mRNA Libraries Research Articles

Abstract 1711: Immunoprofiling of precision-cut cancer tissue slices (PCCTS) as a tool for cancer immunotherapy

Abstract The recent advances in immunotherapies, such as immune checkpoint modulators, bispecific antibodies, and adoptive T-cell transfer, opens new opportunities for the treatment of cancer. Having this broad spectrum of new therapeutic agents available, the demand for predictive and robust preclinical models to minimize translational failures in immuno-oncology is increasing. Indivumed has successfully implemented a model of Precision Cut Cancer Tissue slices (PCCTS) derived from viable human tumor tissue for different applications such as chemotherapeutic agents, small molecules and antibodies. In this study, we investigated the effects of OKT3® on cancer tissue slices especially in respect of gene expression changes and cytokine release. Fresh tumor pieces from three colorectal cancer patients were cut into 400 µm slices using a Krumdieck Tissue Slicer®. PCCTS were incubated for 4 h and 24 h with and without OKT3®, (Muromonab), a therapeutic antibody against CD3. After defined time points, slices were frozen, and the supernatants were collected. In addition, a second set of slices was formalin fixed and paraffin embedded for immunohistochemical analysis of T-cells. RNA isolated from frozen slices was used for library preparation with the TruSeq Stranded mRNA Library Preparation Kit followed by sequencing on a NextSeq 500 device from Illumina. The results generated by different software tools showed a background of gene expression changes caused by cultivation. On the other hand, a very specific up-regulation of genes was detectable after OKT3® treatment. Especially an induction of genes involved in immune related functions, such as IFN gamma and IL-12 signaling was seen. The analysis of cytokines in the supernatants using a ten-plex panel form Meso Scale Discovery confirmed the activation of T-cells after OKT3® treatment. In all three patients a significant increase of IFN gamma, IL-2, and TNF alpha was measured, comparable to a so called “cytokine storm” that is described in literature. The intensity of the effects correlated to the amount of tumor infiltrating T-cells, quantified by anti-CD3 staining in tissue sections. This model of PCCTS represents a unique opportunity to test immune-modulatory compounds in a fully human, patient derived model that is close to the in vivo situation. The data of this study shows that profiling of the immune response by RNA-Seq can be successfully performed. In addition to gene expression changes that occur after treatment, deconvolution based approaches to quantify tumor Infiltrating lymphocytes (TILs) from RNA-Seq data will be a key parameter to personalize the treatment of patients. Citation Format: Nicole Grabinski, Kristina Bernoth, Aljoscha Leusmann, Carolin Fleischhauer-Biermann, Dorte Wendt, Mirja Piller, Moiken Petersen, Nicole Lange, Anna Tiedemann, Monika Schoeppler, Hartmut Juhl, Gerd Helftenbein, Andrea Miegel, Frank Schnieders, Kerstin David. Immunoprofiling of precision-cut cancer tissue slices (PCCTS) as a tool for cancer immunotherapy [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 1711.

Expressed exome capture sequencing: A method for cost-effective exome sequencing for all organisms.

Exome capture is an effective tool for surveying the genome for loci under selection. However, traditional methods require annotated genomic resources. Here, we present a method for creating cDNA probes from expressed mRNA, which are then used to enrich and capture genomic DNA for exon regions. This approach, called "EecSeq," eliminates the need for costly probe design and synthesis. We tested EecSeq in the eastern oyster, Crassostrea virginica, using a controlled exposure experiment. Four adult oysters were heat shocked at 36°C for 1hr along with four control oysters kept at 14°C. Stranded mRNA libraries were prepared for two individuals from each treatment and pooled. Half of the combined library was used for probe synthesis, and half was sequenced to evaluate capture efficiency. Genomic DNA was extracted from all individuals, enriched via captured probes, and sequenced directly. We found that EecSeq had an average capture sensitivity of 86.8% across all known exons and had over 99.4% sensitivity for exons with detectable levels of expression in the mRNA library. For all mapped reads, over 47.9% mapped to exons and 37.0% mapped to expressed targets, which is similar to previously published exon capture studies. EecSeq displayed relatively even coverage within exons (i.e., minor "edge effects") and even coverage across exon GC content. We discovered 5,951 SNPs with a minimum average coverage of 80×, with 3,508 SNPs appearing in exonic regions. We show that EecSeq provides comparable, if not superior, specificity and capture efficiency compared to costly, traditional methods.

Transcriptomic Analysis of Pseudoscorpion Venom Reveals a Unique Cocktail Dominated by Enzymes and Protease Inhibitors.

