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1.
Artículo en Inglés | MEDLINE | ID: mdl-32748760

RESUMEN

BACKGROUND & OBJECTIVE: Nowadays, the interaction between natural products and microRNAs provides a promising field for exploring the chemo preventive agents for various cancers.As a member of microRNAs, the expression of let-7f-5p is universally down regulated in colorectal cancer (CRC). The present study aimed to uncover the function of let-7f-5p in the proliferation of human colon cancer cell line Caco2 and explored chemo preventive agents from natural resources that can prevent the development of CRC. METHODS: Herein, Caco2 cells were transfected with let-7f-5p mimic and inhibitor to manipulate let-7f-5p levels, and the expression of let-7f-5p wasper formed by RT­qPCR. Next, we determined how let-7f-5p regulates Caco2 cell proliferation by using MTT, wound-healing, cell cycle,and colony formation assays.Besides, to further understand the effect of let-7f-5p, we evaluated the protein level of AMER3 and SLC9A9 by using western blotting assays. RESULTS: The results showed a suppressive function of let-7f-5p on Caco2 cell proliferation and then put forward a triterpenoid (rotundic acid, RA) which significant antagonized the effect of cell proliferation, restitution after wounding,and colony formation caused by let-7f-5p. Moreover, the western blot results further indicated that the inhibitory effect of RA might be due to its suppressive role in let-7f-5p-targeted AMER3 and SLC9A9 regulation. CONCLUSION: Our validation study results confirmed that let-7f-5p was a potent tumor suppressor gene of Caco2 cell proliferation,and RA showed as a regulator of the effect oflet-7f-5p on cell proliferation and then could be a potential chemo preventive agent for CRC treatment.

2.
Respir Res ; 21(1): 163, 2020 Jun 29.
Artículo en Inglés | MEDLINE | ID: mdl-32600344

RESUMEN

OBJECTIVE: This study investigated the influence of Coronavirus Disease 2019 (COVID-19) on lung function in early convalescence phase. METHODS: A retrospective study of COVID-19 patients at the Fifth Affiliated Hospital of Sun Yat-sen University were conducted, with serial assessments including lung volumes (TLC), spirometry (FVC, FEV1), lung diffusing capacity for carbon monoxide (DLCO),respiratory muscle strength, 6-min walking distance (6MWD) and high resolution CT being collected at 30 days after discharged. RESULTS: Fifty-seven patients completed the serial assessments. There were 40 non-severe cases and 17 severe cases. Thirty-one patients (54.3%) had abnormal CT findings. Abnormalities were detected in the pulmonary function tests in 43 (75.4%) of the patients. Six (10.5%), 5(8.7%), 25(43.8%) 7(12.3%), and 30 (52.6%) patients had FVC, FEV1, FEV1/FVC ratio, TLC, and DLCO values less than 80% of predicted values, respectively. 28 (49.1%) and 13 (22.8%) patients had PImax and PEmax values less than 80% of the corresponding predicted values. Compared with non-severe cases, severe patients showed higher incidence of DLCO impairment (75.6%vs42.5%, p = 0.019), higher lung total severity score (TSS) and R20, and significantly lower percentage of predicted TLC and 6MWD. No significant correlation between TSS and pulmonary function parameters was found during follow-up visit. CONCLUSION: Impaired diffusing-capacity, lower respiratory muscle strength, and lung imaging abnormalities were detected in more than half of the COVID-19 patients in early convalescence phase. Compared with non-severe cases, severe patients had a higher incidence of DLCO impairment and encountered more TLC decrease and 6MWD decline.


Asunto(s)
Infecciones por Coronavirus/diagnóstico , Infecciones por Coronavirus/epidemiología , Neumonía Viral/diagnóstico , Neumonía Viral/epidemiología , Músculos Respiratorios/fisiopatología , Síndrome Respiratorio Agudo Grave/diagnóstico , Adulto , Anciano , Distribución de Chi-Cuadrado , China/epidemiología , Estudios de Cohortes , Convalecencia , Prueba de Esfuerzo , Femenino , Estudios de Seguimiento , Volumen Espiratorio Forzado , Humanos , Incidencia , Masculino , Persona de Mediana Edad , Fuerza Muscular , Pandemias , Alta del Paciente , Radiografía Torácica/métodos , Pruebas de Función Respiratoria , Estudios Retrospectivos , Medición de Riesgo , Síndrome Respiratorio Agudo Grave/epidemiología , Espirometría/métodos , Centros de Atención Terciaria , Tomografía Computarizada por Rayos X/métodos , Capacidad Vital/fisiología
3.
Arthritis Rheumatol ; 2020 Jun 30.
Artículo en Inglés | MEDLINE | ID: mdl-32602227

