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Characterization of multidrug-resistant enterobacteria
No full textEnterobakterije su Gram negativne bakterije te komenzali probavnog sustava čovjeka i mnogih životinjskih vrsta. Jedna od brojnih enterobakterija je inkapsulirana vrsta Klebsiella pneumoniae koja uzrokuje mnogobrojne bolničke i izvanbolničke infekcije. Rezistencija na antibiotike pojam je koji podrazumijeva mogućnost daljnjeg dijeljenja bakterijskih kolonija, neovisno o koncentraciji antibiotika. Osim s genetskog stajališta, rezistencija na antibiotike može biti gledana i s biokemijskog stajališta što uključuje smanjenu propusnost membrane, inaktivaciju antibiotika te efluks pumpe. Među najvažnije multirezistentne bakterije spadaju enterobakterije koje proizvode beta laktamaze proširenog spektra, vrste bakterijskih enzima koji hidroliziraju beta laktamske antibiotike, poput penicilina, cefalosporina i karbapenema. U radu su korištena 33 izolata K. pneumoniae dobivenih iz Kliničkog bolničkog centra Rijeka. Fenotipske su metode uključivale izradu antibiograma u kojem su rađeni testovi sinergizma dvostrukim diskom te kombinacije diskova, pomoću kojih se na kraju odredila produkcija beta laktamaza proširenog spektra. Molekularne metode korištene u ovom radu su lančana polimerazna reakcija te Sangerovo sekvencioniranje kojima je određeno prisutstvo gena rezistencije OXA-48, TEM, SHV i CTX-M. Najzastupljeniji gen bio je OXA-48, zatim CTX-M, a geni TEM i SHV jednako su bili zastupljeni. Sangerovim sekvencioniranjem uočene su razlike između CTX-M 15 podtipova beta laktamaze proširenog spektra analizirana u dva različita soja gdje je jedan soj bio kromosomski kodiran, a drugi plazmidno s brojnim insercijama.Enterobacteriaceae are Gram-negative bacteria and commensals in the human digestive system, as well as in many animal species. One of Enterobacteriaceae is encapsulated bacterium K. pneumoniae which causes many hospital infections and infections outside of the hospital. Antibiotic resistance is the ability of bacterial division nonetheless of antibiotic concertation. Apart from a genetic point of view, antibiotic resistance can be considered as biochemical mechanisms which include reduced membrane permeability, antibiotic inactivation and efflux pumps. Among the most important multiresistant bacteria are extended-spectrum beta-lactamase-producing Enterobacteriaceae which produce extended beta-lactamases. Extended beta-lactamases are bacterial enzymes with the ability to hydrolyze beta-lactam antibiotics, e.g. penicillin, cephalosporins and carbapenems. In this thesis, 33 K. pneumoniae isolates from Klinički bolnički centar Rijeka were used. Phenotype methods that were used were antibiograms, including a double-disk synergy test and a combination disk test, which, in the end, showed the production of extended-spectrum beta-lactamases. Molecular methods that were used were polymerase chain reaction and Sanger’s sequencing showing the presence of resistance genes OXA-48, TEM, SHV and CTX-M. The most common gene was OXA-48 and then CTX-M, while genes TEM and SHV were equally common. Sanger’s sequencing showed differences between subtype extended-spectrum beta-lactamase, CTX-15, analyzed in two different strains. One of them was chromosomally encoded, while the other one was plasmid with a lot of insertions
Usporedba različitih FYVE domena i anti-PI3P protutijela za praćenje lokalizacije PI3P-a u BALB 3T3 stanicama
No full textPhosphoinositides are phosphorylated membrane lipids that control the intracellular membrane traffic and have a role in cell signaling. Phosphatidylinositol 3-phosphate (PI3P) is a key regulator of membrane and vesicular trafficking localized primarily on the early and late endosomal membranes where it regulates endosomal maturation. The major source of PI3P is the class III phosphoinositide 3-kinase vacuolar protein sorting 34 (Vps34). In the cells, PI3P can be visualized using antibodies and/or protein domains with a specific PI3P-binding property, such as FYVE or PX.
