imagine (Institute of molecular genetics and genetic engineering)
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Probiotička karakterizacija soja Limosilactobacillus fermentum BGHV110 i njegov uticaj na urođeni imunski odgovor kod Caenorhabditis elegans
Get PDFProbiotic lactobacilli exhibit the potential to promote health benefits for the host. Thanks to its numerous beneficial effects on human health, Limosilactobacillus fermentum stood out as an excellent candidate for the development of commercial probiotic preparations aiming to prevent community-acquired infections. In this study, several in vitro tests, including biofilm formation assay, assessment of antibiotic susceptibility, survival in simulated gastrointestinal tract conditions and attachment to intestinal Caco-2 cells, were used to estimate the safety and probiotic potential of L. fermentum BGHV110 strain. Additionally, Caenorhabditis elegans was used as an in vivo model system for the evaluation of L. fermentum BGHV110 influence on the host’s innate immune response. The results revealed that L. fermentum BGHV110 strain showed an excellent capability to survive harsh conditions of the gut, to attach to intestinal Caco-2 cells and to stimulate conserved p38 MAPK immunity pathway and expression of the clc-1 claudin-like gene and antimicrobial peptides in C. elegans in order to enhance the immune response against pathogens. Finally, L. fermentum BGHV110 showed no virulence traits and susceptibility to tested antibiotics, confirming its safety status which enables it to be applied as a future probiotic
Alginate microfibers with immobilized cells as a 3D in vitro model for anticancer drug testing: demonstration in osteosarcoma cell cultures
No full textIn anticancer drug development closing the gap between 2D in vitro and in vivo studies on animals
and further clinical studies is one of the urging aims considering low translation of the results obtained
in these preclinical systems to humans. This is why the development of 3D in vitro systems that would
closely mimic in vivo response to anticancer drugs is of the utmost importance. Here we propose a
simple 3D in vitro culture model based on alginate hydrogel microfibers with immobilized cancer cells.
The microfibers were obtained by manual extrusion of a suspension of murine osteosarcoma cells
(K7M2 wt, 4×106
cells/ml) in a 2 %wt Na-alginate solution through a blunt edge stainless steel needle
(25G) into a gelling solution containing Ca2+ (0.18 M CaCl2×2H2O). After 15 min of gelation the formed
microfibers (diameter ~500 µm) were transferred to T-25 flasks (1.5 g of microfibers in 15 ml culture
medium) and cultured for 7 days. Histological analysis showed that alginate microfibers supported cell
viability and formation of cellular aggregates. In the second series, microfibers were cultured in a 96-
well plate (3 cm of microfibers in 100 µl culture medium per well). The cells immobilized in microfibers
were treated with 0,25-20 µM doxorubicin as a model anticancer drug 24 h post cell immobilization,
while treated cells in 2D served as controls. The results showed significant increase in resistance to
doxorubicin in the 3D culture compared to the 2D: the IC50 value for the 3D was ~3 µM while that for
the 2D culture was ~0.5 µM. The obtained higher resistance to the anticancer drug compared to 2D
cultures is in agreement with the resistance of cancers to treatments in vivo, showing the potentials
of the proposed system as a more relevant in vitro model for anticancer drug testing.Conference Programme Book:Tumour Microenvironment Meeting & 3D Model Workshop, 6-8 September 202
A continuous-flow 3D osteosarcoma model: formation of spheroid-like aggregates under perfusion
No full textOnline Abstract Book: Goodbye Flat Biology: Next Generation Cancer Models, Berlin, Germany, 10-12 October, 202
CONSUMPTION OF EXOPOLYSACCHARIDES FROM LACTIPLANTIBACILLUS PLANTARUM BGAN8 ALTERS THE GUT MICROBIOTA OF DA RAT
Get PDFIntroduction: Exopolysaccharides (EPS) are carbohydrate polymers with important biological activities such
as immunomodulatory, antioxidative and antitumor (1). How EPS affect the gut microbiota, which plays an
important role in maintaining homeostasis in the organism, is poorly understood to date. Therefore, the aim
of this study was to investigate how EPS-AN8, derived from Lactiplantibacillus plantarum BGAN8, affects part
of the gut microbiota in the duodenum of healthy Dark Agouti rats (DA).
Materials and Methods: EPS-AN8 was administered to male DA at low (0.1 mg/mL) and high (1 mg/mL)
dose over a 15-day period. Total duodenal DNA was isolated and PCR amplicon for 16SrRNA was sequenced
on Illumina NovaSeq paired-end platform. Furthermore, we tracked key parameters of oxidative stress and
inflammation in duodenal homogenates.
Results: The higher dose of EPS-AN8 resulted in an increased Shannon's diversity index. The most
significant differences were observed in the increased relative abundance of the genera Ruminococcus,
Dubosiella, Enterohabdus, and Adlercreutzia. At the same time, we demonstrated that neither dosage caused
negative side effects such as inflammation and oxidative stress.
