BMC Research Notes - Latest articles

RNAase footprinting demonstrates antigenomic hepatitis delta virus ribozyme structural rearrangement as a result of self-cleavage reaction

2008-05-16

Background: Hepatitis delta virus (HDV) is a satellite virus of hepatitis B. During viral replication the 1700-nucleotide-long genomic RNA and its complement, the antigenomic RNA, undergo self-cleavage catalyzed by internal ribozyme motifs that are essential for propagation of the virus in vivo. These self-cleavage activities are provided by 85-nucleotide-long sequence elements, the genomic and antigenomic forms of HDV ribozyme. Recently four permuted variants of the antigenomic HDV cis-ribozyme with a self-cleavage site located at the 5' proximity, in the middle, or nearby the 3' end of the molecule were constructed and synthesized. These constructs exhibit equal activity, a bi-phasic kinetics of self-cleavage reaction and reaction products with low and high stability. We have used ribonuclease probing to footprint the structures of uncleaved and post-cleaved forms of the antigenomic HDV ribozymes in solution. Uncleaved ribozymes, associated and individual products of the self-cleavage reaction were analyzed using ribonuclease and Fe(II)-EDTA protection assays to reveal the differences in the structure of pre- and post-cleaved antigenomic HDV ribozyme in solution.FindingsOur findings demonstrate that a significant conformational change accompanies catalysis in the antigenomic HDV ribozyme in solution, in contrast to minor conformational switch observed in crystals of the genomic form. This study indicates that changes in the structure of stem P1 and stem P4 are minor, those of the region ascribed to stem P2, stem P3 and loop l3 are dramatic, while stem P1.1 results from the self-cleavage reaction. Conclusions: Our data agree with the structure of post-cleaved and disagree with that of pre-cleaved forms of HDV ribozyme published elsewhere.

Detection of the transforming AKT1 mutation E17K in non-small cell lung cancer by high resolution melting

2008-05-16

Background: A recurrent somatic mutation, E17K, in the pleckstrin homology domain of the AKT1 gene, has been recently described in breast, colorectal, and ovarian cancers. AKT1 is a pivotal mediator of signalling pathways involved in cell survival, proliferation and growth. The E17K mutation stimulates downstream signalling and exhibits transforming activity in vitro and in vivo. FindingsWe developed a sensitive high resolution melting (HRM) assay to detect the E17K mutation from formalin-fixed paraffin-embedded tumours. We screened 219 non-small cell lung cancer biopsies for the mutation using HRM analysis. Four samples were identified as HRM positive. Subsequent sequencing of those samples confirmed the E17K mutation in one of the cases. A rare single nucleotide polymorphism was detected in each of the remaining three samples. The E17K was found in one of the 14 squamous cell carcinomas. No mutations were found in 141 adenocarcinomas and 39 large cell carcinomas. Conclusions: The AKT1 E17K mutation is very rare in lung cancer and might be associated with tumorigenesis in squamous cell carcinoma. HRM represents a rapid cost-effective and robust screening of low frequency mutations such as AKT1 mutations in clinical samples.

Interaction of the growth and tumour suppressor NORE1A with microtubules is not required for its growth-suppressive function

