DNA Array

Background Rhizotoxic ions in problem soils inhibit nutrient and water acquisition by roots, which in turn leads to reduced crop yields. Previous studies on the effects of rhizotoxic ions on root growth and physiological functions suggested that some mechanisms were common to all rhizotoxins, while others were more specific. To understand this complex system, we performed comparative transcriptomic analysis with various rhizotoxic ions, followed by bioinformatics analysis, in the model plant Arabidopsis thaliana. Results Roots of Arabidopsis were treated with the major rhizotoxic stressors, aluminum (Al) ions, cadmium (Cd) ions, copper (Cu) ions and sodium (NaCl) chloride, and the gene expression responses were analyzed by DNA array technology. The top 2.5% of genes whose expression was most increased by each stressor were compared with identify common and specific gene expression responses induced by these stressors. A number of genes encoding glutathione-S-transferases, peroxidase...

Successful use and reliability of microarray technology is highly dependent on several factors, including surface chemistry parameters and accessibility of cDNA targets to the DNA probes ®xed onto the surface. Here, we show that functionalisation of glass slides with homemade dendrimers allow production of more sensitive and reliable DNA microarrays. The dendrimers are nanometric structures of size-controlled diameter with aldehyde function at their periphery. Covalent attachment of these spherical reactive chemical structures on amino-silanised glass slides generates a reactive ~100 A Ê layer onto which amino-modi®ed DNA probes are covalently bound. This new grafting chemistry leads to the formation of uniform and homogenous spots. Moreover, probe concentration before spotting could be reduced from 0.2 to 0.02 mg/ml with PCR products and from 20 to 5 mM with 70mer oligonucleotides without affecting signal intensities after hybridisation with Cy3-and Cy5-labelled targets. More interestingly, while the binding capacity of captured probes on dendrimer-activated glass surface (named dendrislides) is roughly similar to other functionalised glass slides from commercial sources, detection sensitivity was 2-fold higher than with other available DNA microarrays. This detection limit was estimated to 0.1 pM of cDNA targets. Altogether, these features make dendrimer-activated slides ideal for manufacturing cost-effective DNA arrays applicable for gene expression and detection of mutations.

Zinc deficiency affects hepatic functions and due to the central role of the liver in metabolism, this may contribute to metabolic alterations in other tissues in zinc deficiency. In addition to clinical manifestations of zinc deficiency, we used cDNA- and oligonucleotide-arrays to compare the expression of > 2500 different genes in liver of rats force-fed a zinc-adequate or a zinc-deficient diet for 11 d. Radio- or fluorescence-labeled cDNAs from liver of control and zinc-deficient rats were hybridized to arrays. Approximately 1550 mRNAs were detected above background levels; by comparing expression profiles of the two groups, the mRNA levels of 66 genes were found to be altered by zinc deficiency. Steady-state expression levels of 35 genes were reduced, whereas the mRNA-levels of 31 genes were elevated. Array data were verified by Northern blot analysis for 24 selected genes and 19 were confirmed to be up- or down-regulated. Among those, predominantly gene products that partici...

Plant-derived essential oils with monoterpenoids have been used as antifungal drugs since ancient times, but the mode of action of these natural hydrocarbons at the molecular level is not understood. In order to understand the mechanisms of toxicity of α-terpinene (a cyclic monoterpene), a culture of Saccharomyces cerevisiae was exposed to 0.02% α-terpinene for 2 h and transcript profiles were obtained using yeast DNA arrays. These profiles, when compared with transcript profiles of untreated cultures, revealed that the expression of 793 genes was affected. For 435 genes, mRNA levels in treated cells compared with control cells differed by more than two-fold, whereas for 358 genes, it was <0.5-fold. Northern blots were performed for selected genes to verify the microarray results. Functional analysis of the up-regulated genes indicates that, similar to commonly used antifungal drugs, α-terpinene exposure affected genes involved in ergosterol biosynthesis and sterol uptake. In addition, transcriptional induction of genes related to lipid metabolism, cell wall structure and function, detoxification and cellular transport was observed in response to terpinene toxicity. Notably, the functions of 192 up-regulated genes are still unknown, but their characterization will probably shed light on the mechanisms of drug resistance and sensitivity. Taken together, this study showed that α-terpinene has strong antifungal activities and its modes of action resemble those of presently used antifungal drugs.

In obesity and type 2 diabetes, expression of the GLUT4 glucose transporter is decreased selectively in adipocytes. Adipose-specific Glut4 (also known as Slc2a4) knockout (adipose-Glut4 2 /2 ) mice show insulin resistance secondarily in muscle and liver. Here we show, using DNA arrays, that expression of retinol binding protein-4 (RBP4) is elevated in adipose tissue of adipose-Glut4 2 /2 mice. We show that serum RBP4 levels are elevated in insulin-resistant mice and humans with obesity and type 2 diabetes. RBP4 levels are normalized by rosiglitazone, an insulin-sensitizing drug. Transgenic overexpression of human RBP4 or injection of recombinant RBP4 in normal mice causes insulin resistance. Conversely, genetic deletion of Rbp4 enhances insulin sensitivity. Fenretinide, a synthetic retinoid that increases urinary excretion of RBP4, normalizes serum RBP4 levels and improves insulin resistance and glucose intolerance in mice with obesity induced by a high-fat diet. Increasing serum RBP4 induces hepatic expression of the gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PEPCK) and impairs insulin signalling in muscle. Thus, RBP4 is an adipocytederived 'signal' that may contribute to the pathogenesis of type 2 diabetes. Lowering RBP4 could be a new strategy for treating type 2 diabetes.

