imagine (Institute of molecular genetics and genetic engineering)
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Inhibition of miR-21 promotes cellular senescence in NT2-derived astrocytes
Get PDFAstrocytes are the main homeostatic cells in the central nervous system (CNS) that provide mechanical,
metabolic, and trophic support to neurons. Disruption of their physiological role or acquisition of
senescence-associated phenotype can contribute to the CNS dysfunction and pathology. However, molecular
mechanisms underlying the complex physiology of astrocytes are explored insufficiently. Recent studies have
shown that miRNAs are involved in the regulation of astrocyte function through different mechanisms.
Although miR-21 has been reported as an astrocytic miRNA with an important role in astrogliosis, no link
between this miRNA and cellular senescence of astrocytes has been identified. To address the role of miR-21
in astrocytes, with special focus on cellular senescence, we used NT2/A (astrocytes derived from NT2/D1
cells). Downregulation of miR-21 expression in both immature and mature NT2/A by the antisense
technology induced the arrest of cell growth and premature cellular senescence, as indicated by senescence
hallmarks such as increased expression of cell cycle inhibitors p21 and p53 and augmented senescenceassociated
β-galactosidase activity. Additionally, in silico analysis predicted many of the genes, previously
shown to be upregulated in astrocytes with the irradiation-induced senescence, as miR-21 targets. Taken
together, our results point to miR-21 as a potential regulator of astrocyte senescence. To the best of our
knowledge, these are the first data showing the link between miR-21 and cellular senescence of astrocytes.
Since senescent astrocytes are associated with different CNS pathologies, development of novel therapeutic
strategies based on miRNA manipulation could prevent senescence and may improve the physiological
outcome.BOOK OF ABSTRACTS: 10th JUBILEE INTERNATIONAL CONFERENCE ON RADIATION IN VARIOUS FIELDS OF RESEARCH Jun 13-17, 2022, Herceg Novi - Montenegr
ISOLATION AND CHARACTERIZATION OF TWO NEW LYTIC BACTERIOPHAGES, SJM3 AND LASTA, ACTIVE AGAINST KLEBSIELLA PNEUMONIAE
No full textBACKGROUND
Acquisition of genetic elements for antibiotic resistance and the emergence of hypervirulent strains escalates
the severity of K. pneumoniae infections, not only in immunocompromised but also in healthy individuals.
Bacteriophages are being evaluated as a potential way to combat multidrug resistant pathogens. Phages are
suitable to precisely target the infection-causing bacteria whilst not disturbing the beneficial
microorganisms present in the body.
OBJECTIVES
Isolation of bacteriophages of Klebsiella pneumoniae, their detailed characterization and bioinformatic analysis,
as well as lytic and antibiofilm activity testing.
METHODS
Two lytic phages (SJM3 and LASTA) active against nosocomial carbapenem- and colistin- resistant K. pneumoniae
strains, were isolated from wastewater, purified and concentrated using CsCl gradient ultracentrifugation.
Their genomes were sequenced and analyzed. Adsorption rate, parameters of replication cycle, as well
as virion morphology were determined, too. Bactericidal efficiency of the two phages, applied individually
or as a cocktail, was tested on planktonic and biofilm cells of K. pneumoniae Ni9 strain, and surviving colonies
were tested for resistance.
RESULTS
Bacteriophages SJM3 and LASTA belong to Podoviridae family and have a narrow host range. Within 5 minutes
of incubation, about 90% of LASTA and SJM3 particles adsorb to host cells. Both phages have circular genomes
the size of 62.4 kb and differ by only 13 nucleotide substitutions. SJM3 and LASTA have similar lytic effect
when applied individually or as cocktail. However, no combination could eradicate K. pneumoniae biofilm.
