Halimane Derivatives from Plectranthus ornatus Codd. as Novel Anti-cancer Agents.

The Plectranthus genus is often cited for its medicinal properties. Plectranthus ornatus Codd. is traditionally used in Africa for the treatment of gastric and liver diseases and their leaves are used for their antibiotic action. The main constituent of P. ornatus is the halimane compound, 11 R∗-acetoxyhalima-5,13E-dien-15-oic acid (Hal), described for its antimicrobial and anticancer properties. The objective of this work was to improve the activity of the halimane lead molecule. Further physiochemical characterisation was performed on Hal. To the best of our knowledge, this work constitutes the first published data of the absolute configurations by SCXRD and thermal stability of Hal. Using Hal, reactions with different amines were carried out to afford novel semi-synthetic derivatives and their structural elucidation was completed. The cytotoxicity of the derivatives was assessed against three leukaemia cancer cell lines (CCRF-CEM, K562 and HL-60). The antioxidant activity was investigated using H2O2-induced HGF-1 cells and their anti-inflammatory activity was studied using RT-PCR and ELISA. Our data showed that amide derivatives of Hal presented moderate cytotoxicity and more potent activity when compared to the parent molecule, giving insight into the SAR of Hal. The derivatives also displayed protection against oxidative damage to DNA. Finally, the derivatives possessed anti-inflammatory properties at the level of gene and protein expression for the cytokines IL-1ß, TNF-α and IL-6, induced by LPS in normal HGF-1 cells. Overall, our study provides useful insight into the enhanced biological activities of semi-synthetic Hal derivatives, as a starting point for novel drug formulations in cancer therapy.

Biological Properties of Extracts Obtained from In Vitro Culture of Plectranthus scutellarioides in a Cell Model.

Plectranthus scutellarioides (L.) R.Br. is a medicinal plant that has long been used in traditional medicine to treat conditions such as abscesses, ulcers, and ear and eye infections. It is known to have a wide range of biological properties, such as antibacterial, antioxidant, antifungal, anti-inflammatory, anti-diabetic and anti-cancer effects. In this study, we established in vitro cultures from both the aerial parts and roots of Plectranthus scutellarioides. Subsequently, we compared the basic phytochemical profile of the obtained extracts and conducted a biological analysis to assess their potential for inducing apoptosis in breast (MCF-7) and lung (A549) cancer cells. Phytochemical analysis by HPLC-MS revealed the presence of compounds belonging to phenolic acids (ferulic, syringic, vanillic, rosmarinic, chlorogenic, caffeic, coumaric, dihydroxybenzoic acids), flavonoids (eriodyctiol and cirsimaritin), and terpenes such as 6,11,12,14,16-Pentahydroxy-3,17diacetyl-8,11,13-abietatrien-7-one, 6,11,12,14,16-Pentahydroxy-3,17-diacetyl5,8,11,13-abietatetraen-7-one, and 3,6,12-Trihydroxy-2-acetyl-8,12-abietadien7,11,14-trione. The results show that both extracts have a cytotoxic and genotoxic effect against MCF-7 and A549 cancer cells, with a different degree of sensitivity. It was also shown that both extracts can induce apoptosis by altering the expression of apoptotic genes (Bax, Bcl-2, TP53, Fas, and TNFSF10), reducing mitochondrial membrane potential, increasing ROS levels, and increasing DNA damage. In addition, it has been shown that the tested extracts can alter blood coagulation parameters. Our results indicate that extracts from in vitro cultures of Plectranthus scutellarioides aerial parts and roots have promising therapeutic application, but further research is needed to better understand the mechanisms of their action in the in vitro model.

DNA Damage Induced by T-2 Mycotoxin in Human Skin Fibroblast Cell Line-Hs68.

