Selection of DNA aptamers that bind to influenza A viruses with high affinity and broad subtype specificity

AbstractMany cases of influenza are reported worldwide every year. The influenza virus often acquires new antigenicity, which is known as antigenic shift; this results in the emergence of new virus strains, for which preexisting immunity is not found in the population resulting in influenza pandemics. In the event a new strain emerges, diagnostic tools must be developed rapidly to detect the novel influenza strain. The generation of high affinity antibodies is costly and takes time; therefore, an alternative detection system, aptamer detection, provides a viable alternative to antibodies as a diagnostic tool. In this study, we developed DNA aptamers that bind to HA1 proteins of multiple influenza A virus subtypes by the SELEX procedure. To evaluate the binding properties of these aptamers using colorimetric methods, we developed a novel aptamer-based sandwich detection method employing our newly identified aptamers. This novel sandwich enzyme-linked aptamer assay successfully detected the H5N1, H1N1, and H3N2 subtypes of influenza A virus with almost equal sensitivities. These findings suggest that our aptamers are attractive candidates for use as simple and sensitive diagnostic tools that need sandwich system for detecting the influenza A virus with broad subtype specificities

Gene expression profiles of circulating leukocytes correlate with renal disease activity in IgA nephropathy

BACKGROUND: The goal of these studies was to explore the possibility of using gene expression profiles of circulating leukocytes as a functional fingerprint of nephritic disease activity. METHODS: This feasibility study utilized IgA nephropathy (IgAN) as a model system. Genes differentially expressed in IgAN patients were identified by Affymetrix GeneChip microarrays, and compared with gene expression of focal segmental glomerulosclerosis (FSGS), minimal change disease, antineutrophil cytoplasmic antibody (ANCA) glomerulonephritis, and with healthy volunteers. Of the genes identified, 15 transcriptionally up-regulated were validated in a larger cohort of patients using TaqMan polymerase chain reaction (PCR). To test whether increased expression of these genes correlated with disease activity, cluster analyses were performed utilizing the TaqMan PCR values. Taking a mathematical approach, we tested whether gene expression values were correlative with kidney function, as reflected by serum creatinine and creatinine clearance values. RESULTS: We identified 15 genes significantly correlative with disease activity in IgAN. This gene signature of IgAN patients' leukocytes reflected kidney function. This was demonstrated in that mathematically generated theoretical values of serum creatinine and creatinine clearance correlated significantly with actual IgAN patient values of serum creatinine and creatinine clearance. There was no apparent correlation with hematuria and proteinuria. The expression levels of this same gene set in ANCA glomerulonephritis or Lupus nephritis patients were not correlative with serum creatinine or creatinine clearance values. CONCLUSION: These data indicate that leukocytes carry informative disease-specific markers of pathogenic changes in renal tissue

Altered mRNA expression in renal biopsy tissue from patients with IgA nephropathy

Altered mRNA expression in renal biopsy tissue from patients with IgA nephropathy. Background. Immunoglobulin A (IgA) nephropathy (IgAN) is a renal disease characterized by glomerular deposition of IgA-dominant immune deposits that cause glomerular inflammation and sclerosis. Gene expression changes induced in renal tissues/cells as a result of the disease are largely uncharacterized. Methods. A sensitive differential mRNA display technique, restriction endonucleolytic analysis of differentially expressed sequences (READS®) compared similarly processed normal renal tissue to renal biopsy RNA from patients with IgAN, minimal change disease, and necrotizing crescentic glomerulonephritis. A subset of genes with altered expression in IgAN as identified by the READS® technology was further characterized and expression levels confirmed using real-time quantitative polymerase chain reaction (RT-PCR) analysis (TaqMan®) in all RNA. Results. Initial READS® analysis showed IgAN samples have lower mRNA levels relative to normal renal tissue mRNA samples based upon total RNA as measured by ribosomal RNA. One hundred seventy-five differentially expressed non-redundant fragments were found from 860 initial candidate fragments. Twenty genes were selected for additional TaqMan® analysis, and 13 of 20 genes showed statistically different expression when comparing biopsies from normal individuals and IgAN patients. Expression differences were seen in these genes in biopsies of IgAN of differing clinical activities. Gene expression cluster analysis using the Ward method detailed disease- and gene-related clusters. Detailed examination of the promoter regions of the genes within two gene clusters revealed common gene transcriptional regulatory protein-binding sites. Conclusion. IgAN leads to significant changes in overall mRNA transcription levels within the renal tissue, in addition to gene-specific mRNA level changes. Disease-related patterns of expression were identified and gene-specific clusters suggest common mechanisms of transcriptional alteration

Altered mRNA expression in renal biopsy tissue from patients with IgA nephropathy

Altered mRNA expression in renal biopsy tissue from patients with IgA nephropathy. Background. Immunoglobulin A (IgA) nephropathy (IgAN) is a renal disease characterized by glomerular deposition of IgA-dominant immune deposits that cause glomerular inflammation and sclerosis. Gene expression changes induced in renal tissues/cells as a result of the disease are largely uncharacterized. Methods. A sensitive differential mRNA display technique, restriction endonucleolytic analysis of differentially expressed sequences (READS®) compared similarly processed normal renal tissue to renal biopsy RNA from patients with IgAN, minimal change disease, and necrotizing crescentic glomerulonephritis. A subset of genes with altered expression in IgAN as identified by the READS® technology was further characterized and expression levels confirmed using real-time quantitative polymerase chain reaction (RT-PCR) analysis (TaqMan®) in all RNA. Results. Initial READS® analysis showed IgAN samples have lower mRNA levels relative to normal renal tissue mRNA samples based upon total RNA as measured by ribosomal RNA. One hundred seventy-five differentially expressed non-redundant fragments were found from 860 initial candidate fragments. Twenty genes were selected for additional TaqMan® analysis, and 13 of 20 genes showed statistically different expression when comparing biopsies from normal individuals and IgAN patients. Expression differences were seen in these genes in biopsies of IgAN of differing clinical activities. Gene expression cluster analysis using the Ward method detailed disease- and gene-related clusters. Detailed examination of the promoter regions of the genes within two gene clusters revealed common gene transcriptional regulatory protein-binding sites. Conclusion. IgAN leads to significant changes in overall mRNA transcription levels within the renal tissue, in addition to gene-specific mRNA level changes. Disease-related patterns of expression were identified and gene-specific clusters suggest common mechanisms of transcriptional alteration

