Changes in the Level of DNA Methylation in Candida albicans under the Influence of Physical and Chemical Factors.

The effects of physical factors such as radiation (electromagnetic, microwave, infrared, laser, UVC, and X-ray) and high temperature, as well as chemical factors (controlled atmosphere) on the level of global DNA cytosine methylation in C. albicans ATCC 10231 cells were investigated. Prolonged exposure to each type of radiation significantly increased the DNA methylation level. In addition, the global methylation level in C. albicans cells increased with the incubation temperature. An increase in the percentage of methylated DNA was also noted in C. albicans cells cultured in an atmosphere with reduced O2. In contrast, in an atmosphere containing more than 3% CO2 and in anaerobic conditions, the DNA methylation level decreased relative to the control. This study showed that prolonged exposure to various types of radiation and high temperature as well as reduced O2 in the atmosphere caused a significant increase in the global DNA methylation level. This is most likely a response protecting DNA against damage, which at the same time can lead to epigenetic disorders, and in consequence can adversely affect the functioning of the organism.

The Importance of Visceral Hypersensitivity in Irritable Bowel Syndrome-Plant Metabolites in IBS Treatment.

The visceral stimuli from the digestive tract are transmitted via afferent nerves through the spinal cord to the brain, where they are felt as pain. The overreaction observed in the brain of irritable bowel syndrome (IBS) patients may be due to increased peripheral sensitivity to stimuli from the gastrointestinal tract. Although the exact pathway is uncertain, attenuation of visceral hypersensitivity is still of interest in treating IBS. It has been shown that stress stimulates the sympathetic nervous system while inhibiting the vagus nerve (VN). In addition, stress factors lead to dysbiosis and chronic low-grade inflammation of the intestinal mucosa, which can lead to lower gastrointestinal visceral hypersensitivity. Therefore, an important goal in the treatment of IBS is the normalization of the intestinal microflora. An interesting option seems to be nutraceuticals, including Terminalia chebula, which has antibacterial and antimicrobial activity against various pathogenic Gram-positive and Gram-negative bacteria. Additionally, short-term transcutaneous vagus nerve stimulation can reduce the stress-induced increase in intestinal permeability, thereby reducing inflammation. The conducted studies also indicate a relationship between the stimulation of the vagus nerve (VN) and the activation of neuromodulatory networks in the central nervous system. Therefore, it seems reasonable to conclude that a two-way action through stimulating the VN and using nutraceuticals may become an effective therapy in treating IBS.

Increased Markers of Oxidative Stress and Positive Correlation Low-Grade Inflammation with Positive Symptoms in the First Episode of Schizophrenia in Drug-Naïve Patients.

Schizophrenia is a severe and chronic mental illness usually diagnosed in adolescents and young adults. Many studies indicate that oxidative stress causes membrane dysfunction and cell damage, which is implicated in the pathophysiology of schizophrenia. The purpose of our study was to evaluate oxidative stress markers (the main primary products of lipid peroxidation, lipid hydroperoxides (LOOH), and end products of lipid peroxidation, malondialdehyde (MDA), superoxide dismutase (SOD), glutathione (GSH), and Ferric Reducing Ability of Plasma (FRAP)) in the plasma of patients with the first episode of schizophrenia in drug-naïve patients (22 men and 12 women aged 17-29). The control group (Ctrl) comprised 26 healthy subjects (19 men and 7 women, aged 18-30 years). The Positive and Negative Syndrome Scale (PANSS) was applied to evaluate psychotic symptoms. Analyses of the oxidative stress variables revealed an increased level of SOD (U/mL) in subjects with schizophrenia versus control group. In addition, lipid damage measured as LOOHs µ (mol/L) and MDA was significantly higher in patients with schizophrenia in comparison to control subjects. There was a positive correlation between MDA µmol/L and PANSS P and a positive correlation between C-reactive protein (CRP) and the PANSS P scale. The elevated level of superoxide dismutase in patients with the first episode of schizophrenia can be explained by compensatory mechanisms to counteract oxidative stress. Malondialdehyde can be used as a simple biomarker of low-grade systemic inflammation associated with oxidative stress. A positive correlation between CRP and PANSS P scale and MDA and PANSS P scale may indicate a significant relationship between the development of low-grade inflammation and damage associated with oxidative stress in the development of the first symptoms of schizophrenia.