In this thesis we tested the validity of three FYVE domain-containing probes (GFP-FYVE EEA1, GFP-2xFYVE EEA1 and GFP-2xFYVE Hrs), and anti-PI3P antibody for the detection of PI3P in BALB 3T3 cells. Our results showed that all three probes and the antibody can be used to visualize cytoplasmic PI3P that was mostly confined to the early and late endosomes/lysosomes. In addition to endo-lysosomal PI3P in the cytoplasm, we also detected PI3P in the nucleus and nucleolus. We visualized nuclear PI3P with all of the probes and the antibody, while nucleolar PI3P could be visualized only with GFP-2xFYVE EEA1 and GFP-2xFYVE Hrs which have a higher PI3P-binding affinity. Furthermore, by inhibiting Vps34, we showed that all the probes and the antibody were highly specific for PI3P, and confirmed that Vps34 is a major source of PI3P within the cell. Next, we observed that the simultaneous inhibition of Vps34 and the overexpression of high affinity probes slightly increased heterochromatin levels in the nucleus. Finally, we investigated the expression of nucleolar markers UBF, B23 and fibrillarin in human megakaryoblastic leukemia cell line (Dami) and found that their expression remains mostly unchanged during Dami cells maturation. These data indicate that PI3P may be involved in different nuclear processes. Also, these results open new questions about the role of PI3P in the process of chromatin remodeling, which is a subject of future research.Fosfoinozitidi su fosforilirani membranski lipidi koji imaju ulogu u unutarstaničnom membranskom prometu te staničnoj signalizaciji. Fosfatidilinozitol 3-fosfat (PI3P) je ključni regulator membranskog i vezikularnog prometa lokaliziran uglavnom na membranama ranih i kasnih endosoma gdje kontrolira njihovo sazrijevanje. Glavni izvor PI3P-a je Vps34 kinaza, fosfoinozitid 3-kinaza klase III. Unutar stanica, PI3P može biti vizualiziran korištenjem protutijela i/ili proteinskih domena sa svojstvom specifičnog vezanja na PI3P, kao što su FYVE ili PX domene.
U ovom radu ispitali smo učinkovitost vizualizacije PI3P-a u BALB 3T3 staničnoj liniji korištenjem različitih proba koje sadrže FYVE domenu (GFP-FYVE EEA1, GFP-2xFYVE EEA1 and GFP-2xFYVE Hrs) i anti-PI3P protutijela. Naši rezultati pokazali su da se sve probe i protutijelo mogu upotrijebiti za uspješnu vizualizaciju PI3P-a u citoplazmi te da je lokaliziran na membranama ranih i kasnih endosoma/lizosoma. Uz endolizosomalni PI3P u citoplazmi, vizualizirali smo i PI3P lokaliziran u jezgri te jezgrici stanica. Nuklearni PI3P vizualiziran je ekspresijom svih navedenih proba te protutijelom, dok je PI3P u jezgrici bilo moguće vizualizirati samo s GFP-2xFYVE EEA1 and GFP-2xFYVE Hrs probama koje imaju viši afinitet vezanja za PI3P. Nadalje, inhibicijom Vps34 kinaze pokazali smo da su i probe i protutijelo visoko specifični za PI3P te potvrdili da je Vps34 glavni izvor sinteze PI3P-a. Dodatno, pokazali smo da istodobna inhibicija Vps34 kinaze i ekspresija visoko afinitetnih proba blago povećava razinu heterokromatina u jezgri. Konačno, istražili smo ekspresiju nukleolarnih markera UBF-a, B23 i fibrilarina u ljudskoj megakarioblastičnoj staničnoj liniji (Dami) i uočili da se njihova ekspresija ne mijenja tijekom sazrijevanja Dami stanica. Ovi podaci upućuju na potencijalnu ulogu PI3P-a u različitim procesima u jezgri. Također, ovi rezultati otvaraju nova pitanja o ulozi PI3P-a u procesu kondenzacije kromatina što će biti predmet budućih istraživanja
LC-MS/MS analysis of the fenolic content from olive leaf tea cultivars Istarska bjelica, Leccino and Buža, with the aim of optimization of a fuctional product
No full textPoznato je da fenoli maslinovog ulja imaju širok raspon pozitivnog djelovanja, ali je manje poznato da se te blagotvorne komponente nalaze i u ostalim dijelovima stabla masline. Upravo stoga, ovaj je rad usmjeren na analizu lišća maslina. Nedavna istraživanja sastava maslinovog lišća pokazala su kako se u lišću masline nalazi visoka količina polifenola, čime se može objasniti prethodno poznata antibakterijska, antivirusna i antimikrobna aktivnost. Cilj diplomskog rada bio je utvrđivanje potencijala lišća sorti masline Buža, Istarska Bjelica i Leccino, kao prirodnog izvora polifenola pogodnih za svakodnevnu konzumaciju u obliku čaja.
Specifično se pratio utjecaj sorte masline, vremena i temperature maceracije na sadržaj polifenola. Na temelju dobivenih rezultata provedena je optimizacija pripreme čaja. Razina uspješnosti optimizirane metode utvrđena je s organskom ekstrakcijom u metanolu. Analiza polifenolnog sadržaja provedena je na masenom spektrometru s trostrukim kvadropolom (LC-QQQ). Naposljetku, uz pomoć Design-Expert softvera dizajnirane su mješavine čaja na temelju željenog sastava polifenola.
