Transcript Database Research Articles

Differences in renal cortex transcriptional profiling of wild-type and novel type B cystinuria model rats

Cystinuria is a genetic disorder of cystine transport that accounts for 1–2% of all cases of renal lithiasis. It is characterized by hyperexcretion of cystine in urine and recurrent cystine lithiasis. Defective transport of cystine into epithelial cells of renal tubules occurs because of mutations of the transport heterodimer, including protein b0,+AT (encoded by SLC7A9) and rBAT (encoded by SLC3A1) linked through a covalent disulfide bond. Study generated a novel type B cystinuria rat model by artificially deleting 7 bp of Slc7a9 gene exon 3 using the CRISPR-Cas9 system, and those Slc7a9-deficient rats were proved to be similar with cystinuria in terms of genome, transcriptome, translation, and biologic phenotypes with no off-target editing. Subsequent comparisons of renal histopathology indicated model rats gained typical secondary changes as medullary fibrosis with no stone formation. A total of 689 DEGs (383 upregulated and 306 downregulated) were differentially expressed in the renal cortex of cystinuria rats. In accordance with the functional annotation of DEGs, the potential role of glutathione metabolism processes in the kidney of cystinuria rat model was proposed, and KEGG analysis results showed that knock-out of Slc7a9 gene triggered more biological changes which has not been studied. In short, for the first time, a rat model and its transcriptional database that mimics the pathogenesis and clinical consequences of human type B cystinuria were generated.

Whole-Genome Sequence Resource of Fusarium oxysporum Strain TH15, a Plant Growth Promoting Endophytic Fungus Isolated from Tetrastigma hemsleyanum

Whole-Genome Sequence Resource of <i>Fusarium oxysporum</i> Strain TH15, a Plant Growth Promoting Endophytic Fungus Isolated from <i>Tetrastigma hemsleyanum</i>

Identification and Validation of 7-lncRNA Signature of Epigenetic Disorders by Comprehensive Epigenetic Analysis.

The survival rate of patients with lung adenocarcinoma (LUAD) is low. This study analyzed the correlation between the expression of long noncoding RNA (lncRNA) and epigenetic alterations along with the investigation of the prognostic value of these outcomes for LUAD. Differentially expressed lncRNAs were identified based on multiomic data and positively related genes using DESeq2 in R, differentially histone-modifying genes specific to LUAD based on histone modification data, gene enhancers from information collected from the FANTOM5 (Function Annotation Of The Mammalian Genome-5) (fantom.gsc.riken.jp/5) human enhancer database, gene promoters using the ChIPseeker and the human lincRNAs Transcripts database in R, and differentially methylated regions (DMRs) using Bumphunter in R. Overall survival was estimated by Kaplan-Meier, comparisons were performed among groups using log-rank tests to derive differences between sample subclasses, and epigenetic lncRNAs (epi-lncRNAs) potentially relevant to LUAD prognosis were identified. A total of seven dysregulated epi-lncRNAs in LUAD were identified by comparing histone modifications and alterations in histone methylation regions on lncRNA promoter and enhancer elements, including H3K4me2, H3K27me3, H3K4me1, H3K9me3, H4K20me1, H3K9ac, H3K79me2, H3K27ac, H3K4me3, and H3K36me3. Furthermore, 69 LUAD-specific dysregulated epi-lncRNAs were identified. Moreover, lncRNAs-based prognostic analysis of LUAD samples was performed and explored that seven of these lncRNAs, including A2M-AS1, AL161431.1, DDX11-AS1, FAM83A-AS1, MHENCR, MNX1-AS1, and NKILA (7-EpiLncRNA), showed the potential to serve as markers for LUAD prognosis. Additionally, patients having a high 7-EpiLncRNA score showed a generally more unfavorable prognosis compared with those which scored lower. Seven lncRNAs were identified as markers of prognosis in patients with LUAD. The outcomes of this research will help us understand epigenetically aberrant regulation of lncRNA expression in LUAD in a better way and have implications for research advances in the regulatory role of lncRNAs in LUAD.

