Multi-strategy orthogonal enhancement and analysis of aldo-keto reductase thermal stability.

Given their outstanding efficiency and selectivity, enzymes are integral in various domains such as drug synthesis, the food industry, and environmental management. However, the inherent instability of natural enzymes limits their widespread industrial application. In this study, we underscore the efficacy of enhancing protein thermal stability through comprehensive protein design strategies, encompassing elements such as the free energy of protein folding, internal forces within proteins, and the overall structural design. We also demonstrate the efficiency and precision of combinatorial screening in the thermal stability design of aldo-keto reductase (AKR7-2-1). In our research, three single-point mutations and five combinatorial mutations were strategically introduced into AKR7-2-1, using multiple computational techniques. Notably, the E12I/S235I mutant showed a significant increase of 25.4 °C in its melting temperature (Tm). Furthermore, the optimal mutant, E12V/S235I, maintained 80 % of its activity while realizing a 16.8 °C elevation in Tm. Remarkably, its half-life at 50 °C was increased to twenty times that of the wild type. Structural analysis indicates that this enhanced thermal stability primarily arises from reduced oscillation in the loop region and increased internal hydrogen bonding. The promising results achieved with AKR7-2-1 demonstrate that our strategy could serve as a valuable reference for enhancing the thermal stability of other industrial enzymes.

Ascertaining appropriate measuring methods to determine tire wear particle pollution on driving school grounds in China.

Tire wear particles (TWPs) are garnering increasing attention due to their potential adverse environmental impacts. However, precisely ascertaining TWPs content is challenging due to the complexity and variability of the tire components used in the environment, indicating that more reliable methods to accurately determine TWPs are necessary. In this study, driving school grounds were used as a case study to ascertain an appropriate and reliable method to determine TWPs levels based on a comprehensive comparison between different analytical results using styrene butadiene rubber (SBR), N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine (6PPD), and zinc (Zn) as analytical markers. Thermogravimetric analysis-Gas chromatography mass spectrometry (TGA-GC-MS) method reliability using SBR was verified and applied to measure TWPs levels on driving school grounds. By reliably converting SBR content to TWPs content, the average TWPs content on driving school grounds was measured at 190.13 ± 101.89 mg/g. The highest TWPs content was 281.83 ± 171.44 mg/g under the reverse stall parking driving programs, while the slope start and stop driving programs was lower at 208.36 ± 124.11 mg/g. Our findings highlight the importance of accurately determining TWPs content within specific environments while comprehensively exploring associated patterns of change to better understand the environmental risks of TWPs.

Specific coenzyme preference switching for an aldo-keto reductase that synthesizes the chiral intermediate of duloxetine.

The synthesis of chiral intermediates for the traditional antidepressant duloxetine has gained significant attention as the number of depressed patients continues to grow. S-N, N-Dimethyl-3-hydroxy-3-(2-thienyl)-1-propanamide (S-DHTP) is a critical intermediate in the synthesis of duloxetine, and the chemical synthesis process is complex and environmentally unfriendly. Reduced nicotinamide adenine dinucleotide phosphate (NADPH) is a major cost driver in the biocatalytic production of S-DHTP from N, N-Dimethyl-3-keto-3-(2-thienyl)-1-propanamide (DKTP). Here, we successfully modified the coenzyme preference of an aldo-keto reductase (AKR7-2-1) to use the cheaper reduced nicotinamide adenine dinucleotide (NADH) through a coenzyme preference modification approach. We utilized protein engineering to create a superior mutant, Y53F, which increased the coenzyme specificity of AKR7-2-1 by 875-fold and improved its thermal stability, enhancing its potential for industrial applications. Molecular dynamics simulations were performed to demonstrate the effect of mutations at key sites on the protein, revealing the altered coenzyme preference and increased thermal stability from structural and energetic changes. This study validates the viability of the coenzyme preference modification strategy for aldo-keto reductase, offering valuable insights for fellow researchers and guiding future investigations.

Construction of Immobilized Enzymes with Yeast and Metal-Organic Frameworks for Enhanced Biocatalytic Activities.

