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1.  Update on comparative genome mapping between Malus and Pyrus 
BMC Research Notes  2009;2:182.
Comparative genome mapping determines the linkage between homologous genes of related taxa. It has already been used in plants to characterize agronomically important genes in lesser studied species, using information from better studied species. In the Maloideae sub-family, which includes fruit species such as apple, pear, loquat and quince, genome co-linearity has been suggested between the genera Malus and Pyrus; however map comparisons are incomplete to date.
Genetic maps for the apple rootstocks 'Malling 9' ('M.9') (Malus × domestica) and 'Robusta 5' ('R5') (Malus × robusta), and pear cultivars 'Bartlett' and 'La France' (Pyrus communis) were constructed using Simple Sequence Repeat (SSR) markers developed from both species, including a new set of 73 pear Expressed Sequence Tag (EST) SSR markers. Integrated genetic maps for apple and pear were then constructed using 87 and 131 SSR markers in common, respectively.
The genetic maps were aligned using 102 markers in common, including 64 pear SSR markers and 38 apple SSR markers. Of these 102 markers, 90 anchor markers showed complete co-linearity between the two genomes.
Our alignment of the genetic maps of two Malus cultivars of differing species origin with two Pyrus communis cultivars confirms the ready transferability of SSR markers from one genus to the other and supports a high level of co-linearity within the sub-family Maloideae between the genomes of Malus and Pyrus.
PMCID: PMC2749866  PMID: 19747407
2.  Accelerating epistasis analysis in human genetics with consumer graphics hardware 
BMC Research Notes  2009;2:149.
Human geneticists are now capable of measuring more than one million DNA sequence variations from across the human genome. The new challenge is to develop computationally feasible methods capable of analyzing these data for associations with common human disease, particularly in the context of epistasis. Epistasis describes the situation where multiple genes interact in a complex non-linear manner to determine an individual's disease risk and is thought to be ubiquitous for common diseases. Multifactor Dimensionality Reduction (MDR) is an algorithm capable of detecting epistasis. An exhaustive analysis with MDR is often computationally expensive, particularly for high order interactions. This challenge has previously been met with parallel computation and expensive hardware. The option we examine here exploits commodity hardware designed for computer graphics. In modern computers Graphics Processing Units (GPUs) have more memory bandwidth and computational capability than Central Processing Units (CPUs) and are well suited to this problem. Advances in the video game industry have led to an economy of scale creating a situation where these powerful components are readily available at very low cost. Here we implement and evaluate the performance of the MDR algorithm on GPUs. Of primary interest are the time required for an epistasis analysis and the price to performance ratio of available solutions.
We found that using MDR on GPUs consistently increased performance per machine over both a feature rich Java software package and a C++ cluster implementation. The performance of a GPU workstation running a GPU implementation reduces computation time by a factor of 160 compared to an 8-core workstation running the Java implementation on CPUs. This GPU workstation performs similarly to 150 cores running an optimized C++ implementation on a Beowulf cluster. Furthermore this GPU system provides extremely cost effective performance while leaving the CPU available for other tasks. The GPU workstation containing three GPUs costs $2000 while obtaining similar performance on a Beowulf cluster requires 150 CPU cores which, including the added infrastructure and support cost of the cluster system, cost approximately $82,500.
Graphics hardware based computing provides a cost effective means to perform genetic analysis of epistasis using MDR on large datasets without the infrastructure of a computing cluster.
PMCID: PMC2732631  PMID: 19630950
3.  Ocular changes in primary hypothyroidism 
BMC Research Notes  2009;2:266.
To determine the ocular changes related to hypothyrodism in newly diagnosed patients without orbitopathy.
Thirty-three patients diagnosed to have primary overt hypothyroidism were enrolled in the study. All subjects were assigned to underwent central corneal thickness (CCT), anterior chamber volume, depth and angle measurements with the Scheimpflug camera (Pentacam, Oculus) and cup to disc ratio (C/D), mean retinal thickness and mean retinal nerve fiber layer (RNFL) thickness measurements with optical coherence tomography (OCT) in addition to ophthalmological examination preceeding the replacement therapy and at the 1st, 3rd and 6th months of treatment.
The mean age of the patients included in the study were 40.58 ± 1.32 years. The thyroid hormone levels return to normal levels in all patients during the follow-up period, however the mean intraocular pressure (IOP) revealed no significant change. The mean CCT was 538.05 ± 3.85 μ initially and demonstrated no statistically significant change as the anterior chamber volume, depth and angle measurements did. The mean C/D ratio was 0.29 ± 0.03 and the mean retinal thickness was 255.83 ± 19.49 μ initially and the treatment did not give rise to any significant change. The mean RNFL thickness was also stable during the control visits, so no statistically significant change was encountered.
