exomeSuite: Whole exome sequence variant filtering tool for rapid identification of putative disease causing SNVs/indels.

Exome and whole-genome analyses powered by next-generation sequencing (NGS) have become invaluable tools in identifying causal mutations responsible for Mendelian disorders. Given that individual exomes contain several thousand single nucleotide variants and insertions/deletions, it remains a challenge to analyze large numbers of variants from multiple exomes to identify causal alleles associated with inherited conditions. To this end, we have developed user-friendly software that analyzes variant calls from multiple individuals to facilitate identification of causal mutations. The software, termed exomeSuite, filters for putative causative variants of monogenic diseases inherited in one of three forms: dominant, recessive caused by a homozygous variant, or recessive caused by two compound heterozygous variants. In addition, exomeSuite can perform homozygosity mapping and analyze the variant data of multiple unrelated individuals. Here we demonstrate that filtering of variants with exomeSuite reduces datasets to a fraction of a percent of their original size. To the best of our knowledge this is the first freely available software developed to analyze variant data from multiple individuals that rapidly assimilates and filters large data sets based on pattern of inheritance.

Uptake of abdominal aortic aneurysm screening. A cohort study.

BACKGROUND: Abdominal aortic aneurysms (AAA) are responsible for 1.4% of UK deaths. Deprivation is a risk factor for AAA. Screening reduces AAA related mortality and is cost effective if uptake remains high. The Highland aneurysm screening programme (HASP) began in 2001 offering screening to men in a sparsely populated area. The aim was to identify whether uptake varies with deprivation or rurality, in the context of an established programme. METHODS: Retrospective interrogation of HASP records was performed on all men offered screening from 2001 until 2010. Deprivation and rurality status were derived from postcode of residence (SIMD'09 and URC'08) and the relationships with screening uptake were examined. RESULTS: Mean uptake over the decade was 90.1%. There was a strong association between deprivation and uptake, which ranged from 79.5% in the most deprived population to 97.5% in the least deprived (p < 0.001). The odds of men who were least deprived attending was 10.6 times higher than those who were most deprived (p < 0.001). Higher uptake was observed in more rural areas (p = 0.02). When combined in a logistic regression model, only deprivation remained significant, indicating any apparent effect of rurality was explained by deprivation. No change was observed in the mean aortic diameter of 65-year-old men or the incidence of AAA. CONCLUSION: HASP has a high uptake even in the most deprived and rural populations, demonstrating that programme design has overcome any potential rural disadvantage. A gradient of uptake associated with deprivation remains, although even the most deprived have an uptake of almost 80%.

Studies of the activity of diphtheria toxin. I. Poliovirus replication in intoxicated HeLa cells.

It has been demonstrated that a saturating dose of diphtheria toxin produced a 90% inhibition of polio-virus replication in HeLa cells. This inhibition was reflected in infectious viral RNA synthesis and in mature virus production. Toxin had no direct effect on virus particles or I-RNA, and poliovirus adsorption and eclipse appeared to be carried out normally in intoxicated cells. When toxin was given at various time intervals after infection, the amount of inhibition depended on the time of toxin addition. Toxin given before or immediately after infection gave maximum inhibition, while toxin given several hours after infection had little effect. The data suggest that toxin inhibits viral replication through its effect on protein synthesis. It is likely that a critical step in the viral replication cycle, the production of poliovirus-induced RNA polymerase, is inhibited, and possibly the synthesis of capsid protein. Ammonium salts and the aliphatic amines, glycamine and prolamine, prevented the inhibition of viral replication by toxin. The kinetics of the protective action of ammonium chloride and diphtheria antitoxin are remarkably similar.

Advances in imaging of Stargardt disease.

Stargardt disease (STGD1) is an autosomal-recessively inherited condition often associated with mutations in ABCA4 and characterized by accumulation of autofluorescent lipofuscin deposits in the retinal pigment epithelium (RPE). Non-invasive imaging techniques including fundus autofluorescence (FAF), spectral domain optical coherence tomography (SD-OCT) and adaptive optics scanning laser ophthalmoscopy (AOSLO) have the potential to improve understanding of vision loss in patients with STGD. We describe a comprehensive approach to the study of patients with STGD. Measures of retinal structure and FAF were correlated with visual function including best-corrected visual acuity (BCVA), color vision, kinetic and static perimetry, fundus-guided microperimetry and full-field and multifocal electroretinography. Mutation analysis of the ABCA4 gene was carried out by sequencing the complete coding region. Preliminary data suggest that a combination of imaging modalities may provide a sensitive measure of disease progression and response to experimental therapies in patients with STGD.