Recently we have described the molecular cloning of the genetic determinant coding for the S-fimbrial adhesin (Sfa), a sialic acid-recognizing pilus frequently found among extraintestinal Escherichia coli isolates. Fimbriae from the resulting Sfa+ E. coli K-12 clone were isolated, and an Sfa-specific antiserum was prepared. Western blots indicate that S fimbriae isolated from different uropathogenic and meningitis-associated E. coli strains, including 083:K1 isolates, were serologically related. The Sfa-specific antibodies did not cross-react with P fimbriae, but did cross-react with FlC fimbriae. Furthermore the sfa+ recombinant DNAs and some cloned sfa-flanking regions were used as probes in Southern experiments. Chromosomal DNAs isolated from 018:K1 and 083:K1 meningitis strains with and without S fimbriae and from uropathogenic 06:K+ strains were hybridized against these sfa-specific probes. Only one copy of the sfa determinant was identified on the chromosome of these strains. No sfa-specific sequences were observed on the chromosome of E. coli K-12 strains and an 07:K1 isolate. With the exception of small alterations in the sfa-coding region the genetic determinants for S fimbriae were identical in uropathogenic 06:K+ and meningitis 018:K1 and 083:K1 strains. The sfa determinant was also detected on the chromosome of Kl isolates with an Sfa-negative phenotype, and specific cross-hybridization signals were visible after blotting against FlC-specific DNA. In addition homology among the different strains was observed in the sfa-flanking regions.

Objective: Buyang Huanwu Decoction (BYHW), a famous herbal prescription in traditional Chinese medicine (TCM), has been used for 200 years for treating ischemic heart failure (IHF). This study aims to assess the efficacy and safety of BYHW combined with guidelineguided pharmacotherapy in patients with IHF and explore the biological mechanism by which BYHW exerts its efficacy. Methods: In the multicenter, double-blind, randomized controlled trial, a total of 80 patients with IHF were randomized to receive BYHW or placebo for 3 months. The primary efficacy endpoints were New York Heart Association (NYHA) classification, TCM syndrome scores, N-terminal pro-B-type natriuretic peptide (NT-ProBNP), whereas the mechanism exploration endpoints included energy metabolism parameters and coagulation function parameters. In addition, we performed the proteomic study of the serum of patients after treatment by label-free quantification technology to verify the candidate target proteins and pathways. Results: After 3 months of treatment, the NYHA classification, TCM syndrome scores, and the percentage of subjects with at least 30% reduction in NT-ProBNP were significantly improved in the BYHW group, compared with the control group (p < 0.05); BYHW treatment also significantly regulated blood glucose, blood lipid levels, ameliorated energy metabolism and improved coagulation function parameters. There

The Kidd (JK) blood group system has 3 antigens: Jka, Jkb, and Jk3. The forming antibodies directed against these antigens can lead to hemolytic transfusion reactions due to incompatible blood transfusions. This study aimed to determine the incidence of Jka and Jkb antigens and their phenotypes among blood donors at King Abdulaziz Medical City-Western Region, Saudi Arabia. In this cross-sectional retrospective study, a total of 14,987 blood donors were attended at the blood bank of King Abdulaziz Medical City-Western Region between January 2020 and May 2024. Serological investigation was conducted using a solid-phase technique to identify the Jka and Jkb antigens as well as the phenotypes. Most blood donors were Saudis (n = 12,844, 85.70%), whereas 2143 (14.30%) were non-Saudis. The distributions of Jka and Jkb antigens in Saudis were 87.87% and 53.94%, respectively. The frequencies in the non-Saudi population were 84.10% and 64.39%, respectively. This difference was statistically significant (P < .01). The proportions of the JK phenotypes showed a highly statistically significant difference between Saudis and non-Saudis (P < .01). Among Saudis, the JK phenotypes were 46.04% Jk(a+b-), 41.48% Jk(a+b+), 12.09% Jk(a-b+). and 0.03% Jk(a-bIn In contrast, the JK phenotypes in non-Saudis were 35.60% Jk(a+b-), 48.53% Jk(a+b+), and 15.87% Jk(a-b+), with no incidence of Jk(a-b-) phenotype. This study reported the frequencies of JK blood group among Saudi and non-Saudi blood donors. Based on these findings, incorporation of JK antigen screening into transfusion protocols is recommended to enhance the safety and efficacy of blood transfusions. Abbreviations: HDFN = hemolytic disease of the fetus and newborn, HTRs = hemolytic transfusion reactions, JK = Kidd, KAMC-WR = King Abdulaziz Medical City-Western Region.

Black Aspergilli are second most important cause of contamination of poultry feed and their by-products with ochratoxin-A after Aspergillus ochraceous in Pakistan. A total of 23 black Aspergilli were isolated from poultry feeds. Of these black Aspergilli samples, 12 (52.17%) were toxigenic (Ochratoxin A). A PCR based molecular method of cloning and sequencing of ITS (internal transcribed spacer) regions of fungal DNA was developed to identify these fungi. A universal pair of primers (ITS1 and ITS4) was used and PCR product (600 bp) for A. niger was amplified. These specific universal primers of ITS region were tested for twelve DNA samples of different isolates of Aspergillus species isolated from poultry feeds. The sequencing of ITS-region provided a powerful and reliable tool for identification of black Aspergilli responsible for the production of ochratoxin-A in poultry feed.

RNA interference (RNAi) is implicated in maintaining tandem DNA arrays as constitutive heterochromatin. We used chromatin immunoprecipitation with antibodies to RNA polymerase II (RNAPol-ChIP) to test for transcription of the following repeat arrays in human cells: subtelomeric D4Z4, pericentromeric satellite 2, and centromeric satellite α. D4Z4 has a promoter-like sequence upstream of an ORF in its 3.3-kb repeat unit. A short D4Z4 array at 4q35 is linked to facioscapulohumeral muscular dystrophy (FSHD). By RNAPol-ChIP and RT-PCR, little or no transcription of D4Z4 was detected in FSHD and normal myoblasts; lymphoblasts from an FSHD patient, a control, and a patient with D4Z4 hypomethylation due to mutation of DNMT3B (ICF syndrome); and normal or cancer tissues. However, RNAPol-ChIP assays indicated transcription of D4Z4 in a chromosome 4-containing human-mouse somatic cell hybrid. ChIP and RT-PCR showed satellite DNA transcription in some cancers and lymphoblastoid cell lines, although only at a low level. Given the evidence for the involvement of RNAi in satellite DNA heterochromatinization, it is surprising that, at most, a very small fraction of satellite DNA was associated with RNA Pol II. In addition, our results do not support the previously hypothesized disease-linked differential transcription of D4Z4 sequences in short, FSHD-linked arrays.

