Abstract:The pandemic outbreak of influenza has been started from Mexico in 2009 to 70 countries during 2 months. On 11th of June , WHO announced influenza pandemic alert level rose to the highest level 6, which means the first influenza pandemic in 21st century is coming. Till 6th of July, 94 512 confirmed cases from more than 120 countries and areas were reported, including 429 cases were died. The genetic fragment of swine, poultry sources and human influenza viruses are contained in this strain, A/H1N1 influenza virus, of the pandemic. It is of great significance of studying the genetic reassortment, evolution and its biological characteristics of this virus strain to prevent and control the pandemic. At present, the genetic evolution of strain has been identified, and the potential biological characteristics have been analyzed by genetic traits, however, clinical manifestation should be further concerned, and the tendency of influenza pandemic and genetic changes need to be monitored closely. The complexity of influenza virus ecosystems, mutation of genome, and easy to preserve in “Nature Gene Pool” and reassortment, make the influenza pandemic inevitable. We should face the threat of influenza pandemic, enhance the surveillance of influenza virus in ecosystems, strengthen the epidemiological investigation, develop the vaccines and drugs, and establish an effective public health security system, in order to reduce the destruction of the influenza pandemic.

Abstract:Over the past 28 months, the induced pluripotent stem cells (iPS cells, with characteristics identical to those of embryonic stem cells (ES cells)) directly in vitro reprogrammed from nonembryonic cells and tissues have captured great attentions in both scientific community and general public. Somatic reprogramming, dedifferentiation and the resource of pluripotent stem cells become the research hot points of stem cells and developmental biology. Person-, patient- and disease-specific iPS cells could be very useful in regenerative medicine and drug discovery, etc. iPS cell history, the several key steps in generating iPS cells (including approaches to gene delivery, combined uses of defined factors and small molecules to induce somatic cells into iPS cells, and choice of somatic cell types), patient- and disease-specific iPS cells, in vivo and in vitro differentiation of iPS cells, and the possibility of genetic modification-free iPS cells were discussed.

Abstract:Pathogen inflection could induce specific immune response which will results in immunopathology when overreacted. Severe complications arising from influenza virus infection (pandemic influenza or the highly pathogenic avian H5N1viruses) are often associated with rapid, massive inflammatory cell infiltration, acute respiratory distress, reactive hemophagocytosis and multiple organ involvement. Histological and pathological studies show that a critical role for an excessive host response in mediating this pathology. Generally, the same imflammatory factors mediating tissue damage during the anti-influenza immune response are also essential for efficient elimination of virus. It was discussed how various effector arms of immune system can act deleteriously to initiate or exacerbate pathological damage in this viral infection. It will be helpful to understand the protective mechanism of influenza virus infection and a significant challenge in the design of harmless yet effective therapeutic strategies for tackling influenza virus.

Abstract:Copy number variations (CNVs) refer as a DNA segment that is 1 kb or larger and is presented at a variable copy number in comparison with a reference genome. Classes of CNVs include insertions, deletions, duplications and their complex combinations. Because they widely distributed in the genome with some important characteristics, such as heritable, relative stable and heterogeneity, CNVs are considered as novel genomic polymorphism markers. And the alteration of gene dosage which resulted from CNVs could change phenotype, so a novel CNV genome-wide association analysis (CNV-GWAS) strategy appeared recently and began to used for identifying susceptible genes of complex diseases. It was approved that it could complement the tranditional genome-wide association studies based on single nucleotide polymorphisms. Therefore, genomic structure variances are favorable for revealing the molecular mechanisms and genetic foundation of complex diseases.

Abstract:Previous studies suggest that NGAL (neutrophil gelatinase-associated lipocalin) is involved in the transformation and development of esophageal carcinoma. Alteration of NGAL expression can trigger the change of cellular morphology in esophageal carcinoma cells. However, the mechanisms remain unclear. To get a better understanding of NGAL function in esophageal carcinoma, NGAL protein was expressed in methylotrophic yeast, Pichia pastoris, and purified by chromatography. EC1.71 cells expressed high levels of NGALR (NGAL receptor) and EC109 cells expressed low levels of NGALR were used as cells model. The trafficking and the possible function of NGAL protein were then analyzed in the esophageal carcinoma cells. The results showed that 5-FAM-labeled recombinant NGAL protein could internalize into the EC1.71 and EC109 cells. Furthermore, the internalized NGAL protein could induce the alteration of cellular morphology, resulting in generation of autophagosome, transcriptional up-regulation of genes associated with autophagy and increase of phospho-ERK1/2 (p-ERK1/2). Interestingly, the treatment with the NGAL protein did not affect the intracellular iron level. These data indicate that induced autophagy by exogenous NGAL protein is a mechanism that internalized NGAL plays important roles in esophageal carcinoma cells, independent with NGAL-mediated iron transport process, while ERK1/2 signal pathway is involved in activation of autophagy by exogenous NGAL protein.

