RESUMEN
Immunoglobulin G (IgG) antibodies are major drivers of inflammation during infectious and autoimmune diseases. In pooled serum IgG (IVIg), however, antibodies have a potent immunomodulatory and anti-inflammatory activity, but how this is mediated is unclear. We studied IgG-dependent initiation of resolution of inflammation in cytokine- and autoantibody-driven models of rheumatoid arthritis and found IVIg sialylation inhibited joint inflammation, whereas inhibition of osteoclastogenesis was sialic acid independent. Instead, IVIg-dependent inhibition of osteoclastogenesis was abrogated in mice lacking receptors Dectin-1 or FcγRIIb. Atomistic molecular dynamics simulations and super-resolution microscopy revealed that Dectin-1 promoted FcγRIIb membrane conformations that allowed productive IgG binding and enhanced interactions with mouse and human IgG subclasses. IVIg reprogrammed monocytes via FcγRIIb-dependent signaling that required Dectin-1. Our data identify a pathogen-independent function of Dectin-1 as a co-inhibitory checkpoint for IgG-dependent inhibition of mouse and human osteoclastogenesis. These findings may have implications for therapeutic targeting of autoantibody and cytokine-driven inflammation.
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Artritis Reumatoide , Inmunoglobulinas Intravenosas , Lectinas Tipo C , Receptores de IgG , Animales , Humanos , Ratones , Artritis Reumatoide/tratamiento farmacológico , Artritis Reumatoide/inmunología , Membrana Celular/metabolismo , Inmunoglobulinas Intravenosas/administración & dosificación , Lectinas Tipo C/metabolismo , Ratones Endogámicos C57BL , Osteoclastos/metabolismo , Procesamiento Proteico-Postraduccional , Receptores de IgG/metabolismoRESUMEN
Periosteal stem and progenitor cells (PSPCs) are major contributors to bone maintenance and repair. Deciphering the molecular mechanisms that regulate their function is crucial for the successful generation and application of future therapeutics. Here, we pinpoint Hox transcription factors as necessary and sufficient for periosteal stem cell function. Hox genes are transcriptionally enriched in periosteal stem cells and their overexpression in more committed progenitors drives reprogramming to a naïve, self-renewing stem cell-like state. Crucially, individual Hox family members are expressed in a location-specific manner and their stem cell-promoting activity is only observed when the Hox gene is matched to the anatomical origin of the PSPC, demonstrating a role for the embryonic Hox code in adult stem cells. Finally, we demonstrate that Hoxa10 overexpression partially restores the age-related decline in fracture repair. Together, our data highlight the importance of Hox genes as key regulators of PSPC identity in skeletal homeostasis and repair.
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Células Madre Adultas , Genes Homeobox , Humanos , Adulto , Genes Homeobox/genética , Proteínas de Homeodominio/genética , Células Madre , HuesosRESUMEN
Macroautophagy is a lysosomal degradative pathway essential for neuron survival. Here, we show that macroautophagy requires the Alzheimer's disease (AD)-related protein presenilin-1 (PS1). In PS1 null blastocysts, neurons from mice hypomorphic for PS1 or conditionally depleted of PS1, substrate proteolysis and autophagosome clearance during macroautophagy are prevented as a result of a selective impairment of autolysosome acidification and cathepsin activation. These deficits are caused by failed PS1-dependent targeting of the v-ATPase V0a1 subunit to lysosomes. N-glycosylation of the V0a1 subunit, essential for its efficient ER-to-lysosome delivery, requires the selective binding of PS1 holoprotein to the unglycosylated subunit and the Sec61alpha/oligosaccharyltransferase complex. PS1 mutations causing early-onset AD produce a similar lysosomal/autophagy phenotype in fibroblasts from AD patients. PS1 is therefore essential for v-ATPase targeting to lysosomes, lysosome acidification, and proteolysis during autophagy. Defective lysosomal proteolysis represents a basis for pathogenic protein accumulations and neuronal cell death in AD and suggests previously unidentified therapeutic targets.
