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Background and Objectives: This single-center study aimed to assess the role of laparoscopic greater curvature plication (LGCP) in bariatric surgery. Materials and Methods: Using data from our institution's prospectively maintained database, we identified adult patients with obesity who underwent either laparoscopic sleeve gastrectomy (LSG) or LGCP between January 2012 and July 2017. In total, 280 patients were enrolled in this study. Results: The body mass index was higher in the LSG group than in the LGCP group (39.3 vs. 33.3, p < 0.001). Both groups achieved significant weight loss during the 3-year follow-up (p < 0.001). The weight-reduction rate was higher in the LSG group than in the LGCP group 6, 12, and 24 months postoperatively (p = 0.001, 0.001, and 0.012, respectively). The reoperation rate of the LGCP group was higher than that of the LSG group (p = 0.001). No deaths were recorded in either group. Conclusions: Although both the LGCP and LSG groups achieved significant weight loss over three years, the LGCP group demonstrated a lower weight-reduction rate and a higher reoperation rate than the LSG group. Thus, it is necessary to reassess the role of LGCP in bariatric surgery, particularly when LSG is a feasible alternative.
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Cirugía Bariátrica , Gastroplastia , Laparoscopía , Obesidad Mórbida , Adulto , Índice de Masa Corporal , Gastrectomía , Humanos , Obesidad Mórbida/cirugía , Complicaciones Posoperatorias/cirugía , Estudios Retrospectivos , Resultado del Tratamiento , Pérdida de PesoRESUMEN
Parkinson's disease is a neurodegenerative disorder with a multifactorial aetiology. Nevertheless, the genetic predisposition in many families with multi-incidence disease remains unknown. This study aimed to identify novel genes that cause familial Parkinson's disease. Whole exome sequencing was performed in three affected members of the index family with a late-onset autosomal-dominant parkinsonism and polyneuropathy. We identified a novel heterozygous substitution c.941A>C (p.Tyr314Ser) in the mitochondrial ubiquinol-cytochrome c reductase core protein 1 (UQCRC1) gene, which co-segregates with disease within the family. Additional analysis of 699 unrelated Parkinson's disease probands with autosomal-dominant Parkinson's disease and 1934 patients with sporadic Parkinson's disease revealed another two variants in UQCRC1 in the probands with familial Parkinson's disease, c.931A>C (p.Ile311Leu) and an allele with concomitant splicing mutation (c.70-1G>A) and a frameshift insertion (c.73_74insG, p.Ala25Glyfs*27). All substitutions were absent in 1077 controls and the Taiwan Biobank exome database from healthy participants (n = 1517 exomes). We then assayed the pathogenicity of the identified rare variants using CRISPR/Cas9-based knock-in human dopaminergic SH-SY5Y cell lines, Drosophila and mouse models. Mutant UQCRC1 expression leads to neurite degeneration and mitochondrial respiratory chain dysfunction in SH-SY5Y cells. UQCRC1 p.Tyr314Ser knock-in Drosophila and mouse models exhibit age-dependent locomotor defects, dopaminergic neuronal loss, peripheral neuropathy, impaired respiratory chain complex III activity and aberrant mitochondrial ultrastructures in nigral neurons. Furthermore, intraperitoneal injection of levodopa could significantly improve the motor dysfunction in UQCRC1 p.Tyr314Ser mutant knock-in mice. Taken together, our in vitro and in vivo studies support the functional pathogenicity of rare UQCRC1 variants in familial parkinsonism. Our findings expand an additional link of mitochondrial complex III dysfunction in Parkinson's disease.
