Investigation of Chlamydiaceae in semen and cauda epididymidis and seroprevalence of Chlamydophila abortus in breeding bulls

Background

Reproductive disorders associated with chlamydial infection have been reported worldwide in cattle and there are indications of potential venereal transmission.

Methods

Semen samples from 21 dairy bulls and cauda epididymidis tissue samples from 43 beef bulls were analysed for chlamydial agent by real-time polymerase chain reaction (PCR) including an internal amplification control (mimic). Additionally, presence of antibodies against Chlamydophila (Cp.) abortus among the bulls was investigated with the commercial Pourquier^® ELISA Cp. abortus serum verification kit.

Results

No chlamydial agent was detected by PCR in either the semen samples or in the tissue samples. Additionally, no antibodies against Cp. abortus were detected.

Conclusions

The results suggest that Cp. abortus is very rare, or absent in Swedish bulls and thus the risk for venereal transmission of chlamydial infection through their semen is low. However, because Chlamydophila spp. infection rates seem to differ throughout the world, it is essential to clarify the relative importance of transmission of the infection through semen on cattle fertility.     

Recodon: Coalescent simulation of coding DNA sequences with recombination, migration and demography

Background

Coalescent simulations have proven very useful in many population genetics studies. In order to arrive to meaningful conclusions, it is important that these simulations resemble the process of molecular evolution as much as possible. To date, no single coalescent program is able to simulate codon sequences sampled from populations with recombination, migration and growth.

Results

We introduce a new coalescent program, called Recodon, which is able to simulate samples of coding DNA sequences under complex scenarios in which several evolutionary forces can interact simultaneously (namely, recombination, migration and demography). The basic codon model implemented is an extension to the general time-reversible model of nucleotide substitution with a proportion of invariable sites and among-site rate variation. In addition, the program implements non-reversible processes and mixtures of different codon models.

Conclusion

Recodon is a flexible tool for the simulation of coding DNA sequences under realistic evolutionary models. These simulations can be used to build parameter distributions for testing evolutionary hypotheses using experimental data. Recodon is written in C, can run in parallel, and is freely available from http://darwin.uvigo.es/.     

Re-analysis of the larval testis data on meiotic sex chromosome inactivation revealed evidence for tissue-specific gene expression related to the drosophila X chromosome

Background

Molecular oxygen (O₂) is one of the key metabolites of all obligate and facultative aerobic pro- and eukaryotes. It plays a fundamental role in energy homeostasis whereas oxygen deprivation, in turn, broadly affects various physiological and pathophysiological processes. Therefore, real-time monitoring of cellular oxygen levels is basically a prerequisite for the analysis of hypoxia-induced processes in living cells and tissues.

Results

We developed a genetically encoded F?rster resonance energy transfer (FRET)-based biosensor allowing the observation of changing molecular oxygen concentrations inside living cells. This biosensor named FluBO (fluorescent protein-based biosensor for oxygen) consists of the yellow fluorescent protein (YFP) that is sensitive towards oxygen depletion and the hypoxia-tolerant flavin-binding fluorescent protein (FbFP). Since O₂ is essential for the formation of the YFP chromophore, efficient FRET from the FbFP donor domain to the YFP acceptor domain only occurs in the presence but not in the absence of oxygen. The oxygen biosensor was used for continuous real-time monitoring of temporal changes of O₂ levels in the cytoplasm of Escherichia coli cells during batch cultivation.

Conclusions

FluBO represents a unique FRET-based oxygen biosensor which allows the non-invasive ratiometric readout of cellular oxygen. Thus, FluBO can serve as a novel and powerful probe for investigating the occurrence of hypoxia and its effects on a variety of (patho)physiological processes in living cells.     

EMS mutagenesis and qPCR-HRM prescreening for point mutations in an embryogenic cell suspension of grapevine

Key message

Embryogenic suspension cultures are suitable for EMS mutagenesis in grapevine, and HRM prescreening of EMS-treated somatic embryo clusters allows rapid detection of point mutations before plant regeneration.

