imagine (Institute of molecular genetics and genetic engineering)
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Bacteria in drinking and bathing mineral waters of Serbia with polymer-degrading potential
Get PDFThree mineral water occurrences, captured by wells with a depth of 6.5-442.5 m and used for drinking
and bathing purposes, were sampled and cultivated under conditions favouring the growth of anaerobic,
microaerophilic or CO2 bacteria, in order to capture predominantly anaerobic portion of the bacteriome, which is
dominant in water and soils. Cultivated bacteria were identified by next-generation 16S sequencing and their
biotechnological potential in plastics and lignocellulose degradation was explored. Most abundant genera
detected in examined samples mainly belong to facultative anaerobes that are common representatives of water
and soil environments. In total, 17 genera were detected with a relative abundance over 1% in all three samples,
including Aeromonas, Exiguobacterium, Comamonas and Acinetobacter. Half of the screened isolates demonstrated
growth on at least one plastic or lignocellulosic polymer, with one isolate demonstrating growth on all tested
substrates, one demonstrating carboxymethyl cellulose- and one arabinoxylan-degrading ability. Some of the
representatives of genera identified with high relative abundance in mineral water samples, such as Aeromonas,
Klebsiella, Escherichia, Salmonella, Enterobacter, Pseudomonas and Staphylococcus, have been previously documented
to have pathogenic potential. Due to the use of investigated mineral waters for drinking and bathing, the health
risk from such bacteria in these occurrences needs to be continuously monitored, while, on the other hand,
mineral waters deserve special attention in the future from the aspect of screening for biotechnologically
relevant enzymes.2nd International Conference on Chemo and BioInformatics, ICCBIKG 2023, September 28-29, 2023 Kragujeva
The story of the SOX genes: for better or for worse…
Get PDFThe SOX genes encode a group of transcription factors showing diverse expression patterns during de- velopment and acting as key regulators of diverse cellular processes. SOX transcription factors are in- volved in multiple events from the maintaining of stem cells pluripotency and cell fate decision to driving terminal differentiation of cells into specialized cell types. During adulthood SOX transcription factors control various physiological processes. Mutations in SOX genes have been associated with severe clin- ical disorders, while deregulation of their expressions cause a broad range of pathological condition. Ac- cumulating evidence suggests that SOX proteins act as oncogenes and recent evidence points toward pro-proliferative, pro-survival and/or anti-differentiation roles of the SOX proteins.
The results of long-term research of the structure, regulation of expression and the function of selected SOX genes will be presented. It will include data obtained by studying the roles of SOX genes in in vitro neural differentiation of pluripotent embryonal carcinoma cells, as well as interaction of SOX transcrip- tion factors with signalling pathways active during neurogenesis and oncogenesis. Special focus will be made on ongoing research focused on the roles of SOX genes in promotion of malignant phenotype of cancer cells and maintaining of cancer stem cells. The interplay of SOX transcription factors and mi- croRNAs in the brain under physiological and pathological conditions, along with crosstalk between SOX genes and long non-coding RNAs in glioblastoma will be discussed. The role of SOX transcription factors in ageing and age-related diseases will be outlined
Determination of muscle fiber types expressing ANKRD2
Get PDFIntroduction: Ankyrin Repeat Domain 2 (ANKRD2) is expressed in skeletal muscle, where plays a role in
muscle development, differentiation and adaptation to stress. Human skeletal muscle consists of three
major fiber types: type 1 (slow-twitch, oxidative), type 2A (fast-twitch, oxidative) and type 2X (fast-twitch,
glycolytic). ANKRD2 is reported to be primarily expressed in type 1 myofibers. However, recent findings
on human single myofibers and our study of chicken muscles have shown that this protein may also be
expressed in type 2A fibers. Hence, our objective was to examine whether ANKRD2 is present in human
fast, type 2A muscle fibers using immunohistochemistry.
Methods: Samples of large leg musclessoleus, gastrocnemius, vastusintermedius and vastuslateralis were
obtained from human cadaveric tissue. Serial cryosections were independently stained with anti-ANKRD2
and antibodies for different myosin heavy chain isoforms (6H1 for type 2X, BF35 for type 1 and 2A, antiMHCs for type 1 and anti-MHCf for type 2A and 2X fibers). Immunostained tissues were analyzed by fluorescent microscopy.
