I. INTRODUCTION Induction of osteogenesis is a highly investigated field of regenerative medicine. The suitability of the osteogenic models to form bone-like structures is determined by the cell culture media applied and the type of model used during culturing. Our aim was to investigate the osteogenesis of MG-63 cells cultured in 2D monolayer cultures in contrast to 3D bioprinted models cultured in different media. II. METHODS To investigate the mineralization of MG-63 human osteosarcoma cell line, 3D bioprinted models (BioX Bioprinter, GELMA Bioink, Cellink) were cultured for three weeks in α-MEM cell culture medium and compared to 2D monolayer cells cultured in DMEM or α-MEM cell culture medium. The mineralization of the MG-63 cell layers was visualized by Alizarin Red staining, using light microscopy. Gene expression of osteogenic markers was evaluated by TaqMan arrays. III. RESULTS The results of Alizarin Red staining show an intense level of mineralization in 3D bioprinted MG-63 models. Furthermore, all five investigated osteogenic markers (Col1A1, BGLAP, BMP2, FN1, SP7) showed increased levels of gene expression in the 3D bioprinted MG-63 models after 3 three weeks of incubation in contrast to the 2D cultured MG-63 cell layers. IV. DISCUSSION The study shows significant differences in 2D cultured and 3D bioprinted MG-63 models. While in the 2D cultures the α-MEM medium induced only some osteogenic markers, in the 3D bioprinted model the level of all five osteogenic markers increased dramatically, indicating that the osteogenic differentiation was more successful in the bioprinted MG-63 cell system. Such models will make our further tissue differentiation studies and drug testing experiments possible.

Keywords: 3D printing, cell culture, bioink. 

Using nanoparticles is becoming common in medicine and biotechnology. Moreover, nanoparticles are also present in our environment as nano-pollution which might enter living organisms. Proteins which get into contact with nanoparticles, might undergo structural changes. Aggregation and amyloid formation of proteins and peptides can also be affected by nanoparticles. Our group is interested in investigating the aggregation and amyloid formation of the amyloid-beta (Aβ) peptide which is known for creating amyloid fibrils in the brain in Alzheimer’s disease. We used in-house expressed recombinant Aβ(1-42) to study the structure, aggregation kinetics, and cytotoxicity of the peptide. We characterized nanoparticles of different materials (calcium-fluoride, silica, polystyrene) and studied their effects on Aβ. Based on thioflavin T fluorescence, we found that nanoparticles typically reduce lag time of the aggregation. We used circular dichroism (CD) spectroscopy and the BeStSel method to determine secondary structure composition. Aβ fibrils formed alone have a high content of parallel β-sheets. Nanoparticles, depending on their type, affect the secondary structure composition of Aβ aggregates altering the fractions of the different types of β-sheet structures. Toxicity measurements on mouse hippocampal cells suggest that Aβ is highly cytotoxic, but nanoparticles might reduce cytotoxicity of Aβ if applied in appropriate concentrations. This work was supported by the National Research, Development and Innovation Office of Hungary (grants PD135510, K138937, 2019-2.1.6-NEMZ_KI-2019-00012, and 2019-2.1.11-TÉT-2020-00101), the National Brain Research Program NAP 3.0 of the Hungarian Academy of Sciences (NAP2022-I-3/2022), and the Gedeon Richter Talentum Foundation (grant ’Richter Gedeon Kiválósági PhD Ösztöndíj’).

Keywords: protein secondary structure, amyloid, nanoparticles.

Autoimmune diseases (ADs) pose a growing public health challenge despite advances in diagnosis and treatment. Existing therapies involve broad immunosuppression, often causing severe side effects and patient dissatisfaction. There's a pressing need for more precise, less immunosuppressive immunomodulators to effectively manage ADs. Autoreactive T cells play a central role in ADs. Kv1.3 and KCa3.1 ion channels are expressed differently in T cell subtypes, offering a way to selectively modulate T cell responses. Overactive autoreactive T cells, especially effector memory T (TEM) cells with high Kv1.3 expression, contribute to AD pathogenesis. Kv1.3 inhibition has proven effective in treating ADs in animal models without compromising the protective immune response provided by other T cells.

