imagine (Institute of molecular genetics and genetic engineering)
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Exploring the substrate spectrum of phylogenetically distinct bacterial polyesterases
Get PDFThe rapid escalation of plastic waste accumulation presents a significant threat of the modern world, demanding an immediate solution. Over the last years, utilization of the enzymatic machinery of various microorganisms has emerged as an environmentally friendly asset in tackling this pressing global challenge. Thus, various hydrolases have been demonstrated to effectively degrade polyesters. Plastic waste streams often consist of a variety of different polyesters, as impurities, mainly due to wrong disposal practices, rendering recycling process challenging. The elucidation of the selective degradation of polyesters by hydrolases could offer a proper solution to this problem, enhancing the recyclability performance. Towards this, our study focused on the investigation of four bacterial polyesterases, including DaPUase, IsPETase, PfPHOase, and Se1JFR, a novel PETase-like lipase. The enzymes, which were biochemically characterized and structurally analyzed, demonstrated degradation ability of synthetic plastics. While a consistent pattern of polyesters’ degradation was observed across all enzymes, Se1JFR stood out in the degradation of PBS, PLA, and polyether PU. Additionally, it exhibited comparable results to IsPETase, a benchmark mesophilic PETase, in the degradation of PCL and semi-crystalline PET. Our results point out the wide substrate spectrum of bacterial hydrolases and underscore the significant potential of PETase-like enzymes in polyesters degradation
GLYCOSIDE HYDROLASES FROM FRESHWATER FISH GILL MICROBIOTA AS BIOFILM INHIBITORS FOR ENHANCED FOOD SAFETY
Get PDFThe formation of biofilms by foodborne pathogens
is a constant challenge in the food industry,
leading to an increased risk of contamination and
compromising food safety. Many of the chemicals
commonly used for sanitation in the food industry
are unable to remove biofilms, are harmful
to surfaces and can be toxic. The effectiveness
of disinfectants can be improved using enzymes
that specifically target biofilm components such
as exopolysaccharides, extracellular DNA, or proteins.
In this study we investigated the potential
of glycoside hydrolases originating from the
gill microbiota of freshwater fish to control biofilm
formation in the most common foodborne
pathogens. We demonstrated that β-glucosidase
from Microbacterium sp. BG28 (BglB-BG28) effectively
inhibits cellulose-rich biofilms formed by
Salmonella enteritidis, S. typhimurium, S. infantis,
and Escherichia coli. When these bacteria were cultivated overnight with 200 μL/mL enzyme, up
to 80% less biofilm was formed. By fluorescence
microscopy, we visualised the inhibition of biofilms
on plastic, glass and aluminium, materials
commonly used in the food industry. When used
as a pre-treatment, BglB-BG28 increased the
bactericidal efficacy of Oxicid®S, a commercially
available surface disinfectant. Its effectiveness at
temperatures up to 50 °C and in a pH range from
4 to 8 together with compatibility with non-ionic
detergents and high tolerance to sodium chloride
and glucose give BglB-BG28 advantages in
harsh and diverse industrial environments. Importantly,
no toxicity to Caenorhabditis elegans
was observed at enzyme concentrations of up
to 1 mg/ml. Overall, these results demonstrate
the suitability of the β-glucosidase BglB-BG28 for
the formulation of a novel enzyme-based disinfectant
to be used in food processing facilities.Book of abstract: From biotechnology to human and planetary health XIII congress of microbiologists of Serbia with international participation Mikromed regio 5, ums series 24: 4th – 6th april 2024, Mona Plaza hotel, Belgrade, Serbi
CHEMICAL COMPOSITION AND QUORUM SENSING INHIBITION ACTIVITY OF HORSERADISH (ARMORACIA RUSTICANA) ROOT EXTRACTS
Get PDFDuring the past decades several quorum sensing
inhibitors (QSI) of plant origin have been isolated
and chemically characterized. QSI agents
of plant origin represent potential alternative
or complementary approach to antibiotic treatment
of multidrug-resistant bacteria and infections
caused by bacterial biofilms. The aim of
the current study was to screen QSI activities
of horseradish root extracts obtained using
