Simultaneous Screening of 172 Veterinary Drugs by Modified QuEChERS-LC-MS/MS in TCM Galli Gigerii Endothelium Corneum.

An analytical method was developed for the screening of 172 veterinary drugs in traditional Chinese medicine Galli Gigerii Endothelium Corneum by high-performance liquid chromatography tandem mass spectrometry. The samples were pretreated by a modified QuEChERS method. A Zorbax Eclipse plus C18 column (1.8 µm, 3.0 × 150 mm2, Agilent) was used for the separation of analytes by gradient elution. All analytes were detected by electrospray ionization mass spectrometry with multiple reaction monitoring mode. Good linearity with R ≥ 0.99 was exhibited for all analytes within the respective range. The recoveries of all monitored analytes ranged from 55.4 to 127.6% at three spiked levels (limit of quantitation-LOQ, 2-fold LOQ, 10-fold LOQ), with relative standard deviations <17.8%. The estimated LOQ levels were 0.2-20 µg/kg. The application of this method provides a reference for the safety control of traditional Chinese medicines.

Self-healing hydrogel based on poly (vinyl alcohol)-poly (lysine)-gum arabic accelerates diabetic wound healing under photothermal sterilization.

Diabetic wounds are a significant clinical challenge. Developing effective antibacterial dressings is crucial for preventing wound ulcers caused by bacterial infections. In this study, a self-healing antibacterial hydrogel (polyvinyl alcohol (PVA)-polylysine-gum arabic, PLG hydrogels) with near-infrared photothermal response was prepared by linking PVA and a novel polysaccharide-amino acid compound (PG) through borate bonding combined with freeze-thaw cycling. Subsequently, the hydrogel was modified by incorporating inorganic nanoparticles (modified graphene oxide (GM)). The experimental results showed that the PLGM3 hydrogels (PLG@GM hydrogels, 3.0 wt%) could effectively kill bacteria and promote diabetic wound tissue healing under 808-nm near-infrared laser irradiation. Therefore, this hydrogel system provides a new idea for developing novel dressings for treating diabetic wounds.

An NIR light-driven AgBiS2@ZIF-8 hybrid photocatalyst for rapid bacteria-killing.

Bacterial infection is the most common risk factor that causes the failure of implantation surgery. Therefore, the development of biocompatible implants with excellent antibacterial properties is of utmost importance. In this study, NIR light-driven AgBiS2@ZIF-8 hybrid photocatalysts for rapid bacteria-killing were prepared. AgBiS2@ZIF-8 exhibited excellent photocatalytic activity due to the rapid transfer of photoelectrons from AgBiS2 to ZIF-8, resulting in abundant reactive oxygen species (ROS) to kill bacteria. Meanwhile, AgBiS2@ZIF-8 exhibited a noteworthy photothermal effect, which could effectively convert NIR light into heat. Subsequently, the NIR light-driven antibacterial activity of AgBiS2@ZIF-8/Ti against S. aureus and E. coli was studied. The experimental results showed that AgBiS2@ZIF-8 displayed enhanced photodynamic therapy (PDT) and photothermal therapy (PTT) performance. Under irradiation with 808 nm NIR light for 10 min, AgBiS2@ZIF-8/Ti could effectively eliminate 98.55% of S. aureus in vitro, 99.34% of E. coli in vitro and 95% S. aureus in vivo. At the same time, AgBiS2@ZIF-8/Ti had good biocompatibility. Therefore, AgBiS2@ZIF-8/Ti showed potential as an antibacterial material, which provided a strategy to fight polymicrobial infections.

Simultaneously quantifying hundreds of acylcarnitines in multiple biological matrices within ten minutes using ultrahigh-performance liquid-chromatography and tandem mass spectrometry.

