Enzyme Activity Assays Research Articles

Carcass decay inhibits denitrification indirectly by regulating the microbiota and physicochemical properties in a model water system

Abstract Animal carcass decomposition is involved in nitrogen (N) cycle in the aquatic ecosystems, for example denitrification, volatilization and leaching. The denitrification process is a crucial part of the nitrogen cycle, helping to alleviate biological toxicity resulting from nitrate accumulation. However, it is still unknown whether and how cadaver decay affects the denitrifiers and their activity in river water. In this study, six denitrifiers (encoding napA, narG, nirK, nirS, norB and nosZ, respectively) and denitrifying activity were investigated in a model water system with nitrogen pollution from corpse decomposition using high‐throughput sequencing, real‐time quantitative PCR and denitrifying enzyme activity (DEA) assay. Cadaver decay increased the quantity and altered the composition of denitrifying microbiota. Multiple regression on distance matrices revealed that corpse decay had a significantly greater impact on the denitrification communities than temperature. To our surprise, the DEA decreased in the contaminated water by cadavers. The multiple regression models revealed that the abundance of denitrifying taxa and the gene copies may jointly predict changes in DEA, with highest contribution coming from the norB‐type Hydrogenophaga. Further, the partial least squares path model (PLS‐PM) showed that corpse decay indirectly inhibited DEA via the denitrification community and water physicochemical properties. Synthesis and applications. This study provides important insights into how cadaver decomposition can reduce denitrification activity in water systems. These findings can assist us in scientifically managing and remediating polluted water bodies.

Discovery of α-Glucosidase/Acetylcholinesterase Inhibitors from a Traditional Herbal Prescription of Qi-Li-Qiang-Xin Capsule by Time-Based Fractionation and Enzymatic Activity Assay

Discovery of α-Glucosidase/Acetylcholinesterase Inhibitors from a Traditional Herbal Prescription of Qi-Li-Qiang-Xin Capsule by Time-Based Fractionation and Enzymatic Activity Assay

Toxicological response and bioaccumulation of strontium in Triticum durum (Nachit) and Triticum aestivum (Ibtissam)

The present study examines the toxicological effects and bioaccumulation of strontium (Sr2+) in two Moroccan wheat species, Triticum durum (Nachit) and Triticum aestivum (Ibtissam). Germination rates and seedling growth parameters were evaluated under Sr2+ concentrations of 0, 10, 100, and 1000 mM. Results revealed a significant decline in germination rates as Sr2+ concentrations increased, with T. aestivum showing higher sensitivity compared to T. durum. Low Sr2+ concentrations (10 mM) initially promoted stem growth in T. aestivum, but growth declined sharply at higher concentrations. Both species sustained growth at moderate Sr2+ concentrations (100 mM), but experienced a substantial reduction at 1000 mM, with T. durum demonstrating slightly better tolerance. Elevated Sr2+ concentrations notably affected root length and stem branching, indicating severe phytotoxic effects. Enzyme activity assays showed that at low Sr2+ concentration (10 mM), GST, CAT, POD, and SOD activities were similar to the control. At moderate concentrations (100 mM), GST and POD activities increased, suggesting enhanced detoxification. At high concentrations (1000 mM), all enzyme activities were significantly elevated, reflecting a robust antioxidative defense mechanism. This study provides valuable insights into the toxicological impacts and physiological responses of these wheat species to Sr2+ stress.

