Influence of aromatic replacement in indoline inhibitors of butyrylcholinesterase and mitogen-activated protein kinase p38α

Magistrska naloga obravnava novejši pristop k zdravljenju Alzheimerjeve bolezni (AB) z načrtovanjem in sintezo novih dvojnih zaviralcev, ki so usmerjeni proti dvema ključnima encimoma butirilholin esterazi (BChE) in z mitogenom aktivirani kinazi p38α (p38α MAPK). Alzheimerjeva bolezen je nevrodegenerativna motnja, za katero je značilen upad kognitivnih sposobnosti in je tesno povezana z disregulacijo holinergične nevrotransmisije in nevrovnetnih procesov, ki jih posreduje p38α MAPK. Zaviranje BChE izboljša kognitivne funkcije zaradi povečanja koncentracije živčnega prenašalca acetilholina, zaviranje kinaze p38α MAPK pa zmanjša nevrovnetje in hiperfosforilacijo proteina tau, kar bi lahko izboljšalo klinične znake, ki se kažejo pri AB. Na osnovi selektivnega zaviralca p38 MAPK, spojine ARRY-371797, smo načrtovali in sintetizirali pet novih spojin, označenih s številkami 9, 10, 16, 22 in 28 v šeststopenjski sintezi, pri čemer smo izhajali iz metil 2-fluoro-4-metil-5-nitrobenzoata. Naše končne spojine se razlikujejo od spojine ARRY-371797 v aromatskih substituentih, ki zamenjajo 2,4-difluorofenilno skupino v spojini vodnici. Z novimi substituenti smo želeli ohraniti ali povečati jakost zaviranja p38α MAPK in BChE. Spojine smo očistili, potrdili njihovo strukturo s spektroskopskimi metodami in jim izmerili aktivnost s pomočjo biokemijskih testiranj na rekombinantnih encimih. Izmed vseh zaviralcev se je za najobetavnejšega izkazala spojina 9, ki zavira človeško (h)BChE z IC50 vrednostjo 6,4 µM in p38α MAPK z IC50 = 0,28 µM.This thesis addresses a novel approach to the treatment of Alzheimer\u27s disease (AD) by the design and synthesis of novel dual inhibitors, targeting two key enzymes butyrylcholinesterase (BChE) and mitogen-activated kinase p38α. Alzheimer\u27s disease is a neurodegenerative disorder characterised by cognitive decline and is closely associated with dysregulation of cholinergic neurotransmission and neuroinflammatory processes mediated by p38α MAPK. Inhibition of BChE improves cognitive function by increasing the concentration of the neurotransmitter acetylcholine, inhibition of p38α MAPK kinase reduces neuroinflammation and hyperphosphorylation of tau protein, which could improve the clinical signs manifested in AD. Based on a selective p38 MAPK inhibitor, compound ARRY-371797, we designed six new compounds, labelled as 9, 10, 16, 22, 28. Based on a literature search, we developed a six-step synthesis starting from methyl 2-fluoro-4-methyl-5-nitrobenzoate. Our final compounds differ from compound ARRY-371797 in 4 substituents replacing the 2,4-difluorophenyl group. With the new substituents we wanted to maintain or increase the inhibition potency of p38α MAPK and BChE. The compounds were purified and their structure confirmed by spectroscopic methods and their residual activity determined by biochemical assays on recombinant enzymes. Among all inhibitors, compound 9 was found to be the most promising with an IC50 value of 6.4 µM against human (h)BChE and with and IC50 = 0.28 µM against p38α MAPK

Comparison of extraction of essential oils from orange peels waste with selected organic solvents and natural deep eutectic solvents

