A novel high-throughput screening method is described in which a family of DNA aptamers selected against E. coli outer membrane proteins (OMPs) is subjected to PCR in the presence of fluorophore-dUTP conjugates using Deep Vent® exo- polymerase. The fluorophore-doped aptamers and their complementary strands are then heated to render them single-stranded and screened in filter well microtiter plates for fluorescence resonance energy transfer (FRET) assay potential. Using this system, a superior competitive FRET-aptamer designated EcO 4R was identified and the location of its putative binding pocket was determined by individually testing FRET potential in each of the secondary loop structures. By labeling the binding pocket with Alexa Fluor (AF) 647 and binding the aptamer to heavily Black Hole Quencher-3 (BHQ-3)-labeled E. coli bacteria, detection of as few as 30 live unlabeled E. coli per ml was achieved in a competitive displacement FRET assay format. The far red fluorescence emission enables detection in largely blue-green autofluorescent matrices. In addition, the competitive transfer of AF 647-EcO-4R aptamer to unlabeled E. coli cells after a 15 min equilibration period was verified by fluorescence microscopy. The present study also demonstrated that high aptamer affinity is not well correlated with competitive FRET potential.
A novel assay was developed for the detection of Bacillus thuringiensis (BT) spores. The assay is based on the fluorescence observed after binding an aptamer-quantum dot conjugate to BT spores. The in vitro selection and amplification technique called SELEX (Systematic Evolution of Ligands by EXponential enrichment) was used in order to identify the DNA aptamer sequence specific for BT. The 60 base aptamer was then coupled to fluorescent zinc sulfide-capped, cadmium selenide quantum dots (QD). The assay is semi-quantitative, specific and can detect BT at concentrations of about 1,000 colony forming units/ml.
BackgroundNucleic acid aptamers have long demonstrated the capacity to bind viral envelope proteins and to inhibit the progression of pathogenic virus infections. Here we report on initial efforts to develop and screen DNA aptamers against recombinant envelope proteins or synthetic peptides and whole inactivated viruses from several virulent arboviruses including Chikungunya, Crimean-Congo hemorrhagic fever (CCHF), dengue, tickborne encephalitis and West Nile viruses. We also analyzed sequence data and secondary structures for commonalities that might reveal consensus binding sites among the various aptamers. Some of the highest affinity and most specific aptamers in the down-selected libraries were demonstrated to have diagnostic utility in lateral flow chromatographic assays and in a fluorescent aptamer-magnetic bead sandwich assay. Some of the reported aptamers may also be able to bind viral envelope proteins in vivo and therefore may have antiviral potential in passive immunity or prophylactic applications.ResultsSeveral arbovirus DNA aptamer sequences emerged multiple times in the various down selected aptamer libraries thereby suggesting some consensus sequences for binding arbovirus envelope proteins. Screening of aptamers by enzyme-linked aptamer sorbent assay (ELASA) was useful for ranking relative aptamer affinities against their cognate viral targets. Additional study of the aptamer sequences and secondary structures of top-ranked anti-arboviral aptamers suggest potential virus binding motifs exist within some of the key aptamers and are highlighted in the supplemental figures for this article. One sequence segment (ACGGGTCCGGACA) emerged 60 times in the anti-CCHF aptamer library, but nowhere else in the anti-arbovirus library and only a few other times in a larger library of aptamers known to bind bacteria and rickettsia or other targets. Diagnostic utility of some of the aptamers for arbovirus detection in lateral flow chromatographic assays and a fluorescent sandwich assay on the surface of magnetic microbeads is also demonstrated.ConclusionsThis article catalogues numerous DNA aptamer sequences which can bind various important pathogenic arboviruses and have, in some cases, already demonstrated diagnostic potential. These aptamer sequences are proprietary, patent-pending, and partially characterized. Therefore, they are offered to the scientific community for potential research use in diagnostic assays, biosensor applications or for possible passive immunity and prophylaxis against pathogenic viruses.
