Melatonin Modulates Cell Cycle Dynamics and Promotes Hippocampal Cell Proliferation After Ischemic Injury in Neonatal Rats.

Promoting neural cell proliferation may represent an important strategy for enhancing brain repair after developmental brain injury. The present study aimed to assess the effects of melatonin on cell proliferation after an ischemic injury in the developing hippocampus, focusing on cell cycle dynamics. After in vivo neonatal hypoxia-ischemia (HI), hippocampal cell cycle dynamics were assessed by flow cytometry, together with histological evaluation of dentate gyrus cellularity and proliferation. Melatonin significantly increased the number of proliferating cells in the G2/M phase as well as the proliferating cell nuclear antigen (PCNA) and doublecortin (DCX) labeling reduced by HI. In vivo BrdU labeling revealed a higher BrdU-positivity in the dentate gyrus of ischemic rats treated with melatonin, an effect followed by increased cellularity and preserved hippocampal tissue integrity. These results indicate that the protective effect of melatonin after ischemic injury in neonatal rats may rely on the modulation of cell cycle dynamics of newborn hippocampal cells and increased cell proliferation.

Flow cytometric analysis of hepatopancreatic cells from Armadillidium vulgare highlights terrestrial isopods as efficient environmental bioindicators in ex vivo settings.

Several studies have reported the high bioindication capacity of Isopoda (Crustacea, Oniscidea), which is related to their important ability to accumulate contaminants, usefulness in soil ecotoxicology and bioindication activities. Any change in the isopod population, diversity and life cycle can indicate relevant pollution levels. The analysis of target tissues, such as the hepatopancreas, is another emerging approach (from a cytologic/histological level) to detect contaminant accumulation from different sources. In this study, tissue disaggregation procedures were optimised in the hepatopancreas, and flow cytometry (FC) was applied to detect cell viability and several cell functions. After disaggregation, two hepatopancreatic cell types, small (S) and big (B), were still recognisable: they differed in morphology and behaviour. The analyses were conducted for the first time on isopods from sites under different conditions of ecological disturbance through cytometric re-interpretation of ecological-environmental parameters. Significant differences in cell functional parameters were found, highlighting that isopod hepatopancreatic cells can be efficiently analysed by FC and represent standardisable, early biological indicators, tracing environmental-induced stress through cytologic/histologic analyses.

Clozapine suppresses NADPH oxidase activation, counteracts cytosolic H2O2, and triggers early onset mitochondrial dysfunction during adipogenesis of human liposarcoma SW872 cells.

Long-term treatment of schizophrenia with clozapine (CLZ), an atypical antipsychotic drug, is associated with an increased incidence of metabolic disorders mediated by poorly understood mechanisms. We herein report that CLZ, while slowing down the morphological changes and lipid accumulation occurring during SW872 cell adipogenesis, also causes an early (day 3) inhibition of the expression/nuclear translocation of CAAT/enhancer-binding protein ß and peroxisome proliferator-activated receptor γ. Under the same conditions, CLZ blunts NADPH oxidase-derived reactive oxygen species (ROS) by a dual mechanism involving enzyme inhibition and ROS scavenging. These effects were accompanied by hampered activation of the nuclear factor (erythroid-derived2)-like 2 (Nrf2)-dependent antioxidant responses compared to controls, and by an aggravated formation of mitochondrial superoxide. CLZ failed to exert ROS scavenging activities in the mitochondrial compartment but appeared to actively scavenge cytosolic H2O2 derived from mitochondrial superoxide. The early formation of mitochondrial ROS promoted by CLZ was also associated with signs of mitochondrial dysfunction. Some of the above findings were recapitulated using mouse embryonic fibroblasts. We conclude that the NADPH oxidase inhibitory and cytosolic ROS scavenging activities of CLZ slow down SW872 cell adipogenesis and suppress their Nrf2 activation, an event apparently connected with increased mitochondrial ROS formation, which is associated with insulin resistance and metabolic syndrome. Thus, the cellular events characterised herein may help to shed light on the more detailed molecular mechanisms explaining some of the adverse metabolic effects of CLZ.

Modulation of the Circulating Extracellular Vesicles in Response to Different Exercise Regimens and Study of Their Inflammatory Effects.

