The microRNA miR-152 can mitigate and prevent the toxic effect of benzene on porcine ovarian cells.

Epigenetic methods to prevent the reproductive toxicity of oil-related environmental contaminants are currently unavailable. The present study aimed to examine the ability of the microRNA miR-152 to mitigate the effects of benzene on ovarian cells. Porcine ovarian granulosa cells transfected or not transfected with miR-152 mimics were cultured with or without benzene (0, 10 and 100 ng/ml). The expression of miR-152; viability; proliferation (cell proliferation and expression of mRNAs and accumulation of PCNA and cyclin B1); apoptosis (expression of mRNAs and accumulation of bax and caspase 3; and the proportion of cells with fragmented DNA); and release of progesterone, estradiol and IGF-I were analyzed via RT-qPCR; the Trypan blue exclusion test; quantitative immunocytochemistry; BrdU; XTT; TUNEL assays; and ELISA. Administration of benzene promoted the expression of apoptosis markers and reduced cell viability, all measured markers of proliferation, the release of steroid hormones and IGF-I. Overexpression of miR-152 was associated with increased cell viability, proliferation, progesterone and IGF-I release and reduced apoptosis and estradiol output. Moreover, miR-152 mitigated or prevented the effects of benzene on all the measured parameters in addition to estradiol release. The present observations suggest the toxic effect of benzene and the stimulatory influence of miR-152 on ovarian cell functions. Moreover, this is the first demonstration of the ability of miRNAs to mitigate and prevent the reproductive toxicity of benzene.

Mesenchymal stem cells of Oravka chicken breed: promising path to biodiversity conservation.

Mesenchymal stem cells (MSCs) are multilineage cells able to differentiate into other cell types. MSCs derived from bone marrow or compact bones are the most accessible stem cells used in tissue engineering. Therefore, the aim of this study was to isolate, characterize and cryopreserve MSCs of endangered Oravka chicken breed. MSCs were obtained from compact bones of the femur and tibiotarsus. MSCs were spindle-shaped and were able to differentiate into osteo-, adipo-, and chondrocytes under the specific differentiation conditions. Furthermore, MSCs were positive for surface markers such as CD29, CD44, CD73, CD90, CD105, CD146 and negative for CD34CD45 by flow cytometry. Moreover, MSCs demonstrated high positivity of "stemness" markers aldehyde dehydrogenase, alkaline phosphatase as well as for intracellular markers vimentin, desmin, α-SMA. Subsequently, MSCs were cryopreserved using 10% dimethyl sulfoxide in liquid nitrogen. Based on the results from the viability, phenotype, and ultrastructure assessment we can concluded that the MSCs were not negatively affected by the cryopreservation. Finally, MSCs of endangered Oravka chicken breed were successfully stored in animal gene bank, thus making them a valuable genetic resource.

Involvement of microRNA miR-125b in the control of porcine ovarian cell functions.

The existing knowledge of the involvement of miR-125b in the control of ovarian functions is insufficient. To evaluate the role of miR-125b in the control of basic porcine ovarian granulosa cell functions, we examined the upregulation (using miR-125b mimics) and downregulation (using miR-125b inhibitor) of this miR-125b. Expression levels of miR-125b, viability, proliferation (expression and accumulation of PCNA and cyclin B1), the proportion of proliferative active cells, apoptosis (expression and accumulation of bax and caspase 3), the proportion of cells containing DNA fragmentation, steroid hormones, IGF-I, oxytocin, and prostaglandin E2 release were analysed by RT-qPCR, Trypan blue exclusion test, quantitative immunocytochemistry, XTT and TUNEL assays, and ELISA. Transfection of cells with miR-125b mimics decreased cell viability, proliferation, and the release of progesterone, testosterone, estradiol, and oxytocin, but stimulated apoptosis and prostaglandin E2 output. Transfection of cells with miR-125b inhibitor had the opposite effect. Moreover, it prevented the effects of miR-125b mimics. Our observations suggest that miR-125b is a potent physiological inhibitor of granulosa ovarian cell functions - cell cycle, apoptosis, and secretory activity.

Interrelationships Between miR-34a and FSH in the Control of Porcine Ovarian Cell Functions.