Metal-organic frameworks (MOFs) have become promising host materials for enzyme immobilization and protection. Herein, ZIF-8 nanocubes were successfully self-assembled onto yeast as a biological template to obtain hybrid Y@ZIF-8. The size, morphology, and loading efficiency of ZIF-8 nanoparticles assembled on yeast templates can be well-regulated by adjusting the various synthetic parameters. Particularly, the amount of water significantly affected the particle size of ZIF-8 assembled on yeast. Through using a cross-linking agent, the relative enzyme activity of Y@ZIF-8@t-CAT could be greatly enhanced and remained the highest even after seven consecutive cycles, with improved cycling stability, as compared to that of Y@ZIF-8@CAT. In addition to the effect of the physicochemical properties of Y@ZIF-8 on the loading efficiency, the temperature tolerance, pH tolerance, and storage stability of Y@ZIF-8@t-CAT were also systematically investigated. Importantly, the catalytic activity of free catalase was decreased to 72% by 45 days, while the activity of the immobilized catalase remained above 99%, suggesting good storage stability. The present work demonstrates that yeast-templated ZIF-8 nanoparticles have a high potential to be used as biocompatible immobilization materials and are promising candidates for the preparation of effective biocatalysts in biomedicine applications.

The Differences of Nutrient Components in Edible and Feeding Coix Seed at Different Developmental Stages Based on a Combined Analysis of Metabolomics.

Coix lachryma-jobi L. is an excellent plant resource that has a concomitant function for medicine, foodstuff and forage in China. At present, the commonly used cultivar for both medicine and foodstuff is Xiaobaike, and the cultivar for foraging is Daheishan. However, differences in the internal composition of plants lead to the expression of different phenotypic traits. In order to comprehensively elucidate the differences in nutrient composition changes in Coix seeds, a non-targeted metabolomics method based on ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (LC-Q-TOF-MS) was used to analyze the metabolic changes in Coix seeds at different developmental stages. An edible Coix relative (Xiaobaike) and a feeding Coix relative (Daheishan) were selected as the research subjects. In the metabolome analysis of Coix seed, 314 metabolites were identified and detected, among which organic acids, carbohydrates, lipids, nucleotides and flavonoids were the main components. As an important standard for evaluating the quality of Coix seed, seven lipids were detected, among which fatty acids included not only even-chain fatty acids, but also odd-chain fatty acids, which was the first time detecting a variety of odd-chain fatty acids in Coix seed. The analysis of the compound contents in edible and feeding-type Coix lachryma-jobi L. and the lipid content at the mature stage showed that, among them, arachidic acid, behenic acid, heptadecanoic acid, heneicosanoic acid and pristanic acid may be the key compounds affecting the lipid content. In addition, in the whole process of semen coicis maturation, edible and feeding Coix show similar trends, and changes in the third period show clear compounds in the opposite situation, suggesting that edible and feeding Coix not only guarantee the relative stability of species but also provide raw materials for genetic breeding. This study provides valuable information on the formation of the edible and medicinal qualities of Coix.

A Minireview for Recent Development of Nanomaterial-Based Detection of Antibiotics.

Antibiotics are considered a new type of organic pollutant. Antibiotic residues have become a global issue due to their harm to human health. As the use of antibiotics is increasing in human life, such as in medicine, crops, livestock, and even drinking water, the accurate analysis of antibiotics is very vital. In order to develop rapid and on-site approaches for the detection of antibiotics and the analysis of trace-level residual antibiotics, a high-sensitivity, simple, and portable solution is required. Meanwhile, the rapid nanotechnology development of a variety of nanomaterials has been achieved. In this review, nanomaterial-based techniques for antibiotic detection are discussed, and some reports that have employed combined nanomaterials with optical techniques or electrochemical techniques are highlighted.

Fabrication of supported Pt/CeO2 nanocatalysts doped with different elements for CO oxidation: theoretical and experimental studies.

Supported Pt/CeO2 catalysts have been widely used in carbon monoxide (CO) oxidation; however, the high oxygen vacancy formation energy (Evac) in the process leads to the poor performance of these catalysts. Herein, we explored different element (Pr, Cu, or N) doped CeO2 supports using Ce-based metal-organic frameworks (MOFs) as precursors via calcination treatment. The obtained CeO2 supports were used to load Pt nanoparticles. These catalysts were systematically characterized by various techniques, and they showed superior catalytic activity for CO oxidation compared to undoped catalysts which could be attributed to the formation of Ce3+, and high amounts of Oads/(Oads + Olat) and Ptδ+/Pttotal. Moreover, density functional theory calculations with on-site Coulomb interaction correction (DFT+U) were performed to provide atomic-scale insights into the reaction process by the Mars-van Krevelen (M-vK) mechanism, which revealed that the element-doped catalysts could simultaneously reduce the adsorption energies of CO and lower reaction energy barriers in the *OOCO associative pathway.

