imagine (Institute of molecular genetics and genetic engineering)
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The importance of genomic profiling for differential diagnosis of pediatric lung disease patients with suspected ciliopathies
Get PDFUvod/Cilj Izmenjena funkcija aksonemalne strukture dovodi do ciliopatija (motornih i senzornih), koje su do sada povezane sa brojnim pedijatrijskim poremećajima, uključujući i respiratorne. Primarna cilijarna diskinezija (PCD) najčešća je ciliopatija, koja nastaje kao posledica poremećaja u motornim cilijama. Promenjena struktura i/ ili funkcija motornih cilija dovodi do neonatalnog respiratornog distresa, hroničnog vlažnog kašlja, simptoma nazalne sekrecije, bronhoektazija, hronične upale sinusa i uha, a 50% bolesnika ima i situs inversus. Ovi simptomi su prilično uobičajeni kod male dece i u drugim stanjima; stoga je uspostavljanje precizne dijagnoze otežano. Cilj ovog istraživanja je ukazivanje na značaj genomskog profilisanja bolesnika i dizajniranje strategije za genetičku analizu podataka kod bolesnika suspektnih na ciliopatije sa kliničkom slikom sličnom drugim bolestima pluća. Metode Sproveli smo bioinformatičku analizu podataka dobijenih metodom sekvenciranja nove generacije 21 bolesnika sa potvrđenom ili suspektnom dijagnozom PCD-a. Analizirano je 93 gena: 29 PCD gena, 45 gena asociranih sa pojedinačnim simptomima plućnih bolesti i 19 gena asociranih sa senzornim ciliopatijama. Rezultati Dizajnirani algoritam za genetičku analizu NAM je omogućio da potvrdimo kliničku i uspostavimo genetičku dijagnozu kod 17/21 (80,95%) bolesnika, među kojima je 11/21 (52,38%) PCD bolesnika. Kod 3/21 (14,28%) bolesnika detektovane su monoalelske varijante u PCD genima, kod 6/21 (28,57%) bolesnika detektovane su varijante u genima relevantnim za druga plućna oboljenja, dok je kod 1/21 (4,76%) bolesnika genetička osnovna bolesti ostala nerazjašnjena. Zaključak Dizajniranje strategije za lakše i brže uspostavljanje konačne dijagnoze ciliopatija je obavezno i uključuje i kliničku i genetičku potvrdu bolesti.Introduction/Objective Dysfunction of the axonemal structure leads to ciliopathies. Sensory and mo-tile ciliopathies have been associated with numerous pediatric diseases, including respiratory diseases. Primary ciliary dyskinesia (PCD) is ciliopathy linked to the dysfunction of motile cilia. Motile ciliary dys-function in childhood leads to chronic rhinosinusitis, persistent cough, neonatal respiratory distress, bronchiectasis, and situs inversus (SI) have 50% of patients. These symptoms are common among pediatric lung diseases, which additionally makes it difficult to establish the accurate diagnosis. The aim of the study was to point out the significance of genomic profiling for patients with suspected ciliopathies and to design a strategy for genomic analysis relevant for differential diagnosis of lung disease patients with suspected ciliopathies. Methods We conducted a bioinformatic analysis of data generated by New Generation Sequencing (NGS) approach of 21 patients with final or suspected diagnosis of PCD. It was analyzed 93 genes: 29 PCD genes, 45 genes related to individual symptoms of lung diseases, and 19 genes related to sensory ciliopathies. Results the algorithm we have designed, enabled us to establish the clinical and genetic diagnosis for 17/21 (80.95%) patients, among which 11/21 (52.38%) were PCD patients. In 3/21 (14.28%) patients we detected monoallelic variants in PCD disease-causing genes. In 6/21 (28.57%) patients, variants in genes for other pulmonary diseases were detected, and for one patient, genetic background of disease remained unclear. Conclusion an improved strategy for easier and faster establishment of final diagnosis of ciliopathies is mandatory and includes both, clinical and genetic confirmation of disease