V industriji predelovanja sadja velik problem predstavljajo olupki citrusov, ki sicer vsebujejo biološko aktivne snovi, eterična olja in podobne spojine, ki so sicer na trgu zaželene. A vendar olupki predstavljajo problem pri odlaganju zaradi visoke vsebnosti organskih snovi, prav tako je problematičen nizek pH ekstraktov iz olupkov. Odpadni olupki citrusov predstavljajo odličen vir za pridobivanje eteričnih olj iz flaveda – zunanjega, obarvanega dela olupka. Eterična olja so kompleksne mešanice hlapnih organskih spojin, ki imajo značilen vonj po rastlinah ali njihovih plodovih. V pomarančah večji del eteričnega olja predstavlja monociklični monoterpen limonen, ki ima vonj po limoni. Limonen se lahko iz olupkov pridobiva z ekstrakcijo trdno-tekoče, pri kateri poznamo več različnih tehnik, ki se delijo na tradicionalne in moderne. Tako med tradicionalne tehnike spadajo šaržna ekstrakcija trdno-tekoče, ekstrakcija po Soxhletu, hladno stiskanje in vodna ali parna destilacija. Moderne tehnike so se razvile z namenom, da se pri posameznih tradicionalnih tehnikah izboljša slabe lastnosti oziroma se poudari dobre. Pri vsem tem pa je tudi pomembno, da se zmanjšuje porabo organskih topil ali pa se jih popolnoma nadomesti z bolj zelenimi topili, med katere spadajo naravna globoka evtektična topila (angl. natural deep eutectic solvents, NADES). Slednja so mešanica dveh ali več čistih snovi v optimalnem razmerju, kot rezultat pa je temperatura tališča mešanice nižja od temperature tališča idealne mešanice. Komponente NADES so primarni metaboliti in/ali biološko obnovljive snovi in predstavljajo donorje ali akceptorje vodikovih vezi. Namen magistrskega dela je bila priprava NADES in primerjava z organskimi topili, koliko limonena ekstrahirajo. Skupen sinergijski učinek NADES z limonenom sem želela izkoristiti za aplikacijo v kremah, zato sem za donor vodikove vezi izbrala različne aminokisline, za akceptor vodikove vezi pa holin klorid ali glicerol. Ekstrakcijo z organskimi topili (metanol, etanol, etil acetat in aceton), sem izvajala po Soxhletu in šaržno, medtem ko je ekstrakcija z NADES potekala zgolj šaržno, saj ta niso hlapna. Analiza limonena v ekstraktih je potekala z GC-MS, analiza izluženih proteinov pa z UV-Vis spektrometrom. Ker so proteini kontaminant, sem teh želela ekstrahiranih čim manj. Ugotovila sem, da so izkoristki limonena pri Soxhletovi tehniki višji od izkoristkov pri šaržni tehniki. Ugotovila sem tudi, da noben pripravljen NADES ni ekstrahiral limonena.Citrus peels, which otherwise contain biologically active substances, essential oils and similar compounds that are otherwise desirable on the market, pose a major problem in the fruit processing industry, because the peels themselves pose a disposal problem due to their high content of organic matter, the low pH of extracts the peels is also problematic. Waste citrus peels are an excellent source for obtaining essential oils from the flavedo - the outer, colored part of the peel. Essential oils are complex mixtures of volatile organic compounds that have the characteristic smell of plants or their fruits. In oranges, the major part of the essential oil is the monocyclic monoterpene limonene, which has a lemony fragrance. Limonene can be extracted from peels through solid-liquid extraction, with various techniques falling into traditional and modern ones. Traditional techniques include batch extraction, Soxhlet extraction, cold pressing, and water or steam distillation. Modern Techniques have been developed to improve the drawbacks of individual traditional techniques or to emphasize their strengths. Additionally, it is essential to reduce the use of organic solvents or replace them entirely with greener solvents, including natural deep eutectic solvents (NADES). The latter are mixtures of two or more pure compounds in an optimal ratio, resulting in a melting point lower than the melting point of an ideal mixture. The compounds in NADESs are primary metabolites and/or bio-renewable substances that act as hydrogen bond donors or hydrogen bond acceptors. The aim of master\u27s thesis was preparation of NADESs and compare with organic solvents how much limonene they extract. I wanted to use the common synergistic effect of NADES with limonene for application in creams, that\u27s why I used various amino acids as hydrogen bond donor and choline chloride or glycerol as hydrogen bond acceptor. Extraction with organic solvents (methanol, ethanol, ethyl acetate and acetone) was performed with batch extraction and using Soxhlet method, while extraction with NADESs was performed only with batch extraction since they are non-volatile. For analysis of limonene in extracts GC-MS was used, and for the analysis of extracted proteins UV-Vis spectrometry was used. Since proteins are contaminant I wanted to extract as few of them as possible. Results showed that yields of limonene obtained with the Soxhlet technique were higher than those obtained with the batch extraction. Results also showed that none of the prepared deep eutectic solvents extracted limonene

Development of immunoproteasome inhibitors and modulators of immune signaling pathways with anti-tumor and anti-inflammatory activity

