Antiphospholipid syndrome presenting as extensive skin ulcers on unilateral lower extremity: a case report.

Antiphospholipid syndrome (APS) is an autoimmune disorder characterized by recurrent arterial and venous thrombosis, habitual fetal miscarriages, often accompanied by mild to moderate thrombocytopenia, and persistent moderate-to-high titer positivity for antiphospholipid antibodies (aPLs). However, patients with antiphospholipid antibodies may also present with several nonthrombotic clinical manifestations, such as thrombocytopenia, cardiac valve disease, nephropathy, skin ulcers, or cognitive dysfunction, which are collectively referred to as nonstandard manifestations of APS. Of these, for APS with predominantly cutaneous ulcers, previous reports have focused on APS with combined cutaneous vasculitis, and its medical treatment, rather than cutaneous ulcers with predominantly fatty inflammatory lesions, and the associated surgical treatment. Here, we admitted a relatively rare case of primary APS with extensive skin ulceration of the right lower extremity, without cutaneous vasculitis, in the presence of extensive and severe inflammatory lipoatrophy, carrying anti-ß2-glycoprotein I and lupus anticoagulant, which is reported as follows, with a view to raising awareness of this disease.

Schisandra chinensis Bee Pollen Ameliorates Colitis in Mice by Modulating Gut Microbiota and Regulating Treg/Th17 Balance.

Colitis is a chronic disease associated with alterations in the composition of gut microbiota. Schisandra chinensis bee pollen extract (SCPE) has been proved to be rich in phenolic compounds and effective in modulating gut microbiota, but its effect on colitis and the underlying mechanism remains unclear. This study investigates the relationship between colitis amelioration and the gut microbiota regulation of SCPE via fecal microbial transplantation (FMT). The results showed that administration of 20.4 g/kg BW of SCPE could primely ameliorate colitis induced by dextran sulfate sodium (DSS) in mice, showing as more integration of colon tissue structure and the colonic epithelial barrier, as well as lower oxidative stress and inflammation levels compared with colitis mice. Moreover, SCPE supplement restored the balance of T regulatory (Treg) cells and T helper 17 (Th17) cells. Gut microbiota analysis showed SCPE treatment could reshape the gut microbiota balance and improve the abundance of gut microbiota, especially the beneficial bacteria (Akkermansia and Lactobacillus) related to the production of short-chain fatty acids and the regulation of immunity. Most importantly, the protection of 20.4 g/kg BW of SCPE on colitis can be perfectly transmitted by fecal microbiota. Therefore, the gut microbiota-SCFAS-Treg/Th17 axis can be the main mechanism for SCPE to ameliorate colitis. This study suggests that SCPE can be a new promising functional food for prevention and treatment of colitis by reshaping gut microbiota and regulating gut immunity.

Correction: Mitochondria-targeting nanozyme alleviating temporomandibular joint pain by inhibiting the TNFα/NF-κB/NEAT1 pathway.

Correction for 'Mitochondria-targeting nanozyme alleviating temporomandibular joint pain by inhibiting the TNFα/NF-κB/NEAT1 pathway' by Qian Bai et al., J. Mater. Chem. B, 2023, https://doi.org/10.1039/d3tb00929g.

Mitochondria-targeting nanozyme alleviating temporomandibular joint pain by inhibiting the TNFα/NF-κB/NEAT1 pathway.

