Evolving ω-amine transaminase AtATA guided by substrate-enzyme binding free energy for enhancing activity and stability against non-natural substrates.

In the field of chiral amine synthesis, ω-amine transaminase (ω-ATA) is one of the most established enzymes capable of asymmetric amination under optimal conditions. However, the applicability of ω-ATA toward more non-natural complex molecules remains limited due to its low transamination activity, thermostability, and narrow substrate scope. Here, by employing a combined approach of computational virtual screening strategy and combinatorial active-site saturation test/iterative saturation mutagenesis strategy, we have constructed the best variant M14C3-V5 (M14C3-V62A-V116S-E117I-L118I-V147F) with improved ω-ATA from Aspergillus terreus (AtATA) activity and thermostability toward non-natural substrate 1-acetylnaphthalene, which is the ketone precursor for producing the intermediate (R)-(+)-1-(1-naphthyl)ethylamine [(R)-NEA] of cinacalcet hydrochloride, showing activity enhancement of up to 3.4-fold compared to parent enzyme M14C3 (AtATA-F115L-M150C-H210N-M280C-V149A-L182F-L187F). The computational tools YASARA, Discovery Studio, Amber, and FoldX were applied for predicting mutation hotspots based on substrate-enzyme binding free energies and to show the possible mechanism with features related to AtATA structure, catalytic activity, and stability in silico analyses. M14C3-V5 achieved 71.8% conversion toward 50 mM 1-acetylnaphthalene in a 50 mL preparative-scale reaction for preparing (R)-NEA. Moreover, M14C3-V5 expanded the substrate scope toward aromatic ketone compounds. The generated virtual screening strategy based on the changes in binding free energies has successfully predicted the AtATA activity toward 1-acetylnaphthalene and related substrates. Together with experimental data, these approaches can serve as a gateway to explore desirable performances, expand enzyme-substrate scope, and accelerate biocatalysis.IMPORTANCEChiral amine is a crucial compound with many valuable applications. Their asymmetric synthesis employing ω-amine transaminases (ω-ATAs) is considered an attractive method. However, most ω-ATAs exhibit low activity and stability toward various non-natural substrates, which limits their industrial application. In this work, protein engineering strategy and computer-aided design are performed to evolve the activity and stability of ω-ATA from Aspergillus terreus toward non-natural substrates. After five rounds of mutations, the best variant, M14C3-V5, is obtained, showing better catalytic efficiency toward 1-acetylnaphthalene and higher thermostability than the original enzyme, M14C3. The robust combinational variant acquired displayed significant application value for pushing the asymmetric synthesis of aromatic chiral amines to a higher level.

Efficacy of scleral buckling for the treatment of rhegmatogenous retinal detachment using a novel foldable capsular buckle.

AIM: To evaluate the effectiveness and safety of scleral buckling for the treatment of rhegmatogenous retinal detachment (RRD) using a novel foldable capsular buckle (FCB). METHODS: This was a series of case observation studies. Eighteen patients (18 eyes) who visited our ophthalmology department between August 2020 and August 2022 and were treated for RRD with scleral buckling using FCB were included. The procedure was similar to conventional scleral buckling, while a balloon-like FCB was placed onto the retinal break with balanced salt solution filling for a broad, external indentation instead of the silicone buckle. The retinal reattachment rate, best corrected visual acuity (BCVA), intraocular pressure (IOP), refractive dioptre and astigmatism degree, and complications were evaluated and recorded. RESULTS: There were 7 males and 11 females aged 19-58y. The average time course of RRD was 12d, ranging from 7-20d. The retinal break was located in the superior quadrants in 8 eyes and in the inferior quadrants in 10 eyes, with macula-off detachments in 12 eyes. The patients were followed-up for at least 6mo. The final retinal reattachment rate was 100%. The BCVA was significantly improved compared with the baseline (P<0.05). There was no significant change in refractive dioptre or astigmatism degree at each follow-up (all P>0.05). Three patients had transiently high IOPs within one week after surgery. Mild diplopia occurred in 5 patients after surgery and then disappeared after the balloon fluid was removed. CONCLUSION: The success rate of FCB scleral buckling for RRD is satisfactory. This procedure can be expected to be applied in new, uncomplicated cases of RRD.

