Engineering an (R)-selective transaminase for asymmetric synthesis of (R)-3-aminobutanol.

(R)-selective transaminases show promise as catalysts for the asymmetric synthesis of chiral amines, which are building blocks of various small molecule drugs. However, their application is limited by poor substrate acceptance and low catalytic efficiency. Here, a potential (R)-selective transaminase from Fodinicurvata sediminis (FsTA) was identified through a substrate truncating strategy, and used as starting point for enzyme engineering toward catalysis of 4-hydroxy-2-butanone, a substrate that poses challenges in catalysis. Molecular docking and dynamics simulations revealed Y90 as the key residue responsible for poor substrate binding. Starting from the variant (Y90F, mut1) with initial activity, FsTA was systematically modified to improve substrate-binding through active site reshaping and consensus sequence strategy, yielding three variants (H30R, V152K, and Y156F) with improved activity. A quadruple mutation variant H30R/Y90F/V152K/Y156F (mut4) was also found to show a 7.95-fold greater catalytic efficiency (kcat/KM) than the initial variant mut1. Furthermore, mut4 also enhanced the thermostability of enzyme significantly, with the Tm value increasing by 10 °C. This variant also exhibited significantly improved activity toward a series of ketones that are either not accepted or poorly accepted by the wild-type. This study provides a basis for the rational design of an active to creating variants that can accommodate novel substrates.

Deciphering the molecular and cellular atlas of immune cells in septic patients with different bacterial infections.

BACKGROUND: Sepsis is a life-threatening organ dysfunction caused by abnormal immune responses to various, predominantly bacterial, infections. Different bacterial infections lead to substantial variation in disease manifestation and therapeutic strategies. However, the underlying cellular heterogeneity and mechanisms involved remain poorly understood. METHODS: Multiple bulk transcriptome datasets from septic patients with 12 types of bacterial infections were integrated to identify signature genes for each infection. Signature genes were mapped onto an integrated large single-cell RNA (scRNA) dataset from septic patients, to identify subsets of cells associated with different sepsis types, and multiple omics datasets were combined to reveal the underlying molecular mechanisms. In addition, an scRNA dataset and spatial transcriptome data were used to identify signaling pathways in sepsis-related cells. Finally, molecular screening, optimization, and de novo design were conducted to identify potential targeted drugs and compounds. RESULTS: We elucidated the cellular heterogeneity among septic patients with different bacterial infections. In Escherichia coli (E. coli) sepsis, 19 signature genes involved in epigenetic regulation and metabolism were identified, of which DRAM1 was demonstrated to promote autophagy and glycolysis in response to E. coli infection. DRAM1 upregulation was confirmed in an independent sepsis cohort. Further, we showed that DRAM1 could maintain survival of a pro-inflammatory monocyte subset, C10_ULK1, which induces systemic inflammation by interacting with other cell subsets via resistin and integrin signaling pathways in blood and kidney tissue, respectively. Finally, retapamulin was identified and optimized as a potential drug for treatment of E. coli sepsis targeting the signature gene, DRAM1, and inhibiting E. coli protein synthesis. Several other targeted drugs were also identified in other types of sepsis, including nystatin targeting C1QA in Neisseria sepsis and dalfopristin targeting CTSD in Streptococcus viridans sepsis. CONCLUSION: Our study provides a comprehensive overview of the cellular heterogeneity and underlying mechanisms in septic patients with various bacterial infections, providing insights to inform development of stratified targeted therapies for sepsis.

Responses of soil bacterial and fungal communities to the long-term monoculture of grapevine.

Soil microorganisms are essential for the long-term sustainability of agricultural ecosystems. However, continuous grapevine replanting can disrupt the stability of soil microbial communities. We investigated the bacterial and fungal abundance, diversity, and community composition in rhizosphere soils with continuous grapevine replanting for 5, 6, 7 (Y5, Y6, and Y7; short-term), and 20 (Y20; long-term) years with high-throughput sequencing. Results showed that diversities and abundances of bacterial and fungal communities in Y20 were significantly lower than in other samples. The bacterial and fungal community compositions were markedly affected by the replanting time and planting year. After short-term grapevine replanting, relative abundances of potential beneficial bacteria and harmful fungi in rhizosphere soils were higher compared to long-term planting. Bacterial and fungal communities were significantly correlated with available nitrogen (AN), available phosphorus, available potassium (AK), and pH. AK and AN were the primary soil factors related to the shift of bacterial and fungal communities. Bacterial and fungal co-occurrence patterns were remarkably affected by replanting time, showing that fallow land harbored co-occurrence networks more complex than those in other groups, with the Y20 group showing the lowest complexity. Then, we isolated the dominant fungi in grapevine rhizosphere soil after continuous replanting and verified the harmful effects of three candidate strains through pot experiments. The results showed that 12 days post-treating the soil with fungal spore suspensions significantly inhibited grapevine seedlings' growth, whereas Fusarium solani inhibited plant growth. Overall, we showed that F. solani might be a potentially harmful fungus related to grapevine replant diseases. KEY POINTS: • Continuous grapevine planting reduced soil microbe diversities/abundances. • Beneficial bacteria and harmful fungi increased after short-term replanting. • F. solani may be a harmful fungus related to grapevine replant diseases.

An LC-MS/MS method for simultaneous determination of 1,5-dicaffeoylquinic acid and 1-O-acetylbritannilactone in rat plasma and its application to a pharmacokinetic study.

A selective and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the simultaneous quantitative determination of 1,5-dicaffeoylquinic acid (1,5-DCQA) and 1-O-acetylbritannilactone (1-O-ABL) in rat plasma. Chromatographic separation was performed on a Zorbax Eclipse XDB-C18 column using isocratic mobile phase consisting of methanol-water-formic acid (70:30:0.1, v/v/v) at a flow rate of 0.25 mL/min. The detection was achieved using a triple-quadrupole tandem MS in selected reaction monitoring mode. The calibration curves of all analytes in plasma showed good linearity over the concentration ranges of 0.850-213 ng/mL for 1,5-DCQA, and 0.520-130 ng/mL for 1-O-ABL, respectively. The extraction recoveries were ≥78.5%, and the matrix effect ranged from 91.4 to 102.7% in all the plasma samples. The method was successfully applied for the pharmacokinetic study of the two active components in the collected plasma following oral administration of Inula britannica extract in rats.

Exploring pyrimidine-substituted curcumin analogues: design, synthesis and effects on EGFR signaling.

Epidermal growth factor receptor (EGFR) is an effective molecular target of anti-cancer therapies. Curcumin inhibits cancer cell growth in vitro by suppressing gene expression of EGFR and reduces tumor growth in various animal models. To overcome instable and insoluble properties of curcumin as therapeutics, we designed and synthesized six novel pyrimidine-substituted curcumin analogues with or without a hydroxyl group originally present in curcumin. The cell viability tests indicated that IC50 of the analogues containing hydroxyl group were 3 to 8-fold lower than those of the analogues without hydroxyl group in two colon cancer cell lines tested. Western blot analysis indicates the analogues containing hydroxyl group inhibited expression and tyrosine phosphorylation of EGFR. Further protein analyses showed that the analogues had anti-cellular proliferation, pro-apoptosis, and cell cycle arrest properties associated with suppressed EGFR expression. These results indicate that the hydroxyl groups in curcumin and the analogues were critical for observed biological activities.

MiR-23a Regulates the Vasculogenesis of Coronary Artery Disease by Targeting Epidermal Growth Factor Receptor.

OBJECTIVE: Circulating microRNAs (miRNAs) in patient body fluids have recently been considered to hold the potential of being novel disease biomarkers and drug targets. We aimed to investigate the correlation between the levels of circulating miR-23a and the expression of epidermal growth factor receptor (EGFR) in the pathogenesis of patients with coronary heart disease to further explore the mechanism involved in its vasculogenesis. METHOD: Three different cohorts, including 13 acute myocardial infarction (AMI) patients, 176 angina pectoris patients, and 127 control subjects, were enrolled to investigate the expression levels of circulating miR-23a in patients with myocardial ischemia and also the relationship between plasma miR-23a and severity of coronary stenosis. Plasma miR-23a levels of participants were examined by real-time quantitative PCR. Simultaneously, plasma cardiac troponin I (cTnI) concentrations were measured by ELISAs. We further detected the correlation of miR-23a and EGFR by molecular and animal assays. RESULT: MiR-23a was enriched in not only diseased endothelial progenitor cells (EPCs) but also in the plasma of patients with coronary artery disease (CAD). Besides, we found out miR-23a was able to suppress EGFR expression and EPC activities. Reporter assays confirmed the direct binding and repression of miR-23a to the 3'-UTR of EGFR mRNA. Knockdown of miR-23a not only restored EGFR levels and angiogenic activities of diseased EPCs in vitro, but further promoted blood flow recovery in ischemic limbs of mice. CONCLUSION: Circulating miR-23a may be a new biomarker for CAD and as a potential diagnostic tool. And increased miR-23a level may be used to predict the presence and severity of coronary lesions in patients with CAD.

Isothiouronium modification empowers pyrimidine-substituted curcumin analogs potent cytotoxicity and Golgi localization.

Most of protein post-translational modifications occur in the Golgi and many human diseases are associated with abnormal Golgi function or improper post translational modifications of proteins in the Golgi. In this study, we designed and synthesized 4 × 6 series of novel isothiouronium-modified (E,E)-4,6-bis(styryl)-pyrimidine analogs and found that they localized at the Golgi as visualized by the intrinsic fluorescence of the analogs. The isothiouronium-modified analogs had potent cytotoxicity in both normal (Chinese Hamster Ovary or CHO) and cancer cells. Furthermore, permethylated isothiouronium-modified analogs showed cancer cell-selective cytotoxicity. The molecular mechanisms underlying Golgi localization of isothiouronium-modified compounds were investigated using 7 CHO and 4 human cancer cell lines and the results indicated that the compounds had binding partners in the Golgi. Thus, isothiouronium-modified analogs might be promising anticancer agents, novel Golgi staining reagents, and useful research tools for studying Golgi functions in normal or cancer cells and in Golgi-related human diseases.

Efficient synthesis of jusbetonin, an indolo[3,2-b]quinoline glycoside, and its derivatives.

Jusbetonin, an indolo[3,2-b]quinoline alkaloid glycoside originally isolated from Justicia betonica, and its derivatives were synthesized. The key steps in the synthetic strategy were the construction of indolo[3,2-b]quinoline skeleton and efficient coupling with the saccharides, in which the alpha-D-glycopyranosyl bromides were shown to be effective donors. Primary screening showed that all synthesized compounds possessed moderate proliferation inhibitory activity.

Synthesis and analysis of potential DNA intercalators containing quinoline-glucose hybrids.

A new series of potential DNA bisintercalators have been designed by linking 8-hydroxy-quinoline to the 6-CH(2)OH group of glucose and connecting the 1-OH group of glucose with various linkers, such as quinol, glycol, and triethylene glycol. These new compounds were well-synthesized and fully characterized. Preliminary binding assays of these compounds to calf thymus DNA (CT-DNA) were conducted using UV-absorption and fluorescence spectroscopy analysis. The primary antitumor activity of these compounds was also performed.