Acylcarnitines are metabolic intermediates of fatty acids and branched-chain amino acids having vital biofunctions and pathophysiological significances. Here, we developed a high-throughput method for quantifying hundreds of acylcarnitines in one run using ultrahigh performance liquid chromatography and tandem mass spectrometry (UPLC-MS/MS). This enabled simultaneous quantification of 1136 acylcarnitines (C0-C26) within 10-min with good sensitivity (limit of detection < 0.7 fmol), linearity (correlation coefficient > 0.992), accuracy (relative error < 20%), precision (coefficient of variation (CV), CV < 15%), stability (CV < 15%), and inter-technician consistency (CV < 20%, n = 6). We also established a quantitative structure-retention relationship (goodness of fit > 0.998) for predicting retention time (tR) of acylcarnitines with no standards and built a database of their multiple reaction monitoring parameters (tR, ion-pairs, and collision energy). Furthermore, we quantified 514 acylcarnitines in human plasma and urine, mouse kidney, liver, heart, lung, and muscle. This provides a rapid method for quantifying acylcarnitines in multiple biological matrices.

How the intrinsic functional connectivity patterns of the semantic network support semantic processing.

Semantic processing, a core of language comprehension, involves the activation of brain regions dispersed extensively across the frontal, temporal, and parietal cortices that compose the semantic network. To comprehend the functional structure of this semantic network and how it prepares for semantic processing, we investigated its intrinsic functional connectivity (FC) and the relation between this pattern and semantic processing ability in a large sample from the Human Connectome Project (HCP) dataset. We first defined a well-studied brain network for semantic processing, and then we characterized the within-network connectivity (WNC) and the between-network connectivity (BNC) within this network using a voxel-based global brain connectivity (GBC) method based on resting-state functional magnetic resonance imaging (fMRI). The results showed that 97.73% of the voxels in the semantic network displayed considerably greater WNC than BNC, demonstrating that the semantic network is a fairly encapsulated network. Moreover, multiple connector hubs in the semantic network were identified after applying the criterion of WNC > 1 SD above the mean WNC of the semantic network. More importantly, three of these connector hubs (i.e., the left anterior temporal lobe, angular gyrus, and orbital part of the inferior frontal gyrus) were reliably associated with semantic processing ability. Our findings suggest that the three identified regions use WNC as the central mechanism for supporting semantic processing and that task-independent spontaneous connectivity in the semantic network is essential for semantic processing.

Early Prediction of Wound Healing Outcome Based on Chronic Wound Registry Database.

Chronic wounds cause a number of unnecessary amputations due to a delay in proper treatment. To expedite timely treatment, this paper presents an algorithm which uses a logistic regression classifier to predict whether the wound will heal or not within a specified time. The prediction is made at three time-points: one month, three months, and six months from the first visit of the patient to the healthcare facility. This prediction is made using a systematically collected chronic wound registry and is based entirely on data collected during patients' first visit. The algorithm achieves an area under the receiver operating characteristic curve (AUC) of 0.75, 0.72, and 0.71 for the prediction at the three time-points, respectively.Clinical relevance- Using the proposed prediction model, the clinicians will have an early estimate of the time taken to heal thereby providing appropriate treatments. We hope this will ensure timely treatments and reduce the number of unnecessary amputations.

[Vitamin A and vitamin D nutritional status among children and adolescents aged 6-17 years in Jiangsu Province during 2016-2017].

OBJECTIVE: This study aimed to investigate vitamin A and vitamin D conditions and related factors among children and adolescents aged 6-17 years in Jiangsu Province of China. METHODS: All the data were derived from China Nutrition and Health Surveillance of Children and Lactating Mothers in 2016-2017. By applying multiple stage stratified cluster random sampling method, 3244 children aged 6-17 years were selected from 12 survey sites in Jiangsu Province. Face to face interview, physical measurements and 6 mL blood sample were used to collect the general information, anthropometric information and blood nutritional indexes of the participants. Multivariate Logistic analysis was used for vitamin A and vitamin D conditions to test related factors. RESULTS: The prevalence of vitamin A deficiency and marginal deficiency was 0.8% and 15.8%, respectively.23.2% of the participants had vitamin D deficiency, 54.2% had vitamin D insufficiency and 4.8% had vitamin A insufficiency combined with vitamin D deficiency. Age group, weight levels, screen time and mother's education levels are the relevant factors of vitamin A insufficiency in children and adolescents. The related factors of vitamin D deficiency among children and adolescents are gender, age group, residence, physical activity level, screen time and mother's education levels. Gender, residence, weight levels, screen time and mother's education levels are the related factors of vitamin A insufficiency combined with vitamin D deficiency. CONCLUSION: Vitamin A insufficiency and vitamin D deficiency are at high epidemic levels among children and adolescents in Jiangsu Province in 2016-2017.