Neither hypothyroidism, nor its replacement therapy gave rise to any change of IOP, CCT, anterior chamber parameters, RNFL, retinal thickness and C/D ratio.
PMCID: PMC2813236  PMID: 20040111
4.  Amount of external CME in groups of specialties: a nation-wide survey among Finnish doctors 
BMC Research Notes  2009;2:265.
Continuing medical education (CME) is an integral part of continuing professional development and a prerequisite for good quality in health care. We aimed to describe and analyse the number of days spent in formal CME outside the workplace by specialty among Finnish doctors of working age.
The number of days in formal CME outside the workplace in 2005 reported by specialists was obtained from an annual postal survey, conducted by the Finnish Medical Association in March 2006, of all working-age doctors. Those who had attained their specialist degree before 2005 were included in the study. The 49 specialties were re-categorised into 15 groups. The mean reported number of days and 95% confidence intervals were calculated. Differences were analysed by Poisson regression adjusted for relevant covariates.
The response rate to the question about CME was 70.2% (7,374) among specialists. The median age (interquartile range) of the respondents was 49 years (from 44 to 55 years), and 51.7% (3,810) were female. The mean reported number of days in CME was 8.8 (95% CI 8.7-9.0). Neurologists and surgery specialists participated in CME the most frequently (10.3 and 10.4 days) and ophthalmologists the least (7.6 days). In comparison with anaesthesiology and intensive care specialists, most specialists reported having significantly more formal CME, and no group reported having less.
Significant variation was observed, and we therefore suggest studies seeking to account for this variation.
The results have originally been published in Finnish in the Finnish Medical Journal.
PMCID: PMC2804579  PMID: 20038290
5.  Validation of internal reference genes for quantitative real-time PCR in a non-model organism, the yellow-necked mouse, Apodemus flavicollis 
BMC Research Notes  2009;2:264.
Reference genes are used as internal standards to normalize mRNA abundance in quantitative real-time PCR and thereby allow a direct comparison between samples. So far most of these expression studies used human or classical laboratory model species whereas studies on non-model organism under in-situ conditions are quite rare. However, only studies in free-ranging populations can reveal the effects of natural selection on the expression levels of functional important genes. In order to test the feasibility of gene expression studies in wildlife samples we transferred and validated potential reference genes that were developed for lab mice (Mus musculus) to samples of wild yellow-necked mice, Apodemus flavicollis. The stability and suitability of eight potential reference genes was accessed by the programs BestKeeper, NormFinder and geNorm.
Although the three programs used different algorithms the ranking order of reference genes was significantly concordant and geNorm differed in only one, NormFinder in two positions compared to BestKeeper. The genes ordered by their mean rank from the most to the least stable gene were: Rps18, Sdha, Canx, Actg1, Pgk1, Ubc, Rpl13a and Actb. Analyses of the normalization factor revealed best results when the five most stable genes were included for normalization.
We established a SYBR green qPCR assay for liver samples of wild A. flavicollis and conclude that five genes should be used for appropriate normalization. Our study provides the basis to investigate differential expression of genes under selection under natural selection conditions in liver samples of A. flavicollis. This approach might also be applicable to other non-model organisms.
PMCID: PMC2804578  PMID: 20030847
6.  Students' and teachers' perceptions of clinical assessment program: A qualitative study in a PBL curriculum 
BMC Research Notes  2009;2:263.
"Examinations drive students' learning." This statement refers to what is assumed to be one of the strongest relationships in education. We explored in this research how and why students differ in their approaches to learning, how assessment affects deep learning, and which barriers stand in the way of good assessment and learning in the clinical years of a Problem Based Learning (PBL) graduate entry medical curriculum.
Method: We conducted a qualitative, phenomenological study using semi-structured group interviews with students and semi-structured individual interviews with teachers and students. The transcripts were analyzed, and themes were identified.
Setting: The research was conducted at the King Saud bin Abdulaziz University for Health Sciences, College of Medicine, Riyadh, Saudi Arabia from November 2007 to March 2008.
Results: A total of 28 students participated in 7 focus group interviews. Semi-structured individual interviews were conducted with 12 teachers and 12 students. The analysis yielded four themes: summative assessment, formative assessment, continuous assessment of clinical attachments, and learning objectives.