Contrast-enhanced ultrasound evaluation of renal perfusion before angioplasty and its predictive value for hypertension.

BACKGROUND: Atherosclerotic renal artery stenosis (ARAS) is a common disease in the elderly population. OBJECTIVE: The aim was to develop a contrast-enhanced ultrasound (CEUS)-based model for predicting post-angioplasty improvement in hypertension in patients with severe ARAS. METHODS: Thirty-five patients with severe ARAS (⩾ 70%) were included in this study, and 42 renal arteries received percutaneous transluminal renal arterial stenting. An optimal integral formula was developed from pre-interventional color-coded duplex sonography (CCDS) and CEUS parameters using least absolute shrinkage and selection operator (LASSO) regression and receiver operating characteristic (ROC) curve analysis. A model for predicting short-term hypertension improvement was established using the integral formula and clinical risk factors. Bootstrapping was used for internal validation. RESULTS: Two integral formulas, LASSO.CCDS and LASSO.CEUS, were established. ROC curves of the two integral formulas showed that LASSO.CEUS was the better formula for predicting hypertension improvement (AUC 0.816, specificity 78.6%). Univariate and multivariate regression analyses showed that duration of hypertension (OR 0.841, P= 0.027), diabetes (OR = 0.019, P= 0.010), and LASSO.CEUS (OR 7.641, P= 0.052) were predictors of short-term hypertension improvement after interventional therapy. Using LASSO.CEUS combined with clinical risk factors, the following prediction model was established: logit (short-term improvement in hypertension) = 1.879-0.173 × hypertension duration - 3.961 × diabetes + 2.034 × LASSO.CEUS (AUC 0.939). CONCLUSIONS: The model established using CEUS parameters and clinical risk factors could predict hypertension improvement after interventional therapy, but further research and verification are needed.

Stereoselective synthesis of (R)-(+)-1-(1-naphthyl)ethylamine by ω-amine transaminase immobilized on amino modified multi-walled carbon nanotubes and biocatalyst recycling.

Immobilized enzymes exhibit favorable advantages in biocatalysis, such as high operation stability, feasible reusability, and improved organic solvents tolerance. Herein, an immobilized ω-amine transaminase AtATA@MWCNTs-NH2 is successfully prepared using amino modified multi-walled carbon nanotubes as carrier and glutaraldehyde as crosslinker. Under the optimum immobilization conditions, the activity recovery is 78.7%. Compared with purified enzyme AtATA, AtATA@MWCNTs-NH2 possesses superior stability, even in harsh conditions (e.g., high temperature, acidic or alkali environment, and different kind of organic solvents). To simplify the separation and extraction of products, we choose methanol (10%, v/v) as the cosolvent, replacing DMSO (20%, v/v) in our previous work, for the catalytic reaction of AtATA@MWCNTs-NH2. AtATA@MWCNTs-NH2 can be used for stereoselective synthesis (R)-(+)- 1(1-naphthyl)ethylamine ((R)-NEA) for 15 cycles, with the e.e.p (enantiomeric excess) > 99.5%. The catalytic process of AtATA@MWCNTs-NH2 achieves cycle production of (R)-NEA using methanol as cosolvent.

[Related factors of endogenous erythropoietin and its association with 10-year risks of cardiovascular disease in a community-based Chinese study].

