imagine (Institute of molecular genetics and genetic engineering)
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Exploring Bacterial OMV (Outer Membrane Vesicles)-sRNAs Mediated Interkingdom Communication with Plants and Fungi (ExplOMV)
Get PDFThe role of extracellular small RNA (sRNA) in interkingdom communication between
microorganisms and their eukaryotic hosts has been revealed recently. Extracellular vesicles
(EVs) are a significant delivery route of sRNAs, thus regulating gene expression in the recipient
cell. Such interactions have been shown between fungi and plants as well as bacteria and
animals but, little is known about sRNA-mediated host responses between bacteria and plants
or fungi.
This project aims to study the role of sRNAs in the outer membrane vesicles (OMVs) of the
plant growth promoting bacteria (PGPB) (of genera Paraburkholderia, Pseudomonas and
Enterobacter) in the naturally occurring communication with plant (Arabidopsis thaliana) and
phytopathogenic fungi (Botrytis cinerea). Further, we will explore the ability of engineered
OMV-sRNAs to manipulate fungal genes, as a means to suppress fungal diseases of plants.
To monitor the delivery of the OMV cargo into plant and fungi, OMVs and sRNAs will be
fluorescently labeled and OMVs’ interaction with recipient cells, and RNA delivery, will be
monitored by confocal microscopy. sRNA from OMVs and cells of bacteria, grown separately or
in the interaction with the plant or fungus, will be sequenced and compared to define the
criteria for the selection of sRNAs to be packed into OMVs. To identify sRNAs delivered by
OMVs to recipient cells, sRNA-Seq and bioinformatics analysis of plant and fungal cells upon
exposure to bacteria will be performed. The ability of OMV-sRNAs to modulate the expression
of predicted target genes in the host cells will be tested both for endogenous bacterial and
engineered sRNAs, designed to target recipient’s gene(s).
The main expected results: the visualization of OMV cargo delivery to recipient cells; revealing
the selection criteria for sRNA packaging into OMVs; and evidence of OMV-sRNAs mediated
regulation of host genes will be novel, ground-breaking discoveries, impacting many research
fields , from agriculture to medicine. The project will pave the way for using engineered OMVs-
sRNAs to modulate plant/fungal physiology, encouraging further studies of the application of
this process in biocontrol against phytopathogenic fungi, with a potential to its extrapolation to
animal and human fungal pathogens.Principal Investigator: Dr Dragana NikolićDuration period: 2021-202
Zebrafish ankrd1a as a common player in heart regeneration and skeletal muscle repair - a new prospect for unlocking regenerative potential of human heart (ZEBARR)
Get PDFZEBARR project aims to contribute to better understanding of cellular and molecular
mechanisms of healing injured cardiac and skeletal muscles by investigating a common player
that participates in both processes. As a model organism competent in both regeneration of the
heart and repair of skeletal muscle after injury we will use adult zebrafish. Injured zebrafish
heart gets fully regenerated in 60 days by proliferation of unaffected cardiomyocytes, while
skeletal muscle replace injured tissue by activation of dedicated pool of stem cells called
satellite-like cells, that pass thought transformation to form myofibers. Do these processes
have common players and if they do in which particular molecular event(s) they participate?
Are there any points of intersection? We hypothesized that these players and associated
pathways do exist and might help to unlock endogenous regenerative mechanism of human
heart. In our preliminary experiments, ankrd1a gene, homologue of mammalian pleiotropic
transcriptional regulator ANKRD1, stood out as a common player in these processes, since it
was found activated in both muscle tissues after the injury (cryoinjury of ventricle wall and stab
wound of skeletal muscle). Deciphering precise role of ankrd1a in different pathways involved
in regeneration of zebrafish heart and repair of skeletal muscle and its contribution to the
regenerative potential of different zebrafish muscles will help us to understand these very
important processes in depth. Moreover, ankrd1a might become strong candidate for
consideration when designing novel strategies for healing human heart after myocardial
infraction.
Experimental work will be organized in five work packages:
WP1. Determination of spatio-temporal profile of ankrd1a expression after injury of adult
zebrafish heart and skeletal muscle.
WP2. Investigation of ankrd1a function in regeneration of adult zebrafish heart.
WP3. Investigation of ankrd1a function in repair of adult zebrafish skeletal muscle.