Rezultati su pokazali utjecaj sorte masline i načina pripreme na kvantitativni i kvalitativni sastav polifenola. Najviša količina ukupnih fenola pronađena je u uzorcima čaja Istarske bjelice u čijoj se pripremi koristila kipuća voda, uz maceraciju od 15 min. Uzorci sorti Buža i Leccino, iako su sadržavali manje ukupne količine polifenola, istaknuli su se po raznolikosti polifenolnog sastava. Ovim radom dobili smo važne spoznaje o utjecaju sorte masline na kvantitativni i kvalitativni fenolni sastav. Opis monosortnih uzoraka čaja omogućuje daljnji razvoj funkcionalnog čaja s blagotvorinim učincima na zdravlje.Beneficial properties of polyphenols are widely recognized, and polyphenols-rich olive oil has been identified as one of essential food for healthy effect of Mediterranean diet. However, over the past decade, the olive leaves have been discovered as an alternative rich source of polyphenols as well. Recent studies on olive leaves confirmed a high phenolic content that explain their previously described strong antibacterial, antimicrobial and antiviral activity. Therefore, the major aim of our work was to comprehensively determine olive leaves phenolic content in cultivars Istarska bjelica, Leccino and Buža, as natural source of bioactive compounds suitable for daily consumption in the form of tea.
For this purpose, we examined the influence of olive leaf variety, maceration time and temperature on the final tea phenolic composition. Based on the obtained results, the method optimization was carried out. The level of success of the optimized method was determined with the organic extraction in methanol. Phenolic compounds were analysed by use of liquid chromatography coupled to triple quadrupole mass spectrometer (LC-QQQ). Finally, using the Design-Expert software, tea mixtures where designed based on the desired polyphenol composition.
Expectedly, results revealed significant influence of the olive variety, but also maceration parameters on the quantitative and qualitative phenolic composition. The highest amount of phenolic compounds was obtained for Istarska bjelica after 15 min maceration. Samples of Buža an Leccino, although they contained less total amount of polyphenols, were distinguished by variety of the content. Furthermore, we designed several functional olive leaf teas mixtures with phenolic composition adjusted to the desired health effect. Results pinpoint the role of optimized phenolic composition in design and improvement of olive leaves teas quality
Validation of immunohistochemical markers on Monodelphis domestica cortical tissue cells
No full textKorteks čini najveći dio volumena mozga te se ovdje procesuira većina pristiglih informacija. Istražujući tijek razvoja mozga, nailazi se na veliki broj neodgovorenih pitanja. Bolje razumijevanje tog perioda dalo bi potpuniju sliku o funkcijama i mehanizmima koji se događaju u mozgu, a nova saznanja o neuralnim matičnim stanicama mogu se pokazati iznimno korisna u terapiji neuroloških bolesti i ozljeda. Jedan od načina identifikacije neuralnih matičnih stanica, kao i zrelih stanica, je pomoću imunohistokemijskih markera. U ovom istraživanju testiramo markere SOX2, SOX9, GFAP, NeuN, MBP, Iba1, Ki67 na korteksima novorođenih oposuma (Monodelphis domestica). Testiranje je provedeno koristeći dvije metode, imunohistokemiju (IHC) te izotropni frakcionator (IFR). U sklopu IHC metode, korišteni su tanko narezani poprečni presjeci korteksa, dok je za IFR korištena homogena smjesa jezgri stanica korteksa. U obje metode izvršena su imunooznačavanja sa spomenutim markerima te vizualizacija pomoću fluorescentnog mikroskopa. SOX2+ i SOX9+ stanice kolokalizirane su u području ventrikula. Postoji velika razlika u broju SOX2+ i SOX9+ stanica u ostatku korteksa, u korist SOX2+ stanica. Ta se razlika s vremenom smanjuje, što upućuje na početak astrogeneze. GFAP+ stanice su jasno vidljive tek kod nešto starijih oposuma te su uglavnom koekspimirane sa SOX9, dok kod mlađih oposuma to nije bio slučaj. Mlađi oposumi imali su veliki broj NeuN+/SOX2+ stanica što bi upućivalo da se radi o mladim neuronima kod kojih dolazi po sporijeg utišavanja ekspresije SOX2 kao moguća posljedica produljene neurogeneze oposuma. MBP nije dao informacije o prisutnosti oligodendrocita, također, marker proliferacije, Ki67, nije se pokazao dovoljno specifičan. Suprotno tome, Iba1 marker jasno je označio mikroglije već kod najranije testirane dobi oposuma. IHC metodom je ispitana valjanost markera koji su pomogli u identifikaciji i lokalizaciji stanica korteksa mladih oposuma. IFR metoda je dodatno trebala pomoći u kvantifikaciji stanica, no dobiveni podaci nisu pouzdani. Nema studija koje bi potvrdile točnost tih podataka.The cerebral cortex makes up the largest part of the brain's volume, and most of the information received is processed here. Exploring the course of brain development, one encounters a number of unanswered questions. A better understanding of this period would give a more complete picture of the functions and mechanisms occurring in the brain, and new insights into neural stem cells may prove extremely useful in the treatment of neurological diseases and injuries. One way to identify neural stem cells, as well as mature cells, is by using immunohistochemical markers. In this study, we test the markers SOX2, SOX9, GFAP, NeuN, MBP, Iba1, Ki67 on cortices of newborn opossums (Monodelphis domestica). Testing was performed using two methods, immunohistochemistry (IHC) and isotropic fractionator (IFR). As part of the IHC method, thinly sliced cross sections of the cortex were used, while for IFR a homogeneous mixture of cortex cell nuclei was used. In both methods, immunolabeling