Whole-transcriptome Sequencing Research Articles

Whole transcriptome landscape in HAPE under the stress of environment at high altitudes: new insights into the mechanisms of hypobaric hypoxia tolerance.

High altitude pulmonary edema (HAPE) is an idiopathic, noncardiogenic form of pulmonary edema that occurs at high altitudes. It is characterized by a severe clinical course and carries a significant mortality risk. Despite its clinical relevance, the molecular mechanisms underlying HAPE are not well understood. We conducted whole-transcriptome RNA sequencing on blood samples from 6 pairs of HAPE patients and healthy controls to identify differentially expressed (DE) mRNAs, miRNAs, circRNAs, lncRNAs, along with alternative splicing (AS) events, gene fusions, and novel transcripts. To explore the regulatory dynamics, we constructed ceRNA networks and analyzed immune cell infiltration patterns, further annotating the biological functions of these transcripts. For empirical validation, we selected five circRNAs from the ceRNA network and conducted RT-qPCR on 50 paired samples. Additionally, we assessed the correlations between circRNA expression levels and clinical data to evaluate their diagnostic potential. We observed 2,023 differentially expressed mRNAs (DEmRNAs), 84 DEmiRNAs, 200 DEcircRNAs, and 3,573 DElncRNAs. A total of 139 'A3SS' events, 103 'A5SS' events, 545 'MXE' events, 14 'RI' events, and 1,482 'SE' events were identified in the AS events analysis between the two groups. Two ceRNA networks were constructed. T cells, follicular helper, and Macrophages M1 cells exhibited the strongest positive correlation (R=0.82), while naive B cells and memory B cells demonstrated the strongest negative correlation (R=-0.62). In total, the expression of three circRNAs was significantly different in a larger cohort. Hsa_circ_0058497, hsa_circ_0081006, and hsa_circ_0083220 demonstrated consistent with the RNA sequencing results. These three circRNAs strongly correlate with clinical indicators and exhibit potential as diagnostic biomarkers. Finally, we verified five genes (CXCR4, HSD17B2, ANGPTL4, TIMP3, N4BP3) that were differentially expressed in endothelial cells under normoxia and hypoxia through bioinformatics and RT-qPCR analyses. This study elucidates the differential expression of coding and non-coding RNAs (ncRNAs) in HAPE, identifies new transcripts and genes, and enhances our understanding of the transcriptional characteristics of HAPE. Moreover, it highlights the potential role of circRNAs in advancing the diagnosis and treatment of HAPE.

Whole-transcriptome sequencing revealed the role of noncoding RNAs in susceptibility and resistance of Pekin ducks to DHAV-3

As the most prevalent pathogen of duck viral hepatitis (DVH), duck hepatitis A virus genotype 3 (DHAV-3) has caused huge economic losses to the duck industry in China. Herein, we obtained whole-transcriptome sequencing data of susceptible (S) and resistant (R) Pekin duckling samples at 0 h, 12 h, and 24 h after DHAV-3 infection. We found that DHAV-3 infection induces 5,396 differentially expressed genes (DEGs), 85 differentially expressed miRNAs (DEMs), and 727 differentially expressed lncRNAs (DELs) at 24 hpi in S vs. R ducks, those upregulated genes were enriched in inflammation and cell communications pathways and downregulated genes were related to metabolic processes. Upregulated genes showed high connectivity with the miR-33, miR-193, and miR-11591, and downregulated genes were mainly regulated by miR-2954, miR-125, and miR-146b. With the construction of lncRNA-miRNA-mRNA axis, we further identified a few aberrantly expressed lncRNAs (e.g., MSTRG.36194.1, MSTRG.50601.1, MSTRG.34328.7, and MSTRG.29445.1) that regulate expression of hub genes (e.g., THBD, CLIC2, IL8, ACOX2, GPHN, SMLR1, and HAO1) by sponging those highly connected miRNAs. Altogether, our findings defined a dual role of ncRNAs in immune and metabolic regulation during DHAV-3 infection, suggesting potential new targets for treating DHAV-3 infected ducks.

Whole-Transcriptome Sequencing of Ovary Reveals the ceRNA Regulation Network in Egg Production of Gaoyou Duck.

