Seroprevalence of <Emphasis Type="Italic">Borrelia burgdorferi</Emphasis> sensu lato and <Emphasis Type="Italic">Anaplasma phagocytophilum</Emphasis> in Danish horses

Background

Borrelia burgdorferi sensu lato and Anaplasma phagocytophilum are able to infect horses. However, the extend to which Danish horses are infected and seroconvert due to these two bacteria is unknown. The aim of the present study was to evaluate the seroprevalence of B. burgdorferi sensu lato and A. phagocytophilum in Danish horses.

Methods

A total of 390 blood samples collected from all major regions of Denmark and with a geographical distribution corresponding to the density of the Danish horse population were analyzed. All samples were examined for the presence of antibodies against B. burgdorferi sensu lato and A. phagocytophilum by the use of the SNAP^®4DX ^® ELISA test.

Results

Overall, 29.0% of the horses were seropositive for B. burgdorferi sensu lato whereas 22.3% were seropositive for A. phagocytophilum.

Conclusions

Antibodies against B burgdorferi sensu lato and A. phagocytophilum are commonly found among Danish horses thus showing that Danish horses are frequently infected by these organisms.

     

A densely interconnected genome-wide network of microRNAs and oncogenic pathways revealed using gene expression signatures

MicroRNAs (miRNAs) are important components of cellular signaling pathways, acting either as pathway regulators or pathway targets. Currently, only a limited number of miRNAs have been functionally linked to specific signaling pathways. Here, we explored if gene expression signatures could be used to represent miRNA activities and integrated with genomic signatures of oncogenic pathway activity to identify connections between miRNAs and oncogenic pathways on a high-throughput, genome-wide scale. Mapping >300 gene expression signatures to >700 primary tumor profiles, we constructed a genome-wide miRNA-pathway network predicting the associations of 276 human miRNAs to 26 oncogenic pathways. The miRNA-pathway network confirmed a host of previously reported miRNA/pathway associations and uncovered several novel associations that were subsequently experimentally validated. Globally, the miRNA-pathway network demonstrates a small-world, but not scale-free, organization characterized by multiple distinct, tightly knit modules each exhibiting a high density of connections. However, unlike genetic or metabolic networks typified by only a few highly connected nodes ("hubs"), most nodes in the miRNA-pathway network are highly connected. Sequence-based computational analysis confirmed that highly-interconnected miRNAs are likely to be regulated by common pathways to target similar sets of downstream genes, suggesting a pervasive and high level of functional redundancy among coexpressed miRNAs. We conclude that gene expression signatures can be used as surrogates of miRNA activity. Our strategy facilitates the task of discovering novel miRNA-pathway connections, since gene expression data for multiple normal and disease conditions are abundantly available.     

Statistical methods of translating microarray data into clinically relevant diagnostic information in colorectal cancer

MOTIVATION: It is a common practice in cancer microarray experiments that a normal tissue is collected from the same individual from whom the tumor tissue was taken. The indirect design is usually adopted for the experiment that uses a common reference RNA hybridized both to normal and tumor tissues. However, it is often the case that the test material is not large enough for the experimenter to extract enough RNA to conduct the microarray experiment. Hence, collecting n cases does not necessarily end up with a matched pair sample of size n. Instead we usually have a matched pair sample of size n1, and two independent samples of sizes n2 and n3, respectively, for 'reference versus normal tissue only' and 'reference versus tumor tissue only' hybridizations (n=n1 + n2 + n3). Standard statistical methods need to be modified and new statistical procedures are developed for analyzing this mixed dataset. RESULTS: We propose a new test statistic, t3, as a means of combining all the information in the mixed dataset for detecting differentially expressed (DE) genes between normal and tumor tissues. We employed the extended receiver operating characteristic approach to the mixed dataset. We devised a measure of disagreement between a RT-PCR experiment and a microarray experiment. Hotelling's T2 statistic is employed to detect a set of DE genes and its prediction rate is compared with the prediction rate of a univariate procedure. We observe that Hotelling's T2 statistic detects DE genes more efficiently than a univariate procedure and that further research is warranted on the formal test procedure using Hotelling's T2 statistic. CONTACT: bskim@yonsei.ac.kr.     

In vitro analysis of the chain termination reaction in the synthesis of poly-(R)-beta-hydroxybutyrate by the class III synthase from Allochromatium vinosum

Allochromatium vinosum polyhydroxyalkanoate synthase catalyzes formation of poly-(R)-3-hydroxybutyrate (PHB) from (R)-3-hydroxybutyryl-coenzyme A (HB-CoA). (R)-3-Hydroxybutyryl-N-acetylcysteamine (HB-NAC) is an alternative substrate for this synthase in vitro, with a turnover 1% that of HB-CoA. With HB-NAC, the molecular weight (M(w)) of PHB produced at substrate-to-enzyme ratios of 1500-15 000 is approximately 75 kDa. (1)H NMR shows that PHB produced has hydroxybutyrate at the alcohol end and N-acetylcysteamine (NAC) at the carboxylate end of the polymer. Exogenous NAC has no effect on the M(w) of PHB produced with HB-CoA or HB-NAC in vitro, whereas PHB from a polymerization reaction with both HB-CoA and HB-NAC has intermediate M(w). These results can be accommodated by two models. In the first, NAC liberated at the active site during polymerization acts as a chain transfer agent. In the second, there is a noncovalent polymer intermediate covalently linked to NAC, which can dissociate from the active site.     

