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Photothermal therapy (PTT) is a promising cancer therapeutic approach due to its spatial selectivity and high potency. Indocyanine green (ICG) has been considered a biocompatible PTT agent. However, ICG has several challenges to hinder its clinical use including rapid blood clearance and instability to heat, light, and solvent, leading to a loss of photoactivation property and PTT efficacy. Herein, we leveraged stabilizing components, methyl-ß-cyclodextrin and liposomes, in one nanoplatform (ICD lipo) to enhance ICG stability and the photothermal therapeutic effect against cancer. Compared to ICG, ICD lipo displayed a 4.8-fold reduction in degradation in PBS solvent after 30 days and a 3.4-fold reduction in photobleaching after near-infrared laser irradiation. Moreover, in tumor-bearing mice, ICD lipo presented a 2.7-fold increase in tumor targetability and inhibited tumor growth 9.6 times more effectively than did ICG without any serious toxicity. We believe that ICD lipo could be a potential PTT agent for cancer therapeutics.
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Verde de Indocianina , Liposomas , Terapia Fototérmica , Verde de Indocianina/química , Verde de Indocianina/farmacología , Verde de Indocianina/uso terapéutico , Animales , Ratones , Liposomas/química , Humanos , beta-Ciclodextrinas/química , Línea Celular Tumoral , Neoplasias/terapia , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Femenino , Ratones Endogámicos BALB C , FototerapiaRESUMEN
Extended liver resection carries the risk of post-surgery liver failure involving thrombospondin-1-mediated aggravation of hepatic epithelial plasticity and function. Mesenchymal stromal cells (MSCs), by interfering with thrombospondin-1 (THBS1), counteract hepatic dysfunction, though the mechanisms involved remain unknown. Herein, two-thirds partial hepatectomy in mice increased hepatic THBS1, downstream transforming growth factor-ß3, and perturbation of liver tissue homeostasis. All these events were ameliorated by hepatic transfusion of human bone marrow-derived MSCs. Treatment attenuated platelet and macrophage recruitment to the liver, both major sources of THBS1. By mitigating THBS1, MSCs muted surgery-induced tissue deterioration and dysfunction, and thus supported post-hepatectomy regeneration. After liver surgery, patients displayed increased tissue THBS1, which is associated with functional impairment and may indicate a higher risk of post-surgery complications. Since liver dysfunction involving THBS1 improves with MSC treatment in various animal models, it seems feasible to also modulate THBS1 in humans to impede post-surgery acute liver failure.
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Hepatopatías , Células Madre Mesenquimatosas , Humanos , Ratones , Animales , Hepatectomía , Regeneración Hepática/fisiología , TrombospondinasRESUMEN
Tissue infiltration by circulating leukocytes via directed migration (also referred to as chemotaxis) is a common pathogenic mechanism of inflammatory diseases. G protein-coupled receptors (GPCRs) are essential for sensing chemokine gradients and directing the movement of leukocytes during immune responses. The tumor necrosis factor α-induced protein 8-like (TIPE or TNFAIP8L) family of proteins are newly described pilot proteins that control directed migration of murine leukocytes. However, how leukocytes integrate site-specific directional cues, such as chemokine gradients, and utilize GPCR and TIPE proteins to make directional decisions are not well understood. Using both gene knockdown and biochemical methods, we demonstrated here that 2 human TIPE family members, TNFAIP8 and TIPE2, were essential for directed migration of human CD4+ T cells. T cells deficient in both of these proteins completely lost their directionality. TNFAIP8 interacted with the Gαi subunit of heterotrimeric (α, ß, γ) G proteins, whereas TIPE2 bound to PIP2 and PIP3 to spatiotemporally control immune cell migration. Using deletion and site-directed mutagenesis, we established that Gαi interacted with TNFAIP8 through its C-terminal amino acids, and that TIPE2 protein interacted with PIP2 and PIP3 through its positively charged amino acids on the α0 helix and at the grip-like entrance. We also discovered that TIPE protein membrane translocation (i.e. crucial for sensing chemokine gradients) was dependent on PIP2. Collectively, our work describes a new mechanistic paradigm for how human T cells integrate GPCR and phospholipid signaling pathways to control directed migration. These findings have implications for therapeutically targeting TIPE proteins in human inflammatory and autoimmune diseases.
