Steel silos for particulate solid materials. Part 1, Actions at filling and discharge.

V članku podajamo izraze za membranske sile v osno-simetričnem jeklenem silosu med procesoma polnjenja in praznjenja s sipkim materialom. Izrazi so predstavljeni tudi v grafični obliki, kar omogoča hitro in enostavno oceno velikosti in razporeditve membranskih sil v posameznih delih silosa. Grafi veljajo za poljubno geometrijo silosa in za poljuben material, ki se hrani. Na podlagi teh grafov lahko preverimo, kakšen je vpliv geometrije silosa in shranjenega materiala na razporeditev in velikost membranskih sil. Na koncu podajamo ilustrativen primer izračuna vplivov (pritiskov) in membranskih sil pri polnjenju in praznjenju silosa.In the paper, the expressions for membrane forces in an axisymmetric steel silo structure at filling and discharge with a particulate solid material are presented. Graphical plots of these expressions are given. They can be used for a quick and easy estimate of membrane forces distribution in all parts of a silo structure. The plots are valid for any silo geometry and for any material stored. The influence of silo geometry and stored material properties on the size and distribution of membrane forces can be thus checked. The paper completes with an illustrative example where actions (pressure loads) at filling and discharge on the walls of a silo are computed and the corresponding membrane forces are evaluated

Wild edible plants: Nutritional and toxicological characteristics, retrieval strategies and importance for today's society

Wild edible plants (WEPs) are part of the cultural and genetic heritage of different regions of the world. In times of famine and scarcity, these sources of nutrients and health-promoting compounds have received high importance mainly in rural and suburban areas. Although currently underutilized, WEPs are still consumed traditionally by different communities and are gaining relevance in today's society. However, these foods lack recognition as significant contributors to the human diet in developed areas. This review describes the nutritional value of WEPs from the North-eastern region of Portugal and points out those containing potentially toxic compounds. Several retrieval strategies are presented with the aim of promoting the (re)use, production, commercialization and conservation of WEPs (wild harvested plants and crop wild relatives), and their importance for social, economic and agro-ecological development is highlighted.The authors are grateful to the Foundation for Science and Technology of Portugal and FEDER, under Programme PT2020, for financial support to CIMO (UID/AGR/00690/2013). J. Pinela thanks FCT for his grant (SFRH/BD/92994/2013) funded by European Social Fund and Portuguese Ministry of Education and Science (MEC) through Programa Operacional Capital Humano (POCH).info:eu-repo/semantics/publishedVersio

