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1.
Cell Prolif ; 54(11): e13129, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34585454

RESUMO

OBJECTIVES: Conditioned medium (CM) from 2D cell culture can mitigate the weakened regenerative capacity of the implanted stem cells. However, the capacity of 3D CM to prime dental pulp stem cells (DPSCs) for pulp regeneration and its protein profile are still elusive. We aim to investigate the protein profile of CM derived from 3D tooth germs, and to unveil its potential for DPSCs-based pulp regeneration. MATERIALS AND METHODS: We prepared CM of 3D ex vivo cultured tooth germ organs (3D TGO-CM) and CM of 2D cultured tooth germ cells (2D TGC-CM) and applied them to prime DPSCs. Influences on cell behaviours and protein profiles of CMs were compared. In vivo pulp regeneration of CMs-primed DPSCs was explored using a tooth root fragment model on nude mice. RESULTS: TGO-CM enhanced DPSCs proliferation, migration, in vitro mineralization, odontogenic differentiation, and angiogenesis performances. The TGO-CM group generated superior pulp structures, more odontogenic cells attachment, and enhanced vasculature at 4 weeks post-surgery, compared with the TGC-CM group. Secretome analysis revealed that TGO-CM contained more odontogenic and angiogenic growth factors and fewer pro-inflammatory cytokines. Mechanisms leading to the differential CM profiles may be attributed to the cytokine-cytokine receptor interaction and PI3K-Akt signalling pathway. CONCLUSIONS: The unique secretome profile of 3D TGO-CM made it a successful priming cocktail to enhance DPSCs-based early pulp regeneration.


Assuntos
Meios de Cultivo Condicionados/metabolismo , Polpa Dentária/metabolismo , Regeneração/fisiologia , Células-Tronco/citologia , Dente/citologia , Animais , Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Células Cultivadas , Humanos , Camundongos Nus , Fosfatidilinositol 3-Quinases/metabolismo
2.
Int J Mol Sci ; 22(17)2021 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-34502120

RESUMO

Diabetes mellitus is a main risk factor for delayed fracture healing and fracture non-unions. Successful fracture healing requires stimuli from different immune cells, known to be affected in diabetics. Especially, application of mononuclear cells has been proposed to promote wound and fracture healing. Thus, aim was to investigate the effect of pre-/diabetic conditions on mononuclear cell functions essential to promote osteoprogenitor cell function. We here show that pre-/diabetic conditions suppress the expression of chemokines, e.g., CCL2 and CCL8 in osteoprogenitor cells. The associated MCP-1 and MCP-2 were significantly reduced in serum of diabetics. Both MCPs chemoattract mononuclear THP-1 cells. Migration of these cells is suppressed under hyperglycemic conditions, proposing that less mononuclear cells invade the site of fracture in diabetics. Further, we show that the composition of cytokines secreted by mononuclear cells strongly differ between diabetics and controls. Similar is seen in THP-1 cells cultured under hyperinsulinemia or hyperglycemia. The altered secretome reduces the positive effect of the THP-1 cell conditioned medium on migration of osteoprogenitor cells. In summary, our data support that factors secreted by mononuclear cells may support fracture healing by promoting migration of osteoprogenitor cells but suggest that this effect might be reduced in diabetics.


Assuntos
Meios de Cultivo Condicionados/metabolismo , Diabetes Mellitus/metabolismo , Consolidação da Fratura , Monócitos/metabolismo , Animais , Biomarcadores , Movimento Celular , Células Cultivadas , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Quimiocina CCL8/metabolismo , Quimiocinas/metabolismo , Quimiotaxia de Leucócito/imunologia , Humanos , Hiperglicemia/metabolismo , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Sistema de Sinalização das MAP Quinases , Monócitos/imunologia , Osteoblastos/metabolismo , Osteogênese , Células THP-1
3.
Sci Rep ; 11(1): 17282, 2021 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-34446785

RESUMO

Due to the frequency of biofilm-forming Staphylococcus aureus and Staphylococcus epidermidis in orthopedics, it is crucial to understand the interaction between the soluble factors produced by prokaryotes and their effects on eukaryotes. Our knowledge concerning the effect of soluble biofilm factors (SBF) and their virulence potential on osteogenic differentiation is limited to few studies, particularly when there is no direct contact between prokaryotic and eukaryotic cells. SBF were produced by incubating biofilm from S. aureus and S. epidermidis in osteogenic media. Osteoblasts of seven donors were included in this study. Our results demonstrate that the detrimental effects of these pathogens do not require direct contact between prokaryotic and eukaryotic cells. SBF produced by S. aureus and S. epidermidis affect the metabolic activity of osteoblasts. However, the effect of SBF derived from S. aureus seems to be more pronounced compared to that of S. epidermidis. The influence of SBF of S. aureus and S. epidermidis on gene expression of COL1A1, ALPL, BGLAP, SPP1, RUNX2 is bacteria-, patient-, concentration-, and incubation time dependent. Mineralization was monitored by staining the calcium and phosphate deposition and revealed that the SBF of S. epidermidis markedly inhibits calcium deposition; however, S. aureus shows a less inhibitory effect. Therefore, these new findings support the hypotheses that soluble biofilm factors affect the osteogenic processes substantially, particularly when there is no direct interaction between bacteria and osteoblast.


