imagine (Institute of molecular genetics and genetic engineering)
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The Role of SOX2 and SOX9 Transcription Factors in the Reactivation-Related Functional Properties of NT2/D1-Derived Astrocytes
Get PDFAstrocytes are the main homeostatic cells in the central nervous system, with the unique ability to transform from quiescent into a reactive state in response to pathological conditions by reacquiring some precursor properties. This process is known as reactive astrogliosis, a compensatory response that mediates tissue damage and recovery. Although it is well known that SOX transcription factors drive the expression of phenotype-specific genetic programs during neurodevelopment, their roles in mature astrocytes have not been studied extensively. We focused on the transcription factors SOX2 and SOX9, shown to be re-expressed in reactive astrocytes, in order to study the reactivation-related functional properties of astrocytes mediated by those proteins. We performed an initial screening of SOX2 and SOX9 expression after sensorimotor cortex ablation injury in rats and conducted gain-of-function studies in vitro using astrocytes derived from the human NT2/D1 cell line. Our results revealed the direct involvement of SOX2 in the reacquisition of proliferation in mature NT2/D1-derived astrocytes, while SOX9 overexpression increased migratory potential and glutamate uptake in these cells. Our results imply that modulation of SOX gene expression may change the functional properties of astrocytes, which holds promise for the discovery of potential therapeutic targets in the development of novel strategies for tissue regeneration and recovery
DEGRADATION OF POLYAMIDE/POLYURETHANE TEXTILE BLEND BY STREPTOMYCES SP. R1
Get PDFThe increasing production and utilization of
synthetic polymers in the textile industry over
the past five decades has raised concerns about
the environmental impact of the industry. The
recalcitrant nature of synthetic fibers hampers
the biodegradation of these textiles in the environment
and leads to the accumulation of textile
waste. Effective solutions for recycling and proper
disposal of textile waste are lacking, however,
the use of microorganisms and enzymes has
emerged as a promising approach. The genus
Streptomyces has been well studied as a producer
of different hydrolytic enzymes, several of which
have found use in industrial settings as well. As
an integral part of the soil microbiome, Streptomyces
species have been shown to interact with
different textile materials in soil and may play a
role in the degradation of these materials. This
study aimed to examine the interaction of Streptomyces
sp. R1, isolated from the rhizosphere of
Cotinus coggygria, with polyamide/polyurethane
textile, and identify potential enzymes involved in the biodegradation of synthetic textiles. The
degradation of the textile was tested in liquid
cultures (minimal salt medium) and model compost,
bio-augmented with Streptomyces sp. R1
for 4 months. After the incubation, morphological,
and changes in the functional groups of the
textiles were analysed using scanning electron
microscopy (SEM) and Fourier transform infrared
spectroscopy (FTIR). The surface of the textile
showed noticeable cracks and fissures after
4 months of burial in the bioaugmented model
compost, alongside changes in the functional
groups of the polyamide/polyurethane textile,
which indicates biodegradation of the synthetic
fibers. Searching the genome of Streptomyces sp.
R1, several enzymes involved in the degradation
of synthetic polymers were identified, including
an esterase homologous to highly efficient plastic
degrading depolymerases. Overall, the results
presented here indicate Streptomyces sp. R1 has
the potential for synthetic textile degradation
and bioremediation.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
THE USE OF INTEGRATIVE MULTI-OMICS APPROACH IN CULTIVATION AND CHARACTERIZATION OF GUT BACTERIA RELATED TO MICROBIOTA-GUT-BRAIN AXIS AS A SOURCE FOR NEXT GENERATION PROBIOTICS
Get PDFThere has been an epidemic of various non-communicable
degenerative and autoimmune diseases,
strongly associated with the modern
lifestyle. Among them, neurodegenerative and
psychiatric disorders represent a huge burden on
society. Recently, all these diseases have been associated
with the gut microbiota dysbiosis. Gut
microbiota-host interaction research has been
greatly improved due to development of molecular
high-throughput techniques based on
various ‘omics’ techniques coupled with bioinformatics
and data science developments. However,
the mechanisms of the host–microbiota crosstalk
are still poorly understood. The NextGenBiotics
project proposes an innovative integrative
multi-omics research strategy for deciphering
the mechanism behind the cross-talk among
microbiota and gut-brain-axis. The 118 novel
NGPs candidates belonging to Dorea sp., Blautia
sp., Bacteroides sp., Roseburia sp., Sellimonas
sp., Faecalicatena sp., Phascolarctobacterium faecium,
