Developing a microfluidic-based system to quantify cell capture efficiency

Micro-fabrication technology has substantial potential for identifying molecular markers expressed on the surfaces of tissue cells and viruses. It has been found in several conceptual prototypes that cells with such markers are able to be captured by their antibodies immobilized on microchannel substrates and unbound cells are flushed out by a driven flow. The feasibility and reliability of such a microfluidic-based assay, however, remains to be further tested. In the current work, we developed a microfluidic-based system consisting of a microfluidic chip, an image grabbing unit, data acquisition and analysis software, as well as a supporting base. Specific binding of CD59-expressed or BSA-coupled human red blood cells (RBCs) to anti-CD59 or anti-BSA antibody-immobilized chip surfaces was quantified by capture efficiency and by the fraction of bound cells. Impacts of respective flow rate, cell concentration, antibody concentration and site density were tested systematically. The measured data indicated that the assay was robust. The robustness was further confirmed by capture efficiencies measured from an independent ELISA-based cell binding assay. These results demonstrated that the system developed provided a new platform to effectively quantify cellular surface markers effectively, which promoted the potential applications in both biological studies and clinical diagnoses. Supported by the National Key Basic Research Program of China (Grant No. 2006CB910303), National Natural Science Foundation of China (Grant Nos. 30730032 and 10332060), National High-Tech Research and Development Program of China (Grant No. 2007AA02Z306) and Chinese Academy of Sciences Grant (Grant No.2005-1-16)     

Interspecies transmission and host restriction of avian H5N1 influenza virus

Long-term endemicity of avian H5N1 influenza virus in poultry and continuous sporadic human infections in several countries has raised the concern of another potential pandemic influenza. Suspicion of the avian origin of the previous pandemics results in the close investigation of the mechanism of interspecies transmission. Entry and fusion is the first step for the H5N1 influenza virus to get into the host cells affecting the host ranges. Therefore receptor usage study has been a major focus for the last few years. We now know the difference of the sialic acid structures and distributions in different species, even in the different parts of the same host. Many host factors interacting with the influenza virus component proteins have been identified and their role in the host range expansion and interspecies transmission is under detailed scrutiny. Here we review current progress in the receptor usage and host factors. Supported by the National Basic Research Program of China (Grant Nos. 2005CB523001, 2005CB523002), National Key Technologies Research & Development Program (Grant 2006BAD06A01/2006BAD06A04); US National Institutes of Health (NIH) (Grant 3 U19 AI051915-05S1), the National Natural Science Foundation of China (Grant 30599434). GAO FG is a distinguished young investigator of the NSFC (Grant No. 30525010).     

Characterization of GLUT4-containing vesicles in 3T3-L1 adipocytes by total internal reflection fluorescence microscopy

Insulin-responsive GLUT4 (glucose transporter 4) translocation plays a major role in regulating glucose uptake in adipose tissue and muscle. Whether or not there is a specialized secretory GSV (GLUT4 storage vesicle) pool, and more importantly how GSVs are translocated to the PM (plasma membrane) under insulin stimulation is still under debate. In the present study, we systematically analyzed the dynamics of a large number of single GLUT4-containing vesicles in 3T3-L1 adipocytes by TIRFM (total internal reflection fluorescence microscopy). We found that GLUT4-containing vesicles can be classified into three groups according to their mobility, namely vertical, stable, and lateral GLUT4-containing vesicles. Among these groups, vertical GLUT4-containing vesicles exclude transferrin receptors and move towards the PM specifically in response to insulin stimulation, while stable and lateral GLUT4-containing vesicles contain transferrin receptors and show no insulin responsiveness. These data demonstrate that vertical GLUT4-containing vesicles correspond to specialized secretory GSVs, which approach the PM directly and bypass the constitutive recycling pathway. Contributed equally to this work Supported by the National Natural Science Foundation of China (Grant Nos. 30470448 and 30130230), the National key Basic Research Program of China (Grant No. 2004CB720000), the Knowledge Innovative Program of The Chinese Academy of Sciences (Grant Nos. KSCX2-SW-224 and Y2004018), the Li Foundation and the Sinogerman Scientific Center.     

