Postmortem genetic testing in young individuals: What clinical medical practitioners need to know.

The death of a young person is most often a tragic occurrence, more so when this death was unexpected. Forensic pathologists are mandated to investigate such deaths, and there has been a strong move internationally towards genetic testing as an additional investigative tool. The aim of our article is to bring the advantage of implementing the so-called molecular autopsy in a local setting to the attention of medical practitioners. When a multidisciplinary approach is taken in cases of sudden unexpected death, the benefits to family members, and society as a whole, are irrefutable.

Cardiovascular deaths: What do the genes say?

Cardiovascular deaths: What do the genes say?

A partially randomised trial of pretomanid, moxifloxacin and pyrazinamide for pulmonary TB.

BACKGROUND: Treatment for TB is lengthy and toxic, and new regimens are needed.METHODS: Participants with pulmonary drug-susceptible TB (DS-TB) were randomised to receive: 200 mg pretomanid (Pa, PMD) daily, 400 mg moxifloxacin (M) and 1500 mg pyrazinamide (Z) for 6 months (6Pa200MZ) or 4 months (4Pa200MZ); 100 mg pretomanid daily for 4 months in the same combination (4Pa100MZ); or standard DS-TB treatment for 6 months. The primary outcome was treatment failure or relapse at 12 months post-randomisation. The non-inferiority margin for between-group differences was 12.0%. Recruitment was paused following three deaths and not resumed.RESULTS: Respectively 4/47 (8.5%), 11/57 (19.3%), 14/52 (26.9%) and 1/53 (1.9%) DS-TB outcomes were unfavourable in patients on 6Pa200MZ, 4Pa200MZ, 4Pa100MZ and controls. There was a 6.6% (95% CI -2.2% to 15.4%) difference per protocol and 9.9% (95%CI -4.1% to 23.9%) modified intention-to-treat difference in unfavourable responses between the control and 6Pa200MZ arms. Grade 3+ adverse events affected 68/203 (33.5%) receiving experimental regimens, and 19/68 (27.9%) on control. Ten of 203 (4.9%) participants on experimental arms and 2/68 (2.9%) controls died.CONCLUSION: PaMZ regimens did not achieve non-inferiority in this under-powered trial. An ongoing evaluation of PMD remains a priority.

XANNpred: neural nets that predict the propensity of a protein to yield diffraction-quality crystals.

Production of diffracting crystals is a critical step in determining the three-dimensional structure of a protein by X-ray crystallography. Computational techniques to rank proteins by their propensity to yield diffraction-quality crystals can improve efficiency in obtaining structural data by guiding both protein selection and construct design. XANNpred comprises a pair of artificial neural networks that each predict the propensity of a selected protein sequence to produce diffraction-quality crystals by current structural biology techniques. Blind tests show XANNpred has accuracy and Matthews correlation values ranging from 75% to 81% and 0.50 to 0.63 respectively; values of area under the receiver operator characteristic (ROC) curve range from 0.81 to 0.88. On blind test data XANNpred outperforms the other available algorithms XtalPred, PXS, OB-Score, and ParCrys. XANNpred also guides construct design by presenting graphs of predicted propensity for diffraction-quality crystals against residue sequence position. The XANNpred-SG algorithm is likely to be most useful to target selection in structural genomics consortia, while the XANNpred-PDB algorithm is more suited to the general structural biology community. XANNpred predictions that include sliding window graphs are freely available from http://www.compbio.dundee.ac.uk/xannpred

The Scottish Structural Proteomics Facility: targets, methods and outputs.

The Scottish Structural Proteomics Facility was funded to develop a laboratory scale approach to high throughput structure determination. The effort was successful in that over 40 structures were determined. These structures and the methods harnessed to obtain them are reported here. This report reflects on the value of automation but also on the continued requirement for a high degree of scientific and technical expertise. The efficiency of the process poses challenges to the current paradigm of structural analysis and publication. In the 5 year period we published ten peer-reviewed papers reporting structural data arising from the pipeline. Nevertheless, the number of structures solved exceeded our ability to analyse and publish each new finding. By reporting the experimental details and depositing the structures we hope to maximize the impact of the project by allowing others to follow up the relevant biology.

