An individualized stemness-related signature to predict prognosis and immunotherapy responses for gastric cancer using single-cell and bulk tissue transcriptomes.

BACKGROUND: Currently, many stemness-related signatures have been developed for gastric cancer (GC) to predict prognosis and immunotherapy outcomes. However, due to batch effects, these signatures cannot accurately analyze patients one by one, rendering them impractical in real clinical scenarios. Therefore, we aimed to develop an individualized and clinically applicable signature based on GC stemness. METHODS: Malignant epithelial cells from single-cell RNA-Seq data of GC were used to identify stemness-related signature genes based on the CytoTRACE score. Using two bulk tissue datasets as training data, the enrichment scores of the signature genes were applied to classify samples into two subtypes. Then, using the identified subtypes as criteria, we developed an individualized stemness-related signature based on the within-sample relative expression orderings of genes. RESULTS: We identified 175 stemness-related signature genes, which exhibited significantly higher AUCell scores in poorly differentiated GCs compared to differentiated GCs. In training datasets, GC samples were classified into two subtypes with significantly different survival times and genomic characteristics. Utilizing the two subtypes, an individualized signature was constructed containing 47 gene pairs. In four independent testing datasets, GC samples classified as high risk exhibited significantly shorter survival times, higher infiltration of M2 macrophages, and lower immune responses compared to low-risk samples. Moreover, the potential therapeutic targets and corresponding drugs were identified for the high-risk group, such as CD248 targeted by ontuxizumab. CONCLUSIONS: We developed an individualized stemness-related signature, which can accurately predict the prognosis and efficacy of immunotherapy for each GC sample.

Analysis of Bacterial Diversity and Communities Associated with Tricholoma matsutake Fruiting Bodies by Barcoded Pyrosequencing in Sichuan Province, Southwest China.

Endophytes play an important role in the growth and development of the host. However, the study of endophytes is mostly focused on plants, and reports on bacteria associated with fungi are relatively rare. We studied the bacteria associated with fruiting bodies of Tricholoma matsutake picked from seven main T. matsutake-producing areas in Sichuan, China, by barcoded pyrosequencing. About 8,272 reads were obtained per sample, representing 40 phyla, 103 classes, and 495 genera of bacteria and archaea, and 361-797 operational taxonomic units were observed at a 97% similarity level. The bacterial community was always both more abundant and more diverse than the archaeal community. UniFrac analysis showed there were some difference of bacterial communities among the samples sites. Three bacterial phyla, Proteobacteria, Bacteroidetes, and Firmicutes, were dominant in all samples. Correlation analysis showed there was a significant correlation between some soil properties and bacterial community associated with T. matsutake. This study demonstrated that the bacteria associated with T. matsutake fruiting bodies were diversified. Among these bacteria, we may find some strains that can promote the growth of T. matsutake.

Chemical compositions and volatile compounds of Tricholoma matsutake from different geographical areas at different stages of maturity.

Tricholoma matsutake is a commercially important edible fungus. Volatile compounds, chemical compositions, and nutritional values of fruiting bodies at different stages of maturity from different geographical areas were analyzed. The main volatile compounds in T. matsutake fruiting bodies were (E)-2-octenal, phenylacetaldehyde, 3-octanone, methyl cinnamate, benzaldehyde, and 1- octen-3-ol. Kinds and levels of volatile compounds from different geographical areas varied. As the fruiting bodies aged, levels of methyl cinnamate and 1-octen-3-ol gradually declined. Potassium was the most abundant element in T. matsutake fruiting bodies. Of 17 amino acids detected in fruiting bodies, glutamate was the most abundant. Volatile compounds, chemical compositions, and nutritional values of T. matsutake varied with age and geographical origin and can serve as chemical indicators for classication of T. matsutake from different geographical areas and at different stages of maturity.

Staurosporine from the endophytic Streptomyces sp. strain CNS-42 acts as a potential biocontrol agent and growth elicitor in cucumber.

