Bacterial pseudaminic acid binding to Siglec-10 induces a macrophage interleukin-10 response and suppresses phagocytosis.

Pseudaminic acid (Pse) on pathogenic bacteria exopolysaccharide engages with the sialic acid-binding immunoglobulin-type lectin (Siglec)-10 receptor on macrophages via the critical 7-N-acetyl group. This binding stimulates macrophages to secrete interleukin 10 that suppresses phagocytosis against bacteria, but can be reverted by blocking Pse-Siglec-10 interaction with Pse-binding protein as a promising therapy.

An Immunological Polysaccharide from Tremella fuciformis: Essential Role of Acetylation in Immunomodulation.

The edible fungus Tremella fuciformis was shown to have a high molecular weight (1.87 × 103 kDa) bioactive polysaccharide, denoted as TFP-F1. Monosaccharide composition and NMR analysis of the polysaccharide and its derivatives indicated it contained fucose (Fucp), xylose (Xylp), mannose (Manp), and glucuronic acid (GlcAp) in a ratio of 0.9:1.0:3.2:1.2. Using IR, NMR, and GC-MS spectroscopic data, the structure of TFP-F1 was elucidated as {→3)-[ß-D-GlcAp-(1→2)]-α-D-Manp-(1→3)-α-D-Manp-(1→3)-[α-L-Fucp-(1→2)-ß-D-Xylp-(1→2)]-α-D-Manp-(1→}n, with partial acetylation of C6-OH in mannoses. Furthermore, at a concentration of 1 µg/mL, TFP-F1 was found to stimulate the secretion of TNF-α and IL-6 in J774A.1 macrophage cells in vitro via interaction with toll-like receptor 4 (TLR4). The removal of O-acetyl groups led to the loss of immunomodulatory activities, demonstrating that O-acetyl groups play an essential role in enhancing the production of pro-inflammatory cytokines.

A hexasaccharide from capsular polysaccharide of carbapenem-resistant Klebsiella pneumoniae KN2 is a ligand of Toll-like receptor 4.

Klebsiella pneumoniae serotype KN2 is a carbapenem-resistant strain and leads to the health care-associated infections, such as bloodstream infections. Its capsular polysaccharide (CPS) was isolated and cleaved by a specific enzyme from a bacteriophage into a hexasaccharide-repeating unit. With GC-MS, NMR, and Mass analyses, the structure of KN2 CPS was determined to be {→3)-ß-D-Glcp-(1→3)-[α-D-GlcpA-(1→4)-ß-D-Glcp-(1→6)]-α-D-Galp-(1→6)-ß-D-Galp-(1→3)-ß-D-Galp-(1→}n. We demonstrated that 1 µg/mL CPS could stimulate J774A.1 murine macrophages to release tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in vitro. Also, we proved that KN2 CPS induced the immune response through Toll-like receptor 4 (TLR4) in the human embryonic kidney (HEK)-293 cells. Strikingly, the hexasaccharide alone shows the same immune response as the CPS, suggesting that the hexasaccharide can shape the adaptive immunity to be a potential vaccine adjuvant. The glucuronic acid (GlcA) on other polysaccharides can affect the immune response, but the GlcA-reduced KN2 CPS and hexasaccharide still maintain their immunomodulatory activities.

New Hydroquinone Monoterpenoid and Cembranoid-Related Metabolites from the Soft Coral Sarcophyton tenuispiculatum.

Chemical investigation of the marine soft coral Sarcophyton tenuispiculatum resulted in the isolation of a 1,4-dihydrobenzoquinone, sarcotenuhydroquinone (1), three new cembranoids, sarcotenusenes A‒C (2‒4), and ten previously reported metabolites 5-14. The chemical structures of all isolated metabolites were determined by detailed spectroscopic analyses. In biological assays, anti-inflammatory, cytotoxic, and peroxisome proliferator-activated receptor γ (PPAR-γ) transcription factor assays of all compounds were performed. None of the isolated compounds were found to exhibit activity in the PPAR-γ transcription factor assay. The anti-inflammatory assays showed that (+)-7α,8ß-dihydroxydeepoxysarcophine (13) inhibited the production of IL-1ß to 56 ± 1% at a concentration of 30 µM in lipopolysaccharide (LPS)-stimulated J774A.1 macrophage cells. In addition, 1 and 2 were found to exhibit cytotoxicity towards a panel of cancer cell lines.

