imagine (Institute of molecular genetics and genetic engineering)
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Efficient Degradation of Consumer-Grade PLA by Commercial Savinase: Optimized Conditions and Molecular Dynamics Insights
No full textPoly(lactic acid) (PLA) is the most widely produced biopolymer globally, with 920,000 tons reaching the market in 2024. Although it is a biobased polymer and biodegradable, it currently poses a threat by generating a considerable waste stream in the environment unless its end-of-life options are further developed. Biorecycling of PLA is a promising solution and is critical for PLA to be a truly sustainable alternative to conventional plastics, further enabling the (bio)plastics circular economy. This work investigates the repurposing of commercial Savinase 12T preparation, already produced on a large scale for the detergent industry, for the degradation of consumer-grade PLA. Savinase-degraded postconsumer PLA single-use cups at a rate of 166 mg·day–1·mg enzyme–1 for the lid part (crystallinity (Xc) 22.9%) and 40 mg·day–1·mg enzyme–1 for the body part (Xc 42.0%). The average degradation rate of PLA 3D printer filaments was determined to be 56 mg·day–1·mg enzyme–1 under optimized conditions of pH 8.5 and 42 °C. Lactic acid was identified as the main PLA degradation product of Savinase depolymerizing activity. Molecular modeling revealed Asn155 and Ser125 as key residues involved in PLA tetramer binding
Pd-nanoparticles supported on bacterial nanocellulose as a catalyst for the hydrogenation of polyfunctional reagents containing C=O and C=C bonds
No full textThe bacterial nanocellulose (BNC) was synthesized by cultivating the Komagataeibacter
medellinensis strain in Hestrin-Schramm medium under static conditions.1 Pd nanoparticles were
supported on BNC by impregnating of palladium (II) acetate in acetone, followed by reduction of Pd
ions using NaBH4 in methanol. The 2wt% Pd/BNC and 4wt% Pd/BNC catalysts were synthesized
and characterized by SEM, TEM, FTIR, XPS, and TA. The catalysts were screened in liquid phase
hydrogenation in a batch reactor. Their reactivity in hydrogenation of acetophenone (ACP), furfural
(FU), 5-hydroxymethylfurfural (HMF) and cinnamaldehyde (CAL) was tested under mild conditions
(p = 4–9 bar H2, T = 30–70°C) in various solvents, including water, isopropanol, ethanol, acetonitrile,
and toluene. The catalysts exhibited high selectivity towards 1-phenylethanol (PE) during ACP
hydrogenation in water and especially in isopropanol (ca. 100% PE). During FU hydrogenation,
Pd/BNC primarily facilitated the formation of furfuryl alcohol, followed by saturation of the furan
ring. Surprisingly, Pd/BNC demonstrated low catalytic activity in HMF hydrogenation in both nonpolar
and polar solvents. In CAL hydrogenation, Pd/BNC exhibited 100% conversion and up to 96%
selectivity towards hydrocinnamaldehyde (C=C bond saturation), almost independently of the solvent
(isopropanol/toluene) used. The catalyst demonstrated excellent recyclability in CAL hydrogenation
in isopropanol.Book of Abstracts, Programme and General Information
(International conference)
NanoOstrava 2025 – 9th Nanomaterials and Nanotechnology Meeting
VSB – Technical University of Ostrava, Czech Republic
May 19-22, 202
Towards a 3D cell culture platform for anticancer drug testing
No full textObjectives
The aim of our research is to establish a platform of in vitro systems for cancer cell cultures in three-dimensional (3D) environments that will be sufficiently simple but still capturing some of the main tumor features to be applicable for cancer research and anticancer drug testing. Here we present development of carcinoma and osteosarcoma cell culture models, based on cancer cell lines, alginate-based scaffolds and perfusion bioreactor followed by model validation by application of standard anticancer drugs.
Methods
The carcinoma culture model comprises alginate microfibers produced by extrusion of cell suspensions (human glioblastoma U87 cells, human non-small lung cancer NCI-H460 cells; 2-4 × 106 cell/ml) in Na-alginate solutions (1–3 wt.%) into a gelling bath containing Ca2+ or Ba2+ (45–180 mM) and cultured statically for up to 28 days. The model was tested by application of standard anticancer drugs (i.e. 100 μM temozolomide (TMZ), 0.5-50 µM cisplatin).
