BACKGROUND: Cytokine licensing with pro-inflammatory molecules, such as tumour necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ), has emerged as a promising strategy to enhance the therapeutic potential of multipotent mesenchymal stromal cells (MSCs). While licensing has demonstrated benefits for immunomodulation, its effects on other key MSC functions, including differentiation and paracrine activity, remain incompletely explored. In this study, we evaluated the transcriptomic, metabolomic, and functional changes induced by short-term TNF-α/IFN-γ priming of Wharton's jelly-derived MSCs (WJ-MSCs). METHODS: WJ-MSCs were expanded and exposed to TNF-α and IFN-γ (10 ng/ml each) for 24 h. Transcriptomic analysis was performed using RNA sequencing to identify differentially expressed genes related to immune modulation and lineage commitment. Metabolomic profiling was conducted using high-resolution mass spectrometry to assess changes in metabolic pathways. Functional assays evaluated the effects of cytokine priming on induced differentiation and growth factor secretion. RESULTS: Cytokine licensing induced notable alterations in gene expression, upregulating pathways linked to immune response, inflammation, and cytokine signalling. However, short-term cytokine treatment significantly attenuated the osteogenic and adipogenic differentiation of MSCs, as evidenced by the reduced expression of RUNX2, ALP, CEBPA, and PPARG. The priming had a negligible effect on EGF, FGF-2, HGF, LIF, and SCF secretion. The production of VEGF-A and VEGF-C was elevated, although the levels remained low. Metabolomic analysis revealed enhanced kynurenine pathway activity, indicative of increased tryptophan catabolism, accompanied by elevated levels of fatty acids and polyamines. CONCLUSIONS: Our findings demonstrate that TNF-α/IFN-γ priming reprograms WJ-MSCs by enhancing their immunomodulatory capacity at the expense of differentiation potential. These results highlight the need for tailored strategies to optimize MSC functionality for specific clinical applications.

• MeSH
• Cell Differentiation * drug effects   MeSH
• Cytokines * pharmacology   MeSH
• Immunomodulation * drug effects   MeSH
• Interferon-gamma * pharmacology   MeSH
• Cells, Cultured MeSH
• Humans MeSH
• Mesenchymal Stem Cells * metabolism   cytology   drug effects   immunology   MeSH
• Tumor Necrosis Factor-alpha * pharmacology   MeSH
• Wharton Jelly * cytology   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

OBJECTIVE: Despite availability of an array of antihypertensive drugs, malignant hypertension remains a life-threatening condition, and new therapeutic strategies for the treatment of malignant hypertension and malignant hypertension-associated organ damage are needed. The aim of the present study was to assess the effects of nitric oxide (NO)-independent soluble guanylyl cyclase (sGC) stimulator on the course of malignant hypertension. The second aim was to investigate if the treatment with sodium-glucose cotransporter type 2 (SGLT2) inhibitor would augment the expected beneficial actions of the sGC stimulation on the course of malignant hypertension. METHODS: As a model of malignant hypertension, Ren-2 transgenic rats (TGR) treated with nonspecific NO synthase inhibitor (Nω-nitro- l -arginine methyl ester, l -NAME) was used. Blood pressure (BP) was monitored by radiotelemetry, and the treatment was started 3 days before administration of l -NAME. RESULTS: The treatment with sGC stimulator BAY 41-8543, alone or combined with SGLT2 inhibitor empagliflozin, abolished malignant hypertension-related mortality in TGR receiving l -NAME. These two treatment regimens also prevented BP increases after l -NAME administration in TGR, and even decreased BP below values observed in control TGR, and prevented cardiac dysfunction and malignant hypertension-related morbidity. The treatment with the SGLT2 inhibitor empagliflozin did not further augment the beneficial actions of sGC stimulator on the course of malignant hypertension-related mortality. CONCLUSION: The treatment with NO-independent sGC stimulator displayed marked protective actions on the course of malignant hypertension-related mortality and malignant hypertension-related cardiac damage. This suggests that application of sGC stimulator could be a promising therapeutic means for the treatment of malignant hypertension.

