https://hdl.handle.net/20.500.14178/901 2026-05-11T12:29:46Z 2026-05-11T12:29:46Z Musil, Dominik Krhutová, Markéta Blažková, Kristýna Kramná, Anežka Brázdová, Andrea Výmolová, Barbora Houdová Megová, Magdalena Hadzima, Martin Kryštůfek, Robin Šubr, Vladimír Kostka, Libor Etrych, Tomáš Ormsby, Tereza Sácha, Pavel Ambramson, Jakub Konvalinka, Jan https://hdl.handle.net/20.500.14178/3798 2026-05-08T01:00:20Z 2026-01-01T00:00:00Z

CD64-Targeted Polymer-Drug Conjugates Exploit Cathepsin K-Dependent Payload Release for Selective Elimination of Immunosuppressive Macrophages Musil, Dominik; Krhutová, Markéta; Blažková, Kristýna; Kramná, Anežka; Brázdová, Andrea; Výmolová, Barbora; Houdová Megová, Magdalena; Hadzima, Martin; Kryštůfek, Robin; Šubr, Vladimír; Kostka, Libor; Etrych, Tomáš; Ormsby, Tereza; Sácha, Pavel; Ambramson, Jakub; Konvalinka, Jan Selective depletion of immunosuppressive macrophages in the tumor microenvironment is a promising strategy in cancer therapy. CD64 is broadly expressed on myeloid cells, including both pro-inflammatory M1-like and immunosuppressive M2-like macrophages that resemble tumor-associated macrophages (TAMs), and thus represents an attractive entry receptor for targeted payload delivery. We developed HPMA-based CD64-targeted polymer-drug conjugates (CD64-TPDCs) that combine multivalent receptor engagement with enzyme-responsive payload release. These copolymers are decorated with the CD64-binding cyclic peptide cp33 and carry the cytotoxic payload mertansine (DM1) bound via cathepsin-cleavable peptide linkers. Multivalent cp33 presentation on the polymer markedly increased the apparent affinity for human CD64, resulting in subnanomolar binding and selective recognition of CD64-expressing cells, significantly improving the binding potency of monovalent cp33 peptide. In polarized M2-like human monocyte-derived macrophages (MDMs), we showed that cytotoxic Gly-Phe-Leu-Gly-DM1 CD64-TPDCs selectively induced apoptosis. In contrast, M1-like MDMs were largely spared despite expressing higher levels of CD64. In M2-like MDMs, CD64-TPDCs rapidly accumulated in lysosomes, whereas in M1-like cells, they remained largely confined to endosomes. To elucidate the basis of this selectivity, we profiled expression of cathepsins in polarized MDMs. We found that M2-like MDMs display substantially higher levels of cathepsin K, establishing a model in which cathepsin K is the major protease responsible for Gly-Phe-Leu-Gly linker cleavage and DM1 release in M2-like macrophages. These findings demonstrate that CD64-TPDCs can be engineered to exploit subset-specific trafficking and cathepsin K-dependent linker cleavage for the selective elimination of M2-like macrophages. This work provides a generalizable design principle for stimuli-responsive PDCs that may actively target immunosuppressive myeloid cells in tumors.

2026-01-01T00:00:00Z Chudějová, Kateřina Mattioni Marchetti, Vittoria Zaccaria, Vita Kanova, Stepanka Šrámková, Anna Krůtová, Marcela Ryšková, Lenka Kroneislová, Gabriela Horvathova, Beata Tejkalova, Renata Krejci, Eva Vagnerova, Iva Zemanova, Zuzana Bitar, Ibrahim https://hdl.handle.net/20.500.14178/3743 2026-04-28T01:00:32Z 2026-01-01T00:00:00Z