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Mitocondrias/genética , Trastornos Parkinsonianos/genética , Polineuropatías/genética , Edad de Inicio , Anciano , Animales , Antiparkinsonianos/uso terapéutico , Línea Celular , Aberraciones Cromosómicas , Drosophila , Complejo III de Transporte de Electrones/genética , Femenino , Mutación del Sistema de Lectura , Técnicas de Sustitución del Gen , Genes Dominantes , Humanos , Levodopa/uso terapéutico , Masculino , Ratones , Persona de Mediana Edad , Mutación/genética , Trastornos Parkinsonianos/complicaciones , Trastornos Parkinsonianos/tratamiento farmacológico , Linaje , Polineuropatías/etiología , Secuenciación del ExomaAsunto(s)
Divertículo/diagnóstico , Enfermedades del Yeyuno/diagnóstico , Mesenterio/patología , Enfermedades Peritoneales/patología , Anciano , Divertículo/complicaciones , Humanos , Vólvulo Intestinal/diagnóstico por imagen , Enfermedades del Yeyuno/complicaciones , Masculino , Mesenterio/diagnóstico por imagen , Tomografía Computarizada por Rayos XRESUMEN
Monoclonal antibodies have become the fastest growing protein therapeutics in recent years. The stability and heterogeneity pertaining to its physical and chemical structures remain a big challenge. Tryptophan fluorescence has been proven to be a versatile tool to monitor protein tertiary structure. By modeling the tryptophan fluorescence emission envelope with log-normal distribution curves, the quantitative measure can be exercised for the routine characterization of monoclonal antibody overall tertiary structure. Furthermore, the log-normal deconvolution results can be presented as a two-dimensional plot with tryptophan emission bandwidth vs. emission maximum to enhance the resolution when comparing samples or as a function of applied perturbations. We demonstrate this by studying four different monoclonal antibodies, which show the distinction on emission bandwidth-maximum plot despite their similarity in overall amino acid sequences and tertiary structures. This strategy is also used to demonstrate the tertiary structure comparability between different lots manufactured for one of the monoclonal antibodies (mAb2). In addition, in the unfolding transition studies of mAb2 as a function of guanidine hydrochloride concentration, the evolution of the tertiary structure can be clearly traced in the emission bandwidth-maximum plot.
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Anticuerpos Monoclonales/química , Triptófano/química , Fluorescencia , Guanidina , Desnaturalización Proteica , Estructura Terciaria de Proteína , Espectrometría de Fluorescencia/métodosRESUMEN
UNLABELLED: Vaccinia virus, the prototype of the Orthopoxvirus genus in the family Poxviridae, infects a wide range of cell lines and animals. Vaccinia mature virus particles of the WR strain reportedly enter HeLa cells through fluid-phase endocytosis. However, the intracellular trafficking process of the vaccinia mature virus between cellular uptake and membrane fusion remains unknown. We used live imaging of single virus particles with a combination of various cellular vesicle markers, to track fluorescent vaccinia mature virus particle movement in cells. Furthermore, we performed functional interference assays to perturb distinct vesicle trafficking processes in order to delineate the specific route undertaken by vaccinia mature virus prior to membrane fusion and virus core uncoating in cells. Our results showed that vaccinia virus traffics to early endosomes, where recycling endosome markers Rab11 and Rab22 are recruited to participate in subsequent virus trafficking prior to virus core uncoating in the cytoplasm. Furthermore, we identified WASH-VPEF/FAM21-retromer complexes that mediate endosome fission and sorting of virus-containing vesicles prior to virus core uncoating in the cytoplasm. IMPORTANCE: Vaccinia mature virions of the WR strain enter HeLa cells through fluid phase endocytosis. We previously demonstrated that virus-containing vesicles are internalized into phosphatidylinositol 3-phosphate positive macropinosomes, which are then fused with Rab5-positive early endosomes. However, the subsequent process of sorting the virion-containing vesicles prior to membrane fusion remains unclear. We dissected the intracellular trafficking pathway of vaccinia mature virions in cells up to virus core uncoating in cytoplasm. We show that vaccinia mature virions first travel to early endosomes. Subsequent trafficking events require the important endosome-tethered protein VPEF/FAM21, which recruits WASH and retromer protein complexes to the endosome. There, the complex executes endosomal membrane fission and cargo sorting to the Rab11-positive and Rab22-positive recycling pathway, resulting in membrane fusion and virus core uncoating in the cytoplasm.