and Faecalimonas sp. were cultivated. The
25 NGPs with confirmed safe status and potential
probiotic potential were screened in C. elegans
model for their effects on behavioural and neuronal
activity. The most prominent candidates
with ability to upregulate expression of genes
involved in neurotransmiting are further tested
in EAE (an animal model for MS) and CUMS depression
model. The specific microbiota-derived
metabolites have been identified as potential
neuro- and psycho-biotics. The NextGenBiotics is
highly ambitious project, dedicated to pioneering
work in the field of multi-omics studies related
to the cultivation of novel anaerobic NGPs
and the studying of their effect on MGBA. This
concept enabled studying bidirectional communication
between gut microbiota and brain
on the functional level that will significantly
contribute to the growing body data related to
MGBA. The results obtained during NextGenBiotics
determined the genes/metabolites and the
associated mechanisms involved in health-promoting
effects of NGPs in MGBA beyond stateof-
the-art, broadening the scientific knowledge
and opening up the possible novel therapeutic
approaches in prevention and therapy of neurodegenerative
and psychiatric diseases.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
BACTERIOPHAGES OF MULTIDRUG-RESISTANT NOSOCOMIAL PATHOGENS – BELGRADE EXPERIENCE
Get PDFAntimicrobial resistance (AMR) arises when
bacteria and other microbes stop responding
to medications. AMR is now recognized as one
of serious global health threats, repeatedly
appearing in the World Health Organization’s
(WHO) lists of urgent global health challenges,
including the 2024 list. It is taking a fatal toll
– nearly 5 million deaths globally per year are
associated with AMR, encompassing 1.27 million
directly attributed to AMR. The COVID-19
pandemic paved the way for aggravation of
bacterial AMR – primarily due to enhancement
in unspecific and unjustified prescription and
use of broad-spectrum antibiotics, resulting in
what is now recognized as „silent pandemic of
AMR“. Bacteriophages (phages) are natural and
specific predators of bacteria - viruses that can
infect, replicate inside and lyse arguably any
bacteria. Their therapeutic potential is being
hastily evaluated through different approaches:
in silico, in vitro, ex vivo and in vivo – in laboratory
animals as well as in human case and clinical
studies. Although the results are promising,bacteria rapidly develop resistance against
phages, which why the isolation and research
of new phages is needed. Our work is concentrated
on three bacterial species for which critical
priority by WHO has been declared – carbapenem-
resistant Acinetobacter baumannii,
Pseudomonas aeruginosa and Klebsiella pneumoniae.
Twenty distinct pathogenic strains of
A. baumannii, 6 K. pneumoniae and 6 P. aeruginosa
were used as targets for bacteriophage
isolation, and total of 14, 22 and 8 potentially
distinct phages were collected, respectively. All
strains were nosocomial isolates obtained from
various tissues, including from terminally ill patients.
Six phages were characterized in detail.
In particular, phage vB_AbaM_ISTD was applied
against A. baumannii in zebrafish embryo
model of systemic infection, and demonstrated
powerful therapeutic potential, eradicating the
infection. Interestingly, its DNA was characterized
with highly modified thymidine (amassing
1228 Da), making it the largest non-canonical
deoxynucleoside reported so far.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
HERBAL PRODUCTS AS AN ALTERNATIVE TO ANTIBIOTICS: APPLICATION POSSIBILITIES AND LIMITATIONS
Get PDFAntimicrobial resistance (AMR) has developed as
one of the top 10 global public health threats
facing humanity. As the nosocomial bacterial
strains are being increasingly resistant to most
clinically available antibiotics, there is a constant
need for exploration of new substances
that could kill them or inhibit their growth, or
alternatively inhibit some of their essential virulence
factors to counteract the lack of new antibacterials
and the rise of antibiotic resistance,
plants could represent a potential solution.
Plants produce a variety of bioactive secondary
metabolites that could be used to fuel the future
discovery pipeline. Aim of the present study was
to examine inhibitory activity of the supercritical
extract of J. communis L. green pseudofructus
(7SCO2) against the growth, biofilm production
and several virulence factors of significant nosocomial
bacterial pathogens. The extract was
obtained by fractional extraction with supercritical
CO2, and the qualitative and quantitative
analysis was performed using the GC-FID/MS
method. Clinical isolates of Pseudomonas aeruginosa,Acinetobacter baumannii, Staphylococcus
aureus (methicillin-sensitive-MSSA and methicillin-
resistant - MRSA), Enterococcus faecalis, and
Klebsiella pneumoniae, as well as their antibiotic
resistance profiles, were obtained from the Clinical
Hospital Centre “Dr Dragiša Mišović Dedinje”.
