Specific Probe Sets Research Articles

Improved real-time PCR method for quantification of the abundance of all known ribotypes of the ichthyotoxic dinoflagellate Cochlodinium polykrikoides by comparing 4 different preparation methods

Red tides by the ichthyotoxic dinoflagellate Cochlodinium polykrikoides have caused large scaled mortality of fish and great loss in aquaculture industry in many countries. Detecting and quantifying the abundance of this species are the most critical step in minimizing the loss. The conventional quantitative real-time PCR (qPCR) method has been used for quantifying the abundance of this species. However, when analyzing >500 samples collected during huge C. polykrikoides red tides in South Sea of Korea in 2014, this conventional method and the previously developed specific primer and probe set for C. polykrikoides did not give reasonable abundances when compared with cell counting data. Thus improved qPCR methods and a new specific primer and probe set reflecting recent discovery of 2 new ribotypes have to be developed. A new species-specific primer and probe set for detecting all 3 ribotypes of C. polykrikoides was developed and provided in this study. Furthermore, because the standard curve between cell abundance and threshold cycle value (Ct) is critical, the efficiencies of 4 different preparation methods used to determine standard curves were comparatively evaluated. The standard curves were determined by using the following 4 different preparations: (1) extraction of DNA from a dense culture of C. polykrikoides followed by serial dilution of the extracted DNA (CDD method), (2) extraction of DNA from each of the serially diluted cultures with different concentrations of C. polykrikoides cultures (CCD method), (3) extraction of DNA from a dense field sample of C. polykrikoides collected from natural seawater and then dilution of the extracted DNA in serial (FDD method), and (4) extraction of DNA from each of the serially diluted field samples having different concentrations of C. polykrikoides (FCD method). These 4 methods yielded different results. The abundances of C. polykrikoides in the samples collected from the coastal waters of South Sea, Korea, in 2014–2015, obtained using the standard curves determined by the CCD and the FCD methods, were the most similar (0.93–1.03 times) and the second closest (1.16–1.33 times) to the actual cell abundances obtained by enumeration of cells. Thus, our results suggest that the CCD method is a more effective tool to quantify the abundance of C. polykrikoides than the conventional method, CDD, and the FDD and FCD methods.

Development of a comprehensive detection and identification molecular based system for eight pospiviroids

Pospiviroids cause serious economic damage to solanaceous crops, including tomato (Solanum lycopersicum), and cause symptomless infections in many horticultural plants. Symptomless infection and seed transmission are obstacles in visual inspection for plant quarantine. Therefore, in the present study, a comprehensive detection and identification system for molecular diagnosis of eight pospiviroids was developed, based on real-time reverse transcription polymerase chain reaction (RT-PCR). In this system, two real-time RT-PCR methods based on SYBR Green and TaqMan technologies were combined. First, tomato seed samples were screened for all the eight pospiviroids by the SYBR Green method using three primer pairs, including one pair of universal primers. Samples that were positive using the universal primer set were subjected to secondary screening for the identification to species level of six viroids by TaqMan real-time RT-PCR method, with specific primer and probe sets. This two-phase system could specifically detect a viroid in a sample of 400 seeds containing a single contaminated seed. The developed system could be an effective and simple tool to avoid the introduction of pospiviroids through tomato seeds.

Application of bioinformatics in probe design enables detection of enteroviruses on different taxonomic levels by advanced in situ hybridization technology

BackgroundEnteroviral infections are common, affecting humans across all age groups. RT-PCR is widely used to detect these viruses in clinical samples. However, there is a need for sensitive and specific in situ detection methods for formalin-fixed tissues, allowing for the anatomical localization of the virus and identification of its serotype. ObjectivesThe aim was to design novel enterovirus probes, assess the impact of probe design for the detection and optimize the new single molecule in situ hybridization technology for the detection of enteroviruses in formalin-fixed paraffin-embedded samples. Study designFour enterovirus RNA-targeted oligonucleotide RNA probes – two probes for wide range enterovirus detection and two for serotype-targeted detection of Coxsackievirus B1 (CVB1) – were designed and validated for the commercially available QuantiGene ViewRNA in situ hybridization method. The probe specificities were tested using a panel of cell lines infected with different enterovirus serotypes and CVB infected mouse pancreata. ResultsThe two widely reactive probe sets recognized 19 and 20 of the 20 enterovirus serotypes tested, as well as 27 and 31 of the 31 CVB1 strains tested. The two CVB1 specific probe sets detected 30 and 14 of the 31 CVB1 strains, with only minor cross-reactivity to other serotypes. Similar results were observed in stained tissues from CVB –infected mice. ConclusionsThese novel in-house designed probe sets enable the detection of enteroviruses from formalin-fixed tissue samples. Optimization of probe sequences makes it possible to tailor the assay for the detection of enteroviruses on the serotype or species level.

