See what you eat--broad GMO screening with microarrays.

Despite the controversy of whether genetically modified organisms (GMOs) are beneficial or harmful for humans, animals, and/or ecosystems, the number of cultivated GMOs is increasing every year. Many countries and federations have implemented safety and surveillance systems for GMOs. Potent testing technologies need to be developed and implemented to monitor the increasing number of GMOs. First, these GMO tests need to be comprehensive, i.e., should detect all, or at least the most important, GMOs on the market. This type of GMO screening requires a high degree of parallel tests or multiplexing. To date, DNA microarrays have the highest number of multiplexing capabilities when nucleic acids are analyzed. This trend article focuses on the evolution of DNA microarrays for GMO testing. Over the last 7 years, combinations of multiplex PCR detection and microarray detection have been developed to qualitatively assess the presence of GMOs. One example is the commercially available DualChip GMO (Eppendorf, Germany; http://www.eppendorf-biochip.com), which is the only GMO screening system successfully validated in a multicenter study. With use of innovative amplification techniques, promising steps have recently been taken to make GMO detection with microarrays quantitative.

Highly adherent small-colony variants of Pseudomonas aeruginosa in cystic fibrosis lung infection.

Pseudomonas aeruginosa, an opportunistic human pathogen and ubiquitous environmental bacterium, is capable of forming specialized bacterial communities, referred to as biofilm. The results of this study demonstrate that the unique environment of the cystic fibrosis (CF) lung seems to select for a subgroup of autoaggregative and hyperpiliated P. aeruginosa small-colony variants (SCVs). These morphotypes showed increased fitness under stationary growth conditions in comparison with clonal wild-types and fast-growing revertants isolated from the SCV population in vitro. In accordance with the SCVs being hyperpiliated, they exhibited increased twitching motility and capacity for biofilm formation. In addition, the SCVs attached strongly to the pneumocytic cell line A549. The emergence of these highly adherent SCVs within the CF lung might play a key role in the pathogenesis of P. aeruginosa lung infection, where a biofilm mode of growth is thought to be responsible for persistent infection.

Epidemiologically non-feasible singleton reactors at the final stage of BoHV1 eradication: serological evidence of BoHV2 cross-reactivity.

A voluntary marker-independent Bovine Herpesvirus 1 (BoHV1) eradication program started in 1986; in 1998 it changed to a compulsory one. Certification of free regions in European member states is based on Article 10 of directive 64/432/EEC. According to this rule Bavaria is listed as free of BoHV1 since October 2011. Surveillance of BoHV1-free dairy cattle farms is currently performed with quarterly bulk-milk testing. Non-negative bulk-milk results must be confirmed by blood tests in cattle older than nine months. An increased regional rate of non-negative bulk-milk samples and the subsequent detection of epidemiologically non-feasible singleton BoHV1-reactors by analysis of blood were observed at the final stage of eradication in southwest Bavaria. Nineteen case farms (734 animals) defined by singleton reactors born at least two years after certification of the farms as BoHV1-free, 23 negative control (NC) farms (NC I: 321 animals) from the same region, 11 NC-farms (NC II: 423 animals) from an already-certified Article 10 region in northeast Bavaria and two BoHV1-infected farms (264 animals) were analysed using BoHV1-, BoHV2- and Feline Herpesvirus 1 (FeHV1)-neutralisation tests (NTs), and three commercially available ELISAs supplied by Idexx Laboratories, B.V., The Netherlands: the CHEKIT™ Trachitest 2nd Gen. test for milk or serum (Trachitest), Herdchek™ gB- (gB-ELISA) and Herdchek™ gE-ELISA (gE-ELISA). Significantly increased levels of BoHV2 antibodies were observed on case farms compared to NC I or II farms. Additionally, reactivity by gB-ELISA and the Trachitest was significantly increased for animals with BoHV2 neutralising antibodies. Singleton BoHV1-reactors tested negative by gE-ELISA even if an elevated cut-off of 0.95±0.05 was applied. At this cut-off, the gE-ELISA was as sensitive and specific as the gB-ELISA. Comparative titration of milk samples from seropositive animals from a BoHV1-infected dairy cattle farm and from singleton BoHV1-reactors performed in CHEKIT™ Trachitest 2nd Gen. Milk revealed that the slopes of both groups were distinct; therefore, optimised cut-offs for bulk-milk testing to exclude singleton BoHV1-reactors are proposed.

Evidence for polyadenylated mRNA in Pseudomonas aeruginosa.

In this paper, we report the synthesis of Pseudomonas aeruginosa cDNA in the presence of oligo(dT) primers. Hybridization of oligonucleotide DNA microarrays indicates that under the experimental conditions used, at least 43.7% of the expressed genes from P. aeruginosa PAO1, representing many different functional classes, can be detected by using oligo(dT)-primed cDNAs.

Global regulation of quorum sensing and virulence by VqsR in Pseudomonas aeruginosa.

Pathogenesis of Pseudomonas aeruginosa is controlled to a major extent by the two quorum-sensing systems las and rhl. The previously uncharacterized gene PA2591 was identified as a major virulence regulator, vqsR, in the quorum-sensing hierarchy. vqsR is a member of the LuxR family and possesses a las box in its upstream region. Transposon inactivation of vqsR abrogated the production of N-acylhomoserine lactones and the secretion of exoproducts and diminished bacterial virulence for Caenorhabditis elegans. Cytotoxicity towards macrophages was not affected. vqsR mRNA was expressed more strongly in the presence of human serum and oxidative stress than under standard growth conditions. High-density oligonucleotide microarrays were used to compare the global expression profile of a wild-type strain and a vqsR mutant. One-hundred-and-fifty-one and 113 genes were significantly differentially expressed in the presence of H(2)O(2) and human serum, respectively. The disruption of vqsR repressed the expression of genes that are known to be promoted by quorum sensing and activated the expression of genes that are known to be repressed by quorum sensing. Moreover, the vqsR mutant harboured less mRNA transcript for the production of siderophores and membrane-bound elements of antibiotic resistance. The protein encoded by PA2591 regulates several traits of pathogenicity; hence, the name vqsR ('virulence and quorum-sensing regulator') was assigned to PA2591.

Expression analysis of a highly adherent and cytotoxic small colony variant of Pseudomonas aeruginosa isolated from a lung of a patient with cystic fibrosis.

The heterogeneous environment of the lung of the cystic fibrosis (CF) patient gives rise to Pseudomonas aeruginosa small colony variants (SCVs) with increased antibiotic resistance, autoaggregative growth behavior, and an enhanced ability to form biofilms. In this study, oligonucleotide DNA microarrays were used to perform a genome-wide expression study of autoaggregative and highly adherent P. aeruginosa SCV 20265 isolated from a CF patient's lung in comparison with its clonal wild type and a revertant generated in vitro from the SCV population. Most strikingly, SCV 20265 showed a pronounced upregulation of the type III protein secretion system (TTSS) and the respective effector proteins. This differential expression was shown to be biologically meaningful, as SCV 20265 and other hyperpiliated and autoaggregative SCVs with increased TTSS expression were significantly more cytotoxic for macrophages in vitro and were more virulent in a mouse model of respiratory tract infection than the wild type. The observed cytotoxicity and virulence of SCV 20265 required exsA, an important transcriptional activator of the TTSS. Thus, the prevailing assumption that P. aeruginosa is subject to selection towards reduced cytotoxicity and attenuated virulence during chronic CF lung infection might not apply to all clonal variants.