Gurgem-7 toxicity assessment: Regulation of cell survival or death by traditional Mongolian prescription.

Surgical treatments and chemotherapy are the most commonly used methods of colorectal cancer treatment (CRC), unfortunately, these therapies have many side effects. Moreover, despite advances in primary and adjuvant treatments, the survival time in CRC patients is still unsatisfactory. Treatment options for patients with CRC continue to advance and recent research has shown that colorectal cancer is sensitive to plant-derived substances. The use of natural compounds contained in herbal extracts for the treatment of colon cancer or as adjunctive therapy for CRC gives patients a wide range of treatment options. In this study, we evaluate the potential toxicity of the Mongolian preparation - Gurgem-7 composed of Crocus sativus, Veronica officinalis, Capsella bursa-pastoris, Arctostaphylos uva-ursi, Calendula officinalis, Gentiana lutea, and Terminalia chebula. Therefore, the aim of this study was to determine its biological activities, biochemical and molecular features in vitro and composition analysis by HPLC-ESI-QTOF-MS/MS platform. We identified 18 metabolites and 8 of them were quantified. Majority of the secondary metabolites belonged to the group of phenolic constituents with taxifolin, chlorogenic acids' family, hydroxysafflor yellow A and hydroxybenzoic acid as leading compounds. In turn, our in vitro results suggest that the preparation inhibits cell metabolic activity through oxidative stress, numerous DNA damage and cell cycle arrest. Simultaneously enzymatic and non-enzymatic cell protection mechanisms mediated by TP53/Keap1 and Nrf2/HO-1 pathways may be activated in a cell-specific manner in vitro. In conclusion, we provide preliminary molecular evidence of the toxic properties of Gurgem-7 preparation to Caco-2 and CT26. WT cells related to insufficient action of their repair and adaptive mechanisms to stress conditions.

Fractalkine, sICAM-1 and Kynurenine Pathway in Restrictive Anorexia Nervosa-Exploratory Study.

The link between the kynurenine pathway and immunomodulatory molecules-fractalkine and soluble intercellular adhesion molecule-1 (sICAM-1)-in anorexia nervosa (AN) remains unknown. Fractalkine, sICAM-1, tryptophan (TRP), kynurenine (KYN), neuroprotective kynurenic acid (KYNA), neurotoxic 3-OH-kynurenine (3-OH-KYN), and the expression of mRNA for kynurenine aminotransferases (KAT1-3) were studied in 20 female patients with restrictive AN (mostly drug-free, all during first episode of the disease) and in 24 controls. In AN, serum fractalkine, but not sICAM-1, KYNA, KYN, TRP or 3-OH-KYN, was higher; ratios TRP/KYN, KYN/KYNA, KYN/3-OH-KYN and KYNA/3-OH-KYN were unaltered. The expression of the gene encoding KAT3, but not of genes encoding KAT1 and KAT2 (measured in blood mononuclear cells), was higher in patients with AN. In AN, fractalkine positively correlated with TRP, while sICAM-1 was negatively associated with 3-OH-KYN and positively linked with the ratio KYN/3-OH-KYN. Furthermore, TRP and fractalkine were negatively associated with the body mass index (BMI) in AN. Expression of KAT1, KAT2 and KAT3 did not correlate with fractalkine, sICAM-1 or BMI, either in AN or control. Increased fractalkine may be an independent factor associated with the restrictive type of AN. Excessive physical activity probably underlies increased expression of KAT3 observed among enrolled patients. Further, longitudinal studies on a larger cohort of patients should be aimed to clarify the contribution of fractalkine and KAT3 to the pathogenesis of AN.

Risk of wild fungi treatment failure: Phallus impudicus-induced telomere damage triggers p21/p53 and p16-dependent cell cycle arrest and may contribute to male fertility reduction in vitro.