Transcriptomic and genomic analyses have illuminated the diversity of venoms in three of the four venomous arachnid orders (scorpions, spiders, and ticks). To date, no venom gland transcriptome analysis has been available for pseudoscorpions, the fourth venomous arachnid lineage. To redress this gap, we sequenced an mRNA library generated from the venom glands of the species Synsphyronus apimelus (Garypidae). High-throughput sequencing by the Illumina protocol, followed by de novo assembly, resulted in a total of 238,331 transcripts. From those, we annotated 131 transcripts, which code for putative peptides/proteins with similar sequences to previously reported venom components available from different arachnid species in protein databases. Transcripts putatively coding for enzymes showed the richest diversity, followed by other venom components such as peptidase inhibitors, cysteine-rich peptides, and thyroglobulin 1-like peptides. Only 11 transcripts were found that code for putatively low molecular mass spider toxins. This study constitutes the first report of the diversity of components within pseudoscorpion venom.

Peculiar genes for thermostable bifunctional catalase-peroxidases in Chaetomium thermophilum and their molecular evolution

Catalase-peroxidases represent one important subfamily of ancestral antioxidant enzymes originally evolved in bacteria for the protection against various forms of oxidative stress. KatG genes coding for these bifunctional catalase-peroxidases were during their peculiar evolution transferred from Bacteroidetes to the fungal phylum Ascomycota via a horizontal gene transfer event. Here we analyse a newly discovered fungal katG gene without introns coding for a thermostable catalase-peroxidase from Chaetomium thermophilum var. dissitum and compare it with closely related thermophilic and mesophilic katGs and their translation products. We show that CthediskatG gene resembling its bacterial counterparts has a typical eukaryotic transcription start site and also contains a conserved eukaryotic polyadenylation signal behind its 3′ terminus. Moreover, we have detected polyA tails in corresponding transcripts of katG from two different mRNA libraries of C. thermophilum var. disstum. Although otherwise highly conserved, only in katG genes of two C. thermophilum variants a unique 60 bp long deletion leading in the translated product with high probability to a modified loop and thus access to the prosthetic heme group was observed. We also present an updated molecular phylogeny revealing the evolutionary position of fungal thermostable catalase-peroxidases within a robust phylogenetic tree of the whole KatG subfamily.

Transcriptional profiling of swine mammary gland during the transition from colostrogenesis to lactogenesis using RNA sequencing

BackgroundColostrum and milk are essential sources of antibodies and nutrients for the neonate, playing a key role in their survival and growth. Slight abnormalities in the timing of colostrogenesis/lactogenesis potentially threaten piglet survival. To further delineate the genes and transcription regulators implicated in the control of the transition from colostrogenesis to lactogenesis, we applied RNA-seq analysis of swine mammary gland tissue from late-gestation to farrowing. Three 2nd parity sows were used for mammary tissue biopsies on days 14, 10, 6 and 2 before (−) parturition and on day 1 after (+) parturition. A total of 15 mRNA libraries were sequenced on a HiSeq2500 (Illumina Inc.). The Dynamic Impact Approach and the Ingenuity Pathway Analysis were used for pathway analysis and gene network analysis, respectively.ResultsA large number of differentially expressed genes were detected very close to parturition (−2d) and at farrowing (+ 1d). The results reflect the extraordinary metabolic changes in the swine mammary gland once it enters into the crucial phases of lactogenesis and underscore a strong transcriptional component in the control of colostrogenesis. There was marked upregulation of genes involved in synthesis of colostrum and main milk components (i.e. proteins, fat, lactose and antimicrobial factors) with a pivotal role of CSN1S2, LALBA, WAP, SAA2, and BTN1A1. The sustained activation of transcription regulators such as SREBP1 and XBP1 suggested they help coordinate these adaptations.ConclusionsOverall, the precise timing for the transition from colostrogenesis to lactogenesis in swine mammary gland remains uncharacterized. However, our transcriptomic data support the hypothesis that the transition occurs before parturition. This is likely attributable to upregulation of a wide array of genes including those involved in ‘Protein and Carbohydrate Metabolism’, ‘Immune System’, ‘Lipid Metabolism’, ‘PPAR signaling pathway’ and ‘Prolactin signaling pathway’ along with the activation of transcription regulators controlling lipid synthesis and endoplasmic reticulum biogenesis and stress response.