WP4. Identification of ankrd1a downstream targets and associated pathways involved in
regeneration of heart and repair of skeletal muscle of adult zebrafish
WP5. Testing the mediator role of ankrd1a in the beneficial effect of exercise on regeneration
of heart and repair of skeletal muscle.Principal Investigator: Dr Snezana Kojic, IMGGEDuration period: 2021-202
Bis-Bibenzyls from the Liverwort Pellia endiviifolia and Their Biological Activity
Get PDFBased on previous investigations where bis-bibenzyls isolated from liverworts showed various biological activities (cytotoxic, antimicrobial, and antiviral), we investigated their cytotoxic activity in several human cancer cell lines. From the methylene-chloride/methanol extract of the liverwort Pellia endiviifolia, three bis-bibenzyls of the perrottetin type were isolated, namely perrottetin E, 10 '-hydroxyperrottetin E, and 10,10 '-dihydroxyperrottetin E. The last two were found for the first time in this species. Their structures were resolved using 1D and 2D NMR, as well as by comparison with data in the literature. Cytotoxic activity of the isolated compounds was tested on three human leukemia cell lines, HL-60 (acute promyelocytic leukemia cells), U-937 (acute monocytic leukemia cells), and K-562 (human chronic myelogenous leukemia cells), as well as on human embryonal teratocarcinoma cell line (NT2/D1) and human glioblastoma cell lines A-172 and U-251, and compared to the previously isolated bis-bibenzyls (perrottetins) of similar structure. The isolated compounds exhibited modest activity against leukemia cells and significant activity against NT2/D1 and A-172. Overall, the most active cytotoxic compounds in this investigation were perrottetin E (1), isolated in this work from Pellia endiviifolia, and perrottetin F phenanthrene derivative (7), previously isolated from Lunularia cruciata and added for a comparison of their cytotoxic activity
Reduced Expression of Autophagy Markers and Expansion of Myeloid-Derived Suppressor Cells Correlate With Poor T Cell Response in Severe COVID-19 Patients
Get PDFWidespread coronavirus disease (COVID)-19 is causing pneumonia, respiratory and multiorgan failure in susceptible individuals. Dysregulated immune response marks severe COVID-19, but the immunological mechanisms driving COVID-19 pathogenesis are still largely unknown, which is hampering the development of efficient treatments. Here we analyzed similar to 140 parameters of cellular and humoral immune response in peripheral blood of 41 COVID-19 patients and 16 age/gender-matched healthy donors by flow-cytometry, quantitative PCR, western blot and ELISA, followed by integrated correlation analyses with similar to 30 common clinical and laboratory parameters. We found that lymphocytopenia in severe COVID-19 patients (n=20) strongly affects T, NK and NKT cells, but not B cells and antibody production. Unlike increased activation of ICOS-1+ CD4+ T cells in mild COVID-19 patients (n=21), T cells in severe patients showed impaired activation, low IFN-gamma production and high functional exhaustion, which correlated with significantly down-regulated HLA-DR expression in monocytes, dendritic cells and B cells. The latter phenomenon was followed by lower interferon responsive factor (IRF)-8 and autophagy-related genes expressions, and the expansion of myeloid derived suppressor cells (MDSC). Intriguingly, PD-L1-, ILT-3-, and IDO-1-expressing monocytic MDSC were the dominant producers of IL-6 and IL-10, which correlated with the increased inflammation and accumulation of regulatory B and T cell subsets in severe COVID-19 patients. Overall, down-regulated IRF-8 and autophagy-related genes expression, and the expansion of MDSC subsets could play critical roles in dysregulating T cell response in COVID-19, which could have large implications in diagnostics and design of novel therapeutics for this disease
The FKBP5 genotype and childhood trauma effects on FKBP5 DNA methylation in patients with psychosis, their unaffected siblings, and healthy controls