Psycho-linguistic and educational challenges in Teaching Chinese (Mandarin) Language: voices from None-Chinese teachers of Mandarin language.

BACKGROUND: Chinese/Mandarin language instruction to undergraduate students from non-Chinese speaking countries has become a topic of increasing interest, driven by China's influential role in global political and economic dynamics. With Mandarin being the language spoken by approximately 70% of Chinese speakers, it is not only taught within China but also in institutions around the world. While teaching any second or foreign language presents unique challenges, the specific challenges faced in Mandarin language education have not received comprehensive scrutiny, prompting the need for further investigation. OBJECTIVES: The primary objective of this study was to explore the challenges associated with teaching and learning Mandarin from the perspectives of non-native Mandarin language teachers. By adopting a qualitative (phenomenological) research approach, we aimed to uncover the multifaceted challenges that confront both educators and learners in the context of Mandarin language instruction. METHODOLOGY: The research methodology employed for this study involved the use of semi-structured interviews conducted with 15 Mandarin language teachers, who were chosen through theoretical sampling. These interviews were transcribed into text files and subsequently subjected to qualitative data analysis, which was facilitated by the use of MAQDA software. This approach allowed us to identify and categorize the various challenges and problems encountered in the teaching and learning of Mandarin. FINDINGS: The research findings revealed a range of challenges and problems experienced in Mandarin language education. These challenges were grouped into two primary thematic categories: psycho-linguistic and educational challenges. The linguistic challenges are deeply rooted in the distinctive features of the Mandarin language, presenting obstacles that necessitate innovative teaching strategies and materials. Educational challenges extend beyond native speaker proficiency, emphasizing the requirement for a comprehensive pedagogical skill set among instructors. IMPLICATIONS: The implications of this research extend to various stakeholders in the field of Chinese/Mandarin language education, including policymakers, educators, curriculum designers, and learners. Understanding the linguistic, educational, and psychological challenges can inform the development of more effective teaching methods, curriculum design, and motivation-enhancing strategies, ultimately promoting a more successful and engaging Mandarin language learning experience for non-native speakers.

Can companies get more government subsidies through improving their ESG performance? Empirical evidence from China.

Environmental protection and social obligation fulfillment have become hot subjects as the "dual carbon" approach has been developed and deepened. The ESG system is consistent with China's current policies, abandoning the traditional business philosophy of economic supremacy in favor of comprehensively measuring corporate social responsibility and sustainable development capability across three dimensions: environmental (E), social (S), and corporate governance (G), which receive widespread attention from all sectors of society. Based on observational data from A-share listed businesses in Shanghai and Shenzhen from 2011 to 2020, this study empirically evaluates the influence and mechanism of ESG on government subsidies. The research results indicate that enterprises can receive more government subsidies by improving ESG performance. Mechanism analysis found that corporate transparency plays a positive mediating role in the process of ESG affecting government subsidies. Further research on political affiliation and property rights has found that companies without political affiliation are more inclined to receive more government subsidies by improving ESG performance, and the impact of political affiliation and ESG performance on government subsidies is mutually complementary. Enterprises with different property rights have different strengths of motivation to increase government subsidies by improving ESG performance. State owned enterprises (excluding central enterprises) are the strongest, followed by non-state-owned enterprises, and central enterprises are the weakest. Therefore, enterprises should be further encouraged to strengthen ESG construction, improve the quality of ESG information disclosure, improve resource allocation efficiency, and promote high-quality development of enterprises.

Characterization of natural peptides in Pheretima by integrating proteogenomics and label-free peptidomics.

