RESUMO
PorB is a well-characterized outer membrane protein that is common among Neisseria species and is required for survival. A vaccine candidate, PorB induces antibody responses that are directed against six variable surface-exposed loops that differ in sequence depending on serotype. Although Neisseria meningitidis is naturally competent and porB genetic mosaicism provides evidence for strong positive selection, the sequences of PorB serotypes commonly associated with invasive disease are often conserved, calling into question the interaction of specific PorB loop sequences in immune engagement. In this report, we provide evidence that antibody binding to a PorB epitope can be altered by sequence mutations in non-epitope loops. Through the construction of hybrid PorB types and PorB molecular dynamics simulations, we demonstrate that loops both adjacent and non-adjacent to the epitope loop can enhance or diminish antibody binding, a phenotype that correlates with serum bactericidal activity. We further examine the interaction of PorB with outer membrane-associated proteins, including PorA and RmpM. Deletion of these proteins alters the composition of PorB-containing native complexes and reduces antibody binding and serum killing relative to the parental strain, suggesting that both intramolecular and intermolecular PorB interactions contribute to host adaptive immune evasion.
Assuntos
Neisseria meningitidis Sorogrupo B/metabolismo , Neisseria meningitidis/metabolismo , Porinas/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Anticorpos Antibacterianos/imunologia , Proteínas da Membrana Bacteriana Externa/metabolismo , Epitopos/metabolismo , Heterogeneidade Genética , Neisseria meningitidis/genética , Neisseria meningitidis Sorogrupo B/genética , Porinas/genética , Ligação Proteica , Sorogrupo , Transdução de SinaisRESUMO
The outer membrane (OM) of Gram-negative bacteria is composed of lipopolysaccharide (LPS) in the outer leaflet and phospholipids in the inner leaflet. The outer membrane protein H (OprH) of Pseudomonas aeruginosa provides an increased stability to the OMs by directly interacting with LPS. Here we report the influence of various P. aeruginosa and, for comparison, Escherichia coli LPS environments on the physical properties of the OMs and OprH using all-atom molecular dynamics simulations. The simulations reveal that although the P. aeruginosa OMs are thinner hydrophobic bilayers than the E. coli OMs, which is expected from the difference in the acyl chain length of their lipid A, this effect is almost imperceptible around OprH due to a dynamically adjusted hydrophobic match between OprH and the OM. The structure and dynamics of the extracellular loops of OprH show distinct behaviors in different LPS environments. Including the O-antigen greatly reduces the flexibility of the OprH loops and increases the interactions between these loops and LPS. Furthermore, our study shows that the interactions between OprH and LPS mainly depend on the secondary structure of OprH and the chemical structure of LPS, resulting in distinctive patterns in different LPS environments.
Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Lipopolissacarídeos/metabolismo , Simulação de Dinâmica Molecular , Sequência de Aminoácidos , Proteínas da Membrana Bacteriana Externa/química , Membrana Celular/metabolismo , Resistência Microbiana a Medicamentos , Escherichia coli/citologia , Ligação Proteica , Conformação Proteica , Pseudomonas aeruginosa/citologia , Pseudomonas aeruginosa/efeitos dos fármacos , Água/químicaRESUMO
It remains controversial whether the routes from somatic cells to induced pluripotent stem cells (iPSCs) are related to the reverse order of normal developmental processes. Specifically, it remains unaddressed whether or not the differentiated cells become iPSCs through their original tissue stem cell-like state. Previous studies analyzing the reprogramming process mostly used fibroblasts; however, the stem cell characteristics of fibroblasts made it difficult to address this. Here, we generated iPSCs from mouse astrocytes, a type of glial cells, by three (OCT3/4, KLF4, and SOX2), two (OCT3/4 and KLF4), or four (OCT3/4, KLF4, and SOX2 plus c-MYC) factors. Sox1, a neural stem cell (NSC)-specific transcription factor, is transiently up-regulated during reprogramming, and Sox1-positive cells become iPSCs. The up-regulation of Sox1 is essential for OCT3/4- and KLF4-induced reprogramming. Genome-wide analysis revealed that the gene expression profile of Sox1-expressing intermediate-state cells resembles that of NSCs. Furthermore, the intermediate-state cells are able to generate neurospheres, which can differentiate into both neurons and glial cells. Remarkably, during fibroblast reprogramming, neither Sox1 up-regulation nor an increase in neurogenic potential occurs. Our results thus demonstrate that astrocytes are reprogrammed through an NSC-like state.
