Structural and functional characterization of HMGB1 protein in rat liver during experimentally-induced diabetes type 1

Оксидативни стрес и хронична инфламација сматрају се главним узроцима појаве дијабетичних компликација, међу којима су и оштећења јетре. Важну улогу медијатора ових процеса може имати ендогени протеин HMGB1, који у ванћелијску средину доспева из некротичних, оштећених и активираних ћелија. Како је у дијабетесу понашање HMGB1 протеина слабо изучавано, у овој докторској дисертацији испитиван је допринос HMGB1 оштећењима јетре пацова са стрептозотоцином-изазваним ДТ1. Показано је да ниво оштећења јетре током дијабетесa корелише са присуством ванћелијског HMGB1. Овај протеин, у дијабетичној јетри, бива структурно модификован ацетилацијом, фосфорилацијом и O-GlcNAc гликозилацијом што корелише са његовим изласком из једра ћелија у цитоплазму и повећањем његовог присуства у јетри и серуму. Резултати у вези са снижавањeм нивоа ванћелијског HMGB1 третманом дијабетичних пацова мелатонином или етил пируватом, указују да HMGB1 доприноси оштећењу јетре у дијабетесу одржавањем стања хроничне инфламације, стишавањем антиоксидативне одбране и стишавањем регенерације. Ванћелијски HMGB1 кроз интеракције са TLR4 рецептором активира MAPК/NF-κB p65 и ЈАК1/STAT3 сигналне путеве, доприносећи повећању продукције проинфламацијских цитокина TNF-α и IL-6 и акутно-фазног протеина хаптоглобина. Подстицањем NF-κB p65 инфламацијског пута, HMGB1 делује негативно на цитопротективни одговор у дијабетичној јетри тако што онемогућава активност Nrf2 протеина, одговорног за стишавање инфламације и продукцију антиоксидативних ензима. На стишавање регенеративног потенцијала јетре, активирана HMGB1/TLR4 оса утиче преко увећања присуства негативних регулатора ћелијског циклуса - протеина p53 и p21, и смањењем нивоа циклина D1. Добијени резултати указују на сложеност деловања HMGB1 протеина у дијабетесу и на значај спречавања ослобађања HMGB1 или блокаде HMGB1/TLR4 осе у циљу одлагања настанка оштећења јетре.Oxidative stress and chronic inflammation are considered to be the main causes of diabetic complications, one of which is liver damage. An important mediator of these processes may be the endogenous HMGB1 protein, when released into the extracellular environment from the necrotic, damaged or activated cells. As the HMGB1 role in diabetes was insufficiently studied, in this doctoral dissertation the contribution of HMGB1 to liver damage of streptozotocin-induced diabetic rats was investigated. It has been shown that the level of liver damage in diabetes correlates with the presence of extracellular HMGB1. In diabetic liver, this protein is structurally modified by acetylation, phosphorylation, and O-GlcNAc glycosylation, which correlates with its translocation from the nucleus to the cytoplasm and an increase in its presence in the liver and serum. Reduction of the level of extracellular HMGB1 by melatonin or ethyl pyruvate treatment of diabetic rats, shows that HMGB1 contributes to diabetic liver damage by maintaining a chronic inflammation, by lowering antioxidant defense and by reducing regeneration. Extracellular HMGB1 activates MAPK/NF-κB p65 and JAK1/STAT3 signaling pathways through interactions with the TLR4 receptor, thus contributing increased production of proinflammatory cytokines TNF-α and IL-6 and the acute-phase protein, haptoglobin. By stimulating the NF-κB p65 inflammatory pathway, HMGB1 acts negatively on the cytoprotective response of the diabetic liver, by disabling Nrf2 protein activity, which is responsible for reduction of inflammation and antioxidant enzymes production. Activated HMGB1/TLR4 axis reduces regenerative potential of the liver by increasing the presence of negative cell cycle regulators - proteins p53 and p21, and also by decreasing the level of cyclin D1. The obtained results indicate the complexity of HMGB1 protein action in diabetes and underlines the importance of preventing the release of HMGB1 or blockage of HMGB1/TLR4 axis in order to delay the occurrence of liver damage

Structural and functional characterization of HMGB1 protein in rat liver during experimentally-induced diabetes type 1

