101. The role of focal adhesion kinase in bladder cancer : translation from in vitro to ex vivo human urothelial carcinomasGaja Markovič, Nataša Resnik, Aleksandar Janev, Daša Zupančič, Gašper Grubelnik, Maruša Debeljak, Maja Čemažar, Tanja Jesenko, Maša Omerzel, Tomaž Smrkolj, Mateja Erdani-Kreft, 2025, izvirni znanstveni članek Povzetek: Background: Focal adhesion kinase (FAK), a cytoplasmic tyrosine kinase, plays a crucial role in focal adhesion turnover by interfacing between the extracellular space, transmembrane integrins, and actin filaments. Its significance for the progression of several malignancies, including bladder cancer, has been well-documented. However, its precise role and the implications of its inhibition in bladder cancer tissues and urothelial in vitro models has not been fully explored. This study examined FAK expression and function in human bladder cancer biopsies and in vitro bladder cancer models. Materials and methods: Ex vivo analyses were performed using reverse transcription-quantitative PCR (qRT-PCR), western blotting, and immunohistochemistry to compare FAK expression between bladder cancer tissues and adjacent normal tissues. In vitro, FAK expression was assessed in low-grade (LG) human non-invasive papilloma urothelial cell line RT4 for NMIBC (Ta), high-grade (HG) human muscle-invasive cancer urothelial cell line T24 for MIBC (T2) and normal porcine urothelial (NPU) cells using qRT-PCR and western blotting, as well as flow cytometry for the quantification of FAK-positive RT4 and T24 cells. The role of FAK in cancer cell survival was explored in vitro using microRNA (miRNA) to silence FAK expression. Additionally, we used FAK inhibitors PND-1186, PF-573228 and defactinib to investigate the effects of FAK inhibition on normal compared to cancerous bladder urothelial cells. Results: Ex vivo analyses demonstrated significantly higher FAK expression in bladder cancer tissues compared to adjacent normal tissues. Similarly, in vitro analyses showed significantly higher FAK expression in RT4 and T24 cells than NPU cells. Silencing FAK using anti-FAK plasmids led to increased caspase-3-mediated apoptosis of RT4 and T24 cells and growth reduction of stably transfected T24 cells. Importantly, based on cell viability assays, treatment with 100 μM defactinib for 2 hours per day on 3 consecutive days was identified as a clinically relevant regimen. Under this treatment, the viability of differentiated NPU cells remained high at 108.4 ± 17.1%, while the viability of 2-day RT4 and 2-day T24 cells was drastically reduced to 4.1 ± 2.7% and 7.6 ± 2.9%, respectively. Conclusions: To our knowledge, this is the first report demonstrating the role of FAK and its inhibition across both normal and cancerous bladder urothelial models. This study highlights the critical role of FAK in the progression of human bladder cancer and establishes a foundation for exploring FAK inhibition as a potential therapeutic approach in bladder cancer treatment.
Ključne besede: bladder cancer, defactinib, focal adhesion kinase, human urothelial carcinoma, urothelial cell
Objavljeno v DiRROS: 09.12.2025; Ogledov: 71; Prenosov: 34
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102. Effects of Limosilactobacillus reuteri DSM 17938 in neonates exposed to antibiotics: a randomised controlled trialJana Lozar Krivec, Petra Bratina, Andreja Valcl, Katja Lozar Manfreda, Andraž Petrovčič, Evgen Benedik, Tanja Obermajer, Bojana Bogovič Matijašić, Maja Rupnik, Aleksander Mahnič, Darja Paro Panjan, 2025, izvirni znanstveni članek Povzetek: Perinatal antibiotic exposure potentially leads to gut microbiota dysbiosis, which is associated with functional gastrointestinal disorders (FGIDs). We aimed to investigate the effects of Limosilactobacillus reuteri DSM 17938 supplementation on the development of FGIDs, crying and sleep duration, and the gut microbial composition in infants exposed to antibiotics during the neonatal period. In this randomised, double-blind, placebo-controlled study, we included 89 term neonates treated with antibiotics. Neonates received the study product for six weeks. FGIDs, assessed by the Infant Gastrointestinal Symptom Questionnaire, crying and sleep duration were assessed at four and eight weeks, and six months after enrolment. Faecal samples were collected six weeks and twelve months after enrolment. The gut microbial community composition was analysed using 16S amplicon sequencing and qPCR. The proportion of infants with FGIDs was greater in the control group, although the difference between the groups was significant only six months after enrolment. At all time points, the probiotic group presented a longer sleep duration and shorter crying time than the control group, but the difference was not statistically significant. Probiotic consumption had no significant effect on the gut microbiota composition except for increased L. reuteri DSM 17938 abundance in the probiotic group at six weeks after enrolment. At specific time points after supplementation with L. reuteri DSM 17938, a reduction in the prevalence of FGIDs was observed in the probiotic group. However, no observable effect on the gut microbiota was detected during the intervention. We believe that probiotic supplementation in neonates during and after antibiotic treatment to minimise the negative effects of antibiotics on gut function during this vulnerable period of human development warrants further investigation.
