Chemokine CCL21 determines immunotherapy response in hepatocellular carcinoma by affecting neutrophil polarization.

BACKGROUND: The efficacy of immune checkpoint inhibitors (ICIs) in hepatocellular carcinoma (HCC) is poor and great heterogeneity among individuals. Chemokines are highly correlated with tumor immune response. Here, we aimed to identify an effective chemokine for predicting the efficacy of immunotherapy in HCC. METHODS: Chemokine C-C motif ligand 21 (CCL21) was screened by transcriptomic analysis in tumor tissues from HCC patients with different responses to ICIs. The least absolute shrinkage and selection operator (LASSO) regression analysis was conducted to construct a predictive nomogram. Neutrophils in vitro and HCC subcutaneous tumor model in vivo were applied to explore the role of CCL21 on the tumor microenvironment (TME) of HCC. RESULTS: Transcriptome analysis showed that CCL21 level was much higher in HCC patients with response to immunotherapy. The predictive nomogram was constructed and validated as a classifier. CCL21 could inhibit N2 neutrophil polarization by suppressing the activation of nuclear factor kappa B (NF-κB) pathway. In addition, CCL21 enhanced the therapeutic efficacy of ICIs. CONCLUSION: CCL21 may serve as a predictive biomarker for immunotherapy response in HCC patients. High levels of CCL21 in TME inhibit immunosuppressive polarization of neutrophils. CCL21 in combination with ICIs may offer a novel therapeutic strategy for HCC.

LINC00665 promotes glycolysis in lung adenocarcinoma cells via the let-7c-5p/HMMR axis.

Lung adenocarcinoma (LUAD) is one of the most lethal and common malignancies. The energy metabolism of LUAD is a critical factor affecting its malignant progression, and research on this topic can aid in the development of novel cancer treatment targets. Bioinformatics analysis of the expression of long non-coding RNA (lncRNA) LINC00665 in LUAD was performed. Downstream regulatory molecules of LINC00665 were predicted using the StarBase database. We used quantitative reverse transcription polymerase chain reaction and western blot to measure the expression at mRNA and protein levels, respectively. The effects of the LINC00665/let-7c-5p/HMMR axis on cell viability in vitro were tested by CCK-8 assay. The regulatory effects on glycolysis were analyzed by extracellular acidification rate, oxygen consumption rate, glucose uptake, adenosine triphosphate production, and lactate production. The predicted competitive endogenous RNA mechanism between LINC00665 and let-7c-5p/HMMR was verified by a dual-luciferase reporter gene assay. LINC00665 was upregulated in LUAD. Silencing LINC00665 inhibited tumor proliferation and reduced the glycolytic activity of tumor cells. Additionally, the expression of LINC00665 had a negative correlation with that of let-7c-5p, while the expression of HMMR was remarkably inhibited by let-7c-5p. HMMR could affect the development of LUAD by influencing glycolytic capacity. Mechanistically, LINC00665 acted as a molecular sponge to absorb let-7c-5p and targeted HMMR. Transfection of let-7c-5p inhibitor or overexpression of HMMR plasmid could reverse the inhibition in proliferation and glycolysis of LUAD cells induced by silencing of LINC00665. In summary, this study demonstrated that the LINC00665/let-7c-5p/HMMR regulatory axis promoted the tumorigenesis of LUAD by enhancing aerobic glycolysis, suggesting that this regulatory axis was an effective target for inhibiting LUAD progression and providing theoretical support for the development of new drugs for LUAD.

Sinomenine attenuates pulmonary fibrosis by downregulating TGF-ß1/Smad3, PI3K/Akt and NF-κB signaling pathways.

