Effects of liquid extract from Plinia cauliflora fruits residues on Chinese hamsters biochemical parameters / Efeitos do extrato líquido de resíduos de frutos de Plinia cauliflora sobre os parâmetros bioquímicos de hamsters chineses

Plinia cauliflora (Mart.) Kausel, popularly known as jabuticaba, is rich in polyphenols. Phenolic compounds exhibit several biological properties, which reflect on biomarkers such as biochemical parameters. In the present study, we evaluated the plasmatic levels of glucose, total cholesterol, HDL-cholesterol, triglycerides, and uric acid of Chinese hamsters fed for 45 days with a regular diet or cholesterol-enriched diet supplemented with a liquid extract obtained from P. cauliflora fruits residues standardized in ellagic acid and total phenolic compounds. The results showed that the concentrated extract obtained from jabuticaba residues increased the glycemia of animals fed with a regular diet and reduced the plasmatic uric acid levels of animals fed with a cholesterol-enriched diet. Since hyperuricemia is considered to be a significant risk factor of metabolic disorders and the principal pathological basis of gout, the liquid extract from P. cauliflora fruits residues would be a promising candidate as a novel hypouricaemic agent for further investigation.

Plinia cauliflora (Mart.) Kausel, popularmente conhecida como jabuticaba, é rica em polifenois. Os compostos fenólicos apresentam diversas propriedades biológicas, que refletem em biomarcadores, como os parâmetros bioquímicos. No presente estudo, avaliamos os níveis plasmáticos de glicose, colesterol total, HDL-colesterol, triglicerídeos e ácido úrico em hamsters chineses alimentados por 45 dias com dieta regular ou dieta enriquecida com colesterol suplementada com extrato líquido obtido de resíduos de frutos de P. cauliflora padronizado em ácido elágico e compostos fenólicos totais. Os resultados mostraram que o extrato concentrado obtido dos resíduos de jabuticaba aumentou a glicemia dos animais alimentados com dieta regular e reduziu os níveis plasmáticos de ácido úrico dos animais alimentados com dieta rica em colesterol. Uma vez que a hiperuricemia é considerada um fator de risco significativo de distúrbios metabólicos e a principal base patológica da gota, o extrato líquido dos resíduos de frutas de P. cauliflora seria um candidato promissor como um novo agente hipouricêmico para investigação posterior.

Advances in epigenetic regulation of Chinese hamster ovary cells / 生物工程学报

Chinese hamster ovary (CHO) cells play an irreplaceable role in biopharmaceuticals because the cells can be adapted to grow in suspension cultures and are capable of producing high quality biologics exhibiting human-like post-translational modifications. However, gene expression regulation such as transgene silencing and epigenetic modifications may reduce the recombinant protein production due to the decrease of expression stability of CHO cells. This paper summarized the role of epigenetic modifications in CHO cells, including DNA methylation, histone modification and miRNA, as well as their effects on gene expression regulation.

Knockout of caspase-7 gene improves the expression of recombinant protein in CHO cell line through the cell cycle arrest in G2/M phase

BACKGROUND: Chinese hamster ovary cell line has been used routinely as a bioproduction factory of numerous biopharmaceuticals. So far, various engineering strategies have been recruited to improve the production efficiency of this cell line such as apoptosis engineering. Previously, it is reported that the caspase-7 deficiency in CHO cells reduces the cell proliferation rate. But the effect of this reduction on the CHO cell productivity remained unclear. Hence, in the study at hand the effect of caspase-7 deficiency was assessed on the cell growth, viability and protein expression. In addition, the enzymatic activity of caspase-3 was investigated in the absence of caspase-7. RESULTS: Findings showed that in the absence of caspase-7, both cell growth and cell viability were decreased. Cell cycle analysis illustrated that the CHO knockout (CHO-KO) cells experienced a cell cycle arrest in G2/M phase. This cell cycle arrest resulted in a 1.7-fold increase in the expression of luciferase in CHO-KO cells compared to parenteral cells. Furthermore, in the apoptotic situation the enzymatic activity of caspase-3 in CHO-KO cells was approximately 3 times more than CHO-K1 cells. CONCLUSIONS: These findings represented that; however, caspase-7 deficiency reduces the cell proliferation rate but the resulted cell cycle arrest leads to the enhancement of recombinant protein expression. Moreover, increasing in the caspase-3 enzymatic activity compensates the absence of caspase-7 in the caspase cascade of apoptosis.