Results: In addition to slow, type 1, ANKRD2 wasfound expressed in fast, type 2A myofibers, which both
have oxidative metabolism. Further, we did not observe ANDRD2 expression in glycolytic, type 2X
myiofibers. This pattern of ANKRD2 expression was consistent across all examined muscles.
Conclusion: Our resultsimplicate that the regulatory mechanism of ANKRD2 expression in human skeletal muscle is associated with oxidative metabolism, rather than muscle contraction speed
A novel YtnP lactonase reduces the expression of p. aeruginosa MMA83 quorum sensing andvirulence factors gene expression
Get PDFIntroduction: Quorum quenching (QQ) isthe enzymatic degradation of cell-to-cellsignaling molecules.In this study, the potential of the novel YtnP lactonase, the quorum quenching enzyme derived from S.maltophilia, to reduce P. aeruginosa quorum sensing and virulence factor gene expression was investigated.Methods: MMA83 culture (adjusted to 1.5x105 CFU/ml) was treated with recombinant YtnP lactonase(final concentration 50 μg/ml) at 37°C for 12 hours under aeration. RNA isolation of the treated and untreated MMA83 culture was performed using the RNeasy Mini Kit (Qiagen, Germany) according to theprotocol. Quantitative reverse transcription-polymerase chain reaction (RT-qPCR), was used to analyzethe effect ofYtnP lactonase on the relative mRNA levels of the LasI/LasR, RhiI/RhiR, and PQS signaling network genes of P. aeruginosa MMA83 and virulence factor genes. The rpsL was used as an endogenouscontrol to normalize obtained data following the 2-ΔΔCt method.Results: The QS genes belonging to three QS networks – LasI/LasR, RhiI/RhiR, and PQS of P. aeruginosaMMA83 treated with YtnP lactonase were significantly downregulated. The RT -qPCR results show thattreatment with YtnP-lactonase decreased the relative mRNA levels of genes involved in the productionof elastase (lasB approximately 2-fold), alginate (algK approximately 2.2-fold), pyocyanin (phzM approximately 3.5-fold), pyoverdin (pvdS approximately 2-fold), and rhamnolipid (rhlC approximately 4-fold).These results suggest that YtnP lactonase exerts an antivirulence effect at the transcription level.Conclusion: YtnP lactonase, a quorum quenching (QQ) enzyme, has the potential to be used as an innovative enzyme-based antivirulence therapeutic to combat infections caused by P. aeruginosa
NUDT15 as potential marker for pharmacogenetic-guided 6-mercaptopurine therapy in children with acute lymphoblastic leukemia in Serbia
Get PDFIntroduction: The NUDT15 is new pharmacogene of importance for 6-mercaptopurine therapy, given to
children with acute lymphoblastic leukemia (ALL). The association ofside effectsin children with variants
in NUDT15 are well established in Asian populations, yet the relevance ofthis pharmacogene in European
populationsremainslargely unexplored. The aim of thisstudy wasto identify pharmacogenetic variants
in coding and neighbouring regions of NUDT15 gene and analyse if the expression levels of NUDT15 can
predict the occurence of side effects of 6-mercaptopurine during the maintenance therapy in children
with ALL of Serbian origin.
Methods: The genotyping of coding and neighbouring regions of NUDT15 gene was performed using
PCR and Sangersequencing based technology in 48 children with ALL. NUDT15 expression was analyzed
in mononuclear cells of 24 ALL patients at diagnosis and 6 healthy controls by qRT-PCR, and association
with surogate markers was assessed using adequate statistical methodology.
