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Acute promyelocytic leukemia lacking t(15;17): Molecular evidence of atypical PML/RAR-α transcriptional variant by gene sequencing
Get PDFUvod. Precizno dijagnostikovanje akutne promijelocitne leukemije (APL), ne samo na osnovu morfoloških i kliničkih parametara, već i na molekularnom nivou, veoma je važno radi primene adekvatne ciljane terapije. Prikaz bolesnika. Prikazali smo bolesnicu, staru 62 godine, sa dijagnozom APL. Primenom standardne citogenetičke analize, kao i primenom fluorescentne in situ hibridizacije (FISH), nije bilo potvrđeno prisustvo t(15;17) kod opisane bolesnice. Primenom metode reverzna transkriptazalančana reakcija polimeraze (RT-PCR), identifikovana su dva atipična promyelotic leukemia/retinoic acid receptor alpha (PML/RAR-α) fuziona transkripta. Oba transkripta su predstavljala izoforme. Duži transkript je zadržao "okvir čitanja" i kodirao je funkcionalan PML/RAR-α aberantni protein, dok je kraći transkript bio van "okvira čitanja". Zaključak. Naša studija ukazuje na potrebu za primenom molekularne metodologije u svakodnevnoj kliničkoj praksi. Precizna karakterizacija PML/RAR-α fuzionih transkipta čini osnovu za identifikovanje retkih bolesnika čije lečenje zahteva dodatni oprez. Prema našim saznanjima, ovo je tek peti slučaj opisanog atipičnog PML/RAR-α transkripta koji u sebi sadrži celokupan PML egzon 7a, a među njima jedini koji se nije mogao detektovati primenom citogenetičke i FISH analize. Svi ovde predstavljeni slučajevi su imali smrtni ishod. Zbog toga, naši rezulatati, zajedno sa sličnim slučajevima opisanim u literaturi, naglašavaju značaj detaljne identifikacije atipičnih PML/RAR-α fuzija, ne samo u svrhu prepoznavanja njihove uloge u procesu leukemogeneze, veći i u smislu procene njihovog uticaja na ishod lečenja.Introduction. The accurate diagnosis of acute promyelocytic leukemia (APL), not only on the morphological and clinical, but also on the molecular level, is very important for application of targeted therapies. Case report. A 62year-old woman presented with APL. By using conventional cytogenetic analysis as well as applying the fluorescence in situ hybridization (FISH) analysis it has not been possible to confirm the presence of t(15;17) in the presented patient. Using reverse transcriptase polymerase chain reaction (RT-PCR) two atypical promyelotic leukemia/retinoic acid receptor alpha (PML/RAR-α) fusion transcripts were identified. Both detected transcripts were isoforms. The larger transcript was in-frame, coding for functional aberrant PML/RAR-α protein, while the shorter transcript was an out-of-frame. Conclusion. Our study highlights the need for the application of molecular methodology in daily clinical practice. Precise characterization of PML/RAR-α fusion transcript creates a basis for identifying rare individual cases that require special caution when treating such patients. To our knowledge this is only the fifth case of atypical PML/RAR-α transcript containing full PML exon 7a, and among them the only one that was cytogenetically cryptic and FISH negative. All of the herein presented cases had lethal outcome. Therefore, our findings with the additional review of the literature, emphasizes the importance of detailed identification of atypical PML/RAR-α fusions, not only for the purpose of knowing their role in leukemogenesis, but also for the assessment of the impact that they can have on the outcome of the treatment
Large-scale chromosome flip-flop reversible inversion mediates phenotypic switching of expression of antibiotic resistance in lactococci