different organic solvents and different root
processing methods (drying at 40°C, 60°C or extraction
of fresh material). Common opportunistic
pathogen Pseudomonas aeruginosa MMA83
was used for QSI screen. RT-qPCR was used to
analyze the effect of the extract on the relative
mRNA levels of the genes QS (lasR, lasI, rhlR, rhlI,
mvfR, pqsH) and the genes involved in P. aeruginosa
MMA83 virulence (lasB, phzM, rhlC, algK,
pvdS). Chemical composition of extracts was
determined by UHPLC Q-ToF MS analysis. The
most active extract obtained using fresh roots
and hexane/ethyl acetate (1:1) solvent mixture
was able to significantly reduce content all examined
mRNA. Qualitative chemical analysis
reviled presence of 15 phenolic acids and their
derivatives, 9 flavonoids and 10 glucosinolates
in majority of examined extracts. It is significant
to emphasize that the most active QSI extract
did not contain a single one, out of ten dominant
glucosinolates, which have undergone to
hydrolysis yielding isothiocyanates and other
sulphur-containing compounds responsible for
QSI effects. Our results strongly indicate that
even mild thermal treatment (40°C) of horseradish
roots prior to extraction could lead to severe
reduction or loss of QSI activity.Book of abstract: From biotechnology to human and planetary health XIII congress of microbiologists of Serbia with international participation Mikromed regio 5, ums series 24: 4th – 6th april 2024, Mona Plaza hotel, Belgrade, Serbi
Antimicrobial Rhenium Tricarbonyl Complexes: Accelerating their Discovery by Leveraging Machine Learning Models
No full textThe expanded prevalence of resistant bacteria and the inherent challenges of complicated
infections highlight the urgent need to develop alternative antibiotic options. Through
conventional screening approaches, the discovery of new antibiotics has proven to be
challenging. Anti-infective drugs, including antibacterials, antivirals, antifungals, and
antiparasitics, have become less effective due to the spread of drug resistance. In this work,
we helped define the design of next-generation antibiotic analogs based on metal complexes.
For this purpose, we used artificial intelligence (AI) methods, demonstrating superior ability
to tackle resistance in Gram-positive and Gram-negative bacteria, including multidrugresistant
strains. The existing AI approaches’ bottleneck relies on the current antibiotics’
structural similarities. Herein, we developed a machine learning approach that predicts the
minimum inhibitory concentration (MIC) of Re-complexes towards two S. aureus strains
(ATCC 43300 - MRSA and ATCC 25923 - MSSA). A Multi-layer Perceptron (MLP) was tailored
with the structural features of the Re-complexes to develop the prediction model. Although
our approach is demonstrated with a specific example of rhenium carbonyl complexes, the
predictive model can be readily adjusted to other candidate metal complexes. The work
shows the application of the developed approach in the de novo design of a metal-based
antibiotic with targeted activity against a challenging pathogen.Book of abstracts: 5th Belgrade Bioinformatics Conference, Serbia, Belgrade,17-20 june 2024
Multi-scale modeling uncovers 7q11.23 copy number variation-dependent alterations in ribosomal biogenesis, mTOR and neuronal maturation and excitability
No full textCopy number variation (CNV) at 7q11.23 causes Williams-Beuren (WBS) and 7q
microduplication syndrome (7Dup), neurodevelopmental disorders featuring intellectual
disability accompanied by symmetrically opposite neurocognitive features. Although
significant progress has been made in understanding the molecular mechanisms underlying
7q11.23-related pathophysiology, the propagation of CNV dosage across gene expression
layers and their interplay remains elusive. Here, we uncovered 7q11.23 dosage-dependent
symmetrically opposite dynamics in neuronal differentiation and intrinsic excitability. By
integrating transcriptomics, translatomics and proteomics of patient-derived and isogenic
induced neurons, we found that genes related to neuronal transmission follow 7q11.23
dosage and are transcriptionally controlled, while translational factors and ribosomal
genes are post-transcriptionally buffered. Consistently, we found phospho-RPS6 (pRPS6)
down-regulated in WBS and up-regulated in 7Dup. Surprisingly, phospho-4EBP (p4EBP)
was altered in the opposite direction reflecting dosage-specific changes in the total 4EBP