Phage resistance in Ni9 strain develops as soon as 3 hours post infection and is of transient nature.ELECTRONIC ABSTRACT BOOK: FEMS Conference on Microbiology in association with Serbian Society of Microbiology 30 June - 2 July 2022; Serbi
Effects of Chemotherapy for Metastatic Colorectal Cancer on the TGF-beta Signaling and Related miRNAs hsa-miR-17-5p, hsa-miR-21-5p and hsa-miR-93-5p
No full textMetastatic colorectal cancer (mCRC) patients are treated with standard chemotherapeutic drugs in the form of FOLFOX and FOLFIRI regimens. There are no reliable markers that could predict response to chemotherapy for mCRC. TGF-beta signaling which interacts with microRNA (miRNA) network has important roles in tumor progression and chemotherapy resistance, thus the interplay between TGF-beta signaling and miRNAs could be crucial for treatment response. The aim of this study was to analyze the effect of chemotherapy for mCRC on TGF-beta signaling and related miRNAs. Hsa-miR-17-5p, hsa-miR-21-5p and hsa-miR-93-5p were selected out of 316 miRNAs with multiple targets within the TGF-beta signaling by in silico analysis. SW620 cells were treated with chemotherapeutic drugs for mCRC for 1, 3 and 6 days and expression of selected miRNAs, PAI-1, CDH1 and VIM was measured. Expression of TGF-beta signaling-related hsa-miR-17-5p, hsa-miR-21-5p and hsa-miR-93-5p was time-dependently altered in SW620 cells treated with chemotherapeutics for mCRC. The expression of hsa-miR-93-5p remained downregulated after 6 days under combined treatments FOX and FIRI as well as the hsa-miR-17-5p expression under FIRI. Chemotherapy regimens for mCRC increased expression of a major TGF-beta signaling target gene PAI-1, independently of the selected miRNAs expression. These treatments also increased the expression of epithelial-mesenchymal transition (EMT) markers CDH1 and VIM on day 3 resulting in decrease of mesenchymal-like characteristics. However, their expression returned close to basal level on day 6. In conclusion, after initial response to chemotherapeutic drugs SW620 cells start to return close to the basal mesenchymal state while the long-term downregulated expression pattern of hsa-miR-93-5p and hsa-miR-17-5p makes them candidates worth testing as biomarkers for monitoring combined chemotherapeutic treatments therapy response in mCRC patients
Preeclamptic Women Have Disrupted Placental microRNA Expression at the Time of Preeclampsia Diagnosis: Meta-Analysis
Get PDFIntroduction: Preeclampsia (PE) is a pregnancy-associated, multi-organ, life-threatening disease that appears after the 20th week of gestation. The aim of this study was to perform a systematic review and meta-analysis to determine whether women with PE have disrupted miRNA expression compared to women who do not have PE.Methods: We conducted a systematic review and meta-analysis of studies that reported miRNAs expression levels in placenta or peripheral blood of pregnant women with vs. without PE. Studies published before October 29, 2021 were identified through PubMed, EMBASE and Web of Science. Two reviewers used predefined forms and protocols to evaluate independently the eligibility of studies based on titles and abstracts and to perform full-text screening, data abstraction and quality assessment. Standardized mean difference (SMD) was used as a measure of effect size.Results: 229 publications were included in the systematic review and 53 in the meta-analysis. The expression levels in placenta were significantly higher in women with PE compared to women without PE for miRNA-16 (SMD = 1.51,95%CI = 0.55-2.46), miRNA-20b (SMD = 0.89, 95%CI = 0.33-1.45), miRNA-23a (SMD = 2.02, 95%CI = 1.25-2.78), miRNA-29b (SMD = 1.37, 95%CI = 0.36-2.37), miRNA-155 (SMD = 2.99, 95%CI = 0.83-5.14) and miRNA-210 (SMD = 1.63, 95%CI = 0.69-2.58), and significantly lower for miRNA-376c (SMD = -4.86, 95%CI = -9.51 to -0.20). An increased level of miRNK-155 expression was found in peripheral blood of women with PE (SMD = 2.06, 95%CI = 0.35-3.76), while the expression level of miRNA-16 was significantly lower in peripheral blood of PE women (SMD = -0.47, 95%CI = -0.91 to -0.03). The functional roles of the presented miRNAs include control of trophoblast proliferation, migration, invasion, apoptosis, differentiation, cellular metabolism and angiogenesis.Conclusion: miRNAs play an important role in the pathophysiology of PE. The identification of differentially expressed miRNAs in maternal blood creates an opportunity to define an easily accessible biomarker of PE
Hydroxyl radical scavenging potential of the late embryogenesis abundant proteins (LEA) proteins from Ramonda serbica - in silico approach
No full textRamonda serbica Panc. is a resurrection plant that can survive long desiccation
periods (extreme loss of cellular water). The accumulation of late embryogenesis abundant
proteins (LEAPs) is a crucial step in desiccation tolerance mechanism. Based on in vitro studies,
LEAPs can be involved in antioxidative defense, ion sequestration, structural stabilization
of both membranes and enzymes during freezing or drying, while by forming intracellular
proteinaceous condensates they increase structural integrity and intracellular viscosity of cells
during desiccation. Here we investigated the antioxidative potential of LEAPs identified by de
novo transcriptomics of R. serbica, based on their primary and secondary confirmation. In our
previous work [1], we displayed the antioxidative capacity of 20 free proteogenic amino acids
(FAA) through determining their hydroxyl radical (•
OH, generated in Fenton reaction) scavenging
rate by using electron paramagnetic resonance. These results served as a basis for generating
a model for prediction of •
OH scavenging activity for selected proteins. In addition, the model
was built based on protein primary sequences, hydrophobicity, 3D structure and predicted
solvent accessible area. Manually curated data for peptides and proteins with experimentally
determined •
OH scavenging rate were used for training and testing. The model was fed into
machine learning algorithm and •
OH scavenging potential scale was created using IC50 values.
