Sanghuangporus sanghuang extract inhibits the proliferation and invasion of lung cancer cells in vitro and in vivo.

BACKGROUND/OBJECTIVES: Sanghuangporus sanghuang (SS) has various medicinal effects, including anti-inflammation and anticancer activities. Despite the extensive research on SS, its molecular mechanisms of action on lung cancer are unclear. This study examined the impact of an SS alcohol extract (SAE) on lung cancer using in vitro and in vivo models. MATERIALS/METHODS: Different concentrations of SAE were used to culture lung cancer cells (A549 and H1650). A cell counting kit-8 assay was used to detect the survival ability of A549 and H1650 cells. A scratch assay and transwell cell invasion assay were used to detect the migration rate and invasive ability of SAE. Western blot analysis was used to detect the expression of B-cell lymphoma-2 (Bcl-2), Bcl2-associated X (Bax), cyclin D1, cyclin-dependent kinases 4 (CDK4), signal transducer and activator of transcription 3 (STAT3), and phosphorylated STAT3 (p-STAT3). Lung cancer xenograft mice were used to detect the inhibiting ability of SAE in vivo. Hematoxylin and eosin staining and immunohistochemistry were used to detect the effect of SAE on the structural changes to the tumor and the expression of Bcl-2, Bax, cyclin D1, CDK4, STAT3, and p-STAT3 in lung cancer xenograft mice. RESULTS: SAE could inhibit lung cancer proliferation significantly in vitro and in vivo without cytotoxicity. SAE suppressed the viability, migration, and invasion of lung cancer cells in a dose and time-dependent manner. The SAE treatment significantly decreased the proapoptotic Bcl-2/Bax ratio and the expression of pro-proliferative proteins Cyclin D1 and CDK4 in vitro and in vivo. Furthermore, SAE also inhibited STAT3 expression. CONCLUSIONS: SAE reduced the cell viability and suppressed cell migration and invasion in human lung cancer cells. Moreover, SAE also exhibited anti-proliferation effects in vivo. Therefore, SAE may have benefits in cancer therapy.

Identification of a pathogen causing fruiting body rot of Sanghuangporus vaninii.

Sanghuangporus vaninii is a medicinal macrofungus that is increasingly cultivated in China. During cultivation, it was found that the fruiting body of S. vaninii was susceptible to pathogenic fungi, resulting in significant economic losses to the industry. The symptoms of the disease occur in the initial stage of fruiting body development. The isolate YZB-1 was obtained from the junction of the diseased and healthy areas of the fruiting body. In order to verify the pathogenicity of YZB-1, its purified spore suspension was inoculated into the exposed area nearby the developing fruiting body of S. vaninii. After 10 days, the same disease symptoms appeared in the inoculated area. Morphological identification and molecular analysis of rDNA ITS region confirmed that the isolate YZB-1 was identified as Trichoderma virens. The temperature stability assay revealed that the mycelia of YZB-1 grew the fastest at 25 °C, with growth slowing down gradually as the temperature increased or decreased. Dual-culture tests of T. virens and S. vaninii showed that the inhibition rate of T. virens on S. vaninii mycelium was the highest (79.01 ± 2.79%) at 25 °C, and more green spores were produced at the intersection of T. virens and S. vaninii.

Effects of Cultivation Methods on the Nutritional Content, Active Component Content, and Antioxidant Activity of Fruiting Bodies of Sanghuangporus baumii (Agaricomycetes).

To provide a scientific reference for improving the sawdust cultivation of Sanghuangporus baumii, comparative studies were conducted on the contents of nutritional components and active components and the antioxidant activity of the fruiting bodies of S. baumii cultivated with sawdust and cut logs. The results indicate that, first, cultivation methods had little effect on the contents of crude fat and the measured 16 kinds of amino acids [including total essential amino acids (EAA), total nonessential amino acids (NEAA), EAA/NEAA, and EAA/total amino acid (TAA)], but had a great influence on the contents of crude protein, crude fiber and TAA. These results suggest that the nutritional content under sawdust cultivation was significantly higher than that under cut-log cultivation. Second, the cultivation methods had little effect on the content of triterpenoids but had a great effect on the contents of polysaccharides, total flavonoids and total phenols, which showed that cut-log cultivation was significantly higher than sawdust cultivation. Third, the cultivation methods had a great effect on the antioxidant activities (ABTS and FRAP), which showed that cut-log cultivation was significantly higher than sawdust cultivation. The contents of polysaccharides, total flavonoids, and total phenols and the ABTS and FRAP activities using sawdust cultivation were lower than those using cut-log cultivation, which may be related to the mushroom strains, cultivation medium formula and cultivation technology. The results provide a solid basis for the improvement and promotion of new cultivation technologies for S. baumii.

