Integrative Metabolomic Analysis of Serum and Selected Serum Exosomal microRNA in Metastatic Castration-Resistant Prostate Cancer.

Metastatic castration-resistant prostate cancer (mCRPC) remains a lethal disease due to the absence of effective therapies. A more comprehensive understanding of molecular events, encompassing the dysregulation of microRNAs (miRs) and metabolic reprogramming, holds the potential to unveil precise mechanisms underlying mCRPC. This study aims to assess the expression of selected serum exosomal miRs (miR-15a, miR-16, miR-19a-3p, miR-21, and miR-141a-3p) alongside serum metabolomic profiling and their correlation in patients with mCRPC and benign prostate hyperplasia (BPH). Blood serum samples from mCRPC patients (n = 51) and BPH patients (n = 48) underwent metabolome analysis through 1H-NMR spectroscopy. The expression levels of serum exosomal miRs in mCRPC and BPH patients were evaluated using a quantitative real-time polymerase chain reaction (qRT-PCR). The 1H-NMR metabolomics analysis revealed significant alterations in lactate, acetate, citrate, 3-hydroxybutyrate, and branched-chain amino acids (BCAAs, including valine, leucine, and isoleucine) in mCRPC patients compared to BPH patients. MiR-15a, miR-16, miR-19a-3p, and miR-21 exhibited a downregulation of more than twofold in the mCRPC group. Significant correlations were predominantly observed between lactate, citrate, acetate, and miR-15a, miR-16, miR-19a-3p, and miR-21. The importance of integrating metabolome analysis of serum with selected serum exosomal miRs in mCRPC patients has been confirmed, suggesting their potential utility for distinguishing of mCRPC from BPH.

Sensitive SARS-CoV-2 detection, air travel Covid-19 testing, variant determination and fast direct PCR detection, using ddPCR and RT-qPCR methods.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) monitoring in air traffic is important in the prevention of the virus spreading from abroad. The gold standard for SARS-CoV-2 detection is RT-qPCR; however, for early and low viral load detection, a much more sensitive method, such as droplet digital PCR (ddPCR), is required. Our first step was to developed both, ddPCR and RT-qPCR methods, for sensitive SARS-CoV-2 detection. Analysis of ten swab/saliva samples of five Covid-19 patients in different stages of disease showed positivity in 6/10 samples with RT-qPCR and 9/10 with ddPCR. We also used our RT-qPCR method for SARS-CoV-2 detection without the need of RNA extraction, obtaining results in 90-120 minutes. We analyzed 116 self-collected saliva samples from passengers and airport staff arriving from abroad. All samples were negative by RT-qPCR, while 1 was positive, using ddPCR. Lastly, we developed ddPCR assays for SARS-CoV-2 variants identification (alpha, beta, gamma, delta/kappa) that are more economically advantageous when compared to NGS. Our findings demonstrated that saliva samples can be stored at ambient temperature, as we did not observe any significant difference between a fresh sample and the same sample after 24 hours (p = 0.23), hence, saliva collection is the optimal route for sampling airplane passengers. Our results also showed that droplet digital PCR is a more suitable method for detecting virus from saliva, compared to RT-qPCR. Keywords: COVID-19; RT-PCR; ddPCR; SARS-CoV-2; nasopharyngeal swab; saliva.

Polymorphisms in the gene encoding CYP1A2 influence prostate cancer risk and progression.

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.

Role of Genetic Variations in CDK2, CCNE1 and p27KIP1 in Prostate Cancer.