RESUMEN

OBJECTIVE: SSc-ILD is characterized by variable clinical outcomes, activation of innate immune pattern recognition receptors (PRRs), and accumulation of αSMA expressing myofibroblasts. The association of these entities with mitochondrial DNA (mtDNA), an endogenous ligand for the intracellular DNA-sensing PRRs TLR9 and cGAS-STING, has yet to be determined. METHODS: Human lung fibroblasts from normal donors (NHLFs) and SSc-ILD explants were treated with synthetic CpG DNA and assayed for αSMA expression and extracellular mtDNA using qPCR for the human MT-ATP6 gene. Plasma MT-ATP6 concentrations were evaluated in two independent SSc-ILD cohorts and demographically matched controls. The ability of control and SSc-ILD plasma to induce TLR9 and cGAS-STING activation was evaluated with commercially available HEK 293 reporter cells. Plasma concentrations of type I interferons, IL-6, and oxidized DNA were measured with using electrochemiluminescence and ELISA-based methods, respectively. Extracellular vesicles (EVs) precipitated from plasma were evaluated for MT-ATP6 concentrations and proteomics via liquid chromatography-mass spectrometry. RESULTS: NHLFs and SSc-ILD fibroblasts develop increased αSMA expression and MT-ATP6 release following CpG stimulation. Plasma mtDNA concentrations are increased in two SSc-ILD cohorts, reflective of ventilatory decline, and positively associated with both TLR9 and cGAS-STING activation as well as Type I interferon and IL-6 expression. Plasma mtDNA is not oxidized and is conveyed by EVs displaying a proteomics profile consistent with a multicellular origin. CONCLUSION: These findings demonstrate an unrecognized connection between EV-encapsulated mtDNA, clinical outcomes, and intracellular DNA-sensing PRR activation in SSc-ILD. Further study of these interactions could catalyze novel mechanistic and therapeutic insights into SSc-ILD and related disorders.

4.
J Immunol Res ; 2020: 7285747, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32566688

RESUMEN

Background: Systemic lupus erythematosus (SLE) is a multifactorial autoimmune disease characterized by apoptotic clearance deficiency provoking autoimmune responses and leading to multiple organ damage. PPAR-δ, encoded by the PPARD gene, was induced in macrophages promoting the timely disposal of apoptotic cells. Biological studies had provided solid foundation of PPARD involvement in SLE; it is worthwhile to further explore the genetic contribution of PPARD to SLE. Methods: We performed a discovery-replication genetic association study. The discovery study was based on previous reported GWAS data. And the replication study was conducted in 1003 SLE patients and 815 healthy controls from Henan, Middle East of China. Further, we analyzed the eQTL effect to identify possible functional significance. Results: In the genetic association analysis, we observed significant association between the risk C allele of rs4713853 (p = 0.03, OR 1.167, 95% CI 1.015-1.341) and increased SLE susceptibility. Moreover, individuals with the risk C allele were associated with lower expression of PPARD and DEF6. Our clinical analysis showed that SLE patients with the risk C allele of rs4713853 were more likely to present a higher proportion of anti-Sm antibody presence (CC+CT vs. TT, 20.0% vs. 14.2%, p = 0.039) and higher level of Scr (median inter quarter range CC+CT vs. TT, 56 48-71 vs. 54 46-64 µmol/L, p = 0.002). Conclusions: In conclusion, our study identified a novel association between PPARD rs4713853 and SLE susceptibility in Chinese populations. By integrating multiple layers of analysis, we suggested that PPARD might be a main candidate in the pathogenesis of SLE.

5.
iScience ; 23(6): 101203, 2020 May 28.
Artículo en Inglés | MEDLINE | ID: mdl-32516721

RESUMEN

Leukocyte common antigen-related receptor tyrosine phosphatases (LAR-RPTPs) are evolutionarily conserved presynaptic organizers. The synaptic role of vertebrate LAR-RPTPs in vivo, however, remains unclear. In the current study, we analyzed the synaptic role of PTPσ using newly generated, single conditional knockout (cKO) mice targeting PTPσ. We found that the number of synapses was reduced in PTPσ cKO cultured neurons in association with impaired excitatory synaptic transmission, abnormal vesicle localization, and abnormal synaptic ultrastructure. Strikingly, loss of presynaptic PTPσ reduced neurotransmitter release prominently at excitatory synapses, concomitant with drastic reductions in excitatory innervations onto postsynaptic target areas in vivo. Furthermore, loss of presynaptic PTPσ in hippocampal CA1 pyramidal neurons had no impact on postsynaptic glutamate receptor responses in subicular pyramidal neurons. Postsynaptic PTPσ deletion had no effect on excitatory synaptic strength. Taken together, these results demonstrate that PTPσ is a bona fide presynaptic adhesion molecule that controls neurotransmitter release and excitatory inputs.