To investigate the regulatory mechanism of the competing endogenous RNAs (ceRNAs) on the egg performance of Gaoyou ducks, full transcriptome sequencing was performed to analyze the ovarian tissues in Gaoyou ducks. The ducks were categorized into high- and low-yield groups based on the individual in-cage egg production records and the hematoxylin-eosin (HE) staining results. The differentially expressed genes (DEGs), long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs) were further processed by GO (gene ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) analyses. In total, 72 DEmRNAs; 23 DElncRNAs; 4 DEcircRNAs; and 5 signaling pathways, including the ovarian steroidogenesis, PI3K-Akt, hedgehog, tryptophan metabolism, and oocyte meiosis signaling pathways, were significantly enriched. These results suggest that they could be associated with the Gaoyou duck's ovarian function and affect the total egg production or double-yolked egg production. Furthermore, a coregulation network based on the related candidate ceRNAs across the high- and low-yield egg production groups was constructed. Our findings provide new insights into the mechanisms underlying the molecular regulation of related circRNA/lncRNA-miRNA-mRNA in the egg production and double-yolked egg traits of Gaoyou ducks.

Identification of ceRNA-vsiRNA-mRNA network for exploring the mechanism underlying pathogenesis of sugarcane mosaic virus in resistant and susceptible maize inbred lines

RNA silencing plays an important role in plant antiviral responses, which trigger the production of virus-derived small interfering RNAs (vsiRNAs). The competing endogenous RNA (ceRNA) hypothesis revealed a unique mechanism in which circular RNAs (circRNAs) and long non-coding RNAs (lncRNAs) can interact with small RNAs to regulate the expression of corresponding target mRNAs. Sugarcane mosaic virus (SCMV) infection causes severe economic losses in maize (Zea mays L.) production worldwide. This study compared and analyzed characteristics of vsiRNAs derived from SCMV and their target genes in resistant (Chang7-2) and susceptible (Mo17) maize inbred lines through whole-transcriptome RNA sequencing and degradome sequencing. The results showed that 706 transcripts were targeted by 204 vsiRNAs, including 784 vsiRNA-target gene pairs. Furthermore, ceRNA networks of circRNA/lncRNA-vsiRNA-mRNA in response of maize to SCMV infection were obtained, including 3 differentially expressed (DE) circRNAs, 36 DElncRNAs, 105 vsiRNAs, and 342 DEmRNAs in Mo17 plants, and 3 DEcircRNAs, 35 DElncRNAs, 23 vsiRNAs, and 87 DEmRNAs in Chang7-2 plants. Our results also showed that the transcripts of ZmDCLs, ZmAGOs, and ZmRDRs were differentially accumulated in resistant and susceptible maize inbred lines after SCMV infection. These findings provide valuable insights into the relationship between SCMV-derived vsiRNAs and potential ceRNAs fine-tuning the SCMV-maize interaction and offer novel clues to reveal the mechanism underlying the pathogenesis of SCMV.

Whole-transcriptome characterization and functional analysis of lncRNA-miRNA-mRNA regulatory networks responsive to sugarcane mosaic virus in maize resistant and susceptible inbred lines

Sugarcane mosaic virus (SCMV) is one of the most important pathogens causing maize dwarf mosaic disease, which seriously affects the yield and quality of maize. Currently, the molecular mechanism of non-coding RNAs (ncRNAs) responding to SCMV infection in maize is still uncovered. In this study, a total of 112 differentially expressed (DE)-long non-coding RNAs (lncRNAs), 24 DE-microRNAs (miRNAs), and 1822 DE-messenger RNAs (mRNAs), and 363 DE-lncRNAs, 230 DE-miRNAs, and 4376 DE-mRNAs were identified in maize resistant (Chang7-2) and susceptible (Mo17) inbred lines in response to SCMV infection through whole-transcriptome RNA sequencing, respectively. Moreover, 4874 mRNAs potentially targeted by 635 miRNAs were obtained by degradome sequencing. Subsequently, several crucial SCMV-responsive lncRNA-miRNA-mRNA networks were established, of which the expression levels of lncRNA10865-miR166j-3p-HDZ25/69 (class III homeodomain-leucine zipper 25/69) module, and lncRNA14234-miR394a-5p-SPL11 (squamosal promoter-binding protein-like 11) module were further verified. Additionally, silencing lncRNA10865 increased the accumulations of SCMV and miR166j-3p, while silencing lncRNA14234 decreased the accumulations of SCMV and SPL11 targeted by miR394a-5p. This study revealed the interactions of lncRNAs, miRNAs and mRNAs in maize resistant and susceptible materials, providing novel clues to reveal the mechanism of maize in resistance to SCMV from the perspective of competing endogenous RNA (ceRNA) regulatory networks.

