CoLoC-seq probes the global topology of organelle transcriptomes.

Proper RNA localisation is essential for physiological gene expression. Various kinds of genome-wide approaches permit to comprehensively profile subcellular transcriptomes. Among them, cell fractionation methods, that couple RNase treatment of isolated organelles to the sequencing of protected transcripts, remain most widely used, mainly because they do not require genetic modification of the studied system and can be easily implemented in any cells or tissues, including in non-model species. However, they suffer from numerous false-positives since incompletely digested contaminant RNAs can still be captured and erroneously identified as resident transcripts. Here we introduce Controlled Level of Contamination coupled to deep sequencing (CoLoC-seq) as a new subcellular transcriptomics approach that efficiently bypasses this caveat. CoLoC-seq leverages classical enzymatic kinetics and tracks the depletion dynamics of transcripts in a gradient of an exogenously added RNase, with or without organellar membranes. By means of straightforward mathematical modelling, CoLoC-seq infers the localisation topology of RNAs and robustly distinguishes between genuinely resident, luminal transcripts and merely abundant surface-attached contaminants. Our generic approach performed well on human mitochondria and is in principle applicable to other membrane-bounded organelles, including plastids, compartments of the vacuolar system, extracellular vesicles, and viral particles.

Hooded crows (Corvus cornix) manufacture objects relative to a mental template.

It was recently found that not only tool-specialized New Caledonian crows, but also Goffin cockatoos can manufacture physical objects in accordance with a mental template. That is, they can emulate features of existing objects when they manufacture new items. Both species spontaneously ripped pieces of card into large strips if they had previously learned that a large template was rewarded, and small strips when they previously learned that a small template was rewarded. Among New Caledonian crows, this cognitive ability was suggested as a potential mechanism underlying the transmission of natural tool designs. Here, we tested for the same ability in another non-specialised tool user-Hooded crows (Corvus cornix). Crows were exposed to pre-made template objects, varying first in colour and then in size, and were rewarded only if they chose pre-made objects that matched the template. In subsequent tests, birds were given the opportunity to manufacture versions of these objects. All three crows ripped paper pieces from the same colour material as the rewarded template, and, crucially, also manufactured objects that were more similar in size to previously rewarded, than unrewarded, templates, despite the birds being rewarded at random in both tests. Therefore, we found the ability to manufacture physical objects relative to a mental template in yet another bird species not specialized in using or making foraging tools in the wild, but with a high level of brain and cognitive development.

eIF4G is retained on ribosomes elongating and terminating on short upstream ORFs to control reinitiation in yeast.

Translation reinitiation is a gene-specific translational control mechanism. It is characterized by the ability of short upstream ORFs to prevent full ribosomal recycling and allow the post-termination 40S subunit to resume traversing downstream for the next initiation event. It is well known that variable transcript-specific features of various uORFs and their prospective interactions with initiation factors lend them an unequivocal regulatory potential. Here, we investigated the proposed role of the major initiation scaffold protein eIF4G in reinitiation and its prospective interactions with uORF's cis-acting features in yeast. In analogy to the eIF3 complex, we found that eIF4G and eIF4A but not eIF4E (all constituting the eIF4F complex) are preferentially retained on ribosomes elongating and terminating on reinitiation-permissive uORFs. The loss of the eIF4G contact with eIF4A specifically increased this retention and, as a result, increased the efficiency of reinitiation on downstream initiation codons. Combining the eIF4A-binding mutation with that affecting the integrity of the eIF4G1-RNA2-binding domain eliminated this specificity and produced epistatic interaction with a mutation in one specific cis-acting feature. We conclude that similar to humans, eIF4G is retained on ribosomes elongating uORFs to control reinitiation also in yeast.

Analysis of Aluminum Oxides Submicron Particle Agglomeration in Polymethyl Methacrylate Composites.

Agglomeration of distributed particles is the main problem in polymer composites reinforced with such particles. It leads to a decrease in mechanical performance and its poor reproducibility. Thus, development of methods to address the agglomeration of particles is relevant. Evaluation of the size and concentration of agglomerates is required to select a method to address agglomeration. The paper analyzes aluminum oxide particles agglomeration in particles-reinforced polymethyl methacrylate (PMMA) composites. Quantitative parameters of polystyrene-coated aluminum oxide particles agglomerates are obtained for the first time in this article. Unlike uncoated aluminum oxide particles, when coated aluminum oxide particles are used, agglomerates concentration in polymer composites decreases approx. 10 times. It demonstrates that modification of submicron particles by a polymer coating decreases the number of agglomerates in the polymer composite. The use of transmittance and opacity values to estimate particles agglomerates is reasonable in this article. It is shown that the difference in optical performance of specimens reinforced with coated and the original particles is related to the number and average size of agglomerates in the specimens. For example, when the concentration exceeds 0.2%, transmittance values for the specimens reinforced with coated particles are greater than the ones for the specimens reinforced with the original particles.