levels. This highlights both different dosage-sensitive deregulations of the mTOR pathway
as well as distinct roles of pRPS6 and p4EBP during neurogenesis. Our work demonstrates
the importance of multi-scale disease modeling across molecular and functional layers
and uncovers the pathophysiological relevance of ribosomal biogenesis in a paradigm pair
of complex neurodevelopmental disorders and uncouples the roles of pRPS6 and p4EBPs
as mechanistically actionable relays in neurodevelopmental disorders.Book of abstracts: 5th Belgrade Bioinformatics Conference, Serbia, Belgrade,17-20 june 2024
EFFECTS OF PROTEOLYTICALLY-ACTIVE LACTOBACILLI STRAINS ON SOURDOUGH STARTER FERMENTATION PROCESS
No full textGluten-related disorders have surged in recent years, underscoring the necessity for innovative
approaches to alleviate symptoms and enhance gluten digestion. In this study, we embarked on a
comprehensive exploration of proteolytic activity within a collection of Lactobacillus strains to
uncover their potential in gluten peptide hydrolysis and sourdough fermentation process. We
initiated our investigation by screening 120 Lactobacillus strains for proteolytic activity on gluten
peptides, with a goal of identifying the most potent candidates. Subsequent probiotic
characterization followed, focusing on antimicrobial capabilities against prevalent pathogens.
Safety characterization ensued, including testing for antibiotic resistance, ensuring the suitability of
selected strains for further investigation, ending in the refinement of our selection to 11 candidates.
To assess their applicability in sourdough fermentation, the selected strains were introduced into
khorasan wheat sourdough starters, and their impact on growth kinetics and pH modulation was
monitored. Among the selected strains, only one strain of Lactobacillus brevis (BGZLS30-24)
demonstrated a significant effect on the growth kinetics and pH reduction of the sourdough starter.
Additionally, protein isolation from the mature sourdough starter facilitated the evaluation of
proteolytic activity within the dough inoculated with the selected strain. While a detectable
proteolytic activity was observed, its magnitude appeared to be attenuated compared to the initial
screening test. Furthermore, metagenomic analysis of sourdough starters was conducted to gain
insights into microbial diversity dynamics during the maturation process, revealing that the addition
of the BGZLS30-24 significantly shortens the microbiota maturation time. Textural analysis of
sourdough breads was conducted to elucidate the impact of BGZLS30-24 on the final product,
revealing its contribution to increased bread volume and reduced hardness, indicating
improvements in textural properties. Our findings show the versatile role of selected Lactobacillus
brevis BGZLS30-24 in gluten digestion and sourdough fermentation, hinting at it’s potential as an
adjunct in developing novel strategies for managing gluten-related disorders and enhancing the
quality of bakery products.Book of Abstracts : The 3rd International UNIFood Conference, UNIFood2024 Conference, Belgrade, June 28-29, 202
INVESTIGATING THE GENETIC COMPLEXITY OF NEUTROPENIA IN PEDIATRIC PATIENTS WITH GLYCOGEN STORAGE DISEASE IB: A MODIFIER GENE PERSPECTIVE
No full textBackground: Glycogen Storage Disease Ib (GSD Ib) is a rare metabolic disorder characterized by
deficiency of the glucose-6-phosphate translocase (G6PT), leading to impaired glucose homeostasis. A
major characteristic of GSD Ib is neutropenia, accompanied by metabolic disturbances. The severity
and progression of neutropenia, along with neutrophil dysfunction, have been observed to show
variation among individuals sharing the same genotype. Despite GSD Ib being a monogenic disease,
extensive research and clinical experience have revealed that the relationship between genotype and
phenotype is not straightforward. This study aims to elucidate the role of potential modifier genes in
the context of neutropenia in GSD Ib, with a particular focus on five pediatric patients harboring the
pathogenic homozygous genetic variant c.1042_1043delCT in the SLC37A4 gene. Notably, this group
of patients exhibits a diverse course of neutropenia, with two patients manifesting mild and
intermittent neutropenia, while others experience severe and persistent neutropenia.