Immunogenicity and influencing factors of hepatitis B vaccination based on different vaccination schedules among chronic kidney disease patients / 中华流行病学杂志

Objective: To explore the immunogenicity and influencing factors of hepatitis B vaccination based on different vaccination schedules among chronic kidney disease (CKD) patients. Methods: CKD patients who participated in randomized controlled trials in four hospitals in Shanxi province and completed three doses of 20 µg vaccination (at months 0, 1 and 6) and four doses of 20 µg or 60 µg vaccination (at months 0, 1, 2, and 6) were surveyed from May 2019 to July 2020.According to the ratio of 1∶1∶1, 273 CKD patients were divided into 3 groups randomly. Quantification of the anti-hepatitis B surface antigen-antibody (anti-HBs) in serum samples was performed using chemiluminescent microparticle immunoassay at months 1 and 6 after the entire course of the vaccinations. The positive rate, high-level positive rate, geometric mean concentration (GMC) of anti-HBs, and the influencing factors were analyzed by χ2 tests, analysis of variance, unconditional logistic regression analysis. Results: A total of 273 CKD patitents were participants.The positive rates in the CKD patients with four doses of 20 µg vaccination (92.96%,66/71) or 60 µg vaccination (93.15%, 68/73) were higher than that in the CKD patients with three doses of 20 µg vaccination (81.69%, 58/71) at month one after the full course of the vaccinations (P<0.05). The GMCs of anti-HBs showed similar results (2 091.11 mIU/ml and 2 441.50 mIU/ml vs. 1 675.21 mIU/ml) (P<0.05). The positive rate was higher in the CKD patients with four doses of 60 µg vaccination (94.83%,55/58) than in those with three doses of 20 µg vaccination (78.79%,52/66) (P<0.05) at month six after the full course of the vaccinations. And the GMC of anti-HBs in the patients with four doses of 60 µg vaccination (824.28 mIU/ml) was significantly higher than those in the patients with 3 or 4 doses of 20 µg vaccination (639.74 mIU/ml and 755.53 mIU/ml) (P<0.05). After controlling the confounding factors, the positive rate in the CKD patients with four doses of 60 µg vaccination were 3.19 (95%CI: 1.02-9.96) and 5.32 (95%CI: 1.27-22.19) times higher than those in the patients with three doses of 20 µg vaccination at months 1 and 6 after the full course of the vaccinations, respectively. The positive rate in CKD patients without immune suppression or hormone therapy was 3.33 (95%CI: 1.26-8.80) and 4.78 (95%CI: 1.47-15.57) times higher than those in the patients with such therapy, respectively. Conclusions: Four doses of 20 µg or 60 µg hepatitis B vaccination could improve the immunogenicity in patients with CKD. And four doses of 60 µg vaccination might play a positive role in maintaining anti-HBs in this population. The immunogenicity in the CKD patients with immune suppression or hormone therapy was poor.

Impact of metabolic enzymes overexpression on transient expression of anti-hLAG3 by CHO cells / 生物工程学报

To enhance recombinant protein production by CHO cells, We compared the impact of overexpression of metabolic enzymes, namely pyruvate carboxylase 2 (PYC2), malate dehydrogenase Ⅱ (MDH2), alanine aminotransferase Ⅰ (ALT1), ornithine transcarbamylase (OTC), carbamoyl phosphate synthetase Ⅰ (CPSⅠ), and metabolism related proteins, namely taurine transporter (TAUT) and Vitreoscilla hemoglobin (VHb), on transient expression of anti-hLAG3 by ExpiCHO-S. Overexpression of these 7 proteins could differentially enhance antibody production. OTC, CPSI, MDH2, and PYC2 overexpression could improve antibody titer by 29.2%, 27.6%, 24.1%, and 20.3%, respectively. Specifically, OTC and MDH2 could obviously improve early-stage antibody production rate and the culture period was shortened by 4 days compared with that of the control. In addition, OTC and MDH2 had little impact on the affinity of anti-hLAG3. In most cases, overexpression of these proteins had little impact on the cell growth of ExpiCHO-S. MDH2 and ALT1 overexpression in H293T cells could also improve antibody production. Overall, overexpression of enzymes involved in cellular metabolism is an effective tool to improve antibody production in transient expression system.

