INTRODUCTION: The prevalence of metabolic dysfunction-associated steatotic liver disease (MASLD) may be as high as 38% in the adult population with potential serious complications, multiple comorbidities and a high socioeconomic burden. However, there is a general lack of awareness and knowledge about MASLD and its progressive stages (metabolic dysfunction-associated steatohepatitis (MASH) and fibrosis). Therefore, MASLD is still far underdiagnosed. The 'Global Research Initiative for Patient Screening on MASH' (GRIPonMASH) consortium focuses on this unmet public health need. GRIPonMASH will help (primary) healthcare providers to implement a patient care pathway, as recommended by multiple scientific societies, to identify patients at risk of severe MASLD and to raise awareness. Furthermore, GRIPonMASH will contribute to a better understanding of the pathophysiology of MASLD and improved identification of diagnostic and prognostic markers to detect individuals at risk. METHODS: This is a prospective multicentre observational study in which 10 000 high-risk patients (type 2 diabetes mellitus, obesity, metabolic syndrome or hypertension) will be screened in 10 European countries using at least two non-invasive tests (Fibrosis-4 index and FibroScan). Blood samples and liver biopsy material will be collected and biobanked, and multiomics analyses will be conducted. ETHICS AND DISSEMINATION: The study will be conducted in compliance with this protocol and applicable national and international regulatory requirements. The study initiation package is submitted at the local level. The study protocol has been approved by local medical ethical committees in all 10 participating countries. Results will be made public and published in scientific, peer-reviewed, international journals and at international conferences. REGISTRATION DETAILS: NCT05651724, registration date: 15 Dec 2022.

• MeSH
• Diabetes Mellitus, Type 2 complications   MeSH
• Liver Cirrhosis diagnosis   MeSH
• Humans MeSH
• Metabolic Syndrome complications   MeSH
• Multicenter Studies as Topic MeSH
• Non-alcoholic Fatty Liver Disease * diagnosis   MeSH
• Mass Screening * methods   MeSH
• Prospective Studies MeSH
• Research Design MeSH
• Fatty Liver * diagnosis   epidemiology   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Clinical Trial Protocol MeSH

Epilepsy, affecting over 50 million people globally, presents a significant neurological challenge. Effective prevention of epileptic seizures relies on proper administration and monitoring of Anti-Seizure Medication (ASMs). Therapeutic Drug Monitoring (TDM) ensures optimal dosage adjustment, minimizing adverse effects and potential drug interactions. While traditional venous blood collection for TDM may be stressful, emerging alternative sampling methods, particularly Dried Blood Spot (DBS) or oral fluid offer less invasive way of sampling. This study aimed to develop and validate an analytical method for the determination of lamotrigine in such alternative samples. The sample, either DBS or oral fluid, was subjected to extraction, evaporation, and reconstitution in 15 % acetonitrile containing 0.1 % formic acid. A Kinetex C18 Polar column was used for liquid chromatographic separation and MS in ESI+ mode was used for detection and quantitation of lamotrigine using an isotopically labelled internal standard according to EMA guidelines. The calibration range of the developed method enables the determination of lamotrigine in the concentration range of 1-30 μg/mL in DBS and 0.5-20 μg/mL in oral fluid. Oral fluid and DBS samples from patients treated with lamotrigine analysed by the developed method were compared to plasma concentrations measured by the hospital's accredited laboratory. Preliminary results indicate a promising potential for these alternative matrices in clinical TDM applications. By offering a less invasive sampling approach, this method improves the accessibility and safety of pharmacotherapy for epilepsy patients. The results of this study lay the foundation for further clinical applications by implementing alternative matrix TDM, which may significantly advance personalized care in epilepsy management.