Electrospun Sandwich-like Structure of PVDF-HFP/Cellulose/PVDF-HFP Membrane for Lithium-Ion Batteries.

Cellulose membranes have eco-friendly, renewable, and cost-effective features, but they lack satisfactory cycle stability as a sustainable separator for batteries. In this study, a two-step method was employed to prepare a sandwich-like composite membrane of poly(vinylidene fluoride-co-hexafluoropropylene) (PVDF-HFP)/cellulose/ PVDF-HFP (PCP). The method involved first dissolving and regenerating a cellulose membrane and then electrospinning PVDF-HFP on its surface. The resulting PCP composite membrane exhibits excellent properties such as high porosity (60.71%), good tensile strength (4.8 MPa), and thermal stability up to 160 °C. It also has exceptional electrolyte uptake properties (710.81 wt.%), low interfacial resistance (241.39 Ω), and high ionic conductivity (0.73 mS/cm) compared to commercial polypropylene (PP) separators (1121.4 Ω and 0.26 mS/cm). Additionally, the rate capability (163.2 mAh/g) and cycling performance (98.11% after 100 cycles at 0.5 C) of the PCP composite membrane are superior to those of PP separators. These results demonstrate that the PCP composite membrane has potential as a promising separator for high-powered, secure lithium-ion batteries.

Studies on the interaction mechanism between xanthine oxidase and osmundacetone: Molecular docking, multi-spectroscopy and dynamical simulation.

Xanthine oxidase (XO) is a key enzyme in uric acid production, and its molybdopterin (Mo-Pt) domain is an important catalytic center when xanthine and hypoxanthine are oxidated. It is found that the extract of Inonotus obliquus has an inhibitory effect on XO. In this study, five key chemical compounds were initially identified using liquid chromatography-mass spectrometry (LC-MS), and two compounds, osmundacetone ((3E)-4-(3,4-dihydroxyphenyl)-3-buten-2-one) and protocatechuic aldehyde (3,4-dihydroxybenzaldehyde), were screened as the XO inhibitors by ultrafiltration technology. Osmundacetone bound XO strongly and competitively inhibited XO with a half-maximal inhibitory concentration of 129.08 ± 1.71 µM, and its inhibition mechanism, was investigated. Osmundacetone and XO via static quenching and spontaneously bound with XO with high affinity, primarily via hydrophobic interactions and hydrogen bonds. Molecular docking studies showed that osmundacetone was inserted into the Mo-Pt center and interacted with hydrophobic residues of Phe911, Gly913, Phe914, Ser1008, Phe1009, Thr1010, Val1011, and Ala1079 of XO. In summary, these findings suggest that provide theoretical basis for the research and development of XO inhibitors from Inonotus obliquus.

Anti-gout activity and the interaction mechanisms between Sanghuangporus vaninii active components and xanthine oxidase.

Xanthine oxidase (XO) plays a critical role in the progression of gout. We showed in a previous study that Sanghuangporus vaninii (S. vaninii), a perennial, medicinal, and edible fungus traditionally used to treat various symptoms, contains XO inhibitors. In the current study, we isolated an active component of S. vaninii using high performance countercurrent chromatography and identified it as davallialactone using mass spectrometry with 97.726 % purity. A microplate reader showed that davallialactone had mixed inhibition of XO activity with a half-inhibitory concentration value of 90.07 ± 2.12 µM. In addition, the collision between davallialactone and XO led to fluorescence quenching and conformational changes in XO, which were mainly driven by hydrophobicity and hydrogen bonding. Molecular simulations further showed that davallialactone was located at the center of the molybdopterin (Mo-Pt) of XO and interacted with amino acid residues Phe798, Arg912, Met1038, Ala1078, Ala1079, Gln1194, and Gly1260, suggesting that entering the enzyme-catalyzed reaction was unfavorable for the substrate. We also observed face-to-face π-π interactions between the aryl ring of davallialactone and Phe914. Cell biology experiments indicated that davallialactone reduced the expression of the inflammatory factors, tumor necrosis factor alpha and interleukin-1 beta (P < 0.05), can effectively alleviate cellular oxidative stress. This study showed that davallialactone significantly inhibits XO and has the potential to be developed into a novel medicine to prevent hyperuricemia and treat gout.

Conformation and anticancer activity of a novel mannogalactan from the fruiting bodies of Sanghuangporus sanghuang on HepG2 cells.

