IFITM3 inhibits severe fever with thrombocytopenia syndrome virus entry and interacts with viral Gc protein.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging tick-borne hemorrhagic fever disease with high fatality rate of 10%-20%. Vaccines or specific therapeutic measures remain lacking. Human interferon inducible transmembrane protein 3 (hIFITM3) is a broad-spectrum antiviral factor targeting viral entry. However, the antiviral activity of hIFITM3 against SFTS virus (SFTSV) and the functional mechanism of IFITM3 remains unclear. Here we demonstrate that endogenous IFITM3 provides protection against SFTSV infection and participates in the anti-SFTSV effect of type Ⅰ and Ⅲ interferons (IFNs). IFITM3 overexpression exhibits anti-SFTSV function by blocking Gn/Gc-mediated viral entry and fusion. Further studies showed that IFITM3 binds SFTSV Gc directly and its intramembrane domain (IMD) is responsible for this interaction and restriction of SFTSV entry. Mutation of two neighboring cysteines on IMD weakens IFITM3-Gc interaction and attenuates the antiviral activity of IFITM3, suggesting that IFITM3-Gc interaction may partly mediate the inhibition of SFTSV entry. Overall, our data demonstrate for the first time that hIFITM3 plays a critical role in the IFNs-mediated anti-SFTSV response, and uncover a novel mechanism of IFITM3 restriction of SFTSV infection, highlighting the potential of clinical intervention on SFTS disease.

Quaternization drives spleen-to-lung tropism conversion for mRNA-loaded lipid-like nanoassemblies.

Background: As the overwhelming majority of advanced mRNA delivery systems are preferentially accumulated in the liver, there is an accelerating growth in the demand for the development of non-liver mRNA delivery platforms. Methods: In this study, we prepared cationic lipid-like nanoassemblies through a N-quaternizing strategy. Their physicochemical properties, in vitro mRNA delivery efficiency, and organ tropism in mice were investigated. Results: Introduction of quaternary ammonium groups onto lipid-like nanoassemblies not only enhances their mRNA delivery performance in vitro, but also completely alters their tropism from the spleen to the lung after intravenous administration in mice. Quaternized lipid-like nanoassemblies exhibit ultra-high specificity to the lung and are predominantly taken up by pulmonary immune cells, leading to over 95% of exogenous mRNA translation in the lungs. Such mRNA delivery carriers are stable even after more than one-year storage at ambient temperature. Conclusions: Quaternization provides an alternative method for design of new lung-targeted mRNA delivery systems without incorporation of targeting ligands, which should extend the therapeutic applicability of mRNA to lung diseases.

Chimeric antigen receptor therapy meets mRNA technology.

Genetically engineered immune cells expressing chimeric antigen receptors (CARs) have emerged as a new game changer in cancer immunotherapy. The utility of CAR T cell therapy against hematological malignancies has been validated in clinical practice. Other CAR immune cells are currently under investigation to improve the potency of CAR therapy in solid tumors. As a new class of therapeutic modalities, mRNA-based therapeutics hold enormous potential beyond COVID-19 mRNA vaccines. Arming immune cells with mRNA-encoded CARs represents a new frontier in cancer and beyond, enabling in vivo generation of CAR cells without causing transgene integration. In this review, we summarize recent advances in mRNA-based CAR immunotherapies and highlight their opportunities and challenges for the development of a new generation of living drugs.

JFD, a Novel Natural Inhibitor of Keap1 Alkylation, Suppresses Intracellular Mycobacterium Tuberculosis Growth through Keap1/Nrf2/SOD2-Mediated ROS Accumulation.

