EXPRESSION OF PSA-NCAM IN OPTIC TECTUM OF TELEOSTEI FISH IN RELATION TO OCCUPIED ECOLOGICAL NICHE

PSA-NCAM je postranslacijska modifikacija nastala reverzibilnim vezanjem polisijalinske kiseline naekstracelularne domene neuronske stanične adhezijske molekule. Pod utjecajem brojnih okolišnih stimulacija mozak riba je sposoban za visok stupanj plastičnosti pri čemu višestruku ulogu ima PSA-NCAM molekula. Stoga cilj ovoga rada bio je usporediti ekspresiju PSA-NCAM molekule u optičkom tektumu sedam različitih vrsta riba koštunjača koje zauzimaju različite ekološke niše.PSA-NCAM is a posttranslational modification, a result of reversible binding of polysialic acid on the extracellular domain of the neural cell adhesion molecules. The fish brain has high degree capacity ofplasticity under the influence of a number of environmental stimulation with multiple roles of PSA-NCAM molecules. The goal of this study was to compare the expression of PSA-NCAM molecules inthe optical tectum of seven different teleost species that occupy different ecological niches

Vitamin D Deficiency Among Medical Students in Osijek, Croatia

Aim: This study aimed to evaluate serum levels of 25-OH D3 (calcidiol) among students of the Faculty of Medicine Osijek, Croatia, thereby determining to what extent vitamin D deficiency is present. Methods: The present cross-sectional analysis was based on data collected from 60 participants. Blood sampling was done in March 2021. Concentrations of 25-OH D3 were measured using LC/MS-MS procedure on Shimadzu LCMS 8050 and RECIPE kit for serum levels of 25-OH-D3. Results: The study was conducted on a sample of 60 respondents aged 19 to 28, of whom 16 were men and 44 were women. All subjects had a 25-OH D3 deficiency (0.05). Conclusion: Vitamin D deficiency was detected in all subjects. In addition to the results of several other studies conducted worldwide that evaluated vitamin D status among medical students, this study further highlights the problem affecting this student subgroup

Early spring flora of the Sub-Pannonic steppic grassland (NATURA 2000 site) in Bilje, northeast Croatia

The diversity of early spring vascular flora was studied in the Sub- pannonic steppic grassland in the village of Bilje, north eastern Croatia. In all, 109 plant taxa within 35 families were found. The highest number of taxa belongs to the families Poaceae, Fabaceae, Asteraceae, Caryophyllaceae, Lamiaceae and Rosaceae. Specific habitat conditions, characterized by moderately wet and moderately acidic soil with intermediate fertility and the effects of the continental climate favour the development of different plant life forms. Out of the total recorded plant taxa, hemicryptophytes make up 59.6%, followed by therophytes (22.0%) and geophytes (13.8%). Chorological analysis shows that the most numerous are plants of Eurasian (33.9%), Pontic-Central-Asian (21.1%) and Central European (21.1%) floral elements. According to their status in the Red List, three critically endangered (CR), one vulnerable (VU) and three nearly threatened (NT) plant species were found. Altogether, the steppe-like grassland in Bilje is a unique habitat rich in valuable plants of the Croatian flora, including the critically endangered Doronicum hungaricum, therefore it is of great importance to preserve it. Important management tools include mowing and controlling the spreading of cultivated and invasive plant species

Primjena metformina i liraglutida utječe na strukturu hipokampusnih gangliozida u modelu štakora na dijeti obogaćenoj mastima i ugljikohidratima