T-2 mycotoxin is the most potent representative of the trichothecene group A and is produced by various Fusarium species, including F. sporotrichioides, F. poae, and F. acuminatum. T-2 toxin has been reported to have toxic effects on various tissues and organs, and humans and animals alike suffer a variety of pathological conditions after consumption of mycotoxin-contaminated food. The T-2 toxin's unique feature is dermal toxicity, characterized by skin inflammation. In this in vitro study, we investigated the molecular mechanism of T-2 toxin-induced genotoxicity in the human skin fibroblast-Hs68 cell line. For the purpose of investigation, the cells were treated with T-2 toxin in 0.1, 1, and 10 µM concentrations and incubated for 24 h and 48 h. Nuclear DNA (nDNA) is found within the nucleus of eukaryotic cells and has a double-helix structure. nDNA encodes the primary structure of proteins, consisting of the basic amino acid sequence. The alkaline comet assay results showed that T-2 toxin induces DNA alkali-labile sites. The DNA strand breaks in cells, and the DNA damage level is correlated with the increasing concentration and time of exposure to T-2 toxin. The evaluation of nDNA damage revealed that exposure to toxin resulted in an increasing lesion frequency in Hs68 cells with HPRT1 and TP53 genes. Further analyses were focused on mRNA expression changes in two groups of genes involved in the inflammatory and repair processes. The level of mRNA increased for all examined inflammatory genes (TNF, INFG, IL1A, and IL1B). In the second group of genes related to the repair process, changes in expression induced by toxin in genes-LIG3 and APEX were observed. The level of mRNA for LIG3 decreased, while that for APEX increased. In the case of LIG1, FEN, and XRCC1, no changes in mRNA level between the control and T-2 toxin probes were observed. In conclusion, the results of this study indicate that T-2 toxin shows genotoxic effects on Hs68 cells, and the molecular mechanism of this toxic effect is related to nDNA damage.

Single-Nucleotide Polymorphisms in Genes Maintaining the Stability of Mitochondrial DNA Affect the Occurrence, Onset, Severity and Treatment of Major Depressive Disorder.

One of the key features of major depressive disorder (MDD, depression) is increased oxidative stress manifested by elevated levels of mtROS, a hallmark of mitochondrial dysfunction, which can arise from mitochondrial DNA (mtDNA) damage. Thus, the current study explores possibility that the single-nucleotide polymorphisms (SNPs) of genes encoding the three enzymes that are thought to be implicated in the replication, repair or degradation of mtDNA, i.e., POLG, ENDOG and EXOG, have an impact on the occurrence, onset, severity and treatment of MDD. Five SNPs were selected: EXOG c.-188T > G (rs9838614), EXOG c.*627G > A (rs1065800), POLG c.-1370T > A (rs1054875), ENDOG c.-394T > C (rs2977998) and ENDOG c.-220C > T (rs2997922), while genotyping was performed on 538 DNA samples (277 cases and 261 controls) using TaqMan probes. All SNPs of EXOG and ENDOG modulated the risk of depression, but the strongest effect was observed for rs1065800, while rs9838614 and rs2977998 indicate that they might influence the severity of symptoms, and, to a lesser extent, treatment effectiveness. Although the SNP located in POLG did not affect occurrence of the disease, the result suggests that it may influence the onset and treatment outcome. These findings further support the hypothesis that mtDNA damage and impairment in its metabolism play a crucial role not only in the development, but also in the treatment of depression.

Chitosan Nanoparticles-Preparation, Characterization and Their Combination with Ginkgo biloba Extract in Preliminary In Vitro Studies.

Nanoparticles (NPs), due to their size, have a key position in nanotechnology as a spectrum of solutions in medicine. NPs improve the ability of active substances to penetrate various routes: transdermal, but also digestive (active endocytosis), respiratory and injection. Chitosan, an N-deacetylated derivative of chitin, is a natural biodegradable cationic polymer with antioxidant, anti-inflammatory and antimicrobial properties. Cross-linked chitosan is an excellent matrix for the production of nanoparticles containing active substances, e.g., the Ginkgo biloba extract (GBE). Chitosan nanoparticles with the Ginkgo biloba extract (GBE) were obtained by ion gelation using TPP as a cross-linking agent. The obtained product was characterized in terms of morphology and size based on SEM and Zeta Sizer analyses as well as an effective encapsulation of GBE in nanoparticles-FTIR-ATR and UV-Vis analyses. The kinetics of release of the active substance in water and physiological saline were checked. Biological studies were carried out on normal and cancer cell lines to check the cytotoxic effect of GBE, chitosan nanoparticles and a combination of the chitosan nanoparticles with GBE. The obtained nanoparticles contained and released GBE encapsulated in research media. Pure NPs, GBE and a combination of NPs and the extract showed cytotoxicity against tumor cells, with no cytotoxicity against the physiological cell line.