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Enfermedad de Alzheimer/metabolismo , Autofagia , Lisosomas/metabolismo , Presenilina-1/genética , Presenilina-1/metabolismo , Proteínas/metabolismo , Enfermedad de Alzheimer/patología , Animales , Blastocisto/metabolismo , Línea Celular , Eliminación de Gen , Técnicas de Inactivación de Genes , Glicosilación , Humanos , Hidrólisis , Ratones , Ratones Noqueados , Neuronas/metabolismo , ATPasas de Translocación de Protón Vacuolares/metabolismo , Vacuolas/metabolismoRESUMEN
Quantum dots (QDs) have garnered a significant amount of attention as promising memristive materials owing to their size-dependent tunable bandgap, structural stability, and high level of applicability for neuromorphic computing. Despite these advantageous properties, the development of QD-based memristors has been hindered by challenges in understanding and adjusting the resistive switching (RS) behavior of QDs. Herein, we propose three types of InP/ZnSe/ZnS QD-based memristors to elucidate the RS mechanism, employing a thin poly(methyl methacrylate) layer. This approach not only allows us to identify which carriers (electron or hole) are trapped within the QD layer but also successfully demonstrates QD-based synaptic devices. Furthermore, to utilize the QD memristor as a synapse, long-term potentiation/depression (LTP/LTD) characteristics are measured, resulting in a low nonlinearity of LTP/LTD at 0.1/1. On the basis of the LTP/LTD characteristics, single-layer perceptron simulations were performed using the Extended Modified National Institute of Standards and Technology, verifying a maximum recognition rate of 91.46%.
RESUMEN
Type II toxin-antitoxin (TA) systems are ubiquitously distributed genetic elements in prokaryotes and are crucial for cell maintenance and survival under environmental stresses. The antitoxin is a modular protein consisting of the disordered C-terminal region that physically contacts and neutralizes the cognate toxin and the well-folded N-terminal DNA binding domain responsible for autorepression of TA transcription. However, how the two functional domains communicate is largely unknown. Herein, we determined the crystal structure of the N-terminal domain of the type II antitoxin MazE-mt10 from Mycobacterium tuberculosis, revealing a homodimer of the ribbon-helix-helix (RHH) fold with distinct DNA binding specificity. NMR studies demonstrated that full-length MazE-mt10 forms the helical and coiled states in equilibrium within the C-terminal region, and that helical propensity is allosterically enhanced by the N-terminal binding to the cognate operator DNA. This coil-to-helix transition may promote toxin binding/neutralization of MazE-mt10 and further stabilize the TA-DNA transcription repressor. This is supported by many crystal structures of type II TA complexes in which antitoxins form an α-helical structure at the TA interface. The hidden helical state of free MazE-mt10 in solution, favored by DNA binding, adds a new dimension to the regulatory mechanism of type II TA systems. Furthermore, complementary approaches using X-ray crystallography and NMR allow us to study the allosteric interdomain interplay of many other full-length antitoxins of type II TA systems.
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Antitoxinas , Mycobacterium tuberculosis , Mycobacterium tuberculosis/metabolismo , Antitoxinas/química , Modelos Moleculares , Factores de Transcripción/metabolismo , ADN/metabolismo , Proteínas Bacterianas/metabolismoRESUMEN
An electrochemical sensor based on an electroactive nanocomposite was designed for the first time consisting of electrochemically reduced graphene oxide (ERGO), polyaniline (PANI), and poly(alizarin red S) (PARS) for ciprofloxacin (CIPF) detection. The ERGO/PANI/PARS-modified screen-printed carbon electrode (SPCE) was constructed through a three-step electrochemical protocol and characterized using FTIR, UV-visible spectroscopy, FESEM, CV, LSV, and EIS. The new electrochemical CIPF sensor demonstrated a low detection limit of 0.0021 µM, a broad linear range of 0.01 to 69.8 µM, a high sensitivity of 5.09 µA/µM/cm2, and reasonable selectivity and reproducibility. Moreover, the ERGO/PANI/PARS/SPCE was successfully utilized to determine CIPF in milk with good recoveries and relative standard deviation (< 5%), which were close to those with HPLC analysis.
