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The role of proteomics in determining the proteins involved in the pathophysiology of liver disease
No full textKronična bolest jetre predstavlja veliki i rastući zdravstveni i ekonomski teret u društvu. Bolesti jetre su općenito asimptomatske sve dok ne dođe do očitih komplikacija i do ozbiljnog oštećenja jetre. S obzirom na progresivni i općenito neotkriveni tijek bolesti jetre, od velike važnosti je primjena modernih „omics“ tehnologija u identifikaciji specifičnih proteina koji bi mogli imati uzročnu ulogu u bolesti.
Povezivanjem genetskih čimbenika s bolestima putem specifičnih proteina, omogućava se isticanje potencijalnih terapijskih ciljeva, usklađivanje postojećih lijekova s novim indikacijama bolesti i smanjuje potencijalna sumnja u sigurnost lijekova u razvoju. Razvrstavanje specifičnih proteina bitno je u obradi rezultata proteomičke analize. U ovom radu korišten je sustav klasifikacije PANTHER i baza STRING za predviđanje interakcija proteina. Sustav klasifikacije PANTHER (Protein ANalysis THrough Evolutionary Relationships) je dizajniran je za klasifikaciju proteina (i njihovih gena) kako bi se olakšala analiza velike propusnosti. STRING je baza poznatih i računalno predviđenih interakcija protein-protein iz interakcija združenih iz drugih (primarnih) baza podataka. Za obradu podataka korišteni su rezultati proteomičke analize objavljenih radova u području hepatotoksičnosti.
Korišteni programi razvrstali su proteine prema njihovim funkcijama, procesu u kojima sudjeluju, položaju i putu u stanici, smjestili u izravne i neizravne funkcionalne asocijacije. Time je omogućena obrada rezultata proteomičke analize kroz dubinsku analizu komplementa svih njegovih proteina, proteoma jetre.Chronic liver disease is a major and growing health and economic burden on society. Liver disease is usually asymptomatic until obvious complications and severe liver damage occur. Given the progressive and generally unrecognized course of liver disease, it is important to apply modern "omics" technologies to identify specific proteins that may play a causal role in the disease.
By linking genetic factors to disease via specific proteins, it is possible to highlight potential therapeutic targets, tailor existing drugs to new indications for the disease, and reduce potential doubts about the safety of drugs in development. Classification of specific proteins is essential for processing the results of proteomic analyzes. In this work, the classification system PANTHER and the database STRING were used to predict protein interactions. The classification system PANTHER (Protein ANalysis THrough Evolutionary Relationships) is used to classify proteins (and their genes) to facilitate high throughput analysis. STRING is a database of known and computer-calculated protein-protein interactions compiled from interactions in other (primary) databases. The results of proteomic analysis of published papers in the field of hepatotoxicity were used for data processing.
The programs used sorted the proteins according to their functions, the process they are involved in, their position and pathway in the cell, and classified them into direct and indirect functional relationships. This allows the processing of the results of the proteome analysis through an in-depth analysis of the totality of its proteins, the liver proteome
Macrophages from the tumor microenvironment
No full textTumorski mikrookoliš sastoji se od ne-tumorskih stanica koje okružuju tumor. Tumorski mikrookoliš se znatno razlikuje od zdravog tkiva te zbog toga okolne stanice mogu poprimiti drugačiju funkciju nego li bi to imale u normalnim uvjetima. Tumor također lučenjem citokina i direktnom interakcijom sa okolnim stanicama mijenja svoj mikrookoliš. Jedna od najučestalijih stanica u tumorskom mikrookolišu su makrofazi. Makrofazi iz mikrookoliša tumora (TAM) su stanice koje je tumor prilagodio u svrhu svog preživljavanja i razvijanja. TAM lučenjem raznih citokina i eksprimiranjem različitih receptora i liganada pomaže tumoru tako što omogućuje izjegavanje imunološkog sustava, potiče angiogenezu i limfogenezu tumora te i metastaziranje. TAMovi također negativno utječu na postojeće terapije zbog čega se sve intenzivnije traže lijekovi koji bi inhibirali TAMov utjecaj te čiji su osnovni principi opisani u ovom radu. Tumorski mikrookoliš svakog tumora, a time i funkcija TAMa je različita zbog čega se u ovom radu posebnu pažnju pridalo TAMovima u karcinomu dojke i karcinomu pluća.Tumor microenvironment is contained of non-tumor cells which surround the tumor. Tumor microenvironment is significally different from healthy tissue and because of that surrounding cells can have different function than they would have in healthy tissue. Also, with secretion of cytokines or with direct interaction with surrounding cells, tumor can change its microenvironment. One of the most common cells in tumor microenvironment are macrophages. Tumor associated macrophages (TAM) are cells which are modified by tumor in the purpose of its survival and development. With secreting numerous cytokines and with expressing different receptors and ligands TAM helps tumor by allowing the immune response to be avoided, while it promotes angiogenesis and lymhogenesis and metastasis of tumor. TAMs can also negatively affect on the existing therapies which is why drugs that would inhibit TAM effect and whose basic principles are described in this paper are increasingly being searched for. Tumor microenvironment of every tumor and by that the function of TAM is different and because of that special attention was paid in this paper on TAMs in breast and lung tumors
Influence of chemically functionalized single-walled carbon nanotubes on the characteristics of astrocytes exposed to in vitro traumatic injury
