Introduction Breast cancer is one of the most prevalent pathologies effecting women today. Many therapies are being explored to help fight the disease, and immunotherapy is in the forefront of such efforts. Macrophages have been known to act against tumor cells, and research is being conducted on how to further exploit these tendencies for more efficient treatment. Chimeric Antigen Receptor (CAR) therapy is one such possibility being studied. These receptors, when transduced onto macrophages, help them specifically target the cancer we hope to eliminate, further improving the efficacy of the killing. Aim We examined how cell proliferation of HER2+ tumor cell line (JIMT-1) is altered in co-cultures with CAR macrophages (CAR-Ms), which express HER2 specific extracellular domain and FcgR 2A intracellular, signalization domain. We also analyzed the cytokine production of the macrophages in the presence of the cancer cells. To enhance immunotherapy, the tumor cells were pretreated with the chemotherapeutic agent Epirubicin. Methods JIMT-1 cancer cells expressing mCherry to be used in this study were cultured in the presence of Epirubicin after determining the optimal concentration of the chemotherapeutic agent. Meanwhile, we differentiated monocytes into macrophages and induced their M1 polarization. We then co-incubated the macrophages with cancer cells for 4 and 24 hours, and measured cytokine production (IL-1b and TNFα) via ELISA. Changes in the number of mCherry positive cancer cells were monitored using the high content microscope system, in live cell mode. Results We have concluded that there is a relatively higher cytokine production by the CAR-M as compared to the control macrophages in the presence of tumor cells. Using the confocal microscope, we found that the number of JIMT-1 cells was reduced to a greater extent in the presence of CAR-M compared to the control and this was further enhanced by Epirubicin pretreatment. Discussion Although the CAR system needs further optimization, it is starting to show significant results and its impact can be enhanced in combination with chemotherapy in the fight against cancer.

Témavezető: Dr. Kókai Endre és Vigh Dorottya

Pseudomonas aeruginosa is a significant pathogen in opportunistic and hospital-acquired infections. Its capsule is a critical virulence factor contributing to antibiotic resistance, which poses a major treatment challenge. Bacteriophage therapy offers a promising alternative due to its target specificity and efficacy against biofilms. Isolation and sequencing of bacteriophage DNA is crucial for understanding their genetic composition and therapeutic potential. Sequencing provides information on the phage's lytic capabilities, host specificity, and potential virulence, ensuring its safe use in treatment. This study focuses on the isolation and characterization of bacteriophages from wastewater samples to combat multidrug-resistant P. aeruginosa, as well as optimizing phage DNA extraction methods for long-read sequencing. 10 clinical multidrug-resistant P. aeruginosa strains were used in this study. Antibiotic susceptibility was determined using the standard disc diffusion test for eleven different commonly used antibiotics. The strains were resistant to 8 of the 11 antibiotics, with one strain resistant to all 11. To isolate phages against these strains, we analysed 15 wastewater samples from a wastewater treatment plant in Budapest, Hungary. We detected the phages using the spot method. A total of 28 active phages against six of the original ten strains were isolated and evaluated by host range testing. The host range of the phages ranged from 1 to 6 strains per phage. For long-read sequencing, phage DNA was isolated using the standard method based on chloroform-isoamyl-alcohol. The resulting sequencing with the Oxford Nanopore platform was contaminated with bacterial DNA, leading to the assumption that capsule materials interfere with DNA isolation. We have developed two new DNA isolation methods based on chloroform-isoamyl- alcohol for the extraction of phage DNA. Using Nanodrop, gel electrophoresis, and Qubit, we compared the quality and purity of the extracted DNA. The DNA concentration was low and not pure, it had a high protein concentration. We believe that the new methods could not solve the problem of interfering capsules. This study demonstrates the potential of wastewater-derived bacteriophages as therapeutic agents against multidrug-resistant P. aeruginosa and provides valuable insight into the refinement of phage DNA extraction methods. Future research will focus on refining these techniques to make them more suitable for downstream analysis.

