Gamma rays assisted synthesis of N doped-graphene quantum dots from multiwall carbon nanotubes

Gamma rays are the powerful tool for top-down synthesis of nitrogen doped graphene quantum dots (NGQDs) from multiwall carbon nanotubes. Different doses of gamma rays (100, 200 and 300 kGy) were applied to the multiwall carbon nanotubes suspended in mixture of sulfuric and nitric acid (3:1 ratio). After purification, NGQD were characterized to investigate their structure (morphology, particle size, nanomechanical and nanoelectrical properties, chemical composition, photoluminescence, reactive oxygen species production, antibacterial activity and biocompatibility). Viscoelastic measurements revealed that NGQDs nanoparticles had Young’ modulus of elasticity almost equal to single wall carbon nanotubes (SWCNTs (6,5)). Electrostatic force and scanning tunneling microscopy showed that all types of the NGQDs nanoparticles had negative charge distributed homogeneously. All NGQDs samples produced singlet oxygen and the NGQDs300 sample showed moderate antibacterial activity and good biocompatibility

Transcriptomic profiling of iPSC-derived astrocytes from patients with 22q11.2 deletion syndrome

Background: Neurodevelopmental disorders represent considerable public health challenges.One of the syndromes associated with heightened risk of NDDs is 22q11.2 Deletion Syndrome (22q11.2DS), caused by microdeletion in chromosomal region 22q11.2. However, molecular mechanisms underlying NDDs are largely unidentified. Here we analyzed transcriptomic profiling of 22q11.2DS astrocytes. Material and Methods: Total RNA was isolated from iPSCs-derived astrocytes of two cases with familial 22q11.2DS with 1.5Mb microdeletion and one healthy control. Paired- end RNA-seq was carried out on Illumina NovaSeq 6000 sequencer. Bioinformatic processing of raw data was conducted via NVIDIA platform, while differential gene expression analysis was performed in RStudio using DESeq2 R package. The obtained list of DEGs was used for pathway enrichment analysis by employing EnrichR and WikiPathways. Results: 125 DEGs with lower expression and 287 DEGs with higher expression in 22q11.2DS astrocytes compared to the control were obtained. For genes with lower expression in 22q11.2DS astrocytes, 22q11.2 Copy Number Variation Syndrome and Axon Guidance were the top enriched pathways, while for genes with higher expression in 22q11.2DS astrocytes we did not identify biological pathways that are enriched in DEG lists more than would be expected by chance. We found 178 DEGs with lower expression and 205 DEGs with higher expression in astrocytes of symptomatic child with 22q11.2DS compared to oligosymptomatic mother with 22q11.2DS. Employing EnrichR and WikiPathways we did not identify biological pathways that are enriched in DEG lists more than would be expected by chance. Conclusion: We provide preliminary evidence for an altered transcriptomic landscape of 22q11.2DS astrocytes.Book of absctracts: European Society of Human Genetics (ESHG) 2025 Conference, May 24–27, 2025. Milan, Ital

Degradation of biomaterials by Streptomyces microflavus DG19: depolymerization activity, genome mining, and soil burial assessment

The accumulation of plastic waste remains a significant environmental challenge despite the alarming evidence and public efforts, emphasizing the need for biodegradable alternatives and appropriate remediation strategies. In this study, Streptomyces microflavus DG19 was evaluated for capacity to degrade a selection of biomaterials that are increasingly penetrating market as readily degradable alternatives. S. microflavus DG19 rapidly degraded poly(3-hydroxybutyrate-co-3-hydroxyvalerate) films in liquid culture (96% weight loss in 7 days) and demonstrated activity against poly(ε-caprolactone) in both agar-based and liquid culture experiments and against cellulose in Congo red assay. 3-Hydroxybutyrate and lactic acid were also metabolized. Genomic analysis identified a number of enzymes involved in carbohydrate and bioplastic degradation. A putative extracellular poly(3-hydroxybutyrate) (PHB) depolymerase (SmPHBase) containing a variant substrate binding domain, and other enzymes involved in 3-hydroxybutyrate metabolism, were of special interest. The presence of > 30 biosynthetic gene clusters highlights this strain’s potential for upcycling bioplastic-containing waste. Soil burial tests demonstrated substantial weight loss in pure biomaterial films and multilayer consumable items containing PHB, showcasing the applicability of S. microflavus DG19 as a composting enhancer. Overall, the findings highlight the pertinence of specialized bacterial strains to biomaterial recycling and upcycling