One-year multicenter surveillance of Fosfomycin resistance Enterobacterales: the rise of FosA3-producing P. mirabilis Chudějová, Kateřina; Mattioni Marchetti, Vittoria; Zaccaria, Vita; Kanova, Stepanka; Šrámková, Anna; Krůtová, Marcela; Ryšková, Lenka; Kroneislová, Gabriela; Horvathova, Beata; Tejkalova, Renata; Krejci, Eva; Vagnerova, Iva; Zemanova, Zuzana; Bitar, Ibrahim The global rise of antimicrobial resistance has renewed interest in fosfomycin (FOS), an old antibiotic with activity against multidrug-resistant Enterobacterales . However, resistance to FOS is increasing, driven by impaired drug uptake, target modification, and by fosA -encoded enzymatic inactivation. This study assessed the prevalence and molecular basis of FOS resistance among Enterobacterales collected in Czech tertiary care hospitals in 2024. A total of 211 preliminary FOS-resistant isolates were obtained from nine hospitals across the Czech Republic, predominantly Proteus mirabilis (n=149) and Escherichia coli (n=57). All isolates showed elevated FOS MICs, and the PPF test identified FosA activity in 34/211 isolates. Carbon-source growth testing demonstrated widespread impairment of GlpT and UhpT transporters (93.8 % affecting both). PCR confirmed fosA genes in 14 isolates (9 P. mirabilis , 5 E. coli ). WGS revealed fosA3 as the dominant variant (85.7 %), followed by fosA4 (14.3 %). P. mirabilis isolates primarily belonged to ST185 and ST135, forming two Czech-specific fosA3 clusters with limited relatedness to international genomes. FosA-producing E. coli displayed broader diversity (ST69, ST58, ST550, ST1308). FosA4 was detected exclusively in E. coli . Most fosA -positive strains co-harbored ESBL genes, predominantly bla <inf>CTX-M-65</inf>. SNP-based phylogenies indicated local clonal circulation of fosA3 -positive P. mirabilis ST185, whereas E. coli isolates showed heterogeneous international linkages. Analysis of GlpT/UhpT/MurA identified numerous amino acid substitutions, though only a minority were predicted to affect protein function. This study documents the first broader emergence of plasmid-mediated FOS resistance in Czech Enterobacterales and underscores the importance of continuous genomic surveillance of fosA -mediated resistance.

2026-01-01T00:00:00Z Steiner Mrázová, Lenka Vrbacká, Alena Majer, Filip Stránecký, Viktor Nosková, Lenka Záhoráková, Daniela Májovská, Jitka Bitar, Ibrahim Klempíř, Jiří Šaligová, Jana Majlingová, Stella Giertlová, Mária Drenčáková, Petra Harvanová, Denisa Solařová, Pavla Brůha, Radan Dušek, Petr Kmoch, Stanislav Sikora, Jakub Jedličková, Ivana https://hdl.handle.net/20.500.14178/3741 2026-04-22T01:00:16Z 2026-01-01T00:00:00Z

Analyses of ATP7B mRNA in Nasopharyngeal Swab Samples Increase Yields of Wilson Disease Molecular Genetic Diagnostics Steiner Mrázová, Lenka; Vrbacká, Alena; Majer, Filip; Stránecký, Viktor; Nosková, Lenka; Záhoráková, Daniela; Májovská, Jitka; Bitar, Ibrahim; Klempíř, Jiří; Šaligová, Jana; Majlingová, Stella; Giertlová, Mária; Drenčáková, Petra; Harvanová, Denisa; Solařová, Pavla; Brůha, Radan; Dušek, Petr; Kmoch, Stanislav; Sikora, Jakub; Jedličková, Ivana Wilson disease (WD) is an autosomal recessive disorder of copper transport caused by bi-allelic pathogenic variants in the ATPase copper transporting beta gene (ATP7B). Results of standard genetic diagnostics remain inconclusive in 3%-20% of WD patients in part due to problematic assessment of variants of unknown or conflicting pathogenicity (synonymous variants included). Correct interpretation of potential effects of such variants can be substantially enhanced by RNA analyses. This strategy is, however, of limited utility in WD patients because of predominant liver expression of ATP7B. To avoid invasive bioptic liver collection and increase WD diagnostic yields, we searched for a surrogate tissue sample and identified profiles of ATP7B transcripts in nasopharyngeal swabs that were comparable to liver. Amplicons spanning ATP7B Exons 3-21 were prepared from the swab material and analysed by long-read nanopore sequencing to enable the detection of splicing changes and variant phasing. Diagnostic utility of this novel in vivo methodology was demonstrated by characterization of mRNA splicing abnormalities caused by synonymous ATP7B variants c.1488C>T (p.(Gly496=)), c.2241C>T (p.(Ile747=)), c.2292C>T (p.(Phe764=)), and a nonsense variant c.2336G>A (p.(Trp779Ter)) in four WD patients, who were not genetically resolved by standard techniques. Nasopharyngeal swab sampling is minimally invasive and allows effective analyses of mRNA to detect and/or validate effects of ATP7B variants in WD patients. Conclusive genetic diagnosis attained by this novel technique may facilitate family counselling and substantiate initiation of copper-chelation therapy in presymptomatic individuals.

