Set Of Oligonucleotide Probes Research Articles

Bacterioplankton diversity and community composition in the Southern Lagoon of Venice

The Lagoon of Venice is a large water basin that exchanges water with the Northern Adriatic Sea through three large inlets. In this study, the 16S rRNA approach was used to investigate the bacterial diversity and community composition within the southern basin of the Lagoon of Venice and at one inlet in October 2007 and June 2008. Comparative sequence analysis of 645 mostly partial 16S rRNA gene sequences indicated high diversity and dominance of Alphaproteobacteria, Gammaproteobacteria and Bacteroidetes at the lagoon as well as at the inlet station, therefore pointing to significant mixing. Many of these sequences were close to the 16S rRNA of marine, often coastal, bacterioplankton, such as the Roseobacter clade, the family Vibrionaceae, and class Flavobacteria. Sequences of Actinobacteria were indicators of a freshwater input. The composition of the bacterioplankton was quantified by catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH) with a set of rRNA-targeted oligonucleotide probes. CARD-FISH counts corroborated the dominance of members of the phyla Alphaproteobacteria, Gammaproteobacteria and Bacteroidetes. When assessed by a probe set for the quantification of selected clades within Alphaproteobacteria and Gammaproteobacteria, bacterioplankton composition differed between October 2007 and June 2008, and also between the inlet and the lagoon. In particular, members of the readily culturable copiotrophic gammaproteobacterial genera Vibrio, Alteromonas and Pseudoalteromonas were enriched in the southern basin of the Lagoon of Venice. Interestingly, the alphaproteobacterial SAR11 clade and related clusters were also present in high abundances at the inlet and within the lagoon, which was indicative of inflow of water from the open sea.

Toward an Alzheimer's disease diagnosis via high-resolution blood gene expression

Background There is a significant need for reliable molecular biomarkers to aid in Alzheimer's disease (AD) clinical diagnosis. Methods We performed a genome-wide investigation of the human transcriptome, taking into account the discriminatory power of splice variations from the blood of 80 AD patients and 70 nondemented control (NDC) individuals. Results We characterized a blood RNA signature composed of 170 oligonucleotide probe sets associated with 133 genes that can correctly distinguish AD patients from NDC with a sensitivity of 100% and specificity of 96%. Functionally, this signature highlights genes involved in pathways that were associated with macrophages and lymphocytes within AD patients: Transforming growth factor (TGF-β) signaling, oxidative stress, innate immunity and inflammation, cholesterol homeostasis, and lipid-raft perturbation, whereas other genes may also provide new insights in the biology of AD. Conclusions This study provides proof-of-concept that whole-blood profiling can generate an AD-associated classification signature via the specific relative expression of biologically relevant RNAs. Such a signature will need to be validated with extended patient cohorts, and evaluated to learn whether it can differentiate AD from others types of dementia.

The Research of Phenylhexyl Isothiocyanate's Influence on p16 Gene Methylation of Leukaemia Cell

Objective Aberrant DNA methylation of CpG site is among the earliest and most frequent alterations in cancer. The aim of this study was to study whether phenylhexyl isothiocyanate can reduce the methylation level of P16 gene. This study used a microarray-based method for quantificationally detecting changes of P16 gene methylation in leukemia patient and U266 cell line. And to simply discuss the effect of phenylhexyl isothiocyanate on tumor methylation.Methods This method used bisulfite-modified DNA as a template for PCR amplification, resulting in conversion of unmethylated cytosine, but not methylated cytosine, into thymine within CpG islands of interest. One set of oligonucleotide probes were designed to fabricate a DNA microarray to detect the methylation changes of P16 gene CpG islands. Each set contained a pair of methylated and unmethylated oligonucleotides for interrogating 3 CpG sites in close proximity. By TA cloning, PCR, sequencing, positive and negative DNA targets were required. Next drawing a standard curve by fluorescence intensity. Leukemia samples DNA were abstracted and bisulfite-modified. Sample DNA targets were required by PCR amplification and were hybridized with the microarry. Finally the microarry was scanned with ScanArray Lite microarray analysis systems.Results The linear relationship (R2=0.9660) was established and it could be used to eliminate background noise. The methylation level of U266 is hypermethylation, after cultured with PHI, the level reduce. Eleven patients have P16 gene hypermethylation in thrity. After culture with PHI, seven patients showed level reduce, one patient showed raise, three patients showed remaining.Conclusion PHI can reduce the methylation level of P16 gene in U266 cell line, and also can reduce the methylation level of P16 gene of leukemia patient samples in vitro culture.