• MeSH
• Anticonvulsants * analysis   blood   MeSH
• Chromatography, Liquid methods   MeSH
• Epilepsy drug therapy   MeSH
• Calibration MeSH
• Liquid Chromatography-Mass Spectrometry MeSH
• Lamotrigine * analysis   blood   MeSH
• Humans MeSH
• Limit of Detection MeSH
• Drug Monitoring * methods   MeSH
• Reproducibility of Results MeSH
• Saliva * chemistry   MeSH
• Tandem Mass Spectrometry methods   MeSH
• Dried Blood Spot Testing * methods   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Validation Study MeSH

No study has systematically compared the suitability of DNA methylation (DNAme) profiles in non-invasive samples for the detection of breast cancer (BC). We assess non-tumour DNAme in 1,100 cervical, buccal, and blood samples from BC cases and controls and find that cervical samples exhibit the largest nuber of differentially methylated sites, followed by buccal samples. No sites were significant in blood after FDR adjustment. Deriving DNAme-based classifiers for BC detection in each sample type (WID-buccal-, cervical-, or blood-BC), we achieve validation AUCs of 0.75, 0.66, and 0.51, respectively. Buccal and cervical BC-associated DNAme alterations distinguish between BC cases and controls in both surrogate and breast tissue (AUC > 0.88), yet individual sites and the directionality of methylation changes are not identical between these two sample types, and buccal sample DNAme aligns with breast methylation changes more closely. Pending additional validation, these insights may have the potential to improve non-invasive personalized BC prevention.

• MeSH
• Adult MeSH
• Epigenesis, Genetic * MeSH
• Epigenomics * methods   MeSH
• Middle Aged MeSH
• Humans MeSH
• DNA Methylation * MeSH
• Breast Neoplasms * genetics   diagnosis   MeSH
• Case-Control Studies MeSH
• Mouth Mucosa metabolism   MeSH
• Check Tag
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Female MeSH
• Publication type
• Journal Article MeSH

Invasive fungal disease (IFD) presents a life-threatening condition in immunocompromised patients, thus often prompting empirical administration of antifungal treatment, without adequate mycological evidence. Over the past years, wide use of antifungal prophylaxis resulted in decreased occurrence of IFD but has contributed to changes in the spectrum of fungal pathogens, revealing the occurrence of previously rare fungal genera causing breakthrough infections. The expanding spectrum of clinically relevant fungal pathogens required the implementation of screening approaches permitting broad rather than targeted fungus detection to support timely onset of pre-emptive antifungal treatment. To address this diagnostically important aspect in a prospective setting, we analyzed 935 serial peripheral blood (PB) samples from 195 pediatric and adult patients at high risk for IFD, involving individuals displaying febrile neutropenia during treatment of hematological malignancies or following allogeneic hematopoietic stem cell transplantation. Two different panfungal-PCR-screening methods combined with ensuing fungal genus identification by Sanger sequencing were employed. In the great majority of PB-specimens displaying fungal DNAemia, the findings were transient and revealed fungi commonly regarded as non-pathogenic or rarely pathogenic even in the highly immunocompromised patient setting. Hence, to adequately exploit the diagnostic potential of panfungal-PCR approaches for detecting IFD, particularly if caused by hitherto rarely observed fungal pathogens, it is necessary to confirm the findings by repeated testing and to identify the fungal genus present by ensuing analysis. If applied appropriately, panfungal-PCR-screening can help prevent unnecessary empirical therapy, and conversely, contribute to timely employment of effective pre-emptive antifungal treatment strategies.

• MeSH
• Antifungal Agents therapeutic use   MeSH
• Child MeSH
• DNA, Fungal * analysis   MeSH
• Adult MeSH
• Febrile Neutropenia * microbiology   MeSH
• Hematologic Neoplasms complications   MeSH
• Fungi isolation & purification   genetics   MeSH
• Immunocompromised Host * MeSH
• Invasive Fungal Infections epidemiology   prevention & control   etiology   microbiology   MeSH
• Middle Aged MeSH
• Humans MeSH
• Adolescent MeSH
• Young Adult MeSH
• Child, Preschool MeSH
• Prevalence MeSH
• Prospective Studies MeSH
• Aged MeSH
• Hematopoietic Stem Cell Transplantation adverse effects   MeSH
• Check Tag
• Child MeSH
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Adolescent MeSH
• Young Adult MeSH
• Male MeSH
• Child, Preschool MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Letter MeSH

