Comprehensive Gene Expression Profiling Research Articles

Abstract 3847: Inhibition of GCN2 sensitizes ASNS-downregulated cancer cells to asparaginase by disrupting amino acid response

Abstract General control nonderepressible 2 (GCN2) plays a major role in cellular response to amino acid limitation. Although maintenance of amino acid homeostasis is critical for tumor growth, little is known concerning the contribution of GCN2 to cancer cell proliferation and its potential as a therapeutic target. In this study, we demonstrate that inhibition of GCN2 with small-molecule compounds sensitizes cancer cells to anti-leukemic agent asparaginase (ASNase). We first tested acute lymphoblastic leukemia (ALL) cells and showed that treatment with GCN2 inhibitors rendered ALL cells sensitive to ASNase by preventing the induction of asparagine synthetase (ASNS). The prevention of ASNS expression suppresses asparagine synthesis and thus inhibits protein translation. In addition, comprehensive gene expression profiling revealed that the combined treatment induced stress-activated MAPK pathway and thereby triggering apoptosis. Using cell-panel analyses, we also identified a clear synergy between GCN2 inhibitors and ASNase in acute myelogenous leukaemia (AML) and pancreatic cancer cells besides ALL. Notably, basal ASNS expression levels were significantly correlated with sensitivity to combinatorial treatment. Furthermore, the combination displayed robust antitumor activity in mouse xenograft models of ALL, AML, and pancreatic cancer. These results demonstrate a novel mechanistic insight into a role of GCN2 in amino acid response and provide a rationale for further investigating GCN2 inhibitors for the treatment of cancer. Citation Format: Akito Nakamura, Tadahiro Nambu, Shunsuke Ebara, Yuka Hasegawa, Kosei Toyoshima, Yasuko Tsuchiya, Daisuke Tomita, Jun Fujimoto, Osamu Kurasawa, Chisato Takahara, Ayumi Kawamura, Ryuichi Nishigaki, Yoshinori Satomi, Akito Hata, Takahito Hara. Inhibition of GCN2 sensitizes ASNS-downregulated cancer cells to asparaginase by disrupting amino acid response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3847.

Early responses to dehydration in contrasting wild Arachis species.

Wild peanut relatives (Arachis spp.) are genetically diverse and were selected throughout evolution to a range of environments constituting, therefore, an important source of allelic diversity for abiotic stress tolerance. In particular, A. duranensis and A. stenosperma, the parents of the reference Arachis A-genome genetic map, show contrasting transpiration behavior under limited water conditions. This study aimed to build a comprehensive gene expression profile of these two wild species under dehydration stress caused by the withdrawal of hydroponic nutrient solution. For this purpose, roots of both genotypes were collected at seven time-points during the early stages of dehydration and used to construct cDNA paired-end libraries. Physiological analyses indicated initial differences in gas exchange parameters between the drought-tolerant genotype of A. duranensis and the drought-sensitive genotype of A. stenosperma. High-quality Illumina reads were mapped against the A. duranensis reference genome and resulted in the identification of 1,235 and 799 Differentially Expressed Genes (DEGs) that responded to the stress treatment in roots of A. duranensis and A. stenosperma, respectively. Further analysis, including functional annotation and identification of biological pathways represented by these DEGs confirmed the distinct gene expression behavior of the two contrasting Arachis species genotypes under dehydration stress. Some species-exclusive and common DEGs were then selected for qRT-PCR analysis, which corroborated the in silico expression profiling. These included genes coding for regulators and effectors involved in drought tolerance responses, such as activation of osmosensing molecular cascades, control of hormone and osmolyte content, and protection of macromolecules. This dataset of transcripts induced during the dehydration process in two wild Arachis genotypes constitute new tools for the understanding of the distinct gene regulation processes in these closely related species but with contrasting drought responsiveness. In addition, our findings provide insights into the nature of drought tolerance in wild germoplasm, which might be explored as novel sources of diversity and useful wild alleles to develop climate-resilient crop varieties.

Consensus molecular subtypes classification of colorectal cancer as a predictive factor for chemotherapeutic efficacy against metastatic colorectal cancer.