The osteosarcoma culture model utilizes murine K7M2-wt osteosarcoma cells immobilized in Ca-alginate hydrogels (2 wt.%) with hydroxyapatite particles (2 wt.%) in the form of microfibers to imitate bone extracellular matrix (ECM) composition or macroporous scaffolds (discs 9 mm in diameter, 4 mm thick) to imitate bone structure, too. The microfibers (4 × 106 cell/ml) were cultured statically for up to 21 days, while cell-seeded porous scaffolds (15x106 cells/ml) were cultured under continuous perfusion in bioreactors (“3D Perfuse”, ICTMF, Belgrade, Serbia) for 7 days (40 μm/s superficial velocity). The microfiber model was used to determine the half-maximal inhibitory concentration (IC50) of doxorubicin (DOX; 0.25-20 μg/ml), while the scaffold model was tested by application of 1 μg/ml DOX for 24 h at day 7.
The cultured cells were characterized regarding metabolic activity (MTT; Live/dead assays), morphology, ECM, distribution within carriers (histological and immunohistological analyses) and resistance-related gene expression by qPCR.
Results
Alginate microfibers supported carcinoma cells although extrusion conditions and gelling cation had to be optimized for each cell type. Ca-alginate microfibers supported glioblastoma U87 cells in a 4-week culture, while the TMZ treatment in a clinically relevant regime induced an increase in expression of drug–resistance related genes, significantly higher than that in 2D cell cultures. However, Ca-alginate hydrogel was unstable in the RPMI culture medium so that Ba-alginate microfibers were optimized for NCI-H460 cells. Application of cisplatin showed over 5-fold higher IC50 compared to 2D cultures (25 μM vs. 1-5 μM) and it further increased with the increase in the microfiber preculture period (from 1 to 4 days).
In osteosarcoma cell cultures both environments supported formation of spheroid-like structures with noticeable ECM, particularly promoted by medium perfusion through macroporous scaffolds. Application of DOX yielded IC50 of 2–3 μg/ml for cells in microfibers vs. 0.5 μg/ml for cells in 2D. Nevertheless, 1 μg/ml of DOX applied in perfused cultures slightly impeded cell viability as compared to the non-treated perfused cultures.
Conclusions
Overall, the stepwise approach used in this work to develop 3D cancer cell culture systems provided key parameters for optimization of the culture models, demonstrating potentials for anticancer drug testing.
Topic: Cancer
Subtopic: Cancer models
Type: Poster presentation
TERMIS25_1486 Advancing Metastasis Research: Development and Application of Tumor-on-a-Chip Models for Drug Screening and Translational MedicineJesús Ruiz-Espigares1, Daniel Martinez-Moreno2, Julia López-de Andrés2, Gema Jiménez1, Juan Antonio Marchal 1
1University of Granada, Faculty of Medicine, Insitituto de Investigación Biosanitaria ibs.GRANADA, Granada, Spain; 2University of Granada, Facultad de Medicina, Insitituto de Investigación Biosanitaria ibs.GRANADA, Granada, Spain
Introduction/Objectives
Most deaths caused by cancer are due to the appearance of secondary tumors as a result of a process called metastasis. For this reason, in order to better understand this pathology, researches has been carried out throughout history on the synthesis of models capable of faithfully recreating the tumor microenvironment (TME). Thus, in this review we intend to study new models for the study of metastasis, including tumor-on-a-chip technology. In addition, preliminary results will be presented on the development of a three-dimensional model demonstrating its functionality in drug screening.
Methods
The scientific development has been obtained from the Review “Evolution of Metastasis Study Models toward Metastasis-On-A-Chip: The Ultimate Model?”, already published in the journal Small (D1/IF: 15.153), in which I am first author, where the bibliographic search of 158 articles and scientific reviews in different databases was carried out. In addition, Live/Dead assays, Alamar Blue, migration by cell tracking with Cell Trackers and metalloprotease assays with different types of drug concentrations were performed in MCF-7 and MG-63 cell lines.
Results
In this work we have carried out a literature review on the different phases of metastasis, the unknowns that remain for its complete understanding and the use of new models for the study of this pathology. The aim is to provide an overview of the current landscape and the great potential of these tumor-on-a-chip systems for in vitro translational research of this pathology. Thanks to this research and the new model created, the performance of each drug, palbociclib and doxorubicin, has been observed for each MCF7 and MG63 cell line, respectively. For MCF7, a reduction in proliferation, migration and metalloprotease secretion was observed, while for MG63 a significant decrease in viability, proliferation and metalloprotease secretion was observed.
