Differential expression of NAT1 pharmacogene in hormone receptor positive vs. negative female breast tumors may affect drug treatment.

Studies have reported overexpression of NAT1 gene for xenobiotic metabolizing arylamine N-acetyltransferase type 1 in estrogen receptor positive breast tumors, and this association has been linked to patient chemoresistance and response to tamoxifen. We probed the expression of NAT1, using quantitative reverse transcription PCR to screen clinically characterized breast cancer tissue cDNA arrays. Primers detecting all NAT1 alternative transcripts were used, and the protocol and results are reported according to consensus guidelines. The clinical information about 166 tumor samples screened is provided, including tumor stage, estrogen and progesterone receptor status and HER2 expression. NAT1 was found to be significantly (P < 0.001) upregulated in hormone receptor positive vs. negative tumors. No correlation was apparent between NAT1 and tumor stage or HER2 expression. Our findings demonstrate a strong correlation between the expression of NAT1 and steroid hormone receptors in breast tumors, supporting its possible utility as a pharmacogenetic biomarker or drug target. Of the two polymorphic NAT genes, NAT1 is the one primarily expressed in breast tissue, and is subjected to regulation by two differential promoters and more than one polyadenylation signal. Hormonal factors may enhance NAT1 gene expression at the transcriptional or epigenetic level, and tamoxifen has additionally been shown to inhibit NAT1 enzymatic activity. The outcome of tamoxifen treatment is also more favorable in patients with NAT1 overexpressing tumors. The study adds to the growing body of evidence implicating NAT1 in breast cancer and its pharmacological treatment.

Improving gene expression analysis efficacy from formalin-fixed paraffin embedded tissues.

INTRODUCTION: Improving RNA isolation and cDNA synthesis techniques has emerged due to advancements in the knowledge of molecular basis of most diseases. This in turn increased the need of higher quantity and quality of the extracted genetic material to be used for a variety of diagnostic tests and experiments. AIM: The aim of the study was to compare three modified methods for RNA extraction from formalin-fixed paraffin embedded (FFPE) biopsied tissue and different cDNA synthesis strategies to facilitate study of gene expression. MATERIALS AND METHODS: Compared RNA extraction methods were: lysis buffer, phenol-based extraction, and combination of both with concomitant use of silica-based spin columns. RNA quantity and purity were estimated spectrophotometrically. Different priming strategies for cDNA synthesis were applied: oligo dT, combination of oligo dT and random hexamer, and gene specific primer. Two-step RT-qPCR of ribosomal protein L37A on preamplified and non-preamplified cDNA templates was performed. RESULTS: The combination of lysis buffer with phenol based extraction gave higher RNA yield. By doing cDNA preamplification, the confidence of detection by qPCR was raised, and efficiency was improved. The preamplified template increased the sensitivity of analysis. CONCLUSIONS: Together, the combination of approaches improved substantially the reproducibility and validity of quantitative gene expression analyses from FFPE tissues.

Ferrum phosphoricum D12 Treatment Affects J774A.1 Cell Proliferation, Transcription Levels of Iron Metabolism, Antioxidant Defense, and Inflammation-related Genes.

BACKGROUND: Ferrum phosphoricum (FP) is prescribed as a homeopathic remedy to treat the early stages of fever and inflammation in cases of colds or flu, muscle fatigue and anemia. We aimed to analyze the molecular mechanisms of action of FP D12 on cell proliferation and mRNA expression of iron metabolism, antioxidant defense and inflammation-related genes in mouse J774A.1 macrophages. METHODS: Cell proliferation was examined using the MTT test. RT-qPCR analyses were performed to estimate gene expression changes. Relative gene expression levels were calculated using the 2-ΔΔCt method. The effect of treatment using FP D12 tablets was compared with that using placebo tablets (PT). RESULTS: FP D12 in low concentrations (0.0125 mg/mL to 0.025 mg/mL) significantly stimulated proliferation of J774A.1 cells by up to 11% (p < 0.01) versus control untreated cells and by up to 40% (p < 0.01) versus PT-treated cells in the respective concentration. FP D12 versus PT induced a significant increase in mRNA expression of ferritin light chain (Ftl1) (by 8-fold, p < 0.01), ß-2-microglobulin (B2m) (by 2.5-fold, p < 0.05) and iron-responsive element binding protein 2 (Ireb2) (by 4-fold, p < 0.05), and induced a slight decrease in myosin IE (Myo1e) mRNA expression levels (by 0.4-fold, p < 0.01) in macrophages. A highly significant (r2 = 0.99, p < 0.05) correlation was observed between Ireb2 and B2m transcription levels. Significant stimulation of antioxidant enzyme Gpx-1 (by 1.27-fold, p < 0.01) in cells by 0.025 mg/mL FP D12, but a slight decrease (by 0.12-fold, p < 0.05) in 0.0125 mg/mL-treated cells, was observed. A significant increase in the gene expression of IL-1ß (by 3.5-fold, р < 0.05) in macrophages was also detected. CONCLUSION: Ferrum phosphoricum in D12 dilution potentially exhibits iron retention, antioxidant and immunomodulation activities, possibly by modulating transcription levels of related genes in non-stimulated mouse macrophages.

A mediastinal malignant thyroid paraganglioma: A case report and literature review.

INTRODUCTION AND IMPORTANCE: Thyroid cancer is the most common endocrine malignancy, while primary paraganglioma of the thyroid gland (TP) is an unusual tumour and in rare cases, this disease tends to mimic thyroid cancer. They are rare extra-adrenal neuroendocrine tumours originating from the neural crest, and are found almost exclusively in the head and neck area. CASE PRESENTATION: We present a case of a 53-year-old man, in whom a mediastinal lesion originating from the left lobe of the thyroid gland was found on routine ultrasound and subsequent computed tomography (CT). CLINICAL DISCUSSION: Total thyroidectomy and lymph dissection were performed. A review of the literature was made and a discussion was held regarding the diagnosis, the importance of surgical treatment and further behaviour. CONCLUSION: Surgical removal of the thyroid gland is the main treatment, followed by radiation therapy. The diagnosis and differential diagnosis with other thyroid tumours is extremely important in terms of subsequent behaviour and prognosis.

New Circulating Circular RNAs with Diagnostic and Prognostic Potential in Advanced Colorectal Cancer.

Circular RNAs (circRNAs) are a group of special endogenous long non-coding RNAs which are highly stable in the circulation, and, thus, more suitable as new biomarkers of colorectal cancer (CRC). The aim of our study was to explore the plasma expression levels of four circRNAs: has_circ_0001445, hsa_circ_0003028, hsa_circ_0007915 and hsa_circ_0008717 in patients with CRC and to evaluate their associations with clinicopathological characteristics and the clinical outcome of the patients. CircRNAs were extracted from patients' plasma obtained prior to chemotherapy. Their expression levels were measured by qPCR and calculated applying the 2-ΔΔCt method. The levels of all four circRNAs were significantly increased in the plasma of CRC patients. At the optimal cut-off values hsa_circ_0001445 and hsa_circ_0007915 in plasma could significantly distinguish between patients with or without metastatic CRC with 92.56% sensitivity and 42.86% specificity, and with 86.07% sensitivity and 57.14% specificity, respectively. The mean overall survival (OS) of patients with high/intermediate expression of hsa_circ_0001445 was 30 months, significantly higher in comparison with the mean OS of the patients with low expression-20 months (log-rank test, p = 0.034). In multivariate Cox regression analysis, the low levels of hsa_circ_0001445 were also associated with shorter survival (HR = 1.59, 95% CI: 1.02-2.47, p = 0.040). A prognostic significance of hsa_circ_0001445 for patients with metastatic CRC was established.

Oncogenic Functions and Clinical Significance of Circular RNAs in Colorectal Cancer.

Colorectal cancer (CRC) is ranked as the second most commonly diagnosed disease in females and the third in males worldwide. Therefore, the finding of new more reliable biomarkers for early diagnosis, for prediction of metastasis, and resistance to conventional therapies is an important challenge in overcoming the disease. The current review presents circular RNAs (circRNAs) with their unique features as potential prognostic and diagnostic biomarkers in CRC. The review highlights the mechanism of action and the role of circRNAs with oncogenic functions in the CRC as well as the association between their expression and clinicopathological characteristics of CRC patients. The comprehension of the role of oncogenic circRNAs in CRC pathogenesis is growing rapidly and the next step is using them as suitable new drug targets in the personalized treatment of CRC patients.

Circulating miR-618 Has Prognostic Significance in Patients with Metastatic Colon Cancer.

The present study evaluated the prognostic role of circulating miRNA-618 in patients with metastatic colon cancer (mCC) and whether miR-618 gene rs2682818 single nucleotide polymorphisms (SNP) are associated with colon cancer susceptibility and expression levels of mature miR-618. In total, 104 patients with mCC before starting the chemotherapy were investigated. The expression status of circulating miR-618 in mCC was evaluated by quantitative PCR. TaqMan PCR assay was used for rs2682818 SNP genotyping. miR-618 was overexpressed in serum of mCC patients. Patients with high and intermediate expression of miR-618 had a significantly longer mean overall survival (OS) of 21 months than patients with low expression-16 months. In addition, multivariate Cox regression analysis confirmed the association between high/intermediate levels of miRNA-618 and longer OS, HR = 0.51, 95% CI: 0.30-0.86, p = 0.012. miR-618 rs2682818 SNP significantly decreased the risk of colon cancer susceptibility in both heterozygous codominant (AC vs. CC, OR = 0.39, 95% CI: 0.17-0.88, p = 0.024) and overdominant (AC vs. CC + AA, OR = 0.37, 95% CI: 0.16-0.85, p = 0.018) genetic models. Our data suggest that circulating miRNA-618 could be useful as a prognostic biomarker in mCC. Patients harboring AC rs2682818 genotype have a decreased risk for colon cancer in comparison with patients with CC and AA genotypes.

Effects of sulphur-containing mineral water intake on oxidative status and markers for inflammation in healthy subjects.

CONTEXT: Sulphurous mineral waters (SMW) have a wide range of applications. Sulphur content of mineral waters is considered as possible determinant for their anti-inflammatory or pro-inflammatory effects. OBJECTIVE: To explore the healing properties of Varna basin mineral water by analysing possible antioxidative and anti-inflammatory effects. MATERIALS AND METHODS: An intervention with Varna SMW intake was performed with healthy volunteers. Total thiols, total glutathione and its fractions, reactive oxygen metabolites, malondialdehyde, intracellular adhesion molecule (ICAM-1) and vascular cell adhesion molecule (VCAM-1) were measured. Expression of γ-gluthamyl-cysteinyl ligase (GCL) and sICAM-1 genes was also analysed. RESULTS: A significantly increased total glutathione and total thiols were observed at the end of the intervention. GCL and sICAM-1 gene expressions were increased after the intervention. CONCLUSION: SMW consumption improved redox status of the body. We suggested that these beneficial effects may be attributed to the established high levels of sulphur-containing compounds in Varna mineral water.

Attitudes towards e-Health during the COVID-19 Pandemic: Untangling the Gordian Knot in Greece, Bulgaria and Romania in Healthcare Professionals and Students?

BACKGROUND: Until recent times, healthcare specialists lagged behind other fields in terms of computer networks and software usage, partly because they were uncomfortable with information and communication technologies. The rapid development of personal wearables, their easier connections with smartphones and other similar devices, and the possibilities of online or cloud storage of personal medical data led to improvements of eHealth services in terms of ease of access and reduced costs of delivery. SUBJECTS AND METHODS: The current research aimed to fill a research gap regarding attitudes towards e-Health during the COVID-19 pandemic in three Balkan countries: Greece, Bulgaria, and Romania. A total number of 775 adult participants (healthcare professionals and students in the field) were contacted in the winter of 2020 and asked to complete a questionnaire regarding their e-Health attitudes. RESULTS: The Greek participants had the most negative attitudes toward e-Health services. This may be due to forced use of eHealth applications during the pandemic crisis and the need for mastering the necessary technical skills to use them. CONCLUSIONS: It should be noted that in the scientific literature no research is found on this topic, therefore it is the first study aimed to assessing and comparing attitudes towards eHealth in these countries.

