imagine (Institute of molecular genetics and genetic engineering)
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The role of targeted (epi)genetic modifications in potential diabetes therapy
Get PDFU osnovi dijabetesa se nalazi smanjen broj beta ćelija endokrinog pankreasa, njihovo poremećeno
funkcionisanje ili gubitak identiteta u procesu dediferencijacije. Jedna od aktuelnih strategija za potencijalnu
primenu u terapiji dijabetesa je i direktno ćelijsko reprogramiranje kojim bi se nadomestio
nedostatak funkcionalnih beta ćelija i insulina u organizmu. Ovaj pravac u istraživanjima podrazumeva
transdiferencijaciju somatskih ćelija poreklom iz različitih organa u ćelije koje proizvode insulin
kroz modulaciju ekspresije transkripcionih faktora koji su ključni za održavanje ćelijskog identiteta.
U ovom poglavlju biće predstavljena aktuelna istraživanja koja podrazumevaju ćelijsko reprogramiranje
uz pomoć novih sintetičkih alata koji imaju ulogu da dirigovano uvode izmene u (epi)genom sa
posebnim osvrtom na CRISPR/Cas9 sistem i njegove modifikacije. Alfa ćelije endokrinog pankreasa
predstavljaju atraktivan izvor ćelija za potencijalnu terapiju dijabetesa zato što dele zajedničko poreklo
sa beta ćelijama, imaju visok nivo plastičnosti kao i zbog bliske pozicioniranosti koja obezbeđuje
prirodno okruženje pogodno za njihovo preživljavanje. Jedna od nedavnih studija je podrazumevala
upotrebu EpiCRISPR sistema za ciljano uvođenje metilacije u okviru promotora gena Arx u alfa ćelijama
pankreasa miša u cilju njihove transdiferencijacije. Uvedene izmene na nivou epigenoma su dovele
do pokretanja ekspresije insulina u alfa ćelijama pankreasa miša i inicijacije procesa njihove
transdiferencijacije u ćelije koje produkuju insulin.Diabetes is caused by a reduced number of beta cell mass, impaired functioning, or loss of beta cell
identity through the dedifferentiation process. Direct cellular reprogramming is one of the current
strategies in the potential diabetes therapy, which would replace the lack of functional beta cells and
regulate insulin levels. This research approach involves the transdifferentiation of somatic cells from
several organs into insulin-producing cells by modulating the expression pattern of transcription factors
responsible for maintaining cellular identity. This chapter will present current research involving
cellular reprogramming using the new synthetic tools that have ability to introduce targeted (epi)genetic
modifications. Special attention will be paid to the CRISPR/Cas9 system and its modifications.
Pancreatic alpha cells represent an attractive cell source for potential diabetes therapy because they
share a common origin with beta cells, have a high level of plasticity, and provide a natural environment
suitable for cell survival because of their close placement. One of the recent studies involved the
use of the EpiCRISPR system for targeted DNA methylation within the Arx gene promoter in murine
pancreatic alpha cells. The introduced changes at the epigenetic level led to the initiation of insulin
expression in the alpha cells of the mouse pancreas and the initiation of their transdifferentiation process
into insulin-producing cells
Molekularna karakterizacija mikroorganizama izolovanih iz kontaminirane životne sredine i njihova primena za degradaciju bis(2-hidroksietil) tereftalata
Get PDFPlastika kao izvor zagađenja postaje sve veći globalni problem. Većina plastike je
dizajnirana za jednokratnu upotrebu, što dodatno povećava količinu plastičnog otpada.
Usled preopterećenja zemlje i voda plastičnim otpadom neophodno je razviti procese koji
će omogućiti efikasno uklanjanje plastike iz životne sredine [1]. Polietilen-tereftalat (PET)
spada u najkorišćenije polimere za izradu plastike, te je neophodno naći efikasan način za
njegovu degradaciju. Kao povoljan metod degradacije PET plastike predlaže se enzimska
hidroliza korišćenjem mikroorganizama koji kao izvor ugljenika mogu koristiti PET [2].