VRGT's "Designer Miniprotein" technology identified lead candidates with strong affinity and selectivity for Kv1.3. Our screening system uses phage libraries based on animal toxins and deep sequencing to find potential drugs. VRGT's patentable lead molecule is a high-affinity, highly selective Kv1.3 inhibitor, promising targeted therapy with minimal side effects. Ex vivo experiments demonstrated its effectiveness in preventing TEM cell activation, suggesting a safer profile than small molecules and lower immunogenicity than large biologics. Our lead compound also showed in vivo efficacy in a rat model of contact dermatitis.

This research received funding from the National Research, Development, and Innovation (NRDI) Office under grant ID 2019-1.1.1-PIACI-KFI-2019-00127 and the Cooperative Doctoral Programme-2020 managed by the Ministry for Innovation and Technology (ITM).

Keywords: drug development, Miniproteins, Autoimmune diseases, Autoreactive T-cells, Kv1.3 channel.

The gut microbiome plays a crucial role in human health, and recent advances in next-generation sequencing have enabled exploration of this unique ecosystem. This work aims to identify factors affecting metagenomic analysis pipelines when they are applied for yeasts in the microbiome. One key aspect of metagenomic analysis involves comparing the DNA sequences from a sample with those stored in genomic databases to determine the composition of microbiome populations. The quality and comprehensiveness of the reference genome set used for mapping are critical in ensuring that all microbes in the sample are accurately represented. This task can be particularly challenging for rare or uncultivated microbes(mostly bacteria). Another factor to consider is the position of the target genome within a concatenated reference genome set, as it can impact mapping accuracy, especially due to the heuristic nature of the mapping algorithm. Utilizing a collection of hundreds of Saccharomyces yeast genomes, along with other yeast species, we created an artificial virtual metagenome (with simulated Illumina reads using SimuSCoP) and an artificial virtual ancient metagenome using Gargammel with various coverages of dozens of species. Subsequently we conducted mapping to different sets of concatenated reference genomes and performed allele calling to determine reliability of genomic analysis, down to the level of clade assignment. One of our primary objectives is to investigate the potential impact of the target species' position within the reference genome, particularly in the case of Saccharomyces cerevisiae. We predict that the target species' position within the reference genome set has a substantial impact on mapping accuracy. Choosing the right reference genome set may impact clade assignment and community composition analysis. The research contributes to understanding the role of yeasts in human health and more accurate metagenomic analyses in the context of the gut microbiome.

Keywords: yeast, metagenomic analysis, virtual metagenome, gut microbiome, next-generation sequencing.

Biocontrol yeast has emerged as a promising and environmentally friendly alternative to chemical pesticides in modern agriculture. This approach harnesses the natural antagonistic properties of specific yeast strains to combat plant pathogens, thereby reducing the reliance on harmful chemical agents. In this study, we investigated the gene sg in the dimorphic yeast Schizosaccharomyces japonicus, known for its biocontrol potential. We employed a range of molecular biology techniques, including genomic DNA isolation, PCR reaction, gel electrophoresis, cell growth in gel medium, RNA isolation, cDNA synthesis, and qPCR reaction, to analyze sg expression in both yeast and hyphae cells of Sch. japonicus. Primers were also specifically designed for the purposes of this research. By utilizing sce3 and sce_ver2 as control genes for normalization, we quantified sg expression levels.

In the context of biocontrol, understanding gene expression in both cell types is crucial. Our findings revealed that the sg gene exhibited a significantly higher expression in yeast cells compared to hyphae cells, while still maintaining a notable presence in the latter. This observation suggests that sg gene plays a role in the yeast cell's growth and reproduction, potentially contributing to its biocontrol properties by influencing interactions with other microorganisms. Moreover, in hyphae cells, sg gene is involved in regulating filamentous growth, which is essential for nutrient acquisition and environmental adaptation. The dual expression of sg gene across different cell types highlights the flexibility and adaptability exhibited by Sch. japonicus. These results open new avenues for utilizing fission yeast as biocontrol agents against plant pathogens. This research contributes to sustainable agriculture by providing eco-friendly solutions for crop disease management.