Genotype and Phenotype Correlation of the TPMT∗8 Allele in Thiopurine Metabolism

Thiopurine 6-mercaptopurine (6-MP) is metabolized by thiopurine methyl transferase (TPMT). TPMT genetic variation results in some individuals having reduced or absent TPMT enzyme activity. If these individuals take a full thiopurine dose, life-threatening adverse events can occur. Testing identifies patients with reduced or absent TPMT activity and is recommended prior to initiation of therapy. The TPMT*8 allele, defined by c.644G>A (p.Arg215His), is common among individuals of African ancestry (∼2.3% minor allele frequency), but is not included in genotyping recommendations due to its uncertain function. Here, a clinical TPMT enzyme activity assay was used to assess TPMT activity in red blood cells from 982 patients, including those with *1/*8 (n=22), *3A/*8 (n=1), and *3C/*8 (n=1) TPMT diplotypes. The average 6-MMP production (primary TPMT product measured clinically) was 3.08 ± 0.16 nmol/mL/hr for *1/*8 individuals, compared to 3.77 ± 0.03 nmol/mL/hr for normal metabolizers (p=0.0001) and 2.39 ± 0.06 nmol 6-MMP/mL/hr for intermediate metabolizers (p<0.0001). Individuals with a TPMT*1/*8 diplotype demonstrated reduced 6-MP metabolism between that of normal metabolizers and intermediate metabolizers, suggesting that TPMT*8 is a reduced function allele.

Molecular responses reveal that two glutathione S-transferase CsGSTU8s contribute to detoxification of glyphosate in tea plants (Camellia sinensis)

Tea plant (Camellia sinensis) is an important economical crop that frequently suffers from various herbicides, especially glyphosate. However, the molecular responses and regulatory mechanisms of glyphosate stress in tea plants remain poorly understood. Here, we reported a transcriptome dataset and identified large number of differentially expressed genes (DEGs) under glyphosate exposure. Next, two glutathione S-transferase genes (CsGSTU8–1 and CsGSTU8–2) that upregulated significantly were screened as candidate genes. Tissue-specific expression patterns showed that both CsGSTU8–1 and CsGSTU8–2 had extremely high expression levels in the roots and were predominantly localized in the nucleus and plasma membrane based on subcellular localization. Both were significantly upregulated at different time points under various stressors, including drought, cold, salt, pathogen infections, and SA treatments. An enzymatic activity assay showed that CsGSTU8–1 catalyzes the conjugation of glutathione with 2,4-dinitrochlorobenzene (CDNB). Functional analysis in yeast verified that the two genes significantly contributed to the detoxification of glyphosate, and CsGSTU8–1 had a stronger role in detoxification than CsGSTU8–2. Taken together, these findings provide insights into the molecular responses of tea plants to glyphosate and the functions of CsGSTU8s in glyphosate detoxification, which can be used as a promising genetic resource for improving herbicide resistance in tea cultivars.

Methanolic extracts of litchi (Litchi chinensis Sonn.): A novel approach of targeting glucose-6-phosphate dehydrogenase for liver cancer therapy

Cancer metabolism has emerged as a potential target for innovative therapeutic approaches in the treatment of cancer. Cancer metabolism has received much attention, particularly in relation to glucose metabolism. It has been observed that human malignancies have high levels of glucose-6-phosphate dehydrogenase (G6PD) activity which is an important enzyme of glucose metabolism. This overactivity is associated with the cell death and angiogenesis, highlighting its potential as a viable target for cancer treatment. This study was conducted to examine the methanolic extracts from the seeds, bark and leaves of litchi (Litchi chinensis Sonn.) in order to discover effective compounds targeting G6PD and potentially active entities against liver cancer. Plant extract screening for the target protein was carried out through enzymatic activity assay. The recombinant plasmid pET-24a-HmG6PD was expressed in E. coli (BL21-DE3) strain, then purified and assessed using metal affinity chromatography with Ni-NTA columns and SDS-PAGE. The cytotoxicity of plant extracts against liver cancer HepG2 cells was assessed using the MTT assay. All three extracts demonstrated significant inhibitory effects (>80% inhibition) against G6PD. They were then subjected to testing at various concentrations, and their IC50 values were subsequently determined. The extracts of litchi (leaf, IC50: 1.199 μg/mL; bark, IC50: 2.350 μg/mL; seeds, IC50: 1.238 μg/mL) displayed significant inhibition of G6PD activity at lower concentrations. Subsequently, the leaf extract of litchi was further assessed for its impact on HepG2 cell lines in a dose-dependent manner and exhibited strong potential as an inhibitor of cancer cell progression. Moreover, the results of acute toxicity study in mice revealed nontoxic effects of litchi leaf extract on hepatocytes. The results imply that Litchi chinensis leaf extract could be considered as a promising candidate for safer drug development in the treatment of liver cancer.

