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.

Circulating metabolites in the early stage of breast cancer were not related to cancer stage or subtypes but associated with ki67 level. Promising statistical discrimination from controls.

It was documented that the presence of malignancy in an organism causes metabolomic alterations in blood plasma which applies also to breast cancer. Breast cancer is a heterogeneous disease and there are only limited known relations of plasma metabolomic signatures with the tumour characteristics in early BC and knowing them would be of great advantage in noninvasive diagnostics. In this study, we focused on the metabolic alterations in early BC in blood plasma with the aim to identify metabolomic characteristics of BC subtypes. We used 50 early BC patients (FIGO stage I and II), where no additional metabolomic changes from metastatically changed remote organs were to be expected. We compared plasma levels of metabolites against controls and among various molecular and histological BC subtypes. BC patients showed decreased plasma levels of branched-chain amino acids BCAAs (and related keto-acids), histidine pyruvate and alanine balanced with an increased level of 3-hydroxybutyrate. The levels of circulating metabolites were not related to BC molecular subtypes (luminal A/luminal B), histological finding or grade, eventually stage, which indicate that in early BC, the BC patients share common metabolomics fingerprint in blood plasma independent of grade, stage or molecular subtype of BC. We observed statistically significant correlations between tumour proliferation marker Ki-67 level and circulating metabolites: alanine, citrate, tyrosine, glutamine, histidine and proline. This may point out the metabolites those levels could be associated with tumour growth, and conversely, the rate of tumour proliferation could be potentially estimated from plasma metabolites. When analyzing metabolomic changes in BC, we concluded that some of them could be associated with the metabolomic features of cancer cells, but the other observed alterations in blood plasma are the results of the complex mutual biochemical pathways in the comprehensive inter-organ metabolic exchange and communication. In the end, statistical discrimination against controls performed with AUC >0.91 showed the very promising potential of plasma metabolomics in the search for biomarkers for oncologic diseases.

High-grade serous ovarian carcinoma and detection of inactivated BRCA genes from biopsy material of Slovak patients.

Ovarian cancer is the leading cause of mortality among all gynecological cancers in developed countries and its most common and most lethal type is the high-grade serous ovarian carcinoma (HGSC). At the molecular level, nearly half of all HGSCs exhibit ineffective homologous DNA recombination and disruption of DNA damage/repair pathway inactivation caused often by BRCA1 and BRCA2 gene mutation. Recently, the detection of BRCA1/2 mutations became important for personalized treatment of HGSC patients with the PARP-inhibitors in the defined clinical setting of relapse after positive adjuvant platinum-based chemotherapeutic response. Based on the selection of patients by regional oncologists, we attempted to verify the possibilities of BRCA1/2 mutation testing on archival formalin-fixed paraffin-embedded (FFPE) biopsy material from regional hospitals. In the study we used: a/ FFPE tumor resections of 97 patients sent to our laboratory, originally stored in archives of regional departments for a period of 1-3 years and retrieved on the principle to contain a maximum of non-necrotic tumor tissue, b/ next-generation sequencing (NGS) assay covering all known mutations in the BRCA1/2 genes on MiSeq (Illumina® platform), and c/ Sophia DDM® bioinformatics platform. After processing of FFPE samples, 5 cases were excluded due to the insufficient genomic DNA quantity. Bioinformatics results of NGS analyses of 92 patients' samples indicated 17.39% pathogenic mutations and 32.61% potentially pathogenic mutations in genes BRCA1/2. Overall, 50% pathogenic and potentially pathogenic mutations were detected in the patient's cohort. The relatively high incidence of BRCA1/2 mutations in our series may be influenced by various indicators including the selection of patients based on adjuvant therapy response as well as regional or population heterogeneity in their frequency. Based on the interdisciplinary cooperation, the use of archival biopsy material processed primarily and stored for a longer period in different laboratories without uniformly defined pre-analytical conditions allows identifying the HGSC patients who might better respond to the PARP-inhibition therapy.

Breast Cancer and the Other Non-Coding RNAs.