Témavezető: Krisztina Szarka és Evelin Szotak

The mammalian proteomes contain multiple proteins that are encoded by retrovirus- or retrotransposon- related genes which have been integrated into the host germ cells’ genomes during the early evolution of mammalians. The capsid-like and protease domains of retroviral-like proteins (RVLPs) are homologous to the structural (capsid) and enzymatic (aspartic protease) domains of retroviral and retrotransposon polyproteins. The capsid-like domains of RVLPs can mediate protein-protein interactions and oligomerization to form extracellular virus-like particles, while their protease domain can cleave proteolytically various target proteins. In case of some RVLPs, the protease is encoded by an alternative open reading frame of the gene and expressed via a specific translation mechanism (frameshifting). Examples of these RVLPs include members of paternally expressed gene (PEG), paraneoplastic Ma antigen (PNMA) and retrotransposon Gag-like protein (RTL) families. Several RVLPs have been described so far, but multiple members of this family remained to be characterized, such as the retrotransposon Gag-like protein 3 (RTL3). In this project we aimed the biochemical characterization of mouse RTL3. We designed plasmid vectors for the homologous and heterologous expression of RTL3 in mouse and human cell lines, respectively. We aimed to study the expression, intra- and extracellular localization, and proteolytic activity of RTL3, as well as to identify its interaction partners. Besides the wild-type, mutant forms of RTL3 were also designed and studied. The efficiency of transfection and expression of RTL3 was much higher in human (HEK293T) than in mouse cell lines (C2C12, NIH3T3). After optimizing the protocols for detection of RTL3 we proved the existence of frameshifting in case of RTL3. We observed cytosolic expression for RTL3, but the subcellular localization changed if the protease domain was also expressed. The preliminary studies provided no evidence for the activity of the protease domain, but implied the ability of RTL3 to form extracellular particles. Immunoprecipitation coupled to mass spectrometry was used to identify previously unknown interaction partners of RTL3. Our work is the first which gives insight into the characteristics of RTL3 at protein level. This project was supported by TKP2021-EGA-20 project, by János Bolyai Research Scholarship of MTA (BO/00110/23/5 to J.A.M) and by TalentUD program of University of Debrecen (to Y.W.M).

Témavezető: Dr.János András Mótyán

The emergence of antibiotic resistance has become a global health crisis. Multidrug-resistant pathogens are increasingly becoming more difficult to treat, especially with conventional antibiotics. As the development of new antibiotics is seemingly slowing down, new strategies for dealing with these multidrug-resistant pathogens need to be undertaken. The One Health Approach which emphasizes the connection between human, animal, and environmental health, offers a vital framework for tackling this challenge. This research aimed to investigate the prevalence and genetic basis of extended-spectrum β-lactamase (ESBL)- and AmpC beta-lactamase-producing Enterobacterales found in the gut microbiome of white storks (Ciconia ciconia) that nest in urban environments and forage in landfills in Hungary. Through the One Health approach, this study explores the intersection of wildlife and environmental health in the context of antibiotic resistance. Faecal samples were collected from 81 young storks and cultured on selective cefotaxime-eosin methylene blue and blood agar media. The bacterial isolates were identified as Escherichia coli using the MALDI-TOF mass spectrometry system. Antibiotic susceptibility was performed according to EUCAST guidelines. To identify the genetic background of phenotypic resistance , DNA was isolated from the strains according to the protocol of the Zymo Quick-DNA Fungal/Bacterial Miniprep Kit. The purity of the samples was checked by NanoDrop One spectrophotometer and the concentration of the samples was measured by Qubit 4 Fluorometer. Whole genome sequencing was carried out on an Oxford Nanopore MinION sequencing device. Eighteen samples yielded a positive result on the selective media, and 25 bacterial strains were subsequently isolated, of which 88% (22/25) were ESBL-producing, and 12% (3/25) were AmpC-producing. Based on the analyses of the assembled genomes we identified bla CMY-59 , which is responsible for AmpC resistance, and bla CTX-M-1 , bla CTX-M-14 , bla CTX-M-15 , bla CTX-M-27 , and bla CTX-M-65 genes, which are responsible for ESBL resistance. The obtained Escherichia coli sequence types (STs) were diverse. These findings underscore the potential role of migratory birds as vectors of multidrug-resistant pathogens and highlight the importance of monitoring wildlife in global antibiotic resistance surveillance efforts.

