Andrej Ficek

Wastewater-based surveillance can track community SARS-CoV-2, but RNA signals at wastewater treatment plants are affected by sampling, analytical recovery and transfer to solids. We quantified SARS-CoV-2 RNA in influent and biologically treated effluent from eight Slovak municipal wastewater treatment plants during the period October–December 2020. Twenty-seven 24 h influent composites and seven same-day effluent samples were analysed by reverse-transcription quantitative PCR targeting ORF1b, S, E and RdRp. RNA was detected in 20/27 influents (74%; 95% CI, 54–89%) and 3/7 effluents (43%; 95% CI, 10–82%). Influent loads ranged from 4.66 × 10⁹ to 5.60 × 10¹³ copies day⁻¹; the maximum population-normalised load was 5.84 × 10⁵ copies per population equivalent per day. All seven effluent concentrations were lower than paired influents (exact sign test, p = 0.0156). Apparent log₁₀ reductions were 0.18 and 0.76 for quantifiable effluents, approximately 1.75 for one detection at the operational limit of detection, and ≥1.9–≥2.5 for four non-detects. A one-day process profile at the selected plant detected RNA in activated-sludge supernatant, digested-sludge supernatant and reject water. Because solids, infectivity and whole-process recovery were not measured, the results describe RNA occurrence rather than viral inactivation or a mass balance. These pilot data provide a historical baseline for Slovakia and define requirements for repeated sampling, process controls and paired liquid-solid measurements.

Aims: Chemotherapy resistance remains a major challenge in breast cancer (BC) treatment. This study investigated whether resistance development is associated with DNA methylation changes and assessed the potential of the DNA methyltransferase inhibitor decitabine (DAC) to reverse these alterations and enhance chemosensitivity. Methods: Molecular profiling and functional assays were used to characterize paclitaxel-(PAC) and doxorubicin-(DOX) resistant BC cell lines derived from luminal A (T-47D), triple-negative (MDA-MB-231), and HER2-positive trastuzumab-resistant (JIMT-1) models. Therapeutic responses to DAC and DOX, alone and in combination, were evaluated in MDA-MB-231 xenografts. DNA methylation–associated gene expression changes were analyzed through integrative approaches. Results: Chemoresistant cells exhibited a slow-cycling phenotype, reduced tumorigenicity, and extensive genomic alterations. Upregulation of RELB and downregulation of PPARG were observed across several resistant cell lines, while CDA expression was uniformly elevated in all DOX-resistant models. PAC-resistant xenografts displayed widespread methylation and transcriptomic reprogramming. DAC treatment partially restored aberrant methylation patterns and increased Ki-67 expression, potentially enhancing DOX responsiveness. Conclusions: Chemoresistance in BC involves extensive genomic and epigenetic reprogramming. DAC modulates methylation and tumor phenotype but is insufficient to overcome resistance, highlighting the need for rational combination strategies. Graphical

Background: X-linked myotubular myopathy (XLMTM) is a rare congenital myopathy resulting from dysfunction of the protein myotubularin encoded by the MTM1 gene. XLMTM has a high neonatal and infantile mortality rate due to a severe myopathic phenotype and respiratory failure. However, in a minority of XLMTM cases, patients present with milder phenotypes and achieve ambulation and adulthood. Notable facial dysmorphia is also present. Methods: We investigated the genotype–phenotype correlations in newly diagnosed XLMTM patients in a patients’ cohort (previously published data plus three novel variants, n = 414). Based on the facial gestalt difference between XLMTM patients and unaffected controls, we investigated the use of the Face2Gene application. Results: Significant associations between severe phenotype and truncating variants (p < 0.001), frameshift variants (p < 0.001), nonsense variants (p = 0.006), and in/del variants (p = 0.036) were present. Missense variants were significantly associated with the mild and moderate phenotype (p < 0.001). The Face2Gene application showed a significant difference between XLMTM patients and unaffected controls (p = 0.001). Conclusions: Using genotype–phenotype correlations could predict the disease course in most XLMTM patients, but still with limitations. The Face2Gene application seems to be a practical, non-invasive diagnostic approach in XLMTM using the correct algorithm.

X-linked myotubular myopathy (XLMTM) is a rare congenital myopathy resulting from dysfunction of the protein myotubularin encoded by the MTM1 gene. XLMTM has a high neonatal and infantile mortality rate due to a severe myopathic phenotype and respiratory failure. However, in a minority of XLMTM cases, patients present with milder phenotypes and achieve ambulation and adulthood. Regarding wide phenotypic variability, we investigated the genotype-phenotype correlations in newly diagnosed XLMTM patients in a patients' cohort (previously published data plus three novel variants, n=414). We found a significant association between severe phenotype and truncating variants (p < 0.001), frameshift variants (p < 0.001), nonsense variants (p = 0.006), and in/del variants (p = 0.036). On the contrary, missense variants were significantly associated with the mild and moderate phenotype (p < 0.001). We found no significant association between phenotype and the MTM1-specific functional domain, contrasting with previously published data. Based on the significant facial gestalt difference between XLMTM patients and unaffected controls (p = 0.001), we consider the Face2Gene application as an effective diagnostic tool in XLMTM when using the correct algorithm. The Face2Gene application seems to be a practical, non-invasive diagnostic approach in XLMTM.

Mutations in the voltage-gated sodium channel Na_v1.1 (SCN1A) are linked to various epileptic phenotypes with different severities, however, the consequences of newly identified SCN1A variants on patient phenotype is uncertain so far. The functional impact of nine SCN1A variants, including five novel variants identified in this study, was studied using whole-cell patch-clamp recordings measurement of mutant Na_v1.1 channels expressed in HEK293T mammalian cells. E78X, W384X, E1587K, and R1596C channels failed to produce measurable sodium currents, indicating complete loss of channel function. E788K and M909K variants resulted in partial loss of function by exhibiting reduced current density, depolarizing shifts of the activation and hyperpolarizing shifts of the inactivation curves, and slower recovery from inactivation. Hyperpolarizing shifts of the activation and inactivation curves were observed in D249E channels along with slower recovery from inactivation. Slower recovery from inactivation was observed in E78D and T1934I with reduced current density in T1934I channels. Various functional effects were observed with the lack of sodium current being mainly associated with severe phenotypes and milder symptoms with less damaging channel alteration. In vitro functional analysis is thus fundamental for elucidation of the molecular mechanisms of epilepsy, to guide patients’ treatment, and finally indicate misdiagnosis of SCN1A related epilepsies.