Whole-transcriptome analysis reveals mechanisms underlying antibacterial activity and biofilm inhibition by a malic acid combination (MAC) in Pseudomonas aeruginosa

Background Pseudomonas aeruginosa is a highly prevalent bacterial species known for its ability to cause various infections and its remarkable adaptability and biofilm-forming capabilities. In earlier work, we conducted research involving the screening of 33 metabolites obtained from a commercial source against two prevalent bacterial strains, Escherichia coli and Staphylococcus aureus. Through screening assays, we discovered a novel malic acid combination (MAC) consisting of malic acid, citric acid, glycine, and hippuric acid, which displayed significant inhibitory effects. However, the precise underlying mechanism and the potential impact of the MAC on bacterial biofilm formation remain unknown and warrant further investigation. Methods To determine the antibacterial effectiveness of the MAC against Pseudomonas aeruginosa, we conducted minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) assays. Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) techniques were employed to observe bacterial morphology and biofilm formation. We further performed a biofilm inhibition assay to assess the effect of the MAC on biofilm formation. Whole-transcriptome sequencing and bioinformatics analysis were employed to elucidate the antibacterial mechanism of the MAC. Additionally, the expression levels of differentially expressed genes were validated using the real-time PCR approach. Results Our findings demonstrated the antibacterial activity of the MAC against P. aeruginosa. SEM analysis revealed that the MAC can induce morphological changes in bacterial cells. The biofilm assay showed that the MAC could reduce biofilm formation. Whole-transcriptome analysis revealed 1093 differentially expressed genes consisting of 659 upregulated genes and 434 downregulated genes, in response to the MAC treatment. Mechanistically, the MAC inhibited P. aeruginosa growth by targeting metabolic processes, secretion system, signal transduction, and cell membrane functions, thereby potentially compromising the survival of this human pathogen. This study provides valuable insights into the antibacterial and antibiofilm activities of the MAC, a synergistic and cost-effective malic acid combination, which holds promise as a potential therapeutic drug cocktail for treating human infectious diseases in the future.

The Genomic Landscape of Urothelial Carcinoma with High and Low ERBB2 Expression.

Recent data suggests that HER2-targeted treatment is efficacious in urothelial carcinoma (UC). We investigated the genomic, transcriptomic, and immune landscapes and clinical outcomes in UC segmented by ERBB2 expression. NextGen DNA/RNA sequencing was performed for 4743 UC tumors. A total of 3% (124/4125) of tumors had HER2 IHC and whole transcriptome sequencing (WTS) data. ERRB2-high and -low tumors were defined by ≥75th and <25th percentiles of ERBB2 expression, respectively. PD-L1 (SP142) positive staining was defined as ≥2+ and ≥5%. HER2 (4B5) positive staining was defined as ≥3+ and >10% or 2+ and >10% with positive HER2 in situ hybridization (ISH). Of the patients who were ERBB2-high, 79% (61/77) were HER2 positive via IHC. Tumors from lower tract UC had higher ERBB2 expression compared to upper tract UC (50 v 40 median TPM (mTPM), p < 0.001). ERBB2 expression was similar between primary and metastatic tumors (47 v 47 mTPM, p = 0.95). ERBB2-high tumors had a higher prevalence of pathogenic mutations in pTERT, ERBB2, and ELF3 versus ERBB2-low tumors, p < 0.001. ERBB2-high tumors had higher expressions of ADC target genes NECTIN4 (12 v 8 mTPM) and TACSTD2 (366 v 74 mTPM) versus ERBB2-low (p < 0.001), as well as better overall survival from time of tissue sampling than ERBB2-low (HR 1.71, p < 0.001). Our study demonstrated a high concordance between HER2 expression by IHC and ERBB2 gene expression by WTS in UC. Differences in ADC target expression between ERBB2-high vs. ERBB2-low UC may provide a rationale for combination treatment strategies with HER2-ADC. The association between high ERBB2 expression and survival advantage warrants further investigation.

Identification of FOXP3+ epithelial cells contributing to pancreatic proliferation and angiogenesis.