Results: The genotypig revealed the presence of 5 variantsin NUDT15 (NUDT15(NM_018283.4):c.36A>C,
NUDT15(NM_018283.4):c.158+117C>T,NUDT15(NM_018283.4):c.158+174G>A,NUDT15(NM_018283.4):c.159-
91G>A,NUDT15(NM_018283.4):c.*7G>A), none of them with effects on the expression or the function of
NUDT15 protein. There was no statistically significant association between the expression of NUDT15 at
diagnosis and the surogate markers of side effects (number of episodes of leukopenia (p=0.821), number of weeks without therapy (p=0.507), number of weeks with lower dose (p=0.434), average doses
(p=0.374)) of 6-mercaptopurine during the maintenance therapy.
Conclusion: Presently, NUDT15 cannot be used as a pharmacogene in predicting the toxicity of 6-mercaptopurine terapy in children with ALL in Serbia
Short chain fatty acid producing faecalimonas sp. NGB245 isolated from human gut modulates neurosignaling in Caenorhabditis elegans
Get PDFIntroduction: Gut-brain axis has been identified as an important target for prevention of neurodegenerative and psychiatric disease. To date, specific microbial strains resident in the intestinal ecosystem
have been described to modulate several behaviour-related functions in the host. Faecalimonas sp. is
anaerobic bacteria affiliated with the family Lachnospiraceae, which represents a highly prevalent beneficial bacteria in the human gut and have potential to be used as next generation probiotic.
Methods: Faecalimonas sp. NGB245 was isolated from human fecal material by pre-inoculation in
BACTEC media followed by serial dilutions spreading on Columbia Blood Agar supplemented with cysteine and sodium thioglycolate in Whitley Anaerobic Workstation. Production of short chain fatty acid
(SCFA) was detected after bacterial growth in Columbia broth supplemented with cellobiose by HPLC.
Host response was followed on Caenorhabditis elegans model by evaluated expression of the genes involved in neurosignaling by qPCR.
Results: We showed that Faecalimonassp. NGB245 exhibits high capacity of production of SCFA including acetate (12,17 mM), propionate (3,02 mM) and butyrate (10,33 mM). Moreover, C. elegansfed with Faecalimonas sp. NGB245 showed higher expression of the genes involved in neurotransmitter synthesis
(tph-1, cat-2), neurotransmitter release (unc-64, snb-1, snt-1), neurotransmitter receptor (npr-1) and different classes of neuropeptides(flp-18, flp-21, nlp-28, nlp-29) in comparison to wormsfed with Escherichia
coli OP50, as a standard laboratory food.
Conclusion: The obtained results imply that Faecalimonas sp. NGB245 isolate could be considered as
next generation probiotic to be used in prevention and treatment of neurodegenerative and psychiatric
diseases
AdeABC efflux pump-mediated resistance to tigecycline in Acinetobacter baumannii isolates from Balkan hospitals
Get PDFIntroduction: Multidrug-resistant (MDR) Acinetobacter baumannii has been recognized as one of the
most serious healthcare challenges worldwide. Although tigecycline represents one of the last resort
therapies for MDR A. baumannii, resistance to this antibiotic has been reported and mostly is mediated
by AdeABC efflux pump. The aim of our study was to investigate the molecular mechanism responsible
for tigecycline resistance of thirty-seven A. baumannii isolates from Balkan medical settings (Serbia,
Bosnia and Herzegovina and Montenegro) gathered in 2016 and 2022.
Methods: Minimal inhibitory concentration (MIC) values for tigecycline were determined using microdilution method according to EUCAST guidelines. Inhibition of the efflux of tigecycline wastested by
the same method using a combination of antibiotic and efflux pump inhibitor (CCCP). Amino acid alternations within AdeS and AdeR proteins were detected by comparing to sequences of referent isolates
ATCC19606 and ATCC17978. Expression of the adeB gene ofselected isolates was monitored by RT-qPCR.
Results: All tested isolates were resistant to tigecycline and showed significant decrease in tigecycline
MIC values in presence of CCCP (≥16-fold reduction) indicating that antibiotic efflux is responsible for
tigecycline resistance. The analysis of two-component system AdeRS, regulatory system of RND efflux
pump AdeABC, revealed that most of the isolates have G186V and N268H alternations in AdeS (n=32),
while most common changes in AdeR were V120I and A136V (n=29). In addition, RT-qPCR showed that
selected isolates upregulate expression of the adeB gene (from 1,13- to 3-fold).
