Prostate cancer ranks as the most diagnosed oncological disease in Slovak men and the third most common cause of their cancer related mortality. G-protein coupled estrogen receptor 1 (GPER1) is estrogen receptor mediating fast non-genomic estrogen signaling and shown potential as therapeutic target for later stages of the disease and in chemoprevention. However, little is currently known about the impact of genetic variability in the GPER1 gene on the prostate cancer risk and disease characteristics. Genomic DNA samples isolated from peripheral blood of 701 prostate cancer patients and 659 healthy men were used for the single nucleotide polymorphism (SNP) analysis. The presence of GPER1 SNPs (rs3808350, rs3808351, rs11544331) was determined by ARMS-PCR. GPER1 expression was quantified by semi-quantitative RT-PCR using mRNA isolated from prostate cancer tissues and using benign prostate hyperplasia tissues as a control. The GPER1 SNP rs3808350 was associated with a higher risk of developing prostate cancer, especially in patients with a Gleason score ≥ 7 and with nodal and distant metastases. No association was found between the rs3808351 and rs11544331 and prostate cancer. No significant changes in GPER1 expression were found. The GPER1 rs3808350 was associated with a higher risk of prostate cancer as well as with its characteristics and appears to influence prostate cancer susceptibility and aggressiveness. In the future, these findings could help in personalized treatment and prognosis determination.

Anew in vitromodel for modeling Duchenne muscular dystrophy, derived from pediatric patient with a unique mutation in DMD gene is introduced. Peripheral blood was used as asource for primary cells (mononuclear cells) and subsequently reprogrammed into induced pluripotent stem cells with synthetic Sendai vector. Reprogrammed iPS cells showed the expression of pluripotency factors and the ability to differentiateinto all three germ layers.

The purpose of this study was to investigate association between the aromatase (CYP19A1) rs2414096 polymorphism and the risk of prostate cancer development, progression and aggressiveness in the Slovak population. Aromatase catalyses the final reaction of androgens aromatization to estrogens, irreversible conversion of androstenedione to estrone and testosterone to estradiol. The study population consisted of 721 prostate cancer patients and 660 men in the control group. Genotyping was performed via restriction fragment length polymorphism analysis. The expression levels of CYP19A1 in prostate cancer tissues were compared with those in benign prostatic hyperplasia tissues. The CYP19A1 rs2414096 AA genotype and A allele were significantly associated with an increased risk of prostate cancer development. The significant association between this genotype and PSA level ≥ 10 ng/ml, Gleason score ≥ 7 and with the presence of metastases was observed. Significantly decreased relative expression of CYP19A1 was detected in prostate cancer tissues. Patients with the AA genotype presented higher relative CYP19A1 expression than did those with the GG genotype. Significantly lower circulating testosterone levels and higher serum PSA levels were detected in patients with the AA genotype. The CYP19A1 rs2414096 polymorphism was significantly associated with prostate cancer development and aggressiveness in Slovak patients.

Radioligand therapy with 177 Lu-Prostate Specific Membrane Antigen is a very promising treatment option for patients with prostate cancer. In the past ten years it started to be used as one of the final options in patients with metastatic, castration resistant prostate cancer, after previously exploiting all the common treatments. In Slovakia, this treatment has been available since the beginning of the year 2020. In this time, we have been able to adopt many of the recommendations from the European guidelines and adapt the rest to the clinical and national legislative requirements. In the time period between February 2020 and June 2024, we treated 104 patients and administered total of 362 cycles of the radioligand therapy with 177 Lu-Prostate Specific Membrane Antigen. Although the numbers of our patients suitable for systemic evaluation are for now rather small, our result has so far showed the correlation with some of the largest multicentric trials. More patients will be included in the further evaluation with upcoming months, and more precise data will be available.