It is an effective strategy to treat tuberculosis by enhancing reactive oxygen species- (ROS-) mediated killing of Mycobacterium tuberculosis in macrophages, but there are no current therapeutic agents targeting this pathway. Honeysuckle has been used as the traditional medicine for tuberculosis treatment for 1500 years. Japoflavone D (JFD) is a novel biflavonoid isolated from Honeysuckle promoting ROS accumulation by Nrf2 pathway in hepatocarcinoma cells. However, its activity to kill M. tuberculosis in macrophages and molecular mechanism has not been reported. Our results showed that JFD enhances the M. tuberculosis elimination by boosting ROS levels in THP-1 cells. Moreover, the massive ROS accumulation activates p38 to induce apoptosis. Notably, the mechanism revealed that JFD suppresses the nuclear transport of Nrf2, thereby inhibiting SOD2 transcription, leading to a large ROS accumulation. Further studies showed that JFD disrupts the Keap1 alkylation at specific residues Cys14, Cys257, and Cys319, which is crucial for Nrf2 activation, thereby interrupts the nuclear transport of Nrf2. In pharmacokinetic study, JFD can stay as the prototype for 24 h in mice and can be excreted in feces without any toxicity. Our data reveal for the first time that a novel biflavonoid JFD as a potent inhibitor of Keap1 alkylation can suppress the nuclear transport of Nrf2. And it is the first research of the inhibitor of Keap1 alkylation. Furthermore, JFD robustly promotes M. tuberculosis elimination from macrophages by inhibiting Keap1/Nrf2/SOD2 pathway, resulting in the ROS accumulation. This work identified Keap1 alkylation as a new drug target for tuberculosis and provides a preliminary basis for the development of antituberculosis lead compounds based on JFD.

Discussion on treatment courses of brucellosis with spondylitis - a report of two cases.

Human Brucellosis is a zoonotic contagious disease caused by Brucella infection and is common throughout the world, which can travel through the bloodstream to various organs. Brucellar spondylitis(BS) is the foremost cause of brucellosis's debilitating and disabling complications. We report two sisters with brucellosis complicated by lumbar spondylodiscitis accompanied by cold abscess formation. The diagnosis was based on their symptoms, epidemiological characteristics, laboratory and magnetic resonance imaging(MRI) results. Our therapeutic strategy in these two cases indicate that drug combination and prolongation of the use of antibiotics is a therapeutic strategy worthy of popularizing to reach a greater clearance rate of the infection.

A PEG-lipid-free COVID-19 mRNA vaccine triggers robust immune responses in mice.

COVID-19 mRNA vaccines represent a completely new category of vaccines and play a crucial role in controlling the COVID-19 pandemic. In this study, we have developed a PEG-lipid-free two-component mRNA vaccine (PFTCmvac) by formulating mRNA encoding the receptor binding domain (RBD) of SARS-CoV-2 into lipid-like nanoassemblies. Without using polyethylene glycol (PEG)-lipids, the self-assembled PFTCmvac forms thermostable nanoassemblies and exhibits a dose-dependent cellular uptake and membrane disruption, eventually leading to high-level protein expression in both mammalian cells and mice. Vaccination with PFTCmvac elicits strong humoral and cellular responses in mice, without evidence of significant adverse reactions. In addition, the vaccine platform does not trigger complement activation in human serum, even at a high serum concentration. Collectively, the PEG-lipid-free two-component nanoassemblies provide an alternative delivery technology for COVID-19 mRNA vaccines and opportunities for the rapid production of new mRNA vaccines against emerging infectious diseases.

Tubuloside B, isolated from Cistanche tubulosa, a promising agent against M1 macrophage activation via synergistically targeting Mob1 and ERK1/2.

Targeting macrophage M1 polarization is a promising strategy with fewer detrimental effects in COVID-19 curation. Phenylethanoid glycosides (PhGs) of Cistanche tubulosa are a botanical drug to possess various anti-inflammation-related functions, such as immunomodulating, hepatoprotective or neuroprotective functions, whereas their anti-inflammatory activity is rarely understood. A search into their anti-inflammatory characteristics led to the isolation of 49 PhGs along with 15 new PhGs. Their inhibitory effects against M1 polarization induced by LPS plus IFN-γ were explored in RAW264.7 macrophages. Of these PhGs, tubuloside B (Tub B) exerted substantial NO scavenging effect both in chemical- and cell-based assays, and it inhibited massive production of cytokines and chemokines. Tub B decreased ERK1/2 phosphorylation via direct binding and inhibited the MAPK signaling pathway. Tub B also directly binded to Mob1 protein, thereby increased the stability and level of Mob1 protein by inhibiting ubiquitinated degradation. Mob1 was pivotal for the anti-inflammatory activity of Tub B, and it acted independently of the canonical Hippo-YAP pathway. Moreover, ERK1/2 and Mob1 also had a synergic effect on modulating the inflammatory response. Finally, these effects of Tub B were verified in mice with LPS-induced systemic inflammatory response syndrome. Taken together, these results indicated that Tub B acted as a promising agent against M1 macrophage activation by synergistically targeting ERK1/2 and Mob1, and that it may potentially be a drug candidate to prevent/treat inflammatory diseases, especially in COVID-19.

Genus Lonicera: New drug discovery from traditional usage to modern chemical and pharmacological research.

BACKGROUND: Lonicera Linn. belonging to the family Caprifoliaceae, the largest genus in the plant family, includes about more than 200 species, which are mainly distributed in northern Africa, North America, Europe and Asia. Some species of this genus have been usually used in traditional Chinese medicine as well as functional foods, cosmetics and other applications, such as L. japonica Thunb. Bioactive components and pharmacological activities of the genus Lonicera plants have received an increasing interest from the scientific community. Thus, a comprehensive and systematic review on their traditional usage in China, chemical components, and their pharmacological properties of their whole plants, bioactive extracts, and bioactive isolates including partial structure-activity relationships from the genus is indispensable. METHODS: Information on genus Lonicera of this systematic electronic literature search was gathered via the published articles, patents, clinical trials website (https://clinicaltrials.gov/) and several online bibliographic databases (PubMed, Sci Finder, Research Gate, Science Direct, CNKI, Web of Science and Google Scholar). The following keywords were used for the online search: Lonicera, phytochemical composition, Lonicerae japonica, Lonicera review articles, bioactivities of Lonicera, anti-inflammatory, antiviral, antimicrobial, anticancer, hepatoprotective, antioxidant, neuroprotective, anti-diabetic, and clinical trials. This review paper consists of a total of 225 papers covering the Lonicera genus from 1800 to 2021, including research articles, reviews, patents, and book chapters. RESULTS: In this review (1800s-2021), about 420 components from the genus of Lonicera Linn. including 87 flavonoids, 222 terpenoids, 51 organic acids, and other compounds, together with their pharmacological activities including anti-inflammatory, antiviral, antimicrobial, anticancer, hepatoprotective, antioxidant, neuroprotective, antidiabetic, anti-allergic, immunomodulatory effects, and toxicity were summarized. CONCLUSION: The relationship is discussed among their traditional usage, their pharmacological properties, and their chemical components, which indicate the genus Lonicera have a large prospect in terms of new drug exploitation, especially in COVID-19 treatment.

