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A phospholipase D high producing strain with transphosphatidylation activity that is suitable for phosphatidylserine synthesis was screened by our laboratory and named as Streptomyces cinnamoneum SK43.003. The enzyme structural and biochemical properties were investigated using the molecular biology method. A 1521-bp fragment of the phospholipase D gene from Streptomyces cinnamoneum SK43.003 was amplified by PCR and encoded for 506 amino acids. The primary structure contained two conserved HKD and GG/S motifs. The pld gene was cloned and expressed in Escherichia coli. The purified enzyme exhibited the highest activity at a pH value of 6.0 andtemperature of 60°C. The enzyme was stable within a pH range of 4-7 for 24 h or at temperatures below 50°C. In addition, Triton X-100, Fe2+ , and Al3+ were beneficial to the enzyme activity, whereas Zn2+ and Cu2+ dramatically inhibited its activity. In a two-phase system, the enzyme could convert phosphatidylcholine to phosphatidylserine with a 92% transformation rate.
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Fosfolipase D , Streptomyces , Streptomyces/genética , Fosfolipase D/genética , Fosfolipase D/química , Fosfolipase D/metabolismo , Fosfatidilserinas , Escherichia coli/genéticaRESUMO
BACKGROUND: Alginate lyases (EC 4.4.2.3/4.4.2.11) have been applied to produce alginate oligosaccharides, which have physiological advantages such as prebiotic and antidiabetic effects, and are of benefit in the food and pharmaceutical industries. Extracellular production of recombinant proteins in Escherichia coli presents advantages including simplified downstream processing and high productivity; however, the presence of certain signal peptides does not always ensure successful secretion, which make the extracellular production of alginate lyase in E. coli rarely reported but of great significance. RESULTS: A PL7 family alginate lyase, Aly01, with its native signal peptide from Vibrio natriegens SK42.001, was identified, characterized, and extracellularly expressed in E. coli. The enzyme specifically released trisaccharide from alginate and was strictly NaCl activated. Green fluorescent protein (GFP) was fused with the Aly01 signal peptide and successfully secreted in E. coli to expand the feasibility of using this signal peptide to produce other heterologous proteins extracellularly. Through a synergistic strategy of utilizing Terrific Broth (TB) medium supplemented with 120 mmol L-1 glycine and 10 mmol L-1 calcium, the lag phase of protein secretion was reduced to 3 h from 12 h; meanwhile calcium remedied glycine-related cell growth impairment, leading to further enhancement of overall enzyme productivity, reaching a maximum of 4.55 U mL-1 . CONCLUSION: A new salt-activated alginate lyase, Aly01, was identified and characterized. E. coli employed its signal peptide and extracellularly expressed both Aly01 and a GFP, which indicated the signal peptide of Aly01 could be a powerful tool for extracellular production of other heterologous proteins in E. coli. © 2021 Society of Chemical Industry.
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Escherichia coli/genética , Espaço Extracelular/enzimologia , Polissacarídeo-Liases/química , Polissacarídeo-Liases/genética , Cloreto de Sódio/metabolismo , Alginatos/metabolismo , Sequência de Aminoácidos , Clonagem Molecular , Ativação Enzimática , Estabilidade Enzimática , Escherichia coli/metabolismo , Espaço Extracelular/química , Espaço Extracelular/genética , Expressão Gênica , Concentração de Íons de Hidrogênio , Polissacarídeo-Liases/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Cloreto de Sódio/química , Especificidade por SubstratoRESUMO
Long-term structural health monitoring (SHM) has become an important tool to ensure the safety of infrastructures. However, determining methods to extract valuable information from large amounts of data from SHM systems for effective identification of damage still remains a major challenge. This paper provides a novel effective method for structural damage detection by introduction of space and time windows in the traditional principal component analysis (PCA) technique. Numerical results with a planar beam model demonstrate that, due to the presence of space and time windows, the proposed double-window PCA method (DWPCA) has a higher sensitivity for damage identification than the previous method moving PCA (MPCA), which combines only time windows with PCA. Further studies indicate that the developed approach, as compared to the MPCA method, has a higher resolution in localizing damage by space windows and also in quantitative evaluation of damage severity. Finally, a finite-element model of a practical bridge is used to prove that the proposed DWPCA method has greater sensitivity for damage detection than traditional methods and potential for applications in practical engineering.