Assuntos
Astrócitos/metabolismo , Técnicas de Reprogramação Celular , Reprogramação Celular , Células-Tronco Neurais/metabolismo , Células-Tronco Pluripotentes/metabolismo , Fatores de Transcrição/biossíntese , Animais , Astrócitos/citologia , Células Cultivadas , Fator 4 Semelhante a Kruppel , Camundongos , Células-Tronco Neurais/citologia , Células-Tronco Pluripotentes/citologia , Fatores de Transcrição/genéticaRESUMO
The present study examines the effects of the model dependence, ionic strength, divalent ions, and hydrophobic interaction on the structural organization of the human neurofilament (NF) brush, using canonical ensemble Monte Carlo (MC) simulations of a coarse-grained model with the amino-acid resolution. The model simplifies the interactions between the NF core and the sidearm or between the sidearms by the sum of excluded volume, electrostatic, and hydrophobic interactions, where both monovalent salt ions and solvents are implicitly incorporated into the electrostatic interaction potential. Several important observations are made from the MC simulations of the coarse-grained model NF systems. First, the mean-field type description of monovalent salt ions works reasonably well in the NF system. Second, the manner by which the NF sidearms are arranged on the surface of the NF backbone core has little influence on the lateral extension of NF sidearms. Third, the lateral extension of the NF sidearms is highly affected by the ionic strength of the system: at low ionic strength, NF-M is most extended but at high ionic strength, NF-H is more stretched out because of the effective screening of the electrostatic interaction. Fourth, the presence of Ca(2+) ions induces the attraction between negatively charged residues, which leads to the contraction of the overall NF extension. Finally, the introduction of hydrophobic interaction does not change the general structural organization of the NF sidearms except that the overall extension is contracted.
Assuntos
Íons , Modelos Moleculares , Proteínas de Neurofilamentos/química , Concentração Osmolar , Humanos , Método de Monte Carlo , Conformação ProteicaRESUMO
Glycolipids (such as glycoglycerolipids, glycosphingolipids, and glycosylphosphatidylinositol) and lipoglycans (such as lipopolysaccharides (LPS), lipooligosaccharides (LOS), mycobacterial lipoarabinomannan, and mycoplasma lipoglycans) are typically found on the surface of cell membranes and play crucial roles in various cellular functions. Characterizing their structure and dynamics at the molecular level is essential to understand their biological roles, but systematic generation of glycolipid and lipoglycan structures is challenging because of great variations in lipid structures and glycan sequences (i.e., carbohydrate types and their linkages). To facilitate the generation of all-atom glycolipid/LPS/LOS structures, we have developed Glycolipid Modeler and LPS Modeler in CHARMM-GUI ( http://www.charmm-gui.org ), a web-based interface that simplifies building of complex biological simulation systems. In addition, we have incorporated these modules into Membrane Builder so that users can readily build a complex symmetric or asymmetric biological membrane system with various glycolipids and LPS/LOS. These tools are expected to be useful in innovative and novel glycolipid/LPS/LOS modeling and simulation research by easing tedious and intricate steps in modeling complex biological systems and shall provide insight into structures, dynamics, and underlying mechanisms of complex glycolipid-/LPS-/LOS-containing biological membrane systems.
