imagine (Institute of molecular genetics and genetic engineering)
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In vitro activity of novel cinnamic acids hydrazides against clinically important pathogens
No full textAntimicrobial-resistance (AMR) has become the greatest concern and highly challenging issue when treating nosocomial infections. The exigency to develop new potent compounds continues to increase worldwide, whereby derivatives of natural products are becoming more attractive. In the present pa-per, the microbiological assessment of a series of 12 cinnamide hydrazides, four of them completely novel, against clinically relevant pathogens has discovered several derivatives with promising in vitro activities against Acinetobacter baumannii, one of the most dreaded opportunistic pathogens in hospi-tals. The compounds were synthesized by combining one of three different natural acids (cinnamic, 4-chloro or 4-methoxy) with four monothiocarbohydrazones (MTCHs) -an important class of synthetic organic molecules. Their structure was confirmed by elemental microanalysis, as well as ATR-FTIR, H-1 and C-13 NMR spectra, with the addition of 2D NMR spectra for novel compounds. The hybrids of cinnamic acids and pyridine derivatives are particularly active compounds with the lowest MIC50 value of 10.4 mu M for p-chloro cinnamic acid and acetyl pyridine derivatives. An alignment-independent 3D QSAR model identified pharmacophoric hotspots and suggested several structural modifications that might improve the potency of this class of compounds against A. baumannii. The compounds are strong iron-chelating agents forming complexes with a stability constant between 10 7 and 10 9 . The synthesized derivatives represent a promising class of antibacterial compounds with activities comparable to the commonly used antibiotics
Redesigned pMAL expression vector for easy and fast purification of active native antimicrobial peptides
No full textAims The aim of this study was to construct the improved pMAL expression vector to increase the efficacy of purification of small native peptides and their clear-cut separation from MBP tag. The modifications we introduced can be applied to many expression vectors. Methods and Results To improve the pMAL expression vector, we introduced the His(6) tag and the enterokinase cleavage site (Ek) downstream from the MBP tag and Xa cleavage site on the original vector. For cloning of a desired peptide DNA, the enterokinase site contains a unique BsaBI restriction site adjacent to the original multi-cloning site. This redesigned pMAL vector was optimized for the purification of cytoplasmic (pMALc5HisEk) and periplasmic (pMALp5HisEk) peptides. The purification of native and active peptide (P) was obtained following two-step affinity chromatography. In the first step, the entire MBP-His(6)-Ek-P fusion protein is purified using the Ni-NTA agarose column. This fusion protein was cleaved with active His(6) tagged enterokinase. In the second step, the further purification was performed by column containing the mixture of amylose and Ni-NTA agarose resins. This removes both the MBP-His(6) and His(6)-enterokinase leaving pure native protein in solution. These new vectors and the two-step purification protocol were successfully applied in purification of active native small antimicrobial peptides (AMPs), lactococcin A and human beta-defensin. Conclusions We constructed the improved pMAL expression vectors and established the pipeline and optimal conditions for their use in efficient purification of large amounts of active native small peptides. Significance and Impact of the Study Choice of expression vector impacts on the efficiency of expression and purification of desired proteins. The idea of redesigning pMAL vector was driven by the need for rapid purification of larger amounts of active native AMPs. This newly improved pMAL vector, the cloning strategy, expression conditions and two-step purification protocol represent a unique simple approach which can be applied in every laboratory
Immunomodulatory Properties of Pomegranate Peel Extract in a Model of Human Peripheral Blood Mononuclear Cell Culture
Get PDFPomegranate peel extract (PoPEx) has been shown to have antioxidant and anti-inflammatory properties, but its effect on the adaptive immune system has not been sufficiently investigated. In this study, the treatment of human peripheral blood mononuclear cells (PBMC) with PoPEx (range 6.25-400 mu g/mL) resulted in cytotoxicity at concentrations of 100 mu g/mL and higher, due to the induction of apoptosis and oxidative stress, whereas autophagy was reduced. At non-cytotoxic concentrations, the opposite effect on these processes was observed simultaneously with the inhibition of PHA-induced PBMC proliferation and a significant decrease in the expression of CD4. PoPEx differently modulated the expression of activation markers (CD69, CD25, ICOS) and PD1 (inhibitory marker), depending on the dose and T-cell subsets. PoPEx (starting from 12.5 mu g/mL) suppressed the production of Th1 (IFN-gamma), Th17 (IL-17A, IL-17F, and IL-22), Th9 (IL-9), and proinflammatory cytokines (TNF-alpha and IL-6) in culture supernatants. Lower concentrations upregulated Th2 (IL-5 and IL-13) and Treg (IL-10) responses as well as CD4+CD25hiFoxp3+ cell frequency. Higher concentrations of PoPEx increased the frequency of IL-10- and TGF-beta-producing T-cells (much higher in the CD4+ subset). In conclusion, our study suggested for the first time complex immunoregulatory effects of PoPEx on T cells, which could assist in the suppression of chronic inflammatory and autoimmune diseases
The gene regulation knowledge commons: the action area of GREEKC
Get PDFAs computational modeling becomes more essential to analyze and understand biological regulatory mechanisms, governance of the many databases and knowledge bases that support this domain is crucial to guarantee reliability and interoperability of resources. To address this, the COST Action Gene Regulation Ensemble Effort for the Knowledge Commons (GREEKC, CA15205, www.greekc.org) organized nine workshops in a four-year period, starting September 2016. The workshops brought together a wide range of experts from all over the world working on various steps in the knowledge management process that focuses on understanding gene regulatory mechanisms. The discussions between ontologists, curators, text miners, biologists, bioinformaticians, philosophers and computational scientists spawned a host of activities aimed to standardize and update existing knowledge management workflows and involve end-users in the process of designing the Gene Regulation Knowledge Commons (GRKC). Here the GREEKC consortium describes its main achievements in improving this GRKC
Cobalt(II) and magnesium(II) complexes with 1,3-pdta-type of ligands: influence of an alkyl substituent at 1,3-propanediamine chain on the structural and antimicrobial properties of the complex
No full textTo investigate how modification in the structure of 1,3-propanediamine chain of 1,3-pdta (1,3-propanediamine-N,N,N′,N′-tetraacetate) ligand affects the structural and biological properties of the corresponding metal complexes, two new octahedral complexes, [Co(H2O)5Co(2,2-diMe-1,3-pdta)]·H2O (1) and [Mg(H2O)5Mg(2,2-diMe-1,3-pdta)]·1.5H2O (2) (2,2-diMe-1,3-pdta = 2,2-dimethyl-1,3-propanediamine-N,N,N′,N′-tetraacetate), were synthesized and characterized by IR spectroscopy and single-crystal X-ray diffraction analysis. Additionally, UV-Vis and NMR spectroscopic methods were applied for the characterization of 1 and 2, respectively. Crystallographic data indicate that these complexes contain 2,2-diMe-1,3-pdta coordinated to the metal ion through 2 N and 4 O atoms forming [M(H2O)5M′(2,2-diMe-1,3-pdta)] complex unit (M, M′ = Co(II), Co(II) (1) and M, M′ = Mg(II), Mg(II) (2)), which is composed of [M′(2,2-diMe-1,3-pdta)]2− and [M(H2O)5O]2+ octahedra bridged by one of the axial carboxylate groups. The antimicrobial activities of 1 and 2 were evaluated against different bacteria and Candida spp., while their cytotoxic effect was tested on the normal human lung fibroblasts (MRC-5). The ability of 1 and 2 to inhibit formation of C. glabrata biofilms was also assessed. The obtained structural parameters and biological properties of the two complexes were compared to Co(II) and Mg(II) complexes with 1,3-pdta ligand
Neuroprotective progesterone potential
Get PDFPoremećena moždana cirkulacija, koja je karakteristična za starenje i mnoga cerebrovaskularna i/ili
neurodegenerativna oboljenja, povezuje se sa prooksidativnim i proapoptotskim promenama mnogobrojnih
biomolekula, uključujući lipide, proteine i DNK, i narušavanjem strukture i funkcije ćelija prečeone
moždane kore (PFC) i hipokampusa (HIPP). Do sada je testiran efekat brojnih jedinjenja da bi se ublažile
posledice narušenog protoka krvi kroz moždano tkivo, ali adekvatna terapija još uvek nije dostupna. Ipak,
kao jedan od mogućih terapeutika, izdvaja se steroidni hormon, progesteron (pregn-4-en-3,20-dion, P4),
za kojeg je pokazano da ispoljava zaštitne efekte u mnogim životinjskim eksperimentalnim modelima kojima
se oponašaju pojedini aspekti poremećaja strukture i funkcije moždanih ćelija primećeni kod starijih
ljudi i obolelih od cerebrovaskularnih i/ili neurodegenerativnih bolesti. Stoga, fokus ovog rada je pružanje
mogućnosti boljeg razumevanja molekulskih mehanizama delovanja P4 u mozgu u fiziološkom stanju,
kao i terapijskog potencijala koji iskazuje u animalnim modelima cerebrovaskularnih i neurodegenerativnih
bolesti, sa posebnim osvrtom na trajnu moždanu hipoperfuziju (MH) gde pokazuje regionalno-specifično
dejstvo. Iako se P4, za sada, u humanoj populaciji pokazao kao neadekvatan terapeutik u lečenju
patološkog stanja povezanog sa poremećenom moždanom cirkulacijom, dodatne kliničke studije bi obezbedile
saznanja o efektu ovog potentnog neurosteroida kod obolelih od drugih cerebrovaskularnih i/ili
neurodegenerativnih bolesti, dok bi u slučaju ohrabrujućih rezultata, P4 terapija mogla znatno olakšati
kvalitet života obolelih.Disturbed cerebral circulation, characteristic for aging and many cerebrovascular and/or neurodegenerative
diseases, is associated with prooxidative and proapoptotic changes of various biomolecules, including
lipids, proteins and DNA, and with compromised cell structure and function in prefrontal cortex
(PFC) and hippocampus (HIPP). Numerous compounds, until today, have been tested to alleviate the outcomes
of impaired blood flow through brain tissue, but adequate therapy is not yet available. However, one
of the possible therapeutics is singled out, the steroid hormone, progesterone (pregn-4-en-3,20-dione,
P4), which has been shown to exert protective effects in many animal experimental models that mimic certain
aspects of brain structural and functional changes observed in the elderly and those suffering from
cerebrovascular and/or neurodegenerative diseases. Therefore, this paper is focused on providing a better
understanding of physiological P4-mediated brain molecular mechanisms, as well as its therapeutic
potential in animal models of cerebrovascular and neurodegenerative diseases with the emphasis on permanent
cerebral hypoperfusion (CH) where the regional-specific response is observed. Although P4 is
shown as inadequate for treating a pathological condition associated with disturbed cerebral circulation,
additional clinical studies would provide insights into the potential therapeutic capacity of this potent neurosteroid
in other cerebrovascular and/or neurodegenerative diseases and conditions. In the case of encouraging
results, P4 therapy could significantly improve the quality of patients’ life
Distinctive regulation of different phenolics biosynthesis by high light and UV-B in three basil varieties
Get PDFThree varieties of basil (Ocimum x citriodorum, Ocimum basilicum var. Genovese and Ocimumbasilicum var. purpurascens) were used to examine the effect of different PAR intensities (100, 400and 1400 µmol photons m-2s-1), as well as different UV-B/PAR ratios, on leaf phenolics accumulation, and components of the ascorbate-glutathione cycle. Phenolic compounds represent themost abundant class of secondary metabolites, and their function in plants involves protectionagainst numerous abiotic stresses, role in growth and development, flowering, reproduction andseed dispersion. A preferential accumulation of HCAs over Flav, induction of class III POD activityand decreased ascorbate content were characteristic responses to pro-oxidative effect of the highUV-B/PAR ratio in all three basil varieties. The most remarkable result was the lack of accumulationof epidermal UV absorbing substances, which appeared to be a crucial photoprotective mechanism in sunlight. The contrasting effect of ecologically relevant UV-B radiation on basil plants,pro-oxidative vs. acclimative, was determined by the quality and intensity of the background light.Acclimation of basil to high light comprises a number of processes, among which are the accumulation of epidermal flavonoids and total leaf phenolics, antioxidant response (increase in Asc andGSH) and lack of downregulation of PSII upon increasing light intensity. Among varieties that weused as model system in this study, purple basil, with the highest constitutive amount of anthocyanidins, showed the least induction of epidermal flavonoids and lack of light effect upon transferfrom initial 50 to 250 µmol photons m-2s-1.Book of Abstracts: 4th International Conference on Plant Biology [and] 23rd SPPS Meeting, 6-8 October 2022, Belgrad
Supplementary information for the article: Ljubic, V.; Milosevic, M.; Cvetkovic, S.; Stojanovic, M.; Novovic, K.; Dinic, M.; Popovic, M. The New Exopolysaccharide Produced by the Probiotic Strain L. Reuteri B2: Extraction, Biological Properties, and Possible Application for Ni2+ Ion Removal from the Contaminated Water. Biomass Conversion and Biorefinery 2022. https://doi.org/10.1007/s13399-022-03292-5.
