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The development of unstable carotid atherosclerotic plaques is associated with the induction of neutrophil extracellular traps (NETs) via the activation of diverse inflammatory mediators in the circulating bloodstream. However, the underlying mechanisms through which NETs influence the microenvironment of atherosclerotic plaques and contribute to the development of unstable carotid plaques remain largely elusive. The objective of this study was to elucidate the role of myeloid differentiation protein 1 (MD-1, LY86)-induced NETs underlying the crosstalk between unstable plaque formation and the plaque microenvironment. We employed bioinformatics analysis to identify key genes associated with carotid-unstable plaque, followed by comprehensive validation using various experimental approaches on tissue specimens and plasma samples classified based on pathological characteristics. Patients with carotid-unstable plaques exhibited elevated plasma concentrations of MD-1 (LY86), while patients with stable plaques demonstrated comparatively lower levels. Furthermore, soluble MD-1 was found to induce the formation of NETs through activation of Toll-like receptor signaling pathway. The proliferative and immature vascularization effects of NETs on endothelial cells, as well as their inhibitory impact on cell migration, are directly correlated with the concentration of NETs. Additionally, NETs were found to activate the NF-κB signaling pathway, thereby upregulating ICAM1, VCAM1, MMP14, VEGFA, and IL6 expression in both Human umbilical vein endothelial cells (HUVECs) and HAECs. Subsequently, a significant increase in intraplaque neovascularization by NETs results in poor carotid plaque stability, and NETs in turn stimulate macrophages to produce more MD-1, generating a harmful positive feedback loop. Our findings suggest that soluble MD-1 in the bloodstream triggers the production of NETs through activation of the Toll-like receptor signaling pathway and further indicate NETs mediate a crosstalk between the microenvironment of the carotid plaque and the neovascularization of the intraplaque region. Inhibiting NETs formation or MD-1 secretion may represent a promising strategy to effectively suppress the development of unstable carotid plaques.
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Armadilhas Extracelulares , Placa Aterosclerótica , Transdução de Sinais , Humanos , Armadilhas Extracelulares/metabolismo , Placa Aterosclerótica/metabolismo , Placa Aterosclerótica/patologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Neutrófilos/metabolismo , Neutrófilos/imunologia , Microambiente Celular , Masculino , NF-kappa B/metabolismo , FemininoRESUMO
Two-component systems (TCSs) sensing and responding to various stimuli outside and inside cells are valuable resources for developing biosensors with synthetic biology applications. However, the use of TCS-based biosensors suffers from a limited effector spectrum, hypersensitivity, low dynamic range, and unwanted signal crosstalk. Here, we developed a tailor-made Escherichia coli whole-cell γ-aminobutyric acid (GABA) biosensor by engineering a chimeric GABA chemoreceptor PctC and TCS. By testing different TCSs, the chimeric PctC/PhoQ showed the response to GABA. Chimera-directed evolution and introduction of the insulated chimeric pair PctC/PhoQ*PhoP* produced biosensors with up to 3.50-fold dynamic range and good orthogonality. To further enhance the dynamic range and lower the basal leakage, three strategies, engineering of PhoP DNA binding sites, fine-tuning reporter expression by optimizing transcription/translation components, and a tobacco etch virus protease-controlled protein degradation, were integrated. This chimeric biosensor displayed a low basal leakage, a large dynamic range (15.8-fold), and a high threshold level (22.7 g L-1). Finally, the optimized biosensor was successfully applied in the high-throughput microdroplet screening of GABA-overproducing Corynebacterium glutamicum, demonstrating its desired properties for extracellular signal biosensing.