No full textTraumatska ozljeda mozga (engl. traumatic brain injury, TBI) je jedan od vodećih uzroka smrti i invaliditeta širom svijeta, a posebno u osoba mlađih od 45 godina i predstavlja značajan javnozdravstveni problem današnjice. Astrociti, stanice rasprostranjene širom središnjeg živčanog sustava (engl. central nervous system, CNS), značajno su podložne oštećenju nakon TBI. Dosadašnja su istraživanja potvrdila ulogu astrocita u održavanju strukture, funkcije i aktivnosti CNS-a, ali i u potencijalnoj regeneraciji CNS-a nakon ozljede. Do danas učinkoviti terapijski pristup u liječenju TBI nije otkriven, stoga su se istraživači okrenuli ka novom pristupu - tkivnom inženjeringu, točnije primjeni nanomaterijala. Cilj ovog istraživanja bio je istražiti utječu li jednozidne ugljikove nanocjevčice (engl. single walled carbon nanotubes, SWCNTs), kemijski funkcionalizirane s poli-m-aminobenzen sulfonskom kiselinom (engl. poly-m-aminobenzene sulfonic acid, PABS), na preživljavanje i određene karakteristike astrocita u in vitro modelu teške TBI. Istražen je utjecaj PABS-SWCNTs na ekspresiju kiselog fibrilarnog proteina glije (engl. glial fibrillary acidic protein, GFAP) u ozlijeđenim astrocitima. Nije zamijećena značajna razika u intenzitetu GFAP fluorescencije u traumatiziranim stanicama u odnosu na neozlijeđene, a nije uočena ni promjena ekspresije ovog markera u traumatiziranim stanicama tretiranima s PABS-SWCNTs. U drugom je pokusu praćen utjecaj PABS-SWCNTs na preživljenje astrocita nakon traume in vitro te su rezultati istraživanja pokazali da PABS-SWCNTs ne potiču bolje preživljenje, ali nisu ni citotoksične za ozlijeđene astrocite u kulturi. Kako bi se istražilo postoje li promjene u sadržaju sekretoma astrocita nakon ozljede, odnosno primjene istraživanog nanomaterijala, analiziran je sadržaj citokina u mediju za kulturu stanica. Ozljeda stanica uzrokovala je smanjeno otpuštanje Eotaksina-1 i povećanu razinu SDF-1 alfa u mediju, a primjena PABS-SWCNTs nije značajno utjecala na ove promjene. Aplikacija ovog tipa funkcionaliziranih nanocjevčica bila je praćena povećanim otpuštanjem RANTES-a iz ozlijeđenih astrocita u odnosu i na neozlijeđene i ozlijeđene netretirane stanice.
Zaključno, rezultati ovog rada ukazuju da primjena PABS-SWCNTs ne djeluje na preživljenje astrocita podvrgnutih teškoj in vitro TBI unutar prva 24 sata nakon ozljede, no uzrokuje neke promjene u profilu izlučenih citokina koje bi mogle utjecati na funkcije ove vrste stanica u patofiziološkim procesima nakon TBI.Traumatic brain injury (TBI) is one of the major causes of death and disability all over the world, especially among people under the age of 45, which is why it is considered a major public health problem of the modern age. Astrocytes are cells located in the central nervous system (CNS), which are significantly susceptible to damage after TBI. Previous researches have confirmed the importance of astrocytes in maintaining the structures, functions and activity of the CNS, but also the potential role in regeneration of the CNS, after TBI. So far, the effective therapeutic approach has not been discovered, which is why researchers have turned to a new approach, which includes tissue engineering, with nanomaterials. The aim of this study was to investigate the effect of single-walled carbon nanotubes (SWCNTs), chemically functionalized with poly-m-aminobenzene sulfonic acids (PABS), to better viability and proliferation of an astrocyte in an in vitro model of severe TBI. The effect of PABS-SWCNT on the expression of glial fibrillary acid (GFAP) was investigated in injured astrocytes. There was no significant difference in GFAP fluorescence intensity in injured cells compared to uninjured ones, also there was no change observed in expression of this market, in traumatized cells compared to ones treated with PABS-SWCNTs. In the second case, experiment monitored the effect of PABS-SWCNT on astrocyte survival after in vitro trauma, and the results of the study showed that PABS-SWCNTs did not lead to better survival, but also did not have cytotoxic effect on injured astrocytes in culture. In order to investigate whether there are changes in the content of astrocyte secretome or in the application of the investigated nanomaterial, cytokine content in the culture cell medium was analyzed. Cell injury caused decrease of Eotaxin-1 release and increase in SDF-1 alpha in the medium, and administration of PABS-SWCNT did not affect these changes. Application of this type of functionalized nanotubes was followed by an increased dismissal of RANTES, from injured astrocytes compared to uninjured and injured untreated cells.
In conclusion, the results of this study indicate that the use of PABS-SWCNTs does not affect the survival of astrocytes subjected to severe in vitro TBI within the first 24 hours after injury, but there was some changes in expression profile of cytokines that could affect on function of this type of cells in pathophysiological process after TBI
Novoproizvedeno mišje monoklonsko protutijelo prepoznaje tkivno specifične glikoforme markera izvanstaničnih vezikula CD63 u mišjim tkivima
No full textCD63 is a ubiquitously expressed transmembrane glycoprotein frequently used as a general protein marker of extracellular vesicles (EVs). EVs are cell-secreted and membrane-enclosed nanoparticles which circulate through the blood and other body fluids and thus are available for isolation. EV cargo is very dynamic and reflects the biological status of the originating tissue, which, coupled with their accessibility from body fluids, could make EVs a valuable diagnostic and prognostic tool. However, identification of tissue-specific EVs is still lacking.
