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1.  Comparison of RNA extraction methods from biofilm samples of Staphylococcus epidermidis 
BMC Research Notes  2011;4:572.
Microbial biofilms are communities of bacteria adhered to a surface and surrounded by an extracellular polymeric matrix. Biofilms have been associated with increased antibiotic resistance and tolerance to the immune system. Staphylococcus epidermidis is the major bacterial species found in biofilm-related infections on indwelling medical devices. Obtaining high quality mRNA from biofilms is crucial to validate the transcriptional measurements associated with the switching to the biofilm mode of growth. Therefore, we selected three commercially available RNA extraction kits with distinct characteristics, including those using silica membrane or organic extraction methods, and enzymatic or mechanical cell lysis, and evaluated the RNA quality obtained from two distinct S. epidermidis bacterial biofilms.
RNA extracted using the different kits was evaluated for quantity, purity, integrity, and functionally. All kits were able to extract intact and functional total RNA from the biofilms generated from each S. epidermidis strain. The results demonstrated that the kit based on mechanical lysis and organic extraction (FastRNA® Pro Blue) was the only one that was able to isolate pure and large quantities of RNA. Normalized expression of the icaA virulence gene showed that RNA extracted with PureLink™ had a significant lower concentration of icaA mRNA transcripts than the other kits tested.
When working with complex samples, such as biofilms, that contain a high content extracellular polysaccharide and proteins, special care should be taken when selecting the appropriate RNA extraction system, in order to obtain accurate, reproducible, and biologically significant results. Among the RNA extraction kits tested, FastRNA® Pro Blue was the best option for both S. epidermidis biofilms used.
PMCID: PMC3260333  PMID: 22208502
2.  Distribution of allele frequencies at TTN g.231054C > T, RPL27A g.3109537C > T and AKIRIN2 c.*188G > A between Japanese Black and four other cattle breeds with differing historical selection for marbling 
BMC Research Notes  2011;4:10.
Marbling defined by the amount and distribution of intramuscular fat, so-called Shimofuri, is an economically important trait of beef cattle in Japan. Our previous study detected 3 single nucleotide polymorphisms (SNPs), g.231054C > T, g.3109537C > T and c.*188G > A, respectively, in the 5' flanking region of the titin (TTN), the 5' flanking region of the ribosomal protein L27a (RPL27A) and the 3' untranslated region of the akirin 2 genes (AKIRIN2), which have been considered as positional functional candidates for the genes responsible for marbling, and showed association of these SNPs with marbling in Japanese Black beef cattle. In the present study, we investigated the allele frequency distribution of the 3 SNPs among the 5 cattle breeds, Japanese Black, Japanese Brown, Japanese Shorthorn, Holstein and Brown Swiss breeds.
We genotyped the TTN g.231054C > T, RPL27A g.3109537C > T and AKIRIN2 c.*188G > A SNPs by polymerase chain reaction-restriction fragment length polymorphism method, using 101 sires and 1,705 paternal half sib progeny steers from 8 sires for Japanese Black, 86 sires and 27 paternal half sib progeny steers from 3 sires for Japanese Brown, 79 sires and 264 paternal half sib progeny steers from 14 sires for Japanese Shorthorn, 119 unrelated cows for Holstein, and 118 unrelated cows for Brown Swiss breeds. As compared to the frequencies of the g.231054C > T T, g.3109537C > T T and c.*188G > A A alleles, associated with high marbling, in Japanese Black breed that has been subjected to a strong selection for high marbling, those in the breeds, Japanese Shorthorn, Holstein and Brown Swiss breeds, that have not been selected for high marbling were null or lower. The Japanese Brown breed selected slightly for high marbling showed lower frequency than Japanese Black breed in the g.3109537C > T T allele, whereas no differences were detected between the 2 breeds in the frequencies of the g.231054C > T T and c.*188G > A A alleles.
Based on this finding, we hypothesized that the pressure of the strong selection for high marbling in Japanese Black breed has increased the frequencies of the T, T and A alleles at the TTN g.231054C > T, RPL27A g.3109537C > T and AKIRIN2 c.*188G > A SNPs, respectively. This study, together with the previous association studies, suggested that the 3 SNPs may be useful for effective marker-assisted selection to increase the levels of marbling.
PMCID: PMC3032689  PMID: 21251324
3.  Differences in dietary pattern between obese and eutrophic children 
BMC Research Notes  2011;4:567.
Excessive consumption of energy is a decisive factor of obesity, but a simple quantitative assessment of consumption between obese and eutrophic individuals not always explains the problem, raising questions about the importance of the qualitative aspects of food. Therefore, the purpose of this study was to evaluate the differences in nutrient composition and meal patterns between eutrophic and obese schoolchildren.