Application of reverse dot blot hybridization to simultaneous detection and identification of harmful algae.

Warning and monitoring projects of harmful algal blooms require simple and rapid methods for simultaneous and accurate detection and identification of causative algae present in the environmental samples. Here, reverse dot blot hybridization (RDBH) was employed to simultaneously detect several harmful algae by using five representative bloom-forming microalgae along the Chinese coast. A set of specific probes for RDBH were developed by PCR, cloning, and sequencing of the internal transcribed spacer (ITS), alignment analysis, and probe design. Each probe was oligo (dT)-tailed and spotted onto positively charged nylon membrane to make up a low-density oligonucleotide array. Universal primers designed within the conserved regions were used to amplify the ITS sequences by using genomic DNA of target as templates. The digoxigenin (Dig)-labeled PCR products were denatured and then hybridized to the oligonucleotide array. The array produced a unique hybridization pattern for each target species differentiating them from each other. The preparations of oligonucleotide array and hybridization conditions were optimized. The developed RDBH demonstrated a detection limit up to 10 cells. The detection performance of RDBH was relatively stable and not affected by non-target species and the fixation time of target species over at least 30 days. The RDBH could recover all the target species from the simulated field samples and target species confirmed by the subsequent microscopy examination in the environmental samples. These results indicate that RDBH can be a new technical platform for parallel discrimination of harmful algae and is promising for environmental monitoring of these microorganisms.

Investigation of avian influenza virus in poultry and wild birds due to novel avian-origin influenza A(H10N8) in Nanchang City, China

Multiple reassortment events within poultry and wild birds had resulted in the establishment of another novel avian influenza A(H10N8) virus, and finally resulted in human death in Nanchang, China. However, there was a paucity of information on the prevalence of avian influenza virus in poultry and wild birds in Nanchang area. We investigated avian influenza virus in poultry and wild birds from live poultry markets, poultry countyards, delivery vehicles, and wild-bird habitats in Nanchang. We analyzed 1036 samples from wild birds and domestic poultry collected from December 2013 to February 2014. Original biological samples were tested for the presence of avian influenza virus using specific primer and probe sets of H5, H7, H9, H10 and N8 subtypes by real-time RT-PCR. In our analysis, the majority (97.98%) of positive samples were from live poultry markets. Among the poultry samples from chickens and ducks, AIV prevalence was 26.05 and 30.81%, respectively. Mixed infection of different HA subtypes was very common. Additionally, H10 subtypes coexistence with N8 was the most prevalent agent during the emergence of H10N8. This event illustrated a long-term surveillance was so helpful for pandemic preparedness and response.

CEM-Designer: Design of custom expression microarrays in the post-ENCODE Era

Microarrays are widely used in gene expression studies, and custom expression microarrays are popular to monitor expression changes of a customer-defined set of genes. However, the complexity of transcriptomes uncovered recently make custom expression microarray design a non-trivial task. Pervasive transcription and alternative processing of transcripts generate a wealth of interweaved transcripts that requires well-considered probe design strategies and is largely neglected in existing approaches.We developed the web server CEM-Designer that facilitates microarray platform independent design of custom expression microarrays for complex transcriptomes. CEM-Designer covers (i) the collection and generation of a set of unique target sequences from different sources and (ii) the selection of a set of sensitive and specific probes that optimally represents the target sequences. Probe design itself is left to third party software to ensure that probes meet provider-specific constraints.CEM-Designer is available at http://designpipeline.bioinf.uni-leipzig.de.

