Cudraflavone B induces human glioblastoma cells apoptosis via ER stress-induced autophagy.

BACKGROUND: Glioblastoma (GBM) is the most common malignant intracranial tumor with a low survival rate. However, only few drugs responsible for GBM therpies, hence new drug development for it is highly required. The natural product Cudraflavone B (CUB) has been reported to potentially kill a variety of tumor cells. Currently, its anit-cancer effect on GBM still remains unknown. Herein, we investigated whether CUB could affect the proliferation and apoptosis of GBM cells to show anti-GBM potential. RESULTS: CUB selectively inhibited cell viability and induced cell apoptosis by activating the endoplasmic reticulum stress (ER stress) related pathway, as well as harnessing the autophagy-related PI3K/mTOR/LC3B signaling pathway. Typical morphological changes of autophagy were also observed in CUB treated cells by microscope and scanning electron microscope (SEM) examination. 4-Phenylbutyric acid (4-PBA), an ER stress inhibitor, restored the CUB-caused alteration in signaling pathway and morphological change. CONCLUSIONS: Our finding suggests that CUB impaired cell growth and induced cell apoptosis of glioblastoma through ER stress and autophagy-related signaling pathways, and it might be an attractive drug for treatment of GBM.

Identification of natural compound garcinone E as a novel autophagic flux inhibitor with anticancer effect in nasopharyngeal carcinoma cells.

CONTEXT: Current chemotherapeutic drugs cannot meet the treatment needs of patients with nasopharyngeal carcinoma (NPC), so urgent action is needed to discover novel chemotherapeutic agents. Our previous study revealed that garcinone E (GE) inhibited the proliferation and metastasis of NPC, suggesting that the compound might display promising anticancer activity. OBJECTIVE: To examine the mechanism underlying the anti-NPC activity of GE for the first time. MATERIALS AND METHODS: For MTS assay, NPC cells were treated with 2.5-20 µmol/L GE or dimethyl sulfoxide for 24, 48, and 72 h. Colony formation capacity, cell cycle distribution, and in vivo xenograft experiment of GE were assessed. MDC staining, StubRFP-sensGFP-LC3 observation, LysoBrite Blue staining, and immunofluorescence examined the autophagy of NPC cells after GE exposure. Western blotting, RNA-sequencing, and RT-qPCR measured protein and mRNA levels. RESULTS: GE suppressed cell viability with an IC50 of 7.64, 8.83 and 4.65 µmol/L for HK1, HONE1 and S18 cells. GE inhibited colony formation and cell cycle, increased autophagosome number, and inhibited the autophagic flux partially by blocking lysosome-autophagosome fusion, and repressed S18 xenograft growth. GE dysregulated the expression of autophagy- and cell cycle-related proteins such as Beclin-1, SQSTM1/p62, LC3, CDKs, and Cyclins. Bioinformatics GO and KEGG pathway enrichment analysis of RNA-seq showed that autophagy was enriched in differentially expressed genes upon GE treatment. DISCUSSION AND CONCLUSION: GE acts as an autophagic flux inhibitor, which may have potential chemotherapeutic use for NPC treatment and may have an application in basic research to explore the mechanisms of autophagy.

NCALD affects drug resistance and prognosis by acting as a ceRNA of CX3CL1 in ovarian cancer.

Drug resistance, an impenetrable barrier in the treatment of ovarian cancer (OC), is often associated with poor outcomes. Hence, it is urgent to discover new factors controlling drug resistance and survival. The association between neurocalcin delta (NCALD) and cancer drug resistance is poorly understood. Here, we reveal that NCALD messenger RNA expression, probably regulated by DNA methylation and microRNAs, was significantly downregulated in at least three independent microarrays covering 633 ovarian carcinomas and 16 normal controls, which includes the Cancer Genome Atlas (TCGA) ovarian cohort. In the sub-groups of the TCGA cohort, NCALD was suppressed in 90 platinum-resistant tissues vs in 197 sensitive tissues. It is consistent with the quantitative reverse transcription polymerase chain reaction results revealing gene downregulation in carboplatin-resistant SKOV3 and HeyA8 OC cells as compared with that in controls. Low expression of NCALD predicted poor overall survival (OS) in sub-groups of 1656 patients, progression-free survival (PFS) in 1435 patients, and post-progression survival (PPS) in 782 patients according to Kaplan-Meier plotter covering 1815 OC patients. Comprehensive bioinformatic analyses strongly implicated NCALD in the regulation of drug resistance, probably via competing for endogenous RNA (ceRNA) interactions with CX3CL1 and tumor immune-microenvironment. NCALD acted as a ceRNA for CX3CL1 in 21 different cancers includes OC according to Starbase. These two genes negatively correlated with tumor purity and positively correlated with infiltration levels of neutrophils and dendritic cells in OC. The combined low expression of NCALD and CX3CL1 showed better prognosis potential for OS, PFS, and PPS in the 1815 OC patients than any of the individually tested genes. In summary, NCALD acts as a ceRNA for CX3CL1, and its downregulation may affect drug resistance and prognosis in OC. Thus, NCALD could be a new therapeutic target for anticancer therapy and a new biomarker for survival prediction in OC.