Abstract:Sensory substitution has been reported in recent brain imaging studies with blind people and others with sensory deficits so that sensory cortical regions traditionally considered unimodal respond to stimulation from other sensory modalities. Similar effects are also found for normal sighted people with sensory deprivation (blindfolded), indicative of pre-existing neuronal pathways for multiple sensory interactions. Such pathways are considered latent in that they only become unmasked or potentiated in the event of sensory deafferentation, although whether sensory deprivation is necessary to expose these pathways is unclear due to inconclusive evidence. With a relatively strong power in experimental design, visual cortical activation was observed when normal sighted participants (not blindfolded) judged whether auditorily-presented nouns referred to artificial or natural objects. The results suggest the above mentioned pathways can be exposed without sensory deafferentation and therefore are not totally latent. This establishes a boundary condition constraining theoretical models for the neural basis of multiple sensory interactions.

Abstract:The gamma-secretase complex mediates the final cleavage of APP to generate the principal component of amyloid plaques in the brains of Alzheimer’s disease patients. Four integral membrane proteins (PS, NCT, PEN-2 and APH-1) are essential and sufficient for gamma-secretase activity. To identify the promoter of human nicastrin gene (NCT), its 5′-flanking region has been characterized and a 270 bp fragment containing the TSS(transcription start site) for the promoter activity has been identified. EMSA assays confirmed that all four AP-1 binding sites and two NFAT sites in the NCT promoter region were able to bind relative transcription factors in vitro. Mutations, as well as treatment with PDTC, which adjust the regulatory effect of AP-1 and NFAT, altered NCT promoter activity in both HeLa cells and rat cortical neurons. The results demonstrated that AP-1 and NFAT are involved in the regulation of hNCT transcription and suggest that balanced activation of AP-1 and NFAT ensures a strict temporal and tissue-specific control of NCT transcription.

Abstract:In order to investigate if there exists interaction between mitogen-activated protein kinase (MAPK) and signal transducer and activator of transcription 3 (STAT3) protein, and how the interaction regulates tumor necrosis factor-α (TNF-α) transcription activity, the human p38 and extracellular-signal regulated protein kinase 2 (ERK2) genes were amplified from human flag-p38 and flag-ERK2 by polymerase chain reaction (PCR) and cloned into pcDNA3-HA. Protein expression of the plasmids was examined by Western blotting. Co-immunoprecipitation was used to identify if there exists interaction between MAPK and STAT3 proteins. If the interaction was approved to be true, report gene system was applied to find how the interaction affect transcriptional expression of TNF-α. After STAT3 pathway was inhibited by RNA interfering, the action on TNF-α activity was determined. The results of DNA sequencing and enzyme digestion showed that the cloned p38 and ERK2 genes were correct, to be 1 080 bp or so. p38 and ERK2 proteins were expressed in 293T cell to be approximately 40 ku. Co-immunoprecipitation data showed that p38 and ERK2 proteins integrated with STAT3 protein in vivo. TNF-α reporter gene activity results found that protein complex of p38-STAT3 and ERK2-STAT3 coordinately increased TNF-α activity. After STAT3 was interfered, the TNF-α activity markedly decreased. These data indicated that there exists interaction between p38 and STAT3 protein, ERK2 and STAT3 protein. The complex of the proteins can coordinately regulate TNF-α expression. After interfereing STAT3 pathway, the coordinated action on TNF-α transcription activity might be obviously reduced.