Bis-(2-hidroksietil)-tereftalat (BHET) je intermedijer u izgradnji PET polimera, te je
korišćen kao model sistem za ispitivanje degradacije PET plastike [3].
Cilj ovog rada je bila identifikacija mikroorganizama koji potencijalno razgrađuju
BHET, kao i primena čistih kultura za degradaciju istog.
Tokom studije testirana je osetljivost mikroorganizama na antibiotike (penicillin i
nalidiksinsku kiselinu). Praćena je pojava zona inhibicije 24h nakon zasejavanja
mikroorganizama. Nakon antibiograma rađeno je izolovanje DNK po protokolu za Grampozitivne
i Gram-negativne bakterije. Za svaki soj mikroorganizama umnoženi su geni za
16s rRNK lančanom reakcijom polimeraze (PCR). Rađena je preparativna agarozna
elektroforeza sa PCR produktima i DNK je ekstrahovana iz gela. Uzorci su poslati na
sekvenciranje i dobijene sekvence su spojene u programu BioEdit. Dobijene sekvence su
poređene u NCBI BLAST bazi podataka i identifikovani su rodovi izolovanih
mikroorganizama.
Ispitivana je degradacija BHET-a pomoću čistih kultura mikroorganizama, gde je
korišćena mineralna podloga, a jedan od izvora ugljenika bio je BHET. Inkubacija je trajala
14 dana, a degradacija je praćena u prvom i poslednjem danu inkubacije pomoću tankoslojne
hromatografije (TLC). Korišćen je sistem rastvarača benzen:dioksan:sirćetna kiselina
(90:25:4, v/v/v). Na slici 1 su prikazani rezultati TLC-a gde su korišćeni sojevi
Pseudomonas, Bacillus i Rhodococcus. Kao najefikasniji soj pokazao se Rhodococcus. Kako je pokazano da sami mikroorganizmi mogu da potpuno ili delimično
transformišu BHET, dalji eksperimenti će se odvijati u smeru ispitivanja potpune
degradacije pomoću konzorcijuma mikroorganizama.KNJIGA IZVODA: 9. simpozijum Hemija i zaštita životne sredine Kladovo, 4-7. jun 2023. BOOK OF ABSTRACTS : 9th Symposium Chemistry and Environmental Protection Kladovo, 4-7th June 202
Hypoxia preconditioning reduces the differentiation potential of human pluripotent stem cells and alters the expression of SOX genes and miR-21
Get PDFBrain trauma leads to the induction of neural stem cell proliferation and the migration
of young neurons to injured areas. However, these neurons are insufficient to fully
restore neuronal function due to the limited potential of adult neurogenesis. This study
aimed to investigate the effect of hypoxia, a condition that underlines a wide spectrum
of brain pathologies, on pluripotency and the capacity of stem cells to differentiate
into neural progenitors. We analyzed the expression of SOX genes and microRNAs as
they control a variety of cellular processes during neuronal differentiation, including
cell proliferation and cell fate determination. In vitro neuronal differentiation of
human embryonal carcinoma cell line NT2/D1 and induced pluripotent stem cells
were used as a model system of adult neurogenesis. Cobalt chloride was used to
induce hypoxia.
The results of the analysis showed that, following hypoxia, the efficiency of neuronal
induction was significantly decreased, that coincident with decline in mRNA
expression levels of SOXB and SOXC genes. In contrast to that, the expression level of
miR-21 was significantly increased.