Microarray-based identification of genes associated with prognosis and drug resistance in ovarian cancer.

The outcome for patients with ovarian cancer (OC) is poor because of drug resistance. Therefore, identification of factors that affect drug resistance and prognosis in OC is needed. In the present study, we identified 131 genes significantly dysregulated in 90 platinum-resistant OC tissues compared with 197 sensitive tissues, of which 30 were significantly associated with disease-free survival (DFS; n = 16), overall survival (OS; n = 6), or both (n = 8) in 489 OC patients of the The Cancer Genome Atlas cohort. Of these 30 genes, 17 were significantly upregulated and 13 were downregulated in the 90 resistant tissues, and with one exception, all of the up-/downregulated genes in resistant tissues were predictors of shorter DFS or/and OS. LAX1, MECOM, and PDIA4 were independent risk factors for DFS, and KLF1, SLC7A11, and PDIA4 for OS; combining these genes provided more accurate predictions for DFS and OS than any of the genes used individually. We further verified downregulation of PDIA4 protein in 51 specimens of patients with OC (24 drug resistant's and 27 sensitive's), which confirmed that downregulated PDIA4 predicted DFS and OS. PDIA4 also consistently predicted OS in a larger sample of 1656 patients with OC. These 30 genes, particularly the PDIA4, could be therapeutic targets or biomarkers for managing OC.

Identification of Flower-Specific Promoters through Comparative Transcriptome Analysis in Brassica napus.

Brassica napus (oilseed rape) is an economically important oil crop worldwide. Sclerotinia stem rot (SSR) caused by Sclerotinia sclerotiorum is a threat to oilseed rape production. Because the flower petals play pivotal roles in the SSR disease cycle, it is useful to express the resistance-related genes specifically in flowers to hinder further infection with S. sclerotiorum. To screen flower-specific promoters, we first analyzed the transcriptome data from 12 different tissues of the B. napus line ZS11. In total, 249 flower-specific candidate genes with high expression in petals were identified, and the expression patterns of 30 candidate genes were verified by quantitative real-time transcription-PCR (qRT-PCR) analysis. Furthermore, two novel flower-specific promoters (FSP046 and FSP061 promoter) were identified, and the tissue specificity and continuous expression in petals were determined in transgenic Arabidopsis thaliana with fusing the promoters to ß-glucuronidase (GUS)-reporter gene. GUS staining, transcript expression pattern, and GUS activity analysis indicated that FSP046 and FSP061 promoter were strictly flower-specific promoters, and FSP046 promoter had a stronger activity. The two promoters were further confirmed to be able to direct GUS expression in B. napus flowers using transient expression system. The transcriptome data and the flower-specific promoters screened in the present study will benefit fundamental research for improving the agronomic traits as well as disease and pest control in a tissue-specific manner.

Syntenic quantitative trait loci and genomic divergence for Sclerotinia resistance and flowering time in Brassica napus.

Oilseed rape (Brassica napus) is an allotetraploid with two subgenomes descended from a common ancestor. Accordingly, its genome contains syntenic regions with many duplicate genes, some of which may have retained their original functions, whereas others may have diverged. Here, we mapped quantitative trait loci (QTL) for stem rot resistance (SRR), a disease caused by the fungus Sclerotinia sclerotiorum, and flowering time (FT) in a recombinant inbred line population. The population was genotyped using B. napus 60K single nucleotide polymorphism arrays and phenotyped in six (FT) and nine (SSR) experimental conditions or environments. In total, we detected 30 SRR QTL and 22 FT QTL and show that some of the major QTL associated with these two traits were co-localized, suggesting a genetic linkage between them. Two SRR QTL on chromosome A2 and two on chromosome C2 were shown to be syntenic, suggesting the functional conservation of these regions. We used the syntenic properties of the genomic regions to exclude genes for selection candidates responsible for QTL-associated traits. For example, 152 of the 185 genes could be excluded from a syntenic A2-C2 region. These findings will help to elucidate polyploid genomics in future studies, in addition to providing useful information for B. napus breeding programs.

The high-quality genome of Brassica napus cultivar 'ZS11' reveals the introgression history in semi-winter morphotype.

Allotetraploid oilseed rape (Brassica napus L.) is an agriculturally important crop. Cultivation and breeding of B. napus by humans has resulted in numerous genetically diverse morphotypes with optimized agronomic traits and ecophysiological adaptation. To further understand the genetic basis of diversification and adaptation, we report a draft genome of an Asian semi-winter oilseed rape cultivar 'ZS11' and its comprehensive genomic comparison with the genomes of the winter-type cultivar 'Darmor-bzh' as well as two progenitors. The integrated BAC-to-BAC and whole-genome shotgun sequencing strategies were effective in the assembly of repetitive regions (especially young long terminal repeats) and resulted in a high-quality genome assembly of B. napus 'ZS11'. Within a short evolutionary period (~6700 years ago), semi-winter-type 'ZS11' and the winter-type 'Darmor-bzh' maintained highly genomic collinearity. Even so, certain genetic differences were also detected in two morphotypes. Relative to 'Darmor-bzh', both two subgenomes of 'ZS11' are closely related to its progenitors, and the 'ZS11' genome harbored several specific segmental homoeologous exchanges (HEs). Furthermore, the semi-winter-type 'ZS11' underwent potential genomic introgressions with B. rapa (Ar ). Some of these genetic differences were associated with key agronomic traits. A key gene of A03.FLC3 regulating vernalization-responsive flowering time in 'ZS11' was first experienced HE, and then underwent genomic introgression event with Ar , which potentially has led to genetic differences in controlling vernalization in the semi-winter types. Our observations improved our understanding of the genetic diversity of different B. napus morphotypes and the cultivation history of semi-winter oilseed rape in Asia.

EST-based in silico identification and in vitro test of antimicrobial peptides in Brassica napus.

BACKGROUND: Brassica napus is the third leading source of vegetable oil in the world after soybean and oil palm. The accumulation of gene sequences, especially expressed sequence tags (ESTs) from plant cDNA libraries, has provided a rich resource for genes discovery including potential antimicrobial peptides (AMPs). In this study, we used ESTs including those generated from B. napus cDNA libraries of seeds, pathogen-challenged leaves and deposited in the public databases, as a model, to perform in silico identification and consequently in vitro confirmation of putative AMP activities through a highly efficient system of recombinant AMP prokaryotic expression. RESULTS: In total, 35,788 were generated from cDNA libraries of pathogen-challenged leaves and 187,272 ESTs from seeds of B. napus, and the 644,998 ESTs of B. napus were downloaded from the EST database of PlantGDB. They formed 201,200 unigenes. First, all the known AMPs from the AMP databank (APD2 database) were individually queried against all the unigenes using the BLASTX program. A total of 972 unigenes that matched the 27 known AMP sequences in APD2 database were extracted and annotated using Blast2GO program. Among these unigenes, 237 unigenes from B. napus pathogen-challenged leaves had the highest ratio (1.15 %) in this unigene dataset, which is 13 times that of the unigene datasets of B. napus seeds (0.09 %) and 2.3 times that of the public EST dataset. About 87 % of each EST library was lipid-transfer protein (LTP) (32 % of total unigenes), defensin, histone, endochitinase, and gibberellin-regulated proteins. The most abundant unigenes in the leaf library were endochitinase and defensin, and LTP and histone in the pub EST library. After masking of the repeat sequence, 606 peptides that were orthologous matched to different AMP families were found. The phylogeny and conserved structural motifs of seven AMPs families were also analysed. To investigate the antimicrobial activities of the predicted peptides, 31 potential AMP genes belonging to different AMP families were selected to test their antimicrobial activities after bioinformatics identification. The AMP genes were all optimized according to Escherichia coli codon usage and synthetized through one-step polymerase chain reaction method. The results showed that 28 recombinant AMPs displayed expected antimicrobial activities against E. coli and Micrococcus luteus and Sclerotinia sclerotiorum strains. CONCLUSION: The study not only significantly expanded the number of known/predicted peptides, but also contributed to long-term plant genetic improvement for increased resistance to diverse pathogens of B.napus. These results proved that the high-throughput method developed that combined an in silico procedure with a recombinant AMP prokaryotic expression system is considerably efficient for identification of new AMPs from genome or EST sequence databases.

ocsESTdb: a database of oil crop seed EST sequences for comparative analysis and investigation of a global metabolic network and oil accumulation metabolism.

