain shown by reduced CE-SDS were (98.19±0.06)%, (97.97±0.03)% and (98.57±0.03)%. The main peak area percentages shown by SE-HPLC were (99.59±0.01)%, (99.56±0.01)% and (99.74±0.01)%. The isoelectric points of the main peak shown by iCIEF were (8.70±0.01),(8.26±0.01) and (8.85±0.01). The concentrations of polysorbate 20 were (0.34±0.00),(0.35±0.00) and (0.35±0.01) μg·mL-1, respectively. The glycan mapping analysis was relatively sensitive, and the percentage of fucosylated N linked glycan was less than 0.5%. CONCLUSION: Up-to-date quality control methods for recombinant anti-EBOV mAbs are established in this study, which may be used to ensure the safety, effectiveness and quality controllability of the product. The methods can provide technical assists to Ebola outbreak and be a reference of the quality control of other domestic cocktail monoclonal antibody products.

Development of a novel reporter gene method for determination of ADCC potency of anti-CD20 monoclonal antibody / 药学学报

The biological activity of ADCC by anti-CD20 monoclonal antibody was determined by BioGlo™ Luciferase Assay System using Jurkat/NFAT-luc+FcγRIIIa cell line as effector cell and WIL2-S cell line as target cell. The developed method was verified for specificity, precision and accuracy. Anti-CD20 monoclonal antibody showed a dose-response mode by the developed method, and the determination result complied with the following four-parameter equation: y = (A-D)/[1 + (X/C)(B)] + D. The optimized parameters of the method were determined including the antibodies diluted concentration (18,000 ng·mL(-1)), dilution rate (1:5), the ratio of effector cell and target cell (6:1), and induction time (6 h). The values of eight independent tests have passed a statistical test for curve regression analysis, linear or parallelism, which showed the method possessed good specificity. Four different dilute groups of recovery rates sample were determined for 3 times, and the result showed mean relative potencies of (44.39±3.93)%, (72.74±2.78)%, (128.28±7.01)% and (168.19±2.70)% respectively, with a variation coefficient of less than 10%, and the recoveries of (88.78±7.85)%, (96.99±3.70)%, (102.63±5.61)% and (112.12±1.80)% respectively. A novel reporter gene method for determination of biological activity of ADCC by anti-CD20 monoclonal antibody was successfully developed, which showed strong specificity, good reproducibility and high accuracy, and might be used routinely.

Gene Expression Profile of Persistent Postoperative Hypertension Patients with Aldosterone-producing Adenomas / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Hypertension often persists after adrenalectomy for primary aldosteronism (PA). Many studies have analyzed the outcomes of adrenalectomy for aldosterone-producing adenomas (APA) to identify predictive factors for persistent hypertension. However, differentially expressed genes in persistent postoperative hypertension remain unknown. Our aim was to describe gene expression profile of persistent postoperative hypertension patients with APA.</p><p><b>METHODS</b>In this study, we described and compared gene expression profiles in persistent postoperative hypertension and postoperative normotension in Chinese patients with APA using microarray analysis. Confirmation was performed with quantitative real time-polymerase chain reaction analysis. Bioinformatic analysis (gene ontology analysis, pathway analysis and network analysis) was used for further research.</p><p><b>RESULTS</b>Microarray analysis identified a total of 99 differentially expressed genes, including 18 up-regulated and 81 down-regulated genes. Among the dysregulated genes were fat atypical cadherin 1 as well as fatty acid binding protein 4 and other genes that have not been previously studied in persistent postoperative hypertension with APA. Bioinformatics analysis indicated that differentially expressed genes were associated with lipid metabolic process, metal ion binding, and cell differentiation. Pathway analysis determined that five pathways corresponded to the dysregulated transcripts. The mRNAs-ncRNAs co-expression network was composed of 49 network nodes and 72 connections between 18 coding genes and 31 noncoding genes.</p><p><b>CONCLUSIONS</b>This study revealed differentially expressed genes in persistent postoperative hypertension with APA and provided a resource of candidate genes for exploration of possible drug targets and prognostic markers.</p>