Our findings advance the study of SOX TFs, miR-21, and their possible interplay in
ischemia-related pathologies, establishing them as prospective biomarkers and
possible targets for future diagnostic and therapeutic approaches.BOOK OF ABSTRACTS: 8th CONGRESS OF SERBIAN NEUROSCIENCE SOCIETY with international participation 31 May – 2 June 2023. Belgrade, Serbi
Investigation of Antimicrobial, Anti-Quorum Sensing, and Cytotoxic Activities of Flavonoids Isolated from Pulicaria armena Boiss. & Kotschy ex Boiss. (Asteraceae)
No full textThis is the first report on the separation and biological assessment of all metabolites derived from Pulicaria armena (Asteraceae) which is an endemic species narrowly distributed in the eastern part of Turkey. The phytochemical analysis of P. armena resulted in the identification of one simple phenolic glucoside together with eight flavon and flavonol derivatives whose chemical structures were elucidated by NMR experiments and by the comparison of the spectral data with the relevant literature. The screening of all molecules for their antimicrobial, anti-quorum sensing, and cytotoxic activities revealed the biological potential of some of the isolated compounds. Additionally, quorum sensing inhibitory activity of quercetagetin 5,7,3’ trimethyl ether was supported by molecular docking studies in the active site of LasR which is the primary regulator of this cell-to-cell communication system in bacteria. Lastly, the critical molecular properties indicating drug-likeness of the compounds isolated from P. armena were predicted. As microbial infections can be a serious problem for cancer patients with compromised immune systems, this comprehensive phytochemical research on P. armena with its anti-quorum sensing and cytotoxic compounds can provide a new approach to the treatment
Xемијска карактеризација и антимикробна активност орашастог плода и зелене љуске Juglans nigra l.
Get PDFJuglans nigra (Black walnut) is a source of health-promoting
biologically active compounds used in traditional medicine. The investigation of
bioactive compounds in black walnut could lead to its broader application, as
well as application of its by-products. Therefore, this study aimed to characterize
J. nigra nut and green husk based on chemical analysis of their petroleum ether
and ethanol extracts obtained by ultrasonic and reflux extraction methods,
respectively. Different extract fractions were tested for their antimicrobial
activities using Gram-negative bacteria (Escherichia coli, Pseudomonas
aeruginosa), Gram-positive bacteria (Enterococcus faecalis Staphylococcus
aureus) and yeast (reference strain and clinical isolates of Candida albicans).
Ethanol extracts analysis, performed by High Performance Liquid
Chromatography (HPLC), singled out the ellagic acid as the most dominant
compound in nut (55.0 ± 1.3 × 10-3 kg m-3
) and green husk (114.1 ± 0.5× 10-3 kg
m-3
) extracts. Non-polar compounds were evaluated using Gas Chromatography
(GC) analysis of petroleum ether extracts. Juglans nigra nut and green husk
contained two saturated fatty acids, palmitic acid (C16:0) and stearic acid
(C18:0), then, monounsaturated fatty acids, palmitoleic (C16:1n-7), oleic
(C18:1n-9), and vaccenic acid (C18:1n-7), as well as polyunsaturated fatty acids,
linoleic (C18:2n-6), γ-linolenic (C18:3n-6), and α-linolenic (C18:3n-3) acids.
