imagine (Institute of molecular genetics and genetic engineering)
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Green in situ synthesis of Ag- and Cu-based nanoparticles on viscose fabric using a Punica granatum peel extract
No full textThe demand for medical textiles in various forms with strong antimicrobial activity drastically increased during the COVID19 pandemic. In an attempt to tackle this issue and to develop antimicrobial textiles in more environmentally benign manner, a viscose fabric after coating with biopolymer chitosan has been impregnated with Ag- and Cu-based nanoparticles. Chitosan was applied in the presence and absence of cross-linker 1,2,3,4-butanetetracarboxylic acid (BTCA). In situ green synthesis of nanoparticles was performed using a Punica granatum (pomegranate) peel extract as a reducing and stabilizing agent. Formation of nanoparticles on the fiber surface was confirmed by FESEM. Elemental analysis by XPS showed the synthesized nanoparticles exist as AgCl and a mixture of Cu/CuO/Cu2O in the modified samples. Moreover, these nanoparticles appeared to be present not only on the sample surface but also buried within the fibers, as indicated by XPS mapping and depth profiling measurements. All impregnated fabrics exhibited excellent antifungal activity providing the maximum reduction of yeast Candida albicans colonies. Antibacterial activity was stronger against Gram-negative bacteria Escherichia coli than Gram-positive bacteria Staphylococcus aureus, and it was highly influenced by metal content. The fabrics impregnated with AgCl nanoparticles showed lower cytotoxicity towards human keratinocyte cells
Characterization of the molecular basis of the protective effect of the exopolysaccharides from lactobacilli in the DA rats mod el exposed to prolonged cadmium(ii) exposure
No full textKadmijum, široko rasprostranjen toksičan metal, izaziva brojne negativne zdravstvene efekte zbogčega je neophodno naći adekvatne načine zaštite od dejstva ovog metala. Poznato svojstvo nekihlaktobacila da interaguju sa metalima se sve češće pripisuje prisustvu egzopolisaharida (EPS) napovršini ćelije za koji je pokazano da može da formira komplekse sa jonima kadmijuma u vodenomrastvoru. Međutim, mehanizmi zaštitnog dejstva EPS na organizme nisu poznati. Među obećavajućimkandidatima iz naše laboratorijske kolekcije (BGLMM), se istakao EPS-AN8, izolovan i prečišćen izsoja Lactiplantibacillus plantarum BGAN8. In silico analiza genetičkih determinanti odgovornih zabiosintezu EPS-AN8 i detaljna karakterizacija fizičko-hemijskih osobina je ukazala da je reč oheteropolisaharidu sa potencijalno visokim afinitetom za vezivanje kadmijumovih jona. Ispitivanjain vitro su pokazala da EPS-AN8 štiti intestinalne epitelne ćelije od negativnog dejstvakadmijuma(II), tako što snižava parametre oksidativnog stresa i inflamacije u ćelijama, i čuvameđućelijske veze. S obzirom da su, za opštu populaciju, glavni izvori kadmijuma hrana i piće,ispitivanje in vivo zaštitnih efekta EPS-AN8 je praćeno u pacovima koji su paralelno izloženiproduženom, oralnom unosu niže (5 mg/L) i više (50 mg/L) doze jona kadmijuma. Pri unosu EPS-AN8 zabeleženo je smanjeno deponovanje metala u tkivima, ublažavanje histoloških oštećenjaduodenuma, jetre i bubrega, kao i sniženi parametri oksidativnog stresa i inflamacije u duodenumu.Dodatno, primećene su i slabije izražene promene u sastavu mikrobiote duodenuma karakterističneza oralnu izloženost kadmijumu(II). Pretpostavljeni mehanizmi uspostavljene zaštite mogu bitibazirani na ogromnom kapacitetu EPS-AN8 za vezivanje kadmijumovih jona i/ili njegovim direktnimeffektom na ublažavanje oštećenja izazvanih kadmijumom.Cadmium, a widespread toxic metal, causes numerous negative health effects, leading to the urgentneed to find adequate protection. The known ability of some lactobacilli to interact with metals isincreasingly attributed to the presence of the surface exopolysaccharides (EPS). It has been shownthat EPS could form complexes with cadmium ions in aqueous solution. However, the possiblemechanisms of the EPS-mediated protection in organisms are not known. Among the otherscandidates from our laboratory collection (BGLMM), EPS-AN8, derived from the strainLactiplantibacillus plantarum BGAN8, stood out. In silico analysis of the genetic