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Técnicas Biossensoriais , Proteínas de Escherichia coli , Escherichia coli , Ácido gama-Aminobutírico , Técnicas Biossensoriais/métodos , Técnicas Biossensoriais/instrumentação , Ácido gama-Aminobutírico/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismoRESUMO
Low-dimensional (LD) materials, with atomically thin anisotropic structures, exhibit remarkable physical and chemical properties, prominently featuring piezoelectricity resulting from the absence of centrosymmetry. This characteristic has led to diverse applications, including sensors, actuators, and micro- and nanoelectromechanical systems. While piezoelectric effects are observed across zero-dimensional (0D), one-dimensional (1D), and two-dimensional (2D) LD materials, challenges such as effective charge separation and crystal structure imperfections limit their full potential. Addressing these issues requires innovative solutions, with the integration of LD materials with polymers, ceramics, metals, and other porous materials proving a key strategy to significantly enhance piezoelectric properties. This review comprehensively covers recent advances in synthesizing and characterizing piezoelectric composites based on LD materials and porous materials. The synergistic combination of LD materials with other substances, especially porous materials, demonstrates notable performance improvements, addressing inherent challenges. The review also explores future directions and challenges in developing these composite materials, highlighting potential applications across various technological domains.
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It has been well proved that the introduction of halogen can effectively modify the optoelectronic properties of classic symmetric nonfullerene acceptors (NFAs). However, the relevant studies for asymmetric NFAs are limited, especially the effect of halogen substitution number and position on the photovoltaic performance is not clear. In this work, four asymmetric NFAs with A-D-A1-A2 structure are developed by tuning the number and position of chlorine atoms on the 1,1-dicyanomethylene-3-indanone end groups, namely, A303, A304, A305, and A306. The related NFAs show progressively deeper energy levels and red-shifted absorption spectra as the degree of chlorination increases. The PM6:A306-constructed organic solar cells (OSCs) give a champion power conversion efficiency (PCE) of 13.03%. This is mainly ascribed to the most efficient exciton dissociation and collection, suppressed charge recombination, and optimal morphology. Moreover, by alternating the substitution position, the PM6:A305-based device yielded a higher PCE of 12.53% than that of PM6:A304 (12.05%). This work offers fresh insights into establishing excellent asymmetric NFAs for OSCs.
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The development of blue-emissive ambipolar organic semiconductor is an arduous target due to the large energy gap, but is an indispensable part for electroluminescent device, especially for the transformative display technology of simple-structured organic light-emitting transistor (SS-OLET). Herein, we designed and synthesized two new dibenzothiophene sulfone-based high mobility blue-emissive organic semiconductors (DNaDBSOs), which demonstrate superior optical property with solid-state photoluminescent quantum yield of 46-67 % and typical ambipolar-transporting properties in SS-OLETs with symmetric gold electrodes. Comprehensive experimental and theoretical characterizations reveal the natural of ambipolar property for such blue-emissive DNaDBSOs-based materials is ascribed to a synergistic effect on lowering LUMO level and reduced electron injection barrier induced by the interfacial dipoles effect on gold electrodes due to the incorporation of appropriate DBSO unit. Finally, efficient electroluminescence properties with high-quality blue emission (CIE (0.179, 0.119)) and a narrow full-width at half-maximum of 48â nm are achieved for DNaDBSO-based SS-OLET, showing good spatial control of the recombination zone in conducting channel. This work provides a new avenue for designing ambipolar emissive organic semiconductors by incorporating the synergistic effect of energy level regulation and molecular-metal interaction, which would advance the development of superior optoelectronic materials and their high-density integrated optoelectronic devices and circuits.
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Polyolefin plastics, such as polyethylene (PE) and polystyrene (PS), are the most widely used synthetic plastics in our daily life. However, the chemical structure of polyolefin plastics is composed of carbon-carbon (C-C) bonds, which is extremely stable and makes polyolefin plastics recalcitrant to degradation. The growing accumulation of plastic waste has caused serious environmental pollution and has become a global environmental concern. In this study, we isolated a unique Raoultella sp. DY2415 strain from petroleum-contaminated soil that can degrade PE and PS film. After 60 d of incubation with strain DY2415, the weight of the UV-irradiated PE (UVPE) film and PS film decreased by 8% and 2%, respectively. Apparent microbial colonization and holes on the surface of the films were observed by scanning electron microscopy (SEM). Furthermore, the Fourier transform infrared spectrometer (FTIR) results showed that new oxygen-containing functional groups such as -OH and -CO were introduced into the polyolefin molecular structure. Potential enzymes that may be involved in the biodegradation of polyolefin plastics were analyzed. These results demonstrate that Raoultella sp. DY2415 has the ability to degrade polyolefin plastics and provide a basis for further investigating the biodegradation mechanism.
