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BACKGROUND: The unique size, physical and chemical properties, and ultra-high stability of nanozymes have attracted extensive attentions in sensing, but improvement of catalytic activity of the nanozymes is still an urgent issue. Given the ultra-high simulated enzyme activity of metal nanoparticles and the advantage of multi-enzyme catalysis, an Au-decorated MoS2 nanosheets (MoS2/Au NS) integrating the double peroxidase-like (POD) activity is developed. RESULTS: By optimizing and adjusting the density of AuNPs, as well as its morphology and other parameters, a monodisperse and high-density distribution of AuNPs on MoS2 nanosheets was obtained, which can greatly improve the POD-like activity of MoS2/Au NS. Nafion solution was applied to assist the modification of MoS2/Au NS on the electrode surface so as to improved its stability. An electrochemical H2O2 detection platform was constructed by modifying MoS2/Au NS nanozyme on the SPCE using the conductive Nafion solution. And the negatively charged sulfonic acid group can eliminate negatively charged electroactive substances to improve the specificity. Then ascorbic acid was used to stimulate tumor cells to produce H2O2 as therapeutic model, an ultrasensitive chronocoulometry detection for H2O2 in cell lysate was established. The logarithmically of ΔQ and the logarithmically of H2O2 concentration showed a good linear relationship between 1 µM and 500 mM, with a LOD value of 0.3 µM. SIGNIFICANCE: The developed H2O2 sensor has excellent stability, reproducibility (RSD = 2.3 %, n = 6) and selectivity, realized the quantitative detection of H2O2 in cell lysate. Compared with commercial fluorescence detection kits for H2O2 in cell lysate, it is worth mentioning that the electrochemical H2O2 sensor developed in this study is simpler and faster, with higher sensitivity and lower cost. This provides a potential substitute for disease diagnosis and treatment evaluation based on accurate detection of H2O2.
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Antineoplásicos , Dissulfetos , Técnicas Eletroquímicas , Ouro , Peróxido de Hidrogênio , Nanopartículas Metálicas , Molibdênio , Ouro/química , Molibdênio/química , Peróxido de Hidrogênio/análise , Peróxido de Hidrogênio/química , Dissulfetos/química , Nanopartículas Metálicas/química , Técnicas Eletroquímicas/métodos , Humanos , Antineoplásicos/química , Antineoplásicos/farmacologia , Antineoplásicos/análise , Nanoestruturas/química , Limite de Detecção , Peroxidase/química , Peroxidase/metabolismo , Ensaios de Seleção de Medicamentos AntitumoraisRESUMO
IL-1R-associated kinases (IRAKs) are signal transducers of the TLR/IL-1R-MyD88-TRAF6 pathways. Vertebrates possess two IRAK lineages, IRAK1/2/3 and IRAK4. In mammals, IRAK4/IRAK1 and IRAK4/IRAK2 are pathway enhancers, whereas IRAK3 is a repressor. However, in bony fish, IRAK2 is absent, and it remains elusive how fish IRAK1/3/4 functionally differ from their mammalian counterparts. In this study, we explored this using the zebrafish model. First, we showed that in human 293T cells, zebrafish IRAK1 and IRAK4 were components of the Myddosome (MyD88-IRAK4-IRAK1) complex, with IRAK1 serving as a potent pathway enhancer. Then, we discovered two zebrafish IRAK3 variants: one (IRAK3a) contains an N-terminal Death domain, a middle pseudokinase domain, and a C-terminal TRAF6-binding domain, whereas the other (IRAK3b) lost both the kinase and TRAF6-binding domains. This truncation of IRAK3 variants could be a conserved phenomenon in fish, because it is also observed in trout and grass carp. We proceeded to show that zebrafish IRAK3a acts as a pathway enhancer by binding with MyD88 and TRAF6, but its activity is milder than IRAK1, possibly because it has no kinase activity. Zebrafish IRAK3b, however, plays a sheer negative role, apparently because of its lack of kinase and TRAF6-binding domains. Moreover, zebrafish IRAK3a/3b inhibit the activity of IRAK1/4, not by interacting with IRAK1/4 but possibly by competing for MyD88 and TRAF6. Finally, we have verified the essential activities of zebrafish IRAK1/3a/3b/4 in zebrafish cells and embryos. In summary, to our knowledge, our findings provide new insights into the molecular functions of fish IRAKs and the evolution of the IRAK functional modes in vertebrates.
