1. Discovery of novel biomarkers with extended non-coding RNA interactor networks from genetic and protein biomarkersGregor Jezernik, Damjan Glavač, Pavel Skok, Martina Krušič, Uroš Potočnik, Mario Gorenjak, 2024, izvirni znanstveni članek Opis: Curated online interaction databases and gene ontology tools have streamlined the analysis of highly complex gene/protein networks. However, understanding of disease pathogenesis has gradually shifted from a protein-based core to complex interactive networks where non-coding RNA (ncRNA) is thought to play an essential role. As current gene ontology is based predominantly on protein-level information, there is a growing need to analyze networks with ncRNA. In this study, we propose a gene ontology workflow integrating ncRNA using the NPInter V5.0 database. To validate the proposed workflow, we analyzed our previously published curated biomarker datasets for hidden disease susceptibility processes and pharmacogenomics. Our results show a novel involvement of melanogenesis in psoriasis response to biological drugs in general. Hyperpigmentation has been previously observed in psoriasis following treatment with currently indicated biological drugs, thus calling attention to melanogenesis research as a response biomarker in psoriasis. Moreover, our proposed workflow highlights the need to critically evaluate computed ncRNA interactions within databases and a demand for gene ontology analysis of large miRNA blocks.
Ključne besede: gene ontology, non-coding RNA, disease pathogenesis
Objavljeno v DKUM: 06.12.2024; Ogledov: 0; Prenosov: 1
 Celotno besedilo (9,14 MB)
Gradivo ima več datotek! Več... |
2. Meta-analytic comparison of global RNA transcriptomes of acute and chronic myeloid leukemia cells reveals novel gene candidates governing myeloid malignanciesStaša Jurgec, Gregor Jezernik, Mario Gorenjak, Tomaž Büdefeld, Uroš Potočnik, 2022, izvirni znanstveni članek Opis: Despite advances in the understanding of genetic risk factors and molecular mechanisms underlying acute myeloid leukemia (AML) and chronic myeloid leukemia (CML), clinical outcomes of current therapies in terms of disease relapse and mortality rate pose a great economic and social burden. To overcome this, the identification of new molecular prognostic biomarkers and pharmacological targets is crucial. Recent studies have suggested that AML and CML may share common pathogenic mechanisms and cellular substrates. To this end, in the present study, global transcriptome profiles of AML and CML at the molecular and cellular level were directly compared using a combination of meta-analysis and modern statistics, and novel candidate genes and specific biological processes associated with the pathogenesis of AML and CML were characterized. Our study significantly improves our current understanding of myeloid leukemia and will help develop new therapeutic targets and biomarkers for disease progression, management and treatment response.
Ključne besede: AML, CML, meta-analysis, lincRNA, spliceosome
Objavljeno v DKUM: 05.12.2024; Ogledov: 0; Prenosov: 0
Celotno besedilo (1,04 MB)
Gradivo ima več datotek! Več... |
3. Identification of novel loci involved in adalimumab response in Crohn’s disease patients using integration of genome profiling and isoform-level immune-cell deconvoluted transcriptome profiling of colon tissueMario Gorenjak, Gregor Jezernik, Martina Krušič, Pavel Skok, Uroš Potočnik, 2022, izvirni znanstveni članek Opis: Crohn’s disease is a consequence of dysregulated inflammatory response to the host’s microbiota. Although anti-TNF treatment improves the quality of the patient’s life, a large proportion of patients lose response to the treatment. The past decade of research has led to a continuum of studies showcasing the heterogeneity of anti-TNF response; thus, the aim of the present study was to dissect transcriptome-wide findings to transcript isoform specific levels and combine the analyses with refined information of immune cell landscapes in colon tissue, and subsequently select promising candidates using gene ontology and genomic integration. We enrolled Slovenian Crohn’s disease patients who were naïve with respect to adalimumab treatment. We performed colon tissue RNA sequencing and peripheral blood mononuclear cell DNA genotyping with a subsequent contemporary integrative approach to combine immune cell deconvoluted isoform transcript specific transcriptome analysis, gene ontology layering and genomic data. We identified nine genes (MACF1, CTSE, HDLBP, HSPA9, HLA-DMB, TAP2, LGMN, ANAPC11, ACP5) with 15 transcripts and 16 variants involved in the adalimumab response. Our study identified loci, some of which were previously shown to contribute to inflammatory bowel disease susceptibility, as novel loci involved in adalimumab response in Crohn’s disease patients.
