Strand-specific Libraries Research Articles

Tracking-seq reveals the heterogeneity of off-target effects in CRISPR-Cas9-mediated genome editing.

The continued development of novel genome editors calls for a universal method to analyze their off-target effects. Here we describe a versatile method, called Tracking-seq, for in situ identification of off-target effects that is broadly applicable to common genome-editing tools, including Cas9, base editors and prime editors. Through tracking replication protein A (RPA)-bound single-stranded DNA followed by strand-specific library construction, Tracking-seq requires a low cell input and is suitable for in vitro, ex vivo and in vivo genome editing, providing a sensitive and practical genome-wide approach for off-target detection in various scenarios. We show, using the same guide RNA, that Tracking-seq detects heterogeneity in off-target effects between different editor modalities and between different cell types, underscoring the necessity of direct measurement in the original system.

Influence of postruminal casein infusion and exogenous glucagon-like peptide 2 administration on the jejunal mucosal transcriptome in cattle.

We previously demonstrated that postruminal casein infusion and exogenous glucagon-like peptide 2 (GLP-2) administration independently stimulated growth and carbohydrase activity of the pancreas and jejunal mucosa in cattle. The objective of the current study was to profile the jejunal mucosal transcriptome of cattle using next-generation RNA sequencing in response to postruminal casein infusion and exogenous GLP-2. Twenty-four Holstein steers [250 ± 23.1 kg body weight (BW)] received a continuous abomasal infusion of 3.94 g raw corn starch/kg of BW combined with either 0 or 1.30 g casein/kg of BW for 7 d. Steers received subcutaneous injections at 0800 and 2000 h to provide either 0 or 100 μg GLP-2/kg of BW per day. At the end of the 7-d treatment period, steers were slaughtered for collection of the jejunal mucosa. Total RNA was extracted from jejunal mucosal tissue, strand-specific cDNA libraries were prepared, and RNA sequencing was conducted to generate 150-bp paired-end reads at a depth of 40 M reads per sample. Differentially expressed genes (DEG), KEGG pathway enrichment, and gene ontology enrichment were determined based on the FDR-corrected P-value (padj). Exogenous GLP-2 administration upregulated (padj < 0.05) 667 genes and downregulated 1,101 genes of the jejunal mucosa. Sphingolipid metabolism, bile secretion, adherens junction, and galactose metabolism were among the top KEGG pathways enriched with upregulated DEG (padj < 0.05) in response to exogenous GLP-2 administration. The top gene ontologies enriched with upregulated DEG (padj < 0.05) in response to exogenous GLP-2 administration included nutrient metabolic processes, brush border and bicellular tight junction assembly, and enzyme and transporter activities. Exogenous GLP-2 administration increased or tended to increase (padj < 0.10) brush border carbohydrase (MGAM, LCT, TREH), hexose transporter (SLC5A1, SLC2A2), and associated transcription factor (HNF1, GATA4, KAT2B) mRNA expression of the jejunal mucosa. Gene ontologies and KEGG pathways that were downregulated (padj < 0.05) in response to exogenous GLP-2 were related to genetic information processing. Postruminal casein infusion downregulated (padj < 0.05) 7 jejunal mucosal genes that collectively did not result in enriched KEGG pathways or gene ontologies. This study highlights some of the transcriptional mechanisms associated with increased growth, starch assimilation capacity, and barrier function of the jejunal mucosa in response to exogenous GLP-2 administration.

A High-Efficiency Capture-Based NGS Approach for Comprehensive Analysis of Mitochondrial Transcriptome.

The transcription of the mitochondrial genome is pivotal for maintenance of mitochondrial functions, and the deregulated mitochondrial transcriptome contributes to various pathological changes. Despite substantial progress having been achieved in uncovering the transcriptional complexity of the nuclear transcriptome, many unknowns and controversies remain for the mitochondrial transcriptome, partially owing to the lack of a highly efficient mitochondrial RNA (mtRNA) sequencing and analysis approach. Here, we first comprehensively evaluated the influence of essential experimental protocols, including strand-specific library construction, two RNA enrichment strategies, and optimal rRNA depletion, on accurately profiling mitochondrial transcriptome in whole-transcriptome sequencing (WTS) data. Based on these insights, we developed a highly efficient approach specifically suitable for targeted sequencing of whole mitochondrial transcriptome, termed capture-based mtRNA seq (CAP), in which strand-specific library construction and optimal rRNA depletion were applied. Compared with WTS, CAP has a great decrease of required data volume without affecting the sensitivity and accuracy of detection. In addition, CAP also characterized the unannotated mt-tRNA transcripts whose expression levels are below the detection limits of conventional WTS. As a proof-of-concept characterization of mtRNAs, the transcription initiation sites and mtRNA cleavage ratio were accurately identified in CAP data. Moreover, CAP had very reliable performance in plasma and single-cell samples, highlighting its wide application. Altogether, the present study has established a highly efficient pipeline for targeted sequencing of mtRNAs, which may pave the way toward functional annotation of mtRNAs and mtRNA-based diagnostic and therapeutic strategies in various diseases.