Breast cancer is very heterogenous and the most common gynaecological cancer, with various factors affecting its development. While its impact on human lives and national health budgets is still rising in almost all global areas, many molecular mechanisms affecting its onset and development remain unclear. Conventional treatments still prove inadequate in some aspects, and appropriate molecular therapeutic targets are required for improved outcomes. Recent scientific interest has therefore focused on the non-coding RNAs roles in tumour development and their potential as therapeutic targets. These RNAs comprise the majority of the human transcript and their broad action mechanisms range from gene silencing to chromatin remodelling. Many non-coding RNAs also have altered expression in breast cancer cell lines and tissues, and this is often connected with increased proliferation, a degraded extracellular environment, and higher endothelial to mesenchymal transition. Herein, we summarise the known abnormalities in the function and expression of long non-coding RNAs, Piwi interacting RNAs, small nucleolar RNAs and small nuclear RNAs in breast cancer, and how these abnormalities affect the development of this deadly disease. Finally, the use of RNA interference to suppress breast cancer growth is summarised.

Pathway Analysis of Selected Circulating miRNAs in Plasma of Breast Cancer Patients: A Preliminary Study.

MicroRNAs in the circulation of breast cancer (BC) patients have great potential for the early diagnosis, treatment and monitoring of breast cancer. The aim of this preliminary study was to obtain the expression profile of selected miRNAs in the plasma of BC patients that could discriminate BC patients from healthy volunteers and may be useful in early detection of BC. Significantly deregulated miRNAs were evaluated by pathway analysis with the prediction of potential miRNA targets. The study enrolled plasma samples from 65 BC patients and 34 healthy volunteers. Selected miRNAs were screened in pilot testing by the real-time PCR (qPCR) method, and the most appropriate reference genes were selected for normalisation by the geNorm algorithm. In the final testing, we detected miR-99a, miR-130a, miR-484 and miR-1260a (p < 0.05) as significantly up-regulated in the plasma of BC patients. Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway analysis revealed that all significantly deregulated miRNAs are involved in the Hippo and Transforming Growth Factor-beta (TGF-beta) signalling pathways. Our study confirmed a different profile of selected circulating miRNAs in the plasma of BC patients with an emphasis on some critical points in the analysis process.

Monosomy 3 Influences Epithelial-Mesenchymal Transition Gene Expression in Uveal Melanoma Patients; Consequences for Liquid Biopsy.

Despite outstanding advances in diagnosis and the treatment of primary uveal melanoma (UM), nearly 50% of UM patients develop metastases via hematogenous dissemination, driven by the epithelial-mesenchymal transition (EMT). Despite the failure in UM to date, a liquid biopsy may offer a feasible non-invasive approach for monitoring metastatic disease progression and addressing protracted dormancy. To detect circulating tumor cells (CTCs) in UM patients, we evaluated the mRNA expression of EMT-associated transcription factors in CD45-depleted blood fraction, using qRT-PCR. ddPCR was employed to assess UM-specific GNA11, GNAQ, PLCß4, and CYSLTR2 mutations in plasma DNA. Moreover, microarray analysis was performed on total RNA isolated from tumor tissues to estimate the prognostic value of EMT-associated gene expression. In total, 42 primary UM and 11 metastatic patients were enrolled. All CD45-depleted samples were negative for CTC when compared to the peripheral blood fraction of 60 healthy controls. Tumor-specific mutations were detected in the plasma of 21.4% patients, merely, in 9.4% of primary UM, while 54.5% in metastatic patients. Unsupervised hierarchical clustering of differentially expressed EMT genes showed significant differences between monosomy 3 and disomy 3 tumors. Newly identified genes can serve as non-invasive prognostic biomarkers that can support therapeutic decisions.

Cold Atmospheric Pressure Plasma (CAP) as a New Tool for the Management of Vulva Cancer and Vulvar Premalignant Lesions in Gynaecological Oncology.