Inflammatory cytokines that are secreted into the spinal trigeminal nucleus caudalis (Sp5C) may augment inflammation and cause pain associated with temporomandibular joint disorders (TMD). In a two-step process, we attached triphenylphosphonium (TPP) to the surface of a cubic liposome metal-organic framework (MOF) loaded with ruthenium (Ru) nanozyme. The design targeted mitochondria and was designated Mito-Ru MOF. This structure scavenges free radicals and reactive oxygen species (ROS) and alleviates oxidative stress. The present study aimed to investigate the effects and mechanisms by which Mito-Ru MOF ameliorates TMD pain. Intra-temporomandibular joint (TMJ) injections of complete Freund's adjuvant (CFA) induced inflammatory pain for ≥10 d in the skin areas innervated by the trigeminal nerve. Tumor necrosis factor-alpha (TNF-α), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), long non-coding RNA nuclear paraspeckle assembly transcript 1 (lncRNA NEAT1), and ROS also have been proved to be significantly upregulated in the Sp5C of TMD mice. Moreover, a single Mito-Ru MOF treatment alleviated TMD pain for 3 d and downregulated TNF-α, NF-κB, lncRNA NEAT1, and ROS. NF-κB knockdown downregulated NEAT1 in the TMD mice. Hence, Mito-Ru MOF inhibited the production of ROS and alleviated CFA-induced TMD pain via the TNF-α/NF-κB/NEAT1 pathway. Therefore, Mito-Ru MOF could effectively treat the pain related to TMD and other conditions associated with severe acute inflammatory activation.

Association between trajectories in cardiac damage and clinical outcomes after transcatheter aortic valve replacement.

BACKGROUND: There is little evidence of evolution in cardiac damage after transcatheter aortic valve replacement (TAVR) in aortic stenosis (AS) patients. Less is known about the prognostic value and potential utility of different cardiac damage trajectories following TAVR. OBJECTIVES: This study aims to investigate the cardiac damage trajectories following TAVR and explore their association with subsequent clinical outcomes. METHODS: AS patients undergoing TAVR were enrolled and classified into five cardiac damage stages (0-4) based on the echocardiographic staging classification retrospectively. They were further grouped into early stage (stage 0-2) and advanced stage (stage 3-4). The cardiac damage trajectories in TAVR recipients were evaluated according to their trend between baseline and 30 days after TAVR. RESULTS: A total of 644 TAVR recipients were enrolled, with four distinct trajectories identified. Compared to patients with early-early trajectory, patients with early-advanced trajectory were at 30-fold risk of all-cause death (HR 30.99, 95% CI 13.80-69.56; p < 0.001). In multivariable analyses, early-advanced trajectory was associated with higher 2-year all-cause death (HR 24.08, 95% CI 9.07-63.90; p < 0.001), cardiac death (HR 19.34, 95% CI 3.06-122.34; p < 0.05), and cardiac rehospitalization (HR 4.19, 95% CI 1.49-11.76; p < 0.05) after TAVR. CONCLUSIONS: This investigation provided insight into four cardiac damage trajectories in TAVR recipients and confirmed the prognostic value of distinct trajectories. Early-advanced trajectory was associated with poor clinical prognosis following TAVR.

NT-proBNP trajectory after transcatheter aortic valve replacement and its association with 5-year clinical outcomes.

Background: There are only limited reports on the trends of NT-proBNP after transcatheter aortic valve replacement (TAVR) in aortic stenosis (AS) and even fewer report on the prognostic value of the NT-proBNP trajectory following TAVR. Objectives: This study aims to investigate short-term NT-proBNP trajectory following TAVR and explore its association with clinical outcomes in TAVR recipients. Methods: Aortic stenosis patients undergoing TAVR were included if they had NT-proBNP levels recorded at baseline, prior to discharge, and within 30 days after TAVR. We used latent class trajectory models to identify NT-proBNP trajectories based on their trends over time. Results: Three distinct NT-proBNP trajectories were identified from 798 TAVR recipients, which were named class 1 (N = 661), class 2 (N = 102), and class 3 (N = 35). Compared to those with trajectory class 1, patients with trajectory class 2 had a more than 2.3-fold risk of 5-year all-cause death and 3.4-fold risk of cardiac death, while patients with trajectory class 3 had a more than 6.6-fold risk of all-cause death and 8.8-fold risk of cardiac death. By contrast, the groups had no differences in 5-year hospitalization rates. In multivariable analyses, the risk of 5-year all-cause mortality was significantly higher in patients with trajectory class 2 (HR 1.90, 95% CI 1.03-3.52, P = 0.04) and class 3 (HR 5.70, 95% CI 2.45-13.23, P < 0.01). Conclusion: Our findings implied different short-term evolution of NT-proBNP levels in TAVR recipients and its prognostic value for AS patients following TAVR. NT-proBNP trajectory may have further prognostic value, in addition to its baseline level. This may aid clinicians with regards to patient selection and risk prediction in TAVR recipients.