PHA-accumulating microorganisms in full-scale wastewater treatment plants

A study was conducted to clarify phylogenetic affiliations of polyhydroxyalkanoate (PHA)-accumulating microorganisms in full-scale activated sludge processes. Activated sludge samples obtained from three full-scale activated sludge processes were aerobically incubated with excess acetate to increase their PHA content. The buoyant density separation method was applied to selectively collect PHA-accumulating cells, which were then analysed by the group-level FISH and the PCR-DGGE-sequencing methods, and possible PHA-accumulating microbial groups were screened. A set of oligonucleotide probes targeting the microbial groups suspected to accumulate PHA was introduced, and seven oligonucleotide probes were newly designed for this purpose. PHA accumulation of probe-positive cells was confirmed by the post-FISH PHA staining method, wherein PHA staining with Nile Blue A (NBA) was applied after FISH. As a result, the following seven bacterial groups were found to have PHA: Dechloromonas, Accumulibacter, Thauera, Zoogloea, Comamonas, Competibacter and a novel cluster in Beta-proteobacteria. Based on the results of the post-FISH PHA staining method, these seven bacterial groups were estimated to account for around four-tenths to two-thirds of total PHA-accumulating microorganisms.

Locus-specific microsatellite markers in common bean (Phaseolus vulgaris L.): isolation and characterization

This study aimed at the development of microsatellite markers for Phaseolus vulgaris L. by two techniques: hybridization-based detection, and transfer of microsatellite markers developed for chickpea to the common bean. Small-insert genomic libraries of common bean were screened for simple sequence repeat (SSR)-containing fragments with a set of microsatellite-specific oligonucleotide probes. Twenty-five (GA)10 positive clones detected by a dinucleotide repeat probe were selected for sequencing. Sixteen of 18 primer pairs, complementary to the SSR-flanking regions and tested in eight P. vulgaris L. accessions, are polymorphic at an intra-specific level. Mendelian inheritance of the sequence-tagged microsatellite site (STMS) markers was demonstrated using a set of recombinant inbred lines (RILs) and their parents. A total of 46 chickpea STMS markers of 447 different primer combinations amplified loci in the genome of common bean. Sequencing of amplified products from Phaseolus with these primer combinations demonstrated that the sequence of microsatellite marker TA 176s proved to be significantly similar to transcription factor SCOF1 of soy bean, controlling the response to cold stress, and also to transcription factor zinc finger protein (a Kruppel-like zinc finger protein, or TFIIIA), coordinating the reaction to osmotic stress in Medicago truncatula. Sequence analysis of this site in the two common bean parental lines BAT 477 and DOR 364 revealed that the sequence of BAT 477 was 5 bp longer than the corresponding sequence of DOR 364. Amplification of this fragment in six RILs of common bean detected the existence of sequence polymorphisms between the different lines.

An 86-Probe Gene Expression Signature Can Predict Survival in AML with Normal Karyotype Independently of FLT3 ITD and NPM1 Mutation Status - A Collaborative Study from the AMLCG and CALGB Study Groups.

