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Reconstitution of non-carrier, heterozygous and homozygous prothrombin belgrade mutation carrier plasma using recombinant proteins
Get PDFIntroduction: The prothrombin Belgrade variant (c.1787G>A, p.Arg596Gln) is a rare mutation found in
Serbia, Japan, China, America, India and leads to antithrombin resistance. Prothrombin Belgrade mutation influencesthrombin-antithrombin interactions and leadsto impaired inactivation of mutated thrombin. Also, it affectssodium binding site in thrombin, which isimportant forswitching from fast thrombin
configuration (coagulant properties) to slow configuration (anticoagulant properties). It has only been
found in a heterozygous state, which could mean that homozygous carriers are incompatible with life.
By using prothrombin (FII) deficient plasma, we could reconstitute plasma of wild type, heterozygous and
homozygous carrier, which could give more insight into the mechanism of this mutation.
Methods: Recombinant wild type and mutated prothrombin were generated by transient transfection
in HEK293T cell line. Western blot analysis was performed to test the efficiency of transfection. Human
Prothrombin ELISA (Nordic BioSite, Sweden) was used in order to measure recombinant prothrombin
concentration. Overall Hemostasis Potential (OHP) assay was performed to assess recombinant protein
activity. Recombinant wild type and mutated prothrombin were added to FII deficient plasma (Siemens,
Germany) in order to create reconstituted plasma, in the final concentration of 0.1 mg/mL, as it is approximately the level of prothrombin in human plasma.
Results: Reconstituted plasma samples that correspond to non-carrier, heterozygous carrier, and homozygous mutation carrier plasma were reconstructed. Recombinant proteinstested by OHP assay were
functional.
Conclusion: Reconstituted plasma samples allow us to examine the mechanism of prothrombin Belgrade mutation in various assays and in homozygous form as well
PB1917: EXPRESSION OF THE LONG NON-CODING RNA MALAT1 IN CHRONIC LYMPHOCYTIC LEUKEMIA
Get PDFBackground: The long non-coding RNA (lncRNA) MALAT1 (metastasis-associated lung adenocarcinoma transcript 1) dysregulated expression and prognostic significance have been reported in a variety of cancers, including hematological malignancies, but have been poorly investigated in chronic lymphocytic leukemia (CLL). Acting through regulation of gene expression at transcriptional and post-transcriptional level, lncRNA MALAT1 is involved in many cellular processes such as proliferation, apoptosis, migration and drug resistance. However, its role as either an oncogene or a tumor-supressor is still controversial, and clearly tumor type-dependent. Aims: To analyze the expression pattern of lncRNA MALAT1 in CLL, and evaluate its prognostic relevance. Methods: This study enrolled 114 unselected CLL patients (pts) and 20 healthy controls (hcs). Clinical and laboratory characteristics of pts were determined at diagnosis, while genetic analyses were performed during the period prior to first treatment. The expression of MALAT1 was analyzed in peripheral blood mononuclear cells by RQ-PCR, using TaqMan chemistry and GAPDH as endogenous control; relative quantification was made by comparative ddCt method, using hcs as calibrator. Results: CLL cohort consisted of 81 males and 33 females (male/female=2.45), with median age at diagnosis of 59 years (range 33-80). Hcs group consisted of 15 males and 5 females (male/female=3), with median age at diagnosis of 71 years (range 65-85). Distribution of Binet stages (112/114 pts) was as follows: A-46.4%, B-39.3%, C-14.3%. Del13q, normal karyotype, trisomy12, del11q and del17p were detected by FISH in 33%, 35%, 9.3%, 10.3% and 12.4% of pts, respectively (97/114 pts). CD38 status (85/114 pts) was negative in 70.6% and positive in 29.4% of pts. Regarding IGHV mutational status (114 pts), 41.2% of pts were mutated, and 58.8% unmutated. Median follow-up was 72 months (range 1-360). LncRNA MALAT1 was overexpressed in CLL pts compared to hcs (p<0.001). Median value of MALAT1 expression was used to