The "LLQY" Motif on SARS-CoV-2 Spike Protein Affects S Incorporation into Virus Particles.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) glycoprotein mediates viral entry and membrane fusion. Its cleavage at S1/S2 and S2' sites during the biosynthesis in virus producer cells and viral entry are critical for viral infection and transmission. In contrast, the biological significance of the junction region between both cleavage sites for S protein synthesis and function is less understood. By analyzing the conservation and structure of S protein, we found that intrachain contacts formed by the conserved tyrosine (Y) residue 756 (Y756) with three α-helices contribute to the spike's conformational stability. When Y756 is mutated to an amino acid residue that can provide hydrogen bonds, S protein could be expressed as a cleaved form, but not vice versa. Also, the L753 mutation linked to the Y756 hydrogen bond prevents the S protein from being cleaved. Y756 and L753 mutations alter S protein subcellular localization. Importantly, Y756 and L753 mutations are demonstrated to reduce the infectivity of the SARS-CoV-2 pseudoviruses by interfering with the incorporation of S protein into pseudovirus particles and causing the pseudoviruses to lose their sensitivity to neutralizing antibodies. Furthermore, both mutations affect the assembly and production of SARS-CoV-2 virus-like particles in cell culture. Together, our findings reveal for the first time a critical role for the conserved L753-LQ-Y756 motif between S1/S2 and S2' cleavage sites in S protein synthesis and processing as well as virus assembly and infection. IMPORTANCE The continuous emergence of SARS-CoV-2 variants such as the delta or lambda lineage caused the continuation of the COVID-19 epidemic and challenged the effectiveness of the existing vaccines. Logically, the spike (S) protein mutation has attracted much concern. However, the key amino acids in S protein for its structure and function are still not very clear. In this study, we discovered for the first time that the conserved residues Y756 and L753 at the junction between the S1/S2 and S2' sites are very important, like the S2' cleavage site R815, for the synthesis and processing of S protein such as protease cleavage, and that the mutations severely interfered with the incorporation of S protein into pseudotyped virus particles and SARS-CoV-2 virus-like particles. Consequently, we delineate the novel potential target for the design of broad-spectrum antiviral drugs in the future, especially in the emergence of SARS-CoV-2 variants.

Identifying Potential Neoantigens for Cervical Cancer Immunotherapy Using Comprehensive Genomic Variation Profiling of Cervical Intraepithelial Neoplasia and Cervical Cancer.

Cervical cancer (CC) is one of the most common gynecological malignant tumors. The 5-year survival rate remains poor for the advanced and metastatic cervical cancer for the lack of effective treatments. Immunotherapy plays an important role in clinical tumor therapy. Neoantigens derived from tumor-specific somatic mutations are prospective targets for immunotherapy. Hence, the identification of new targets is of great significance for the treatment of advanced and metastatic cervical cancer. In this study, we performed whole-exome sequencing in 70 samples, including 25 cervical intraepithelial neoplasia (CINs) with corresponding blood samples and 10 CCs along with paired adjacent tissues to identify genomic variations and to find the potential neoantigens for CC immunotherapy. Using systematic bioinformatics pipeline, we found that C>T transitions were in both CINs and CCs. In contrast, the number of somatic mutations in CCs was significantly higher than those in CINs (t-test, P = 6.60E-04). Meanwhile, mutational signatures analysis revealed that signature 6 was detected in CIN2, CIN3, and CC, but not in CIN1, while signature 2 was only observed in CCs. Furthermore, PIK3CA, ARHGAP5 and ADGRB1 were identified as potential driver genes in this report, of which ADGRB1 was firstly reported in CC. Based on the genomic variation profiling of CINs and CCs, we identified 2586 potential neoantigens in these patients, of which 45 neoantigens were found in three neoantigen-related databases (TSNAdb, IEDB, and CTDatabase). Our current findings lay a solid foundation for the study of the pathogenesis of CC and the development of neoantigen-targeted immunotherapeutic measures.

Secreted Expression of mRNA-Encoded Truncated ACE2 Variants for SARS-CoV-2 via Lipid-Like Nanoassemblies.

The transfer of foreign synthetic messenger RNA (mRNA) into cells is essential for mRNA-based protein-replacement therapies. Prophylactic mRNA COVID-19 vaccines commonly utilize nanotechnology to deliver mRNA encoding SARS-CoV-2 vaccine antigens, thereby triggering the body's immune response and preventing infections. In this study, a new combinatorial library of symmetric lipid-like compounds is constructed, and among which a lead compound is selected to prepare lipid-like nanoassemblies (LLNs) for intracellular delivery of mRNA. After multiround optimization, the mRNA formulated into core-shell-structured LLNs exhibits more than three orders of magnitude higher resistance to serum than the unprotected mRNA, and leads to sustained and high-level protein expression in mammalian cells. A single intravenous injection of LLNs into mice achieves over 95% mRNA translation in the spleen, without causing significant hematological and histological changes. Delivery of in-vitro-transcribed mRNA that encodes high-affinity truncated ACE2 variants (tACE2v mRNA) through LLNs induces elevated expression and secretion of tACE2v decoys, which is able to effectively block the binding of the receptor-binding domain of the SARS-CoV-2 to the human ACE2 receptor. The robust neutralization activity in vitro suggests that intracellular delivery of mRNA encoding ACE2 receptor mimics via LLNs may represent a potential intervention strategy for COVID-19.