The consensus molecular subtypes (CMS) classification is one of the most robust colorectal cancer (CRC) classifications based on comprehensive gene expression profiles. This study aimed to clarify whether the CMS is a predictive factor for therapeutic effects of standard chemotherapies for metastatic CRC (mCRC). We retrospectively enrolled 193 patients with mCRCs, and using comprehensive gene expression data, classified them into 4 subtypes: CMS1–CMS4. The associations between the subtypes and treatment outcomes were analyzed. Regarding first-line chemotherapy, irinotecan (IRI)-based chemotherapy was significantly superior to oxaliplatin (OX)-based chemotherapy for progression-free survival (PFS; hazard ratio [HR] = 0.31, 95% confidence interval [CI] 0.13–0.64) and overall survival (OS; HR = 0.45, 95% CI 0.19–0.99) in CMS4. Regarding the anti-epidermal growth factor receptor (anti-EGFR) therapy, CMS1 showed particularly worse PFS (HR = 2.50, 95% CI 1.31–4.39) and OS (HR = 4.23, 95% CI 1.83–9.04), and CMS2 showed particularly good PFS (HR = 0.67, 95% CI 0.44–1.01) and OS (HR = 0.49, 95% CI 0.27–0.87) compared with the other subtypes. The biological characteristics of CMS may influence the efficacy of chemotherapy. CMS might be a new predictive factor for the efficacy of chemotherapy against mCRCs.

Genome-wide identification and characterization of cysteine-rich polycomb-like protein (CPP) family genes in cucumber (Cucumis sativus) and their roles in stress responses

Cysteine-rich polycomb-like protein (CPP) transcription factors are a small gene family involved in the regulation of plant growth, development and stress response. Genome-wide identification and characterization of CPP gene family have been carried out in several plant species. However, no comprehensive phylogenetic analysis or expression profiling of CPP genes in Cucumis sativus has been reported. In this study, we characterized the CPP family genes in C. sativus, and 5 CPP candidate genes were found in cucumber genome. CPP proteins from cucumber and other plant species were classified into two groups, which were further divided into five subgroups based on the phylogenetic distribution. Most CPP genes in the same subgroup shared similar gene structures and conserved motifs. Transcriptome data revealed that CsCPP genes were expressed in leaves, ovaries, flowers, stems, roots, and tendril tissues. qRT-PCR expression analysis showed that many CsCPP genes exhibited different expression patterns in cucumber leaves under abiotic stresses including salt, cold, drought, and ABA. These results demonstrate that cucumber CPP gene family may play critical roles in plant development as well as in the responses to environmental stresses.

Effect of CTLA-4 overexpression on response to ipilimumab in melanoma.

190 Background: CD8 positive tumor infiltrating lymphocytes (TILS) are highly associated with immune response and prognosis, and are also under investigation as a marker of response to checkpoint inhibitors. Given lack of predictive biomarkers for ipilimumab, growing number of trials for new indications for combination ipilimumab + nivolumab, and evidence to support therapeutic target overexpression as markers of response, we examined the role of CTLA-4 expression and TILS in response to ipilimumab and combination ipilimumab + nivolumab in melanoma. Methods: Formalin-fixed paraffin embedded melanoma samples taken prior to treatment by ipilimumab (n = 36) or combination ipilimumab + nivolumab (n = 10) were evaluated by a comprehensive immune gene expression profile to establish the relationship between CTLA-4 and CD8 and therapeutic response (RECIST v1.1). Results: Increased CTLA-4 expression was moderately associated with increased TILS (r2= .41, p = .004). This was observed in the monotherapy group (r2= .38, p = .02), and was higher in the smaller combination therapy group, though not statistically significant (r2= .59, p = .06). Higher levels of TILS were observed in responders who achieved clinical benefit from either regimen within 6 months (n = 20). No significant difference was observed between responders (M = 57.1, SD = 30.2) and nonresponders (M = 48.6, SD = 32.9); t(44) = -.895, p = .376. Lower levels of CTLA-4 were observed in responders who achieved clinical benefit from either regimen within 6 months. No significant difference was observed between responders (M = 54, SD = 35) and nonresponders (M = 38.7, SD = 26.8); t(44) = 1.70, p = .09. The ratio of TILS to CTLA-4 expression was higher in responders who achieved clinical benefit within 6 months (n = 20).No significant difference was observed between responders (M = 5.2, SD = 14.0) and nonresponders (M = 1.4, SD = 2.7); t(41) = -1.2, p = .212. Conclusions: While not statistically significant, CTLA-4 expression in melanoma patients treated with either ipilimumab or combination ipilimumab + nivolumab was lower in responders compared to nonresponders. This analysis does not support the concept that over-expression of CTLA-4 is a biomarker of response to anti-CTLA-4 therapy.