The goal of this study was to examine the tissue specificity of CD63 protein using a novel murine monoclonal antibody mCD63.07 produced at the Center for Proteomics, University of Rijeka, Faculty of Medicine, Croatia. Western blot analysis of salivary gland, brain, lung, liver, kidney, muscle, bone marrow and spleen tissue obtained from wild type (wt) mice revealed that the expression levels and protein forms of CD63 varied among murine tissues. Molecular weight of detected CD63 forms ranged from 25 to 170 kDa, depending on the tissue, whereby some of CD63 forms were tissue-specific. A comparison with corresponding knockout tissues confirmed the high specificity of mCD63.07 antibody. PNG-ase F deglycosylation treatment of proteins from salivary gland, kidney, bone marrow, liver, muscle and spleen wt samples resulted in merging of higher molecular weight protein bands into one band at approximately 24 kDa. Thus the observed differences in detected CD63 forms arise from tissue-specific N-glycosylation of the CD63 protein.
The findings indicate that CD63 glycoforms are tissue-specific and therefore might be used for isolation of tissue-specific EVs. Future characterization of human tissue-specific glycoforms of CD63 could enable the development of new EV-related blood-based diagnostic methods.CD63 je ubikvitarno eksprimirani transmembranski glikoprotein, najčešće korišten kao opći proteinski marker izvanstaničnih vezikula (IV). IV-e su membranom obavijene nanočestice koje stanice izlučuju u krv i druge tjelesne tekućine, što ih čini lako dostupnima za izolaciju. Sadržaj IV-a je vrlo dinamičan i odražava biološki status tkiva iz kojih potječu, što u kombinaciji s njihovom lako dostupnošću može učiniti IV-e vrijednim dijagnostičkim i prognostičkim alatom. Međutim, identifikacija tkivno-specifičnih IV-a još uvijek nije moguća.
Cilj ove studije bio je ispitati tkivnu specifičnost proteina CD63 pomoću novog mišjeg monoklonskog antitijela mCD63.07 proizvedenog u Centru za proteomiku Medicinskog fakulteta u Rijeci. Western blot analiza tkiva žlijezde slinovnice, mozga, pluća, jetre, bubrega, mišića, koštane srži i slezene izoliranih iz miševa divljeg tipa otkrila je da se razina ekspresije i proteinski oblici CD63 razlikuju u mišjim tkivima. Molekularna masa detektiranih oblika CD63 kretala se u rasponu od 25 do 170 kDa, ovisno o tkivu, te su neki od oblika pokazali tkivno-specifičnu ekspresiju. Usporedba s odgovarajućim knockout tkivima potvrdila je visoku specifičnost antitijela mCD63.07. Deglikozilacijski tretman proteina iz žlijezde slinovnice, bubrega, koštane srži, jetre, mišića i slezine rezultirao je spajanjem proteinskih bendova veće molekularne mase u jedan bend od približno 24 kDa, što potvrđuje da uočene razlike u opaženim oblicima CD63 proteina potječu iz tkivno-specifične N-glikozilacije proteina CD63.
Ovi rezultati pokazuju da su glikoforme proteina CD63 tkivno-specifične te se stoga mogu koristiti za izolaciju tkivno-specifičnih IV-a. Buduća karakterizacija glikoformi CD63 specifičnih za ljudska tkiva mogla bi omogućiti razvoj novih dijagnostičkih metoda temeljenih na izolaciji IV-a iz krvi
Mechanochemical synthesis and characterization of supramolecular complexes of cyclodextrin and biologically active ferrocene derivatives
No full textCiklodekstrini su spojevi koji mogu vezati lipofilne molekule lijeka ili lipofilni dio molekule lijeka u svoju centralnu šupljinu te na taj način stvoriti inkluzijske komplekse topljive u vodi što predstavlja oslonac za pripravu novih formulacija i njihovu farmaceutsku primjenu. U ovom radu opisana je sinteza i karakterizacija inkluzijskog kompleksa β-ciklodekstrina s lipofilnim biološki aktivnim derivatom ferocena i jod-uracila (5-jod-N-1,N-3-di{4-[1-(1-ferocenilmetil)-1,2,3-triazol-4-il]metil}-pirimidin-2,4-dion, L1) s dokazanim protutumorskim djelovanjem. Mehanokemijskom sintezom u kugličnom mlinu dobiveni su kompleksi α- i β-ciklodekstrina s ferocenskom kiselinom kao modelni spojevi te ciljani inkluzijski kompleks spoja L1 i β-ciklodekstrina. Pomoću rezultata UV-Vis spektrofotometrijske titracije određena je konstanta stabilnosti inkluzijskog kompleksa u vodi. Za analize kompleksa spojeva nastalih mehanokemijskim reakcijama korištene su termogravimetrijska (TGA) analiza i NMR spektroskopija. Rezultati istraživanja pokazuju kako navedenim postupcima dolazi do stvaranja inkluzijskog kompleksa β-ciklodekstrina i modelnog spoja ferocenske kiseline u