An optimized secretory expression system and immunogenicity evaluation for glycosylated gp90 of avian reticuloendotheliosis virus.

Reticuloendotheliosis is an important immunosuppressive disease, associated with avian reticuloendotheliosis virus (REV) infection, and causes notable economic losses worldwide. Glycoprotein gp90 is an important structural protein of REV, and considered to be the most important immunogenic antigen, which can induce neutralizing antibodies against REV. In this study, an optimized suspension culture system was developed and applied to secretory express the immunogenic surface antigen gp90. To achieve an optimal glycosylation, the gp90 was designed to secretory expressed into the supernatant of the cell culture, which also occurs in the natural protein maturation procedure of REV. Serum-free culture medium was introduced to simplify the purification process and reduce the production costs. Based on the purified glycosylated gp90, an oil-emulsion subunit REV vaccine candidate was developed and evaluated in chickens. The subunit gp90-based vaccine induced fast immune responses, high levels of antibodies (REV-specific antibody, gp90-specific antibody, and neutralizing antibody against REV), and preferential T helper 2 (Th2) (interleukin-4 secretion) not Th1 (interferon-γ secretion) response. Furthermore, the viremia induced by REV infection was significantly reduced in chickens immunized with the glycosylated gp90. Overall, an optimized secretory expression system for glycosylated gp90 was developed, and the glycosylated gp90 obtained in this study retained good immunogenicity and could be an attractive vaccine candidate to protect chickens against REV horizonal infection.

[Genetic analysis of a pedigree affected with tuberous sclerosis complex caused by a novel mutation of TSC1].

OBJECTIVE: To analyze the clinical phenotype of a Chinese pedigree affected with Tuberous sclerosis complex (TSC) and explore pathogenic mutations of TSC1 and TSC2 gene. METHODS: Unique clinical phenotypes，the results of imaging, examination of the proband and special family history, collectively, made the constellation of features of TSC. Genomic DNA was obtained from six affected and eight unaffected members of the family and potential mutations of the TSC1 and TSC2 genes were detected by PCR-amplification of the exons and exon-intron boundaries and direct sequencing. A total of 150 normal unrelated individuals were used as controls. RESULTS: Genetic analysis documented the presence of a heterozygous mutation, c.1781_1782delTG (p.Val594GlyfsX11), in the exon 15 of TSC1 gene within all the patients of the family. This mutation was not observed in the eight unaffected family members or in the 150 unrelated control subjects from the same population , or the Human Gene Mutation Database (HGMD) and had completely co-segregated with the disease phenotype in the family. CONCLUSION: The c.1781_1782delTG mutation of TSC1 gene may be responsible for the tuberous sclerosis complex in this family. The data presented in the present study are of significance to clinicians, as well as genetic counselors, and may provide new clues for molecular diagnosis of this disease．.

First cephalosomatic anastomosis in a human model.

BACKGROUND: Cephalosomatic anastomosis (CSA) has never been attempted before in man as the transected spinal cords of the body donor and body recipient could not be "fused" back together. Recent advances made this possible. Here, we report on the surgical steps necessary to reconnect a head to a body at the cervical level. METHODS: Full rehearsal of a CSA on two recently deceased human cadavers was performed at Harbin Medical University, Harbin, China. RESULTS: The surgery took 18 hours to complete within the time frame planned for this surgery. Several advances resulted from this rehearsal, including optimization of the surgical steps, sparing of the main nerves (phrenics, recurrent laryngeal nerves), and assessment of vertebral stabilization. CONCLUSION: Several specialties are involved in a full-scale CSA, including neck surgery, vascular surgery, orthopedic surgery, plastic surgery, gastrointestinal surgery, and neurosurgery, as well as the operating staff. This rehearsal confirmed the surgical feasibility of a human CSA and further validated the surgical plan. Education and coordination of all the operating teams and coordination of the operative staff was achieved in preparation for the live human CSA.

Downregulation of B7-H4 in the MHCC97-H hepatocellular carcinoma cell line by arsenic trioxide.

