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Porphyrins and related macrocycles: Combining photosensitization with radio- or optical- imaging for next generation theranostic agents
No full textThis review summarises recent research into combining the photosensitizing properties of porphyrins with imaging techniques such as PET and NIR fluorescence for so called “theranostic” applications, which combine biomedical imaging and therapeutic potential into a single administered substance. The photophysical mechanisms of both the therapeutic and diagnostic properties of porphyrins are discussed, as well as key characteristics that are required in order to deliver the most effective treatment
Protein misassembly and aggregation as potential convergence points for non-genetic causes of chronic mental illness
No full textChronic mental illnesses (CMI), such as schizophrenia or recurrent affective disorders, are complex conditions with both genetic and non-genetic elements. In many other chronic brain conditions, including Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis and frontotemporal dementia, sporadic instances of the disease are more common than gene-driven familial cases. Yet, the pathology of these conditions can be characterized by the presence of aberrant protein homeostasis, proteostasis, resulting in misfolded or aggregated proteins in the brains of patients that predominantly do not derive from genetic mutations. While visible deposits of aggregated protein have not yet been detected in CMI patients, we propose the existence of more subtle protein misassembly in these conditions, which form a continuum with the psychiatric phenotypes found in the early stages of many neurodegenerative conditions. Such proteinopathies need not rely on genetic variation. In a similar manner to the established aberrant neurotransmitter homeostasis in CMI, aberrant homeostasis of proteins is a functional statement that can only partially be explained by, but is certainly complementary to, genetic approaches. Here, we review evidence for aberrant proteostasis signatures from post mortem human cases, in vivo animal work, and in vitro analysis of candidate proteins misassembled in CMI. The five best-characterized proteins in this respect are currently DISC1, dysbindin-1, CRMP1, TRIOBP-1, and NPAS3. Misassembly of these proteins with inherently unstructured domains is triggered by extracellular stressors and thus provides a converging point for non-genetic causes of CMI
Ochratoxin A in the blood of wild boars (Sus scrofa)
No full textOkratoksin A (OTA) je sekundarni metabolit nekih vrsta plijesni iz rodova Aspergillus i Penicillium. Jedan je od najčešće nađenih mikotoksina u žitaricama koje se koriste kao hrana za životinje. Dokazano je nefrotoksičan, imunotoksičan i teratogen u svim do sada ispitivanim pokusnim i domaćim životinjama. Zbog svojeg kancerogenog potencijala Međunarodna agencija za istraživanje raka svrstala je ovaj mikotoksin u skupinu 2B – potencijalno kancerogen za ljude. Budući da u lovištima lovoovlaštenici prihranjuju divlje životinje kukuruzom koji je tijekom skladištenja izložen različitim vremenskim uvjetima koji mogu pogodovati razvoju plijesni, divlje svinje ali i druge divlje životinje mogu biti izložene ovom mikotoksinu.
Istraživanje opisano u ovom radu provedeno je na 36 uzoraka seruma divljih svinja prikupljenih nakon odstrela iz različitih lovišta u 11 županija u Republici Hrvatskoj. Najveći broj uzoraka sakupljen je u Vukovarsko-srijemskoj županiji. OTA je iz seruma ekstrahiran tekućinsko-tekućinskom ekstrakcijom, te analiziran metodom visokoučinske tekućinske kromatografije s fluorescentnim detektorom. U svim ispitivanim uzorcima pronađen je OTA. Njegova srednja vrijednost koncentracije iznosila je 3,02±1,83 ng/mL (min 1,29; max 8,90).