with the mentioned markers and visualization using a fluorescent microscope were performed. SOX2+ and SOX9+ cells are colocalized in the ventricular area. There is a large difference in the number of SOX2+ and SOX9+ cells in the rest of the cortex, in favor of SOX2+ cells. This difference decreases over time, suggesting the onset of astrogenesis. GFAP+ cells are clearly visible only in slightly older opossums and are mostly coexpressed with SOX9, while in younger opossums this was not the case. Younger opossums had a large number of NeuN+/SOX2+ cells, which could indicate that these are young neurons that have a slower silencing of SOX2 expression as a possible consequence of prolonged neurogenesis of the opossum. MBP did not provide information on the presence of oligodendrocytes, also, the proliferation marker, Ki67, did not prove to be specific enough. In contrast, the Iba1 marker clearly marked microglia already at the earliest tested age of the opossum. The validity of markers that helped in the identification and localization of cortex cells of young opossums was examined by the IHC method. The IFR method was supposed to further aid in cell quantification, but the data obtained is not reliable. There are no studies to confirm the accuracy of this data
Causes of nitrosamine contamination (N-Nitrosodimethylamine, N-Nitrosodiethylamine) in drugs and their impact on the supply of the market in the Republic of Croatia
No full textU lipnju 2018. godine regulatorne agencije su postale svjesne pojave onečišćenja N-nitrozodimetilaminom (NDMA) u djelatnoj tvari valsartan. Nakon početnih analiza, utvrđeno je da je ovim onečišćenjem obuhvaćena skupina lijekova blokatori angiotenzin II receptora koji u svojoj strukturi imaju tetrazolni prsten. Kako je NDMA mogući ljudski kancerogen, došlo je do povlačenja određenih serija lijekova s djelatnom tvari valsartan u svijetu i u Republici Hrvatskoj. Sve povučene serije lijekova su imale djelatnu tvar proizvođača Zhejiang Huahai Pharmaceuticals (ZHP), Linhai, Kina. Kasnije je pokazano da je promjena proizvodnog postupka za djelatnu tvar dovela do onečišćenja s NDMA, što je bio početak preispitivanja dosadašnje farmaceutske prakse i ocjene sintetskih procesa. Nedugo nakon otkrivanja NDMA, N-nitrozodietilamin (NDEA) i drugi nitrozamini su u različitim koncentracijama detektirani u pojedinim serijama lijekova iz skupine sartana te u pojedinim serijama metformina i ranitidina. Stoga je bilo potrebno utvrditi moguće uzroke nastanka nitrozamina i rizik kojemu su pacijenti bili potencijalno izloženi. Objavljene su dozvoljene maksimalne razine detekcije i kvantifikacije te razvijene metode analize nitrozaminskih onečišćenja, dan je rok proizvođačima djelatnih tvari koje su zahvaćene i nositeljima odobrenja za stavljanje lijeka u promet unutar kojeg moraju provesti procjenu rizika i osigurati nove strategije provjere kakvoće proizvedenih serija djelatne tvari. U ovom radu su navedeni potencijalni načini nastanka nitrozamina u lijekovima prema informacijama dostupnim na stranicama regulatornih agencija kao što su EMA, nacionalnih agencija država članica EU, FDA, Swissmedic i drugih. Prikazano je i utjecaj nitrozaminskih onečišćenja na opskrbu tržišta Republike Hrvatske te objašnjeni pojmovi bitni za razumijevanje rada.In June 2018, regulatory agencies became aware of contamination of the active substance valsartan with N-nitrosodimethylamine (NDMA). After initial analyzes was discovered thath the group of drugs found to be affected is angiotensin II receptor blockers, which have a tetrazole ring in their structure. As NDMA is a possible human carcinogen, a series of drugs has been withdrawn worldwide, as well as in the Republic of Croatia. What the withdrawn batches of drugs had in the first moments in common is active pharmaceutical ingredient manufactured by Zhejiang Huahai Pharmaceuticals (ZHP), Linhai, China. It was later shown that the change in their manufacture process led to contamination with NDMA, but the mentioned event was the beginning of a review of the current pharmaceutical practice and evaluation of synthetic processes. Shortly after the discovery of NDMA, N-nitrosodiethylamine (NDEA) and other nitrosamines were detected in various concentrations in individual batches of sartans, metformin, and ranitidine. Therefore, it was necessary to determine the possible causes of nitrosamine and the risk to which patients were potentially exposed. Permitted maximum levels of detection and quantification have been published, and methods for the analysis of nitrosamine contaminants have been developed, a deadline is given for the manufacturers of the active pharmaceutical substances concerned and the marketing authorization holders within which they must review to carry out a risk assessment and ensure the existence of batch control strategies for the active pharmaceutical substance.This paper lists potential ways of creating nitrosamines in drugs according to the information available on the websites of regulatory agencies such as the EMA, FDA, Swissmedic and so on. The impact of nitrosamine contaminants on the supply of the market of the Republic of Croatia is also presented, and the terms important for understanding the paper are explained
Inhibicija JAK/STAT signalnog puta u liječenju astme
No full textAstma je kronična opstruktivna bolest dišnih puteva karakterizirana simptomima: teškog disanja („zviždanja“), gubitka daha, kašalja, stezanja u prsima i proizvodnji sputuma. Astma je globalna bolest koja neravnomjerno pogađa ljude neovisno o rasi, dobi, spolu etničkoj pripadnosti ili geografskoj lokaciji. Procjenjuje se da između 2-18% populacije u svijetu boluje od astme.