Get PDFBacteria can gain resistance to antimicrobials by acquiring and expressing genetic elements that encode resistance determinants such as efflux pumps and drug-modifying enzymes, thus hampering treatment of infection. Previously we showed that acquisition of spectinomycin resistance in a lactococcal strain was correlated with a reversible genomic inversion, but the precise location and the genes affected were unknown. Here we use long read whole-genome sequencing to precisely define the genomic inversion and we use quantitative PCR to identify associated changes in gene expression levels. The boundaries of the inversion fall within two identical copies of a prophage-like sequence, located on the left and right replichores; this suggests possible mechanisms for inversion through homologous recombination or prophage activity. The inversion is asymmetrical in respect of the axis between the origin and terminus of the replication and modulates the expression of a SAM-dependent methyltransferase, whose heterologous expression confers resistance to spectinomycin in lactococci and that is up regulated on exposure to spectinomycin. This study provides one of the first examples of phase variation via large-scale chromosomal inversions that confers a switch in antimicrobial resistance in bacteria and the first outside of Staphylococcus aureus
Streptomyces sp. BV410 isolate from chamomile rhizosphere soil efficiently produces staurosporine with antifungal and antiangiogenic properties
Get PDFApplying a bioactivity-guided isolation approach, staurosporine was separated and identified as the active principle in the culture extract of the new isolate Streptomyces sp. BV410 collected from the chamomile rhizosphere. The biotechnological production of staurosporine by strain BV410 was optimized to yield 56 mg/L after 14 days of incubation in soy flour-glucose-starch-mannitol-based fermentation medium (JS). The addition of FeSO4 significantly improved the staurosporine yield by 30%, while the addition of ZnSO4 significantly reduced staurosporine yield by 62% in comparison with the starting conditions. Although staurosporine was first isolated in 1977 from Lentzea albida (now Streptomyces staurosporeus) and its potent kinase inhibitory effect has been established, here, the biological activity of this natural product was assessed in depth in vivo using a selection of transgenic zebrafish (Danio rerio) models, including Tg(fli1:EGFP) with green fluorescent protein-labeled endothelial cells allowing visualization and monitoring of blood vessels. This confirmed a remarkable antiangiogenic activity of the compound at doses of 1 ng/ml (2.14 nmol/L) which is below doses inducing toxic effects (45 ng/ml; 75 nmol/L). A new, efficient producing strain of commercially significant staurosporine has been described along with optimized fermentation conditions, which may lead to optimization of the staurosporine scaffold and its wider applicability
Increased Incidence of Clostridium-like Species and Lower Diversity of Common Commensal Bacteria in Gut Microbiota of Children with Neurodevelopmental Disorders
No full textNitrate inhibits primary root growth by reducing accumulation of reactive oxygen species in the root tip in Medicago truncatula
Get PDFIn Medicago truncatula, nitrate, acting as a signal perceived by NITRATE TRANSPORTER1/PEPTIDE TRANSPORTER FAMILY 6.8 (MtNPF6.8), inhibits primary root growth through a reduction of root cell elongation. Since reactive oxygen species (ROS) produced and converted in root tip (O-2(center dot-) - gt H2O2 - gt (OH)-O-center dot) have been reported to control cell elongation, the impact of nitrate on the distribution of these ROS in the primary root of M. truncatula was analyzed. We found that nitrate reduced the content of O-2(center dot-), H2O2 and (OH)-O-center dot in the root tip of three wild type genotypes sensitive to nitrate (R108, DZA, A17), inhibition of root growth and O-2(center dot-) accumulation being highly correlated. Nitrate also modified the capacity of R108 root tip to produce or remove ROS. The ROS content decrease observed in R108 in response to nitrate is linked to changes in peroxidase activity (EC1.11.1.7) with an increase in peroxidative activity that scavenge H2O2 and a