RESUMO
Porcine reproductive and respiratory syndrome virus (PRRSV) causes a highly transmissible disease of significant concern in the pig industry. Previous studies have demonstrated that the XM-2020 strain (a lineage 1.8 PRRSV IA/2012/NADC30) can induce special hemorrhagic injury in the small intestines. However, the specific mechanism underlying this injurious effect remains incompletely understood. In this study, we examined the pathogenic properties of XM-2020 and YC-2020 strains (a lineage 1.5 PRRSV IA/2014/NADC34) in piglets. Animal pathogenic tests revealed that with either Lineage 1 PRRSVs strains XM-2020 or YC-2020 demonstrated pronounced intestinal hemorrhage and suppression of peripheral immunological organs, comparing to JXA1 infection. Transcriptome analysis of diseased small intestines unveiled that PRRSV infection stimulated oxidative and inflammatory reactions. Remarkably, we also observed activation of the complement system alongside a notable down-regulation of complement and coagulation cascade pathways in the Lineage 1 PRRSVs infection group. Based on these findings, we propose that the primary mechanism driving the hemorrhagic injury of the small intestine caused by Lineage 1 PRRSVs is the suppression of complement and coagulation cascades resulting from immunosuppression. This discovery deepens our understanding of the pathogenicity of PRRSV in the small intestine and provides promising ways out for the development of innovative strategies aimed at controlling PRRSV.
Assuntos
Proteínas do Sistema Complemento , Síndrome Respiratória e Reprodutiva Suína , Vírus da Síndrome Respiratória e Reprodutiva Suína , Animais , Suínos , Proteínas do Sistema Complemento/imunologia , Proteínas do Sistema Complemento/metabolismo , Vírus da Síndrome Respiratória e Reprodutiva Suína/patogenicidade , Síndrome Respiratória e Reprodutiva Suína/virologia , Síndrome Respiratória e Reprodutiva Suína/patologia , Coagulação Sanguínea , Intestino Delgado/virologia , Intestino Delgado/patologia , Intestinos/virologia , Intestinos/patologia , Perfilação da Expressão Gênica , HemorragiaRESUMO
Siglec-9 is an MHC-independent inhibitory receptor expressed on a subset of natural killer (NK) cells. Siglec-9 restrains NK cytotoxicity by binding to sialoglycans (sialic acid-containing glycans) on target cells. Despite the importance of Siglec-9 interactions in tumor immune evasion, their role as an immune evasion mechanism during HIV infection has not been investigated. Using in vivo phenotypic analyses, we found that Siglec-9+ CD56dim NK cells, during HIV infection, exhibit an activated phenotype with higher expression of activating receptors and markers (NKp30, CD38, CD16, DNAM-1, perforin) and lower expression of the inhibitory receptor NKG2A, compared to Siglec-9- CD56dim NK cells. We also found that levels of Siglec-9+ CD56dim NK cells inversely correlate with viral load during viremic infection and CD4+ T cell-associated HIV DNA during suppressed infection. Using in vitro cytotoxicity assays, we confirmed that Siglec-9+ NK cells exhibit higher cytotoxicity towards HIV-infected cells compared to Siglec-9- NK cells. These data are consistent with the notion that Siglec-9+ NK cells are highly cytotoxic against HIV-infected cells. However, blocking Siglec-9 enhanced NK cells' ability to lyse HIV-infected cells, consistent with the known inhibitory function of the Siglec-9 molecule. Together, these data support a model in which the Siglec-9+ CD56dim NK subpopulation is highly cytotoxic against HIV-infected cells even whilst being restrained by the inhibitory effects of Siglec-9. To harness the cytotoxic capacity of the Siglec-9+ NK subpopulation, which is dampened by Siglec-9, we developed a proof-of-concept approach to selectively disrupt Siglec/sialoglycan interactions between NK and HIV-infected cells. We achieved this goal by conjugating Sialidase to several HIV broadly neutralizing antibodies. These conjugates selectively desialylated HIV-infected cells and enhanced NK cells' capacity to kill them. In summary, we identified a novel, glycan-based interaction that may contribute to HIV-infected cells' ability to evade NK immunosurveillance and developed an approach to break this interaction.