Imunski sistem ima ključno, a kompleksno vlogo pri zaščiti organizma. V določenih okoliščinah lahko postane prekomerno aktiven ali premalo odziven, kar vodi v razvoj kroničnih vnetij, avtoimunskih bolezni in rakavih obolenj. Z zaviranjem pretiranega imunskega odziva lahko zdravimo avtoimunske bolezni, medtem ko lahko z imunomodulatorji, ki dodatno aktivirajo imunski sistem, okrepimo odziv proti okužbam ali rakavim celicam. V našem delu smo se osredotočili na razvoj novih imunomodulatornih učinkovin, s katerimi smo ciljali različne farmakološke tarče vpletene v patogenezo omenjenih obolenj: TLR7 in 8, TAK1 ter imunoproteasom. Načrtovanja in optimizacije novih učinkovin smo se lotili z različnimi pristopi. Sintetizirali smo serijo spojin z izoksazolo[5,4-d]pirimidinskim skeletom, kjer so najbolje delujoči analogi izkazali selektivno agonistično delovanje na TLR7 v nizko mikromolarnih koncentracijah ter povečali sproščanje vnetnih citokinov. Z načrtovanjem na osnovi fragmentov smo pripravili obsežno knjižnico 2-cianobenzotiazolov, benzimidazolov ter 1-metilbenzimidazolov z različnimi elektron privlačnimi ter elektron donorskimi skupinami vezanimi na obroč. Določili smo reaktivnost in stabilnost sintetiziranih derivatov ter razvili analoge, ki bodo koristni za prihodnje aplikacije v biokonjugaciji in kemijski biologiji. Pripravljeni analogi so izkazali selektivno zaviralno aktivnost do podenote ß5i imunoproteasoma z vrednostmi IC50 v nizkem mikromolarnem območju. Prav tako so izkazali sposobnost znižanja sproščanja citokinov ter selektivno citotoksično delovanje na celice multiplega mieloma. Kot prvi smo načrtovali, sintetizirali ter biološko ovrednotili himerne razgrajevalce (PROTAC) TAK1, ki so povzročili razgradnjo TAK1 v s TNF-? stimulirani celični liniji raka dojke ter monocitni levkemični celični liniji. Spojini, ki sta izzvali najvišjo razgradnjo TAK1, sta izkazovali signifikanten vpliv na zmanjšanje živosti omenjenih celičnih linij stimuliranih s TNF-?, ter izzvali celično apoptozo. Selektivno delovanje molekul PROTAC le pod vnetnimi pogoji predstavlja velik potencial za nadaljnji razvoj selektivnih terapij za različna obolenja povezana z neustreznim imunskim odzivom. Na vseh treh področjih smo dosegli zadane cilje in razvili nove selektivne modulatorje z aktivnostmi v nizko mikromolarnih ter nanomolarnih koncentracijah.The immune system plays an important but complex role in protecting the organism against patogens. Under certain circumstances, it can become overactive or underactive, which can lead to the development of chronic inflammation, autoimmune diseases, allergies, and cancer. Autoimmune diseases can be treated by suppressing an excessive immune response, while immunomodulators, which additionally activate the immune system, can strengthen the response against infections or cancer cells. In our work, we have focused on the development of new immunomodulatory agents for different pharmacological targets involved in the development of autoimmune diseases and cancers: TLR7 and 8, TAK1 and the immunoproteasome. We synthesized a series of compounds with an isoxazolo[5,4-d]pyrimidine scaffold, with the most potent analogs showing selective agonist activity on TLR7 at low micromolar concentrations and increasing the secretion of inflammatory cytokines. Using fragment-based design, we prepared a large library of 2-cyanobenzothiazoles, benzimidazoles and 1-methylbenzimidazoles with different electron-withdrawing and electron-donating substituents. We determined the reactivity and stability of synthesized derivatives and developed analogs that will be useful for future applications in bioconjugation and chemical biology. The prepared analogs showed selective inhibitory activity against the β5i-subunit of the immunoproteasome with IC50 values in the low micromolar range. They also showed a reduction in the release of cytokines and selective cytotoxicity on multiple myeloma cells. We were the first to develop, synthesize, and biologically study chimeric TAK1 degraders (TAK1 PROTACs), which induced TAK1 degradation in a TNF-α-stimulated breast cancer cell line and a monocytic leukemia cell line. The compounds that induced the strongest degradation of TAK1 showed a significant effect on reducing the viability of the aforementioned TNF-α-stimulated cell lines and caused cell apoptosis. The selective effect of PROTACs only under inflammatory conditions represents great potential for the further development of selective therapies for various conditions related to the improper immune response. To conclude, we achieved our goals in all three areas, and developed new selective modulators with activities at low micromolar and nanomolar concentrations

Development and validation of a new method for determining trace elements in nail and hair samples

