Steponaitienė, Rūta

Late diagnosis and the lack of effective early detection techniques contribute to the poor prognosis of pancreatic ductal adenocarcinoma (PDAC). To address this challenge, we develop ¹H NMR-based metabolomics-AI platforms employing customized multilayer support vector machine (SVM), AutoGluon, and Tabular Foundation Model (TabPFN) frameworks. These platforms integrate serum metabolomic profiles-including small-molecule metabolites and lipoproteins-with clinical/biochemical parameters (age, CA19-9) and Activin A, derived from 902 participants (424 high-risk controls and 478 PDAC cases). Our TabPFN-based algorithm, PanMETAI, outperform state-of-the-art models. In the Taiwanese training and validation cohort, the model achieved an impressive AUC of 0.99 (95% CI: 0.98-0.99). Its robustness is further confirmed in a Lithuanian external validation cohort (n = 322), which yields an AUC of 0.93 (0.90-0.95). Notably, it identifies key signature patterns that improve early-stage (I/II) PDAC diagnosis and perform well with small sample sizes (n = 50). TabPFN-PanMETAI offers a rapid, accurate, and non-invasive tool for early PDAC detection, with strong potential for clinical application.

Pratarmė. Mieli studentai, Gastroenterologija yra viena įdomiausių ir greičiausiai besiplėtojančių medicinos sričių. Dėl mokslo ir technologijų pažangos XXI a. gastroenterologija apima vis platesnį kepenų ir virškinamojo kanalo ligų, gydymo metodų spektrą. Tai neabejotinai viena įvairiapusiškiausių specialybių, kur akivaizdi klinikinių įgūdžių, molekulinių ir ultragarsinių tyrimų bei sudėtingų endoskopinių intervencijų sąsaja. Uždegiminės žarnyno ligos, retos virškinamojo kanalo ligos, mikrobiotos ir kepenų transplantacija yra tik nedidelė dalis sričių, kurios yra patikėtos gydytojams gastroenterologams. Šis Lietuvos sveikatos mokslų universiteto Medicinos akademijos Medicinos fakulteto Gastroenterologijos klinikos (toliau - Gastroenterologijos klinika) kolektyvo parengtas vadovėlis yra skirtas studentams, siekiantiems susipažinti su gastroenterologijos pagrindais. Jau beveik 30 metų Gastroenterologijos klinika aktyviai dalyvauja rengiant tarptautines diagnostikos ir gydymo gaires, yra prestižinių tarptautinių mokslo projektų ir konsorciumų dalyvė, o 2020 m. klinika tapo ir asocijuota Europos retų kepenų ligų tinklo nare. Mūsų klinikos mokslinių tyrimų rezultatai yra publikuojami prestižiniuose mokslo leidiniuose: Nature, Lancet, New England Journal of Medicine, Nature Genetics ir kituose. Rengdami šį vadovėlį stengėmės perteikti visą klinikos sukauptą ilgametę pedagoginę ir klinikinę patirtį bei naujausius mokslo pasiekimus. Tikimės, kad šis vadovėlis ne tik suteiks Jums pagrindinių žinių apie gastroenterologiją, bet ir paskatins rinktis šią specialybę rezidentūros studijose. Autorių vardu prof. Juozas Kupčinskas

Šio tyrimo tikslas nustatyti gliomoms specifines iRNR N6-metiladenozino modifikacijas gliomos kamieninėse ląstelėse ir navikuose, siekiant atrinkti naujus, kliniškai reikšmingus gliomų molekulinius žymenis. Uždaviniai: 1. Nustatyti gliomos kamieninių ląstelių (NCH421k) iRNR N6-metiladenozino (m6A) modifikacijų profilį lyginant su glioblastomos ląstelėmis (U87-MG) ir atrinkti potencialias, m6A metilintas iRNR, susijusias su gliomos kamienišku ir progresavimu. 2. Apibūdinti iRNR molekulių rinkinį su būdingomis m6A epitranskriptominėmis modifikacijomis, susijusiomis su gliomos patogeneze ir paciento prognoze. 3. Ištirti gliomos kamieninėms ląstelėms specifinius m6A metilintus iRNR transkriptus gliomos navikuose, siekiant įvertinti jų poveikį naviko patologijai bei pacientų klinikinėms charakteristikoms. Tyrimai rodo, kad m6A modifikacijos galėtų būti potencialus taikinys gliomų atveju, o terapinis epitranskriptomo modifikacijų reguliavimas gliomos kamieninėse ląstelėse galėtų padėti kontroliuoti jų augimą, atsinaujinimą ir naviko vystymąsi.