Conclusion: This study revealed that AdeABC overexpression is the main mechanism of tigecycline resistance in A. baumannii isolated in Balkan hospitals
Unveiling therapeutic potential of bacteriophage treatment in acinetobacter baumannii-infected zebrafish embryo model
Get PDFIntroduction: There is an urgent demand for the development of new therapeutic approachesto combat multidrug-resistant Acinetobacter baumannii, and bacteriophages appear to be a highly promising
solution. Phages are suitable to precisely target the infection-causing bacteria without disrupting the
beneficial microbiota. The zebrafish (Danio rerio) embryo model represents an insightful animal model
for preclinical studying of various infectious diseases and for discovery of novel safe and effective antimicrobial drugs.
Methods: Systemic bacterial infection was established by microinjection of 2000 cells of nosocomial
carbapenem-resistant A. baumannii strain 6077/12 into the bloodstream of 48 hour old zebrafish embryos. Infected embryos were treated by parenteral administration of 4 different doses (10, 50, 100, 500
PFU) of bacteriophage vB_AbaM_ISTD at 6 hours after infection (hpi). Efficacy of treatment was evaluated according to embryo survival, morphological malformations and bacterial burden (CFU) over a 3-
day period.
Results: A. baumannii-infected embryos treated with bacteriophage resulted with 100% survival rate,
while 70% of untreated embryos survived to 24 hpi and none to the end of the experiment. Viable bacterial cell count and embryo morphology observations indicated that the administered phage effectively reduced A. baumanii infection in vivo. The most effective dose was 500 PFU, decreasing the bacterial
load by 3.09 log units during 24 hpi, while lower bacteriophage doses(10, 50 and 100 PFU) produced less
prominent, but also significant bacterial reduction of 2.10, 2.19 and 2.67 log units, respectively.
Conclusion: Parentheral administration of phage ISTD demonstrated potent therapeutic activity against
A. baumannii infection in every investigated dose
Uloga EPS-AN8 u zaštiti DA pacova izloženih oralnom unosu kadmijuma(II)
Get PDFКадмијум је глобално присутан токсични метал који изазива бројне штетне ефекте у организму и заузима седмо место на листи приоритетних супстанци од којих је неопходно пронаћи оптималне начине заштите. Наше пређашње студије су показале да егзополисахарид изолован из соја Lactiplantibacillus plantarum BGAN8 (EPS-AN8) показује висок афинитет за везивање јона кадмијума у воденом раствору и остварује значајан ниво in vitro заштите Caco-2 ћелија од његових токсичних ефеката. Имајући у виду да је за општу популацију најзаступљенији унос кадмијума исхраном, у овој студији је праћен паралелни ефекат уноса кадмијума (кроз воду) и EPS-AN8 (кроз храну) у DA пацовима. Након 30 дана третмана, утврђено је да је унос EPS-AN8 допринео сниженом нивоу депонованог метала у бубрезима, јетри и крви, а повећаном садржају у фекалном материјалу. Такође, микрографије дуоденума, бубрега и јетре су указале на ниже степене кадмијумом индукованих оштећења. Праћењем активности ензима који учествују у антиоксидативној заштити (CAT, GST), терминалних производа липидне пероксидације (MDA) и продукције проинфламаторних цитокина (IL-1β, TNFα, IFN-γ) у хомогенатима дуоденума, утврђен је нижи ниво оксидативног стреса и инфламације, што представља додатни показатељ заштите коју остварује EPS-AN8. С обзиром да орални унос кадмијума доводи до значајних промена у цревној микробиоти, изоловали смо тоталну DNK из дуоденума и секвенцирали 16S rDNK ампликон на Illumina NovaSeq платформи. Примећено је да унос EPS-AN8 ублажава појаву промена у релативној заступљености бактеријских родова и врста карактерисичних за излагање кадмијуму, попут пораста броја опортунистичких патогених бактерија и