Synergistic Antitumor Effect of Grifola frondose Polysaccharide-Protein Complex in Combination with Cyclophosphamide in H22 Tumor-Bearing Mice.

Hepatocellular carcinoma (HCC) is the most common type of liver malignancy and remains a global health threat. The objective of the current study was to determine whether the combination of a cold-water extracted polysaccharide-protein complex from Grifolia frondosa (GFG) and cyclophosphamide (CTX) could inhibit tumor growth by suppressing the expression of angiogenesis-related proteins in H22 tumor-bearing mice. The results showed that the inhibition rate of GFG combined with CTX on H22 tumors was 65.29%, which was significantly higher than that of GFG treatment alone (24.82%). GFG combined with CTX significantly increased the expression levels of vascular endothelial growth factor, basic fibroblast growth factor, matrix metalloproteinase 2, and matrix metalloproteinase 9. Additionally, thymus index, spleen index, natural killer (NK) cell activity, interferon-γ (IFN-γ), interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α) and interleukin-2 (IL-2) levels increased significantly after GFG treatment, especially after high-doses of GFG combined with CTX treatment (p < 0.05). The thymus index, spleen index, NK cell activity, IFN-γ, IL-1ß, TNF-α, and IL-2 levels were 1.90, 1.46, 1.30, 2.13, 1.64, 2.03, and 1.24 times of those treated with CTX alone. Thus, we proposed that GFG can alleviate the side effects of CTX by relieving the immunosuppressive effect, liver/renal injury, and oxidative stress. In conclusion, the combination of GFG and CTX for cancer treatment may be a promising strategy, and GFG is expected to be a potential adjuvant alternative for the treatment of HCC.

A cold-water extracted polysaccharide-protein complex from Grifola frondosa exhibited anti-tumor activity via TLR4-NF-κB signaling activation and gut microbiota modification in H22 tumor-bearing mice.

Grifola frondosa polysaccharide-protein complex (G. frondosa PPC) is a polymer which consists of polysaccharides and proteins/peptides linked by covalent bonds. In our previous ex vivo research, it has been demonstrated that a cold-water extracted G. frondosa PPC has stronger antitumor activity than a G. frondosa PPC extracted from boiling water. The main purpose of the current study was to further evaluate the anti-hepatocellular carcinoma and gut microbiota regulation effects of two PPCs isolated from G. frondosa at 4 °C (GFG-4) and 100 °C (GFG-100) in vivo. The results exhibited that GFG-4 remarkably upregulated the expression of related proteins in TLR4-NF-κB and apoptosis pathway, thereby inhibiting the development of H22 tumors. Additionally, GFG-4 increased the abundance of norank_f__Muribaculaceae and Bacillus and reduced the abundance of Lactobacillus. Short chain fatty acids (SCFAs) analysis suggested that GFG-4 promoted SCFAs production, particularly butyric acid. Conclusively, the present experiments revealed GFG-4 has the potential of anti-hepatocellular carcinoma growth via activating TLR4-NF-κB pathway and regulating gut microbiota. Therefore, G. frondosa PPCs could be considered as safe and effective natural ingredient for treatment of hepatocellular carcinoma. The present study also provides a theoretical foundation for the regulation of gut microbiota by G. frondosa PPCs.

The Interaction between Mushroom Polysaccharides and Gut Microbiota and Their Effect on Human Health: A Review.

Mushroom polysaccharides are a kind of biological macromolecule extracted from the fruiting body, mycelium or fermentation liquid of edible fungi. In recent years, the research on mushroom polysaccharides for alleviating metabolic diseases, inflammatory bowel diseases, cancers and other symptoms by changing the intestinal microenvironment has been increasing. Mushroom polysaccharides could promote human health by regulating gut microbiota, increasing the production of short-chain fatty acids, improving intestinal mucosal barrier, regulating lipid metabolism and activating specific signaling pathways. Notably, these biological activities are closely related to the molecular weight, monosaccharide composition and type of the glycosidic bond of mushroom polysaccharide. This review aims to summarize the latest studies: (1) Regulatory effects of mushroom polysaccharides on gut microbiota; (2) The effect of mushroom polysaccharide structure on gut microbiota; (3) Metabolism of mushroom polysaccharides by gut microbiota; and (4) Effects of mushroom polysaccharides on gut microbe-mediated diseases. It provides a theoretical basis for further exploring the mechanism of mushroom polysaccharides for regulating gut microbiota and gives a reference for developing and utilizing mushroom polysaccharides as promising prebiotics in the future.