Minimum inhibitory concentrations (MICs) of
the 7SCO2 were determined by broth-microdilution
method. Examination of the anti-adhesive
effect of the extract was carried out using the
spectrophotometric method. The pyocyanin
production of Pseudomonas aeruginosa was determined
by the method described by Rampioni
et al. Most significant findings of this study
are potent antivirulence activity of the 7SCO2
against P. aeruginosa through the inhibition of
pyocyanin production. In addition, the biofilm
production of A. baumannii was inhibited by the
7SCO2 in concentration 50 μg/mL. Finally, notable
antivirulence activity of the 7SCO2 against
E. faecalis and S. aureus was detected, since it
significantly inhibited collagen and laminin adhesion
of these pathogens.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
Plastic waste up-cycling potential of Streptomyces spp.: a genomic examination
Get PDFThe accumulation of plastic waste has become an ever-growing global problem, with world
production of plastic materials reaching >380 million tons annually and only predicted to
increase in the coming years. An efficient means of disposing and recycling plastic waste is
urgently needed. Due to environmental risk factors and high energy consumption of
mechanical and chemical recycling methods research focus has shifted towards biological
means of recycling. Biocatalysis offers an environmentally friendly and potentially very
efficient strategy for plastic waste degradation and valorization by utilizing the reaction
products in downstream biosynthetic reactions (up-cycling) (1).
Streptomyces spp. are highly regarded as bioactive secondary metabolite producers,
however, the genus proved a promising source of industrially relevant enzymes as well (2).
Leveraging this unique combination of biosynthetic and biocatalytic capabilities a collection
of Streptomyces strains was screened for their plastic-degrading potential using different
polyester-based polymers. Strains that could degrade and utilize plastic polymers and
monomers as the sole carbon source were sequenced and the genomes searched for
homologs of known plastic-degrading enzymes and biosynthetic clusters for bioactive
compounds. Enzymes capable of degrading both conventional petrochemical and bioplastics
were detected in the genomes of all tested strains. Interestingly, enzymes closely related to
highly active poly(ethylene terephthalate) degrading enzymes were found in most strains. As
expected, analysis of the biosynthetic potential yielded numerous gene clusters associated
with polyketide, non-ribosomal peptide and lassopeptide synthesis. Finally, the ability to
convert plastics to biologically active metabolites was confirmed using Streptomyces sp.
PM1. When grown on polyurethanes as the sole carbon source this strain showed
antimicrobial activity against Staphylococcus aureus.
In conclusion, this work highlights the potential of Streptomyces strains to biotransform and
up-cycle a variety of plastics into bioactive molecules while underlying mechanisms can be
elucidated by genome mining.Book of abstracts: 6th Symposium on Biotransformations for Pharmaceutical and Cosmetic Industry June 17-21, 2024, Kraków, Polan
On the prediction of protein dynamics: should one be optimistic?
No full textProtein dynamics are key to protein functions, with action modes ranging from subtle motions
impacting thermodynamics, to large amplitude conformational changes involved in complex
multi-body mechanisms. While the prediction of (well) folded structures may be taken as
an achievement in the deep learning era with Alphafold2 and the like, predicting dynamics
essentially remains an open problem.
This talk will review recent work in this realm, based on novel insights on loop closure
techniques coupling kinematic models in high dimensional dihedral angle spaces, and Monte
Carlo Markov Chain sampling techniques of the Hit-and-Run type. Along the way, I will discuss
connexions with other problems, including high dimensional volumes and densities of states,
as well as mixture models in flat tori to capture couplings between torsion angles.
These ingredients will make us ponder on the opportunity to be optimistic regarding the
accurate and fast prediction of protein dynamics.Book of abstracts: 5th Belgrade Bioinformatics Conference, Serbia, Belgrade,17-20 june 2024
Analysis of unlikely (and rare) local protein conformations
No full textThree-dimensional (3D) protein structures underpin the biological functions that are
essential to life. Access to this 3D information is of great interest for both basic and applied
research. Traditionally, structures are analyzed by assigning secondary structures (helices,
sheets and loops). However, this description does not allow loops to be properly described
and does not provide accurate details of the fine structure of repetitive structures.