Conclusion: Considering the existing trend to market foods with additional health benefits, our results
suggest that EPS-AN8 could be a good candidate for functional food supplementation.International Society of Microbiota
10th ISM World Congress on
Targeting Microbiota
October 17-19, 2023 – Venice, Ital
From waste streams to biotherapeutics: making a connection using bacteria
Get PDFMicroorganisms, our planet’s original inhabitants discovered with the invention of the
first microscope in the 17th century, have consistently facilitated our daily life. However,
our modern life generates enormous amounts of wastes, such as plastic, food, and
chemicals from the pharmaceutical industry. Bacterial natural products hold an
important position in this industry, as drug leads in synthetic chemistry and biology,
essential for the discovery of effective agents against a variety of human diseases. If the
existing waste is used as a nutrient source for microbial production of valuable
biomolecules, that concept is called “waste to value” or “upcycling”.
This concept was explored using bacterial biopigment prodigiosin (PG, Fig. 1) as part of
the BioECOLogics project. This proof of concept demonstrates how the bacteria Serratia
marcescens ATCC 27117 can use a waste stream from the food industry as a carbon
source to grow and produce its bioactive secondary metabolite PG. The unique structure
of this molecule was changed through green chemical [1] and biopolymer formulation
[2] approaches, as well as metal complexation. Finally, these sustainable biotherapeutics
were validated in vitro (antimicrobial, anticancer) and in vivo (nematode Caenorhabditis
elegans and zebrafish Danio rerio).Book of Abstracts: 9th Conference of Young Chemists of Serbia
Novi Sad, 4th November 202
Mutation rates of 22 autosomal STR LOCI in a European population from central Balkan, Republic of Serbia
Get PDFAnalysis of short tandem repeats (STRs) is a tool for human DNA identification, including paternity
and criminal investigations. Locus-specific STR mutation rates are critical for interpretation of DNA
identification in practice. Accumulating evidence suggests that STR mutation rates are populationspecific.
However, STR mutation rates based on trios have not been analyzed in European population
yet. In this study, mutation rates of 20 CODIS and two additional autosomal STR loci (Penta E and
Penta D) were determined from 1279 cases of paternity testing in the Serbian population, and the
relationships between STR mutation rates and sex, allele length, and heterozygosity were investigated.
A total of 63 mutations were observed at 18 loci in a total of 28278 allele transmissions, including 62
(98.4%) single-step and 1 (1.6%) two-step mutations. The average mutation rate was 1.7×10-3 (95%
CI: 1.3-2.3×10-3), whereas locus-specific mutation rates ranged from 5.6×10-3 (D12S391) to 0.6×10-3
(D2S1338, D5S818, and D21S11). No mutation was observed at the TPOX, TH01, D16S539, and
D22S1045 loci. The mutation rate of paternal origin (2.4×10-3) was eight times higher than that of
maternal origin (0.3×10-3), and long alleles mutated more frequently than short and medium alleles
(χ2=4.436 and χ2=4.646, respectively, p<0.05). No differences were found when analyzing overall
expansion versus contraction (χ2=0.999, p=0.317). Among short alleles two expansions and no
contraction were detected, while among long ones three expansions and nine contractions were
observed. Furthermore, we found no correlation between locus-specific mutation rates and
corresponding heterozygosity (rs=-0.188, p=0.559). Comparisons between populations revealed
statistically significant differences in locus-specific mutation rates of 13 CODIS STRs between Serbian
population and Chinese or Brazilian. Our results show that STR mutation rates depend on sex, allele
size and population, and provide useful data on STR mutation rate based on trios for human DNA
identification in European populations.Book of abstracts: International Conference of Biochemists and Molecular Biologists in Bosnia and Herzegovina - ABMBBIH May, 202
Antioxidative and antiinflamatory effects of walnut supplementation (Juglans regia L.)on heart with fructose-rich diet-induced metabolic syndrome
Get PDFPovećana konzumacija fruktoze kroz industrijsku hranu doprinosi razvoju metaboličkog sindroma (MetS)
koji prati hronična inflamacija niskog intenziteta, oksidativni stres i aktivacija renin-angiotenzin sistema (RAS).
Orasi su bogat izvor antioksidanasa i polinezasićenih n-3 masnih kiselina sa antiinflamatornim efektima.
Na životinjskom modelu MetS-a je pokazano da suplementacija orasima ima zaštitni efekat na srce kroz
smanjenje odnosa n-6/n-3 masnih kiselina i povećanje nivoa angiotenzin konvertujućeg enzima 2 (ACE2),
kardioprotektivne komponente RAS-a. Orasi u srcu deluju protektivno i na antioksidativnu osu SIRT1-
FoxO3a-MnSOD/katalaza, koja je kompromitovana ishranom bogatom fruktozom. Ulogu negativnih regulatora
ove ose, Nox4 i ChREBP, u srcu treba dodatno ispitati kako bi se utvrdio njihov potencijal u nutritivnoj
terapiji.
