Significant Variation In Expression Research Articles

Panitumumab and cetuximab affect differently miRNA expression in colorectal cancer cells.

Background &aim: Resistance to anti-EGFR monoclonal antibodiesin metastatic colorectal cancer (CRC)is frequent and prognostic biomarkers are lacking. MicroRNAs (miR) are good candidates in this context. We aimed to characterize cetuximab and panitumumab exposure influence on miR expression in colorectal cancer cells to identify those regulating the EGFR pathway and implicated in resistance to treatment. Finally, we aimed to identify miR expression in serum of patients with advanced CRC treated with cetuximab or panitumumab. Results: Cetuximab and panitumumab exposure induced significant expression variations of 17 miR out of a miRnome panel of 752. Six of those miR interacted with at least one downstream element of the EGFR pathway. Conclusion: After the bioinformatics two-phase process, five miR rarely described before could be potential actors of anti-EGFR monoclonal antibodyresistance: miR-95-3p, miR-139-5p, miR-145-5p, miR-429 and miR-1247-5p. In vivo, we detected the expression of miR-139-5p and miR-145-5p in serum of patients with metastatic CRC.

Neuroendocrine regulation of reproduction in Atlantic cod (Gadus morhua): Evidence of Eya3 as an integrator of photoperiodic cues and nutritional regulation to initiate sexual maturation

Evidence from mammals and aves alludes to a possibly conserved seasonal photoperiod induced neuroendocrine cascade which stimulates subsequent sexual maturation however our understanding of this mechanism in teleosts is lacking. Unlike all teleosts studied to date, the Atlantic cod (Gadus morhua) is a short day breeder with the reduction in day-length from the summer solstice stimulating gametogenesis. Cod specific orthologues of eya3, tshβ and dio2 were identified and their expression was monitored in the brain and pituitary of cod held under either stimulated or inhibited photoperiod conditions. While no differential expression was apparent in brain dio2 &tshβ and pituitary tshβ, there was significant temporal variation in expression of pituitary eya3 under the SNP treatment, with expression level elevating in association with active gametogenesis. Under the LL treatment, sexual maturation was inhibited and there was a corresponding suppression of eya3 expression. In a second study the impact of size/energetic status on the initiation of sexual maturation was investigated. In the feed restricted population maturation was significantly suppressed (5% sexually mature) compared to the ab libitum fed stock (95% sexually mature) with there being a concomitant significant suppression in pituitary eya3 expression. Overall, these results suggest that pituitary eya3 has the potential to act as an integrator of both environmental and energetic regulation of sexual maturation of cod. Being the first account of eya3 induction in a short day breeding teleost, the conserved association with stimulation of reproduction and not seasonal state indicates that the upstream drivers which initiate the pathway differ among vertebrates according to their breeding strategies, but the pathway itself and its role in the reproductive cascade appears to be conserved across the vertebrate clade.

Adrenomedullin Expression Characterizes Leukemia Stem Cells and Associates With an Inflammatory Signature in Acute Myeloid Leukemia.

Adrenomedullin (ADM) is a hypotensive and vasodilator peptide belonging to the calcitonin gene-related peptide family. It is secreted in vitro by endothelial cells and vascular smooth muscle cells, and is significantly upregulated by a number of stimuli. Moreover, ADM participates in the regulation of hematopoietic compartment, solid tumors and leukemias, such as acute myeloid leukemia (AML). To better characterize ADM involvement in AML pathogenesis, we investigated its expression during human hematopoiesis and in leukemic subsets, based on a morphological, cytogenetic and molecular characterization and in T cells from AML patients. In hematopoietic stem/progenitor cells and T lymphocytes from healthy subjects, ADM transcript was barely detectable. It was expressed at low levels by megakaryocytes and erythroblasts, while higher levels were measured in neutrophils, monocytes and plasma cells. Moreover, cells populating the hematopoietic niche, including mesenchymal stem cells, showed to express ADM. ADM was overexpressed in AML cells versus normal CD34+ cells and in the subset of leukemia compared with hematopoietic stem cells. In parallel, we detected a significant variation of ADM expression among cytogenetic subgroups, measuring the highest levels in inv(16)/t(16;16) or complex karyotype AML. According to the mutational status of AML-related genes, the analysis showed a lower expression of ADM in FLT3-ITD, NPM1-mutated AML and FLT3-ITD/NPM1-mutated cases compared with wild-type ones. Moreover, ADM expression had a negative impact on overall survival within the favorable risk class, while showing a potential positive impact within the subgroup receiving a not-intensive treatment. The expression of 135 genes involved in leukemogenesis, regulation of cell proliferation, ferroptosis, protection from apoptosis, HIF-1α signaling, JAK-STAT pathway, immune and inflammatory responses was correlated with ADM levels in the bone marrow cells of at least two AML cohorts. Moreover, ADM was upregulated in CD4+ T and CD8+ T cells from AML patients compared with healthy controls and some ADM co-expressed genes participate in a signature of immune tolerance that characterizes CD4+ T cells from leukemic patients. Overall, our study shows that ADM expression in AML associates with a stem cell phenotype, inflammatory signatures and genes related to immunosuppression, all factors that contribute to therapy resistance and disease relapse.