Forkhead box protein 3 (FOXP3), traditionally recognized as a specific transcription factor for regulatory T cells (Tregs), has also been identified in various tumor epithelial cells (named as cancer-FOXP3, c-FOXP3). However, the natural state and functional role of FOXP3 positive tumor epithelial cells remain unknown. Monoclonal cells expressing varying levels of c-FOXP3 were isolated from established PANC-1 cells using limited dilution. Whole transcriptome sequencing and weighted gene co-expression network analysis (WGCNA) were conducted on these subsets, followed by in vitro and in vivo functional investigations. In addition, we identified c-FOXP3+E-cadherin- epithelial cells in human pancreatic cancer tissues after radical resection by immunofluorescence co-staining. We also investigated the connection between c-FOXP3+E-cadherin- epithelial cells and their clinicopathological features. Our study uncovered a distinct subset of c-FOXP3+ tumor epithelial cells characterized by reduced E-cadherin expression. C-FOXP3+E-cadherin- cells displayed significant proliferation potential and pro-angiogenic effect through the expression of chemokines, including C-X-C motif ligand 1 (CXCL1), C-X-C motif ligand 5 (CXCL5), and C-X-C motif ligand 8 (CXCL8). Notably, higher counts of c-FOXP3+E-Cadherin- cells correlated with poorer prognosis, lower tumor differentiation, lymph node metastasis, and vascular invasion in pancreatic ductal adenocarcinoma (PDAC). In conclusion, this work revealed the stable expression of FOXP3 in tumor epithelial cells, marking a distinct subset. C-FOXP3+E-cadherin- epithelial cells exhibit active proliferation and promote angiogenesis in a vascular endothelial growth factor A (VEGFA) independent manner. These findings provide novel insights into PDAC prognosis and therapeutic avenues.NEW & NOTEWORTHY In this study, we revealed a novel c-FOXP3+ tumor epithelial cell subset marked by diminished E-cadherin and stable FOXP3 expression. These subpopulations not only show robust proliferation and drive angiogenesis via CXCL1, CXCL5, and CXCL8, bypassing VEGFA pathways, but their heightened presence also correlates with adverse PDAC outcomes. By challenging traditional epithelial cell definitions and extending lymphocyte markers to these cells, our findings present innovative targets for PDAC treatment and enrich our understanding of cell biology.

#277 : Transcriptome Analysis Revealed a Delayed and Attenuated Estrogen Response in IUA Patients

Background and Aims: Intrauterine adhesion (IUA) is characterized by the formation of scar tissue in the endometrium following a variety of insults such as endometrial damage, miscarriage, and infection. Despite current management with intrauterine adhesion separation and adjuvant therapies such as estrogen supplementation, a high recurrence rate and inadequate therapeutic response persist in some patients. More importantly, compromised endometrial proliferation in these patients lead to subsequent infertility. However, effective therapies to improve the impaired response to estrogen have yet to be identified. This study seeks to elucidate the molecular basis for the adverse estrogen response in IUA patients and to identify novel therapeutic targets. Method: Whole-Transcriptome sequencing: proliferative endometrium of normal and IUA patients. Results: By performing differential and enrichment analysis of transcriptomic data, we observed a considerable downregulation of estrogen-relatedgenesets in IUA patients. The expression of enhancer RNAs (eRNAs) serves as a hallmark of transcriptional activation. By identifying eRNAs in transcriptomic data and co-analyzing with ERa ChIPseq and H3K27ac ChIPseq datasets, we identified a series of downregulated eRNAs, which is mediated by estrogen. We further confirmed the downregulation of eRNAs and target genes. Interestingly, the expression of ESR1 is similar in both groups, we performed immunofluorescence analysis and demonstrated a considerable reduction of ERa-positive cells with a loss of nuclear localization in IUA. This finding provides further evidence of the insensitivity of IUA patients to estrogen and suggests a loss of estrogen genomic effects. Furthermore, we found that prolonging estrogen exposure time and increasing estrogen concentration partially enhanced estrogen effects in IUA patients, yet remained compromised, indicating the need for alternative therapeutic strategies. Conclusion: The compromised response to estrogen in IUA patients is attributed to the attenuation of estrogen receptor alpha (ERa)-mediated genomic effect.

