On the Chemical Purity and Oxygen Isotopic Composition of α-Cellulose Extractable from Higher Plants and the Implications for Climate, Metabolic, and Physiological Studies.

The 18O/16O ratio of α-cellulose in land plants has proved of interest for climate, environmental, physiological, and metabolic studies. Reliable application of such a ratio may be compromised by the presence of hemicellulose impurities in the α-cellulose product obtainable with current extraction methods, as the impurities are known to be isotopically different from that of the α-cellulose. We first compared the quality of hydrolysates of "α-cellulose products" obtained with four representative extraction methods (Jayme and Wise; Brendel; Zhou; Loader) and quantified the hemicellulose-derived non-glucose sugars in the α-cellulose products from 40 land grass species using gas chromatography-mass spectrometry (GC/MS). Second, we performed compound-specific isotope analysis of the hydrolysates using GC/Pyrolysis/IRMS. These results were then compared with the bulk isotope analysis using EA/Pyrolysis/IRMS of the α-cellulose products. We found that overall, the Zhou method afforded the highest purity α-cellulose as judged by the minimal presence of lignin and the second-lowest presence of non-glucose sugars. Isotopic analysis then showed that the O-2-O-6 of the α-cellulose glucosyl units were all depleted in 18O by 0.0-4.3 mUr (average, 1.9 mUr) in a species-dependent manner relative to the α-cellulose products. The positive isotopic bias of using the α-cellulose product instead of the glucosyl units stems mainly from the fact that the pentoses that dominate hemicellulose contamination in the α-cellulose product are relatively enriched in 18O (compared to hexoses) as they inherit only the relatively 18O-enriched O-2-O-5 moiety of sucrose, the common precursor of pentoses and hexoses in cellulose, and are further enriched in 18O by the (incomplete) hydrolysis.

Discovery of the Caged-Vibsane Norditerpenoids with Unprecedented Chemical Architectures and Exploration of Their Various Acid Tolerances.

Cyclovibsanones A-D (1-4, respectively), featuring unprecedented caged tricyclo[5.4.1.05,9]dodecane and bicyclo[4.2.1]hexane cores, were isolated from the leaves of Viburnum odoratissimum. Their structures as well as that of one chemical derivative (5), which was transformed from 2, were determined by spectroscopic data, theoretical calculations, and the ML-JDP4/MAEΔΔδ methods. In addition, compounds 1 and 2 were found to possess dissimilarities in acid tolerance during nuclear magnetic resonance (NMR) experiments. The potential mechanism was consequently postulated and further supported through NMR analysis and mechanistic calculations. Biologically, chemical derivative 5 exerted antiproliferative activity against HepG2 cells.

Deoxyelephantopin, a germacrane-type sesquiterpene lactone from Elephantopus scaber, induces mitochondrial apoptosis of hepatocarcinoma cells by targeting Hsp90α in vitro and in vivo.

Hepatocellular carcinoma has been known as the most frequent subtype of liver cancer with a high rate of spread, metastases, and recurrence, also dismal treatment effects. However, effective therapies for HCC are still required. Nowadays, natural products have been known as a valuable source for drug discovery. In this research, 44 sesquiterpene lactones isolated from the Elephantopus scaber Linn. (Asteraceae) were tested by MTT assay for the antitumor activities. Deoxyelephantopin (DET) was found to exert significant cytotoxicity on HepG2 and Hep3B cells. Moreover, we found that DET treatment markedly reduced the growth of HCC cells in a concentration-dependent manner, which was better than sorafenib. Furthermore, DET induced mitochondrial dysfunction, oxidative stress, and cellular apoptosis. Additionally, we found that DET and sorafenib synergistically induced apoptosis and mitochondrial dysfunction in HCC cells. DET combined with sorafenib was also efficacious in tumor xenograft model. Molecular docking experiments revealed that DET had a potentially high binding affinity with Hsp90α. Moreover, Drug Affinity Responsive Target Stability assay suggested that DET could directly target Hsp90α. Additionally, the expression of Hsp90α was both decreased in vitro and in vivo. Altogether, this study revealed that DET might be a promising agent for HCC therapy by targeting Hsp90α.

Semisynthesis and Non-Small-Cell Lung Cancer Cytotoxicity Evaluation of Germacrane-Type Sesquiterpene Lactones from Elephantopus scaber.