Comparative Transcriptome Analysis of Key Genes and Pathways Activated in Response to Fat Deposition in Two Sheep Breeds With Distinct Tail Phenotype.

Fat tail in sheep presents a valuable energy reserve that has historically facilitated adaptation to harsh environments. However, in modern intensive and semi-intensive sheep industry systems, breeds with leaner tails are more desirable. In the present study, RNA sequencing (RNA-Seq) was applied to determine the transcriptome profiles of tail fat tissues in two Chinese sheep breeds, fat-rumped Altay sheep and thin-tailed Xinjiang fine wool (XFW) sheep, with extreme fat tail phenotype difference. Then the differentially expressed genes (DEGs) and their sequence variations were further analyzed. In total, 21,527 genes were detected, among which 3,965 displayed significant expression variations in tail fat tissues of the two sheep breeds (P < 0.05), including 707 upregulated and 3,258 downregulated genes. Gene Ontology (GO) analysis disclosed that 198 DEGs were related to fat metabolism. In Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, the majority of DEGs were significantly enriched in “adipocytokine signaling,” “PPAR signaling,” and “metabolic pathways” (P < 0.05); moreover, some genes were involved in multiple pathways. Among the 198 DEGs, 22 genes were markedly up- or downregulated in tail fat tissue of Altay sheep, indicating that these genes might be closely related to the fat tail trait of this breed. A total of 41,724 and 42,193 SNPs were detected in the transcriptomic data of tail fat tissues obtained from Altay and XFW sheep, respectively. The distribution of seven SNPs in the coding regions of the 22 candidate genes was further investigated in populations of three sheep breeds with distinct tail phenotypes. In particular, the g.18167532T/C (Oar_v3.1) mutation of the ATP-binding cassette transporter A1 (ABCA1) gene and g.57036072G/T (Oar_v3.1) mutation of the solute carrier family 27 member 2 (SLC27A2) gene showed significantly different distributions and were closely associated with tail phenotype (P < 0.05). The present study provides transcriptomic evidence explaining the differences in fat- and thin-tailed sheep breeds and reveals numerous DEGs and SNPs associated with tail phenotype. Our data provide a valuable theoretical basis for selection of lean-tailed sheep breeds.

Combining Everolimus and Ku0063794 Promotes Apoptosis of Hepatocellular Carcinoma Cells via Reduced Autophagy Resulting from Diminished Expression of miR-4790-3p.

It is challenging to overcome the low response rate of everolimus in the treatment of patients with hepatocellular carcinoma (HCC). To overcome this challenge, we combined everolimus with Ku0063794, the inhibitor of mTORC1 and mTORC2, to achieve higher anticancer effects. However, the precise mechanism for the synergistic effects is not clearly understood yet. To achieve this aim, the miRNAs were selected that showed the most significant variation in expression according to the mono- and combination therapy of everolimus and Ku0063794. Subsequently, the roles of specific miRNAs were determined in the processes of the treatment modalities. Compared to individual monotherapies, the combination therapy significantly reduced viability, increased apoptosis, and reduced autophagy in HepG2 cells. The combination therapy led to significantly lower expression of miR-4790-3p and higher expression of zinc finger protein225 (ZNF225)—the predicted target of miR-4790-3p. The functional study of miR-4790-3p and ZNF225 revealed that regarding autophagy, miR-4790-3p promoted it, while ZNF225 inhibited it. In addition, regarding apoptosis, miR-4790-3p inhibited it, while ZNF225 promoted it. It was also found that HCC tissues were characterized by higher expression of miR-4790-3p and lower expression of ZNF225; HCC tissues were also characterized by higher autophagic flux. We, thus, conclude that the potentiated anticancer effect of the everolimus and Ku0063794 combination therapy is strongly associated with reduced autophagy resulting from diminished expression of miR-4790-3p, as well as higher expression of ZNF225.

Gene expression profiles of oral soft tissue-derived fibroblast from healing wounds: correlation with clinical outcome, autophagy activation and fibrotic markers expression.

