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1.
Nat Biotechnol ; 42(1): 99-108, 2024 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-37012448

RESUMEN

RNA velocity provides an approach for inferring cellular state transitions from single-cell RNA sequencing (scRNA-seq) data. Conventional RNA velocity models infer universal kinetics from all cells in an scRNA-seq experiment, resulting in unpredictable performance in experiments with multi-stage and/or multi-lineage transition of cell states where the assumption of the same kinetic rates for all cells no longer holds. Here we present cellDancer, a scalable deep neural network that locally infers velocity for each cell from its neighbors and then relays a series of local velocities to provide single-cell resolution inference of velocity kinetics. In the simulation benchmark, cellDancer shows robust performance in multiple kinetic regimes, high dropout ratio datasets and sparse datasets. We show that cellDancer overcomes the limitations of existing RNA velocity models in modeling erythroid maturation and hippocampus development. Moreover, cellDancer provides cell-specific predictions of transcription, splicing and degradation rates, which we identify as potential indicators of cell fate in the mouse pancreas.


Asunto(s)
ARN , Análisis de la Célula Individual , Animales , Ratones , ARN/genética , Análisis de Secuencia de ARN/métodos , Análisis de la Célula Individual/métodos , Simulación por Computador , Redes Neurales de la Computación , Perfilación de la Expresión Génica/métodos
2.
Eur J Med Chem ; 258: 115590, 2023 Oct 05.
Artículo en Inglés | MEDLINE | ID: mdl-37406381

RESUMEN

Although traditional EGFR-TKIs have advanced the treatment landscape of NSCLC with sensitive driver mutations (del19 or L858R), some NSCLC patients with EGFR exon 20 insertion mutations have been left with few effective therapies. The development of novel TKIs is still in progress. Herein, we describe the structure-guided design of a novel selective and orally bioavailable inhibitor, YK-029A, which could overcome both the T790 M mutations and exon 20 insertion of EGFR. YK-029A inhibited EGFR signaling, suppressed sensitive mutations and ex20ins of EGFR-driven cell proliferation, and was largely effective with oral administration in vivo. Furthermore, YK-029A exhibited significant antitumor activity in EGFRex20ins-driven patients-derived xenograft (PDX) models, preventing tumor progression or causing tumor regression at well-tolerated dosages. Based on the outcomes of preclinical efficacy and safety studies, YK-029A will enter phase Ⅲ clinical trials for the treatment of EGFRex20ins NSCLC.


Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Animales , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Mutagénesis Insercional , Receptores ErbB/genética , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , Mutación , Exones , Modelos Animales de Enfermedad , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico
3.
J Phys Chem B ; 127(13): 2900-2908, 2023 04 06.
Artículo en Inglés | MEDLINE | ID: mdl-36977372

RESUMEN

We aim to elucidate the molecular mechanism of the reciprocal relation of calmodulin's (CaM) target binding and its affinity for calcium ions (Ca2+), which is central to decoding CaM-dependent Ca2+ signaling in a cell. We employed stopped-flow experiments and coarse-grained molecular simulations that learn the coordination chemistry of Ca2+ in CaM from first-principle calculations. The associative memories as part of the coarse-grained force fields built on known protein structures further influence CaM's selection of its polymorphic target peptides in the simulations. We modeled the peptides from the Ca2+/CaM-binding domain of Ca2+/CaM-dependent kinase II (CaMKII), CaMKIIp (293-310) and selected distinctive mutations at the N-terminus. Our stopped-flow experiments have shown that the CaM's affinity for Ca2+ in the bound complex of Ca2+/CaM/CaMKIIp decreased significantly when Ca2+/CaM bound to the mutant peptide (296-AAA-298) compared to that bound to the wild-type peptide (296-RRK-298). The coarse-grained molecular simulations revealed that the 296-AAA-298 mutant peptide destabilized the structures of Ca2+-binding loops at the C-domain of CaM (c-CaM) due to both loss of electrostatic interactions and differences in polymorphic structures. We have leveraged a powerful coarse-grained approach to advance a residue-level understanding of the reciprocal relation in CaM, that could not be possibly achieved by other computational approaches.