Two series of germacrane-type sesquiterpene lactones were produced by semisynthetic modulation of scaberol C, which was prepared by a standard chemical transformation from an Elephantopus scaber extract. Their inhibition activities against non-small-cell lung cancer cells were screened, and preliminary structure-activity relationships were also established. Among them, monomeric analog 1u and dimeric analog 3d exhibited superior anti-non-small-cell lung cancer cytotoxic potencies with IC50 values of 4.3 and 0.7 µM against A549 cells, respectively, and were more active than cisplatin and the standard sesquiterpene lactones, parthenolide and scabertopin. Further studies revealed that compounds 1u and 3d cause G2/M phase arrest and induce apoptosis through the activation of mitochondrial pathways in A549 cells. Collectively, the results obtained suggest that compounds 1u and 3d are promising anti-non-small-cell lung cancer lead compounds.

Sesquiterpene lactones from Elephantopus scaber exhibit cytotoxic effects on glioma cells by targeting GSTP1.

Sesquiterpene lactones possess excellent anti-tumor activity in multiple cancer cell lines, including glioma, the most common type of malignant brain tumor with high mortality. However, the detailed mechanism of this type of constituent, especially the potential target for anti-glioma effect, is still unclear. Here, we collected 52 sesquiterpene lactones from Elephantopus scaber Linn. for network pharmacology analysis. The results demonstrated that the targets of the active components were markedly enriched on the pathways in cancer, which were closely related to cell proliferation regulation. Next, the Gene Expression Omnibus (GEO) and DisGeNET were analyzed by bioinformatics, and 429 glioma-related targets were obtained. Furtherly, 34 common targets of compounds and glioma were revealed, and they were significantly enriched in MAPK signaling pathway. Subsequently, we constructed a common target-compound network, and glutathione S-transferase Pi 1 (GSTP1) had the highest degree value, which explained its significance in the network. Therefore, we speculated that the compounds might exert an anti-glioma effect by targeting GSTP1. To verify the above results, we obtained part of sesquiterpene lactones isolated from E. scaber in our laboratory and evaluated their activities against glioma U87 cells. Among these sesquiterpene lactones (1-27), compounds 1 (elephantopinolide A), 2 (cis-scabertopin) and 3 (elephantopinolide F) exhibited the strongest inhibitory effect, and the IC50 values were 4.22 ± 0.14 µM, 4.28 ± 0.21 µM and 1.79 ± 0.24 µM, respectively. The results from molecular docking, cellular thermal shift assay (CETSA), as well as RT-PCR and Western blot analysis suggested that the compounds exerted an inhibitory effect by targeting GSTP1. Meanwhile, the compounds also activated JNK/STAT3 signaling pathway. Furthermore, we found that 1, 2 and 3 could suppress cell proliferation and also induce mitochondrial dysfunction as well as oxidative stress, eventually leading to cellular apoptosis. Taken together, this study revealed that sesquiterpene lactones from E. scaber could be a promising therapeutic strategy for the treatment of glioma by targeting GSTP1.

Chamaejasmenin E from Stellera chamaejasme induces apoptosis of hepatocellular carcinoma cells by targeting c-Met in vitro and in vivo.

Hepatocellular carcinoma (HCC), the most prevalent liver cancer, is considered one of the most lethal malignancies with a dismal outcome. There is an urgent need to find novel therapeutic approaches to treat HCC. At present, natural products have served as a valuable source for drug discovery. Here, we obtained five known biflavones from the root of Stellera chamaejasme and evaluated their activities against HCC Hep3B cells in vitro. Chamaejasmenin E (CE) exhibited the strongest inhibitory effect among these biflavones. Furthermore, we found that CE could suppress the cell proliferation and colony formation, as well as the migration ability of HCC cells, but there was no significant toxicity on normal liver cells. Additionally, CE induced mitochondrial dysfunction and oxidative stress, eventually leading to cellular apoptosis. Mechanistically, the potential target of CE was predicted by database screening, showing that the compound might exert an inhibitory effect by targeting at c-Met. Next, this result was confirmed by molecular docking, cellular thermal shift assay (CETSA), as well as RT-PCR and Western blot analysis. Meanwhile, CE also reduced the downstream proteins of c-Met in HCC cells. In concordance with above results, CE is efficacious and non-toxic in tumor xenograft model. Taken together, our findings revealed an underlying tumor-suppressive mechanism of CE, which provided a foundation for identifying the target of biflavones.

Leaf transition from heterotrophy to autotrophy is recorded in the intraleaf C, H and O isotope patterns of leaf organic matter.

