Extracellular RNA Induces Neutrophil Recruitment Via Toll-Like Receptor 3 During Venous Thrombosis After Vascular Injury.

BACKGROUND: Venous thromboembolism is associated with endothelial cell activation that contributes to the inflammation-dependent activation of the coagulation system. Cellular damage is associated with the release of different species of extracellular RNA (eRNA) involved in inflammation and coagulation. TLR3 (toll-like receptor 3), which recognizes (viral) single-stranded or double-stranded RNAs and self-RNA fragments, might be the receptor of these species of eRNA during venous thromboembolism. Here, we investigate how the TLR3/eRNA axis contributes to venous thromboembolism. METHODS AND RESULTS: Thrombus formation and size in wild-type and TLR3 deficient (-/-) mice were monitored by ultrasonography after venous thrombosis induction using the ferric chloride and stasis models. Mice were treated with RNase I, with polyinosinic-polycytidylic acid, a TLR3 agonist, or with RNA extracted from murine endothelial cells. Gene expression and signaling pathway activation were analyzed in HEK293T cells overexpressing TLR3 in response to eRNA or in human umbilical vein endothelial cells transfected with a small interference RNA against TLR3. Plasma clot formation on treated human umbilical vein endothelial cells was analyzed. Thrombosis exacerbated eRNA release in vivo and increased eRNA content within the thrombus. RNase I treatment reduced thrombus size compared with vehicle-treated mice (P<0.05). Polyinosinic-polycytidylic acid and eRNA treatments increased thrombus size in wild-type mice (P<0.01 and P<0.05), but not in TLR3-/- mice, by reinforcing neutrophil recruitment (P<0.05). Mechanistically, TLR3 activation in endothelial cells promotes CXCL5 (C-X-C motif chemokine 5) secretion (P<0.001) and NFκB (nuclear factor kappa-light-chain-enhancer of activated B cells) activation (P<0.05). Finally, eRNA triggered plasma clot formation in vitro (P<0.01). CONCLUSIONS: We show that eRNA and TLR3 activation enhance venous thromboembolism through neutrophil recruitment possibly through secretion of CXCL5, a potent neutrophil chemoattractant.

Genome-wide enhancer RNA profiling adds molecular links between genetic variation and human cancers.

BACKGROUND: Dysregulation of enhancer transcription occurs in multiple cancers. Enhancer RNAs (eRNAs) are transcribed products from enhancers that play critical roles in transcriptional control. Characterizing the genetic basis of eRNA expression may elucidate the molecular mechanisms underlying cancers. METHODS: Initially, a comprehensive analysis of eRNA quantitative trait loci (eRNAQTLs) was performed in The Cancer Genome Atlas (TCGA), and functional features were characterized using multi-omics data. To establish the first eRNAQTL profiles for colorectal cancer (CRC) in China, epigenomic data were used to define active enhancers, which were subsequently integrated with transcription and genotyping data from 154 paired CRC samples. Finally, large-scale case-control studies (34,585 cases and 69,544 controls) were conducted along with multipronged experiments to investigate the potential mechanisms by which candidate eRNAQTLs affect CRC risk. RESULTS: A total of 300,112 eRNAQTLs were identified across 30 different cancer types, which exert their influence on eRNA transcription by modulating chromatin status, binding affinity to transcription factors and RNA-binding proteins. These eRNAQTLs were found to be significantly enriched in cancer risk loci, explaining a substantial proportion of cancer heritability. Additionally, tumor-specific eRNAQTLs exhibited high responsiveness to the development of cancer. Moreover, the target genes of these eRNAs were associated with dysregulated signaling pathways and immune cell infiltration in cancer, highlighting their potential as therapeutic targets. Furthermore, multiple ethnic population studies have confirmed that an eRNAQTL rs3094296-T variant decreases the risk of CRC in populations from China (OR = 0.91, 95%CI 0.88-0.95, P = 2.92 × 10-7) and Europe (OR = 0.92, 95%CI 0.88-0.95, P = 4.61 × 10-6). Mechanistically, rs3094296 had an allele-specific effect on the transcription of the eRNA ENSR00000155786, which functioned as a transcriptional activator promoting the expression of its target gene SENP7. These two genes synergistically suppressed tumor cell proliferation. Our curated list of variants, genes, and drugs has been made available in CancereRNAQTL ( http://canernaqtl.whu.edu.cn/#/ ) to serve as an informative resource for advancing this field. CONCLUSION: Our findings underscore the significance of eRNAQTLs in transcriptional regulation and disease heritability, pinpointing the potential of eRNA-based therapeutic strategies in cancers.

