Project description:Spodoptera frugiperda is a notorious agricultural pest in its caterpillar form. As most Lepidoptera, its genome is organized in 31 chromosomes with dispersed centromeres instead of a single one per chromosome. This property is called holocentrism and is found in several animal groups as well as some plants. In the best known monocentric species, the pericentromeric region is abundant in repeat elements and is usually constituted of a large heterochromatin block. Thus we wanted to address the localization of euchromatin and heterochromatin in Sf9 cell line by using 2 markers corresponding to a tri-methylation of H3 on the lysine 4 (H3K4me3) or di-methylation on lysine 9 (H3K9me2). ChIP-seq on Sf9 cells have been performed using specific antibodies and compared to an Input sample.
Project description:Nematodes such as Steinernema carpocapsae are used as organic pesticides because of their ability to prey on live insects. They do so thanks to their symbiotic bacteria, that they release within the hemocoel of insects. In this study we wanted to study how Spodoptera frugiperda, a Lepidoptera pest of crops becoming invasive around the world, defend themselves against the nematodes. We infested S. frugiperda larvae with nematodes and dissected three tissues: the midgut, the fat body and the hemocytes at two time-points: 8 h and 15 h after infestation. We performed single-end RNA-seq on these samples to study the tissue specificity of S. fru response, as well as its dynamic.
Project description:We developed a protein microarray to identify autoantigens in Systemic Lupus Erythematosus (SLE). Baculovirus-Sf9 cell expression system was used to create a protein microarray with 1543 full-length human proteins expressed with a biotin carboxyl carrier protein (BCCP) folding tag. We assayed sera from UK and USA SLE individuals (total n=277), age/ancestry matched control cohorts (n=280) and a confounding disease cohort (n=92).
Project description:MicroRNAs (miRNAs) as small non-coding RNAs play important roles in many biological processes such as development, cell signalling and immune response. Studies also suggest that miRNAs are important in host–virus interactions where the host limits virus infection by differentially expressing miRNAs that target essential viral genes. Here, we identified conserved and new miRNAs from Spodoptera frugiperda cells (Sf9) using a combination of deep sequencing and bioinformatics as well as experimental approaches. S. frugiperda miRNAs share common features of miRNAs in other organisms, such as uracil (U) at the 59 end of miRNA. The 59 ends of the miRNAs were more conserved than the 39 ends, revealing evolutionary protection of the seed region in miRNAs. The predominant miRNAs were found to be conserved among arthropods. The majority of homologous miRNAs were found in Bombyx mori, with 76 of the 90 identified miRNAs. We found that seed shifting and arm switching have happened in this insect’s miRNAs. Expression levels of the majority of miRNAs changed following baculovirus infection. Results revealed that baculovirus infection mainly led to an overall suppression of cellular miRNAs. We found four different genes being regulated by sfr-miR-184 at the post-transcriptional level. The data presented here further support conservation of miRNAs in insects and other organisms. In addition, the results reveal a differential expression of host miRNAs upon baculovirus infection, suggesting their potential roles in host–virus interactions. Seed shifting and arm switching happened during evolution of miRNAs in different insects and caused miRNA diversification, which led to changes in the target repository of miRNAs. Identification of miRNA and other small non coding RNA in NPV infected Sf9 cells
Project description:PTEN is frequently mutated in a wide range of malignancies. Beyond suppressing tumorigenesis, PTEN is involved in multiple biological processes, and the complexity of PTEN function is partially attributed to PTEN family members including PTENα and PTENβ. Here, we report the identification of PTENε (also named as PTEN5), a novel N-terminal-extended PTEN isoform that suppresses tumor invasion and metastasis. We show that the translation of PTENε is initiated from the CUG816 codon within the 5’UTR region of PTEN mRNA. PTENε mainly localizes in the cell membrane, and physically associates with and dephosphorylates VASP and ACTR2, which govern filopodia formation and cell motility. We found that endogenous depletion of PTENε promotes filopodia formation and enhances the metastasis capacity of tumor cells. Overall, we identify a new isoform of PTEN with distinct localization and function compared to the known members of the PTEN family. These findings enrich the PTEN family constitution and advance our current understanding of the importance and diversity of PTEN functions.
Project description:Human papilloma (HPV) virus-like particle (VLP) vaccines were recently licensed. Though neutralizing antibody titers are thought to be the main effectors of protection against infection, early predictors of long-term efficacy are not yet defined and a comprehensive understanding of innate and adaptive immune responses to vaccination is still lacking. Here, microarrays were used to compare the gene expression signature in HPV-16 L1 VLP-stimulated PBMC from 17 vaccine and 4 placebo recipients before vaccination, and 1 month after receiving the second immunization. Vaccination with HPV-16 L1 VLP was associated with modulation of genes involved in the inflammatory/defense response, cytokine, interferon and cell cycle pathways in VLP-stimulated PBMC. In addition, there was up-regulation of probesets associated with cytotoxic (GZMB, TNFSF10) and regulatory (INDO, CTLA4) activities. The strongest correlations with neutralizing antibody titers were found for cyclin d2 (CCND2) and galectin (LGALS2). Twenty-two differentially expressed probesets were selected for confirmation by RT-PCR in an independent sample set. Agreement with the microarray data was seen for over two-thirds of these probesets. Up-regulation of immune/defense response genes by VLP, in particular interferon-induced genes was observed in PBMC collected prior to vaccination, with many of these genes being further induced following vaccination. In conclusion, we used gene expression profiling to identify important innate and adaptive response related- genes induced by vaccination with HPV VLP. Further studies are needed to identify gene expression signatures of immunogenicity and long-term protection with potential utility in prediction of long-term HPV vaccination outcomes in clinical trials. Experiment Overall Design: Microarrays (Affymetrix Human Focus) were used to compare the gene expression signature in PBMCs stimulated for 3 days with media alone, Sf9/baculovirus insect cell lysate, and HPV-16 L1 VLP expressed from baculovirus-infected Sf9 insect cells from 17 vaccine and 4 placebo recipients before vaccination, and 1 month after receiving the second immunization (2 months after the initial immunization. Post-vaccination baculovirus sample for Pt078 and pre-vaccination baculovirus sample for Pt082 failed QC.