Structure-Based Design of Active-Site-Directed, Highly Potent, Selective, and Orally Bioavailable Low-Molecular-Weight Protein Tyrosine Phosphatase Inhibitors.

Protein tyrosine phosphatases constitute an important class of drug targets whose potential has been limited by the paucity of drug-like small-molecule inhibitors. We recently described a class of active-site-directed, moderately selective, and potent inhibitors of the low-molecular-weight protein tyrosine phosphatase (LMW-PTP). Here, we report our extensive structure-based design and optimization effort that afforded inhibitors with vastly improved potency and specificity. The leading compound inhibits LMW-PTP potently and selectively (Ki = 1.2 nM, >8000-fold selectivity). Many compounds exhibit favorable drug-like properties, such as low molecular weight, weak cytochrome P450 inhibition, high metabolic stability, moderate to high cell permeability (Papp > 0.2 nm/s), and moderate to good oral bioavailability (% F from 23 to 50% in mice), and therefore can be used as in vivo chemical probes to further dissect the complex biological as well as pathophysiological roles of LMW-PTP and for the development of therapeutics targeting LMW-PTP.

A cold-water polysaccharide-protein complex from Grifola frondosa exhibited antiproliferative activity via mitochondrial apoptotic and Fas/FasL pathways in HepG2 cells.

Grifola frondosa (G. frondosa) is widely known for its anti-tumor potential, which has been demonstrated by numerous scientific researches. In this study, two water soluble polysaccharide-protein complexes were extracted from G. frondosa at 4 °C (GFG-4) and 100 °C (GFG-100) and purified. Compared with GFG-100, GFG-4 had a higher protein content and molecular weight. The main monosaccharides of GFG-4 and GFG-100 were rhamnose, glucose, and galactose, with an approximate ratio of 3.00: 1.00: 0.86 and 2.85: 1.00: 0.94, respectively. The Fourier transform infrared spectra indicated that the two polysaccharide-protein complexes displayed characteristic functional groups of polysaccharides and proteins, and mainly contain pyranose ring with α-glycosidic linkage. Atomic force microscope images showed that both GFG-4 and GFG-100 exhibited straight chains, and GFG-4 possessed a relatively abundant fraction of branched chains. Intriguingly, GFG-4 showed a stronger antiproliferative activity against HepG2 cells than GFG-100. The mechanisms were further investigated by quantitative real-time PCR and western blot, it found that GFG-4 inhibited the proliferation of HepG2 cells mainly through the intrinsic activation of mitochondrial pathway and the Fas/FasL-mediated Caspase-8/-3 pathway. Conclusively, G. frondosa cold-water extracted polysaccharide-protein complexes could be used as a functional food for preventing or treating hepatocellular carcinoma.

The Toxoplasma glucan phosphatase TgLaforin utilizes a distinct functional mechanism that can be exploited by therapeutic inhibitors.

Toxoplasma gondii is an intracellular parasite that generates amylopectin granules (AGs), a polysaccharide associated with bradyzoites that define chronic T. gondii infection. AGs are postulated to act as an essential energy storage molecule that enable bradyzoite persistence, transmission, and reactivation. Importantly, reactivation can result in the life-threatening symptoms of toxoplasmosis. T. gondii encodes glucan dikinase and glucan phosphatase enzymes that are homologous to the plant and animal enzymes involved in reversible glucan phosphorylation and which are required for efficient polysaccharide degradation and utilization. However, the structural determinants that regulate reversible glucan phosphorylation in T. gondii are unclear. Herein, we define key functional aspects of the T. gondii glucan phosphatase TgLaforin (TGME49_205290). We demonstrate that TgLaforin possesses an atypical split carbohydrate-binding-module domain. AlphaFold2 modeling combined with hydrogen-deuterium exchange mass spectrometry and differential scanning fluorimetry also demonstrate the unique structural dynamics of TgLaforin with regard to glucan binding. Moreover, we show that TgLaforin forms a dual specificity phosphatase domain-mediated dimer. Finally, the distinct properties of the glucan phosphatase catalytic domain were exploited to identify a small molecule inhibitor of TgLaforin catalytic activity. Together, these studies define a distinct mechanism of TgLaforin activity, opening up a new avenue of T. gondii bradyzoite biology as a therapeutic target.

