imagine (Institute of molecular genetics and genetic engineering)
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Spatial profile of ankrd1a activation during regeneration of zebrafish heart
Get PDFIntroduction: In contrast to humans, zebrafish have a remarkable ability to regenerate injured heart
through a complex and highly orchestrated processinvolving all cardiac structures. The majorsource of
new myocardial cells are resident cardiomyocytes, which dedifferentiate and reinitiate proliferation, invading the area of injury to replace the lost myocardium. The response of the myocardium and coronary
vasculature is preceded by activation of epi- and endocardium, which form active scaffolds to guide regeneration. The aim of thisstudy wasto identify cardiac structuresin which ankrd1a gene is activated during zebrafish heart regeneration.
Methods: We crossed several zebrafish reporter lines: TgBAC(ankrd1a:EGFP) (to identify cells expressing
ankrd1a), Tg(myl7:nls-dsRedExpress) (for labeling cardiomyocyte nuclei) and Tg(kdrl:RAS-mCherry) (for labeling endocardial/endothelial cells). Zebrafish hearts were cryoinjured and left to regenerate for 3 and
7 days. Dedifferentiating cardiomyocytes and epicardial cells were immunostained with anti-MYH7 and
anti-caveolin1 antibody, respectively. Cells labeled with transgenes and immunostaining were visualized on tissue cryosections by fluorescent microscopy.
Results: Zebrafish ankrd1a was activated in the injury border zone cardiomyocytes, located between
the injured and remote myocardium. Its expression preceded that of a dedifferentiation marker, MYH7.
The TgBAC(ankrd1a:EGFP) transgene was not detected in epicardial or endocardial cells of regenerating
zebrafish heart.
Conclusion: Activation of ankrd1a during regeneration of zebrafish heart is restricted to borderzone
cardiomyocytes, implicating this gene in dedifferentiation and proliferation of cardiomyocytes. The absence of ankrd1a expression in epicardium and endocardium indicatesthat this gene does not contribute
to the regeneration process occuring in these layers of the heart
Investigation of the role of the glucose-6-phosphate translocase in the activation of autophagy and glycogen-selective autophagy in glycogen storage disease type IB patients
Get PDFIntroduction: Glycogen storage disease type Ib (GSD-Ib) is characterized by a deficiency of glucose-6-
phosphate translocase (G6PT) encoded by the SLC37A4 gene, affecting glucose homeostasis and disrupting autophagy. Recent findings suggest that G6PT may also play a role in autophagy and
glycogen-selective autophagy (glycophagy) activation independent of its transport function. To investigate this hypothesis, two groups of GSD-Ib patients carrying variants with different effects on G6PT
transport activity and stability (p.Asn27Lys and p.Leu348Valfs*53), were compared to the control group
of subjects.
Methods: The relative expression levels of SLC37A4 gene, autophagy (mTOR, ULK1, PRKAG1), and glycophagy markers(GABARAPL1, GAA, STBD1) were assessed in mononuclear cells of GSD Ib patients(four
carrying p.Asn27Lys and four carrying p.Leu348Valfs*53 variant) compared to control group using RTqPCR. Statistical analysis was performed using one-way ANOVA followed by a post-hoc t-test.
Results: The p.Asn27Lys group exhibited 1.5-2.5 times higher expression of SLC37A4 and autophagy
markers, while the p.Leu348Valfs*53 group showed downregulation by approximately 50% compared to
the control group. Glycophagy markers were increased twofold in both patient groups, except for GAA,
which had similar expression levels as the control group.