RATIONALE: Quantitatively relating 13 C/12 C, 2 H/1 H and 18 O/16 O ratios of plant α-cellulose and 2 H/1 H of n-alkanes to environmental conditions and metabolic status should ideally be based on the leaf, the plant organ most sensitive to environmental change. The fact that leaf organic matter is composed of isotopically different heterotrophic and autotrophic components means that it is imperative that one be able to disentangle the relative heterotrophic and autotrophic contributions to leaf organic matter. METHODS: We tackled this issue by two-dimensional sampling of leaf water and α-cellulose, and specific n-alkanes from greenhouse-grown immature and mature and field-grown mature banana leaves, taking advantage of their large areas and thick waxy layers. Leaf water, α-cellulose and n-alkane isotope ratios were then characterized using elemental analysis isotope ratio mass spectrometry (IRMS) or gas chromatography IRMS. A three-member (heterotrophy, autotrophy and photoheterotrophy) conceptual linear mixing model was then proposed for disentangling the relative contributions of the three trophic modes. RESULTS: We discovered distinct spatial leaf water, α-cellulose and n-alkane isotope ratio patterns that varied with leaf developmental stages. We inferred from the conceptual model that, averaged over the leaf blade, only 20% of α-cellulose in banana leaf is autotrophically laid down in both greenhouse-grown and field-grown banana leaves, while approximately 60% and 100% of n-alkanes are produced autotrophically in greenhouse-grown and field-grown banana leaves, respectively. There exist distinct lateral (edge to midrib) gradients in autotrophic contributions of α-cellulose and n-alkanes. CONCLUSIONS: Efforts to establish quantitative isotope-environment relationships should take into account the fact that the evaporative leaf water 18 O and 2 H enrichment signal recorded in autotrophically laid down α-cellulose is significantly diluted by the heterotrophically formed α-cellulose. The δ2 H value of field-grown mature banana leaf n-alkanes is much more sensitive than α-cellulose as a recorder of the growth environment. Quantitative isotope-environment relationship based on greenhouse-grown n-alkane δ2 H values may not be reliable.

The effect of processing medium on the 2 H/1 H of carbon-bound hydrogen in α-cellulose extracted from higher plants.

RATIONALE: Although the 2 H/1 H ratio of the carbon-bound hydrogens (C-Hs) in α-cellulose extracted from higher plants has long been used successfully for climate, environmental and metabolic studies, the assumption that bleaching with acidified NaClO2 to remove lignin before pure α-cellulose can be obtained does not alter the 2 H/1 H ratio of α-cellulose C-Hs has nonetheless not been tested. METHODS: For reliable application of the 2 H/1 H ratio of α-cellulose C-H, we processed plant materials representing different phytochemistries and photosynthetic carbon assimilation modes in isotopically contrasting bleaching media (with an isotopic difference of 273 mUr). All the isotope ratios were measured by elemental analyzer/isotope ratio mass spectrometry (EA/IRMS). RESULTS: Our results show that H from the bleaching medium does appear in the final pure α-cellulose product, although the isotopic alteration to the C-H in α-cellulose due to the incorporation of processing H from the medium is small if isotopically "natural" water is used to prepare the processing medium. However, under prolonged bleaching such an isotope effect can be significant, implying that standardizing the bleaching process is necessary for reliable 2 H/1 H measurement. CONCLUSIONS: The currently adopted method for removing lignin for α-cellulose extraction from higher plant materials with acidified NaClO2 bleaching is considered acceptable in terms of preserving the isotopic fidelity if isotopically "natural" water is used to prepare the bleaching solution.

Progress in Core-Shell Magnetic Mesoporous Materials for Enriching Post-Translationally Modified Peptides.

Endogenous peptides, particularly those with post-translational modifications, are increasingly being studied as biomarkers for diagnosing various diseases. However, they are weakly ionizable, have a low abundance in biological samples, and may be interfered with by high levels of proteins, peptides, and other macromolecular impurities, resulting in a high limit of detection and insufficient amounts of post-translationally modified peptides in real biological samples to be examined. Therefore, separation and enrichment are necessary before analyzing these biomarkers using mass spectrometry. Mesoporous materials have regular adjustable pores that can eliminate large proteins and impurities, and their large specific surface area can bind more target peptides, but this may result in the partial loss or destruction of target peptides during centrifugal separation. On the other hand, magnetic mesoporous materials can be used to separate the target using an external magnetic field, which improves the separation efficiency and yield. Core-shell magnetic mesoporous materials are widely utilized for peptide separation and enrichment due to their biocompatibility, efficient enrichment capability, and excellent recoverability. This paper provides a review of the latest progress in core-shell magnetic mesoporous materials for enriching glycopeptides and phosphopeptides and compares their enrichment performance with different types of functionalization methods.