• MeSH
• Benzhydryl Compounds pharmacology   MeSH
• Sodium-Glucose Transporter 2 Inhibitors MeSH
• Glucosides pharmacology   therapeutic use   MeSH
• Hypertension, Malignant * prevention & control   drug therapy   MeSH
• Blood Pressure drug effects   MeSH
• Rats MeSH
• Morpholines MeSH
• NG-Nitroarginine Methyl Ester pharmacology   MeSH
• Rats, Transgenic MeSH
• Pyrazoles * pharmacology   therapeutic use   MeSH
• Pyrimidines * therapeutic use   pharmacology   MeSH
• Soluble Guanylyl Cyclase * metabolism   MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Male MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

Sekvenování genů 16S, 18S a ITS regionů se stalo jedním z nejdůležitějších nástrojů v molekulární diagnostice, zejména v oblasti mikrobiologie, patologie a soudního lékařství. Výše zmíněné geny, obsahující konzervované i variabilní oblasti, jsou hojně využívány pro taxonomické zařazení bakterií a eukaryot. Sekvenování 16S rDNA umožňuje detekci bakteriálních infekcí, zatímco sekvenace ITS regionů a 18S rDNA je využívána při identifikaci mykotických, případně parazitárních infekcí, a to především v případech, kdy tradiční metody selhávají. Tento článek se zaměřuje na rozšířené možnosti těchto metod, jejich uplatnění v klinické diagnostice a výzkumu, zkoumá výhody a nevýhody, a diskutuje potenciální budoucí vývoj v oblasti technologie sekvenování nové generace (NGS).

Gene sequencing of 16S, 18S, and ITS regions is a crucial tool in molecular diagnostics, especially in microbiology, pathology and forensic medicine. These genes contain conserved and variable regions and are widely used for the taxonomic classification of bacteria and eukaryotes. Sequencing of 16S rDNA helps detect bacterial infections, while sequencing of ITS regions and 18S rDNA is used to identify fungal or parasitic infections, especially when traditional methods are ineffective. This article focuses on the expanded possibilities of these methods, their application in clinical diagnostics and research, their advantages and disadvantages, and discusses potential future developments in the field of next-generation sequencing (NGS) technology.

• Keywords
• Internal Transcribed Spacers, 16S rDNA, 18S rDNA,
• MeSH
• Molecular Diagnostic Techniques classification   methods   MeSH
• DNA, Bacterial analysis   genetics   classification   MeSH
• DNA analysis   genetics   classification   ultrastructure   MeSH
• Communicable Diseases * diagnosis   genetics   MeSH
• Humans MeSH
• High-Throughput Nucleotide Sequencing * classification   methods   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Review MeSH

For patients with advanced stage non-small cell lung cancer (NSCLC), treatment strategies have changed significantly due to the introduction of targeted therapies and immunotherapy. In the last few years, we have seen an explosive growth of newly introduced targeted therapies in oncology and this development is expected to continue in the future. Besides primary targetable aberrations, emerging diagnostic biomarkers also include relevant co-occurring mutations and resistance mechanisms involved in disease progression, that have impact on optimal treatment management. To accommodate testing of pending biomarkers, it is necessary to establish routine large-panel next-generation sequencing (NGS) for all patients with advanced stage NSCLC. For cost-effectiveness and accessibility, it is recommended to implement predictive molecular testing using large-panel NGS in a dedicated, centralized expert laboratory within a regional oncology network. The central molecular testing center should host a regional Molecular Tumor Board and function as a hub for interpretation of rare and complex testing results and clinical decision-making.

• Publication type
• Journal Article MeSH
• Review MeSH

BACKGROUND/AIM: Targeted therapy has become increasingly important in treating lung adenocarcinoma, the most common subtype of lung cancer. Next-generation sequencing (NGS) enables precise identification of specific genetic alterations in individual tumor tissues, thereby guiding targeted therapy selection. This study aimed to analyze mutations present in adenocarcinoma tissues using NGS, assess the benefit of targeted therapy and evaluate the progress in availability of targeted therapies over last five years. PATIENTS AND METHODS: The study included 237 lung adenocarcinoma patients treated between 2018-2020. The Archer FusionPlex CTL panel was used for NGS analysis. RESULTS: Gene variants covered by the panel were detected in 57% patients and fusion genes in 5.9% patients. At the time of the study, 34 patients (14.3% of patients) were identified with a targetable variant. Twenty-five patients with EGFR variants, 8 patients with EML4-ALK fusion and one patient with CD74-ROS1 fusion received targeted therapy. Prognosis of patients at advanced stages with EGFR variants treated by tyrosine kinase inhibitors and patients with EML4-ALK fusion treated by alectinib was significantly favorable compared to patients without any targetable variant treated by chemotherapy (p=0.0172, p=0.0096, respectively). Based on treatment guidelines applicable in May 2023, the number of patients who could profit from targeted therapy would be 64 (27.0% of patients), this is an increase by 88% in comparison to recommendations valid in 2018-2020. CONCLUSION: As lung adenocarcinoma patients significantly benefit from targeted therapy, the assessment of mutational profiles using NGS could become a crucial approach in the routine management of oncological patients.