Our aim was to evaluate gene expression profiling of fibroblasts from human alveolar mucosa (M), buccal attached gingiva (G) and palatal (P) tissues during early wound healing, correlating it with clinical response. M, G and P biopsies were harvested from six patients at baseline and 24hr after surgery. Clinical response was evaluated through Early wound Healing Score (EHS). Fibrotic markers expression and autophagy were assessed on fibroblasts isolated from those tissues by Western blot and qRT-PCR. Fibroblasts from two patients were subjected to RT2 profiler array, followed by network analysis of the differentially expressed genes. The expression of key genes was validated with qRT-PCR on all patients. At 24hr after surgery, EHS was higher in P and G than in M. In line with our clinical results, no autophagy and myofibroblast differentiation were observed in G and P. We observed significant variations in mRNA expression of key genes: RAC1, SERPINE1 and TIMP1, involved in scar formation; CDH1, ITGA4 and ITGB5, contributing to myofibroblast differentiation; and IL6 and CXCL1, involved in inflammation. We identified some genes involved in periodontal soft tissue clinical outcome, providing novel insights into the molecular mechanisms of oral repair (ClinicalTrial.gov-NCT04202822).

Abstract PO035: Identification and validation of endometrial cancer specific miRNA biomarkers in endometrial fluid collected using saline infused sonohysterography techniques

Abstract Abnormal uterine bleeding is a common benign gynecologic complaint around the time of menopause, and is also the most common symptom of endometrial cancer. This complaint prompts in-office endometrial biopsy to exclude malignancy. Unfortunately, because only a limited portion of the lining tissue is sampled, the endometrial biopsy is not always accurate which can lead to delays in diagnosis and the potential for worsening of the disease and prognosis. Biomolecules have proven to be useful tools in cancer screening and early diagnosis. Liquid biopsy has been widely accepted as a less- or non-invasive way to obtain samples from patients, including blood, urine, saliva and other types of body fluids. However, no circulating biomarkers have been easily identified in the patient blood of endometrial cancer. MicroRNA are a class of small molecules and has shown promise in cancer detection. MicroRNAs may be detected in the biopsy tissue or by testing the fluid that cells secrete. Endometrial tissue is inherently difficult to sample in the office. Although direct sampling from the endometrium has limitations, but the fluid secreted by the lining endometrial epithelial cells can be easily sampled using a technique adapted from an in-office procedure known as a sonohysterography. This procedure is used in the evaluation of abnormal uterine bleeding by introducing saline solution into the endometrial cavity as a contrast material during transvaginal ultrasound. The infused saline is normally discarded, but can be collected for microRNA analysis. In this study, we report the preliminary results of potential miRNA biomarkers identified in 60 patient endometrial fluid collected using sonohystography technique. Patients were recruited who planned to undergo hysterectomy for either benign indication or for a known endometrial cancer diagnosis. The endometrial fluid was collected prior to surgery using a technique similar to that used in sonohysterography. The miRNA was extracted and quantitated. Following the reverse transcription, each sample was amplified on a customized microarray plate with known specific miRNA target primer printed in each well (miScrpt miRNA PCR Array). Patient demographic and health information was collected to be used in the final analysis along with the final pathology results of their surgery. In total, 60 patient samples have been collected and processed (30 cancer and 30 controls). Significant variations in expression were identified in several miRNA markers. Markers with at least a two-fold increase (e.g. miR-429, miR-146a-5p, miR-34b-3p) or decrease (e.g. miR-296-5p, miR-204-5p) in expression were considered candidates for validation. Twelve miRNA markers with the greatest variation in expression were chosen to be used on an additional set of patients to further validate the results. The results will be presented. The method developed here will provide a non-invasive way in early diagnosis and screening for endometrial cancer and therefore impact on improvement of women’s health. Citation Format: Jianing Yang, Joel Barkley, Kameron Firouzi, Bikash Bhattarai, Dean Coonrod, Frederic Zenhausern. Identification and validation of endometrial cancer specific miRNA biomarkers in endometrial fluid collected using saline infused sonohysterography techniques [abstract]. In: Proceedings of the AACR Virtual Special Conference: Endometrial Cancer: New Biology Driving Research and Treatment; 2020 Nov 9-10. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(3_Suppl):Abstract nr PO035.

Hippophae rhamnoides mediate gene expression profiles against keratinocytes infection of Staphylococcus aureus.

