Secretome of stem cells from human exfoliated deciduous teeth (SHED) and its extracellular vesicles improves keratinocytes migration, viability, and attenuation of H2 O2 -induced cytotoxicity.

Therapies for wound healing using the secretome and extracellular vesicles (EVs) of mesenchymal stem/stromal cells have been shown to be successful in preclinical studies. This study aimed to characterise the protein content of the secretome from stem cells from human exfoliated deciduous teeth (SHED) and analyse the in vitro effects of SHED-conditioned medium (SHED-CM) and SHED extracellular vesicles (SHED-EVs) on keratinocytes. EVs were isolated and characterised. The keratinocyte viability and migration of cells treated with SHED-EVs and conditioned medium (CM) were evaluated. An HaCaT apoptosis model induced by H2 O2 in vitro was performed with H2 O2 followed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and live/dead assays. Finally, the expression of vascular endothelial growth factor (VEGF) in keratinocytes treated with secretome and EVs was evaluated by immunofluorescence staining and confirmed with RT-qPCR. SHED-EVs revealed a cup-shaped morphology with expression of the classical markers for exosomes CD9 and CD63, and a diameter of 181 ± 87 nm. The internalisation of EVs by HaCaT cells was confirmed by fluorescence microscopy. Proteomic analysis identified that SHED-CM is enriched with proteins related to stress response and development, including cytokines (CXCL8, IL-6, CSF1, CCL2) and growth factors (IGF2, MYDGF, PDGF). The results also indicated that 50% CM and 0.4-0.6 µg/mL EVs were similarly efficient for improving keratinocyte viability, migration, and attenuation of H2 O2 -induced cytotoxicity. Additionally, expression of VEGF on keratinocytes increased when treated with SHED secretome and EVs. Furthermore, VEGF gene expression in keratinocytes increased significantly when treated with SHED secretome and EVs. Both SHED-CM and SHED-EVs may therefore be promising therapeutic tools for accelerating re-epithelialization in wound healing.

Temperature influence on NiFeMo nanoparticles magnetic properties and their viability in biomedical applications.

NiFeMo alloy nanoparticles were synthesized by co-precipitation in the presence of organic additives. Nanoparticles thermal evolution shows that there is a significant increase in the average size (from 28 to 60 nm), consolidating a crystalline structure of the same type as the Ni3 Fe phase but with lattice parameter a = 0.362 nm. Measurements of magnetic properties follow this morphological and structural evolution increasing saturation magnetization (Ms) by 578% and reducing remanence magnetization (Mr) by 29%. Cell viability assays on as-synthesized revealed that nanoparticles (NPs) are not cytotoxic up to a concentration of 0.4 µg/mL for both non-tumorigenic (fibroblasts and macrophages) and tumor cells (melanoma).

BDE-99 (2,2',4,4',5 - pentain polybrominated diphenyl ether) induces toxic effects in Oreochromis niloticus after sub-chronic and oral exposure.

PBDEs are toxic, lipophilic, hydrophobic, and persistent artificial chemicals, characterized by high physical and chemical stability. Although PBDEs are known to disturb hormone signaling, many effects of 2,2',4,4',5 - pentain polybrominated diphenyl ethers (BDE-99) in fish remain unclear. The current study investigates the effects of BDE-99 in Oreochromis niloticus where sixty-four juvenile fish were orally exposed to 0.294, 2.94, 29.4 ng g-1 of BDE-99, every 10 days, during 80 days. The results showed histopathological findings in liver and kidney, increasing acetylcholinesterase activity in muscle, disturbs in the antioxidant system in liver and brain and decreasing the plasmatic levels of vitellogenin in females. According to multivariate analysis (IBR), the higher doses are related to the interaction of oxidative and non-oxidative enzymes. The present study provided evidence of deleterious effects after sub-chronic exposure of BDE 99 to O. niloticus, increasing the knowledge about its risk of exposure in fish.

Functionalized Hydrogels for Cartilage Repair: The Value of Secretome-Instructive Signaling.