Progress in the evaluation of transgenic fish for possi-ble ecological risk and its containment strategies

Genetically improved transgenic fish possess many beneficial economic traits; however, the commercial aquaculture of transgenic fish has not been performed till date. One of the major reasons for this is the possible ecological risk associated with the escape or release of the transgenic fish. Using a growth hormone transgenic fish with rapid growth characteristics as a subject, this paper analyzes the following: the essence of the potential ecological risks posed by transgenic fish; ecological risk in the current situation due to transgenic fish via one-factor phenotypic and fitness analysis, and mathematical model deduction. Then, it expounds new ideas and the latest findings using an artificially simulated ecosystem for the evaluation of the ecological risks posed by transgenic fish. Further, the study comments on the strategies and principles of controlling these ecological risks by using a triploid approach. Based on these results, we propose that ecological risk evaluation and prevention strategies are indispensable important components and should be accompanied with breeding research in order to provide enlightments for transgenic fish breeding, evaluation of the ecological risks posed by transgenic fish, and development of containment strategies against the risks. Supported by the Development Plan of the State Key Fundamental Research of China (Grant Nos. 2007CB109205 and 2007CB109206), the National Natural Science Foundation of China (Grant No. 30430540), and the ‘863’ High Technology Project (Grant No. 2006AA10Z141)     

tRNAs as regulators in gene expression

Transfer RNAs (tRNAs) hold a central place in protein synthesis by interpreting the genetic information stored in DNA into the amino acid sequence of protein, thus functioning as “adaptor” molecules. In recent years, however, various studies have shown that tRNAs have additional functions beyond participating in protein synthesis. When suffering from certain nutritional stresses, tRNAs change the level of aminoacylation to became uncharged, and these uncharged tRNAs act as effector molecules to regulate global gene expression, so that the stressed organism copes with the adverse environmental stresses. In budding yeast and certain mammalian cells, the retrograde movement of mature tRNAs from cytoplasm to nucleus serves as a mechanism for the surveillance system within the nucleus to continue monitoring the integrity of tRNAs. On the other hand, this retrograde action effectively reduces the global protein synthesis level under conditions of nutritional starvation. Quite recently, various publications have shown that tRNAs are not stable molecules in an absolute sense. Under certain physiological or environmental stresses, they are specifically cleaved into fragments of different lengths in the anticodon loop or anticodon left arm. These cleavages are not a meaningless random degradation phenomenon. Instead, a novel class of signal molecules such as tRNA halves or sitRNAs may be produced, which are closely correlated with the modulation of global gene expression. Investigation of the regulatory functions of tRNAs is a frontier, which seeks to reveal the structural and functional diversity of tRNAs as well as their vital functions during the expression of genetic information. Supported by National Natural Science Foundation of China (Grant Nos. 30870530 and 30570398) and the National Key Basic Research Program of China (Grant No. 2005CB724600)     

Rapid characterization of the binding property of HtrA2/Omi PDZ domain by validation screening of PDZ ligand library

HtrA2/Omi is a mammalian mitochondrial serine protease, and was found to have dual roles in mammalian cells, not only acting as an apoptosis-inducing protein but also maintaining mitochondrial homeostasis. PDZ domain is one of the most important protein-protein interaction modules and is involved in a variety of important cellular functions, such as signal transduction, degradation of proteins,and formation of cytoskeleton. Recently, it was reported that the PDZ domain of HtrA2/Omi might regulate proteolytic activity through its interactions with ligand proteins. In this study, we rapidly characterized the binding properties of HtrA2/Omi PDZ domain by validation screening of the PDZ ligand library with yeast two-hybrid approach. Then, we predicted its novel ligand proteins in human proteome and reconfirmed them in the yeast two-hybrid system. Finally, we analyzed the smallest networks bordered by the shortest path length between the protein pairs of novel interactions to evaluate the confidence of the identified interactions. The results revealed some novel binding properties of HtrA2/Omi PDZ domain. Besides the reported Class Ⅱ PDZ motif, it also binds to Class Ⅰ and Class Ⅲ motifs, and exhibits restricted variability at P-3, which means that the P-3 residue is selected according to the composition of the last three residues. Seven novel ligand proteins of HtrA2/Omi PDZ domain were discovered, providing significant clues for further clarifying the roles of HtrA2/Omi.Moreover, this study proves the high efficiency and practicability of the newly developed validation screening of candidate ligand library method for binding property characterization of peptide-binding domains.     