Mitochondrial Damage Induced by T-2 Mycotoxin on Human Skin-Fibroblast Hs68 Cell Line.

T-2 toxin is produced by different Fusarium species and belongs to the group of type A trichothecene mycotoxins. T-2 toxin contaminates various grains, such as wheat, barley, maize, or rice, thus posing a risk to human and animal health. The toxin has toxicological effects on human and animal digestive, immune, nervous and reproductive systems. In addition, the most significant toxic effect can be observed on the skin. This in vitro study focused on T-2 toxicity on human skin fibroblast Hs68 cell line mitochondria. In the first step of this study, T-2 toxin's effect on the cell mitochondrial membrane potential (MMP) was determined. The cells were exposed to T-2 toxin, which resulted in dose- and time-dependent changes and a decrease in MMP. The obtained results revealed that the changes of intracellular reactive oxygen species (ROS) in the Hs68 cells were not affected by T-2 toxin. A further mitochondrial genome analysis showed that T-2 toxin in a dose- and time-dependent manner decreased the number of mitochondrial DNA (mtDNA) copies in cells. In addition, T-2 toxin genotoxicity causing mtDNA damage was evaluated. It was found that incubation of Hs68 cells in the presence of T-2 toxin, in a dose- and time-dependent manner, increased the level of mtDNA damage in both tested mtDNA regions: NADH dehydrogenase subunit 1 (ND1) and NADH dehydrogenase subunit 5 (ND5). In conclusion, the results of the in vitro study revealed that T-2 toxin shows adverse effects on Hs68 cell mitochondria. T-2 toxin induces mitochondrial dysfunction and mtDNA damage, which may cause the disruption of adenosine triphosphate (ATP) synthesis and, in consequence, cell death.

Anticancer Properties of Plectranthus ornatus-Derived Phytochemicals Inducing Apoptosis via Mitochondrial Pathway.

Since cancer treatment by radio- and chemotherapy has been linked to safety concerns, there is a need for new and alternative anticancer drugs; as such, compounds isolated from plants represent promising candidates. The current study investigates the anticancer features of halimane (11R*,13E)-11-acetoxyhalima-5,13-dien-15-oic acid (HAL) and the labdane diterpenes 1α,6ß-diacetoxy-8α,13R*-epoxy-14-labden-11-one (PLEC) and forskolin-like 1:1 mixture of 1,6-di-O-acetylforskolin and 1,6-di-O-acetyl-9-deoxyforskolin (MRC) isolated from Plectranthus ornatus in MCF7 and FaDu cancer cell lines. Cytotoxicity was assessed by MTT assay, ROS production by Di-chloro-dihydro-fluorescein diacetate assay (DCFH) or Red Mitochondrial Superoxide Indicator (MitoSOX) and Mitochondrial Membrane Potential (MMP) by fluorescent probe JC-1 (5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide). In addition, the relative amounts of mitochondrial DNA (mtDNA) were determined using quantitative Real-Time-PCR (qRT-PCR) and damage to mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) by semi-long run quantitative Real-Time-PCR (SLR-qRT-PCR). Gene expression was determined using Reverse-Transcription-qPCR. Caspase-3/7 activity by fluorescence was assessed. Assessment of General In Vivo Toxicity has been determined by Brine Shrimp Lethality Bioassay. The studied HAL and PLEC were found to have a cytotoxic effect in MCF7 with IC50 = 13.61 µg/mL and IC50 = 17.49 µg/mL and in FaDu with IC50 = 15.12 µg/mL and IC50 = 32.66 µg/mL cancer cell lines. In the two tested cancer cell lines, the phytochemicals increased ROS production and mitochondrial damage in the ND1 and ND5 gene regions and reduced MMP (ΔΨm) and mitochondrial copy numbers. They also changed the expression of pro- and anti-apoptotic genes (Bax, Bcl-2, TP53, Cas-3, Cas-8, Cas-9, Apaf-1 and MCL-1). Studies demonstrated increase in caspase 3/7 activity in tested cancer cell lines. In addition, we showed no toxic effect in in vivo test for the compounds tested. The potential mechanism of action may have been associated with the induction of apoptosis in MCF7 and FaDu cancer cells via the mitochondrial pathway; however, further in vivo research is needed to understand the mechanisms of action and potential of these compounds.