Asunto(s)
Calcio , Calmodulina , Calmodulina/química , Secuencia de Aminoácidos , Calcio/química , Unión Proteica , Simulación por Computador , Sitios de Unión
4.
Artículo en Chino | MEDLINE | ID: mdl-35822361

RESUMEN

Objective:To investigate the correlation between the facial auditory nerve and anterior inferior cerebellar artery vascular loop in MRI of the internal auditory meatus and idiopathic sudden sensorineural hearing loss. Methods:This retrospective study enrolled 144 patients with idiopathic sudden sensorineural hearing loss(SSNHL)(experimental group) and 36 healthy subjects with 72 ears(control group), who attended the Department of Otolaryngology Head and Neck Surgery, the 940th Hospital of the Joint Logistics Support Unit of the Chinese PLA from January, 2019 to January, 2021. The magnetic resonance imaging(MRI) data of the internal auditory meatus and clinical data were collected and compared between the two groups. Results:The distance between the auditory nerve and the peripheral vessels in the unilateral SSNHL-affected ear was significantly different from that in the contralateral ears and that in the healthy ears of the control group (P<0.05). The distance between the auditory nerve and the peripheral vessels in both ear affected by bilateral SSNHL was significantly different from that in the healthy ears of the control group (P<0.05). There was no significant difference in radiological grading of vascular loops between the ears affected by unilateral SSNHL and the contralateral ears and the healthy ear of the control group (P>0.05). No statistically significant differences in radiological grading of vascular loops were found between both ears with bilateral SSNHL and the healthy ears in the control group(P>0.05). The severity of hearing loss, audiometric configuration, radiological grading of vascular loops and the distance between the facial auditory nerve and peripheral vessels were not significantly different between the affected ears in unilateral SSNHL and both ears in bilateral SSNHL (P>0.05). Conclusion:SSNHL is associated with the distance between the auditory nerve and the nearest peripheral vessel. SSNHL may occur when the vessel compresses the auditory nerve.


Asunto(s)
Pérdida Auditiva Sensorineural , Pérdida Auditiva Súbita , Arterias , Nervio Coclear , Pérdida Auditiva Sensorineural/diagnóstico por imagen , Pérdida Auditiva Sensorineural/patología , Pérdida Auditiva Súbita/diagnóstico por imagen , Humanos , Imagen por Resonancia Magnética , Estudios Retrospectivos
5.
Sci Data ; 9(1): 111, 2022 03 29.
Artículo en Inglés | MEDLINE | ID: mdl-35351915

RESUMEN

Rabbits have been widely used for studying ocular physiology and pathology due to their relatively large eye size and similar structures with human eyes. Various rabbit ocular disease models, such as dry eye, age-related macular degeneration, and glaucoma, have been established. Despite the growing application of proteomics in vision research using rabbit ocular models, there is no spectral assay library for rabbit eye proteome publicly available. Here, we generated spectral assay libraries for rabbit eye compartments, including conjunctiva, cornea, iris, retina, sclera, vitreous humor, and tears using fractionated samples and ion mobility separation enabling deep proteome coverage. The rabbit eye spectral assay library includes 9,830 protein groups and 113,593 peptides. We present the data as a freely available community resource for proteomic studies in the vision field. Instrument data and spectral libraries are available via ProteomeXchange with identifier PXD031194.


Asunto(s)
Córnea , Proteoma , Retina , Animales , Córnea/metabolismo , Proteómica , Conejos , Retina/metabolismo
6.
J Phys Chem Lett ; 13(3): 763-769, 2022 Jan 27.
Artículo en Inglés | MEDLINE | ID: mdl-35040657

RESUMEN

Organic photovoltaics (OPV) is an emerging solar cell technology that offers vast advantages such as low-cost manufacturing, transparency, and solution processability. However, because the performance of OPV devices is still disappointing compared to their inorganic counterparts, better understanding of how controlling the molecular-level morphology can impact performance is needed. To this end, one has to overcome significant challenges that stem from the complexity and heterogeneity of the underlying electronic structure and molecular morphology. In this Letter, we address this challenge in the context of the DBP/C70 OPV system by employing a modular workflow that combines recent advances in electronic structure, molecular dynamics, and rate theory. We show how the wide range of interfacial pairs can be classified into four types of interfacial donor-acceptor geometries and find that the least populated interfacial geometry gives rise to the fastest charge transfer (CT) rates.