Abstract:hCLEC-2(human C-type lectin-like receptor-2) is a novel identified typeⅡ transmembrane receptor protein. It is found to be closely associated with virus infection, platelet aggregation, tumor metastasis and signal transduction. To study the biological function of hCLEC-2 and signaling pathways it is involved in, a specific antibody against the extracellular domain of hCLEC-2 was prepared. Then, a pET23b-CRD recombinant plasmid was constructed and transformed into BL21 for protein expression. After the induction with IPTG, hCLEC-2-CRD-His was found to be expressed in the inclusion body. The fusion protein in inclusion body was dissolved in 6 mol/L guanidine hydrochloride, purified using Ni-agarose, refolded and dialyzed against PBS. The purified hCLEC-2-CRD-His was used as an antigen to prepare polyclonal antiserum in rabbits, which was subjected to affinity purification with Protein G Sepharose. The specificity of anti-CRD antibody was identified by Western blot analysis of the ectopic expressed GFP-hCLEC-2 and GFP-CRD in comparison with that of GFP antibody. By using this specific antibody, the endogenous hCLEC-2 was revealed to be down-regulated in human monocyte THP-1 cells treated with PMA and IL-4. This preliminary result suggests a correlation between the expression of hCLEC-2 and the differentiation of monocyte. Collectively, the studies here provide a favorable tool for further investigation of hCLEC-2 associated biological functions.

Abstract:In the past 30 years, the research and application of transgenic technology in gene expression of animal had become a noticeable advancement in experimental biology and applied biology. Microinjection，retrovirus mediated method and embryonic stem cells method were all the traditional methods of producing transgenic animals. But these methods had defects and limited application in the research of transgenic animals in the future. Exogenous gene was transferred into germ cells of male mice, in order to study efficiency of transgenic mice in different spermiogenesis stages. The green fluorescence protein expression plasmid(pIRES2-EGFP) and liposomes were mixed and injected into male mouse testis and epididymis. Then the intratesticular mice were mated with female at day 7, 16, 30 and 42 after infection. Polymerase chain reaction(PCR) and Southern blot were applied to identify transgenic mice. The positive ratio was 6.82%, 0, 56.86%, 42.86% by PCR analysis and 6.82%, 0, 47.06%, 34.69% by Southern blot analysis . The transgenic mice showed green fluorescence in fluorescence imager and fluorescent microscope under EGFP excitation light (488 nm). Through comparing efficiency of transgenic mice in different spermiogenesis stages, it could provided an important theoretic foundation for efficient production of transgenic animals by testis mediated gene transfer.

Abstract:With thousands of sequenced 16 S rRNA genes available, and advancements in oligonucleotide microarray technology, the detection of microorganisms in microbial communities consisting of hundreds of species may be possible. The existing algorithms developed for sequence-specific probe design are not suitable for applications in large-scale bacteria detection due to the lack of coverage, flexibility and efficiency. Many other strategies developed for group-specific probe design focus on how to find a unique group-specific probe that can specifically detect all target sequences of a group. Unique group-specific probe for each group can not always be found. Hence, it is necessary to design non-unique probes. Each probe can specifically detect target sequences of a different subgroup. Combination of multiple probes can achieve higher coverage. However, it is a time-consuming task to evaluate all possible combinations. A feasible algorithm using relative entropy and genetic algorithm (GA) to design group-specific non-unique probes was presented.

Abstract:Based on the characteristic of nucleotide distribution in nucleosome positioning and inhibiting sequences, the method of Increment of Diversity with Quadratic Discriminant (IDQD) was applied to the classification of these two types of sequences. The mean area under ROC curve archives 0.958. By using this model, the nucleosome formation potential was analyzed in the regions around the splice sites (GT/AG). The results show that coding regions have a high potential to form the nucleosome and the primary RNA transcripts are rigid, while DNA sequences corresponding to the splice sites and their adjacent intron regions tend to be nucleosome free and the primary transcripts from these regions are relative flexible. Moreover, the negative correlation between nucleosome positioning/inhibiting of DNA sequences and RNA flexibility/rigidity is demonstrated around the splice sites, providing a mechanism for understanding the correlation between the nucleosome positioning of DNA and the splicing of transcribed RNA sequences.