Introduction The European Water Framework Directive aims to achieve a good ecological and chemical status in surface water of European rivers by the year 2015. Since sediments and particulate matter act as secondary sources for pollutants, applied sediment toxicology is perceived to play a major role for obtaining new knowledge that can contribute to successful attainment of the goal. However, the existing bioassays for sediment toxicity analyses do not provide sufficient data concerning bioavailability of environmental pollutants. In this regard, there is an urgent need to combine sediment contact assays with gene expression analysis to investigate mechanism-specific sediment toxicity. Purpose The aim of the novel joint research project is to develop a eukaryotic test system, which can be used to investigate the ecotoxicological effects of contaminated sediments on gene expression level (DNA-array and RT-PCR). Current ecotoxicological research customarily involves a battery of bioassays to cover different toxicological endpoints (e.g., teratogenicity, genotoxicity, mutagenicity, Ah-receptormediated toxicity, neurotoxicity). In contrast, methods that detect alterations in gene expression offer deeper insight by elucidating how chemical exposure and/or environmental challenge affect multiple metabolic pathways leading to these particular kinds of toxic response. Gene expression profiles reflect the way cells and organisms adapt or respond to a changing environment. Conclusion The present project aspires to increase the fundamental molecular and physiological knowledge concerning the mode of action of environmental toxicants in zebrafish (Danio rerio). By working with partners from the academic and research institutions as well as from industry and waterway regulations, the success of this basic research-driven joint project in terms of development and implementation of novel sediment toxicity methods will be realized.

Background: Complex allergenic sources such as moulds, foods and mites contain complex panels of IgE-binding molecules which need to be cloned, produced and characterized in order to mimic the entire allergenicity of whole extracts reconstituted by mixing single standardized recombinant allergens. Methods: Phage surface display of cDNA libraries selectively enriched for allergen-expressing clones using IgE from allergic patients allows rapid isolation of large panels of allergens. For the characterization of all different clones present in enriched cDNA libraries in a fast and cost-effective way, high-throughput screening technology is required. Results: The combination of selective enrichment of cDNA libraries based on biopanning against serum IgE from sensitized patients and automated robot technology for picking and high-density gridding of clones onto filter membranes, followed by hybridization, enables fast identification of all the different clones present in an enriched libra...

We have demonstrated that the dynamics of nucleic acid hybridization in microarrays depend on the physical structure of immobilized probes. We have immobilized oligonucleotide-3 0 -phosphates with and without stem-loop structure on epoxylated glass surface, followed by hybridization under different conditions, viz., hybridization buffer, pH condition, temperature and ionic strength. In a comparative study, we have established that array constructed using probes with stem-loop structure displayed $2.2 times higher hybridization signals than the probes without it. The stem-loop DNA array format is simple and flexible in design and thus potentially useful in various DNA diagnostic tests.

Reduction in the background problems to improve the efficiency of nonradioactive labeling and detection procedure has been the focus of attention in the recent past for wider acceptance of the technique in nucleic acid research. We have achieved success in obtaining a relatively background free detection of single copy genes such as, T-cell receptor-delta (TCR-delta) and interleukin-2 receptor (L-2r) in human genome by using optimized concentrations of the digoxigenin labeled probes. Inclusion of such an optimization step for each probe before carrying out the actual hybridization experiment did not require any further modification in hybridization conditions and detection protocols as suggested earlier [Anal Biochem, 210 (1993) 235; Colloq Boehringer Mannheim, 2 (1991) 4.]

Lassa virus (LASV) is a zoonotic virus endemic to western Africa that can cause a potentially lethal and hemorrhagic disease, Lassa fever (LF). Survivors suffer a myriad of sequelae, most notably sudden onset sensorineural hearing loss (SNHL), the mechanism of which remains unclear. Unfortunately, studies aiming to identify the mechanism of these sequelae are limited due to the biosafety level 4 (BSL4) requirements of LASV itself. ML29, a reassortant virus proposed as an experimental vaccine candidate against LASV, is potentially an ideal surrogate model of LF in STAT1-/-mice due to similar phenotype in these animals. We intended to better characterize ML29 pathogenesis and potential sequelae in this animal model. Our results indicate that while both CD4 and CD8 T cells are responsible for acute disease in ML29 infection, ML29 induces significant hearing loss in a mechanism independent of either CD4 or CD8 T cells. We believe that this model could provide valuable information for viral-associated hearing loss in general.

Extraintestinal pathogenic E. coli (ExPEC) cause an array of diseases, including sepsis, neonatal meningitis, and urinary tract infections. Many putative virulence factors that might modulate ExPEC pathogenesis have been identified through sequencing efforts, epidemiology, and gene expression profiling, but few of these genes have been assigned clearly defined functional roles during infection. Using zebrafish embryos as surrogate hosts, we have developed a model system with the ability to resolve diverse virulence phenotypes and niche-specific restrictions among closely related ExPEC isolates during either localized or systemic infections. In side-by-side comparisons of prototypic ExPEC isolates, we observed an unexpectedly high degree of phenotypic diversity that is not readily apparent using more traditional animal hosts. In particular, the capacity of different ExPEC isolates to persist and multiply within the zebrafish host and cause disease was shown to be variably dependent upon two secreted toxins, a-hemolysin and cytotoxic necrotizing factor. Both of these toxins appear to function primarily in the neutralization of phagocytes, which are recruited in high numbers to sites of infection where they act as an essential host defense against ExPEC as well as less virulent E. coli strains. These results establish zebrafish as a valuable tool for the elucidation and functional analysis of both ExPEC virulence factors and host defense mechanisms.

