[Early physical growth and disease analysis among children born delivered by HBsAg-positive mothers].

Objective: To estimate the early physical growth and disease in children born to HBsAg-positive mothers. Methods: This was a retrospective cohort study. Three areas as Xihu in Hangzhou, Lanxi in Jinhua, and Haiyan in Jiaxing in Zhejiang province were selected by cluster sampling. The growth outcomes of children born to HBsAg-positive mothers (exposure group) and matched 1∶1 women uninfected with HBV (control group) in 2014 were investigated and compared at birth, 6, 9, 12, and 18 months, respectively. There were totally 342 children in each group. Results: The incidences of low birth weight (LBW) for children born to exposure and control group were 1.8% (6/342), and 2.6% (9/342), respectively (P=0.433); and, rates of preterm birth were 2.3% (8/342), and 2.0% (7/342), respectively (P=0.794). The mean birth weight of children born to mothers without HBV infection (3.4±0.4) kg was dramatically higher than children in exposure group (3.3±0.4) kg (P=0.019). At 18 months, the average head circumference was significantly greater among children in control group (47.3±1.3) cm than children in exposure group (47.0±2.0) cm (P=0.038). Additional, mean birth weeks, height, weight, increases in height/weight/head circumference each month, weight/height/head circumference for age Z scores, proportion of growth retardation and low weight, disease prevalence were not observed statistically differences between two groups (P>0.05). All children born to HBsAg-positive mothers were received three-dose HBV vaccination. The rate of hepatitis B immunoglobulin for births born to HBsAg-positive was 98.8% (338/342). Mother to children transmission of HBV at 18 months was 1.0% (1/97). Conclusion: No significant differences in growth development and disease prevalence were found among children born to HBsAg-positive women and women without HBV infection.

[Relationship between ATP-binding cassette subfamily B member 1 and cytochrome P450 2C19 polymorphisms and the effect of clopidogrel post percutaneous coronary intervention in patients with acute coronary syndrome].

OBJECTIVE: To observe the relationship between ATP-binding cassette subfamily B member 1 (ABCB1) and cytochrome P450 (CYP)2C19 polymorphisms and the effect of clopidogrel post percutaneous coronary intervention in patients with coronary artery disease. METHODS: A total of 300 consecutive patients with acute coronary syndrome undergoing selected percutaneous coronary intervention in General Hospital of the People's Liberation Army from October 2010 to August 2012 and treated with clopidogrel were enrolled and retrospectively analyzed. Antiplatelet responsiveness of clopidogrel was estimated by thrombelastograph. The patients were divided into 3 groups: remarkable efficacy group (adenosine diphosphate pathway inhibition rate >80%, 105 cases), effective group (adenosine diphosphate pathway inhibition rate of 50%-80%, 100 cases), and poor responsiveness group (adenosine diphosphate pathway inhibition rate <50%, 95 cases). CYP2C19 and ABCB1 polymorphisms were detected by PCR combined with restrictive fragment length polymorphism (PCR-RELP) method in all patients. A total of 200 patients were performed by high performance liquid chromatography with electrospray tandem mass spectrum methods (HTLC-MS/MS), which was applied for determining the plasma concentration level of clopidogrel metabolites between remarkable efficacy group and poor responsiveness group. Major adverse cardiovascular events and bleeding events were observed through follow-up. RESULTS: (1) There were significantly differences in gender, smoking and alanine transaminase level among 3 groups(P<0.01 or 0.05). (2)There was no significant difference in the ratio of TT, CC and CT genotype of ABCB1 gene among 3 groups(P>0.05). There was significant difference in the ratio of poor, middle and strong metabolizer genotype of CYP2C19 gene (P<0.05). (3)Recurrent angina rates were 8.6%(9/105), 6.0%(6/100) and 18.9%(18/95) (P<0.05), and bleeding events rates were 1.0% (1/105), 1.0%(1/100) and 8.4%(8/95)respectively (P<0.01) in remarkable efficacy group, effective group and poor responsiveness group during the 1 year follow up. There were no significant difference in rates of myocardial infarction, heart failure, ischemic stroke and death among 3 groups (all P>0.05) during follow up. Rates of major adverse cardiovascular events and bleeding events were similar in patients with TT, CC and CT genotype of ABCB1 (14.6%(13/89), 12.8(19/148)and 11.6%(5/43), P>0.05). Rates of major adverse cardiovascular events and bleeding events were 9.5%(2/21), 17.8(27/152) and 7.5%(8/107) in poor, middle and strong metabolizer genotype of CYP2C19 gene patients (P<0.05). (4) Plasma concentration of clopidogrel was significantly lower and relative concentration of acid metabolites was significantly higher in poor responsiveness group than in remarkable efficacy group(P<0.01 or 0.05). There was no significantly different in plasma relative concentration of 2-oxo-clopidogrel between remarkable efficacy group and poor responsiveness group. CONCLUSION: ABCB1 gene polymorphism is not but CYP2C19 gene polymorphisms is related with antiplatelet responsiveness of clopidogrel and clinical cardiovascular disease events in patients with acute coronary syndrome undergoing selected percutaneous coronary intervention.