NCAPG promotes the oncogenesis and progression of non-small cell lung cancer cells through upregulating LGALS1 expression

BackgroundNumerous common oncogenic driver events have been confirmed in non-small cell lung cancer (NSCLC). Although targeted therapy has revolutionized NSCLC treatment, some patients still do not respond. NCAPG, also known as non-SMC condensin I complex subunit G, was positively associated with proliferation and migration in several tumor types.MethodsWe used transcriptional sequencing and TCGA database analysis to identify NCAPG as a new therapeutic target for NSCLC. The oncogenic roles of NCAPG in NSCLC tumor growth and metastasis were detected in vitro and in vivo. Ncapg+/+ or Ncapg+/− mice with urethane treatment were analyzed for oncogenesis of NSCLC.ResultsWe investigated NCAPG as a new oncogenic driver which promoted NSCLC tumorigenesis and progression. We used transcriptome sequencing and the Cancer Genome Atlas (TCGA) database analysis to screen and found that NCAPG was negatively correlated with NSCLC survival. Using immunohistochemistry, we demonstrated that NCAPG overexpression was an independent risk factor for NSCLC survival. Functionally, NCAPG knockdown inhibited proliferation, migration, and invasion of NSCLC cells in vitro and in vivo. We exposed wildtype or Ncapg+/− mice to urethane and discovered that urethane-induced lung tumors were reduced in Ncapg+/− mice. Mechanistically, the function of NCAPG in promoting initiation and progression of NSCLC was closely related to LGALS1, which was also upregulated in NSCLC and might interact directly with NCAPG.ConclusionsThis study indicates that NCAPG is one of the essential factors for NSCLC oncogenesis and progression, providing a new target for prognosis prediction and treatment of NSCLC.

Large-scale analyses of heat shock transcription factors and database construction based on whole-genome genes in horticultural and representative plants.

Heat shock transcription factor (Hsf) plays a critical role in regulating heat resistance. Here, 2950 Hsf family genes were identified from 111 horticultural and representative plants. More Hsf genes were detected in higher plants than in lower plants. Based on all Hsf genes, we constructed a phylogenetic tree, which indicated that Hsf genes of each branch evolved independently after species differentiation. Furthermore, we uncovered the evolutionary trajectories of Hsf genes by motif analysis. There were only six motifs (M1–M6) in lower plants, and then four novel motifs (M7–M10) appeared in higher plants. However, the motifs of some Hsf genes were lost in higher plants, indicating that Hsf genes have undergone sequence variation during their evolution. The number of Hsf genes lost was greater than the number of genes that were duplicated after whole-genome duplication in higher plants. The heat response network was constructed using 24 Hsf genes and 2421 downstream and 222 upstream genes of Arabidopsis. Further enrichment analysis revealed that Hsf genes and other transcription factors interacted with each other in the response to heat stress. Global expression maps were illustrated for Hsf genes under various abiotic and biotic stresses and several developmental stages in Arabidopsis. Syntenic and phylogenetic analyses were conducted using Hsf genes of Arabidopsis and the pan-genome of 18 Brassica rapa accessions. We also performed expression pattern analysis of Hsf and six Hsp family genes using expression values from different tissues and heat treatments in B. rapa. The interaction network between the Hsf and Hsp gene families was constructed in B. rapa, and several core genes were detected in the network. Finally, we constructed an Hsf database (http://hsfdb.bio2db.com) for researchers to retrieve Hsf gene family information. Therefore, our study will provide rich resources for the study of the evolution and function of Hsf genes.

Transcriptomic analysis using dual RNA sequencing revealed a Pathogen–Host interaction after Edwardsiella anguillarum infection in European eel (Anguilla anguilla)

Many studies have explored differentially expressed genes (DEGs) between some pathogens and hosts, but no study has focused on the interaction of DEGs between Edwardsiella anguillarum (Ea) and Anguilla anguilla (Aa). In this study, we examined the interactions of DEGs during Ea infection and Aa anti-infection processes by dual RNA sequencing. Total RNA from in vitro and in vivo (Aa liver) Ea culture was extracted. Using high-throughput transcriptomics, significant DEGs that were expressed between Ea cultured in vitro versus in vivo and those in the liver of the infected group versus control group were identified. Protein–protein interactions between the pathogen and host were explored using Cytoscape according to the HPIDB 3.0 interaction transcription database. The results showed that the liver in the infection group presented with severe bleeding and a large number of thrombi in the hepatic vessels. We found 490 upregulated and 398 downregulated DEGs of Ea in vivo versus Ea cultured in vitro, and 2177 upregulated and 970 downregulated genes in the liver of the infected eels. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of the pathogen DEGs revealed that the upregulated genes were mainly enriched in migration, colonization, biofilm formation, and significantly enriched in ABC transport and quorum sensing; the downregulated genes were mainly involved in metabolism, information transduction, organelle formation, enzyme catalysis, molecular transport, and binding. GO of the host DEGs showed that metabolic process, catalytic activity, single organism metabolic process, small molecule binding, nucleotide binding, nucleotide phosphate binding, and anion binding were markedly enriched. Finally, we found that 79 Ea and 148 Aa proteins encoded by these DEGs were involved in an interaction network, and some pathogen (DegP, gcvP, infC, carB, rpoC, trpD, sthA, and FhuB) and host proteins (MANBA, STAT1, ETS2, ZEP1, TKT1, NMI and RBPMS) appear to play crucial roles in infection. Thus, determining the interaction networks revealed crucial molecular mechanisms underlying the process of pathogenic infection and host anti-infection.

