S100P as a potential biomarker for immunosuppressive microenvironment in pancreatic cancer: a bioinformatics analysis and in vitro study.

BACKGROUND: Immunosuppression is a significant factor contributing to the poor prognosis of cancer. S100P, a member of the S100 protein family, has been implicated in various cancers. However, its role in the tumor microenvironment (TME) of pancreatic cancer remains unclear. This study aimed to investigate the potential impact of S100P on TME characteristics in patients with pancreatic cancer. METHODS: Multiple data (including microarray, RNA-Seq, and scRNA-Seq) were obtained from public databases. The expression pattern of S100P was comprehensively evaluated in RNA-Seq data and validated in four different microarray datasets. Prognostic value was assessed through Kaplan-Meier plotter and Cox regression analyses. Immune infiltration levels were determined using the ESTIMATE and ssGSEA algorithms and validated at the single-cell level. Spearman correlation test was used to examine the correlation between S100P expression and immune checkpoint genes, and tumor mutation burden (TMB). DNA methylation analysis was performed to investigate the change in mRNA expression. Reverse transcription PCR (RT-PCR) and immunohistochemical (IHC) were utilized to validate the expression using five cell lines and 60 pancreatic cancer tissues. RESULTS: This study found that S100P was differentially expressed in pancreatic cancer and was associated with poor prognosis (P < 0.05). Notably, S100P exhibited a significant negative-correlation with immune cell infiltration, particularly CD8 + T cells. Furthermore, a close association between S100P and immunotherapy was observed, as it strongly correlated with TMB and the expression levels of TIGIT, HAVCR2, CTLA4, and BTLA (P < 0.05). Intriguingly, higher S100P expression demonstrated a negative correlation with methylation levels (cg14323984, cg27027375, cg14900031, cg14140379, cg25083732, cg07210669, cg26233331, and cg22266967), which were associated with CD8 + T cells. In vitro RT-PCR validated upregulated S100P expression across all five pancreatic cancer cell lines, and IHC confirmed high S100P levels in pancreatic cancer tissues (P < 0.05). CONCLUSION: These findings suggest that S100P could serve as a promising biomarker for immunosuppressive microenvironment, which may provide a novel therapeutic way for pancreatic cancer.

Diversity, antibacterial activity and chemical analyses of gut-associated fungi isolated from the Crocothemis servilia.

Insect-associated fungi are a potentially rich source of novel natural products with antibacterial activity. Here, we investigated the community composition and phylogenetic diversity of gut-associated fungi of the dragonfly (Crocothemis Servilia) using a combination of culture-dependent and culture-independent methods. A total of 42 fungal isolates were obtained from the guts of the dragonfly, which belonged to four classes and thirteen different genera. Amplicon sequencing analyses revealed that the fungal communities were more diverse, and a total of 136 genera were identified and dominated by the genera Wojnowiciella and Phoma. The antibacterial bioassay showed that five fungal crude extracts of representative isolates have shown antibacterial activities. Among them, the extract of Phoma sp. QTH17 showed the best antibacterial activities against Escherichia coli, Micrococcus tetragenus, and Staphylococcus aureus with the disc diameter of inhibition zone diameter (IZD) of 6.50, 10.80, and 8.70 mm, respectively. Chemical analysis of Phoma sp. QTH17 led to the discovery of five known compounds, including ergosterol (1), 3-Chlorogentisyl alcohol (2), epoxydon (3), epoxydon 6-methylsalicylate ester (4) and mannitol (5). Among them, the compound 3 exhibited potent antibacterial activities against E. coli, M. tetragenus, and S. aureus with the IZD of 7.00, 14.00, and 12.50 mm, respectively, which were slightly weaker than those of the positive gentamicin sulfate with the IZD of 11.13, 18.30, and 12.13 mm, respectively. In conclusion, our results confirmed that the diversity of gut-associated fungi of C. Servilia could be expected to explore the resource of new species and antibacterial substances.