Staphylococcus aureus Panton-Valentine Leukocidin triggers an alternative NETosis process targeting mitochondria.

Panton-Valentine Leukocidin (PVL) is a bicomponent leukotoxin produced by 3%-10% of clinical Staphylococcus aureus (SA) strains involved in the severity of hospital and community-acquired infections. Although PVL was long known as a pore-forming toxin, recent studies have challenged the formation of a pore at the plasma membrane, while its endocytosis and the exact mode of action remain to be defined. In vitro immunolabeling of human neutrophils shows that Neutrophil Extracellular Traps (NETosis) is triggered by the action of purified PVL, but not by Gamma hemolysin CB (HlgCB), a structurally similar SA leukotoxin. PVL causes the ejection of chromatin fibers (NETs) decorated with antibacterial peptides independently of the NADPH oxidase oxidative burst. Leukotoxin partially colocalizes with mitochondria and enhances the production of reactive oxygen species from these organelles, while showing an increased autophagy, which results unnecessary for NETs ejection. PVL NETosis is elicited through Ca2+ -activated SK channels and Myeloperoxidase activity but is abolished by Allopurinol pretreatment of neutrophils. Moreover, massive citrullination of the histone H3 is performed by peptidyl arginine deiminases. Inhibition of this latter enzymes fails to abolish NET extrusion. Unexpectedly, PVL NETosis does not seem to involve Src kinases, which is the main kinase family activated downstream the binding of PVL F subunit to CD45 receptor, while the specific kinase pathway differs from the NADPH oxidase-dependent NETosis. PVL alone causes a different and specific form of NETosis that may rather represent a bacterial strategy conceived to disarm and disrupt the immune response, eventually allowing SA to spread.

YBEY is an essential biogenesis factor for mitochondrial ribosomes.

Ribosome biogenesis requires numerous trans-acting factors, some of which are deeply conserved. In Bacteria, the endoribonuclease YbeY is believed to be involved in 16S rRNA 3'-end processing and its loss was associated with ribosomal abnormalities. In Eukarya, YBEY appears to generally localize to mitochondria (or chloroplasts). Here we show that the deletion of human YBEY results in a severe respiratory deficiency and morphologically abnormal mitochondria as an apparent consequence of impaired mitochondrial translation. Reduced stability of 12S rRNA and the deficiency of several proteins of the small ribosomal subunit in YBEY knockout cells pointed towards a defect in mitochondrial ribosome biogenesis. The specific interaction of mitoribosomal protein uS11m with YBEY suggests that the latter helps to properly incorporate uS11m into the nascent small subunit in its late assembly stage. This scenario shows similarities with final stages of cytosolic ribosome biogenesis, and may represent a late checkpoint before the mitoribosome engages in translation.

CT and MRI Imaging of Theranostic Bimodal Fe3O4@Au NanoParticles in Tumor Bearing Mice.

Gold-containing nanoparticles are proven to be an effective radiosensitizer in the radiotherapy of tumors. Reliable imaging of nanoparticles in a tumor and surrounding normal tissues is crucial both for diagnostics and for nanoparticle application as radiosensitizers. The Fe3O4 core was introduced into gold nanoparticles to form a core/shell structure suitable for MRI imaging. The aim of this study was to assess the in vivo bimodal CT and MRI enhancement ability of novel core/shell Fe3O4@Au theranostic nanoparticles. Core/shell Fe3O4@Au nanoparticles were synthesized and coated with PEG and glucose. C57Bl/6 mice bearing Ca755 mammary adenocarcinoma tumors received intravenous injections of the nanoparticles. CT and MRI were performed at several timepoints between 5 and 102 min, and on day 17 post-injection. Core/shell Fe3O4@Au nanoparticles provided significant enhancement of the tumor and tumor blood vessels. Nanoparticles also accumulated in the liver and spleen and were retained in these organs for 17 days. Mice did not show any signs of toxicity over the study duration. These results indicate that theranostic bimodal Fe3O4@Au nanoparticles are non-toxic and serve as effective contrast agents both for CT and MRI diagnostics. These nanoparticles have potential for future biomedical applications in cancer diagnostics and beyond.

