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Novi pristup u identifikaciji supresora dss1 sekvenciranjem celih genoma Ustilago maydis
No full textGljiva Ustilago maydis (DeCandole) Corda biotrofni je patogen kukuruza (Zea mays). Svrstava
se u razdeo Basidiomycetes i red Ustilaginales, čiji predstavnici inficiraju veliki broj ekonomski
značajnih monokotiledonih i dikotiledonih biljaka (Banuett, 1995b). Tokom svog tronedeljnog životnog ciklusa, Ustilago maydis, iako okarakterisan kao dimorfna
gljiva, prolazi kroz 3 forme:
1. haploidnu – jednoćelijski, saprofitski, nepatogeni stadijum koji se razmnožava
pupljenjem (označava se i kao sporidijum);
2. dikariotsku – filamentozni, parazitski (domaćin je neophodan za razvoj ove forme) i
patogeni stadijum (indukuje stvaranje tumora ili gala), nastao sparivanjem haploidnih ćelija
suprotnog pola i može se propagirati na podlogama koje sadrže aktivni ugalj;
3. diploidnu – nastaje fuzijom jedara (kariogamijom) i prethodi formiranju teliospora u
završnoj fazi formiranja tumora (Feldbrügge i drugi, 2004)
Late-stage diversification of bacterial natural products through biocatalysis
Get PDFBacterial natural products (BNPs) have been and still are very important sources of leads for drug development and chemical novelty. The possibility to perform late-stage diversification of these biomolecules is an attractive approach to obtain novel structures by means other than total chemical synthesis or metal complexation reactions. Although biocatalysis is gaining popularity within the context of green chemistry, a vast majority of the sequenced genome data aimed at deciphering metabolic pathways for BNP biosynthesis, possibly containing additional tailoring enzymes, remains underexplored. In this review, we report a systematic overview of biotransformations of 21 molecules, which include derivatization by halogenation, esterification, reduction, oxidation, alkylation and nitration reactions, as well as degradation products as their sub-derivatives. These BNPs were grouped based on their biological activities into antibacterial (5), antifungal (5), anticancer (5), immunosuppressive (2) and quorum sensing modulating (4) compounds. This study summarized 73 derivatives and 16 degradation sub-derivatives originating from 12 BNPs. The remaining 9 BNPs have not been reported as substrates in biotransformation reactions. The highest number of 28 biocatalytic reactions was reported for the antibacterial drug vancomycin, followed by 18 reactions reported for the immunosuppressive drug rapamycin, suggesting that research focus to date was more on BNPs that were already in clinical use. The most common biocatalysts include oxidoreductases, transferases, lipases, isomerases and haloperoxidases. This review highlights biocatalytic routes for the late-stage diversification reactions of BNPs, which potentially help to recognize the structural optimizations of bioactive scaffolds for the generation of new biomolecules, eventually leading to drug development
Silver and silicon doped βTCP scaffolds with gentamicin or ceftazidime loaded P(3HB) coatings as multifunctional biomaterials for bone regeneration
No full textThe risk of bacterial infections is a significant challenge faced frequently in the use of implants or scaffolds for bone regeneration. Therefore, this study focusses on the development and characterisation of novel β tricalcium phosphate (βTCP) scaffolds co-doped with silver and silicon, along with composites coated with antibiotic-loaded poly(3-hydroxybutyrate) (P(3HB)) layers. The successful incorporation of silver and silicon dopants while maintaining the formation of βTCP phase was confirmed using X-ray fluorescence (XRF), X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FTIR), Raman spectroscopy, and scanning electron microscopy/energy-dispersive X-ray spectroscopy (SEM/EDS) analysis. The developed materials demonstrated comparable total and open porosity (∼64–70 vol%), suggesting high interconnectivity between pores conducive to nutrient transport and tissue repair. The increase in compressive strength was achieved for both doped (4.73 ± 0.79 MPa) and P(3HB) coated (5.79 ± 0.92 MPa) scaffolds due to the fine bioceramic microstructure and polymeric coating. Silver and silicon-modified βTCP demonstrated enhanced growth inhibition of Gram-negative (Pseudomonas aeruginosa, Escherichia coli) and Gram-positive (Staphylococcus aureus) bacterial strains in vitro compared to the pure βTCP. P(3HB) coatings, enriched with gentamicin or ceftazidime, exhibited burst and sustained release of the antibiotics from the scaffolds up to 120 h further intensifying the bacteria-killing capability, with evident inhibition zones observed in vitro. Moreover, the composites exhibited apatite-forming ability, suggesting their bioactive potential. In vivo evaluation using Caenorhabditis elegans demonstrated the lack of toxicity of the tested materials. The simultaneous incorporation of the dopants and antibiotic-loaded P(3HB) coatings not only offer a dual antibacterial approach but may also facilitate bone regeneration. However, further in vitro and in vivo investigations are needed to assess their potential in clinical application