Background and Objectives: Immune checkpoint inhibitors such as PD-1 and TIM-3 play an important role in regulating the host immune response and are proposed as potential prognostic markers and therapeutic targets in severe cases of COVID-19. We evaluated the expression of PD-1 and TIM-3 on T cells, as well as the concentration of sPD-1 in plasma, to clarify the role of these molecules in patients infected with SARS-CoV-2. Materials and Methods: In this retrospective observational study, we analysed the expression of PD-1 and TIM-3 on CD4+ and CD8+ T cells upon admission and after 7 days of hospitalisation in 770 adult patients. We also evaluated sPD-1 levels in the plasma of 145 patients at different stages of COVID-19 and of 11 control subjects. Molecules were determined using conventional flow cytometry and ELISA and the data were statistically processed. Results: We observed a significantly higher expression of PD-1 on CD4+ cells in deceased patients than in those with mild-to-moderate disease. All patients with COVID-19 exhibited a significantly higher expression of TIM-3 on both CD4+ and CD8+ T cells compared to controls. After 1 week of hospitalisation, there was no significant change in PD-1 or TIM-3 expression on CD4+ or CD8+ T cells across the studied groups. sPD-1 concentrations were not significantly different between survivors and non-survivors. Plasma sPD-1 levels did not correlate with PD-1 expression on T cells, but a significant correlation was observed between CD4+ PD-1 and CD8+ PD-1. Using machine-learning algorithms, we supported our observations and confirmed immunological variables capable of predicting survival, with AUC = 0.786. Conclusions: Analysis of the immune response may be useful for monitoring and predicting the course of COVID-19 upon admission. However, it is essential to evaluate complex immune parameters in conjunction with other key clinical and laboratory indicators.

BACKGROUND:Glial fibrillary acidic protein (GFAP) and ubiquitin C-terminal hydrolase-L1 (UCH-L1) are blood biomarkers that able to aid in the assessment of mild traumatic brain injury (mTBI) patients and reduce computed tomography (CT) overuse. OBJECTIVES:The aim of this study was to evaluate the predictive performance of individual biomarkers and their combination (i.e. mTBI assay) in detecting clinically significant intracranial injuries in mTBI. Furthermore, the influence of older age on the predictive performance of individual biomarkers and their combination was investigated. METHODS:This prospective multicenter study was conducted in 12 European healthcare centers. Adults with suspected mTBI presenting to the emergency department (ED) of each participating healthcare center within 12 h of head trauma were enrolled. GFAP and UCH-L1 were determined in blood samples collected from each participant. Head CT was considered as reference standard for the presence of intracranial injury. RESULTS:The mTBI assay yielded the highest sensitivity [95.5%, 95% confidence interval (CI): 89.9-98.5] and the highest negative predictive value (NPV) value (97.3%, 95% CI: 93.9-98.9) for the exclusion of intracranial lesions in mTBI. The sensitivities and NPVs of individual biomarkers were lower compared with the mTBI assay. In adults over 65 years, the individual biomarkers and the mTBI assay displayed the weakest diagnostic performances. After optimizing cutoff values for the mTBI assay for older adults, the following diagnostic accuracy measures were obtained: sensitivity 87.7%, 95% CI: 77.2-94.5 and NPV: 94.4%, 95% CI: 89.6-97.0 ( P  < 0.001). CONCLUSION:The mTBI assay yielded high sensitivity and NPV for the exclusion of significant intracranial injuries in mTBI patients presenting to the ED within 12 h from injury, performing better than individual biomarkers. A significant age-dependent influence on the predictive performances of the individual biomarkers and the mTBI assay was demonstrated.

Vitamin D deficiency is involved in the pathogenesis of multiple sclerosis (MS), a severe autoimmune demyelinating disease of the central nervous system. The gene polymorphism Cdx-2 (rs11568820, G/A) seriously influences the trancriptional activity of the vitamin D receptor (VDR) that binds the vitamin D responsive elements of target genes including HLA-DRB1*15. The aim of the present study in Slovaks was to analyse the association of Cdx-2 variants with the risk of MS and disability progression, and to assess the DRB1*15:01 allele as a possible confounding factor. In total, 493 MS patients and 417 healthy controls were involved in this study. The genotyping of Cdx-2 was performed using restriction analysis; DRB1*15:01 positivity was determined by a high-resolution melting analysis of its surrogate marker rs3135388 (G/A). Our results did not prove any allelic association between Cdx-2 and a risk of MS (minor allele A - 0.181 in patients vs. 0.161 in controls, OR = 1.15, .95 CI = 0.90-1.47, p = 0.289). The logistic regression analysis, adjusted for sex and age, showed no differences in Cdx-2 genotype counts when using an additive, dominant or recessive genetic model (p = 0.351, 0.150, 0.240 respectively). The Cdx-2 variants were also not associated with disease disability progression, evaluated using the Multiple Sclerosis Severity Score. The HLA-DRB1*15:01 allele was found to strongly increase the risk of MS in our study (0.300 in patients vs. 0.101 in controls, OR = 3.83, .95 CI = 2.94-4.99, p = 1.016 × 10-26, dominant genetic model OR = 4.62, .95 CI = 3.40-6.26, p = 9.1 × 10-23). In summary, we found the Cdx-2 as a single genetic marker not to be associated with MS development or progression in Slovaks, independently of HLA-DRB1*15:01 status.