BACKGROUND/AIM: Our aim was to investigate possible influences of genetic variants in genes involved in the G1/S transition [cyclin-dependent kinase-2 (CDK2), cyclin E1 (CCNE1) and cyclin-dependent kinase inhibitor 1B (p27KIP1)] on the expression/activity of their corresponding proteins and to assess the functional impact of these variants on the risk of prostate cancer. MATERIALS AND METHODS: We genotyped 530 cases and 562 healthy controls for two relevant single nucleotide polymorphisms (CDK2 rs2069408 and CCNE1 rs997669) by TaqMan genotyping assay. p27KIP1 rs2066827 polymorphisms were studied by polymerase chain reaction-restriction fragment length polymorphism assay. In addition, the expression of CDK2, CCNE1 and p27KIP1 was evaluated by quantitative real-time-polymerase chain reaction and western blotting in 44 prostate cancer tissues and 31 benign prostatic hyperplasia tissues. RESULTS: No association was found between CDK2 rs2069408, CCNE1 rs997669 or p27KIP1 rs2066827 polymorphisms and an increased risk of prostate cancer development. Higher CDK2 expression was more prevalent in those with rs2069408 GG genotype than in AA carriers (p>0.05). We also noted reduced p27KIP1 protein expression in those with the p27KIP1 G109 allele. No difference was observed for CCNE1 expression in relation to the risky genotype (CC). A significant association was detected between CCNE1 mRNA overexpression and development of higher-grade carcinomas (Gleason score >7, p<0.05). CONCLUSION: Polymorphisms CDK2 rs2069408, CCNE1 rs997669 and p27KIP1 rs2066827 have no significant impact on prostate cancer risk nor on the gene and protein expression of CDK2, CCNE1 and p27KIP1, although high CCNE1 expression was significantly associated with a higher tumour grade in patients with prostate cancer.

Proton magnetic resonance spectroscopy changes in the brainstem in patients after mild traumatic brain injury with loss of consciousness.

INTRODUCTION: Loss of consciousness (LOC) is used as a diagnostic feature of mild traumatic brain injury (MTBI). However, only 10% of concussions result in LOC. There are only a limited number of in-vivo studies dealing with unconsciousness and structural and functional integrity of the brainstem in patients with MTBI. The aim of our pilot study was to assess the sensitivity of proton magnetic resonance spectroscopy (1H-MRS) to detect metabolic changes in the brainstem in patients after MTBI with unconscioussness. METHODS: Twenty-four patients (12 with LOC, and 12 without LOC) within 3 days of MTBI and 19 healthy controls were examined. All subjects underwent single-voxel 1H-MRS examination of the upper brainstem. Spectra were evaluated using LCModel software. Ratios of total N-acetylaspartate (tNAA), total choline-containing compounds (tCho) and glutamate plus glutamine (Glx) to total creatine (tCre) were used for calculations. RESULTS: We found a significant decrease in tNAA/tCre and tCho/tCre ratios in the patient group with LOC when compared with the control group of healthy volunteers (P=0.002 and P=0.041, respectively), and a significant decrease in the tNAA/tCre ratio in the LOC group when compared with patients without LOC (P=0.04). Other metabolite ratios in the brainstem did not show any significant group differences. CONCLUSION: Our findings indicate that decrease of tNAA/tCre ratio in the upper brainstem using single-voxel 1H-MRS may provide a potential biomarker for MTBI associated with LOC.

Association of MDM2 T309G (rs2279744) Polymorphism and Expression Changes With Risk of Prostate Cancer in the Slovak Population.

BACKGROUND/AIM: The aim of this study was to evaluate the relationship between MDM2 T309G polymorphism and prostate cancer risk in the Slovak population and the association of this polymorphism with MDM2 expression and clinicopathological features. MATERIALS AND METHODS: The MDM2 T309G polymorphism was determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis in 506 prostate cancer patients and 592 controls. Quantitative real-time (RT)-PCR and western blot analysis were applied to examine MDM2 expression in 47 prostate cancer tissues and 43 benign prostatic hyperplasia (BPH) tissues. RESULTS: A decreased risk of prostate cancer in men carrying the GG genotype in comparison with the TT genotype was found. A decrease in the relative MDM2 mRNA and protein levels was found in prostate cancer tissues among patients with the MDM2 GG genotype. CONCLUSION: There is a potentially protective effect of the MDM2 GG genotype on the risk of prostate cancer in the Slovak male population.

Androgen receptor and soy isoflavones in prostate cancer.

