Biotin-labeled Oligonucleotide Research Articles

Development of polymerase chain reaction-lateral flow dipstick assay for detection of Mycoplasma bovis in cattle

Mycoplasma bovis (M. bovis) is capable of causing a range of diseases in cattle, encompassing calf pneumonia, arthritis, conjunctivitis, meningitis, and mastitis. It is widely recognized as one of the predominant pathogens posing a significant threat to the global cattle industry. Therefore, accurate and sensitive methods are urgently needed to detect M. bovis. This study aims to detect M. bovis by combining colloidal gold with biotin-labeled oligonucleotides to improve detection sensitivity and form a chromogenic detection probe based on signal amplification technology. Here, we developed a sensitive and specific polymerase chain reaction-lateral flow dipstick assay (PCR-LFD) strip for efficient nucleic acid detection of M. bovis. A pair of specific primers with 5’ ends labeled with biotin and digoxigenin probes was designed for PCR experiments. Colloidal gold particles-labeled anti-digoxigenin IgG coated gold-labeled test strip was prepared, streptavidin was used as the detection probe, and nitrocellulose membrane coated goat anti-mouse IgG was used as the control line. Our results showed that the detection limit of the PCR-LFD was 89 fg/µL for the M. bovis DNA. The results from the test strip were highly consistent with those from real-time qPCR. This assay were highly specific for M. bovis, as there were no cross-reactions with other microorganisms tested and the detection sensitivity of the test was also relatively high (97.67%). The novel strips present a promising tool for the cost-effective and sensitive diagnosis of M. bovis.

Alignment of helicases on single-stranded DNA increases activity.

Helicases function in most biological processes that utilize RNA or DNA nucleic acids including replication, recombination, repair, transcription, splicing, and translation. They are motor proteins that bind ATP and then catalyze hydrolysis to release energy which is transduced for conformational changes. Different conformations correspond to different steps in a process that results in movement of the enzyme along the nucleic acid track in a unidirectional manner. Some helicases such as DEAD-box helicases do not translocate, but these enzymes transduce chemical energy from ATP hydrolysis to unwind secondary structure in DNA or RNA. Some helicases function as monomers while others assemble into defined structures, either dimers or higher order oligomers. Dda helicase from bacteriophage T4 and NS3 helicase domain from the hepatitis C virus are examples of monomeric helicases. These helicases can bind to single-stranded DNA in a manner that appears like train engines on a track. When monomeric helicases align on DNA, the activity of the enzymes increases. Helicase activity can include the rate of duplex unwinding and the total number of base pairs melted during a single binding event or processivity. Dda and NS3h are considered as having low processivity, unwinding fewer than 50 base pairs per binding event. Here, we report fusing two molecules of NS3h molecules together through genetically linking the C-terminus of one molecule to the N-terminus of a second NS3h molecule. We observed increased processivity relative to NS3h possibly arising from the increased probability that at least one of the helicases will completely unwind the DNA prior to dissociation. The dimeric enzyme also binds DNA more like the full-length NS3 helicase. Finally, the dimer can displace streptavidin from biotin-labeled oligonucleotide, whereas monomeric NS3h cannot.

DNA immobilization and detection using DNAbinding proteins.

The immobilization of sensing bioreceptors is a critical feature affecting the final performance of a biosensor. For DNA detection, the (strept)avidin-biotin affinity interaction is often used for the immobilization of biotin-labeled oligonucleotides or PCR amplicons. Herein, DNAbinding proteins are proposed as alternative universal anchors for both DNA immobilization and detection, based on the strong and specific affinity interaction between certain DNAbinding proteins and their respective dsDNA binding sites. These binding sites can be incorporated in the target DNA molecule during synthesis and by PCR, eliminating the need for post-synthesis chemical modification and resulting in lower costs. When scCro DNAbinding protein was immobilized on microplates and nitrocellulose membrane, both ssDNA and dsDNA targets were successfully detected. The detection limits achieved were similar to those obtained with the streptavidin-biotin system. However, the scCro system resulted in higher signals while using less amount of protein. The adsorption properties of scCro were superior to streptavidin's, making scCro a viable alternative as an anchor biomolecule for the development of DNA assays and biosensors. Finally, a nucleic acid lateral flow assay based solely on two different DNAbinding proteins, scCro and dHP, was developed for the detection of a PCR amplicon. Overall, the proposed system appears to be very promising and with potential use for multiplex detection using various DNAbinding proteins with different sequence specificities. Further work is required to better understand the adsorption properties of these biomolecules on nitrocellulose, optimize the assays comprehensively, and achieve improved sensitivities.