Afterglow Electrochemiluminescence from Nitrogen-Deficient Graphitic Carbon Nitride.

Almost all current electrochemiluminescent reagents require real-time electrochemical stimulation to emit light. Here, we report a novel electrochemiluminescent reagent, nitrogen-deficient graphitic carbon nitride (CNx), that can emit afterglow electrochemiluminescence (ECL) after cessation of electric excitation. CNx obtained by post-thermal treatment of graphitic carbon nitride (CN) with KSCN has a cyanamide group and a nitrogen vacancy, which created defects to trap electrically injected electrons. The trapped electrons can slowly release and react with coreactants to emit light with longevity. The cathodic afterglow ECL lasts for 70 s after pulsing the CNx nanosheet (CNxNS-1.6)-modified glassy carbon electrode at -1.0 V for 20 s in 2.0 M PBS containing 1 mM K2S2O8. The afterglow ECL mechanism is revealed by investigation of its influencing factors and ECL wavelength. The discovery of afterglow ECL may open a new doorway for new significant applications of the ECL technique and provide a deeper understanding of the structure-property relationships of CN.

Tire wear particles: Trends from bibliometric analysis, environmental distribution with meta-analysis, and implications.

Tire wear particles (TWPs), as one of pristine microplastics and non-exhaust emission pollutants, have received extensive attention from scholars worldwide in recent years. In the context of the increasing number of related research results, this study evaluated the current status of TWPs research based on bibliometric analysis and meta-analysis and then discussed in-depth the environmental implications involving transport, transformation of released additives in potential and combined pollution with other microplastics in TWPs researches. Results showed that the regional layout of TWPs research was mainly concentrated in Europe and North America, but with specific countries of the United States, Germany, China, the United Kingdom, and Sweden. Thus, Asia and Africa should timely carry out related research on TWPs considering their large vehicle ownerships. In addition, keyword co-occurrence analysis based on CiteSpace showed that biotoxicity, environmental distribution and human health risks are the current research hotspots. Furthermore, the content of TWPs varied greatly by country and environmental media according to the meta-analysis. It is warranted to be urgently investigated on the distribution, quantitative analysis, migration, additives transformation with toxic effects and control measures of TWPs under the influence of various complex factors such as energy innovation and smart driving. The obtained findings can help understand the developing status of TWPs and then promoting their related investigations in future.

Machine learning application in forecasting tire wear particles emission in China under different potential socioeconomic and climate scenarios with tire microplastics context.

Little information is available on different contribution of TMPs from tire wear particles (TWPs), recycled tire crumbs (RTCs) and tire repair-polished Debris (TRDs) in the environment at national scale and their potential tendency. In this study, the TWPs were predicted using machine learning method of CNN (Convolutional Neural Networks) algorithms under different potential socioeconomic and climate scenarios based on the estimation of TMPs in China. Results showed that TWPs emission exhibited the most important part of TMPs, followed by RTCs and TRDs in China. The three mentioned tire microplastics largely distributed in Chinese coastal provinces. After machine learning applied in CNN using the dataset of estimated emission of TWPs from 2008 to 2018, the express delivery volume and education funding at the current increased rate would not have significant impacts on TWPs emissions; Additionally, TWPs emissions were also sensitive to changes of economic and transportation development; Low temperature conditions would further promote TWPs emissions. Accordingly, the rational development of logistics and green economy, the equilibrium improvement of education quality, and the increase of public traffic with new energy would be helpful to mitigate TWPs emissions. The obtained findings can enhance the understanding TMPs emission at particular scale and their corresponding precise management.

A simple integrated microfluidic platform for the research of hydrogels containing gradients in cell density induced breast cancer electrochemotherapy.