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Complejos Multiproteicos/fisiología , Virus Vaccinia/fisiología , Proteínas de Transporte Vesicular/metabolismo , Transporte Biológico/fisiología , Proteínas Portadoras/metabolismo , Cartilla de ADN/genética , Fluorescencia , Vectores Genéticos/genética , Células HeLa , Humanos , Proteínas de Microfilamentos/metabolismo , Microscopía Confocal , Mutagénesis , ARN Interferente Pequeño/genética , Proteínas de Transporte Vesicular/genética , Virión/metabolismo , Proteínas de Unión al GTP rab/metabolismoRESUMEN
Therapeutic antibodies play an important role in the public healthcare system to treat patients with a variety of diseases. Protein characterization using an array of analytical tools provides in-depth information for drug quality, safety, efficacy, and the further understanding of the molecule. A therapeutic antibody candidate MAB1 exhibits unique binding properties to both cation and anion exchange columns at neutral pH. This uniqueness disrupts standard purification processes and necessitates adjustments in manufacturing. This study identifies that the charge heterogeneity of MAB1 is primarily due to the N-terminal cyclization of glutamine to pyroglutamine and, to a lesser extent, succinimide intermediate, deamidation, and C-terminal lysine. Using three approaches, i.e., deferential chemical labeling, H/D exchange, and molecular modeling, the binding to anion exchange resins is attributed to negatively charged patches on the antibody's surface, involving specific carboxylic acid residues. The methodologies shown here can be extended to study protein binding orientation in column chromatography.
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Objectives: Staying physically active is a cost-efficient strategy for disease prevention during a pandemic. The purposes of this study were to explore precautionary behaviors, psychological factors associated with physical activity and sedentary behavior, and impacts of active and sedentary lifestyles on the quality of life in the early stage of the coronavirus disease 2019 (COVID-19) outbreak. Methods: Participants were community-dwelling adults aged over 20 years who had not been infected with COVID-19 and who lived in the United States. A study with a cross-sectional design was conducted between July and October 2020. Quantitative data were collected by a self-reported questionnaire. Results: In total, 467 valid responses were obtained. Participants who engaged in an active lifestyle had significantly higher scores on all domains of quality of life compared to those who engaged in an inactive lifestyle. Participants with a non-sedentary lifestyle had significantly higher scores of psychological and social domains of quality of life than those with a sedentary lifestyle. Conclusion: Engaging in an active lifestyle and avoiding a sedentary lifestyle are recommended when facing future, unpredictable pandemics similar to COVID-19.
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COVID-19 , Adulto , Humanos , COVID-19/epidemiología , Conducta Sedentaria , Calidad de Vida , Estudios Transversales , Ejercicio Físico , Estilo de Vida , Brotes de EnfermedadesRESUMEN
BACKGROUND: Mitochondrial membrane proteinâassociated neurodegeneration (MPAN) is a rare genetic disease characterized by progressive neurodegeneration with brain iron accumulations combined with neuronal α-synuclein and tau aggregations. Mutations in C19orf12 have been associated with both autosomal recessive and autosomal dominant inheritance patterns of MPAN. METHODS: We present clinical features and functional evidence from a Taiwanese family with autosomal dominant MPAN caused by a novel heterozygous frameshift and nonsense mutation in C19orf12, c273_274 insA (p.P92Tfs*9). To verify the pathogenicity of the identified variant, we examined the mitochondrial function, morphology, protein aggregation, neuronal apoptosis, and RNA interactome in p.P92Tfs*9 mutant knock-in SH-SY5Y cells created with CRISPR-Cas9 technology. RESULTS: Clinically, the patients with the C19orf12 p.P92Tfs*9 mutation presented with generalized dystonia, retrocollis, cerebellar ataxia, and cognitive decline, starting in their mid-20s. The identified novel frameshift mutation is located in the evolutionarily conserved region of the last exon of C19orf12. In vitro studies revealed that the p.P92Tfs*9 variant is associated with impaired mitochondrial function, reduced ATP production, aberrant mitochondria interconnectivity and ultrastructure. Increased neuronal α-synuclein and tau aggregations, and apoptosis were observed under conditions of mitochondrial stress. Transcriptomic analysis revealed that the expression of genes in clusters related to mitochondrial fission, lipid metabolism, and iron homeostasis pathways was altered in the C19orf12 p.P92Tfs*9 mutant cells compared to control cells. CONCLUSION: Our findings provide clinical, genetic, and mechanistic insight revealing a novel heterozygous C19orf12 frameshift mutation to be a cause of autosomal dominant MPAN, further strengthening the importance of mitochondrial dysfunction in the pathogenesis of MPAN.