Role for carbohydrate response element-binding protein (ChREBP) in high glucose-mediated repression of long noncoding RNA Tug1

Long noncoding RNAs (lncRNAs) have been shown to play key roles in a variety of biological activities of the cell. However, less is known about how lncRNAs respond to environmental cues and what transcriptional mechanisms regulate their expression. Studies from our laboratory have shown that the lncRNA Tug1 (taurine upregulated gene 1) is crucial for the progression of diabetic kidney disease, a major microvascular complication of diabetes. Using a combination of proximity labeling with the engineered soybean ascorbate peroxidase (APEX2), ChIP-qPCR, biotin-labeled oligonucleotide pulldown, and classical promoter luciferase assays in kidney podocytes, we extend our initial observations in the current study and now provide a detailed analysis on a how high-glucose milieu downregulates Tug1 expression in podocytes. Our results revealed an essential role for the transcription factor carbohydrate response element binding protein (ChREBP) in controlling Tug1 transcription in the podocytes in response to increased glucose levels. Along with ChREBP, other coregulators, including MAX dimerization protein (MLX), MAX dimerization protein 1 (MXD1), and histone deacetylase 1 (HDAC1), were enriched at the Tug1 promoter under high-glucose conditions. These observations provide the first characterization of the mouse Tug1 promoter's response to the high-glucose milieu. Our findings illustrate a molecular mechanism by which ChREBP can coordinate glucose homeostasis with the expression of the lncRNA Tug1 and further our understanding of dynamic transcriptional regulation of lncRNAs in a disease state.

The Arabidopsis lncRNA ASCO modulates the transcriptome through interaction with splicing factors.

Alternative splicing (AS) is a major source of transcriptome diversity. Long noncoding RNAs (lncRNAs) have emerged as regulators of AS through different molecular mechanisms. In Arabidopsis thaliana, the AS regulators NSRs interact with the ALTERNATIVE SPLICING COMPETITOR (ASCO) lncRNA. Here, we analyze the effect of the knock-down and overexpression of ASCO at the genome-wide level and find a large number of deregulated and differentially spliced genes related to flagellin responses and biotic stress. In agreement, ASCO-silenced plants are more sensitive to flagellin. However, only a minor subset of deregulated genes overlaps with the AS defects of the nsra/b double mutant, suggesting an alternative way of action for ASCO. Using biotin-labeled oligonucleotides for RNA-mediated ribonucleoprotein purification, we show that ASCO binds to the highly conserved spliceosome component PRP8a. ASCO overaccumulation impairs the recognition of specific flagellin-related transcripts by PRP8a. We further show that ASCO also binds to another spliceosome component, SmD1b, indicating that it interacts with multiple splicing factors. Hence, lncRNAs may integrate a dynamic network including spliceosome core proteins, to modulate transcriptome reprogramming in eukaryotes.

