Exposure to 'farming' and objective markers of atopy: a systematic review and meta-analysis.

There is growing interest in the 'farm effect' on the spectrum of allergy. Evidence concerning the farm effect on asthma, eczema, and allergic rhinitis has been systematically synthesized, but without a specific focus on objective markers of sensitization. This focus is important, as farm exposures may be related to allergy but not to non-allergic phenotypes of disease. We aimed to systematically review and meta-analyse literature that has investigated associations between farm exposure at any age and objective measures of atopy, that is serum IgE or skin prick tests results. Using predefined inclusion and exclusion criteria, we identified 29 articles for review. IgE levels were measured in either childhood or adulthood by eighteen studies, while skin prick testing was performed in sixteen studies. Newcastle-Ottawa Scale quality assessments indicated that the majority of these studies demonstrated a representative sample of selected participants. Due to significant heterogeneity in study measures and methodology between studies, only few were meta-analysed. This meta-analysis showed a significant protective effect of farm exposure before 1 year of life on allergic sensitization (OR = 0.60 [0.52-0.70]). Farm exposure during childhood was also associated with a reduced risk of sensitization to cat or timothy (OR = 0.60 [0.51-0.70]; OR=0.46 [0.41-0.51]). Studies investigating the effect of farm exposure in adult life could not be meta-analysed, and their results were inconsistent. Insufficient studies investigated food sensitization as an outcome to allow synthesis. The majority of studies included in this review investigated childhood farm exposure, finding evidence to support a protective childhood 'farm effect' against subsequent atopy. There is inconsistent evidence on the association between farm exposure in adulthood and allergic sensitization. Further studies are needed to tease out the exact exposures and timing associated with farming environments that protect against allergic disease.

Bioinformatics meets parasitology.

The advent and integration of high-throughput '-omics' technologies (e.g. genomics, transcriptomics, proteomics, metabolomics, glycomics and lipidomics) are revolutionizing the way biology is done, allowing the systems biology of organisms to be explored. These technologies are now providing unique opportunities for global, molecular investigations of parasites. For example, studies of a transcriptome (all transcripts in an organism, tissue or cell) have become instrumental in providing insights into aspects of gene expression, regulation and function in a parasite, which is a major step to understanding its biology. The purpose of this article was to review recent applications of next-generation sequencing technologies and bioinformatic tools to large-scale investigations of the transcriptomes of parasitic nematodes of socio-economic significance (particularly key species of the order Strongylida) and to indicate the prospects and implications of these explorations for developing novel methods of parasite intervention.

Bioinformatic analysis of abundant, gender-enriched transcripts of adult Ascaris suum (Nematoda) using a semi-automated workflow platform.

Expressed sequence tag (EST) data representing transcripts with a high level of differential hybridization in suppressive-subtractive hybridization (SSH)-based microarray analysis between adult female and male Ascaris suum were subjected to detailed bioinformatic analysis. A total of 361 ESTs clustered into 209 sequences, of which 52 and 157 represented transcripts that were enriched in female and male A. suum, respectively. Thirty (57.7%) of the 'female' subset of 52 sequences had orthologues/homologues in other parasitic nematodes and/or Caenorhabditis elegans, 13 (25%) exclusively in other parasitic nematodes and nine (17.3%) had no match in any other organism for which sequence data are currently available; the C. elegans orthologues encoded molecules involved in reproduction as well as embryonic and gamete development, such as vitellogenins and chitin-binding proteins. Of the 'male' subset of 157 sequences, 73 (46.5%) had orthologues/homologues in other parasitic nematodes and/or C. elegans, 57 (37.5%) in other parasitic nematodes only, and 22 (14.5%) had no significant similarity match in any other organism; the C. elegans orthologues encoded predominantly major sperm proteins (MSPs), kinases and phosphatases, actins, myosins and an Ancylostoma secreted protein-like molecule. The findings of the present study should support further genomic investigations of A. suum.

A portrait of the "SCP/TAPS" proteins of eukaryotes--developing a framework for fundamental research and biotechnological outcomes.