Determination of drug antibody ratio in an antibody-drug conjugate / 药学学报

This paper reports the determination of the drug antibody ratio in an antibody-drug conjugate with two methods, i.e. LC-MS and UV/VIS, and to provide a reliable method to scientifically evaluate and effectively control the drug antibody ratio. Deglycosylated sample was analyzed with C4 column followed by MS, and the number of conjugated drugs in the antibody was determined by the molecular weight increase due to the addition of different number of drugs to the antibody, and then drug antibody ratio was calculated by weighted average of different number of drugs conjugated to the antibody. Optical density at 252 and 280 nm was measured with UV/VIS, and due to the difference of extinction coefficients between the antibody and the drug, the drug antibody ratio was calculated from linear equation with two unknowns. The drug antibody ratio was 3.21 and 3.25 respectively measured by the two methods, and the results were similar with the two methods. Our study indicated that both methods, LC-MS and UV/VIS, could be applied to the analysis of drug antibody ratio of the antibody drug conjugate.

The impact of fluoride on in vitro cultured human chondrocytes / 中国地方病学杂志

Objective To study the cell vitality and ultra-structure of in vitro cultured fetus chondrocytes exposed to different doses of fluoride.Methods Primary chondrocytes were obtained from articular cartilage of the 24-27 weeks,aborted and dead fetuses.The third generation of primary cultured chondmcytes were exposed to concentrations of 0,10-2,5 × 10-3,10-3,10-4,10-5,10-6,10-7 and 10-8 mol/L fluoride for 24,48 and 72 h.Cell vitality was detected with Cell Counting Kit-8 (CCK-8) and ultra-structure of chondrocytes was observed by transmission electron microscope.Results The cell vitalities of chondrocytes exposed to doses of fluoride (10-2,5 ×10-3,10-3,10-4,10-5,10-6,10-7 and 10-8 moL/L) for 24,48 and 72 h were(15.04 ± 0.55)％,(62.53 ± 1.03)％,(100.34 ± 5.19)％,(111.40 ± 3.69)％,(121.47 + 6.09)％,(129.95 ± 4.96)％,(121.81 ± 4.97)％,(111.00 ± 1.63)％;(10.35 ± 0.64)％,(35.23 ± 2.41)％,(110.30 ± 2.07)％,(113.66 ± 6.98)％,(120.36 ± 6.23)％,(133.40 ± 5.80)％,(126.06 ± 5.40)％,(115.62 ± 7.33)％; (6.19 ± 0.16)％,(18.44 ± 0.21)％,(120.83 ± 4.93)％,(123.77 ± 4.82)％,(129.09 ± 5.21)％,(140.44 + 4.18)％,(131.99 ± 7.00)％,(124.10 ± 3.68)％,respectively.The cell vitalities of 10-2,5 × 10-3 mol/L fluoride groups were significantly lower than that of the control group (all P ＜ 0.05).The cell vitality of 10-2 mol/L group was significantly lower than that of the 5 × 10-3 mol/L group (P ＜ 0.05).Doses of fluoride (10-2,5 × 10-3 mol/L) could inhibit the cell vitality and promote the apoptosis of chondrocytes in vitro with increasing doses and prolonged time.The cell vitalities of 10-3,10-4,10-5,10-6,10-7,10-8 mol/L of fluoride groups were significantly higher than that of the control group (except the 24 h 10-3 mol/L,P ＜ 0.05).Between 10-4 and 10-3 mol/L groups(the vitalities of 48 h and 72 h were higher,but not significantly); 10-5 and 10-4 mol/L groups (the vitality of 72 h was higher,but not significantly); 10-6 and 10-5 mol/L groups,the cell vitalities were significantly higher than that of the control group(all P ＜ 0.05).Between 10-7 and 10-6 mol/L groups,10-8 and 10-7 mol/L groups (the vitality of 72 h was lower,but not significantly),the cell vitalities were significantly lower than that of the control group(all P ＜ 0.05).Doses of fluoride(10-3-10-8 mol/L) could promote the cell vitality of chondrocytes in vitro with prolonged time.The optimal concentration for the promotion was 10-6 mol/L.The cells of the control group were characterized as regular morphology,the abnormal surface microvillis,abundant cytoplasm and mitochondrial,abundant and slightly expanded rough endoplasmic reticulums and low electron-dense materials.The cells of 10-6 mol/L fluoride group had the following changes,increased and swell mitochondrial,hypertrophy and expanded rough endoplasmic reticulums.The cells of 5 × 10-3 mol/L fluoride group had the following changes,decreased microvillis,invaginated cell membrane,pyknosis and apoptotic body.Conclusion Doses of fluoride (10-3-10-8 mol/L) can promote the proliferation of human chondrocytes cultured in vitro.Doses of fluoride (10-2,5 × 10-3 mol/L) can promote the apoptosis of human chondrocytes cultured in vitro.

