Investigation of xenotropic murine leukemia virus-related virus (XMRV) in human and other cell lines

Xenotropic murine leukemia virus-related virus (XMRV) was discovered in human prostate tumors and later in some chronic fatigue syndrome (CFS) patients. However, subsequent studies have identified various sources of potential contamination with XMRV and other murine leukemia virus (MLV)-related sequences in test samples. Biological and nucleotide sequence analysis indicates that XMRV is distinct from known xenotropic MLVs and has a broad host range and cell tropism including human cells. Therefore, it is prudent to minimize the risk of human exposure to infection by evaluating XMRV contamination in cell lines handled in laboratory research and particularly those used in the manufacture of biological products. Nested DNA PCR assays were optimized for investigating XMRV gag and env sequences in various cell lines, which included MRC-5, Vero, HEK-293, MDCK, HeLa, and A549, that may be used in the development of some vaccines and other cell lines broadly used in research. The sensitivity of the DNA PCR assays was <10 copies in approximately 1.8 x 10⁵ cells equivalent of human DNA. The results indicated the absence of XMRV in the cell lines tested; although in some cases DNA fragments identified as cellular sequences were seen following the first round of PCR amplification with the env primer pair.     

Life or Death? A Physiogenomic Approach to Understand Individual Variation in Responses to Hemorrhagic Shock

Severe hemorrhage due to trauma is a major cause of death throughout the world. It has often been observed that some victims are able to withstand hemorrhage better than others. For decades investigators have attempted to identify physiological mechanisms that distinguish survivors from nonsurvivors for the purpose of providing more informed therapies. As an alternative approach to address this issue, we have initiated a research program to identify genes and genetic mechanisms that contribute to this phenotype of survival time after controlled hemorrhage. From physiogenomic studies using inbred rat strains, we have demonstrated that this phenotype is a heritable quantitative trait, and is therefore a complex trait regulated by multiple genes. Our work continues to identify quantitative trait loci as well as potential epigenetic mechanisms that might influence survival time after severe hemorrhage. Our ultimate goal is to improve survival to traumatic hemorrhage and attendant shock via regulation of genetic mechanisms and to provide knowledge that will lead to genetically-informed personalized treatments.     

Fat maintenance is a predictor of the murine lifespan response to dietary restriction

Dietary restriction (DR), one of the most robust life-extending manipulations, is usually associated with reduced adiposity. This reduction is hypothesized to be important in the life-extending effect of DR, because excess adiposity is associated with metabolic and age-related disease. Previously, we described remarkable variation in the lifespan response of 41 recombinant inbred strains of mice to DR, ranging from life extension to life shortening. Here, we used this variation to determine the relationship of lifespan modulation under DR to fat loss. Across strains, DR life extension correlated inversely with fat reduction, measured at midlife (males, r= -0.41, P<0.05, n=38 strains; females, r= -0.63, P<0.001, n=33 strains) and later ages. Thus, strains with the least reduction in fat were more likely to show life extension, and those with the greatest reduction were more likely to have shortened lifespan. We identified two significant quantitative trait loci (QTLs) affecting fat mass under DR in males but none for lifespan, precluding the confirmation of these loci as coordinate modulators of adiposity and longevity. Our data also provide evidence for a QTL previously shown to affect fuel efficiency under DR. In summary, the data do not support an important role for fat reduction in life extension by DR. They suggest instead that factors associated with maintaining adiposity are important for survival and life extension under DR.     

<Emphasis Type="Italic">Cis</Emphasis>-regulation of <Emphasis Type="Italic">IRF5</Emphasis>expression is unable to fully account for systemic lupus erythematosus association: analysis of multiple experiments with lymphoblastoid cell lines

Introduction  

Interferon regulatory factor 5 gene (IRF5) polymorphisms are strongly associated with several diseases, including systemic lupus erythematosus (SLE). The association includes risk and protective components. They could be due to combinations of functional polymorphisms and related to cis-regulation of IRF5 expression, but their mechanisms are still uncertain. We hypothesised that thorough testing of the relationships between IRF5 polymorphisms, expression data from multiple experiments and SLE-associated haplotypes might provide useful new information.     

Proteomic analysis of heterosis during maize seed germination

Heterosis is observed for most phenotypic traits and developmental stages in many plants. In this study, the embryos, from germinating seeds after 24 h of soaking, for five elite maize hybrids and their parents were selected to unravel the genetic basis of heterosis using 2‐D proteomic method. In total, 257 (80.06%), 363 (58.74%), 351 (79.95%), 242 (54.50%), and 244 (46.30%) nonadditively expressed proteins were identified in hybrids Zhengdan 958, Nongda 108, Yuyu 22, Xundan 20, and Xundan 18, respectively. The nonadditive proteins were divided into above high‐parent (++; 811, 55.66%), high‐parent (+; 121, 8.30%), partial dominance (+?; 249, 17.09%), low‐parent (?; 30, 2.06%), below low‐parent (? ?; 62, 4.26%), and D (different; 184, 12.63%) expression patterns. The observed patterns indicate the important roles of dominance, partial dominance, and overdominance in regulating seed germination in maize. Additionally, 54 different proteins were identified by mass spectrometry and classified into nine functional groups: metabolism (9), cell detoxification (8), unknown functional proteins (8), chaperones (7), signal transduction (6), development process (5), other (5), transporter (3), and stress response (3). Of these, the most interesting are those involved with germination‐related hormone signal transduction and the abscisic acid and gibberellin regulation networks.     

重组PAC1-EC1(N)对表达不同PAC1变体的细胞系细胞活性的影响

垂体腺苷酸环化酶激活多肽(pituitary adenylate cyclase-activating polypeptide,PACAP)特异受体PAC1(normal型)N端胞外域[简称PAC1-EC1(N)]具有调控PAC1活性的作用.为研究PAC1-EC1(N)对表达不同PAC1变体的细胞系活性的影响,利用基因...     

Planthopper‐rice interactions: unequal stresses on pure‐line and hybrid rice under similar experimental conditions

Hybrid and pure‐line (inbred) rice [Oryza sativa L. (Poaceae)] varieties have distinct physiologies, particularly as related to their nutrient requirements. These differences could confound the results and interpretation of experiments that compare rice varieties grown in pots for their resistance and responses to herbivores. In this study, a series of experiments was conducted to identify potentially confounding interactions between pot size (soil volume), fertilizer regime, and the use of acetate insect cages with rice line type (hybrid, fertile parental inbred, and male sterile inbred) during bioassays with the brown planthopper, Nilaparvata lugens (Stål) (Hemiptera: Delphacidae). The growth of hybrid rice was often limited (relatively low biomass and low grain production) compared to fertile inbred lines even in large pots (7 200 ml) and when grown without added fertilizer. Several interactions between the effects of growth conditions and line type were detected. Acetate cages caused a significant reduction in grain yield in hybrids, but not in inbreds, mainly resulting from a cage‐induced decrease in grain weight (smaller grains). Hybrids and male sterile lines often had higher root or above‐ground biomass in the largest caged pots compared with fertile inbred lines, but biomass was similar in smaller pots, indicating that the large pots allowed longer unimpeded growth of fertile inbreds, but not other line types. There were no interactions between the presence or absence of planthoppers with line type or experimental conditions. All line types were equally susceptible to planthoppers. Often the effects of planthopper feeding on plant fitness (i.e., tolerance) were apparent when plants were grown in large pots but not in small pots, particularly for hybrid lines and under high nitrogen regimes. On the basis of these results we recommend that researchers ensure that plants are not unequally stressed by inadequate growth conditions during comparative studies with herbivores on physiologically distinct rice varieties or rice species. We recommend the use of large pots (soil volume) and lower fertilizer levels with young, non‐reproductive plants during comparative bioassays with planthoppers. Field cages are recommended for hybrid‐inbred comparisons during older plant stages but these are subject to a range of other confounding variables.     