A Facile Procedure for One-Pot Stable Conjugation of Two Proglucagon Cysteine-Containing Peptide Analogs.

Optimization of peptides for therapeutic purposes often includes chemical conjugation or modification with substituents that serve to broaden pharmacology or improve pharmacokinetics. We report a convenient and rapid procedure for one-pot, site-specific conjugation of two cysteine-containing peptides that utilizes a bivalent linker comprising maleimide and iodoacetyl functional groups. Following maleimide-mediated peptide conjugation the linker was converted from an unstable thiosuccinimide to a stable thioether bond suitable for biological study by mild aqueous hydrolysis. The procedure is exemplified by peptide-peptide, peptide-small molecule, and peptide-fatty acid conjugations. The method provides a facile approach to search for enhanced biological outcomes through additive and sustained peptide pharmacology unencumbered by the prospect of chemical rearrangement in the course of biological study.

CB1 and GLP-1 Receptors Cross Talk Provides New Therapies for Obesity.

Glucagon-like peptide 1 receptor (GLP-1R) agonists effectively improve glycemia and body weight in patients with type 2 diabetes and obesity but have limited weight-lowering efficacy and minimal insulin sensitizing action. In preclinical models, peripherally restricted cannabinoid receptor type 1 (CB1R) inhibitors, which are devoid of the neuropsychiatric adverse effects observed with brain-penetrant CB1R blockers, ameliorate obesity and its multiple metabolic complications. Using mouse models with genetic loss of CB1R or GLP-1R, we demonstrate that these two metabolic receptors modulate food intake and body weight via reciprocal functional interactions. In diet-induced obese mice, the coadministration of a peripheral CB1R inhibitor with long-acting GLP-1R agonists achieves greater reduction in body weight and fat mass than monotherapies by promoting negative energy balance. This cotreatment also results in larger improvements in systemic and hepatic insulin action, systemic dyslipidemia, and reduction of hepatic steatosis. Thus, peripheral CB1R blockade may allow safely potentiating the antiobesity and antidiabetic effects of currently available GLP-1R agonists.

Dynamic replacement of H3.3 affects nuclear reprogramming in early bovine SCNT embryos.

The histone variant H3.3 is an important maternal factor in fertilization of oocytes and reprogramming of somatic cell nuclear transfer (SCNT) embryos. As a crucial replacement histone, maternal H3.3 is involved in chromatin remodeling and zygote genome activation. Litte is, however, known about the replacement of H3.3 in the bovine SCNT embryos. In this study, the maternal H3.3 in mature ooplasm was labeled with HA tag and the donor cells H3.3 was labeled with Flag tag, in order to observe the replacement of H3.3 in the bovine SCNT embryos. Meanwhile, maternal H3.3 knockdown was performed by microinjecting two different interfering fragments before nucleus transfer. It was showed that the dynamic replacement between maternal- and donor nucleus-derived H3.3 was detected after SCNT. And it could be observed that the blastocyst development rate of the cloned embryos decreased from 22.3% to 8.2-10.3% (P < 0.05), the expression of Pou5f1 and Sox2 was down-regulated and the level of H3K9me3 was increased in the interfered embryos. In summary, H3.3 replacement impacted on the process of reprogramming, including embryonic development potential, activation of pluripotency genes and epigenetic modification in bovine SCNT embryos.

Molecular mechanisms of bioactive polysaccharides from Ganoderma lucidum (Lingzhi), a review.

