In Vitro ATP Regeneration from Polyphosphate and AMP by Polyphosphate:AMP Phosphotransferase and Adenylate Kinase from Acinetobacter johnsonii 210A

In vitro enzyme-based ATP regeneration systems are important for improving yields of ATP-dependent enzymatic reactions for preparative organic synthesis and biocatalysis. Several enzymatic ATP regeneration systems have been described but have some disadvantages. We report here on the use of polyphosphate:AMP phosphotransferase (PPT) from Acinetobacter johnsonii strain 210A in an ATP regeneration system based on the use of polyphosphate (polyP) and AMP as substrates. We have examined the substrate specificity of PPT and demonstrated ATP regeneration from AMP and polyP using firefly luciferase and hexokinase as model ATP-requiring enzymes. PPT catalyzes the reaction polyP_n + AMP → ADP + polyP_n−1. The ADP can be converted to ATP by adenylate kinase (AdK). Substrate specificity with nucleoside and 2′-deoxynucleoside monophosphates was examined using partially purified PPT by measuring the formation of nucleoside diphosphates with high-pressure liquid chromatography. AMP and 2′-dAMP were efficiently phosphorylated to ADP and 2′-dADP, respectively. GMP, UMP, CMP, and IMP were not converted to the corresponding diphosphates at significant rates. Sufficient AdK and PPT activity in A. johnsonii 210A cell extract allowed demonstration of polyP-dependent ATP regeneration using a firefly luciferase-based ATP assay. Bioluminescence from the luciferase reaction, which normally decays very rapidly, was sustained in the presence of A. johnsonii 210A cell extract, MgCl₂, polyP_n=35, and AMP. Similar reaction mixtures containing strain 210A cell extract or partially purified PPT, polyP, AMP, glucose, and hexokinase formed glucose 6-phosphate. The results indicate that PPT from A. johnsonii is specific for AMP and 2′-dAMP and catalyzes a key reaction in the cell-free regeneration of ATP from AMP and polyP. The PPT/AdK system provides an alternative to existing enzymatic ATP regeneration systems in which phosphoenolpyruvate and acetylphosphate serve as phosphoryl donors and has the advantage that AMP and polyP are stabile, inexpensive substrates.     

Use of chromomycin A3 staining in bovine sperm cells for detection of protamine deficiency

Sperm chromatin integrity is essential for accurate transmission of male genetic information, and normal sperm chromatin structure is important for fertilization. Protamine is a nuclear protein that plays a key role in sperm DNA integrity, because it is responsible for sperm DNA stability and packing until the paternal genome is delivered into the oocyte during fertilization. Our aim was to investigate protamine deficiency in sperm cells of Bos indicus bulls (Nelore) using chromomycin A₃ (CMA₃) staining. Frozen semen from 14 bulls were thawed, then fixed in Carnoy's solution. Smears were prepared and analyzed by microscopy. As a positive control of CMA₃ staining, sperm from one bull was subjected to deprotamination of nuclei. The percentage of CMA₃-positive bovine sperm did not vary among batches. Only two bulls showed a higher percentage of CMA₃-positive sperm cells compared to the others. CMA₃ is a simple and useful tool for detecting sperm protamine deficiency in bulls.     

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.     

Electrochemical disinfection of dental implants--a proof of concept

Background

Peri-implantitis has gained significant clinical attention in recent years. This disease is an inflammatory reaction to microorganisms around dental implants. Due to the limited accessibility, non-invasive antimicrobial strategies are of high interest. An unexpected approach to implant disinfection may evolve from electrolysis. Given the electrical conductivity of titanium implants, alkalinity or active oxidants can be generated in body fluids. We investigated the use of dental titanium implants as electrodes for the local generation of disinfectants. Our hypothesis was that electrolysis can reduce viable counts of adhering bacteria, and that this reduction should be greater if active oxidative species are generated.

Methodology/Principal Findings

As model systems, dental implants, covered with a mono-species biofilm of Escherichia coli C43, were placed in photographic gelatin prepared with physiological saline. Implants were treated by a continuous current of 0 - 10 mA for 15 minutes. The reduction of viable counts was investigated on cathodes and anodes. In separate experiments, the local change in pH was visualized using color indicators embedded in the gelatin. Oxidative species were qualitatively detected by potassium iodide-starch paper. The in situ generated alkaline environment around cathodic implants caused a reduction of up to 2 orders of magnitude in viable E. coli counts. On anodic implants, in contrast to cathodic counterparts, oxidative species were detected. Here, a current of merely 7.5 mA caused complete kill of the bacteria.

Conclusions/Significance

This laboratory study shows that electrochemical treatment may provide access to a new way to decontaminate dental implants in situ.     

Genomic selection of purebred animals for crossbred performance in the presence of dominant gene action

Background

Genomic selection is an appealing method to select purebreds for crossbred performance. In the case of crossbred records, single nucleotide polymorphism (SNP) effects can be estimated using an additive model or a breed-specific allele model. In most studies, additive gene action is assumed. However, dominance is the likely genetic basis of heterosis. Advantages of incorporating dominance in genomic selection were investigated in a two-way crossbreeding program for a trait with different magnitudes of dominance. Training was carried out only once in the simulation.

Results

When the dominance variance and heterosis were large and overdominance was present, a dominance model including both additive and dominance SNP effects gave substantially greater cumulative response to selection than the additive model. Extra response was the result of an increase in heterosis but at a cost of reduced purebred performance. When the dominance variance and heterosis were realistic but with overdominance, the advantage of the dominance model decreased but was still significant. When overdominance was absent, the dominance model was slightly favored over the additive model, but the difference in response between the models increased as the number of quantitative trait loci increased. This reveals the importance of exploiting dominance even in the absence of overdominance. When there was no dominance, response to selection for the dominance model was as high as for the additive model, indicating robustness of the dominance model. The breed-specific allele model was inferior to the dominance model in all cases and to the additive model except when the dominance variance and heterosis were large and with overdominance. However, the advantage of the dominance model over the breed-specific allele model may decrease as differences in linkage disequilibrium between the breeds increase. Retraining is expected to reduce the advantage of the dominance model over the alternatives, because in general, the advantage becomes important only after five or six generations post-training.

