Changes in plasma PLAC-1 concentration and its expression during early-mid pregnancy in bovine placental tissues - a pilot study.

BACKGROUND: Placenta-specific protein 1 (PLAC1) is a small secreted protein considered to be a molecule with a significant role in the development of the placenta and the establishment of the mother-foetus interface. This study aimed to confirm the presence of bovine PLAC1 and to examine its profile in the placenta and plasma in the first six months of pregnancy. The expression pattern of PLAC1 was analysed by RT-qPCR and Western Blotting. Quantitative evaluation was carried out using ELISA. RESULTS: PLAC1 concentrations in the plasma of pregnant cows were significantly higher (p < 0.05) than those obtained from non-pregnant animals. PLAC1 protein concentrations in the placental tissues of the foetal part were significantly (p < 0.05) higher than in the tissues of the maternal part of the placenta. PLAC1 transcripts were detected in both placental tissue samples and epithelial cell cultures. CONCLUSIONS: In conclusion, the results of the present preliminary study suggest that PLAC1 is involved in the development of bovine placenta. The presence of this protein in the plasma of pregnant animals as early as the first month may make it a potential candidate as a pregnancy marker in cows. Further studies on exact mechanisms of action of PLAC1 in bovine placenta are necessary.

Analysis of multiple biomarkers revealed the size matters of Cu particles for barley response under foliar exposure.

The impact of particle size of engineered nanoparticles (ENPs) on plant response has marginally been investigated under the foliar application so far. Concerning the significance of particle diameter for their properties and interaction with plants, the effect of size should be considered in the analysis of the effect of micronutrient-based ENPs on plants. It is of particular importance for ENPs containing Cu due to plants needing a relatively low amount of this element, thus there is a risk of overdosing during application as a fertilizer or pesticide. Here, we examined the biochemical and transcriptional response of barley (Hordeum vulgare L.) to Cu nanoparticles (nano-Cu) with different diameters (25 nm, 50 nm, 70 nm), microparticles (micro-Cu), and chelated Cu (EDTA-Cu). The plants suffering from Cu deficiency were foliar sprayed with Cu compounds at 1000 mg/L during the tillering stage. 1- and 7-day plants were analyzed in terms of biomass, Cu content, the activity of enzymes involved with antioxidant response, the content of low molecular weight compounds, and the expression of genes regulated metal homeostasis, aquaporins, and defense. The results showed that the Cu leaf level was differentiated over time and after 7 days it was higher under exposure to the smallest nano-Cu than other particulate Cu. Regardless of the duration of exposure, the Cu content was highest in plants treated with Cu-EDTA. The cluster analysis of all markers revealed a clear distinct response to the smallest nano-Cu and other particulate and ionic treatments. The bigger nano-Cu, depending on the markers, caused the medium effects between the nano-Cu 25 nm and micro-Cu and Cu-EDTA. The found size thresholds at the nanoscale will be useful for the fabrication of safe-by-design agrochemicals to provide crop security and attenuate environmental impact.

Transcriptional response of Cu-deficient barley (Hordeum vulgare L.) to foliar-applied nano-Cu: Molecular crosstalk between Cu loading into plants and changes in Cu homeostasis genes.

For safe and effective nutrient management, the cutting-edge approaches to plant fertilization are continuously developed. The aim of the study was to analyze the transcriptional response of barley suffering from Cu deficiency to foliar application of nanoparticulate Cu (nano-Cu) and its ionic form (CuSO4) at 100 and 1000 mg L-1 for the examination of their supplementing effect. The initial interactions of Cu-compounds with barley leaves were analyzed with spectroscopic (ICP-OES) and microscopic (SEM-EDS) methods. To determine Cu cellular status, the impact of Cu-compounds on the expression of genes involved in regulating Cu homeostasis (PAA1, PAA2, RAN1, COPT5), aquaporins (NIP2.1, PIP1.1, TIP1.1, TIP1.2) and antioxidant defense response (SOD CuZn, SOD Fe, SOD Mn, CAT) after 1 and 7 days of exposure was analyzed. Although Cu accumulation in plant leaves was detected overtime, the Cu content in leaves exposed to nano-Cu for 7 days was 44.5% lower than in CuSO4 at 100 mg L-1. However, nano-Cu aggregates remaining on the leaf surface indicated a potential difference between measured Cu content and the real Cu pool present in the plant. Our study revealed significant changes in the pattern of gene expression overtime depending on Cu-compound type and dose. Despite the initial puzzling patterns of gene expression, after 7 days all Cu transporters showed significant down-regulation under Cu-compounds exposure to prevent Cu excess in plant cells. Conversely, aquaporin gene expression was induced after 7 days, especially by nano-Cu and CuSO4 at 100 mg L-1 due to the stimulatory effect of low Cu doses. Our study revealed that the gradual release of Cu ions from nano-Cu at a lower rate provided a milder molecular response than CuSO4. It might indicate that nano-Cu maintained better metal balance in plants than the conventional compounds, thus may be considered as a long-term supplier of Cu.

Reference genes expression stability in Avena sativa L. during compatible and incompatible interactions with Puccinia graminis.

A reliable qPCR experiment requires the selection of reference genes with a stable level of expression in a given experimental system. This study attempts to determine the reference genes (RGs) for the A. sativa-P. graminis experimental setup. We evaluated nine candidate reference genes in A. sativa (oat line Pg4 and the cultivar Kasztan) during compatible and incompatible interactions with different pathotypes of Puccinia graminis f. sp. avenae in six time points post-inoculation. The identification of genes with high expression stability was performed by four algorithms (geNorm, NormFinder, BestKeeper and ΔCt method). We found that the most appropriate combination of RGs for RT-qPCR data normalization were HNR (heterogeneous nuclear ribonucleoprotein 27C) + EF1A (elongation factor 1-alpha) + EIF4A (eukaryotic initiation factor 4A-3). The worst candidates for normalization in this dataset were CYP (cyclophilin) and TUA (alpha tubulin). Identified reference genes are suitable candidates for the standardization of gene expression studies in the A. sativa-P. graminis interaction system and potentially other related pathogens. To date, this is the first report of RGs selection in this pathosystem.

Effect of Sex Steroids and PGF2α on the Expression of Their Receptors and Decorin in Bovine Caruncular Epithelial Cells in Early-Mid Pregnancy.

Changes in the expression of various genes, including pregnancy-associated hormone receptors and extracellular matrix proteins, have been suggested to play a significant role in bovine placental development. This study aimed to examine the influence of sex steroids and PGF2α on decorin (DCN) expression in the epithelial cells of bovine caruncle in early−mid pregnancy in cows. The expression patterns of DCN, PTGFR, PGR and ESR1 were analyzed by RT-qPCR and Western blotting in primary caruncular epithelial cell cultures (PCECC) and placental tissue homogenates derived from the 2nd and 4th months of pregnancy. PCECC were found to express DCN, PTGFR, PGR and ESR1. The intensity of PGR staining was higher in cells derived from the 4th month of pregnancy (p < 0.05). The 17ß-estradiol, progesterone and PGF2α have not been shown to affect DCN expression. PGF2α decreased PTGFR expression in cells derived from the 4th month of gestation (p < 0.05). In conclusion, the results of the present preliminary study showed that the expression of the PTGFR, ESR1, PGR and DCN in PCECC does not vary throughout early−mid pregnancy. Further studies should be carried out to observe the relationship between hormonal status and cellular adhesion to determine their importance for properly developing placentation and pregnancy in cows.

One Host-Multiple Applications: Zebrafish (Danio rerio) as Promising Model for Studying Human Cancers and Pathogenic Diseases.

In recent years, zebrafish (ZF) has been increasingly applied as a model in human disease studies, with a particular focus on cancer. A number of advantages make it an attractive alternative for mice widely used so far. Due to the many advantages of zebrafish, modifications can be based on different mechanisms and the induction of human disease can take different forms depending on the research goal. Genetic manipulation, tumor transplantation, or injection of the pathogen are only a few examples of using ZF as a model. Most of the studies are conducted in order to understand the disease mechanism, monitor disease progression, test new or alternative therapies, and select the best treatment. The transplantation of cancer cells derived from patients enables the development of personalized medicine. To better mimic a patient's body environment, immune-deficient models (SCID) have been developed. A lower immune response is mostly generated by genetic manipulation but also by irradiation or dexamethasone treatment. For many studies, using SCID provides a better chance to avoid cancer cell rejection. In this review, we describe the main directions of using ZF in research, explain why and how zebrafish can be used as a model, what kind of limitations will be met and how to overcome them. We collected recent achievements in this field, indicating promising perspectives for the future.

Validation of reference genes as an internal control for studying Avena sativa-Puccinia coronata interaction by RT-qPCR.

In this study we evaluated eleven candidate reference genes in Avena sativa during compatible and incompatible interactions with two different pathotypes of Puccinia coronata f. sp. avenae in six time points post-inoculation. The identification of genes with high expression stability was performed by four algorithms (geNorm, NormFinder, BestKeeper and ΔCt method). The results obtained confirmed that the combination of two genes would be sufficient for reliable normalization of the expression data. In general, the most stable in the tested plant-pathogen system were HNR (heterogeneous nuclear ribonucleoprotein 27C) and EF1A (elongation factor 1-alpha). ARF (ADP-ribosylation factor) and EIF4A (eukaryotic initiation factor 4A-3) could also be considered as exhibiting high expression stability. CYP (cyclophilin) was shown by all assessment methods to be the worst candidate for normalization in this dataset. To date, this is the first report of reference genes selection in A. sativa-P. coronata interaction system. Identified reference genes enable reliable and comprehensive RT-qPCR analysis of oat gene expression in response to crown rust infection. Understanding the molecular mechanisms involved in the host-pathogen interactions may expand knowledge of durable resistance strategies beneficial to modern oat breeding.

Reference gene selection in bovine caruncular epithelial cells under pregnancy-associated hormones exposure.

Examination of transcriptional regulation occurring during pregnancy establishment and maintenance requires the identification of endogenous reference genes characterized by high expression stability. Since the expression of some reference genes may be modulated by pregnancy-associated hormones, the goal of our study was to identify suitable reference genes unaffected by hormonal treatment. In our study bovine caruncular epithelial cells were subjected to progesterone, estrogen and prostaglandin F2α treatment. Ten candidate reference genes (ACTR1A, CNOT11, HDAC1, HPRT1, RPL19, RPS9, SDHA, SUZ12, UXT and ZNF131) were evaluated with the use of four approaches (geNorm, NormFinder, BestKeeper, delta Ct). We found that RPS9 and SUZ12 displayed the highest expression stability in the tested material. Moreover, HPRT1 and SDHA were found inappropriate for RT-qPCR data normalization as they demonstrated the highest expression variability out of all candidates analysed. Hence geNorm calculations shown that the use of just two best-performing genes would be sufficient for obtaining reliable results, we propose that RPS9 and SUZ12 be used as suitable endogenous controls in future studies investigating gene expression in normal and compromised pregnancies.

Carex muskingumensis and Osmotic Stress: Identification of Reference Genes for Transcriptional Profiling by RT-qPCR.

Carex muskingumensis is a highly valued perennial ornamental grass cultivated worldwide. However, there is limited genetic data regarding this species. Selection of proper reference genes (RGs) for reverse transcription quantitative PCR (RT-qPCR) data normalization has become an essential step in gene expression analysis. In this study, we aimed to examine expression stability of nine candidate RGs in C. muskingumensis plants, subjected to osmotic stress, generated either by salinity or PEG treatment. The identification of genes exhibiting high expression stability was performed by four algorithms (geNorm, NormFinder, BestKeeper and deltaCt method). The results showed that the combination of two genes would be sufficient for reliable expression data normalization. ADP (ADP-ribosylation factor) and TBP (TATA-box-binding protein) were identified as the most stably expressed under salinity treatment, while eIF4A (eukaryotic initiation factor 4A) and TBP were found to show the highest stability under PEG-induced drought. A set of three genes (ADP, eIF4A and TBP) displayed the highest expression stability across all experimental samples tested in this study. To our best knowledge, this is the first report regarding RGs selection in C. muskingumensis. It will provide valuable starting point information for conducting further analyses in this and related species concerning their responses to water shortage and salinity stress.

