Conditional knockout mice to study alternative splicing in vivo

Analysis of genomes has revealed that the total number of human genes is comparable to those of simpler organisms, and thus, the number of genes does not correlate with the complexity and functional diversity of different organisms. Multiple mechanisms, including alternative splicing, are believed to contribute to the molecular complexity in higher eukaryotes. Given the fact that more than half of human genes undergo alternative splicing, however, little is known about the biological relevance of most alternative splicing events and their regulatory mechanisms. Recent work has highlighted the power of reverse genetic approaches in addressing regulated splicing in animal models. Here, we focus on the conditional knockout approach adapted for splicing research with the intention to provide a general guide to the generation of mouse models to study regulated splicing in development and disease.     

Expression and purification of human endostatin from Hansenula polymorpha A16

Endostatin (EDN), an endogenous angiogenesis inhibitor of 20 kDa, was originally isolated from the supernatant of culture murine hemangioendothelioma cell line. Interest in EDN arises from its therapeutic potential as anti-tumor and anti-angiogenesis agents. However, it is difficult to obtain sufficient quantities of native EDN from its natural resources. We report here the construction of a pMIRH-type vector pLRG29 by introducing the 25s rDNA of Hansenua wingei and the Km(R) gene into the YIp vector pSML12 and the EDN expression vector pLRG-EDN by cloning the human EDN cDNA into pLRG29. Human EDN was expressed in Hansenula polymorpha (H.p.) A16 (pLRG-EDN) as a secreted soluble protein. The yield of the secreted EDN was 65 mg/L in shake flask. The secreted EDN was purified to a purity of 98 % by the use of SP Sepharose FF ion-exchange chromatography and Sepharose-heparin Hi Trap affinity chromatography. The MTT and chicken embryo chorioallantoic membrane assay demonstrated that the human EDN produced from H. polymorpha inhibited in vitro the proliferation of human umbilical vein endothelial cells and in vivo the neovascularization induced by bFGF.     

Identification and characterisation of a homolog of an activation gene for the recombination activating gene 1 (RAG 1) in amphioxus

Expression of recombination activating genes (RAG) involved in the V (D) J recombination is regulated by the RAG1 gene activator (RGA) in mammals. The sequence of a cDNA clone from an amphioxus cDNA library was found to be homologous to that of RGA from mouse stromal cells with 45% identity. The full-length cDNA sequence comprises 1119 bp and encodes a putative protein of 210 amino acid residues. Characterisation of the amino acid sequence revealed that two MtN3 domains and seven transmembrane spans are present in this protein, indicating a potential role as a plasma membrane protein. This gene is expressed in many tissues and at differential developmental stages. A high expression level of RGA is detected in gonad tissues, and gastrula embryo and adult stages. The presence of the RGA gene in amphioxus suggests that the signal pathway required for the expression of RAG could exist in this primitive protochordate. It also implies that in the related molecules, primitive adaptive immunity may have existed in cephalochordate although the complete machinery of VDJ rearrangement may not be formed.     

Bayesian models for the analysis of genetic structure when populations are correlated

MOTIVATION: Population allele frequencies are correlated when populations have a shared history or when they exchange genes. Unfortunately, most models for allele frequency and inference about population structure ignore this correlation. Recent analytical results show that among populations, correlations can be very high, which could affect estimates of population genetic structure. In this study, we propose a mixture beta model to characterize the allele frequency distribution among populations. This formulation incorporates the correlation among populations as well as extending the model to data with different clusters of populations. RESULTS: Using simulated data, we show that in general, the mixture model provides a good approximation of the among-population allele frequency distribution and a good estimate of correlation among populations. Results from fitting the mixture model to a dataset of genotypes at 377 autosomal microsatellite loci from human populations indicate high correlation among populations, which may not be appropriate to neglect. Traditional measures of population structure tend to overestimate the amount of genetic differentiation when correlation is neglected. Inference is performed in a Bayesian framework. CONTACT: fur@ohsu.edu.     

Epidemiological evaluations of the efficacy of slow-released praziquantel-medicated bars for dogs in the prevention and control of cystic echinococcosis in man and animals

To assess the epidemiological efficacy of type SRP III slow-released praziquantel-medicated bars for dogs in the prevention and control of cystic echinococcosis in man and livestock, praziquantel-medicated bars were implanted subcutaneously in over 90% of dogs in villages in north Xinjiang, China, where cystic echinococcosis is highly endemic. After implantation, infection rate of Echinococcus granulosus in dogs, specific antibodies in children and prevalence of echinococcosis in one-year-old lambs were observed for 3 years. Coproantigen of E. granulosus was positive in 41.2% of the dogs at the start of experiment. In the second and third year after medicated-bar implantation, coproantigen was undetectable in any dogs examined, while 3.0% of dogs were positive at the end of the fourth year. The antibody positive rate in 7-year-old pupils, that was 41.2% before the experiment, declined gradually and it was 5.4% in the fourth year, while children in the non-intervention control area showed 30.6% seropositivity. The prevalence of hydatid disease in children aged 7–16 years also declined significantly. The prevalence of hydatidosis in lambs one year of age was 44.8% in the first year, dropping to 10.7% in the fourth year, while in the non-intervention control area the level of infection was 46.4%. These results demonstrated not only that the slow released praziquantel-medicated bars efficiently blocked reinfection in dogs at least for 2 years, but also the measure was effective in preventing transmission of cystic echinococcosis to both man and livestock.     