The diet of 83 children (42 obese and 41 eutrophic), aged between 7 and 11 years of age, was assessed by two non-consecutive dietary recalls. After the software analysis of macro and micronutrients composition, the different types and amount of legumes, fruits and vegetables were analyzed to verify the dietary patterns.
No differences were verified in energy consumption between the groups (eutrophic = 1934.2 ± 672.7 kcal, obese = 1835.8 ± 621.2 kcal). In general, children showed consumption within the recommended ranges of carbohydrates, lipids and proteins. The average consumption of fiber was higher in the eutrophic group (20.7 g) when compared to the obese group (14.8 g). The dietary fiber was strongly correlated with the number of servings of beans (r = 0.77), when compared to fruits (r = 0.44) and leafy vegetables (r = 0.13). It was also observed that the higher the consumption of fiber and beans, the lower the proportion of dietary fat (r = -0.22) in the diet. Generally, there was a low consumption of fiber (20.7 g = eutrophic group/14.8 g = obese group), beans (1.1 portions in the eutrophic and obese groups), fruits (0.7 portions eutrophic group and 0.6 obese group) and vegetables (1.3 eutrophic group and 1.1 obese group).
It is concluded that the obesity was more related to a dietary pattern of low intake of dietary fiber than excessive energy consumption and macronutrients imbalance.
PMCID: PMC3339399  PMID: 22206728
4.  Real-time PCR quantification of the canine filaggrin orthologue in the skin of atopic and non-atopic dogs: a pilot study 
BMC Research Notes  2011;4:554.
Canine atopic dermatitis (AD) is a common inflammatory skin disease associated with defects in the epidermal barrier, particularly in West Highland white terriers (WHWTs). It shares many similarities with human AD, and so may be a useful animal model for this disease. Epidermal dysfunction in human AD can be caused by mutations in the gene encoding the epidermal protein filaggrin (FLG) and, in some atopic patients, be associated with altered FLG mRNA and protein expression in lesional and/or non-lesional skin. In experimental models of canine AD, mRNA expression of the orthologous canine filaggrin gene may be reduced in non-lesional skin compared with healthy controls. However, there is no published data on canine filaggrin mRNA expression in the skin of dogs with naturally-occurring AD. Hence, the aim of this pilot study was to develop a reverse transcriptase real-time PCR assay to compare filaggrin mRNA expression in the skin of atopic (n = 7) and non-atopic dogs (n = 5) from five breeds, including eight WHWTs.
Overall, filaggrin mRNA expression in non-lesional atopic skin was decreased compared to non-lesional non-atopic skin (two fold change); however this difference was only statistically significant in the subgroup of WHWTs (P = 0.03).
Although limited by the small sample size, these results indicate that, comparable to some cases of human AD, altered filaggrin mRNA expression may exist in the skin of some atopic dogs with naturally-occurring disease. Additional studies, including larger sample numbers, will be necessary to confirm this finding and to investigate whether mutations in the filaggrin gene exist and contribute to epidermal lesions of AD in dogs.
PMCID: PMC3339370  PMID: 22188733
5.  Effect of controlled and uncontrolled cooling rate on motility parameters of cryopreserved ram spermatozoa 
BMC Research Notes  2011;4:547.
Ram spermatozoa are sensitive to extreme changes in temperature during the freeze-thaw process. The degree of damage depends on a combined effect of various factors including freezing temperature. The aim of this study was to determine the effects of two cooling method (controlled-rate and uncontrolled-rate) on pre-freezing and post-thaw sperm motility parameters.
Ejaculates were collected using the artificial vagina from four Chal rams and three replicates of the ejaculates were diluted with a Tris-based extender and packed in 0.25 ml straws. Then, sample processed according to the two methods. Method 1: straws cooled from 37 to 5°C, at a liner rate of -0.3°C/min in a controlled-rate cooling machine (custom-built) and equilibrated at 5°C for 80 min, then the straws were frozen at rate of -0.3°C/min from 5°C to -10°C and -25°C/min from -10°C to -150°C and plunged into liquid nitrogen for storage. Method 2: straws were transferred to refrigerator and maintained at 5°C for 3 h, then the straws were frozen in liquid nitrogen vapor, 4 cm above the liquid nitrogen for 15 min and plunged into liquid nitrogen. Computer-assisted sperm motility analysis was used to analyze sperm motion characteristics.
Controlled rate of freezing (Method 1) significantly improve the pre-freezing and post-thaw total and progressive motility compared to uncontrolled rate (Method 2). In specific kinetic parameters, Method 1 gives significantly higher value for VSL and VCL in comparison with Method 2. There are no significant differences between the two methods for VAP and LIN. In conclusion, controlled rate of cooling conferred better cryopreserving ability to ram spermatozoa compared to uncontrolled rate of cooling prior to programmable freezing.