Background and Objectives: Neuropeptide Y (NPY) family peptides and dipeptidyl peptidase-4 (DPP-4) are involved in gastrointestinal regulation and may contribute to obesity and type 2 diabetes mellitus (T2DM) pathophysiology. This study investigates their expression in jejunal muscular tissue and associations with gastrointestinal symptoms in patients with obesity, with (OB+/DM+) and without T2DM (OB+/DM−). Materials and Methods: This cross-sectional study includes forty-four patients undergoing laparoscopic Roux-en-Y gastric bypass divided based on T2DM status. Gastrointestinal symptoms were assessed using the Gastrointestinal Symptom Rating Scale (GSRS) questionnaire, and jejunal tissue samples were analyzed for DPP-4, NPY, peptide YY (PYY), and pancreatic polypeptide (PP) mRNA and protein levels. Results: DPP-4, NPY, PYY, and PP gene expression in jejunal muscular tissue was similar between groups. In the OB+/DM+ group, PP protein was higher, while DPP-4 and PYY were lower compared to the OB+/DM− group. Significant positive correlations between DPP-4 and NPY, PYY, and PP were found in the OB+/DM− group, while only DPP-4 and PYY correlated in the OB+/DM+ group. Gastrointestinal symptoms in the OB+/DM− group showed positive correlations with PP (abdominal pain), DPP-4 (indigestion), and NPY (constipation). Conclusions: The study demonstrates significant differences in DPP-4, PYY, and PP protein expression between patients with obesity, with or without T2DM. Peptide correlations with gastrointestinal symptoms in non-diabetic patients suggest distinct regulatory mechanisms, warranting further research.

Introduction: Ulcerative colitis (UC) is an inflammatory bowel disease marked by chronic inflammation of the colon’s inner lining. Its pathogenesis involves a multifactorial interplay between genetic and environmental factors, dysregulated immune responses, and alterations in the gut microbiome. MicroRNAs (miRNAs) have emerged as critical regulators of gene expression implicated in UC pathogenesis.Understanding the differential expression of miRNAs in colonic epithelial cells and their extracellular vesicles (EVs) during bacterial exposure could offer potential insights into the interplay between gut microbiota and epithelial cell-mediated inflammatory responses.Aims & Methods: The objective of this study was to investigate the expression of UC-associated miRNAs in colonic epithelial cells and their EVs,using UC patients and control subjects-derived colonic epithelial cells co-cultures with Escherichia coli and Phocaeicola vulgatus bacteria.Colonic stem cells from healthy controls (HC) (n=10) and patients with active UC (n=10) were used to generate 3D colonic epithelial organoids,which subsequently served as a cell source for constructing polarized monolayers of colonic epithelial cells. These monolayers were co-cultured with commensal E. coli or P. vulgatus bacteria for 2 hours, washed, replenished with fresh culture medium, and further incubated for an additional 24 hours. EVs from cell-conditioned media were isolated using precipitation method and characterized by ELISA (CD63, HSP70, Apo-A1), Dynamic Light Scattering and cryo-TEM. RNAs from colonic epithelial cells and precipitated EVs were isolated using silica-column based method. MiRNAs expression was evaluated by TaqMan qPCR. Statistical analysis and visualization of the results were performed using R studio.Results: Although statistically insignificant, but the expression of miR-183-5p and miR-135b-5p in epithelial cells of HC group was tended to de -crease after contact with E. coli compared to untreated cells (resp. FC=0.84(p=0.574) and FC=0.87 (p=0.798)), while miR-146a-5p increased (FC=1.37(p=0.798)). In the EVs samples of HC, the same trends were observed.Interestingly, in the UC patients’ cells, after incubation with E. coli, the expression of all studied miRNAs - miR-183-5p, miR-135b-5p, miR-146a-5p - tended to increase (resp. FC=1.53 (p=0.730), FC=1.71 (p=0.666), andFC=2.39 (p=0.258)).Similar expression changes were observed in the EVs of colonic epithelial cells from UC patients following stimulation with E. coli (resp. FC=1.32(p=0.730) (miR-183-5p), FC=1.32 (p=0.666) (miR-135b-5p), and FC=2.07(p=0.258) (miR-146a-5p)).After culturing colonic epithelial cells with P. vulgatus the expression of all investigated miRNAs, compared to unstimulated cells, remained almost unchanged, both in HC and in UC patients’ monolayers. Meanwhile, in both the HC and UC groups, a decrease in miR-183-5p and miR-135b-5p expression was observed in EVs following contact with P. vulgatus, compared to control conditions (in the HC group resp. FC=0.59 (p=0.382) andFC=0.51 (p=0.234); in the UC group resp. FC=0.70 (p=0.605) and FC=0.73(p=0.489)). However, the expression of miR-146a showed a decreasing trend only in the HC group compared to unstimulated cells (FC=0.53(p=0.161)).Conclusion: Due to the considerable variation in the response between study subjects, the observed expression changes did not reach statistical significance. The experimental study revealed a tendency that the expression of miRNAs associated with UC can be modulated by commensal gut bacteria, particularly E. coli, both in colonic epithelial cells and their EVs.

