imagine (Institute of molecular genetics and genetic engineering)
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Many plant pathogenic Pseudomonas savastanoi pv glycinea isolates possess an inactive quorum sensing ahlR gene via a point mutation
No full textMany plant bacterial pathogens monitor their group behaviour and their population density via production of N-acyl homoserine lactone signals which regulate the expression of several genes via the LuxI/R homologs. This regulatory network, termed quorum sensing (QS), is present in the soybean bacterial pathogen Pseudomonas savastanoi pv glycinea (Psg). The sequenced genomes of two strains of Psg, race 4 and B076, contain an N-acyl homoserine lactone (AHL) based LuxI/R QS system named AhlI/R. While studying the QS system of Psg strains race 4 and B076 isolated in USA, LMG5066 in New Zealand and IBSBF355 in Brazil, we found that B076, LMG5066 and IBSBF355 possess a point mutation in the ahlR gene that causes a frameshift resulting in a truncated AhlR protein. Psg race 4 does not possess the mutation in ahlR and the QS system is functional. The same mutation in the ahlR genewas found to be also present in 9 of 19 Psg strains isolated from diseased soybean in Illinois. Phenotypic analysis of strains showed that swarming motility is repressed whereas phosphate solubilisation was activated by QS in Psg. Analysing the secretome, we also found that four proteins were under QS regulation
Evaluation of toxicity and antioxidative effects of Tussilago farfara and Verbascum thapsus water extracts in zebrafish and in bronchial epithelial cells
Get PDFTussilago farfara (coltsfoot) and Verbascum thapsus (mullein) have been used as folk remedies for treating respiratory disorders. The aim of this study was to test the toxicity of the water extracts of T. farfara and V. thapsus in vivo in zebrafish and in vitro in BEAS 2B epithelial bronchial cells. To the best of our knowledge, this is the first study to investigate the antioxidative properties of T. farfara and V. thapsus extracts in cell culture. Our results show that the T. farfara leaf extract does not produce toxic effects on zebrafish embryos or BEAS 2B cells, and that it has a protective effect in BEAS 2B after induction of oxidative stress. The water extract from V. thapsus displayed pronounced toxic effects on zebrafish embryos and BEAS 2B cells and did not exhibit a significant antioxidative effect on BEAS 2B cells exposed to oxidative stress. Our results suggest that the use of T. farfara water leaf extract is potentially safe and effective in treating respiratory disorders, whereas the use of V. thapsus needs further investigation
Wild edible onions - Allium flavum and Allium carinatum - successfully prevent adverse effects of chemotherapeutic drug doxorubicin
Get PDFThe objective of this study was to evaluate potential of two chemically characterized edible wild onion species, Allium flavum and Allium carinatum, to reduce side effects of cytostatic doxorubicin (Dox). Since Dox application is mainly limited due to its high cardiotoxicity, while there are no approved cardioprotective agents for the prevention of Dox adverse effects, new co-treatments are urgently needed. Here, we showed that methanol extracts expressed high antioxidant activity and synergistically increased Dox anticancer activity against human hepatoma (HepG2) and lung carcinoma (A549) cells, while protected normal human fibroblasts (MRC-5) from Dox cytotoxicity. Analysis of the antioxidative enzymes level (catalase and superoxide dismutases) showed that the catalase level was differently altered in cancer cells compared to normal cells upon applied treatments. In vivo toxicity evaluation in the zebrafish model revealed significantly lower toxicity of extracts compared to Dox, and no teratogenic effects at applied doses. We found that extracts successfully rescued the Dox-treated embryos of life-threating cardiomyopathy, while at the same time reduced developmental toxicity and neutropenia. Further analysis demonstrated that extracts had higher anti-angiogenic activity than sunitinib or auranofin, clinically used anti-angiogenic drugs. In addition, angiogenesis was markedly more suppressed in Dox-extract cotreatments than upon single treatments