Cell density is important for tumour metastasis, treatment and prognosis. Characterizing changes in cell density for electrochemotherapy (ECT) can reveal sub-populations in pathological states, and adjust treatment program. In this work, a simple and convenient microfluidic platform was developed to study the effect cell density on ECT by integrating the improved cell gradient generator, cell culture chamber and indium tin oxide interdigital electrodes. Agarose, as extracellular matrix (ECM), was used to 3D cell culture to imitate in vivo microenvironment. The precision and reproducibility of cell density gradient with agarose solution were achieved because the hydrophobic modification of microchannels surface resulted in reducing cell adhesion and residue. ECT cytotoxicity assay with difference in cell densities was studied. The results showed that tumour cell density is one of the most factors for ECT treatment and ECT cytotoxicity has a certain of cell density-depended. But only electroporation on low cell density level, ECM would be one of the most key factors for ECT cytotoxicity, which would provide a new idea for chip-based cell assay in clinical diagnosis and drug screening in ordinary laboratories.

Effect of the problem-based learning method on student electroencephalograms and microcirculatory blood perfusion in the teaching of sports physiology / Efecto del método de aprendizaje basado en problemas en electroencefalogramas y perfusión sanguínea microcirculatoria de estudiantes en la enseñanza de la fisiología del deporte / Efeito do método de aprendizagem baseada em problemas em eletroencefalogramas e perfusão sanguínea microcirculatória de estudantes no ensino de fisiologia do esporte

ABSTRACT Introduction Applying the problem-based learning (PBL) method to the teaching of sports physiology. Objective This study explored the mechanism of the PBL method to improve the interest and learning effectiveness of students. Methods Twenty male students at the Physical Education College of Hubei Minzu University were randomly divided into a PBL group (10) and a traditional teaching method group (TTM). During the test, the subjects in the TTM group sat quietly listening to the experienced teacher, while the subjects in PBL group sat quietly and participated in a 20-minute group discussion under the guidance of the experienced teacher. Transcutaneous partial pressure of oxygen (TcPO2), microcirculatory blood perfusion (MBP), and alpha- and beta-band power were monitored at the beginning of and during the test. Results The mean of the PBL-group quiz score was significantly higher than that of the TTM group. In the PBL group, the alpha power of the students decreased statistically in the F3, T3, P3, and O1 channels and the beta power of the students increased statistically in the F7, F3, T3, C3, P3, and O1 as compared to the baseline values. Conclusion PBL can be an effective learning mechanism, since the students are actively engaged in the teaching of sports physiology. Level of Evidence I; Therapeutic studies - Investigating treatment results.

RESUMEN Introducción Aplicación del método de Aprendizaje Basado en Problemas (PBL), a la enseñanza de la fisiología del deporte. Objetivos Este estudio exploró el mecanismo del método PBL para ampliar el interés y la eficacia del aprendizaje de los estudiantes. Métodos Veinte estudiantes varones de la Facultad de Educación Física de la Universidad de Hubei Minzu fueron divididos aleatoriamente en el grupo PBL (10) y en un grupo de método de enseñanza tradicional (TTM). Durante la prueba, los participantes del grupo TTM permanecieron sentados y escuchando en silencio al profesor experimentado, mientras que los del grupo PBL permanecieron sentados y participaron en un debate de grupo de 20 minutos de acuerdo con la orientación del mismo profesor. La presión parcial de oxígeno transcutáneo (TcpO2), la perfusión sanguínea microcirculatoria (MBP) y la potencia de las bandas alfa y beta se monitorizaron al inicio y durante la prueba. Resultados La puntuación media del cuestionario del grupo PBL fue significativamente mayor que la del grupo TTM. En el grupo PBL, la potencia alfa de los estudiantes disminuyó estadísticamente en los canales F3, T3, P3 y O1 y la potencia beta de los estudiantes aumentó en los canales F7, F3, T3, C3, P3 y O1 en comparación con los valores de referencia. Conclusión El PBL puede ser un mecanismo de aprendizaje eficaz, ya que los estudiantes participan activamente en la enseñanza de la fisiología del deporte. Nivel de evidencia I; Estudios terapéuticos - Investigación de los resultados del tratamiento.

