Mouse DNA contamination in human tissue tested for XMRV

Background

We used a PCR-based approach to study the prevalence of genetic sequences related to a gammaretrovirus, xenotropic murine leukemia virus-related virus, XMRV, in human prostate cancer. This virus has been identified in the US in prostate cancer patients and in those with chronic fatigue syndrome. However, with the exception of two patients in Germany, XMRV has not been identified in prostate cancer tissue in Europe. Most putative associations of new or old human retroviruses with diseases have turned out to be due to contamination. We have looked for XMRV sequences in DNA extracted from formalin-fixed paraffin- embedded prostate tissues. To control for contamination, PCR assays to detect either mouse mitochondrial DNA (mtDNA) or intracisternal A particle (IAP) long terminal repeat DNA were run on all samples, owing to their very high copy number in mouse cells.

Results

In general agreement with the US prevalence, XMRV-like sequences were found in 4.8% of prostate cancers. However, these were also positive, as were 21.5% of XMRV-negative cases, for IAP sequences, and many, but not all were positive for mtDNA sequences.

Conclusions

These results show that contamination with mouse DNA is widespread and detectable by the highly sensitive IAP assay, but not always with less sensitive assays, such as murine mtDNA PCR. This study highlights the ubiquitous presence of mouse DNA in laboratory specimens and offers a means of rigorous validation for future studies of murine retroviruses in human disease.     

Context-specific metabolic networks are consistent with experiments

Reconstructions of cellular metabolism are publicly available for a variety of different microorganisms and some mammalian genomes. To date, these reconstructions are “genome-scale” and strive to include all reactions implied by the genome annotation, as well as those with direct experimental evidence. Clearly, many of the reactions in a genome-scale reconstruction will not be active under particular conditions or in a particular cell type. Methods to tailor these comprehensive genome-scale reconstructions into context-specific networks will aid predictive in silico modeling for a particular situation. We present a method called Gene Inactivity Moderated by Metabolism and Expression (GIMME) to achieve this goal. The GIMME algorithm uses quantitative gene expression data and one or more presupposed metabolic objectives to produce the context-specific reconstruction that is most consistent with the available data. Furthermore, the algorithm provides a quantitative inconsistency score indicating how consistent a set of gene expression data is with a particular metabolic objective. We show that this algorithm produces results consistent with biological experiments and intuition for adaptive evolution of bacteria, rational design of metabolic engineering strains, and human skeletal muscle cells. This work represents progress towards producing constraint-based models of metabolism that are specific to the conditions where the expression profiling data is available.     

Multiple sources of contamination in samples from patients reported to have XMRV infection

Xenotropic murine leukemia virus (MLV)-related retrovirus (XMRV) was reported to be associated with prostate cancer by Urisman, et al. in 2006 and chronic fatigue syndrome (CFS) by Lombardi, et al. in 2009. To investigate this association, we independently evaluated plasma samples from 4 patients with CFS reported by Lombardi, et al. to have XMRV infection and from 5 healthy controls reported to be XMRV uninfected. We also analyzed viral sequences obtained from supernatants of cell cultures found to contain XMRV after coculture with 9 clinical samples from 8 patients. A qPCR assay capable of distinguishing XMRV from endogenous MLVs showed that the viral sequences detected in the CFS patient plasma behaved like endogenous MLVs and not XMRV. Single-genome sequences (N = 89) from CFS patient plasma were indistinguishable from endogenous MLVs found in the mouse genome that are distinct from XMRV. By contrast, XMRV sequences were detected by qPCR in 2 of the 5 plasma samples from healthy controls (sequencing of the qPCR product confirmed XMRV not MLV). Single-genome sequences (N = 234) from the 9 culture supernatants reportedly positive for XMRV were indistinguishable from XMRV sequences obtained from 22Rv1 and XMRV-contaminated 293T cell-lines. These results indicate that MLV DNA detected in the plasma samples from CFS patients evaluated in this study was from contaminating mouse genomic DNA and that XMRV detected in plasma samples from healthy controls and in cultures of patient samples was due to cross-contamination with XMRV (virus or nucleic acid).     