Molecular Mechanism of the Regulatory Effect of Schisandrol A on the Immune Function of Mice Based on a Transcription Factor Regulatory Network.

The molecular mechanism of the regulatory effed of schizandrol A (SA) on the immune function of cyclophosphamide-induced immunosuppressive mice was explored in this study. On the basis of 1619 differentially expressed genes related to the regulatory effect of SA on the immune function of mice screened in our previous study, transcription factors and their corresponding target genes were screened in the Transcriptional Regulatory Element Database (TRED), and a transcription factor target gene regulatory network was constructed. The key nodes of the network were statistically analyzed to clarify the role of transcription factors in the regulatory network. The correlation of network genes with diseases was analyzed with an online annotation tool through the Database for Annotation, Visualization and Integrated Discovery (DAVID). Finally, the key factors related to the regulatory effect of SA on the immune function of mice were screened and verified by animal experiments and the detection of related protein expression by western blot analysis. The results showed that SA could alleviate the immunosuppression induced by cyclophosphamide in mice and regulate the protein expression of Jun, Trp53, and Creb1 in the spleen tissue of mice, together with the transcription factors Atf4 and E2f2. SA may thus play a role in the alleviation of some immunity-related diseases (such as cancer) by regulating the immune function of mice through multiple genes and their transcription factors.

Flavonoid biosynthesis in four Dendrobium species based on transcriptome sequencing and metabolite analysis.

Dendrobium is a genus of plants used as traditional Chinese herbal medicines, with high economic and medicinal value. To reveal the mechanism of flavonoid biosynthesis in Dendrobium, the metabolites and transcriptomes of four Dendrobium species (D. chrysotoxum, D. nobile, D. fimbriatum, and D. denneanum) were analyzed comprehensively. Ultra-high-performance liquid chromatography-tandem mass spectrometry analysis revealed ten flavonoid compounds in Dendrobium. In total, 100,096 unigenes were obtained from the transcript database of the four Dendrobium species. Among the identified differentially expressed genes, 51 were associated with flavonoid biosynthesis, and 670 differentially expressed transcription factors were predicted, including 194 MYB, 87 bHLH, and 100 WRKY family transcription factors, respectively. Transcriptome analysis showed that the expression levels of structural genes such as chalcone synthase (CHS), cinnamate-4-hydroxylase (C4H), and flavonoid 3'-hydroxylase (F3'H) were lower in D. chrysotoxum, D. nobile, and D. fimbriatum than those in D. denneanum, which may be the main reason for the low flavonoid contents in D. chrysotoxum, D. nobile, and D. fimbriatum. The expression level of structural genes corresponded to the accumulation level of flavonols in the different Dendrobium species. The results deepen the understanding of the molecular mechanism of flavonoid biosynthesis in Dendrobium and provide novel insights into the synthesis and accumulation of flavonoids in Dendrobium.

Threshold based Support Vector Machine Learning Algorithm for Sequential Patterns

Now a days the pattern recognition is the major challenge in the field of data mining. The researchers focus on using data mining for wide variety of applications like market basket analysis, advertisement, and medical field etc., Here the transcriptional database is used for all the conventional algorithms, which is based on daily usage of object and/or performance of patients. Here the proposed research work uses sequential pattern mining approach using classification technique of Threshold based Support Vector Machine learning (T-SVM) algorithm. The pattern mining is to give the variable according to the user’s interest by statistical model. Here this proposed research work is used to analysis the gene sequence datasets. Further, the T-SVM technique is used to classify the dataset based on sequential pattern mining approach. Especially, the threshold-based model is used for predicting the upcoming state of interest by sequential patterns. Because this makes deeper understanding about sequential input data and classify the result by providing threshold values. Therefore, the proposed method is efficient than the conventional method by getting the value of achievable classification accuracy, precision, False Positive rate, True Positive rate and it also reduces operating time. This proposed model is performed in MATLAB in the adaptation of 2018a.

