A novel Gateway®-compatible binary vector allows direct selection of recombinant clones in Agrobacterium tumefaciens

Background

In vivo detection of protein-bound genomic regions can be achieved by combining chromatin-immunoprecipitation with next-generation sequencing technology (ChIP-seq). The large amount of sequence data produced by this method needs to be analyzed in a statistically proper and computationally efficient manner. The generation of high copy numbers of DNA fragments as an artifact of the PCR step in ChIP-seq is an important source of bias of this methodology.

Results

We present here an R package for the statistical analysis of ChIP-seq experiments. Taking the average size of DNA fragments subjected to sequencing into account, the software calculates single-nucleotide read-enrichment values. After normalization, sample and control are compared using a test based on the ratio test or the Poisson distribution. Test statistic thresholds to control the false discovery rate are obtained through random permutations. Computational efficiency is achieved by implementing the most time-consuming functions in C++ and integrating these in the R package. An analysis of simulated and experimental ChIP-seq data is presented to demonstrate the robustness of our method against PCR-artefacts and its adequate control of the error rate.

Conclusions

The software ChIP-seq Analysis in R (CSAR) enables fast and accurate detection of protein-bound genomic regions through the analysis of ChIP-seq experiments. Compared to existing methods, we found that our package shows greater robustness against PCR-artefacts and better control of the error rate.     

Olfactory recognition: a simple memory system

Mice have an olfactory (pheromone) recognition memory located at the first relay in the sensory system. It is acquired with one-trial learning, contingent upon norepinephrine activation at mating, and lasts for several weeks. The mechanism involves Hebbian (association-dependent) changes in synaptic efficacy at dendrodendritic synapses in the accessory olfactory bulb. As a result of this memory, males made familiar by mating are recognized by the females, thereby mitigating pregnancy block. Such a memory function is biologically important to the female, as it is required to sustain pregnancy in the presence of her stud male's odors.     

High selfing rate inferred for white fonio [<Emphasis Type="Italic">Digitaria exilis</Emphasis> (Kippist.) Stapf] reproductive system opens up opportunities for breeding programs

In a context of the global major changes, it is mandatory to enlarge the range of crops supporting food security and pay great attention to neglected and underutilized species. However, basic knowledge of the biology of many neglected and underutilized species is still lacking to increase their yields. In this study, the mating system of white fonio [Digitaria exilis (Kippist.) Stapf], a West African minor and promising cereal, is assessed. Progenies arrays from both homozygous and heterozygous mothers were genotyped with microsatellites markers. The rate of genotyping errors in the experiments was assessed and a likelihood framework was used to determine the probability of different mating systems: outcrossing, self-fertilization and apomixis. The results suggested that white fonio has a highly selfing reproductive system with a possible outcrossing rate of 1.7%. Understanding the reproduction system of white fonio opens up opportunities for more effective breeding programs and a wider use of this cereal for food security improvement.     

Multivariate model for the assessment of risk of fetal loss in goat herds

The observational study was carried out in a population of Polish breeding goats in 2007 to determine the prevalence of fetal loss and identify risk factors contributing to its occurrence. The multivariate model allowing to predict the risk of the occurrence of fetal loss in a herd in a study population was developed. Data on the occurrence of fetal loss, as well as of 28 hypothesized risk factors were collected from goat owners using standardized questionnaire during face-to-face reviews on farms. Moreover, data on the herd-level seroprevalence of four abortifacient infections--Chlamydophila abortus, Leptospira spp., BVDV-1 and Neospora caninum--were included in the final analysis. Fetal loss was reported as occurring often in 12 of 49 goat herds (24.5%). The relationship between the hypothesized risk factors and the occurrence of fetal loss was verified in the multivariate logistic regression (alpha=0.05). Final analysis yielded four risk factors: regular veterinary supervision at least twice a year (OR 0.188; CI 95% 0.054 - 0.656), frequent occurrence of injuries and fractures (OR 3.172; CI 95% 1.081 - 9.310), frequent occurrence of respiratory signs in adult goats (OR 4.848; CI 95% 1.353 - 17.377) and presence of antibodies to C. abortus in a herd (OR 58.116; CI 95% 1.369 - 2466.438). The accuracy of the multivariate model was analyzed using receiver operating characteristic (ROC) curve technique. Area under the curve was 0.895 (CI 95% 0.801-0.981). For optimal cut-off value of 0.20-0.35 the multivariate model had sensitivity of 75.00% and specificity of 89.19% in predicting fetal loss in a herd.     