ISAAC program je prva globalna studija prevalencije astme čiji su zabrinjavajući podaci doveli do stavljanja velikog fokusa na astmu početkom 21.st. Etiologija astme ovisi o dva glavna čimbenika: genetici i okolišu. Njihovo preplitanje daje široku vanjsku i unutarnju fenotipizaciju bolesti kao i kompliciranu kasifikaciju težine kliničke slike. Postoji niz obrazaca GINA-e za dijagnosticiranje pojedinog tipa astme kao i propisane dozirane terapije. Današnja dijagnostika astme temelji se na funkcijskim plućnim testovima i širokom spektru bronhoprovokacijskih testova uz korištenje modernih alata poput biomarkera i OMIC metoda u identificiranju specifičnog patofiziološkog mehanizma upale. Najčešći vanjski uzročnici astme su: alergeni, polutanti te mikroorganizmi (posebno virusi i bakterije). Alergijska astma najrašireniji je tip astme s najvećim trendom rasta oboljelih (osobito djece). Genetika astme proučavana je: studijama povezanosti, asocijacijskim studijama (GWAS) i epigenetskim studijama. Astmatska upala dijeli se na Th1 i Th2 tip odgovora. Dok Th1 odogovor nije toliko poznat, Th2 odgovor je proučen s etiološke i
terapeutske strane. U Th2 odgovoru sudjeluje mnoštvo imunoloških stanica, a najveći utjecaj imaju: T-limfociti, eozinofili i mastociti. Stanice glatkog mišićja, epitela i subepitela dišnih puteva najpogođenija su tkiva u astmatskoj upali u kojima sudjeluju proupalni citokini što za posljedicu ima zatvaranje dišnih puteva i limitirani protok zraka. JAK/STAT signalni put je od iznimne
važnosti u astmatskim upalama. Vezanjem liganda za receptor pokreće se inicijacijski signal-multimerizacijom receptora, pokreće se kaskadni signalni put fosforilacije JAK kinaze koji uzrokuje aktivaciju STAT transkripcijskog čimbenika ključnog u transkripcijskoj ekspresiji citokin-inducirajućih gena. Poremećaj ovog signala u astmi dovodi do snažne migracije imunoloških
stanica i proizvodnje citokina u bronhima. Jak-inhibitorima nastoji se spriječiti inicijacija upale uzrokovane aktivacijom JAK/STATsignalnog puta. Taj pristup je drugačiji, od dosadašnjih terapija koje su uglavnom riješavale simptome.
Trenutno poznati Jak inhibitori u kliničkim, pretkliničkim ili patentnim aplikacijama su: Theravance: TD-8236, Rigel/AstraZeneca: R256/AZD0449, AstraZeneca: „Example 35“, Almirall: LAS194046 , Vectura: VR588, Merck: iJAK-001 i IJC-1, Genentech: iJAK-381 i GDC-0214 te PM-43I kao jedini STAT inhibitor.Asthma is a chronic obstructive airways disease characterized by symptoms: shortness of breath ("whistling"), loss of breath, coughing, chest tightness and sputum production. Asthma is a global disease that affects people unevenly
regardless of race, age, gender, ethnicity, or geographic location. It is estimated that between 2-18% of the world's population suffers from asthma. The ISAAC program is the first global study of asthma prevalence whose worrying data led to a major focus on asthma in the early 21st century. The etiology of asthma depends on two main factors: genetics and the
environment. Their intertwining gives a broad external and internal phenotyping of the disease as well as a complicated classification of the severity of asthma. There are a number of GINA patterns for diagnosing a particular type of asthma as well as the prescribed dosage therapy for it. Today’s diagnosis of asthma is based on functional lung tests and a wide range
of bronchoprovocation tests with the assistance of modern tools such as biomarkers and OMIC methods in identifying the specific pathophysiological mechanism of inflammation. The most common external causes of asthma are: allergens, pollutants and microorganisms (especially viruses and bacteria). Allergic asthma is the most common type of asthma with the highest growth trend in patients (especially children). Asthma genetics have been studied in: association studies, association studies (GWAS), and epigenetic studies. Asthmatic inflammation is divided into Th1 and Th2 type responses.
While the Th1 response is not so well known, the Th2 response has been studied from the etiological and therapeutic side. Many immune cells participate in the Th2 response, and the most influential are: T-lymphocytes, eosinophils and mast cells. Smooth muscle, epithelial, and subepithelial cells of the airways are the most affected tissues in asthmatic inflammation,
mediated by proinflammatory cytokines, causing airway closure and limited airflow.