Cartilage repair has been a challenge in the medical field for many years. Although treatments that alleviate pain and injury are available, none can effectively regenerate the cartilage. Currently, regenerative medicine and tissue engineering are among the developed strategies to treat cartilage injury. The use of stem cells, associated or not with scaffolds, has shown potential in cartilage regeneration. However, it is currently known that the effect of stem cells occurs mainly through the secretion of paracrine factors that act on local cells. In this review, we will address the use of the secretome-a set of bioactive factors (soluble factors and extracellular vesicles) secreted by the cells-of mesenchymal stem cells as a treatment for cartilage regeneration. We will also discuss methodologies for priming the secretome to enhance the chondroregenerative potential. In addition, considering the difficulty of delivering therapies to the injured cartilage site, we will address works that use hydrogels functionalized with growth factors and secretome components. We aim to show that secretome-functionalized hydrogels can be an exciting approach to cell-free cartilage repair therapy.

Biological Synthesis of Low Cytotoxicity Silver Nanoparticles (AgNPs) by the Fungus Chaetomium thermophilum-Sustainable Nanotechnology.

Fungal biotechnology research has rapidly increased as a result of the growing awareness of sustainable development and the pressing need to explore eco-friendly options. In the nanotechnology field, silver nanoparticles (AgNPs) are currently being studied for application in cancer therapy, tumour detection, drug delivery, and elsewhere. Therefore, synthesising nanoparticles (NPs) with low toxicity has become essential in the biomedical area. The fungus Chaetomium thermophilum (C. thermophilum) was here investigated-to the best of our knowledge, for the first time-for application in the production of AgNPs. Transmission electronic microscopy (TEM) images demonstrated a spherical AgNP shape, with an average size of 8.93 nm. Energy-dispersive X-ray spectrometry (EDX) confirmed the presence of elemental silver. A neutral red uptake (NRU) test evaluated the cytotoxicity of the AgNPs at different inhibitory concentrations (ICs). A half-maximal concentration (IC50 = 119.69 µg/mL) was used to predict a half-maximal lethal dose (LD50 = 624.31 mg/kg), indicating a Global Harmonized System of Classification and Labelling of Chemicals (GHS) acute toxicity estimate (ATE) classification category of 4. The fungus extract showed a non-toxic profile at the IC tested. Additionally, the interaction between the AgNPs and the Balb/c 3T3 NIH cells at an ultrastructural level resulted in preserved cells structures at non-toxic concentrations (IC20 = 91.77 µg/mL), demonstrating their potential as sustainable substitutes for physical and chemically made AgNPs. Nonetheless, at the IC50, the cytoplasm of the cells was damaged and mitochondrial morphological alteration was evident. This fact highlights the fact that dose-dependent phenomena are involved, as well as emphasising the importance of investigating NPs' effects on mitochondria, as disruption to this organelle can impact health.

Pathological findings and morphologic correlation of the lungs of autopsied patients with SARS-CoV-2 infection in the Brazilian Amazon using transmission electron microscopy

Abstract INTRODUCTION: Electron microscopy (EM) is a rapid and effective tool that can be used to create images of a whole spectrum of virus-host interactions and, as such, has long been used in the discovery and description of viral mechanisms. METHODS: Electron microscopy was used to evaluate the pulmonary pathologies of postmortem lung sections from three patients who died from infection with SARS-associated coronavirus 2 (SARS-CoV-2), a new member of the Coronaviridae family. RESULTS: Diffuse alveolar damage (DAD) was predominant in all three patients. The early exudative stage was characterized principally by edema and extravasation of red blood cells into the alveolar space with injury to the alveolar epithelial cells; this was followed by detachment, apoptosis, and necrosis of type I and II pneumocytes. The capillaries exhibited congestion, exposure of the basement membrane from denuded endothelial cells, platelet adhesion, fibrin thrombi, and rupture of the capillary walls. The proliferative stage was characterized by pronounced proliferation of type II alveolar pneumocytes and multinucleated giant cells. The cytopathic effect of SARS-CoV-2 was observed both in degenerated type II pneumocytes and freely circulating in the alveoli, with components from virions, macrophages, lymphocytes, and cellular debris. CONCLUSIONS: Viral particles consistent with the characteristics of SARS-CoV-2 were observed mainly in degenerated pneumocytes, in the endothelium, or freely circulating in the alveoli. In the final stage of illness, the alveolar spaces were replaced by fibrosis.