• MeSH
• Adenocarcinoma of Lung * drug therapy   genetics   MeSH
• ErbB Receptors genetics   MeSH
• Humans MeSH
• Mutation MeSH
• Lung Neoplasms * drug therapy   genetics   pathology   MeSH
• Proto-Oncogene Proteins genetics   MeSH
• Receptor Protein-Tyrosine Kinases genetics   MeSH
• Protein-Tyrosine Kinases genetics   MeSH
• High-Throughput Nucleotide Sequencing MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

Micro-electromembrane extraction (μ-EME) was presented for the selective extraction of four main β-lactam antibiotics (penicillin, phenoxypenicillin, ampicillin, and amoxicillin) from complex samples. A volatile solvent (ethyl acetate or chloroform) was sandwiched between a plug of the complex sample and another plug of an aqueous acceptor solution in a transparent polymeric tube and formed the so-called free liquid membrane (FLM). The use of the FLM eliminated the evaporation of the solvent and enabled the μ-EME of the antibiotics, which was carried out by the application of DC voltage to the terminal aqueous solutions. The drugs in the complex sample were selectively transferred through the FLM to the acceptor solution, which was directly used for their determination by micellar electrokinetic chromatography with ultraviolet detection (MEKC-UV). The μ-EME was characterized by sub-μA electric currents, high elimination of matrix components, high stability of operational solutions, and suitability for extracting undiluted complex samples. The μ-EME/MEKC-UV method yielded good analytical repeatability (RSDs of peak areas ≤5%), extraction recoveries (40-84%), accuracy (92-105%) and linearity over one and a half order of magnitude (R2 ≥ 0.9998), and was applied to the determination of the four β-lactam antibiotics in human serum and waste water at clinically and environmentally relevant concentration levels. Further improvement in the method sensitivity was achieved by changing the μ-EME tube geometry (conical shape) and increasing the complex sample volume (100 μL). The analytes were enriched by factors of 7.6-11.5, the limits of detection dropped down to less than 18 ng/mL, and the modified μ-EME/MEKC-UV method enabled the trace determination of β-lactam antibiotics in complex samples.

• MeSH
• Anti-Bacterial Agents MeSH
• beta-Lactams MeSH
• Electricity * MeSH
• Humans MeSH
• Membranes, Artificial * MeSH
• Solvents MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

Epidemie onemocnění COVID-19 znamená globální krizi veřejného zdraví. Proto je věnována pozornost preventivní opatřením, která by mohla přispět k redukci rizika infekce a příznivě ovlivnit průběh onemocnění. Aktuálně je věnována pozornost využití nutraceutik, především vitaminu D, minerálům a beta glukanům. V této studii sledujeme vztah hladin vitaminu D u imunodeficitních pacientů k riziku vzniku onemocnění COVID-19. V souboru 71 pacientů nacházíme gradaci vzniku onemocnění a jeho průběhu u pacientů s hodnotami vitaminu D nižšími než 30 ng/ml. U jedinců s hladinou vitaminu D vyšší než 40 ng/ml zjišťujeme vysokou míru protekce a příznivý průběh klinických projevů po aplikaci minerálů a beta glukanů.

The COVID-19 outbreak marks a global public health crisis. Therefore, consideration is given to preventative measures that could contribute to reducing the risk of infection and positively influence the course of the disease. Attention is currently being paid to the use of nutraceuticals, mainly vitamin D, minerals and beta glucans. In this study, we monitor the relationship of vitamin D levels in immunodeficient patients to the risk of developing COVID-19. In a set of 71 patients, we find gradation of disease onset and progression in patients with values less than 30 ng/mL. In individuals with vitamin D levels above 40 ng/mL, we find a high level of protection, and a beneficial course of clinical manifestation stemming from the application of minerals and beta glucans.

