imagine (Institute of molecular genetics and genetic engineering)
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Point-of-Need DNA Testing for Detection of Foodborne Pathogenic Bacteria
Get PDFFoodborne pathogenic bacteria present a crucial food safety issue. Conventional diagnostic methods are time-consuming and can be only performed on previously produced food. The advancing field of point-of-need diagnostic devices integrating molecular methods, biosensors, microfluidics, and nanomaterials offers new avenues for swift, low-cost detection of pathogens with high sensitivity and specificity. These analyses and screening of food items can be performed during all phases of production. This review presents major developments achieved in recent years in point-of-need diagnostics in land-based sector and sheds light on current challenges in achieving wider acceptance of portable devices in the food industry. Particular emphasis is placed on methods for testing nucleic acids, protocols for portable nucleic acid extraction and amplification, as well as on the means for low-cost detection and read-out signal amplification
VARIANT C.454-397T>C OF ESTROGEN RECEPTOR Α INFLUENCES RESULTS OF LUPUS ANTICOAGULANT SCREENING AND FIBRINOGEN MEASURMENT IN PATIENTS WITH ACUTE MYOCARDIAL INAFARCTION - A PILOT STUDY
No full textEstrogens are steroid hormones which, beside reproductive organs, influence other
physiological systems i.e. cardiovascular system, skeleton, cognition, etc. Their effects
are achieved via estrogen receptors (ER) - α and β - both being polymorphic.
Previously, a possible association was assessed between variants of ERα gene and
antiphospholipid syndrome (APS), an autoimmune condition, predisposing to acute
myocardial infarction (AMI). Lupus anticoagulant (LA) are one of the most frequently
found antibodies in APS and fibrinogen level can be considered as an indicator of
inflammation accompanying AMI. Our objective was to examine the influence of ERα
c.454-397T>C variant on results of LA screening and fibrinogen measurement in
patients with AMI. Study involved 120 patients (54 males and 66 females) with AMI.
Commercially available tests were employed for LA screening and fibrinogen
measurement. ERα genotyping performed using PCR-RFLP analysis. Results were
analyzed using Student's t-test. On average, higher results of LA1 test were encountered
in patients who were homozygote for Erα c. 454-397C allele (46.4±16.07 s vs
39.9±6.41 s; P=0.008). LA2 results in patients with Erα c. 454-397CC genotype
(51.8±13.82 s) were significantly above (P=0.018) values in patients with other
genotypes (39.9±6.43). Higher fibrinogen concentrations were measured in carriers of
Erα c. 454-397C allele (5.4±1.34 g/L vs 6.3±1.90 g/L; P=0.007). Also, homozygosity
for Erα c. 454-397C allele was associated with difference in certain demographic
characteristics i.e. higher body mass (82.7±16.38 kg vs 74.8±14.75 kg; P= 0.030) and
height (175±7.8 cm vs 168±9.4 cm; P=0.002). Gender related differences were not
present for either of the reported parameters. Our pilot results indicate higher values of
LA screening tests and fibrinogen measurement in the presence of the ERα c. 454-397C
allele. Further studies are necessary to comprehensively evaluate the causes and
significance of these associations.Abstract book: Immunology at the confluence of multidisciplinary approaches, Hotel Mona Plaza Belgrade December 6th-8th, 201
Genomic profiling of glycogen storage diseases: from NGS method to the CRISPR/CAS9 technology
No full textGlycogen storage diseases (GSD) are inherited disorders of glycogen synthesis or degradation,
which primarily affect the liver, kidneys and intestinal mucosa. Since the consequences of
these disorders are serious and irreversible, molecular-genetic testing is essential for precise
diagnosis and optimal medical treatment. We analyzed 41 patients with clinical suspicion of
GSD and 75 control subjects from Serbia using Sanger and next-generation sequencing (NGS).
Pathogenicity of novel variants was determined based on expressional, computational analysis
and patient’s phenotype. The genomic profiling of analyzed patients revealed 5 patients with
GSD Ia and 31 patients with GSD Ib. Using the NGS method we identified patients with GSD
III, VI, IXa, cholesteryl ester storage disease and Shwachman-Diamond syndrome. In SLC37A4
gene of GSD Ib patients we detected 4 novel variants: p.Gly83Glu, p.Gly135Asp, p.Pro191Ser
and p.Ser263Glyfs*33. The CRISPR/Cas9 knockin method was used to introduce p.Gly83Glu
variant in SLC37A4 gene of HEK293 cell line in order to establish the new in vitro model
system for the GSD Ib and to functionaly characterize this variant. Computational, expression
analysis and clinical presentation in patients confirmed the pathogenic effect of the novel
variants (p.Gly83Glu and p.Ser263Glyfs*33) in SLC37A4 gene. Comprehensive moleculargenetic
analysis of patients with clinical suspicion of GSD from Serbia achieved 100%
mutation detection rate, allowing early application of proper therapy specific to the patient’s
genotype. The results of this work supported the usefulness of NGS for correct diagnostics
of GSD and differential diagnosis in patients with overlapping phenotypes. Furthermore, we
established a novel human kidney cell model for GSD Ib which lacks SLC37A4 expression
and therefore could be a useful tool to study the pathogenesis of the GSD Ib and to test the
molecular therapeutics designed to correct metabolic abnormalities related to the GSD Ib.Abstracts of the 6th CONGRESS OF THE SERBIAN GENETIC SOCIETY Vrnjačka Banja; October 13 to 17, 2019
Inflammatory and apoptotic genetic markers associate with specific Crohn’s disease phenotypes
No full textObjectives: NIPT continues to expend and offer the most advanced testing option. High
coverage sequencing NIPT extended the availability of clinically relevant information.
This case series examines some complex positive results and and important clinical and
technical points raised.
Methods: Maternal blood samples submitted to Sequenom laboratories for MaterniTGenome
testing, were subjected to DNA extraction, library preparation, and whole genome massively
parallel sequencing as described by Jensen et al.
Sequencing data were analysed using novel algoritam to detect genome wide structural
changes as described by Lefkowits et al.
Results: MaterniTGenome identified loss of chromosome 9 (p24-p23) and gain of chromosome
4 (q32-q35) and suggested unbalanced translocation in anomalous fetus. Amniocentesis
confirmed result.
Amniocentesis showed unspecified change on chromosome 7 without precise definition of
breaking points. MaterniTGenome identified structural change suggestive of a deletion 5.95
Mb in size in the region q36.2-q36.3 of chromosome 7 in anomalous fetus. Amniocentesis
showed mosaic structural change 46,XX/46,XX, del (3) (pter-p14.2).
MaterniTGenome did not identified any change in chromosomal representation. Postanatal
karyotype was normal 46XX.
Conclusions: High coverage sequencing NIPT (MaterniTGenome by Sequenom) delivers
information regarding chromosome conditions beyond other conventional NIPTs. These
results showed 100% of accuracy and may have clinical relevance and may provide further
insight into the etiology of fetal structural chromosomal abnormalities.Abstracts of the 6th CONGRESS OF THE SERBIAN GENETIC SOCIETY Vrnjačka Banja; October 13 to 17, 2019
Dynamic conservation of Picea omorika populations in the republic of Srpska, Bosnia and Herzegovina
No full textSerbian spruce Picea omorika (Panč.) Purk. is a rare, IUCN red-listed European conifer endemic
to the refugial Balkan region. Current rigid conservation (without any intervention allowed)
and designation of seven Genetic Conservation Units (GCUs) included into pan-European
network for dynamic conservation of forest genetic resources (four natural populations
and three planted stands) are not based on genetic data. We carried out a comprehensive
field survey in the Republic of Srpska, Bosnia and Herzegovina (RS-BH), and genotyped 689
individuals from 14 populations with ten highly informative nuclear EST-SSRs. Seed sources
for the re-establishment of populations at sites fully burned over the past 100 years, were
identified and we applied analytical methods for prioritizing populations for conservation
based on their contribution to the geographical structuring of genetic diversity. The levels
of genetic diversity of studied Serbian spruce populations (Ae= 2.524, HO=0.465, HE=0.451)
are slightly lower than those found in eastern populations analysed previously with the
same molecular markers, and effective population size is generally ≥15. Populations are
highly genetically differentiated [Hedrick’s G’ST=0.186 (± 0.044); Jost’s D=0.097 (±0.040)]
and comprise ten distinct gene pools. Although wildfires contribute to admixture of gene
pools within populations, re-establishment from seeds from extirpated populations has likely
