A Multi-Biomarker Approach to Increase the Accuracy of Diagnosis and Management of Coronary Artery Disease.

Non-invasive possibilities of predicting cardiovascular risk and monitoring the treatment and progression of coronary artery disease (CAD) are important subjects of cardiovascular research. Various inflammatory markers have been identified as potential biomarkers of CAD, including interleukin-6 (IL-6), lipocalin-2 (LCN-2), growth differentiation factor 15 (GDF-15), and T cell immunoglobulin and mucin domain-3 (TIM-3). This research aims to identify their utility in the investigation of CAD severity and progression. The basic anthropometric parameters, as well as the levels of urea, creatinine, CRP, leukocytes, fibrinogen, and biomarkers of inflammation, were measured in 130 patients who underwent coronary angiography. In male patients, divided according to findings on coronary angiography, we observed an increasing expression of GDF-15 with increasing stenosis (with worsening findings). In females, we observed increasing fibrinogen expression with increasing stenosis, i.e., findings on coronary angiography. Correlation analysis did not confirm the relationship between TIM-3, LCN and 2, IL-6 and the severity of findings obtained by coronary angiography; however, the correlation of TIM-3 and LCN-2 expression was positive with the finding, and the correlation of IL-6 with the finding was surprisingly negative. Understanding the role of these inflammatory markers in CAD can be helpful in risk stratification, guiding therapeutic strategies, and monitoring treatment responses in patients with CAD.

Non-Invasive Endometrial Cancer Screening through Urinary Fluorescent Metabolome Profile Monitoring and Machine Learning Algorithms.

Endometrial cancer is becoming increasingly common, highlighting the need for improved diagnostic methods that are both effective and non-invasive. This study investigates the use of urinary fluorescence spectroscopy as a potential diagnostic tool for endometrial cancer. Urine samples were collected from endometrial cancer patients (n = 77), patients with benign uterine tumors (n = 23), and control gynecological patients attending regular checkups or follow-ups (n = 96). These samples were analyzed using synchronous fluorescence spectroscopy to measure the total fluorescent metabolome profile, and specific fluorescence ratios were created to differentiate between control, benign, and malignant samples. These spectral markers demonstrated potential clinical applicability with AUC as high as 80%. Partial Least Squares Discriminant Analysis (PLS-DA) was employed to reduce data dimensionality and enhance class separation. Additionally, machine learning models, including Random Forest (RF), Logistic Regression (LR), Support Vector Machine (SVM), and Stochastic Gradient Descent (SGD), were utilized to distinguish between controls and endometrial cancer patients. PLS-DA achieved an overall accuracy of 79% and an AUC of 90%. These promising results indicate that urinary fluorescence spectroscopy, combined with advanced machine learning models, has the potential to revolutionize endometrial cancer diagnostics, offering a rapid, accurate, and non-invasive alternative to current methods.

The Pivotal Role of the Key Angiogenic Factors in the Development of Endometrioid Pathologies of the Uterus and Ovary.

A characteristic feature of uterine pathologies is a specific change in cell metabolism, which predominantly manifests as a shift in the need for nutrients, thereby directing cells to engage in different angiogenic marker activities. Angiogenesis is one of the main signals supporting the survival and development of cells and tissues not only under physiological conditions. Therefore, it is necessary that we understand pathological hyperactivation in all uterine diseases, from endometriosis through ovarian endometrioid adenocarcinoma to malignant transformed cells of the uterine epithelium and body. This work presents the gene expression results of selected angiogenesis targets (VEGF-A, TGF-ß1, ANG1/2, and HIF-1α), cell migration, and cell-cell interaction determined in vitro. Our results suggest that angiogenesis varies in the tested pathological conditions (ectopic endometriosis-12Z; ovarian endometrioid adenocarcinoma-A2780; tumors-SK-UT-1 and RL-95-2) compared to physiological angiogenesis (HME1). The differential expression of angiogenic factors may contribute (or is a contributing factor) to the observed differences to acknowledge an inherent variability in angiogenesis among cell lines. Determining the genomic phenomena responsible for processes associated with inadequate angiogenesis in the pelvic region could help us to develop individual treatment strategies and explain resistance to treatment.