P68.04 Molecular Mechanism of MK2 Promoting Lung Adenocarcinoma Progression by Phosphorylating Transcription Regulator CRABP2

The morbidity and mortality of lung cancer rank the first among malignant tumors in China. EGFR-TKIs has achieved good efficacy in advanced lung adenocarcinoma with EGFR-positive, however, drug resistance and rapid progress of some patients are still inevitable. Therefore, it is of great significance to find new therapeutic targets. Abnormally activated transcriptional regulators are involved in many biological processes of the development of malignant tumors, further exploration of their functional mechanisms is expected to provide a new direction for targeted tumor therapy. TRRUST and CHIPBase databases of transcriptional regulatory factor and TCGA databases were integrated to screen the transcriptional regulatory factors with significantly different expression in lung adenocarcinoma. The prediction of interacting molecules was performed on the Linkedomics, and preliminary verification was performed using WB. The clinicopathological data of lung adenocarcinoma patients with survival data from GEO and TCGA databases were downloaded to analyze the effect of CRABP2 and MK2 expression on survival prognosis. Transcription and protein expression of CRABP2 were significantly up-regulated in lung adenocarcinoma (Figure 1). Bioinformatics analysis showed that MK2 interacted with CRABP2, and CRABP2 protein decreased significantly after knockdown of MK2 protein expression. The overall survival of lung adenocarcinoma patients with high CRABP2 and MK2 expressions were worse than that of the low expression group (P = 3.4e-04 and 6.5e-05, respectively) (Figure 2).View Large Image Figure ViewerDownload Hi-res image Download (PPT) MK2 may phosphorylate CRABP2 to mediate the progression of lung adenocarcinoma, and the MK2-CRABP2 signaling pathway is expected to become a biomarker or a new target for therapy of lung adenocarcinoma. However, further molecular experiments are needed for further exploration and verification.

Identification of Novel Kinase–Transcription Factor–mRNA–miRNA Regulatory Network in Nasopharyngeal Carcinoma by Bioinformatics Analysis

PurposeNasopharyngeal carcinoma (NPC) is one of the most common malignant tumors of the head and neck. This study aimed to investigate the crucial genes and regulatory networks involved in the carcinogenesis of NPC using a bioinformatics approach.MethodsFive mRNA and two miRNA expression datasets were downloaded from the Gene Expression Omnibus (GEO) database. Differentially expressed genes (DEGs) and miRNAs (DEMs) between NPC and normal samples were analyzed using R software. The WebGestalt tool was used for functional enrichment analysis, and protein–protein interaction (PPI) network analysis of DEGs was performed using STRING database. Transcription factors (TFs) were predicted using TRRUST and Transcriptional Regulatory Element Database (TRED). Kinases were identified using X2Kgui. The miRNAs of DEGs were predicted using miRWalk database. A kinase–TF–mRNA–miRNA integrated network was constructed, and hub nodes were selected. The hub genes were validated using NPC datasets from the GEO and Oncomine databases. Finally, candidate small-molecule agents were predicted using CMap.ResultsA total of 122 DEGs and 44 DEMs were identified. DEGs were associated with the immune response, leukocyte activation, endoplasmic reticulum stress in GO analysis, and the NF-κB signaling pathway in KEGG analysis. Four significant modules were identified using PPI network analysis. Subsequently, 26 TFs, 73 kinases, and 2499 miRNAs were predicted. The predicted miRNAs were cross-referenced with DEMs, and seven overlapping miRNAs were selected. In the kinase–TF–mRNA–miRNA integrated network, eight genes (PTGS2, FN1, MMP1, PLAU, MMP3, CD19, BMP2, and PIGR) were identified as hub genes. Hub genes were validated with consistent results, indicating the reliability of our findings. Finally, six candidate small-molecule agents (phenoxybenzamine, luteolin, thioguanosine, reserpine, blebbistatin, and camptothecin) were predicted.ConclusionWe identified DEGs and an NPC regulatory network involving kinases, TFs, mRNAs, and miRNAs, which might provide promising insight into the pathogenesis, treatment, and prognosis of NPC.

Abstract 1534: Mesothelin isoform 2 is a novel target for allogenic CARγδT cell therapy in solid tumors

Abstract Mesothelin (MSLN) is a tumor-associated antigen over-expressed on the cell surface of various malignant tumor cells, including majority of mesothelioma and ovarian cancer. MSLN-directed therapy has been intensively studied in preclinical and clinical settings. However, the clinical efficacy and safety of mesothelin-targeted therapy remains to be demonstrated. To identify a safe and effective target for CAR-T cell therapy in solid tumors, we used artificial intelligent platform developed with public and private RNA transcription database to identify cancer-associated antigens caused by alternative spicing. One of interesting targets is an alternative spicing isoform from MSLN. Human MSLN transcript has at least three isoforms. The isoform 1 is the predominant transcript detected in normal and tumor tissues and has been a promising target for cancer immunotherapy. The isoform 2 is the minor transcript using alternatively spliced exons producing 8 additional amino acids insertion compared to isoform 1. The isoform 3 produces a truncated and soluble MSLN. Our data demonstrated that MSLN isoform 2 is specifically expressed in ovarian cancers but not normal tissues, confirmed by Q-PCR studies. Furthermore, we generated mouse hybridoma antibodies specifically targeting MSLN isoform 2. The antibody specificity was screened and confirmed by ELISA-based reaction to MSLN isoform 2-specific peptide and flow cytometry-based binding to 293T cells overexpressing MSLN isoform 2 but not isoform 1 proteins. We further detected the endogenous MSLN isoform 2 expression in human mesothelioma cell line NCI H226 and further confirmed MSLN isoform 2 expression on human primary mesothelioma and ovarian cancer tissue but not normal tissues by immunohistochemistry staining. To study if targeting MSLN isoform 2 with chimeric antigen receptor (CAR) can control the tumor growth, we made CAR retrovirus construct and showed that genetically modified allogenic gamma-delta T cells expressing CAR for MSLN isoform 2 can kill human mesothelioma cells in vitro and in vivo. In summary, we have demonstrated that MSLN isoform 2 is a tumor-specific antigen, which can be targeted for CAR-T cell therapy. Citation Format: Xiaohong Wang, Yibin Chen, Anupama Gopisetty, Leonardo Mirandola, Lucia Piccotti, Quynh Nguyen, Maurizio Chiriva-Internati. Mesothelin isoform 2 is a novel target for allogenic CARγδT cell therapy in solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1534.

Development of the GABAergic and glutamatergic neurons of the lateral hypothalamus

In the last few years we assist to an unexpected deluge of genomic data on hypothalamic development and structure. Perhaps most surprisingly, the Lateral Zone has received much attention too. The new information focuses first of all on transcriptional heterogeneity. Many already known and a number of hitherto unknown lateral hypothalamic neurons have been described to an enormous degree of detail. Maybe the most surprising novel discoveries are two: First, some restricted regions of the embryonic forebrain neuroepithelium generate specific LHA neurons, either GABAergic or glutamatergic. Second, evidence is mounting that supports the existence of numerous kinds of “bilingual” lateral hypothalamic neurons, expressing (and releasing) glutamate and GABA both as well as assorted neuropeptides. This is not accepted by all, and it could be that genomic researchers need a common set of rules to interpret their data (sensitivity, significance, age of analysis). In any case, some of the new results appear to confirm hypotheses about the ability of the hypothalamus and in particular its Lateral Zone to achieve physiological flexibility on a fixed connectivity (“biochemical switching”). Furthermore, the results succinctly reviewed here are the basis for future advances, since the transcriptional databases generated can now be mined e.g. for adhesion genes, to figure out the causes of the peculiar histology of the Lateral Zone; or for ion channel genes, to clarify present and future electrophysiological data. And with the specific expression data about small subpopulations of neurons, their connections can now be specifically labeled, revealing novel relations with functional significance.