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Petróleo , Poliestirenos , Poliestirenos/metabolismo , Polietileno/química , Solo , Enterobacteriaceae , Biodegradação Ambiental , Carbono , Plásticos/metabolismoRESUMO
FeNC catalysts demonstrate remarkable activity and stability for the oxygen reduction reaction (ORR) in polymer electrolyte membrane fuel cells and Zn-air batteries (ZABs). The local coordination of Fe single atoms in FeNC catalysts strongly impacts ORR activity. Herein, FeNC catalysts containing Fe single atoms sites with FeN3 , FeN4 , and FeN5 coordinations are synthesized by carbonization of Fe-rich polypyrrole precursors. The FeN5 sites possess a higher Fe oxidation state (+2.62) than the FeN3 (+2.23) and FeN4 (+2.47) sites, and higher ORR activity. Density functional theory calculations verify that the FeN5 coordination optimizes the adsorption and desorption of ORR intermediates, dramatically lowering the energy barrier for OH- desorption in the rate-limiting ORR step. A primary ZAB constructed using the FeNC catalyst with FeN5 sites demonstrates state-of-the-art performance (an open circuit potential of 1.629 V, power density of 159 mW cm-2 ). Results confirm an intimate structure-activity relationship between Fe coordination, Fe oxidation state, and ORR activity in FeNC catalysts.
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BACKGROUND: Heme has attracted much attention because of its wide applications in medicine and food. The products of genes hemBCDEFY convert 5-aminolevulinic acid to protoporphyrin IX (PPIX; the immediate precursor of heme); protoporphyrin ferrochelatase (FECH) inserts Fe2+ into PPIX to generate heme. Biosynthesis of heme is limited by the need for optimized expression levels of multiple genes, complex regulatory mechanisms, and low enzymatic activity; these problems need to be overcome in metabolic engineering to improve heme synthesis. RESULTS: We report a heme biosensor-guided screening strategy using the heme-responsive protein HrtR to regulate tcR expression in Escherichia coli, providing a quantifiable link between the intracellular heme concentration and cell survival in selective conditions (i.e., the presence of tetracycline). This system was used for rapid enrichment screening of heme-producing strains from a library with random ribosome binding site (RBS) variants and from a FECH mutant library. Through up to four rounds of iterative evolution, strains with optimal RBS intensities for the combination of hemBCDEFY were screened; we obtained a PPIX titer of 160.8 mg/L, the highest yield yet reported in shaken-flask fermentation. A high-activity FECH variant was obtained from the saturation mutagenesis library. Fed-batch fermentation of strain SH20C, harboring the optimized hemBCDEFY and the FECH mutant, produced 127.6 mg/L of heme. CONCLUSION: We sequentially improved the multigene biosynthesis pathway of PPIX and performed in vivo directed evolution of FECH, based on a heme biosensor, which demonstrated the effectiveness of the heme biosensor-based pathway optimization strategy and broadens our understanding of the mechanism of heme synthesis.
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Background: The prognosis of gallbladder cancer (GBC) is dismal. This study aimed to compare the outcomes of adjuvant chemoradiotherapy (ACR) with those of surgery alone (S) and adjuvant chemotherapy (AC). Method: The Surveillance, Epidemiology, and End Results (SEER) Program database was used to identify patients diagnosed with GBC and undergoing surgery between 2004 and 2015. The patients were divided into the S, AC, and ACR groups according to their treatment. Categorical variables were compared by Pearson's chi-square test, and a 1:1:1 propensity score matching analysis (PSM) was performed. Overall survival was assessed by Kaplan-Meier curves with log-rank tests. Subgroup analyses were conducted. Result: A total of 5451 patients were identified in the SEER database. After PSM, the two-year survival among patients who received S, AC, and ACR was 36%, 39%, and 45%, respectively. ACR was associated with improved two-year survival (p < 0.001), while the survival rates were similar in the AC and S groups (p = 0.127) but better in the ACR group than in the AC group (p = 0.012). Subgroup analyses indicated that while the two-year survival rates did not differ significantly in stage II GBC patients between the groups (all p > 0.05), ACR was associated with significantly improved two-year survival in stage â ¢a (p = 0.008), â ¢b (p < 0.001), and â £b (p < 0.001) GBC patients. Conclusion: The combination of surgery and ACR as the treatment modality provided greater survival benefits for GBC patients, particularly for those with advanced tumor staging.
