Myeloid-Mas Signaling Modulates Pathogenic Crosstalk among MYC+CD63+ Endothelial Cells, MMP12+ Macrophages, and Monocytes in Acetaminophen-Induced Liver Injury.

Acetaminophen overdose is a leading cause of acute liver failure (ALF). Despite the pivotal role of the inflammatory microenvironment in the progression of advanced acetaminophen-induced liver injury (AILI), a comprehensive understanding of the underlying cellular interactions and molecular mechanisms remains elusive. Mas is a G protein-coupled receptor highly expressed by myeloid cells; however, its role in the AILI microenvironment remains to be elucidated. A multidimensional approach, including single-cell RNA sequencing, spatial transcriptomics, and hour-long intravital imaging, is employed to characterize the microenvironment in Mas1 deficient mice at the systemic and cell-specific levels. The characteristic landscape of mouse AILI models involves reciprocal cellular communication among MYC+CD63+ endothelial cells, MMP12+ macrophages, and monocytes, which is maintained by enhanced glycolysis and the NF-κB/TNF-α signaling pathway due to myeloid-Mas deficiency. Importantly, the pathogenic microenvironment is delineated in samples obtained from patients with ALF, demonstrating its clinical relevance. In summary, these findings greatly enhance the understanding of the microenvironment in advanced AILI and offer potential avenues for patient stratification and identification of novel therapeutic targets.

Prognostic value of virtual portal pressure gradient response in compensated cirrhotic patients treated with carvedilol.

AIM: This study aimed to assess the prognostic significance of virtual portal pressure gradient (vPPG) response to carvedilol in patients with compensated cirrhosis (CC). METHODS: Compensated cirrhosis patients with high-risk varices were prospectively enrolled to receive carvedilol for prevention of first variceal hemorrhage (VH) and followed up for 1 year. The vPPG response was defined as a reduction of vPPG >10% from baseline after 1-month therapy. Logistic and Cox regression analyses were performed to identify independent predictors for vPPG response and first decompensation, respectively. Competitive risk models were constructed to predict disease progression, and validated using the C-index, Kaplan-Meier analysis, competitive risk analysis, and calibration curves. RESULTS: A total of 129 patients completed this study, of whom 56 (43.4%) achieved vPPG response and were referred as vPPG responders. Baseline vPPG, red color sign, Model for End-stage Liver Disease score, serum monocyte chemoattractant protein-1 (MCP-1), and laminin levels significantly correlated with vPPG response, which itself was further documented as an independent predictor of VH, ascites, and overall decompensation events in CC. Moreover, the red color sign or Child-Turcotte-Pugh score effectively predicted VH, while ascites correlated well with portal flow velocity or MCP-1. The predictive models for VH and ascites showed a good discrimination with C-index values of 0.747 and 0.689 respectively, and the high consistency on calibration curves. CONCLUSION: The vPPG response could be used as a noninvasive tool for prediction of disease progression in patients with CC.

Dual-Function Meta-Grating Based on Tunable Fano Resonance for Reflective Filter and Sensor Applications.

Localized surface plasmon resonance (LSPR)-based sensors exhibit enormous potential in the areas of medical diagnosis, food safety regulation and environmental monitoring. However, the broadband spectral lineshape of LSPR hampers the observation of wavelength shifts in sensing processes, thus preventing its widespread applications in sensors. Here, we describe an improved plasmonic sensor based on Fano resonances between LSPR and the Rayleigh anomaly (RA) in a metal-insulator-metal (MIM) meta-grating, which is composed of silver nanoshell array, an isolation grating mask and a continuous gold film. The MIM configuration offers more freedom to control the optical properties of LSPR, RA and the Fano resonance between them. Strong couplings between LSPR and RA formed a series of narrowband reflection peaks (with a linewidth of ~20 nm in full width at half maximum (FWHM) and a reflectivity nearing 100%) within an LSPR-based broadband extinction window in the experiment, making the meta-grating promising for applications of high-efficiency reflective filters. A Fano resonance that is well optimized between LSPR and RA by carefully adjusting the angles of incident light can switch such a nano-device to an improved biological/chemical sensor with a figure of merit (FOM) larger than 57 and capability of detecting the local refractive index changes caused by the bonding of target molecules on the surface of the nano-device. The figure of merit of the hybrid sensor in the detection of target molecules is 6 and 15 times higher than that of the simple RA- and LSPR-based sensors, respectively.