DDX6 Helicase Behavior and Protein Partners in Human Adipose Tissue-Derived Stem Cells during Early Adipogenesis and Osteogenesis.

DDX6 helicase is an RNA-binding protein involved in different aspects of gene expression regulation. The roles played by DDX6 depend on the complexes associated with it. Here, for the first time, we characterize the protein complexes associated with DDX6 in human adipose tissue-derived stem cells (hASCs) and analyze the dynamics of this helicase under different conditions of translational activity and differentiation. The results obtained demonstrated that the DDX6 helicase is associated with proteins involved in the control of mRNA localization, translation and metabolism in hASCs. DDX6 complexes may also assemble into more complex structures, such as RNA-dependent granules, the abundance and composition of which change upon inhibited translational activity. This finding supports the supposition that DDX6 is possibly involved in the regulation of the mRNA life cycle in hASCs. Although there was no significant variation in the protein composition of these complexes during early adipogenic or osteogenic induction, there was a change in the distribution pattern of DDX6: the number of DDX6 granules per cell was reduced during adipogenesis and was enhanced during osteogenesis.

Visible-light reduced silver nanoparticles' toxicity in Allium cepa test system.

Silver nanoparticles (AgNPs) are widely used in consumer products due to their antibacterial property; however, their potential toxicity and release into the environment raises concern. Based on the limited understanding of AgNPs aggregation behavior, this study aimed to investigate the toxicity of uncoated (uc-AgNP) and coated with polyvinylpyrrolidone (PVP-AgNP), at low concentrations (0.5-100 ng/mL), under dark and visible-light exposure, using a plant test system. We exposed Allium cepa seeds to both types of AgNPs for 4-5 days to evaluate several toxicity endpoints. AgNPs did not cause acute toxicity (i.e., inhibition of seed germination and root development), but caused genotoxicity and biochemical alterations in oxidative stress parameters (lipid peroxidation) and activities of antioxidant enzymes (superoxide dismutase and catalase) in light and dark conditions. However, the light exposure decreased the rate of chromosomal aberration and micronuclei up to 5.60x in uc-AgNP and 2.01x in PVP-AgNP, and 2.69x in uc-AgNP and 3.70x in PVP-AgNP, respectively. Thus, light exposure reduced the overall genotoxicity of these AgNPs. In addition, mitotic index alterations and morphoanatomical changes in meristematic cells were observed only in the dark condition at the highest concentrations, demonstrating that light also reduces AgNPs cytotoxicity. The light-dependent aggregation of AgNPs may have reduced toxicity by reducing the uptake of these NPs by the cells. Our findings demonstrate that AgNPs can be genotoxic, cytotoxic and induce morphoanatomical and biochemical changes in A. cepa roots even at low concentrations, and that visible-light alters their aggregation state, and decreases their toxicity. We suggest that visible light can be an alternative treatment to remediate AgNP residues, minimizing their toxicity and environmental risks.

Cell cycle genes are downregulated after adipogenic triggering in human adipose tissue-derived stem cells by regulation of mRNA abundance.

The adipogenic process is characterized by the expression of adipocyte differentiation markers that lead to changes in cell metabolism and to the accumulation of lipid droplets. Moreover, during early adipogenesis, cells undergo a strong downregulation of translational activity with a decrease in cell size, proliferation and migration. In the present study, we identified that after 24 hours of adipogenic induction, human adipose tissue-derived stem cells (hASCs) undergo a G1-cell cycle arrest consistent with reduced proliferation, and this effect was correlated with a shift in polysome profile with an enrichment of the monosomal fraction and a reduction of the polysomal fraction. Polysome profiling analysis also revealed that this change in the monosomal/polysomal ratio was related to a strong downregulation of cell cycle and proliferation genes, such as cyclins and cyclin-dependent kinases (CDKs). Comparing total and polysome-associated mRNA sequencing, we also observed that this downregulation was mostly due to a reduction of cell cycle and proliferation transcripts via control of total mRNA abundance, rather than by translational control.