An epigenomic landscape of cervical intraepithelial neoplasia and cervical cancer using single-base resolution methylome and hydroxymethylome.

BACKGROUND: Cervical cancer (CC) is the second leading cause of cancer death among women worldwide. Epigenetic regulation of gene expression through DNA methylation and hydroxymethylation plays a pivotal role during tumorigenesis. In this study, to analyze the epigenomic landscape and identify potential biomarkers for CCs, we selected a series of samples from normal to cervical intra-epithelial neoplasia (CINs) to CCs and performed an integrative analysis of whole-genome bisulfite sequencing (WGBS-seq), oxidative WGBS, RNA-seq, and external histone modifications profiling data. RESULTS: In the development and progression of CC, there were genome-wide hypo-methylation and hypo-hydroxymethylation, accompanied by local hyper-methylation and hyper-hydroxymethylation. Hydroxymethylation prefers to distribute in the CpG islands and CpG shores, as displayed a trend of gradual decline from health to CIN2, while a trend of increase from CIN3 to CC. The differentially methylated and hydroxymethylated region-associated genes both enriched in Hippo and other cancer-related signaling pathways that drive cervical carcinogenesis. Furthermore, we identified eight novel differentially methylated/hydroxymethylated-associated genes (DES, MAL, MTIF2, PIP5K1A, RPS6KA6, ANGEL2, MPP, and PAPSS2) significantly correlated with the overall survival of CC. In addition, no any correlation was observed between methylation or hydroxymethylation levels and somatic copy number variations in CINs and CCs. CONCLUSION: Our current study systematically delineates the map of methylome and hydroxymethylome from CINs to CC, and some differentially methylated/hydroxymethylated-associated genes can be used as the potential epigenetic biomarkers in CC prognosis.

Exploring and exploiting plant cyclic peptides for drug discovery and development.

Ever since the discovery of insulin, natural peptides have become an important resource for therapeutic development. Decades of research has led to the discovery of a long list of peptide drugs with broad applications in clinics, from antibiotics to hypertension treatment to pain management. Many of these US FDA-approved peptide drugs are derived from microorganisms and animals. By contrast, the great potential of plant cyclic peptides as therapeutics remains largely unexplored. These macrocyclic peptides typically have rigid structures, good bioavailability and membrane permeability, making them appealing candidates for drug development and engineering. In this review, we introduce the three major classes of plant cyclic peptides and summarize their potential medical applications. We discuss how we can leverage the genome information of many different plants to quickly search for new cyclic peptides and how we can take advantage of the insights gained from their biosynthetic pathways to transform the process of production and drug development. These recent developments have provided a new angle for exploring and exploiting plant cyclic peptides, and we believe that many more peptide drugs derived from plants are about to come.

MiR-122-5p knockdown protects against APAP-mediated liver injury through up-regulating NDRG3.