Koncentracija OTA u serumu divljih svinja u ovom istraživanju malo je niža od koncentracije izmjerene u krvi divljih svinja u istraživanjima provedenim u drugim europskim zemljama. Iako je izmjerena koncentracija OTA niska, konzumacija mesa divljih svinja može utjecati na ukupnu izloženosti ljudi ovom mikotoksinu.Ochratoxin A is a secondary metabolite produced by fungi of the Aspergillus and Penicillium genus. It often contaminates food and feed, especially wheat and maize. It is nephrotoxic, immunotoxic and teratogenic in all experimental and domestic animals. The International Agency for Research on Cancer classified OTA as a Group 2 B - possible human carcinogen. Maize is an important part of a wild boar’s diet due to its availability at supplemental feeding sites and the wild boar’s opportunistic feeding habits. This supplemental maize is often of poor quality to begin with and is further adulterated by weather conditions.
In the present study 36 wild boar serum samples were collected from 11 Croatian counties. The largest number of collected samples was from the Vukovarsko-srijemska country. OTA was extracted using liquid-liquid extraction method, immunoaffinity columns and analysed using high performance liquid chromatography with a fluorescence detector. OTA was found in all of the tested samples. Average concentration was 3.02±1.83 ng/mL (min 1.29; max 8.90). This concentration is comparable with similar studies from Europe. Although the measured OTA concentrations were low, they could contribute to overall OTA exposure in humans
Construction of vectors for heterologous expression of ICP34.5 - the main neurovirulence factor of herpes simplex virus 1
No full textHerpes simpleks virus (HSV) je dvolančani DNA virus i važan ljudski patogen koji uzrokuje razne bolesti, od klinički gotovo beznačajnih do po život opasnog encefalitisa. Pokazano je da je virusni protein ICP34.5 potreban za neurovirulenciju u miševa i neurološke bolesti u ljudi. ICP34.5 je uključen u inhibiciju obrane domaćina, inhibiciju autofagije i poremećaj aktivacije IRF3. Iako intenzivno proučavan, funkcija ICP34.5 još uvijek nije potpuno objašnjena. Glavni cilj ovog istraživanja je kloniranje i ekspresija proteina ICP34.5 u bakterijama te afinitetno pročišćavanje proteina u svrhu generiranja visoko specifičnih monoklonalnih antitijela. U tu svrhu, koristeći se tehnikama molekularne biologije klonirali smo gen ICP34.5 koji nosi biljeg FLAG iz eukariotskog ekspresijskog plazmida u bakterijski ekspresijski sustav pET32 koji nosi šest-histidinski biljeg aminokiselina nužnih za afinitetno pročišćavanje. Uspješnost kloniranja i potvrda ispravne DNA sekvence potvrđena je sekvenciranjem. Ekspresija proteina provedena je u bakterijama DE3 te je dobiven proteinski produkt očekivane molekularne mase, međutim razina proteina je bila relativno slaba. Posljedično, afinitetno pročišćavanje rezultiralo je malom količinom proteina. Kako bismo povećali efikasnost proizvodnje proteina ICP34.5 sekvencu gena smo prilagodili prema bakterijskom sustavu (kodon optimizacija) što je značajno utjecalo na razinu proizvedenog proteina. Osim toga, ispitali smo unutarstaničnu lokalizaciju proteina ICP34.5 eksprimiranog s eukariotskog plazmidnog vektora. Iznenađujuće, iako se protein nalazi u citoplazmi - staničnom odjeljku u kojem je opisana glavnina funkcija proteina ICP34.5, većinu proteina uočili smo lokaliziranog u jezgri i strukturama nalik jezgricama. Našim radom generirali smo neophodne vektore i optimizirali heterologni sustav za proizvodnju
proteina ICP34.5 u sklopu šireg projekta generiranja antitijela za ovaj protein, a što će u konačnici rezultirati boljim razumijevanjem biologije ICP34.5.Herpes simplex virus (HSV) is a double-stranded DNA virus and an important human pathogen that causes various illnesses, from clinically almost insignificant to life-threatening encephalitis. It has been shown that viral protein ICP34.5 is required for neurovirulence in mice and neurological diseases in humans. ICP34.5 is involved in the inhibition of host defense, inhibition of autophagy and disruption of IRF3 activation. Although intensively studied, the function of ICP34.5 is still not completely understood.