Abstract

Somatic embryogenesis is an excellent system for induced mutagenesis and clonal propagation in woody plants. Our work was focused on establishing a procedure for inducing ethyl methanesulfonate (EMS) mutagenesis in grapevine. Embryogenic cell aggregates (ECAs) growing in liquid medium were treated with increasing concentrations of EMS. We found that EMS dramatically affects the viability of ECAs at concentrations above 20 mM (25.5 ± 2.9 % survival), whereas concentrations above 10 mM affect embryogenic potential (22.1 ± 1.7 % of ECAs gave rise to embryos). Embryo masses generated from EMS-treated embryogenic cell aggregates were prescreened by quantitative PCR-High Resolution Melting (qPCR-HRM) to detect single nucleotide polymorphisms (SNPs) in a 1,000-bp VvNCED1-encoding DNA fragment, which served as the target gene. Detected mutations were verified in regenerated plants by PCR and sequencing. qPCR-HRM analysis of the difference plots for the fluorescence signals allowed detection of a mutation in a sample from an embryogenic aggregate treated with 10 mM EMS. To confirm the nature of the mutation, embryos from this aggregate were recovered and germinated, and leaves were collected for PCR and sequencing analysis. The alignment of sequences from regenerated plants with the wild-type sequence revealed a transitional mutation (G/C to A/T) in the 1,000-bp VvNCED1-encoding region. To our knowledge, this is the first time that EMS mutagenesis has been performed using an embryogenic cell suspension of grapevine.     

Mutation analysis of the WNT4 gene in Han Chinese women with premature ovarian failure

Background

The WNT4 gene plays an important role in female sex determination and differentiation. It also contributes to maintaining of the ovaries and the survival of follicles.

Methods

We sequenced the coding region and splice sites of WNT4 in 145 Han Chinese women with premature ovarian failure (POF) and 200 healthy controls.

Results

Only one novel variation, in Exon 2 (195C > T), was detected among the women with POF. However, this synonymous variation did not result in a change in amino acid sequence (65 Asp > Asp). No further variants were found in any of the samples.

Conclusion

Although we cannot provide any evidence that it is a possible disease-causing gene, this study is the first attempt to investigate the possible role of WNT4 in Han Chinese women with POF.     

A nucleotide binding rectification Brownian ratchet model for translocation of Y-family DNA polymerases

Background

Self-organization is a fundamental feature of living organisms at all hierarchical levels from molecule to organ. It has also been documented in developing embryos.

Methods

In this study, a scale-invariant power law (SIPL) method has been used to study self-organization in developing embryos. The SIPL coefficient was calculated using a centro-axial skew symmetrical matrix (CSSM) generated by entering the components of the Cartesian coordinates; for each component, one CSSM was generated. A basic square matrix (BSM) was constructed and the determinant was calculated in order to estimate the SIPL coefficient. This was applied to developing C. elegans during early stages of embryogenesis. The power law property of the method was evaluated using the straight line and Koch curve and the results were consistent with fractal dimensions (fd). Diffusion-limited aggregation (DLA) was used to validate the SIPL method.

Results and conclusion

The fractal dimensions of both the straight line and Koch curve showed consistency with the SIPL coefficients, which indicated the power law behavior of the SIPL method. The results showed that the ABp sublineage had a higher SIPL coefficient than EMS, indicating that ABp is more organized than EMS. The fd determined using DLA was higher in ABp than in EMS and its value was consistent with type 1 cluster formation, while that in EMS was consistent with type 2.     

Human T-cell leukemia virus type 2 Tax protein induces interleukin 2-independent growth in a T-cell line

Background

Evolutionary analysis may serve as a useful approach to identify and characterize host defense and viral proteins involved in genetic conflicts. We analyzed patterns of coding sequence evolution of genes with known (TRIM5 α and APOBEC3G) or suspected (TRIM19/PML) roles in virus restriction, or in viral pathogenesis (PPIA, encoding Cyclophilin A), in the same set of human and non-human primate species.