Practical aspects of equine parasite control: a review based upon a workshop discussion consensus.

Development of resistance of several important equine parasites to most of the available anthelmintic drug classes has led to a reconsideration of parasite control strategies in many equine establishments. Routine prophylactic treatments based on simple calendar-based schemes are no longer reliable and veterinary equine clinicians are increasingly seeking advice and guidance on more sustainable approaches to equine parasite control. Most techniques for the detection of equine helminth parasites are based on faecal analysis and very few tests have been developed as diagnostic tests for resistance. Recently, some molecular and in vitro based diagnostic assays have been developed and have shown promise, but none of these are currently available for veterinary practice. Presently, the only reliable method for the detection of anthelmintic resistance is a simple faecal egg count reduction test, and clinicians are urged to perform such tests on a regular basis. The key to managing anthelmintic resistance is maintaining parasite refugia and this concept is discussed in relation to treatment strategies, drug rotations and pasture management. It is concluded that treatment strategies need to change and more reliance should now be placed on surveillance of parasite burdens and regular drug efficacy tests are also recommended to ensure continuing drug efficacy. The present review is based upon discussions held at an equine parasite workshop arranged by the French Equine Veterinary Association (Association Vétérinaire Equine Française, AVEF) in Reims, France, in October 2008.

Effect of streptolysin O on erythrocyte membranes, liposomes, and lipid dispersions. A protein-cholesterol interaction.

The effect of the bacterial cytolytic toxin, streptolysin O (SLO), on rabbit erythrocyte membranes, liposomes, and lipid dispersions was examined. SLO produced no gross alterations in the major erythrocyte membrane proteins or lipids. However, when erythrocytes were treated with SLO and examined by electron microscopy, rings and "C"-shaped structures were observed in the cell membrane. The rings had an electron-dense center, 24 nm in diameter, and the overall diameter of the structure was 38 nm. Ring formation also occurred when erythrocyte membranes were fixed with glutaraldehyde and OsO4 before the addition of toxin. In contrast, rings were not seen when erythrocytes were treated with toxin at 0 degrees C, indicating that adsorption of SLO to the membrane is not sufficient for ring formation since toxin is known to bind to erythrocytes at that temperature. The ring structures were present on lecithin-cholesterol-dicetylphosphate liposomes after SLO treatment, but there was no release of the trapped, internal markers, K2CrO4 or glucose. The crucial role of cholesterol in the formation of rings and C's was demonstrated by the fact that these structures were present in toxin-treated cholesterol dispersions, but not in lecithin-dicetylphosphate dispersions nor in the SLO preparations alone. The importance of cholesterol was also shown by the finding that no rings were present in membranes or cholesterol dispersions which had been treated with digitonin before SLO was added. Although rings do not appear to be "holes" in the membrane, a model is proposed which suggests that cholesterol molecules are sequestered during ring and C-structure formation, and that this process plays a role in SLO-induced hemolysis.

Unusual hindgut malrotation and associated mesenteric vascular abnormality presenting as large bowel obstruction in an adult.

We report an unusual and not previously described congenital hindgut malrotation presenting as large bowel obstruction in an adult.

Light and inherited retinal degeneration.

Light deprivation has long been considered a potential treatment for patients with inherited retinal degenerative diseases, but no therapeutic benefit has been demonstrated to date. In the few clinical studies that have addressed this issue, the underlying mutations were unknown. Our rapidly expanding knowledge of the genes and mechanisms involved in retinal degeneration have made it possible to reconsider the potential value of light restriction in specific genetic contexts. This review summarises the clinical evidence for a modifying role of light exposure in retinal degeneration and experimental evidence from animal models, focusing on retinitis pigmentosa with regional degeneration, Oguchi disease, and Stargardt macular dystrophy. These cases illustrate distinct pathophysiological roles for light, and suggest that light restriction may benefit carefully defined subsets of patients.

Effect of streptolysin S on liposomes. Influence of membrane lipid composition on toxin action.