Assuntos
Antígenos CD/metabolismo , Antígeno CD56/imunologia , Infecções por HIV/patologia , HIV/fisiologia , Células Matadoras Naturais/imunologia , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico/metabolismo , Carga Viral , Viremia/patologia , Antígenos CD/genética , Infecções por HIV/imunologia , Infecções por HIV/metabolismo , Infecções por HIV/virologia , Humanos , Células Matadoras Naturais/metabolismo , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico/genética , Viremia/imunologia , Viremia/metabolismo , Viremia/virologiaRESUMO
In this study, an NADC34-like strain of porcine reproductive and respiratory syndrome virus (PRRSV), YC-2020, was isolated from a pig farm in Yuncheng, Shanxi Province, China. Phylogenetic and molecular evolutionary analysis showed that the genome sequence of YC-2020 was very similar to those of NADC34-like PRRSV strains in the ORF2-7 region. However, it was more closely related to NADC30-like PRRSV and highly pathogenic (HP) PRRSV in the NSP2 and NSP3-9 coding regions, respectively, suggesting that recombination had occurred between viruses belonging to lineages 1 and 8. Piglets infected with YC-2020 exhibited mild clinical signs, but they had severe histopathological lesions in their lungs. These findings reveal novel genetic and pathogenic features of this isolate.
Assuntos
Síndrome Respiratória e Reprodutiva Suína , Vírus da Síndrome Respiratória e Reprodutiva Suína , Animais , Suínos , Vírus da Síndrome Respiratória e Reprodutiva Suína/genética , Síndrome Respiratória e Reprodutiva Suína/genética , Filogenia , Genoma Viral , China , Variação GenéticaRESUMO
BACKGROUND: Chronic kidney disease (CKD) is a common cause of morbidity and mortality in captive wildlife species. However, CKD has been rarely documented in giant pandas. CASE PRESENTATION: The following report describes a case of an eight-year-old female giant panda showing clinical signs of epistaxis, bloody diarrhea, polyuria, azotemia and anemia. The animal died despite of supportive treatments. Necropsy was performed. Grossly, both kidneys were shrunken and scarred with pallor. Subcutis edema and petechia on the epicardium of the heart were observed. The tissue samples were made into paraffin sections and stained by H.E and special staining including Periodic Acid-Schiff (PAS), von Kossa, Masson's trichrome, Phosphotungstic acid-hematoxylin (PTAH), and Congo red. Histopathology examination revealed severe chronic tubulointerstitial nephritis with marked interstitial fibrosis, glomerulosclerosis, tubular atrophy and calcification in kidneys, and acute necrotizing hemorrhagic myocarditis with calcification in heart. Other lesions included intestinal hemorrhage, hepatic fatty degeneration and necrosis with hemosiderin, and splenic hemosiderin. CONCLUSIONS: In summary, chronic kidney disease was finally diagnosed based on the association of clinical, gross, and histopathological findings. Heart failure secondary to CKD is the leading cause of death in this giant panda. The potential cause of CKD in this animal is possibly due to long term and uncontrolled hypertension. Blood pressure monitoring is essential in establishing the diagnosis and management of hypertension in giant panda.
Assuntos
Hipertensão , Insuficiência Renal Crônica , Ursidae , Animais , Feminino , Hemossiderina , Insuficiência Renal Crônica/veterinária , Rim , Hipertensão/veterináriaRESUMO
Photoactivatable fluorophores are emerging optical probes for biological applications. Most photoactivatable fluorophores are relatively large in size and need to be activated by ultraviolet light; this dramatically limits their applications. To introduce photoactivatable fluorophores into proteins, recent investigations have explored several protein-labeling technologies, including fluorescein arsenical hairpin (FlAsH) Tag, HaloTag labeling, SNAPTag labeling, and other bioorthogonal chemistry-based methods. However, these technologies require a multistep labeling process. Here, by using genetic code expansion and a single sulfur-for-oxygen atom replacement within an existing fluorescent amino acid, we have site-specifically incorporated the photoactivatable fluorescent amino acid thioacridonylalanine (SAcd) into proteins in a single step. Moreover, upon exposure to visible light, SAcd can be efficiently desulfurized to its oxo derivatives, thus restoring the strong fluorescence of labeled proteins.