BACKGROUND: Since COVID-19 became a global epidemic disease in 2019, pulmonary fibrosis (PF) has become more prevalent among persons with severe infections, with IPF being the most prevalent form. In traditional Chinese medicine, various disorders are treated using Sinomenine (SIN). The SIN's strategy for PF defense is unclear. METHODS: Bleomycin (BLM) was used to induce PF, after which inflammatory factors, lung histological alterations, and the TGF-/Smad signaling pathway were assessed. By administering various dosages of SIN and the TGF- receptor inhibitor SB-431,542 to human embryonic lung fibroblasts (HFL-1) and A549 cells, we were able to examine proliferation and migration as well as the signaling molecules implicated in Epithelial-Mesenchymal Transition (EMT) and Extra-Cellular Matrix (ECM). RESULTS: In vivo, SIN reduced the pathological changes in the lung tissue induced by BLM, reduced the abnormal expression of inflammatory cytokines, and improved the weight and survival rate of mice. In vitro, SIN inhibited the migration and proliferation by inhibiting TGF-ß1/Smad3, PI3K/Akt, and NF-κB pathways, prevented the myofibroblasts (FMT) of HFL-1, reversed the EMT of A549 cells, restored the balance of matrix metalloenzymes, and reduced the expression of ECM proteins. CONCLUSION: SIN attenuated PF by down-regulating TGF-ß/Smad3, PI3K/Akt, and NF-κB signaling pathways, being a potential effective drug in the treatment of PF.

Multivalent Targeting of the Asialoglycoprotein Receptor by Virus-Like Particles.

The asialoglycoprotein receptor (ASGPR) is expressed in high density on hepatocytes. Multivalent variants of galactosyl carbohydrates bind ASGPR with high affinity, enabling hepatic delivery of ligand-bound cargo. Virus-like particle (VLP) conjugates of a relatively high-affinity ligand were efficiently endocytosed by ASGPR-expressing cells in a manner strongly dependent on the nature and density of ligand display, with the best formulation using a nanomolar-, but not a picomolar-level, binder. Optimized particles were taken up by HepG2 cells with greater efficiency than competing small molecules or the natural multigalactosylated ligand, asialoorosomucoid. Upon systemic injection in mice, these VLPs were rapidly cleared to the liver and were found in association with sinusoidal endothelial cells, Kupffer cells, hepatocytes, dendritic cells, and other immune cells. Both ASGPR-targeted and nontargeted particles were distributed similarly to endothelial and Kupffer cells, but targeted particles were distributed to a greater number and fraction of hepatocytes. Thus, selective cellular trafficking in the liver is difficult to achieve: even with the most potent ASGPR targeting available, barrier cells take up much of the injected particles and hepatocytes are accessed only approximately twice as efficiently in the best case.

Lipid Microparticles Show Similar Efficacy With Lipid Nanoparticles in Delivering mRNA and Preventing Cancer.

PURPOSE: Messenger RNA (mRNA) has shown great promise for vaccine against both infectious diseases and cancer. However, mRNA is unstable and requires a delivery vehicle for efficient cellular uptake and degradation protection. So far, lipid nanoparticles (LNPs) represent the most advanced delivery platform for mRNA delivery. However, no published studies have compared lipid microparticles (LMPs) with lipid nanoparticles (LNPs) in delivering mRNA systematically, therefore, we compared the impact of particle size on delivery efficacy of mRNA vaccine and subsequent immune responses. METHODS: Herein, we prepared 3 different size lipid particles, from nano-sized to micro-sized, and they loaded similar amounts of mRNA. These lipid particles were investigated both in vitro and in vivo, followed by evaluating the impact of particle size on inducing cellular and humoral immune responses. RESULTS: In this study, all mRNA vaccines showed a robust immune response and lipid microparticles (LMPs) show similar efficacy with lipid nanoparticles (LNPs) in delivering mRNA and preventing cancer. In addition, immune adjuvants, either toll like receptors or active molecules from traditional Chinese medicine, can improve the efficacy of mRNA vaccines. CONCLUSIONS: Considering the efficiency of delivery and endocytosis, besides lipid nanoparticles with size smaller than 150 nm, lipid microparticles (LMPs) also have the potential to be an alternative and promising delivery system for mRNA vaccines.

Wrinkle-bioinspired silver nanowire surface enhanced Raman scattering sensors for pesticide molecule detection.