Staphylococcus aureus causes a wide range of skin diseases such as bacterial keratitis, follicles, psoriasis, cellulitis and atopic dermatitis. This study aims to investigate the S. aureus mediated molecular modulation, and the effect of HR in reversing the deleterious impact of S. aureus in keratinocytes. Human keratinocyte (HaCaT) cells were cultured in DMEM, supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Subcultures were divided into three flasks: control with no S. aureus and extract (C), S. aureus infected (SA) and S. aureus infected cells and extract (SE). RNA was isolated and microarray analysis was performed. The data was annotated using GO functional analysis and DAVID functional annotation. For each comparison group, significant probes were filtered out at significant cut off by fold change (P < 0.05 and/or > twofold change). For SA vs control, SE vs control, and SE vs SA, 204, 9369, 9900 probes were filtered respectively. In SA vs control, TNF signaling, NOD-like receptor and chemokine receptor signaling pathways were upregulated with key genes such as CCL2, CCL20 and BIRC3. The SE vs SA, showed significant expression variations of a number of important genes. Molecular pathways associated with ILs, TNFs, TGFs, IFNs, FGFs, MAPKs, MMPs, caspases and Wnts were either up regulated or downregulated. This effect was reversed by the extract, possibly through downregulating various proinflammatory cytokines and apoptotic pathways. Our study reveals that S. aureus inserts a negative impact on the regulation of various key genes which is apparently reversed by the HR extract.

3D printing novel in vitro cancer cell culture model systems for lung cancer stem cell study

Two-dimensional (2D) in vitro cell cultures and laboratory animals have been used traditionally as the gold-standard preclinical cancer model systems. However, for cancer stem cell (CSC) studies, they exhibit notable limitations on simulating native environment, which depreciate their translatability for clinical development purposes. In this study, different three-dimensional (3D) printing platforms were used to establish novel 3D cell cultures enriched in CSCs from non-small cell lung cancer (NSCLC) patients and cell lines. Rigid scaffolds with an elevated compressive modulus and uniform pores and channels were produced using different filaments. Hydrogel-based scaffolds were printed with a more irregular distribution of pores and a lower compressive modulus. As a 3D model of reference, suspension spheroid cultures were established. Therein, cancer cell lines exhibited enhanced proliferation profiles on rigid scaffolds compared to the same cells grown on either hydrogel scaffolds or tumor spheres. Meanwhile, primary cancer cells grew considerably better on hydrogel scaffolds or in tumor sphere culture, compared to cells grown on rigid scaffolds. Gene expression analysis confirmed that tumor spheres and cells seeded on hydrogel scaffolds significantly overexpress most of stemness and invasion promoters tested compared to control cells grown in 2D culture. A different phenomenon was observed within cells growing on the rigid scaffolds, where fewer significant variations in gene expression were detected. Our findings provide strong evidence for the advantageous usage of 3D printed models, especially those which use GelMA-PEGDA hydrogels as the primary scaffold material, for studying lung CSCs. The results demonstrated that the 3D printed scaffolds were better to mimic tumor complexity and regulate cancer cell behavior than in vivo 2D culture models.

Using RNentropy to Detect Significant Variation in Gene Expression Across Multiple RNA-Seq or Single-Cell RNA-Seq Samples.

RNA-Seq has become the de facto standard technique for characterization and quantification of transcriptomes, and a large number of methods and tools have been proposed to model and detect differential gene expression based on the comparison of transcript abundances across different samples. However, state-of-the-art methods for this task are usually designed for pairwise comparisons, that is, can identify significant variation of expression only between two conditions or samples. We describe the use of RNentropy, a methodology based on information theory, devised to overcome this limitation. RNentropy can thus detect significant variations of gene expression in RNA-Seq data across any number of samples and conditions, and can be applied downstream of any analysis pipeline for the quantification of gene expression from raw sequencing data. RNentropy takes as input gene (or transcript) expression values, defined with any measure suitable for the comparison of transcript levels across samples and conditions. The output consists of genes (or transcripts) exhibiting significant variation of expression across the conditions studied, together with the samples in which they result to be over- or underexpressed. RNentropy is implemented as an R package and freely available from the CRAN repository. We provide a detailed guide to the functions and parameters of the package and usage examples to demonstrate the software capabilities, alsoshowing how it can be applied to the analysis of single-cell RNA sequencing data.

Screening for sheath blight resistance lines of rice (0ryza sativa L.) variety swarna by using of chemical mutagen

The present investigation was undertaken to identify the resistant lines amongst mutated rice population of the variety swarna induced by the sodium azide. The variability in disease reaction was observed among mutated rice lines. Out of the total 1000 mutant plants, 47 plants were screened that show low disease index, ranged between 0 to 5 were selected for next M generation. In M2 generation the selected genotypes show differences for disease index, only 12 lines shows resistance in M2 generation in field condition and in humidity chamber condition only 8 lines shows resistance. The number of resistance lines were decreases in humidity condition. Non significant variation in morphological expression and in yield attributes among the selected mutants was also reported. Thus, these lines could be considered a potential source for disease resistance against the sheath blight of rice and could be used further in the crossing programme for development of sheath blight resistant rice variety.