OBJECTIVE: To investigate the associated factors of endogenous erythropoietin (EPO) and its association with 10-year risks of atherosclerotic cardiovascular disease in a Chinese community-based general population. METHODS: The participants of this study were from an atherosclerosis cohort survey which was established by the Department of Cardiology, Peking University First Hospital in 2011. The cohort survey was performed in the Gucheng and Pingguoyuan communities of Shijingshan district in Beijing, China. The inclusion criteria of this study were: (1) endogenous EPO was measured; (2) health questionnaire data and other clinical data were complete; (3) participatants who had cardiovascular or cerebrovascular diseases (defined as self-reported coronary heart disease, stroke or transient ischemic attack) or anemia or estimated glomerular filtration rate (eGFR) < 60 mL/(min·1.73 m2) at baseline were excluded. Multivariate linear regression model was used to examine the associated factors of endogenous EPO. The participants were grouped into low (< 5%), moderate (5%-10%) and high risk (≥10%) groups, based on predicted 10-year cardiovascular disease risk using the prediction for atherosclerotic cardiovascular disease risk in China (China-PAR) equations. RESULTS: A total of 4 013 participants were included. Mean age of them was (55.9±8.2) years, 62.2% (n=2 496) of them were female, and 46.3% (n=1 859), 70.9% (n=2 845), 21.9% (n=879) had hypertension, dyslipidemia and diabetes, individually. The average body mass index was (26.1±3.3) kg/m2. The median of EPO level was 12.8 (9.3-17.4) IU/L and 25.1% (n=998) were at high 10-years risk of cardiovascular disease. Hemoglobin (ß=-0.05, 95%CI: -0.07 to -0.04) and eGFR ≥90 mL/(min·1.73 m2) (ß=-0.05, 95%CI: -0.07 to -0.04) were associated with lower in transformed EPO levels while hypertension (ß=0.08, 95%CI: 0.05 to 0.12) and obesity (ß=0.14, 95%CI: 0.09 to 0.18) were associated with higher in transformed EPO levels in multivariate linear regression analyses. Ten-year cardiovascular disease risks were positively associated with in transformed EPO levels (ß=0.07, 95%CI: 0.05 to 0.09). The participants at moderate and high cardiovascular disease risks had significant higher EPO levels than the low risk group (all P < 0.05). CONCLUSION: In community-based Beijing populations, endogenous EPO was associated with hemoglobin, renal function, obesity and hypertension. Individuals at high 10-years cardiovascular disease risks have higher endogenous EPO levels. Endogenous EPO may be a potential risk marker of cardiovascular disease.

Thirty-day outcomes of in-hospital multi-vessel versus culprit-only revascularization strategy for ST-segment elevation myocardial infarction with multivessel coronary disease.

BACKGROUND: Many studies have demonstrated the benefit of complete multivessel revascularization versus culprit-only intervention in patients of ST-segment elevation myocardial infarction (STEMI) and multivessel coronary artery disease. However, only a few single-center retrospective studies were performed on small Chinese cohorts. Our study aims to demonstrate the advantage of multivessel percutaneous intervention (PCI) strategy on 30-day in-hospital outcomes to patients with STEMI and multivessel disease in larger Chinese population. METHODS: From the Improving Care for Cardiovascular Disease in China-Acute Coronary Syndrome (CCC-ACS) project, 5935 patients with STEMI and multivessel disease undergoing PCI and hospitalized for fewer than 30 days were analyzed. After 5: 1 propensity score matching, 3577 patients with culprit-only PCI and 877 with in-hospital multivessel PCI were included. The primary outcome was major adverse cardiovascular and cerebrovascular event (MACCE), defined as a composite of myocardial infarction, all-cause death, stent thrombosis, heart failure, and stroke. RESULTS: Multivariable logistic regression analysis revealed that in-hospital multivessel PCI was associated with lower risk of 30-day MACCE (adjusted OR = 0.75, 95% CI: 0.57-0.98, P = 0.032) than culprit-only PCI and conferred no increased risk of all-cause death, myocardial infarction, stent thrombosis, stroke, or bleeding. Subgroup analysis showed that MACCE reduction was observed more often from patients with trans-femoral access (OR = 0.34, 95% CI: 0.15-0.74) than with trans-radial access (OR = 0.87, 95% CI: 0.66-1.16, P for interaction = 0.017). CONCLUSIONS: The in-hospital multivessel PCI strategy was associated with a lower risk of 30-day MACCE than culprit-only PCI in patients with STEMI and multivessel coronary artery disease.

Comparison of the efficacy and safety of ultrasonic cycloplasty vs valve implantation and anti-VEGF for the treatment of fundus disease-related neovascular glaucoma.