prevailed. As much as 14% of alleles are not preserved in the extant network of four GCUs
(natural populations only) which does not include eastern, genetically distinct and more
diverse populations. Seven populations positively contribute to within-population genetic
diversity, four to genetic differentiation, and two are globally important in terms of diversity
and differentiation. The high conservation value of two populations was not evident from
nuclear but from available knowledge on mitochondrial diversity.Abstracts of the 6th CONGRESS OF THE SERBIAN GENETIC SOCIETY Vrnjačka Banja; October 13 to 17, 2019
Bioinformatics prediction of mitochondrial and chloroplast transcripts possibly involved in maize abiotic stress signaling
No full textIn plants, the expression of mitochondrial and chloroplast genomes is continuously changing
to enable adequate functional adjustments related to overall metabolic changes needed for
plant adaptation to fluctuating environmental conditions. In order to identify mitochondrial
and chloroplast transcripts expressed prior to stresses, we have analyzed mitochondrial and
chloroplast transcriptomes of 46 maize inbred lines chosen from three main heterotic groups
(BSSS, Lancaster, Iowa dent) regularly used in elite maize breeding programs. Organelles’
data were extracted from transcriptome data obtained by next generation sequencing of
total leaf transcriptomes of 46 maize inbred lines grown under the optimal conditions in the
greenhouse. All data manipulation and differential gene expression analysis were performed
by using Galaxy software (https://usegalaxy.eu/). Detected organellar complex transcripts
milieus were expected to contain candidates that modulate responses of their genomes
by activating certain signaling pathways upon exposure to abiotic stresses. With the aim
to identify such candidates we focused on selecting transcripts that were differentially
expressed between 46 maize inbred lines grown under the optimal breeding conditions and
also annotated as important for abiotic stresses response in different plant species. Selected
transcripts/genes were then subjected to freely available tools for gene networking analysis
with the aim to depict possible anterograde and retrograde signaling between nucleus and
mitochondria/chloroplasts under the abiotic stresses. Obtained results are promising in terms
of planning biological experiments for testing the roles of genes that were bioinformatically
predicted as novel members of abiotic stress signaling network between nucleus and
organelles in maize.Abstracts of the 6th CONGRESS OF THE SERBIAN GENETIC SOCIETY Vrnjačka Banja; October 13 to 17, 2019
CRISPR/Cas9 mutagenesis as a tool to gain insight Into the roles of highly homologous plant DSS1 genes
No full textDSS1 is a small protein, highly conserved across different species. As a member of
intrinsically disordered protein family, DSS1 interacts with different protein partners,
thus forming complexes involved in diverse biological functions. For instance, human
DSS1 is a mediator in cellular processes such as: DNA repair; regulation of protein
homeostasis; mRNA biogenesis, splicing and export, etc.
Yet, DSS1 involvement in maintenance of genome integrity through homologous
recombination is its only function well studied in Arabidopsis. None of the other
known functions of DSS1 was investigated in plants. Also, the fact that Arabidopsis
thaliana genome contains two highly homologous AtDSS1(I) i AtDSS1(V) genes,
suggests a question of whether or not these gene paralogs share similar functions.
Using CRISPR/Cas9-mediated target mutagenesis, we obtained two stable lines
of Arabidopsis containing mutations in either DSS1(I) or DSS1(V) gene. Here we
present efficient strategies for the selection of targets, design of single-guide RNA,
vector construction and analysis of transgenic lines. High resolution melting method
followed by Sanger sequencing enabled us to select plants with desired mutations
in DSS1 genes. The mutants with 2 nt substitution in AtDSS1(V) and 1nt gene
insertion in AtDSS1(I) gene were chosen for further study. The former mutation led
to altered ORFs and the latter caused premature stop codon. Morphological analysis
of the single dss1 mutant plants reveled differences in rosette shape, stem length
and branching pattern. In addition, the dss1(V)-/dss1(V)- lines showed increased
sensitivity to oxidative stress as compared to the wild type plants.
In conclusion, as a very precise method for mutagenesis CRISPR technology proved to
be a beneficial and supreme method to explore the roles of duplicated genes sharing
highly similar DNA sequences in plants.Abstracts of the 6th CONGRESS OF THE SERBIAN GENETIC SOCIETY Vrnjačka Banja; October 13 to 17, 2019
Metabolic syndrome in inflammatory bowel disease: Association with genetic markers of obesity and inflammation
No full textMetabolic syndrome (MS) and inflammatory bowel disease (IBD) share common
pathophysiological features including chronic inflammation in visceral adipose tissue.