Témavezető: Bubán Réka Zsófia

Detecting antimicrobial-resistant (AMR) bacteria is an increasing concern for public health. A significant aspect of this issue involves extended-spectrum beta-lactamase (ESBL)-producing Enterobacterales. Understanding the mechanisms behind this resistance and developing strategies to prevent its spread remain subjects of active discussion. It affects not only humans but also animals, particularly pets, which share close contact with their owners. Additionally, the environment plays a crucial role in this interconnected system. This study investigates the prevalence of ESBL-producing Enterobacterales in dog faeces collected from urban green spaces in two major Hungarian cities, Budapest and Debrecen. Of the 399 samples analysed, 24 isolates of ESBL- producing Enterobacterales were identified. Among these, 88% (22/24) demonstrated resistance to three or more antimicrobial classes, though all isolates remained susceptible to amikacin and ertapenem. The genomic DNA of the bacterial isolates was analysed using polymerase chain reaction (PCR) and whole genome sequencing (WGS) in order to identify antimicrobial resistance genes. As a result, blaCTX-M-1 was the most dominant ESBL-encoding gene, followed by blaCTX-M-15, blaCTX-M-27, blaCTX-M-55, blaCTX-M-14, blaCTX-M-3, and blaCTX-M-32, most of which were plasmid-mediated. Phylogenetic groups, virulence genes and multi locus sequence types (MLSTs) were also identified, revealing a genetically diverse set of isolates. Common sequence types included ST10, ST58, and ST155, followed by ST69, ST93, ST448, and ST553. The most prevalent type, ST58, was identified in three samples collected from both Budapest and Debrecen. This research aims to characterise ESBL-producing Enterobacterales in Hungarian cities, highlighting urban green spaces as potential reservoirs of AMR and indicators of public health risks.

Témavezető: Benmazouz Isma és Cseri Karolina

The spike protein (S) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) plays a critical role in viral attachment and entry, making it a central target for vaccination strategies. Currently marketed vaccines predominantly rely on the expression of the S protein to elicit humoral and, to some extent, cellular immunity, potentially protecting against infection. In our study, we aimed to investigate the effects of the S protein on macrophage polarization and the cytokine milieu. To achieve this, primary macrophages and macrophages derived from THP-1 cells were transfected with constructs coding for the S protein. Subsequently, the cells were polarized into M1 (pro-inflammatory) and M2 (anti-inflammatory) macrophages. In parallel experiments, cells were polarized into M1 and M2 phenotypes prior to transfection with the constructs to compare the effects of the spike protein on pre- differentiated versus post-differentiated macrophages. Fluorescence-activated cell sorting (FACS) was employed to analyze polarization using CD80 and CD206 markers. Additionally, cytokine analysis was performed on the cell culture supernatant. Preliminary results indicate that in both polarized primary cells and THP-1-derived macrophages, the spike protein did not alter macrophage polarization compared to the mock vector in either the M1 or M2 subtypes. However, cytokine analysis revealed changes in the secretion profile, consistent with anticipated immunological responses. Notably, there was a distinct cytokine profile in cells treated with spike-coding mRNA vaccine. Further experiments are ongoing to elucidate the broader impact of the S protein on the immune system.