Assessment of global capacity to conduct tuberculosis drug development trials: do we have what it takes?

OBJECTIVE: To assess clinical trial sites and associated mycobacteriology laboratories for their capacity to conduct registration-standard tuberculosis (TB) drug trials and develop a database of assessed sites and laboratories. SETTING: Assessments of clinical trial sites and associated mycobacteriology laboratories were conducted in 39 countries from 2006 to 2008. DESIGN: Sites were interviewed using a set of questionnaires to assess the clinical site, pharmacy, data management, regulatory, ethics and importation requirements and mycobacteriology laboratory. Each site and laboratory was rated as able to conduct TB drug registration trials within 0-6 months, >6-12 months, >1-2 years and >2 years. RESULTS: Eighty-four clinical trial sites and associated mycobacteriology laboratories in 39 countries were assessed. Of the clinical trial sites, 50% were judged capable of being ready within 6 months, 32.1% in 6-12 months and 14.3% in 1-2 years. Three sites would be ready in more than 2 years. Of the 72 mycobacteriology laboratories, 27.8% could be made ready within 6 months, 37.5% within 6-12 months and 27.8% within 1-2 years. CONCLUSION: This survey indicates that developing adequate capacity to fully evaluate the compounds now in the clinical phases of development will require significant capacity-building efforts.

TarO: a target optimisation system for structural biology.

TarO (http://www.compbio.dundee.ac.uk/taro) offers a single point of reference for key bioinformatics analyses relevant to selecting proteins or domains for study by structural biology techniques. The protein sequence is analysed by 17 algorithms and compared to 8 databases. TarO gathers putative homologues, including orthologues, and then obtains predictions of properties for these sequences including crystallisation propensity, protein disorder and post-translational modifications. Analyses are run on a high-performance computing cluster, the results integrated, stored in a database and accessed through a web-based user interface. Output is in tabulated format and in the form of an annotated multiple sequence alignment (MSA) that may be edited interactively in the program Jalview. TarO also simplifies the gathering of additional annotations via the Distributed Annotation System, both from the MSA in Jalview and through links to Dasty2. Routes to other information gateways are included, for example to relevant pages from UniProt, COG and the Conserved Domains Database. Open access to TarO is available from a guest account with private accounts for academic use available on request. Future development of TarO will include further analysis steps and integration with the Protein Information Management System (PIMS), a sister project in the BBSRC 'Structural Proteomics of Rational Targets' initiative.

Expression of transmembrane protein tyrosine phosphatase gamma (PTPgamma) in normal and neoplastic human tissues.

AIMS: To establish the conditions for protein tyrosine phosphatase gamma (PTPgamma) detection in paraffin tissues using two antibodies raised against its NH(2)- (anti-P4) and COOH-termini (gammaTL1); to analyse its expression in normal tissues and to perform an initial screening of neoplastic tissues. METHODS AND RESULTS: Membranous and/or cytoplasmic PTPgamma expression was detected in the majority of epithelial cell types and in endocrine cells, with the highest expression in adrenal medulla, endocrine cells of the gastrointestinal tract and pancreatic islets. Both antibodies stained the thyroid follicular epithelium, but only anti-P4 antibody stained the colloid matrix, suggesting shedding/secretion of the PTPgamma extracellular domain. Marked loss of PTPgamma immunoreactivity was detected in subsets of ovarian (21%), breast (56%) and lung (80%) neoplasms. Conversely, cytoplasmic positivity was found in 37% of lymphomas, mainly of high-grade histotypes, while normal lymphocytes were negative. Brain tissue showed PTPgamma expression in a few neuronal and glial elements and PTPgamma was overexpressed in the majority of high-grade astrocytomas. CONCLUSIONS: We have analysed PTPgamma expression in archival paraffin-embedded tissues for the first time, demonstrating particularly high expression in endocrine cells and both down- and up-regulation in neoplasia, the latter possibly reflecting the undifferentiated state of the neoplastic cells, suggesting a complex role for this phosphatase.