The main aim of this study is to clone and express ICP34.5 protein from bacteria and to affinity purificate the protein for purpose of generating highly specific antibodies. For this purpose, using molecular biology techniques, we cloned ICP34.5 gene carrying the FLAG marker from the eukaryotic expression plasmid to the bacterial expression system pET32 carrying the six-histidine bilayer of the amino acids necessary for affinity purification. Success of cloning and validation of the correct DNA sequence was confirmed by sequencing. Protein expression was performed in DE3 bacteria and the resulting protein product of the expected molecular weight was obtained, however the protein level was relatively weak. Consequently, affinity purification resulted in a small amount of protein. To increase the efficiency of protein production, the ICP34.5 gene sequence was adapted to the bacterial system (codon optimiozation), which significantly affected the level of protein produced. In addition, we investigated the intracellular localization of the ICP34.5 protein expressed from the eukaryotic plasmid vector. Surprisingly, though the protein is found in the cytoplasm - cell division that describes most of the ICP34.5 protein function, most of the proteins have been found localized in nuclei and nucleolus-like structures. Our work generated the necessary vectors and optimized the heterologous ICP34.5 protein production system as
part of a broader antibody-generating project for this protein, and ultimately result in a better understanding of ICP34.5 biology
Biološki učinak makrolidnih konjugata
No full textAntimikrobno djelovanje makrolida prepoznato je u 20.-tom stoljeću kada su se počeli primjenjivati u liječenju infekcija uzrokovanih bakterijama i gljivicama, a također i liječenju raka. Prvi 15-člani makrolid koji je pokazao terapijske učinke bio je azitromicin. Od postavljanja azitromicina na tržište do danas, otkriven je veliki broj drugih makrolida i makrolidnih struktura različitog spektra djelovanja. No, zbog kontinuiranog korištenja makrolida i nepravilne primjene pojavio se problem rezistentnosti mikroorganizama i na tu grupu antimikrobnih spojeva. Kako bi se riješio taj problem započeta su istraživanja makrolidnih konjugata, odnosno derivata makrolida s ciljem kombiniranja povoljnih karakteristika makrolida uvođenjem dodatnih kemijskih skupina poput esterske, amidne, ureidne, karbamatne, sulfonamidne i dr. Makrolid se također može modificirati na način da se s kinolonom spaja u jedinstvenu cjelinu povezivanjem premosnicom određene duljine. Takav spoj se zove makrolon i pokazao se vrlo uspješnim u liječenju bakterijskih infekcija. Danas se u literaturi može pronaći veliki broj makrolidnih konjugata specifične aktivnosti u određenom terapijskom području, koje nismo zbog opsežnosti materije obuhvatili ovim radom. Cilj je ovog rada obuhvatiti biološki najučinkovitije makrolidne konjugate s antibakterijskim, protutumorskim i antivirusnim učinkom.Antimicrobial effect of macrolides has been recognized in the 20th century when they were used for treatment of infections caused by bacteria and fungi, as well as cancer. Azithromycin was the first fifteen-membered macrolide that showed therapeutic effect. Since placement of azithromycin on market, a large number of other macrolides and macrolide structures with different biological effects was discovered. However, continuous and inadequate usage of macrolides has resulted in antibiotic resistance of microorganisms, even for new generations of antimicrobial compounds. With the aim of addressing that issue, research of macrolide conjugates or macrolide derivates was initiated. The research was steered to combine beneficial macrolide characteristics with additional chemical groups such as ester, amide, ureido, carbamate, sulfonamide, and others. Macrolide can also be modified by attaching quinolone via a linker of specific length. Such compound is named macrolone and it has been proven to be effective for treatment of bacterial infections. Although a large number of macrolide conjugates with specific activities can be found in the literature not all were mentioned within this paper because of the immensity of the data. The main aim was to present only biologically most effective macrolide conjugates with antibacterial, antitumor or antiviral activity