The interaction mechanisms of tetrahydrocurcumin on alcohol dehydrogenase: Insights from enzymatic activity assays, spectroscopic methods and computational techniques

Tetrahydrocurcumin (THC), a major metabolite of curcumin, is renowned for its potent antioxidant and anti-inflammatory properties, including preventing liver injury from alcohol. Alcohol dehydrogenase (ADH), an essential enzyme in alcohol metabolism, is significant in the development of alcohol-induced diseases. Hence, enzymatic activity assays, spectroscopic methods, and computational techniques were employed in this study to explore their interaction. The results indicated that THC can competitively inhibit ADH activity, as inferred from the kinetic parameter values. Meanwhile, THC spontaneously entered the substrate-binding cavity of ADH to form a binary complex, and surrounded by Thr178, Leu57, Ile318, Leu141, Glu68, Leu116, Phe93, Val294, and Asp49. The Ka value of this binding was 1.724 ± 0.017 × 104 M−1 at 298 K. This binding process was driven by hydrogen bonds and van der Waals forces; additionally, the π electrons in THC were significant for the stability of the ADH-THC complex. The presence of THC led to a decrease in the α-helix content of ADH, resulting in a looser secondary structure. Furthermore, molecular dynamic simulations revealed that Glu68, Leu116, Phe93, Val294, and Asp49 made significant contributions to the binding process. The inhibition of ADH catalytic activity may be primarily attributed to the interaction between THC and the Val294 residue.

Characterization of an engineered ACE2 protein for its improved biological features and its transduction into MSCs: A novel approach to combat COVID-19 infection

Transduced MSCs that express engineered ACE2 could be highly beneficial to combat COVID-19. Engineered ACE2 can act as decoy targets for the virus, preventing its entry into healthy lung cells. To this end, genetic engineering techniques were used to integrate the ACE2 gene into the MSCs genome. The MSCs were evaluated for proper expression and functionality. The mutated form of ACE2 was characterized using various techniques such as protein expression analysis, binding affinity against spike protein, thermal stability assessment, and enzymatic activity assays. The functionality of the mACE2 was assessed on SARS-CoV-2 using the virus-neutralizing test. The obtained results indicated that by introducing specific mutations in the ACE2 gene, the resulting mutant ACE2 had enhanced interaction with viral spike protein, its thermal stability was increased, and its enzymatic function was inhibited as a decoy receptor. Moreover, the mACE2 protein showed higher efficacy in the neutralization of the SARS-CoV-2. In conclusion, this study proposes a novel approach with potential benefits such as targeted drug delivery and reduced side effects on healthy tissues. These transduced MSCs can also be used in combination with other anti-COVID-19 treatments. Design of similar engineered biomolecules with desired properties could also be used to target other diseases.

HSPB1 alleviates acute-on-chronic liver failure via the P53/Bax pathway.