Development and mapping of SSR markers linked to resistance-gene homologue clusters in common bean

Common bean is an important but often a disease-susceptible legume crop of temperate, subtropical and tropical regions worldwide. The crop is affected by bacterial, fungal and viral pathogens. The strategy of resistance-gene homologue (RGH) cloning has proven to be an efficient tool for identifying markers and R (resistance) genes associated with resistances to diseases. Microsatellite or SSR markers can be identified by physical association with RGH clones on large-insert DNA clones such as bacterial artificial chromosomes (BACs). Our objectives in this work were to identify RGH-SSR in a BAC library from the Andean genotype G19833 and to test and map any polymorphic markers to identify associations with known positions of disease resistance genes. We developed a set of specific probes designed for clades of common bean RGH genes and then identified positive BAC clones and developed microsatellites from BACs having SSR loci in their end sequences. A total of 629 new RGH-SSRs were identified and named BMr (bean microsatellite RGH-associated markers). A subset of these markers was screened for detecting polymorphism in the genetic mapping population DOR364×G19833. A genetic map was constructed with a total of 264 markers, among which were 80 RGH loci anchored to single-copy RFLP and SSR markers. Clusters of RGH-SSRs were observed on most of the linkage groups of common bean and in positions associated with R-genes and QTL. The use of these new markers to select for disease resistance is discussed.

An immunomagnetic separation-real-time PCR system for the detection of Alicyclobacillus acidoterrestris in fruit products

Alicyclobacillus acidoterrestris is the most important spoilage species within the Alicyclobacillus genus and has become a major issue in the pasteurized fruit juice industry. The aim of this study was to develop a method combining immunomagnetic separation (IMS) with real-time PCR system (IMS-PCR) for rapid and specific detection of A. acidoterrestris in fruit products. A real-time PCR with the TaqMan system was designed to target the 16S rDNA genes with specific primer and probe set. The specificity of the assay was confirmed using 9 A. acidoterrestris strains and 21 non-A. acidoterrestris strains. The results indicated that no combination of the designed primers and probe was found in any Alicyclobacillus genus except A. acidoterrestris. The detection limit of the established IMS-PCR was less than 10CFU/mL and the testing process was accomplished in 2–3h. For the three types of samples (sterile water, apple juice and kiwi juice), the correlation coefficient of standard curves was greater than 0.991, and the calculated PCR efficiencies were from 108% to 109%. As compared with the standard culture method performed concurrently on the same set of samples, the sensitivity, specificity and accuracy of IMS-PCR for 196 naturally contaminated fruit products were 90.0%, 98.3% and 97.5%, respectively. The results exhibited that the proposed IMS-PCR method was effective for the rapid detection of A. acidoterrestris in fruit products.

A Sorghum bicolorexpression atlas reveals dynamic genotype-specific expression profiles for vegetative tissues of grain, sweet and bioenergy sorghums

BackgroundEffective improvement in sorghum crop development necessitates a genomics-based approach to identify functional genes and QTLs. Sequenced in 2009, a comprehensive annotation of the sorghum genome and the development of functional genomics resources is key to enable the discovery and deployment of regulatory and metabolic genes and gene networks for crop improvement.ResultsThis study utilizes the first commercially available whole-transcriptome sorghum microarray (Sorgh-WTa520972F) to identify tissue and genotype-specific expression patterns for all identified Sorghum bicolor exons and UTRs. The genechip contains 1,026,373 probes covering 149,182 exons (27,577 genes) across the Sorghum bicolor nuclear, chloroplast, and mitochondrial genomes. Specific probesets were also included for putative non-coding RNAs that may play a role in gene regulation (e.g., microRNAs), and confirmed functional small RNAs in related species (maize and sugarcane) were also included in our array design. We generated expression data for 78 samples with a combination of four different tissue types (shoot, root, leaf and stem), two dissected stem tissues (pith and rind) and six diverse genotypes, which included 6 public sorghum lines (R159, Atlas, Fremont, PI152611, AR2400 and PI455230) representing grain, sweet, forage, and high biomass ideotypes.ConclusionsHere we present a summary of the microarray dataset, including analysis of tissue-specific gene expression profiles and associated expression profiles of relevant metabolic pathways. With an aim to enable identification and functional characterization of genes in sorghum, this expression atlas presents a new and valuable resource to the research community.

OP0188 Tocilizumab and Rituximab, but Not Adalimumab, Display Highly Concordant Molecular Effects in the Rheumatoid Arthritis Synovium

ObjectivesThe clinical effects of biologics in patients with rheumatoid arthritis (RA) are well known. By contrast, little is known about their molecular effects in the RA synovium, and how administration...

