KDM7A-DT induces genotoxic stress, tumorigenesis, and progression of p53 missense mutation-associated invasive breast cancer.

Stress-induced promoter-associated and antisense lncRNAs (si-paancRNAs) originate from a reservoir of oxidative stress (OS)-specific promoters via RNAPII pausing-mediated divergent antisense transcription. Several studies have shown that the KDM7A divergent transcript gene (KDM7A-DT), which encodes a si-paancRNA, is overexpressed in some cancer types. However, the mechanisms of this overexpression and its corresponding roles in oncogenesis and cancer progression are poorly understood. We found that KDM7A-DT expression is correlated with highly aggressive cancer types and specific inherently determined subtypes (such as ductal invasive breast carcinoma (BRCA) basal subtype). Its regulation is determined by missense TP53 mutations in a subtype-specific context. KDM7A-DT transcribes several intermediate-sized ncRNAs and a full-length transcript, exhibiting distinct expression and localization patterns. Overexpression of KDM7A-DT upregulates TP53 protein expression and H2AX phosphorylation in nonmalignant fibroblasts, while in semi-transformed fibroblasts, OS superinduces KDM7A-DT expression in a TP53-dependent manner. KDM7A-DT knockdown and gene expression profiling in TP53-missense mutated luminal A BRCA variant, where it is abundantly expressed, indicate its significant role in cancer pathways. Endogenous over-expression of KDM7A-DT inhibits DNA damage response/repair (DDR/R) via the TP53BP1-mediated pathway, reducing apoptosis and promoting G2/M checkpoint arrest. Higher KDM7A-DT expression in BRCA is associated with KDM7A-DT locus gain/amplification, higher histologic grade, aneuploidy, hypoxia, immune modulation scores, and activation of the c-myc pathway. Higher KDM7A-DT expression is associated with relatively poor survival outcomes in patients with luminal A or Basal subtypes. In contrast, it is associated with favorable outcomes in patients with HER2+ER- or luminal B subtypes. KDM7A-DT levels are coregulated with critical transcripts and proteins aberrantly expressed in BRCA, including those involved in DNA repair via non-homologous end joining and epithelial-to-mesenchymal transition pathway. In summary, KDM7A-DT and its si-lncRNA exhibit several intrinsic biological and clinical characteristics that suggest important roles in invasive BRCA and its subtypes. KDM7A-DT-defined mRNA and protein subnetworks offer resources for identifying clinically relevant RNA-based signatures and prospective targets for therapeutic intervention.

Acquired Triazole Resistance Alters Pathogenicity-Associated Features in Candida auris in an Isolate-Dependent Manner.

Fluconazole resistance is commonly encountered in Candida auris, and the yeast frequently displays resistance to other standard drugs, which severely limits the number of effective therapeutic agents against this emerging pathogen. In this study, we aimed to investigate the effect of acquired azole resistance on the viability, stress response, and virulence of this species. Fluconazole-, posaconazole-, and voriconazole- resistant strains were generated from two susceptible C. auris clinical isolates (0381, 0387) and compared under various conditions. Several evolved strains became pan-azole-resistant, as well as echinocandin-cross-resistant. While being pan-azole-resistant, the 0381-derived posaconazole-evolved strain colonized brain tissue more efficiently than any other strain, suggesting that fitness cost is not necessarily a consequence of resistance development in C. auris. All 0387-derived evolved strains carried a loss of function mutation (R160S) in BCY1, an inhibitor of the PKA pathway. Sequencing data also revealed that posaconazole treatment can result in ERG3 mutation in C. auris. Despite using the same mechanisms to generate the evolved strains, both genotype and phenotype analysis highlighted that the development of resistance was unique for each strain. Our data suggest that C. auris triazole resistance development is a highly complex process, initiated by several pleiotropic factors.

Dataset of bulged G-quadruplex forming sequences in the human genome.