N6-methyladenosine-modified circRIMS2 mediates synaptic and memory impairments by activating GluN2B ubiquitination in Alzheimer's disease

BackgroundSynaptic degeneration occurs in the early stage of Alzheimer's disease (AD) before devastating symptoms, strongly correlated with cognitive decline. Circular RNAs (circRNAs) are abundantly enriched in neural tissues, and aberrant expression of circRNAs precedes AD symptoms, significantly correlated with clinical dementia severity. However, the direct relationship between circRNA dysregulation and synaptic impairment in the early stage of AD remains poorly understood.MethodsHippocampal whole-transcriptome sequencing was performed to identify dysregulated circRNAs and miRNAs in 4-month-old wild-type and APP/PS1 mice. RNA antisense purification and mass spectrometry were utilized to unveil interactions between circRIMS2 and methyltransferase 3, N6-adenosine-methyltransferase complex catalytic subunit (METTL3). The roles of circRIMS2/miR-3968 in synaptic targeting of UBE2K-mediated ubiquitination of GluN2B subunit of NMDA receptor were evaluated via numerous lentiviruses followed by morphological staining, co-immunoprecipitation and behavioral testing. Further, a membrane-permeable peptide was used to block the ubiquitination of K1082 on GluN2B in AD mice.ResultscircRIMS2 was significantly upregulated in 4-month-old APP/PS1 mice, which was mediated by METTL3-dependent N6-methyladenosine (m6A) modification. Overexpression of circRIMS2 led to synaptic and memory impairments in 4-month-old C57BL/6 mice. MiR-3968/UBE2K was validated as the downstream of circRIMS2. Elevated UBE2K induced synaptic dysfunction of AD through ubiquitinating K1082 on GluN2B. Silencing METTL3 or blocking the ubiquitination of K1082 on GluN2B with a short membrane-permeable peptide remarkably rescued synaptic dysfunction in AD mice.ConclusionsIn conclusion, our study demonstrated that m6A-modified circRIMS2 mediates the synaptic and memory impairments in AD by activating the UBE2K-dependent ubiquitination and degradation of GluN2B via sponging miR-3968, providing novel therapeutic strategies for AD.

A Nasal Swab Classifier to Evaluate the Probability of Lung Cancer in Patients With Pulmonary Nodules

Accurate assessment of the probability of lung cancer (pCA) is critical in patients with pulmonary nodules (PNs) to help guide decision-making. We sought to validate a clinical-genomic classifier developed using whole-transcriptome sequencing of nasal epithelial cells from patients with a PN≤ 30mm who smoke or have previously smoked. Can the pCA in individuals with a PN and a history of smoking be predicted by a classifier that uses clinical factors and genomic data from nasal epithelial cells obtained by cytologic brushing? Machine learning was used to train a classifier using genomic and clinical features on 1,120 patients with PNs labeled as benign or malignant established by a final diagnosis or a minimum of 12months of radiographic surveillance. The classifier was designed to yield low-, intermediate-, and high-risk categories. The classifier was validated in an independent set of 312 patients, including 63 patients with a prior history of cancer (other than lung cancer), comparing the classifier prediction with the known clinical outcome. In the primary validation set, sensitivity and specificity for low-risk classification were 96%and 42%, whereas sensitivity and specificity for high-risk classification was 58%and 90%, respectively. Sensitivity was similar across stages of non-small cell lung cancer, independent of subtype. Performance compared favorably with clinical-only risk models. Analysis of 63 patients with prior cancer showed similar performance as did subanalyses of patients with light vsheavy smoking burden and those eligible for lung cancer screening vsthose who were not. The nasal classifier provides an accurate assessment of pCA in individuals with a PN≤ 30mm who smoke or have previously smoked. Classifier-guided decision-making could lead to fewer diagnostic procedures in patients without cancer and more timely treatment in patients with lung cancer.

Diffuse Gliomas with FGFR3-TACC3 Fusions: Oncogenic Mechanisms, Hallmarks, and Therapeutic Perspectives

In 2012, whole-transcriptome sequencing analysis led to the discovery of recurrent fusions involving the FGFR3 and TACC3 genes as the main oncological driver in a subset of human glioblastomas. Since then, FGFR3-TACC3 fusions have been identified in several other solid cancers. Further studies dissected the oncogenic mechanisms of the fusion protein and its complex interplay with cancer cell metabolism. FGFR3-TACC3 fusion-driven gliomas emerged as a defined subgroup with specific clinical, histological, and molecular features. Several FGFR inhibitors were tested in FGFR3-TACC3 fusion-positive gliomas and proved some efficacy, although inferior to the results seen in other FGFR3-TACC3 fusion-driven cancers. In this review, we summarize and discuss the state-of-the-art knowledge resulting from a 10-year research effort in the field, its clinical implications for glioma patients, the potential reasons for targeted therapy failures, and the perspective of emerging treatments.