Vulvar cancer (VC) is a specific form of malignancy accounting for 5-6% of all gynaecologic malignancies. Although VC occurs most commonly in women after 60 years of age, disease incidence has risen progressively in premenopausal women in recent decades. VC demonstrates particular features requiring well-adapted therapeutic approaches to avoid potential treatment-related complications. Significant improvements in disease-free survival and overall survival rates for patients diagnosed with post-stage I disease have been achieved by implementing a combination therapy consisting of radical surgical resection, systemic chemotherapy and/or radiotherapy. Achieving local control remains challenging. However, mostly due to specific anatomical conditions, the need for comprehensive surgical reconstruction and frequent post-operative healing complications. Novel therapeutic tools better adapted to VC particularities are essential for improving individual outcomes. To this end, cold atmospheric plasma (CAP) treatment is a promising option for VC, and is particularly appropriate for the local treatment of dysplastic lesions, early intraepithelial cancer, and invasive tumours. In addition, CAP also helps reduce inflammatory complications and improve wound healing. The application of CAP may realise either directly or indirectly utilising nanoparticle technologies. CAP has demonstrated remarkable treatment benefits for several malignant conditions, and has created new medical fields, such as "plasma medicine" and "plasma oncology". This article highlights the benefits of CAP for the treatment of VC, VC pre-stages, and postsurgical wound complications. There has not yet been a published report of CAP on vulvar cancer cells, and so this review summarises the progress made in gynaecological oncology and in other cancers, and promotes an important, understudied area for future research. The paradigm shift from reactive to predictive, preventive and personalised medical approaches in overall VC management is also considered.

Liquid Biopsy as a Tool for Differentiation of Leiomyomas and Sarcomas of Corpus Uteri.

Utilization of liquid biopsy in the management of cancerous diseases is becoming more attractive. This method can overcome typical limitations of tissue biopsies, especially invasiveness, no repeatability, and the inability to monitor responses to medication during treatment as well as condition during follow-up. Liquid biopsy also provides greater possibility of early prediction of cancer presence. Corpus uteri mesenchymal tumors are comprised of benign variants, which are mostly leiomyomas, but also a heterogenous group of malignant sarcomas. Pre-surgical differentiation between these tumors is very difficult and the final description of tumor characteristics usually requires excision and histological examination. The leiomyomas and malignant leiomyosarcomas are especially difficult to distinguish and can, therefore, be easily misdiagnosed. Because of the very aggressive character of sarcomas, liquid biopsy based on early diagnosis and differentiation of these tumors would be extremely helpful. Moreover, after excision of the tumor, liquid biopsy can contribute to an increased knowledge of sarcoma behavior at the molecular level, especially on the formation of metastases which is still not well understood. In this review, we summarize the most important knowledge of mesenchymal uterine tumors, the possibilities and benefits of liquid biopsy utilization, the types of molecules and cells that can be analyzed with this approach, and the possibility of their isolation and capture. Finally, we review the typical abnormalities of leiomyomas and sarcomas that can be searched and analyzed in liquid biopsy samples with the final aim to pre-surgically differentiate between benign and malignant mesenchymal tumors.

Aberrant Methylation Status of Tumour Suppressor Genes in Ovarian Cancer Tissue and Paired Plasma Samples.

Ovarian cancer is a highly heterogeneous disease and its formation is affected by many epidemiological factors. It has typical lack of early signs and symptoms, and almost 70% of ovarian cancers are diagnosed in advanced stages. Robust, early and non-invasive ovarian cancer diagnosis will certainly be beneficial. Herein we analysed the regulatory sequence methylation profiles of the RASSF1, PTEN, CDH1 and PAX1 tumour suppressor genes by pyrosequencing in healthy, benign and malignant ovarian tissues, and corresponding plasma samples. We recorded statistically significant higher methylation levels (p < 0.05) in the CDH1 and PAX1 genes in malignant tissues than in controls (39.06 ± 18.78 versus 24.22 ± 6.93; 13.55 ± 10.65 versus 5.73 ± 2.19). Higher values in the CDH1 gene were also found in plasma samples (22.25 ± 14.13 versus 46.42 ± 20.91). A similar methylation pattern with positive correlation between plasma and benign lesions was noted in the CDH1 gene (r = 0.886, p = 0.019) and malignant lesions in the PAX1 gene (r = 0.771, p < 0.001). The random forest algorithm combining methylation indices of all four genes and age determined 0.932 AUC (area under the receiver operating characteristic (ROC) curve) prediction power in the model classifying malignant lesions and controls. Our study results indicate the effects of methylation changes in ovarian cancer development and suggest that the CDH1 gene is a potential candidate for non-invasive diagnosis of ovarian cancer.