MicroRNAs of the mesothorax in Qinlingacris elaeodes, an alpine grasshopper showing a wing polymorphism with unilateral wing form.

MicroRNAs (miRNAs) are now recognized as key post-transcriptional regulators in regulation of phenotypic diversity. Qinlingacris elaeodes is a species of the alpine grasshopper, which is endemic to China. Adult individuals have three wing forms: wingless, unilateral-winged and short-winged. This is an ideal species to investigate the phenotypic plasticity, development and evolution of insect wings because of its case of unilateral wing form in both the sexes. We sequenced a small RNA library prepared from mesothoraxes of the adult grasshoppers using the Illumina deep sequencing technology. Approximately 12,792,458 raw reads were generated, of which the 854,580 high-quality reads were used only for miRNA identification. In this study, we identified 49 conserved miRNAs belonging to 41 families and 69 species-specific miRNAs. Moreover, seven miRNA*s were detected both for conserved miRNAs and species-specific miRNAs, which were supported by hairpin forming precursors based on polymerase chain reaction. This is the first description of miRNAs in alpine grasshoppers. The results provide a useful resource for further studies on molecular regulation and evolution of miRNAs in grasshoppers. These findings not only enrich the miRNAs for insects but also lay the groundwork for the study of post-transcriptional regulation of wing forms.

N-acetylcysteine attenuates subcutaneous administration of bleomycin-induced skin fibrosis and oxidative stress in a mouse model of scleroderma.

BACKGROUND: Several lines of evidence suggest that the generation of reactive oxygen species (ROS) is of major importance in the pathogenesis of scleroderma, and thus antioxidant therapy may be useful for patients with an impaired oxidative defence mechanism. AIM: To examine the effect of N-acetylcysteine (NAC) on skin fibrosis and oxidative stress in a bleomycin (BLM)-induced mouse model of scleroderma. METHODS: We used this mouse model to evaluate the effect of NAC on skin fibrosis and oxidative stress. Skin fibrosis was evaluated by histopathological examination and hydroxyproline content. To measure lipid peroxidation, we used a thiobarbituric acid-reactive species, malondialdehyde (MDA). Oxidative protein damage (carbonyl content) and the activities of catalase (CAT) and superoxide dismutase (SOD) were determined to evaluate oxidative stress in the skin tissue. RESULTS: Treatment with NAC attenuated the skin fibrosis induced by BLM, significantly reducing the MDA and protein carbonyl content in these mice. SOD activity in BLM-only mice and BLM plus NAC-treated mice was increased compared with control mice. However, there was no significant difference in skin SOD activity of mice treated with both BLM and NAC compared with those treated with BLM only. In addition, CAT activity was not altered in the BLM plus NAC mice. CONCLUSIONS: NAC treatment attenuates skin fibrosis in a BLM-induced mouse model of scleroderma, and this is associated with diminished oxidative stress. The results suggest that NAC may be a potential therapeutic agent for patients with scleroderma.

