imagine (Institute of molecular genetics and genetic engineering)
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Decreased TSPAN14 Expression Contributes to NSCLC Progression
Get PDFTspan14 is a transmembrane protein of the tetraspanin (Tspan) protein family. Different members of the Tspan family can promote or suppress tumor progression. The exact role of Tspan14 in tumor cells is unknown. Earlier, mutational inactivation of the TSPAN14 gene has been proposed to coincide with a low survival rate in NSCLC patients. This study aimed to investigate the correlation of TSPAN14 lack of function with clinicopathological features of NSCLC patients, and to elucidate the role TSPAN14 might have in NSCLC progression. TSPAN14 expression was lower in tumor cells than non-tumor cells in NSCLC patients' samples. The decreased gene expression was correlated with a low survival rate of patients and was more frequent in patients with aggressive, invasive tumor types. Additionally, the role of decreased TSPAN14 expression in the metastatic potential of cancer cells was confirmed in NSCLC cell lines. The highly invasive NSCLC cell line (NCI-H661) had the lowest TSPAN14 gene and protein expression, whereas the NSCLC cell line with the highest TSPAN14 expression (NCI-H460) had no significant metastatic potential. Finally, silencing of TSPAN14 in these non-metastatic cancer cells caused an increased expression of matrix-degrading enzymes MMP-2 and MMP-9, followed by an elevated capacity of cancer cells to degrade gelatin. The results of this study propose TSPAN14 expression as an indicator of NSCLC metastatic potential and progression
Degradable 2-Hydroxyethyl Methacrylate/Gelatin/Alginate Hydrogels Infused by Nanocolloidal Graphene Oxide as Promising Drug Delivery and Scaffolding Biomaterials
Get PDFThe design and evaluation of novel 2-hydroxyethyl methacrylate/gelatin/alginate/graphene oxide hydrogels as innovative scaffolding biomaterials, which concurrently are the suitable drug delivery carrier, was proposed. The hydrogels were prepared by the adapted porogen leaching method; this is also the first time this method has been used to incorporate nanocolloidal graphene oxide through the hydrogel and simultaneously form porous structures. The effects of a material's composition on its chemical, morphological, mechanical, and swelling properties, as well as on cell viability and in vitro degradation, were assessed using Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), measurements of Young's modulus, gravimetric method and MTT test, respectively. The engineered hydrogels show good swelling capacity, fully hydrophilic surfaces, tunable porosity (from 56 to 76%) and mechanical properties (from 1.69 to 4.78 MPa), curcumin entrapment efficiency above 99% and excellent curcumin release performances. In vitro cytotoxicity on healthy human fibroblast (MRC5 cells) by MTT test reveal that the materials are nontoxic and biocompatible, proposing novel hydrogels for in vivo clinical evaluation to optimize tissue regeneration treatments by coupling the hydrogels with cells and different active agents to create material/biofactor hybrids with new levels of biofunctionality
Isolation, Characterization and Draft Genome Analysis of Bacteriophages Infecting Acidovorax citrulli
Get PDFBacterial fruit blotch and seedling blight, caused by Acidovorax citrulli, is one of the most destructive diseases of melon and watermelon in many countries. Pathogen-free seed and cultural practices are major pillars of the disease control. However, use of bacteriophages as natural biocontrol agents might also contribute to the disease management. Therefore, we isolated 12 bacteriophages specific to A. citrulli, from phyllosphere and rhizosphere of diseased watermelon plants. The phage strains were characterized based on their host range, plaque and virion morphology, thermal inactivation point, adsorption rate, one step growth curve, restriction fragment length polymorphism (RFLP), and genomic analysis. Transmission electron microscopy of three phage strains indicated that they belong to the order Caudovirales, family Siphoviridae. All phages lysed 30 out of 32 tested A. citrulli strains isolated in Serbia, and did not lyse other less related bacterial species. They produced clear plaques, 2 mm in diameter, on bacterial lawns of different A. citrulli strains after 24 h of incubation. The thermal inactivation point was 66 or 67 degrees C. They were stable at pH 5-9, but were sensitive to chloroform and inactivated in either 5 or 10 min exposure to ultraviolet (UV) light. RFLP analysis using EcoRI, BsmI and BamHI enzymes did not show genetic differences among the tested phages. Adsorption rate and one step growth curve were determined for the Acidovorax phage ACF1. Draft genome sequence of the ACF1 phage was 59.377 bp in size, with guanine-cytosine (GC) content 64.5%, including 89 open reading frames. This phage shared a very high genomic identity with Acidovorax phage ACPWH, isolated in South Korea. Evaluation of systemic nature of ACF1 strain showed that it can be absorbed by roots and translocated to upper parts of watermelon plants where it survived up to 10 days