Biophysical insights from a single chain camelid antibody directed against the Disrupted-in-Schizophrenia 1 protein
Get PDFAccumulating evidence suggests an important role for the Disrupted-in-Schizophrenia 1 (DISC1) protein in neurodevelopment and chronic mental illness. In particular, the C-terminal 300 amino acids of DISC1 have been found to mediate important protein-protein interactions and to harbor functionally important phosphorylation sites and disease-associated polymorphisms. However, long disordered regions and oligomer-forming subdomains have so far impeded structural analysis. VHH domains derived from camelid heavy chain only antibodies are minimal antigen binding modules with appreciable solubility and stability, which makes them well suited for the stabilizing proteins prior to structural investigation. Here, we report on the generation of a VHH domain derived from an immunized Lama glama, displaying high affinity for the human DISC1 C region (aa 691±836), and its characterization by surface plasmon resonance, size exclusion chromatography and immunological techniques. The VHH-DISC1 (C region) complex was also used for structural investigation by small angle X-ray scattering analysis. In combination with molecular modeling, these data support predictions regarding the three-dimensional fold of this DISC1 segment as well as its steric arrangement in complex with our VHH antibody
Adverse drug reactions of nonsteroidal anti-inflammatory drugs reported in the period of 2015 to 2018
No full textNesteroidni protuupalni lijekovi (NSAIL-i), osim dobro poznatih korisnih učinaka, mogu uzrokovati ozbiljne nuspojave. Najčešće ozbiljne nuspojave koje uzrokuju klasični NSAIL-i su: nuspojave probavnog sustava, bubrežne i kardiovaskularne nuspojave.
Cilj ovog diplomskog rada je analizirati učestalost i karakteristike prijavljenih nuspojava NSAIL-a te ih usporediti s nuspojavama ostalih lijekova.
Provedena je retrospektivna opservacijska studija nuspojava NSAIL-a prijavljenih HALMED-u u razdoblju od 2015. do 2018. Nuspojave su analizirane s obzirom na dob i spol bolesnika, očekivanost, ozbiljnost, vrstu, ishod, pripadnost nuspojava prema klasifikaciji organskih sustava prema MedDRA-i te prijavitelja, s osvrtom na učestalost nuspojava nesteroidnih protuupalnih lijekova u ukupnom broju prijavljenih nuspojava svih lijekova u promatranom razdoblju. Analiza svih podataka provedena je u programu Excel, a podaci su obrađeni deskriptivnom statistikom.
U razdoblju od 1. siječnja 2015. do 31. prosinca 2017., zaprimljeno je ukupno 512 sumnji na nuspojave NSAIL-a. Najveći broj prijava zaprimljen je za ibuprofen 34,4% (176/512). Udio ozbiljnih nuspojava NSAIL-a iznosio je 25,8% (132/512) u odnosu na sve prijave sumnji na nuspojave NSAIL-a.
Manje od trećine svih nuspojava NSAIL-a ocijenjeno je ozbiljnim, što je podudarno s brojem ozbiljnih nuspojava prijavljenih za sve lijekove. Obzirom na ozbiljnost nuspojava ove skupine lijekova i njihovu široku primjenu, budući da se znatan broj lijekova iz skupine NSAIL izdaje bez recepta, potreban je povećan oprez pri njihovoj uporabi, naročito kod pacijenata starije životne dobi. Također, kako bi se povećala svijest o važnosti prijavljivanja nuspojava te o opasnostima koje predstavlja neadekvatna primjena lijekova, potrebna je stalna edukacija zdravstvenih djelatnika i pacijenata.Nonsteroidal anti-inflammatory drugs (NSAIDs), apart from well known useful effects, could cause serious adverse drug reactions (ADRs). The most common serious ADRs caused by traditional NSAIDs are: gastrointestinal, renal and cardiovascular ADRs.
The aim of this master thesis is to analyse frequency and characteristics of reported NSAIDs' ADRs and to compare them with ADRs of other drugs.