An Evaluation of the Novel Biological Properties of Diterpenes Isolated from Plectranthus ornatus Codd. In Vitro and In Silico.

Plectranthus ornatus Codd, the genus Plectranthus of the Lamiaceae family, has been used as traditional medicine in Africa, India and Australia. Pharmacological studies show the use of this plant to treat digestive problems. In turn, leaves were used for their antibiotic properties in some regions of Brazil to treat skin infections. The present study examines the anti-inflammatory, antioxidant and cytotoxic effects of the halimane and labdane diterpenes (11R*,13E)-11-acetoxyhalima-5,13-dien-15-oic acid (HAL) and 1α,6ß-diacetoxy-8α,13R*-epoxy-14-labden-11-one (PLEC) and the forskolin-like 1:1 mixture of 1,6-di-O-acetylforskolin and 1,6-di-O-acetyl-9-deoxyforskolin (MRC) isolated from P. ornatus on lung (A549) and leukemia (CCRF-CEM) cancer cell lines, and on normal human retinal pigment epithelial (ARPE-19) cell line in vitro. Additionally, molecular docking and computational approaches were used. ADMET properties were analysed through SwissADME and proTox-II-Prediction. The results indicate that all tested compounds significantly reduced the viability of the cancer cells and demonstrated no cytotoxic effects against the non-neoplastic cell line. The apoptosis indicators showed increased ROS levels for both the tested A549 and CCRF-CEM cancer cell lines after treatment. Furthermore, computational studies found HAL to exhibit moderate antioxidant activity. In addition, selected compounds changed mitochondrial membrane potential (MMP), and increased DNA damage and mitochondrial copy number for the CCRF-CEM cancer cell line; they also demonstrated anti-inflammatory effects on the ARPE-19 normal cell line upon lipopolysaccharide (LPS) treatment, which was associated with the modulation of IL-6, IL-8, TNF-α and GM-CSF genes expression. Docking studies gave indication about the lowest binding energy for 1,6-di-O-acetylforskolin docked into IL-6, TNF-α and GM-CSF, and 1,6-di-O-acetyl-9-deoxyforskolin docked into IL-8. The ADMET studies showed drug-likeness properties for the studied compounds. Thus, halimane and labdane diterpenes isolated from P. ornatus appear to offer biological potential; however, further research is necessary to understand their interactions and beneficial properties.

Agomelatine Changed the Expression and Methylation Status of Inflammatory Genes in Blood and Brain Structures of Male Wistar Rats after Chronic Mild Stress Procedure.

The preclinical research conducted so far suggest that depression development may be influenced by the inflammatory pathways both at the periphery and within the central nervous system. Furthermore, inflammation is considered to be strongly connected with antidepressant treatment resistance. Thus, this study explores whether the chronic mild stress (CMS) procedure and agomelatine treatment induce changes in TGFA, TGFB, IRF1, PTGS2 and IKBKB expression and methylation status in peripheral blood mononuclear cells (PBMCs) and in the brain structures of rats. Adult male Wistar rats were subjected to the CMS and further divided into matched subgroups to receive vehicle or agomelatine. TaqMan gene expression assay and methylation-sensitive high-resolution melting (MS-HRM) were used to evaluate the expression of the genes and the methylation status of their promoters, respectively. Our findings confirm that both CMS and antidepressant agomelatine treatment influenced the expression level and methylation status of the promoter region of investigated genes in PBMCs and the brain. What is more, the present study showed that response to either stress stimuli or agomelatine differed between brain structures. Concluding, our results indicate that TGFA, TGFB, PTGS2, IRF1 and IKBKB could be associated with depression and its treatment.

Variation of genes encoding nitric oxide synthases and antioxidant enzymes as potential risks of multiple sclerosis development: a preliminary study.