vodenoj otopini i mehanokemijski do stvaranja kompleksa β-ciklodekstrina i spoja L1 što je dokazano TGA analizom i NMR spektroskopijom.Cyclodextrins are compounds that can bind lipophilic drug molecules or a lipophilic part of a drug molecule into their central cavity, and thus form water-soluble inclusion complexes, which is the basis for the preparation of new formulations and their pharmaceutical application. This paper describes the synthesis and characterization of the inclusion complex of β-cyclodextrin with a lipophilic biologically active derivative of ferrocene and iodouracil (5-iodo-N-1,N-3-di{4-[1-(1-ferrocenylmethyl)-1,2,3-triazol-4-yl]methyl}-pyrimidine-2,4-dione, L1) with proven antitumor activity. Mechanochemical synthesis in a ball mill yielded complexes of α- and β-cyclodextrin with ferrocenic acid as model compounds, and a target inclusion complex of compound L1 and β-cyclodextrin. The stability constant of the inclusion complex in water was determined by the results of UV-Vis spectrophotometric titration. Thermogravimetric (TGA) analysis and NMR spectroscopy were used to analyze complexes of compounds formed by mechanochemical reactions. The research results show that these methods lead to the formation of an inclusion complex of β-cyclodextrin and a model compound of ferrocenic acid in aqueous solution, and mechanochemically to the formation of a complex of β-cyclodextrin and L1 compound, which was proven by TGA analysis and NMR spectroscopy
Cytotoxic effects of capers (Capparis spinosa L.) on different human cancer cell lines
No full textKarcinomi su skup različitih bolesti koje se međusobno razlikuju prema svojoj etiologiji, biologiji i kliničkoj slici te posljedično i po načinu liječenja. Progresivno povećanje nekontrolirane stanične diobe razlog je što se karcinom klinički najčešće očituje kao nakupina velikog broja zloćudno preobraženih stanica. Karcinom je drugi vodeći uzrok smrti u svijetu, a odgovoran je za 9,6 milijuna smrtnih slučajeva u 2018. godini. Karcinom mokraćnog mjehura je deveti najčešći oblik maligniteta i 13. najčešći uzrok smrti od karcinoma diljem svijeta. Rak dojke najčešći je tumor u žena u razvijenim zemljama svijeta. Godišnje u Hrvatskoj oboli oko 2600 žena zbog čega smo u skupini zemalja s visokom incidencijom. Cilj ovog diplomskog rada je ispitati citotoksično djelovanje kapara (Capparis spinosa L.) na staničnim linijama humanog karcinoma dojke (MDA-MB-231) i karcinoma mokraćnog mjehura (T24). Pretpostavka je kako će se broj karcinomskih stanica smanjiti, posljedično izlaganjem stanica ekstraktu kapare koji je dobiven različitim metodama izolacije, u odnosu na kontrolnu skupinu. MTT testom je ispitano citotoksično djelovanje na staničnim linijama MDA-MB-231 i T24. Postotak preživljenja karcinomskih stanica dobili smo uspoređivanjem apsorbancije formazana kod stanica tretiranih ekstraktom C. spinosa dobivenim različitim metodama izolacije i apsorbancije netretiranih stanica. Vremena inkubacije za test citotoksičnosti bila su 4, 24, 48 i 72 sata. Rezultati su prikazani grafički, gdje je prikazana ovisnost vremena inkubacije i postotka metaboličkih aktivnih stanica. Citotoksično djelovanje kapare na stanice karcinoma mokraćnog mjehura T24 značajan je pri koncentracijama 50 µg/mL i 100 µg/mL. Najveći citotoksični učinak imala je kapara s Brača čiji je ekstrakt dobiven mikrovalnom destilacijom pri koncentraciji 50 µg/mL u vremenu inkubacije od 72 sata. Najznačajniji citotoksični učinci ekstrakta kapare na karcinom dojke bili su vidljivi pri većim koncentracijama 50 µg/mL i 100 µg/mL. Najznačajniji učinak na staničnu liniju MDA-MB-231 ostvaren je s kaparom s Brača čiji je ekstrakt dobiven mikrovalnom ekstrakcijom pri koncentraciji 10 µg/mL u vremenu 72 h inkubacije. In vitro izlaganje stanica karcinoma dojke (MDA-MB-231) i mokraćnog mjehura (T24) ekstraktima kapare (Capparis spinosa L.) izoliranih različitim metodama izolacije dovodi do smanjenog preživljenja karcinomskih stanica. Ispitivani ekstrakti kapare ostvaruju citotoksičan učinak koji ovisi o koncentraciji i vremenu inkubacije. Citotoksični učinak kapara (Capparis spinosa L.) je potvrđen, što je ujedno i hipoteza ovog rada. S obzirom na dobivene rezultate citotoksičnog učinka kapara na MDA-MB-231 i T24 stanične linije, bilo bi od velikog interesa nastaviti istraživanja ovih biljaka na karcinome u in vivo pokusima, kako bi pokazala eventualna korisnost ovih biljaka u liječenju karcinoma.