The Application of Trichoderma Strains or Metabolites Alters the Olive Leaf Metabolome and the Expression of Defense-Related Genes

Biocontrol fungal strains of the genus Trichoderma can antagonize numerous plant pathogens and promote plant growth using different mechanisms of action, including the production of secondary metabolites (SMs). In this work we analyzed the effects of repeated applications of selected Trichoderma strains or SMs on young olive trees on the stimulation of plant growth and on the development of olive leaf spot disease caused by Fusicladium oleagineum. In addition, metabolomic analyses and gene expression profiles of olive leaves were carried out by LC–MS Q-TOF and real-time RT-PCR, respectively. A total of 104 phenolic compounds were detected from olive leave extracts and 20 were putatively identified. Targeted and untargeted approaches revealed significant differences in both the number and type of phenolic compounds accumulated in olive leaves after Trichoderma applications, as compared to water-treated plants. Different secoiridoids were less abundant in treated plants than in controls, while the accumulation of flavonoids (including luteolin and apigenin derivatives) increased following the application of specific Trichoderma strain. The induction of defense-related genes, and of genes involved in the synthesis of the secoiridoid oleuropein, was also analyzed and revealed a significant variation of gene expression according to the strain or metabolite applied.

Identification of QTLs and Candidate Genes Associated with Leaf Angle and Leaf Orientation Value in Maize (Zea mays L.) Based on GBS

Leaf angle (LA) and leaf orientation value (LOV) are two crucial traits constituting the plant architecture (PA) of maize. Screening of quantitative trait loci (QTLs) and candidate genes (CGs) related to leaf angle and leaf orientation value provides the molecular basis for the improvement of maize plant architecture. In this study, we utilized genotyping by sequencing (GBS) on 179 maize recombined inbred lines (RILs) obtained by crossing two inbred lines with significant variation in leaf angle and leaf orientation value. A total of 4235 markers were used for the construction of the genetic linkage map. The total length of the genetic map was 1514.57 cM with the average genetic distance between each marker of 0.36 cM. On association of this map with phenotypic data from four environments, nine QTLs associated with leaf angle and nine QTLs associated with leaf orientation value were identified, explaining 78.89% and 59.71% of the phenotypic variation of leaf angle and leaf orientation value, respectively. Two QTLs explained greater than 10% of the phenotypic variation and three QTLs controlling both leaf angle and leaf orientation value were screened. Three candidate genes were identified as the most possible genes associated with leaf angle and leaf orientation value by screening the annotations of genes underlying these crucial QTLs. These were Zm00001d019053 might be associated with leaf angle, Zm00001d028164 and Zm00001d025352 might be associated with leaf angle and leaf orientation value, respectively. Expression analysis of candidate genes from parents and subsets also confirmed the significant variation in expression. Differences in DNA of the inbred lines with different phenotype were identified based on analysis of allelic variations in the candidate genes. These results not only enrich QTLs and candidate genes associated with leaf angle and leaf orientation value of maize, but also provide references for the improvement of plant architecture and molecular marker-assisted breeding.

Quantification of Allele-Specific HLA Expression with Nanopore Long-Read Sequencing