Effects of the pore-forming nystatin on the cell-sized lipid vesicles

Nistatin spada v skupino makrolidnih polienskih antimikotikov. Podobno kot amfotericin B ima širok spekter antimikotinega delovanja, vendar se navkljub velikemu potencialu uporablja skoraj izkljuno topikalno, saj je ob sistemski uporabi nefrotoksien.Biološka aktivnost nistatina je povezana predvsem z njegovo zmožnostjo tvorbe transmembranskih kanalkov v plazemskih membranah odzivnih celic, ki prepušajo razline ione in celo manjše molekule, kar na koncu privede do porušenja elektrokemijskih gradientov ter nazadnje do celine smrti. Klinina uporabnost polienskih antimikotikov temelji na dejstvu, da imajo precej izrazitejši uinek na celice gliv, katerih membrane vsebujejo kot glavni sterol ergosterol, kot pa na loveške celice, za katere je znailen holesterol. Mehanizem delovanja polienov na ravni membrane še ni v celoti pojasnjen, pogledi raziskovalcev pa se razlikujejo zlasti glede pomena in naina vplivanja sterolov. Temeljni cilj raziskav v okviru disertacije je bila torej dopolnitev znanja o mehanizmu delovanja nistatina na ravni celine membrane, kar predstavlja osnovo za njegovo uinkovitejšo uporabo pri zdravljenju okužb ter zmanjševanje toksinosti za loveške celice. Pridobljeno znanje o porotvornem delovanju nistatina pomeni tudi prispevek k razvoju reguliranega prenosa zdravilnih snovi v celice. Membransko delovanje nistatina smo sprva preuevali na fosfolipidnih mehurkih celinih velikosti iz 1-palmitoil-2-oleil-glicero-3-fosfoholina (POPC), pripravljenih po elektroformacijski metodi. V ta namen smo z uporabo mikromanipulacijskega sistema in mikropipet prenašali mehurke, napolnjene s saharozno raztopino, v izomolarno glukozno raztopino z dodanim nistatinom v koncentracijah od 10 do 900 μmol/L ter jih opazovali pod faznokontrastnim mikroskopom. Uporabljena metodologija, ki predstavlja novost na podroju preuevanja polienov, je omogoila spremljanje oblikovnih sprememb membran in osmotskih pojavov na posaminih mehurkih. Eksperimenti z brezsterolnimi mehurki so razkrili tri osnovne vzorce odzivov mehurkov, ki so prehajali en v drugega ob višanju koncentracij nistatina v merilni raztopini. Pri nižjih koncentracijah nistatina (150–250 mol/L) je prišlo do sprememb oblik mehurkov ter do nastanka razlino oblikovanih izrastkov z znailnimi asovnimi vzorci spreminjanja oblik in dolžin. Slednje je posledica spreminjanja ravnovesne razlike v površinah med zunanjim in notranjim fosfolipidnim monoslojem (A0), zato smo z natannim spremljanjem omenjenih asovnih vzorcev lahko pojasnili zaporedje korakov pri vgrajevanju nistatina v membrane. V obmoju srednje visokih koncentracij nistatina (250–400 mol/L) so se 2 pojavljale kratkotrajne napetostne pore ter s tem povezano bledenje mehurkov, ki je bilo vse hitrejše z višanjem koncentracij nistatina. Pri najvišjih koncentracijah (> 400 mol/L) pa je prišlo do bolj obsežnih in stabilnejših napetostnih por, to je poasnih in hitrih pokov. Pojav napetostnih por smo uspeli razložiti z osmotskimi procesi in dvigom lateralne napetosti membrane zaradi tvorbe transmembranskih nistatinskih kanalkov z vejo prepustnostjo za glukozo kot za saharozo. Zviševanje površinske gostote nistatinskih kanalkov pospeši osmotske procese, kar vodi v edalje vejo obstojnost napetostnih por ter na koncu povzroi eksplozije. S teoretinim modelom smo lahko zadovoljivo opisali posamezne tipe osmotskih pojavov ter ocenili, kako se spreminja površinska gostota nistatinskih kanalkov v membranah v odvisnosti od koncentracij nistatina v merilni raztopini. Dokazali smo torej, da je porotvorno delovanje nistatina možno tudi v brezsterolnih membranah, glede esar doslej ni bilo konsenza. Nato smo v membrane dodajali holesterol in ergosterol v razlinih molarnih deležih. Eksperimenti s POPC-holesterolnimi mehurki so namre osnova za preuevanje toksinosti nistatina za loveške celice, tisti s POPCergosterolnimi pa za preuevanje delovanja nistatina na tarne mikrobne patogene, to je glive in tripanosomatidne kinetoplastide. Izkazalo se je, da ergosterol v membranah izrazito pospeši vgrajevanje nistatina vanje ter njegovo porotvorno delovanje, o emer pria pomik znailnih morfoloških sekvenc in osmotskih pojavov, katerih osnovno sosledje ostane v osnovi enako, k nižjim koncentracijam. Za POPC-ergosterolne mehurke so bile znailne tudi dolgotrajne napetostne pore, ki predstavljajo mehanizem, ki je morda pomemben tudi v in vivo pogojih. Po drugi strani je prisotnost holesterola v molarnem deležu, tipinem za sesalske celice (45 mol%), blago zavrla delovanje nistatina. Pokazali smo tudi, da je uinkovanje nistatina v kompleksnem, nelinearnem odnosu z deležem ergosterola v membranah. Primerjave so pokazale, da ti rezultati lahko pomenijo prvo potrditev teoretino napovedanih sterolnih supermrež v primeru mehurkov celinih velikosti. Z uporabo teoretinega modela smo ugotovili, da zaznanih osmotskih pojavov ni možno razložiti zgolj z vplivi sterolov na mehanske lastnosti lipidnih membran, temve da moramo upoštevati predvsem njihove neposredne vplive na vgrajevanje in porotvorno aktivnost nistatina. Temeljne toksikološke raziskave delovanja nistatina na holesterol vsebujoe mehurke smo dopolnili s preuevanjem uinkov nistatina na ovarijske epitelijske celice kitajskih hrkov (celicah CHO) z uporabo faznokontrastne in fluorescenne mikroskopije. Tudi pri celicah je prišlo do nabrekanja ter pojava napetostnih por zaradi osmotskih procesov sprio porotvorne aktivnosti nistatina. Po drugi strani pa smo pri celicah opazili tudi odzive, ki se razlikujejo od tistih pri mehurkih. Pri nizkih koncentracijah nistatina nismo opazili nitastih izrastkov, kar smo pripisali splošenju izvihkov notranjega monosloja, t. i. kaveol, kar bi lahko uravnotežilo vgrajevanje nistatinskih molekul v zunanji monosloj. Pri višjih koncentracijah nistatina pa so se na celinih membranah razvili sferini izrastki (blebi), ki so se z višanjem koncentracij postopno veali in združevali, vse do pojava nabreklih celinih oblik, imenovanih celini mehurki, ki so pri najvišjih koncentracijah pokali. Omenjene osmotske pojave pri celicah smo primerjali s tistimi pri mehurkih in jih opisali z dopolnjenim teoretinim modelom. S študijami celine viabilnosti ter s prikazom razgradnje aktinskega citoskeleta, oznaenega z zeleno fibrilarno beljakovino, pa smo jih uspeli povezati tudi s procesom celine smrti.Nystatin belongs to a group of polyene macrolide antimicotics. Although