Pheretima, also called "earthworms", is a well-known animal-derived traditional Chinese medicine that is extensively used in over 50 Chinese patent medicines (CPMs) in Chinese Pharmacopoeia (2020 edition). However, its zoological origin is unclear, both in the herbal market and CPMs. In this study, a strategy for integrating in-house annotated protein databases constructed from close evolutionary relationship-sourced RNA sequencing data from public archival resources and various sequencing algorithms (restricted search, open search, and de novo) was developed to characterize the phenotype of natural peptides of three major commercial species of Pheretima, including Pheretima aspergillum (PA), Pheretima vulgaris (PV), and Metaphire magna (MM). We identified 10,477 natural peptides in the PA, 7,451 in PV, and 5,896 in MM samples. Five specific signature peptides were screened and then validated using synthetic peptides; these demonstrated robust specificity for the authentication of PA, PV, and MM. Finally, all marker peptides were successfully applied to identify the zoological origins of Brain Heart capsules and Xiaohuoluo pills, revealing the inconsistent Pheretima species used in these CPMs. In conclusion, our integrated strategy could be used for the in-depth characterization of natural peptides of other animal-derived traditional Chinese medicines, especially non-model species with poorly annotated protein databases.

Phytosterol conversion into C9 non-hydroxylated derivatives through gene regulation in Mycobacterium fortuitum.

Androst-4-ene-3,17-dione (AD) and 22-hydroxy-23,24-bisnorchol-4-ene-3-one (4-HBC) are important drug intermediates that can be biosynthesized from phytosterols. However, the C9 hydroxylation of steroids via 3-ketosteroid 9α-hydroxylase (KSH) limits AD and 4-HBC accumulation. Five active KshAs, the oxidation component of KSH, were identified in Mycobacterium fortuitum ATCC 35855 for the first time. The deletion of kshAs indicated that the five KshA genes were jointly responsible for C9 hydroxylation during phytosterol biotransformation. MFKDΔkshA, the five KshAs deficient strain, blocked C9 hydroxylation and produced 5.37 g/L AD and 0.55 g/L 4-HBC. The dual function reductase Opccr knockout and 17ß-hydroxysteroid dehydrogenase Hsd4A enhancement reduced 4-HBC content from 8.75 to 1.72% and increased AD content from 84.13 to 91.34%, with 8.24 g/L AD being accumulated from 15 g/L phytosterol. In contrast, hsd4A and thioesterase fadA5 knockout resulted in the accumulation of 5.36 g/L 4-HBC from 10 g/L phytosterol. We constructed efficient AD (MFKDΔkshAΔopccr_hsd4A) and 4-HBC (MFKDΔkshAΔhsd4AΔfadA5) producers and provided insights for further metabolic engineering of the M. fortuitum ATCC 35855 strain for steroid productions. KEY POINTS: • Five active KshAs were first identified in M. fortuitum ATCC 35855. • Deactivation of all five KshAs blocks the steroid C9 hydroxylation reaction. • AD or 4-HBC production was improved by Hsd4A, FadA5, and Opccr modification.

Targeted trace ingredients coupled with chemometric analysis for consistency evaluation of Panax notoginseng saponins injectable formulations.

Evaluating the consistency of herb injectable formulations could improve their product quality and clinical safety, particularly concerning the composition and content levels of trace ingredients. Panax notoginseng Saponins Injection (PNSI), widely used in China for treating acute cardiovascular diseases, contains low-abundance (10%-25%) and trace saponins in addition to its five main constituents (notoginsenoside R1, ginsenoside Rg1, ginsenoside Re, ginsenoside Rb1, and ginsenoside Rd). This study aimed to establish a robust analytical method and assess the variability in trace saponin levels within PNSI from different vendors and formulation types. To achieve this, a liquid chromatography-triple quadrupole mass spectrometry (LC-MS/MS) method employing multiple ions monitoring (MIM) was developed. A "post-column valve switching" strategy was implemented to eliminate highly abundant peaks (NR1, Rg1, and Re) at 26 min. A total of 51 saponins in PNSI were quantified or relatively quantified using 18 saponin standards, with digoxin as the internal standard. This study evaluated 119 batches of PNSI from seven vendors, revealing significant variability in trace saponin levels among different vendors and formulation types. These findings highlight the importance of consistent content in low-abundance and trace saponins to ensure product control and clinical safety. Standardization of these ingredients is crucial for maintaining the quality and effectiveness of PNSI in treating acute cardiovascular diseases.