No full textHypothalamic-pituitary-adrenal (HPA) axis activity mediates the relationship between childhood trauma (CT) and psychosis. The FKBP5 gene, one of the key regulators of HPA axis activity after stress exposure, has been found associated with psychosis. Allele-specific and CT related FKBP5 demethylation in intron 7 was revealed in different psychiatric disorders. However, no studies have investigated FKBP5 methylation in subjects with different genetic liability for psychosis. A total of 144 participants were included in the study: 48 patients with psychotic disorders, 50 unaffected siblings, and 46 healthy controls. CT was assessed by Childhood Trauma Questionnaire. The FKBP5 rs1360780 was genotyped and FKBP5 methylation analyses were performed using bisulfite conversion followed by Sanger sequencing at three CpG sites in intron 7. Mixed linear model was used to assess group differences depending on rs1360780 T allele and CT. Results showed a significant T allele-dependent decrease of FKBP5 methylation in patients compared to unaffected siblings and controls. Effect of interaction between T allele and CT exposure on FKBP5 demethylation was found in controls. No effect of both risk factors (T allele and CT) on FKBP5 methylation level was found in unaffected siblings. We confirmed previous evidence of the association between the FKBP5 rs1360780 T allele, CT, and decreased FKBP5 methylation in intron 7. Allele-specific FKBP5 demethylation found in patients could shed a light on altered HPA axis activity in a subgroup of patients related to stress-induced psychosis. FKBP5 methylation and potential protective mechanisms in unaffected siblings after trauma exposure require further investigation
Genetic analysis of SMAD4 C-terminal domain in patients with microsatellite stable early- age onset colorectal cancer
Get PDFSMAD4 protein loss is a relatively common feature of sporadic
colorectal cancers (CRC), and it was observed to be even more frequent in early-age onset CRC
patients and microsatellite stable (MSS) tumors. Pathogenic variants in the SMAD4 gene are
usually missense or nonsense mutations, and they are more frequent in the C-terminal domain. The
aim of this study was to perform genetic analysis of SMAD4 C-terminal domain of MSS early-age
onset CRC patients. This pilot study was conducted with a purpose of investigating if such genetic
screening strategy would be useful for diagnostic purposes in this specific subgroup of CRC
patients.On June 24 and 25, 2021, Fight Colorectal Cancer (Fight CRC) and Dr. José Perea from Fundación Jiménez Díaz University Hospital in Madrid, Spain, virtually co-hosted the third annual early-age onset colorectal cancer (EAO CRC) international symposium, Rally on Research: EAO CR
Characterization of the late embryogenesis abundant (LEA) proteins family in hydrated and desiccated Ramonda serbica Panc. leaves
Get PDFEndemic plant species, Ramonda serbica is a resurrection plant that can tolerate extreme
dehydration (desiccation, loss of 95% of cellular water) even over months. The
accumulation of late embryogenesis abundant proteins (LEAPs) is a crucial step in the
mechanism of desiccation tolerance. The role of LEAPs is not completely resolved, but
they are accepted as intrinsically disordered proteins (IDPs). Based on previously
established de novo transcriptome database of R. serbica leaves we identify around 160
members of LEA gene family. Identified LEAPs were classified into six groups: LEA 1-5
and seed maturation proteins (SMPs) according to protein family (Pfam) database. Based
on multiple sequence alignment, secondary structure prediction and 3D structure modeling,
we conducted LEA protein structure analysis. We showed that more than 50% of identified
LEAPs exhibited a high propensity to form α-helices. As predicted by several
bioinformatic tools, more than 70% of identified LEAPs were found to be highly
disordered. Thus, these proteins are predicted to be disordered in solution, but they acquire
a secondary, predominantly α-helical structure during drying, in contrast to globular
proteins, which most often causes the loss of structure upon dehydration. By using
molecular dynamic simulations, we identified the most favorable conformations of
representative LEAPs and we have studied conformational transitions driven by the water
scarcity. Structural characterization of LEAPs is a key to understand their function and
regulation of their intrinsic structural disorder-to-order transition during desiccation as a
requirement for biological function, in order to promote development of new therapeutic
strategies in neurodegenerative disorders, cell preservation technology and the
improvement of crop drought tolerance.Serbian Biochemical Society, 10th Conference, Kragujevac, Serbia, 24.09.202
Application of crISPr/cas9 technology in the discovery of new molecular therapeutics
Get PDFCRISPR/Cas9 (eng. Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein 9) je prirodni
alat za editovanje genoma usvojen iz prokariotskog adaptivnog imunskog odbrambenog sistema.