Development and optimization of perfusion process for mammalian cell culture / 生物工程学报

In recent years, the demand of biologics has increased rapidly. Cell culture process with perfusion mode has become more and more popular due to its high productivity, good quality and high efficiency. In this paper, the unique operation and the details of process optimization for perfusion culture mode are discussed by comparing with traditional batch culture process. Meanwhile, the progress and strategies in the development and optimization of perfusion culture process in recent years are summarized to provide reference for the future development of mammalian cell perfusion culture technology.

Numerical simulation and optimization of impeller combination used in stirred bioreactor / 生物工程学报

Bioreactors have been central in monoclonal antibodies and vaccines manufacturing by mammalian cells in suspension culture. Numerical simulation of five impeller combinations in a stirred bioreactor was conducted, and characteristics of velocity vectors, distributions of gas hold-up, distributions of shear rate in the bioreactor using 5 impeller combinations were numerically elucidated. In addition, genetically engineered CHO cells were cultivated in bioreactor installed with 5 different impeller combinations in fed-batch culture mode. The cell growth and antibody level were directly related to the maximum shear rate in the bioreactor, and the highest viable cell density and the peak antibody level were achieved in FBMI3 impeller combination, indicating that CHO cells are sensitive to shear force produced by impeller movement when cells were cultivated in bioreactor at large scale, and the maximum shear rate would play key roles in scaling-up of bioreactor at industrial scale.

Caspase-7 deficiency in Chinese hamster ovary cells reduces cell proliferation and viability

BACKGROUND: Chinese hamster ovary (CHO) cells are the most commonly used mammalian host cell In the commercial-scale production of biopharmaceutical proteins. Modification of genes involved in apoptosis may improve the productivity of CHO cells. Executive caspases, including caspases 3 and 7, play critical roles in apoptosis. The effects of the ablation of the caspase 7 gene on proliferation and viability of CHO cells remains unknown. In this study, we applied clustered regularly interspaced short palindromic repeat (CRISPR/Cas9) to target caspase 7 gene of CHO K1 cell via all in one and homology targeted integration strategies. Consequently, the effect of caspase 7 deficiency on cell proliferation, viability, and apoptosis was studied by MTT assay and flow cytometry. RESULTS: Findings of gel electrophoresis, western blotting, and sequencing confirmed the caspase 7 gene silencing in CHO cells (CHO-KO). Proliferation assay revealed that caspase 7 deficiency in CHO cells resulted in the reduction of proliferation in various CHO-KO clones. Besides, the disruption of caspase 7 had negative effects on cell viability in exposure with NaBu which confirmed by MTT assay. Results of flow cytometry using Anexin V/PI demonstrated that Nabu treatment (11 mM) declined the percentage of live CHO-K1 and CHO-KO cells to 70.3% and 5.79%. These results verified that the CHO-K1 cells were more resistant to apoptosis than CHO-KO, however most of CHO-KO cells undergone early apoptosis (91.9%) which seems to be a fascinating finding. CONCLUSION: These results reveal that caspase 7 may be involved in the cell cycle progression of CHO cells. Furthermore, it seems that targeting caspase 7 is not the ideal route as it had previously been imagined within the prevention of apoptosis but the relation between caspase 7 deficiency, cell cycle arrest, and the occurrence of early apoptosis will require more investigation.