Development and benchmarking of machine learning models to classify patients suitable for outpatient lower extremity joint arthroplasty.

STUDY OBJECTIVE: Performing hip or knee arthroplasty as an outpatient surgery has been shown to be operationally and financially beneficial for selected patients. By applying machine learning models to predict patients suitable for outpatient arthroplasty, health care systems can better utilize resources efficiently. The goal of this study was to develop predictive models for identifying patients likely to be discharged same-day following hip or knee arthroplasty. DESIGN: Model performance was assessed with 10-fold stratified cross-validation, evaluated over baseline determined by the proportion of eligible outpatient arthroplasty over sample size. The models used for classification were logistic regression, support vector classifier, balanced random forest, balanced bagging XGBoost classifier, and balanced bagging LightGBM classifier. SETTING: The patient records were sampled from arthroplasty procedures at a single institution from October 2013 to November 2021. PATIENTS: The electronic intake records of 7322 knee and hip arthroplasty patients were sampled for the dataset. After data processing, 5523 records were kept for model training and validation. INTERVENTIONS: None. MEASUREMENTS: The primary measures for the models were the F1-score, area under the receiver operating characteristic curve (ROCAUC), and area under the precision-recall curve. To measure feature importance, the SHapley Additive exPlanations value (SHAP) were reported from the model with the highest F1-score. RESULTS: The best performing classifier (balanced random forest classifier) achieved an F1-score of 0.347: an improvement of 0.174 over baseline and 0.031 over logistic regression. The ROCAUC for this model was 0.734. Using SHAP, the top determinant features of the model included patient sex, surgical approach, surgery type, and body mass index. CONCLUSIONS: Machine learning models may utilize electronic health records to screen arthroplasty procedures for outpatient eligibility. Tree-based models demonstrated superior performance in this study.

Hepatocyte-specific Mas activation enhances lipophagy and fatty acid oxidation to protect against acetaminophen-induced hepatotoxicity in mice.

BACKGROUND & AIMS: Acetaminophen (APAP) is the most common cause of drug-induced liver injury (DILI); however, treatment options are limited. Mas is a G protein-coupled receptor whose role in APAP-induced hepatotoxicity has not yet been examined. METHODS: Intrahepatic Mas expression was determined in both human and mouse DILI models. Mas1-/-, AlbcreMas1f/f, Ppara-/-, Mas1-/-Ppara-/- and wild-type mice were challenged with APAP for the in vivo analyses of Mas-AKT-FOXO1 axis-dependent lipophagy and fatty acid oxidation (FAO), using pharmacological compounds and genetic tools. Liver samples were collected for RNA-sequencing, proteomics, metabolomics, lipidomics, and metabolic flux analysis. Live-imaging of liver and histological, biochemical, and molecular studies were performed to evaluate APAP-induced hepatotoxicity in mice. Primary hepatocytes and hepatic cell lines were exposed to APAP for in vitro analysis. RESULTS: Intrahepatic Mas expression was significantly upregulated in human and mouse DILI models. Mice with systemic, liver-specific, or hepatocyte-specific Mas1 deficiency were vulnerable to APAP-induced hepatotoxicity. They exhibited substantially impaired lipophagy and downstream FAO, which was accompanied by the activation of AKT and suppression of FOXO1. In addition, the prophylactic activation of Mas conferred strong protection against APAP challenge in mice, with remarkably enhanced lipophagy and FAO dependent on the AKT-FOXO1 axis. Moreover, the protective effects of AVE0991 were substantially diminished by the inhibition of either lipophagy or FAO. CONCLUSIONS: The activation of Mas on hepatocytes enhanced AKT-FOXO1-dependent lipophagy and downstream FAO, protecting mice from APAP-induced hepatotoxicity and indicating that hepatocyte-specific Mas might be a novel therapeutic target for DILI. IMPACT AND IMPLICATIONS: Mas signalling arises as a novel therapeutic target for patients with APAP-induced liver injury. The Mas-AKT/FOXO1-fatty acid degradation pathway could be critical for the development of treatment strategies for APAP overdose. When Mas signalling is targeted, the extent of liver injury should be considered at the time of administration. These findings obtained from APAP-challenged mice still need to be confirmed in a clinical context.