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Sistemas de Mensajero Secundario , Transducción de Señal , Humanos , Animales , Ratones , Quimiocinas , Aminoácidos , Lípidos , Péptidos y Proteínas de Señalización IntracelularRESUMEN
Tight regulation of Toll-like receptor (TLR)-mediated inflammatory responses is important for innate immunity. Here, we show that T-cell death-associated gene 51 (TDAG51/PHLDA1) is a novel regulator of the transcription factor FoxO1, regulating inflammatory mediator production in the lipopolysaccharide (LPS)-induced inflammatory response. TDAG51 induction by LPS stimulation was mediated by the TLR2/4 signaling pathway in bone marrow-derived macrophages (BMMs). LPS-induced inflammatory mediator production was significantly decreased in TDAG51-deficient BMMs. In TDAG51-deficient mice, LPS- or pathogenic Escherichia coli infection-induced lethal shock was reduced by decreasing serum proinflammatory cytokine levels. The recruitment of 14-3-3ζ to FoxO1 was competitively inhibited by the TDAG51-FoxO1 interaction, leading to blockade of FoxO1 cytoplasmic translocation and thereby strengthening FoxO1 nuclear accumulation. TDAG51/FoxO1 double-deficient BMMs showed significantly reduced inflammatory mediator production compared with TDAG51- or FoxO1-deficient BMMs. TDAG51/FoxO1 double deficiency protected mice against LPS- or pathogenic E. coli infection-induced lethal shock by weakening the systemic inflammatory response. Thus, these results indicate that TDAG51 acts as a regulator of the transcription factor FoxO1, leading to strengthened FoxO1 activity in the LPS-induced inflammatory response.
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Escherichia coli , Lipopolisacáridos , Ratones , Animales , Proteínas 14-3-3 , Factores de Transcripción/genética , Mediadores de InflamaciónRESUMEN
Inflammatory bowel disease (IBD), including ulcerative colitis and Crohn's disease, is a group of chronic inflammatory diseases of the gastrointestinal tract. Although the multifactorial etiology of IBD pathogenesis is relatively well documented, the regulatory factors that confer a risk of IBD pathogenesis remain less explored. In this study, we report that T-cell death-associated gene 51 (TDAG51/PHLDA1) is a novel regulator of the development of dextran sulfate sodium (DSS)-induced colitis in mice. TDAG51 expression was elevated in the colon tissues of DSS-induced experimental colitis mice. TDAG51 deficiency protected mice against acute DSS-induced lethality and body weight changes and disease severity. DSS-induced structural damage and mucus secretion in colon tissues were significantly reduced in TDAG51-deficient mice compared with wild-type mice. We observed similar results in a DSS-induced chronic colitis mouse model. Finally, we showed that the production of inflammatory mediators, including proinflammatory enzymes, molecules and cytokines, was decreased in DSS-treated TDAG51-deficient mice compared with DSS-treated wild-type mice. Thus, we demonstrated that TDAG51 deficiency plays a protective role against DSS-induced colitis by decreasing the production of inflammatory mediators in mice. These findings suggest that TDAG51 is a novel regulator of the development of DSS-induced colitis and is a potential therapeutic target for IBD.