When several continuous guanine runs are present closely in a nucleic acid sequence, a secondary structure called G-quadruplex can form (G4s). Such structures in the genome could serve as structural and functional regulators in gene expression, DNA-protein binding, epigenetic modification, and genotoxic stress. Several types of G4-forming DNA sequences exist, including bulged G4-forming sequences (G4-BS). Such bulges occur due to the presence of non-guanine bases in specific locations (G-runs) in the G4-forming sequences. At present, search algorithms do not identify stable G4-BS conformations, making genome-wide studies of G4-like structures difficult. Data provided in this study are related to a published article "Stable bulged G-quadruplexes in the human genome: Identification, experimental validation and functionalization" published by Nucleic Acids Research [DIO.org/10.193/nar/gkad252]. Based on our studies in vitro and G4-seq and G4 CUT&Tag data analysis, we have specified and validated three pG4-BS models. In this article, a large collection of 'raw' (unfiltered) dataset is presented, which includes three subfamilies of pG4-BS. For each of pG4-BS, we provide strand-specific genomic boundaries. Data on pG4-BS might be useful in elucidating their structural, functional, and evolutionary roles. Furthermore, they may provide insight into the pathobiology of G4-like structures and their potential therapeutic applications.

Hard nut to crack: Solving the disulfide linkage pattern of the Neosartorya (Aspergillus) fischeri antifungal protein 2.

As a consequence of the fast resistance spreading, a limited number of drugs are available to treat fungal infections. Therefore, there is an urgent need to develop new antifungal treatment strategies. The features of a disulfide bond-stabilized antifungal protein, NFAP2 secreted by the mold Neosartorya (Aspergillus) fischeri render it to be a promising template for future protein-based antifungal drug design, which requires knowledge about the native disulfide linkage pattern as it is one of the prerequisites for biological activity. However, in the lack of tryptic and chymotryptic proteolytic sites in the ACNCPNNCK sequence, the determination of the disulfide linkage pattern of NFAP2 is not easy with traditional mass spectrometry-based methods. According to in silico predictions working with a preliminary nuclear magnetic resonance (NMR) solution structure, two disulfide isomers of NFAP2 (abbacc and abbcac) were possible. Both were chemically synthesized; and comparative reversed-phase high-performance liquid chromatography, electronic circular dichroism and NMR spectroscopy analyses, and antifungal susceptibility and efficacy tests indicated that the abbcac is the native pattern. This knowledge allowed rational modification of NAFP2 to improve the antifungal efficacy and spectrum through the modulation of the evolutionarily conserved γ-core region, which is responsible for the activity of several antimicrobial peptides. Disruption of the steric structure of NFAP2 upon γ-core modification led to the conclusions that this motif may affect the formation of the biologically active three-dimensional structure, and that the γ-core modulation is not an efficient tool to improve the antifungal efficacy or to change the antifungal spectrum of NFAP2.

Stable bulged G-quadruplexes in the human genome: identification, experimental validation and functionalization.

DNA sequence composition determines the topology and stability of G-quadruplexes (G4s). Bulged G-quadruplex structures (G4-Bs) are a subset of G4s characterized by 3D conformations with bulges. Current search algorithms fail to capture stable G4-B, making their genome-wide study infeasible. Here, we introduced a large family of computationally defined and experimentally verified potential G4-B forming sequences (pG4-BS). We found 478 263 pG4-BS regions that do not overlap 'canonical' G4-forming sequences in the human genome and are preferentially localized in transcription regulatory regions including R-loops and open chromatin. Over 90% of protein-coding genes contain pG4-BS in their promoter or gene body. We observed generally higher pG4-BS content in R-loops and their flanks, longer genes that are associated with brain tissue, immune and developmental processes. Also, the presence of pG4-BS on both template and non-template strands in promoters is associated with oncogenesis, cardiovascular disease and stemness. Our G4-BS models predicted G4-forming ability in vitro with 91.5% accuracy. Analysis of G4-seq and CUT&Tag data strongly supports the existence of G4-BS conformations genome-wide. We reconstructed a novel G4-B 3D structure located in the E2F8 promoter. This study defines a large family of G4-like sequences, offering new insights into the essential biological functions and potential future therapeutic uses of G4-B.

The Membrane Activity of the Amphibian Temporin B Peptide Analog TB_KKG6K Sheds Light on the Mechanism That Kills Candida albicans.