Cartilage Formation In Vivo Using High Concentration Collagen-Based Bioink with MSC and Decellularized ECM Granules.

The aim of this study was to verify the applicability of high-concentration collagen-based bioink with MSC (ADSC) and decellularized ECM granules for the formation of cartilage tissue de novo after subcutaneous implantation of the scaffolds in rats. The printability of the bioink (4% collagen, 2.5% decellularized ECM granules, derived via 280 µm sieve) was shown. Three collagen-based compositions were studied: (1) with ECM; (2) with MSC; (3) with ECM and MSC. It has been established that decellularized ECM granules are able to stimulate chondrogenesis both in cell-free and MSC-laden scaffolds. Undesirable effects have been identified: bone formation as well as cartilage formation outside of the scaffold area. The key perspectives and limitations of ECM granules (powder) application have been discussed.

Synthesis, Anti-Influenza H1N1 and Anti-Dengue Activity of A-Ring Modified Oleanonic Acid Polyamine Derivatives.

A series of sixteen A-ring modified (2,3-indolo-, 2-benzylidene) oleanonic acid derivatives, holding some cyclic amines, linear polyamines and benzylaminocarboxamides at C28, has been synthesized and screened for antiviral activity against influenza A/PuertoRico/8/34 (H1N1) and Dengue virus serotypes of DENV-1, -2, -3, -4. It was found that 28-homopiperazine 2 and 3-N-phthalyl 22 amides of oleanonic acid demonstrated high potency with selectivity index SI 27 (IC50 21 µM) and 42 (IC50 12 µM). Oleanonic acid aminoethylpiperazine amide 6 and C-azepano-erythrodiol 23 appeared to be the most effective compounds against DENV-1 (IC50's 67 and 107 µM) and -2 (IC50's 86 and 68 µM correspondingly) serotypes.

Telomere Length in Chromosomally Normal and Abnormal Miscarriages and Ongoing Pregnancies and Its Association with 5-hydroxymethylcytosine Patterns.

The present study investigates telomere length (TL) in dividing chorionic cytotrophoblast cells from karyotypically normal and abnormal first trimester miscarriages and ongoing pregnancies. Using Q-FISH, we measured relative TLs in the metaphase chromosomes of 61 chorionic villous samples. Relative TLs did not differ between karyotypically normal samples from miscarriages and those from ongoing pregnancies (p = 0.3739). However, among the karyotypically abnormal samples, relative TLs were significantly higher in ongoing pregnancies than in miscarriages (p < 0.0001). Relative TLs were also significantly higher in chorion samples from karyotypically abnormal ongoing pregnancies than in those from karyotypically normal ones (p = 0.0018) in contrast to miscarriages, where relative TL values were higher in the karyotypically normal samples (p = 0.002). In the karyotypically abnormal chorionic cytotrophoblast, the TL variance was significantly lower than in any other group (p < 0.05). Assessed by TL ratios between sister chromatids, interchromatid TL asymmetry demonstrated similar patterns across all of the chorion samples (p = 0.22) but significantly exceeded that in PHA-stimulated lymphocytes (p < 0.0001, p = 0.0003). The longer telomere was predominantly present in the hydroxymethylated sister chromatid in chromosomes featuring hemihydroxymethylation (containing 5-hydroxymethylcytosine in only one sister chromatid)-a typical sign of chorionic cytotrophoblast cells. Our results suggest that the phenomena of interchromatid TL asymmetry and its association to 5hmC patterns in chorionic cytotrophoblast, which are potentially linked to telomere lengthening through recombination, are inherent to the development programme. The TL differences in chorionic cytotrophoblast that are associated with karyotype and embryo viability seem to be determined by heredity rather than telomere elongation mechanisms. The inheritance of long telomeres by a karyotypically abnormal embryo promotes his development, whereas TL in karyotypically normal first-trimester embryos does not seem to have a considerable impact on developmental capacity.

Can Mitochondrial DNA be CRISPRized: Pro and Contra.