A wide range of proteins belonging to the SCP/TAPS "family" has been described for various eukaryotic organisms, including plants and animals (vertebrates and invertebrates, such as helminths). Although SCP/TAPS proteins have been proposed to play key roles in a number of fundamental biological processes, such as host-pathogen interactions and defence mechanisms, there is a paucity of information on their genetic relationships, structures and functions, and there is no standardised nomenclature for these proteins. A detailed analysis of the relationships of members of the SCP/TAPS family of proteins, based on key protein signatures, could provide a foundation for investigating these areas. In this article, we review the current state of knowledge of key SCP/TAPS proteins of eukaryotes, with an emphasis on those from parasitic helminths, and undertake a comprehensive, systematic phylogenetic analysis of currently available full-length protein sequence data (considering characteristic protein signatures or motifs) to infer relationships and provide a framework (based on statistical support) for the naming of these proteins. This framework is intended to guide genomic and molecular biological explorations of key SCP/TAPS molecules associated with infectious diseases of plants and animals. In particular, fundamental investigations of these molecules in parasites and the integration of structural and functional data could lead to new and innovative approaches for the control of parasitic diseases, with important biotechnological outcomes.

Exploring transcriptional conservation between Ancylostoma caninum and Haemonchus contortus by oligonucleotide microarray and bioinformatic analyses.

In this study, we identified, using an established oligonucleotide microarray platform for the parasitic nematode Haemonchus contortus, transcripts that are 'conserved' between serum-activated and non-activated L3s of Ancylostoma caninum (aL3 and L3, respectively) and H. contortus by cross-species hybridization (CSH) at high stringency and conducted extensive bioinformatic analyses of the cross-hybridizing expressed sequence tags (ESTs). The microarray analysis revealed significant differential hybridization between aL3 and L3 for 32 molecules from A. caninum, of which 29 were shown to have homologues/orthologues in the free-living nematode Caenorhabditis elegans and/or A. caninum and the other three molecules had no homologues in current gene databases. 'Non-wildtype' RNAi phenotypes were recorded for 13 of the C. elegans homologues. A subset of 16 C. elegans homologues/orthologues (i.e. genes abce-1, act-2, C08H9.2, C55F2.1, calu-1, col-181, cpr-6, elo-2, asp-1, K07E3.4, rpn-2, sel-9, T28C12.4, hsb-1, Y57G11C.15 and ZK593.1) were predicted to interact genetically with a total of 156 (range 1-88) other genes. Gene ontology (GO) analysis of the interacting genes revealed that the most common subcategories were signal transduction (7%), intracellular protein transport and glycolysis (6.2%) within 'biological process'; nuclear (25.7%) and intracellular (19.8%) within 'cellular component'; and ATP-binding (14.4%) and protein-binding (8.4%) within 'molecular function'. The potential roles of key molecules in the two blood-feeding parasitic nematodes are discussed in relation to the known roles of their homologues/orthologues in C. elegans. The CSH approach used may provide a tool for the screening of genes conserved across a range of different taxa of parasites for which DNA microarray platforms are not available.

Molecular and phylogenetic characterization of cytochromes c from Haemonchus contortus and Trichostrongylus vitrinus (Nematoda: Trichostrongylida).

Although cytochrome c genes (cyt c) and proteins (CYT C) have been relatively well studied in mammals, very little is known about them in parasitic helminths. In the present study, we investigated this group of molecules in Haemonchus contortus (barber's pole worm) and Trichostrongylus vitrinus (black scour worm), two parasitic nematodes of small ruminants. The cyt c gene (512 bp) of H. contortus had one intron and encoded a transcript of 345 nucleotides, whilst that of T. vitrinus (792 bp) had two introns and encoded a transcript of 360 nucleotides. The transcription of cyt c in T. vitrinus was substantially greater in adult males compared with females, although no such gender-enrichment was evident in adults of H. contortus. These findings were supported at the protein level by immunoblot analyses. The inferred proteins (designated Hc-CYT C and Tv-CYT C, respectively) shared nucleotide and amino acid identities of 78% and 85%, respectively. The alignment of these and other CYT C sequences from nematodes, flatworms, insects and mammals identified conserved motifs associated with CYT C oxidase- and reductase- as well as haem-binding. One residue (histidine-26) was conserved for mammals, whereas this residue was absent from all nematodes; the functional significance of this difference is not yet known. Both phylogenetic analysis and protein modelling revealed that CYT C proteins of nematodes are structurally distinct from those of mammals and other organisms, suggesting their potential as targets for parasite intervention.