Assuntos
Quinases Associadas a Receptores de Interleucina-1 , Fator 88 de Diferenciação Mieloide , Transdução de Sinais , Fator 6 Associado a Receptor de TNF , Proteínas de Peixe-Zebra , Peixe-Zebra , Animais , Quinases Associadas a Receptores de Interleucina-1/metabolismo , Quinases Associadas a Receptores de Interleucina-1/genética , Fator 88 de Diferenciação Mieloide/metabolismo , Fator 88 de Diferenciação Mieloide/genética , Fator 6 Associado a Receptor de TNF/metabolismo , Fator 6 Associado a Receptor de TNF/genética , Humanos , Transdução de Sinais/imunologia , Células HEK293 , Proteínas de Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genéticaRESUMO
The IL-17 pathway displays remarkably diverse functional modes between different subphyla, classes, and even orders, yet its driving factors remains elusive. Here, we demonstrate that the IL-17 pathway originated through domain shuffling between a Toll-like receptor (TLR)/IL-1R pathway and a neurotrophin-RTK (receptor-tyrosine-kinase) pathway (a Trunk-Torso pathway). Unlike other new pathways that evolve independently, the IL-17 pathway remains intertwined with its donor pathways throughout later evolution. This intertwining not only influenced the gains and losses of domains and components in the pathway but also drove the diversification of the pathway's functional modes among animal lineages. For instance, we reveal that the crustacean female sex hormone, a neurotrophin inducing sex differentiation, could interact with IL-17Rs and thus be classified as true IL-17s. Additionally, the insect prothoracicotropic hormone, a neurotrophin initiating ecdysis in Drosophila by binding to Torso, could bind to IL-17Rs in other insects. Furthermore, IL-17R and TLR/IL-1R pathways maintain crosstalk in amphioxus and zebrafish. Moreover, the loss of the Death domain in the pathway adaptor connection to IκB kinase and stress-activated protein kinase (CIKSs) dramatically reduced their abilities to activate nuclear factor-kappaB (NF-κB) and activator protein 1 (AP-1) in amphioxus and zebrafish. Reinstating this Death domain not only enhanced NF-κB/AP-1 activation but also strengthened anti-bacterial immunity in zebrafish larvae. This could explain why the mammalian IL-17 pathway, whose CIKS also lacks Death, is considered a weak signaling activator, relying on synergies with other pathways. Our findings provide insights into the functional diversity of the IL-17 pathway and unveil evolutionary principles that could govern the pathway and be used to redesign and manipulate it.
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Interleucina-17 , Transdução de Sinais , Receptores Toll-Like , Animais , Interleucina-17/metabolismo , Receptores Toll-Like/metabolismo , Fatores de Crescimento Neural/metabolismo , Fatores de Crescimento Neural/genética , Receptores de Interleucina-1/metabolismo , Receptores de Interleucina-1/genética , Evolução Molecular , Receptores de Interleucina-17/metabolismo , Receptores de Interleucina-17/genéticaRESUMO
The accurate and rapid detection of specific antibodies in blood is very important for efficient diagnosis and precise treatment. Conventional methods often suffer from time-consuming operations and/or a narrow detection range. In this work, for the rapid determination of bevacizumab in plasma, a series of chimeric hairpin DNA aptamer-based probes were designed by the modification, labeling and theoretical computation of an original aptamer. Then, the dissociation constant of the modified hairpin DNA to bevacizumab was measured and screened using microscale thermophoresis. The best chimeric hairpin DNA aptamer-based probe was then selected, and a one-step platform for the rapid determination of bevacizumab was constructed. This strategy has the advantages of being simple, fast and label-free. Because of the design and screening of the hairpin DNA, as well as the optimization of the concentration and electrochemical parameters, a low detection limit of 0.37 pM (0.054 ng mL-1) with a wide linear range (1 pM-1 µM) was obtained. Finally, the rationally constructed biosensor was successfully applied to the determination of bevacizumab in spiked samples, and it showed good accuracy and precision. This method is expected to truly realize accurate and rapid detection of bevacizumab and provides a new idea for the precise treatment of diseases.
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Aptâmeros de Nucleotídeos , Técnicas Biossensoriais , Bevacizumab , Técnicas Biossensoriais/métodos , DNA , Sondas de DNA/genética , Limite de Detecção , Técnicas EletroquímicasRESUMO
Alzheimer's disease (AD), a prevalent multiple neurodegenerative disease, has gained attention, particularly in the aging population. However, presently available therapies merely focus on alleviating the symptoms of AD and fail to slow disease progression significantly. Traditional Chinese medicine (TCM) has been used to ameliorate symptoms or interfere with the pathogenesis of aging-associated diseases for many years based on disease-modifying in multiple pathological roles with multi-targets, multi-systems and multi-aspects. Mahonia species as a TCM present potential for anti-inflammatory activity, antioxidant activity, anti-acetylcholinesterase activity, and antiamyloid- beta activity that was briefly discussed in this review. They are regarded as promising drug candidates for AD therapy. The findings in this review support the use of Mahonia species as an alternative therapy source for treating AD.