A System for Global Analysis of Correlation between Protein Expression and mRNA

The influence of mRNA abundance on protein expression continues to be a topic of significant interest and debate. In various systems, the correlations between these molecules has been reported to be anywhere from 20–70%. Many of these studies have been conducted in complex, in vivo systems, and assayed just a fraction of the genes present due to difficulties in quantitation of many proteins simultaneously. Our approach is to examine this relationship as the result of a single translational event, in a global manner, and without the additional factors introduced by in vivo systems. We created a protein library whose constituents can be decoded by next generation sequencing using cDNA display techniques, a synthetic, bacterial based, translation system, and an expression library derived from an mRNA population. This data, along with sequencing data from the precursor mRNA library, allow for a correlation analysis across all genes contained within the mRNA library. Preliminary results indicates that in this system, ~60% of the variance in protein expression can be explained by mRNA abundance. To determine the stability of the correlation, we modified the translation sequences in the gene library and reanalyzed the protein to mRNA relationship; variations in the correlation reflect the influence of the translational sequences on the protein expression. This system can be utilized to explore the mRNA and protein relationship for wild‐type translational systems, or to evaluate the effects of modifications or synthetic sequences.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract P2-10-05: The impact of TP53 mutation and pathological response to neoadjuvant chemotherapy on triple negative breast cancer outcomes

Abstract Background: Triple Negative Breast Cancer (TNBC) accounts for 15-20% of breast cancers, and has a poor prognosis compared with hormone receptor-positive or HER2-amplified disease. There is currently no targeted therapy to salvage chemotherapy resistant TNBC confirming a need for predictive biomarkers of sensitivity to therapy. This study aimed to evaluate the impact of TP53 mutations, common in TNBC, on sensitivity to therapy and patient outcomes. Methods: TNBC samples (pretreatment biopsies and surgical specimens, n=106) had annotated response to standardized anthracycline/taxane based neoadjuvant chemotherapy (NACT) and 3-year survival data, by which time most recurrences occur in TNBC. RNAseq was performed using Illumina HiSeq with mRNA library preparation protocol to identify TP53 mutations, which were then scored using the Evolutionary Action Score (EAS), a novel computational model shown to be successful at identifying deleterious mutations. This work was supported by an ASCO Conquer Cancer Foundation Young Investigator Award grant. Results: 106 TNBC patients were enrolled on this study, 14 had stage I, 65 stage II, and 24 had stage III disease. 64 patient samples have been analyzed to date, of which 39 (61%) harbored a TP53 mutation, and 24 (38%) were TP53 wildtype. Pathologic complete response (pCR) and survival Of 47 patients who received NACT, 14 (30%) achieved a pCR, and 33 (70%) had residual disease (RD). Patients who achieved a pCR had a significantly better 3-year recurrence free survival (RFS) (100% vs 59%, p=0.011) and overall survival (OS) (100% vs 62%, p-0.01) compared to patients who had residual disease. TP53 status and pCR The rate of pCR was significantly higher in the TP53 mutant group (11/29, 38%) compared to TP53 wildtype (2/17, 12%) TNBC (OR = 4.6, (0.9, 24), p = 0.046). Patients with wild-type and mutant TP53 TNBC who achieved a pCR had excellent 3-year RFS and OS at 100%. Among patients with RD, TP53 wild-type TNBC exhibited a non-significant trend towards higher 3-year RFS (72% vs 49%,HR = 2.5 (0.8, 8.0) p = 0.12) and OS (79% vs 48%,HR = 2.6 (0.8, 8.4) p = 0.11) compared to TP53 mutant TNBC. TP53 status and survival The 3-year OS for TP53 mutant TNBC was 72%, compared to 86% for TP53 wildtype TNBC (HR = 1.5 (0.5, 4.3), p = 0.48). There was no difference in 3-year RFS between the two groups. TP53 EAS and survival Among 39 non-silent TP53 mutant cases, EAS ranged from 42.3 to 100 with a median score of 87.6. The 3-year OS for patients with EAS ≥86.7 was 73%, compared to 74% for patients with EAS <86.7 (HR 0.8 (0.2, 3.0), p=0.78). When separated into tertiles, the 3-year OS rates were 75%, 68%, and 77% for the lower, middle, and upper thirds respectively. The 3-year OS rates for TNBC with EAS = 0 (TP53 wildtype or silent TP53 mutation) was 87%. Conclusion: TP53 mutations were associated with a higher chance of achieving a pCR; NACT Patients who achieved a pCR to NACT had excellent outcomes, regardless of TP53 mutation status. In patients with RD following NACT, TP53 mutant TNBC had a trend towards worse outcomes compared to TP53 wild-type TNBC. Larger cohorts are required to further evaluate the impact of TP53 mutations and TP53 EAS on survival outcomes in TNBC. Citation Format: Mitri Z, Lin C-H, Hess KR, Thompson A, Lichtarge O, Hubbard R, Fu C, Symmans WF, Moulder-Thompson SL. The impact of TP53 mutation and pathological response to neoadjuvant chemotherapy on triple negative breast cancer outcomes [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-10-05.