Community composition, bacterial symbionts, antibacterial and antioxidant activities of honeybee-associated fungi.

BACKGROUND: Fungi associated with insects represent one potentially rich source for the discovery of novel metabolites. However, a comprehensive understanding of the fungal communities of Apis mellifera ligustica remains elusive. RESULTS: Here, we investigated the phylogenetic diversity and community composition of honeybee-associated fungi using combination of culture-dependent and culture-independent approaches. A total of forty-five fungi were isolated and purified from the Apis mellifera ligustica, royal jelly, and honeycomb, which belonged to four classes and eleven different genera. Furthermore, 28 bacterial 16S rRNA gene sequences were obtained by PCR from the fungal metagenome. High-throughput sequencing analyses revealed that the fungal communities were more diverse, a total of 62 fungal genera were detected in the honeybee gut by culture-independent method, whereas only 4 genera were isolated by culture-dependent method. Similarly, 247 fungal genera were detected in the honeycomb, whereas only 4 genera were isolated. In addition, we assessed the antibacterial and antioxidant activities of fungal isolates. Most fungal crude extracts obtained from the cultivation supernatant exhibited antioxidant activities. Only two fungal crude extracts displayed moderate activity against Escherichia coli and Staphylococcus aureus. Chemical analysis of Chaetomium subaffine MFFC22 led to the discovery of three known compounds, including cochliodinol (1), emodin (2), chrysophanol (3). Among them, cochliodinol (1) showed intense DPPH radical scavenging activity with the 50% inhibitory concentration (IC50) of 3.06 µg/mL, which was comparable to that of the positive ascorbic acid (IC50 = 2.25 µg/mL). Compound 2 displayed weak inhibitory activities against Micrococcus tetragenus and S. aureus. CONCLUSIONS: This research provided a fundamental clue for the complex interactions among honeybees, fungi, bacterial symbionts, and the effects on the honeybee. Furthermore, the diversity of honeybee-associated fungi had great potential in finding the resource of new species and antioxidants.

Diversity and antibacterial potential of the Actinobacteria associated with Apis mellifera ligustica.

Insect-associated Actinobacteria are a potentially rich source of novel natural products with antibacterial activity. Here, the community composition of Actinobacteria associated with Apis mellifera ligustica was investigated by integrated culture-dependent and independent methods. A total of 61 strains of Streptomyces genera were isolated from the honeycomb, larva, and different anatomical parts of the honeybee's body using the culture-dependent method. Amplicon sequencing analyses revealed that the actinobacterial communities were dominated by the family of Bifidobacteriaceae and Microbacteriaceae in the honeybee gut, and Nocardiaceae and Pseudonocardiaceae in the honeycomb, whereas only Streptomyces genera were isolated by the culture-dependent method. Culture-independent analyses showed more diverse actinobacterial communities than those of culture-dependent methods. The antibacterial bioassay showed that most crude extracts of representative isolates exhibited antibacterial activities. Among them, the crude extract of Streptomyces sp. FCF01 showed the best antibacterial activities against Staphylococcus aureus, Micrococcus tetragenus, and Pseudomonas syringae pv. actinidiae (Psa) with the disc diameter of inhibition zone diameter (IZD) of 23.00, 15.00, and 13.33 mm, respectively. Chemical analysis of Streptomyces sp. FCF01 led to the isolation of three secondary metabolites, including mayamycin (1), mayamycin B (2), and N-(2-Hydroxyphenyl) acetamide (3). Among them, compound 1 displayed strong antibacterial activity against S. aureus, M. tetragenus, and Psa with minimum inhibitory concentrations (MIC) values of 6.25, 12.5, and 6.25 µg/ml, respectively. In addition, two novel derivative compounds 1a and 1b were synthesized by acetylation of compound 1. Both compounds 1a and 1b displayed similar antibacterial activities with those of metabolite 1. These results indicated that Streptomyces species associated with honeybees had great potential in finding antibiotics.