• MeSH
• COVID-19 * prevention & control   MeSH
• Humans MeSH
• Vitamin D Deficiency MeSH
• Immunologic Deficiency Syndromes drug therapy   MeSH
• Vitamin D * analysis   metabolism   therapeutic use   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Review MeSH

Oxidative stress is an important toxicity and genotoxicity mechanism of many chronic adverse health outcomes. This study developed a sensitive extraction method for urine matrix (based on lyophilization, without the need for pre-cleaning by solid phase extraction), coupled to LC-MS/MS analysis of the biomarker 8-hydroxy-2'-deoxyguanosine (8-OHdG). The methodology was validated in urine samples from a cohort of Spanish pregnant women collected during the first, second and third trimester of pregnancy, and urine samples collected within 24 h after delivery (n = 85). A detection and quantification limit of 0.01 and 0.05 μg/L, respectively, were established. The median 8-OHdG concentration was 2.18 μg/L (range 0.33-7.79); and the corresponding creatinine-adjusted concentrations ranged from 1.04 to 13.12 with median of 4.48 μg 8-OHdG/g creatinine. The concentrations of non-adjusted 8-OHdG significantly decreased (p < 0.05) in the 3rd trimester and post-delivery urine samples when compared to the 1st trimester levels. 8-OHdG concentrations were further studied in placenta samples matching the same urine samples (n = 26), with a median value of 1.3 ng 8-OHdG/g of tissue. Placental 8-OHdG concentrations were correlated with urinary levels of non-adjusted 8-OHdG in the 3rd trimester. Considering the small cohort size, results must be interpreted with caution, however statistical analyses revealed elevated urinary non-adjusted 8-OHdG levels in the 1st trimester of mothers that delivered boys compared to those who delivered girls (p < 0.01). Increased urinary non-adjusted 8-OHdG concentrations at the time of delivery were significantly associated with clinical records (any type of clinical record during pregnancy; p < 0.05). The novel extraction and analytical method for the assessment of 8-OHdG is applicable for sensitive analysis of multiple analytes or biomarkers in urine matrix. This method could also be applied for other matrices such as blood or tissues. Our findings show that 8-OHdG in urine of pregnant women could predict oxidative stress in placenta and can be related to characteristics such as maternal obesity, mode of delivery and newborn sex.

• MeSH
• 8-Hydroxy-2'-Deoxyguanosine MeSH
• Biomarkers urine   MeSH
• Chromatography, Liquid methods   MeSH
• Deoxyguanosine * urine   MeSH
• Creatinine urine   MeSH
• Humans MeSH
• Infant, Newborn MeSH
• Oxidative Stress MeSH
• Placenta MeSH
• DNA Damage MeSH
• Tandem Mass Spectrometry methods   MeSH
• Pregnancy MeSH
• Pregnant People * MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Infant, Newborn MeSH
• Pregnancy MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

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

Gaussian and exponentially modified Gaussian functions were incorporated into integrating algorithms used by an open-source, cross-platform tool called CycloBranch. The quantitation is demonstrated on bacterial pyoverdines separated by fine isotope features. Using our algorithm, we can separate the m/z values 694.25802 and 694.26731 (a 0.009 Da difference), where the former belongs to the most intense peak of pyoverdine D (PvdD), and the latter to the second most intense peak of pyoverdine E (PvdE) in the respective isotopic clusters of [M + Fe-H]2+ ions. The areas under chromatographic curves of standards were analyzed for the limit of detection (LOD), limit of quantitation (LOQ), and regression coefficient calculations. The quantitative module returned a LOD and LOQ of 1.4 and 4.3 ng/mL, respectively, for both PvdD and PvdE in human urine. If present and detected in mass spectra, the intensities of user-defined [M + H]+, [M + Na]+, [M + K]+, [M + Fe-H]2+, or other ion types, can be accumulated and used for quantitation. The quantitation result is returned by CycloBranch in seconds or minutes, contrary to an hours-long manual approach, prone to user-born errors originating from necessary copying among various software environments. Native Bruker, Waters, Thermo, txt, mgf, mzML, and mzXML data formats are supported in CycloBranch, which is freely available at https://ms.biomed.cas.cz/cyclobranch.

• MeSH
• Algorithms * MeSH
• Chromatography, Liquid methods   MeSH
• Mass Spectrometry methods   MeSH
• Isotopes MeSH
• Humans MeSH
• Software * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH