A real-time PCR for specific detection of the Legionella pneumophila serogroup 1 ST1 complex.

OBJECTIVE: Legionella pneumophila serogroup 1 (Lp1) sequence type (ST) 1 is globally widespread in the environment and accounts for a significant proportion of Legionella infections, including nosocomial Legionnaires' disease (LD). This study aimed to design a sensitive and specific detection method for Lp ST1 that will underpin epidemiological investigations and risk assessment. METHODS: A total of 628 Lp genomes (126 ST1s) were analyzed by comparative genomics. Interrogation of more than 900 accessory genes revealed seven candidate targets for specific ST1 detection and specific primers and hydrolysis probes were designed and evaluated. The analytical sensitivity and specificity of the seven primer and probe sets were evaluated on serially diluted DNA extracted from the reference strain CIP107629 and via qPCR applied on 200 characterized isolates. The diagnostic performance of the assay was evaluated on 142 culture-proven clinical samples from LD cases and a real-life investigation of a case cluster. RESULTS: Of seven qPCR assays that underwent analytical validation, one PCR target (lpp1868) showed higher sensitivity and specificity for ST1 and ST1-like strains. The diagnostic performance of the assay using respiratory samples corresponded to a sensitivity of 95% (19/20) (95% CI (75.1-99.9)) and specificity of 100% (122/122) (95% CI (97-100)). The ST1 PCR assay could link two out of three culture-negative hospitalized LD cases to ST1 during a real-time investigation. CONCLUSION: Using whole genome sequencing (WGS) data, we developed and validated a sensitive and specific qPCR assay for the detection of Lp1 belonging to the ST1 clonal complex by amplification of the lpp1868 gene. The ST1 qPCR is expected to deliver an added value for Lp control and prevention, in conjunction with other recently developed molecular assays.

A bioinformatics tool for ensuring the backwards compatibility of Legionella pneumophila typing in the genomic era.

OBJECTIVES: Whole genome sequencing (WGS) has revolutionized the subtyping of Legionella pneumophila but calling the traditional sequence-based type from genomic data is hampered by multiple copies of the mompS locus. We propose a novel bioinformatics solution for rectifying that limitation, ensuring the feasibility of WGS for cluster investigation. METHODS: We designed a novel approach based on the alignment of raw reads with a reference sequence. With WGS, reads originating from either of the two mompS copies cannot be differentiated. Therefore, when non-identical copies were present, we applied a read-filtering strategy based on read alignment to a reference sequence via unique 'anchors'. If minimal read coverage was achieved after filtration (≥3X), a consensus sequence was built based on mapped reads followed by calling the sequence-based typing allele. The entire procedure was implemented using a Perl script. RESULTS: The method was validated using a diverse sample of 265 L. pneumophila genomes, consisting of 59 different sequence types (STs) and 23 mompS variants; 57 of the 265 (22%) had non-identical mompS copies. In 237 of the 265 samples (89.4%), mompS calling was successful and no erroneous calling occurred. A 98.1% success was recorded among 109 samples meeting quality requirements. The method was superior to alternative approaches. CONCLUSIONS: As WGS becomes more accessible, technical difficulties in routine clinical and surveillance work will arise. The case of mompS in L. pneumophila serves as an example for such limitations that necessitate the development of novel computational solutions that meet end-user demands.

Pollution-affected fish hepatic transcriptome and its expression patterns on exposure to cadmium.