Abstract:Many previous studies have shown that auditory cortical neurons are sensitive to sound spatial information, however, the mechanism of sound spatial coding is still not fully understood. Until now, detail studies on sound spatial coding have not been reported in the rat primary auditory cortex. Using electrophysiological technique, spatial response areas of 151 neurons in the rat primary auditory cortex were investigated. The relationships between spike counts and average first-spike latencies in the spatial response areas were analyzed. The results showed that, the majority (52.32%) of cortical neurons exhibited contralateral preference in the frontal auditory space whereas other neurons exhibited ipsilateral preference (18.54%) and midline preference (18.54%), and only a few neurons were included in the category of omnidirection (3.31%) and complex (7.28%). For the majority of cortical neurons, the arithmetic center of the preferred spatial area were distributed in the up and middle portion of the contralateral space relative to the recording side. Most neurons responded strongly to stimuli from their preferred space with shorter average first-spike latencies, and responded weakly to stimuli from non-preferred space with longer average latencies. In the spatial response area, the spike counts were negatively correlated with average first-spike latencies. The auditory cortex might use the information of both spike counts and average response time to code sound spatial information.

Abstract:To date, deep brain stimulation (DBS) is the most effective clinical treatment for Parkinson’s disease (PD). However the mechanism of action of DBS is still under investigation which hampers the further improvement of DBS and study of the pathogenesis of PD. In past decade, kinds of experiment apparatuses and means were applied to explore the mechanism of action of high frequency stimulation (HFS) on target nucleus, such as subthalamic nucleus (STN). Among these, the change of neural spiking activity of STN during ongoing HFS can reflect precisely the influence of HFS on nucleus, thus, is the better method to determine the mechanism. However, large stimulus artifacts (SA) have greatly restrained the application of this technique. An online filter method based on template subtraction was developed and evaluated for the removal of stimulus artifacts and their harmonics from extracellular neural recording. The data presented here illustrated that although this algorithm is simple and low computation intensive, it can well recover spikes for low SNR signal, even spikes completely submerged by SA. Utilizing this effective algorithm, the relation between the type of neural response and the frequency and amplitude of stimulation was determined; meanwhile effect of HFS with clinically effective parameters on mean firing rats was studied and analyzed. Experiment results illustrated that the percentage of STN neurons which showed inhibition to HFS rise with the increment of frequency and amplitude of stimulus. In addition, Experiment results showed that the change of mean firing rat may not be directly involved with the symptoms of Parkinson’s disease (PD), and the increase of burst neural activity should more likely be the neurophysiologic basis of PD. Furthermore, it was found that inhibition of spontaneous neural activity leading to reduction of mean firing rate and burst activity may be the part of mechanism of action of deep brain stimulation.

Abstract:Poly aspartic acid or its salts is a kind of newly innocuous, environmental friendly biodegradable polymer, recognized as green material, and widely applied in such areas as agriculture, medicine, commodity, water treatment. In order to prepare new poly aspartic acid-metronidazole (PASP-MTI) nanoprodrug, observe its trichomonacidal effect in vitro, and explore its probable mechanism, the nanoprodrug PASP-MTI was synthesized by DL-aspartic acid and metronidazole in a relatively mild condition. Its structural characteristics were attested by means of infrared spectrum (IR), ¹H nuclear magnetic resonance (¹H-NMR), transmission electron microscope(TEM) etc measurements. The release behavior of metronidazole in PASP-MTI nanoprodrug was investigated by the dialysis method. The MTT assay was employed to test PASP-MTI’s trichomonacidal effect in vitro and the apoptotic-like processes of trichomonas were detected by fluorescence microscope and flow cytometer (FCM). The IR and ¹H-NMR showed that the metronidazole was conjugated to poly aspartic acid high molecular chains by ester bonds. Synthesized PASP-MTI nanoprodrug was globular and had a mean diameter 404.8 nm with favorable dispersity. The drug loading was about 30% and the cumulative release profile was delayed (about 38.01% of MTI loading in the PASP-MTI nanoprodrug was released within the first 24 h and 86.64% in 30 d, while the release of free metronidazole was almost complete by 24 h). The trichomonacidal activity of PASP-MTI nanoprodrug increased remarkably{IC₅₀ of PASP-MTI and free metronidazole was 1.22 mg/L and 2.85 mg/L, respectively}. Observed by fluorescence microscope, the nuclei of trichomonas presented a series of changes similar to apoptosis such as chromatin agglutination, nuclear fragmentation, etc. The FCM study indicated that the apoptotic rate in the PASP-MTI group was the highest of all groups, up to 35.69%. It is concluded that PASP can be effectively used in drug delivery systems, the synthetic novel poly aspartic acid-metronidazole nanoprodrug has higher drug loading, better the role of prolonged-release and higher trichomonacidal effect in vitro. Its mechanism may be related with enhancing the polymer prodrug’s internalization and inducing the apoptosis of trichomonas.

