Upregulation of LENK and VIP in paracervical ganglion neurons supplying the urinary bladder of tetrodotoxin- and resiniferatoxin-treated female pigs

Both resiniferatoxin (RTX) and tetrodotoxin (TTX) have been reported to be effective in several clinical trials aiming to cure urinary bladder dysfunction. The goal of this experiment was to study the effect of intravesical administration of RTX and TTX on the chemical coding of paracervical ganglion (PCG) neurons that supply the urinary bladder in pigs. The vasoactive intestinal peptide (VIP) and the opioid family member Leu5-enkephalin (LENK) are both known for their regulatory effects in the function of the porcine genitourinary tract. The PCG neurons innervating the urinary bladder were identified by application of the retrograde tracer Fast Blue (FB), injected into the bladder wall prior to intravesical RTX or TTX administration. Immunocytochemical detection of LENK and VIP expression in the FB-labelled perikarya revealed that in the control group 25.15% of the FB-positive PCG neurons contained LENK, and 9.22% of them expressed VIP. Intravesical infusion of RTX resulted in an increase in the number of LENKIR neurons to 48.19% and VIP-IR perikarya to 11.25%. Optional treatment with TTX induced increase of LENK-IR neurons up to 81.67% and VIP-IR population to 16.46% of the FB-positive PCG cells. The present results show that both neurotoxins affect the chemical coding of PCG nervous cells supplying the porcine urinary bladder and that they stimulate both LENK and VIP expression. Furthermore, the results indicate a possible involvement of LENK and VIP neurons in the mechanisms of action of RTX and TTX in the therapy of overactive bladder disorder.     

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.     

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.

     

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.     

Modelling of the effect of dry periods on yielding of spring barley

The paper presents a weather-yield model developed for the purpose of estimating spring barley yield on the basis of dry spells occurring in individual periods between the phenological phases of that plant. For that purpose research material on spring barley, originating from the years 1976-1997, was used as well as diurnal sums of precipitation. Five periods were considered in the analysis: sowing-emergence, emergence-tillering, tillering-heading, heading-milk ripeness and milk ripeness-full ripeness. In the study a model of changes in the amount of water available for plant during rainless periods was used. Five measures were adopted for characterisation of the approximation error: correlation coefficient, mean relative error, relative root mean square error, model efficiency and coefficient of residual mass. The analyses performed demonstrated that yield reduction is significantly influenced by rainless periods that occur in the sowing-emergence and tillering-heading inter-phase periods. The adopted criteria for yield reduction estimation show considerable similarity for the emergence-tillering and heading-milk ripeness inter-phase periods. At the same time, their influence on yield reduction is three-fold lower than during the sowing-emergence and tillering-heading inter-phase periods. Analyses performed with the use of the developed model indicate that yield size is affected by rainless periods of duration longer than 30% of the inter-phase period.     

Breeding value of selected blackcurrant (<Emphasis Type="Italic">Ribes nigrum</Emphasis> L.) genotypes for early-age fruit yield and its quality

The study was conducted in the years from 2011 to 2013 at the Research Institute of Horticulture in Skierniewice, Poland. The aim of the research was to assess the breeding value, based on the effects of general and specific combining abilities (GCA and SCA), of 15 parental genotypes of blackcurrant in terms of fruit yield and quality. The plant material consisted of F₁ generation seedlings obtained by crossing, in a factorial mating design of twelve maternal (‘Bona’, ‘Big Ben’, ‘Chereshnieva’, Kupoliniai’, ‘Gofert’, ‘Tines’, ‘Sofievskaia’, ‘Tihope’, ‘Ores’, ‘Ruben’, ‘Titania’ and D13B/11) and three paternal cultivars (‘Ceres’, ‘Foxendown’ and ‘Saniuta’). It was found that the cultivars ‘Ruben’, ‘Big Ben’, ‘Gofert’ and D13B/11 had significant positive effects of GCA on fruit yield at least in two consecutive years. ‘Ruben’, ‘Saniuta’ and D13B/11 had positive GCA effects on fruit weight. For ‘Chereshnieva’, ‘Gofert’, ‘Tines’, ‘Sofievskaia’, ‘Tihope’, ‘Titania’, ‘Ceres’ and Saniuta’ positive GCA effects were estimated for soluble solids content, whereas ‘Ruben’, ‘Ceres’, ‘Gofert’ and ‘Ores’ had positive GCA effects on the ascorbic acid content in fruit. This indicated a high breeding value of these parental genotypes in terms of the evaluated traits. The significantly positive values of SCA, estimated for the crossing combinations: ‘Big Ben’?×?‘Saniuta’, ‘Ruben’?×?‘Foxendown’, ‘Titania’?×?‘Ceres’, ‘Kupoliniai’?×?‘Saniuta’, ‘Gofert’?×?‘Foxendown’, ‘Gofert’?×?‘Saniuta’, ‘Tines’?×?‘Ceres’ and ‘Tihope’?×?‘Foxendown’ for at least two traits describing fruit yield and quality, were evidence of the interaction of both these parental genotypes in the creation of new dessert-type cultivars of blackcurrant.     

