Molecular insights into the differences between cyanobacterial and plant LPORs and prolamellar body formation: In vitro studies.

Light-dependent protochlorophyllide oxidoreductase (LPOR) has captivated the interest of the research community for decades. One reason is the photocatalytic nature of the reaction catalyzed by the enzyme, and the other is the involvement of LPOR in the formation of a paracrystalline lattice called a prolamellar body (PLB) that disintegrates upon illumination, initiating a process of photosynthetic membrane formation. In this paper, we have integrated three traditional methods previously employed to study the properties of the enzyme: molecular biology, spectroscopy, and electron microscopy. We found that for cyanobacterial LPOR, substrates binding appears to be independent of lipids, with membrane interaction primarily affecting the enzyme post-reaction, with MGDG and PG having opposite effects on SynPOR. In contrast, plant isoforms exhibit sequence alterations, rendering the enzyme effective in substrate binding mainly in the presence of anionic lipids, depending on residues at positions 122, 312, and 318. Moreover, we demonstrated that the interaction with MGDG could initially serve as enhancement of the substrate specificity towards monovinyl-protochlorophyllide (Pchlide). We have shown that the second LPOR isoforms of eudicots and monocots accumulated mutations that made these variants less and more dependent on anionic lipids, respectively. Finally, we have shown that in the presence of Pchlide, NADP+, and the lipids, plant but not cyanobacterial LPOR homolog remodel membranes into the cubic phase. The cubic phase is preserved if samples supplemented with NADP + are enriched with NADPH. The results are discussed in the evolutionary context, and the model of PLB formation is presented.

Studies on Autophagy and Apoptosis of Fibrosarcoma HT-1080 Cells Mediated by Chalcone with Indole Moiety.

This study demonstrated the anticancer efficacy of chalcones with indole moiety (MIPP, MOMIPP) in fibrosarcoma cells for the first time. The results showed that MIPP and MOMIPP reduced the viability of HT-1080 cells in a concentration-dependent manner. MOMIPP was more active than MIPP in HT-1080 cells, showing lower IC50 values (3.67 vs. 29.90 µM). Both compounds at a concentration of 1 µM induced apoptosis in HT-1080 cells, causing death strictly related to caspase activation, as cell viability was restored when the caspase inhibitor Z-VAD was added. Reactive oxygen species production was approximately 3-fold higher than in control cells, and cotreatment with the inhibitor of mitochondrial ATPase oligomycin diminished this effect. Such effects were also reflected in mitochondrial dysfunction, including decreased membrane potential. Interestingly, the compounds that were studied caused massive vacuolization in HT-1080 cells. Immunocytochemical staining and TEM analysis showed that HT-1080 cells exhibited increased expression of the LC3-II protein and the presence of autophagosomes with a double membrane, respectively. Both compounds induced apoptosis, highlighting a promising link between autophagy and apoptosis. This connection could be a new target for therapeutic strategies to overcome chemoresistance, which is a significant cause of treatment failure and tumour recurrence in fibrosarcoma following traditional chemotherapy.

Reengineering of an Artificial Protein Cage for Efficient Packaging of Active Enzymes.

Protein cages that readily encapsulate active enzymes of interest present useful nanotools for delivery and catalysis, wherein those with programmable disassembly characteristics serve as particularly attractive platforms. Here, a general guest packaging system based on an artificial protein cage, TRAP-cage, the disassembly of which can be induced by the addition of reducing agents, is established. In this system, TRAP-cage with SpyCatcher moieties in the lumen is prepared using genetic modification of the protein building block and assembled into a cage structure with either monovalent gold ions or molecular crosslinkers. The resulting protein cage can efficiently capture guest proteins equipped with a SpyTag by simply mixing them in an aqueous solution. This post-assembly loading system, which circumvents the exposure of guests to thiol-reactive crosslinkers, enables the packaging of enzymes possessing a catalytic cysteine or a metal cofactor while retaining their catalytic activity.

Structural features of Prussian Blue-related iron complex - FeT of activity to peroxidate unsaturated fatty acids.