Abstract 221: Development of a rapid, highly sensitive cell-free RNA NGS assay for the detection of gene fusion and splicing variants in cancers

Abstract Background: Utility of cell-free nucleic acids (cf-NAs) for early detection and surveillance of tumor represents a new path in personalized medicine. Gene fusions, which usually arise from chromosomal rearrangement, play pivotal roles in numerous human malignancies and have been exploited for disease diagnosis and therapeutics. RNA sequencing, which is not influenced by the complication of introns, is considered superior to DNA sequencing for the identification of fusion genes and splicing variants, and has emerged as a critical tool in cancer liquid biopsy. Methods: We developed a novel approach of preparing strand-specific RNA-Seq libraries. The cDNA produced from the cell-free RNA (cfRNA) samples were labelled with unique molecular identifiers (UMI) during library construction. The libraries were enriched using hybrid capture and the sequencing data were analyzed using Predicine in-house analysis pipeline. To assess the performance of the newly developed cfRNA assay, we compared it with a variety of commercial RNA library kits. Results: The new RNA library construction workflow was streamlined, and it reduces the library preparation time from 2 days to 6 hours with less than 1 h of hands-on time. The yield of the library prepared by the new method was higher than those of the commonly used commercial kits on the market. Sequencing analysis and Droplet Digital PCR (ddPCR) measurement both revealed that our assay was more sensitive in detecting gene fusions and splicing variants in cfRNA than other commercial kits. The assay can reliably detect fusion/splicing variants down to the level of &amp;lt;10 copies of input cfRNA with high specificity. Conclusions: We developed a new RNA library preparation method that produced strand-specific libraries from low input quantities of cfRNA in a fast, simple, and robust manner. This RNA-Seq assay has demonstrated superior performance in known fusion detection and novel fusion discovery in cfRNA. Citation Format: fang liu, xiangying sun, jianrong dong, kemin zhou, yong huang, shidong jia, pan du, Binggang xiang, shujun luo. Development of a rapid, highly sensitive cell-free RNA NGS assay for the detection of gene fusion and splicing variants in cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 221.

Role of long non-coding RNA in regulatory network response to Candidatus Liberibacter asiaticus in citrus.

Long non-coding RNAs (lncRNAs) serve as crucial regulators in plant response to various diseases, while none have been systematically identified and characterized in response to citrus Huanglongbing (HLB) caused by Candidatus Liberibacter asiaticus (CLas) bacteria. Here, we comprehensively investigated the transcriptional and regulatory dynamics of the lncRNAs in response to CLas. Samples were collected from leaf midribs of CLas- and mock-inoculated HLB-tolerant rough lemon (Citrus jambhiri) and HLB-sensitive sweet orange (C. sinensis) at week 0, 7, 17, and 34 following inoculation using CLas+ budwood of three biological replicates in the greenhouse. A total of 8,742 lncRNAs, including 2,529 novel lncRNAs, were identified from RNA-seq data with rRNA-removed from strand-specific libraries. Genomic variation analyses of conserved lncRNAs from 38 citrus accessions showed that 26 single nucleotide polymorphisms (SNPs) were significantly correlated with HLB. In addition, lncRNA-mRNA weighted gene co-expression network analysis (WGCNA) showed a significant module correlated with CLas-inoculation in rough lemon. Notably, the most significant LNC_28805 and multiple co-expressed genes related to plant defense in the module were targeted by miRNA5021, suggesting that LNC28805 might compete with endogenous miR5021 to maintain the homeostasis of immune gene expression levels. Candidate WRKY33 and SYP121 genes targeted by miRNA5021 were identified as two key hub genes interacting with bacteria pathogen response genes based on the prediction of protein-protein interaction (PPI) network. These two genes were also found within HLB-associated QTL in linkage group 6. Overall, our findings provide a reference for a better understanding of the role of lncRNAs involved in citrus HLB regulation.

Competing endogenous RNA network construction based on long non-coding RNAs, microRNAs, and mRNAs related to fat deposition in Songliao black swine.

China has a long history of pig breeding and a number of local breeds. The Songliao Black pig, bred in China in 2009, shows high variation in backfat thickness and therefore is well-suited to fat deposition research. Fat deposition is a complex trait, and the underlying regulatory factors are not fully characterized. In this study, the molecular basis of fat deposition traits was evaluated by comparisons between three individuals with extremely high-backfat thickness and three with extremely low-backfat thickness selected from 53 gilts. Subcutaneous adipose tissues of the back were collected for strand-specific library RNA sequencing (RNA-seq) and small RNA-seq. We identified 13 184 mRNAs, 2046 long non-coding (lnc)RNAs, and 494 micro (mi)RNAs by high-throughput sequencing. Furthermore, we detected 150 differentially expressed mRNAs, 66 differentially expressed lncRNAs, and eight differentially expressed miRNAs. A functional enrichment analysis indicated that these genes are involved in multiple fat metabolism-related pathways, including positive regulation of fat cell differentiation, and fat digestion and absorption. We used various algorithms (miRanda, TargetScan, and RNAhybrid) to predict targeting relationships and constructed a competing endogenous RNA network containing seven lncRNAs, three miRNAs, and six mRNAs. All these genes were differentially expressed between the extremely high and low backfat thickness groups or enriched in pathways related to fat metabolism. Our results provide insight into the regulatory mechanisms by which non-coding RNAs and their target genes influence backfat deposition in pigs. Furthermore, our newly constructed competing endogenous RNA (lncRNA-miRNA-mRNA) network provides a basis for further exploration of fat deposition traits and non-coding RNA functions.

Identification of long non-coding RNAs involved in floral scent of Rosa hybrida.