The results of this study confirm that assessment affects students' perceptions of learning and how they learn. These effects are not uniformly positive. According to the students, the predominantly summative assessment program offers little inducement to engage in deep learning. They express a clear preference for formative assessment, which may foster a deeper approach to learning. Efforts to achieve more clinically relevant assessment with adequate balance between the various types of assessment are required. Research is needed to decide this balance.
PMCID: PMC2804577  PMID: 20030842
7.  Sphingobacterium respiratory tract infection in patients with cystic fibrosis 
BMC Research Notes  2009;2:262.
Bacteria that belong to the genus Sphingobacterium are Gram-negative, non-fermentative bacilli, ubiquitous in nature and rarely involved in human infections. The aims of this study were to evaluate the epidemiology of infection by Sphingobacterium in a cohort of patients affected by Cystic Fibrosis (CF), the antibiotic susceptibility and the DNA fingerprinting of the isolated strains and to analyze some clinical outcomes of the infected patients.
Between January 2006 and June 2008, patients (n = 332) attending the Regional CF Unit in Naples, Italy, were enrolled.
Sputum samples were processed for microscopic, cultural, phenotypic identification and antibiotic susceptibility testing. DNA fingerprinting was performed by pulsed-field gel electrophoresis (PFGE). A total of 21 strains of Sphingobacterium were isolated from 7 patients (13 of S. spiritovorum, 8 of S. multivorum). S. multivorum isolates were more resistant than those of S. spiritovorum. PFGE profiles were in general heterogeneous, which suggested independent circulation.
This is the first Italian report about respiratory tract infections by Sphingobacterium in CF patients. In our cohort, these infections were not associated with a deterioration of pulmonary function during the follow-up period. Although the exact role of this microorganism in CF lung disease is unknown and the number of infected patients was small, this study could represent an important starting-point for understanding the epidemiology and the possible pathogenic role of Sphingobacterium in CF patients.
PMCID: PMC2805677  PMID: 20030840
8.  Characterization of some Brucella species from Zimbabwe by biochemical profiling and AMOS-PCR 
BMC Research Notes  2009;2:261.
Bovine brucellosis caused by Brucella abortus is endemic in most large commercial and smallholder cattle farms of Zimbabwe, while brucellosis in other domestic animals is rare. The diagnosis of brucellosis is mainly accomplished using serological tests. However, some Brucella spp. have been isolated from clinical cases in the field and kept in culture collection but their biochemical profiles were not documented. We report biochemical profiling and AMOS-PCR characterization of some of these field isolates of Brucella originating from both commercial and smallholder cattle farming sectors of Zimbabwe.
Fourteen isolates of Brucella from culture collection were typed using biochemical profiles, agglutination by monospecific antisera, susceptibility to Brucella-specific bacteriophages and by AMOS-PCR that amplifies species- specific IS711. The results of the biochemical profiles for B. abortus biovar 1 (11 isolates) and biovar 2 (2 isolates) were consistent with those of reference strains. A single isolate from a goat originating from a smallholder mixed animal farm was identified as B. melitensis biovar 1. The AMOS-PCR produced DNA products of sizes 498 bp and 731 bp for B. abortus (biovar 1 and 2) and B. melitensis biovar 1, respectively.
We concluded that the biochemical profiles and AMOS-PCR characterization were consistent with their respective species and biovars. B. abortus biovar 1 is likely to be the predominant cause of brucellosis in both commercial and smallholder cattle farms in Zimbabwe.
PMCID: PMC2807434  PMID: 20028545
9.  Evaluation of self-reported ethnicity in a case-control population: the stroke prevention in young women study 
BMC Research Notes  2009;2:260.
Population-based association studies are used to identify common susceptibility variants for complex genetic traits. These studies are susceptible to confounding from unknown population substructure. Here we apply a model-based clustering approach to our case-control study of stroke among young women to examine if self-reported ethnicity can serve as a proxy for genetic ancestry.
A population-based case-control study of stroke among women aged 15-49 identified 361 cases of first ischemic stroke and 401 age-comparable control subjects. Thirty single nucleotide polymorphisms (SNPs) throughout the genome unrelated to stroke risk and with established ancestry-based allele frequency differences were genotyped in all participants. The Structure program was used to iteratively evaluate for K = 1 to 5 potential genetic-based subpopulations. Evaluating the population as a whole, the Structure output plateaued at K = 2 clusters. 98% of self-reported Caucasians had an estimated probability ≥50% of belonging to Cluster 1, while 94% of self-reported African-Americans had an estimated probability ≥50% of belonging to Cluster 2. Stratifying the participants by self-reported ethnicity and repeating the analyses revealed the presence of two clusters among Caucasians, suggesting that potential substructure may exist.
