imagine (Institute of molecular genetics and genetic engineering)
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Pomegranate Peel Extract Differently Modulates Gene Expression in Gingiva-Derived Mesenchymal Stromal Cells under Physiological and Inflammatory Conditions
Get PDFPomegranate has shown a favorable effect on gingivitis/periodontitis, but the mechanisms involved are poorly understood. The aim of this study was to test the effect of pomegranate peel extract (PoPEx) on gingiva-derived mesenchymal stromal cells (GMSCs) under physiological and inflammatory conditions. GMSC lines from healthy (H) and periodontitis (P) gingiva (n = 3 of each) were established. The lines were treated with two non-toxic concentrations of PoPEX (low—10; high—40 µg/mL), with or without additional lipopolysaccharide (LPS) stimulation. Twenty-four genes in GMSCs involved in different functions were examined using real-time polymerase chain reaction (RT-PCR). PoPEx (mostly at higher concentrations) inhibited the basal expression of IL-6, MCP-1, GRO-α, RANTES, IP-10, HIF-1α, SDF-1, and HGF but increased the expression of IL-8, TLR3, TGF-β, TGF-β/LAP ratio, IDO-1, and IGFB4 genes in H-GMSCs. PoPEx increased IL-6, RANTES, MMP3, and BMP2 but inhibited TLR2 and GRO-α gene expression in P-GMSCs. LPS upregulated genes for proinflammatory cytokines and chemokines, tissue regeneration/repair (MMP3, IGFBP4, HGF), and immunomodulation (IP-10, RANTES, IDO-1, TLR3, COX-2), more strongly in P-GMSCs. PoPEx also potentiated most genes’ expression in LPS-stimulated P-GMSCs, including upregulation of osteoblastic genes (RUNX2, BMP2, COL1A1, and OPG), simultaneously inhibiting cell proliferation. In conclusion, the modulatory effects of PoPEx on gene expression in GMSCs are complex and dependent on applied concentrations, GMSC type, and LPS stimulation. Generally, the effect is more pronounced in inflammation-simulating conditions
Antibiotic potential of the Ambigol Cyanobacterial natural product class and simplified synthetic analogs
No full textThe ambigols are cyanobacterial natural products characterized by three polychlorinated aromatic building blocks connected by biaryl and biaryl ether bridges. All ambigols known to date possess promising biological activities. Most significantly, ambigol A was reported to have antibacterial activity against Gram-positive bacteria, such as Bacillus megaterium and B. subtilis. We established a diverse compound library for in-depth biological evaluation building on our previous bio- and total synthetic research on this natural product family. To explore the antimicrobial potential in detail and to determine initial structure–activity relationships of this product class, a large set of dimeric and trimeric compounds were screened against selected bacterial and Candida target strains. Our results reveal exceptional antibiotic activity of the ambigols, especially against challenging clinical isolates
Asocijacija varijanti u genima PRMT6, PEX10 I SOX5 sa idiopatskim muškim sterilitetom: dokazi iz populacije Severne Makedonije i ažurirana meta-analiza
Get PDFPRMT6, PEX10 and SOX5 genetic variants were identified as male infertility-associated loci in a genome-wide association study and further validated in various populations. Still, the results of previous case-control studies varied, which could be due to differences in participants’ ethnic backgrounds. The main purpose of the present study was to evaluate the supposed association of these variants with idiopathic male infertility in North Macedonian population. Furthermore, we aimed to conduct the systematic quantitative data synthesis which includes the results of previous studies on the same issue in other European and non-European populations. A total of 137 men from North Macedonia diagnosed with idiopathic infertility and 130 age-matched fertile controls were included in the present case-control study. PCR-RFLP method was used for genotyping. Meta-analysis was performed by OpenMeta-analyst statistical software. Variants rs10842262 in SOX5, rs2477686 in PEX10 and rs12097821 in PRMT6 showed the lack of statistically significant differences in genotype distributions between men diagnosed with idiopathic infertility and the control group. Still, rs10842262 allele G frequency was