The effect of copper nanoparticles and copper (II) salt on redox reactions and epigenetic changes in a rat model

The aim of the study was to evaluate the effects of a diet containing different levels of Cu in two different chemical forms (carbonate and nanoparticles) on redox reactions and epigenetic changes in a rat model. For 4 weeks, five experimental groups (eight rats in each) were fed diets with two dosages of added Cu (standard—6.5 mg/kg or half of the standard dosage—3.25 mg/kg, and as a negative control no additional Cu in the mineral mixture) in two forms (standard—CuCO₃ and copper nanoparticles). Addition of Cu nanoparticles resulted in higher Cp (ceruloplasmin) activity and LOOH (lipid peroxides) and MDA (malondialdehyde) content, as well as decrease the CAT (catalase) activity and level of PC (protein carbonyl), 3‐NT (3‐nitrotyrosine), 8‐OHdG (8‐hydroxydeoxyguanosine), GSH + GSSG (total glutathione) and DNA methylation. Reducing the dose of copper resulted in a decrease in the level of LOOH and GSH + GSSG as well as CAT activity, but increased the level of PC and methylated DNA. Based on these evidence, we concluded that addition of copper nanoparticles in the diet reduces protein oxidation and nitration as well as DNA oxidation and methylation. Lowering the level of Cu in the diet increases the oxidation of proteins and DNA methylation.     

Decreases in urine specific gravity and urinary creatinine in dogs with different diseases but with normal renal function

The subject of this study was to determine urine specific gravity (USg) and urinary creatinine (UCrn) in dogs with different diseases but with normal renal function. Sick dogs with different diseases were divided into nine groups. Dogs suffering from polyuria/polydipsia, vomits, diarrhoea and females in oestrus or pregnant were excluded from the studies. The healthy dogs served as a control group. Over a three-year period, a total of 267 dogs were examined clinically as well as using imaging and laboratory diagnostics methods. In sick dogs, USg and UCrn were found to be essentially decreased (except animals with neurological and uterine diseases, and neurological diseases, respectively), as compared with normal dogs. In clinically healthy animals of the control group, UCrn and USg did not significantly differ between the females and males. As for the control group, no correlation between UCrn/USg and the body weight/age was found, either.     

A simple and efficient CRISPR/Cas9 platform for induction of single and multiple,heritable mutations in barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.)

Background

Genome editing of monocot plants can be accomplished by using the components of the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat/CRISPR associated Cas9) technology specifically optimized for these types of plants. Here, we present the development of RNA-guided Cas9 system for simplex and multiplex genome editing in barley.

Results

We developed a set of customizable RNA-guided Cas9 binary vectors and sgRNA modules for simplex and multiplex editing in barley. To facilitate the design of RNA-guided Cas9 constructs, the pBract derived binary vectors were adapted to Gateway cloning and only one restriction enzyme was required for construction of the sgRNA. We designed a synthetic, codon optimized Cas9 gene containing the N terminal SV40 nuclear localization signal and the UBQ10 Arabidopsis 1st intron. Two different sgRNAs were constructed for simplex editing and one polycistronic tRNA-gRNA construct (PTG) for multiplex editing using an endogenous tRNA processing system. The RNA-guided Cas9 constructs were validated in transgenic barley plants produced by Agrobacterium-mediated transformation. The highest mutation rate was observed in simplex editing of the cytokinin oxidase/dehydrogenase HvCKX1 gene, where mutations at the hvckx1 locus were detected in 88% of the screened T₀ plants. We also proved the efficacy of the PTG construct in the multiplex editing of two CKX genes by obtaining 9 plants (21% of all edited plants) with mutations induced in both HvCKX1 and HvCKX3. Analysis of the T₁ lines revealed that mutations in the HvCKX1 gene were transmitted to the next generation of plants. Among 220 screened T₁ plants we identified 85 heterozygous and 28 homozygous mutants, most of them bearing frameshift mutations in the HvCKX1 gene. We also observed independent segregation of mutations and the Cas9-sgRNA T-DNA insert in several T₁ plants. Moreover, the knockout mutations of the Nud gene generated phenotype mutants with naked grains, and the phenotypic changes were identifiable in T₀ plants.