Quantitative analysis and dietary risk assessment of aflatoxins in Chinese post-fermented dark tea.

Recently, mycotoxins safety in Chinese post-fermented teas has attracted much attention because it is still controversial whether environmental fungi in the teas are able to produce aflatoxins. In this study, a rapid and selective analytical method based on immunoaffinity column (IAC) cleaning coupled with liquid chromatography-mass spectrometry (LC-MS) was developed to quantify four aflatoxins (B1, B2, G1 and G2) in post-fermented teas. Recoveries ranged from 80.8 to 92.2% with relative standard deviation less than 3%. The limits of detection and quantification were 0.109-0.348 µg kg-1 and 0.364-1.159 µg kg-1, respectively. Two out of 158 samples were positive for aflatoxins examination (occurrence rate 1.27%). The deterministic assessment for the maximal aflatoxins exposure under upper bound was 9.19 × 10-6 µg kg-1 day-1 from heavy exposure consumers (≥50 year age group), but the exposure was lower than the JECFA acceptable value of 1.0 ng kg-1 day-1 on liver risk. Probabilistic assessment showed that the upper bound 95th percentile carcinogenic risk value for the 318 consumers (Yunnan China and Ulan Bator Mongolia) was 1.75 × 10-7, and for heavy exposure consumers was 2.4 × 10-7, and the values equally below the acceptable carcinogenic risk level.

Determination of six groups of mycotoxins in Chinese dark tea and the associated risk assessment.

Chinese dark tea is widely enjoyed for its multiple health-promoting effects and pleasant taste. However, its production involves fermentation by microbiota in raw tea, some of which are filamentous fungi and thus potential mycotoxin producers. Accordingly, whether mycotoxins pose health risk on dark tea consumption has become a public concern. In this study, a cleaning method of multi-functional column (MFC) and immunoaffinity column (IAC) in tandem combined to HPLC detection was developed and validated for determining ten mycotoxins of six groups (i.e., aflatoxins of B1, B2, G1 and G2, ochratoxin A, zearalenone, deoxynivalenol, fumonisins of B1, B2, and T-2) in dark teas. The interferences from secondary metabolites were effectively reduced, and the sensitivities and recoveries of the method were qualified for tea matrices. Six groups mycotoxins were determined in 108 samples representing the major Chinese dark teas by using the new method. Subsequently, the dietary exposure and health risks were evaluated for different age and gender groups in Kunming and Pu'er in China and Ulan Bator in Mongolia. The occurrence of zearalenone was 4.63% and that of ochratoxin A was 1.85%, with the other four groups mycotoxins were below the limits of quantification. The hazard index values for the five groups' non-carcinogenic mycotoxins were far below 1.0. The deterministic risk assessment indicated no non-carcinogenic risks for dark tea consumption in the three areas. Probabilistic estimation showed that the maximum value of 95th percentile carcinogenic risk value for the aflatoxins was 2.12 × 10-8, which is far below the acceptable carcinogenic risk level (10-6). Hereby, six groups mycotoxins in Chinese dark tea showed no observed risk concern to consumers.

Simultaneous Determination of Four Aflatoxins in Dark Tea by Multifunctional Purification Column and Immunoaffinity Column Coupled to Liquid Chromatography Tandem Mass Spectrometry.