Lasje in nohti so lahko pomembni biološki vzorci za oceno pretekle izpostavljenosti elementom v sledovih, zato se vse pogosteje uporabljajo v kliničnih in epidemioloških raziskavah, v katerih ocenjujejo vpliv okoljskih dejavnikov na zdravje ljudi. Koncentracije elementov v sledovih v Laboratoriju za analitiko encimov in elementov v sledovih, Univerzitetnega kliničnega centra Ljubljana (UKCL), določamo v vzorcih krvi in urina. V okviru magistrske naloge smo razvili in validirali metodo za kvantitativno določanje koncentracij 26 elementov v sledovih v vzorcih las in nohtov, vključno s 7 esencialnimi in 19 neesencialnimi elementi. Na podlagi pregleda literature smo razvili metodo za določanje elementov v sledovih, ki vključuje predanalitske postopke (spiranje, sušenje in kislinski razklop v mikrovalovni pečici) ter analizo tekočih vzorcev z uporabo induktivno sklopljene plazme z masno spektrometrijo (ICP-MS). Metodo smo validirali po smernicah Inštituta za klinične in laboratorijske standarde (CLSI), pri čemer smo določili zmogljivost zaznavanja, natančnost, pravilnost, linearnost in delovno območje metode. Na podlagi validacijskih parametrov smo ugotovili, da metoda izpolnjuje zahteve za predvideno uporabo pri določanju elementov v sledovih v nohtih in laseh. Na vzorcih 140 zdravih oseb obeh spolov smo določili 95 % referenčne intervale za esencialne in neesencialne elemente, ki so bili primerljivi z literaturo, čeprav nekoliko ožji. V nadaljevanju smo ocenili vpliv eksogenih in endogenih dejavnikov ter življenjskih navad in razvad na koncentracije elementov v nohtih na nogi in laseh. Določili smo razlike v koncentracijah V, Mn, Co, Ni, Cu, Zn, As, Se, Sr, Mo, Ag, Cd, Sn med spoloma. S starostjo so se koncentracije večine elementov v vzorcih las in nohtov rahlo nižale. Koncentracije Hg v Obalno-kraški in Goriški regiji so bile povišane, pri ostalih elementih nismo določili pomembnih odstopanj med regijami. Uživanje prehranskih dodatkov je povečalo vsebnost Se v nohtih, uživanje morske hrane pa koncentracije Hg v nohtih in laseh. Kadilci so imeli višje koncentracije Cd in Pb v laseh in nohtih, koncentracije omenjenih elementov pa so korelirale tudi s številom pokajenih cigaret na dan. Lakiranje nohtov ni pokazalo statistično značilnih razlik v koncentraciji elementov, medtem ko je barvanje las vplivalo na koncentracije nekaterih elementov. Mn, Co, Ni, Sr in Ba so bili v umetno barvanih laseh višji, Cu, Zn, As, Se in Mo pa nižji glede na nebarvane. Nazadnje smo primerjali koncentracije elementov v sledovih v nohtih in laseh. Šibko pozitivne korelacije smo določili za elemente Mn, Co, Cu, Se, Al, Ga, As, Sn, Cs, Au V in Pb, močno pozitivno korelacijo pa za element Hg. Nova metoda določanja elementov v sledovih v nohtih in laseh je zanesljiva, vendar so potrebne dodatne usmeritve v standardizacijo in primerljivost rezultatov med različnimi laboratoriji ter opredelitev vpliva barvanja las ter endogenih dejavnikov na večjem številu vzorcev, saj so dosedanji rezultati med študijami nekonsistentni.Hair and nails can serve as important biological samples for assessing past exposure to trace elements, making them increasingly valuable in clinical and epidemiological studies evaluating the effects of environmental factors on human health. In the Laboratory for Enzyme and Trace Element Analytics at the University Medical Centre Ljubljana (UMCL), concentrations of trace elements are typically determined in blood and urine samples. In this master\u27s thesis, we developed and validated a method for the quantitative determination of concentrations of 26 trace elements in hair and nail samples, including 7 essential and 19 non-essential elements. Based on a literature review, we developed a method for trace element determination that incorporates pre-analytical procedures (washing, drying, and acid digestion using a microwave) and liquid sample analysis using inductively coupled plasma mass spectrometry (ICP-MS). The method was validated according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI), assessing detection capability, precision, accuracy, linearity, and working range. Based on the validation parameters, we determined that the method meets the requirements for its intended use in determining trace elements in nails and hair. In samples from 140 healthy individuals of both sexes, we established 95% reference intervals for both essential and non-essential elements, which were comparable to those in the literature, albeit somewhat narrower. We further evaluated the impact of exogenous and endogenous factors on trace element concentrations in toenails and hair. We observed differences in the concentrations of V, Mn, Co, Ni, Cu, Zn, As, Se, Sr, Mo, Ag, Cd, and Sn between genders. With age, the concentrations of most elements in hair and nail samples slightly decreased. Hg concentrations were elevated in the Coastal-Karst and Goriška regions, while no significant regional differences were observed for other elements. Supplement intake increased Se content in nails, and seafood consumption raised Hg concentrations in both nails and hair. Smokers had higher concentrations of Cd and Pb in their hair and nails, with these elements correlating with the number of cigarettes smoked per day. Nail polish did not show statistically significant differences in element concentrations, whereas hair dyeing affected the concentrations of certain elements. Mn, Co, Ni, Sr, and Ba were higher in dyed hair, while Cu, Zn, As, Se, and Mo were lower compared to undyed hair. Finally, we compared trace element concentrations in nails and hair. Weak positive correlations were found for Mn, Co, Cu, Se, Al, Ga, As, Sn, Cs, Au, V, and Pb, while a strong positive correlation was observed for Hg. The newly developed method for determining trace elements in nails and hair is reliable, although further efforts are needed to standardize and harmonize results between different laboratories, as well as to better understand the influence of hair dyeing and endogenous factors based on a larger number of samples, as current study results have been inconsistent