Individuals of the fish Lithognathus mormyrus were exposed to a series of pollutants including: benzo[a]pyrene, pp-DDE, Aroclor 1254, perfluorooctanoic acid, tributyl-tin chloride, lindane, estradiol, 4-nonylphenol, methyl mercury chloride, and cadmium chloride. Five mixtures of the pollutants were injected. Each mixture included one to three compounds. A microarray was constructed using 4608 L. mormyrus hepatic cDNAs cloned from the pollutant-exposed fish. Most clones (4456) were sequenced and assembled into 1494 annotated unique clones. The constructed microarray was used to identify changes in hepatic gene expression profile on exposure to cadmium administered to the fish by feeding or injections. Thirty-one unique clones showed altered expression levels on exposure to cadmium. Prominently differentially expressed genes included elastase 4, carboxypeptidase B, trypsinogen, perforin, complement C31, cytochrome P450 2K5, ceruloplasmin, carboxyl ester lipase, and metallothionein. Twelve sequences have no available annotation. Most genes (23) were downregulated and hypothesized to be affected by general toxicity due to the intensive cadmium exposure regime. The concept of an operational multigene cDNA microarray, aimed at routine and fast biomonitoring of multiple environmental threats, is outlined and the cadmium exposure experiment has been used to demonstrate functional and methodological aspects of the biomonitoring tool. The components of the outlined system include: (1) spotted array, composed of both pollution-affected and constitutively expressed genes, the latter are used for normalization; (2) standard, repeatable labeling procedure of a reference transcript population; and (3) biomarker indices derived from the profile of expression ratio across the pollution-affected genes, between the field-sampled transcript populations and the reference.

Hepatopancreatic multi-transcript expression patterns in the crayfish Cherax quadricarinatus during the moult cycle.

Alterations of hepatopancreatic multi-transcript expression patterns, related to induced moult cycle, were identified in male Cherax quadricarinatus through cDNA microarray hybridizations of hepatopancreatic transcript populations. Moult was induced by X-organ sinus gland extirpation or by repeated injections of 20-hydroxyecdysone. Manipulated males were sacrificed at premoult or early postmoult, and a reference population was sacrificed at intermoult. Differentially expressed genes among the four combinations of two induction methods and two moult stages were identified. Biologically interesting clusters revealing concurrently changing transcript expressions across treatments were selected, characterized by a general shift of expression throughout premoult and early postmoult vs. intermoult, or by different premoult vs. postmoult expressions. A number of genes were differentially expressed in 20-hydroxyecdysone-injected crayfish vs. X-organ sinus gland extirpated males.

Glycolytic enzymes associated with the cell surface of Streptococcus pneumoniae are antigenic in humans and elicit protective immune responses in the mouse.

Streptococcus pneumoniae is a leading cause of otitis media, sinusitis, pneumonia, bacteraemia and meningitis worldwide. The drawbacks associated with the limited number of various capsular polysaccharides that can be included in the polysaccharide-based vaccines focuses much attention on pneumococcal proteins as vaccine candidates. We extracted an enriched cell wall fraction from S. pneumoniae WU2. Approximately 150 soluble proteins could be identified by 2D gel electrophoresis. The proteins were screened by 2D-Western blotting using sera that were obtained longitudinally from children attending day-care centres at 18, 30 and 42 months of age and sera from healthy adult volunteers. The proteins were further identified using matrix-assisted laser desorption ionization-time of flight mass spectrometry. Seventeen proteins were antigenic in children and adults, of which 13 showed an increasing antibody response with age in all eight children analysed. Two immunogenic proteins, fructose-bisphosphate aldolase (FBA) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and a control protein with known low immunogenicity, heat shock protein 70 (DnaK), were expressed in Escherichia coli, purified and used to immunize mice. Mouse antibodies elicited to the recombinant (r) FBA and rGAPDH were cross-reactive with several genetically unrelated strains of different serotypes and conferred protection to respiratory challenge with virulent pneumococci. In addition, the FBA used in this study (NP_345117) does not have a human ortholog and warrants further investigation as a candidate for a pneumococcal vaccine. In conclusion, the immunoproteomics based approach utilized in the present study appears to be a suitable tool for identification of novel S. pneumoniae vaccine candidates.

Harvesting the human genome: the Israeli perspective.

The post-genome era is at our door, and soon the complete human genome sequence will be available for the next set of goals. Israel is well equipped and skilled to join the worldwide harvest of the human genome, but additional massive government investment is required. This will affect various domains of activity, including the fields of diagnostics and therapeutics. The technologies and know-how described above constitute the basis for future human genome applications in Israel.

GeneCards: a novel functional genomics compendium with automated data mining and query reformulation support.