A novel water-soluble mannogalactan (SSPS1) with an average molecular weight of 2.04 × 104 Da was obtained from the fruiting bodies of the Sanghuangporus sanghuang. It revealed that SSPS1 was composed of d-galactose, d-mannose, l-fucose, 3-O-methylgalactose and d-glucose in a ratio of 6.2:3.9:3.1:2.1:1.0. The structural elucidation of SSPS1 consisted of 1, 6-linked α-D-Galp, 1, 6-linked α-D-Manp and 1, 6-linked 3-O-methyl-α-D-Galp backbone with branching at O-2 of 1, 6-α-D-mannosyl residues by α-L-Fucp and α-D-Glcp units. The conformational parameters suggested that a flexible chain conformation of SSPS1 in solution based on light scattering and atomic force microscopy imaging. Intriguingly, it presented potent anticancer activity on HepG2 cell with Rq and Ra values increased dramatically up to 73.93 nm and 53.92 nm compared with the control. The analysis of flow cytometry indicated SSPS1 could induce the apoptosis of HepG2 cells and arrest them via S phase. Western blot assay further uncovered that apoptosis process was triggered by SSPS1 via a mitochondria-mediated signaling pathway, which was evidenced by an increased ratio of Bax/Bcl-2, the release of cytochrome c and the strong activation of caspase-3 and 9. Taken together, these results suggested that SSPS1 might be applied in functional food as an anticancer agent.

Identifying Quantitative Trait Loci and Candidate Genes Conferring Resistance to Soybean Mosaic Virus SC7 by Quantitative Trait Loci-Sequencing in Soybean.

Soybean mosaic virus (SMV) is detrimental to soybean (Glycine max) breeding, seed quality, and yield worldwide. Improving the basic resistance of host plants is the most effective and economical method to reduce damage from SMV. Therefore, it is necessary to identify and clone novel SMV resistance genes. Here, we report the characterization of two soybean cultivars, DN50 and XQD, with different levels of resistance to SMV. Compared with XQD, DN50 exhibits enhanced resistance to the SMV strain SC7. By combining bulked-segregant analysis (BSA)-seq and fine-mapping, we identified a novel resistance locus, R SMV -11, spanning an approximately 207-kb region on chromosome 11 and containing 25 annotated genes in the reference Williams 82 genome. Of these genes, we identified eleven with non-synonymous single-nucleotide polymorphisms (SNPs) or insertion-deletion mutations (InDels) in their coding regions between two parents. One gene, GmMATE68 (Glyma.11G028900), harbored a frameshift mutation. GmMATE68 encodes a multidrug and toxic compound extrusion (MATE) transporter that is expressed in all soybean tissues and is induced by SC7. Given that MATE transporter families have been reported to be linked with plant disease resistance, we suggest that GmMATE68 is responsible for SC7 resistance in DN50. Our results reveal a novel SMV-resistance locus, improving understanding of the genetics of soybean disease resistance and providing a potential new tool for marker-assisted selection breeding in soybean.

Characterization, immunostimulatory and antitumor activities of a ß-galactoglucofurannan from cultivated Sanghuangporus vaninii under forest.

A biomacromolecule, named as ß-galactoglucofurannan (SVPS2), was isolated from the cultivated parts of Sanghuangporus vaninii under the forest. Its primary and advanced structure was analyzed by a series of techniques including GC-MS, methylation, NMR, MALS as well as AFM. The results indicated that SVPS2 was a kind of 1, 5-linked ß-Glucofurannan consisting of ß-glucose, ß-galactose and α-fucose with 23.4 KDa. It exhibited a single-stranded chain with an average height of 0.72 nm in saline solution. The immunostimulation test indicated SVPS2 could facilitate the initiation of the immune reaction and promote the secretion of cytokines in vitro. Moreover, SVPS2 could mediate the apoptosis of HT-29 cells by blocking them in S phase. Western blot assay revealed an upregulation of Bax, Cytochrome c and cleaved caspase-3 by SVPS2, accompanied by a downregulation of Bcl-2. These results collectively demonstrate that antitumor mechanism of SVPS2 may be associated with enhancing immune response and inducing apoptosis of tumor cells in vitro. Therefore, SVPS2 might be utilized as a promising therapeutic agent against colon cancer and functional food with immunomodulatory activity.

Geography-Driven Evolution of Potato Virus A Revealed by Genetic Diversity Analysis of the Complete Genome.