BACKGROUND: Oil crop seeds are important sources of fatty acids (FAs) for human and animal nutrition. Despite their importance, there is a lack of an essential bioinformatics resource on gene transcription of oil crops from a comparative perspective. In this study, we developed ocsESTdb, the first database of expressed sequence tag (EST) information on seeds of four large-scale oil crops with an emphasis on global metabolic networks and oil accumulation metabolism that target the involved unigenes. DESCRIPTION: A total of 248,522 ESTs and 106,835 unigenes were collected from the cDNA libraries of rapeseed (Brassica napus), soybean (Glycine max), sesame (Sesamum indicum) and peanut (Arachis hypogaea). These unigenes were annotated by a sequence similarity search against databases including TAIR, NR protein database, Gene Ontology, COG, Swiss-Prot, TrEMBL and Kyoto Encyclopedia of Genes and Genomes (KEGG). Five genome-scale metabolic networks that contain different numbers of metabolites and gene-enzyme reaction-association entries were analysed and constructed using Cytoscape and yEd programs. Details of unigene entries, deduced amino acid sequences and putative annotation are available from our database to browse, search and download. Intuitive and graphical representations of EST/unigene sequences, functional annotations, metabolic pathways and metabolic networks are also available. ocsESTdb will be updated regularly and can be freely accessed at http://ocri-genomics.org/ocsESTdb/ . CONCLUSION: ocsESTdb may serve as a valuable and unique resource for comparative analysis of acyl lipid synthesis and metabolism in oilseed plants. It also may provide vital insights into improving oil content in seeds of oil crop species by transcriptional reconstruction of the metabolic network.

Genome-wide comparative analysis of NBS-encoding genes between Brassica species and Arabidopsis thaliana.

BACKGROUND: Plant disease resistance (R) genes with the nucleotide binding site (NBS) play an important role in offering resistance to pathogens. The availability of complete genome sequences of Brassica oleracea and Brassica rapa provides an important opportunity for researchers to identify and characterize NBS-encoding R genes in Brassica species and to compare with analogues in Arabidopsis thaliana based on a comparative genomics approach. However, little is known about the evolutionary fate of NBS-encoding genes in the Brassica lineage after split from A. thaliana. RESULTS: Here we present genome-wide analysis of NBS-encoding genes in B. oleracea, B. rapa and A. thaliana. Through the employment of HMM search and manual curation, we identified 157, 206 and 167 NBS-encoding genes in B. oleracea, B. rapa and A. thaliana genomes, respectively. Phylogenetic analysis among 3 species classified NBS-encoding genes into 6 subgroups. Tandem duplication and whole genome triplication (WGT) analyses revealed that after WGT of the Brassica ancestor, NBS-encoding homologous gene pairs on triplicated regions in Brassica ancestor were deleted or lost quickly, but NBS-encoding genes in Brassica species experienced species-specific gene amplification by tandem duplication after divergence of B. rapa and B. oleracea. Expression profiling of NBS-encoding orthologous gene pairs indicated the differential expression pattern of retained orthologous gene copies in B. oleracea and B. rapa. Furthermore, evolutionary analysis of CNL type NBS-encoding orthologous gene pairs among 3 species suggested that orthologous genes in B. rapa species have undergone stronger negative selection than those in B .oleracea species. But for TNL type, there are no significant differences in the orthologous gene pairs between the two species. CONCLUSION: This study is first identification and characterization of NBS-encoding genes in B. rapa and B. oleracea based on whole genome sequences. Through tandem duplication and whole genome triplication analysis in B. oleracea, B. rapa and A. thaliana genomes, our study provides insight into the evolutionary history of NBS-encoding genes after divergence of A. thaliana and the Brassica lineage. These results together with expression pattern analysis of NBS-encoding orthologous genes provide useful resource for functional characterization of these genes and genetic improvement of relevant crops.