Impact of early changes in cardiac damage following transcatheter aortic valve implantation.

BACKGROUND: The staging classification of aortic stenosis (AS) which characterises the extent of cardiac damage has been validated in patients undergoing transcatheter aortic valve implantation (TAVI). Short-term changes in cardiac damage after TAVI and their association with long-term prognosis remain unknown. AIMS: This study aims to investigate the early evolution of cardiac damage after TAVI and the association of residual cardiac damage with clinical outcomes in TAVI recipients. METHODS: AS patients undergoing TAVI were consecutively enrolled and classified into five stages of cardiac damage (0-4). Early change in cardiac damage was defined as any change of stage at 30 days (Δcardiac damage between baseline pre-TAVI and 30 days post-TAVI). RESULTS: Within 30 days post-TAVI, the baseline cardiac damage stage had changed in 22.2% of 644 TAVI recipients, accompanied by improvements in the degree of dyspnoea and left ventricular ejection fraction (LVEF). Two-year mortality was associated with residual cardiac damage within 30 days post-TAVI (hazard ratio [HR] 2.97, 95% confidence interval [CI]: 2.07-4.25; p<0.001). Compared to unchanged cardiac damage post-TAVI, further cardiac damage within 30 days was associated with a higher crude risk of 2-year mortality (HR 22.04, 95% CI: 9.87-49.20; p<0.001). Cardiac deterioration within 30 days post-TAVI was an independent risk factor for 2-year mortality (HR 19.564, 95% CI: 8.047-47.565; p<0.001). CONCLUSIONS: This investigation provided insight into the early evolution of cardiac damage in TAVI recipients and confirmed the predictive value of both residual and early changes in cardiac damage post-TAVI. Cardiac deterioration within 30 days is associated with poor clinical prognosis.

MicroRNA-455-3p accelerate malignant progression of tumor by targeting H2AFZ in colorectal cancer.

Colorectal cancer (CRC) becomes the second leading cause of cancer-related deaths in 2020. Emerging studies have indicated that microRNAs (miRNAs) play a key role in tumorigenesis and progression. The dysfunctions of miR-455-3p are observed in many cancers. However, its biological function in CRC remains to be confirmed. By sequencing serum sample, miR-455-3p was found to be up-regulated in CRC patients. RT-qPCR demonstrated that the miR-455-3p expression was both higher in the serum and tumor tissues of CRC patients. Furthermore, it indicated that miR-455-3p had the ability in promoting cell proliferation, suppressing cell apoptosis, and stimulating cell migration. In vivo experiments also showed that miR-455-3p promoted tumor growth. Additionally, H2AFZ was proved as the direct gene target of miR-455-3p by dual-luciferase assay. Taken together, miR-455-3p functioned as a tumor promoter in CRC development by regulating H2AFZ directly. Thus, it has enormous potential as a biomarker in the diagnosis of CRC.

Stabilization improvement of the squeezed optical fields using a high signal-to-noise ratio bootstrap photodetector.

High-precision cavity locking is crucial for squeezing optical fields. Here, a bootstrapped low-noise photodetector is utilized in the generation process of the squeezed state of light. This process is based on a combination of a modified trans-impedance amplifier (TIA) circuit and a two-stage bootstrap amplifier circuit. This not only achieves high-precision and long-term stable locking of the optical cavity, but it also improves the degree to which the light field is squeezed. The experiment results show that the detector has a high signal-to-noise ratio (SNR) of 26.7 dB at the analysis frequency of 3 MHz when measuring the shot noise with an injection optical power of 800 µW, and the equivalent optical power noise level is lower than 2.4 pW/Hz in the frequency range of 1-30 MHz. Moreover, the squeezing degree of the quadrature amplitude squeezed state light field can be improved by more than 34.9% when the detector is used for optical cavity locking. The photodetector is useful in continuous variable (CV) quantum information research.