Patients with acute myeloid leukemia and a normal karyotype (NK AML) comprise 50% of all AML cases and show heterogeneous treatment outcomes and survival. We used gene expression profiling to develop a prognostic gene signature that predicts survival in this clinically relevant AML subgroup. Our analysis was based on data from 163 patients with newly diagnosed NK AML treated in the German multicenter AMLCG 2000 trial, for whom pretreatment gene expression profiles were obtained using Affymetrix HG-U133 microarrays. We used supervised principal component analysis to identify 86 oligonucleotide probesets (corresponding to 66 different genes and ESTs) that were correlated with overall survival (OS), and to define a prognostic score based on these probesets. When applied to an independent test cohort of 79 NK AML cases from the same AMLCG trial, the continuous prognostic score was predictive of OS (P=0.002, hazard ratio [HR] for a change in prognostic score equal to the difference between the 75th and 25th percentiles of the score = 1.94) and event-free survival (EFS) (P = 0.001, HR=1.70). The score based on our gene signature showed a strong correlation with the presence of the FLT3 internal tandem duplication (ITD), but retained its prognostic value for OS in the test cohort even after adjustment for FLT3 ITD, NPM1 status and age (P=0.037, HR=1.65). When we defined a cut-off value in the training population and used it to dichotomize the gene expression score values in the test cohort, the resulting two subgroups had significantly different OS (median, 259 days vs. not reached, P=0.002) and event-free survival (EFS) (median, 72 vs. 300 days, P = 0.015). We subsequently confirmed our findings in a group of 64 NK AML patients (Blood 2006; 108:1677–83) treated on CALGB 9621. In this validation cohort, our continuous gene expression score was predictive of OS (P < 0.001, HR=4.11) and EFS (P < 0.001, HR=2.90). In multivariate analyses that adjusted for age, NPM1 and FLT3 ITD status, the gene expression score remained significant for OS (P = 0.007, HR=3.40). When we used the prognostic score to split the CALGB validation cohort into two groups, based on the same cut-off value as in the AMLCG test population, the two resulting subgroups differed in their OS (median, 375 days vs. not reached, P < 0.001) and EFS (median, 258 vs. 728 days, P = 0.027). In summary, we present a novel and robust gene expression signature that offers independent prognostic information for patients with normal karyotype AML.

Microarray-based method to analyze methylation status of E-cadherin gene in leukemia

BackgroundAberrant DNA methylation of the CpG sites of the tumor suppressor gene is closely associated with carcinogenesis. Recently, several studies have indicated the aberrant methylation of E-cadherin gene could be a potential marker for leukemic patients. MethodWe used bisulfite-modified DNA as a template for PCR amplification, resulting in conversion of unmethylated, but not methylated, cytosine into thymine within CpG islands of interest. The amplified product containing a pool of DNA fragments with altered nucleotide sequences was then hybridized with an oligonucleotide-based microarray. Five sets of oligonucleotide probes were designed to detect the methylation patterns of E-cadherin gene CpG islands in leukemia samples. The results were further validated by methylation-specific PCR (MSP). ResultsWe found that all leukemia samples were methylated at different levels within the target sequences. The specific regions (the CpG sites #16–19 and #20–22) were revealed as hotspots for methylation in leukemic patients. These results showed that the microarray assay could successfully detect methylation changes of E-cadherin gene in leukemia quantitatively. ConclusionThe oligonucleotide-based microarray can be a quick and reliable tool to map methylation status in CpG islands. This established microarray could be potentially useful for clinical researches and diagnosis.

Stored Packed Red Blood Cell Transfusion Up-regulates Inflammatory Gene Expression in Circulating Leukocytes

The transfusion of more than 6 units of packed red blood cells (PRBCs) within the first 12 hours of injury is the strongest independent predictor of multiple organ failure (MOF). This suggests that stored blood contains bioactive factors that may modify the immunoinflammatory response. To simulate postinjury major transfusions ex vivo, we obtained whole blood from 4 healthy adults and divided it into four 7-mL groups (I-IV). Group I was not diluted. Group II had 7 mL of 0.9% sterile saline (SS) added. Group III received 3.5 mL each of leuko-reduced stored PRBC and SS (simulating a major transfusion). Group IV received 3.5 mL each of SS and a hemoglobin-based oxygen carrier (PolyHeme) to evaluate the effects of hemoglobin alone. The hemoglobin content in groups III and IV was measured to be equal. Total leukocyte RNA was purified, and its gene array profiles were obtained. Of the 56,475 oligonucleotide probe sets interrogated, 415 were statistically different (P < 0.001). Fourteen of the 415 probe sets were inflammatory-related. The PRBC group had a significantly different expression profile compared with the others and included up-regulation of the interleukin-8, toll-like receptor 4, cryropyrin, prostaglandin-endoperoxide synthase-2, and heparinase genes. PRBCs activate inflammatory genes in circulating leukocytes, which may be central to the pathogenesis of the adverse inflammatory responses that lead to postinjury MOF.