Why the Gold Standard Approach by Mammography Demands Extension by Multiomics? Application of Liquid Biopsy miRNA Profiles to Breast Cancer Disease Management.

In the global context, the epidemic of breast cancer (BC) is evident for the early 21st century. Evidence shows that national mammography screening programs have sufficiently reduced BC related mortality. Therefore, the great utility of the mammography-based screening is not an issue. However, both false positive and false negative BC diagnosis, excessive biopsies, and irradiation linked to mammography application, as well as sub-optimal mammography-based screening, such as in the case of high-dense breast tissue in young females, altogether increase awareness among the experts regarding the limitations of mammography-based screening. Severe concerns regarding the mammography as the "golden standard" approach demanding complementary tools to cover the evident deficits led the authors to present innovative strategies, which would sufficiently improve the quality of the BC management and services to the patient. Contextually, this article provides insights into mammography deficits and current clinical data demonstrating the great potential of non-invasive diagnostic tools utilizing circulating miRNA profiles as an adjunct to conventional mammography for the population screening and personalization of BC management.

miRNA in a multiomic context for diagnosis, treatment monitoring and personalized management of metastatic breast cancer.

Metastatic breast cancer is characterized by aggressive spreading to distant organs. Despite huge multilevel research, there are still several important challenges that have to be clarified in the management of this disease. Therefore, recent investigations have implemented a modern, multiomic approach with the aim of identifying specific biomarkers for not only early detection but also to predict treatment responses and metastatic spread. Specific attention is paid to short miRNAs, which regulate gene expression at the post-transcriptional level. Aberrant miRNA expression could initiate cancer development, cell proliferation, invasion, migration, metastatic spread or drug resistance. An miRNA signature is, therefore, believed to be a promising biomarker and prediction tool that could be utilized in all phases of carcinogenesis. This article offers comprehensive information about miRNA profiles useful for diagnostic and treatment purposes that may sufficiently advance breast cancer management and improve individual outcomes in the near future.

Vasomotor, urogenital, psychological, and somatic symptoms in association with CYP1B1 polymorphisms in Slovak women of different menopausal status.

OBJECTIVES: The aim of this study was to examine if the Arg48Gly, Ala119Ser, Leu432Val, and Asn453Ser polymorphisms in the CYP1B1 estrogen-metabolizing gene are associated with menopausal symptom experience in healthy Slovak women aged 40-60 years. We also investigated the possible association of other factors with menopausal symptoms, including health status, physical activity, reproductive history, psychological status, and smoking. METHODS: The total sample consisted of 367 women (mean age 49.11 ± 5.86 years), encompassing 180 premenopausal (mean age 45.06 ± 3.81 years), 29 peri-menopausal (mean age 49.41 ± 3.94 years), and 158 postmenopausal (mean age 53.71 ± 4.54 years) women. The research comprised anthropometric and bioelectrical impedance analysis measurements (BIA), blood or saliva samples collected for DNA analysis, and a specific menopausal questionnaire. RESULTS: CYP1B1 Arg48Gly is significantly associated with vasomotor, psychological, and somatic symptoms. It appears that the Gly/Gly genotype is a risk factor during the postmenopause and protective in the pre- and peri-menopause. CYP1B1 Ala119Ser was associated with all menopausal symptoms, with the Ser/Ser genotype increasing risk in the premenopause and offering protection in the peri- and postmenopause. Polymorphisms Leu432Val and Asn453Ser gave unequivocal results; independent of menopausal status, the Leu/Leu genotype was associated with increasing risk of vasomotor, urogenital, and psychological symptoms and the Asn/Asn genotype provided a protective effect against psychological symptoms. CONCLUSIONS: Our results suggest possible associations of CYP1B1 polymorphisms with the occurrence and manifestation of particular menopausal symptoms in healthy mid-life Slovak women.