Ključne besede: Crohn’s disease, adalimumab, transcriptome, isoforms, deconvolution
Objavljeno v DKUM: 05.12.2024; Ogledov: 0; Prenosov: 0
Celotno besedilo (1,65 MB)
Gradivo ima več datotek! Več... |
4. Isoform-level transcriptome analysis of peripheral blood mononuclear cells from breast cancer patients identifies a disease-associated RASGEF1A isoformHelena Sabina Čelešnik, Mario Gorenjak, Martina Krušič, Bojana Crnobrnja, Monika Sobočan, Iztok Takač, Darja Arko, Uroš Potočnik, 2024, izvirni znanstveni članek Opis: Breast cancer (BC) comprises multiple subtypes with distinct molecular features, which differ in their interplay with host immunity, prognosis, and treatment. Non-invasive blood analyses can provide valuable insights into systemic immunity during cancer. The aim of this study was to analyze the expression of transcriptional isoforms in peripheral blood mononuclear cells (PBMCs) from BC patients and healthy women to identify potential BC immune biomarkers. Methods: RNA sequencing and isoform-level bioinformatics were performed on PBMCs from 12 triple-negative and 13 luminal A patients. Isoform expression validation by qRT-PCR and clinicopathological correlations were performed in a larger cohort (156 BC patients and 32 healthy women). Results: Transcriptional analyses showed a significant (p < 0.001) decrease in the ENST00000374459 RASGEF1A isoform in PBMCs of BC compared to healthy subjects, indicating disease-related expression changes. The decrease was associated with higher ctDNA and Ki-67 values. Conclusions: The levels of the RASGEF1A transcriptional isoform ENST00000374459 may have the potential to distinguish between BC and healthy subjects. The downregulation of ENST00000374459 in breast cancer is associated with higher proliferation and ctDNA shedding. Specialized bioinformatics analyses such as isoform analyses hold significant promise in the detection of biomarkers, since standard RNA sequencing analyses may overlook specific transcriptional changes that may be disease-associated and biologically important.
Ključne besede: breast cancer, peripheral blood, isoform-level RNA-seq, RASGEF1A ENST00000374459, ctDNA, Ki-67
Objavljeno v DKUM: 26.11.2024; Ogledov: 0; Prenosov: 0
Celotno besedilo (1,52 MB)
Gradivo ima več datotek! Več... |
5. Izražanje izoform gena DDB2 v mononuklearnih celicah periferne krvi pri različnih podtipih raka dojkeKarin Rajh, 2024, diplomsko delo Opis: Rak dojke (RD) je najpogosteje diagnosticiran rak pri ženskah in je glavni vzrok smrti zaradi raka pri ženskah po vsem svetu. Gre za heterogeno bolezen, ki se razlikuje na molekularni, histološki in klinični ravni. Ločimo več podtipov RD, ki jih delimo na podlagi histopatoloških in molekularnih značilnosti. Podtipa trojno negativni RD in HER2-pozitivni RD (TNRD in HER2+) sta na splošno bolj imunogena in povezana z boljšo odzivnostjo na imunoterapijo kot luminalna podtipa (luminalni A in B).
Analize krvi omogočajo vpogled v sistemski imunski odziv na tumorske celice. Odvzem krvi je lahko dostopna metoda pridobivanja bioloških vzorcev, ki lahko pripomore k zgodnjemu odkrivanju RD. V Centru za humano genetiko in farmakogenomiko Medicinske fakultete v Mariboru so z metodo RNA-seq opravili sekvenciranje transkriptoma imunskih celic iz periferne krvi (mononuklearnih celic periferne krvi, PBMC) pri 25 bolnicah z RD, od tega 13 s podtipom luminalni A in 12 s podtipom TNRD. Z bioinformatsko analizo transkripcijskih izoform so identificirali izoformo ENST00000378603 gena DDB2 kot potencialno pomembno, saj je pokazala značilno diferencialno izražanje med tema dvema podtipoma RD. V nasprotju z omenjeno izoformo pa so se druge izoforme gena DDB2, na primer izoforma ENST00000256996, enako izražale pri podtipih TNRD in luminalni A.
Namen te diplomske naloge je bila validacija ter razširitev teh ugotovitev z analizo izoform gena DDB2 z metodo RT-qPCR na večjem številu vzorcev, ki so vključevali 182 vzorcev bolnic z RD vseh podtipov in 37 vzorcev zdravih oseb. Da bi lahko ovrednotili izražanje izoform gena DDB2 z metodo RT-qPCR, je bilo najprej potrebno optimizirati reakcijske pogoje s specifičnimi oligonukleotidnimi začetniki za obe izoformi. Po optimizaciji reakcijskih pogojev smo z RT-qPCR izmerili izražanje izoforme ENST00000256996 pri bolnicah z RD in zdravih osebah. V nasprotju pa je bila optimizacija reakcijskih pogojev za izoformo ENST00000378603 neuspešna.