Pt-induced crosslinks promote target enrichment and protection from serum nucleases

Identifying the interactions of small molecules with biomolecules in complex cellular environments is a significant challenge. As one important example, despite being widely used for decades, much is still not understood regarding the cellular targets of Pt(II)-based anticancer drugs. In this study we introduce a novel method for isolation of Pt(II)-bound biomolecules using a DNA hybridization pull-down approach. Using a modified Pt reagent, click-ligation of a DNA oligonucleotide to both a Pt(II)-bound DNA hairpin and bovine serum albumin (BSA) are demonstrated. Subsequent hybridization to a biotin-labeled oligonucleotide allows for efficient isolation of Pt(II)-bound species by streptavidin pulldown. We also find that platinated bovine serum albumin readily crosslinks to DNA in the absence of click ligation, and that a fraction of BSA-bound Pt(II) can transfer to DNA over time. Interestingly, in in vitro studies, fragmented mammalian DNA that is crosslinked to BSA through Pt(II) exhibits significantly increased protection from degradation by serum nucleases.

Electrochemiluminescent Imaging for Multi-immunoassay Sensitized by Dual DNA Amplification of Polymer Dot Signal.

A true-color electrochemiluminescent (ECL) imaging strategy was designed for a multi-immunoassay of proteins by coupling highly efficient polymer dots (Pdots) with dual DNA amplification. The Pdots were prepared by nanoprecipitation of poly[(9,9-dioctylfuorenyl-2,7-diyl)- alt-co-(1,4-benzo-{2,1',3}-thiadiazole)] in the presence of poly(styrene- co-maleic anhydride) and functionalized with DNA1 that hybridized with black hole quencher-labeled DNA2 to self-quench the ECL emission. The Pdots modified Au/ITO electrode showed 100-fold stronger ECL emission than the Pdots modified ITO electrode. After the capture antibody immobilized on Au/ITO slide recognized the target protein and then reacted with biotin-labeled antibody, streptavidin and biotin-labeled oligonucleotide, respectively, a large number of DNA1 functionalized Pdots could be introduced onto the slide surface by rolling circle amplification of the oligonucleotide to trigger the enzymatically cyclic release of the Pdots from the self-quenched probes to solution in the presence of Exo III. The dual DNA amplification produced a greatly amplified ECL signal for true-color ECL imaging. Using carcinoembryonic antigen, cytokeratin-19-fragment, and neuron-specific enolase as a lung cancer-specific biomarker panel, the ECL imaging-based multi-immunoassay exhibited excellent performance with a linear range of 1 pg mL-1 to 500 ng mL-1 and limits of detection of 0.17, 0.12, and 0.22 pg mL-1, respectively. The proposed method could accurately detect these biomarkers in clinical human serum samples for lung cancer screening. The Pdots-based true-color ECL imaging approach possessed the advantages of visual analysis along with wide detection range and high sensitivity and thus has great potential in clinical application.

Sensitive mutant detection by concentrating mutant DNA with allele-specific capture and its application to analysis of contaminated grains in rice.

We developed a method for detection of mutants in a large number of plants, and found this method to be applicable to detection of a mutant allele at a concentration of 1/1000. Many techniques for SNP analysis have been developed, but most of these techniques are not so sensitive to be used for detection of mutants in a large number of plants. Although some highly sensitive methods of SNP analysis have been reported, they are costly. In the present study, a method for concentrating mutant DNA was examined for sensitive detection of an SNP allele in a bulked DNA sample. PCR products of mutant alleles were captured by biotin-labeled oligonucleotide conjugated with streptavidin-coated magnetic beads. By repeated captures of each strand and combining both strands, mutant alleles with a concentration of 1/1000 in wild-type alleles were detectable by CAPS or dCAPS analysis. Indirect capture of a mutant allele was possible, but efficiency was slightly lower than that of the direct capture. The developed method was applied to detection of contamination of rice grains by grains of a different cultivar. Possible applications of this method are discussed.

Liquid Hybridization and Solid Phase Detection: A Highly Sensitive and Accurate Strategy for MicroRNA Detection in Plants and Animals.

