Ewelina Kałużna

Atrial fibrillation (AF) is a common cardiac arrhythmia increasing stroke risk. Current treatments show limited effectiveness and can cause serious side effects. At the moment there is a lack of well-established human models recapitulating complex cardiac microenvironment for studying potential AF therapies. To address the limitations of existing models we developed 3D in vitro human Engineered Heart Tissue (EHT) model using atrial and ventricular human-induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) with either human atrial fibroblasts (HAF) or human ventricular fibroblasts (HVF).These chamber-specific EHTs exhibit differences in gene expression, ion channels and physiological responses, enabling studying Frank-Starling mechanisms and Ca ²⁺ response. Using K _Ca 2 channel inhibitor AP14145, we observed increased contraction force and prolonged relaxation time, suggesting negative modulation of K _Ca 2.2/K _Ca 2.3 channels and increased atrial-effective refractory period (AERP). The EHT model provides a valuable platform for examining K _Ca 2 channel inhibitors impact and evaluating chamber-specific drug effects in controlled in vitro settings.

Understanding the molecular mechanisms of severe respiratory syndrome coronavirus 2 (SARS‐CoV‐2) infection is essential for the successful development of therapeutic strategies against the COVID‐19 pandemic. Numerous studies have focused on the identification of host factors and cellular pathways involved in the viral replication cycle. The speed and magnitude of hijacking the translation machinery of host mRNA, and shutting down host transcription are still not well understood. Since SARS‐CoV‐2 relies on host RNA‐binding proteins for the infection progression, several efforts have been made to define the SARS‐CoV‐2 RNA‐bound proteomes (RNA–protein interactomes). Methodologies that enable the systemic capture of protein interactors of given RNA in vivo have been adapted for the identification of the SARS‐CoV‐2 RNA interactome. The obtained proteomic data aided by genome‐wide and targeted CRISPR perturbation screens, revealed host factors with either pro‐ or anti‐viral activity and highlighted cellular processes and factors involved in host response. We focus here on the recent studies on SARS‐CoV‐2 RNA–protein interactomes, with regard to both the technological aspects of RNA interactome capture methods and the obtained results. We also summarize several related studies, which were used in the interpretation of the SARS‐CoV‐2 RNA–protein interactomes. These studies provided the selection of host factors that are potentially suitable candidates for antiviral therapy. Finally, we underscore the importance of RNA–protein interactome studies in regard to the effective development of antiviral strategies against current and future threats.This article is categorized under: RNA Interactions with Proteins and Other Molecules > Protein‐RNA Interactions: Functional Implications RNA in Disease and Development > RNA in Disease RNA Methods > RNA Analyses in Cells

The Picornaviridae are a diverse family of positive-strand RNA viruses that includes numerous human and veterinary pathogens¹. Among these, hepatitis A virus (HAV), a common cause of acute hepatitis in humans, is unique in that it is hepatotropic and is released from hepatocytes without lysis in small vesicles that resemble exosomes^2,3. These quasi-enveloped virions are infectious and are the only form of virus that can be detected in the blood during acute infection². By contrast, non-enveloped naked virions are shed in faeces and stripped of membranes by bile salts during passage through the bile ducts to the gut⁴. How these two distinct types of infectious hepatoviruses enter cells to initiate infection is unclear. Here, we describe a genome-wide forward screen that shows that glucosylceramide synthase and other components of the ganglioside synthetic pathway are crucial host factors that are required for cellular entry by hepatoviruses. We show that gangliosides—preferentially disialogangliosides—function as essential endolysosome receptors that are required for infection by both naked and quasi-enveloped virions. In the absence of gangliosides, both virion types are efficiently internalized through endocytosis, but capsids fail to uncoat and accumulate within LAMP1⁺ endolysosomes. Gangliosides relieve this block, binding to the capsid at low pH and facilitating a late step in entry involving uncoating and delivery of the RNA genome to the cytoplasm. These results reveal an atypical cellular entry pathway for hepatoviruses that is unique among picornaviruses.

The aim of this study is to describe the prevalence of single single-nucleotide polymorphisms (SNPs) as well as their combinations in genes encoding proteins involved in the immune response in children with bacterial meningitis. The prospective study group consisted of 39 children with bacterial meningitis and 49 family members surveyed between 2012 and 2016. Eleven SNPs in five genes involved in immune response were analysed. The mean number of minor frequency alleles (MAF) of studied SNPs was lowest in the control group and highest in patients with pneumococcal meningitis. We found that carrying ≥6 MAF of studied SNPs was associated with an increased risk of pneumococcal meningitis. The prevalence of risky variants was noted to be higher in patients with pneumococcal meningitis as compared to the control group. In conclusion, genetic factors are a relevant factor in determining the susceptibility to bacterial meningitis. A statistically significant cumulative effect of mutated variants on increasing the risk of bacterial meningitis was detected. Combining all three SNPs in MBL2 improves the prediction of susceptibility to pneumococcal meningitis. Analysis of risky alleles can help indicate people prone to the disease who are ‘gene-immunocompromised’.

