Whole-genome Transcriptome Analysis Research Articles

Bacterial peptidoglycan serves as a critical modulator of the gut-immune-brain axis in Drosophila

Metabolites and compounds derived from gut-associated bacteria can modulate numerous physiological processes in the host, including immunity and behavior. Using a model of oral bacterial infection, we previously demonstrated that gut-derived peptidoglycan (PGN), an essential constituent of the bacterial cell envelope, influences female fruit fly egg-laying behavior by activating the NF-κB cascade in a subset of brain neurons. These findings underscore PGN as a potential mediator of communication between gut bacteria and the brain in Drosophila, prompting further investigation into its impact on all brain cells. Through high-resolution mass spectrometry, we now show that PGN fragments produced by gut bacteria can rapidly reach the central nervous system. In Addition, by employing a combination of whole-genome transcriptome analyses, comprehensive genetic assays, and reporter gene systems, we reveal that gut bacterial infection triggers a PGN dose-dependent NF-κB immune response in perineurial glia, forming the continuous outer cell layer of the blood–brain barrier. Furthermore, we demonstrate that persistent PGN-dependent NF-κB activation in perineurial glial cells correlates with a reduction in lifespan and early neurological decline. Overall, our findings establish gut-derived PGN as a critical mediator of the gut-immune-brain axis in Drosophila.

Exploring the potential role of four Rhizophagus irregularis nuclear effectors: opportunities and technical limitations.

Arbuscular mycorrhizal fungi (AMF) are obligate symbionts that interact with the roots of most land plants. The genome of the AMF model species Rhizophagus irregularis contains hundreds of predicted small effector proteins that are secreted extracellularly but also into the plant cells to suppress plant immunity and modify plant physiology to establish a niche for growth. Here, we investigated the role of four nuclear-localized putative effectors, i.e., GLOIN707, GLOIN781, GLOIN261, and RiSP749, in mycorrhization and plant growth. We initially intended to execute the functional studies in Solanum lycopersicum, a host plant of economic interest not previously used for AMF effector biology, but extended our studies to the model host Medicago truncatula as well as the non-host Arabidopsis thaliana because of the technical advantages of working with these models. Furthermore, for three effectors, the implementation of reverse genetic tools, yeast two-hybrid screening and whole-genome transcriptome analysis revealed potential host plant nuclear targets and the downstream triggered transcriptional responses. We identified and validated a host protein interactors participating in mycorrhization in the host.S. lycopersicum and demonstrated by transcriptomics the effectors possible involvement in different molecular processes, i.e., the regulation of DNA replication, methylglyoxal detoxification, and RNA splicing. We conclude that R. irregularis nuclear-localized effector proteins may act on different pathways to modulate symbiosis and plant physiology and discuss the pros and cons of the tools used.

Abstract 576: The pan-quadruplex drug QN-302 selectively down-regulates key pathway targets in pancreatic cancer cells that are up-regulated in human pancreatic cancer

Abstract We have previously disclosed (Ahmed et al, ACS Med Chem Lett, 2020) a novel small molecule G-quadruplex binding compound, QN-302, that is highly potent in pancreatic ductal adenocarcinoma (PDAC) cells and in several in vivo models for this disease. QN-302 was invented at University College London UK and has been subsequently developed by Qualigen Therapeutics Inc. It was cleared by the FDA in August 2023 for Phase 1a clinical trials in USA and is currently under clinical evaluation. RNA-seq whole-genome transcriptome analysis revealed that QN-302 selectively targets G-quadruplexes in cancer-related genes, notably by forming stable G-quadruplex complexes with appropriate promoter sequences. This results in the transcriptional down regulation of affected genes, which map to for example, the hedgehog, axon guidance, WNT/β-catenin and P38 signal transduction pathways. In many instances individual expression changes are modest, though statistically significant: cumulatively, they affect whole pathways. We have revisited the RNA-seq data for QN-302 in PDAC MIA-PACA2 cells (GSE151741 at https://www.ncbi.nlm.nih.gov/geo/) in detail and now identify a small group of eleven down-regulated genes, all with >3-fold expression changes. Interestingly all these genes and their genes products have been previously identified as playing a role in human PDAC (they also have varying roles in other cancer types). Some have been validated as anticancer targets. 10/11 of these genes (https://www.proteinatlas.org/) have been previously reported as being over-expressed in significant fractions of large (>150) groups of human cancer patients from The Cancer Genome Atlas (TCGA) project. Cellular transcriptome data on these eleven genes is presented here, which together with earlier reports on them, supports the designation of QN-302 as a pan-G-quadruplex agent targeting a group of major genes involved in PDAC maintenance and metastasis. Data from a xenograft model for pancreatic cancer is also available for four of these genes, which concurs with the cellular data. We suggest that the identification of these 11 genes may also provide a basis for biomarker selection and possible patient stratification based on transcriptome data. Citation Format: Stephen Neidle, Ahmed A. Ahmed, Tariq Arshad. The pan-quadruplex drug QN-302 selectively down-regulates key pathway targets in pancreatic cancer cells that are up-regulated in human pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 576.