The multifunctional characteristics of Phallus impudicus extract encourage to conduct research for its potential use in medical applications. Well, science is constantly seeking new evidence for the biological activity of extracts of natural origin. Drugs of natural origin should not cause any side effects on the physiological functions of the human body; however, this is not always successful. In this study, we used in vitro approach to evaluate the toxicity of alcohol Phallus impudicus extract on spermatogenic cells. We show, for the first time, cytotoxic properties of Phallus impudicus treatment associated with a decrease in cellular metabolic activity, dysregulation of redox homeostasis and impairment of selected antioxidant cell protection systems. As a consequence, p53/p21- and p16-mediated cell cycle arrest followed by p27 activation is initiated. The observed changes were associated with telomere shortening and numerous DNA damage at the chromosome ends (altered expression pattern of TRF1 and TRF2 proteins), as well as upregulation of cleaved caspase-3 with a decrease in Bcl-2 expression, suggesting induction of apoptotic death. Therefore, these results provide molecular evidence for mechanistic pathways and novel adverse outcomes linked to the Phallus impudicus treatment towards men's health and fertility reduction.

Influence of the Treatment Used in Inflammatory Bowel Disease on the Protease Activities.

INTRODUCTION: There is growing evidence that intestinal proteases have a role in the pathogenesis of gastrointestinal inflammatory diseases. Inflammatory bowel disease (IBD), which includes Crohn's disease (CD) and ulcerative colitis (UC), has an additional source of proteases represented by infiltrated and activated inflammatory cells. The aim of our study was to determine proteolytic system activity in patients with CD and UC. We limited the number of proteases tested by determining proteases active in acidic, neutral and alkaline pH. MATERIALS AND METHODS: The study included 40 patients with IBD - 20 CD patients and 20 UC patients. The control group consisted of 20 healthy subjects. Among the 20 CD patients, 17 were treated with aminosalicylates, 14 with azathioprine, and 4 with corticosteroids, while 8 patients were undergoing biological treatment. Among the 20 UC patients, 19 were treated with aminosalicylates, 8 with azathioprine, and 3 with corticosteroids. The total protein concentration was assayed by the Lowry method. The optimal pH was assayed in pH from 2.2 to 12.8, separated by 0.2 intervals. Proteolytic activities were determined against different substrates (gelatine, haemoglobin, ovalbumin, albumin, cytochrome C, and casein), and haemoglobin was the optimal substrate. Protease activities were determined according to Anson method. Determination of the activities of natural inhibitors of acidic, neutral and alkaline proteases is based on the Lee and Lin method. RESULTS: Decreases were observed in the activities of acid proteases (pH 5), alkaline proteases (pH 7), and neutral proteases (pH 7.6 and 8.6) in the groups of CD patients in remission in comparison with the active phase. In the group of patients with biologically treated CD patients, acid protease activity (pH 5.0) was lower than in CD patients not receiving biological treatment. Activities of neutral (pH 7.0) and alkaline (pH 7.6 and 8.6) proteases in the plasma of patients with UC in remission were lower in comparison to the active phase. Activities of acid (pH 5.0) and alkaline (8.6) protease inhibitors were higher in CD patients in the active phase in comparison to remission. In UC patients with exacerbation of the disease, the activity of alkaline (pH 8.6) protease inhibitors was increased compared to remission. CONCLUSION: 1. Our research may suggest that the immunomodulatory treatment used in IBD, aimed at reducing the level of leukocytes and reduction of inflammation, may contribute to a reduction in protease activity. 2. The decrease of protease activities in patients with CD and UC in remission may be a marker suggesting the patients' response to the treatment.

Different regulation of PARP1, PARP2, PARP3 and TRPM2 genes expression in acute myeloid leukemia cells.