The effect of the bacterial cytolytic toxin, streptolysin S, on liposomes composed of various phospholipids was investigated. Large unilamellar vesicles containing [14C]sucrose were prepared by reverse-phase evaporation, and membrane damage produced by the toxin was measured by following the release of labeled marker. The net charge of the liposomes had little or no effect on their susceptibility to steptolysin S and the toxin was about equally effective on liposomes composed of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and phosphatidylglycerol. Experiments with liposomes composed of synthetic phospholipids showed that the ability of the toxin to produce membrane damage depended on the degree of unsaturation of the fatty acyl chains. The order of sensitivity was C18 : 2 phosphatidylcholine greater than C18: I phosphatidylcholine greater than C18 : 0 phosphatidylcholine = C16 : 0 phosphatidylcholine. Liposomes containing the latter two phospholipids were virtually unaffected by streptolysin S, and experiments with C18 : 0 phosphatidylcholine suggested that toxin activity does not bind to liposomes composed of phospholipids with saturated fatty acyl chains. The inclusion of 40 mol% cholesterol in C16 : 0 phosphatidylcholine and C18 : 0 phosphatidylcholine liposomes made these vesicles sensitive to streptolysin S. Egg phosphatidylcholine liposomes, which were unaffected at 0 degrees C and 4 degrees C became susceptible to the toxin at these temperatures when cholesterol was included. Liposomes composed of C14 : 0 phosphatidylcholine were unaffected by streptolysin S at temperatures below the chain-melting transition temperature (23 degrees C) of this phospholipid, but became increasingly susceptible above this temperature. The results suggest that the fluidity of the phospholipid hydrocarbon chains in the membrane is important in streptolysin S action.

Approximate dimensions of membrane lesions produced by streptolysin S and streptolysin O.

Membrane lesions produced by the streptococcal membranolysins streptolysin S and streptolysin O were investigated. Escape of labeled marker molecules of various sizes from resealed sheep erythrocyte ghosts treated with the toxins for 30 min allowed estimation of the sizes of the primary channels formed. Streptolysin S formed lesions ranging in size up to 45 A in diameter, and even high toxin concentrations did not result in larger channels. The lesions produced by streptolysin O exceeded 128 A in diameter. Kinetics experiments demonstrated that the primary streptolysin O lesions were formed rapidly (1-2 min), but release of marker molecules from streptolysin S-treated vesicles began only after a 5-15-min lag period. Label release from large unilamellar liposomes treated with streptolysin S suggested that membrane fluidity does not affect the size of the streptolysin S lesions.

Resistance to streptolysin O in mammalian cells treated with oxygenated derivatives of cholesterol. Cholesterol content of resistant cells and recovery of streptolysin O sensitivity.

Cultures of L cells and HeLa cells were made resistant to the cytolytic toxin, streptolysin O, by incubating them in the presence of 20 alpha-hydroxycholesterol or 25-hydroxycholesterol. Such cells were also found to be more resistant to the cytotoxic effects of saponin and digitonin, agents known to interact with membrane cholesterol. Sterol synthesis in L cells that had been treated with either of the oxygenated derivatives of cholesterol was reduced by almost 90%, and the free cholesterol content of streptolysin O-resistant HeLa and L cells fell to approx. 50% of control cell levels. Significant recovery of sensitivity to streptolysin O occurred in about 6 h when refractory L cells were incubated in serum or cholesterol. Partial recovery was observed when the cultures were incubated for 24 h in mevalonate or lipid-depleted serum. The results provide further support for the role of membrane cholesterol in the cytotoxic action of streptolysin O on mammalian cells.

Children's surgery performed by adult general surgeons in Scotland: the present and future.

OBJECTIVE: The aim of this study was to determine the extent and the future of paediatric surgery performed in Scotland outside of the designated surgical paediatric centres. MATERIALS AND METHODS: An anonymous questionnaire was sent to all 111 Scottish members of the Association of Surgeons of Great Britain and Ireland. There was a response rate of 69%. RESULTS: Overall, 45% of responders operated on children. This was independent of the surgeon's age but was related to the type of hospital that the surgeon worked in. Eighty-four per cent of responders had a lower age limit under which they would not operate and 94% stated that there were specific circumstances where they would not operate. A mean of 18.5 elective procedures (range 0-250, median two) and six emergency procedures (range 0-30, median five) were carried out by each surgeon operating on children under the age of five per annum. Only 13% of responders thought that their successor would operate on children. CONCLUSIONS: Non-specialist paediatric surgery in Scotland is currently provided by a significant number of surgeons whose successors will not continue to provide a comparative paediatric service. This has implications for local provision of care, emergency management and capacity of existing children's hospitals in the future.