The etiology of acute lymphoblastic leukemia (ALL) is complex, linked with both environmental exposures and genetic factors. Functional variants of the methylenetetrahydrofolate reductase (MTHFR) gene result in disturbance in folate metabolism and may affect susceptibility to cancer. The study was performed to evaluate whether MTHFR C677T and A1298C polymorphisms, analyzed separately and together, are associated with the development of ALL in a population under 18 years of age of Caucasian ancestry. The study included 117 pediatric patients (59% males, mean age at diagnosis 7.4 ± 5.2 years) with ALL, confirmed by conventional immunophenotyping surface-marker analysis and 404 healthy control subjects (48.5% men, mean age 37.7 ± 11.3 years). The MTHFR C677T and A1298C genotypes were analyzed using allele discrimination tests with Taq-Man fluorescent probes. The MTHFR 677TT genotype was related to a 2-fold increase in risk of ALL (P = .014). The 677T-1298C haplotype was found in ALL patients but not in controls (frequency 0.598%; P <.0001). The observed frequency of carriers of this rare haplotype was 12%, including 677CT/1298CC (1.7%), 677TT/1298AC (6.0%), and 677CT/1298AC (4.3%) genotypes. The MTHFR 677T allele alone or in combination with the MTHFR 1298C allele significantly increases the risk of development of ALL in Polish population under 18 years of age. Further studies of haplotype composition in subjects with the 677CT/1298AC genotype are necessary to assess the risk of childhood ALL.

The prognosis concerning the treatment of patients with chronic hepatitis C (CHC) is closely related to the genotype of the virus as well as to the factors dependent on the patient. It was proved that polymorphisms of the gene encoding interleukin 28B (IL28B) are associated with sustained viral response, which in the case of profitable variants of IL28B polymorphisms may reach up to 87% of the patients. The aim of the study is to determine the prevalence of alleles and distribution of IL28B polymorphisms genotypes in the examined group of patients with CHC in Wielkopolska Province. A total of 710 people with diagnosed hepatitis C virus were examined in order to determine the distribution of polymorphisms of gene IL28B rs12979860, rs8099917 and rs12980275. The polymorphisms were evaluated by sequencing of PCR products. The most often noted profitable variant was genotype TT for polymorphism rs8099917 present in 43.5% of the patients, next was AA rs12980275 in 22.5%. The rarest was the profitable variant CC of the polymorphism rs12979860 present in 17.5% of the patients. An occurrence of at least 2 IL28B polymorphisms in the preferred variants (homozygote CC, TT, AA) was found in 239 out of 710 (34%) patients, among which 117 patients had favorable genotypes for all 3 examined polymorphisms. The SNP distribution of gene IL28 with fixed prognostic value in the population of patients with chronic hepatitis C is different from the general population, and shows the need to evaluate polymorphisms prior to treatment.

To evaluate the association of IFNL3 (IL28B) SNP rs4803217 with severity of disease and treatment outcome in chronic hepatitis C (CHC). The study enrolled 196 CHC Polish patients (82 women and 114 men in age 20-64) infected with hepatitis C virus (HCV) genotype 1. They were treatment naïve and qualified to pegylated interferon alpha (PEG-IFN-α) and ribavirin (RBV) therapy. The analyzed baseline parameters included: degree of inflammation, stage of fibrosis, viral load as well as alanine aminotransferase (ALT), asparagine aminotransferase (AST) and total bilirubin (TBIL). The analysis of response to therapy included: sustained virological response (SVR), defined as undetectable serum HCV RNA level six month after completion of 48-wk therapy, and relapse, defined as achieving undetectable viral load at the end of treatment but not SVR. HCV genotyping and HCV RNA quantification were performed using commercially available tests. DNA was isolated from peripheral blood mononuclear cells or from buccal cell swabs. In addition to rs4803217, also single nucleotide polymorphisms (SNPs) (rs12979860, rs8099917 and rs12980275) of known significance in predicting of HCV clearance were analyzed. SNPs were determined by high resolution melt analysis and confirmed by sequencing of amplicons. Frequency of rs4803217 genotypes in studied group was as follows: 27.55%; 54.59% and 17.86% for CC, CA and AA, respectively. The rs4803217 SNP, similar to other analyzed SNPs, was not associated with severity of CHC (grade of inflammation, stage of fibrosis, baseline viral load as well as biochemical parameters: ALT, AST, TBIL). It was demonstrated that the rs4803217C allele is associated with SVR (C vs A: P < 0.0001; dose of C allele: P = 0.0002) and non-relapse (C vs A: P = 0.001; dose of C allele: P = 0.002). Moreover, it was found that patients with CC genotype have significantly higher response rates as compared with CA/AA patients (P < 0.0001), whereas patients carrying A allele are significantly predisposed to relapse after treatment (P = 0.0007). Moreover, the association of rs4803217 with SVR was comparable to that of rs12979860 and stronger as observed for rs12980275 and rs8099917. Association of rs4803217 with relapse, was the strongest as compared with the other SNPs. The analysis of combined rs4803217 and rs8099917 genotypes demonstrated that additional genotyping of rs8099917 had no significant impact on the prediction of SVR. Multivariate analysis revealed that among analyzed SNPs only rs4803217 is an independent predictor of SVR (P = 0.016) and relapse (P = 0.024). The rs4803217 SNP is a strong, independent and superior predictor of SVR and relapse in HCV genotype 1 infected CHC patients treated with PEG-IFN-α and RBV.