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Compuestos de Anilina , Antraquinonas , Carbono , Ciprofloxacina , Técnicas Electroquímicas , Electrodos , Grafito , Límite de Detección , Leche , Grafito/química , Leche/química , Compuestos de Anilina/química , Técnicas Electroquímicas/métodos , Técnicas Electroquímicas/instrumentación , Animales , Ciprofloxacina/análisis , Carbono/química , Antraquinonas/química , Reproducibilidad de los Resultados , Contaminación de Alimentos/análisis , Antibacterianos/análisisRESUMEN
Self-assemblies (i.e., nanoclusters) of the RAS GTPase on the membrane act as scaffolds that activate downstream RAF kinases and drive MAPK signaling for cell proliferation and tumorigenesis. However, the mechanistic details of nanoclustering remain largely unknown. Here, size-tunable nanodisc platforms and paramagnetic relaxation enhancement (PRE) analyses revealed the structural basis of the cooperative assembly processes of fully processed KRAS, mutated in a quarter of human cancers. The cooperativity is modulated by the mutation and nucleotide states of KRAS and the lipid composition of the membrane. Notably, the oncogenic mutants assemble in nonsequential pathways with two mutually cooperative 'α/α' and 'α/ß' interfaces, while α/α dimerization of wild-type KRAS promotes the secondary α/ß interaction sequentially. Mutation-based interface engineering was used to selectively trap the oligomeric intermediates of KRAS and probe their favorable interface interactions. Transiently exposed interfaces were available for the assembly. Real-time NMR demonstrated that higher-order oligomers retain higher numbers of active GTP-bound protomers in KRAS GTPase cycling. These data provide a deeper understanding of the nanocluster-enhanced signaling in response to the environment. Furthermore, our methodology is applicable to assemblies of many other membrane GTPases and lipid nanoparticle-based formulations of stable protein oligomers with enhanced cooperativity.
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Proteínas Proto-Oncogénicas p21(ras) , Transducción de Señal , Humanos , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Proteínas ras/química , Quinasas raf/metabolismo , DimerizaciónRESUMEN
The E6 and E7 proteins of specific subtypes of human papillomavirus (HPV), including HPV 16 and 18, are highly associated with cervical cancer as they modulate cell cycle regulation. The aim of this study was to investigate the potential antitumor effects of a messenger RNA-HPV therapeutic vaccine (mHTV) containing nononcogenic E6 and E7 proteins. To achieve this, C57BL/6j mice were injected with the vaccine via both intramuscular and subcutaneous routes, and the resulting effects were evaluated. mHTV immunization markedly induced robust T cell-mediated immune responses and significantly suppressed tumor growth in both subcutaneous and orthotopic tumor-implanted mouse model, with a significant infiltration of immune cells into tumor tissues. Tumor retransplantation at day 62 postprimary vaccination completely halted progression in all mHTV-treated mice. Furthermore, tumor expansion was significantly reduced upon TC-1 transplantation 160 days after the last immunization. Immunization of rhesus monkeys with mHTV elicited promising immune responses. The immunogenicity of mHTV in nonhuman primates provides strong evidence for clinical application against HPV-related cancers in humans. All data suggest that mHTV can be used as both a therapeutic and prophylactic vaccine.
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Proteínas Oncogénicas Virales , Infecciones por Papillomavirus , Vacunas contra Papillomavirus , Neoplasias del Cuello Uterino , Humanos , Femenino , Animales , Ratones , Virus del Papiloma Humano , Proteínas Oncogénicas Virales/genética , Infecciones por Papillomavirus/prevención & control , ARN Mensajero/genética , Proteínas E7 de Papillomavirus/genética , Ratones Endogámicos C57BL , Vacunación/métodos , Inmunización , Neoplasias del Cuello Uterino/prevención & controlRESUMEN
Aging is associated with impaired tissue regeneration. Stem cell number and function have been identified as potential culprits. We first demonstrate a direct correlation between stem cell number and time to bone fracture union in a human patient cohort. We then devised an animal model recapitulating this age-associated decline in bone healing and identified increased cellular senescence caused by a systemic and local proinflammatory environment as the major contributor to the decline in skeletal stem/progenitor cell (SSPC) number and function. Decoupling age-associated systemic inflammation from chronological aging by using transgenic Nfkb1KO mice, we determined that the elevated inflammatory environment, and not chronological age, was responsible for the decrease in SSPC number and function. By using a pharmacological approach inhibiting NF-κB activation, we demonstrate a functional rejuvenation of aged SSPCs with decreased senescence, increased SSPC number, and increased osteogenic function. Unbiased, whole-genome RNA sequencing confirmed the reversal of the aging phenotype. Finally, in an ectopic model of bone healing, we demonstrate a functional restoration of regenerative potential in aged SSPCs. These data identify aging-associated inflammation as the cause of SSPC dysfunction and provide mechanistic insights into its reversal.