Novel strategies to mine alcoholism-related haplotypes and genes by combining existing knowledge framework

High-throughout single nucleotide polymorphism detection technology and the existing knowledge provide strong support for mining the disease-related haplotypes and genes. In this study, first, we apply four kinds of haplotype identification methods (Confidence Intervals, Four Gamete Tests, Solid Spine of LD and fusing method of haplotype block) into high-throughout SNP genotype data to identify blocks, then use cluster analysis to verify the effectiveness of the four methods, and select the alcoholism-related SNP haplotypes through risk analysis. Second, we establish a mapping from haplotypes to alcoholism-related genes. Third, we inquire NCBI SNP and gene databases to locate the blocks and identify the candidate genes. In the end, we make gene function annotation by KEGG, Biocarta, and GO database. We find 159 haplotype blocks, which relate to the alcoholism most possibly on chromosome 1∼22, including 227 haplotypes, of which 102 SNP haplotypes may increase the risk of alcoholism. We get 121 alcoholism-related genes and verify their reliability by the functional annotation of biology. In a word, we not only can handle the SNP data easily, but also can locate the disease-related genes precisely by combining our novel strategies of mining alcoholism-related haplotypes and genes with existing knowledge framework. Supported by the National Natural Science Foundation of China (Grant Nos. 30570424, 60601010 and 30600367), the National High-Tech Research and Development Program of China, (Grant No.2007AA02Z329), the Key Science and Technology Program of Heilongjiang Province(Grant No.GB03C602-4), Natural Science Foundation of Heilongjiang Province (Grant No. F2008-02), Youth Science Foundation of Harbin Medical University (Grant No. 060045) and Science Foundation of Heilongjiang Province Education Department (Grant Nos. 11531113 and 1152hq28).     

MicroRNA in cell differentiation and development

The regulation of gene expression by microRNAs (miRNAs) is a recently discovered pattern of gene regulation in animals and plants. MiRNAs have been implicated in various aspects of animal development and cell differentiation, such as early embryonic development, neuronal development, muscle development, and lymphocyte development, by the analysis of genetic deletions of individual miRNAs in mammals. These studies show that miRNAs are key regulators in animal development and are potential causes of human diseases. Here we review some recent discoveries about the functions of miRNAs in cell differentiation and development. Supported by National Key Basic Research and Development Program of China (Grant No. 2005CB724602) and Knowledge Innovation Project of the Chinese Academy of Sciences (Grant Nos. KSCX2-YW-R-096, KSCX1-YW-R-64)     

Co-expression with RadA and the characterization of stRad55B, a RadA paralog from the hyperthermophilic crenarchaea Sulfolobus tokodaii

ST0838 (designed stRad55B) is one of the four RadA paralogs (or Rad55 homologues) in the genome of the hyperthermophilic crenarchaeon Sulfolobus tokodaii. The gene is induced by UV irradiation, suggesting that it is involved in DNA recombinational repair in this organism. However, this protein could not be expressed normally in vitro. In this study, thermostable and soluble stRad55B was obtained by co-expression with S. tokodaii RadA (stRadA) in E. coli, and the enzymatic properties were examined. It was found that stRad55B bound ssDNA preferentially and had a very weak ATPase activity that was not stimulated by DNA. The recombinant protein inhibited the strand exchange activity promoted by stRadA, indicating that stRad55B might be an inhibitor to the homologous recombination in this archaeon. The results will be helpful for further functional and interaction analysis of RadA paralogs and for the understanding of the mechanism of recombinational repair in archaea. Supported by the National Basic Research Program of China (Grant No. 2004CB719604) and National Natural Science Foundation of China (Grant Nos. 30470386 and 30700011)     