Mitochondria represent a chimera of macromolecules encoded either in the organellar genome, mtDNA, or in the nuclear one. If the pathway of protein targeting to different sub-compartments of mitochondria was relatively well studied, import of small noncoding RNAs into mammalian mitochondria still awaits mechanistic explanations and its functional issues are often not understood thus raising polemics. At the same time, RNA mitochondrial import pathway has an obvious attractiveness as it appears as a unique natural mechanism permitting to address nucleic acids into the organelles. Deciphering the function(s) of imported RNAs inside the mitochondria is extremely complicated due to their relatively low abundance, which suggests their regulatory role. We previously demonstrated that mitochondrial targeting of small noncoding RNAs able to specifically anneal with the mutant mitochondrial DNA led to a decrease of the mtDNA heteroplasmy level by inhibiting mutant mtDNA replication. We then demonstrated that increasing level of expression of such antireplicative recombinant RNAs increases significantly the antireplicative effect. In this report, we present a new data investigating the possibility to establish a CRISPR-Cas9 system targeting mtDNA exploiting of the pathway of RNA import into mitochondria. Mitochondrially addressed Cas9 versions and a set of mitochondrially targeted guide RNAs were tested in vitro and in vivo and their effect on mtDNA copy number was demonstrated. So far, the system appeared as more complicated for use than previously found for nuclear DNA, because only application of a pair of guide RNAs produced the effect of mtDNA depletion. We discuss, in a critical way, these results and put them in a broader context of polemics concerning the possibilities of manipulation of mtDNA in mammalians. The findings described here prove the potential of the RNA import pathway as a tool for studying mtDNA and for future therapy of mitochondrial disorders. © The Authors. IUBMB Life published by Wiley Periodicals, Inc. on behalf of International Union of Biochemistry and Molecular Biology, 70(12):1233-1239, 2018.

Evidence for New Light-Independent Pathways for Generation of the Endogenous Aryl Hydrocarbon Receptor Agonist FICZ.

Activation of the aryl hydrocarbon receptor (AhR), a conserved transcription factor best known as a target for highly toxic halogenated substances such as dioxin, under normal xenobiotic-free conditions is of considerable scientific interest. We have demonstrated previously that a photoproduct of tryptophan, 6-formylindolo[3,2-b]carbazole (FICZ), fulfills the criteria for an endogenous ligand for this receptor and proposed that this compound is the enigmatic mediator of the physiological functions of AhR. Here, we describe novel light-independent pathways by which FICZ can be formed. The oxidant H2O2 was shown to convert tryptophan to FICZ on its own in the absence of light. The enzymatic deamination of tryptamine yielded indole-3-acetaldehyde (I3A), which then rearranged to FICZ and its oxidation product, indolo[3,2-b]carbazole-6-carboxylic acid (CICZ). Indole-3-pyruvate (I3P) also produced I3A, FICZ, and CICZ. Malassezia yeast species, which constitute a part of the normal skin microbiota, produce a number of AhR activators from tryptophan. We identified both FICZ and CICZ among those products. Formation of FICZ from tryptophan or I3P produces a complex mixture of indole derivatives, some of which are CYP1A1 inhibitors. These can hinder the cellular clearance of FICZ and thereby increase its power as an AhR agonist. We present a general molecular mechanism involving dehydrogenations and oxidative coupling for the formation of FICZ in which I3A is the important precursor. In conclusion, our results suggest that FICZ is likely to be formed systemically.

Water loss from litchi (Litchi chinensis) and longan (Dimocarpus longan) fruits is biphasic and controlled by a complex pericarpal transpiration barrier.

MAIN CONCLUSION: In litchi and longan fruits, a specialised pericarp controls water loss by a protective system consisting of two resistances in series and two water reservoirs separated by a barrier. In the fruits of litchi (Litchi chinensis) and longan (Dimocarpus longan), the pericarp is solely a protective structure lacking functional stomata and completely enclosing the aril that is the edible part. Maintaining a high water content of the fruits is crucial for ensuring the economic value of these important fruit crops. The water loss rates from mature fruits were determined and analysed in terms of the properties of the pericarps. Water loss kinetics and sorption isotherms were measured gravimetrically. The pericarps were studied with microscopy, and cuticular waxes and cutin were analysed with gas chromatography and mass spectrometry. The kinetics of fruit water loss are biphasic with a high initial rate and a lower equilibrium rate lasting for many hours. The outer and inner surfaces of the pericarps are covered with cuticles. Litchi and longan fruits have a unique type of transpiration barrier consisting of two resistances in series (endo- and exocarp cuticles) and two reservoirs of water (aril and mesocarp). The exocarp permeability controls the water loss from fresh fruits while in fruits kept for an extended time at low relative humidity it is determined by the endo- and exocarp permeabilities. Permeances measured are within the range for typical fruit cuticles. The findings may be used to design optimal postharvest storage strategies for litchi and longan fruits.