RNA-Seq of Arabidopsis Pollen Uncovers Novel Transcription and Alternative Splicing

Pollen grains of Arabidopsis (Arabidopsis thaliana) contain two haploid sperm cells enclosed in a haploid vegetative cell. Upon germination, the vegetative cell extrudes a pollen tube that carries the sperm to an ovule for fertilization. Knowing the identity, relative abundance, and splicing patterns of pollen transcripts will improve our understanding of pollen and allow investigation of tissue-specific splicing in plants. Most Arabidopsis pollen transcriptome studies have used the ATH1 microarray, which does not assay splice variants and lacks specific probe sets for many genes. To investigate the pollen transcriptome, we performed high-throughput sequencing (RNA-Seq) of Arabidopsis pollen and seedlings for comparison. Gene expression was more diverse in seedling, and genes involved in cell wall biogenesis were highly expressed in pollen. RNA-Seq detected at least 4,172 protein-coding genes expressed in pollen, including 289 assayed only by nonspecific probe sets. Additional exons and previously unannotated 5' and 3' untranslated regions for pollen-expressed genes were revealed. We detected regions in the genome not previously annotated as expressed; 14 were tested and 12 were confirmed by polymerase chain reaction. Gapped read alignments revealed 1,908 high-confidence new splicing events supported by 10 or more spliced read alignments. Alternative splicing patterns in pollen and seedling were highly correlated. For most alternatively spliced genes, the ratio of variants in pollen and seedling was similar, except for some encoding proteins involved in RNA splicing. This study highlights the robustness of splicing patterns in plants and the importance of ongoing annotation and visualization of RNA-Seq data using interactive tools such as Integrated Genome Browser.

Modeling formamide denaturation of probe-target hybrids for improved microarray probe design in microbial diagnostics.

Application of high-density microarrays to the diagnostic analysis of microbial communities is challenged by the optimization of oligonucleotide probe sensitivity and specificity, as it is generally unfeasible to experimentally test thousands of probes. This study investigated the adjustment of hybridization stringency using formamide with the idea that sensitivity and specificity can be optimized during probe design if the hybridization efficiency of oligonucleotides with target and non-target molecules can be predicted as a function of formamide concentration. Sigmoidal denaturation profiles were obtained using fluorescently labeled and fragmented 16S rRNA gene amplicon of Escherichia coli as the target with increasing concentrations of formamide in the hybridization buffer. A linear free energy model (LFEM) was developed and microarray-specific nearest neighbor rules were derived. The model simulated formamide melting with a denaturant m-value that increased hybridization free energy (ΔG°) by 0.173 kcal/mol per percent of formamide added (v/v). Using the LFEM and specific probe sets, free energy rules were systematically established to predict the stability of single and double mismatches, including bulged and tandem mismatches. The absolute error in predicting the position of experimental denaturation profiles was less than 5% formamide for more than 90 percent of probes, enabling a practical level of accuracy in probe design. The potential of the modeling approach for probe design and optimization is demonstrated using a dataset including the 16S rRNA gene of Rhodobacter sphaeroides as an additional target molecule. The LFEM and thermodynamic databases were incorporated into a computational tool (ProbeMelt) that is freely available at http://DECIPHER.cee.wisc.edu.

Assessment of Aspergillus fumigatus in Guinea Pig Bronchoalveolar Lavages and Pulmonary Tissue by Culture and Realtime Polymerase Chain Reaction Studies

In this study we pursued a diagnostic target in Aspergillus fumigatus (AF) by using qualitative Realtime PCR combined with proprietary DNA primers and a hydrolysis probe specific for this fungal target. Qualitative Realtime PCR is a diagnostic tool that utilizes Realtime PCR technology and detects the presence or absence target specific DNA within a predetermined detection range. Respiratory tissue and fluids from experimentally infected guinea pigs were tested by extracting DNA from the samples which were amplified and detected using AF specific DNA primers and probe. This study included qualitative evaluations of all specimens for the presence of the DNA of AF. The findings in the tissues after AF infection were compared to the numbers of spores in aerosolized samples used to inoculate the animals. Results demonstrated that the specific probe and primer set could detect the presence or absence of AF DNA in the sample. The qualitative detection limit of the assay ranged from 6 × 104 copies to 6 copies. Since blood cultures are rarely positive for Aspergillosis, our data indicate that qualitative Realtime PCR, in combination with the appropriate DNA primers and probe can serve as an effective diagnostic tool in the early detection of fungal infections.