Conclusions

We demonstrated the effectiveness of an optimized RNA-guided Cas9 system that can be used for generating homozygous knockout mutants in the progeny of transgenic barely plants. This is also the first report of successful multiplex editing in barley using a tRNA processing system.

     

Occurrence of reproductive disorders in pig herds with and without Chlamydia suis infection – statistical analysis of breeding parameters

Chlamydiae are frequently encountered Gram‐negative intracellular eubacteria that can cause clear manifestations or clinically asymptomatic disorders. C. suis and other chlamydia are primarily isolated in cases of reproductive disorders. This study was performed to estimate the impact of Chlamydia suis infection on reproduction in sows by analyzing reproduction rates and breeding parameters. The test was conducted on first generation (F1) pigs from Polish Landrace (PL) × Polish Large White (PLW). Sixty‐four herds were investigated and 500 vaginal swabs were collected. Isolation of DNA was carried out directly from the swabs. All samples were analyzed for Chlamydia suis by real‐time PCR with a locked nucleic acid (LNA)‐containing probe. To analyze the impact of chlamydia infection on reproductive parameters, evaluation questionnaires were used. Reproductive problems were found in 77.3% of the farms tested. The receiver operating characteristic curve indicated that in the farms with 10 up to 120 sows, there were higher reproductive problems with chlamydia infection than in smaller and bigger pig farms. The most common problems were estrus repetition, which was reported by 57.81% of the surveyed farms, and the birth of dead piglets, which was reported by 31.25% of the investigated pig farms. Abortions, which were reported by 28.12% of the surveyed farms, were the least common reproductive disorders.     

Identification of genome‐wide selection signatures in the Limousin beef cattle breed

The study is aimed at identifying selection footprints within the genome of Limousin cattle. With the use of Extended Haplotype Homozygosity test, supplemented with correction for variation in recombination rates across the genome, we created map of selection footprints and detected 173 significant (p < 0.01) core haplotypes being potentially under positive selection. Within these regions, a number of candidate genes associated inter alia with skeletal muscle growth (GDF15, BMP7, BMP4 and TGFB3) or postmortem proteolysis and meat maturation (CAPN1 and CAPN5) were annotated. Noticeable clusters of selection footprints were detected on chromosomes 1, 4, 8 and 14, which are known to carry several quantitative trait loci for growth traits and meat quality. The study provides information about the genes and metabolic pathways potentially modified under the influence of directional selection, aimed at improving beef production characteristics in Limousin cattle.     

Detection of equine X chromosome mosaicism in a mare using an equine X whole chromosome painting probe (WCPP)--a case report

An infertile mare with hypoplastic ovaries was subjected to cytogenetic analysis. Fluorescence in situ hybridisation (FISH) using the equine X whole chromosome painting probe (WCPP) was carried out on a chromosome preparation obtained from blood lymphocyte culture. The number of analysed spreads was high (235) and in the X chromosome aneuploidy in mosaic form was diagnosed. The karyotype formula was 63,X / 64,XX / 65,XXX. The ratio of the three lines was 15%, 82% and 3%, respectively. The application of the FISH technique with WCPP is discussed.     

Biological and chemical hazards occurring in fish and fishery products

It is common knowledge that fish are a nutritious component of a human diet, as they constitute a valuable and desired source of protein and polyunsaturated fatty acids. However, they are likely to pose a risk to consumer health. The presence of pathogenic bacteria or their toxins, parasites, biogenic amines and toxins or chemical residues may be a causal agent of foodborne illnesses in humans, sometimes even with fatal effect. Usually, infections are a result of insufficient thermal treatment or consumption of raw fish dishes. There are also risk factors that cannot be eliminated by such treatments as cooking, salting or freezing.     