divide the cohort into MALAT1low and MALAT1high groups, and association with clinical and biological features at diagnosis was assessed. In both pts and hcs MALAT1 expression was not associated with age but, unlike hcs, MALAT1high status was significantly associated with male sex in CLL (p=0.003). Regarding laboratory parameters, MALAT1 expression showed no correlation with leukocyte, lymphocyte and platelet counts, and serum β2-microglobulin, but exerted a positive correlation with hemoglobin level (r=0.315, p=0.003) and a negative correlation with lactate dehydrogenase (LDH) level (r=-0.303, p=0.004). MALAT1 expression was higher in Binet A and B pts vs. Binet C pts (p=0.037). There was also a trend toward higher MALAT1 expression in pts with favorable (del13q) and intermediate (normal karyotype, trisomy12) cytogenetics in comparison to pts with unfavorable (del11q and del17p) cytogenetics (p=0.059). In addition, MALAT1high status was associated with CD38-negative status (p=0.017), but not with IGHV mutational status. Finally, while the association of MALAT1 expression with the time to first treatment was not detected, longer median overall survival (OS) in MALAT1high vs. MALAT1low group was observed (142 vs. 82 months, log rank p=0.032). Summary/Conclusion: LncRNA MALAT1 is up-regulated in CLL. However, high MALAT1 expression is associated with several favorable prognostic markers (high hemoglobin, low LDH, early clinical stages, negative CD38 status), as well as longer OS. The exact mechanisms of MALAT1 function in CLL pathogenesis and/or progression remain to be determined
Preanalyzed data for the article: Lukić J, Gyalog G, Horváth Z, et al. Evaluation of Post-Larval Diets for Indoor Weaned Largemouth Bass (Micropterus salmoides). Animals. 2023;13(20):3179. doi:10.3390/ani13203179
No full text***Dataset contents*** Preanalyzed data_10.3390_ani13203179.xlsx (287.0Kb)
13.06..cvs (9.610Kb)
21.06..cvs (30.19Kb)
Alkaline phosphatase.cvs (29.37Kb)
Amylase.cvs (15.51Kb)
CCK.cvs (12.53Kb)
Chymotrypsin.cvs (30.49Kb)
Feed analysis.cvs (15.78Kb)
Kn_b.cvs (42.45Kb)
Lipase.cvs (28.56Kb)
PLA2.cvs (25.07Kb)
Primer_efficiency.cvs (11.81Kb)
Proteins_PBS.cvs (10.35Kb)
Proteins_Tris.cvs (10.17Kb)
qPCR_2.cvs (13.53Kb)
qPCR_3.cvs (14.72Kb)
qPCR_4.cvs (14.76Kb)
qPCR_5.cvs (15.67Kb)
qPCR_6.cvs (15.05Kb)
qPCR_7.cvs (15.10Kb)
qPCR_8.cvs (13.74Kb)
qPCR_summary.cvs (21.57Kb)
qPCR1.cvs (12.74Kb)
SGR.cvs (26.01Kb)
T3.cvs (14.12Kb)
T4.cvs (13.90Kb)
Trypsin.cvs (28.98Kb)Preanalyzed data for:[ https://doi.org/10.3390/ani13203179]Related to published version:[https://imagine.imgge.bg.ac.rs/handle/123456789/2107
Genetic analysis and allele-specific expression of SMAD7 3′UTR variants in human colorectal cancer reveal a novel somatic variant exhibiting allelic imbalance
No full textConsidering the impact of SMAD7 deregulation in colorectal cancer (CRC) progression and the
significance of single nucleotide variant (SNV)-mediated disruptions of microRNA (miRNA)-dependent regulation
for cancer susceptibility, our study aimed to analyze genetic variation in the SMAD7 3′ untranslated region (
3′UTR) in CRC, measure differences in allelic mRNA expression, and evaluate its interference with miRNAmediated
post-transcriptional regulation.
Patients and Methods: This study included 80 patients with different CRC stages and six human colon cancer cell
lines of various histological origins. SMAD7 3′ UTR was analyzed by direct sequencing, followed by the relative
quantification of differential allelic expression of detected variants by allele-specific qRT-PCR. In silico tools were
employed for predictions of regulatory consequences of detected variants.
Results: A total of four different SNVs in one cell line and nine patients were found, among which were a novel
somatic point variant and three already known germline variants (rs16950113, rs1050799536, and
rs1043778717). All evaluated SNVs exhibited variable extents of allelic imbalance in expression. In silico analysis
predicted significant effects of SNVs on miRNA binding efficiency, with each SNV disrupting existing and
creating new target sites for one or more miRNAs.