Insulin resistance has many deleterious effects on the central nervous system, including the initiation and potentiation of neurodegeneration. While the pathogenesis of Alzheimer’s disease has been extensively researched with many insights into the effects of amyloids and neurofibrillary tangles, the connection between the two pathogenic entities has not yet been fully elucidated. Gangliosides are commonly found in neuronal membranes and myelin, specifically in lipid rafts that have been linked to pathological amyloidogenesis. In this study, 64 Sprague Dawley rats with equal sex distribution were separated into four sex-specific groups, as follows: control group on standard diet; group on high-fat, high-sugar diet (HFHSD); group on HFHSD treated with metformin; and group on HFHSD treated with liraglutide. Free-floating immunohistochemistry of the rat hippocampi was performed to analyze group-specific and sex-specific changes in the composition of the four most common gangliosides found in neuronal membranes and myelin sheaths, GM1, GD1a, GD1b and GT1b. The groups on HFHSD showed glucose tolerance impairment and body weight increase at the end of the experiment, whereas the groups treated with pharmacotherapeutics had better insulin sensitivity and decreases in body weight by the end of the experiment. Most changes were observed for GM1 and GD1b. Positive immunoreactivity for GM1 was observed in the male group treated with liraglutide in regions where it is not physiologically found. The changes observed following HFHSD and liraglutide treatment were suggestive of ganglioside restructuring that might have implications on pathological amyloidogenesis. Metformin treatment did not significantly alter the hippocampal ganglioside composition in either sex.Inzulinska rezistencija ima negativan učinak na središnji živčani sustav, uključujući razvoj i potencijaciju neurodegenerativnih promjena. Iako postoje brojna saznanja o patogenezi Alzheimerove bolesti u kontekstu amiloidnih depozita i neurofibrilarnih vlakana, veza između inzulinske rezistencije i Alzheimerove bolesti još uvijek nije u potpunosti razjašnjena. Gangliozidi su prisutni u membranama neurona i mijelinskim ovojnicama, osobito u sklopu lipidnih splavi, koje su identificirane kao mjesto patološke amiloidogeneze. Ukupno je 64 Sprague Dawley štakora jednake spolne distribucije podijeljeno u četiri spolno specifične skupine: kontrolnu skupinu na standardnoj dijeti, skupinu na dijeti obogaćenoj mastima i ugljikohidratima (HFHSD), skupinu na HFHSD uz liječenje metforminom te skupinu na HFHSD uz liječenje liraglutidom. Provedena je free-floating imunohistokemijska analiza promjena četiri najzastupljenija gangliozida u neuronskim membranama i mijelinskim ovojnicama: GM1, GD1a, GD1b i GT1b. Skupine na HFHSD pokazale su poremećenu toleranciju glukoze te povećanje tjelesne mase na kraju eksperimenta, dok su skupine koje su primale farmakoterapeutike pokazale poboljšanje tolerancije glukoze te smanjenje tjelesne mase na kraju eksperimenta. Najviše promjena zabilježeno je kod gangliozida GM1 i GD1b. Pozitivna imunoreaktivnost za GM1 zabilježena je kod mužjaka koji su liječeni liraglutidom i u regijama hipokampusa gdje u fiziološkim uvjetima nisu prisutni. Promjene nakon HFHSD i liječenja liraglutidom upućuju na restrukturiranje hipokampusnih gangliozida, što može imati implikacije za patološku amiloidogenezu. Liječenje metforminom nije značajno utjecalo na hipokampusnu strukturu gangliozida u oba spola

Retro is the new modern: Contemporary application of gold impregnation staining on brain cryosections for digital image analysis

Background and purpose: Since the times of Golgi and Cajal, impregnation with gold or silver has been used to visualize microscopic details of the nervous tissue. Although immunohistochemistry has largely replaced impregnation techniques, they are still used, and there is a growing interest in combining them with modern image analysis methods for quantitative studies in neuroscience. The aim of this research was to modify the gold chloride impregnation method published by Schmued to improve consistency of staining, to be adequate for digital image analysis. Materials and methods: Brains of 8 six-month-old female Wistar rats were fixed in 4% PFA, cryoprotected in sucrose and flash‑frozen in liquid nitrogen. Neighboring sets of coronal sections were chosen for gold impregnation, Nissl staining and MAP2 immunohistochemistry. Whole-slide images and images of specific regions were taken for analysis. Results: Myelinated fibers were stained dark reddish to brown on goldstained sections, and other tissue was yellowish, which gave an excellent contrast for digital image analysis. Gold staining was consistent in all regions, and no major artifacts were noticed. When compared to Nissl and MAP2, only myelinated structures were stained with gold impregnation. Conclusions: Modified gold impregnation method is an alternative that’s on par with traditional myelin staining methods. The new, modified gold impregnation method gives a consistent and reproducible staining suitable for digital image analysis. It can be useful in morphometric evaluation of nervous tissue and investigation of neuropathological changes in nervous tissue, especially for quantitative studies