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Escarabajos , Colitis , Enfermedades Inflamatorias del Intestino , Ratones , Animales , Sulfato de Dextran/toxicidad , Colitis/inducido químicamente , Mediadores de InflamaciónRESUMEN
Osteoclasts (OCs) are clinically important cells that resorb bone matrix. Accelerated bone destruction by OCs is closely linked to the development of metabolic bone diseases. In this study, we screened novel chemical inhibitors targeting OC differentiation to identify drug candidates for metabolic bone diseases. We identified that 1,3-dibenzyl-5-fluorouracil, also named OCI-101, is a novel inhibitor of osteoclastogenesis. The formation of multinucleated OCs is reduced by treatment with OCI-101 in a dose-dependent manner. OCI-101 inhibited the expression of OC markers via downregulation of receptor activator of NF-κB ligand and M-CSF signaling pathways. Finally, we showed that OCI-101 prevents ovariectomy-induced bone loss by suppressing OC differentiation in mice. Hence, these results demonstrated that OCI-101 is a good drug candidate for treating metabolic bone diseases.
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BACKGROUND: eHealth and telehealth play a crucial role in assisting older adults who visit hospitals frequently or who live in nursing homes and can benefit from staying at home while being cared for. Adapting to new technologies can be difficult for older people. Thus, to better apply these technologies to older adults' lives, many studies have analyzed the acceptance factors for this particular population. However, there is not yet a consensual framework that can be used in further development and to search for solutions. OBJECTIVE: This paper aims to present an integrated acceptance framework (IAF) for older users' acceptance of eHealth based on 43 studies selected through a systematic review. METHODS: We conducted a 4-step study. First, through a systematic review in the field of eHealth from 2010 to 2020, the acceptance factors and basic data for analysis were extracted. Second, we conducted a thematic analysis to group the factors into themes to propose an integrated framework for acceptance. Third, we defined a metric to evaluate the impact of the factors addressed in the studies. Finally, the differences among the important IAF factors were analyzed according to the participants' health conditions, verification time, and year. RESULTS: Through a systematic review, 731 studies were found in 5 major databases, resulting in 43 (5.9%) selected studies using the PRISMA (Preferred Reporting Item for Systematic Reviews and Meta-Analyses) methodology. First, the research methods and acceptance factors for eHealth were compared and analyzed, extracting a total of 105 acceptance factors, which were grouped later, resulting in an IAF. A total of 5 dimensions (ie, personal, user-technology relational, technological, service-related, and environmental) emerged, with a total of 23 factors. In addition, we assessed the quality of evidence and then conducted a stratification analysis to reveal the more appropriate factors depending on the health condition and assessment time. Finally, we assessed the factors and dimensions that have recently become more important. CONCLUSIONS: The result of this investigation is a framework for conducting research on eHealth acceptance. To elaborately analyze the impact of the factors of the proposed framework, the criteria for evaluating the evidence from the studies that have the extracted factors are presented. Through this process, the impact of each factor in the IAF has been presented, in addition to the framework proposal. Moreover, a meta-analysis of the current status of research is presented, highlighting the areas where specific measures are needed to facilitate eHealth acceptance.
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Telemedicina , Anciano , Análisis Factorial , Humanos , Casas de Salud , Proyectos de Investigación , Tecnología , Telemedicina/métodosRESUMEN
Mucosal surfaces are continuously exposed to, and challenged by, numerous commensal and pathogenic organisms. To guard against infections, a majority of the thymus-derived T lymphocytes are deployed at the mucosa. Although chemokines are known to be involved in the mucosal lymphocyte deployment, it is not clear whether lymphocytes enter the mucosa through directed migration or enhanced random migration. Here we report that TIPE (tumor necrosis factor-α-induced protein 8 (TNFAIP8)-like) proteins mediate directed migration of T lymphocytes into lung mucosa, and they are crucial for mucosal immune defense against Streptococcus pneumoniae infection. Knockout of both Tnfaip8 and Tipe2, which encode polarity proteins that control the directionality of lymphocyte migration, significantly reduced the numbers of T lymphocytes in the lung of mice. Compared with wild-type mice, Tnfaip8-/- Tipe2-/- mice also developed more severe infection with more pathogens entering blood circulation upon nasal Streptococcus pneumoniae challenge. Single-cell RNA-sequencing analysis revealed that TIPE proteins selectively affected mucosal homing of a unique subpopulation of T cells, called "T cells-2", which expressed high levels of Ccr9, Tcf7, and Rag1/2 genes. TNFAIP8 and TIPE2 appeared to have overlapping functions since deficiency in both yielded the strongest phenotype. These data demonstrate that TIPE family of proteins are crucial for lung mucosal immunity. Strategies targeting TIPE proteins may help develop mucosal vaccines or treat inflammatory diseases of the lung.