Colorectal cancer (CRC) is the second most prevalent cancer type worldwide, which highlights the urgent need for non-invasive biomarkers for its early detection and improved prognosis. We aimed to investigate the patterns of long non-coding RNAs (lncRNAs) in small extracellular vesicles (sEVs) collected from low-volume blood serum specimens of CRC patients, focusing on their potential as diagnostic biomarkers. Our research comprised two phases: an initial exploratory phase involving RNA sequencing of sEVs from 76 CRC patients and 29 healthy controls, and a subsequent validation phase with a larger cohort of 159 CRC patients and 138 healthy controls. Techniques such as dynamic light scattering, transmission electron microscopy, and Western blotting were utilized for sEV characterization. Optimized protocol for sEV purification, RNA isolation and preamplification was applied to successfully sequence the RNA content of sEVs and validate the results by RT-qPCR. We successfully isolated sEVs from blood serum and prepared sequencing libraries from a low amount of RNA. High-throughput sequencing identified differential levels of 460 transcripts between CRC patients and healthy controls, including mRNAs, lncRNAs, and pseudogenes, with approximately 20% being lncRNAs, highlighting several tumor-specific lncRNAs that have not been associated with CRC development and progression. The validation phase confirmed the upregulation of three lncRNAs (NALT1, AL096828, and LINC01637) in blood serum of CRC patients. This study not only identified lncRNA profiles in a population of sEVs from low-volume blood serum specimens of CRC patients but also highlights the value of innovative techniques in biomolecular research, particularly for the detection and analysis of low-abundance biomolecules in clinical samples. The identification of specific lncRNAs associated with CRC provides a foundation for future research into their functional roles in cancer development and potential clinical applications.

• MeSH
• Biomarkers MeSH
• Extracellular Vesicles * genetics   MeSH
• Colorectal Neoplasms * diagnosis   genetics   MeSH
• Humans MeSH
• RNA, Long Noncoding * genetics   MeSH
• Neoplasms, Second Primary * MeSH
• Serum MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

Přestože adukty s globinem jsou v preventivní medicíně široce využívány jako biomarkery dlouhodobé expozice reaktivním látkám, jejich další osud v organismu byl poprvé prozkoumán až v našem předchozím projektu. Prokázali jsme, že hydrolytickým štěpením globinových aduktů u potkanů vznikají adukty s aminokyselinami nebo s dipeptidy, vylučované močí. Nyní bude studováno vylučování těchto štěpných produktů u člověka. Budou vypracovány a do hygienické praxe v ČR zavedeny nové strategie biologického monitorování expozice průmyslovým škodlivinám ethylenoxidu a N,N-dimethylformamidu, kdy dosavadní odběry krve budou nahrazeny analýzou moče. Ze získaných údajů bude odvozen obecný matematicko-statistický model umožňující interpretovat nálezy štěpných produktů odvozených od dalších látek včetně karcinogenů, na něž se v projektu též zaměříme. Zavedení zcela nové kategorie biomarkerů specifických pro výchozí látky a vhodných pro hodnocení dlouhodobých expozic pomocí neinvazivního odběru vzorků bude významným přínosem pro prevenci poškození zdraví účinkem chemických látek v mezinárodním měřítku.; Although the adducts with globin have been widely used in preventive medicine as biomarkers of long-term exposures to reactive chemicals, we were the first to explore their subsequent fate in the organism. We found in rats that hydrolytic cleavage of the globin adducts produces amino acid or dipeptide adducts that are excreted in urine. In our current project we will investigate urinary excretion of these cleavage products in humans. First, we plan to develop and implement into public health practice new strategies of biomonitoring of industrial toxicants ethylene oxide and N,N-dimethylformamide, based on analysis of urine instead of blood. Obtained data will be used to create a general statistical model allowing to interpret urinary levels of cleavage products from other chemicals including carcinogens. Implementation of a novel promising category of biomarkers that combine specificity for the parent xenobiotic, monitoring of long-term exposures, and non-invasive sampling will significantly contribute to prevention of health damage due to exposures to chemicals.

