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Ferroptosis is a pattern of cell death caused by iron-dependent accumulation of lipid peroxides and is closely associated with the occurrence and development of multiple diseases. Acrolein (ACR), one of the final metabolites of lipid peroxidation, is a reactive carbonyl species with strong biotoxicity. Effective detection of ACR is important for understanding its role in the progression of ferroptosis and studying the specific mechanisms of ferroptosis-mediated diseases. However, visualization detection of ACR during ferroptosis has not yet been reported. In this work, the first ratiometric fluorescent probe (HBT-SH) based on 2-(2'-hydroxyphenyl) benzothiazole (HBT) was designed for tracing endogenous ACR with an unprecedented regiospecific ACR-induced intramolecular cyclization strategy, which employs 2-aminoethanethiol as an ACR-selective recognition receptor. The experimental results showed that HBT-SH has excellent selectivity, high sensitivity (LOD = 0.26 µM) and good biocompatibility. More importantly, the upregulation of ACR levels was observed during ferroptosis in HeLa cells and zebrafish, indicating that ACR may be a specific active molecule that plays an essential biological role during ferroptosis or may serve as a potential marker of ferroptosis, which has great significance for studying the pathological process and treatment options of ferroptosis-related diseases.
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Acroleína , Ferroptose , Corantes Fluorescentes , Peixe-Zebra , Ferroptose/efeitos dos fármacos , Acroleína/química , Acroleína/metabolismo , Corantes Fluorescentes/química , Corantes Fluorescentes/síntese química , Humanos , Células HeLa , Animais , Regulação para Cima/efeitos dos fármacos , Imagem Óptica , Estrutura MolecularRESUMO
The static and dynamic structures of DNA duplexes affected by 5S-Tg (Tg, Thymine glycol) epimers were studied using MD simulations and Markov State Models (MSMs) analysis. The results show that the 5S,6S-Tg base caused little perturbation to the helix, and the base-flipping barrier was determined to be 4.4 kcal mol-1 through the use of enhanced sampling meta-eABF calculations, comparable to 5.4 kcal mol-1 of the corresponding thymine flipping. Two conformations with the different hydrogen bond structures between 5S,6R-Tg and A19 were identified in several independent MD trajectories. The 5S,6R-Tg:O6HO6â¢â¢â¢N1:A19 hydrogen bond is present in the high-energy conformation displaying a clear helical distortion, and near barrier-free Tg base flipping. The low-energy conformation always maintains Watson-Crick base pairing between 5S,6R-Tg and A19, and 5S-Tg base flipping is accompanied by a small barrier of ca. 2.0 KBT (T = 298 K). The same conformations are observed in the MSMs analysis. Moreover, the transition path and metastable structures of the damaged base flipping are for the first time verified through MSMs analysis. The data clearly show that the epimers have completely different influence on the stability of the DNA duplex, thus implying different enzymatic mechanisms for DNA repair.
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Reparo do DNA , DNA , Pareamento de Bases , DNA/química , Dano ao DNA , Ligação de Hidrogênio , Conformação de Ácido Nucleico , TermodinâmicaRESUMO
Generally, different isoforms of proteins exert separate biological functions. However, due to similar structures and identical catalysis functions, distinguishing isoforms is challenging. Summarizing a molecular design strategy has great significance in developing a protein-specific fluorescent probe. Usually, recognition of a group was deemed to be the key to a protein isoform-specific response. However, some novel literature reported that fluorophore could play a vital role in the protein isoform-specific response. It means that any part of the fluorescent probe could affect the detected properties. In this work, we report the generation of the first probe to specifically recognize HexA(ß-N-acetylhexosaminidase A), Hex-C4, by adjusting the length of the linker. Hex-C4 exhibits specific recognition of HexA both in vitro and in living cells. The integration of the fluorescent spectrum and the MD (molecular dynamics) results provide two factors for the molecular design of isoform-specific fluorescent probes. One is the interaction between tetraphenyl ethylene (AIE fluorogen) and amino acid residues, and the other is the interaction between amino acid residues and the binding group. In this work, a powerful tool to detect HexA in living cells is reported for the first time. Further, a workable molecular design strategy for protein isoform-specific fluorescent probes is summarized.
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Acetilglucosaminidase , Corantes Fluorescentes , Isoformas de Proteínas , Aminoácidos , CatáliseRESUMO
Nitroxyl (HNO) and endoplasmic reticulum (ER) stress are considered to play important effects in the administration of many pathological processes of Parkinson's disease (PD). However, the intricate relationship between the neurotoxicity of HNO and ER stress in the processes of PD is still unknown. To completely comprehend the pathogenic activity of HNO during ER stress and achieve early diagnosis of PD, developing sensitive tools for HNO sensing in vivo is essential. In this work, a two-photon fluorescent probe (KD-HNO) was developed with highly selective and sensitive (7.93 nM) response for HNO in vitro. Then, utilizing KD-HNO, we found that HNO levels were distinctly increased in tunicamycin-stimulated PC12 cells, which are characterized by ER stress and PD features. Most importantly, we detected a considerable increase in HNO levels in the brains of PD-model mice, indicating a positive correlation between PD and HNO levels for the first time. Collectively, these findings revealed that KD-HNO is an excellent tool not only for understanding the biological effects of HNO in pathological processes of PD but also for early PD diagnosis.
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Doença de Parkinson , Ratos , Camundongos , Animais , Doença de Parkinson/diagnóstico , Corantes Fluorescentes , Óxidos de Nitrogênio , Retículo EndoplasmáticoRESUMO
Despite their great success in recognizing small molecules in vitro, nucleic acid aptamers are rarely used in clinical settings. This is partially due to the lack of structure-based mechanistic information. In this work, atomistic molecular dynamics simulations are used to study the static and dynamic supramolecular structures relevant to the process of the wild-type (wt) nucleic acid aptamer recognition and binding of ATP. The effects brought about by mutation of key residues in the recognition site are also explored. The simulations reveal that the aptamer displays a high degree of rigidity and is structurally very little affected by the binding of ATP. Interaction energy decomposition shows that dispersion forces from π-stacking between ATP and the G6 and A23 nucleobases in the aptamer binding site plays a more important role in stabilizing the supramolecular complex, compared to hydrogen-bond interaction between ATP and G22. Moreover, metadynamics simulations show that during the association process, water molecules act as essential bridges connecting ATP with G22, which favors the dynamic stability of the complex. The calculations carried out on three mutated aptamer structures confirm the crucial role of the hydrogen bonds and π-stacking interactions for the binding affinity of the ATP nucleic acid aptamer.
Assuntos
Trifosfato de Adenosina/química , Aptâmeros de Nucleotídeos/química , Simulação de Dinâmica Molecular , Aptâmeros de Nucleotídeos/genética , Pareamento de Bases , Ligação de Hidrogênio , MutaçãoRESUMO
SQLE (squalene epoxidase) is a cell membrane-bound enzyme. It is a target of fungicides and may become a new target for cancer therapy. Therefore, monitoring the content and distribution of the key enzyme in living cells is very challenging. To achieve this goal, tetraphenyl ethylene-Ter (TPE-Ter) was first designed as a new fluorescent probe to SQLE based on its active cavity. Spectral experiments discovered that SQLE/TPE-Ter shows stronger emission with fast response time and low interference from other analytes. Molecular dynamics simulation clearly confirmed the complex structure of SQLE/TPE-Ter, and the key residues contribute to restriction of TPE-Ter single-molecular motion in the cavity. TPE-Ter-specific response to SQLE is successfully demonstrated in living cells such as LO2, HepG2, and fungi. Imaging of TPE-Ter-treated fungi indicates that it can be used to rapidly assess antifungal drug susceptibility (30 min at least). The present work provides a powerful tool to detect content and distribution of SQLE in living cells.
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Corantes Fluorescentes , Esqualeno Mono-Oxigenase , Antifúngicos , Linhagem Celular TumoralRESUMO
BACKGROUND: Porcine reproductive and respiratory syndrome (PRRS) is one of the most economically devastating diseases affecting the swine industry globally. Evaluation of antibody responses and neutralizing antibody titers is the most effective method for vaccine evaluation. In this study, the B cell line epitopes of PRRSV M protein were predicted, and two peptide ELISA assays were established (M-A110-129 ELISA, M-A148-174 ELISA) to detect antibodies against PRRSV M protein. Field serum samples collected from pig farms were used to validate the peptide ELISA and compare it with an indirect immunofluorescence assay. RESULTS: The sensitivity and specificity of M-A110-129 ELISA and M-A148-174 ELISA were (111/125) 88.80%, (69/70) 98.57% and (122/125) 97.60%, (70/70) 100%, relative to indirect immunofluorescence assay. This peptide ELISA could detect antibodies against different genotypes of PRRSV including type 1 PRRSV, classical PRRSV, HP-PRRSV, and NADC30 like PRRSV, but not antibodies against other common swine viruses. The results of ROC analysis showed that the area under the curve (AUC) of the M-A110-129 ELISA and M-A148-174 ELISA were 0.967 and 0.996, respectively. Compared the concordance of results using two peptide ELISA assays, the IDEXX PRRSV X3 Ab ELISA and a virus neutralization test, were assessed using a series of 147 sera from pigs vaccinated with the NADC30-like PRRSV inactivated vaccine. The M-A148-174 ELISA had the best consistency, with a Cohen's kappa coefficient of 0.8772. The concordance rates of the Hipra PRRSV ELISA kit, M-A110-129 ELISA and M-A148-174 ELISA in the field seropositive detection results were 91.08, 86.32 and 95.35%, relative to indirect immunofluorescence assay. CONCLUSIONS: In summary, compared with M-A110-129 ELISA, the PRRSV M-A148-174 ELISA is of value for detecting antibodies against PRRSV and the evaluation of the NADC30-like PRRSV inactivated vaccine, but the advantage is insufficient in serological early diagnosis.
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Ensaio de Imunoadsorção Enzimática/veterinária , Síndrome Respiratória e Reprodutiva Suína/imunologia , Vacinas de Produtos Inativados/imunologia , Proteínas da Matriz Viral/imunologia , Animais , Anticorpos Neutralizantes , Ensaio de Imunoadsorção Enzimática/métodos , Técnica Indireta de Fluorescência para Anticorpo/veterinária , Síndrome Respiratória e Reprodutiva Suína/diagnóstico , Vírus da Síndrome Respiratória e Reprodutiva Suína/imunologia , Sensibilidade e Especificidade , SuínosRESUMO
Since the working environment of Multiple Unmanned Surface Vehicles (MUSVs) is accompanied by a large number of uncertainties and various hazards, in order to ensure the collision avoidance capability of MUSVs in complex marine environments, the perception of complex marine environments by MUSVs is the first problem that needs to be solved. A cooperative perception framework with uncertain event detection, cooperative collision avoidance pattern recognition and environmental ontology model is proposed to realize the cooperative perception process of MUSVs using ontology and Bayesian network theory. The cooperative perception approach was validated by simulating experiments. Results show the effectiveness of cooperative perception approach.
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The reaction of HO⢠radical with DNA is intensively studied both mechanistically and analytically for lesions formation. Several aspects related to the reaction paths of purine moieties with the formation of 5',8-cyclopurines (cPu), 8-oxopurines (8-oxo-Pu), and their relationship are not well understood. In this study, we investigated the reaction of HO⢠radical with a 21-mer double-stranded oligodeoxynucleotide (ds-ODNs) in γ-irradiated aqueous solutions under various oxygen concentrations and accurately quantified the six purine lesions (i.e., four cPu and two 8-oxo-Pu) by LC-MS/MS analysis using isotopomeric internal standards. In the absence of oxygen, 8-oxo-Pu lesions are only â¼4 times more than cPu lesions. By increasing oxygen concentration, the 8-oxo-Pu and the cPu gradually increase and decrease, respectively, reaching a gap of â¼130 times at 2.01 × 10-4 M of O2. Kinetic treatment of the data allows to estimate the C5' radical competition between cyclization and oxygen trapping in ds-ODNs, and lastly the rate constants of the four cyclization steps. Tailored computational studies by means of dispersion-corrected DFT calculations were performed on the CGC and TAT in their double-strand models for each cPu diastereoisomer along with the complete reaction pathways of the cyclization steps. Our findings reveal unheralded reaction mechanisms that resolve the long-standing issues with C5' radical cyclization in purine moieties of DNA sequences.
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Selective C-H oxidation is thought to be a highly suitable strategy for building synthetic blocks and generating bioactive compounds. Noncovalent DNA catalysis for C-H bond cleavage is studied for the first time in order to delineate the so-called 'oxidation enhancement effect' on oxidatively generated damage in DNA duplex structures. Herein, DFT methods have been used to gain insight into the reactivity of the 5-hydroxy-6-peroxyl-5,6-dihydrothymine radical using ten single-stranded and duplex DNA models. Reliable M06-2X/6-31+G(d,p) calculations indicate that hydrogen bonding between the complementary base pairs significantly enhances the reactivity of the thymine peroxyl radical in duplex DNA models towards the C1'-H1' bond. An excellent linear relationship of the reaction activation barrier vs. the difference between the bond dissociation free energies (BDFE) of the C-H and O-H bonds is observed. With the noted role of charge transfer from LPO4' on 2-deoxyribose to its adjacent C1'-H1' anti-bonding orbital, a hyperconjugation effect is proposed to explain the reason why the barrier heights are close to each other for the studied duplex DNA models. The difference in the reactivity of the thymine peroxyl radical in the duplex and related single-strand DNA models is rationalized in terms of the preparatory energy and the optimal σC1'-H1' and oxyl-p based π*-orbital interactions.
Assuntos
DNA/química , Teoria da Densidade Funcional , Peróxidos/química , Pirimidinas/química , Estrutura MolecularRESUMO
Enzymes contain several subunits to maintain different biological functions. However, it remains a great challenge for specific discrimination of one subunit over another. Toward this end, the fluorescent probe TPEMA is now presented for highly specific detection of the B subunit of cytosolic creatine (CK) kinase isoenzyme (CK-B). Owing to its aggregation-induced emission property, TPEMA shows highly boosted emission toward CK-B with a fast response time and very low interference from other analytes, including the M subunit of CK (CK-M). With the aid of a Job plot assay, ITC assay and molecular dynamics simulation, it was directly confirmed that the remarkably enhanced fluorescence of TPEMA in the presence of CK-B results from the restriction of single molecular motion in the cavity. Selective wash-free fluorescence imaging of CK-B in macrophages under different treatments was successfully demonstrated.
Assuntos
Enzimas/ultraestrutura , Corantes Fluorescentes , Creatina Quinase/ultraestrutura , Macrófagos/enzimologia , Macrófagos/ultraestrutura , Simulação de Dinâmica Molecular , Imagem Molecular , Movimento (Física) , Imagem ÓpticaRESUMO
As some stimuli utilized in conventional drug delivery systems can also be found in normal cells, it is inevitable that encapsulated drugs escape from carriers into normal cells. Based on mutual interactions among proteins, polyphenol compounds, and metal ions, we developed a serial-stimuli-responsive drug delivery system. With multi-crosslinking structure, nanocapsules can maintain the integrity of the framework, even with a certain amount of stimuli present, and eventually reach tumor cells to initiate apoptosis, and protect normal cells from being damaged. Meanwhile, the fluorescence of DOX will be quenched when encapsulated in nanocapsules. This property means that the DOX that is released from nanocapsules can be monitored in real-time based on the recovery of fluorescence. These versatile nanocapsules exhibit great potentials to treat cancer.
Assuntos
Doxorrubicina/química , Nanocápsulas/química , Animais , Bovinos , Sobrevivência Celular/efeitos dos fármacos , Doxorrubicina/metabolismo , Doxorrubicina/farmacologia , Liberação Controlada de Fármacos , Células Hep G2 , Humanos , Metais/química , Polifenóis/química , Soroalbumina Bovina/químicaRESUMO
Fluorinated carbonates have attracted increasing attention in high-voltage lithium ion battery applications. Under free and electric fields, their structure-related solvent dynamic properties such as charge and discharge rate, however, are rarely reported. Herein, solutions including linear fluorinated carbonates have been simulated using joint MD/DFT calculations. For the first time, the dielectric constants of the four pure fluorinated carbonates have been predicted to be ca. 5.4 (4: ethyl-2,2,2-trifluoroethyl carbonate)-12.1 (7: di-1,1,2,2,2-pentafluoroethyl carbonate), which are dependent on the number of fluorine atoms. Minor difference in the solvation free energies and few contact ion associations were observed in the ETFEC and DTFEC solvents. Their comparable viscosity values were found through calculations of ion diffusivity and conductivity. In the electric fields, the Li+ mobilities in the two solutions have similar values, showing that the dynamic properties of the electrolytic solutions are almost independent of the number of fluorine atoms on these carbonates. There emerges an oriented and ordered arrangement of the solvent molecules and thus the largely decreased dielectric constants under the electric fields, even though the field strength is very low. These interesting phenomena should be relevant in the formation of contact and aggregate ion pairs, leading to unexpected reduction of the charge-discharge rate-related ion mobility in mixed solvents.
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Oxidative damage to RNA has been linked to change or loss of RNA function and development of many human age-related diseases. However, knowledge on the nature of RNA oxidative damage is relatively limited. In this study, oxidative damage to RNA is investigated under anaerobic and aerobic conditions by exploring the properties and reactions of 5-hydroxyl-2'-uridin-6-yl and its peroxyl diastereoisomers in the RNA strand, respectively. Selective addition of OH to the nucleic base from the 5'-end is studied at the molecular level for the first time, explaining the large number of the 5S-isomer available for further reactions. Our results provide clear evidence that the efficiency of C2'-H2' bond activation in the peroxyl isomers is lower than in the carbon radical species. An exception is observed for the isomer cis-(5S,6R)-A1, whose internucleotidyl H2'-abstraction barrier is far smaller than that in the corresponding C6-yl radical. However, analysis of the equilibrium species distribution reveals that the amount of cis-(5S,6R)-A1 is very small among the peroxyl diastereoisomers, and hence the resulting products from direct strand scission should be a less important component in RNA oxidative damage. The species with maximum distribution is the cis-(5S,6R)-B1 isomer, which is derived from cis-(5S,6R)-A1 and has a moderate intranucleotidyl H2'-abstraction barrier. More importantly, the reaction is mildly exothermic. These results show that the main fraction of the intranucleotidyl H2'-abstraction intermediates can be formed from the cis-(5S,6R)-B1 isomer. The absolute reduction potentials, the hydrogen atom binding energies, and the key structural parameters of the C6-peroxyl species are used to understand the diverse reactivity of the cis-(5S,6R) diastereoisomers toward the C2'-H2' bonds activation. The present study shows that in addition to the selectivity of the OH radical addition, there is a strong correlation between the conformation of the modified uracil base and its reactivity in RNA oxidative damage.
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RNA/química , Aerobiose , Oxirredução , Teoria QuânticaRESUMO
This paper is concerned with the distributed field estimation problem using a sensor network, and the main purpose is to design a local filter for each sensor node to estimate a spatially-distributed physical process using the measurements of the whole network. The finite element method is employed to discretize the infinite dimensional process, which is described by a partial differential equation, and an approximate finite dimensional linear system is established. Due to the sparsity on the spatial distribution of the source function, the â 1 -regularized H ∞ filtering is introduced to solve the estimation problem, which attempts to provide better performance than the classical centralized Kalman filtering. Finally, a numerical example is provided to demonstrate the effectiveness and applicability of the proposed method.
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The endoplasmic reticulum (ER) has a central role in the fine-tuning of environmental and internal stimuli. We herein report a ratiometric fluorescent probe, α-Naph, capable of determining basal H2O2 in the ER. The probe specifically responds to H2O2. The limit of detection of the probe is as low as 38 nM, making it a feasible sensor to image intracellular basal H2O2. In addition, utilizing its ratiometric property, we are able to measure the concentration of H2O2 in the ER quantitatively, eliminating the error caused by the probe concentration and environment. The intracellular concentration of H2O2 in the ER is calculated to be 0.692 µM under normal conditions and 1.26 µM under the stimulation of phorbol myristate acetate.
Assuntos
Retículo Endoplasmático/metabolismo , Corantes Fluorescentes/química , Peróxido de Hidrogênio/análise , Naftalimidas/química , Corantes Fluorescentes/síntese química , Células HeLa , Humanos , Limite de Detecção , Microscopia Confocal/métodos , Microscopia de Fluorescência/métodos , Naftalimidas/síntese químicaRESUMO
Herein, binding of the ß-cyclodextrin (ß-CD) host to the unstable aryl pentazole guest has been confirmed experimentally and theoretically. After the confinement of aryl pentazole, electron density reorganization was studied by M06-2X dispersion-corrected DFT and further reflected in the characteristic shift in the NMR spectra. Among the host-guest complexes, the inclusion complex is favored with the phenyl ring expectedly encapsulated within the cavity through noncovalent interactions such as hydrogen bonding, C-Hπ, and the special Csp2-HH-Csp3 bonding discovered by the NBO, QTAIM, and NCI-RDG theories. The host-guest binding renders the enhancement of the nitrogen-ring aromatic character; this has been analyzed by employing nucleus-independent chemical shift (NICS)-based profiles. The non-covalent interaction largely enhances the thermal stability of the guest through a change of the decomposition reaction path whether the guest is encapsulated or not by the host. By comparison of the structures of B1-B4, the enhancement could be assigned to the ion-type transition structure stabilized by the C-H bonds of the host.
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The reactivity of thymine peroxy radicals in DNA and their fate are studied using the reliable DFT methods. The most accessible H1' abstraction by the C6-peroxyl once reported experimentally is effectively competitive to the crosslinking reaction between the C6-peroxyl and the C5 or C6 on the 5'-adjacent thymine base. The rare transfer of the ObH1' group to the C1' radical from the formed hydroperoxide happens with a very strong heat release. Afterwards, the parallel reactions including the H1' and H2' abstractions by the C6-alkoxyl in an inter-nucleotidyl manner lead to direct formation of thymine glycol. After the H1' abstraction by the C6-alkoxyl, the apyrimidinic site can be formed on C1' through effective N1-glycosidic bond rupture. The geometric rearrangements and the orbital interaction between the H donor and the σ-type H acceptor are used to explain the difference of the H2' abstraction barriers by C6-alkoxyl. Hence, new radical reaction paths for the formation of DNA oxidation products are suggested, which are strongly different from the previously suggested paths with the tetraoxide intermediate.
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DNA/química , Radicais Livres/química , Gases/química , Oxirredução , Termodinâmica , Timina/análogos & derivados , Timina/química , Água/químicaRESUMO
The crystal structure of 2,4,6,8,10,12-hexanitro-2,4,6,8,10,12-hexaazaiso-wurtzitane (CL-20) p-xylene solvate, and the solvent effects on the crystal faces of CL-20 were studied through a combined experimental and theoretical method. The properties were analyzed by thermogravimetry-differential scanning calorimetry (TG-DSC), Fourier transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD).The growth morphology of CL-20p-xylene solvate crystal was predicted with a modified attachment energy model. The crystal structure of CL-20p-xylene solvate belonged to the Pbca space group with the unit cell parameters, a=8.0704(12) Å, b=13.4095(20) Å, c=33.0817(49) Å, and Z=4, which indicated that the p-xylene solvent molecules could enter the crystal lattice of CL-20 and thus the CL-20 p-xylene solvate is formed. According to the solvent-effected attachment energy calculations, (002) and (11-1) faces should not be visible at all, while the percentage area of the (011) face could be increased from 7.81% in vacuum to 12.51% in p-xylene solution. The predicted results from the modified attachment energy model agreed very well with the observed morphology of crystals grown from p-xylene solution.
Assuntos
Compostos Aza/química , Compostos Heterocíclicos/química , Modelos Moleculares , Xilenos/química , Cristalografia por Raios X , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
The transportation sector is a significant contributor to greenhouse gas (GHG) emissions in China. However, real-world GHG emissions from in-use light-duty diesel trucks (LDDTs) are largely uncertain due to data paucity. In this study, we have conducted real driving emission (RDE) tests of real-world CO2, N2O, and CH4 emissions from 12 in-use LDDTs in China. Results reveal that China's CH4 emission rates from LDDTs are overestimated by 57.71 ± 39.15 % if using the previous ratio method of CO2:CH4. Notably, under real-world driving conditions, such as speeds exceeding 60 km/h, maximum exhaust gas temperatures are reached, potentially impacting urea decomposition catalyst temperatures and subsequently influencing N2O production, which is highly sensitive to system temperature. Moreover, uphill roads can increase CO2 emissions by 51.93 % compared to downhill roads. Despite the tightening of vehicle emission standards, CO2 and N2O emissions from the LDDTs have not decreased linearly. However, LDDTs meeting the China VI standard exhibit the lowest average CO2, N2O and CH4 emission factors (EFs) of 335.26 ± 21.72 g/km, 2.7 ± 0.69 mg/km and 3.50 ± 0.70 mg/km, respectively. At last, the uncertainties in the GHG EFs for the tested LDDTs through RDE tests were (-39 %, 82 %) in our study, while a significantly higher uncertainty (-85 %, 182 %) for GHG EFs of LDDTs were found in our study and other reported literature in China, largely due to the application of different non-native vehicle emission factor models and testing methods, as well as different vehicles of control emission standards. Our study highlights more urgent needs for direct RDE tests and the importance of considering real driving conditions, such as road grades, in special geographical regions when undertaking carbon accounting work in the transportation sector.