Brevibacillus laterosporus strains BGSP7, BGSP9 and BGSP11 isolated from silage produce broad spectrum multi-antimicrobials
Get PDFBacteria active against multi-drug resistant pathogens, isolated by direct selection of colonies from clover silage samples, produce zones of inhibition against two Gram-negative (Klebsiella pneumoniae Ni9 and Pseudomonas aeruginosa MMA83) and two Gram-positive (Staphylococcus aureus ATCC25923 and Listeria monocytogenes ATCC19111) pathogens. Isolates BGSP7, BGSP9, BGSP11 and BGSP12 produced the largest zones of inhibition against all four pathogens when grown in LB broth with aeration at 37 degrees C. Isolates BGSP7, BGSP9, BGSP11 and BGSP12 were identified as Brevibacillus laterosporus and pulsed field gel electrophoresis and extracellular protein profiles showed that three different strains (BGSP7, BGSP9 and BGSP11) were isolated. A semi-native SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis) gel overlay assay showed that BGSP7 and BGSP9 produce small antimicrobial molecules of about 1.5 kDa, while BGSP11 produces antimicrobial molecules of 1.5 and 6 kDa active against S. aureus ATCC25923. Amino acid analysis of two antimicrobial molecules (1583.73 Da; from BGSP7 and 1556.31 Da; from BGSP11) revealed that they have a similar composition and differ only by virtue of the presence of a methionine which is present only in BGSP11 molecule. Genome sequencing of the three isolates revealed the presence of gene clusters associated with the production of non-ribosomally synthesized peptides (brevibacillin, bogorol, gramicidin S, plipastatin and tyrocin) and bacteriocins (laterosporulin, a lactococcin 972-like bacteriocin, as well as putative linocin M18, sactipeptide, UviB and lantipeptide-like molecules). Ultimately, the purification of a number of antimicrobial molecules from each isolate suggests that they can be considered as potent biocontrol strains that produce an arsenal of antimicrobial molecules active against Gram-positive and Gram-negative multi-resistant pathogens, fungi and insects
Probiotic potential of Lactobacillus fermentum G-4 originating from the meconium of newborns
Get PDFThe present study was dedicated to determining probiotic potential of a human isolate G-4, originated from meconium. The isolate was identified using morphological, physiological and biochemical assays and molecular method based on 16S rRNA gene sequencing. In order to evaluate its probiotic properties in vitro tests were performed: the survival in simulated gastrointestinal conditions, adhesion to hexadecane, and antimicrobial activity. Safety aspects of the isolate were examined by testing toxicity, gastrointestinal tolerance and bacterial translocation in vivo, as well as hemolytic activity in vitro. The isolate G-4, identified as Lactobacillus fermentum, showed viability in artificial gastric and intestinal juice (low degree of cell viability reduction for 0.69 and 1.30 logCFU mL(-1) units, respectively), moderate adhesion to hexadecane (39 +/- 2.1 %), and antimicrobial activity against Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Salmonella enterica subsp. enterica serotype Abony and Clostridium sporogenes, due to production of lactic acid (9.80 g L-1). No signs of toxicity, bacterial translocation, hemolytic activity, were observed
In vitro digestion of meat- and cereal-based food matrix enriched with grape extracts: How are polyphenol composition, bioaccessibility and antioxidant activity affected?
No full textThe aim of this study was to evaluate the effect of enriching a complex food matrix (FM) with grape extracts on polyphenol content, composition, bioaccessibility and antioxidant activity during digestion. The grape extracts and FM were separately tested under the same conditions as controls. The FM by itself contains a significant amount of phenolic acids and flavonols, influencing the final recovery of polyphenols from grape extracts. The FM significantly increased the total recovery of polyphenols after digestion of grape seed extracts compared to those digested without the FM; however, a low recovery of proantocyanidins and total flavonoids was observed. Digestive fluids and FM compounds significantly increased the total polyphenol content of grape digests and significantly contributed to their ABTS(center dot+) scavenging activity and ferrous-ion-chelating capacity. The present study suggested that enrichment of meat-and cereal-based products with grape polyphenol extracts could be a good strategy to formulate a healthier diet.This is the peer-reviewed version of the article:
[https://imagine.imgge.bg.ac.rs/handle/123456789/1609][https://www.sciencedirect.com/science/article/abs/pii/S030881461930175X?via%3Dihub
Antiplasmodial Activity and In Vivo Bio-Distribution of Chloroquine Molecules Released with a 4-(4-Ethynylphenyl)-Triazole Moiety from Organometallo-Cobalamins
Get PDFWe have explored the possibility of using organometallic derivatives of cobalamin as a scaffold for the delivery of the same antimalarial drug to both erythro- and hepatocytes. This hybrid molecule approach, intended as a possible tool for the development of multi-stage antimalarial agents, pivots on the preparation of azide-functionalized drugs which, after coupling to the vitamin, are released with a 4-(4-ethynylphenyl)-triazole functionality. Three chloroquine and one imidazolopiperazine derivative (based on the KAF156 structure) were selected as model drugs. One hybrid chloroquine conjugate was extensively studied via fluorescent labelling for in vitro and in vivo bio-distribution studies and gave proof-of-concept for the design. It showed no toxicity in vivo (zebrafish model) as well as no hepatotoxicity, no cardiotoxicity or developmental toxicity of the embryos. All 4-(4-ethynylphenyl)-triazole derivatives of chloroquine were equally active against chloroquine-resistant (CQR) and chloroquine-sensitive (CQS) Plasmodium falciparum strains
Presence of thrombophilia and levels of coagulation factors, coagulation inhibitors and TAFI do not affect global haemostasis or bleeding phenotype in patients with haemophilia A
No full textThe extent of recent material transfers and past demographic history in Norway spruce inferred from genomic data
No full textThe transfer of forest reproductive material for afforestation and improvement, which has
been very common in Europe especially over the last two centuries, may bias inferences on
past population movements in response to the last glacial maximum (LGM), some 18,000
years ago. In the present study, we genotyped 1,672 individuals belonging to three Picea
species (P. abies, P. obovata, and P. omorika) at 400,000 SNPs using exome capture to infer
the past demographic history of Norway spruce (P. abies) and estimate the amount of recent
introduction used to establish the Norway spruce breeding program in southern Sweden.
Most of these trees belong to P. abies and originate from the base populations of the Swedish
breeding program. Others originate from populations across the natural ranges of the three
species. Of the 1,499 individuals stemming from the breeding program, a large proportion
corresponds to recent introductions from mainland Europe. The split of P. omorika occurred
23 million years ago (mya), while the divergence between P. obovata and P. abies began 17.6
mya. Demographic inferences retrieved the same main clusters within P. abies as previous
studies, that is, a vast northern domain ranging from Norway to central Russia, where the
species is progressively replaced by Siberian spruce (P. obovata) and two smaller domains,
an Alpine domain and a Carpathian one, but also revealed further subdivision and gene flow
among clusters. The three main domains divergence was ancient (15 mya), and all three
went through a bottleneck corresponding to the LGM. Approximately 17% of P. abies Nordic
domain migrated from P. obovata ~0.103 mya, when both species had much larger effective
population sizes. Our analysis of genome-wide polymorphism data thus revealed the complex
demographic history of Picea genus in Western Europe and highlighted the importance of
material transfer in Swedish breeding program.Abstracts of the 6th CONGRESS OF THE SERBIAN GENETIC SOCIETY Vrnjačka Banja; October 13 to 17, 2019
Exome-scale variation data in a serbian cohort study
No full textAlthough the importance of population diversity in genomic research has long been
recognized, smaller populations, like the Serbian population, are under-represented in
genetic data repositories. In order to respond to this need, to fill in the sparse zones of
the global genomic map, we analyzed exome data obtained using the next generation
sequencing (NGS) platform TruSightOne (Ilumina), in a cohort of unrelated individuals from
Serbia, sequenced as part of disease-specific genetic studies. Variant calling was performed
by using the GATK (Genome Analysis ToolKit). The over-represented variants are annotated
with population allele frequencies and public resources such as GnomAD, 1000G and ClinVar
and effect predictions tools, ISTREE and the Ensembl Variant Effect Predictor (VEP). Here we
present preliminary findings on genetic variants specific for a Serbian cohort such as those
in genes RP1L1, KRT4 and SHANK3. The obtained results will improve the characterization of
population-specific, germline and somatic disease associated variants in a wider context. This
is the first-ever report on exome-scale data in the thus far under-studied Serbian population.