6.
Mol Brain ; 13(1): 94, 2020 Jun 17.
Artículo en Inglés | MEDLINE | ID: mdl-32552840

RESUMEN

Members of the leukocyte common antigen-related receptor protein tyrosine phosphatase (LAR-RPTP) family, comprising PTPσ, PTPδ and LAR, are key hubs for presynaptic assembly and differentiation in vertebrate neurons. However, roles of individual LAR-RPTP members have not been investigated using member-specific conditional knockout mice. Here, we show that loss of PTPδ had no overt effect on synapse development in mouse cultured hippocampal neurons. Moreover, loss of PTPδ in presynaptic CA1 hippocampal neurons did not influence neurotransmitter release in subicular pyramidal neurons, suggesting that PTPδ is not critical for presynaptic function in vivo. Our results demonstrate that PTPδ is not essential for synapse maintenance or transmission, at least in the mouse hippocampus, and underscore the importance of using sophisticated genetic approaches to confirm the roles of synaptic proteins.

7.
Arterioscler Thromb Vasc Biol ; 40(6): 1510-1522, 2020 06.
Artículo en Inglés | MEDLINE | ID: mdl-32349535

RESUMEN

OBJECTIVE: Endothelial Cav-1 (caveolin-1) expression plays a relevant role during atherogenesis by controlling NO production, vascular inflammation, LDL (low-density lipoprotein) transcytosis, and extracellular matrix remodeling. Additional studies have identified cholesterol-rich membrane domains as important regulators of autophagy by recruiting ATGs (autophagy-related proteins) to the plasma membrane. Here, we investigate how the expression of Cav-1 in the aortic endothelium influences autophagy and whether enhanced autophagy contributes to the atheroprotective phenotype observed in Cav-1-deficient mice. Approach and Results: To analyze the impact of Cav-1 deficiency on regulation of autophagy in the aortic endothelium during the progression of atherosclerosis, we fed Ldlr-/- and Cav-1-/-Ldlr-/- mice a Western diet and assessed autophagy in the vasculature. We observe that the absence of Cav-1 promotes autophagy activation in athero-prone areas of the aortic endothelium by enhancing autophagic flux. Mechanistically, we found that Cav-1 interacts with the ATG5-ATG12 complex and influences the cellular localization of autophagosome components in lipid rafts, which controls the autophagosome formation and autophagic flux. Pharmacological inhibition of autophagy attenuates the atheroprotection observed in Cav-1-/- mice by increasing endothelial inflammation and macrophage recruitment, identifying a novel molecular mechanism by which Cav-1 deficiency protects against the progression of atherosclerosis. CONCLUSIONS: These results identify Cav-1 as a relevant regulator of autophagy in the aortic endothelium and demonstrate that pharmacological suppression of autophagic flux in Cav-1-deficient mice attenuates the atheroprotection observed in Cav-1-/- mice. Additionally, these findings suggest that activation of endothelial autophagy by blocking Cav-1 might provide a potential therapeutic strategy for cardiovascular diseases including atherosclerosis.


Asunto(s)
Aterosclerosis/prevención & control , Autofagia/fisiología , Caveolina 1/deficiencia , Endotelio Vascular/fisiopatología , Vasculitis/prevención & control , Adenina/análogos & derivados , Adenina/farmacología , Animales , Aorta/patología , Aorta/fisiopatología , Aorta/ultraestructura , Aterosclerosis/etiología , Autofagia/efectos de los fármacos , Caveolina 1/análisis , Caveolina 1/fisiología , Dieta Occidental , Células Endoteliales/química , Células Endoteliales/fisiología , Células Endoteliales/ultraestructura , Endotelio Vascular/química , Endotelio Vascular/ultraestructura , Femenino , Humanos , Masculino , Microdominios de Membrana/química , Microdominios de Membrana/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Células 3T3 NIH , Receptores de LDL/deficiencia
8.
Nutrients ; 12(4)2020 Apr 18.
Artículo en Inglés | MEDLINE | ID: mdl-32325708

