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1.
Int J Mol Sci ; 24(3)2023 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-36769362

RESUMO

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.


Assuntos
Vesículas Extracelulares , MicroRNAs , Humanos , MicroRNAs/genética , Macrófagos , Exercício Físico
2.
Biol Chem ; 403(3): 345-360, 2022 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-34883001

RESUMO

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.


Assuntos
Antineoplásicos , Dendrímeros , Neoplasias , Antineoplásicos/química , Antineoplásicos/farmacologia , Dendrímeros/química , Dendrímeros/metabolismo , Dendrímeros/farmacologia , Espectroscopia de Ressonância de Spin Eletrônica , Humanos , Leucócitos Mononucleares , Naftalimidas/farmacologia , Poliaminas
3.
Int J Mol Sci ; 23(22)2022 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-36430575

RESUMO

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.


Assuntos
Vesículas Extracelulares , MicroRNAs , Vesículas Extracelulares/metabolismo , MicroRNAs/metabolismo , Anti-Inflamatórios/farmacologia , Comunicação Celular , Exercício Físico
4.
Int J Mol Sci ; 24(1)2022 Dec 28.
Artigo em Inglês | MEDLINE | ID: mdl-36613943

RESUMO

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.


Assuntos
Toxinas Bacterianas , Campylobacter jejuni , Humanos , Toxinas Bacterianas/farmacologia , Toxinas Bacterianas/metabolismo , Células CACO-2 , Campylobacter jejuni/metabolismo , Proliferação de Células , Bactérias Gram-Negativas/metabolismo , Células U937
5.
Int J Mol Sci ; 23(6)2022 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-35328491

RESUMO

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.


Assuntos
Antineoplásicos , Neoplasias da Mama , Nanopartículas , Antineoplásicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Doxorrubicina/química , Portadores de Fármacos/química , Sistemas de Liberação de Medicamentos/métodos , Feminino , Humanos , Mitocôndrias , Células Mieloides , Nanopartículas/química , Dióxido de Silício/química
6.
Int J Mol Sci ; 23(23)2022 Nov 23.
Artigo em Inglês | MEDLINE | ID: mdl-36498922

RESUMO

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.


Assuntos
Anafilaxia , COVID-19 , Hipersensibilidade , Humanos , Teste de Degranulação de Basófilos , Vacinas contra COVID-19 , Reprodutibilidade dos Testes , Basófilos , Hipersensibilidade/diagnóstico , Testes Cutâneos , Polietilenoglicóis/efeitos adversos
7.
Biol Chem ; 402(10): 1225-1237, 2021 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-34090314

RESUMO

The fluorescent probes represent an important tool in the biological study, in fact characterization of cellular structures and organelles are an important tool-target for understanding the mechanisms regulating most biological processes. Recently, a series of polyamino-macrocycles based on 1,4,7,10-tetraazacyclododecane was synthesized, bearing one or two NBD units (AJ2NBD·4HCl) useful as sensors for metal cations and halides able to target and to detect apolar environment, as lipid membranes. In this paper, we firstly illustrate the chemical synthesis of the AJ2NBD probe, its electronic absorption spectra and its behavior regarding pH of the environment. Lack of any cellular toxicity and an efficient labelling on fresh, living cells was demonstrated, allowing the use of AJ2NBD in biological studies. In particular, this green fluorescent probe may represent a potential dye for the compartments involved in the endosomal/autophagic pathway. This research's field should benefit from the use of AJ2NBD as a vesicular tracer, however, to ensure the precise nature of vesicles/vacuoles traced by this new probe, other more specific tests are needed.


Assuntos
Corantes Fluorescentes , Lisossomos , Autofagia , Endossomos
8.
J Pineal Res ; 71(1): e12747, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34085316

RESUMO

Mitochondrial dysfunction is considered one of the hallmarks of ischemia/reperfusion injury. Mitochondria are plastic organelles that undergo continuous biogenesis, fusion, and fission. They can be transferred between cells through tunneling nanotubes (TNTs), dynamic structures that allow the exchange of proteins, soluble molecules, and organelles. Maintaining mitochondrial dynamics is crucial to cell function and survival. The present study aimed to assess the effects of melatonin on mitochondrial dynamics, TNT formation, and mitochondria transfer in HT22 cells exposed to oxygen/glucose deprivation followed by reoxygenation (OGD/R). The results showed that melatonin treatment during the reoxygenation phase reduced mitochondrial reactive oxygen species (ROS) production, improved cell viability, and increased the expression of PGC1α and SIRT3. Melatonin also preserved the expression of the membrane translocase proteins TOM20 and TIM23, and of the matrix protein HSP60, which are involved in mitochondrial biogenesis. Moreover, it promoted mitochondrial fusion and enhanced the expression of MFN2 and OPA1. Remarkably, melatonin also fostered mitochondrial transfer between injured HT22 cells through TNT connections. These results provide new insights into the effect of melatonin on mitochondrial network reshaping and cell survival. Fostering TNTs formation represents a novel mechanism mediating the protective effect of melatonin in ischemia/reperfusion injury.