Methods: Whole genome sequencing was conducted on five subjects from unrelated nonconsanguineous
families, all presenting with previously identified pathogenic homozygous variant in
the SLC37A4 gene. Advanced bioinformatics tools were employed to analyze the genomic data and
explore potential associations between genetic variations and the observed clinical variations in
neutropenia. We performed parameterized variant filtering, pathogenicity score-based prioritization
(based on scores from pathogenicity scoring tools), and functional association to identify the modifier
variant(s) (based on functionally annotated ontologies and knowledgebase).
Results: Our study reveals a complex interplay of potential modifier genes in these subjects, providing
insights into the phenotypic heterogeneity observed in GSD Ib patients with the specific
c.1042_1043delCT variant. The distinct neutropenic courses, with two patients exhibiting mild and
intermittent neutropenia, and others with severe and persistent neutropenia, highlight the importance
of further investigation into the genetic factors influencing disease presentation.
Conclusion: This research underscores the potential significance of modifier genes, particularly within
the context of the identified pathogenic variant in the SLC37A4 gene, in shaping the diverse course of
neutropenia in GSD Ib. Understanding potential genetic modifiers can provide valuable insights into
the molecular base of the disease and guide future research focused on developing customized
therapeutic approaches for the specific neutropenic phenotype.32nd Meeting of the European Society of Paediatric Clinical Research (ESPCR) in Opatija, Croatia, May 24-25, 2024
Da li farmakogenetika ima uticaj na ishod lečenja odraslih pacijenata sa akutnom mijeloidnom leukemijom lečenih primenom citarabina i antraciklina? - srpsko iskustvo
No full textBackground: Cytarabine-anthracycline-based induction chemotherapy remains the standard of care for remission induction among patients with newly diagnosed acute myeloid leukaemia (AML). There are remarkable differences in therapy response among AML patients. This fact could be partly explained by the patients' genetic variability related to the metabolic paths of cytarabine and anthracyclines. This study aims to evaluate the effect of variants in pharmacogenes SLC29A1, DCK, ABCB1, GSTM1, and GSTT1, as well as laboratory and AML-related parameters on clinical outcomes in adult AML patients. Methods: A total of 100 AML patients were included in the study. Pharmacogenetic variants SLC29A1 rs9394992, DCK rs12648166, ABCB1 rs2032582, and GSTM1 and GSTT1 gene deletions were detected by methodology based on PCR, fragment analysis and direct sequencing. The methods of descriptive and analytic statistics were used. Survival analysis was done using the Kaplan-Meier method using the Log-Rank test. Results: This is the first study of adult AML pharmacogenetics in the Serbian population. Clinical outcomes in our cohort of AML patients were not impacted by analysed variants in SLC29A1, DCK, ABCB1 and GSTT1, and GSTM1 genes, independently or in combinations. Achievement of complete remission was identified as an independent prognostic indicator of clinical outcome. Conclusions: The population-specific genomic profile has to be considered in pharmacogenetics. Since the data on AML pharmacogenetics in European populations is limited, our results contribute to knowledge in this field and strongly indicate that a high-throughput approach must be applied to find particular pharmacogenetic markers of AML in the European population.Uvod: Indukciona terapija zasnovana na citarabinu i antraciklinu standard je lečenja novodijagnostikovanih odraslih pacijenata sa akutnom mijeloidnom leukemijom (AML). Ishodi lečenja među obolelima od AML značajno se razlikuju. Ove razlike bi se delimično mogle objasniti genetičkim varijabilitetom metaboličkih puteva citarabina i antraciklina. Cilj ovog istraživanja bilo je ispitivanje uticaja varijanti u farmakogenima SLC29A1, DCK, ABCB1, GSTM1 i GSTT1, kao i laboratorijskih i parametara vezanih za AML na ishode lečenja odraslih bolesnika sa AML. Metode: Ukupno 100 bolesnika sa AML je uključeno u studiju. Farmakogenetičke varijante SLC29A1 rs9394992, DCK