Témavezető: Dr. Mohamed Mahdi és Aya Muffti

Transforming Growth Factor-β Inhibited Membrane-Associated Protein (TIMAP) is a regulatory subunit of Protein Phosphatase 1 (PP1), predominantly expressed in endothelial cells, as well as lung and brain tissues. It has been implicated in various cellular processes, including differentiation and signaling. Previously, we demonstrated that TIMAP is downregulated during neuronal differentiation; however, its role in neuronal cells remains largely uncharacterized. Our mRNA-seq results comparing the transcriptome of control and TIMAP-silenced SH-SY5Y cells revealed several overrepresented cellular mechanisms and signaling pathways potentially involving TIMAP. Most notably, the bone morphogenic protein (BMP) pathway stood out, prompting us to investigate TIMAP’s role in this signaling cascade. Functional assays were performed to examine TIMAP’s effect on BMP signaling pathway by comparing the response to recombinant BMP-7 treatment in control and TIMAP-silenced SH-SY5Y cells. We optimized BMP activation through time- and concentration-dependent experiments and analyzed SMAD phosphorylation using Western blotting. The deactivation of the signaling pathway was studied by monitoring SMAD dephosphorylation at an 8-hour interval post-BMP treatment. TIMAP-depleted cells exhibited altered BMP-SMAD signaling dynamics, with SMAD1 phosphorylation persisting longer, suggesting TIMAP's involvement in modulating BMP signaling. Pull-down experiments using tagged PP1c and TIMAP recombinant proteins were conducted to identify protein-protein interactions involving the PP1c-TIMAP complex and BMP signaling components. These analyses confirmed interactions between the PP1c-TIMAP complex and phosphorylated SMAD proteins, indicating that TIMAP may influence BMP pathway activity by regulating SMAD dephosphorylation. Our findings demonstrate that TIMAP plays a critical role in neuronal cellular mechanisms by acting as a regulatory component of the BMP signaling pathway. These results provide novel insights into the molecular mechanisms underlying neuroblastoma differentiation and establish TIMAP as a potential modulator of neuronal development. Funding: This research was funded by the National Research, Development and Innovation Fund (NKFI) under grant number FK135384.

Témavezető: Dr Anita Boratkó és Márton Fonódi

Transglutaminase 2 (TG2) is a diverse, ubiquitous, multifunctional protein involved in various biological functions. Owing to its four domains organization, TG2 acts as a GTPase, kinase, protein disulfide isomerase and a scaffolding factor. It mediates varied cellular processes such as; apoptosis, angiogenesis, autophagy, wound healing, transmembrane signalling, mitochondrial functions and gene expression regulation. TG2 is present in almost all cellular compartments and is associated with diseases such as neurodegenerative diseases, inflammatory diseases, celiac diseases and many types of cancers. Evidence from our previous study depicts TG2 binds RNA, potentially playing a role in post-translational regulations. RNA-pull-down experiment corroborated that TG2 binds RNA only in its open conformation. Based on our experiments and molecular modelling, we hypothesized that the RNA binding site could either overlap with the GTP/GDP binding site or present on C-terminal unordered positively charged surface patches of TG2. The objective of this study was to localise better this novel RNA-binding property of TG2. We cloned C-terminal beta-barrel domain truncated (TG2/1-582) and mutant (W241A, R580A, K173E/F174A, 173KFIKN177 to 173EASED177 and 173KFIKNIP179 to 173EASEDSA179) TG2 constructs using ligase independent cloning and site-directed mutagenesis, respectively. The suitable clones were checked using restriction enzyme digestion and Sanger sequencing. Then recombinant TG2 proteins were produced in E. coli Rosetta 2 (DE3) competent cells upon IPTG induction and purified by Ni-NTA affinity chromatography. SDS-PAGE and Western blot showed more than 90% purity of recombinant TG2 proteins, and the retained transglutaminase activity suggested the maintenance of their native conformation. The RNA-binding properties of the TG2 mutants were tested using magnetic RNA-protein pull-down experiments and Biolayer Interferometry (Blitz). The truncated TG2 protein maintained its RNA-binding, while GTP binding mutants demonstrated impaired RNA-binding properties suggesting that the GTP binding site within TG2 contributes to TG2-RNA interaction. Our results provide additional evidence about TG2 RNA-binding ability and its potential regulation by nucleotide binding, which could be involved in the mechanism of essential cellular processes.

