development of a new reference standard for microarray experiments

Often microarray studies require a reference to indirectly compare the samples under observation. References based on pooled RNA from different cell lines have already been described (here referred to as RNA-R), but they usually do not exhaustively represent the set of genes printed on a chip, thus requiring many adjustments during the analyses. A reference could also be generated in vitro transcribing the collection of cDNA clones printed on the microarray in use (here referred to as T3-R). Here we describe an alternative and simpler PCR-based methodology to construct a similar reference (Chip-R), and we extensively test and compare it to both RNA-R and T3-R. The use of both Chip-R and T3-R dramatically increases the number of signals on the slides and gives more reproducible results than RNA-R. Each reference preparation is also evaluated in a simple microarray experiment comparing two different RNA populations. Our results show that the introduction of a reference always interferes with the analysis. Indeed, the direct comparison is able to identify more up- or down-regulated genes than any reference-mediated analysis. However, if a reference has to be used, Chip-R and T3-R are able to guarantee more reliable results than RNA-R

Effective antiprotease-antibiotic treatment of experimental anthrax

BACKGROUND: Inhalation anthrax is characterized by a systemic spread of the challenge agent, Bacillus anthracis. It causes severe damage, including multiple hemorrhagic lesions, to host tissues and organs. It is widely believed that anthrax lethal toxin secreted by proliferating bacteria is a major cause of death, however, the pathology of intoxication in experimental animals is drastically different from that found during the infectious process. In order to close a gap between our understanding of anthrax molecular pathology and the most prominent clinical features of the infectious process we undertook bioinformatic and experimental analyses of potential proteolytic virulence factors of B. anthracis distinct from lethal toxin. METHODS: Secreted proteins (other than lethal and edema toxins) produced by B. anthracis were tested for tissue-damaging activity and toxicity in mice. Chemical protease inhibitors and rabbit immune sera raised against B. anthracis proteases were used to treat mice challenged with B. anthracis (Sterne) spores. RESULTS: B. anthracis strain delta Ames (pXO1(-), pXO2(-)) producing no lethal and edema toxins secrets a number of metalloprotease virulence factors upon cultivation under aerobic conditions, including those with hemorrhagic, caseinolytic and collagenolytic activities, belonging to M4 and M9 thermolysin and bacterial collagenase families, respectively. These factors are directly toxic to DBA/2 mice upon intratracheal administration at 0.5 mg/kg and higher doses. Chemical protease inhibitors (phosphoramidon and 1, 10-phenanthroline), as well as immune sera against M4 and M9 proteases of B. anthracis, were used to treat mice challenged with B. anthracis (Sterne) spores. These substances demonstrate a substantial protective efficacy in combination with ciprofloxacin therapy initiated as late as 48 h post spore challenge, compared to the antibiotic alone. CONCLUSION: Secreted proteolytic enzymes are important pathogenic factors of B. anthrasis, which can be considered as effective therapeutic targets in the development of anthrax treatment and prophylactic approaches complementing anti-lethal toxin therapy

Validation of endogenous reference genes for qRT-PCR analysis of human visceral adipose samples

<p>Abstract</p> <p>Background</p> <p>Given the epidemic proportions of obesity worldwide and the concurrent prevalence of metabolic syndrome, there is an urgent need for better understanding the underlying mechanisms of metabolic syndrome, in particular, the gene expression differences which may participate in obesity, insulin resistance and the associated series of chronic liver conditions. Real-time PCR (qRT-PCR) is the standard method for studying changes in relative gene expression in different tissues and experimental conditions. However, variations in amount of starting material, enzymatic efficiency and presence of inhibitors can lead to quantification errors. Hence the need for accurate data normalization is vital. Among several known strategies for data normalization, the use of reference genes as an internal control is the most common approach. Recent studies have shown that both obesity and presence of insulin resistance influence an expression of commonly used reference genes in omental fat. In this study we validated candidate reference genes suitable for qRT-PCR profiling experiments using visceral adipose samples from obese and lean individuals.</p> <p>Results</p> <p>Cross-validation of expression stability of eight selected reference genes using three popular algorithms, <it>GeNorm</it>, <it>NormFinder </it>and <it>BestKeeper </it>found <it>ACTB </it>and <it>RPII </it>as most stable reference genes.</p> <p>Conclusions</p> <p>We recommend <it>ACTB </it>and <it>RPII </it>as stable reference genes most suitable for gene expression studies of human visceral adipose tissue. The use of these genes as a reference pair may further enhance the robustness of qRT-PCR in this model system.</p