• Keywords
• biologické monitorování, globinové adukty, štěpné produkty, ethylenoxid, N,N-dimethylformamid, biological monitoring, globin adducts, cleavage products, ethylene oxide, N,N-dimethylformamide,

• NML Publication type
• závěrečné zprávy o řešení grantu AZV MZ ČR

This paper focuses on non-invasive blood glucose determination using photoplethysmographic (PPG) signals, which is crucial for managing diabetes. Diabetes stands as one of the world’s major chronic diseases. Untreated diabetes frequently leads to fatalities. Current self-monitoring techniques for measuring diabetes require invasive procedures such as blood or bodily fluid sampling, which may be very uncomfortable. Hence, there is an opportunity for non-invasive blood glucose monitoring through smart devices capable of measuring PPG signals. The primary goal of this research was to propose methods for glycemic classification into two groups (low and high glycemia) and to predict specific glycemia values using machine learning techniques. Two datasets were created by measuring PPG signals from 16 individuals using two different smart devices – a smart wristband and a smartphone. Simultaneously, the reference blood glucose levels were invasively measured using a glucometer. The PPG signals were preprocessed, and 27 different features were extracted. With the use of feature selection, only 10 relevant features were chosen. Numerous machine learning models were developed. Random Forest (RF) and Support Vector Machine (SVM) with the radial basis function (RBF) kernel performed best in classifying PPG signals into two groups. These models achieved an accuracy of 76% (SVM) and 75% (RF) on the smart wristband test dataset. The functionality of the proposed models was then verified on the smartphone test dataset, where both models achieved similar accuracy: 74% (SVM) and 75% (RF). For predicting specific glycemia values, RF performed best. Mean Absolute Error (MAE) was 1.25 mmol/l on the smart wristband test dataset and 1.37 mmol/l on the smartphone test dataset.

Biomonitoring of human exposure to reactive electrophilic chemicals such as ethylene oxide (EO) has been commonly based on the determination of adducts with N-terminal valine in blood protein globin, but a systematic search has also been undertaken to find surrogate markers enabling non-invasive sampling. Recently, N-(2-hydroxyethyl)-L-valyl-L-leucine (HEVL) has been identified as an ultimate cleavage product of EO-adducted globin in the urine of occupationally exposed workers. Herein, full validation of the analytical procedure consisting of solid-phase extraction of HEVL from urine samples (2 mL) followed by high-performance liquid chromatography-electrospray ionization-high-resolution mass spectrometry determination using deuterium-labeled HEVL as an internal standard (IS) is described. Method limit of quantitation is 0.25 ng/mL, and its selectivity is excellent as demonstrated by the invariable ratio of the qualifier and quantifier ion intensities across diverse urine samples and synthetic standard. The linear calibration model was applicable over the whole concentration range tested (0.25-10 ng/mL). The method accuracy assessed as a recovery of HEVL using a spiking experiment was 98-100%. Within-day precision of the method ranged from 1.8% to 3.0%, while the results from consecutive analytical runs conducted within 1 week or within 10-150 weeks differed in the range of 2.2-9.7%. The stability study on urine samples (-20°C up to 3 years, freeze-and-thaw up to 10 cycles) as well as on aqueous solutions (5°C up to 4 months) indicated no relevant changes in HEVL concentration (≤4%) over the time tested. Analytical responses of both HEVL and IS correlated with urinary creatinine as an index of matrix composition, but this matrix effect was mostly eliminated using the HEVL/IS peak area ratio, attaining the IS-normalized relative matrix effect <3%. In conclusion, the method complied successfully with the bioanalytical method validation criteria, making it a reliable tool for HEVL determination in human biomonitoring.