Biofilm prevention and quorum sensing interference via surface-bound peptoid

The emergence of antibiotic resistance has ushered in a post-antibiotic era, highlighting the urgent need for alternative, cytocompatible antimicrobial strategies. Among these, antimicrobial peptides (AMPs) are promising to overcome antibacterial resistance being at the same time cytocompatible, but they are limited by fast enzymatic degradation. Peptoids are synthetic and bio-mimetic biomolecules that overcome the limitations of AMPs with resistance to proteolytic degradation. This study examined the antibacterial and cytocompatible peptoid GN2-Npm9 to reduce the risk of infection in titanium implants. Ti6Al4V samples were chemically pre-treated (CT) to favour osteointegration and functionalization. The zeta potential titration curves evidenced a mechanism of electrostatic attraction between the peptoid and CT substrate on the functionalized samples (CT_GN2-Npm9). XPS analysis and fluorescence microscopy confirmed the presence of the peptoid on CT_GN2-Npm9 and evidenced a uniform distribution. The peptoid was released in water with slow kinetics for at least 9 days (HPLC analyses). CT and CT_GN2-Npm9 specimens were subjected to biological assays against oral plaque collected from patients affected by periodontitis, showing a direct biofilm reduction of 60 % in comparison to CT and a specific effect towards pathogens as evidenced by proteomics studies. For investigating the mechanism of biofilm prevention, a culture of Pseudomonas aeruginosa was performed by conditioning the culture medium with the supernatant from the plaque test. It was observed that the biofilm of P. aeruginosa was significantly reduced due to a peptoid’s indirect effect demonstrated by the expression of genes involved in the quorum sensing network and elastase gene (lasB) that resulted in down-regulation only by the supernatants from CT-GN2-Npm9 specimens

Može li se zamrznuti gruš koristiti u proizvodnji bijelih sireva u salamuri?

This study explored the potential of overcoming the seasonal nature of caprine milk by using frozen curds from late lactation, frozen at two different pressing stages, to produce a 14-day ripened white brined cheese. Frozen curds present notable advantages over frozen milk as a raw material, including reduced storage space requirements, the elimination of whey production, and lower water consumption compared to powdered milk. Textural analysis of the resulting cheeses demonstrated significantly reduced firmness by the 14th day of ripening in brine. Microstructural examination using Scanning Electron Microscopy (SEM) on the 1st day of ripening revealed that the experimental cheeses exhibited a disordered and less compact structure compared to the control samples. By the 14th day, the experimental cheeses disintegrated in brine, due to increased water absorption associated primarily with structural changes of the cheese matrix. To address these challenges, the study proposes two viable strategies for successful white brined cheese production: achieving a lower pH through prolonged traditional salting methods or shortening the maturation period in brine.Ova je studija ispitivala rješavanje problema sezonske prirode kozjeg mlijeka za proizvodnju bijelog sira u salamuri s 14 dana zrenja upotrebom zamrznutih gruševa s kraja laktacije, zamrznutih u dvije različite faze prešanja. Zamrznuti gruš ima značajne prednosti u odnosu na zamrznuto mlijeko radi smanjenih zahtjeva za prostorom skladištenja, eliminaciju sirutke, kao i manju potrošnju vode u usporedbi s korištenjem mlijeka u prahu. Analiza teksture eksperimentalnih sireva pokazala je značajno smanjenu čvrstoću do 14. dana zrenja u salamuri. Skenirajućom elektronskom mikroskopijom (SEM) prvog dana zrenja otkriveno je da eksperimentalni sirevi imaju neuređenu i manje kompaktnu mikrostrukturu u odnosu na kontrolne uzorke. Do 14. dana, eksperimentalni sirevi su se dezintegrirali u salamuri uslijed povećane apsorpcije vode, pretežno zbog strukturnih promjena u sirnom matriksu. Kako bi se ovi izazovi svladali, studija predlaže dvije strategije za uspješnu proizvodnju bijelog sira u salamuri: postizanje niže inicijalne pH vrijednosti kroz produžene tradicionalne metode soljenja ili skraćenje perioda zrenja u salamuri