Rad je financiran od Hrvatske zaklade za znanost projektom IP-2016-06-1316.Cancers are a set of different diseases that differ from each other in their etiology, biology and clinical picture, and consequently in the method of treatment. Cancer is most often clinically manifested as a cluster of a large number of malignant transformed cells whose number is progressively increasing due to uncontrolled cell division. Cancer is the second leading cause of death in the world, and is responsible for 9.6 million of deaths in 2018. Bladder cancer is the 9th most common form of malignancy and the 13th most common cause of death from cancer worldwide. Breast cancer is the most common tumor in women in the developed countries of the world. In Croatia, it is also in first place in terms of incidence, because about 2,600 women get sick in Croatia every year. The aim of this thesis is to examine the cytotoxic effect of capers (Capparis spinosa L.) on human breast cancer cell line (MDA-MB-231) and bladder cancer cell line (T24). It is assumed that the number of cancer cells will decrease, consequently by exposing the cells to capers extract obtained by different isolation methods, relative to the control group. The MTT test was performed by a cytotoxic assay on MDA-MB-231 and T24 cell lines. The survival rate of cancer cells was obtained by comparing the absorbance of formazan in cells treated with C. spinosa extract obtained by different methods of isolation and the absorbance of untreated cells. Incubation times for the cytotoxicity test were 4, 24, 48, and 72 hours. The presentation of the results is graphical where the dependence of the incubation time and the percentage of metabolically active cells is shown. The cytotoxic effect of capers on T24 bladder cancer cells is significant at concentrations of 50 µg/mL and 100 µg/mL. The greatest cytotoxic effect was exerted by a capers from Brač whose extract was obtained by microwave distillation at a concentration of 50 µg/mL during an incubation time of 72 hours. The most significant cytotoxic effects of caper extract on breast cancer were seen at higher concentrations of 50 µg/mL and 100 µg/mL. The most significant effect on MDA-MB-231 cell line was achieved with a caper from Brač whose extract was obtained by microwave extraction at a concentration of 10 µg/mL during the 72h incubation period. In vitro exposure of breast cancer cells (MDA-MB-231) and bladder cancer cells (T24) to caper extracts (Capparis spinosa L.) isolated by various isolation methods leads to reduced cancer cell survival. The tested caper extracts achieve a cytotoxic effect that depends on the concentration and incubation time. The cytotoxic effect of caper (Capparis spinosa L.) has been confirmed, which is also the hypothesis of this paper. Since there is no such study, i.e. the cytotoxic effect of capers on MDA-MB-231 and T24 cell lines, it would be good to repeat the experiment to confirm the results and, if necessary, upgrade the obtained results.
This research has been fully supported by the Croatian Science Foundation under the project (HRZZ-IP-06-2016-1316 "Plants as a source of bioactive sulphur compounds and their ability to hyperaccumulate metals")
Effects of aegerolysin proteins from Pleurotus mushrooms on bees Apis mellifera
No full textEgerolizinski proteini ostreolizin A (OlyA), pleurotolizin A2 (PlyA2) i erilizin fosfoetanolamin (CPE), glavni membranski sfingolipid beskralježnjaka. Kada su vezani na CPE, ovi egerolizini regrutiraju partnerski protein pleurotolizin B (PlyB) sa MACPF domenom (eng. membrane-attack complex/perforin), sa kojim formiraju bikomponentne transmembranske komplekse, odnosno transmembranske pore. Kompleksi OlyA6/PlyB, PlyA2/PlyB i EryA/PlyB pokazali su jaki selektivni toksični učinak za važne
štetne insekte kukuruznu zlaticu i krumpirovu zlaticu te stoga predstavljaju nove kandidate u razvoju biopesticida. U ovom istraživanju testirali smo toksičnost kompleksa egerolizin/PlyB za pčelu Apis melifera carnica koja je ekološki i ekonomski važan oprašivač i proizvođač meda. Lipide izolirane iz crijeva pčela razdvojili smo pomoću tankoslojne kromatografije zajedno sa
standardnim lipidom CPE-om te smo ih prenijeli na PVDF membrane i inkubirali sa otopinom OlyA6. Ovim metodama potvrdili smo prisutnost CPEa u staničnim membranama crijeva pčela, kao i vezanje OlyA6 na CPE među lipidima iz crijeva pčela. Metoda površinske rezonancije plazmona i test otpuštanja kalceina pokazali su da su interakcije svih egerolizina sa
liposomima izrađenim od nepolarnih lipida izoliranih iz crijeva pčela značajno jače u prisutnosti PlyB, kao i njihova litička aktivnost. U usporedbi sa OlyA6 i PlyA2, EryA ostvaruje slabije interakcije sa liposomima te posljedično ima manju litičku aktivnost. Na kraju smo istražili utjecaj OlyA6/PlyB, PlyA2/PlyB i EryA/PlyB na hranjenje i preživljavanje pčela. Dok
EryA/PlyB nije utjecao na hranjenje niti preživljavanje pčela, kompleksi OlyA6/PlyB i PlyA2/PlyB imali su toksičan učinak na pčele. Srednja letalna koncentracija (LC50) koja se ispoljava u toku 48 sati iznosi 0,22 mg/mL za OlyA6/PlyB i 0,39 mg/mL za PlyA2/PlyB.Aegerolysin proteins ostreolysin A6 (OlyA6), pleurotolysin A2 (PlyA2) and erylysin A (EryA) produced by the mushroom genus Pleurotus bind strongly to CPE, the main sphingolipid in invertebrate cell membranes. Upon binding, these aegerolysins can recruit a protein partner pleurotolysin B (PlyB) that has a membrane-attack complex/perforin (MACPF) domain to form bicomponent transmembrane pore complexes. OlyA6/PlyB, PlyA2/PlyB and EryA/PlyB complexes show selective toxicity toward two important pests: the western corn rootworm and the Colorado potato beetle. Due to their
specific interactions with their membrane lipid receptor CPE, these complexes might represent new candidates for development of biopesticides. In this study, we evaluated the toxicities of these aegerolysin/PlyB complexes to honeybees (Apis mellifera carnica) that are ecologically and economically very important as crop pollinators and honey producers. For this purpose, we separated lipids extracted from honeybee midgut using thin-layer chromatography together with standard lipid CPE. We transferred the separated lipids to PVDF membranes and incubated them with the solution of OlyA6 . These assays confirmed the presence of CPE in honeybee midgut cell membranes as well as binding of OlyA6 to CPE
in honeybee midgut lipids. Surface plasmon resonance and calcein release assay showed that the interactions of all three aegerolysins with the nonpolar liposomes made of honeybee midgut lipids were considerably stronger in presence of PlyB, as well as their lytic activity. Compared to OlyA6 and PlyB, EryA showed weaker interaction with these liposomes and
consequently a lower lytic activity. Lastly, we investigated the effects of OlyA6/PlyB, PlyA2/PlyB and EryA/PlyB on the feeding and survival rates of honey bees. While EryA/PlyB complex did not affect honeybee survival and
feeding rates, OlyA6/PlyB and PlyA2/PlyB were toxic to honeybees with 48 h mean lethal concentrations (LC50) of 0.22 and 0.39 mg/mL, respectively
DISC1 I FUS: KOAGREGACIJA PROTEINA POVEZANIH S MENTALNIM I NEURODEGENERATIVNIM BOLESTIMA
No full textChronic mental illnesses (CMI) such as schizophrenia, bipolar disorder or depression have overlapping genetic backgrounds and symptoms. These illnesses are manifested as disruptions of mental health (cognitive, emotional and behavioural) from various etiological factors. As current diagnostic methods are almost entirely dependent on psychiatric evaluation, and given the complexity of the background, it is hard to develop new approaches to research these illnesses.
Neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS) or Alzheimer’s disease are incurable conditions that result in progressive degeneration of nerve cells. These illnesses are well known to be associated with the occurrence of protein aggregation. Recent studies suggest that this same biological state, in which misfolded proteins aggregate, may be found in mental illnesses as well.
The Disrupted in Schizophrenia 1 (DISC1) protein has been identified in the insoluble fraction of the brain tissue of patients suffering from schizophrenia, bipolar disorder and depression, implying that DISC1 aggregated. The Fused in Sarcoma protein (FUS) has been found in in the insoluble fraction of the brain tissue of patients suffering from various neurodegenerative illnesses, including ALS and frontotemporal dementia (FTD). Mutations of FUS have been described as contributing to the progress of illnesses.
We investigated the interaction of DISC1 with FUS in protein aggregates. We compared wild-type and mutated FUS protein with DISC1 in cultured mammalian cells. This interaction provides information on the biological link between mental illnesses and neurodegenerative disorders.
Microscopic examination of cells overexpressing these two proteins showed the co-aggregation of mutant type FUS with DISC1. Additionally, co-aggregation of fragments of DISC1 with the mutant type (mutation on C-terminal tyrosine) FUS protein was examined to determine which domains of this protein are involved in co-aggregation. One of DISC1 region has been seen aggregating in previous researches. We confirmed the importance of this region for aggregation in a different cell line – human neuroblastoma cell line, because it is of greater biological relevance compared to previously seen results in human embryonic kidney cells. The S>C region is another region of DISC1, which showed no aggregates in cells. Combined with FUS protein, it has been seen that the D>I region does not co-aggregate with FUS protein, while the results of the S>C region suggest potential coaggregation, but for now this remains uncertain.
The results obtained here, and further research on these proteins could help explain biological connections between neurodegenerative disorders and mental illnesses, overlap in symptoms of these illnesses and the difficulty of making the diagnose.Kronične mentalne bolesti, kao što su shizofrenija, bipolarni poremećaj ili depresija imaju zajedničku genetsku pozadinu i simptome. Ova oboljenja manifestiraju se kao poremećaj mentalnog zdravlja (kognitivnog, emotivnog i bihevioralnog) zbog različitih etioloških faktora. Budući da su dijagnostičke metode gotovo u potpunosti ovisne o psihijatrijskoj evaluaciji, a sama pozadina bolesti je kompleksna, teško je razviti nove pristupe.
Neurodegenerativne bolesti, kao što su amiotrofična lateralna skleroza (ALS) i Alzheimerova bolest, neizlječiva su stanja koja rezultiraju progresivnom degeneracijom živčanih stanica. Ove bolesti poznate su po pojavi proteinskih agregata. Nedavne studije pretpostavljaju da se ovo isto biološko stanje, u kojem nepravilno formirani proteini agregiraju, može naći i u mentalnim bolestima.
Disrupted-in-Schizophrenia 1 (DISC1) je protein identificiran u netopivim frakcijama moždanih tkiva pacijenata oboljelih od shizofrenije, bipolarnog poremećaja i depresije, što implicira da DISC1, u ovim stanjima, stvara agregate. Fused in Sarcoma protein (FUS) je identificiran u netopivim frakcijama moždanih tkiva pacijenata oboljelih od različitih neurodegenerativnih bolesti poput ALS-a i frontotemporalne demencije (FTD). U ovom slučaju, mutacije FUS proteina povezane su napredovanjem bolesti.
Istraživali smo interakciju proteina DISC1 i FUS u proteinskim agregatima. Uspoređivali smo wild-type i FUS mutant sa DISC1 proteinom u kultiviranim stanicama. Ova interakcija nudi informacije o biološkoj poveznici između mentalnih i neurodegenerativnih bolesti.
Mikroskopska analiza stanica s povećanom ekpresijom ova dva proteina je rezultirala u koagregaciji DISC1 proteina i FUS mutanta. Uz to, ispitivali smo koagregaciju fragmenata DISC1 proteina sa FUS mutantom (mutacija na C-terminalnom tirozinu) kako bismo utvrdili koja domena DISC1 je uključena u koagregaciju. Jedna od ovih regija pokazala je agregaciju u prethodnim istraživanjima. Važnost ove regije za proces agregacije potvrdili smo u boljoj staničnoj liniji – humane stanice neuroblastoma, zbog veće biološke relevantnosti za rezultate u usporedbi s prethodno korištenim humanim embrionalnim stanicama bubrega. S>C regija nije do sada agregirala u stanicama. Zajedno sa FUS proteinom, vidjeli smo kako D>I regija ne koagregira sa FUS mutantom, dok rezultati za S>C regiju pretpostavljaju potencijalnu koagregaciju, no za sad, to ostaje nedokazano.
Rezultati koje smo do sada prikupili i buduća istraživanja na ovim proteinima, mogli bi objasniti biološku pozadinu između neurodegenerativnih i mentalnih bolesti, preklapanje u simptomima kao i poteškoće prilikom postavljanja dijagnoze
Historical data in control mice and rats from preclinical toxicity studies
No full textTijekom pretkliničkog istraživanja i razvoja iz pojedinih studija na laboratorijskim životinjama, prikupljaju se različiti podaci o određenim vrstama ili sojevima laboratorijskih životinja, posebice kontrolnih grupa, sa svrhom utvrđivanja razlikovnosti pojedinih parametara u odnosu na testne grupe, kao i u odnosu na literaturno dostupne podatke. Rezultat razvoja toksikologije, kao i svih njenih grana (klinička patologija, patohistologija, toksokinetika, itd.) daje utemeljen značaj toksikološkim studijama, što ujedno pridonosi pretkliničkom razvoju. Kako bi se shvatilo kako će novo testirana supstancija utjecati na ljudsko tijelo i zdravlje,
prije provedbe kliničkih ispitivanja na ljudima, potrebno je provesti pretkliničko istraživanje na životinjama, prvenstveno glodavcima, a u kasnijoj fazi istraživanja i razvoja supstancije i na drugim životinjskim vrstama. Škodljivost testnih supstancija na laboratorijske životinje u toksikološkim studijama pratimo kroz niz parametara, a među njima su i praćenje promjena parametara hematologije, biokemije i težine organa. Cilj rada bio je sakupiti podatke iz više toksikoloških studija, točnije
rezultate njihovih kontrolnih grupa na mužjacima i ženkama Sprague Dawley štakora starima osam tjedana i mužjacima CD-1 (Charles River) miševa starima osam tjedana, kako bi mi isti kasnije mogli koristiti za lakše razumijevanje i tumačenje dobivenih rezultata (parametara), istih ili sličnih toksikoloških studija. Dobivene rezultate usporedio sam s dostupnim literaturnim podacima. Prema protokolu, a po završetku toksikoloških studija životinje sam vagao, uzorkovao krv za hematološke i biokemijske analize te su nakon autopsije uzorkovani određeni organi. Ti su parametri od značaja za ovaj rad. Nakon analize krvi na automatskom
hematološkom analizatoru Sysmex XT-2000iV i analizatoru kliničke kemije Olympus AU480, dobiveni su hematološki i biokemijski parametri. Dobiveni podaci u skladu su s objavljenim literaturnim podacima što potkrjepljuje opravdanost rada. Također, značajni su za svaku ustanovu koja se bavi toksikološkim studijama, radi lakšeg tumačenja rezultata budućih studija. Cilj je nadopunjavati do sada dobivene rezultate s podacima novih studija (kontrolnih grupa) radi veće značajnosti i
vjerodostojnosti istih.During preclinical research and development from individual studies on laboratory animals, different data are collected on certain species or strains of laboratory animals, especially control groups, in order to determine the differentiation of individual parameters in relation to test groups and literature available. The result of the development of
toxicology, as well as all its branches (clinical pathology, pathohistology, toxokinetics, etc.) gives grounded importance to toxicological studies, which also contributes to preclinical development. In order to understand how the newly tested substance will affect the human body and health, before conducting clinical trials in humans, it is necessary to conduct
preclinical research on animals, primarily rodents, and at a later stage of research and development on other animal species. Harmfulness of test substances to laboratory animals in toxicological studies is monitored through a number of parameters, and among them are the monitoring of changes in the parameters of hematology, biochemistry and organ
weight. The aim of the study was to collect data from several toxicological studies, more precisely the results of their control groups on eight-weekold male and female Sprague Dawley rats and eight-week-old male CD-1 (Charles River) mice, so that they could be used later for easier understanding and interpretation of the obtained results (parameters),