Common inbred strains of the laboratory mouse that are susceptible to infection by mouse xenotropic gammaretroviruses and the human-derived retrovirus XMRV

Laboratory mouse strains carry endogenous copies of the xenotropic mouse leukemia viruses (X-MLVs), named for their inability to infect cells of the laboratory mouse. This resistance to exogenous infection is due to a nonpermissive variant of the XPR1 gammaretrovirus receptor, a resistance that also limits in vivo expression of germ line X-MLV proviruses capable of producing infectious virus. Because laboratory mice vary widely in their proviral contents and in their virus expression patterns, we screened inbred strains for sequence and functional variants of the XPR1 receptor. We also typed inbred strains and wild mouse species for an endogenous provirus, Bxv1, that is capable of producing infectious X-MLV and that also contributes to the generation of pathogenic recombinant MLVs. We identified the active Bxv1 provirus in many common inbred strains and in some Japanese Mus molossinus mice but in none of the other wild mouse species that carry X-MLVs. Our screening for Xpr1 variants identified the permissive Xpr1(sxv) allele in 7 strains of laboratory mice, including a Bxv1-positive strain, F/St, which is characterized by lifelong X-MLV viremia. Cells from three strains carrying Xpr1(sxv), namely, SWR, SJL, and SIM.R, were shown to be infectable by X-MLV and XMRV; these strains carry different alleles at Fv1 and vary in their sensitivities to specific X/P-MLV isolates and XMRV. Several strains with Xpr1(sxv) lack the active Bxv1 provirus or other endogenous X-MLVs and may provide a useful model system to evaluate the in vivo spread of these gammaretroviruses and their disease potential in their natural host.     

Patterns of bird invasion are consistent with environmental filtering

Predicting invasion potential has global significance for managing ecosystems as well as important theoretical implications for understanding community assembly. Phylogenetic relationships of introduced species to the extant community may be predictive of establishment success because of the opposing forces of competition/shared enemies (which should limit invasions by close relatives) versus environmental filtering (which should allow invasions by close relatives). We examine here the association between establishment success of introduced birds and their phylogenetic relatedness to the extant avifauna within three highly invaded regions (Florida, New Zealand, and Hawaii). Published information on both successful and failed introductions, as well as native species, was compiled for all three regions. We created a phylogeny for each avifauna including all native and introduced bird species. From the estimated branch lengths on these phylogenies, we calculated multiple measurements of relatedness between each introduced species and the extant avifauna. We used generalized linear models to test for an association between relatedness and establishment success. We found that close relatedness to the extant avifauna was significantly associated with increased establishment success for exotic birds both at the regional (Florida, Hawaii, New Zealand) and sub‐regional (islands within Hawaii) levels. Our results suggest that habitat filtering may be more important than interspecific competition in avian communities assembled under high rates of anthropogenic species introductions. This work also supports the utility of community phylogenetic methods in the study of vertebrate invasions.     

Identification of diverse BoLA DQA3 genes consistent with non-allelic sequences

Genetic diversity within the DQA genes of the major histocompatibility complex (Mhc) of cattle is characterised by multiple polymorphic loci that can vary in number between haplotypes. Previous analysis of the second exon sequences derived from genomic BoLA DQA3 genes identified two distinct families, DQA3*01 and DQA3*02 . In this report, we describe the nucleotide and predicted amino acid sequences of the entire coding region of three transcribed BoLA DQA3 genes representing each of these families. These data provide additional evidence that the BoLA DQA3 locus is distinct from BoLA DQA1 and BoLA DQA2 . In addition, the amino acid sequence of DQA3 genes from the two families is shown to differ by 35 out of the 254 amino acids. Putative locus-specific amino acid sequence motifs within the transmembrane and intracytoplasmic domains of DQA genes are shown to differ between the DQA3*01 and DQA3*02 genes. Phylogenetic analysis reveals a genetic distance that is considerably larger than that seen between orthologous Mhc allelic families. These data are consistent with either an extremely divergent family of DQA3 genes or an allele at an additional BoLA DQA4 locus.     