EFFECTS OF CHANGES IN GLYCOLIPID CELL MEMBRANE COMPOSITION ON THE COMPOSITION OF LIPID RAFTS IN SH-SY5Y HUMAN NEUROBLASTOMA CELL LINE

Cilj: Gangliozidi su važni za stabilizaciju i organizaciju membranskih mikrodomena koji služe kao komunikacijska središta za prenošenje inzulinske signalizacije u stanicu. Ometanje sinteze gangliozida trebalo bi poremetiti inzulinsku signalizaciju pojačavajući je ili snižavajući, ovisno o tome koji je enzim poremećen. To bi pak trebalo uzrokovati promjene u staničnoj neuroplastičnosti, morfologiji i toleranciji na inzulin. Ustroj studije: Stanična linija humanog neuroblastoma SH-SY5Y diferencirana je 10 dana retinoičnom kiselinom i tretirana 48 sati s dvije izoforme P4 inhibitora sinteze gangliozida, miglustat inhibitorom sinteze gangliozida i inhibitorom razgradnje glikolipida - conduritol B epoksidom. Materijali i metode: Za MTT stanice su uzgajane, diferencirane i tretirane u pločicama s 96 jažica presvučenih kolagenom. Nakon tretmana utvrđena je održivost stanica. Stanice su uzgajane i diferencirane na pokrovnim staklima presvučenim kolagenom za imunocitokemiju i morfologiju i obojane specifičnim antitijelima za epitope od interesa te FITC bojom za morfologiju. Za Aneksin V, western blot i MALDI-TOF analize, stanice su uzgajane u šest bioloških replika za svaku koncentraciju u pločicama sa 6 jažica, nakon što su tretirane stanice sastrugane, homogenirane i pripremljene za daljnju analizu protočnom citometrijom, western blotom ili MALDI- TOF MS. Rezultati: ometanje biosinteze glikolipida uzrokuje o dozi ovisnu staničnu smrt i promjenu morfologije. Miglustat je uzrokovao očekivane morfološke promjene u obliku produljenja neurita, dok je CBE u sufektivnim koncentracijama imao reverzni učinak na staničnu morfologiju. Put signalizacije inzulina bio je pojačan u svim tretmanima. Stanice su pokazale promjene u neuroplastičnosti i različit odgovor na inducirani metabolički stres, ovisno o tome koji je inhibitor primijenjen. Zaključak: Ometanje sastava glikolipida ometat će inzulinsku signaliziraciju, uzrokovati morfološke promjene i poremetiti neuroplastičnost stanica.Objectives: Gangliosides are essential for stabilizing and organization of the membrane microdomains, which serve as communication hubs for relaying insulin signaling into the cell. Interfering with ganglioside synthesis should disrupt insulin signaling by downregulation or upregulation, depending on which enzyme is disrupted. This, in turn, should cause changes in cell neuroplasticity, morphology, and tolerance to insulin challenge. Study design: SH-SY5Y human neuroblastoma cell line was differentiated for 10 days with retinoic acid and treated for 48 hours with two isoforms of P4 inhibitor of ganglioside synthesis, miglustat inhibitor of ganglioside synthesis, and inhibitor of glycolipid degradation – conduritol B epoxide. Material and methods: For MTT, cells were grown, differentiated, and treated in collagen-coated 96 healthy plates. After treatment, cell viability was determined. Cells were grown and differentiated on collagen-coated glass slides for immunocytochemistry and morphology and stained with specific antibodies for epitopes of interest, and FITC dye for morphology. For Annexin V, Western blots, and MALDI-TOF, cells were grown in six biological replicas for each treatment concentration and protocol in 6-well plates. After treatment cells were scraped, homogenated, and prepared for further analysis by flow cytometry, Western blot, or MALDI-TOF MS. Results: Interfering with glycolipid biosynthesis caused dose-dependent cell death and change in morphology. Miglustat caused expected morphology changes in the form of neurite elongation, whilst CBE in sub-effective concentrations had the reverse effect on cell morphology. The insulin signaling pathway was upregulated with all treatments. Cells demonstrated changes in neuroplasticity and different responses to induced metabolic stress depending on which inhibitor was applied. Conclusion: Disrupting glycolipid composition will interfere with insulting signaling, cause morphological changes, and disrupt the neuroplasticity of the cells