Enhancer demethylation-regulated gene score identified molecular subtypes, inspiring immunotherapy or CDK4/6 inhibitor therapy in oesophageal squamous cell carcinoma.

BACKGROUND: The 5-year survival rate of oesophageal squamous cell carcinoma (ESCC) is approximately 20%. The prognosis and drug response exhibit substantial heterogeneity in ESCC, impeding progress in survival outcomes. Our goal is to identify a signature for tumour subtype classification, enabling precise clinical treatments. METHODS: Utilising pre-treatment multi-omics data from an ESCC dataset (n = 310), an enhancer methylation-eRNA-target gene regulation network was constructed and validated by in vitro experiments. Four machine learning methods collectively identified core target genes, establishing an Enhancer Demethylation-Regulated Gene Score (EDRGS) model for classification. The molecular function of EDRGS subtyping was explored in scRNA-seq (n = 60) and bulk-seq (n = 310), and the EDRGS's potential to predict treatment response was assessed in datasets of various cancer types. FINDINGS: EDRGS stratified ESCCs into EDRGS-high/low subtypes, with EDRGS-high signifying a less favourable prognosis in ESCC and nine additional cancer types. EDRGS-high exhibited an immune-hot but immune-suppressive phenotype with elevated immune checkpoint expression, increased T cell infiltration, and IFNγ signalling in ESCC, suggesting a better response to immunotherapy. Notably, EDRGS outperformed PD-L1 in predicting anti-PD-1/L1 therapy effectiveness in ESCC (n = 42), kidney renal clear cell carcinoma (KIRC, n = 181), and bladder urothelial carcinoma (BLCA, n = 348) cohorts. EDRGS-low showed a cell cycle-activated phenotype with higher CDK4 and/or CDK6 expression, demonstrating a superior response to the CDK4/6 inhibitor palbociclib, validated in ESCC (n = 26), melanoma (n = 18), prostate cancer (n = 15) cells, and PDX models derived from patients with pancreatic cancer (n = 30). INTERPRETATION: Identification of EDRGS subtypes enlightens ESCC categorisation, offering clinical insights for patient management in immunotherapy (anti-PD-1/L1) and CDK4/6 inhibitor therapy across cancer types. FUNDING: This study was supported by funding from the National Key R&D Program of China (2021YFC2501000, 2020YFA0803300), the National Natural Science Foundation of China (82030089, 82188102), the CAMS Innovation Fund for Medical Sciences (2021-I2M-1-018, 2022-I2M-2-001, 2021-I2M-1-067), the Fundamental Research Funds for the Central Universities (3332021091).

Identification of key eRNAs for intervertebral disc degeneration by integrated multinomial bioinformatics analysis.

BACKGROUND: Intervertebral disc degeneration (IVDD) is a common degenerative condition leading to abnormal stress distribution under load, causing intervertebral stenosis, facet joint degeneration, and foraminal stenosis. Very little is known about the molecular mechanism of eRNAs in IVDD. METHODS: Gene expression profiles of 38 annulus disc samples composed of 27 less degenerated discs (LDs) and 11 more degenerated discs (MDs) were retrieved from the GEO database. Then, differentially expressed enhancer RNAs (DEeRNAs), differentially expressed target genes (DETGs), and differentially expressed transcription factors (DETFs), hallmark of cancer signalling pathways according to GSVA; the types and quantity of immune cells according to CIBERSORT; and immune gene sets according to ssGSEA were analysed to construct an IVDD-related eRNA network. Then, multidimensional validation was performed to explore the interactions among DEeRNAs, DETFs and DEGs in space. RESULTS: A total of 53 components, 14 DETGs, 15 DEeRNAs, 3 DETFs, 5 immune cells, 9 hallmarks, and 7 immune gene sets, were selected to construct the regulatory network. After validation by online multidimensional databases, 21 interactive DEeRNA-DEG-DETF axes related to IVDD exacerbation were identified, among which the C1S-CTNNB1-CHD4 axis was the most significant. CONCLUSION: Based upon the results of our study, we theorize that the C1S-CTNNB1-CHD4 axis plays a vital role in IVDD exacerbation. Specifically, C1S recruits CTNNB1 and upregulates the expression of CHD4 in IVDD, and subsequently, CHD4 suppresses glycolysis and activates oxidative phosphorylation, thus generating insoluble collagen fibre deposits and leading to the progression of IVDD. Overall, these DEeRNAs could comprise promising therapeutic targets for IVDD due to their high tissue specificity.