RESUMO Introdução Aplicação do método de aprendizagem baseada em problemas (PBL) ao ensino da fisiologia do esporte. Objetivos Este estudo explorou o mecanismo do método PBL para ampliar o interesse e a eficácia da aprendizagem dos estudantes. Métodos Vinte estudantes do sexo masculino da Faculdade de Educação Física da Universidade Hubei Minzu foram divididos randomicamente em um grupo PBL (10) e um grupo de método de ensino tradicional (TTM). Durante o teste, os participantes do grupo TTM ficaram sentados e em silêncio ouvindo o professor experiente, enquanto os do grupo PBL ficaram sentados e participaram de uma discussão em grupo de 20 minutos de acordo com a orientação do mesmo professor. A pressão parcial do oxigênio transcutâneo (TcPO2), a perfusão sanguínea microcirculatória (MPB) e a potência das bandas alfa e beta foram monitoradas no início e durante o teste. Resultados A média do escore do questionário do grupo PBL foi significativamente maior do que a do grupo TTM. No grupo PBL, o poder alfa dos estudantes diminuiu em termos estatísticos nos canais F3, T3, P3 e O1 e o poder beta dos estudantes aumentou nos canais F7, F3, T3, C3, P3 e O1 em comparação com os valores basais. Conclusão O PBL pode ser um mecanismo eficaz de aprendizagem, uma vez que os estudantes ficam ativamente engajados no ensino da fisiologia do esporte. Nível de Evidência I; Estudos terapêuticos - Investigação dos resultados do tratamento.

Hierarchically Annular Mesoporous Carbon Derived from Phenolic Resin for Efficient Removal of Antibiotics in Wastewater.

Antibiotics have become a new type of environmental pollutant due to their extensive use. High-performance adsorbents are of paramount significance for a cost-effective and environmentally friendly strategy to remove antibiotics from water environments. Herein, we report a novel annular mesoporous carbon (MCN), prepared by phenolic resin and triblock copolymer F127, as a high-performance adsorbent to remove penicillin, streptomycin, and tetracycline hydrochloride from wastewater. The MCNs have high purity, rich annular mesoporosity, a high surface area (605.53 m2/g), and large pore volume (0.58 cm3/g), improving the adsorption capacity and facilitating the efficient removal of penicillin, streptomycin, and tetracycline hydrochloride from water. In the application of MCNs to treat these three kinds of residual antibiotics, the adsorption amounts of tetracycline hydrochloride were higher than penicillin and streptomycin, and the adsorption capacity was up to 880.6 mg/g. Moreover, high removal efficiency (99.6%) and excellent recyclability were achieved. The results demonstrate that MCN adsorbents have significant potential in the treatment of water contaminated with antibiotics.

CD109 Is a Critical Determinant of EGFR Expression and Signaling, and Tumorigenicity in Squamous Cell Carcinoma Cells.

(1) Background: Squamous cell carcinoma (SCC) is one of the leading causes of cancer-related deaths worldwide. CD109 is overexpressed in many cancers including SCC. Although a pro-tumorigenic role for CD109 has been shown in non-SCC cancers, and in one type of SCC, the mechanisms and signaling pathways reported are discrepant. (2) Methods: The CD109-EGFR interaction and CD109-mediated regulation of EGFR expression, signaling, and stemness were studied using microarray, immunoblot, immunoprecipitation, qPCR, immunofluorescence, and/or spheroid formation assays. The role of CD109 in tumor progression and metastasis was studied using xenograft tumor growth and metastatic models. (3) Results: We establish the in vivo tumorigenicity of CD109 in vulvar SCC cells and demonstrate that CD109 is an essential regulator of EGFR expression at the mRNA and protein levels and of EGFR/AKT signaling in vulvar and hypopharyngeal SCC cells. Furthermore, we show that the mechanism involves EGFR-CD109 heteromerization and colocalization, leading to the stabilization of EGFR levels. Additionally, we demonstrate that the maintenance of epithelial morphology and in vitro tumorigenicity of SCC cells require CD109 localization to the cell surface. (4) Conclusions: Our study identifies an essential role for CD109 in vulvar SCC progression. We demonstrate that CD109 regulates SCC cellular stemness and epithelial morphology via a cell-surface CD109-EGFR interaction, stabilization of EGFR levels and EGFR/AKT signaling.

Tumor Cell-Specific and Lipase-Responsive Delivery of Hydrogen Sulfide for Sensitizing Chemotherapy of Pancreatic Cancer.

