Peanut (Arachis hypogaea) Expressed Sequence Tag Project: Progress and Application

Many plant ESTs have been sequenced as an alternative to whole genome sequences, including peanut because of the genome size and complexity. The US peanut research community had the historic 2004 Atlanta Genomics Workshop and named the EST project as a main priority. As of August 2011, the peanut research community had deposited 252,832 ESTs in the public NCBI EST database, and this resource has been providing the community valuable tools and core foundations for various genome-scale experiments before the whole genome sequencing project. These EST resources have been used for marker development, gene cloning, microarray gene expression and genetic map construction. Certainly, the peanut EST sequence resources have been shown to have a wide range of applications and accomplished its essential role at the time of need. Then the EST project contributes to the second historic event, the Peanut Genome Project 2010 Inaugural Meeting also held in Atlanta where it was decided to sequence the entire peanut genome. After the completion of peanut whole genome sequencing, ESTs or transcriptome will continue to play an important role to fill in knowledge gaps, to identify particular genes and to explore gene function

DPHL: A DIA Pan-human Protein Mass Spectrometry Library for Robust Biomarker Discovery

To address the increasing need for detecting and validating protein biomarkers in clinical specimens, mass spectrometry (MS)-based targeted proteomic techniques, including the selected reaction monitoring (SRM), parallel reaction monitoring (PRM), and massively parallel data-independent acquisition (DIA), have been developed. For optimal performance, they require the fragment ion spectra of targeted peptides as prior knowledge. In this report, we describe a MS pipeline and spectral resource to support targeted proteomics studies for human tissue samples. To build the spectral resource, we integrated common open-source MS computational tools to assemble a freely accessible computational workflow based on Docker. We then applied the workflow to generate DPHL, a comprehensive DIA pan-human library, from 1096 data-dependent acquisition (DDA) MS raw files for 16 types of cancer samples. This extensive spectral resource was then applied to a proteomic study of 17 prostate cancer (PCa) patients. Thereafter, PRM validation was applied to a larger study of 57 PCa patients and the differential expression of three proteins in prostate tumor was validated. As a second application, the DPHL spectral resource was applied to a study consisting of plasma samples from 19 diffuse large B cell lymphoma (DLBCL) patients and 18 healthy control subjects. Differentially expressed proteins between DLBCL patients and healthy control subjects were detected by DIA-MS and confirmed by PRM. These data demonstrate that the DPHL supports DIA and PRM MS pipelines for robust protein biomarker discovery. DPHL is freely accessible at https://www.iprox.org/page/project.html?id=IPX0001400000

Acetyl-CoA Synthetase 2 Promotes Cell Migration and Invasion of Renal Cell Carcinoma by Upregulating Lysosomal-Associated Membrane Protein 1 Expression

Background/Aims: Reprogramming energy metabolism is an emerging hallmark of many cancers, and this alteration is especially evident in renal cell carcinomas (RCCs). However, few studies have been conducted on lipid metabolism. This study investigated the function and mechanism of lipid metabolism-related acetyl-CoA synthetase 2 (ACSS2) in RCC development, cell migration and invasion. Methods: Quantitative real-time PCR (qRT-PCR) was used to determine the expression of ACSS2 in cancer tissue and adjacent tissue. The inhibition of ACSS2 expression was achieved by RNA interference, which was confirmed by qRT-PCR and Western blotting. Cell proliferation and apoptosis were detected by a CCK8 assay and a flow cytometry analysis, respectively. Cell migration and invasion were determined by the scratch and transwell assays. Following the knockdown of ACSS2 expression, the expression of the autophagy-related factor LAMP1 was measured by qRT-PCR and Western blotting. Results: Compared to adjacent tissues, ACSS2 expression was upregulated in RCC cancer tissues and positively correlated with metastasis. Inhibition of ACSS2 had no effect on RCC cell proliferation or apoptosis. However, decreased ACSS2 expression was found to inhibit RCC cell migration and invasion. ACSS2 was determined to promote the expression of LAMP1, which can also promote cell migration. This pathway may be considered a potential mechanism through which ACSS2 participates in RCC development. Conclusion: These data suggest that ACSS2 is an important factor for promoting RCC development and is essential for cell migration and invasion, which it promotes by increasing the expression of LAMP1. Taken together, these findings reveal a potential target for the diagnosis and treatment of RCC