Chinese medicinal plants and their surrounding rhizospheric soil serve as promising sources of actinobacteria. A total of 180 actinobacteria strains were isolated from the rhizosphere soil, leaves, stems, and roots of nine selected plants and have been identified as potential biocontrol agents against Fusarium oxysporum f. sp. cucumerinum. An endophytic strain CNS-42 isolated from Alisma orientale showed the largest zone of inhibition demonstrating a potent effect against F. oxysporum f. sp. cucumerinum and a broad antimicrobial activity against bacteria, yeasts, and other pathogenic fungi. The in vivo biocontrol assays showed that the disease severity index was significantly reduced (P < 0.05), and plant shoot fresh weight and height increased greatly (P < 0.05) in plantlets treated with strain CNS-42 compared to the negative control. This isolate was identified as Streptomyces sp. based on cultural, physiological, morphological characteristics, and 16S rRNA gene analysis. Further bioassay-guided isolation and purification revealed that staurosporine was responsible for its antifungal and plant growth promoting activities and the latter property of staurosporine is reported for the first time. The in vivo assay was further performed and indicated that staurosporine showed good growth promoting effect on the plant shoot biomass of cucumber. This is the first critical evidence identifying CNS-42 as a biocontrol agent for the soil borne pathogen, F. oxysporum f. sp. cucumerinum.

[Community structure and diversity of entophytic bacteria in Tricholoma matsutake in Sichuan Province, Southwest China].

In order to understand the diversity and community structure of endophytic bacteria in Tricholoma matsutake, 14 Tricholoma matsutake samples were collected from 7 main production counties of Xiaojin, Yajiang, Muli, Yanyuan, Yanbian, Huidong and Mianning. The entophytic bacterial community structure and diversity were investigated by PCR denaturing gradient gel electrophoresis (PCR-DGGE). The results showed that the diversity and community structure of endophytic bacteria in T. matsutake varied significantly, while those from the environments alike had high similarity. In addition, each T. matsutake sample contained more than 15 species of endophytes, with that from Yanyuan having the highest endophytes diversity index and abundance, while those from Xiaojin were the lowest. Phylogenetic tree showed that endophytic bacterial species in T. matsutake were abundant. Dominant bacterial population in T. matsutake collected from different places varied, and Pseudomonas, Ewingella and Bacillus were distributed in all samples and retained a certain advantage. Alcaligenes and Sphingobacterium distributed in most samples, while Duganella and Lysinibacillus only in certain ones. This study demonstrated that the endophytic bacteria in T. matsutake were abundant and diversified, which could be in favor of searching the dominant bacteria promoting the growth of T. matsutake.

[Endophytic bacterial diversity in Codonopsis pilosula, Ephedra sinica, and Lamiophlomis rotate: a study with LH-PCR].

To investigate the endophytic bacterial diversity in the three medicinal plant species Codonopsis pilosula, Ephedra sinica, and Lamiophlomis rotata in Ganzi of Sichuan, Southwest China, the total DNA of the three species were extracted by stringent surface sterilization, and studied with length heterogeneity-PCR (LH-PCR) method. For the same plant species, their root-, stem-, and leaf LH-PCR profiles were in a high level of similarity, with little differences in band richness. However, there existed great differences in the LH-PCR profiles among different plant species. C. pilosula had the biggest band richness, followed by E. sinica, and L. rotata. In the three plant species, the endophytic bacteria with an approximately 474 bp DNA length were dominant. The endophytic bacterial diversity of the plants was negatively correlated with rhizosphere soil available phosphorus content, but positively correlated with rhizosphere soil pH. Elevation and rhizosphere soil total nitrogen content were the important environmental factors affecting the distribution of enophytic bacteria in these plant species. The information of population diversity obtained from LH-PCR could more intuitively reflect the differences of bacterial diversity among different plant species, and thus, LH-PCR would be available to be used for analyzing the endophytic bacterial diversity in medicinal plants, providing information and guidance for the further isolation of microbial resources.

[Active ingredients and efficacies of Ganoderma lucidum cultivated on non-medicinal parts of Chinese medicinal herbs].