Conclusions
These new tumor-on-a-chip models make it possible to recapitulate in vivo the physiology and pathophysiology at the organ level, mimicking in vitro tissue structures and functions, which will allow progress towards personalized medicine, generating chips from patient samples that mimic the original tumor, in order to perform a precise pharmacological screening to establish the most appropriate treatment protocol. Likewise, the functionality of the developed model has been observed by observing the different effects of each drug in the three-dimensional model, reflecting the clinical performance of the drug in this in vitro model
Amino Acid Substitutions in Bacteriocin Lactolisterin BU Reveal Functional Domains Involved in Biological Activity Against Staphylococcus aureus
No full textThe emergence of multidrug-resistant pathogens has driven the development of novel antimicrobial peptides (AMPs) as therapeutic alternatives. Lactolisterin LBU (LBU) is a bacteriocin with promising activity against Gram-positive bacteria, including Staphylococcus aureus. In this study, we designed and evaluated a panel of amino acid variants of LBU to investigate domain–activity relationships and improve activity. Peptides were commercially synthesized, and their effect was evaluated for minimal inhibitory concentration (MIC), minimal bactericidal concentration (MBC), hemolytic activity, cytotoxicity, in vivo toxicity, and virulence modulation. AlphaFold3 structural prediction of LBU revealed a four-helix topology with amphipathic and hydrophobic segments. Helical wheel projections identified helices I and IV as amphipathic, suggesting their potential involvement in membrane interaction and activity. Glycine-to-alanine substitutions at helix I markedly increased antimicrobial activity but altered toxicity profiles. In contrast, changes at helix junctions and kinks reduced antimicrobial activity. We also showed differential regulation of virulence genes upon sub-MIC treatment. Overall, rational substitution enabled identification of residues critical for activity and toxicity, providing insights into therapeutic tuning of lactolisterin-based peptides
Metagenomic analysis of the airborne antibiotic resistome and mobilome in Belgrade metropolitan area during fall season
No full textBackground and objectives: Antimicrobial resistance (AMR) has been identified as one of the top
three threats to public health in the 21st century. The role of the environment in the spread of
antimicrobial resistance is increasingly recognized. The airborne route for the transmission of antibiotic
resistance genes (ARGs) is particularly important as bioaerosols carrying ARGs can travel long
distances and remain in the atmosphere for long periods of time. In addition, mobile genetic elements
(MGEs) can facilitate the spread of ARGs in the environment. Considering that Serbia is a country
struggling with overuse and misuse of antibiotics, a high percentage of multidrug-resistant bacterial
isolates and poor air quality, the Serbian capital Belgrade was recognized as an interesting research
model for studying the airborne transmission of AMR.
Methods: Outdoor air was sampled with hydrophobic polypropylene membrane filters (air volume of
90 m3) at seven differently categorized locations: industrial (Veliki Crljeni and Barajevo), traffic (Borča,
Despota Stefana, Leštane, Banovo Brdo) and background (Zeleno brdo) during the fall season 2024.The DNA samples from the air were subjected to shotgun metagenome sequencing (Illumina Novaseq
X plus) and bioinformatic analysis (Novogene, UK). ARGs and MGEs were annotated using CARD,
Integrall, Isfinder and Plasmid databases, respectively.
Results: Metagenomic analysis revealed that the air sampled in Barajevo had the highest relative
abundance of ARGs, with antibiotic transformation/deactivation being the predominant resistance
mechanism, while the lowest value was observed in the Borča sample. In addition, the ARG profile
varied between samples, although Zeleno Brdo/Borča and Banovo Brdo/Veliki Crljeni showed similar
ARG patterns. The most abundant ARG in almost all samples was blaTEM. Among the MGEs, over 20
insertion sequences were revealed when analysed with ISfinder, with 5 out of 7 sites showing similar
patterns, while Barajevo and Despota Stefana fell out of the group. The same observation was made
when the plasmid profile of the samples was compared using the Plasmid database. In contrast,
integron-related sequences differ significantly among the samples.
Conclusions: This study revealed that the air in Belgrade could be a potential dissemination route for
antimicrobial resistance.Abstract book: 10th Symposium on Antimicrobial Resistance in Animals and the Environment (ARAE), 30 June –2 July 2025 Berlin, German
Resistome dynamics in soil exposed to the herbicide Smetolachlor
No full textSoil microbiota is essential for maintaining soil health. Pesticides that disrupt microbial
activities impair soil quality and harm the environment. Bacteria play a significant role in
metabolizing chloroacetanilide pesticides. However, herbicide exposure induces
environmental stress, triggering adaptive mechanisms such as horizontal gene transfer,
which accelerate the establishment and spread of resistance. Surviving in pesticidecontaminated
fields, bacteria become highly tolerant to pesticides/heavy metals and
simultaneously resistant to antibiotics.