Androgens and androgen receptor (AR) play a critical role not only in normal prostate development, but also in prostate cancer. For that reason, androgen deprivation therapy (ADT) is the primary treatment for prostate cancer. However, the majority of patients develop castration-resistant prostate cancer, which eventually leads to mortality. Novel therapeutic approaches, including dietary changes, have been explored. Soy isoflavones have become a focus of interest because of their positive health benefits on numerous diseases, particularly hormone-related cancers, including prostate and breast cancers. An important strategy for the prevention and/or treatment of prostate cancer might thus be the action of soy isoflavones on the AR signaling pathway. The current review article provides a detailed overview of the anticancer potential of soy isoflavones (genistein, daidzein and glycitein), as mediated by their effect on AR.

Detection of BRAFV600E Mutation in Melanoma Patients by Digital PCR of Circulating DNA.

AIMS: About 50% of melanomas have the BRAFV600E mutation. This mutation is an attractive therapeutic target. The aims of our study were to detect BRAFV600E mutations within circulating cell-free DNA in plasma ("liquid biopsy") by a droplet digital PCR (ddPCR) method, and to investigate how well the Breslow-Clark score can be predicted by ddPCR. MATERIALS AND METHODS: We analyzed 113 patients with malignant melanoma. ddPCR was performed using the QX200 system (BIO-RAD®, Hercules). All samples were tested in duplicate. Besides the results of the liquid biopsy, we have collected data on gender and age of the patients, as well as the mitotic activity of the tumor; the tumor subtype and localization, and the Breslow-Clark score. The limit of detection (LoD) was determined by the method of Tzonev. The LoD was found to be five events per well. RESULTS: The BRAFV600E mutation was detected in 37 of 113 samples. A moderate predictive accuracy of the Breslow-Clark score can be attained with the mitotic activity and the type of melanoma as the most important predictors. CONCLUSION: Our results show that ddPCR is a highly sensitive method and could be used for a routine laboratory detection of the BRAFV600E mutation as well as for follow-up monitoring to determine the treatment response in patients with malignant melanomas.

Involvement of calcium regulating ion channels in contractility of human isolated urinary bladder.

This study specified the role of several key calcium-operating ion channels in contraction/relaxation of human detrusor muscle as possible target for overactive bladder (OAB) treatment. Detrusor samples, obtained from 18 males (average age 61.5 ± 5.9 years), were investigated by organ tissue bath method with following agents: diltiazem for L-type voltage-gated calcium channels; 3-fluropyridine-4-carboxylic acid (FPCA) for Orai-STIM channels; SKF 96365-hydrochloride for transient receptor potential (TRP) channels, T-type channels and Orai-STIM channels; 2- aminoethoxydiphenyl borate (2-APB) for inositol-triphosphate receptors (IP3Rs) and Orai-STIM channels. Oxybutynin and mirabegron were tested under the same conditions as controls. Mirabegron, 2-APB and FPCA exhibited the best suppressive effect on carbachol-induced detrusor contractility. As expressed by area under the contractile curve (AUCC), 2-APB, FPCA and mirabegron have similar AUCC: 1.79, 1.73, 1.73. The highest AUCC was 3.64 for diltiazem+SKF, followed by 3.21 for diltiazem, 3.16 for SKF and 2.94 for oxybutynin. The lowest median amplitude and contraction variability is for 2-APB followed by mirabegron and FPCA. There were significant differences between: 2-APB/FPCA vs.: ditiazem, diltiazem+SKF and SKF. Summary of results suggested the principal role of IP3Rs, Orai-STIM coupling and large-conductance calcium-activated potassium channels in detrusor contraction and pointed on Orai-STIM channels as possible targets for OAB pharmacotherapy.

The safety and efficacy of Heparin and Nadroparin compared to placebo in acute ischemic stroke - pilot study.