Star-shaped poly(epsilon-caprolactones) with well-defined architecture as potential drug carriers
Get PDFThe present study reports the potential application of star-shaped poly(epsilon-caprolactones) with different number of arms as new drug delivery matrix. Linear and star-shaped PCL ibuprofen loaded microspheres were prepared using oil-in-water (o/w) solvent evaporation technique and characterized with FTIR, DSC, XRD and SEM analysis. High yield, encapsulation efficiency and drug loadings were obtained for all microspheres. FTIR analysis revealed the existence of interactions between polymer matrix and drug, while the DSC analysis suggested that drug was encapsulated in an amorphous form. SEM analysis confirmed that regular, spherical in shape star-shaped microspheres, with diameter between 80 and 90 mu n, were obtained, while quite larger microspheres, 110 mu m, were prepared from linear PCL. The advantage of using starshaped PCL microspheres instead of linear PCL was seen from drug release profiles which demonstrated higher amount of drug released from star-shaped polymer matrix as a consequence of their branched, flexible structure. Microspheres prepared from the polymers with the most branched structure showed the highest amount of the released drug after 24 h. Finally, cytotoxicity tests, performed using normal human fibroblasts (MRCS), indicated the absence of cytotoxicity at lower concentrations of microspheres proving the great potential of star-shaped PCL systems in comparison to linear ones
A polyesterase from the Antarctic bacterium Moraxella sp. degrades highly crystalline synthetic polymers
Get PDFThe uncontrolled release of plastics in the environment has rendered them ubiquitous around the planet, threatening the wildlife and human health. Biodegradation and valorization of plastics has emerged as an ecofriendly alternative to conventional management techniques. Discovery of novel polymer-degrading enzymes with diversified properties is hence an important task in order to explore different operational conditions for plastic-waste upcycling. In the present study, a barely studied psychrophilic enzyme (MoPE) from the Antractic bacterium Moraxella sp. was heterologously expressed, characterized and its potential in polymer degradation was further investigated. Based on its amino acid composition and structure, MoPE resembled PET-degrading enzymes, sharing features from both mesophilic and thermophilic homologues. MoPE hydrolyzes nonbiodegradable plastics, such as polyethylene terephthalate and polyurethane, as well as biodegradableThis is the peer reviewed version of the paper: Nikolaivits, E., Taxeidis, G., Gkountela, C., Vouyiouka, S., Maslak, V., Nikodinovic-Runic, J., & Topakas, E. (2022). A polyesterase from the Antarctic bacterium Moraxella sp. Degrades highly crystalline synthetic polymers. Journal of Hazardous Materials, 434, 128900.[ https://doi.org/10.1016/j.jhazmat.2022.128900]Published version: [https://imagine.imgge.bg.ac.rs/handle/123456789/1564
Silver(I) Complexes with Clinically Used Azoles: Synthesis, Structural Characterization and Antimicrobial Evaluation
Get PDFRecently, we synthesized silver(I) complex with the antifungal agent itraconazole, which showed improved anti-Candida potential and therapeutic safety in comparison to itraconazole and rescued zebrafish embryos affected by lethal C. albicans infection, when used in safe doses. Inspired by these results, in the present study, three new silver(I) complexes with clinically used azoles, econazole (ecz), clotrimazole (ctz) and voriconazole (vcz), [Ag(ecz)2]SbF6 (Ag1), [Ag(ctz)2]SbF6 (Ag2) and {[Ag(vcz)2]SbF6}n (Ag3) were synthesized and structurally characterized by elemental microanalysis, mass spectrometry, spectroscopy (1H NMR, IR and UV-Vis), cyclic voltammetry, molar conductivity measurements, and single crystal X-ray diffraction analysis. The spectroscopic and crystallographic results revealed that, in the synthesized silver(I) complexes, azole ligands are monodentately coordinated to the Ag(I) ion through the nitrogen atom forming [Ag(azole)2]+ complex cation. The antimicrobial effect of complexes and azole ligands was evaluated against different Candida species, as well as Gram-positive and Gram-negative bacteria. The synthesized complexes Ag1-3 exhibited good to moderate antimicrobial activity being, in most cases, more active than the corresponding azole ligands. Complexes Ag2 and Ag3 also showed strong inhibitory activity against C. albicans biofilm formation and strong inhibition of C. albicans filamentation at subinhibitory concentrations