MicroRNAs (miRNAs) play important roles in nearly every aspect of biology, including physiological, biochemical, developmental and pathological processes. Therefore, a highly sensitive and accurate method of detection of miRNAs has great potential in research on theory and application, such as the clinical approach to medicine, animal and plant production, as well as stress response. Here, we report a strategic method to detect miRNAs from multicellular organisms, which mainly includes liquid hybridization and solid phase detection (LHSPD); it has been verified in various species and is much more sensitive than traditional biotin-labeled Northern blots. By using this strategy and chemiluminescent detection with digoxigenin (DIG)-labeled or biotin-labeled oligonucleotide probes, as low as 0.01–0.25 fmol [for DIG-CDP Star (disodium2-chloro-5-(4-methoxyspiro{1,2-dioxetane-3,2′-(5′-chloro)tricyclo[3.3.1.13,7]decan}-4-yl)phenyl phosphate) system], 0.005–0.1 fmol (for biotin-CDP Star system), or 0.05–0.5 fmol (for biotin-luminol system) of miRNA can be detected and one-base difference can be distinguished between miRNA sequences. Moreover, LHSPD performed very well in the quantitative analysis of miRNAs, and the whole process can be completed within about 9 h. The strategy of LHSPD provides an effective solution for rapid, accurate, and sensitive detection and quantitative analysis of miRNAs in plants and animals.

Chilo iridescent virus (CIV) ORF 012L encodes a protein with both exonuclease and endonuclease functions.

Chilo iridescent virus (CIV) is the type member of the genus Iridovirus within the family Iridoviridae. The virions of CIV contain a single linear dsDNA molecule that is circularly permuted and terminally redundant. The genome of CIV contains an open reading frame (ORF 012L) encoding a protein homologous to exonuclease II of Schizosaccharomyces pombe. In this study, we focused on the characterization of CIV ORF 012L. The target ORF was cloned into the pET28a vector, expressed in E. coli strain BL21 (DE3) pLysS with an N-terminal His tag and purified to homogeneity by using Ni-NTA affinity chromatography. Biochemical characterization of the purified CIV 012L confirmed that this viral protein is a functional 5'-3' exonuclease that digests 3'-biotin-labelled oligonucleotides and linear double-stranded DNA (dsDNA) molecules from their 5' termini in a highly processive manner. CIV 012L also has a potent endonuclease activity on dsDNA in vitro. In addition, CIV 012L converted supercoiled plasmid DNA (replicative form I, RFI) into the open circular form (RFII) and then open circular form into linear form (RFIII). Endonuclease activity of CIV 012L was optimal in the presence of 10 mM Mg(2+) or 30mM Mn(2+) ions and at 150mM NaCl or KCl salt concentrations. The highest endonuclease activity was obtained at pH 8, and it reached a maximum at 55°C. The CIV 012L protein showed deficiencies for both double- and single-stranded RNAs.

Definition of a Bidirectional Activity-Dependent Pathway Involving BDNF and Narp.

One of the cardinal features of neural development and adult plasticity is the contribution of activity-dependent signaling pathways. However, the interrelationships between different activity-dependent genes are not well understood. The immediate early gene neuronal-activity-regulated pentraxin (NPTX2 or Narp) encodes a protein that has been associated with excitatory synaptogenesis, AMPA receptor aggregation, and the onset of critical periods. Here, we show that Narp is a direct transcriptional target of brain-derived neurotrophic factor (BDNF), another highly regulated activity-dependent gene involved in synaptic plasticity. Unexpectedly, Narp is bidirectionally regulated by BDNF. Acute BDNF withdrawal results in downregulation of Narp, whereas transcription of Narp is greatly enhanced by BDNF. Furthermore, our results show that BDNF directly regulates Narp to mediate glutamatergic transmission and mossy fiber plasticity. Hence, Narp serves as a significant epistatic target of BDNF to regulate synaptic plasticity during periods of dynamic activity.

Human Obesity Associated with an Intronic SNP in the Brain-Derived Neurotrophic Factor Locus.