2.54 Targeting Refractory CLL Lacking ATM Function with Sapacitabine-Containing Therapy

Cytogenetic abnormalities are common, and have prognostic implications, in chronic lymphocytic leukemia (CLL). In about 7080% CLL cases, chromosomal abnormalities can be identified by fluorescence in-situ hybridization (FISH) using a disease specific probe set. The most common genomic aberrations include deletion 13q, trisomy 12, and deletion in 11q, 17p and 6q. Approximately 10% of previously untreated patients with CLL exhibit a substantial deletion in the q arm of chromosome 11, the site of the ATM gene at (11q22.3). This lesion occurs at increased incidence of about 40% in patients relapsing on fludarabine-cyclophosphamide-rituximab (FCR) chemoimmunotherapy. The residual ATM allele is often mutated, suggesting that the region containing ATM may be important for response to chemoimmunotherapy. Sapacitabine is the orally bioavailable prodrug of the nucleoside analog CNDAC. It is undergoing clinical trials and has presented favorable clinical activity in advanced acute leukemias and myelodysplastic syndromes. CNDAC induces cell death by a unique mechanism of action. Incorporation of CNDAC triphosphate into DNA results in a single-strand break (SSB), which is converted to a one-ended double-strand break (DSB) upon collapse of the DNA replication fork. We have demonstrated that CNDAC-induced DSBs, which are lethal if not repaired, are repaired predominantly through homologous recombination (HR), relative to other repair pathways, such as transcription-coupled nucleotide excision (TC-NER) and non-homologous end-joining (NHEJ). CNDAC greatly sensitizes the viability of cells lacking HR components, such as ATM, Rad51D, Xrcc3 and Brca2. Based on this evidence, we hypothesize that CLL patients with 11q deletion that lack ATM function will be selectively sensitized to sapacitabinecontaining therapy. In order to distinguish ATM non-functional and functional individuals among del(11q22-23) patients characterized by FISH, we have established an assay of ATM activity. ATM is activated in response to irradiation and will further phosphorylate its downstream targets. We have shown that phosphorylation of KAP1 and Smc1 is dependent on the activity of ATM. In this assay, samples from eight patients with normal FISH karyotype were collected. These samples were mock treated or irradiated at dose of 10 Gy and protein lysates were harvested. The lysates were then combined as a positive pool comparator for the 11q deletion CLL samples, and both total and phosphorylated proteins were quantitated in immunoblots. The averaged phosphorylation level of the KAP1 and Smc1 proteins among the CLL patient samples was calculated and compared with those of the positive pool. The value of the samples to the positive pool of 30% was used as indicative of deficient ATM function. Using this cutoff, we identified five ATM non-functional individuals among ten del(11q22-23) CLL samples isolated from archived stocks. A phase II clinical trial of sapacitabine in combination with cyclophosphamide and rituximab (SCR) for previously treated patients with CLL having del(11q22-23) is open for patient recruitment. We will extend clinical investigations of sapacitabine to these CLL patients, focusing on the comparisons of responses of patients defined by the loss of ATM function. The results will test the hypothesis that loss of ATM function will sensitize the disease to sapacitabine therapy and prevent disease progression.

HPV Genotype Detection Using Hybrid Capture Sample Preparation Combined with Whole Genome Amplification and Multiplex Detection with Luminex XMAP

Infection with a high-risk carcinogenic type of human papillomavirus (HPV) is necessary for the development of cervical cancer. The digene HC2 HPV Test (HC2) is an important screening tool but lacks genotyping capability. To address this issue, we developed an assay for the rapid genotyping of HPV in cervical specimens. The three steps of this assay include Hybrid Capture target enrichment, whole-genome amplification, and Luminex XMAP detection. The assay includes the simultaneous detection of two genomic regions from each of 17 high-risk and two low-risk HPV types most associated with disease. The assay performance was tested on HPV plasmids as well as clinical specimens. An analytical limit of detection of 100 copies or less was demonstrated for linear, circular, and integrated HPV DNA. This finding is at least 1 log lower than the HC2 assay limit of detection. There was no cross-reactivity among the HPV types up to 1,000,000 copies. There was also no substantial assay interference from substances in cervical specimens. Although the clinical performance of the assay was not formally tested, the assay had good agreement (Cohen's kappa equal to 0.72) with both a PCR-based HPV genotyping assay (n = 131) and the HC2 assay (n = 502) using representative cervical specimens. This assay may be easy to automate and could be applied for the detection of other targets in future studies.