Aim: FeT is a complex of Fe3+, ferricyanide and tartrate, similar in structure to Prussian Blue. Its synthesis was planned to produce a potential antiproliferative drug. Methods: Dynamic light scattering was applied to study nanostructures formed by FeT complexes, while their biological activity was tested following changes in cell proliferation using cultured T24 human bladder cancer cells. Results: The antiproliferative activity of FeT derived from its ability to peroxidate unsaturated fatty acids, which can cause cell death through oxidative stress and/or ferroptosis. FeT molecules associate into drop-like nanostructures in water solutions, between 10-130 nm, which can bind albumin. Conclusion: Fatty acid peroxidation is significantly activated by light. The characteristics and reactivity of FeT represent a prospective application in medicine.

Induced pluripotent stem cell-derived extracellular vesicles enriched with miR-126 induce proangiogenic properties and promote repair of ischemic tissue.

Emerging evidence suggests that stem cell-derived extracellular vesicles (EVs) may induce pro-regenerative effects in ischemic tissues by delivering bioactive molecules, including microRNAs. Recent studies have also shown pro-regenerative benefits of EVs derived from induced pluripotent stem (iPS) cells. However, the underlying mechanisms of EV benefits and the role of their transferred regulatory molecules remain incompletely understood. Accordingly, we investigated the effects of human iPS-derived EVs (iPS-EVs) enriched in proangiogenic miR-126 (iPS-miR-126-EVs) on functional properties of human endothelial cells (ECs) in vitro. We also examined the outcomes following EV injection in a murine model of limb ischemia in vivo. EVs were isolated from conditioned media from cultures of unmodified and genetically modified human iPS cells overexpressing miR-126. The iPS-miR-126-EVs were enriched in miR-126 when compared with control iPS-EVs and effectively transferred miR-126 along with other miRNAs to recipient ECs improving their functional properties essential for ischemic tissue repair, including proliferation, metabolic activity, cell survival, migration, and angiogenic potential. Injection of iPS-miR-126-EVs in vivo in a murine model of acute limb ischemia promoted angiogenesis, increased perfusion, and enhanced functional recovery. These observations corresponded with elevated expression of genes for several proangiogenic factors in ischemic tissues following iPS-miR-126-EV transplantation. These results indicate that innate pro-regenerative properties of iPS-EVs may be further enhanced by altering their molecular composition via controlled genetic modifications. Such iPS-EVs overexpressing selected microRNAs, including miR-126, may represent a novel acellular tool for therapy of ischemic tissues in vivo.

Dysregulated iron homeostasis in dystrophin-deficient cardiomyocytes: correction by gene editing and pharmacological treatment.

AIMS: Duchenne muscular dystrophy (DMD)-associated cardiomyopathy is a serious life-threatening complication, the mechanisms of which have not been fully established, and therefore no effective treatment is currently available. The purpose of the study was to identify new molecular signatures of the cardiomyopathy development in DMD. METHODS AND RESULTS: For modelling of DMD-associated cardiomyopathy, we prepared three pairs of isogenic control and dystrophin-deficient human induced pluripotent stem cell (hiPSC) lines. Two isogenic hiPSC lines were obtained by CRISPR/Cas9-mediated deletion of DMD exon 50 in unaffected cells generated from healthy donor and then differentiated into cardiomyocytes (hiPSC-CM). The latter were subjected to global transcriptomic and proteomic analyses followed by more in-depth investigation of selected pathway and pharmacological modulation of observed defects. Proteomic analysis indicated a decrease in the level of mitoNEET protein in dystrophin-deficient hiPSC-CM, suggesting alteration in iron metabolism. Further experiments demonstrated increased labile iron pool both in the cytoplasm and mitochondria, a decrease in ferroportin level and an increase in both ferritin and transferrin receptor in DMD hiPSC-CM. Importantly, CRISPR/Cas9-mediated correction of the mutation in the patient-derived hiPSC reversed the observed changes in iron metabolism and restored normal iron levels in cardiomyocytes. Moreover, treatment of DMD hiPSC-CM with deferoxamine (DFO, iron chelator) or pioglitazone (mitoNEET stabilizing compound) decreased the level of reactive oxygen species in DMD hiPSC-CM. CONCLUSION: To our knowledge, this study demonstrated for the first time impaired iron metabolism in human DMD cardiomyocytes, and potential reversal of this effect by correction of DMD mutation or pharmacological treatment. This implies that iron overload-regulating compounds may serve as novel therapeutic agents in DMD-associated cardiomyopathy.