Our study aimed to elucidate the effect of miR-34a mimics and miR-34a inhibitor and their combination on basic functions of ovarian cells cultured with and without FSH, and effect of FSH on expression of endogenous miR-34a. Viability, proliferation, proportion of proliferative active cells, apoptosis, proportion of DNA fragmented cells, accumulation of FSHR, steroid hormones, IGF-I, oxytocin, and prostaglandin E2 release, and expression levels of miR-34a were analysed. MiR-34a mimics decreased proliferation, apoptosis, testosterone, and estradiol output, stimulated release of progesterone, IGF-I, oxytocin, and occurrence of FSHR. MiR-34a inhibitor had an opposite effect and prevented effects of miR-34a mimics. FSH promoted expression of miR-34a, viability, proliferation, steroid hormones, IGF-I, oxytocin, and prostaglandin E2 output, and reduced apoptosis. Furthermore, miR-34a mimics supported effect of FSH on viability, apoptosis, progesterone, and IGF-I and inverted FSH action on proliferation, testosterone, and estradiol output. Our observations suggest that miR-34a can be involved in control of basic ovarian functions and that miR-34a and FSH are synergists in their actions on ovarian cell functions. Ability of FSH to promote miR-34a expression and ability of miR-34a mimics to increase occurrence of FSHR and to modify FSH effects suggest the existence of self-stimulating FSH-miR-34a axis, and that miR-34a can mediate actions of FSH on ovarian cells.

Comprehensive Flow-Cytometric Quality Assessment of Ram Sperm Intended for Gene Banking Using Standard and Novel Fertility Biomarkers.

Flow cytometry becomes a common method for analysis of spermatozoa quality. Standard sperm characteristics such as viability, acrosome and chromatin integrity, oxidative damage (ROS) etc. can be easily assess in any animal semen samples. Moreover, several fertility-related markers were observed in humans and some other mammals. However, these fertility biomarkers have not been previously studied in ram. The aim of this study was to optimize the flow-cytometric analysis of these standard and novel markers in ram semen. Ram semen samples from Slovak native sheep breeds were analyzed using CASA system for motility and concentration and were subsequently stained with several fluorescent dyes or specific antibodies to evaluate sperm viability (SYBR-14), apoptosis (Annexin V, YO-PRO-1, FLICA, Caspases 3/7), acrosome status (PNA, LCA, GAPDHS), capacitation (merocyanine 540, FLUO-4 AM), mitochondrial activity (MitoTracker Green, rhodamine 123, JC-1), ROS (CM-H2DCFDA, DHE, MitoSOX Red, BODIPY), chromatin (acridine orange), leukocyte content, ubiquitination and aggresome formation, and overexpression of negative biomarkers (MKRN1, SPTRX-3, PAWP, H3K4me2). Analyzed semen samples were divided into two groups according to viability as indicators of semen quality: Group 1 (viability over 60%) and Group 2 (viability under 60%). Significant (p < 0.05) differences were found between these groups in sperm motility and concentration, apoptosis, acrosome integrity (only PNA), mitochondrial activity, ROS production (except for DHE), leukocyte and aggresome content, and high PAWP expression. In conclusion, several standard and novel fluorescent probes have been confirmed to be suitable for multiplex ram semen analysis by flow cytometry as well as several antibodies have been validated for the specific detection of ubiquitin, PAWP and H3K4me2 in ram spermatozoa.

Basic ß-1,3-Glucanase from Drosera binata Exhibits Antifungal Potential in Transgenic Tobacco Plants.

The basic ß-1,3-glucanase of the carnivorous plant Drosera binata was tested as a purified protein, as well as under the control of a double CaMV35S promoter in transgenic tobacco for its capability to inhibit the growth of Trichoderma viride, Rhizoctonia solani, Alternaria solani, and Fusarium poae in an in-vitro assay. The purified protein inhibited tested phytopathogens but not the saprophytic fungus T. viride. Out of the analysed transgenic plants, lines 13, 16, 19, and 22 exhibited high DbGluc1 transcript abundance normalised to the actin transcript. Because of DbGluc1 transgene expression, lines 13 and 16 showed a 1.7-fold increase and lines 19 and 22 showed more than a 2-fold increase in total ß-1,3-glucanase activity compared to the non-transgenic control. In accordance with the purified ß-1,3-glucanase in-vitro antifungal assay, crude protein extracts of lines 19 and 22 significantly inhibited the growth of phytopathogens (14-34%). Further analyses revealed that the complementary action of transgenic ß-1,3-glucanase and 20% higher activity of endogenous chitinase(s) in these lines were crucial for maximising the antifungal efficiency of crude protein extracts.

