Abstract:Microfluidic chip, which is one of the most fascinating analytical technologies in recent years, has made striking progress in chemistry, biology, medicine and other research fields. Several unique properties of microfluidics, such as high-throughput, miniaturization and multifunctional integration, make them ideal for their applications in biomedical research. More and more efforts have been dedicated to the research of Caenorhabditis elegans on a microfluidic platform for the past ten years. The related progress for biomedical purpose of Caenorhabditis elegans was reviewed. In particular, advances on microfluidic-based researches have been focused. The automated immobilization, behaviors, development, neurology, drug screening and gene screening of Caenorhabditis elegans were summarized. The prospects for the applications of microfluidic device are also discussed.

Abstract:Language development in preterm children is quite special. Behavioral studies found that preterm children were lagging behind their full term peers in areas such as vocabulary, syntax, and semantic verbal fluency. The effect of preterm birth on language development may last till early adulthood，and the degrees of such lags were influenced by biological and social factors. With the development of brain imaging, studies began to examine the brain development of premature children. Researchers have found group differences in white matter (WM) structures, subcortical gray matter (GM), and the cerebellum among preterm adolescents and their full term peers; yet the brain mechanism of language development in preterm children needs further researches to confirm. The paper describes the latest progress of behavior and neuron studies on preterm children's language development, thus to explore the law of language development and cognitive neuroscience mechanism in preterm children. Research suggests that behavior study and brain research should be combined to extend their advantages, thus to explore the mechanism of the language development of premature children, and to provide unique evidence of language acquirement of normal children.

Abstract:CrRLK1-L receptor-like kinases(RLKs) possess novel motifs with unknown functions in the extracellular domain and the typical Ser/Thr kinase domain in the intracellular region. CrRLK1-L subfamily spread broadly in angiosperm, however, no homologs of CrRLK1-L proteins exist in animals and microorganisms. CrRLK1-L RLKs express extensively in most of tissues, and mainly involve in signal exchanging between male and female gametophytes, the sensing of cell-wall-integrality and cell elongation control of the vegetative organs, and also in the response of cell to diverse biotic stress, etc. CrRLK1-L RLKs commonly localize on plasm-membrane, and their kinase activity is essential for their biological functions. Therefore, CrRLK1-L RLKs can function as cell surface sensor for extracellular signal and initiate a signal transduction that largely independent of the known phytohormone signaling pathways. Detailed study of CrRLK1-L RLKs gene functions will be helpful for our understanding the molecular mechanisms in the special biological processes in plant, especially in sexual reproduction in that optimal utilization will benefit the agriculture production in future.

Abstract:The 2011 Nobel Prize in Physiology or Medicine is announced. Bruce A. Beutler and Jules A. Hoffmann received one-half of the 2011 Nobel Prize in Physiology or Medicine, for their discoveries concerning the activation of innate immunity; the other half went to Ralph M. Steinman for his discovery of the dendritic cell and its role in adaptive immunity. Three winners' work has revolutionized our understanding of the immune system. And it has a bright prospect in the applied domain of anti-infections immunity, organ transplantation, and treatment of autoimmune diseases.

Abstract:In order to evaluate whether APP-Ab injection to hippocampus influence Morris water maze behavior and neuronal degeneration and to further investigate the potential mechanisms, rats were anaesthetized and fixed on a stereotaxic instrument and bilateral injection 1 μl of anti-APP antibody (10 g/L) was made using microsyringe. Meanwhile, NS or IgG-intrahippocampal-injected (1 μl; 10 g/L) rats served as vehicle controls. Antibodies were injected into the hippocampus (AP: -3.0; L and R: 2.0; V: 3.5 mm). The Morris water maze test was performed to test animals' learning and memory ability. After APP-Ab injection, cresyl violet and Fluoro-Jade B staining were used to investigate neuronal degeneration. Immunohistochemistry staining was used to detect MAP-2 and phosphorylated paxillin and tau distribution at hippocampus. APP-Ab injection to hippocampus could prolong the escape latency to find hidden platform and decreased the exploratory time and crossing numbers in the training quadrant. APP-Ab injection was also shown to cause neuronal cell death and degeneration by cresyl violet (CV) staining and Fluoro Jade-B (FJB) staining. Moreover, decreased MAP2 immunoreactivity, increased phosphorylated paxillin and phosphorylated tau immunostaining were observed in the pyramidal cells. It can be concluded that intrahippocampus injection of APP-Ab could induce cognitive deficits and neurodegenerative changes. APP-Ab injection also affected the distribution of MAP2, paxillin and tau protein.