Assuntos
Biofilmes/crescimento & desenvolvimento , Diferenciação Celular/fisiologia , Osteoblastos/microbiologia , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/fisiologia , Staphylococcus epidermidis/fisiologia , Adulto , Idoso , Biofilmes/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Meios de Cultivo Condicionados/metabolismo , Meios de Cultivo Condicionados/farmacologia , Feminino , Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Pessoa de Meia-Idade , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteogênese/fisiologia , Especificidade da Espécie , Infecções Estafilocócicas/genética , Staphylococcus aureus/metabolismo , Staphylococcus aureus/patogenicidade , Staphylococcus epidermidis/metabolismo , Staphylococcus epidermidis/patogenicidade , Virulência
4.
Am J Physiol Cell Physiol ; 321(3): C535-C548, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34288724

RESUMO

Extracellular vesicles (EVs) contain biological molecules and are secreted by cells into the extracellular milieu. The endothelial sodium channel (EnNaC) plays an important role in modulating endothelial cell stiffness. We hypothesized EVs secreted from human aortic endothelial cells (hAoECs) positively regulate EnNaC in an autocrine-dependent manner. A comprehensive lipidomic analysis using targeted mass spectrometry was performed on multiple preparations of EVs isolated from the conditioned media of hAoECs or complete growth media of these cells. Cultured hAoECs challenged with EVs isolated from the conditioned media of these cells resulted in an increase in EnNaC activity when compared with the same concentration of media-derived EVs or vehicle alone. EVs isolated from the conditioned media of hAoECs but not human fibroblast cells were enriched in MARCKS-like protein 1 (MLP1). The pharmacological inhibition of the negative regulator of MLP1, protein kinase C, in cultured hAoECs resulted in an increase in EV size and release compared with vehicle or pharmacological inhibition of protein kinase D. The MLP1-enriched EVs increased the density of actin filaments in cultured hAoECs compared with EVs isolated from human fibroblast cells lacking MLP1. We quantified 141 lipids from glycerolipids, glycerophospholipids, and sphingolipids in conditioned media EVs that represented twice the number found in control media EVs. The concentrations of sphingomyelin, lysophosphatidylcholine and phosphatidylethanolamine were higher in conditioned media EVs. These results provide the first evidence for EnNaC regulation in hAoECs by EVs and provide insight into a possible mechanism involving MLP1, unsaturated lipids, and bioactive lipids.


Assuntos
Proteínas de Ligação a Calmodulina/genética , Meios de Cultivo Condicionados/farmacologia , Células Endoteliais/metabolismo , Vesículas Extracelulares/metabolismo , Lisofosfatidilcolinas/metabolismo , Proteínas dos Microfilamentos/genética , Fosfatidiletanolaminas/metabolismo , Esfingomielinas/metabolismo , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/ultraestrutura , Aorta/citologia , Aorta/metabolismo , Comunicação Autócrina , Proteínas de Ligação a Calmodulina/metabolismo , Meios de Cultivo Condicionados/química , Meios de Cultivo Condicionados/metabolismo , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Vesículas Extracelulares/química , Expressão Gênica , Glicerofosfolipídeos/metabolismo , Humanos , Lipidômica/métodos , Lisofosfatidilcolinas/farmacologia , Proteínas dos Microfilamentos/metabolismo , Fosfatidiletanolaminas/farmacologia , Cultura Primária de Células , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais , Esfingomielinas/farmacologia
5.
Nat Commun ; 12(1): 4228, 2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34244484

RESUMO

Homozygous deletion of methylthioadenosine phosphorylase (MTAP) in cancers such as glioblastoma represents a potentially targetable vulnerability. Homozygous MTAP-deleted cell lines in culture show elevation of MTAP's substrate metabolite, methylthioadenosine (MTA). High levels of MTA inhibit protein arginine methyltransferase 5 (PRMT5), which sensitizes MTAP-deleted cells to PRMT5 and methionine adenosyltransferase 2A (MAT2A) inhibition. While this concept has been extensively corroborated in vitro, the clinical relevance relies on exhibiting significant MTA accumulation in human glioblastoma. In this work, using comprehensive metabolomic profiling, we show that MTA secreted by MTAP-deleted cells in vitro results in high levels of extracellular MTA. We further demonstrate that homozygous MTAP-deleted primary glioblastoma tumors do not significantly accumulate MTA in vivo due to metabolism of MTA by MTAP-expressing stroma. These findings highlight metabolic discrepancies between in vitro models and primary human tumors that must be considered when developing strategies for precision therapies targeting glioblastoma with homozygous MTAP deletion.


Assuntos
Neoplasias Encefálicas/genética , Encéfalo/patologia , Desoxiadenosinas/metabolismo , Glioblastoma/genética , Purina-Núcleosídeo Fosforilase/deficiência , Tionucleosídeos/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Encéfalo/efeitos dos fármacos , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Meios de Cultivo Condicionados/metabolismo , Desoxiadenosinas/análise , Feminino , Secções Congeladas , Glioblastoma/tratamento farmacológico , Glioblastoma/patologia , Homozigoto , Humanos , Metabolômica , Metionina Adenosiltransferase/metabolismo , Terapia de Alvo Molecular/métodos , Medicina de Precisão/métodos , Proteína-Arginina N-Metiltransferases/metabolismo , Purina-Núcleosídeo Fosforilase/genética , Deleção de Sequência , Tionucleosídeos/análise , Ensaios Antitumorais Modelo de Xenoenxerto
6.
Front Immunol ; 12: 679839, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34276668