AIM: To compare the clinical efficacy and safety of ultrasonic cycloplasty (UCP) vs Ahmed glaucoma drainage valve implantation (ADV) in addition to intravitreal anti-vascular endothelial growth factor (VEGF) for treatment of fundus disease-related neovascular glaucoma (NVG). METHODS: A total of 43 patients (45 eyes) with NVG secondary to fundus diseases underwent anti-VEGF combined with UCP or ADV from August 2020 to March 2022 were enrolled in this retrospective cohort study. Of them, 14 patients (15 eyes) were treated with both UCP and anti-VEGF as the UCP group and 29 patients (30 eyes) treated with both ADV and anti-VEGF as the ADV group. The success of the treatment was defined as intraocular pressure (IOP) between 11-20 mm Hg with or without using IOP-lowering drugs. IOP measurement, IOP lowering drugs at baseline and follow-ups period and complications were recorded. RESULTS: The average age was 63.03±9.95 and 52.27±12.89y in ADV and UCP groups, respectively (P=1.947). The fundus pathology included proliferative diabetic retinopathy in 42 eyes and retinal vein occlusion in 3 eyes. All eyes in both groups achieved successful treatment at 3mo. While the success rate was 90.0% (27/30) in the ADV group and 86.7% (13/15) in the UCP group at the last follow-up of 6mo (P>0.05). IOP was significantly lower with reduction of drug use than the baseline in both groups (both P<0.05). And the ADV group needed fewer anti-glaucoma drops than the UCP group from 1d to 3mo. The comfort scores of patients in the ADV group were significantly lower than those in the UCP group in the first week after the operation (P<0.05). CONCLUSION: UCP is an alternative to the ADV with the same efficacy but non-invasive for the treatment of NVG.

Enhancing the organic solvent resistance of ω-amine transaminase for enantioselective synthesis of (R)-(+)-1(1-naphthyl)-ethylamine.

BACKGROUND: Biocatalysis in high-concentration organic solvents has been applied to produce various industrial products with many advantages. However, using enzymes in organic solvents often suffers from inactivation or decreased catalytic activity and stability. An R-selective ω-amine transaminase from Aspergillus terreus (AtATA) exhibited activity toward 1-acetylnaphthalene. However, AtATA displayed unsatisfactory organic solvent resistance, which is required to enhance the solubility of the hydrophobic substrate 1-acetylnaphthalene. So, improving the tolerance of enzymes in organic solvents is essential. MAIN METHODS AND RESULTS: The method of regional random mutation combined with combinatorial mutation was used to improve the resistance of AtATA in organic solvents. Enzyme surface areas are structural elements that undergo reversible conformational transitions, thus affecting the stability of the enzyme in organic solvents. Herein, three surface areas containing three loops were selected as potential mutation regions. And the "best" mutant T23I/T200K/P260S (M3) was acquired. In different concentrations of dimethyl sulfoxide (DMSO), the catalytic efficiency (kcat /Km ) toward 1-acetylnaphthalene and the stability (half-life t1/2 ) were higher than the wild-type (WT) of AtATA. The results of decreased Root Mean Square Fluctuation (RMSF) values via 20-ns molecular dynamics (MD) simulations under 15%, 25%, 35%, and 45% DMSO revealed that mutant M3 had lower flexibility, acquiring a more stable protein structure and contributing to its organic solvents stability than WT. Furthermore, M3 was applied to convert 1-acetylnaphthalene for synthesizing (R)-(+)-1(1-naphthyl)-ethylamine ((R)-NEA), which was an intermediate of Cinacalcet Hydrochloride for the treatment of secondary hyperthyroidism and hypercalcemia. Moreover, in a 20-mL scale-up experiment, 10 mM 1-acetylnaphthalene can be converted to (R)-NEA with 85.2% yield and a strict R-stereoselectivity (enantiomeric excess (e.e.) value >99.5%) within 10 h under 25% DMSO. CONCLUSION: The beneficial mutation sites were identified to tailor AtATA's organic solvents stability via regional random mutation. The "best" mutant T23I/T200K/P260S (M3) holds great potential application for the synthesis of (R)-NEA.

Diagnosis and Treatment of Water-Contaminated Severe Legionella Pneumonia with Digestive Symptoms as the First Symptom: A Case Report and Review of the Literature.