Conclusion: Individuals carrying the p.Asn27Lys variant display the presence of SLC37A4 transcript in
their cells, which correlates with autophagy activation. Conversely, in patients with the p.Leu348Valfs*53
variant SLC37A4 is downregulated, indicating compromised autophagy activation. These findings support the role of G6PT in autophagy activation, independent of itstransport activity. Furthermore, the elevated expression of glycophagy markers observed in both patient groups can be attributed to the
accumulated glycogen
Characterization of outer membrane vesicles of plant growth promoting bacteria Paraburkholderia phytofirmans PsJN
Get PDFPlant growth promoting bacteria (PGPB) are microorganisms present in plant
rhizosphere that stimulate plant growth, as well as prime plants against abiotic
and biotic stress. PGPB are also located in plant intercellular space and can directly
interfere with plant pathogens. Many different mechanisms of interaction
between plants and PGPB has been shown, but the interaction through outer
membrane vesicles (OMVs) has only been proposed. OMVs are membrane-enclosed
nanostructures produced by Gram-negative bacteria. Size of OMVs can
range from 20 to 300 nm, depending on the strain. In this research, we determined
optimal method for isolation of OMVs produced by Paraburkholderia
phytofirmans PsJN, characterized the size range of OMVs, their concentration
and origin. OMVs from PGPB PsJN have been isolated us\ing two methodological
approaches: 1) differential centrifugation, ultrafiltration, and purification
using Optiprep density gradient, and 2) OMV isolation by the commercial
ExoBacteria OMV Isolation Kit that uses an affinity-based column system. Size
and concentration of isolated OMVs were analyzed using Nanoparticle Tracking
Analysis (NTA). As the OMVs are derived from outer membranes of bacteria, their
origin has been proven using an essay for detection of lipopolysaccharides. In
addition, protein profiles of OMV from PsJN were visualized by SDS-PAGE. Comparing
different methods for isolation and purification of vesicles from PsJN we
concluded that for our PGPB strain the commercial ExoBacteria OMV Isolation
Kit provides the highest yield. Quantification of OMV using NTA of OMVs produced
by P. phytofirmans PsJN, isolated with the commercial kit, showed a mean
particle numeration of 4.05*109 particles/ml. Size of OMVs isolated from PsJN
ranged from 50 to 200 nm. Detection of lipopolysaccharides confirmed that
isolated vesicles originated from outer membranes of PsJN. These findings are
the important first step in our research of the role of OMVs in PGPB interaction
with plants.Book of abstract: ICGEB WORKSHOP; Trends in microbial solutions for sustainable agriculture, 13 – 15 September 2023. Belgrade, Serbi
PHENYLBUTYRIC ACID REDUCES MOLECULAR MARKERS OF ER STRESS-INDUCED APOPTOSIS IN GLYCOGEN STORAGE DISEASE TYPE IB IN VITRO MODEL SYSTEM
Get PDFBackground: The current therapy for glycogen
storage disease Ib (GSD Ib) fails to prevent the
development of renal dysfunction, and hepatocellular/
renal carcinoma in many patients. Therefore,
new therapies for the treatment of life-threatening
complications of GSD Ib are of great interest. Recent
studies revealed that chronic endoplasmic reticulum
(ER) stress and increased apoptosis are
involved in pathogenesis of GSD Ib, whereas small
molecule phenylbutyrate (4-PBA) showed the capability
of reducing ER stress-induced apoptosis.
Methods: To analyze the function of 4-PBA
as ER stress inhibitor, we created a G6PT-deficient
FlpInHEK293 cell line using the CRISPR/Cas9
knockout method and tested if 4-PBA could decrease
chronic metabolic stress and prevent cell
death. We analyzed molecular markers of unfolded
protein response (ATF4, DDIT3, HSPA5, XBP1s),
and apoptosis (BCL2/BAX, CASP3, CASP7) in
G6PT-deficient cells before and upon the treatment
using RT-qPCR method.Results: Treatment with the most effective
dose of 1 mM 4-PBA reduced the expression of
executioner caspases (CASP3, CASP7) and increased
the BCL2/BAX ratio, indicating a reduced
apoptosis level. Additionally, 4-PBA decreased
UPR marker expression in G6PT-deficient cells.
Our results proved the concept that 4-PBA could
alleviate markers of ER stress detected in the GSD
Ib in vitro model system and prevent cell death.