A retrospective observational study of NSAIDs' ADRs, reported to HALMED in the period of 2015 to 2018, was performed. The data were analysed with regard to age and gender of a patient, expectedness, seriousness, type, outcome, distribution to MedDRA system organ class (SOC) and reporter of the ADRs, including the assessment of the NSAIDs' ADRs in reference to the total number of the reported ADRs. Data were analysed using Excel and descriptive statistics.
In the period from 1 January 2015 to 31 December 2017, a total of 512 reports of NSAIDs’ ADRs were received. The highest number of the reports was received for ibuprofen 34,4% (176/512). Percentage of serious NSAIDs’ ADRs was 25,8% (132/512) in relation to the total number of all ADRs reported for NSAIDs.
Of all reported NSAIDs' ADRs, less than a third were rated as serious, which is in accordance with the number of serious ADRs reported for all drugs. Considering the severity of ADRs caused by this group of drugs, its wide usage and their mainly over-the-counter status, an increased vigilance is needed, especially in elderly patients. Also, to raise awareness levels of ADR reporting and the risks associated with inappropriate drug use, continuing education of healthcare professionals and patients is required
Synthesis of 4,5-dihydrobenzo[b]thieno[2,3-d]oxepine derivatives for their anti-inflammatory activity evaluation
No full textU sklopu ovog rada provedena je sinteza 4,5-dihidrobenzo[b]tieno[2,3-d]oksepinskih analoga uključujući amide 6a i b, kiselinu 7, alkohol 8 te pet različitih aminoalkoksi derivata 9a-e. Ključni intermedijari u sintezi ovih spojeva su dva metoksi regioizomera 4,5-dihidrobenzo[b]tieno[2,3-d]oksepinskih estera (5a i 5b). Strukture svih spojeva razjašnjene su na temelju njihovih 1H i 13C NMR spektara. Ispitat će se protuupalna učinkovitost svih heterocikličkih spojeva sa zavinutom strukturom, posebice inhibicija TNF-α i protuupalnih citokina poput IL2 i IFNγ. Dobivena aktivnost će se usporediti sa aktivnošću sličnih spojeva opisanih u literaturi i analozima prethodno dobivenim u našem laboratoriju.Within this thesis synthesis of different 4,5-dihydrobenzo[b]thieno[2,3-d]oxepine derivatives including amides 6a, b, acid 7, alcohol 8 and five different aminoalkoxy derivatives 9a-e was performed. A key intermediates for these compounds were two methoxy regioisomers of 4,5-dihydrobenzo[b]thieno[2,3-d]oxepine esters (5a and 5b). The structures of all prepared compounds were elucidated based on their 1H and 13C NMR spectra. All angular heterocyclic compounds are aimed for testing their anti-inflammatory activity, particularly inhibition of TNF-α and pro-inflammatory cytokines such as IL2 and IFNγ and comparison to the inhibitory activity of similar compounds described in the literature and analogs previously obtained in our group
Analysis of suspected adverse reactions reports from the National base for antihistamines
No full textAntihistaminici za sistemsku primjenu smanjuju ili blokiraju djelovanje histamina kompetitivnim vezanjem za H1-receptor. Najčešće se koriste u liječenju reakcija preosjetljivosti tipa I. Svrha ovog rada je analiza prijava sumnji na nuspojave antihistaminika za sistemsku primjenu (ATK R06) koje su zaprimljene u hrvatskoj Agenciji za lijekove i medicinske proizvode (HALMED) u razdoblju od 1. siječnja 2015. do 31. prosinca 2017. godine. Podaci su analizirani prema: dobi i spolu pacijenta, očekivanosti, ozbiljnosti, povezanosti, vrsti, ishodu, pripadnosti nuspojava prema klasifikaciji organskih sustava Medicinskog rječnika za regulatorne poslove (MedDRA) te prijavitelju s osvrtom na njihovu učestalost u ukupnom broju prijavljenih nuspojava. Od ukupno 161 prijave sumnji na nuspojave najveći broj prijavljen je od strane liječnika i ljekarnika za pacijente ženskog spola te dobnu skupinu od 18 do 44 godine. Od 372 utvrđene nuspojave antihistaminika za sistemsku primjenu najviše ih je zabilježeno za organski sustav (SOC) Opći poremećaji i reakcije na mjestu primjene, SOC Poremećaji živčanog sustava te SOC Ozljede, trovanja i proceduralne komplikacije, a somnolencija i glavobolja su najčešće od njih. 26,1% nuspojava antihistaminika za sistemsku primjenu ocijenjeno je kao ozbiljne, a najučestalije od njih su pokušaj samoubojstva i namjerno predoziranje primjenom loratadina ili desloratadina. Najveći broj neočekivanih nuspojava zabilježen je za loratadin, desloratadin i feksofenadin. U analiziranom razdoblju, sigurnosni signal antihistaminika za sistemsku primjenu (ATK R06) nije detektiran. Rezultati dobiveni ovim istraživanjem u skladu su sa već otprije zabilježenim podacima iz europske (EudraVigilance) i svjetske (VigiAccess) baze nuspojava. Analizom baza podataka uz liječnike, ljekarnici su česti prijavitelji sumnji na nuspojave zbog velikog broja bezreceptnih lijekova prisutnih na tržištu. Iako broj prijava sumnji na nuspojave antihistaminika, ali i drugih lijekova, raste iz godine u godinu potrebno je uložiti dodatan napor kako bi se podigla svijest o važnosti spontanog prijavljivanja nuspojava. Stoga je rad HALMED-a, ali i ostalih