Rat bone marrow derived mesenchymal progenitor cells support mouse ES cell growth and germ-like cell differentiation

Mouse embryonic fibroblasts (MEFs) have been used as feeder cells to support the growth of mouse embryonic stem cell (mESC) and primordial germ cells (PGC) in culture for many years. However, MEF preparation is a complex and tedious task. Recently, there are reports indicating that the microenvironment provided by bone marrow stromal cells could support the survival of embryonic-like stem cells in bone marrow. In this report, rat bone marrow derived mesenchymal progenitor cells (MPC) were used as feeder cells to culture mouse Oct4-GFP ES cell and ES cell derived germ cells. FACS results show that similar to MEF, rat MPC could efficiently support growth of the mouse Oct4-GFP ES cell line in culture (MPC 85.5 +/- 5.1\% vs MEF 84.1 +/- 6.2\%). ES cells could be subcultured for > 15 passages without losing morphological characteristics. The cultured cells expressed stem cell marker alkaline phosphatase, Oct4, Sox2, and SSEA-1. Furthermore, rat MPC cells were able to support survival of germ cells isolated from mouse Oct4-GFP ES cell formed embryoid bodies (EB). After induction by retinoic acid for 7 days, some isolated cells differentiated to spermatogonial stem-like cells, expressing Mvh, Stra-8, Hsp90-alpha, integrin beta 1 and alpha 6. Compared with traditional MEF culture systems, the rat MPC culture system is effective in supporting ES cell growth and is easy to prepare. (C) 2009 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved

Identification and characteristics of a novel testis-specific gene, Tsc24, in human and mice

Testis-specific genes are essential for the spermatogenesis in mammalian male reproduction. We have identified a novel gene, Tsc24, from the results of the Affymetrix Genechip analysis in the developmental stage of days 4, 9, 18, 35, 54 and 6 months of postnatal Balb/C mouse testis. The scaling signal intensities of Tsc24 in the six stages of mouse testis showed that the expression of Tsc24 was not detected on day 4, 9 or 18 but on day 35, 54 and 6 months. The full cDNA length of mouse Tsc24 was 899 bp, with a 624 bp open reading frame encoding a 207 amino acid protein with a predicted molecular weight of 23.997 kDa. The results of semi-quantitative RTPCR showed that the expression of mouse Tsc24 can only be detected after the mouse was 35 d old and the expressing level increased gradually from day 35 to 6 months. Of the eight tissues (liver, spleen, heart, lung, brain, kidney, epididymis, and testis) examined in mice, and of the 12 tissues (liver, kidney, muscle, brain, spleen, adipose, lung, heart, epididymis, testis, ovary and uterus) examined in human, Tsc24 was exclusively expressed in the testis, but in none of the other studied tissues. The result of subcellular localization of GFP-Tsc24 fusion protein in Cos-7 cells supports that Tsc24 protein is expressed in nuclear. Our study should be a basis for function characterization of the Tsc24 gene, leading to the elucidation of the molecular events underlying mammalian male reproduction

Ascorbate antagonizes nickel ion to regulate JMJD1A expression in kidney cancer cells

Abnormal expression of histone demethylase Jumonji domain-containing protein 1A (JMJD1A) is associated with many kinds of cancers. JMJD1A is also a hypoxic response gene and its expression is regulated by hypoxia-inducible factor-1 (HIF-1). In this study, we determined the role of JMJD1A in development and hypoxia pathway. We also measured the expression of JMJD1A and two hypoxia factors glucose transporter 1 (GLUT1) and vascular endothelial growth factor (VEGF) in 786-0 and HEK293 cells treated with different concentrations of NiCl2 (2.5100 M) for 24 h, and found that JMJD1A mRNA and protein were up-regulated with increased concentrations of NiCl2. We then observed that ascorbate could retard the up-regulated effect of NiCl2-induced JMJD1A expression in a dose-dependent manner through decreasing the stability of HIF-1 protein. Immunohistochemical analysis further demonstrated ascorbate antagonized Ni-2-induced up-regulation of JMJD1A expression in 786-0, HEK293, and OS-RC-2 cells. These findings suggest that both Ni-2 and ascorbate can regulate the expression of histone demethylase JMJD1A, which is important for cancer development or inhibition