Phagocytosis enhancement, endotoxin tolerance, and signal mechanisms of immunologically active glucuronoxylomannan from Auricularia auricula-judae.

Glucuronoxylomannan (AAPS) from the edible wood ear mushroom Auricularia auricula-judae has been demonstrated to exhibit immunostimulatory properties through its binding to TLR4. However, the mechanisms of immune modulation by AAPS in mammalian cells remains unclear. In the present study, we demonstrated that AAPS induced immunostimulatory effects were regulated by reactive oxygen species, mitogen-activated protein kinases, protein kinase C-α and NF-κB. AAPS remarkably increased the phagocytosis and bactericidal activity of macrophages. In lipopolysaccharide-activated macrophages, AAPS induced endotoxin tolerance like effect characterized by the downregulation of nitric oxide, interleukin-6 and TNF-α via the downregulation of NF-κB activation. Our findings provide firm scientific evidences for the immunoenhancing properties of wood ear mushroom, and the potential of AAPS to be strong candidates for the development of new carbohydrate-based nutraceutical supplements in the management of immunity related disorders in the future.

New Biscembranoids Sardigitolides A-D and Known Cembranoid-Related Compounds from Sarcophyton digitatum: Isolation, Structure Elucidation, and Bioactivities.

Chemical examination from the cultured soft coral Sarcophyton digitatum resulted in the isolation and structural identification of four new biscembranoidal metabolites, sardigitolides A-D (1-4), along with three previously isolated biscembranoids, sarcophytolide L (5), glaucumolide A (6), glaucumolide B (7), and two known cembranoids (8 and 9). The chemical structures of all isolates were elucidated on the basis of 1D and 2D NMR spectroscopic analyses. Additionally, in order to discover bioactivity of marine natural products, 1-8 were examined in terms of their inhibitory potential against the upregulation of inflammatory factor production in lipopolysaccharide (LPS)-stimulated murine macrophage J774A.1 cells and their cytotoxicities against a limited panel of cancer cells. The anti-inflammatory results showed that at a concentration of 10 µg/mL, 6 and 8 inhibited the production of IL-1ß to 68 ± 1 and 56 ± 1%, respectively, in LPS-stimulated murine macrophages J774A.1. Furthermore, sardigitolide B (2) displayed cytotoxicities toward MCF-7 and MDA-MB-231 cancer cell lines with the IC50 values of 9.6 ± 3.0 and 14.8 ± 4.0 µg/mL, respectively.

Multimeric TAT peptides are effective in vitro inhibitors of Staphylococcus saprophyticus.

TAT (48-60) is a tridecapeptide from the envelope protein of HIV that was previously shown to possess cell-penetrating properties and antibacterial activity, making it a potential drug delivery agent for anticancer drugs and as antibacterial compound. Previous reports indicated that dimerization enhances the desired bioactivity of TAT; hence, we sought to synthesize multimeric TAT peptides. Herein, we describe the effects of multimerization on the antibacterial activity and secondary structure of the peptide. Terminal modifications such as N-acetylation and C-amidation were employed in the design. TATp monomer, dimer, and tetramer were synthesized using solid-phase peptide synthesis, purified by reversed-phase HPLC, and then characterized by mass spectrometry. Multimerization of the peptide did not change the secondary structure conformation. The CD analysis revealed a polyproline-II conformation for all peptide designs. Thus, this study provides a method of increasing the biological activity of the peptide by multimerization while retaining the secondary conformation of its monomeric unit. Furthermore, the bacteria Staphylococcus saprophyticus was found to be susceptible to the dimer and tetramer, with MIC50 of 12.50 µm and <1.56 µm, respectively. This suggests a structure-activity relationship whereby the antibacterial activity increases with increase in valency.

Cyanine dye mediated mitochondrial targeting enhances the anti-cancer activity of small-molecule cargoes.

Organelle-specific delivery systems are of significant clinical interest. We demonstrate the use of common cyanine dyes Cy3 and Cy5 as vectors for targeting and delivering cargoes to mitochondria in cancer cells. Specifically, conjugation to the dyes can increase cytotoxicity by up to 1000-fold.

H-Phosphonate Synthesis and Biological Evaluation of an Immunomodulatory Phosphoglycolipid from Thermophilic Bacteria.