significantly increased in men with poor sperm concentration (P= 0.024, OR = 2.10, 95%CI 1.08-4.06). Meta-analysis further showed the association of rs10842262 and rs12097821 with the risk of idiopathic male infertility. Our results obtained in North Macedonian population supported the previous reports on the involvement of rs10842262 in the genetic basis of male infertility. The meta-analysis confirmed the association of rs10842262 and rs12097821 with male infertility occurrence. Still, additional studies are needed to support the present findings.Asocijacija varijanti u genima PRMT6, PEX10 i SOX5 sa muškim sterilitetom identifikovana je u studiji genetičke asocijacije na čitavom genomu i kasnije analizirana u studijama slučajeva i kontrola u različitim populacijama. Rezultati prethodnih studija su pokazali značajnu varijabilnost, što može biti posledica razlika u etničkom poreklu studijskih grupa. Osnovni cilj ovog istraživanja je analiza asocijacije navedenih genetičkih varijanti sa rizikom za pojavu idiopatskog muškog steriliteta u populaciji Severne Makedonije. Takođe, naš cilj je bio i sprovođenje sistematske kvantitativne sinteze podataka iz studija sa istom ili sličnom temom istraživanja sprovedenim u drugim evropskim i neevropskim populacijama. Ukupno 137 muškaraca sa idiopatskim sterilitetom iz Severne Makedonije i 130 fertilnih kontrola slične starosti uključeno je u studiju slučajeva i kontrola. Genotipizacaija je vršena PCR-RFLP metodom, dok je za meta-analizu korišćen statistički softver OpenMeta-analyst. Za varijante rs10842262 u SOX5, rs2477686 u PEX10 i rs12097821 u PRMT6 nije utvrđena statistički značajna razlika u distribucijama genotipova između grupe ispitanika sa idiopatskim sterilitetom i kontrolne grupe. Međutim, učestalost alela G varijante rs10842262 bila je značajno povećana kod muškaraca sa niskom koncentracijom spermatozoida (P= 0.024, OR = 2.10, 95%CI 1.08– 4.06). Meta-analizom pokazana je asocijacija rs10842262, ali i rs12097821, sa rizikom za razvoj idiopatskog muškog steriliteta. Naši rezultati ustanovljeni u populaciji Severne Makedonije idu u prilog prethodnim navodima o učešću rs10842262 u genetičkoj osnovi muškog steriliteta. Ipak, dodatne studije su neophodne kako bi potvrdile značaj rezultata ovog istraživanja
Functional characterization of DSS1(I) and DSS1(V) in response to oxidative stress in Arabidopsis thaliana
Get PDFDSS1 je mali, visoko konzervisan protein i pripada familiji prirodno neuređenih proteina. Novija
istraživanja su otkrila ulogu DSS1 u procesu DSSilacije, koja podrazumeva specifično označavanje
oksidovanih proteina za uklanjanje putem proteazomnog sistema. Genom Arabidopsis thaliana sadrži
dva visoko homologna gena DSS1(I) i DSS1(V). Najbolje proučena uloga DSS1 u biljnom svetu je u
održavanju integriteta DNK kroz homolognu rekombinaciju (HR). Cilj ovog rada je bio da se
identifikuju funkcionalne različitosti između proteina DSS1 sa fokusom na njihovu potencijalnu
ulogu u oksidativnom stresu. Istraživanje je zasnovano na ispitivanju različitih dss1 mutantnih linija
u model sistemu A. thaliana, kroz uporednu analizu sa biljkama divljeg tipa, u normalnim fiziološkim
uslovima i uslovima oksidativnog stresa. Pokazano je da t-dss1(V) T-DNK insercioni mutanati imaju
brže klijanje, dok u kasnijim fazama zaostaju u razviću. Dodatno, uspostavljene su pojedinačne linije
mutanata dss1(I).19 i dss1(V).20 sa velikim indelima pomoću precizne tehnologije CRISPR/Cas9.
Kod ovih mutanata su uočene razlike u dužini korena, stabla i površini rozete. Utvrđeno je da su
mutanati t-dss1(V) (knockdown) i dss1(V).20 (knockout) visoko osetljivi na oksidativni stres. Takođe,
nemogućnost generisanja dss1 duplih mutanata ukazuje na njihovu esencijalnu ulogu u biološkim
procesima. Pored toga, pokazano je da samo overekspresija AtDSS1(I) kroz test funkcionalne
komplementacije dovodi do povećanja rezistencije Δdss1 mutanta Ustilago maydis na ispitivane
genotoksične agense, što ukazuje na evolutivnu očuvanost njegove uloge u popravci DNK putem HR.
U skladu sa navedenim, postoji funkcionalna razlika između biljnih gena DSS1 i može se smatrati da
je protein DSS1(V) ključna komponenta u biljnom odgovoru na oksidativni stres.DSS1 is a small, highly conserved protein and a member of the intrinsically disordered protein family.
