Colocalization of Serum Amyloid A with Microtubules in Human Coronary Artery Endothelial Cells

Serum amyloid A (SAA) acts as a major acute phase protein and represents a sensitive and accurate marker of inflammation. Besides its hepatic origin, as the main source of serum SAA, this protein is also produced extrahepatically. The mRNA levels of SAA become significantly elevated following proinflammatory stimuli, as well as, are induced through their own positive feedback in human primary coronary artery endothelial cells. However, the intracellular functions of SAA are so far unknown. Colocalization of SAA with cytoskeletal filaments has previously been proposed, so we analyzed the colocalization of SAA with all three cytoskeletal elements: actin filaments, vimentin filaments, and microtubules. Immunofluorescent double-labeling analyses confirmed by PLA method revealed a strict colocalization of SAA with microtubules and a very infrequent attachment to vimentin while the distribution of actin filaments appeared clearly separated from SAA staining. Also, no significant colocalization was found between SAA and endomembranes labeled with the fluorescent lipid stain DiO6. However, SAA appears to be located also unbound in the cytosol, as well as inside the nucleus and within nanotubes extending from the cells or bridging neighboring cells. These different locations of SAA in endothelial cells strongly indicate multiple potential functions of this protein

The role of cholesterol-sphingomyelin membrane nanodomains in the stability of intercellular membrane nanotubes

Intercellular membrane nanotubes (ICNs) are highly curved tubular structures that connect neighboring cells. The stability of these structures depends on the inner cytoskeleton and the cell membrane composition. Yet, due to the difficulty in the extraction of ICNs, the cell membrane composition remains elusive. In the present study, a raft marker, ostreolysin, revealed the enrichment of cholesterol-sphingomyelin membrane nanodomains along ICNs in a T24 (malignant) urothelial cancer cell line. Cholesterol depletion, due to the addition of methyl-β-cyclodextrin, caused the dispersion of cholesterol-sphingomyelin membrane nanodomains and the retraction of ICNs. The depletion of cholesterol also led to cytoskeleton reorganization and to formation of actin stress fibers. Live cell imaging data revealed the possible functional coupling between the change from polygonal to spherical shape, cell separation, and the disconnection of ICNs. The ICN was modeled as an axisymmetric tubular structure, enabling us to investigate the effects of cholesterol content on the ICN curvature. The removal of cholesterol was predicted to reduce the positive spontaneous curvature of the remaining membrane components, increasing their curvature mismatch with the tube curvature. The mechanisms by which the increased curvature mismatch could contribute to the disconnection of ICNs are discussed

Comparative lipidomic study of urothelial cancer models: association with urothelial cancer cell invasiveness

A joint NMR/LC-MS approach allows to establish significant differences in the lipidoma of invasive urothelial carcinoma cells (T24) with respect to noninvasive urothelial cells (RT4)

The impact of the COVID-19 pandemic on the mental health of pregnant women

COVID-19 se je hitro razširil po vsem svetu in povzročil pandemijo, ki je prinesla mnoge ukrepe varovanja javnega zdravja in spremenila življenje tudi nosečnicam. Vsled tega je namen zaključnega dela raziskati vpliv pandemije COVID-19 na duševno zdravje nosečnic. Zaključno delo temelji na raziskovalni metodi dela analize in sinteze podatkov. Izveden je bil »scooping« pregled (pregled obsega) znanstvene literature. Literaturo smo iskali v podatkovnih bazah Cinahl, MEDLINE, SAGE Journals in Web of Science glede na vključitvene in izključitvene kriterije. Potek iskanja virov smo prikazali s PRISMA diagramom. Kakovost izbranih člankov smo ocenjevali in jih uvrstili v nivo s pomočjo hierarhije dokazov. Rezultate smo pridobili na osnovi vsebinske analize in sinteze zbranih podatkov. V analizo in pregled smo vključili 34 člankov. Ugotovili smo, da so negotovosti in spremembe v zdravstveni praksi v času pandemije ustvarile dodatno zaskrbljenost ter negativno vplivale na duševno zdravje nosečnic. Nosečnice tako tvegajo razvoj duševnih težav, kot so depresija, tesnoba in simptomi posttravmatskega stresa. Raziskovalci poudarjajo psihosocialno podporo in pomen spremljanja perinatalnega duševnega zdravja med pandemijo za zaščito duševnega zdravja nosečnic. Pregled literature na temo duševnega zdravja nosečnic v času pandemije COVIDA-19 pri slednjih izkazuje strah, zaskrbljenost, depresijo in anksioznost. Kako razsežen je psihološki vpliv pandemije, ki še vedno traja, še strokovnjaki sami ne vedo, zato bodo v prihodnje potrebne nove raziskave.COVID-19 spread rapidly around the world and caused a pandemic that brought many measures to protect public health and changed the lives of pregnant women as well. Consequently, the purpose of the final work is to investigate the impact of the COVID-19 pandemic on the mental health of pregnant women. The diploma paper is based on the research method of data analysis and synthesis. A scooping review of the scientific literature was performed. We searched for literature in the databases Cinahl, MEDLINE, SAGE Journals and Web of Science, according to the inclusion and exclusion criteria. The process of searching for sources was presented with a PRISMA diagram. The quality of the selected articles was assessed and ranked using a hierarchy of evidence. The results were obtained on the basis of content analysis and synthesis of collected data. We analyzed and examined 34 articles. We found out, that uncertainties and changes in health practices during the pandemic created additional concerns, and negatively affected on the mental health of pregnant women. Pregnant women are thus at risk of developing mental problems such as depression, anxiety and post-traumatic stress symptoms. The researchers emphasize psychosocial support and the importance of monitoring perinatal mental health during a pandemic to protect the mental health of pregnant women. A review of the literature on the mental health of pregnant women during the COVID-19 pandemic shows fear, anxiety, depression and anxiety in the latter. The extent of the psychological impact of the pandemic, which is still ongoing, is not known to experts yet, that is why further researches in the future are necessary

