Genetic Mapping of Prince Rupprecht's Larch (Larix principis-rupprechtii Mayr) by Specific-Locus Amplified Fragment Sequencing.

A high-density genetic linkage map is essential for plant genetics and genomics research. However, due to the deficiency of genomic data and high-quality molecular markers, no genetic map has been published for Prince Rupprecht's larch (Larix principis-rupprechtii Mayr), a conifer species with high ecological and commercial value in northern China. In this study, 145 F1 progeny individuals from an intraspecific cross between two elite clones of L. principis-rupprechtii and their parents were employed to construct the first genetic map in this important tree species using specific-locus amplified fragment sequencing (SLAF-seq). After preprocessing, the procedure yielded 300.20 Gb of raw data containing 1501.22 M pair-end reads. A total of 324,352 SNP markers were detected and 122,785 of them were polymorphic, with a polymorphism rate of 37.86%. Ultimately, 6099 SNPs were organized into a genetic map containing 12 linkage groups, consistent with the haploid chromosome number of larch and most other species in the Pinaceae family. The linkage map spanned 2415.58 cM and covered 99.6% of the L. principis-rupprechtii genome with an average of 0.4 cM between adjacent markers. To the best of our knowledge, this map is the first reference map for L. principis-rupprechtii, as well as the densest one obtained in larch species thus far. The genome-wide SNPs and the high-resolution genetic map will provide a foundation for future quantitative trait loci mapping, map-based cloning, marker-assisted selection, comparative genomics, and genome sequence assembly for larch trees.

MTHFR C677T polymorphism analysis: A simple, effective restriction enzyme-based method improving previous protocols.

BACKGROUND: 5,10-Methylentetrahydrofolate reductase (MTHFR) C677T polymorphism is one of the most studied genetic variations in the human genome. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) is one of the most used techniques to characterize the point mutations in genomic sequences because of its suitability and low cost. The most widely used method for the MTHFR C677T polymorphism characterization was developed by Frosst et al. (1995) but appears to have some technical limitations. The aim of this study was to propose a novel PCR-RFLP method for the detection of this polymorphism. METHODS: In order to retrieve all published articles possibly describing any PCR-RFLP methods useful to analyze MTHFR C677T polymorphism, we performed systematic queries on PubMed, using a combination of Boolean operators (AND/OR) and MeSH terms. Amplify software was used in order to design a new primer pair following the optimal standard criteria. Primer-BLAST software was used to check primer pair's biological specificity. RESULTS: The analysis of previous literature showed that PCR-RFLP method remains the most used technique. None of the 108 primer pairs described was ideal with regard to main accepted primer pair biochemical technical parameters. The new primer pair amplifies a DNA-fragment of 513 base pair (bp) that, in the presence of the polymorphism, is cut by Hinf I enzyme in two pieces of 146 bp and 367 bp and clearly visible on 2% agarose gel. The level of expertise and the materials required are minimal and the protocol takes one day to carry out. CONCLUSION: Our original PCR-RFLP strategy, specifically designed to make the analysis optimal with respect to PCR primers and gel analysis, fits the ideal criteria compared to the widely used strategy by Frosst et al (1995) as well as any other PCR-RFLP strategies proposed for MTHFR C677T polymorphism genotyping to date.

Association of IL-8 and eNOS polymorphisms with clinical outcomes in bevacizumab-treated breast cancer patients: an exploratory analysis

Background. The role of bevacizumab in metastatic breast cancer is controversial. Identification of predictive biomarkers could help to select patients who really benefit from it. We evaluated the association of angiogenesis-related gene polymorphisms with the treatment outcome of bevacizumab in metastatic breast cancer patients. Patients and methods. eNOS-786T/C and -894G/T, IL-8-251T/A genomic polymorphisms were assessed in 31 metastatic breast cancer patients treated with bevacizumab plus chemotherapy in the first-line setting. Testing for association between each polymorphism and treatment outcome was performed. Results. Patients with IL-8 251 AA genotype showed a significantly lower progression-free survival in each combination comparison: "TT" vs "AA" (13 vs 8 months; p = 0.008); TT vs TA vs AA (13 vs 11 vs 8 months; p = 0.02); TT vs TA +AA (13 vs 11 months; p = 0.01); TT + TA vs AA (12 vs 8 months; p = 0.01) and a lower overall survival when compared with TT +TA genotype (26 vs 51 months, p = 0.04). Patients carrying eNOS 894 TT genotype showed a statistically significant lower progression-free survival than patients with GG genotype (11.5 vs 26.5 months; p = 0.04) with no differences in the overall survival. No association with response rate was found with any of the polymorphisms analyzed. Conclusion. These findings suggest that IL-8 251T/A and eNOS-894 G/T polymorphisms might have a role in predicting treatment outcome of bevacizumab in metastatic breast cancer. Our results are hypothesis generating and need to be confirmed in larger clinical trials (AU)