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Envejecimiento/metabolismo , Curación de Fractura , Fracturas Óseas/metabolismo , Osteogénesis , Células Madre/metabolismo , Envejecimiento/genética , Envejecimiento/patología , Animales , Femenino , Fracturas Óseas/patología , Humanos , Inflamación/genética , Inflamación/metabolismo , Inflamación/patología , Masculino , Ratones , Ratones Noqueados , Subunidad p50 de NF-kappa B/genética , Subunidad p50 de NF-kappa B/metabolismo , Células Madre/patologíaRESUMEN
Exosomes are membrane vesicles containing proteins, lipids, DNA, mRNA, and micro RNA (miRNA). Exosomal miRNA from donor cells can regulate the gene expression of recipient cells. Here, Ri chickens were divided into resistant (Mx/A; BF2/B21) and susceptible (Mx/G; BF2/B13) trait by genotyping of Mx and BF2 genes. Then, Ri chickens were infected with H5N1, a highly pathogenic avian influenza virus (HPAIV). Exosomes were purified from blood serum of resistant chickens for small RNA sequencing. Sequencing data were analysed using FastQCv0.11.7, Cutadapt 1.16, miRBase v21, non-coding RNA database, RNAcentral 10.0, and miRDeep2. Differentially expressed miRNAs were determined using statistical methods, including fold-change, exactTest using edgeR, and hierarchical clustering. Target genes were predicted using miRDB. Gene ontology analysis was performed using gProfiler. Twenty miRNAs showed significantly different expression patterns between resistant control and infected chickens. Nine miRNAs were up-regulated and 11 miRNAs were down-regulated in the infected chickens compared with that in the control chickens. In target gene analysis, various immune-related genes, such as cytokines, chemokines, and signalling molecules, were detected. In particular, mitogen-activated protein kinase (MAPK) pathway molecules were highly controlled by differentially expressed miRNAs. The result of qRT-PCR for miRNAs was identical with sequencing data and miRNA expression level was higher in resistant than susceptible chickens. This study will help to better understand the host immune response, particularly exosomal miRNA expression against HPAIV H5N1 and could help to determine biomarkers for disease resistance.
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Pollos , Exosomas/genética , Gripe Aviar/virología , MicroARNs/genética , Enfermedades de las Aves de Corral/virología , Animales , Subtipo H5N1 del Virus de la Influenza A/fisiologíaRESUMEN
Programmed cell death 5 (PDCD5) has been associated with human cancers as a regulator of cell death; however, the role of PDCD5 in the endothelium has not been revealed. Thus, we investigated whether PDCD5 regulates protein kinase B (PKB/AKT)-endothelial nitric oxide synthase (eNOS)-dependent signal transduction in the endothelium and affects atherosclerosis. Endothelial-specific PDCD5 knockout mice showed significantly reduced vascular remodeling compared with wild-type (WT) mice after partial carotid ligation. WT PDCD5 competitively inhibited interaction between histone deacetylase 3 (HDAC3) and AKT, but PDCD5L6R, an HDAC3-binding-deficient mutant, did not. Knockdown of PDCD5 accelerated HDAC3-AKT interaction, AKT and eNOS phosphorylation, and nitric oxide (NO) production in human umbilical vein endothelial cells. Moreover, we found that serum PDCD5 levels reflect endothelial NO production and are correlated with diabetes mellitus, high-density lipoprotein cholesterol, and coronary calcium in human samples obtained from the cardiovascular high-risk cohort. Therefore, we conclude that PDCD5 is associated with endothelial dysfunction and may be a novel therapeutic target in atherosclerosis.