MiR-122-5p serves as a novel biomarker for drug-induced liver injury (DILI), but its function in DILI remains unclear. The present study, therefore, explored the function and potential mechanism of miR-122-5p in DILI. Sprague-Dawley (SD) rats were treated with miR-122-5p antagomir, and then DILI was induced in the rats by acetaminophen (APAP). To determine the effect of miR-122-5p on DILI in vivo, liver injury was examined by HE staining and TUNEL assays, and the levels of serum ALT and AST were determined using an automated clinical chemistry analyzer. To further reveal the mechanism of miR-122-5p in DILI, THLE-2 (normal liver cell line) cells were transfected with miR-122-5p mimic and inhibitor, NDRG3, and siNDRG3, and then injured by APAP. The relationship between miR-122-5p and NDRG3 was determined by TargetScan, luciferase reporter assay, and Western blot. The viability and apoptosis of THLE-2 cells were detected by CCK-8 and flow cytometry, respectively. The levels of mRNA and protein in vivo and in vitro were measured by qRT-PCR and Western blot, respectively. APAP induced liver injury and increased the levels of ALT, AST, and miR-122-5p in DILI rats. However, these effects of APAP were attenuated by miR-122-5p antagomir. MiR-122-5p negatively regulated NDRG3 expression. APAP decreased cell viability, apoptosis resistance, and Bcl-w and Bcl-2 levels whereas increased Bax level in THLE-2 cells. However, these effects of APAP on THLE-2 cells were promoted by miR-122-5p up-regulation but inhibited by miR-122-5p knockdown. MiR-122-5p knockdown protects against APAP-mediated liver injury through up-regulating NDRG3.

Topical Application of Exosomes Derived from Human Umbilical Cord Mesenchymal Stem Cells in Combination with Sponge Spicules for Treatment of Photoaging.

PURPOSE: The topical application of exosomes secreted by mesenchymal stem cells (MSC-Exos) on the skin is a very new and interesting topic in the medical field. In this study, we aimed to investigate whether marine sponge Haliclona sp. spicules (SHSs) could effectively enhance the skin delivery of human umbilical cord-derived MSC-Exos (hucMSC-Exos), and further evaluate the topical application of hucMSC-Exos combined with SHSs in rejuvenating photoaged mouse skin. MATERIALS AND METHODS: SHSs were isolated from the explants of sponge Haliclona sp. with our proprietary method, and hucMSC-Exos were prepared from the conditioned medium of hucMSCs using ultracentrifugation. The effects of SHSs on the skin penetration of fluorescently labeled hucMSC-Exos were determined using confocal microscopy in vitro (porcine skin) and in vivo (mouse skin). The therapeutic effects of hucMSC-Exos coupled with SHSs against UV-induced photoaging in mice were assessed by using microwrinkles analysis, pathohistological examination and real-time RT-PCR. We also tested the skin irritation caused by the combination of hucMSC-Exos and SHSs in guinea pigs. RESULTS: In vitro results showed that hucMSC-Exos could not readily penetrate through porcine skin by themselves. However, SHSs increased the skin absorption of exosomes by a factor of 5.87 through creating microchannels. Similar penetration enhancement of hucMSC-Exos was observed after SHSs treatment in mice. The combined use of hucMSC-Exos and SHSs showed significant anti-photoaging effects in mice, including reducing microwrinkles, alleviating histopathological changes, and promoting the expression of extracellular matrix constituents, whereas hucMSC-Exos alone produced considerably weaker effects. Skin irritation test showed that the combination of hucMSC-Exos and SHSs caused slight irritation, and the skin recovered shortly. CONCLUSION: SHSs provide a safe and effective way to enhance the skin delivery of MSC-Exos. Moreover, the combination of MSC-Exos and SHSs may be of much use in the treatment of photoaging.

4,5-di-O-caffeoylquinic acid methyl ester isolated from Lonicera japonica Thunb. targets the Keap1/Nrf2 pathway to attenuate H2O2-induced liver oxidative damage in HepG2 cells.