Brain-derived neurotrophic factor (BDNF) plays a key role in energy balance. In population studies, SNPs of the BDNF locus have been linked to obesity, but the mechanism by which these variants cause weight gain is unknown. Here, we examined human hypothalamic BDNF expression in association with 44 BDNF SNPs. We observed that the minor C allele of rs12291063 is associated with lower human ventromedial hypothalamic BDNF expression (p < 0.001) and greater adiposity in both adult and pediatric cohorts (p values < 0.05). We further demonstrated that the major T allele for rs12291063 possesses a binding capacity for the transcriptional regulator, heterogeneous nuclear ribonucleoprotein D0B, knockdown of which disrupts transactivation by the T allele. Binding and transactivation functions are both disrupted by substituting C for T. These findings provide a rationale for BDNF augmentation as a targeted treatment for obesity in individuals who have the rs12291063 CC genotype.

In situ hybridization for fungal ribosomal RNA sequences in paraffin-embedded tissues using biotin-labeled locked nucleic acid probes.

Ribosomal RNAs (rRNA) are conserved, abundant species-specific sequences that are used for phylogenetically classifying organisms. Due to their abundance and species specificity, rRNA sequences have been established as optimal targets for in situ hybridization (ISH). ISH for rRNA sequences using DNA oligonucleotide probes has been utilized to detect a variety of fungi in paraffin tissues. However, ISH with some oligonucleotide DNA probes produces weak signals, and applications for nucleotide modification may be useful to enhance hybridization signal. ISH with LNA probes has been shown to result in improved ISH signal. A protocol for LNA ISH with biotin-labeled LNA oligonucleotide probes is described.

Early Growth Response-1 (EGR-1) and Nuclear Factor of Activated T Cells (NFAT) Cooperate to Mediate CD40L Expression in Megakaryocytes and Platelets

Increasing evidence implicates circulating platelets as mediators of chronic inflammatory and autoimmune diseases via the expression and release of CD40L, an important modulator of inflammation and adaptive immune responses traditionally associated with activated T cells. Emerging evidence suggests that platelet CD40L is dynamically regulated in several chronic inflammatory and autoimmune diseases and may mediate progression and secondary pathology associated with those disease states. The present study identifies NFATc2 as a key transcriptional modulator of CD40L expression in megakaryocytes and inflammatory activity of platelets. Furthermore, the current data show that EGR-1, a member of the early growth response family of zinc finger transcription factors, modulates NFATc2-dependent regulation of CD40L expression in megakaryocytes. Our novel demonstration that in vivo biochemical or genetic inhibition of NFATc2 activity in megakaryocyte diminishes platelet CD40L implicates the NFATc2/EGR-1 axis as a key regulatory pathway of inflammatory and immunomodulatory activity in platelets and represents a target for the development of therapeutics for the potential treatment of chronic inflammatory and autoimmune diseases.

A Quantitative Assay to Study Protein:DNA Interactions, Discover Transcriptional Regulators of Gene Expression, and Identify Novel Anti-tumor Agents

Many DNA-binding assays such as electrophoretic mobility shift assays (EMSA), chemiluminescent assays, chromatin immunoprecipitation (ChIP)-based assays, and multiwell-based assays are used to measure transcription factor activity. However, these assays are nonquantitative, lack specificity, may involve the use of radiolabeled oligonucleotides, and may not be adaptable for the screening of inhibitors of DNA binding. On the other hand, using a quantitative DNA-binding enzyme-linked immunosorbent assay (D-ELISA) assay, we demonstrate nuclear protein interactions with DNA using the RUNX2 transcription factor that depend on specific association with consensus DNA-binding sequences present on biotin-labeled oligonucleotides. Preparation of cells, extraction of nuclear protein, and design of double stranded oligonucleotides are described. Avidin-coated 96-well plates are fixed with alkaline buffer and incubated with nuclear proteins in nucleotide blocking buffer. Following extensive washing of the plates, specific primary antibody and secondary antibody incubations are followed by the addition of horseradish peroxidase substrate and development of the colorimetric reaction. Stop reaction mode or continuous kinetic monitoring were used to quantitatively measure protein interaction with DNA. We discuss appropriate specificity controls, including treatment with non-specific IgG or without protein or primary antibody. Applications of the assay are described including its utility in drug screening and representative positive and negative results are discussed.

