The potential antitumor effect of chrysophanol in relation to cervical cancer cells.

Chrysophanol is an anthraquinone with proven antitumor activity against several tumor cell lines. However, its effect on cervical cancer cells is still unknown. Therefore, HeLa cells were exposed to various concentrations of chrysophanol and then subjected to biochemical, ultrastructural, and morphological analysis. It has been shown using flow cytometry and MTT reduction assay that chrysophanol has been shown to inhibit cell viability and arrest cells in the G2/M phase of the cell cycle. Using Annexin V/propidium iodide staining, a significant increase in apoptosis was found after chrysophanol treatment on HeLa cells, and this process was mediated by caspases 3/7 with a clear inactivation of the antiapoptotic Bcl-2 family protein. However, the demonstrated increased number of cells with double-stranded DNA breaks suggests that chrysophanol also causes DNA damage. By means of electron and fluorescence microscopy, a clear effect of chrysophanol on the intensification of degradation processes, on changes in the structure of the nucleus, endoplasmic reticulum and mitochondria was demonstrated. The changes visible in the mitochondria may be related to the increase in the level of free radicals induced by chrysophanol, which induces apoptosis, inter alia, by increasing the permeability of mitochondrial membranes. The range of observed changes depended on the concentration of anthraquinone was tested.

The intensity of physiological and behavioral responses of horses to predator vocalizations.

BACKGROUND: Predatory attacks on horses can become a problem in some parts of the world, particularly when considering the recovering gray wolf populations. The issue studied was whether horses transformed by humans and placed in stable-pasture environments had retained their natural abilities to respond to predation risk. The objective of the study was to determine the changes in cardiac activity, cortisol concentrations, and behavior of horses in response to the vocalizations of two predators: the gray wolf (Canis lupus), which the horses of the breed studied had coevolved with but not been exposed to recently, and Arabian leopard (Panthera pardus nimr), from which the horses had been mostly isolated. In addition, we hypothesized that a higher proportion of Thoroughbred (TB) horse ancestry in the pedigree would result in higher emotional excitability in response to predator vocalizations. Nineteen horses were divided into groups of 75%, 50% and 25% TB ancestry. The auditory test conducted in a paddock comprised a 10-min prestimulus period, a 5-min stimulus period when one of the predators was heard, and a 10-min poststimulus period without any experimental stimuli. RESULTS: The increase in heart rate and saliva cortisol concentration in response to predator vocalizations indicated some level of stress in the horses. The lowered beat-to-beat intervals revealed a decrease in parasympathetic nervous system activity. The behavioral responses were less distinct than the physiological changes. The responses were more pronounced with leopard vocalizations than wolf vocalizations. CONCLUSIONS: The horses responded with weak signs of anxiety when exposed to predator vocalizations. A tendency towards a stronger internal reaction to predators in horses with a higher proportion of TB genes suggested that the response intensity was partly innate. The more pronounced response to leopard than wolf may indicate that horses are more frightened of a threatening sound from an unknown predator than one known by their ancestors. The differing response can be also due to differences in the characteristic of the predators' vocalizations. Our findings suggested that the present-day horses' abilities to coexist with predators are weak. Hence, humans should protect horses against predation, especially when introducing them into seminatural locations.

MIX2: A Novel Natural Multi-Component Modulator of Multidrug-Resistance and Hallmarks of Cancer Cells.

Multidrug resistance is one of the key barriers suppressing the effectiveness of drug therapies of malignant tumors. Here, we report a study on the effect of a mix of natural extracts (MIX2) prepared from fresh fruits of Prunus spinosa, Crataegus monogyna, Sorbus aucuparia, and Euonymus europaeus on the classic hallmarks of cancer cells and the expression of multidrug resistance proteins. In the studies, HeLa and T98G cell lines, and classic methods of molecular biology, including RT-qPCR, Western blot, flow cytometry, and confocal imaging, were used. Additionally, migration, adhesion, and proliferation assays were performed. The obtained results indicate that the MIX2 cocktail presents strong anti-cancer properties. MIX2 is not toxic, but at the same time significantly alters the migration, proliferation, and adhesion of tumor cells. Furthermore, it was found that cells exposed to the mixture presented a significantly reduced expression level of genes associated with MDR, including ABCB1, which encodes for glycoprotein P. In vitro data showed that MIX2 effectively sensitizes tumor cells to doxorubicin. We postulate that modulation of the multidrug resistance phenotype of tumors with the use of MIX2 may be considered as a safe and applicable tool in sustaining drug delivery therapies of malignancies.

Selected CD molecules and the phagocytosis of microvesicles released from erythrocytes ex vivo.

BACKGROUND AND OBJECTIVES: The accumulation of microvesicles in erythrocyte concentrates during storage or irradiation may be responsible for clinical symptoms such as inflammation, coagulation and immunization. Our aim was to determine whether any of the cluster of differentiation (CD) molecules responsible for important functions are present on microvesicles, and if their expression level is dependent on the storage period of erythrocyte concentrates. MATERIAL AND METHODS: Erythrocyte microvesicles were isolated from 'fresh' (2nd day) and 'old' (42nd day) stored erythrocyte concentrates. Qualitative cytometric analysis of 0·5 µm, erythrocyte-derived, PS-exposing vesicles was performed using the annexin V-FITC, anti-CD235a-PE antibody and calibrated beads. The microvesicles were also visualized under a confocal microscope. The expression of the molecules CD235a, CD44, CD47, CD55, CD59 and of phosphatidylserine (PS) was compared using flow cytometry. Measurements of microvesicle phagocytosis by human monocytes were carried out using a flow cytometer and a confocal microscope. RESULTS: The analysis of the microvesicles with calibration beads allowed us to identify these structures with a diameter of about 0·5 µm in the 'fresh' and 'old' samples. At day 2, the microvesicles had elevated expression levels of CD47, reduced expression levels of PS, CD55 and CD59. The phagocytosis index was higher for the microvesicles isolated from the 42-day-old erythrocyte concentrates. CONCLUSION: This research may bring us closer to understanding the factors responsible for erythrocyte ageing and to evaluate the quality of stored red blood concentrates intended for transfusion.

Molecular Signature of Prospero Homeobox 1 (PROX1) in Follicular Thyroid Carcinoma Cells.

The prospero homeobox 1 (PROX1) transcription factor is a product of one of the lymphangiogenesis master genes. It has also been suggested to play a role in carcinogenesis, although its precise role in tumour development and metastasis remains unclear. The aim of this study was to gain more knowledge on the PROX1 function in thyroid tumorigenesis. Follicular thyroid cancer-derived cells-CGTH-W-1-were transfected with PROX1-siRNA (small interfering RNA) and their proliferation, cell cycle, apoptosis and motility were then analysed. The transcriptional signature of PROX1 depletion was determined using RNA-Sequencing (RNA-Seq) and the expression of relevant genes was further validated using reverse transcriptase quantitative PCR (RT-qPCR), Western blot and immunocytochemistry. PROX1 depletion resulted in a decreased cell motility, with both migratory and invasive potential being significantly reduced. The cell morphology was also affected, while the other studied cancer-related cell characteristics were not significantly altered. RNA-seq analysis revealed significant changes in the expression of transcripts encoding genes involved in both motility and cytoskeleton organization. Our transcriptional analysis of PROX1-depleted follicular thyroid carcinoma cells followed by functional and phenotypical analyses provide, for the first time, evidence that PROX1 plays an important role in the metastasis of thyroid cancer cells by regulating genes involved in focal adhesion and cytoskeleton organization in tumour cells.

Systematic annotation of celiac disease loci refines pathological pathways and suggests a genetic explanation for increased interferon-gamma levels.

Although genome-wide association studies and fine mapping have identified 39 non-HLA loci associated with celiac disease (CD), it is difficult to pinpoint the functional variants and susceptibility genes in these loci. We applied integrative approaches to annotate and prioritize functional single nucleotide polymorphisms (SNPs), genes and pathways affected in CD. CD-associated SNPs were intersected with regulatory elements categorized by the ENCODE project to prioritize functional variants, while results from cis-expression quantitative trait loci (eQTL) mapping in 1469 blood samples were combined with co-expression analyses to prioritize causative genes. To identify the key cell types involved in CD, we performed pathway analysis on RNA-sequencing data from different immune cell populations and on publicly available expression data on non-immune tissues. We discovered that CD SNPs are significantly enriched in B-cell-specific enhancer regions, suggesting that, besides T-cell processes, B-cell responses play a major role in CD. By combining eQTL and co-expression analyses, we prioritized 43 susceptibility genes in 36 loci. Pathway and tissue-specific expression analyses on these genes suggested enrichment of CD genes in the Th1, Th2 and Th17 pathways, but also predicted a role for four genes in the intestinal barrier function. We also discovered an intricate transcriptional connectivity between CD susceptibility genes and interferon-γ, a key effector in CD, despite the absence of CD-associated SNPs in the IFNG locus. Using systems biology, we prioritized the CD-associated functional SNPs and genes. By highlighting a role for B cells in CD, which classically has been described as a T-cell-driven disease, we offer new insights into the mechanisms and pathways underlying CD.

3D visualization and quantitative analysis of human erythrocyte phagocytosis.

Since the erythrophagocytosis of opsonized erythrocytes is investigated mainly by calculating the phagocytic index using subjective light microscopy evaluation, we present methods for the quantitative and qualitative analysis of human cell erythrophagocytosis. Erythrocytes from two storage periods were used. Using Imaris software, we were able to create a three-dimensional model of erythrophagocytosis. The use of microscopy instead of cytometry revealed a significantly higher number of monocytes and erythrocytes that appeared active in phagocytosis. Spatial reconstruction allowed for detailed analysis of the process by precisely locating erythrocytes in phagocytes. Additionally, a technique of sequential image registration using Nis Elements software allowed for observation of the course of phagocytosis over a range of time intervals. This in vitro research may be helpful for understanding the cellular interactions between monocytes and erythrocytes. The cytometric method-being relatively rapid, sensitive, and specific-can serve as an alternative technique to microscopy in the quantitative analysis of erythrophagocytosis. This allows us to avoid counting the erythrocytes nonspecifically attached to monocytes and gives objective results.

Mycophenolic acid attenuates the tumour necrosis factor-α-mediated proinflammatory response in endothelial cells by blocking the MAPK/NF-κB and ROS pathways.

BACKGROUND: Mycophenolate mofetil (MMF) has beneficial effects in cardiac transplant patients beyond the suppression of tissue rejection. Moreover, mycophenolic acid (MPA), its active metabolite, has been associated with positive effects on atherosclerosis in animal models. The attachment of leukocytes to the vascular endothelium and the subsequent migration of these cells into the vessel wall are early events in inflammation and atherosclerosis. The aim of this study was to investigate the effects of MPA on tumour necrosis-α (TNF-α)-induced, endothelial cell proinflammatory responses and the underlying mechanisms. METHODS AND RESULTS: Human aortic endothelial cells (HAECs) were treated with different concentrations (primarily 50 µM) of MPA before treatment with TNF-α. The surface protein and mRNA expressions of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) were determined by flow cytometry and real-time RT-PCR, respectively. Adhesion of leukocytes to TNF-α-treated HAECs was evaluated by an adhesion assay. Activation of mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) was evaluated by measuring the levels of their phosphorylation using flow cytometry. NF-κB p65 translocation was detected by Western blotting. The production of reactive oxygen species (ROS) was determined by reduction in fluorescent 2',7'-dichlorofluorescein diacetate (H2 DCFH-DA). MPA significantly inhibits TNF-α-induced ICAM-1, VCAM-1 surface protein and mRNA expression as well as adhesion of mononuclear leukocytes to HAEC. ICAM-1 and VCAM-1 expressions were also reduced by antioxidants such as pyrrolidine dithiocarbamate, diphenylene iodonium and apocynin. MPA inhibited TNF-α-stimulated ROS generation similarly to apocynin. TNF-α increased ICAM-1 and VCAM-1 expression via c-Jun NH2 -terminal kinase (JNK), extracellular signal-regulated kinase (ERK1/2) and p38 MAPK. MPA and apocynin inhibited TNF-α-induced phosphorylation of all three MAP kinases. Furthermore, TNF-α-induced NF-κB activation was attenuated by SP600125 (JNK inhibitor), PD98059 (ERK1/2 inhibitor, SB203580 (p38 MAPK inhibitor) and MPA. MPA also inhibited TNF-α-induced nuclear translocation of NF-κB p65. CONCLUSION: These results suggest that, in addition to the prevention of rejection, MPA may be a promising approach for the treatment of inflammatory vascular disease.

Feto-maternal haemorrhage assessment in a woman with a large population of red blood cells containing fetal haemoglobin.

BACKGROUND: FMH quantification is necessary to calculate an individual dose of prophylactic anti-RhD immunoglobulin and to diagnose fetal anaemia causes. We encountered a healthy woman with a numerous RBCs containing fetal haemoglobin (HbF). AIMS: To investigate the cause of this sign and the correct evaluation of fetal RBCs in maternal circulation. MATERIALS AND METHODS: Patients samples and artificial mixtures were tested by microscopic Kleihaur-Betke (KB) and flow cytometric (FC) tests with anti-HbF + anti-CA (carbonic anhydrase), and with anti-D. The patient's blood count with reticulocyte parameters, and concentration of bilirubin, haptoglobin, iron, transferrin, ferritin, hepcidin, sTR, HbF, HbA2 were measured. Genes coding the beta- and gamma-globin were sequenced. RESULTS: It was impossible to distinguish the population of fetal and maternal HbF positive cells using KBT and FC with anti-HbF. Application of anti-CA and anti-D allowed to separate them. Maternal blood haematological and biochemical parameters were normal but HbF was 3.3% of total Hb concentration (normal < 1%). There were no mutations in the beta- and gamma-globin genes, but Xmn I polymorphism at -158 position in gamma-globin gene was detected in the homozygous state. CONCLUSION: A very large population of HbF positive cells sometimes can be detect in a healthy woman. Implementation of the various procedures for FMH assessment is necessary in the such case, otherwise, the detection of fetal erythrocytes may not be possible or can give false results.

Potential biomarkers for multiple sclerosis stage from targeted proteomics and microRNA sequencing.

Multiple sclerosis is a chronic demyelinating disease of the central nervous system. There is a need for new circulating biomarkers for multiple sclerosis, in particular, markers that differentiate multiple sclerosis subtypes (relapsing-remitting, secondary progressive and primary progressive multiple sclerosis), as this can help in making treatment decisions. In this study, we explore two classes of potential multiple sclerosis biomarkers-proteins and microRNAs-circulating in the cerebrospinal fluid and serum. Targeted medium-throughput proteomics (92 proteins) and microRNA sequencing were performed on serum samples collected in a cross-sectional case-control cohort (cohort I, controls n = 30, multiple sclerosis n = 75) and a prospective multiple sclerosis cohort (cohort II, n = 93). For cohort I, we also made these measurements in paired cerebrospinal fluid samples. In the cohort I cerebrospinal fluid, we observed differences between multiple sclerosis and controls for 13 proteins, including some previously described to be markers for multiple sclerosis [e.g. CD27, C-X-C motif chemokine 13 (CXCL13) and interleukin-7 (IL7)]. No microRNAs were significantly differentially expressed between multiple sclerosis and controls in the cerebrospinal fluid. In serum, 10 proteins, including angiopoietin-1 receptor (TIE2), and 16 microRNAs were significantly different between relapsing-remitting multiple sclerosis and secondary progressive multiple sclerosis after performing a meta-analysis combining both cohorts. In the prospective part of the study, participants with relapsing-remitting multiple sclerosis were followed for around 3 years, during which time 12 participants converted to secondary progressive multiple sclerosis. In these longitudinally collected serum samples, we observed a peak in granzyme B, A and H proteins around the time of conversion. Single-sample enrichment analysis of serum microRNA profiles revealed that the peak in granzyme B levels around conversion coincides with enrichment for microRNAs that are enriched in CD4+, CD8+ and natural killer cells (e.g. miRNA-150). We identified several proteins and microRNAs in serum that represent potential biomarkers for relapsing-remitting and secondary progressive multiple sclerosis. Conversion to secondary progressive disease is marked by a peak in granzyme B levels and enrichment for immune-related microRNAs. This indicates that specific immune cell-driven processes may contribute to the conversion of relapsing-remitting multiple sclerosis to secondary progressive multiple sclerosis.

Variation of Physiological and Behavioural Parameters during the Oestrous Cycle in Mares.

The behaviour of oestrous mares is well-known in horse breeding. However, alterations in their physiological and behavioural indices during the whole oestrous cycle are scarcely known. The objective of the study was to analyse changes in cardiac activity variables, rectal and superficial temperatures, behaviour towards humans and conspecifics, and the time of standing and locomotor activity in mares during their oestrous cycle. Fifteen adult mares in oestrus were examined in the morning and evening (six successive days) and in dioestrus (five days-once every third day). The oscillation of physiological and behavioural variables accompanies changes in mares' sexual behaviour. Most physiological variables studied in oestrus indicate the elevated activity of the adrenergic nervous system and, opposite to that, both behaviour towards humans and conspecifics and the time of standing relate to a relaxed state. The end of oestrus, manifested by a rapid decrease in most of the physiological variables studied, is followed by changes of behavioural variables at the beginning of dioestrus. The time of locomotor activity arises at the end of oestrus. The outcomes may contribute to the knowledge of, among others, mare owners who evaluate the oestrus by mares' sexual behaviours without regarding other rhythmically changing variables.

Role of α5ß1 and αvß3 integrins in relation to adhesion and spreading dynamics of prostate cancer cells interacting with fibronectin under in vitro conditions.

Interaction of cell integrins with the ECM (extracellular matrix) proteins is commonly assumed to be associated with cell dissemination and tumour metastases. Since these processes depend on the mechanism of cell-protein interaction, we have attempted to show the contribution of α5ß1 and αvß3 integrins of the prostate cancer PC-3 cells in in vitro interaction with FN (fibronectin) adsorbed on defined polystyrene surfaces. Cell adhesion, spreading and cytoskeleton organization were studied using antibodies against integrins or a GRGDSP (Gly-Arg-Gly-Asp-Ser-Pro) peptide. The results show that blocking the α5ß1 integrin causes: (i) a decrease in the number of the adherent cells in the early phase of adhesion and (ii) a decrease in the dynamics of cell spreading and cell shape changes, and weaker reorganization of cytoskeletal proteins than in the control cells. Conversely, the blocking of the αvß3 integrin: (i) causes no observable effect on the number of the adhered cells; however, (ii) causes an increase in the dynamics of cell spreading and cell shape changes, and stronger reorganization of cytoskeletal proteins than in the control cells. Interestingly, the blocking of integrins with a GRGDSP peptide strongly decreases the number of the adhered cells, and a complete inhibition of cell spreading. Our results strongly suggest that the α5ß1 integrin plays the main role in the adhesion and spreading of PC-3 cells interacting with FN, whereas the αvß3 integrin seems to regulate other receptors in the spreading process. Moreover, integrin-FN interaction through the RGD sequence evidently curbed the cell adhesion and spreading.

Horses' Response to a Novel Diet: Different Herbs Added to Dry, Wet or Wet-Sweetened Oats.

The commercial horse feed industry uses palatants to mask undesirable tastes of feeds and enhance product acceptance. However, an unknown odour or taste may also hinder feed intake, due to, inter alia, novelty. The objective of the study was to assess the horses' response to novel diet: five different herbs added alternately to dry, wet or wet-sweetened oats. Twenty adult horses were given different diet combinations of a feed presentation and a herb: field mint, common yarrow, common chamomile, common sage and common nettle, consecutively, once daily. The response to novelty was assessed regarding traits showing the willingness to consume: times of olfaction and consumption, times and numbers of intervals in consumption and drinking water, and the mass of leftovers. The results show that properties of the herbs studied did not hinder the consumption and only the odour of the dry common sage delayed the intake. Wetting or wetting and sweetening the diet accelerated the intake. In conclusion, herbs in small amounts do not significantly affect the willingness to consume feed. Although wet and wet-sweetened diet presentations may be novel to horses, they increase the feed palatability and can be suggested for use when preparing horse diets.

PROM1, CXCL8, RUNX1, NAV1 and TP73 genes as independent markers predictive of prognosis or response to treatment in two cohorts of high-grade serous ovarian cancer patients.

Considering the vast biological diversity and high mortality rate in high-grade ovarian cancers, identification of novel biomarkers, enabling precise diagnosis and effective, less aggravating treatment, is of paramount importance. Based on scientific literature data, we selected 80 cancer-related genes and evaluated their mRNA expression in 70 high-grade serous ovarian cancer (HGSOC) samples by Real-Time qPCR. The results were validated in an independent Northern American cohort of 85 HGSOC patients with publicly available NGS RNA-seq data. Detailed statistical analyses of our cohort with multivariate Cox and logistic regression models considering clinico-pathological data and different TP53 mutation statuses, revealed an altered expression of 49 genes to affect the prognosis and/or treatment response. Next, these genes were investigated in the validation cohort, to confirm the clinical significance of their expression alterations, and to identify genetic variants with an expected high or moderate impact on their products. The expression changes of five genes, PROM1, CXCL8, RUNX1, NAV1, TP73, were found to predict prognosis or response to treatment in both cohorts, depending on the TP53 mutation status. In addition, we revealed novel and confirmed known SNPs in these genes, and showed that SNPs in the PROM1 gene correlated with its elevated expression.

Physcion Induces Potential Anticancer Effects in Cervical Cancer Cells.

BACKGROUND: The extent of morphological and ultrastructural changes in HeLa cells was assessed by optical, fluorescence and electron microscopy after exposure to various concentrations of physcion, taking into account the biological properties of the test compound. METHODS: Cell viability was assessed by MTT assay, while the cell cycle, LC3 expression, apoptosis, change of mitochondrial potential, Bcl-2 protein expression level and the level of reactive oxygen species were analyzed by flow cytometry. RESULTS: As a result of physcion encumbrance, concentration-dependent inhibition of HeLa cell viability and the G0/G1 phase of the cell cycle was observed. Activation of the lysosomal system was also revealed, which was expressed by an increased number of lysosomes, autophage vacuoles and increased expression of the LC3 protein, a marker of the autophagy process. Transmission electron microscopy and fluorescence microscopy showed that physcion induced clear changes in cervical cancer cells, especially in the structure of the nucleus and mitochondria, which correlated with the production of reactive oxygen species by the test compound and indicated the induction of the oxidative process. At the same time, the pro-apoptotic effect of physcion was demonstrated, and this mechanism was dependent on the activation of caspases 3/7 and the reduction in Bcl-2 protein expression. CONCLUSION: The obtained results indicate an antitumor mechanism of action of physcion, based on the induction of oxidative stress, autophagy and apoptosis.

Behavioural and Physiological Changes in a Herd of Arabian Mares after the Separation of Individuals Differently Ranked within the Dominance Hierarchy.

Horses in a herd develop and maintain a dominance hierarchy between all individuals. There are many situations in riding facilities and studs in which horses have to be separated out of a group. The aim of the study was to determine the rate of behaviours, level of locomotor activity and cardiac activity variables in a herd of horses during a short social separation of individuals differently ranked in the dominance hierarchy. Twelve adult Arabian mares were involved. A behavioural test had been performed before the main experiment to determine the rank order of the mares in this social herd. Three tests were performed when a dominant, mixed and submissive three-member group of mares was separated for 10 min. The response of the remaining herd was determined by a rate of behaviours, time of locomotor activity and cardiac parameters. The results of the experiment reveal evident changes towards emotional arousal in the social herd elicited by a short separation of some conspecifics. The herd created by humans preserves the sensitivity to a temporary loss of its members. The response of the remaining herd does not depend strictly on the composition of the separated mares regarding their rank in the dominance hierarchy.

Horse Preferences for Insolation, Shade or Mist Curtain in the Paddock under Heat Conditions: Cardiac and Behavioural Response Analysis.

The horse's welfare and, consequently, the emotional arousal may be connected with stressful environmental conditions. This study aimed to determine whether horses show behavioural or physiological symptoms of thermal discomfort and if their behaviour and cardiac parameters are related to freely chosen insolated (IS), shaded (SH), or water sprayed (with a mist curtain (MC)) areas in a paddock under heat conditions (29-32 °C, 42.0 ± 1.5% humidity). Twelve adult horses freely moving in the paddock were studied during a 45 min solitary turnout. Six cardiac variables, locomotor, and non-locomotor activities as well as rectal temperature before and after the test were monitored with regard to the area of staying. Horses did not show clear preferences regarding the time spent in IS, SH, and MC, although preferences of particular horses differed considerably. When staying under IS and MC conditions, the horses showed a higher level of relaxation compared to SH. Horses did not exhibit symptoms of thermal discomfort while staying in the sun. Free choice between the three areas differing in environmental conditions could be a crucial factor in maintaining body temperature as well as emotional arousal at similar levels. Thus, the provision of a shade and mist curtain in paddocks seems to be reasonable.

Circulating miRNAs as Potential Biomarkers for Celiac Disease Development.

Background & Aims: Celiac disease (CeD), an immune-mediated disease with enteropathy triggered by gluten, affects ~1% of the general European population. Currently, there are no biomarkers to predict CeD development. MicroRNAs (miRNAs) are short RNAs involved in post-transcriptional gene regulation, and certain disease- and stage-specific miRNA profiles have been found previously. We aimed to investigate whether circulating miRNAs can predict the development of CeD. Methods: Using next-generation miRNA-sequencing, we determined miRNAs in >200 serum samples from 53 participants of the PreventCD study, of whom 33 developed CeD during follow-up. Following study inclusion at 3 months of age, samples were drawn at predefined ages, diagnosis (first anti-transglutaminase antibody (TGA) positivity or diagnostic biopsy) and after the start of a gluten-free diet (GFD). This allowed identification of circulating miRNAs that are deregulated before TGA positivity. For validation of the biomarkers for CeD and GFD response, two additional cohorts were included in subsequent meta-analyses. Additionally, miRNAs were measured in duodenal biopsies in a case-control cohort. Results: 53 circulating miRNAs were increased (27) or decreased (26) in CeD versus controls. We assessed specific trends in these individual miRNAs in the PreventCD cohort by grouping the pre-diagnostic samples of the CeD patients (all had negative TGA) by how close to seroconversion (first sample positive TGA) the samples were taken. 8/53 miRNAs differed significantly between controls and samples taken <1 year before TGA positivity: miR-21-3p, miR-374a-5p, 144-3p, miR-500a-3p, miR-486-3p let-7d-3p, let-7e-5p and miR-3605-3p. 6/26 downregulated miRNAs reconstituted upon GFD, including miR-150-5p/-3p, whereas no upregulated miRNAs were downregulated upon GFD. 15/53 biomarker candidates also differed between CeD biopsies and controls, with a concordant direction, indicating that these circulating miRNAs might originate from the intestine. Conclusions: We identified 53 circulating miRNAs that are potential early biomarkers for CeD, of which several can be detected more than a year before TGA positivity and some start to normalize upon GFD.

[Role of Nrf2 transcription factor in cellular response to oxidative stress]. / Rola czynnika transkrypcyjnego Nrf2 w odpowiedzi komórkowej na stres oksydacyjny.

Nrf2 transcription factor plays a crucial role in protection of cells against oxidative stress. Under normal conditions Nrf2 is sequestered in cytoplasm by a cytoskeletal protein, Keapl. Situation is changed under stressful conditions,when electrophiles and/or reactiveoxygen species (ROS) cause dissociation of Nrf2 from Keap1. As a consequence, Nrf2 is translocated to the nucleus, that leads to activation of cytoprotective genes involved in electrophile conjugation, excretion of xenobiotics, ROS scavenging and stabilization of cellular redox potential. Amongst xenobiotics, there are many mutagenic and cancerogenic factors. Phase I and II enzymes are responsible for inactivation and removal of such compounds. It has been shown that induction of phase II enzymes confers protection upon insult by reactive metabolites of cancerogens or ROS. Since Nrf2 is an inductor of those enzymes it can be considered as a potential target for cancer chemoprevention. Similarly, in case of neurodegenerative disorders, which pathogenesis is connected to oxidative stress, Nrf2 may be important for therapeutical and preventive strategies

[The new face of factors induced by hypoxia--HIF-1 and HIF-2 and oxidative stress]. / Nowe oblicza czynników indukowanych przez hipoksje--HIF-1 i HIF-2 a stres oksydacyjny.

Studies on the mechanisms of adaptation to adverse hypoxic conditions led to the discovery of hypoxia inducible factors, HIF-1 and HIF-2. These factors regulate the expression of many genes which allow cells to adapt to changes in oxygen concentration and counteract the effects of oxidative stress developing in hypoxia. Regulation of HIF activity is dependent on the prolyl hydroxylases activity and results in its degradation under normoxic conditions by the ubiquitin-dependent proteasome pathway. Recent studies indicate a specific role of reactive oxygenspecies (ROS) generated by the mitochondrial respiratory chain in regulation of HIF stability. ROS affect also the level of nitric oxide (NO), leading to a reduction in its concentration by forming reactive nitrogen species (RNS), which may cause the increase in oxidative and nitrosative stress. Regulation of HIF activity by ROS, NO and RNS is currently the subject of many studies and seems to be a mechanism dependent on conditions (e.g., normoxia/hypoxia), or concentrations of individual stimulators (e.g. NO donor used).