it has a wide spectrum of activity towards pathogenic fungi, similar to amphotericin B, it is used mainly topically because of its nephrotoxicity when applied systemically. The biological target for polyene action is the plasma membrane of sensitive cells, where they can form membranespanning channels, permeable to different ions and even smaller molecules, resulting in the disturbance of the cellular electrochemical gradients, and ultimately cell death. The fungal cells whose principal membrane sterol is ergosterol are much more susceptible to polyenes than cholesterol-containing human cells, which is the basis of their clinical use. The consensus regarding the mechanism of the polyene membrane activity, especially the role of sterols, has not been reached yet. Therefore, the principal intention of the doctoral research has been to bring new insights about the mode of action of nystatin at the membrane level, which could improve the efficiency of its use in different infections as well as reduce its toxicity to human cells. In addition, the knowledge about the mechanism of the nystatin poreforming activity is essential for the development of the regulated delivery of drugs into cells. Firstly, we studied the membrane activity of nystatin in vesicles with dimensions comparable to the size of an average human cell (giant unilamellar vesicles – GUVs), which were prepared from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) using the electroformation method. For this purpose GUVs filled with a sucrose solution were transferred using the micromanipulation system and micropipettes into an isomolar glucose solution with nystatin concentrations of 10 mol/L to 900 mol/L, and observed using the phase contrast microscopy. This novel methodological approach enabled us to study the osmotic phenomena and morphological responses of individual GUVs exposed to nystatin. Three characteristic concentration-dependent responses of sterol-free GUVs were detected experimentally in three different, partially overlapping, nystatin concentration ranges. At low nystatin concentrations (150–250 mol/L) vesicle shape changes and various membrane formations with distinct temporal shape and length evolution patterns were observed. These patterns can be explained by the changes in the equilibrium leaflet-area difference (A0), thereby revealing the steps of the nystatin membrane partition. The characteristic feature for the medium nystatin concentrations (250–400 mol/L) were transient tension pores associated with vesicle fading, which was intensified with increasing nystatin concentrations. At the highest nystatin concentrations (> 400 mol/L), two basic types of vesicle ruptures were encountered on the basis of the intensity of the osmotic process, i.e., the slow and fast ruptures (explosions). The occurrence of tension pores was attributed to osmotic processes leading to an increase of the lateral membrane tension as a consequence of the formaton of the transmembrane nystatin channels being more permeable to glucose compared to sucrose. As the surface density of nystatin channels increases, the tension pores become more durable (slow ruptures) and, eventually, explosions occur. Using a theoretical model, the dependency of the surface density of channels to the nystatin concentration was established and successfully correlated to different vesicle behavior. Therefore, we demonstrated that nystatin is able to form pores in the absence of sterols, which was debated untill now. Afterwards, we conducted experiments with GUVs containing cholesterol and ergosterol in different molar fractions. POPC-cholesterol GUVs namely enable the study of the toxic effects of nystatin on human cells, while POPC-ergosterol GUVs serve as model membranes for microbial fungal and trypanosomal kinetoplastid pathogens. It was shown that the nystatin partition and pore-forming activity are significantly increased in the ergosterol-containing membranes. This was demonstrated by the shift of the characteristic vesicle responses towards lower nystatin concentrations, while the basic concentrationdependent succession of phenomena was conserved. Furthermore, for POPC-ergosterol GUVs the long-lasting tension pores were characteristic and might represent an important mechanism also in in vivo conditions. In contrast, the addition of cholesterol into membranes in the molar fraction typical fot mammal cells (45 mol%) was found to be inhibitory for the nystatin action. It was also demonstrated that the nystatin activity is in a complex, nonlinear relation to the ergosterol membrane’s molar fraction. The comparison of the results to those of other studies in the field revealed that this could be the first confirmation of the existence of previously mathematically predicted sterol superlattices in the case of cell-sized vesicles. The theoretical modelling has shown that the sterol-induced changes of the osmotic phenomena could not be explained adequately on the basis of the altered membarne mechanical characteristics, and were therefore interpreted mainly by the direct influences of the sterols on the membrane partition and the channel-formation process of nystatin. The basic toxicological research of the nystatin effects on the POPC-cholesterol GUVs were complemented by the study of the responses of Chinese hamster ovary (CHO) epithelial cells to the pore-forming agent nystatin, which were investigated using phase-contrast and fluorescence microscopy. As in the case of GUVs, the pore-forming activity of nystatin induced the osmotic processes resulting in the cell swelling and in tension pores. However, certain responses of CHO cells were different compared to GUVs. Firstly, no membrane protrusions were observed at lower nystatin concentration, which can be explained by the flattening of the invaginated membrane structures, such as caveolae, thereby compensating the increase of the surface of the outer cell membrane monolayer as a result of nystatin insertion. With increasing nystatin concentrations, the formation of spherically shaped blebs, gradually growing and coalescing into “cell-vesicles”, and finally cell ruptures, were observed. These osmotic phenomena in cells were compared to those in GUVs and interpreted using an extended theoretical model. By using the cell viability studies and the demonstration of the GFP-tagged actin cytoskleton degradation we were able to link them to the process of cell death