Microsurgical treatment of ruptured anterior communicating aneurysms: a clinical analysis of 35 cases / 中华神经医学杂志

Objective To investigate the methods and efficacy of microscopic surgical treatment in patients with anterior communicating artery aneurysm. Methods The clinical data of 35 patients with ruptured anterior communicating artery aneurysm, admitted to our hospital from January 2008 to January 2011 and performed microneurosurgical treatment via pterional approch, were retrospective analyzed; their surgical techniques and efficacy were concluded. Results All the aneurysms in 35 patients were successfully clipped. Follow-up was performed for 3-6 months: complete recovery was achieved in 19 patients, good recovery in 11, and mild disability in 3 (including 1 with psychiatric symptom); death appeared in 2 patients. Conclusion The microsurgery via pterional approach is an effective method in treating anterior communicating artery aneurysms,resulting from its full exposure of aneurysms and few complications; adequate dissection and exposure of the entire H shaped complex body of anterior communicating artery are the keys to a successful clipping of aneurysm neck; and the selection of aneurysm clip suitable for the aneurysm and the preservation of the perforating arteries should be specially emphasized.

Multiplex ligation-dependent probe amplification for detecting AZF microdeletions on the Y chromosome in infertile men with azoospermia or severe oligozoospermia / 中华男科学杂志

<p><b>OBJECTIVE</b>To investigate the possibility of applying multiplex ligation-dependent probe amplification (MLPA) to the detection of azoospermia factor (AZF) microdeletion on the Y chromosome in infertile men with azoospermia or severe oligozoospermia.</p><p><b>METHODS</b>DNA samples were obtained from 147 azoospermia or severe oligozoospermia patients and 154 normal controls. After denatured at 95 degrees C, the samples were hybridized to the specific probes designed for the AZF region. With the ligase, the hybrid products were amplified by a pair of universal primers labeled with FAM fluorescence, and then separated by capillary electrophoresis for data analysis. Meanwhile all the samples were subjected to multiplex-PCR (mPCR) analysis for sequence-tagged sites (STS) in the AZF region.</p><p><b>RESULTS</b>STS deletion was detected in 22 (15.0%) of the 147 patients but not in the normal controls. By MLPA, 40 (27.2%) of the patients were found with specific probe omission in the AZF region, as compared with 20 cases in the control group.</p><p><b>CONCLUSION</b>Compared with mPCR, MLPA has a better sensitivity in detecting AZF microdeletions, and it provides more precise genetic information on the AZF regions, which may contribute to in-depth exploration into the etiological mechanism of impaired spermatogenesis.</p>

Genetic counseling and clinical outcome of fetus with de novo chromosomal aberrations / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To analyze the chromosome rearrangements and clinical outcome in fetus detected at prenatal diagnosis, and provide information for genetic counseling about de novo chromosomal aberrations.</p><p><b>METHODS</b>From January 2006 to December 2009, we found 12 cases of de novo chromosomal aberrations in 2 583 cases of prenatal cytogenetic analyses and reviewed the karyotypes, other experimental analyses data, fetal ultrasound findings and clinical outcomes.</p><p><b>RESULTS</b>Out of the 12 de novo chromosomal aberrations, 10 had unbalanced translocations and 2 had balanced reciprocal translocations. Eight of the 10 unbalanced translocation cases were terminated therapeutically, and 2 were delivered with full term. Neonates were phenotypically normal in the 2 cases with unbalanced translocations, but 1 had language retardation when followed up. The two balanced translocation cases were delivered with full term, and the neonates were phenotypically normal and clinical examinations were normal too.</p><p><b>CONCLUSION</b>Detailed cytogenetic and molecular study will be adjunctive tools for predicting the phenotype of fetus with de novo chromosomal aberrations. Fetal ultrasound examination will provide convincible demonstration to determine the outcome of pregnancy.</p>

Effect of fluoride on the expression of bcl-2 and Bax proteins in chondrocyte / 中国地方病学杂志