In vitro evaluation of immunomodulatory effect of Lacticaseibacillus casei extracellular vesicles

Probiotiki so skupki živih mikroorganizmov, ki imajo ob apliciranju v zadostnih količinah koristne učinke na zdravje gostitelja. Predstavljajo eno izmed ključnih strategij za zdravljenje disbioze, stanja neravnovesja med koristnimi in potencialno patogenimi bakterijami, ki je povezano z različnimi zdravstvenimi stanji. Njihova uporaba se je v zadnjih letih zelo razširila. Kljub splošno priznani varnosti pri uporabi so zabeleženi primeri neželenih učinkov, pri čemer so najresnejše okužbe zlasti pri ranljivih skupinah, kot so kritično bolni, nedonošenčki in posamezniki z oslabljenim imunskim sistemom. Mehanizem delovanja probiotikov še ni povsem pojasnjen. Znano je, da lahko krepijo črevesno pregrado, izpodrivajo patogene organizme in proizvajajo protimikrobne snovi. Izkazujejo tudi širok spekter encimskih aktivnosti in imajo sposobnost moduliranja imunskega sistema. Postbiotiki so definirani kot pripravki neživih mikroorganizmov in/ali njihovih komponent, ki prinašajo zdravstveno korist za gostitelja. V primerjavi s probiotiki prinašajo številne prednosti, kot so zmanjšanje tveganja za mikrobno translokacijo, okužbo in prenos genov, odpornih na antibiotike, daljši rok trajanja, lažje shranjevanje in enostavnejšo standardizacijo. Med postbiotike sodijo tudi zunajcelični vezikli (ZV). To so sferični, dvoslojni proteolipidi, dimenzij od 20 do 300 nm, ki jih bakterije sproščajo v zunajcelični prostor. ZV vsebujejo bioaktivne proteine, metabolite, lipide in nukleinske kisline in so pomemben dejavnik v medcelični komunikaciji. Molekule na površini ZV se lahko pripnejo na zunajcelične proteine ali specifične receptorje gostiteljske celice in sprožijo primarno reakcijo. ZV nosijo tudi skupine molekul, imenovane s patogeni povezani molekularni vzorci (angl. pathogen-associated molecular patterns – PAMPs), ki jih prepoznajo vzorčno prepoznavni receptorji (angl. pattern recognition receptors – PRRs) in sprožijo različne signalne poti. L. casei je po Gramu pozitivna bakterija, široko uporabljena v fermentiranih živilih in probiotičnih izdelkih, skupaj z drugimi koristnimi bakterijami. Številne študije nakazujejo, da bi bakterija L. casei lahko olajšala številne bolezni, med drugim srčno-žilne bolezni, debelost, delovala kot zaščita pred nastopom sladkorne bolezni tipa 2 in blažila škodo, povzročeno zaradi črevesnih bolezni. Prav tako je dokazano, da zmanjša pojavnost in trajanje driske pri otrocih in zdravih mladih odraslih. Več študij nakazuje, da imajo specifični sevi L. casei imunomodulatorne lastnosti in lahko vplivajo na izločanje citokinov. Hipoteza te študije je, da sev L. casei proizvaja zunajcelične vezikle, ki bi lahko imeli pomembno vlogo pri imunomodulatornem učinku seva. Cilji raziskav so zato vključevali preverjanje, ali sev L. casei proizvaja zunajcelične vezikle, karakterizacijo le-teh in ocenitev vpliva seva L. casei ter njegovih ZV na dveh različnih in vitro modelih. Specifični sev, ki je uporabljen v tej študiji, je trenutno v procesu pridobitve patenta, zato polnega imena seva trenutno ni mogoče razkriti. Prvi ključni korak je bila ekstrakcija ZV iz celične kulture. Po uspešni kultivaciji seva L. casei smo bakterije in celične ostanke odstranili s pomočjo centrifugiranja, filtriranja in ultracentrifugiranja. Tako prečiščene ZV smo nato kvantificirali s pomočjo Bradfordove metode, ki temelji na barvni reakciji med hidrofobnim barvilom in proteini. ZV smo vizualizirali z uporabo visoko ločljivega transmisijskega elektronskega mikroskopa. Porazdelitev velikosti ZV smo določili z uporabo Zetasizer Nano ZSP. Za in vitro študije smo uporabili dve celični liniji, in sicer RAW 264 (mišji makrofagi) in Caco-2 (humane epitelijske celice kolorektalnega raka). Preden smo se lotili raziskovanja imunomodulatornih učinkov ZV, smo s testom celične viabilnosti preverili, ali so ZV ali sev L. casei citotoksični. Celice smo inkubirali z ZV različnih koncentracij in sevom L. casei. Po inkubaciji smo celično viabilnost določili z uporabo testa proliferacije celic CellTiter 96® AQueous One Solution. Reagent vsebuje tetrazolijevo sol (3-(4,5-dimetiltiazol-2-il)-5-(3-karboksimetoksifenil)-2-(4-sulfofenil))-2H-tetrazolijevo sol (MTS), ki jo presnovno aktivne celice reducirajo do topnega formazana, pri čemer pride do obarvanja, ki ga je mogoče izmeriti spektrofotometrično. Količina nastalega produkta je neposredno sorazmerna številu živih celic v kulturi. V obeh celičnih linijah so se ZV in L. casei izkazali kot necitoksični. Nadaljevali smo s testi za dokazovanje imunomodulatornega učinka L. casei in ZV izbranih koncentracij. Zanimalo nas je, ali ZV in L. casei lahko zavrejo celično proizvodnjo dušikovega oksida (NO) v stimuliranih celicah RAW 264. NO deluje kot vnetni mediator in igra ključno vlogo pri kroničnem vnetju. Izvedli smo Griessov test, ki predstavlja