A panel of real-time PCR assays for specific detection of three phytoplasmas from the apple proliferation group

We report here on the development of combination of assays for fast, reliable, specific and sensitive detection and discrimination of ‘ Candidatus Phytoplasma mali’, ‘ Ca. P. prunorum’ and ‘ Ca. P. pyri’ from the 16Sr-X (apple proliferation – AP) group. These phytoplasmas are causal agents of diseases of fruit trees within the family Rosaceae, namely apple proliferation (AP), European stone fruit yellows (ESFY) and pear decline (PD). The designed panel of assays uses TaqMan ® minor groove binder probes (MGB). It comprises the same set of primers and specific probes for species-specific amplification within the 16S–23S rRNA intergenic spacer region, a set of primers and probes for amplification of the 16S ribosomal DNA region for the universal phytoplasma detection, and an additional set of primers and probe for 18S rRNA as an endogenous quality control of DNA extraction. The performance characteristics of the panel were evaluated. The advantages of new assays were shown in a comparative study with the conventional PCR, which proved their higher sensitivity combined with three-fold shorter time of testing process; and in comparison with two reported multiplex real-time PCR assays for detection of ‘ Ca. P. mali’ or ‘ Ca. P. pyri’. New panel of assays were tested on the DNA samples of ‘ Ca. P. mali’, ‘Ca. P. prunorum’, ‘ Ca. P. pyri’, other phytoplasmas and other bacteria isolated from plant material. Additionally, 198 symptomatic and asymptomatic fruit tree field samples collecting during several growing seasons were tested with new assays as well. The results of this study indicate that the combination of three specific assays may be applied in routine phytoplasma surveys and in the certification programs.

ALK gene rearrangements in unselected caucasians with non-small cell lung carcinoma (NSCLC).

10533 Background: Transforming rearrangements of the ALK gene have been reported in a subset of NSCLC, commonly fusing the ALK kinase domain with EML4 promoter but rarer fusion partners (TFG and KIF5B) have also been described. ALK is a valid clinical target based on phase I clinical data with dual ALK/MET inhibitor. We investigated the cytogenetic patterns of ALK rearrangements using FISH and the molecular and clinical characteristics associated with ALK+ in an unselected NSCLC population. Methods: Cohort included 447 NSCLC with previous EGFR and MET copy number gain (CNG) available; median age at diagnosis = 66 years, 83% male, 52% former and 35% current smokers; 52% of tumors were adenocarcinoma/BAC, stage I to IV was 37%, 22%, 27%, and 14%, respectively. Dual color FISH analyses were performed with ALK break-apart probe (Abbott Molecular) and 3 novel fusion probe sets specific for EML4-ALK, KIF5B-ALK and TFG-ALK. Results: Using the break apart probe, 12 patients (2.68%) were identified as ALK+. Multiple different cytogenetic patterns were observed, but virtually all carried the EML4-ALK fusion when specific probe sets were used. Using logistic regression analyses, higher odds were verified for ALK+ among males vs. females (p = 0.03), never vs. ever smokers (p = 0.005), adenocarcinoma vs. other histologies (p = 0.0013), and younger vs. older age (p = 0.08). No association was found between ALK+ and clinical stage, EGFR or MET CNG. There was no association between survival and ALK+ after adjusting for patient's gender, smoking history, histology and age (HR = 0.70; 95% CI 0.0256, 1.913, p = 0.48). Conclusions: In this large study of unselected Caucasian NSCLC patients, ALK+ was associated with male gender, never smoking status and adenocarcinoma histology suggesting these factors could be used to enrich ALK screened populations. The specific FISH probe sets identified EML4- ALK in different FISH patterns observed with the ALK rearrangement probe. ALK+ is distinct from EGFR and MET CNG, suggesting independent molecular mechanisms. ALK+ showed no significant prognostic impact, the observed favorable effect was unstable most likely due to the low number of ALK+. No significant financial relationships to disclose.