AIMS: This study aimed to compare the efficacy and safety of heparin and nadroparin in order to provide an additional therapeutic option for patients with acute ischemic stroke in, whom systemic thrombolysis was excluded, or thrombectomy could not be performed. METHODS: We describe a prospective randomized double-blind placebo-controlled pilot study in acute ischemic stroke. The therapeutic window was between 4.5 and 24 h after the onset of stroke. During the first 24 h of treatment, the patients divided into 3 groups received placebo, heparin or nadroparin (in therapeutic doses). During the following 48 h, each patient received nadroparin in the therapeutic dose. 24 h after start of treatment they began taking 100 mg aspirin daily. The primary safety indicator was incidence of complications such as intracerebral or systemic hemorrhage, or death. Efficacy was primarily monitored by the neurological modified Rankin Scale (mRS) at 90 days. RESULTS: There were no signs of intracerebral or systemic bleeding in the cohort of 87 patients. Two patients died - one (3.7%) in the heparin and one (3.8%) in the placebo group due to causes not connected with the treatment. There was a statistically significant difference in mRS on the 90th day between the heparin and placebo groups (21 (80%) vs 13 (50%), P=0.0350) and between the nadroparin and placebo groups (29 (85%) vs 13 (50%), P=0.0031). CONCLUSION: The results show that the treatment with heparin and nadroparin is safe and effective. TRIAL REGISTRATION: Trial is registered in ClinicalTrials.gov: NCT01862978.

Polymorphism of the SRD5A2 gene and the risk of prostate cancer.

Androgens are actively involved in the development of the prostate gland and appear to be essential for prostate carcinogenesis. The product of the SRD5A2 gene, membrane­bound steroid 5­α­reductase, type II enzyme, is key in testosterone metabolism. The present study explored the association between the SRD5A2 V89L gene polymorphism and the risk of developing prostate cancer. The study cohort consisted of 456 male Slovak patients, including 260 cases with histologically confirmed prostate cancer and 196 age­matched controls without any clinically suspected infections of the prostate. Polymerase chain reaction-restriction fragment length polymorphism analysis was used to detect the SRD5A2 polymorphism on codon 89. Odds ratios (ORs) with corresponding 95% confidence intervals (95% CIs) for different allele variants were calculated in order to determine the association between the SRD5A2 V89L gene polymorphism and prostate cancer. The distribution of V89L variants in the control group was consistent with the Hardy­Weinberg equilibrium (χ2 test, P=0.266) with a significant deviation in the case group (χ2 test, P=0.04). However, no association between the SRD5A2 polymorphism and an increased risk of developing prostate cancer was identified. When the wild type VV variant was used as a reference, the ORs for different allele variants ranged from 1.11 (95% CI 0.66­1.87, P=0.70) for the LL genotype to 0.99 (95% CI 0.68­1.46, P=0.99) for the LL + VL genotypes. No particular allele variant was identified to exhibit an increased capacity to promote the development of highly aggressive prostate cancer (Gleason ≥7) or induce carcinogenesis at an earlier onset (<65 years of age). It was confirmed that in the population studied, the SRD5A2 V89L polymorphism was not associated with the risk of prostate cancer and SRD5A2 was not shown to be a key gene involved in prostate cancer development. Published data indicate that a combination of multiple genetic changes are required for prostate cancer development, rather than a single gene change. Therefore, it was hypothesized that high-throughput genotyping may be more effective than single nucleotide polymorphism detection.

The expression profile of acid-sensing ion channel (ASIC) subunits ASIC1a, ASIC1b, ASIC2a, ASIC2b, and ASIC3 in the esophageal vagal afferent nerve subtypes.