Discovery of the ARP2 protein as a determining molecule in tumor cell death

Cancer is a multifactorial disease that constitutes a serious public health problem worldwide. Prostate cancer advanced stages are associated with the development of androgen-independent tumors and an apoptosis-resistant phenotype that progresses to metastasis. By studying androgen-independent lymphoid nodule carcinoma of the prostate (LNCaP) cells induced to apoptosis by serum elimination, we identified the activation of a non-selective cationic channel of 23pS conductance that promotes incoming Ca2+ currents, as well as apoptosis final stages. arp2cDNA was isolated and identified to be of the same cell type, and mRNA was expressed in Xenopus laevis oocytes, which was found to be associated with the activation of incoming Ca2+ currents and induction to apoptosis. cDNA, which encodes the ARP2 protein, was overexpressed in LNCaP cells and Chinese hamster ovary cells, which induced apoptosis. Our evidence suggests that protein ARP2 overexpression and transit to the cell membrane allows an increased Ca2+ incoming current that initiates the apoptosis process in epithelial-type cells whose phenotype shows resistance to programmed cell death.

Increasing transgenic expression in recombinant Chinese hamster ovary cells using introns in different directions / 生物工程学报

The aim of this study is to investigate the effect of the chimeric intron in different directions on the expression of the nerve growth factor (NGF) in recombinant Chinese hamster ovary (CHO) cells. The chimeric intron that contained the splice sequence of the first intron of the human β-globin and the human immunoglobulin heavy chain variable region intron was used. NGF gene was cloned into the expression vectors containing the chimeric intron in the forward or reverse direction, followed by transfecting into CHO cells, and screened under G418 to produce the stable transfected CHO cells. Fluorescence quantitative PCR, ELISA, and Western blotting were performed to detect the recombinant NGF gene expression in CHO cells. The results showed that the chimeric introns could significantly enhance the expression of NGF in recombinant CHO cells. Moreover, the enhancing effect on NGF expression level by the intron in the forward direction showed stronger than that of the reverse direction both at mRNA and protein level. In conclusion, the chimeric intron could increase NGF expression in stably transfected CHO cells and the effect is associated with the direction of the intron insertion.

Effect of Amino Acid Motifs in Integrin β3 Cytoplasmic Tail on αⅡbβ3-Mediated Cell function in 293T cell models / 中国实验血液学杂志

OBJECTIVE@#To establish 293T cell lines stably expressing Calpain-cleavage related α3 cytoplasmic tail mutants, and to explore the effect of amino acid motifs in integrin β3 cytoplasmic tail on αⅡbβ3-mediated cell function.@*METHODS@#293T cell lines stably co-expressing human wild type integrin αⅡb and full length β3 or mutant β3, including β3-ΔNITY (β3 cytoplasmic tail NITY motif deleted), β3-Δ754 (β3 cytoplasmic tail TNITYRGT motif deleted) and β3-Δ759 (β3 cytoplasmic tail RGT motif deleted) were established. Spreading and adhesion of these stable cell lines on immobilized fibrinogen were tested.@*RESULTS@#293T-αⅡbβ3ΔNITY, 293T-αⅡbβ3Δ754, 293T-αⅡbβ3Δ759 and 293T-αⅡbβ3 cell lines were successfully established. Compared with the 293T cells, 293T-αⅡbβ3 cells which expressed full β3, possessed well adhesion and spread ability on immobilized fibrinogen, suggesting it can be as a surrogate for platelet. Compared with 293T-αⅡbβ3 cells, the 293T-αⅡbβ3ΔNITY cells showed a partial impairment of adhesion and spreadability on immobilized fibrinogen. while the 293T-αⅡbβ3Δ754 cells and 293T-αⅡbβ3Δ759 cells failed to adhere or spread on immobilized fibrinogen.@*CONCLUSION@#To the cell spreading function mediated by integrin β3, RGT motif is vital, while NITY can be dispensable. These established 293T cell lines stably expressing different β3 mutants provide a solid basis for a further analysis of mass spectrometry.