Long non-coding RNAs (lncRNAs) were found to play important roles in transcriptional, post-transcriptional, and epigenetic gene regulation in various biological processes. However, lncRNAs and their regulatory roles remain poorly studied in horticultural plants. Rose is economically important not only for their wide use as garden and cut flowers but also as important sources of natural fragrance for perfume and cosmetics industry, but presently little was known about the regulatory mechanism of the floral scent production. In this paper, a RNA-Seq analysis with strand-specific libraries, was performed to rose flowers in different flowering stages. The scented variety ‘Tianmidemeng’ (Rosa hybrida) was used as plant material. A total of 13,957 lncRNAs were identified by mining the RNA-Seq data, including 10,887 annotated lncRNAs and 3070 novel lncRNAs. Among them, 10,075 lncRNAs were predicted to possess a total of 29,622 target genes, including 54 synthase genes and 24 transcription factors related to floral scent synthesis. 425 lncRNAs were differentially expressed during the flowering process, among which 19 were differentially expressed among all the three flowering stages. Using weighted correlation network analysis (WGCNA), we correlate the differentially-expressed lncRNAs to synthesis of individual floral scent compounds. Furthermore, regulatory function of one of candidate lncRNAs for floral scent synthesis was verified using VIGS method in the rose. In this study, we were able to show that lncRNAs may play important roles in floral scent production in the rose. This study also improves our understanding of how plants regulate their secondary metabolism by lncRNAs.

RNA-seq Sample Preparation Kits Strongly Affect Transcriptome Profiles of a Gas-Fermenting Bacterium.

ABSTRACTTranscriptome analysis via RNA sequencing (RNA-seq) has become a standard technique employed across various biological fields of study. The rapid adoption of the RNA-seq approach has been mediated, in part, by the development of different commercial RNA-seq library preparation kits compatible with standard next-generation sequencing (NGS) platforms. Generally, the essential steps of library preparation, such as rRNA depletion and first-strand cDNA synthesis, are tailored to a specific group of organisms (e.g., eukaryotes versus prokaryotes) or genomic GC content. Therefore, the selection of appropriate commercial products is of crucial importance to capture the transcriptome of interest as closely to the native state as possible without introduction of technical bias. However, researchers rarely have the resources and time to test various commercial RNA-seq kits for their samples. This work reports a side-by-side comparison of RNA-seq data from Clostridium autoethanogenum obtained using three commercial rRNA removal and strand-specific library construction products of NuGEN Technologies, Qiagen, and Zymo Research and assesses their performance relative to published data. While all three vendors advertise their products as suitable for prokaryotes, we found significant differences in their performance regarding rRNA removal, strand specificity, and most importantly, transcript abundance distribution profiles. Notably, RNA-seq data obtained with Qiagen products were most similar to published data and delivered the best results in terms of library strandedness and transcript abundance distribution range. Our results highlight the importance of finding appropriate organism-specific workflows and library preparation products for RNA-seq studies.IMPORTANCE RNA-seq is a powerful technique for transcriptome profiling while involving elaborate sample processing before library sequencing. We show that RNA-seq library preparation kits can strongly affect the outcome of an RNA-seq experiment. Although library preparation benefits from the availability of various commercial kits, choosing appropriate products for the specific samples can be challenging for new users or for users working with unconventional organisms. Evaluating the performance of different commercial products requires significant financial and time investments infeasible for most researchers. Therefore, users are often guided in their choice of kits by published data involving similar input samples. We conclude that important consideration should be given to selecting sample processing workflows for any given organism.

Gypsy retrotransposon-derived maize lncRNA GARR2 modulates gibberellin response.

Long non-coding RNAs (lncRNAs) mediate diverse biological events mainly through the modulation of transcriptional hierarchy. The phytohormone gibberellin (GA) is essential for various aspects of plant growth and development. However, the roles of lncRNAs in the regulation of the GA response remain largely unknown. Through sequencing multiple strand-specific and ribosomal-depleted RNA libraries, we delineated the landscape of lncRNAs in maize (Zea mays). Out of identified lncRNAs, 445 GIBBERELLIN-RESPONSIVE lncRNAs (GARRs) were differentially expressed upon GA application. By the intersection of GARRs from normal-height and dwarf plants from an advanced backcross population, four shared GARRs (GARR1 to GARR4) were identified. Out of these four shared GARRs, GARR2 was derived from a Gypsy LTR retrotransposon. GA-responsive element P-boxes were identified upstream of GARR2. GARR2-edited lines exhibited a GA-induced phenotype. Editing of GARR2 resulted in changes in the transcriptional abundance of GA pathway components and endogenous GA contents. Besides GA, GARR2 affected the primary auxin response. An RNA pull-down assay revealed the HECT ubiquitin-protein ligase family member ZmUPL1 as a potential interaction target of GARR2. GARR2 influenced the abundance of ZmUPL1 in the GA response. Our study uncovers lncRNA players involved in the modulation of the GA response and guides the development of plant height ideotype driven by knowledge of the phytohormone GA.