The ability of microRNAs to regulate the immune response in ischemia/reperfusion inflammatory pathways.

MicroRNAs play a crucial role in regulating the immune responses induced by ischemia/reperfusion injury. Through their ability to modulate gene expression, microRNAs adjust immune responses by targeting specific genes and signaling pathways. This review focuses on the impact of microRNAs on the inflammatory pathways triggered during ischemia/reperfusion injury and highlights their ability to modulate inflammation, playing a critical role in the pathophysiology of ischemia/reperfusion injury. Dysregulated expression of microRNAs contributes to the pathogenesis of ischemia/reperfusion injury, therefore targeting specific microRNAs offers an opportunity to restore immune homeostasis and improve patient outcomes. Understanding the complex network of immunoregulatory microRNAs could provide novel therapeutic interventions aimed at attenuating excessive inflammation and preserving tissue integrity.

p.L1795F LRRK2 variant is a common cause of Parkinson's disease in Central Europe.

Pathogenic variants in LRRK2 are one of the most common genetic risk factors for Parkinson's disease (PD). Recently, the lesser-known p.L1795F variant was proposed as a strong genetic risk factor for PD, however, further families are currently lacking in literature. A multicentre young onset and familial PD cohort (n = 220) from 9 movement disorder centres across Central Europe within the CEGEMOD consortium was screened for rare LRRK2 variants using whole exome sequencing data. We identified 4 PD cases with heterozygous p.L1795F variant. All 4 cases were characterised by akinetic-rigid PD phenotype with early onset of severe motor fluctuations, 2 receiving LCIG therapy and 2 implanted with STN DBS; all 4 cases showed unsatisfactory effect of advanced therapies on motor fluctuations. Our data also suggest that p.L1795F may represent the most common currently known pathogenic LRRK2 variant in Central Europe compared to the more studied p.G2019S, being present in 1.81% of PD cases within the Central European cohort and 3.23% of familial PD cases. Together with the ongoing clinical trials for LRRK2 inhibitors, this finding emphasises the urgent need for more ethnic diversity in PD genetic research.

Serum trace element levels and activity of enzymes associated with oxidative stress in endometriosis and endometrial cancer.

Endometriosis and endometrial cancer are closely related to oxidative stress. However, the direct relationship between copper and zinc levels and oxidative stress in the extracellular and intracellular space remains unclear. The presented study is focused on the determination of serum Zn and Cu levels, glutathione concentration and enzyme activity in three groups: patients diagnosed with endometrial cancer (EC), patients diagnosed with endometriosis (EM), and a healthy control group. Spectrophotometric determination of trace elements revealed that levels of zinc and copper were lower in blood plasma of patients with endometriosis as compared with the other groups; however, there were no significant differences in the Cu/Zn ratio. Furthermore, significantly increased blood serum glutathione levels were detected in both EM and EC groups compared with the control group. While the activity of superoxide dismutase (SOD) was similar across the studied groups, we observed differences in the activity of other enzymes associated with oxidative stress, including glutathione peroxidase (GPx), glutathione reductase (GR) and glutathione S-transferase (GST), between the control group and the EM and EC patients. Additionally, analysis of gene expression based on free circulating mRNA indicated significant differences in the expression of SOD isoenzymes between the patient groups and the control group; expression of GPx isoenzymes was also altered. Obtained results may have potential application in diagnostics as well as monitoring of endometriosis and endometrial cancer.

Visualization of the composition of the urinary fluorescent metabolome. Why is it important to consider initial urine concentration?