Intracellular tacrolimus concentration correlates with impaired renal function through regulation of the IS-AHR-ABC transporter in peripheral blood mononuclear cells

BackgroundsTacrolimus (TAC) concentration in peripheral blood mononuclear cells (PBMCs) is regarded as a better predictor of its immunosuppressive effect than the TAC concentration in whole blood. However, whether the exposure of TAC in PBMCs or WB was altered in post-transplant recipients with renal impairment remains unclear. MethodsWe investigated the relationship of trough TAC concentration in WB and PBMCs with renal functions in post-transplant recipients. The pharmacokinetic profiles of TAC in PBMCs and WB in the two chronic kidney disease (CKD) rat models were examined using UPLC-MS/MS. Western blotting and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) were used to analyze the expression of proteins and mRNAs related to TAC metabolism and transport, respectively. In addition, the effects of uremic toxins on human PBMCs were investigated using whole-transcriptome sequencing (RNA sequencing [RNA-seq]). ResultsWe observed a decrease in the trough TAC concentration in PBMCs in the recipients with estimated glomerular filtration rate (eGFR) < 90 mL/min, compared with those of recipients with eGFR > 90 mL/min, but there was no difference in blood based on TAC concentrations (C0Blood). In a 150-patient post-transplant cohort, no significant relationship was observed between PBMCs and WB concentrations of TAC, and the eGFR value was correlated with TAC C0PBMCs but not with TAC C0Blood. In two CKD rat models, the TAC pharmacokinetic profile in the PBMCs was significantly lower than that in the control group; however, the blood TAC pharmacokinetic profiles in the two groups were similar. Transcriptome results showed that co-incubation of human PBMCs with uremic toxins upregulated the expression of AHR, ABCB1, and ABCC2. Compared to control rats, plasma IS increased by 1.93- and 2.26-fold and the expression of AHR, P-gp, and MRP2 in PBMCs was higher in AD and 5/6 nephrectomy (NX) rats, without modifying the expression of other proteins related to TAC exposure. ConclusionThe pharmacokinetics of TAC in PBMCs changed with a decline in renal function. Uremic toxins accumulate during renal insufficiency, which activates AHR, upregulates the expression of P-gp and MRP2, and affects their intracellular concentrations. Our findings suggest that monitoring TAC concentrations in PBMCs is more important than monitoring WB concentrations in post-transplant recipients with renal impairment.

Whole-Transcriptome RNA Sequencing Uncovers the Global Expression Changes and RNA Regulatory Networks in Duck Embryonic Myogenesis.

Duck meat is pivotal in providing high-quality protein for human nutrition, underscoring the importance of studying duck myogenesis. The regulatory mechanisms governing duck myogenesis involve both coding and non-coding RNAs, yet their specific expression patterns and molecular mechanisms remain elusive. To address this knowledge gap, we performed expression profiling analyses of mRNAs, lncRNAs, circRNAs, and miRNAs involved in duck myogenesis using whole-transcriptome RNA-seq. Our analysis identified 1733 differentially expressed (DE)-mRNAs, 1116 DE-lncRNAs, 54 DE-circRNAs, and 174 DE-miRNAs when comparing myoblasts and myotubes. A GO analysis highlighted the enrichment of DE molecules in the extracellular region, protein binding, and exocyst. A KEGG analysis pinpointed pathways related to ferroptosis, PPAR signaling, nitrogen metabolism, cell cycle, cardiac muscle contraction, glycerolipid metabolism, and actin cytoskeleton. A total of 51 trans-acting lncRNAs, including ENSAPLT00020002101 and ENSAPLT00020012069, were predicted to participate in regulating myoblast proliferation and differentiation. Based on the ceRNAs, we constructed lncRNA-miRNA-mRNA and circRNA-miRNA-mRNA ceRNA networks involving five miRNAs (miR-129-5p, miR-133a-5p, miR-22-3p, miR-27b-3p, and let-7b-5p) that are relevant to myogenesis. Furthermore, the GO and KEGG analyses of the DE-mRNAs within the ceRNA network underscored the significant enrichment of the glycerolipid metabolism pathway. We identified five different DE-mRNAs, specifically ENSAPLG00020001677, ENSAPLG00020002183, ENSAPLG00020005019, ENSAPLG00020010497, and ENSAPLG00020017682, as potential target genes that are crucial for myogenesis in the context of glycerolipid metabolism. These five mRNAs are integral to ceRNA networks, with miR-107_R-2 and miR-1260 emerging as key regulators. In summary, this study provides a valuable resource elucidating the intricate interplay of mRNA-lncRNA-circRNA-miRNA in duck myogenesis, shedding light on the molecular mechanisms that govern this critical biological process.

