Oksüdatiivse stressi roll Wolframi sündroom 1 ja hüpotermia korral

Väitekirja elektrooniline versioon ei sisalda publikatsiooneWolframi sündroom on haruldane autosomaalne retsessiivne haigus, mida iseloomustavad juveniilne diabeet (magediabeet, tüüp I suhkrudiabeet), nägemisnärvi kahjustus, kuulmishäired, progressiivne neurodegeneratsioon, endokriinsed kahjustused ja psühhiaatrilised probleemid. Wolframi sündroom on põhjustatud mõlemas alleelis esinevatest mutatsioonidest WFS1 geenis, mille tõttu geeni produkt, wolframiin, ei oma enam tavapärast funktsiooni. Mutantse wolframiini puhul kuhjuvad voltumata valgud endoplasmaatilise retiikulumi luumenisse ning põhjustavad endoplasmaatilise retiikulumi stressi (ka oksüdatiivset stressi) ja sealse homoöstaasi häirumist ja apoptootilise raja käivitumist. Antud töö peaeesmärgiks oli kirjeldada Wfs1-defektiga (KO) hiire metaboloomi ning oksüdatiivse stressi taset erinevates kudedes (maks, süda, neerud ja pankreas) ja biovedelikes (veri ja uriin) enne ja pärast antioksüdantide manustamist. Lisaks analüüsida missugune on hüpotermia mõju glutatiooni (GSH) süsteemile erinevates rakuliinides. Selgus, et nooremad KO hiired kasutavad energia saamiseks eelkõige glükoosi, glükoneogeneesi ja anaeroobset glükolüüsi, kuid hilisemas vanuses kui haigus rohkem progresseerunud, eelistatult lipolüüsi. Lisaks esines noorematel KO hiirtel glükosuuria, mis tüüpiliselt diabeedi varajases staadiumis ei esine. Näitasime, et redutseeritud GSH kontsentratsioon on üldiselt KO hiirte kudedes madalam kui metsiktüüpi (WT) liigikaaslastel. Maksakoes siiski täheldasime mõningast GSH taseme tõusu noortel hiirtel, mis viitab GSH sünteesi intensiivistumisele stressitingimustes. Antioksüdantse ensüümi GSH peroksüdaasi aktiivsus oli südames ja maksas KO hiirtel kõrgem ja GSH reduktaasi aktiivsus madalam võrreldes WT hiirtega. Neerukoes oli mõlema ensüümi aktiivsus KO hiirtel kõrgem. Antioksüdantite manustamine parandas eelkõige GSH taset südames ja maksakoes ning suurendas vanematel ja vähendas noorematel hiirtel GSH süsteemi ensüümide aktiivsust. Hüpotermia-indutseeritud rakkudes on kõrgem totaalse GSH kontsentratsioon metsik-tüüpi hiire embrüonaalsetes fibroblastides ja HeLa rakkudes, kusjuures esimestes vähenes oksüdeeritud GSH tase ja teises jäi see muutumatuks.The deficiency in WFS1 gene causes Wolfram syndrome (WS), which represents a valuable disease model currently available for identifying markers associated with endoplasmic reticulum (ER) stress, juvenile-onset diabetes and neurodegeneration. Another important factor is that WS arises from mutation of a single gene, which makes it a good model for teasing out the mechanisms of ER dysfunction than other multifactorial conditions like diabetes and oxidative stress. Studying oxidative stress and metabolic profiling of Wfs1-deficient mice under hyperglycemic conditions to find therapies aimed at reducing stress in patients or those at risk for developing diabetes. Also this might give new insight of the association between the Wfs1 and its functions. The metabolomic characterization of Wfs1-deficient mice (KO) revealed a broad spectrum of metabolic complications and affected glutathione redox status in the knock-out mice. At the whole organism level, the glucose use, gluconeogenesis and anaerobic glycolysis appear to be increased in the early stages of the disease, but later the energy demand is satisfied by intensified lipolysis. Furthermore, in the blood and liver tissue of KO mice, the progression of the WS exceeds hypouricemia into hyperuricemia. In the pancreas and heart tissue young mice, glycosuria preceded hyperglycemia, which implied to kidney dysfunction. The concentration of reduced glutathione (GSH) was generally decreased in Wfs1-deficient mice, but a slight upregulation of GSH in the liver is probably an attempt to control ER stress. In the liver and heart the activity of glutathione peroxidase was increased and the activity of glutathione reductase was decreased in KO mice compared to wild-type littermates. In the kidneys KO mice, the activity of both enzymes increased. The antioxidants had the highest effect improving the glutathione status in the liver and heart tissue of Wfs1-deficient mice. In the liver tissue, the studied antioxidants mainly reduced the acitivity of glutathione reductase and peroxidase in older mice and inversely in the younger littermates. Hypothermia induced the highest level of total glutathione in wild-type mouse embryonic fibroblasts and HeLa cells, whereas the concentration of oxidized glutathione was decreased or remained unchanged, respectively.https://www.ester.ee/record=b522645