Drug treatment of Alzheimer's disease patients leads to expression changes in peripheral blood cells

Background Increasing cholinergic activity has been the primary mechanism for treating dementia due to Alzheimer's disease. However, the effectiveness of cholinesterase inhibitors (ChEIs) is still widely debated. The identification of specific biomarkers capable of identifying patients more likely to respond to these treatments could potentially provide specific evidence to clearly address this controversy through patient stratification. The goal of this study was to determine the feasibility of discovering biomarkers specific for the treatment of Alzheimer's disease. Methods Peripheral blood was collected from a cohort of patients treated with different ChEIs. Total RNA was isolated and profiled on the human Genome-Wide SpliceArray (GWSA) to test the feasibility of discriminating the different treatment subgroups of subjects based on the expression patterns generated from the Genome-Wide SpliceArray. Results Specific expression differences were identified for the various treatment groups that lead to a clear separation between patients treated with ChEIs versus naïve patients when Principal Component Analysis was performed on probe sets selected for differential expression. In addition, specific probe sets were identified to be dependent on the inhibitor used among the treated patients. Conclusions Distinct separation between non-treated, galantamine, donepezil, and rivastigmine-treated patients was clearly identified based on small sets of expression probes. The ability to identify drug-specific treatment expression differences strengthens the potential for using peripheral gene signatures for the identification of individuals responding to drug treatment.

Development of multitarget real-time PCR for the rapid, specific, and sensitive detection of Yersinia pestis in milk and ground beef.

Real-time PCR has been used previously to detect Yersinia pestis; this study applies this rapid, specific, and sensitive nucleic acid-based method to the detection and quantitation of Y. pestis specifically in food. Five sets of primers and corresponding TaqMan dual-labelled fluorogenic hybridization probes for Y. pestis were designed and optimized for specificity testing using genomic DNA from 71 bacterial strains. Four Y. pestis -specific primer and probe sets were developed, based on the virulence plasmid targets, and used to distinguish this bacterium from the various Yersinia and other bacterial species tested. An additional primer and probe set, based on a chromosomal gene target, distinguished Y. pestis and Yersinia pseudotuberculosis from the various Yersinia and other bacterial species tested. With optimized conditions, the quantitative detection limit of the probes for Y. pestis pure cultures ranged from 13 to 220 CFU. Standard curves were generated for the probes and used to determine the amplification efficiencies. The primers and probes demonstrated high amplification efficiencies, and their performance was evaluated using spiked milk and ground beef samples. The quantitative detection limit was 10(1) to 10(3) CFU/ml in milk and 10(2) to 10(5) CFU/g in ground beef without any preenrichment step. Testing the hybridization probes on food samples demonstrated the detection of Y. pestis in a foodborne application; this is the first such report, to our knowledge.

LXR ligand lowers LDL cholesterol in primates, is lipid neutral in hamster, and reduces atherosclerosis in mouse

Liver X receptors (LXRs) are ligand-activated transcription factors that coordinate regulation of gene expression involved in several cellular functions but most notably cholesterol homeostasis encompassing cholesterol transport, catabolism, and absorption. WAY-252623 (LXR-623) is a highly selective and orally bioavailable synthetic modulator of LXR, which demonstrated efficacy for reducing lesion progression in the murine LDLR(-/-) atherosclerosis model with no associated increase in hepatic lipogenesis either in this model or Syrian hamsters. In nonhuman primates with normal lipid levels, WAY-252623 significantly reduced total (50-55%) and LDL-cholesterol (LDLc) (70-77%) in a time- and dose-dependent manner as well as increased expression of the target genes ABCA1/G1 in peripheral blood cells. Statistically significant decreases in LDLc were noted as early as day 7, reached a maximum by day 28, and exceeded reductions observed for simvastatin alone (20 mg/kg). Transient increases in circulating triglycerides and liver enzymes reverted to baseline levels over the course of the study. Complementary microarray analysis of duodenum and liver gene expression revealed differential activation of LXR target genes and suggested no direct activation of hepatic lipogenesis. WAY-252623 displays a unique and favorable pharmacological profile suggesting synthetic LXR ligands with these characteristics may be suitable for evaluation in patients with atherosclerotic dyslipidemia.