Investigating public health impacts of deer in a protected drinking water supply watershed.

The World Health Organisation's (WHO) Water Safety Plans highlight the need for preventative risk management when managing water contamination risks. As part of this approach, a management framework incorporating multiple barriers is necessary and there is a need to validate those barriers through scientific evidence. This paper reports on a study undertaken to validate the effectiveness, in terms of pathogen numbers, of having protected watersheds. The study aimed to determine if the deer population in a protected watershed carried Cryptosporidium and whether or not it was human infectious. Deer faecal samples were collected from the protected watersheds over a 12 month period and analysed using a new method, developed as part of this project, for genotyping Cryptosporidium. Early results showed the presence of Cryptosporidium, but following a refinement in the method no human infectious Cryptosporidium was detected. The results give some confidence that having protected watersheds is an effective barrier against pathogen contamination. They do not, however, imply that continued monitoring and management of the deer should cease. To maintain compliance with the Water Safety Plans, continual validation of barrier effectiveness is required.

Cryptosporidium--biotechnological advances in the detection, diagnosis and analysis of genetic variation.

Cryptosporidiosis is predominantly a gastrointestinal disease of humans and other animals, caused by various species of protozoan parasites representing the genus Cryptosporidium. This disease, transmitted mainly via the faecal-oral route (in water or food), is of major socioeconomic importance worldwide. The diagnosis and genetic characterization of the different species and population variants (usually recognised as "genotypes" or "subgenotypes") of Cryptosporidium is central to the prevention, surveillance and control of cryptosporidiosis, particularly given that there is presently no broadly applicable treatment regimen for this disease. Although traditional phenotypic techniques have had major limitations in the specific diagnosis of cryptosporidiosis, there have been major advances in the development of molecular analytical and diagnostic tools. This article provides a concise account of Cryptosporidium and cryptosporidiosis, and focuses mainly on recent advances in nucleic acid-based approaches for the diagnosis of cryptosporidiosis and analysis of genetic variation within and among species of Cryptosporidium. These advances represent a significant step toward an improved understanding of the epidemiology as well as the prevention and control of cryptosporidiosis.

Hydrolysis of organophosphorus insecticides by in vitro modified carboxylesterase E3 from Lucilia cuprina.

Resistance of the blowfly, Lucilia cuprina, to organophosphorus (OP) insecticides is due to mutations in LcalphaE7, the gene encoding carboxylesterase E3, that enhance the enzyme's ability to hydrolyse insecticides. Two mutations occur naturally, G137D in the oxyanion hole of the esterase, and W251L in the acyl binding pocket. Previous in vitro mutagenesis and expression of these modifications to the cloned gene have confirmed their functional significance. G137D enhances hydrolysis of diethyl and dimethyl phosphates by 55- and 33-fold, respectively. W251L increases dimethyl phosphate hydrolysis similarly, but only 10-fold for the diethyl homolog; unlike G137D however, it also retains ability to hydrolyse carboxylesters in the leaving group of malathion (malathion carboxylesterase, MCE), conferring strong resistance to this compound. In the present work, we substituted these and nearby amino acids by others expected to affect the efficiency of the enzyme. Changing G137 to glutamate or histidine was less effective than aspartate in improving OP hydrolase activity and like G137D, it diminished MCE activity, primarily through increases in Km. Various substitutions of W251 to other smaller residues had a broadly similar effect to W251L on OP hydrolase and MCE activities, but at least two were quantitatively better in kinetic parameters relating to malathion resistance. One, W251G, which occurs naturally in a malathion resistant hymenopterous parasitoid, improved MCE activity more than 20-fold. Mutations at other sites near the bottom of the catalytic cleft generally diminished OP hydrolase and MCE activities but one, F309L, also yielded some improvements in OP hydrolase activities. The results are discussed in relation to likely steric effects on enzyme-substrate interactions and future evolution of this gene.