Urine is a highly complex fluorescent system, the fluorescence of which can be affected by many factors, including the often-ignored initial urine concentration in comprehensive fluorescent urine analysis. In this study, a total urine fluorescent metabolome profile (uTFMP) was created as a three-dimensional fluorescence profile of serial synchronous spectra of urine diluted by geometric progression. uTFMP was generated using software designed for this purpose after recalculating the 3D data concerning the initial urine concentration. It can be presented as a contour map (top view) or as a more illustrative and straightforward simple curve, thus usable in various medicinal applications.

Stage-Dependent Levels of Brain-Derived Neurotrophic Factor and Matrix Metalloproteinase 9 in the Prognosis of Colorectal Cancer.

PURPOSE: The development of sensitive and non-invasive biomarkers for the early detection of CRC and determination of their role in the individual stages of CRC. METHODS: MMP-9 expression in serum and tissue, and BDNF expression in plasma were detected using the ELISA method. MMP-9 and BDNF in the tissue were also determined by immunohistochemical staining. RESULTS: To assess the balance between changes in survival and tumor progression, we compared BDNF/MMP-9 ratios in tissues of living and deceased individuals. The tissue BDNF/MMP-9 ratio (evaluated immunohistochemically) decreased significantly with the progression of the disease in living patients. The BDNF/MMP-9 ratio was statistically significantly reduced in stages II and III compared to the benign group. However, in deceased individuals, the ratio showed an opposite tendency. CONCLUSION: The determination of the tissue BDNF/MMP9 ratio can be used as a prognostic biomarker of CRC.

Small Non-Coding RNAs as New Biomarkers to Evaluate the Quality of the Embryo in the IVF Process.

The increased interest in assisted reproduction through in vitro fertilization (IVF) leads to an urgent need to identify biomarkers that reliably highly predict the success of pregnancy. Despite advances in diagnostics, treatment, and IVF approaches, the 30% success rate of IVF seems insurmountable. Idiopathic infertility does not have any explanation for IVF failure especially when a patient is treated with a healthy competitive embryo capable of implantation and development. Since appropriate intercellular communication is essential after embryo implantation, the emergence of the investigation of embryonic secretome including short non-coding RNA (sncRNA) molecules is crucial. That's why biomarker identification, sncRNAs secreted during the IVF process into the blastocyst's cultivation medium, by the implementation of artificial intelligence opens the door to a better understanding of the bidirectional communication between embryonic cells and the endometrium and so the success of the IVF. This study presents a set of promising new sncRNAs which are revealed to predictively distinguish a high-quality embryo, suitable for an embryo transfer in the IVF process, from a low-quality embryo with 86% accuracy. The identified exact combination of miRNAs/piRNAs as a non-invasively obtained biomarker for quality embryo determination, increasing the likelihood of implantation and the success of pregnancy after an embryo transfer.

Tryptophan: Its Metabolism along the Kynurenine, Serotonin, and Indole Pathway in Malignant Melanoma.

(1) Background: Tryptophan metabolism is known to be one of the important mechanisms used by cancer to evade immune surveillance. Altered tryptophan metabolism was studied in patients with pigmented malignant melanoma confirmed histologically by the anatomic stage grouping for cutaneous melanoma using clinical staging on the basis of the Breslow thickness of the melanoma, the degree of spread to regional lymph nodes, and by the presence of distant metastasis. (2) Methods: Urinary tryptophan metabolites were detected by RP-HPLC method. (3) Results: In the present work, we provided evidence of altered metabolism of all tryptophan pathways in melanoma patients. (4) Conclusions: Knowledge of the shifted serotonin pathway toward DHICA formation and kynurenine pathway shifted toward NAD+ production could serve in the early detection of the disease and the initiation of early treatment of malignant melanoma.

Derivative Three-Dimensional Synchronous Fluorescence Analysis of Tear Fluid and Their Processing for the Diagnosis of Glaucoma.