Produkcija biofilma i rezistencija na antiseptike kod multirezistentnih kliničkih izolata Acinetobacter baumannii
No full textAcinetobacter baumannii is a notorious pathogen known for its extensive drug resistance and ability to form biofilms, making infections difficult to treat and control. This study investigated antibiotic resistance profiles, biofilm production, and environmental adaptability of 32 A. baumannii clinical isolates. Predominantly hospital-derived, the isolates showed a high proportion of antimicrobial drug resistance, with 93.75% classified as extensively drug-resistant (XDR), and the rest as multidrug-resistant (MDR). Notably, isolates demonstrated high resistance to amikacin and meropenem (MIC 50 >4096 mg/mL and 64 mg/mL, respectively). Biofilm production analysis revealed 13 strong producers, 14 moderate, 4 weak, and 1 non-producer. Strong and moderate biofilm producers exhibited higher antibiotic resistance on average. The most favorable conditions for biofilm formation proved to be in glucose-supplemented BHI and at room temperature. Six selected strong biofilm producers displayed significant variability in biofilm production across different media and temperatures. In antiseptic and topical antibiotic persistence tests, isolates showed varied survival and biofilm production, with some thriving and enhancing biofilm in saline and boric acid. The findings emphasize the adaptability and resilience of A. baumannii in clinical settings, highlighting the challenges in treating biofilm-associated infections.Acinetobacter baumannii je opasan patogen poznat po svojoj opsežnoj rezistenciji na lekove i sposobnosti da formira biofilmove, čineći infekcije teškim za lečenje i kontrolu. U ovoj studiji ispitani su profili rezistencije na antibiotike, produkcija biofilma i prilagodljivost različitim uticajima spoljašnjih faktora 32 klinička izolata A. baumannii. Izolati su poticali pretežno iz bolničkih sredina i pokazali su visoku rezistenciju na antimikrobne lekove, pri čemu je 93,75% klasifikovano kao ekstenzivno rezistentno (XDR), dok su ostali bili multi-rezistentni (MDR). Kao posebno značajna istakla se visoka rezistencija na amikacin i meropenem (MIC 50 >4096 mg/mL i 64 mg/mL, respektivno). Analizom produkcije biofilma pokazano je da 13 testiranih izolata formira jak biofilm, 14 umereno formira biofilm, dok su 4 izolata slabi proizvođači biofilma, a samo 1 izolat nije proizvodio biofilm. Jaki i umereni proizvođači biofilma su u proseku ispoljili veću rezistenciju na antibiotike. Najpovoljniji uslovi za formiranje biofilma bili su BHI medijum sa dodatkom glukoze i sobna temperatura. Šest odabranih jakih proizvođača biofilma pokazalo je značajnu varijabilnost u produkciji biofilma u različitim medijumima i pri različitoj temperaturi kultivacije. U testovima perzistencije u rastvorima antiseptika i antibiotika za topikalnu primenu, izolati su pokazali različit nivo preživljavanja i produkcije biofilma, pri čemu se kod nekih broj kolonija i biofilm povećao, u fiziološkom rastvoru i u bornoj kiselini. Ovi rezultati ukazuju na izuzetnu prilagodljivost i otpornost A. baumannii u bolničkim uslovima, ističući izazove u lečenju infekcija povezanih sa biofilmom
Probiotici kao prirodna inovativna i jedinstvena strategija za usporavanje starenja ćelija
No full textAccording to the World Health Organization (WHO),
the number of people worldwide over 60 years of age
was estimated to be 1 billion in 2019, with an
expected rise to 2.1 billion by 2050. This global phenomenon is described with the metaphor «silver
tsunami« used by Forbes.com and The Economist,
which vividly connects population ageing with its
huge impact on the economy, healthcare systems
and people’s quality of life. Therefore, one of the
goals of the United Nations Decade of Healthy
Ageing Initiative (2021-2030), is to prevent the
onset of age-related diseases in the elderly population by improving the research on «healthy ageing«.
