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Naltrexone (NTX), a homolog of the opiate antidote naloxone, is an orally active long-acting general opioid receptor antagonist used in the treatment of opiate dependence. NTX is also found to relieve craving for alcohol and is one of few FDA-approved medications for treatment of alcohol use disorder (AUD). While it was early on established that NTX acts by blocking the binding of endogenous opioid peptide ligands released by alcohol, experimental evidence emerged that could not be fully accounted for by this explanation alone, suggesting that NTX may have additional modes of action. Mu- and kappa-opioid receptors (MOP and KOP, respectively) are structurally related G-protein-coupled receptors (GPCRs), but they are anatomically differently distributed and functionally distinct, often mediating opposite responses, with MOP typically promoting euphoria and reward, while KOP is associated with dysphoria and aversive states. While the actions of NTX on MOP are extensively characterized, the interactions with KOP are not. Here, we used sensitive fluorescence-based methods with single-molecule sensitivity to study in live cells the influence of alcohol (ethanol, EtOH) on KOP and the interaction between KOP and NTX. Our data show that alcohol, at relevant concentrations (10-40 mM), alters KOP interactions with the lipid environment in the plasma membrane. The counteracting effects of NTX are exerted by both its canonical action on KOP and its hitherto unrevealed effects on the lateral dynamics and organization of lipids in the plasma membrane. The KOP-specific antagonist LY2444296, in clinical trial for major depressive disorder (MDD), blocks KOP but does not show the full action profile of NTX. The therapeutic effect of NTX treatment in AUD may in part be due to direct actions on KOP and in part due to its effect on the surrounding lipid environment., Competing Interests: Competing interests: The authors declare no competing interests. Ethics approval and consent to participate: All experiments were done in the laboratory and did not involve humans or animals. The experiments did not involve tissues from humans or other vertebrates. All experiments were done in accordance with scientific standards., (© 2024. The Author(s).)

RBCs deformability plays a crucial role in maintaining proper blood flow and oxygen delivery throughout the body. Conventional ektacytometry fails to differentiate between variations in deformability of RBC subpopulations as the averaging measurement process obscures these differences. In this study, we introduced an approach that integrates label-free optics-based techniques (flow cytometry, phase-contrast, and two-photon excitation fluorescent microscopy) with ektacytometry to evaluate subpopulations that exhibit decreased RBCs deformability upon an in vitro oxidation using 0.5 mM TBHP, as a low-level oxidative agent. We found that flow cytometry can easily detect rigidified and oxidized subpopulations based on FSC/SSC light distribution, as well as RBCs fluorescence intensity and peak area likely originating from hemoglobin photo and/or degradation products. Two-photon excitation fluorescence microscopy proved altered morphology and spatial location of fluorescence intensity signal near the membrane of oxidized RBCs, when compared to control RBCs, indicating a link with the reduced deformability. The proposed label-free optics-based methodology, which combines established techniques with more sophisticated microscopy, emerges as a promising tool for detecting mechano-biological changes in different RBC subpopulations induced by oxidative stress. The findings suggest potential applications in clinical practice for monitoring pathological conditions influenced by physical or environmental stress and as a quality control measure for stored RBCs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Hemoglobin (Hb), a life-sustaining and highly abundant erythrocyte protein, is not readily fluorescent. A few studies have already reported Two-Photon Excited Fluorescence (TPEF) of Hb, however, the mechanisms through which Hb becomes fluorescent upon interaction with ultrashort laser pulses are not completely understood. Here, we characterized photophysically this interaction on Hb thin film and erythrocytes using fluorescence spectroscopy upon single-photon/two-photon absorption, and UV-VIS single-photon absorption spectroscopy. A gradual increase of the fluorescence intensity, ending up with saturation, is observed upon prolonged exposure of Hb thin layer and erythrocytes to ultrashort laser pulses at 730 nm. When compared to protoporphyrin IX (PpIX) and oxidized Hb by H 2 O 2 , TPEF spectra from a thin Hb film and erythrocytes showed good mutual agreement, broad peaking at 550 nm, supporting hemoglobin undergoes degradation and that same fluorescent specie(s) originating from the heme moiety are generated. The uniform square shaped patterns of the fluorescent photoproduct exhibited the same level of the fluorescence intensity even after 12 weeks from the formation, indicating high photoproduct stability. We finally demonstrated the full potential of the formed Hb photoproduct with TPEF scanning microscopy towards spatiotemporally controlled micropatterning in HTF and single human erythrocyte labelling and tracking in the whole blood., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