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With the continuous emergence and spread of drug-resistant and multi-drug-resistant Staphylococcus aureus, traditional antibiotic treatment has gradually lost its effect. There is an urgent need to develop and study new and effective bio-green inhibitors to control S. aureus. In this study, the S. aureus phage DZ25 was isolated from milk and the lysin LysDZ25 with excellent tolerance to serum and NaCl solution was identified. Subsequently, to improve the lytic activity and thermal stability of LysDZ25, RoseTTAFold was used to construct three-dimensional (3D) structures, molecular dynamics (MD) simulation was used for conformational acquisition, and the MDL strategy previously developed in our lab was used to rationally design variants. After two rounds of rational design, the optimal variant with improved thermal stability, S333V/N245R/D299L, was obtained, and its half-life time was 4.0-fold that of wild-type LysDZ25. At 37, 40, 45, and 50 °C, the lytic activity of the optimal triple-point variant S333V/N245R/D299L was increased by 17.3-, 26.7-, 20.2-, and 50.1-fold compared with that of the wild-type LysDZ25, respectively. Finally, cell count was used to evaluate the lytic activity, and the results showed that the optimal variant S333V/N245R/D299L could drop about 3.5â¯logâ¯10 values compared with the control and about 2.6â¯logâ¯10 values compared with the wild-type LysDZ25.
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Staphylococcus aureus Resistente à Meticilina , Infecções Estafilocócicas , Humanos , Staphylococcus aureus , Fagos de Staphylococcus/genética , Antibacterianos/farmacologiaRESUMO
Polyurethanes (PU) are one of the most used categories of plastics and have become a significant source of environmental pollutants. Degrading the refractory PU wastes using environmentally friendly strategies is in high demand. In this study, three microbial consortia from the landfill leachate were enriched using PU powder as the sole carbon source. The consortia efficiently degraded polyester PU film and accumulated high biomass within 1 week. Scanning electron microscopy, Fourier transform infrared spectroscopy, and contact angle analyses showed significant physical and chemical changes to the PU film after incubating with the consortia for 48 h. In addition, the degradation products adipic acid and butanediol were detected by high-performance liquid chromatography in the supernatant of the consortia. Microbial composition and extracellular enzyme analyses revealed that the consortia can secrete esterase and urease, which were potentially involved in the degradation of PU. The dominant microbes in the consortia changed when continuously passaged for 50 generations of growth on the PU films. This work demonstrates the potential use of microbial consortia in the biodegradation of PU wastes. KEY POINTS: ⢠Microbial consortia enriched from landfill leachate degraded polyurethane film. ⢠Consortia reached high biomass within 1 week using polyurethane film as the sole carbon source. ⢠The consortia secreted potential polyurethane-degrading enzymes.
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Poliuretanos , Poluentes Químicos da Água , Poliuretanos/metabolismo , Consórcios Microbianos , Microbiologia do Solo , Biodegradação Ambiental , Instalações de Eliminação de ResíduosRESUMO
Background and objective: Acupuncture is a promising non-pharmacological therapy for patients with prolonged disorder of consciousness (PDOC); however, its underlying mechanism remains uncertain. This study aimed to reveal the modulatory effects of acupuncture on the cerebral cortex activity among patients with PDOC. Materials and methods: Twenty-eight PDOC patients were randomly assigned to the treatment (n = 14) or control (n = 14) group. The treatment group received one session of acupuncture, while the control group received one session of sham acupuncture. All patients underwent evaluation of the functional connectivity and activation response of the dorsolateral prefrontal cortex (DLPFC), primary motor cortex (M1), and primary somatosensory cortex (S1) via functional near-infrared spectroscopy. We further explored the potential correlation of the consciousness level and activation response/functional connectivity with acupuncture. Results: Compared to the control group, a single session of acupuncture significantly tended to enhance resting-state functional connectivity (rsFC) in DLPFC-M1, DLPFC-M1, and S1-S1. And the activation level of the DLPFC (both sides) in the acupuncture group is significantly higher than those in sham acupuncture group. However, no significant correlation was found between the consciousness level and activation response/functional connectivity. Conclusion: One session of acupuncture has a significant modulation of rsFC and activation in the DLPFC, M1, and S1 with PDOC patients. Acupuncture-evoked effect may have some functional significance in PDOC patients. This is an important step toward exploring the acupuncture effects on PDOC patients.