Engineered 3D osteosarcoma microenvironment model: Bridging in vitro - in vivo gap in cancer research and anticancer drug screening

Introduction Current treatments for osteosarcoma typically include surgical excision followed by neoadjuvant and adjuvant chemotherapy. Research attempts to streamline and improve these treatments, but progress is slow mostly due to limited translation of in vitro to in vivo studies. The aim of this work was to develop and validate an engineered three-dimensional (3D) osteosarcoma model based on macroporous composite scaffolds, as cell carriers, and biomimetic perfusion bioreactor for osteosarcoma research and anticancer drug screening. Material and method The scaffolds (4 mm thick discs, 9 mm in diameter) were produced by controlled gelation of hydroxyapatite (HAP) suspension in Na-alginate solution (2 wt.% alginate and 2 wt.% HAP) followed by freeze-drying and rehydration in the culture medium. Murine osteosarcoma K7M2-wt cells were seeded onto the scaffolds (15x106 cells cm-3 scaffold volume) and cultivated for 7 days in "3D Perfuse" bioreactors under continuous medium superficial velocity of 40 μm s-1, while static cultures served as a control. To evaluate this model for anticancer drug screening, bioreactor cultures were treated with doxorubicin (1 μg cm-3), on day 1 (first study) or on day 7 (second study) and lasted for 1 day, while untreated bioreactor culture served as a control. The scaffolds were assessed regarding the cell metabolic activity by MTT, morphology and distribution by histological and scanning electron microscopy analyses. Masson-trichrome and reticulin staining were used for extracellular matrix (ECM) analysis, while quantitative real-time PCR (qRTPCR) assessed osteosarcoma marker expression.Result and discussion After short-term cultures, biological assessment showed that the cells stayed viable and metabolically active, produced ECM, expressed osteosarcoma markers and spontaneously formed aggregates under both culture conditions. However, cells in the bioreactor culture exhibited higher metabolic activity, while the cell aggregates were slightly larger (~1.2-fold), more compact with higher amounts of reticular fibers, more numerous and more uniformly distributed throughout the scaffold compared to the static culture. These results could be explained by positive effects of flow on cells due to enhanced mass transport and adequate hydrodynamic shear stresses. Evaluation of the model for anticancer drug screening has shown a negligible effect of doxorubicin on individual cells as well as cell aggregates implying that the developed model more closely mimics in vivo drug responses than 2D cultures. Conclusion This study has shown potentials of engineered 3D osteosarcoma microenvironment model based on macroporous composite scaffolds, and perfusion bioreactor for relevant and reliable osteosarcoma research and anticancer drug screening.EACR 2025 Congress Abstract

Effect of doxorubicin and quercetin combined treatment on osteosarcoma model systems