We demonstrate wrinkle-bioinspired flexible SERS sensor covered by silver nanowire (AgNWs) network for the detection of pesticide molecules. Compared with the silver film deposition substrates, the wrinkle-bioinspired AgNWs SERS substrates show stronger SERS effect, which were attributed to the electromagnetic field enhancement posed by the relatively high density "hot spots" of AgNWs. In order to investigate the adsorption performance of wrinkle-bioinspired flexible sensors, we measured the corresponding contact angles of AgNWs on the substrate surfaces before and after plasma treatment and found that the AgNWs treated with plasma are more hydrophilic than that without plasma treatment. Additionally, the wrinkle-bioinspired SERS sensors show different SERS activities under different tensile strain, and the 10-6 mol/L concentration of Rhodamine dye (R6G) molecules can be detected by portable Raman spectra, reducing greatly detection cost. The surface plasmon resonance of AgNWs is induced by adjusting the deformation of AgNWs substrate, enhancing the SERS signal. This reliability of wrinkle-bioinspired SERS sensors is further verified by in situ detection of pesticide molecules.

Production of Gypenoside XVII from Ginsenoside Rb1 by Enzymatic Transformation and Their Anti-Inflammatory Activity In Vitro and In Vivo.

The enzymatic transformation of the sugar moiety of the gypenosides provides a new way to obtain more pharmacologically active components. A gene encoding a family 1 glycosyl hydrolase from Bifidobacterium dentium was cloned and expressed in Escherichia coli. The recombinant enzyme was purified, and its molecular weight was approximately 44 kDa. The recombinant BdbglB exhibited an optimal activity at 35 °C and pH 5.4. The purified recombinant enzyme, exhibiting ß-glucosidase activity, was used to produce gypenoside XVII (Gyp XVII) via highly selective and efficient hydrolysis of the outer glucose moiety linked to the C-3 position in ginsenoside Rb1 (G-Rb1). Under the optimal reaction conditions for large scale production of gypenoside XVII, 40 g ginsenoside Rb1 was transformed by using 45 g crude enzyme at pH 5.4 and 35 °C for 10 h with a molar yield of 100%. Furthermore, the anti-inflammatory effects of the product gypenoside XVII and its conversion precursor ginsenoside Rb1 were evaluated by using lipopolysaccharide (LPS)-induced murine RAW 264.7 macrophages and the xylene-induced acute inflammation model of mouse ear edema, respectively. Gypenoside XVII showed improved anti-inflammatory activity, which significantly inhibited the generation of TNF-α and IL-6 more effectively than its precursor ginsenoside Rb1. In addition, the swelling inhibition rate of gypenoside XVII was 80.55%, while the rate of its precursor was 40.47%, the results also indicated that gypenoside XVII had better anti-inflammatory activity than ginsenoside Rb1. Hence, this enzymatic method would be useful in the large-scale production of gypenoside XVII, which may become a new potent anti-inflammatory candidate drug.

Epoxide Electroreduction.

Selective hydrogenation of epoxides would be a direct and powerful approach for alcohol synthesis, but it has proven to be elusive. Here, electrochemically epoxide hydrogenation using electrons and protons as reductants is reported. A wide range of primary, secondary, and tertiary alcohols can be achieved through selective Markovnikov or anti-Markovnikov ring opening in the absence of transition metals. Mechanistic investigations revealed that the regioselectivity is controlled by the thermodynamic stabilities of the in situ generated benzyl radicals for aryl-substituted epoxides and the kinetic tendency for Markovnikov selective ring opening for alkyl-substituted epoxides.

Switchable Radical Carbonylation by Philicity Regulation.

Carbonylation reactions involving CO as readily available C1 synthons have become one of the most important tools for the construction of carbonyl compounds from feedstock chemicals. Despite numerous catalytic methods for carbonylation reactions proceeding via ionic or radical pathways, an inherent limitation to these methods is the need to control switchable single and double carbonylative formation of value-added products from the same and simple starting materials. Here, we describe a new strategy that exploits photoredox catalysis to regulate the philicity of amine coupling partners to drive switchable radical carbonylation reactions. In double carbonylation, amines were first transformed into nitrogen radical cations by single-electron transfer-oxidation and coupled with CO to form carbamoyl radicals, which further underwent radical cross-coupling with the incipient cyanoalkyl acyl radicals to afford the double carbonylation products. Upon the addition of stoichiometric 4-dimethylaminopyridine (DMAP), DMAP competitively traps the initially formed cyanoalkyl acyl radical to form the relatively stabilized cyanoalkyl acyl-DMAP salts that engaged in the subsequent substitution with the nucleophilic amines to produce the single carbonylation products. The reaction proceeded smoothly with excellent selectivity in the presence of various amine nucleophiles at room temperature, generating valuable amides and α-ketoamides in a versatile and controlled fashion. Combined experimental and computational studies provided mechanistic insights into the possible pathways.

