Enrichment of DNRA bacteria in a continuous culture

Denitrification and dissimilatory nitrate reduction to ammonium (DNRA) are competing microbial nitrate-reduction processes. The occurrence of DNRA has been shown to be effected qualitatively by various parameters in the environment. A more quantitative understanding can be obtained using enrichment cultures in a laboratory reactor, yet no successful DNRA enrichment culture has been described. We showed that a stable DNRA-dominated enrichment culture can be obtained in a chemostat system. The enrichment was based on the hypothesis that nitrate limitation is the dominant factor in selecting for DNRA. First, a conventional denitrifying culture was enriched from activated sludge, with acetate and nitrate as substrates. Next, the acetate concentration in the medium was increased to obtain nitrate-limiting conditions. As a result, conversions shifted from denitrification to DNRA. In this selection of a DNRA culture, two important factors were the nitrate limitation and a relatively low dilution rate (0.026 h⁻¹). The culture was a highly enriched population of Deltaproteobacteria most closely related to Geobacter lovleyi, based on 16S rRNA gene sequencing (97% similarity). We established a stable and reproducible cultivation method for the enrichment of DNRA bacteria in a continuously operated reactor system. This enrichment method allows to further investigate the DNRA process and address the factors for competition between DNRA and denitrification, or other N-conversion pathways.     

Mutation in TERMINAL FLOWER1 reverses the photoperiodic requirement for flowering in the wild strawberry Fragaria vesca

Photoperiodic flowering has been extensively studied in the annual short-day and long-day plants rice (Oryza sativa) and Arabidopsis (Arabidopsis thaliana), whereas less is known about the control of flowering in perennials. In the perennial wild strawberry, Fragaria vesca (Rosaceae), short-day and perpetual flowering long-day accessions occur. Genetic analyses showed that differences in their flowering responses are caused by a single gene, SEASONAL FLOWERING LOCUS, which may encode the F. vesca homolog of TERMINAL FLOWER1 (FvTFL1). We show through high-resolution mapping and transgenic approaches that FvTFL1 is the basis of this change in flowering behavior and demonstrate that FvTFL1 acts as a photoperiodically regulated repressor. In short-day F. vesca, long photoperiods activate FvTFL1 mRNA expression and short days suppress it, promoting flower induction. These seasonal cycles in FvTFL1 mRNA level confer seasonal cycling of vegetative and reproductive development. Mutations in FvTFL1 prevent long-day suppression of flowering, and the early flowering that then occurs under long days is dependent on the F. vesca homolog of FLOWERING LOCUS T. This photoperiodic response mechanism differs from those described in model annual plants. We suggest that this mechanism controls flowering within the perennial growth cycle in F. vesca and demonstrate that a change in a single gene reverses the photoperiodic requirements for flowering.     

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.     

Genomic selection of purebreds for crossbred performance

Background

One of the main limitations of many livestock breeding programs is that selection is in pure breeds housed in high-health environments but the aim is to improve crossbred performance under field conditions. Genomic selection (GS) using high-density genotyping could be used to address this. However in crossbred populations, 1) effects of SNPs may be breed specific, and 2) linkage disequilibrium may not be restricted to markers that are tightly linked to the QTL. In this study we apply GS to select for commercial crossbred performance and compare a model with breed-specific effects of SNP alleles (BSAM) to a model where SNP effects are assumed the same across breeds (ASGM). The impact of breed relatedness (generations since separation), size of the population used for training, and marker density were evaluated. Trait phenotype was controlled by 30 QTL and had a heritability of 0.30 for crossbred individuals. A Bayesian method (Bayes-B) was used to estimate the SNP effects in the crossbred training population and the accuracy of resulting GS breeding values for commercial crossbred performance was validated in the purebred population.

Results

Results demonstrate that crossbred data can be used to evaluate purebreds for commercial crossbred performance. Accuracies based on crossbred data were generally not much lower than accuracies based on pure breed data and almost identical when the breeds crossed were closely related breeds. The accuracy of both models (ASGM and BSAM) increased with marker density and size of the training data. Accuracies of both models also tended to decrease with increasing distance between breeds. However the effect of marker density, training data size and distance between breeds differed between the two models. BSAM only performed better than AGSM when the number of markers was small (500), the number of records used for training was large (4000), and when breeds were distantly related or unrelated.