Introduction: Although Legionella is not the most common pathogen of community-acquired pneumonia, the epidemiological distribution of pneumonia pathogens has changed in recent years, with a gradual increase in some rare pathogens. For example, pneumonia that occurs after water source contamination is mostly caused by Legionella infection. This paper reports the diagnosis and treatment process of a patient after Legionella infection, who had misdiagnosis at the beginning, rapidly progressed to severe disease and combined with fungal infection. This article focuses on the timely and effective treatment of rapidly progressing Legionella pneumonia, in anticipation of a better understanding of the diagnosis and treatment of the disease. Case Presentation: Here, we report a case of legionella infection with the nausea, vomiting as the first symptoms accompanied by weakness, chills, dizziness, abdominal discomfort in a 75-year-old female. The patient had a history of type 2 diabetes for 30 years, diabetic peripheral neuropathy for more than 20 years, arterial hypertension for 10 years, bone hyperplasia for more than 5 years, resection of right-sided thyroid cystadenoma in 1990. The patient had firstly been diagnosed with cholecystitis and gallbladder neck stones, diet abstinence, metronidazole, cefoperazone sulbactam, and rehydration were given. The patient responded poorly to these empiric treatments. The patient was given moxifloxacin in combination with azithromycin after the onset of respiratory symptoms, but the condition continued to deteriorate, and tigecycline was subsequently added. After the mechanical ventilation and the treatment plan adjusting, she improved significantly. Conclusion: Immunocompromised patient combined with underlying diseases are more susceptible to infection in an environment contaminated with Legionella, and the rapid onset and atypical respiratory symptoms make it easy to misdiagnose the disease, thus delaying treatment and leading to further deterioration. Timely diagnosis, early mechanical ventilation and rational drug administration were fundamental to treat Legionella pneumonia.

[Extraction of Extracelluar Vesicles Derived from Mycobacterium tuberculosis and Their Effect on the Production of Reactive Oxygen Species and Expression of Inflammatory Factors in Mouse Bone Marrow-Derived Dendritic Cells].

Objective: To isolate extracellular vesicles (EVs) from Mycobacterium tuberculosis ( Mtb), to examine their morphology, particle size, and distribution, to study the effect of EVs derived from Mtb ( Mtb-EVs) on intracellular reactive oxygen species (ROS) production and cytokine secretion in dendritic cells (DCs), and to make preliminary exploration of Mtb-EVs' effect on the immune regulation of DCs. Methods: Mtb-EVs were obtained by ultrafiltration concentration and the protein concentration was determined by BCA assay. The morphology of Mtb-EVs was observed through negative staining electron microscopy (EM). The particle size distribution and concentration of Mtb-EVs were determined by nanoparticle tracking analysis (NTA). Mouse bone marrow was isolated through sterile procedures and mice myeloid DCs were induced and amplified by the combined use of recombinant mouse granulocyte-macrophage colony-stimulating factor (rm GM-CSF) and recombinant mouse interleukin-4 (rm IL-4). Then, morphological and immunophenotypic characterization was performed. After that, the DCs were treated with Mtb-EVs at different concentrations and CCK-8 assay was done to measure their effect on the survival rate of DCs and to identify the appropriate stimulation concentration for subsequent experimental procedures. The intracellular ROS levels of DCs were evaluated with DCFH-DA fluorescence probe and the cytokine secretion of DCs was determined by ELISA. Results: EM observation showed that Mtb-EVs isolated by ultrafiltration concentration were spherical vesicles of varied sizes, all being approximately 100 nm in diameter and displaying typical morphology. NTA results from NanoSight nanoparticle tracker showed that the peak particle size was 98.5 nm, that the average particle size was 110.2 nm, and that the particle size was mainly distributed between 68.4-155.7 nm. Mtb-EVs that were smaller than 250 nm accounted for 98.39% of the total. Mouse myeloid DCs directionally induced and amplified in vitro displayed typical DC phenotype and morphology, and the purity exceeded 85%. EM verified the abundance of microvilli and radial protuberance on the surface of DCs, which had uniform cytoplasm and clear nuclear membrane. Loaded with Mtb-EVs at different concentrations, including 10 2, 10 3, and 10 4 particles/cell, the DCs had significantly upregulated levels of intracellular ROS ( P<0.05). In addition, Mtb-EVs induced the release of IL-1ß and IL-6 in a dose-dependent manner ( P<0.05). Conclusion: We established in the study a technical process for the extraction of Mtb-EVs by ultrafiltration concentration and obtained Mtb-EVs with sound morphology, high purity, and concentrated particle size distribution. Furthermore, Mtb-EVs can upregulate the intracellular ROS level in DCs and induce the release of IL-1ß and IL-6 in a dose-dependent manner.