The Western-type diet characterized by a high daily
intake of saturated fats and refined carbohydrates
leads to disturbance in gut microbiota and prolonged
low-grade inflammation, linked with cognitive impairment, emotional disorders, and dementia, but also
with inflammatory bowel disease (10 million people
worldwide, CDC), irritable bowel syndrome (between
25 and 45 million people in USA, 11% of global population, CDC), cardiovascular diseases (the leading cause of death globally, taking an estimated 17.9
million lives each year, WHO), cancer (globally, 18
million cases diagnosed in 2020, WCRF), and finally
accelerated ageing. New data suggest that modulation of gut microbiome composed of complex microbial communities could influence host ageing.
Metagenomics studies report the differences in the
composition of gut microbiota between young and
old subjects showing age-related dysbiosis reflecting
a decrease in the short-chain fatty acids and g-amino
butyric acid (GABA) core producers. We have tested
several carefully selected natural isolates of lactic
acid bacteria, originating from artisanal dairy products from specific geographical locations in the
Balkan peninsula for the ability to decelerate cellaging process. Our results revealed that these strains
possess exciting probiotic features: i) strengthen the
epithelial intestinal barrier through stimulation of
autophagy, ii) upregulate the tight junctions between
the epithelial cells of the intestine which prevent the
passage of harmful substances from the intestine to
other organs, iii) activate the antimicrobial defense,
iv) show high adhesion capability to intestinal cells,
without producing harmful effects on their viability, v)
exhibit excellent antioxidant activity, as well as vi)
exceptional anti-inflammatory effects reflected in
reduced levels of LPS-induced pro-inflammatory
cytokines in cell culture, and vii) extend the lifespan
of Caenorhabditis elegans via autophagy activation,
making them great candidates for probiotic starter
cultures for functional dairy food.Prema podacima Svetske zdravstvene organizacije
(SZO) broj ljudi {irom sveta starijih od 60 godina procenjen je na 1 milijardu 2019. godine, sa o~ekivanim
porastom na 2,1 milijarde do 2050. godine. Ovaj
globalni fenomen je opisan metaforom »srebrni
cunami«, koju su prvi upotrebili Forbes.com i The
Economist, koji slikovito povezuje starenje stanov -
ni{tva sa ogromnim uticajem na ekonomiju,
zdravstveni sistem i kvalitet `ivota ljudi. Stoga je jedan
od ciljeva Inicijative Ujedinjenih nacija Dekada
zdravog starenja (2021–2030) da se unapredi
istra`ivanje o »zdravom starenju« i spre~i nastanak
bolesti povezanih sa starenjem kod starije populacije.