Оксидативни стрес и хронична инфламација сматрају се главним узроцима појаве дијабетичних компликација, међу којима су и оштећења јетре. Важну улогу медијатора ових процеса може имати ендогени протеин HMGB1, који у ванћелијску средину доспева из некротичних, оштећених и активираних ћелија. Како је у дијабетесу понашање HMGB1 протеина слабо изучавано, у овој докторској дисертацији испитиван је допринос HMGB1 оштећењима јетре пацова са стрептозотоцином-изазваним ДТ1. Показано је да ниво оштећења јетре током дијабетесa корелише са присуством ванћелијског HMGB1. Овај протеин, у дијабетичној јетри, бива структурно модификован ацетилацијом, фосфорилацијом и O-GlcNAc гликозилацијом што корелише са његовим изласком из једра ћелија у цитоплазму и повећањем његовог присуства у јетри и серуму. Резултати у вези са снижавањeм нивоа ванћелијског HMGB1 третманом дијабетичних пацова мелатонином или етил пируватом, указују да HMGB1 доприноси оштећењу јетре у дијабетесу одржавањем стања хроничне инфламације, стишавањем антиоксидативне одбране и стишавањем регенерације. Ванћелијски HMGB1 кроз интеракције са TLR4 рецептором активира MAPК/NF-κB p65 и ЈАК1/STAT3 сигналне путеве, доприносећи повећању продукције проинфламацијских цитокина TNF-α и IL-6 и акутно-фазног протеина хаптоглобина. Подстицањем NF-κB p65 инфламацијског пута, HMGB1 делује негативно на цитопротективни одговор у дијабетичној јетри тако што онемогућава активност Nrf2 протеина, одговорног за стишавање инфламације и продукцију антиоксидативних ензима. На стишавање регенеративног потенцијала јетре, активирана HMGB1/TLR4 оса утиче преко увећања присуства негативних регулатора ћелијског циклуса - протеина p53 и p21, и смањењем нивоа циклина D1. Добијени резултати указују на сложеност деловања HMGB1 протеина у дијабетесу и на значај спречавања ослобађања HMGB1 или блокаде HMGB1/TLR4 осе у циљу одлагања настанка оштећења јетре.Oxidative stress and chronic inflammation are considered to be the main causes of diabetic complications, one of which is liver damage. An important mediator of these processes may be the endogenous HMGB1 protein, when released into the extracellular environment from the necrotic, damaged or activated cells. As the HMGB1 role in diabetes was insufficiently studied, in this doctoral dissertation the contribution of HMGB1 to liver damage of streptozotocin-induced diabetic rats was investigated. It has been shown that the level of liver damage in diabetes correlates with the presence of extracellular HMGB1. In diabetic liver, this protein is structurally modified by acetylation, phosphorylation, and O-GlcNAc glycosylation, which correlates with its translocation from the nucleus to the cytoplasm and an increase in its presence in the liver and serum. Reduction of the level of extracellular HMGB1 by melatonin or ethyl pyruvate treatment of diabetic rats, shows that HMGB1 contributes to diabetic liver damage by maintaining a chronic inflammation, by lowering antioxidant defense and by reducing regeneration. Extracellular HMGB1 activates MAPK/NF-κB p65 and JAK1/STAT3 signaling pathways through interactions with the TLR4 receptor, thus contributing increased production of proinflammatory cytokines TNF-α and IL-6 and the acute-phase protein, haptoglobin. By stimulating the NF-κB p65 inflammatory pathway, HMGB1 acts negatively on the cytoprotective response of the diabetic liver, by disabling Nrf2 protein activity, which is responsible for reduction of inflammation and antioxidant enzymes production. Activated HMGB1/TLR4 axis reduces regenerative potential of the liver by increasing the presence of negative cell cycle regulators - proteins p53 and p21, and also by decreasing the level of cyclin D1. The obtained results indicate the complexity of HMGB1 protein action in diabetes and underlines the importance of preventing the release of HMGB1 or blockage of HMGB1/TLR4 axis in order to delay the occurrence of liver damage

Ultrastructural morphometry of neurosecretory granules in the neuroblastomas of paediatric patients

Introduction: Peripheral neuroblastic tumours are among the most frequently detected tumours in paediatric patients. A correlation between the aggressiveness of these tumours and their clinical prognosis has been discovered. Neurosecretory granules containing neurotransmitters, an ultrastructural feature of neurons, were found in the cells of peripheral neuroblastic tumours, as well. Aim: The aim of this study was to compare the number of neurosecretory granules found in cells of tumours, with a favourable prognosis to the corresponding number in cells of tumours and with an unfavourable one with the use of transmission electron microscopy (TEM). Material and Methods: Ten tissue biopsies were obtained from paediatric patients diagnosed with peripheral neuroblastic tumours. The tumour samples were first frozen, then fixed in glutaraldehyde, and embedded in epoxy resin. Ultrastructural morphometric analysis was performed on ultrathin sections. Results: When observed under a transmission electron microscope (TEM), neurosecretory granules appear as round structures of a small diameter, with an electron dense centre surrounded by an electron lucent, peripheral halo. It was found that the cells of tumours with an unfavourable prognosis have more neurosecretory granules (141.2 ± 89.18 per 60 cells per sample, on average) in their cytoplasm than the cells of tumours with a favourable one (37.2 ± 41.17 per 60 cells per sample, on average). Conclusion: The larger number of neurosecretory granules found in cells of tumours with an unfavourable prognosis could potentially shed some light on the role these structures play in tumourigenesis of peripheral neuroblastic tumours