Sickle Cell Hemoglobin Induces Autophagy in Human Macrophages

BACKGROUND: Autophagy plays an important role in multiple cell processes including elimination of misfolded proteins and damaged organelles, clearance of intracellular microbes and regulation of innate immunity. Autophagic activity is significantly up regulated in diseases characterized by proteins aggregation such as Alzheimer's, Huntington's, and Parkinson's diseases. Sickle cell anemia (SCA) is associated with E6V mutation in beta-globin gene that induces hemoglobin (Hb) polymerization under low oxygen or acidic conditions. SCA is characterized by chronic presence of low-level plasma Hb (3-10 µM). Hb is cleared from circulation by macrophage's endocytosis, resulting in degradation of Hb in lysosomes. Acidic lysosome environment may induce Hb polymerization, lysosome dysregulation and autophagy activation.HYPOTHESIS: We hypothesize that endocytosis of HbS induces autophagy in human macrophages.METHODS: The study was approved by Howard University review board (IRB) and all subjects provided written inform consent prior the sample collection. Whole blood samples were obtained from three SCA patients and healthy individuals. Human THP-1 promonocytic cells were differentiated into macrophages with 25 nM PMA for 72 hrs and treated with either purified HbS or HbA (5 µM, Sigma-Aldrich). Total RNA was depleted of cytoplasmic and mitochondrial ribosomal RNA, and strand-specific libraries were constructed using the Illumina ® TruSeq Stranded Total RNA Gold kit. Libraries were sequenced on an Illumina ® NextSeq 500 using 75 bp paired-end sequencing on two v2.5 150 cycle High-Output kits, generating 40-50 million paired-end reads per sample. The sequencing data were mapped to human reference genome using Dragen RNA v.3.8.4 software (Illumina). Comparisons were conducted using Dragen differential expression software v. 3.6.3 software (Illumina). Transmission electron microscopy (TEM) imaging was performed at Talos 200X transmission electron microscope (Thermo Fisher Scientific). CYTO-ID Autophagy detection kit (Enzo Life Science) was used for detection of autophagy in live cells by fluorescence microscopy, and fluorescent microplate assay.RESULTS: In THP-1-derived macrophages treated with HbS compared to HbA, 28362 genes were detected including 322 differentially expressed genes (1.1%, 1.5-fold difference, 187 down and 135 up) at 5% FDR. In THP-1-differentiated macrophages treated with SCD RBC lysate compared to control RBC lysate, 30840 genes were detected including 256 differentially expressed genes (0.83%, 1.5-fold difference, 117 down and 139 up) at 5% FDR.We focused our analysis on 33 autophagy-related genes. Among these 33 genes, 30 genes were up-regulated (1.2-1.7-fold) and only 3 genes were down-regulated in macrophages treated with HbS compared to HbA (Fig.1). These changes were statistically significant for 20 genes (p<0.01). No significant differences were found in expression of autophagy-related genes in macrophages treated either with HbS or SCD RBC lysate.Autophagy activation was validated by CYTO-ID Autophagy detection kit, demonstrating significant autophagy activation in HbS treated macrophages compared to HbA treatment (1.2-fold, p=0.0019). Transmission electron microscopy demonstrated accumulation of autophagosomes in the cells treated with HbS compared to non-treated cells and cells treated with HbA.CONCLUSION: Treatment of macrophages with sickle cell hemoglobin or lysate of red blood cells collected from SCD patients up-regulates expression of autophagy-related genes and increased autophagosome formation.ACKNOWLEDGMENTS: We thank Drs. Castle Raley and Keith Crandall, George Washington University SPH Genomics Core, for sequencing and guidance in data analysis; Dr. Christine Brantner, GW Nanofabrication and Imaging Center, for TEM imaging; and students Kavita Pandya, Gretchen Whitney High School, Cerritos, CA, and Tharindi Jayatilake, Montgomery Blair High School, Silver Spring, MD, for help in analysis of autophagy-related gene expression. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Upregulation of Antiviral Factors That Inhibit HIV-1 Infection in Sickle Cell Disease

IntroductionPatients with Sickle cell disease (SCD) have lower risk for HIV-1 infection. We showed that ex vivo HIV-1 replication is blocked in SCD PBMCs in part because of the increased expression of ferroportin (FPN) and activation of SAMHD1, a host antiviral restriction factor. We hypothesized that rupture of sickling red blood cells releases sickle cell hemoglobin (HbS) that is phagocyted by macrophages leading to upregulation of innate antiviral response and inhibition of HIV-1 replication. We accessed changes in antiviral gene expression in PBMCs obtained from SCD patients compared to healthy controls. We also analyzed antiviral gene expression in macrophages treated with HbS compared to the HbA treatment.MethodsThe study was approved by Howard University review board (IRB) and all subjects consented to sample collection. Whole blood was collected from 9 SCA patients and 9 age and gender- matched healthy controls. PBMCs were activated with PHA (0.5 μg/ml) for 24-48 hrs followed by IL-2 (10 U/ml) for 24 hrs. Human THP-1 cells were differentiated into macrophages with PMA (25nM) for 72 hrs and treated with purified HbS or HbA (5µM). RNA strand-specific libraries were constructed using TruSeq Stranded Total RNA Gold kit (Illumina) and sequenced on an Illumina NextSeq 500 using 75 bp paired-end sequencing on two v2.5 150 cycle High-Output kits, generating 40-50 million paired-end reads per sample. The sequencing data were mapped using Dragen RNA and compared using Dragen differential expression software (Illumina). Ingenuity Pathway analysis (IPA, Qiagen) was used for pathway analysis.ResultsIn activated SCD PBMCs compared to control PBMCs, 40432 genes were detected including 2230 differentially expressed genes (5.5%, 1.5-fold difference, 287 down and 1943 up) at 5% false discovery rate. In non-activated SCD PBMCs compared to control PBMCs, 33119 genes were detected including 5299 differentially expressed genes (16%, 923 down and 4376 up). In THP-1-differentiated macrophages treated with HbS versus HbA, 28362 genes were detected including 322 differentially expressed genes (1.1%, 187 down and 135 up).We focused our analysis on 61 genes including viral restriction factors and iron regulatory genes. In activated SCD PBMCs, four genes had highest upregulation: APOBEC3A (23-fold, p=2 x 10 -5), CH25H (11-fold, p=4 x 10 -5), heme oxygenase-1 (HMOX1, 13-fold, p=1.5 x 10 -12) and FPN (SLC40A1, 5-fold, p = 9 x 10 -8) (Fig.1). Several additional genes were upregulated with 1.5-3-fold increase and high significance including APOBEC3B, BRD4, CD40, CDKN1A (p21), GDR1, IFIT3, IFITM3 and SAMHD1. In non-activated SCD PBMCs, the most upregulated gene was PKR (EIF2AK2, 15-fold, p=1 x 10 -11) and genes with 1.5-3 fold upregulation included APOBEC3B, BST2, CPSF6, IFI16, IFITM3, ISG15, LGALS3BP, PML and RTF1 (Fig.1). Of these genes, only IFITM3 overlapped between activated and non-activated PBMCs.To test whether circulating HbS leads to the upregulation of antiviral response, we analyzed HbS-treated macrophages and found upregulation of several antiviral restriction factors (1.5-2.3 fold): IFIT3, LGALS3BP, MX2 and RTF1 (Fig.1). Unsupervised IPA showed upregulation of IRF-7 signaling pathway and down regulation of viral infection and replication (Fig.2). We validated the CH25 and HO-1 antiviral role in activated SCD PBMCs using small molecule inhibitors. We also confirmed overexpression of CH25H and HO-1 by western blot and ELISA. We observed higher levels of IRF7 in the activated SCD PBMCs confirming that it may play a role in the induction of antiviral response.ConclusionWe propose that HbS released by hemolysis and uptaken by macrophages leads to the IRF-7-triggered induction of antiviral state in macrophages that will induced antiviral state in non-activated circulating PBMCs likely though the cytokines and interferons secretion known to be elevated in SCD patients. Upregulation of PKR (EIFAK2) levels in non-activated PBMCs strongly argue toward this possibility. Upon activation of PBMCs, additional factors are expressed including CH25H, HO-1, APOBEC3A and FPN that facilitated stronger and more robust anti-HIV-1 effect and block viral replication. Taken together, our study point to novel mechanism of upregulation of antiviral factors mediated by sickle cell hemoglobin that included induction of antiviral, heme- and iron- regulatory pathways. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Integrated analysis of competing endogenous RNA networks in peripheral blood mononuclear cells of systemic lupus erythematosus