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Doença de Alzheimer , Mahonia , Doenças Neurodegenerativas , Medicina Tradicional Chinesa , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/prevenção & controle , Antioxidantes/farmacologia , Antioxidantes/uso terapêuticoRESUMO
Small ubiquitin-like modifier (SUMO) regulates various biological processes, including the MyD88/TICAMs-IRAKs-TRAF6-NF-κB pathway, one of the core immune pathways. However, its functions are inconsistent between invertebrates and vertebrates and have rarely been investigated in lower chordates, including amphioxus and fishes. Here, we investigated the SUMOylation gene system in the amphioxus, a living basal chordate. We found that amphioxus has a SUMOylation system that has a complete set of genes and preserves several ancestral traits. We proceeded to study their molecular functions using the mammal cell lines. Both amphioxus SUMO1 and SUMO2 were shown to be able to attach to NF-κB Rel and to inhibit NF-κB activation by 50-75% in a dose-dependent fashion. The inhibition by SUMO2 could be further enhanced by the addition of the SUMO E2 ligase UBC9. In comparison, while human SUMO2 inhibited RelA, human SUMO1 slightly activated RelA. We also showed that, similar to human PIAS1-4, amphioxus PIAS could serve as a SUMO E3 ligase and promote its self-SUMOylation. This suggests that amphioxus PIAS is functionally compatible in human cells. Moreover, we showed that amphioxus PIAS is not only able to inhibit NF-κB activation induced by MyD88, TICAM-like, TRAF6 and IRAK4 but also able to suppress NF-κB Rel completely in the presence of SUMO1/2 in a dose-insensitive manner. This suggests that PIAS could effectively block Rel by promoting Rel SUMOylation. In comparison, in humans, only PIAS3, but not PIAS1/2/4, has been reported to promote NF-κB SUMOylation. Taken together, the findings from amphioxus, together with those from mammals and other species, not only offer insights into the functional volatility of the animal SUMO system, but also shed light on its evolutionary transitions from amphioxus to fish, and ultimately to humans.
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Anfioxos , NF-kappa B , Humanos , Animais , NF-kappa B/genética , NF-kappa B/metabolismo , Ubiquitina , Fator 88 de Diferenciação Mieloide/metabolismo , Fator 6 Associado a Receptor de TNF/genética , Fator 6 Associado a Receptor de TNF/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Anfioxos/genética , Anfioxos/metabolismo , Mamíferos/metabolismo , Chaperonas Moleculares , Proteínas Inibidoras de STAT Ativados/genéticaRESUMO
The chitin-based peritrophic matrix (PM) is a structure critical for both gut immunity and digestion in invertebrates. PM was traditionally considered lost in all vertebrates, but a PM-like chitinous membrane (CM) has recently been discovered in fishes, which may increase the knowledge on vertebrate gut physiology and structural evolution. Here, we show that in zebrafish, the CM affects ingestion behavior, microbial homeostasis, epithelial renewal, digestion, growth, and longevity. Young mutant fish without CM appear healthy and are able to complete their life cycle normally, but with increasing age they develop gut inflammation, resulting in gut atrophy. Unlike mammals, zebrafish have no visible gel-forming mucin layers to protect their gut epithelia, but at least in young fish, the CM is not a prerequisite for the antibacterial gut immunity. These findings provide new insights into the role of the CM in fish prosperity and its eventual loss in tetrapods. These findings may also help to improve fish health and conservation, as well as to advance the understanding of vertebrate gut physiology and human intestinal diseases.
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Quitina , Peixe-Zebra , Animais , Humanos , Membranas , Inflamação , Estágios do Ciclo de Vida , MamíferosRESUMO
Water management in the catalyst layers (CLs) of proton-exchange membrane fuel cells is crucial for its commercialization and popularization. However, the high experimental or computational cost in obtaining water distribution and diffusion remains a bottleneck in the existing experimental methods and simulation algorithms, and further mechanistic exploration at the nanoscale is necessary. Herein, we integrate, for the first time, molecular dynamics simulation with our customized analysis framework based on a multiattribute point cloud dataset and an advanced deep learning network. This was achieved through our workflow that generates simulated transport data of water molecules in the CLs as the training and test dataset. Deep learning framework models the multibody solid-liquid system of CLs on a molecular scale and completes the mapping from the Pt/C substrate structure and Nafion aggregates to the density distribution and diffusion coefficient of water molecules. The prediction results are comprehensively analyzed and error evaluated, which reveals the highly anisotropic interaction landscape between 50,000 pairs of interacting nanoparticles and explains the structure and water transport property relationship in the hydrated Nafion film on the molecular scale. Compared to the conventional methods, the proposed deep learning framework shows computational cost efficiency, accuracy, and good visual display. Further, it has a generality potential to model macro- and microscopic mass transport in different components of fuel cells. Our framework is expected to make real-time predictions of the distribution and diffusion of water molecules in CLs as well as establish statistical significance in the structural optimization and design of CLs and other components of fuel cells.
