Anhong Ning

De novo characterization of Lentinula edodes C91-3 transcriptome by deep Solexa sequencing

Lentinula edodes, has been utilized as food, as well as, in popular medicine, moreover, its extract isolated from its mycelium and fruiting body have shown several therapeutic properties. Yet little is understood about its genes involved in these properties, and the absence of L.edodes genomes has been a barrier to the development of functional genomics research. However, high throughput sequencing technologies are now being widely applied to non-model species. To facilitate research on L.edodes, we leveraged Solexa sequencing technology in de novo assembly of L.edodes C(91-3) transcriptome. In a single run, we produced more than 57 million sequencing reads. These reads were assembled into 28,923 unigene sequences (mean size=689bp) including 18,120 unigenes with coding sequence (CDS). Based on similarity search with known proteins, assembled unigene sequences were annotated with gene descriptions, gene ontology (GO) and clusters of orthologous group (COG) terms. Our data provides the first comprehensive sequence resource available for functional genomics studies in L.edodes, and demonstrates the utility of Illumina/Solexa sequencing for de novo transcriptome characterization and gene discovery in a non-model mushroom.

A Novel Apoptosis Correlated Molecule: Expression and Characterization of Protein Latcripin-1 from Lentinula edodes C91–3

An apoptosis correlated molecule—protein Latcripin-1 of Lentinula edodes C91–3—was expressed and characterized in Pichia pastoris GS115. The total RNA was obtained from Lentinula edodes C91–3. According to the transcriptome, the full-length gene of Latcripin-1 was isolated with 3′-Full Rapid Amplification of cDNA Ends (RACE) and 5′-Full RACE methods. The full-length gene was inserted into the secretory expression vector pPIC9K. The protein Latcripin-1 was expressed in Pichia pastoris GS115 and analyzed by Sodium Dodecylsulfonate Polyacrylate Gel Electrophoresis (SDS-PAGE) and Western blot. The Western blot showed that the protein was expressed successfully. The biological function of protein Latcripin-1 on A549 cells was studied with flow cytometry and the 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyl-tetrazolium Bromide (MTT) method. The toxic effect of protein Latcripin-1 was detected with the MTT method by co-culturing the characterized protein with chick embryo fibroblasts. The MTT assay results showed that there was a great difference between protein Latcripin-1 groups and the control group (p < 0.05). There was no toxic effect of the characterized protein on chick embryo fibroblasts. The flow cytometry showed that there was a significant difference between the protein groups of interest and the control group according to apoptosis function (p < 0.05). At the same time, cell ultrastructure observed by transmission electron microscopy supported the results of flow cytometry. The work demonstrates that protein Latcripin-1 can induce apoptosis of human lung cancer cells A549 and brings new insights into and advantages to finding anti-tumor proteins.

Expression and functional analysis of novel molecule — Latcripin-13 domain from Lentinula edodes C91-3 produced in prokaryotic expression system

The shiitake mushroom Lentinula edodes has health benefits and is used to treat various diseases due to its immunomodulatory and antineoplastic properties. In the present study, the Latcripin-13 domain, isolated from L. edodes, was expressed in Escherichia coli Rosetta-gami(DE3) in the form of inclusion bodies. The Latcripin-13 domain was purified by Ni-His affinity chromatography with high purity and refolded by urea gradient dialysis. The product showed biological activity in A549 cells, a human lung cancer cell line, by flow cytometry and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) method. The MTT assay and the flow cytometry results revealed that there was a great difference between the Latcripin-13 domain-treated group and the control group (p<0.05). Similarly, cell apoptosis observed by transmission electron microscopy (TEM) supported the flow cytometry results. This work demonstrated that the Latcripin-13 domain can induce apoptosis of A549 cells, which will bring new insights into the development of new antitumor drugs in the future.

In vitro antitumor activity of Latcripin-15 regulator of chromosome condensation 1 domain protein

Cancer is one of the most significant health problems worldwide and thus the development of novel therapeutic agents with fewer side effects is required. The present study investigated the in vitro anticancer effects of a newly isolated fungal protein. In this study, Latcripin-15 (LP-15) regulator of chromosome condensation 1 (RCC1) domain protein, which is obtained from the Lentinula edodes C91-3 fungal strain, was identified, cloned, expressed, purified and re-folded to assess the in vitro antitumor activity of the protein. LP-15 RCC1 full-length cDNA was isolated from Lentinula edodes using 3′ and 5′-rapid amplification of cDNA ends and then cloned, expressed, purified and re-folded in vitro. In addition, the effects of the isolated LP-15 RCC1 proteins functional domain on the viability and apoptosis of human lung cancer A549 cells were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, transmission electron microscopy, flow cytometry and Hoechst 33258 staining. The LP-15 RCC1 functional domain protein was successfully expressed, purified and re-folded in vitro. Treatment with the LP-15 RCC1 functional domain protein significantly reduced tumor cell viability and induced apoptosis in A549 cells. The results of the present study indicate that the LP-15 RCC1 functional domain requires further investigation as a novel therapeutic agent for cancer therapy.