Correlation of lung cancer mutational profile with immune profile.

146 Background: The association between neoplasm mutational and immune profiles has not been well-characterized. Methods: We collected 26 lung cancer formalin-fixed paraffin embedded (FFPE) samples which had been tested with a comprehensive mutation profile to detect clinically actionable mutations, and a comprehensive immune gene expression profile, which interrogates PD-L1 immunohistochemistry (IHC), PD-L1/2 copy number, CD3/CD8 IHC, microsatellite instability status, mutational burden, and the expression profile of 54 immune-related genes. The ranking of gene expression and 7 immune phenotypes was compared to a reference population. Six cases were positive for an activating KRAS mutation and 2 cases were positive for an activating EGFR mutation. Principal component analysis was performed to determine the association of EGFR/KRAS mutations with the measured immune landscape. Results: The proinflammatory immune phenotype was significantly correlated with KRAS mutation positive samples (first principal component, R squared = 0.53, p < 0.05). Similarly, CD38 expression was correlated with KRAS mutation (R squared = 0.47, p < 0.05). CD137, KLRD1, and DDX58 expression was significantly correlated with EGFR positive samples (second principal component, R squared = 0.47, 0.37, 0.35 respectively, p < 0.05 in all cases). Unsupervised hierarchical clustering of the samples resulted in three distinct clusters, EGFR positive, KRAS positive, and EGFR negative/KRAS negative. In the KRAS positive cluster, high proinflammatory immune phenotype, VISTA moderate expression, presence of CD3/CD8 tumor infiltrating lymphocytes (TILs), and low KLRD1 were overrepresented (p < 0.05), while low VISTA and proinflammatory moderate immune phenotype were significantly underrepresented (p < 0.05). In the EGFR positive cluster DDX58 high, very low TILs, and very low CD8 were significantly (p < 0.05) overrepresented. Conclusions: KRAS mutation positivity is significantly associated with a proinflammatrory immune phenotype and CD3/CD8 infiltration. KRAS positive, EGFR positive, and KRAS/EGFR negative clusters are immunophenotypically distinct. A higher number of specimens is necessary to verify and expand these findings.

Immune response-associated gene profiling in Japanese melanoma patients using multi-omics analysis.

Project High-tech Omics-based Patient Evaluation (HOPE), including comprehensive whole-exome sequencing (WES) and gene expression profiling (GEP) using freshly resected tumor specimens, has been in progress since its implementation in 2014. Among a total of 1,685cancer patients, 13melanoma patients were registered in the HOPE Project and were characterized using multi-omics analyses. Among the 13melanoma patients, 4were deceased, and 9were alive. The mean overall survival (OS) and relapse‑free survival (RFS) times of the melanoma patients were 16.9and14.7months, respectively. Previously, we developed an immune response‑associated gene list, which consisted of 164genes in Project HOPE, for evaluating the immunological status. In the present study, the association of immune response‑associated gene expression with immunological parameters, such as programmed death-ligand1 (PD-L1) and CD8 expression levels, single nucleotide variant (SNV) number, and Vogelstein driver gene mutation number, was investigated. With respect to PD-L1 expression, both immuno-suppression and immuno-stimulation-related genes were upregulated in PD-L1-positive melanomas. In contrast, regarding Vogelstein driver mutations, several T-cell activation-related genes were significantly downregulated in the high driver gene mutation group. In addition, many T-cell activation-related genes were upregulated in the CD8-positive melanomas. The correlation of immune response-associated gene expression with the survival time of the melanoma patients was investigated. Eight specific genes were commonly identified as genes that were significantly correlated for both the overall OS and RFS time, which could be possible prognostic factors for melanoma patients. These results revealed that an immune response-associated gene panel could be an informative tool for evaluating the immunological status prior to clinical immunotherapy in the upcoming era of genomic cancer medicine.