Probable laboratory contamination of clinical specimens with Leptospira meyeri

Leptospira meyeri were isolated from the blood or the pleural effusion cultures of four patients at a commercial clinical laboratory. All isolates were identical in their 16S rDNA sequences and NotI or SfiI restriction profiles of the chromosome DNA on pulsed-field gel electrophoresis. However, intraperitoneal inoculation of the isolate (NIID1) failed to kill hamsters and none of the patients' sera reacted with L. meyeri strains isolated, indicating leptospiral contamination occurred during laboratory investigation.     

Rare coding sequence changes are consistent with Ecdysozoa, not Coelomata

There is growing interest in the use of alternative, more slowly-evolving RGCs (rare genomic changes). Recently, Rogozin and coauthors (Rogozin et al. 2007) proposed a novel phylogenetic method employing rare amino acid changes, RGC-CAMs (rare genomic changes-conserved amino acids-multiple substitutions). They applied their method to 694 sets of eukaryotic orthologs in order to distinguish the relationship between nematodes, arthropods and deuterostomes. They concluded that such rare amino acid changes were consistent with the Coelomata hypothesis, which groups arthropods and deuterostomes to the exclusion of nematodes. Here we use newly available genomic sequences from Nematostella vectensis, a basal metazoan, and from Brugia malayi, an additional nematode. We show that the apparent support for Coelomata is likely to be the result of the rapid rate of evolution leading to Caenorhabditis nematodes. Including the additional species paints a very different picture, with 13 remaining characters consistent with Ecdysozoa versus only 1 consistent with Coelomata.     

Disease-associated mutations in SOD1 are impervious to dominant positive or negative effects

The familial form of amyotrophic lateral sclerosis is caused by mutations in the SOD1 gene encoding the cytosolic antioxidant enzyme Cu,Zn superoxide dismutase. Although there is no clear correlation between disease and dismutating catalytic activity among the various disease-associated SOD1 alleles, all of the known missense mutations significantly alter the half-life of the encoded polypeptides. Using transient transfection studies in mammalian cells, it was demonstrated that a frameshift mutation in SOD1 which results in a truncated polypeptide is similarly destabilized. Using an epitope-tagging strategy to discriminate between mutant and wild-type SOD1 polypeptides, no evidence for dominant effects on polypeptide stability was detected, including that of a positive effect of the wild-type on mutant SOD1 polypeptides or that of a negative effect of mutant on wild-type SOD1 polypeptides. These experiments thus favor a non-catalytic role of mutant forms of SOD1 in disease progression.     

Adenoviruses with nonidentical terminal sequences are viable.

Adenovirus genomes consist of linear DNA molecules containing inverted terminal repeat sequences (ITRs) of 100 to 200 base pairs. The importance of identical termini for viability of adenoviruses was investigated. The viral strains used in this study were wild-type adenovirus type 5 (Ad5) and a variant Ad2 strain with termini which were distinct from those of all other human adenoviruses sequenced to date. A hybrid virus (sub54), obtained by recombination between Ad2 and Ad5, derived the left 42 to 52% of its genome from Ad2 and the right 58 to 48% from Ad5. Southern blotting analysis with labeled oligodeoxynucleotides indicated that both Ad2 and Ad5 ITRs were present in sub54 viral DNA preparations, and successive plaque purifications of sub54 demonstrated that viruses with nonidentical terminal sequences were viable but were rapidly converted to viruses with identical ends. Cloning of the sub54 genome as a bacterial plasmid supported the observations made by analysis of sub54 virion DNA. A plasmid, pFG154, was isolated which contained the entire adenovirus genome with an Ad2 ITR at the left terminus covalently linked to an Ad5 ITR at the right terminus. Upon transfection of mammalian cells with pFG154, viral progeny were obtained which had all possible combinations of termini, thus confirming that molecules with nonidentical termini are viable. Pure populations of viruses with nonidentical termini could not be isolated, suggesting efficient repair of one end with the opposite terminus used as a template. A model for this process is proposed involving strand displacement replication and emphasizing the importance of panhandle formation (annealing of terminal sequences) as a replicative intermediate.     