Identification and characterization of putative enhancer regions that direct Il6 transcription in murine macrophages.

Interleukin-6 (IL-6) plays a crucial role in various cellular functions, including innate and adaptive immune responses. Dysregulated expression of IL-6 is associated with hyperinflammation and chronic inflammatory diseases. In this study, we aimed to identify the enhancer regions responsible for robust Il6 mRNA expression in murine macrophages. Through comprehensive genome-wide ChIP- and ATAC-seq analyses, we identified two distinct clusters, termed E1 and E2 regions, located at -144 to -163 kb relative to the Il6 transcription start site in lipopolysaccharide (LPS)-activated murine macrophages. These clusters exhibited an accumulation of histone modification marks (H3K27ac and H3K4me1), as well as open chromatin, and were found to contain binding sites for the transcription factors PU.1, NF-κB, C/EBPß, and JunB. Upregulation of non-coding RNA (ncRNA) transcripts from the E1 and E2 regions was observed upon LPS stimulation, and repression of these ncRNAs resulted in abrogation of Il6 expression. Additionally, deletion of either E1 or E2 region significantly impaired Il6 expression, while CRISPR/dCas9 activation-mediated recruitment of the co-activator p300 to the E1 and E2 regions facilitated Il6 expression. Collectively, our findings suggest that the E1 and E2 regions serve as putative enhancers for Il6 expression.

Enhancer switching in cell lineage priming is linked to eRNA, Brg1's AT-hook, and SWI/SNF recruitment.

RNA transcribed from enhancers, i.e., eRNA, has been suggested to directly activate transcription by recruiting transcription factors and co-activators. Although there have been specific examples of eRNA functioning in this way, it is not clear how general this may be. We find that the AT-hook of SWI/SNF preferentially binds RNA and, as part of the esBAF complex, associates with eRNA transcribed from intronic and intergenic regions. Our data suggest that SWI/SNF is globally recruited in cis by eRNA to cell-type-specific enhancers, representative of two distinct stages that mimic early mammalian development, and not at enhancers that are shared between the two stages. In this manner, SWI/SNF facilitates recruitment and/or activation of MLL3/4, p300/CBP, and Mediator to stage-specific enhancers and super-enhancers that regulate the transcription of metabolic and cell lineage priming-related genes. These findings highlight a connection between ATP-dependent chromatin remodeling and eRNA in cell identity and typical- and super-enhancer activation.

Identification and Validation of eRNA as a Prognostic Indicator for Cervical Cancer.

The survival of CESC patients is closely related to the expression of enhancer RNA (eRNA). In this work, we downloaded eRNA expression, clinical, and gene expression data from the TCeA and TCGA portals. A total of 7936 differentially expressed eRNAs were discovered by limma analysis, and the relationship between these eRNAs and survival was analyzed by univariate Cox hazard analysis, LASSO regression, and multivariate Cox hazard analysis to obtain an 8-eRNA model. Risk score heat maps, KM curves, ROC analysis, robustness analysis, and nomograms further indicate that this 8-eRNA model is a novel indicator with high prognostic performance independent of clinicopathological classification. The model divided patients into high-risk and low-risk groups, compared pathway diversity between the two groups through GSEA analysis, and provided potential therapeutic agents for high-risk patients.

An overlooked source of false positives in eDNA-based biodiversity assessment and management.