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Cancer metastasis accounts for nearly 90% of all cancer deaths. Metastatic cancer progression requires both cancer cell migration to the site of the metastasis and subsequent proliferation after colonization. However, it has long been recognized that cancer cell migration and proliferation can be uncoupled; but the mechanism underlying this paradox is not well understood. Here we report that TNFAIP8 (tumor necrosis factor-α-induced protein 8), a "professional" transfer protein of phosphoinositide second messengers, promotes cancer cell migration or metastasis but inhibits its proliferation or cancer growth. TNFAIP8-deficient mice developed larger tumors, but TNFAIP8-deficient tumor cells completely lost their ability to migrate toward chemoattractants and were defective in colonizing lung tissues as compared to wild-type counterparts. Mechanistically, TNFAIP8 served as a cellular "pilot" of tumor cell migration by locally amplifying PI3K-AKT and Rac signals on the cell membrane facing chemoattractant; at the same time, TNFAIP8 also acted as a global inhibitor of tumor cell growth and proliferation by regulating Hippo signaling pathway. These findings help explain the migration-proliferation paradox of cancer cells that characterizes many cancers.
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Proteínas Reguladoras de la Apoptosis/metabolismo , Fibrosarcoma/patología , Neoplasias Pulmonares/patología , Neoplasias Cutáneas/patología , Animales , Proteínas Reguladoras de la Apoptosis/genética , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Dietilnitrosamina/efectos adversos , Femenino , Fibrosarcoma/inducido químicamente , Fibrosarcoma/genética , Fibrosarcoma/metabolismo , Regulación Neoplásica de la Expresión Génica , Vía de Señalización Hippo , Humanos , Neoplasias Pulmonares/inducido químicamente , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Masculino , Metilcolantreno/efectos adversos , Ratones , Fosfatidilinositol 3-Quinasas/metabolismo , Neoplasias Cutáneas/inducido químicamente , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/metabolismoRESUMEN
Paired box protein 5 (Pax5) is a crucial transcription factor responsible for B-cell lineage specification and commitment. In this study, we identified a negative role of Pax5 in osteoclastogenesis. The expression of Pax5 was time-dependently downregulated by receptor activator of nuclear factor kappa B (RANK) ligand (RANKL) stimulation in osteoclastogenesis. Osteoclast (OC) differentiation and bone resorption were inhibited (68.9% and 48% reductions, respectively) by forced expression of Pax5 in OC lineage cells. Pax5 led to the induction of antiosteoclastogenic factors through downregulation of B lymphocyte-induced maturation protein 1 (Blimp1). To examine the negative role of Pax5 in vivo, we generated Pax5 transgenic (Pax5Tg) mice expressing the human Pax5 transgene under the control of the tartrate-resistant acid phosphatase (TRAP) promoter, which is expressed mainly in OC lineage cells. OC differentiation and bone resorption were inhibited (54.2-76.9% and 24.0-26.2% reductions, respectively) in Pax5Tg mice, thereby contributing to the osteopetrotic-like bone phenotype characterized by increased bone mineral density (13.0-13.6% higher), trabecular bone volume fraction (32.5-38.1% higher), trabecular thickness (8.4-9.0% higher), and trabecular number (25.5-26.7% higher) and decreased trabecular spacing (9.3-10.4% lower) compared to wild-type control mice. Furthermore, the number of OCs was decreased (48.8-65.3% reduction) in Pax5Tg mice. These findings indicate that Pax5 plays a negative role in OC lineage specification and commitment through Blimp1 downregulation. Thus, our data suggest that the Pax5-Blimp1 axis is crucial for the regulation of RANKL-induced osteoclastogenesis.