Tortuosity of branch retinal artery is more associated with the genesis and progress of diabetic retinopathy.

Objective: The purpose of this study is to investigate the potential of using the tortuosity of branch retinal artery as a more promising indicator for early detection and accurate assessment of diabetic retinopathy (DR). Design and method: The diagnoses, consisting of whether DR or not as well as DR severity, were given by ophthalmologists upon the assessment of those fundus images from 495 diabetic patients. Meanwhile, benefiting from those good contrast and high optical resolution fundus images taken by confocal scanning laser ophthalmoscope, the branch arteries, branch veins, main arteries and main veins in retina can be segmented independently, and the tortuosity values of them were further extracted to investigate their potential correlations with DR genesis and progress based on one-way ANOVA test. Results: For both two comparisons, i.e., between non-DR group and DR group as well as among groups with different DR severity levels, larger tortuosity increments were always observed in retinal arteries and the increments in branch retinal vessels were even larger. Furthermore, it was newly found that branch arterial tortuosity was significantly associated with both DR genesis (p=0.030) and DR progress (p<0.001). Conclusion: Based on this cohort study of 495 diabetic patients without DR and with different DR severity, the branch arterial tortuosity has been found to be more closely associated with DR genesis as well as DR progress. Therefore, the branch arterial tortuosity is expected to be a more direct and specific indicator for early detection of DR as well as accurate assessment of DR severity, which can further guide timely and rational management of DR to prevent from visual impairment or even blindness resulting from DR.

RNA sequencing profiling of mRNAs, long noncoding RNAs, and circular RNAs in Trigeminal Ganglion following Temporomandibular Joint inflammation.

Patients with temporomandibular joint disorders (TMD) have high levels of inflammatory pain-related disability, which seriously affects their physical and mental health. However, an effective treatment is yet to be developed. Both circular RNAs (circRNAs) and long noncoding RNAs (lncRNAs) contribute to regulating pain conduction. In our current study, we report the expression profiles of circRNAs, lncRNAs, and mRNAs in the trigeminal ganglion (TG) associated with complete Freund's adjuvant (CFA)-induced TMD inflammation pain. The collected TGs from the experimental (CFA) and control (saline) groups were processed for deep RNA sequencing. Overall, 1078,909,068 clean reads were obtained. A total of 15,657 novel lncRNAs were identified, where 281 lncRNAs were differentially expressed on CFA3D and 350 lncRNAs were differentially expressed on CFA6D. In addition, a total of 55,441 mRNAs and 27,805 circRNAs were identified, where 3,914 mRNAs and 91 circRNAs were found differentially expressed, between the CFA3D and saline groups, while 4,232 mRNAs and 98 DE circRNAs were differentially expressed between the CFA6D and saline groups. Based on functional analyses, we found that the most significant enriched biological processes of the upregulated mRNAs were involved in the immunity, neuron projection, inflammatory response, MAPK signaling pathway, Ras signaling pathway, chemokine signaling pathway, and inflammatory response in TG. Further analyses of Gene Ontology and the Kyoto Encyclopedia of Genes and Genomes pathway suggest the involvement of dysregulated genes in the pain occurrence mechanism. Our findings provide a resource for expression patterns of gene transcripts in regions related to pain. These results suggest that apoptosis and neuroinflammation are important pathogenic mechanisms underlying TMD pain. Some of the reported differentially expressed genes might be considered promising therapeutic targets. The current research study revealed the expression profiles of circRNAs, lncRNAs, and mRNAs during TMD inflammation pain and sheds light on the roles of circRNAs and lncRNAs underlying the pain pathway in the trigeminal system of TMD inflammation pain.