• MeSH
• Dipeptides * MeSH
• Ethylene Oxide * MeSH
• Globins MeSH
• Leucine MeSH
• Humans MeSH
• Reproducibility of Results MeSH
• Chromatography, High Pressure Liquid MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

BACKGROUND/AIM: Non-invasive circulating tumor biomarkers in liquid biopsy, such as microRNAs (miRNA), provide for better personalization of treatment strategies. The aim of our study was to assess the prognosis of patients with melanoma undergoing tumor resection with curative intent based on analysis of selected circulating miRNAs. PATIENTS AND METHODS: A total of 22 patients with stage I to III melanoma were enrolled into this prospective study. Plasma samples were obtained pre-surgery and early post-surgery from peripheral blood draws. A panel of 23 candidate miRNAs was designed and expression of miRNAs were analyzed by reverse transcription-quantitative polymerase chain reaction with exogenous reference control cel-miR-39-3p. RESULTS: Higher preoperative expression levels of miR-99a (p=0.008), miR-320 (p=0.009), miR-1908 (p=0.001), miR-494 (p=0.018) and miR-4487 (p=0.048) were associated with a shorter disease-free interval. Similarly, higher preoperative plasma levels of miR-99a (p=0.017), miR-221 (p=0.026), miR-320 (p=0.016), miR-494 (p=0.009), miR-1260 (p=0.026) and miR-1908 (p=0.024) were associated with worse overall survival. No significant differences between pre- and postoperative plasma miRNA levels were observed. CONCLUSION: Liquid biopsy is a minimally-invasive approach which can lead to a better understanding of cancer behavior and offers the possibility of precise patient prognosis, allowing selection of the most appropriate treatment. Our study showed that preoperative plasma levels of miR-99a, miR-221, miR-320, miR-494, miR-1908 and miR-4487 were associated with disease-free interval and overall survival of patients with early-stage melanoma. This approach may help in decision-making about the appropriateness of modern adjuvant treatment administration in patients with resectable melanoma.

• MeSH
• Circulating MicroRNA * MeSH
• Humans MeSH
• Melanoma * genetics   surgery   MeSH
• MicroRNAs * metabolism   MeSH
• Biomarkers, Tumor genetics   MeSH
• Prognosis MeSH
• Prospective Studies MeSH
• Gene Expression Profiling MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

Analysis of dried urine spots (DUSs) is becoming an emerging technique in clinical, toxicological, and forensic chemistry due to the fully non-invasive collection, facile transportation, and simple storage of DUS samples. Correct DUS collection and elution is of the utmost importance because inadequate DUS sampling/processing may have direct consequences on quantitative DUS analyses and these aspects were, for the first time, comprehensively investigated in this contribution. Various groups of endogenous and exogenous species were selected as model analytes and their concentrations were monitored in DUSs collected on standard cellulose-based sampling cards. Strong chromatographic effects were observed for most analytes having a crucial impact on their distribution within the DUSs during sampling. Concentrations of target analytes were up to 3.75-fold higher in the central DUS sub-punch in comparison to the liquid urine. Consequently, substantially reduced concentrations of these analytes were determined in peripheral DUS sub-punches demonstrating that sub-punching, often applied to dried material spots, is not acceptable for quantitative DUS analyses. Hence, a simple, rapid, and user-friendly procedure was suggested, which employed an in-vial collection of a known urine volume on a pre-punched sampling disc (using a low-cost micropipette designed for patient-centric clinical sampling) and in-vial processing of the whole DUS. Excellent accuracy (0.20%) and precision (0.89%) of liquid transfers were achieved by the micropipette, which was also applied to remote DUS collection by laic and expert users. The resulting DUS eluates were analysed by capillary electrophoresis (CE) for the determination of endogenous urine species. The CE results demonstrated no significant differences between the two user groups, elution efficiencies of 88-100% (in comparison to the liquid urine), and precision better than 5.5%.

• MeSH
• Humans MeSH
• Specimen Handling * MeSH
• Body Fluids * MeSH
• Dried Blood Spot Testing methods   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH