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Broadening of signals from atoms at interfaces can often be a limiting factor in applying solution NMR to the structure determination of complexes. Common contributors to such problems include exchange between free and bound states and the increased molecular weight of complexes relative to the free components, but another cause that can be more difficult to deal with occurs when conformational dynamics within the interface takes place at an intermediate rate on the chemical shift timescale. In this work we show how a carefully chosen mutation in the protein HMG-D rescued such a situation, making possible high-resolution structure determination of its complex with a dA2 bulge DNA ligand designed to mimic a natural DNA bend, and thereby revealing a new spatial organization of the complex.
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BACKGROUND: Extracellular adenosine triphosphate (ATP) is an important signal molecule. In previous studies, intensive research had revealed the crucial roles of family with sequence similarity 3 member A (FAM3A) in controlling hepatic glucolipid metabolism, islet ß cell function, adipocyte differentiation, blood pressure, and other biological and pathophysiological processes. Although mitochondrial protein FAM3A plays crucial roles in the regulation of glucolipid metabolism via stimulating ATP release to activate P2 receptor pathways, its mechanism in promoting ATP release in hepatocytes remains unrevealed. METHODS: db/db, high-fat diet (HFD)-fed, and global pannexin 1 (PANX1) knockout mice, as well as liver sections of individuals, were used in this study. Adenoviruses and adeno-associated viruses were utilized for in vivo gene overexpression or inhibition. To evaluate the metabolic status in mice, oral glucose tolerance test (OGTT), pyruvate tolerance test (PTT), insulin tolerance test (ITT), and magnetic resonance imaging (MRI) were conducted. Protein-protein interactions were determined by coimmunoprecipitation with mass spectrometry (MS) assays. RESULTS: In livers of individuals and mice with steatosis, the expression of ATP-permeable channel PANX1 was increased (P < 0.01). Hepatic PANX1 overexpression ameliorated the dysregulated glucolipid metabolism in obese mice. Mice with hepatic PANX1 knockdown or global PANX1 knockout exhibited disturbed glucolipid metabolism. Restoration of hepatic PANX1 rescued the metabolic disorders of PANX1-deficient mice (P < 0.05). Mechanistically, ATP release is mediated by the PANX1-activated protein kinase B-forkhead box protein O1 (Akt-FOXO1) pathway to inhibit gluconeogenesis via P2Y receptors in hepatocytes. PANX1-mediated ATP release also activated calmodulin (CaM) (P < 0.01), which interacted with c-Jun N-terminal kinase (JNK) to inhibit its activity, thereby deactivating the transcription factor activator protein-1 (AP1) and repressing fatty acid synthase (FAS) expression and lipid synthesis (P < 0.05). FAM3A stimulated the expression of PANX1 via heat shock factor 1 (HSF1) in hepatocytes (P < 0.05). Notably, FAM3A overexpression failed to promote ATP release, inhibit the expression of gluconeogenic and lipogenic genes, and suppress gluconeogenesis and lipid deposition in PANX1-deficient hepatocytes and livers. CONCLUSIONS: PANX1-mediated release of ATP plays a crucial role in maintaining hepatic glucolipid homeostasis, and it confers FAM3A's suppressive effects on hepatic gluconeogenesis and lipogenesis.
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Trifosfato de Adenosina , Conexinas , Gluconeogênese , Lipogênese , Fígado , Proteínas do Tecido Nervoso , Animais , Conexinas/metabolismo , Camundongos , Gluconeogênese/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/genética , Trifosfato de Adenosina/metabolismo , Lipogênese/fisiologia , Fígado/metabolismo , Camundongos Knockout , Masculino , Humanos , Dieta Hiperlipídica/efeitos adversos , CitocinasRESUMO
AlphaFold2 and related computational tools have greatly aided studies of structural biology through their ability to accurately predict protein structures. In the present work, we explored AF2 structural models of the 17 canonical members of the human PARP protein family and supplemented this analysis with new experiments and an overview of recent published data. PARP proteins are typically involved in the modification of proteins and nucleic acids through mono or poly(ADP-ribosyl)ation, but this function can be modulated by the presence of various auxiliary protein domains. Our analysis provides a comprehensive view of the structured domains and long intrinsically disordered regions within human PARPs, offering a revised basis for understanding the function of these proteins. Among other functional insights, the study provides a model of PARP1 domain dynamics in the DNA-free and DNA-bound states and enhances the connection between ADP-ribosylation and RNA biology and between ADP-ribosylation and ubiquitin-like modifications by predicting putative RNA-binding domains and E2-related RWD domains in certain PARPs. In line with the bioinformatic analysis, we demonstrate for the first time PARP14's RNA-binding capability and RNA ADP-ribosylation activity in vitro. While our insights align with existing experimental data and are probably accurate, they need further validation through experiments.
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Inibidores de Poli(ADP-Ribose) Polimerases , Poli(ADP-Ribose) Polimerases , Humanos , Poli(ADP-Ribose) Polimerases/metabolismo , Domínios Proteicos , ADP-Ribosilação , RNA/metabolismoRESUMO
TRIM proteins are the largest family of E3 ligases in mammals. They include the intracellular antibody receptor TRIM21, which is responsible for mediating targeted protein degradation during Trim-Away. Despite their importance, the ubiquitination mechanism of TRIM ligases has remained elusive. Here we show that while Trim-Away activation results in ubiquitination of both ligase and substrate, ligase ubiquitination is not required for substrate degradation. N-terminal TRIM21 RING ubiquitination by the E2 Ube2W can be inhibited by N-terminal acetylation, but this doesn't prevent substrate ubiquitination nor degradation. Instead, uncoupling ligase and substrate degradation prevents ligase recycling and extends functional persistence in cells. Further, Trim-Away degrades substrates irrespective of whether they contain lysines or are N-terminally acetylated, which may explain the ability of TRIM21 to counteract fast-evolving pathogens and degrade diverse substrates.
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Lisina , Ubiquitina-Proteína Ligases , Animais , Lisina/metabolismo , Ubiquitinação , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Processamento de Proteína Pós-Traducional , Proteólise , Mamíferos/metabolismoRESUMO
PARP-1 is a key early responder to DNA damage in eukaryotic cells. An allosteric mechanism links initial sensing of DNA single-strand breaks by PARP-1's F1 and F2 domains via a process of further domain assembly to activation of the catalytic domain (CAT); synthesis and attachment of poly(ADP-ribose) (PAR) chains to protein sidechains then signals for assembly of DNA repair components. A key component in transmission of the allosteric signal is the HD subdomain of CAT, which alone bridges between the assembled DNA-binding domains and the active site in the ART subdomain of CAT. Here we present a study of isolated CAT domain from human PARP-1, using NMR-based dynamics experiments to analyse WT apo-protein as well as a set of inhibitor complexes (with veliparib, olaparib, talazoparib and EB-47) and point mutants (L713F, L765A and L765F), together with new crystal structures of the free CAT domain and inhibitor complexes. Variations in both dynamics and structures amongst these species point to a model for full-length PARP-1 activation where first DNA binding and then substrate interaction successively destabilise the folded structure of the HD subdomain to the point where its steric blockade of the active site is released and PAR synthesis can proceed.
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Poli(ADP-Ribose) Polimerase-1/química , Regulação Alostérica , Amidas/química , Domínio Catalítico , Cristalografia por Raios X , Dano ao DNA , Ativação Enzimática , Modelos Moleculares , Mutação , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Poli(ADP-Ribose) Polimerase-1/genética , Poli(ADP-Ribose) Polimerase-1/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases/química , Domínios ProteicosRESUMO
VARP and TBC1D5 are accessory/regulatory proteins of retromer-mediated retrograde trafficking from endosomes. Using an NMR/X-ray approach, we determined the structure of the complex between retromer subunit VPS29 and a 12 residue, four-cysteine/Zn++ microdomain, which we term a Zn-fingernail, two of which are present in VARP. Mutations that abolish VPS29:VARP binding inhibit trafficking from endosomes to the cell surface. We show that VARP and TBC1D5 bind the same site on VPS29 and can compete for binding VPS29 in vivo. The relative disposition of VPS29s in hetero-hexameric, membrane-attached, retromer arches indicates that VARP will prefer binding to assembled retromer coats through simultaneous binding of two VPS29s. The TBC1D5:VPS29 interaction is over one billion years old but the Zn-fingernail appears only in VARP homologues in the lineage directly giving rise to animals at which point the retromer/VARP/TBC1D5 regulatory network became fully established.
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Evolução Molecular , Fatores de Troca do Nucleotídeo Guanina/química , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/metabolismo , Zinco/metabolismo , Microscopia Crioeletrônica , Cisteína/química , Proteínas Ativadoras de GTPase/química , Proteínas Ativadoras de GTPase/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Células HeLa , Humanos , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Conformação Proteica , Proteínas de Transporte Vesicular/genética , Dedos de ZincoRESUMO
The anti-cancer drug target poly(ADP-ribose) polymerase 1 (PARP1) and its close homologue, PARP2, are early responders to DNA damage in human cells1,2. After binding to genomic lesions, these enzymes use NAD+ to modify numerous proteins with mono- and poly(ADP-ribose) signals that are important for the subsequent decompaction of chromatin and the recruitment of repair factors3,4. These post-translational modifications are predominantly serine-linked and require the accessory factor HPF1, which is specific for the DNA damage response and switches the amino acid specificity of PARP1 and PARP2 from aspartate or glutamate to serine residues5-10. Here we report a co-structure of HPF1 bound to the catalytic domain of PARP2 that, in combination with NMR and biochemical data, reveals a composite active site formed by residues from HPF1 and PARP1 or PARP2 . The assembly of this catalytic centre is essential for the addition of ADP-ribose moieties after DNA damage in human cells. In response to DNA damage and occupancy of the NAD+-binding site, the interaction of HPF1 with PARP1 or PARP2 is enhanced by allosteric networks that operate within the PARP proteins, providing an additional level of regulation in the induction of the DNA damage response. As HPF1 forms a joint active site with PARP1 or PARP2, our data implicate HPF1 as an important determinant of the response to clinical PARP inhibitors.
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ADP-Ribosilação , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Dano ao DNA , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Poli(ADP-Ribose) Polimerase-1/química , Poli(ADP-Ribose) Polimerase-1/metabolismo , Poli(ADP-Ribose) Polimerases/química , Poli(ADP-Ribose) Polimerases/metabolismo , Regulação Alostérica , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Biocatálise , Proteínas de Transporte/genética , Domínio Catalítico , Células HEK293 , Humanos , Modelos Moleculares , Mutação , NAD/metabolismo , Ressonância Magnética Nuclear Biomolecular , Proteínas Nucleares/genética , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Anêmonas-do-MarRESUMO
The cytosolic antibody receptor TRIM21 possesses unique ubiquitination activity that drives broad-spectrum anti-pathogen targeting and underpins the protein depletion technology Trim-Away. This activity is dependent on formation of self-anchored, K63-linked ubiquitin chains by the heterodimeric E2 enzyme Ube2N/Ube2V2. Here we reveal how TRIM21 facilitates ubiquitin transfer and differentiates this E2 from other closely related enzymes. A tri-ionic motif provides optimally distributed anchor points that allow TRIM21 to wrap an Ube2N~Ub complex around its RING domain, locking the closed conformation and promoting ubiquitin discharge. Mutation of these anchor points inhibits ubiquitination with Ube2N/Ube2V2, viral neutralization and immune signalling. We show that the same mechanism is employed by the anti-HIV restriction factor TRIM5 and identify spatially conserved ionic anchor points in other Ube2N-recruiting RING E3s. The tri-ionic motif is exclusively required for Ube2N but not Ube2D1 activity and provides a generic E2-specific catalysis mechanism for RING E3s.
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Lisina/metabolismo , Ribonucleoproteínas/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitinação/fisiologia , Motivos de Aminoácidos/genética , Fatores de Restrição Antivirais , Biocatálise , Cristalografia por Raios X , Células HEK293 , Células HeLa , Humanos , Modelos Moleculares , Mutação , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica/genética , Ribonucleoproteínas/química , Ribonucleoproteínas/genética , Proteínas com Motivo Tripartido/metabolismo , Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/química , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Clathrin-mediated endocytosis (CME) is key to maintaining the transmembrane protein composition of cells' limiting membranes. During mammalian CME, a reversible phosphorylation event occurs on Thr156 of the µ2 subunit of the main endocytic clathrin adaptor, AP2. We show that this phosphorylation event starts during clathrin-coated pit (CCP) initiation and increases throughout CCP lifetime. µ2Thr156 phosphorylation favors a new, cargo-bound conformation of AP2 and simultaneously creates a binding platform for the endocytic NECAP proteins but without significantly altering AP2's cargo affinity in vitro. We describe the structural bases of both. NECAP arrival at CCPs parallels that of clathrin and increases with µ2Thr156 phosphorylation. In turn, NECAP recruits drivers of late stages of CCP formation, including SNX9, via a site distinct from where NECAP binds AP2. Disruption of the different modules of this phosphorylation-based temporal regulatory system results in CCP maturation being delayed and/or stalled, hence impairing global rates of CME.
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Complexo 2 de Proteínas Adaptadoras/genética , Subunidades alfa do Complexo de Proteínas Adaptadoras/genética , Endocitose/genética , Nexinas de Classificação/genética , Complexo 2 de Proteínas Adaptadoras/metabolismo , Clatrina/genética , Clatrina/metabolismo , Vesículas Revestidas por Clatrina/genética , Vesículas Revestidas por Clatrina/metabolismo , Invaginações Revestidas da Membrana Celular/genética , Invaginações Revestidas da Membrana Celular/metabolismo , Humanos , Fosforilação/genética , Ligação Proteica/genéticaRESUMO
Drug-loading often suffers from tedious procedures, limited loading efficiency, slow release, and therefore a low curative effect. Cancer easily recurs and metastasizes even after a solid tumor is removed. Herein, we report a simple strategy with multi-drug co-loading and burst drug release for a high curative effect and anti-recurrence/metastasis. CuS nanoparticles, protoporphyrin IX, and doxorubicin were added to the precursors of ZIF-8 with one-pot co-loading during the formation of ZIF-8 for chemo-, photothermal-, and photodynamic-therapy to eliminate solid tumors. Negative CpG, as a kind of immune adjuvant, was adsorbed on the positive surface of ZIF-8 to inhibit the recurrence and metastasis of tumors with its long-term immune response. Precision treatment with one-pot multi-drug co-loading, controllable drug delivery, and multi-modality therapy may be anticipated by this versatile strategy.
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BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) is one of the most common chronic liver diseases with a high prevalence in the general population. The association between NAFLD and cardiovascular disease has been well addressed in previous studies. However, whether NAFLD is associated with carotid artery disease in a community-based Chinese population remained unclear. The aim of this study was to investigate the association between NAFLD and carotid artery disease. METHODS: A total of 2612 participants (1091 men and 1521 women) aged 40 years and older from Jidong of Tangshan city (China) were selected for this study. NAFLD was diagnosed by abdominal ultrasonography. The presence of carotid stenosis or plaque was evaluated by carotid artery ultrasonography. Logistic regression was used to analyze the association between NAFLD and carotid artery disease. RESULTS: Participants with NAFLD have a higher prevalence of carotid stenosis (12.9% vs. 4.6%) and carotid plaque (21.9% vs. 15.0%) than those without NAFLD. After adjusting for age, gender, smoking status, income, physical activity, diabetes, hypertension, triglyceride, waist-hip ratio, and high-density lipoprotein, NAFLD is significantly associated with carotid stenosis (odds ratio [OR]: 2.06, 95% confidence interval [CI]: 1.45-2.91), but the association between NAFLD and carotid plaque is not statistically significant (OR: 1.10, 95% CI: 0.8-1.40). CONCLUSION: A significant association between NAFLD and carotid stenosis is found in a Chinese population.
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Doenças das Artérias Carótidas/complicações , Espessura Intima-Media Carotídea , Hepatopatia Gordurosa não Alcoólica/complicações , Adulto , China , Estudos Transversais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Prevalência , Fatores de RiscoRESUMO
Cell surface Fc receptors activate inflammation and are tightly controlled to prevent autoimmunity. Antibodies also simulate potent immune signalling from inside the cell via the cytosolic antibody receptor TRIM21, but how this is regulated is unknown. Here we show that TRIM21 signalling is constitutively repressed by its B-Box domain and activated by phosphorylation. The B-Box occupies an E2 binding site on the catalytic RING domain by mimicking E2-E3 interactions, inhibiting TRIM21 ubiquitination and preventing immune activation. TRIM21 is derepressed by IKKß and TBK1 phosphorylation of an LxxIS motif in the RING domain, at the interface with the B-Box. Incorporation of phosphoserine or a phosphomimetic within this motif relieves B-Box inhibition, promoting E2 binding, RING catalysis, NF-κB activation and cytokine transcription upon infection with DNA or RNA viruses. These data explain how intracellular antibody signalling is regulated and reveal that the B-Box is a critical regulator of RING E3 ligase activity.
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Regulação da Expressão Gênica , Processamento de Proteína Pós-Traducional , Receptores Fc/metabolismo , Ribonucleoproteínas/metabolismo , Transdução de Sinais , Animais , Linhagem Celular , Humanos , Quinase I-kappa B/metabolismo , Camundongos , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismoRESUMO
Ratiometric fluorescence detection attracts much attention because of its decreased environmental influence and easy-to-differentiate color and intensity change. Herein, a guest-encapsulation metal-organic framework (MOF), Ru@MIL-NH2, is prepared with 2-aminoterephthalic acid, AlCl3, and Ru(bpy)32+ by a simple one-pot method for ratiometric fluorescence sensing of water in organic solvents. The rational selection of the excitation wavelength provides dual emission at 465 and 615 nm from Ru@MIL-NH2 under a single excitation of 300 nm. High sensitivity, low detection limit (0.02% v/v), wide response range (0-100%), and fast response (less than 1 min) are obtained for ratiometric fluorescence sensing of water under single excitation with Ru@MIL-NH2 as the probe. Moreover, the result of water content is independent of the concentration of Ru@MIL-NH2 as the merit of ratiometric fluorescence detection. The response mechanism reveals that the protonation of the nitrogen atom of the MIL-NH2, the π-conjugation system, and the stable fluorescence of Ru(bpy)32+ achieve the ratiometric fluorescence. The analysis of real spirit samples confirms the proposed method. A test strip is prepared with Ru@MIL-NH2 for convenient use. We believe that such turn-on ratiometric host-guest MOFs and the rational selection of excitation wavelength will offer guidance for ratiometric fluorescence detection with wide applications.
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Homeodomain proteins control the developmental transition between the haploid and diploid phases in several eukaryotic lineages, but it is not known whether this regulatory mechanism reflects the ancestral condition or, instead, convergent evolution. We have characterized the mating-type locus of the amoebozoan Dictyostelium discoideum, which encodes two pairs of small proteins that determine the three mating types of this species; none of these proteins display recognizable homology to known families. We report that the nuclear magnetic resonance structures of two of them, MatA and MatB, contain helix-turn-helix folds flanked by largely disordered amino- and carboxyl-terminal tails. This fold closely resembles that of homeodomain transcription factors, and, like those proteins, MatA and MatB each bind DNA characteristically using the third helix of their folded domains. By constructing chimeric versions containing parts of MatA and MatB, we demonstrate that the carboxyl-terminal tail, not the central DNA binding motif, confers mating specificity, providing mechanistic insight into how a third mating type might have originated. Finally, we show that these homeodomain-like proteins specify zygote function: Hemizygous diploids, formed in crosses between a wild-type strain and a mat null mutant, grow and differentiate identically to haploids. We propose that Dictyostelium MatA and MatB are divergent homeodomain proteins with a conserved function in triggering the haploid-to-diploid transition that can be traced back to the last common ancestor of eukaryotes.
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Proteínas de Ligação a DNA/metabolismo , Dictyostelium/fisiologia , Diploide , Haploidia , Proteínas de Homeodomínio/metabolismo , Sequência de Aminoácidos , Sequência Conservada , Proteínas de Ligação a DNA/química , Proteínas de Homeodomínio/química , Estágios do Ciclo de Vida , Espectroscopia de Ressonância Magnética , Modelos Biológicos , Modelos Moleculares , Conformação Proteica , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Relação Estrutura-AtividadeRESUMO
The combination of multidrug chemotherapy and photothermal therapy (PTT) enhances cancer therapeutic efficacy. Herein, we develop a simple and smart pH/NIR dual-stimulus-responsive degradable mesoporous CoFe2O4@PDA@ZIF-8 sandwich nanocomposite. The mesoporous CoFe2O4 core acts as T2-weighted magnetic resonance (MR) imaging probe, PTT agent, and loading platform of hydrophilic doxorubicin (DOX). A polydopamine (PDA) layer is used to avoid the premature leakage of DOX before arriving at tumor site, enhance PTT efficiency, and facilitate the integration of ZIF-8 (a kind of metal-organic framework). The ZIF-8 shell serves to encapsulate hydrophobic camptothecin (CPT) and as the switch for the pH and NIR stimulation-responsive release of the two drugs. Therefore, T2-weighted MR imaging-guided multidrug chemotherapy and PTT synergistic treatment is achieved. Two kinds of anticancer drugs, hydrophilic DOX and hydrophobic CPT, are successfully loaded in CoFe2O4 and ZIF-8, respectively, so no mutual interference between the two drugs exists. A unique two-stage stepwise release process is exhibited for CPT and DOX with an interval of 12 h to improve the anticancer efficacy under the acidic microenvironment of tumor tissue. NIR irradiation achieves the burst drug-release and PTT after laser stimulation, simultaneously. With this smart design, high drug concentration is achieved at the tumor site by quick release, especially for the therapeutic drugs that show nonlinear pharmacokinetics, and PTT is integrated efficiently. Furthermore, negligible biotoxicity and a remarkable synergic antitumor effect of the hybrid nanocomposites are validated by HepG2 cells and tumor-bearing mice as models. Our multidrug delivery-releasing composite improves tumor therapeutic efficiency significantly compared with a single-drug chemotherapy system. The simple multifunctional composite system can be applied as an effective platform for personal nanomedicine with diagnosis, smart drug delivery, and cancer treatment through its remarkable photothermal property and controllable multidrug release.