The identification of informative parameters in the flow cytometric analysis of breast carcinoma.

DNA ploidy and the measurement of proliferation or S-phase fraction are both of prognostic significance in breast cancer, yet clinical use is minimal in the U.K. Immunohistochemistry is, however, used to aid diagnosis, so a panel of antibodies were analysed by flow cytometry to assess their predictive value for prognosis, tumour stage and grade. Of 10 parameters tested on 202 breast tumour samples, tumour cell proliferation and DNA ploidy were the two most informative; cytokeratin staining, natural killer and B-cell infiltration also proved to be of value but there was no prognostic value in measuring tumour infiltrating monocytes, helper/suppressor T-cell ratios, tumour cell reactivity with carcinoembryonic antigen or human milk fat globulin antibodies. For each of the informative parameters, scores numerically weighted towards a poorer prognosis were derived which when combined, correlated with tumour grade, stage and prognosis. Such data interpretation is objective, and can be transposed to other human tumours.

Bacteria as solid phase in a concentration fluorescence immunoassay analysis of antibodies to surface antigens.

A modified solid-phase fluorescence immunoassay was developed using bacterial cells as the solid phase to screen antibodies produced against surface antigens from a clinical isolate of Escherichia coli, strain 1-149. The bacterial solid phase was used to analyze both polyclonal and monoclonal antibodies. The bacterial concentration fluorescence immunoassay (BCFIA) showed up to 50-fold greater sensitivity in bacterial cell detection as compared to ELISA (enzyme-linked immunosorbent assay). Moreover, BCFIA was considerably faster than ELISA with uniform reproducibility. This paper demonstrates the utility of using bacteria and their surface antigens as solid-phase matrices for antibody characterization in a FIA.

Macular pigment and lutein supplementation in retinitis pigmentosa and Usher syndrome.

PURPOSE: To determine macular pigment (MP) in patients with inherited retinal degeneration and the response of MP and vision to supplementation of lutein. METHODS: Patients with retinitis pigmentosa (RP) or Usher syndrome and normal subjects had MP optical density profiles measured with heterochromatic flicker photometry. Serum carotenoids, visual acuity, foveal sensitivity, and retinal thickness (by optical coherence tomography [OCT]) were quantified. The effects on MP and central vision of 6 months of lutein supplementation at 20 mg/d were determined. RESULTS: MP density in the patients as a group did not differ from normal. Among patients with lower MP, there was a higher percentage of females, smokers, and light-colored irides. Disease expression tended to be more severe in patients with lower MP. Inner retinal thickness by OCT correlated positively with MP density in the patients. After supplementation, all participants showed an increase in serum lutein. Only approximately half the patients showed a statistically significant increase in MP. Retinal nonresponders had slightly greater disease severity but were otherwise not distinguishable from responders. Central vision was unchanged after supplementation. CONCLUSIONS: Factors previously associated with lower or higher MP density in normal subjects showed similar associations in RP and Usher syndrome. In addition, MP in patients may be affected by stage of retinal disease, especially that leading to abnormal foveal architecture. MP could be augmented by supplemental lutein in many but not all patients. There was no change in central vision after 6 months of lutein supplementation, but long-term influences on the natural history of these retinal degenerations require further study.

Protective immunisation of guinea pigs against Dictyocaulus viviparus using excretory/secretory products of adult parasites.

Parasite preparations were examined for their ability to induce protective immunity against Dictyocaulus viviparus in guinea pigs. Dunkin-Hartley strain guinea pigs were immunised with somatic extracts of adult parasites, somatic extracts of third stage larvae or excretory/secretory (ES) products from adult parasites. The groups were immunised twice with Freund's adjuvant four weeks apart and challenged with 6000 infective L3. Significant levels of protective immunity were observed only in the adult ES-immunised animals. The antibody responses of the different groups were compared following analysis by ELISA and immunoprecipitation. To examine the protective role of antibody, guinea pigs were passively immunised with serum from animals immunised with adult ES products or serum from guinea pigs exposed to experimental D. viviparus infection. Following challenge with infective L3, lung-worm burdens of these groups were significantly lower than in guinea pigs which received normal sera. The results suggest that D. viviparus adult ES products contain protective antigens and that antibody-mediated mechanisms contribute to immune protection.