Results and conclusion

This analysis revealed previously unidentified clusters of positively selected sites in APOBEC3G and TRIM5 α that may delineate new virus-interaction domains. In contrast, our evolutionary analyses suggest that PPIA is not under diversifying selection in primates, consistent with the interaction of Cyclophilin A being limited to the HIV-1M/SIVcpz lineage. The strong sequence conservation of the TRIM19/PML sequences among primates suggests that this gene does not play a role in antiretroviral defense.     

Distinct signal transduction processes by IL-4 and IL-13 and influences from the Q551R variant of the human IL-4 receptor alpha chain

Background

Pseudomonas aeruginosa and Burkholderia cepacia infections of cystic fibrosis patients' lungs are often resistant to conventional antibiotic therapy. Protegrins are antimicrobial peptides with potent activity against many bacteria, including P. aeruginosa. The present study evaluates the correlation between protegrin-1 (PG-1) sensitivity/resistance and protegrin binding in P. aeruginosa and B. cepacia.

Methods

The PG-1 sensitivity/resistance and PG-1 binding properties of P. aeruginosa and B. cepacia were assessed using radial diffusion assays, radioiodinated PG-1, and surface plasmon resonance (BiaCore).

Results

The six P. aeruginosa strains examined were very sensitive to PG-1, exhibiting minimal active concentrations from 0.0625–0.5 μg/ml in radial diffusion assays. In contrast, all five B. cepacia strains examined were greater than 10-fold to 100-fold more resistant, with minimal active concentrations ranging from 6–10 μg/ml. When incubated with a radioiodinated variant of PG-1, a sensitive P. aeruginosa strain bound considerably more protegrin molecules per cell than a resistant B. cepacia strain. Binding/diffusion and surface plasmon resonance assays revealed that isolated lipopolysaccharide (LPS) and lipid A from the sensitive P. aeruginosa strains bound PG-1 more effectively than LPS and lipid A from resistant B. cepacia strains.

Conclusion

These findings support the hypothesis that the relative resistance of B. cepacia to protegrin is due to a reduced number of PG-1 binding sites on the lipid A moiety of its LPS.     

Boron delays dehydration and stimulates root growth in Eucalyptus urophylla (Blake,S.T.) under osmotic stress

Background

Water and nutritional restrictions are limiting factors for the growth of Eucalyptus trees in tropical climates. In the dry season, boron (B) uptake is severely affected.

Aims

The objectives of this study were to evaluate the phloem mobility of B and whether its deficiency can increase plant sensitivity to osmotic stress. It was also tested to what extent foliar application of B could mitigate the negative effects of drought under low B supply.

Methods

Seedlings of a drought tolerant Eucalyptus urophylla (Blake, S. T.) clone were grown in nutrient solution, subjected to low availability of B for 25 days, and then submitted to a progressive osmotic stress. After imposition of osmotic stress, B was applied to young or mature leaves.

Results

B applications, mainly to mature leaf, stimulated root growth and delayed dehydration under osmotic stress and led to an increased B translocation and carbon isotopic composition. The expression of B transporters and pectin metabolism genes were also increased in water-stressed plants supplied with B by foliar application.

Conclusions

B deficiency led to increased plant dehydration and decreased root growth under osmotic stress. The application of B to mature leaf of water-stressed plants proved effective in mitigating the negative effects of water deficit in root growth.     

