imagine (Institute of molecular genetics and genetic engineering)
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Mimicking the enzymatic plant cell wall hydrolysis mechanism for the degradation of polyethylene terephthalate
No full textPlastic pollution presents a global challenge, impacting ecosystems, wildlife, and economies. Polyethylene terephthalate (PET), widely used in products like bottles, significantly contributes to this issue due to poor waste collection. In recent years, there has been increasing interest in plant biomass-degrading enzymes for plastic breakdown, due to the structural and physicochemical similarities between natural and synthetic polymers. Filamentous fungi involved in hemicellulose degradation have developed a complex mode of action that includes not only enzymes but also biosurfactants; surface-active molecules that facilitate enzyme-substrate interactions. For this reason, this study aimed to mimic the mechanism of biomass degradation by repurposing plant cell wall degrading enzymes including a cutinase and three esterases to cooperatively contribute to PET degradation. Surfactants of different charge were also introduced in the reactions, as their role is similar to biosurfactants, altering the surface tension of the polymers and thus improving enzymes’ accessibility. Notably, Fusarium oxysporum cutinase combined with anionic surfactant exhibited a 2.3- and 1.6-fold higher efficacy in hydrolyzing amorphous and semi-crystalline PET, respectively. When cutinase was combined with either of two ferulic acid esterases, it resulted in complete conversion of PET intermediate products to TPA, increasing the overall product release up to 1.9– fold in presence of surfactant. The combination of cutinase with a glucuronoyl esterase demonstrated significant potential in plastic depolymerization, increasing degradation yields in semi-crystalline PET by up to 1.4-fold. The approach of incorporating enzyme cocktails and surfactants emerge as an efficient solution for PET degradation in mild reaction conditions, with potential applications in eco-friendly plastic waste management
Bacterial nanocellulose: From bacteria to breakthrough biomaterials
No full textRecent advances in natural resource exploration have driven the development of highperformance biomedical materials using green chemistry, in order to minimize adverse effects
on the environment. Bacterial nanocellulose (BNC), a sustainable material obtained from
bacteria, offers exceptional properties like high mechanical strength, crystallinity,
biodegradability, and tunable surface chemistry. Its biocompatibility makes it valuable in
medicine and pharmacy. In our study, BNC was utilized for controlled drug delivery of
actinomycin D. The structure of BNC was significantly modified by oxidation using TEMPO,
in order to adjust the release kinetics. Oxidized BNC showed improved chemical and physical
properties, and enhanced compound release control. In another project, we developed a twostep process to produce edible BNC, obtained from lignocellulose coming from wood waste,
functionalized by the adsorption of black raspberry extract using simple soaking. This approach
converts plant biomass as a highly abundant renewable resource is converted into high-valueadded sustainable product.Book of abstracts: The 3
rd Serbian Conference on Materials
Application and Technology; SCOM 2024, Belgrade, Serbia, 16
th
- 18st of October 202
The Effect of the Violacein-Doxorubicin Combination on Human iPSC-Derived Cardiomyocytes
No full textInvestigations of natural compounds with selective toxicity to tumor cells aim to improve the efficacy
of available therapy. Our prior research demonstrated the anti-tumor activity of the bacterial pigment
violacein on RD, HS-729, and SJRH30 cell lines, originating from rhabdomyosarcoma (RMS), the most
common soft tissue malignancy in children. Additionally, we observed a synergistic cytotoxic effect
when violacein was combined with doxorubicin, particularly on RD cells. However, doxorubicin, a
standard chemotherapeutic agent in RMS treatment, is known for its significant cardiotoxicity. Given
this, we investigated the effect of the violacein-doxorubicin combination on the viability and
contractile function of human-induced pluripotent stem cell (iPSC)-derived cardiomyocytes. The
cardiomyocytes were exposed to 1 μM doxorubicin in conjunction with the IC50 or IC25
concentration of violacein, previously determined for RD cell line. Violacein alone did not impact
cardiomyocyte viability. However, when combined with doxorubicin, a significant reduction in cell
viability was observed at 24 and 48 hours for the IC50 concentration, and at 48 hours for the IC25
concentration. Regarding contractility, violacein did not alter key parameters such as beating rate,
contraction duration, and relaxation duration. However, at the IC50 concentration, violacein reduced
contraction and relaxation velocities in two out of four iPSC cell lines examined. Importantly, the
combination of violacein with doxorubicin did not exacerbate the adverse effects of doxorubicin on
cardiomyocyte contractility. In conclusion, while the violacein-doxorubicin combination demonstrated
enhanced cytotoxicity against RMS cell lines, the cardiotoxic effects associated with doxorubicin