Identification of EHMT2 As a New Target in T-Cell Acute Lymphoblastic Leukemia

▪Acute lymphoblastic leukemia (ALL) constitutes a family of genetically heterogeneous lymphoid neoplasms and is the most common cancer in childhood. T-cell acute lymphoblastic leukemia/lymphoma (T-ALL) is an aggressive subtype of ALL and accounts for approximately 15-20% of all cases. Despite remarkable improvement in the prognosis of childhood ALL over the past few decades, many patients will require additional therapy for relapsed/refractory disease, and ALL remains the second leading cause of cancer-related death in children. Genomic studies have identified recurrent somatic alterations in genes involved in DNA methylation and post-translational histone modifications in ALL, suggesting emerging opportunities for novel therapeutic interventions.To identify new potential targets in T-ALL we tested a collection of inhibitors of epigenetic modifiers, including inhibitors of the histone acetyltransferase (HAT) p300 (CTPB and C646) and inhibitors of protein methyltransferases (PMTs), including targeting EZH2 (GSK126), DOT1L (SGC0946), and G9a (EHMT2)/G9a-like (GLP) (EHMT1) (UNC0642 and BIX01294). We demonstrated that the G9a/GLP inhibitors significantly impaired cell viability compared to other molecules as measured by the reduction of cellular ATP content.G9a/EHMT2 and GLP/EHMT1 are conserved protein lysine methyltransferases that contain a Su(Var), Enhancer of Zeste, Trithorax (SET) domain. G9a localizes in euchromatin regions and regulates gene expression and chromosome structure through de novo mono- and di- methylation of histone H3 lysine 9 (H3K9me1/2). In particular, di- and trimethylation of H3K9 in gene promoters have been associated with transcriptional repression. Despite recent evidence that G9a is involved in different tumor types, the role of the G9a/GLP complex has not been systematically studied in T-ALL.Because cancer driver genes and response to targeted therapy are frequently correlated with activating somatic mutations and/or expression levels we analyzed several transcriptional databases, including the Differentiation Map (DMAP), Cancer Cell Line Encyclopedia, and primary T-ALL datasets. Collectively, the results revealed that EHMT2 is a preferential marker of lymphoid differentiation and is highly expressed in T-ALL compared to other cancer subtypes or normal bone marrow cells. We next validated the effects of UNC0642 and extended testing to BRD4770, another selective G9a/GLP inhibitor. Competitive inhibition of G9a with both molecules impaired cell growth and triggered starvation-induced autophagic activity as shown by the detection of LC3-II by immunoblotting and formation of cytoplasmic autophagic vacuoles. Moreover, di-methylated histone H3 lysine 9 (H3K9me2) levels were decreased as measured by western blotting after histone extraction, supporting the notion that phenotypic changes upon small molecule treatment may be due to H3K9me2 loss and epigenetic reprogramming.To genetically validate G9a as a potential new target in T-ALL we intersected our chemical screening results with a previously published short hairpin RNA (shRNA) screen targeting nearly 350 chromatin regulator genes (Knoechel B. et al., Cancer Cell, 2014). EHMT2 scored among the top 20 hits that impaired the viability of T-ALL cells, further supporting our initial observation of the dependency of T-ALL on EHMT2. Because shRNA-based screens are limited by the occurrence of off-target effects and potentially the incomplete repression of target genes, we took a CRISPR/Cas9 approach and designed sgRNA guides targeting the SET domain of EHMT2. Consistently, genetic knockout of EHMT2 in T-ALL cell lines impaired cell growth and induced apoptosis measured by Annexin V/ Propidium iodide staining and quantified by flow cytometry.In conclusion, the intersection of multiple chemical, genetic, and genomic approaches led to the identification of G9a/EHMT2 as a druggable candidate in T-ALL. Our preliminary data strongly support the further investigation of EHMT2 in this aggressive subset of leukemia. DisclosuresStegmaier:Novartis: Consultancy, Research Funding.

Full-length sequencing of Ginkgo biloba L. reveals the synthesis of terpenoids during seed development

Full-length transcriptome sequencing based on the PacBio sequencing platform could significantly optimize the annotation of gene structures. As an ancient relic gymnosperm in the monotypic order Ginkgoales, Ginkgo biloba L. contains rich terpenoids that are medicinally valuable. The seeds have abundant edible endosperm, which is delicious and of high nutritional value. However, existing molecular studies on the developmental process of ginkgo seeds are relatively weak, and the biosynthesis of terpenoids in seeds has received little attention. Therefore, single-molecule real-time (SMRT) technology and Illumina sequencing were combined to sequence six tissues related to the reproductive growth and development of ginkgo in order to generate a high-quality full-length transcription database. In total, 20.98 Gb of clean reads containing 178,548 full-length non-chimeric (FLNC) sequences were obtained. From these data, 4019 novel genes and 22,845 novel isoforms were predicted, 52.32 % of the novel genes were annotated, and three novel isoforms were annotated in terpene synthesis related pathways. The enrichment analysis of differentially expressed genes (DEGs) showed that, 95 genes were enriched into 21 categories related to seed development, and 47 DEGs were enriched in the skeletal pathway of terpene synthesis. Combined with the real-time quantitative reverse transcription PCR (qRT-PCR), the phosphosynthase family members synthesizing terpene precursors have diverse and complex expression trends during seed development. Our findings confirm the advantages of SMRT, which facilitated the construction a rich transcript data-set for research on the development of ginkgo seeds, enriching the annotation of the ginkgo genome, and enhancing our understanding of gene regulation of terpene biosynthesis in ginkgo seeds.