Microexon gene transcriptional profiles and evolution provide insights into blood processing by the Schistosoma japonicum esophagus

BackgroundAdult schistosomes have a well-developed alimentary tract comprising an oral sucker around the mouth, a short esophagus and a blind ending gut. The esophagus is not simply a muscular tube for conducting blood from the mouth to gut but is divided into compartments, surrounded by anterior and posterior glands, where processing of ingested blood is initiated. Self-cure of rhesus macaques from a Schistosoma japonicum infection appears to operate by blocking the secretory functions of these glands so that the worms cease feeding and slowly starve to death. Here we use subtractive RNASeq to characterise the genes encoding the principal secretory products of S. japonicum esophageal glands, preparatory to evaluating their relevance as targets of the self-cure process.Methodology/Principal findingsThe heads and a small portion of the rear end of male and female S. japonicum worms were separately enriched by microdissection, for mRNA isolation and library construction. The sequence reads were then assembled de novo using Trinity and those genes enriched more than eightfold in the head preparation were subjected to detailed bioinformatics analysis. Of the 62 genes selected from the male heads, more than one third comprised MEGs encoding secreted or membrane-anchored proteins. Database searching using conserved motifs revealed that the MEG-4 and MEG-8/9 families had counterparts in the bird schistosome Trichobilharzia regenti, indicating an ancient association with blood processing. A second group of MEGs, including a MEG-26 family, encoded short peptides with amphipathic properties that most likely interact with ingested host cell membranes to destabilise them. A number of lysosomal hydrolases, two protease inhibitors, a secreted VAL and a putative natterin complete the line-up. There was surprisingly little difference between expression patterns in males and females despite the latter processing much more blood.Significance/ConclusionsThe mixture of approximately 40 proteins specifically secreted by the esophageal glands is responsible for initiating blood processing in the adult worm esophagus. They comprise the potential targets for the self-cure process in the rhesus macaque, and thus represent a completely new cohort of secreted proteins that can be investigated as vaccine candidates.

Discovery of Transcription Factors Novel to Mouse Cerebellar Granule Cell Development Through Laser-Capture Microdissection.

Laser-capture microdissection was used to isolate external germinal layer tissue from three developmental periods of mouse cerebellar development: embryonic days 13, 15, and 18. The cerebellar granule cell-enriched mRNA library was generated with next-generation sequencing using the Helicos technology. Our objective was to discover transcriptional regulators that could be important for the development of cerebellar granule cells-the most numerous neuron in the central nervous system. Through differential expression analysis, we have identified 82 differentially expressed transcription factors (TFs) from a total of 1311 differentially expressed genes. In addition, with TF-binding sequence analysis, we have identified 46 TF candidates that could be key regulators responsible for the variation in the granule cell transcriptome between developmental stages. Altogether, we identified 125 potential TFs (82 from differential expression analysis, 46 from motif analysis with 3 overlaps in the two sets). From this gene set, 37 TFs are considered novel due to the lack of previous knowledge about their roles in cerebellar development. The results from transcriptome-wide analyses were validated with existing online databases, qRT-PCR, and in situ hybridization. This study provides an initial insight into the TFs of cerebellar granule cells that might be important for development and provide valuable information for further functional studies on these transcriptional regulators.

Purification of Exosomes from Primary Schwann Cells, RNA Extraction, and Next-Generation Sequencing of Exosomal RNAs.

Exosomes are small (30-150nm) vesicles of endosomal origin secreted by most cell types. Exosomes contain proteins, lipids, and RNA species including microRNA, mRNA, rRNA, and long noncoding RNAs. The mechanisms associated with exosome synthesis and cargo loading are still poorly understood. A role for exosomes in intercellular communication has been reported in physiological and pathological conditions both in vitro and in vivo. Previous studies have suggested that Schwann cell-derived exosomes regulate neuronal functions, but the mechanisms are still unclear. Here, we describe protocols to establish rat neonatal Schwann cell cultures and to isolate exosomes from the conditioned medium of these cultures by differential ultracentrifugation. To analyze the RNA content of Schwann cell-derived exosomes, we detail protocols for RNA extraction and next-generation sequencing using miRNA and mRNA libraries. The protocol also includes RNA sequencing of Schwann cells, which allows the comparison between RNA content from cells and the secreted exosomes. Identification of RNAs present in Schwann cell-derived exosomes is a valuable tool to understand novel roles of Schwann cells in neuronal function in health and disease.