EFFECTS OF CHANGES IN GLYCOLIPID CELL MEMBRANE COMPOSITION ON THE COMPOSITION OF LIPID RAFTS IN SH-SY5Y HUMAN NEUROBLASTOMA CELL LINE

Cilj: Gangliozidi su važni za stabilizaciju i organizaciju membranskih mikrodomena koji služe kao komunikacijska središta za prenošenje inzulinske signalizacije u stanicu. Ometanje sinteze gangliozida trebalo bi poremetiti inzulinsku signalizaciju pojačavajući je ili snižavajući, ovisno o tome koji je enzim poremećen. To bi pak trebalo uzrokovati promjene u staničnoj neuroplastičnosti, morfologiji i toleranciji na inzulin. Ustroj studije: Stanična linija humanog neuroblastoma SH-SY5Y diferencirana je 10 dana retinoičnom kiselinom i tretirana 48 sati s dvije izoforme P4 inhibitora sinteze gangliozida, miglustat inhibitorom sinteze gangliozida i inhibitorom razgradnje glikolipida - conduritol B epoksidom. Materijali i metode: Za MTT stanice su uzgajane, diferencirane i tretirane u pločicama s 96 jažica presvučenih kolagenom. Nakon tretmana utvrđena je održivost stanica. Stanice su uzgajane i diferencirane na pokrovnim staklima presvučenim kolagenom za imunocitokemiju i morfologiju i obojane specifičnim antitijelima za epitope od interesa te FITC bojom za morfologiju. Za Aneksin V, western blot i MALDI-TOF analize, stanice su uzgajane u šest bioloških replika za svaku koncentraciju u pločicama sa 6 jažica, nakon što su tretirane stanice sastrugane, homogenirane i pripremljene za daljnju analizu protočnom citometrijom, western blotom ili MALDI- TOF MS. Rezultati: ometanje biosinteze glikolipida uzrokuje o dozi ovisnu staničnu smrt i promjenu morfologije. Miglustat je uzrokovao očekivane morfološke promjene u obliku produljenja neurita, dok je CBE u sufektivnim koncentracijama imao reverzni učinak na staničnu morfologiju. Put signalizacije inzulina bio je pojačan u svim tretmanima. Stanice su pokazale promjene u neuroplastičnosti i različit odgovor na inducirani metabolički stres, ovisno o tome koji je inhibitor primijenjen. Zaključak: Ometanje sastava glikolipida ometat će inzulinsku signaliziraciju, uzrokovati morfološke promjene i poremetiti neuroplastičnost stanica.Objectives: Gangliosides are essential for stabilizing and organization of the membrane microdomains, which serve as communication hubs for relaying insulin signaling into the cell. Interfering with ganglioside synthesis should disrupt insulin signaling by downregulation or upregulation, depending on which enzyme is disrupted. This, in turn, should cause changes in cell neuroplasticity, morphology, and tolerance to insulin challenge. Study design: SH-SY5Y human neuroblastoma cell line was differentiated for 10 days with retinoic acid and treated for 48 hours with two isoforms of P4 inhibitor of ganglioside synthesis, miglustat inhibitor of ganglioside synthesis, and inhibitor of glycolipid degradation – conduritol B epoxide. Material and methods: For MTT, cells were grown, differentiated, and treated in collagen-coated 96 healthy plates. After treatment, cell viability was determined. Cells were grown and differentiated on collagen-coated glass slides for immunocytochemistry and morphology and stained with specific antibodies for epitopes of interest, and FITC dye for morphology. For Annexin V, Western blots, and MALDI-TOF, cells were grown in six biological replicas for each treatment concentration and protocol in 6-well plates. After treatment cells were scraped, homogenated, and prepared for further analysis by flow cytometry, Western blot, or MALDI-TOF MS. Results: Interfering with glycolipid biosynthesis caused dose-dependent cell death and change in morphology. Miglustat caused expected morphology changes in the form of neurite elongation, whilst CBE in sub-effective concentrations had the reverse effect on cell morphology. The insulin signaling pathway was upregulated with all treatments. Cells demonstrated changes in neuroplasticity and different responses to induced metabolic stress depending on which inhibitor was applied. Conclusion: Disrupting glycolipid composition will interfere with insulting signaling, cause morphological changes, and disrupt the neuroplasticity of the cells

