Identification and characterization of long non-coding RNAs in porcine granulosa cells exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin

Background: Long non-coding RNAs(lncRNAs) may regulate gene expression in numerous biological processes including cellular response to xenobiotics.The exposure of living organisms to 2,3,7,8-tetrachlorodibenzo-p-dioxin(TCDD),a persistent environmental contaminant,results in reproductive defects in many species including pigs.The aims of the study were to identify and characterize lncRNAs in porcine granulosa cells as well as to examine the effects of TCDD on the lncRNA expression profile in the cells.Results: One thousand six hundred sixty-six lncRNAs were identified and characterized in porcine granulosa cells.The identified lncRNAs were found to be shorter than mRNAs.In addition,the number of exons was lower in lncRNAs than in m RNAs and their exons were longer.TCDD affected the expression of 22 lncRNAs(differentially expressed lncRNAs [DELs]; log2 fold change ≥ 1,P-adjusted 0.05) in the examined cells.Potential functions of DELs were indirectly predicted via searching their target cis-and trans-regulated protein-coding genes.The coexpression analysis revealed that DELs may influence the expression of numerous genes,including those involved in cellular response to xenobiotics,dioxin metabolism,endoplasmic reticulum stress and cell proliferation.Aryl hydrocarbon receptor(AhR) and cytochrome P450 1 A1(CYP1 A1) were found among the trans-regulated genes.Conclusions: These findings indicate that the identified lncRNAs may constitute a part of the regulatory mechanism of TCDD action in granulosa cells.To our knowledge,this is the first study describing lncRNAs in porcine granulosa cells as well as TCDD effects on the lncRNA expression profile.These results may trigger new research directions leading to better understanding of molecular processes induced by xenobiotics in the ovary.     

MALDI-TOF MS identification of Prototheca algae associated with bovine mastitis

We evaluated the use of MALDI-TOF MS for the identification of 3 major, dairy-associated Prototheca species, namely, Prototheca bovis (formerly P. zopfii genotype 2), P. blaschkeae, and P. ciferrii (formerly P. zopfii genotype 1). The MALDI-TOF MS spectra established for those species were introduced into the reference spectra library of the Bruker Biotyper MALDI-TOF MS analysis software. Next, 31 Prototheca isolates from Holstein cows with mastitis, from herds located in the midwestern area of São Paulo State, Brazil, were subjected to MALDI-TOF MS profiling. MALDI-TOF MS allowed identification of 22 of 27 P. bovis and 3 of 4 P. blaschkeae isolates with scores >2.0, with 5 of 27 P. bovis and 1 of 4 P. blaschkeae isolates identified only to the genus level. With our extended algae database, MALDI-TOF MS can contribute to quick and effective speciation of Prototheca from mastitis cases.     

Induction of defense mechanisms in seedlings of oilseed winter rape inoculated with Phoma lingam (Leptosphaeria maculans)

The aim of this study was to find the biochemical markers of blackleg, evoked by the fungus Leptosphaeria maculans in seedlings of oilseed winter rape. Analyses of antioxidant enzymes activity and reactive oxygen species detection were performed 24, 48 and 72 h after inoculation. Moreover, measurements of the total pool of phenolic compounds, trans-cinnamic acid and ferulic acid were carried out. The results proved the virulence of Ph L5 isolate towards the rape seedlings. An increase in total phenolic content, including trans-cinnamic acid, was observed 24 h after inoculation. High activity of phenylalanine ammonia-lyase is probably another symptom of the defense mechanisms, despite the fact that it was not accompanied by elevated phenolics level, which were probably rapidly exhausted during Phoma lingam inoculation. A drop in phenolics content was balanced by higher activity of SOD, CAT and non-specific peroxidases. Enhanced activity of these enzymes was a response to the reactive oxygen species overproduction. ROS accumulation in indirectly inoculated first true leaves after inoculation may suggest a systemic defense response.     