Molecular and clinical attributes of uterine leiomyomas.

Uterine leiomyomas, also called uterine fibroids or myomas, represent one of the most common benign tumour types in women of a fertile age. Leiomyomas arise due to transformation of the layer of smooth muscle cells of corpus uteri - the myometrium. Despite frequent occurrence of this disease, the molecular mechanisms behind the origin and development of leiomyomas are still relatively unknown. Most predisposed are obese women and women of African origin. In more than half of cases, leiomyomas remain asymptomatic. Genetic factors also have an important impact on the development of these hormone-dependent tumours. However, the clinical and molecular characteristics of familiar and sporadic leiomyomas can widely differ. The main reason is the heterogeneity of this disease and the abundance of factors which can underlie their tumourigenesis. Clinical diagnosis of uterine leiomyomas without surgical interference can be hindered in the case of small, mostly submucosal leiomyomas or if it is necessary to avoid potential malignancy of tumour. Also, medical treatment of uterine leiomyomas cannot be nowadays considered sufficient with many medical agents still being tested only within clinical research. The main goal of this article is to summarise known facts about the aetiology of leiomyomas, risk factors that contribute to their development, known molecular-genetic aberrations connected with the presence of leiomyomas as well as the possibilities of their diagnosis and treatment.

Association of single nucleotide polymorphisms in FGF-RAS/MAP signalling cascade with breast cancer susceptibility.

The fibroblast growth factor receptors (FGFRs) and Ras/mitogen activated protein (RAS/MAP) signalling cascades are the main molecular pathways involved in breast carcinogenesis. This study aims to determine the association between FGF10 (rs4415084 C>T), FGFR2 (rs2981582 C>T) and MAP3K1 (rs889312 A>C) gene polymorphisms and breast cancer, to analyse the discriminative ability of each SNP and to test the accuracy of the predictive breast cancer risk model which includes all SNPs. We conducted a case-control study of 170 women (57.06 ± 11.60 years) with histologically confirmed breast cancer and 146 controls (50.24 ± 10.69 years). High resolution melting (HRM) method with Sanger sequencing validation was used in analyses. We have revealed significant association of FGFR2 and MAP3K1 polymorphisms with breast cancer. The odds ratio of FGFR2 T allele was 1.897 (95% CI 1.231-2.936, p = 0.004) and MAP3K1 C allele 1.804 (95% CI 1.151-2.845, p = 0.012). FGFR2 polymorphism achieved the best discriminative ability (41.95%). The Random Forest algorithm selected FGFR2, MAP3K1 and age as important breast cancer predictors. The accuracy of this prediction model approached moderate accuracy (70%), with 35.9% sensitivity and 88.6% specificity.

Droplet digital PCR analysis of CDH13 methylation status in Slovak women with invasive ductal breast cancer.

Identifying novel epigenetic biomarkers is a promising way to improve the clinical management of patients with breast cancer. Our study aimed to determine the methylation pattern of 25 tumor suppressor genes (TSG) and select the best methylation biomarker associated with clinicopathological features in the cohort of Slovak patients diagnosed with invasive ductal carcinoma (IDC). Overall, 166 formalin-fixed, paraffin-embedded (FFPE) tissues obtained from patients with IDC were included in the study. The methylation status of the promoter regions of 25 TSG was analyzed using semiquantitative methylation-specific MLPA (MS-MLPA). We identified CDH13 as the most frequently methylated gene in our cohort of patients. Further analysis by ddPCR confirmed an increased level of methylation in the promoter region of CDH13. A significant difference in CDH13 methylation levels was observed between IDC molecular subtypes LUM A versus HER2 (P = 0.0116) and HER2 versus TNBC (P = 0.0234). In addition, significantly higher methylation was detected in HER2+ versus HER2- tumors (P = 0.0004) and PR- versus PR+ tumors (P = 0.0421). Our results provide evidence that alteration in CDH13 methylation is associated with clinicopathological features in the cohort of Slovak patients with IDC. In addition, using ddPCR as a methylation-sensitive method represents a promising approach characterized by higher precision and technical simplicity to measure the methylation of target CpGs in CDH13 compared to other conventional methods such as MS-MLPA.