Stress Conditions Affect the Immunomodulatory Potential of Candida albicans Extracellular Vesicles and Their Impact on Cytokine Release by THP-1 Human Macrophages.

Human immune cells possess the ability to react complexly and effectively after contact with microbial virulence factors, including those transported in cell-derived structures of nanometer sizes termed extracellular vesicles (EVs). EVs are produced by organisms of all kingdoms, including fungi pathogenic to humans. In this work, the immunomodulatory properties of EVs produced under oxidative stress conditions or at host concentrations of CO2 by the fungal pathogen Candida albicans were investigated. The interaction of EVs with human pro-monocytes of the U-937 cell line was established, and the most notable effect was attributed to oxidative stress-related EVs. The immunomodulatory potential of tested EVs against human THP-1 macrophages was verified using cytotoxicity assay, ROS-production assay, and the measurement of cytokine production. All fungal EVs tested did not show a significant cytotoxic effect on THP-1 cells, although a slight pro-oxidative impact was indicated for EVs released by C. albicans cells grown under oxidative stress. Furthermore, for all tested types of EVs, the pro-inflammatory properties related to increased IL-8 and TNF-α production and decreased IL-10 secretion were demonstrated, with the most significant effect observed for EVs released under oxidative stress conditions.

Candida albicans Biofilm-Derived Extracellular Vesicles Are Involved in the Tolerance to Caspofungin, Biofilm Detachment, and Fungal Proteolytic Activity.

It has been repeatedly reported that the cells of organisms in all kingdoms of life produce nanometer-sized lipid membrane-enveloped extracellular vesicles (EVs), transporting and protecting various substances of cellular origin. While the composition of EVs produced by human pathogenic fungi has been studied in recent decades, another important challenge is the analysis of their functionality. Thus far, fungal EVs have been shown to play significant roles in intercellular communication, biofilm production, and modulation of host immune cell responses. In this study, we verified the involvement of biofilm-derived EVs produced by two different strains of Candida albicans-C. albicans SC5314 and 3147 (ATCC 10231)-in various aspects of biofilm function by examining its thickness, stability, metabolic activity, and cell viability in the presence of EVs and the antifungal drug caspofungin. Furthermore, the proteolytic activity against the kininogen-derived antimicrobial peptide NAT26 was confirmed by HPLC analysis for C. albicans EVs that are known to carry, among others, particular members of the secreted aspartic proteinases (Saps) family. In conclusion, EVs derived from C. albicans biofilms were shown to be involved in biofilm tolerance to caspofungin, biofilm detachment, and fungal proteolytic activity.

Anticancer and antibacterial properties of carbon nanotubes are governed by their functional groups.

Due to their high strength, low weight, and biologically-inspired dimensions, carbon nanotubes have found wide interest across all of medicine. In this study, four types of highly dispersible multi-walled carbon nanotubes (CNTs) of similar dimensions, but slightly different chemical compositions, were compared with an unmodified material to verify the impact their surface chemistry has on cytocompatibility, anticancer, inflammation, and antibacterial properties. Minute changes in the chemical composition were found to greatly affect the biological performance of the CNTs. Specifically, the CNTs with a large number of carbon atoms with a +2 coordination number induced cytotoxicity in macrophages and melanoma cells, and had a moderate antibacterial effect against Gram-positive (S. aureus) and Gram-negative (E. coli) bacteria strains, all while being cytocompatible towards human dermal fibroblasts. Moreover, substituting some of the OH groups with ammonia diminished their cytotoxicity towards macrophages while still maintaining the aforementioned positive qualities. At the same time, CNTs with a large number of carbon atoms with a +3 coordination number had a high innate cytocompatibility towards normal healthy cells but were toxic towards cancer cells and bacteria. The latter was further boosted by reacting the CNTs' carboxyl groups with ammonia. Although requiring further analyses, the results of this study, thus, introduce new CNTs that without drugs can treat cancer, inflammation, and/or infection while still remaining cytocompatible with mammalian cells.