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Proteínas Reguladoras de la Apoptosis/metabolismo , Endotelio Vascular/metabolismo , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Remodelación Vascular , Animales , Proteínas Reguladoras de la Apoptosis/genética , Aterosclerosis/genética , Aterosclerosis/metabolismo , Aterosclerosis/patología , HDL-Colesterol/genética , HDL-Colesterol/metabolismo , Diabetes Mellitus/genética , Diabetes Mellitus/metabolismo , Diabetes Mellitus/patología , Endotelio Vascular/patología , Histona Desacetilasas/genética , Histona Desacetilasas/metabolismo , Células Endoteliales de la Vena Umbilical Humana/patología , Humanos , Ratones , Ratones Noqueados , Proteínas de Neoplasias/genética , Óxido Nítrico Sintasa de Tipo III/genética , Óxido Nítrico Sintasa de Tipo III/metabolismo , Fosforilación/genética , Proteínas Proto-Oncogénicas c-akt/genéticaRESUMEN
A mandibular advancement device (MAD) is a commonly used treatment modality for patients with mild-to-moderate obstructive sleep apnea. Although MADs have excellent therapeutic efficacy, dental side effects were observed with long-term use of MADs. The aim of this study was to analyze the force distribution on the entire dentition according to the materials and design of the MADs. Three types of MADs were applied: model 1 (single layer of polyethylene terephthalate glycol (PETG)), model 2 (double layer of PETG + thermoplastic polyurethane (TPU)), and model 3 (core-reinforced multilayer). In the maxilla, regardless of the model, the incisors showed the lowest force distribution. In most tooth positions, the force distribution was lower in models 2 and 3 than in model 1. In the mandible, the mandibular second molar showed a significantly lower force in all models. The mandibular incisors, canines, and molars showed the highest force values in model 1 and the lowest values in model 3. Depending on the material and design of the device, the biomechanical effect on the dentition varies, and the core-reinforced multilayered MAD can reduce the force delivered to the dentition more effectively than the conventional single- or double-layer devices.
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Avance Mandibular , Apnea Obstructiva del Sueño , Humanos , Ferulas Oclusales , Resultado del TratamientoRESUMEN
The absence of functional peripheral myelin protein 22 (PMP22) is associated with shortened lifespan in rodents and severe peripheral nerve myelin abnormalities in several species including humans. Schwann cells and nerves from PMP22 knock-out (KO) mice show deranged cholesterol distribution and aberrant lipid raft morphology, supporting an unrecognized role for PMP22 in cellular lipid metabolism. To examine the mechanisms underlying these abnormalities, we studied Schwann cells and nerves from male and female PMP22 KO mice. Whole-cell current-clamp recordings in cultured Schwann cells revealed increased membrane capacitance and decreased membrane resistance in the absence of PMP22, which was consistent with a reduction in membrane cholesterol. Nerves from PMP22-deficient mice contained abnormal lipid droplets, with both mRNA and protein levels of apolipoprotein E (apoE) and ATP-binding cassette transporter A1 (ABCA1) being highly upregulated. Despite the upregulation of ABCA1 and apoE, the absence of PMP22 resulted in reduced localization of the transporter to the cell membrane and diminished secretion of apoE. The absence of PMP22 also impaired ABCA1-mediated cholesterol efflux capacity. In nerves from ABCA1 KO mice, the expression of PMP22 was significantly elevated and the subcellular processing of the overproduced protein was aberrant. In wild-type samples, double immunolabeling identified overlapping distribution of PMP22 and ABCA1 at the Schwann cell plasma membrane and the two proteins were coimmunoprecipitated from Schwann cell and nerve lysates. Together, these results reveal a novel role for PMP22 in regulating lipid metabolism and cholesterol trafficking through functional interaction with the cholesterol efflux regulatory protein ABCA1.SIGNIFICANCE STATEMENT Understanding the subcellular events that underlie abnormal myelin formation in hereditary neuropathies is critical for advancing therapy development. Peripheral myelin protein 22 (PMP22) is an essential peripheral myelin protein because its genetic abnormalities account for â¼80% of hereditary neuropathies. Here, we demonstrate that in the absence of PMP22, the cellular and electrophysiological properties of the Schwann cells' plasma membrane are altered and cholesterol trafficking and lipid homeostasis are perturbed. The molecular mechanisms for these abnormalities involve a functional interplay among PMP22, cholesterol, apolipoprotein E, and the major cholesterol-efflux transporter protein ATP-binding cassette transporter A1 (ABCA1). These findings establish a critical role for PMP22 in the maintenance of cholesterol homeostasis in Schwann cells.