Molecular Profiling and Gene Banking of Rabbit EPCs Derived from Two Biological Sources.

Endothelial progenitor cells (EPCs) have been broadly studied for several years due to their outstanding regenerative potential. Moreover, these cells might be a valuable source of genetic information for the preservation of endangered animal species. However, a controversy regarding their characterization still exists. The aim of this study was to isolate and compare the rabbit peripheral blood- and bone marrow-derived EPCs with human umbilical vein endothelial cells (HUVECs) in terms of their phenotype and morphology that could be affected by the passage number or cryopreservation as well as to assess their possible neuro-differentiation potential. Briefly, cells were isolated and cultured under standard endothelial conditions until passage 3. The morphological changes during the culture were monitored and each passage was analyzed for the typical phenotype using flow cytometry, quantitative real-time polymerase chain reaction (qPCR) and novel digital droplet PCR (ddPCR), and compared to HUVECs. The neurogenic differentiation was induced using a commercial kit. Rabbit cells were also cryopreserved for at least 3 months and then analyzed after thawing. According to the obtained results, both rabbit EPCs exhibit a spindle-shaped morphology and high proliferation rate. The both cell lines possess same stable phenotype: CD14-CD29+CD31-CD34-CD44+CD45-CD49f+CD73+CD90+CD105+CD133-CD146-CD166+VE-cadherin+VEGFR-2+SSEA-4+MSCA-1-vWF+eNOS+AcLDL+ALDH+vimentin+desmin+α-SMA+, slightly different from HUVECs. Moreover, both induced rabbit EPCs exhibit neuron-like morphological changes and expression of neuronal markers ENO2 and MAP2. In addition, cryopreserved rabbit cells maintained high viability (>85%) and endothelial phenotype after thawing. In conclusion, our findings suggest that cells expanded from the rabbit peripheral blood and bone marrow are of the endothelial origin with a stable marker expression and interesting proliferation and differentiation capacity.

Phenotypical Characterization and Neurogenic Differentiation of Rabbit Adipose Tissue-Derived Mesenchymal Stem Cells.

Although the rabbit is a frequently used biological model, the phenotype of rabbit adipose-derived mesenchymal stem cells (rAT-MSCs) is not well characterized. One of the reasons is the absence of specific anti-rabbit antibodies. The study aimed to characterize rAT-MSCs using flow cytometry and PCR methods, especially digital droplet PCR, which confirmed the expression of selected markers at the mRNA level. A combination of these methods validated the expression of MSCs markers (CD29, CD44, CD73, CD90 and CD105). In addition, cells were also positive for CD49f, vimentin, desmin, α-SMA, ALDH and also for the pluripotent markers: NANOG, OCT4 and SOX2. Moreover, the present study proved the ability of rAT-MSCs to differentiate into a neurogenic lineage based on the confirmed expression of neuronal markers ENO2 and MAP2. Obtained results suggest that rAT-MSCs have, despite the slight differences in marker expression, the similar phenotype as human AT-MSCs and possess the neurodifferentiation ability. Accordingly, rAT-MSCs should be subjected to further studies with potential application in veterinary medicine but also, in case of their cryopreservation, as a source of genetic information of endangered species stored in the gene bank.

The toxic influence of silver and titanium dioxide nanoparticles on cultured ovarian granulosa cells.

The application of metal nanoparticles in modern society is growing, but there is insufficient data concerning their influence on reproductive processes and comparison of their biological activity. The present experiments aimed to compare the effects of silver and titanium dioxide nanoparticles (AgNPs and TiO2NPs) on ovarian granulosa cell functions. AgNPs and TiO2NPs were added to culture of porcine granulosa cells at doses 0, 0.01, 0.1, 1 or 10 µg/mL. The mRNAs for proliferating cell nuclear antigen (PCNA), cyclin B1, bax and caspase 3 were quantified by RT-PCR; release of progesterone was analyzed by ELISA. It was shown that both AgNPs and TiO2NPs significantly reduced all the measured parameters. ED50 of the inhibitory influence of AgNPs on the main ovarian cell parameters was higher than ED50 of TiO2NPs. The ability of AgNPs and TiO2NPs to suppress ovarian granulosa cell functions should be taken into account by their application.

Combined approach for characterization and quality assessment of rabbit bone marrow-derived mesenchymal stem cells intended for gene banking.