Germline mutations in predisposition genes account for only 20% of all familial colorectal cancers (CRC) and the remaining genetic burden may be due to rare high- to moderate-penetrance germline variants that are not explored. With the aim of identifying such potential cancer-predisposing variants, we performed whole exome sequencing on three CRC cases and three unaffected members of a Polish family and identified two novel heterozygous variants: a coding variant in APC downregulated 1 gene (APCDD1, p.R299H) and a non-coding variant in the 5′ untranslated region (UTR) of histone deacetylase 5 gene (HDAC5). Sanger sequencing confirmed the variants segregating with the disease and Taqman assays revealed 8 additional APCDD1 variants in a cohort of 1705 familial CRC patients and no further HDAC5 variants. Proliferation assays indicated an insignificant proliferative impact for the APCDD1 variant. Luciferase reporter assays using the HDAC5 variant resulted in an enhanced promoter activit...

Komagataella phaffii, formerly Pichia pastoris (P. pastoris), is a promising methylotrophic yeast used in industry to produce recombinant protein and valuable metabolites. In this study, a genome-scale metabolic model (GEMs) was reconstructed and used to assess P. pastoris' metabolic capabilities for the production of S-adenosyl-l-methionine (AdoMet or SAM or SAMe) from individual carbon sources along with the addition of l-methionine. In a model-driven P. pastoris strain, the well-established genome-scale metabolic model iAUKM can be implemented to predict high valuable metabolite production. The model, iAUKM, was created by merging the previously published iMT1026 model and the draught model generated using Raven toolbox from the KEGG database which covered 2309 enzymatic reactions associated with 1033 metabolic genes and 1750 metabolites. The highly curated model was successful in capturing P. pastoris growth on various carbon sources, as well as AdoMet production under various growth conditions. Many overexpression gene targets for increasing AdoMet accumulation in the cell have been predicted for various carbon sources. Inorganic phosphatase (IPP) was one of the predicted overexpression targets as revealed from simulations using iAUKM. When IPP gene was integrated into P. pastoris, we found that AdoMet accumulation increased by 16% and 14% using glucose and glycerol as carbon sources, respectively. Our in silico results shed light on the factors limiting AdoMet production, as well as key pathways for rationalized engineering to increase AdoMet yield.

Las dificultades derivadas de la gran complejidad del genoma humano, debido a la gran cantidad de genes que lo constituyen, y las dificultades a la hora de realizar un correcto diagnóstico molecular han requerido la utilización de tecnologías avanzadas, como las matrices de ADN. Por medio de esta tecnología es posible analizar en un solo experimento grandes secuencias de regiones genómicas, detectar mutaciones o polimorfismos característicos de cada individuo, así como analizar variaciones de expresión de genes implicados en diferentes enfermedades o en la utilización de fármacos. Del mismo modo, la información derivada de las matrices de ADN permite un gran avance en la medicina personalizada aplicada a cada paciente, pues se evitan los efectos adversos no deseados y se obtiene el mejor rendimiento para cada fármaco utilizado. Palabras clave: Matrices de ADN. ARN. Expresión diferencial. SNP. Resecuenciación. Diagnóstico molecular. ADN arrays: a general overview and specific applications Difficulties derived from the big complexity of the human genome (because of the great amount of genes) and difficulties at the time of performing a correct molecular diagnosis have led to the utilization of advanced technologies such as DNA arrays. By this technique, it is possible to analyze in a single experiment great sequences of genomic regions, to detect mutations or polymorphisms (characteristic of each individual), and to analyze variations in the expression of genes implicated in different disorders or in the utilization of drugs. Similarly, the use of DNA arrays-derived information has led to a considerable advance in the personalized medicine that is applied to every patient, avoiding unwanted adverse effects and the consecution of the best performance effectiveness for each drug.

We aimed to clarify the prime role of recurrence in stage I lung cancer. To determine the expression profiles, quantitative RT-PCR and real-time PCR were performed subsequently to evaluate the validity of meaningful molecules identified by 0.12 K c-DNA array experiment surveys. In all, 10 lung cancer patients presenting with recurrence of stage IB were selected and compared with 10 stage IB lung cancer patients without recurrence since biopsied 3 years previously. On c-DNA microarray data analysis using pairs of recurred and the corresponding nonrecurred patients, the following genes were found to be upregulated in the recurred cases: matrix metalloproteinase (MMP)-10 in five cases, MMP-12 in two cases, MMP-11, MMP-14, MMP-15, fos, cyclin E2, E 2 F3, TGF-a in each one case. The most frequently upregulated genes in recurred lung cancers were MMP-10 (stromelysin-2) and MMP-12 (macrophage elastase). On transcriptional assay by quantitative RT-PCR and real-time RT-PCR analysis to validate those molecules, both transcripts of MMP-10 and MMP-12 were significantly more upregulated in recurred stage IB lung cancer than in the non-recurred stage IB lung cancer (P ¼ 0.004). Transcript levels were identical to c-DNA array data. The protein levels of these entities were also evaluated by immunohistochemistry of archival slides. By immunohistochemistry, MMP-10 monoclonal antibody showed more intense immunoreactivity in the recurred stage IB lung cancer than in the nonrecurred stage IB lung cancer (P ¼ 0.0313). Our approach revealed that MMP-10 plays an important role in the recurrence in stage IB lung cancer, irrespective of the histologic type.