Effect of cell cycle inhibitor p19ARF on senescence of human diploid cell

To investigate the effect of cell cycle inhibitor p19ARF on replicative senescence of human diploid cell, recombinant p19ARF eukaryotic expression vector was constructed and p19ARF gene was transfected into human diploid fibroblasts (WI-38 cells) by liposome-mediated transfection for overexpression. Then, the effects of p19ARF on replicative senescence of WI-38 cells were observed. The results re- vealed that, compared with control cells, the WI-38 cells in which p19ARF gene was introduced showed significant up-regulation of p53 and p21 expression level, decrease of cell generation by 10 12 generations, decline of cell growth rate with cell cycle being arrested at G1 phase, increase of positive rate of senescent marker SA-β-gal staining, and decrease of mitochondrial membrane potential. The morphology of the transfected fibroblasts presented the characteristics changes similar to senescent cells. These results indicated that high expression of p19ARF may promote the senescent process of human diploid cells.     

Novel associations between rhizobial populations and legume species within the genera Lathyrus and O xytropis grown in the temperate region of China

Fifty rhizobial isolates of Lathyrus and Oxytropis collected from northern regions of China were studied in their genotypic characterization based upon analyses of ARDRA, 16S-23S IGS PCR-RFLP, TP-RAPD, MLEE, sequences of 16S rDNA gene and housekeeping genes of atpD, recA and glnII. The results demonstrated that most of the Lathyrus rhizobia belonged to Rhizobium and most of the Oxytropis rhizobia belonged to Sinorhizobium. A novel group of Rhizobium sp. I and S. meliloti were identified as the main microsymbionts respectively associated with Lathyrus and Oxytropis species in the collection area, which were new associations between rhizobia and the mentioned hosts. This study also provides new evidence for biogeography of rhizobia. Supported by the National Program for Basic S&T Platform Construction (Grant No. 2005DKA21201-1), the National Natural Science Foundation of China (Grant No. 30670001), and the National Basic Research Program of China (Grant No. 2006CB100206)     

Analysis of the essential DNA region for OsEBP-89 promoter in response to methyl jasmonic acid

In rice, the characterization of OsEBP-89 is inducible by various stress-or hormone-stimuli, including ethylene, abscisic acid (ABA), jasmonate acid (JA), drought and cold. Here, we report the investigation of essential DNA region within OsEBP-89 promoter for methyl jasmonic acid (MeJA) induction. PLACE analysis indicates that this promoter sequence contains multiple potential elements in response to various stimuli. First, we fused this promoter with GUS gene and analyzed its expression under MeJA treatment through Agrobacterium infiltration mediating transient expression in tobacco leaves. Our results revealed that this chimeric gene could be inducible by MeJA in tobacco leaves. To further determine the crucial sequences responsible for MeJA induction, we generated a series of deletion promoters which were fused with GUS reporter gene respectively. The results of transient expression of GUS gene driven by these mutant promoters show that the essential region for MeJA induction is positioned in the region between −1200 and −800 in OsEBP-89 promoter containing a G-box (−1127), which is distinct from the essential region containing ERE (−562) for ACC induction. In all, our finding is helpful in understanding the molecular mechanism of OsEBP-89 expression under different stimuli. OsEBP-89, essential DNA region, methyl jasmonic acid, transient assay, promoter, tobacco leaves Contributed equally to this work Supported by the National Basic Research Program of China (Grant No. 2006CB101700) and the National Natural Science Foundation of China (Grant Nos. 30671135, 30525034 and 30730060)     

Chromosomal localization of silkworm (Bombyx mori) sericin gene 1 and chymotrypsin inhibitor 13 using fluorescence in situ hybridization

The chromosomal locations of two single-copy genes, Ser-1 and CI-13, in silkworm (Bombyx mori) were detected at the molecular cytogenetics level by fluorescence in situ hybridization in the study. The results showed that Ser-1 is located near the distal end of the 11th linkage group, relatively at the 12.5±1.4 position in pachytene; and that CI-13 has been mapped near the distal end of the 2nd linkage group, relatively at the 8.2±1.2 position in pachytene. Furthermore, their location model map-FISH map on silkworm chromosome was drawn. The FISH technique and its application to silkworm are also discussed in this paper.     