BackgroundSystemic lupus erythematosus (SLE) is an autoimmune disease with a complicated pathogenesis, and its aetiology has not been clearly unveiled. The lack of effective diagnosis and treatment methods makes it necessary to explore the molecular mechanism of SLE. We aimed to identify some critical signalling pathways and key competing endogenous RNAs (ceRNAs) underlying the molecular mechanism of SLE and to map out the systematic signalling networks by integrating the data on different kinds of RNAs.MethodsPeripheral blood mononuclear cells (PBMCs) were collected from both SLE patients and healthy subjects, RNA was extracted from the PBMCs, and RNA libraries including ribosomal RNA-depleted strand-specific libraries and small RNA libraries were built for deep RNA sequencing (RNA-seq). RNA-seq yielded differential expression profiles of lncRNAs/circRNAs/miRNAs/mRNAs related to SLE. The DAVID database (v. 6.8) was employed for Gene Ontology (GO) and KEGG pathway analysis. ceRNA networks (circRNA/lncRNA-miRNA-mRNA) were constructed and visualized using Cytoscape software (v. 3.5.0). The TargetScan and miRanda databases were used to predict target relationships in ceRNA networks. qRT-PCR was used to verify our data.ResultsDifferential expression of ceRNAs related to SLE was detected in SLE patients’ PBMCs: 644 mRNAs (384 upregulated, 260 downregulated), 326 miRNAs (223 upregulated, 103 downregulated), 221 lncRNAs (79 upregulated, 142 downregulated), and 31 circRNAs (21 upregulated, 10 downregulated). We drew ceRNA signalling networks made up of the differentially expressed mRNAs/miRNAs/lncRNAs/circRNAs mentioned above, and the hub genes included IRF5, IFNAR2, TLR7, IRAK4, STAT1, STAT2, C2, and Tyk2. These hub genes were involved in ceRNA signalling pathways, such as the IL-17 signalling pathway and type I interferon signalling pathway.ConclusionsWe explored the differential expression profiles of various kinds of ceRNAs and integrated signalling networks constructed by ceRNAs. Our findings offer new insights into the pathogenesis of SLE and hint at therapeutic strategies.

Checkpoint blockade combined with neoadjuvant chemotherapy (NACT) in advanced-stage epithelial ovarian cancer (EOC): preliminary results from a phase II clinical trial

<h3>Objectives:</h3> Standard of care treatment for advanced stage EOC is a combination of chemotherapy and surgery. Despite this approach, the majority of women remain incurable, underscoring the need for novel therapeutics. This study assessed the pathologic objective response rate (pORR), historically reported as 6-10% and tumor transcriptomic changes in women with advanced stage EOC treated with neoadjuvant pembrolizumab, paclitaxel, and carboplatin. <h3>Methods:</h3> This was an open-label, single-arm, phase II trial for women with newly diagnosed biopsy proven EOC dispositioned to receive carboplatin, paclitaxel and pembrolizumab 200mg/m² every 21 days. Patients received 3-4 cycles prior to interval debulking surgery followed by another 3-4 cycles of adjuvant therapy. A planned interim analysis to assess futility (based on reported historic response with NACT, required 1 pORR) was completed after enrollment of 20 of planned 40 evaluable patients. Transcriptomic analyses of bulk RNA to identify functional genes and significant pathways from matched specimens from treatment naïve tumor, and tumor after 3-4 cycles of NACT plus immune checkpoint inhibitor, (ICI) were completed. Strand-specific RNA libraries were sequenced using single-end reads (50 bp, average 40 million reads per sample) on the DNBS<i>eq platform.</i> Differentially expressed genes (DEG) with a false discovery rate q < 0.05 and a fold change of > 1.5 were used. <h3>Results:</h3> Interim data for the first 20 patients evaluable for pORR are reported. Most women were White (95%) and had high grade serous histology (100%). In 11 cases (55%), R0 (complete) cytoreduction was achieved. There have been no surgical complications related to combination treatment. Treatment was generally well tolerated, with mostly grade 1/2 toxicity: 30% pruritic rash, 10% thyroid abnormalities. One patient had grade 3 nephritis that recovered with treatment. A total of 6 patients achieved a pathologic complete response (pCR, 30%) and another 4 (25%) pathologic partial response (pPR) for a 55% pORR. There were 4 pts with deleterious genetic mutation in the homologous recombination pathway (20%) and 2 patients with Fanconi anemia somatic mutations (10%). There were 863 DEGs between the chemonaïve and ICI+NACT matched tumors. ICI+NACT induced activation of PI3K-Akt pathway, myeloid differentiation, AGE-RAGE signaling, and focal adhesion kinase pathways which included upregulation of FXR, NLRP11 and FOXL2 expression, known regulators of inflammation and immune responses. <h3>Conclusions:</h3> Interim analysis demonstrates an increase in pORR compared to previously reported in literature (6-10%) and is generally well tolerated. Transcriptomic analyses revealed specific ICI induced gene signatures when combined with NACT. Further characterization of complete and partial responders are needed to improve patient selection for ICI therapies.