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The Apextrin C-terminal (ApeC) domain is a new protein domain largely specific to aquatic invertebrates. In amphioxus, a short-form ApeC-containing protein (ACP) family is capable of binding peptidoglycan (PGN) and agglutinating bacteria via its ApeC domain. However, the functions of ApeC in other phyla remain unknown. Here we examined 130 ACPs from gastropods and bivalves, the first and second biggest mollusk classes. They were classified into nine groups based on their phylogenetics and architectures, including three groups of short-form ACPs, one group of apextrins and two groups of ACPs of complex architectures. No groups have orthologs in other phyla and only four groups have members in both gastropods and bivalves, suggesting that mollusk ACPs are highly diversified. We selected one bivalve ACP (CgACP1; from the oyster Crossostrea gigas) and one gastropod ACP (BgACP1; from the snail Biomphalaria glabrata) for functional experiments. Both are highly-expressed, secreted short-form ACPs and hence comparable to the amphioxus ACPs previously reported. We found that recombinant CgACP1 and BgACP1 bound with yeasts and several bacteria with different affinities. They also agglutinated these microbes, but showed no inhibiting or killing effects. Further analyses show that both ACPs had high affinities to the Lys-type PGN from S. aureus but weak or no affinities to the DAP-type PGN from Bacillus subtilis. Both recombinant ACPs displayed weak or no affinities to other microbial cell wall components, including lipopolysaccharide (LPS), lipoteichoic acid (LTA), zymosan A, chitin, chitosan and cellulose, as well as to several PGN moieties, including muramyl dipeptide (MDP), N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc). Besides, CgACP1 had the highest expression in the gill and could be greatly up-regulated quickly after bacterial challenge. This is reminiscent of the amphioxus ACP1/2 which serve as essential mucus lectins in the gill. Taken together, the current findings from mollusk and amphioxus ACPs suggest several basic common traits for the ApeC domains, including the high affinity to Lys-type PGN, the bacterial binding and agglutinating capacity, and the role as mucus proteins to protect the mucosal surface.
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Quitosana , Anfioxos , Animais , Peptidoglicano/farmacologia , Lipopolissacarídeos , Acetilmuramil-Alanil-Isoglutamina/farmacologia , Staphylococcus aureus/metabolismo , Acetilglucosamina/química , Zimosan , Anfioxos/metabolismo , Bactérias/metabolismo , Parede Celular/metabolismo , Lectinas , Moluscos , CeluloseRESUMO
Background: As adjunctive therapy, puerarin injection has been widely applied for the treatment of unstable angina pectoris (UAP) in China during the past decades. However, the efficacy of puerarin injection as adjunctive therapy for UAP has not been well confirmed. The purpose of this meta-analysis was to summarize the available evidence to estimate the efficacy of puerarin injection in treating UAP. Objective: A systematic review and meta-analysis with subgroup analysis and sensitivity analysis according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) principle were carried out to evaluate the efficacy of puerarin injection as adjunctive therapy in treating UAP. Methods: To obtain the published randomized controlled trials (RCTs) on puerarin injection, databases, namely, China National Knowledge Infrastructure (CNKI), Wanfang Database, Chinese Biomedical Literature Database, Sino-Med, PubMed, China Science and Technology Journal Database (VIP), Medline, Google Scholar, Cochrane Library, Chinese Science Citation Database, and Embase were systematically searched until June 2021. In this meta-analysis, Review Manager version 5.3 software and Stata version 12.0 software were employed to analyze the collected data. Based on the methodological quality, years of publications, sample size and dosages, sensitivity analysis, and subgroup analysis were performed. The GRADE assessment was conducted by the software GRADEpro version 3.6 software. Results: A total of 17 RCTs involving 1,459 patients were included in this meta-analysis. Results indicated that puerarin injection as adjunctive therapy was more superior than conventional Western medicine alone in reducing angina symptoms [risk ratio (RR) = 1.22, 95% CI 1.16 to 1.28, Z = 8.11, p < 0.00001] and improving ECG (RR = 1.32, 95% CI 1.20 to 1.44, Z = 6.00, p < 0.00001), meanwhile reducing the frequency of angina attack [mean difference (MD) = -2.22, 95% CI -2.53 to -1.90, Z = 13.97, p < 0.00001] and the duration of angina attack (MD = -2.00, 95% CI -2.39 to -1.61, Z = 9.99, p < 0.00001) for the treatment of UAP. Results from the GRADE assessment suggested that the comprehensive quality of this evidence was low. Conclusion: This meta-analysis indicated that puerarin injection was more effective than using conventional Western medicine alone in the treatment of UAP. However, because of the low methodological quality of the included RCTs, more evidence was still needed to verify the efficacy of puerarin injection.