Transcriptome profile analysis of young floral buds of fertile and sterile plants from the self-pollinated offspring of the hybrid between novel restorer line NR1 and Nsa CMS line in Brassica napus.

BACKGROUND: The fertile and sterile plants were derived from the self-pollinated offspring of the F1 hybrid between the novel restorer line NR1 and the Nsa CMS line in Brassica napus. To elucidate gene expression and regulation caused by the A and C subgenomes of B. napus, as well as the alien chromosome and cytoplasm from Sinapis arvensis during the development of young floral buds, we performed a genome-wide high-throughput transcriptomic sequencing for young floral buds of sterile and fertile plants. RESULTS: In this study, equal amounts of total RNAs taken from young floral buds of sterile and fertile plants were sequenced using the Illumina/Solexa platform. After filtered out low quality data, a total of 2,760,574 and 2,714,441 clean tags were remained in the two libraries, from which 242,163 (Ste) and 253,507 (Fer) distinct tags were obtained. All distinct sequencing tags were annotated using all possible CATG+17-nt sequences of the genome and transcriptome of Brassica rapa and those of Brassica oleracea as the reference sequences, respectively. In total, 3231 genes of B. rapa and 3371 genes of B. oleracea were detected with significant differential expression levels. GO and pathway-based analyses were performed to determine and further to understand the biological functions of those differentially expressed genes (DEGs). In addition, there were 1089 specially expressed unknown tags in Fer, which were neither mapped to B. oleracea nor to B. rapa, and these unique tags were presumed to arise basically from the added alien chromosome of S. arvensis. Fifteen genes were randomly selected and their expression levels were confirmed by quantitative RT-PCR, and fourteen of them showed consistent expression patterns with the digital gene expression (DGE) data. CONCLUSIONS: A number of genes were differentially expressed between the young floral buds of sterile and fertile plants. Some of these genes may be candidates for future research on CMS in Nsa line, fertility restoration and improved agronomic traits in NR1 line. Further study of the unknown tags which were specifically expressed in Fer will help to explore desirable agronomic traits from wild species.

Comprehensive analysis of RNA-seq data reveals the complexity of the transcriptome in Brassica rapa.

BACKGROUND: The species Brassica rapa (2n=20, AA) is an important vegetable and oilseed crop, and serves as an excellent model for genomic and evolutionary research in Brassica species. With the availability of whole genome sequence of B. rapa, it is essential to further determine the activity of all functional elements of the B. rapa genome and explore the transcriptome on a genome-wide scale. Here, RNA-seq data was employed to provide a genome-wide transcriptional landscape and characterization of the annotated and novel transcripts and alternative splicing events across tissues. RESULTS: RNA-seq reads were generated using the Illumina platform from six different tissues (root, stem, leaf, flower, silique and callus) of the B. rapa accession Chiifu-401-42, the same line used for whole genome sequencing. First, these data detected the widespread transcription of the B. rapa genome, leading to the identification of numerous novel transcripts and definition of 5'/3' UTRs of known genes. Second, 78.8% of the total annotated genes were detected as expressed and 45.8% were constitutively expressed across all tissues. We further defined several groups of genes: housekeeping genes, tissue-specific expressed genes and co-expressed genes across tissues, which will serve as a valuable repository for future crop functional genomics research. Third, alternative splicing (AS) is estimated to occur in more than 29.4% of intron-containing B. rapa genes, and 65% of them were commonly detected in more than two tissues. Interestingly, genes with high rate of AS were over-represented in GO categories relating to transcriptional regulation and signal transduction, suggesting potential importance of AS for playing regulatory role in these genes. Further, we observed that intron retention (IR) is predominant in the AS events and seems to preferentially occurred in genes with short introns. CONCLUSIONS: The high-resolution RNA-seq analysis provides a global transcriptional landscape as a complement to the B. rapa genome sequence, which will advance our understanding of the dynamics and complexity of the B. rapa transcriptome. The atlas of gene expression in different tissues will be useful for accelerating research on functional genomics and genome evolution in Brassica species.

A novel PCR-based method for high throughput prokaryotic expression of antimicrobial peptide genes.