Hypoxia enhances anti-fibrotic properties of extracellular vesicles derived from hiPSCs via the miR302b-3p/TGFß/SMAD2 axis.

BACKGROUND: Cardiac fibrosis is one of the top killers among fibrotic diseases and continues to be a global unaddressed health problem. The lack of effective treatment combined with the considerable socioeconomic burden highlights the urgent need for innovative therapeutic options. Here, we evaluated the anti-fibrotic properties of extracellular vesicles (EVs) derived from human induced pluripotent stem cells (hiPSCs) that were cultured under various oxygen concentrations. METHODS: EVs were isolated from three hiPSC lines cultured under normoxia (21% O2; EV-N) or reduced oxygen concentration (hypoxia): 3% O2 (EV-H3) or 5% O2 (EV-H5). The anti-fibrotic activity of EVs was tested in an in vitro model of cardiac fibrosis, followed by a detailed investigation of the underlying molecular mechanisms. Sequencing of EV miRNAs combined with bioinformatics analysis was conducted and a selected miRNA was validated using a miRNA mimic and inhibitor. Finally, EVs were tested in a mouse model of angiotensin II-induced cardiac fibrosis. RESULTS: We provide evidence that an oxygen concentration of 5% enhances the anti-fibrotic effects of hiPS-EVs. These EVs were more effective in reducing pro-fibrotic markers in activated human cardiac fibroblasts, when compared to EV-N or EV-H3. We show that EV-H5 act through the canonical TGFß/SMAD pathway, primarily via miR-302b-3p, which is the most abundant miRNA in EV-H5. Our results show that EV-H5 not only target transcripts of several profibrotic genes, including SMAD2 and TGFBR2, but also reduce the stiffness of activated fibroblasts. In a mouse model of heart fibrosis, EV-H5 outperformed EV-N in suppressing the inflammatory response in the host and by attenuating collagen deposition and reducing pro-fibrotic markers in cardiac tissue. CONCLUSIONS: In this work, we provide evidence of superior anti-fibrotic properties of EV-H5 over EV-N or EV-H3. Our study uncovers that fine regulation of oxygen concentration in the cellular environment may enhance the anti-fibrotic effects of hiPS-EVs, which has great potential to be applied for heart regeneration.

Anti-inflammatory, Anti-fibrotic and Pro-cardiomyogenic Effects of Genetically Engineered Extracellular Vesicles Enriched in miR-1 and miR-199a on Human Cardiac Fibroblasts.

RATIONALE: Emerging evidence indicates that stem cell (SC)- derived extracellular vesicles (EVs) carrying bioactive miRNAs are able to repair damaged or infarcted myocardium and ameliorate adverse remodeling. Fibroblasts represent a major cell population responsible for scar formation in the damaged heart. However, the effects of EVs on cardiac fibroblast (CFs) biology and function has not been investigated. OBJECTIVE: To analyze the biological impact of stem cell-derived EVs (SC-EVs) enriched in miR-1 and miR-199a on CFs and to elucidate the underlying molecular mechanisms. METHODS AND RESULTS: Genetically engineered human induced pluripotent stem cells (hiPS) and umbilical cord-derived mesenchymal stem cells (UC-MSCs) expressing miR-1 or miR-199a were used to produce miR-EVs. Cells and EVs were thoughtfully analyzed for miRNA expression using RT-qPCR method. Both hiPS-miRs-EVs and UC-MSC-miRs-EVs effectively transferred miRNAs to recipient CFs, however, hiPS-miRs-EVs triggered cardiomyogenic gene expression in CFs more efficiently than UC-MSC-miRs-EVs. Importantly, hiPS-miR-1-EVs exhibited cytoprotective effects on CFs by reducing apoptosis, decreasing levels of pro-inflammatory cytokines (CCL2, IL-1ß, IL-8) and downregulating the expression of a pro-fibrotic gene - α-smooth muscle actin (α-SMA). Notably, we identified a novel role of miR-199a-3p delivered by hiPS-EVs to CFs, in triggering the expression of cardiomyogenic genes (NKX2.5, TNTC, MEF2C) and ion channels involved in cardiomyocyte contractility (HCN2, SCN5A, KCNJ2, KCND3). By targeting SERPINE2, miR-199a-3p may reduce pro-fibrotic properties of CFs, whereas miR-199a-5p targeted BCAM and TSPAN6, which may be implicated in downregulation of inflammation. CONCLUSIONS: hiPS-EVs carrying miR-1 and miR-199a attenuate apoptosis and pro-fibrotic and pro-inflammatory activities of CFs, and increase cardiomyogenic gene expression. These finding serve as rationale for targeting fibroblasts with novel EV-based miRNA therapies to improve heart repair after myocardial injury.