Molecular Cloning, Genomic Structure, and Expression Analysis of MUC20, a Novel Mucin Protein, Up-regulated in Injured Kidney

Immunoglobulin A nephropathy (IgAN) is the most common primary glomerulonephritis in the world. Here, we identify a cDNA encoding a novel mucin protein, shown previously to be up-regulated in IgAN patients, from a human kidney cDNA library. This protein contains a mucin tandem repeat of 19 amino acids consisting of many threonine, serine, and proline residues and likely to be extensively O-glycosylated; thus, this gene was classified in the mucin family and named MUC20. The human MUC20 gene contains at least four exons and is localized close to MUC4 on chromosome 3q29. We found variations in repeat numbers in the mucin tandem domain, suggesting polymorphism of this region. Northern blot and reverse transcription-PCR analyses revealed that human MUC20 mRNA was expressed most highly in kidney and moderately in placenta, colon, lung, prostate, and liver. Immunohistochemical analysis of human kidney revealed that MUC20 protein was localized in the proximal tubules. Immunoblotting analysis of MUC20 proteins produced in Madin-Darby canine kidney and HEK293 cells indicated the localization of MUC20 protein in a membrane fraction and extensive posttranslational modification. Immunoelectron microscopy of MUC20-producing Madin-Darby canine kidney cells demonstrated that MUC20 protein was localized on the plasma membrane. Expression of MUC20 mRNA in a human kidney cell line was up-regulated by tumor necrosis factor-alpha, phorbol 12-myristate 13-acetate, or lipopolysaccharide. Two species of MUC20 mRNA (hMUC20-L and hMUC20-S), resulting from alternative transcription, were identified in human tissue, whereas only one variant was observed in mouse tissues. Mouse MUC20 mRNA was expressed in the epithelial cells of proximal tubules, and the expression increased dramatically with the progression of lupus nephritis in the kidney of MRL/MpJ-lpr/lpr mice. Moreover, the expression of mouse MUC20 was augmented in renal tissues acutely injured by cisplatin or unilateral ureteral obstruction. These characteristics suggest that the production of MUC20 is correlated with development and progression of IgAN and other renal injuries

Molecular evolution of versatile derivatives from a GFP-like protein in the marine copepod Chiridius poppei.

Fluorescent proteins are now indispensable tools in molecular research. They have also been adapted for a wide variety of uses in cases involving creative applications, including textiles, aquarium fish, and ornamental plants. Our colleagues have previously cloned a yellow GFP-like protein derived from the marine copepod Chiridius poppei (YGFP), and moreover, succeeded in generating transgenic flowers with clearly visible fluorescence, without the need for high-sensitivity imaging equipment. However, due to the low Stokes shift of YGFP (10 nm), it is difficult to separate emitted light of a labeled object from the light used for excitation; hence, limitations for various applications remain. In this study, which was aimed at developing YGFP mutants with increased Stokes shifts, we conducted stepwise molecular evolution experiments on YGFP by screening random mutations at three key amino acids, based on their fluorescent characteristics and structural stabilities, followed by optimization of their fluorescence output by DNA shuffling of the entire coding sequence. We successfully identified an eYGFPuv that had an excitation maximum in UV wavelengths and a 24-fold increase in fluorescence intensity compared to the previously reported YGFP mutant (H52D). In addition, eYGFPuv exhibited almost 9-fold higher fluorescence intensity compared to the commercially available GFPuv when expressed in human colon carcinoma HCT116 cells and without any differences in cytotoxicity. Thus, this novel mutant with the desirable characteristics of bright fluorescence, long Stokes shift, and low cytotoxity, may be particularly well suited to a variety of molecular and biological applications

An Aptamer-Based Biosensor for Direct, Label-Free Detection of Melamine in Raw Milk

Melamine, a nitrogen-rich compound, has been used as a food and milk additive to falsely increase the protein content. However, melamine is toxic, and high melamine levels in food or in milk can cause kidney and urinary problems, or even death. Hence, the detection of melamine in food and milk is desirable, for which numerous detection methods have been developed. Several methods have successfully detected melamine in raw milk; however, they require a sample preparation before the analyses. This study aimed to develop an aptamer-DNAzyme conjugated biosensor for label-free detection of melamine, in raw milk, without any sample preparation. An aptamer-DNAzyme conjugated biosensor was developed via screening using microarray analysis to identify the candidate aptamers followed by an optimization, to reduce the background noise and improve the aptamer properties, thereby, enhancing the signal-to-noise (S/N) ratio of the screened biosensor. The developed biosensor was evaluated via colorimetric detection and tested with raw milk without any sample preparation, using N-methylmesoporphyrin IX for fluorescence detection. The biosensor displayed significantly higher signal intensity at 2 mM melamine (S/N ratio, 20.2), which was sufficient to detect melamine at high concentrations, in raw milk

Summary of cell viability and FP transcription levels in HCT116 cells expressing various FPs.

<p>Summary of cell viability and FP transcription levels in HCT116 cells expressing various FPs.</p