Dry tea matrix contains an abundance of caffeine and polyphenols which are different from the food matrix (e.g., protein, lipid, and carbohydrates), and only a few studies have tried aflatoxins determination with tea samples. Here, a specific, accurate, and sensitive method was developed and validated for the simultaneous determination of aflatoxin B1, B2, G1, and G2 in dark teas. Aflatoxins were extracted by acetonitrile/water, press-filtered, and cleaned by multifunctional purification column (MFC) and immunoaffinity column (IAC) in tandem. The cleaned extract was analyzed by liquid chromatography tandem mass spectrometry. The matrix interference was effectively reduced by MFC-IAC cleaning method. Recoveries at the spiking concentrations of 5-60 µg/kg ranged from 77.5 to 93%, with relative standard deviations <11.0%. The correlation coefficients of aflatoxins standard were >0.998. The limits of detection were 0.024-0.21 µg/kg and the limits of quantification were 0.08-0.74 µg/kg. The intra- and interday accuracy ranged from 74 to 87%, and the intra- and interday precisions ranged from 0.4 to 3.1%. After the method validation, the aflatoxins contaminations in 100 collected dark teas were detected, and the results were compared with those of other methods.

Detoxification of U(VI) by Paecilomyces catenlannulatus investigated by batch, XANES and EXAFS techniques.

Paecilomyces catenlannulatus (P. catenlannulatus) as a genus of entomogenous fungus presented a variety of surface reactive groups by batch characterizations. The detoxification of U(VI) by P. catenlannulatus was investigated under different water chemistry (pH, incubation time, foreign anions and U(VI) concentration) by batch techniques. Approximately 75% of U(VI) was reduced to U(IV) (i.e., U(IV)O2(s)) by P. catenlannulatus at pH 5.5 and 7 days under glovebox conditions, therefore the formation of precipitates decreased the toxicity of U(VI) for P. catenlannulatus. In addition, phosphate facilitate the U(VI) reduction, whereas carbonate and sulfate inhibited the U(VI) reduction. The activities of catalase (CAT), superoxide dismutase (SOD) and glutathione (GSH) level were stimulated exposure to 1-30 mg/L U(VI), indicating that CAT, SOD and GSH were antagonized for the oxidant stress derived from U(VI) at low concentrations. According to XPS and XANES analysis, the occurrence of U(IV) revealed the reduction of adsorbed U(VI) to U(IV) by P. catenlannulatus. The results of EXAFS analysis indicated that the fitting of U-O and U-U shell for U-loaded P. catenlannulatus was similar to that of U(IV)O2(s)). The formation of U-bearing precipitates decreased the toxicity of U(VI) for P. catenlannulatus. These findings indicated that P. catenlannulatus is capable to detoxify U(VI) by extracellar/intracellar defense systems. Therefore, P. catenlannulatus can be utilized as a promising bioadsorbents for remediation of uranium-contaminated wastewater in environmental cleanup.

[Anti-tumor effects of a novel cyclophosphamide derivate 9b in vivo and in vitro].

This study is to investigate the anti-tumor activities of a novel cyclophosphamide derivate 4, 6-diphenyl cyclophosphamide (9b) in vivo and in vitro, and its possible mechanism of action. The inhibitory effects of 9b on human hepatoma cell line HepG2, human breast carcinoma cell line MCF-7 and human myeloid leukemia cell line K562 were measured by MTT assay in vitro. Cell cycle distribution and apoptotic rate were evaluated by flow cytometry. To evaluate the anti-tumor effect of 9b in vivo, mouse model bearing inoculated H22 tumor was established. The results indicated that 9b could inhibit the proliferation of HepG2, MCF-7 and K562 cells in a dose and time dependent manner. The ICo50 values of 9b were 32.34 micromol.L-1 to HepG2 cells, 87.07 micromol.L-1 to MCF-7 cells and 149.10 micromol.L-1 to K562 cells after incubation for 48 h. The results of flow cytometry indicated that after being treated for 48 h with different concentrations of 9b, the ratios of HepG2, MCF-7 cells at the Go/G1 phase and K562 cells at the G0/Gl phase and G2/M phase increased significantly compared with control group, and the apoptotic rate increased with the increase of the concentration of 9b. 9b could significantly reduce tumor weight of H22 solid tumor mouse model in vivo. To summarize, 9b showed significantly anti-tumor activity in vivo and in vitro, of which the mechanism might be associated with the change of cell cycle distribution and induction of tumor cell apoptosis.