BAC libraries construction from the ancestral diploid genomes of the allotetraploid cultivated peanut

Background

Cultivated peanut, Arachis hypogaea is an allotetraploid of recent origin, with an AABB genome. In common with many other polyploids, it seems that a severe genetic bottle-neck was imposed at the species origin, via hybridisation of two wild species and spontaneous chromosome duplication. Therefore, the study of the genome of peanut is hampered both by the crop's low genetic diversity and its polyploidy. In contrast to cultivated peanut, most wild Arachis species are diploid with high genetic diversity. The study of diploid Arachis genomes is therefore attractive, both to simplify the construction of genetic and physical maps, and for the isolation and characterization of wild alleles. The most probable wild ancestors of cultivated peanut are A. duranensis and A. ipaënsis with genome types AA and BB respectively.

Results

We constructed and characterized two large-insert libraries in Bacterial Artificial Chromosome (BAC) vector, one for each of the diploid ancestral species. The libraries (AA and BB) are respectively c. 7.4 and c. 5.3 genome equivalents with low organelle contamination and average insert sizes of 110 and 100 kb. Both libraries were used for the isolation of clones containing genetically mapped legume anchor markers (single copy genes), and resistance gene analogues.

Conclusion

These diploid BAC libraries are important tools for the isolation of wild alleles conferring resistances to biotic stresses, comparisons of orthologous regions of the AA and BB genomes with each other and with other legume species, and will facilitate the construction of a physical map.

     

Local spread of classical swine fever upon virus introduction into The Netherlands: Mapping of areas at high risk

Background

In the recent past, the introduction of Classical Swine Fever Virus (CSFV) followed by between-herd spread has given rise to a number of large epidemics in The Netherlands and Belgium. Both these countries are pork-exporting countries. Particularly important in these epidemics has been the occurrence of substantial "neighborhood transmission" from herd to herd in the presence of base-line control measures prescribed by EU legislation. Here we propose a calculation procedure to map out "high-risk areas" for local between-herd spread of CSFV as a tool to support decision making on prevention and control of CSFV outbreaks. In this procedure the identification of such areas is based on an estimated inter-herd distance dependent probability of neighborhood transmission or "local transmission". Using this distance-dependent probability, we derive a threshold value for the local density of herds. In areas with local herd density above threshold, local transmission alone can already lead to epidemic spread, whereas in below-threshold areas this is not the case. The first type of area is termed 'high-risk' for spread of CSFV, while the latter type is termed 'low-risk'.

Results

As we show for the case of The Netherlands, once the distance-dependent probability of local transmission has been estimated from CSFV outbreak data, it is possible to produce a map of the country in which areas of high-risk herds and of low-risk herds are identified. We made these maps even more informative by estimating border zones between the two types of areas. In these border zones the risk of local transmission of infection to a nearby high-risk area exceeds a certain level.

Conclusion

The risk maps provide an easily understandable visualization of the spatial heterogeneities in transmission risk. They serve as a tool for area-specific designs of control strategies, and possibly also for spatial planning of areas where livestock farming is allowed. Similar risk maps can in principle be constructed for other highly-transmissible livestock infections that spread via neighborhood transmission.

     

Insights from Streptococcus pneumoniae glucose kinase structural model

Streptococcus pneumonia is the common cause of sepsis and meningitis. Emergence of multiple antibiotic resistant strains in the community‐acquired bacterium is catastrophic. Glucose kinase (GLK) is a regulatory enzyme capable of adding phosphate group to glucose in the first step of streptomycin biosynthesis. The activity of glucose kinase was regulated by the Carbon Catabolite Repression (CCR) system. Therefore, it is important to establish the structure‐function relation of GLK in S. pneumoniae. However, a solved structure for S. pneumoniae GLK is not available at the protein data bank (PDB). Therefore, we created a model of GLK from S. pnemoniae using the X‐ray structure of Glk from E. faecalis as template with MODELLER (a comparative modeling program). The model was validated using protein structure checking tools such as PROCHECK, WHAT IF and ProSA for reliability. The active site amino acid Asp114 in the template is retained in S. pneumoniae GLK model (Asp115). Solvent accessible surface area (ASA) analysis of the GLK model showed that known key residues playing important role in active site for ligand binding and metal ion binding are buried and hence not accessible to solvent. The information thus discussed provides insight to the molecular understanding of glucose kinase in S. pneumoniae.     

Neurotoxic,cytotoxic, apoptotic and antiproliferative effects of some marine algae extracts on the NA2B cell line

Oxidative stress contributes to cancer pathologies and to apoptosis. Marine algae exhibit cytotoxic, antiproliferative and apoptotic effects; their metabolites have been used to treat many types of cancer. We investigated in culture extracts of Petalonia fascia, Jania longifurca and Halimeda tuna to determine their effects on mouse neuroblastoma cell line, NA2B. NA2B cells were treated with algae extracts, and the survival and proliferation of NA2B cells were assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The effects of algae extracts on oxidative stress in NA2B cells also were investigated using nitric oxide synthase (NOS) immunocytochemistry and apoptosis was assessed using terminal deoxynucleotidyl transferase dUTP nick end labeling. We observed significant neurite inhibition with moderate damage by the neurotoxicity-screening test (NST) at IC₅₀ dilutions of the extracts. MTT demonstrated that J. longifurca extracts were more toxic than P. fascia and H. tuna extracts. We found an increase of endothelial and inducible NOS immunostaining for oxidative stress and TUNEL analysis revealed increased apoptosis after application of extract. Our findings suggest that the algae we tested may have potential use for treatment of cancer.     