RESUMO

Background: It is highly desirable to develop new strategies based on secretomics to more accurately selection of embryos with the highest developmental potential for transfer. Granulocyte-macrophage colony-stimulating factor (GM-CSF) has been reported to promote embryo development and pregnancy establishment. However, the predictive value of GM-CSF in single blastocyst selection remains unclear. This study is to determine the concentration of GM-CSF in human single-blastocyst conditioned medium (SBCM) and to evaluate its association with embryo quality and pregnancy outcome. Methods: The patients with ≤38 years of age receiving the first cycle of assisted reproductive therapy were included in this study. The patients who had <4 top-quality embryos formed by the fertilized two pronuclear zygotes on day 3 were excluded. A total of 126 SBCM samples (SBCMs) were included, of which blastocysts from 77 SBCMs were later transferred in subsequent frozen-thawed embryo transfer. The concentrations of GM-CSF were detected by single-molecule array (SIMOA) and analyzed for their possible association with embryo quality and pregnancy outcomes. The top-quality embryo (TQ), positive HCG (HP), clinical pregnancy (CP), and ongoing pregnancy (OP) rates were determined and compared between groups divided based on GM-CSF concentrations. Results: The detection rate of GM-CSF was found to be 50% in all SBCMs. There were significant differences in TQ rate, HP rate, CP rate and OP rate among high concentration group, medium concentration group and low concentration group. Both GM-CSF alone or GM-CSF combined with the morphological score (MS) had a greater AUC of ROC curve than that of MS alone to predict the pregnancy outcome, and GM-CSF combined with MS had the highest AUC. Conclusions: The concentration of GM-CSF in SBCM was detected at fg/ml levels, which was associated with embryo quality and pregnancy outcome. Collectively, GM-CSF may be used as a biomarker for prediction of pregnancy outcome and selection of embryos with high developmental potential for transfer in assisted reproductive technology (ART).


Assuntos
Biomarcadores , Blastocisto/metabolismo , Meios de Cultivo Condicionados/metabolismo , Implantação do Embrião , Fator Estimulador de Colônias de Granulócitos e Macrófagos/biossíntese , Embrião de Mamíferos , Feminino , Humanos , Gravidez , Resultado da Gravidez , Curva ROC , Técnicas de Reprodução Assistida
7.
Cancer Genomics Proteomics ; 18(4): 569-578, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34183389

RESUMO

BACKGROUND/AIM: Mesenchymal stem cell-based tumor therapy is still limited due to the insufficient secretion of effectors and discrepancies between their in vitro and in vivo efficacy. We investigated whether genetically engineered adipose tissue-derived mesenchymal stem cells (ASCs) overexpressing tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) had inhibitory effects on H460 tumor growth both in vitro and in an H460 xenograft model. MATERIALS AND METHODS: Genetically engineered adipose tissue-derived mesenchymal stem cells (ASCs) overexpressing tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) were obtained from plasmid transfection with pCMV3-TRAIL and -interferon (IFN)-ß (producing ASC-TRAIL and ASC-IFN-ß, respectively). Death of H460 cells co-cultured with ASCs, ASC-TRAIL, and ASC-IFN-ß or exposed to their conditioned medium was evaluated via apoptosis and cytotoxicity assays. In addition, in an H460 xenograft model (n=10 per group), the antitumor potential of TRAIL-overexpressing, and IFN-ß-overexpressing ASCs was investigated. RESULTS: Conditioned medium obtained from ASC-IFN-ß increased apoptosis of H460 cells more than did ASC-TRAIL. Additionally, in H460 xenograft models, while native ASCs promoted tumor growth, ASC-TRAIL and ASC-IFN-ß both dramatically suppressed tumor growth. Interestingly, in the context of ASC-IFN-ß, tumors were detected only in 20% of nude mice, with smaller sizes and lower weights than those of the control group. CONCLUSION: TRAIL-overexpressing ASCs can be used to treat tumors; ASC-IFN-ß in particular secrete a higher level of TRAIL.


Assuntos
Tecido Adiposo/metabolismo , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Tecido Adiposo/citologia , Animais , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Meios de Cultivo Condicionados/metabolismo , Meios de Cultivo Condicionados/farmacologia , Humanos , Interferon beta/genética , Interferon beta/metabolismo , Camundongos , Camundongos Nus , Neoplasias Experimentais/patologia , Neoplasias Experimentais/terapia , Ligante Indutor de Apoptose Relacionado a TNF/genética , Ensaios Antitumorais Modelo de Xenoenxerto
8.
J Vis Exp ; (171)2021 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-34096925