Ethanol extracts of both J. nigra nut and green husk showed antimicrobial
activity against C. albicans which is the most common cause of yeast infections.Juglans nigra (црни орах) као извор биолошки активних једињења користе се у
традиционалној медицини. Истраживање биоактивних једињења присутних у црном ораху
довело би до шире примене његових производа. Зато ова студија има за циљ да
окарактерише орашаст плод и зелену љуску Ј. nigra на основу хемијске анализе њихових
петролетарских и етанолних екстраката, добијених ултразвучним и рефлукс методама
екстракције. Антимикробна активност различитих фракција екстракта је тестирана
коришћењем Грам-негативних бактерија (Escherichia coli, Pseudomonas aeruginosa), Грамозитивних бактерија (Enterococcus faecalis Staphylococcus aureus) и квасница (референтни
сој и клинички изолати Candida albicans). Анализа течном хроматографијом високих
перформанси, издвојила је елагинску киселину као најдоминантније једињење у етанолним
екстрактима орашастог плода (55,0 ± 1,3 × 10-3 kg m-3) и зелене љуске (114,1 ± 0,5 × 10-3
g m-3). Неполарна једињења су процењена применом гасне хроматографске анализе
петролетарских екстракта. Орашаст плод и зелена љуска садрже две засићене масне
киселине, палмитинску (C16:0) и стеаринску киселину (C18:0), затим мононезасићене
масне киселине, палмитолеинску (C16:1n-7), олеинску (C18:1n-9), и вакценску киселину
(C18:1n-7), као и полинезасићене масне киселине, линолну (C18:2n-6), γ-линоленску
(C18:3n-6) и α-линоленску (C18: 3n-3) киселину. Етанолни екстракти орашастог плода и
зелене љуске показали су антимикробну активност нa сојевима C. albicans која је најчешћи
узрочник гљивичних инфекција
Biodegradable and active zein-gelatin-based electrospun mats and solvent-cast films incorporating sage extract: Formulation and comparative characterization
No full textThis study aimed to develop active, biodegradable materials for food packaging by incorporating sage extract (SE) within a zein-gelatin blend by electrospinning and solvent casting. The fabrication techniques, SE incorporation, and its content (5, 10% w/w) determined the materials’ properties. Electrospinning produced 0.36–0.53 mm thick, non-transparent fibrous mats (mean fiber diameter 1.12–1.36 µm). Solvent casting generated 0.34–0.41 mm thick, transparent continuous films. The analysis indicated the constituents’ compatibility, homogenous dispersion, and efficient SE incorporation without strong chemical interactions and phase separation. The solvent-cast films presented more ordered structures, higher mechanical resistance, elongation, and water vapor barrier performance than the electrospun mats. The SE-incorporating formulations showed phenolics’ delivery ability to food simulants influenced by structure, SE content, and media polarity. The electrospun mats expressed higher DPPH• radicals’ inhibition, while the solvent-cast films showed stronger Staphylococcus aureus and Escherichia coli growth inhibition, increased by SE incorporation. All formulations showed rapid complete bio-disintegration in compost (18–25 days).Relation to accepted version: [https://imagine.imgge.bg.ac.rs/handle/123456789/1921
Preparation of Juglans nigra L. husk extracts by applying an ultrasonic-assisted extraction method and process optimization of minerals extraction using response surface methodology
No full textThe aims of this research were preparation of Juglans nigra husk extracts by employing an ultrasonic-assisted extraction method and process optimization of minerals extraction using response surface methodology. Extracts from J. nigra L. fresh husk were prepared using ethanol (70%, v/v) as a solvent, at different extraction temperatures: 15, 30 and 40 °C, and extraction times: 5, 15, 30, 60 and 80 min. The content of minerals in ethanol extracts was determined using inductively coupled plasma optical emission spectrometry (ICP-OES). The response surface methodology (RSM) was developed to evaluate the effect of two extraction factors as well as to optimize the extraction variables for each mineral individually. The RSM models pointed out 40 °C temperature and extraction time in the range 77–80 min as optimal conditions for extraction of macro- and micro-elements. Results of RSM optimization showed the relative order of maximum obtained yield of macro-elements: K>P>S>Ca>Mg and maximum yield of micro-elements was Si>Zn>Sr>Cu.Relation to accepted version: [https://imagine.imgge.bg.ac.rs/handle/123456789/1922
Metagenomic Analysis of Bacterial Community and Isolation of Representative Strains from Vranjska Banja Hot Spring, Serbia