Effect of wildfires on soil respiration in three typical Mediterranean forest ecosystems in Madrid, Spain

Background and Aims

Mediterranean forests are vulnerable to numerous threats including wildfires due to a combination of climatic factors and increased urbanization. In addition, increased temperatures and summer drought lead to increased risk of forest fires as a result of climate change. This may have important consequences for C dynamics and balance in these ecosystems. Soil respiration was measured over 2 successive years in Holm oak (Quercus ilex subsp. ballota; Qi); Pyrenean Oak (Quercus pyrenaica Willd; Qp); and Scots pine (Pinus sylvestris L.; Ps) forest stands located in the area surrounding Madrid (Spain), to assess the long term effects of wildfires on C efflux from the soil, soil properties, and the role of soil temperature and soil moisture in the variation of soil respiration.

Methods

Soil respiration, soil temperature, soil moisture, fine root mass, microbial biomass, biological and chemical soil parameters were compared between non burned (NB) and burned sites (B).

Results

The annual C losses through soil respiration from NB sites in Qi, Qp and Ps were 790, 1010, 1380 gCm^?2?yr^?1, respectively, with the B sites emitting 43 %, 22 % and 11 % less in Qi, Qp and Ps respectively. Soil microclimate changed with higher soil temperature and lower soil moisture in B sites after fire. Exchangeable cations and the pH also decreased. The total SOC stocks were not significantly altered, but 6–8 years after wildfires, there was still measurably lower fine root and microbial biomass, while SOC quality changed, indicated by lower the C/N ratio and the labile carbon and a relative increase in refractory SOC forms, which resulted in lower Q₁₀ values.

Conclusions

We found long term effects of wildfires on the physical, chemical and biological soil characteristics, which in turn affected soil respiration. The response of soil respiration to temperature was controlled by moisture and changed with ecosystem type, season, and between B and NB sites. Lower post-burn Q₁₀ integrated the loss of roots and microbial biomass, change in SOC quality and a decrease in soil moisture.     

EEVD motif of heat shock cognate protein 70 contributes to bacterial uptake by trophoblast giant cells

Background

The uptake of abortion-inducing pathogens by trophoblast giant (TG) cells is a key event in infectious abortion. However, little is known about phagocytic functions of TG cells against the pathogens. Here we show that heat shock cognate protein 70 (Hsc70) contributes to bacterial uptake by TG cells and the EEVD motif of Hsc70 plays an important role in this.

Methods

Brucella abortus and Listeria monocytogenes were used as the bacterial antigen in this study. Recombinant proteins containing tetratricopeptide repeat (TPR) domains were constructed and confirmation of the binding capacity to Hsc70 was assessed by ELISA. The recombinant TPR proteins were used for investigation of the effect of TPR proteins on bacterial uptake by TG cells and on pregnancy in mice.

Results

The monoclonal antibody that inhibits bacterial uptake by TG cells reacted with the EEVD motif of Hsc70. Bacterial TPR proteins bound to the C-terminal of Hsc70 through its EEVD motif and this binding inhibited bacterial uptake by TG cells. Infectious abortion was also prevented by blocking the EEVD motif of Hsc70.

Conclusions

Our results demonstrate that surface located Hsc70 on TG cells mediates the uptake of pathogenic bacteria and proteins containing the TPR domain inhibit the function of Hsc70 by binding to its EEVD motif. These molecules may be useful in the development of methods for preventing infectious abortion.     

CapsID: a web-based tool for developing parsimonious sets of CAPS molecular markers for genotyping

Background

The neuronal ceroid lipofuscinoses (NCL) are a heterogenous group of inherited progressive neurodegenerative diseases in different mammalian species. Tibetan Terrier and Polish Owczarek Nizinny (PON) dogs show rare late-onset NCL variants with autosomal recessive inheritance, which can not be explained by mutations of known human NCL genes. These dog breeds represent animal models for human late-onset NCL. In mice the chloride channel 3 gene (Clcn3) encoding an intracellular chloride channel was described to cause a phenotype similar to NCL.