RESUMO

Metabolic dysfunction-associated fatty liver disease (MAFLD), previously known as non-alcoholic fatty liver disease (NAFLD), is the most prevalent liver disease worldwide due to its relationship with obesity, diabetes type 2, and dyslipidemia. Hepatic steatosis, the accumulation of lipid droplets in the liver parenchyma, is a key feature of the disease preceding the inflammation observed in steatohepatitis, fibrosis, and end-stage liver disease. Lipid accumulation in hepatocytes might interfere with proper metabolism of xenobiotics and endogenous molecules, as well as to induce cellular processes leading to the advance of the disease. Although the experimental study of steatosis can be performed in vivo, in vitro approaches to the study of steatosis are complementary tools with different advantages. Hepatocyte culture in lipid overload-conditioned medium is an excellent reproducible option for the study of hepatic steatosis allowing the identification of cellular processes related to lipid accumulation, such as oxidative and reticular stresses, autophagia, proliferation, cell death, etcetera, as well as other testing including drug effectiveness, and toxicological testing, among many other possible applications. Here, it was aimed to describe the methodology of hepatocyte cell culture in lipid overload-conditioned medium. HepG2 cells were cultured in RMPI 1640 medium conditioned with sodium palmitate and sodium oleate. Importantly, the ratio of these two lipids is crucial to favor lipid droplet accumulation, while maintaining cell proliferation and a moderate mortality rate, as occurs in the liver during the disease. The methodology, from the preparation of the lipid solution stocks, mixture, addition to the medium, and hepatocyte culture is shown. With this approach, it is possible to identify lipid droplets in the hepatocytes that are readily observable by Oil-red O staining, as well as curves of proliferation/mortality rates.


Assuntos
Técnicas de Cultura de Células , Hepatócitos , Metabolismo dos Lipídeos , Hepatopatia Gordurosa não Alcoólica , Meios de Cultivo Condicionados/metabolismo , Células Hep G2 , Humanos , Fígado/citologia , Fígado/metabolismo , Ácido Palmítico/metabolismo
9.
Methods Mol Biol ; 2348: 285-304, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34160815

RESUMO

During the last years, the study of extracellular vesicles (EVs) and its cargo has gained interest in the scientific media. EVs have been found in all biofluids and it is postulated that all cells are capable to secrete a wide variety of these vesicles, which play a key role in different cell-to-cell communication processes as well as in the microenvironment modulation. In the EV cargo, DNA, protein, and RNA molecules can be found, including long noncoding RNAs (lncRNAs). Several authors consider the study of EV lncRNAs an ideal source of biomarkers due to the easy sampling of EVs in different biofluids and the high specificity of the lncRNA expression pattern.In the present chapter, a detailed explanation of the EV isolation workflow followed by RNA isolation and lncRNA gene expression study is provided for two sample sources: blood plasma and cell culture conditioned media. EVs from both plasma samples and cell cultured media are isolated using sequential ultracentrifugation method (UC), which has been reported as one of the best methods available to date in terms of purity. UC is followed by RNA extraction based on the combination of phenol/guanidine-based lysis of samples with silica-membrane-based purification of total RNA. LncRNA quantification is performed by qRT-PCR. This chapter includes detailed discussion on lncRNA quantification using hydrolysis probes, recommended housekeeping genes and evaluation of methods for comparing lncRNA levels between EVs and its parental cells. In summary, we describe here the main steps for a successful isolation of the EVs-lncRNA cargo, paying attention to how overcome the different challenges found in the experimental procedure and in the data analysis of lncRNA expression from this source.


Assuntos
Ácidos Nucleicos Livres , Meios de Cultivo Condicionados/metabolismo , Vesículas Extracelulares/metabolismo , RNA Longo não Codificante/genética , Biomarcadores , Fracionamento Químico/métodos , Exossomos/metabolismo , Humanos , Biópsia Líquida/métodos , Reação em Cadeia da Polimerase , RNA Longo não Codificante/sangue , RNA Longo não Codificante/isolamento & purificação , RNA Longo não Codificante/metabolismo , Reprodutibilidade dos Testes
10.
J Med Microbiol ; 70(6)2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-34170218

RESUMO

Introduction. This study describes the identification and partial characterization of persistence-inducing factors (PIFs) from staphylococci.Hypothesis/Gap Statement. Increases in persisters during mid-log phase growth indicate that quorum-sensing factors might be produced by staphylococci.Aim. To identify and partially characterize PIFs from Staphylococcus epidermidis RP62A and Staphylococcus aureus SH1000.Methodology. Others have demonstrated a significant increase in persister numbers during mid-log phase. Inducers of this mid-log increase have yet to be identified in staphylococci. Optical density at 600 nm (OD600) was used instead of time to determine when persister numbers increased during logarithmic growth. Concentrated culture filtrates (CCFs) from S. epidermidis and S. aureus were obtained at various OD600s and following incubation at 16 h. The CCFs were used to develop a PIF assay. The PIF assay was used to partially characterize PIF from S. epidermidis and S. aureus for sizing of PIF activity, temperature and protease sensitivity and inter-species communications.Results. The optimal OD600s for S. epidermidis and S. aureus PIF assays were 2.0 and 0.5, respectively. The highest PIF activity for both species was from CCF following incubation overnight (16 h). S. epidermidis' PIF activity was decreased by storage at 4 oC but not at 20 oC (16 h), 37 oC (1 h) or 100 oC (15 min). S. aureus' PIF activity was decreased following storage at 4 oC (2 weeks) and after boiling at 100 oC for 5 min but not after incubation at 37 oC (1 h). PIF activity from both species went through a 3000 molecular weight cutoff ultrafilter. Proteinase K treatment of S. aureus PIF decreased activity but did not decrease the PIF activity of S. epidermidis. PIF from S. epidermidis did not increase persisters when used to treat S. aureus cells and nor did PIF from S. aureus increase persisters when used to treat S. epidermidis cells.Conclusions. Attempts to discover PIFs for staphylococci were unsuccessful due to the time-based means used to identify mid-log. Both staphylococcal species produce extracellular, low-molecular-weight inducers of persistence when assayed using an OD600 -based PIF assay.