Integrated analysis of genes encoding ATP-dependent chromatin remodellers identifies CHD7 as a potential target for colorectal cancer therapy.

BACKGROUND: Genes participating in chromatin organization and regulation are frequently mutated or dysregulated in cancers. ATP-dependent chromatin remodelers (ATPCRs) play a key role in organizing genomic DNA within chromatin, therefore regulating gene expression. The oncogenic role of ATPCRs and the mechanism involved remains unclear. METHODS: We analyzed the genomic and transcriptional aberrations of the genes encoding ATPCRs in The Cancer Genome Atlas (TCGA) cohort. A series of cellular experiments and mouse tumor-bearing experiments were conducted to reveal the regulatory function of CHD7 on the growth of colorectal cancer cells. RNA-seq and ATAC-seq approaches together with ChIP assays were performed to elucidate the downstream targets and the molecular mechanisms. RESULTS: Our data showed that many ATPCRs represented a high frequency of somatic copy number alterations, widespread somatic mutations, remarkable expression abnormalities, and significant correlation with overall survival, suggesting several somatic driver candidates including chromodomain helicase DNA-binding protein 7 (CHD7) in colorectal cancer. We experimentally demonstrated that CHD7 promotes the growth of colorectal cancer cells in vitro and in vivo. CHD7 can bind to the promoters of target genes to maintain chromatin accessibility and facilitate transcription. We found that CHD7 knockdown downregulates AK4 expression and activates AMPK phosphorylation, thereby promoting the phosphorylation and stability of p53 and leading to the inhibition of the colorectal cancer growth. Our muti-omics analyses of ATPCRs across large-scale cancer specimens identified potential therapeutic targets and our experimental studies revealed a novel CHD7-AK4-AMPK-p53 axis that plays an oncogenic role in colorectal cancer.

Effects of Different Pesticides on the Brewing of Wine Investigated by GC-MS-Based Metabolomics.

The application of pesticides is critical during the growth of high-quality grape for wine making. However, pesticide residues have significant influence on the wine flavor. In this study, gas chromatography-mass spectrometry (GC-MS) was performed and the obtained datasets were analyzed with multivariate statistical methods to investigate changes in flavor substances in wine during fermentation. The principal component analysis (PCA) score plot showed significant differences in the metabolites of wine treated with various pesticides. In trials using five pesticides (hexaconazole, difenoconazole, flutriafol, tebuconazole, and propiconazole), more than 86 metabolites were changed. Most of these metabolites were natural flavor compounds, like carbohydrates, amino acids, and short-chain fatty acids and their derivatives, which essentially define the appearance, aroma, flavor, and taste of the wine. Moreover, the five pesticides added to grape pulp exhibited different effects on the metabolic pathways, involving mainly alanine, aspartate and glutamate metabolism, butanoate metabolism, arginine, and proline metabolism. The results of this study will provide new insight into the potential impact of pesticide residues on the metabolites and sensory profile of wine during fermentation.

The optimized biocatalytic synthesis of (S)-methyl 2-chlorobutanoate by Acinetobacter sp. lipase.

Epilepsy is a chronic disease caused by sudden abnormal discharge of brain neurons, leading to transient brain dysfunction. Levetiracetam, developed by the UCB company in Belgium, is an effective drug for the treatment of epilepsy. (S)-Methyl 2-chlorobutanoate is an important chiral building block of levetiracetam, which has attracted a great deal of attention. In this study, a strain of lipase-produced Acinetobacter sp. zjutfet-1 was screened from soil samples. At optimized conditions for fermentation and biocatalysis, the bacterial lipase exhibited high catalytic activity for hydrolysis and stereoselectivity toward racemic methyl 2-chlorobutanoate. When the enzymatic reaction was carried out in 6% of racemic substrate, the enantiomeric excess (e.e.s ) reached more than 95%, with a yield of over 86%. Therefore, this lipase can efficiently resolve racemic methyl 2-chlorobutanoate and obtain (S)-methyl 2-chlorobutanoate, which presents great potential in the industrial production of levetiracetam.