The role of the cytochrome P450 1A2 (CYP1A2) rs2472299, rs2470890 and rs11072508 polymorphisms in prostate cancer risk, disease progression and tumour development remains unclear. The potential associations of these three CYP1A2 polymorphisms and haplotypes with prostate cancer susceptibility and its clinicopathological characteristics were therefore investigated. The present case-control study consisted of 522 patients with prostate cancer and 554 healthy controls. High-resolution melting analysis was used to determine the CYP1A2 polymorphisms. No significant association in prostate cancer risk was seen for CYP1A2 rs2472299 and rs11072508. However, a significantly decreased risk of prostate cancer was found for CYP1A2 rs2470890 [odds ratio (OR), 0.67; P=0.02] in the recessive model. After analysis of the associations of clinical status and these three CYP1A2 polymorphisms, the CYP1A2 rs2470890 and rs11072508 polymorphisms showed a positive association with a higher Gleason score (rs2470890 OR, 1.36, P=0.04 in the allelic model; rs11072508 OR, 1.37, P=0.04 in the allelic model and OR, 1.60, P=0.03 in the dominant model). All three polymorphisms showed a significant positive association with pathological T stage in the additive, allelic and dominant genetic models (P<0.05). Haplotype analysis revealed that the most common haplotypes 'GTT' and 'ACC' were significantly associated with pathological T stages 3 and 4 (OR, 0.62; P=0.02 and OR, 1.54; P=0.03, respectively). A significant association was found between the 'GTT' haplotype and the Gleason score (OR, 0.71; P=0.03). In conclusion, these CYP1A2 polymorphisms and haplotypes have the potential to predict prostate cancer disease progression.

Pyruvate carboxylase (PC) is an enzyme catalyzing the carboxylation of pyruvate to oxaloacetate. The enzymatic generation of oxaloacetate, an intermediate of the Krebs cycle, could provide the cancer cells with the additional anaplerotic capacity and promote their anabolic metabolism. Recent studies revealed that several types of cancer cells express PC. The gained anaplerotic capability of cells mediated by PC correlates with their expedited growth, higher aggressiveness, and increased metastatic potential. By immunohistochemical staining and immunoblotting analysis, we investigated PC expression among samples of different types of human brain tumors. Our results show that PC is expressed by the cells in glioblastoma, astrocytoma, oligodendroglioma, and meningioma tumors. The presence of PC in these tumors suppose that PC could support the anabolic metabolism of their cellular constituents by its anaplerotic capability.

Pyruvate carboxylase (PC) is a mitochondrial enzyme catalyzing the ATP-dependent reaction of pyruvate prolongation with bicarbonate ion to oxaloacetate. The synthesis of oxaloacetate by PC, an intermediate of the Krebs cycle, is recently recognized as a significant anaplerotic reaction that supports the biosynthetic capability, growth, aggressiveness, and even viability of several cancer cell types. PC expression was confirmed in several types of cancer cells and tumors. To evaluate the possibility that prostate tumor-forming cells are also exploiting the anaplerotic role of PC, we applied immunoblotting analysis to estimate its presence. Our results revealed that PC is present among the lysate proteins derived from prostate cancer and benign prostatic hyperplasia samples. The expression of PC in cells of prostate tumors and benign prostatic hyperplasia supposes that PC could facilitate the formation of oxaloacetate in situ and enhance the autonomy of their biosynthetic metabolism from the availability of extracellular substrates by increasing the cellular anaplerotic capability (Tab. 1, Fig. 1, Ref. 30). Keywords: pyruvate carboxylase, prostate cancer, cancer metabolism, anaplerosis.

Leucine is an essential, ketogenic amino acid with proteinogenic, metabolic, and signaling roles. It is readily imported from the bloodstream into the brain parenchyma. Therefore, it could serve as a putative substrate that is complementing glucose for sustaining the metabolic needs of brain tumor cells. Here, we investigated the ability of cultured human cancer cells to metabolize leucine. Indeed, cancer cells dispose of leucine from their environment and enrich their media with the metabolite 2-oxoisocaproate. The enrichment of the culture media with a high level of leucine stimulated the production of 3-hydroxybutyrate. When 13C6-leucine was offered, it led to an increased appearance of the heavier citrate isotope with a molar mass greater by two units in the culture media. The expression of 3-methylcrotonyl-CoA carboxylase (MCC), an enzyme characteristic for the irreversible part of the leucine catabolic pathway, was detected in cultured cancer cells and human tumor samples by immunoprobing methods. Our results demonstrate that these cancer cells can catabolize leucine and furnish its carbon atoms into the tricarboxylic acid (TCA) cycle. Furthermore, the release of 3-hydroxybutyrate and citrate by cancer cells suggests their capability to exchange these metabolites with their milieu and the capability to participate in their metabolism. This indicates that leucine could be an additional substrate for cancer cell metabolism in the brain parenchyma. In this way, leucine could potentially contribute to the synthesis of metabolites such as lipids, which require the withdrawal of citrate from the TCA cycle.