from the same or similar toxicological studies. I compared the obtained results with the available literature data. According to the protocol, and after the completion of toxicological studies, the animals were weighed, blood was sampled for hematological and biochemical analyzes, and certain organs were sampled after the autopsy. These parameters are
relevant to this paper. After blood analysis on an automatic hematology analyzer Sysmex XT-2000iV and a clinical chemistry analyzer Olympus AU480, hematological and biochemical parameters were obtained. The obtained data are in accordance with the published literature data, which supports the justification of my thesis. They are also important for any
institution that deals with toxicological studies, in order to facilitate the interpretation of the results of future studies. The aim is to supplement the previously obtained results with the data of new studies (control groups) to greatly increase their significance and credibility
Analysis of the features of the maximum permitted wholesale prices of medical product that act on thenervous system
No full textRegulatorni mehanizmi kontrole cijena lijekova koriste se u većini država članica Europske Unije. Jedan su od elemenata vladinih strategija za ograničavanje javnih izdataka za zdravstvenu zaštitu. Sustav reguliranja cijena lijekova u Republici Hrvatskoj obuhvaća lijekove koji se izdaju na recept. HALMED određuje najvišu dozvoljenu cijenu lijeka na veliko i iznimno višu od najviše dozvoljene cijene lijeka na veliko. Svrha istraživanja je analiza značajki najviše dozvoljene cijene lijeka na veliko za
lijekove koji djeluju na živčani sustav. Prvi cilj je testirati hipotezu kojom se pretpostavlja da se određivanjem najviše dozvoljene cijene lijeka na veliko postiže smanjenje iste za dio lijekova obuhvaćenih postupkom u 2020. godini. Drugi cilj je testirati hipotezu kojom se pretpostavlja da je smanjenje cijene na listi lijekova HZZO-a posljedica smanjenja najviše dozvoljene cijene lijeka na veliko iz godišnjeg izračuna cijena u 2020. godini. Metode istraživanja bazirane su na statističkoj analizi podataka spomenutih lijekova u Microsoft Excel programu, verzija 2016. godina. Kao izvor numeričkih podataka korišteni su izračuni najviših dozvoljenih cijena lijekova na veliko prema jedinstvenom pravilniku za 2019. i 2020. godinu ustupljeni od strane HALMED-a i HZZO-a. Literaturne podatke čine znanstveni članci, knjige i web stranice na temu vanjskog referentnog sustava cijena u Republici Hrvatskoj i svijetu, općeg sustava za regulaciju cijena lijekova u zdravstvenom sustavu, ATK klasifikacije lijekova, lijekova koji djeluju na živčani sustav i dr. Rezultati istraživanja ukazuju na značajan utjecaj najviše dozvoljene cijene lijeka na veliko na smanjenje cijena lijekova s djelovanjem na živčani sustav koji se nalaze i koji se ne nalaze na listi lijekova HZZO-a. Zaključno, vanjski referentni sustav cijena dugoročno možda neće dovesti do zadovoljavajućeg rezultata smanjenja
cijena lijekova ili pak proširene pristupačnosti lijekova krajnjim korisnicima ukoliko regulatorni okviri nisu oblikovani u cilju povećanja uštede javnih izdataka i pristupu učinkovitim terapijama za sve pacijente bez negativnog utjecaja na zdravstveni sustav.Regulatory mechanisms for controlling the prices of medicines are used in most Member States of the European Union. They are one of elements government strategies to limit public expenditure on health care. The system of regulating the prices of medicines in the Republic of Croatia includes prescription medicines. HALMED sets the maximum permitted
wholesale price of a medicine and exceptionally higher than the maximum permitted wholesale price of the medicine. The purpose of the study was to analyze the characteristics of the maximum permitted wholesale price of a medicine for medicines that act on the nervous system. The first aim is to test the hypothesis which assumes that the determination of the maximum permitted wholesale price of a medicine achieves a reduction of the price for some of the medicines covered by the procedure in 2020. The second aim is to test the hypothesis which assumes that the price reduction on the HZZO list of medicines is a consequence of the reduction of the maximum permitted wholesale price of the medicine from the
annual price calculation in 2020. The research methods are based on statistical analysis of data on the mentioned group of medicines in the Microsoft Excel program, version 2016. As a source of numerical data, calculations of the maximum
permitted wholesale prices of medicines according to the unified ordinance for 2019 and 2020, provided by HALMED and HZZO, were used. Literature data include scientific articles, books and websites on the topic of external reference pricing system in the Republic of Croatia and in the world, general system for regulation of drug prices in the health system, ATK
classification of drugs, drugs acting on the nervous system, etc. The research results indicate significant impact of the maximum permitted wholesale price of a medicine on the reduction of the prices of drugs with an effect on the nervous system which are on and aren't on the HZZO list. In conclusion, an external reference pricing system may not lead to a
satisfactory result in lowering drug prices or expanding drug affordability to end-users in the long run if regulatory frameworks are not designed to increase public expenditures and access to effective therapies for all patients without adversely affecting the health system