Viral sequences are associated with many histocompatibility genes

A C57BL/6By 5.5 kb Pvu II polymorphic restriction fragment which hybridizes with a spleen focus-forming env probe and maps in the H-30 region has been cloned, and a 358 by subfragment subcloned. Hybridization and sequencing studies show that the 358 by fragment is encoded by the region of the pol gene of murine retrovirus which codes for an endonuclease critical for viral integration. Hybridizations of digested murine genomic DNAs with the 358 by probe generate 31 restriction fragment length polymorphisms (RFLPs); 16 of these can be placed near the following 15 minor histocompatibility (H) loci: H-3, H-4, H-7, H-13, H-15, H-16, H-17, H-19, H-22, H-24, H-27, H-30, H-34, H-36, and H-38. We suggest that the proximity of viral sequences to H loci is probably evolutionarily and functionally significant and that the closeness of viral sequences and minor H loci can probably be utilized to facilitate the cloning of minor H genes. During the course of these studies, it has become possible to tentatively assign H-17, H-34, and H-38 to chromosome 12. In addition, it was observed that several H-2 congenic strains retain portions of chromosome 12 from the parental donor strains used in their derivation.     

Image-based patient-specific flow simulations are consistent with stroke in pediatric cerebrovascular disease

Biomechanics and Modeling in Mechanobiology - Moyamoya disease (MMD) is characterized by narrowing of the distal internal carotid artery and the circle of Willis (CoW) and leads to recurring...     

Egg laying decisions in Drosophila are consistent with foraging costs of larval progeny

Decision-making is defined as selection amongst options based on their utility, in a flexible and context-dependent manner. Oviposition site selection by the female fly, Drosophila melanogaster, has been suggested to be a simple and genetically tractable model for understanding the biological mechanisms that implement decisions. Paradoxically, female Drosophila have been found to avoid oviposition on sugar which contrasts with known Drosophila feeding preferences. Here we demonstrate that female Drosophila prefer egg laying on sugar, but this preference is sensitive to the size of the egg laying substrate. With larger experimental substrates, females preferred to lay eggs directly on sugar containing media over other (plain, bitter or salty) media. This was in contrast to smaller substrates with closely spaced choices where females preferred non-sweetened media. We show that in small egg laying chambers newly hatched first instar larvae are able to migrate along a diffusion gradient to the sugar side. In contrast, in contexts where females preferred egg laying directly on sugar, larvae were unable to migrate to find the sucrose if released on the sugar free side of the chamber. Thus, where larval foraging costs are high, female Drosophila choose to lay their eggs directly upon the nutritious sugar substrate. Our results offer a powerful model for female decision-making.     

The kinetics of bovine growth hormone folding are consistent with a framework model