Investigating the potential effects of α-synuclein aggregation on susceptibility to chronic stress in a mouse Parkinson's disease model.

BACKGROUND: Parkinson's disease (PD) is a motor disorder characterized by the degeneration of dopaminergic neurons, putatively due to the accumulation of α-synuclein (α-syn) in Lewy bodies (LBs) in Substantia Nigra. PD is also associated with the formation of LBs in brain areas responsible for emotional and cognitive regulation such as the amygdala and prefrontal cortex, and concurrent depression prevalence in PD patients. The exact link between dopaminergic cell loss, α-syn aggregation, depression, and stress, a major depression risk factor, is unclear. Therefore, we aimed to explore the interplay between sensitivity to chronic stress and α-syn aggregation. METHODS: Bilateral injections of α-syn preformed fibrils (PFFs) into the striatum of C57Bl/6 J mice were used to induce α-syn aggregation. Three months after injections, animals were exposed to chronic social defeat stress. RESULTS: α-syn aggregation did not affect stress susceptibility but independently caused increased locomotor activity in the open field test, reduced anxiety in the light-dark box test, and increased active time in the tail suspension test. Ex vivo analysis revealed modest dopaminergic neuron loss in the substantia nigra and reduced dopaminergic innervation in the dorsal striatum in PFFs injected groups. α-Syn aggregates were prominent in the amygdala, prefrontal cortex, and substantia nigra, with minimal α-syn aggregation in the raphe nuclei and locus coeruleus. CONCLUSIONS: Progressive bilateral α-syn aggregation might lead to compensatory activity increase and alterations in emotionally regulated behavior, without affecting stress susceptibility. Understanding how α-syn aggregation and degeneration in specific brain structures contribute to depression and anxiety in PD patients requires further investigation.

Isolation and Characteristics of Extracellular Vesicles Produced by Probiotics: Yeast Saccharomyces boulardii CNCM I-745 and Bacterium Streptococcus salivarius K12.

Numerous probiotic microorganisms have repeatedly been shown to produce nanometer-sized structures named extracellular vesicles (EVs). Recently, it has been suggested that similarly to whole microbial cells, EVs produced by probiotics may also demonstrate health benefits to the host, while their application does not involve the risk of infection caused by live microorganisms. In this work, we isolated EVs from two probiotic species originating from different taxonomic domains - yeast Saccharomyces boulardii CNCM I-745 and bacterium Streptococcus salivarius K12. The diameters of S. boulardii EVs were about 142 nm and for S. salivarius EVs about 123 nm. For S. boulardii EVs, 1641 proteins and for S. salivarius EVs, 466 proteins were identified with a liquid chromatography-coupled tandem mass spectrometry and then functionally classified. In both microbial species, metabolic proteins significantly contributed to the cargo of EVs comprising 25% and 26% of all identified vesicular proteins for fungi and bacteria, respectively. Moreover, enzymes associated with cell wall rearrangement, including enzymatically active glucanases, were also identified in EVs. Furthermore, probiotic EVs were shown to influence host cells and stimulate the production of IL-1ß and IL-8 by the human monocytic cell line THP-1, and, at the same time, did not cause any remarkable reduction in the survival rate of Galleria mellonella larvae in this invertebrate model commonly used to evaluate microbial EV toxicity. These observations suggest that the EVs produced by the investigated probiotic microorganisms may be promising structures for future use in pro-health applications.

Canalised and plastic components of melanin-based colouration: a diet-manipulation experiment in house sparrows.