Genome-wide DNA methylome and whole-transcriptome landscapes of spontaneous intraductal papilloma in tree shrews.

BackgroundBreast intraductal papilloma (IP) is mainly caused by the abnormal proliferation of ductal epithelial cells. Tree shrews have potential as an animal model for the study of breast tumours; however, little is known regarding the transcriptome and DNA methylome landscapes of breast IP in tree shrews. In this research, we conducted whole-genome DNA methylation and transcriptome analyses of breast IP and normal mammary glands in tree shrews.MethodsDNA methylation profiles were generated from the whole-genome bisulfite sequencing and whole-transcriptome landscapes of IP and control groups of tree shrews through strand-specific library construction and RNA sequencing. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes functional enrichment analyses and gene set enrichment analysis were performed. Spearman’s correlation analysis was used to identify statistical relationships between gene expression and DNA methylation.ResultsA genome-wide perspective of the epigenetic regulation of protein-coding genes in breast IP in tree shrews was obtained. The methylation levels at CG sites were considerably higher than those at CHG or CHH sites, and were highest in gene body regions. In total, 3,486, 82 and 361 differentially methylated regions (DMRs) were identified in the context of CG, CHG, and CHH, respectively, and 701 differentially methylated genes (DMGs) were found. Further, through transcriptomic analysis, 62 differentially expressed genes, 50 long noncoding RNAs, and 32 circular RNAs were identified in breast IP compared to normal mammary glands. Correlation analysis between the DNA methylation and transcriptome data revealed that 25 DMGs were also differentially expressed genes, among which the expression levels of 9 genes were negatively correlated with methylation levels in gene body regions. Importantly, integrated analysis identified 3 genes (PDZ domain-containing 1, ATPase plasma membrane Ca2+ transporting 4 and Lymphocyte cytosolic protein 1) that could serve as candidates for further study of breast IP in tree shrews.ConclusionsThis research has unearthed the comprehensive landscape of the transcriptome and DNA methylome of spontaneous IP in tree shrews, as well as candidate tumorigenesis related genes in IP. These results will contribute to the use of tree shrews in animal models of breast tumours.

Sequential Study By RNA-Seq Shows a Reduction in Inflammation and a Cell Cycle Activation in T-Lymphocytes from Myelodysplastic Syndrome with Del(5q) after Lenalidomide Treatment in Patients Included in the “Sintra-REV” Clinical Trial

Introduction: Lenalidomide is a potent drug with pleiotropic effects in patients with myelodysplastic syndrome (MDS) with deletion of the long arm of chromosome 5 [del(5q)]. The clinical efficacy of lenalidomide in MDS patients has been extensively reviewed and although the mechanisms of action in del(5q) clone have been previously described, in vivo sequential studies of modulatory effect on T lymphocytes are lacking. Our study was conducted in patients included in the Sintra-REV Clinical Trial: Lenalidomide (Revlimid) phase III, multicenter, randomized, double-blind study versus placebo in patients with low-risk MDS (low and intermediate IPSS-1) with del(5q), with anemia (HB≤12gr/dl) and without transfusion needs. Aim: The aim of this study was to explore the effect of lenalidomide in T-lymphocytes in MDS patients with del(5q) and without transfusion dependence. Materials and Methods: Sequential study was carried out in 26 samples from 13 paired MDS patients with del (5q). Seven out 13 were treated with lenalidomide and achieved a major erythroid and cytogenetic response. Peripheral blood (PB) samples were collected before and one month after treatment in treated-patients and at the same time points for non-treated patients. CD3+ cells were collected from PB samples and total RNA was isolated. SureSelect Strand Specific RNA library (Agilent Technologies) was applied to study changes in RNA levels. Raw reads were aligned against the Human genome GRCh37 using the STAR aligner. Counts were assigned to Ensembl gene IDs through HTseq using its UNION version. Differential gene expression was determined with DESeq2, considering as statistically significant those genes with FDR &amp;lt; 0.05. Pathway over-representation analysis (ORA) was conducted in the Webgestalt suite. Results: 332 genes were differentially expressed in CD3+ lymphocytes one month after lenalidomide treatment in our cohort of patients; 199 of them were over-expressed after the administration of this drug (Fig 1a). Of note, none of them were observed in non-treated patients after one month. The ORA revealed significant differences in the gene expression profile of sixteen cytokines and enrichment of genes of the cell cycle pathway (35 genes). The most relevant up-regulated cytokines were: IL10, TNFSF10, IFNGand IL6. These data explain lenalidomide-induced activation of an antileukemic immune response and secretion of anti-inflammatory cytokines. Although lenalidomide has been reported to reduce the expression of IL6 secreted by myeloid cell derived from MDS clon, we have observed upregulation of this gene in T-lymphocytes. Moreover, our study showed a downregulation of MBP6 that may help to correct the anemia and also attenuate inflammation signaling in MDS patients with del(5q) (Fig 1b). In addition, the most represented up-regulated genes related to cell cycle pathway were: cyclines (CCNB1, CCNB2, CDK1), centromere genes (CENPE, CENPM, CENPU), kinesin family members (KIF18A, KIF23, KIF2C), BUB1 mitotic checkpoint genes (BUB1, BUB1B), and genes involved in cell division (CDC6, CDC7,CDC25A). It has been described that lenalidomide inhibits CDC25A selectively in the del(5q) clone resulting in G2/M arrest and apoptosis. By contrast, our study showed that this gene was upregulated in T-lymphocytes promoting cell cycle and proliferation of these cells (Fig 1b). Conclusions: The immunomodulatory properties of lenalidomide can be summarized in two: a) regulation of antileukemic and anti-inflammatory cytokines production, b) activation of cell cycle and proliferation in T cells. To our knowledge, this is the first report describing RNA expression profiles in PB CD3+ lymphocytes collected from lenalidomide-treated del(5q) patients, contributing to overall understanding of lenalidomide action. Disclosures Sanz: Abbvie Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; LaHoffman Roche Ltd.: Membership on an entity's Board of Directors or advisory committees; Takeda Pharmaceutical Ltd.: Membership on an entity's Board of Directors or advisory committees; Helsinn: Membership on an entity's Board of Directors or advisory committees. Fenaux:Abbvie: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Jazz: Honoraria, Research Funding; BMS: Honoraria, Research Funding. Diez-Campelo:Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene-BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. OffLabel Disclosure: Lenalidomide was administered in anemic but not transfusion-dependence patients with low-risk MDS and del(5q)