Paradoxical embolism and acute arterial occlusion: rare or unsuspected?

PURPOSE: The high prevalence of clinically silent venous thrombosis and the presence of a patent foramen ovale (PFO) in up to 35% of the general population suggests that paradoxical emboli may be the cause of an ischemic stroke or a peripheral thromboembolic occlusion more often than is presently considered. This study was undertaken to review our experience with presumed paradoxical embolism. METHODS: Hospital records were reviewed for all patients diagnosed with both a documented PFO and a thromboembolic event between January 1970 and June 1993. Patients with a ventricular or an atrial septal defect or a probable pulmonary arteriovenous fistula were excluded. RESULTS: The presumptive diagnosis of paradoxical embolism was made in seven patients. There were five men and two women, with a median age of 43 years. Four patients were admitted with an acute cerebral ischemic event, and in three others hospitalization was prompted by the development of an acutely ischemic limb (two upper extremity; one lower extremity). In none was there evidence of angiographically significant peripheral or extracranial atherosclerotic occlusive disease. Symptoms suggestive of pulmonary emboli were noted in two patients, and in only one patient was there evidence on physical examination of a deep venous thrombosis. Before 1988 the diagnosis of paradoxical embolism had been made in only one patient after postmortem examination. All six patients who were discharged were available for follow-up (mean 20 months; range 6 to 60 months). There was one late death from lung cancer. Recurrent paradoxical emboli have not been documented during the follow-up period. CONCLUSIONS: The incidence of presumed paradoxical embolism has increased dramatically in the recent past as a consequence of our improved ability to unequivocally detect PFO with associated physiologic shunting. The suspicion of this heretofore "rare" event should be raised, particularly in the young or middle-aged adult diagnosed with an acute thromboembolic event. Until the risk of recurrent ischemic events in the presence of a PFO is better defined, we currently recommend closure of the foramen ovale after a significant or recurrent paradoxical embolus. Otherwise, the selective use of intracaval filters, antiplatelet therapy, and oral anticoagulation remain undefined.

Bicyclic peptides. VI. Synthesis, conformation, and ion-binding of two bicyclic nonapeptides.

Two related homodetic bicyclic nonapeptides (cyclo Glu-X-Pro-Gly-Lys-X-Pro-Gly)-cyclo (l gamma----5 epsilon), X = Ala(BCP2), X = Leu(BCP3) have been synthesized using conventional solution phase methods involving mixed anhydride coupling reactions starting with appropriately protected naturally occurring amino acids. The conformation and ion binding properties of BCP2 have been studied by nuclear magnetic resonance and circular dichroism techniques. The results of these studies have been compared to those of BCP3. The presence of Ala caused both Ala-Pro bonds to be trans in free BCP2. This characteristic imparted subtle differences to the ion-binding properties of BCP2 as compared to free BCP3 which has one cis Leu-Pro bond and one trans Leu-Pro bond.

The sonographically solid appearing benign cystic teratoma.

The sonographic appearance of 6 solid benign cystic teratomas (in 5 patients) was reviewed. Three of these tumors were difficult to identify due to the presence of acoustical shadowing obscuring the posterior portions of the tumors. This appearance was mistaken ultrasonically for bowel gas. The tumors were diagnosed by abdominal radiographs showing calcifications, teeth and/or radiolucency due to fat. With the presence of a clinically palpable pelvic mass in a young woman, and an apparently normal sonogram, an abdominal radiograph should be considered to look for evidence of a benign cystic teratoma.

Laser-EXAFS: Fast Extended X-ray Absorption Fine Structure Spectroscopy with a Single Pulse of Laser-Produced X-rays.

The extended x-ray absorption fine structure (EXAFS) spectrum of aluminum has been measured with a nanosecond pulse of soft x-rays generated by a laser-produced plasma. This technique provides a practical alternative to synchrotorn radiation for the acquisition of EXAFS data. It also provides a unique capability for the analysis of molecular structure in highly transient chemical species.