PMCID: PMC3349620  PMID: 22185483
6.  Clinical outcome of skin yaws lesions after treatment with benzathinebenzylpenicillin in a pygmy population in Lobaye, Central African Republic 
BMC Research Notes  2011;4:543.
Yaws is a bacterial skin and bone infectious disease caused by Treponema pallidum pertenue. It is endemic, particularly among pygmies in Central African Republic. To assess the clinical cure rate after treatment with benzathinepenicillin in this population, we conducted a cohort survey of 243 patients in the Lobaye region.
Findings and conclusion
The rate of healing of lesions after 5 months was 95.9%. This relatively satisfactory level of therapeutic response implies that yaws could be controlled in the Central African Republic. Thus, reinforcement of the management of new cases and of contacts is suggested.
PMCID: PMC3309964  PMID: 22171605
Yaws; Treatment; Central African Republic
7.  Heart rate variability analysis in sheep affected by transmissible spongiform encephalopathies 
BMC Research Notes  2011;4:539.
The function of the autonomic nervous system can be assessed by determining heart rate variability (HRV), which is impaired in some brainstem diseases in humans. Transmissible spongiform encephalopathies (TSEs) in sheep are diseases characterised by accumulation of disease-associated prion protein in the brainstem, including nuclei of the parasympathetic nervous system. This study was undertaken to assess whether analysis of HRV can be used as an aid in the diagnosis of TSEs in clinically affected, naturally or experimentally infected sheep.
When HRV indices were compared between 41 clinical TSE cases (18 sheep infected with scrapie and 23 sheep infected with bovine spongiform encephalopathy), 11 control sheep and six sheep reported as scrapie suspects or dosed with BSE brain homogenate, which were not confirmed as TSE cases by postmortem tests, no significant differences were found between the groups. Median heart rate was significantly different but only when sheep were grouped by gender: it was higher in female TSE cases than in control sheep and higher in female than castrated male ovine classical BSE cases.
HRV analysis was not useful as a diagnostic aid for TSEs of sheep.
PMCID: PMC3265556  PMID: 22168827
8.  Clustering of classical swine fever virus isolates by codon pair bias 
BMC Research Notes  2011;4:521.
The genetic code consists of non-random usage of synonymous codons for the same amino acids, termed codon bias or codon usage. Codon juxtaposition is also non-random, referred to as codon context bias or codon pair bias. The codon and codon pair bias vary among different organisms, as well as with viruses. Reasons for these differences are not completely understood. For classical swine fever virus (CSFV), it was suggested that the synonymous codon usage does not significantly influence virulence, but the relationship between variations in codon pair usage and CSFV virulence is unknown. Virulence can be related to the fitness of a virus: Differences in codon pair usage influence genome translation efficiency, which may in turn relate to the fitness of a virus. Accordingly, the potential of the codon pair bias for clustering CSFV isolates into classes of different virulence was investigated.
The complete genomic sequences encoding the viral polyprotein of 52 different CSFV isolates were analyzed. This included 49 sequences from the GenBank database (NCBI) and three newly sequenced genomes. The codon usage did not differ among isolates of different virulence or genotype. In contrast, a clustering of isolates based on their codon pair bias was observed, clearly discriminating highly virulent isolates and vaccine strains on one side from moderately virulent strains on the other side. However, phylogenetic trees based on the codon pair bias and on the primary nucleotide sequence resulted in a very similar genotype distribution.
Clustering of CSFV genomes based on their codon pair bias correlate with the genotype rather than with the virulence of the isolates.
PMCID: PMC3341591  PMID: 22126254
9.  Psychometric assessment of the Wagnild and Young's resilience scale in Kano, Nigeria 
BMC Research Notes  2011;4:509.
Resilience seemed to lie at the core of the recent promotion of positive mental health and wellbeing. This concept has been well studied in western countries and less in developing countries, particularly Nigeria. The aim of the study is therefore, to demonstrate the internal consistency and concurrent validity of the Resilience Scale (RS) and its 14-item short version (RS-14) in a Nigerian sample.