indirektno metodo za določanje NO preko nitrita (NO2–), enega od dveh primarnih, stabilnih in nehlapnih razgradnih produktov NO. Griessov reagent vsebuje sulfanilno kislino in N-[1-naftil]etilendiamin. V kislem okolju sulfanilna kislina reagira z NO2–, pri čemer nastane diazonijeva sol, ki se nato združi z N-[1-naftil]etilendiaminom v visoko obarvano azo spojino, ki jo lahko zaznamo spektrofotometrično. Celice RAW 264 smo stimulirali z lipopolisaharidom (LPS), ki je močan imunostimulator in v celicah sproži vnetni odziv, ter tretirali z ZV različnih koncentracij in sevom L. casei. Po 24 urah smo zbrali supernatante in izvedli test. Sledila je analiza genskega izražanja. Zanimalo nas je, ali ZV in L. casei vplivajo na gensko izražanje interlevkina 6 (Il-6) ali dejavnika tumorske nekroze alfa (Tnf-α), ki igrata pomembno vlogo v imunskem odzivu. Celice RAW 264 smo predinkubirali z ZV in sevom L. casei ter stimulirali z LPS. Kot kontrolo smo uporabili celice brez tretiranja in stimulacije, kot pozitivno kontrolo pa celice brez predhodnega tretiranja, stimulirane z LPS. Zatem je bila izvedena ekstrakcija RNA z uporabo reagenta NucleoZOL® in reverzna transkripcija za sintezo komplementarne DNA. Nato smo izvedli amplifikacijo tarčnih genov z uporabo kvantitativne verižne reakcije s polimerazo (qPCR) s fluoroforom SYBR GREEN. Za izvedbo statističnih analiz smo uporabili GraphPad 8.4.2. Študija je pokazala, da je sev L. casei zmožen proizvajati zunajcelične vezikle. Ugotovili smo, da nobena od preučevanih koncentracij ZV ni citotoksična za Caco-2 celice, niti za celice RAW 264. Rezultati te študije kažejo, da ZV lahko modulirajo imunski sistem. Sev L. casei in njegovi ZV so zmanjšali proizvodnjo NO v celicah RAW 264, stimuliranih z LPS-om, kar nakazuje, da so ZV lahko koristni pri zdravstvenih stanjih, kjer je prisotna prekomerna proizvodnja NO. Prikazali smo tudi zmanjšano izražanje Il-6 in Tnf-α mRNA v LPS-stimuliranih celicah RAW 264, tretiranih z ZV, kar nakazuje, da bi ZV lahko bili koristni pri obvladovanju bolezni, ki jih spremlja prekomerno vnetje. Čeprav lahko to študijo štejemo kot preliminarno, bi ZV lahko igrali ključno vlogo pri imunomodulatornih lastnostih L. casei in bi jih lahko obravnavali kot obetavno terapevtsko orodje v primerih, ko žive probiotične bakterije niso priporočljive. Pomembno je omeniti, da so potrebne nadaljnje raziskave, vendar je ta študija odprla novo smer raziskav, ki vzbuja veliko zanimanje. ZV, pridobljeni iz L. casei, bi lahko postali del terapevtske strategije za različna stanja, pri katerih prevladuje vnetni proces.Probiotics are live microorganisms that, when administered in adequate amounts, confer a health benefit on the host. They are a prevailing strategy to treat dysbiosis, i.e., an imbalance between beneficial and potentially pathogenic bacteria in the gut, which is associated with various health conditions. Probiotics have gained popularity over the past years and are widely used for their health benefits. However, it is important to note that they also have some drawbacks, including short shelf life and potential side effects, most notably infection in more vulnerable patients. A postbiotic is a preparation of inanimate microorganisms and/or their components that confers a health benefit on the host. These postbiotics offer a promising solution to address some of the associated drawbacks. Probiotic-derived extracellular vesicles are considered postbiotics and are spherical, bilayered proteolipids containing specific subsets of bioactive molecules. Lacticaseibacillus casei has demonstrated significant potential in treating various diseases, including cardiovascular diseases, intestinal diseases, and cancer. The specific strain of L. casei used in this study is currently undergoing the patent process, and therefore, the full name cannot be disclosed at this time. The hypothesis of this study is that the L. casei strain is capable of producing extracellular vesicles (EVs), which may play a crucial role in its immunomodulatory effects. Thus, the objectives were to isolate EVs and evaluate their immunomodulatory activity in vitro. EVs were extracted through centrifugation, characterized using transmission electron microscopy (TEM) and dynamic light scattering (DLS) with Zetasizer Nano ZSP, and utilized as a treatment, along with the L. casei strain, in Caco-2 and RAW 264 cell lines. The results demonstrated that L. casei EVs had no effect on cell viability, decreased the production of nitric oxide (NO) in RAW 264 cells stimulated with lipopolysaccharides (LPS), and reduced the expression of interleukin-6 and tumor necrosis factor-α in LPS-stimulated RAW 264 cells. Based on these results, we can conclude that L. casei can produce EVs, which display immunomodulatory effects that may play an important role in beneficial activity to the host, thereby opening new avenues for promising future treatments