Keywords: biocontrol, fission yeast, dimorphism, filamentous growth, sustainable agriculture, gene expression.

In the breadmaking industry, sourdough represents a special microenvironment where lactic acid and other bacteria and yeasts (mainly Saccharomyces cerevisiae) coexist, each contributing to the formation of a dynamic microbial community. Their cohabitation ensures the unique properties that have been exploited in the process of bread preparation for centuries. Analysis of sourdough samples from different regions provides insight into the origin and uniqueness of microbes that are found in local sourdough starters. Moreover, an investigation of those microbial communities and their genomic properties provides particular information on how these microbes coexist and evolve. With the help of third-generation sequencing technologies, such as Oxford Nanopore’s long-read-based metagenomics/metabarcoding and whole genome sequencing solutions, we were able to gather information about various Hungarian sourdough samples. To our knowledge, we are the first to analyze the bacterial and yeast community from local sourdoughs of the country along with a detailed phylogenomic analysis of S. cerevisiae isolates found in them. We determined that local Hungarian sourdoughs contain industrial, polyploid baker’s yeasts instead of yeasts of the traditional sourdough clade, and they form tetraploid and triploid populations. Other yeast species recovered included Pichia kudriavzevii and P. membranifaciens. Bacterial communities were dominated by Weissella and Lactobacilli.

Keywords: metabarcoding, phylogenomics, yeasts, sourdough.

In this work, different oil-industrial wastewater samples supplied by the MOL group were treated by various environmentally friendly anaerobic biodegradation techniques. These samples were rich in organic compounds (e.g., volatile organic chemicals, long-chain alcohols) and could contain other toxic impurities (e.g., Zn, Mn, Mo, etc.). The main goal was to generate consumable bioenergy during the purification of the mixture. Among biological purification techniques, the well-known anaerobic digestion, the dark fermentation of hydrogen, and the developing bioelectrochemical system were investigated. This work focused on the treatment, utilization opportunities, and energy recovery strategies of these methods.

Keywords: anaerob biodegradation, bioelectrochemical system, biogas, biohydrogen, wastewater treatment, dark fermentation, bioenergetics.

Introduction: Pituitary adenylate cyclase-activating polipeptide (PACAP) signaling is involved in various inflammatory processes. A common manifestation of systemic inflammation is fever, which is usually induced in animal models with the administration of bacterial lipopolysaccharide (LPS). A role for PACAP signaling was suggested in LPS-induced fever, but the underlying mechanisms of how PACAP contributes to febrile response have remained unclarified.

Methods: We administered LPS (120 µg/kg, intraperitoneally) to mice with the Pacap gene, i.e., the gene encoding the PACAP protein, either present (Pacap+/+) (n=15) or absent (Pacap−/−) (n=14) and measured their thermoregulatory responses, serum cytokine levels, and tissue cyclooxygenase-2 (COX-2) expression.

Results: We found that the LPS-induced febrile response was attenuated in Pacap−/− mice compared to their Pacap+/+ littermates starting from ~120 min postinfusion. Administration of LPS resulted in amplification of COX-2 mRNA expression in the lungs, liver, and brain of the mice in both genotypes at 210 min postinfusion. Serum concentration of the pyrogenic cytokines interleukin (IL)-1α and β were significantly increased in Pacap+/+ mice in response to LPS compared with saline, whereas the change was not significant between the treatment groups in Pacap−/− mice. In case of IL-1α and β, the intergenotype difference between the LPS-treated groups was also significant. The rise in IL-10 was significantly attenuated in Pacap−/− mice compared to Pacap+/+ mice.

Conclusion: Our results suggest that PACAP contributes to the later phases of LPS-induced fever by modulation of COX-2 protein expression in the periphery and the brain, as well as by augmentation of pyrogenic cytokine levels in the circulation. These findings advance the understanding of the crosstalk between PACAP signaling and the “cytokine-COX-2” axis in systemic inflammation, thereby open up the possibilities for new therapeutic approaches.

Keywords: thermoregulation, PACAP.