Transcriptome Analysis of Liver Tissue of Cipangopaludina cathayensis Under Hypoxic Stress

Cipangopaludina cathayensis is a snail species unique to China. Domestic research on C. cathayensis has mainly focused on aquaculture technology, especially the paddy field breeding method, where water quality plays a critical role in the cultivation of C. cathayensis. Dissolved oxygen is one of the most important factors in the aquatic environment because it impacts a series of biological activities such as the growth and development, metabolism, reproduction, and breeding of aquatic animals. When the dissolution of oxygen in water is less than 2 mg/L, the water is in a low oxygen or anoxic state. Hypoxia can slow the growth and development of aquatic animals, reduce their disease resistance and reproductive ability, and, in serious cases, can lead to their death. In recent years, research on C. cathayensis has mainly focused on the anti-tumor mechanism, immune response, nutritional value evaluation, and antibiotic resistance. However, there is currently no report on the regulation of response to hypoxia in C. cathayensis either domestically or internationally. The purpose of this study was to explore the differential expression of genes in the liver of C. cathayensis under hypoxic stress.In this study, healthy C. cathayensis without mechanical damage were cultured in a hypoxia stress group (2.5 mg/L) and a normoxia (control) group (6.9 mg/L), with 90 C. cathayensis in each group and 3 replicates. For the low oxygen stress treatment, dissolved oxygen was decreased from 6.9 mg/L to 2.5 mg/L within 1 h and maintained for 24 h. The liver tissue was taken as the experimental material in both the hypoxia stress group and normoxic group. The total RNA was extracted and an mRNA library was constructed. The liver tissue samples of C. cathayensis from both groups were sequenced and analyzed using an Illumina HiSeq-2500 technology platform, and unigenes were compared and annotated in GO, KOG, Nr, and KEGG databases. The differentially expressed genes were analyzed using DESeq. Bioinformatics analysis was performed on the function of GO and KEGG of differentially expressed genes, and the key differentially expressed genes were further validated by qPCR.Transcriptome analysis results showed that 500 584 transcripts were assembled from the original data and 23 379 unigenes were obtained by sequencing, with an average length of 686.65 bp and N50 of 1 127 bp. Among the unigenes, 26 636 were found to be homologous to genes in the Nr protein database. Additionally, 22 907 unigenes were annotated in the GO database, 13 290 in the KOG database, and at least 4 179 in the KEGG database. Compared with the control group, 176 differentially expressed genes were screened in the hypoxia stress group, among which 64 and 112 were up- and down-regulated, respectively. Further, GO functional enrichment analysis found that the differential genes were mainly enriched in chitin metabolic and glucosamine-containing compound metabolic processes in the biological process. Differential genes were also enriched in collagen trimer in the cellular component and chitin binding in the molecular function. The enrichment analysis results of the KEGG pathway mainly focused on four pathway categories, namely environmental information processing, genetic information processing, metabolism, and organismal systems. Finally, the qPCR results of six key differentially expressed genes were obtained by RT-qPCR. Among the up-regulated genes under hypoxic stress were the heat shock proteins 70B2 and beta-6, and the down-regulated genes were chitinase-like protein 4, collagen alpha-1 chain (XIV), collagen alpha-4 chain (XIV), and phosphatase-related protein type 5, which confirmed the reliability of the transcriptome sequencing results.Studies have shown that, through transcriptome sequencing, the expression information of relevant functional genes in C. cathayensis liver tissues under hypoxic stress can be obtained. Among them, the expression of heat shock protein genes were up-regulated, indicating that hypoxic stress activated the physiological activities of C. cathayensis to adapt to hypoxia and protected the body from hypoxic damage. In addition, the down-regulated expression of related genes in the metabolic pathway indicates that the growth of C. cathayensis is affected under hypoxic environments. In conclusion, the results of this study provide basic data and a theoretical basis for the in-depth study of the regulatory mechanism of C. cathayensis in response to hypoxic stress