A multi-array multi-SNP genotyping algorithm for Affymetrix SNP microarrays

Modern strategies for mapping disease loci require efficient genotyping of a large number of known polymorphic sites in the genome. The sensitive and high-throughput nature of hybridization-based DNA microarray technology provides an ideal platform for such an application by interrogating up to hundreds of thousands of single nucleotide polymorphisms (SNPs) in a single assay. Similar to the development of expression arrays, these genotyping arrays pose many data analytic challenges that are often platform specific. Affymetrix SNP arrays, e.g. use multiple sets of short oligonucleotide probes for each known SNP, and require effective statistical methods to combine these probe intensities in order to generate reliable and accurate genotype calls. We developed an integrated multi-SNP, multi-array genotype calling algorithm for Affymetrix SNP arrays, MAMS, that combines single-array multi-SNP (SAMS) and multi-array, single-SNP (MASS) calls to improve the accuracy of genotype calls, without the need for training data or computation-intensive normalization procedures as in other multi-array methods. The algorithm uses resampling techniques and model-based clustering to derive single array based genotype calls, which are subsequently refined by competitive genotype calls based on (MASS) clustering. The resampling scheme caps computation for single-array analysis and hence is readily scalable, important in view of expanding numbers of SNPs per array. The MASS update is designed to improve calls for atypical SNPs, harboring allele-imbalanced binding affinities, that are difficult to genotype without information from other arrays. Using a publicly available data set of HapMap samples from Affymetrix, and independent calls by alternative genotyping methods from the HapMap project, we show that our approach performs competitively to existing methods. R functions are available upon request from the authors.

Functional bacterial and archaeal community structures of major trophic groups in a full-scale anaerobic sludge digester

Functional Bacteria and Archaea community structures of a full-scale anaerobic sludge digester were investigated by using a full-cycle 16S rRNA approach followed by microautoradiography (MAR)–fluorescent in situ hybridization (FISH) technique and micromanipulation. FISH analysis with a comprehensive set of 16S and 23S rRNA-targeted oligonucleotide probes based on 16S rRNA clone libraries revealed that the Gram-positive bacteria represented by probe HGC69A-hybridized Actinobacteria (8.5±1.4% of total 4′, 6-diamidino-2-phenylindole (DAPI)-stained cells) and probe LGC354-hybridized Firmicutes (3.8±0.8%) were the major phylogenetic bacterial phyla, followed by Bacteroidetes (4.0±1.2%) and Chloroflexi (3.7±0.8%). The probe MX825-hybridized Methanosaeta (7.6±0.8%) was the most abundant archaeal group, followed by Methanomicrobiales (2.8±0.6%) and Methanobacteriaceae (2.7±0.4%). The functional community structures (diversity and relative abundance) of major trophic groups were quantitatively analyzed by MAR–FISH. The results revealed that glucose-degrading microbial community had higher abundance (ca. 10.6±4.9% of total DAPI-stained cells) and diversity (at least seven phylogenetic groups) as compared with fatty acid-utilizing microbial communities, which were more specialized to a few phylogenetic groups. Despite the dominance of Betaproteobacteria, members of Chloroflexi, Smithella, Syntrophomonas and Methanosaeta groups dominated the [ 14C]glucose-, [ 14C]propionate-, [ 14C]butyrate- and [ 14C]acetate-utilizing microorganism community, and accounted for 27.7±4.3%, 29.6±7.0%, 34.5±7.6% and 18.2±9.5%, respectively. In spite of low abundance (ca. 1%), the hitherto unknown metabolic functions of Spirochaeta and candidate phylum of TM7 as well as Synergistes were found to be glucose and acetate utilization, respectively.