Long noncoding RNAs emerge from transposon-derived antisense sequences and may contribute to infection stage-specific transposon regulation in a fungal phytopathogen

BackgroundThe genome of the obligate biotrophic phytopathogenic barley powdery mildew fungus Blumeria hordei is inflated due to highly abundant and possibly active transposable elements (TEs). In the absence of the otherwise common repeat-induced point mutation transposon defense mechanism, noncoding RNAs could be key for regulating the activity of TEs and coding genes during the pathogenic life cycle.ResultsWe performed time-course whole-transcriptome shotgun sequencing (RNA-seq) of total RNA derived from infected barley leaf epidermis at various stages of fungal pathogenesis and observed significant transcript accumulation and time point-dependent regulation of TEs in B. hordei. Using a manually curated consensus database of 344 TEs, we discovered phased small RNAs mapping to 104 consensus transposons, suggesting that RNA interference contributes significantly to their regulation. Further, we identified 5,127 long noncoding RNAs (lncRNAs) genome-wide in B. hordei, of which 823 originated from the antisense strand of a TE. Co-expression network analysis of lncRNAs, TEs, and coding genes throughout the asexual life cycle of B. hordei points at extensive positive and negative co-regulation of lncRNAs, subsets of TEs and coding genes.ConclusionsOur work suggests that similar to mammals and plants, fungal lncRNAs support the dynamic modulation of transcript levels, including TEs, during pivotal stages of host infection. The lncRNAs may support transcriptional diversity and plasticity amid loss of coding genes in powdery mildew fungi and may give rise to novel regulatory elements and virulence peptides, thus representing key drivers of rapid evolutionary adaptation to promote pathogenicity and overcome host defense.

Key pathways and genes that are altered during treatment with hyperbaric oxygen in patients with sepsis due to necrotizing soft tissue infection (HBOmic study)

BackgroundFor decades, the basic treatment strategies of necrotizing soft tissue infections (NSTI) have remained unchanged, primarily relying on aggressive surgical removal of infected tissue, broad-spectrum antibiotics, and supportive intensive care. One treatment strategy that has been proposed as an adjunctive measure to improve patient outcomes is hyperbaric oxygen (HBO2) treatment. HBO2 treatment has been linked to several immune modulatory effects; however, investigating these effects is complicated due to the disease's acute life-threatening nature, metabolic and cell homeostasis dependent variability in treatment effects, and heterogeneity with respect to both patient characteristics and involved pathogens. To embrace this complexity, we aimed to explore the underlying biological mechanisms of HBO2 treatment in patients with NSTI on the gene expression level.MethodsWe conducted an observational cohort study on prospective collected data, including 85 patients admitted to the intensive care unit (ICU) for NSTI. All patients were treated with one or two HBO2 treatments and had one blood sample taken before and after the intervention. Total RNAs from blood samples were extracted and mRNA purified with rRNA depletion, followed by whole-transcriptome RNA sequencing with a targeted sequencing depth of 20 million reads. A model for differentially expressed genes (DEGs) was fitted, and the functional aspects of the obtained set of genes was predicted with GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of genes and Genomes) enrichment analyses. All analyses were corrected for multiple testing with FDR.ResultsAfter sequential steps of quality control, a final of 160 biological replicates were included in the present study. We found 394 protein coding genes that were significantly DEGs between the two conditions with FDR < 0.01, of which 205 were upregulated and 189 were downregulated. The enrichment analysis of these DEGs revealed 20 GO terms in biological processes and 12 KEGG pathways that were significantly overrepresented in the upregulated DEGs, of which the term; “adaptive immune response” (GO:0002250) (FDR = 9.88E-13) and “T cell receptor signaling pathway” (hsa04660) (FDR = 1.20E-07) were the most significant. Among the downregulated DEGs two biological processes were significantly enriched, of which the GO term “apoptotic process” (GO:0006915) was the most significant (FDR = 0.001), followed by “Positive regulation of T helper 1 cell cytokine production” (GO:2000556), and “NF-kappa B signaling pathway” (hsa04064) was the only KEGG pathway that was significantly overrepresented (FDR = 0.001).ConclusionsWhen one or two sessions of HBO2 treatment were administered to patients with a dysregulated immune response and systemic inflammation due to NSTI, the important genes that were regulated during the intervention were involved in activation of T helper cells and downregulation of the disease-induced highly inflammatory pathway NF-κB, which was associated with a decrease in the mRNA level of pro-inflammatory factors.Trial registration: Biological material was collected during the INFECT study, registered at ClinicalTrials.gov (NCT01790698).