backgroundresponsible for EPS-AN8 biosynthesis and characterization of the physicochemical propertiesindicated that EPS-AN8 is the heteropolysaccharide with a putative high affinity for bindingcadmium. In vitro assays showed that EPS-AN8 protects intestinal epithelial cells from thedeleterious effect of cadmium, by lowering the parameters of oxidative stress and inflammation incells, and maintaining intercellular junctions. For the general population, the main source of cadmiumis diet. Orally given EPS-AN8, and exposure of rats to prolonged, oral intake of lower (5 mg/L) andhigher (50 mg/L) dose of cadmium(II) resulted in decreased body burden of this metal, lowered levelof histological damages in the duodenum, the liver and the kidneys and reduced parameters ofoxidative stress and inflammation. Additionally, EPS-AN8 led to less pronounced cadmium inducedchanges of the duodenal microbiota composition. The possible mechanisms of the EPS-AN8established protection may be based on its enormous capacity to adsorb cadmium ions and/or its directeffect to mitigate cadmium-provoked damages
Brain organoids as a model for studying altered translation in autism
No full textIntroduction:
The complexity of autism spectrum disorder (ASD) is manifested by the extensive heterogeneity among patients, both in terms of the assortment of comorbidities and genetic causes. In neurons, with their uniquely polarized morphology, tight and highly localized translational regulation of specific mRNAs plays a major role in activity-dependent changes in synaptic structure and function. Accordingly, dysregulation of mRNA localization and translation has been found in a number of neuronal disorders. The contribution of aberrant protein synthesis to pathomechanisms of ASD was originally uncovered through highly penetrant monogenic forms of ASD (Tuberous sclerosis, Cowden syndrome and Fragile X Syndrome, caused by mutations in TSC1/TSC2, PTEN, and FMR1 respectively), and was later suggested as a hallmark of the disease regardless of etiology. The Consortium ALTRUISM (Altered Translation in Autism) aims to understand the pathophysiology of ASD downstream of clinically-relevant mutations in FMR1, PTEN and TSC2, which impinge on protein synthesis. Elucidating the molecular underpinnings of ASD is essential for devising efficient patient stratification strategies and identifying predictive biomarkers for a possible drug repurposing of already existing therapies targeting translational regulators.
Methods:
We have employed a systems biology approach based on OMICs technologies for the quantification of transcriptomes (including single-cell), translatomes, miRNAs and proteomes along with morphological studies in iPSC-derived brain organoids (Trujillo et al., 2019, Cell Stem Cell) engineered to harbor patient-relevant mutations in FMR1, PTEN and TSC2.
Results:
We generated three series of isogenic iPSC lines, composed of control (electroporated with the empty vector used for CRSIPR-Cas9 editing) and TSC2, PTEN and FMR1 mutant lines. All 12 lines were successfully differentiated into cortical brain organoids. The TSC2 and PTEN mutant organoids showed increased budding compared to control lines. We are currently performing immunofluorescence analysis to further elucidate morphological differences caused by selected mutations. In parallel, we profiled organoids at the level of transcriptome (single-cell and bulk), proteome and miRNAs. The acquired data is currently being analyzed.
In addition, we observed an important heterogeneity in the size of organoids originating from the same line. To investigate this heterogeneity, we selected organoids from the same line and dish, but with different sizes, and profiled them using single-cell RNAseq and morphological analysis. We found that organoid size was strongly associated with significant differences in cell composition, maturity and morphology, and therefore we further optimized the Trujillo protocol to obtain more reproducible organoid sizes.
Conclusion:
Analyzes of the acquired data and cross-layer integration will lead to the identification of deregulated pathways and potential biomarkers that are specific to the disease (mutation) or shared among them. The putative biomarkers will then be validated in a cohort of 150 deeply phenotyped ASD patients.