The goal of this study was to determine real-life impact of chloroacetanilide herbicide Smetolachlor
on antibiotic resistance gene (ARG) abundance in herbicide-treated and
untreated soils.
The soil from experimental fields sown with maize was sampled 1 week before and 4
weeks after spring spraying. The herbicide content in the soil was determined by LC-MS.
Total metagenomic DNA was isolated from the treated and control samples and shortread
shotgun sequencing was performed on the DNBSEQ-G400 platform (MGI). After the
bioinformatics processing of sequences assembled contigs were checked against the
CARD database with ResFinder/RGI.
Preliminary analyses suggest notable differences in ARGs distribution before and after
herbicide treatment, as well as between treated and control plots. These differences
likely reflect the mobility and persistence of the herbicide under real-life agricultural
conditions.
Our study highlights the potential of herbicide to influence soil microbiome by disrupting
microbial community’s structure and potentially promoting antibiotic resistance and
selecting resistant genes. This interaction raises concerns about the widespread use of
herbicides in agriculture, as it may undermine antibiotic effectiveness, posing a threat to
public health and global efforts to combat resistance.Book of abstract: FEMS MICRO 2025: Congress & Exhibition, taking place in Milan, Italy, from 14-17 July 2025
Development and characterization of a temozolomide-resistant human glioblastoma cell line
No full textGlioblastoma is the most lethal form of brain cancer, with a median survival of only 14 months.
The poor prognosis is largely associated with development of resistance to temozolomide (TMZ),
the first-line chemotherapy and standard of care for glioblastoma patients. Therefore, reliable
preclinical models of TMZ resistance are essential for developing strategies translatable to clinical
practice. To develop a clinically relevant in vitro resistance model reflecting the cyclic TMZ
dosing used in the Stupp protocol (5-day treatment/3-week break), we adapted a culturecompatible
regimen. We exposed the TMZ-sensitive A-172 human glioblastoma cell line to 10 μM
TMZ for two 3-day treatment cycles, each followed by a 3-week recovery. We assessed TMZ
sensitivity in parental and treated cells using the MTT assay after one, two, and three days of TMZ
exposure. Additionally, we performed wound healing and EGFR immunostaining assays.
Compared to the parental line, IC₅₀ values increased progressively: from 136.2 to 1582 μM (single
treatment), 60.1 to 377.2 μM (double treatment), and 32.74 to 206 μM (triple treatment), indicating
a gradual resistance development. In the wound healing assay, the resistant line showed slower
gap closure: after 72 h, the sensitive line nearly closed the wound, while the resistant line exhibited
2-fold slower closure kinetics. EGFR immunostaining indicated a slight increase in expression in
resistant cells. The cyclic TMZ regimen adapted for in vitro use successfully induced resistance in
A-172 cells, generating a cell line with reduced migratory capacity and slightly elevated EGFR
expression.BeCELS 2025: Belgrade Conference for Early-Career Life Scientists, taking place on Friday, September 5, 2025, at the Institute of Molecular Genetics and Genetic Engineering (IMGGE) in Belgrad
Characterisation of T3SS-positive non-pathogenic Pseudomonas strains: plant growth promotion, induced systemic resistance in sugar beet (Beta vulgaris L.) and effector repertoires
No full textThe intricate interplay between plants and microorganisms in agricultural ecosystems offers significant opportunities to improve crop productivity and resilience in the context of climate change and increasing pathogen challenges. This study addresses the functional dynamics of the type 3 secretion system (T3SS) of non-pathogenic Pseudomonas strains associated with sugar beet (Beta vulgaris L.). From the collection consisted of plant-beneficial Pseudomonas strains isolated from sugar beet T3SS-positive isolates were identified by detecting conserved hrcRST genes of the T3SS operon. Genetic diversity and T3SS expression profiles were characterized. The plant growth-promoting activity and induction of systemic resistance were tested on sugar beet of the variety Heston (Maribo, Denmark). The effector repertoires were predicted using Effectidor machine learning model. Our results show that T3SS is widely distributed among sugar beet - associated Pseudomonas, with different T3SS gene sequences and expression patterns. In planta experiments showed that isolate P. marginalis OL141 had the central role in stimulating sugar beet growth, while the P. marginalis Orh26 improved resistance to Pseudomonas syringae pv. aptata infection. Customised bioinformatical pipeline resulted in the detection of effector proteins specific to non-pathogenic Pseudomonas strains and showed that the majority of strains had less than 10 effectors and that this number is lower than in pathogenic strains, where the number could reach 30. Future research aims to explore the precise mechanisms that control T3SS-mediated interactions between plants and microbes. This will contribute to the understanding and development of a wide range of implications for sustainable agriculture and global food security.BeCELS 2025: Belgrade Conference for Early-Career Life Scientists, taking place on Friday, September 5, 2025, at the Institute of Molecular Genetics and Genetic Engineering (IMGGE) in Belgrad
Designing synthetic bacterial consortia from tomato seed endophytes for improved drought resilience and growth promotion
No full textBacteria naturally exist in complex communities shaped by antagonistic, synergistic, and
competitive interactions, influenced by both internal and external factors. Understanding these
dynamics is essential for designing stable synthetic consortia for biofertilizers, as their
performance depends on microbial stability. Tailored consortia offer a promising strategy for
enhancing drought resilience and plant growth by integrating complementary plant growthpromoting
traits such as stress tolerance, phytohormone production, and nutrient mobilization.