persisted, which does not speak in favor of violacein’s potential as a candidate for combination therapy
in RMS treatment.Book of abstract: “HDIR-7: Advances in Cancer Research &
Treatment”
The 7th Meeting of the Croatian Association for Cancer
Research with International Participation
November 7 & 8, 2024
Hotel International, Zagreb, Croati
Development of a Biomimetic Platform for 3D Cancer Cell Cultures
No full textIntroduction
Biomimetic systems for three-dimensional (3D) cell cultures are recognized as a possible tool to overcome
limitations of current preclinical methods for characterization of new drugs relying on studies in cell
monolayers (i.e. two-dimensional (2D) cell cultures) followed by in vivo studies on animals. In specific, due
to inherent limitations of the 2D cell cultures such as changed cell morphology, metabolism and responses to
drugs, the results of these studies often do not correspond being even contradictory to the results obtained in
studies in vivo, which is known as in vitro-in vivo gap. On the other hand, studies in animals are expensive,
complicated and ethically problematic as the relevance of the obtained results to humans is low. This problem
is especially recognized in development of anticancer drugs stressing the need for more reliable in vitro tumor
models. One of the approaches in this direction is tumor engineering based on the use of biomaterial scaffolds
as artificial extracellular matrices (ECM) and biomimetic bioreactors providing efficient mass transport so to
support longer term cell cultures. In the project BioengineeredTumor we aim to develop such systems using a
methodical, bottom-up approach starting from well defined cancer cell lines in alginate based scaffolds
cultured in perfusion bioreactors. By systematically defining the key parameters of these culture systems, the
goal is to build a sufficiently simple platform for the use by scientists without technical expertise but still
capturing some of the main tumor features. The envisioned applications are in cancer research and anticancer
drug screening with the prospect of extension to other cancer cell types and cells isolated from patients. Here
we showcase certain steps in development and optimization of two model systems, for cultures of carcinoma
and osteosarcoma cells.Book of proceedings: BIOMATERIALS AND NOVEL TECHNOLOGIES FOR
HEALTHCARE 4th Biennal International Conference BioMaH, October 15th-18th, 2024
Rome, Ital
Biocontrol potential of Streptomycetes isolated from three soil samples of various origin
No full textStreptomyces is the largest group of actinomycetes widespread in terrestrial ecosystems. Members of this genus are interesting because of their ability to produce wide-range of bioactive secondary metabolites. The aim of this work was to search for the isolates that could be used as biocontrol agents in tomato protection. The experimental plan was to isolate Streptomyces species from cultivated agricultural soil from open field and greenhouse, as well as from non-cultivated forest soil collected from a depth of 20 cm in the area of Smederevska Palanka. Based on their growth characteristics on Mannitol Soy Flour (MSF) agar media, 30 different Streptomyces spp. isolates were extracted based on morphological characteristics and 16S RNA sequence: 9 from the agricultural soil (7 from open field and 2 from greenhouse), and 21 from the non-agricultural (forest) soil. Biological activity of these isolates against tomato soil-borne pathogens - Fusarium oxysporum f. sp. lycopersici (FOL) and Ralstonia solanacearum (RS) was tested in vitro using dual cultivation method. In RS biocontrol experiment, agar plug of a 7-day-old streptomycete culture was placed on the center of Mueller Hinton agar plate preinoculated with RS nutrient broth culture. FOL biocontrol experiment was set up by streaking and growth of tested streptomyces isolate on one side of potato dextrose agar plate, while an agar plug of a 5-days-old FOL culture was placed on the opposite side of the medium. Each treatment was performed in three repetitions. The pathogen cultures on the corresponding media in Petri dishes were used as control treatments. Inhibition of RS growth was evaluated by measuring diameter (mm) of the zone of inhibition around the streptomyces disc after 24h of incubation, while the inhibition of FOL was shown in the percentage of growth inhibition after 7 days of incubation. From 30 investigated Streptomyces spp. isolates, 16 showed biological activity, 12 affected only one pathogen (6 on FOL and 6 on RS), and 4 showed an antagonistic effect on both pathogens. The obtained results were statistically processed by analysis of variance and tested with the LSD and Tukey test in the program IBM SPSS Statistics, version 26.0. All 16 biologically active isolates showed statistically significant activity compared to the control. Antagonistic reaction to FOL was shown by 10 tested isolates. The isolate BJO11 had the highest average percentage of growth inhibition - 26.67%, while BJO1 had the lowest - 17.43%. Other isolates had an average growth inhibition between 18.46% and 24.62%. Growth of RS was inhibited by 10 Streptomyces spp. isolates. The isolate BJS19b, caused the largest average diameter of the zone of inhibition - 29 mm, while the weakest biocontrol activity was shown by the isolate BJS23 - 10 mm. The other isolates inhibited RS growth from 11 to 19 mm. The results indicated that abundant population of Streptomyces spp. is present in our agricultural and especially in non-agricultural soils. These isolates vary in their biocontrol activity against FOL and RS. The active ones may have potential in control of the tomato soil borne pathogens