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Transportador 1 de Casete de Unión a ATP/metabolismo , Membrana Celular/metabolismo , Colesterol/metabolismo , Metabolismo de los Lípidos , Proteínas de la Mielina/metabolismo , Células de Schwann/metabolismo , Animales , Transporte Biológico , Células Cultivadas , Femenino , Regulación de la Expresión Génica , Masculino , Potenciales de la Membrana , Ratones Noqueados , Proteínas de la Mielina/genética , Neuronas/metabolismoRESUMEN
A common form of hereditary autosomal dominant demyelinating neuropathy known as Charcot-Marie-Tooth disease type 1A (CMT1A) is linked with duplication of the peripheral myelin protein 22 (PMP22) gene. Although studies from animal models have led to better understanding of the pathobiology of these neuropathies, there continues to be a gap in the translation of findings from rodents to humans. Because PMP22 was originally identified in fibroblasts as growth arrest specific gene 3 (gas3) and is expressed broadly in the body, it was tested whether skin cells from neuropathic patients would display the cellular pathology observed in Schwann cells from rodent models. Dermal fibroblasts from two CMT1A pedigrees with confirmed PMP22 gene duplication were studied. Samples from age-matched non-neuropathic individuals were used as controls. CMT1A patient-derived cultures contain approximately 1.5-fold elevated levels of PMP22 mRNA, exhibit reduced mitotic potential, and display intracellular protein aggregates as compared to cells from unaffected individuals. The presence of cytosolic PMP22 coincides with a decrease in proteasome activity and an increase in autophagy-lysosomal proteins, including LC3-II and LAMP1. These results indicate that the abnormalities in the subcellular processing of excess PMP22 elicit a detectable response in human CMT1A fibroblasts, a phenotype that resembles Schwann cells from neuropathic mice. These findings support the use of human CMT1A fibroblasts as a platform for therapy testing.
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Enfermedad de Charcot-Marie-Tooth/metabolismo , Fibroblastos/metabolismo , Proteínas de la Mielina/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Piel/metabolismo , Adolescente , Adulto , Proliferación Celular/fisiología , Enfermedad de Charcot-Marie-Tooth/patología , Fibroblastos/patología , Duplicación de Gen , Humanos , Persona de Mediana Edad , Piel/patología , Adulto JovenRESUMEN
Colorectal cancer (CRC) is the third leading cause of cancer-related death worldwide. The prognosis and overall survival of CRC are known to be significantly correlated with the overexpression of PD-L1. Since combination therapies can significantly improve therapeutic efficacy, we constructed doxorubicin (DOX) conjugated and anti-PD-L1 targeting gold nanoparticles (PD-L1-AuNP-DOX) for the targeted chemo-photothermal therapy of CRC. DOX and anti-PD-L1 antibody were conjugated to the α-terminal end group of lipoic acid polyethylene glycol N-hydroxysuccinimide (LA-PEG-NHS) using an amide linkage, and PD-L1-AuNP-DOX was constructed by linking LA-PEG-DOX, LA-PEG-PD-L1, and a short PEG chain on the surface of AuNP using thiol-Au covalent bonds. Physicochemical characterizations and biological studies of PD-L1-AuNP-DOX were performed in the presence of near-infrared (NIR) irradiation (biologic studies were conducted using cellular uptake, apoptosis, and cell cycle assays in CT-26 cells). PD-L1-AuNP-DOX (40.0 ± 3.1 nm) was successfully constructed and facilitated the efficient intracellular uptake of DOX as evidenced by pronounced apoptotic effects (66.0%) in CT-26 cells. PD-L1-AuNP-DOX treatment plus NIR irradiation significantly and synergistically suppressed the in vitro proliferation of CT-26 cells by increasing apoptosis and cell cycle arrest. The study demonstrates that PD-L1-AuNP-DOX in combination with synergistic targeted chemo-photothermal therapy has a considerable potential for the treatment of localized CRC.