Rabbit mesenchymal stem cells (rMSCs) are promising agents for the preservation of genetic biodiversity in domestic rabbit breeds. However, rMSCs must meet certain requirements to be used for cryopreservation in animal gene banks. Currently, there are numerous discrepancies in the published data regarding the rMSC phenotype, which may complicate efforts to evaluate their purity and suitability for reuse after cryopreservation in gene and tissue banks. We propose a combined approach (flow cytometry, PCR, differentiation and ultrastructure studies) for the characterization and recovery of rMSCs after cryopreservation. Flow cytometric analyses of rMSCs confirmed the expression of CD29, CD44, vimentin, desmin and α-SMA. RT-PCR revealed the expression of other markers at the mRNA level (SSEA-4, CD73, CD90, CD105, CD146 and CD166). rMSCs showed efficient multilineage differentiation into adipo-, chondro- and osteogenic lineages, SOX2 expression (pluripotency) and typical MSC morphology and ultrastructure. The confirmed rMSCs were subsequently used for cryopreservation. Efficient recovery of rMSCs after cryogenic freezing was demonstrated by high cell viability, normal ultrastructure of reseeded rMSCs, high expression of CD29 and CD44 and lineage differentiation capacity. The proposed combined approach could be used for characterization, cryopreservation and recovery of rMSCs as genetic resources for native rabbit breeds.

Quercetin directly inhibits basal ovarian cell functions and their response to the stimulatory action of FSH.

Plants, fruits, and vegetables containing the bioflavonoid quercetin are widely used in food, beverages, and medicines; however, the effects of quercetin on reproductive processes and the possible mechanisms of quercetin action require extensive investigation. The aim of our study was to examine the direct effects of quercetin on basic ovarian cell functions and their response to follicle-stimulating hormone (FSH) and insulin-like growth factor I (IGF-I), known hormonal stimulators of reproduction. We analyzed the effects of quercetin alone (0, 1, 10, and 100 ng/ml) on cultured porcine ovarian granulosa cells or isolated ovarian follicles; or of quercetin (10 ng/ml) in combination with FSH (0, 0.01, 0.1, or 1 IU/ml) or IGF-I (0, 1, 10, or 100 ng/ml) on cultured porcine granulosa cells. The expression of proliferative (PCNA, cyclin B1) and apoptotic (BAX) markers, as well as markers for release of progesterone (P4), testosterone (T), and leptin (L), were measured by quantitative immunocytochemistry, Western immunoblotting, RT-qPCR, and EIA/RIA. Addition of quercetin reduced the accumulation of PCNA and cyclin B1, as well as their transcript levels, promoted the accumulation of BAX, decreased the release of P4 and L, and increased the release of T in cultured granulosa cells. In ovarian follicles, quercetin reduced the levels of both P4 and T. Exposure to FSH stimulated PCNA and decreased BAX accumulation, and increased the release of P4, T, and L. Quercetin inhibited and even reversed the effects of FSH. Like FSH, IGF-I also promoted granulosa cell proliferation and suppressed apoptosis. Quercetin did not modify IGF-I effects. These data suggest that the plant molecule quercetin can directly down-regulate basal ovarian cell functions (proliferation, apoptosis, and release of ovarian steroid and peptide hormones) and their response to the stimulatory activity of the upstream hormonal stimulator FSH.

Survivability of rabbit amniotic fluid-derived mesenchymal stem cells post slow-freezing or vitrification.

This work aimed to evaluate the effect of two distinct cryopreservation procedures - conventional slow-freezing and vitrification, on survivability and mesenchymal marker expression stability of rabbit amniotic fluid-derived mesenchymal stem cells (rAF-MSCs). Cells at passage 2 were slowly frozen, using 10% of dimethylsulfoxide, or vitrified, using 40% of ethylene glycol, 0.5 M sucrose and 18% Ficoll 70. After three months storage in liquid nitrogen, viability, chromosomal stability, ultrastructure, surface and intracellular marker expression and differentiation potential of cells were evaluated immediately post-thawing/warming and after additional culture for 48-72 h. Our results showed decreased (P ≤ 0.05) viability of cells post-thawing/warming. However, after additional culture, the viability was similar to those in fresh counterparts in both cryopreserved groups. Increase (P ≤ 0.05) in the population doubling time of vitrified cells was observed, while doubling time of slow-frozen cells remained similar to non-cryopreserved cells. No changes in karyotype (chromosomal numbers) were observed in frozen/vitrified AF-MSCs, and histological staining confirmed similar differentiation potential of fresh and frozen/vitrified cells. Analysis of mesenchymal marker expression by qPCR showed that both cryopreservation approaches significantly affected expression of CD73 and CD90 surface markers. These changes were not detected using flow cytometry. In summary, the conventional slow-freezing and vitrification are reliable and effective approaches for the cryopreservation of rabbit AF-MSCs. Nevertheless, our study confirmed affected expression of some mesenchymal markers following cryopreservation.