decrease in hydroxylic activity that converts H2O2 into (OH)-O-center dot. These changes impair the accumulation of H2O2 and then the accumulation of (OH)-O-center dot, the species responsible for cell wall loosening and cell elongation. Accordingly, nitrate inhibitory effect was abolished by externally added H2O2 or mimicked by KI, an H2O2 scavenger. In contrast, nitrate has no effect on ROS production or removal capacities in npf6.8-2, a knockdown line insensitive to nitrate, affected in the nitrate transporter MtNPF6.8 (in R108 background) by RNAi. Altogether, our data show that ROS are mediators acting downstream of MtNPF6.8 in the nitrate signaling pathway.This is the peer reviewed version of the paper: Zang, L., Morère-Le Paven, M.-C., Clochard, T., Porcher, A., Satour, P., Mojović, M., Vidović, M., Limami, A. M., & Montrichard, F. (2020). Nitrate inhibits primary root growth by reducing accumulation of reactive oxygen species in the root tip in Medicago truncatula. Plant Physiology and Biochemistry, 146, 363–373.[https://doi.org/10.1016/j.plaphy.2019.11.006]Published version: [https://imagine.imgge.bg.ac.rs/handle/123456789/1368
Fatty acid composition of rosehip seed oil
Get PDFRosehip is a pseudo-fruit of the rose plant, one of the most widespread wild species of the Rosa genus in Serbia. Due to its nutritional and sensory properties, rosehip is widely utilized for the production of jams, marmalades, juices, teas, etc. On the other hand, rosehip seeds are waste material from the food industry, which represent are rich source of fatty acids. The aim of this study was to assess the fatty acids profile of rosehip seeds by applying two extraction methods: ultrasound-assisted extraction combined with organic solvent extraction (UAE/OSCE) and organic solvent conventional extraction (OSCE). The identification and quantification of fatty acids (FA), in the form of methyl esters, were performed by gas chromatography with a flame ionization detector (GC/FID). Based on the obtained results, it was observed that the composition of fatty acids and their relative amounts were influenced by the applied method, as well as by the solvent-to-sample ratio. The analysis revealed the presence of unsaturated fatty acids (UFA) as the dominant ones in most studied samples, whereas the most abundant fatty acids were, in descending order, stearic acid (48.11%), linoleic acid (35.38%), palmitoleic acid (33.78%) and eicosadienoic acid (30.57%).Шипурак је псеудо-плод биљке руже, једне од најраспрострањенијих дивљих врста из рода Rosa у Србији. Захваљујући
нутритивним и сензорним својствима, шипурак има широку примену у производњи џемова, мармелада, сокова, чајева
итд. Семенке шипурка су отпадни материјал у прехрамбеној индустрији који представља добар извор масних киселина.
Циљ овог рада био је да се процени профил масних киселина у семенкама шипурка применом две методе екстракције:
ултразвучнеекстракције у комбинацији са екстракцијом органским растварачем (UZE/KEOR) и конвенционалне
екстракције органским растварачем (KEOR). Идентификација масних киселина (MK), у форми метил естара, извршена је
методом гасне хроматографије са пламено-јонизујућим детектором (GC/FID). На основу добијених резултата, уочено је
да су примењене методе екстракције, као и однос растварача и масе узорка, имали утицај на састав масних киселина и
њихову релативну обилност. Незасићене масне киселине (NMK) идентификоване су као доминантне у већини
испитиваних узорака. Најзаступљеније масне киселине су биле стеаринска (48,11%), линолна (35,38%), палмитолеинска
(33,78%) и еикосадиеноинска киселина (30,57%)Šipurak je pseudo-plod biljke ruže, jedne od najrasprostranjenijih divljih vrsta iz roda Rosa u Srbiji. Zahvaljujući
nutritivnim i senzornim svojstvima, šipurak ima široku primenu u proizvodnji džemova, marmelada, sokova, čajeva
itd. Semenke šipurka su otpadni materijal u prehrambenoj industriji koji predstavlja dobar izvor masnih kiselina.