Assuntos
Isquemia Encefálica/patologia , Estruturas da Membrana Celular/efeitos dos fármacos , Melatonina/farmacologia , Mitocôndrias/efeitos dos fármacos , Neurônios/ultraestrutura , Animais , Linhagem Celular , Hipocampo/efeitos dos fármacos , Hipocampo/patologia , Hipocampo/ultraestrutura , Camundongos , Mitocôndrias/metabolismo , Nanotubos , Neurônios/efeitos dos fármacos , Neurônios/patologia , Traumatismo por Reperfusão/patologia
9.
Mol Pharm ; 17(7): 2691-2702, 2020 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-32484691

RESUMO

Copper(II) carbosilane metallodendrimers are promising nanosized anticancer metallodrugs. The precise control on their design enables an accurate structure-to-activity study. We hypothesized that different structural features, such as the dendrimer generation and metal counterion, modulate the interaction with tumor cells, and subsequently, the effectivity and selectivity of the therapy. A computer-aided analysis of the electron paramagnetic resonance (EPR) spectra allowed us to obtain dynamical and structural details on the interactions over time between the dendrimers and the cells, the myeloid U937 tumor cells and peripheral blood mononuclear cells (PBMC). The intracellular fate of the metallodendrimers was studied through a complete in vitro evaluation, including cytotoxicity, cytostaticity, and sublethal effects regarding mitochondria function, lysosomal compartments, and autophagic organelle involvement. EPR results confirmed a higher membrane stabilization for chloride dendrimers and low generation complexes, which ultimately influence the metallodrug uptake and intracellular fate. The in vitro evaluation revealed that Cu(II) metallodendrimers are cytostatic and moderate cytotoxic agents for U937 tumor cells, inducing death processes through the mitochondria-lysosome axis as well as autophagic vacuole formation, while barely affecting healthy monocytes. The study provided valuable insight into the mechanism of action of these nanosized metallodrugs and relevant structural parameters affecting the activity.


Assuntos
Cobre/química , Citotoxinas/administração & dosagem , Dendrímeros/administração & dosagem , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Leucócitos Mononucleares/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Silanos/química , Autofagia , Linhagem Celular Tumoral , Citotoxinas/química , Citotoxinas/toxicidade , Dendrímeros/química , Dendrímeros/metabolismo , Dendrímeros/toxicidade , Humanos , Lisossomos/efeitos dos fármacos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/fisiologia
10.
Int J Mol Sci ; 21(6)2020 Mar 23.
Artigo em Inglês | MEDLINE | ID: mdl-32210050

RESUMO

The Gram-negative Campylobacter jejuni is a major cause of foodborne gastroenteritis in humans worldwide. The cytotoxic effects of Campylobacter have been mainly ascribed to the actions of the cytolethal distending toxin (CDT): it is mandatory to put in evidence risk factors for sequela development, such as reactive arthritis (ReA) and Guillain-Barré syndrome (GBS). Several researches are directed to managing symptom severity and the possible onset of sequelae. We found for the first time that rapamycin (RM) is able to largely inhibit the action of C. jejuni lysate CDT in U937 cells, and to partially avoid the activation of specific sub-lethal effects. In fact, we observed that the ability of this drug to redirect lysosomal compartment, stimulate ER-remodeling (highlighted by ER-lysosome and ER-mitochondria contacts), protect mitochondria network, and downregulate CD317/tetherin, is an important component of membrane microdomains. In particular, lysosomes are involved in the process of the reduction of intoxication, until the final step of lysosome exocytosis. Our results indicate that rapamycin confers protection against C. jejuni bacterial lysate insults to myeloid cells.


Assuntos
Antígeno 2 do Estroma da Médula Óssea/metabolismo , Campylobacter jejuni/fisiologia , Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Exocitose , Lisossomos/metabolismo , Biomarcadores , Morte Celular/efeitos dos fármacos , Proliferação de Células , Células Cultivadas , Retículo Endoplasmático/efeitos dos fármacos , Estresse do Retículo Endoplasmático , Exocitose/efeitos dos fármacos , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proibitinas , Transdução de Sinais/efeitos dos fármacos , Sirolimo/farmacologia , Células U937/metabolismo , Células U937/microbiologia
11.
Int J Mol Sci ; 21(21)2020 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-33114229

RESUMO

Extracellular vesicles (EVs) play a crucial role in the intercellular crosstalk. Mesenchymal stem cell-derived EVs (MSC-EVs), displaying promising therapeutic roles, contribute to the strong rationale for developing EVs as an alternative therapeutic option. EV analysis still represents one of the major issues to be solved in order to translate the use of MSC-EV detection in clinical settings. Even if flow cytometry (FC) has been largely applied for EV studies, the lack of consensus on protocols for FC detection of EVs generated controversy. Standard FC procedures, based on scatter measurements, only allows the detection of the "tip of the iceberg" of all EVs. We applied an alternative FC approach based on the use of a trigger threshold on a fluorescence channel. The EV numbers obtained by the application of the fluorescence triggering resulted significantly higher in respect to them obtained from the same samples acquired by placing the threshold on the side scatter (SSC) channel. The analysis of EV concentrations carried out by three different standardized flow cytometers allowed us to achieve a high level of reproducibility (CV < 20%). By applying the here-reported method highly reproducible results in terms of EV analysis and concentration measurements were obtained.


Assuntos
Vesículas Extracelulares/metabolismo , Citometria de Fluxo/métodos , Células-Tronco Mesenquimais/citologia , Animais , Células Cultivadas , Difusão Dinâmica da Luz , Separação Imunomagnética , Células-Tronco Mesenquimais/metabolismo
12.
Molecules ; 25(13)2020 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-32635622

RESUMO

The burden of neoplastic diseases is widely recognized as a severe cause of mortality. The clinical inadequacy of most anticancer therapeutics urgently prompted intense drug discovery efforts toward the identification of new chemical entities endowed with a potent and safe antitumor profile. In this scenario, targeting cancer cells apoptosis machinery has emerged as a relevant strategy, useful for tackling the emergence of drug resistance. On this basis, a small library of naturally inspired hybrid molecules was obtained by combining, through a click chemistry approach, "privileged" synthons such as curcumin scaffold and 1,2,3-triazole building block. Compound 1, bearing a para-fluoro phenyl moiety, showed low-micromolar potency against T acute lymphoblastic leukemia cell growth. More in-depth biologic studies demonstrated, for this analog, cell death-inducing properties associated with its capability to simultaneously activate both the receptor and the mitochondrial apoptosis cascades. This peculiar behavior offers promises for achieving an expanded anticancer effect, namely intense cytotoxic response coupled with reduced predisposition of chemoresistance insurgence. Altogether, this study allowed the identification of compound 1 as a lead compound worth to be progressed as an anticancer drug candidate.


Assuntos
Antineoplásicos/farmacologia , Apoptose , Curcumina/farmacologia , Leucemia de Células T/patologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Triazóis/química , Antineoplásicos/química , Proliferação de Células , Curcumina/química , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Leucemia de Células T/tratamento farmacológico , Estrutura Molecular , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Relação Estrutura-Atividade , Células Tumorais Cultivadas
13.
Bioconjug Chem ; 28(2): 524-538, 2017 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-28068077

RESUMO

Poly(propyleneimine) glycodendrimers fully modified with maltose units were administered to different cancer cell lines and their effect on cell viability was evaluated by using MTS assay and flow cytometry. The mechanism of dendrimer-cell interactions was investigated by the electron paramagnetic resonance (EPR) technique by using a new nitroxide-conjugated glycodendrimer. The nitroxide groups did not modify both the biological properties (cell viability and apoptosis degree) of the dendrimers in the presence of the cells and the dendrimer-cell interactions. Since this class of dendrimers is already known to be biocompatible for human healthy cells, noncancer cells such as human peripheral blood mononuclear cells (PBMCs) and macrophages were also treated with the glycodendrimer, and EPR spectra of the nitroxide-conjugated glycodendrimer were compared for cancer and noncancer cells. It was found that this dendrimer selectively affects the cell viability of tumor cells, while, surprisingly, PBMC proliferation is induced. Moreover, H-bond-active glycodendrimer-cell interactions were different for the different cancer cell lines and noncancer cells. The nitroxide-conjugated glycodendrimer was able to interact with the cell membrane and eventually cross it, getting in contact with cytosol antioxidants. This study helps to clarify the potential anticancer effect of this class of dendrimers opening to future applications of these macromolecules as new antitumor agents.