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Multiwalled carbon nanotube/polymer composites with aligned and isotropic micropores are constructed by a facile ice-templated freeze-drying method in a wide density range, with controllable types and contents of the nanoscale building blocks, in order to tune the shielding performance together with the considerable mechanical and electrical properties. Under the mutual promotion of the frame and porous structure, the lightweight high-performance shielding is achieved: a 2.3 mm thick sample can reach 46.7 and 21.7 dB in the microwave X-band while the density is merely 32.3 and 9.0 mg cm-3 , respectively. The lowest density corresponds to a value of shielding effectiveness divided by both the density and thickness up to 104 dB cm2 g-1 , far beyond the conductive polymer composites with other fillers ever reported. The shielding mechanism of the flexible porous materials is further demonstrated by an in situ compression experiment.
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Alginate lyases (ALys) whose degrading products, alginate oligosaccharides, exhibit various outstanding biochemical activities have aroused increasing interest of researchers in the marine bioresource field. However, their predominant sourcing from marine bacteria, with limited yields and unclear genetic backgrounds, presents a challenge for industrial production. In this study, ALys (Aly01) from Vibrio natriegens SK 42.001 was expressed in Bacillus subtilis (B. subtilis), a nonpathogenic microorganism recognized as generally safe (GRAS). This accomplishment was realized through a comprehensive strategy involving vector and host selection, promoter and signal peptide screening, and engineering of the ribosome binding site (RBS) and the N-terminal coding sequence (NCS). The optimal combination was identified as the pP43NMK and B. subtilis WB600. Among the 19 reported strong promoters, PnprE exhibited the best performance, showing intracellular enzyme activities of 4.47 U/mL. Despite expectations, dual promoter construction did not yield a significant increase. Further, SPydhT demonstrated the highest extracellular activity (1.33 U/mL), which was further improved by RBS/NCS engineering, reaching 4.58 U/mL. Finally, after fed-batch fermentation, the extracellular activity reached 18.01 U/mL, which was the highest of ALys with a high molecular weight expressed in B. subtilis. These findings are expected to offer valuable insights into the heterologous expression of ALys in B. subtilis.
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Bacillus subtilis , Proteínas de Bactérias , Polissacarídeo-Liases , Regiões Promotoras Genéticas , Sinais Direcionadores de Proteínas , Bacillus subtilis/genética , Bacillus subtilis/enzimologia , Bacillus subtilis/metabolismo , Polissacarídeo-Liases/genética , Polissacarídeo-Liases/metabolismo , Polissacarídeo-Liases/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Sinais Direcionadores de Proteínas/genética , Vibrio/genética , Vibrio/enzimologia , Vetores Genéticos/metabolismo , Vetores Genéticos/genética , Vetores Genéticos/química , Iniciação Traducional da Cadeia PeptídicaRESUMO
BACKGROUND: Accidental impact on a player's head by a powerful soccer ball may lead to brain injuries and concussions during games. It is crucial to assess these injuries promptly and accurately on the field. However, it is challenging for referees, coaches, and even players themselves to accurately recognize potential injuries and concussions following such impacts. Therefore, it is necessary to establish a list of minimum ball velocity thresholds that can result in concussions at different impact locations on the head. Additionally, it is important to identify the affected brain regions responsible for impairments in brain function and potential clinical symptoms. METHODS: By using a full human finite element model, dynamic responses and brain injuries caused by unintentional soccer ball impacts on six distinct head locations (forehead, tempus, crown, occiput, face, and jaw) at varying ball velocities (10, 15, 20, 25, 30, 35, 40, and 60 m/s) were simulated and investigated. Intracranial pressure, Von-Mises stress, and first principal strain were analyzed, the ball velocity thresholds resulting in concussions at different impact locations were evaluated, and the damage evolution patterns in the brain tissue were analyzed. RESULTS: The impact on the occiput is most susceptible to induce brain injuries compared to all other impact locations. For a conservative assessment, the risk of concussion is present once the soccer ball reaches 17.2 m/s in a frontal impact, 16.6 m/s in a parietal impact, 14.0 m/s in an occipital impact, 17.8 m/s in a temporal impact, 18.5 m/s in a facial impact or 19.2 m/s in a mandibular impact. The brain exhibits the most significant dynamic responses during the initial 10-20 ms, and the damaged regions are primarily concentrated in the medial temporal lobe and the corpus callosum, potentially causing impairments in brain functions. CONCLUSIONS: This work offers a framework for quantitatively assessing brain injuries and concussions induced by an unintentional soccer ball impact. Determining the ball velocity thresholds at various impact locations provides a benchmark for evaluating the risks of concussion. The examination of brain tissue damage evolution introduces a novel approach to linking biomechanical responses with possible clinical symptoms.