Increased concentration of plasma homocysteine (Hcy) is an independent risk factor of cardiovascular disease, yet the mechanism by which hyperhomocysteinemia (HHcy) causes cardiac dysfunction is largely unknown. The aim of present study was to investigate the contribution of sarcoplasmic reticulum to impaired cardiac contractile function in HHCy. HHcy-induced by subcutaneous injection of Hcy (0.45 μmol/g of body weight) twice a day for a period of 2 weeks resulted in significant decrease in developed left ventricular pressure and maximum rate of ventricular relaxation. Our results show that abundances of SR Ca2+-handling proteins, Ca2+-ATPase (SERCA2), calsequestrin and histidine-rich calcium-binding protein are significantly reduced while the content of phospholamban is unchanged. Moreover, we found that increased PLN:SERCA2 ratio results in the inhibition of SERCA2 activity at low free Ca2+ concentrations. We further discovered that HHcy is not associated with increased oxidative stress in SR. Taken together, these findings suggest that disturbances in SR Ca2+ handling, caused by altered protein contents but not oxidative damage, may contribute to impaired cardiac contractility in HHcy.

Neurodegenerative disorders, including Alzheimer's disease (AD), Parkinson's disease (PD), or systemic amyloidosis, are characterized by the specific protein transformation from the native state to stable insoluble deposits, e.g., amyloid plaques. The design of potential therapeutic agents and drugs focuses on the destabilization of the bonds in their beta-rich structures. Surprisingly, ferritin derivatives have recently been proposed to destabilize fibril structures. Using atomic force microscopy (AFM) and fluorescence spectrophotometry, we confirmed the destructive effect of reconstructed ferritin (RF) and magnetoferritin (MF) on lysosome amyloid fibrils (LAF). The presence of iron was shown to be the main factor responsible for the destruction of LAF. Moreover, we found that the interaction of RF and MF with LAF caused a significant increase in the release of potentially harmful ferrous ions. Zeta potential and UV spectroscopic measurements of LAF and ferritin derivative mixtures revealed a considerable difference in RF compared to MF. Our results contribute to a better understanding of the mechanism of fibril destabilization by ferritin-like proteins. From this point of view, ferritin derivatives seem to have a dual effect: therapeutic (fibril destruction) and adverse (oxidative stress initiated by increased Fe2+ release). Thus, ferritins may play a significant role in various future biomedical applications.

Magnetite mineralization in human tissue is associated with various pathological processes, especially neurodegenerative disorders. Ferritin's mineral core is believed to be a precursor of magnetite mineralization. Magnetoferritin (MF) was prepared with different iron loading factors (LFs) as a model system for pathological ferritin to analyze its MRI relaxivity properties compared to those of native ferritin (NF). The results revealed that MF differs statistically significantly from NF, with the same LF, for all studied relaxation parameters at 7 T: r(1), r(2), r(2)*, r(2)/r(1), r(2)*/r(1). Distinguishability of MF from NF may be useful in non-invasive MRI diagnosis of pathological processes associated with iron accumulation and magnetite mineralization (e.g., neurodegenerative disorders, cancer, and diseases of the heart, lung and liver). In addition, it was found that MF samples possess very strong correlation and MF's relaxivity is linearly dependent on the LF, and the transverse and longitudinal ratios r(2)/r(1) and r(2)*/r(1) possess complementary information. This is useful in eliminating false-positive hypointensive artefacts and diagnosis of the different stages of pathology. These findings could contribute to the exploitation of MRI techniques in the non-invasive diagnosis of iron-related pathological processes in human tissue.

COVID-19 (Coronavirus Disease) is an infectious disease caused by the coronavirus SARS-CoV-2 (Severe acute respiratory syndrome Coronavirus 2), which belongs to the genus Betacoronavirus. It was first identified in patients with severe respiratory disease in December 2019 in Wuhan, China. It mainly affects the respiratory system, and in severe cases causes serious lung infection or pneumonia, which can lead to the death of the patient. Clinical studies show that SARS-CoV-2 infection in critical cases causes acute tissue damage due to a pathological immune response. The immune response to a new coronavirus is complex and involves many processes of specific and non-specific immunity. Analysis of available studies has shown various changes, especially in the area of specific cellular immunity, including lymphopenia, decreased T cells (CD3+, CD4+ and CD8+), changes in the T cell compartment associated with symptom progression, deterioration of the condition and development of lung damage. We provide a detailed review of the analyses of immune checkpoint molecules PD-1, TIM-3, LAG-3 CTLA-4, TIGIT, BTLA, CD223, IDO-1 and VISTA on exhausted T cells in patients with asymptomatic to symptomatic stages of COVID-19 infection. Furthermore, this review may help to better understand the pathological T cell immune response and improve the design of therapeutic strategies for patients with SARS-CoV-2 infection.