Zbog svoje visoke efikasnosti i preciznosti, protein Cas9 koji pripada CRISPR sistemu klase II, pronašao je
primenu u mnogim poljima nauke. Editovanje genoma zasnovano na tehnologiji CRISPR/Cas9 predstavlja
jedan od najperspektivnijih alata za lečenje humanih bolesti sa genetičkom osnovom, uključujući kardiovaskularne
bolesti, neurodegenerativne poremećaje i različite vrste tumora. Genska terapija zasnovana na
CRISPR/Cas9 opsežno se proučava u pretkliničkim i kliničkim studijama. Editovanje genoma CRISPR/Cas9 je
takođe robustan alat za stvaranje in vitro ćelijskih i životinjskih model sistema za istraživanje i lečenje genetičkih
bolesti, posebno bolesti povezanih sa tačkastim promenama. U ovom radu, osvrnućemo se kratko na
istoriju i mehanizam sistema CRISPR/Cas9, različite metodološke pristupe, napraviti pregled primena u biomedicini
i opisati njegovu ulogu u razvoju novih molekularnih terapeutika za retke nasledne bolesti.Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein 9 (CRISPR/Cas9) is a
naturally occurring genome editing tool adopted from the prokaryotic adaptive immune defense system.
Due to its high efficiency and precision, the Cas9 protein derived from the type II CRISPR system, has found
applications in many fields of science. Currently, CRISPR/Cas9-based genome editing has become one of
the most promising tools for treating human genetic diseases, including cardiovascular diseases, neurodegenerative
disorders, and various types of tumors. CRISPR/Cas9-based gene therapy is extensively studied
in preclinic and clinic treatments. CRISPR/Cas9 genome editing is also a robust tool to create in vitro cellular
and animal model systems for investigating and treating human genetic disorders, particularly diseases
associated with point mutations. Therefore, in this review, we will present a brief history and mechanism of
the CRISPR/Cas9 system. We will also describe the different methodological approaches, review applications
in biomedicine and describe its role in the development of new molecular therapeutics for rare inherited
diseases
The molecular basis of monogenic diabetes
Get PDFMonogenski dijabetes je heterogen oblik dijabetesa u čiji nastanak je uključen veliki broj gena. Promene u
genima uzročnicima monogenskog dijabetesa narušavaju funkciju β-ćelije pankreasa dovodeći do smanjene
ili oštećene sekrecije insulina i, posledično, hiperglikemije kod pacijenata. Produkte ovih gena uglavnom
čine transkripcioni faktori, zatim membranski kanali i proteini sa specifičnom funkcijom u ćeliji. Mnogi
transkripcioni faktori monogenskog dijabetesa imaju plejotropno dejstvo, te se kod nosioca promena u
ovim genima mogu uočiti ne samo poremećaji u funkcionisanju β-ćelija, već i defekti u drugim organima ili
multisistemski poremećaji. Dva glavna oblika monogenskog dijabetesa su neonatalni dijabetes, koji se javlja
u prvih 6 meseci života, i mnogo češći, MODY dijabetes koji nastaje u mlađem odraslom dobu. Najčešći geni
uzročnici neonatalnog dijabetesa su geni KCNJ11, ABCC8 i INS kao i lokus 6q24, dok se u slučaju MODY
dijabetesa izdvajaju dva dominantna gena HNF1A i GCK. Iako je monogenski dijabetes veoma redak, njegovo
prepoznavanje među rasprostranjenijim oblicima dijabetesa je od izuzetnog značaja, s obzirom na to da su
terapija, klinička prezentacija, subklasifikacija i prognoza toka bolesti specifični prema genu uzročniku.
Savremene tehnologije sekvenciranja (NGS) su pronašle svoje mesto u dijagnozi monogenskog dijabetesa,
budući da je ova metoda nezamenjiva kada je u pitanju analiza velikog broja gena i heterogen fenotip koji
se sreće kod ovog tipa dijabetesa. Rastuća saznanja o genima uzročnicima monogenskog dijabetesa su
izdvojila ovaj oblik dijabetesa kao dobrog kandidata za implementaciju relativno novog koncepta, precizne
medicine dijabetesa, čiji bi krajnji cilj bio postizanje boljeg kvaliteta života pacijenata.Monogenic diabetes is a heterogeneous form of diabetes resulting from defects in a single gene. Defects in
monogenic diabetes-related genes disrupt the β-cells function, leading to reduced or impaired insulin secretion
and, consequently, hyperglycemia in patients. Products of these genes are mainly transcription factors,
followed by membrane channels and proteins with a specific cell function. Most transcription factors
involved in monogenic diabetes have a pleiotropic effect expanding the β-cells disfunction with defects in
other organs or multisystem disorders. Two main forms of monogenic diabetes are neonatal diabetes, appearing
in the first 6 months of life, and the more common, MODY diabetes, that occurs in young adulthood.