Antidepressant drug paroxetine blocks the open pore of Kv3.1 potassium channel

In patients with epilepsy, depression is a common comorbidity but difficult to be treated because many antidepressants cause pro-convulsive effects. Thus, it is important to identify the risk of seizures associated with antidepressants. To determine whether paroxetine, a very potent selective serotonin reuptake inhibitor (SSRI), interacts with ion channels that modulate neuronal excitability, we examined the effects of paroxetine on Kv3.1 potassium channels, which contribute to highfrequency firing of interneurons, using the whole-cell patch-clamp technique. Kv3.1 channels were cloned from rat neurons and expressed in Chinese hamster ovary cells. Paroxetine reversibly reduced the amplitude of Kv3.1 current, with an IC₅₀ value of 9.43 µM and a Hill coefficient of 1.43, and also accelerated the decay of Kv3.1 current. The paroxetine-induced inhibition of Kv3.1 channels was voltage-dependent even when the channels were fully open. The binding (k₊₁) and unbinding (k₋₁) rate constants for the paroxetine effect were 4.5 µM⁻¹s⁻¹ and 35.8 s⁻¹, respectively, yielding a calculated K(D) value of 7.9 µM. The analyses of Kv3.1 tail current indicated that paroxetine did not affect ion selectivity and slowed its deactivation time course, resulting in a tail crossover phenomenon. Paroxetine inhibited Kv3.1 channels in a usedependent manner. Taken together, these results suggest that paroxetine blocks the open state of Kv3.1 channels. Given the role of Kv3.1 in fast spiking of interneurons, our data imply that the blockade of Kv3.1 by paroxetine might elevate epileptic activity of neural networks by interfering with repetitive firing of inhibitory neurons.

The treatment effect of novel hGHRH homodimer to male infertility hamster

Extra-hypothalamic growth hormone-releasing hormone (GHRH) plays an important role in reproduction. To study the treatment effect of Grin (a novel hGHRH homodimer), the infertility models of 85 male Chinese hamsters were established by intraperitoneally injecting 20 mg/kg of cyclophosphamide once in a week for 5 weeks and the treatment with Grin or human menopausal gonadotropin (hMG) as positive control was evaluated by performing a 3-week mating experiment. 2–8 mg/kg of Grin and 200 U/kg of hMG showed similar effect and different pathological characteristics. Compared to the single cyclophosphamide group (0%), the pregnancy rates (H-, M-, L-Grin 26.7, 30.8, 31.3%, and hMG 31.3%) showed significant difference, but there was no difference between the hMG and Grin groups. The single cyclophosphamide group presented loose tubules with pathologic vacuoles and significant TUNEL positive cells. Grin induced less weight of body or testis, compactly aligned tubules with little intra-lumens, whereas hMG caused more weight of body or testis, enlarging tubules with annular clearance. Grin presented a dose-dependent manner or cell differentiation-dependentincrease in testicular GHRH receptor, and did not impact the levels of blood and testicular GH, testosterone. Grin promotes fertility by proliferating and differentiating primitive cells through up-regulating testicular GHRH receptor without triggering GH secretion, which might solve the etiology of oligoasthenozoospermia.

Chigger Mite (Acari: Trombiculidae) Survey of Rodents in Shandong Province, Northern China

Chigger mites are parasites of rodents and other vertebrates, invertebrates, and other arthropods, and are the only vectors of scrub typhus, in addition to other zoonoses. Therefore, investigating their distribution, diversity, and seasonal abundance is important for public health. Rodent surveillance was conducted at 6 districts in Shandong Province, northern China (114–112°E, 34–38°N), from January to December 2011. Overall, 225/286 (78.7%) rodents captured were infested with chigger mites. A total of 451 chigger mites were identified as belonging to 5 most commonly collected species and 3 genera in 1 family. Leptotrombidium scutellare and Leptotrombidium intermedia were the most commonly collected chigger mites. L. scutellare (66.2%, 36.7%, and 49.0%) was the most frequently collected chigger mite from Apodemus agrarius, Rattus norvegicus, and Microtus fortis, respectively, whereas L. intermedia (61.5% and 63.2%) was the most frequently collected chigger mite from Cricetulus triton and Mus musculus, respectively. This study demonstrated a relatively high prevalence of chigger mites that varied seasonally in Shandong Province, China.