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Anticuerpos/uso terapéutico , Antígeno B7-H1/inmunología , Neoplasias Colorrectales/tratamiento farmacológico , Doxorrubicina/uso terapéutico , Oro/química , Nanopartículas del Metal/química , Fotoquimioterapia/métodos , Animales , Anticuerpos/administración & dosificación , Apoptosis/efectos de los fármacos , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Terapia Combinada/métodos , Sistemas de Liberación de Medicamentos/métodos , Ratones , Polietilenglicoles/química , Especies Reactivas de Oxígeno/metabolismo , Succinimidas/química , Ácido Tióctico/químicaRESUMEN
An important feature of the mammary gland is its ability to undergo profound morphological, physiological, and intracellular changes to establish and maintain secretory function. During this process, key polarity proteins and receptors are recruited to the surface of mammary epithelial cells (MECs), and the vesicle transport system develops and matures. However, the intracellular mechanisms responsible for the development of secretory function in these cells are unclear. The vesicular zinc (Zn2+) transporter ZnT2 is critical for appropriate mammary gland architecture, and ZnT2 deletion is associated with cytoplasmic Zn2+ accumulation, loss of secretory function and lactation failure. The underlying mechanisms are important to understand as numerous mutations and non-synonymous genetic variation in ZnT2 have been detected in women that result in severe Zn2+ deficiency in exclusively breastfed infants. Here we found that ZnT2 deletion in lactating mice and cultured MECs resulted in Zn2+-mediated degradation of phosphatase and tensin homolog (PTEN), which impaired intercellular junction formation, prolactin receptor trafficking, and alveolar lumen development. Moreover, ZnT2 directly interacted with vacuolar H+-ATPase (V-ATPase), and ZnT2 deletion impaired vesicle biogenesis, acidification, trafficking, and secretion. In summary, our findings indicate that ZnT2 and V-ATPase interact and that this interaction critically mediates polarity establishment, alveolar development, and secretory function in the lactating mammary gland. Our observations implicate disruption in ZnT2 function as a modifier of secretory capacity and lactation performance.
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Proteínas de Transporte de Catión/metabolismo , Proteínas de Transporte de Catión/fisiología , ATPasas de Translocación de Protón Vacuolares/metabolismo , Animales , Transporte Biológico , Proteínas Portadoras , Polaridad Celular/fisiología , Células Cultivadas , Células Epiteliales/metabolismo , Femenino , Homeostasis , Lactancia/genética , Glándulas Mamarias Animales/metabolismo , Ratones , Ratones Noqueados , Fosfohidrolasa PTEN/metabolismo , Vías Secretoras , ATPasas de Translocación de Protón Vacuolares/fisiología , Zinc/metabolismoRESUMEN
Pompe disease is a systemic metabolic disorder characterized by lack of acid-alpha glucosidase (GAA) resulting in ubiquitous lysosomal glycogen accumulation. Respiratory and ambulatory dysfunction are prominent features in patients with Pompe yet the mechanism defining the development of muscle weakness is currently unclear. Transgenic animal models of Pompe disease mirroring the patient phenotype have been invaluable in mechanistic and therapeutic study. Here, we demonstrate significant pathological alterations at neuromuscular junctions (NMJs) of the diaphragm and tibialis anterior muscle as prominent features of disease pathology in Gaa knockout mice. Postsynaptic defects including increased motor endplate area and fragmentation were readily observed in Gaa(-/-) but not wild-type mice. Presynaptic neuropathic changes were also evident, as demonstrated by significant reduction in the levels of neurofilament proteins, and alterations in axonal fiber diameter and myelin thickness within the sciatic and phrenic nerves. Our data suggest the loss of NMJ integrity is a primary contributor to the decline in respiratory and ambulatory function in Pompe and arises from both pre- and postsynaptic pathology. These observations highlight the importance of systemic phenotype correction, specifically restoration of GAA to skeletal muscle and the nervous system for treatment of Pompe disease.