Differential responsiveness to immunoablative therapy in refractory rheumatoid arthritis is associated with level and avidity of anti-cyclic citrullinated protein autoantibodies: a case study

In order to identify pathogenic correlates of refractory rheumatoid arthritis (RA), antibodies against anti-cyclic citrullinated protein (ACPAs) were investigated in RA patients in whom the dysregulated immune system had been ablated by high-dose chemotherapy (HDC) and autologous haematopoietic stem cell transplantation (HSCT). Six patients with refractory RA were extensively characterized in terms of levels of total immunoglobulins, RA-specific autoantibodies (ACPAs and rheumatoid factor) and antibodies against rubella, tetanus toxoid (TT) and phosphorylcholine before and after HDC plus HSCT. Additionally, the avidity of ACPAs was measured before and after treatment and compared with the avidity of TT antibodies following repeated immunizations. Synovial biopsies were obtained by arthroscopy before HDC plus HSCT, and analyzed by immunohistochemistry. In the three patients with clinically long-lasting responses to HDC plus HSCT (median 423 days), significant reductions in ACPA-IgG levels after therapy were observed (median level dropped from 215 to 34 arbitrary units/ml; P = 0.05). In contrast, stable ACPA-IgG levels were observed in three patients who relapsed shortly after HDC plus HSCT (median of 67 days). Clinical responders had ACPA-IgG of lower avidity (r = 0.75; P = 0.08) and higher degree of inflammation histologically (r = 0.73; P = 0.09). Relapse (after 38 to 530 days) in all patients was preceded by rising levels of low avidity ACPA-IgG (after 30 to 388 days), in contrast to the stable titres of high avidity TT antibodies. In conclusion, humoral autoimmune responses were differentially modulated by immunoablative therapy in patients with synovial inflammation and low avidity ACPA-IgG autoantibodies as compared with patients with high levels of high avidity ACPA-IgG. The distinct clinical disease course after immunoablative therapy based on levels and avidity of ACPA-IgG indicates that refractory RA is not a single disease entity.     

Reference genes for quantitative reverse transcription-polymerase chain reaction expression studies in wild and cultivated peanut

Background

Molecular genetic studies on rare tumour entities, such as bone tumours, often require the use of decalcified, formalin-fixed, paraffin-embedded tissue (dFFPE) samples. Regardless of which decalcification procedure is used, this introduces a vast breakdown of DNA that precludes the possibility of further molecular genetic testing. We set out to establish a robust protocol that would overcome these intrinsic hurdles for bone tumour research.

Findings

The goal of our study was to establish a protocol, using a modified DNA isolation procedure and quality controls, to select decalcified samples suitable for array-CGH testing. Archival paraffin blocks were obtained from 9 different pathology departments throughout Europe, using different fixation, embedding and decalcification procedures, in order to preclude a bias for certain lab protocols. Isolated DNA samples were subjected to direct chemical labelling and enzymatic labelling systems and were hybridised on a high resolution oligonucleotide chip containing 44,000 reporter elements. Genomic alterations (gains and losses) were readily detected in most of the samples analysed. For example, both homozygous deletions of 0.6 Mb and high level of amplifications of 0.7 Mb were identified.

Conclusions

We established a robust protocol for molecular genetic testing of dFFPE derived DNA, irrespective of fixation, decalcification or sample type used. This approach may greatly facilitate further genetic testing on rare tumour entities where archival decalcified, formalin fixed samples are the only source.     

Breeding value prediction for production traits in layer chickens using pedigree or genomic relationships in a reduced animal model

Background

Genomic selection involves breeding value estimation of selection candidates based on high-density SNP genotypes. To quantify the potential benefit of genomic selection, accuracies of estimated breeding values (EBV) obtained with different methods using pedigree or high-density SNP genotypes were evaluated and compared in a commercial layer chicken breeding line.

Methods

The following traits were analyzed: egg production, egg weight, egg color, shell strength, age at sexual maturity, body weight, albumen height, and yolk weight. Predictions appropriate for early or late selection were compared. A total of 2,708 birds were genotyped for 23,356 segregating SNP, including 1,563 females with records. Phenotypes on relatives without genotypes were incorporated in the analysis (in total 13,049 production records).The data were analyzed with a Reduced Animal Model using a relationship matrix based on pedigree data or on marker genotypes and with a Bayesian method using model averaging. Using a validation set that consisted of individuals from the generation following training, these methods were compared by correlating EBV with phenotypes corrected for fixed effects, selecting the top 30 individuals based on EBV and evaluating their mean phenotype, and by regressing phenotypes on EBV.

Results

Using high-density SNP genotypes increased accuracies of EBV up to two-fold for selection at an early age and by up to 88% for selection at a later age. Accuracy increases at an early age can be mostly attributed to improved estimates of parental EBV for shell quality and egg production, while for other egg quality traits it is mostly due to improved estimates of Mendelian sampling effects. A relatively small number of markers was sufficient to explain most of the genetic variation for egg weight and body weight.