Assuntos
Fatores Biológicos/metabolismo , Staphylococcus aureus/fisiologia , Staphylococcus epidermidis/fisiologia , Antibacterianos/farmacologia , Fatores Biológicos/química , Fatores Biológicos/farmacologia , Meios de Cultivo Condicionados/metabolismo , Meios de Cultivo Condicionados/farmacologia , Viabilidade Microbiana/efeitos dos fármacos , Peso Molecular , Peptídeo Hidrolases/metabolismo , Especificidade da Espécie , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/crescimento & desenvolvimento , Staphylococcus aureus/metabolismo , Staphylococcus epidermidis/efeitos dos fármacos , Staphylococcus epidermidis/crescimento & desenvolvimento , Staphylococcus epidermidis/metabolismo , Temperatura
11.
Nat Commun ; 12(1): 3655, 2021 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-34135341

RESUMO

RNA in extracellular vesicles (EVs) are uptaken by cells, where they regulate fundamental cellular functions. EV-derived mRNA in recipient cells can be translated. However, it is still elusive whether "naked nonvesicular extracellular mRNA" (nex-mRNA) that are not packed in EVs can be uptaken by cells and, if so, whether they have any functions in recipient cells. Here, we show the entrance of nex-mRNA in the nucleus, where they exert a translation-independent function. Human nex-interleukin-1ß (IL1ß)-mRNA outside cells proved to be captured by RNA-binding zinc finger CCCH domain containing protein 12D (ZC3H12D)-expressing human natural killer (NK) cells. ZC3H12D recruited to the cell membrane binds to the 3'-untranslated region of nex-IL1ß-mRNA and transports it to the nucleus. The nex-IL1ß-mRNA in the NK cell nucleus upregulates antiapoptotic gene expression, migration activity, and interferon-γ production, leading to the killing of cancer cells and antimetastasis in mice. These results implicate the diverse actions of mRNA.


Assuntos
Núcleo Celular/metabolismo , Espaço Extracelular/metabolismo , RNA Mensageiro/metabolismo , Regiões 3' não Traduzidas , Animais , Antineoplásicos/farmacologia , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Meios de Cultivo Condicionados/metabolismo , Endorribonucleases/metabolismo , Humanos , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Células Matadoras Naturais/metabolismo , Camundongos , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/farmacologia , Proteínas de Ligação a RNA/metabolismo
12.
Int J Mol Sci ; 22(11)2021 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-34072546

RESUMO

Non-targeted effects (NTE) of ionizing radiation may initiate myeloid neoplasms (MN). Here, protein mediators (I) in irradiated human mesenchymal stromal cells (MSC) as the NTE source, (II) in MSC conditioned supernatant and (III) in human bone marrow CD34+ cells undergoing genotoxic NTE were investigated. Healthy sublethal irradiated MSC showed significantly increased levels of reactive oxygen species. These cells responded by increasing intracellular abundance of proteins involved in proteasomal degradation, protein translation, cytoskeleton dynamics, nucleocytoplasmic shuttling, and those with antioxidant activity. Among the increased proteins were THY1 and GNA11/14, which are signaling proteins with hitherto unknown functions in the radiation response and NTE. In the corresponding MSC conditioned medium, the three chaperones GRP78, CALR, and PDIA3 were increased. Together with GPI, these were the only four altered proteins, which were associated with the observed genotoxic NTE. Healthy CD34+ cells cultured in MSC conditioned medium suffered from more than a six-fold increase in γH2AX focal staining, indicative for DNA double-strand breaks, as well as numerical and structural chromosomal aberrations within three days. At this stage, five proteins were altered, among them IQGAP1, HMGB1, and PA2G4, which are involved in malign development. In summary, our data provide novel insights into three sequential steps of genotoxic signaling from irradiated MSC to CD34+ cells, implicating that induced NTE might initiate the development of MN.


Assuntos
Células da Medula Óssea/metabolismo , Diferenciação Celular , Dano ao DNA , Células-Tronco Mesenquimais/metabolismo , Proteoma , Transdução de Sinais , Idoso , Antígenos CD34/metabolismo , Biomarcadores , Células da Medula Óssea/citologia , Diferenciação Celular/genética , Diferenciação Celular/efeitos da radiação , Sobrevivência Celular/genética , Instabilidade Cromossômica , Meios de Cultivo Condicionados/metabolismo , Feminino , Histonas/metabolismo , Humanos , Masculino , Células-Tronco Mesenquimais/citologia , Modelos Biológicos , Proteômica/métodos , Radiação Ionizante , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos da radiação
13.
Cells ; 10(5)2021 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-34068340

RESUMO

Here we report the use of a microfluidic system to assess the differential metabolomics of murine embryos cultured with endometrial cells-conditioned media (CM). Groups of 10, 1-cell murine B6C3F1 × B6D2F1 embryos were cultured in the microfluidic device. To produce CM, mouse uterine epithelial cells were cultured in potassium simplex optimized medium (KSOM) for 24 h. Media samples were collected from devices after 5 days of culture with KSOM (control) and CM, analyzed by reverse phase liquid chromatography and untargeted positive ion mode mass spectrometry analysis. Blastocyst rates were significantly higher (p < 0.05) in CM (71.8%) compared to control media (54.6%). We observed significant upregulation of 341 compounds and downregulation of 214 compounds in spent media from CM devices when compared to control. Out of these, 353 compounds were identified showing a significant increased abundance of metabolites involved in key metabolic pathways (e.g., arginine, proline and pyrimidine metabolism) in the CM group, suggesting a beneficial effect of CM on embryo development. The metabolomic study carried out in a microfluidic environment confirms our hypothesis on the potential of uterine epithelial cells to enhance blastocyst development. Further investigations are required to highlight specific pathways involved in embryo development and implantation.