Biodiversity conservation and management in urban aquatic ecosystems is crucial to human welfare, and environmental DNA (eDNA)-based methods have become popular in biodiversity assessment. Here we report a highly overlooked source of significant false positives for eDNA-based biodiversity assessment in urban aquatic ecosystems supplied with treated wastewater - eDNA pollution originating from treated wastewater represents a noteworthy source of false positives. To investigate whether eDNA pollution is specific to a certain treatment or prevalent across methods employed by wastewater treatment plants, we conducted tests on effluent treated using three different secondary processes, both before and after upgrades to tertiary treatment. We metabarcoded eDNA collected from effluent immediately after full treatment and detected diverse native and non-native, commercial and ornamental fishes (48 taxa) across all treatment processes before and after upgrades. Thus, eDNA pollution occurred irrespective of the treatment processes applied. Release of eDNA pollution into natural aquatic ecosystems could translate into false positives for eDNA-based analysis. We discuss and propose technical solutions to minimize these false positives in environmental nucleic acid-based biodiversity assessments and conservation programs.

Treatment with RNase alleviates brain injury but not neuroinflammation in neonatal hypoxia/ischemia.

There is a need for new treatments to reduce brain injuries derived from neonatal hypoxia/ischemia. The only viable option used in the clinic today in infants born at term is therapeutic hypothermia, which has a limited efficacy. Treatments with exogenous RNase have shown great promise in a range of different adult animal models including stroke, ischemia/reperfusion injury, or experimental heart transplantation, often by conferring vascular protective and anti-inflammatory effects. However, any neuroprotective function of RNase treatment in the neonate remains unknown. Using a well-established model of neonatal hypoxic/ischemic brain injury, we evaluated the influence of RNase treatment on RNase activity, gray and white matter tissue loss, blood-brain barrier function, as well as levels and expression of inflammatory cytokines in the brain up to 6 h after the injury using multiplex immunoassay and RT-PCR. Intraperitoneal treatment with RNase increased RNase activity in both plasma and cerebropinal fluids. The RNase treatment resulted in a reduction of brain tissue loss but did not affect the blood-brain barrier function and had only a minor modulatory effect on the inflammatory response. It is concluded that RNase treatment may be promising as a neuroprotective regimen, whereas the mechanistic effects of this treatment appear to be different in the neonate compared to the adult and need further investigation.

An Emerging Role for Enhancer RNAs in Brain Disorders.

Noncoding DNA undergoes widespread context-dependent transcription to produce noncoding RNAs. In recent decades, tremendous advances in genomics and transcriptomics have revealed important regulatory roles for noncoding DNA elements and the RNAs that they produce. Enhancers are one such element that are well-established drivers of gene expression changes in response to a variety of factors such as external stimuli, cellular responses, developmental cues, and disease states. They are known to act at long distances, interact with multiple target gene loci simultaneously, synergize with other enhancers, and associate with dynamic chromatin architectures to form a complex regulatory network. Recent advances in enhancer biology have revealed that upon activation, enhancers transcribe long noncoding RNAs, known as enhancer RNAs (eRNAs), that have been shown to play important roles in enhancer-mediated gene regulation and chromatin-modifying activities. In the brain, enhancer dysregulation and eRNA transcription has been reported in numerous disorders from acute injuries to chronic neurodegeneration. Because this is an emerging area, a comprehensive understanding of eRNA function has not yet been achieved in brain disorders; however, the findings to date have illuminated a role for eRNAs in activity-driven gene expression and phenotypic outcomes. In this review, we highlight the breadth of the current literature on eRNA biology in brain health and disease and discuss the challenges as well as focus areas and strategies for future in-depth research on eRNAs in brain health and disease.

A therapeutically targetable positive feedback loop between lnc-HLX-2-7, HLX, and MYC that promotes group 3 medulloblastoma.