The mortality rate of acute-on-chronic liver failure (ACLF) remains significantly elevated; hence, this study aimed to investigate the impact of heat shock protein family B (small) member 1 (HSPB1) on ACLF in vivo and in vitro and the underlying mechanism. This study used the ACLF mouse model, and liver damage extent was studied employing Masson trichrome, hematoxylin and eosin (H&E), Sirius red staining, and serum biochemical indices. Similarly, hepatocyte injury in lipopolysaccharide (LPS)-induced L02 cells was evaluated using cell counting kit-8 assay, enzymatic activity, flow cytometry, and TUNEL assay, while the underlying mechanism was investigated using western blot. Results showed that the morphology of liver tissue in ACLF mice was changed and was characterized by cirrhosis, fibrosis, collagen fiber deposition, inflammatory cell infiltration, and elevated liver injury indices. Moreover, HSPB1 was upregulated in both ACLF patients and mice, where overexpressing HSPB1 was found to inhibit ACLF-induced liver damage. Similarly, the HSPB1 expression in LPS-treated L02 cell lines was also increased, where overexpressing HSPB1 was found to promote cell viability, inhibit liver injury-related enzyme activity, and suppress apoptosis. Mechanistic investigations revealed that HSPB1 was responsible for inhibiting p-P53 and Bax protein levels, where activated P53 counteracted HSPB1's effects on cellular behaviors. In conclusion, HSPB1 attenuated ACLF-induced liver injury in vivo and inhibited LPS-induced hepatocyte damage in vitro, suggesting that HSPB1 may be a novel target for ACLF therapy.

Monitoring Enzymatic Reaction Kinetics and Activity Assays in Confined Nanospace.

Enzyme-mediating biotransformations commonly occur in micro- and nanospace, which is crucial to maintain the essential biochemical processes and physiological functions in living systems. Probing enzyme-catalytic reactions in a biomimetic fashion remains challenging due to the lack of competent tools and methodology. Here, we show that studying enzymatic reaction kinetics can be readily achieved by a well-designed solid-state nanopore. Using tyrosine as a classical substrate, we quantitatively characterize the catalytic activity of tyrosinase (TYR) and tyrosine decarboxylase (TDC) in a nanoconfined space. Tyrosine was first immobilized in the nanopipette, wherein the active sites of tyrosine were left unoccupied. When successively exposed to TYR and TDC, a two-step cascade reaction can spontaneously take place. In this process, the surface wettability and charge of the nanopipette stemming from the catalytic products can sensitively regulate ion transport and ionic current rectification behavior, which were monitored by ionic current signal. In this biomimetic scenario, we obtained the enzymatic reaction kinetics of monophenyl oxidase that were not previously actualized in the conventional macroenvironment. Significantly, TYR showed higher enzyme activity, with a Km value of 1.59 mM, which was lower than that measured in a free and open space (with a Km of 3.01 mM). This suggests that tyrosine should be the most appropriate substrate of TYR, thus improving our understanding of tyrosine-associated biochemical reactions. This work offers an applicable technical platform to mimic enzyme-mediated biotransformations and biometabolisms.

Bacteroides fragilis alleviates necrotizing enterocolitis through restoring bile acid metabolism balance using bile salt hydrolase and inhibiting FXR-NLRP3 signaling pathway

ABSTRACT Necrotizing enterocolitis (NEC) is a leading cause of morbidity and mortality in premature infants with no specific treatments available. We aimed to identify the molecular mechanisms underlying NEC and investigate the therapeutic effects of Bacteroides fragilis on NEC. Clinical samples of infant feces, bile acid-targeted metabolomics, pathological staining, bioinformatics analysis, NEC rat model, and co-immunoprecipitation were used to explore the pathogenesis of NEC. Taxonomic characterization of the bile salt hydrolase (bsh) gene, enzyme activity assays, 16S rRNA sequencing, and organoids were used to explore the therapeutic effects of B. fragilis on NEC-related intestinal damage. Clinical samples, NEC rat models, and in vitro experiments revealed that total bile acid increased in the blood but decreased in feces. Moreover, the levels of FXR and other bile acid metabolism-related genes were abnormal, resulting in disordered bile acid metabolism in NEC. Taurochenodeoxycholic acid accelerated NEC pathogenesis and taurodeoxycholate alleviated NEC. B. fragilis displayed bsh genes and enzyme activity and alleviated intestinal damage by restoring gut microbiota dysbiosis and bile acid metabolism abnormalities by inhibiting the FXR-NLRP3 signaling pathway. Our results provide valuable insights into the therapeutic role of B. fragilis in NEC. Administering B. fragilis may substantially alleviate intestinal damage in NEC.