Relational thinking and the cultural conditionality of human understanding

For Western researchers, the confrontation and understanding of so-called “non-Western” cultural traditions are intricately bound up with linguistic, cultural, historical, and socializational disparity issues. Indeed, the interpretation of various cultures is a process where not only the subject of the interpretation, i.e., the culture, is important, but also interpreter-related factors, e.g., in terms of geography, politics, and economy, play a role. A basic assumption of the present chapter is that Western epistemology embodies only one of the numerous diverse models of human thought and our perception of reality. This chapter presents a specific epistemology, which differs in its characteristics from the norms derived from Euro-American epistemological discourses. The basic paradigms of this model, which I call “relational epistemology,” have been developed in Sinic, especially Chinese, intellectual traditionsit is predicated on the idea of seeing the world as a complex network of interconnections. Such models for interpreting questions of knowledge were thus based on the compatibility of the structures of external reality on the one hand and the structure of the human mind on the other

Density–nematic coupling in isotropic solution of DNA

Monte Carlo simulations of isotropic solutions of double-stranded DNA (deoxyribonucleic acid) are performed using the well-established oxDNA model. By comparing the fluctuation amplitudes with theoretical predictions, the parameters of a generic macroscopic model of an isotropic linear polymer solution/melt are determined. A multiscale continuum field model is thus obtained, corresponding to the full specificity of the isotropic phase of double-stranded DNA in the usual B-form as perceived at the macroscopic level. Present research is particularly focused on the coupling between spatial concentration/density variations of the polymer and the emerging nematic orientation order of the chains. This rather unfamiliar, only recently described phenomenon, inherent to linear polymers, is outlined and interpreted. Quantitative predictions are provided for the degree of nematic order induced by concentration gradients in isotropic solutions of double-stranded DNA