The framework model of protein folding requires the hydrogen-bonded secondary structure to be formed early in folding (i.e. the formation of secondary structure precedes the tertiary structure) (Kim, P. S., and Baldwin, R. L. (1982) Annu. Rev. Biochem. 51, 459-489). To test the framework model directly the kinetics of bovine growth hormone (bGH) folding were compared utilizing two methods of detection, one that measures the secondary structure (far ultraviolet circular dichroism) and another that measures the tertiary structure (near ultraviolet absorbance). The results demonstrate that, under identical folding conditions, the kinetics observed by far ultraviolet circular dichroism are faster than those observed by ultraviolet absorption. The faster kinetics observed by circular dichroism indicate the existence of a helix-containing intermediate which is consistent with the framework model. The effect of protein concentration and denaturant concentration on the kinetics of refolding were studied. The rate of refolding measured by absorbance and circular dichroism was dependent on protein concentration. The protein concentration dependence on refolding is due to the transient formation of an associated intermediate. The concentration dependence of folding is taken as evidence that folding is a sequential process with partially folded monomers responsible for the observed association effect. At dilute protein concentrations the refolding can be studied independent of the association phenomena. The growth hormones utilized in this study were derived from Escherichia coli through recombinant DNA technology and from bovine pituitaries. The pituitary-derived bGH has been shown to be heterogeneous at the NH2 terminus (Lorenson, M. F., and Ellis, S. (1975) Endocrinology 96, 833-838), whereas the recombinant DNA-derived bGH contains a single NH2 terminus. No differences in the folding kinetics between the recombinant DNA and pituitary derived-bGH were observed. It is concluded that the heterogeneity of the NH2 terminus of growth hormone obtained from bovine pituitaries does not affect the observed in vitro folding kinetics.     

Phylogenetic relationships of Loxosceles and Sicarius spiders are consistent with Western Gondwanan vicariance

The modern geographic distribution of the spider family Sicariidae is consistent with an evolutionary origin on Western Gondwana. Both sicariid genera, Loxosceles and Sicarius are diverse in Africa and South/Central America. Loxosceles are also diverse in North America and the West Indies, and have species described from Mediterranean Europe and China. We tested vicariance hypotheses using molecular phylogenetics and molecular dating analyses of 28S, COI, 16S, and NADHI sequences. We recover reciprocal monophyly of African and South American Sicarius, paraphyletic Southern African Loxosceles and monophyletic New World Loxosceles within which an Old World species group that includes L. rufescens is derived. These patterns are consistent with a sicariid common ancestor on Western Gondwana. North American Loxosceles are monophyletic, sister to Caribbean taxa, and resolved in a larger clade with South American Loxosceles. With fossil data this pattern is consistent with colonization of North America via a land bridge predating the modern Isthmus of Panama.     

Low plasma levels of 2-hydrdxyestrone are consistent with its rapid metabolic clearance

Published plasma levels of the catechol estrogen 2-hydroxyestrone (2-OHE₁) are comparable to those of estrone and estradiol. In light of the very high (40,000 L/d) metabolic clearance rate of 2-OHEi, these concentrations imply unreasonable production rates. We therefore re-examined plasma 2-OHE₁ levels using a modified radiotnnmmoassay procedure. Plasma samples are extracted with ethyl acetate and passed over a short column of LH-20 Sephadex before equilibration with an antiserum directed against a 2-hydroxyestrone-17-(O-carboxymethyl)oxime-bovine serum albumin conjugate. Plasma 2-OHE₁ concentrations are indistinguishable from blank (< 15 pg/ml) in men and non-pregnant women, but rise to ~ 200 pg/ml during pregnancy. These values for 2-OHE₁ levels are consistent with the rapid metabolic clearance of this catechol estrogen.     

In silico predictions of Escherichia coli metabolic capabilities are consistent with experimental data

A significant goal in the post-genome era is to relate the annotated genome sequence to the physiological functions of a cell. Working from the annotated genome sequence, as well as biochemical and physiological information, it is possible to reconstruct complete metabolic networks. Furthermore, computational methods have been developed to interpret and predict the optimal performance of a metabolic network under a range of growth conditions. We have tested the hypothesis that Escherichia coli uses its metabolism to grow at a maximal rate using the E. coli MG1655 metabolic reconstruction. Based on this hypothesis, we formulated experiments that describe the quantitative relationship between a primary carbon source (acetate or succinate) uptake rate, oxygen uptake rate, and maximal cellular growth rate. We found that the experimental data were consistent with the stated hypothesis, namely that the E. coli metabolic network is optimized to maximize growth under the experimental conditions considered. This study thus demonstrates how the combination of in silico and experimental biology can be used to obtain a quantitative genotype-phenotype relationship for metabolism in bacterial cells.