Trends in innovative pediatric drug development in China based on clinical trial registration data.

In China, the focus of drug research and development has gradually shifted from generic to innovative drugs. Using the Chinese Clinical Trials Registry and Information Transparency Platform, we retrospectively analyzed clinical trials of innovative pediatric drugs conducted in mainland China over the last decade. The goal of this work was to better understand the characteristics of and historical changes in innovative pediatric drug research and development (R&D) in China and to provide effective data support for policy makers and other stakeholders. This study included 198 innovative pediatric drug clinical trials. The data showed that, although some progress has been made in the R&D of innovative pediatric drugs in China, many factors limiting this progress still exist, such as concentrated R&D areas, inadequate pediatric participants, and unbalanced source distributions. The level of innovative pediatric drug R&D in China currently lags behind the global level and has not kept pace with anti-neoplastic drug R&D in China. To promote the innovative development of pediatric drugs in China, the Chinese government must develop an R&D supervision framework, improve the motivation and innovation capabilities of pharmaceutical companies, and optimize the source distribution between regions.

Silk fibroin-based coating with pH-dependent controlled release of Cu2+ for removal of implant bacterial infections.

The implantation of medical devices is frequently accompanied by the invasion of bacteria, which may lead to implant failure. Therefore, an intelligent and responsive coating seems particularly essential in hindering implant-associated infections. Herein, a self-defensive antimicrobial coating, accompanied by silk fibroin as a valve, was successfully prepared on the titanium (Ti-Cu@SF) for pH-controlled release of Cu2+. The results showed that the layer could set free massive Cu2+ to strive against E. coli and S. aureus for self-defense when exposed to a slightly acidic condition. By contrary, a little Cu2+ was released in the physiological situation, which could avoid damage to the normal cells and showed excellent in vitro pH-dependent antibiosis. Besides, in vivo experiment confirmed that Ti-Cu@SF could work as an antibacterial material to kill S. aureus keenly and display negligible toxicity in vivo. Consequently, the design provided support for endowing the layer with outstanding biocompatibility and addressing the issue of bacterial infection during the implantation of Ti substrates.

Isolation and identification of a novel vardenafil analogue, propoxy-vardenafil, found as an adulterant in a health supplement.

Background: A novel vardenafil analogue was detected from a health wine claimed to be anti-impotence during a special inspection in an online store. Methods: The unknown compound was found by using ultra-high performance liquid chromatography coupled to quadrupole time of flight mass spectrometry (UHPLC/Q-TOF MS). The characteristic product ions were similar to those of vardenafil. The UV spectrum of the compound closely mirrored that of vardenafil. The analogue underwent purification by semi-preparative HPLC and structurally identified by FT-IR and NMR analysis. Results: Based on the data, The structure of the analogue was characterized as 2-[2-propyloxy-5-(4-ethylpiperazin-1-yl)sulfonylphenyl]-5-methyl-7-propyl-3H-imidazo [5,15,1-f] [1,2,4]triazin-4-one, simplified as propoxy-vardenafil. Conclusion: To the best of our knowledge, the analogue has not been reported and is even only ninth vardenafil analogue, which was confirmed that a n-propyloxy group had replaced the ethoxy group on the aromatic ring of vardenafil. Therefore, It is essential to pay more attention to vardenafil analogues in the routine inspection of health supplements.

Hepatoprotective effect of Artemisia Argyi essential oil on bisphenol A-induced hepatotoxicity via inhibition of ferroptosis in mice.