New amperometric and potentiometric immunosensors based on gold nanoparticles/tris(2,2'-bipyridyl)cobalt(III) multilayer films for hepatitis B surface antigen determinations

Two generic, fast, sensitive and novel electrochemical immunosensors have been developed. Initially, a layer of plasma-polymerized Nafion film (PPF) was deposited on the platinum electrode surface, then positively charged tris(2,2'-bipyridyl)cobalt(III) (Co(bpy)(3)(3+)) and negatively charged gold nanoparticles were assembled on the PPF-modified Pt electrode by layer-by-layer technique. Finally, hepatitis B surface antibody (HBsAb) was electrostatically adsorbed on the gold nanoparticles surface. Electrochemical behavior of the {Au/Co(bpy)(3)(3+)}(n) multilayer film-modified electrodes was studied. Cyclic voltammetry, electrochemical impedance spectroscopy (EIS) were adopted to monitor the regular growth of the multilayer films. The performance and factors influencing the performance of the resulting immunosensors were studied in detail. The multilayer film-modified immunosensor was used for hepatitis B surface antigen (HBsAg) determination via the amperometric and potentiometric immunosensor systems, and both systems provided the same linear ranges from 0.05 to 4.5 microg/mL with different detection limits for the amperometric system 0.005 microg/mL and for the potentiometric system 0.015 microg/mL. The immunosensors were used to analyse HBsAg in human serum samples. Analytical results of clinical samples show that the developed immunoassay is comparable with the enzyme-linked immunosorbent assays (ELISAs) method, implying a promising alternative approach for detecting HBsAg in the clinical diagnosis. In addition, the multilayer films also showed better stability for 1 month at least.     

An electrochemical stripping metalloimmunoassay based on silver-enhanced gold nanoparticle label

A novel, sensitive electrochemical immunoassay has been developed based on the precipitation of silver on colloidal gold labels which, after silver metal dissolution in an acidic solution, was indirectly determined by anodic stripping voltammetry (ASV) at a glassy-carbon electrode. The method was evaluated for a noncompetitive heterogeneous immunoassay of an immunoglobulin G (IgG) as a model. The influence of relevant experimental variables, including the reaction time of antigen with antibody, the dilution ratio of the colloidal gold-labeled antibody and the parameters of the anodic stripping operation, upon the peak current was examined and optimized. The anodic stripping peak current depended linearly on the IgG concentration over the range of 1.66 ng ml(-1) to 27.25 microg ml(-1) in a logarithmic plot. A detection limit as low as 1 ng ml(-1) (i.e., 6 x 10(-12) M) human IgG was achieved, which is competitive with colorimetric enzyme linked immuno-sorbent assay (ELISA) or with immunoassays based on fluorescent europium chelate labels. The high performance of the method is attributed to the sensitive ASV determination of silver (I) at a glassy-carbon electrode (detection limit of 5 x 10(-9) M) and to the catalytic precipitation of a large number of silver on the colloidal gold-labeled antibody.     

Semiconductor nanocrystals for biological imaging

Conventional organic fluorophores suffer from poor photo stability, narrow absorption spectra and broad emission spectra. Semiconductor nanocrystals, however, are highly photo-stable with broad absorption spectra and narrow size-tunable emission spectra. Recent advances in the synthesis of these materials have resulted in the generation of bright, sensitive, extremely photo-stable and biocompatible semiconductor fluorophores. Commercial availability facilitates their application in a variety of unprecedented biological experiments, including multiplexed cellular imaging, long-term in vitro and in vivo labeling, deep tissue structure mapping and single particle investigation of dynamic cellular processes. Semiconductor nanocrystals are one of the first examples of nanotechnology enabling a new class of biomedical applications.     

Postglacial colonization of the Tibetan plateau inferred from the matrilineal genetic structure of the endemic red-necked snow finch, Pyrgilauda ruficollis

Most phylogeographical studies of postglacial colonization focus on high latitude locations in the Northern Hemisphere. Here, we studied the phylogeographical structure of the red-necked snow finch Pyrgilauda ruficollis, an endemic species of the Tibetan plateau. We analysed 879 base pairs (bp) of the mitochondrial cytochrome b gene and 529 bp of the control region in 41 birds from four regional groups separated by mountain ranges. We detected 34 haplotypes, 31 of which occurred in a single individual and only three of which were shared among sampling sites within regional groups or among regional groups. Haplotype diversity was high (h = 0.94); nucleotide diversity was low (eth = 0.00415) and genetic differentiation was virtually non-existent. Analyses of mismatch distributions and geographically nested clades yielded results consistent with contiguous range expansion, and the expansion times were estimated as 0.07-0.19 million years ago (Ma). Our results suggest that P. ruficollis colonized the Tibetan plateau after the extensive glacial period (0.5-0.175 Ma), expanding from the eastern margin towards the inner plateau. Thus, in contrast to many of the post-glacial phylogeographical structures known at high latitudes, this colonization occurred without matrilineal population structuring. This might be due to the short glacial cycles typical of the Tibetan plateau, adaptation of P. ruficollis to cold conditions, or refugia and colonized habitat being semicontinuous and thus promoting population mixing.