Recent studies revealed the role of DSS1 in a process called DSSylation, which represents the specific
marking of oxidatively damaged proteins for removal by the proteasome system. The Arabidopsis
genome contains two highly homologous genes DSS1(I) and DSS1(V) and role in maintaining DNA
integrity through homologous recombination is the only well-studied function in plants. The aim of
this work was to identify functional differences between DSS1 proteins by focusing on their potential
roles in oxidative stress and considering that plants are sessile organisms exposed to harmful
environmental influences. This study is based on examining various dss1 mutant lines in the model
system A. thaliana through comparative analysis with wild-type plants, under normal physiological
conditions and oxidative stress. It was shown that t-dss1(V) T-DNA insertion mutants have faster
germination, while having developmental delay in further stages. Additionally, single dss1(I).19 and
dss1(V).20 mutant lines with large indels were generated using precision CRISPR/Cas9 technology.
The characterization of these mutant lines revealed differences in root and stem lengths, and rosette
area size. t-dss1(V) (knockdown) and dss1(V).20 (knockout) mutants showed an increased sensitivity
to oxidative stress. In this work, the dss1 double mutant was not obtained. Also, only overexpressing
AtDSS1(I) through functional complementation test increases resistance of the Δdss1 mutant of U.
maydis to the exposed genotoxic agents. This indicates its conserved role in DNA repair via HR. Our
study showed functional differences between plants DSS1 genes and a potentially important
component DSS1(V) in plant oxidative stress response
Bryophyte extracts as immunomodulators
Get PDFBriofite (mahovine, jetrenjače i rožnjače) predstavljaju drugu najveću grupu kopnenih biljaka posle cvetnica,
sa više od 20 000 vrsta rasprostranjenih širom sveta. Iako se od davnina koriste u tradicionalnoj medicini,
tek je u skorije vreme počelo intenzivnije istraživanje njihovog hemijskog sastava, kao i potencijalnih
bioloških aktivnosti. Među mnogobrojnim aktivnim komponentama briofita, polifenoli i triterpeni predstavljaju
najvažnije grupe jedinjenja, koja su ujedno i nosioci njihove biološke aktivnosti. Antioksidativni,
anti-inflamatorni, antitumorski, antineurodegenerativni, antimikrobni, antidijabetični i proregenerativni
potencijal ekstrakata različitih briofita dokazan je u velikom broju studija. Ovaj pregledni rad ima za cilj da
predstavi najnovija istraživanja o hemijskom sastavu i biološkimaktivnostima ekstrakata briofita sa posebnim
osvrtom na mahovine Hypnum cupressiforme i Hedwigia ciliata i njihovoj potencijalnoj upotrebi kao
imunomodulatora u prevenciji i/ili terapiji različitih bolesti čoveka.Bryophytes (mosses, liverworts, and hornworts) are the second largest group of land plants after flowering
plants, with more than 20,000 species distributed worldwide. Although they have been used in traditional
medicine since ancient times, their chemical composition and potential biological activities have
only recently begun to be studied more intensively. Among the numerous bioactive constituents of moss
plants, polyphenols and triterpenes represent the most important groups of compounds, which are also
carriers of their biological activity. The antioxidant, anti-inflammatory, antitumor, antineurodegenerative,
antimicrobial, antidiabetic, and proregenerative potential of various moss extracts have been confirmed
in a large number of studies. This review presents the latest research on the chemical composition and biological
activity of bryophyte extracts with special reference to the mosses Hypnum cupressiforme and
Hedwigia ciliata and their potential use as immunomodulators in the prevention and/or therapy of various
human diseases
Importance of pharmacogenetics for ischemic stroke therapy with recombinant tissue plasminogen activator
Get PDFIshemijski moždani udar (IMU) je praćen visokom incidencijom invaliditeta i mortaliteta širom sveta. Trombolitička
terapija rekombinovanim tkivnim aktivatorom plazminogena (rtPA) predstavlja jedinu odobrenu
terapiju u lečenju akutnog IMU (AIMU). Pokazano je da primena rtPA terapije dovodi do boljeg funkcionalnog
oporavka pacijenata nakon AIMU. Međutim, kod određenog broja pacijenata, rtPA terapija je praćena
neželjenim efektima, što značajno utiče na njihov oporavak.