Cilj ovog rada bio je da se proceni profil masnih kiselina u semenkama šipurka primenom dve metode ekstrakcije:
ultrazvučneekstrakcije u kombinaciji sa ekstrakcijom organskim rastvaračem (UZE/KEOR) i konvencionalne
ekstrakcije organskim rastvaračem (KEOR). Identifikacija masnih kiselina (MK), u formi metil estara, izvršena je
metodom gasne hromatografije sa plameno-jonizujućim detektorom (GC/FID). Na osnovu dobijenih rezultata, uočeno je
da su primenjene metode ekstrakcije, kao i odnos rastvarača i mase uzorka, imali uticaj na sastav masnih kiselina i
njihovu relativnu obilnost. Nezasićene masne kiseline (NMK) identifikovane su kao dominantne u većini
ispitivanih uzoraka. Najzastupljenije masne kiseline su bile stearinska (48,11%), linolna (35,38%), palmitoleinska
(33,78%) i eikosadienoinska kiselina (30,57%
Identification and characterization of biogenic silencers of Pseudomonas aeruginosa quorum sensing
Get PDFNemogućnost kontrole pojave i širenja rezistencije na antibiotike usmerila je istraživanja u poslednje dve dekade ka iznalaženju novih terapeutskih opcija, sa ciljem tretmana infekcija izazvanih patogenim bakterijama rezistentnim na veći broj kliniĉki znaĉajnih antibiotika. Uzevši u obzir ĉinjenicu da je antibiotska rezistencija kompleksan, multifaktorijalni fenomen, rešenje ovog problema ukljuĉuje niz pristupa usmerenih na kontrolu faktora koji olakšavaju nastanak i širenje rezistencije. Jedan od tih pristupa sastoji se u razvoju novih terapeutika koji bi delovali mehanizmima razliĉitim od trenutno dostupnih antibiotika. U tom pogledu, antivirulentna terapija zamišljena je kao obećavajuća alternativa sa ciljem kontrole virulencije specifiĉne za odreĊene patogene, bez vršenja snažnog selektivnog pritiska na bakterijske ćelije. Imajući u vidu da je Pseudomonas aeruginosa jedan od vodećih uzroĉnika unutarbolniĉkih infekcija širom sveta, leĉenje infekcija izazvanih ovim patogenom predstavlja veliki terapeutski izazov. P. aeruginosa svoj patogeni potencijal ostvaruje zahvaljujući mnogobrojnim uroĊenim, steĉenim i adaptivnim mehanizmima rezistencije. Pored toga, posedovanje sistema meĊućelijske komunikacije (eng. quorum sensing, QS) ovom patogenu omogućava fleksibilnost u regulaciji ekspresije gena ukljuĉenih u virulenciju, formiranje biofilma, produkciju sekundarnih metabolita i faktora koji imaju ulogu u zaštiti od imunskog sistema domaćina. Stoga bi primena antivirulentne terapije zasnovane na utišavanju meĊućelijske komunikacije bakterija mogla poslužiti kao obećavajuće oruĊe u kontroli infekcija izazvanih predstavnicima P. aeruginosa za koje ne postoji adekvatna terapija trenutno dostupnim antibioticima. U skladu sa time, predmet prouĉavanja ove teze bila je potraga za novim utišivaĉima meĊućelijske komunikacije bakterija (eng. quorum quenching, QQ) produkovanih od strane kliniĉkih izolata koji tokom infekcija dele istu ekološku nišu sa P. aeruginosa. Analizirana je kolekcija Gramnegativnih kliniĉkih izolata Laboratorije za molekularnu mikrobiologiju, Instituta za molekularnu genetiku i genetiĉko inženjerstvo, Univerziteta u Beogradu sa ciljem pronalaženja sojeva koji produkuju QQ molekule. Nakon odabira sojeva nosioca QQ fenotipa, usledila je njihova identifikacija kao i funkcionalna karakterizacija na model sistemu kliniĉkog izolata P. aeruginosa MMA83. Upotrebom Chromobacterium violaceum CV026 biosenzora, selektovano je 19 sojeva (od ukupno 633 analiziranih izolata) koji su ispoljavali QQ fenotip, od kojih su dva izolata Delftia sp. 11304 i Burkholderia sp. BCC4135, na osnovu najznaĉajnije QQ aktivnosti izdvojena za detaljniju karakterizaciju. UtvrĊeno je da Delftia sp. 11304 soj pripada Delftia tsuruhatensis vrsti, dok Burkholderia sp. BCC4135 pripada Burkholderia cepacia vrsti sa novim sekvencnim tipom ST1485. In silico analizom genomskih sekvenci ustanovljeno je da oba odabrana soja poseduju izuzetan potencijal virulencije i rezistencije na