OBJECTIVE: Ganoderma lucidum was cultivated on non-medicinal parts of Salvia miltiorrhiza, Chrysanthemum morifolium, Ptatycodgn grandlfiorum, as all are Chinese traditional herbal medicines. We studied the changes of active ingredients and efficacies of the Ganoderma lucidum fruit bodies. METHODS: The agronomic characters, polysaccharide and terpene contents, acute toxicity and efficacy of Ganoderma lucidum grown on the non-medicinal part of the three materials were compared with that grown on the ordinary formula group (OF. G) which was composed of corn cob, cotton seed shell. RESULTS: Biological conversion efficiencies of the Ganoderma lucidum fruit body using non-medicinal parts were higher than that of using the ordinary formula group (OF. G), though growth periods became longer; Contents of active ingredients were all improved except that the terpene content of the Salvia miltiorrhiza group was decreased. Both polysaccharide and terpene from the Chrysanthemum morifolium group were the highest, contents of which were respectively 2.47% and 0.79%; Acute toxicity test showed that Ganoderma lucidum fruit bodies were all with low toxicities. Mice maximum tolerance dose were 100 g/kg weight. In hemolysin test and sleeping promotion test, the Chrysanthemum morifolium group showed better effect than the ordinary formula group (OF. G). In anti-fatigue test, only the ordinary formula group (OF. G) proved to be more effective. CONCLUSION: It's feasible to cultivate Ganoderma lucidum and active ingredients and efficacies of Ganoderma lucidum have been changed using the non-medicinal parts of Chinese medicinal herbs.

Isolation of a wild Morchella spp. strain and the effects of its extract on ethanol-induced gastric mucosal lesions in rats.

A Morchella spp. strain was isolated from a wild morel mushroom, and the effects of its mycelia extract on the ethanol-induced gastric mucosal lesions of rats were investigated in vivo. Sequence analysis of internal transcribed spacer suggested that this Morchella spp. strain (strain No. M1) was clustered together with M. conica in the phylogenetic tree. The superoxide dismutase (SOD) activity increased significantly compared to the control. However, the malondialdehyde (MDA) level and myeloperoxidase (MPO) activity decreased significantly compared to the control. These results indicated that M1 is one member of M. conica and the protective effects of M1 extract against the ethanol-induced gastric lesions may be related to the increased SOD activity and decreased MDA level and MPO activity in rats.

An assessment of the genetic diversity within Ganoderma strains with AFLP and ITS PCR-RFLP.

Ganoderma lucidum is one of the most important medicinal materials and plant pathogens. Because of its specific interhybridization, the genetic background, however, is relatively unclear. It made identification of Ganoderma strains, especially closely related strains difficulty. Amplified fragment length polymorphism (AFLP) using 14 primer combinations and internal transcribed spacer (ITS) PCR-RFLP were used in a comparative study which was designed to investigate the closely related Ganoderma strains genetic relations at molecular level. The analysis of 37 Ganoderma strains showed there were 177 polymorphic AFLP markers and 12 ITS PCR-RFLP markers, and all accessions could be uniquely identified. Among the Ganoderma accessions, similarity coefficients ranged from 0.07692 to 0.99194 in AFLP. The Ganoderma strains formed a tight cluster in nine groups in AFLP whereas seven groups in ITS PCR-RFLP. The cluster analysis revealed that the taxonomical system of subgenus Ganoderma is composed of Sect. Ganoderma and Sect. Phaeonema, and the strain 22 should be a variant form of strain 21. All methods delineated the Ganoderma strains from the different regions seeming to show a greater level of genetic diversity. It indicated that the genotype study at molecular level is a useful complement method to the current classification system of Ganoderma strains based on morphological traits. The congruency of the experiments was analyzed using the biostatistical software DPS V3.01.

[Discrimination of Ganoderma based on high performance liquid chromatographic fingerprints combined with chemometrics methods].

High performance liquid chromatography (HPLC)-based system was utilized for generating chemical fingerprints of 11 Ganoderma strains. The data were statistically evaluated by using chemometric methods in order to classify the samples. The similarities of all the 11 samples and the relative peak areas of 13 common peaks were newly calculated separately. Then different chemometrics methods including hierarchical cluster analysis (HCA), principal component analysis (PCA) and discriminate analysis (DA) were applied to classify the G. lucidum samples. Consistent results show that the application of HPLC fingerprint coupled with powerful chemometrics analysis in the discrimination and classification of Ganoderma is a reliable and scientific approach.