Exercise-released extracellular vesicles (EVs) are emerging as a novel class of exerkines that promotes systemic beneficial effects. However, slight differences in the applied exercise protocols in terms of mode, intensity and duration, as well as the need for standardized protocols for EV isolation, make the comparison of the studies in the literature extremely difficult. This work aims to investigate the EV amount and EV-associated miRNAs released in circulation in response to different physical exercise regimens. Healthy individuals were subjected to different exercise protocols: acute aerobic exercise (AAE) and training (AT), acute maximal aerobic exercise (AMAE) and altitude aerobic training (AAT). We found a tendency for total EVs to increase in the sedentary condition compared to trained participants following AAE. Moreover, the cytofluorimetric analysis showed an increase in CD81+/SGCA+/CD45- EVs in response to AAE. Although a single bout of moderate/maximal exercise did not impact the total EV number, EV-miRNA levels were affected as a result. In detail, EV-associated miR-206, miR-133b and miR-146a were upregulated following AAE, and this trend appeared intensity-dependent. Finally, THP-1 macrophage treatment with exercise-derived EVs induced an increase of the mRNAs encoding for IL-1ß, IL-6 and CD163 using baseline and immediately post-exercise EVs. Still, 1 h post-exercise EVs failed to stimulate a pro-inflammatory program. In conclusion, the reported data provide a better understanding of the release of circulating EVs and their role as mediators of the inflammatory processes associated with exercise.

Extracellular Vesicles as New Players in Drug Delivery: A Focus on Red Blood Cells-Derived EVs.

The article is divided into several sections, focusing on extracellular vesicles' (EVs) nature, features, commonly employed methodologies and strategies for their isolation/preparation, and their characterization/visualization. This work aims to give an overview of advances in EVs' extensive nanomedical-drug delivery applications. Furthermore, considerations for EVs translation to clinical application are summarized here, before focusing the review on a special kind of extracellular vesicles, the ones derived from red blood cells (RBCEVs). Generally, employing EVs as drug carriers means managing entities with advantageous properties over synthetic vehicles or nanoparticles. Besides the fact that certain EVs also reveal intrinsic therapeutic characteristics, in regenerative medicine, EVs nanosize, lipidomic and proteomic profiles enable them to pass biologic barriers and display cell/tissue tropisms; indeed, EVs engineering can further optimize their organ targeting. In the second part of the review, we focus our attention on RBCEVs. First, we describe the biogenesis and composition of those naturally produced by red blood cells (RBCs) under physiological and pathological conditions. Afterwards, we discuss the current procedures to isolate and/or produce RBCEVs in the lab and to load a specific cargo for therapeutic exploitation. Finally, we disclose the most recent applications of RBCEVs at the in vitro and preclinical research level and their potential industrial exploitation. In conclusion, RBCEVs can be, in the near future, a very promising and versatile platform for several clinical applications and pharmaceutical exploitations.

Flow Cytometric Identification and Enumeration of Monocyte Subsets in Bovine and Water Buffalo Peripheral Blood.

Monocytes are innate immune system key players with pivotal roles during infection and inflammation. They migrate into tissues and differentiate into myeloid effect cells (macrophages, dendritic cells) which orchestrate inflammatory processes and are interfaces between the innate and adaptive immune responses. Their clinical relevance to health and disease of cattle (Bos taurus) and water buffalo (Bubalus bubalis), two of the most important livestock species, has been highlighted in physiologic (pregnancy) and pathologic (mastitis, metritis, and viral infections) conditions. The existence of three different monocyte subsets in cattle was established by flow cytometry (FC), as follows: classical (cM; CD14++ CD16-/low ), intermediate (intM; CD14++/+ CD16+ ), and non-classical (ncM; CD14-/low CD16++ ) monocytes. FC applications for studying the immune system of cattle and water buffalo still have significant limitations. In this article, we describe some practical approaches to overcome these limitations and, in particular, allow the identification and enumeration of cM, intM, and ncM subpopulations in cattle and buffalo peripheral blood. Indeed, we propose the new procedure lyse/wash/no-centrifugation (L/W/NC) that can be combined with the FC absolute counting procedures and can overcome specific issues of the lyse/no-wash protocols (L/NW). Finally, for the first time, we demonstrated the existence of cM, intM, and ncM monocyte subsets also in the water buffalo, showing some interesting differences with cattle, such as the bubaline cM are mainly CD14+/++ /CD16+ . These subtle differences may influence inflammatory disease regulation in, for example, mastitis and metritis. The upregulation of CD16 expression on cM may reveal different monocyte priming, leading to different functional features of macrophages/dendritic cells in tissues after infection. © 2023 Wiley Periodicals LLC. Basic Protocol: Absolute count of cM, intM, and ncM without compensation Alternate Protocol: Absolute count of cM, intM, and ncM for single laser platform Support Protocol 1: In-house monoclonal antibody labeling using a Pacific Blue™ kit Support Protocol 2: In-house monoclonal antibody labeling using an Alexa Fluor® 647 kit Support Protocol 3: Titration of fluorochrome-conjugated antibodies.