BACKGROUND: Sensitive and rapid diagnosis of the early stages of glaucoma from tear fluid is a great challenge for researchers. METHODS: Tear fluid was analyzed using three-dimensional synchronous fluorescence spectroscopy (3D-SFS). Our previously published results briefly describe the main methods which applied the second derivative to a selected synchronous spectrum Δλ = 110 nm in distinguishing between healthy subjects (CTRL) and patients with glaucoma (POAG). RESULTS: In this paper, a novel strategy was used to evaluate three-dimensional spectra from the tear fluid database of our patients. A series of synchronous excitation spectra were processed as a front view and presented as a single curve showcasing the overall fluorescence profile of the tear fluid. The second derivative spectrum provides two parameters that can enhance the distinction between CTRL and POAG tear fluid. CONCLUSIONS: Combining different types of 3D-SFS data can offer interesting and useful diagnostic tools and it can be used as input for machine learning and process automation.

The possible role of machine learning in detection of increased cardiovascular risk patients - KSC MR Study (design).

Introduction: Currently, just a few major parameters are used for cardiovascular (CV) risk quantification to identify many of the high-risk subjects; however, they leave a lot of them with an underestimated level of CV risk which does not reflect the reality. Material and methods: The submitted study design of the Kosice Selective Coronarography Multiple Risk (KSC MR) Study will use computer analysis of coronary angiography results of admitted patients along with broad patients' characteristics based on questionnaires, physical findings, laboratory and many other examinations. Results: Obtained data will undergo machine learning protocols with the aim of developing algorithms which will include all available parameters and accurately calculate the probability of coronary artery disease. Conclusions: The KSC MR study results, if positive, could establisha base for development of proper software for revealing high-risk patients, as well as patients with suggested positive coronary angiography findings, based on the principles of personalised medicine.

Successful correction of hyperglycemia is critical for weight loss and a decrease in cardiovascular risk in obese patients.

Weight loss is recommended for obese patients with cardiovascular risk; however, it remains questionable how hyperglycemia affects this process. To address this problem, we aimed to determine the association between weight loss, lipid profile, and body mass parameters in obese normoglycemic and hyperglycemic patients. Obese (body mass index30 kg/m2) normoglycemic and hyperglycemic volunteers were placed on a weight reduction program that included a balanced, low-calorie diet and moderate exercise for 6 months. Participants were assessed for serum glucose, ß-cell functions, insulin resistance, lipid metabolism, lipoprotein profile, and body mass parameters. This weight reduction program fully normalized serum glucose levels only in a subpopulation of patients. These individuals also exhibited a significant reduction in body weight, and significant improvement in serum lipid profile and insulin resistance. In contrast, the patients that remained hyperglycemic were characterized by persistent insulin resistance, increased levels of atherogenic fractions of LDL and HDL lipoproteins, and elevated values of a modified Atherogenic Index of Plasma. Correlation analysis indicated a strong positive association between the modified Atherogenic Index of Plasma with atherogenic lipid profile, insulin resistance, and body mass parameters, indicating its usefulness in clinical studies in obese patients. Overall, our data indicate that successful treatment of hyperglycemia facilitates weight loss and improves the composition of blood lipids, while persisting hyperglycemia negatively affects the weight loss process and maintains an atherogenic lipid profile. Because hyperglycemia predisposes to cardiovascular disorders, its correction should be the primary goal during weight reduction therapy.

Loss of Skeletal Muscle Mass and Intracellular Water as Undesired Outcomes of Weight Reduction in Obese Hyperglycemic Women: A Short-Term Longitudinal Study.