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In this study, data-driven deep learning methods were applied in order to model and predict the treatment of real municipal wastewater using anaerobic membrane bioreactors (AnMBRs). Based on the one-year operating data of two AnMBRs, six parameters related to the experimental conditions (temperature of reactor, temperature of environment, temperature of influent, influent pH, influent COD, and flux) and eight parameters for wastewater treatment evaluation (effluent pH, effluent COD, COD removal efficiency, biogas composition (CH4, N2, and CO2), biogas production rate, and oxidation-reduction potential) were selected to establish the data sets. Three deep learning network structures were proposed to analyze and reproduce the relationship between the input parameters and output evaluation parameters. The statistical analysis showed that deep learning closely agrees with the AnMBR experimental results. The prediction accuracy rate of the proposed densely connected convolutional network (DenseNet) can reach up to 97.44%, and the single calculation time can be reduced to within 1 s, suggesting the high performance of AnMBR treatment prediction with deep learning methods.
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Aprendizado Profundo , Águas Residuárias , Anaerobiose , Reatores Biológicos , Membranas Artificiais , Eliminação de Resíduos LíquidosRESUMO
Background: Ligustrazine injection has been widely used as adjunctive therapy in the treatment of acute cerebral infarction (ACI) during the past decades in China, but its clinical efficacy is not yet well confirmed. This study aims to evaluate the efficacy of ligustrazine injection as adjunctive therapy for ACI. Methods: Databases including China National Knowledge Infrastructure (CNKI), China Science and Technology Journal Database (VIP), PubMed, Medline, Google Scholar, Chinese Biomedical Literature Database, Cochrane Library, Embase, Sino-Med, Wanfang Database, and Chinese Science Citation Database were systematically searched for the published randomized controlled trials (RCTs) on ligustrazine injection in the treatment of ACI until November 2020. Meta-analysis was performed on the primary outcome measure (i.e., clinical effective rate) and the secondary outcome measure [i.e., neurological deficit score (NDS), fibrinogen, low shear blood viscosity (LBV), and high shear blood viscosity (HBV)]. The quality of the included RCTs was assessed according to the M scoring system (the refined Jadad scale). Sensitivity analysis and subgroup analysis were conducted according to the methodological quality, years of publication, and sample size. Results: Nineteen RCTs, containing 2022 patients, were included in this study. Meta-analysis indicated that ligustrazine injection combined with Western medicine could achieve a better effect in the treatment of ACI than using Western medicine alone in terms of clinical effective rate (RR = 1.24; 95% CI, 1.19-1.29), NDS (MD = -3.88; 95%CI, -4.51 to -3.61), fibrinogen (MD = -0.59; 95% CI, -0.76 to -0.42), LBV (MD = -2.11; 95% CI, -3.16 to -1.06), and HBV (MD = -0.88; 95% CI, -1.20 to -0.55). Conclusions: This research indicated that ligustrazine injection as adjunctive therapy seemed to be more effective than using western medicine alone in treating ACI. However, more evidence is required to confirm the efficacy of ligustrazine injection due to the low methodological quality of the included RCTs.
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The apextrin C-terminal (ApeC) domain is a class of newly discovered protein domains with an origin dating back to prokaryotes. ApeC-containing proteins (ACPs) have been found in various marine and aquatic invertebrates, but their functions and the underlying mechanisms are largely unknown. Early studies suggested that amphioxus ACP1 and ACP2 bind to bacterial cell walls and have a role in immunity. Here we identified another two amphioxus ACPs (ACP3 and ACP5), which belong to the same phylogenetic clade with ACP1/2, but show distinct expression patterns and sequence divergence (40-50% sequence identities). Both ACP3 and ACP5 were mainly expressed in the intestine and hepatic cecum, and could be up-regulated after bacterial challenge. Both prokaryotic-expressed recombinant ACP3 and ACP5 could bind with several species of bacteria and yeasts, showing agglutinating activity but no microbicidal activity. ELISA assays suggested that their ApeC domains could interact with peptidoglycan (PGN), but not with lipoteichoic acid (LTA), lipopolysaccharides (LPS) and zymosan A. Furthermore, they can only bind to Lys-type PGN from Staphylococcus aureus, but not to DAP-type PGN from Bacillus subtilis and not to moieties of PGN such as MDPs, NAMs and NAGs. This recognition spectrum is different from that of ACP1/2. We also found that when expressed in mammalian cells, ACP3 could interact with TRAF6 via a conserved non-ApeC region, which inhibited the ubiquitination of TRAF6 and hence suppressed downstream NF-κB activation. This work helped define a novel subfamily of ACPs, which have conserved structures, and have related yet diversified molecular functions. Its members have dual roles, with ApeC as a lectin and a conserved unknown region as a signal transduction regulator. These findings expand our understanding of the ACP functions and may guide future research on the role of ACPs in different animal clades.