The RS, RS-14, the Hospital Anxiety Depression Scale (HADS) and two screening questions on experience of recent and upcoming distress were administered to 70 clinical students who consented to participate after a major professional examination. Internal consistency and convergent validity were assessed. The participants mean age was 22.50 years (SD = 0.60). The mean score of RS and RS-14 were 130.23 (SD = 17.08) and 74.17 (SD = 10.14) respectively. Cronbach's alpha coefficient for the RS was 0.87 and that of the RS-14 was 0.81. The mean RS score by gender was 132.04 (SD = 19.08) and 126.52 (SD = 11.50) for males and females respectively and the difference was significant (t = 2.50; p = 0.012). The correlation of RS with RS-14 (r = 0.97; p = 0.000), the HADS depression (r = -0.28; p = 0.017) and anxiety (r = -0.26; p = 0.028) subscales, were significant. The corresponding t-test values for the means of RS and RS-14 scores for both cases and non-cases as determined by HADS, were significant at p < 0.05 and p < 0.01 for the depression and anxiety subscales respectively. The difference between RS means of those who experienced distress (38/125.69) to those that did not (32/134.05) from the recent clinical examination was also significant (t = 2.01; p = 0.045).
The study confirms that the RS and RS-14 may be potentially useful instruments to measure resilience in Nigerians.
PMCID: PMC3261834  PMID: 22112503
10.  Studies of the transmissibility of the agent of bovine spongiform encephalopathy to the domestic chicken 
BMC Research Notes  2011;4:501.
Transmission of the prion disease bovine spongiform encephalopathy (BSE) occurred accidentally to cattle and several other mammalian species via feed supplemented with meat and bone meal contaminated with infected bovine tissue. Prior to United Kingdom controls in 1996 on the feeding of mammalian meat and bone meal to farmed animals, the domestic chicken was potentially exposed to feed contaminated with the causal agent of BSE. Although confirmed prion diseases are unrecorded in avian species a study was undertaken to transmit BSE to the domestic chicken by parenteral and oral inoculations. Transmissibility was assessed by clinical monitoring, histopathological examinations, detection of a putative disease form of an avian prion protein (PrP) in recipient tissues and by mouse bioassay of tissues. Occurrence of a progressive neurological syndrome in the primary transmission study was investigated by sub-passage experiments.
No clinical, pathological or bioassay evidence of transmission of BSE to the chicken was obtained in the primary or sub-passage experiments. Survival data showed no significant differences between control and treatment groups. Neurological signs observed, not previously described in the domestic chicken, were not associated with significant pathology. The diagnostic techniques applied failed to detect a disease associated form of PrP.
Important from a risk assessment perspective, the present study has established that the domestic chicken does not develop a prion disease after large parenteral exposures to the BSE agent or after oral exposures equivalent to previous exposures via commercial diets. Future investigations into the potential susceptibility of avian species to mammalian prion diseases require species-specific immunochemical techniques and more refined experimental models.
PMCID: PMC3341577  PMID: 22093239
11.  Allyl isothiocyanate induced stress response in Caenorhabditis elegans 
BMC Research Notes  2011;4:502.
Allyl isothiocyanate (AITC) from mustard is cytotoxic; however the mechanism of its toxicity is unknown. We examined the effects of AITC on heat shock protein (HSP) 70 expression in Caenorhabditis elegans. We also examined factors affecting the production of AITC from its precursor, sinigrin, a glucosinolate, in ground Brassica juncea cv. Vulcan seed as mustard has some potential as a biopesticide.
An assay to determine the concentration of AITC in ground mustard seed was improved to allow the measurement of AITC release in the first minutes after exposure of ground mustard seed to water. Using this assay, we determined that temperatures above 67°C decreased sinigrin conversion to AITC in hydrated ground B. juncea seed. A pH near 6.0 was found to be necessary for AITC release. RT-qPCR revealed no significant change in HSP70A mRNA expression at low concentrations of AITC (< 0.1 μM). However, treatment with higher concentrations (> 1.0 μM) resulted in a four- to five-fold increase in expression. A HSP70 ELISA showed that AITC toxicity in C. elegans was ameliorated by the presence of ground seed from low sinigrin B. juncea cv. Arrid.
• AITC induced toxicity in C. elegans, as measured by HSP70 expression.
• Conditions required for the conversion of sinigrin to AITC in ground B. juncea seed were determined.
• The use of C. elegans as a bioassay to test AITC or mustard biopesticide efficacy is discussed.
PMCID: PMC3471387  PMID: 22093285
Brassica; myrosinase; glucosinolate; HSP70; toxicity; ELISA
12.  Constitutive expression of a grapevine polygalacturonase-inhibiting protein affects gene expression and cell wall properties in uninfected tobacco 
BMC Research Notes  2011;4:493.
Polygalacturonase-inhibiting proteins (PGIPs) directly limit the effective ingress of fungal pathogens by inhibiting cell wall-degrading endopolygalacturonases (ePGs). Transgenic tobacco plants over-expressing grapevine (Vitis vinifera) Vvpgip1 have previously been shown to be resistant to Botrytis infection. In this study we characterized two of these PGIP over-expressing lines with known resistance phenotypes by gene expression and hormone profiling in the absence of pathogen infection.