Desmosome Assembly and Cell-Cell Adhesion Are Membrane Raft-dependent Processes

The aim of our study was to investigate the association of desmosomal proteins with cholesterol-enriched membrane domains, commonly called membrane rafts, and the influence of cholesterol on desmosome assembly in epithelial Madin-Darby canine kidney cells (clone MDc-2). Biochemical analysis proved an association of desmosomal cadherin desmocollin 2 (Dsc2) in cholesterol-enriched fractions that contain membrane raft markers caveolin-1 and flotillin-1 and the novel raft marker ostreolysin. Cold detergent extraction of biotinylated plasma membranes revealed that ∼60% of Dsc2 associates with membrane rafts while the remainder is present in nonraft and cholesterol-poor membranes. The results of immunofluorescence microscopy confirmed colocalization of Dsc2 and ostreolysin. Partial depletion of cholesterol with methyl-β-cyclodextrin disturbs desmosome assembly, as revealed by sequential recordings of live cells. Moreover, cholesterol depletion significantly reduces the strength of cell-cell junctions and partially releases Dsc2 from membrane rafts. Our data indicate that a pool of Dsc2 is associated with membrane rafts, particularly with the ostreolysin type of membrane raft, and that intact membrane rafts are necessary for desmosome assembly. Taken together, these data suggest cholesterol as a potential regulator that promotes desmosome assembly

Selective targeting of lectins and their macropinocytosis in urothelial tumours

Urinary bladder cancer can be treated by intravesical application of therapeutic agents, but the specific targeting of cancer urothelial cells and the endocytotic pathways of the agents are not known. During carcinogenesis, the superficial urothelial cells exhibit changes in sugar residues on the apical plasma membranes. This can be exploited for selective targeting from the luminal side of the bladder. Here we show that the plant lectins Jacalin (from Artocarpus integrifolia), ACA (from Amaranthus caudatus) and DSA (from Datura stramonium) selectively bind to the apical plasma membrane of low- (RT4) and high-grade (T24) cancer urothelial cells in vitro and urothelial tumours ex vivo. The amount of lectin binding was significantly different between RT4 and T24 cells. Endocytosis of lectins was observed only in cancer urothelial cells and not in normal urothelial cells. Transmission electron microscopy analysis showed macropinosomes, endosome-like vesicles and multivesicular bodies filled with lectins in RT4 and T24 cells and also in cells of urothelial tumours ex vivo. Endocytosis of Jacalin and ACA in cancer cells was decreased in vitro after addition of inhibitor of macropinocytosis 5-(N-ethyl-N-isopropyl) amiloride (EIPA) and increased after stimulation of macropinocytosis with epidermal growth factor (EGF). Clathrin, caveolin and flotillin did not colocalise with lectins. These results confirm that the predominant mechanism of lectin endocytosis in cancer urothelial cells is macropinocytosis. Therefore, we propose that lectins in combination with conjugated therapeutic agents are promising tools for improved intravesical therapy by targeting cancer cells