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BACKGROUND: Biliary tract cancers (BTCs) are a series of heterogeneous malignancies that are broadly grouped based on the anatomical site where they arise into subtypes including intrahepatic cholangiocarcinoma (ICC), extrahepatic cholangiocarcinoma (ECC), gallbladder cancer (GBC), and ampulla of Vater cancer (AVC). METHODS AND RESULTS: The present study provides an overview of the epidemiology of the various BTCs based on data from the National Cancer Institute's Surveillance, Epidemiology, and End Results (SEER) database from 2000 to 2018. Distinct differences in both incidence and mortality rates were observed for these BTCs as a function of age, sex, ethnicity, and calendar year. In 2018, BTCs emerged as the fifth most prevalent form of alimentary tract cancer in the USA. While the incidence and mortality of ICC appear to be increasing, the incidence rates of GBC, ECC, and AVC have remained stable, as have the corresponding mortality rates. The most common and deadliest BTCs in 2018 were ICC and GBC among males and females, respectively. The ethnic groups exhibiting the highest incidence rates of these different BTCs were American Indians and Alaska Natives for GBC, and Asian and Pacific Islanders for ICC, ECC, and AVC. The incidence of all of these forms of BTC rose with age. There were some variations in BTCs in terms of staging, locoregional surgical treatments, adjuvant therapies, and prognostic outcomes from 2000 to 2018. CONCLUSIONS: The epidemiological characteristics, staging, locoregional surgical treatments, adjuvant therapies, and prognostic outcomes were distinct for each of these BTCs.
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Neoplasias dos Ductos Biliares , Neoplasias do Sistema Biliar , Colangiocarcinoma , Neoplasias da Vesícula Biliar , Masculino , Feminino , Humanos , Estados Unidos/epidemiologia , Neoplasias dos Ductos Biliares/epidemiologia , Neoplasias dos Ductos Biliares/patologia , Neoplasias do Sistema Biliar/epidemiologia , Neoplasias do Sistema Biliar/patologia , Colangiocarcinoma/epidemiologia , Colangiocarcinoma/patologia , Neoplasias da Vesícula Biliar/epidemiologia , Neoplasias da Vesícula Biliar/patologia , Ductos Biliares Intra-HepáticosRESUMO
BACKGROUND: ß-Farnesene is a sesquiterpene with versatile industrial applications. The production of ß-farnesene from waste lipid feedstock is an attractive method for sustainable production and recycling waste oil. Yarrowia lipolytica is an unconventional oleaginous yeast, which can use lipid feedstock and has great potential to synthesize acetyl-CoA-derived chemicals. RESULTS: In this study, we engineered Y. lipolytica to produce ß-farnesene from lipid feedstock. To direct the flux of acetyl-CoA, which is generated from lipid ß-oxidation, to ß-farnesene synthesis, the mevalonate synthesis pathway was compartmentalized into peroxisomes. ß-Farnesene production was then engineered by the protein engineering of ß-farnesene synthase and pathway engineering. The regulation of lipid metabolism by enhancing ß-oxidation and eliminating intracellular lipid synthesis was further performed to improve the ß-farnesene synthesis. As a result, the final ß-farnesene production with bio-engineering reached 35.2 g/L and 31.9 g/L using oleic acid and waste cooking oil, respectively, which are the highest ß-farnesene titers reported in Y. lipolytica. CONCLUSIONS: This study demonstrates that engineered Y. lipolytica could realize the sustainable production of value-added acetyl-CoA-derived chemicals from waste lipid feedstock.