Human leukocyte antigen (HLA) typing plays a critical role in evaluating donor-recipient compatibility prior to hematopoietic cell transplantation (HCT) to minimize the risk of rejection and graft versus host disease (GVHD). Compared to traditional sequence-based methods for HLA typing, next-generation sequencing offers significant advantages in terms of accuracy, turnaround time, and cost (Weimer et al., JMD, 2016). Nevertheless, an intrinsic limitation of DNA-based typing is that it does not quantify HLA gene expression, which has been implicated in clinical outcomes (Petersdorf et al., Blood, 2014; Petersdorf et al., NEJM, 2015). Previously, we demonstrated simultaneous HLA class I genotyping and gene-level expression analysis by RNA-seq using nanopore long-read sequencing (Montgomery et al., JMD, 2020). Given that mismatches in both class I and class II HLA genes-as well as the relative expression of individual alleles-impact donor-recipient compatibility, we sought to build on our previous work by quantifying allele-specific expression of both class I and class II HLA loci in donor lymphocytes. For this study, mRNA was isolated from peripheral blood lymphocytes from 12 donors. Barcoded cDNA libraries were prepared and sequenced on MinION flow cells (R9.4.1) using MinKNOW (v3.1.13) to a median depth of 1.6x106reads. Basecalling and demultiplexing were performed with Albacore (v2.3.4) or Guppy (v2.3.1), and adapter trimming was performed with Porechop (v0.2.3). Processed reads were aligned to the international ImMunoGeneTics project (IMGT) HLA database (v3.41.0) using minimap2 (v2.17). Reads mapping to individual HLA loci were realigned to allele-specific references using subject HLA types determined by Athlon (v1.0) or Illumina sequencing. In parallel, library size factors were estimated by aligning reads to GRCh38, counting reads in genes with HTseq (v0.12.4), and using trimmed mean of M-values normalization. As shown in Fig. 1, we observed higher expression of HLA class I genes compared to class II (median 593 vs. 150, p &amp;lt; 0.001, Mann-Whitney U test), a pattern consistent with a mixture of primarily T cells, which express class I genes, as well as B cells, which express both class I and II. Within class I genes, we observed the highest expression of HLA-B, followed by HLA-A, and HLA-C (median 663, 578, and 459, respectively). Within class II, we observed the highest expression of HLA-DPB1, followed by HLA-DRB1, and HLA-DQB1 (median 281, 266, and 104, respectively). Importantly, we observed significant variation in expression both between and within alleles of individual HLA genes, suggesting that HLA type alone does not accurately predict HLA expression. We next analyzed HLA-DPB1 specifically, given reports that the risk of GVHD in HCT recipients with HLA-DPB1mismatched donors is modulated by HLA-DPB1 expression (Petersdorf et al., NEJM, 2015). Of note, HLA-DPB1 expression is linked to a single nucleotide polymorphism, rs9277534, which can be imputed from HLA-DPB1 type (Meurer et al., Front Immunol, 2018). Accordingly, we analyzed HLA-DPB1 expression conditioned on rs9277534 genotype. Although we observed lower HLA-DPB1 expression for the 'A' allele compared to 'G' (median 220 vs. 265), consistent with the reported association, this difference was not statistically significant (p = 0.22, Mann-Whitney U test). Furthermore, we observed significant variation in expression among 'A' alleles, with normalized counts ranging from 57 to 408 (vs. 191 to 367 for 'G' alleles). In this study, we demonstrate the feasibility of quantifying allele-specific expression of both class I and class II HLA genes with nanopore long-read sequencing. Taken together, our results reveal extensive variation in the expression of class I and class II HLA loci, even after accounting for individual allele types and known markers of expression. These results emphasize the potential value of methods, such as nanopore sequencing, for directly quantifying allele-specific HLA expression to develop improved risk prediction models that can inform the evaluation of donor-recipient immunocompatibility. Disclosures No relevant conflicts of interest to declare.

Transcriptome profile of drought responsive candidate genes in varieties of citrus rootstocks with different tolerance strategies

Sunki Maravilha' and 'Sunki Tropical' are citrus plants with different drought tolerance strategies. `Sunki Tropical' is considered as another rootstock alternative for use in drought prone areas, however, its genetic expression profiles regarding response to water deficit, are still unknown. In this study, the transcriptome of the ‘Sunki Maravilha’ and ‘Sunki Tropical’ rootstocks under control were analyzed by RNA-seq technique using the Illumina HiSeq-2500. A total of 109,114.164 reads were produced and mapped against the Citrus sinensis reference genome (version 1.1). 3.607 transcripts showed significant variation in expression between the water deficit and control treatments with 739 genes up-regulated and 2868 down-regulated during water deficit imposition. The differentially expressed genes (DEGs) identified are involved in more than 100 biological processes and molecular functions whereas most enriched for the function of oxidoreductase, protein phosporilation, transcriptional regulation, ligation processes and response to oxidative stress. DEGs related to the biosynthesis of ABA (Abscisic Acid), redox system, cell wall mechanism, root development, transcription factors (TFs) and kinase proteins (KPs), were also identified. This work lays the basic foundation for further investigation of the specific functions of genes in citrus rootstocks, Sunki Maravilha’ and Sunki Tropical’, with different strategies of survival under water deficit and also provides candidate genes for engineering or breeding of citrus plants aiming drought tolerance.

MicroRNA expression profiling reveals potential roles for microRNA in the liver during pigeon (Columba livia) development

The liver is the central organ for metabolism and influence the growth and development of the animals. To date, little is known about the microRNA (miRNA) in pigeon livers, particularly in different developmental stages. A comprehensive investigation into miRNA transcriptomes in livers across 3 pigeon developmental stages (1, 14, 28 d old) and an adult stage (2 y old) was performed by small RNA sequencing. We identified 312 known miRNA, 433 conserved miRNA, and 192 novel miRNA in pigeon livers. A set of differentially expressed (DE) miRNA in livers were screened out during pigeon development. This set of miRNA might be involved in hepatospecific phenotype and liver development. A Short Time-series Expression Miner analysis indicated significant expression variations in DE miRNA during liver development of pigeons. These DE miRNA with different expression patterns might play essential roles in response to growth factor, cell morphogenesis, and gland development, etc. Protein–protein interaction network and Molecular Complex Detection analysis identified several vital target genes (e.g., TNRC6B, FRS2, PTCH1, etc.) of DE miRNA, which is closely linked in liver development and enriched in PI3K cascade and regulation of growth. Our results expanded the repertoire of pigeon miRNA and may be of help in better understanding the mechanism of squab's rapid development from the perspective of liver development.