Tebuconazole exacerbates co-occurrence and horizontal transfer of antibiotic resistance genes

As one of the most widely used pesticides in the global fungicide market, tebuconazole has become heavily embedded in soil along with antibiotic resistance genes (ARGs). However, it remains unclear whether the selective pressure produced by tebuconazole affects ARGs and their horizontal transfer. In this experiment, we simulated a tebuconazole-contaminated soil ecosystem and observed changes in the abundance of ARGs and mobile genetic element (MGEs) due to tebuconazole exposure. We also established a plasmid RP4-mediated conjugative transfer system to investigate in depth the impact of tebuconazole on the horizontal transfer of ARGs and its mechanism of action. The results showed that under tebuconazole treatment at concentrations ranging from 0 to 10 mg/L, there was a gradual increase in the frequency of plasmid conjugative transfer, peaking at 10 mg/L which was 7.93 times higher than that of the control group, significantly promoting horizontal transfer of ARGs. Further analysis revealed that the conjugative transfer system under tebuconazole stress exhibited strong ability to form biofilm, and the conjugative transfer frequency ratio of biofilm to planktonic bacteria varied with the growth cycle of biofilm. Additionally, scanning electron microscopy and flow cytometry demonstrated increased cell membrane permeability in both donor and recipient bacteria under tebuconazole stress, accompanied by upregulation of ompA gene expression controlling cell membrane permeability. Furthermore, enzyme activity assays indicated significant increases in CAT, SOD activity, and GSH content in recipient bacteria under tebuconazole stress. Moreover, expression levels of transmembrane transporter gene trfAp as well as genes involved in oxidative stress and SOS response were found to be correlated with the frequency of plasmid conjugative transfer.

Enzymatic activity assays and functional component profiling reveals coordinated defense response of mungbean against Cercospora canescens during leaf spot disease

Enzymatic activity assays and functional component profiling reveals coordinated defense response of mungbean against Cercospora canescens during leaf spot disease

Integrating UHPLC-Q-TOF-MS/MS, network pharmacology, bioinformatics and experimental validation to uncover the anti-cancer mechanisms of TiaoPi AnChang decoction in colorectal cancer

Ethnopharmacological relevanceThe TiaoPi AnChang Decoction (TPACD), a Traditional Chinese Medicine (TCM) prescription based on Xiangsha Liujunzi Decoction, has demonstrated clinical efficacy as an adjuvant therapy for colorectal cancer (CRC) patients. However, its specific ingredients and potential mechanisms of action remain unclear. Aim of the studyTo identify the primary active ingredients of TPACD, their molecular targets, and potential mechanisms underlying the efficacy of TPACD in CRC treatment. Materials and methodsThis study investigated the clinically validated TCM formula TPACD. In vitro and in vivo experiments were used to demonstrate TPACD's regulatory effects on various malignant phenotypes of tumors, providing basic research support for its anti-cancer activity. To understand its pharmacodynamic basis, we utilized ultra-high performance liquid chromatography-quadrupole-time-of-flight-mass spectrometry/mass spectrometry (UHPLC-Q-TOF-MS/MS) to analyze TPACD constituents present in the bloodstream. Network pharmacology and bioinformatics analyses were used to identify potential active components and their molecular targets for TPACD's therapeutic effects in CRC. Subsequent experiments further elucidated its pharmacological mechanism. ResultsTPACD inhibits various malignant cellular processes, such as cell proliferation, apoptosis, migration, and invasion, and has shown potential anti-CRC activities both in vitro and in vivo. Following the identification of 109 constituents absorbed into the blood from TPACD, network pharmacology analysis predicted 42 potential anti-CRC targets. Clinical analyses highlighted three genes as prognostic key genes of TPACD's therapeutic action: C-X-C motif chemokine ligand 8 (CXCL8), fatty acid binding protein 4 (FABP4), and matrix metallopeptidase 3 (MMP3). Drug sensitivity analyses, molecular docking simulations and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) identified MMP3 as the most promising target for TPACD's anti-CRC action. Enzyme activity assays confirmed that TPACD inhibits MMP3 enzyme activity. Surface plasmon resonance (SPR) characterized the binding affinity between MMP3 and effective active components of TPACD, including luteolin, quercetin, kaempferol, and liensinine. ConclusionsTPACD exhibits anti-CRC activity in vitro and in vivo, with MMP3 identified as a critical target. The active compounds, including luteolin, quercetin, kaempferol, and liensinine, absorbed into the bloodstream, contribute to TPACD's efficacy by targeting MMP3.