Genske varijante, ne samo da utiču na predispoziciju za IMU, već i na terapijski odgovor i klinički ishod
same bolesti. Usled velikih interindividualnih varijacija u odgovoru na primljenu trombolitičku terapiju,
farmakogenetika se sve više fokusira na ispitivanje uticaja genskih varijanti nakon primene pomenute terapije
za lečenje AIMU, imajući u vidu da su već neke polimorfne varijante povezane sa različitim odgovorom
na rtPA terapiju, tokom same bolesti, ali i javljanjem rtPA-indukovanih hemoragijskih komplikacija.
U poslednjih 20 godina, farmakogenetičke rtPA studije su pokušavale da pronađu genske faktore rizika
povezane sa odgovorom na rtPA terapiju. Većina ovih studija koristila je studije ispitivanja gena kandidata.
Međutim, nedavne sudije asocijacije kompletnog genoma su ukazale na to da bi genska predispozicija
mogla da utiče na rtPA odgovor.
Cilj ovog rada je prikaz dosadašnjih rezultata o genima i genskim varijantama povezanih sa hemoragijskim
komplikacijama i stepenom rekanalizacije nakon rtPA terapije kod pacijenata sa AIMU, kao i doprinosa
doktorske distertacije pod nazivom ,,Ispitivanje povezanosti polimorfizama gena koji regulišu fibrinolizu
i integritet vanćelijskog matriksa sa efektima terapije ishemijskog moždanog udara rekombinovanim tkivnim
aktivatorom plazminogena’’ postojećim znanjima o uticaju genskih varijanti na oporavak pacijenata
nakon AIMU lečenog rtPA terapijom.Ischemic stroke (IS) is a major global health concern with high rates of disability and mortality. The approved
treatment for acute ischemic stroke is thrombolytic therapy, which involves using recombinant
tissue plasminogen activator (rtPA). While rtPA therapy has been shown to improve functional recovery in
patients, it also has drawbacks and can lead to adverse effects that hinder patient recovery.
Genetic variations not only affect the susceptibility to ischemic stroke but also play a role in determining
the response to rtPA treatment and clinical outcomes. To better understand the individual variability in response
to thrombolytic therapy, pharmacogenetic studies are focusing on investigating the influence of
genetic variations. These studies aim to identify specific genetic variants associated with different responses
to rtPA therapy and the occurrence of rtPA-induced hemorrhagic complications.
Traditionally, candidate gene strategies have been used in pharmacogenetic studies of rtPA. However, recent
advancements in genome-wide association studies (GWAS) have suggested that genetic predisposition
may have an impact on the response to rtPA.
This study aims to present current findings on genes and genetic variations linked to hemorrhagic transformation
and recanalization rates following rtPA therapy in patients with acute ischemic stroke. It also
highlights insights provided by a doctoral dissertation titled “Analysis of the association between polymorphisms
within genes which regulate fibrinolysis and extracellular matrix integrity with the effects of
ischemic stroke therapy with recombinant tissue plasminogen activator”. The research contributes to our
understanding of how gene variants can affect patient recovery after acute ischemic stroke treated with
rtPA therap
Hybridosomes from spruce needle homogenate
Get PDFIntroduction: Being of compatible structure with biomembranes, lipid–based nanoparticles are considered
as convenient platforms for drug delivery systems. In the proposed work we considered formation of lipid
nanovesicles associated with bioactive phytochemicals from spruce needle homogenate (here called
hybridosomes). We formed hybridosomes by mixing appropriate amounts of lecithin, supernatant of isolation
of extracellular particles from spruce needle homogenate and glycerol.
Methods: We visualized hybridosomes by light microscopy and cryogenic transmission electron microscopy
and assessed them by flow cytometry, dynamic light scattering, ultraviolet–visual spectroscopy and
interferometric microscopy.
Results: We found that the particles consisted of a bilayer membrane and a fluid-like interior. Flow cytometry
and interferometric light microscopy measurements showed that the majority of the particles were
nano-sized. Dynamic light scattering and interferometric light microscopy measurements agreed well with the
determined average hydrodynamic radius of the particles Rh (between 140 and 180 nm) while their number
densities were in the range between 10^13 and 10^14/mL indicating that hybridosomes present about 2/3 of the
mixture, excluding solvent and other small molecules.