Assuntos
Glicolipídeos/química , Lipopolissacarídeos/química , Proteínas de Bactérias/química , Antígenos CD59/química , Campylobacter jejuni/química , Membrana Celular/química , Simulação por Computador , Escherichia coli/química , Glicosilfosfatidilinositóis/química , Humanos , Simulação de Dinâmica Molecular , Interface Usuário-ComputadorRESUMO
AIM: To introduce and evaluate the efficacy and technical aspects of endoscopic submucosal dissection (ESD) using a novel device, the Fork knife. METHODS: From March 2004 to April 2008, ESD was performed on 265 gastric lesions using a Fork knife (Endo FS) (group A) and on 72 gastric lesions using a Flexknife (group B) at a single tertiary referral center. We retrospectively compared the endoscopic characteristics of the tumors, pathological findings, and sizes of the resected specimens. We also compared the en bloc resection rate, complete resection rate, complications, and procedure time between the two groups. RESULTS: The mean size of the resected specimens was 4.27 +/- 1.26 cm in group A and 4.29 +/- 1.48 cm in group B. The en bloc resection rate was 95.8% (254/265 lesions) in group A and 93.1% (67/72) in group B. Complete ESD without tumor cell invasion of the resected margin was obtained in 81.1% (215/265) of group A and in 73.6% (53/72) of group B. The perforation rate was 0.8% (2/265) in group A and 1.4% (1/72) in group B. The mean procedure time was 59.63 +/- 56.12 min in group A and 76.65 +/- 70.75 min in group B (P < 0.05). CONCLUSION: The Fork knife (Endo FS) is useful for clinical practice and has the advantage of reducing the procedure time.
Assuntos
Dissecação/instrumentação , Endoscópios Gastrointestinais/normas , Endoscopia Gastrointestinal/métodos , Mucosa Intestinal/cirurgia , Idoso , Feminino , Humanos , Injeções/instrumentação , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Irrigação Terapêutica/instrumentação , Fatores de TempoRESUMO
The outer membrane (OM) of Gram-negative bacteria is a unique asymmetric lipid bilayer containing lipopolysaccharides (LPS) in the outer leaflet and phospholipids in the inner leaflet. Due to the lack of porins with large pores, the substrate-specific outer membrane proteins play a crucial role in the uptake of small molecules in Pseudomonas aeruginosa. In this study, we report the influences of OM compositions on the ion permeation properties of OccK5 (also known as OpdH), such as ion selectivity and diffusion constants, using all-atom molecular dynamics simulations. The simulations indicate that OccK5 shows a remarkable anion selectivity independent of the OM composition and effective cation concentration. Intriguingly, the outer core and O-antigens of LPS sterically occlude the channel entrance and decrease the diffusion constants of ions approaching the channel, which was also observed in our previous OmpF porin simulations in Escherichia coli OMs. Our results emphasize the role of native membranes in fine-tuning the functional properties of membrane channels.
Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Canais Iônicos/metabolismo , Bicamadas Lipídicas/metabolismo , Pseudomonas aeruginosa/química , Proteínas da Membrana Bacteriana Externa/química , Cálcio/química , Cálcio/metabolismo , Cloretos/química , Cloretos/metabolismo , Difusão , Glicerofosfolipídeos/química , Glicerofosfolipídeos/metabolismo , Ativação do Canal Iônico , Canais Iônicos/química , Transporte de Íons , Bicamadas Lipídicas/química , Lipopolissacarídeos/química , Lipopolissacarídeos/metabolismo , Simulação de Dinâmica Molecular , Potássio/química , Potássio/metabolismo , Ligação ProteicaRESUMO
The process of cell reprogramming has been characterized considerably since the successful generation of induced pluripotent stem cells. However, the importance of cell-cell communications for cellular reprogramming remains largely unknown. Secreted factors, which are expressed and secreted during reprogramming, may influence the reprogramming efficiency. Here, we have identified Sostdc1, Glb1l2, Fetub, Dpp4, Gdf3, Trh, and Tdgf1 as prominently upregulated secreted factors during reprogramming. Our detailed analysis reveals that these seven factors may be categorized into four groups based on their expression patterns in relation to the reprogramming stages. Remarkably, knockdown of Sostdc1, which is the most prominently upregulated factor and which is expressed earlier than the other six factors, results in reduced reprogramming efficiency, suggesting its involvement in the reprogramming process.