Assuntos
Antineoplásicos/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Dendrímeros/farmacologia , Óxidos de Nitrogênio/farmacologia , Polipropilenos/farmacologia , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Dendrímeros/química , Espectroscopia de Ressonância de Spin Eletrônica , Humanos , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/efeitos dos fármacos , Maltose/análogos & derivados , Maltose/farmacologia , Neoplasias/tratamento farmacológico , Óxidos de Nitrogênio/química , Polipropilenos/química
15.
Amino Acids ; 48(8): 1897-911, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-26724921

RESUMO

A growing body of scientific reports indicates that the role of creatine (Cr) in cellular biochemistry and physiology goes beyond its contribution to cell energy. Indeed Cr has been shown to exert multiple effects promoting a wide range of physiological responses in vitro as well as in vivo. Included in these, Cr promotes in vitro neuron and muscle cell differentiation, viability and survival under normal or adverse conditions; anabolic, protective and pro-differentiative effects have also been observed in vivo. For example Cr has been shown to accelerate in vitro differentiation of cultured C2C12 myoblasts into myotubes, where it also induces a slight but significant hypertrophic effect as compared to unsupplemented cultures; Cr also prevents the anti-differentiation effects caused by oxidative stress in the same cells. In trained adults, Cr increases the mRNA expression of relevant myogemic factors, protein synthesis, muscle strength and size, in cooperation with physical exercise. As to neurons and central nervous system, Cr favors the electrophysiological maturation of chick neuroblasts in vitro and protects them from oxidative stress-caused killing; similarly, Cr promotes the survival and differentiation of GABA-ergic neurons in fetal spinal cord cultures in vitro; in vivo, maternal Cr supplementation promotes the morpho-functional development of hippocampal neurons in rat offsprings. This article, which presents also some new experimental data, focuses on the trophic, pro-survival and pro-differentiation effects of Cr and examines the ensuing preventive and therapeutic potential in pathological muscle and brain conditions.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Creatina/farmacologia , Citoproteção/efeitos dos fármacos , Modelos Biológicos , Estresse Oxidativo/efeitos dos fármacos , Animais , Diferenciação Celular/fisiologia , Creatina/metabolismo , Citoproteção/fisiologia , Camundongos , Fibras Musculares Esqueléticas/metabolismo , Mioblastos Esqueléticos/metabolismo , Neurônios/metabolismo , Estresse Oxidativo/fisiologia
16.
Arch Microbiol ; 197(6): 773-80, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25868793

RESUMO

In Saccharomyces cerevisiae, a typical apoptotic phenotype is induced by some stress factors such as sugars, acetic acid, hydrogen peroxide, aspirin and age. Nevertheless, no data have been reported for apoptosis induced by puromycin, a damaging agent known to induce apoptosis in mammalian cells. We treated S. cerevisiae with puromycin to induce apoptosis and evaluated the percentage of dead cells by using Hoechst 33342 staining, transmission electron microscopy (TEM) and Annexin V flow cytometry (FC) analysis. Hoechst 33342 fluorescence images were processed to acquire parameters to use for multiparameter analysis [and perform a principal component analysis, (PCA)]. Cell viability was evaluated by Rhodamine 123 (Rh 123) and Acridine Orange microscope fluorescence staining. The results show puromycin-induced apoptosis in S. cerevisiae, and the PCA analysis indicated that the increasing percentage of apoptotic cells delineated a well-defined graph profile. The results were supported by TEM and FC. This study gives new insights into yeast apoptosis using puromycin as inducer agent, and PCA analysis may complement molecular analysis facilitating further studies to its detection.