Introduction Osteosarcoma (OS) is a highly aggressive bone tumor primarily affecting pediatric patients. Standard treatments include surgical resection, chemotherapy, and radiation for tumors that cannot be surgically removed. Although the 5–year survival rate is 65.5%, patients with metastases and recurrence have a significantly lower survival rate of ~30%. Despite this concerning statistic, the treatment for OS has remained largely unchanged over the past three decades. This stagnation in treatment innovation highlights the urgent need for further research and development in therapies tailored specifically for OS. Material and method We identified the DEGs between bone (7 samples) and OS (27 samples). To conduct in-depth study of the obtained upregulated DEGs, we constructed a PPI network and identified the most significant gene cluster. We investigated the effects of the combined treatment with doxorubicin and quercetin on SAOS-2 osteosarcoma cells in 2D condition and immobilized in alginate microbeads in 3D condition. We assessed the effects of treatments on cell viability using MTT and the expression of genes using qPCR. Result and discussion We have analysed DEGs between bone and human osteosarcoma samples and identified 630 upregulated genes. We extended the networks with information from DrugBank to identify potential therapeutics for osteosarcoma focusing on the top 10% of interconnected genes in cluster due to their important biological functions. The identified cluster had enrichment in biological processes connected to oxidative phosphorylation and we found quercetin as a promising candidate for treating OS. We analysed quercetin’s effect utilizing the Saos-2 in 2D and 3D on viability and gene expression, alone or in combination with doxorubicin. Following treatment, we assessed cell viability and the expression of genes. Our results have shown that the combined treatment statistically significantly decreased the viability of SAOS-2 cells cultured in 2D and 3D conditions compared to cells treated with doxorubicin. We analyzed the expression of genes associated with poor prognosis in patients such as pluripotency genes, an OS marker, and a resistance-related gene. Collectively our results show different responses to the combined treatment depending on the model system used. Conclusion The combined treatment substantially reduced cell viability in 2D and 3D models and decreased expression of genes associated with poor prognosis compared to doxorubicin alone in 3D models. We can hypothesize that microenvironment-based mechanisms modulate cell sensitivity to therapy and increase resistance to treatment of osteosarcoma cells cultured in 3D condition. Understanding the molecular mechanisms will significantly contribute to the development and enhancement of existing therapies, thereby facilitating advancements in the treatment of osteosarcoma.EACR 2025 Congress Abstract

DEVELOPMENT OF NOVEL FLUOROGENIC SUBSTRATES FOR RAPID AND SENSITIVE ASSESSMENT OF PHA DEPOLYMERASE ACTIVITY

Polyhydroxyalkanoates (PHAs) are microbial polyesters synthesized naturally and commercialized as biodegradable plastics, yet their biodegradation in the environment was shown to be slow (1). PHA depolymerases (PHAses) are essential for their degradation. Currently known PHA depolymerases lack the efficiency for larger-scale processing, highlighting the need for discovering and engineering more efficient variants. The lack of a reproducible, high-throughput methodology hinders straightforward and reliable monitoring and assessment of PHA depolymerases' activity (2). In our previous research, we reported the synthesis of two chromogenic compounds derived from polyhydroxyoctanoate (PHO) and their successful application in a continuous, quantitative spectrophotometric assay. This assay enables the rapid evaluation of PHO depolymerase activities within just 10 minutes at temperatures exceeding 45 °C, streamlining the comparison of PHAses and advancing the analysis of enzymatic degradation processes (3). To further improve sensitivity and enable measurements at lower substrate concentrations, we designed and synthesized for the first time three novel fluorogenic substrates: 4-methylumbelliferone esters (4-mU) of 3-hydroxy butyric acid (4-mUHB), 3-hydroxyoctanoic (4-mUHO) acid and octanoic acid (4-mUO). These substrates were fully characterized using NMR, IR, and LC/MS, confirming their structure and suitability for enzymatic assays. Novel fluorogenic substrates were then used in the enzymatic activity assay with the benchmark PHO depolymerase from Pseudomonas fluorescens GK13. This activity was compared to commercially available Candida antarctica lipase B (CALB). A clear distinction in activity was observed between PHOase and CALB lipase when tested with these substrates. These assays were rapid and highly sensitive, requiring low substrate concentrations facilitating the discovery and optimization of more efficient PHA depolymerases for industrial and environmental applications.BOOK OF ABSTRACTS: 17th International Symposium on Biocatalysis and Biotransformations Basel, Switzerland, June 29-July 3, 202

Association Between Hypertension, Dipping Status, and ACE and AGTR1 Gene Polymorphisms in Adolescents with Type 1 Diabetes

Objectives: This study aims to show the distribution of angiotensin-converting enzyme (ACE) rs1799752 (I>D) gene insertion/deletion (I/D) polymorphism and angiotensin II receptor type 1 (AGTR1) rs5186 (A>C) gene polymorphism in adolescents with hypertension (HT) and type 1 diabetes (T1D), as well as its association with hypertension and the diurnal variation of mean blood pressure (dipping phenomenon). Methods: A cross-sectional study was conducted involving 118 adolescents diagnosed with T1D who underwent clinical and laboratory investigations, genetic analyses, and 24 h ambulatory blood pressure monitoring. The genotype frequencies were compared between adolescents with HT and those with normal blood pressure. Additionally, the genotype frequencies were compared between dippers and non-dippers. Results: Patients with HT were more likely to be female and exhibited significantly poorer glycemic control and higher triglycerides, along with increased body mass index and daily insulin dosage. The prevalence of ACE rs1799752 genotypes in the hypertensive group was 20% II, 66.7% ID, and 13.3% DD, which did not significantly differ from the normal blood pressure group with 29.1% II, 53.4% ID, and 17.5% DD (p = 0.625). The prevalence of AGTR1 rs5186 genotypes in the hypertensive group was 53.3% AC, 40% AA, and 6.7% CC, which also did not significantly differ from the normal blood pressure group with 39.8% AC, 52.4% AA, and 7.8% CC (p = 0.608). A total of 46% of the patients exhibited non-dipping phenomena. The prevalence of non-dippers among the ACE genotypes was 13% DD, 33.3% II, and 53.7% ID (p = 0.369), while for the AGTR1 genotypes, it was 50% AA, 42.6% AC, and 7.4% CC (p = 0.976). Conclusions: Our results indicate that in our adolescents with T1D, clinical and metabolic factors such as higher body mass index, triglycerides, suboptimal glycemic control, and female gender are more indicative of the development of hypertension than ACE and AGTR1 gene polymorphisms. A potential reason for this finding could be the young age of the patients or the relatively small size of the study group. Future research involving larger sample sizes is needed to further investigate the genetic predisposition for the development of hypertension

GENE EXPRESSION PROFILING OF IPSC-DERIVED NEURAL PROGENITORS FROM PATIENTS WITH 22Q11.2 DUPLICATION SYNDROME

The molecular mechanisms underlying neurodevelopmental disorders (NDDs) are understudied. 22q11.2 Duplication Syndrome (22q11.2DupS) is associated with an increased risk of NDDs, including autism spectrum disorders, developmental delay, and intellectual disability, while potentially having protective effect against schizophrenia. Here we performed transcriptomic profiling of 22q11.2DupS neural progenitors (NPCs). Total RNA was isolated from iPSCs-derived NPCs of a family trio consisting of a child with 22q11.2DupS, a carrier unaffected mother, and a healthy control father. Paired-end RNA-sequencing was carried out by commercial services. FASTQ files were processed on an NVIDIA platform and differential gene expression analysis was carried out in RStudio using the DESeq2 R package. The resulting lists of differentially expressed genes (DEGs) was utilized for pathway and gene ontology enrichment analysis using clusterProfiler in RStudio. 60 DEGs with lower expression and 58 DEGs with higher expression were obtained in 22q11.2DupS NPCs compared to both carrier and control NPCs. For genes with lower expression in 22q11.2DupS NPCs, “miRNA targets in ECM and membrane receptors” was the top enriched pathway. On the other hand, for genes with higher expression in 22q11.2DupS NPCs, no biological pathways were identified as being significantly enriched in the DEG list beyond what would be expected by chance. However, for genes with higher expression in 22q11.2DupS NPCs, we obtained statistically significant enrichment of molecular functions such as Histone demethylase activity. 22q11.2DupS NPCs exhibit altered signaling pathways and molecular functions, providing preliminary insights into the impact of this microduplication on neural differentiation.Abstract book: FENS Regional Meeting 2025, Oslo, Norway, 16-19 June 202