Anna Moshnikova, Sergey Kuznetsov and Andrei V Khokhlatchev — 2008-05-15

Background: The NORE1 protein was identified in a yeast two-hybrid screen as a Ras effector that binds Ras protein in a GTP-dependent manner. NORE1A is a growth and tumour suppressor that is inactivated in a variety of cancers. In transformed human cells, both full-length NORE1A protein and its effector domain alone (amino acids 191-363) are localized to microtubules and centrosomes. However, the mechanism by which NORE1A associates with these cytoskeletal elements is not known; furthermore, whether centrosomally-associated or microtubule-associated NORE1A suppresses tumour cell growth has not been yet established.FindingsWe have shown that purified NORE1A fails to bind to microtubules in vitro suggesting that other protein(s) mediate NORE1A-microtubule association. Using mass-spectrometry, we identified the Microtubule-Associated Protein 1B (MAP1B) and its homologue C19ORF5 as NORE1A interaction partners. Suppression of C19ORF5 expression by RNA interference (RNAi) and immunodepletion of C19ORF5 protein from cell extracts showed that binding of NORE1A to microtubules is not dependent on C19ORF5. Conversely, RNAi suppression of MAP1B revealed that MAP1B is required for association of NORE1A with microtubules. RNAi-mediated depletion of C19ORF5 or MAP1B did not prevent centrosomal localization of NORE1A. Moreover, the depletion of C19ORF5 or MAP1B did not prevent NORE1A's ability to suppress tumour cell growth. Conclusions: The interaction of NORE1A with microtubules is mediated by MAP1B, but not C19ORF5 protein. Interaction of NORE1A with centrosomes is not dependent on C19ORF5 or MAP1B, and appears to involve a different mechanism independent of binding to microtubules. The NORE1A microtubular localization is not required for growth suppression.

Comparison of palpation- versus ultrasound-guided fine-needle aspiration biopsies in the evaluation of thyroid nodules

Ahmet Selcuk Can and Kamil Peker — 2008-05-15

Background: The aim of this study was to compare the results of palpation- versus ultrasound-guided thyroid fine-needle aspiration (FNA) biopsies.FindingsClinical data, cytology and histopathology results were retrospectively analyzed on all patients who underwent thyroid FNA biopsy in our outpatient endocrinology clinic between January 1998 and April 2003. The same investigators performed all thyroid FNAs (ASC) and cytological evaluations (KP). Subjects in the ultrasound-guided group were older, otherwise there were no differences in baseline characteristics (gender, thyroid function, the frequency of multinodular goiter, nodule diameter and nodule location) between groups. Cytology results in nodules aspirated by palpation (n=202) versus ultrasound guidance (n=184) were as follows: malignant 2.0% versus 2.7% (p=0.74), benign 69.8% versus 79.9% (p=0.02), indeterminate 1.0% versus 4.9% (p=0.02), inadequate 27.2% versus 12.5% (p<0.01). Malignant results were compared with Fisher's exact test. Other cytology categories were compared with chi-square test. Eighteen patients from the palpation- and 23 from ultrasound-guided group underwent surgery. In the palpation-guided group, the sensitivity of FNA was 100%, specificity 94%, positive predictive value 67% and negative predictive value 100%. In the ultrasound-guided group, the sensitivity of FNA was 100%, specificity 80%, positive predictive value 73% and negative predictive value 100%. Conclusions: We demonstrate that ultrasound guidance for thyroid FNA significantly decreases inadequate for evaluation category. We also confirm the high sensitivity and specificity of thyroid FNA biopsy in the diagnosis of thyroid cancer. Where available, we recommend universal application of ultrasound guidance for thyroid FNA biopsy as a standard component of this diagnostic technique.

Survival of Burkholderia pseudomallei in Water

Richard A Moore, Apichai Tuanyok and Donald E Woods — 2008-05-07

Background: The ability of Burkholderia pseudomallei to survive in water likely contributes to its environmental persistence in endemic regions. To determine the physiological adaptations which allow B. pseudomallei to survive in aqueous environments, we performed microarray analyses of B. pseudomallei cultures transferred from Luria broth (LB) to distilled water. Findings. Increased expression of a gene encoding for a putative membrane protein (BPSL0721) was confirmed using a lux-based transcriptional reporter system, and maximal expression was noted at approximately 6 hrs after shifting cells from LB to water. A BPSL0721 deficient mutant of B. pseudomallei was able to survive in water for at least 90 days indicating that although involved, BPSL0721 was not essential for survival. BPSL2961, a gene encoding a putative phosphatidylglycerol phosphatase (PGP), was also induced when cells were shifted to water. This gene is likely involved in cell membrane biosynthesis. We were unable to construct a PGP mutant suggesting that the gene is not only involved in survival in water but is essential for cell viability. We also examined mutants of polyhydroxybutyrate synthase (phbC), lipopolysaccharide (LPS) oligosaccharide and capsule synthesis, and these mutations did not affect survival in water. LPS mutants lacking outer core were found to lose viability in water by 200 days indicating that an intact LPS core provides an outer membrane architecture which allows prolonged survival in water. Conclusions. The results from these studies suggest that B. pseudomallei survival in water is a complex process that requires an LPS molecule which contains an intact core region.