Assuntos
Blastocisto/metabolismo , Técnicas de Cultura Embrionária/instrumentação , Células Epiteliais/metabolismo , Dispositivos Lab-On-A-Chip , Metaboloma , Metabolômica , Técnicas Analíticas Microfluídicas/instrumentação , Comunicação Parácrina , Útero/metabolismo , Animais , Células Cultivadas , Meios de Cultivo Condicionados/metabolismo , Desenvolvimento Embrionário , Feminino , Espectrometria de Massas , Camundongos , Transdução de Sinais , Útero/citologia
14.
Cells ; 10(5)2021 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-34069578

RESUMO

Corneal injuries are among the leading causes of blindness and vision impairment. Trauma, infectious keratitis, thermal and chemical (acids and alkali burn) injuries may lead to irreversible corneal scarring, neovascularization, conjunctivalization, and limbal stem cell deficiency. Bilateral blindness constitutes 12% of total global blindness and corneal transplantation remains a stand-alone treatment modality for the majority of end-stage corneal diseases. However, global shortage of donor corneas, the potential risk of graft rejection, and severe side effects arising from long-term use of immunosuppressive medications, demands alternative therapeutic approaches. Umbilical cord-derived mesenchymal stem cells can be isolated in large numbers using a relatively less invasive procedure. However, their role in injury induced corneal repair is largely unexplored. Here, we isolated, cultured and characterized mesenchymal stem cells from human umbilical cord, and studied the expression of mesenchymal (CD73, CD90, CD105, and CD34), ocular surface and epithelial (PAX6, WNT7A, and CK-8/18) lineage markers through immunofluorescence. The cultured human limbal and corneal epithelial cells were used as controls. Scratch assay was used to study the corneal epithelial repair potential of umbilical cord-derived mesenchymal stem cells, in vitro. The in vitro cultured umbilical cord-derived mesenchymal stem cells were plastic adherent, showed trilineage differentiation and expressed: mesenchymal markers CD90, CD105, CD73; epithelial marker CK-8/18, and ocular lineage developmental markers PAX6 and WNT-7A. Our findings suggest that umbilical cord-derived mesenchymal stem cells promote repair of the injured corneal epithelium by stimulating the proliferation of corneal epithelial cells, in vitro. They may serve as a potential non-ocular source of stem cells for treating injury induced bilateral corneal diseases.


Assuntos
Células Epiteliais/metabolismo , Epitélio Corneano/metabolismo , Células-Tronco Mesenquimais/metabolismo , Comunicação Parácrina , Cordão Umbilical/citologia , Cicatrização , Adulto , Animais , Linhagem da Célula , Movimento Celular , Proliferação de Células , Células Cultivadas , Cricetinae , Meios de Cultivo Condicionados/metabolismo , Células Epiteliais/patologia , Epitélio Corneano/patologia , Feminino , Humanos , Queratina-18/metabolismo , Queratina-8/metabolismo , Pessoa de Meia-Idade , Fator de Transcrição PAX6/metabolismo , Reepitelização , Transdução de Sinais , Proteínas Wnt/metabolismo , Adulto Jovem
15.
Cells ; 10(5)2021 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-34067928

RESUMO

In patients undergoing coronary artery bypass grafting (CABG), ischemia/reperfusion injury (IRI) is the main contributor to organ dysfunction. Aging-induced vascular damage may be further aggravated during CABG. Favorable effects of conditioned medium (CM) from mesenchymal stem cells (MSCs) have been suggested against IRI. We hypothesized that adding CM to saline protects vascular grafts from IRI in rats. We found that CM contains 28 factors involved in apoptosis, inflammation, and oxidative stress. Thoracic aortic rings from 15-month-old rats were explanted and immediately mounted in organ bath chambers (aged group) or underwent 24 h of cold ischemic preservation in saline-supplemented either with vehicle (aged-IR group) or CM (aged-IR+CM group), prior to mounting. Three-month-old rats were used as referent young animals. Aging was associated with an increase in intima-to-media thickness, an increase in collagen content, higher caspase-12 mRNA levels, and immunoreactivity compared to young rats. Impaired endothelium-dependent vasorelaxation to acetylcholine in the aged-IR group compared to the aged-aorta was improved by CM (aged 61 ± 2% vs. aged-IR 38 ± 2% vs. aged-IR+CM 50 ± 3%, p < 0.05). In the aged-IR group, the already high mRNA levels of caspase-12 were decreased by CM. CM alleviates endothelial dysfunction following IRI in 15-month-old rats. The protective effect may be related to the inhibition of caspase-12 expression.