Ganoderma lucidum, commonly known as "Lingzhi" in Chinese, are well-known medicinal mushrooms. Lingzhi has been used in traditional Chinese herbal medicines for more than two thousand years. G. lucidum polysaccharides (GLPs) are present at high levels in G. lucidum cells and GLPs have molecular weights ranging from thousands to millions. GLPs have been widely studied for their various biological activities, such as antioxidant, antitumor, anti-inflammatory, antiviral, anti-diabetes, and immunomodulatory activities. The methods for GLPs extraction and characterization are mature, but the comprehensive research on the relationship between GLPs structure (i.e., molecular weight, tertiary structure, branching, substituents, and monosaccharide composition) and function is still quite limited. The aim of this review is to update and summarize the mechanisms of the various bioactive polysaccharides extracted from G. lucidum. The information presented on these bio-mechanisms should be valuable in the research and development of GLPs-derived therapeutics.

Stepwise Construction of Disulfides in Peptides.

The disulfide bond plays an important role in biological systems. It defines global conformation, and ultimately the biological activity and stability of the peptide or protein. It is frequently present, singly or multiply, in biologically important peptide hormones and toxins. Numerous disulfide-containing peptides have been approved by the regulatory agencies as marketed drugs. Chemical synthesis is one of the prerequisite tools needed to gain deep insights into the structure-function relationships of these biomolecules. Along with the development of solid-phase peptide synthesis, a number of methods of disulfide construction have been established. This minireview will focus on the regiospecific, stepwise construction of multiple disulfides used in the chemical synthesis of peptides. We intend for this article to serve a reference for peptide chemists conducting complex peptide syntheses and also hope to stimulate the future development of disulfide methodologies.

Peptide Conjugates with Small Molecules Designed to Enhance Efficacy and Safety.

Peptides constitute molecular diversity with unique molecular mechanisms of action that are proven indispensable in the management of many human diseases, but of only a mere fraction relative to more traditional small molecule-based medicines. The integration of these two therapeutic modalities offers the potential to enhance and broaden pharmacology while minimizing dose-dependent toxicology. This review summarizes numerous advances in drug design, synthesis and development that provide direction for next-generation research endeavors in this field. Medicinal studies in this area have largely focused upon the application of peptides to selectively enhance small molecule cytotoxicity to more effectively treat multiple oncologic diseases. To a lesser and steadily emerging extent peptides are being therapeutically employed to complement and diversify the pharmacology of small molecule drugs in diseases other than just cancer. No matter the disease, the purpose of the molecular integration remains constant and it is to achieve superior therapeutic outcomes with diminished adverse effects. We review linker technology and conjugation chemistries that have enabled integrated and targeted pharmacology with controlled release. Finally, we offer our perspective on opportunities and obstacles in the field.

Clearance kinetics of the VGF-derived neuropeptide TLQP-21.

TLQP-21 is a multifunctional neuropeptide and a promising new medicinal target for cardiometabolic and neurological diseases. However, to date its clearance kinetics and plasma stability have not been studied. The presence of four arginine residues led us to hypothesize that its half-life is relatively short. Conversely, its biological activities led us to hypothesize that the peptide is still taken up by adipose tissues effectively. [125I]TLQP-21 was i.v. administered in rats followed by chasing the plasma radioactivity and assessing tissue uptake. Plasma stability was measured using LC-MS. In vivo lipolysis was assessed by the palmitate rate of appearance. RESULTS: A small single i.v. dose of [125I]TLQP-21 had a terminal half-life of 110 min with a terminal clearance rate constant, kt, of 0.0063/min, and an initial half-life of 0.97 min with an initial clearance rate constant, ki, of 0.71/min. The total net uptake by adipose tissue accounts for 4.4% of the entire dose equivalent while the liver, pancreas and adrenal gland showed higher uptake. Uptake by the brain was negligible, suggesting that i.v.-injected peptide does not cross the blood-brain-barrier. TLQP-21 sustained isoproterenol-stimulated lipolysis in vivo. Finally, TLQP-21 was rapidly degraded producing several N-terminal and central sequence fragments after 10 and 60 min in plasma in vitro. This study investigated the clearance and stability of TLQP-21 peptide for the first time. While its pro-lipolytic effect supports and extends previous findings, its short half-life and sequential cleavage in the plasma suggest strategies for chemical modifications in order to enhance its stability and therapeutic efficacy.

Genome-wide analysis of ATP-binding cassette (ABC) transporters in the sweetpotato whitefly, Bemisia tabaci.