The environmental pollutant bisphenol A (BPA), used in the manufacture of plastic packaging materials for various diets, is widely distributed in the environment and causes severe hepatotoxicity by inducing oxidative stress. Artemisia argyi essential oil (AAEO), a volatile oil component isolated from Artemisia argyi H.Lév. & Vaniot, has pharmacological effects, especially for hepatoprotective actions. However, the potential effect of AAEO in BPA induced hepatotoxicity has not been characterized. First, we analyzed the chemical composition in AAEO by gas chromatography-mass spectrometry. Herein, we investigated the effect of AAEO on hepatic metabolic changes in mice exposed to BPA. Results showed that compared with the BPA group, AAEO could reduce the level of liver function enzymes in BPA mice serum, and ameliorate hepatic lesions and fibrosis. Additionally, 20 differential metabolites screened by metabolomics were mainly involved in the reprogramming of glutathione metabolism, purine metabolism, and polyunsaturated fatty acid synthesis. Moreover, AAEO could reduce hepatic ferroptosis induced by BPA, as demonstrated by reducing xanthine oxidase activity, up-regulating the activities of glutathione peroxidase 4 (GPX4), superoxide dismutase, and catalase and the expression of SLC7A11 to promote the glutathione synthetic, while inhibiting transferrin receptor 1 (TFR1) expression to reduce the accumulation of Fe2+ in cells. Therefore, our study identified AAEO as a hepatic protectant against BPA-induced hepatotoxicity by reversing the occurrence of ferroptosis.

Rational design of artificial Lewis pairs coupling with polyethylene glycol for efficient electrochemical ammonia synthesis.

Ammonia (NH3) synthesis at mild conditions by electrocatalytic nitrogen reduction (eNRR) has received more attention and has been regarded as a promising alternative to the traditional Haber-Bosch process. Lewis acid-base pairs (LPs) can chemisorb and react with nitrogen by electronic interaction, while the tuning of the microenvironment near electrode can hinder hydrogen evolution reaction (HER) thus improving the selectivity of the eNRR. Herein, the FeOOH nanorod coupled with LPs on the surface (i.e., Fe, Fe-O) was synthesized, which could effectively drive eNRR. Meanwhile, polyethylene glycol (PEG) was introduced to serve as a local non-aqueous electrolyte system to inhibit HER. The prepared FeOOH-150 catalyst achieved outstanding eNRR performance with an NH3 yield rate of 118.07 µg h-1mgcat-1 and a Faradaic efficiency of 51.4 % at -0.6 V vs. RHE in 0.1 M LiClO4 + 20 % PEG. Both the experiment and DFT calculations revealed that the interaction of PEG with Lewis base sites could optimize nitrogen adsorption configuration and activation.

Artemisia argyi H.Lév. & Vaniot essential oil induces ferroptosis in pancreatic cancer cells via up-regulation of TFR1 and depletion of γ-glutamyl cycle.

Artemisia argyi H.Lév. & Vaniot, a traditional Chinese medicine with a history spanning over two millennia, has been extensively used in folk medicine to treat dysmenorrhea, uterine bleeding and inflammation. Recent studies have demonstrated that the essential oil extracted from Artemisia argyi H.Lév. & Vaniot, known as AAEO, exhibits significant anti-tumor properties against liver and lung cancers. There is a scarcity of research on the potential impact of AAEO on pancreatic cancer (PC) cells. In this study, UPLC-MS/MS-based metabolomics method was established to evaluate the effect of AAEO on the proliferation of PC cells. The differential compounds included 5-oxoproline, glutamate, γ-glutamylcysteine, glutathione, arachidonic acid, adrenal acid and linoleic acid were detected by metabolomics, enriching in the γ-glutamyl cycle and polyunsaturated fatty acid metabolism, which were closely related to ferroptosis. Meanwhile, AAEO dramatically increased the levels of intracellular iron ion via up-regulation of TFR1, augmented reactive oxygen species and malondialdehyde in a dose-dependent manner by down-regulation of γ-glutamyl cycle through decreasing expressions of SLC7A11. Additionally, ß-caryophyllene oxide, one of the main components of AAEO, could covalently bind to Cys in SW1990 cells to form a conjugate Cpo-Cys, resulting in the inhibition of glutathione synthesis. Importantly, the ferroptosis inhibitor deferoxamine significantly blocked the inhibitory effect of AAEO on SW1990 cells. Meanwhile, ß-caryophyllene oxide, dihydro-ß-ionone and α-bisabolol had strong binding force with GPX4, SLC7A11 and TFR1, respectively. These findings showed that AAEO induced ferroptosis via regulation of γ-glutamyl cycle by SLC7A11 and iron disorders by TFR1. Our study discovered AAEO as a potential therapeutic approach to induce ferroptosis to prevent or treat PC.