Assuntos
Alanina/química , Corantes Fluorescentes/química , Alanina/análogos & derivados , Luz , Estrutura Molecular , Processos FotoquímicosRESUMO
Antibodies, particularly of the immunoglobulin G (IgG) isotype, are a group of biomolecules that are extensively used as affinity reagents for many applications in research, disease diagnostics, and therapy. Most of these applications require antibodies to be modified with specific functional moieties, including fluorophores, drugs, and proteins. Thus, a variety of methodologies have been developed for the covalent labeling of antibodies. The most common methods stably attach functional molecules to lysine or cysteine residues, which unavoidably results in heterogeneous products that cannot be further purified. In an effort to prepare homogeneous antibody conjugates, bioorthogonal handles have been site-specifically introduced via enzymatic treatment, genetic code expansion, or genetically encoded tagging, followed by functionalization using bioorthogonal conjugation reactions. The resulting homogeneous products have proven superior to their heterogeneous counterparts for both in vitro and in vivo usage. Nevertheless, additional chemical treatment or protein engineering of antibodies is required for incorporation of the bioorthogonal handles, processes that often affect antibody folding, stability, and/or production yield and cost. Accordingly, concurrent with advances in the fields of bioorthogonal chemistry and protein engineering, there is growing interest in site-specifically labeling native (nonengineered) antibodies without chemical or enzymatic treatments. In this review, we highlight recent strategies for producing site-specific native antibody conjugates and provide a comprehensive summary of the merits and disadvantages of these strategies.
Assuntos
Código Genético , Imunoconjugados , Engenharia de ProteínasRESUMO
Site-specific antibody conjugates with a well-defined structure and superb therapeutic index are of great interest for basic research, disease diagnostics, and therapy. Here, we develop a novel proximity-induced antibody conjugation strategy enabling site-specific covalent bond formation between functional moieties and native antibodies without antibody engineering or additional UV/chemical treatment. A high conjugation efficiency and specificity was achieved with IgGs from different species and subclasses. The utility of this approach was demonstrated by site-specific conjugation of the small-molecule fluorophore to a native antibody and in vitro characterization of its activities.
Assuntos
Corantes Fluorescentes/química , Imunoconjugados/química , Imunoglobulina G/química , Animais , Antineoplásicos Imunológicos/química , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Humanos , Camundongos , Engenharia de Proteínas , Trastuzumab/química , Raios UltravioletaRESUMO
To promote glucose uptake into fat and muscle cells, insulin causes the translocation of GLUT4 glucose transporters from intracellular vesicles to the cell surface. Previous data support a model in which TUG traps GLUT4-containing vesicles and tethers them intracellularly in unstimulated cells and in which insulin mobilizes this pool of vesicles by releasing this tether. Here we show that TUG undergoes site-specific endoproteolytic cleavage, which separates a GLUT4-binding, N-terminal region of TUG from a C-terminal region previously suggested to bind an intracellular anchor. Cleavage is accelerated by insulin stimulation in 3T3-L1 adipocytes and is highly dependent upon adipocyte differentiation. The N-terminal TUG cleavage product has properties of a novel 18-kDa ubiquitin-like modifier, which we call TUGUL. The C-terminal product is observed at the expected size of 42 kDa and also as a 54-kDa form that is released from membranes into the cytosol. In transfected cells, intact TUG links GLUT4 to PIST and also binds Golgin-160 through its C-terminal region. PIST is an effector of TC10α, a GTPase previously shown to transmit an insulin signal required for GLUT4 translocation, and we show using RNAi that TC10α is required for TUG proteolytic processing. Finally, we demonstrate that a cleavage-resistant form of TUG does not support highly insulin-responsive GLUT4 translocation or glucose uptake in 3T3-L1 adipocytes. Together with previous results, these data support a model whereby insulin stimulates TUG cleavage to liberate GLUT4 storage vesicles from the Golgi matrix, which promotes GLUT4 translocation to the cell surface and enhances glucose uptake.