BACKGROUND: 4,5-di-O-caffeoylquinic acid methyl ester (4,5-CQME) is a caffeoylquinic acid (CQA) isolated from Lonicera japonica Thunb., a traditional Chinese medicine. To date, the biological activity of 4,5-CQME has not been fully investigated. PURPOSE: The aim of the current study was to explore the anti-oxidative activity and the underlying mechanism of 4,5-CQME. METHODS: MTT assay was used to evaluate the cytoprotective effect of 4,5-CQME. DCFH-DA was used as a fluorescence probe to detect intracellular ROS. The mitochondrial membrane potential was detected using the fluorescent probe JC-1. MDA and GSH levels were measured using MDA and GSH commercial kits, respectively. Apoptosis assay was performed using the Annexin V-FITC/PI method. The functional mechanism of 4,5-CQME was investigated by analyzing relative signaling pathways through immunofluorescent staining, quantitative PCR and western blot analysis. RESULTS: HepG2 cells were incubated with different concentrations of 4,5-CQME for 12 h before exposure to 500 µM H2O2 for 3 h. 4,5-CQME attenuated H2O2-induced oxidative damage and had a higher cytoprotective effect than 3-caffeoylquinic acid, 3-caffeoylquinic acid methyl ester, or 4,5-di-O-caffeoylquinic acid. 4,5-CQME also reduced ROS and MDA levels and rescued GSH depletion. Western blots demonstrated that 4,5-CQME decreased Bax/Bcl-2 and Bak levels. A mechanistic study confirmed that 4,5-CQME significantly suppressed H2O2-induced MAPKs phosphorylation but had little effect on MAPKs phosphorylation under normal conditions. By contrast, 4,5-CQME induced AKT phosphorylation in the presence or absence of H2O2. 4,5-CQME also regulated the Keap1/Nrf2 signaling pathway and enhanced both the mRNA and protein expressions of HO-1 and NQO1. The anti-oxidative effect of 4,5-CQME was greatly abolished by co-incubation with the Nrf2 inhibitor ML385 or PI3K inhibitor wortmannin. CONCLUSIONS: Taken together, these results showed that 4,5-CQME offered significant protection against H2O2-induced oxidative stress, and its effect was in part due to the modulation of the Keap1/Nrf2 pathway.

3,4,5-Tri-O-caffeoylquinic acid methyl ester isolated from Lonicera japonica Thunb. Flower buds facilitates hepatitis B virus replication in HepG2.2.15 cells.

Caffeoylquinic acids are well known for their prominent antiviral activities. Beyond our expectations, we initially found 3,4,5-Tri-O-caffeoylquinic acid methyl ester (3,4,5-CQME) from L. japonica can facilitate HBV DNA and antigens secretion. This study aimed to investigate its underlying molecular mechanism. The results indicate that 3,4,5-CQME signally increased intracellular and secreted HBsAg levels by more than two times in HepG2.2.15 cells and HepAD38 cells. Furthermore, levels of HBeAg, HBV DNA and RNA were significantly enhanced by 3-day 3,4,5-CQME treatment; it didn't directly affect intracellular cccDNA amount, although it slightly increased cccDNA accumulation as a HBV DNA replication feedback. In addition, treatment with 3,4,5-CQME significantly induced HBx protein expression for viral replication. We utilized a phospho-antibody assay to profile the signal transduction change by 3,4,5-CQME to illuminate its molecular mechanism. The results indicate that treatment with 3,4,5-CQME activated AKT/mTOR, MAPK and NF-κB pathways verified by immunoblot. Moreover, 3,4,5-CQME upregulated the expression of nuclear transcriptional factors PGC1α and PPARα. In short, 3,4,5-CQME promotes HBV transcription and replication by upregulating HBx expression and activating HBV transcriptional regulation-related signals. As caffeoylquinic acids are widely present in traditional Chinese medicines, the risk of intaking caffeoylquinic acids-containing herbs for hepatitis B treatment requires more evaluation and further research.

Surface-Confined Macrocyclization via Dynamic Covalent Chemistry.

Surface-confined synthesis is a promising approach to build complex molecular nanostructures including macrocycles. However, despite the recent advances in on-surface macrocyclization under ultrahigh vacuum, selective synthesis of monodisperse and multicomponent macrocycles remains a challenge. Here, we report on an on-surface formation of [6 + 6] Schiff-base macrocycles via dynamic covalent chemistry. The macrocycles form two-dimensional crystalline domains on the micrometer scale, enabled by dynamic conversion of open-chain oligomers into well-defined ∼3.0 nm hexagonal macrocycles. We further show that by tailoring the length of the alkyl substituents, it is possible to control which of three possible products-oligomers, macrocycles, or polymers-will form at the surface. In situ scanning tunneling microscopy imaging combined with density functional theory calculations and molecular dynamics simulations unravel the synergistic effect of surface confinement and solvent in leading to preferential on-surface macrocyclization.