Inappropriately low aldosterone concentrations in adults with AIDS-related diarrhoea in Zambia: a study of response to fluid challenge

Trevor Kaile, Isaac Zulu, Ruth Lumayi, Neil Ashman and Paul Kelly — 2008-04-17

Background: Chronic diarrhoea is one of the most debilitating consequences of HIV infection in sub-Saharan Africa and it carries a high mortality rate. We report unexpectedly low concentrations of circulating aldosterone in 12 patients (6 men, 6 women) in the University Teaching Hospital, Lusaka, who all had diarrhoea for over one month. Changes in serum electrolytes, blood pressure, Karnofsky score and serum aldosterone concentration were being monitored during a short study of responses to saline infusion (3 litres/24h) over 72 hours. FindingsAt baseline, 9/12 (75%) of the patients were hyponatraemic, 10/11 (91%) were hypokalaemic, and 6/12 (50%) had undetectable aldosterone concentrations. Blood pressure and Karnofsky score rose and creatinine concentration fell in response to the infusion. Conclusions: Circulating aldosterone concentrations were inappropriately low and complicate the profound electrolyte deficiencies resulting from chronic diarrhoea. Management of these deficiencies needs to be more aggressive than is currently practised and consideration should be given to a formal clinical trial of mineralocorticoid replacement in these severely ill patients. If the inappropriately low aldosterone reflects a general adrenal failure, it may explain a considerable proportion of the high mortality seen both before and after initiation of anti-retroviral therapy.

MarkerSet: a marker selection tool based on markers location and informativity in experimental designs

Olivier Demeure and Frederic Lecerf — 2008-03-26

Background: The recent sequencing of full genomes has led to the availability of many SNP markers which are very useful for the mapping of complex traits. In livestock production, there are still no commercial arrays and many studies use home-made sets of SNPs. Thus, the current methodologies for SNP genotyping are still expensive and it is a crucial step to select the SNPs to use. Indeed, the main factors affecting the power of the linkage analyses are the density of the genetic map and the heterozygosity of markers in tested animal parents. FindingsThis is why we have developed a PERL program selecting a defined number of markers based on their locations on the genome and their informativity in specific experimental designs. As an option, different experimental designs can be combined in order to select the best possible common marker set. The program has been tested using different conditions of marker informativity and density with both real and simulated datasets. The results show the efficiency of our program to select the most informative markers even if there is a wide range of informativity for whole genome scan mapping analyses. In case of combination of different experimental crosses, the multidesign mode can optimize the SNP markers selection. Conclusions: Written in PERL, it assures a maximum portability to other operating systems (OS) and the source code availability for user modifications. Except for the simulation mode which could be time consuming, MarkerSet can compute results in a very short time.