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Plastic waste is rapidly accumulating in the environment and becoming a huge global challenge. Many studies have highlighted the role of microbial metabolic engineering for the valorization of polyethylene terephthalate (PET) waste. In this study, we proposed a new conceptual scheme for upcycling of PET. We constructed a multifunctional Pseudomonas putida KT2440 to simultaneously secrete PET hydrolase LCC, a leaf-branch compost cutinase, and synthesize muconic acid (MA) using the PET hydrolysate. The final product MA and extracellular LCC can be separated from the supernatant of the culture by ultrafiltration, and the latter was used for the next round of PET hydrolysis. A total of 0.50 g MA was produced from 1 g PET in each cycle of the whole biological processes, reaching 68% of the theoretical conversion. This new conceptual scheme for the valorization of PET waste should have advantages over existing PET upcycling schemes and provides new ideas for the utilization of other macromolecular resources that are difficult to decompose, such as lignin.
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Pseudomonas putida , Hidrolases/genética , Hidrolases/metabolismo , Lignina/metabolismo , Plásticos/metabolismo , Polietilenotereftalatos , Pseudomonas putida/genética , Pseudomonas putida/metabolismo , Ácido Sórbico/análogos & derivadosRESUMO
Background: The purpose of this meta-analysis was to evaluate the efficacy of lymph node dissection in patients with intrahepatic cholangiocarcinoma (ICC). Methods: The literature from January 2009 to December 2021 was searched to determine the comparative study of lymph node dissection and non-lymph node dissection in patients with ICC. Results: Seventeen studies were included in the analysis. There were no significant differences in 1-, 3-, and 5-year overall survival (OR = 0.80, p = 0.10; OR = 0.93, p = 0.71; OR = 0.80, p = 0.21) and 1-, 3-, and 5-year disease-free survival (OR = 0.89, p = 0.73; OR = 0.92, p = 0.81; OR = 0.85, p = 0.62). Conclusions: Lymph node dissection does not seem to have a positive effect on the overall survival and disease-free survival.
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Background: Due to limited time windows and technical requirements, only a small percentage of patients can receive reperfusion therapy for acute ischemic stroke (AIS). Previous studies have shown that LongShengZhi (LSZ) capsule can improve neurological outcomes in patients after AIS, yet those results have not been finally verified through rigorous randomized controlled trials. Thus, this trial was designed to further clarify the efficacy and safety of LSZ capsule for patients with AIS. Methods: LSZ capsule on Functional Recovery after Acute Ischemic Stroke (LONGAN) trial is a prospective, multicenter, randomized, placebo-controlled, double-blind, parallel-group, superiority trial that enrolls patients from stroke and rehabilitation units in China. We will enroll 1,376 patients aged 18 years or older with AIS within 7 days of symptom onset and a National Institute of Health Stroke Scale (NIHSS) score of 4-15. Eligible patients will be randomized to receive either 2 g LSZ capsules three times a day or placebo LSZ capsules for 90 days. The primary outcome is the proportion of patients with favorable outcomes, as measured by the modified Rankin Scale (mRS) 90 days after randomization. The main safety outcome is the proportion of severe adverse events. Conclusion: This study will be the first randomized, double-blind trial to evaluate the efficacy and safety of LSZ capsule in patients with AIS. In order to improve the transparency and reproducibility of the trial, the data will be analyzed in accordance with this pre-specified plan for statistical analysis to reduce bias due to selective analysis and reporting. This trial aims to provide high-quality evidence for the efficacy and safety of LSZ capsule for AIS.