Multiple sclerosis (MS) is a neurodegenerative disease characterized by a variable clinical course and diverse pathophysiology, including nitrative and oxidative stresses as well as inflammation. We aimed to detect the potential association between five selected single-nucleotide polymorphisms (SNPs) in genes encoding nitric oxide synthetases as well as antioxidant enzymes and the development of MS in a Polish population. Genomic DNA was isolated from peripheral blood collected from 142 MS patients and 140 controls. Using Taq-Man® probes, we genotyped the following SNPs: rs1879417 in NOS1, and rs2297518 in NOS2 as well as rs4880 in SOD2, rs7943316 in CAT, rs713041 in GPX4. In the case of rs2297518, the C/C genotype and C allele SNP were associated with an enhanced occurrence of MS, while the C/T, T/T genotypes, and T allele of the same polymorphism reduced this risk. Moreover, the C/C homozygote and C allele of the rs4880 SNP reduced MS risk, while the T allele increased the risk. In addition, the A/T heterozygote of rs7943316 polymorphism was associated with an increased risk of MS occurrence. We also detected that the C/C genotype and C allele of rs713041 decreased the risk of MS, whereas the T/T genotype and T allele increased this risk. In conclusion, the results of our study suggest some links between polymorphic variability in the nitrative/oxidative stress-related genes and the risk of MS development in the Polish population.

Dysregulation in the Expression of Platelet Surface Receptors in Acute Coronary Syndrome Patients-Emphasis on P2Y12.

The pathological conditions caused by blood platelet activation constitute a fundamental core in the pathogenesis of Acute Coronary Syndrome (ACS). The hyperactivity of platelets in ACS is well-documented, but there is still little research into the molecular basis of phenotypic changes in platelet functionality. To expand the knowledge of this phenomenon, we analyzed the disturbances in the expression of several key platelet receptors and the aspect of regulating potential abnormalities. Platelet surface receptors are responsible for maintaining the hemostatic balance, platelet interaction with immune cells, and support of the coagulation cascade leading to occlusion of the vessel lumen. Due to their prominent role, platelet receptors constitute a major target in pharmacological treatment. Our work aimed to identify the molecular alteration of platelet surface receptors, which showed augmented mRNA expression of P2Y12, GP1BB, ITGA2B, and ITGB3 and increased protein concentrations of P2Y12 and GP IIb/IIIa in ACS. The upregulation of the P2Y12 level was also confirmed by confocal and cytometric visualization. Furthermore, we evaluated the expression of two microRNAs: miR-223-3p and miR-126-3p, which were suggested to regulate platelet P2Y12 expression. Results of our study present new insight into the molecular background of ACS.

Direct T-2 Toxicity on Human Skin-Fibroblast Hs68 Cell Line-In Vitro Study.

T-2 toxin is produced by different Fusarium species, and it can infect crops such as wheat, barley, and corn. It is known that the T-2 toxin induces various forms of toxicity such as hepatotoxicity, nephrotoxicity, immunotoxicity, and neurotoxicity. In addition, T-2 toxin possesses a strong dermal irritation effect and can be absorbed even through intact skin. As a dermal irritant agent, it is estimated to be 400 times more toxic than sulfur mustard. Toxic effects can include redness, blistering, and necrosis, but the molecular mechanism of these effects still remains unknown. This in vitro study focused on the direct toxicity of T-2 toxin on human skin-fibroblast Hs68 cell line. As a result, the level of toxicity of T-2 toxin and its cytotoxic mechanism of action was determined. In cytotoxicity assays, the dose and time-dependent cytotoxic effect of T-2 on a cell line was observed. Bioluminometry results showed that relative levels of ATP in treated cells were decreased. Further analysis of the toxin's impact on the induction of apoptosis and necrosis processes showed the significant predominance of PI-stained cells, lack of caspase 3/7 activity, and increased concentration of released Human Cytokeratin 18 in treated cells, which indicates the necrosis process. In conclusion, the results of an in vitro human skin fibroblast model revealed for the first time that the T-2 toxin induces necrosis as a toxicity effect. These results provide new insight into the toxic T-2 mechanism on the skin.

Variations in the Gene Expression Profile in Atherosclerotic Patients with Non-Fatal ACS: A Preliminary Study.