смањења бројности лактобацила. Остварени заштитни ефекти указују на снажан потенцијал примене EPS-AN8 у условима високе контаминације кадмијумом.Kadmijum je globalno prisutan toksični metal koji izaziva brojne štetne efekte u organizmu i zauzima sedmo mesto na listi prioritetnih supstanci od kojih je neophodno pronaći optimalne načine zaštite. Naše pređašnje studije su pokazale da egzopolisaharid izolovan iz soja Lactiplantibacillus plantarum BGAN8 (EPS-AN8) pokazuje visok afinitet za vezivanje jona kadmijuma u vodenom rastvoru i ostvaruje značajan nivo in vitro zaštite Caco-2 ćelija od njegovih toksičnih efekata. Imajući u vidu da je za opštu populaciju najzastupljeniji unos kadmijuma ishranom, u ovoj studiji je praćen paralelni efekat unosa kadmijuma (kroz vodu) i EPS-AN8 (kroz hranu) u DA pacovima. Nakon 30 dana tretmana, utvrđeno je da je unos EPS-AN8 doprineo sniženom nivou deponovanog metala u bubrezima, jetri i krvi, a povećanom sadržaju u fekalnom materijalu. Takođe, mikrografije duodenuma, bubrega i jetre su ukazale na niže stepene kadmijumom indukovanih oštećenja. Praćenjem aktivnosti enzima koji učestvuju u antioksidativnoj zaštiti (CAT, GST), terminalnih proizvoda lipidne peroksidacije (MDA) i produkcije proinflamatornih citokina (IL-1β, TNFα, IFN-γ) u homogenatima duodenuma, utvrđen je niži nivo oksidativnog stresa i inflamacije, što predstavlja dodatni pokazatelj zaštite koju ostvaruje EPS-AN8. S obzirom da oralni unos kadmijuma dovodi do značajnih promena u crevnoj mikrobioti, izolovali smo totalnu DNK iz duodenuma i sekvencirali 16S rDNK amplikon na Illumina NovaSeq platformi. Primećeno je da unos EPS-AN8 ublažava pojavu promena u relativnoj zastupljenosti bakterijskih rodova i vrsta karakterističnih za ilaganje kadmijumu, poput porasta broja oportunističkih patogenih bakterija i smanjenja brojnosti laktobacila. Ostvareni zaštitni efekti ukazuju na snažan potencijal primene EPS-AN8 u uslovima visoke kontaminacije kadmijumom.Naučni skup Svetski dan imunologije 2023; 2023 Apr 27; Belgrade, Serbia. Belgrade: Serbian Academy of Sciences and Arts; 2023
Genotypes and virulence factors in Staphylococcus aureus isolated from dairy cows with subclinical mastitis in Serbia
No full textThis study aimed to determine the prevalence of Staphylococcus aureus subclinical mastitis and to genotype the S. aureus isolates using the 16S–23S rRNA intergenic spacer (RS-PCR) method. In addition, the genes responsible for adherence, biofilm formation, host evasion, tissue necrosis, methicillin resistance, and enterotoxin production of S. aureus were investigated. The overall prevalence of S. aureus subclinical mastitis in lactating cows was 5.4% (95% confidence interval, CI=4.7–6.1%). An increased risk of S. aureus intramammary infection was observed on small family farms (odds ratio, OR=4.2, 95% CI=2.6–6.6, P < 0.001) and medium-sized farms (OR=3.5, 95% CI=2.2–5.7, P < 0.001). The RS-PCR analysis revealed 44 genotypes and genotype variants, of which 15 new genotypes and five new variants were detected within small and medium-sized farms. S. aureus isolates of new genotypes and genotype variants carried the clfA gene responsible for adherence at a lower frequency (64.8%) and enterotoxin-producing genes sea (20.4%), seb (14.8%) and sec (14.8%) at a higher frequency than the other known genotypes (P < 0.001), and were confirmed to carry the sej and sep genes. The spa gene was detected in all S. aureus isolates, whereas none harbored bap, ser, or tsst-1 genes. Methicillin-resistant strains of S. aureus (MRSA) were also detected, with a higher prevalence (19.2%) on large farms with more than 50 cows (P < 0.001). Using molecular techniques as diagnostic tools provides a better understanding of intramammary staphylococcal infections’ occurrence, spread, and eradication