antimikrobna jedinjenja. Priroda QQ molekula koje produkuju ovi sojevi bila je razliĉita; konstatovano je da soj 11304 produkuje male molekule neproteinske prirode (QS inhibitore, QSI), dok BCC4135 produkuje QQ enzime. Pored toga, kod BCC4135 izolata utvrĊeno je prisustvo i QS fenomena. Ukupni etil-acetatni ekstrakt supernatanta soja D. tsuruhatensis 11304 (QSI ekstrakt) ostvario je znaĉajan antivirulentni potencijal na model sistemu kliniĉkog izolata P. aeruginosa MMA83...finding novel therapeutic options to combat multidrug-resistant pathogenic bacteria during the last two decades. Given the fact that antimicrobial resistance is a complex, multifactorial phenomenon, the solution to this problem comprises a range of approaches focused on monitoring the factors that facilitate the emergence and spread of resistance. One of proposed strategies consists of developing novel therapeutics that operate under different principles to the currently available antibiotics. In this respect, antivirulence therapy has been conceived as a promising alternative to control virulence in a pathogen-specific manner, without exerting strong selective pressure on the bacterial cells. Having in mind that Pseudomonas aeruginosa has been considered as a leading cause of nosocomial infections worldwide, the treatment of infections caused by this pathogen represents a major therapeutic challenge. The pathogenic potential of P. aeruginosa has been accomplished due to a numerous innate, acquired, and adaptive resistance mechanisms. In addition, the presence of cell-to-cell communication system (quorum sensing, QS) allows this pathogen the flexibility in the regulation of virulence gene expression responsible for biofilm development, production of secondary metabolites, and immuneevasive factors. Therefore, the use of antivirulence therapy based on the silencing of bacterial communication could serve as a promising tool in the control of infections caused by P. aeruginosa for which there is no adequate therapy with currently available antibiotics. Accordingly, the subject of this thesis was discovery and characterization of novel quenchers of bacterial cell-to-cell communication produced by clinical isolates that share the same ecological niche with P. aeruginosa during infections. A collection of Gram-negative clinical isolates from the Laboratory for Molecular Microbiology Institute of Molecular Genetics and Genetic Engineering University of Belgrade was analyzed in order to select the strains that produce quorum quenching (QQ) molecules. After the selection of strains carrying QQ phenotype, the identification of QQ molecules as well as their functional characterization on the model system of P. aeruginosa MMA83 clinical isolate was performed. Using Chromobacterium violaceum CV026 biosensor, 19 strains (out of a total of 633 analyzed isolates) with QQ phenotype were discovered, among which two clinical isolates Delftia sp. 11304 and Burkholderia sp. BCC4135 have been selected as strongest producers of QQ molecules for detailed characterization. According to the genomic sequence analysis, Delftia sp. 11304 was identified as a Delftia tsuruhatensis species while Burkholderia sp. BCC4135 was unveiled as Burkholderia cepacia with novel sequence type ST1485. In silico analysis of genomic sequences indicated that both selected strains possess significant antimicrobial resistance and virulence potential. The nature of the QQ molecules produced by these strains was determined as different; strain 11304 was found to produce small molecules of non-proteinaceous nature (QS inhibitors, QSI), while BCC4135 produced QQ enzymes. Besides, the presence of the QS phenomenon was determined in BCC4135 isolate. The total ethyl acetate extract of the D. tsuruhatensis 11304 culture supernatant (QSI extract) showed significant antivirulence potential on the model system of clinical isolate P. aeruginosa MMA83..