The pathophysiology of atherosclerosis and acute coronary syndrome (ACS) is related to interactions between immune cells, endothelium, and blood platelets. An increasing number of reports confirm the link between excessive immune activation and cellular cross-talk with ACS incidence. Our genetic and proteomic analysis was performed on strictly selected atherosclerotic patients with non-fatal ACS without typical risk factors and healthy donors. Results showed changes in the gene expression levels of the various inflammatory factors derived from the peripheral blood cells that drive the over-activation of the immune system. The enhanced activation of the immune system may lead to the overexpression of the pro-inflammatory mediators, which causes self-perpetuating machinery of processes associated with thrombosis. In our preliminary study, we confirmed an altered expression of genes associated with the inflammation and overall interaction of the vascular microenvironment. Furthermore, 5 of 92 analyzed genes, CCL2, CCR2, CSF2, GZMB, and ICOS, were expressed only in patients with ACS. In conclusion, the augmented expression of the pro-inflammatory genes from the peripheral blood cells may be a crucial genetic factor leading to the occurrence of acute inflammation and thus be significant in ACS pathogenesis.

Chronic Mild Stress and Venlafaxine Treatment Were Associated with Altered Expression Level and Methylation Status of New Candidate Inflammatory Genes in PBMCs and Brain Structures of Wistar Rats.

Preclinical studies conducted to date suggest that depression could be elicited by the elevated expression of proinflammatory molecules: these play a key role in the mediation of neurochemical, neuroendocrine and behavioral changes. Thus, this study investigates the effect of chronic mild stress (CMS) and administration of venlafaxine (SSRI) on the expression and methylation status of new target inflammatory genes: TGFA, TGFB, IRF1, PTGS2 and IKBKB, in peripheral blood mononuclear cells (PMBCs) and in selected brain structures of rats. Adult male Wistar rats were subjected to the CMS and further divided into matched subgroups to receive vehicle or venlafaxine. TaqMan gene expression assay and methylation-sensitive high-resolution melting (MS-HRM) were used to evaluate the expression of the genes and the methylation status of their promoters, respectively. Our results indicate that both CMS and chronic treatment with venlafaxine were associated with changes in expression of the studied genes and their promoter methylation status in PMBCs and the brain. Moreover, the effect of antidepressant administration clearly differed between brain structures. Summarizing, our results confirm at least a partial association between TGFA, TGFB, IRF1, PTGS2 and IKBKB and depressive disorders.

Relationship between Oxidative Stress and Imatinib Resistance in Model Chronic Myeloid Leukemia Cells.

Chronic myeloid leukemia (CML) develops due to the presence of the BCR-ABL1 protein, a target of tyrosine kinase inhibitors (TKIs), such as imatinib (IM), used in a CML therapy. CML eradication is a challenge due to developing resistance to TKIs. BCR-ABL1 induces endogenous oxidative stress leading to genomic instability and development of TKI resistance. Model CML cells susceptible or resistant to IM, as well as wild-type, non-cancer cells without the BCR-ABL1 protein were treated with IM, hydrogen peroxide (H2O2) as a model trigger of external oxidative stress, or with IM+H2O2. Accumulation of reactive oxygen species (ROS), DNA damage, activity of selected antioxidant enzymes and glutathione (GSH), and mitochondrial potential (MMP) were assessed. We observed increase in ROS accumulation in BCR-ABL1 positive cells and distinct levels of ROS accumulation in IM-susceptible cells when compared to IM-resistant ones, as well as increased DNA damage caused by IM action in sensitive cells. Depletion of GSH levels and a decreased activity of glutathione peroxidase (GPx) in the presence of IM was higher in the cells susceptible to IM. IM-resistant cells showed an increase of catalase activity and a depletion of MMP. BCR-ABL1 kinase alters ROS metabolism, and IM resistance is accompanied by the changes in activity of GPx, catalase, and alterations in MMP.

Single-Nucleotide Polymorphisms in Oxidative Stress-Related Genes and the Risk of a Stroke in a Polish Population-A Preliminary Study.