Recent studies suggest that long non-coding RNAs (lncRNAs) contribute to medulloblastoma (MB) formation and progression. We have identified an lncRNA, lnc-HLX-2-7, as a potential therapeutic target in group 3 (G3) MBs. lnc-HLX-2-7 RNA specifically accumulates in the promoter region of HLX, a sense-overlapping gene of lnc-HLX-2-7, which activates HLX expression by recruiting multiple factors, including enhancer elements. RNA sequencing and chromatin immunoprecipitation reveal that HLX binds to and activates the promoters of several oncogenes, including TBX2, LIN9, HOXM1, and MYC. Intravenous treatment with cerium-oxide-nanoparticle-coated antisense oligonucleotides targeting lnc-HLX-2-7 (CNP-lnc-HLX-2-7) inhibits tumor growth by 40%-50% in an intracranial MB xenograft mouse model. Combining CNP-lnc-HLX-2-7 with standard-of-care cisplatin further inhibits tumor growth and significantly prolongs mouse survival compared with CNP-lnc-HLX-2-7 monotherapy. Thus, the lnc-HLX-2-7-HLX-MYC axis is important for regulating G3 MB progression, providing a strong rationale for using lnc-HLX-2-7 as a therapeutic target for G3 MBs.

Larger particle size distribution of environmental RNA compared to environmental DNA: a case study targeting the mitochondrial cytochrome b gene in zebrafish (Danio rerio) using experimental aquariums.

Environmental RNA (eRNA) analysis is conventionally expected to infer physiological information about organisms within their ecosystems, whereas environmental DNA (eDNA) analysis only infers their presence and abundance. Despite the promise of eRNA application, basic research on eRNA characteristics and dynamics is limited. The present study conducted aquarium experiments using zebrafish (Danio rerio) to estimate the particle size distribution (PSD) of eRNA in order to better understand the persistence state of eRNA particles. Rearing water samples were sequentially filtered using different pore-size filters, and the resulting size-fractioned mitochondrial cytochrome b (CytB) eDNA and eRNA data were modeled with the Weibull complementary cumulative distribution function (CCDF) to estimate the parameters characterizing the PSDs. It was revealed that the scale parameter (α) was significantly higher (i.e., the mean particle size was larger) for eRNA than eDNA, while the shape parameter (ß) was not significantly different between them. This result supports the hypothesis that most eRNA particles are likely in a protected, intra-cellular state, which mitigates eRNA degradation in water. Moreover, these findings also imply the heterogeneous dispersion of eRNA relative to eDNA and suggest an efficient method of eRNA collection using a larger pore-size filter. Further studies on the characteristics and dynamics of eRNA particles should be pursued in the future.

Comparative evaluation for the performance of environmental DNA and RNA analyses targeting mitochondrial and nuclear genes from ayu (Plecoglossus altivelis).

Environmental DNA and RNA (eDNA and eRNA; collectively eNA) analyses have the potential for non-invasive and cost-efficient biomonitoring compared with traditional capture-based surveys. Although various types of eNA particles, including not only mitochondrial eDNA but also nuclear eDNA and their transcripts, are present in the water, performances of eNA detection and quantification have not yet been evaluated sufficiently across multiple mitochondrial and nuclear genes. We conducted a tank experiment with ayu (Plecoglossus altivelis) to compare the detection sensitivity, yields per water sample, and quantification variability between replicates of each type of eNAs. The assay targeting the multi-copy nuclear gene exhibited a higher sensitivity than the assay targeting the mitochondrial gene, and both the target eDNA and eRNA concentrations per water sample were higher for the nuclear gene. On the contrary, variation in eRNA quantifications per sample does not necessarily correspond to that in eDNA, and the intra-sample quantification variability (represented as the CVs between PCR replicates) tended to be larger for eRNA than eDNA. Our results suggested that, even if suitable to the sensitive detection of species occurrence, the use of eRNA particularly derived from multi-copy nuclear gene may not be necessarily appropriate for the reliable assessment of species abundance. The findings in this study would help optimize eNA analyses for making biomonitoring and stock assessment in aquatic environments more efficient and reliable.

Identification and characterization of CHD4-associated eRNA as a novel modulator of fetal hemoglobin levels in ß-thalassemia.

