AIM Due to increasing use of mitochondrial DNA (mtDNA) sequencing in both forensic practice and clinical disease research, this study explores the optimization of the next-generation sequencing (NGS) method for whole mitochondrial genome analysis on the Illumina MiSeq platform. METHODS Initial attempts using pre-made commercial primers were unsuccessful, leading to the design of novel custom-designed primers in our laboratory and optimization of sequencing chemistry and protocols. A comprehensive protocol was developed, involving long-range amplification, enzymatic fragmentation, and the use of IDT® for Illumina DNA/RNA UD Indexes and MiSeq Reagent Nano Kit v2 (300 cycles), whereby DNA extraction, quantification, and library preparation were all performed according to optimized protocols. RESULTS Successful amplification was confirmed using gel electrophoresis and Agilent Bioanalyzer, with optimized conditions yielding clear, specific amplicons 9.8 and 8.5 kb in length. Sequencing results demonstrated high-quality reads with an average coverage depth of 742x and a GC content of 43-45%. The study highlights the efficiency of custom primers and individual library normalization for reliable mtDNA sequencing. CONCLUSION These findings advance the application of NGS in forensic and clinical settings by enhancing the detection of rare mutations and mitochondrial heteroplasmy, paving the way for routine mtDNA analysis using NGS technology.

In this reflection, I share my journey from being a participant in the first Connect Symposium in 2017 to returning as a junior organizer and lecturer in 2025. What began as a formative experience during my undergraduate studies became a turning point in my academic path, guiding me through a PhD and deeper engagement with the European research landscape. The symposium offered far more than lectures — it introduced me to the importance of science communication, regional cooperation, and networking, all of which shaped my career. Eight years later, I return to Neum alongside a student colleague I met during that first Connect, now ready to pass on what we once received. As we focus this year on “Towards the EU Research Area,” I hope our stories and experiences will inspire a new generation to see science not just as a profession, but as a shared, cross-border endeavor.

PURPOSE Accurate lung cancer TNM staging depends on macroscopic and microscopic tumor evaluation of resection specimens. However, small nodules (<1 cm) are difficult to extract and correlate with in vivo imaging. We investigated whether microCT could better localize lesions or guide pathology to otherwise undetected abnormalities. MATERIALS AND METHODS Paired ex vivo CT and microCT were performed after inflating and freezing surgically removed lung lobes (resolution 80 to 120 µm). Rigorous matching between CT, microCT, and histopathology was performed on areas containing abnormalities on microCT. RESULTS A total of 57 lobectomy specimens were analyzed. MicroCT-guided microscopic examination led to 2 additional primary carcinomas, 2 separate tumor nodules from the primary lung tumor, and 1 atypical adenomatous hyperplasia lesion that were not evident before surgery. For both patients with separate tumor nodules, the cT1 stage was upgraded to a pT3. In addition, the microCT provided insight into underlying structural disease (ie, emphysema and fibrosis). CONCLUSIONS In 5 out of 57 resection specimens (9%), microCT showed additional (pre-)cancerous lesions. This explorative study suggests that lobar microCT could serve as a valuable guide for pathologists by pointing them toward areas that may warrant further investigation. In this way, it is a practical and beneficial tool, capable of facilitating a more precise TNM classification in tumor resection specimens, which needs further validation in a prospective study.

Glioblastoma (GBM) remains a major clinical challenge due to limited therapeutic success despite standard treatments including surgery, radiotherapy, and temozolomide (TMZ). Recent evidence links hyperglycemia to cancer progression, and altered glucose metabolism has emerged as a key factor in GBM development. Metformin, an antidiabetic drug, has shown promise in improving survival in GBM patients, possibly due to its ability to cross the blood-brain barrier and target metabolic pathways involved in tumor growth. Preclinical studies suggest metformin may enhance TMZ efficacy by acting on glioma stem cells and overcoming resistance mechanisms. Its activation of AMPK and modulation of Wnt signaling further support its therapeutic potential. However, while early studies and clinical trials have explored metformin’s safety and efficacy, its direct impact on GBM survival remains unclear. Ongoing research aims to clarify its mechanisms and identify responsive patient subgroups. Novel strategies, including PPARγ agonists and nanoerythrosome-based drug delivery systems, are also under investigation to improve metformin’s therapeutic profile. Rigorous clinical trials and mechanistic studies are essential to determine the role of metformin as adjunct therapy in GBM treatment.