From Plant to Yeast-Advances in Biosynthesis of Artemisinin.

Malaria is a life-threatening disease. Artemisinin-based combination therapy (ACT) is the preferred choice for malaria treatment recommended by the World Health Organization. At present, the main source of artemisinin is extracted from Artemisia annua; however, the artemisinin content in A. annua is only 0.1-1%, which cannot meet global demand. Meanwhile, the chemical synthesis of artemisinin has disadvantages such as complicated steps, high cost and low yield. Therefore, the application of the synthetic biology approach to produce artemisinin in vivo has magnificent prospects. In this review, the biosynthesis pathway of artemisinin was summarized. Then we discussed the advances in the heterologous biosynthesis of artemisinin using microorganisms (Escherichia coli and Saccharomyces cerevisiae) as chassis cells. With yeast as the cell factory, the production of artemisinin was transferred from plant to yeast. Through the optimization of the fermentation process, the yield of artemisinic acid reached 25 g/L, thereby producing the semi-synthesis of artemisinin. Moreover, we reviewed the genetic engineering in A. annua to improve the artemisinin content, which included overexpressing artemisinin biosynthesis pathway genes, blocking key genes in competitive pathways, and regulating the expression of transcription factors related to artemisinin biosynthesis. Finally, the research progress of artemisinin production in other plants (Nicotiana, Physcomitrella, etc.) was discussed. The current advances in artemisinin biosynthesis may help lay the foundation for the remarkable up-regulation of artemisinin production in A. annua through gene editing or molecular design breeding in the future.

A non-invasive model based on the virtual portal pressure gradient to predict the first variceal hemorrhage in cirrhotic patients.

BACKGROUND AND AIMS: This study aimed to establish a non-invasive model based on the virtual portal pressure gradient (vPPG) to predict the first variceal hemorrhage (VH) in patients with cirrhosis. METHODS: This single-center study prospectively enrolled cirrhotic patients as the training and validation cohorts during different time periods. The PPG-detection software (PPGS 1.0) was used to perform vPPG calculation, which involves 2 steps including three-dimensional (3D) reconstruction of portal vein tree and subsequent application of computational fluid dynamics. All patients were given standard primary prophylaxis against VH and followed up for 2 years. Data from the training cohort were assessed using univariate and multivariate Cox regression and Kaplan-Meier analyses, by which a nomogram with its dynamic form was developed to estimate the probability of VH. RESULTS: In the training cohort (n = 128), 37 (28.9%) experienced VH during 2-year follow-up. Four variables including vPPG ≥ 10.5 mmHg (p < 0.001), PLT < 56 × 109/L (p = 0.048), albumin < 32 g/L (p < 0.001) and INR ≥ 1.2 (p = 0.022) were identified as independent risk factors of VH, among which vPPG showed the best diagnostic performance (AUC 0.875). Subsequently, these predictors were incorporated into the nomogram, of which C-indexes were 0.891 and 0.926 for the training and validation cohorts, respectively. Calibration curves demonstrated a great calibration ability of the model. At the threshold probabilities of 0.1-0.6 (1 year) and 0.1-1.0 (2 years), this nomogram could offer more net benefits in decision curve analysis. CONCLUSIONS: The vPPG-based nomogram could be used for risk stratification of the first VH in patients with cirrhosis.

Hardware-Assisted Security Monitoring Unit for Real-Time Ensuring Secure Instruction Execution and Data Processing in Embedded Systems.