High Impact Gene Discovery: Simple Strand-Specific mRNA Library Construction and Differential Regulatory Analysis Based on Gene Co-Expression Network.

Plant transcription factors have potential to behave as hubs in gene regulatory networks through altering the expression of many downstream genes, and identification of such hub transcription factors strongly enhances our understating of biological processes. Transcriptome analysis has become a staple of gene expression analyses. In addition to current advances in Next Generation Sequencing (NGS) technology, various methods for mRNA library construction and downstream data analyses have been enthusiastically developed. Here, we describe Breath Adapter Directional sequencing (BrAD-seq), a simple strand-specific mRNA library preparation for the Illumina platform, allowing easy scaling of transcriptome experiments with low reagent and labor costs. This protocol includes our recent modifications and the detailed practical procedure for BrAD-seq. Because extracting biological meanings from large-scale transcriptome data presents a significant challenge, we also describe a new analytical method that goes beyond differential expression. Differential regulatory analysis (DRA) is based on a gene co-expression network to address which regulatory factor or factors have the ability to predict the abundance of differentially expressed genes between two groups or conditions. This protocol provides a ready-to-use informatics pipeline from raw sequence data to DRA for plant transcriptome datasets.

Abstract A60: Full-length transcripts identification from deep transcriptomic analysis of Bothrops jararaca venom gland

Abstract Introduction: Head and neck cancer is a collective term defined by topographic and anatomic features to describe malignant tumors of the upper aerodigestive tract. Despite the different strategies used in the treatment of head and neck squamous cell carcinoma (HNSCC), the overall survival rate is around 50% and the main reason for treatment failure is the frequent development of loco-regional recurrences, which affect around 30% of HNSCC patients. The increase in cancer diagnosis makes it necessary to search for alternative sources to inhibit tumor progression, as well as the development of more efficient drugs. Identification of natural resources as therapeutic potential may lead to the development of new antitumor treatments. Objectives: The aim was to identify transcripts expressed in venom glands of the Bothrops jararaca snake with therapeutic potential for head and neck squamous cell carcinoma (HNSCC) tumors. Materials and Methods: The mRNA library from B. jararaca was sequenced using Illumina NextSeq 500 platform (76 bp unpaired-end) and NextSeq 500/550 Mid Output v2 kit (150 cycles). Library was constructed using TruSeq Stranded mRNA LT Sample Prep Kit (Illumina). De novo assembly of the B. jararaca transcriptome was performed using Velvet software to assemble the transcriptome contigs. Results: The RNA-seq of B. jararaca venom gland generated 4,401,692 raw reads. The de novo Velvet assembly produced 3,498,133 contigs, which matched against NCBI-Blastn transcripts. From these contigs were identified 3,638 full-length transcripts, from which were selected two potential therapeutic genes, Fosfolipases A2 1 and Fosfolipases A2 2. Conclusions: In conclusion, this transcriptome sequencing identified two potential therapeutic transcripts, Fosfolipases A2 1 and Fosfolipases A2 2, which will be analyzed in HNSCC cell lines. Citation Format: Elisa Alves Messias, Flavia Coltri Eguchi, Bruna Pereira Sorroche, Angela Das Neves Oliveira, Lidia Maria Rebolho Batista Arantes, Ana Carolina De Carvalho, Anita mitico tanaka Azevedo, Kathleen Fernandes Grego, Matias Eliseo Melendez, Andre Lopes Carvalho. Full-length transcripts identification from deep transcriptomic analysis of Bothrops jararaca venom gland [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr A60.