As the leading cause of global mortality, cardiovascular diseases demand improved and innovative strategies for early detection and risk assessment to enhance prevention and timely treatment. This comprehensive review examines the potential of combining high-sensitivity cardiac troponins (hs-cTns) and homocysteine (Hcy) as complementary biomarkers for enhanced cardiovascular risk prediction. hs-cTn assays have revolutionized cardiovascular diagnostics by enabling the detection of minimal myocardial injury, improving early diagnosis of acute coronary syndrome, and providing robust prognostic information in both symptomatic and asymptomatic populations. Hcy, while established as a marker of vascular dysfunction, presents an interpretative challenge due to multiple confounding factors and inconsistent therapeutic responses. Emerging evidence demonstrates significant correlations between elevated Hcy and troponins across various clinical conditions, suggesting that their combined assessment—reflecting both myocardial injury and vascular dysfunction—may improve cardiovascular risk stratification. While initial findings are promising, additional studies are required to validate the clinical value of the combined marker approach. Future development of personalized interpretation algorithms, and multi-marker panels incorporating these biomarkers, may significantly advance cardiovascular medicine and enable more effective population-specific risk management strategies.

The aim of this study was to determine differences in hydration status before and after a judo training session. The sample of participants included boys (N = 60) and girls (N = 35) aged 11–14 years. The variables used as indicators of hydration status were body weight (BW), body mass index (BMI), fat mass (FM), and total body water (TBW). Based on mean values obtained after training, body weight decreased by 0.78 kg in girls and 0.54 kg in boys. Body mass index decreased by 0.39 in girls and 0.45 in boys, while fat mass decreased by 0.52 kg in girls and 0.18 kg in boys. Finally, the reduction in total body water amounted to 0.72 in boys and 0.28 in girls. Statistical significance was confirmed using a t-test for changes in body weight, body mass index, and total body water. Based on the findings of this study, it can be concluded that the introduction of scheduled rehydration breaks during training may reduce fluid loss and help preserve muscular strength in young judo athletes.

Mammalian cells, particularly human cell culture models, are essential for studying disease pathophysiology and producing cell-based therapeutic products. Monitoring and controlling cell culture conditions accurately is essential for optimal cell growth and health, as even minor variations can significantly influence cell behavior. The presence of viruses, bacteria, and their by-products are key indicators of cell culture contamination. Conventional assays for quantifying cellular health and microbial contaminants such as endotoxins are end-point assays that are often laborious, require specialized equipment, and typically detect contamination only at advanced stages. For example, the chromogenic Limulus amoebocyte lysate assay, used for quantifying endotoxin, a bacte rial by-product, is often susceptible to interference from serum proteins in the culture medium. In this work, we present a simple and sensitive aptamer-based biosensor designed to detect bacterial-secreted endotoxins in various complex cell culture media. As a proof of concept, human induced pluripotent stem cells (hiPSCs) were deliberately contaminated with Escherichia coli (E. coli), and the biosensor's response to endotoxins released by the bacteria was monitored over a 24-h period. The biosensor demonstrated a reliable linear response with a detection limit of 0.33 ± 0.06 pg/mL in DMEM and 0.142 ± 0.025 pg/mL in StemFlex medium. Its performance in complex sample matrices suggests the potential for integration with industrial-scale cell culture systems for real-time contamination detection, providing a cost-effective, efficient, and timely method to monitor cell health and ensure sterile conditions for therapeutic cell cultivation.

Lava tubes, a common volcanic feature on terrestrial planets, offer critical insights into lava flow processes and may serve as future potential habitats for space crews and other facilities on the Moon and Mars. Seismic detection of these features is challenging as the irregular morphology and rough cave ceilings and walls generate complex seismic wavefields dominated by strong scattering and reverberation rather than pure reflections. Here we present observations of enhanced backscattering in seismic data collected above terrestrial lava tubes. We show that the spatial and frequency characteristics of wavefield intensity can be related to the dimensions of the lava tubes. Our findings suggest that, when geological indicators such as collapse pits are present, this method would enable mapping lava tubes on Earth and, by proxy, on the Moon and Mars, and that the approach is readily adaptable for future planetary exploration.