MOTIVATION: Modern biology is shifting from the 'one gene one postdoc' approach to genomic analyses that include the simultaneous monitoring of thousands of genes. The importance of efficient access to concise and integrated biomedical information to support data analysis and decision making is therefore increasing rapidly, in both academic and industrial research. However, knowledge discovery in the widely scattered resources relevant for biomedical research is often a cumbersome and non-trivial task, one that requires a significant amount of training and effort. RESULTS: To develop a model for a new type of topic-specific overview resource that provides efficient access to distributed information, we designed a database called 'GeneCards'. It is a freely accessible Web resource that offers one hypertext 'card' for each of the more than 7000 human genes that currently have an approved gene symbol published by the HUGO/GDB nomenclature committee. The presented information aims at giving immediate insight into current knowledge about the respective gene, including a focus on its functions in health and disease. It is compiled by Perl scripts that automatically extract relevant information from several databases, including SWISS-PROT, OMIM, Genatlas and GDB. Analyses of the interactions of users with the Web interface of GeneCards triggered development of easy-to-scan displays optimized for human browsing. Also, we developed algorithms that offer 'ready-to-click' query reformulation support, to facilitate information retrieval and exploration. Many of the long-term users turn to GeneCards to quickly access information about the function of very large sets of genes, for example in the realm of large-scale expression studies using 'DNA chip' technology or two-dimensional protein electrophoresis. AVAILABILITY: Freely available at http://bioinformatics.weizmann.ac.il/cards/ CONTACT: cards@bioinformatics.weizmann.ac.il

Kinetic analysis in mixed micelles of partially purified rat brain phospholipase D activity and its activation by phosphatidylinositol 4,5-bisphosphate.

A partially purified rat brain membrane phospholipase D (PLD) activity was characterized in a mixed micellar system consisting of 1-palmitoyl-2-[6-N-(7-nitrobenzo-2-oxa-1,3-diazol-4-yl)-amino]capr oyl-phosphatidylcholine (NBD-PC) and Triton X-100, under conditions where Triton X-100 has a surface dilution effect on PLD activity and the catalytic rate is dependent on the surface concentration (expressed in terms of molar ratio) of NBD-PC. PLD activity was specifically activated by phosphatidylinositol 4,5-bisphosphate (PIP2), and the curve of activation versus PIP2 molar ratio fitted a Michaelis-Menten equation with a K(act) value between molar ratios of 0.001-0.002. Maximal activation was observed at a PIP2 molar ratio of 0.01. Similar values were obtained when activities of partially purified PLD as well as membrane-bound PLD were determined towards pure NBD-PC micelles. In the mixed micellar system PIP2 was shown to elevate by 6-22 fold the specificity constant of PLD towards NBD-PC (K(A), which is proportional to Vmax/Km). Kinetic analysis of PLD trans-phosphatidylation activity towards ethanol, 1-propanol and 1-butanol revealed a Michaelis-Menten type dependence on alcohol concentration up to 1000, 200 and 80 mM, respectively. While Vmax values were similar towards all three alcohols, enzyme affinity increased as the alcohol was longer, and Km values for ethanol, 1-propanol and 1-butanol were 291, 75 and 16 mM (respectively). PLD specificity constants (K(A)) towards ethanol, 1-propanol and 1-butanol were shown to be respectively 260, 940 and 5,920 times higher than to water, the competing substrate. 1-Propanol and 1-butanol inhibited PLD activity above 400 and 100 mM, respectively. The present results indicate that partially purified PLD obeys surface dilution kinetics with regard to its phospholipid substrate PC and its cofactor PIP2, and that in the presence of alcohols, its transphosphatidylation activity may be analyzed as a competitive reaction to the hydrolysis reaction.

Enzymology of mammalian phospholipases D: in vitro studies.

The existence of multiple forms of phopholipase D was clearly established in a large number of biochemical studies that described and characterized the enzymological properties of the different PLD activities. This review summarizes the in vitro evidence showing differential subcellular localization and chromatographic properties of putative PLD isozymes, their phospholipid and alcohol substrate specificities, their modulation by various divalent cations, small G proteins and protein kinase c isozymes, and the role of phosphatidylinositol 4,5-bisphosphate as a cofactor of phospholipase D.