Lipid Rafts: The Maestros of Normal Brain Development

Lipid rafts, specialised microdomains within cell membranes, play a central role in orchestrating various aspects of neurodevelopment, ranging from neural differentiation to the formation of functional neuronal networks. This review focuses on the multifaceted involvement of lipid rafts in key neurodevelopmental processes, including neural differentiation, synaptogenesis and myelination. Through the spatial organisation of signalling components, lipid rafts facilitate precise signalling events that determine neural fate during embryonic development and in adulthood. The evolutionary conservation of lipid rafts underscores their fundamental importance for the structural and functional complexity of the nervous system in all species. Furthermore, there is increasing evidence that environmental factors can modulate the composition and function of lipid rafts and influence neurodevelopmental processes. Understanding the intricate interplay between lipid rafts and neurodevelopment not only sheds light on the fundamental mechanisms governing brain development but also has implications for therapeutic strategies aimed at cultivating neuronal networks and addressing neurodevelopmental disorders

A Lack of GD3 Synthase Leads to Impaired Renal Expression of Connexins and Pannexin1 in St8sia1 Knockout Mice

The aim of this study was to determine the effects of altered ganglioside composition on the expression of Cx37, Cx40, Cx43, Cx45, and Panx1 in different kidney regions of St8sia1 gene knockout mice (St8sia1 KO) lacking the GD3 synthase enzyme. Experiments were performed in twelve male 6-month-old mice: four wild-type (C57BL/6-type, WT) and eight St8sia1 KO mice. After euthanasia, kidney tissue was harvested, embedded in paraffin wax, and processed for immunohistochemistry. The expression of connexins and Panx1 was determined in different regions of the kidney: cortex (CTX.), outer stripe of outer medulla (O.S.), inner stripe of outer medulla (IN.S.), and inner medulla (IN.MED.). We determined significantly lower expression of Cx37, Cx40, Cx45, and Panx1 in different parts of the kidneys of St8sia1 KO mice compared with WT. The most consistent decrease was found in the O.S. where all markers (Cx 37, 40, 45 and Panx1) were disrupted in St8si1 KO mice. In the CTX. region, we observed decrease in the expression of Cx37, Cx45, and Panx1, while reduced expression of Cx37 and Panx1 was more specific to IN.S. The results of the present study suggest that deficiency of GD3 synthase in St8sia1 KO mice leads to disruption of renal Cx expression, which is probably related to alteration of ganglioside composition