De novo transcriptome assembly and comparative analysis between male and benzyladenine-induced female inflorescence buds of Plukenetia volubilis

Plukenetia volubilis is a promising oilseed crop due to its seeds being rich in unsaturated fatty acids, especially alpha-linolenic acid. P. volubilis is monoecious, with separate male and female flowers on the same inflorescence. We previously reported that male flowers were converted to female flowers by exogenous cytokinin (6-benzyladenine, 6-BA) treatment in P. volubilis. To identify candidate genes associated with floral sex differentiation of P. volubilis, we performed de novo transcriptome assembly and comparative analysis on control male inflorescence buds (MIB) and female inflorescence buds (FIB) induced by 6-BA using Illumina sequencing technology. A total of 57,664 unigenes with an average length of 979 bp were assembled from 104.1 million clean reads, and 45,235 (78.45%) unigenes were successfully annotated in the public databases. Notably, Gene Ontology analyses revealed that 4193 and 3880 unigenes were enriched in the categories of reproduction and reproductive processes, respectively. Differential expression analysis identified 1385 differentially expressed unigenes between MIB and FIB, of which six unigenes related to cytokinin and auxin signaling pathways and 16 important transcription factor (TF) genes including MADS-box family members were identified. In particular, several unigenes encoding important TFs, such as homologs of CRABS CLAW, RADIALIS-like 1, RADIALIS-like 2, HECATE 2, WUSCHEL-related homeobox 9, and SUPERMAN, were expressed at higher levels in FIB than in MIB. The expression patterns of the 36 selected unigenes revealed by transcriptome analysis were successfully validated by quantitative real-time PCR. This study not only provides comprehensive gene expression profiles of P. volubilis inflorescence buds, but also lays the foundation for research on the molecular mechanism of floral sex determination in P. volubilis and other monoecious plants.

Transcriptome Analysis of Peripheral Blood Mononuclear Cells From Chronic Hepatitis B and Hepatocellular Carcinoma Patients: A Network-Based Attitude

Aim: The aim of the study was constructing a protein–protein interaction network for chronic hepatitis B (CHB) and hepatocellular carcinoma (HCC) patients. Materials & methods: Comprehensive gene expression profile of peripheral blood mononuclear cells of CHB and HCC were obtained from Gene Expression Omnibus/NCBI database. Differentially expressed genes (DEGs) of samples were analyzed using GEO2R web application. Results: The majority of DEGs in both CHB and HCC has been enriched in immune system responses. However, there was a significant disparity between the enrichment of these genes (especially genes associated with Toll-like receptor-and-TNF) in CHB-HCC compared with normal-CHB. Conclusion: The transcriptome properties of peripheral blood mononuclear cells are changed in patients with HBV-HCC. The immune response genes are the most deregulated genes in HCC patients. [Formula: see text]

Single-Cell Transcriptome Analyses Reveal Taxol Resistant Subpopulations in Esophageal Squamous Cancer Cells

Taxol resistance is a main problem in the chemotherapy for many different cancers, including esophageal squamous cell carcinoma (ESCC). The functional heterogeneity of gene expression within the taxol resistant cancer cells correlates with its taxol resistance. However, the mechanisms of taxol resistance remains poorly understood. Single-cell RNA-seq can provide comprehensive gene expression profiles in single cancer cells, which will allow us to gain better understanding the mechanism responsible for taxol resistance in ESCC. Through low dose and long-term treatment of taxol, we successfully induced taxol resistance in ESCC cell line KYSE 30. Gene expression profiles were measured utilizing population RNA-seq. Furthermore, single-cell RNA-seq was utilized to investigate the heterogeneity of taxol resistant cancer cells. 37 single cells from KYSE 30 cells and 73 single cells from taxol resistant KYSE 30 cells were subjected to single-cell RNA-seq for gene expression profiling. Except previously reported cell cycles, cell death, and cell proliferation, we found that genes in cell movement are significantly up-regulated in taxol resistant cancer cells. In canonical pathways, decreased EIF2 signaling pathway and increased epithelial adherens junctions are functionally involved in taxol resistance. With single-cell RNA-seq, taxol resistant cancer cells were clustered into two major subclones, which shown unexpected progressive dynamics of taxol resistance. Comparison of these two subpopulations, we found 307 specific genes in subclone 1 and 85 specific genes in subclone 2. The subclone 1 specific genes which were significantly enriched in the biosynthesis of amino acids and cell-cell adhesion, can be identified in population RNA-seq and single-cell RNA-seq, while the subclone 2 specific genes which were enriched in the RNA splicing and RNA stabilization, only can be identified in the single-cell RNA-seq. Single-cell RNA-seq reveals taxol resistance subpopulations in the resistant cancer cells. And the subclone specific genes may function as a real taxol resistance related genes, which could provide putative targets against drug-resistant for clinical cancer therapy.

Transcriptome profiling of fully open flowers in a frost-tolerant almond genotype in response to freezing stress.

Spring frost is a major limiting abiotic stress for the cultivation of almonds [Prunus dulcis (Mill.)] in Mediterranean areas or the Middle East. Spring frost, in particular, damages almond fully open flowers, resulting to significant reduction in yield. Little is known about the genetic factors expressed after frost stress in Prunus spp. as well as in almond fully open flowers. Here, we provide the molecular signature of pistils of fully open flowers from a frost-tolerant almond genotype. The level of frost tolerance in this genotype was determined for all three flowering stages and was confirmed by comparing it to two other cultivars using several physiological analyses. Afterwards, comprehensive expression profiling of genes expressed in fully open flowers was performed after being exposed to frost temperatures (during post-thaw period). Clean reads, 27,104,070 and 32,730,772, were obtained for non-frost-treated and frost-treated (FT) libraries, respectively. A total of 62.24Mb was assembled, generating 50,896 unigenes and 66,906 transcripts. Therefore, 863 upregulated genes and 555 downregulated genes were identified in the FT library. Functional annotation showed that most of the upregulated genes were related to various biological processes involved in responding to abiotic stress. For the first time, a highly expressed cold-shock protein was identified in the reproductive organ of fruit trees. The expression of six genes was validated by RT-PCR. As the first comprehensive analysis of open flowers in a frost-tolerant almond genotype, this study represents a key step toward the molecular breeding of fruit tree species for frost tolerance.

Comprehensive gene and microRNA expression profiling on cardiovascular system in zebrafish co-exposured of SiNPs and MeHg

Air pollution has been shown to increase cardiovascular diseases. However, little attention has been paid to the combined effects of PM and air pollutants on the cardiovascular system. To explore this, a high-throughput sequencing technology was used to determine combined effects of silica nanoparticles (SiNPs) and MeHg in zebrafish. Our study demonstrated that SiNPs and MeHg co-exposure could cause significant changes in mRNA and miRNA expression patterns in zebrafish. The differentially expressed (DE) genes in profiles 17 and 26 of STC analysis suggest that SiNPs and MeHg co-exposure had more proinflammatory and cardiovascular toxicity in zebrafish than single exposure. Major gene functions associated with cardiovascular system in the co-exposed zebrafish were discerned from the dynamic-gene-network, including stxbp1a, celf4, ahr1b and bai2. In addition, the prominently expressed pathway of cardiac muscle contraction was targeted by 3 DE miRNAs identified by the miRNA-pathway-network (dre-miR-7147, dre-miR-26a and dre-miR-375), which included 23 DE genes. This study presents a global view of the combined SiNPs and MeHg toxicity on the dynamic expression of both mRNAs and miRNAs in zebrafish, and could serve as fundamental research clues for future studies, especially on cardiovascular system toxicity.

Systematic analysis of gene expression patterns associated with postmortem interval in human tissues

Postmortem mRNA degradation is considered to be the major concern in gene expression research utilizing human postmortem tissues. A key factor in this process is the postmortem interval (PMI), which is defined as the interval between death and sample collection. However, global patterns of postmortem mRNA degradation at individual gene levels across diverse human tissues remain largely unknown. In this study, we performed a systematic analysis of alteration of gene expression associated with PMI in human tissues. From the Genotype-Tissue Expression (GTEx) database, we evaluated gene expression levels of 2,016 high-quality postmortem samples from 316 donors of European descent, with PMI ranging from 1 to 27 hours. We found that PMI-related mRNA degradation is tissue-specific, gene-specific, and even genotype-dependent, thus drawing a more comprehensive picture of PMI-associated gene expression across diverse human tissues. Additionally, we also identified 266 differentially variable (DV) genes, such as DEFB4B and IFNG, whose expression is significantly dispersed between short PMI (S-PMI) and long PMI (L-PMI) groups. In summary, our analyses provide a comprehensive profile of PMI-associated gene expression, which will help interpret gene expression patterns in the evaluation of postmortem tissues.