No full textUsing the universal primers (UNI16Sfw and UNI16Srev) [1], the representative isolates were identified by 16S rDNA sequencing. Amplification was carried out in a thermal cycler (Applied Biosystems, ThermoFisher Scientific) and DNA fragments were amplified as follows: initial denaturation at 94 °C for 5 min, followed by 30 cycles consisting of denaturation at 94 °C for 1 min, annealing at 55°C for 1 min, and polymerization at 72°C for 1 min, and a final extension at 72°C for 7 min. The expected length was 1549 bp. Aliquots (5 µl) of the amplified products were subjected to electrophoresis in 1% agarose gel (ThermoFisher Scientific) in TAE buffer (40 mM Tris acetate, 1 mM EDTA, pH 8.2). Gels were stained with ethidium bromide (500 ng/mL) and visualized under UV light (BioDoc Analyze). All amplicons were eluted and purified using GeneJet PCR Purification Kit (ThermoScientific) by following the manufacturer`s protocol. The PCR products that we obtained were sequenced by the Macrogen Sequencing Service (Macrogen, Amsterdam, The Netherlands) and analyzed by using BLAST algorithm (http://www.ncbi.nlm.nih.gov/index.html). Selected isolates were identified as follows: isolate B2 - Lacotbacillus reuteri, isolate H10 - Lactobacillus murinus, and isolate J7 - Klebsiella oxytoca [2]. The most numerous colonies belong to isolate B2, hence it was chosen for further characterization as a potential source for exopolysaccharide (EPS) production.Related to the published version: [https://imagine.imgge.bg.ac.rs/handle/123456789/1523]Supplementary material for: [https://doi.org/10.1007/s13399-022-03292-5
In vitro colistin susceptibility of pandrug-resistant Ac. baumannii is restored in the presence of selenium nanoparticles
No full textAims: To investigate the synergistic activity of colistin and selenium nanoparticles (SeNPs) against pandrug-resistant (PDR) Ac. baumannii. Methods and Results: Chequerboard and time-kill assays were employed to ex- plore the potential synergistic interactions between colistin and SeNPs against Ac. baumannii isolates (8), previously determined as colistin-resistant (MIC range 16– 256 μg ml−1 ). Also, whole-genome sequencing (WGS) and gene expression analyses were used to elucidate the mechanisms of colistin resistance. Exceptionally strong synergistic activity (FICI range 0.004–0.035) of colistin and SeNPs against colistin- resistant isolates was revealed. Colistin (0.5 or 1 μg ml −1 ) used in combination with SeNPs (0.5 μg ml−1 ) was able to reduce initial inoculum during the first 4 h of incuba- tion, in contrast to colistin (0.5, 1 or 2 μg ml−1 ) alone. Conclusions: These findings propose colistin/SeNPs combination as a new option to fight PDR Ac. baumannii, the therapeutic possibilities of which should be proved in future in vivo studies. Significance and Impact of Study: Here we present the first evidence of synergy between colistin and selenium compounds against bacteria in general. Also, WGS and gene expression analyses provide some new insights into Ac. baumannii colistin resistance mechanisms.This is the peer reviewed version of the following article: Ušjak, Dušan, Novović, Katarina, Filipić, Brankica, Kojić, Milan, Filipović, Nenad, Stevanović, Magdalena, Milenković, Marina, "In vitro colistin susceptibility of pandrug-resistant Ac. baumannii is restored in the presence of selenium nanoparticles" in Journal of Applied Microbiology, 133, no. 3 (2022):1197-1206, [https://doi.org/10.1111/jam.15638]Published version:[https://imagine.imgge.bg.ac.rs/handle/123456789/1600