Conclusion: Imbalance observed in the expression of SNV alleles altering miRNA binding suggests that all
investigated SNVs are potential contributing factors impacting SMAD7 expression regulation in CRC that further
studies should investigate
New approach to thrombophilia screening
Get PDFThrombophilia represents multifactorial disease associated with an increased predisposition toward
development of arterial or venous thrombosis. However, in ~50% patients episodes of thrombosis occur
without previously identified risk factors associated with thrombophilia. Project entitled „New approach
to thrombophilia screening“ aims to solve a problem of inavailability of diagnostic assays for detection of
potentially life-threatening thrombotic disorder – the antithrombin resistance, by development of the
antithrombin resistance detection test, in the form of diagnostic kit for rapid screening in clinical
laboratories.Principal Investigator: Dr. Valentina Djordjevic, IMGGE
Duration period: 2023-2024Duration period: 2023-202
Cabbage (Brassica oleracea var. capitata L.) grown under the conditions of the life cycle of winter oilseed rape (Brassica napus L.) in order to achieve a stable seed yield
Get PDFThe expression of genes that induce the transformation of meristems into the reproductive stage in oilseed rape is realized inconditions of low positive temperatures for a certain period of time. Such a flowering process is called the vernalization pathway.A four-factor field trial with 6 genotypes of head cabbage was set up at the Institute of Vegetable Crops in Smederevska Palanka,of which three parental genotypes were divergent by geographical origin: Scc, B and N, and three more F1 hybrids were selectedby diallel crossing: Scc x B, Scc x N and B x N. In order to achieve a different vegetative stage, seedlings were sown at three sowingdates: August 15th, September 1st and September 15th. Transplanting was done on October 20th. The results of sowing headcabbage within the sowing period for oilseed rape were the induction of the flower mechanism, the absence of the head formationphenophase, and the realization of a stable seed yield. The experiment was performed in vivo in the control version and in thetreatment with gibberellic acid – GA3. The influence of all four factors: season, genotype, sowing date and GA3 treatment showedstatistical significance for the yield components as well as for the yield itself and seed quality. The three seasons in which theexperiment was evaluated differed in temperature during overwintering: 2010/2011 was moderately cold, 2011/2012 wasextremely cold, while 2012/2013 was warm. In the cold season, the seed yield was low, and reduced to the biologicalmaintenance of the species, while the highest seed yield was achieved in the third – warm (2012/2013) season in the first sowingperiod. The experiment also confirmed the existence of an identical flower mechanism in the species Brassica napus L. andBrassica oleracea var. capitata L.Експресија гена који индукују трансформацију меристема у репродуктивни стадијум код уљане репице остварује се уусловима ниских позитивних температура у извесном трајању. Такав процес цветања назива се вернализациони пут. УИнституту за повртарство из Смедеревске Паланке постављен је четворофакторски пољски оглед са 6 генотипова купусаглавичара, од којих су три родитељска генотипа дивергентна по географском пореклу: Scc, B и N, а од њих је диалелнимукрштањем селекционисано још три F1 хибрида: Scc x B, Scc x N, и B x N. У циљу постизања различитог вегетативногстадијума расада, сетва је вршена у три рока сетве: 15. август, 1. септембар и 15. септембар. Расађивање је вршено 20.октобра. Резултат сетве купуса главичара у року сетве за уљану репицу била је индукција цветног механизма, изостанакфенофазе формирања главице и реализација стабилног приноса семена. Оглед је постављен in vivo у контролној верзијии у третману гиберелинском киселином – GA3. Утицај сва четири фактора: сезоне, генотипа, рока сетве и третмана GA3показао је статистичку значајност на особине компоненте приноса, као и сам принос и квалитет семена. Три сезоне укојима је оглед евалуиран биле су температурно различите у периоду презимљавања: 2010/2011 је била умеренохладна, 2011/2012 је била изразито хладна, док је 2012/2013 била топла. У температурно хладној сезони принос семенабио је низак, сведен на биолошко одржавање врсте, док је највећи принос семена остварен у трећој – топлој (2012/2013)сезони истраживања у првом року сетве. Оглед је потврдио и постојање идентичног цветног механизма код врстаBrassica napus L. и Brassica oleracea var. capitata L.Кључне речи: купус главичар, уљана репица, вернализациј
GTF2I dosage regulates neuronal differentiation and social behavior in 7q11.23 neurodevelopmental disorders
Get PDFCopy number variations at 7q11.23 cause neurodevelopmental disorders with shared and opposite manifestations.