As a result, more systematic approaches to describing 3D structures have been developed,
known as Structural Alphabets (SA). Within this framework, Protein Blocks (PBs) is the SA
that has had the most success and application. The 16 PBs, named from PB a to PB p, are
pentapeptides that can finely approximate the entire 3D structure. They have a strong
sequence-structure relationship and form a grammar in which certain PBs preferentially
follows a PB. There are so highly preferential transitions. Some PBs are strongly directed
to two or three PBs. However, there are also rare but present transitions, i.e. present with
a frequency less than 1%.
The work carried out here involved analyzing data from the Protein Data Bank to see which
transitions are very common and which are rare. Secondly, the amino acid frequencies of
the PBs involved in these rare transitions were compared with the frequencies classically
expected to answer this simple question: Are these rare, and therefore unexpected,
transitions linked to different amino acid compositions to those observed in PBs in
general. Finally, a similar analysis was carried out using AlphaFold2 models of the human
proteome. This work highlights the specific behavior of a number of PBs and amino acids.Book of abstracts: 5th Belgrade Bioinformatics Conference, Serbia, Belgrade,17-20 june 2024
PHARMACOGENOMICS AS A BASIS FOR PERSONALIZED MEDICINE
No full textEven with the same diagnosis, for many diseases, treatment should not be the same for each
patient. A need for optimizing therapies based on patient’s unique clinical features and genetic
background is recognized in almost every patient. Pharmacogenomics is a basis for personalized
medicine. It studies the human response to drugs determined by the unique DNA signature in
the genes responsible for the metabolism of a particular drug. Knowing pharmacogenomics
markers before the therapy administration could help apply therapy protocols that fit to
individual patients according to their genetic background. In that way patients receive adequate
therapy (the right drug, the right dose at the right time) with optimal management of the drug
efficacy and avoidance of adverse drug reactions. Population pharmacogenomics research has
pointed out that pharmacogenomics markers are population-specific, with frequencies varying
across different ethnic groups. Guidelines for the use of pharmacogenomics tests, the
interpretation of results and drug dosage recommendations according to pharmacogenomics
marker identified in a patient, are issued, curated and updated by relevant agencies and
consortia. Awareness about the importance of the application of pharmacogenomics testing is
rising in the scientific community as well as in the general population. The immense
development and application of new generation sequencing technologies has opened up the
possibility for genome-scale research in pharmacogenomics filed and speed up the translation
of knowledge and its implementation from bed to bedside. With the support of bioinformatics
and artificial intelligence tools a door for using pharmacogenomics in personalized medicine
are wide open.VII Congress of the Serbian Genetic Society Zlatibor; October 2 to 5, 2024
ESTABLISHMENT OF A MODEL SYSTEM FOR STUDYING NEURODEVELOPMENTAL DISORDERS USING INDUCED PLURIPOTENT STEM CELLS DERIVED FROM PATIENTS WITH 22Q11.2 DELETION SYNDROME
No full text22q11.2 Deletion Syndrome is the most common microdeletion syndrome in humans. It is
associated with elevated risk for neurodevelopmental psychiatric disorders and thus represents
a powerful genetics-first approach to delineate molecular mechanisms underlying these
disorders. Although many animal models mimic human diseases, only limited success has been
achieved in revealing molecular mechanisms underlying human brain diseases. Our goal was to
establish patient-specific induced pluripotent stem cells (iPSCs) since they represent a powerful
tool for establishing in vitro models of disorders.
Peripheral blood mononuclear cells of control subjects and patients with 22q11.2 microdeletion
were reprogrammed using CytoTune™-iPS 2.0 Sendai Reprogramming Kit. Generated iPSC
cell lines were characterized by analyzing their morphology, pluripotency, genomic integrity
and the ability to differentiate into three germ layers. iPSCs were differentiated into neural
progenitor cells and neurons using Dual-SMAD inhibition method and 3D cerebral organoids
in order to analyze neural differentiation in patient-specific background. RNA sequencing was
performed to determine differentially expressed gene sets and dysregulated pathways in neural
cells derived from patients with 22q11.2 microdeletion.
We successfully generated iPSC lines from patients with 22q11.2 microdeletion and healthy
individuals, characterized them and differentiated into neural progenitors, neurons and cerebral
organoids. List of differentially expressed genes was determined. Generated patient-specific
iPSCs represent a powerful model system for studying molecular mechanisms underlying
NDDs.VII Congress of the Serbian Genetic Society Zlatibor; October 2 to 5, 2024