Global gene expression was performed by a cross-species microarray approach using a potato cDNA microarray. The degree of potential cross-hybridization between probes was modeled by a novel computational workflow designed in-house. Probe annotations were updated by predicting probe-to-transcript hybridizations and combining information derived from other plant species. Comparing uninfected Vvpgip1-overexpressing lines to wild-type (WT), 318 probes showed significant change in expression. Functional groups of genes involved in metabolism and associated to the cell wall were identified and consequent cell wall analysis revealed increased lignin-levels in the transgenic lines, but no major differences in cell wall-derived polysaccharides. GO enrichment analysis also identified genes responsive to auxin, which was supported by elevated indole-acetic acid (IAA) levels in the transgenic lines. Finally, a down-regulation of xyloglucan endotransglycosylase/hydrolases (XTHs), which are important in cell wall remodeling, was linked to a decrease in total XTH activity.
This evaluation of PGIP over-expressing plants performed under pathogen-free conditions to exclude the classical PGIP-ePG inhibition interaction indicates additional roles for PGIPs beyond the inhibition of ePGs.
PMCID: PMC3339426  PMID: 22078230
13.  RNA isolation for transcriptomics of human and mouse small skin biopsies 
BMC Research Notes  2011;4:438.
Isolation of RNA from skin biopsies presents a challenge, due to the tough nature of skin tissue and a high presence of RNases. As we lacked the dedicated equipment, i.e. homogenizer or bead-beater, needed for the available RNA from skin isolation methods, we adapted and tested our zebrafish single-embryo RNA-isolation protocol for RNA isolation from skin punch biopsies.
We tested our new RNA-isolation protocol in two experiments: a large-scale study with 97 human skin samples, and a small study with 16 mouse skin samples. Human skin was sampled with 4.0 mm biopsy punches and for the mouse skin different punch diameter sizes were tested; 1.0, 1.5, 2.0, and 2.5 mm. The average RNA yield in human samples was 1.5 μg with an average RNA quality RIN value of 8.1. For the mouse biopsies, the average RNA yield was 2.4 μg with an average RIN value of 7.5. For 96% of the human biopsies and 100% of the mouse biopsies we obtained enough high-quality RNA. The RNA samples were successfully tested in a transcriptomics analysis using the Affymetrix and Roche NimbleGen platforms.
Using our new RNA-isolation protocol, we were able to consistently isolate high-quality RNA, which is apt for further transcriptomics analysis. Furthermore, this method is already useable on biopsy material obtained with a punch diameter as small as 1.5 mm.
PMCID: PMC3221605  PMID: 22023775
14.  Pyrosequencing assay for rapid identification of Mycobacterium tuberculosis complex species 
BMC Research Notes  2011;4:423.
Identification of the Mycobacterium tuberculosis complex organisms to the species level is important for diagnostic, therapeutic and epidemiologic perspectives. Indeed, isolates are routinely identified as belonging to the M. tuberculosis complex without further discrimination in agreement with the high genomic similarity of the M. tuberculosis complex members and the resulting complex available identification tools.
We herein develop a pyrosequencing assay analyzing polymorphisms within glpK, pykA and gyrB genes to identify members of the M. tuberculosis complex at the species level. The assay was evaluated with 22 M. tuberculosis, 21 M. bovis, 3 M. caprae, 3 M. microti, 2 M. bovis BCG, 2 M. pinnipedii, 1 M. canettii and 1 M. africanum type I isolates. The resulted pyrograms were consistent with conventional DNA sequencing data and successfully identified all isolates. Additionally, 127 clinical M. tuberculosis complex isolates were analyzed and were unambiguously identified as M. tuberculosis.
We proposed a pyrosequencing-based scheme for the rapid identification of M. tuberculosis complex isolates at the species level. The assay is robust, specific, rapid and can be easily introduced in the routine activity.
PMCID: PMC3214197  PMID: 22011383
Mycobacterium tuberculosis complex; pyrosequencing; identification
15.  Analytical variables influencing the performance of a miRNA based laboratory assay for prediction of relapse in stage I non-small cell lung cancer (NSCLC) 
BMC Research Notes  2011;4:424.
Laboratory assays are needed for early stage non-small lung cancer (NSCLC) that can link molecular and clinical heterogeneity to predict relapse after surgical resection. We technically validated two miRNA assays for prediction of relapse in NSCLC. Total RNA from seventy-five formalin-fixed and paraffin-embedded (FFPE) specimens was extracted, labeled and hybridized to Affymetrix miRNA arrays using different RNA input amounts, ATP-mix dilutions, array lots and RNA extraction- and labeling methods in a total of 166 hybridizations. Two combinations of RNA extraction- and labeling methods (assays I and II) were applied to a cohort of 68 early stage NSCLC patients.