We have developed DNA microarrays containing stem-loop DNA probes with short single-stranded overhangs immobilized on a Packard HydroGel chip, a 3-dimensional porous gel substrate. Microarrays were fabricated by immobilizing self-complementary single-stranded oligonucleotides, which adopt a partially duplex structure upon denaturing and reannealing. Hybridization of single-stranded DNA targets to such arrays is enhanced by contiguous stacking interactions with stem-loop probes and is highly sequence specific. Subsequent enzymatic ligation of the targets to the probes followed by stringent washing further enhances the mismatched base discrimination. We demonstrate here that these microarrays provide excellent specificity with signalto-background ratios of from 10-to 300-fold. In a comparative study, we demonstrated that HydroGel arrays display 10-30 times higher hybridization signals than some solid surface DNA microarrays. Using Sanger sequencing reactions, we have also developed a method for preparing nested 3′-deletion sets from a target and evaluated the use of stem-loop DNA arrays for detecting p53 mutations in the deletion set. The stem-loop DNA array format is simple, robust and flexible in design, thus it is potentially useful in various DNA diagnostic tests.

Hantaviruses cause two human diseases: hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). Hantaviruses infect human endothelial cells but cause little or no damage to the infected endothelium. We analyzed with Affymetrix DNA Arrays (Santa Clara, CA) the endothelial cell transcriptional responses directed by hantaviruses associated with HPS [New York-1 virus (NY-1V)], HFRS [Hantaan virus (HTNV)], or by a hantavirus not associated with human disease [Prospect Hill virus (PHV)]. Hantavirus infections induced 117 cellular genes and repressed 25 genes by &gt;3-fold, 4 days postinfection (p.i.). Although &gt;80% of cells were infected by each virus 1 day p.i., PHV induced or repressed 67 genes at this early time compared with three genes altered by HTNV or NY-1V. The early high-level induction of 24 IFN-stimulated genes by PHV (4- to 229-fold) represents a fundamental difference in the temporal regulation of cellular responses by pathogenic and nonpathog...

Almtraet-1. Hatchling snapping turtles (Chelydra serpentina) that were placed on platforms of dry styrofoam survived for 7 h without freezing at temperatures below-4°C. 2. Turtles that were confined to artificial nests in frozen soil were unable to withstand exposure to temperatures much lower than-2°C. 3. Animals that were held in dry surroundings and then cooled to-2°C froze rapidly when they were brought into contact with crystals of ice. 4. These observations indicate that turtles which die from cold exposure when they are forced to overwinter in nests in the field probably succumb to inoculative freezing caused by contact with ice in the soil and not to spontaneous nucleation caused by body fluids reaching their limit of supercooling.

P ainted turtles (Chrysemys picta) are common residents of shallow lakes and marshes across much of North America east of the Rocky Mountains-so common, in fact, that the species has become a "model organism" for studies on the ecology and evolution of chelonians (Wilbur and Morin 1988). The natural history of painted turtles differs from that of most other species in an important respect, however. Whereas neonates of other freshwater turtles usually emerge from their subterranean nest in late summer or autumn and move to a nearby marsh, lake, or stream to spend their first winter, hatchling painted turtles typically remain inside their shallow (8-14 cm) nest throughout their first winter and do not emerge above the ground until the following spring (Ernst et al. 1994). This behavior commonly causes neonatal painted turtles in northern regions-from Nebraska (Packard 1997, Packard et al. 1997a), northern Illinois (Weisrock and Janzen 1999), and New Jersey (DePari 1996) northward to the limit of distribution in southern Canada (Storey et al. 1988)-to be exposed during winter to ice and cold, with temperatures in some nests dipping below-10°C (Figure 1). Many of these hatchlings withstand such extremes and emerge from the nest when the soil finally thaws in the spring (Table 1).

Two-class neutral zone classifiers were recently proposed for use in microbial community profiling applications. These classifiers allow a region of neutrality for cases where probe hybridization outcomes are too ambiguous to have adequate confidence in assigning a "binding" or "no binding" result. In this paper, we generalize the idea of neutral zone classifiers to an arbitrary number of classes and apply it to improve the process of microbial community profiling by considering a third class for the outcome of probe hybridization experiments, "partial binding." We introduce a family of class distributions that uses a mixture of Gaussian distributions as a model for a Box-Cox power transformation of the raw intensity measurements. Stratified cross-validation analyses are used to assess the efficacy of the proposed three-class neutral zone classifier. This article has supplementary material online.

In Escherichia coli, the H-NS protein plays an important role in the structure and the functioning of bacterial chromosome. A homologous protein has also been identified in several enteric bacteria and in closely related organisms such as Haemophilus influenzae. To get information on their structure and their function, we identified H-NS-like proteins in various microorganisms by different procedures. In silico analysis of their amino acid sequence and/or in vivo experiments provide evidence that more than 20 proteins belong to the same class of regulatory proteins. Moreover, large scale technologies demonstrate that, at least in E. coli, the loss of motility in hns mutants results from a lack of flagellin biosynthesis, due to the in vivo repression of flagellar gene expression. In contrast, several genes involved in adaptation to low pH are strongly induced in a H-NS deficient strain, resulting in an increased resistance to acidic stress. Finally, expression profiling and phenotypic analysis suggest that, unlike H-NS, its paralogous protein StpA does not play any role in these processes.

