Integrated Single-Cell and Transcriptome Sequencing Analyses Develop a Ubiquitination-Associated Signature in Gastric Cancer and Identified OTULIN as a Novel Biomarker.

BACKGROUND: Gastric cancer (GC) is the most commonly diagnosed digestive system malignancy with a dismal survival outcome. The prognostic value of ubiquitination-related genes (URGs) in GC has yet to be discovered. METHODS: Two GC cohort datasets were obtained from the Cancer Genome Atlas Program (TCGA) and Gene Expression Omnibus (GEO) databases. Stepwise Cox analysis was employed to generate a signature. Then, we applied unsupervised clustering analysis to determine subclusters in GC based on URGs. Single-cell analysis was carried out to depict the cellular location of model genes. The CIBERSORT method was performed to estimate the immune landscape. Finally, preliminary wet lab work was utilized to disclose the potential effect of OTULIN. RESULTS: Our proposed signature was set up based on five URGs (OTULIN, UBE2C, USP1, USP2, and MAPT) which could serve as a risk classifier to categorize GC cases. In addition, it was demonstrated that the ubiquitination-associated model could depict the immune landscape and forecast immunotherapy response for GC patients. Furthermore, in vitro experiments determined the function and effect of OUTLIN in GC. We observed that the knockdown of OUTLIN could suppress cell viability and metastatic ability of GC cell lines. CONCLUSIONS: Our data lays the groundwork for a comprehensive investigation into the role of URGs in GC and determined OTULIN as a candidate GC biomarker.

Transcriptome profiling reveals insertional mutagenesis suppressed the expression of candidate pathogenicity genes in honeybee fungal pathogen, Ascosphaera apis.

Chalkbrood disease is caused by Ascosphaera apis which severely affects honeybee brood. Spore inoculation experiments shown pathogenicity varies among different strains and mutants, however, the molecular mechanism of pathogenicity is unclear. We sequenced, assembled and annotated the transcriptomes of wild type (SPE1) and three mutants (SPE2, SPE3 and SPE4) with reduced pathogenicity that were constructed in our previous study. Illumina sequencing generated a total of 394,910,604 clean reads and de novo Trinity-based assembled into 12,989 unigenes, among these, 9,598 genes were successfully annotated to known proteins in UniProt database. A total of 172, 3,996, and 650 genes were up-regulated and 4,403, 2,845, and 3,016 genes were down-regulated between SPE2-SPE1, SPE3-SPE1, and SPE4-SPE1, respectively. Overall, several genes with a potential role in fungal pathogenicity were detected down-regulated in mutants including 100 hydrolytic enzymes, 117 transcriptional factors, and 47 cell wall related genes. KEGG pathway enrichment analysis reveals 216 genes involved in nine pathways were down-regulated in mutants compared to wild type. The down-regulation of more pathways involved in pathogenicity in SPE2 and SPE4 than SPE3 supports their lower pathogenicity during in-vitro bioassay experiment. Expression of 12 down-regulated genes in mutants was validated by quantitative real time PCR. This study provides valuable information on transcriptome variation caused by mutation for further functional validation of candidate pathogenicity genes in A. apis.

Significant transcriptional changes in mature daughter Varroa destructor mites during infestation of different developmental stages of honeybees.

BACKGROUND: Varroa destructor is considered a major cause of honeybee (Apis mellifera) colony losses worldwide. Although V. destructor mites exhibit preference behavior for certain honeybee lifecycle stages, the mechanism underlying host finding and preference remains largely unknown. RESULTS: By using a de novo transcriptome assembly strategy, we sequenced the mature daughter V. destructor mite transcriptome during infestation of different stages of honeybees (brood cells, newly emerged bees and adult bees). A total of 132 779 unigenes were obtained with an average length of 2745 bp and N50 of 5706 bp. About 63.1% of the transcriptome could be annotated based on sequence homology to the predatory mite Metaseiulus occidentalis proteins. Expression analysis revealed that mature daughter mites had distinct transcriptome profiles after infestation of different honeybee stages, and that the majority of the differentially expressed genes (DEGs) of mite infesting adult honeybees were down-regulated compared to that infesting the sealed brood cells. Gene ontology and KEGG pathway enrichment analyses showed that a large number of DEGs were involved in cellular process and metabolic process, suggesting that Varroa mites undergo metabolic adjustment to accommodate the cellular, molecular and/or immune response of the honeybees. Interestingly, in adult honeybees, some mite DEGs involved in neurotransmitter biosynthesis and transport were identified and their levels of expression were validated by quantitative polymerase chain reaction (qPCR). CONCLUSION: These results provide evidence for transcriptional reprogramming in mature daughter Varroa mites during infestation of honeybees, which may be relevant to understanding the mechanism underpinning adaptation and preference behavior of these mites for honeybees. © 2020 Society of Chemical Industry.