Wolframi sündroom 1 geeni defektiga hiire metaboloomi kirjeldus

Ligipääs piiratud kuni 01.01.20162016-01-0

On the methodological limitations of detecting oxidative stress: effects of paraquat on measures of oxidative status in greenfinches

Oxidative stress (OS) is widely believed to be responsible for the generation of trade-offs in evolutionary ecology by means of constraining investment into a number of components of fitness. Yet, progress in understanding the true role of OS in ecology and evolution has remained elusive. Interpretation of current findings is particularly hampered by the scarcity of experiments demonstrating which of the many available parameters of oxidative status respond most sensitively to and are relevant for measuring OS. We addressed these questions in wild-caught captive greenfinches (Carduelis chloris) by experimental induction of OS by administration of the pro-oxidant compound paraquat with drinking water. Treatment induced 50% mortality, a significant drop in body mass and an increase in oxidative DNA damage and glutathione levels in erythrocytes among the survivors of the high paraquat (0.2 g l(-1) over 7 days) group. Samples taken 3 days after the end of paraquat treatment showed no effect on the peroxidation of lipids (plasma malondialdehyde), carbonylation of proteins (in erythrocytes), parameters of plasma antioxidant protection (total antioxidant capacity and oxygen radical absorbance), uric acid or carotenoids. Our findings of an increase in one marker of damage and one marker of protection from the multitude of measured variables indicate that detection of OS is difficult even under the most stringent experimental induction of oxidative insult. We hope that this study highlights the need for reconsideration of over-simplistic models of OS and draws attention to the limitations of detection of OS due to time-lagged and hormetic upregulation of protective mechanisms. This study also underpins the diagnostic value of measurement of oxidative damage to DNA bases and assessment of erythrocyte glutathione levels

Modification of the linker amino acid in the cell-penetrating peptide NickFect55 leads to enhanced pDNA transfection for in vivo applications

Despite numerous efforts over the last three decades, nucleic acid-based therapeutics still lack delivery platforms in the clinical stage. Cell-penetrating peptides (CPPs) may offer solutions as potential delivery vectors. We have previously shown that designing a “kinked” structure in the peptide backbone resulted in a CPP with efficient in vitro transfection properties. Further optimization of the charge distribution in the C-terminal part of the peptide led to potent in vivo activity with the resultant CPP NickFect55 (NF55). Currently, the impact of the linker amino acid was further investigated in the CPP NF55, with the aim to discover potential transfection reagents for in vivo application. Taking into account the expression of the delivered reporter in the lung tissue of mice, and the cell transfection in the human lung adenocarcinoma cell line, the new peptides NF55-Dap and NF55-Dab* have a high potential for delivering nucleic acid-based therapeutics to treat lung associated diseases, such as adenocarcinoma.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)19/20907-72022/3056-

<i>Pseudomonas putida</i> Responds to the Toxin GraT by Inducing Ribosome Biogenesis Factors and Repressing TCA Cycle Enzymes

The potentially self-poisonous toxin-antitoxin modules are widespread in bacterial chromosomes, but despite extensive studies, their biological importance remains poorly understood. Here, we used whole-cell proteomics to study the cellular effects of the Pseudomonas putida toxin GraT that is known to inhibit growth and ribosome maturation in a cold-dependent manner when the graA antitoxin gene is deleted from the genome. Proteomic analysis of P. putida wild-type and &#916;graA strains at 30 &#176;C and 25 &#176;C, where the growth is differently affected by GraT, revealed two major responses to GraT at both temperatures. First, ribosome biogenesis factors, including the RNA helicase DeaD and RNase III, are upregulated in &#916;graA. This likely serves to alleviate the ribosome biogenesis defect of the &#916;graA strain. Secondly, proteome data indicated that GraT induces downregulation of central carbon metabolism, as suggested by the decreased levels of TCA cycle enzymes isocitrate dehydrogenase Idh, &#945;-ketoglutarate dehydrogenase subunit SucA, and succinate-CoA ligase subunit SucD. Metabolomic analysis revealed remarkable GraT-dependent accumulation of oxaloacetate at 25 &#176;C and a reduced amount of malate, another TCA intermediate. The accumulation of oxaloacetate is likely due to decreased flux through the TCA cycle but also indicates inhibition of anabolic pathways in GraT-affected bacteria. Thus, proteomic and metabolomic analysis of the &#916;graA strain revealed that GraT-mediated stress triggers several responses that reprogram the cell physiology to alleviate the GraT-caused damage