Discussion: Simple and low-cost preparation method, non-demanding saving process and efficient
formation procedure suggest that large scale production of hybridosomes from lipids and spruce needle
homogenate is feasible.Small New World 2.0 4-5 September 2023., Graz, Austri
Extracellular vesicles from blood plasma as mediators of anti-inflammatory effects, oxidative stress and angiogenesis in HUVEC
Get PDFIntroduction: Blood plasma is used in regenerative medicine for more than 30 years as it was found to have
impact on angiogenesis and proliferation of endothelial cells. This effect was initially ascribed to growth factors
and cytokines, however, particularly in the last decade researchers are focusing on platelet-derived
extracellular vesicles (PEVs) that are also present in the platelet rich plasma.
Methods: We prepared platelet and extracellular vesicles-rich plasma (PVRP) by centrifugation of the
human blood, and differential (ultra) centrifugation of PVRP to isolate PEVs. We exposed Human Umbilical Vein
Endothelial Cells (HUVEC) to 5% PEVs for 24 hours and assessed inflammation markers (Interleukin(IL)-1beta,
IL-6 and Tumor necrosis factor (TNF)-alpha using ELISA tests), oxidative stress markers (Cholinesterase
(ChE) and glutathione S-transferase (GST) activity by spectrophotometry, as well reactive oxygen species
(ROS) and lipid droplets (LD) by flow cytometry). We observed morphological changes in HUVEC indicating
angiogenesis by using optical microscopy.
Results: We found that after 24 hours caused a decrease of the concentration of IL-6, IL-1beta and TNF-alpha.
Also, ChE and GST activity and ROS count were decreased. LD production, which is triggered in need to protect
the cells from free radicals and oxidative stress damage, was however higher. Treatment of the cells had
impact on cell morphology with progressed formation of the tubes and cell connecting, which is regarded as the
beginning of HUVEC angiogenesis process.
Discussion: Beneficial effect of PVRP in healing and regeneration may include suppression of inflammation
and oxidative stress by PEVs.Small New World 2.0 4-5 September 2023., Graz, Austri
Germline Variants in Cancer Predisposition Genes in Pediatric Patients with Central Nervous System Tumors
Get PDFCentral nervous system (CNS) tumors comprise around 20% of childhood malignancies. Germline variants in cancer predisposition genes (CPGs) are found in approximately 10% of pediatric patients with CNS tumors. This study aimed to characterize variants in CPGs in pediatric patients with CNS tumors and correlate these findings with clinically relevant data. Genomic DNA was isolated from the peripheral blood of 51 pediatric patients and further analyzed by the next-generation sequencing approach. Bioinformatic analysis was done using an “in-house” gene list panel, which included 144 genes related to pediatric brain tumors, and the gene list panel Neoplasm (HP:0002664). Our study found that 27% of pediatric patients with CNS tumors have a germline variant in some of the known CPGs, like ALK, APC, CHEK2, ELP1, MLH1, MSH2, NF1, NF2 and TP53. This study represents the first comprehensive evaluation of germline variants in pediatric patients with CNS tumors in the Western Balkans region. Our results indicate the necessity of genomic research to reveal the genetic basis of pediatric CNS tumors, as well as to define targets for the application and development of innovative therapeutics that form the basis of the upcoming era of personalized medicine
DNA/BSA binding affinity of pyocyanin produced by Pseudomonas aeruginosa
Get PDFPyocyanin (PYO) is a green blue pigment that is produced extracellularly by the Gram- negative bacteria Pseudomonas aeruginosa. Its color depends on pH value. It exists in blue zwitterion form at neutral and alkaline conditions, while in an acidic environment, it becomes pink after protonation. PYO has shown the antibacterial activity, as well as the ability to inhibit the growth of fungi like Aspergillus fumigatus and Candida albicans. Moreover, it shows the high cytotoxic effect against the human pancreatic cancer cells by inducing their apoptosis. To evaluate the possible mechanism of antimicrobial activity of PYO, in the present study, we have investigated its interactions with calf thymus DNA (ct-DNA) and bovine serum albumin (BSA) by fluorescence emission spectroscopy. The obtained value of binding constant to BSA is relatively high (KA = 5.3 × 10^6 M^-1 s^-1), showing the ability of PYO to bind to this transport protein. We have also used synchronous fluorescence spectroscopy to explore the structural changes in BSA in the presence of the studied biopigment. In contrast with the mentioned results for binding to BSA, PYO has shown a low affinity to ct-DNA, what can be seen from the value of its binding constant (KA = 7.8 × 10^3 M^-1 s^-1).Presented at the 9th International Electronic Conference on Medicinal Chemistry, 1–30 November 2023; 13
Available online: [https://ecmc2023.sciforum.net/