BACKGROUND: To facilitate the screening of large quantities of new antimicrobial peptides (AMPs), we describe a cost-effective method for high throughput prokaryotic expression of AMPs. EDDIE, an autoproteolytic mutant of the N-terminal autoprotease, Npro, from classical swine fever virus, was selected as a fusion protein partner. The expression system was used for high-level expression of six antimicrobial peptides with different sizes: Bombinin-like peptide 7, Temporin G, hexapeptide, Combi-1, human Histatin 9, and human Histatin 6. These expressed AMPs were purified and evaluated for antimicrobial activity. RESULTS: Two or four primers were used to synthesize each AMP gene in a single step PCR. Each synthetic gene was then cloned into the pET30a/His-EDDIE-GFP vector via an in vivo recombination strategy. Each AMP was then expressed as an Npro fusion protein in Escherichia coli. The expressed fusion proteins existed as inclusion bodies in the cytoplasm and the expression levels of the six AMPs reached up to 40% of the total cell protein content. On in vitro refolding, the fusion AMPs was released from the C-terminal end of the autoprotease by self-cleavage, leaving AMPs with an authentic N terminus. The released fusion partner was easily purified by Ni-NTA chromatography. All recombinant AMPs displayed expected antimicrobial activity against E. coli, Micrococcus luteus and S. cerevisia. CONCLUSIONS: The method described in this report allows the fast synthesis of genes that are optimized for over-expression in E. coli and for the production of sufficiently large amounts of peptides for functional and structural characterization. The Npro partner system, without the need for chemical or enzymatic removal of the fusion tag, is a low-cost, efficient way of producing AMPs for characterization. The cloning method, combined with bioinformatic analyses from genome and EST sequence data, will also be useful for screening new AMPs. Plasmid pET30a/His-EDDIE-GFP also provides green/white colony selection for high-throughput recombinant AMP cloning.

Gene expression profiling of Sinapis alba leaves under drought stress and rewatering growth conditions with Illumina deep sequencing.

Sinapis alba has many desirable agronomic traits including tolerance to drought. In this investigation, we performed the genome-wide transcriptional profiling of S. alba leaves under drought stress and rewatering growth conditions in an attempt to identify candidate genes involved in drought tolerance, using the Illumina deep sequencing technology. The comparative analysis revealed numerous changes in gene expression level attributable to the drought stress, which resulted in the down-regulation of 309 genes and the up-regulation of 248 genes. Gene ontology analysis revealed that the differentially expressed genes were mainly involved in cell division and catalytic and metabolic processes. Our results provide useful information for further analyses of the drought stress tolerance in Sinapis, and will facilitate molecular breeding for Brassica crop plants.

USF1/CD90 signaling in maintaining glioblastoma stem cells and tumor-associated macrophages adhesion.

BACKGROUND: Glioblastoma stem cells (GSCs) and their interplay with tumor-associated macrophages (TAMs) are responsible for malignant growth and tumor recurrence of glioblastoma multiforme (GBM), but the underlying mechanisms are largely unknown. METHODS: Cell viability, stemness, migration, and invasion were measured in GSCs after the knockdown of upstream stimulating factor 1 (USF1). Luciferase assay and chromatin immunoprecipitation qPCR were performed to determine the regulation of CD90 by USF1. Immunohistochemistry and immunofluorescent staining were used to examine the expression of USF1 and GSC markers, as well as the crosstalk between GSCs and TAMs. In addition, the interaction between GSCs and TAMs was confirmed using in vivo GBM models. RESULTS: We show that USF1 promotes malignant glioblastoma phenotypes and GSCs-TAMs physical interaction by inducing CD90 expression. USF1 predicts a poor prognosis for glioma patients and is upregulated in patient-derived GSCs and glioblastoma cell lines. USF1 overexpression increases the proliferation, invasion, and neurosphere formation of GSCs and glioblastoma cell lines, while USF1 knockdown exerts an opposite effect. Further mechanistic studies reveal that USF1 promotes GSC stemness by directly regulating CD90 expression. Importantly, CD90 of GSCs functions as an anchor for physical interaction with macrophages. Additionally, the USF1/CD90 signaling axis supports the GSCs and TAMs adhesion and immunosuppressive feature of TAMs, which in turn enhance the stemness of GSCs. Moreover, the overexpression of CD90 restores the stemness property in USF1 knockdown GSCs and its immunosuppressive microenvironment. CONCLUSIONS: Our findings indicate that the USF1/CD90 axis might be a potential therapeutic target for the treatment of glioblastoma.

Analysis of expression sequence tags from a full-length-enriched cDNA library of developing sesame seeds (Sesamum indicum).