Savremeni na~in `ivota i ishrane, koju karakteri{e
visok dnevni unos zasi}enih masti i rafinisanih
ugljenih hidrata, dovodi do poreme}aja mikrobiote
creva i hroni~nnih upalnih procesa u organizmu,
povezanih sa kognitivnim o{te}enjem, emocionalnim
poreme}ajima i demencijom, ali i sa inflamatornom
bole{}u creva (10 miliona ljudi {irom sveta, CDC),
sindromom iritabilnog creva (izme|u 25 i 45 miliona
ljudi u SAD, 11% svetske populacije, CDC), kardio vaskularnim bolestima (vode}i uzrok smrti u svetu,
koji godi{nje oduzimaju oko 17,9 miliona `ivota,
SZO), kancerom (globalno 18 miliona slu~ajeva
dijagnostikovanih 2020. godine, VCRF), i ubrzanim
starenjem. Novi podaci sugeri{u da bi modulacija
mikrobioma creva sastavljenog od slo`enih mikrobnih zajednica mogla uticati na starenje organizma.
Metagenomske studije ukazuju na razlike u sastavu
crevne mikrobiote izme|u mladih i starih osoba koji
pokazuju disbiozu povezanu sa uzrastom koja se
ogleda u smanjenju kratkolan~anih masnih kiselina i
proizvo|a~a g-amino buterne kiseline (GABA). Testi -
rali smo nekoliko pa`ljivo odabranih prirodnih izolata
bakterija mle~ne kiseline, poreklom od tradicionalnih
mle~nih proizvoda proizvedenih u doma}instvima na
specifi~nim geografskim lokalitetima na Balkanskom
poluostrvu na sposobnost usporavanja procesa
starenja }elija. Na{i rezultati su pokazali da ovi sojevi
poseduju izuzetne probioti~ke karakteristike: i)
oja~avaju epitelnu crevnu barijeru putem stimulacije
autofagije, ii) reguli{u tesne veze izme|u epitelnih
}elija creva koje spre~avaju prolazak {tetnih supstanci
iz creva u druge organe, iii) aktiviraju antimikrobnu
odbranu, iv) pokazuju visoku sposobnost adhezije na
crevne }elije, bez {tetnog uticaja na njihovu vitalnost,
v) pokazuju odli~nu antioksidativnu aktivnost, kao i vi)
izuzetne antiinflamatorne efekte koji se ogledaju u
smanjenom nivou LPS-indukovanih proinflamatornih
citokina u }elijskoj kulturi, i vii) produ`avaju `ivotni
vek Caenorhabditis elegans putem aktivacije autofagije, {to ih ~ini odli~nim kandidatima za probioti~ke
starter kulture za funkcionalne mle~ne proizvode.Book of abstract: XIII International Serbian Congress of Medical Biochemistry and Laboratory Medicine. 16-18 September 2024, Belgrade, Serbia
Seven-Year Longitudinal Study: Clinical Evaluation of Knee Osteoarthritic Patients Treated with Mesenchymal Stem Cells
No full textBackground/Objectives: Numerous studies have demonstrated the safety and efficacy of intraarticular stem cell injections for treating osteoarthritic knee joints, reporting symptom reduction and pain relief within a few months of treatment. Here, we report the results of a 7-year follow-up after a single intraarticular injection of 0.5–1 × 107 autologous adipose tissue-derived mesenchymal stem cells in patients with OA (Kellgren-Lawrence grade 2 to 4). Methods: Nine patients were treated, and two patients had bilateral disease. Patients were evaluated clinically and radiologically using X-ray and MRI. A comprehensive statistical analysis was undertaken to evaluate the obtained results. Results: All clinical scores and range of motion significantly improved within the first six months after injection. At the 18-month time point, a significant improvement in cartilage structure was observed on MRI while X-ray showed no changes in subchondral bone of distal femur and proximal tibia. At the 60-month time point, the clinical scores were still improved compared to baseline, except for the range of motion, which decreased almost back to the baseline level. At 84 months, the clinical scores decreased significantly toward the baseline level, but the MRI structural characteristics of cartilage still remained significantly better than those measured at baseline. Conclusions: Adipose tissue-derived stem cell therapy has substantial long-term clinical effects on patients with knee osteoarthritis
EXPLORING VARIATION IN ADHESION G PROTEIN-COUPLED RECEPTOR GENES: INSIGHTS FROM GENOMIC DATASETS OF PEDIATRIC RARE DISEASE CASES IN SERBIA
No full textBackground: Adhesion G protein-coupled receptors (aGPCRs) represent the second largest family in
the GPCR superfamily of receptors. The aGPCR family is comprised by 32+1 members which have been
shown to be involved in important physiological processes, such as brain development and immune
responses. Their role in pathophysiological processes such as neurological disorders, inflammation,
angiogenesis and male infertility has been noted. Also, genetic association of aGPCRs with several
diseases has identified them as potential drug targets. Systematic analysis and annotation of aGPCR
variants is still lacking. With the emergence of high-throughput technologies and bioinformatic tools
to extract this information from genomic data, this objective becomes achievable. We explored the
entire aGPCR gene family using data obtained from whole exome sequencing of 113 pediatric cases
with various clinical phenotypes. Our aim was to describe the genetic variant repertoire of the coding
regions of each gene, in terms of type, damaging effect and affected receptor domains in order to
identify variants of potential clinical significance.