Temporin B (TB) is a 13-amino-acid-long, cationic peptide secreted by the granular glands of the European frog Rana temporaria. We recently showed that the modified TB peptide analog TB_KKG6K rapidly killed planktonic and sessile Candida albicans at low micromolar concentrations and was neither hemolytic nor cytotoxic to mammalian cells in vitro. The present study aimed to shed light into its mechanism of action, with a focus on its fungal cell membrane activity. We utilized different fluorescent dyes to prove that it rapidly induces membrane depolarization and permeabilization. Studies on model membrane systems revealed that the TB analog undergoes hydrophobic and electrostatic membrane interactions, showing a preference for anionic lipids, and identified phosphatidylinositol and cardiolipin as possible peptide targets. Fluorescence microscopy using fluorescein isothiocyanate-labeled TB_KKG6K in the presence of the lipophilic dye FM4-64 indicated that the peptide compromises membrane integrity and rapidly enters C. albicans cells in an energy-independent manner. Peptide-treated cells analyzed by cryo-based electron microscopy exhibited no signs of cell lysis; however, subcellular structures had disintegrated, suggesting that intracellular activity may form part of the killing mechanism of the peptide. Taken together, this study proved that TB_KKG6K compromises C. albicans membrane function, which explains the previously observed rapid, fungicidal mode of action and supports its great potential as a future anti-Candida therapeutic. IMPORTANCE Fungal infections with the opportunistic human pathogen C. albicans are associated with high mortality rates in immunocompromised patients. This is partly due to the yeast's ability to rapidly develop resistance toward currently available antifungals. Small, cationic, membrane-active peptides are promising compounds to fight against resistance development, as many of them effectuate rapid fungal cell death. This fast killing is believed to hamper the development of resistance, as the fungi do not have sufficient time to adapt to the antifungal compound. We previously reported that the synthetic variant of the amphibian TB peptide, TB_KKG6K, rapidly kills C. albicans. In the current study, the mechanism of action of the TB analog was investigated. We show that this TB analog is membrane-active and impairs cell membrane function, highlighting its potential to be developed as an attractive alternative anti-C. albicans therapeutic that may hinder the development of resistance.

Functionalized Mesoporous Silica Nanoparticles for Drug-Delivery to Multidrug-Resistant Cancer Cells.

Background: Multidrug resistance is a common reason behind the failure of chemotherapy. Even if the therapy is effective, serious adverse effects might develop due to the low specificity and selectivity of antineoplastic agents. Mesoporous silica nanoparticles (MSNs) are promising materials for tumor-targeting and drug-delivery due to their small size, relatively inert nature, and extremely large specific surfaces that can be functionalized by therapeutic and targeting entities. We aimed to create a fluorescently labeled MSN-based drug-delivery system and investigate their internalization and drug-releasing capability in drug-sensitive MCF-7 and P-glycoprotein-overexpressing multidrug-resistant MCF-7 KCR cancer cells. Methods and Results: To track the uptake and subcellular distribution of MSNs, particles with covalently coupled red fluorescent Rhodamine B (RhoB) were produced (RhoB@MSNs). Both MCF-7 and MCF-7 KCR cells accumulated a significant amount of RhoB@MSNs. The intracellular RhoB@MSN concentrations did not differ between sensitive and multidrug-resistant cells and were kept at the same level even after cessation of RhoB@MSN exposure. Although most RhoB@MSNs resided in the cytoplasm, significantly more RhoB@MSNs co-localized with lysosomes in multidrug-resistant cells compared to sensitive counterparts. To examine the drug-delivery capability of these particles, RhoB@Rho123@MSNs were established, where RhoB-functionalized nanoparticles carried green fluorescent Rhodamine 123 (Rho123) - a P-glycoprotein substrate - as cargo within mesopores. Significantly higher Rho123 fluorescence intensity was detected in RhoB@Rho123@MSN-treated multidrug-resistant cells than in free Rho123-exposed counterparts. The exceptional drug-delivery potential of MSNs was further verified using Mitomycin C (MMC)-loaded RhoB@MSNs (RhoB@MMC@MSNs). Exposures to RhoB@MMC@MSNs significantly decreased the viability not only of drug-sensitive but of multidrug-resistant cells and the elimination of MDR cells was significantly more robust than upon free MMC treatments. Conclusion: The efficient delivery of Rho123 and MMC to multidrug-resistant cells via MSNs, the amplified and presumably prolonged intracellular drug concentration, and the consequently enhanced cytotoxic effects envision the enormous potential of MSNs to defeat multidrug-resistant cancer.

The combination of Neosartorya (Aspergillus) fischeri antifungal proteins with rationally designed γ-core peptide derivatives is effective for plant and crop protection.