By applying our model, we classified 164 LEAPs according to their potential for •
OH scavenging.
Further work will focus on the experimental evaluation of the obtained model by measuring of
the rate of •
OH scavenging in the presence of recombinantly produced LEAPs.
Keywords: desiccation tolerance; electron paramagnetic resonance (EPR); intrinsically disordered
proteins; machine learning; resurrection plants; secondary structure
Machine Learning Modeling from Omics Data as Prospective Tool for Improvement of Inflammatory Bowel Disease Diagnosis and Clinical Classifications
Get PDFResearch of inflammatory bowel disease (IBD) has identified numerous molecular players involved in the disease development. Even so, the understanding of IBD is incomplete, while disease treatment is still far from the precision medicine. Reliable diagnostic and prognostic biomarkers in IBD are limited which may reduce efficient therapeutic outcomes. High-throughput technologies and artificial intelligence emerged as powerful tools in search of unrevealed molecular patterns that could give important insights into IBD pathogenesis and help to address unmet clinical needs. Machine learning, a subtype of artificial intelligence, uses complex mathematical algorithms to learn from existing data in order to predict future outcomes. The scientific community has been increasingly employing machine learning for the prediction of IBD outcomes from comprehensive patient data-clinical records, genomic, transcriptomic, proteomic, metagenomic, and other IBD relevant omics data. This review aims to present fundamental principles behind machine learning modeling and its current application in IBD research with the focus on studies that explored genomic and transcriptomic data. We described different strategies used for dealing with omics data and outlined the best-performing methods. Before being translated into clinical settings, the developed machine learning models should be tested in independent prospective studies as well as randomized controlled trials
Planar printed electrodes for electroporation with high EM field homogeneity
No full textUp to date, several designs of planar electroporation (EP) electrodes have been reported. We propose a novel planar general array-type electrode design, which can be optimized for a desired area of exposure, electric field magnitude and high field homogeneity (uniformity). Unlike other designs that mostly use interdigitated electrodes with alternating potentials, in this design the same polarity electric potentials are used on all elements of the electrode array, with a circular ground electrode surrounding the electrode array. Thereby, an exposure area can be increased and the electric field depth is increased, as well. We describe the procedures used for the design optimization, applicable in general to this type of arrays. Following the initial theoretical assessment, we use full-wave numerical simulations for the design optimization. The electric field measurements on printed circuit board prototypes are included to validate the numerical calculations. Two designs (type A/type B) are presented. Field homogeneity with less than 10% variation in the majority of points of interest is observed, for the designed area of exposure sufficient to place a standard Petri dish bottom (35 mm diameter), and field levels comparable with those obtained in cuvettes. We perform EP experiments in order to confirm the expected EP efficiency. Results confirm high EP efficiency as well as possible easy adaptation of this electrode type for various design specifications. The proposed electrode design is low-cost, scalable, it allows flexible adjustment of the exposure area by adding additional array elements, and both in vivo and in vitro utilization is envisioned with somewhat different applicator mountings
Different Functions of Recombinantly Expressed Domains of Tenascin-C in Glial Scar Formation
Get PDFExtracellular matrix glycoprotein tenascin-C (TnC) is highly expressed in vertebrates during embryonic development and thereafter transiently in tissue niches undergoing extensive remodeling during regeneration after injury. TnC's different functions can be attributed to its multimodular structure represented by distinct domains and alternatively spliced isoforms. Upon central nervous system injury, TnC is upregulated and secreted into the extracellular matrix mainly by astrocytes. The goal of the present study was to elucidate the role of different TnC domains in events that take place after spinal cord injury (SCI). Astrocyte cultures prepared from TnC-deficient (TnC-/-) and wild-type (TnC+/+) mice were scratched and treated