Analogical reasoning in amazons.

Two juvenile orange-winged amazons (Amazona amazonica) were initially trained to match visual stimuli by color, shape, and number of items, but not by size. After learning these three identity matching-to-sample tasks, the parrots transferred discriminative responding to new stimuli from the same categories that had been used in training (other colors, shapes, and numbers of items) as well as to stimuli from a different category (stimuli varying in size). In the critical testing phase, both parrots exhibited reliable relational matching-to-sample (RMTS) behavior, suggesting that they perceived and compared the relationship between objects in the sample stimulus pair to the relationship between objects in the comparison stimulus pairs, even though no physical matches were possible between items in the sample and comparison pairs. The parrots spontaneously exhibited this higher-order relational responding without having ever before been trained on RMTS tasks, therefore joining apes and crows in displaying this abstract cognitive behavior.

Deficiency in a very-long-chain fatty acid ß-ketoacyl-coenzyme a synthase of tomato impairs microgametogenesis and causes floral organ fusion.

Previously, it was shown that ß-ketoacyl-coenzyme A synthase ECERIFERUM6 (CER6) is necessary for the biosynthesis of very-long-chain fatty acids with chain lengths beyond C28 in tomato (Solanum lycopersicum) fruits and C26 in Arabidopsis (Arabidopsis thaliana) leaves and the pollen coat. CER6 loss of function in Arabidopsis resulted in conditional male sterility, since pollen coat lipids are responsible for contact-mediated pollen hydration. In tomato, on the contrary, pollen hydration does not rely on pollen coat lipids. Nevertheless, mutation in SlCER6 impairs fertility and floral morphology. Here, the contribution of SlCER6 to the sexual reproduction and flower development of tomato was addressed. Cytological analysis and cross-pollination experiments revealed that the slcer6 mutant has male sterility caused by (1) hampered pollen dispersal and (2) abnormal tapetum development. SlCER6 loss of function provokes a decrease of n- and iso-alkanes with chain lengths of C27 or greater and of anteiso-alkanes with chain lengths of C28 or greater in flower cuticular waxes, but it has no impact on flower cuticle ultrastructure and cutin content. Expression analysis confirmed high transcription levels of SlCER6 in the anther and the petal, preferentially in sites subject to epidermal fusion. Hence, wax deficiency was proposed to be the primary reason for the flower fusion phenomenon in tomato. The SlCER6 substrate specificity was revisited. It might be involved in elongation of not only linear but also branched very-long-chain fatty acids, leading to production of the corresponding alkanes. SlCER6 implements a function in the sexual reproduction of tomato that is different from the one in Arabidopsis: SlCER6 is essential for the regulation of timely tapetum degradation and, consequently, microgametogenesis.

Pharmacokinetic Parameters of Recombinant Human Cyclophilin A in Mice.

BACKGROUND AND OBJECTIVE: Cyclophilin A (CypA) is an isomerase that functions as a chaperone, housekeeping protein, and cyclosporine A (CsA) ligand. Secreted CypA is a proinflammatory factor, chemoattractant, immune regulator, and factor of antitumor immunity. Experimental data suggest clinical applications of recombinant human CypA (rhCypA) as a biotherapeutic for cancer immunotherapy, stimulation of tissue regeneration, treatment of brain pathologies, and as a supportive treatment for CsA-based therapies. The objective of this study is to analyze the pharmacokinetics of rhCypA in a mouse model. METHODS: rhCypA was isotope-labeled with 125I and injected intraperitoneally (i.p.) or subcutaneously (s/c) into female mice as a single dose of 100 µg per mouse, equivalent to the estimated first-in-human dose. Analysis of 125I-rhCypA biodistribution and excretion was performed by direct radiometry of the blood, viscera, and urine of mice 0.5-72 h following its administration. RESULTS: rhCypA showed rapid and even tissue-organ distribution, with the highest tropism (fT = 1.56) and accumulation (maximum concentration, Cmax = 137-167 µg/g) in the kidneys, its primary excretory organ. rhCypA showed the lowest tropism to the bone marrow and the brain (fT = 0.07) but the longest retention in these organs [mean retention time (MRT) = 25-28 h]. CONCLUSION: This study identified promising target organs for rhCypA's potential therapeutic effects. The mode of rhCypA accumulation and retention in organs could be primarily due to the expression of its receptors in them. For the first time, rhCypA was shown to cross the blood-brain barrier and accumulate in the brain. These rhCypA pharmacokinetic data could be extrapolated to humans as preliminary data for possible clinical trials.