Conclusion

In conclusion, GS can be conducted in crossbred population and models that fit breed-specific effects of SNP alleles may not be necessary, especially with high marker density. This opens great opportunities for genetic improvement of purebreds for performance of their crossbred descendents in the field, without the need to track pedigrees through the system.     

Mapping carcass and meat quality QTL on Sus Scrofa chromosome 2 in commercial finishing pigs

Quantitative trait loci (QTL) affecting carcass and meat quality located on SSC2 were identified using variance component methods. A large number of traits involved in meat and carcass quality was detected in a commercial crossbred population: 1855 pigs sired by 17 boars from a synthetic line, which where homozygous (A/A) for IGF2. Using combined linkage and linkage disequilibrium mapping (LDLA), several QTL significantly affecting loin muscle mass, ham weight and ham muscles (outer ham and knuckle ham) and meat quality traits, such as Minolta-L* and -b*, ultimate pH and Japanese colour score were detected. These results agreed well with previous QTL-studies involving SSC2. Since our study is carried out on crossbreds, different QTL may be segregating in the parental lines. To address this question, we compared models with a single QTL-variance component with models allowing for separate sire and dam QTL-variance components. The same QTL were identified using a single QTL variance component model compared to a model allowing for separate variances with minor differences with respect to QTL location. However, the variance component method made it possible to detect QTL segregating in the paternal line (e.g. HAMB), the maternal lines (e.g. Ham) or in both (e.g. pHu). Combining association and linkage information among haplotypes improved slightly the significance of the QTL compared to an analysis using linkage information only.     

Heat Shock Protein-Derived T-Cell Epitopes Contribute to Autoimmune Inflammation in Pediatric Crohn's Disease

Pediatric Crohn''s disease is a chronic auto inflammatory bowel disorder affecting children under the age of 17 years. A putative etiopathogenesis of Crohn''s disease (CD) is associated with disregulation of immune response to antigens commonly present in the gut microenvironment. Heat shock proteins (HSP) have been identified as ubiquitous antigens with the ability to modulate inflammatory responses associated with several autoimmune diseases. The present study tested the contribution of immune responses to HSP in the amplification of autoimmune inflammation in chronically inflamed mucosa of pediatric CD patients. Colonic biopsies obtained from normal and CD mucosa were stimulated with pairs of Pan HLA-DR binder HSP60-derived peptides (human/bacterial homologues). The modulation of RNA and protein levels of induced proinflammatory cytokines were measured. We identified two epitopes capable of sustaining proinflammatory responses, specifically TNF〈 and IFN© induction, in the inflamed intestinal mucosa in CD patients. The responses correlated positively with clinical and histological measurements of disease activity, thus suggesting a contribution of immune responses to HSP in pediatric CD site-specific mucosal inflammation.     

The Fe-Rich Clay Microsystems in Basalt-Komatiite Lavas: Importance of Fe-Smectites for Pre-Biotic Molecule Catalysis During the Hadean Eon

During the Hadean to early Archean period (4.5–3.5 Ga), the surface of the Earth’s crust was predominantly composed of basalt and komatiite lavas. The conditions imposed by the chemical composition of these rocks favoured the crystallization of Fe-Mg clays rather than that of Al-rich ones (montmorillonite). Fe-Mg clays were formed inside chemical microsystems through sea weathering or hydrothermal alteration, and for the most part, through post-magmatic processes. Indeed, at the end of the cooling stage, Fe-Mg clays precipitated directly from the residual liquid which concentrated in the voids remaining in the crystal framework of the mafic-ultramafic lavas. Nontronite-celadonite and chlorite-saponite covered all the solid surfaces (crystals, glass) and are associated with tiny pyroxene and apatite crystals forming the so-called “mesostasis”. The mesostasis was scattered in the lava body as micro-settings tens of micrometres wide. Thus, every square metre of basalt or komatiite rocks was punctuated by myriads of clay-rich patches, each of them potentially behaving as a single chemical reactor which could concentrate the organics diluted in the ocean water. Considering the high catalytic potentiality of clays, and particularly those of the Fe-rich ones (electron exchangers), it is probable that large parts of the surface of the young Earth participated in the synthesis of prebiotic molecules during the Hadean to early Archean period through innumerable clay-rich micro-settings in the massive parts and the altered surfaces of komatiite and basaltic lavas. This leads us to suggest that Fe,Mg-clays should be preferred to Al-rich ones (montmorillonite) to conduct experiments for the synthesis and the polymerisation of prebiotic molecules.     