Structure, biochemistry, and gene expression patterns of the proline biosynthetic enzyme pyrroline-5-carboxylate reductase (PYCR), an emerging cancer therapy target.

Proline metabolism features prominently in the unique metabolism of cancer cells. Proline biosynthetic genes are consistently upregulated in multiple cancers, while the proline catabolic enzyme proline dehydrogenase has dual, context-dependent pro-cancer and pro-apoptotic functions. Furthermore, the cycling of proline and Δ1-pyrroline-5-carboxylate through the proline cycle impacts cellular growth and death pathways by maintaining redox homeostasis between the cytosol and mitochondria. Here we focus on the last enzyme of proline biosynthesis, Δ1-pyrroline-5-carboxylate reductase, known as PYCR in humans. PYCR catalyzes the NAD(P)H-dependent reduction of Δ1-pyrroline-5-carboxylate to proline and forms the reductive half of the proline metabolic cycle. We review the research on the three-dimensional structure, biochemistry, inhibition, and cancer biology of PYCR. To provide a global view of PYCR gene upregulation in cancer, we mined RNA transcript databases to analyze differential gene expression in 28 cancer types. This analysis revealed strong, widespread upregulation of PYCR genes, especially PYCR1. Altogether, the research over the past 20 years makes a compelling case for PYCR as a cancer therapy target. We conclude with a discussion of some of the major challenges for the field, including developing isoform-specific inhibitors, elucidating the function of the long C-terminus of PYCR1/2, and characterizing the interactome of PYCR.

Combining single-molecule sequencing and next-generation sequencing to provide insight into the complex response of Iris halophila Pall. to Pb exposure

Iris halophila Pall., a Pb accumulator with high ecological and industrial values, has enormous application potential for the phytoremediation of Pb-contaminated soil. However, the lack of full-length transcriptome information obstructs understanding of the molecular regulatory network of I. halophila response to Pb. To construct the full-length transcript database and identify the molecular mechanisms underlying I. halophila response to Pb, the shoots, roots, flowers, and Pb-treated seedlings of I. halophila were subjected to single-molecular real-time sequencing and next-generation sequencing. A total of 40,034 complete coding sequences, 2028 alternative splices, 4649 long non-coding RNAs (lncRNAs) were predicted. Among them, 2614 annotated differentially expressed genes (DEGs) and 19 differentially expressed lncRNAs (DELs) were identified in Pb-treated I. halophila shoot, while 7242 annotated DEGs and 57 DELs were identified in root under Pb conditions. Biological function enrichment analysis of DEGs revealed the key roles of hormone signaling transduction, GSH metabolism, metal ion transporters in I. halophila response to Pb. LncRNA play crucial roles in I. halophila resistance to Pb by regulating the key downstream metabolic pathways. These results not only enrich the genetic information of Iris. L plants, but also enhance the understanding of molecular responses to Pb in I. halophila, and will lay an important foundation for future research on Pb phytoremediation or low-Pb food crop and development of I. halophila for industrial uses.

Effects of Chronic Nicotine Exposure and 24h Withdrawal on Synaptosomal Protein Expression in the Mouse Ventral Hippocampus