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Regulación hacia Abajo/genética , Osteogénesis , Factor de Transcripción PAX5/metabolismo , Factor 1 de Unión al Dominio 1 de Regulación Positiva/genética , Animales , Huesos/patología , Linaje de la Célula , Regulación hacia Abajo/efectos de los fármacos , Expresión Génica , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutación/genética , Osteoclastos/metabolismo , Osteogénesis/genética , Osteopetrosis/genética , Osteopetrosis/patología , Factor de Transcripción PAX5/genética , Fenotipo , Ligando RANK/farmacología , Células RAW 264.7 , TransgenesRESUMEN
The nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ) is the master transcriptional regulator in adipogenesis. PPARγ forms a heterodimer with another nuclear receptor, retinoid X receptor (RXR), to form an active transcriptional complex, and their transcriptional activity is tightly regulated by the association with either coactivators or corepressors. In this study, we identified T-cell death-associated gene 51 (TDAG51) as a novel corepressor of PPARγ-mediated transcriptional regulation. We showed that TDAG51 expression is abundantly maintained in the early stage of adipogenic differentiation. Forced expression of TDAG51 inhibited adipocyte differentiation in 3T3-L1 cells. We found that TDAG51 physically interacts with PPARγ in a ligand-independent manner. In deletion mutant analyses, large portions of the TDAG51 domains, including the pleckstrin homology-like, glutamine repeat and proline-glutamine repeat domains but not the proline-histidine repeat domain, are involved in the interaction with the region between residues 140 and 506, including the DNA binding domain, hinge, ligand binding domain and activation function-2 domain, in PPARγ. The heterodimer formation of PPARγ-RXRα was competitively inhibited in a ligand-independent manner by TDAG51 binding to PPARγ. Thus, our data suggest that TDAG51, which could determine adipogenic cell fate, acts as a novel negative regulator of PPARγ by blocking RXRα recruitment to the PPARγ-RXRα heterodimer complex in adipogenesis.
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Adipogénesis , PPAR gamma/metabolismo , Multimerización de Proteína , Receptor alfa X Retinoide/metabolismo , Linfocitos T/citología , Linfocitos T/metabolismo , Factores de Transcripción/genética , Células 3T3-L1 , Adipocitos/citología , Adipocitos/metabolismo , Animales , Muerte Celular , Proteínas de Unión a Ácidos Grasos/genética , Proteínas de Unión a Ácidos Grasos/metabolismo , Ratones , Regiones Promotoras Genéticas/genética , Unión Proteica , Factores de Transcripción/metabolismoRESUMEN
Globally, the percentage of older people in the general population is growing. Smart homes have the potential to help older adults to live independently and healthy, improving their quality of life, and relieving the pressure on the healthcare and social care systems. For that, we need to understand how older adults live and their needs. Thus, this study aims to analyze the residentially-based lifestyles (RBL) of older adults and segment them to compare and analyze the real needs of smart home functions for each group. To identify a person's RBL, a questionnaire was designed to include questions about activities at home, social events, quality of life, etc. This study surveyed 271 older Koreans. As a result of the survey on RBL, five groups with different characteristics were clustered. Finally, each groups' features and the differences in their needs for smart home functions were compared and analyzed. The priority of needed functions for each group was found to be significantly different. In a total of 26 smart home functions, there were meaningful differences in the needs for 16 functions among the groups. This study presents the results in South Korea, according to older adults' RBL and their smart home needs.