Genome-wide profiling of Hfq-bound RNAs reveals the iron-responsive small RNA RusT in Caulobacter crescentus.

The alphaproteobacterium Caulobacter crescentus thrives in oligotrophic environments and is able to optimally exploit minimal resources by entertaining an intricate network of gene expression control mechanisms. Numerous transcriptional activators and repressors have been reported to contribute to these processes, but only few studies have focused on regulation at the post-transcriptional level in C. crescentus. Small RNAs (sRNAs) are a prominent class of regulators of bacterial gene expression, and most sRNAs characterized today engage in direct base-pairing interactions to modulate the translation and/or stability of target mRNAs. In many cases, the ubiquitous RNA chaperone, Hfq, contributes to the establishment of RNA-RNA interactions. Although the deletion of the hfq gene is associated with a severe loss of fitness in C. crescentus, the RNA ligands of the chaperone have remained largely unexplored. Here we report on the identification of coding and non-coding transcripts associated with Hfq in C. crescentus and demonstrate Hfq-dependent post-transcriptional regulation in this organism. We show that the Hfq-bound sRNA RusT is transcriptionally controlled by the NtrYX two-component system and induced in response to iron starvation. By combining RusT pulse expression with whole-genome transcriptome analysis, we determine 16 candidate target transcripts that are deregulated, many of which encode outer membrane transporters. We hence suggest RusT to support remodeling of the C. crescentus cell surface when iron supplies are limited.IMPORTANCEThe conserved RNA-binding protein Hfq contributes significantly to the adaptation of bacteria to different environmental conditions. Hfq not only stabilizes associated sRNAs but also promotes inter-molecular base-pairing interactions with target transcripts. Hfq plays a pivotal role for growth and survival, controlling central metabolism and cell wall synthesis in the oligotroph Caulobacter crescentus. However, direct evidence for Hfq-dependent post-transcriptional regulation and potential oligotrophy in C. crescentus has been lacking. Here, we identified sRNAs and mRNAs associated with Hfq in vivo, and demonstrated the requirement of Hfq for sRNA-mediated regulation, particularly of outer membrane transporters in C. crescentus.

Integration of genomic and transcriptomic data of inbred mouse models for polygenic obesity and leanness revealed “obese” and “lean” candidate alleles in polyadenylation signals

Most mammalian genes have multiple polyadenylation (PA) sites, and alternative polyadenylation (APA) has been linked to diseases such as obesity. Studies have shown that changes in the polyadenylation signal (PAS) can influence the efficiency of cleavage and affect disease susceptibility and phenotype. In our recent study we used inbred mouse models of polygenic obesity and leanness and identified single-nucleotide polymorphisms in PAS (PAS-SNPs) within several obesity candidate genes, including five with differential expression. Nevertheless, to date, there has been no systematic, whole-genome-level approach aiming to prioritise PAS-SNPs potentially affecting APA. Therefore, in this study, we build upon our previous work by integrating existing genomics data with transcriptomics. DEGs were identified in nine tissues by Affymetrix GeneChip. PA and PAS sites were from the PolyASite 2.0 portal. Prioritisation of candidate PAS-SNPs was performed based on whether they were located in DEG, the type of PAS changes they caused, locations of PA sites relative to PAS, and location(s) and expression(s) of Affymetrix probes within a given gene in various tissues. For the candidates, potential consequences due to the alteration in APA events were investigated using bioinformatics databases and tools. The analysis found 127 PAS-SNPs in 101 DEGs across different tissues and identified 12 high-priority and 7 moderate-priority PAS-SNP candidates in 10 and 7 DEGs, respectively. Candidate PAS-SNPs were in 3′ UTR of 12 protein-coding genes (Lean line: Edil3, Eif2s1, Fbxl3, Hlf, Hsf2bp, Knop1, Lair1, Nmrk1; Fat line: Ehd1, Rpl14, Spon1, Txndc9), introns of four protein-coding genes (Lean line: Abi3bp, Prr16; Fat line: Agmo, Itga7) and intron of one lncRNA (Lean line: 1700086O06Rik). The integration of whole-genome sequencing and transcriptome analyses in this study has identified potential genome-wide candidate SNPs that could affect APA by altering/disrupting PAS motifs and be related to obesity in mice. The data provides a foundation for further research into these PAS-SNPs, their genes, and their contribution to the obesity/leanness phenotype, and contributes a part in explaining missing heritability commonly observed in complex traits.