Basophil Activation Test with Different Polyethylene Glycols in Patients with Suspected PEG Hypersensitivity Reactions.

Allergic reactions to COVID-19 vaccine components are rare but should be considered. Polyethylene glycol (PEG) is responsible for anaphylaxis in mRNA vaccines. Skin tests have been used in the allergological work-up programs for COVID-19 vaccine evaluation. However, the reproducibility of the skin prick test is time-dependent and the reactivity declines over time. Therefore, we combined the administration of the skin tests with the basophil activation test (BAT) using PEG2000, PEG4000 and DMG-PEG2000, where the BAT was considered positive when the percentage of activated basophils was higher than 6%, 5% and 6.5%, for PEG 4000, PEG2000 and DMG-PEG2000, respectively. To this end, among the subjects that underwent allergy counseling at the Allergy Unit of our Institution during the 2020/2021 vaccination campaign, 13 patients had a suggested medical history of PEG/drug hypersensitivity and were enrolled together with 10 healthy donors. Among the enrolled patients 2 out of 13 tested patients were positive to the skin test. The BAT was negative in terms of the percentages of activated basophils in all analyzed samples, but the stimulation index (SI) was higher than 2.5 in 4 out of 13 patients. These data evidenced that, when the SI is higher than 2.5, even in the absence of positivity to BAT, the BAT to PEG may be a useful tool to be coupled to skin tests to evidence even low-grade reactions.

Extracellular Vesicles as Players in the Anti-Inflammatory Inter-Cellular Crosstalk Induced by Exercise Training.

Extracellular Vesicles (EVs) are circulating particles surrounded by a plasma membrane carrying a cargo consisting of proteins, lipids, RNAs, and DNA fragments, stemming from the cells from which they originated. EV factors (i.e., miRNAs) play relevant roles in intercellular crosstalk, both locally and systemically. As EVs increasingly gained attention as potential carriers for targeted genes, the study of EV effects on the host immune response became more relevant. It has been demonstrated that EVs regulate the host immune response, executing both pro- and anti-inflammatory functions. It is also known that physical exercise triggers anti-inflammatory effects. This review underlines the role of circulating EVs as players in the anti-inflammatory events associated with the regulation of the host's immune response to physical exercise.

Melatonin Attenuates Ischemic-like Cell Injury by Promoting Autophagosome Maturation via the Sirt1/FoxO1/Rab7 Axis in Hippocampal HT22 Cells and in Organotypic Cultures.

Dysfunctional autophagy is linked to neuronal damage in ischemia/reperfusion injury. The Ras-related protein 7 (Rab7), a member of the Rab family of small GTPases, appears crucial for the progression of the autophagic flux, and its activity is strictly interconnected with the histone deacetylase Silent information regulator 1 (Sirt1) and transcription factor Forkhead box class O1 (FoxO1). The present study assessed the neuroprotective role of melatonin in the modulation of the Sirt1/FoxO1/Rab7 axis in HT22 cells and organotypic hippocampal cultures exposed to oxygen-glucose deprivation followed by reoxygenation (OGD/R). The results showed that melatonin re-established physiological levels of autophagy and reduced propidium iodide-positive cells, speeding up autophagosome (AP) maturation and increasing lysosomal activity. Our study revealed that melatonin modulates autophagic pathways, increasing the expression of both Rab7 and FoxO1 and restoring the Sirt1 expression affected by OGD/R. In addition, the Sirt1 inhibitor EX-527 significantly reduced Rab7, Sirt1, and FoxO1 expression, as well as autolysosomes formation, and blocked the neuroprotective effect of melatonin. Overall, our findings provide, for the first time, new insights into the neuroprotective role of melatonin against ischemic injury through the activation of the Sirt1/FoxO1/Rab7 axis.