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Viviendas para Ancianos , Estilo de Vida , Microcomputadores , Calidad de Vida , Anciano , Anciano de 80 o más Años , Atención a la Salud , Femenino , Viviendas para Ancianos/normas , Viviendas para Ancianos/tendencias , Humanos , Masculino , República de Corea , Apoyo SocialRESUMEN
P2X5 is a member of the P2X purinergic receptor family of ligand-gated cation channels and has recently been shown to regulate inflammatory bone loss. In this study, we report that P2X5 is a protective immune regulator during Listeria monocytogenes infection, as P2X5-deficient mice exhibit increased bacterial loads in the spleen and liver, increased tissue damage, and early (within 3-6 d) susceptibility to systemic L. monocytogenes infection. Whereas P2X5-deficient mice experience normal monocyte recruitment in response to L. monocytogenes, P2X5-deficient bone marrow-derived macrophages (BMMs) exhibit defective cytosolic killing of L. monocytogenes We further showed that P2X5 is required for L. monocytogenes-induced inflammasome activation and IL-1ß production and that defective L. monocytogenes killing in P2X5-deficient BMMs is substantially rescued by exogenous IL-1ß or IL-18. Finally, in vitro BMM killing and in vivo L. monocytogenes infection experiments employing either P2X7 deficiency or extracellular ATP depletion suggest that P2X5-dependent anti-L. monocytogenes immunity is independent of the ATP-P2X7 inflammasome activation pathway. Together, our findings elucidate a novel and specific role for P2X5 as a critical mediator of protective immunity.
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Inflamasomas/inmunología , Listeria monocytogenes/inmunología , Listeriosis/inmunología , Macrófagos/inmunología , Monocitos/inmunología , Receptores Purinérgicos P2X5/deficiencia , Adenosina Trifosfato/genética , Adenosina Trifosfato/inmunología , Animales , Susceptibilidad a Enfermedades , Inflamasomas/genética , Interleucina-18/genética , Interleucina-18/inmunología , Interleucina-1beta/genética , Interleucina-1beta/inmunología , Listeriosis/genética , Listeriosis/patología , Macrófagos/patología , Ratones , Ratones Noqueados , Monocitos/patología , Receptores Purinérgicos P2X5/inmunologíaRESUMEN
Osteoclasts are multinucleated, giant cells derived from myeloid progenitors. While receptor activator of NF-κB ligand (RANKL) stimulation is the primary driver of osteoclast differentiation, additional signaling further contributes to osteoclast maturation. Here, we demonstrate that immunoglobulin superfamily member 11 (IgSF11), whose expression increases during osteoclast differentiation, regulates osteoclast differentiation through interaction with postsynaptic density protein 95 (PSD-95), a scaffold protein with multiple protein interaction domains. IgSF11 deficiency in vivo results in impaired osteoclast differentiation and bone resorption but no observed defect in bone formation. Consequently, IgSF11-deficient mice exhibit increased bone mass. Using in vitro osteoclast culture systems, we show that IgSF11 functions through homophilic interactions. Additionally, we demonstrate that impaired osteoclast differentiation in IgSF11-deficient cells is rescued by full-length IgSF11 and that the IgSF11-PSD-95 interaction requires the 75 C-terminal amino acids of IgSF11. Our findings reveal a critical role for IgSF11 during osteoclast differentiation and suggest a role for IgSF11 in a receptor- and signal transduction molecule-containing protein complex.
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Purinergic signaling plays important roles in bone. P2X5, a member of ligand-gated ion channel receptors, has been demonstrated to regulate osteoclast maturation. However, the molecular mechanism of P2X5-mediated osteoclast regulation remains unclear. Here, we identified methylosome protein 50 (MEP50), a critical cofactor of the protein arginine methyltransferase 5 (PRMT5), as a P2X5-associating molecule. RNAi-mediated knockdown of MEP50 results in decreased formation of mature osteoclasts. MEP50 associates with P2X5, and this association requires the C-terminal intracellular region of P2X5. Additionally, impaired maturation of P2X5-deficient osteoclasts could be restored by transduction of full-length P2X5, but not a C-terminal deletion mutant of P2X5. These results indicate that P2X5 associates with MEP50 and suggest a link between the PRMT5 complex and P2X5 signaling in osteoclast maturation.