The synthesis of a library of bacterial phosphoglycolipid, PGL-1, is described. Key features of the synthesis include regioselective esterification of the primary alcohol of the diacylglycerol moiety and an H-phosphonate method to install the phosphate in PGL-1 in comparison with earlier reported procedures. A representative set of PGL-1 analogues was prepared and evaluated for their biological activities. Results showed that the immunological activity of PGL-1 is dependent on the chain lengths of the fatty acids.

Ergosterol peroxide from marine fungus Phoma sp. induces ROS-dependent apoptosis and autophagy in human lung adenocarcinoma cells.

As part of our ongoing search for novel therapeutic structures from microorganism, the chemical examination of marine fungus Phoma sp. resulted in the isolation of ergosterol, ergosterol peroxide (EP), and 9,11-dehydroergosterol peroxide (DEP). The bioassay results demonstrated that the three isolates reduced the viability of various cancer cells, with EP being highest in human lung cancer cell line A549 cells. EP induced caspase-dependent apoptosis through mitochondrial damage in A549 cells. Additionally, EP-induced ROS generation and apoptosis were attenuated by ROS-generating enzymes inhibitors and antioxidant N-acetylcysteine, indicated that ROS played an important role in EP-mediated apoptosis in A549 cells. Furthermore, it was observed that EP induced ROS-dependent autophagy, which attenuated apoptosis in A549 cells. On the other hand, EP reduced the LPS/ATP-induced proliferation and migration of A549 cells through attenuated NLRP3 inflammasome activity. Additionally, EP showed synergistic cytotoxic effect with antitumor drug Sorafenib in A549 cell viability inhibition. Furthermore, Micro-Western Array and Western blot analyses demonstrated that the protein levels of EGFR, HSP27, MEK5, AKT1, mTOR, Smad2, Smad3, TAB1, NF-κB, and HIF1-α decreased, while the levels of p-p38α, p-ERK1/2, p-JNK, fibronectin and p27 increased. Collectively, the results of this study demonstrated that EP might be useful to develop a therapeutic candidate for lung cancer complications.

Antrodia cinnamomea Galactomannan Elicits Immuno-stimulatory Activity Through Toll-like Receptor 4.

Antrodia cinnamomea (A. cinnamomea) is a medicinal fungus used in traditional Chinese medicine to treat different kinds of ailments, including liver diseases, abdominal pain, drug intoxication, diarrhea, itchy skin, hypertension, and cancer. Polysaccharides have been identified as one of the major pharmacologically active ingredients present in A. cinnamomea. The present study aims to investigate the immunoenhancing activity of galactomannan isolated from A. cinnamomea. The cold water-soluble polysaccharide (galactomannan-repeated; MW>70 kDa; named ACP) of A. cinnamomea was isolated, and immunostimulatory properties were studied through different immune cell models including mouse macrophages and human dendritic cells. Through Toll-like receptor 4, ACP stimulated tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in J774A.1 mouse macrophages, mouse peritoneal macrophages and human dendritic cells. It was further identified that ACP elicited its immunostimulatory activity through protein kinase C-α (PKC-α) and mitogen activated protein kinases (MAPK) phosphorylation. Furthermore, ACP exerted the endotoxin tolerance-like effect through NF-κB inhibition. These findings demonstrate the potential of A. cinnamomea galactomannan as an immunostimulator or an adjuvant in immunotherapy and vaccination.

Carboxylic and O-acetyl moieties are essential for the immunostimulatory activity of glucuronoxylomannan: a novel TLR4 specific immunostimulator from Auricularia auricula-judae.

This study established the comprehensive repeating unit structure of immunologically active glucuronoxylomannan (AAPS) from wood ear mushroom, Auricularia auricula-judae. We identified Toll-like receptor 4 (TLR4) as a critical receptor involved in AAPS-induced macrophage activation to secrete pro-inflammatory cytokines. Molecular modeling data and chemical modifications of AAPS revealed that both carboxylic and acetyl moieties of AAPS are equally essential in TLR4 binding to exert in vitro immunostimulatory activity.

Affinity-Driven Covalent Modulator of the Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) Cascade.