Objective: Ulcerative colitis (UC) is characterized by disrupted mucosal barrier and microbial composition. Yet, it remains uncertain if commensal gut bacteria can influence the maintenance of the colon’s protective lining. Therefore, we aimed to assess how colonic epithelial cells from UC patients respond to commensal bacteria. Materials and Methods: Co-culture model of colonic epithelial organoid-derived monolayers of UC patients (n=9) and non-IBD controls (n=8) and Escherichia coli (ATCC25922) and Phocaeicola vulgatus (ATCC8482) bacteria type strains were used for evaluation of epithelial barrier integrity (ZO1), pathogen recognition (TLR4) and stress induction (HSPA1A, HSPB1) gene expression, as well as miRNA (miR183-5p, miR-135b-5p, miR-146a-5p) expression in host epithelial cells and extracellular vesicles (EVs). Results: E. coli and P. vulgatus did not trigger pathogen-pattern recognition or stress responses in colonic epithelial cells but showed a tendency to increase ZO1 expression in non-IBD cells (FC = 3.59 and FC = 2.27, respectively, p ≥ 0.05), while decreasing it in UC cells (FC = 0.67 and FC = 0.75, respectively, p ≥ 0.05). The studied miRNAs – miR-183-5p, miR-135b-5p, miR-146a-5p – tended to increase in the EVs of UC colonic epithelial cells after E. coli stimulation (FC=1.32, FC=1.32, and FC=2.07, respectively, p ≥ 0.05). Conversely, cultivation with P. vulgatus tended to decrease miR-183-5p (FC=0.70, FC=0.59, respectively, p ≥ 0.05) and miR-135b-5p (FC=0.73, FC=0.51, respectively, p ≥ 0.05) expression in both UC and non-IBD groups. Conclusions: E. coli and P. vulgatus contibute in compromising mucosal barrier integrity and influencing miRNA expression in extracellular vesicles.

Introduction. Recent findings uncover the importance of gut microbiota and its impairment in the course of ulcerative colitis (UC) (1). However, the unaltered fecal microbiota and the potential involvement of its constituents in the modulation of colonic epithelial cell responses during UC is usually left out of focus. Aim The aim of the study was to identify which fecal microbiota components remain unaltered during UC and assess the interplay of these bacteria with intestinal epithelial cells.

Patient-derived human intestinal organoids are becoming an indispensable tool for the research of digestive system in health and disease. However, very little is still known about the long-term culturing effect on global genomic methylation level in colonic epithelial organoids derived from healthy individuals as well as active and quiescent ulcerative colitis (UC) patients. In this study, we aimed to evaluate the epigenetic stability of these organoids by assessing the methylation level of LINE-1 during prolonged culturing. We found that LINE-1 region of both healthy control and UC patient colon tissues as well as corresponding epithelial organoids is highly methylated (exceeding 60%). We also showed that long-term culturing of colonic epithelial organoids generated from stem cells of healthy and diseased (both active and quiescent UC) individuals results in decrease of LINE-1 (up to 8%) methylation level, when compared to tissue of origin and short-term cultures. Moreover, we revealed that LINE-1 methylation level in sub-cultured organoids decreases at different pace depending on the patient diagnosis (healthy control, active or quiescent UC). Therefore, we propose LINE-1 as a potential and convenient biomarker for reliable assessment of global methylation status of patient-derived intestinal epithelial organoids in routine testing of ex vivo cultures.