Host genetic variants associated with COVID-19 reconsidered in a Slovak cohort.

We present the results of an association study involving hospitalized coronavirus disease 2019 (COVID-19) patients with a clinical background during the 3rd pandemic wave of COVID-19 in Slovakia. Seventeen single nucleotide variants (SNVs) in the eleven most relevant genes, according to the COVID-19 Host Genetics Initiative, were investigated. Our study confirms the validity of the influence of LZTFL1 and 2'-5'-oligoadenylate synthetase (OAS)1/OAS3 genetic variants on the severity of COVID-19. For two LZTFL1 SNVs in complete linkage disequilibrium, rs17713054 and rs73064425, the odds ratios of baseline allelic associations and logistic regressions (LR) adjusted for age and sex ranged in the four tested designs from 2.04 to 2.41 and from 2.05 to 3.98, respectively. The OAS1/OAS3 haplotype 'gttg' carrying a functional allele G of splice-acceptor variant rs10774671 manifested its protective function in the Delta pandemic wave. Significant baseline allelic associations of two DPP9 variants in all tested designs and two IFNAR2 variants in the Omicron pandemic wave were not confirmed by adjusted LR. Nevertheless, adjusted LR showed significant associations of NOTCH4 rs3131294 and TYK2 rs2304256 variants with severity of COVID-19. Hospitalized patients' reported comorbidities were not correlated with genetic variants, except for obesity, smoking (IFNAR2), and hypertension (NOTCH4). The results of our study suggest that host genetic variations have an impact on the severity and duration of acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Considering the differences in allelic associations between pandemic waves, they support the hypothesis that every new SARS-CoV-2 variant may modify the host immune response by reconfiguring involved pathways.

Power of biomarkers and their relative contributions to metabolic syndrome in Slovak adult women.

BACKGROUND: Metabolic syndrome (MetS) comprises a cluster of risk components which pre-dispose individuals to cardiovascular mortality. AIM: The purpose of this study is to investigate the variability of biochemical and anthropometric characteristics, apolipoprotein E (APOE) and angiotensin converting enzyme (ACE) genes and their contribution to MetS manifestation. SUBJECTS AND METHODS: A total of 438 adult women were recruited from different localities in Slovakia. All data was established by standard anthropometric, biochemical and genetic methods. RESULTS: The logarithm of the ratio of plasma concentration of triglycerides to HDL-cholesterol [log(TG-to-HDL-C)], waist circumference, systolic blood pressure, apolipoprotein A1, glucose and alanin aminotransferase accounted for most of the differences in MetS manifestation. Logistic regression showed that participants with risk values of the atherogenic index log(TG-to-HDL-C) had a 15.62-fold higher risk of MetS compared to those with lower values for this index (95% CI = 8.3-29.1). Women with hyperglycaemia (or formerly diagnosed diabetes mellitus) had an 8.82-times higher risk of MetS (95%CI = 3.22-24.16). Women with hyper-uricaemia had the same risk of MetS incidence as women with abdominal obesity, Exp (B) = 4.05.Hypercholesterolaemia, ACE and APOE genotypes did not influence MetS. CONCLUSION: MetS may involve many risk factors that can cause serious disorders in multiple organs. However, women with risk values involving plasma atherogenic index log (TG-to-HDL-C) experienced the highest risk of developing MetS.

Underexplored reciprocity between genome-wide methylation status and long non-coding RNA expression reflected in breast cancer research: potential impacts for the disease management in the framework of 3P medicine.