Project description:Cervical cancer (CC) is the fourth leading cause of deaths in gynecological malignancies. Although the etiology of CC has been extensively investigated, the exact pathogenesis of CC remains incomplete. Recently, single-cell technologies demonstrated advantages in exploring intra-tumoral diversification among various tumor cells. However, single-cell transcriptome (scRNA-seq) analysis of CC cells and microenvironment has not been conducted. In this study, a total of 6 samples (3 CC and 3 adjacent normal tissues) were examined by scRNA-seq. Here, we performed single-cell RNA sequencing (scRNA-seq) to survey the transcriptomes of 57,669 cells derived from three CC tumors with paired normal adjacent non-tumor (NAT) samples. Single-cell transcriptomics analysis revealed extensive heterogeneity in malignant cells of human CCs, wherein epithelial subpopulation exhibited different genomic and transcriptomic signatures. We also identified cancer-associated fibroblasts (CAF) that may promote tumor progression of CC, and further distinguished inflammatory CAF (iCAF) and myofibroblastic CAF (myCAF). CD8+ T cell diversity revealed both proliferative (MKI67+) and non-cycling exhausted (PDCD1+) subpopulations at the end of the trajectory path. We used the epithelial signature genes derived from scRNA-seq to deconvolute bulk RNA-seq data of CC, identifying four different CC subtypes, namely hypoxia (S-H subtype), proliferation (S-P subtype), differentiation (S-D subtype), and immunoactive (S-I subtype) subtype. Our results lay the foundation for precision prognostic and therapeutic stratification of CC.
Project description:Cervical cancer (CC) is one of the most common malignancy in women worldwide. It is characterized by a natural continuous phenomenon, that is, it is in the initial stage of HPV infection, progresses to intraepithelial neoplasia, and then develops into invasion and metastasis. Determining the complexity of tumor microenvironment (TME) can deepen our understanding of lesion progression and provide novel therapeutic strategies for CC. We performed the single-cell RNA sequencing on the normal cervix, intraepithelial neoplasia, primary tumor and metastatic lymph node tissues to describe the composition, lineage, and functional status of immune cells and mesenchymal cells at different stages of CC progression. A total of 59913 single cells were obtained and divided into 9 cellular clusters, including immune cells (T/NK cells, macrophages, B cells, plasma cells, mast cells and neutrophils) and mesenchymal cells (endothelial cells, smooth muscle cells and fibroblasts). Our results showed that there were distinct cell subpopulations in different stages of CC. High-stage intraepithelial neoplasia (HSIL) tissue exhibited a low, recently activated TME, and it was characterized by high infiltration of tissue-resident CD8 T cell, effector NK cells, Treg, DC1, pDC, and M1-like macrophages. Tumor tissue displayed high enrichment of exhausted CD8 T cells, resident NK cells and M2-like macrophages, suggesting immunosuppressive TME. Metastatic lymph node consisted of naive T cell, central memory T cell, circling NK cells, cytotoxic CD8+ T cells and effector memory CD8 T cells, suggesting an early activated phase of immune response. This study is the first to delineate the transcriptome profile of immune cells during CC progression using single-cell RNA sequencing. Our results indicated that HSIL exhibited a low, recently activated TME, tumor displayed immunosuppressive statue, and metastatic lymph node showed early activated phase of immune response. Our study enhanced the understanding of dynamic change of TME during CC progression and has implications for the development of novel treatments to inhibit the initiation and progression of CC.
Project description:RNA-Seq has been increasingly used for the quantification and characterization of transcriptomes. The ongoing development of the technology promises the more accurate measurement of gene expression. However, its benefits over widely accepted microarray technologies have not been adequately assessed, especially in toxicogenomics studies. The goal of this study is to enhance the scientific community's understanding of the advantages and challenges of RNA-Seq in the quantification of gene expression by comparing analysis results from RNA-Seq and microarray data on a toxicogenomics study. A typical toxicogenomics study design was used to compare the performance of an RNA-Seq approach (Illumina Genome Analyzer II) to a microarray-based approach (Affymetrix Rat Genome 230 2.0 arrays) for detecting differentially expressed genes (DEGs) in the kidneys of rats treated with aristolochic acid (AA), a carcinogenic and nephrotoxic chemical most notably used for weight loss. We studied the comparability of the RNA-Seq and microarray data in terms of absolute gene expression, gene expression patterns, differentially expressed genes, and biological interpretation. We found that RNA-Seq was more sensitive in detecting genes with low expression levels, while similar gene expression patterns were observed for both platforms. Moreover, although the overlap of the DEGs was only 40-50%, the biological interpretation was largely consistent between the RNA-Seq and microarray data. RNA-Seq maintained a consistent biological interpretation with time-tested microarray platforms while generating more sensitive results. However, there is clearly a need for future investigations to better understand the advantages and limitations of RNA-Seq in toxicogenomics studies and environmental health research. Eight rats were randomly divided into two groups: four rats were administered with aristolochic acid (AA), and four rats were treated with the control vehicle. RNA samples were extracted from the kidney tissue of each rat and were independently assayed with both the NGS (Illumina Genome Analyzer II) and the microarray (Affymetrix Rat Genome 230 2.0) platforms. The RNA-Seq and microarray data were compared in terms of absolute gene expression, gene expression patterns, differentially expressed genes, and biological interpretation.