"Guidelines prescribed by General Practitioners to patients with acute low back pain regarding ""return to work """

BackgroundThe traditional treatment protocol for acute low back pain (ALBP) primarily used by healthcare professionals has in the past decade been strict bed rest; corsets; traction and `back schools'. However; current research has led to dramatic changes in the traditional treatment protocol. The literature suggests that the protocol should be replaced by parsimonious imaging; early return to normal activities and greater emphasis on exercise to prevent recurrences of ALBP and to treat chronic pain. The aim of this study was to investigate the guidelines prescribed by general practitioners (GPs) to patients with acute low back pain (ALBP) regarding `return to work'.MethodsA systematic sample of 212 GPs; selected from a list supplied by the Health Professions Council of South Africa (HPCSA); was selected to complete questionnaires. The highest qualifications of the GPs were MBChB or MFamMed; and all of them practise in the Bloemfontein area.ResultsSixty-three respondents stated that 40of ALBP patients returned for follow-up consultations. Of the 63 respondents; eight GPs had not consulted ALBP patients in the preceding two years; and thus were excluded from the final number of respondents. Bed rest is still prescribed by 67.27of GPs and; although 47.27of the GPs were aware of the change in protocol; only 9prescribe `return to work'. A total of 18.18are aware of evidence-based guidelines and 10of the GPs prescribe these. Among the guidelines defined by the GPs are lifestyle changes; rest and stabilisation. Only 18.18of ALBP patients are referred to occupational therapy for treatment.ConclusionOnly 47.27of the GPs knew about the new ALBP protocol; and even fewer had any knowledge of the content of the new protocol. Also; the guidelines prescribed by the GPs concerning `return to work' were indefinite. The researchers hypothesised that the reasons for this were a lack of awareness of the change in the acute low back pain protocol suggested by the Agency for Healthcare Research and Quality; as well as a lack of knowledge of the evidence-based guidelines suggested for their profession

Effects of triamcinolone on quality of life in patients with persistent allergic rhinitis.

OBJECTIVE: To study the effects of triamcinolone acetonide on health-related quality of life (HRQL) in adult patients with allergic rhinitis. METHODS: This study was conducted in South Africa as a placebo-controlled, multicenter, randomized, double-blind study. Following a 7-day baseline run-in, patients were treated for 28 days with either triamcinolone or placebo. Interviewer-assisted quality-of-life assessments were conducted using the Juniper Rhinoconjunctivitis Quality of Life Questionnaire (RQLQ). Total symptom scores, including nasal congestion, were measured using daily diary cards. RESULTS: A total of 337 patients were recruited and 253 patients completed the study per protocol, of which 55 had seasonal allergic rhinitis (SAR) and 198 had persistent allergic rhinitis (PAR). Improvements in the mean scores per area of the RQLQ were significantly better with triamcinolone compared with placebo for the entire study group for activities (P = .04 at visit 4) and sleep, nasal symptoms, emotional problems, and overall score (P = .002, P = .04, P = .03, and P = .04, respectively, at visit 3). When the patients with SAR were separated from the patients with PAR in the analysis, improvement with triamcinolone was better than placebo only in the PAR patients. The overall investigator and patient assessments of relief favored triamcinolone. CONCLUSIONS: Triamcinolone given for 4 weeks improves symptom scores and HRQL in patients with allergic rhinitis. The ability of triamcinolone to relieve nasal congestion symptoms in PAR patients was correlated with improvements in HRQL.

Reduced expression of protein tyrosine phosphatase gamma in lung and ovarian tumors.