Dosadašnja istraživanja su potvrdila korisne efekte konzumacije oraha na kardiometaboličko zdravlje i
predložila molekularne mehanizme u srcu koji leže u osnovi njihovih antiinflamatornih i antioksidativnih
efekata koji mogu poništiti štetne efekte ishrane bogate fruktozom. Translacioni potencijal istraživanja
ukazuje na mogućnost razvoja novih, nefarmakoloških pristupa u terapiji kardiometaboličkih bolesti.Increased fructose intake via industrial food is associated with metabolic syndrome (MetS) which is characterized
by chronic low-intensity inflammation, oxidative stress and activation of the renin-angiotensin
system (RAS). Walnuts are a rich source of antioxidants and polyunsaturated n-3 fatty acids that exert antiinflammatory
effects.
Walnut supplementation in an animal model of MetS has been shown to have a protective effect on the
heart through a reduction of n-6/n-3 fatty acid ratio and an increase of angiotensin-converting enzyme 2
(ACE2) level, a cardioprotective component of the RAS. Walnut consumption also showed a protective effect
on the antioxidative axis SIRT1-FoxO3a-MnSOD/catalase in the heart, which was compromised by
fructose-rich diet. The role of the negative regulators of this axis, Nox4 and ChREBP, in the heart should be
further investigated to determine their nutritional therapy potential.
Previous studies confirmed the beneficial effects of walnut consumption on cardiometabolic health and
proposed the molecular mechanisms underlying their anti-inflammatory and antioxidative effects in the
heart, which could reverse the harmful effects of fructose-rich diet. The translational potential of the research
indicates the possibility for development of novel, nutritional, non-pharmacological approaches in
cardiometabolic disease therapy
Alpha-1 antitrypsin expression is upregulated in multidrug-resistant cancer cells
No full textIdentification of the signature molecular profiles involved in therapy resistance is of vital importance in developing new strategies for treatments and disease monitoring. Protein alpha-1 antitrypsin (AAT, encoded by SERPINA1 gene) is an acute-phase protein, and its high expression has been linked with unfavorable clinical outcome in different types of cancer; however, data on its involvement in therapy resistance are still insufficient. We analyzed SERPINA1 mRNA expression in three different multidrug-resistant (MDR) cell lines—U87-TxR, NCI-H460/R, and DLD1-TxR—and in U87 cells grown in alginate microfibers as a 3D cellular model of glioblastoma. Expression of IL-6 as a major modulator of SERPINA1 was also analyzed. Additionally, AAT protein expression in MDR cells was analyzed by immunofluorescence. SERPINA1 gene expression and AAT protein expression were significantly upregulated in all the tested MDR cell lines compared with their sensitive counterparts. Moreover, SERPINA1 was significantly upregulated in 3D models of glioblastoma, previously found to have upregulated drug-resistance-related gene expression compared with 2D cells. With the exception of NCI-H460/R, in all cell lines as well as in a 3D model of U87 cells, increase in SERPINA1 expression correlated with the increase in IL-6 expression. Our results indicate that AAT could be utilized as a biomarker of therapy resistance in cancer; however, further studies are needed to elucidate the mechanisms driving AAT upregulation in therapy resistance and its biological significance in this process
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
Development of nystatin-based antifungal, biodegradable polymer composite materials for food packaging via. melt processing approach
Get PDFNovel bio-based, biodegradable packaging material was developed with multi-functional properties of good mechanical strength, potent biocompatibility, and antifungal attributes, predominantly against fungi vital in food spoilage. Biodegradable polymer composites were prepared with a natural antifungal agent, nystatin (Nyst), by melt processing technique for the first time. Initially, Polycaprolactone (PCL) was melt-mixed with various percentages of nystatin to produce nystatin-encapsulated PCL composites (PCL/Nyst). The as-prepared PCL/Nyst composites were melt-mixed with polylactic acid (PLA) to produce nystatin PLA/PCL blend composites. Subsequently, the prepared composites were compression molded in the form of films (1 mm) for further characterization. The composite's structural properties were evaluated by SEM, FTIR, mechanical and thermal studies. In addition, the composites were tested in vitro against a panel of pathogenic fungi and for antibiofilm attributes, specifically against three Candida species (C. albicans ATCC10231, C. parapsilosis ATCC22019, and C. glabrata ATCC2001), and foodborne Penicillium sp. All the composites containing 2 to 20 wt% nystatin displayed good activity and sustained nystatin release for up to 4 days. Thus, the overall study demonstrates the potential application of natural antifungal agents in biodegradable polymers to produce novel composite films for antimicrobial packaging without inducing any toxicity, judged from the toxicity assay using nematode Caenorhabditis elegans