Peroxisome Proliferator-activated Receptor γ Agonists Induce Cell Cycle Arrest through Transcriptional Regulation of Krüppel-like Factor 4 (KLF4)

Peroxisome proliferator-activated receptor γ (PPARγ), a subgroup of ligand-activated nuclear receptors, plays critical roles in cell cycle regulation, differentiation, apoptosis, and invasion. PPARγ is involved in tumorigenesis and is a potent target for cancer therapy. PPARγ transactivation of KLF4 has been demonstrated in various studies; however, how PPARγ regulates KLF4 expression is not clear. In this study, we reveal that PPARγ regulates the expression of KLF4 by binding directly to the PPAR response element (PPRE) within the KLF4 promoter. The PPRE resides at -1657 to -1669 bp upstream of the KLF4 ATG codon, which is essential for the transactivation of troglitazone-induced KLF4 expression. Furthermore, we found that stable silencing of KLF4 obviously suppressed the G(1)/S arrest and anti-proliferation effects induced by PPARγ ligands. Taken together, our data indicate that up-regulation of KLF4 upon PPARγ activation is mediated through the PPRE in the KLF4 promoter, thus providing further insights into the PPARγ signal transduction pathway as well as a novel cancer therapeutic strategy.

Phosphatidylinositol 4-Phosphate 5-Kinase α Facilitates Toll-like Receptor 4-mediated Microglial Inflammation through Regulation of the Toll/Interleukin-1 Receptor Domain-containing Adaptor Protein (TIRAP) Location*

Phosphatidylinositol (PI) 4,5-bisphosphate (PIP(2)), generated by PI 4-phosphate 5-kinase (PIP5K), regulates many critical cellular events. PIP(2) is also known to mediate plasma membrane localization of the Toll/IL-1 receptor domain-containing adaptor protein (TIRAP), required for the MyD88-dependent Toll-like receptor (TLR) 4 signaling pathway. Microglia are the primary immune competent cells in brain tissue, and TLR4 is important for microglial activation. However, a functional role for PIP5K and PIP(2) in TLR4-dependent microglial activation remains unclear. Here, we knocked down PIP5Kα, a PIP5K isoform, in a BV2 microglial cell line using stable expression of lentiviral shRNA constructs or siRNA transfection. PIP5Kα knockdown significantly suppressed induction of inflammatory mediators, including IL-6, IL-1β, and nitric oxide, by lipopolysaccharide. PIP5Kα knockdown also attenuated signaling events downstream of TLR4 activation, including p38 MAPK and JNK phosphorylation, NF-κB p65 nuclear translocation, and IκB-α degradation. Complementation of the PIP5Kα knockdown cells with wild type but not kinase-dead PIP5Kα effectively restored the LPS-mediated inflammatory response. We found that PIP5Kα and TIRAP colocalized at the cell surface and interacted with each other, whereas kinase-dead PIP5Kα rendered TIRAP soluble. Furthermore, in LPS-stimulated control cells, plasma membrane PIP(2) increased and subsequently declined, and TIRAP underwent bi-directional translocation between the membrane and cytosol, which temporally correlated with the changes in PIP(2). In contrast, PIP5Kα knockdown that reduced PIP(2) levels disrupted TIRAP membrane targeting by LPS. Together, our results suggest that PIP5Kα promotes TLR4-associated microglial inflammation by mediating PIP(2)-dependent recruitment of TIRAP to the plasma membrane.