Ključne besede: probiotic, infantile colic, functional gastrointestinal disorders, gut microbiota
Objavljeno v DiRROS: 09.12.2025; Ogledov: 76; Prenosov: 38
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105. Development and performance evaluation of a clinical metagenomics approach for identifying pathogens in the whole blood from patients with undifferentiated feverJan Slunečko, Rok Kogoj, Samo Zakotnik, Alen Suljič, Nataša Knap, Martin Bosilj, Franc Strle, Tatjana Avšič-Županc, Petra Bogovič, Miša Korva, 2025, izvirni znanstveni članek Povzetek: Introduction: Blood culture is the cornerstone of microbiological diagnostics for patients with acute undifferentiated fever and no obvious localization of infection; however, up to 50% of cases remain undiagnosed. Infections caused by arboviruses, fastidious or even uncultivable bacteria, or parasites often go undiagnosed without the use of target-specific molecular methods. These are typically performed in a stepwise manner, increasing cost and delaying results. Metagenomic next-generation sequencing (mNGS) has recently gained recognition as a potential universal pathogen detection tool for such cases. Our study aimed to develop a streamlined mNGS workflow for simultaneous detection of intracellular and cell-free pathogens within a single sequencing library. Methods: Total nucleic acid was isolated separately from 200 EDTA blood samples. The plasma isolate was processed with DNase, followed by the depletion of host ribosomal and messenger RNA, reverse transcription, and sequence-independent single primer amplification (SISPA). The whole blood isolate was only reverse transcribed, with no other pre-processing manipulation. Finally, the two fractions were combined prior to library preparation and sequencing using either Oxford Nanopore Technologies or Illumina. Following established bioinformatics analysis, we developed a mathematical ranking approach (ClinSeq score) that enabled quick identification of relevant pathogens in approximately one hour. Results: The mNGS workflow reached 79.5% (159/200) overall sensitivity. For bacteria the sensitivity was 88.6% (70/79), DNA viruses, 66.7% (10/15) and for RNA viruses 73.8% (76/103). Pathogen detections by individual sequencing methods showed overall sensitivity of Illumina and ONT to be 80.0% (76/95) and 79.1% (83/105) respectively. The ClinSeq score correctly highlighted the pathogen in 126/200 (63.0%) samples effectively with a Cohen’s kappa (κ) agreement of 0.61 with manual analysis. Conclusion: Developed comprehensive mNGS workflow detects a wide range of pathogens in patients with acute undifferentiated fever. The unified workflow improves sensitivity for intracellular bacteria and RNA viruses, reduces time, cost and complexity by eliminating the need for separate library preparations, enabling faster turnaround suitable for clinical settings. The ClinSeq score effectively differentiates true pathogen signals from background noise, reducing false positives and manual interpretation time. Overall, the workflow demonstrates flexible, and efficient pathogen detection, supporting its potential for clinical diagnostics and improved patient management.
Ključne besede: mNGS, clinical metagenomics, molecular diagnostics, universal pathogen detection, enhanced RNA virus detection
Objavljeno v DiRROS: 09.12.2025; Ogledov: 92; Prenosov: 47
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