No disponible

Association between CC motif chemokine ligand 5 (CCL5) polymorphisms and asthma risk: an updated meta-analysis

Background: Findings regarding the associations between the CC motif chemokine ligand 5 (CCL5) -403G/A and -28C/G polymorphisms and asthma risk are controversial. We performed a meta-analysis to determine whether CCL5 polymorphisms are associated with asthma risk. Methods: We searched the Pubmed, Embase, Chinese National Knowledge Infrastructure (CNKI), and Wanfang databases for studies published before June 2013. The strength of associations was calculated using ORs with 95% CIs. Results: Twenty case-control studies were included in this meta-analysis. We did not observe a significant association between the CCL5 -403G/A polymorphism and asthma risk (OR, 1.10; 95% CI, 0.93-1.30; P=.25). The CCL5 -28C/G polymorphism, however, was associated with a significantly elevated asthma risk (OR, 1.17; 95% CI, 1.02-1.33; P=.02). Subgroup analyses found that the CCL5 -28C/G polymorphism was significantly associated with asthma risk in Asians (OR, 1.16; 95% CI, 1.01-1.33; P=.04) and children (OR, 1.29; 95% CI, 1.03-1.63; P=.03). Conclusions: This meta-analysis suggests that the CCL5 -28C/G polymorphism is a risk factor for asthma (AU)

Introducción: Existen discrepancias entre la asociación del riesgo de padecer asma y diferentes polimorfismos del ligando de la quimiocina CC5 (CCL5). En este trabajo se ha realizado un meta-análisis para determinar si los polimorfismos CCL5-403G / A y CCL5-28C / G se asocian con el riesgo de asma bronquial. Métodos: Se utilizaron diversas bases de datos para realizar las búsquedas de estudios publicados antes de junio de 2013, incluyendo: PubMed, EMBASE, CNKI (Infraestructura del Conocimiento Nacional Chino) y Wanfang Se calcularon los odd ratios combinados (OR) con intervalos de confianza del 95% (IC). Resultados: Se incluyeron un total de 20 estudios de casos y controles. No se encontró una asociación significativa entre el polimorfismo CCL5-403G / A y el riesgo de asma (OR = 1,10, IC del 95%: 0,93 a 1,30, p = 0,25). Por el contrario, el polimorfismo CCL5-28C / G, se asoció con un riesgo significativamente elevado de asma (OR = 1,17, IC del 95%: 1,02 a 1,33, p = 0,02). En los análisis de subgrupos, el riesgo de asma fue significativamente mayor en los asiáticos con el polimorfismo CCL5-28C / G (OR = 1.16, 95% IC 1,01-1,33, P = 0,04) y los niños (OR = 1.29, 95% IC 1,03-1,63, P = 0,03). Conclusiones: Este meta-análisis sugiere que el polimorfismo CCL5-28C / G es un factor de riesgo significativo para padecer asma bronquial (AU)

Automated masking of AFLP markers improves reliability of phylogenetic analyses.

The amplified fragment length polymorphisms (AFLP) method has become an attractive tool in phylogenetics due to the ease with which large numbers of characters can be generated. In contrast to sequence-based phylogenetic approaches, AFLP data consist of anonymous multilocus markers. However, potential artificial amplifications or amplification failures of fragments contained in the AFLP data set will reduce AFLP reliability especially in phylogenetic inferences. In the present study, we introduce a new automated scoring approach, called "AMARE" (AFLP MAtrix REduction). The approach is based on replicates and makes marker selection dependent on marker reproducibility to control for scoring errors. To demonstrate the effectiveness of our approach we record error rate estimations, resolution scores, PCoA and stemminess calculations. As in general the true tree (i.e. the species phylogeny) is not known, we tested AMARE with empirical, already published AFLP data sets, and compared tree topologies of different AMARE generated character matrices to existing phylogenetic trees and/or other independent sources such as morphological and geographical data. It turns out that the selection of masked character matrices with highest resolution scores gave similar or even better phylogenetic results than the original AFLP data sets.