RESUMEN

The study investigated the protective effect of walnut oligopeptides (WOPs) against ethanol-induced gastric injury using Sprague-Dawley (SD) rats. Rats were randomly divided into seven groups based on body weight (10/group), normal group, ethanol group, whey protein group (220 mg/kg body weight), omeprazole group (20 mg/kg body weight), and three WOPs groups (220, 440, 880 mg/kg body weight). After 30 days of treatment with WOPs, rats were given 5 ml/kg absolute ethanol by gavage to induce gastric mucosal injury. Gastric ulcer index (GUI) were determined and the following measured; gastric content pH, gastric mucin, endogenous pepsinogens (PG), prostaglandin E2 (PGE2), inflammatory cytokines, oxidative stress indicators, and the expression of apoptosis-related proteins were measured to evaluate the gastroprotective effect of WOPs. The results showed that the administration with WOPs markedly mitigated the hemorrhagic gastric lesions caused by ethanol in rats, and decreased the GUI, the gastric content pH, PG1, PG2, and NO levels, enhanced mucin and PGE2. Also, WOPs repressed gastric inflammation through the reduction of TNF-α, IL-6, IL-1ß and increase IL-10 levels, and revealed antioxidant properties with the enhancement of superoxide dismutase, glutathione, and catalase activity, while reduction of malondialdehyde. Moreover, WOPs treatment significantly down-regulated Bax, caspase-3 and nuclear factor-κB p65 (NF-κB p65) expression, while up-regulating the expression of Bcl-2 and inhibitor kappa Bα (IκBα) protein. These results indicated that WOPs have protective effects against ethanol-induced gastric mucosal injury in rats through anti-inflammatory, anti-oxidation, and anti-apoptosis mechanisms.

9.
J Clin Invest ; 130(3): 1233-1251, 2020 Mar 02.
Artículo en Inglés | MEDLINE | ID: mdl-32039915

RESUMEN

Smooth muscle cell (SMC) proliferation has been thought to limit the progression of thoracic aortic aneurysm and dissection (TAAD) because loss of medial cells associates with advanced disease. We investigated effects of SMC proliferation in the aortic media by conditional disruption of Tsc1, which hyperactivates mTOR complex 1. Consequent SMC hyperplasia led to progressive medial degeneration and TAAD. In addition to diminished contractile and synthetic functions, fate-mapped SMCs displayed increased proteolysis, endocytosis, phagocytosis, and lysosomal clearance of extracellular matrix and apoptotic cells. SMCs acquired a limited repertoire of macrophage markers and functions via biogenesis of degradative organelles through an mTOR/ß-catenin/MITF-dependent pathway, but were distinguishable from conventional macrophages by an absence of hematopoietic lineage markers and certain immune effectors even in the context of hyperlipidemia. Similar mTOR activation and induction of a degradative SMC phenotype in a model of mild TAAD due to Fbn1 mutation greatly worsened disease with near-uniform lethality. The finding of increased lysosomal markers in medial SMCs from clinical TAAD specimens with hyperplasia and matrix degradation further supports the concept that proliferation of degradative SMCs within the media causes aortic disease, thus identifying mTOR-dependent phenotypic modulation as a therapeutic target for combating TAAD.

10.
Int J Prosthodont ; 33(1): 39-47, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-31860912

RESUMEN

PURPOSE: To systematically review the current literature on the influence of abutment material (metal vs ceramic) and soft tissue thickness on peri-implant soft tissue discoloration in partially edentulous patients restored with implant-supported single crowns. METHODS: An electronic MEDLINE search was performed to identify randomized controlled clinical trials (RCTs) up to and including March 2017. The search was complemented by a manual search of related bibliographies. Selection of studies was made independently by two reviewers based on the inclusion criteria. Spectrophotometric data (ΔE values) and soft tissue thickness values were extracted, and, whenever applicable, a meta-analysis using a random-effects approach was performed. RESULTS: The search resulted in 208 titles and 30 abstracts. Full-text analysis was performed for 13 articles, resulting in 6 included RCTs. Meta-analysis of a total of 266 abutments revealed significantly lower ΔE values for ceramic abutments when compared to the overall metal abutments (z test value = 1.99, P = .05), with a mean difference of 1.41 (95% CI 0.02, 2.80). Nonsignificant differences were found between titanium and zirconia (z test value = 1.59, P = .11). Limited information on the correlation between soft tissue thickness and ΔE values was found. Hence, it was not possible to perform a meta-analysis of this question. CONCLUSION: The color outcome of the peri-implant soft tissue might be influenced by the abutment material. Ceramic abutments appear to provide an improved color matching between peri-implant soft tissues and soft tissues around natural teeth when compared to metallic abutments. These findings support the preference for all-ceramic or "white" abutments in esthetically demanding cases.