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Concussão Encefálica , Futebol , Humanos , Futebol/lesões , Concussão Encefálica/fisiopatologia , Fenômenos Biomecânicos , Análise de Elementos Finitos , Lesões Encefálicas/fisiopatologia , Traumatismos em Atletas/fisiopatologia , Simulação por Computador , Encéfalo/fisiopatologia , Aceleração , Pressão Intracraniana/fisiologiaRESUMO
Although the finite element head model (FEHM) has been widely utilized to analyze injury locations and patterns in traumatic brain injury, significant controversy persists regarding the selection of a mechanical injury variable and its corresponding threshold. This paper aims to determine an objective injury threshold for maximum principal strain (MPS) through a novel data-driven method, and to validate and apply it. We extract the peak responses from all elements across 100 head impact simulations to form a dataset, and then determine the objective injury threshold by analyzing the relationship between the combined injury degree and the threshold according to the stationary value principle. Using an occipital impact case from a clinical report as an example, we evaluate the accuracy of the injury prediction based on the new threshold. The results show that the injury area predicted by finite element analysis closely matches the main injury area observed in CT images, without the issue of over- or underestimating the injury due to an unreasonable threshold. Furthermore, by applying this threshold to the finite element analysis of designed occipital impacts, we observe, for the first time, supra-tentorium cerebelli injury, which is related to visual memory impairment. This discovery may indicate the biomechanical mechanism of visual memory impairment after occipital impacts reported in clinical cases.
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BACKGROUND: Although the Head Injury Criteria (HIC) has been widely applied to assess head impact injuries, it faces two outstanding problems: 1) HIC is affected strongly by the cut-off frequency when processing acceleration signals. And these cut-off frequencies are experiential and lack unified guidelines; 2) If the head was impacted on a different part, should the corresponding HIC threshold be the same? If these problems are not resolved, it could potentially lead to a critical misinterpretation of the safety assessment. METHODS: Finite element method was used to reconstruct head impacts. The head model includes tissues like skull, brainstem, cerebrospinal fluid, etc. The head model was impacted in the frontal, occipital, parietal or lateral direction with different impact velocities. Acceleration signals of the head model were extracted directly from the skull and the head centroid node. To obtain a robust HIC, the filtering class of acceleration signals were analyzed carefully. Then, the relation between rigid body HIC and the centroid node HIC were studied systematically. RESULTS: When the filtering class of rigid body acceleration and centroid node acceleration reached the cut-off frequency, the corresponding derivative of HIC tended to change smoothly. Using these cut-off frequencies, robust HICs were obtained. The rigid body HIC far exceeded that of centroid node HIC, such as 8, 9, 14 and 31 times exceeded in the frontal, occipital, parietal and lateral impact conditions, respectively. Moreover, approximate linear relations were found between the rigid body HIC and the centroid node HIC in different impact directions, respectively. From these relations, the injury thresholds of rigid body HIC of various directions were given quantitatively. CONCLUSIONS: The rational filtering class like CFC 800 and CFC 700 were given for rigid body HIC and centroid node HIC, respectively. The rigid body HIC had a significant discrepancy from the centroid node HIC. Linear relations between the rigid body HIC and centroid node HIC were found, and their slopes changed with impact directions. From these relations, we can adjust the injury thresholds reasonably if the head receives different impacts. These findings can effectively enhance the applicability of HIC.
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Aceleração , Traumatismos Craniocerebrais , Análise de Elementos Finitos , Humanos , Traumatismos Craniocerebrais/fisiopatologia , Fenômenos Biomecânicos , Simulação por Computador , Acidentes de TrânsitoRESUMO
BACKGROUND: Fibrosis is a tissue damage repair response caused by multiple pathogenic factors which could occur in almost every apparatus and leading to the tissue structure damage, physiological abnormality, and even organ failure until death. Up to now, there is still no specific drugs or strategies can effectively block or changeover tissue fibrosis. JNKs, a subset of mitogen-activated protein kinases (MAPK), have been reported that participates in various biological processes, such as genetic expression, DNA damage, and cell activation/proliferation/death pathways. Increasing studies indicated that abnormal regulation of JNK signal pathway has strongly associated with tissue fibrosis. AIM OF REVIEW: This review designed to sum up the molecular mechanism progresses in the role of JNK signal pathway in organ fibrosis, hoping to provide a novel therapy strategy to tackle tissue fibrosis. KEY SCIENTIFIC CONCEPTS OF REVIEW: Recent evidence shows that JNK signaling pathway could modulates inflammation, immunoreaction, oxidative stress and Multiple cell biological functions in organ fibrosis. Therefore, targeting the JNK pathway may be a useful strategy in cure fibrosis.
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Fms-like tyrosine kinase 3 (FLT3) has been validated as a therapeutic target for acute myeloid leukemia (AML). While a number of FLT3 kinase inhibitors have been approved for AML treatment, the clinical data revealed that they cannot achieve complete and sustained suppression of FLT3 signaling at the tolerated dose. Here we report a series of new, potent and selective FLT3 proteolysis targeting chimera degraders. The optimal compound LWY713 potently induced the degradation of FLT3 with a DC50 value of 0.64 nM and a Dmax value of 94.8% in AML MV4-11 cells with FLT3-internal tandem duplication (ITD) mutation. Mechanistic studies demonstrated that LWY713 selectively induced FLT3 degradation in a cereblon- and proteasome-dependent manner. LWY713 potently inhibited FLT3 signaling, suppressed cell proliferation, and induced cell G0/G1-phase arrest and apoptosis in MV4-11 cells. Importantly, LWY713 displayed potent in vivo antitumor activity in MV4-11 xenograft models.
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Leucemia Mieloide Aguda , Tirosina Quinase 3 Semelhante a fms , Humanos , Tirosina Quinase 3 Semelhante a fms/genética , Linhagem Celular Tumoral , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Mutação , Proliferação de Células , Apoptose , Leucemia Mieloide Aguda/patologiaRESUMO
Selective degradation of cyclin-dependent kinases 12 and 13 (CDK12/13) emerges as a new potential therapeutic approach for triple-negative breast cancer (TNBC) and other human cancers. While several proteolysis-targeting chimera (PROTAC) degraders of CDK12/13 were reported, none are orally bioavailable. Here, we report the discovery of ZLC491 as a potent, selective, and orally bioavailable CDK12/13 PROTAC degrader. The compound effectively degraded CDK12 and CDK13 with DC50 values of 32 and 28 nM, respectively, in TNBC MDA-MB-231 cells. Global proteomic assessment and mechanistic studies revealed that ZLC491 selectively induced CDK12/13 degradation in a cereblon- and proteasome-dependent manner. Furthermore, the molecule efficiently suppressed transcription and expression of long genes, predominantly a subset of genes associated with DNA damage response, and significantly inhibited proliferation of multiple TNBC cell lines. Importantly, ZLC491 achieved an oral bioavailability of 46.8% in rats and demonstrated potent in vivo degradative effects on CDK12/13 in an MDA-MB-231 xenografted mouse model.
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Antineoplásicos , Disponibilidade Biológica , Quinases Ciclina-Dependentes , Proteólise , Humanos , Animais , Quinases Ciclina-Dependentes/antagonistas & inibidores , Quinases Ciclina-Dependentes/metabolismo , Linhagem Celular Tumoral , Antineoplásicos/farmacologia , Antineoplásicos/química , Antineoplásicos/farmacocinética , Ratos , Proteólise/efeitos dos fármacos , Administração Oral , Proliferação de Células/efeitos dos fármacos , Camundongos , Feminino , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/patologia , Descoberta de Drogas , Ensaios Antitumorais Modelo de Xenoenxerto , Ratos Sprague-Dawley , Camundongos Nus , Relação Estrutura-Atividade , Proteína Quinase CDC2RESUMO
Cyclin-dependent kinases 12/13 play pivotal roles in orchestrating transcription elongation, DNA damage response, and maintenance of genomic stability. Biallelic CDK12 loss has been documented in various malignancies. Here, we develop a selective CDK12/13 PROTAC degrader, YJ9069, which effectively inhibits proliferation in subsets of prostate cancer cells preferentially over benign immortalized cells. CDK12/13 degradation rapidly triggers gene-length-dependent transcriptional elongation defects, leading to DNA damage and cell-cycle arrest. In vivo, YJ9069 significantly suppresses prostate tumor growth. Modifications of YJ9069 yielded an orally bioavailable CDK12/13 degrader, YJ1206, which exhibits comparable efficacy with significantly less toxicity. To identify pathways synthetically lethal upon CDK12/13 degradation, phosphorylation pathway arrays were performed using cell lines treated with YJ1206. Interestingly, degradation or genetic knockdown of CDK12/13 led to activation of the AKT pathway. Targeting CDK12/13 for degradation, in conjunction with inhibiting the AKT pathway, resulted in a synthetic lethal effect in preclinical prostate cancer models.
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Quinases Ciclina-Dependentes , Proteínas Proto-Oncogênicas c-akt , Mutações Sintéticas Letais , Humanos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Masculino , Quinases Ciclina-Dependentes/metabolismo , Quinases Ciclina-Dependentes/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Transdução de Sinais/efeitos dos fármacos , Camundongos , Proliferação de Células/efeitos dos fármacos , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/patologia , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Administração Oral , Proteólise/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Camundongos Nus , Dano ao DNA/efeitos dos fármacos , Disponibilidade Biológica , Proteína Quinase CDC2RESUMO
Biallelic loss of cyclin-dependent kinase 12 (CDK12) defines a unique molecular subtype of metastatic castration-resistant prostate cancer (mCRPC). It remains unclear, however, whether CDK12 loss per se is sufficient to drive prostate cancer development-either alone, or in the context of other genetic alterations-and whether CDK12-mutant tumors exhibit sensitivity to specific pharmacotherapies. Here, we demonstrate that tissue-specific Cdk12 ablation is sufficient to induce preneoplastic lesions and robust T cell infiltration in the mouse prostate. Allograft-based CRISPR screening demonstrated that Cdk12 loss is positively associated with Trp53 inactivation but negatively associated with Pten inactivation-akin to what is observed in human mCRPC. Consistent with this, ablation of Cdk12 in prostate organoids with concurrent Trp53 loss promotes their proliferation and ability to form tumors in mice, while Cdk12 knockout in the Pten-null prostate cancer mouse model abrogates tumor growth. Bigenic Cdk12 and Trp53 loss allografts represent a new syngeneic model for the study of androgen receptor (AR)-positive, luminal prostate cancer. Notably, Cdk12/Trp53 loss prostate tumors are sensitive to immune checkpoint blockade. Cdk12-null organoids (either with or without Trp53 co-ablation) and patient-derived xenografts from tumors with CDK12 inactivation are highly sensitive to inhibition or degradation of its paralog kinase, CDK13. Together, these data identify CDK12 as a bona fide tumor suppressor gene with impact on tumor progression and lends support to paralog-based synthetic lethality as a promising strategy for treating CDK12-mutant mCRPC.