Soil erosion as a source of sediment and phosphorus in rivers and reservoirs - Watershed analyses using WaTEM/SEDEM.

Spatially distributed modelling of sediment and phosphorus fluxes on a scale of thousands of square kilometers always involves a compromise between the quality of the data input and the complexity of the model that can be applied. WaTEM/SEDEM offers an approach that allows us to target on spatially focused outputs that can easily be implemented in the decision-making process for effective watershed control. The results for a study area covering the watersheds of 58 large reservoirs threatened by eutrophication within the Czech Republic are presented here as an example of the available analyses. The total area of the watersheds is 27,472 km2. After building a complex river topology scheme and estimating the trap efficiencies in all reservoirs within the river networks, we are able to estimate the total transport efficiency of each river unit for any outlet point (terminal reservoir). The sources of the greatest amounts of sediment (phosphorus) can be identified on the scale of single parcels. According the model, the total soil loss in the study area is 7487 Gg year-1 (2.73 Mg ha-1 year-1). The total sediment entry into the river systems in the target area is 1705 Gg year-1 (15.2% of the total soil loss). The total deposition in the 9890 water reservoirs of various sizes in the target area is 1139 Gg year-1. This means that the deposition in the landscape is 5.1× higher than the deposition in the reservoirs within the study area. The mean annual sediment transport by all watershed outlets is 566 Gg year-1. The cost of dredging the sediment would be about 12.8 million EUR year-1. There is great spatial variability in the deposition and transport processes, but it is imperative to provide strengthened soil protection directly on-site, especially in watersheds where the sediment delivery ratio is much higher than the average value. Phosphorus transported by water erosion is an important element in the balances of phosphorus sources in basins. Sewage waters usually play the predominant role in triggering the eutrophication effect, but there are also reservoirs where erosion-based phosphorus plays a major role.

Risk to residents, infrastructure, and water bodies from flash floods and sediment transport.

Intense rainfall-runoff events and subsequent soil erosion can cause serious damage to the infrastructure in residential areas in Europe countries and all over the world. In the Czech Republic, the Ministry of the Interior has supported an analysis dealing with the risks to residents, infrastructure, and water bodies from flash floods and sediment transport. A total of more than 150,000 risk points were identified by GIS morphology and land-use analysis. The threat, the vulnerability, and the resulting risk category were determined for each of these points. The WaTEM/SEDEM model was used to assess the threat with 10-m data resolution. The summarized vulnerability of real objects on individual runoff trajectories was combined with the threat of sediment transport, resulting in the overall risk represented by a 5-degree scale, from lowest (1) to highest (5). The output of the project lies stored in the WEB application. Nineteen percent of the sites in the Czech Republic, i.e., more than 23,000 sites, have been assigned to categories 4 and 5, with a high level of risk. Thirty-four percent of cadastral units are classified as the high risky (4416 cadasters, with a total area 24,707 km2). Approximately 30% of the population of the Czech Republic lives in high-risk cadastral areas. Four scenarios of protection were modeled. To reduce the high-risk and very high-risk sites (categories 4 and 5), the most effective solution is the implementation of technical measures or conversion to grassland within the contributing watersheds. This could reduce the number of high-risk sites from 23,400 to 3700.Methods of sediment transport modeling and risk evaluation, based on presented USLE input data and documented WaTEM/SEDEM model, can be used worldwide. Especially in post-soviet union countries with shared arable land development and erosion consequences.

Critical assessment of the efficiency of CD34 and CD133 antibodies for enrichment of rabbit hematopoietic stem cells.