Abstract 370: T-PLL cells resemble memory-type T-cells with aberrant effector functions implicating a leukemogenic cooperation of TCL1A and TCR signaling

Abstract The pathogenesis of the rare and aggressive T-cell prolymphocytic leukemia (T-PLL) is poorly understood, which particularly applies to a mechanistic concept around its hallmark oncogene TCL1A. Existing data implicate TCL1A as a catalytic enhancer of the oncogenic kinase AKT, a central node in a T-cell’s antigen receptor (TCR) signaling cascade, which mediates proliferation and differentiation. The levels and role of TCR activation in T-PLL’s pathogenesis are not known. To first clarify which physiological T-cell subset T-PLL cells most resemble, we performed comprehensive global gene expression profiling and immunophenotyping of primary T-PLL (n=79) in comparison to healthy-donor derived T-cell populations. Principle component analyses and gene signature alignments revealed a high similarity of T-PLL cells to (central) memory T-lymphocytes over naïve T-cells. Surface markers revealed a spectrum of memory-type differentiation (n=69/79; 87%) with predominant central-memory stages (n=35/79; 44%). The usually TCR and/or CD28-coreceptor positive T-PLL cells revealed no restrictions to genetic or surface TCR-clonotypes. The abnormally high basal activation levels (surface CD25, CD38, CD69) correlated in their degree with inferior clinical outcomes (med. survival 20.8 vs 58.3 mo.; p=0.0012). In parallel, T-PLL cells lost expression of negative-regulatory TCR-co-receptors (e.g. CTLA-4, LAG3). Fittingly, TCR engagement of primary T-PLL cells revealed a trend to hyperactive intracellular responses and interleukin(IL)-2 release alongside a prominent Th1-cytokine program. T-PLL cells also showed a robust resistance to stimulation-induced cell death and agonistic CD95 ligation. TCR-derived signals (phospho-kinase induction, IL-2 release) were enhanced in vitro by the modulated presence of TCL1A with kinetics indicative of a sensitizer relationship, mainly in the CD3 axis as opposed to the CD28 branch. A mouse model with TCL1A-initiated protracted development of T-PLL (Lckpr-TCL1Atg) revealed congruent findings with the aberrant T-cell phenotype of human T-PLL. TCL1A expressing T-cells of this model, that were further equipped with monoclonal epitope-defined TCRs against ovalbumine or a chimeric-antigen-receptor (CAR) against carcinoembryonic antigen, gained a pre-leukemic growth advantage in scenarios of pulsed or continuous low-level receptor stimulation. Overall, we establish that T-PLL cells resemble antigen-experienced memory T-cells. Retention of functional effector responses to TCR stimulation and loss of restricting activation regulators underlie a highly activated phenotype and a marked resistance to death-inducing signals. TCL1A proactively enhances TCR responses and we postulate that this leukemogenic cooperation drives accumulation of memory-type cells that utilize amplified, hence permissive, low-level cognate antigen input. Note: This abstract was not presented at the meeting. Citation Format: Alexandra Schrader, Kathrin Warner, Sebastian Oberbeck, Giuliano Crispatzu, Petra Mayer, Sabine Pützer, Hans Diebner, Stephan Stilgenbauer, Georg Hopfinger, Jan Dürig, Torsten Haferlach, Mark Lanasa, Ingo Roeder, Michael Hallek, Dimitrios Mougiakakos, Michael von Bergwelt-Baildon, Monika Brüggemann, Sebastian Newrzela, Hinrich Abken, Marco Herling. T-PLL cells resemble memory-type T-cells with aberrant effector functions implicating a leukemogenic cooperation of TCL1A and TCR signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 370. doi:10.1158/1538-7445.AM2017-370

Transcriptome profiling of Cucumis metuliferus infected by Meloidogyne incognita provides new insights into putative defense regulatory network in Cucurbitaceae

Root-knot nematodes (RKN) represent extensive challenges to Cucurbitaceae crops. However, Cucumis metuliferus (Cm) is known to be resistant to Meloidogyne incognita (Mi) infections. Thus, analysis of differentially expressed genes may lead to a comprehensive gene expression profiling of the incompatible Cm-Mi interaction. In this study, the time-course transcriptome of Cm against Mi infection was monitored using RNA-Seq. More than 170000 transcripts were examined in Cm roots, and 2430 genes were subsequently identified as differentially expressed in response to Mi infection. Based on function annotation and orthologs finding, the potential mechanism of transcriptional factor, cytoskeleton, pathogen-related genes and plant hormone were assessed at the transcription level. A comparison of gene expression levels between Mi-infected Cm and cucumber plants revealed that cytoskeleton-related genes are key regulators of Cm resistance to Mi. We herein discuss the dual nature of cytoskeleton-related genes in the susceptibility and resistance of plant hosts to Mi. Our observations provide novel insights into the responses of Cm to Mi at the transcriptome level. The data generated in this study may be useful for elucidating the mechanism underlying resistance to RKNs in cucurbitaceous crops.