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Biallelic loss of cyclin-dependent kinase 12 (CDK12) defines a metastatic castration-resistant prostate cancer (mCRPC) subtype. It remains unclear, however, whether CDK12 loss drives prostate cancer (PCa) development or uncovers pharmacologic vulnerabilities. Here, we show Cdk12 ablation in murine prostate epithelium is sufficient to induce preneoplastic lesions with lymphocytic infiltration. In allograft-based CRISPR screening, Cdk12 loss associates positively with Trp53 inactivation but negatively with Pten inactivation. Moreover, concurrent Cdk12/Trp53 ablation promotes proliferation of prostate-derived organoids, while Cdk12 knockout in Pten-null mice abrogates prostate tumor growth. In syngeneic systems, Cdk12/Trp53-null allografts exhibit luminal morphology and immune checkpoint blockade sensitivity. Mechanistically, Cdk12 inactivation mediates genomic instability by inducing transcription-replication conflicts. Strikingly, CDK12-mutant organoids and patient-derived xenografts are sensitive to inhibition or degradation of the paralog kinase, CDK13. We therein establish CDK12 as a bona fide tumor suppressor, mechanistically define how CDK12 inactivation causes genomic instability, and advance a therapeutic strategy for CDK12-mutant mCRPC.
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Quinases Ciclina-Dependentes , Neoplasias da Próstata , Mutações Sintéticas Letais , Masculino , Animais , Humanos , Quinases Ciclina-Dependentes/metabolismo , Quinases Ciclina-Dependentes/genética , Camundongos , Mutações Sintéticas Letais/genética , Neoplasias da Próstata/patologia , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteína Supressora de Tumor p53/genética , Progressão da Doença , PTEN Fosfo-Hidrolase/metabolismo , PTEN Fosfo-Hidrolase/genética , Instabilidade Genômica , Transcrição Gênica , Organoides/patologia , Organoides/metabolismo , Neoplasias de Próstata Resistentes à Castração/patologia , Neoplasias de Próstata Resistentes à Castração/genética , Neoplasias de Próstata Resistentes à Castração/metabolismo , Proliferação de Células/genética , Replicação do DNA/genética , Camundongos Knockout , Linhagem Celular Tumoral , Camundongos Endogâmicos C57BL , Proteína Quinase CDC2RESUMO
The analysis of exhaled breath has opened up new exciting avenues in medical diagnostics, sleep monitoring, and drunk driving detection. Nevertheless, the detection accuracy is greatly affected due to high humidity in the exhaled breath. Here, we propose a regulation method to solve the problem of humidity adaptability in the ethanol-monitoring process by building a heterojunction and hollow-out nanostructure. Therefore, large specific surface area hollow-out Fe2O3-loaded NiO heterojunction nanorods assembled by porous ultrathin nanosheets were prepared by a well-tailored interface reaction. The excellent response (51.2 toward 10 ppm ethanol at 80% relative humidity) and selectivity to ethanol under high relative humidity with a lower operating temperature (150 °C) were obtained, and the detection limit was as low as 0.5 ppb with excellent long-term stability. The superior gas-sensing performance was attributed to the high surface activity of the heterojunction and hollow-out nanostructure. More importantly, GC-MS, diffuse reflectance Fourier transform infrared spectroscopy, and DFT were utilized to analyze the mechanisms of heterojunction sensitization, ethanol-sensing reaction, and high-humidity adaptability. Our integrated low-power MEMS Internet of Things (IoT) system based on Fe2O3@NiO successfully demonstrates the functional verification of ethanol detection in human exhalation, and the integrated voice alarm and IoT positioning functions are expected to solve the problem of real-time monitoring and rapid initial screening of drunk driving. Overall, this novel method plays a vital role in areas such as control of material morphology and composition, breath analysis, gas-sensing mechanism research, and artificial olfaction.