Fetal-to-adult hemoglobin switching is controlled by programmed silencing of γ-globin while the re-activation of fetal hemoglobin (HbF) is an effective strategy for ameliorating the clinical severity of ß-thalassemia and sickle cell disease. The identification of enhancer RNAs (eRNAs) related to the fetal (α2γ2) to adult hemoglobin (α2ß2) switching remains incomplete. In this study, the transcriptomes of GYPA+ cells from six ß-thalassemia patients with extreme HbF levels were sequenced to identify differences in patterns of noncoding RNA expression. It is interesting that an enhancer upstream of CHD4, an HbF-related core subunit of the NuRD complex, was differentially transcribed. We found a significantly positive correlation of eRNA-CHD4 enhancer-gene interaction using the public database of FANTOM5. Specifically, the eRNA-CHD4 expression was found to be significantly higher in both CD34+ HSPCs and HUDEP-2 than those in K562 cells which commonly expressed high level of HbF, suggesting a correlation between eRNA and HbF expression. Furthermore, prediction of transcription binding sites of cis-eQTLs and the CHD4 genomic region revealed a putative interaction site between rs73264846 and ZNF410, a known transcription factor regulating HbF expression. Moreover, in-vitro validation showed that the inhibition of eRNA could reduce the expression of HBG expression in HUDEP-2 cells. Taken together, the findings of this study demonstrate that a distal enhancer contributes to stage-specific silencing of γ-globin genes through direct modulation of CHD4 expression and provide insights into the epigenetic mechanisms of NuRD-mediated hemoglobin switching.

New prospects of environmental RNA metabarcoding research in biological diversity, ecotoxicological monitoring, and detection of COVID-19: a critical review.

Ecosystems are multifaceted and complex systems and understanding their composition is crucial for the implementation of efficient conservation and management. Conventional approaches to biodiversity surveys can have limitations in detecting the complete range of species present. In contrast, the study of environmental RNA (eRNA) offers a non-invasive and comprehensive method for monitoring and evaluating biodiversity across different ecosystems. Similar to eDNA, the examination of genetic material found in environmental samples can identify and measure many species, including ones that pose challenges to traditional methods. However, eRNA is degraded quickly and therefore shows promise in detection of living organisms closer to their actual location than eDNA methods. This method provides a comprehensive perspective on the well-being of ecosystems, facilitating the development of focused conservation approaches to save at-risk species and uphold ecological equilibrium. Furthermore, eRNA has been recognized as a valuable method for the identification of COVID-19 in the environment, besides its established uses in biodiversity protection. The SARS-CoV-2 virus, which is accountable for the worldwide epidemic, releases RNA particles into the surrounding environment via human waste, providing insights into the feasibility of detecting it in wastewater and other samples taken from the environment. In this article, we critically reviewed the recent research activities that use the eRNA method, including its utilization in biodiversity conservation, ecological surveillance, and ecotoxicological monitoring as well as its innovative potential in identifying COVID-19. Through this review, the reader can understand the recent developments, prospects, and challenges of eRNA research in ecosystem management and biodiversity conservation.

Super enhancer loci of EGFR regulate EGFR variant 8 through enhancer RNA and strongly associate with survival in HNSCCs.

Epidermal growth factor receptor (EGFR) has been shown to be overexpressed in human cancers due to mutation, amplification, and epigenetic hyperactivity, which leads to deregulated transcriptional mechanism. Among the eight different EGFR isoforms, the mechanism of regulation of full-length variant 1 is well-known, no studies have examined the function & factors regulating the expression of variant 8. This study aimed to understand the function of EGFR super-enhancer loci and its associated transcription factors regulating the expression of EGFR variant 8. Our study shows that overexpression of variant 8 and its transcription was more prevalent than variant 1 in many cancers and positively correlated with the EGFR-AS1 expression in oral cancer and HNSCC. Notably, individuals overexpressing variant 8 showed shorter overall survival and had a greater connection with other clinical traits than patients with overexpression of variant 1. In this study, TCGA enhancer RNA profiling on the constituent enhancer (CE1 and CE2) region revealed that the multiple enhancer RNAs formed from CE2 by employing CE1 as a promoter. Our bioinformatic analysis further supports the enrichment of enhancer RNA specific chromatin marks H3K27ac, H3K4me1, POL2 and H2AZ on CE2. GeneHancer and 3D chromatin capture analysis showed clustered interactions between CE1, CE2 loci and this interaction may regulates expression of both EGFR-eRNA and variant 8. Moreover, increased expression of SNAI2 and its close relationship to EGFR-AS1 and variant 8 suggest that SNAI2 could regulates variant 8 overexpression by building a MegaTrans complex with both EGFR-eRNA and EGFR-AS1. Our findings show that EGFR variant 8 and its transcriptional regulation & chromatin modification by eRNAs may provide a rationale for targeting RNA splicing in combination with targeted EGFR therapies in cancer.