Abstract A61: Identification of therapeutic proteins in venom gland of Ectatomma opaciventre for head and neck tumor therapy

Abstract Background: Head and neck cancer is the sixth most common cancer site worldwide and is associated with low survival and high mortality rates, when diagnosed in advanced stages. The pursuit for potential therapeutic targets in natural resources may lead to the development of new antineoplastic agents. Several compounds coming from venomous animals are being tested in clinical trials due to their high selectivity and different physiologic activities. In this way, ant-venom glands are composed of bioactive molecules, such as peptides/proteins and allergens, which may be considered as potential therapeutic agents for oncology. Objectives: The aim was to identify potential therapeutic proteins expressed in venomous glands of Ectatomma opaciventre ants, for the treatment of head and neck squamous cell carcinoma (HNSCC) tumors. Materials and Methods: The mRNA library of Ectatomma opaciventre was sequenced using Illumina NextSeq 500 platform (76 bp unpaired-end) and NextSeq 500/550 Mid Output v2 kit (150 cycles). Libraries were constructed using TruSeq Stranded mRNA LT Sample Prep Kit (Illumina). A de novo assembly of the Ectatomma opaciventre transcriptome was performed using Velvet software. Results: The RNA-seq of E. opaciventre venom gland generated 55,131,904 raw reads. The de novo Velvet assembly produced 23,254 contigs, which matched against 12,061 NCBI-Blastn transcripts. From these contigs, 437 full-length transcripts were identified, from which two potential therapeutic genes were selected: SPARC and Phospholipase B. Both SPARC and Phospholipase B genes have been previously described as having antitumor properties. Conclusions: In conclusion, this deep transcriptome sequencing may have identified two potential therapeutic proteins, which are being analyzed in HNSCC cell lines. Citation Format: Ângela das Neves Oliveira, Lucas Ian Veloso Correia, Lídia Maria Rebolho Batista Arantes, Ana Carolina de Carvalho, Elisa Alves Messias, Bruna Pereira Sorroche, Flávia Coltri Eguchi, Renata Santos Rodrigues, Matias Eliseo Melendez, André Lopes Carvalho. Identification of therapeutic proteins in venom gland of Ectatomma opaciventre for head and neck tumor therapy [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr A61.

Integrated analysis of microRNA and mRNA expression profiles during the sex-differentiation sensitive period in oriental river prawn, Macrobrachium nipponense

Male oriental river prawns (Macrobrachium nipponense) grow faster than females, and therefore, reach larger sizes by harvest time. Histological observations have indicated that the sex-differentiation sensitive period (which includes the formation of the androgenic gland, the testis, and the ovary) is from post-larvae (PL) developmental stage for M. nipponense. In this study, we prepared four microRNA (miRNA) and mRNA libraries using samples collected from sex-differentiation sensitive period (PL7 to PL16) to perform RNA-sequencing for identifying sex-related candidate miRNAs, genes, and metabolic pathways. A total of nine intersection miRNAs were identified, of which three were highly expressed in the androgenic gland, and their expression was verified by quantitative Real-Time PCR (qPCR). These three miRNAs and their 11 predicted target genes may be strong candidates for sex-related miRNAs and sex-related genes in M. nipponense. Five vital sex-related metabolic pathways were also identified that may regulate other sex-differentiation and sex-determination mechanisms. Finding of the study provide important insights to enhance our understanding on sex-differentiation and sex-determination mechanisms for M. nipponense.

Identification of pterygium-related mRNA expression profiling by microarray analysis.

PurposePterygium is a common degenerative and proliferative disease of the ocular surface. In some severe cases, pterygium may lead to irregular corneal astigmatism and corneal stromal scarring with visual impairment. The proliferative capacities of pterygial cells make them appear similar to tumorigenesis. Although considerable progress has been made towards understanding the etiology of the disease, the pathogenesis of pterygium has not been completely understood. In our previous study, we constructed pterygium-related lncRNA libraries by using microarray to investigate the potential roles of lncRNAs in pterygium. In this study, our objective was to explore the role of mRNA in pterygium.Patients and methodsIn this study, we constructed pterygium-related mRNA libraries by using microarray to investigate the potential roles of mRNAs in pterygium. Quantitative real-time PCR (qRT-PCR) was performed to validate some of the deregulated mRNAs in 10 patients.ResultsA total of 1485 upregulated and 2978 downregulated mRNAs were identified in pterygium tissues compared with paired adjacent normal conjunctival tissues (log fold change>2.0). qRT-PCR was performed to validate four overregulated and two underregulated mRNAs in 10 patients.ConclusionsOur results reveal differentially expressed mRNAs in pterygium and suggest that mRNAs may be the novel molecular targets for therapy of pterygium.