Improved N-phenylpyrrolamide inhibitors of DNA gyrase as antibacterial agents for high-priority bacterial strains
No full textIn this work, we describe an improved series of N-phenylpyrrolamide inhibitors that exhibit potent activity against DNA gyrase and are highly effective against high-priority gram-positive bacteria. The most potent compounds show low nanomolar IC50 values against Escherichia coli DNA gyrase, and in addition, compound 7c also inhibits E. coli topoisomerase IV in the nanomolar concentration range, making it a promising candidate for the development of potent dual inhibitors for these enzymes. All tested compounds show high selectivity towards the human isoform DNA topoisomerase IIα. Compounds 6a, 6d, 6e and 6f show MIC values between 0.031 and 0.0625 μg/mL against vancomycin-intermediate S. aureus (VISA) and Enterococcus faecalis strains. Compound 6g shows an inhibitory effect against the methicillin-resistant S. aureus strain (MRSA) with a MIC of 0.0625 μg/mL and against the E. faecalis strain with a MIC of 0.125 μg/mL. In a time-kill assay, compound 6d showed a dose-dependent bactericidal effect on the MRSA strain and achieved bactericidal activity at 8 × MIC after 8 h. The duration of the post-antibiotic effect (PAE) on the MRSA strain for compound 6d was 2 h, which corresponds to the PAE duration for ciprofloxacin. The compounds were not cytotoxic at effective concentrations, as determined in an MTS assay on the MCF-7 breast cancer cell line
Elucidating the role of cellular parabiosis in determining the preferential site of metastasis
No full textAlthough primary cancer can affect any part of the body, there is a striking variability
in cancer prevalence across different organs. Moreover, metastases are non-randomly
distributed among organs, with lung, liver, lymph nodes and bone acting as metastatic
hotspots. The reasons for such inter-organ variability in the prevalence of primary cancers
and metastases are insufficiently understood. We aim to understand the impact of cellular
parabiosis in carcinogenesis with specific focus on its role in determining the preferential
host tissue for metastasis. Our main hypothesis is that malignant phenotype can be
suppressed and delayed via the paradigm of cellular parabiosis as healthy surrounding
cells would complement metastatic cell and thus the „healthy homeostasis“ could be
maintained. To address this question we follow several research lines with aim to: 1)
identify intrinsic features within a healthy organ in the absence of cancer that would
predetermine such organ to become a primary cancer or metastatic host; 2) test the role
of the cellular environment in the metastatic nesting, specifically, we aim to predict the
preferential site of metastasis based on gene expression patterns; and 3) perform the most
comprehensive and up-to-date systematic review and meta-analysis on the prevalence
of metastatic sites from different primary cancers. We show that susceptibility of organs
to a primary cancer or a metastasis is linked with their distinctive intrinsic features in the
healthy state. In particular, while susceptibility of an organ to primary cancer is associated
with the abundance of endothelial cells and atypical gene expression, susceptibility to
metastases correlates with high content of immune cells and high expression of immune
genes. These data shed light on some fundamental aspects of cancer biology and pave
new avenues for mitigation of cancer.Book of abstracts: 5th Belgrade Bioinformatics Conference, Serbia, Belgrade,17-20 june 2024
A pipeline for the identification of disease-specific genetic biomarkers using NGS sequencing data of cfDNAs in human plasma
No full textCirculating cell-free DNAs are DNA fragments released into the blood by different tissues.
They can be isolated from a routine blood draw that is a cost-effective, fast and noninvasive
procedure. While their presence is detectable under healthy conditions, there
are solid evidences of their association with various clinical conditions. This explains
the great interest for cell-free DNAs in clinical settings, because of their potential role
as biomarkers. Furthermore, the availability of a higher number of samples and highthroughput
sequencing technologies have enabled the production of massive amounts
of complex genomic data. As consequence, the potentiality of cell-free DNAs can be
exploited only if supported by a computational platform that streamlines the execution
of the analysis and makes it reproducible and shareable across different platforms. The
proposed computational pipeline addresses the complexity of this analysis. The pipeline
starts with the raw data pre-processing necessary to remove residual adapters and filter
the low-quality reads. It follows the composition analysis where the average sample
composition is calculated and expressed in terms of specific target regions of human DNA.