However, the interplay of these two pathologies is still unraveled. This study aimed to
analyze MS, obesity and dyslipidemia, in association with IBD occurrence and disease activity
markers. For the first time, obesity related FTO rs9939609 genetic variant was examined as
potential risk factor of IBD.
The study encompassed 104 IBD patients, of which 50 were diagnosed as Crohn’s disease
(CD) and 54 as ulcerative colitis (UC), as well as 45 non-IBD controls. Participants’ medical
records were examined for body mass index (BMI), waist circumference (WC), total
cholesterol (TCh), low and high density lipoprotein cholesterols (LDL-C, HDL-C), triglycerides,
disease and endoscopic activity scores, C-reactive protein and fecal calprotectin. Expression
levels of IL17A, IL17F, IL23A and TLR9 genes were measured in paired non-inflamed and
inflamed intestinal biopsies of each patient, collected during the colonoscopy. Additionally,
total of 94 CD, 98 UC and 91 control subjects were genotyped for FTO variant rs9939609.
Results showed association of MS with CD, while MS components, central obesity and
low HDL-C, were associated with both CD and UC. IBD lipid profile was characterized with
significantly decreased TCh and HDL-C, while LDL-C was significantly reduced only in CD.
Negative correlations were detected between the level of TCh and Crohn’s disease activity
index. WC negatively correlated with both IL17A and IL17F mRNA levels in inflamed colonic
CD mucosa. We showed that FTO rs9939609 AA genotype carriers had increased risk for CD
development.
This study demonstrates that MS, central obesity and altered lipid profile are significant
factors involved in IBD pathogenesis. Association of FTO rs9939609 variant with CD could
direct further nutrigenomic studies in IBD research.Abstracts of the 6th CONGRESS OF THE SERBIAN GENETIC SOCIETY Vrnjačka Banja; October 13 to 17, 2019
Solid state treatment with Lactobacillus paracasei subsp. paracasei BGHN14 and Lactobacillus rhamnosus BGT10 improves nutrient bioavailability in granular fish feed
Get PDFThe aim of this research was to improve nutritive value of fishmeal-based feed by lactobacilli in order to achieve satisfactory nutrient availability needed to support fish development. Feed was solid-state treated at a laboratory scale with the combination of Lactobacillus paracaseisubsp. paracasei BGHN14 and Lactobacillus rhamnosus BGT10 in different experimental settings, which included the variation of strain ratio, total lactobacilli concentration, percentage of moisture and duration of incubation. Short peptides, soluble proteins, phospho-, neutral and unsaturated lipids were quantified. Differences among treated and control feeds were evaluated by Student t-test, while Gaussian process regression (GPR) modeling was employed to simulate the incubation process and define the optimal treatment combination in the context of overall feed nutritional profile. Treatment duration was shown to be the critical determinant of final outcome, either as single factor or via interaction with strain ratio. Optimal nutrient balance was achieved with 12 h incubation period, 260% moisture, 75:25 and 50:50 BGHN14:BGT10 ratios and 200 mg of lactobacilli per g of dry feed. This study should serve as the basis for large-scale tests which would simulate on-farm production of both fishmeal-based and unconventional, lower cost aquafeed with added value
Serbian spruce and climate change: Possible outcomes and conservation strategy
No full textSerbian spruce is an old, relict and currently rare and endangered spruce species particularly susceptible to climate change that attracts a lot of attention in the academic community since its discovery in 1875. Today, Serbian spruce is limited to the area of ~100 km2 localized around the mid-course of the Drina River in the mountainous central Balkans, at the border of Republic of Serbia and Republic of Srpska, Bosnia and Herzegovina. Within this small area, ~30 remnant populations of various sizes are scattered at north-to-northwest orientated slopes of hills. Given the current state of Serbian spruce populations as well as extreme climate events and poor natural regeneration in this species, applied “do not touch” conservation approach is severely questioned, and some alternative protection and in situ and ex situ conservation actions have been put forward. In situ actions should involve: (1) facilitation of natural regeneration by selective removal of individual competitor trees, (2) assisted natural regeneration by planting high quality seedlings throughout the species current natural range or at nearby suitable sites, and (3) assisted natural regeneration by direct seeding. Although in situ actions may provide short-term persistence of Serbian spruce, ex situ actions, i.e., assisted migration, achieved by both assisted range expansion and assisted species migration, are inevitable, since it is very likely that Serbian spruce will disappear from its natural habitats in the near future due to the rapid climate change