Gut Microbiota Dysbiosis Associated With Altered Production of Short Chain Fatty Acids in Children With Neurodevelopmental Disorders
Get PDFWhile gut microbiota dysbiosis has been linked with autism, its role in the etiology of other neurodevelopmental disorders (NDD) is largely underexplored. To our knowledge this is the first study to evaluate gut microbiota diversity and composition in 36 children from the Republic of Serbia diagnosed with NDD and 28 healthy children. The results revealed an increased incidence of potentially harmful bacteria, closely related to Clostridium species, in the NDD patient group compared to the Control group: Desulfotomaculum guttoideum (P lt 0.01), Intestinibacter bartlettii (P lt 0.05), and Romboutsia ilealis (P lt 0.001). On the other hand, significantly lower diversity of common commensal bacteria in the NDD group of patients was noticed. Enterococcus faecalis (P lt 0.05), Enterococcus gallinarum (P lt 0.01), Streptococcus pasteurianus (P lt 0.05), Lactobacillus rhamnosus (P lt 0.01) and Bifidobacteria sp. were detected in lower numbers of patients or were even absent in some NDD patients. In addition, butyrate-producing bacteria Faecalibacterium prausnitzii (P lt 0.01), Butyricicoccus pullicaecorum (P lt 0.05), and Eubacterium rectale (P = 0.07) were less frequent in the NDD patient group. In line with that, the levels of fecal short chain fatty acids (SCFAs) were determined. Although significant differences in SCFA levels were not detected between NDD patients and the Control group, a positive correlation was noted between number of rDNA amplicons obtained with universal primers and level of propionic acid, as well as a trend for levels of total SCFAs and butyric acid in the Control group. This correlation is lost in the NDD patient group, indicating that NDD patients' microbiota differs from the microbiota of healthy children in the presence or number of strong SCFA-producing bacteria. According to a range-weighted richness index it was observed that microbial diversity was significantly lower in the NDD patient group. Our study reveals that the intestinal microbiota from NDD patients differs from the microbiota of healthy children. It is hypothesized that early life microbiome might have an impact on GI disturbances and accompanied behavioral problems frequently observed in patients with a broad spectrum of NDD
Pharmacogenomic Markers of Methotrexate Response in the Consolidation Phase of Pediatric Acute Lymphoblastic Leukemia Treatment
Get PDFMethotrexate (MTX) is one of the staples of pediatric acute lymphoblastic leukemia (ALL) treatment. MTX targets the folate metabolic pathway (FMP). Abnormal function of the enzymes in FMP, due to genetic aberrations, leads to adverse drug reactions. The aim of this study was to investigate variants in pharmacogenes involved in FMP and their association with MTX pharmacokinetics (MTX elimination profile) and toxicity in the consolidation therapy phase of pediatric ALL patients. Eleven variants in the thymidylate synthetase (TYMS), methylenetetrahydrofolate reductase (MTHFR), dihydrofolate reductase (DHFR), SLC19A1 and SLCO1B genes were analyzed in 148 patients, using PCR- and sequencing-based methodology. For the Serbian and European control groups, data on allele frequency distribution were extracted from in-house and public databases. Our results show that the A allele of SLC19A1 c.80 variant contributes to slow MTX elimination. Additionally, the AA genotype of the same variant is a predictor of MTX-related hepatotoxicity. Patients homozygous for TYMS 6bp deletion were more likely to experience gastrointestinal toxicity. No allele frequency dissimilarity was found for the analyzed variants between Serbian and European populations. Statistical modelling did not show a joint effect of analyzed variants. Our results indicate that SLC19A1 c.80 variant and TYMS 6bp deletion are the most promising pharmacogenomic markers of MTX response in pediatric ALL patients
Non-cytotoxic photostable monomethine cyanine platforms: Combined paradigm of nucleic acid staining and in vivo imaging
No full textCyanine based chemosensing platforms have successfully been employed over the past couple of decades in various fields of biomolecular sciences. Still a substantial number of recent advances and improvements on this class of compounds are reported in the art. This paper presents our latest work on the improved synthetic approach, study on photophysical properties, and biosensing applicability of monomethine cyanine dyes. The series of mono-, di- and tricationic dyes showed up to 5-fold enhanced resistance against photobleaching compared to the commercially available Thiazole Orange (TO). The title compounds were studied as potential molecular probes for the detection of deoxyribonucleic acid, demonstrating their capacity as excellent fluorescent labeling agents. Depending on the dye chemical structure, current Cl-TO compounds exhibit up to 834-fold enhanced fluorescence emission and form stable complexes with Calf Thymus-DNA. The calculated binding constants were found to be higher than several conventional fluorogenic dyes for nucleic acid detection. All studied derivatives appeared as less cytotoxic than the Thiazole Orange. IC50 concentrations in human fibro-blasts MRCS cell line were calculated up to 50 mu M for the synthesized Cl-TO dyes, and 0.5 mu M for the parental Thiazole Orange. Two of the dyes were found very competent in post-electrophoretic visualization of DNA. As demonstrated by the agarose gel electrophoresis, the staining efficiency and detection limits of the dyes were comparable to the widely used Ethidium Bromide. The tricationic dye revealed great potential for cell cycle analysis in G1, S and G2 phases. The chlorinated TO derivatives readily stain human cells in vivo, while they can effectively be applied for eukaryotic and microbial cell staining