The JAK / STAT signaling pathway is crucial for asthmatic inflammation. The receptor-ligand initiation signal initiates receptor multimerization, consequetnly activating a cascade pathway of JAK kinase phosphorylation that causes activation of the STAT transcription key in the transcriptional expression of cytokine-inducing genes. Disruption of this signal in asthma leads to strong migration of immune cells and production of cytokines in the bronchi. Jak-inhibitors in asthma aim to prevent the very initiation of JAK / STAT inflammation, unlike previous symptomatic therapies. Currently known strong inhibitors in clinical, preclinical or patent applications are: Theravance: TD-8236, Rigel / AstraZeneca: R256 / AZD0449, AstraZeneca: "Example 35", Almirall: LAS194046, Vectura: VR588, Merck: iJAK-001 and IJC- 1, Genentech: iJAK-381 and GDC-0214 and PM-43I as the sole STAT inhibitor
Optineurin u neurodegenerativnim bolestima
No full textOd otkrića optineurina do danas pronađene su brojne mutacije u optineurinu koje se povezuju s nastankom nekoliko različitih bolesti: glaukomom, amiotrofičnom lateralnom sklerozom (ALS) i frontotemporalnom demencijom (FTD). Nadalje, poliformizmi koji dovode do smanjene ekspresije optineurina povezani su s Pagetovom bolesti kostiju i upalnom bolešću crijeva. Optineurin sudjeluje u brojnim staničnim procesima u kojima mu se pridaje protektivna uloga. Ovim radom bit će sažeta uloga optineurina u neuroinflamaciji, autofagiji, procesima staničnog transporta i nekroptozi, s istaknutim fokusom na utjecaj mutacija u optineurinu na neurodegeneraciju, odnosno povezanost s mehanizmima nastanka ALS-a i glaukoma. Mutacije u optineurinu dostatne su da naruše homeostazu staničnih procesa u kojima sudjeluje. Zanimljivo je da ALS i FTD uzrokuju iste, a glaukom različite mutacije u optineurinu, sugerirajući preklapanje patogeneze ALS-a i FTD-a te različitu patogenezu glaukoma.Since the discovery of optineurin, numerous mutations in optineurin have been found to be associated with the onset of several different diseases: glaucoma, amyotrophic lateral sclerosis (ALS), and frontotemporal dementia (FTD). Furthermore, polymorphisms leading to decreased optineurin expression have been associated with Paget’s disease of bone and inflammatory bowel disease. Optineurin participates in a number of cellular processes in which it was assigned a protective role. This paper summarizes the role of optineurin in neuroinflammation, autophagy, cell trafficking and necroptosis. A prominent focus is the impact of mutations in optineurin on neurodegeneration, or their relationship with pathogenesis of ALS and glaucoma. Mutations in optineurin are sufficient to disrupt the homeostasis of cellular processes in which it participates. Interestingly, ALS and FTD are caused by the same mutations, whereas glaucoma is caused by different mutations in optineurin, suggesting an overlapping pathogenesis of ALS and FTD, and distinct pathogenesis of glaucoma
Macrolides in treatment of bacterial and viral infections
No full textMakrolidni antibiotici su makrociklički spojevi koji u svojoj strukturi sadrže jedan ili dva šećera. Gotovo svi makrolidni antibiotici koje danas koristimo za liječenje su derivati prirodno izoliranih makrolida iz biljaka, gljiva, aktinobakterija i kukaca. Danas postoje brojni derivati makrolida koji s obzirom na modifikacije pripadaju različitim skupinama makrolida, ali klinički najznačajniji makrolidi su oni sa 14-, 15- ili 16- članim makrocikličkim prstenom. Makrolidi su općenito pokazali široki spektar djelovanja, od protuupalnog, imunomodularnog, antifungalnog, antiparazitskog i antitumorskog, ali daleko najizraženijim antibakterijskim djelovanjem. Antibakterijsko djelovanje makrolidnih antibiotika se ispoljava vezanjem unutar izlaznog tunela peptida 50S ribosomske podjedinice čime se ometa sinteza proteina bakterija. Uz antibakterijsko djelovanje, makrolidni antibiotici su se pokazali klinički korisnima i u borbi protiv virusa. Tako je na primjer azitromicin pokazao in vitro antivirusno djelovanje protiv zika- i rinovirusa. Time je azitromicin postao zanimljiv kao jedno od mogućih rješenja za aktualnu pandemiju uzrokovanu SARs-CoV-2 koronavirusom. Iako antivirusno djelovanje azitromicina protiv SARs-CoV-2 koronavirusa nije potvrđeno, postoje naznake kako bi u kombinaciji s hidroksiklorokinom mogao biti učinkovit. U ovome radu nastojali smo reakcijom transamidacije derivata