7.
Front Mol Biosci ; 8: 661322, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-34504868

RESUMEN

Calmodulin (CaM) is a calcium-binding protein that transduces signals to downstream proteins through target binding upon calcium binding in a time-dependent manner. Understanding the target binding process that tunes CaM's affinity for the calcium ions (Ca2+), or vice versa, may provide insight into how Ca2+-CaM selects its target binding proteins. However, modeling of Ca2+-CaM in molecular simulations is challenging because of the gross structural changes in its central linker regions while the two lobes are relatively rigid due to tight binding of the Ca2+ to the calcium-binding loops where the loop forms a pentagonal bipyramidal coordination geometry with Ca2+. This feature that underlies the reciprocal relation between Ca2+ binding and target binding of CaM, however, has yet to be considered in the structural modeling. Here, we presented a coarse-grained model based on the Associative memory, Water mediated, Structure, and Energy Model (AWSEM) protein force field, to investigate the salient features of CaM. Particularly, we optimized the force field of CaM and that of Ca2+ ions by using its coordination chemistry in the calcium-binding loops to match with experimental observations. We presented a "community model" of CaM that is capable of sampling various conformations of CaM, incorporating various calcium-binding states, and carrying the memory of binding with various targets, which sets the foundation of the reciprocal relation of target binding and Ca2+ binding in future studies.

8.
J Chem Phys ; 154(21): 214108, 2021 Jun 07.
Artículo en Inglés | MEDLINE | ID: mdl-34240998

RESUMEN

In this paper, we present CTRAMER (Charge-Transfer RAtes from Molecular dynamics, Electronic structure, and Rate theory)-an open-source software package for calculating interfacial charge-transfer (CT) rate constants in organic photovoltaic (OPV) materials based on ab initio calculations and molecular dynamics simulations. The software is based on identifying representative donor/acceptor geometries within interfacial structures obtained from molecular dynamics simulation of donor/acceptor blends and calculating the corresponding Fermi's golden rule CT rate constants within the framework of the linearized-semiclassical approximation. While the methods used are well established, the integration of these state-of-the-art tools originating from different disciplines to study photoinduced CT processes with explicit treatment of the environment, in our opinion, makes this package unique and innovative. The software also provides tools for investigating other observables of interest. After outlining the features and implementation details, the usage and performance of the software are demonstrated with results from an example OPV system.

9.
J Chem Phys ; 154(12): 124104, 2021 Mar 28.
Artículo en Inglés | MEDLINE | ID: mdl-33810667

RESUMEN

It is challenging to parameterize the force field for calcium ions (Ca2+) in calcium-binding proteins because of their unique coordination chemistry that involves the surrounding atoms required for stability. In this work, we observed a wide variation in Ca2+ binding loop conformations of the Ca2+-binding protein calmodulin, which adopts the most populated ternary structures determined from the molecular dynamics simulations, followed by ab initio quantum mechanical (QM) calculations on all 12 amino acids in the loop that coordinate Ca2+ in aqueous solution. Ca2+ charges were derived by fitting to the electrostatic potential in the context of a classical or polarizable force field (PFF). We discovered that the atomic radius of Ca2+ in conventional force fields is too large for the QM calculation to capture the variation in the coordination geometry of Ca2+ in its ionic form, leading to unphysical charges. Specifically, we found that the fitted atomic charges of Ca2+ in the context of PFF depend on the coordinating geometry of electronegative atoms from the amino acids in the loop. Although nearby water molecules do not influence the atomic charge of Ca2+, they are crucial for compensating for the coordination of Ca2+ due to the conformational flexibility in the EF-hand loop. Our method advances the development of force fields for metal ions and protein binding sites in dynamic environments.