The immunogenicity of the acetylcholinesterases of the cattle lungworm Dictyocaulus viviparus.

Somatic extracts and excretory/secretory (ES) products of the adult stage of the cattle nematode, Dictyocaulus viviparus, were examined for acetylcholinesterase (AChE) activity. Both were found to contain activity which had an optimum pH of 9.5, however, the adult ES products contained over 200 times more AChE activity per unit protein. Gel electrophoresis and specific enzyme staining revealed 5 migratory isoforms of AChE which were common to adult ES products and adult homogenates. Comparison of L3 with L4 and adult extracts indicated that the AChE were only produced by later developmental stages of this parasite. The antigenicity of D. viviparus AChE was demonstrated by binding to serum IgG from naturally and experimentally infected calves but the enzymes were not recognized by calves vaccinated twice with 400 Gy-irradiated larvae. This is the first report of helminth AChE release by a parasitic nematode in a pulmonary location. The presence of these enzymes in such high amounts in the ES products, along with their immunogenicity, suggests that they might have an important role to play in the immunobiology of D. viviparus in the lungs.

The repeatability of faecal egg counts, peripheral eosinophil counts, and plasma pepsinogen concentrations during deliberate infections with Ostertagia circumcincta.

Faecal egg counts, peripheral blood eosinophil counts and plasma pepsinogen concentrations were monitored during 2 successive, deliberate infections in 24 Scottish Blackface sheep. For all 3 techniques, the repeatability of replicate counts or of measurements made at short intervals were high which suggests that all 3 assays were reliable. Within an infection the repeatability of different samples from the same animal decreased as the interval between samples increased. The repeatability between infections was only moderate for faecal egg counts but high for peripheral eosinophil counts and plasma pepsinogen concentrations. Of the 3 variables, faecal egg count was the most strongly associated with the worm burden. Together, the three variables accounted for, in a statistical sense, one half of the variation in worm burden. The three variables, if measured concurrently, should provide a more effective identification of resistant and susceptible lambs.

Immunisation of guinea pigs against Dictyocaulus viviparus using adult ES products enriched for acetylcholinesterases.

The adult ES products of Dictyocaulus viviparus are a source of protective antigens against challenge in the guinea pig laboratory model. High levels of acetylcholinesterase (AChE) activity are present in these products and these enzymes are immunogenic in infected cattle. Here, the potential role of these enzymes in protective immunity was investigated using a fraction enriched for AChE to immunise guinea pigs. The antibody response stimulated by immunisation with AChE-enriched ES products and the worm burdens obtained following challenge with infective larvae were compared with those in animals immunised with whole ES products and challenge controls. The AChE-enriched preparation stimulated high levels of enzyme-specific antibody in immunised animals, which was not the case for those which received unfractionaed ES products. Worm burdens of guinea pigs which received the AChE-enriched fraction were significantly lower than those obtained in adjuvant controls. The animals which received the unfractionated ES products were not significantly protected against challenge. These results suggest that AChEs may be potential candidates for incorporation in a sub-unit vaccine against D. viviparus.

An ovine major histocompatibility complex DRB1 allele is associated with low faecal egg counts following natural, predominantly Ostertagia circumcincta infection.

Infection with Ostertagia circumcincta is a major constraint on sheep production in temperate areas of the world. A potential control strategy is the use of genetically resistant sheep. Therefore we examined the association between MHC-DRB1 alleles and faecal egg counts following natural, predominately O. circumcincta infection in a flock of Scottish Blackface sheep. Nineteen DRB1 alleles were identified by a combination of variation in the length of simple repetitive sequences within the intron between exons 2 and 3 and hybridisation of selected oligonucleotides to polymorphisms within exon 2. Faecal samples were taken from 200 lambs from one to six months of age at intervals of 4 weeks. Genetic effects were strongest at 6 months of age. Least-squares analysis indicated that substitution of the most common allele (I) by allele G2 would result in a 58-fold reduction in faecal egg counts in 6-month-old lambs and a 22-fold reduction in 5-month-old lambs. These results suggest that the major histocompatibility complex plays an important role in the development of resistance to O. circumcincta.