Capping the hydroxyl groups (-OH) of α-cellulose to reduce Hy-groscopicity for accurate 18O/16O measurement by EA/Py/IRMS.

Obtaining an accurate measurement of 18O/16O at natural abundance level for land plants-derived α-cellulose with the currently popular EA/Py/IRMS (elemental analysis/pyrolysis/isotope ratio mass spectrometry) method is a challenge due to the hygroscopic nature of the exposed hydroxyl groups, as the 18O/16O of adsorbed moisture is usually different from that of the α-cellulose and the relative amount of adsorbed moisture is sample- and relative humidity-dependent. To minimize the hygroscopicity-related measurement error, we capped the hydroxyl groups of α-cellulose by benzylation to various degrees and found that the 18O/16O ratio of α-cellulose increased with the degree of benzyl substitution (DS), consistent with the theoretical prediction that a reduced presence of exposed hydroxyl groups should lead to a more accurate (and therefore more reliable) α-cellulose 18O/16O measurement. We propose the establishment of a moisture adsorption-degree of substitution or percentage of oxygen-18O/16O ratio equation, based on the measurement of C%, O% and δ18O of variably capped α-cellulose, so that a robust correction can be made in a plant species- and laboratory conditions-specific manner. Failure to do so will lead to an average underestimate of α-cellulose δ18O by 3.5 mUr under "average" laboratory conditions.

SCP-7, a germacrane-type sesquiterpene lactone derivative, induces ROS-mediated apoptosis in NSCLC cells in vitro and in vivo.

Scabertopin (SCP), an abundant germacrane-type sesquiterpene lactone (SLC) isolated from Elephantopus scaber, was selected as a reference compound for modification and evaluation as anticancer agents for non-small cell lung cancer (NSCLC) treatment. All derivatives (SCP-1-SCP-13) except for SCP-3 showed potential inhibitory effect (IC50 5.2-9.7 µM) against A549 cells. The most promising compound SCP-7 also showed good cytotoxic activity against another two NSCLC cell lines (H1299 and H460), with IC50 value of 4.4 and 8.9 µM, respectively. Furthermore, SCP-7 could induce apoptotic cell death that was associated with the increased reactive oxygen species (ROS) generation, the loss of mitochondrial membrane potential, Bcl-2 family proteins modulation, caspases-3 and PARP cleavage. In addition, SCP-7 also inhibited cell growth by increasing Bax expression and reducing the Ki-67 positive cells in vivo, but there were no obvious toxic and side effects on internal organs. Mechanistically, PharmMapper, molecular docking and Western blot analysis revealed that SCP-7 might interact with the epidermal growth factor receptor (EGFR) and inhibit its expression in lung cancer cells. Together, above results suggest further effective application of SCP-7 as a potential anti-tumor agent in the treatment of NSCLC.

Small Molecule Accurate Recognition Technology accelerated isolation of structurally diverse sesquiterpenes from Litsea lancilimba Merr.

Four undescribed sesquiterpenes (1-4) and 20 known sesquiterpenes (5-24) were obtained from the fruits of Litsea lancilimba Merr. by Small Molecule Accurate Recognition Technology (SMART). The gross structures and the relative configurations of the new compounds were deduced by spectroscopic data analysis. The absolute configurations were defined by comparison of their experimental and calculated electronic circular dichroism (ECD) spectra data. All compounds were screened for their neuroprotective effects against H2O2-induced SH-SY5Y cells injury. Compounds 2, 3, 7, 8 and 13 exhibited comparable neuroprotective activity to the positive trolox at 50 µM.

GP73 is a glucogenic hormone contributing to SARS-CoV-2-induced hyperglycemia.

Severe cases of infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are associated with elevated blood glucose levels and metabolic complications. However, the molecular mechanisms for how SARS-CoV-2 infection alters glycometabolic control are incompletely understood. Here, we connect the circulating protein GP73 with enhanced hepatic gluconeogenesis during SARS-CoV-2 infection. We first demonstrate that GP73 secretion is induced in multiple tissues upon fasting and that GP73 stimulates hepatic gluconeogenesis through the cAMP/PKA signaling pathway. We further show that GP73 secretion is increased in cultured cells infected with SARS-CoV-2, after overexpression of SARS-CoV-2 nucleocapsid and spike proteins and in lungs and livers of mice infected with a mouse-adapted SARS-CoV-2 strain. GP73 blockade with an antibody inhibits excessive glucogenesis stimulated by SARS-CoV-2 in vitro and lowers elevated fasting blood glucose levels in infected mice. In patients with COVID-19, plasma GP73 levels are elevated and positively correlate with blood glucose levels. Our data suggest that GP73 is a glucogenic hormone that likely contributes to SARS-CoV-2-induced abnormalities in systemic glucose metabolism.