This study investigates the engineering of tomato-seed endophytic bacterial consortia, aiming to
develop a novel bioinoculant for improving drought tolerance in tomato, sugar beet, and pepper
through synergistic plant-microbe interactions. From 91 initial strains, six were selected based on
their ability to promote tomato seed germination and seedling growth, excluding one due to
antagonism. Their biocontrol potential against plant pathogens and mutual antagonistic
interactions were assessed, leading to the assembly of four synthetic consortia, O3, M1, M2, and
M3, combining complementary drought-alleviating traits. Their growth dynamics were monitored
over a 48-hour coculture period. Finally, the effect of each consortium on tomato seed germination
was analyzed. The selected strains demonstrated biocontrol potential against seven species of plant
pathogens. Coculture experiments with the four consortia showed stable coexistence of the strains
over 48 hours, with O3 and M1 notably promoting tomato seed germination. Additionally, we
employed MIMIC, a computational tool for modeling bacterial community dynamics, to validate
these synthetic consortia for plant treatment. Future work will assess the impact of these consortia
on drought resilience in planta.BeCELS 2025: Belgrade Conference for Early-Career Life Scientists, taking place on Friday, September 5, 2025, at the Institute of Molecular Genetics and Genetic Engineering (IMGGE) in Belgrad
Investigation of novel clusters of bacterial head-to-tail cyclized peptides from the Bacillus pumilus genome
No full textBacterial headtotail cyclized peptides are a group of ribosomally synthesized and post -
translationally modified peptides (RiPPs) of relatively large size. During maturation, the peptide
undergoes cyclization, forming a peptide bond between its Cterminus and Nterminus. Compared
to other cyclic RiPPs bacterial cyclized peptides are distinguished by their larger cyclic structure
and likely by their cyclization mechanism. The sequences of these peptides are highly diverse, and
are not similar to each other. However, they share key properties: remarkable resistance to high
temperatures, proteases, and pH fluctuations. These features make them promising candidates for
applications in food preservation and medicine, for example, as antimicrobial agents. All these
peptides are hydrophobic, and their mechanism of action is thought to involve disrupting the target
organism’s membrane. It is believed that they form pores in the membrane, altering ion
permeability and ultimately leading to cell death. However, information on their mode of action
remains scarce, and the mechanisms for specific representat ives of this group are not fully
understood. Recently, our research team identified two novel clusters of bacterial headtotail
cyclized peptides in the genome of Bacillus pumilus. The clusters differ from each other in both
the primary sequence of the precursor peptide and the number of genes within the cluster.
Moreover, one of the clusters contains two precursor peptides, which is highly unusual for a cluster
of this type. We tested the antibacterial activity of this strain against a range of Gram positive and
Gram negative bacteria, observing zones of growth inhibition likely linked to the production of
cyclized peptides. To further investigate their antibacterial activity, functions of genes, and
cyclization mechanism, we constructed a recombinant shuttle vector based on the pHT01 plasmid
for heterologous expression of these clusters in another Bacillus strain.BeCELS 2025: Belgrade Conference for Early-Career Life Scientists, taking place on Friday, September 5, 2025, at the Institute of Molecular Genetics and Genetic Engineering (IMGGE) in Belgrad