Identification of SNPs and candidate genes associated with growth using GWAS and transcriptome analysis in Coilia nasus

The improvement of growth trait can foster the sustained and stable development of the aquaculture industry and is generally regarded as a primary objective in the breeding projects for economic fish. Coilia nasus, a migratory fish species naturally inhabiting in the middle and lower reaches of the Yangtze River and offshore waters of China, exhibits significant culturing potential and economic value. However, due to genetic degradation, there is a pronounced trend towards miniaturization and exaggerated growth variations within the cultured population. In order to facilitate the healthy development of the aquaculture industry of C. nasus, whole-genome association analysis (GWAS) and transcriptome analysis (RNA-seq) have been employed to identify molecular markers associated with growth traits and elucidate genetic foundations. A total of 234 individuals of C. nasus were subjected to whole-genome resequencing for genotyping, resulting in the identification of 2,306,254 high-quality SNPs. Fourteen SNPs were proved to have suggestive association with body length (BL) or body weight (BW), with the explained phenotypic variation ranging from 2.21% to 2.71%. We obtained two shared significant SNP peaks for BL and BW on LG6 and LG9, and bmp8a and uqcrfs1 were identified as their causal genes. Comparative transcriptome analysis based on extreme body size obtained 54 and 20 overlapping differentially expressed genes (DEGs) between different sexes in brain and muscle, respectively. Combining the results of GWAS and RNA-seq, we identified five candidate genes and one pathway associated with growth, including uqcrfs1, bmp8a, gabrb4, tshz3a, ahcyl1, and the mTORC1 signaling pathway. Our research findings provide molecular markers for genetic selection in C. nasus growth, and contribute to understand the molecular mechanisms of population growth variations.

Reactive gliosis in traumatic brain injury: a comprehensive review.

Traumatic brain injury (TBI) is one of the most common pathological conditions impacting the central nervous system (CNS). A neurological deficit associated with TBI results from a complex of pathogenetic mechanisms including glutamate excitotoxicity, inflammation, demyelination, programmed cell death, or the development of edema. The critical components contributing to CNS response, damage control, and regeneration after TBI are glial cells-in reaction to tissue damage, their activation, hypertrophy, and proliferation occur, followed by the formation of a glial scar. The glial scar creates a barrier in damaged tissue and helps protect the CNS in the acute phase post-injury. However, this process prevents complete tissue recovery in the late/chronic phase by producing permanent scarring, which significantly impacts brain function. Various glial cell types participate in the scar formation, but this process is mostly attributed to reactive astrocytes and microglia, which play important roles in several brain pathologies. Novel technologies including whole-genome transcriptomic and epigenomic analyses, and unbiased proteomics, show that both astrocytes and microglia represent groups of heterogenic cell subpopulations with different genomic and functional characteristics, that are responsible for their role in neurodegeneration, neuroprotection and regeneration. Depending on the representation of distinct glia subpopulations, the tissue damage as well as the regenerative processes or delayed neurodegeneration after TBI may thus differ in nearby or remote areas or in different brain structures. This review summarizes TBI as a complex process, where the resultant effect is severity-, region- and time-dependent and determined by the model of the CNS injury and the distance of the explored area from the lesion site. Here, we also discuss findings concerning intercellular signaling, long-term impacts of TBI and the possibilities of novel therapeutical approaches. We believe that a comprehensive study with an emphasis on glial cells, involved in tissue post-injury processes, may be helpful for further research of TBI and be the decisive factor when choosing a TBI model.

Whole-genome resequencing and transcriptome analyses of four generation mutants to reveal spur-type and skin-color related genes in apple (Malus domestica Borkh. Cv. Red delicious)

BackgroundBud sport is a kind of somatic mutation that usually occurred in apple. ‘Red Delicious’ is considered to be a special plant material of bud sport, whereas the genetic basis of plant mutants is still unknown. In this study, we used whole-genome resequencing and transcriptome sequencing to identify genes related to spur-type and skin-color in the ‘Red Delicious’ (G0) and its four generation mutants including ‘Starking Red’ (G1), ‘Starkrimson’ (G2), ‘Campbell Redchief’ (G3) and ‘Vallee Spur’ (G4).ResultsThe number of single nucleotide polymorphisms (SNPs), insertions and deletions (InDels) and structural variations (SVs) were decreased in four generation mutants compared to G0, and the number of unique SNPs and InDels were over 9-fold and 4-fold higher in G1 versus (vs.) G2 and G2 vs. G3, respectively. Chromosomes 2, 5, 11 and 15 carried the most SNPs, InDels and SVs, while chromosomes 1 and 6 carried the least. Meanwhile, we identified 4,356 variation genes by whole-genome resequencing and transcriptome, and obtained 13 and 16 differentially expressed genes (DEGs) related to spur-type and skin-color by gene expression levels. Among them, DELLA and 4CL7 were the potential genes that regulate the difference of spur-type and skin-color characters, respectively.ConclusionsOur study identified potential genes associated with spur-type and skin-color differences in ‘Red Delicious’ and its four generation mutants, which provides a theoretical foundation for the mechanism of the apple bud sport.