agencija u svijetu, od ključne važnosti za razvoj sigurnije primjene lijekova te sprječavanja nastanka nuspojava. Cilj razvoja antihistaminika za sistemsku primjenu jest povećanje efikasnosti njihove primjene za određenu indikaciju te smanjenje broja nuspojava.Antihistamines for systemic use reduce or block the activity of histamine by competitive binding to the H1 receptor. They are the most commonly used in treating type I hypersensitivity reactions. The purpose of this thesis is to analyze the suspected adverse reactions of antihistamines for systemic use (ATK R06) reported to the Agency for Medicinal Products and Medical Devices of Croatia (HALMED) in the period from 1 January 2015 to 31 December 2017. The data were analysed with regard to age and gender of a patient, type, seriousness, expectedness, outcome, distribution to System organ class (SOC) and reporter of the adverse reactions of antihistamines for systemic use including comparison with the total number of the reported adverse reactions to medicinal products. There were 161 reports of suspected adverse reactions in the analyzed period and the highest number was received from doctors and pharmacists for female patients and age group from 18 to 44 years. Of the 372 identified adverse reactions of antihistamines for systemic use, most have been reported for the System organ class (SOC) General disorders and administration site conditions, SOC Nervous system disorders and SOC Injury, poisoning and procedural complications. Somnolence and headache were the most common adverse reactions. 26,1% of adverse reactions of antihistamines for systemic use were evaluated as serious. Most of them were suicidal attempts and intentionally overdose with loratadine or desloratadine. The highest number of unexpected adverse reactions were reported for loratadine, desloratadine and fexofenadine. During analyzed period, the safety signal of antihistamines for systemic use (ATK R06) was not detected. The results from this study are in accordance with the previously recorded data from european (EudraVigilance) and the worldwide (VigiAccess) base of adverse reactions. According to the databases pharmacists are frequent reporters of adverse reactions due to the high number of nonprescription medicinal products which are present on the market. Although the number of suspected adverse reactions to antihistamines, as well as other medicinal
products, is growing year after year, additional efforts are needed to raise awareness of the importance of spontaneous reporting. Therefore HALMED's work, as well as work of other agencies in the world, is of crucial importance for the development to safer use of medicinal products and the prevention of adverse reactions. The development of antihistamines for systemic use will increase the efficiency of their application for a specific indication and reduce the number of adverse reactions
Autophagy analysis in Neuro 2a cell line with CRISPR/Cas9 mediated optineurin deletion
No full textAutofagija je eukariotski stanični proces kojim se uklanja nepotreban i toksičan unutarstanični sadržaj. Optineurin je ubikvitin vezujući adaptorski protein koji u procesu autofagije veže stanični sadržaj te omogućuje njegovu lizosomalnu razgradnju. Nekoliko istraživanja pokazalo je da nedostatak optineurina u stanicama uzrokuje poremećaj autofagije te time omogućuje nakupljanje proteinskih agregata. Budući da su proteinski agregati obilježje neurodegenerativnih bolesti poput amiotrofične lateralne skleroze (ALS), predloženo je da mutacije optineurina u bolesnika s ALS-om doprinose razvoju bolesti zbog poremećaja autofagije. Međutim, točan mehanizam patogeneze ALS-a kod mutacija optineurina nije poznat jer je potonji multifunkcionalni adaptor u više koraka autofagije, ali i drugim procesima poput regulacije prijenosa upalnih signala i prometa staničnih vezikula.