Study of Streptavidin-Modified Quantum Dots by Capillary Electrophoresis

Great boom of nanotechnologies impacts almost all areas of science and therefore detail understanding of the properties of nanomaterials as well as their interaction abilities is required. Surface modification and functionalization of nanoparticles is of a great interest due to the wide range of applications in the area of nanomedicine, nanobiology, and/or biochemistry. In this study, CdTe QDs were synthesized using microwave reactor and their surface was modified by streptavidin to ensure further suitability for bioconjugation with biotin-labelled oligonucleotides. For characterization of the synthesized QDs and for monitoring of the interaction with the oligonucleotide, capillary and gel electrophoresis was used. Moreover, complementary advantages of absorption (CE–UV) and laser-induced fluorescence detection (CE–LIF) were exploited. Comparison the electrophoretic mobilities obtained for streptavidin-modified QDs by CE–LIF (−9.87 × 10−9 m2/V/s) and by CE–UV (−10.02 × 10−9 m2/V/s) was in a good agreement enabling us to identify the peak of streptavidin-modified QDs in the CE–UV electropherogram containing also the peak of unreacted streptavidin. Subsequent conjugation of streptavidin-modified QDs with two model biotinylated oligonucleotides (BCL-2 and HBV) led to formation of the complex represented in the electropherograms as a very sharp peak. This peak height increased with time for 15.5 and 27 mAU using BCL-2 oligonucleotide and HBV oligonucleotide, respectively during 30 min interaction.

P057 Cytokine mediated regulation of PPARg activity in human macrophages

Introduction PPAR gamma has a well-defined role in atherosclerosis and diabetes and also serves as a link between metabolism and inflammation by modulating lipid uptake in macrophages. Therefore, determining the molecular mechanism of PPARg tissue specificity is critical for avoiding adverse effects arising from therapies targeting PPARg. Methods Our central hypothesis is that a cytokine induced mechanism might be responsible for the cell type specific function of PPARg in macrophages, thus we sought to identify novel PPARg co-regulators by combining biochemical and proteomic approaches. For this, nuclei of Thp-1 cells treated with Rosiglitazone and IL-4 was incubated with a biotin-labeled oligonucleotide encoding PPARg and STAT6 consensus sites, then the complex submitted to MS analysis. Results We identified several co-factor candidates and are in the process of validating these results. We also plan to confirm the in vivo binding of the candidates by ChIP assays and test their functional role on PPARg activity by siRNA mediated knock down experiments. Conclusions Our results suggest that PPARg constitutively binds DNA and it is the interaction with other transcription factors that fine-tunes its activity in macrophages. We expect that our studies will uncover some of the mechanisms by which PPARg exerts its cell-type specific functions in certain subsets of immune cells.

Detection of Vibrio parahaemolyticus in shellfish using polymerase chain reaction-enzyme-linked immunosorbent assay

This study evaluated the application of polymerase chain reaction-enzyme-linked immunosorbent assay (PCR-ELISA) for the detection of Vibrio parahaemolyticus in shellfish. The PCRs were selected to amplify a species-specific sequence region. In particular, internal tl biotin-labelled oligonucleotide probe was used to capture the DIG-labelled PCR products. Next, the probe PCR product hybrids, immobilized on a streptavidin-coated microtiter plate, were detected with peroxidase-conjugated anti-digoxigenin antibody (anti-DIG-POD) and the colorimetric peroxidase substrate ABTS [2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid)] using an ELISA plate reader. The PCR-ELISA system described is a feasible, sensitive method for the direct and specific detection of V. parahaemolyticus in shellfish samples. Compared with gel-based detection methods, PCR-ELISA in this study increased sensitivity by 100-fold for V. parahaemolyticus. The PCR-ELISA described may be used for potential rapid detection in routine shellfish analysis for the seafood industry. The sector requires simultaneous large-scale sample screenings to monitor contamination levels in processing plants and evaluate the performance of the hazard analysis and critical control point (HACCP) system. PCR-ELISA also proved to be economical, with a cost of about 9 Euros per sample, and the quick assay taking 8 h to complete starting from DNA extraction.