Development and in vitro characterization of a humanized scFv against fungal infections.

The resistance and the birth of new intrinsic and multidrug-resistant pathogenic species like C. auris is creating great concern in the antifungal world. Given the limited drug arsenal and the lack of effectiveness of the available compounds, there is an urgent need for innovative approaches. The murine mAb 2G8 was humanized and engineered in silico to develop a single-chain fragment variable (hscFv) antibody against ß-1,3-glucans which was then expressed in E. coli. Among the recombinant proteins developed, a soluble candidate with high stability and affinity was obtained. This selected protein is VL-linker-VH oriented, and it is characterized by the presence of two ubiquitin monomers at the N-terminus and a His tag at the C-terminus. This construct, Ub2-hscFv-His, guaranteed stability, solubility, efficient purification and satisfactory recovery of the recombinant product. HscFv can bind ß-1,3-glucans both as coated antigens and on C. auris and C. albicans cells similarly to its murine parental and showed long stability and retention of binding ability when stored at 4°, -20° and -80° C. Furthermore, it was efficient in enhancing the antifungal activity of drugs caspofungin and amphotericin B against C. auris. The use of biological drugs as antifungals is limited; here we present a promising hscFv which has the potential to be useful in combination with currently available antifungal drugs.

A microfluidic approach to fabricate sucrose decorated liposomes with increased uptake in breast cancer cells.

Developing targeted drug delivery systems is an urgent need to decrease the side effects and increase the drug's efficiency. Most cancer cells show an increased sugar consumption compared to healthy cells due to the deregulation of sugar transporters. Consequently, liposomes, as a biocompatible nanocarrier, could be surface decorated by sugars to enhance drug targeting into cancer cells. Our work outlines a new strategy to easily manufacture sucrose decorated liposomes using sucrose stearate, a biocompatible and biodegradable non-ionic surfactant, with a scalable microfluidic approach. Sucrose decorated liposomes were loaded with berberine hydrochloride, a well-known phytochemical compound to investigate its effects on triple-negative breast cancer cells (MDA-MB-231). Using the microfluidic manufacturing system, we prepared berberine-loaded liposomes using a mixture of phosphatidylcholine and cholesterol with and without sucrose stearate with a size up to 140 nm and narrow polydispersity. Stability was confirmed for 90 days, and the in vitro release profile was evaluated. The formulations showed acceptable in vitro biocompatibility and significantly higher anti-proliferative effect on MDA-MB-231 cell line. These results have been confirmed by an increased uptake evaluated by flow cytometry and confocal microscopy. Taken together, our findings represent an innovative, easy, and scalable approach to obtain sugar decorated liposomal formulations without any surface-chemistry reactions. They can be potentially used as an anticancer targeted drug delivery system.

Ceramide Aminoethylphosphonate as a New Molecular Target for Pore-Forming Aegerolysin-Based Protein Complexes.