Significance of UGT1A1*28 genotype in patients with advanced liver injury caused by chronic hepatitis C
Get PDFUvod: Hronični hepatitis C (HHC) je značajan uzročnik morbiditeta i mortaliteta u svetu. Značaj genetskih faktora u patogenezi HHC još uvek nije u potpunosti razjašnjen. Varijacije UGT1A1 gena su najčeša uzrok nasledne nekonjugovane hiperbilirubinemije - Zilberovog sindroma. Ovo je prva studija koja se bavi ispitivanjem učesta I osti TA ponavljanja u promotorskoj regiji UGT1A1 gena i analizom povezanosti UGT1A1*28 genotipa sa stepenom fibroze, viremijom i biohemijskim markerima kod pacijenata sa teškim oštećenjem jetre izazvanim HHC i virusološkim relapsom. Metode: Analizirana su TA ponavljanja u promotorskoj regiji UGT1A1 gena, kod 42 pacijenta sa teškom fibrozom i cirozom izazvanom HHC, koji postigli stabilan virusološki odgovor i 42 ispitanika u kontrolnoj grupi zdravih dobro - voljnih davalaca krvi. Pacijenti sa HHC su dodatno analizirani kliničkim pregledima, laboratorijskim analizama (hematološki, biohemijski i virusološki) i fibroskenom jetre. Rezultati: UGT1A1*28 genotip (7/7 TA ponavljanja) je bio prisutan kod 23.8% pacijenata sa HHC i 16,7% zdravih ispitanika, ali bez statistički značajne razlike (p= 0,49). Nivoi feritina i ukupnog bilirubina su bili u korelaciji sa prisustvom UGT1A1*28 pre primene antivirusne terapije, što sugeriše prediktivnu ulogu ovog genotipa. U ovoj studiji nije bilo korelacije UGT1A1*28 genotipa sa stepenom fibroze i viremijom. Prisustvo UGT1A1*28 genotipa nije uticalo na terapijske prekide i redukcije doze antivirusnih lekova, ishod lečenja niti pojavu kasnog virusološkog res lapsa kod pacijenata sa HHC i teškim oštećenjem jetre. Zaključak: Učestalost UGT1A1*28 genotipa je visoka među srpskim zdravim ispitanicima i pacijentima sa HHC infekcijom. Ispitivani genotip se ne dovodi u vezu sa nes željenim efektima ribavirina niti ima uticaj na ishod lečenja i dugoročnu prognozu pacijenata sa HHC.Background: Chronic hepatitis C (CHC) is a significant cause of liver related morbidity and mortality worldwide. The role of genetics in the host response to hepatitis C virus is not elucidated. Genetic variations in UGT1A1 gene are the most common cause of hereditary unconjugated hyperbilirubinemia-Gilbert syndrome. This is the first study investigating the association of UGT1A1 TA repeats promoter genotypes with the degree of liver injury, viremia and biochemical markers in CHC patients with advanced liver injury and late virological relapse. Methods: Genetic testing of UGT1A1 TA repeats promoter genotypes was performed in 42 CHC patients with advanced fibrosis and cirrhosis who achieved sustained virological response and 42 healthy blood donors. CHC patients were evaluated for clinical findings, laboratory tests and imaging. Results: UGT1A1*28 genotype (7/7 TA repeats) was observed in 23.8% CHC patients and 16.7% healthy controls with no significant difference in genotype frequencies (p= 0.49). Pretreatment levels of ferritin and bilirubin were associated with the presence of U G T1A1*28 genotype, indicating its potential as a predictive marker. However, in our study, there was no correlation of U G T1A 1*28 genotype with the degree of fibrosis or viremia. During antiviral treatment, dose reductions and treatment interruptions, as well as treatment success and occurrence of late virological relapse were not related to the presence of U G T1A 1*28 genotype in CHC patients with severe liver injury. Conclusions: Frequencies of U G T1A 1*28 genotype are high in both Serbian CHC patients and healthy subjects. The presence of U G T1A 1*28 genotype was not associated with ribavirin-related adverse effects and had no effect on long term outcome in CHC patients
Prothrombin expression in cancer-derived cell lines