Next, a random forest classifier algorithm searches for a subset of the target regions that
perform best at predicting the health outcome. The statistical significance of the output
is validated by the MANOVA test. Finally, the pipeline runs through the original set of
sequencing data to retrieve what sequences best match the composition represented by
the pool of target regions deriving from the previous step. The ultimate result is a list of
nucleotide sequences that have been identified as the best performing indicators of a
clinical condition based on the analysis described above. Thanks to the containerization
technology and workflows managers this pipeline can be shared and executed with the
same functionalities across different platforms, and its installation process is automated.Book of abstracts: 5th Belgrade Bioinformatics Conference, Serbia, Belgrade,17-20 june 2024
Analysis of AlphaFold2 Predicted Structures of Aggregation Factors in Lactic Acid Bacteria
No full textimportant for colonization, kin and kind recognition, and bacterial survival.
There is a group of aggregation factors in lactic acid bacteria that have several distinctive
features: these are large proteins with a molecular mass greater than 150 kDa, containing an
N-terminal signal sequence and an LPXTG-like cell wall anchor domain, as well as a different
number of repetitive domains. These aggregation-promoting proteins are also called/known
as Snow-flake Forming Collagen Binding Aggregation Factors (SFCBAF) due to their unique
aggregation phenotype (PMIDs: 22182285, 29018422, 25955159, 30027759, 38014957).
Predictions for different members of this group (predicted by the InterPro program) indicate
a varying number and, in some cases, different compositions of repetitive domains.
Comparison of the predicted structures of known aggregation factors (AggL from Lactococcus
lactis; AggE from Enterococcus faecium; AggLb from Lacticaseibacillus paracasei; AggLr from
Lactococcus raffinolactis; and AggA from Tetragenococcus halophilus) using AlphaFold2
revealed structural similarities, which may explain the similar phenotype despite low identity
(e.g., AggLb is identical to AggA at 21.73% and AggL at 41.14%, while AggA is identical to AggL
at 32.43%). The structure itself resembles a shoe-like structure: with a heel and a sole in
the form of a loop, consisting of 6-7 adhesion domains superfamily (InterPro: IPR008966).
The most structurally dissimilar among them is AggLb, which is the largest of the described
aggregation factors of this type and has a greater number of repeats in the second half
of the protein compared to other members of this group. The calculation of electrostatic
potential shows that the protein surfaces predominantly have negative potential, which is
consistent with previously shown data for the AggLb protein, where the strain producing
AggLb demonstrated higher affinities for chloroform and a lower percentage of adhesion
to ethyl acetate, indicating that AggLb is able to provide strong electron-donor and weaker
electron-acceptor features to the bacteria.
In summary, this research enhances our comprehension of the structure of aggregation
factors in lactic acid bacteria.Book of abstracts: 5th Belgrade Bioinformatics Conference, Serbia, Belgrade,17-20 june 2024
Efficient Large Scale Multimodal Image Registration
No full textMultimodal imaging refers to the capturing of complementary information about a
specimen by different imaging techniques (modalities). Such complementary information
allows reaching deeper understanding and improved analysis and diagnostics performance.
Multimodal imaging combined with correlated analysis of the acquired data can be very
useful for both human and AI-based decision making. For successful correlation and fusion
of the heterogeneous information, acquired images need to be accurately aligned – a task
which is far from easy, given the great diversity of imaging modalities and specimens,
combined with the typically very large size of medical and biomedical images.
In this work we present a computationally efficient method that reaches a state-ofart
performance for multimodal image registration. The method is based on computing
the cross-mutual information function (CMIF) through efficient evaluation of mutual
information in the Fourier domain for every possible discrete translation. Utilizing the
power of GPU-based processing and performing a search over a limited set of rotation
angles, the approach facilitates accurate rigid alignment at high speed.
We demonstrate how this approach can be used for improved deep learning-driven
oral cancer detection by practically enabling information fusion from different imaging
modalities on whole slide image data, overcoming problems originating from stitching
artefacts and microscope drift.Book of abstracts: 5th Belgrade Bioinformatics Conference, Serbia, Belgrade,17-20 june 2024