Asunto(s)
Calcio/química , Calcio/metabolismo , Calmodulina/metabolismo , Animales , Sitios de Unión , Calmodulina/química , Bovinos , Motivos EF Hand , Humanos , Simulación de Dinámica Molecular , Unión Proteica , Teoría Cuántica , Electricidad Estática , Agua/química
10.
J Chem Theory Comput ; 16(10): 6481-6490, 2020 Oct 13.
Artículo en Inglés | MEDLINE | ID: mdl-32997944

RESUMEN

We present a comprehensive analysis of the interplay between the choice of an electronic structure method and the effect of using polarizable force fields vs. nonpolarizable force fields when calculating solution-phase charge-transfer (CT) rates. The analysis is based on an integrative approach that combines inputs from electronic structure calculations and molecular dynamics simulations and is performed in the context of the carotenoid-porphyrin-C60 molecular triad dissolved in an explicit tetrahydrofuran (THF) liquid solvent. Marcus theory rate constants are calculated for the multiple CT processes that occur in this system based on either polarizable or nonpolarizable force fields, parameterized using density functional theory (DFT) with either the B3LYP or the Baer-Neuhauser-Livshits (BNL) density functionals. We find that the effect of switching from nonpolarizable to polarizable force fields on the CT rates is strongly dependent on the choice of the density functional. More specifically, the rate constants obtained using polarizable and nonpolarizable force fields differ significantly when B3LYP is used, while much smaller changes are observed when BNL is used. It is shown that this behavior can be traced back to the tendency of B3LYP to overstabilize CT states, thereby pushing the underlying electronic transitions to the deep inverted region, where even small changes in the force fields can lead to significant changes in the CT rate constants. Our results demonstrate the importance of combining polarizable force fields with an electronic structure method that can accurately capture the energies of excited CT states when calculating charge-transfer rates.

11.
Biophys J ; 118(7): 1665-1678, 2020 04 07.
Artículo en Inglés | MEDLINE | ID: mdl-32145192

RESUMEN

We have developed a computational method of atomistically refining the structural ensemble of intrinsically disordered peptides (IDPs) facilitated by experimental measurements using circular dichroism spectroscopy (CD). A major challenge surrounding this approach stems from the deconvolution of experimental CD spectra into secondary structure features of the IDP ensemble. Currently available algorithms for CD deconvolution were designed to analyze the spectra of proteins with stable secondary structures. Herein, our work aims to minimize any bias from the peptide deconvolution analysis by implementing a non-negative linear least-squares fitting algorithm in conjunction with a CD reference data set that contains soluble and denatured proteins (SDP48). The non-negative linear least-squares method yields the best results for deconvolution of proteins with higher disordered content than currently available methods, according to a validation analysis of a set of protein spectra with Protein Data Bank entries. We subsequently used this analysis to deconvolute our experimental CD data to refine our computational model of the peptide secondary structure ensemble produced by all-atom molecular dynamics simulations with implicit solvent. We applied this approach to determine the ensemble structures of a set of short IDPs, that mimic the calmodulin binding domain of calcium/calmodulin-dependent protein kinase II and its 1-amino-acid and 3-amino-acid mutants. Our study offers a, to our knowledge, novel way to solve the ensemble secondary structures of IDPs in solution, which is important to advance the understanding of their roles in regulating signaling pathways through the formation of complexes with multiple partners.


Asunto(s)
Proteínas Intrínsecamente Desordenadas , Simulación de Dinámica Molecular , Dicroismo Circular , Péptidos , Conformación Proteica , Estructura Secundaria de Proteína , Proteínas
12.
Phys Chem Chem Phys ; 19(34): 22969-22980, 2017 Aug 30.
Artículo en Inglés | MEDLINE | ID: mdl-28815237