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Diferenciación Celular , Osteoclastos/metabolismo , Receptores Purinérgicos P2X5/metabolismo , Factores de Transcripción/metabolismo , Animales , Sitios de Unión , Células HEK293 , Humanos , Ratones , Osteoclastos/citología , Unión Proteica , Proteína-Arginina N-Metiltransferasas/metabolismo , Receptores Purinérgicos P2X5/química , Transducción de Señal , Factores de Transcripción/genéticaRESUMEN
Postpartum depression is a severe emotional and mental disorder that involves maternal care defects and psychiatric illness. Postpartum depression is closely associated with a combination of physical changes and physiological stress during pregnancy or after parturition in stress-sensitive women. Although postpartum depression is relatively well known to have deleterious effects on the developing fetus, the influence of genetic risk factors on the development of postpartum depression remains unclear. In this study, we discovered a novel function of T cell death-associated gene 51 (TDAG51/PHLDA1) in the regulation of maternal and depressive-like behavior. After parturition, TDAG51-deficient dams showed impaired maternal behavior in pup retrieving, nursing and nest building tests. In contrast to the normal dams, the TDAG51-deficient dams also exhibited more sensitive depressive-like behaviors after parturition. Furthermore, changes in the expression levels of various maternal and depressive-like behavior-associated genes regulating neuroendocrine factor and monoamine neurotransmitter levels were observed in TDAG51-deficient postpartum brain tissues. These findings indicate that TDAG51 plays a protective role against maternal care defects and depressive-like behavior after parturition. Thus, TDAG51 is a maternal care-associated gene that functions as a crucial regulator of maternal and depressive-like behavior after parturition.
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Trastorno Depresivo/genética , Conducta Materna , Parto/genética , Factores de Transcripción/genética , Animales , Encéfalo/metabolismo , Trastorno Depresivo/fisiopatología , Femenino , Regulación de la Expresión Génica/genética , Humanos , Ratones , Ratones Noqueados , Neurotransmisores/genética , Parto/fisiología , EmbarazoRESUMEN
B lymphocytes are produced from hematopoietic stem cells (HSCs) through the highly ordered process of B lymphopoiesis, which is regulated by a complex network of cytokines, chemokines and cell adhesion molecules derived from the hematopoietic niche. Primary osteoblasts function as an osteoblastic niche (OBN) that supports in vitro B lymphopoiesis. However, there are significant limitations to the use of primary osteoblasts, including their relative scarcity and the consistency and efficiency of the limited purification and proliferation of these cells. Thus, development of a stable osteoblast cell line that can function as a biomimetic or artificial OBN is necessary. In this study, we developed a stable osteoblastic cell line, designated OBN4, which functions as an osteoblast-based artificial niche that supports in vitro B lymphopoiesis. We demonstrated that the production of a B220+ cell population from Lineage- (Lin-) Sca-1+ c-Kit+ hematopoietic stem and progenitor cells (HSPCs) was increased ~1.7-fold by OBN4 cells relative to production by primary osteoblasts and OP9 cells in coculture experiments. Consistently, OBN4 cells exhibited the highest production of B220+ IgM+ cell populations (6.7±0.6-13.6±0.6%) in an IL-7- and stromal cell-derived factor 1-dependent manner, with higher production than primary osteoblasts (3.7±0.5-6.4±0.6%) and OP9 cells (1.8±0.6-3.9±0.5%). In addition, the production of B220+ IgM+ IgD+ cell populations was significantly enhanced by OBN4 cells (15.4±1.1-18.9±3.2%) relative to production by primary osteoblasts (9.5±0.6-14.6±1.6%) and OP9 cells (9.1±0.5-10.3±1.8%). We conclude that OBN4 cells support in vitro B lymphopoiesis of Lin- Sca-1+ c-Kit+ HSPCs more efficiently than primary osteoblasts or OP9 stromal cells.