Assuntos
Adipócitos/metabolismo , Proteínas de Transporte/metabolismo , Transportador de Glucose Tipo 4/metabolismo , Glucose/metabolismo , Células 3T3-L1 , Adipócitos/citologia , Adipócitos/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Proteínas de Transporte/genética , Glucose/farmacocinética , Transportador de Glucose Tipo 4/genética , Complexo de Golgi/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Immunoblotting , Insulina/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Microscopia de Fluorescência , Dados de Sequência Molecular , Mutação , Transporte Proteico/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Interferência de RNA , Homologia de Sequência de AminoácidosRESUMO
Despite the rapid evolution of therapeutic antibodies, their clinical efficacy in the treatment of bone tumors is hampered due to the inadequate pharmacokinetics and poor bone tissue accessibility of these large macromolecules. Here, we show that engineering therapeutic antibodies with bone-homing peptide sequences dramatically enhances their concentrations in the bone metastatic niche, resulting in significantly reduced survival and progression of breast cancer bone metastases. To enhance the bone tumor-targeting ability of engineered antibodies, we introduced varying numbers of bone-homing peptides into permissive sites of the anti-HER2 antibody, trastuzumab. Compared to the unmodified antibody, the engineered antibodies have similar pharmacokinetics and in vitro cytotoxic activity, but exhibit improved bone tumor distribution in vivo. Accordingly, in xenograft models of breast cancer metastasis to bone sites, engineered antibodies with enhanced bone specificity exhibit increased inhibition of both initial bone metastases and secondary multiorgan metastases. Furthermore, this engineering strategy is also applied to prepare bone-targeting antibody-drug conjugates with enhanced therapeutic efficacy. These results demonstrate that adding bone-specific targeting to antibody therapy results in robust bone tumor delivery efficacy. This provides a powerful strategy to overcome the poor accessibility of antibodies to the bone tumors and the consequential resistance to the therapy.
RESUMO
In most bacteria, cell division is centrally organized by the FtsZ protein, which assembles into dynamic filaments at the division site along the cell membrane that interact with other key cell division proteins. In gammaproteobacteria such as Escherichia coli, FtsZ filaments are anchored to the cell membrane by two essential proteins, FtsA and ZipA. Canonically, this interaction was believed to be mediated solely by the FtsZ C-terminal peptide (CTP) domain that interacts with these and several other regulatory proteins. However, we now provide evidence of a second interaction between FtsZ and ZipA. Using site-specific photoactivated cross-linking, we identified a noncanonical FtsZ-binding site on ZipA on the opposite side from the FtsZ CTP-binding pocket. Cross-linking at this site was unaffected by the truncation of the FtsZ linker and CTP domains, indicating that this noncanonical site must interact directly with the globular core domain of FtsZ. Mutations introduced into either the canonical or noncanonical binding sites on ZipA disrupted photo-cross-linking with FtsZ and normal ZipA function in cell division, suggesting that both binding modes are important for normal cell growth and division. One mutation at the noncanonical face was also found to suppress defects of several other canonical and noncanonical site mutations in ZipA, suggesting there is some interdependence between the two sites. Taken together, these results suggest that ZipA employs a two-pronged FtsZ-binding mechanism. IMPORTANCE The tubulin homolog FtsZ plays a central early role in organizing bacterial cell division proteins at the cytoplasmic membrane. However, FtsZ does not directly interact with the membrane itself, instead relying on proteins such as FtsA to tether it to the membrane. In gammaproteobacteria, ZipA serves as a second essential membrane anchor along with FtsA. Although FtsA has a unique role in activating synthesis of the cell division septum, and ZipA may in turn activate FtsA, it was thought that both proteins interacted only with the conserved C terminus of FtsZ and were essentially interchangeable in their ability to tether FtsZ to the membrane. Here we challenge this view, providing evidence that ZipA directly contacts both the C terminus and the core domain of FtsZ. Such a two-pronged interaction between ZipA and FtsZ suggests that ZipA and FtsA may serve distinct membrane-anchoring roles for FtsZ.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas do Citoesqueleto/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/citologia , Escherichia coli/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Transporte/química , Proteínas de Transporte/genética , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Divisão Celular , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/genética , Escherichia coli/química , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Mutação , Ligação Proteica , Domínios ProteicosRESUMO