Assuntos
Reprogramação Celular/genética , Regulação da Expressão Gênica , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas Morfogenéticas Ósseas/genética , Proteínas Morfogenéticas Ósseas/metabolismo , Células Cultivadas , Dipeptidil Peptidase 4/genética , Dipeptidil Peptidase 4/metabolismo , Fator de Crescimento Epidérmico/genética , Fator de Crescimento Epidérmico/metabolismo , Fetuína-B/genética , Fetuína-B/metabolismo , Fibroblastos/metabolismo , Citometria de Fluxo , Fator 3 de Diferenciação de Crescimento/genética , Fator 3 de Diferenciação de Crescimento/metabolismo , Immunoblotting , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Análise em Microsséries , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Treatment with several Wnt/ß-catenin signaling pathway regulators can change the cellular reprogramming efficiency; however, the dynamics and role of endogenous Wnt/ß-catenin signaling in reprogramming remain largely unanswered. Here we identify the upregulation of WNT2 and subsequent ß-catenin nuclear accumulation as key events in reprogramming. Transient nuclear accumulation of ß-catenin occurs early in MEF reprogramming. Wnt2 is strongly expressed in the early stage of reprogramming. Wnt2 knockdown suppresses the nuclear accumulation of ß-catenin and reduces the reprogramming efficiency. WNT2 overexpression promotes ß-catenin nuclear accumulation and enhances the reprogramming efficiency. WNT2 contributes to the promotion of cell proliferation. Experiments with several drugs that control the Wnt pathway also indicate the importance of ß-catenin nuclear accumulation in reprogramming. Our findings reveal the role of WNT2/ß-catenin signaling in reprogramming.
Assuntos
Núcleo Celular/metabolismo , Reprogramação Celular , Células-Tronco Embrionárias/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Proteína Wnt2/genética , beta Catenina/genética , Animais , Caderinas/genética , Caderinas/metabolismo , Núcleo Celular/genética , Proliferação de Células , Embrião de Mamíferos , Células-Tronco Embrionárias/citologia , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Expressão Gênica , Células HEK293 , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Camundongos , Camundongos Endogâmicos ICR , Proteína Homeobox Nanog/genética , Proteína Homeobox Nanog/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Fatores de Transcrição da Família Snail/genética , Fatores de Transcrição da Família Snail/metabolismo , Antígenos Thy-1/genética , Antígenos Thy-1/metabolismo , Proteína Wnt2/metabolismo , beta Catenina/metabolismoRESUMO
The role of secreted molecules in cellular reprogramming has been poorly understood. Here we identify a truncated form of ephrin receptor A7 (EPHA7) as a key regulator of reprogramming. Truncated EPHA7 is prominently upregulated and secreted during reprogramming. EPHA7 expression is directly regulated by OCT3/4. EphA7 knockdown results in marked reduction of reprogramming efficiency, and the addition of truncated EPHA7 is able to restore it. ERK activity is markedly reduced during reprogramming, and the secreted, truncated EPHA7 is responsible for ERK activity reduction. Remarkably, treatment of EphA7-knockdown MEFs with the ERK pathway inhibitor restores reprogramming efficiency. Analyses show that truncated EPHA7-induced ERK activity reduction plays an important role in the middle phase of reprogramming. Thus, our findings uncover the importance of secreted EPHA7-induced ERK activity reduction in reprogramming.
Assuntos
Reprogramação Celular , Fibroblastos/citologia , Sistema de Sinalização das MAP Quinases , Receptor EphA7/metabolismo , Animais , Células Cultivadas , Fibroblastos/metabolismo , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos ICR , Receptor EphA7/genéticaRESUMO
We report colonoscopic features of an intussuscepted Meckel's diverticulum, presenting with hematochezia. A 35-year-old woman presented to the emergency room with acute onset, transient, sharp, severe epigastric pain that began 6 h earlier. Colonoscopy revealed a reddish, soft, fist-sized polypoid lesion in the terminal ileum. The lesion was misinterpreted as a hematoma by an inexperienced endoscopist. The patient began to complain of intermittent, severe periumbilical pain following the colonoscopic examination. Subsequent computed tomography showed an enteric intussusception. An exploratory laparotomy revealed an intussuscepted Meckel's diverticulum, with transmural infarction. Colonoscopy was of little use in assessing the intussusception. However, colonoscopic examination may be performed initially, especially in an intussuscepted Meckel's diverticulum presenting with hematochezia. Endoscopists should note the endoscopic features of an intussuscepted Meckel's diverticulum.