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Mutación del Sistema de Lectura , Neuroblastoma , Humanos , Mutación del Sistema de Lectura/genética , alfa-Sinucleína/genética , Linaje , Proteínas Mitocondriales/genética , Mitocondrias/genética , Mitocondrias/metabolismo , Mutación , Proteínas de la Membrana/genética , Hierro/metabolismoRESUMEN
TET2 is a member of the Ten-eleven translocation (Tet) family of DNA dioxygenases that regulate gene expression by promoting DNA demethylation (enzymatic activity) and partnering with chromatin regulatory complexes (nonenzymatic functions). TET2 is highly expressed in the hematopoietic lineage, where its molecular functions are the subject of continuous investigations because of the prevalence of TET2 mutations in hematologic malignancies. Previously, we have implicated Tet2 catalytic and noncatalytic functions in the regulation of myeloid and lymphoid lineages, respectively. However, the impact of these functions of Tet2 on hematopoiesis as the bone marrow ages remains unclear. Here, we conducted comparative transplantations and transcriptomic analyses of 3-, 6-, 9-, and 12-month-old Tet2 catalytic mutant (Mut) and knockout (KO) bone marrow. Tet2 Mut bone marrow of all ages exclusively caused hematopoietic disorders of the myeloid lineage. In contrast, young Tet2 KO bone marrow developed both lymphoid and myeloid diseases, whereas older Tet2 KO bone marrow predominantly elicited myeloid disorders with shorter latency than age-matched Tet2 Mut bone marrow. We identified robust gene dysregulation in Tet2 KO Lin- cells at 6 months that involved lymphoma and myelodysplastic syndrome and/or leukemia-causing genes, many of which were hypermethylated early in life. There was a shift from lymphoid to myeloid gene deregulation in Tet2 KO Lin- cells with age, underpinning the higher incidence of myeloid diseases. These findings expand on the dynamic regulation of bone marrow by Tet2 and show that its catalytic-dependent and -independent roles have distinct impacts on myeloid and lymphoid lineages with age.
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Dioxigenasas , Enfermedades Hematológicas , Neoplasias Hematológicas , Síndromes Mielodisplásicos , Humanos , Lactante , Médula Ósea/metabolismo , Neoplasias Hematológicas/genética , Síndromes Mielodisplásicos/metabolismo , Hematopoyesis/genética , Enfermedades Hematológicas/genética , Dioxigenasas/genética , Dioxigenasas/metabolismo , MutaciónRESUMEN
The aim of current single-center study was to compare the short-term outcome of suction and gauze wiping alone versus the irrigation and suction technique for peritoneal decontamination among patients who underwent laparoscopic repair of PPU. Using data from our institution's prospectively maintained database, 105 patients who underwent laparoscopic repair were enrolled in this study. The participants were further divided into the group who received peritoneal irrigation (irrigation group, n = 67) and group who received gauze wiping and suction only (suction only group, n = 38). The irrigation group had a longer operative time (140 vs. 113 min, p = 0.0001), higher number of drainage tubes (38.8% vs. 0%, p < 0.0001) and a higher incidence of intra-abdominal abscess (10.4% vs. 0%, p = 0.0469) than the suction only group. Peritoneal irrigation may be associated with a prolonged operative time and a higher number of abdominal drains. Meanwhile, gauze wiping and suction may be sufficient for peritoneal decontamination during the laparoscopic repair of PPU as further infectious complications are not observed.
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Laparoscopía , Úlcera Péptica Perforada , Humanos , Succión/métodos , Descontaminación , Resultado del Tratamiento , Laparoscopía/métodos , Úlcera Péptica Perforada/complicaciones , Complicaciones Posoperatorias/etiologíaRESUMEN
Pathogenic variants of the WFS1 gene can cause recessive-inherited Wolfram syndrome or dominant-inherited Wolfram-like syndrome with optic atrophy and hearing impairment. Using the Sendai virus delivery system, we generated induced pluripotent stem cells from the peripheral blood mononuclear cells of a female patient with the WFS1 pathogenic variant c.2051C > T (p.Ala684Val). The resulting induced pluripotent stem cells exhibited a normal karyotype and pluripotency, as confirmed using immunofluorescence staining, and differentiated into three germ layers in vivo. This cellular model provides a useful platform for investigating the pathogenic mechanisms of both blindness and deafness related to WFS1 variants.