BACKGROUND: ABC transporter superfamily is one of the largest and ubiquitous groups of proteins. Because of their role in detoxification, insect ABC transporters have gained more attention in recent years. In this study, we annotated ABC transporters from a newly sequenced sweetpotato whitefly genome. Bemisia tabaci Q biotype is an emerging global invasive species that has caused extensive damages to field crops as well as ornamental plants. RESULTS: A total of 55 ABC transporters containing all eight described subfamilies (A to H) were identified in the B. tabaci Q genome, including 8 ABCAs, 3 ABCBs, 6 ABCCs, 2 ABCDs, 1 ABCE, 3 ABCFs, 23 ABCGs and 9 ABCHs. In comparison to other species, subfamilies G and H in both phloem- and blood-sucking arthropods are expanded. The temporal expression profiles of these 55 ABC transporters throughout B. tabaci developmental stages and their responses to imidacloprid, a neonicotinoid insecticide, were investigated using RNA-seq analysis. Furthermore, the mRNA expression of 24 ABC transporters (44% of the total) representing all eight subfamilies was confirmed by the quantitative real-time PCR (RT-qPCR). Furthermore, mRNA expression levels estimated by RT-qPCR and RNA-seq analyses were significantly correlated (r = 0.684, p < 0.01). CONCLUSIONS: It is the first genome-wide analysis of the entire repertoire of ABC transporters in B. tabaci. The identification of these ABC transporters, their temporal expression profiles during B. tabaci development, and their response to a neonicotinoid insecticide lay the foundation for functional genomic understanding of their contribution to the invasiveness of B. tabaci.

Inhibition of Low Molecular Weight Protein Tyrosine Phosphatase by an Induced-Fit Mechanism.

The low molecular weight protein tyrosine phosphatase (LMW-PTP) is a regulator of a number of signaling pathways and has been implicated as a potential target for oncology and diabetes/obesity. There is significant therapeutic interest in developing potent and selective inhibitors to control LMW-PTP activity. We report the discovery of a novel class of LMW-PTP inhibitors derived from sulfophenyl acetic amide (SPAA), some of which exhibit greater than 50-fold preference for LMW-PTP over a large panel of PTPs. X-ray crystallography reveals that binding of SPAA-based inhibitors induces a striking conformational change in the LMW-PTP active site, leading to the formation of a previously undisclosed hydrophobic pocket to accommodate the α-phenyl ring in the ligand. This induced-fit mechanism is likely a major contributor responsible for the exquisite inhibitor selectivity.

Mycobacterial Protein Tyrosine Phosphatases A and B Inhibitors Augment the Bactericidal Activity of the Standard Anti-tuberculosis Regimen.

Novel drugs are required to shorten the duration of treatment for tuberculosis (TB) and to combat the emergence of drug resistance. One approach has been to identify and target Mycobacterium tuberculosis (Mtb) virulence factors, which promote the establishment of TB infection and pathogenesis. Mtb produces a number of virulence factors, including two protein tyrosine phosphatases (PTPs), mPTPA and mPTPB, to evade the antimicrobial functions of host macrophages. To assess the therapeutic potential of targeting the virulent Mtb PTPs, we developed highly potent and selective inhibitors of mPTPA (L335-M34) and mPTPB (L01-Z08) with drug-like properties. We tested the bactericidal activity of L335-M34 and L01-Z08 alone or together in combination with the standard antitubercular regimen of isoniazid-rifampicin-pyrazinamide (HRZ) in the guinea pig model of chronic TB infection, which faithfully recapitulates some of the key histological features of human TB lesions. Following a single dose of L335-M34 50mg/kg and L01-Z08 20 mg/kg, plasma levels were maintained at levels 10-fold greater than the biochemical IC50 for 12-24 hours. Although neither PTP inhibitor alone significantly enhanced the antibacterial activity of HRZ, dual inhibition of mPTPA and mPTPB in combination with HRZ showed modest synergy, even after 2 weeks of treatment. After 6 weeks of treatment, the degree of lung inflammation correlated with the bactericidal activity of each drug regimen. This study highlights the potential utility of targeting Mtb virulence factors, and specifically the Mtb PTPs, as a strategy for enhancing the activity of standard anti-TB treatment.

Tissue-specific Proteogenomic Analysis of Plutella xylostella Larval Midgut Using a Multialgorithm Pipeline.