Whether melanin-based plumage colouration accurately reflects a bird's quality is still controversial. To better understand potential mechanisms behind the observed variation in plumage colouration, we shifted our attention from a high-level expression of colour to low-level physiological phenomena by targeting the microstructure and pigment content of the feather. In a well-studied model system, the house sparrow (Passer domesticus), we combined an experimental manipulation of birds' physiological condition and availability of resources that are key to the production of the studied colouration (phenylalanine and tyrosine (PT). We found that feathers from sparrows fed with the control diet had noticeably lower values of brightness, suggesting a higher quality of the ornamental "blackness" in comparison to those sampled from birds fed with a PT-reduced diet. Electron paramagnetic resonance (EPR) spectroscopy detected higher melanin concentrations in samples from the control than the PT-reduced group. Our multi-level analysis excluded mechanisms such as barbule density and melanosomes' distribution, clearly pointing to the finest-level proxy of colour: the concentration of melanin in melanosomes themselves. Despite melanins being manufactured by birds endogenously, the efficiency of melanogenesis can be noticeably limited by diet. As a result, the birds' plumage colouration is affected, which may entail consequences in social signalling.

Insight Into the Properties and Immunoregulatory Effect of Extracellular Vesicles Produced by Candida glabrata, Candida parapsilosis, and Candida tropicalis Biofilms.

Currently, non-albicans Candida species, including C. tropicalis, C. glabrata, and C. parapsilosis, are becoming an increasing epidemiological threat, predominantly due to the distinct collection of virulence mechanisms, as well as emerging resistance to antifungal drugs typically used in the treatment of candidiasis. They can produce biofilms that release extracellular vesicles (EVs), which are nanometric spherical structures surrounded by a lipid bilayer, transporting diversified biologically active cargo, that may be involved in intercellular communication, biofilm matrix production, and interaction with the host. In this work, we characterize the size and protein composition of these structures for three species of non-albicans Candida fungi forming biofilm, indicating considerable heterogeneity of the investigated population of fungal EVs. Examination of the influence of EVs on cytokine production by the human monocytic cell line THP-1 differentiated into macrophage-like cells revealed that the tested vesicles have a stimulating effect on the secretion of tumor necrosis factor α and interleukin 8, while they reduce the production of interleukin 10. This may indicate the proinflammatory nature of the effect of EVs produced by these species on the host immune cells. Moreover, it has been indicated that vesicles may be involved in C. tropicalis biofilm resistance to fluconazole and caspofungin. This reveals the important role of EVs not only in the physiology of C. tropicalis, C. glabrata, and C. parapsilosis fungi but also in the pathogenesis of infections associated with the production of fungal biofilm.

Dopamine D2 and Serotonin 5-HT1A Dimeric Receptor-Binding Monomeric Antibody scFv as a Potential Ligand for Carrying Drugs Targeting Selected Areas of the Brain.

Targeted therapy uses multiple ways of ensuring that the drug will be delivered to the desired site. One of these ways is an encapsulation of the drug and functionalization of the surface. Among the many molecules that can perform such a task, the present work focused on the antibodies of single-chain variable fragments (scFvs format). We studied scFv, which specifically recognizes the dopamine D2 and serotonin 5-HT1A receptor heteromers. The scFvD2-5-HT1A protein was analyzed biochemically and biologically, and the obtained results indicated that the antibody is properly folded and non-toxic and can be described as low-immunogenic. It is not only able to bind to the D2-5-HT1A receptor heteromer, but it also influences the cAMP signaling pathway and-when surfaced on nanogold particles-it can cross the blood-brain barrier in in vitro models. When administered to mice, it decreased locomotor activity, matching the effect induced by clozapine. Thus, we are strongly convinced that scFvD2-5-HT1A, which was a subject of the present investigation, is a promising targeting ligand with the potential for the functionalization of nanocarriers targeting selected areas of the brain.

Chemically induced protein cage assembly with programmable opening and cargo release.

Engineered protein cages are promising tools that can be customized for applications in medicine and nanotechnology. A major challenge is developing a straightforward strategy for endowing cages with bespoke, inducible disassembly. Such cages would allow release of encapsulated cargoes at desired timing and location. Here, we achieve such programmable disassembly using protein cages, in which the subunits are held together by different molecular cross-linkers. This modular system enables cage disassembly to be controlled in a condition-dependent manner. Structural details of the resulting cages were determined using cryo­electron microscopy, which allowed observation of bridging cross-linkers at intended positions. Triggered disassembly was demonstrated by high-speed atomic force microscopy and subsequent cargo release using an encapsulated Förster resonance energy transfer pair whose signal depends on the quaternary structure of the cage.