The most common genes involved in neonatal diabetes are KCNJ11, ABCC8 and INS, followed by the
6q24 lokus, while in the case of MODY diabetes the two dominant genes HNF1A and GCK stand out. Although
monogenic diabetes is very rare, its recognition among the more common forms of diabetes is of
great importance, since therapy, clinical subclassification and presentation, as well as disease prognosis are
gene-specific. Modern sequencing technologies (NGS) have found their place in the diagnosis of monogenic
diabetes, as this method is irreplaceable when it comes to the analysis of a large number of genes
and the heterogeneous phenotype encountered in these patients. Growing knowledge about monogenic
diabetes-related genes has singled out this form of diabetes as a good candidate for the implementation of
a relatively new concept, the precision diabetes medicine, whose ultimate goal would be achieving a better
quality of life for the patients
Differential diagnosis of eosinophilic infiltrate in esophageal mucosa by applying molecular biology method
Get PDFĆelije imunskog sistema imaju važnu ulogu u očuvanju barijere gastrointestinalnog trakta. Savremeni način
života, izloženost različitim alergenima i genetska predispozicija mogu da dovedu do narušavanja odnosa i
broja ćelija imunskog sistema, čime se remeti homeostaza na nivou crevne sluznice i omogućava razvoj bolesti.
Povećanje broja eozinofilnih leukocita u sluznici i narušavanje barijere gastrointestinalnog trakta karakteristika
je različitih primarnih i sekundarnih eozinofilnih gastrointestinalnih bolesti (EGIB). Usled čestog preklapanja
u histološkom nalazu klinički različitih entiteta (kao što su eozinofilni ezofagitis, gastroezofagusna
refluksna bolest i eozinofilni gastroentritis) postoji konstantna potreba za otkrivanjem novih markera diferencijacije,
kao i veze između kliničkih karakteristika i patofizioloških mehanizama bolesti.
Identifikacija EGIB transkriptoma i razlučivanje imunopatogeneze različitih EGIB oboljenja omogućava identifikaciju
novih molekularnih markera. Molekularno profilisanje gena specifičnih za određeno oboljenje
poboljšava dijagnozu i kliničko praćenje, omogućava brz odabir adekvatnog terapijskog pristupa, kao i personalizovani
medicinski pristup obolelima. Iako postoji značajan broj studija koje se bave validacijom novih
lekova kod EGIB obolelih, većina njih je još uvek u vidu preliminarnih istraživanja, te je neophodna dalja verifikacija
efekata ispitivanih lekova u kliničkoj praksi.The cells of the immune system play an important role in preserving the barrier of the gastrointestinal tract.
Modern lifestyle, exposure to various allergens and genetic predisposition can lead to a disruption of the relationship
and the number of cells of the immune system, which disturb homeostasis at the level of the intestinal
mucosa and enables the development of the disease.
An increase in the number of eosinophilic leukocytes in the mucosa and disruption of the gastrointestinal
tract barrier is a characteristic of various primary and secondary eosinophilic gastrointestinal diseases (EGIB).
Due to the frequent overlap in the histological findings of clinically different entities (such as eosinophilic
esophagitis, gastroesophageal reflux disease and eosinophilic gastroentritis), there is a constant need to
discover new markers of differentiation, as well as the relationship between clinical characteristics and pathophysiological
mechanisms of the disease.
Identification of EGIB transcriptoma and differentiation of immunopathogenesis of different EGIB diseases
enables identification of new molecular markers. Molecular profiling of disease-specific genes improves diagnosis
and clinical monitoring, enables rapid selection of an adequate therapeutic approach, as well as a personalized
medical approach to patients. Although there are a significant number of studies regarding the
validation of new drugs in EGIB patients, most of them are still in the form of preliminary research, and further
verification of the effects of the tested drugs in clinical practice is necessary