Profiling of drought-responsive microRNA and mRNA in tomato using high-throughput sequencing

BackgroundAbiotic stresses cause severe loss of crop production. Among them, drought is one of the most frequent environmental stresses, which limits crop growth, development and productivity. Plant drought tolerance is fine-tuned by a complex gene regulatory network. Understanding the molecular regulation of this polygenic trait is crucial for the eventual success to improve plant yield and quality. Recent studies have demonstrated that microRNAs play critical roles in plant drought tolerance. However, little is known about the microRNA in drought response of the model plant tomato. Here, we described the profiling of drought-responsive microRNA and mRNA in tomato using high-throughput next-generation sequencing.ResultsDrought stress was applied on the seedlings of M82, a drought-sensitive cultivated tomato genotype, and IL9–1, a drought-tolerant introgression line derived from the stress-resistant wild species Solanum pennellii LA0716 and M82. Under drought, IL9–1 performed superior than M82 regarding survival rate, H2O2 elimination and leaf turgor maintenance. A total of four small RNA and eight mRNA libraries were constructed and sequenced using Illumina sequencing technology. 105 conserved and 179 novel microRNAs were identified, among them, 54 and 98 were differentially expressed upon drought stress, respectively. The majority of the differentially-expressed conserved microRNAs was up-regulated in IL9–1 whereas down-regulated in M82. Under drought stress, 2714 and 1161 genes were found to be differentially expressed in M82 and IL9–1, respectively, and many of their homologues are involved in plant stress, such as genes encoding transcription factor and protein kinase. Various pathways involved in abiotic stress were revealed by Gene Ontology and pathway analysis. The mRNA sequencing results indicated that most of the target genes were regulated by their corresponding microRNAs, which suggested that microRNAs may play essential roles in the drought tolerance of tomato.ConclusionIn this study, numerous microRNAs and mRNAs involved in the drought response of tomato were identified using high-throughput sequencing, which will provide new insights into the complex regulatory network of plant adaption to drought stress. This work will also help to exploit new players functioning in plant drought-stress tolerance.

Effect of TCHL-based therapy on immune cell content in on-treatment, neoadjuvant-treated HER2-positive breast cancer patients.

583 Background: In the TCHL trial (NCT01485926) 78 women with HER2-positive breast cancer (BC) underwent neo-adjuvant treatment with either TCH (Docetaxel, Carboplatin, Trastuzumab) or TCHL (TCH + Lapatinib) therapy. Of the 78 patients, 24 consented to an optional on-treatment biopsy 20 days after 1 cycle of therapy. We analysed the impact of tumour infiltrating lymphocytes (TILs) on pathological complete response (pCR) and also determined the impact of TCH/TCHL therapy on immune cell modulation after 20 days of treatment. Methods: We assessed TIL and stromal lymphocytes (SL) counts using immunohistochemical staining with Haemotoxalyin+Eosin, AE1/AE3 and CD45 in formalin fixed paraffin embedded (FFPE) baseline biopsy samples and in fresh frozen (FF) biopsies taken 20-days post cycle 1 (Day-20) of TCH/TCHL. RNA libraries were generated, using the Truseq mRNA library prep kit on the Neoprep platform and sequenced on the NextSeq 500. We measured the transcriptomic profile of 8 pre and on-treatment sample pairs and then used the Microenvironment Cell Populations (MCP)-counter method to measure the abundance of 10 immune cell populations (T cells, CD8 T cells, cytotoxic lymphocytes, NK cells, B lineage, myeloid dendritic cells, neutrophils, endothelial cells and fibroblasts). Results: We found that higher baseline levels of TILs (p = 0.045) but not SL were associated with an increased likelihood of a patient achieving a pCR to TCH/L based therapy. We found in day 20 on-treatment biopsies of women that subsequently went onto have a pCR that levels of SLs but not TILs were significantly higher (p = 0.049) than in those women who did not have a pCR. Finally we found significant increases in the level of monocytes (p = 0.05) and fibroblasts (p = 0.01), but not other immune cell populations, in the day 20 on-treatment biopsies in comparison with the mutated pre-treatment biopsies. Conclusions: In our study baseline TILs but not SLs have a predictive role in the likelihood of a patient achieving a pCR. We also found that TCHL based therapy significantly altered both monocytes and fibroblasts, indicating a possible role for these immune subtypes in response to TCHL therapy.

Salt stress affects mRNA editing in soybean chloroplasts.