Downregulation of the protein synthesis machinery is a major regulatory event during early adipogenic differentiation of human adipose-derived stromal cells.

Commitment of adult stem cells involves the activation of specific gene networks regulated from transcription to protein synthesis. Here, we used ribosome profiling to identify mRNAs regulated at the translational level, through both differential association to polysomes and modulation of their translational rates. We observed that translational regulation during the differentiation of human adipose-derived stromal cells (hASCs, also known as adipose-derived mesenchymal stem cells), a subset of which are stem cells, to adipocytes was a major regulatory event. hASCs showed a significant reduction of whole protein synthesis after adipogenic induction and a downregulation of the expression and translational efficiency of ribosomal proteins. Additionally, focal adhesion and cytoskeletal proteins were downregulated at the translational level. This negative regulation of the essential biological functions of hASCs resulted in a reduction in cell size and the potential of hASCs to migrate. We analyzed whether the inactivation of key translation initiation factors was involved in this observed major repression of translation. We showed that there was an increase in the hypo phosphorylated forms of 4E-BP1, a negative regulator of translation, during early adipogenesis. Our results showed that extensive translational regulation occurred during the early stage of the adipogenic differentiation of hASCs.

The Protein Content of Extracellular Vesicles Derived from Expanded Human Umbilical Cord Blood-Derived CD133+ and Human Bone Marrow-Derived Mesenchymal Stem Cells Partially Explains Why both Sources are Advantageous for Regenerative Medicine.

Adult stem cells have beneficial effects when exposed to damaged tissue due, at least in part, to their paracrine activity, which includes soluble factors and extracellular vesicles (EVs). Given the multiplicity of signals carried by these vesicles through the horizontal transfer of functional molecules, human mesenchymal stem cell (hMSCs) and CD133+ cell-derived EVs have been tested in various disease models and shown to recover damaged tissues. In this study, we profiled the protein content of EVs derived from expanded human CD133+ cells and bone marrow-derived hMSCs with the intention of better understanding the functions performed by these vesicles/cells and delineating the most appropriate use of each EV in future therapeutic procedures. Using LC-MS/MS analysis, we identified 623 proteins for expanded CD133+-EVs and 797 proteins for hMSCs-EVs. Although the EVs from both origins were qualitatively similar, when protein abundance was considered, hMSCs-EVs and CD133+-EVs were different. Gene Ontology (GO) enrichment analysis in CD133+-EVs revealed proteins involved in a variety of angiogenesis-related functions as well proteins related to the cytoskeleton and highly implicated in cell motility and cellular activation. In contrast, when overrepresented proteins in hMSCs-EVs were analyzed, a GO cluster of immune response-related genes involved with immune response-regulating factors acting on phagocytosis and innate immunity was identified. Together our data demonstrate that from the point of view of protein content, expanded CD133+-EVs and hMSCs-EVs are in part similar but also sufficiently different to reflect the main beneficial paracrine effects widely reported in pre-clinical studies using expanded CD133+ cells and/or hBM-MSCs.

Manifestações eletrofisiológicas em adultos com HIV/AIDS submetidos e não-submetidos à terapia anti-retroviral / Electrophysiological manifestations in adults with HIV/AIDS submitted and not submitted to antiretroviral therapy

BACKGROUND: auditory evoked potentials (AEP) assess the neuroelectric activity on the auditory pathway -from the auditory nerve to the cerebral cortex - in response to an acoustic stimulus or event. Studies have demonstrated electrophysiological abnormalities in individuals with HIV/AIDS. AIM: to characterize the hearing electrophysiological manifestations in adults with HIV/AIDS by comparing the results obtained in the group exposed to antiretroviral therapy with those obtained in the group not exposed to such treatment. METHOD: electrophysiological evaluation of hearing (Auditory Brainstem Response - ABR, Auditory Middle Latency Reponse - AMLR and P300) was conducted in 56 individuals with HIV/AIDS: 24 participants composed group I (not exposed to antiretroviral treatment) and 32 participants composed group II (exposed to treatment). RESULTS: alterations in every AEP were observed in individuals with HIV/ AIDS, especially in the ABR. Indeed, the group exposed to antiretroviral treatment presented more alterations. CONCLUSION: individuals with HIV/AIDS may present alterations on the central and peripheral auditory nervous system. The group exposed to antiretroviral therapy presents more alterations on the brainstem auditory pathway.