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Fosfatase Ácida/metabolismo , Interações entre Hospedeiro e Microrganismos , Domínios e Motivos de Interação entre Proteínas , Transdução de Sinais , Fator 6 Associado a Receptor de TNF/metabolismo , Fosfatase Ácida/química , Fosfatase Ácida/genética , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Parede Celular/imunologia , Parede Celular/metabolismo , Clonagem Molecular , Biologia Computacional/métodos , Bases de Dados Genéticas , Expressão Gênica , Perfilação da Expressão Gênica , Humanos , Invertebrados , Ligação Proteica , Staphylococcus aureus/imunologia , Staphylococcus aureus/metabolismoRESUMO
Amphioxus (e.g., Branchiostoma belcheri, Bb) has recently emerged as a new model for studying the origin and evolution of vertebrate immunity. Mammalian lymphocyte-specific tyrosine kinase (Lck) plays crucial roles in T cell activation, differentiation and homeostasis, and is reported to phosphorylate both the ITIM and ITSM of PD-1 to induce the recruitment of phosphatases and thus the inhibitory function of PD-1. Here, we identified and cloned the amphioxus homolog of human Lck. By generating and using an antibody against BbLck, we found that BbLck is expressed in the amphioxus gut and gill. Through overexpression of BbLck in Jurkat T cells, we found that upon TCR stimulation, BbLck was subjected to tyrosine phosphorylation and could partially rescue Lck-dependent tyrosine phosphorylation in Lck-knockdown T cells. Mass spectrometric analysis of BbLck immunoprecipitates from immunostimulants-treated amphioxus, revealed a BbLck-associated membrane-bound receptor LRR (BbLcLRR). By overexpressing BbLcLRR in Jurkat T cells, we demonstrated that BbLcLRR was tyrosine phosphorylated upon TCR stimulation, which was inhibited by Lck knockdown and was rescued by overexpression of BbLck. By mutating single tyrosine to phenylalanine (Y-F), we identified three tyrosine residues (Y539, Y655, and Y690) (3Y) of BbLcLRR as the major Lck phosphorylation sites. Reporter gene assays showed that overexpression of BbLcLRR but not the BbLcLRR-3YF mutant inhibited TCR-induced NF-κB activation. In Lck-knockdown T cells, the decline of TCR-induced IL-2 production was reversed by overexpression of BbLck, and this reversion was inhibited by co-expression of BbLcLRR but not the BbLcLRR-3YF mutant. Sequence analysis showed that the three tyrosine-containing sequences were conserved with the tyrosine-based inhibition motifs (ITIMs) or ITIM-like motifs. And TCR stimulation induced the association of BbLcLRR with tyrosine phosphatases SHIP1 and to a lesser extent with SHP1/2. Moreover, overexpression of wild-type BbLcLRR but not its 3YF mutant inhibited TCR-induced tyrosine phosphorylation of multiple signaling proteins probably via recruiting SHIP1. Thus, we identified a novel immunoreceptor BbLcLRR, which is phosphorylated by Lck and then exerts a phosphorylation-dependent inhibitory role in TCR-mediated T-cell activation, implying a mechanism for the maintenance of self-tolerance and homeostasis of amphioxus immune system and the evolutionary conservatism of Lck-regulated inhibitory receptor pathway.
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Receptores Coestimuladores e Inibidores de Linfócitos T/metabolismo , Anfioxos/metabolismo , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Animais , Biomarcadores , Clonagem Molecular , Receptores Coestimuladores e Inibidores de Linfócitos T/genética , Bases de Dados Genéticas , Ensaio de Imunoadsorção Enzimática , Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imunofenotipagem , Interleucina-2/biossíntese , Células Jurkat , Anfioxos/genética , Ativação Linfocitária , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/genética , Fosforilação , Coelhos , Receptores de Antígenos de Linfócitos T/metabolismo , Análise de Sequência de DNA , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismoRESUMO
In this study, a high-performance anti-fouling coating based on poly adenine (polyAn) as well as a highly specific cluster of differentiation 20 (CD20) epitope mimetic peptide (CN14) were employed to synergistically construct a facile biosensor for the rapid and sensitive determination of rituximab in lymphoma patients' plasma. The well-designed and optimized polyAn coating displayed excellent stability, hydrophilicity, thanks to its intrinsic affinity with gold surface and thoroughly exposed hydrophilic phosphate groups. Moreover, the proposed strategy avoids the necessity to modify binding groups (e.g. thiol), making it more facile, repeatable and efficient. When dealing with complex clinical plasma samples, the polyAn coating demonstrated better anti-fouling performance and lower background signal in comparison with mercaptan and bovine serum albumin coatings. The dissociation constant (~60 nM) between CN14 and rituximab was measured by microscale thermophoresis and their binding mechanism was further explained using computer simulation. The constructed GE/CN14/polyA20 biosensor displayed satisfactory performance with detection limit of 35.26 ng/mL. Finally, the proposed biosensor was successfully applied for rapidly determining rituximab in lymphoma patients' plasma, and exhibited comparable accuracy to the commercial ELISA, but has advantages including a shorter detection time, wider detection range and lower cost. It's worth noting that the anti-fouling polyAn coating can be tailored according to the surface property of sensing interface and can be easily expanded to other gold electrode related biosensors.