Mitochondrial damage-associated molecular patterns stimulate reactive oxygen species production in human microglia

Microglia are the resident innate immune cells of the central nervous system and exert functions of host defense and maintenance of normal tissue homeostasis, along with support of neuronal processes in the healthy brain. Chronic and dysregulated microglial cell activation has increasingly been linked to the status of neuroinflammation underlying many neurodegenerative diseases, including multiple sclerosis (MS). However, the stimulus (or stimuli) and mechanisms by which microglial activation is initiated and maintained MS are still debated. The purpose of our research was to investigate whether the endogenous mitochondrial (mt)-derived damage-associated molecular patterns (MTDs) mtDNA, N-formyl peptides and cardiolipin (CL) contribute to these phenomena. We characterized the effects of the abovementioned MTDs on microglia activation in vitro (i.e. using HMC3 cells) by evaluating the expression of gene coding for proteins involved in their binding and coupled to downstream signaling pathways, the up-regulation of markers of activation on the cell surface and the production of pro-inflammatory cytokines and reactive oxygen species. At the transcriptional level, significant variations in the mRNA relative expression of five of eleven selected genes were observed in response to stimulation. No changes in activation of antigenic profile or functional properties of HMC3 cells were observed; there was no up-regulation of HLA-DR expression or increased secretion of tumor necrosis factor-α and interleukin-6. However, after stimulation with mtDNA and CL, an increase in cellular oxidative stress, but not in the mt ROS O2−, compared to control cells, were observed. There were no effects on cell viability. Overall, our data suggest that MTDs could cause a failure in microglial activation toward a pro-inflammatory phenotype, possibly triggering an endogenous regulatory mechanism for the resolution of neuroinflammation. This could open a door for the development of drugs selectively targeting microglia and modulating its functionality to treat MS and/or other neurodegenerative conditions in which MTDs have a pathogenic relevance.

Abstract 190: Prognostic significance of esterase gene expression in multiple myeloma

Abstract Introduction: The esterase enzyme family is a subclass of the hydrolase enzyme superfamily that functions to split ester bonds. Several esterases have been identified, which differ in substrate specificity and biological function, and the expression of specific esterases may be dysregulated in cancer. Limited information is available regarding the expression of esterases in multiple myeloma (MM), despite potential for exploitation in novel therapeutic approaches such as peptide-drug conjugates (e.g. melflufen). We evaluated the biological significance of esterase enzyme expression in MM. Methods: For gene expression profiling, bone marrow aspirates were obtained from MM patients (pts) or healthy donors after obtaining written informed consent and following approved protocols in compliance with the Declaration of Helsinki. CD138+ plasma cells were enriched and used for RNA preparation. Illumina-compatible RNA sequencing libraries were prepared and sequenced. A cut-off value of &amp;gt;1 Reads Per Kilobase of transcript per Million mapped reads (RPKM) was used to filter expressed genes. The Welch two-sample t-test was used to identify significant variation in gene expression. Contribution of esterase gene expression to survival outcome was estimated by Kaplan-Meier analysis; significance between two groups was deduced using a Mantel-Cox log rank test. Results: Of 123 pts with MM, bone marrow aspirates were collected from 41 pts with newly diagnosed (ND) MM, 81 pts with relapsed/refractory (RR) MM, and 1 pt with stable disease. Samples were also obtained from two healthy donors. The most abundant esterases in MM samples were OVCA2, PAFAH1B2, NXPE3, UCHL3, LIPA, ABHD10, UCHL5, CASD1, ABHD13, and USP4 (median log2[RPKM] range: 3.9 to 2.1). The least abundant esterases were NLGN1, NLGN2, PON1, CEL, PON3, NLGN4Y, IL17A, AADAC, CES5A, and ASPG (median log2[RPKM]: -5.1 to -10.7). The expression levels of OVCA2, PAFAH1B2, NXPE3, UCHL5, SIAE, ESD, PNPLA6, and PAFAH1B3 were significantly higher (P&amp;lt;0.05), and of PCED1B significantly lower (P&amp;lt;0.05), in RRMM versus NDMM. The expression level of all these genes was comparatively higher in the two healthy donor samples. High expression of OVCA2, PAFAH1B3, CES4A, or PNPLA6 was associated with poorer prognosis (overall survival [OS]) versus low expression samples (median OS for high vs low expression [months]: OVCA2: 55 vs 122; PAFAH1B3: 55 vs 124; CES4A: 55 vs not reached; PNPLA6: 89 vs 127; all P≤0.01), whereas low expression of NXPE4 or GZMA was associated with poorer prognosis versus high expression samples (median OS for high vs low expression [months]: NXPE4: 120 vs 55; GZMA: 120 vs 74; both P≤0.03). Conclusion: Esterase expression levels in MM change on progression from NDMM to RRMM. High expression of OVCA2, PAFAH1B3, CES4A, and PNPLA6, and low expression of NXPE4 and GZMA, were identified as poor prognostic markers in pts with MM, suggesting a role for these esterases in myeloma biology. Citation Format: Romika Kumari, M. Mamun Majumder, Juha Lievonen, Raija Silvennoinen, Pekka Anttila, Nina N. Nupponen, Fredrik Lehmann, Caroline A. Heckman. Prognostic significance of esterase gene expression in multiple myeloma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 190.