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Imageamento por Ressonância Magnética , Animais , Doxorrubicina , Sistemas de Liberação de Medicamentos , Liberação Controlada de Fármacos , Concentração de Íons de Hidrogênio , CamundongosRESUMO
For predicting the temperature distribution within skin tissue in 980-nm laser-evoked potentials (LEPs) experiments, a five-layer finite element model (FEM-5) was constructed based on Pennes bio-heat conduction equation and the Lambert-Beer law. The prediction results of the FEM-5 model were verified by ex vivo pig skin and in vivo rat experiments. Thirty ex vivo pig skin samples were used to verify the temperature distribution predicted by the model. The output energy of the laser was 1.8, 3, and 4.4 J. The laser spot radius was 1 mm. The experiment time was 30 s. The laser stimulated the surface of the ex vivo pig skin beginning at 10 s and lasted for 40 ms. A thermocouple thermometer was used to measure the temperature of the surface and internal layers of the ex vivo pig skin, and the sampling frequency was set to 60 Hz. For the in vivo experiments, nine adult male Wistar rats weighing 180 ± 10 g were used to verify the prediction results of the model by tail-flick latency. The output energy of the laser was 1.4 and 2.08 J. The pulsed width was 40 ms. The laser spot radius was 1 mm. The Pearson product-moment correlation and Kruskal-Wallis test were used to analyze the correlation and the difference of data. The results of all experiments showed that the measured and predicted data had no significant difference (P > 0.05) and good correlation (r > 0.9). The safe laser output energy range (1.8-3 J) was also predicted. Using the FEM-5 model prediction, the effective pain depth could be accurately controlled, and the nociceptors could be selectively activated. The FEM-5 model can be extended to guide experimental research and clinical applications for humans.
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Análise de Elementos Finitos , Lasers , Dor/patologia , Temperatura Cutânea/efeitos da radiação , Temperatura , Adulto , Animais , Humanos , Masculino , Ratos Wistar , Reprodutibilidade dos Testes , Pele/efeitos da radiação , Sus scrofa , CaudaRESUMO
Faithful DNA replication is essential to all forms of life and depends on the action of 3'-5' exonucleases that remove misincorporated nucleotides from the newly synthesized strand. However, how the DNA is transferred from the polymerase to the exonuclease active site is not known. Here we present the cryo-EM structure of the editing mode of the catalytic core of the Escherichia coli replisome, revealing a dramatic distortion of the DNA whereby the polymerase thumb domain acts as a wedge that separates the two DNA strands. Importantly, NMR analysis of the DNA substrate shows that the presence of a mismatch increases the fraying of the DNA, thus enabling it to reach the exonuclease active site. Therefore the mismatch corrects itself, whereas the exonuclease subunit plays a passive role. Hence, our work provides unique insights into high-fidelity replication and establishes a new paradigm for the correction of misincorporated nucleotides.
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DNA Polimerase III/química , Replicação do DNA , Pareamento de Bases , Sequência de Bases , Domínio Catalítico , Microscopia Crioeletrônica , Reparo de Erro de Pareamento de DNA , DNA Bacteriano/química , DNA Bacteriano/genética , Escherichia coli/enzimologia , Escherichia coli/genética , Conformação de Ácido Nucleico , Ligação Proteica , Conformação Proteica em alfa-Hélice , Estrutura Quaternária de Proteína , Subunidades Proteicas/químicaRESUMO
In this study, we report the synthesis and application of mesoporous CoFe2O4@MIL-100(Fe) hybrid magnetic nanoparticles (MNPs) for the simultaneous removal of inorganic arsenic (iAs). The hybrid adsorbent had a core-shell and mesoporous structure with an average diameter of 260 nm. The nanoscale size and mesoporous character impart a fast adsorption rate and high adsorption capacity for iAs. In total, 0.1 mg L-1 As(V) and As(III) could be adsorbed within 2 min, and the maximum adsorption capacities were 114.8 mg g-1 for As(V) and 143.6 mg g-1 for As(III), higher than most previously reported adsorbents. The anti-interference capacity for iAs adsorption was improved by the electrostatic repulsion and size exclusion effects of the MIL-100(Fe) shell, which also decreased the zero-charge point of the hybrid absorbent for a broad pH adsorption range. The adsorption mechanisms of iAs on the MNPs are proposed. An Fe-O-As structure was formed on CoFe2O4@MIL-100(Fe) through hydroxyl substitution with the deprotonated iAs species. Monolayer adsorption of As(V) was observed, while hydrogen bonding led to the multi-layer adsorption of neutral As(III) for its high adsorption capacity. The high efficiency and the excellent pH- and interference-tolerance capacities of CoFe2O4@MIL-100(Fe) allowed effective iAs removal from natural water samples, as validated with batch magnetic separation mode and a portable filtration strategy.