Methods: Whole exome sequencing was performed (Illumina DNA Prep with Exome 2.0 Plus
Enrichment). Sequencing data was processed using an in-house developed bioinformatics pipeline for
germline short variant discovery. For each gene, coding region variants were annotated using the
ANNOVAR tool as either synonymous, missense, stop gain, in-frame indel or frameshift indel. Possible
impact of the identified variants on the structure and function of proteins was computationally
inferred by SIFT and PolyPhen-2.
Results: The majority of variants identified in coding regions of aGPCR genes were either synonymous
or missense benign. As anticipated, more deleterious variants were comparatively rare. Genetic
variability in each gene was calculated as the number of detected variants per coding nucleotide count.
We identified two members of aGPCR family, ADGRE1 and ADGRF3, as being among the genes with
the highest genetic variability rate. A detailed look at the ADGRE1 and ADGRF3 genes showed that
more common non-synonymous variants are confined to the N-terminal domain.
Conclusion: In this pilot study, we investigated the spectrum of aGPCR genetic variants present in the
Serbian pediatric cohort, which is important for defining the aGPCR genomic repertoire in various rare
diseases. Also, development of bioinformatics tools capable of automatically and accurately
annotating genetic variants is crucial for the field of aGPCRs.32nd Meeting of the European Society of Paediatric Clinical Research (ESPCR) in Opatija, Croatia, May 24-25, 2024
CAN NUDT15 BE PHARMACOGENETIC OR PHARMACOTRANSRIPTOMIC MARKER FOR 6- MERCAPTOPURINE IN CHILDREN WITH ACUTE LYMPHOBLASTIC LEUKEMIA IN SERBIA
No full textBackground: 6-mercaptopurine is a drug used in therapeutic protocol for the treatment of children
with acute lymphoblastic leukemia (ALL), mostly during the phase of maintenance therapy. The side
effects related to this drug could be partly attributed to pharmacogenetic variants in the TPMT gene.
Recently, the NUDT15 gene has been recognized as a new gene of interest for the optimization of 6-
mercaptopurine therapy. The association of side effects in children with variants in NUDT15 is well
established in Asian populations, yet the relevance of this pharmacogene in European populations
remains largely unexplored. The aim of this study was to identify pharmacogenetic variants in the
coding and neighboring regions of the NUDT15 gene and analyze if the expression levels of the NUDT15
gene can be of use as a pharmacotranscriptomic marker, to predict the occurrence of side effects of 6-
mercaptopurine during the maintenance therapy in children with ALL of Serbian origin.
Methods: The genotyping of coding and neighboring regions of the NUDT15 gene was performed using
PCR and Sanger sequencing based technology in 48 children with ALL. NUDT15 expression was
analyzed in mononuclear cells of 24 children with ALL before the start of the therapy and 6 healthy
individuals by qRT-PCR. The association of genotyping and expression analysis data with surrogate
markers of toxicity was assessed using adequate statistical methodology.