BACKGROUND: Sesame (Sesamum indicum) is one of the most important oilseed crops with high oil contents and rich nutrient value. However, genetic improvement efforts in sesame could not get benefit from molecular biology technology due to poor DNA and RNA sequence resources. In this study, we carried out a large scale of expressed sequence tags (ESTs) sequencing from developing sesame seeds and further conducted analysis on seed storage products-related genes. RESULTS: A normalized and full-length enriched cDNA library from 5 ~ 30 days old immature seeds was constructed and randomly sequenced, leading to generation of 41,248 expressed sequence tags (ESTs) which then formed 4,713 contigs and 27,708 singletons with 44.9% uniESTs being putative full-length open reading frames. Approximately 26,091 of all these uniESTs have significant matches to the counterparts in Nr database of GenBank, and 21,628 of them were assigned to one or more Gene ontology (GO) terms. Homologous genes involved in oil biosynthesis were identified including some conservative transcription factors regulating oil biosynthesis such as LEAFY COTYLEDON1 (LEC1), PICKLE (PKL), WRINKLED1 (WRI1) and majority of them were found for the first time in sesame seeds. One hundred and 17 ESTs were identified possibly involved in biosynthesis of sesame lignans, sesamin and sesamolin. In total, 9,347 putative functional genes from developing seeds were identified, which accounts for one third of total genes in the sesame genome. Further analysis of the uniESTs identified 1,949 non-redundant simple sequence repeats (SSRs). CONCLUSIONS: This study has provided an overview of genes expressed during sesame seed development. This collection of sesame full-length cDNAs covered a wide variety of genes in seeds, in particular, candidate genes involved in biosynthesis of sesame oils and lignans. These EST sequences enriched with full length will contribute to comparative genomic studies on sesame and other oilseed plants and serve as an abundant information platform for functional marker development and functional gene study.

Real-time measurement of repetitive micro bulk motion vector and motion noise removal in optical coherence tomography angiography.

In this work, we developed a motion estimation and correction method which real-time obtained the direction and displacement of repetitive micro bulk motion (such as cardiac and respiratory motion) on an SS-OCT system without additional tracking hardware, and reduced the motion noise in optical coherence tomography angiography (OCTA). In the approach, the direction of repetitive micro bulk motion was considered fixed, and proportional relationships between the motion components in three directions were determined; Then we performed one-dimension cross-correlation to obtain depth displacement which was further used to obtain other two motion components, and greatly reduced the computation; The processing speed on a graphic processing unit was 478 pairs of B-Scans per second, and the measurement range was larger than the range of the angiogram-based methods. Lastly, corrupt angiograms were recovered by adaptive scan protocol, and reduced acquisition time in comparison with the previous work.

Low Expression of SLC7A11 Confers Drug Resistance and Worse Survival in Ovarian Cancer via Inhibition of Cell Autophagy as a Competing Endogenous RNA.

Drug resistance is the main cause of chemotherapy failure in ovarian cancer (OC), and identifying potential druggable targets of autophagy is a novel and promising approach to overcoming drug resistance. In this study, 131 genes associated with autophagy were identified from three autophagy-related databases, and of these, 14 were differentially expressed in 90 drug-resistant OC tissues versus 197 sensitive tissues according to the Cancer Genome Atlas ovarian cancer cohort. Among these 14 genes, SLC7A11 was significantly decreased in two paclitaxel-resistant OC cells (HeyA8-R and SKOV3-R) and in 90 drug-resistant tissues compared with their controls. In vitro overexpression of SLC7A11 significantly increased the sensitivity of HeyA8-R cells to paclitaxel, inhibited colony formation, induced apoptosis, and arrested cell cycle. Further, low SLC7A11 expression was correlated with poor overall survival (OS), progression-free survival (PFS), and post-progression survival (PPS) in 1815 OC patients. Mechanistically, SLC7A11 strongly regulated cell autophagy as a competing endogenous RNA (ceRNA) based on pan-cancer analyses of 32 tumor types. Specifically, as a ceRNA for autophagy genes STX17, RAB33B, and UVRAG, SLC7A11 was strongly and positively co-expressed with these three genes in 20, 12, and 12 different tumors, respectively, in 379 OC tissues and in 90 drug-resistant OC tissues, and the former two were significantly upregulated in SLC7A11-overexpressed HeyA8-R cells. Further, SLC7A11 induced the protein expression of other autophagy genes, such as LC3, Atg16L1, and Atg7, and the expression of the respective proteins was further increased when the cells were treated with paclitaxel. The results strongly suggest that SLC7A11 regulates autophagy via ceRNA interactions with the three abovementioned genes in pan-cancer and in drug-resistant OC. Moreover, low expression of STX17 and UVRAG also significantly predicted low OS, PFS, and PPS. The combination of SLC7A11 with STX17 was more predictive of OS and PFS than either individually, and the combination of SLC7A11 with UVRAG was highly predictive of OS and PPS. The above results indicated that decreased SLC7A11 resulted in drug resistance and effected low rates of survival in OC patients, probably via ceRNA interactions with autophagy genes, and thus the gene could serve as a therapeutic target and potential biomarker in OC.