Among our combined sample of African-American and Caucasian participants there is no large unknown subpopulation and self-reported ethnicity can serve as a proxy for genetic ancestry. Ethnicity-specific analyses indicate that population substructure may exist among the Caucasian participants indicating that further studies are warranted.
PMCID: PMC2801514  PMID: 20021678
10.  Suppression of MMP activity in bovine cartilage explants cultures has little if any effect on the release of aggrecanase-derived aggrecan fragments 
BMC Research Notes  2009;2:259.
Progressive loss of articular cartilage is a central hallmark in many joint disease, however, the relative importance of individual proteolytic pathways leading to cartilage erosion is at present unknown. We therefore investigated the time-dependant release ex vivo of MMP- and aggrecanase-derived fragments of aggrecan and type II collagen into the supernatant of bovine cartilage explants cultures using neo-epitope specific immunoassays, and to associate the release of these fragments with the activity of proteolytic enzymes using inhibitors.
Bovine cartilage explants were cultured in the presence or absence of the catabolic cytokines oncostatin M (OSM) and tumor necrosis factor alpha (TNFα). In parallel, explants were co-cultured with protease inhibitors such as GM6001, TIMP1, TIMP2 and TIMP3. Fragments released into the supernatant were determined using a range of neo-epitope specific immunoassays; (1) sandwich 342FFGVG-G2 ELISA, (2) competition NITEGE373ELISA (3) sandwich G1-NITEGE373 ELISA (4) competition 374ARGSV ELISA, and (5) sandwich 374ARGSV-G2 ELISA all detecting aggrecan fragments, and (6) sandwich CTX-II ELISA, detecting C-telopeptides of type II collagen. We found that (1) aggrecanase-derived aggrecan fragments are released in the early (day 2-7) and mid phase (day 9-14) into the supernatant from bovine explants cultures stimulated with catabolic cytokines, (2) the release of NITEGE373 neo-epitopes are delayed compared to the corresponding 374ARGSV fragments, (3) the MMP inhibitor GM6001 did not reduce the release of aggrecanase-derived fragment, but induced a further delay in the release of these fragments, and finally (4) the MMP-derived aggrecan and type II collagen fragments were released in the late phase (day 16-21) only.
Our data support the model, that aggrecanases and MMPs act independently in the processing of the aggrecan molecules, and furthermore that suppression of MMP-activity had little if any effect on the quantity of aggrecanase-derived fragments released from explants cultures.
PMCID: PMC2803187  PMID: 20021645
11.  TILLING in the two-rowed barley cultivar 'Barke' reveals preferred sites of functional diversity in the gene HvHox1 
BMC Research Notes  2009;2:258.
The economic importance of cereals such as barley, and the demand for improved yield and quality require a better understanding of the genetic components that modulate biologically and commercially relevant traits. While Arabidopsis thaliana is the premiere model plant system, the spectrum of its traits cannot address all of the fundamental questions of crop plant development. Unlike Arabidopsis, barley is both a crop and a model system for scientific research, and it is increasingly being used for genetic and molecular investigations into the conserved biological processes of cereals. A common challenge in genetic studies in plants with large genomes arises from the very time-consuming work of associating mutant phenotypes with gene sequence information, especially if insertion mutagenesis is not routine, as in barley. Reverse genetics based on chemical mutagenesis represents the best solution to this obstacle.
In barley, we generated a new TILLING (Targeting Local Lesions IN Genomes) resource comprising 10,279 M2 mutants in the two-rowed malting cultivar 'Barke,' which has been used in the generation of other genomic resources in barley (~150,000 ESTs, DH mapping population). The value of this new resource was tested using selected candidate genes. An average frequency of approximately one mutation per 0.5 Mb was determined by screening ten fragments of six different genes. The ethyl methanesulphonate (EMS)mutagenesis efficiency was studied by recording and relating the mutagenesis-dependent effects found in the three mutant generations (M1-M3). A detailed analysis was performed for the homeodomain-leucine-zipper (HD-ZIP) gene HvHox1. Thirty-one mutations were identified by screening a 1,270-bp fragment in 7,348 M2 lines. Three of the newly identified mutants exhibited either a six-rowed or an intermedium-spike phenotype, and one mutant displayed a significantly altered spikelet morphology compared to that of the 'Barke' wild type. Our results indicate a bias in the frequency of independent functional mutations at specific base pair (bp) positions within the gene HvHox1.