RESUMEN

The light-harvesting molecular triad consisting of carotenoid polyene (C), diaryl-porphyrin (P) and pyrrole-fullerene (C60) is a donor-acceptor molecule capable of absorbing incident light in the visible range. Its ability to convert solar energy to electrical excitation and charge separation energy suggests a great potential in real-world applications. The ensemble of its conformations under ambient conditions varies widely according to its electronic state. In previous work, we applied a non-polarizable model to study the conformational distribution of the molecular triad in the ground and charge separated states. However, due to the lack of polarization, which imparts subtle changes in the charge distribution on atoms, molecular simulations fail to produce accurate average dipole moments. We developed the first polarizable model for a molecular triad to investigate the structural and dynamic properties of a molecular triad in the ground state in an explicit organic solvent, tetrahydrofuran (THF). We performed first-principles electronic structure calculations of the individual components in the triad as well as THF and then fit the partial atomic charges to the electrostatic potential using the i-RESP methodology. We validated these force field parameters by comparing the thermodynamic and dynamic properties obtained from molecular dynamics simulations with those from experiments. We enhanced the sampling of the triad conformations with replica exchange molecular dynamics simulations. We characterized the effects of induced polarization on the structural stability of the triad by analyzing the free energy landscapes constructed with polarizable force fields. Furthermore, by using principal component analysis, we found that the molecular triad conformations adopted a small range of torsional angles with induced polarization. The triad conformation solvated in polar solvent with a polarizable force field qualitatively agrees with that obtained from nuclear magnetic resonance spectroscopy.

13.
Biophys J ; 112(6): 1105-1119, 2017 Mar 28.
Artículo en Inglés | MEDLINE | ID: mdl-28355539

RESUMEN

We investigated the impact of bound calmodulin (CaM)-target compound structure on the affinity of calcium (Ca2+) by integrating coarse-grained models and all-atomistic simulations with nonequilibrium physics. We focused on binding between CaM and two specific targets, Ca2+/CaM-dependent protein kinase II (CaMKII) and neurogranin (Ng), as they both regulate CaM-dependent Ca2+ signaling pathways in neurons. It was shown experimentally that Ca2+/CaM (holoCaM) binds to the CaMKII peptide with overwhelmingly higher affinity than Ca2+-free CaM (apoCaM); the binding of CaMKII peptide to CaM in return increases the Ca2+ affinity for CaM. However, this reciprocal relation was not observed in the Ng peptide (Ng13-49), which binds to apoCaM or holoCaM with binding affinities of the same order of magnitude. Unlike the holoCaM-CaMKII peptide, whose structure can be determined by crystallography, the structural description of the apoCaM-Ng13-49 is unknown due to low binding affinity, therefore we computationally generated an ensemble of apoCaM-Ng13-49 structures by matching the changes in the chemical shifts of CaM upon Ng13-49 binding from nuclear magnetic resonance experiments. Next, we computed the changes in Ca2+ affinity for CaM with and without binding targets in atomistic models using Jarzynski's equality. We discovered the molecular underpinnings of lowered affinity of Ca2+ for CaM in the presence of Ng13-49 by showing that the N-terminal acidic region of Ng peptide pries open the ß-sheet structure between the Ca2+ binding loops particularly at C-domain of CaM, enabling Ca2+ release. In contrast, CaMKII peptide increases Ca2+ affinity for the C-domain of CaM by stabilizing the two Ca2+ binding loops. We speculate that the distinctive structural difference in the bound complexes of apoCaM-Ng13-49 and holoCaM-CaMKII delineates the importance of CaM's progressive mechanism of target binding on its Ca2+ binding affinities.


Asunto(s)
Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/química , Calcio/metabolismo , Calmodulina/metabolismo , Neurogranina/química , Fragmentos de Péptidos/metabolismo , Secuencia de Aminoácidos , Apoproteínas/química , Apoproteínas/metabolismo , Calmodulina/química , Modelos Moleculares , Fragmentos de Péptidos/química , Unión Proteica , Conformación Proteica
14.
Am J Otolaryngol ; 36(2): 242-8, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-25554003