Rabbits have many hereditary diseases common to humans and are therefore a valuable model for regenerative disease and hematopoietic stem cell (HSC) therapies. Currently, there is no substantial data on the isolation and/or enrichment of rabbit HSCs. This study was initiated to evaluate the efficiency of the commercially available anti-CD34 and anti-CD133 antibodies for the detection and potential enrichment of rabbit HSCs from peripheral blood. PBMCs from rabbit and human blood were labelled with different clones of anti-human CD34 monoclonal antibodies (AC136, 581, and 8G12) and rabbit polyclonal CD34 antibody (pCD34) and anti-human CD133 monoclonal antibodies (AC133 and 293C3). Flow cytometry showed a higher percentage of rabbit CD34+ cells labelled by AC136 in comparison to the clone 581 and pCD34 (P < 0.01). A higher percentage of rabbit CD133+ cells were also detected by 293C3 compared to the AC133 clone (P < 0.01). Therefore, AC136 clone was used for the indirect immunomagnetic enrichment of rabbit CD34+ cells using magnetic-activated cell sorting (MACS). The enrichment of the rabbit CD34+ cells after sorting was low in comparison to human samples (2.4% vs. 39.6%). PCR analyses confirmed the efficient enrichment of human CD34+ cells and the low expression of CD34 mRNA in rabbit positive fraction. In conclusion, the tested antibodies might be suitable for detection, but not for sorting the rabbit CD34+ HSCs and new specific anti-rabbit CD34 antibodies are needed for efficient enrichment of rabbit HSCs. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 2018 © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:1278-1289, 2018.

[Parallel investigations of blood and alveolar air in the diagnosis of lethal carbon monoxide poisoning]. / Súbezné vysetrovanie krvi a alveolárneho vzduchu v diagnostike letálnych intoxikácií oxidom uholnatým.

Carbon monoxide poisonings are the most frequent among fatal gas and volatile substances intoxications. The authors present diagnostic options of fatal carbon monoxide poisoning by parallel blood investigations for the content of karbonylhaemoglobin and determining of the concentration of carbon monoxide in the alveolar air. The analysis of 160 cases of lethal poisonings with carbon monoxide over a period of 20 years was carried out. The cases were divided into subgroups according to the place of death to poisonings in flats, garages, bathrooms, in fires, road traffic accidents and mining accidents. Tabular cases were divided into poisonings with a dominant share of carbon monoxide; the lethal concentration (0.075 % volume percentage or more) in the alveolar air was found in 75 % of cases. By pairs of victims different concentrations of carbon monoxide in the alveolar air were found. It confirms the fact that the course of intoxication and time of death also depends on the state of health of an individual. In 25 % of cases composite action with other toxic substances and factors (cyanide ions, ethanol, carbon dioxide, smoke inhalation solids, burns etc.) was detected. The obtained results point to an important and irreplaceable role of the toxicological - chemical analysis of the alveolar air in the context of additional laboratory investigations at autopsy in the diagnosis of fatal carbon monoxide poisoning. Investigation of the alveolar air should be the standard methodological procedure in the diagnosis of fatal poisonings by gases and volatile substances.

Different RNA and protein expression of surface markers in rabbit amniotic fluid-derived mesenchymal stem cells.

Over the years, there has been much confusion in defining molecular markers of mesenchymal stem cells (MSCs) for other than human species due to a lack of species-specific antibodies. Therefore, the aim of our study was to define rabbit amniotic fluid-derived mesenchymal stem cells (rAF-MSCs) and to reflect upon the current identification of AF-MSCs by providing a summary of detected surface markers in different species. The expression of rAF-MSC surface markers was analyzed at the protein and mRNA level. Flow cytometry analyses showed that rAF-MSCs were positive for CD29 and CD44, low positive for CD90, but negative for CD73, CD105, and CD166. Interestingly, RT-PCR (reverse transcription-polymerase chain reaction) exposed a discprepancy between transcribed mRNA and protein expression, as the cells expressed mRNA of all MSC markers: CD29, CD44, CD73, CD90, CD105, and CD166. Our results also confirmed the mesenchymal nature of isolated cells by morphology, ultrastructure, and intracellular marker expression profile. In addition, the expression of few pluripotency markers was also detected. We also found that passaging did not affect apoptosis and viability. Similarly, changes in karyotype were not observed during passaging. In conclusion, the provided characteristics may be used as a comprehensive set of criteria to define and characterize rAF-MSCs, required for the identification of these cells in preclinical investigations. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1601-1613, 2017.

Direct effect of curcumin on porcine ovarian cell functions.