Normobaric hypoxia shows enhanced FOXO1 signaling in obese mouse gastrocnemius muscle linked to metabolism and muscle structure and neuromuscular innervation.

Skeletal muscle relies on mitochondria for sustainable ATP production, which may be impacted by reduced oxygen availability (hypoxia). Compared with long-term hypoxia, the mechanistic in vivo response to acute hypoxia remains elusive. Therefore, we aimed to provide an integrated description of the Musculus gastrocnemius response to acute hypoxia. Fasted male C57BL/6JOlaHsd mice, fed a 40en% fat diet for six weeks, were exposed to 12% O2 normobaric hypoxia or normoxia (20.9% O2) for six hours (n = 12 per group). Whole-body energy metabolism and the transcriptome response of the M. gastrocnemius were analyzed and confirmed by acylcarnitine determination and Q-PCR. At the whole-body level, six hours of hypoxia reduced energy expenditure, increased blood glucose and tended to decreased the respiratory exchange ratio (RER). Whole-genome transcriptome analysis revealed upregulation of forkhead box-O (FOXO) signalling, including an increased expression of tribbles pseudokinase 3 (Trib3). Trib3 positively correlated with blood glucose levels. Upregulated carnitine palmitoyltransferase 1A negatively correlated with the RER, but the significantly increased in tissue C14-1, C16-0 and C18-1 acylcarnitines supported that β-oxidation was not regulated. The hypoxia-induced FOXO activation could also be connected to altered gene expression related to fiber-type switching, extracellular matrix remodeling, muscle differentiation and neuromuscular junction denervation. Our results suggest that a six-hour exposure of obese mice to 12% O2 normobaric hypoxia impacts M. gastrocnemius via FOXO1, initiating alterations that may contribute to muscle remodeling of which denervation is novel and warrants further investigation. The findings support an early role of hypoxia in tissue alterations in hypoxia-associated conditions such as aging and obesity.

BCL3 rearrangements in B-cell lymphoid neoplasms occur in two breakpoint clusters associated with different diseases.

The t(14;19)(q32;q13) often juxtaposes BCL3 with immunoglobulin heavy chain (IGH) resulting in overexpression of the gene. In contrast to other oncogenic translocations, BCL3 rearrangement (BCL3-R) has been associated with a broad spectrum of lymphoid neoplasms. Here we report an integrative whole-genome sequence, transcriptomic, and DNA methylation analysis of 13 lymphoid neoplasms with BCL3-R. The resolution of the breakpoints at single base-pair revealed that they occur in two clusters at 5' (n=9) and 3' (n=4) regions of BCL3 associated with two different biological and clinical entities. Both breakpoints were mediated by aberrant class switch recombination of the IGH locus. However, the 5' breakpoints (upstream) juxtaposed BCL3 next to an IGH enhancer leading to overexpression of the gene whereas the 3' breakpoints (downstream) positioned BCL3 outside the influence of the IGH and were not associated with its expression. Upstream BCL3-R tumors had unmutated IGHV, trisomy 12, and mutated genes frequently seen in chronic lymphocytic leukemia (CLL) but had an atypical CLL morphology, immunophenotype, DNA methylome, and expression profile that differ from conventional CLL. In contrast, downstream BCL3-R neoplasms were atypical splenic or nodal marginal zone lymphomas (MZL) with mutated IGHV, complex karyotypes and mutated genes typical of MZL. Two of the latter four tumors transformed to a large B-cell lymphoma. We designed a novel fluorescence in situ hybridization assay that recognizes the two different breakpoints and validated these findings in 17 independent tumors. Overall, upstream or downstream breakpoints of BCL3-R are mainly associated with two subtypes of lymphoid neoplasms with different (epi)genomic, expression, and clinicopathological features resembling atypical CLL and MZL, respectively.

Human Adult Renal Progenitor Cells Prevent Cisplatin-Nephrotoxicity by Inducing CYP1B1 Overexpression and miR-27b-3p Down-Regulation through Extracellular Vesicles.