Our study represents the first step towards an ethnically relevant reference dataset, which
would be subsequently integrated into precision medical initiatives relevant to individuals of
Serbian and Western Balkan descent worldwide.Abstracts of the 6th CONGRESS OF THE SERBIAN GENETIC SOCIETY Vrnjačka Banja; October 13 to 17, 2019
Leaf total transcriptome analysis enriches insight into maize abiotic stress signaling network
No full textAbiotic stresses cause great losses in crop production worldwide. Due to climatic changes
temperatures higher than the average are frequently observed in temperate areas in the
course of maize flowering and grain filling period, displaying detrimental effect on maize yield.
In order to overcome this problem development of drought tolerant maize lines, but also
cold tolerant maize lines that would allow earlier sowing and hence avoidance of extremely
high temperatures during sensitive developmental phases in maize, became important goal
of maize breeding programs in temperate areas. A set of 46 maize inbred lines was chosen
from three main heterotic groups (BSSS, Lancaster, Iowa dent) important for commercial
breeding programs. Plants were grown under the optimal conditions in the greenhouse and
sampled upon entering V5 growth stage. A total RNA isolated from leaves of three plants of
each inbred line was used for DNA library preparation by Illumina TruSeq Stranded RNA LT
kit. Pair-end sequencing was performed on MiSeq Illumina sequencer using MiSeq Reagent
kit v2 (2x150bp). For data manipulation and analysis a custom made bioinformatics pipeline
was used. Differential gene expression analysis revealed a minimum of 21 genes that were
strongly statistically supported for differential expression between the inbred lines and also
annotated as involved in abiotic stress responses in other plant species. We tested 10 of
these genes in maize by using qPCR on the subset of 8 inbred lines grown under optimal, cold
and drought conditions. Real-time PCR results revealed significant change in the expression
level of tested genes under abiotic stresses, supporting bioinformatics prediction. Verified
genes were then submitted to free online gene networking tools and after determining
relationships between predicted networks and inbred line phenotypes, possible novel gene
candidates for enriching maize abiotic stress signaling network resolution were proposed.Abstracts of the 6th CONGRESS OF THE SERBIAN GENETIC SOCIETY Vrnjačka Banja; October 13 to 17, 2019
Electronic cigarette liquids show toxic effects in vitro and in vivo
No full textThe use of electronic cigarettes is on the rise with a public perceiving them as a safer alternative to conventional cigarettes. However, their safety has only recently started being studied to a greater extent.
The aim of this study was to test the toxicity of commercially available electronic cigarette liquids (ECL) with and without nicotine in vitro and in vivo.
Two cell lines originating from the lung tissue were used - A549 and BEAS-2B. Using MTT assay, we have shown that there is a dose dependant decrease in cell viability in case of both tested liquids, and in both cell lines. We also detected cell-free mitochondrial DNA in culture medium of treated cells, indicating necrotic cell death. In order to investigate if cells undergone necroptosis we used pre-treatment with necrostatin-1 (Nec-1), a specific RIP-1 kinase inhibitor. The addition of Nec-1 did not affect cell survival under tested conditions suggesting that the cells do not go through the process of controlled necrosis.
In vivo toxicity of different concentrations of ECL was assessed by the analysis of survival, hatching and malformations of zebrafish embryos. The effects of ECL with nicotine were comparable to those when embryos were treated with pure nicotine at the same concentration, while the ECL without nicotine did not have toxic effects in used concentrations. The embryo mortality was increased when treated with ECL with 20µg/mL of nicotine and several embryo malformations (pericardial oedema, body curvature, shortening of the body and decreased movements) were present at 10µg/mL concentration.
Our result highlight the need for further testing the molecular effects and safety of electronic cigarettes