[Selection of reference genes for quantitative real-time PCR in six oil-tea camellia based on RNA].

qRT-PCR is becoming a routine tool in molecular biology to study gene expression. It is nec- essary to find stable reference genes when performing qRT-PCR. The expression of genes cloned in oil-tea camellia currently can't be accurately analyzed because of a lack of suitable reference genes. We collected different tissues (including roots, stems, leaves, flowers and seeds) from six oil-tea camellia species to determine stable reference genes. Five novel and ten traditional reference gene sequences were selected from the RNA-seq database of Camellia oleifera C. Abel seeds and specific PCR primers were designed for each. Cycle threshold (Ct) data were obtained from each reaction for all samples. Three different software tools, geNorm, NormFinder and BestKeeper were applied to calculate the expression stability of the candidate reference genes according to the Ct values. The results were similar between analyzed by the three software packages, and indicated that the traditional gene TUBa-3, AC17a and the novel gene CESA were relatively stable in all species and tissues. However, no genes were sufficiently stable across all species and tissues, thus the optimal number of reference genes required for accurate normalization varied from two to six. Finally, the relative expression ofsqualene synthase (SQS) and squalene epoxidase (SQE) genes related to important ingredients squalene and tea saponin in oil-tea camellia seeds were compared by using stable to less stable reference genes. The comparison results validated the selection of reference genes in the current study. In summary, different optimal numbers of suitable reference genes were found for the different tissues of six oil-tea camellia species.

Evaluation of potential habitat with an integrated analysis of a spatial conservation strategy for David's deer, Elaphurus davidians.

How to assess the potential habitat integrating landscape dynamics and population research, and how to reintroduce animals to potential habitats in environments highly human disturbed are still questions to be answered in conservation biology. According to behavioral research on Elaphurus davidians, we have developed a suitability index and a risk index to evaluate the potential habitats for the deer. With these indices, we conducted two transect assessments to evaluate the gradient change of the target region. Then, taking rivers as border lines, we tabulated the forest areas, high grassland area and total area and then compared the forest and high grassland area in each subregion. Furthermore, we computed the land use transfer matrix for the whole Yancheng coast during 1987-2000. We also computed human modified index (HMI) in six subregions. Lastly with a geographical information system support we obtained the spatial distribution of the indices and evaluation of the whole potential habitats from a neighborhood analysis. The transect assessment showed that the suitability of the coastal area was higher than that of the inland area for the deer, while the southern area was higher than the northern. Landscape metrics and HMI analysis showed that different landscape patterns and different anthropogenic disturbance existed within the region, and the increasing human disturbance was the key factor causing the pattern dynamics. The evaluation of potential habitats showed that there was an estimated carrying capacity of no more than 10,000 for David's deer reintroduction into the natural area. Also the reintroduction strategy was discussed. This integrated approach linked the population research and the landscape metrics, and the dataset with different scale; thus, it is an approach likely to be useful for the protection of other large animal in a landscape highly disturbed by humans.

Vascular endothelial growth factor and its receptor Flk-1 are expressed in the hippocampus following entorhinal deafferentation.

Vascular endothelial growth factor (VEGF) has been thought of as a mitogen that promotes proliferation of endothelial cells and as a neurotrophic factor that stimulates neurogenesis and axonal growth in both peripheral and central nervous systems. To investigate the potential involvement of VEGF in the lesion-induced reorganization in the brain, the expression changes of VEGF and its receptor Flk-1 were analyzed in the mouse hippocampus after transections of the entorhinal afferents. In situ hybridization and immunohistochemistry showed the time-dependent expression upregulation of VEGF mRNA and protein in the entorhinally denervated hippocampal stratum lacunosum-moleculare and dentate outer molecular layer, which initiated by 3 days postlesion, reached its maximum at 7-15 days postlesion, still persisted by 30 days postlesion for protein, and recovered to the normal levels at 30 days postlesion for mRNA and at 60 days postlesion for protein. Double labeling of VEGF and glial fibrillary acidic protein revealed that VEGF-expressing cells in the denervated areas were reactive astrocytes. Semi-quantitative RT-PCR analysis showed that VEGF receptor Flk-1 mRNA was also time-dependently upregulated in the deafferented hippocampus with its maximal elevation at 7-15 days postlesion while the Flt-1 mRNA levels remained unchanged at any time point we examined. Immunohistochemistry analysis also displayed the upregulation of Flk-1 protein in the denervated stratum lacunosum-moleculare and outer molecular layer with a time course similar to that of VEGF mRNA upregulation. Flk-1 receptors were found to be expressed not only by reactive astrocytes but also by neurites, which most likely belong to sprouting axons by 7 days postlesion and regrowing dendrites by 15-30 days postlesion. From these data we suggest that the spatiotemporal upregulation of VEGF and Flk-1 in the hippocampus is induced by entorhinal deafferentation and that VEGF may be involved in the structural reorganization in the deafferented hippocampus via directly or indirectly promoting neurite growth.