The current study is focused on the influence of hyperglycemia on weight loss in obese premenopausal women. Specifically, the study evaluated the impact of a six-month individualized low-calorie diet combined with moderate exercise on weight reduction and glucose metabolism in obese women with normoglycemia compared to obese women with moderate hyperglycemia. The results indicated that patients with normoglycemia achieved a successful weight loss, which was connected to a decrease in adipose tissue and reflected by diminished content of visceral fat area (VFA) and percent body fat. In contrast, weight reduction in patients with hyperglycemia was connected not only to the loss of VFA but also to undesired decrease in skeletal muscle mass as well as intracellular and total body water. These unfavorable outcomes were observed despite normalization of glucose metabolism reflected by statistically significant lowering glucose, fructosamine, advanced glycation end-products, and HOMA-IR levels. Overall, the obtained results indicate the importance of the measurement of the carbohydrate profile in obese women and the need for an early introduction of weight reduction strategies before the development of hyperglycemia.

The Effect of Oestrogen Supplementation on Antioxidant Enzymes and Mitochondrial Respiratory Function After Myocardial Infarction of Ovariectomized Rats.

ABSTRACT: Acute myocardial infarction (MI) is the leading cause of mortality worldwide with premenopausal women showing a lower incidence of cardiovascular disease compared with men of the same age. After menopause, this advantage disappears, suggesting that sex hormones play a cardioprotective role. This study was aimed to assess on the activity of antioxidant enzymes in plasma and the respiratory function of isolated heart mitochondria after the induction of MI in rats after ovariectomy and estradiol benzoate supplementation. Sprague-Dawley female rats were ovariectomized 3 months before the induction of MI and supplemented/not supplemented with oestrogen 3 months before/7 days after the induction of MI. No significant differences in glutathione peroxidase activities were found in any group. Differences between values were only significant in the ovariectomized not supplemented group (P < 0.01) for the glutathione reductase activity and glutathione concentrations. In isolated mitochondria (7 days after MI), the decline in respiration was observed comparing the ovariectomized and nonovariectomized group. Respiratory functions did not show significant differences between animals supplemented with oestrogen before MI or treated with oestrogen after MI. Ovariectomy worsened mitochondrial dysfunction after MI, and oestrogen supplementation before or after the induction of MI did not improve mitochondrial function.

Zinc(II) niflumato complex effects on MMP activity and gene expression in human endometrial cell lines.

Endometriosis is a chronic inflammatory disease which increasingly affects young women under 35 years of age and leads to subfertility even infertility. Analysis of the cytotoxic effect of zinc(II) niflumato complex with neocuproine ([Zn(neo)(nif)2] or Zn-Nif) on immortalized human endometriotic cell line (12Z) and on control immortalized human endometrial stromal cell line (hTERT) was performed using xCELLigence technology for approximately 72 h following the treatment with Zn-Nif as well as cell viability Trypan Blue Assay. 12Z cell line proliferated more slowly compared to unaffected cells, whereas hTERT cells did not show similar behavior after treatment. The complex probably reduces the effect of pro-inflammatory pathways due to the effect of NSAID, while presence of zinc might reduce the level of ROS and regulate ER2 levels and MMP activity. The observed effects and high selectivity for rapidly proliferating cells with increased inflammatory activity suggest a good prognosis of successful decrease of endometriosis stage with this complex.

Selected In Situ Hybridization Methods: Principles and Application.

We are experiencing rapid progress in all types of imaging techniques used in the detection of various numbers and types of mutation. In situ hybridization (ISH) is the primary technique for the discovery of mutation agents, which are presented in a variety of cells. The ability of DNA to complementary bind is one of the main principles in every method used in ISH. From the first use of in situ techniques, scientists paid attention to the improvement of the probe design and detection, to enhance the fluorescent signal intensity and inhibition of cross-hybrid presence. This article discusses the individual types and modifications, and is focused on explaining the principles and limitations of ISH division on different types of probes. The article describes a design of probes for individual types of in situ hybridization (ISH), as well as the gradual combination of several laboratory procedures to achieve the highest possible sensitivity and to prevent undesirable events accompanying hybridization. The article also informs about applications of the methodology, in practice and in research, to detect cell to cell communication and principles of gene silencing, process of oncogenesis, and many other unknown processes taking place in organisms at the DNA/RNA level.