Assuntos
Apoptose/efeitos dos fármacos , Inibidores da Síntese de Proteínas/farmacologia , Puromicina/farmacologia , Saccharomyces cerevisiae/efeitos dos fármacos , Citometria de Fluxo , Microscopia Eletrônica de Transmissão , Análise de Componente Principal , Saccharomyces cerevisiae/fisiologia
17.
J Pineal Res ; 56(4): 382-97, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24650016

RESUMO

Among the numerous functions of melatonin, the control of survival and differentiation of mesenchymal stem cells (MSCs) has been recently proposed. MSCs are a heterogeneous population of multipotent elements resident in tissues such as bone marrow, muscle, and adipose tissue, which are primarily involved in developmental and regeneration processes, gaining thus increasing interest for tissue repair and restoration therapeutic protocols. Receptor-dependent and receptor-independent responses to melatonin are suggested to occur in these cells. These involve antioxidant or redox-dependent functions of this indolamine as well as secondary effects resulting from autocrine and paracrine responses. Inflammatory cytokines and adipokines, proangiogenic/mitogenic stimuli, and other mediators that influence the differentiation processes may affect the survival and functional integrity of these mesenchymal precursor cells. In this scenario, melatonin seems to regulate signaling pathways that drive commitment and differentiation of MSC into osteogenic, chondrogenic, adipogenic, or myogenic lineages. Common pathways suggested to be involved as master regulators of these processes are the Wnt/ß-catenin pathway, the MAPKs and the, TGF-ß signaling. In this respect melatonin emerges a novel and potential modulator of MSC lineage commitment and adipogenic differentiation. These and other aspects of the physiological and pharmacological effects of melatonin as regulator of MSC are discussed in this review.


Assuntos
Adipogenia/fisiologia , Diferenciação Celular/fisiologia , Sistema de Sinalização das MAP Quinases/fisiologia , Melatonina/metabolismo , Células-Tronco Mesenquimais/metabolismo , Via de Sinalização Wnt/fisiologia , Adipocinas/metabolismo , Animais , Antioxidantes/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Células-Tronco Mesenquimais/citologia , Fator de Crescimento Transformador beta/metabolismo , Proteínas Wnt/metabolismo
18.
Mol Neurobiol ; 2024 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-38358438

RESUMO

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.

19.
Int J Biol Macromol ; 275(Pt 2): 133722, 2024 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-38977053

RESUMO

The valorization of discarded wool from dairy sheep breeding is a challenging issue. The most proposed strategies lie in the processing of keratin extracted from wool without reducing the molecular weight of the protein chains (the high molecular weight-HMW keratin). Here, the HMW keratin has been spun for the first time by solution blow spinning. A screening study of the process carried out with a 2-level full factorial design revealed that keratin filaments can be obtained by using the polyethylene oxide at 900 kDa, a 2 bar air pressure, and a 30 cm needle-collector distance. An annealing at 80 °C for 15 min, at pH 3.5 with citric acid contributes to increasing the viscosity of the keratin solutions thereby allowing the production of defect-free and water-stable filaments having diameters from 1 to 6 µm. A negligible toxic effect was observed after 24 and 48 h on HT29 epithelial cells and normal blood cells displayed behavior similar to the control demonstrating that the patches are hemocompatible. Therefore, the developed SBS process of keratin aqueous solutions could represent a valuable platform for developing patches that need to be blood-contacting and deposited in-situ.

20.
Sci Rep ; 14(1): 14610, 2024 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-38918594

RESUMO

Extracellular vesicles (EVs) are promising natural nanocarriers for the delivery of therapeutic agents. As with any other kind of cell, red blood cells (RBCs) produce a limited number of EVs under physiological and pathological conditions. Thus, RBC-derived extracellular vesicles (RBCEVs) have been recently suggested as next-generation delivery systems for therapeutic purposes. In this paper, we show that thanks to their unique biological and physicochemical features, RBCs can be efficiently pre-loaded with several kinds of molecules and further used to generate RBCEVs. A physical vesiculation method, based on "soft extrusion", was developed, producing an extremely high yield of cargo-loaded RBCEV mimetics. The RBCEVs population has been deeply characterized according to the new guidelines MISEV2023, showing great homogeneity in terms of size, biological features, membrane architecture and cargo. In vitro preliminary results demonstrated that RBCEVs are abundantly internalized by cells and exert peculiar biological effects. Indeed, efficient loading and delivery of miR-210 by RBCEVs to HUVEC has been proven, as well as the inhibition of a known mRNA target. Of note, the bench-scale process can be scaled-up and translated into clinics. In conclusion, this investigation could open the way to a new biomimetic platform for RNA-based therapies and/or other therapeutic cargoes useful in several diseases.


Assuntos
Eritrócitos , Vesículas Extracelulares , Células Endoteliais da Veia Umbilical Humana , MicroRNAs , Humanos , Vesículas Extracelulares/metabolismo , Eritrócitos/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Sistemas de Liberação de Medicamentos , Biomimética/métodos , RNA/metabolismo
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