Up-regulation of cystatin C expression in the murine hippocampus following perforant path transections.

Cystatins are endogenous cysteine protease inhibitors that modulate the turnover of intracellular and extracellular proteins. These inhibitors are strongly implicated in a variety of pathological processes such as tumor metastasis and many degenerating CNS disorders. Here we report the expression of cystatin C, a major cysteine protease inhibitor of mammalian animals, in the murine hippocampus at 3, 7, 15 and 30 days following perforant path transections. Northern blot analysis showed that cystatin C transcripts were up-regulated in a transient manner with a significant increase at 7 and 15 days post-lesion (219% and 185% of control, respectively) in the rat hippocampus after entorhinal deafferentation. In situ hybridization and immunohistochemistry confirmed the time-dependent up-regulation of both cystatin C mRNA and protein expressions in a mouse model which initiated at 3 days post-lesion, reached maximal levels 7-15 days post-lesion, and remained slightly elevated by day 30 post-lesion. The modulation of cystatin C expression was observed to occur specifically in the entorhinally denervated zones: the stratum lacunosum-moleculare of the hippocampus and the outer molecular layer of the dentate gyrus. Double labeling by either a combination of in situ hybridization for cystatin C with immunohistochemistry for glial fibrillary acidic protein or double immunofluorescence staining for both proteins in mouse hippocampus at 7 and 15 days post-lesion revealed that most cystatin C-expressing cells are astrocytes. From these results we suggest that the spatiotemporal up-regulation of cystatin C in the hippocampus is induced by entorhinal deafferentation and that cystatin C may be involved in the astroglia-mediated neural plasticity events in the hippocampus following perforant path transections.

Efficient propagation of human herpesvirus 6B in a T-cell line derived from a patient with adult T-cell leukemia/lymphoma.

The efficient propagation of the OK strain of the B variant of human herpesvirus 6 (HHV-6B) was demonstrated in a line of T cells, TaY, established from the peripheral blood lymphocytes of a patient with adult T-cell leukemia/lymphoma (ATL). Growth of TaY cells depends on the presence of IL-2 and the cells harbor HTLV-I genomes. A severe cytopathic effect (CPE) was observed in many HHV-6B(OK)-infected TaY cells one week after infection. The release of virus from HHV-6B(OK)-infected TaY cells [TaY(OK)] was first detected after three days and increased rapidly for up to seven days after infection, as demonstrated by PCR. The titer of HHV-6B(OK) in the supernatant was comparable to the value of 10(3.5) TCID50/ml obtained with PHA-activated cord blood lymphocytes (CBL) that had been infected with HHV-6B(OK). The replication of the virus was shown to depend to a considerable extent on cell viability. Electron microscopy revealed many herpesvirus-type capsid- and enveloped-viruses in the nuclei and cytoplasm of degenerated cells in TaY(OK) cultures. The U1102 strain of HHV-6A and the Z29 strain of HHV-6B also infected TaY cells productively, as detected by PCR and an immunofluorescence test. These results suggest that the activation of CD4+ T lymphocytes with mitogens such as PHA or IL-2 and the expression of some cellular gene or the HTLV-I gene might be essential for efficient propagation of HHV-6B. TaY cells should play an important role in future investigations of cell-virus interactions and genetic variations or cell tropism of HHV-6 isolates since no cell line that shows propagation of both HHV-6A and HHV-6B has been reported to date.

Quantification of human herpesvirus 6 in healthy volunteers and patients with lymphoproliferative disorders by PCR-ELISA.