Effect of <Emphasis Type="Italic">Silene vulgaris</Emphasis> and Heavy Metal Pollution on Soil Microbial Diversity in Long-Term Contaminated Soil

In this study, we analysed the impact of heavy metals and plant rhizodeposition on the structure of indigenous microbial communities in rhizosphere and bulk soil that had been exposed to heavy metals for more than 150 years. Samples of the rhizosphere of Silene vulgaris and non-rhizosphere soils 250 and 450 m from the source of emission that had different metal concentrations were collected for analyses. The results showed that soils were collected 250 m from the smelter had a higher number of Cd-resistant CFU compared with the samples that were collected from 450 m, but no significant differences were observed in the number of total and oligotrophic CFU or the equivalent cell numbers between rhizosphere and non-rhizosphere soils that were taken 250 and 450 m from the emitter. Unweighted pair group method with arithmetic mean (UPGMA) cluster analysis of the denaturing gradient gel electrophoresis (DGGE) profiles, as well as a cluster analysis that was generated on the phospholipid fatty acid (PLFA) profiles, showed that the bacterial community structure of rhizosphere soils depended more on the plant than on the distance and metal concentrations. The sequencing of the 16S rDNA fragments that were excised from the DGGE gel revealed representatives of the phyla Bacteroidetes, Acidobacteria, Gemmatimonadetes, Actinobacteria and Betaproteobacteria in the analysed soil with a predominance of the first three groups. The obtained results demonstrated that the presence of S. vulgaris did not affect the number of CFUs, except for those of Cd-resistant bacteria. However, the presence of S. vulgaris altered the soil bacterial community structure, regardless of the sampling site, which supported the thesis that plants have a higher impact on soil microbial community than metal contamination.     

Effects of thermal-time artificial scarification on the germination dynamics of black locust (<Emphasis Type="Italic">Robinia pseudoacacia</Emphasis> L.) seeds

The aim of this study was to determine the efficiency of methods for the artificial scarification of black locust seeds based on the influence of the air and liquid nitrogen temperatures. Three populations were selected for the analyses: a managed tree stand, a selected seed stand and a seed orchard. Samples of 200 seeds (4 × 50) were randomly selected. Before scarification, the seeds were stored at ?70 °C for 24 h. Nine different scarification treatments were evaluated. To calculate the germination capacity and the dynamics of germination, observations were made on days 3, 5, 7, 10 and 14 after treatment. For statistical analyses, we used repeated measures analysis of variance. The obtained results indicated that the highest scarification efficiency for black locust seeds occurred in the treatment in liquid nitrogen and in an air temperature range of 85–90 °C. The provenance of seeds had a significant effect on the dynamics of germination. Thermal methods and the use of liquid nitrogen can be alternatives to the labour intensive methods of cutting the seeds or using measures that require specialised equipment.     

Physiological Responses of <Emphasis Type="Italic">Rosa rubiginosa</Emphasis> to Saline Environment

The aim of this work was to analyse the response of Rosa rubiginosa to salinity induced by different concentrations of sodium chloride and calcium chloride (0, 25, 50, 100, 150 and 200 mM). Besides salt accumulation and pH changes, other parameters were investigated including photosynthetic activity, leaf water content, the dynamics of necrosis and chlorosis appearance and leaf drying. The study was complemented with microscopic analysis of changes in leaf anatomy. R. rubiginosa was more sensitive to the salinity induced by calcium chloride than by sodium chloride. Plant response to salinity differed depending of the salt concentration. These differences were manifested by higher dynamics of necrosis and chlorosis appearance and leaf drying. CaCl₂ showed greater inhibition of the photosynthetic apparatus and photosynthetic activity. Treatment with CaCl₂ caused more visible deformation of palisade cells, reduction in their density and overall reduction in leaf thickness. The study demonstrated higher accumulation of CaCl₂ in the soil, and thus greater limitations in water availability resulting in reduced leaf water content and quicker drying of leaves as compared with NaCl-treated plants.     