Traditional medicines provide a fertile ground to explore potent lead compounds, yet their transformation into modern drugs is fraught with challenges in deciphering the target that is mechanistically valid for its biological activity. Herein we reveal that (Z)-(+)-isochaihulactone (1) exhibited significant inhibition against multiple-drug-resistant (MDR) cancer cell lines and mice xenografts. NMR spectroscopy showed that 1 resisted an off-target thiolate, thus indicating that 1 was a target covalent inhibitor (TCI). By identifying the pharmacophore of 1 (α,ß-unsaturated moiety), a probe derived from 1 was designed and synthesized for TCI-oriented activity-based proteome profiling. By MS/MS and computer-guided molecular biology approaches, an affinity-driven Michael addition of the noncatalytic C247 residue of GAPDH was found to control the "ON/OFF" switch of apoptosis through non-canonically nuclear GAPDH translocation, which bypasses the common apoptosis-resistant route of MDR cancers.

A GalNAc/Gal-specific lectin from the sea mussel Crenomytilus grayanus modulates immune response in macrophages and in mice.

A GalNAc/Gal-specific lectin (CGL) from the edible mussel Crenomytilus grayanus has been demonstrated to exhibit antibacterial properties. However, the mechanism of immune modulation by CGL in mammalian cells remains unclear. Here, we demonstrated that CGL can activate immune responses in macrophages and in mice. In the in vitro cell models, CGL induced tumour necrosis factor-α and interleukin-6 secretion in mouse RAW264.7 macrophages, mouse bone marrow-derived macrophages, human THP-1 macrophages, human peripheral blood mononuclear cells and human blood monocyte-derived macrophages. The CGL-mediated cytokine production was regulated by reactive oxygen species, mitogen-activated protein kinases, protein kinase C-α/δ and NF-κB. Interestingly, in lipopolysaccharide-activated macrophages, CGL induced endotoxin tolerance (characterized by the downregulation of nitric oxide, inducible nitric oxide synthase, interleukin-6 and cyclooxygenase II) via the downregulation of IRAK2 expression, JNK1/2 phosphorylation and NF-κB activation. CGL also slightly increased the bactericidal activity of macrophages and induced cytokine production in mouse models. Overall, our data indicate that CGL has the potential to be used as an immune modulator in mammals.

Galactomannan from Antrodia cinnamomea Enhances the Phagocytic Activity of Macrophages.

Galactomannan with an octasaccharide-repeating unit (ACP) was isolated from Taiwan medicinal mushroom Antrodia cinnamomea, and its chemical structure was determined herein. ACP significantly enhanced the phagocytosis and bactericidal activity of J774A.1 murine macrophages against Escherichia coli, with prospects for developing a new immunomodulatory compound or adjuvant in immunotherapy and vaccination.

Sinularin induces oxidative stress-mediated G2/M arrest and apoptosis in oral cancer cells.

Soft corals-derived natural product, sinularin, was antiproliferative against some cancers but its effect and detailed mechanism on oral cancer cells remain unclear. The subject of this study is to examine the antioral cancer effects and underlying detailed mechanisms in terms of cell viability, oxidative stress, cell cycle analysis, and apoptosis analyses. In MTS assay, sinularin dose-responsively decreased cell viability of three oral cancer cells (Ca9-22, HSC-3, and CAL 27) but only little damage to oral normal cells (HGF-1). This cell killing effect was rescued by the antioxidant N-acetylcysteine (NAC) pretreatment. Abnormal cell morphology and induction of reactive oxygen species (ROS) were found in sinularin-treated oral cancer Ca9-22 cells, however, NAC pretreatment also recovered these changes. Sinularin arrested the Ca9-22 cells at G2/M phase and dysregulated the G2/M regulatory proteins such as cdc2 and cyclin B1. Sinularin dose-responsively induced apoptosis on Ca9-22 cells in terms of flow cytometry (annexin V and pancaspase analyses) and western blotting (caspases 3, 8, 9) and poly (ADP-ribose) polymerase (PARP). These apoptotic changes of sinularin-treated Ca9-22 cells were rescued by NAC pretreatment. Taken together, sinularin induces oxidative stress-mediated antiproliferation, G2/M arrest, and apoptosis against oral cancer cells and may be a potential marine drug for antioral cancer therapy.

Identification of a new class of WNT1 inhibitor: Cancer cells migration, G-quadruplex stabilization and target validation.