The present preliminary case-control study was undertaken to detect the potential association of six single nucleotide polymorphisms (SNPs) in oxidative stress-related genes: SOD2 (c.47T > C; rs4880), CAT (c.-89A > T; rs7943316), GPX4 (c.660T > A; rs713041), NOS1 (g.117803515C > T; rs1879417) and NOS2 (c.1823C > T; rs2297518 and c.-227G > C; rs10459953) and the occurrence of a stroke. The SNPs were determined using the TaqMan® Allelic Discrimination Assay in 107 patients with strokes and 107 age- and sex-matched individuals who had not experienced cerebrovascular accidents. The T alleles of the rs4880 were positively correlated with a stroke (bootstrap OR 1.31; 1.07-1.59 95% CI). In the case of the rs713041, an association with the T allele was found (bootstrap OR 1.36; 1.12-1.67). In addition, the occurrence of a stroke was associated with the presence of the C allele of the rs1879417 (bootstrap OR 1.32; 1.09-1.61). We also found that the C/C genotype and C allele of the rs2297518 increased the risk of a stroke (bootstrap ORs 7.00; 4.34-11.29 and 4.96; 3.88-6.34, respectively). Moreover, the C allele of the rs10459953 was associated with an increased occurrence of this disease (bootstrap OR 1.31; 1.08-1.60). These results indicated that genetics variants in the SOD2, GPX4, NOS1 and NOS2 might be associated with susceptibility to strokes in the Polish population.

The Effect of Chronic Mild Stress and Escitalopram on the Expression and Methylation Levels of Genes Involved in the Oxidative and Nitrosative Stresses as Well as Tryptophan Catabolites Pathway in the Blood and Brain Structures.

Previous studies suggest that depression may be associated with reactive oxygen species overproduction and disorders of the tryptophan catabolites pathway. Moreover, one-third of patients do not respond to conventional pharmacotherapy. Therefore, the study investigates the molecular effect of escitalopram on the expression of Cat, Gpx1/4, Nos1/2, Tph1/2, Ido1, Kmo, and Kynu and promoter methylation in the hippocampus, amygdala, cerebral cortex, and blood of rats exposed to CMS (chronic mild stress). The animals were exposed to CMS for two or seven weeks followed by escitalopram treatment for five weeks. The mRNA and protein expression of the genes were analysed using the TaqMan Gene Expression Assay and Western blotting, while the methylation was determined using methylation-sensitive high-resolution melting. The CMS caused an increase of Gpx1 and Nos1 mRNA expression in the hippocampus, which was normalised by escitalopram administration. Moreover, Tph1 and Tph2 mRNA expression in the cerebral cortex was increased in stressed rats after escitalopram therapy. The methylation status of the Cat promoter was decreased in the hippocampus and cerebral cortex of the rats after escitalopram therapy. The Gpx4 protein levels were decreased following escitalopram compared to the stressed/saline group. It appears that CMS and escitalopram influence the expression and methylation of the studied genes.

Autophagy Genes for Wet Age-Related Macular Degeneration in a Finnish Case-Control Study.

Age-related macular degeneration is an eye disease that is the main cause of legal blindness in the elderly in developed countries. Despite this, its pathogenesis is not completely known, and many genetic, epigenetic, environmental and lifestyle factors may be involved. Vision loss in age-related macular degeneration (AMD) is usually consequence of the occurrence of its wet (neovascular) form that is targeted in the clinic by anti-VEGF (vascular endothelial growth factor) treatment. The wet form of AMD is associated with the accumulation of cellular waste in the retinal pigment epithelium, which is removed by autophagy and the proteosomal degradation system. In the present work, we searched for the association between genotypes and alleles of single nucleotide polymorphisms (SNPs) of autophagy-related genes and wet AMD occurrence in a cohort of Finnish patients undergoing anti-VEGF therapy and controls. Additionally, the correlation between treatment efficacy and genotypes was investigated. Overall, 225 wet AMD patients and 161 controls were enrolled in this study. Ten SNPs (rs2295080, rs11121704, rs1057079, rs1064261, rs573775, rs11246867, rs3088051, rs10902469, rs73105013, rs10277) in the mTOR (Mechanistic Target of Rapamycin), ATG5 (Autophagy Related 5), ULK1 (Unc-51-Like Autophagy Activating Kinase 1), MAP1LC3A (Microtubule Associated Protein 1 Light Chain 3 α), SQSTM1 (Sequestosome 1) were analyzed with RT-PCR-based genotyping. The genotype/alleles rs2295080-G, rs11121704-C, rs1057079-C and rs73105013-T associated with an increased, whereas rs2295080-TT, rs2295080-T, rs11121704-TT, rs1057079-TT, rs1057079-T, rs573775-AA and rs73105013-C with a decreased occurrence of wet AMD. In addition, the rs2295080-GG, rs2295080-GT, rs1057079-TT, rs11246867-AG, rs3088051-CC and rs10277-CC genotypes were a positively correlated cumulative number of anti-VEGF injections in 2 years. Therefore, variability in autophagy genes may have an impact on the risk of wet AMD occurrence and the efficacy of anti-VEGF treatment.