Introduction Reperfusion failure (RF) describes a condition in which patients suffering a large vessel occlusion (LVO) stroke present insufficient tissue reperfusion and recovery despite optimal mechanical thrombectomy (MT) results. Approximately 50% of patients suffering from LVO are affected. Our current understanding of the underlying pathomechanisms is limited and mostly based on rodent models. The goal of this study was to further characterize RF by applying advanced multimodal hemodynamic imaging in stroke patients. Methods Patients from the IMPreST study with LVO stroke and successful recanalization [corresponding to thrombolysis in cerebral ischemia grade (TICI) 2b-3] were included. Follow-ups with blood oxygenation-level dependent cerebrovascular reactivity (BOLD-CVR) and non-invasive optimal vessel analysis (NOVA) imaging were performed (<72 h, 7 days and 90 days). Demographic and clinical data (NIHSS and mRS) were collected. Results Of the 49 patients included in IMPreST, 18 patients met the inclusion criteria. Based on the perfusion weighted imaging (PWI) of the affected area compared to the contralateral side after MT, patients were stratified into three groups: hypoperfusion (n = 3), normalization (n = 8), and hyperperfusion (n = 7). The hyperperfusion group tended to show poorest clinical outcome (mRS 3 months: 2.5 [Q1–Q3 2.0–3.0] vs. normalization: 1 [Q1–Q3 0.75–3.0], p = 0.169) and had significantly lower BOLD-CVR values at visit one and two compared to hypoperfusion and normalization groups, indicating impaired cerebrovascular reactivity (visit1 hyperperfusion group −0.01 [Q1–Q3–0.02 – 0.07], normalization group 0.12 [0.09, 0.19], hypoperfusion group, 0.09 [0.09, 0.11] p = 0.049, visit2 hyperperfusion group 0.07 [Q1–Q3 0.03–0.10], normalization group 0.17 [0.16, 0.18], hypoperfusion group 0.10 [0.09, 0.11], p = 0.014). Discussion We found three patterns of reperfusion after successful MT of LVO stroke: normalization, hypo- and hyperperfusion of the ischemic area on days at < 72 h after stroke. There was substantial inhomogeneity in perfusion and clinical outcomes between the three groups. Next to poorest clinical outcome, the hyperperfusion-group showed poorest cerebrovascular reserve, reflecting findings of RF in rodent models. Thus, we suggest that RF includes both hypo- as well as hyperperfusion. Early detection using advanced imaging would allow a better identification of patients at risk for poor clinical outcome. Clinical trial registration http://clinicaltrials.gov, Identifier (NCT04035746).

Gestational diabetes mellitus (GDM) is associated with various maternal and fetal complications, including long-term cardiovascular risks for affected women. This study aimed to investigate the relationship between GDM and arterial stiffness during pregnancy and 2 months postpartum. A cross-sectional study was conducted with 100 pregnant women of 28–40 weeks of gestation and divided into 2 groups. The groups were divided based on oral glucose tolerance test (oGTT) results: 50 women with GDM and 50 controls with normal oGTT. All participants in the GDM group had a form of GDM that was successfully managed by diet only. Arterial stiffness was assessed using a non-invasive oscillometric device, the Arteriograph® (TensioMedTM Kft, Budapest, Hungary), by measuring the right brachial artery. The results showed that during pregnancy the pulse wave velocity (PWV) was unexpectedly significantly lower in the GDM group compared to controls (8.10 m/s vs. 8.65 m/s, P T< 0.05). Central and brachial augmentation index (AIx) values showed no significant differences between the groups. No differences in PWV or AIx were observed between GDM and control groups 2 months postpartum; however, within the GDM group, central (0.50 % vs. 11.45 %) and brachial (–73.35 % vs. –51.75 %) AIx significantly increased after delivery, indicating postpartum vascular effects of GDM. These findings suggest that even diet-managed forms of GDM can induce vascular changes postpartum, underscoring the importance of early detection and management. Further research is needed to explore the mechanisms underlying these changes and their possible long-term implications for cardiovascular health in post-GDM women.

Pseudomonas aeruginosa is an opportunistic pathogen that frequently causes infections in immunocompromised patients and is involved in outbreaks of hospital-acquired infections with a high mortality rate. Aminoglycosides are a large category of antibiotics that bind specifically to 16S rRNA in 30S ribosomal subunits and disturb protein translation. This antibiotic class plays a significant bactericidal role against a wide range of Gram-negative bacteria such as P. aeruginosa. Among different aminoglycoside resistance mechanisms, inactivation of drugs by plasmid-encoded aminoglycoside-modifying enzymes (AMEs) is a common determinant of aminoglycoside resistance in  P. aeruginosa. These plasmids are spread worldwide, and they are transferred to a wide range of different species. This study aims to detect resistance mechanisms and  identify the most prevalent aminoglycoside resistance genes in P. aeruginosa clinical isolates, collected from the University Clinical Centre Tuzla. This study included a total of 230 clinical P. aeruginosa isolates. Antimicrobial susceptibility tests were performed using the disk diffusion method and the Vitek2 system. Isolates displaying increased MIC values for aminoglycoside antibiotics were included in the multiplex PCR reaction, for the detection of aminoglycoside-modifying enzyme genes. The most prevalent genotype among isolates was aac (6')-I. All aac (6')-I genotyped isolates also displayed a high rate of resistance to other classes of antibiotics, and they were characterized as multidrug-resistant (MDR) or extensively drug-resistant (XDR). Results indicate that the aminoglycoside-resistance genes are highly prevalent and could easily spread among P. aeruginosa strains.