Influence of trap type on the captures of Lymantria dispar L. (Lepidoptera: Erebidae)

The spongy moth, Lymantria dispar L. (Lepidoptera: Eribidae), is a serious pest of deciduous forests and causes widespread defoliation. Despite this, few studies have evaluated the wide-ranging surveillance of adult male L. dispar using different types of pheromone-baited traps. We evaluated the effect of trap type on captures of adult male L. dispar at 18 sites in Europetwo in Slovenia, two in Spain, 12 in Greece, one in Hungary, and one in Croatia. Seven different trap types, G trap and eGymer 1–6, were evaluated June–September 2022 and 2023. Generally, captures of L. dispar started in late June and lasted until mid-August. Trap type affected captures. The G trap (consisting of a dark brown plastic rectangular parallel-piped body) caught significantly more L. dispar than other trap types in many instances, particularly when the peak of the flight period occurred. Captures of L. dispar in pairs of different trap types showed a significant correlation in trap catch in most investigations, suggesting that most detected comparable fluctuations in L. dispar abundance. We recommend that the G trap be used for wide-ranging surveillance of L. dispar in Europe

Žabkar dance center - conceptual design for the revitalisation of Žabkar\u27s factory in Ljubljana

Prenova industrijske dediščine v Ljubljani je vedno znova ponavljajoč se problem, ki večinoma rezultira v izgubi teh dragocenih fragmentov naše identitete. Na račun kapitala je Ljubljana izgubila že skoraj vso industrijsko dediščino in tudi Žabkarjevi tovarni se obeta rušitev. Projekt predstavlja alternativo in pokaže, da se z revitalizacijo stare železolivarne lahko ohrani ta fragment dediščine. Prenova Žabkarjeve tovarne temelji na želji po ohranitvi raznolikosti, ki bogati in s tem ohranja naše mesto svojstveno. Ob izpostavitvi pomanjkljivosti trenutnega stanja objekta in urbanističnih prvin ožje okolice se obenem razjasnijo tudi priložnosti za izboljšave, ki so možne brez izbrisa zgodovine našega prostora. Zasnova plesnega centra se tako geometrijsko kot programsko odziva na okolico, ki s svojo raznolikostjo in specifiko vzpostavlja različne robne pogoje. Ključnega pomena so obstoječi nastavki prostora in osi, ki narekujejo potek zasnove, širši cilj pa je povezava in oživitev degradirane cone, na kar se navezuje tudi programska zasnova. Z adaptacijo prostorov stare tovarne v nov plesni center se dopolni že obstoječe kulturno središče, ki ga sestavljata Gospodarsko razstavišče in že predvidena dozidava Baragovega semenišča, namenjena Slovenskemu mladinskemu gledališču. Povezava z Gospodarskim razstaviščem omogoča dogodke večjega tipa, medtem ko je plesni center zato lahko zasnovan kot skupek manjših studiev in dvoran, ki so del vsakodnevne vadbe in rednih manjših dogodkov. Izbira mešanja javnih programov in plesnih prostorov temelji na želji oživitve prostora na vsakodnevni ravni in na vzpostavitvi javnega roba med razstaviščem in mestom.The issue of the renovation of Ljubljana’s industrial heritage is a recurring problem, which mostly results in the loss of these precious fragments of our identity. At the expense of capital, Ljubljana has already lost almost all of its industrial heritage, and the Žabkar factory is also facing demolition. The project presents an alternative and shows that by revitalising the old ironworks factory, this fragment of heritage can be preserved. The renovation of the Žabkar factory is based on the desire to preserve the layered nature of the space that enriches our area and thus keeps our city distinctive. While highlighting the shortcomings of the current state of the building and the urban elements of the surroundings, it also highlights the opportunities for improvements that are possible without erasing the history of our space. The design of the dance centre responds both geometrically and with its programme to the surroundings, which, with their diversity and specificity, establish different boundary conditions. The existing spatial settings and axes that dictate the course of the design are key, but the broader aim is to connect and revitalise the degraded zone, which is also linked to the programme design. The adaptation of the old factory premises into a new dance centre complements the existing cultural centre, which consists of the Exhibition and Convention Centre (Gospodarsko razstavišče) and the already planned extension of the Barag Seminary for the theatre (Slovensko mladinsko gledališče). The connection to the Gospodarsko razstavišče allows for larger events, while the dance centre can therefore be designed as a collection of smaller studios and halls that are part of the daily rehearsals and regular smaller events. The choice of mixing public programmes and dance spaces is based on the desire to revitalise the space on a day-to-day level and to create a public edge between the exhibition centre and the city