Asunto(s)
Pilares Dentales , Implantes Dentales de Diente Único , Coronas , Encía , Humanos , Ensayos Clínicos Controlados Aleatorios como Asunto , Espectrofotometría , Titanio , Circonio
11.
Int J Prosthodont ; 33(1): 63-73, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-31860915

RESUMEN

PURPOSE: To evaluate the influence of different cements on the color outcomes of CAD/CAM lithium-disilicate implant crowns cemented to titanium-base abutments utilizing spectrophotometric analysis. MATERIALS AND METHODS: A clinical situation with a missing lateral incisor was mimicked using a maxillary plastic model. Titanium-base-supported monolithic lithium disilicate crowns with identical designs were fabricated using a laboratory CAD/CAM system. The crowns were cemented with three provisional cements and with six definitive cements on both nonsandblasted and sandblasted titanium-base abutments for a total of 15 test groups. As a control group, identical crowns were attached with try-in paste on composite die abutments that duplicated the shape of the titanium-base abutments. The colors of the labial surfaces of the crowns and the peri-implant artificial soft tissue were measured with a spectrophotometer and recorded in CIE L*a*b* parameters. Color differences between the test and control groups were calculated as: ΔE = ([ΔL*]2 + [Δa*]2 + [Δb*]2)1/2. Kruskal-Wallis test was used to compare ΔE values across different groups. RESULTS: The median ΔE values reported for crowns cemented with different definitive cements on titanium-base abutments ranged from 1.4 to 2.9 for the crown surface and from 1.7 to 1.9 on the peri-implant artificial soft tissue; when the titanium-base abutments were sandblasted, the respective median ΔE values ranged from 0.8 to 4.0 and from 1.4 to 2.2. Ceramic crowns cemented with Multilink HO 0 cement presented significantly (P < .01) lower ΔE values than the other cement types for the crown surface independent of sandblasting and for the artificial soft tissue surface when the titanium abutments were sandblasted (P = .011). CONCLUSION: Within the limitations of this study, Multilink HO 0 (Ivoclar Vivadent) cement showed the most favorable masking ability and the most favorable color outcome among the evaluated definitive cements. Cements of more opaque shades appeared in general to be more favorable in terms of masking the gray color of the titanium-base abutments.


Asunto(s)
Implantes Dentales , Titanio , Color , Coronas , Pilares Dentales , Porcelana Dental , Circonio
12.
Sensors (Basel) ; 20(1)2019 Dec 21.
Artículo en Inglés | MEDLINE | ID: mdl-31877704

RESUMEN

Catching cancer at an early stage is necessary to make it easier to treat and to save people's lives rather than just extending them. Reactive oxygen species (ROS) have sparked a huge interest owing to their vital role in various biological processes, especially in tumorigenesis, thus leading to the potential of ROS as prognostic biomarkers for cancer. Herein, a non-enzymatic biosensor for the dynamic monitoring of intracellular hydrogen peroxide (H2O2), the most important ROS, via an effective electrode composed of poly (diallyldimethylammonium chloride) (PDDA)-capped reduced graphene oxide (RGO) nanosheets with high loading trimetallic AuPtAg nanoalloy, is proposed. The designed biosensor was able to measure H2O2 released from different cancerous cells promptly and precisely owing to the impressive conductivity of RGO and PDDA and the excellent synergistic effect of the ternary alloy in boosting the electrocatalytic activity. Built upon the peroxidase-like activity of the nanoalloy, the developed sensor exhibited distinguished electrochemical performance, resulting in a low detection limit of 1.2 nM and a wide linear range from 0.05 µM to 5.5 mM. Our approach offers a significant contribution toward the further elucidation of the role of ROS in carcinogenesis and the effective screening of cancer at an early stage.