Introduction: Extracellular vesicles (EVs) play an essential role in cell and infection biology and have the potential to act as biomarkers for novel diagnostic tools. EVs are membrane-enclosed nanoparticles secreted by the majority of cells. They contain various types of biomolecules, including proteins, lipids, and non-coding RNAs such as miRNAs. It has been indicated that miRNAs found in EVs affect mRNA stability of recipient cells, thereby playing a crucial role in diseases progression. However, there is no clear consensus as to whether the packaging of miRNAs into EVs occurs randomly or selectively. Lipopolysaccharide (LPS) is an endotoxin of gram-negative bacteria known to initiate the response of immune system and was associated with pathogenesis of inflammatory bowel diseases and cancer. Fundamental mechanisms of molecular packaging into EVs and specificity remain unclear and need to be studied further. Aims & Methods: The aim of this study was to assess the miRNA profile of extracellular vesicles compared to the miRNA profile of their host cells (colon adenocarcinoma and healthy colon cells), after stimulation with lipopolysaccharide.Methods: Experiments were conducted using primary human colonic epithelial cells (HCoEpiC) and adenocarcinoma cells (Caco-2, HCT116) (5 pas-sages per cell line). Cells were treated with bacterial LPS from Salmonella enterica (Serotype typhimurium) (Sigma-Aldrich) (10 μg/ml) 24 hours and after 24, 48, 72 hours their EVs were collected using polyethylene-glycol-based method. RNA was purified from cells and EVs using commercial kits. Small RNA sequencing was performed using HiSeq 2500 platform (Illumina, CA, USA). Bioinformatic analysis performed using R package and algorithms for small sequencing data (DEseq2 and etc.). Results: After 24 hours LPS treatment, miR-146 and miR-451 were overexpressed in HCT116, Caco-2 and HCoEpic cells. The profile of deregulated miRNAs found in EVs of these cell lines was as follows: i. miR-122-5p, miR-451a, miR-30b-5p, 381-3p, 99a-5p were upregulated and miR-20a-5p, miR-17-5p, miR-93-5p, miR-335-5p, miR-378c were down-regulated in Caco-2 cell line produced EVs, ii. miR-215-5p was downregulated in HcoEpic cell line produced EVs, and; iii. miRNAs expression was not deregulated in HCT116 cell line EVs. After 48- and 72-hours LPS treatment miRNA expression was deregulated only in HCOepic cell line: miR-451a, miR-147b-3p, miR-146a-5p, but not in the EVs secreted by this cell line. This research shows that host cells hold a sorting mechanism that guides specific miRNAs into exosomes. It is important to mention that we noticed that certain miRNAs sequences were found only in EVs of cells, but not in the cells, and some of them (even the most overexpressed) had not been packaged into EVs.Conclusion: The miRNA profile of extracellular vesicles differs from miRNA profile of the host cells after stimulation with lipopolysaccharide.

Introduction: Diet is one of the most important factors contributing to the multistep process of carcinogenesis. The functional role of exogenous xe-no-microRNAs (miRNAs) in this context is poorly understood. Recently, we reported the detectability of food-derived xeno miRNAs in various foods and in the gastrointestinal and colonic mucosa. However, little is known about the translational role of xeno-miRNA in the GI mucosa and specifically in GI diseases. Aims & Methods: In this work, we aimed to evaluate the potential clinical relevance of the xeno-miRNA miR-168 in the gastric mucosa along the preneoplastic conditions and gastric carcinogenesis. To this end, we performed quantitative analysis of miR-168 in different settings: 1. Samples from patients with normal mucosa (N), chronic non-atrophic (CNAG) and atrophic gastritis (CAG) and intestinal metaplasia (IM) (n=72); 2. Matched non-tumorous (NT-) and tumorous (T-) gastric cancer (GC) tissues (n=82) and 3) matched NT- and T-colorectal cancer (CRC) tissues (n=40). Survival analysis was performed using Kaplan-Meyer analysis. Results: MiR-168 was reproducibly detectable in the samples examined, with higher levels in stomach compared to colon tissue. In the stomach, a significantly higher level of miR-168 was observed in NT-GC compared to N, CNAG, AG/IM samples (p<0.01 each), but there was no difference related to H. pylori positivity or inflammation grade. Interestingly, miR-168 was higher in patients with moderate or severe AG/IM or OLGIM 3/4. Despite a significant correlation of miR-168 expression, paired sample analysis revealed a higher level of miR-168 in NT-GC compared to T-GC, with the highest level observed in the cardia and the lowest in the gastric mucosa of the antrum. Survival analysis showed only a small trend towards worse overall survival for patients with highest to lowest miR-168 levels, mostly during the first 3 years, but no long-term difference regardless of Lauren’s classification. MiR-168 levels in normal mucosa were not associated with overall survival. In correlation with matched GC samples, NT-CRC showed higher miR-168 levels compared to T-CRC, although overall there was a positive correlation between matched tissues. No survival difference was observed for higher or lower miR-168 levels in CRC. Conclusion: Food-derived xeno-miRNA are present in the gastric and colonic mucosa, with the highest levels in the stomach compared to the colon and in non-tumour compared to tumour tissues. While the potential impact of miR-168 on overall survival was weak, the higher level of miR-168 in patients with moderate and severe IM deserves further attention and further functional analyses are needed to better understand the functional role of xeno-miRNA in the GI tract.