Urban soil resources of medium-sized cities in Poland: a comparative case study of Toruń and Zielona Góra

Purpose

Despite the many studies of urban soils, a comparative analysis for cities of a similar size has not yet been conducted. Thus, the aim of this review paper was to compare the soil distribution patterns in the area of two medium-sized Polish cities (Toruń and Zielona Góra). The authors attempted to answer the question of how natural and technogenic factors contributed to the transformation of urban soils and what the similarities and differences are between these two studied cities.

Materials and methods

First, both the natural and the human-related (including historical) factors influencing the soil formation in the studied cities were analysed. Then, a comparison of the degree of transformation of the urban soil environment was presented. The data obtained by the authors during nearly two decades of research (over 200 soil profiles) were used.

Results and discussion

Intensive development of the built-up areas in Toruń brought heavy and long-term transformations of soils, which demonstrate the typical properties of Urbic Technosols, Ekranic Technosols and other technogenic soils. Zielona Góra showed a similar state of soil transformation over a considerably smaller area. Currently, the differences in the soil properties in many built up areas have been blurred, despite the habitat and historical base. The similarities of the soil properties concerned, in particular, a high content of skeletal remains (from a few to over 30%), elevated pH (in KCl) values (even above 8.0) and the artificial soil horizons formation. Both cities struggle with similar problems regarding the changes in the land use within the areas covered by these soils.

Conclusions

It was found that, despite the significant habitat and historical differences between the two studied cities, most of the urban soils, especially Urbic Technosols, Ekranic Technosols and Regosols (Relocatic and Technic), are characterised by similar morphology and properties. The most important differences are the time and scale of the area transformation, which influence the extent of Technosols and Anthrosols within the city borders. The most distinct differences concern the natural and slightly transformed soils, which are the results of various soil-forming factors.

     

How the deadwood of different tree species in various stages of decomposition affected nutrient dynamics?

Purpose

The aim of the study was to estimate how the deadwood of different tree species in various stages of decomposition affected nutrient dynamics.

Materials and methods

The deadwood of eight species (common alder, common aspen, common ash, silver fir, pedunculate oak, Norway spruce, common hornbeam and silver birch) was selected. Three logs from each species in the third, fourth and fifth decay classes were chosen for analysis. Wood in the third decay class was characterised by larger hard fragments, fragmented bark and no branches; in the fourth decay class, it was characterised by small pieces and a fragmented bark; and in the fifth decay class, it was characterised by a soft texture and no bark. The investigation was carried out in the Czarna Rózga Reserve in Central Poland. Tension lysimeters were installed under each log in the humus horizon. The water samples collected from tension lysimeters were chemically analysed in the laboratory. The water chemistry was analysed by means of ion chromatography using a DIONEX ICS 5000 unit.

Results and discussion

The leachate obtained from different tree species contained different ion concentrations. The high similarity of the concentration of total anions and statistically significant differences in the content of total cations were determined in the leachate from the wood of coniferous and deciduous species. The concentration of the cations increases with the advancement of the decomposition level. A general linear model analysis demonstrates that wood species and the decomposition classes are of equal importance in defining the ion composition of the filtrate leaching from deadwood.

Conclusions

Wood at the highest decomposition stage releases more ions to the surface soil layers than wood at the lower decay class. An exception from the rule is the wood of birch, which in the III decay class releases more ions than in its higher decay classes. When comparing the ionic composition of leachate released from wood of coniferous and deciduous tree species, the latter are characterised by higher cation concentrations in comparison with coniferous species. Among the deciduous species, wood of such species as ash, hornbeam, aspen, birch and alder has the most favourable effects on the soil surface horizons through its supply with ionic substances. The ash wood releases high amounts of calcium, hornbeam wood releases magnesium and sodium, and aspen releases calcium, potassium and nitrate anion. From the analysed coniferous species, fir wood has a more favourable effect in terms of ion release to soil than spruce wood.

     

Optimisation of sodium and potassium concentrations and pH in the artificial seminal plasma of common carp <Emphasis Type="Italic">Cyprinus carpio</Emphasis> L.

The effect of sodium and potassium concentrations as well as optimal pH on the motility of common carp Cyprinus carpio L. sperm during short-term storage in artificial seminal plasma (ASP) was investigated. Sperm was collected from individual males (n?=?5) and each sample diluted tenfold (1:9) in ASP (sperm:extender) containing 2 mM CaCl₂, 1 mM Mg₂SO₄ and 20 mM Tris at pH 8.0 and supplemented by the following concentrations of sodium and potassium (mM/mM): 0/150, 20/130, 40/110, 75/75, 110/40, 130/20 and 150/0. The osmolality of all ASP variants was set at 310 mOsm kg^?1. Sperm motility was measured using a CASA system during 72 h of storage. Immediately after dilution, sperm motility was high (90%) both in each variant and in the control group (fresh sperm). After 72-h storage, the highest sperm motility was noted in ASP containing 110 mM NaCl and 40 mM KCl. No differences were found in the motility of samples preserved within the pH range of 7.0–9.0. Our data suggest that for the short-term storage of common carp sperm, whereas the pH of the solution does not play a crucial role, a specific potassium concentration of around 40 mM is required.