Access to large patient cohort data and biobanked resources is a catalyst for progress in genomics and biomedical research, increasing statistical power, and unlocking deeper insights—especially in areas like rare diseases and mental health. Responsible research necessitates maintenance of data privacy, regulatory compliance, and research standardization. It can appear that these guiding principles oppose each other and present barriers to responsible Open science. To address these critical challenges, we developed MINDDS-Connect, a federated data collaboration platform that integrates a web-based interface with decentralized Docker instances via a REST API. This architecture allows registered users to securely query samples across the platform’s network, and offers a tool to facilitate the formation of virtual multi-centric meta-cohorts and research collaboration. MINDDS-Connect allows institutions to retain data control while enabling collaborative research and meta-cohort analysis through standardized metadata fields. Its implementation across five European centers enhanced the accessibility of 900 samples, demonstrating its effectiveness in enabling cohort construction and promoting collaborative research. The platform provides a secure, open-source solution consistent with EU Open Science policies, advancing large-scale mental health research.

ABSTRACT This study demonstrates the usage of primarily discarded waste – pomegranate peel as secondary raw material – biosorbent for broader applications. The focus was on the optimisation of key cationic dye methylene blue (MB) – pomegranate peel (PP) adsorption process parameters, as well as cost analysis assessing the possibility of scaling up. The optimal values of the key parameters were pH 6, biosorbent mass 100 mg, contact time of 50 min, and initial analyte concentration 100 mg/L for MB removal from aqueous solutions providing high removal efficiency values > 88%. Fourier-transform infrared spectroscopy (FTIR) showed that MB adsorption on PP was presumably via hydrogen bonds with the OH groups present in PP. Changes noted via elemental composition analysis given by electron dispersive spectroscopy (EDS) confirmed the sorption of MB. Biosorption occurred mainly as a pseudo-second-order kinetic reaction combined with phase III of the intraparticle diffusion model (both R2 ≥ 0.92). Through a simple and fast batch MB sorption process with many advantages compared to literature data, a maximum sorption capacity of 384.61 mg/g could be achieved. Pomegranate peel was identified as a low-cost adsorbent with excellent potential for MB removal, economically viable (0.74 $/mol), demonstrating great possibilities for industrial application. Highlights Biosorption of phenothiazine dye on novel waste material from pomegranate peel in its native form. A univariate general procedure was performed, FTIR, SEM, and EDS characterisation of biosorbents. An optimal pH value was determined to be 6, while the optimal mass was 100 mg. A maximum biosorption capacity of 384.61 mg/g could be achieved. The mechanism of adsorption is best obeyed by the Langmuir and Freundlich models. The total analysis expenses for the entire procedure were just 0.74 $/mol. GRAPHICAL ABSTRACT

B-1 cells are innate-like immune cells abundant in serosal cavities with antibodies enriched in bacterial recognition, yet their existence in humans has been controversial1, 2–3. The CD5+ B-1a subset expresses anti-inflammatory molecules including IL-10, PDL1 and CTLA4 and can be immunoregulatory4, 5–6. Unlike conventional B cells that are continuously replenished, B-1a cells are produced early in life and maintained through self-renewal7. Here we show that the transcription factors TCF1 and LEF1 are critical regulators of B-1a cells. LEF1 expression is highest in fetal and bone marrow B-1 progenitors, whereas the levels of TCF1 are higher in splenic and peritoneal B-1 cells than in B-1 progenitors. TCF1–LEF1 double deficient mice have reduced B-1a cells and defective B-1a cell maintenance. These transcription factors promote MYC-dependent metabolic pathways and induce a stem-like population upon activation, partly via IL-10 production. In the absence of TCF1 and LEF1, B-1 cells proliferate excessively and acquire an exhausted phenotype with reduced IL-10 and PDL1 expression. Furthermore, adoptive transfer of B-1 cells lacking TCF1 and LEF1 fails to suppress brain inflammation. These transcription factors are also expressed in human chronic lymphocytic leukaemia B cells and in a B-1-like population that is abundant in pleural fluid and circulation of some patients with pleural infection. Our findings define a TCF1–LEF1-driven transcriptional program that integrates stemness and regulatory function in B-1a cells. The transcription factors TCF1 and LEF1 promote self-renewal and regulatory functions in B-1a cells.