The hardware security of embedded systems is raising more and more concerns in numerous safety-critical applications, such as in the automotive, aerospace, avionic, and railway systems. Embedded systems are gaining popularity in these safety-sensitive sectors with high performance, low power, and great reliability, which are ideal control platforms for executing instruction operation and data processing. However, modern embedded systems are still exposing many potential hardware vulnerabilities to malicious attacks, including software-level and hardware-level attacks; these can cause program execution failure and confidential data leakage. For this reason, this paper presents a novel embedded system by integrating a hardware-assisted security monitoring unit (SMU), for achieving a reinforced system-on-chip (SoC) on ensuring program execution and data processing security. This architecture design was implemented and evaluated on a Xilinx Virtex-5 FPGA development board. Based on the evaluation of the SMU hardware implementation in terms of performance overhead, security capability, and resource consumption, the experimental results indicate that the SMU does not lead to a significant speed degradation to processor while executing different benchmarks, and its average performance overhead reduces to 2.18% on typical 8-KB I/D-Caches. Security capability evaluation confirms the monitoring effectiveness of SMU against both instruction and data tampering attacks. Meanwhile, the SoC satisfies a good balance between high-security and resource overhead.

IFN-γ facilitates liver fibrogenesis by CD161+CD4+ T cells through a regenerative IL-23/IL-17 axis in chronic hepatitis B virus infection.

OBJECTIVES: This study aimed to determine the role of CD161+CD4+ T cells in chronic hepatitis B virus (HBV) infection. METHODS: A total of 94 patients with chronic hepatitis B (CHB), 73 with liver cirrhosis (LC) and 28 healthy controls were enrolled to determine frequency, cytokine production and chemokine receptor expression of circulating CD161+CD4+ T cells. Among these, 50 CHB and 34 LC patients were followed up for a period of 52-week entecavir monotherapy to assess the association of CD161+CD4+ T cells with seroconversion of HBV e antigen (HBeAg). In addition, 15 patients with hepatocellular carcinoma (HCC) and 15 with hepatic haemangioma (HHA) were enrolled to compare the paired circulating and intrahepatic CD161+CD4+ T cells. RESULTS: CD161+CD4+ T cells were found to accumulate in the circulation of HBV cohorts, which showed a significant correlation with the clinical parameters of disease progression. In addition, higher numbers of circulating CD161+CD4+ T cells were associated with an improved serological response of HBeAg to antiviral treatment. Moreover, CD161+CD4+ T cells as compared to homologous CD161-CD4+ T cells produced more pro-inflammatory cytokines including interleukin (IL)-17 and interferon (IFN)-γ and expressed higher levels of liver-homing chemokine receptors including CCR6, CXCR6 and CX3CR1. Notably, a significant enrichment of CD161+CD4+ T cell subsets co-expressing IFN-γ and IL-17 was observed in HBV-associated cirrhotic livers. During in vitro co-cultures, circulating CD161+CD4+ T cells in the chronic HBV setting exhibited prominent pro-fibrogenic effects by regulating primary hepatic stellate cells through a regenerative IFN-γ/IL-23/IL-17 axis. CONCLUSIONS: In chronic HBV infection, CD161+CD4+ T cells play antiviral, pro-inflammatory and pro-fibrogenic roles.

Engineering Promiscuous Alcohol Dehydrogenase Activity of a Reductive Aminase AspRedAm for Selective Reduction of Biobased Furans.

Catalytic promiscuity is a promising starting point for improving the existing enzymes and even creating novel enzymes. In this work, site-directed mutagenesis was performed to improve promiscuous alcohol dehydrogenase activity of reductive aminase from Aspergillus oryzae (AspRedAm). AspRedAm showed the cofactor preference toward NADPH in reductive aminations, while it favored NADH in the reduction reactions. Some key amino acid residues such as N93, I118, M119, and D169 were identified for mutagenesis by molecular docking. Variant N93A showed the optimal pH and temperature of 8 and 30°C, respectively, in the reduction of 5-hydroxymethylfurfural (HMF). The thermostability was enhanced upon mutation of N93 to alanine. The catalytic efficiency of variant N93A (k cat/K m, 23.6 mM-1 s-1) was approximately 2-fold higher compared to that of the wild-type (WT) enzyme (13.1 mM-1 s-1). The improved catalytic efficiency of this variant may be attributed to the reduced steric hindrance that stems from the smaller side chain of alanine in the substrate-binding pocket. Both the WT enzyme and variant N93A had broad substrate specificity. Escherichia coli (E. coli) cells harboring plain vector enabled selective reduction of biobased furans to target alcohols, with the conversions of 35-95% and the selectivities of >93%. The introduction of variant N93A to E. coli resulted in improved substrate conversions (>98%) and selectivities (>99%).