The diagnostic and prognostic usage of circulating tumor DNA in operable hepatocellular carcinoma.

Circulating tumor DNA (ctDNA) is a promising noninvasive biomarker for hepatocellular carcinoma (HCC). In this study, we aimed to assess the diagnostic and prognostic value of ctDNA in HCC. Twenty-six operable HCC, 10 hepatitis and 10 cirrhosis patients were enrolled in this study. Treatment-naïve blood samples were collected from all patients, nevertheless resected tissue and postoperative blood samples were only collected from HCC patients. A custom-designed sequencing panel covering 354 genes was used to identify somatic mutations. Collectively, we identified 139 somatic mutations from 25 HCC baseline plasma samples (96.2%). TP53 (50.00%) was the most common mutant gene, and R249S was the most recurrent mutation (19.2%). Twenty-three patients (88.5%) carried at least one ctDNA mutation validated in matched tissue, and the driver mutations exhibited an advanced concordance than non-driver mutations (67.6% vs. 33.8%, P = 0.0002). For HCC patients, the number of mutations in ctDNA (R2 = 0.1682, P = 0.0375), maximal variant allele frequency (VAF) in ctDNA (R2 = 0.4974, P < 0.0001) and ctDNA concentration (R2 = 0.2676, P = 0.0068) were linearly correlated with tumor size. Multiple circulating cell-free DNA (cfDNA) parameters could be used in differentiating malignant lesions from benign lesions, and the performance was no less than blood alpha-fetoprotein (AFP). HCC patients with detectable mutation in postoperative plasma had a poor DFS than those without (17.5 months vs. 6.7 months, HR = 7.655, P < 0.0001), and postoperative cfDNA status (HR = 10.293, P < 0.0001) was an independent risk factors for recurrence. In conclusion, ctDNA profiling is potentially valuable in differential diagnosis and prognostic evaluation of HCC.

Chemical constituents from Lonicera japonica flower buds and their anti-hepatoma and anti-HBV activities.

Three new naturally occurring monoterpenoids, japopenoid A (1), japopenoid B (23) japopenoid C (24), and one new caffeoylquinic acid derivative (28), together with thirty-one known compounds (2-22, 25-27, 29-35), were isolated and identified from the flower buds of Lonicera japonica Thunb. Their structures were determined by extensive 1D and 2D NMR spectroscopic methods, high-resolution mass spectrometry, and the absolute configurations of 1, 23, 24 were determined by comparison of their electronic circular dichroism (ECD) spectrum with literature and theoretical calculation. Structurally, compound 1 is a monoterpenoid featured with an unusual tricyclic skeleton. All compounds (1-35) were evaluated for their cytotoxicities against human liver cancer cell lines (HepG 2 and SMMC-7721). Compound 12 exhibited the most potent activity with IC50 values of 26.54 ±â€¯1.95 and 8.72 ±â€¯1.57 µg/ml against HepG 2 and SMMC-7721, and the IC50 values of compound 13 were 26.54 ±â€¯1.95 and 12.35 ±â€¯1.43 µg/ml, respectively. Western blot results further proved that compound 13 induces hepatoma cell apoptosis via the intrinsic apoptosis pathway. In addition, most terpenoids showed inhibitory activity against HBsAg and HBeAg secretion, and HBV DNA replication. In particular, 25 µg/mlof compound 11 inhibits HBsAg and HBeAg secretion, and HBV DNA replication by 39.39 ±â€¯5.25, 15.64 ±â€¯1.25, and 16.13 ±â€¯4.10% compared to the control (p < 0.05). These results indicated that L. japonica flower buds could be served as functional food for anti-hepatoma and anti-HBV activities.