Abstract:Temporins are a kind of small, hydrophobic and C-terminus amidated antimicrobial peptides from Rana species. They are effective against bacteria, fungi, yeast, protozoa and viruses. Two novel temporins named as temporin-La(LLRHVVKILEKYL_amide) and temporin-Lb(LFRHVVKIFEKYL_amide) were cloned from Lithobates catesbeianus. Synthetic peptides of temporin-La and temporin-Lb showed strong antimicrobial activities against bacteria tested, especially Gram-positive bacteria. Besides, temporin-La showed no haemolytic activity to rabbit erythrocytes at the concentration of 250 mg/L while temporin-Lb showed weak haemolytic activity(LC₅₀ ≈ 230 μmol/L). Transmission electron microscopy showed that temporin-La and temporin-Lb induced different effects on bacterial structure of Staphylococcus aureus.

Abstract:To construct expression vectors of small hairpin RNA aimed at N-acetylglucosaminyltransferase Ⅴ(GnT-Ⅴ) gene, and to investigate effects of GnT-Ⅴ shRNA on proliferation, adhesion, migration and invasion of LoVo cell line. siRNAs were designed according to the coding sequence of GnT-Ⅴ gene, shRNA expression vectors were constructed and transfected into LoVo cell line, cell lines which stably expressed low level of GnT-Ⅴwere established by G418 screening. The mRNA and protein expression of GnT-Ⅴwere measured by semi- quantitative reverse transcription polymerase chain reaction(RT-PCR) and Western blot analysis, respectively. The effects of pGPU6/GFP/Neo GnT-Ⅴ shRNA vectors on proliferation, adhesion, migration and invasion of LoVo cell line were evaluated by CCK-8 assay, heterogenous adhesion, wound closure assay, chemotactic migration and cell invasive experiment, respectively. GnT-Ⅴ shRNA expression plasmid was constructed successfully and pGPU6/GFP/Neo GnT-Ⅴ shRNA down-regulated expression of GnT-Ⅴ dramatically in LoVo cell. Expression of LoVo GnT-Ⅴ/1564 and LoVo GnT-Ⅴ/2224 dereased by 82%, 71.5% respectively at mRNA level, and 68%, 56% respectively at protein level. The more effective interfered cell line, LoVo GnT-Ⅴ/1564, was chosen to do further experiment. CCK-8 assay showed proliferation of LoVo GnT-Ⅴ/1564 was suppressed obviously, compared to proliferation of negative control group cell (P < 0.001),especially in 72 hours; down-regulation of GnT-Ⅴ expression can enhance adhesive ability(t=-3.357, P < 0.01) and inhibit chemotactic migration(t=44.051, P < 0.001) in LoVo cell line; quantitative analysis of the wound closure assay also indicated that down-regulation of GnT-Ⅴ expression can significantly prolong wound heal hours of LoVo cell line; cell invasive experiment using Matrigel gel showed that penetrative cell numbers of LoVo GnT-Ⅴ/1564 and LoVo GnT-Ⅴ/NC were 5.10±1.25 and 39.55±2.16 respectively, penetrative cell numbers of LoVo GnT-Ⅴ/1564 cell was reduced obviously, compared to negative control group cell (t=61.626, P < 0.001). The shRNA aimed at GnT-Ⅴ gene could reduce the expression of GnT-Ⅴ both in the level of mRNA and protein. By this way, it can inhibit proliferation, migration and invasion of LoVo cell line, so the sequence of RNA interference against GnT-Ⅴ may be a valid target to treat colorectal cancer.

Abstract:The ripeness of tomato is regulated by multi-genes. Transcripts of mRNA extracted from tomato at breaker stage were normalized, and cDNA with pTRV was constructed after reverse transcription. In order to construct a normalized cDNA silencing library, a novel procedure was adopted to reduce the abundant copy number and increase the rare copy number. Then the normalized cDNA was put into pTRV vector to construct a cDNA library and this cDNA library was screened with the technology of virus induced gene silencing. PDS, which was related about synthesis of lycopene, was used as marker gene to establish the model of screening. Among one hundred mixed agrobateria, PDS was successfully screened in the first stage of model establishment.