Genome wide identification, phylogeny, and expression of bone morphogenetic protein genes in tetraploidized common carp (Cyprinus carpio)

Bone morphogenetic proteins (Bmps) are a group of signaling molecules known to play important roles during formation and maintenance of various organs, not only bone, but also muscle, blood and so on. Common carp (Cyprinus carpio) is one of the most intensively studied fish due to its economic and environmental importance. Besides, common carp has encountered an additional round of whole genome duplication (WGD) compared with many closely related diploid teleost, which make it one of the most important models for genome evolutionary studies in teleost. Comprehensive genome resources of common carp have been developed recently, which facilitate the thorough characterization of bmp gene family in the tetraploidized common carp genome. We identified a total of 44 bmps from the common carp genome, which are twice as many as that of zebrafish. Phylogenetic analysis revealed that most of bmps are highly conserved. Comparative analysis was performed across six typical vertebrate genomes. It appeared that all the bmp genes in common carp were duplicated. Obviously, the expansion of the bmp gene family in common carp was due to the latest additional round of whole genome duplication and made it more abundant than other diploid teleosts. Expression signatures were assessed in major tissues, including gill, intestine, liver, spleen, skin, heart, gonad, muscle, kidney, head kidney, brain and blood, which demonstrated the comprehensive expression profiles of bmp genes in the tetraploidized genome. Significant gene expression divergences were observed which revealed substantial functional divergences of those duplicated bmp genes post the latest WGD event. The conserved synteny blocks of bmp5s revealed the genome rearrangement of common carp post the 4R WGD. The whole set of bmp gene family in common carp provides insight into gene fate of tetraploidized common carp genome post recent WGD.

Identification of putative carboxylesterase and aldehyde oxidase genes from the antennae of the rice leaffolder, Cnaphalocrocis medinalis (Lepidoptera: Pyralidae)

In insects, odorant-degrading enzymes (ODEs) play essential roles in the degradation of volatile odorants and maintenance of olfactory sensitivity. ODEs include several enzyme families with detoxification functions, such as carboxylesterases (CXEs) and aldehyde oxidases (AOXs). The rice leaffolder, Cnaphalocrocismedinalis (Lepidoptera: Pyralidae), is a serious rice insect pest in Asia. In this study, 18 putative CXE genes and four AOX genes were identified from the antennae of C. medinalis by retrieving a previously released transcriptome dataset. BLASTX searching and phylogenetic analyses showed that these genes are closely related to their respective orthologs in other lepidopteran species. Expression patterns of these genes were determined by reverse transcription-quantitative PCR (RT-qPCR). Four candidate genes, including three CmedCXEs (CmedCXE17, CmedCXE20 and CmedCXE24) and one CmedAOX (CmedAOX2) were antenna-enriched and considered potentially involved in odorant degradation. Our findings provide a comprehensive sequence resource and expression profiles of CXE and AOX genes in C. medinalis antennae, which may facilitate further studies of the odorant degradation mechanisms in this insect species.

Defining the transcriptomic landscape of the developing enteric nervous system and its cellular environment