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Pérdida Auditiva , Células Madre Pluripotentes Inducidas , Síndrome de Wolfram , Humanos , Femenino , Células Madre Pluripotentes Inducidas/patología , Leucocitos Mononucleares/patología , Pérdida Auditiva/genética , Síndrome de Wolfram/genética , Síndrome de Wolfram/patología , MutaciónRESUMEN
The Inhibitor of disheveled and axin (Idax) and its ortholog the Retinoid inducible nuclear factor (Rinf) are DNA binding proteins with nuclear and cytoplasmic functions. Rinf is expressed in embryonic stem cells (ESCs) where it regulates transcription of the Ten-eleven translocation (Tet) enzymes, promoting neural and suppressing mesendoderm/trophectoderm differentiation. Here, we find that Idax, which is not expressed in ESCs, is induced upon differentiation. Like Rinf, Idax facilitates neural and silences trophectodermal programs. Individual or combined loss of Idax and Rinf led to downregulation of neural and upregulation of trophectoderm markers during differentiation of ESCs to embryoid bodies as well as during directed differentiation of ESCs to neural progenitor cells (NPCs) and trophoblast-like cells. These defects resemble those of Tet-deficient ESCs. Consistently, Tet genes are direct targets of Idax and Rinf, and loss of Idax and Rinf led to downregulation of Tet enzymes during ESC differentiation to NPCs and trophoblast-like cells. While Idax and Rinf single and double knockout (DKO) mice were viable and overtly normal, DKO embryos had reduced expression of several NPC markers in embryonic forebrains and deregulated expression of selected trophoblast markers in placentas. NPCs derived from DKO forebrains had reduced self-renewal while DKO placentas had increased junctional zone and reduced labyrinth layers. Together, our findings establish Idax and Rinf as regulators of Tet enzymes for proper differentiation of ESCs.
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Proteínas de Unión al ADN/metabolismo , Células-Madre Neurales , Animales , Diferenciación Celular/genética , Proteínas de Unión al ADN/genética , Cuerpos Embrioides/metabolismo , Células Madre Embrionarias/metabolismo , Ratones , Células-Madre Neurales/metabolismoRESUMEN
Pathogenic variants of OPA1 have been associated with autosomal dominant optic atrophy (DOA), leading to optic, auditory, and other sensorineural neuropathies and myopathies. Using the Sendai virus delivery system, we generated induced pluripotent stem cells from the peripheral blood mononuclear cells of a female patient with the OPA1 pathogenic variant c.1468T>C (p.Cys490Arg). The resulting induced pluripotent stem cells exhibited a normal karyotype and pluripotency, as confirmed using immunofluorescence staining, and differentiated into three germ layers in vivo. This cellular model is a useful platform for investigating the pathogenic mechanisms of both blindness and deafness related to OPA1 variants.
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Pérdida Auditiva , Células Madre Pluripotentes Inducidas , Atrofia Óptica Autosómica Dominante , Humanos , Femenino , Células Madre Pluripotentes Inducidas/patología , Leucocitos Mononucleares/patología , Mutación , Atrofia Óptica Autosómica Dominante/genética , Atrofia Óptica Autosómica Dominante/patología , Pérdida Auditiva/genética , GTP Fosfohidrolasas/genéticaRESUMEN
Single-molecule microscopy has evolved into the ultimate-sensitivity toolkit to study systems from small molecules to living cells, with the prospect of revolutionizing the modern biosciences. Here we survey the current state of the art in single-molecule tools including fluorescence spectroscopy, tethered particle microscopy, optical and magnetic tweezers, and atomic force microscopy. We also provide guidelines for choosing the right approach from the available single-molecule toolkit for applications as diverse as structural biology, enzymology, nanotechnology and systems biology.
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Microscopía de Fuerza Atómica/métodos , Microscopía/métodos , Espectrometría de Fluorescencia/métodos , Animales , Biotecnología/métodos , Diseño de Equipo , Humanos , Procesamiento de Imagen Asistido por Computador , Microscopía Fluorescente/métodos , Modelos Estadísticos , Nanotecnología/métodos , Pinzas Ópticas , Sensibilidad y Especificidad , Programas InformáticosRESUMEN
Neurodegeneration with brain iron accumulation (NBIA) is a genetically and phenotypically heterogeneous group of inherited neurodegenerative disorder characterized by basal ganglia iron deposition. Mutations in Pantothenate Kinase 2 (PANK2) are major genetic causes for patients with NBIA. The location of PANK2 in the mitochondria suggests mutant PANK2 causing mitochondrial dysfunction in the pathogenesis of NBIA. Here, we used the Sendai virus delivery system to generate induced pluripotent stem cells (iPSCs) from peripheral blood mononuclear cells of a female patient having compound heterozygous mutations in PANK2. This cellular model could provide a platform for pathophysiological studies of NBIA in the future.