RNA input amount and RNA extraction- and labeling methods affected signal intensity and the number of detected probes and probe sets, and caused large variation, whereas different ATP-mix dilutions and array lots did not. Leave-one-out accuracies for prediction of relapse were 63% and 73% for the two assays. Prognosticator calls ("no recurrence" or "recurrence") were consistent, independent on RNA amount, ATP-mix dilution, array lots and RNA extraction method. The calls were not robust to changes in labeling method.
In this study, we demonstrate that some analytical conditions such as RNA extraction- and labeling methods are important for the variation in assay performance whereas others are not. Thus, careful optimization that address all analytical steps and variables can improve the accuracy of prediction and facilitate the introduction of microRNA arrays in the clinic for prediction of relapse in stage I non-small cell lung cancer (NSCLC).
PMCID: PMC3221722  PMID: 22011393
18.  The RICORDO approach to semantic interoperability for biomedical data and models: strategy, standards and solutions 
BMC Research Notes  2011;4:313.
The practice and research of medicine generates considerable quantities of data and model resources (DMRs). Although in principle biomedical resources are re-usable, in practice few can currently be shared. In particular, the clinical communities in physiology and pharmacology research, as well as medical education, (i.e. PPME communities) are facing considerable operational and technical obstacles in sharing data and models.
We outline the efforts of the PPME communities to achieve automated semantic interoperability for clinical resource documentation in collaboration with the RICORDO project. Current community practices in resource documentation and knowledge management are overviewed. Furthermore, requirements and improvements sought by the PPME communities to current documentation practices are discussed. The RICORDO plan and effort in creating a representational framework and associated open software toolkit for the automated management of PPME metadata resources is also described.
RICORDO is providing the PPME community with tools to effect, share and reason over clinical resource annotations. This work is contributing to the semantic interoperability of DMRs through ontology-based annotation by (i) supporting more effective navigation and re-use of clinical DMRs, as well as (ii) sustaining interoperability operations based on the criterion of biological similarity. Operations facilitated by RICORDO will range from automated dataset matching to model merging and managing complex simulation workflows. In effect, RICORDO is contributing to community standards for resource sharing and interoperability.
PMCID: PMC3192696  PMID: 21878109
19.  Severe anaemia is associated with a higher risk for preeclampsia and poor perinatal outcomes in Kassala hospital, eastern Sudan 
BMC Research Notes  2011;4:311.
Anaemia during pregnancy is major health problem. There is conflicting literature regarding the association between anaemia and its severity and maternal and perinatal outcomes.
This is a retrospective case-control study conducted at Kassala hospital, eastern Sudan. Medical files of pregnant women with severe anaemia (haemoglobin (Hb) < 7 g/dl, n = 303) who delivered from January 2008 to December 2010 were reviewed. Socio-demographic and obstetric data were analysed and compared with a similar number of women with mild/moderate anaemia (Hb = 7-10.9 g/dl, n = 303) and with no anaemia (Hb > 11 g/dl, n = 303). Logistic regression analysis was performed separately for each of the outcome measures: preeclampsia, eclampsia, preterm birth, low birth weight (LBW) and stillbirth.
There were 9578 deliveries at Kassala hospital, 4012 (41.8%) women had anaemia and 303 (3.2%) had severe anaemia. The corrected risk for preeclampsia increased only in severe anaemia (OR = 3.6, 95% CI: 1.4-9.1, P = 0.007). Compared with women with no anaemia, the risk of LBW was 2.5 times higher in women with mild/moderate anaemia (95% CI: 1.1-5.7), and 8.0 times higher in women with severe anaemia (95% CI: 3.8-16.0). The risk of preterm delivery increased significantly with the severity of anaemia (OR = 3.2 for women with mild/moderate anaemia and OR = 6.6 for women with severe anaemia, compared with women with no anaemia). The corrected risk for stillbirth increased only in severe anaemia (OR = 4.3, 95% CI: 1.9-9.1, P < 0.001).
The greater the severity of the anaemia during pregnancy, the greater the risk of preeclampsia, preterm delivery, LBW and stillbirth. Preventive measures should be undertaken to decrease the prevalence of anaemia in pregnancy.
PMCID: PMC3224576  PMID: 21867566
20.  Simple parametric survival analysis with anonymized register data: A cohort study with truncated and interval censored event and censoring times 
BMC Research Notes  2011;4:308.
To preserve patient anonymity, health register data may be provided as binned data only. Here we consider as example, how to estimate mean survival time after a diagnosis of metastatic colorectal cancer from Norwegian register data on time to death or censoring binned into 30 day intervals. All events occurring in the first three months (90 days) after diagnosis were removed to achieve comparability with a clinical trial. The aim of the paper is to develop and implement a simple, and yet flexible method for analyzing such interval censored and truncated data.