makrolida (24) koji sadrži aminopropilni lanac i derivata glukoze (26) sa povoljno smještenom amidnom skupinom pripraviti novi derivat makrolida (27) uvođenjem glukoze u položaj N(9a) makrocikličkog prstena.Macrolide antibiotics are macrocyclic compounds that contain one or two sugars in their structure. Almost all macrolide antibiotics that are curently used in clinics are derivatives of naturally isolated macrolides from plants, fungi, actinobacteria and insects. Numerous macrolide derivatives are reported so far in the literature but clinically most significant macrolides are those having a 14-, 15- or 16- membered macrocyclic ring. Macrolides have generally shown a wide range of biological activity, such as anti-inflammatory, immunomodulary, antifungal, antiparasitic and antitumor, but the most pronounced is definitely antibacterial activity. Macrolide antibiotics exert their antimicrobial activity through binding within the peptide exit tunnel of the bacterial 50S ribosomal subunit peptide and interfering with bacterial protein synthesis. In addition to bacterial activity, macrolides have also shown to be clinically useful in fighting virus pathogens. For example, azithromycin
showed in vitro antiviral activity against Zika and Rhino viruses. Thus, azithromycin has become interesting in assessing its potential to treat infections caused by SARs-CoV-2 coronavirus. Although the antiviral acitivity of azithromycin against SARs-CoV-2 coronavirus has not been confirmed, there are indications that it may be effective in combination
with hydroxychloroquine. In this paper, we tried to prepare a new structuraly unique macrolide derivative (27) by introducing a glucose to a N(9a) position of the macrocyclic ring using the reaction of transamidation of a macrolide derivative (24) containing an aminopropyl chain and a glucose derivative (26) with an incorporated amide group
In vitro karakterizacija TNF-om posredovane programirane stanične smrti u stanicama s deficijencijom optineurina
No full textIn recent years neuroinflammation was found to be a major contributor to disease progression in neurodegenerative disorders such as amyotrophic laterals sclerosis (ALS). Inflammatory cytokines like tumor necrosis factor (TNF) can push cells into programmed cell death, such as apoptosis or necroptosis, which are mediated by receptor-interacting protein kinase 1 (RIPK1). It is not entirely clear what pushes cells to different forms of cell death. A recent study proposed that RIPK1 turnover is mediated by an adaptor protein optineurin, which is included in various cellular signaling and trafficking processes. Loss of optineurin was reported to be associated with enhanced motor neuron death and axonal degeneration, both of which are part of ALS pathology. However, this was not consistently reported in other studies, so the exact mechanism of action of optineurin in neuroprotection is still debated. Here, we focused on characterizing TNF-mediated programmed cell death in various optineurin-deficient cell lines. To address the role of optineurin in apoptosis and necroptosis, we first generated a novel mouse fibroblast (L929) optineurin knockout (Optn KO) cell line using CRISPR/Cas9 technique. L929 cells have been reported to be permissive to necroptotic cell death, so they were used to set up the assays. We induced necroptosis and apoptosis in wild-type (WT) and Optn KO L929 cells to determine the effect Optn KO on cell survival. Furthermore, we tested if cell types of the central nervous system (CNS), such as microglial (BV2) cells and neuronal (Neuro2A) cells, are dying by necroptosis. Our study confirmed that L929 cells are highly susceptible to necroptotic death. However, the survival rate of WT and Optn KO L929 cells was similar, which differs from previously published results. In BV2 and Neuro2A cells we detected a significant decrease in cell survival upon stimulating apoptosis and necroptosis, which was not rescued by caspase or RIPK1 inhibition. Notably, we did not detect any difference between WT and Optn KO BV2 cells, suggesting that optineurin deficiency does not sensitize microglia to TNF-mediated apoptosis and/or necroptosis. In addition, our data suggest that another TNF-mediated mechanism may push cells to death, which is independent of caspases or RIPK1.Tijekom posljednjih godina je utvrđeno da neuroinflamacija snažno utječe na progresiju neurodegenerativnih bolesti poput amiotrofične lateralne skleroze (ALS). Upalni citokini poput faktora tumorske nekroze (TNF) mogu usmjeriti stanice u različite oblike stanične smrti uključujući apoptozu i nekroptozu, koje su posredovane protein-kinazom RIPK1 (prema engl. receptor-interacting protein kinase 1). Nije još u potpunosti razjašnjeno što tjera stanice u različite oblike smrti. Nedavna istraživanja predlažu da adaptorski protein optineurin, koji je uključen u niz signalnih i transportnih procesa u stanicama, regulira obrtaj RIPK1. Nedavno je objavljeno da je gubitak optineurina povezan s pojačanom smrti motorničkih neurona i aksonalnom degeneracijom, procesima koji su dio patologije ALS-a. Međutim, ti rezultati su opovrgnuti u nekoliko drugih studija i točan mehanizam optineurina u neuroprotekciji je još nejasan. U ovom radu, naglasak je stavljen na karakterizaciju