Enhancing regioselectivity of sucrose phosphorylase by loop engineering for glycosylation of L-ascorbic acid.

Sucrose phosphorylase (SPase) has a remarkable capacity to synthesize numerous glucosides from abundantly available sucrose under mild conditions but suffers from specificity and regioselectivity issues. In this study, a loop engineering strategy was introduced to enhance the regioselectivity and substrate specificity of SPase for the efficient synthesis of 2-O-α-D-glucopyranosyl-L-ascorbic acid (AA-2G) via L-ascorbic acid (L-AA). P134, L341, and L343 were identified as "hotspots" for modulating the flexibility of loops, which significantly influenced the H-bonding network of L-AA in the active site, as well as the entrance of the substrate channel, thereby altering the regioselectivity and substrate specificity. Finally, the mutant L341V/L343F, with near-perfect control of the selectivity synthesis of the 2-OH group of L-AA (> 99%), was obtained. The AA-2G production by the mutant reached 244 g L-1 in a whole-cell biotransformation system, and the conversion rate of L-AA reached 64%, which is the highest level reported to date. Our work also provides a successful loop engineering case for modulating the regioselectivity and specificity of sucrose phosphorylase. KEY POINTS: • "Hotspots" were identified in the flexible loops of sucrose phosphorylase. • Mutants exhibited improved regioselectivity and specificity against L-ascorbic acid. • Synthesized AA-2G with high yield and regioselectivity by whole-cell of mutant.

Design, synthesis and antitumor evaluation of novel 1H-indole-2-carboxylic acid derivatives targeting 14-3-3η protein.

In this work, a series of novel 1H-indole-2-carboxylic acid derivatives targeting 14-3-3η protein were designed and synthesized for treatment of liver cancer. After structural optimization for several rounds, C11 displayed a relatively better affinity with 14-3-3η, as well as the best inhibitory activities against several typical human liver cancer cell lines, including Bel-7402, SMMC-7721, SNU-387, Hep G2 and Hep 3B cells. Compound C11 also displayed best inhibitory activity against chemotherapy-resistant Bel-7402/5-Fu cells. Besides, C11 was rather safe against hERG and possessed moderate T1/2 and CL values in liver microsomes. In anti-proliferation, trans-well and cell apoptosis assays, C11 also showed its huge potential as a potent antitumor agent. Then, Western blot assay was conducted, following analyzed by molecular docking, the anti-proliferative mechanisms of this small-molecule inhibitor were revealed. Moreover, C11 was demonstrated to induce G1-S phase cell cycle arrest in liver cancer cells.

Traditional Chinese Medicine Constitution Identification Based on Objective Facial and Tongue Features: A Delphi Study and a Diagnostic Nomogram for Blood Stasis Constitution.