Get PDFThe link between thrombotic disorders and cancer has been known for over 150 years, although the precise mechanism of this relationship has not yet been resolved. Current data show that thrombin has a significant role in cancer metabolism, invasiveness, adhesion and survival. However, data regarding the expression of the thrombin precursor prothrombin in various cancer cell lines are scarce. Therefore, it was our objective to determine whether common cancer-derived cell lines (Caco-2, MCF-7, SK-BR-3, U-87 and U-251) express prothrombin. The prothrombin RNA expression level was assessed by qPCR, and the presence of prothrombin was analyzed by Western blot analysis. Our results show that Caco-2 cells originating from colorectal adenocarcinoma express prothrombin, whereas other analyzed cell lines do not. Our results provide a background for further research into the role of (pro) thrombin in cancer etiopathology
CRISPR/Cas9 genome editing of SLC37A4 gene elucidates the role of molecular markers of endoplasmic reticulum stress and apoptosis in renal involvement in glycogen storage disease type Ib
No full textGlycogen storage disease type Ib (GSD Ib) is an autosomal recessive disorder, caused by a deficiency of ubiquitously expressed SLC37A4 protein. Deficiency of SLC37A4 leads to abnormal storage of glycogen in the liver and kidneys, resulting in long-term complications of renal disease and hepatocellular adenomas, whose mechanisms are poorly understood. Molecular markers of the adaptive responses to the metabolic stress caused by a deficiency of SLC37A4, such as markers related to the endoplasmic reticulum (ER) stress and unfolded protein response (UPR), have not been extensively studied. The aim of this study was to investigate the expression of molecular markers of the UPR response and apoptosis related to a deficiency of SLC37A4 in kidney cells. For that purpose, we intended to establish a human kidney cell model system for GSD Ib. The novel variant c.248G gt A, found in GSD Ib patients, was introduced into the Flp-In (TM) T-REx (TM)-293 cell line using CRISPR/Cas9-mediated precise gene editing method, resulting in significant decrease of SLC37A4 gene expression. In this model system we used RT-qPCR analysis to investigate the expression of molecular markers of the UPR response (ATF4, DDIT3, HSPA5, and XBP1s) and apoptosis (BCL2, BAX). We demonstrated that under chronic metabolic stress conditions caused by SLC37A4 deficiency, the ER stress-induced UPR was triggered, resulting in suppression of the UPR molecular markers and cell survival promotion (decreased expression levels of ATF4, DDIT3, HSPA5, with the exception of XHE1s). However, persistent metabolic stress overrides an adaptation and induces apoptosis through increased expression of pro-apoptotic markers (decreased ratio of BCL2/BAX genes). We established a cellular model system characterized by a deficiency of SLC37A4, which presents pathological manifestations of GSD Ib in the kidney. Expression analysis in a novel model system supports the hypothesis that renal dysfunction in the GSD Ib is partly due to the ER stress and increased apoptosis
Controlled drug release carriers based on PCL/PEO/PCL block copolymers
No full textIn order to create a new drug delivery system, the ibuprofen-loaded triblock copolymer PCL/PEO/PCL (PCEC) microspheres with a low PEO content ( lt 2 wt%) were prepared by oil in water (o/w) solvent evaporation technique. The influence of PEO content, molecular weight of a polymer matrix and drug loading on the ibuprofen release profiles were evaluated. The interactions between polymer matrix and ibuprofen were detected by FTIR analysis. The presence of hydrophilic PEO segment in PCL chains caused the decrease in particle size, which further had a great impact on the drug release kinetics, i.e., initially faster release and significantly higher quantity of released drug compared to neat PCL. Ibuprofen release behavior from polymer matrix was governed by a diffusion process. In vitro cytotoxicity tests revealed that empty PCL and PCEC microspheres were not toxic at low concentrations, while ibuprofen-loaded microspheres exhibited cytotoxicity correlated with amounts of incorporated drug
Genomic profiling of pediatric patients with primary ciliary dyskinesia: genotype-phenotype correlation and functional characterization of novel genetic variants
Get PDFPrimarna cilijarna diskinezija (PCD) predstavlja retku bolest koja se nasleđuje na autozomno recesivan način ili je nasleđivanje vezano za hromozom X, i predominantno utiče na funkcionisanje pluća, reproduktivnih organa i na lateralnost unutrašnjih organa. PCD je klinički i genetički veoma heterogen poremećaj koji se javlja odmah po rođenju, a karakteriše se promenama na motornim cilijama koje su posledica patogenih varijanti u genima koji kodiraju za proteine koji su neophodni za pravilnu strukturu i funkciju ovih organela. Do sada je opisano 38 gena uzročnika bolesti koje su odgovorne za nastanak PCD-a, a smatra se da je taj broj mnogo veći s obzirom da 2500 proteina učestvuje u izgradnji i pravilnom funkcionisanju cilija. Velika raznolikost kliničke slike PCD pacijenata često dovodi do odloženog uspostavljanja precizne dijagnoze ove bolesti, a nije retkost i da pacijenti sa drugim bolestima pluća budu okarakterisani kao PCD pacijenti. Genetički profil ove bolesti kod pacijenata sa teritorije Srbije do sada nije utvrđen, pa je identifikacija gena uzročnika neophodna. Stoga je u okviru ove teze urađeno genomsko profilisanje pacijenata sa primarnom cilijarnom diskinezijom u cilju razvoja genetičkog algoritma koji bi, osim opisanih patogenih genetičkih varijanti i gena uzročnika, obuhvatio i novootkrivene varijante u kodirajućim regionima gena uzročnika PCD-a i gena kandidata za PCD. Sve ovo omogućava razvoj strategije za diferencijalnu dijagnozu PCD-a i drugih bolesti pluća suspektnih na ciliopatije koji se klinički manifestuju kao PCD. Takođe, pristupljeno je funkcionalnoj karakterizaciji novootkrivenih patogenih varijanti u već poznatim genima koji su odgovorni za razvoj PCD-a i/ili u genima koji do sada nisu asocirani sa PCD-om, a koji bi mogli da dovedu do karakterističnog fenotipa kod pacijenta. Jedna od glavnih odlika PCD pacijenata je strukturna i/ili funkcionalna promena na cilijama, pa su one nepokretne, slabo pokretne, promenjen im je obrazac kretanja ili odsustvuju...Primary ciliary dyskinesia (PCD) is a rare disease that is inherited in autosomal recessive manner or the inheritance is X-linked, and predominantly affects the functioning of the lungs, reproductive organs and the laterality of the internal organs. PCD is clinically and genetically very heterogeneous disorder that occurs immediately after birth, and is characterized by alterations in motor cilia due to pathogenic variants in genes encoding proteins that are necessary for the proper structure and function of these organelles. So far, there have been described 38 PCD-causative genes, and this number is considered to be much higher given that 2500 proteins participate in the formation and functioning of the cilia. Clinical heterogeneity of PCD patients often leads to the delayed establishment of a precise diagnosis of the disease, and it is not unusual that patients with other lung diseases are classified as PCD patients. The genetic background of the disease in patients with Serbian descent has not been established so far, so the mutational profile involved in the pathogenesis of PCD is necessary. Therefore, within this thesis, genomic profiling of patients with primary ciliary dyskinesia was performed in order to develop a genetic algorithm that, in addition to the described disease-causing pathogenic genetic variants and genes, would include novel variants in the coding regions of PCD-causative and candidate genes. This approach allows the establishment of a strategy for the differential diagnosis of PCD and other lung diseases suspected to ciliopathies that are clinically manifested as PCD. Functional characterization of a novel potentially pathogenic variant in PCD disease-causing genes and genes that have not been associated with PCD so far, but could be associated with the characteristic phenotype of PCD, was performed. One of the main features of PCD is ultrastructural defects of cilia leading to ciliary immotility, abnormal motility, or their absence..
Examination of probiotic and immunomodulatory characteristics of natural isolates of enterococci on in vitro and in vivo models
Get PDFEnterokoke pripadaju grupi mleĉnokiselinskih bakterija koje su široko rasprostranjene u prirodi. Predominantno se nalaze u gastrointestinalnom traktu životinja, od insekata do ĉoveka i preko fecesa se oslobaĊaju u spoljašnju sredinu, nakon ĉega naseljavaju razliĉita staništa. Imaju kontroverzni status zbog uloge u bolniĉkim infekcijama i širenja gena odgovornih za rezistenciju na antibiotike. S druge strane, neki sojevi se koriste kao probiotici za životinje i ĉoveka, kao i starter kulture u mlekarskoj industriji. Da bi se enterokoke koristile kao probiotici neophodno je da budu bezbedne za korišćenje, da nemaju hemolitiĉku i želatinoznu aktivnost i da nisu otporne na antibiotike. Enterokoke sintetišu antimikrobne proteine, bakteriocine, i na taj naĉin regulišu brojnost drugih bakterija i modulišu odgovor domaćina na infekciju. One mogu da sintetišu i biomolekule koji interaguju sa ćelijama domaćina i na taj naĉin mogu da menjaju razliĉite signalne puteve unutar ćelija domaćina. Biomolekuli sintetisani od strane probiotiĉkih enterokoka nazivaju se postbiotici. Primena postbiotika predstavlja bezbednu alternativu korišćenja enterokoka u biomedicini. Ova doktorska disertacija imala ja za cilj da ispita probiotiĉka i imunomodulatorna svojstva prirodnih sojeva enterokoka izolovanih iz fermentisanih mleĉnih proizvoda sa podruĉja Zapadnog Balkana. Definisana su tri cilja: 1. da se pronaĊu sojevi bezbedni za korišćenje u biomedicini; 2. da se ispitaju probiotiĉke karakteristike tih sojeva u modelima in vitro; i 3. da se ispita imunomodulatorni efekat odabranog soja u modelima in vitro i in vivo. U cilju ispitivanja bezbedne upotrebe enterokoka kao probiotika testirano je ukupno 75 sojeva iz fermentisanih mleĉnih proizvoda koje pripadaju vrstama: Enterococcus durans (50 sojeva), Enterococcus faecium (15 sojeva), Enterococcus faecalis (6 sojeva), Enterococcus italicus (3 soja) i Enterococcus hirae (1 soj). Hemolitiĉku aktivnost pokazalo je 18,7% (14/75), dok je želatinaznu aktivnost imalo 6,7% (5/75) testiranih sojeva. Na osnovu rezultata testa mikrodilucije pokazana je visoka uĉestalost rezistencije na ciprofloksacin 48,2% (27/56), dok su u manjoj meri testirani sojevi rezistentni na gentamicin 10,7% (6/56). Na osnovu rezultata sposobnosti formiranja biofilma u primenjenim uslovima, devet sojeva nema sposobnost formiranja biofilma, 11 sojeva ima mogućnost formiranja slabog biofilma, BGGO9-28 ima sposobnost formiranja jakog biofilma, dok sojevi BGTRK4-42 i BGZLM1-5 pokazuju veoma jaku sposobnost formiranja...are predominantly found in the gastrointestinal tract of animals, from insects to humans and through faeces they are released into the environment, where they colonize different habitats. They have a controversial status according to the role as causative agents of hospital infections and the spread of genetic determinants of antibiotic resistance. In contrary, some strains have been used used as probiotics for animals and humans, as well as a starter cultures in the dairy industry. In order to use enterococci as probiotics, it is necessary to determine their safety, i.e. absence of hemolytic and gelatinase activity and susceptiblity to clinicaly relevant antibiotics. Enterococci synthesize antimicrobial proteins, bacteriocins, by which way they regulate the number of other bacteria and modulate the host response to the infection. They can also synthesize biomolecules that interact with the host cells, and change the different signalling pathways within host cells. Biomolecules synthesized by probiotic enterococci are called postbiotics. The use of postbiotic represents a safety alternative to the enterococci application in biomedicine. The aim of this dissertation was examination of probiotic and immunomodulatory characteristics of enterococci isolated from fermented dairy products from the Western Balkans countries. Three objectives were defined: 1. to find strains safe for use in biomedicine; 2. to examine the probiotic characteristics on in vitro models, and 3. to examine the immunomodulatory effect of the selected strain on in vitro and in vivo models. In order to analyse the safe use of enterococci as a probiotics, a total of 75 isolates from fermented dairy products were tested: Enterococcus durans (50 isolates), En. faecium (15 isolates), En. faecalis (6 isolates), En. italicus (3 isolates) and En. hirae (1 isolate). 18.7% (14/75) strains showed hemolytic activity, while 6.7% (5/75) had gelatinase activity. Based on the results of the microdilution test, 48.2% (27/56) of strains were ciprofloxacin resistance, while 10.7% (6/56) strains were resistant to gentamicin. According to the analysis of selected enterococci genomes, high incidence of adhesin encoding genes could be noticed. It was found that 30.4% of strains have three genes encoding different virulence factors, 21.7% of strains having two or four genes, 17.4% having five genes, and 4.3% of studied enterococci containing 6 or 7 genes which encode virulence factors, while the presence or absence of gene within the fsr operon varies from the analyzed strains..