Based on LOH studies protein tyrosine phosphatasegamma (PTPgamma) has been suggested as a candidate tumor suppressor gene involved in the oncogenesis of lung and renal cancers. In order to assess the involvement of PTPgamma in tumor development we developed a PTPgamma-specific monoclonal antibody (gammaTL1) (IgM isotype) by immunization with a synthetic peptide of 15 amino acids corresponding to the amino acid sequence nos. 1423-1438 just outside the phosphatase domain-II. In line with the fact that the antibody was raised to an intracellular domain of the PTPgamma molecule the antibody labeled the cell membrane of fixed cells but did not stain the outside of the cell membrane in the immunofluorescence assay. The Mab gammaTL1 recognized a full-length baculovirus recombinant PTPgamma protein of 185 kDa, in addition to putative cleavage products of 120 kDa, 114/110 kDa and 80 kDa, on Western blots of lysates of PTPgamma-gene transfected Sf9 insect cells but not of tumor cell lysates. Based on immunoperoxidase and immunofluorescence assays on cryostat sections, however, PTPgamma was expressed in more than 90% of both normal, human tissue samples and in the (non-) tumor cells of carcinoma samples. However, PTPgamma was not found in 28% of the overall lung tumor samples, i.e. in 50% of the lung adenocarcinoma samples, while the expression was weak and heterogeneous in 71% of squamous lung cell carcinomas. PTPgamma was not suppressed in the normal cells between the lung carcinoma cells. The presence of PTPgamma, assayed by immunofluorescence in lung tumor cell lines (H69, H128, H82, C3) was confirmed by RT-PCR assay. Interestingly, the 90% expression score of PTPgamma protein in normal ovarian tissue samples was reduced dramatically to 44 and 38% in both the non-tumorous and tumorous cells, respectively, in ovarian tumor samples. PTPgamma was absent in the HT29 human colon carcinoma cell line both by immunofluorescence and RT-PCR assay. In summary, we have developed a PTPgamma-specific monoclonal antibody, that demonstrated that the expression of PTPgamma is severely reduced (>50%) in lung tumors and ovarian tumors.

Continuous in vitro cultivation of Babesia caballi in serum-free medium.

Experiments were undertaken to develop a serum-free medium for the in vitro cultivation of Babesia caballi, a tick-borne hemoprotozoan parasite, one of the causative agents of equine piroplasmosis. A modified HL-1 medium supplemented with horse serum, L-glutamine, antibiotics, and hypoxanthine was used. B. caballi organisms were continuously cultivated at 37 degrees C in microaerophilous stationary-phase culture in a humidified atmosphere containing 5% CO2 in air before exposure to serum-free culture conditions. For serum-free propagation, lipid-rich bovine serum albumin (LR-BSA), alone or with chemically defined lipids (CDL), were added instead of serum. Media containing LR-BSA alone or LR-BSA and CDL in various amounts supported the in vitro propagation of B. caballi. Growth was maintained for more than 6 months. The growth rates obtained in serum-free media were similar to those previously obtained in traditional serum-containing medium.

The effect of dietary protein on reproduction in the mare. V. Endocrine changes and conception during the early post partum period.

Pregnant Anglo-Arab and Thoroughbred mares (n = 24) were divided randomly according to age and breed into 4 groups of 6 mares each from approximately 6 weeks before their expected foaling date. Diets received by the 4 groups varied in essential amino-acid and total protein contents. Serum progestagen, FSH and LH concentrations were determined from the day of parturition until foal heat and during the 1st oestrous cycle following foal heat. Serum progestagen, FSH and LH concentrations did not differ between the treatment groups. Progestagen concentrations were high (mean = 7.0: 5.2-16.4 ng/ml) at parturition but decreased rapidly within 48 h. As progestagen concentrations decreased LH concentrations increased from Days 3-6 post partum to reach maximum values at, or the day after ovulation. FSH concentrations declined 3-4 d after parturition and increased 2-3 d before ovulation at foal heat. The duration of elevated progestagen concentrations during the luteal phase of the subsequent oestrous cycle affected the interovulatory period. A 12-14 d FSH cyclical releasing pattern occurred. Season/photoperiod affected the resumption of normal oestrous cyclicity during the post partum period. The duration of the 1st oestrous cycle after foal heat in mares fed a low-quality protein diet showed a greater range (13-30 d) compared to mares fed a high-quality protein diet (18-26 d).