Purification of Chinese Sacbrood Virus (CSBV), Gene Cloning and Prokaryotic Expression of its Structural Protein VP1.

The aim of this study was to purify the Chinese Sacbrood Virus Beijing Miyun (BJMY-CSBV) from infected Apis cerana larvae, clone structural protein gene VP1 (named BJMY-CSBV-VP1), and investigate its biological information. The result indicated that the capsid of CSBV is of spherical shape. Gene clone experiment showed that the BJMY-CSBV-VP1 gene sequence comprised 945 bp, encoding 315 amino acids with relative molecular weight of 35.59 kDa and isoelectric point 9.38 pI. Phylogenetic analysis of amino acid sequences showed that the BJMY-CSBV-VP1 and LNDD_2015 were grouped together. Protein secondary structure prediction showed that the gene contained two α-helices, thirteen ß-folds, six polypeptide binding sites, and no disulfide bridge. Simultaneously, the BJMY-CSBV-VP1 was ligated to the expression vector pET32a(+) and then transformed into the Escherichia coli BL21 (DE3) for prokaryotic expression. The optimal expression experiment revealed that the protein was found in the inclusion body. The recombinant protein was successfully purified by washing buffer combined with supersonic fragmentation. In this study, we obtained the purified BJMY-CSBV particles, cloned BJMY-CSBV-VP1 gene, investigated the detailed information of the gene by analyzing the sequence, and obtained the purified recombinant protein, which could help for further understanding of the function of the structural protein gene VP1.

Elemene Induces Apoptosis of Human Gastric Cancer Cell Line BGC-823 via Extracellular Signal-Regulated Kinase (ERK) 1/2 Signaling Pathway.

BACKGROUND Elemene is extracted from a traditional herbal medicine and is commonly used in the treatment of cancer in China. However, its effect on gastric cancer cells remains unknown. The goal of this study was to investigate its effect on human gastric cancer cells. MATERIAL AND METHODS Human gastric cancer BGC-823 cells and a tumor-bearing mouse model were employed to be divided into 4 groups: control group, elemene group, PD98059 group (an ERK 1/2 signaling pathway inhibitor), and the combined group (elemene plus PD98059). The tumor size, cell proliferation, expression of ERK 1/2 and phosphorylated ERK 1/2 (p-ERK 1/2), Bcl-2 mRNA, and Bax mRNA were measured. Moreover, cell apoptosis was detected and the apoptosis index was calculated. RESULTS Elemene and PD98059 each significantly inhibited the proliferation of gastric cancer cells BGC-823, and their combination showed higher synergistic inhibitory effect (P<0.05). We also found increased expression levels of p-ERK l/2 protein and Bax mRNA, but reduced level of Bcl-2 mRNA expression (P<0.05). Elemene presented higher apoptosis rate in a dose-dependent manner (P<0.05). Furthermore, the injection of elemene decreased the weight of transplanted tumors. CONCLUSIONS Elemene can inhibit the proliferation and induce the apoptosis of gastric cancer cells associated with the ERK 1/2 signaling pathway and expression levels of Bax mRNA and Bcl-2 mRNA.

Curcumin Enhances the Effects of 5-Fluorouracil and Oxaliplatin in Inducing Gastric Cancer Cell Apoptosis Both In Vitro and In Vivo.

Despite the efficacy of fluoropyrimidines and oxaliplatin-based chemotherapy for patients, this treatment leads to significant patient inconvenience, toxicity, and cost. This study aims to validate a nontoxic agent, curcumin, to the current chemotherapeutic regimen. In in vitro experiments, curcumin induced apoptosis in gastric cancer cell line BGC-823. Synergistic antitumor effects of curcumin were observed in combination with 5-fluorouracil (5-FU) and oxaliplatin. These effects were accompanied by downregulation of the expression of Bcl-2 protein and mRNA and upregulation of the expression of Bax and caspase 3, 8, and 9. In addition, the in vivo study showed that the combination of curcumin and 5-FU/oxaliplatin exhibited potent growth inhibition of BGC-823 xenograft tumors. Furthermore, compared with the control group, no significant difference was observed in the body weight of curcumin-treated nude mice. In conclusion, curcumin may act synergistically with the chemotherapeutic regimen FOLFOX in gastric cancer in vitro and in vivo by inducing apoptosis via Bcl/Bax-caspase 8,9-caspase 3 pathway.