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Enfermedad del Almacenamiento de Glucógeno Tipo II/patología , Glicoproteínas de Membrana/metabolismo , Músculo Esquelético/patología , Unión Neuromuscular/patología , Nervio Frénico/patología , Animales , Diafragma/metabolismo , Diafragma/patología , Modelos Animales de Enfermedad , Enfermedad del Almacenamiento de Glucógeno Tipo II/genética , Enfermedad del Almacenamiento de Glucógeno Tipo II/metabolismo , Humanos , Glicoproteínas de Membrana/genética , Ratones , Ratones Noqueados , Músculo Esquelético/metabolismo , Unión Neuromuscular/metabolismo , Nervio Frénico/metabolismo , Tibia/metabolismo , Tibia/patologíaRESUMEN
Hepatic steatosis prevails each year. Autophagy is integral in mitochondrial quality control and lipid homeostasis in the liver. No pharmacological strategies are currently available to reduce hepatic steatosis, but exercise has been known to improve clinical outcomes of chronic liver disease, particularly nonalcoholic fatty liver disease (NAFLD). Recent studies suggest that exercise may improve NAFLD through enhancing autophagy.
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Autofagia/fisiología , Ejercicio Físico/fisiología , Enfermedad del Hígado Graso no Alcohólico/fisiopatología , Humanos , Metabolismo de los Lípidos , Hígado/metabolismo , Mitocondrias Hepáticas/metabolismo , Músculo Esquelético/metabolismo , Enfermedad del Hígado Graso no Alcohólico/metabolismoRESUMEN
It has been hypothesized that α-synuclein (αS) misfolding may begin in peripheral nerves and spread to the central nervous system (CNS), leading to Parkinson disease and related disorders. Although recent data suggest that αS pathology can spread within the mouse brain, there is no direct evidence for spread of disease from a peripheral site. In the present study, we show that hind limb intramuscular (IM) injection of αS can induce pathology in the CNS in the human Ala53Thr (M83) and wild-type (M20) αS transgenic (Tg) mouse models. Within 2-3 mo after IM injection in αS homozygous M83 Tg mice and 3-4 mo for hemizygous M83 Tg mice, these animals developed a rapid, synchronized, and predictable induction of widespread CNS αS inclusion pathology, accompanied by astrogliosis, microgliosis, and debilitating motor impairments. In M20 Tg mice, starting at 4 mo after IM injection, we observed αS inclusion pathology in the spinal cord, but motor function remained intact. Transection of the sciatic nerve in the M83 Tg mice significantly delayed the appearance of CNS pathology and motor symptoms, demonstrating the involvement of retrograde transport in inducing αS CNS inclusion pathology. Outside of scrapie-mediated prion disease, to our knowledge, this findiing is the first evidence that an entire neurodegenerative proteinopathy associated with a robust, lethal motor phenotype can be initiated by peripheral inoculation with a pathogenic protein. Furthermore, this facile, synchronized rapid-onset model of α-synucleinopathy will be highly valuable in testing disease-modifying therapies and dissecting the mechanism(s) that drive αS-induced neurodegeneration.
Asunto(s)
Sistema Nervioso Central/metabolismo , Sistema Nervioso Central/patología , Actividad Motora , alfa-Sinucleína/administración & dosificación , alfa-Sinucleína/metabolismo , Animales , Sistema Nervioso Central/fisiopatología , Humanos , Inyecciones Intramusculares , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Fenotipo , Análisis de SupervivenciaRESUMEN
The zinc transporter ZnT2 (SLC30A2) imports zinc into vesicles in secreting mammary epithelial cells (MECs) and is critical for zinc efflux into milk during lactation. Recent studies show that ZnT2 also imports zinc into mitochondria and is expressed in the non-lactating mammary gland and non-secreting MECs, highlighting the importance of ZnT2 in general mammary gland biology. In this study we used nulliparous and lactating ZnT2-null mice and characterized the consequences on mammary gland development, function during lactation, and milk composition. We found that ZnT2 was primarily expressed in MECs and to a limited extent in macrophages in the nulliparous mammary gland and loss of ZnT2 impaired mammary expansion during development. Secondly, we found that lactating ZnT2-null mice had substantial defects in mammary gland architecture and MEC function during secretion, including fewer, condensed and disorganized alveoli, impaired Stat5 activation, and unpolarized MECs. Loss of ZnT2 led to reduced milk volume and milk containing less protein, fat, and lactose compared with wild-type littermates, implicating ZnT2 in the regulation of mammary differentiation and optimal milk production during lactation. Together, these results demonstrate that ZnT2-mediated zinc transport is critical for mammary gland function, suggesting that defects in ZnT2 not only reduce milk zinc concentration but may compromise breast health and increase the risk for lactation insufficiency in lactating women.