Low molecular weight metabolites as possible new non-invasive tool for selecting bovine in vitro produced embryos

Selecting high quality preimplantation embryo for transfer has been the most difficult task when producing embryos in vitro. To date the most used non-invasive method is based on visual observation. Developing a non-invasive method for embryo assessment is essential to have a profitable in vitro embryo production (IVP) and embryo transfer system. Molecular characterization of embryo growth media has been proposed as an complementary method to visual assessment of embryo morphology. In this study we are demonstrating a novel method, allowing sample collection at different embryo development stages, without compromising embryo quality, to determine potential viability markers for bovine IVP. Single bovine embryos were cultured in 60µl SOF+0.4% BSA droplets under mineral oil. Twenty µl of culture media was removed at day 2, 5 and 8 post-fertilization. A total of 58 samples were analyzed using liquid chromatography-mass spectrometry (Q-Trap 3200), followed by principal component analysis. Our results indicate that there are significant differences (p<0,00001) in concentrations for proline (m/z = 116), inositol (m/z of sodium adduct = 203) and citrate (m/z of sodium adduct = 215) also in the amino acid group of leucine and isoleucine (m/z = 132), phenylalanine (m/z = 165) and arginine (m/z = 211) between the normally developed and retarded in development embryo culture media. Platelet activating factor (m/z = 524) (PAF) was roughly 3 fold increased in day 5 to day 8 embryo culture media. Unfortunately the increase of PAF was not statistically significant between normally developing and retarded embryos. These results demonstrate that it is possible to remove culture media samples from droplets and not significantly affect embryo development. Applying this method for embryo selection provides a possibility to identify well-developing embryos and provides an opportunity for improving the herds genetic value

Cell-Penetrating Peptide and siRNA-Mediated Therapeutic Effects on Endometriosis and Cancer In Vitro Models

Gene therapy is a powerful tool for the development of new treatment strategies for various conditions, by aiming to transport biologically active nucleic acids into diseased cells. To achieve that goal, we used highly potential delivery vectors, cell-penetrating peptides (CPPs), as oligonucleotide carriers for the development of a therapeutic approach for endometriosis and cancer. Despite marked differences, both of these conditions still exhibit similarities, like excessive, uncoordinated, and autonomous cellular proliferation and invasion, accompanied by overlapping gene expression patterns. Thus, in the current study, we investigated the therapeutic effects of CPP and siRNA nanoparticles using in vitro models of benign endometriosis and malignant glioblastoma. We demonstrated that CPPs PepFect6 and NickFect70 are highly effective in transfecting cell lines, primary cell cultures, and three-dimensional spheroids. CPP nanoparticles are capable of inducing siRNA-specific knockdown of therapeutic genes, ribonucleotide reductase subunit M2 (RRM2), and vascular endothelial growth factor (VEGF), which results in the reduction of in vitro cellular proliferation, invasion, and migration. In addition, we proved that it is possible to achieve synergistic suppression of endometriosis cellular proliferation and invasion by combining gene therapy and hormonal treatment approaches by co-administering CPP/siRNA nanoparticles together with the endometriosis-drug danazol. We suggest a novel target, RRM2, for endometriosis therapy and as a proof-of-concept, we propose a CPP-mediated gene therapy approach for endometriosis and cancer

In vitro culture and non-invasive metabolic profiling of single bovine embryos

Selecting high-quality embryos for transfer has been a difficult task when producing bovine embryos in vitro. The most used non-invasive method is based on visual observation. Molecular characterisation of embryo growth media has been proposed as a complementary method. In this study we demonstrate a culture medium sampling method for identifying potential embryonic viability markers to predict normal or abnormal embryonic development. During single embryo culture, 20 µL culture media was removed at Days 2, 5 and 8 after fertilisation from the same droplet (60 µL). In all, 58 samples were analysed using liquid chromatography–mass spectrometry. We demonstrate that it is possible to remove samples from the same culture medium droplets and not significantly affect blastocyst rate (25.2%). Changes in any single low molecular weight compound were not predictive enough. Combining multiple low molecular weight signals made it possible to predict Day 2 and 5 embryo development to the blastocyst stage with an accuracy of 64%. Elevated concentrations of lysophosphatidylethanolamines (m/z = 453, 566, 588) in the culture media of Day 8 well-developing embryos were observed. Choline (104 m/z) and citrate (215 m/z) concentrations were increased in embryos in which development was retarded. Metabolic profiling provides possibilities to identify well-developing embryos before transfer, thus improving pregnancy rates and the number of calves born