The early organoid formation is a stochastic process, often leading to organoids of different sizes, maturity and morphology. We demonstrated that the size of organoids reflects differences in cell composition, maturity and morphology. These results should be taken into consideration to avoid spurious results when comparing organoids across conditions.BaCell 3D - Building Advanced multiCellular systems in 3D from 8 May 2023 to 9 May 202
Differences in MB-COMT DNA methylation in monozygotic twins on phenotypic indicators of impulsivity
No full textEpigenetic modifications of the membrane bound catechol-O-methyltransferase (MB-COMT) gene may affect the enzymatic degradation of dopamine, and consequently, human behavior. This study investigated the association between membrane bound catechol-O-methyltransferase DNA methylation (DNAm) differences in 92 monozygotic (MZ) twins with phenotypic manifestations of cognitive, behavioral, and personality indicators associated with reward-related behaviors and lack of control. We used pyrosequencing to determine DNAm of the regulatory region of membrane bound catechol-O-methyltransferase in saliva DNA. Results of intrapair differences in the percentage of membrane bound catechol-O-methyltransferase DNAm at each of five CpG sites show that there are associations between phenotypic indicators of lack of control and membrane bound catechol-O-methyltransferase DNAm differences on CpG1, CpG2 and CpG4, suggesting the common epigenetic patterns for personality traits, cognitive functions, and risk behaviors
Supplementary data for the article:Bačić J, Pavlović M, Kušić-Tišma J, Širca S, Theuerschuh M, Gerič Stare B. First Report of the Root-Knot Nematode Meloidogyne luci on Tomato in Serbia. Plant Disease. 2023;107(8):2554. doi:10.1094/PDIS-01-23-0164-PDN
No full textSupplementary material for:[https://doi.org/10.1094/PDIS-01-23-0164-PDN]Related to published version: [https://imagine.imgge.bg.ac.rs/handle/123456789/2762]This work was financially supported by the Serbian Plant Protection 12 Directorate of MAFWM in the frame of Program of Measures in Plant Health in 2021, the 13 Slovenian Research Agency in the frame of Research Programme Agrobiodiversity (P4-0072) 14 and the Ministry of Agriculture, Forestry and Food of the Republic of Slovenia in the frame of 15 Expert work in the field of plant protection (C2337)
A Comparison of MGMT Testing by MSP and qMSP in Paired Snap-Frozen and Formalin-Fixed Paraffin-Embedded Gliomas
Get PDFComparative analysis of the conventional methylation-specific PCR (MSP) vs. the quantitative MSP (qMSP) assessment of the O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation status in 34 snap-frozen (SF) glioma samples was performed. The accuracy of the semi-quantitative MSP was compared with the corresponding qMSP semi-quantitative values using two semi-quantitative cut-off values (0—unmethylated and 1—weakly methylated) to discriminate methylated from unmethylated samples. In the case of the cut-off value 0, MSP test showed 80.0% sensitivity and 78.9% specificity compared to the reference qMSP analysis. However, when using the cut-off value 1, the diagnostic accuracy of the MSP test was significantly higher (85.7% sensitivity, 85.2% specificity). Fleiss’ Kappa statistical analyses indicated moderate agreement (Fleiss’ Kappa Coefficient = 0.509; 70.59% agreement) between MSP and qMSP semi-quantitative measurements of MGMT promoter methylation in glioma patients, justifying the conventional MSP use in diagnostics and confirming its high reliability. Further, we aimed to compare the validity of SF and formalin-fixed paraffin-embedded (FFPE) glioma samples for MGMT testing. Statistical analyses indicated moderate overall agreement of FFPE glioma samples and SF MSP semi-quantitative measurements (Fleiss’ Kappa Coefficient = 0.516/0.509; 70.0% agreement) and emphasized their low reliability in the assessment of highly methylated MGMT promoter samples
Exploring the antibacterial potential of Lactococcus lactis subsp. lactis bv. diacetylactis BGBU1-4 by genome mining, bacteriocin gene overexpression, and chemical protein synthesis of lactolisterin BU variants
No full textLactic acid bacterium Lactococcus lactis BGBU1-4 produces 43 amino acids (aa) long bacteriocin, lactolisterin BU (LBU), a 5.161 kDa peptide with potent antibacterial activity against many Gram-positive pathogens. In addition, BGBU1-4 produces an additional unknown product of 3.642 kDa with antibacterial activity. Here, we determined that the significant amount of naturally produced LBU breaks down to create a 3.642 kDa truncated form of LBU bacteriocin consisting of 31 N-terminal aa (LBU1-31) that exhibits 12.5% the antibacterial activity of the full-length LBU. We showed that chemically synthesized LBU is stable and 50% less active than native LBU, and so we used the synthetic peptides of LBU and its variants to further study their activities and antibacterial potential. Deletion analysis of LBU revealed that the 24 N-terminal aa of LBU (LBU1-24) are responsible for antibacterial activity, while downstream aa (25–43) determine the species-specific effectiveness of LBU. Although LBU1-31 contains aa 1–24, the truncation at position 31 is predicted to change the structure within aa 15–31 and might impact on antibacterial activity. Intriguingly, whole genome sequencing and genome mining established that BGBU1-4 is abundant in genes that encode potential antibacterials, but produces LBU and its breakdown product LBU1-31 exclusively