The inability of small molecule drugs to diffuse into tumor interstitium is responsible for the relatively low effectiveness of chemotherapy. Herein, a hydrogen sulfide (H2S) gas-involved chemosensitization strategy is proposed for pancreatic cancer treatment by developing a tumor-specific lipase-responsive nanomedicine based on aptamer-conjugated DATS/Dox co-loaded PCL-b-PEO micelle (DA/D@Ms-A). After receptor-mediated endocytosis and subsequent digestion of PCL blocks by intracellular lipase, the nanomedicine releases Dox and DATS, which then react with intracellular glutathione to produce H2S. The cytotoxicity result indicates that H2S can enhance Dox chemotherapy efficiency owing to the synergetic therapeutic effect of Dox and H2S. Moreover, the nanomedicine is featured with well tumor penetration capability benefitting from the targeting ability of aptamers and high in vivo biocompatibility due to the high density of PEO and biodegradable PCL. The nanomedicine capable of synergetic gas-chemotherapy holds great potential for pancreatic cancer treatment.

Visible light-induced selenylative spirocyclization of biaryl ynones toward the formation of selenated spiro[5.5]trienones.

A visible-light induced dearomative cascade cyclization of biaryl ynones with diselenides under photocatalyst and external additive-free conditions has been explored, giving a series of selenated spiro[5.5]trienones in moderate to good yields. The Se-Se bond in diselenides could be cleaved to generate arylselenyl radicals under visible light irradiation in the absence of a photocatalyst. This protocol provides a facile and green method for the synthesis of spiro[5.5]trienones.

Enhancing the biocatalytic synthesis of chiral drug intermediate by rational design an aldo-keto reductase from Bacillus megaterium YC4-R4.

In recent years, with the increasing number of patients with depression, the efficient synthesis of the first-line antidepressant drug duloxetine intermediate (S-N,N-dimethyl-3-hydroxy-3-(2-thienyl)-1-propanamine, S-DHTP) has attracted great attention. The wild-type AKR3-2-9 from Bacillus megaterium YC4-R4 exhibits high application potential of catalyzing N,N-dimethyl-3-keto-3-(2-thienyl)-1-propanamine (DKTP) to prepare S-DHTP, but there is still much room for improvement. In this work, rational design was carried out to enhance the catalytic potential of AKR3-2-9. Notably, compared to the wild-type AKR3-2-9, three mutants (Ile189Val, Asn256Asp, and Ile189Val + Asn256Asp) were obtained, and their catalytic efficiencies were increased by 1.3 times, 2.3 times, and 1.31 times, respectively. Besides, the thermal stability and organic solvent resistance were improved. More importantly, when the concentration of the substrate DKTP was 0.5 g/L, the catalytic yields of Ile189Val, Asn256Asp and Ile189Val + Asn256Asp were increased by 1.45 times, 1.86 times, and 2.05 times, respectively. Besides, the corresponding optical purities of the three mutants were 92.7 %, 94.3 % and 93.8 %. The above results indicated that the rational design of the AKR of Bacillus megaterium YC4-R4 enhanced its potential for biocatalytic preparation of S-DHTP.

CXCR4 knockdown enhances sensitivity of paclitaxel via the PI3K/Akt/mTOR pathway in ovarian carcinoma.

Epithelial ovarian cancer (EOC) is the deadliest gynecological malignancy. EOC control remains difficult, and EOC patients show poor prognosis regarding metastasis and chemotherapy resistance. The aim of this study was to estimate the effect of CXCR4 knockdown-mediated reduction of cancer stem cells (CSCs) and epithelial-mesenchymal transition (EMT) stemness and enhancement of chemotherapy sensitivity in EOC. Mechanisms contributing to these effects were also explored. Our data showed distinct contribution of CXCR4 overexpression by dependent PI3K/Akt/mTOR signaling pathway in EOC development. CXCR4 knockdown resulted in a reduction in CSCs and EMT formation and enhancement of chemotherapy sensitivity in tumor cells, which was further advanced by blocking CXCR4-PI3K/Akt/mTOR signaling. This study also documented the critical role of silencing CXCR4 in sensitizing ovarian CSCs to chemotherapy. Thus, targeting CXCR4 to suppress EOC progression, specifically in combination with paclitaxel (PTX) treatment, may have clinical application value.