Arsenic trioxide (As2O3; ATO), a compound which is characterized by its ability to function as a potent anticancer agent, has been investigated in a variety of carcinomas. B7­H4, a transmembrane protein, may inhibit the function of the T cell effector, and therefore, may be useful in investigating different types of tumor therapies. However, few studies have been published previously associated with the roles of ATO and B7­H4 in human hepatocellular carcinoma (HCC). The aim of the present study was to investigate the anti­invasive role of ATO in HCC, to determine the effect of ATO treatment on the expression of B7­H4 and to further assess the possible underlying mechanisms. Following treatment of the cells with 2, 4 and 8 µM ATO for 48 h, cell counting kit­8 (CCK­8), Transwell and western blot assays were used to determine the extent of human MHCC97­H HCC cell proliferation, apoptosis, invasion and B7­H4 expression, respectively. The results revealed that 1 µM ATO markedly decreased cellular proliferation, and ATO administered at concentrations of 0.1, 0.2 and 0.5 µM markedly inhibited the migration and invasion of the human MHCC97­H HCC cell line. The expression of B7­H4 in the treatment groups was markedly reduced. Signal transduction mediated via the Janus kinase 2/signal transducers and activators of transcription 3 pathway was inhibited upon treatment with 0.1, 0.2 and 0.5 µM ATO. Additionally, the protein expression levels of matrix metalloproteinase 2 and vascular endothelial growth factor were markedly reduced in HCC cells upon treatment with ATO. In conclusion, ATO may reduce the protein expression levels of B7­H4 in MHCC97­H HCC cells, and further affected HCC tumorigenesis and progression. ATO may be a putative agent for the development of therapeutic strategies against human liver cancer.

Serum miR-335 Level is Associated with the Treatment Response to Trans-Arterial Chemoembolization and Prognosis in Patients with Hepatocellular Carcinoma.

AIM: To identify the role of serum MicroRNA-335 (miR-335) in determining the treatment response to Trans-arterial chemoembolization (TACE) in patients with hepatocellular carcinoma (HCC) and their prognosis after TACE. METHODS: A total of 125 HCC patients were enrolled in this study. All these patients underwent TACE and the treatment response was evaluated. All patients were followed for prognosis analyses. Serum miR-335 levels immediate before and 30 days after TACE were determined. RESULTS: HCC patients had significantly lower miR-335 levels than hepatitis patients and healthy controls. Lower serum miR-335 levels were closely associated with more progressive clinical features, including a higher mean serum AFP level, more vascular invasion, cirrhosis and larger tumor size. Response rates were higher in patients with high miR-335 compared to those with low miR-335 level. Patients with lower serum miR-335 levels had significantly poorer prognosis than patients with higher serum miR-335 levels. CONCLUSION: Our data suggest that serum miR-335 can be used as a molecular marker to predict the treatment response and clinical outcome in HCC patients receiving TACE.

A recombinant avian leukosis virus subgroup j for directly monitoring viral infection and the selection of neutralizing antibodies.

Avian leukosis virus subgroup J (ALV-J) has induced serious clinical outbreaks and has become a serious infectious disease of chickens in China. We describe here the creation of a recombinant ALV-J tagged with the enhanced green fluorescent protein (named rHPRS-103EGFP). We successfully utilize the rHPRS-103EGFP to visualize viral infection and for development of a simplified serum-neutralization test.

Two novel mutations in the DSRAD gene in two Chinese pedigrees with dyschromatosis symmetrica hereditaria.

BACKGROUND: Dyschromatosis symmetrica hereditaria (DSH) is a highly penetrant autosomal dominant cutaneous disorder characterized by a mixture of hyperpigmented and hypopigmented macules localized on the back of the extremities. Genetic studies have identified pathogenic mutations in the double-stranded RNA-specific adenosine deaminase (DSRAD) gene as responsible for this disorder. OBJECTIVES: To identify additional gene mutations of DSRAD in patients with DSH. METHODS: Two Chinese families with typical DSH were subjected to mutation detection in DSRAD. All DSRAD exons and their flanking intronic sequences were amplified and sequenced. RESULTS: Two novel mutations in the functional domains of the DSRAD gene were identified in two pedigrees. The c. 3140G>A(p.G1047D) mutation was found in all patients but not in the healthy individuals from the same family (I) and c.1760 A>G(p.Y587C) mutation was found in all the patients but not in the healthy family members (II). CONCLUSIONS: Two novel DSRAD mutations, p.G1047D and p.Y587C, were found in Chinese patients with DSH and our data add new variants to the knowledge of DSRAD mutations in DSH.

Prevalence and associated factors of knee osteoarthritis in a community-based population in Heilongjiang, Northeast China.