Employing Gamma-Ray-Modified Carbon Quantum Dots to Combat a Wide Range of Bacteria
Get PDFNowadays, it is a great challenge to develop new medicines for treating various infectious diseases. The treatment of these diseases is of utmost interest to further prevent the development of multi-drug resistance in different pathogens. Carbon quantum dots, as a new member of the carbon nanomaterials family, can potentially be used as a highly promising visible-light-triggered antibacterial agent. In this work, the results of antibacterial and cytotoxic activities of gamma-ray-irradiated carbon quantum dots are presented. Carbon quantum dots (CQDs) were synthesized from citric acid by a pyrolysis procedure and irradiated by gamma rays at different doses (25, 50, 100 and 200 kGy). Structure, chemical composition and optical properties were investigated by atomic force microscopy, transmission electron microscopy, X-ray photoelectron spectroscopy, Fourier transform infrared spectroscopy, Raman spectroscopy, UV-Vis spectrometry and photoluminescence. Structural analysis showed that CQDs have a spherical-like shape and dose-dependent average diameters and heights. Antibacterial tests showed that all irradiated dots had antibacterial activity but CQDs irradiated with dose of 100 kGy had antibacterial activity against all seven pathogen-reference bacterial strains. Gamma-ray-modified CQDs did not show any cytotoxicity toward human fetal-originated MRC-5 cells. Moreover, fluorescence microscopy showed excellent cellular uptake of CQDs irradiated with doses of 25 and 200 kGy into MRC-5 cells.This article belongs to the Special Issue Novel Antimicrobial Agents: Design, Synthesis and Biological EvaluationSupplementary data: [https://imagine.imgge.bg.ac.rs/handle/123456789/1895
Drugst.One - A plug-and-play solution for online systems medicine and network-based drug repurposing
Get PDFIn recent decades, the development of new drugs has become increasingly expensive
and inefficient, and the molecular mechanisms of pharmaceuticals often remain poorly
understood. In response, numerous computational systems and network medicine tools have
been developed to prioritize drug repurposing candidates. However, such tools often require
local installation and configuration or lack follow-up visual network mining capabilities. To
address these challenges and simplify network exploration and drug repurposing candidate
prediction, we have developed Drugst.One. It is a customizable plug-and-play solution with
its own data warehousing system integrating multiple interaction databases to enable
interactive modeling and analysis of the associations between proteins, drugs, and diseases.
With just three lines of code, it has the capacity to convert any systems medicine software into
an interactive web tool for identifying drug repurposing candidates, thus providing a powerful
and accessible resource for advancing drug discovery efforts. To demonstrate the utility of
Drugst.One’s low-code approach, we have integrated it with 20 existing computational
systems medicine tools of various types, with the intent to expand the Drugst.One Initiative
with additional collaboration partners.
Drugst.One is, to our knowledge, the first approach to unify and simplify web-based networkbased
visualization and drug repurposing, posing a valuable resource for the research
community. Learn more about Drugst.One and the Drugst.One Initiative at https://drugst.one.Book of abstract: 4th Belgrade Bioinformatics Conference, June 19-23, 202
Online in silico validation of disease and gene sets, clusterings or subnetworks with DIGEST
Get PDFGiven the constraints faced in the development of new drugs, the importance of drug repurposing
has reached unprecedented levels. A key aspect of effective drug repurposing lies in the discovery
of disease mechanisms and the identification of clusters of diseases with shared mechanistic
characteristics. While various methods exist for computing candidate disease mechanisms
and clusters, the absence of ground truth presents challenges in validating these predictions
through in silico means. This obstacle significantly impedes the widespread adoption of in silico
prediction tools, as experimentalists often hesitate to conduct wet-lab validations without
clearly quantified initial plausibility.
To address this issue, we introduce DIGEST (in silico validation of disease and gene sets,
clusterings or subnetworks). DIGEST is a Python-based validation tool that offers multiple
avenues for utilization. It is accessible as a web interface through https://digest-validation.net, as
a stand-alone package, or via a REST API. DIGEST streamlines the process of in silico validation
by providing fully automated pipelines. These pipelines encompass critical components such as
disease and gene ID mapping, enrichment analysis, comparisons of shared genes and variants,
and background distribution estimation. Additionally, DIGEST incorporates functionality to
automatically update the external databases utilized by the pipelines. By employing DIGEST,
users gain the ability to assess the statistical significance of candidate mechanisms in terms
of functional and genetic coherence. The tool enables the computation of empirical P-values
with ease, requiring only a few simple clicks. With its comprehensive and user-friendly features,
DIGEST greatly facilitates the evaluation of candidate mechanisms, empowering researchers to
quantify the plausibility of predicted mechanisms in a robust and efficient manner.Book of abstract: 4th Belgrade Bioinformatics Conference, June 19-23, 202