Simultaneous Viral Whole-Genome Sequencing and Differential Expression Profiling in Respiratory Syncytial Virus Infection of Infants.

Targeted metagenomics using strand-specific libraries with target enrichment is a sensitive, generalized approach to pathogen sequencing and transcriptome profiling. Using this method, we recovered 13 (76%) complete human respiratory syncytial virus (RSV) genomes from 17 clinical respiratory samples, reconstructed the phylogeny of the infecting viruses, and detected differential gene expression between 2 RSV subgroups, specifically, a lower expression of the P gene and a higher expression of the M2 gene in RSV-A than in RSV-B. This methodology can help to relate viral genetics to clinical phenotype and facilitate ongoing population-level RSV surveillance and vaccine development. Clinical Trials Registration. NCT03627572 and NCT03756766.

Identification and characterization of long noncoding RNAs provide insight into the regulation of gene expression in response to heat stress in rainbow trout (Oncorhynchus mykiss)

Rainbow trout are typical cold-water fish species. However, with the intensification of global warming, high temperatures have severely restricted the development of aquaculture during the summer. Understanding the molecular regulatory mechanisms of rainbow trout responses to heat stress will be beneficial for alleviating heat stress-related damage. In this study, we performed RNA-seq of liver tissues from rainbow trout under heat stress (24 °C) and control conditions (18 °C) to identify lncRNAs and target genes by strand-specific library. Changes in nonspecific immune parameters revealed that a strong stress response occurred in rainbow trout at 24 °C. More than 658 million filtered reads and 5916 lncRNAs were identified from six libraries. A total of 927 novel lncRNAs were identified, and 428 differentially expressed lncRNAs were screened with stringent thresholds. The RNA-seq results were verified by RT-qPCR. In addition, a regulatory network of lncRNA-mRNA functional interactions was constructed, and the potential antisense, cis and trans targets of lncRNAs were predicted. GO and KEGG enrichment analyses showed that many target genes involved in maintenance of homeostasis or adaptation to stress and stimuli were highly induced under heat stress. Several regulatory pathways were also found to be involved in heat stress, including the thyroid hormone signaling pathway, the PI3K-Akt signaling pathway, and the estrogen signaling pathway, among others. These results broaden our understanding of lncRNAs associated with heat stress and provide new insights into the lncRNA mediated regulation of the rainbow trout heat stress response.

Hypoxia Dependent and Independent Dysregulation of the Transcriptome in Sickle Cell Anemia