Antibody-based therapies have proved to be of great value in cancer treatment. Despite the clinical success of these biopharmaceuticals, reaching targets in the bone microenvironment has proved to be difficult due to the relatively low vascularization of bone tissue and the presence of physical barriers. Here, we have used an innovative bone-targeting (BonTarg) technology to generate a first-in-class bone-targeting antibody. Our strategy involves the use of pClick antibody conjugation technology to chemically couple the bone-targeting moiety bisphosphonate to therapeutic antibodies. Bisphosphonate modification of these antibodies results in the delivery of higher conjugate concentrations to the bone metastatic niche, relative to other tissues. In xenograft mice models, this strategy provides enhanced inhibition of bone metastases and multiorgan secondary metastases that arise from bone lesions. Specific delivery of therapeutic antibodies to the bone, therefore, represents a promising strategy for the treatment of bone metastatic cancers and other bone diseases.
Assuntos
Neoplasias Ósseas , Animais , Anticorpos/uso terapêutico , Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/patologia , Neoplasias Ósseas/secundário , Osso e Ossos , Difosfonatos/uso terapêutico , Xenoenxertos , Humanos , Camundongos , Metástase Neoplásica/patologia , Microambiente TumoralRESUMO
Rationale: Therapeutic antibody conjugates allow for the specific delivery of cytotoxic agents or immune cells to tumors, thus enhancing the antitumor activity of these agents and minimizing adverse systemic effects. Most current antibody conjugates are prepared by nonspecific modification of antibody cysteine or lysine residues, inevitably resulting in the generation of heterogeneous conjugates with limited therapeutic efficacies. Traditional strategies to prepare homogeneous antibody conjugates require antibody engineering or chemical/enzymatic treatments, processes that often affect antibody folding and stability, as well as yield and cost. Developing a simple and cost-effective way to precisely couple functional payloads to native antibodies is of great importance. Methods: We describe a simple proximity-induced antibody conjugation method (pClick) that enables the synthesis of homogeneous antibody conjugates from native antibodies without requiring additional antibody engineering or post-synthesis treatments. A proximity-activated crosslinker is introduced into a chemically synthesized affinity peptide modified with a bioorthogonal handle. Upon binding to a specific antibody site, the affinity peptide covalently attaches to the antibody via spontaneous crosslinking, yielding an antibody molecule ready for bioorthogonal conjugation with payloads. Results: We have prepared well-defined antibody-drug conjugates and bispecific small molecule-antibody conjugates using pClick technology. The resulting conjugates exhibit excellent in vitro cytotoxic activity against cancer cells and, in the case of bispecific conjugates, superb antitumor activity in mouse xenograft models. Conclusions: Our pClick technology enables efficient, simple, and site-specific conjugation of various moieties to the existing native antibodies. This technology does not require antibody engineering or additional UV/chemical/enzymatic treatments, therefore providing a general, convenient strategy for developing novel antibody conjugates.
Assuntos
Química Click/métodos , Imunoconjugados/química , Imunotoxinas/química , Animais , Anticorpos Biespecíficos/química , Anticorpos Biespecíficos/farmacologia , Anticorpos Monoclonais/uso terapêutico , Antígenos , Antineoplásicos/farmacologia , Linhagem Celular , Humanos , Imunoconjugados/farmacologia , Imunotoxinas/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos NOD , Neoplasias/tratamento farmacológicoRESUMO
Genetically site-specific introduction of noncanonical amino acids (ncAAs) for protein conjugation generally requires incorporation through exogenous feeding of chemically synthesized ncAAs. We developed a p-amino-phenylalanine (pAF)-based relay system that enables site-specific functionalization of proteins without chemical synthesis of the building blocks. pAF was biosynthesized under optimized conditions, followed by site-specific incorporation into a specific protein residue. The resulting protein was ready for functionalization using an oxidative conjugation reaction. We demonstrated the use of this relay system by preparing a fluorophore-labeled anti-HER2 single-chain variable fragment antibody for fluorescent imaging.