The effect of dietary protein on reproduction in the mare. VI. Serum progestagen concentrations during pregnancy.

Sixty-four Thoroughbred and Anglo-Arab mares aged 6-12 years were used, of which 40 were non-lactating and 24 lactating. Foals from these 24 mares were weaned at the age of 6 months. Non-lactating and lactating mares were divided into 4 dietary groups each. The total daily protein intake and the protein quality (essential amino-acid content) differed in the 4 groups of non-lactating and 4 groups of lactating mares. The mares were covered and the effect of the quantity and quality of dietary protein on serum progestagen concentrations during pregnancy was studied. A sharp decline in serum progestagen concentrations was recorded in all dietary groups from Days 18 to 40 of pregnancy, with some individual mares reaching values of less than 4 ng/ml. Serum progestagen concentrations recorded in some of the non-lactating mares on the low-quality protein diet increased to higher values (p < 0.05) than those of mares in the other 3 dietary groups at 35-140 days of pregnancy. A similar trend was observed for the lactating mares on a low-quality protein diet at 30-84 days of pregnancy. No such trends were observed in any of the other dietary groups. High-quality protein supplementation increased serum progestagen concentrations during the 1st 30 days of pregnancy. Lactation depressed serum progestagen concentrations until after the foals were weaned.

The effect of dietary protein on reproduction in the mare. VII. Embryonic development, early embryonic death, foetal losses and their relationship with serum progestagen.

Sixty-four Thoroughbred and Anglo-Arab mares aged 6-12 years were randomly allocated to 4 dietary groups and fed diets that differed in the total protein content and quality (essential amino-acids). Forty mares were non-lactating and 24 lactating. Eight mares were withdrawn from the investigation owing to injuries or gynaecological pathology. An overall conception rate of 94.6% and a foaling rate of 80% was achieved. Five of 14 (35.7%) mares (Group 1) fed a low-quality protein diet suffered from early embryonic loss before 90 days of pregnancy compared to 3 of 41 (7.3%) mares in the remaining groups that received the higher-quality protein in their diets. Serum progestagen concentrations of mares in Group 1 that suffered foetal loss were indicative of luteal function insufficiency during the 1st 40 days post-ovulation. Non-lactating mares in all 4 groups gained on average approximately 30 kg in mass during the 90 days before the breeding period. Lactating mares in Group 1 (low-quality protein) lost on average 25 kg in mass during lactation, with no weight loss observed among the lactating mares in the other 3 groups. No difference in the diameter of the embryonic vesicle was found between dietary groups until Day 35 of pregnancy.

The effect of dietary protein on reproduction in the mare. I. The composition and evaluation of the digestibility of dietary protein from different sources.

Four rations that differed in their crude protein and essential amino-acid content were compiled. Digestibility of the crude protein and essential amino-acid contents were determined biologically in a feeding trial using 4 Anglo-Arab stallions. Their respective daily diets were: Diet 1:2 kg cubes, 5 kg tef hay (Eragrostis tef); Diet 2:2 kg cubes, 5 kg lucerne hay (Medicago sativa); Diet 3:2 kg cubes, 5 kg tef hay, 200 g fishmeal; Diet 4:2 kg cubes, 5 kg lucerne hay, 200 g fishmeal. The concentrations of the amino-acids threonine, iso-leucine, leucine and arginine were increased in the total ration when lucerne hay replaced the tef hay while fishmeal supplementation increased the methionine and lysine contents, which provided a wide-range of concentrations of digestible amino-acids in each of the 4 rations.

The effect of dietary protein on reproduction in the mare. II. Growth of foals, body mass of mares and serum protein concentration of mares during the anovulatory, transitional and pregnant periods.