Developing the Wnt pathway inhibitors has been considered as a therapeutic approach for cancers and other Wnt-related diseases. Previously we found that the G-rich sequence of WNT1 promoter is capable of forming G-quadruplex structure and stabilizing agents for Wnt1-mediated signaling pathway. Using a established cell-based drug screen system that enabled the evaluation of WNT1 expression activity in a G-quadruplex structure dependent manner, we evaluated a series of 6-substituted 9-chloro-11H-indeno[1,2-c]quinolin-11-one derivatives that potentially inhibit the Wnt1-mediated signaling pathway. The most potent compound SJ26 showed repression of WNT1 activity in a G-quadruplex structure-dependent manner. Moreover, compound SJ26 inhibited the WNT1-mediated downstream signaling pathway and suppressed migration activity of cancer cells. Thus, we have identified a tetracyclic azafluorenone, SJ26, that is capable of binding to G-quadruplex DNA structure, repressing WNT1 expression, and inhibiting cell migration.

Phosphoproteomic analysis of Methanohalophilus portucalensis FDF1(T) identified the role of protein phosphorylation in methanogenesis and osmoregulation.

Methanogens have gained much attention for their metabolic product, methane, which could be an energy substitute but also contributes to the greenhouse effect. One factor that controls methane emission, reversible protein phosphorylation, is a crucial signaling switch, and phosphoproteomics has become a powerful tool for large-scale surveying. Here, we conducted the first phosphorylation-mediated regulation study in halophilic Methanohalophilus portucalensis FDF1(T), a model strain for studying stress response mechanisms in osmoadaptation. A shotgun approach and MS-based analysis identified 149 unique phosphoproteins. Among them, 26% participated in methanogenesis and osmolytes biosynthesis pathways. Of note, we uncovered that protein phosphorylation might be a crucial factor to modulate the pyrrolysine (Pyl) incorporation and Pyl-mediated methylotrophic methanogenesis. Furthermore, heterologous expression of glycine sarcosine N-methyltransferase (GSMT) mutant derivatives in the osmosensitive Escherichia coli MKH13 revealed that the nonphosphorylated T68A mutant resulted in increased salt tolerance. In contrast, mimic phosphorylated mutant T68D proved defective in both enzymatic activity and salinity tolerance for growth. Our study provides new insights into phosphorylation modification as a crucial role of both methanogenesis and osmoadaptation in methanoarchaea, promoting biogas production or reducing future methane emission in response to global warming and climate change.

Down-regulated and Commonly mutated ALPK1 in Lung and Colorectal Cancers.

The ALPK1 gene located in the 4q25 region encodes a newly explored protein kinase which could phosphorylate the amino acid of a domain full of α-helices. Recently, several studies have indicated that the expression of ALPK1 is related to inflammation and various diseases; therefore, the purpose of this investigation was to determine whether the expression of ALPK1 has an influence on tumorigenesis and to further scrutinize its gene polymorphism in order to better understand its clinical importance. In lung and colorectal cancer tissues, the ALPK1 RNA level of the normal part was higher than that of the tumor part using the RT-qPCR analysis. Moreover, differences in HRM melting curves could effectively separate the known mutation sites and be used to identify the two novel variants that might cause the bio-dysfunctions of ALPK1 found in silico predictions. Additionally, in both Lovo colorectal and A549 lung cancer cells with enhanced and depleted expression of ALPK1, the encoded ALPK1 could exert its activity on cell migration without interfering with cell viability. Taken together, these findings suggested that ALPK1 might play a vital role in cancer development and that the newly explored SNPs are found in a Taiwanese cohort.

Structural Basis for the Magnesium-Dependent Activation and Hexamerization of the Lon AAA+ Protease.

The Lon AAA+ protease (LonA) plays important roles in protein homeostasis and regulation of diverse biological processes. LonA behaves as a homomeric hexamer in the presence of magnesium (Mg(2+)) and performs ATP-dependent proteolysis. However, it is also found that LonA can carry out Mg(2+)-dependent degradation of unfolded protein substrate in an ATP-independent manner. Here we show that in the presence of Mg(2+) LonA forms a non-secluded hexameric barrel with prominent openings, which explains why Mg(2+)-activated LonA can operate as a diffusion-based chambered protease to degrade unstructured protein and peptide substrates efficiently in the absence of ATP. A 1.85 Å crystal structure of Mg(2+)-activated protease domain reveals Mg(2+)-dependent remodeling of a substrate-binding loop and a potential metal-binding site near the Ser-Lys catalytic dyad, supported by biophysical binding assays and molecular dynamics simulations. Together, these findings reveal the specific roles of Mg(2+) in the molecular assembly and activation of LonA.