Abstract:A series of recombinant viruses were engineered by gene replacements or gene insertions into an infectious cDNA clone of Tobacco necrosis virus A Chinese isolate (TNV-A^C). TNV-A^C-based virus induced gene silencing (VIGS) containing different structural arrangements of sequences targeting specific genes and variations in the positions of exogenous fragment insertions were evaluated along with temperature effects on inoculated plants. Replacement of the coat protein (CP) gene by exogenous gene fragments could not be applied to TNV-A^C because the chimeric TNV-A^C constructs were unable to induce silencing of corresponding endogenous gene and derivatives lacking the CP could not move systematically in Nicotiana benthamiana. We found that the optimal position for insertion of exogenous gene fragments was the region immediately behind the stop codon of the CP gene, and the maximum lengths of fragments that could be accommodated within virions was around 120 nt. An NbPDS fragment inserted into TNV-A^C as an inverted repeat produced a VIGS derivative that could silence endogenous NbPDS in N. benthamiana with extremely high efficiency. We also found that temperature could significantly affect gene silencing efficiency of inoculated plants and the stability of heterologous gene fragments. Plants grown at 18℃ exhibited substantially higher gene silencing efficiency compared with 24℃, and the duration of silencing persisted for more than 110 days. The TNV-A^C-based VIGS vectors were also able to induce efficient silencing of endogenous Su and ChlH genes in N. benthamiana. Taken together, our results show that TNV-A^C has the potential for development into a novel VIGS vector suitable for functional genomics of N. benthamiana.

Abstract:The neuraminidase (NA) of influenza virus is a receptor-destroying enzyme, removing sialic acid from carbohydrate chains attached to HA, and releasing the viruses from infected cells. The NA genes of the 2009 A H1N1 were sequenced, then the B-cell epitopes were predicted, screened and assessed based on immunoinformatics. Two peptides SE8 and RE6 (amino acid residues 168～175 and 428～433) of NA protein were synthesized and immunized to raise antisera in rabbits. Two antisera are capable of eliciting neutralizing antibodies against 2009 A H1N1 in the in vitro microneutralization assay, furthermore, the anti-releasing effects of hemagglutination existed in the antisera. Alignment with databases showed that the amino acid residues of two epitope peptides are highly conserved amongst the NA sequences of the strains isolated from the world. These findings indicate that SE8 and RE6 represents an attractive candidate for an effective synthetic peptide-based vaccine against 2009 A H1N1 viruses.

Abstract:Murine gammaherpesvirus-68 (MHV-68), a close homologue of Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus, can infect many cell lines efficiently and is able to infect laboratory mice, and has emerged as an excellent model for studying its human counterparts. The replication and transcriptional activator (RTA), mainly encoded by open read frame 50 (ORF50), is a conserved immediate-early protein in gammaherpesviruses. RTA initiates the viral lytic cycle by activating the expression of downstream viral lytic genes. Therefore，it is important to investigate the mechanism by which RTA regulates downstream gene expression. Two RTA dependent promoters of MHV-68 had previously been reported, however, the mechanism of the interaction between RTA and the promoters was not characterized. Our group has previously identified a new RTA responsive element (named RRE-B) in MHV-68 and characterized in detail the mechanisms involved. In the study, we first analyzed the MHV-68 genome and identified a sequence homologous to RRE-B in the promoter region of OFR9, and designated it as ORF9p-RRE. Dual luciferase reporter assay showed that RTA could activate the ORF9 promoter and this activation was dependent on ORF9p-RRE. Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation assay (ChIP) further demonstrated that RTA bound to ORF9p-RRE in vitro and in vivo. In summary, we showed here that the promoter of MHV-68 ORF9 is responsive to RTA activation through ORF9p-RRE. The study provides support to further investigate and understand the role of RTA in regulating viral gene expression and viral life cycle.