Soybean, a crop known by its economic and nutritional importance, has been the subject of several studies that assess the impact and the effective plant responses to abiotic stresses. Salt stress is one of the main environmental stresses and negatively impacts crop growth and yield. In this work, the RNA editing process in the chloroplast of soybean plants was evaluated in response to a salt stress. Bioinformatics approach using sRNA and mRNA libraries were employed to detect specific sites showing differences in editing efficiency. RT-qPCR was used to measure editing efficiency at selected sites. We observed that transcripts of NDHA, NDHB, RPS14 and RPS16 genes presented differences in coverage and editing rates between control and salt-treated libraries. RT-qPCR assays demonstrated an increase in editing efficiency of selected genes. The salt stress enhanced the RNA editing process in transcripts, indicating responses to components of the electron transfer chain, photosystem and translation complexes. These increases can be a response to keep the homeostasis of chloroplast protein functions in response to salt stress.

An efficient field and laboratory workflow for plant phylotranscriptomic projects.

Premise of the study:We describe a field and laboratory workflow developed for plant phylotranscriptomic projects that involves cryogenic tissue collection in the field, RNA extraction and quality control, and library preparation. We also make recommendations for sample curation.Methods and Results:A total of 216 frozen tissue samples of Caryophyllales and other angiosperm taxa were collected from the field or botanical gardens. RNA was extracted, stranded mRNA libraries were prepared, and libraries were sequenced on Illumina HiSeq platforms. These included difficult mucilaginous tissues such as those of Cactaceae and Droseraceae.Conclusions:Our workflow is not only cost effective (ca. $270 per sample, as of August 2016, from tissue to reads) and time efficient (less than 50 h for 10–12 samples including all laboratory work and sample curation), but also has proven robust for extraction of difficult samples such as tissues containing high levels of secondary compounds.

Abstract A14: The transcriptomic landscape of the two emblematic colorectal cancer cell lines HCT116 and HT29

Abstract Colorectal cancer (CRC) is the third leading cause of malignant mortality worldwide, accounting for more than 13% of all cancer death. CRC harbors several alterations, mainly, two major classes of these instability were described: 1/microsatellite instability (MSI) caused by the inactivation and loss of expression of the mismatch repair genes (MMR), and 2/chromosome instability or microsatellite stable (MSS). CRC Cell lines are invaluable biomedical research tools to study these alterations. Two emblematic cell lines for CRC were extensively used, HT29 for MSS and HCT116 for MSI. Short and long reads, high coverage whole transcriptome sequencing of these two cell lines and two normal cell lines, enable us to establish the signaling pathways altered in these two cancer cell lines. Method: Total RNA were extracted from four cell lines HT29, HCT116, CR1780 and CRL18 using RNeasy Mini Kit (Qiagen). mRNA libraries were generated using TruSeq Stranded mRNA Library Prep Kit (Illumina), and high-throughput sequencing were performed using Illumina Systems. Pair-end reads of 100bp and 300bp were generated by Hiseq and Miseq, respectively. All reads were filtered for high-quality reads (≥Q30, adapters trimming and broken pair-reads removing). Reads alignment and genes profiling analysis were done with CLCbio software. Genes upregulated and downregulated with p-value <0.05, in cancer cell lines versus normal cell lines and between the two cancer cell lines were analyzed by Ingenuity Pathways Analysis (IPA). Results: The transcriptome analysis reveals 11,564 different splice variants are differentially expressed in both cancer cell lines HT29 and HCT116, when compared to the two normal cell lines (p-value <0.05). Among which, 9041 transcripts are upregulated and 2523 downregulated. The comparison between HT29 and HCT116 reveals only 333 splice variants differentially expressed. The canonical signaling pathways analysis demonstrates extensive molecular heterogeneity in cancer cell lines. in which, two major pathways clearly appear, mTOR signaling pathway (mTORsp) and estrogen receptor signaling pathway (ERsp). Half or more the number of molecules, which constitute these two pathways, has altered transcription expression, 96 genes among 194 molecules of mTORsp, and 66 genes among 127 molecules of ERsp. Interestingly, when comparing cancer cell lines with each other, 47 genes among 194 of mTORsp have altered expression and no gene from ERsp was found. Conclusion: Our finding established the signaling pathways altered in HT29 and HCT116 and shows the importance of mTOR and estrogen receptor signaling pathways in these two cancer cell lines. Citation Format: Hicham Mansour, Noureddine BOUAROUROU, Jean-Pierre Roperch, Faouzi Habib, Mourad Said. The transcriptomic landscape of the two emblematic colorectal cancer cell lines HCT116 and HT29. [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer: From Initiation to Outcomes; 2016 Sep 17-20; Tampa, FL. Philadelphia (PA): AACR; Cancer Res 2017;77(3 Suppl):Abstract nr A14.