Sickle cell anemia (SCA) is associated with an increased hypoxic response from anemia and vaso-occlusion-impaired tissue perfusion. The effects of hypoxia are mediated by hypoxia transcription factors (HIFs). Chuvash erythrocytosis (CE) is an inherited condition due to homozygosity for the missense mutation in VHL gene (VHLR200W) that impairs interactions of VHL with HIF-α subunits, thereby augmenting transcription of HIF-regulated genes. CE and SCA share increased expression of erythropoietin (EPO) and other HIF target genes. As HIF-regulation of transcription is tissue and differentiation-stage specific, in this study we used reticulocytes, which are easily accessible and purified peripheral blood erythroid cells. We compared the transcriptomes of SCA and CE reticulocytes to differentiate HIF-mediated dysregulation from non-hypoxic dysregulation of SCA transcripts. Our study revealed reticulocyte gene expression changes that are common to both diseases as well as SCA-specific changes. The reticulocytes were purified from 5 HbSS and 5 age- and gender-matched HbAA African-American individuals, and from 17 VHLR200W homozygotes from Chuvashia and 13 wild type Chuvash individuals. Total RNA was depleted of ribosomal RNA and globin transcripts, and reverse transcribed. Strand-specific libraries were constructed for 100 or 125 bp paired-end sequencing to 30-45 million reads using Illumina HiSeq 2500 or 4000 platform. The sequencing data were mapped to human reference genome version GRCh37 using the splice-aware aligner STAR and analyzed using DESeq2. In separate analyses of the two diseases, we identified 1435 genes differentially expressed in SCA among 6965 analyzed genes, 848 increased and 587 decreased in SCA relative to control individuals. We also identified 1498 genes differentially expressed in CE among 8989 analyzed genes, 862 increased and 636 decreased in CE relative to control individuals. Across all analyzed genes, there was a moderate correlation (r=0.30) of expression changes between the two diseases. Among genes differentially expressed, 258 up-regulated and 155 down-regulated genes overlapped between the two diseases, representing a 1.4-fold enrichment. In a combined analysis of the two diseases, we identified 1228 genes among 6924 analyzed genes that shared altered regulation in both diseases. The 693 genes increased in both diseases were enriched (adjusted P &lt; 0.05) in multiple metabolic, inflammatory, and oxidative pathways. The 535 genes decreased in both diseases were enriched in a cell cycle pathway. Among the commonly increased genes, the expression level of ERFE encoding erythroferrone was increased by 9.4-fold in CE and by 4.3-fold in SCA, suggesting markedly altered iron regulation in CE and SCA. This would be expected as both SCA and CE share upregulated erythropoiesis that is associated with augmented erythroferrone. Among the commonly decreased genes, RPL3L encoding Ribosomal Protein L3 Like was decreased by 68% in CE and by 93% in SCA. These common expression changes reflect hypoxic regulation related to chronic anemia in SCA. To assess disease-specific gene expression change, we tested the disease (SCA versus CE) by genotype (mutation versus wildtype) interaction effect. We identified 822 genes that showed disease-specific expression changes among 6924 analyzed genes. Of these disease-specific genes, 304 were increased and 153 decreased in SCA (adjusted P &lt;0.05) but not in CE (nominal P &gt;0.05). For example, SLC16A1, encoding Solute Carrier Family 16 Member 1 and associated with an erythrocyte lactate transporter defect, was increased in SCA by 11-fold whereas PPBP encoding Pro-Platelet Basic Protein was decreased in SCA by 92%; both genes showing no change in CE. The 304 genes with SCA-specific increased expression were enriched in "Thyroid hormone signaling" (5.1 fold) and "Glioma" (7.0-fold) pathways. The 153 genes with SCA-specific decreased expression were enriched in "Ribosome" (12-fold) pathway. Among the disease-specific genes, only 34 increased and 28 decreased in CE but not in SCA. Our study demonstrates high HIF transcriptional activity in both CE and SCA reticulocytes but also reveals hypoxia-independent gene expression changes in SCA reticulocytes. These results suggest that HIF might be a therapeutic target of SCA. These data also shed light on the different molecular mechanisms underlying SCA complications. Disclosures Gordeuk: Pfizer: Research Funding; Modus Therapeutics: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Emmaus: Consultancy, Honoraria; Global Blood Therapeutics: Consultancy, Honoraria, Research Funding; Inctye: Research Funding; CSL Behring: Consultancy, Honoraria, Research Funding; Ironwood: Research Funding; Imara: Research Funding.

R-ChIP for genome-wide mapping of R-loops by using catalytically inactive RNASEH1.

Nascent RNA may form a three-stranded structure with DNA, called an R-loop, which has been linked to fundamental biological processes such as transcription, replication and genome instability. Here, we provide a detailed protocol for a newly developed strategy, named R-ChIP, for robust capture of R-loops genome-wide. Distinct from R-loop-mapping methods based on the monoclonal antibody S9.6, which recognizes RNA-DNA hybrid structures, R-ChIP involves expression of an exogenous catalytically inactive RNASEH1 in cells to bind RNA-DNA hybrids but not resolve them. This is followed by chromatin immunoprecipitation (ChIP) of the tagged RNASEH1 and construction of a strand-specific library for deep sequencing. It takes ~3 weeks to establish a stable cell line expressing the mutant enzyme and 5 more days to proceed with the R-ChIP protocol. In principle, R-ChIP is applicable to both cell lines and animals, as long as the catalytically inactive RNASEH1 can be expressed to study the dynamics of R-loop formation and resolution, as well as its impact on the functionality of the genome. In our recent studies with R-ChIP, we showed an intimate spatiotemporal relationship between R-loops and RNA polymerase II pausing/pause release, as well as linking augmented R-loop formation to DNA damage response induced by driver mutations of key splicing factors associated with myelodysplastic syndrome (MDS).

Strand-specific Single-stranded DNA Sequencing (4S-seq) of E. coli genomes.

Most bacterial genomes have biased nucleotide composition, and the asymmetry is considered to be caused by a single-stranded DNA (ssDNA) deamination arising from the bacterial replication machinery. In order to evaluate the relationship experimentally, the position and frequency of ssDNA formed during replication must be verified clearly. Although many ssDNA detection technologies exist, almost all methods have been developed for eukaryotic genomes. To apply these to bacterial genomes, which harbor a smaller amount of DNA than those of eukaryotes, more efficient, new methods are required. Therefore, we developed a novel strand-specific ssDNA sequencing method, called 4S-seq, for the bacterial genome. The 4S-seq method enriches ssDNA in the extracted genomic DNA by a dsDNA-specific nuclease and implements a strand-specific library using a biotin label with a customized tag. As a result, the 4S-seq is able to calculate the ssDNA content in each strand (Watson/Crick) at each position of the genome efficiently.