Bioinspired Cooperative Photobiocatalytic Regeneration of Oxidized Nicotinamide Cofactors for Catalytic Oxidations.

Invited for this month's cover is the group of Ning Li at South China University of Technology. The image shows an efficient photobiocatalytic system to regenerate oxidized nicotinamide cofactors for dehydrogenase-mediated oxidations. The Communication itself is available at 10.1002/cssc.202100184.

Bioinspired Cooperative Photobiocatalytic Regeneration of Oxidized Nicotinamide Cofactors for Catalytic Oxidations.

Inspired by water-forming NAD(P)H oxidases, a cooperative photobiocatalytic system has been designed to aerobically regenerate the oxidized nicotinamide cofactors. Photocatalysts enable NAD(P)H oxidation with O2 under visible-light irradiation, producing H2 O2 as a byproduct, which is subsequently used as an oxidant by the horseradish peroxidase mediator system (PMS) to oxidize NAD(P)H. The photobiocatalytic system shows a turnover frequency of 8800 min-1 in the oxidation of NAD(P)H. Photobiocatalytic NAD(P)H oxidation proceeds smoothly at pH 6-9. In addition to natural NAD(P)H, synthetic biomimetics are also good substrates for this regeneration system. Total turnover numbers of up to 180000 are obtained for the cofactor when the photobiocatalytic regeneration system is coupled with dehydrogenase-catalyzed oxidations. It may be a promising protocol to recycle the oxidized cofactors for catalytic oxidations.

Significance of MR/OPN/HMGB1 axis in NAFLD-associated hepatic fibrogenesis.

AIMS: The activation of hepatic stellate cells (HSCs) plays a central role in liver fibrosis, however non-alcoholic fatty liver disease (NAFLD) associated liver fibrogenesis have been poorly understood. We aimed to determine the significance of mineralocorticoid receptor (MR)/osteopontin (OPN)/high-mobility group box-1 (HMGB1) axis in this setting. MAIN METHODS: Liver specimens were collected from NAFLD patients and murine NAFLD models established with 12-week high fat diet (HFD) for analysis of both upstream signals of MR and intrahepatic MR/OPN/HMGB1 axis. The in vitro cell model of NAFLD-associated liver fibrogenesis was established by treating LX-2 (a cell line of human HSCs) with free fatty acids (FFA). The effects of MR signaling were evaluated using with ALD (MR activator) or eplerenone (Ep, MR antagonist). Moreover, the in vitro loss- and gain- of function approaches were applied to confirm the upstream and downstream relationships of mediators contained in the intracellular MR/OPN/HMGB1 axis of LX-2. KEY FINDINGS: In NAFLD condition, both human and mouse liver tissue samples demonstrated a significant up-regulation of MR/OPN/HMGB1 axis simultaneously with enhanced expression of pro-fibrogenic markers, including ACTA2, TIMP1, TGFB1 and COL1A1. Besides, enhanced production of serum aldosterone (ALD) was also observed in mouse NAFLD models. Moreover, the in vitro data demonstrated MR play an essential role in FFA-induced HSCs fibrogenesis. Meanwhile, MR acts as the upstream effector mediator of OPN and shares downstream HMGB1 with OPN. SIGNIFICANCE: The MR/OPN/HMGB1 axis could be therapeutically targeted to treat NAFLD associated hepatic fibrogenesis.

Osteopontin acts as a negative regulator of autophagy accelerating lipid accumulation during the development of nonalcoholic fatty liver disease.