Ugotovili smo, da se izoforma ENST00000256996 statistično značilno bolj izraža v vzorcih zdravih oseb kot v vzorcih pacientk z RD, kar je v skladu z našo hipotezo, saj je produkt gena DDB2 udeležen pri popravljanju poškodb DNA, njegova odsotnost pa je povezana s kopičenjem DNA-napak in kancerogenezo. Rezultati so potrdili tudi, da se analizirana izoforma enako izraža v podtipih RD TNRD in luminalni A.
Ključne besede: Rak dojke, TNRD, PBMC, DDB2, transkripcijske izoforme, biooznačevalci
Objavljeno v DKUM: 20.09.2024; Ogledov: 0; Prenosov: 18
Celotno besedilo (2,34 MB) |
6. Optimizacija in vitro stimulacije limfocitov s fitohemaglutininom (PHA) in lipopolisaharidi (LPS) : diplomsko delo univerzitetnega študijskega programa I. stopnjePatricija Počivavšek, 2024, diplomsko delo Opis: Stimulacija limfocitov s fitohemaglutininom(PHA) in lipopolisaharidi(LPS) je ključna metoda za preučevanje imunskih odzivov v eksperimentalnih pogojih. Optimizacija koncentracij omenjenih imunostimulatorjev je bistvena za zagotavljanje fiziološko relevantnih in natančnih rezultatov. V diplomski nalogi smo preučevali odziv limfocitov na različne koncentracije PHA in LPS, ki inducirajo 70% maksimalnega odziva. Pri tem smo prav tako želeli ugotoviti, če najvišje koncentracije PHA in LPS povzročijo celično smrt. Limfocite smo izolirali iz mononuklearnih celic periferne krvi(PBMC) zdravih darovalcev in jih gojili v prisotnosti različnih koncentracij PHA in LPS. Proliferacijo celic smo merili s pomočjo barvila alamarBlue in določili celično viabilnost z uporabo pretočne citometrije. Rezultati so pokazali, da je PHA pri vmesni koncentraciji 1-2,5 μg/mL učinkovit stimulator za doseg 70% maksimalnega odziva limfocitov, hkrati pa najvišje koncentracije PHA povzročajo celično smrt. Nasprotno je bil LPS neuspešen zaradi velike variabilnosti med donorji. Naše ugotovitve prispevajo k boljši standardizaciji eksperimentalnih protokolov pri imunoloških raziskavah.
Ključne besede: limfociti, stimulacija, pretočna citometrija, PHA, LPS, optimizacija
Objavljeno v DKUM: 19.09.2024; Ogledov: 0; Prenosov: 18
Celotno besedilo (1,51 MB) |
7. Optimizacija metode obogatitve fosforiliranih peptidov za analizo z masno spektrometrijo : magistrsko deloDoroteja Golob, 2024, magistrsko delo Opis: Dinamična fosforilacija in defosforilacija proteinov sta esencialna regulatorna mehanizma, ki regulirata večino celične fiziologije, zato ni presenetljivo, da so motnje v njunem delovanju pogosto vzrok za nastanek in napredovanje številnih bolezni. Fosfoproteomika je biološko pomembno področje, ki za analizo fosfoproteinov pogosto uporablja tekočinsko kromatografijo, sklopljeno s tandemsko masno spektrometrijo (LC MS/MS). Pred analizo z LC-MS/MS je ključnega pomena obogatitev fosforiliranih peptidov, ki zviša relativno količino ciljnih molekul in s tem poveča občutljivost metode.
V magistrski nalogi smo optimizirali metodo obogatitve fosforiliranih peptidov za analizo z masno spektrometrijo, pri čemer smo izhajali iz nezadovoljivega protokola proizvajalca. Na voljo smo imeli dva obogatitvena materiala: PureCube Fe-NTA MagBeads in PureCube Ti-NTA MagBeads, katerih kapaciteto smo primerjali. Ugotovili smo, da je v naši izvedbi eksperimenta veliko obetavnejša obogatitev s Ti-NTA magnetnimi kroglicami, ki so že na začetku optimizacije ponujale zadovoljiv delež obogatenih fosfopeptidov, medtem ko bi za uporabo Fe-NTA morali bistveno spremeniti sestavo vezavnega pufra. Optimizirali smo razmerje med količino Ti-NTA in maso vhodnih peptidov ter delež glikolne kisline (GA) v vezavnem pufru. Rezultati kažejo, da z večjo količino afinitetnega materiala obogatimo več fosforiliranih peptidov, brez zmanjšanega deleža le-teh v vzorcu, zato smo kot optimalno določili dvakratno količino Ti-NTA glede na prvotni protokol. Nadalje smo ugotovili, da dodatek GA občutno vpliva na specifičnost vezave, vendar večji delež le-te hkrati obogati manj fosfopeptidov. Kompromis med zvišanjem specifičnosti in večjim številom fosfopeptidov smo tako dosegli pri uporabi vezavnega pufra z 0,2 % deležem GA. Na podlagi alternativnih protokolov in teorije smo prilagodili tudi inkubacijski čas vezave in elucije fosfopeptidov. Nadalje smo ugotavljali minimalno maso vhodnih peptidov, iz katere še dobimo zanesljivo kvantifikacijo fosfopeptidov. Ugotovili smo, da je ta lahko veliko nižja kot navaja proizvajalec; minimalno maso smo postavili pri 50 g, saj nižje mase dajejo zelo variabilne in s tem nezanesljive rezultate.