Demencia por cuerpos de Lewy / Dementia with Lewy bodies

La demencia por cuerpos de Lewy (DCL) es una enfermedad neurodegenerativa, que supone en torno al 10-25% de todas las demencias de la población general y es la segunda causa de demencia degenerativa, en el anciano, tras la enfermedad de Alzheimer. Se caracteriza clínicamente por deterioro cognitivo, con rasgos de demencia frontal, acompañado de: fluctuaciones, parkinsonismo y alucinaciones visuales. Su confirmación diagnóstica se realiza por anatomía patológica posmortem: presencia de abundantes cuerpos de Lewy (CL) en las neuronas de la corteza y en otras zonas cerebrales. Los CL son inclusiones intracitoplasmáticas eosinófilas, esféricas y están constituidos por más de 20 componentes proteicos. Se han definido, en el año 2005, unos criterios diagnósticos para facilitar su reconocimiento y poder etiquetar a los pacientes de posible o probable DCL. La causa de la DCL es desconocida; se supone que, al igual que en la enfermedad de Parkinson, influyen factores ambientales y genéticos. No hay ninguna prueba complementaria ni marcador biológico específico de esta enfermedad, pero la combinación de biomarcadores, los tests neuropsicológicos y las pruebas de neuroimagen son de especial ayuda en el diagnóstico. El tratamiento de la enfermedad es complejo; porque los fármacos que mejoran unos síntomas pueden empeorar otros. En cualquier caso solo hay posibilidad de tratamiento sintomático (AU)

Dementia with Lewy bodies (DLB) is a neurodegenerative disease, accounting for about 10-25% of all dementias in the general population and is the second most common type of degenerative dementia in elderly people after Alzheimer's disease. Clinically, it is characterized by cognitive impairment (with features of frontal dementia) accompanied by fluctuating cognition, parkinsonism and visual hallucinations. Diagnostic confirmation is made by pathologic autopsy: abundant presence of Lewy bodies (LB) in the neurons of the cortex and other areas in the brain. The LB are eosinophilic intracytoplasmic inclusions, spherical and consist of more than 20 protein components. Diagnostic criteria to facilitate recognition of patients with possible or probable DLB were defined in 2005. The etiology of DLB is unknown, although is assumed that, environmental factors and genetic influence, like in Parkinson's disease. There are not additional evidence or specific biological markers of this disease, but neuropsychological and neuroimaging tests are helpful in diagnosis. The treatment of the disease is difficult, there is no cure, only symptomatic. Some drugs may improve some symptoms but also can worse others (AU)

Examining the genetic variation of reference microbial cultures used within food and environmental laboratories using fluorescent amplified fragment length polymorphism analysis.

Fluorescent amplified fragment length polymorphism (FAFLP) analysis was applied to genetically fingerprint 'working culture control strains' used by accredited food microbiology laboratories. A working culture control strain is defined as a subculture from a strain initially obtained from an authenticated source [such as the National Collection of Type Cultures (NCTC)] that is maintained for use with routine testing within the laboratory. Working culture control strains from eight food examination laboratories, representing four bacterial species, were analysed by FAFLP; these were Salmonella Nottingham, Staphylococcus aureus, Listeria monocytogenes and Bacillus cereus. The resultant FAFLP profiles of the eight working culture control strains for each of these species were compared against the appropriate freeze-dried ampoules obtained directly from NCTC. FAFLP results demonstrated that within 50% of working cultures analysed, several laboratories were routinely using working cultures that were genetically different from the original reference NCTC strains. This study highlights the need for laboratories to review the protocols used to process and maintain control strains and working cultures, with a potential view to utilize single-use quality control materials.

Using large-scale epidemiological evidence to help evaluate biomarkers in cardiovascular disease

No disponible

No disponible

Terminal restriction fragment length measurement errors are affected mainly by fragment length, G+C nucleotide content and secondary structure melting point.