Abstract:To prepare human antibody to rabies virus with high neutralizing potency using ribosome display technology. The immunoglobulin heavy and light chain variable (VH, VL) genes were prepared with the peripheral blood lymphocytes from three volunteers immunized with rabies virus vaccine by PCR. The genes encoding scFv fragments were prepared by randomly combining VH and VL genes by SOE PCR. Rabies virus glycoprotein (RVGp) specific scFv genes were selected over five cycles of ribosome display. The isolated scFv genes were cloned into pET22b(+)/BL21(DE3), from which soluble scFv fragments were prepared. The expressed products of selected clones were analyzed by ELISA for isolating the positive clones. To improve the stability of obtained scFvs, VH-Lc-VK were constructed and characterized. A human scFv gene library with 6.2×10¹² numbers used for ribosome display was constructed. Among the 180 selected clones, four clones RB24, RB71, RB109 and RB156 which exhibited the highest ELISA signals were isolated. The analysis of their sequences showed that they were new human immunoglobulin V genes to RVGp. And the reconstructed VH-Lc-VK antibody fragments can recognize RVGp specifically and antagonize the cytolytic effect of rabies virus on Vero cells. The prepared VH-Lc-VK fragments to RVGp will be useful for preparing engineering antibodies with high affinity against rabies virus. Ribosome display is a rapid means of generating fully human antibody fragments in vitro.

Abstract:Fibroblast growth factor(FGF)-21 is a new member of the fibroblast growth family. Recent studies show that FGF-21 is a novel regulator for glycemic control in various type 2 DM models. However, its potential to treat type 1 diabetes mellitus has not been explored. An evaluation of the function and mechanism of FGF-21 was made in hepatic glucose metabolism of type 1 diabetes mouse model with slow-onset of the impaired glucose tolerance induced by STZ. The mice were administrated with FGF-21, the mRNA expression of GLUT1 and 4 in the liver were detected by the real-time PCR and glycogen synthesis was examined by the anthrone method. The results showed that FGF-21could lower the blood glucose level of the type 1 diabetes model in a dose-depend manner. The blood glucose level of the experimental animals maintained at normal level by injection of the FGF-21 once a day. For the first time, the synergistic effect of FGF-21 with insulin in the animal model was found. Unlike insulin that stimulated GLUT4 expression, FGF-21 could increase the mRNA expression of GLUT1, co-treatment (CT) with insulin and FGF-21 could increase the mRNA expression of both GLUT1 and 4. After long term treatment, the same as insulin, FGF-21 also stimulated glycogen synthesis of the model mice. The results suggested that FGF-21 can regulate glucose metabolism through GLUT1 expression, stimulate glycogen synthesis and improve glucose metabolism in type 1 diabetes model. Moreover, CT administration could increase both GLUT1 and 4 expressions. These data provide the first evidence for clinic application of FGF-21 for treatment of type 1 diabetes patients.

Abstract:The effect of silver colloid concentration was explored for fastest delivery of silver nanoparticles into living C666 cells (a human nasopharyngeal carcinoma cell line) for intracellular surface enhanced raman scattering (SERS) by Electroporation. SERS spectra, the integrated SERS intensity and the reproducibility of spectrum were compared in 6 electroporation experiments of the same electric pulse parameters but different concentration of silver colloid. The silver nanoparticle concentration is best under the condition of 500 μl electroporation buffer containing 50 μl silver colloid using 875 V/cm, 1 ms rectangular electric pulses for electroporation twice. Increasing the concentration of silver nanoparticle could improve the SERS intensity while decrease the reproducibility of the spectrum. Moreover, the observed SERS bands of living C666 cell were tentatively assigned. This work is promising for the practical application of SERS technology, such as real time detection and analysis of the biochemical substance in living cells.

Abstract:To investigate the effect of 50 Hz 1.8 mT sinusoidal, triangular, serrate and square wave electromagnetic field(EMFs) on proliferation, differentiation and gene expression of osteoblasts in vitro, the newborn rat calvarial osteoblasts were isolated by enzyme digestion and randomly divided into 5 groups after one passage．The EMFs treatment group is exposed to 50 Hz 1.8 mT sinusoidal, triangular, serrate and square wave of EMFs and controls without EMFs treatment. The osteoblasts were exposed to the EMFs for 30 min/(time·day). The cells were observed under the contrast phase microscope each day. The EMFs groups were arranged in spiral appearance after 4～8 days of the EMFs treatment. MTT result showed that sinusoidal wave significantly inhibited osteoblast proliferation, whereas square wave enhanced the osteoblast proliferation. The ALP activity in triangular and sinusoidal wave groups significantly increased (P < 0.01) compared with that in control. The number of calcified nodules, ALP stained result and Collagen-Ⅰimmunohistochemistry tested result are according to the result of ALP activity. There was a common result that sinusoidal and triangular wave significantly increased the Igf, Opg and Runx-2 mRNA expression. The four different wave of EMFs at 50 Hz 1.8 mT enhances the osteoblasts maturation, mineralization and Opg and Runx-2 mRNA expression of the osteoblasts, while sinusoidal and triangular have the strongest effect.