A New Approach to the Non-unique Probe Selection Problem

Identification of biological agents in a sample is a relevant problem in medicine. A model to this problem consists of selecting optimal oligonucleotide probe sets for use in hybridization experiments in which target viruses or bacteria are to be identified in biological samples. In such an experiment the presence or absence of these targets is determined by observing whether selected probes bind to their corresponding sequences. The problem is to select a probe set that is able to uniquely identify targets while containing a minimal number of probes. In this paper we describe a heuristic algorithm that produced feasible solution sets that for large, real data sets contain significantly fewer probes than those obtained using other methods. A description of the problem, our approach, and the results are presented. We developed a C++ program and a GUI (Graphical User Interface) to run real and simulated instances of the problem.

Microarray-Based Method for Quantificationally Detecting Methylation Changes of E-Cadherin Gene in Acute Leukemia.

Aberrant DNA methylation of CpG sites is among the earliest and most frequent alterations in cancer. Several studies suggest that aberrant methylation of the CpG sites of the tumor suppressor gene is closely associated with carcinogenesis. So methylation detection is very important. The aim of this study was to describe a microarray-based method for quantificationally detecting changes of E-cadherin methylation in acute leukemia and to simply discuss the effect of microarray on detecting tumor methylation.This method used bisulfite-modified DNA as a template for PCR amplification, resulting in conversion of unmethylated cytosine, but not methylated cytosine, into thymine within CpG islands of interest. Five sets of oligonucleotide probes were designed to fabricate a DNA microarray to detect the methylation changes of E-cadherin gene CpG islands in acute leukemia. Each set contained a pair of methylated and unmethylated oligonucleotides for interrogating 3 or 4 CpG sites in close proximity. By TA cloning, PCR, sequencing, positive and negative DNA targets were obtained. A series of microarray hybridization were performed with mixtures of Cy3 labeled positive and negative DNA targets at different proportions. Next draw a standard curve by fluorescence intensity. Then leukemia samples DNA were abstracted and bisulfite-modified. Sample DNA targets were obtained by PCR amplification and were hybridized with the microarry. Finally the microarry was scanned with ScanArray Lite microarray analysis systems.Five linear relationships(R2=0.9660~0.9963) was established, which said the accuracy and reproducibility of the probes designed for microarray hybridization was good and could be used to eliminate background noise. The experimental results showed that the microarray assay could successfully detect five regions methylation changes of E-cadherin gene in every acute leukemia sample quantificationally. There were different degree of methylation in five acute leukemia samples and the hypermethylation region was the same. The test result was validated by gene sequencing.In tumors E-cadherin expression frequently is reduced, an event that contributes to tumor invasion and metastasis. The methylation of E-cadherin gene promoter is one important reason resulting in the silencing of expression. In normal peripheral blood mononuclear cells and bone marrow, E-cadherin is completely unmethylated. In our results, the methylation of E-cadherin is related to acute leukemia. And the same hypermethylation region may be the critical sites of methylation sites. As shown in this study, the use of a simple control system could test the accuracy and reproducibility of the probes designed for microarray hybridization. This control system can also be used to calibrate the levels of methylation changes detected in the investigated samples by microarray assay. With more and more methylated genes are found, microarray assay could be applied as a useful tool for mapping methylation changes in multiple CpG loci. It's more time-saving and more labor-saving than gene sequencing and can be readily used to high throughput analysis of DNA methylation. It will contribute significant information to our understanding of CpG island methylation in acute leukemia.