From Inducing Autophagy to Programmed Cell Death? The PI3K Functional Domain Study of Protein Latcripin-1 from Lentinula edodes C91-3

The functional study of the edible mycoprotein is important for elucidating the efficacy mechanism of fungi. There are more broad prospects on the field of antitumor mycoprotein. The expression map of Lentinula edodes C91-3 was sequenced and analyzed on the previous work. A novel antitumor mycoprotein—Latcripin-1 (LP1) was found at the first time. In order to clarify the mechanism of its inducing programmed cell death in lung cancer cells, protein LP1 PI3K (phosphatidylinositol 3-kinase) functional domain peptide was obtained by the Rosetta gami prokaryotic expression and purification system. Western blot was used to identify the functional domain and the autophagosomal marker—microtubule associated protein light chain 3. Transmission electron microscopy technology was used to analysed the ultrastructure of the treated cell. Flow cytometry and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) methods were used to detect the biological functions of domain PI3K to lung cancer cell. This work demonstrates that the functional domain PI3K of LP1 maybe induces lung cancer A549 to programmed cell death through autophagy pathway. The functional domain PI3K in L. edodes C91-3 should be ascribed to class III PI3K. Though further study is needed, it will shed new light into developing new anti-tumor polypeptide drugs.

Antibacterial and anti-biofilm activity of the lipid extract from Mantidis ootheca on Pseudomonas aeruginosa

The aim of this study is to assess the antibacterial and anti-biofilm properties of the lipid extract from Mantidis ootheca against the gentamycin resistant Pseudomonas aeruginosa. The chemical composition of the lipid extract and its relative proportion were determined using the technique of gas chromatography coupled with mass spectrometry (GC-MS). Antibacterial susceptibility tests were performed using a disc diffusion assay and the minimum inhibition concentration (MIC) was determined by way of the agar dilution method. The anti-biofilm test was carried out with crystal violet staining and scanning electron microscopy (SEM). There were 16 compounds detected, and the most abundant components were sesquiterpenoids, monoterpenes, and trace aromatic compounds. The MIC for P. aeruginosa was 4 mg/ml and the eradication effect on preformed biofilms was established and compared with a ciprofloxacin control. The results of our study indicated that a lipid extract from M. ootheca could be used as a topical and antibacterial agent with anti-biofilm activity in the future.中文概要目的探究桑螵蛸脂类提取物的成分及对铜绿假单胞菌 的抗菌和抗生物膜作用。创新点中国传统中药桑螵蛸一直广泛应用于肾病的治 疗,在抗菌领域未见报道,本实验首次证明桑螵 蛸脂类提取物对铜绿假单胞菌有明显的抗菌和 抗生物膜作用。方法采用气相色谱-质谱联用技术,测定桑螵蛸脂质 提取物的化学成分及其相对比例。采用纸片扩散 法和琼脂平板稀释法观察桑螵蛸脂类提取物对 铜绿假单胞菌的抑菌效应并测定最小抑制浓度 (MIC)。采用结晶紫染色法和扫描电镜(SEM) 进行抑制生物被膜的试验。结论桑螵蛸脂类提取物中含有16 种化合物,最丰富 的成分分别是倍半萜类化合物、单萜和微量芳香 族化合物。桑螵蛸脂类提取物对铜绿假单胞菌的 MIC 为4 mg/ml,对铜绿假单胞菌生物被膜的抑 制作用明显。

LP1 from Lentinula edodes C91-3 Induces Autophagy, Apoptosis and Reduces Metastasis in Human Gastric Cancer Cell Line SGC-7901