Acceleration of epithelial cell syndecan-1 shedding by anthrax hemolytic virulence factors

BACKGROUND: It has been recently reported that major pathogens Staphylococcus aureus and Pseudomonas aeruginosa accelerate a normal process of cell surface syndecan-1 (Synd1) ectodomain shedding as a mechanism of host damage due to the production of shedding-inducing virulence factors. We tested if acceleration of Synd1 shedding takes place in vitro upon treatment of epithelial cells with B. anthracis hemolysins, as well as in vivo during anthrax infection in mice. RESULTS: The isolated anthrax hemolytic proteins AnlB (sphingomyelinase) and AnlO (cholesterol-binding pore-forming factor), as well as ClnA (B. cereus homolog of B. anthracis phosphatidyl choline-preferring phospholipase C) cause accelerated shedding of Synd1 and E-cadherin from epithelial cells and compromise epithelial barrier integrity within a few hours. In comparison with hemolysins in a similar range of concentrations, anthrax lethal toxin (LT) also accelerates shedding albeit at slower rate. Individual components of LT, lethal factor and protective antigen are inactive with regard to shedding. Inhibition experiments favor a hypothesis that activities of tested bacterial shedding inducers converge on the stimulation of cytoplasmic tyrosine kinases of the Syk family, ultimately leading to activation of cellular sheddase. Both LT and AnlO modulate ERK1/2 and p38 MAPK signaling pathways, while JNK pathway seems to be irrelevant to accelerated shedding. Accelerated shedding of Synd1 also takes place in DBA/2 mice challenged with Bacillus anthracis (Sterne) spores. Elevated levels of shed ectodomain are readily detectable in circulation after 24 h. CONCLUSION: The concerted acceleration of shedding by several virulence factors could represent a new pathogenic mechanism contributing to disruption of epithelial or endothelial integrity, hemorrhage, edema and abnormal cell signaling during anthrax infection

Pro-apoptotic and antiproliferative activity of human KCNRG, a putative tumor suppressor in 13q14 region

Deletion of 13q14.3 and a candidate gene KCNRG (potassium channel regulating gene) is the most frequent chromosomal abnormality in B-cell chronic lymphocytic leukemia and is a common finding in multiple myeloma (MM). KCNRG protein may interfere with the normal assembly of the K+ channel proteins causing the suppression of Kv currents. We aimed to examine possible role of KCNRG haploinsufficiency in chronic lymphocytic leukemia (CLL) and MM cells. We performed detailed genomic analysis of the KCNRG locus; studied effects of the stable overexpression of KCNRG isoforms in RPMI-8226, HL-60, and LnCaP cells; and evaluated relative expression of its transcripts in various human lymphomas. Three MM cell lines and 35 CLL PBL samples were screened for KCNRG mutations. KCNRG exerts growth suppressive and pro-apoptotic effects in HL-60, LnCaP, and RPMI-8226 cells. Direct sequencing of KCNRG exons revealed point mutation delT in RPMI-8226 cell line. Levels of major isoform of KCNRG mRNA are lower in DLBL lymphomas compared to normal PBL samples, while levels of its minor mRNA are decreased across the broad range of the lymphoma types. The haploinsufficiency of KCNRG might be relevant to the progression of CLL and MM at least in a subset of patients

Iron-Binding Compounds Produced by Wood-Decaying Basidiomycetes

The chrome azurol-S universal siderophore assay and the rapid paper electrophoresis siderophore assay were used to screen 10 wood-decaying basidiomycete isolates for the formation of iron-chelating compounds. All 10 isolates were positive for chrome azurol-S reactivity on solid plating medium and in liquid cultures, and 9 of the 10 isolates produced fluorescent iron-binding compounds in the paper electrophoresis assay

Evidence for Inhibition of Termite (Isoptera: Rhinotermitidae) Feeding by Extracellular Metabolites of a Wood Decay Fungus

Siderophores are iron-chelating extracellular fungal metabolites which may be involved in initiating wood decay. A purified siderophore extract isolated from the brown-rot decay fungus Gloeophyllum trabeum (Pers. ex Fr.) Murr. (Basidiomycetes: Polyporaceae) was found to deter feeding by Formosan subterranean termites, Coptotermes formosanus Shiraki (Isoptera: Rhinotermitidae). This fungus has previously been associated with preferential feeding on decayed wood by subterranean termites, and solvent extracts have been reported to induce termite trail-following, arrestment, and/or aggregation. This is the first report of G. trabeum metabolites or fungal siderophores having a negative behavioral effect on subterranean termites