Role of protein kinase B in breast cancer

Breast cancer is the most common cancer in women and is increasing in both the developed and developing countries. There is an urgent need to understand the precise mechanisms of tumour development in breast cancer, to develop new treatment strategies and to identify predictive markers for tumour aggressiveness and therapy resistance. A protein called protein kinase B (PKB, also called Akt) is frequently elevated in breast cancers and has been implicated as a key player in breast cancer development and progression. The activation level of PKB is also thought to correlate with patient outcome. However, the function of the three isoforms of PKB in mediating the crucial responses is unknown. We have developed a set of antisense phosphorothioate oligonucleotide probes that target antisense-active regions in PKB and that enable >90% knockdown of all three known PKB isoforms (alpha, beta and gamma), either individually or in various combinations, including removal of all three isoforms together. We have demonstrated that these agents specifically and potently prevent the growth of breast cancer cells. Application of these antisense agents offers a unique opportunity to understand how PKB works and contributes to breast cancer, and to provide insight into the role of signalling by individual PKB isoforms in breast cancer cells. Such work may also identify clinically relevant markers of disease, thereby enabling better predictors of patient outcome, and provide the necessary intellectual framework for the development of PKB-isoform selective inhibitors (for example, antisense oligonucleotides, small chemical inhibitors) as novel therapeutic agents.

Herbaspirillum hiltneri sp. nov., isolated from surface-sterilized wheat roots

The genus Herbaspirillum of the Betaproteobacteria mainly comprises diazotrophic bacteria with a potential for endophytic and systemic colonization of a variety of plants. The plant-associated bacterial isolates N3(T), N5 and N9 were derived from surface-sterilized wheat roots. After phylogenetic analysis of 16S rRNA gene sequence data the isolates could be allocated to the genus Herbaspirillum, and 99.9 % similarity to the sequence of Herbaspirillum lusitanum P6-12(T) was found. A set of 16S rRNA gene-targeted oligonucleotide probes was developed for the identification of the three novel isolates and H. lusitanum (Hhilu446), and for the specific detection of several other Herbaspirillum species described recently. For higher phylogenetic resolution, the 23S rRNA gene sequences of all members of the genus was sequenced and used to construct a phylogenetic tree. Isolates N3(T), N5 and N9 formed a group that was distinct from all other Herbaspirillum species. In addition, isolate N3(T) and H. lusitanum P6-12(T) exhibited a DNA-DNA hybridization value of only 25 %. The value for DNA-DNA hybridization between N3(T) and other members of the genus Herbaspirillum was between 14 and 32 %; DNA-DNA hybridization between strain N3(T) and isolates N5 and N9 produced values above 95 %. This places the three isolates as representatives of a novel species within the genus Herbaspirillum. A Biolog GN2 assay supported this conclusion. The major fatty acids were C(16 : 1)omega7c, C(16 : 0) and C(18 : 1)omega7c, and the DNA G+C content ranged from 60.9 to 61.5 mol%. Therefore these three isolates should be classified within a novel species, for which the name Herbaspirillum hiltneri sp. nov. is proposed. The type strain is N3(T) (=DSM 17495(T)=LMG 23131(T)).

On-chip non-equilibrium dissociation curves and dissociation rate constants as methods to assess specificity of oligonucleotide probes

Nucleic acid hybridization serves as backbone for many high-throughput systems for detection, expression analysis, comparative genomics and re-sequencing. Specificity of hybridization between probes and intended targets is always critical. Approaches to ensure and evaluate specificity include use of mismatch probes, obtaining dissociation curves rather than single temperature hybridizations, and comparative hybridizations. In this study, we quantify effects of mismatch type and position on intensity of hybridization signals and provide a new approach based on dissociation rate constants to evaluate specificity of hybridized signals in complex target mixtures. Using an extensive set of 18mer oligonucleotide probes on an in situ synthesized biochip platform, we demonstrate that mismatches in the center of the probe are more discriminating than mismatches toward the extremities of the probe and mismatches toward the attached end are less discriminating than those toward the loose end. The observed destabilizing effect of a mismatch type agreed in general with predictions using the nearest neighbor model. Use of a new parameter, specific dissociation temperature (Td-w, temperature of maximum specific dissociation rate constant), obtained from probe–target duplex dissociation profiles considerably improved the evaluation of specificity. These results have broad implications for hybridization data obtained from complex mixtures of nucleic acids.