Plant pathogenic fungi are responsible for enormous crop losses worldwide. Overcoming this problem is challenging as these fungi can be highly resistant to approved chemical fungicides. There is thus a need to develop and introduce fundamentally new plant and crop protection strategies for sustainable agricultural production. Highly stable extracellular antifungal proteins (AFPs) and their rationally designed peptide derivatives (PDs) constitute feasible options to meet this challenge. In the present study, their potential for topical application to protect plants and crops as combinatorial biofungicides is supported by the investigation of two Neosartorya (Aspergillus) fischeri AFPs (NFAP and NFAP2) and their γ-core PDs. Previously, the biofungicidal potential of NFAP, its rationally designed γ-core PD (γNFAP-opt), and NFAP2 was reported. Susceptibility tests in the present study extended the in vitro antifungal spectrum of NFAP2 and its γ-core PD (γNFAP2-opt) to Botrytis, Cladosporium, and Fusarium spp. Besides, in vitro additive or indifferent interactions, and synergism were observed when NFAP or NFAP2 was applied in combination with γNFAP-opt. Except for γNFAP2-opt, the investigated proteins and peptides did not show any toxicity to tomato plant leaves. The application of NFAP in combination with γNFAP-opt effectively inhibited conidial germination, biofilm formation, and hyphal extension of the necrotrophic mold Botrytis cinerea on tomato plant leaves. However, the same combination only partially impeded the B. cinerea-mediated decay of tomato fruits, but mitigated the symptoms. Our results highlight the feasibility of using the combination of AFP and PD as biofungicide for the fungal infection control in plants and crops. Supplementary Information: The online version contains supplementary material available at 10.1007/s10526-022-10132-y.

The effect of antifungal resistance development on the virulence of Candida species.

In recent years, the relevance of diseases associated with fungal pathogens increased worldwide. Members of the Candida genus are responsible for the greatest number of fungal bloodstream infections every year. Epidemiological data consistently indicate a modest shift toward non-albicans species, albeit Candidaalbicans is still the most recognizable species within the genus. As a result, the number of clinically relevant pathogens has increased, and, despite their distinct pathogenicity features, the applicable antifungal agents remained the same. For bloodstream infections, only three classes of drugs are routinely used, namely polyenes, azoles and echinocandins. Antifungal resistance toward all three antifungal drug classes frequently occurs in clinical settings. Compared with the broad range of literature on virulence and antifungal resistance of Candida species separately, only a small portion of studies examined the effect of resistance on virulence. These studies found that resistance to polyenes and echinocandins concluded in significant decrease in the virulence in different Candida species. Meanwhile, in some cases, resistance to azole type antifungals resulted in increased virulence depending on the species and isolates. These findings underline the importance of studies aiming to dissect the connections of virulence and resistance in Candida species.

ABI1-based expression signature predicts breast cancer metastasis and survival.

Despite the current standard of care, breast cancer remains one of the leading causes of mortality in women worldwide, thus emphasizing the need for better predictive and therapeutic targets. ABI1 is associated with poor survival and an aggressive breast cancer phenotype, although its role in tumorigenesis, metastasis, and the disease outcome remains to be elucidated. Here, we define the ABI1-based seven-gene prognostic signature that predicts survival of metastatic breast cancer patients; ABI1 is an essential component of the signature. Genetic disruption of Abi1 in primary breast cancer tumors of PyMT mice led to significant reduction of the number and size of lung metastases in a gene dose-dependent manner. The disruption of Abi1 resulted in deregulation of the WAVE complex at the mRNA and protein levels in mouse tumors. In conclusion, ABI1 is a prognostic metastatic biomarker in breast cancer. We demonstrate, for the first time, that lung metastasis is associated with an Abi1 gene dose and specific gene expression aberrations in primary breast cancer tumors. These results indicate that targeting ABI1 may provide a therapeutic advantage in breast cancer patients.

Kynurenic Acid and Its Analogue SZR-72 Ameliorate the Severity of Experimental Acute Necrotizing Pancreatitis.