Temporal epidemiology of microfilaraemia among migrant workers entering Kuwait

Saeed Akhtar, Hameed GH. H. Mohammad and Edwin Michael — 2008-03-19

Background: There is paucity of published data on the microfilarial infection among migrants from endemic countries entering Kuwait. The primary objectives of this study were to use routine health surveillance data to i) to estimate the prevalence of microfilarial infection in migrant workers to Kuwait and ii) to determine the occurrence of any time trends in the proportions of microfilaria positives among these workers over the recent past. Findings Methods: Monthly aggregates of microfilaria thick slide test results obtained from routine health examinations of migrant workers conducted at the Ports and Border Division of Ministry of Health, Kuwait between January 1, 1992 and December 31, 2006, were available for trend analysis of these time series data. Results: During the study period, the prevalence (per 100,000) of microfilaraemia positive migrant workers was 48 (1169/2449360). A third-order polynomial regression model of monthly proportions of microfilaraemic workers revealed a significant initial increase ( = 2.976 (+/- 0.157); P < 0.001), followed by a significant declining trend ( = - 0.0358 (+/- 0.002); P < 0.001) and a slight but significant upward trend ( = 0.0001 (+/- < 0.001); P < 0.001) towards the end of study period. Conclusion: This study showed a recent steady but apparently asymptotic decline in the prevalence of microfilarial infection in migrant workers from filarial endemic countries to Kuwait. This may reflect either changes in the socio-economic backgrounds of recent migrants or the effects of recently initiated mass drug administration programs carried out in the endemic countries of origin.

Prevalence of malnutrition among settled pastoral Fulani children in Southwest Nigeria

Uwem F Ekpo, Akin M Omotayo and Morenike A Dipeolu — 2008-03-12

Background: There is a dearth of information on the health of pastoral Fulani children living in south western Nigeria. These are fully settled pastoralists whose economy are centred on cattle and farming. In other to monitor and plan appropriate nutritional intervention for their children, a cross-sectional study was carried out to determine the prevalence of malnutrition of pastoral Fulani children. FindingsFulani's children aged 6 months to 15 years, living in 61 settlements in Kwara, Ogun and Oyo States in South western Nigeria participated in the study. Heights and weights of 164 girls and 167 boys were measured. Their anthropometric indices, height-for-age (HA), weight-for-height (WH), and weight-for-age (WA) Z-scores determined. The prevalence of stunting (HAZ < -2), wasting (WHZ < -2) and underweight (WAZ < -2) was 38.7%, 13.6%, and 38.7%, respectively when compared to the reference NCHS/WHO standard used for defining stunting, wasting and underweight. Boys were more malnourished than the girls, but this was not significant (stunting: chi square=0.36; df=1; P=0.54); (underweight: chi square=1.10; df=1; P=0.29); and (wasting: chi square=0.00; df=1; P=0.98) The mean of Z-scores of Height-for-age, Weight-for-age and Weight-for-height in children were -1.502, -1.634 and -0.931 respectively. The SD was 1.52, 1.09 and 1.20 respectively. Using WHO Malnutrition Classification systems, 38.7% of the children were found to be malnourished. Conclusion: These results indicate high prevalence of malnutrition among settled pastoral Fulani children, possibly due to changes food habits and lifestyle occasion by the transition from nomadic to sedentary living. We suggest the inclusion of Fulani's settlements in nutritional intervention for these areas.

Risk-communication capability for public health emergencies varies by community diversity.

2008-03-07

Background: Public health emergencies heighten several challenges in risk-communication: providing trustworthy sources of information, reaching marginalized populations, and minimizing fear and public confusion. In emergencies, however, information may not diffuse equally among all social groups, and gaps in knowledge may increase. Such knowledge gaps vary by social structure and the size, socioeconomic status, and diversity of the population. This study explores the relationship between risk-communication capabilities, as perceived by public officials participating in emergency tabletop exercises, and community size and diversity. FindingsFor each of the three communication functions tested, risk-communication capabilities are perceived to be greater in communities with fewer then 10% of the population speaking a language other than English at home, decreasing as the percentage grows to 20% (ANOVA P a 0.02). With respect to community size, however, we found an N-shaped relationship between perceived risk communication capabilities and population size. Capabilities are perceived highest in the largest communities and lowest in the smallest, but lower in communities with 20,000-49,999 inhabitants compared to those with 2,500-19,999. Conclusions: The results of this study suggest the need to factor population diversity into risk communication plans and the need for improved state or regional risk-communication capabilities, especially for communities with limited local capacity.