Potato virus A (PVA), a member of the genus Potyvirus, is an important potato pathogen that causes 30%-40% yield reduction to global potato production. Knowledge on the genetic structure and the evolutionary forces shaping the structure of this pathogen is limited but vital in developing effective management strategies. In this study, we investigated the population structure and molecular evolution of PVA by analyzing novel complete genomic sequences from Chinese isolates combined with available sequences from Europe, South America, Oceania, and North America. High nucleotide diversity was discovered among the populations studied. Pairwise F ST values between geographical populations of PVA ranged from 0.22 to 0.46, indicating a significant spatial structure for this pathogen. Although purifying selection was detected at the majority of polymorphic sites, significant positive selection was identified in the P1, NIa, and NIb proteins, pointing to adaptive evolution of PVA. Further phylogeny-trait association analysis showed that the clustering of PVA isolates was significantly correlated with geographic regions, suggesting that geography-driven adaptation may be an important determinant of PVA diversification.

Genome assembly of primitive cultivated potato Solanum stenotomum provides insights into potato evolution.

Genetic diversity is the raw material for germplasm enhancement. Landraces and wild species relatives of potato, which contain a rich gene pool of valuable agronomic traits, can provide insights into the genetic diversity behind the adaptability of the common potato. The diploid plant, Solanum stenotomum (Sst), is believed to have an ancestral relationship with modern potato cultivars and be a potential source of resistance against disease. Sequencing of the Sst genome generated an assembly of 852.85 Mb (N50 scaffold size, 3.7 Mb). Pseudomolecule construction anchored 788.75 Mb of the assembly onto 12 pseudochromosomes, with an anchor rate of 92.4%. Genome annotation yielded 41,914 high-confidence protein-coding gene models and comparative analyses with closely related Solanaceae species identified 358 Sst-specific gene families, 885 gene families with expansion along the Sst lineage, and 149 genes experiencing accelerated rates of protein sequence evolution in Sst, the functions of which were mainly associated with defense responses, particularly against bacterial and fungal infection. Insights into the Sst genome and the genomic variation of cultivated potato taxa are valuable in elaborating the impact of potato evolution in early landrace diploid and facilitate modern potato breeding.

Screening anti-gout compounds from Phellinus igniarius by ultrafiltration liquid chromatography mass spectrometry based on evaluation of an in vitro method combined with enzymatic reaction.

In the present study, the anti-inflammation effect of Phellinus igniarius extract was detected on an in vitro model of RAW 264.7 cells stimulated using sodium urate. In this cell model, the content changes of inflammatory cytokines, intercellular adhesion molecule-1, and interleukin-1 beta, in cell culture supernatants were detected using an enzyme-linked immunosorbent assay. The xanthine oxidase inhibitory activity of P. igniarius extracts was determined using a microplate reader. Furthermore, in order to identify the active compounds of P. igniarius, ultrafiltration liquid chromatography mass spectrometry was utilized to screen xanthine oxidase inhibitors from the extract. Our results showed that in the presence of P. igniarius extract, the expressions of interleukin-1 beta and intercellular adhesion molecule-1 decreased (p < 0.01 and p < 0.05, respectively) compared to that in the control group. The extract effective inhibited the xanthine oxidase activity. Finally, seven compounds were screened and identified as potential xanthine oxidase inhibitors from P. igniarius. Taken together, these results demonstrate a potential anti-inflammation bioactivity of P. igniarius in vitro, providing a basis for further in vivo research for the prevention and treatment of gout.

Structural characterization and hypoglycemic activity of an intracellular polysaccharide from Sanghuangporus sanghuang mycelia.

A neutral polysaccharide (SSIPS1) was isolated and purified from cultured mycelia of Sanghuangporus sanghuang by DEAE Sepharose Fast Flow and Sephacryl S-100 columns. Basic monosaccharide composition indicated that SSIPS1 was mainly composed of d-glucose. The results of methylation and 2D-NMR analysis suggested that the glycosidic linkages of SSIPS1 were elucidated to consisted of 1,4-linked α-d-glucopyranose (Glcp) residues with two branched points at O-6. The two branches were composed of 1,4-linked α-D-Glcp terminated with α-D-Glcp, 1,4-linked α-D-Glcp and 1,4-linked ß-Galp terminated by α-D-Glcp. Moreover, its chain conformation was revealed to present a flexible chain conformation in 0.1 NaNO3 with a hydrodynamic radius and radius of gyration of 3.26 and 6.45 nm by multi-angle laser light scattering, with a single chain of 0.559 nm observed by atomic force microscopy. Further, SSIPS1 exhibited a potential inhibitory activity against α-amylase and α-glucosidase, and it had hypoglycemic effects on in vitro insulin resistance of HepG2 cells as well.

Conformational properties and biological activities of α-D-mannan from Sanghuangporus sanghuang in liquid culture.