Asunto(s)
Aleaciones/química , Técnicas Electroquímicas/métodos , Grafito/química , Peróxido de Hidrógeno/análisis , Nanocompuestos/química , Polietilenos/química , Compuestos de Amonio Cuaternario/química , Línea Celular Tumoral , Electrodos , Oro/química , Humanos , Límite de Detección , Platino (Metal)/química , Reproducibilidad de los Resultados , Plata/química
13.
J Acquir Immune Defic Syndr ; 82(5): 514-522, 2019 Dec 15.
Artículo en Inglés | MEDLINE | ID: mdl-31714431

RESUMEN

BACKGROUND: Neurocognitive dysfunction remains prevalent among people living with HIV (PLWH), even after viral suppression on combination antiretroviral therapy (cART). We investigated associations between neuropsychological performance (NP) and patterns of circulating exosomal microRNA (exo-miRNA) expression in PLWH on cART. SETTING: A cross-sectional examination of plasma exo-miRNA among PLWH on cART with systemic viral suppression and volunteers without HIV infection. METHODS: Thirty-one PLWH who started cART during early infection (n = 19) or chronic infection (n = 12) participated in phlebotomy and an 11-test neuropsychological battery after >1 year on treatment. NP higher- or lower-performing participants were categorized based on normalized neuropsychological scores. Total RNA was extracted from purified exosomes of 31 PLWH and 5 volunteers without HIV and subject to small RNA sequencing. Differential expression of exo-miRNAs was examined and biological functions were predicted. RESULTS: Eleven exo-miRNAs were up-regulated in NP lower-performing (n = 18) relative to higher-performing PLWH (n = 13). A high proportion of the differentiating exo-miRNA target the axon guidance KEGG pathway and neurotrophin tyrosine receptor kinase signaling Gene Ontology pathway. Differential expression analysis of exo-miRNAs between NP lower- (n = 7) and higher-performing (n = 12) PLWH within the early infection group alone confirmed largely consistent findings. CONCLUSIONS: Plasma exo-miRNA content differed between NP higher- and lower-performing PLWH. Several differentially expressed exo-miRNAs were predicted to be involved in inflammation and neurodegeneration pathways. Exo-miRNA in plasma may indicate cross-talk between the circulation and central nervous system and thus may be clinically relevant for neurocognitive dysfunction in PLWH.

14.
Opt Express ; 27(18): 25265-25279, 2019 Sep 02.
Artículo en Inglés | MEDLINE | ID: mdl-31510401

RESUMEN

Object motion can introduce unknown phase shift and thus measurement error in multi-image phase-shifting methods of fringe projection profilometry. This paper presents a new method to estimate the unknown phase shifts and reduce the motion-induced error by using three phase maps computed over a multiple measurement sequence and calculating the difference between phase maps. The pixel-wise estimation of the motion-induced phase shifts permits phase-error compensation for non-homogeneous surface motion. Experiments demonstrated the ability of the method to reduce motion-induced error in real-time, for shape measurement of surfaces with high depth variation, and moving and deforming surfaces.

15.
Biochemistry ; 58(36): 3735-3743, 2019 Sep 10.
Artículo en Inglés | MEDLINE | ID: mdl-31424194

RESUMEN

The conserved structural motif D is an important determinant of the speed and fidelity of viral RNA-dependent RNA polymerases (RdRps). Structural and computational studies have suggested that conformational changes in the motif-D loop that help to reposition the catalytic lysine represent critical steps in nucleotide selection and incorporation. Conformations of the motif-D loop in the poliovirus RdRp are likely controlled in part by noncovalent interactions involving the motif-D residue Glu364. This residue swivels between making interactions with Lys228 and Asn370 to stabilize the open and closed loop conformations, respectively. We show here that we can rationally control the motif-D loop conformation by breaking these interactions. The K228A variant favors a more active closed conformation, leading to increased nucleotide incorporation rates and decreased nucleotide selectivity, and the N370A variant favors a less active open conformation, leading to decreased nucleotide incorporation rates and increased nucleotide selectivity. Similar competing interactions likely control nucleotide incorporation rates and fidelity in other viral RdRps. Rational engineering of these interactions may be important in the generation of live, attenuated vaccine strains, considering the established relationships between RdRp function and viral pathogenesis.