Conclusion: We demonstrated, for the first
time, the potential of 4-PBA to be repurposed for
patients with GSD Ib and open perspectives for
translational research that could contribute to a
knowledge of GSD Ib treatments and other genetic
diseases where chronic ER stress-induced apoptosis
contribute to the disease pathology.ABSTRACT BOOK: “Genetic Diseases from Diagnostics to Prevention and Therapy” October 05-14th Balkan Congress of Human Genetics & 9th Rare Disease SEE Meeting 2023; Skopje, October 05-07, 202
Red-Light Transmittance Changes in Variegated Pelargonium zonale—Diurnal Variation in Chloroplast Movement and Photosystem II Efficiency
Get PDFChloroplast movement rapidly ameliorates the effects of suboptimal light intensity by accumulating along the periclinal cell walls, as well as the effects of excess light by shifting to the anticlinal cell walls. These acclimation responses are triggered by phototropins located at the plasma membrane and chloroplast envelope. Here, we used a recently developed non-invasive system sensitive to very small changes in red light leaf transmittance to perform long-term continuous measurements of dark–light transitions. As a model system, we used variegated Pelargonium zonale leaves containing green sectors (GS) with fully developed chloroplasts and achlorophyllous, white sectors (WS) with undifferentiated plastids, and higher phototropin expression levels. We observed biphasic changes in the red-light transmittance and oscillations triggered by medium intensities of white light, described by a transient peak preceded by a constant decrease in transmittance level. A slight change in red-light transmittance was recorded even in WS. Furthermore, the chloroplast position at lower light intensities affected the rapid light curves, while high light intensity decreased saturated electron transport, maximum quantum efficiency of photosystem II, and increased non-photochemical quenching of chlorophyll fluorescence and epidermal flavonoids. Our results extend the knowledge of light-dependent chloroplast movements and thus contribute to a better understanding of their role in regulating photosynthesis under fluctuating light conditions
Alginate microfibers with immobilized cells as a 3D in vitro model for anticancer drug testing: demonstration in osteosarcoma cell cultures
No full textIn anticancer drug development closing the gap between 2D in vitro and in vivo studies on animals
and further clinical studies is one of the urging aims considering low translation of the results obtained
in these preclinical systems to humans. This is why the development of 3D in vitro systems that would
closely mimic in vivo response to anticancer drugs is of the utmost importance. Here we propose a
simple 3D in vitro culture model based on alginate hydrogel microfibers with immobilized cancer cells.
The microfibers were obtained by manual extrusion of a suspension of murine osteosarcoma cells
(K7M2 wt, 4×106
cells/ml) in a 2 %wt Na-alginate solution through a blunt edge stainless steel needle
(25G) into a gelling solution containing Ca2+ (0.18 M CaCl2×2H2O). After 15 min of gelation the formed
microfibers (diameter ~500 µm) were transferred to T-25 flasks (1.5 g of microfibers in 15 ml culture
medium) and cultured for 7 days. Histological analysis showed that alginate microfibers supported cell
viability and formation of cellular aggregates. In the second series, microfibers were cultured in a 96-
well plate (3 cm of microfibers in 100 µl culture medium per well). The cells immobilized in microfibers
were treated with 0,25-20 µM doxorubicin as a model anticancer drug 24 h post cell immobilization,
while treated cells in 2D served as controls. The results showed significant increase in resistance to
doxorubicin in the 3D culture compared to the 2D: the IC50 value for the 3D was ~3 µM while that for
the 2D culture was ~0.5 µM. The obtained higher resistance to the anticancer drug compared to 2D
cultures is in agreement with the resistance of cancers to treatments in vivo, showing the potentials
of the proposed system as a more relevant in vitro model for anticancer drug testing.Conference Programme Book:Tumour Microenvironment Meeting & 3D Model Workshop, 6-8 September 202
A continuous-flow 3D osteosarcoma model: formation of spheroid-like aggregates under perfusion
No full textOnline Abstract Book: Goodbye Flat Biology: Next Generation Cancer Models, Berlin, Germany, 10-12 October, 202
CONSUMPTION OF EXOPOLYSACCHARIDES FROM LACTIPLANTIBACILLUS PLANTARUM BGAN8 ALTERS THE GUT MICROBIOTA OF DA RAT
Get PDFIntroduction: Exopolysaccharides (EPS) are carbohydrate polymers with important biological activities such
as immunomodulatory, antioxidative and antitumor (1). How EPS affect the gut microbiota, which plays an
important role in maintaining homeostasis in the organism, is poorly understood to date. Therefore, the aim
of this study was to investigate how EPS-AN8, derived from Lactiplantibacillus plantarum BGAN8, affects part
of the gut microbiota in the duodenum of healthy Dark Agouti rats (DA).