TNF-om posredovane stanične smrti u različitim staničnim linijama u kojima je deletiran optineurin. Kako bismo analizirali ulogu optineurina u apoptozi i nekroptozi, prvo smo stvorili novu mišju fibroblastnu (L929) optineurin deficijentnu (Optn KO, prema engl. knockout) staničnu liniju pomoću CRISPR/Cas9 tehnologije. L929 stanice su sklone nekroptozi te su iz toga razloga korištene kao pozitivna kontrola. Potaknuli smo nekroptozu i apoptozu u stanicama divljeg tipa (WT) i Optn KO stanicama te promatrali preživljenje stanica. Osim toga, zanimalo nas je da li stanice bitne za središnji živčani sustav umiru nekroptozom, poput mikroglijalne (BV2) i WT neuronalne (Neuro2A) stanične linije. Potvrdili smo da su L929 stanice sklone nekroptotskoj smrti. Međutim, postotak preživljenja WT i Optn KO stanica bio je sličan, suprotno prije objavljenim rezultatima. U BV2 i Neuro2A stanicama smo primijetili značajno smanjeno preživljenje stanica nakon stimulacije apoptoze i nekroptoze, koje se nije poboljšalo nakon inhibicije kaspaza ili RIPK1. Značajno je da nismo otkrili razliku između WT i Optn KO BV2 stanica, što upućuje da deficijencija optineurina ne senzibilizira mikrogliju prema TNF-om posredovanoj apoptozi i/ili nekroptozi. Štoviše, naši rezultati otvaraju mogućnost da postoji još jedan TNF-om posredovan mehanizam koji tjera stanice u smrt, neovisan o kaspazama ili RIPK1
Structure-based design of novel ubiquitin thioesterase OTUB1 inhibitors using supercomputer
No full textZnačaj: Ubikvitinacija je modifikacija u kojoj se na protein kovalentno veže ubikvitin (Ub). Deubikvitinacija je reverzibilni proces ubikvitinaciji, modifikacija u kojoj se uklanja ubikvitin sa proteina. Ravnoteža tih dvaju procesa održava razinu i aktivnost proteina u stanici, a njihova disfunkcija dovodi do nastanka različitih patoloških stanja. Deubikvitinaciju u stanici provode deubikvitinirajući enzimi, kao što je protein ubikvitin tioesteraza OTUB1. Ubikvitin tioesteraza regulira aktivnost proteina p53 i ubikvitinaciju RAS proteina. Zbog povezanosti sa proteinima koji sudjeluju u nastanku tumora, inhibicija ubikvitin tioesteraze u stanici bi mogla biti potencijalna meta u razvoju protutumorskih lijekova.
Rezultati: Virtualnim probirom pronalaze se spojevi koji su komercijalno dostupni. Odabrani spojevi mogu kompetitivno inhibirati ubikvitin tioesterazu zbog svoje strukture i načina vezanja na vezno mjesto ubikvitina na proteinu. Vezno mjesto ubikvitina na proteinu ima aminokiselinske ostatke fenilalanina. Poželjno je da spoj kandidat ima aromatske prstene u strukturi, da bi se na protein mogao vezati π-π stacking interakcijama. Simulacijama molekularne dinamike prikazuje se inhibicija proteina ubikvitin tioesteraze. Koliko je dobra inhibicija proteina ovisi o interakcijama između spojeva i veznog mjesta na proteinu. Računalnim metodama određuju se ADME svojstva spojeva kandidata te pronalaze strukture slične biološke aktivnosti (bioizosteri).
Zaključak: Određeni komercijalno dostupni spojevi mogu inhibirati deubikvitinirajući enzim ubikvitin tioesterazu OTUB1. Inhibitori ubikvitin tioesteraze mogli bi se koristiti kao protutumorski lijekovi. Molekularna dinamika može se koristiti za razvoj takvih kandidata za lijekove.Background: Ubiquitination modifies proteins by covalent attachment of ubiquitin (Ub). Deubiquitination, the reversible process of ubiquitination, removes ubiquitin from the proteins. Balance between these two processes regulates cellular protein levels and activity. Disfunction of ubiquitination and deubiquitination leads to development of pathological conditions in the cell. Deubiquitinases are enzymes that carry out protein deubiquitination. Deubiquitinase ubiquitin thioesterase OTUB1 regulates activity of p53 protein and RAS protein ubiquitination. Cellular inhibition of ubiquitin thioesterase could be a potential target in development of antitumour agents.
Results: Virtual screening of comercially available compounds can be performed using computer aided drug design. Some compounds can competitively inhibit ubiquitin thioesterase OTUB1. Ubiquitin binding site on protein OTUB1 has phenylalanine residues. Therefore, it is preferable that compound has an aromatic ring in its structure. That way it can bind on ubiquitin binding site with π-π stacking interactions. Molecular dynamics simulations show ubiquitin thioesterase inhibition. Interactions between drug candidates and binding site determine how good is the protein inhibition. ADME properties can be defined using computer methods, as well as the chemical compounds with similar structures (bioisosteres).
Conclusion: Certain commercially available compounds can inhibit deubiquitinase ubiquitin thioesterase OTUB1. Inhibitors of ubiquitin thioesterase could be used as antitumour agents. Molecular dynamics can be used to develop such drug candidates