16.
Sci Rep ; 9(1): 12465, 2019 08 28.
Artículo en Inglés | MEDLINE | ID: mdl-31462665

RESUMEN

The unique stacked morphology of the Golgi apparatus had been a topic of intense investigation among the cell biologists over the years. We had previously shown that the two Golgin tethers (GM130 and Golgin45) could, to a large degree, functionally substitute for GRASP-type Golgi stacking proteins to sustain normal Golgi morphology and function in GRASP65/55-double depleted HeLa cells. However, compared to well-studied GM130, the exact role of Golgin45 in Golgi structure remains poorly understood. In this study, we aimed to further characterize the functional role of Golgin45 in Golgi structure and identified Golgin45 as a novel Syntaxin5-binding protein. Based primarily on a sequence homology between Golgin45 and GM130, we found that a leucine zipper-like motif in the central coiled-coil region of Golgin45 appears to serve as a Syntaxin5 binding domain. Mutagenesis study of this conserved domain in Golgin45 showed that a point mutation (D171A) can abrogate the interaction between Golgin45 and Syntaxin5 in pull-down assays using recombinant proteins, whereas this mutant Golgin45 binding to Rab2-GTP was unaffected in vitro. Strikingly, exogenous expression of this Syntaxin5 binding deficient mutant (D171A) of Golgin45 in HeLa cells resulted in frequent intercisternal fusion among neighboring Golgi cisterna, as readily observed by EM and EM tomography. Further, double depletion of the two Syntaxin5-binding Golgin tethers also led to significant intercisternal fusion, while double depletion of GRASP65/55 didn't lead to this phenotype. These results suggest that certain tether-SNARE interaction within Golgi stack may play a role in inhibiting intercisternal fusion among neighboring cisternae, thereby contributing to structural integrity of the Golgi stack.

17.
Cell Rep ; 28(3): 759-772.e10, 2019 07 16.
Artículo en Inglés | MEDLINE | ID: mdl-31315053

RESUMEN

Mechanisms coordinating pancreatic ß cell metabolism with insulin secretion are essential for glucose homeostasis. One key mechanism of ß cell nutrient sensing uses the mitochondrial GTP (mtGTP) cycle. In this cycle, mtGTP synthesized by succinyl-CoA synthetase (SCS) is hydrolyzed via mitochondrial PEPCK (PEPCK-M) to make phosphoenolpyruvate, a high-energy metabolite that integrates TCA cycling and anaplerosis with glucose-stimulated insulin secretion (GSIS). Several strategies, including xenotopic overexpression of yeast mitochondrial GTP/GDP exchanger (GGC1) and human ATP and GTP-specific SCS isoforms, demonstrated the importance of the mtGTP cycle. These studies confirmed that mtGTP triggers and amplifies normal GSIS and rescues defects in GSIS both in vitro and in vivo. Increased mtGTP synthesis enhanced calcium oscillations during GSIS. mtGTP also augmented mitochondrial mass, increased insulin granule number, and membrane proximity without triggering de-differentiation or metabolic fragility. These data highlight the importance of the mtGTP signal in nutrient sensing, insulin secretion, mitochondrial maintenance, and ß cell health.

18.
EMBO J ; 38(16): e99266, 2019 08 15.
Artículo en Inglés | MEDLINE | ID: mdl-31271236

RESUMEN

During MHC-I-restricted antigen processing, peptides generated by cytosolic proteasomes are translocated by the transporter associated with antigen processing (TAP) into the endoplasmic reticulum, where they bind to newly synthesized MHC-I molecules. Dendritic cells and other cell types can also generate MHC-I complexes with peptides derived from internalized proteins, a process called cross-presentation. Here, we show that active proteasomes within cross-presenting cell phagosomes can generate these peptides. Active proteasomes are detectable within endocytic compartments in mouse bone marrow-derived dendritic cells. In TAP-deficient mouse dendritic cells, cross-presentation is enhanced by the introduction of human ß2 -microglobulin, which increases surface expression of MHC-I and suggests a role for recycling MHC-I molecules. In addition, surface MHC-I can be reduced by proteasome inhibition and stabilized by MHC-I-restricted peptides. This is consistent with constitutive proteasome-dependent but TAP-independent peptide loading in the endocytic pathway. Rab-GTPase mutants that restrain phagosome maturation increase proteasome recruitment and enhance TAP-independent cross-presentation. Thus, phagosomal/endosomal binding of peptides locally generated by proteasomes allows cross-presentation to generate MHC-I-peptide complexes identical to those produced by conventional antigen processing.