Assuntos
Corantes Fluorescentes/química , Imagem Óptica , Fenilalanina/análogos & derivados , Coloração e Rotulagem , Tirosina-tRNA Ligase/química , Methanocaldococcus/enzimologia , Estrutura Molecular , Fenilalanina/química , Fenilalanina/genética , Tirosina-tRNA Ligase/metabolismoRESUMO
The phosphorylation and regulation of the proapoptotic Bcl-2 family protein BAD by c-Jun NH2-terminal kinase (JNK) is controversial. JNK can suppress interleukin-3 withdrawal-induced apoptosis via phosphorylation of BAD at Thr201. However, it has also been reported that JNK promotes apoptosis through phosphorylation of BAD at Ser128. Here, we report that JNK is not a BAD Ser128 kinase. JNK phosphorylates murine BAD (mBAD), but not human BAD (hBAD), in which Ser91 is equivalent to Ser128 in mBAD. In contrast, Cdc2, which phosphorylates Ser128, phosphorylates both mBAD and hBAD. Replacement of Ser128 by alanine has no effects on BAD phosphorylation by JNK in vitro and in vivo. Two-dimensional phosphopeptide mapping in combination with phosphoamino acid analysis reveals that JNK does not phosphorylate BAD at Ser128. Elimination of Ser128 phosphorylation has no effects on the proapoptotic activity of BAD in apoptosis induced by UV via JNK or growth factor withdrawal. Thus, our results show that Ser128 is not phosphorylated by JNK for promoting cell death.
Assuntos
Apoptose/fisiologia , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Proteína de Morte Celular Associada a bcl/metabolismo , Alanina/genética , Alanina/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Eritropoetina/farmacologia , Fibroblastos , Células HeLa , Humanos , Interleucina-3/farmacologia , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Camundongos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fosforilação , Serina/metabolismo , Especificidade da Espécie , Treonina/genética , Treonina/metabolismo , Transfecção , Proteína de Morte Celular Associada a bcl/genéticaRESUMO
The GLUT4-regulating protein, TUG, functions to retain GLUT4-containing membrane vesicles intracellularly and, in response to insulin stimulation, releases these vesicles to the cellular exocytic machinery for translocation to the plasma membrane. As part of our on going effort to describe the molecular basis for TUG function, we have determined the tertiary structure and characterized the backbone dynamics for an N-terminal ubiquitin-like domain (TUG-UBL1) using NMR spectroscopy. A well-ordered conformation is observed for residues 10-83 of full-length TUG, and confirms a beta-grasp or ubiquitin-like topology. Although not required for in vitro association with GLUT4, the functional role of the TUG-UBL1 domain has not yet been described. We undertook a limited literature review of similar N-terminal UBL domains and noted that a majority participate in protein-protein interactions, generally functioning as adaptor modules to physically associate the over all activity of the protein with a specific cellular process, such as the ubiquitin-proteasome pathway. In consistent fashion, TUG-UBL1 is not expected to participate in a covalent protein modification reaction as it lacks the characteristic C-terminal diglycine ("GG") motif required for conjugation to an acceptor lysine, and also lacks the three most common acceptor lysine residues involved in polyubiquitination. Additionally, analysis of the TUG-UBL1 molecular surface reveals a lack of conservation of the "Ile-44 hydrophobic face" typically involved in ubiquitin recognition. Instead, we speculate on the possible significance of a concentrated area of negative electrostatic potential with increased backbone mobility, both of which are features suggestive of a potential protein-protein interaction site.