The structure and function of catalytic RNAs

Before the discovery of ribozymes, RNA had been proposed to function as a catalyst, based on the discovery that RNA folded into high-ordered structures as protein did. This hypothesis was confirmed in the 1980s, after the discovery of Tetrahymena group I intron and RNase P ribozyme. There have been about ten ribozymes identified during the past thirty years, as well as the fact that ribosomes function as ribozymes. Advances have been made in understanding the structures and functions of ribozymes, with numerous crystal structures resolved in the past years. Here we review the structure-function relationship of both small and large ribozymes, especially the structural basis of their catalysis. ribozyme, structure, catalysis Supported by National Natural Science Foundation of China (Grant No. 30330170) and National Key Basic Research and Development Program of China (Grant No. 2005CB724604)     

Discovery and identification of Serum Amyloid A pro-tein elevated in lung cancer serum

Two hundred and eighteen serum samples from 175 lung cancer patients and 43 healthy individuals were analyzed by using Surface Enhaced Laser Desorption/Ionization Time of Flight Mass Spectrome-try (SELDI-TOF-MS). The data analyzed by both Biomarker Wizard™ and Biomarker Patterns™ software showed that a protein peak with the molecular weight of 11.6 kDa significantly increased in lung cancer. Meanwhile,the level of this biomarker was progressively increased with the clinical stages of lung cancer. The candidate biomarker was then obtained from tricine one-dimensional sodium dodecyl sul-fate-polyacrylamide gel electrophoresis by matching the molecular weight with peaks on WCX2 chips and was identified as Serum Amyloid A protein (SAA) by MALDI/MS-MS and database searching. It was further validated in the same serum samples by immunoprecipitation with commercial SAA antibody. To confirm the SAA differential expression in lung cancer patients, the same set of serum samples was measured by ELISA assay. The result showed that at the cutoff point 0.446（OD value）on the Receiver Operating Characteristic (ROC) curve, SAA could better discriminate lung cancer from healthy indi-viduals with sensitivity of 84.1% and specificity of 80%. These findings demonstrated that SAA could be characterized as a biomarker related to pathological stages of lung cancer.     

Non-coding RNAs and the acquisition of genomic imprinting in mammals

Genomic imprinting, representing parent-specific expression of alleles at a locus, is mainly evident in flowering plants and placental mammals. Most imprinted genes, including numerous non-coding RNAs, are located in clusters regulated by imprinting control regions (ICRs). The acquisition and evolution of genomic imprinting is among the most fundamental genetic questions. Discoveries about the transition of mammalian imprinted gene domains from their non-imprinted ancestors, especially recent studies undertaken on the most ancient mammalian clades — the marsupials and monotremes from which model species genomes have recently been sequenced, are of high value. By reviewing and analyzing these studies, a close connection between non-coding RNAs and the acquisition of genomic imprinting in mammals is demonstrated. The evidence comes from two observations accompanied with the acquisition of the imprinting: (i) many novel non-coding RNA genes emerged in imprinted regions; (ii) the expressions of some conserved non-coding RNAs have changed dramatically. Furthermore, a systematical analysis of imprinted snoRNA (small nucleolar RNA) genes from 15 vertebrates suggests that the origination of imprinted snoRNAs occurred after the divergence between eutherians and marsupials, followed by a rapid expansion leading to the fixation of major gene families in the eutherian ancestor prior to the radiation of modern placental mammals. Involved in the regulation of imprinted silencing and mediating the chromatins epigenetic modification may be the major roles that non-coding RNAs play during the acquisition of genomic imprinting in mammals. Supported by National Natural Science Foundation of China (Grant No. 30830066), the Ministry of Education of China and Natural Science Foundation of Guangdong Province (Grant No. IRT0447, NSF-05200303) and National Key Basic Research and Development Program of China (Grant No. 2005CB724600)