Asunto(s)
Células Dendríticas/inmunología , Antígenos de Histocompatibilidad Clase I/química , Complejo de la Endopetidasa Proteasomal/inmunología , Microglobulina beta-2/metabolismo , Animales , Presentación de Antígeno , Células Cultivadas , Reactividad Cruzada , Células Dendríticas/citología , Endocitosis , Humanos , Ratones , Fagosomas/inmunología , Proteolisis , Microglobulina beta-2/genética
19.
J Neurosci ; 39(35): 6992-7005, 2019 Aug 28.
Artículo en Inglés | MEDLINE | ID: mdl-31235642

RESUMEN

APP, whose mutations cause familial Alzheimer's disease (FAD), modulates neurotransmission via interaction of its cytoplasmic tail with the synaptic release machinery. Here we identified an intravesicular domain of APP, called intraluminal SV-APP interacting domain (ISVAID), which interacts with glutamatergic, but not GABAergic, synaptic vesicle proteins. ISVAID contains the ß- and α-secretase cleavage sites of APP: proteomic analysis of the interactome of ISVAID suggests that ß- and α-secretase cleavage of APP cuts inside the interaction domain of ISVAID and destabilizes protein-protein interactions. We have tested the functional significance of the ISVAID and of ß-/α-secretase-processing of APP using various ISVAID-derived peptides in competition experiments on both female and male mouse and rats hippocampal slices. A peptide encompassing the entire ISVAID facilitated glutamate, but not GABA, release acting as dominant negative inhibitor of the functions of this APP domain in acute hippocampal slices. In contrast, peptides representing the product of ß-/α-secretase-processing of ISVAID did not alter excitatory neurotransmitter release. These findings suggest that cleavage of APP by either ß- or α-secretase may inactivate the ISVAID, thereby enhancing glutamate release. Our present data support the notion that APP tunes glutamate release, likely through intravesicular and extravesicular interactions with synaptic vesicle proteins and the neurotransmitter release machinery, and that ß-/α cleavage of APP facilitates the release of excitatory neurotransmitter.SIGNIFICANCE STATEMENT Alzheimer's disease has been linked to mutations in APP. However, the biological function of APP is poorly understood. Here we show that an intravesicular APP domain interacts with the proteins that control the release of glutamate, but not GABA. Interfering with the function of this domain promotes glutamate release. This APP domain contains the sites cleaved by ß- and α-secretases: our data suggest that ß-/α cleavage of APP inactivates this functional APP domain promoting excitatory neurotransmitter release.

20.
Circulation ; 140(3): 225-239, 2019 07 16.
Artículo en Inglés | MEDLINE | ID: mdl-31154825

RESUMEN

BACKGROUND: Atherosclerosis is driven by synergistic interactions between pathological, biomechanical, inflammatory, and lipid metabolic factors. Our previous studies demonstrated that absence of caveolin-1 (Cav1)/caveolae in hyperlipidemic mice strongly inhibits atherosclerosis, which was attributed to activation of endothelial nitric oxide (NO) synthase (eNOS) and increased production of NO and reduced inflammation and low-density lipoprotein trafficking. However, the contribution of eNOS activation and NO production in the athero-protection of Cav1 and the exact mechanisms by which Cav1/caveolae control the pathogenesis of diet-induced atherosclerosis are still not clear. METHODS: Triple-knockout mouse lacking expression of eNOS, Cav1, and Ldlr were generated to explore the role of NO production in Cav1-dependent athero-protective function. The effects of Cav1 on lipid trafficking, extracellular matrix remodeling, and vascular inflammation were studied both in vitro and in vivo with a mouse model of diet-induced atherosclerosis. The expression of Cav1 and distribution of caveolae regulated by flow were analyzed by immunofluorescence staining and transmission electron microscopy. RESULTS: We found that absence of Cav1 significantly suppressed atherogenesis in Ldlr-/-eNOS-/- mice, demonstrating that athero-suppression is independent of increased NO production. Instead, we find that the absence of Cav1/caveolae inhibited low-density lipoprotein transport across the endothelium and proatherogenic fibronectin deposition and disturbed flow-mediated endothelial cell inflammation. Consistent with the idea that Cav1/caveolae may play a role in early flow-dependent inflammatory priming, distinct patterns of Cav1 expression and caveolae distribution were observed in athero-prone and athero-resistant areas of the aortic arch even in wild-type mice. CONCLUSIONS: These findings support a role for Cav1/caveolae as a central regulator of atherosclerosis that links biomechanical, metabolic, and inflammatory pathways independently of endothelial eNOS activation and NO production.


Asunto(s)
Aterosclerosis/metabolismo , Caveolina 1/fisiología , Endotelio Vascular/metabolismo , Lipoproteínas LDL/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , Transcitosis/fisiología , Animales , Aterosclerosis/patología , Aterosclerosis/prevención & control , Células Cultivadas , Perros , Endotelio Vascular/patología , Activación Enzimática/fisiología , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos
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