Фармакогеномика – основа персонализоване медицине
Get PDFPersonalizovana medicina koristi znanja o genetičkoj osnovi bolesti da bi se
individualizovalo lečenje svakog pacijenta. Farmakogenomika je jedan od
najvažnijih osnova za primenu personalizovane medicine. Ona proučava odgovor
čoveka na lekove uslovljen DNK zapisom u genima odgovornim za apsorpciju,
distribuciju, metabolizam i ekskreciju određenog leka. Cilj farmakogenomike je
identifikacija specifičnih gena i genskih produkata koji mogu biti meta za
nove terapeutike, kao i identifikacija gena i alelskih varijanti koji utiču na
odgovor na lekove koji se već koriste u terapiji. Za više od 170 lekova su poznati
farmakogenomski markeri a za 64 postoje uputstva za doziranje na osnovu
genotipa. Primena farmakogenomskih testova pre započinjanja terapije omogućuje
da pacijenti dobiju adekvatnu terapiju. Time se smanjuje pojava komplikacija i
neželjenih efekata zbog kojih se terapija prekida, skraćuje se vreme lečenja i
štedi na bolničkim danima i neadekvatnim lekovima. Populaciona
farmakogenomika ukazuje da je poznavanje farmakogenomskih markera
karakterističnih za populaciju ili etničku zajednicu značajno. Zdravstveni
sistem bi ostvario uštede kada bi se sprovodilo prediktivno farmakogenomsko
testiranje bazirano na genotipu populacije. Farmakogenomski testovi su
dostupni i koriste se u kliničkoj praksi u Srbiji. Utvrđeni su i
farmakogenomski markeri koji su zastupljeni u našoj populaciji sa visokom
učestalošću. Farmakogenomika je dala doprinos individualizaciji lečenja i u
KOVID pandemiji.Персонализована медицина користи знања о генетичкој основи болести да би се
индивидуализовало лечење сваког пацијента. Фармакогеномика је један од
најважнијих основа за примену персонализоване медицине. Она проучава одговор
човека на лекове условљен ДНК записом у генима одговорним за апсорпцију,
дистрибуцију, метаболизам и екскрецију одређеног лека. Циљ фармакогеномике је
идентификација специфичних гена и генских продуката који могу бити мета за
нове терапеутике, као и идентификација гена и алелских варијанти који утичу на
одговор на лекове који се већ користе у терапији. За више од 170 лекова су познати
фармакогеномски маркери а за 64 постоје упутства за дозирање на основу
генотипа. Примена фармакогеномских тестова пре започињања терапије омогућује
да пацијенти добију адекватну терапију. Тиме се смањује појава компликација и
нежељених ефеката због којих се терапија прекида, скраћује се време лечења и
штеди на болничким данима и неадекватним лековима. Популациона
фармакогеномика указује да је познавање фармакогеномских маркера
карактеристичних за популацију или етничку заједницу значајно. Здравствени
систем би остварио уштеде када би се спроводило предиктивно фармакогеномско
тестирање базирано на генотипу популације. Фармакогеномски тестови су
доступни и користе се у клиничкој пракси у Србији. Утврђени су и
фармакогеномски маркери који су заступљени у нашој популацији са високом
учесталошћу. Фармакогеномика је дала допринос индивидуализацији лечења и у
КОВИД пандемији.Knjiga sažetaka: Treći Kongres biologa Srbije, Zlatibor, Srbija 21 - 25. 9. 2022