Breast cancer (BC) is the most common female malignancy reaching a pandemic scale worldwide. A comprehensive interplay between genetic alterations and shifted epigenetic regions synergistically leads to disease development and progression into metastatic BC. DNA and histones methylations, as the most studied epigenetic modifications, represent frequent and early events in the process of carcinogenesis. To this end, long non-coding RNAs (lncRNAs) are recognized as potent epigenetic modulators in pathomechanisms of BC by contributing to the regulation of DNA, RNA, and histones' methylation. In turn, the methylation status of DNA, RNA, and histones can affect the level of lncRNAs expression demonstrating the reciprocity of mechanisms involved. Furthermore, lncRNAs might undergo methylation in response to actual medical conditions such as tumor development and treated malignancies. The reciprocity between genome-wide methylation status and long non-coding RNA expression levels in BC remains largely unexplored. Since the bio/medical research in the area is, per evidence, strongly fragmented, the relevance of this reciprocity for BC development and progression has not yet been systematically analyzed. Contextually, the article aims at:consolidating the accumulated knowledge on both-the genome-wide methylation status and corresponding lncRNA expression patterns in BC andhighlighting the potential benefits of this consolidated multi-professional approach for advanced BC management. Based on a big data analysis and machine learning for individualized data interpretation, the proposed approach demonstrates a great potential to promote predictive diagnostics and targeted prevention in the cost-effective primary healthcare (sub-optimal health conditions and protection against the health-to-disease transition) as well as advanced treatment algorithms tailored to the individualized patient profiles in secondary BC care (effective protection against metastatic disease). Clinically relevant examples are provided, including mitochondrial health control and epigenetic regulatory mechanisms involved.

Analysis of HIF-1α expression and genetic polymorphisms in human clear cell renal cell carcinoma.

Introduction: Clear cell renal cell carcinoma (ccRCC) is mostly diagnosed incidentally and has relatively high recurrence rates. Alterations in VHL/HIF and mTOR pathways are commonly present in ccRCC. The present study attempted to identify potential diagnostic markers at the biochemical and molecular level. Methods: In total, 54 subjects (36 patients with ccRCC and 18 cancer-free controls) were enrolled. ELISA was used to measure the levels of HIF-1α in the tumor and healthy kidney tissue. The association between five selected SNPs (rs779805, rs11549465, rs2057482, rs2295080 and rs701848) located in genes of pathologically relevant pathways (VHL/HIF and mTOR) and the risk of ccRCC in the Slovak cohort was studied using real-time PCR. Results: Significant differences in HIF-1α tissue levels were observed between the tumor and healthy kidney tissue (p < 0.001). In the majority (69%) of cases, the levels of HIF-1α were higher in the kidney than in the tumor. Furthermore, the concentration of HIF-1α in the tumor showed a significant positive correlation with CCL3 and IL-1ß (p (R2) 0.007 (0.47); p (R2) 0.011 (0.38). No relationship between intratumoral levels of HIF-1α and clinical tumor characteristics was observed. Rs11549465, rs2057482 in the HIF1A gene did not correlate with the expression of HIF-1α either in the tumor or in the normal kidney. None of the selected SNPs has influenced the susceptibility to ccRCC. Conclusion: More research is neccesary to elucidate the role of HIF-1α in the pathogenesis of ccRCC and the association between selected SNPs and susceptibility to this cancer.

Changes in the Urine Metabolomic Profile in Patients Recovering from Severe COVID-19.

Metabolomics is a relatively new research area that focuses mostly on the profiling of selected molecules and metabolites within the organism. A SARS-CoV-2 infection itself can lead to major disturbances in the metabolite profile of the infected individuals. The aim of this study was to analyze metabolomic changes in the urine of patients during the acute phase of COVID-19 and approximately one month after infection in the recovery period. We discuss the observed changes in relation to the alterations resulting from changes in the blood plasma metabolome, as described in our previous study. The metabolome analysis was performed using NMR spectroscopy from the urine of patients and controls. The urine samples were collected at three timepoints, namely upon hospital admission, during hospitalization, and after discharge from the hospital. The acute COVID-19 phase induced massive alterations in the metabolic composition of urine was linked with various changes taking place in the organism. Discriminatory analyses showed the feasibility of successful discrimination of COVID-19 patients from healthy controls based on urinary metabolite levels, with the highest significance assigned to citrate, Hippurate, and pyruvate. Our results show that the metabolomic changes persist one month after the acute phase and that the organism is not fully recovered.