Aim: Kappa free light chains (kappa-fLC) in cerebrospinal fluid (CSF) were suggested as promising novel markers of intrathecal immunoglobulin (Ig) synthesis in MS patients, with reported discrepant reference values. We aimed to analyse the diagnostic accuracy of kappa-fLC (CSF concentration, quotient, index) and routinely used markers (IgG index, IgG(Reibe)(r)) in MS diagnostics, in prediction of oligoclonal bands (OCB) positivity and in OCB negative patients. Methods: Serum/CSF paired samples were analysed from 46 MS patients and 63 controls with non-inflammatory and non-MS inflammatory diseases of CNS. The concentrations of kappa-fLC were measured immunoturbidimetrically (SPA PLUS (R), Freelite (R)). The ROC curves, diagnostic sensitivities and specificities were analysed with respect to the cut-offs, which were determined using Youden's index. Results: The optimal cut-off values were defined as: 0.76 for IgG index; 0.89 mg/L for IgG(Reiber); 1.08 mg/L for CSF kappa-fLC level; 0.0994 for kappa-fLC quotient; 18.15 for kappa-fLC index. To distinguish MS patients from controls, the highest combined sensitivities/specificities were observed for kappa-fLC index (0.76/0.98), followed by kappa-fLC quotient (0.76/0.97), CSF kappa-fLC level (0.76/0.95), IgG(Reibe)(r) (0.70/0.91) and IgG index (0.65/0.89). The OCB detection showed the sensitivity 0.83 and the specificity 1.00. In prediction of OCB positivity, the novel kappa-fLC markers showed excellent diagnostic accuracy with sensitivities/specificities: 0.92/0.99 for kappa-fLC index, 0.92/0.97 for kappa-fLC quotient, and 0.92/0.96 for CSF kappa-fLC level, compared to 0.76/0.89 for IgG index and 0.76/0.86 for IgG(Reiber). Applying our cut-offs in OCB negative MS patients, the highest diagnostic sensitivity (038) was found for IgG(Re)(iber), followed by 0.13 for IgG index, and surprisingly by 0.00 for all kappa-fLC markers. Conclusion: In MS diagnostics and in prediction of OCB positivity, all novel kappa-fLC markers showed better sensitivities and specificities than routinely used IgG index and IgG mbe , but did not reach those of OCBs. To proove the intrathecal Ig synthesis in patients with suspected MS, we recommend to use kappa-fLC index and quotient along with OCB, especially if OCB findings are equivocal or negative.

The incidence of Alzheimer's disease (AD) increases significantly following chronic stress and brain ischemia which, over the years, cause accumulation of toxic amyloid species and brain damage. The effects of global 15-min ischemia and 120-min reperfusion on the levels of expression of the amyloid precursor protein (APP) and its processing were investigated in the brain cortex (Cx) of male Wistar rats. Additionally, the levels of expression of the amyloid-degrading enzymes neprilysin (NEP), endothelin-converting enzyme-1 (ECE-1), and insulin-degrading enzyme (IDE), as well as of some markers of oxidative damage were assessed. It was shown that the APP mRNA and protein levels in the rat Cx were significantly increased after the ischemic insult. Protein levels of the soluble APP fragments, especially of sAPPβ produced by β-secretase, (BACE-1) and the levels of BACE-1 mRNA and protein expression itself were also increased after ischemia. The protein levels of APP and BACE-1 in the Cx returned to the control values after 120-min reperfusion. The levels of NEP and ECE-1 mRNA also decreased after ischemia, which correlated with the decreased protein levels of these enzymes. However, we have not observed any changes in the protein levels of insulin-degrading enzyme. Contents of the markers of oxidative damage (di-tyrosine and lysine conjugates with lipid peroxidation products) were also increased after ischemia. The obtained data suggest that ischemia shifts APP processing towards the amyloidogenic β-secretase pathway and accumulation of the neurotoxic Aβ peptide as well as triggers oxidative stress in the cells. These results are discussed in the context of the role of stress and ischemia in initiation and progression of AD.