BACKGROUND: Acute myeloid leukemia (AML) is a heterogenic lethal disorder characterized by the accumulation of abnormal myeloid progenitor cells in the bone marrow which results in hematopoietic failure. Despite various efforts in detection and treatment, many patients with AML die of this cancer. That is why it is important to develop novel therapeutic options, employing strategic target genes involved in apoptosis and tumor progression. METHODS: The aim of the study was to evaluate PARP1, PARP2, PARP3, and TRPM2 gene expression at mRNA level using qPCR method in the cells of hematopoietic system of the bone marrow in patients with acute myeloid leukemia, bone marrow collected from healthy patients, peripheral blood of healthy individuals, and hematopoietic stem cells from the peripheral blood after mobilization. RESULTS: The results found that the bone marrow cells of the patients with acute myeloid leukemia (AML) show overexpression of PARP1 and PARP2 genes and decreased TRPM2 gene expression. In the hematopoietic stem cells derived from the normal marrow and peripheral blood after mobilization, the opposite situation was observed, i.e. TRPM2 gene showed increased expression while PARP1 and PARP2 gene expression was reduced. We observed positive correlations between PARP1, PARP2, PARP3, and TRPM2 genes expression in the group of mature mononuclear cells derived from the peripheral blood and in the group of bone marrow-derived cells. In AML cells significant correlations were not observed between the expression of the examined genes. In addition, we observed that the reduced expression of TRPM2 and overexpression of PARP1 are associated with a shorter overall survival of patients, indicating the prognostic significance of these genes expression in AML. CONCLUSIONS: Our research suggests that in physiological conditions in the cells of the hematopoietic system there is mutual positive regulation of PARP1, PARP2, PARP3, and TRPM2 genes expression. PARP1, PARP2, and TRPM2 genes at mRNA level deregulate in acute myeloid leukemia cells.

Increased expression of kynurenine aminotransferases mRNA in lymphocytes of patients with inflammatory bowel disease.

BACKGROUND: Complex interaction of genetic defects with environmental factors seems to play a substantial role in the pathogenesis of inflammatory bowel disease (IBD). Accumulating data implicate a potential role of disturbed tryptophan metabolism in IBD. Kynurenic acid (KYNA), a derivative of tryptophan (TRP) along the kynurenine (KYN) pathway, displays cytoprotective and immunomodulating properties, whereas 3-OH-KYN is a cytotoxic compound, generating free radicals. METHODS: The expression of lymphocytic mRNA encoding enzymes synthesizing KYNA (KAT I-III) and serum levels of TRP and its metabolites were evaluated in 55 patients with IBD, during remission or relapse [27 patients with ulcerative colitis (UC) and 28 patients with Crohn's disease (CD)] and in 50 control individuals. RESULTS: The increased expression of KAT1 and KAT3 mRNA characterized the entire cohorts of patients with UC and CD, as well as relapse-remission subsets. Expression of KAT2 mRNA was enhanced in patients with UC and in patients with CD in remission. In the entire cohorts of UC or CD, TRP levels were lower, whereas KYN, KYNA and 3-OH-KYN were not altered. When analysed in subsets of patients with UC and CD (active phase-remission), KYNA level was significantly lower during remission than relapse, yet not versus control. Functionally, in the whole groups of patients with UC or CD, the TRP/KYN ratio has been lower than control, whereas KYN/KYNA and KYNA/3-OH-KYN ratios were not altered. The ratio KYN/3-OH-KYN increased approximately two-fold among all patients with CD; furthermore, patients with CD with relapse, manifested a significantly higher KYNA/3-OH-KYN ratio than patients in remission. CONCLUSION: The presented data indicate that IBD is associated with an enhanced expression of genes encoding KYNA biosynthetic enzymes in lymphocytes; however, additional mechanisms appear to influence KYNA levels. Higher metabolic conversion of serum TRP in IBD seems to be followed by the functional shift of KYN pathway towards the arm producing KYNA during exacerbation. We propose that KYNA, possibly via interaction with aryl hydrocarbon receptor or G-protein-coupled orphan receptor 35, may serve as a counter-regulatory mechanism, decreasing cytotoxicity and inflammation in IBD. Further longitudinal studies evaluating the individual dynamics of TRP and KYN pathway in patients with IBD, as well as the nature of precise mechanisms regulating KYNA synthesis, should be helpful in better understanding the processes underlying the observed changes.