Protoplast patch-clamping using an upright microscope with a movable stage
Get PDFInvestigating membrane properties of plants is a challenging task, considering that success ofexperiments is highly dependent on the possibility to isolate metabolically active protoplasts thatcan withstand membrane current recordings. The aim of the present work is to obtain viableprotoplasts derived from root cells of Pisum sativum that can be used for the whole-cell patchclamp. We designed the procedure of the pea protoplasts isolation that delivers stable protoplastswith preserved membrane integrity suitable for electrophysiological experiments. We applied acustom approach for patch-clamping protoplasts using a microscope with a movable microscopestage. We recorded prominent inward and prominent outward types of membrane current profiles ofprotoplasts. Obtained data indicate that optimized isolation protocol and custom system for patchclamping, can be applied to study membrane properties of root protoplasts.Čupić Ž, Anić S, editors. Proceedings: Physical Chemistry 2022, Vol. 1.: 16th International Conference on Fundamental and Applied Aspects of Physical Chemistry; 2022 Sep 26-30; Belgrade, Serbia. Belgrade: Society of Physical Chemists of Serbia; 2022. p. 271-4
Optimization of cell culture conditions for neural differentiation of NT2/D1 cells in alginate microfibers
No full textHuman pluripotent embryonal carcinoma NT2/D1 cell line respresents well established and
widely used model system of human in vitro neurogenesis. 3D model systems mimic in vivo
cell growth thus providing better insights into the human tissue dynamics during
development. In order to study early phases of human neurogenesis in 3D model system we
optimized condition for immobilisation of NT2/D1 cells in microalginate fibers, their
propagation and induction of neural differentiation. We analyzed morphological
characteristics, viability, proliferation and expression of specific markers of both, pluripotent
NT2/D1 cells and retinoic acid induced NT2/D1 early neural progenitors. Our results
indicate that the immobilization in microalginate fibers affected viability of NT2/D1 cells but
did not impair the ability of surviving cells to adhere and proliferate. In obtained 3D system
NT2/D1 cells preserved neural differentiation capacity upon induction with retinoic acid.
Induction of NT2/D1 cells immobilized in microalginate fibers, by retinoic acid, represents
exellent 3D model system for studying human neurogenesis and could be used as platform
for screening the effects of drugs and bioactive compounds on initiation and progression of
neural differentiation.Book of Abstracts: Twentieth Young Researchers Conference – Materials Science and Engineering
November 30 – December 2, 2022, Belgrade, Serbi
Наследни фактори ризика за тромбофилију – од тачкастих мутација до примене вештачке интелигенције
Get PDFTrombofilija je patofiziološko stanje povećanog rizika za nastanak
hiperkoagulacije, koja može dovesti do začepljenja krvnog suda (tromboze).
Faktori rizika za nastanak ove multifaktorijalne bolesti mogu biti sredinski,
uzrokovani načinom života, i nasledni (genetski). Do sada je identifikovan
veliki broj naslednih faktora rizika, uglavnom tačkastih mutacija u genima za
proteine hemostaznog sistema. Iako se ove mutacije analiziraju u okviru
rutinskih kliničkih testova, kod značajnog broja bolesnika i nakon sprovedenih
dijagnostičkih procedura, uzrok trombotičkog događaja ostaje nepoznat, što
implicira postojanje neidentifikovanih naslednih faktora rizika. U cilju
njihove identifikacije, vrše se dalje genske analize, asocijativne studije,
karakterizacije potencijalnih faktora rizika u in vitro i in vivo studijama na
različitim model sistemima. U našem dosadašnjem radu detektovano je više
varijanti u kodirajućem i nekodirajućem regionu gena, za koje je karakterizacijom
utvrđeno da utiču na ekspresiju i/ili funcionalnost koagulacionih proteina.