Conclusion

Under dominance and without retraining, genomic selection based on the dominance model is superior to the additive model and the breed-specific allele model to maximize crossbred performance through purebred selection.     

Mutagenesis of varicella-zoster virus glycoprotein I (gI) identifies a cysteine residue critical for gE/gI heterodimer formation, gI structure, and virulence in skin cells

Varicella-zoster virus (VZV) is the alphaherpesvirus that causes chicken pox (varicella) and shingles (zoster). The two VZV glycoproteins gE and gI form a heterodimer that mediates efficient cell-to-cell spread. Deletion of gI yields a small-plaque-phenotype virus, ΔgI virus, which is avirulent in human skin using the xenograft model of VZV pathogenesis. In the present study, 10 mutant viruses were generated to determine which residues were required for the typical function of gI. Three phosphorylation sites in the cytoplasmic domain of gI were not required for VZV virulence in vivo. Two deletion mutants mapped a gE binding region in gI to residues 105 to 125. A glycosylation site, N116, in this region did not affect virulence. Substitution of four cysteine residues highly conserved in the Alphaherpesvirinae established that C95 is required for gE/gI heterodimer formation. The C95A and Δ105-125 (with residues 105 to 125 deleted) viruses had small-plaque phenotypes with reduced replication kinetics in vitro similar to those of the ΔgI virus. The Δ105-125 virus was avirulent for human skin in vivo. In contrast, the C95A mutant replicated in vivo but with significantly reduced kinetics compared to those of the wild-type virus. In addition to abolished gE/gI heterodimer formation, gI from the C95A or the Δ105-125 mutant was not recognized by monoclonal antibodies that detect the canonical conformation of gI, demonstrating structural disruption of gI in these viruses. This alteration prevented gI incorporation into virus particles. Thus, residues C95 and 105 to 125 are critical for gI structure required for gE/gI heterodimer formation, virion incorporation, and ultimately, effective viral spread in human skin.     

Accuracies of genomic breeding values in American Angus beef cattle using K-means clustering for cross-validation

Background

Genomic selection is a recently developed technology that is beginning to revolutionize animal breeding. The objective of this study was to estimate marker effects to derive prediction equations for direct genomic values for 16 routinely recorded traits of American Angus beef cattle and quantify corresponding accuracies of prediction.

Methods

Deregressed estimated breeding values were used as observations in a weighted analysis to derive direct genomic values for 3570 sires genotyped using the Illumina BovineSNP50 BeadChip. These bulls were clustered into five groups using K-means clustering on pedigree estimates of additive genetic relationships between animals, with the aim of increasing within-group and decreasing between-group relationships. All five combinations of four groups were used for model training, with cross-validation performed in the group not used in training. Bivariate animal models were used for each trait to estimate the genetic correlation between deregressed estimated breeding values and direct genomic values.

Results

Accuracies of direct genomic values ranged from 0.22 to 0.69 for the studied traits, with an average of 0.44. Predictions were more accurate when animals within the validation group were more closely related to animals in the training set. When training and validation sets were formed by random allocation, the accuracies of direct genomic values ranged from 0.38 to 0.85, with an average of 0.65, reflecting the greater relationship between animals in training and validation. The accuracies of direct genomic values obtained from training on older animals and validating in younger animals were intermediate to the accuracies obtained from K-means clustering and random clustering for most traits. The genetic correlation between deregressed estimated breeding values and direct genomic values ranged from 0.15 to 0.80 for the traits studied.

Conclusions

These results suggest that genomic estimates of genetic merit can be produced in beef cattle at a young age but the recurrent inclusion of genotyped sires in retraining analyses will be necessary to routinely produce for the industry the direct genomic values with the highest accuracy.     

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.     

Influence of temperature and high acetate concentrations on methanogenesis in lake sediment slurries

Methanogenesis from main methane precursors H(2)/CO(2) and acetate was investigated in a temperature range of 2-70 degrees C using sediments from Lake Baldegg, Switzerland. Psychrophilic, psychrotrophic, mesophilic, and thermophilic methanogenic microbial communities were enriched by incubations for 1-3 months of nonamended sediment slurries at 5, 15, 30, and 50 degrees C. Isotope experiments with slurries amended with (14)C-labeled bicarbonate and (14)C-2-acetate showed that in the psychrophilic community (enriched at 5 degrees C), about 95% of methane originated from acetate, in contrast to the thermophilic community (50 degrees C) where up to 98% of methane was formed from bicarbonate. In the mesophilic community (30 degrees C), acetate was the precursor of about 80% of the methane produced. When the hydrogen-carbon dioxide mixture (H(2)/CO(2)) was used as a substrate, it was directly converted to methane under thermophilic conditions (70 and 50 degrees C). Under mesophilic conditions (30 degrees C), both pathways, hydrogenotrophic and acetoclastic, were observed. At low temperatures (5 and 15 degrees C), H(2)/CO(2) was converted into methane by a two-step process; first acetate was formed, followed by methane production from acetate. When slurries were incubated at high partial pressures of H(2)/CO(2), the high concentrations of acetate produced of more than 20 mM inhibited acetoclastic methanogenesis at a temperature below 15 degrees C. However, slow adaptation of the psychrophilic microbial community to high acetate concentrations was observed.