Endothelin-1 (ET-1) is a vasoactive peptide that modulates bone metabolism via regulatory effects on osteoblasts, chondrocytes, and osteoclasts. While ET-1 may circulate in the blood stream, tissue-specific expression of this peptide is more physiologically relevant. In the present study we measured ET-1 synthesis in sections of fetal rat calvaria (FRC) and in cultured FRC osteoblasts. Regulation of ET-1 synthesis in FRC osteoblasts by bone morphogenetic protein-7 (BMP-7) and transforming growth factor-beta1 (TGF-beta1) also was examined. Immunohistochemical analysis revealed ET-1 staining in calvarial osteoblasts, endothelial cells, and osteocytes. ET-1 mRNA expression was detected in cultured FRC cells and ET-1 peptide was present in conditioned media. During long-term culture of FRC cells (26 days) ET-1 peptide production rose sharply and peaked during the time of cellular proliferation (Days 0-3) then returned to baseline levels by Day 18, when mineralized nodules were forming. Treatment of FRC cells with BMP-7 enhanced ET-1 levels by three-fold on Day 3 and enhanced nodule formation by 15-fold on Day 26. To determine whether ET-1 was involved in an autocrine manner in BMP-7-induced nodule formation, cells were cultured in the presence of BMP-7 and BQ-123, an ET(A) receptor antagonist. BQ-123 had no effect on nodule formation in control or BMP-7-treated cells, indicating that osteoblast-derived ET-1 regulates other cell types in vivo during the bone formation process.
ABSTRACT. The geographical distribution of Bacillus anthracis strains and isolates bearing some of the same genetic markers as the Amerithrax Ames isolate was examined and evaluated. At least one mechanism for the horizontal movement of genetic markers was shown amongst isolates and closely related species and the effect of such mixing was demonstrated on phenotype. The results provided potential mechanisms by which attempts to attribute isolates of Bacillus anthracis to certain geographical and isolate sources may be disrupted.Bacillus anthracis appears to be a bacterium that has unique qualities that might allow using the genotype alone to determine the geographical distribution of genetic markers. It is extremely genetically homogeneous across its worldwide distributed strains (Keim et al. 1997(Keim et al. , 2000(Keim et al. , 2002. However, there are enough single-nucleotide polymorphisms (SNPs) and variable tandem repeat sequence number differences among various isolates to appear to distinguish B. anthracis from various locales. This lack of substantial variability may be attributed to the low-passage number of natural anthrax bacteria and their relative isolation to very limited areas because they are purported to reside in soil as spores for decades before infecting a host and completing another cycle of replication (Keim et al. 1997). The laboratory mutation rate of B. anthracis is ≈2 × 10 -5 (Keim et al. 2001) and the natural mutation rate is ≈2 × 10 -10 (Van Ert et al. 2007), assuming little or no recombination, which may not be the case (Ko et al. 2004); however, we have previously shown that nitration conditions in vitro easily generate profound mutations . Such conditions mimic the inflammatory activity of nitric-oxide synthase, NADPH oxidase and peroxidase in leukocytes against microbes in vivo. The sheer number of organisms produced in the terminal infection of a large animal host, such as a cow, should easily produce mutations well represented in the final spore yield based on the number of spores produced alone. A 550-kg cow could yield 60 mL/kg blood containing ≈10 8 spores per mL (Swartz 2001), totaling 3.3 × 10 12 spores per carcass. This yield could contain from 3.3 × 10 2 to 3.3 × 10 7 mutants. MATERIALS AND METHODSMicrobiological methods. β-Hemolysis was observed on tryptic soy agar plates with 5 % sheep blood (BAP; Remel) after 18 h of growth at 37 °C. Cherry γ phage spot tests were performed on host lawns derived from tryptic soy broth (TSB) cultures, incubated for 18 h at 37 °C with shaking (150 rpm). Plates were swabbed bidirectionally with host lawn, incubated for 2 h at 37 °C, spot-inoculated with 20 μL of undiluted Cherry γ phage, and incubated for 18 h at 37 °C. Antibiotic disk sensitivities were performed with disks placed directly on lawns omitting the 2-h incubation step used for the phage tests. Cherry γ phage rescue used 500-μL aliquots of host culture (host-inoculated TSB incubated for 18 h at 37 °C with shaking) seeded with 20 μL of serially diluted Cherry γ phage (10 0 , 1...
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