Scots Pine (Pinus sylvestris L.) Growth Suppression and Adverse Effects on Human Health Due to Air Pollution in the Upper Silesian Industrial District (USID), Southern Poland

Air pollution emissions were not continually monitored in the Upper Silesian Industrial District (USID), southern Poland, and data is only available for the last 20?years. Long-lasting and severe tree ring reductions in pines growing 5-20 km north of the USID area recorded particularly high levels of air pollution emissions in the period 1950-1990. Especially high amounts of reductions and many missing rings were found in the period 1964-1981. At the same time, pines growing 60?km west of the USID do not record deep ring reductions; this proves that the phenomenon is of a regional nature. Increases in infant mortality and lung, bronchial, and tracheal cancer morbidity rates among males were also recorded in the USID during periods of high air pollution. Infant mortality rates increased several years after the tree ring reductions. Therefore, it may be possible to use tree ring reductions as an early indicator of the occurrence of adverse effects on human health.     

Profiles of seroconversion to porcine circovirus type 2 in herds affected and not affected by postweaning multisystemic wasting syndrome

The aim of the present study was to explore the usefulness of serological methods in the diagnosis of postweaning multisystemic wasting syndrome (PMWS). The study was carried out in 4 PMWS-affected and 6 control farms. Based on the serological profiles, infection with porcine circovirus type 2 (PCV2) was determined to take place at 3-7 weeks of age in the PMWS-affected and at 3-11 weeks of age in the control farms. To compare the dynamics of seroconversion to PCV2 among farms, cross-sectional serological profiles were normalised in relation to the inferred age of infection. The results indicated that the proportion of seropositive pigs increased significantly slower in the affected herds. The most pronounced difference was observed about 4 weeks after infection, when the proportion of seropositive pigs ranged from 0 to 53.3% and from 70 to 100% in PMWS-affected and control herds, respectively. Mean antibody titres at that age were also significantly lower in the affected farms. These observations suggest a delay in the production of PCV2-specific antibodies and indicate that serological methods may be helpful in identifying herds with a high risk of PMWS.     

Genotyping of bovine Prototheca mastitis isolates from Poland

Bovine mastitis due to unicellular, achlorophyllous algae of the genus Prototheca is a serious and complex ailment that accounts for high economic losses in the dairy industry. Bovine protothecal mastitis has been almost exclusively associated with only one species Prototheca zopfii. Recently, based on the 18S rDNA sequence analysis, P. zopfii has been divided into three genotypes (1-3), of which P. zopfii genotype 3 has been given the status of a new species Prototheca blaschkeae sp. nov. The aim of this study was to investigate the genotypic composition of the population of P. zopfii bovine mastitis isolates from Poland, according to that threefold classification. Of the 44 isolates tested, 43 (98%) were identified as P. zopfii genotype 2. Only one isolate was assigned to the newly established P. blaschkeae species. These findings clearly show the predominance of the P. zopfii genotype 2 in the etiology of bovine mammary protothecosis in Poland, but also underline the involvement of P. blaschkeae in the disease. The study is the first to provide molecular characterization of Polish P. zopfii mastitis isolates, collected from different regions of Poland.     

Characteristics of capercaillie (Tetrao urogallus) semen analysed with flow cytometry combined with fertility results

In order to increase the reproductive indices of capercaillie kept in closed breeding facilities, it is necessary to constantly expand the methods of better understanding the characteristics of sperm and their fertilizing potency. The aim of the study was to analyse selected features of capercaillie sperm using flow cytometry and their connection with fertility results. The study included five males, three of which were kept in a family group with eight females and two were kept alone. For sperm viability, acrosome integrity, mitochondrial potential and DNA defragmentation were assessed. Paternity analyses were performed in order to confirm the paternity of the individual and to link the evaluated semen traits with reproductive success. Analyses carried out in the flow cytometer showed any significant differences between males in sperm characteristics. In the semen of male No. 101, the father of all chicks from the analysed family group, 91.3% of live sperm, 91.5% with intact acrosome, 83.6% with active mitochondria and 2.0% with DNA defragmentation were observed. The average fertility rate was 71.0%, and chick hatchability was 100%. Using flow cytometry in the analysis of capercaillie semen and its connection with the results of natural mating, we were able to obtain deeper knowledge about new sperm characteristics that were not examined before and which in the future may be helpful in selecting males for the reproductive flocks and developing assisted reproduction techniques.