A new TILLING population was developed as a resource for high-throughput gene discovery in an alternative barley germplasm. Pilot screening demonstrated a similar or even slightly higher mutation frequency when compared to previously published barley TILLING populations that should allow for the identification of diverse allelic variation. Partial phenotypic evaluation of the M2 and M3 generations has revealed the presence of a wide spectrum of morphological diversity that highlights the great potential of this resource for use in forward genetic screens. Altogether, our study shows the efficiency of screening and the applicability of the new TILLING population for genetic studies in the barley crop model system.
PMCID: PMC2803498  PMID: 20017921
12.  Impact of genome assembly status on ChIP-Seq and ChIP-PET data mapping 
BMC Research Notes  2009;2:257.
ChIP-Seq and ChIP-PET can potentially be used with any genome for genome wide profiling of protein-DNA interaction sites. Unfortunately, it is probable that most genome assemblies will never reach the quality of the human genome assembly. Therefore, it remains to be determined whether ChIP-Seq and ChIP-PET are practicable with genome sequences other than a few (e.g. human and mouse).
Here, we used in silico simulations to assess the impact of completeness or fragmentation of genome assemblies on ChIP-Seq and ChIP-PET data mapping.
Most currently published genome assemblies are suitable for mapping the short sequence tags produced by ChIP-Seq or ChIP-PET.
PMCID: PMC2804576  PMID: 20015379
13.  Identification of horse chestnut coat color genotype using SNaPshot® 
BMC Research Notes  2009;2:255.
The Cantabrian Coast horse breeds of the Iberian Peninsula have mainly black or bay colored coats, but alleles responsible for a chestnut coat color run in these breeds and occasionally, chestnut horses are born. Chestnut coat color is caused by two recessive alleles, e and ea, of the melanocortin-1 receptor gene, whereas the presence of the dominant, wild-type E allele produces black or bay coat horses. Because black or bay colored coats are considered as the purebred phenotype for most of the breeds from this region, it is important to have a fast and reliable method to detect alleles causing chestnut coat color in horses.
In order to assess coat color genotype in reproductive animals with a view to avoiding those bearing chestnut alleles, we have developed a reliable, fast and cost-effective screening device which involves Single Nucleotide Polymorphism (SNP) detection based on SNaPshot® (Applied Biosystems) methodology. We have applied this method to four native breeds from the Iberian Cantabrian Coast: Pottoka and Jaca Navarra pony breeds, in which only black or bay coats are acceptable, and Euskal Herriko Mendiko Zaldia and Burguete heavy breeds, in which chestnut coats are acceptable. The frequency of the chestnut alleles ranged between f = 0.156-0.322 in pony breeds and between f = 0.604-0.716 in heavy breeds.
This study demonstrates the usefulness of the DNA methodology reported herein as a device for identifying chestnut alleles; the methodology constitutes a valuable tool for breeders to decrease the incidence of chestnut animals among Cantabrian Coast pony breeds.
PMCID: PMC2805676  PMID: 20015355
14.  Inferring Drosophila gap gene regulatory network: pattern analysis of simulated gene expression profiles and stability analysis 
BMC Research Notes  2009;2:256.
Inference of gene regulatory networks (GRNs) requires accurate data, a method to simulate the expression patterns and an efficient optimization algorithm to estimate the unknown parameters. Using this approach it is possible to obtain alternative circuits without making any a priori assumptions about the interactions, which all simulate the observed patterns. It is important to analyze the properties of the circuits.
We have analyzed the simulated gene expression patterns of previously obtained circuits that describe gap gene dynamics during early Drosophila melanogaster embryogenesis. Using hierarchical clustering we show that amplitude variation and defects observed in the simulated gene expression patterns are linked to similar circuits, which can be grouped. Furthermore, analysis of the long-term dynamics revealed four main dynamical attractors comprising stable patterns and oscillatory patterns. In addition, we also performed a correlation analysis on the parameters showing an intricate correlation pattern.
The analysis demonstrates that the obtained gap gene circuits are not unique showing variable long-term dynamics and highly correlating scattered parameters. Furthermore, although the model can simulate the pattern up to gastrulation and confirms several of the known regulatory interactions, it does not reproduce the transient expression of all gap genes as observed experimentally. We suggest that the shortcomings of the model may be caused by overfitting, incomplete model description and/or missing data.
PMCID: PMC2808311  PMID: 20015372
15.  Demographic characteristics of patients using a fully integrated psychosocial support service for cancer patients 
BMC Research Notes  2009;2:253.