A variety of commercial DNA arrays specific for humans and rodents are widely available; however, microarrays containing well-characterized genes to study pathway-specific gene expression are not as accessible for domestic animals, such as cattle, sheep and pigs. Therefore, a small-scale application-targeted bovine immune-endocrine cDNA array was developed to evaluate genetic pathways involved in the immune-endocrine axis of cattle during periods of altered homeostasis provoked by physiological or environmental stressors, such as infection, vaccination or disease. For this purpose, 167 cDNA sequences corresponding to immune, endocrine and inflammatory response genes were collected and categorized. Positive controls included 5 housekeeping genes (glyceraldehydes-3-phosphate dehydrogenase, hypoxanthine phosphoribosyltransferase, ribosomal protein L19, b-actin, b2-microglobulin) and bovine genomic DNA. Negative controls were a bacterial gene (Rhodococcus equi 17-kDa virulence-associated protein) and a partial sequence of the plasmid pACYC177. In addition, RNA extracted from un-stimulated, as well as superantigen (Staphylococcus aureus enterotoxin-A, S. aureus Cowan Pansorbin Cells) and mitogen-stimulated (LPS, ConA) bovine blood leukocytes was mixed, reverse transcribed and PCR amplified using gene-specific primers. The endocrine-associated genes were amplified from cDNA derived from un-stimulated bovine hypothalamus, pituitary, adrenal and thyroid gland tissues. The array was constructed in 4 repeating grids of 180 duplicated spots by coupling the PCR amplified 213-630 bp gene fragments onto poly-L-lysine coated glass slides. The bovine immune-endocrine arrays were standardized and preliminary gene expression profiles generated using Cy3 and Cy5 labelled cDNA from un-stimulated and ConA (5 mg/ml) stimulated PBMC of 4 healthy Holstein cows (2-4 replicate arrays/cow) in a time course study. Mononuclear cell-derived cytokine and chemokine (IL-2, IL-1a, TNFa, IFN-g, TGFb-1, MCP-1, MCP-2 and MIP-3a) mRNA exhibited a repeatable and consistently low expression in un-stimulated cells and at least a twofold increased expression following 6 and 24 h ConA stimulation as compared to 0 h un-stimulated controls. In contrast, expression of antigen presenting molecules, MHC-DR, MHC-DQ and MHC-DY, were consistently at least twofold lower following 6 and 24 h ConA stimulation. The only endocrine gene with differential expression following ConA stimulation was prolactin. Additionally, due to the high level of genetic homology between ovine, swine and bovine genes, RNA similarly acquired from sheep and pigs was evaluated and similar gene expression patterns were noted. These data demonstrate that this application-targeted array containing a set of well characterized genes

Characterisation of breakpoints in disease-associated balanced chromosome rearrangements (DBCRs), which disrupt or inactivate specific genes, has facilitated the molecular elucidation of a wide variety of genetic disorders. However, conventional methods for mapping chromosome breakpoints, such as in situ hybridisation with fluorescent dye-labelled bacterial artificial chromosome clones (BAC-FISH), are laborious, time consuming and often with insufficient resolution to unequivocally identify the disrupted gene. By combining DNA array hybridisation with chromosome sorting, the efficiency of breakpoint mapping has dramatically improved. However, this can only be applied when the physical properties of the derivative chromosomes allow them to be flow sorted. To characterise the breakpoints in all types of balanced chromosome rearrangements more efficiently and more accurately, we performed massively parallel sequencing using Illumina 1G analyser and ABI SOLiD systems to generate short sequencing reads from both ends of DNA fragments. We applied this method to four different DBCRs, including two reciprocal translocations and two inversions. By identifying read pairs spanning the breakpoints, we were able to map the breakpoints to a region of a few hundred base pairs that could be confirmed by subsequent PCR amplification and Sanger sequencing of the junction fragments. Our results show the feasibility of paired-end sequencing of systematic breakpoint mapping and gene finding in patients with disease-associated chromosome rearrangements.

Plant-derived essential oils with monoterpenoids have been used as antifungal drugs since ancient times, but the mode of action of these natural hydrocarbons at the molecular level is not understood. In order to understand the mechanisms of toxicity of α-terpinene (a cyclic monoterpene), a culture of Saccharomyces cerevisiae was exposed to 0.02% α-terpinene for 2 h and transcript profiles were obtained using yeast DNA arrays. These profiles, when compared with transcript profiles of untreated cultures, revealed that the expression of 793 genes was affected. For 435 genes, mRNA levels in treated cells compared with control cells differed by more than twofold , whereas for 358 genes, it was <0.5-fold. Northern blots were performed for selected genes to verify the microarray results. Functional analysis of the up-regulated genes indicates that, similar to commonly used antifungal drugs, α-terpinene exposure affected genes involved in ergosterol biosynthesis and sterol uptake. In addition, transcriptional induction of genes related to lipid metabolism, cell wall structure and function, detoxification and cellular transport was observed in response to terpinene toxicity. Notably, the functions of 192 up-regulated genes are still unknown, but their characterization will probably shed light on the mechanisms of drug resistance and sensitivity. Taken together, this study showed that α-terpinene has strong antifungal activities and its modes of action resemble those of presently used antifungal drugs.

A single injection of ML29 reassortant vaccine for Lassa fever induces low, transient viremia, and low or moderate levels of ML29 replication in tissues of common marmosets depending on the dose of the vaccination. The vaccination elicits specific immune responses and completely protects marmosets against fatal disease by induction of sterilizing cell-mediated immunity. DNA array analysis of human peripheral blood mononuclear cells from healthy donors exposed to ML29 revealed that gene expression patterns in ML29-exposed PBMC and control, media-exposed PBMC, clustered together confirming safety profile of the ML29 in non-human primates. The ML29 reassortant is a promising vaccine candidate for Lassa fever.