Limited RNA Editing in Exons of Mouse Liver and Adipose

Several studies have investigated RNA–DNA differences (RDD), presumably due to RNA editing, with conflicting results. We report a rigorous analysis of RDD in exonic regions in mice, taking into account critical biases in RNA-Seq analysis. Using deep-sequenced F1 reciprocal inbred mice, we mapped 40 million RNA-Seq reads per liver sample and 180 million reads per adipose sample. We found 7300 apparent hepatic RDDs using a multiple-site mapping procedure, compared with 293 RDD found using a unique-site mapping procedure. After filtering for repeat sequence, splice junction proximity, undirectional strand, and extremity read bias, 63 RDD remained. In adipose tissue unique-site mapping identified 1667 RDD, and after applying the same four filters, 188 RDDs remained. In both tissues, the filtering procedure increased the proportion of canonical (A-to-I and C-to-U) editing events. The genomic DNA of 12 RDD sites among the potential 63 hepatic RDD was tested by Sanger sequencing, three of which proved to be due to unreferenced SNPs. We validated seven liver RDD with Sequenom technology, including two noncanonical, Gm5424 C-to-I(G) and Pisd I(G)-to-A RDD. Differences in diet, sex, or genetic background had very modest effects on RDD occurrence. Only a small number of apparent RDD sites overlapped between liver and adipose, indicating a high degree of tissue specificity. Our findings underscore the importance of properly filtering for bias in RNA-Seq investigations, including the necessity of confirming the DNA sequence to eliminate unreferenced SNPs. Based on our results, we conclude that RNA editing is likely limited to hundreds of events in exonic RNA in liver and adipose.     

Establishment,characterization and viral susceptibility of two cell lines derived from leopard wrasse Macropharyngodon geoffroy

Two new fish cell lines were established from skin (LWSK) and fin (LWFN) of leopard wrasse Macropharyngodon geoffroy. These cells grew optimally at 25° C in Leibovitz‐15 medium supplemented with 10% foetal bovine serum. Proliferation of M. geoffroy cells remained serum dependent up to cell passage 16, and cell‐plating efficiency ranged from 12 to 16%. Karyotypic analysis of these new cell lines at cell passage 8 indicated that both cell lines remained diploid with a peak chromosomal count of 144. PCR amplification of 16S mitochondrial DNA and the subsequent analysis confirmed that these cell lines were indeed derived from M. geoffroy. Results of viral challenge assays revealed that both LWSK and LWFN shared patterns of viral susceptibility similar to that of six fish viruses tested: LWSK and LWFN cells were highly permissive to channel catfish virus, spring viremia carp virus and snakehead rhabdovirus with high‐yield virus production ranging from 10^7·18±0·17 to 10^8·37±0·16 TCID₅₀ ml^?1 (mean ± s.d .). These newly established cell lines would be useful in attempts to isolate and study aquatic viruses, particularly the viral aetiology of green turtle fibropapilloma as M. geoffroy is known to be one of the common cleaner fish of green sea turtles.     

Determination of variability in monosporidial lines of Tilletia indica by RAPD analysis

Genetic variability in 23 monosporidial lines developed from five isolates of Tilletia indica causing Karnal bunt of wheat isolated from four wheat growing states of India was determined by using 19 rapid amplified polymorphic DNA (RAPD) markers. Amplification profile generated with all the 19 primers produced 3–16 numbers of bands of 1.5–5 kb size. High level of polymorphism (95.2%) suggested wide range of variability. Maximum Jaccard's similarity coefficient (80%) was observed between KB2MsB and KB2MsC followed by KB5MsC and KB5MsE with 75% similarity, whereas it was minimum between KB3MsA and Kb4MsB (47%). The dendrogram derived from the fingerprint analysis with 19 RAPD primers by using UPGMA showed different levels of genetic similarity among monosporidial lines. At 35% genetic similarity, the monosporidial lines were grouped in two clusters. Some primers, viz., OPN-1, OPN-6, OPN-9, OPN-12, OPN-13, OPN-18, OPM-2, OPM-8, OPM-10, OPB-8, OPB-17 and OPB-20 showed 100% polymorphism. The RAPD fingerprint generated by OPN-1 and OPM-3 were analysed and showed high range of variation in genetic make-up of monosporidial lines.