ProPhenol Derived Ligands to Simultaneously Coordinate a Main-Group Metal and a Transition Metal: Application to a Zn-Cu Catalyzed Reaction.

A new bifunctional ligand bearing chiral N-heterocyclic carbene (NHC) and prolinol moieties is presented. Utilizing the designed ligand, an in situ formed Cu/Zn hetero-bimetallic complex unlocks the asymmetric allylic alkylation reactions of allyl phosphates with zinc keto-homoenolates, leading to the formation of various γ-vinyl ketones with good regio- and enantio-selectivity. DF sT calculation supports that the chelation of allyl phosphates with catalyst promotes the SN 2' addition and the ligand-substrate steric interactions account for the stereoselective outcome.

An mALBI-Child-Pugh-based nomogram for predicting post-hepatectomy liver failure grade B-C in patients with huge hepatocellular carcinoma: a multi-institutional study.

OBJECTIVE: Post-hepatectomy liver failure (PHLF) is a severe complication in patients with hepatocellular carcinoma (HCC) who underwent hepatectomy. This study aims to develop a nomogram of PHLF grade B-C in patients with huge HCC (diameter ≥ 10 cm). METHODS: We retrospectively collected clinical information of 514 and 97 patients who underwent hepatectomy for huge HCC at two medical centers between 2016 and 2021. Univariate and multivariate analysis were carried out to screen the independent risk factors of PHLF grade B-C, which were visualized as a nomogram. RESULTS: Three Hundred Forty Three Thousand One Hundred Seventy One  and 97 HCC patients were included in the training cohort, internal validation cohort, and external validation cohort, with probabilities of PHLF grade B-C of 15.1%, 12.9%, and 22.7%, respectively. Pre-operative modified albumin-bilirubin (mALBI) grade (p < 0.001), Child-Pugh classification (p = 0.044), international normalized ratio (INR) (p = 0.005), cirrhosis (p = 0.019), and intraoperative blood loss (p = 0.004) were found to be independently associated with PHLF grade B-C in the training cohort. All the five independent factors were considered in the establishment of the nomogram model. In the internal validation cohort and external validation cohort, the area under receiver operating characteristic curve for the nomogram in PHLF grade B-C prediction reached 0.823 and 0.740, respectively. Divided into different risk groups according to the optimal cut-off value, patients in the high-risk group reported significantly higher frequency of PHLF grade B-C than those in the low-risk group, both in the training cohort and the validation cohort (p < 0.001). CONCLUSIONS: The proposed noninvasive nomogram based on mALBI-Child-Pugh and three other indicators achieved optimal prediction performance of PHLF grade B-C in patients with huge HCC.

From Esters to Ketones via a Photoredox-Assisted Reductive Acyl Cross-Coupling Strategy.

A method was developed for ketone synthesis via a photoredox-assisted reductive acyl cross-coupling (PARAC) using a nickel/photoredox dual-catalyzed cross-electrophile coupling of two different carboxylic acid esters. A variety of aryl, 1°, 2°, 3°-alkyl 2-pyridyl esters can act as acyl electrophiles while N-(acyloxy)phthalimides (NHPI esters) act as 1°, 2°, 3°-radical precursors. Our PARAC strategy provides an alternative and reliable way to synthesize various sterically congested 3°-3°, 3°-2°, and aryl-3° ketones under mild and highly unified conditions, which have been otherwise difficult to access. The combined experimental and computational studies identified a Ni0 /NiI /NiIII pathway for ketone formation.

Electrochemical Palladium-Catalyzed Oxidative Sonogashira Carbonylation of Arylhydrazines and Alkynes to Ynones.

Oxidative carbonylation using carbon monoxide has evolved as an attractive tool to valuable carbonyl-containing compounds, while mixing CO with a stoichiometric amount of a chemical oxidant especially oxygen is hazardous and limits its application in scale-up synthesis. By employing anodic oxidation, we developed an electrochemical palladium-catalyzed oxidative carbonylation of arylhydrazines with alkynes, which is regarded as an alternative supplement of the carbonylative Sonogashira reaction. Combining an undivided cell with constant current mode, oxygen-free conditions avoids the explosion hazard of CO. A diversity of ynones are efficiently obtained using accessible arylhydrazines and alkynes under copper-free conditions. A possible mechanism of the electrochemical Pd(0)/Pd(II) cycle is rationalized based upon cyclic voltammetry, kinetic studies, and intermediates experiments.