RESUMEN

OBJECTIVE: Although histone deacetylase (HDAC) inhibition has been shown to protect against gentamicin (GM)-induced hearing loss in vitro, its protective effect has not been proven in vivo. In the present study, the aim was to investigate the protective effect of sodium butyrate (NaB), a specific HDAC inhibitor, on GM-induced ototoxicity in vivo. METHODS: Forty 8-week-old albino guinea pigs were divided into two experimental groups. Group 1 (n=10) underwent bilateral ear surgery to place sponges (0.3mm(3)) permeated with NaB (10µl, 100mg/ml) and physiological saline (10µl; control) in the right and left round window niches, respectively. The sponges were left in place for 15days to evaluate the effects of NaB at the applied concentration. Group 2 (n=30) underwent the same bilateral ear surgery described for Group 1, except three days after surgery, the animals received intramuscular GM injections (200mg/kg/day) for 5 consecutive days. Seven days after the final GM injection, the protective effects of NaB were examined. RESULTS: After 15days of NaB treatment (10µl, 100mg/ml), an increase in histone acetylation was detected in Corti organ samples. Auditory brainstem response (ABR) threshold shifts and hair cell loss were also reduced in NaB-treated ears after GM administration. Furthermore, GM treatment increased HDAC1 expression in outer hair cells (OHCs) in vivo, and NaB blocked this action. CONCLUSION: GM increases HDAC1 expression in OHCs, and NaB is able to block this action. Thus, it appears that the HDAC inhibitor, NaB, attenuates GM-induced hearing loss in guinea pigs.


Asunto(s)
Potenciales Evocados Auditivos del Tronco Encefálico/efectos de los fármacos , Gentamicinas/toxicidad , Pérdida Auditiva/inducido químicamente , Pérdida Auditiva/tratamiento farmacológico , Inhibidores de Histona Desacetilasas/farmacología , Análisis de Varianza , Animales , Western Blotting , Modelos Animales de Enfermedad , Femenino , Técnica del Anticuerpo Fluorescente Indirecta , Gentamicinas/farmacología , Cobayas , Células Ciliadas Auditivas/efectos de los fármacos , Masculino , Distribución Aleatoria , Valores de Referencia
15.
J Mol Recognit ; 28(2): 74-86, 2015 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-25622562

RESUMEN

Calmodulin (CaM) is a primary calcium (Ca(2+) )-signaling protein that specifically recognizes and activates highly diverse target proteins. We explored the molecular basis of target recognition of CaM with peptides representing the CaM-binding domains from two Ca(2+) -CaM-dependent kinases, CaMKI and CaMKII, by employing experimentally constrained molecular simulations. Detailed binding route analysis revealed that the two CaM target peptides, although similar in length and net charge, follow distinct routes that lead to a higher binding frustration in the CaM-CaMKII complex than in the CaM-CaMKI complex. We discovered that the molecular origin of the binding frustration is caused by intermolecular contacts formed with the C-domain of CaM that need to be broken before the formation of intermolecular contacts with the N-domain of CaM. We argue that the binding frustration is important for determining the kinetics of the recognition process of proteins involving large structural fluctuations.


Asunto(s)
Calmodulina/química , Calmodulina/metabolismo , Péptidos/química , Animales , Sitios de Unión , Proteína Quinasa Tipo 1 Dependiente de Calcio Calmodulina/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Modelos Moleculares , Unión Proteica , Estructura Terciaria de Proteína
16.
J Cell Physiol ; 230(3): 535-45, 2015 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-23526568

RESUMEN

The mechanisms underlying doxorubicin (Dox) resistance in colon cancer cells are not fully understood. MicroRNA (miRNA) play important roles in tumorigenesis and drug resistance. However, the relationship between miRNA and Dox resistance in colon cancer cells has not been previously explored. In this study, we utilized microRNA array and real-time PCR to verify that miR-127, miR-195, miR-22, miR-137 were significantly down-regulated, while miR-21, miR-592 were up-regulated in both HT29/DOX and LOVO/DOX cell lines. In vitro cell viability assay showed that knockdown of miR-195 in HT29 and LOVO cells caused a marked inhibition of Dox-induced cytotoxicity. Moreover, we explored that miR-195 is involved in repression of BCL2L2 expression through targeting its 3'-untranslated region, especially the first binding site within its mRNA. Furthermore, down-regulation of miR-195 conferred DOX resistance in parental cells and reduced cell apoptosis activity, while over-expression of miR-195 sensitized resistant cells to DOX and enhanced cell apoptosis activity, all of which can be partly rescued by BCL2L2 siRNA and cDNA expression. These results may have implications for therapeutic strategies aiming to overcome colon cancer cell resistance to Dox.