Assuntos
Aorta Torácica/metabolismo , Meios de Cultivo Condicionados/metabolismo , Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Células-Tronco Mesenquimais/metabolismo , Traumatismo por Reperfusão/prevenção & controle , Vasodilatação , Fatores Etários , Animais , Aorta Torácica/patologia , Aorta Torácica/fisiopatologia , Caspase 12/genética , Caspase 12/metabolismo , Células Cultivadas , Isquemia Fria , Colágeno/metabolismo , Estresse do Retículo Endoplasmático , Células Endoteliais/patologia , Endotélio Vascular/patologia , Endotélio Vascular/fisiopatologia , Fibrose , Técnicas In Vitro , Masculino , Ratos Endogâmicos Lew , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Traumatismo por Reperfusão/fisiopatologia , Fatores de Tempo
16.
Int J Mol Sci ; 22(9)2021 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-33946667

RESUMO

Transplantation of various types of stem cells as a possible therapy for stroke has been tested for years, and the results are promising. Recent investigations have shown that the administration of the conditioned media obtained after stem cell cultivation can also be effective in the therapy of the central nervous system pathology (hypothesis of their paracrine action). The aim of this study was to evaluate the therapeutic effects of the conditioned medium of hiPSC-derived glial and neuronal progenitor cells in the rat middle cerebral artery occlusion model of the ischemic stroke. Secretory activity of the cultured neuronal and glial progenitor cells was evaluated by proteomic and immunosorbent-based approaches. Therapeutic effects were assessed by overall survival, neurologic deficit and infarct volume dynamics, as well as by the end-point values of the apoptosis- and inflammation-related gene expression levels, the extent of microglia/macrophage infiltration and the numbers of formed blood vessels in the affected area of the brain. As a result, 31% of the protein species discovered in glial progenitor cells-conditioned medium and 45% in neuronal progenitor cells-conditioned medium were cell type specific. The glial progenitor cell-conditioned media showed a higher content of neurotrophins (BDNF, GDNF, CNTF and NGF). We showed that intra-arterial administration of glial progenitor cells-conditioned medium promoted a faster decrease in neurological deficit compared to the control group, reduced microglia/macrophage infiltration, reduced expression of pro-apoptotic gene Bax and pro-inflammatory cytokine gene Tnf, increased expression of anti-inflammatory cytokine genes (Il4, Il10, Il13) and promoted the formation of blood vessels within the damaged area. None of these effects were exerted by the neuronal progenitor cell-conditioned media. The results indicate pronounced cytoprotective, anti-inflammatory and angiogenic properties of soluble factors secreted by glial progenitor cells.


Assuntos
Meios de Cultivo Condicionados/metabolismo , Meios de Cultivo Condicionados/farmacologia , AVC Isquêmico/terapia , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Animais , Células Cultivadas , Modelos Animais de Doenças , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Infarto da Artéria Cerebral Média/metabolismo , Infarto da Artéria Cerebral Média/terapia , Infusões Intra-Arteriais , AVC Isquêmico/metabolismo , AVC Isquêmico/patologia , Masculino , Neuroglia/citologia , Neuroglia/metabolismo , Ratos , Ratos Wistar
17.
Methods Mol Biol ; 2228: 293-306, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33950499

RESUMO

Cells secrete proteins to communicate with their environment. Therefore, it is interesting to characterize the proteins which are released from cells under certain experimental conditions the so-called secretome. Here, often proteins from conditioned medium of cultured cells are analyzed, but these additionally might include also contaminating proteins of serum that have not been sufficiently removed or proteins from dying cells. To provide high-quality secretome data and minimize potential contaminants, we describe a quantitative comparison of conditioned medium and the cellular proteome. The described workflow comprises cell cultivation, sample preparation, and final data analysis which is based on the comparison of data from label-free mass spectrometric quantification of proteins from the conditioned medium with corresponding cellular proteomes enabling the detection of bona fide secreted proteins.


Assuntos
Proteínas de Neoplasias/análise , Proteoma , Proteômica , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em Tandem , Células A549 , Animais , Cromatografia Líquida de Alta Pressão , Meios de Cultivo Condicionados/metabolismo , Humanos , Projetos de Pesquisa , Via Secretória , Fluxo de Trabalho
18.
J Microbiol Biotechnol ; 31(7): 990-998, 2021 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-33958510

RESUMO

Melanin is a natural skin pigment produced by specialized cells called melanocytes via a multistage biochemical pathway known as melanogenesis, involving the oxidation and polymerization of tyrosine. Melanogenesis is initiated upon exposure to ultraviolet (UV) radiation, causing the skin to darken, which protects skin cells from UVB radiation damage. However, the abnormal accumulation of melanin may lead to the development of certain skin diseases, including skin cancer. In this study, the antioxidant and antimelanogenic activities of the cell-free supernatant (CFS) of twenty strains were evaluated. Based on the results of 60% 2,2-diphenyl-1-picrylhydrazyl scavenging activity, 21% 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) scavenging capacity, and a 50% ascorbic acid equivalent ferric reducing antioxidant power value, Limosilactobacillus fermentum JNU532 was selected as the strain with the highest antioxidant potential. No cytotoxicity was observed in cells treated with the CFS of L. fermentum JNU532. Tyrosinase activity was reduced by 16.7% in CFStreated B16F10 cells (but not in the cell-free system), with >23.2% reduction in melanin content upon treatment with the L. fermentum JNU532-derived CFS. The inhibitory effect of the L. fermentum JNU532-derived CFS on B16F10 cell melanogenesis pathways was investigated using quantitative reverse transcription polymerase chain reaction and western blotting. The inhibitory effects of the L. fermentum JNU532-derived CFS were mediated by inhibiting the transcription of TYR, TRP-1, TRP-2, and MITF and the protein expression of TYR, TRP-1, TRP-2, and MITF. Therefore, L. fermentum JNU532 may be considered a potentially useful, natural depigmentation agent.