Do the existing staging systems for primary liver cancer apply to combined hepatocellular carcinoma-intrahepatic cholangiocarcinoma?

BACKGROUND: The incidence of combined hepatocellular carcinoma-intrahepatic cholangiocarcinoma (cHCC-ICC) is relatively low, and the knowledge about the prognosis of cHCC-ICC remains obscure. In the study, we aimed to screen existing primary liver cancer staging systems and shed light on the prognosis and risk factors for cHCC-ICC. METHODS: We retrospectively reviewed 206 cHCC-ICC patients who received curative surgical resection from April 1999 to March 2017. The correlation of survival measures with the histological types or with tumor staging systems was determined and predictive values of tumor staging systems with cHCC-ICC prognosis were compared. RESULTS: The histological type was not associated with overall survival (OS) (P = 0.338) or disease-free survival (DFS) (P = 0.843) of patients after curative surgical resection. BCLC, TNM for HCC, and TNM for ICC stages correlated with both OS and DFS in cHCC-ICC (all P < 0.05). The predictive values of TNM for HCC and TNM for ICC stages were similar in terms of predicting postoperative OS (P = 0.798) and DFS (P = 0.191) in cHCC-ICC. TNM for HCC was superior to BCLC for predicting postoperative OS (P = 0.022) in cHCC-ICC. CONCLUSION: The TNM for HCC staging system should be prioritized for clinical applications in predicting cHCC-ICC prognosis.

Dissociative Bioorthogonal Reactions.

Bioorthogonal reactions that proceed readily under physiological conditions without interference from biomolecules have found widespread application in the life sciences. Complementary to the bioorthogonal reactions that ligate two molecules, reactions that release a molecule or cleave a linker are increasingly attracting interest. Such dissociative bioorthogonal reactions have a broad spectrum of uses, for example, in controlling bio-macromolecule activity, in drug delivery, and in diagnostic assays. This review article summarizes the developed bioorthogonal reactions linked to a release step, outlines representative areas of the applications of such reactions, and discusses aspects that require further improvement.

Tuning Isonitrile/Tetrazine Chemistry for Accelerated Deprotection and Formation of Stable Conjugates.

The isocyano group is a valuable functionality for bioorthogonal reactions because it rapidly reacts with tetrazines to either form stable conjugates or release payloads from 3-isocyanopropyl groups. Here we provide mechanistic insights into the dissociative steps that follow the initial cycloaddition and analyze how structural modifications affect these processes. Three main outcomes of this study have important implications for designing such groups for bioorthogonal applications. First, anion-stabilizing substituents at C-2 of the 3-isocyanopropyl group promote ß-elimination and accelerate deprotection. Second, tetrazines with bulky substituents form stable imine conjugates even with primary isonitriles that are otherwise rapidly hydrolyzed. Third, the elimination step is independent from hydrolysis to the aldehyde and instead can occur directly from the imine intermediate. These findings will allow tuning the structures of tetrazine and isonitrile reactants for application in bioorthogonal ligation and release chemistry.

Bioorthogonal Removal of 3-Isocyanopropyl Groups Enables the Controlled Release of Fluorophores and Drugs in Vivo.

Dissociative bioorthogonal reactions allow for chemically controlling the release of bioactive agents and reporter probes. Here we describe 3-isocyanopropyl substituents as masking groups that can be effectively removed in biological systems. 3-Isocyanopropyl derivatives react with tetrazines to afford 3-oxopropyl groups that eliminate diverse functionalities. The study shows that the reaction is rapid and can liberate phenols and amines near-quantitatively under physiological conditions. The reaction is compatible with living organisms as demonstrated by the release of a resorufin fluorophore and a mexiletine drug in zebrafish embryos implanted with tetrazine-modified beads. The combined benefits of synthetic ease, rapid kinetics, diversity of leaving groups, high release yields, and structural compactness, make 3-isocyanopropyl derivatives attractive chemical caging moieties for uses in chemical biology and drug delivery.