The diamondback moth, Plutella xylostella (L.), is the major cosmopolitan pest of brassica and other cruciferous crops. Its larval midgut is a dynamic tissue that interfaces with a wide variety of toxicological and physiological processes. The draft sequence of the P. xylostella genome was recently released, but its annotation remains challenging because of the low sequence coverage of this branch of life and the poor description of exon/intron splicing rules for these insects. Peptide sequencing by computational assignment of tandem mass spectra to genome sequence information provides an experimental independent approach for confirming or refuting protein predictions, a concept that has been termed proteogenomics. In this study, we carried out an in-depth proteogenomic analysis to complement genome annotation of P. xylostella larval midgut based on shotgun HPLC-ESI-MS/MS data by means of a multialgorithm pipeline. A total of 876,341 tandem mass spectra were searched against the predicted P. xylostella protein sequences and a whole-genome six-frame translation database. Based on a data set comprising 2694 novel genome search specific peptides, we discovered 439 novel protein-coding genes and corrected 128 existing gene models. To get the most accurate data to seed further insect genome annotation, more than half of the novel protein-coding genes, i.e. 235 over 439, were further validated after RT-PCR amplification and sequencing of the corresponding transcripts. Furthermore, we validated 53 novel alternative splicings. Finally, a total of 6764 proteins were identified, resulting in one of the most comprehensive proteogenomic study of a nonmodel animal. As the first tissue-specific proteogenomics analysis of P. xylostella, this study provides the fundamental basis for high-throughput proteomics and functional genomics approaches aimed at deciphering the molecular mechanisms of resistance and controlling this pest.

Cadmium exposure to murine macrophages decreases their inflammatory responses and increases their oxidative stress.

Cadmium (Cd) is an environmental contaminant that poses serious risks to human and wildlife health. The oxidative stress and inflammatory responses induced by Cd were evaluated in RAW264.7 cells. A significant decrease in the cell viability was observed in the group treated with 3 µM Cd for 24 h. The mRNA levels of tumor necrosis factor-α (TNFα), interleukin-6 (IL6), interleukin-1α (IL1α) and Interleukin-1ß (IL1ß) were generally increased or decreased by Cd exposure for 6 and 24 h, respectively. Moreover, pretreatment of the RAW264.7 cells with Cd for 24 h inhibited the transcriptional status of TNFα, IL6, IL1α and IL1ß and the release of these cytokines in response to a 6-h lipopolysaccharide (LPS) treatment in a dose-dependent manner. Furthermore, the Cd exposure elicited oxidative stress not only by disturbing the transcriptional status of genes including superoxide dismutase (Sod), catalase (Cat), glutathione peroxidase(Gpx), glutathione S-transferase 1 a (Gst1a), NAD(P)H: quinone oxidoreductase 1(Nqo1), heme oxygenase 1(Ho-1) but also the enzyme activities of SOD, CAT and glutathione S-transferase (GST). The effects of Cd on the mRNA levels and activities of anti-oxidative enzymes were dependent on the exposure period and dose. These results suggested that Cd exposure generated oxidative stress and decreased the inflammatory responses in a murine macrophage cell line. Furthermore, oxidative stress may be a possible mechanism to explain the dysregulation of the immune function caused by heavy metals in this in vitro system.

Cefsulodin Inspired Potent and Selective Inhibitors of mPTPB, a Virulent Phosphatase from Mycobacterium tuberculosis.

mPTPB is a virulent phosphatase from Mycobacterium tuberculosis and a promising therapeutic target for tuberculosis. To facilitate mPTPB-based drug discovery, we identified α-sulfophenylacetic amide (SPAA) from cefsulodin, a third generation ß-lactam cephalosporin antibiotic, as a novel pTyr pharmacophore for mPTPB. Structure-guided and fragment-based optimization of SPAA led to the most potent and selective mPTPB inhibitor 9, with a K i of 7.9 nM and more than 10,000-fold preference for mPTPB over a large panel of 25 phosphatases. Compound 9 also exhibited excellent cellular activity and specificity in blocking mPTPB function in macrophage. Given its novel structure, modest molecular mass, and extremely high ligand efficiency (0.46), compound 9 represents an outstanding lead compound for anti-TB drug discovery targeting mPTPB.