Salivary microbiome signatures of Poles and Serbians and its potential for prediction of biogeographic ancestry

Biogeographical ancestry analysis is valuable in forensic investigations, especially in missing person cases or crimes without eyewitnesses, as it helps to infer geographic origins from genetic markers. This approach enhances forensic efforts by providing essential clues for identifying individuals with limited direct evidence. Slavic-speaking populations are poorly distinguishable based on human genome variability. However, recent studies show that even populations with close biogeographic origin could be differentiated based on salivary microbiomes. Nevertheless, the salivary microbiomes of Slavs have not been characterized yet. Therefore, this study aimed to compare the composition of the salivary microbiomes of Western and Southern Slavs’ representatives. 16S rRNA libraries from salivary microbiomes of 40 Poles (Western Slavs) and 40 Serbians (Southern Slavs) were prepared via PCR and sequenced on the MiSeq FGx platform (Illumina), giving approximately 100,000 reads per sample. Bioinformatic and statistical analyses were performed to assess the alpha and beta diversity of microbiomes and determine the differences in the abundance of bacterial genera between the groups studied. Analyses of alpha (ACE, Chao1, Shannon, and Simpson) and beta (Jaccard and Bray-Curtis dissimilarity) diversities in the salivary microbiomes clearly distinguished between Poles and Serbians. Alpha and beta diversity metrics were significantly higher in the Serbian population. Fusobacterium, Lautropia, Porphyromonas, Actinobacillus, Capnocytophaga, and Kingella were the most significantly increased genera in Serbians, whereas Veillonella, Selenomonas, Megasphaera, and Atopobium were more prevalent in Poles. In summary, our study identified significant differences in the salivary microbiomes of Poles and Serbians, with distinct microbial signatures associated with each population. These findings highlight the potential of salivary microbiome analysis as a tool for predicting biogeographic ancestry. Nevertheless, further analysis extended to other Slavicspeaking populations is necessary to clarify this issue

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

Sustainable eco-friendly active packaging from food processing waste: Recycled starch and pomegranate peel extract for biodegradable solutions

Reducing plastic usage and improving food preservation are critical steps toward a more sustainable future, and converting food waste into eco-friendly food packaging offers a promising solution to both challenges [1]. Food packaging is essential for safeguarding food from spoilage and microbial growth, with active packaging technologies providing an effective means of extending shelf life. While plastic packaging is commonly chosen for its affordability and strong mechanical and barrier properties, its environmental persistence and limited biodegradability pose significant sustainability issues. In response, there has been growing interest in biopolymers as an alternative to synthetic plastics, offering both environmental benefits and the potential to retain desirable packaging qualities. This study focuses on developing advanced, sustainable food packaging materials derived from food processing waste. Specifically, recycled starch from potato chip factory waste and poly(vinyl alcohol) (PVA) were combined to create an active food packaging material. To enhance the packaging's functionality, pomegranate peel extract (PPE), typically discarded during fruit juice production, was incorporated for its natural antioxidant and antimicrobial properties. Additionally, in a fermentation process conducted in an 8-channel bioreactor, recycled starch served as a growth medium for Streptomyces anulatus BV365 producing actinomycin D (ActD)2, a bioactive compound with antimicrobial effects, to further boost the material’s effectiveness. The study produced several novel packaging materials with varying PPE concentrations (5% and 10%), along with a control sample without PPE, using glycerol as a plasticizer. The incorporation of PPE and ActD enhanced the packaging’s ability to extend food shelf life by imparting antioxidant and antimicrobial properties. Meanwhile, the starch-PVA blend, contributes to the biodegradability of the packaging. The materials were characterized using a range of analytical techniques, including Fourier-transform infrared (FTIR) spectroscopy, scanning electron microscopy (SEM), differential scanning calorimetry (DSC), X-ray diffraction (XRD), wettability and water contact angle (WCA) measurements. FTIR analysis confirmed the successful incorporation of PPE and ActD, while the addition of PPE improved the mechanical strength and swelling behavior of the starch-PVA based materials at both 5 and 10 wt% concentration. These findings demonstrate that the combination of enhanced functionality with environmental sustainability makes these bio-based packaging materials a promising alternative for reducing plastic waste while still preserving food quality.E-book: 19th International Conference on Chemistry and the Environment ICCE 2025 Belgrade 8-12 June 202

Development of Bioactive Cotton, Wool, and Silk Fabrics Functionalized with Origanum vulgare L. for Healthcare and Medical Applications: An In Vivo Study

Background: This study presents an innovative approach to developing bioactive natural fabrics for healthcare and medical applications. Methods: An ethanol extract of Origanum vulgare L. (in further text: OE), exhibiting exceptional antioxidant (100%) and antibacterial activity (>99% against E.coli and S.aureus), was employed to biofunctionalize cotton, wool, and silk fabrics. Results: All biofunctionalized fabrics demonstrated strong antioxidant activity (>99%), while antibacterial efficacy varied by fabric: cotton > 54%, wool > 99%, and silk > 89%. OE-biofunctionalized wool possessed the highest release of OE’s bioactive compounds, followed by silk and cotton, indicating substrate-dependent release behavior. This tunable fabrics’ OE release profile, along with their unique bioactivity, supports targeted applications: OE-functionalized silk for luxury or prolonged therapeutic use (skin-care textiles, post-surgical dressings, anti-aging products), cotton for disposable or short-term use (protective wipes, minor wound coverings), and wool for wound dressings. The biocompatibility and cytotoxicity of OE-biofunctionalized wool were evaluated via in vitro assays using healthy human keratinocytes and in vivo testing in Wistar albino male rats. The obtained results revealed that OE-functionalized wool significantly accelerated wound closure (97.8% by day 14), enhanced collagen synthesis (6.92 µg/mg hydroxyproline), and improved tissue and systemic antioxidant defense while reducing oxidative stress markers in skin and blood samples of rats treated with OE-biofunctionalized wool. Conclusions: OE-biofunctionalized wool demonstrates strong potential as an advanced natural solution for managing chronic wounds. Further clinical validation is recommended to confirm its performance in real-world healthcare settings. This work introduces an entirely new application of OE in textile biofunctionalization, offering alternatives for healthcare and medical textiles