Results

Two full-length cDNA splice variants of the canine CLCN3 gene are reported. The current canine whole genome sequence assembly was used for gene structure analyses and revealed 13 coding CLCN3 exons in 52 kb of genomic sequence. Sequence analysis of the coding exons and flanking intron regions of CLCN3 using six NCL-affected Tibetan terrier dogs and an NCL-affected Polish Owczarek Nizinny (PON) dog, as well as eight healthy Tibetan terrier dogs revealed 13 SNPs. No consistent CLCN3 haplotype was associated with NCL.

Conclusion

For the examined animals we excluded the complete coding region and adjacent intronic regions of canine CLCN3 to harbor disease-causing mutations. Therefore it seems to be unlikely that a mutation in this gene is responsible for the late-onset NCL phenotype in these two dog breeds.     

Molecular Epidemiology of Brucella abortus in Northern Ireland—1991 to 2012

Background

Brucellosis is the most common bacterial zoonoses worldwide. Bovine brucellosis caused by Brucella abortus has far reaching animal health and economic impacts at both the local and national levels. Alongside traditional veterinary epidemiology, the use of molecular typing has recently been applied to inform on bacterial population structure and identify epidemiologically-linked cases of infection. Multi-locus variable number tandem repeat VNTR analysis (MLVA) was used to investigate the molecular epidemiology of a well-characterised Brucella abortus epidemic in Northern Ireland involving 387 herds between 1991 and 2012.

Results

MLVA identified 98 unique B. abortus genotypes from disclosing isolates in the 387 herds involved in the epidemic. Clustering algorithms revealed the relatedness of many of these genotypes. Combined with epidemiological information on chronology of infection and geographic location, these genotype data helped to identify 7 clonal complexes which underpinned the outbreak over the defined period. Hyper-variability of some VNTR loci both within herds and individual animals led to detection of multiple genotypes associated with single outbreaks. However with dense sampling, these genotypes could still be associated with specific clonal complexes thereby permitting inference of epidemiological links. MLVA- based epidemiological monitoring data were congruent with an independent classical veterinary epidemiology study carried out in the same territory.

Conclusions

MLVA is a useful tool in ongoing disease surveillance of B. abortus outbreaks, especially when combined with accurate epidemiological information on disease tracings, geographical clustering of cases and chronology of infection.     

In vitro generation of somaclonal variant plants of sugarcane for tolerance to Fusarium sacchari

Key message

A combination of in vitro culture and mutagenesis using ethyl methanesulfonate (EMS) followed by culture filtrate-mediated selection produced variant sugarcane plants tolerant and resistant to Fusarium sacchari.

Abstract

Eldana saccharina is a destructive pest of the sugarcane crop in South Africa. Fusarium sacchari PNG40 (a fungal strain harmful to E. saccharina) has the potential to be an endophytic biological control agent of the stalk borer. However, the fungus causes Fusarium stalk rot in sugarcane. In the current study, sugarcane plants tolerant and resistant to F. sacchari PNG40 were produced by exposing embryogenic calli to the chemical mutagen ethyl methanesulfonate (EMS), followed by in vitro selection during somatic embryogenesis and plantlet regeneration on media containing F. sacchari culture filtrates (CF). The incorporation of 100 ppm CF in the culture media at the embryo maturation stage, at germination, or at both, resulted in callus necrosis and consequent reduced plantlet yield. Subsequent trimming of the roots of regenerated plants and their exposure to 1,500 ppm CF served as a further selection treatment. Plants produced from EMS-treated calli displayed improved root re-growth in the presence of CF pressure compared with those from non-treated calli. The tolerance of CF-selected plants was confirmed in greenhouse tests by inoculation with F. sacchari PNG40, re-isolation of Fusarium spp. from undamaged tissue of asymptomatic plants and establishment of the identity of fungal isolates as PNG40 using molecular analysis. The restriction of PNG40 presence to the inoculation lesion in some plants suggested their resistance to the fungus. Genotypes exhibiting symptomless endophytic colonization by PNG40 were identified and will be utilised for testing biological control strategies against E. saccharina.