Cisplatin is one of the most effective chemotherapeutic agents strongly associated with nephrotoxicity. Tubular adult renal progenitor cells (tARPC) can regenerate functional tubules and participate in the repair processes after cisplatin exposition. This study investigated the molecular mechanisms underlying the protective effect of tARPC on renal epithelium during cisplatin nephrotoxicity. By performing a whole-genome transcriptomic analysis, we found that tARPC, in presence of cisplatin, can strongly influence the gene expression of renal proximal tubular cell [RPTEC] by inducing overexpression of CYP1B1, a member of the cytochrome P450 superfamily capable of metabolizing cisplatin and of hypoxia/cancer-related lncRNAs as MIR210HG and LINC00511. Particularly, tARPC exerted renoprotection and regeneration effects via extracellular vesicles (EV) enriched with CYP1B1 and miR-27b-3p, a well-known CYP1B1 regulatory miRNA. The expression of CYP1B1 by tARPC was confirmed by analyzing biopsies of cisplatin-treated renal carcinoma patients that showed the colocalization of CYP1B1 with the tARPC marker CD133. CYP1B1 was also overexpressed in urinary EV purified from oncologic patients that presented nephrotoxicity episodes after cisplatin treatment. Interestingly CYP1B1 expression significantly correlated with creatinine and eGFR levels. Taken together, our results show that tARPC are able to counteract cisplatin-induced nephrotoxicity via CYP1B1 release through EV. These findings provide a promising therapeutic strategy for nephrotoxicity risk assessment that could be related to abundance of renal progenitors.

PRDM16 regulates arterial development and vascular integrity.

Proper vascular formation is regulated by multiple signaling pathways. The vascular endothelial growth factor (VEGF) signaling promotes endothelial proliferation. Notch and its downstream targets act to lead endothelial cells toward an arterial fate through regulation of arterial gene expression. However, the mechanisms of how endothelial cells (ECs) in the artery maintain their arterial characteristics remain unclear. Here, we show that PRDM16 (positive regulatory domain-containing protein 16), a zinc finger transcription factor, is expressed in arterial ECs, but not venous ECs in developing embryos and neonatal retinas. Endothelial-specific deletion of Prdm16 induced ectopic venous marker expression in the arterial ECs and reduced vascular smooth muscle cell (vSMC) recruitment around arteries. Whole-genome transcriptome analysis using isolated brain ECs show that the expression of Angpt2 (encoding ANGIOPOIETIN2, which inhibits vSMC recruitment) is upregulated in the Prdm16 knockout ECs. Conversely, forced expression of PRDM16 in venous ECs is sufficient to induce arterial gene expression and repress the ANGPT2 level. Together, these results reveal an arterial cell-autonomous function for PRDM16 in suppressing venous characteristics in arterial ECs.

Integration of ARTP Mutation and Adaptive Laboratory Evolution to Reveal 1,4-Butanediol Degradation in Pseudomonas putida KT2440.

Biotransformation of plastics or their depolymerization monomers as raw materials would offer a better end-of-life solutions to the plastic waste dilemma. 1,4-butanediol (BDO) is one of the major depolymerization monomers of many plastics polymers. BDO valorization presents great significance for waste plastic up-recycling and fermenting feedstock exploitation. In the present study, atmospheric pressure room temperature plasma (ARTP)-induced mutation combined with adaptive laboratory evolution (ALE) was used to improve the BDO utilization capability of Pseudomonas putida KT2440. The excellent mutant P. putida NB10 was isolated and stored in the China Typical Culture Preservation Center (CCTCC) with the deposit number M 2021482. Whole-genome resequencing and transcriptome analysis revealed that the BDO degradation process consists of β-oxidation, glyoxylate carboligase (GCL) pathway, glyoxylate cycle and gluconeogenesis pathway. The imbalance between the two key intermediates (acetyl-CoA and glycolyl-CoA) and the accumulation of cytotoxic aldehydes resulted in the weak metabolism performance of KT2440 in the utilization of BDO. The balance of the carbon flux and enhanced tolerance to cytotoxic intermediates endow NB10 with great BDO degradation capability. This study deeply revealed the metabolic mechanism behind BDO degradation and provided an excellent chassis cell for BDO further up-cycling to high-value chemicals. IMPORTANCE Plastic waste represents not only a global pollution problem but also a carbon-rich, low-cost, globally renewable feedstock for industrial biotechnology. BDO is the basic material for polybutylene terephthalate (PBT), poly butylene adipate-co-terephthalate (PBAT), poly (butylene succinate) (PBS), etc. Herein, the construction of BDO valorization cell factory presents great significance for waste plastic up-recycling and novel fermentation feedstock exploitation. However, BDO is hard to be metabolized and its metabolic pathway is unclear. This study presents a P. putida mutant NB10, obtained through the integration of ARTP and ALE, displaying significant growth improvement with BDO as the sole carbon source. Further genome resequencing, transcriptome analysis and genetic engineering deeply revealed the metabolic mechanism behind BDO degradation in P. putida, this study offers an excellent microbial chassis and modification strategy for plastic waste up-cycling.