Ostreolysin A6 (OlyA6) is a 15 kDa protein produced by the oyster mushroom (Pleurotus ostreatus). It belongs to the aegerolysin family of proteins and binds with high affinity to the insect-specific membrane sphingolipid, ceramide phosphoethanolamine (CPE). In concert with its partnering protein with the membrane-attack-complex/perforin domain, pleurotolysin B (PlyB), OlyA6 can form bicomponent 13-meric transmembrane pores in artificial and biological membranes containing the aegerolysin lipid receptor, CPE. This pore formation is the main underlying molecular mechanism of potent and selective insecticidal activity of OlyA6/PlyB complexes against two economically important coleopteran plant pests: the western corn rootworm and the Colorado potato beetle. In contrast to insects, the main sphingolipid in cell membranes of marine invertebrates (i.e., molluscs and cnidarians) is ceramide aminoethylphosphonate (CAEP), a CPE analogue built on a phosphono rather than the usual phosphate group in its polar head. Our targeted lipidomic analyses of the immune cells (hemocytes) of the marine bivalve, the mussel Mytilus galloprovincialis, confirmed the presence of 29.0 mol% CAEP followed by 36.4 mol% of phosphatidylcholine and 34.6 mol% of phosphatidylethanolamine. Further experiments showed the potent binding of OlyA6 to artificial lipid vesicles supplemented with mussel CAEP, and strong lysis of these vesicles by the OlyA6/PlyB mixture. In Mytilus haemocytes, short term exposure (max. 1 h) to the OlyA6/PlyB mixture induced lysosomal membrane destabilization, decreased phagocytic activity, increased Annexin V binding and oxyradical production, and decreased levels of reduced glutathione, indicating rapid damage of endo-lysosomal and plasma membranes and oxidative stress. Our data suggest CAEP as a novel high-affinity receptor for OlyA6 and a target for cytolytic OlyA6/PlyB complexes.

3ß-Hydroxy-5ß-hydroxy-B-norcholestane-6ß-carboxaldehyde (SEC-B) Induces Proinflammatory Activation of Human Endothelial Cells Associated with Nitric Oxide Production and Endothelial Nitric Oxide Synthase/Caveolin-1 Dysregulation.

Oxysterols are a family of 27-carbon cholesterol oxidation derivatives found in low-density lipoproteins (LDLs) and atherosclerotic plaques where they trigger several biological responses involved in the initiation and progression of atherosclerosis. Several pieces of evidence suggest that oxysterols contribute to endothelial dysfunction (ED) due to their ability to alter membrane fluidity and cell permeability leading to inflammation, oxidative stress and apoptosis. The present study aimed to investigate the molecular events occurring in human microvascular endothelial cells (HMEC-1) in response to autoxidation-generated 3ß-hydroxy-5ß-hydroxy-B-norcholestane-6ß-carboxaldehyde (SEC-B) exposure. Our results highlight that SEC-B rapidly activates HMEC-1 by inducing oxidative stress, nitric oxide (NO) production and pro-inflammatory cytokine release. Exposure to SEC-B up to 24 h results in persistent accumulation of the vasodilator NO paralleled by an upregulation of the endothelial nitric oxide synthase (eNOS) enzyme and downregulation of Caveolin-1 (Cav-1) protein levels. Moreover, reduced expression and extracellular release of the vasoconstrictor factor endothelin-1 (ET-1) are observed. Furthermore, SEC-B stimulates the expression of the cytokines interleukin-6 (IL-6) and tumor necrosis factor-like weak inducer of apoptosis (TWEAK). This proinflammatory state leads to increased monocyte recruitment on activated HMEC-1 cells. Our findings add new knowledge on the role of SEC-B in ED and further support its potential implication in atherosclerosis.

Automated-Mechanical Procedure Compared to Gentle Enzymatic Tissue Dissociation in Cell Function Studies.

The first step to obtain a cellular suspension from tissues is the disaggregation procedure. The cell suspension method has to provide a representative sample of the different cellular subpopulations and to maximize the number of viable functional cells. Here, we analyzed specific cell functions in cell suspensions from several rat tissues obtained by two different methods, automated-mechanical and enzymatic disaggregation. Flow cytometric, confocal, and ultrastructural (TEM) analyses were applied to the spleen, testis, liver and other tissues. Samples were treated by an enzymatic trypsin solution or processed by the Medimachine II (MMII). The automated-mechanical and enzymatic disaggregation procedures have shown to work similarly in some tissues, which displayed comparable amounts of apoptotic/necrotic cells. However, cells obtained by the enzyme-free Medimachine II protocols show a better preservation lysosome and mitochondria labeling, whereas the enzymatic gentle dissociation appears to constantly induce a lower amount of intracellular ROS; nevertheless, lightly increased ROS can be recognized as a complimentary signal to promote cell survival. Therefore, MMII represents a simple, fast, and standardized method for tissue processing, which allows to minimize bias arising from the operator's ability. Our study points out technical issues to be adopted for specific organs and tissues to obtain functional cells.