Objective To study the effect of fluoride on the expression of bcl-2 and Bax in chondrocyte in vitro, and investigate the mechanism of action of chondrocyte apoptosis induced by fluoride. Methods Articular chondrocytes of neonate rat were cultured in vitro and treated with 0(control),5,20,40 mg/L of fluoride,respectively, for 10 days. Then observed the u]trastructure of chondrocytes under eletronicmicroscope, and tested the expression of bcl-2 and Bax in chondrocyte in different groups by Western blotting. Results Abundant rough endoplasmic reticulums (RERs) and complete structure of mitochondria membranes were presented in globular chondrocytes in the control group and 5 mg/L group; but more lipid droplets and vacuoles were seen in the cytoplasm, and the structure of intracellular membranes became incomplete, and some shrieked chromatin and pyknosis were seen in the chondrocytes of the 20,40 mg/L groups. The expression of bcl-2 markedly decreased in 20 mg/L group(0.626 ± 0.042) and 40 mg/L group(0.531± 0.039) compared to the control group(0.876 ± 0.035,all P ＜ 0.01 ). And the expression of Bax significantly increased in 20 mg/L group(0.966 ± 0.047) and 40 mg/Lgroup ( 1 .289 ± 0.156) compared to the control group(0.642 ± 0.050, all P ＜ 0.01). But there was no statistical significant difference of the expression of bcl-2 or Bax between 5 mg/L group(0.885 ± 0.065,0.657 ± 0.045) and control group (all P ＞ 0.05 ). However there were statistical differences of expressions of bcl-2 and Bax between 20 and 40 mg/L groups(all P ＜ 0.01 ). Conclusions Twenty and 40 mg/L fluoride can cause damage to the ultrastructure of chondrocyte, and fluoride possibly promotes chondrocyte apoptosis by reducing the expression of antiapoptotic factor bcl-2 and increasing the expression of Bax.

Arsenic trioxide up-regulates Fas expression in human osteosarcoma cells / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Osteosarcoma is a common primary malignant tumor of bone with a poor prognosis due to its propensity for metastasis. The prognosis of patients is highly dependent on the presence or absence of lung metastasis and on the effectiveness of treatment against it. It has been reported that low level expression of Fas protein in human osteosarcoma cell is closely associated with lung metastasis. A large number of studies have shown that arsenic trioxide (ATO) can inhibit proliferation and induce apoptosis of many cancer cell lines; however, its effects on human osteosarcoma cells (Saos-2 cell line) remains unknown. The aim of this study was to investigate the effects of ATO on Saos-2 cells and to characterize its mechanism of Fas-expressing.</p><p><b>METHODS</b>A group of Saos-2 cells was treated with or without 0.5, 1, 2, 4 and 8 micromol/L ATO for three successive days, and the cytotoxicity of ATO was determined by an 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. Morphological changes in cells were studied by acridine orange/ethidium bromide (AO/EB) double staining. Flow cytometry (FCM) was used to assay cell DNA distribution. Another group of cells was pretreated with 10 nmol/L matrix metalloproteinase 7 (MMP-7) for 3 hours. They were then incubated with or without 2 micromol/L ATO for 24, 48 and 72 hours. Cytotoxicity, Fas protein and mRNA levels were systematically studied using MTT, Western blotting and real-time PCR, respectively. Cell proliferation, cell cycle progression and apoptosis were examined in this study.</p><p><b>RESULTS</b>Proliferation of Saos-2 cells was inhibited by ATO in both a dose- and time-dependent manner. The IC(50) values at 24, 48 and 72 hours were 9.30, 5.54 and 3.49 micromol/L, respectively. The survival rate of Saos-2 cells in the MMP-7 and ATO co-treated group was significantly higher than the ATO group, but it was lower than the control group. ATO induced G(1) phase arrest of the cell cycle and very efficiently stimulated apoptosis in Saos-2 cells, as evidenced by flow cytometric detection of sub-G(1) DNA content and AO/EB staining. Western blotting results indicated that Fas (FasL) protein expression in osteosarcoma cultures markedly increases in a time dependent manner after exposure to ATO. Compared with control, treatment with ATO 2 micromol/L and 4 micromol/L for 48 hours, resulted in increase of Fas gene expression to 28.31% and 56.74%, respectively. Our results indicated that ATO induced-apoptosis of Saos-2 cells may be mediated through the Fas pathway.</p><p><b>CONCLUSIONS</b>ATO suppressed cell proliferation of Saos-2 cell in a dose- and time-dependent manner and increased Fas protein expression. However, Fas-mediated apoptosis was incompletely interrupted by MMP-7, which suggested that other molecular mechanisms may mediate this process.</p>