Abstract 1332: Extracted mRNA from frozen buffy coat samples shows promise for use in downstream sequencing analyses

Abstract Frozen blood samples are routinely collected and stored in medical biobanks across the world, often for periods of several decades, and later used for medical research such as biomarker studies. Some common biomarker targets have included DNA alterations, protein or antibody levels or metabolomics. However, despite the potential importance of gene-expression alterations in circulating immune cells, transcriptional studies on blood samples from biobanks have been limited due to preanalytical technical difficulties of extracting high-quality RNA. In the absence of an RNA preservative, RNA degrades rapidly during the thawing process. Our aim was, therefore, to investigate the possibility of using biobanked blood samples for transcriptomics analyses. First, we used a CryoXtract CXT 350 to establish a protocol capable of isolating high-quality RNA from frozen core aliquots of buffy coat without thawing the entire sample tube. Then, we performed RNA sequencing on RNA extracted from 20 pre-diagnostic buffy coat samples from colorectal cancer patients and matched controls included in the population-based, prospective Northern Sweden Health and Disease Study (NSHDS). For all samples, two different library preparation methods were used (Illumina TruSeq Stranded mRNA poly-A selection and Illumina Stranded Total RNA with Ribo-Zero Globin). We obtained RNA yields of more than 1 µg from a majority of the NSHDS samples (mean=2.57 µg), and more than 92% of samples had RIN values of 6 or higher, indicating suitability for downstream analyses. Preliminary random forest analyses showed expected discriminatory ability between men and women based on data generated from each of the two library preparation methods. In conclusion, our newly established RNA extraction method enables extraction of RNA from frozen, biobanked buffy coat samples, collected in standard tubes, that is of sufficient quantity and quality for use in downstream whole-genome transcriptomic analyses, such as RNA sequencing. Citation Format: Erik Bovinder Ylitalo, Linda Vidman, Sophia Harlid, Bethany Van Guelpen. Extracted mRNA from frozen buffy coat samples shows promise for use in downstream sequencing analyses [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 1332.

PD17-05 GENOMIC FEATURES OF RENAL CELL CARCINOMA DEVELOPED DURING END-STAGE RENAL DISEASE AND DIALYSIS

You have accessJournal of UrologyCME1 Apr 2023PD17-05 GENOMIC FEATURES OF RENAL CELL CARCINOMA DEVELOPED DURING END-STAGE RENAL DISEASE AND DIALYSIS Shigekatsu Maekawa, Todd Johnson, Masashi Fujita, Ryo Takata, Daiki Ikarashi, Tomohiko Matsuura, Renpei Kato, Mitsugu Kanehira, Jun Sugimura, Takaya Abe, Hikewaki Nakagawa, and Wataru Obara Shigekatsu MaekawaShigekatsu Maekawa More articles by this author , Todd JohnsonTodd Johnson More articles by this author , Masashi FujitaMasashi Fujita More articles by this author , Ryo TakataRyo Takata More articles by this author , Daiki IkarashiDaiki Ikarashi More articles by this author , Tomohiko MatsuuraTomohiko Matsuura More articles by this author , Renpei KatoRenpei Kato More articles by this author , Mitsugu KanehiraMitsugu Kanehira More articles by this author , Jun SugimuraJun Sugimura More articles by this author , Takaya AbeTakaya Abe More articles by this author , Hikewaki NakagawaHikewaki Nakagawa More articles by this author , and Wataru ObaraWataru Obara More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003272.05AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Patients with end-stage renal disease (ESRD) or receiving dialysis have a much higher risk for renal cell carcinoma (RCC), but carcinogenic mechanisms and genomic features remain little explored and undefined. This study’s goal was to identify the genomic features of ESRD RCC and characterize them for associations with tumor histology and dialysis exposure. In this study, we obtained 33 RCCs, with various histological subtypes, that developed in ESRD patients receiving dialysis and performed whole-genome sequencing and transcriptome analyses. METHODS: Frozen kidney samples were obtained from 48 patients who were undergoing dialysis treatment and developed RCC at Iwate Medical University Hospital. Among them, we selected 38 subjects for study from whom matched tumor and adjacent non-tumor tissue samples could be obtained and from which DNA (n=38) or RNA (n=26) could be isolated. We performed whole genome sequencing of genomic DNA using MGIEasy FS DNA Library Prep Set and short read sequencer DNBSEQ-G400. mRNA Sample were sequenced using HiSeq. Driver events, copy-number alteration (CNA) analysis, and mutational signature profiling was performed using an analysis pipeline that integrated data from germline and somatic SNVs, Indels, and structural variants as well as CNAs, while transcriptome data was analyzed for differentially expressed genes and through gene set enrichment analysis. RESULTS: ESRD related clear cell RCCs' driver genes and mutations mirrored those in sporadic ccRCCs. Longer dialysis periods significantly correlated with a rare mutational signature SBS23, whose etiology is unknown, and increased mitochondrial copy number. All ACD (acquired cystic disease)-RCCs, which developed specifically in ESRD patients, showed chromosome 16q amplification. Gene expression analysis suggests similarity between certain ACD-RCCs and papillary RCCs and in TCGA papillary RCCs with chromosome 16 gain identified enrichment for genes related to DNA repair, as well as pathways related to reactive oxygen species, oxidative phosphorylation, and targets of Myc. CONCLUSIONS: This analysis suggests that ESRD or dialysis could induce types of cellular stress that impact some specific types of genomic damage leading to oncogenesis. Source of Funding: none © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e497 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Shigekatsu Maekawa More articles by this author Todd Johnson More articles by this author Masashi Fujita More articles by this author Ryo Takata More articles by this author Daiki Ikarashi More articles by this author Tomohiko Matsuura More articles by this author Renpei Kato More articles by this author Mitsugu Kanehira More articles by this author Jun Sugimura More articles by this author Takaya Abe More articles by this author Hikewaki Nakagawa More articles by this author Wataru Obara More articles by this author Expand All Advertisement PDF downloadLoading ...