Discriminating gastric cancer and gastric ulcer using human plasma amino acid metabolic profile.

Patients with gastric ulcer (GU) have a significantly higher risk of developing gastric cancer (GC), especially within 2 years after diagnosis. The main way to improve the prognosis of GC is to predict the tumorigenesis and metastasis in the early stage. The objective of this study was to demonstrate the ability of human plasma amino acid metabolic profile for discriminating GC and GU. In this study, we first used liquid chromatography-tandem mass spectrometry technique to characterize the plasma amino acid metabolism in GC and GU patients. Plasma samples were collected from 84 GC patients and 82 GU patients, and 22 amino acids were detected in each patient. Partial least squares-discriminant analysis model was performed to analyze the data of these amino acids. We observed seven differential amino acids between GC and GU. A regression analysis model was established using these seven amino acids. Finally, a panel of five differential amino acids, including glutamine, ornithine, histidine, arginine and tryptophan, was identified for discriminating GC and GU with good specificity and sensitivity. The receiver operating characteristic curve was used to evaluate diagnostic ability of the regression model and area under the curve was 0.922. In conclusion, this study demonstrated the potential values of plasma amino acid metabolic profile and metabolomic analysis technique in assisting diagnosis of GC. More studies are needed to highlight the theoretical strengths of metabolomics to understand the potential metabolic mechanisms in GC. © 2018 IUBMB Life, 70(6):553-562, 2018.

Association of plasma arginine with breast cancer molecular subtypes in women of Liaoning province.

Arginine is one of the human nonessential amino acids critical for the growth of human cancers. The aim of this study is to investigate the variation of arginine between breast cancer (BC) patients and benign mammary gland disease (control) patients to determine its value in predicting the risk of BC. We also explore the associations between arginine levels and breast cancer subtypes. Preoperative blood samples were obtained from 267 patients (102 BC and 165 controls) in 2015. Plasma arginine values were determined for all preoperative blood samples using liquid chromatography-tandem mass spectrometry (LC-MS/MS) to analyse differences in arginine levels between BC patients and control patients and the correlations between arginine and clinicopathologic parameters in BC. The arginine levels of BC patients were significantly lower than those of control patients (5.96 [3.76-12.47] vs. 12.54 [7.14-24.94], P = 0.000). The area under the curve (AUC) for arginine was 0.721 (95% CI, 0.660-0.782, P < 0.0001). The concentration of arginine was significantly different among different molecular BC subtypes (P = 0.030). Our results suggested that plasma arginine was associated with breast cancer molecular subtypes. © 2016 IUBMB Life, 68(12):980-984, 2016.

Identification of arginine and its "Downstream" molecules as potential markers of breast cancer.

Breast cancer (BC) is the most commonly diagnosed cancer in women worldwide. Arginine is a semiessential amino acid in humans and is essential for several biological pathways in malignant and normal cells, such as ornithine and N1, N12-diacetylspermine (DiAcSpm). This study aimed to determine the role of arginine and these downstream molecules in BC. Plasma arginine, ornithine, and arginine-to-ornithine ratio (AOR) were analysed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Urine samples were measured by the colloid gold aggregation to test determination of urinary diAcSpm. A principal component analysis was performed to evaluate the results observed between breast tumor and control characteristics. Differences in individual metabolite concentrations between BC patients and controls were tested by receiver operating characteristics (ROC) analyses. Student's t tests were used to detect the differences between two groups of normally distributed variables, and Wilcoxon sign rank tests were performed for asymmetrically distributed variables. As we analyzed, BC patients had lower plasma arginine and arginine/ornithine level, and higher plasma ornithine and urinary DiAcSpm concentrations as compared with control patients (P = 0.028, 0.020, 0.002, and 0.011, respectively). And the ROC curve was drawn and the area under the curve of the metabolites was calculated to be 0.659 (P = 0.028), 0.645 (P = 0.045), 0.7233 (P = 0.002), 0.683 (P = 0.011), respectively. In addition, our analysis showed that arginine concentrations and AOR had a positive correlation with ER status, while ornithine had a negative correlation with T stage (P = 0.042, 0.023, respectively).In conclusion, arginine and these downstream molecules were biomarkers for BC. More studies are needed to highlight the theoretical strengths. © 2016 IUBMB Life, 68(10):817-822, 2016.