Accessing the position-specific 18O/16O ratios of lignin monomeric units from higher plants with highly selective hydrogenolysis followed by GC/Py/IRMS analysis.

The 18O/16O of lignin at bulk, molecular and positional levels can be used to extract valuable information about climate, plant growth environment, plant physiology, and plant metabolism. Access to the individual oxygen isotope compositions (δ18O) in the lignin monomeric units is, however, challenging as depolymerization of lignin to release the monomeric units may cause isotope fractionation. We have developed a novel method to measure the δ18O of the three oxygens (O-3, O-4 and O-5) attached to the aromatic ring of the monomeric units (bearing no oxygen in their side chains) releasable by highly selective W2C/AC (tungsten carbide supported by activated carbon)-catalyzed hydrogenolysis of lignin. O-4 is obtained by measuring the δ18O of H-type monomeric unit, while O-3 and O-5 can be calculated following isotope mass balance between H, G and S-type monomeric units measurable simultaneously with GC/Py/IRMS (gas chromatography-pyrolysis-isotope ratio mass spectrometry). The measurement precisions are better than 1.15 mUr and 4.15 mUr at molecular and positional levels, respectively. It was shown that there were a δ18OH > Î´18OG > Î´18OS isotopic order in the herbaceous plant lignin and an (inclusive) opposite order in woody plant lignin. Such differences in isotopic order is likely to be caused by the fact that both L-tyrosine, which carries an 18O-enriched leaf water signal, and L-phenylalanine, which carries mainly a molecular O2 isotopic signal, serve as the precursors for lignin biosynthesis in herbaceous plants while only the latter serves as precursor for lignin biosynthesis in woody plants. We have highlighted the potential application of such molecular and positional levels isotopic signals in plant physiological, metabolic, lignin biosynthetic and climate studies.

The Mitochondrial Protein MAVS Stabilizes p53 to Suppress Tumorigenesis.

Recent reports have shown the critical role of the mitochondrial antiviral signaling (MAVS) protein in virus-induced apoptosis, but the involvement of MAVS in tumorigenesis is still poorly understood. Herein, we report that MAVS is a key regulator of p53 activation and is critical for protecting against tumorigenesis. We find that MAVS promotes p53-dependent cell death in response to DNA damage. MAVS interacts with p53 and mediates p53 mitochondrial recruitment under genotoxic stress. Mechanistically, MAVS inhibits p53 ubiquitination by blocking the formation of the p53-murine double-minute 2 (MDM2) complex, leading to the stabilization of p53. Notably, compared with their wild-type littermates, MAVS knockout mice display decreased resistance to azoxymethane (AOM) or AOM/dextran sulfate sodium salt (DSS)-induced colon cancer. MAVS expression is significantly downregulated in human colon cancer tissues. These results unveil roles for MAVS in DNA damage response and tumor suppression.

HDL-scavenger receptor B type 1 facilitates SARS-CoV-2 entry.

Responsible for the ongoing coronavirus disease 19 (COVID-19) pandemic, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infects host cells through binding of the viral spike protein (SARS-2-S) to the cell-surface receptor angiotensin-converting enzyme 2 (ACE2). Here we show that the high-density lipoprotein (HDL) scavenger receptor B type 1 (SR-B1) facilitates ACE2-dependent entry of SARS-CoV-2. We find that the S1 subunit of SARS-2-S binds to cholesterol and possibly to HDL components to enhance viral uptake in vitro. SR-B1 expression facilitates SARS-CoV-2 entry into ACE2-expressing cells by augmenting virus attachment. Blockade of the cholesterol-binding site on SARS-2-S1 with a monoclonal antibody, or treatment of cultured cells with pharmacological SR-B1 antagonists, inhibits HDL-enhanced SARS-CoV-2 infection. We further show that SR-B1 is coexpressed with ACE2 in human pulmonary tissue and in several extrapulmonary tissues. Our findings reveal that SR-B1 acts as a host factor that promotes SARS-CoV-2 entry and may help explain viral tropism, identify a possible molecular connection between COVID-19 and lipoprotein metabolism, and highlight SR-B1 as a potential therapeutic target to interfere with SARS-CoV-2 infection.