To determine whether actual numbers of human herpesvirus 6 (HHV-6) genome in hematologic neoplasias are associated with disease condition, we developed a quantitative PCR-ELISA for detection of HHV-6. The amount of viral DNA was determined using externally amplified known amounts of the plasmid DNA containing the viral target sequences. First, we determined a viral burden in peripheral blood leukocytes obtained from 23 healthy volunteers and four specimens of lymph nodes with reactive hyperplasia. Using 1 microg of DNA, the prevalence of HHV-6 was 43.4% (10/23), ranging from 0 to 100 HHV-6 genomes in blood obtained from healthy volunteers. The amounts of HHV-6 genomes were < 10 in four non-neoplastic lymph node specimens. We next examined the amount of viral DNA in 21 blood specimens and 19 lymph node specimens obtained from patients with lymphoproliferative diseases (LPD) at the time of diagnosis. The number of HHV-6 genomes in most of the B-cell lymphoma was < 5 in both blood and lymph node specimens, however, two lymph node specimens obtained from immunoblastic lymphadenopathy (IBL) and T-cell lymphoma had very high levels of HHV-6 viral DNA (3705 and 810, respectively). We also found that HHV-6 genomes in peripheral blood were more than 1000 in two patients with chronic lymphocytic leukemia. For all LPD patients combined, there were significantly higher levels of viral DNA (200.6 +/- 654.8 HHV-6 genomes per 1 microg purified DNA) compared to those in healthy volunteers (10.0 +/- 21.0 HHV-6 genomes per 1 microg purified DNA) (P < 0.05). This study demonstrates that a high level of HHV-6 viral DNA is occasionally associated with LPD patients. Although it is still uncertain whether HHV-6 is related to the pathogenesis in LPD or not, our results suggest that measurement of HHV-6 genomes using PCR-ELISA may be useful not only to understand the mechanism of HHV-6 infection in hemopoietic neoplasia but also to manage the care of immnocompromised patients such as bone marrow transplant patients.

Polymorphism of ADH and ALDH genes among four ethnic groups in China and effects upon the risk for alcoholism.

The alcohol dehydrogenases (ADHs) and aldehyde dehydrogenases (ALDHs) that metabolize ethanol are polymorphic. Different alleles encode subunits of the enzymes that differ in their rate of metabolizing ethanol. These polymorphisms are distributed differently among populations and have been shown to influence the risk for alcoholism in some Asian populations. We have examined the allele frequencies at the ADH2, ADH3, and ALDH2 loci in four populations from China (Han, Mongolian, Korean, and Elunchun) and in alcoholics within each population. The four populations differ in allele frequencies, with the Elunchun having a much lower frequency of ADH2*2 alleles, and the Mongolian and Elunchun having a much lower frequency of ALDH2*2 alleles. Within each population, alleles at one or more of these three loci are protective against alcoholism, although the populations differ in which loci play significant roles. The protective allele at each locus (ALDH2*2, ADH2*2, and ADH3*1) encodes a subunit that either metabolizes ethanol to acetaldehyde more rapidly or slows the conversion of acetaldehyde to acetate. Taken as a whole, data demonstrate that genetic differences in the enzymes that metabolize alcohol can substantially affect the risk for alcoholism.

[Expression of foreign tyrosine hydroxylase gene in the rat model of Parkinson's disease].

A total of 75 adult rats were used to produce hemiparkinson's disease (PD) model by unilateral injection of 6-hydroxydopamine (6-OHDA) into the right substantial nigra. Four weeks following the lesions, rats were tested for apomorphine (APO) -induced asymmetric rotation. Only rats that showed more than seven turns per minute were selected as PD models for transplantation. A tyrosine hydroxylase expression plasmid (pSVK3-TH) mixed with lipofectin was transplanted into the right striatum of 15 PD rats. Another 20 PD rats transplanted either with lipofectin or with pSVK3-TH served as controls. Every three days following transplantation, the performance of asymmetric rotation were tested and stained with the TH-immunohistochemistry. Only the animals grafted with pSVK3-TH mixed with lipofectin were found to show the decreased rotational behavior and a few TH-positive cells in the right striatum at the postgraft day 3-12, but not day 18. The results indicate that there exists a correlation between amelioration of asymmetric rotation and the TH gene expression in the denervated striatum. It is suggested that the lipofectin may mediate foreign TH gene expression in the PD rat brain.