The effect of 4 different diets, in terms of protein quantity and quality, on total serum protein (TSP), albumin and globulin was investigated. Non-pregnant mares that were not lactating (n = 36), pregnant mares that had foaled (n = 24) and their foals (n = 24) were used in this study. Daily total protein intake had no effect on blood protein concentrations in the mares. Total protein intake and quality (available essential amino-acids) did affect the body mass of mares during lactation. When mares were fed the minimum recommended (National Research Council 1989) total daily protein, foal mass decreased by approximately 25% at weaning compared to the foals whose dams were on a higher level of protein intake. The TSP concentrations of foals at birth were on average 10 g/l lower than those of the mares. Albumin concentrations of foals during the first 60 days of life were on average 2-3 g/l lower than those of the mares. Globulin concentrations of foals were approximately 5 g/l lower than those of mares at weaning.

The effect of dietary protein on reproduction in the mare. III. Ovarian and uterine changes during the anovulatory, transitional and ovulatory periods in the non-pregnant mare.

In the main experiment the total daily protein intake and quality (essential amino-acids) was varied in 4 groups of mares. The incidence of oestrus in mares during the transitional period was unaffected by protein nutrition. Ovarian activity, as evaluated by follicular development and size of the ovaries, was affected. Mares that received low-quality protein (Groups 1 and 2) had a higher number of smaller follicles (< 10 mm) that developed during the transitional period compared to mares on a high-quality protein intake (Groups 3 and 4). The mares that received the high quality protein ovulated 2-3 weeks earlier in the breeding season in a synchronised period of 4-5 weeks compared to a period of 6-8 weeks in Groups 1 and 2. The duration of the subsequent oestrous cycles was not affected. There was no difference in the diameter of the largest follicle of mares between groups on the day before ovulation. In a separate experiment, 5 maiden Anglo-Arab mares, 4-5 years of age, were slaughtered at different stages during the anovulatory, transitional and ovulatory periods of the breeding cycle. The morphology of the ovaries and uteri of these mares was described and photographed for use as guidelines when comparing ovarian changes and follicular activity of mares.

Differentiation margins of ovarian tumor pathology: first incidences of epithelial ovarian tumors monitored by marker antibodies.

The first incidence of ovarian tumors in The Netherlands was analyzed during the PALGA data. The first incidences of benign epithelial ovarian tumors reach a plateau, at a level of 60 to 65 cases per 100,000 women beyond the age of 40 years. The borderline malignant epithelial ovarian tumors account for 10 per 100,000 women aged 30 to 85, while the ovarian carcinomas reach a plateau level of 25 to 35 per 100,000 women after the age of 50. Despite the long lag period (+/- 10 years) between benign and malignant ovarian tumors, the question of whether or not all epithelial ovarian cancers develop via an intermediate step of cystadenomas is still unanswered. Therefore, we examined whether the expression pattern of intermediate filaments and tumor antigens in normal, benign, and malignant ovarian tissues might contribute to this question. The following changes in expression pattern were observed: generally, all tumor cells retained the keratin profile of the corresponding original cell type. However, in a limited number of tumor samples ectopic keratin types, such as nos. 4, 10, 13, and 14, became expressed additionally. Most epithelial ovarian tumors and mesothelial cells coexpressed vimentin. The panepithelial marker BW495/36 clearly distinguished between negatively stained normal ovarian surface mesothelium and the positively stained (inclusion) cystic epithelium. TAG-72 as well as OV-632 marked a subsequent tumor stage by discriminating between negative serous adenomas and positive serious carcinomas. TAG-72, however, stained both mucinous adenomas and carcinomas. The ovarian tumor markers (OC125, OV-TL 33, OV-TL16, MOv18), all showed an increasing expression level in the sequence order from normal cells to benign and malignant ovarian tumors. Both our epidemiological and our immunohistochemical data have shown that in the Dutch population there is a lag period of at least 10 years between the plateau levels of benign and malignant ovarian tumors. The early transformation of mesothelial cells to benign and/or malignant tumors is clearly marked by a switch on of the BW495/36 marker. Although no general transformation or progression marker from adenomas to carcinomas emerged from this study, TAG-72 might be considered a (progression) marker between the subgroup of benign and malignant serous ovarian tumors.