Identification of stable reference genes for qPCR studies in common wheat (Triticum aestivum L.) seedlings under short-term drought stress.

BACKGROUND: Quantitative PCR (qPCR) is one of the most common and accurate methods of gene expression analysis. However, the biggest challenge for this kind of examinations is normalization of the results, which requires the application of dependable internal controls. The selection of appropriate reference genes (RGs) is one of the most crucial points in qPCR data analysis and for correct assessment of gene expression. Because of the fact that many reports indicate that the expression profiles of typically used RGs can be unstable in certain experimental conditions, species or tissues, reference genes with stable expression levels should be selected individually for each experiment. In this study, we analysed a set of ten candidate RGs for wheat seedlings under short-term drought stress. Our tests included five 'traditional' RGs (GAPDH, ACT, UBI, TUB, and TEF1) and five novel genes developed by the RefGenes tool from the Genevestigator database. RESULTS: Expression stability was assessed using five different algorithms: geNorm, NormFinder, BestKeeper, RefFinder and the delta Ct method. In the final ranking, we identified three genes: CJ705892, ACT, and UBI, as the best candidates for housekeeping genes. However, our data indicated a slight variation between the different algorithms that were used. We revealed that the novel gene CJ705892, obtained by means of in silico analysis, showed the most stable expression in the experimental tissue and condition. CONCLUSIONS: Our results support the statement, that novel genes selected for certain experimental conditions have a more stable level of expression in comparison to routinely applied RGs, like genes encoding actin, tubulin or GAPDH. Selected CJ705892 gene can be used as a housekeeping gene in the expression analysis in wheat seedlings under short-term drought. The results of our study will be useful for subsequent analyses of gene expression in wheat tissues subjected to drought.

Antioxidative system response of pedunculate oak (Quercus robur L.) seedlings to Cd exposure.

The use of pedunculate oak (Quercus robur L.), along with other tree species, for the afforestation of heavy metal contaminated lands is an attractive prospect. Little, however, is known of Q. robur tolerance and its antioxidative system response to heavy metal exposure. The main objective of the study was to determine the cadmium-induced changes in antioxidative system of pedunculate oak in an attempt to identify molecular mechanisms underlying Cd tolerance. This may be of great importance in respect of using Q. robur for phytoremediation purposes. As the response of the antioxidative system to heavy metal contamination can vary within species, the research was conducted on oak seedlings from two different regions of origin. Differences in antioxidative system response of seedlings derived from tested regions of origin were noticed both at the transcript and enzyme activity levels. The obtained results indicate that ascorbate peroxidase (APX; EC 1.11.1.11) and superoxide dismutase (SOD; EC 1.15.1.1) play a first barrier role in oak seedlings response to the oxidative stress caused by Cd exposure. Catalase (CAT; EC 1.11.1.6) is involved in reducing the negative effects of prolonged Cd treatment.

Is it useful to use several "omics" for obtaining valuable results?

The integration of cell communication and the transfer of signals from stimuli via transcription to translation and further to activation of new protein is crucial for appropriate metabolism and function of living organisms. The overall elucidation and the examination of these complex processes require multistep laboratory approaches in order to obtain results which will not only detect particular stage but also indicate the mechanisms lying upon this process. Such results will be reliable because they will cover multidirectional methods and approaches. The analysis of currently available results already provided with the conclusion that often single omics approach does not correspond with other expected information and may bring misinterpretations. That is why the integration of several "omics" is useful for searching entire explanations and answers as well as appropriate interpretation of obtained complex results. The hypothesis was stated that "from transcriptomics can not be concluded to proteomics". This review focuses on the reasons for the integration of transcriptomic, proteomic and other-omics analysis. Moreover it also describes the examples of clinical meanings and mentions some methods used in these approaches.