Several methods of molecular analysis of microbial diversity, including terminal restriction fragment length polymorphism (T-RFLP) analysis are based on measurement of the DNA fragment length. Significant variation between sequence-determined and measured length of restriction fragments (drift) has been observed, which can affect the efficiency of the identification of microorganisms in the analyzed communities. In the past, this variation has been attributed to varying fragment length and purine content. In this study, principal component analysis and multiple regression analysis were applied to find the contributions of those and several other fragment characteristics. We conclude that secondary structure melting point and G+C nucleotide content, besides the fragment length, contribute to the variation observed, whereas the contribution of purine content is less important. Incomplete denaturation of the sample at the start of electrophoretic separation of fragments has been excluded as a major cause of the variation observed. Our regression model explains the observed drift variation by approximately 56%, with standard deviation of the prediction equal to approximately 1.3 bp.

Selection criteria for scoring amplified fragment length polymorphisms (AFLPs) positively affect the reliability of population genetic parameter estimates.

A reliable data set is a fundamental prerequisite for consistent results and conclusions in population genetic studies. However, marker scoring of genetic fingerprints such as amplified fragment length polymorphisms (AFLPs) is a highly subjective procedure, inducing inconsistencies owing to personal or laboratory-specific criteria. We applied two alternative marker selection algorithms, the newly developed script scanAFLP and the recently published AFLPScore, to a large AFLP genome scan to test how population genetic parameters and error rates were affected. These results were confronted with replicated random selections of marker subsets. We show that the newly developed marker selection criteria reduced the mismatch error rate and had a notable influence on estimates of genetic diversity and differentiation. Both effects are likely to influence biological inference. For example, genetic diversity (HS) was 29% lower while genetic differentiation (FST) was 8% higher when applying scanAFLP compared with AFLPScore. Likewise, random selections of markers resulted in substantial deviations of population genetic parameters compared with the data sets including specific selection criteria. These randomly selected marker sets showed surprisingly low variance among replicates. We conclude that stringent marker selection and phenotype calling reduces noise in the data set while retaining patterns of population genetic structure.

Statistical assessment of variability of terminal restriction fragment length polymorphism analysis applied to complex microbial communities.

The variability of terminal restriction fragment polymorphism analysis applied to complex microbial communities was assessed statistically. Recent technological improvements were implemented in the successive steps of the procedure, resulting in a standardized procedure which provided a high level of reproducibility.

[Assessment of performance of "amplified Mycobacterium tuberculosis direct test" in pulmonary and extrapulmonary specimens]. / Akciger ve akciger disi orneklerde "amplifiye Mycobacterium tuberculosis direk test" in güvenilirliginin degerlendirilmesi.

Since rapid diagnosis is critical in control of tuberculosis, nucleic acid amplification techniques have been widely used. The purpose of the present study was to assess the performance of Amplified Mycobacterium tuberculosis Direct Test (Amplified MTD Test, Gen-Probe) for the diagnosis of pulmonary and extrapulmonary tuberculosis in our laboratory. A total of 267 specimens (170 pulmonary and 97 extrapulmonary) were tested in the Clinical Mycobacteriology Laboratory of Manisa (a province located in Aegean part of Turkey) University Hospital from September 2001 to March 2005. When Amplified MTD (AMTD) test results were compared to the culture results taken as the gold standard, the sensitivity, specificity, positive and negative predictive values (PPV, NPV) for pulmonary specimens were found to be 84%, 96%, 73%, and 98%, respectively. When AMTD test positive, culture negative discrepant results were evaluated against the clinical history of the patients, these rates were detected as; 88%, 100%, 100%, and 98%, respectively. For 97 extrapulmonary specimens, sensitivity, specificity, PPV and NPV of AMTD test were 60%, 100%, 100%, and 98%, respectively. In conclusion, the results of the AMTD assay were reliable for the rapid diagnosis of pulmonary tuberculosis; if the results were evaluated together with the clinical status of patients, the performance of the test would be increased. However, even though the culture positive extrapulmonary specimens were sparse in our study (5%), the sensitivity of the AMTD test in extrapulmonary specimens was found less than that in pulmonary specimens. Therefore it is thought that AMTD test results should be evaluated carefully for the diagnosis of extrapulmonary tuberculosis.