Gene coexpression network analysis and tissue-specific profiling of gene expression in jute (Corchorus capsularis L.)

BackgroundJute (Corchorus spp.), belonging to the Malvaceae family, is an important natural fiber crop, second only to cotton, and a multipurpose economic crop. Corchorus capsularis L. is one of the only two commercially cultivated species of jute. Gene expression is spatiotemporal and is influenced by many factors. Therefore, to understand the molecular mechanisms of tissue development, it is necessary to study tissue-specific gene expression and regulation. We used weighted gene coexpression network analysis, to predict the functional roles of gene coexpression modules and individual genes, including those underlying the development of different tissue types. Although several transcriptome studies have been conducted on C. capsularis, there have not yet been any systematic and comprehensive transcriptome analyses for this species.ResultsThere was significant variation in gene expression between plant tissues. Comparative transcriptome analysis and weighted gene coexpression network analysis were performed for different C. capsularis tissues at different developmental stages. We identified numerous tissue-specific differentially expressed genes for each tissue, and 12 coexpression modules, comprising 126 to 4203 genes, associated with the development of various tissues. There was high consistency between the genes in modules related to tissues, and the candidate upregulated genes for each tissue. Further, a gene network including 21 genes directly regulated by transcription factor OMO55970.1 was discovered. Some of the genes, such as OMO55970.1, OMO51203.1, OMO50871.1, and OMO87663.1, directly involved in the development of stem bast tissue.ConclusionWe identified genes that were differentially expressed between tissues of the same developmental stage. Some genes were consistently up- or downregulated, depending on the developmental stage of each tissue. Further, we identified numerous coexpression modules and genes associated with the development of various tissues. These findings elucidate the molecular mechanisms underlying the development of each tissue, and will promote multipurpose molecular breeding in jute and other fiber crops.

Thioredoxin System Protein Expression Is Associated with Poor Clinical Outcome in Adult and Paediatric Gliomas and Medulloblastomas

The thioredoxin (Trx) system is an important enzyme family that regulates cellular redox homeostasis. Protein expression of Trx system family members has been assessed in various cancers and linked to various clinicopathological variables, disease progression, treatment response and survival outcomes but information is lacking in brain tumours. Expression of the system was therefore examined, by immunohistochemistry in different brain tumour types, adult and paediatric cases, to determine if expression was of importance to clinical outcome. Trx system proteins were expressed, to variable levels, across all brain tumour types with significant variations in expression between different tumour types/grades/regions. High Trx reductase (TrxR) expression was linked to worse prognosis across all cohorts. High cytoplasmic TrxR expression was significantly associated with adverse overall survival (OS) in adult glioblastoma (P = 0.027) and paediatric low-grade glioma (LGG) patients (P = 0.012). High expression of nuclear TrxR, cytoplasmic and nuclear Trx and Trx-interacting protein (TxNIP) was associated with improved OS in paediatric LGGs (P = 0.031, P < 0.001, P = 0.044 and P = 0.018, respectively). For patients with high-grade gliomas, both high cytoplasmic TrxR and Trx expression were associated with poor OS (P = 0.002 and P = 0.007, respectively). In medulloblastoma, high expression of cytoplasmic TrxR and Trx and nuclear Trx was associated with worse prognosis (P = 0.013, P = 0.033 and P = 0.007, respectively); with cytoplasmic TrxR and nuclear Trx remaining so in multivariate analysis (P = 0.009 and P = 0.013, respectively). The consistent finding that high levels of cytoplasmic TrxR are associated with a worse prognosis across all cohorts suggests that TrxR is an important therapeutic target in brain cancers.