Objective: The aim of this study was to systematically summarize and form an expert consensus on the theoretical experience of tongue and facial features for the identification of nine types of traditional Chinese medicine (TCM) constitution. Additionally, we sought to explore the feasibility of TCM constitution identification through objective tongue and facial features. Methods: We used Delphi method to investigate the opinions of experts on facial and tongue feature items for identifying TCM constitution. We developed and validated a diagnostic nomogram for blood stasis constitution (BSC) based on objective facial and tongue features to demonstrate the reliability of expert consultation. Results: Eleven experts participated in two rounds of expert consultation. The recovery rates of the two rounds of expert consultation were 100.0% and 90.9%. After the first round, 39 items were screened out from 147 initial items, and 2 items were supplemented by experts. In the second round, 7 items were eliminated, leaving 34 items for 8 types of TCM constitution. The coefficient of variation in the first round was 0.11-0.49 for the 147 items and 0.11-0.29 for the included items. The coefficient of variation in the second round was 0.10-0.27 for the 41 items and 0.10-0.20 for the included items. The W value was 0.548 (P < 0.001) in the first round and 0.240 (P < 0.001) in the second round. Based on expert consultation, we selected BSC as an example and developed and validated a diagnostic nomogram consisting of six indicators: sex, hair volume, lip color-dark purple, susceptibility-facial pigmentation/chloasma/ecchymosis, zygomatic texture-red blood streaks, and sublingual vein-varicose and dark purple. The nomogram showed good discrimination (AUC: 0.917 [95% confidence interval [CI], 0.877-0.956] for the primary dataset, 0.902 [95% CI, 0.828-0.976] for the validation dataset) and good calibration. Decision curve analysis demonstrated that the nomogram was clinically useful. Conclusion: This is the first study to systematically summarize the existing knowledge and clinical experience to form an expert consensus on the tongue and facial features of nine types of TCM constitution. Our results will provide important prior knowledge and expert experience for future constitution identification research. Based on expert consultation, this study presents a nomogram for BSC that incorporates objective facial and tongue features, which can be conveniently used to facilitate the individualized identification of BSC.

Identification of Process-Related Impurities and Corresponding Control Strategy in Biocatalytic Production of 2-O-α-d-Glucopyranosyl-l-ascorbic Acid Using Sucrose Phosphorylase.

2-O-α-d-Glucopyranosyl-l-ascorbic acid (AA-2G) is an ideal substitute for l-ascorbic acid because of its remarkable stability and improved biological activity, which can be easily applied in cosmetic, food, and medicine fields. However, impurity identification and control are significant procedures during the manufacturing of AA-2G. This study assessed a manufacturing routine of AA-2G synthesized by sucrose phosphorylase (SPase). First, three unknown process-related impurities were observed, which were further identified as 3-O-α-d-glucopyranosyl- l-ascorbic acid (impurity I), 2-O-α-d-glucopyranosyl-l-dehydroascorbic acid (impurity II), and 13-O-α-d-glucopyranosyl-2-O-α-d-glucopyranosyl-l-ascorbic acid (impurity III), respectively. Second, a comprehensive formation pathway of impurities was elucidated, and specific strategies corresponding to controlling each impurity were also proposed. Specifically, the content of impurity I can be reduced by 50% by fine tuning reaction conditions. The impurity II-free purification process was also achieved by applying a low concentration of alkali. Finally, a semi-rational design was introduced, and a single mutant L343F was obtained by site-directed mutagenesis, which reduced impurities I and III by 63.9 and 100%, respectively, without affecting the transglycosylation activity. It is expected that the reported impurity identification and control strategies during the AA-2G production will facilitate its industrial production.

Improving the Transglycosylation Activity of α-Glucosidase from Xanthomonas campestris Through Semi-rational Design for the Synthesis of Ethyl Vanillin-α-Glucoside.

The α-glucosidase (EC 3.2.1.20) XgtA produced by Xanthomonas campestris shows high α-glucosyl transfer activity toward alcoholic and phenolic hydroxyl groups. Ethyl vanillin-α-glucoside, a precursor-aroma compound with improved water solubility and thermal stability, can be synthesized through the transglycosylation of ethyl vanillin by XgtA. However, its low ethyl vanillin-α-glucoside yield and ability to hydrolyze ethyl vanillin-α-glucoside limits for industrial applications. Rational design and site-directed mutagenesis were employed to generate three variants of X. campestris α-glucosidase, L145I, S272T, and L145I/S272T, with improved transglycosylation activity toward EV. The highest yield is up to 52.41% by L145I/S272T, which also displayed remarkably lower hydrolysis activity toward the glycoside product EVG compared to XgtA. These results also showed that the mutation in sugar-binding subsite + 1 is more effective than subsite -1 for enhancing the ratio of transglycosylation/hydrolysis for the α-glucosidase XgtA.