Impact of Methyl Jasmonate on Terpenoid Biosynthesis and Functional Analysis of Sesquiterpene Synthesis Genes in Schizonepeta tenuifolia.

This study investigates the impact of methyl jasmonate (MeJA) on the volatile oil composition of Schizonepeta tenuifolia and elucidates the function of the StTPS45 gene, a key player in terpenoid biosynthesis. The effect of different concentrations of MeJA (0, 50, 100, 200, and 300 μmol/L) on the growth of S. tenuifolia adventitious bud clusters was analyzed over a 20 d period. Using gas chromatography-mass spectrometry (GC-MS), 17 compounds were identified from the adventitious bud clusters of S. tenuifolia. Significant changes in the levels of major monoterpenes, including increased contents of (+)-limonene and (+)-menthone, were observed, particularly at higher concentrations of MeJA. Analysis of transcriptome data from three groups treated with 0, 100, and 300 μmol/L MeJA revealed significant changes in the gene expression profiles following MeJA treatment. At 100 μmol/L MeJA, most terpene synthase (TPS) genes were overexpressed. Additionally, gene expression and functional predictions suggested that StTPS45 acts as germacrene D synthase. Therefore, StTPS45 was cloned and expressed in Escherichia coli, and enzyme activity assays confirmed its function as a germacrene D synthase. Molecular docking and structural prediction of StTPS45 further suggested specific interactions with farnesyl diphosphate (FPP), aligning with its role in the terpenoid synthesis pathway. These findings provide valuable insights into the modulation of secondary metabolite pathways by jasmonate signaling and underscore the potential of genetic engineering approaches to enhance the production of specific terpenoids in medicinal plants.

Mechanistic insights into the conversion of flavin adenine dinucleotide (FAD) to 8-formyl FAD in formate oxidase: a combined experimental and in-silico study

Formate oxidase (FOx), which contains 8-formyl flavin adenine dinucleotide (FAD), exhibits a distinct advantage in utilizing ambient oxygen molecules for the oxidation of formic acid compared to other glucose-methanol-choline (GMC) oxidoreductase enzymes that contain only the standard FAD cofactor. The FOx-mediated conversion of FAD to 8-formyl FAD results in an approximate 10-fold increase in formate oxidase activity. However, the mechanistic details underlying the autocatalytic formation of 8-formyl FAD are still not well understood, which impedes further utilization of FOx. In this study, we employ molecular dynamics simulation, QM/MM umbrella sampling simulation, enzyme activity assay, site-directed mutagenesis, and spectroscopic analysis to elucidate the oxidation mechanism of FAD to 8-formyl FAD. Our results reveal that a catalytic water molecule, rather than any catalytic amino acids, serves as a general base to deprotonate the C8 methyl group on FAD, thus facilitating the formation of a quinone-methide tautomer intermediate. An oxygen molecule subsequently oxidizes this intermediate, resulting in a C8 methyl hydroperoxide anion that is protonated and dissociated to form OHC-RP and OH−. During the oxidation of FAD to 8-formyl FAD, the energy barrier for the rate-limiting step is calculated to be 22.8 kcal/mol, which corresponds to the required 14-hour transformation time observed experimentally. Further, the elucidated oxidation mechanism reveals that the autocatalytic formation of 8-formyl FAD depends on the proximal arginine and serine residues, R87 and S94, respectively. Enzymatic activity assay validates that the mutation of R87 to lysine reduces the kcat value to 75% of the wild-type, while the mutation to histidine results in a complete loss of activity. Similarly, the mutant S94I also leads to the deactivation of enzyme. This dependency arises because the nucleophilic OH− group and the quinone-methide tautomer intermediate are stabilized through the noncovalent interaction provided by R87 and S94. These findings not only explain the mechanistic details of each reaction step but also clarify the functional role of R87 and S94 during the oxidative maturation of 8-formyl FAD, thereby providing crucial theoretical support for the development of novel flavoenzymes with enhanced redox properties.