Endogenous synaptogenesis in the deafferented dentate gyrus does not exclude synapse formation by embryonic entorhinal transplants.

After partial deafferentation postsynaptic sites are reinnervated by local sprouting of remaining axons. We have investigated whether this process is sufficient to prevent new synapses being formed by transplanted embryonic tissue. We find that after unilateral entorhinal ablation endogenous sprouting by local axons is unable to reinnervate all the postsynaptic sites in the denervated outer dentate molecular layer. Axons from embryonic entorhinal tissue transplanted adjacent to the denervated area are able to reclaim a further proportion of the denervated postsynaptic sites. Thus, after a large lesion, endogenous sprouting is insufficient to preclude reinnervation by axons from embryonic transplants.

[Role of neural cell adhesion molecule and polysialic acid on the neuronal development and regeneration].

The formation of nervous system depends on intercellular adhesion. This review covered the role of neural cell adhesion molecule (NCAM) and its polysialic acid (PSA) moiety on the neuronal development and regeneration. Mediation of cell adhesion is the fundamental role of NCAM, while the existence of PSA on NCAM decreases cell adhesion by its specific structure. It is known that during the development of chick embryo, the expression of PSA at three critical phases determines whether motoneurons can accurately innervate muscle. Following the peripheral nerve lesions of adult rats, the expression of NCAM is regulated by the state of innervation of muscle. In the adult rat brain, the disconnection of entorhinal cortex and hippocampus results in the elevation of PSA in the outer molecular layer of the dentate gyrus, with this increased expression remains for at least 60 days after lesion. Existing data strongly suggest that the reexpression of PSA in the denervated area may promote axonal outgrowth of transplanted neurons and reconstruct synaptic connection with host.

Gene therapy of Parkinson disease model rat by direct injection of plasmid DNA-lipofectin complex.

Lipofectin-mediated gene transfer was used to introduce plasmid harboring the tyrosine hydroxylase (TH) gene into the striatum of rats with lesions of the nigrostriatal pathway. The rotational asymmetry of Parkinson disease model rat was reduced quickly and significantly, suggesting that plasmid-DNA-transfected brain cells can generate L-dopa locally in the striatum in quantities sufficient to compensate partially for the loss of intrinsic striatal dopaminergic input. Immunohistochemical staining and reverse transcription polymerase chain reaction (RT-PCR) also confirm that striatal cells can express exogenous TH gene. Such in vivo plasmid DNA transfer strategy may be useful in other neurologic disease therapy, especially acute brain insults.

[Fate of human fetal dopamine neurons transplanted into rhesus monkey model of Parkinson's disease: a tyrosine hydroxylase immunocytochemical study].

To predicate the value of human fetal substantia nigra transplantation in clinical treatment of Parkinson's disease (PD), dissociated cells of substantia nigra from 8-12 week old abortive human fetus were grafted into the neostriatum of 5 adult rhesus monkeys with hemiparkinsonism induced by unilateral injection of MPTP. At 2, 5 and 12 months after transplanting the monkeys were sacrificed for tyrosine hydroxylase (TH) immunocytochemistry to examine the survival and possible synaptic contact of transplanted dopamine (DA) neurons. Transplanted TH immunoreactive cells took a pattern of patches scattered in the neostriatum. Each of the cell patches consisted of 3-10 cells. The TH immunoreactive fiber network was seen in the neostriatum. Electron microscopic survey revealed that TH+ buttons arising from grafted DA neurons formed symmetric or asymmetric synapses with TH- dendritic shafts/spines, and TH+ dendrites were seen to form synapses with TH- axons of the host. Additionally, there were a few synapses formed by TH+ axonal terminals with negative buttons. The results suggest that DA neurons from 8-12 week old abortive human fetus are able to survive grafting into the neostriatum of monkey, a species phylogenetically very close to human, and to establish reciprocal synaptic connectivity with the host even at 2 months post-transplanting. It is, therefore, inferable that embryonic human DA neurons transplanted into human neostriatum may have the same fate as in monkeys.