Efficacy of a flock-specific pseudotuberculosis vaccine in goats

In a flock with 33 adult goats repeated abscesses due to Pseudotuberculosis occurred. For the owners refused a sanitation program based on testing and removing, the goats were vaccinated with a flock specific, whole-cells lysate vaccine of Corynebacterium pseudotuberculosis. Partly simultaneously or 14 days later vaccinations against Clostridiosis (Covexin8) and against Erysipelas (Erysorb plus) were performed at different application sites. After a twofold basal vaccination in a 4 week interval the goats were revaccinated in half year intervals. The local (swelling at the injection site) and systemic reactions (temperature elevation, antibody activity) after vaccination and the clinical status of the flock (core body temperature, adspection, abscesses, lymph nodes) were investigated over a period of two years. Due to the vaccination program the rate of new cases of clinical pseudotuberculosis were reduced significantly. In the last 3 months of the investigation no clinical cases occurred. The vaccination with the C. pseudotuberculosis-vaccine induced a significant increase in antibody activities detected by ELISA. Due to interaction of maternal antibodies with the antigen of the vaccine lambs should be vaccinated at the earliest at an age of 9 weeks. Vaccinating the C. pseudotuberculosis-vaccine separately in time to Covexin8 and Erysorb plus induced with increasing numbers of revaccinations higher antibody activities against C. pseudotuberculosis than simultaneous vaccinations with all the 3 vaccines. But this difference does not justify the additional expense of separate vaccinations in time. Despite considerable local and systemic reaction the C. pseudotuberculosis-vaccine is recommended to support sanitation programs.     

Effects of nitric oxide and tumor necrosis factor-alpha on production of prostaglandin F2alpha and E2 in bovine endometrial cells

The purpose of this study was to determine whether nitric oxide (NO) mediates tumor necrosis factor (TNF)alpha influence on the bovine endometrium. TNFalpha influence on the bovine endometrium is limited to the stromal cells. Therefore, it was interesting to find out whether NO production by the stromal cells, stimulated by TNFalpha might influence the endometrial epithelium. Moreover, we investigated the intracellular mechanisms of TNFalpha- and NO-regulated prostaglandin (PG) F(2alpha) and PGE(2) synthesis. Epithelial and stromal cells from the bovine endometrium (Days 2-5 of the oestrous cycle) were separated by means of enzymatic dispersion and cultured for 6-7 days in 48-well plates. The confluent endometrial cells were exposed to a NO donor (S-NAP; 1-1000 microM) for 24 h. S-NAP strongly stimulated PGE(2) production in both bovine endometrial cell types (P<0.001). The effect of SNAP on PGF(2alpha) production was limited only to the stromal cells (P<0.05). To study the intracellular mechanisms of TNFalpha and NO action, stromal cells were incubated for 24 h with TNFalpha or S-NAP and with NO synthase (NOS) inhibitor (L-NAME; 10 microM) or an inhibitor of phosphodiesterase (IBMX; 10 microM). When the cells were exposed to TNFalpha in combination with NOS inhibitor (L-NAME), TNFalpha-stimulated PGs production was reduced (P<0.05). The inhibition of enzymatic degradation of cGMP by IBMX augmented the actions of S-NAP and TNFalpha on PGs production (P<0.05). The overall results suggest that TNFalpha augments PGs production by bovine endometrial stromal cells partially via induction of NOS with subsequent stimulation of NO-cGMP formation. NO also stimulates PGE(2) production in epithelial cells.     

Contextualized re-calculation of enteric methane emission factors for small ruminants in sub-humid Western Africa is far lower than previous estimates

Tropical Animal Health and Production - Given the projected growth of methane emission by ruminants in developing countries, there is a clear need for reliable estimates of their contribution to...