Plant secondary metabolism significantly contributes to defensive measures against adverse abiotic and biotic cues. To investigate stress-induced, transcriptional alterations of underlying effector gene families, which encode enzymes acting consecutively in secondary metabolism and defense reactions, a DNA array (MetArray) harboring gene-specific probes was established. It comprised complete sets of genes encoding 109 secondary product glycosyltransferases and 63 glutathione-utilizing enzymes along with 62 cytochrome P450 monooxygenases and 26 ABC transporters. Their transcriptome was monitored in different organs of unstressed plants and in shoots in response to herbicides, UV-B radiation, endogenous stress hormones, and pathogen infection. A principal component analysis based on the transcription of these effector gene families defined distinct responses and crosstalk. Methyl jasmonate and ethylene treatments were separated from a group combining reactions towards two sulfonylurea herbicides, salicylate and an avirulent strain of Pseudomonas syringae pv. tomato. The responses to the herbicide bromoxynil and UV-B radiation were distinct from both groups. In addition, these analyses pinpointed individual effector genes indicating their role in these stress responses. A small group of genes was diagnostic in differentiating the response to two herbicide classes used. Interestingly, a subset of genes induced by P. syringae was not responsive to the applied stress hormones. Small groups of comprehensively induced effector genes indicate common defense strategies. Furthermore, homologous members within branches of these effector gene families displayed differential expression patterns either in both organs or during stress responses arguing for their non-redundant functions.

One major concern regarding long-term manned space missions is the effect of accumulative doses of space radiation (the mean daily dose is 0.2 ~ 1 mSv). Such missions will inevitably expose astronauts to significant doses, and thus are expected to increase the risks of radiation-related carcinogenicity, neurotoxicity, and lifespan changes. These effects occur by nuclear and cytosol dysfunction, mitochondrial damage, and particular changes in signal transduction or protein synthesis. In addition, microgravity may affect cellular metabolisms, signal transduction, etc., and might lead to synergistic effects with space radiation, which could result in further risk. Because these effects will almost certainly involve cellular signaling, transcriptomics and proteomics will be powerful tools in their analysis. In order to suitably respond to all these risks, both protective measures based on physical and biological principles, and effective intra-flight assessment of the levels of radiation exposure will be required. In this article, the categories of cell death-i.e., apoptosis, necrosis and autophagy-and the recent studies on these mechanisms are reviewed. ©2009 Jpn. Soc. Biol.

One major concern regarding long-term manned space missions is the effect of accumulative doses of space radiation (the mean daily dose is 0.2 ~ 1 mSv). Such missions will inevitably expose astronauts to significant doses, and thus are expected to increase the risks of radiation-related carcinogenicity, neurotoxicity, and lifespan changes. These effects occur by nuclear and cytosol dysfunction, mitochondrial damage, and particular changes in signal transduction or protein synthesis. In addition, microgravity may affect cellular metabolisms, signal transduction, etc., and might lead to synergistic effects with space radiation, which could result in further risk. Because these effects will almost certainly involve cellular signaling, transcriptomics and proteomics will be powerful tools in their analysis. In order to suitably respond to all these risks, both protective measures based on physical and biological principles, and effective intra-flight assessment of the levels of radiation exposure will be required. In this article, the categories of cell death-i.e., apoptosis, necrosis and autophagy-and the recent studies on these mechanisms are reviewed. ©2009 Jpn. Soc. Biol.

SummaryThe covalently linked cell wall protein Ccw12p of Saccharomyces cerevisiae is a GPI‐anchored protein (V. Mrsa et al., 1999, J Bacteriol 181: 3076–3086). Although only 121 amino acids long, the haemagglutinin‐tagged protein released by laminarinase from the cell wall possesses an apparent molecular mass of &gt; 300 kDa. A membrane‐bound form with an apparent molecular mass of 58 kDa is highly O‐ and N‐glycosylated and contains the GPI anchor. With a half‐life of 2 min, the membrane form is transformed to the &gt; 300 kDa form. The deletion mutant ccw12Δ grows slower than the wild type, is highly sensitive to Calcofluor white and contains 2.5 times more chitin. Further, compared with wild‐type yeast, significantly more proteins are released from intact cells when treated with dithiothreitol. Interestingly, these defects become less pronounced when further GPI‐anchored cell wall proteins are deleted. Mutant ΔGPI (simultaneous deletion of CCW12, CCW13/DAN1, CCW14, TIP1 and CWP1) ...

When a process is dominated by few important frequencies the observations of this process can be modelled by a harmonic process ). If the amplitudes of these dominating frequencies vary over time their dominance may not be apparent during the whole process. To discriminate between frequencies relevant for such a process we determine the distribution of the periodogram ordinates, and use this distribution to derive a procedure to assess the relevance of the frequencies. This procedure uses the standardized median (Gather and Schultze (1999)) to determine the variance of the error process. In a simulation study we show that this procedure is very efficient even under difficult conditions such as a low signal-to-noise ratio or AR(1) disturbances. Furthermore, we show that the necessary transformation to estimate the amplitudes from periodogram ordinates leads to a good normality approximation which makes it especially easy to model the development of the amplitudes from these estimates.

An algorithm based on a modified sticker model accompanied with an advanced MEMS-based microarray technology is demonstrated to solve SAT problem, which has long served as a benchmark in DNA computing. Unlike conventional DNA computing algorithms needing an initial data pool to cover correct and incorrect answers and further executing a series of separation procedures to destroy the unwanted ones, we built solutions in parts to satisfy one clause in one step, and eventually solve the entire Boolean formula through steps. No time-consuming sample preparation procedures and delicate sample applying equipment were required for the computing process. Moreover, experimental results show the bound DNA sequences can sustain the chemical solutions during computing processes such that the proposed method shall be useful in dealing with large-scale problems.