Application of recombinant GPⅢa combined Luminex beads for the detection of HPA-1a antibody / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To generate recombinant GPⅢa as an alternative source for HPA-1a antigen and combine it with Luminex xMAP beads for the detection of HPA-1a-specific alloantibody.</p><p><b>METHODS</b>The full coding region of ITGB3 gene was amplified and ligated with pcDNA3.1. The recombinant plasmid was transfected into CHO cells, and those with stable expression were screened with G418. Expressed protein was identified and coupled with Luminex xMAP beads, which were then reacted with sera samples. Subsequently, phycoerythrin-labeled anti-species IgG antibody was added to the reaction wells and the median fluorescence was determined on a Luminex-100 analyzer.</p><p><b>RESULTS</b>DNA sequencing confirmed that the cloned ITGB3 gene was HPA-1aa. The recombinant GPⅢa was coupled with Luminex xMAP beads. The sensitivity of Luminex beads assay to detect HPA-1a antibody was dilution 1/32 (3.125 U/mL). The Luminex beads assay could specifically identify the HPA-1a antibody from the test sera, and the results were consistent with that of monoclonal antibody-specific immobilization of platelet antigens (MAIPA) technology. Cross-reactivity was not observed with the samples containing HLA, ABO and other HPA antibodies (HPA-3a and HPA-5b). The results illustrated that to detect HPA antibody with Luminex xMAP beads technology is feasible.</p><p><b>CONCLUSION</b>Recombinant GPⅢa was successfully obtained and used to establish a Luminex technology-based method for the detection of HPA antibodies.</p>

Characterization and Expression Analysis of Peroxiredoxin Genes in NNK-induced V79 Cells / 生物医学与环境科学（英文）

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent and prevalent nitrosamine procarcinogen found in cigarette smoke. The aim of this work is to study alterations in peroxiredoxin (Prx) expression induced by NNK during carcinogenesis. Characterization of Prx genes from hamster was performed using bioinformatics. V79 cells were induced with different concentrations of NNK (0.1-0.4 mg/mL), and the expression levels of six Prx genes (Prx1-Prx6) were measured by qRT-PCR 24 h following NNK treatment. Prx gene expression was induced by NNK stress, and the highest transcription levels were induced by over 20.42-fold relative to that of the control. NNK induced alterations in Prx expression over the course of lung cancer, which means Prxs may play important roles in ROS detoxification under NNK stress and their functions are complementary.

Protective Effect of an Isoflavone, Tectorigenin, Against Oxidative Stress-induced Cell Death via Catalase Activation

BACKGROUND: Isoflavones are biologically active compounds that occur naturally in a variety of plants, with relatively high levels in soybean. Tectorigenin, an isoflavone, protects against hydrogen peroxide (H2O2)-induced cell damage. However, the underlying mechanism is unknown. METHODS: The MTT assay was performed to determine cell viability. Catalase activity was assessed by determining the amount of enzyme required to degrade 1 μM H2O2. Protein expression of catalase, phospho-extracellular signal-regulated kinase (ERK), IκB-α, and NF-κB were evaluated by Western blot analysis. A mobility shift assay was performed to assess the DNA-binding ability of NF-κB. Transient transfection and a NF-κB luciferase assay were performed to assess transcriptional activity. RESULTS: Tectorigenin reduced H2O2-induced death of Chinese hamster lung fibroblasts (V79-4). In addition, tectorigenin increased the activity and protein expression of catalase. Blockade of catalase activity attenuated the protective effect of tectorigenin against oxidative stress. Furthermore, tectorigenin enhanced phosphorylation of ERK and nuclear expression of NF-κB, while inhibition of ERK and NF-κB attenuated the protective effect of tectorigenin against oxidative stress. CONCLUSIONS: Tectorigenin protects cells against oxidative damage by activating catalase and modulating the ERK and NF-κB signaling pathway.