Few data exist concerning the prevalence of knee OA and associated factors in Northeast China. This study was undertaken to estimate the prevalence of radiographic and symptomatic knee OA among community residents and to elucidate relevant risk factors. Unmatched case-control study was adopted to study risk factors of knee OA. Radiographic OA was evaluated according to the Kellgren and Lawrence grading scheme. Statistical analyses included tests and logistic model regressions. A total of 1,196 people aged 40-84 years participated in the community-based health survey in Northeast China in 2005. Survey participants completed an interviewer-based questionnaire. The standardized prevalence of symptomatic knee OA was 16.05% and it was significantly higher in women than in men (19.87% vs. 11.91%, = 13.76, P < 0.001). There was also an increased tendency with age in both sex (men: x (2) = 29.67, P (trend) < 0.001; women: x (2) = 40.26, P (trend) < 0.001). The prevalence of symptomatic knee OA was significantly higher than that in Beijing and Shantou, while lower than that in Wuchuan county of inner Mongolia with nonsignificant difference. Logistic regressions revealed that age, sex, BMI, and work status might be risk factors for knee OA in urban residents, whereas age, BMI, and smoking habits might be risk factors in rural dwellers. Symptomatic knee OA is extremely common with preponderance for elderly women and constitutes a major public health problem. The findings will be useful to guide the distribution of future health care resources and preventive strategies.

Arterial smooth muscle injury causes blood tissue factor elevation predicting restenosis after pci.

OBJECTIVE: The predictability of the whole-blood tissue factor levels for restenosis after coronary angioplasty is uncertain. We first probed in depth the association between plasma tissue factor concentrations and the development of restenosis after coronary intervention with an animal pathological model. METHODS AND RESULTS: Thirty pigs were used and their coronary arteries were injured for the dilatation of balloons. Morphological measurements include neointimal area, injury score and the extent of area stenosis. Whole-blood tissue factor levels were measured before and after intervention. The circulating tissue factor levels increased significantly after intervention (baseline value, 328.54 ± 47.46 pg/ml; at 30th minute, 618.96 ± 119.08 pg/ml; at 24th hour, 639.34 ± 116.21 pg/ml) (p < 0.01), and the degrees of tissue factor changes correlated positively to the neointimal hyperplasty (r(30th min) = 0.751, r(24th hour) = 0.72, p < 0.01). There was no significant difference with the baseline whole blood tissue factor (TF) levels between restenotic and non-restenotic cases (330.83 ± 47.32 vs. 325.1 ± 49.57 pg/ml) (p > 0.05). The injury of media led to the most distinctive changes of blood tissue factor (p < 0.01). CONCLUSION: Higher values of whole-blood tissue factor may be a predictor of restenosis, and the damaged media might be the main reason of the tissue factor increase.

[Mutation analysis of KRT10 gene in a patient with bullous congenital ichthyosiform erythroderma].

OBJECTIVE: To investigate the gene mutation in one sporadic case of bullous congenital ichthyosiform erythroderma (BCIE), and to explore the relationship between the genotype and phenotype. METHODS: DNA was extracted from the blood samples of the patient with BCIE, unaffected members of the pedigree, and 50 unrelated healthy controls. PCR was used to amplify the hot spot fragment of keratin 1 (KRT1) and keratin 10 (KRT10) gene. The PCR products were directly sequenced to detect the mutations. RESULTS: A heterozygous 467G>A mutation was found in the patient, resulting in the substitution of arginine (R) by histidine (H) in codon 156 (R156H) in the 1A domain of the KRT10 protein but not in the healthy individuals from the family and the 50 unrelated individuals. CONCLUSION: The mutation of 467G>A in exon 1 of KRT10 gene identified may play a major role in the pathogenic mechanism of this case of BCIE.

[Keratin 17 mutation in pachyonychia congenita type 2 in a Chinese Han family].

OBJECTIVE: To investigate the keratin 17 gene (KRT17) mutation in a pedigree with pachyonychia congenita type 2 (PC-II). METHODS: DNA was extracted from the blood samples of the patients, unaffected members of the pedigree, and 100 unrelated healthy controls. PCR was performed to amplify the hot spots in KRT17 gene. PCR products were directly sequenced to detect mutation. RESULTS: A heterozygous 296T-->C mutation was found in all the affected members of this family, which resulted in the substitution of leucine by proline in codon 99 (L99P) in the 1A domain of the KRT17, but not in the healthy individuals from the family and the 100 unrelated controls. CONCLUSION: The mutation of KRT17 may play a major role in the pathogenesis of this pedigree with pachyonychia congenita type 2.

[A comparative study on the prevalence of osteoarthritis in middle and old-aged people from the urban and the rural area in Heilongjiang province].