Considering interval censoring a missing data problem, we implement a simple multiple imputation strategy that allows flexible sensitivity analyses with respect to the shape of the censoring distribution. To allow identification of appropriate parametric models, a χ2-goodness-of-fit test--also imputation based--is derived and supplemented with diagnostic plots. Uncertainty estimates for mean survival times are obtained via a simulation strategy. The validity and statistical efficiency of the proposed method for varying interval lengths is investigated in a simulation study and compared with simpler alternatives.
Mean survival times estimated from the register data ranged from 1.2 (SE = 0.09) to 3.2 (0.31) years depending on period of diagnosis and choice of parametric model. The shape of the censoring distribution within intervals did generally not influence results, whereas the choice of parametric model did, even when different models fit the data equally well. In simulation studies both simple midpoint imputation and multiple imputation yielded nearly unbiased analyses (relative biases of -0.6% to 9.4%) and confidence intervals with near-nominal coverage probabilities (93.4% to 95.7%) for censoring intervals shorter than six months. For 12 month censoring intervals, multiple imputation provided better protection against bias, and coverage probabilities closer to nominal values than simple midpoint imputation.
Binning of event and censoring times should be considered a viable strategy for anonymizing register data on survival times, as they may be readily analyzed with methods based on multiple imputation.
PMCID: PMC3748025  PMID: 21867515
21.  Lay beliefs of TB and TB/HIV co-infection in Addis Ababa, Ethiopia: a qualitative study 
BMC Research Notes  2011;4:277.
Knowledge about lay beliefs of etiology, transmission and treatment of TB, and lay perceptions of the relationship between TB and HIV is important for understanding patients' health seeking behavior and adherence to treatment. We conducted a study to explore lay beliefs about TB and TB/HIV co-infection in Addis Ababa, Ethiopia.
We conducted a qualitative study using in-depth interviews with 15 TB/HIV co-infected patients and 9 health professionals and focus group discussions with 14 co-infected patients in Addis-Ababa, Ethiopia. We found that a predominant lay belief was that TB was caused by exposure to cold. Excessive sun exposure, exposure to mud, smoking, alcohol, khat and inadequate food intake were also reported as causes for TB. Such beliefs initially led to self-treatment. The majority of patients were aware of an association between TB and HIV. Some reported that TB could transform into HIV, while others said that the body could be weakened by HIV and become more susceptible to illnesses such as TB. Some patients classified TB as either HIV-related or non-HIV-related, and weight loss was a hallmark for HIV-related TB. The majority of patients believed that people in the community knew that there was an association between TB and HIV, and some feared that this would predispose them to HIV-related stigma.
There is a need for culturally sensitive information and educational efforts to address misperceptions about TB and HIV. Health professionals should provide information about causes and treatment of TB and HIV to co-infected patients.
PMCID: PMC3161876  PMID: 21813004
22.  CANGS DB: a stand-alone web-based database tool for processing, managing and analyzing 454 data in biodiversity studies 
BMC Research Notes  2011;4:227.
Next generation sequencing (NGS) is widely used in metagenomic and transcriptomic analyses in biodiversity. The ease of data generation provided by NGS platforms has allowed researchers to perform these analyses on their particular study systems. In particular the 454 platform has become the preferred choice for PCR amplicon based biodiversity surveys because it generates the longest sequence reads. Nevertheless, the handling and organization of massive amounts of sequencing data poses a major problem for the research community, particularly when multiple researchers are involved in data acquisition and analysis. An integrated and user-friendly tool, which performs quality control, read trimming, PCR primer removal, and data organization is desperately needed, therefore, to make data interpretation fast and manageable.
We developed CANGS DB (Cleaning and Analyzing Next Generation Sequences DataBase) a flexible, stand alone and user-friendly integrated database tool. CANGS DB is specifically designed to organize and manage the massive amount of sequencing data arising from various NGS projects. CANGS DB also provides an intuitive user interface for sequence trimming and quality control, taxonomy analysis and rarefaction analysis. Our database tool can be easily adapted to handle multiple sequencing projects in parallel with different sample information, amplicon sizes, primer sequences, and quality thresholds, which makes this software especially useful for non-bioinformaticians. Furthermore, CANGS DB is especially suited for projects where multiple users need to access the data. CANGS DB is available at
CANGS DB provides a simple and user-friendly solution to process, store and analyze 454 sequencing data. Being a local database that is accessible through a user-friendly interface, CANGS DB provides the perfect tool for collaborative amplicon based biodiversity surveys without requiring prior bioinformatics skills.