Fluorescent Silica Nanoparticles Targeting Mitochondria: Trafficking in Myeloid Cells and Application as Doxorubicin Delivery System in Breast Cancer Cells.

Fluorescent silica nanoparticles (SiNPs) appear to be a promising imaging platform, showing a specific subcellular localization. In the present study, we first investigated their preferential mitochondrial targeting in myeloid cells, by flow cytometry, confocal microscopy and TEM on both cells and isolated mitochondria, to acquire knowledge in imaging combined with therapeutic applications. Then, we conjugated SiNPs to one of the most used anticancer drugs, doxorubicin (DOX). As an anticancer agent, DOX has high efficacy but also an elevated systemic toxicity, causing multiple side effects. Nanostructures are usually employed to increase the drug circulation time and accumulation in target tissues, reducing undesired cytotoxicity. We tested these functionalized SiNPs (DOX-NPs) on breast cancer cell line MCF-7. We evaluated DOX-NP cytotoxicity, the effect on the cell cycle and on the expression of CD44 antigen, a molecule involved in adhesion and in tumor invasion, comparing DOX-NP to free DOX and stand-alone SiNPs. We found a specific ability to release a minor amount of CD44+ extracellular vesicles (EVs), from both CD81 negative and CD81 positive pools. Modulating the levels of CD44 at the cell surface in cancer cells is thus of great importance for disrupting the signaling pathways that favor tumor progression.

Extracellular Vesicles from Campylobacter jejuni CDT-Treated Caco-2 Cells Inhibit Proliferation of Tumour Intestinal Caco-2 Cells and Myeloid U937 Cells: Detailing the Global Cell Response for Potential Application in Anti-Tumour Strategies.

Cytolethal distending toxin (CDT) is produced by a range of Gram-negative pathogenic bacteria such as Campylobacter jejuni. CDT represents an important virulence factor that is a heterotrimeric complex composed of CdtA, CdtB, and CdtC. CdtA and CdtC constitute regulatory subunits whilst CdtB acts as the catalytic subunit exhibiting phosphatase and DNase activities, resulting in cell cycle arrest and cell death. Extracellular vesicle (EV) secretion is an evolutionarily conserved process that is present throughout all kingdoms. Mammalian EVs play important roles in regular cell-to-cell communications but can also spread pathogen- and host-derived molecules during infections to alter immune responses. Here, we demonstrate that CDT targets the endo-lysosomal compartment, partially evading lysosomal degradation and exploiting unconventional secretion (EV release), which is largely involved in bacterial infections. CDT-like effects are transferred by Caco-2 cells to uninfected heterologous U937 and homologous Caco-2 cells. The journey of EVs derived from CDT-treated Caco-2 cells is associated with both intestinal and myeloid tumour cells. EV release represents the primary route of CDT dissemination, revealing an active toxin as part of the cargo. We demonstrated that bacterial toxins could represent suitable tools in cancer therapy, highlighting both the benefits and limitations. The global cell response involves a moderate induction of apoptosis and autophagic features may play a protective role against toxin-induced cell death. EVs from CDT-treated Caco-2 cells represent reliable CDT carriers, potentially suitable in colorectal cancer treatments. Our data present a potential bacterial-related biotherapeutic supporting a multidrug anticancer protocol.

The effects of Acyclovir administration to NCI-H1975 non-small cell lung cancer cells.

The biochemical mechanisms by which the antiviral drug Acyclovir (ACV) may induce anticancer effects even without detecting human herpesviruses (HHVs) are still poorly understood. Herein, we investigated for the first time how NCI-H1975 non-small cell lung cancer cells responded in vitro to ACV administration by exploring mitochondrial damage and apoptosis induction. We confirmed ACV ability to cause the inhibition of cancer cell growth even without detecting intracellular HHVs; the drug also significantly inhibited the colony formation capacity of NCI-H1975 cells. Cell cycle analysis revealed an increase of the sub-G1 hypodiploid peak after ACV treatment; the activation of caspase-3 and the presence of DNA laddering sustained the capacity of the drug to induce apoptotic cell death. Regarding mitochondrial toxicity, a reduction of mitochondrial membrane potential, altered mitochondrial size and shape, and mtDNA damage were found after ACV administration. Furthermore, an increment of intracellular reactive oxygen species levels as well as the upregulation of NudT3 involved in DNA repair mechanisms were observed. Altogether, these findings suggest that mitochondria may be possible initial targets and/or sites of ACV cytotoxicity within cancer cells in the absence of intracellular HHVs.