Psychosocial support services are an important component of modern cancer treatment. A major challenge for all psychosocial services is the achievement of equity of use. Previous studies in the UK have found that women of higher socio-economic status with breast cancer were over-represented amongst those accessing support services. People with other cancer diagnoses, those from socio-economically deprived areas, and men, were under-represented.
The Oncology Health Service, Kingston Upon Hull, UK, delivers fully integrated psychosocial support and interventions. To assess equity of access in this service, a cross-sectional study of all patients with cancer accessing the service during a 5 day period was carried out. One hundred and forty-five patients attended. Forty four percent were male, and the types of cancer were broadly in the proportions expected on the basis of population prevalence (breast cancer 22%, colorectal cancer 21%, lung cancer 16%). Sixty six percent came from the three most deprived quintiles of the Townsend deprivation Index.
The fully integrated Oncology Health Service in Hull is accessed by a more diverse range of patients than previously reported for other services, and is an example of a model of service by which socially equitable use of psychosocial support in the National Health Service might be achieved.
PMCID: PMC2799437  PMID: 20003474
16.  Informed consent from patients participating in medical education: a survey from a university hospital in Jamaica 
BMC Research Notes  2009;2:252.
Medical students at the University of the West Indies receive clinical training by passing through a series of hospital rotations at the University Hospital of the West Indies (UHWI). Many of these patients are unaware that medical students may be involved in their care. We performed this study to determine patient awareness and their willingness to participate in research and teaching activities.
All consecutive patients admitted to the UHWI between May 1, 2006 and May 29, 2006 who required elective or emergency surgical procedures were prospectively identified These patients were interviewed using a standardised pre-tested questionnaire about their knowledge and willingness to have medical students participate in the delivery of their hospital care. Data was analyzed using SPSS Version 12.0.
There were 83 (39.5%) males and 127 (60.5%) females interviewed. The patients were unaware of the grade of the medical professional performing their interview/examination at admission in 157 (74.8%) cases or the grade of medical professional performing their operations in 101 (48.1%) cases.
Only 14 (6.7%) patients were specifically asked to allow medical students to be present during their clinical evaluation and care. When specifically asked, 1 patient declined. Had they been asked, 196 (93.3%) patients would have voluntarily allowed medical student involvement.
Only 90 (42.9%) patients were made aware that they were admitted to an academic centre with research interests. Only 6 (6.7%) patients declined. Had they been asked, 84 (93.3%) patients would be willing to participate in teaching or research projects.
As medical educators, we are responsible to adhere to ethical and legal guidelines when we interact with patients. It is apparent that there is urgent need for policy development at the UWI to guide clinicians and students on their interactions with patients.
PMCID: PMC2803186  PMID: 20003471
17.  Evidence of the adaptive evolution of immune genes in chicken 
BMC Research Notes  2009;2:254.
The basis for understanding the characteristics of gene functional categories in chicken has been enhanced by the ongoing sequencing of the zebra finch genome, the second bird species to be extensively sequenced. This sequence provides an avian context for examining how variation in chicken has evolved since its divergence from its common ancestor with zebra finch as well as well as a calibrating point for studying intraspecific diversity within chicken. Immune genes have been subject to many selective processes during their evolutionary history: this gene class was investigated here in a set of orthologous chicken and zebra finch genes with functions assigned from the human ortholog. Tests demonstrated that nonsynonymous sites at immune genes were highly conserved both in chicken and on the avian lineage. McDonald-Kreitman tests provided evidence of adaptive evolution and a higher rate of selection on fixation of nonsynonymous substitutions at immune genes compared to that at non-immune genes. Further analyses showed that GC content was much higher in chicken than in zebra finch genes, and was significantly elevated in both species' immune genes. Pathogen challenges are likely to have driven the selective forces that have shaped variation at chicken immune genes, and continue to restrict diversity in this functional class.
PMCID: PMC2804575  PMID: 20003477
18.  Crystallographic structure of ubiquitin in complex with cadmium ions 
BMC Research Notes  2009;2:251.
Ubiquitination plays a critical role in regulating many cellular processes, from DNA repair and gene transcription to cell cycle and apoptosis. It is catalyzed by a specific enzymatic cascade ultimately leading to the conjugation of ubiquitin to lysine residues of the target protein that can be the ubiquitin molecule itself and to the formation of poly-ubiquitin chains.