No full textThe hot spring Vranjska Banja is the hottest spring on the Balkan Peninsula with a water temperature of 63–95 °C and a pH value of 7.1, in situ. According to the physicochemical analysis, Vranjska Banja hot spring belongs to the bicarbonated and sulfated hyperthermal waters. The structures of microbial community of this geothermal spring are still largely unexplored. In order to determine and monitor the diversity of microbiota of the Vranjska Banja hot spring, a comprehensive culture-independent metagenomic analysis was conducted in parallel with a culture-dependent approach for the first time. Microbial profiling using amplicon sequencing analysis revealed the presence of phylogenetically novel taxa, ranging from species to phyla. Cultivation-based methods resulted in the isolation of 17 strains belonging to the genera Anoxybacillus, Bacillus, Geobacillus, and Hydrogenophillus. Whole-genome sequencing of five representative strains was then performed. The genomic characterization and OrthoANI analysis revealed that the Vranjska Banja hot spring harbors phylogenetically novel species of the genus Anoxybacillus, proving its uniqueness. Moreover, these isolates contain stress response genes that enable them to survive in the harsh conditions of the hot springs. The results of the in silico analysis show that most of the sequenced strains have the potential to produce thermostable enzymes (proteases, lipases, amylases, phytase, chitinase, and glucanase) and various antimicrobial molecules that can be of great importance for industrial, agricultural, and biotechnological applications. Finally, this study provides a basis for further research and understanding of the metabolic potential of these microorganisms.Related to accepted version: [https://imagine.imgge.bg.ac.rs/handle/123456789/1931
Seed priming enhances tolerance and accumulation of heavy metals in cd hyperaccumulator silene sendtneri through changes in gene expression
Get PDFTo improve our understanding of the molecular mechanisms underlying seed priming, RNA
transcriptome analysis was performed using primed and non-primed seeds of Silene sendtneri. Seed
priming was performed by submerging the seeds in different priming agents for 24h at 4°C, followed
by rinsing with sterilized water and desiccation to the original moisture content. Silene sendtneri is a
species with no sequenced genome and annotation of de novo assembly of the transcriptome was done
using different species for the reference genome. Gene ontology (GO) analysis indicated that genes
related to heavy metal transporters and heat shock proteins are differentially expressed after priming in
relation to the used priming agent. Within these gene categories, genes such as heavy metal-associated
isoprenylated plant protein 26-like (log2fold -8.79) were downregulated, while others such as heavy
metal ATPase 5 (log2fold 6.46), heat shock factor protein HSF30-like isoform X1 (log2fold 5,98) were
upregulated. Some gene categories are commonly highly expressed after seed priming regardless of the
used agent, but some specific gene categories have been recorded to be expressed in relation to the used
agent.Book of abstracts: International Conference of Biochemists and Molecular Biologists in Bosnia and Herzegovina - ABMBBIH May, 202
Preparation of Juglans nigra L. husk extracts by applying an ultrasonic-assisted extraction method and process optimization of minerals extraction using response surface methodology
No full textThe aims of this research were preparation of Juglans nigra husk extracts by employing an ultrasonic-assisted extraction method and process optimization of minerals extraction using response surface methodology. Extracts from J. nigra L. fresh husk were prepared using ethanol (70%, v/v) as a solvent, at different extraction temperatures: 15, 30 and 40 °C, and extraction times: 5, 15, 30, 60 and 80 min. The content of minerals in ethanol extracts was determined using inductively coupled plasma optical emission spectrometry (ICP-OES). The response surface methodology (RSM) was developed to evaluate the effect of two extraction factors as well as to optimize the extraction variables for each mineral individually. The RSM models pointed out 40 °C temperature and extraction time in the range 77–80 min as optimal conditions for extraction of macro- and micro-elements. Results of RSM optimization showed the relative order of maximum obtained yield of macro-elements: K>P>S>Ca>Mg and maximum yield of micro-elements was Si>Zn>Sr>Cu.This is the peer-reviewed version of the article: [Milić, P., Rajković, K., Nikolić, G., Jeremić, S.,& Đurašević, M.. (2023). Preparation of Juglans nigra L. husk extracts by applying an ultrasonic-assisted extraction method and process optimization of minerals extraction using response surface methodology. in Chemical Engineering Research and Design, 191, 439-445. https://doi.org/10.1016/j.cherd.2023.01.051