When is a person with dementia in need of palliative care? Opinions of healthcare professionals and university students from three South East European countries.

BACKGROUND: Recommendations of the European Association for Palliative Care have been proposed to strengthen the provision of palliative care and it is clear that people with dementia can benefit from palliative care, but no research so far addresses the beliefs of university students and professionals in the social, medical and other science fields in South East Europe regarding the need from palliative care. The objective of this study is to explore when students and professionals consider a person with dementia in need of palliative care and to compare their opinions in three South East European countries. SUBJECTS AND METHODS: An anonymized questionnaire was used based on van Riet Paap et al. (2015) case-vignette. A sample of 1287 participants (student nurses, professional nurses, psychology students, professional psychologists, student doctors, professional doctors, and students and professionals from other non-relevant to medical and social care sciences) was examined in Greece, Bulgaria and Romania. RESULTS: The open responses to the "when" question revealed the categories: after her diagnosis/after the first symptoms; when she and her family cannot cope; at the advance of symptoms; all the time; when she behaves aggressively; I am not sure/there is no specific point. Unexpectedly, age, professional status and field of studies were not found to be significant predictors, but only the country of origin was found to contribute to the differences in the participants' answers regarding the start of palliative care. CONCLUSION: Although professional-student status and relevancy of subject did not predict opinions, the country of origin predicted controversies for the proposed time point of considering a person with dementia in need of palliative care.

Light-dependent processes on the cathode enhance the electrical outputs of sediment microbial fuel cells.

In this study, we explored in details the influence of the light irradiation on the SMFCs electrical outputs. The experiments at both natural and artificial illumination firmly show that during the photoperiods the current grows up. The intensity of the current increase depends on the duration of the photoperiod as well as on the wavelength of the monochromatic light source applied. The highest influence of the light irradiation has been obtained at wavelengths, corresponding to the absorption peaks of essential pigments in the light-harvesting system of oxygenic photosynthesizing microorganisms. The decreased values as well as the discontinued fluctuations of the current as a result of suppressed illumination or substitution of the biocathode with a new one suggest that photosynthesizing microorganisms, co-existing in the cathodic biofilm consortium, contribute to the overall SMFC performance. The microscopic observations confirm the existence of chlorophyll-containing microorganisms on the cathode surface. Though the performed metagenomics DNA analysis has not certified a dominance of photosynthesizing microorganisms, all other results support the hypothesis that the current enhance during the photoperiods is due to the in situ bio-oxygen production on the cathode surface, thus lowering the mass transport limitations for the oxygen reduction reaction.

Gene-gene interaction of µ-opioid receptor and GluR5 kainate receptor subunit is associated with smoking behavior in a Greek population: presence of a dose allele effect.

BACKGROUND: Components of nicotine reward system can potentially influence smoking behavior. The µ-opioid receptor (OPRM1) binds the endogenous opioid peptide ß-endorphin and mediates the reinforcing effects of nicotine, while the GluR5 kainate receptor subunit (encoded by GRIK1 gene), a binding site for known mediators of glutamate neurotransmission, potentially affects the glutaminergic system that is also indirectly implicated in the reward system. METHODS: In the present study, OPRM1 A118G and GRIK1 rs2832407C>A polymorphisms and their interactions were analyzed in 132 smoking initiators (SI) and 144 non-initiators (NI) of Greek origin, using the PCR-RFLP method. RESULTS: No differences were found in the genotype or allele distribution of OPRM1 A118G and GRIK1 rs2832407C>A between SI and NI. However, we found a significant interaction of OPRM1 A118G and GRIK1 rs2832407C>A genotypes associated with smoking initiation in a model adjusted for age, sex, BMI and type 2 diabetes mellitus (odds ratio=1.341, 95% CI 1.024-1.755, p=0.033). A dose effect of OPRM1 and GRIK1 variant alleles was present. Increased number of variant alleles (from 0 to 4) was associated with smoking initiation in the same adjusted model (odds ratio=1.537, 95% CI 1.030-2.293, p=0.036). CONCLUSIONS: Smoking phenotype is a complex interaction of genetic and environmental factors. In the present study, we have shown that gene-gene interaction of components of different systems associated with nicotine reinforcing effects, such as OPRM1 and GRIK1, rather than one gene polymorphism, is associated with smoking behavior.