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Técnicas Biossensoriais , Rituximab , Simulação por Computador , Epitopos , Humanos , Peptídeos , Plasma , Poli A , Rituximab/análiseRESUMO
The rapidly increasing applications of monoclonal antibodies (mAbs) in therapy have necessitated the development of mAb production and purification technologies for both academic and industrial usage. Herein, a histidine-tagged cyclic peptide (HHHHHHGSGSGSDC*AWHLGELVWC*T, the disulfide-bonded cysteines of which are indicated by asterisks, named HT25-cyclopeptide) functionalized monolithic material was developed by the metal ion chelation-based approach. The resulting material possessed suitable affinity and peptide ligand density (13.8 mg peptide ligand per mL of material), good porosity (67.1 %), acceptable specific surface area (52.95 m2/g), and lots of macropores (4.13 µm). Moreover, excellent antibody-specific selectivity, comparable or even better binding capacity (for dried material, maximum static binding capacity and dynamic binding capacity are about 119.3 mg/g and 17.05 mg/g, respectively) for antibody compared to previously developed affinity materials, acceptable resistance to trypsin digestion, and negligible nonspecific protein adsorption, were also achieved on this novel monolithic material. Compared with the corresponding cyclic peptide-based sepharose material, milder elution conditions were employed for the HT25-cyclopeptide-based monolithic material, which could effectively prevent the aggregation and denaturation of the enriched antibodies. This novel material was then successfully applied to the affinity enrichment and purification of mAbs (including infliximab and rituximab) in different cell culture media or IgG in human serum.
Assuntos
Anticorpos Monoclonais/isolamento & purificação , Cromatografia de Afinidade/métodos , Histidina/metabolismo , Peptídeos Cíclicos/química , Adsorção , Quelantes , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/isolamento & purificação , Ligantes , Peptídeos/metabolismo , Peptídeos Cíclicos/metabolismo , SefaroseRESUMO
Apextrin C-terminal (ApeC) is a novel protein domain with unknown functions, although early studies suggest that some ApeC-containing proteins (ACPs) bind to carbohydrates and have a role in development and immunity. Here we investigated the taxonomic distribution, sequence diversification and origin of ACPs in Metazoa. Most ACPs are present in invertebrates from aquatic or moist environments, including cnidarians, mollusks, echinoderms, cephalochordates, flatworms, water bears, nematodes and annelids. However, ACPs are absent in vertebrates and in most arthropod lineages (e.g. insects and crustaceans) except arachnids. ACPs apparently undergo rapid turnover and diversification, hence no orthologs could be found between (sub)phyla. ApeC can function either as a standalone domain or as a partner domain. It has been found to pair up with over ten different domain types in different ACPs. The partner domains are related to immunity, extracellular matrix, protein-protein and protein-carbohydrate interactions. Notably, the domain pair with the widest taxonomic distribution is MACPF/perforin-ApeC, which represent a classic group of ACPs called apextrins. ApeC also frequently pairs up with itself to form dual-ApeC modules in different phyla. Notably, in parasite flatworms, dual-ApeCs are present in 70% of ACPs and all inherited from a common ancestor. The broad distribution of MACPF-ApeC and dual-ApeC suggest their conserved yet unknown functions. We also discovered distant ApeC homologs in bacteria, hence tracing the origin of ApeC back to prokaryotes. Our findings show that ApeC has an ancient origin and is able to function alone or in complex domain architectures, though it is less prevalent than other versatile domains such as immunoglobulin domains and C-type lectin domains. This work provides a foundation for further functional study of this novel domain type.
Assuntos
Variação Genética , Filogenia , Proteínas/química , Sequência de Aminoácidos , Animais , Bactérias/metabolismo , Evolução Molecular , Invertebrados/metabolismo , Domínios Proteicos , Vertebrados/metabolismoRESUMO
There is an urgent need for developing advanced purification techniques with the merits of low cost and satisfactory capacity in order to meet the challenges in the current downstream purification of monoclonal antibodies (mAbs). Herein, a simple and inexpensive nitrogen heterocycle molecule, 1-vinylimidazole (VIM), was proposed as the capture ligand of antibodies for the first time. The corresponding VIM-based non-affinity polymeric material (polyVIM) was then fabricated via a one-step polymerization for use in the highly selective purification of antibodies. Compared to the previously reported materials, this novel material exhibited many advantages without clearly sacrificing selectivity, such as a simpler and faster fabrication (within 1.5 h), comparable or even higher binding capacity (saturated static adsorption capacity > 190 mg/g polymer, dynamic binding capacity about 31.62 mg/g polymer), lower non-specific protein adsorption, and much lower cost. Notably, the polyVIM can effectively purify the antibodies from multiple biological sources with high purity (95.4% for mAbs in the cell culture medium, 93.3% for hIgG in the human serum), with an acceptable recovery (91.6% for mAbs, 77.0% for hIgG), and good reusability (> 10 times). Moreover, the target ELISA binding assay and NFAT-luc reporter gene assay demonstrated that the enriched antibodies can well maintain their binding activity and bioactivity during the whole purification process. The excellent performance of the polyVIM material may be attributed to the high recognition ability of VIM for antibodies, as well as the biocompatible and antifouling properties of the porous polymer. This study provides a promising alternative material for the purification of mAbs in downstream processes and the enrichment of hIgG in human serum.