We present the crystal structure at 3.0 Å resolution of bovine ubiquitin crystallized in presence of cadmium ions. Two molecules of ubiquitin are present in the asymmetric unit. Interestingly this non-covalent dimeric arrangement brings Lys-6 and Lys-63 of each crystallographically-independent monomer in close contact with the C-terminal ends of the other monomer. Residues Leu-8, Ile-44 and Val-70 that form a hydrophobic patch at the surface of the Ub monomer are trapped at the dimer interface.
The structural basis for signalling by poly-Ub chains relies on a visualization of conformations of alternatively linked poly-Ub chains. This arrangement of ubiquitin could illustrate how linkages involving Lys-6 or Lys-63 of ubiquitin are produced in the cell. It also details how ubiquitin molecules can specifically chelate cadmium ions.
PMCID: PMC2804574  PMID: 20003470
19.  Dual agarose magnetic (DAM) ChIP 
BMC Research Notes  2009;2:250.
Chromatin immunoprecipitation (ChIP) has become a very popular technique to study epigenetic regulation because it can be used to identify proteins and protein modifications present at specific locations in chromatin. While techniques have been developed to investigate epigenetic modifications present in chromatin during a specific biological function such as transcription, they depend upon the ability of the ChIP to analyze two epitopes on the same chromatin and are generally time consuming, difficult to perform, and not very sensitive. The Dual Agarose Magnetic (DAM) ChIP procedure described here is designed to address these shortcomings.
Protein A agarose and protein G magnetic beads bound with different IgGs have been combined in a single Chromatin Immunoprecipitation (ChIP) assay to analyze for the presence of two epitopes on the same chromatin at the same time. This procedure has been used with non-immune rabbit IgG bound to either the agarose or beads in order to include an internal negative control for non-specific binding of chromatin. The procedure has also been used with various antibodies including those targeting RNA Polymerase II and replication protein A 70 to determine whether specific forms of modified histones are present in either transcribing or replicating forms of SV40 minichromosomes respectively.
The DAM ChIP procedure is a rapid, simple, and sensitive technique to characterize two epitopes located in the same chromatin. It should be particularly useful for the rapid screening of epigenetic modifications present in biologically active chromatin.
PMCID: PMC2799436  PMID: 20003449
20.  Application of hybridization control probe to increase accuracy on ligation detection or minisequencing diagnostic microarrays 
BMC Research Notes  2009;2:249.
Nucleic acid detection based on ligation reaction or single nucleotide extension of ssDNA probes followed by tag microarray hybridization provides an accurate and sensitive detection tool for various diagnostic purposes. Since microarray quality is crucial for reliable detection, these methods can benefit from correcting for microarray artefacts using specifically adapted techniques.
Here we demonstrate the application of a per-spot hybridization control oligonucleotide probe and a novel way of computing normalization for tag array data. The method takes into account the absolute value of the detection probe signal and the variability in the control probe signal to significantly alleviate problems caused by artefacts and noise on low quality microarrays.
Diagnostic microarray platforms require experimental and computational tools to enable efficient correction of array artefacts. The techniques presented here improve the signal to noise ratio and help in determining true positives with better statistical significance and in allowing the use of arrays with poor quality that would otherwise be discarded.
PMCID: PMC2799435  PMID: 20003444
21.  Cross-species amplification of 36 cyprinid microsatellite loci in Phoxinus phoxinus (L.) and Scardinius erythrophthalmus (L.) 
BMC Research Notes  2009;2:248.
To conduct phylogeographic or population genetic studies, an adequate number of DNA markers for the focal species are required. Due to severe unavailability of genotype markers of any kind for the species Eurasian minnow (Phoxinus phoxinus L.) and rudd (Scardinius erythrophthalmus L.), we set out to attempt cross-amplification of a set of microsatellite loci from related species.
We tested 36 cyprinid microsatellite loci for cross-species amplification in minnow and rudd. Fifteen species-locus combinations produced amplifications in minnow, seven being polymorphic, while 18 combinations amplified in rudd, nine of these being polymorphic.
The positive cross-species amplifications present potential contributions to the establishment of genetic marker sets for population genetics studies of the two focal species.
PMCID: PMC2801513  PMID: 20003440
22.  Presentation and outcome in cancer patients with extensive spread to the brain 
BMC Research Notes  2009;2:247.
Controversy exists around the preferred management of patients with brain metastases and limited survival expectation, e.g. because of extensive brain involvement. Few studies have focused on this particular group of patients.