Fluorescence In Situ Hybridization with Specific Oligonucleotide rRNA Probes Distinguishes the Sibling Species Stylonychia lemnae and Stylonychia mytilus (Ciliophora, Spirotrichea)

Based on morphological and morphogenetic characters alone, the sibling species Stylonychia lemnae and Stylonychia mytilus, members of the Stylonychia mytilus complex, can hardly be distinguished. However, biochemical investigations of the isoenzyme pattern of different enzymes showed a distinct differentiation between these two species. In the last few years, fluorescence in situ hybridization (FISH) techniques have become a suitable and reliable tool for identification and differentiation of closely related species of protozoa, such as ciliates. To distinguish the sibling species, a set of specific oligonucleotide probes were developed. In the present study, the SSU rDNA of 7 clones of Stylonychia lemnae and 13 clones of Stylonychia mytilus, isolated from different geographic regions, were sequenced. Comparing all SSU rDNA sequences of both species, only one single difference within the whole gene was detected. Based on this difference, a set of two oligonucleotide probes, targeting the SSU rRNA of each species (Stylonychia mytilus and Stylonychia lemnae) was designed. These probes were successfully tested by applying the FISH techniques on preserved cells of different clones of both species.

237. Gene Expression Profiles in Diabetic Cardiomyopathy Following SERCA2a Gene Transfer

Diabetes is associated with impaired cardiac function in both humans and animals. As a consequence of myocardial dysfunction, the expression of several genes within the heart is altered. Our aim is to investigate the changes in gene expression profiles accompanying diabetic cardiomyopathy and to identify molecular and cellular pathways and genes that may contribute to cardiac remodeling as a result of the disease and/or its phenotypic rescue by restoration of the sarcoplasmic reticulum calcium ATPase pump (SERCA2a) through adenoviral gene transfer. Using Otsuka Long-Evans Tokushima Fatty (OLETF) rat model of type II diabetes and the Agilent rat chip, which contains oligonucleotide probe sets for 22,000 rat genes, we performed analyses of gene expression by comparing differential changes in age and sex-matched control versus diabetic hearts and diabetic hearts that received gene transfer of SERCA2a (which we previously showed to induce functional improvement in heart failure). Microarray results of selected genes were verified with quantitative real-time PCR and immunoblotting. After normalization and filtration of the data and using a 2-fold cut- off, we found that diabetes has differentially induced the expression of 743 genes (216 up- and 527 down-regulated) which take part in many cellular processes. Our analysis indicates that diabetic cardiomyopathy appears to be, in general, associated with a downregulation of transcripts. Diabetic cardiomyopathic hearts have reduced levels of SERCA2a. Adenoviral gene transfer targeted to these hearts differentially induced the expression of 76 genes and appears, in general, to reverse the transcriptional profile induced by diabetes. Functional gene ontology classification of these genes indicated that SERCA2a restoration is associated with global changes in the cytoskeleton, in cellular energetics and metabolism, in calcium cycling, and in intracellular signaling and transcription regulation pathways. There are also 17 genes of unknown function that are specifically regulated by SERCA overexpression in diabetic hearts. The biological role of some of these novel genes are currently being pursued in other projects. Altogether this investigation provided valuable insight into the pathophysiology of cardiac remodeling and the role of SERCA2a normalization of multiple pathways in diabetes.