2026-01-01T00:00:00Z Gil-Korilis, Adrián Ergui-Arbizu, Jorge Hanák, Petr Danešová, Natálie Tomášová, Kristýna Valíčková, Anna Horák, Josef Gentiluomo, Manuel Levý, Miroslav Křivonosková, Soňa Král, Jan Jungwirth, Jiří Vodičková, Ľudmila Vymetálková, Veronika Azqueta, Amaya Campa, Daniele Vodička, Pavel Vodenková, Soňa https://hdl.handle.net/20.500.14178/3733 2026-04-14T01:00:14Z 2026-01-01T00:00:00Z

Unraveling the telomere-mitochondrial axis in colorectal cancer: Results from a prospectively followed cohort Gil-Korilis, Adrián; Ergui-Arbizu, Jorge; Hanák, Petr; Danešová, Natálie; Tomášová, Kristýna; Valíčková, Anna; Horák, Josef; Gentiluomo, Manuel; Levý, Miroslav; Křivonosková, Soňa; Král, Jan; Jungwirth, Jiří; Vodičková, Ľudmila; Vymetálková, Veronika; Azqueta, Amaya; Campa, Daniele; Vodička, Pavel; Vodenková, Soňa Background Telomere shortening and mitochondrial dysfunction are well-known independent contributors to many diseases, but emerging evidence suggests a reciprocal relationship between the two processes. The role of the so-called telomere-mitochondrial axis in colorectal cancer (CRC) remains largely unknown. Methods This prospective cohort study screened CRC patients who underwent surgery, from whom peripheral blood, intestinal mucosa, and tumor samples were collected. Colonoscopically confirmed cancer- and adenoma-free healthy individuals were screened as controls, from whom peripheral blood and intestinal mucosa samples were obtained. Relative mitochondrial DNA copy number (mtDNA-CN) and relative telomere length (RTL) were measured in all samples by real-time quantitative polymerase chain reaction and were further compared and correlated considering clinical data. Relative mtDNA-CN was quantified using both TaqMan probes and SYBR Green to compare both methods. Finally, multivariable analyses were conducted to investigate the association between both biomarkers and the risk of tumor recurrence and mortality. Results A total of 166 CRC patients and 61 healthy individuals were included in the study. In TNM stage I patients, relative mtDNA-CN and RTL were negatively correlated with each other in intestinal mucosa (ρ = -0.77, p < 0.0001), tumor tissue (ρ = -0.41, p = 0.032), and the tumor-to-intestinal mucosa ratio (ρ = -0.39, p = 0.046). However, these associations disappeared with increasing TNM stage, suggesting a dysregulation of the telomere-mitochondrial axis in advanced disease. Higher relative mtDNA-CN in blood was associated with a lower risk of disease recurrence even after adjusting for multiple covariates (HR = 0.43, 95% CI 0.20-0.97, p = 0.041), highlighting its potential use as a prognostic tool. The quantification of mtDNA-CN performed by both methods -TaqMan probes and SYBR Green- was shown to be positively correlated (p < 0.01). Relative mtDNA-CN and RTL were found to be tissue-dependent in both CRC patients and healthy controls. Conclusions This study provides a novel contribution to the understanding of the almost unexplored telomere-mitochondrial axis in CRC, highlighting its potential role in disease progression and prognosis.

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