There is an abundance of plastic materials used in the widest range of applications, such as packaging, machine parts, biomedical devices and components, etc. However, most materials used today are non-decomposable in the environment, producing a huge burden on ecosystems. The search for better, safer alternatives is still on. Here we present a detailed analysis of a simple, cheap, non-toxic, even edible, eco-friendly material, which can be easily manufactured, laser patterned and used for the fabrication of complex structures. The base substance is gelatin which is made photoresponsive by adding plasticizers and sensitizers. The resulting films were analyzed with respect to their optical, thermal and mechanical properties, which can be modified by a slight variation of chemical composition. The material is optimized for rapid laser-manufacturing of elastic microstructures (lenses, gratings, cantilevers, etc.) without any waste or residues. Overall, the material properties were tailored to increase photothermal responsivity, improve the surface quality and achieve material homogeneity, transparency and long-term stability (as verified using electron microscopy, infrared spectroscopy and differential scanning calorimetry).

High salt (HS) dietary intake leads to impaired vascular endothelium-dependent responses to various physiological stimuli, some of which are mediated by arachidonic acid (AA) metabolites. Transgenic Tff3 -/- gene knockout mice ( Tff3 -/- / C57BL/6N) have changes in lipid metabolism which may affect vascular function and outcomes of stroke. We aimed to study the effects of one week of HS diet (4% NaCl) on vascular function and stroke induced by transient occlusion of middle cerebral artery in Tff3 -/- and wild type (WT/C57BL/6N) mice. Flow-induced dilation (FID) of carotid artery was reduced in WT-HS mice, but not affected in Tff3 -/- - HS mice. Nitric oxide (NO) mediated FID. NO production was decreased with HS diet. On the contrary, acetylcholine-induced dilation was significantly decreased in Tff3 -/- mice on both diets and WT-HS mice. HS intake and Tff3 gene depletion affected the structural components of the vessels. Proteomic analysis revealed a significant effect of Tff3 gene deficiency on HS diet-induced changes in neuronal structural proteins and acute innate immune response proteins' expression and Tff3 depletion, but HS diet did not increase the stroke volume, which is related to proteome modification and upregulation of genes involved mainly in cellular antioxidative defense. In conclusion, Tff3 depletion seems to partially impair vascular function and worsen the outcomes of stroke, which is moderately affected by HS diet.

Magnetic resonance imaging (MRI) of superparamagnetic iron oxide-labeled cells can be used as a non-invasive technique to track stem cells after transplantation. The aim of this study was to (1) evaluate labeling efficiency of D-mannose-coated maghemite nanoparticles (D-mannose(γ-Fe 2 O 3 )) in neural stem cells (NSCs) in comparison to the uncoated nanoparticles, (2) assess nanoparticle utilization as MRI contrast agent to visualize NSCs transplanted into the mouse brain, and (3) test nanoparticle biocompatibility. D-mannose(γ-Fe 2 O 3 ) labeled the NSCs better than the uncoated nanoparticles. The labeled cells were visualized by ex vivo MRI and their localization subsequently confirmed on histological sections. Although the progenitor properties and differentiation of the NSCs were not affected by labeling, subtle effects on stem cells could be detected depending on dose increase, including changes in cell proliferation, viability, and neurosphere diameter. D-mannose coating of maghemite nanoparticles improved NSC labeling and allowed for NSC tracking by ex vivo MRI in the mouse brain, but further analysis of the eventual side effects might be necessary before translation to the clinic.