rs12648166, ABCB1 rs2032582 i delecije gena GSTM1 i GSTT1 određivane su metodologijom zasnovanom na PCR-u, analizom fragmenata i direktnim sekvenciranjem. Korišćene su metode deskriptivne i analitičke statistike. Analiza preživljavanja je sprovedena prema Kaplan-Majerovom metodu upotrebom Log-Rank testa. Rezultati: Ovo je prva farmakogenetička studija odraslih bolesnika sa AML u srpskoj populaciji. Varijante u genima SLC29A1, DCK, ABCB1, GSTT1 i GSTM1 nisu uticale na ishode lečenja u našoj kohorti obolelih od AML, samostalno ili u međusobnim kombinacijama. Međutim, postizanje kompletne remisije bolesti istaklo se kao nezavisni prediktor ishoda lečenja. Zaključak: Prilikom farmakogenetičkih istraživanja neophodno je razmotriti jedinstveni genetički profil ispitivane populacije. Kako su farmakogenetički podaci o AML u evropskim populacijama oskudni, naši rezultati doprinose proširenju saznanja u ovoj oblasti i ukazuju na značaj primena tehnika sekvenciranja nove generacije u cilju otkrivanja posebnih farmakogenetičkih markera kod obolelih od AML u evropskim populacijama
Structural characterization and cytotoxicity of silver(I) complexes with N-methylphenothiazine
No full textIn the reaction of equimolar amounts of N-methylphenothiazine (N-Mephtz) and silver(I) salts,
(AgSbF6 for 1 and AgPF6 for 2) carried out in ethanol under reflux for 2 h, two mononuclear silver(I)
complexes were obtained, [Ag(N-Mephtz)4)]SbF6 (1) and [Ag(N-Mephtz)4]PF6 (2). The synthesized
complexes were structurally characterized by spectroscopic (IR, NMR, UV-Vis) methods, while their
crystal structure was determined by single-crystal X-ray diffraction analysis. In these complexes, four
N-Mephtz ligands are monodentately coordinated to the Ag(I) ion through the sulphur atom, forming
a catonic [Ag(N-Mephtz)4]+ species with [SbF6]– and [PF6]– acting as counter anions for 1 and 2,
respectively. The cytotoxicity of the silver(I) complexes was evaluated on human fibroblasts
(MRC5), human colorectal carcinoma (HCT116) and human lung carcinoma (A549) cell lines. The
interactions of complexes 1 and 2 with calf thymus DNA (ct-DNA) and bovine serum albumin (BSA)
were studied to evaluate their binding affinity toward these biomolecules. In this study, we have also
performed fluorescence competition experiments with site markers for BSA to locate the bindig site
of the investigated complexes to this protein.Book of abstract: 52nd Conference Synthesis and Analysis of Drugs (SAL 2024), Hradec Králové (Czech Republic
Assessing Transcriptomic Responses to Oxidative Stress: Contrasting Wild-Type Arabidopsis Seedlings with dss1(I) and dss1(V) Gene Knockout Mutants
No full textOxidative stress represents a critical facet of the array of abiotic stresses affecting crop growth and yield. In this paper, we investigated the potential differences in the functions of two highly homologous Arabidopsis DSS1 proteins in terms of maintaining genome integrity and response to oxidative stress. In the context of homologous recombination (HR), it was shown that overexpressing AtDSS1(I) using a functional complementation test increases the resistance of the Δdss1 mutant of Ustilago maydis to genotoxic agents. This indicates its conserved role in DNA repair via HR. To investigate the global transcriptome changes occurring in dss1 plant mutant lines, gene expression analysis was conducted using Illumina RNA sequencing technology. Individual RNA libraries were constructed from three total RNA samples isolated from dss1(I), dss1(V), and wild-type (WT) plants under hydrogen peroxide-induced stress. RNA-Seq data analysis and real-time PCR identification revealed major changes in gene expression between mutant lines and WT, while the dss1(I) and dss1(V) mutant lines exhibited analogous transcription profiles. The Kyoto Encyclopedia of Genes and Genomes enrichment analysis revealed significantly enriched metabolic pathways. Notably, genes associated with HR were upregulated in dss1 mutants compared to the WT. Otherwise, genes of the metabolic pathway responsible for the synthesis of secondary metabolites were downregulated in both dss1 mutant lines. These findings highlight the importance of understanding the molecular mechanisms of plant responses to oxidative stress