Study on Tungsten Metallization and Interfacial Bonding of Silicon Nitride High-Temperature Co-Fired Ceramic Substrates.

For the first time, Si3N4 HTCC has been prepared using W as the metal phase by high-temperature co-firing (1830 °C/600 KPa/2 h) as a potential substrate candidate in electronic applications. It was discovered that the addition of Si3N4 to the W paste has a significant impact on thermal expansion coefficient matching and dissolution wetting. As the Si3N4 content increased from 0 to 27.23 vol%, the adhesion strength of W increased continuously from 2.83 kgf/mm2 to 7.04 kgf/mm2. The interfacial bonding of the Si3N4 ceramic and the conduction layer was discussed. SEM analysis confirmed that the interface between Si3N4 and W exhibited an interlocking structure. TEM, HRTEM and XRD indicated the formation of W2C and W5Si3 due to the interface reactions of W with residual carbon and Si3N4, respectively, which contributed to the reactive wetting and good adhesion strength between the interface. Suitable amounts of Si3N4 powder and great interfacial bonding were the main reasons for the tough interfacial matching between the Si3N4 ceramic and the conduction layer.

Whole-Genome Analysis of Mycobacterium neoaurum DSM 1381 and the Validation of Two Key Enzymes Affecting C22 Steroid Intermediates in Sterol Metabolism.

Mycobacterium neoaurum DSM 1381 originated from Mycobacterium neoaurum ATCC 25790 by mutagenesis screening is a strain of degrading phytosterols and accumulating important C22 steroid intermediates, including 22-hydroxy-23, 24-bisnorchola-4-en-3-one (4-HP) and 22-hydroxy-23, 24-bisnorchola-1,4-dien-3-one (HPD). However, the metabolic mechanism of these C22 products in M. neoaurum DSM 1381 remains unknown. Therefore, the whole-genome sequencing and comparative genomics analysis of M. neoaurum DSM 1381 and its parent strain M. neoaurum ATCC 25790 were performed to figure out the mechanism. As a result, 28 nonsynonymous single nucleotide variants (SNVs), 17 coding region Indels, and eight non-coding region Indels were found between the genomes of the two strains. When the wild-type 3-ketosteroid-9α-hydroxylase subunit A1 (KshA1) and ß-hydroxyacyl-CoA dehydrogenase (Hsd4A) were overexpressed in M. neoaurum DSM 1381, the steroids were transformed into the 4-androstene-3, 17- dione (AD) and 1,4-androstadiene-3,17-dione (ADD) instead of C22 intermediates. This result indicated that 173N of KshA1 and 171K of Hsd4A are indispensable to maintaining their activity, respectively. Amino acid sequence alignment analysis show that both N173D in KshA1 and K171E in Hsd4A are conservative sites. The 3D models of these two enzymes were predicted by SWISS-MODEL and AlphaFold2 to understand the inactivation of the two key enzymes. These results indicate that K171E in Hsd4A may destroy the inaction between the NAD+ with the NH3+ and N173D in KshA1 and may disrupt the binding of the catalytic domain to the substrate. A C22 steroid intermediates-accumulating mechanism in M. neoaurum DSM 1381 is proposed, in which the K171E in Hsd4A leads to the enzyme's inactivation, which intercepts the C19 sub-pathways and accelerates the C22 sub-pathways, and the N173D in KshA1 leads to the enzyme's inactivation, which blocks the degradation of C22 intermediates. In conclusion, this study explained the reasons for the accumulation of C22 intermediates in M. neoaurum DSM 1381 by exploring the inactivation mechanism of the two key enzymes.