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Surface chemical modification of carbon nanotubes can enhance the compatibility with polymers and improve flame retardancy performances. In this work, the double bond active sites were constructed on the surface of carbon nanotubes modified by the γ-methacryloyloxypropyl trimethoxysilane (KH570). Glycidyl methacrylate (GMA) was further grafted onto the surface of carbon nanotubes via free radical polymerization. Finally, the flame retardant melamine polyphosphate (MPP) was bonded to the surface of carbon nanotubes by the ring-opening reaction. This modification process was proved to be achieved by infrared spectroscopy and thermogravimetric test. The carbon nanotubes modified by flame retardant were added into the epoxy matrix and cured to prepare flame retardant and thermal conductive composites. The flame retardancy of composites were studied by cone calorimetry, UL94 vertical combustion test and limiting oxygen index. The thermal conductivity of composites was characterized by laser thermal conductivity instrument. The results showed that when the addition amount of flame retardant MPP-modified carbon nanotubes in composites was 10 wt%, the flame retardant level of UL94 reached to V2, the limiting oxygen index increased from 25.1 of pure epoxy resin to 28.3, the PHRR of pure epoxy resin was reduced from 800 kW/m2 to 645 kW/m2 of composites and thermal conductivity of composites was enhanced from 0.21 W/m·K-1 of pure epoxy resin to 0.42 W/m·K-1 of the composites.
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With the increasingly serious drug resistance of Acinetobacter baumannii, there is an increasingly urgent need for new antibacterial drugs. Phage lysin PlyAB1 has a bactericidal effect on drug-resistant A. baumannii, which has the potential to replace antibiotics to fight infection caused by A. baumannii. However, its application is limited by its thermal stability and lytic activity. To solve these problems, molecular dynamics (MD) simulations combined with Hotspot wizard 3.0 were used to identify key residue sites affecting thermal stability, and evolutionary analysis combined with multiple sequence alignment was used to identify key residue sites affecting lytic activity. Four single-point variants with significantly increased thermal stability and four single-point variants with significantly lytic activity were obtained, respectively. Furthermore, by superimposing mutations, we obtained three double-point variants, G100Q/K69R, G100R/K69R, and G100K/K69R, with significantly improved thermal stability and improved lytic activity. At 45°C, the lytic activity and half-life of the optimal variant G100Q/K69R were 1.51- and 24-fold higher than those of the wild PlyAB1, respectively. These results deepen our understanding of the structure and function of phage lysin and contribute to the application of phage lysin in antibiotic substitution.
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Acinetobacter baumannii , Bacteriófagos , Antibacterianos/farmacologia , Bacteriófagos/genética , Mucoproteínas/farmacologiaRESUMO
Heterotrophic bacteria and human mitochondria often use sulfide: quinone oxidoreductase (SQR) and persulfide dioxygenase (PDO) to oxidize sulfide to sulfite and thiosulfate. Bioinformatic analysis showed that the genes encoding RHOD domains were widely presented in annotated sqr-pdo operons and grouped into three types: fused with an SQR domain, fused with a PDO domain, and dissociated proteins. Biochemical evidence suggests that RHODs facilitate the formation of thiosulfate and promote the reaction between inorganic polysulfide and glutathione to produce glutathione polysulfide. However, the physiological roles of RHODs during sulfide oxidation by SQR and PDO could only be tested in an RHOD-free host. To test this, 8 genes encoding RHOD domains in Escherichia coli MG1655 were deleted to produce E. coli RHOD-8K. The sqrCp and pdoCp genes from Cupriavidus pinatubonensis JMP134 were cloned into E. coli RHOD-8K. SQRCp contains a fused RHOD domain at the N-terminus. When the fused RHOD domain of SQRCp was inactivated, the cells oxidized sulfide into increased thiosulfate with the accumulation of cellular sulfane sulfur in comparison with cells containing the intact sqrCp and pdoCp. The complementation of dissociated DUF442 minimized the accumulation of cellular sulfane sulfur and reduced the production of thiosulfate. Further analysis showed that the fused DUF442 domain modulated the activity of SQRCp and prevented it from directly passing the produced sulfane sulfur to GSH. Whereas, the dissociated DUF442 enhanced the PDOCp activity by several folds. Both DUF442 forms minimized the accumulation of cellular sulfane sulfur, which spontaneously reacted with GSH to produce GSSG, causing disulfide stress during sulfide oxidation. Thus, RHODs may play multiple roles during sulfide oxidation.