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Células Madre Pluripotentes Inducidas , Neurodegeneración Asociada a Pantotenato Quinasa , Encéfalo/diagnóstico por imagen , Femenino , Humanos , Trastornos del Metabolismo del Hierro , Leucocitos Mononucleares , Mutación , Distrofias Neuroaxonales , Fosfotransferasas (Aceptor de Grupo Alcohol)/genéticaRESUMEN
BACKGROUND: The c.G6055A (p.G2019S) mutation in leucine-rich repeat kinase 2 (LRRK2) is the most prevalent genetic cause of Parkinson's disease (PD). CRISPR/Cas9-mediated genome editing by homology-directed repair (HDR) has been applied to correct the mutation but may create small insertions and deletions (indels) due to double-strand DNA breaks. Adenine base editors (ABEs) could convert targeted A·T to G·C in genomic DNA without double-strand breaks. However, the correction efficiency of ABE in LRRK2 c.G6055A (p.G2019S) mutation remains unknown yet. This study aimed to compare the mutation correction efficiencies and off-target effects between HDR and ABEs in induced pluripotent stem cells (iPSCs) carrying LRRK2 c.G6055A (p.G2019S) mutation. METHODS: A set of mutation-corrected isogenic lines by editing the LRRK2 c.G6055A (p.G2019S) mutation in a PD patient-derived iPSC line using HDR or ABE were established. The mutation correction efficacies, off-target effects, and indels between HDR and ABE were compared. Comparative transcriptomic and proteomic analyses between the LRRK2 p.G2019S iPSCs and isogenic control cells were performed to identify novel molecular targets involved in LRRK2-parkinsonism pathways. RESULTS: ABE had a higher correction rate (13/53 clones, 24.5%) than HDR (3/47 clones, 6.4%). Twenty-seven HDR clones (57.4%), but no ABE clones, had deletions, though 14 ABE clones (26.4%) had off-target mutations. The corrected isogenic iPSC-derived dopaminergic neurons exhibited reduced LRRK2 kinase activity, decreased phospho-α-synuclein expression, and mitigated neurite shrinkage and apoptosis. Comparative transcriptomic and proteomic analysis identified different gene expression patterns in energy metabolism, protein degradation, and peroxisome proliferator-activated receptor pathways between the mutant and isogenic control cells. CONCLUSIONS: The results of this study envision that ABE could directly correct the pathogenic mutation in iPSCs for reversing disease-related phenotypes in neuropathology and exploring novel pathophysiological targets in PD.
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Células Madre Pluripotentes Inducidas , Enfermedad de Parkinson , Neuronas Dopaminérgicas , Edición Génica , Humanos , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/genética , Mutación , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/terapia , Fenotipo , ProteómicaRESUMEN
Immune checkpoint inhibitors demonstrate clinical activity in many tumor types, however, only a fraction of patients benefit. Combining CD137 agonists with these inhibitors increases anti-tumor activity preclinically, but attempts to translate these observations to the clinic have been hampered by systemic toxicity. Here we describe a human CD137xPD-L1 bispecific antibody, MCLA-145, identified through functional screening of agonist- and immune checkpoint inhibitor arm combinations. MCLA-145 potently activates T cells at sub-nanomolar concentrations, even under suppressive conditions, and enhances T cell priming, differentiation and memory recall responses. In vivo, MCLA-145 anti-tumor activity is superior to immune checkpoint inhibitor comparators and linked to recruitment and intra-tumor expansion of CD8 + T cells. No graft-versus-host-disease is observed in contrast to other antibodies inhibiting the PD-1 and PD-L1 pathway. Non-human primates treated with 100 mg/kg/week of MCLA-145 show no adverse effects. The conditional activation of CD137 signaling by MCLA-145, triggered by neighboring cells expressing >5000 copies of PD-L1, may provide both safety and potency advantages.