Synthesis and bioevaluation of water-soluble ß-cyclodextrin-diterpene acid conjugates.

A series of ß-cyclodextrin (ß-CD)-conjugates were prepared by combining three abietane-type diterpene acids with two azide-functionalized ß-CDs via click chemistry, and the antiviral activity against wild-type and omicron SARS-CoV-2 spike pseudovirus as well as the antibacterial activity against Escherichia coli were investigated. All the synthesised conjugates exhibited no significant cytotoxicity to BHK-21-hACE2 cells with cell viability over 80% at concentration of 15 µM. Among the conjugates, the heptavalent ß-CD-dehydroabietic acid conjugate 6b exhibited higher anti-SARS-CoV-2 activity against the omicron variant compared to the other conjugates. This study suggested that the multivalent diterpene acid derivatives may have potential application against coronaviruses as entry inhibitors.

Stem-loop induced ribosome queuing in the uORF2/ATF4 overlap fine-tunes stress-induced human ATF4 translational control.

ATF4 is a master transcriptional regulator of the integrated stress response leading cells towards adaptation or death. ATF4's induction under stress was thought to be mostly due to delayed translation reinitiation, where the reinitiation-permissive uORF1 plays a key role. Accumulating evidence challenging this mechanism as the sole source of ATF4 translation control prompted us to investigate additional regulatory routes. We identified a highly conserved stem-loop in the uORF2/ATF4 overlap, immediately preceded by a near-cognate CUG, which introduces another layer of regulation in the form of ribosome queuing. These elements explain how the inhibitory uORF2 can be translated under stress, confirming prior observations, but contradicting the original regulatory model. We also identified two highly conserved, potentially modified adenines performing antagonistic roles. Finally, we demonstrate that the canonical ATF4 translation start site is substantially leaky-scanned. Thus, ATF4's translational control is more complex than originally described underpinning its key role in diverse biological processes.

Stem-loop-induced ribosome queuing in the uORF2/ATF4 overlap fine-tunes stress-induced human ATF4 translational control.

Activating transcription factor 4 (ATF4) is a master transcriptional regulator of the integrated stress response, leading cells toward adaptation or death. ATF4's induction under stress was thought to be due to delayed translation reinitiation, where the reinitiation-permissive upstream open reading frame 1 (uORF1) plays a key role. Accumulating evidence challenging this mechanism as the sole source of ATF4 translation control prompted us to investigate additional regulatory routes. We identified a highly conserved stem-loop in the uORF2/ATF4 overlap, immediately preceded by a near-cognate CUG, which introduces another layer of regulation in the form of ribosome queuing. These elements explain how the inhibitory uORF2 can be translated under stress, confirming prior observations but contradicting the original regulatory model. We also identified two highly conserved, potentially modified adenines performing antagonistic roles. Finally, we demonstrated that the canonical ATF4 translation start site is substantially leaky scanned. Thus, ATF4's translational control is more complex than originally described, underpinning its key role in diverse biological processes.

Biological Efficacy of Ionizing Radiation Sources on 3D Organotypic Tissue Slices Assessed by Fluorescence Microscopy.

OBJECTIVE: Traditional cell-based radiobiological methods are inadequate for assessing the toxicity of ionizing radiation exposure in relation to the microstructure of the extracellular matrix. Organotypic tissue slices preserve the spatial organization observed in vivo, making the tissue easily accessible for visualization and staining. This study aims to explore the use of fluorescence microscopy of physiologically compatible 3D tissue cultures to assess the effects of ionizing radiation. METHODS: Organotypic tissue slices were obtained by vibratome, and their mechanical properties were studied. Slices were exposed by two ionizing radiation sources; electron beams (80 Gy and 4 Gy), and soft gamma irradiation (80 Gy and 4 Gy). Two tissue culture protocols were used: the standard (37°C), and hypothermic (30°C) conditions. A qualitative analysis of cell viability in organotypic tissue slices was performed using fluorescent dyes and standard laser confocal microscopy. RESULTS: Biological dosimetry is represented by differentially stained 200-µm thick organotypic tissue sections related to living and dead cells and cell metabolic activity. CONCLUSION: Our results underscore the ability of fluorescence laser scanning confocal microscopy to rapidly assess the radiobiological effects of ionizing radiation in vitro on 3D organotypic tissue slices.