Nanoplastics composite norfloxacin induced changes in conformation and function of lysozyme and differential effects of co-exposure contamination

The concurrent environmental contamination by nanoplastics (NPs) and norfloxacin (NOR) is a burgeoning concern, with significant accumulations in various ecosystems and potential ingress into the human body via the food chain, posing threats to both public health and ecological balance. Despite the gravity of the situation, studies on the co-exposure contamination effects of these substances are limited. Moreover, the response mechanisms of key functional proteins to these pollutants are yet to be fully elucidated. In this work, we conducted a comprehensive assessment of the interaction mechanisms of NPs and NOR with lysozyme under both single and co-exposure condition, utilizing dynamic light scattering, ζ-potential measurements, multi-spectroscopy methods, enzyme activity assays and molecular docking, to obtain a relationship between the compound effects of NPs and NOR. Our results indicate that NPs adsorb NOR on their surface, forming more stable aggregates. These aggregates influence the conformation, secondary structure (α-Helix ratio decreased by 3.1 %) and amino acid residue microenvironment of lysozyme. And changes in structure affect the activity of lysozyme (reduced by 39.9 %) with the influence of composited pollutants exerting stronger changes. Molecular simulation indicated the key residues Asp 52 for protein function located near the docking site, suggesting pollutants preferentially binds to the active center of lysozyme. Through this study, we have found the effect of increased toxicity on lysozyme under the compounded conditions of NPs and NOR, confirming that the increased molecular toxicity of NPs and NOR is predominantly realized through the increase in particle size and stability of the aggregates under weak interactions, as well as induction of protein structural looseness. This study proposes a molecular perspective on the differential effects and mechanisms of NPs-NOR composite pollution, providing new insights into the assessment of in vitro responses to composite pollutant exposure.

Whole Genome Sequencing Analysis of Cellulose-degrading Bacterium DC11 Isolated from Silkworm Excrement and Characterization of Its Key Cellulase Gene ytoP: Running Head: Characterization of strain DC11 and its key gene ytoP

In recent years, the microbial degradation of cellulose has emerged in addressing environmental pollution and acting as a substitute for scarce animal feeds. This study conducted a whole-genome sequencing analysis of a cellulose-degrading bacterium known as Bacillus subtilis DC11, previously isolated from the silkworm excrement. A critical cellulase gene was identified, namely ytoP. The ytoP gene was successfully cloned and expressed in E.coli using genetic engineering. ytoP is a segment of the endoglucanase gene in Bacillus subtilis DC11 and was found to be 1074 bp in size and encoded 357 amino acids. This study effectively constructed the cellulase expression vector and achieved successful expression of the ytoP gene from strain DC11 in E. coli BL21 (DE3). SDS-PAGE electrophoresis revealed that the protein had an approximate size range of 40–50 KDa and a concentration of around 3.675 mg/mL. An assay of enzyme activity demonstrated that the purified protein, with a concentration of approximately 100 μg/mL, exhibited a maximum activity of 12.980 U/mL. Through the integration of whole-genome sequencing and genetic engineering techniques, the critical cellulase gene ytoP from Bacillus subtilis DC11 has been successfully cloned and expressed, achieving highly efficient cellulase production. This study lays the foundation for large-scale applications of microbial cellulose degradation in the future.