Molecular and serological detection of A. centrale- and A. marginale-infected cattle grazing within an endemic area

A reverse line blot hybridization (RLB) one-stage nested PCR (nPCR) for Anaplasma centrale and a nested PCR for Anaplasma marginale were used to detect infected cattle grazing within an endemic region in Israel. A novel set of PCR primers and oligonucleotide probes based on a 16S ribosomal RNA gene was designed for RLB detection of both Anaplasma species, and the performance of the molecular assays compared. The immunofluorescent antibody test (IFA) was used to detect antibodies to both Anaplasma species, whereas, a highly sensitive and specific competitive enzyme-linked immunosorbent assay (cELISA) was used to detect antibodies in A. centrale-vaccinated cattle. The RLB and the nested PCR procedures showed bacteremia with sensitivity of 50 infected erythrocytes per milliliter. Up to 93% of the A. centrale vaccinates carried specific antibodies that were detected by cELISA, and up to 71% of the vaccinated cattle were found to be naturally infected with A. marginale according to the PCR and the RLB assays. Nevertheless, no severe outbreaks of A. marginale infection occurred among vaccinated herds in this endemic region. It appears that both, molecular tools and serology are useful for evaluation of the vaccine efficacy. In the light of wide natural field infection with A. marginale, strong recommendations to continue the A. centrale vaccination program regime will continue until a new generation of non-blood-based vaccine will be developed.

Evaluation of the Phylogenetic Diversity of Prokaryotic Microorganisms in Sphagnum Peat Bogs by Means of Fluorescence In Situ Hybridization (FISH)

The microbial population of sphagnum peat bogs of northern Russia was analyzed with respect to the presence and cell numbers of representatives of particular phylogenetic groups of prokaryotes by means of in situ hybridization with fluorescently labeled group-specific rRNA-targeted oligonucleotide probes with broad detection spectra. The total number of cells that hybridized with universal Archaea- and Bacteria-specific probes varied, in peat samples of different bogs, from 45 to 83% of the number of cells revealed by DAPI staining. Down the bog profiles, the total number of prokaryotes and the fraction of archaea among them increased. Application of a set of oligonucleotide probes showed that the number of microorganisms belonging to such phylogenetic lineages of the domain Bacteria as the phyla Proteobacteria, Bacteroidetes, Actinobacteria, Firmicutes, Acidobacteria, and Planctomycetes constituted, in total, 14.0–26.5% of the number of eubacteria detected in the samples. Among the bacteria identified in the peat samples, the most abundant were representatives of the classes Alphaproteobacteria and Betaproteobacteria and the phyla Acidobacteria, Bacteroidetes, and Actinobacteria.

Long versus short oligonucleotide microarrays for the study of gene expression in nonhuman primates

The high degree of sequence similarity between human and nonhuman primate (NHP) genomic DNA suggests that human genome sequence-based DNA microarrays may be used effectively to study gene expression in NHP disease models. In the present study, two distinct commercially available human genome microarray platforms, the Affymetrix HG U133A GeneChip ® System utilizing Human Genome U133A GeneChips ® and the Applied Biosystems Expression Array System utilizing the Human Genome Survey Microarray, were used to identify and characterize gene expression changes in the anterior cerebellum of a macaque monkey model of human alcoholism. The Affymetrix microarray consists of eleven short oligonucleotide probe sets for each gene while the Applied Biosystems Microarray uses a single long oligonucleotide per gene. Cross-mapping of probes revealed a total of 11,542 genes that are represented on both microarray platforms. Absolute measures of gene expression (“present calls”) from the cerebellum RNA samples were 65–70% (Applied Biosystems Expression Array System) and 27–30% (AffymetrixGeneChip ® System) among these common gene targets. Analysis of variance (ANOVA; p < 0.05; >1.2 fold change; detected on at least 50% of the arrays) indicated 932 and 515 differentially expressed genes for the Applied Biosystems and Affymetrix microarrays, respectively. Significance analysis of microarrays (SAM) identified 255 significant genes at 5% false discovery rate (FDR) for the Applied Biosystems data set and five significant genes at 60% FDR (minimum FDR) for the Affymetrix data set. TaqMan ® assay-based real-time PCR validation of a number of differentially-expressed genes yielded results that agreed well with the array data in the majority of comparisons. This study demonstrates that human sequence-based DNA arrays can be used effectively to detect differential gene expression in an NHP disease model and provides evidence that the use of this long oligonucleotide-based microarray platform may be more suitable for cross-species gene expression studies than a short oligonucleotide-based system.