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Nanoestruturas , Nanotubos , Humanos , Umidade , Expiração , Etanol/análise , Nanoestruturas/químicaRESUMO
Alginate lyases with strict substrate specificity possess potential in directed production of alginate oligosaccharides with specific composition. However, their poor thermostability hampered their applications in industry. In this study, an efficient comprehensive strategy including sequence-based analysis, structure-based analysis, and computer-aid ΔΔGfold value calculation was proposed. It was successfully performed on alginate lyase (PMD) with strict poly-ß-D-mannuronic acid substrate specificity. Four single-point variants A74V, G75V, A240V, and D250G with increased Tm of 3.94 °C, 5.21 °C, 2.56 °C, and 4.80 °C, respectively, were selected out. After ordered combined mutations, a four-point mutant (M4) was finally generated which displayed remarkable increase on thermostability. The Tm of M4 increased from 42.25 °C to 51.59 °C and its half-life at 50 °C was about 58.9-fold of PMD. Meanwhile, there was no obvious loss of enzyme activity (more than 90% retained). Molecular dynamics simulation analysis insisted that the improvement of thermostability might be attribute to the rigidified region A which might be caused by the newly formed hydrogen bonds and salt bridges introduced by mutations, the lower distance of original hydrogen bonds, and the more compact overall structures.
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Alginatos , Polissacarídeo-Liases , Polissacarídeo-Liases/química , Oligossacarídeos , Especificidade por Substrato , Temperatura , Estabilidade EnzimáticaRESUMO
Background: The imbalance of gut microbiota (GM) is associated with a higher risk of thrombosis in patients with atrial fibrillation (AF). Oral anticoagulants (OACs) have been found to significantly reduce the risk of thromboembolism and increase the risk of bleeding. However, the OAC-induced alterations in gut microbiota in patients with AF remain elusive. Methods: In this study, the microbial composition in 42 AF patients who received long-term OAC treatment (AF-OAC group), 47 AF patients who did not (AF group), and 40 volunteers with the risk of AF (control group) were analyzed by 16S rRNA gene sequencing of fecal bacterial DNA. The metagenomic functional prediction of major bacterial taxa was performed using the Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) software package. Results: The gut microbiota differed between the AF-OAC and AF groups. The abundance of Bifidobacterium and Lactobacillus decreased in the two disease groups at the genus level, but OACs treatment mitigated the decreasing tendency and increased beneficial bacterial genera, such as Megamonas. In addition, OACs reduced the abundance of pro-inflammatory taxa on the genus Ruminococcus but increased certain potential pathogenic taxa, such as genera Streptococcus, Escherichia-Shigella, and Klebsiella. The Subgroup Linear discriminant analysis effect size (LEfSe) analyses revealed that Bacteroidetes, Brucella, and Ochrobactrum were more abundant in the anticoagulated bleeding AF patients, Akkermansia and Faecalibacterium were more abundant in the non-anticoagulated-bleeding-AF patients. The neutrophil-to-lymphocyte ratio (NLR) was lower in the AF-OAC group compared with the AF group (P < 0.05). Ruminococcus was positively correlated with the NLR and negatively correlated with the CHA2DS2-VASc score (P < 0.05), and the OACs-enriched species (Megamonas and Actinobacteria) was positively correlated with the prothrombin time (PT) (P < 0.05). Ruminococcus and Roseburia were negatively associated with bleeding events (P < 0.05). Conclusions: Our study suggested that OACs might benefit AF patients by reducing the inflammatory response and modulating the composition and abundance of gut microbiota. In particular, OACs increased the abundance of some gut microbiota involved in bleeding and gastrointestinal dysfunction indicating that the exogenous supplementation with Faecalibacterium and Akkermansia might be a prophylactic strategy for AF-OAC patients to lower the risk of bleeding after anticoagulation.