Sanghuang is a well-known edible and medicinal fungus consumed in Asia for centuries. In the present study, a novel exopolysaccharide named as mannan (SSEPS2) was isolated from the liquid culture broth of Sanghuangporus sanghuang by using ethanol precipitation, anion-exchange chromatography, and size-exclusion chromatography, which was exclusively composed of D-mannose with the molecular weight 9.43 × 104 g/mol. The structural analysis suggested that SSEPS2 consisted of 1,3-linked and 1,2-linked α-D-mannopyranose (Manp), with substitution at O-6 of 1,2-linked α-D-Manp by 1,6-linked α-D-Manp residues and terminal α-D-Manp residues. The exponent of α (0.58) indicated that SSEPS2 existed in a flexible chain conformation, with a coil-like structure in 0.1 M NaNO3 at 25 °C, which was further confirmed by the atomic force microscopy images and a three-dimensional molecular model. It exhibited potential antitumor activity against the growth of HepG2 (human hepatoma) and MCF-7 cell lines in vitro. The IC50 values for inhibiting HepG-2 cells and MCF-7 cells proliferation were 365 and 340 µg/mL, which showed its potential application in the development of anticancer therapeutics.

Granzyme B and perforin are important for regulatory T cell-mediated suppression of tumor clearance.

Granzyme B is important for the ability of NK cells and CD8(+) T cells to kill their targets. However, we showed here that granzyme B-deficient mice clear both allogeneic and syngeneic tumor cell lines more efficiently than do wild-type (WT) mice. To determine whether regulatory T (Treg) cells utilize granzyme B to suppress immune responses against these tumors, we examined the expression and function of granzyme B in Treg cells. Granzyme B was not expressed in naive Treg cells but was highly expressed in 5%-30% of CD4(+)Foxp3(+) Treg cells in the tumor environment. Adoptive transfer of WT Treg cells, but not granzyme B- or perforin-deficient Treg cells, into granzyme B-deficient mice partially restored susceptibility to tumor growth; Treg cells derived from the tumor environment could induce NK and CD8(+) T cell death in a granzyme B- and perforin-dependent fashion. Granzyme B and perforin are therefore relevant for Treg cell-mediated suppression of tumor clearance in vivo.

Bioluminescence imaging reveals systemic dissemination of herpes simplex virus type 1 in the absence of interferon receptors.

Herpes simplex virus type 1 (HSV-1) can produce disseminated, systemic infection in neonates and patients with AIDS or other immunocompromising diseases, resulting in significant morbidity and mortality in spite of antiviral therapy. Components of host immunity that normally limit HSV-1 to localized epithelial and neuronal infection remain incompletely defined. We used in vivo bioluminescence imaging to determine effects of type I and II interferons (IFNs) on replication and tropism of HSV-1 infection in mice with genetic deficiency of type I, type II, or both type I and II IFN receptors. Following footpad or ocular infection of mice lacking type I IFN receptors, HSV-1 spread to parenchymal organs, including lung, liver, spleen, and regional lymph nodes, but mice survived. Deletion of type I and II IFN receptors produced quantitatively greatest and most widespread dissemination of virus to visceral organs and the nervous system, and these mice invariably died after ocular or footpad infection. Type II receptor knockout and wild-type mice had comparable viral replication and localization, with no systemic spread of HSV-1 or lethality. Therefore, while isolated deficiency of type II IFN receptors did not affect pathogenesis, loss of these receptors in combination with genetic deletion of type I receptors had a profound effect on susceptibility to HSV-1. These data demonstrate different effects of type I and II IFNs in limiting systemic dissemination of HSV-1 and further validate the use of bioluminescence imaging for studies of viral pathogenesis.

Imaging 26S proteasome activity and inhibition in living mice.

The ubiquitin-proteasome pathway is the central mediator of regulated proteolysis in cells, and defects in this pathway are associated with cancer and neurodegenerative diseases. To assess 26S proteasome function in living animals, we developed a ubiquitin-luciferase reporter for bioluminescence imaging. The reporter was degraded rapidly under steady-state conditions and stabilized in a dose- and time-dependent manner in response to proteasome inhibitors. Using bioluminescence imaging after one dose of the chemo-therapeutic proteasome inhibitor bortezomib (PS-341), proteasome function in tumor xenografts was blocked within 30 min and returned to nearly baseline by 46 h. After a 2-week regimen of bortezomib, however, imaging of target tumors showed significantly enhanced proteasome inhibition that no longer returned to baseline. The ubiquitin-luciferase reporter enables repetitive tissue-specific analysis of 26S proteasome activity in vivo and should facilitate development and validation of proteasome inhibitors in mouse models, as well as investigations of the ubiquitin-proteasome pathway in disease pathogenesis.