Roles of Multiple Acetoacetyl Coenzyme A Reductases in Polyhydroxybutyrate Biosynthesis in Ralstonia eutropha H16

The bacterium Ralstonia eutropha H16 synthesizes polyhydroxybutyrate (PHB) from acetyl coenzyme A (acetyl-CoA) through reactions catalyzed by a β-ketothiolase (PhaA), an acetoacetyl-CoA reductase (PhaB), and a polyhydroxyalkanoate synthase (PhaC). An operon of three genes encoding these enzymatic steps was discovered in R. eutropha and has been well studied. Sequencing and analysis of the R. eutropha genome revealed putative isologs for each of the PHB biosynthetic genes, many of which had never been characterized. In addition to the previously identified phaB1 gene, the genome contains the isologs phaB2 and phaB3 as well as 15 other potential acetoacetyl-CoA reductases. We have investigated the roles of the three phaB isologs by deleting them from the genome individually and in combination. It was discovered that the gene products of both phaB1 and phaB3 contribute to PHB biosynthesis in fructose minimal medium but that in plant oil minimal medium and rich medium, phaB3 seems to be unexpressed. This raises interesting questions concerning the regulation of phaB3 expression. Deletion of the gene phaB2 did not result in an observable phenotype under the conditions tested, although this gene does encode an active reductase. Addition of the individual reductase genes to the genome of the ΔphaB1 ΔphaB2 ΔphaB3 strain restored PHB production, and in the course of our complementation experiments, we serendipitously created a PHB-hyperproducing mutant. Measurement of the PhaB and PhaA activities of the mutant strains indicated that the thiolase reaction is the limiting step in PHB biosynthesis in R. eutropha H16 during nitrogen-limited growth on fructose.Polyhydroxyalkanoates (PHAs) are natural polyesters synthesized by a wide range of bacteria as carbon and energy reserves. PHAs are typically stored when organisms are in an environment in which carbon is plentiful but the lack of another nutrient limits normal cell growth. It has been found that in environments with fluctuating carbon levels, PHA producers have crucial advantages over rival species (14). In addition to their importance in the microbial world, these polymers have been studied for their potential uses in biodegradable consumer goods (12) and medical products (22) and as chemical precursors (4). Although many PHA monomers have been discovered, the most common are 3-hydroxyalkanoates (32). Common PHAs are typically characterized by their constituent monomers as short-chain-length polymers (SCL-PHA; C₄ and C₅ monomers) or medium-chain-length polymers (MCL-PHA; C₆ and longer monomers).The model organism used to study PHA biosynthesis is the Gram-negative bacterium Ralstonia eutropha. This organism accumulates a high percentage of its cell dry weight (CDW) as SCL-PHA under nutrient limitation. When grown on sugars or plant oils, R. eutropha makes poly(3-hydroxybutyrate) (PHB) almost exclusively, although the addition of precursors such as propionate to the growth medium can lead to incorporation of 3-hydroxyvalerate into the polymer chain as well (2). An operon of biosynthetic genes from R. eutropha encoding enzymes sufficient for synthesis of PHB from acetyl coenzyme A (acetyl-CoA), which consisted of phaC-phaA-phaB, was discovered in the late 1980s (25, 26, 36). In this pathway, two molecules of acetyl-CoA are condensed by a β-ketothiolase (PhaA) and the resulting acetoacetyl-CoA is reduced by a reductase (PhaB) to form (R)-3-hydroxybutyryl-CoA (HB-CoA), which is the substrate for the PHA synthase (PhaC). Sequencing and analysis of the R. eutropha genome revealed the existence of putative isologs for each of the PHA synthetic genes (29). While the existence of alternate β-ketothiolases was already known (39), most of the potential isologs identified had never been characterized.Our group wanted to better understand how acetoacetyl-CoA reduction occurs in R. eutropha. In addition to the earlier-identified phaB gene, now referred to as phaB1 (GeneID, 4249784), the genes phaB2 (GeneID, 4249785) and phaB3 (GeneID, 4250155) were discovered on R. eutropha chromosome 1. Fifteen other potential isologs were also found to encode amino acid sequences that could potentially indicate acetoacetyl-CoA reductase activity (29). The roles of the newly discovered genes in PHB biosynthesis were unclear, especially given the results of an earlier biochemical study that suggested there was a single NADPH-dependent acetoacetyl-CoA reductase in R. eutropha (10). In order to determine the roles of the reductase genes in R. eutropha, we deleted phaB1, phaB2, and phaB3 from the genome both individually and in combination. In addition to characterizing these newly discovered genes, we also hoped to eliminate or diminish formation of HB-CoA by stopping the reduction reaction. Efforts to purify the PHA synthase from R. eutropha have been complicated by the high levels of PHB made by this organism (7). Studying formation and growth of PHB granules is difficult because PHB accumulates at a high rate, causing individual granules to coalesce and become indistinct (44). We therefore believed that an R. eutropha strain with decreased HB-CoA synthesis would be a useful experimental tool and could also serve as a platform for engineering new PHA synthesis pathways into R. eutropha.     

Glioblastoma cell death induced by asiatic acid

Asiatic acid (AA), a triterpene, is known to be cytotoxic to several tumor cell lines. AA induces dose- and time-dependent cell death in U-87 MG human glioblastoma. This cell death occurs via both apoptosis and necrosis. The effect of AA may be cell type-specific as AA-induced cell death was mainly apoptotic in colon cancer RKO cells. AA-induced glioblastoma cell death is associated with decreased mitochondrial membrane potential, activation of caspase-9 and -3, and increased intracellular free Ca²⁺. Although treatment of glioblastoma cells with the caspase inhibitor zVAD-fmk completely abolished AA-induced caspase activation, it did not significantly block AA-induced cell death. AA-induced cell death was significantly prevented by an intracellular Ca²⁺ inhibitor, BAPTA/AM. Taken together, these results indicate that AA induces cell death by both apoptosis and necrosis, with Ca²⁺-mediated necrotic cell death predominating.