To the Editor: Methadone maintenance is the primary treatment modality in the US for opioid dependence, with doses typically titrated to therapeutic response such as improvement of withdrawal symptoms or abstinence from drug use. Serum methadone concentrations are used to guide dosing in

Background: Patients with familial hypercholesterolemia (FH) have a high risk of premature cardiovascular disease (PCVD). Mutations in the LDL receptor (LDLR) gene and the R3500Q mutation in the apolipoprotein B (APOB) gene are known to cause FH, but lack of high-throughput methods makes routine genetic diagnosis difficult. The objective of this work was to develop a DNA array for large-scale identification of mutant LDLR alleles. Methods: We developed a low-density oligonucleotide microarray to identify 118 DNA sequence variations (117 for the LDLR gene and 1 for the APOB gene). We verified specificity and sensitivity by analyzing 1180 previously sequenced DNA samples, and conducted a blind study screening 407 Spanish patients with a clinical diagnosis of FH. Results: The DNA array confirmed the previous genotyping results in almost all cases. In the blind study, the microarray detected at least 1 mutation in 51% of the patients for whom clinical diagnosis was classified as certain according to Dutch FH-MEDPED criteria; it also identified mutations in 37% of those with a diagnosis of probable/possible FH, thus giving a definite diagnosis. Patients harboring null mutations had shorter PCVDfree survival times and higher relative risk of PCVD than patients with a missense mutation. Conclusions: The proposed DNA array allows largescale population screening and provides molecular information regarding mutation type and its correlation with clinical severity of FH, which can be used to develop therapeutic strategies.

Circadian rhythms of cell and organismal physiology are controlled by an autoregulatory transcription-translation feedback loop that regulates the expression of rhythmic genes in a tissue-specific manner. Recent studies have suggested that components of the circadian pacemaker, such as the Clock and Per2 gene products, regulate a wide variety of processes, including obesity, sensitization to cocaine, cancer susceptibility, and morbidity to chemotherapeutic agents. To identify a more complete cohort of genes that are transcriptionally regulated by CLOCK and/or circadian rhythms, we used a DNA array interrogating the mouse protein-encoding transcriptome to measure gene expression in liver and skeletal muscle from WT and Clock mutant mice. In WT tissue, we found that a large percentage of expressed genes were transcription factors that were rhythmic in either muscle or liver, but not in both, suggesting that tissue-specific output of the pacemaker is regulated in part by a transcriptio...

S136 17th ECCMID / 25th ICC, Posters gene of Helicobacter pylori, so called thermophilic helicase-dependent isothermal DNA amplification, (tHDA). Methods: Like PCR, the tHDA reaction selectively amplifies a target sequence defined by two primers. However, unlike PCR, tHDA uses an additional enzyme called thermophilic helicase to separate DNA rather than heat. Since, this DNA amplification is an isothermal technique, as an advantage it does not need any thermocycler. The accuracy of the technique was checked on DNA extracted from pure culture of Helicobacter pylori. Results: We obtained same results when several experiments were performed on specimens prepared from infected gastric biopsies. All the results were shown the equal specificity and sensitivity for this technique in comparison to PCR. Conclusion: THDA can be used for molecular detection of Helicobacter pylori more cost-effectively than PCR in developing countries. Further studies are under taken to develop this technique using ELISA and realtime formats.

Recently, a new type of hybrid resulting from the hybridization between Saccharomyces cerevisiae and Saccharomyces kudriavzevii was described. These strains exhibit physiological properties of potential biotechnological interest. A preliminary characterization of these hybrids showed a trend to reduce the S. kudriavzevii fraction of the hybrid genome. We characterized the genomic constitution of several wine S. cerevisiae × S. kudriavzevii strains by using a combined approach based on the restriction fragment length polymorphism analysis of gene regions, comparative genome hybridizations with S. cerevisiae DNA arrays, ploidy analysis, and gene dose determination by quantitative real-time PCR. The high similarity in the genome structures of the S. cerevisiae × S. kudriavzevii hybrids under study indicates that they originated from a single hybridization event. After hybridization, the hybrid genome underwent extensive chromosomal rearrangements, including chromosome losses and the ge...

DNA microarrays and sensors have become essential tools in the functional analysis of sequence information. Recently we reported that chimeric hexitol (HNA) and altritol (ANA) nucleotide monomers with an anhydrohexitol sugar moiety are easily available and proved their chemistry to be compatible with DNA and RNA synthesis. In this communication we describe a novel analytical platform based on HNA and ANA units to be used as synthetic oligonucleotide arrays on a glass solid support for match/mismatch detection of DNA and RNA targets. Arrays were fabricated by immobilization of diene-modified oligonucleotides on maleimido-activated glass slides. To demonstrate the selectivity and sensitivity of the HNA/ANA arrays and to compare their properties with regular DNA arrays, sequences in the reverse transcriptase gene (codon 74) and the protease gene of HIV-1 (codon 10) were selected. Both, the relative intensity of the signal and match/mismatch discrimination increased up to fivefold for DNA targets and up to 3-3.5-fold for RNA targets applying HNA or ANA arrays (ANA > HNA > DNA). Certainly in the new field of miRNA detection, ANA arrays could prove very beneficial and their properties should be investigated in more detail.

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Paracoccidioiddes brasiliensis is a thermodimorphic fungus endemic to Latin America, where it causes the most prevalent systemic mycosis, paracoccidioidomycosis (PCM). DNA microarray technology has been used to identify patterns of gene expression when a microbe is confronted with conditions of interest, such as in vitro and/or ex vivo interaction with specific cells. P. brasiliensis is one organism that has benefited from this approach. Even though its genome has not been sequenced yet, much has been discovered from its transcriptome and DNA array analyses. In this review, we will outline the current knowledge in P. brasiliensis transcriptome, with focus on differential expression analysis in vitro and on the discussion of the genes that are controlled during the host-pathogen interaction ex vivo in order to give insights into the pathobiology of this fungus. In vitro experiments enabled the delineation of whole metabolic pathways; the description of differential metabolism between mycelium and yeast cells and of the mainly signaling pathways controlling dimorphism, high temperature growth, thermal and oxidative stress, and virulence/ pathogenicity. Recent ex vivo experiments provided advances on the comprehension of the plasticity of response and indicate that P. brasiliensis is not only able to undergo fast and dramatic expression profile changes but can also discern subtle differences, such as whether it is being attacked by a macrophage or submitted to the bloodstream route conditions.