Materials and Methods: EPS-AN8 was administered to male DA at low (0.1 mg/mL) and high (1 mg/mL)
dose over a 15-day period. Total duodenal DNA was isolated and PCR amplicon for 16SrRNA was sequenced
on Illumina NovaSeq paired-end platform. Furthermore, we tracked key parameters of oxidative stress and
inflammation in duodenal homogenates.
Results: The higher dose of EPS-AN8 resulted in an increased Shannon's diversity index. The most
significant differences were observed in the increased relative abundance of the genera Ruminococcus,
Dubosiella, Enterohabdus, and Adlercreutzia. At the same time, we demonstrated that neither dosage caused
negative side effects such as inflammation and oxidative stress.
Conclusion: Considering the existing trend to market foods with additional health benefits, our results
suggest that EPS-AN8 could be a good candidate for functional food supplementation.International Society of Microbiota
10th ISM World Congress on
Targeting Microbiota
October 17-19, 2023 – Venice, Ital
From waste streams to biotherapeutics: making a connection using bacteria
Get PDFMicroorganisms, our planet’s original inhabitants discovered with the invention of the
first microscope in the 17th century, have consistently facilitated our daily life. However,
our modern life generates enormous amounts of wastes, such as plastic, food, and
chemicals from the pharmaceutical industry. Bacterial natural products hold an
important position in this industry, as drug leads in synthetic chemistry and biology,
essential for the discovery of effective agents against a variety of human diseases. If the
existing waste is used as a nutrient source for microbial production of valuable
biomolecules, that concept is called “waste to value” or “upcycling”.
This concept was explored using bacterial biopigment prodigiosin (PG, Fig. 1) as part of
the BioECOLogics project. This proof of concept demonstrates how the bacteria Serratia
marcescens ATCC 27117 can use a waste stream from the food industry as a carbon
source to grow and produce its bioactive secondary metabolite PG. The unique structure
of this molecule was changed through green chemical [1] and biopolymer formulation
[2] approaches, as well as metal complexation. Finally, these sustainable biotherapeutics
were validated in vitro (antimicrobial, anticancer) and in vivo (nematode Caenorhabditis
elegans and zebrafish Danio rerio).Book of Abstracts: 9th Conference of Young Chemists of Serbia
Novi Sad, 4th November 202
Expression of BCL11A in chronic lymphocytic leukaemia
No full textIntroduction The B-cell lymphoma/leukaemia 11A (BCL11A) gene encodes a Kruppel-like transcription factor involved in lymphocyte development during normal haematopoiesis. Aberrant expression of BCL11A has been observed in several haematological malignancies, including chronic lymphocytic leukaemia (CLL). However, its functions in the regulatory networks of malignant B lymphocytes are poorly understood, as are the relations to clinical course and outcome of B-cell malignancies, particularly CLL. Methods The expression of BCL11A was analysed in peripheral blood mononuclear cells of 87 newly-diagnosed CLL patients by quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR), and association with clinical and molecular variables was assessed. Results BCL11A was significantly overexpressed in CLL samples compared to control samples (p lt 0.001). BCL11A expression level exhibited no association with age, sex, leukocyte, lymphocyte and platelet counts, haemoglobin level, serum beta 2-microglobulin, CD38 status and cytogenetic abnormalities. On the other hand, high BCL11A expression was associated with low serum lactate dehydrogenase (p = 0.031), Binet A stage (p = 0.047) and mutated IGHV (p = 0.028). In addition, a positive correlation with BCL2/BAX mRNA ratio was observed (r = 0.36; p lt 0.001). Regarding the association with the time to first treatment (TTFT), a trend towards longer median TTFT in BCL11A high- versus BCL11A low-expressing cases was detected (21 vs. 6 months; p = 0.164). Conclusion The results of this study show that BCL11A is upregulated in CLL patients, and that high BCL11A expression at diagnosis may be associated with better prognosis. These data are consistent with the role of BCL11A expression in CLL biology, and imply its potential prognostic relevance