Curcuma longa Linn (L.) is a plant widely used in cooking (in curry powder a.o.) and in folk medicine, but its action on reproductive processes and its possible mechanisms of action remain to be investigated. The objective of this study was to examine the direct effects of curcumin, the major Curcuma longa L. molecule, on basic ovarian cell functions such as proliferation, apoptosis, viability and steroidogenesis. Porcine ovarian granulosa cells were cultured with and without curcumin (at doses of 0, 1, 10 and 100µg/ml of medium). Markers of proliferation (accumulation of PCNA) and apoptosis (accumulation of bax) were analyzed by immunocytochemistry. The expression of mRNA for PCNA and bax was detected by RT-PCR. Cell viability was detected by trypan blue exclusion test. Release of steroid hormones (progesterone and testosterone) was measured by enzyme immunoassay (EIA). It was observed that addition of curcumin reduced ovarian cell proliferation (expression of both PCNA and its mRNA), promoted apoptosis (accumulation of both bax and its mRNA), reduced cell viability, and stimulated both progesterone and testosterone release. These observations demonstrate the direct suppressive effect of Curcuma longa L./curcumin on female gonads via multiple mechanisms of action - suppression of ovarian cell proliferation and viability, promotion of their apoptosis (at the level of mRNA transcription and subsequent accumulation of promoters of genes regulating these activities) and release of anti-proliferative and pro-apoptotic progesterone and androgen. The potential anti-gonadal action of curcumin should be taken into account by consumers of Curcuma longa L.-containing products.

Structural and functional characterisation of a class I endochitinase of the carnivorous sundew (Drosera rotundifolia L.).

MAIN CONCLUSION: Chitinase gene from the carnivorous plant, Drosera rotundifolia , was cloned and functionally characterised. Plant chitinases are believed to play an important role in the developmental and physiological processes and in responses to biotic and abiotic stress. In addition, there is growing evidence that carnivorous plants can use them to digest insect prey. In this study, a full-length genomic clone consisting of the 1665-bp chitinase gene (gDrChit) and adjacent promoter region of the 698 bp in length were isolated from Drosera rotundifolia L. using degenerate PCR and a genome-walking approach. The corresponding coding sequence of chitinase gene (DrChit) was obtained following RNA isolation from the leaves of aseptically grown in vitro plants, cDNA synthesis with a gene-specific primer and PCR amplification. The open reading frame of cDNA clone consisted of 978 nucleotides and encoded 325 amino acid residues. Sequence analysis indicated that DrChit belongs to the class I group of plant chitinases. Phylogenetic analysis within the Caryophyllales class I chitinases demonstrated a significant evolutionary relatedness of DrChit with clade Ib, which contains the extracellular orthologues that play a role in carnivory. Comparative expression analysis revealed that the DrChit is expressed predominantly in tentacles and is up-regulated by treatment with inducers that mimick insect prey. Enzymatic activity of rDrChit protein expressed in Escherichia coli was confirmed and purified protein exhibited a long oligomer-specific endochitinase activity on glycol-chitin and FITC-chitin. The isolation and expression profile of a chitinase gene from D. rotundifolia has not been reported so far. The obtained results support the role of specific chitinases in digestive processes in carnivorous plant species.

[Post mortem investigation of alveolar air in forensic practice]. / Vysetrovanie alveolárneho vzduchu post mortem v súdnolekárskej praxi.

Exhaled air from biological materials is used for the purpose of toxicologico-chemical analysis particularly in detecting of alcohol influence in traffic or within a working process. Similarly, still a more and more actual requirement seems to be a necessity to analyse alveolar air from necroptic material. This necessity is emphasised not only by the fact, that inhalant intoxications in the form of poisoning represent their substantial part, but also a demand to evidence exactly a noxious agent both specifically and also in at least two materials. And particularly this is sometimes a subject to search for evidence in alveolar air. The authors present their authors certificates for Isolation of alveolar air from autopsy material, issued by the Office for Patents and Inventions in Prague, verified in practice, both by destruction of lung tissue, both the evacuation of alveolar air into a defined volume, hermetically closed space that is used for the toxicological analysis of gaseous and volatile xenobiotics. They propose experimentally verified procedures and conditions obtained by time-consuming examination. To isolation procedures were subjected an adequate number of lung lobes from lungs removed in 201 autopsy cases. The authors practically tested the presupposed amount of alveolar air in individuals according to gender and age. Gradually they have validated various prototypes and optimization methods and their application in solving particular inhaled lethal intoxications and deaths in the irrespirable environment.