PMCID: PMC3146855  PMID: 21718534
23.  Pathological features of Breast Cancer seen in Northwestern Tanzania: a nine years retrospective study 
BMC Research Notes  2011;4:214.
Breast cancer is more common in Western Countries compared to African populations. However in African population, it appears that the disease tends to be more aggressive and occurring at a relatively young age at the time of presentation. The aim of this study was to describe the trend of Breast Cancer in Northwestern Tanzania.
This was a retrospective study which involved all cases of breast cancer diagnosed histologically at Bugando Medical Center from 2002 to 2010. Histological results and slides were retrieved from the records in the Pathology department, clinical information and demographic data for patients were retrieved from surgical wards and department of medical records. Histology slides were re-evaluated for the histological type, grade (By modified Bloom-Richardson score), and presence of necrosis and skin involvement. Data was entered and analyzed by SPSS computer software version 15.
There were 328 patients histologically confirmed to have breast cancer, the mean age at diagnosis was 48.7 years (+/- 13.1). About half of the patients (52.4%) were below 46 years of age, and this group of patients had significantly higher tendency for lymph node metastasis (p = 0.012). The tumor size ranged from 1 cm to 18 cm in diameter with average (mean) of 5.5 cm (+/- 2.5), and median size of 6 cm. Size of the tumor (above 6 cm in diameter) and presence of necrosis within the tumor was significantly associated with high rate of lymph node metastasis (p = 0.000). Of all patients, 64% were at clinical stage III (specifically IIIB) and 70.4% had lymph node metastasis at the time of diagnosis. Only 4.3% of the patients were in clinical stage I at the time of diagnosis. Majority of the patients had invasive ductal carcinoma (91.5%) followed by mucinous carcinoma (5.2%), Invasive lobular carcinoma (3%) and in situ ductal carcinoma (0.3%). In all patients, 185 (56.4%) had tumor with histological grade 3.
Breast cancer in this region show a trend towards relative young age at diagnosis with advanced stage at diagnosis and high rate of lymph node metastasis. Poor Referral system, lack of screening programs and natural aggressive biological behavior of tumor may contribute to advanced disease at the time of diagnosis.
PMCID: PMC3155835  PMID: 21696617
Breast Cancer; North Western Tanzania
24.  Tattoo removal in the typical adolescent 
BMC Research Notes  2011;4:209.
Although popular tattoos are often regretted later on for different reasons. Nevertheless, tattoo removal is a complicated and costly procedure seldom providing satisfactory results. The aim of this study was to investigate the awareness of the implications of tattoo removal among a substantial sample of Italian secondary school adolescents.
Students were recruited by a stratified convenience sample and surveyed by a self administered questionnaire. Logistic regression analysis was performed, reporting adjusted Odds Ratios (OR), with 95% Confidence Interval (CI).
4,277 pupils returned a usable questionnaire. Piercings were more frequently undertaken than tattoos. Only 40% of the respondents were aware of the issues related to tattoo removal. Males and pupils with younger fathers were less likely to be aware, whereas students satisfied with their physical appearance and those with a positive attitude towards body art were more likely to be aware.
Male adolescents with younger fathers can be regarded as the ideal target of corporate health education programs driven by school counsellors and primary care physicians.
PMCID: PMC3145586  PMID: 21693015
survey; epidemiology; tattoo removal; piercing; secondary school adolescents; health education
25.  The importance of RT-qPCR primer design for the detection of siRNA-mediated mRNA silencing 
BMC Research Notes  2011;4:148.
The use of RNAi to analyse gene function in vitro is now widely applied in biological research. However, several difficulties are associated with its use in vivo, mainly relating to inefficient delivery and non-specific effects of short RNA duplexes in animal models. The latter can lead to false positive results when real-time RT-qPCR alone is used to measure target mRNA knockdown.
We observed that detection of an apparent siRNA-mediated knockdown in vivo was dependent on the primers used for real-time RT-qPCR measurement of the target mRNA. Two siRNAs specific for RRM1 with equivalent activity in vitro were administered to A549 xenografts via intratumoural injection. In each case, apparent knockdown of RRM1 mRNA was observed only when the primer pair used in RT-qPCR flanked the siRNA cleavage site. This false-positive result was found to result from co-purified siRNA interfering with both reverse transcription and qPCR.
Our data suggest that using primers flanking the siRNA-mediated cleavage site in RT-qPCR-based measurements of mRNA knockdown in vivo can lead to false positive results. This is particularly relevant where high concentrations of siRNA are introduced, particularly via intratumoural injection, as the siRNA may be co-purified with the RNA and interfere with downstream enzymatic steps. Based on these results, using primers flanking the siRNA target site should be avoided when measuring knockdown of target mRNA by real-time RT-qPCR.
PMCID: PMC3123217  PMID: 21612654

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