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Fibrilação Atrial , Microbioma Gastrointestinal , Acidente Vascular Cerebral , Humanos , Fibrilação Atrial/complicações , Fibrilação Atrial/tratamento farmacológico , Fibrilação Atrial/induzido quimicamente , Filogenia , RNA Ribossômico 16S/genética , Fatores de Risco , Anticoagulantes/uso terapêutico , Hemorragia/induzido quimicamente , Hemorragia/complicações , Administração Oral , Medição de RiscoRESUMO
Various catalysts are developed to improve the performance of metal oxide semiconductor gas sensors, but achieving high selectivity and response intensity in chemiresistive gas sensors (CGSs) remains a significant challenge. In this study, an in situ-annealing approach to synthesize Cu catalytic sites on ultrathin WO2.72 nanowires for detecting toluene at ultralow concentrations (Ra /Rg = 1.9 at 10 ppb) with high selectivity is developed. Experimental and molecular dynamic studies reveal that the Cu single atoms (SAs) act as active sites, promoting the oxidation of toluene and increasing the affinity of Cu single-atom catalysts (SACs)-containing sensing materials for toluene while weakening the association with carbon dioxide or water vapor. Density functional theory studies show that the selective binding of toluene to Cu SAs is due to the favorable binding sites provided by Cu SAs for toluene molecules over other gaseous species, which aids the adsorption of toluene on WO2.72 nanowires. This study demonstrates the successful atomic-level interface regulation engineering of WO2.72 nanowire-supported Cu SAs, providing a potential strategy for the development of highly active and durable CGSs.
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The demand for gas sensors that can detect gases selectively at low temperatures has increased steadily over recent years. Most devices use semiconducting metal oxides as sensing materials which often require high operation temperatures and suffer from a lack of selectivity. Semiconducting metal sulfides were found to be a reasonable alternative for the application in sensing devices at low temperatures. Since metal sulfides are a relatively new class of materials applied in gas sensors, there is little work on sensing mechanisms and overall sensing characteristics of these materials. In this work, the authors investigated the sensing performance of Bi2S3 nanorods operated at 50 °C in the presence of several target gases and found a selective response to oxidizing gases. With the help of DC resistance measurements, diffuse reflectance infrared Fourier transform spectroscopy and work function measurements in a Kelvin Probe setup, the NO2 and O3 sensing mechanisms of Bi2S3 nanorods were revealed. While initially sulfur vacancies were the predominant reaction sites, the formation of nitrates became the key reaction in higher NO2 concentrations. Additionally, it was found that the reaction with O3 healed sulfur vacancies effectively inhibiting the reaction with NO2.
RESUMO
Alginate lyases (ALyases) have been widely applied in enzymatically degrading alginate for the preparation of alginate oligosaccharides (AOS), which possess a range of excellent physiological benefits including immunoregulatory, antivirus, and antidiabetic properties. Among the characterized ALyases, the number of ALyases with strict substrate specificity which possess potential in directed preparation of AOS is quite small. ALyases of polysaccharides lyase (PL) 5 family have been reported to perform poly-ß-D-mannuronic acid (Poly-M) substrate specificity. However, there have been fewer studies with a comprehensive characterization and comparison of PL 5 family ALyases. In this study, a putative PL 5 family ALyase PMD was cloned from Pseudomonas mendocina and expressed in Escherichia coli. The novel ALyase presented maximum activity at 30 °C and pH 7.0. PMD displayed pH stability properties under the range of pH 5 to pH 9, which retained more than 80% relative activity, even when incubated for 48 h. Product analysis indicated that PMD might be an endolytic ALyase with strict Poly M substrate specificity and yield disaccharide and trisaccharide as main products. In addition, residues K58, R66, Y248, and R344 were proposed to be the potential key residues for catalysis via site-directed mutation. Detailed characterization of PMD and comprehensive comparisons could supply some different information about properties of PL 5 ALyases which might be helpful for its application in the directed production of AOS.