Deletion causes Williams-Beuren syndrome featuring hypersociability, while duplication causes 7q11.23
microduplication syndrome (7Dup), frequently exhibiting autism spectrum disorder (ASD). Converging evidence
indicates GTF2I as key mediator of the cognitive-behavioral phenotypes, yet its role in cortical development
and behavioral hallmarks remains largely unknown.We integrated proteomic and transcriptomic profiling
of patient-derived cortical organoids, including longitudinally at single-cell resolution, to dissect 7q11.23
dosage–dependent and GTF2I-specific disease mechanisms. We observed dosage-dependent impaired dynamics
of neural progenitor proliferation, transcriptional imbalances, and highly specific alterations in neuronal
output, leading to precocious excitatory neuron production in 7Dup, which was rescued by restoring physiological
GTF2I levels. Transgenic mice with Gtf2i duplication recapitulated progenitor proliferation and neuronal differentiation
defects alongside ASD-like behaviors. Consistently, inhibition of lysine demethylase 1 (LSD1), a
GTF2I effector, was sufficient to rescue ASD-like phenotypes in transgenic mice, establishing GTF2I-LSD1 axis
as a molecular pathway amenable to therapeutic intervention in ASD
Waste to value: From bugs to drugs
Get PDFOur food, cosmetics, clothes, and other products and
consumables across our lives need to get leaner and
greener and biotechnology solutions can contribute
towards responding to this need. Microorganisms are
the most abundant group of organisms on Earth.
While invisible to the naked eye and thus somewhat
intangible, their abundance and diversity underlie
their role in maintaining a healthy global ecosystem.
Microorganisms have key roles in carbon and nutrient
cycling, human, animal and plant health and are also
a source of various products with applications across
all major industries, including pharmaceutical,
chemical, food, environmental, and the agriculture.
Microbial diversity provides a massive pool of
inimitable chemicals, which nowadays become a
treasured source for innovative biotechnology. In this
context, metabolic engineering is a key enabling
technology for transforming microorganisms into
efficient cell factories for these compounds and
materials. Microorganisms can provide economic and
environmental value via bio-upcycling of variety of
waste streams to obtain next generation eco-friendly
therapeutics.
The novel eco-sustainable routes towards valueadded
biologics through biotechnology will be
presented on a set of bacterially derived natural
products (pyocyanin, prodigiosin, actinomycin and
staurosporine) with proven bioactivities (i.e.
anticancer, antifungal, antibiofilm, antiviral). Their
greener production, processing and, formulation
using innovative techniques such as fermentative
bioprocess intensification, structural optimization via
biocatalysis and formulations using metals, as well as
biopolymeric drug carriers will be highlighted. In this
way, both environmental and biomedical problems of
human are once more addressed by microorganisms.10th International Conference of MIKROBIOKOSMOS, Larissa from 30 Novewmber to 2 December 2023
Exploring the enzymatic ability of strains isolated from plastic-polluted environments for enhancing synthetic and natural biopolymers’ biodegradation
Get PDFBackground: Renowned oil-based polymers represent
an environmental nuisance, due to their well-known
permanency in nature, known to disrupt ecosystems.
In response, bio-based polymers have emerged as an
alternative, providing a sustainable blueprint for
plastic circularity1. However, despite being classified
as biodegradable, these polymers can still show
resistance to biodegradation when not presented
with optimal environmental conditions such as
specific temperature and pH, amongst other key
factors in their breakdown. Due to this, microbial
degradation results in an interesting path to promote
biodegradation of such polymers.2
Objective: This study aims to evaluate the ability of
selected strains isolated from polluted environments
to improve the rate of biopolymers’ biodegradation
such as Polyhydroxy butyrate (PHB), Polylactic acid
(PLA), and Bacterial cellulose (BC) when exposed to
strains with specific enzymatic tools to aid in its
degradation.
Methods: Selected strains were previously isolated
from plastic-polluted soil samples. Strains were
transferred to Mineral Salt media (MSM) biopolymersupplemented
plates (7d, 30℃) (MSM (15 g/l Agar, 9
g/l Na2HPO4 x 12H2O, 1.5 g/l KH2PO4, 1 g/l NH4Cl,
0.2 g/l MgSO4 x 7H2O, 0.2 g/l CaCl2 x 2H2O,
Fe(III)NH4-citrate 0.0012 g/l), (1-3%) biopolymers
(PHB, BC, and PLA) growth and clear-zone method
were used to determine strains’ possible enzymatic
activity. Selected strains were transferred to flasks
containing PHB, PLA, and BC films, respectively, in
MSM and incubated for 20 days at 30℃, 120 rpm.
Samples were washed and dried and their level of
degradation was assessed by FTIR, weight loss, and
scanning electron microscopy (SEM). The ability of the
best performer strain to degrade PHB was
additionally assessed using Respirometer (Echo
Instruments) to determine the biodegradability of
said films under a controlled temperature (25°C) with
a flow rate of 500ml/min.