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Linfocitos B/citología , Linfocitos B/metabolismo , Diferenciación Celular , Linfopoyesis , Animales , Biomarcadores , Línea Celular , Separación Celular , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Inmunofenotipificación , Masculino , Ratones , Osteoblastos/citología , Osteoblastos/metabolismo , Nicho de Células MadreRESUMEN
BACKGROUND: Human prolidase has weak hydrolytic activity for toxic organophosphorus compounds including diisopropyl fluorophosphates (DFP), chemical warfare nerve agents and pesticides. OBJECTIVES: In order to use human prolidase as a catalytic bioscavenger against toxic organophosphorus compound exposure, protein engineering is an important issue to improve the catalytic activity of human prolidase towards the hydrolysis of toxic organophosphorus compounds. METHOD: We developed two human prolidase mutants, A252R and P365R, with a single amino acid substitution using in silico analysis based on the sequence, protein structure and stability to improve the catalytic activity of human prolidase towards DFP hydrolysis. RESULTS: Our results showed that the catalytic efficiencies of A252R and P365R towards DFP hydrolysis were 1.23- and 1.36-fold increases, respectively, than that of the wild type, while the prolidase activities of A252R and P365R towards Leu-Pro hydrolysis were 0.88- and 0.78-fold decreases that of the wild type, respectively, indicating that substitution mutations of A252R and P365R in human prolidase show improved hydrolytic activity for toxic organophosphorus compounds. CONCLUSION: We report here that by introducing either the A252R or P365R substitution mutation, the structural changes affecting catalytic turnover rate and substrate binding affinity are valuable in improving the catalytic activity of human prolidase towards toxic organophosphorus compound hydrolysis.
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Catálisis , Dipeptidasas/química , Proteínas Mutantes/química , Sustitución de Aminoácidos , Sustancias para la Guerra Química/química , Sustancias para la Guerra Química/toxicidad , Dipeptidasas/genética , Humanos , Hidrólisis , Proteínas Mutantes/genética , Compuestos Organofosforados/química , Compuestos Organofosforados/toxicidad , Ingeniería de ProteínasRESUMEN
Many pesticides and chemical warfare nerve agents are highly toxic organophosphorus compounds (OPs), which inhibit acetylcholinesterase activity. Human paraoxonase 1 (PON1) has demonstrated significant potential for use as a catalytic bioscavenger capable of hydrolyzing a broad range of OPs. However, there are several limitations to the use of human PON1 as a catalytic bioscavenger, including the relatively difficult purification of PON1 from human plasma and its dependence on the presence of hydrophobic binding partners to maintain stability. Therefore, research efforts to efficiently produce recombinant human PON1 are necessary. In this study, we developed a Drosophila S2 stable cell line expressing recombinant human PON1. The recombinant human PON1 was fused with the human immunoglobulin Fc domain (PON1-hFc) to improve protein stability and purification efficiency. We purified the recombinant human PON1-hFc from the S2 stable cell line and characterized its enzymatic properties for OP hydrolysis. We purified the recombinant human PON1-hFc from the S2 stable cell line and characterized its enzymatic properties for OP hydrolysis compared with those of the recombinant human PON1 derived from E. coli. We observed that the recombinant human PON1-hFc is functionally more stable for OP hydrolyzing activities compared to the recombinant human PON1. The catalytic efficiency of the recombinant PON1-hFc towards diisopropyl fluorophosphate (DFP, 0.26 × 106 M-1 min-1) and paraoxon hydrolysis (0.015 × 106 M-1 min-1) was 1.63- and 1.24-fold higher, respectively, than the recombinant human PON1. Thus, we report that the recombinant PON1-hFc exerts hydrolytic activity against paraoxon and DFP.