[Effects of Armillariella tabescens polysaccharide IPS-B2 on activity of mouse peritoneal macrophages and transcription of related gene].

OBJECTIVE: To observe the effect of IPS-B2 on mouse peritoneal macrophages and the transcription of IL-1beta, IL-6, TNF-alpha and iNOS. METHOD: ELISA method and Griess method were used to detect the effect of mouse peritoneal macrophages produce cytokines IL-1beta, IL-6, TNF-alpha and cytotoxic effectors NO. The transcription of IL-1beta, IL-6, TNF-alpha and iNOS was detected by real-time RT-PCR method. RESULT: IPS-B2 could not promote mouse peritoneal macrophage production, but it could significantly improve the IL-1beta, IL-6, TNF-alpha content in mouse peritoneal macrophages culture supernatant, and increase the gene expression of IL-1beta, IL-6, TNF-alpha and iNOS. CONCLUSION: IPS-B2 can enhance the ability of peritoneal macrophages to excrete bioactive substances and promote the transcription of bioactive substances to antitumor.

Molecular diversity of Auricularia polytricha revealed by inter-simple sequence repeat and sequence-related amplified polymorphism markers.

Due to unsatisfying attempts to fingerprint Auricularia polytricha, two different molecular maker systems--Inter-Simple Sequence Repeats (ISSR) and Sequence-related amplified polymorphism (SRAP)--were established and tested to quantify molecular diversity among 19 strains of this fungus. A total of 202 (99.0%) and 459 (95.9%) polymorphic bands were detected by 13 ISSR primers and 14 SRAP primer combinations, respectively. By parsimony method, a phylogenetic tree was constructed based on each analysis; the two trees show that 19 A. polytricha strains were distributed into five or four groups. These results demonstrated that both methods were suitable for discriminating among strains of A. polytricha, and the novel SRAP markers are more efficient and preferable. The result also indicated the high level of genetic diversity of A. polytricha and their relationship between each other. These findings would benefit future research in A. polytricha, especially in breeding and medicine development. It also gives a useful method for fingerprinting of other fungi.

Characterisation and immunostimulatory activity of an α-(1→6)-d-glucan from the cultured Armillariella tabescens mycelia.

IPS-B2, an intracellular polysaccharide with anti-tumor effect, was isolated from cultured Armillariella tabescens mycelia by hot water extraction, anion-exchange and gel chromatography. Based on the results of paper chromatography (PC), gas chromatography (GC), infra-red (IR) spectroscopy and (13)C NMR, IPS-B2 was characterized as an α-(1→6)-d-glucan with a molecular weight of 49.5kDa. The effects of IPS-B2 on murine peritoneal macrophages were further investigated. The results demonstrated that IPS-B2 induced nitric oxide (NO) and cytokines (TNF-α, IL-1ß and IL-6) production in macrophages. The outcomes of real-time reverse transcription-polymerase chain reaction (RT-PCR) proved that the transcribing level of inducible NO synthase (iNOS), TNF-α, IL-1ß and IL-6 mRNA in the peritoneal macrophages have been augmented by IPS-B2. These data suggest that the anti-tumor activity of the polysaccharide from A. tabescens may due to activation of macrophage.

[AFLP analysis for genetic diversity of Ganoderma].

OBJECTIVE: To investigate the genetic diversity of Ganoderma cultivars provided for the genuineness study, germ-plasm resource identification, genetic relationship study, breeding, introduction and cultivante of Ganoderma strains. METHOD: With the software of NTSYSpc 2. 1, 24 materials, of G. lucidum and G. sinense, were studied using AFLP to construct the dendrogram. RESULT: There were 177 polymorphic bands with 14 primer combinations. And all materials could be identified with AFLP. CONCLUSION: There actually existed much genetic diversity at the molecular level among the germplasm resources of Ganoderma strains, and all the strains were clustered into G. lucidum group and G. sinense group at the similarity coefficient 0. 676.