The pathophysiology of acute pancreatitis (AP) is not well understood, and the disease does not have specific therapy. Tryptophan metabolite L-kynurenic acid (KYNA) and its synthetic analogue SZR-72 are antagonists of the N-methyl-D-aspartate receptor (NMDAR) and have immune modulatory roles in several inflammatory diseases. Our aims were to investigate the effects of KYNA and SZR-72 on experimental AP and to reveal their possible mode of action. AP was induced by intraperitoneal (i.p.) injection of L-ornithine-HCl (LO) in SPRD rats. Animals were pretreated with 75-300 mg/kg KYNA or SZR-72. Control animals were injected with physiological saline instead of LO, KYNA and/or SZR-72. Laboratory and histological parameters, as well as pancreatic and systemic circulation were measured to evaluate AP severity. Pancreatic heat shock protein-72 and IL-1ß were measured by western blot and ELISA, respectively. Pancreatic expression of NMDAR1 was investigated by RT-PCR and immunohistochemistry. Viability of isolated pancreatic acinar cells in response to LO, KYNA, SZR-72 and/or NMDA administration was assessed by propidium-iodide assay. The effects of LO and/or SZR-72 on neutrophil granulocyte function was also studied. Almost all investigated laboratory and histological parameters of AP were significantly reduced by administration of 300 mg/kg KYNA or SZR-72, whereas the 150 mg/kg or 75 mg/kg doses were less or not effective, respectively. The decreased pancreatic microcirculation was also improved in the AP groups treated with 300 mg/kg KYNA or SZR-72. Interestingly, pancreatic heat shock protein-72 expression was significantly increased by administration of SZR-72, KYNA and/or LO. mRNA and protein expression of NMDAR1 was detected in pancreatic tissue. LO treatment caused acinar cell toxicity which was reversed by 250 µM KYNA or SZR-72. Treatment of acini with NMDA (25, 250, 2000 µM) did not influence the effects of KYNA or SZR-72. Moreover, SZR-72 reduced LO-induced H2O2 production of neutrophil granulocytes. KYNA and SZR-72 have dose-dependent protective effects on LO-induced AP or acinar toxicity which seem to be independent of pancreatic NMDA receptors. Furthermore, SZR-72 treatment suppressed AP-induced activation of neutrophil granulocytes. This study suggests that administration of KYNA and its derivative could be beneficial in AP.

A Tale of Loops and Tails: The Role of Intrinsically Disordered Protein Regions in R-Loop Recognition and Phase Separation.

R-loops are non-canonical, three-stranded nucleic acid structures composed of a DNA:RNA hybrid, a displaced single-stranded (ss)DNA, and a trailing ssRNA overhang. R-loops perform critical biological functions under both normal and disease conditions. To elucidate their cellular functions, we need to understand the mechanisms underlying R-loop formation, recognition, signaling, and resolution. Previous high-throughput screens identified multiple proteins that bind R-loops, with many of these proteins containing folded nucleic acid processing and binding domains that prevent (e.g., topoisomerases), resolve (e.g., helicases, nucleases), or recognize (e.g., KH, RRMs) R-loops. However, a significant number of these R-loop interacting Enzyme and Reader proteins also contain long stretches of intrinsically disordered regions (IDRs). The precise molecular and structural mechanisms by which the folded domains and IDRs synergize to recognize and process R-loops or modulate R-loop-mediated signaling have not been fully explored. While studying one such modular R-loop Reader, the Fragile X Protein (FMRP), we unexpectedly discovered that the C-terminal IDR (C-IDR) of FMRP is the predominant R-loop binding site, with the three N-terminal KH domains recognizing the trailing ssRNA overhang. Interestingly, the C-IDR of FMRP has recently been shown to undergo spontaneous Liquid-Liquid Phase Separation (LLPS) assembly by itself or in complex with another non-canonical nucleic acid structure, RNA G-quadruplex. Furthermore, we have recently shown that FMRP can suppress persistent R-loops that form during transcription, a process that is also enhanced by LLPS via the assembly of membraneless transcription factories. These exciting findings prompted us to explore the role of IDRs in R-loop processing and signaling proteins through a comprehensive bioinformatics and computational biology study. Here, we evaluated IDR prevalence, sequence composition and LLPS propensity for the known R-loop interactome. We observed that, like FMRP, the majority of the R-loop interactome, especially Readers, contains long IDRs that are highly enriched in low complexity sequences with biased amino acid composition, suggesting that these IDRs could directly interact with R-loops, rather than being "mere flexible linkers" connecting the "functional folded enzyme or binding domains". Furthermore, our analysis shows that several proteins in the R-loop interactome are either predicted to or have been experimentally demonstrated to undergo LLPS or are known to be associated with phase separated membraneless organelles. Thus, our overall results present a thought-provoking hypothesis that IDRs in the R-loop interactome can provide a functional link between R-loop recognition via direct binding and downstream signaling through the assembly of LLPS-mediated membrane-less R-loop foci. The absence or dysregulation of the function of IDR-enriched R-loop interactors can potentially lead to severe genomic defects, such as the widespread R-loop-mediated DNA double strand breaks that we recently observed in Fragile X patient-derived cells.