Small RNA analysis of Potato spindle tuber viroid infected Phelipanche ramosa.

Plants defend themselves against virus/viroid infection by induction of a mechanism of viral RNA degradation or translation inhibition. This is achieved by the production of small RNAs referred to as small interfering RNAs and microRNA, the key molecules in establishment of RNA directed silencing. Potato Spindle Tuber Viroid (PSTVd) was the first viroid species to be identified as naturally infecting potato, and it was found to infect many other crop species, wild and ornamental plants. Recently the experimental host range of PSTVd was extended with the root non-photosynthetic parasitic weed--Phelipanche ramosa (L) Pomel. Here we examined the small RNA population in P. ramosa infected with PSTVd and we observed the presence of PSTVd derived small RNAs. The hotspot regions for production of those PSTVd specific small RNAs were defined by their mapping on the viroid genome sequence. Further, we evaluated the expression levels of selected conserved microRNA families in the viroid infected P. ramosa. Upon infection, two members of miRNA395 family were significantly accumulated, while several members of miRNA390, miRNA396, miRNA319, miRNA166, miRNA167 and miRNA159 were strongly down-regulated. All these findings imply the involvement of various small RNA classes in the P. ramosa response to PSTVd infection.

Detection of Potato spindle tuber viroid sequence variants derived from PSTVd-infected Phelipanche ramosa in flower organs of tomato plants.

Potato spindle tuber viroid (PSTVd) is an infectious small, circular, non-coding single-stranded RNA that induces disease on many crop species, ornamental plants, weeds and parasitic plants. PSTVd propagate in their host as a population of closely related but non-identical RNA variants referred to as quasispecies. Recently, we have described three de novo arising PSTVd variants in the parasitic plant Phelipanche ramosa after mechanical inoculation with the PSTVd KF440-2 isolate. These P. ramosa derived mutants were designated as G241-C, C208-U and C227-U PSTVd variants. Each of these variants carries a single-nucleotide substitution compared to the PSTVd KF440-2 sequence from which they are considered to have evolved. Here we complement our previous studies on these mutants by exploring their potential to infect the floral organs of tomato plants. We found that the PSTVd G241-C and C208-U variants were able to replicate in systemic leaves and floral organs of tomato plants, while the PSTVd C227-U variant did not develop systemic infection. Furthermore, we analysed the progeny of these PSTVd variants in sepals and petals of tomato plants for retention of the specific mutations.

Identification of RNA-dependent DNA-methylation regulated promoters in Arabidopsis.

RNA-dependent DNA methylation (RdDM) is an important regulatory event involved in repressive epigenetic modifications that can trigger transcriptional gene silencing (TGS). The criteria we used to pick out promoter sequences targeted by RdDM in Arabidopsis thaliana were the main RdDM hallmark properties: 24nt siRNAs as inducers of DNA methylation and transposable elements (TE) as one of the major targets of RdDM. Those genes whose promoters comprised overlapping sites for 24nt siRNA hits, TE and DNA methylation (siRNA/TE/Methylation overlapping regions), were defined as candidates that might be silenced by RdDM. On this basis two gene sets were created which include abiotic and biotic stress-responsive genes whose promoters may be silenced by RdDM. The DNA methylation status of the At3g50770 (CML41) promoter - one of the selected candidates, was experimentally assayed, and it showed dependence on the RdDM-associated Polymerase IV and Polymerase V. A publicly available 24nt siRNA-centered database called starPRO was developed that allows users easily to discover whether a particular promoter sequence is related to RdDM-associated features such as 24nt siRNA-target sites, TE, tandem repeats and DNA methylation.