Assuntos
Anticorpos Monoclonais/isolamento & purificação , Imidazóis/química , Polímeros/química , Adsorção , Animais , Bevacizumab/isolamento & purificação , Células CHO , Calorimetria , Cricetinae , Cricetulus , Humanos , Imunoglobulina G/sangue , Ligantes , Camundongos , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
IL-1R-associated kinases (IRAK) are important regulators in the TLR/IL-1R pathways, but their function appears inconsistent between Drosophila, bony fishes, and vertebrates. This causes a difficulty to understand the IRAK functions. As a step to reveal the evolution of IRAKs, in this study, we performed comparative and functional analysis of IRAKs by exploiting the amphioxus, a pivotal taxon connecting invertebrates and vertebrates. Sequence and phylogenetic analysis indicated three major IRAK lineages: IRAK1/2/3 is a vertebrate-specific lineage, IRAK4 is an ancient lineage conserved between invertebrate and vertebrates, and Pelle is another ancient lineage that is preserved in protostomes and invertebrate deuterostomes but lost in vertebrate deuterostomes. Pelle is closer neither to IRAK4 nor to IRAK1/2/3, hence suggesting no clear functional analogs to IRAK1/2/3 in nonvertebrates. Functional analysis showed that both amphioxus IRAK4 and Pelle could suppress NF-κB activation induced by MyD88 and TRAF6, which are unlike mammalian and Drosophila IRAKs, but, surprisingly, similar to bony fish IRAK4. Also unlike Drosophila IRAKs, no interaction was detected between amphioxus IRAK4 and Pelle, although both of them were shown capable of binding MyD88. These findings, together with previous reports, show that unlike other signal transducers in the TLR/IL-1R pathways, such as MyD88 and TRAF6, the functions of IRAKs are highly variable during evolution and very specialized in different major animal taxa. Indeed, we suggest that the functional variability of IRAKs might confer plasticity to the signal transduction of the TLR/IL-1R pathways, which in return helps the species to evolve against the pathogens.
Assuntos
Evolução Biológica , Fator 88 de Diferenciação Mieloide/imunologia , Proteínas Serina-Treonina Quinases/imunologia , Transdução de Sinais/imunologia , Fator 6 Associado a Receptor de TNF/imunologia , Animais , Anfioxos , FilogeniaRESUMO
Chitin is a very important and widely-used biopolymer in fungi and lower metazoans, but mysteriously disappears in mammals. Recent studies reveal that at least lower vertebrates have chitin synthases (CS) and use them to synthesize endogenous chitin. Amphioxus, a basal chordate, therefore becomes critical to understand the evolution of CS, as it occupies the transitional position from invertebrates to vertebrates, and is considered as a good proxy to the chordate ancestor. Here, by exploiting multiple genome assemblies, high-depth RNA-seq data and synteny relations, we identify 11-12 CS genes for each amphioxus species. It represents the largest CS gene pool ever found in eukaryotes so far. As comparison, most metazoans have one or two CSs. Amphioxus is the only chordate that has both the very ancient type-I CS family and the more broadly distributed type-II CS family. Specifically, amphioxus has only one type-II CS but 10-11 type-I CSs, which means that amphioxus is the only metazoan with a greatly expanded type-I CS family. Further analysis suggests that the chordate ancestor have at least one type-II CS and an expanded of type-I CS family. We hypothesize that: these ancient CSs are mostly retained in amphioxus; but the whole type-I CS family was lost in urochordates and vertebrates; the type-II CS was later duplicated into two lineages in vertebrates and followed by stochastic losses, till all type-II CSs were eventually lost in birds and mammals. Finally, our expression profiling and preliminary gene knockout analysis suggest that amphioxus CSs could have highly diverse but mildly overlapping functions in various tissues and organs. Taken together, these findings not only provide insights into the evolution of chordate CSs, lay a foundation for further functional study of the chordate CSs. After all, it is mysterious that our chordate ancestor needed so many isoenzymes for chitin formation.