A group of 24 patients with a large number of brain metastases, defined as 10 or more on computed tomography scans, who were managed with palliative whole-brain radiotherapy (WBRT), typically 30 Gy in 10 fractions, were analyzed. The median number of lesions was 14. The patient characteristics were comparable to those of studies in the general population with brain metastases, except for the fact that all patients had active sites of extracranial disease. Clinical benefit, imaging response and overall survival were lower than expected. Median survival, for example was 2 months. Trends towards better survival were found in patients with brain metastases detected at first cancer diagnosis (synchronous manifestation, treatment naïve) and those with better prognostic features according to the graded prognostic assessment (GPA) score.
The benefit of WBRT did not meet the expectations, suggesting that consideration should be given to best supportive care including corticosteroid administration, especially if a patient belongs to the lowest GPA class.
PMCID: PMC2797814  PMID: 20003374
23.  Selection of reference genes for quantitative real-time PCR in equine in vivo and fresh and frozen-thawed in vitro blastocysts 
BMC Research Notes  2009;2:246.
Application of reverse transcription quantitative real-time polymerase chain reaction is very well suited to reveal differences in gene expression between in vivo and in vitro produced embryos. Ultimately, this may lead to optimized equine assisted reproductive techniques. However, for a correct interpretation of the real-time PCR results, all data must be normalized, which is most reliably achieved by calculating the geometric mean of the most stable reference genes. In this study a set of reliable reference genes was identified for equine in vivo and fresh and frozen-thawed in vitro embryos.
The expression stability of 8 candidate reference genes (ACTB, GAPDH, H2A/I, HPRT1, RPL32, SDHA, TUBA4A, UBC) was determined in 3 populations of equine blastocysts (fresh in vivo, fresh and frozen-thawed in vitro embryos). Application of geNorm indicated UBC, GAPDH, ACTB and HPRT1 as the most stable genes in the in vivo embryos and UBC, RPL32, GAPDH and ACTB in both in vitro populations. When in vivo and in vitro embryos were combined, UBC, ACTB, RPL32 and GAPDH were found to be the most stable. SDHA and H2A/I appeared to be highly regulated.
Based on these results, the geometric mean of UBC, ACTB, RPL32 and GAPDH is to be recommended for accurate normalization of quantitative real-time PCR data in equine in vivo and in vitro produced blastocysts.
PMCID: PMC2797813  PMID: 20003356
24.  Optimized PCR with sequence specific primers (PCR-SSP) for fast and efficient determination of Interleukin-6 Promoter -597/-572/-174Haplotypes 
BMC Research Notes  2009;2:245.
Interleukin-6 (IL-6) promoter polymorphisms at positions -597(G→A), -572(G→C) and -174(G→C) were shown to have a clinical impact on different major diseases. At present PCR-SSP protocols for IL-6 -597/-572/-174haplotyping are elaborate and require large amounts of genomic DNA.
We describe an improved typing technique requiring a decreased number of PCR-reactions and a reduced PCR-runtime due to optimized PCR-conditions.
This enables a fast and efficient determination of IL-6 -597/-572/-174haplotypes in clinical diagnosis and further evaluation of IL-6 promoter polymorphisms in larger patient cohorts.
PMCID: PMC2797812  PMID: 20003261
25.  Comparison of the eSwab collection and transportation system to an amies gel transystem for Gram stain of clinical specimens 
BMC Research Notes  2009;2:244.
The first step in routine microbiology laboratory procedures is the collection and safe transportation of swab samples. This can be accomplished using ESwab Collection and Transport System (Copan Italia, Brescia -Italy). The aim of the present study was to compare the results of microscopic examination of gram stain smears prepared directly from clinical specimens, collected and transported in the ESwab, with those obtained using Amies Agar gel Transystem without charcoal (Copan).
Specimens were collected from 80 patients (32 vaginal swabs, 27 cervical swabs, 11 urethral swabs and 10 wound swabs). Two swabs were in random order collected from each patient, one using the conventional Amies gel Transystem, the other using ESwab. One slide was prepared for each specimen using the conventional swab and two sets of slides were prepared from the specimens collected with the ESwab: one using 100 μl and one using 50 μl of the Amies medium. All slides were gram stained using an automated Gram stainer. Microscopic examination of 240 slides (80 with conventional and 160 with ESwab) showed that the quality of smear preparation from the ESwab system, allowed for easier identification of human cells and identification of greater number of microorganisms. Microscopic examination of additional slides prepared from ESwab at 24 or 72 hours after initial collection were equivalent to those prepared when received in the laboratory within 2 hours of collection.
Microscopic examination performed using ESwab, especially when preparing the slides with 100 μl, shows superior results to those obtained using the Amies gel Transystem.
PMCID: PMC2797518  PMID: 20003247

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