Phenylephrine preconditioning in embryonic heart H9c2 cells is mediated by up-regulation of SUR2B/Kir6.2:A first evidence for functional role of SUR2B in sarcolemmal KATP channels and cardioprotection

AbstractATP-sensitive K+ (KATP) channels were originally described in cardiomyocytes, where physiological levels of intracellular ATP keep them in a closed state. Structurally, these channels are composed of pore-forming inward rectifier, Kir6.1 or Kir6.2, and a regulatory, ATP-binding subunit, SUR1, SUR2A or SUR2B. SUR1 and Kir6.2 form pancreatic type of KATP channels, SUR2A and Kir6.2 form cardiac type of KATP channels, SUR2B and Kir6.1 form vascular smooth muscle type of KATP channels. The presence of SUR2B has been described in cardiomyocytes, but its functional significance and role has remained unknown. Pretreatment with phenylephrine (100nM) for 24h increased mRNA levels of SUR2B and Kir6.2, without affecting those levels of SUR1, SUR2A and Kir6.1 in embryonic heart H9c2 cells. Such increase was associated with increased K+ current through KATP channels and Kir6.2/SUR2B protein complexes as revealed by whole cell patch clamp electrophysiology and immunoprecipitation/Western blotting respectively. Pretreatment with phenylephrine (100nM) generated a cellular phenotype that acquired resistance to chemical hypoxia induced by 2,4-dinitrophenol (DNP; 10mM), which was accompanied by increased in K+ current in response to DNP (10mM). Cytoprotection afforded by phenylephrine (100nM) was abolished by infection of H9c2 cells with adenovirus containing Kir6.2AFA, a mutant form of Kir6.2 with largely reduced K+ conductance. Taking all together, the present findings demonstrate that the activation of α1-adrenoceptors up-regulates SUR2B/Kir6.2 to confer cardioprotection. This is the first account of possible physiological role of SUR2B in cardiomyocytes

Anti-melanoma effects of ingenanes isolated from Euphorbia species

In this research, from two species, E. palustris and E. lucida, four ingenane derivatives were isolated. Their anticancer effects were evaluated in the human melanoma – 518A2 cell line and compared with the effects of ingenolmebutate. Selectivity towards human melanoma cells was determined using normal human keratinocytes – HaCaT.11th Conference on Medicinal and Aromatic Plants of Southeast European Countries, (11th CMAPSEEC), Ohrid, North Macedonia, 6–10 October 202

Kvantificiranje fiziološkega stresa v prazgodovini s pomočjo metode anulacije zobnega cementa (TCA):: preliminarni rezultati iz osrednjega Balkana

The Neolithic way of life was accompanied by an increase in various forms of physiological stress (e.g. disease, malnutrition). Here we use the method of tooth cementum annulation (TCA) analysis in order to detect physiological stress that is probably related to calcium metabolism. The TCA method is applied to a sample of teeth from three Mesolithic and five Neolithic individuals from the Central Balkans. The average number of physiological stress episodes is higher in the Neolithic group – but the statistical significance of this result cannot be evaluated due to the small sample size, therefore these results should be taken as preliminary.Življenje v neolitiku je spremljal porast različnih oblik fiziološkega stresa (npr. bolezni, podhranjenost). Predstavljamo uporabo analitske metode anulacije zobnega cementa (TCA), s katero lahko odkrivamo fiziološki stres, ki je verjetno povezan s presnovo kalcija. Metodo smo uporabili pri analizah vzorcev treh mezolitskih in petih neolitskih posameznikov iz osrednjega Balkana. Povprečno število fizioloških epizodnih stresov je v neolitski skupini večje – vendar zaradi majhnega števila vzorcev tega rezultata statistično ne moremo ovrednotiti in ga predstavljamo kot preliminarnega

Primary Teeth Bite Marks Analysis on Various Materials: A Possible Tool in Children Health Risk Analysis and Safety Assessment

Background: All objects put into a child's mouth could be hazardous in terms of trauma and toxic substance exposure. The aims of this study were to evaluate morphological characteristics of the primary teeth bite marks inflicted on various materials and to assess material wear using experimental model. Methods: Bite marks were analyzed on five materials: rubber, plastic, foil, wood, and silicone. In order to mimic children mouthing behavior an experimental setup has been designed using primary teeth placed in dentures and children's equipment specimens. Results: Deciduous teeth make visible and recognizable traces when using physiological forces on all investigated materials. The most significant material loss was revealed in silicone samples, but it has been observed in all material groups, while mouthing with incisors using higher mastication forces were identified as significant predictors for material wear. There were no significant differences between type, species, and morphological-morphometric characteristics of the bite marks which are made by incisors, canines, and molars. Conclusions: In the range of physiological bite forces, deciduous teeth lead to wear of material from which toys are made while the analysis of bite marks in children equipment could give some information regarding the risk of trauma and exposure