Identification of disease-related aberrantly spliced transcripts in myeloma and strategies to target these alterations by RNA-based therapeutics

Novel drug discoveries have shifted the treatment paradigms of most hematological malignancies, including multiple myeloma (MM). However, this plasma cell malignancy remains incurable, and novel therapies are therefore urgently needed. Whole-genome transcriptome analyses in a large cohort of MM patients demonstrated that alterations in pre-mRNA splicing (AS) are frequent in MM. This manuscript describes approaches to identify disease-specific alterations in MM and proposes RNA-based therapeutic strategies to eradicate such alterations. As a “proof of concept”, we examined the causes of aberrant HMMR (Hyaluronan-mediated motility receptor) splicing in MM. We identified clusters of single nucleotide variations (SNVs) in the HMMR transcript where the altered splicing took place. Using bioinformatics tools, we predicted SNVs and splicing factors that potentially contribute to aberrant HMMR splicing. Based on bioinformatic analyses and validation studies, we provided the rationale for RNA-based therapeutic strategies to selectively inhibit altered HMMR splicing in MM. Since splicing is a hallmark of many cancers, strategies described herein for target identification and the design of RNA-based therapeutics that inhibit gene splicing can be applied not only to other genes in MM but also more broadly to other hematological malignancies and solid tumors as well.

Characterization of the innate immune response to Streptococcus pneumoniae infection in zebrafish

Streptococcus pneumoniae (pneumococcus) is one of the most frequent causes of pneumonia, sepsis and meningitis in humans, and an important cause of mortality among children and the elderly. We have previously reported the suitability of the zebrafish (Danio rerio) larval model for the study of the host-pathogen interactions in pneumococcal infection. In the present study, we characterized the zebrafish innate immune response to pneumococcus in detail through a whole-genome level transcriptome analysis and revealed a well-conserved response to this human pathogen in challenged larvae. In addition, to gain understanding of the genetic factors associated with the increased risk for severe pneumococcal infection in humans, we carried out a medium-scale forward genetic screen in zebrafish. In the screen, we identified a mutant fish line which showed compromised resistance to pneumococcus in the septic larval infection model. The transcriptome analysis of the mutant zebrafish larvae revealed deficient expression of a gene homologous for human C-reactive protein (CRP). Furthermore, knockout of one of the six zebrafish crp genes by CRISPR-Cas9 mutagenesis predisposed zebrafish larvae to a more severe pneumococcal infection, and the phenotype was further augmented by concomitant knockdown of a gene for another Crp isoform. This suggests a conserved function of C-reactive protein in anti-pneumococcal immunity in zebrafish. Altogether, this study highlights the similarity of the host response to pneumococcus in zebrafish and humans, gives evidence of the conserved role of C-reactive protein in the defense against pneumococcus, and suggests novel host genes associated with pneumococcal infection.

Transcriptomic Analysis Reveals JAK2/MPL-Independent Effects of Calreticulin Mutations in a C. elegans Model.