OBJECTIVE: To investigate the prevalence of osteoarthritis in inhabitants aged 40 years old and above from urban and rural areas in Heilongjiang province. METHODS: Through multistage stratified cluster random sampling methods, residents aged 40 years and above were selected. All subjects were given a standardized questionnaire and were conducted a radiographic examination on hands, knees, neck spine and lumbar spine after informed consent. All statistics were performed by SPSS13.0. RESULTS: A total of 1196 residents were surveyed, which including 573 males and 623 female subjects. The prevalence of osteoarthritis in cervical spine, lumbar spine, knee and hand for men were 26.00%, 31.20%, 11.87%, 15.53%, respectively and that were 34.80%, 30.20%, 20.06%, 27.93% for women respectively. The prevalence of osteoarthritis increased with aging both in men and women. Prevalence in 60 - 70 age group achieved the peak. The prevalence rates became relatively low among those over the 70 years old than expected. The most common sites of osteoarthritis were knees and hands (16.10%), followed by cervical and lumbar spine (12.40%). CONCLUSION: The prevalence of osteoarthritis was generally high in middle and old-aged people in Heilongjiang province.

Arsenic trioxide and promyelocytic leukemia protein-adenovirus synergistically inhibit in vitro and in vivo growth of a hepatoma cell line.

The present study investigated the in vitro and in vivo growth-inhibitory effects of combination therapy with arsenic trioxide (As2O3) and an adenovirus expressing promyelocytic leukemia protein (Ad-PML). Growth of HepG2 cells in culture was not inhibited by As2O3 at concentrations below 5 micromol/L (p > 0.05). However, growth was inhibited by Ad-PML alone and synergistic growth inhibition was observed following combined treatments (p < 0.05). Flow cytometry analyses demonstrated an increase in apoptosis following combined treatment with As2O3 and Ad-PML for 24 h, which was correlated with increased p53 and decreased Bcl-2 expression. To examine treatment effects on in vivo cell growth, control HepG2 cells and cells treated with As2O3, Ad-PML, or both therapies were subcutaneously injected in nude mice. After 6 weeks, tumor volumes were 0.097 +/- 0.031 and 0.083 +/- 0.005 cm3 in the control and As2O3 alone groups, respectively (p > 0.05), but were undetectable in the Ad-PML alone or Ad-PML plus As2O3 groups. Finally, established HepG2 tumors in nude mice were injected with PBS, Ad-PML, As2O3, or Ad-PML plus As2O3, the tumor volumes were measured by ultrasound, and the therapeutic effects were compared. As2O3 alone had no effect at concentrations below 5 micromol/L (p > 0.05), while Ad-PML alone at a multiplicity of infection of 20 or As2O3 plus Ad-PML significantly decreased tumor volumes (p < 0.05). Thus, the combination of As2O3 and Ad-PML has synergistic inhibitory effects on hepatocellular carcinoma (HCC), possibly resulting from regulation of apoptotic gene expression enhanced HCC apoptosis.

Identification of novel electrophilic metabolites of piper methysticum Forst (Kava).

Dietary supplements containing Piper methysticum Forst. (kava) have been implicated in multiple cases of liver injury in humans, including 10 recently reviewed cases in which patients required liver transplantation following the usage of kava-containing products (Centers for Disease Control and Prevention, reprinted. (2003) J. Am. Med. Assoc. 289, 36-37). To investigate a possible mechanism(s) of kava-induced hepatotoxicity, an extract of kava was incubated in vitro with hepatic microsomes, NADPH, and GSH. Electrophilic intermediates that were generated via metabolic activation were trapped as GSH conjugates and removed from the protein mixture using ultrafiltration. Positive ion electrospray LC-MS/MS with precursor ion scanning was used for the selective detection of GSH conjugates, and LC-MS(n) product ion scanning was used to elucidate their structures. Using this in vitro MS-based screening assay, two novel electrophilic metabolites of kava, 11,12-dihydroxy-7,8-dihydrokavain-o-quinone and 11,12-dihydroxykavain-o-quinone, were identified. Mercapturic acids of these quinoid species were not detected in the urine of a human volunteer following ingestion of a dietary supplement that contained kava; instead, the corresponding catechols were metabolized extensively to glucuronic acid and sulfate conjugates. These observations indicate that quinoid metabolites, under most circumstances, are probably not formed in substantial quantities following the ingestion of moderate doses of kava. However, the formation of electrophilic quinoid metabolites by hepatic microsomes in vitro suggests that such metabolites might contribute to hepatotoxicity in humans when metabolic pathways are altered (e.g., because of a drug interaction, genetic difference in enzyme expression, etc.) or if conjugation pathways become saturated.