TEMA: os potenciais evocados auditivos (PEA) avaliam a atividade neuroelétrica na via auditiva, desde o nervo auditivo até o córtex cerebral, em resposta a um estímulo ou evento acústico. Estudos demonstram anormalidades eletrofisiológicas em indivíduos com HIV/AIDS. OBJETIVO: caracterizar as manifestações eletrofisiológicas da audição em adultos com HIV/AIDS, comparando os resultados obtidos no grupo exposto a tratamento anti-retroviral com os obtidos no grupo não exposto a tratamento. MÉTODO: realizada avaliação eletrofisiológica da audição (PEATE, PEAML e P300) em 56 indivíduos portadores do HIV/AIDS, sendo 24 do Grupo I (não expostos ao tratamento anti-retroviral) e 32 do Grupo II (expostos ao tratamento). RESULTADOS: foram encontradas alterações em todos os PEA nos indivíduos com HIV/AIDS, principalmente no PEATE; sendo que neste, o grupo exposto ao tratamento antiretroviral apresentou mais alterações. CONCLUSÃO: indivíduos com HIV/AIDS podem apresentar alterações no sistema nervoso auditivo periférico e central, sendo que o grupo exposto a tratamento anti-retroviral apresenta mais alterações na via auditiva em tronco encefálico.

Avaliação auditiva na síndrome da imunodeficiência adquirida / Hearing evaluation in acquired immunodeficiency syndrome

OBJETIVO: Caracterizar as manifestações audiológicas em adultos com HIV/AIDS e comparar os resultados de indivíduos expostos e não expostos ao tratamento anti-retroviral. MÉTODOS: Foram submetidos à avaliação audiológica 56 indivíduos com HIV/AIDS de ambos os gêneros, entre 18 e 58 anos de idade, divididos em dois grupos: GEI - composto por 24 indivíduos não expostos ao tratamento anti-retroviral; e GEII - composto por 32 indivíduos expostos ao tratamento anti-retroviral. RESULTADOS: Verificou-se ocorrência de resultados alterados na avaliação audiológica em ambos os grupos, principalmente na audiometria de altas frequências. Em ambos os grupos a principal alteração encontrada foi a perda auditiva neurossensorial. O GEII apresentou mais resultados alterados em todos os procedimentos realizados e maior ocorrência de resultados sugestivos de alterações na orelha média, quando comparado ao GEI. CONCLUSÃO: Indivíduos com HIV/AIDS apresentam alterações auditivas periféricas, sendo estas mais acentuadas no grupo exposto ao tratamento anti-retroviral.

PURPOSE: To characterize audiological manifestations in adults with HIV/AIDS and to compare the results of subjects exposed and not exposed to antiretroviral treatment. METHODS: Fifty six individuals with HIV/AIDS of both genders, with ages between 18 and 58 years, were submitted to audiological evaluation. Subjects were divided into two groups: GEI - composed of 24 individuals not exposed to antiretroviral treatment; and GEII - composed of 32 individuals exposed to antiretroviral treatment. RESULTS: It was verified occurrence of altered results on audiological evaluation in both groups, mainly in the high frequencies audiometry. In both groups, the main alteration found was sensorineural hearing loss. The GEII showed more altered results in all procedures performed, and higher occurrence of findings suggestive of alterations in middle ear, when compared to GEI. CONCLUSION: Individuals with HIV/AIDS in this study had peripheral hearing impairment, more pronounced in the group exposed to antiretroviral treatment.