Accumulating evidence links osteopontin (OPN), a pro-fibrogenic extracellular matrix protein, to the pathogenesis of non-alcoholic fatty liver disease (NAFLD). In this study, liver tissues isolated from non-alcoholic steatohepatitis (NASH) patients expressed higher OPN than those of controls. However, the exact mechanism(s) for this phenomenon is yet to be clarified. Autophagy is the natural, regulated degradation and recycling of a cell's dysfunctional components, in order to maintain homeostasis. Increasing evidence supports that autophagy can constitute an effective Defence mechanism against NAFLD conditions. Herein, we constructed NAFLD mice model by high-fat (HF) and methionine-choline-deficient (MCD) diet and found that OPN is upregulated in livers of NAFLD mice. Besides, secreted OPN inhibited autophagosome-lysosome fusion via binding with its receptors integrin αVß3 and αVß5 in HepG2 cells supplemented with free fatty acids (FFA) and the livers of NAFLD mice. Silencing of OPN attenuated autophagy impairment and reduced lipid accumulation, while supplementation of OPN exhibited the opposite effect. Furthermore, treatment with anti-OPN Ab significantly attenuated steatosis as well as autophagy impairment in the liver. Our findings indicated that OPN plays a vital role in the pathogenesis of the development of NAFLD via autophagy impairment, which might represent a potential new therapeutic target for the treatment of NAFLD.

One-Pot Enzyme Cascade for Controlled Synthesis of Furancarboxylic Acids from 5-Hydroxymethylfurfural by H2 O2 Internal Recycling.

Furancarboxylic acids are promising biobased building blocks in pharmaceutical and polymer industries. In this work, dual-enzyme cascade systems composed of galactose oxidase (GOase) and alcohol dehydrogenases (ADHs) are constructed for controlled synthesis of 5-formyl-2-furancarboxylic acid (FFCA) and 2,5-furandicarboxylic acid (FDCA) from 5-hydroxymethylfurfural (HMF), based on the catalytic promiscuity of ADHs. The byproduct H2 O2 , which is produced in GOase-catalyzed oxidation of HMF to 2,5-diformylfuran (DFF), is used for horseradish peroxidase (HRP)-mediated regeneration of the oxidized nicotinamide cofactors for subsequent oxidation of DFF promoted by an ADH, thus implementing H2 O2 internal recycling. The desired products FFCA and FDCA are obtained with yields of more than 95 %.

Dehydrogenase-Catalyzed Oxidation of Furanics: Exploitation of Hemoglobin Catalytic Promiscuity.

The catalytic promiscuity of hemoglobin (Hb) was explored for regenerating oxidized nicotinamide cofactors [NAD(P)+ ]. With H2 O2 as oxidant, Hb efficiently oxidized NAD(P)H into NAD(P)+ within 30 min. The new NAD(P)+ regeneration system was coupled with horse liver alcohol dehydrogenase (HLADH) for the oxidation of bio-based furanics such as furfural and 5-hydroxymethylfurfural (HMF). The target acids (e.g., 2,5-furandicarboxylic acid, FDCA) were prepared with moderate-to-good yields. The enzymatic regeneration method was applied in l-glutamic dehydrogenase (DH)-mediated oxidative deamination of lglutamate and for l-lactic-DH-mediated oxidation of l-lactate, which furnished α-ketoglutarate and pyruvate in yields of 97 % and 81 %, respectively. A total turnover number (TTON) of up to approximately 5000 for cofactor and an E factor of less than 110 were obtained in the bi-enzymatic cascade synthesis of α-ketoglutarate. Overall, a proof-of-concept based on catalytic promiscuity of Hb was provided for in situ regeneration of NAD(P)+ in DH-catalyzed oxidation reactions.

Effect of milk on antibacterial activity of tetracycline against Escherichia coli and Staphylococcus aureus isolated from bovine mastitis.

The susceptibility of mastitis-causing Escherichia coli and Staphylococcus aureus to two commonly used antibiotics, tetracycline and penicillin G, was tested in raw milk and in Muller-Hinton (MH) broth by introducing a pH indicator, bromocresol purple, which was shown to be a simple, sensitive, and rapid method. The minimum inhibitory concentration (MIC) of penicillin G in milk was the same as those in MH broth, whereas the MIC of tetracycline in milk was 4 to 32 times that in MH. An irreversible binding between tetracycline and large molecules of milk, which might be due to a hydrophobic interaction, was demonstrated by a dialysis test, suggesting the observed impairing effect was due to the action of milk on the tetracycline being tested. Further investigation revealed that much of the reduction of tetracycline's activity in milk was attributable to the milk protein casein, while other heat-sensitive components in milk also play some roles.