Primenom sekvenciranja nove generacije i bioinformatičke obrade, omogućena je
sveobuhvatnija analiza celokupnog genoma i identifikacija klastera gena koji su
povezani sa kompleksnom kliničkom slikom tromboza. Kombinovanjem velikog
broja podataka o genetskim i sredinskim faktorima, primenom veštačke
inteligencije, otvara se mogućnost kompletnijeg sagledavanja mehanizama
trombofilije i multifaktorijalnih bolesti uopšte.Тромбофилија је патофизиолошко стање повећаног ризика за настанак
хиперкоагулације, која може довести до зачепљења крвног суда (тромбозе).
Фактори ризика за настанак ове мултифакторијалне болести могу бити средински,
узроковани начином живота, и наследни (генетски). До сада је идентификован
велики број наследних фактора ризика, углавном тачкастих мутација у генима за
протеине хемостазног система. Иако се ове мутације анализирају у оквиру
рутинских клиничких тестова, код значајног броја болесника и након спроведених
дијагностичких процедура, узрок тромботичког догађаја остаје непознат, што
имплицира постојање неидентификованих наследних фактора ризика. У циљу
њихове идентификације, врше се даље генске анализе, асоцијативне студије,
карактеризације потенцијалних фактора ризика у in vitro и in vivo студијама на
различитим модел системима. У нашем досадашњем раду детектовано је више
варијанти у кодирајућем и некодирајућем региону гена, за које је карактеризацијом
утврђено да утичу на експресију и/или фунционалност коагулационих протеина.
Применом секвенцирања нове генерације и биоинформатичке обраде, омогућена је
свеобухватнија анализа целокупног генома и идентификација кластера гена који су
повезани са комплексном клиничком сликом тромбоза. Комбиновањем великог
броја података о генетским и срединским факторима, применом вештачке
интелигенције, отвара се могућност комплетнијег сагледавања механизама
тромбофилије и мултифакторијалних болести уопште.Knjiga sažetaka: Treći Kongres biologa Srbije, Zlatibor, Srbija 21 - 25. 9. 2022
Synthesis and characterization of a collagen-based composite material containing selenium nanoparticles
No full textMultidrug-resistant bacterial strains represent an emerging global health threat and a great obstacle for bone tissue
engineering. One of the major components of the extracellular matrix of the bone is a collagen protein, while selenium is an
element that has antimicrobial potential, and is also important for bone metabolism and bone health. Here we represent the
incorporation of selenium nanoparticles (SeNPs) synthesized by the green chemical reduction method into collagen gels to
produce a composite material, collagen/SeNPs, with antimicrobial properties. The samples were comprehensively
characterized by zeta potential measurements, dynamic light scattering inductively coupled plasma-mass spectrometry
(ICP-MS), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), optical microscopy, field-emission
scanning electron microscopy (FE-SEM), and differential scanning calorimetry The cytotoxicity of the SeNPS, as well as
collagen/SeNPs, was tested on the MRC-5 cells. It was revealed that collagen/SeNPS expressed a lower cytotoxic effect.
Collagen/SeNPs showed significant antibacterial activity against all tested Gram-positive strains, the major causative agents
of orthopedic infections as well as Candida albicans. Furthermore, three-dimensional β-tricalcium phosphate (3D-TCP)
scaffolds were fabricated by a well-established 3D printing (lithography) method, and afterward preliminary coated by
newly-synthesized SeNPs or collagen/SeNPs. In addition, uncoated 3D-TCP scaffolds as well as coated by collagen/SeNPs
were subjected to biofilm formation. The production of Staphylococcus aureus biofilm on coated scaffolds by collagen/SeNPs
was significantly reduced compared to the uncoated ones.Supplementary material: [https://imagine.imgge.bg.ac.rs/handle/123456789/1774