Assessment of patient flow and optimized use of lean thinking transformation from the perspective of graph theory and spectral graph theory: A case study.

BACKGROUND: Hospital re-engineering initiatives aiming to meet the requirement for patient-centered care often face significant barriers. Opportunities from the optimization of patient flow logistics are often overlooked due to the perception that patient transport related services are ancillary. OBJECTIVES: To reorganize patient pathways by optimizing inpatient assignment and outpatient unit relocation. METHODS: Our analysis was conducted in a campus-based hospital hosting 1694 inpatient beds. Patient flow data was used for algorithm-based optimization to minimize the sum of the distances due to visits to outpatient units and visits by consulting physicians. Inpatients were reordered and outpatient units were relocated to minimize transport need. Optimized schemes were analyzed using graph- and spectral graph theory. RESULTS: Both optimizations yielded an altered hospital layout in which the need for patient transfers decreased (over 30% and 23% in terms of total distance and transfer episodes, respectively). The optimized systems gave rise to buildings with greater specialization, higher importance in terms of contributing to the network architecture, greater synchronization and robustness. CONCLUSIONS: The top-down algorithm-based optimization scheme yielded a system in which the need for cross-building patient transfer decreased. We suggest that network analysis may be a useful tool for capacity planning.

Acute stroke awareness of family physicians: translation of policy to practice.

BACKGROUND: Translating clinical guidelines into routine clinical practice is mandatory to achieve population level improvement of health. Emergence of specific therapy for acute stroke yielded the 'time is brain' concept introducing the need for emergency treatment, pointing to the need for increasing stroke awareness of the general population. General practitioners (GPs) manage chronic diseases and could hence catalyse stroke awareness. In our study, the knowledge of general practitioners toward accurate identification of acute stroke candidacy was investigated. METHODS: GPs and residents in training for family medicine participated in a survey on a voluntary basis using supervised self-administration between the 1st of February 2018 and 31st July 2018. Two clinical cases of acute stroke that differed only regarding the patient's eligibility for intravenous thrombolysis were presented. Participants answered two open-ended questions. Text analysis was performed using NVIVO software. RESULTS: Of the 127 respondents, 69 (54.3%) were female. The median age was 49 (34-62) years. The median time spent working after graduation was 14.5 (2-22.5) years. Board-certified GPs made up 77.2% of the sample. Qualitative analysis revealed stroke as the most frequent diagnosis for both cases. Territorial localization and possible aetiology were also established. Respondents properly identified eligibility for thrombolysis. Quantitative assessment showed that having the practice closer to the stroke centre increases the likelihood of adequate diagnosis for acute stroke. CONCLUSIONS: Our results show that GPs properly diagnose acute stroke and identify intravenous thrombolysis candidates. Moreover, we found that a vigorous acute stroke triage system facilitates the translation of theory into practice.

Triazole Evolution of Candida parapsilosis Results in Cross-Resistance to Other Antifungal Drugs, Influences Stress Responses, and Alters Virulence in an Antifungal Drug-Dependent Manner.

The number of invasive infections caused by Candida species is increasing worldwide. The incidence of candidiasis cases caused by non-albicans Candida species, such as Candida parapsilosis, is also increasing, and non-albicans Candida species are currently responsible for more invasive infections than C. albicans Additionally, while the development of azole resistance during invasive disease with C. albicans remains uncommon, azole-resistant C. parapsilosis strains are frequently isolated in the hospital setting. In this study, we applied direct selection to generate azole-adapted and azole-evolved C. parapsilosis strains in order to examine the effect of azole resistance development on fungal viability and pathogenesis progression. Depending on the drug applied, the different evolved strains developed distinct cross-resistance patterns: the fluconazole-evolved (FLUEVO) and voriconazole-evolved (VOREVO) strains gained resistance to fluconazole and voriconazole only, while posaconazole evolution resulted in cross-resistance to all azoles and the posaconazole-evolved (POSEVO) strains showed higher echinocandin MIC values than the FLUEVO and VOREVO strains. Whole-genome sequencing results identified the development of different resistance mechanisms in the evolved strains: the FLUEVO and VOREVO strains harbored amino acid substitutions in Mrr1p (A808T and N394Y, respectively), and the POSEVO strain harbored an amino acid change in Erg3p (D14Y). By revealing increased efflux pump activity in both the FLUEVO and the VOREVO strains, along with the altered sterol composition of the POSEVO strain, we now highlight the impact of the above-mentioned amino acid changes in C. parapsilosis azole resistance development. We further revealed that the virulence of this species was only slightly or partially affected by fluconazole and voriconazole adaptation, while it significantly decreased after posaconazole adaptation. Our results suggest that triazole adaptation can result in azole cross-resistance and that this process may also result in virulence alterations in C. parapsilosis, depending on the applied drug.IMPORTANCECandida parapsilosis causes life-threatening fungal infections. In the last 2 decades, the increasing number of azole-resistant C. parapsilosis clinical isolates has been attributable to the overuse and misuse of fluconazole, the first-line antifungal agent most commonly used in several countries. To date, the range of applicable antifungal drugs is limited. As a consequence, it is essential to understand the possible mechanisms of antifungal resistance development and their effect on virulence in order to optimize antifungal treatment strategies in the clinical setting. Our results revealed that the prolonged exposure to azoles resulted not only in azole resistance but also in cross-resistance development. Our data further indicate that resistance development may occur through different mechanisms that can also alter the virulence of C. parapsilosis These results highlight the consequences of prolonged drug usage and suggest the need for developing alternative antifungal treatment strategies in clinical practice.