Results: This study showed the presence of 5 variants in the NUDT15 gene in the study group
(rs61746486, rs79687000, rs45465203, rs41284205, and rs61973267). However, none of the identified
variants has shown an effect on the expression or function of the NUDT15 protein. This study showed
no statistically significant association between the expression of NUDT15 in mononuclear cells at
diagnosis and the surrogate 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) and average
doses (p=0.374)) of 6-mercaptopurine during the maintenance therapy.
Conclusion: The data obtained in this study do not support the hypothesis that NUDT15 can be used
as a pharmacogenetic or pharmacotranscriptomic marker in predicting the side effects of 6-
mercaptopurine therapy in children with ALL in Serbia. In the future, more comprehensive methods
on a larger study sample should be performed.32nd Meeting of the European Society of Paediatric Clinical Research (ESPCR) in Opatija, Croatia, May 24-25, 2024
Functionalization of silk with actinomycins from Streptomyces anulatus BV365 for biomedical applications
No full textSilk, traditionally acclaimed as the “queen of fiber,” has been widely used thanks to its brilliant performance such as gentleness, smoothness and comfortableness. Owing to its mechanical characteristics and biocompatibility silk has a definitive role in biomedical applications, both as fibroin and fabric. In this work, the simultaneous dyeing and functionalization of silk fabric with pigments from Streptomyces anulatus BV365 were investigated. This strain produced high amounts of orange extracellular pigments on mannitol-soy flour agar, identified as actinomycin D, C2 and C3. The application of purified actinomycins in the dyeing of multifiber fabric was assessed. Actinomycins exhibited a high affinity towards protein fibers (silk and wool), but washing durability was maintained only with silk. Acidic condition (pH5) and high temperature (65°C) facilitated the silk dyeing. The morphologies and chemical components of the treated silk fabrics were analyzed using scanning electron microscopy and Fourier transform infrared spectroscopy. The results showed the pigments bind to the silk through interaction with the carbonyl group in silk fibroin rendering the functionalized, yet surface that does not cause skin irritation. The treated silk exhibited a remarkable antibacterial effect, while the biocompatibility test performed with 3D-reconstructed human epidermis model indicated safe biological properties, paving the way for future application of this material in medicine
Antimicrobial and antitubercular activity of silver(I) and gold(III) complexes with miconazole
No full textNew silver(I) and gold(III) complexes with the antifungal drug miconazole (mcz), [Ag(NO3)(mcz)2]
(1) and [AuCl3(mcz)] (2), were synthesized by the reaction of AgNО3 and K[AuCl4] with an
equimolar amount of mcz in ethanol. The synthesized complexes 1 and 2 were characterized by mass
spectrometry, IR, UV-Vis and 1H NMR spectroscopy. Complex 1 contains two mcz and one
monodentately coordinated nitrate anion. In the case of complex 2, mcz is a monodentate ligand
coordinated to the Au(III) ion via the triazole nitrogen atom, while the remaining coordination sites
of this metal ion are occupied by chloride anions leading to the square-planar arrangement. The
antimicrobial activity of complexes 1 and 2 was investigated against different bacterial and fungal
strains, as well as their cytotoxic activity on the normal human lung fibroblast cell line (MRC-5).
These complexes were assessed for their in vitro antimycobacterial activity against Mycobacterium
tuberculosis (MTB) H37Rv (ATCC 27294) strain. Coordination of mcz to Ag(I) and Au(III) ions led
to enhancement of its activity against Gram-negative Escherichia coli and Pseudomonas aeruginosa
strains, while against the panel of Staphylococcus aureus and Candida species, only complex 2 has
improved activity in respect to mcz. Both complexes 1 and 2 demonstrated good antitubercular
activity, whereby 1 is twice potent than parent mcz drug.Book of abstract: 52nd Conference Synthesis and Analysis of Drugs (SAL 2024), Hradec Králové (Czech Republic