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Ligando 4-1BB/agonistas , Anticuerpos Biespecíficos/farmacología , Antígeno B7-H1/antagonistas & inhibidores , Linfocitos T CD8-positivos/efectos de los fármacos , Inhibidores de Puntos de Control Inmunológico/farmacología , Ligando 4-1BB/inmunología , Animales , Anticuerpos Biespecíficos/inmunología , Antígeno B7-H1/inmunología , Linfocitos T CD8-positivos/inmunología , Epítopos , Humanos , Inhibidores de Puntos de Control Inmunológico/inmunología , Tolerancia Inmunológica/efectos de los fármacos , Memoria Inmunológica/efectos de los fármacos , Inmunoterapia , Activación de Linfocitos/efectos de los fármacosRESUMEN
Vaccinia virus A26 protein is an envelope protein of the intracellular mature virus (IMV) of vaccinia virus. A mutant A26 protein with a truncation of the 74 C-terminal amino acids was expressed in infected cells but failed to be incorporated into IMV (W. L. Chiu, C. L. Lin, M. H. Yang, D. L. Tzou, and W. Chang, J. Virol 81:2149-2157, 2007). Here, we demonstrate that A27 protein formed a protein complex with the full-length form but not with the truncated form of A26 protein in infected cells as well as in IMV. The formation of the A26-A27 protein complex occurred prior to virion assembly and did not require another A27-binding protein, A17 protein, in the infected cells. A26 protein contains six cysteine residues, and in vitro mutagenesis showed that Cys441 and Cys442 mediated intermolecular disulfide bonds with Cys71 and Cys72 of viral A27 protein, whereas Cys43 and Cys342 mediated intramolecular disulfide bonds. A26 and A27 proteins formed disulfide-linked complexes in transfected 293T cells, showing that the intermolecular disulfide bond formation did not depend on viral redox pathways. Finally, using cell fusion from within and fusion from without, we demonstrate that cell surface glycosaminoglycan is important for virus-cell fusion and that A26 protein, by forming complexes with A27 protein, partially suppresses fusion.
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Proteínas Portadoras/metabolismo , Fusión Celular , Disulfuros/metabolismo , Glicosaminoglicanos/metabolismo , Virus Vaccinia/metabolismo , Proteínas Virales de Fusión/metabolismo , Línea Celular , Cisteína/metabolismo , Humanos , Proteínas de la Membrana , Mutagénesis Sitio-Dirigida , Unión ProteicaRESUMEN
A novel missense mutation, c.941A > C (p.Y314S), in mitochondrial ubiquinol-cytochrome c reductase core protein I (UQCRC1) gene, was recently identified in a family with autosomal-dominant late-onset parkinsonism and polyneuropathy. UQCRC1 encodes a mitochondria respiratory complex III protein. Mutant UQCRC1 cells showed decreased mitochondrial activity and neurite degeneration. Here, we used the Sendai virus delivery system to generate induced pluripotent stem cells (iPSCs) from peripheral blood mononuclear cells of a male patient having a heterozygous UQCRC1 p.Y314S mutation. The established iPSCs could differentiate into three germ layers in vivo. This cellular model provides a platform for further studies of UQCRC1-related parkinsonism and polyneuropathy.
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Células Madre Pluripotentes Inducidas , Trastornos Parkinsonianos , Polineuropatías , Humanos , Leucocitos Mononucleares , Masculino , Mutación , Trastornos Parkinsonianos/genéticaRESUMEN
Vaccinia virus is a large DNA virus that infects many cell cultures in vitro and animal species in vivo. Although it has been used widely as a vaccine, its cell entry pathway remains unclear. In this study, we showed that vaccinia virus intracellular mature virions bound to the filopodia of HeLa cells and moved toward the cell body and entered the cell through an endocytic route that required a dynamin-mediated pathway but not a clathrin- or caveola-mediated pathway. Moreover, virus penetration required a novel cellular protein, vaccinia virus penetration factor (VPEF). VPEF was detected on cell surface lipid rafts and on vesicle-like structures in the cytoplasm. Both vaccinia virus and dextran transiently colocalized with VPEF, and, importantly, knockdown of VPEF expression blocked vaccinia virus penetration as well as intracellular transport of dextran, suggesting that VPEF mediates vaccinia virus entry through a fluid uptake endocytosis process in HeLa cells. Intracellular VPEF-containing vesicles did not colocalize with Rab5a or caveolin but partially colocalized with Rab11, supporting the idea that VPEF plays a role in vesicle trafficking and recycling in HeLa cells. In summary, this study characterized the mechanism by which vaccinia virus enters HeLa cells and identified a cellular factor, VPEF, that is exploited by vaccinia virus for cell entry through fluid phase endocytosis.