The role of physical activity and miRNAs in the vascular aging and cardiac health of dialysis patients.

Cardiovascular comorbidities are independent risk factors for mortality in dialysis patients. MicroRNA signaling has an important role in vascular aging and cardiac health, while physical activity is a primary nonpharmacologic treatment for cardiovascular comorbidities in dialysis patients. To identify the relationships between muscle function, miRNA signaling pathways, the presence of vascular calcifications and the severity of cardiovascular comorbidities, we initially enrolled 90 subjects on hemodialysis therapy and collected complete data from 46 subjects. A group of 26 subjects inactiv group (INC) was monitored during 12 weeks of physical inactivity and another group of 20 patients exercise group (EXC) was followed during 12 weeks of intradialytic, moderate intensity, resistance training intervention applied three times per week. In both groups, we assessed the expression levels of myo-miRNAs, proteins, and muscle function (MF) before and after the 12-week period. Data on the presence of vascular calcifications and the severity of cardiac comorbidities were collected from the patients' EuCliD® records. Using a full structural equitation modelling of the total study sample, we found that the higher the increase in MF was observed in patients, the higher the probability of a decrease in the expression of miR-206 and TRIM63 and the lower severity of cardiac comorbidities. A reduced structural model in INC patients showed that the higher the decrease in MF, the higher the probability of the presence of calcifications and the higher severity of cardiac comorbidities. In EXC patients, we found that the higher the increase in MF, the lower the probability of higher severity of cardiovascular comorbidities.

Effect of hypoxia factors gene silencing on ROS production and metabolic status of A375 malignant melanoma cells.

The innate response of melanocytes to exogenous or endogenous stress stimuli like extreme pH and temperature, metabolite and oxygen deficiency or a high UV dose initiates a cellular stress response. This process activates adaptive processes to minimize the negative impact of the stressor on the pigment cell. Under physiological conditions, a non-cancer cell is directed to apoptosis if the stressor persists. However, malignant melanoma cells will survive persistent stress thanks to distinct "cancerous" signaling pathways (e.g. MEK) and transcription factors that regulate the expression of so-called "survival genes" (e.g. HIF, MITF). In this survival response of cancer cells, MEK pathway directs melanoma cells to deregulate mitochondrial metabolism, to accumulate reduced species (NADH), and to centralize metabolism in the cytosol. The aim of this work was to study the effect of gene silencing in malignant melanoma A375 cells on metabolic processes in cytosol and mitochondria. Gene silencing of HIF-1α, and miR-210 in normoxia and pseudohypoxia, and analysis of its effect on MITF-M, and PDHA1 expression. Detection of cytosolic NADH by Peredox-mCherry Assay. Detection of OCR, and ECAR using Seahorse XF96. Measurement of produced O2•- with MitoTracker Red CMXRos. 1H NMR analysis of metabolites present in cell suspension, and medium. By gene silencing of HIF-1α and miR-210 the expression of PDHA1 was upregulated while that of MITF-M was downregulated, yielding acceleration of mitochondrial respiratory activity and thus elimination of ROS. Hence, we detected a significantly reduced A375 cell viability, an increase in alanine, inositol, nucleotides, and other metabolites that together define apoptosis. Based on the results of measurements of mitochondrial resipiratory activity, ROS production, and changes in the metabolites obtained in cells under the observed conditions, we concluded that silencing of HIF-1α and miR-210 yields apoptosis and, ultimately, apoptotic cell death in A375 melanoma cells.

Strong Dependence between Tryptophan-Related Fluorescence of Urine and Malignant Melanoma.

Urine autofluorescence at 295 nm is significantly higher in patients with malignant melanoma at each clinical stage compared to the healthy group. The largest difference is in the early-stages and without metastases. With increasing stage, the autofluorescence at 295 nm decreases. There is also a significant negative correlation between autofluorescence and Clark classification. Based on our results, it is assumed that the way malignant melanoma grows also affects urinary autofluorescence.