Results: Significant weight loss was observed in
samples exposed to strains, meaning biodegradability
was achieved in an important percentage, proving
their capability to degrade the proposed
biopolymers, compared to samples lacking microbial
presence.10th International Conference of MIKROBIOKOSMOS, Larissa from 30 Novewmber to 2 December 2023
Mitochondrial gene pool variability of the residents of the Republic of Serbia
Get PDFMitohondrijska DNK (mtDNK) se odlikuje nizom osobina koje je čine pogodnom za istraživanja evolutivne
istorije ljudskih populacija koja se zasniva na molekularnim markerima ženske linije nasleđivanja. Tokom
poslednje decenije publikovano je više naučnih radova u kojima je analizirana varijabilnost mtDNK u populaciji
Srbije primenom markera različite rezolucije uključujući i kompletne genome. U skladu sa očekivanjima
zasnovanim na istorijskim, arheološkim i drugim izvorima koji govore u prilog veoma kompleksne
istorije populacija na Balkanskom poluostrvu, mtDNK podaci su potvrdili da se srpska populacija odlikuje
visokim nivoom raznovrsnosti mtDNK koji je posledica izuzetno složene dinamike ove populacije tokom
vremena. Današnji mtDNK profil populacije Srbije ne odstupa od matrilinealnog profila karakterističnog
za druge evropske populacije, a genetičke distance pokazuju da ova populacija zauzima centralnu poziciju
unutar grupe južnoslovenskih populacija koje se odlikuju visokom heterogenošću. Srpska populacija
deli najveći procenat mtDNK haplotipova sa geografski bliskim populacijama Balkanskog poluostrva koje
pripadaju južnoslovenskoj grupi, gde su uočeni i potencijalno privatni haplotipovi. Na osnovu filogenetske
i filogeografske analize kompletnih mitogenoma u srpskoj populaciji detektovane su retke mtDNK linije,
karakteristične za druge regione, poput Bliskog istoka (N1b, HV2), istočne Azije (D4) i Afrike (L2a1), kao i
one koje su potencijalno specifične za Balkansko poluostrvo, poput K1a13a1, U4c1b1 i H6a2b. Pored toga,
srpska populacija deli određeni broj mtDNK podhaplogrupa sa istočno- i zapadnoslovenskim populacijama
kao i sa germanskim populacijama severne i srednje Evrope. Istraživanja varijabilnosti mtDNK su pokazala
da se izuzetno velika raznovrsnost mtDNK savremene populacije Srbije može objasniti genetičkim doprinosom
kako slovenskih i germanskih, tako i pre-slovenskih populacija koje su naseljavale Balkansko poluostrvo
pre Velike seobe naroda.The mitochondrial DNA (mtDNA) is characterized by a number of features that make it suitable for studying
the evolutionary history of human populations based on molecular markers with the female-specific
line of inheritance. During the last decade, several scientific papers were published in which the mtDNA
variability in the population of Serbia was analyzed using markers of different resolution including complete
mitogenomes. In accordance with expectations based on historical, archaeological and other sources
that speak in favor of a very complex history of populations on the Balkan Peninsula, mtDNA data confirmed
that Serbian population is characterized by a high level of mtDNA diversity, which is a consequence
of the exceptionally complex dynamics of this population over time. Today’s mtDNA profile of the Serbian
population does not differ from the matrilineal landscape characteristic of other European populations,
and according to genetic distances, this population occupies a central position within the group of South-
Slavic populations characterized by high heterogeneity. The Serbian population shares the highest percentage
of mtDNA haplotypes with the geographically close populations of the Balkan Peninsula
belonging to the South-Slavic group, where potentially private haplotypes were also observed. Phylogenetic
and phylogeographic analysis of complete mitogenomes in the Serbian population revealed rare
mtDNA lineages, characteristic of other regions, such as the Middle East (N1b, HV2), East Asia (D4) and
Africa (L2a1), as well as those that are potentially specific for Balkan Peninsula, like K1a13a1, U4c1b1 and
H6a2b. In addition, Serbian population shares a certain number of mtDNA subhaplogroups with East- and
West-Slavic populations as well as with the Germanic populations of Northern and Central Europe. Studies
of mtDNA variability have shown that the exceptionally high mtDNA diversity in contemporary Serbian
population may be associated with the genetic contribution of both Slavic and Germanic, as well as pre-
Slavic populations that inhabited the Balkan Peninsula before the Great Migration