The Penicillium chrysogenum Q176 Antimicrobial Protein PAFC Effectively Inhibits the Growth of the Opportunistic Human Pathogen Candida albicans.

Small, cysteine-rich and cationic antimicrobial proteins (AMPs) from filamentous ascomycetes promise treatment alternatives to licensed antifungal drugs. In this study, we characterized the Penicillium chrysogenum Q176 antifungal protein C (PAFC), which is phylogenetically distinct to the other two Penicillium antifungal proteins, PAF and PAFB, that are expressed by this biotechnologically important ascomycete. PAFC is secreted into the culture broth and is co-expressed with PAF and PAFB in the exudates of surface cultures. This observation is in line with the suggested role of AMPs in the adaptive response of the host to endogenous and/or environmental stimuli. The in silico structural model predicted five ß-strands stabilized by four intramolecular disulfide bonds in PAFC. The functional characterization of recombinant PAFC provided evidence for a promising new molecule in anti-Candida therapy. The thermotolerant PAFC killed planktonic cells and reduced the metabolic activity of sessile cells in pre-established biofilms of two Candidaalbicans strains, one of which was a fluconazole-resistant clinical isolate showing higher PAFC sensitivity than the fluconazole-sensitive strain. Candidacidal activity was linked to severe cell morphology changes, PAFC internalization, induction of intracellular reactive oxygen species and plasma membrane disintegration. The lack of hemolytic activity further corroborates the potential applicability of PAFC in clinical therapy.

Novel coronavirus epidemic in the Hungarian population, a cross-sectional nationwide survey to support the exit policy in Hungary.

After months of restrictive containment efforts to fight the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) epidemic, European countries are planning to reopen. To support the process, we conducted a cross-sectional survey among the Hungarian population to estimate the prevalence of infectious cases and prior SARS-CoV-2 exposure. A representative sample (n = 17,787) for the Hungarian population of 14 years or older living in private households (n = 8,283,810) was selected. The study was performed within 16 days after 50 days of restrictions, when the number of confirmed cases was stable low. Naso- and oropharyngeal smears and blood samples were collected for PCR and antibody testing. The testing was accompanied by a questionnaire about symptoms, comorbidities, and contacts. Design-based prevalence estimates were calculated. In total, 10,474 individuals (67.7% taken into account a sample frame error of 2315) of the selected sample participated in the survey. Of the tested individuals, 3 had positive PCR and 69 had positive serological test. Population estimate of the number of SARS-CoV-2 infection and seropositivity were 2421 and 56,439, respectively, thus active infection rate (2.9/10,000) and the prevalence of prior SARS-CoV-2 exposure (68/10,000) was low. Self-reported loss of smell or taste and body aches were significantly more frequent among those with SARS-CoV-2. In this representative, cross-sectional survey of the Hungarian population with a high participation rate, the overall active infection rate was low in sync with the prevalence of prior SARS-CoV-2 exposure. We demonstrated a potential success of containment efforts, supporting an exit strategy. NCT04370067, 30.04.2020.

Size-dependent activity of silver nanoparticles on the morphological switch and biofilm formation of opportunistic pathogenic yeasts.