Témavezető: Dr. Király Róbert és Csaholczi Bianka

In our laboratory, we aimed to improve stem cell-derived dendritic cell (DC) development and immunogenicity by transcription factor-directed cell differentiation. Chemically (doxycycline) inducible, transgenic mouse embryonic stem cell (ESC) lines were generated and tested for this purpose. Previous observations highlighted phenotypic and developmental changes upon the ectopic expression of various transcription factors. However, we observed that the transgenic transcription factor inducibility was reduced during the advanced phase of the ex vivo DC differentiation. To monitor the transgene expression upon differentiation in live cells, I used two ESC clones (C1 and C8), which carry inducible EGFP (enhanced green fluorescent protein) transgene. EGFP is a widely utilized molecular marker for tracking gene expression and transgene induction. In this study, I investigated the dynamics of EGFP expression during the differentiation of mouse ESC-derived DCs and their progenitors. ESCs were differentiated to DCs using an embryoid body (EB) based protocol. For this, the EGFP inducible ESCs were first differentiated for 6 days to form EBs. Subsequently, EBs were disaggregated and further cultured for 6 days in a GM-CSF-containing medium. On day 6, half the cells were treated with doxycycline for EGFP induction and the other half as untreated controls. On day 12, CD45+ blood cells were separated using MACS and further cultured in a GM-CSF containing RPMI medium. On day 18, the medium was refreshed, and half of the cells were treated with LPS to induce DC maturation. Flow cytometry analyses were conducted on days 12 and 19. My results demonstrate that EGFP expression was strongly induced in the presence of doxycycline during the early phase of the differentiation. At day 12, around 60% of the differentiated cells were GFP+. However, the percentage of EGFP-expressing cells decreased significantly in terminally differentiated DCs (approximately 20%), and this was unaltered with or without LPS treatment. We still do not understand the molecular mechanism of transgene silencing, but these new data sets provide insights into the temporal regulation of transgene expression during DC differentiation and highlight the utility of EGFP as a marker in developmental biology research.

Témavezető: István Szatmári

Two members of the Runt-related transcription factor family (RUNX1 and RUNX3) have a critical role in regulation of hematopoiesis during the early stages of mammalian embryonic development. In our laboratory it was uncovered that forced expression of RUNX3 stimulated the formation of the murine embryonic stem cells (ESCs) derived early blood cell progenitors. In this study I have further investigated the role of RUNX3 on the early blood cell formation using an ex vivo ESC differentiation protocol. RUNX3-inducible transgenic mouse ESCs were cultured on gelatin- coated plates for 4 days, followed by trypsinization and disaggregation using 0.25% trypsin-EDTA solution (ESC expansion and harvesting). ESCs were differentiated for five days in a special tissue culture medium to form embryoid bodies (EBs). Then EBs were disaggregated and further differentiated in a GM-CSF containing medium for additional 2, 3 or 4 days. Half of the EB-derived cells were treated with doxycycline for RUNX3 induction, while the other half served as control without doxycycline. Cells were harvested on 7, 8, and 9 days and flow cytometry analyses were performed to monitor the hematopoietic development. I examined the cell surface expression of CD41, CD45 and c-Kit markers. My flow cytometric analysis revealed that elevated percent of c-Kit/CD41 double positive blood cell progenitors were detected in the doxycycline treated cells at day 7, 8 and 9. This suggests that RUNX3 induction enhances the early hematopoietic progenitor formation of the ESC derived ex vivo differentiated cells. On the other hand, the percentage of the pan hematopoietic marker (CD45) expression was continuously increased upon the ESC-derived cell differentiation. These results suggest that over-expression of RUNX3 can selectively upregulate the c-Kit/CD41 blood cell precursor production.

Témavezető: István Szatmári és Chahra Fareh