Validating post-enrichment steps in environmental RNA analysis for improving its availability from water samples.

Environmental RNA (eRNA) analysis is expected to inclusively provide the physiological information of a population and community without individual sampling, having the potential for the improved monitoring of biodiversity and ecosystem function. Protocol development for maximizing eRNA availability is crucial to interpret its detection and quantification results with high accuracy and reliability, but the methodological validation and improvement of eRNA collection and processing methods are scarce. In this study, the technical steps after eRNA extraction, including genomic DNA (gDNA) removal and reverse transcription, were focused on and their performances were compared by zebrafish (Danio rerio) aquarium experiments. Additionally, this study also focused on the eRNA quantification variabilities between replicates and compared them between the PCR and sample levels. Results showed that (i) there was a trade-off between gDNA removal approaches and eRNA yields and an excess gDNA removal could lead to the false-negative eRNA detection, (ii) the use of the gene-specific primers for reverse transcription could increase the eRNA yields for multiple mitochondrial and nuclear genes compared with the random hexamer primers, and (iii) the coefficient of variation (CV) values of eRNA quantifications between PCR replicates were substantially lower for those between samples. Including the study, further knowledge for the sensitive and precise detection of macro-organismal eRNA should be needed for increasing the reliability and robustness of eRNA-based biomonitoring.

Understanding blood development and leukemia using sequencing-based technologies and human cell systems.

Our current understanding of human hematopoiesis has undergone significant transformation throughout the years, challenging conventional views. The evolution of high-throughput technologies has enabled the accumulation of diverse data types, offering new avenues for investigating key regulatory processes in blood cell production and disease. In this review, we will explore the opportunities presented by these advancements for unraveling the molecular mechanisms underlying normal and abnormal hematopoiesis. Specifically, we will focus on the importance of enhancer-associated regulatory networks and highlight the crucial role of enhancer-derived transcription regulation. Additionally, we will discuss the unprecedented power of single-cell methods and the progression in using in vitro human blood differentiation system, in particular induced pluripotent stem cell models, in dissecting hematopoietic processes. Furthermore, we will explore the potential of ever more nuanced patient profiling to allow precision medicine approaches. Ultimately, we advocate for a multiparameter, regulatory network-based approach for providing a more holistic understanding of normal hematopoiesis and blood disorders.

Environmental transcriptomics under heat stress: Can environmental RNA reveal changes in gene expression of aquatic organisms?

To safeguard biodiversity in a changing climate, taxonomic information about species turnover and insights into the health of organisms are required. Environmental DNA approaches are increasingly used for species identification, but cannot provide functional insights. Transcriptomic methods reveal the physiological states of macroorganisms, but are currently species-specific and require tissue sampling or animal sacrifice, making community-wide assessments challenging. Here, we test whether broad functional information (expression level of the transcribed genes) can be harnessed from environmental RNA (eRNA), which includes extra-organismal RNA from macroorganisms along with whole microorganisms. We exposed Daphnia pulex as well as phytoplankton prey and microorganism colonizers to control (20°C) and heat stress (28°C) conditions for 7 days. We sequenced eRNA from tank water (after complete removal of Daphnia) as well as RNA from Daphnia tissue, enabling comparisons of extra-organismal and organismal RNA-based gene expression profiles. Both RNA types detected similar heat stress responses of Daphnia. Using eRNA, we identified 32 Daphnia genes to be differentially expressed following heat stress. Of these, 17 were also differentially expressed and exhibited similar levels of relative expression in organismal RNA. In addition to the extra-organismal Daphnia response, eRNA detected community-wide heat stress responses consisting of distinct functional profiles and 121 differentially expressed genes across eight taxa. Our study demonstrates that environmental transcriptomics based on extra-organismal eRNA can noninvasively reveal gene expression responses of macroorganisms following environmental changes, with broad potential implications for the biomonitoring of health across the trophic chain.