<Emphasis Type="Italic">α</Emphasis>-Amylase inhibitor-1 gene from <Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> expressed in <Emphasis Type="Italic">Coffea arabica</Emphasis>plants inhibits α-amylases from the coffee berry borer pest

Background  

Coffee is an important crop and is crucial to the economy of many developing countries, generating around US70 billion per year. There are 115 species in the < i > Coffea < /i > genus, but only two, < i > C. arabica < /i > and < i > C. canephora < /i > , are commercially cultivated. Coffee plants are attacked by many pathogens and insect-pests, which affect not only the production of coffee but also its grain quality, reducing the commercial value of the product. The main insect-pest, the coffee berry borer ( < i > Hypotheneumus hampei < /i > ), is responsible for worldwide annual losses of around US70 billion per year. There are 115 species in the Coffea genus, but only two, C. arabica and C. canephora, are commercially cultivated. Coffee plants are attacked by many pathogens and insect-pests, which affect not only the production of coffee but also its grain quality, reducing the commercial value of the product. The main insect-pest, the coffee berry borer (Hypotheneumus hampei), is responsible for worldwide annual losses of around US500 million. The coffee berry borer exclusively damages the coffee berries, and it is mainly controlled by organochlorine insecticides that are both toxic and carcinogenic. Unfortunately, natural resistance in the genus Coffea to H. hampei has not been documented. To overcome these problems, biotechnological strategies can be used to introduce an α-amylase inhibitor gene (α-AI1), which confers resistance against the coffee berry borer insect-pest, into C. arabica plants.     

Primary cell culture of Echinococcus granulosus developed from the cystic germinal layer: Biological and functional characterization

Cell cultures of parasitic helminths are an invaluable tool for investigations of basic biological processes, as well as for development of improved chemotherapeutic agents and molecular interactions between host and parasite. We carried out a simple and efficient methodology to isolate Echinococcus granulosus germinal cells which were maintained for at least 4 months while cultivated in the presence of reducing agents and hormones. Microscopic analysis of the primary cell culture revealed the presence of cells with similar Echinococcus germinal cell morphology and behaviour. Population doubling time was estimated at 48 h, showing a rapid division rate. To discard possible host contamination, the specificity of the primary culture was tested by nested PCR, analyzing mdh gene expression and obtaining only one product with the expected size. We also studied the expression of specific E. granulosus proteins in primary cell culture. The novel and systematized method described here constitutes a powerful tool for investigations in cystic echinococcosis on biochemical and biological aspects related to the life cycle of the parasite and mechanisms of host-parasite interactions. This method also constitutes a powerful tool for the design of more efficient therapeutic alternatives.     

Simultaneous HPLC–UV analysis of rufinamide,zonisamide, lamotrigine,oxcarbazepine monohydroxy derivative and felbamate in deproteinized plasma of patients with epilepsy

We present an implementation of a method we previously reported allowing the newer antiepileptic drugs (AEDs) rufinamide (RFN) and zonisamide (ZNS) to be simultaneously determined with lamotrigine (LTG), oxcarbazepine's (OXC) main active metabolite monohydroxycarbamazepine (MHD) and felbamate (FBM) in plasma of patients with epilepsy using high performance liquid chromatography (HPLC) with UV detection. Plasma samples (250 μL) were deproteinized by 1 mL acetonitrile spiked with citalopram as internal standard (I.S.). HPLC analysis was carried out on a Synergi 4 μm Hydro-RP, 250 mm × 4.6 mm I.D. column. The mobile phase was a mixture of potassium dihydrogen phosphate buffer (50 mM, pH 4.5), acetonitrile and methanol (65:26.2:8.8, v/v/v) at an isocratic flow rate of 0.8 mL/min. The UV detector was set at 210 nm. The chromatographic run lasted 19 min. Commonly coprescribed AEDs did not interfere with the assay. Calibration curves were linear for both AEDs over a range of 2–40 μg/mL for RFN and 2–80 μg/mL for ZNS. The limit of quantitation was 2 μg/mL for both analytes and the absolute recovery ranged from 97% to 103% for RFN, ZNS and the I.S. Intra- and interassay precision and accuracy were lower than 10% at all tested concentrations. The present study describes the first simple and validated method for RFN determination in plasma of patients with epilepsy. By grouping different new AEDs in the same assay the method can be advantageous for therapeutic drug monitoring (TDM).