BackgroundMotility and the coordination of moving food through the gastrointestinal tract rely on a complex network of neurons known as the enteric nervous system (ENS). Despite its critical function, many of the molecular mechanisms that direct the development of the ENS and the elaboration of neural network connections remain unknown. The goal of this study was to transcriptionally identify molecular pathways and candidate genes that drive specification, differentiation and the neural circuitry of specific neural progenitors, the phox2b expressing ENS cell lineage, during normal enteric nervous system development. Because ENS development is tightly linked to its environment, the transcriptional landscape of the cellular environment of the intestine was also analyzed.ResultsThousands of zebrafish intestines were manually dissected from a transgenic line expressing green fluorescent protein under the phox2b regulatory elements [Tg(phox2b:EGFP)w37]. Fluorescence-activated cell sorting was used to separate GFP-positive phox2b expressing ENS progenitor and derivatives from GFP-negative intestinal cells. RNA-seq was performed to obtain accurate, reproducible transcriptional profiles and the unbiased detection of low level transcripts. Analysis revealed genes and pathways that may function in ENS cell determination, genes that may be identifiers of different ENS subtypes, and genes that define the non-neural cellular microenvironment of the ENS. Differential expression analysis between the two cell populations revealed the expected neuronal nature of the phox2b expressing lineage including the enrichment for genes required for neurogenesis and synaptogenesis, and identified many novel genes not previously associated with ENS development. Pathway analysis pointed to a high level of G-protein coupled pathway activation, and identified novel roles for candidate pathways such as the Nogo/Reticulon axon guidance pathway in ENS development.ConclusionWe report the comprehensive gene expression profiles of a lineage-specific population of enteric progenitors, their derivatives, and their microenvironment during normal enteric nervous system development. Our results confirm previously implicated genes and pathways required for ENS development, and also identify scores of novel candidate genes and pathways. Thus, our dataset suggests various potential mechanisms that drive ENS development facilitating characterization and discovery of novel therapeutic strategies to improve gastrointestinal disorders.

Illumina-sequencing based transcriptome study of coat color phenotypes in domestic goats

This study performed a comprehensive expression profiling of genes expressed in the skin of goats with three different coat colors by Illumina Sequencing. A total of 91 significantly expressed genes were detected when comparing gray skin to white skin library and these included 74 up-regulated and 17 down-regulated genes in gray skin. There were 67 differentially expressed genes between brown skin and white skin libraries, 23 of which were up-regulated and 44 were down-regulated in brown skin. When we compared brown and gray libraries, 154 differentially expressed genes were found, of which 33 showed higher expression and 121 showed lower expression in brown skin. To our surprise, MC1R, MITF, TYR and KIT showed no significant difference in expression between the goats with three skin colors, whereas ASIP was detected in white skin but not in dark skins. In this study, PMEL, TRPM1, TYRP1 and DCT were significantly up-regulated in brown goat skin compare with gray and white skins. PMEL showed higher expression in gray goat skin compared with white goat skin, whereas there were no significant differences in the expression of TYRP1, TRPM1 and DCT between gray and white skin samples. In addition, ELOVL3 showed higher expression in gray goat skin than in brown and white skins, whereas there was no significant differences in the expression of ELOVL3 between brown and white skin samples. These results expand our understanding of the complex molecular mechanisms of skin physiology and melanogenesis in goat and provide a foundation for future studies.

Whole-Genome DNA Methylation Analyses Revealed Epigenetic Instability in Tumorigenic Human iPS Cell-Derived Neural Stem/Progenitor Cells.

Although human induced pluripotent stem cell (hiPSC) derivatives are considered promising cellular resources for regenerative medicine, their tumorigenicity potentially limits their clinical application in hiPSC technologies. We previously demonstrated that oncogenic hiPSC-derived neural stem/progenitor cells (hiPSC-NS/PCs) produced tumor-like tissues that were distinct from teratomas. To gain insight into the mechanisms underlying the regulation of tumorigenicity in hiPSC-NS/PCs, we performed an integrated analysis using the Infinium HumanMethylation450 BeadChip array and the HumanHT-12 v4.0 Expression BeadChip array to compare the comprehensive DNA methylation and gene expression profiles of tumorigenic hiPSC-NS/PCs (253G1-NS/PCs) and non-tumorigenic cells (201B7-NS/PCs). Although the DNA methylation profiles of 253G1-hiPSCs and 201B7-hiPSCs were similar regardless of passage number, the methylation status of the global DNA methylation profiles of 253G1-NS/PCs and 201B7-NS/PCs differed; the genomic regions surrounding the transcriptional start site of the CAT and PSMD5 genes were hypermethylated in 253G1-NS/PCs but not in 201B7-NS/PCs. Interestingly, the aberrant DNA methylation profile was more pronounced in 253G1-NS/PCs that had been passaged more than 15 times. In addition, we identified aberrations in DNA methylation at the RBP1 gene locus; the DNA methylation frequency in RBP1 changed as 253G1-NS/PCs were sequentially passaged. These results indicate that different NS/PC clones have different DNA methylomes and that DNA methylation patterns are unstable as cells are passaged. Therefore, DNA methylation profiles should be included in the criteria used to evaluate the tumorigenicity of hiPSC-NS/PCs in the clinical setting. Stem Cells 2017;35:1316-1327.