Development of an effective meropenem/KPC-2 inhibitor combination to combat infections caused by carbapenem-resistant Klebsiella pneumoniae

The global public health threat of antibiotic resistance continues to escalate, and necessitates the implementation of urgent measures to expand the arsenal of antimicrobial drugs. This study identified a benzoxaborane compound, namely 5-chloro-1,3-dihydro-1-hydroxy-2,1-benzoxaborole (AN2178), which can inhibit the catalytic activity of the Klebsiella pneumoniae carbapenemase (KPC-2) enzyme effectively. The efficacy of AN2718 as an inhibitor for the KPC-2 enzyme was verified through various assays, including enzyme activity assays and isothermal titration calorimetry. Results of multiple biochemical assays, minimum inhibitory concentration assays and time-killing assays also showed that binding of AN2718 to KPC-2 enabled restoration of the bactericidal effect of meropenem. The survival rate of mice infected with carbapenem-resistant, high-virulence strains increased significantly upon treatment with AN2718. Most importantly, the meropenem and AN2718 combination was effective on KPC-2 mutations such as KPC-33, which evolved clinically and exhibited resistance to ceftazidime-avibactam after clinical use for a couple of years. Comprehensive safety tests both in vitro and in vivo, such as cytotoxicity, haemolytic activity and cytochrome P450 inhibition assays, demonstrated that AN2718 was safe for clinical use. These promising data indicate that AN2718 has high potential for approval for the treatment of drug resistant-bacterial infections, including those caused by ceftazidime-avibactam-resistant strains. AN2718 can be regarded as a valuable addition to the current antimicrobial armamentarium, and a promising tool to combat antimicrobial resistance.

Rapid Screening of Chemical Components in Salvia miltiorrhiza with the Potential to Inhibit Skin Inflammation.

Hyaluronidase possesses the capacity to degrade high-molecular-weight hyaluronic acid into smaller fragments, subsequently initiating a cascade of inflammatory responses and activating dendritic cells. In cases of bacterial infections, substantial quantities of HAase are generated, potentially leading to severe conditions such as cellulitis. Inhibiting hyaluronidase activity may offer anti-inflammatory benefits. Salvia miltiorrhiza Bunge, a traditional Chinese medicine, has anti-inflammatory properties. However, its effects on skin inflammation are not well understood. This study screened and evaluated the active components of S. miltiorrhiza that inhibit skin inflammation, using ligand fishing, enzyme activity assays, drug combination analysis, and molecular docking. By combining magnetic nanomaterials with hyaluronidase functional groups, we immobilized hyaluronidase on magnetic nanomaterials for the first time in the literature. We then utilized an immobilized enzyme to specifically adsorb the ligand; two ligands were identified as salvianolic acid B and rosmarinic acid by HPLC analysis after desorption of the dangling ligands, to complete the rapid screening of potential anti-inflammatory active ingredients in S. miltiorrhiza roots. The median-effect equation and combination index results indicated that their synergistic inhibition of hyaluronidase at a fixed 3:2 ratio was enhanced with increasing concentrations. Kinetic studies revealed that they acted as mixed-type inhibitors of hyaluronidase. Salvianolic acid B had Ki and Kis values of 0.22 and 0.96 μM, respectively, while rosmarinic acid had values of 0.54 and 4.60 μM. Molecular docking revealed that salvianolic acid B had a higher affinity for hyaluronidase than rosmarinic acid. In addition, we observed that a 3:2 combination of SAB and RA significantly decreased the secretion of TNF-α, IL-1, and IL-6 inflammatory cytokines in UVB-irradiated HaCaT cells. These findings identify salvianolic acid B and rosmarinic acid as key components with the potential to inhibit skin inflammation, as found in S. miltiorrhiza. This research is significant for developing skin inflammation treatments. It demonstrates the effectiveness and broad applicability of the magnetic nanoparticle-based ligand fishing approach for screening enzyme inhibitors derived from herbal extracts.