BACKGROUND: Dimorphism and biofilm formation are important virulence factors of some opportunistic human pathogenic yeasts. Such species commensally colonize skin or mucosal surfaces generally in yeast form, but under particular circumstances, convert into virulent hyphae and disseminate internal organs or cause mucocutaneous infections. The yeast-to-hypha shape-conversion promotes the development of a biofilm, a thick extracellular matrix with sessile cells within. The biofilm is capable to prevent the penetration of antifungal drugs, rendering the surviving biofilm-resident cells intrinsic sources of recurrent infections. The aim of this study was to evaluate the ability of silver nanoparticles (AgNPs) to attenuate the morphological switch and biofilm formation of several opportunistic pathogenic yeasts and to determine whether this feature depends on the nanoparticle size. RESULTS: AgNPs in three different sizes were prepared by chemical reduction approach and characterized by transmission electron microscopy, ultraviolet-visible spectroscopy and dynamic light scattering. The antifungal activity was evaluated by the microdilution method, the inhibitory capacity on biofilm formation and the biofilm degradation ability of differently sized AgNPs was assessed by viability assay. The morphological state of opportunistic pathogenic yeast cells in monoculture and in co-culture with human keratinocytes in the presence of AgNPs was examined by flow cytometry and scanning electron microscopy. All the three AgNPs inhibited the growth of the examined opportunistic pathogenic yeasts, nevertheless, AgNPs with the smallest diameter exhibited the most prominent toxic activities. AgNPs attenuated the biofilm formation in a nanoparticle size-dependent manner; however, their biofilm destruction capacity was negligible. AgNPs with the smallest size exerted the most significant effect on suppressing the morphological change of pathogens in monoculture as well as in a co-culture with keratinocytes. CONCLUSIONS: Our results confirm that AgNPs are capable to hinder yeast-to-hypha morphological conversion and biofilm formation of opportunistic pathogens and this biological effect of AgNPs is size-dependent.

Identification and Characterization of a Neutral Locus for Knock-in Purposes in C. parapsilosis.

Invasive fungal infections caused by Candida species affect approximately 700,000 people worldwide resulting in 300,000 deaths annually. Besides Candida albicans, other members of the genus have gained relevance in the last two decades, including C. parapsilosis whose incidence is particularly high amongst low birth weight neonates. To investigate the virulence properties of this pathogen several techniques have been developed for generating knock-out mutants, however, no target locus for knock-in approaches have been published so far. Here we report CpNEUT5L (N5L), an intergenic locus in C. parapsilosis, and introduce an integrative GatewayTM and a classical ligation based replacement plasmid to target it with. As a proof of principle, we fluorescently tagged laboratory and prototroph strains and established that this locus is also suitable for reintegration purposes. We concluded that GFP-expressing constructs integrated into this region provide strong, homogenous fluorescent signals while alteration of this locus affects neither the growth of the mutants in liquid or on solid media, even in the presence of different stressors, nor their basic virulence properties. Hence, our findings demonstrate that N5L is a highly effective neutral locus for knock-in approaches in C. parapsilosis.

Accuracy and Precision of the Receptorial Responsiveness Method (RRM) in the Quantification of A1 Adenosine Receptor Agonists.

The receptorial responsiveness method (RRM) is a procedure that is based on a simple nonlinear regression while using a model with two variables (X, Y) and (at least) one parameter to be determined (cx). The model of RRM describes the co-action of two agonists that consume the same response capacity (due to the use of the same postreceptorial signaling in a biological system). While using RRM, uniquely, an acute increase in the concentration of an agonist (near the receptors) can be quantified (as cx), via evaluating E/c curves that were constructed with the same or another agonist in the same system. As this measurement is sensitive to the implementation of the curve fitting, the goal of the present study was to test RRM by combining different ways and setting options, namely: individual vs. global fitting, ordinary vs. robust fitting, and three weighting options (no weighting vs. weighting by 1/Y2 vs. weighting by 1/SD2). During the testing, RRM was used to estimate the known concentrations of stable synthetic A1 adenosine receptor agonists in isolated, paced guinea pig left atria. The estimates were then compared to the known agonist concentrations (to assess the accuracy of RRM); furthermore, the 95% confidence limits of the best-fit values were also considered (to evaluate the precision of RRM). It was found that, although the global fitting offered the most convenient way to perform RRM, the best estimates were provided by the individual fitting without any weighting, almost irrespective of the fact whether ordinary or robust fitting was chosen.