Despite cigarette smoking being the leading cause of preventable death in the United States 20% of Americans continue to smoke. We now know that 80% of smokers desire to quit; however, less than 5% of them are successful. The low success rate of smoking cessation is partially due to current pharmacotherapies only addressing a subset of nicotine withdrawal symptoms, which is the primary driver of smoking relapse. A prominent, yet unaddressed, symptom of nicotine withdrawal is affective dysfunction. Affective symptoms, such as increased anxiety and reduced emotional regulation, are underpinned by discrete disruption of select brain circuits. For example, the ventral hippocampus (VHIPP) has a well-described role sub-serving the interface between contextual learning and affective responding, suggesting it as a potential circuitry target for examination of molecular alterations during nicotine withdrawal. Therefore, we explored this by treating 18-week-old mice for two weeks with either saline, nicotine (18 mg/kg/day), or 24h withdrawal from nicotine (18 mg/kg/day) via osmotic minipumps (Alzet 2002 model). Following treatment, the subjects were sacrificed and the VHIPP was collected and synaptosomes were prepared. These samples were analyzed via liquid chromatography-mass spectrometry to quantify changes in synaptosomal protein expression. These results were then analyzed using Enrichr pathway and transcriptional factor databases. Our results indicate changes in the axon guidance, vesicle-mediated transport, and transmission across chemical synapses pathways in both nicotine versus saline and withdraw versus saline synaptosomal protein comparisons. However, upon qPCR analysis mRNA expression of key genes associated with the pathways of interest remained unchanged for each treatment group. Further experiments will utilize western blotting to validate changes in total and phosphorylated proteins of interest in the aforementioned pathways.

Copper promoting oyster larval growth and settlement: Molecular insights from RNA-seq

As a cofactor of key enzymes, Cu is required in living organisms, although Cu levels in the natural environment are typically low. In this study, the promotion of growth and settlement on the larvae of oyster Crassostrea angulata was observed at an environmentally relevant concentration (10 μg/L Cu). Interestingly, Cu accumulation in the soft tissue of oyster larvae increased during the larval development and exhibited a sharp increase at the late pelagic stage. With the help of RNA-seq, we constructed a high-quality transcriptional database of the oyster C. angulata larvae (24,257 genes with an average length of 1594 bp) via de novo assembly, which provided the basic molecular changes during the larval development. Network analysis of five early developmental stages and differential expression under Cu exposure were integrated to examine the roles of Cu in oyster larvae. Our molecular analysis demonstrated that both ion channels and organic transporters contributed to Cu internalization from the external environment, including zinc transporters and amino acid transporters. The followed distribution of Cu across cells was achieved by ATP7A, the circulatory system, and the Cu transporters (CTRs). Cu exposure enhanced the ribosome and the calcium binding proteins with a higher rate of translation and shell formation, giving rise to faster growth of oyster larvae. Furthermore, Cu facilitated the settling process by upregulating the chitin binding genes and then promoting the formation of the proteinaceous matrix between larvae and substrate. Our study presents the molecular basis for Cu promotion (i.e., hormesis) effects on oyster larval growth and settlement.

Analysis of temporal gene regulation of Listeria monocytogenes revealed distinct regulatory response modes after exposure to high pressure processing

BackgroundThe pathogen Listeria (L.) monocytogenes is known to survive heat, cold, high pressure, and other extreme conditions. Although the response of this pathogen to pH, osmotic, temperature, and oxidative stress has been studied extensively, its reaction to the stress produced by high pressure processing HPP (which is a preservation method in the food industry), and the activated gene regulatory network (GRN) in response to this stress is still largely unknown.ResultsWe used RNA sequencing transcriptome data of L. monocytogenes (ScottA) treated at 400 MPa and 8∘C, for 8 min and combined it with current information in the literature to create a transcriptional regulation database, depicting the relationship between transcription factors (TFs) and their target genes (TGs) in L. monocytogenes. We then applied network component analysis (NCA), a matrix decomposition method, to reconstruct the activities of the TFs over time. According to our findings, L. monocytogenes responded to the stress applied during HPP by three statistically different gene regulation modes: survival mode during the first 10 min post-treatment, repair mode during 1 h post-treatment, and re-growth mode beyond 6 h after HPP. We identified the TFs and their TGs that were responsible for each of the modes. We developed a plausible model that could explain the regulatory mechanism that L. monocytogenes activated through the well-studied CIRCE operon via the regulator HrcA during the survival mode.ConclusionsOur findings suggest that the timely activation of TFs associated with an immediate stress response, followed by the expression of genes for repair purposes, and then re-growth and metabolism, could be a strategy of L. monocytogenes to survive and recover extreme HPP conditions. We believe that our results give a better understanding of L. monocytogenes behavior after exposure to high pressure that may lead to the design of a specific knock-out process to target the genes or mechanisms. The results can help the food industry select appropriate HPP conditions to prevent L. monocytogenes recovery during food storage.