Antibodies Enhance the Suppressive Activity of Extracellular Vesicles in Mouse Delayed-Type Hypersensitivity.

Previously, we showed that mouse delayed-type hypersensitivity (DTH) can be antigen-specifically downregulated by suppressor T cell-derived miRNA-150 carried by extracellular vesicles (EVs) that target antigen-presenting macrophages. However, the exact mechanism of the suppressive action of miRNA-150-targeted macrophages on effector T cells remained unclear, and our current studies aimed to investigate it. By employing the DTH mouse model, we showed that effector T cells were inhibited by macrophage-released EVs in a miRNA-150-dependent manner. This effect was enhanced by the pre-incubation of EVs with antigen-specific antibodies. Their specific binding to MHC class II-expressing EVs was proved in flow cytometry and ELISA-based experiments. Furthermore, by the use of nanoparticle tracking analysis and transmission electron microscopy, we found that the incubation of macrophage-released EVs with antigen-specific antibodies resulted in EVs' aggregation, which significantly enhanced their suppressive activity in vivo. Nowadays, it is increasingly evident that EVs play an exceptional role in intercellular communication and selective cargo transfer, and thus are considered promising candidates for therapeutic usage. However, EVs appear to be less effective than their parental cells. In this context, our current studies provide evidence that antigen-specific antibodies can be easily used for increasing EVs' biological activity, which has great therapeutic potential.

Circadian Changes of Dendritic Spine Geometry in Mouse Barrel Cortex.

The circadian rhythmicity changes the density and shape of dendritic spines in mouse somatosensory barrel cortex, influencing their stability and maturation. In this study, we analyzed the main geometric parameters of dendritic spines reflecting the strength of synapses located on these spines under light/dark (12:12) and constant darkness conditions, in order to distinguish between endogenously regulated and light-driven parameters. Using morphological analysis of serial electron micrographs, as well as three-dimensional reconstructions, we found that the light induces elongation of single-synapse spine necks and increases in the diameter of double-synapse spine necks, increasing and decreasing the isolation of synapses from the parent dendrite, respectively. During the subjective night of constant darkness, we observed an enlargement of postsynaptic density area in inhibitory synapses and an increase in the number of polyribosomes inside double-synapse spines. The results show that both endogenous effect (circadian clock/locomotor activity) and light affect the morphological parameters of single- and double-synapse spines in the somatosensory cortex: light reduces the efficiency of excitatory synapses on single-synapse spines, increases the effect of synaptic transmission in double-synapse spines, and additionally masks the endogenous clock-driven enlargement of inhibitory synapses located on double-synapse spines. This indicates a special role of double-synapse spines and their inhibitory synapses in the regulation of synaptic transmission during both circadian and diurnal cycles in the mouse somatosensory cortex.

CRY-dependent plasticity of tetrad presynaptic sites in the visual system of Drosophila at the morning peak of activity and sleep.

Tetrad synapses are formed between the retina photoreceptor terminals and postsynaptic cells in the first optic neuropil (lamina) of Drosophila. They are remodelled in the course of the day and show distinct functional changes during activity and sleep. These changes result from fast degradation of the presynaptic scaffolding protein Bruchpilot (BRP) by Cryptochrome (CRY) in the morning and depend on BRP-170, one of two BRP isoforms. This process also affects the number of synaptic vesicles, both clear and dense-core, delivered to the presynaptic elements. In cry01 mutants lacking CRY and in brpΔ170, the number of synaptic vesicles is lower in the morning peak of activity than during night-sleep while in wild-type flies the number of synaptic vesicles is similar at these two time points. CRY may also set phase of the circadian rhythm in plasticity of synapses. The process of synapse remodelling stimulates the formation of clear synaptic vesicles in the morning. They carry histamine, a neurotransmitter in tetrad synapses and seem to be formed from glial capitate projections inside the photoreceptor terminals. In turn dense-core vesicles probably carry synaptic proteins building the tetrad presynaptic element.