PPFIBP1 induces glioma cell migration and invasion through FAK/Src/JNK signaling pathway.

Glioblastoma multiforme (GBM) is the most aggressive brain tumor, with a 5-year survival ratio <5%. Invasive growth is a major determinant of the poor prognosis in GBM. In this study, we demonstrate that high expression of PPFIA binding protein 1 (PPFIBP1) correlates with remarkable invasion and poor prognosis of GBM patients. Using scratch and transwell assay, we find that the invasion and migration of GBM cells are promoted by overexpression of PPFIBP1, while inhibited by knockdown of PPFIBP1. Then, we illustrate that overexpression of PPFIBP1 facilitates glioma cell infiltration and reduces survival in xenograft models. Next, RNA-Seq and GO enrichment analysis reveal that PPFIBP1 regulates differentially expressed gene clusters involved in the Wnt and adhesion-related signaling pathways. Furthermore, we demonstrate that PPFIBP1 activates focal adhesion kinase (FAK), Src, c-Jun N-terminal kinase (JNK), and c-Jun, thereby enhancing Matrix metalloproteinase (MMP)-2 expression probably through interacting with SRCIN1 (p140Cap). Finally, inhibition of phosphorylation of Src and FAK significantly reversed the augmentation of invasion and migration caused by PPFIBP1 overexpression in GBM cells. In conclusion, these findings uncover a novel mechanism of glioma invasion and identify PPFIBP1 as a potential therapeutic target of glioma.

Brassica GLABRA2 genes: analysis of function related to seed oil content and development of functional markers.

Regulation of seed oil accumulation in oilseed rape (Brassica napus) has important economic significance. However, few genes have been characterized that affect final seed oil content. Through a mutant identification, the class IV homeodomain-ZIP transcription factor GLABRA2 (GL2) has been found to regulate seed oil accumulation in Arabidopsis, in addition to its role in trichome development. In this study, we isolated four distinct orthologues of GL2 from B. napus (AC-genome), B. rapa (A) and B. oleracea (C), using an overlapping-PCR strategy. The four GL2 orthologues were very similar, with 96.10-99.69% identity in exon regions, 75.45-93.84% in intron regions, 97.34-99.87% in amino acid sequences. Alignments of the four genes revealed that the A-genome sequences of BnaA.GL2.a from B. napus and BraA.GL2.a from B. rapa are more similar than the others, and likewise the C-genome sequences of BnaC.GL2.b from B. napus and BolC.GL2.a from B. oleracea are more similar. BnaA.GL2.a and BraA.GL2.a from the A-genome are highly expressed in roots, whilst BnaC.GL2.b and BolC.GL2.a from the C-genome are preferentially expressed in seeds. Transgenic ectopic overexpression and suppression of BnaC.GL2.b in Arabidopsis allowed further investigation of the effect on seed oil content. Overexpression generated two phenotypes: the wild-type-like and the gl2-mutant-like (an Arabidopsis glabrous mutant of gl2-2), with increases in seed oil content of 3.5-5.0% in the gl2-mutant-like transgenic plants. Suppression resulted in increases of 2.5-6.1% in seed oil content, and reduced trichome number at the leaf margins. These results suggest that BnaC.GL2.b can negatively regulate oil accumulation in Arabidopsis seeds. As a result of comparing the four GL2 genes, three A/C-genome-specific primer sets were developed and a C-genome-specific EcoRV cleavage site was identified, which can be used as functional markers to distinguish these orthologues within Brassica species. The genes identified and their molecular markers developed in this study will be valuable both for oilseed rape breeding focusing on improvement of seed oil content, and for detecting gene flow between populations.