Za ispitivanje posljedice nedostatka optineurina na tijek bazalne autofagije i autofagije inducirane različitim stimulansima koristili smo staničnu liniju Neuro 2a, u kojoj je pomoću CRISPR/Cas9 metode napravljena delecija gena za optineurin. Pokazali smo kako optineurin nije nužan za induciranje autofagije gladovanjem ili tretmanom s interferonom-β (IFN-β), ali da u stanicama s delecijom optineurina dolazi do blagog nakupljanja markera autofagije p62 i LC3-II te autofagosoma što sugerira da njegov nedostatak može uzrokovati djelomični blok tijeka autofagije. Također, pokazali smo kako nedostatak optineurina uzrokuje nakupljanje LC3-II u nestimuliranim stanicama, što sugerira na djelomični blok tijeka bazalne autofagije. Pritom smo opazili da u stanicama divljeg soja u bazalnoj autofagiji ne dolazi do razgradnje optineurina, što potencijalno sugerira da optineurin nije selektivni adaptor autofagije te da u autofagiji sudjeluje
putem drugih mehanizama. Naši rezultati sugeriraju da optineurin ima ulogu u pokretanju i nesmetanom odvijanju bazalne i inducirane autofagiji te da bi njegov nedostatak mogao biti jedan od uzroka poremećene autofagije i nakupljanja agregiranih proteina u neurodegenerativnim bolestima.Autophagy is a eukaryotic cellular process that removes unnecessary and toxic cellular content. Optineurin is a ubiquitin-binding protein that binds cellular content in the autophagy process and allows its lysosomal degradation. Lack of optineurin causes autophagy related disorders and possible protein aggregation. Since protein aggregates are a hallmark of neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), it is suggested that optineurin mutations in ALS patients lead to disease due to loss of autophagy. However, the exact mechanism of ALS pathogenesis in patients with optineurin mutations is not known. Optineurin is a multifunctional autophagy adaptor, but also has a role in other processes such as inflammation and vesicle trafficking.
To investigate the role of optineurin in basal and induced autophagy, we used Neuro 2a cell line in which optineurin gene was deleted by CRISPR/Cas9 method. We have shown that optineurin is not necessary for inducing autophagy by starvation or IFN-β stimulation, but there was a mild accumulation of autophagy markers p62, LC3-II and autophagosomes in optineurin knockout cells, suggesting that optineurin deficiency can cause partial block of autophagy flow. However, we have shown that lack of optineurin caused the accumulation of LC3-II in non-stimulated cells, suggesting a partial block of basal autophagy flow. In addition, we noticed that there was no optineurin depletion in the wild type cells in basal autophagy, which potentially suggests that optineurin is not a selective autophagy adaptor and that it participates in autophagy through other mechanisms. Our results suggest that optineurin has a role in inducing and proper functioning of basal and induced autophagy and that its deficiency could be one of the causes of disrupted autophagy and the protein aggregation in neurodegenerative diseases