with different recombinantly generated TnC fragments. Gap closure, cell proliferation and expression of GFAP and cytokines were determined in these cultures. Gap closure in vitro was found to be delayed by TnC fragments, an effect mainly mediated by decreasing proliferation of astrocytes. The most potent effects were observed with fragments FnD, FnA and their combination. TnC-/- astrocyte cultures exhibited higher GFAP protein and mRNA expression levels, regardless of the type of fragment used for treatment. Application of TnC fragments induced also pro-inflammatory cytokine production by astrocytes in vitro. In vivo, however, the addition of FnD or Fn(D+A) led to a difference between the two genotypes, with higher levels of GFAP expression in TnC+/+ mice. FnD treatment of injured TnC-/- mice increased the density of activated microglia/macrophages in the injury region, while overall cell proliferation in the injury site was not affected. We suggest that altogether these results may explain how the reaction of astrocytes is delayed while their localization is restricted to the border of the injury site to allow microglia/macrophages to form a lesion core during the first stages of glial scar formation, as mediated by TnC and, in particular, the alternatively spliced FnD domain
Dysfunction of oligodendrocyte inwardly rectifying potassium channel in a rat model of amyotrophic lateral sclerosis
No full textAmyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease caused by the death of motor neurons in the spinal cord and the brain. Although this disease is characterized by motoneuron degeneration, non-neuronal cells such as oligodendrocytes play an important role in the disease onset and progression. The aim of our study was to examine functional properties of oligodendrocytes in the SOD1(G93A) rat model of ALS with a particular focus on the inwardly rectifying potassium channel Kir4.1 that is abundantly expressed in these glial cells and plays a role in the regulation of extracellular K+. First, we demonstrate that the expression of Kir4.1 is diminished in the spinal cord oligodendrocytes of the SOD1(G93A) rat. Moreover, our data show an elevated number of dysmorphic oligodendrocytes in the ALS spinal cord that is indicative of a degenerative phenotype. In order to assess physiological properties of oligodendrocytes, we prepared cell cultures from the rat spinal cord. Oligodendrocytes isolated from the SOD1(G93A) spinal cord display similar ramification of the processes as the control but express a lower level of Kir4.1. We further demonstrate an impairment of oligodendrocyte functional properties in ALS. Remarkably, whole-cell patch-clamp recordings revealed compromised membrane biophysical properties and diminished inward currents in the SOD1(G93A) oligodendrocytes. In addition, the Ba2+-sensitive Kir currents were decreased in ALS oligodendrocytes. Altogether, our findings provide the evidence of impaired Kir4.1 expression and function in oligodendrocytes of the SOD1(G93A) spinal cord, suggesting oligodendrocyte Kir4.1 channel as a potential contributor to the ALS pathophysiology
Colistin Resistance in Environmental Isolates ofAcinetobacter baumannii
No full textAlthough the molecular mechanisms of carbapenem resistance of environmental isolates ofAcinetobacter baumanniiare well described, data on the mechanisms of colistin resistance are scarce. In this study, we report the molecular mechanisms of colistin resistance in environmental isolates ofA. baumannii. Seven clinically relevant isolates ofA. baumanniibelonging to ST-2(Pasteur)were recovered from hospital wastewater and wastewater treatment plant. The phenotypic resistance to colistin was confirmed by broth microdilution with minimum inhibitory concentration values ranging from 20 to 160 mg/L. Colistin sulfate and colistimethate sodium showed bactericidal activity against two colistin-heteroresistant isolatesin vitro, but substantially recovery of population was observed after prolonged incubation.In silicogenome analysis revealed nucleotide variations resulting in amino acid changes in LpxC (N286D), LpxD (E117K), PmrB (A138T, R263S, L267W, Q309P, and A444V), and EptA (F166L, I228V, R348K, A370S, and K531T). According to reverse transcription quantitative PCR, all isolates had increased levels ofeptAmRNA and decreased levels oflpxAandlpxDmRNA. Isolates expressed low hydrophobicity, biofilm, and pellicle formation, but showed excellent survival in river water during 50 days of monitoring. Colistin- and pandrug-resistantA. baumanniidisseminated in the environment could represent the source for the occurrence of serious community-acquired infections