Optimizirano metodo smo testirali na peptidih iz celic, gojenih v različnih pogojih. Analizirali smo fosfoproteom celic, tretiranih s kemoterapevtikom doksorubicin, napram celicam v normalnih rastnih pogojih. Statistična analiza je pokazala, da se je ob izpostavljenosti doksorubicinu količina številinih fosforiliranih peptidov statistično značilno povečala. Uporabljen masni spektrometer se je izkazal za dovolj občutljivega, da te spremembe zazna, optimizirana metoda pa kot učinkovita. Metoda je tako primerna za proučevanje fosfoproteoma, ko imamo opravka z manjšim številom vzorcev, pred širšo uporabo pa bo potrebna dodatna optimizacija.
Ključne besede: proteomika, fosforilacija, obogatitev fosfopeptidov, masna spektrometrija
Objavljeno v DKUM: 12.09.2024; Ogledov: 21; Prenosov: 20
Celotno besedilo (3,68 MB) |
8. Expert meeting report : towards a joint European roadmap to address the unmet needs and priorities of paediatric asthma patients on biologic therapyKornel Golebski, Uroš Potočnik, 2021, pregledni znanstveni članek Opis: Biologics use in severe paediatric asthma
The global prevalence of severe asthma among adolescents ranges from 4% to 11%; and up to 7% of children with asthma display an uncontrolled and severe form that is often associated with a substantial burden on the quality of life of patients and their families, and increasing costs of healthcare [1, 2]. “Childhood asthma” is an umbrella term describing a heterogeneous disease comprising different phenotypes and a wide range of symptoms [3–5].
Despite decades of basic and clinical research, tailored strategies to modify the natural course of asthma, prevent severe exacerbations and inhibit lung function decline are still lacking. In addition, clinical phenotypes are only moderately reliable in the prediction of treatment responses and our current understanding of asthma endotypes is limited. Most asthma endotypes involve concomitant inflammatory pathways and distorted immune parameters. Advances in understanding severe paediatric asthma pathophysiological mechanisms and immunological pathways mediating the airway inflammation would allow better characterisation of these patients as well as optimised intervention, guided by treatable traits and biomarkers [6, 7].
Recent studies have demonstrated the effectiveness of monoclonal antibodies (mAbs), also known as biologics, targeting type 2 inflammation in controlling the symptoms of severe asthma. Currently, four human mAbs are approved for use in children: mAbs that target interleukin (IL)-5 or IL-5 receptor (R) (mepolizumab and benralizumab), mAbs that target IL-4R (dupilumab), and mAbs that target immunoglobulin E (omalizumab). Omalizumab was the first biologic approved to treat moderate-to-severe allergic asthma (≥6 years of age). Mepolizumab and dupilumab have been approved for severe eosinophilic asthma (≥6 and ≥12 years of age, respectively), while benralizumab has been approved in the USA to treat children (≥12 years of age) with severe eosinophilic asthma [8–13].
The introduction of mAb agents in asthma treatment is a milestone in the application of personalised medicine. However, comparative studies and standardised algorithms for the management of paediatric severe asthma to guide the best therapeutic option for paediatric patients with severe asthma are lacking [14]. More personalised medicine approaches may benefit the patient by better matching patients with the most appropriate therapy. Risk stratification, remote monitoring and the integration of multiple data sources could help tailor management for the individual child with severe asthma.
A digital multidisciplinary European expert meeting took place on 9 July 2020. In this workshop, we brought together European respiratory/allergy paediatricians, immunologists, epidemiologists and basic scientists to identify the unmet needs of paediatric severe asthma patients, and set the priorities for clinical and research activities ahead. The participants discussed ongoing initiatives and knowledge gaps, and formulated proposals on how to address these challenges. In this report, we describe the main findings of this expert meeting.
Ključne besede: asthma, paediatric asthma, severe asthma, children, biologics, monoclonal antibodies, biologic therapy, therapy
Objavljeno v DKUM: 14.08.2024; Ogledov: 84; Prenosov: 6
Celotno besedilo (695,54 KB)
Gradivo ima več datotek! Več... |
9. |
10. |