Assuntos
Proteínas de Transporte/química , Modelos Moleculares , Ubiquitinas/química , Sequência de Aminoácidos , Animais , Proteínas de Transporte/metabolismo , Transportador de Glucose Tipo 4/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Estrutura Terciária de Proteína , Alinhamento de Sequência , SoluçõesRESUMO
A facilely prepared fluorescence sensor was developed for dopamine (DA) determination based on polyindole/graphene quantum dots molecularly imprinted polymers (PIn/GQDs@MIPs). The proposed sensor exhibits a high sensitivity with a linear range of 5 × 10(-10) to 1.2 × 10(-6) M and the limit of detection as low as 1 × 10(-10) M in the determination of DA, which is probably due to the tailor-made imprinted cavities for binding DA thought hydrogen bonds between amine groups of DA and oxygen-containing groups of the novel composite. Furthermore, the prepared sensor can rebind DA in dual-type: a low affinity type (noncovalent interaction is off) and a high affinity type (noncovalent interaction is on), and the rebinding interaction can be adjusted by tuning the pH, which shows a unique potential for adjusting the binding interaction while keeping the specificity, allowing for wider applications.
Assuntos
Técnicas Biossensoriais , Dopamina/isolamento & purificação , Grafite/química , Pontos Quânticos , Dopamina/química , Técnicas Eletroquímicas , Fluorescência , Humanos , Limite de Detecção , Impressão Molecular , Polímeros/químicaRESUMO
A facilely prepared fluorescent sensor was developed for dopamine (DA) detection with high sensitivity and selectivity based on polypyrrole/graphene quantum dots (PPy/GQDs) core/shell hybrids. The composites exhibit strong fluorescence emission, which is dramatically enhanced as high as three times than pristine GQDs. The prepared sensor allows a highly sensitive determination of DA by fluorescent intensity decreasing with the addition of DA and presents a good linearity in range of 5-8000 nM with the detection limit of 10 pM (S/N = 3). Furthermore, the application of the proposed approach have been demonstrated in real samples and showed promise in diagnostic purposes.
Assuntos
Técnicas Biossensoriais , Dopamina/isolamento & purificação , Pontos Quânticos/química , Fluorescência , Grafite/química , Limite de Detecção , Polímeros/química , Pirróis/químicaRESUMO
This paper considers a comprehensive naturalistic driving experiment to collect driving data under potential threats on actual Chinese roads. Using acquired real-world naturalistic driving data, a near-crash database is built, which contains vehicle status, potential crash objects, driving environment and road types, weather condition, and driver information and actions. The aims of this study are summarized into two aspects: (1) to cluster different driving-risk levels involved in near-crashes, and (2) to unveil the factors that greatly influence the driving-risk level. A novel method to quantify the driving-risk level of a near-crash scenario is proposed by clustering the braking process characteristics, namely maximum deceleration, average deceleration, and percentage reduction in vehicle kinetic energy. A classification and regression tree (CART) is employed to unveil the relationship among driving risk, driver/vehicle characteristics, and road environment. The results indicate that the velocity when braking, triggering factors, potential object type, and potential crash type exerted the greatest influence on the driving-risk levels in near-crashes.
Assuntos
Acidentes de Trânsito/estatística & dados numéricos , Condução de Veículo/estatística & dados numéricos , Medição de Risco/métodos , China , Mineração de Dados , Bases de Dados Factuais , Humanos , Modelos TeóricosRESUMO
Highly dispersed polypyrrole nanowires are decorated on reduced graphene oxide sheets using a facile in situ synthesis route. The prepared composites exhibit high dispersibility, large effective surface area, and high electric conductivity. All-solid-state flexible supercapacitors are assembled based on the prepared composites, which show excellent electrochemical performances with a specific capacitance of 434.7 F g(-1) at a current density of 1 A g(-1). The as-fabricated supercapacitor also exhibits excellent cycling stability (88.1% capacitance retention after 5000 cycles) and exceptional mechanical flexibility. In addition, outstanding power and energy densities were obtained, demonstrating the significant potential of prepared material for flexible and portable energy storage devices.