Acid-sensing ion channels (ASICs) have been implicated in esophageal acid sensing and mechanotransduction. However, insufficient knowledge of ASIC subunit expression profile in esophageal afferent nerves hampers the understanding of their role. This knowledge is essential because ASIC subunits form heteromultimeric channels with distinct functional properties. We hypothesized that the esophageal putative nociceptive C-fiber nerves (transient receptor potential vanilloid 1, TRPV1-positive) express multiple ASIC subunits and that the ASIC expression profile differs between the nodose TRPV1-positive subtype developmentally derived from placodes and the jugular TRPV1-positive subtype derived from neural crest. We performed single cell RT-PCR on the vagal afferent neurons retrogradely labeled from the esophagus. In the guinea pig, nearly all (90%-95%) nodose and jugular esophageal TRPV1-positive neurons expressed ASICs, most often in a combination (65-75%). ASIC1, ASIC2, and ASIC3 were expressed in 65-75%, 55-70%, and 70%, respectively, of both nodose and jugular TRPV1-positive neurons. The ASIC1 splice variants ASIC1a and ASIC1b and the ASIC2 splice variant ASIC2b were similarly expressed in both nodose and jugular TRPV1-positive neurons. However, ASIC2a was found exclusively in the nodose neurons. In contrast to guinea pig, ASIC3 was almost absent from the mouse vagal esophageal TRPV1-positive neurons. However, ASIC3 was similarly expressed in the nonnociceptive TRPV1-negative (tension mechanoreceptors) neurons in both species. We conclude that the majority of esophageal vagal nociceptive neurons express multiple ASIC subunits. The placode-derived nodose neurons selectively express ASIC2a, known to substantially reduce acid sensitivity of ASIC heteromultimers. ASIC3 is expressed in the guinea pig but not in the mouse vagal esophageal TRPV1-positive neurons, indicating species differences in ASIC expression.

The role of p21 3'UTR gene polymorphism in the risk of prostate cancer: a pilot study.

The cell cycle regulator p21 plays an important role in regulating critical cell activities including cell cycle control, DNA repair and apoptosis. Consequently, it may affect the efficacy of the response to DNA damage and tumor development. The aim of our study was to evaluate the frequencies of the p21 C70T polymorphism, the association between this genetic variant and smoking status, and the serum prostate-specific antigen (PSA) levels and Gleason score in 118 prostate cancer patients and 130 males routinely screened for prostate cancer in the Slovak population. Blood samples were collected from all individuals for DNA isolation, used for subsequent genotyping assays via PCR-RFLP methods. Overall, we did not observe any significant association between this polymorphism and prostate cancer risk. An analysis of the association between the p21 genotypes and smoking was then conducted. Among smokers, CC and CT genotypes were associated with a non­significant increased risk (OR=1.48; 95% CI, 0.80-2.76 and OR=1.15; 95% CI, 0.27­4.77, respectively; p>0.05) in comparison to non-smokers with the CC genotype. Patients with a CT genotype and serum PSA levels≥10 ng/ml had an 84% decrease in prostate cancer risk (OR=0.16; 95% CI, 0.03-0.75; p<0.05) compared to cases with serum PSA levels <10 ng/ml and the CC genotype. No significant association was detected between Gleason score and prostate cancer risk. Based on these results, we concluded that the p21 C70T polymorphism is associated with decreased risk of prostate cancer in Slovak men. To confirm these findings, a systematic approach is required to identify sequence variants in this and other related genes, and subsequently, to test for an association between such variants, smoking status and tumor-specific clinicopathological characteristics in large samples.

Effect of CYP17 and PSA gene polymorphisms on prostate cancer risk and circulating PSA levels in the Slovak population.

Cytochrome P-450c17α (CYP17) and prostate-specific antigen (PSA) genes, which are involved in the androgen metabolism cascade, have been studied as possible candidates for genetic influences on prostate cancer development. Contradictory results prompted us to evaluate the frequencies of polymorphisms in the CYP17 and PSA genes as well as the association between these genetic variants and serum PSA levels in prostate cancer patients and men routinely screened for prostate cancer with PSA in the Slovak male population. The CYP17 and PSA polymorphisms were determined by the PCR-RFLP analysis in 197 Caucasian prostate cancer patients and 256 Caucasian controls. We did not find any association between the CYP17 and PSA genotypes and prostate cancer risk overall, or by grade. Also the total serum PSA levels in the cases with the AG or AA genotype were not significantly higher than in the men with the GG genotype (P > 0.05). Our study did not provide support for the hypothesized relationship between CYP17 and PSA gene polymorphisms and prostate cancer in the Slovak male population.