Influence of altitude on adult emergence and dynamics ofKaltenbachiola strobi (Winnertz) (Diptera: Cecidomyiidae) populations and its parasites in the Tatra National Park, Poland

A field study onKaltenbachiola strobi (Winnertz) (Diptera: Cecidomyiidae), was carried out between 1987 and 1993 in the highest mountain range in the Western Carpathians located within the Tatra National Park, in southern Poland. Rearing and analysis of 5 780 cones ofPicea abies (L.)Karst., collected from trees growing at different altitudes, yielded 30,478 individuals ofKaltenbachiola strobi, which infested 89.10% of the cones. In case of 29.81% of cones there were more than 20 larvae ofK. strobi per cone. It was shown thatK. strobi in the Tatra Mts. is more abundant in spruce stands of the upper mountain forest zone. Significant connections between the time of cone infestation byK. strobi and its parasitoids and characteristic phenophases in spruce foliage and cone development, and also the development of selected vegetation of the forest floor at different heights above sea level were determined. It was shown that the population dynamics ofK. strobi had tendency to alternate the increase and decrease in numbers every two years. It was also discovered that every two years spruce cones were inhabited by the population ofK. strobi with increased percentage of individuals having prolonged diapause. In total, the parasitoids reduced the population ofK. strobi by about 16–22.63%. Parasitism ofK. strobi in the lower mountain forest zone was higher than in the upper zone.Triplatygaster contorticornis Ratz. (Hymenoptera: Platygasteridae),Torymus azureus Boheman (Hymenoptera: Torymidae) andTetrastichus strobilanae Ratz. (Hymenoptera: Eulophidae) were most effiecient in reduction of the population ofK. strobi, and in the lower mountain forest zoneT. azureus was the dominant parasitoid species, while in the upper zoneT. contorticornis andT. strobilanae. It was shown that a considerable part of the populations ofT. azureus, T. contorticornis andT. strobilanae have their diapause prolonged and highly synchronized with the diapause of their host.     

Aphanomyces astaci presence in Japan: a threat to the endemic and endangered crayfish species Cambaroides japonicus?

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Complementary use of natural and artificial wetlands by waterbirds wintering in Doñana,south‐west Spain

• 1. The Doñana wetland complex (SW Spain) holds more wintering waterfowl than any other wetland in Europe.
• 2. This study focused on the use made by 12 common waterbirds (eight ducks and four waders) of the natural seasonal marshes in Doñana National Park (DNP) and the adjacent Veta la Palma (VLP) fish ponds created in the early 1990s. Data used were from aerial and terrestrial surveys collected between October and February during six consecutive winters from 1998/99 to 2003/04. Changes in distribution of each bird taxon were related to changes in the extent of flooded marshes within DNP. Up to 295 000 ducks were counted in VLP during dry periods, and up to 770 000 in DNP when it was flooded.
• 3. The timing and extent of flooding in DNP was highly variable, but there was a consistent pattern in which ducks concentrated in VLP during dry months and winters but redistributed to DNP as more of it was flooded. This refuge effect was also strong for black‐tailed godwits Limosa limosa, but much less so for other waders. Waders feed mainly on invertebrates, and invertebrate biomass in VLP was found to be higher than in DNP. Ducks feed mainly on seeds and plant material, which are more abundant in DNP when flooded.
• 4. When water levels in DNP were stable over the course of a winter, or controlled for in multivariate models, the numbers of ducks at VLP declined over time, probably due to reduced availability of plant foods. In contrast, numbers of waders at VLP were more stable, and their invertebrate prey became more abundant over time, at least in the winter 2003/4.
• 5. In this extremely important wetland complex, the value of natural and artificial wetlands for wintering waterbirds are complementary, providing suitable habitat for different species and for different conditions in a highly variable Mediterranean environment.

Copyright © 2009 John Wiley & Sons, Ltd.