Asunto(s)
Proteínas Reguladoras de la Apoptosis/biosíntesis , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/genética , MicroARNs/genética , Apoptosis/efectos de los fármacos , Proteínas Reguladoras de la Apoptosis/genética , Sitios de Unión , Neoplasias del Colon/patología , Doxorrubicina/administración & dosificación , Resistencia a Antineoplásicos/genética , Regulación Neoplásica de la Expresión Génica , Humanos , ARN Mensajero/efectos de los fármacos
17.
Neurosci Lett ; 578: 111-6, 2014 Aug 22.
Artículo en Inglés | MEDLINE | ID: mdl-24993301

RESUMEN

Spiral ganglion neuron (SGN) injury is a generally accepted precursor of auditory neuropathy. Receptor-interacting protein 3 (RIP3) has been reported as an important necroptosis pathway mediator that can be blocked by necrostatin-1 (Nec-1). In our study, we sought to identify whether necroptosis participated in SGN injury. Ouabain was applied to establish an SGN injury model. We measured the auditory brain-stem response (ABR) threshold shift as an indicator of the auditory conditions. Positive ß3-tubulin immunofluorescence staining indicated the surviving SGNs. RIP3 expression was evaluated using immunofluorescence, quantitative real-time polymerase chain reaction and western blot. SGN injury promoted an increase in RIP3 expression that could be suppressed by application of the necroptosis inhibitor Nec-1. A decreased ABR threshold shift and increased SGN density were observed when Nec-1 was administered with apoptosis inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD). These results demonstrated that necroptosis is an indispensable pathway separately from apoptosis leading to SGN death pathway, in which RIP3 plays an important role.


Asunto(s)
Neuronas/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Ganglio Espiral de la Cóclea/metabolismo , Animales , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Muerte Celular/fisiología , Modelos Animales de Enfermedad , Potenciales Evocados Auditivos del Tronco Encefálico/efectos de los fármacos , Imidazoles/farmacología , Indoles/farmacología , Neuronas/efectos de los fármacos , Oligopéptidos/farmacología , Ouabaína/toxicidad , Ratas , Ratas Sprague-Dawley , Ganglio Espiral de la Cóclea/efectos de los fármacos , Ganglio Espiral de la Cóclea/lesiones
18.
Neurosci Lett ; 561: 140-5, 2014 Feb 21.
Artículo en Inglés | MEDLINE | ID: mdl-24394908

RESUMEN

Neural stem cell (NSC) transplantation into the cochlea has been tested as a treatment for spiral ganglion neuron (SGN) degenerative disease and injury in various animal models. A recent study has shown evidence of functional recovery after transplantation of the stem cells into a degenerated-SGN model. Chemokine stromal cell-derived factor-1 (SDF-1, or known as CXC chemokine ligand-12, CXCL-12) signaling through CXCR4 has previously been identified as a key step in the homing of the stem cells within the injury areas; meanwhile, studies have revealed that the SDF-1/CXCR4 axis is also involved in axon guidance and pathfinding. A study found that transplanted neural precursor cells can migrate to the root of the auditory nerve when animals are subjected to an augmented acoustic environment (AAE). In accordance with these studies, we hypothesize that AAE will up-regulate the expression of SDF-1 in acoustic nerves. We tested our hypothesis by examining the expression of SDF-1 in different acoustic environments, and the results were confirmed by the auditory brainstem response (ABR), immunohistochemical and RT-PCR analyses. The results showed that SDF-1 was expressed at a relatively low level in the SGNs under normal animal unit acoustic conditions (40-50 dB). Moreover, it was significantly up-regulated in the SGNs under the 75 dB (augmented physiological process without hearing loss) and 90 dB AAE (pathological process with light hearing loss) conditions; however, under the 115 dB AAE (pathological process with severe hearing loss) condition, the expression of SDF-1 was not up-regulated. The results confirmed that appropriately augmented acoustical stimuli lead to the up-regulation of SDF-1, which may assist in the migration of the transplanted cells and the subsequent establishment of essential synaptic contacts between the exogenous cells and the host auditory pathway.