Imbalance of Controlled Death in Peripheral Blood Lymphocytes in Crohn's Disease and Ulcerative Colitis.

Background and objectives: Inflammatory bowel disease (IBD) mainly includes Crohn's disease (CD) and ulcerative colitis (UC). Both conditions are associated with an exacerbated intestinal immune response to harmless stimuli, leading to upregulation of pro-inflammatory mediators. Materials and Methods: The subjects of the study were 55 patients with IBD. The control group consisted of 35 healthy subjects. The researched material consisted of peripheral blood lymphocytes collected from the subjects. Expression of the genes BAX, BCL2, CASP3 and CASP9 was assessed at the mRNA level in the peripheral blood lymphocytes of patients with ulcerative colitis and Crohn's disease relative to the healthy subjects. The expression of the genes was determined by rtPCR using TaqMan probes specific for these genes. Results: The group of patients diagnosed with CD had statistically significantly higher expression of the genes BAX (p = 0.012), BCL2 (p = 0.022), CASP3 (p = 0.003) and CASP9 (p = 0.029) than healthy subjects. Expression of BAX, BCL2, CASP3 and CASP9 in UC patients in the active phase of the disease was significantly lower than in patients in remission: BAX (p = 0.001), BCL2 (p = 0.038) and CASP9 (p = 0.007). In patients with UC, the BAX/BCL2 ratio was significantly correlated (r = 0.473) with the duration of the disease. In the group of CD patients treated biologically, a significantly lower BAX/BCL2 ratio was demonstrated than in patients that were not biologically treated. Conclusions: Our research has shown a simultaneous increase in the expression of the anti-apoptotic BCL2 gene and the proapoptotic BAX gene, which suggests the dysregulation of apoptosis mechanisms in IBD. Significantly higher expression of BAX and BCL2 in UC patients in remission as compared to CD may suggest differences in these diseases in terms of prognosis and treatment. Our results may suggest that an underlying imbalance in factors controlling apoptosis in peripheral blood lymphocytes may be the response of the immune system to inflammation of the intestinal mucosa. Modulation of apoptosis may become an important therapeutic mechanism in IBD.

Potential Involvement of BIRC5 in Maintaining Pluripotency and Cell Differentiation of Human Stem Cells.

The BIRC5 gene encodes a survivin protein belonging to class III of inhibitors of apoptosis, IAP. This protein serves a dual role. First, it regulates cell death, and second, it is an important regulator of mitosis progression, although its physiological regulatory function has not been fully understood. Many studies have shown and confirmed that survivin is practically absent in mature tissues in nature, while its overexpression has been reported in many cancerous tissues. There is little information about the significance of BIRC5 expression in normal adult human stem cells. This paper presents the study and analysis of survivin expression at the transcription level using qPCR method, in hematopoietic stem cells from peripheral blood mobilized with a granulocyte growth factor, adherent cells derived from the umbilical cord, and normal bone marrow stem cells. The expression of this gene was also examined in the blood of normal healthy individuals. The results of the analysis have shown that the more mature the cells are, the lower the expression of the BIRC5 gene is. The lowest expression has been found in peripheral blood cells, while the highest in normal bone marrow cells. The more the CD34+ and CD105 cells in the tested material are, the higher the BIRC5 expression is. Stem cells from cell culture show higher BIRC5 expression. The study confirms the involvement of BIRC5 from the IAP family in many physiological processes apart from apoptosis inhibition. The possible effect of BIRC5 on cell proliferation; involvement in cell cycle, cell differentiation, survival, and maintenance of stem cells; and the possible effect of IAP on the antineoplastic properties of mesenchymal stem cells have been demonstrated. Our research suggests that BIRC5 may be responsible for the condition of stem cell pluripotency and its high expression may also be responsible for the dedifferentiation of tumor cells.

Analysis of the expression of BAX, BCL2, BIRC6, CASP3, CASP9 apoptosis genes during the first episode of schizophrenia. / Analiza ekspresji genów apoptozy: BAX, BCL2, BIRC6, CASP3, CASP9 w pierwszym epizodzie schizofrenii.