The Impact of Chronic Mild Stress and Agomelatine Treatment on the Expression Level and Methylation Status of Genes Involved in Tryptophan Catabolic Pathway in PBMCs and Brain Structures.

Depression is the serious mental disorder. Previous studies suggest that the development mechanism of depression may be associated with disorders of the tryptophan catabolic pathway (TRYCAT). Thus, this study investigates the effect of agomelatine treatment on the expression and methylation status of genes involved in TRYCAT in the brain and blood of rats exposed to a chronic mild stress (CMS). Separate groups of rats were exposed to CMS for two or seven weeks; the second group received vehicle or agomelatine for five weeks. After completion of both stress conditions and treatment, the expression levels of messenger RNA (mRNA) and protein, as well as the methylation status of promoters, were measured in peripheral blood mononuclear cells (PBMCs) and in brain structures with the use of TaqMan Gene Expression Assay, Western blot, and methylation-sensitive high-resolution melting techniques. In PBMCs, Kmo mRNA expression increased in the group after CMS, while this effect was normalized by agomelatine therapy. In brain, KatI and KatII expression changed following CMS exposure. Moreover, CMS decreased the methylation status of the second Tdo2 promoter in the amygdala. Protein expression of Tph1, Tph2, Ido1, and KatII changed in the group after CMS and agomelatine administration, most prominently in the basal ganglia, cerebral cortex, hippocampus, and amygdala. The results indicate that CMS and agomelatine affect the mRNA and protein expression, as well as the methylation of promoters of genes involved in the tryptophan catabolic pathway.

Oxidative Damage of Blood Platelets Correlates with the Degree of Psychophysical Disability in Secondary Progressive Multiple Sclerosis.

The results of past research studies show that platelets are one of the main sources of reactive oxygen species (ROS) and reactive nitrogen species (RNS) to be found in the course of many pathological states. The aim of this study was to determine the level of oxidative/nitrative stress biomarkers in blood platelets obtained from multiple sclerosis (MS) patients (n = 110) and to verify their correlation with the clinical parameters of the psychophysical disability of patients. The mitochondrial metabolism of platelets was assessed by measuring the intracellular production of ROS using the fluorescence method with DCFH-DA dye and by identification of changes in the mitochondrial membrane potential of platelets using the JC-1 dye. Moreover, we measured the mRNA expression for the gene encoding the cytochrome c oxidase subunit I (MTCO-1) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in platelets and megakaryocytes using the RT-qPCR method, as well as the concentration of NADPH oxidase (NOX-1) by the ELISA method. Our results proved an increased level of oxidative/nitrative damage of proteins (carbonyl groups, 3-nitrotyrosine) (p < 0.0001) and decreased level of -SH in MS (p < 0.0001) and also a pronounced correlation between these biomarkers and parameters assessed by the Expanded Disability Status Scale and the Beck's Depression Inventory. The application of fluorescence methods showed mitochondrial membrane potential disruption (p < 0.001) and higher production of ROS in platelets from MS compared to control (p < 0.0001). Our research has also confirmed the impairment of red-ox metabolism in MS, which was achieved by increasing the relative mRNA expression in platelets for the genes studied (2-fold increase for the MTCO-1 gene and 1.5-fold increase in GAPDH gene, p < 0.05), as well as the augmented concentration of NOX-1 compared to control (p < 0.0001). Our results indicate that the oxidative/nitrative damage of platelets is implicated in the pathophysiology of MS, which reflects the status of the disease.