Asunto(s)
Quimiocina CXCL12/metabolismo , Neuronas/metabolismo , Ruido , Ganglio Espiral de la Cóclea/metabolismo , Estimulación Acústica , Animales , Quimiocina CXCL12/genética , Potenciales Evocados Auditivos del Tronco Encefálico , Pérdida Auditiva/metabolismo , Pérdida Auditiva/patología , Pérdida Auditiva/fisiopatología , Masculino , Neuronas/citología , ARN Mensajero/metabolismo , Ratas Sprague-Dawley , Ganglio Espiral de la Cóclea/citología
19.
Cell Transplant ; 23(6): 747-60, 2014 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-23809337

RESUMEN

Although neural stem cell (NSC) transplantation is widely expected to become a therapy for nervous system degenerative diseases and injuries, the low neuronal differentiation rate of NSCs transplanted into the inner ear is a major obstacle for the successful treatment of spiral ganglion neuron (SGN) degeneration. In this study, we validated whether the local microenvironment influences the neuronal differentiation of transplanted NSCs in the inner ear. Using a rat SGN degeneration model, we demonstrated that transplanted NSCs were more likely to differentiate into microtubule-associated protein 2 (MAP2)-positive neurons in SGN-degenerated cochleae than in control cochleae. Using real-time quantitative PCR and an immunofluorescence assay, we also proved that the expression of Wnt1 (a ligand of Wnt signaling) increases significantly in Schwann cells in the SGN-degenerated cochlea. We further verified that NSC cultures express receptors and signaling components for Wnts. Based on these expression patterns, we hypothesized that Schwann cell-derived Wnt1 and Wnt signaling might be involved in the regulation of the neuronal differentiation of transplanted NSCs. We verified our hypothesis in vitro using a coculture system. We transduced a lentiviral vector expressing Wnt1 into cochlear Schwann cell cultures and cocultured them with NSC cultures. The coculture with Wnt1-expressing Schwann cells resulted in a significant increase in the percentage of NSCs that differentiated into MAP2-positive neurons, whereas this differentiation-enhancing effect was prevented by Dkk1 (an inhibitor of the Wnt signaling pathway). These results suggested that Wnt1 derived from cochlear Schwann cells enhanced the neuronal differentiation of transplanted NSCs through Wnt signaling pathway activation. Alterations of the microenvironment deserve detailed investigation because they may help us to conceive effective strategies to overcome the barrier of the low differentiation rate of transplanted NSCs.


Asunto(s)
Diferenciación Celular , Degeneración Nerviosa/terapia , Células-Madre Neurales/citología , Células de Schwann/metabolismo , Trasplante de Células Madre , Proteína Wnt1/metabolismo , Animales , Células Cultivadas , Nervio Coclear/metabolismo , Modelos Animales de Enfermedad , Femenino , Vectores Genéticos/metabolismo , Células HEK293 , Humanos , Lentivirus/genética , Ratones , Ratones Endogámicos C57BL , Degeneración Nerviosa/patología , Neuronas/citología , Ratas , Ratas Sprague-Dawley , Células de Schwann/citología , Ganglio Espiral de la Cóclea/metabolismo , Ganglio Espiral de la Cóclea/patología , Proteína Wnt1/genética
20.
Proc Natl Acad Sci U S A ; 110(51): 20545-50, 2013 Dec 17.
Artículo en Inglés | MEDLINE | ID: mdl-24297894

RESUMEN

Protein-protein interactions drive most every biological process, but in many instances the domains mediating recognition are disordered. How specificity in binding is attained in the absence of defined structure contrasts with well-established experimental and theoretical work describing ligand binding to protein. The signaling protein calmodulin presents a unique opportunity to investigate mechanisms for target recognition given that it interacts with several hundred different targets. By advancing coarse-grained computer simulations and experimental techniques, mechanistic insights were gained in defining the pathways leading to recognition and in how target selectivity can be achieved at the molecular level. A model requiring mutually induced conformational changes in both calmodulin and target proteins was necessary and broadly informs how proteins can achieve both high affinity and high specificity.


Asunto(s)
Proteína Quinasa Tipo 1 Dependiente de Calcio Calmodulina/química , Calmodulina/química , Modelos Moleculares , Péptidos/química , Animales , Mamíferos , Unión Proteica , Conformación Proteica
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