OBJECTIVES: The aim of the study was the analysis of (1) the level of BAX,BCL2,BIRC6,CASP3, CASP9 apoptosis genes expression in schizophrenic patients and in the control group, and (2) the relationships between the genes expression level and the morphological and biochemical parameters, as well as the severity of psychopathological symptoms. METHODS: The study included 21 patients diagnosed with schizophrenia according to ICD-10 and 26 healthy subjects. The following parameters were assessed: genes expression in peripheral blood lymphocytes, laboratory parameters and severity of psychopathological symptoms (using the PANSS). RESULTS: The expression of the BCL2 gene and the CASP3 gene was significantly higher in schizophrenic patients compared to the controls. A significant positive correlation was found between the BAX gene expression level and neutrophil, lymphocyte and monocyte counts as well as the severity of negative symptoms (PANSS-N), between BCL2 and CASP9 genes expression and the monocyte count, and between the CASP3 gene expression and the lymphocyte count. CONCLUSIONS: (1) Significantly higher expression of BCL2 and CASP3 genes in schizophrenic patients compared to the controls proves the intensification of the apoptosis process, fitting in the theory of increased apoptosis in the pathophysiology of schizophrenia. (2) Significant correlations between the BAX gene expression and differential blood count parameters (leucocyte, neutrophil, lymphocyte, monocyte count) in the group of schizophrenic patients suggest a relationship with immune dysregulation and confirm the presence of apoptosis in peripheral blood lymphocytes. (3) The BAX gene expression may be indicative of the severity of negative symptoms in schizophrenia. (4) The analysis of the intercorrelation of studied genes expression indicates that BAX and CASP3 genes were the most active in the apoptosis process in the study group.

Oxidative Stress and Effect of Treatment on the Oxidation Product Decomposition Processes in IBD.

Oxidative stress plays an important role in IBD because chronic intestinal inflammation is associated with the overproduction of reactive oxygen species (ROS) leading to oxidative stress, which has been implicated in IBD. Many lines of evidence suggest that IBD is associated with an imbalance between ROS and antioxidant activity which generates oxidative stress as the result of either ROS overproduction or a decrease in antioxidant activity. Our study was to evaluate the influence of oxidative stress and antioxidants on the course of the disease and treatment of IBD patients. Our results show that an increase of LOOH levels positively correlates with an increase in MDA levels; therefore, MDA may be a marker indicating lipid peroxidation. Also, being the decomposition product of oxidation processes, MDA may be applied as a useful biomarker for identifying the effect of endogenous oxidative stress in Crohn's disease patients. The anti-inflammatory efficacy of AZA drugs may be the result of a reduction of the amount of lipid peroxides in the intestinal mucosa cells in CD patients and facilitate mucosal healing.

Single Nucleotide Polymorphisms in Selected Genes in Inflammatory Bowel Disease.

INTRODUCTION: Inflammatory bowel disease (IBD) is a complicated, multifunctional disorder characterized by chronic, recurring inflammation of the digestive tract. The two main types of IBD are ulcerative colitis (UC) and Crohn's disease (CD). The aim of the study was to determine single nucleotide polymorphism in fragments of the genes CARD15/NOD2 and DLG5 in patients from the Lublin Voivodeship. PATIENTS AND METHODS: The study was carried out in Lublin (Poland) in 2016. 27 individuals participated in the research. The research group comprised 9 patients with a diagnosis of Crohn's disease and 9 with ulcerative colitis, aged 20 to 48, and 9 healthy volunteers. RESULTS: No SNPs were confirmed for the CARD15/NOD2 gene fragment, but a substitution (T>C) was found in the DLG5 gene in a Crohn's disease patient. CONCLUSION: Absence of extraintestinal symptoms in patients with Crohn's disease may be associated with the absence of CARD15/NOD2 SNPs. The study suggests that SNPs (T>C substitution) affect the function of the DLG5 protein and thus play a role in the development of IBD, in particular Crohn's disease. The analysis presented is a pilot study due to the small number of samples.