Assuntos
Antioxidantes/metabolismo , Alimentos e Bebidas Fermentados/microbiologia , Lactobacillaceae/metabolismo , Melaninas/antagonistas & inibidores , Ácidos/metabolismo , Animais , Antioxidantes/farmacologia , Ácidos e Sais Biliares/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Meios de Cultivo Condicionados/metabolismo , Meios de Cultivo Condicionados/farmacologia , Oxirredutases Intramoleculares/genética , Oxirredutases Intramoleculares/metabolismo , Melaninas/metabolismo , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Fator de Transcrição Associado à Microftalmia/genética , Fator de Transcrição Associado à Microftalmia/metabolismo , Monofenol Mono-Oxigenase/genética , Monofenol Mono-Oxigenase/metabolismo , Oxirredutases/genética , Oxirredutases/metabolismo , Probióticos
19.
J Adv Res ; 30: 103-112, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-34026290

RESUMO

Introduction: The dermal papilla (DP) represents the major regulatory entity within the hair follicle (HF), inducing hair formation and growth through reciprocal interactions with epithelial cells. However, human DP cells rapidly lose their hair inductive ability when cultured in an epithelium-deficient environment. Objectives: To determine if the conditioned medium collected from interfollicular keratinocytes (KCs-CM) is capable of improving DP cell native properties and inductive phenotype. Methods: DP cells were cultured with KCs-CM both in 2D and 3D culture conditions (spheroids). Further, the hair-inductive capacity of DP cells precultured with KCs-CM was tested in a hair reconstitution assay, after co-grafting with human keratinocytes in nude mice. Results: We demonstrate that KCs-CM contributes to restore the inductivity of cultured human DP cells in a more effective mode than the conventional 3D-cultures. This is supported by the higher active alkaline phosphatase (ALP) levels in DP cells, the improved self-aggregative capacity and the reduced expression of α-SMA and the V1-isoform of versican. Moreover, DP cells cultured with KCs-CM displayed a secretome profile (VEGF, BMP2, TGF- ß1, IL-6) that matches the one observed during anagen. KCs-CM also enhanced DP cell proliferation, while preventing cells to undergo morphological changes characteristic of high passage cells. In opposition, the amount of collagenous and non-collagenous proteins deposited by DP cells was lower in the presence of KCs-CM. The improvement in ALP activity was maintained in 3D spheroidal cultures, even after KCs-CM retrieval, being superior to the effect of the gold-standard culture conditions. Moreover, DP cells cultured with KCs-CM and grafted with human keratinocytes supported the formation of HF- and sebaceous gland-like structures in mice. Conclusion: The proposed strategy encourages future cell-based strategies for HF regeneration not only in the context of hair-associated disorders, but also in the management of wounds to aid in restoring critical skin regulatory appendages.


Assuntos
Folículo Piloso/citologia , Folículo Piloso/metabolismo , Cabelo/fisiologia , Queratinócitos/metabolismo , Regeneração , Animais , Técnicas de Cultura de Células , Proliferação de Células , Células Cultivadas , Colágeno/metabolismo , Meios de Cultivo Condicionados/metabolismo , Derme/metabolismo , Epitélio/metabolismo , Humanos , Camundongos , Camundongos Nus , Fenótipo , Pele/metabolismo , Esferoides Celulares/citologia
20.
Methods Mol Biol ; 2271: 317-330, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33908017

RESUMO

Glycan "node" analysis is the process by which pooled glycans within complex biological samples are chemically deconstructed in a way that facilitates the analytical quantification of uniquely linked monosaccharide units (glycan "nodes"). It is based on glycan methylation analysis (a.k.a. linkage analysis) that has historically been applied to pre-isolated glycans. Thus, when using glycan node analysis, unique glycan features within whole biospecimens such as "core fucosylation," "α2-6 sialylation," "ß1-6 branching," "ß1-4 branching," and "bisecting GlcNAc," are captured as single analytical signals by GC-MS. Here we describe the use of this methodology in cell culture supernatant and in the analysis of IgG (alpha-1 antitrypsin) glycans. The effect of IL-6 and IL-1ß cytokines on secreted hepatocyte protein glycan features is demonstrated; likewise, the impact of neuraminidase treatment of IgG is illustrated. For the majority of glycan nodes, the assay is consistent and reproducible on a day-to-day basis; because of this, relatively subtle shifts in the relative abundance of glycan features can be captured using this approach.


Assuntos
Cromatografia Gasosa-Espectrometria de Massas , Glicômica , Glicoproteínas/análise , Hepatócitos/metabolismo , Imunoglobulina G/análise , Polissacarídeos/análise , Processamento de Proteína Pós-Traducional , Meios de Cultivo Condicionados/metabolismo , Glicosilação , Células Hep G2 , Hepatócitos/efeitos dos fármacos , Humanos , Interleucina-1beta/farmacologia , Interleucina-6/farmacologia , Metilação , Neuraminidase/metabolismo , Projetos de Pesquisa , Via Secretória , Fluxo de Trabalho
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