Micronucleus in vitro induced by inhalable particulate matters in moxa smoke / 中国针灸

<p><b>OBJECTIVE</b>To investigate whether inhalable particulate matters can cause the damage of chromosome or mitotic apparatus to produce micronucleus, and to evaluate genetic toxicology of moxa smoke on chromosome.</p><p><b>METHODS</b>By MTT method, the 24 h half maximal inhibitory concentration (IC50) of moxa smoke condensation (MSC) on Chinese hamster ovary (CHO) cells was 0.087 mg/mL. CHO cells, which were cultured in vitro, were divided into a solvent control group, a positive control group (cyclophosphamide as solvent), a low concentration group, a moderate concentration group and a high concentration group. The low concentration group, moderate concentration group and high concentration group were set approximately 1/8, 1/4, 1/2 of IC50, respectively. Whether micronucleus had dose-effect response induced by the damage of chromosome or mitotic apparatus was observed after CHO cells were contaminated by MSC in the low concentration group, moderate concentration group and high concentration group.</p><p><b>RESULTS</b>The rate of micronucleus induced by MSC in the low concentration group, moderate concentration group and high concentration group was higher than that in the solvent control group (all P < 0.05), which presented dosage-effect response. The experiment was repeated 3 times, indicating it was repeatable with statistical significance.</p><p><b>CONCLUSION</b>High concentration of MSC shows toxicity to induce chromosome damage, which disappears at low concentration. The genetic toxicology is also dependent on concentration, and the concentration of moxa smoke is essential. In clinical treatment, it is noted to control the level of moxa smoke, while the clinical safety standard of moxa smoke concentration is in need of further study.</p>

Hepatitis B Immunoprophylactic Failure and Characteristics of the Hepatitis B Virus Gene in Mother-Infant Pairs in Parts of China / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To determine the hepatitis B immunoprophylactic failure rate in infants born to hepatitis B virus (HBV) infected mothers and to characterize HBV genes.</p><p><b>METHODS</b>HBV-serological testing was conducted for pregnant women and infants. The complete genomes of 30 HBV isolates were sequenced, and genetic characteristics were analyzed using MEGA 5 software.</p><p><b>RESULTS</b>The immunoprophylactic failure rate for infants who had completed the scheduled hepatitis B vaccination program was 5.76% (32/556). High sequence homology (99.8%-100%) was observed in 8 of the 10 mother-infant pairs. We identified 19 subgenotype C2 strains, 9 subgenotype B2 strains, and 2 subgenotype C1 strains. Three serotypes were detected: adr (19/30), adw (9/30), and ayw (2/30). The frequency of amino acid mutation of the 'a' determinant region was 16.67% (5/30), including that of Q129H, F134Y, S136Y, and G145E. We detected 67 amino acid mutations in the basal core promoter, precore, and core regions of the genome.</p><p><b>CONCLUSION</b>The immunoprophylactic failure rate in infants born to HBV-infected mothers is low in the regions of China examined during this study. Moreover, HBV mutation in the 'a' determinant region could not account for immunoprophylactic failure for all infants.</p>

Study of in vitro expression of human platelet ITGB3 gene nonsense mutation c.1476G>A / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To explore the function of a novel nonsense mutation c.1476G>A of ITGB3 gene using an in vitro expression system.</p><p><b>METHODS</b>An eukaryotic expression vector containing ITGB3 c.1476G>A cDNA was generated by site-directed mutagenesis and transformed into E.coli. Plasmid DNA was extracted and sequenced to confirm the target mutations. Wild-type and mutant recombination plasmids were transfected into Chinese hamster ovarian cancer (CHO) cells by nonliposome method, and the stable expression cells were harvested by G418 screening. The ITGB3 gene mRNA transcription and GPIIIa expression level in CHO cells were detected with real-time quantitative PCR, Western blotting and flow cytometry, respectively.</p><p><b>RESULTS</b>The eukaryotic expression vectors of wild ITGB3 cDNA and c.1476G>A mutant were successfully constructed. CHO cells with stable expression were obtained after transfection and screening. Compared with the wild-type transfected cells, the amount of CD61 antigen expression was 37% and mRNA transcription level was only 6% in the mutant-transfected cells. Full length GPIIIa protein was found only in the stably wild-type-transfected cells, but not in mutant-transfected cells by Western blotting analysis.</p><p><b>CONCLUSION</b>The ITGB3 c.1476G>A mutation can decrease the transcription level and further affect GPIIIa synthesis and CD61 antigen expression.</p>