Characterization of a fluorescent 1,8-naphthalimide-functionalized PAMAM dendrimer and its Cu(ii) complexes as cytotoxic drugs: EPR and biological studies in myeloid tumor cells.

The activity and interacting ability of a polyamidoamine (PAMAM) dendrimer modified with 4-N-methylpiperazine-1,8-naphthalimide units (termed D) and complexed by Cu(ii) ions, towards healthy and cancer cells were studied. Comparative electron paramagnetic resonance (EPR) studies of the Cu(ii)-D complex are presented: coordination mode, chemical structure, flexibility and stability of these complexes, in the absence and presence of myeloid cancer cells and peripheral blood mononuclear cells (PBMC). The interactions of Cu(ii) ions in the biological media at different equilibrium times were studied, highlighting different stability and interacting conditions with the cells. Furthermore, flow cytometry and confocal analysis, trace the peculiar properties of the dendrimers in PBMC and U937 cells. Indeed, a new probe (Fly) was used as a potential fluorescent tool for biological imaging of Cu(ii). The study highlights that dendrimer and, mainly, the Cu(ii) metallodendrimer are cytotoxic agents for the cells, specifically for U937 tumor cells, inducing mitochondrial dysfunction, ROS increase and lysosome involvement. The metallodendrimer shows antitumor selectivity, fewer affecting healthy PBMC, inducing a massive apoptotic cell death on U937 cells, in line with the high stability of this complex, as verified by EPR studies. The results underline the potentiality of this metallodendrimer to be used as anticancer drug.

A new humanized antibody is effective against pathogenic fungi in vitro.

Invasive fungal infections mainly affect patients undergoing transplantation, surgery, neoplastic disease, immunocompromised subjects and premature infants, and cause over 1.5 million deaths every year. The most common fungi isolated in invasive diseases are Candida spp., Cryptococcus spp., and Aspergillus spp. and even if four classes of antifungals are available (Azoles, Echinocandins, Polyenes and Pyrimidine analogues), the side effects of drugs and fungal acquired and innate resistance represent the major hurdles to be overcome. Monoclonal antibodies are powerful tools currently used as diagnostic and therapeutic agents in different clinical contexts but not yet developed for the treatment of invasive fungal infections. In this paper we report the development of the first humanized monoclonal antibody specific for ß-1,3 glucans, a vital component of several pathogenic fungi. H5K1 has been tested on C. auris, one of the most urgent threats and resulted efficient both alone and in combination with Caspofungin and Amphotericin B showing an enhancement effect. Our results support further preclinical and clinical developments for the use of H5K1 in the treatment of patients in need.

Lock, Stock and Barrel: Role of Renin-Angiotensin-Aldosterone System in Coronavirus Disease 2019.

Since the end of 2019, the medical-scientific community has been facing a terrible pandemic caused by a new airborne viral agent known as SARS-CoV2. Already in the early stages of the pandemic, following the discovery that the virus uses the ACE2 cell receptor as a molecular target to infect the cells of our body, it was hypothesized that the renin-angiotensin-aldosterone system was involved in the pathogenesis of the disease. Since then, numerous studies have been published on the subject, but the exact role of the renin-angiotensin-aldosterone system in the pathogenesis of COVID-19 is still a matter of debate. RAAS represents an important protagonist in the pathogenesis of COVID-19, providing the virus with the receptor of entry into host cells and determining its organotropism. Furthermore, following infection, the virus is able to cause an increase in plasma ACE2 activity, compromising the normal function of the RAAS. This dysfunction could contribute to the establishment of the thrombo-inflammatory state characteristic of severe forms of COVID-19. Drugs targeting RAAS represent promising therapeutic options for COVID-19 sufferers.