There is growing evidence that Ph-negative myeloproliferative neoplasms (MPNs) are disorders in which multiple molecular mechanisms are significantly disturbed. Since their discovery, CALR driver mutations have been demonstrated to trigger pathogenic mechanisms apart from the well-documented activation of JAK2/MPL-related pathways, but the lack of experimental models harboring CALR mutations in a JAK2/MPL knockout background has hindered the research on these non-canonical mechanisms. In this study, CRISPR/Cas9 was performed to introduce homozygous patient-like calreticulin mutations in a C. elegans model that naturally lacks JAK2 and MPL orthologs. Whole-genome transcriptomic analysis of these worms was conducted, and some of the genes identified to be associated with processes involved in the pathogenesis of MPNs were further validated by qPCR. Some of the transcriptomic alterations corresponded to typically altered genes and processes in cancer and Ph-negative MPN patients that are known to be triggered by mutant calreticulin without the intervention of JAK2/MPL. However, interestingly, we have also found altered other processes described in these diseases that had not been directly attributed to calreticulin mutations without the intervention of JAK2 or MPL. Thus, these results point to a new experimental model for the study of the JAK2/MPL-independent mechanisms of mutant calreticulin that induce these biological alterations, which could be useful to study unknown non-canonical effects of the mutant protein. The comparison with a calreticulin null strain revealed that the alteration of all of these processes seems to be a consequence of a loss of function of mutant calreticulin in the worm, except for the dysregulation of Hedgehog signaling and flh-3. Further analysis of this model could help to delineate these mechanisms, and the verification of these results in mammalian models may unravel new potential therapeutic targets in MPNs. As far as we know, this is the first time that a C. elegans strain with patient-like mutations is proposed as a potential model for leukemia research.

Integrated genomics, QTL mapping, and co-expression analyses identifying candidates of low-temperature tolerance in Brassica napus L.

Low-temperature is a major environmental factor that limits agricultural productivity of over-wintering crops such as rapeseed (Brassica napus L.). Low-temperature tolerance is a complex quantitative trait that is susceptible to environmental conditions. To understand the genetic basis of low-temperature tolerance in rapeseed, we applied whole-genome sequencing, genetic mapping, and transcriptomic analyses. A total of twenty-eight quantitative trait loci for frost damage index were detected across the different environments using whole-genome sequencing in a recombinant inbred line population derived from GZ hui (cold-tolerant) and 10B (cold-sensitive). Compared to the genetic variants of the parentals and the quantitative trait loci scanned by simple sequence repeat markers among an F2 population, one major interval, qFDI.C02.1 in the 0.5–5.9 Mb physical position on C02 was a co-detected locus which was related to low-temperature tolerance. Moreover, genome wide identification of the homoeologous exchange events showed that qFDI.C02.1 overlapped with a homoeologous exchange of the genome, suggesting that chromosome structure variation was important for the low-temperature responses. Present/absent variation (PAV) markers were developed to distinguish the A and C subgenomes, and narrowed qFDI.C02.1 to PAV11-PAV14 approximately 1.6 Mb physical region. Combining with the results from RNA-seq and co-expression network analyses, seven cold-inducing genes were proposed as candidate genes in the qFDI.C02.1, including BnaC02g03120D (E2F_TDP family), BnaC02g03470D (Pseudo ARR-B family), BnaC02g02380D (chalcone-flavonone isomerase 3), BnaC02g03620D (Phosphopantothenate-cysteine ligase 1), BnaC02g03190D (Membrane magnesium transporter), BnaC02g03680D (L-type lectin-domain containing receptor kinase), and BnaC02g03590D (Haloacid dehalogenase-like hydrolase). This study will lay a foundation for exploring low-temperature tolerance genes of GZ hui and breeding cold-tolerance rapeseed varieties.

Whole-Genome Sequencing and Transcriptome Analysis of Ganoderma lucidum Strain Yw-1-5 Provides New Insights into the Enhanced Effect of Tween80 on Exopolysaccharide Production.

Ganoderma lucidum is an important medicinal mushroom widely cultured in Asian countries. Exopolysaccharides are bioactive compounds of G. lucidum with health benefits. Limited exopolysaccharide content hinders its extraction from G. lucidum. The addition of Tween80 had an enhanced effect on G. lucidum exopolysaccharide production in submerged fermentation. However, the mechanism of this effect remains unclear. In this study, we report on a high-quality assembly of G. lucidum strain yw-1-5 to lay the foundation for further transcriptome analysis. The genome sequence was 58.16 Mb and consisted of 58 scaffolds with an N50 of 4.78 Mb. A total of 13,957 protein-coding genes were annotated and Hi-C data mapped to 12 pseudo-chromosomes. Genes encoding glycosyltransferases and glycoside hydrolases were also obtained. Furthermore, RNA-seq was performed in a Tween80-treated group and control group for revealing the enhanced effect of Tween80 on exopolysaccharide production. In total, 655 genes were identified as differentially expressed, including 341 up-regulated and 314 down-regulated. Further analysis of differentially expressed genes showed that groups of MAPK, amino sugar and nucleotide sugar metabolism, autophagy, ubiquitin-mediated proteolysis, peroxisome, starch and sucrose metabolism, TCA cycle, glycolysis/gluconeogenesis KEGG pathway, glycosyltransferases and glycoside hydrolases played important roles in the enhanced effect of Tween80 on exopolysaccharide production. This work provides a valuable resource for facilitating our understanding of the synthesis of polysaccharides and accelerating the breeding of new strains with a high content of exopolysaccharides.