Present study aimed to elucidate the anticancer effect and the possible molecular mechanism underlying the action of Latcripin 1 (LP1), from the mushroom Lentinula edodes strain C91-3 against gastric cancer cell lines SGC-7901 and BGC-823. Cell viability was measured by Cell Counting Kit-8 (CCK-8); morphological changes were observed by phase contrast microscope; autophagy was determined by transmission electron microscope and fluorescence microscope. Apoptosis and cell cycle were assessed by flow cytometer; wound-healing, transwell migration and invasion assays were performed to investigate the effect of LP1 on gastric cancer cell’s migration and invasion. Herein, we found that LP1 resulted in the induction of autophagy by the formation of autophagosomes and conversion of light chain 3 (LC3I into LC3II. LP1 up-regulated the expression level of autophagy-related gene (Atg7, Atg5, Atg12, Atg14) and Beclin1; increased and decreased the expression level of pro-apoptotic (Bax) and anti-apoptotic (Bcl-2) proteins respectively, along with the activation of Caspase-3. At lower-doses, LP1 have shown to arrest cells in the S phase of the cell cycle and decreased the expression level of matrix metalloproteinase MMP-2 and MMP-9. In addition, it has also been shown to regulate the phosphorylation of one of the most hampered gastric cancer pathway, that is, protein kinase B/mammalian target of rapamycin (Akt/mTOR) channel and resulted in cell death. These findings suggested LP1 as a potential natural anti-cancer agent, for exploring the gastric cancer therapies and as a contender for further in vitro and in vivo investigations.

Polysaccharides Extracted from Rhizoma Pleionis Have Antitumor Properties In Vitro and in an H22 Mouse Hepatoma Ascites Model In Vivo

Malignant ascites is a highly severe and intractable complication of advanced or recurrent malignant tumors that is often immunotherapy-resistant. Rhizoma Pleionis is widely used in traditional medicine as an antimicrobial and anticancer agent, but its effectiveness in treating malignant ascites is unclear. In the current study, we investigated the effect of polysaccharides isolated from Rhizoma Pleionis (PRP) on murine hepatocarcinoma H22 cells in an ascites model. We have found that the main components of PRP, that presented a relative molecular weight of 383.57 kDa, were mannose and glucose. We also found that PRP reduced the occurrence of abdominal ascites and increased survival in our mouse model. An immune response in the ascites tumor model was observed by performing a lymphocytes proliferation experiment and an E-rosette test. The ratios of CD8+ cytotoxic T cells and NK cells in the spleen were examined by flow cytometry, and the mRNA expression of Foxp3+in CD4+CD25+ (T regulatory Tregs) was measured by RT-PCR (reverse transcription-polymerase chain reaction). The levels of the cytokines TNF-α (tumor necrosis factor), VEGF (vascular endothelial growth factor), IL-2 (interleukin), and IFN-γ (interferon) in the serum and ascites supernatants were measured by ELISA. The expression of Foxp3 and Stat3 in peritoneal cells in the mouse model was measured by immunocytochemistry. The results indicated that PRP increased H22 tumor cell apoptosis in vivo by activating and enhancing the immune response. Furthermore, the effects of PRP on the proliferation of H22 cells were assessed by the CCK8 assay, Hoechest 33258, and TUNEL staining in vitro. We found that PRP suppressed the proliferation of H22 tumor cells but had no effect on BRL (Big rat liver) -3A rat hepatoma normal cells in vitro. Next, we investigated the underlying immunological mechanism by which PRP inhibits malignant ascites. PRP induced tumor cell apoptosis by inhibiting the Jak1–Stat3 pathway and by activating Caspase-3 and Caspase-8 to increase the Bax/Bcl-2 ratio. Collectively, our results indicate that PRP exhibits significant antitumor properties in H22 cells in vivo and in vitro, indicating that PRP may be used as a new therapeutic drug for cancer treatment.

Combined effect of linolenic acid and tobramycin on Pseudomonas aeruginosa biofilm formation and quorum sensing

Pseudomonas aeruginosa is a ubiquitous Gram negative opportunistic pathogen capable of causing severe nosocomial infections in humans, and tobramycin is currently used to treat P. aeruginosa associated lung infections. Quorum sensing regulates biofilm formation which allows the bacterium to result in fatal infections forcing clinicians to extensively use antibiotics to manage its infections leading to emerging multiple drug resistant strains. As a result, tobramycin is also becoming resistant. Despite extensive studies on drug discovery to alleviate microbial drug resistance, the continued microbial evolution has forced researchers to focus on screening various phytochemicals and dietary compounds for antimicrobial potential. Linolenic acid (LNA) is an essential fatty acid that possesses antimicrobial actions on various microorganisms. It was hypothesized that LNA may affect the formation of biofilm on P. aeruginosa and improve the potency of tobramycin. The present study demonstrated that LNA interfered with cell-to-cell communication and reduced virulence factor production. It further enhanced the potency of tobramycin and synergistically inhibited biofilm formation through P. aeruginosa quorum sensing systems. Therefore, LNA may be considered as a potential agent for adjunctive therapy and its utilization may decrease tobramycin concentration in combined treatment thereby reducing aminoglycoside adverse effects.