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.

Construction and analysis of the tapping panel dryness-related lncRNA/circRNA-miRNA-mRNA ceRNA network in latex of Hevea brasiliensis

Tapping panel dryness (TPD) results in a severe reduction in latex yield in Hevea brasiliensis. However, the molecular regulatory mechanisms of TPD occurrence are still largely unclear. In this study, whole-transcriptome sequencing was carried out on latex from TPD and healthy trees. In total, 7078 long noncoding RNAs (lncRNAs), 3077 circular RNAs (circRNAs), 4956 miRNAs, and 25041 mRNAs were identified in latex, among which 435 lncRNAs, 68 circRNAs, 320 miRNAs, and 1574 mRNAs were differentially expressed in the latex of TPD trees. GO and KEGG analyses indicated that plant hormone signal transduction, MAPK signaling pathway, and ubiquitin-mediated proteolysis were the key pathways associated with TPD onset. Phytohormone profiling revealed significant changes in the contents of 28 hormonal compounds, among which ACC, ABA, IAA, GA, and JA contents were increased, while SA content was reduced in TPD latex, suggesting that hormone homeostasis is disrupted in TPD trees. Furthermore, we constructed a TPD-related competitive endogenous RNA (ceRNA) regulatory network of lncRNA/circRNA-miRNA-mRNA with 561 edges and 434 nodes (188 lncRNAs, 5 circRNAs, 191 miRNAs, and 50 mRNAs) and identified two hub lncRNAs (MSTRG.11908.1 and MSTRG.8791.1) and four hub miRNAs (hbr-miR156, miR156-x, miRf10477-y, and novel-m0452-3p). Notably, the lncRNA-miR156/157-SPL module containing three hubs probably plays a crucial role in TPD onset. The expression of network hubs and the lncRNA-miR156/157-SPL module were further validated by qRT-PCR. Our results reveal the TPD-associated ceRNA regulatory network of lncRNA/circRNA-miRNA-mRNA in latex and lay a foundation for further investigation of molecular regulatory mechanisms for TPD onset in H. brasiliensis.

Can Whole Genome and Whole Transcriptome Sequencing Replace Standard Procedures in CLL Diagnostics?

Background: The advantages of genome-wide sequencing approaches over conventional methods in CLL diagnostics are a matter of debate and exact guidelines for the application of next-generation sequencing in a diagnostic context are currently missing. Aim: Compare the accuracy of whole genome sequencing (WGS) and whole transcriptome sequencing (WTS) in determining the presence of point mutations, IGHV mutational status (IGHVms), and chromosomal aberrations to conventional procedures in a clinical setting. Patients and Methods: The cohort comprised 317 CLL patients. Diagnosis was established following WHO guidelines. WGS (100x, 2x151bp) and WTS (5x10 7 reads, 2x101bp) data were generated on NovaSeq instruments. For routine diagnostics, sequencing was carried out by Sanger or targeted panel sequencing (1500x, 2x101bp). Chromosome banding analysis (CBA) and FISH were available for 231 patients. WGS data were analyzed for chromosomal aberrations and copy number alterations using Manta and GATK. IGHVms was determined by Sanger sequencing and/or fragment analysis for 248 patients. Analysis of IGHVms was carried out using IgCaller (WGS) and MiXCR (WTS). Results: Comparing copy number variants (CNV) and balanced structural variants (SV) between CBA and WGS revealed a high concordance. In detail, 297 CNV were detected by both techniques while 32 and 52 were detected only by CBA (small subclones) or by WGS, respectively. 33 balanced SV were detected by both while 8 and 3 were detected only by CBA and WGS, respectively. A complex karyotype (≥ 3 abnormalities) was observed by both techniques in 55 cases, while 5 and 10 cases were categorized as complex by CBA only or WGS only, respectively. Survival analysis of cases with 3 to 5 chromosomal aberrations predicted by routine or WGS revealed similar hazard ratios that were positively correlated with the number of aberrations. For comparing SNV calls from routine and WGS, we investigated genes with validated mutations in at least ten patients in routine diagnostics. This criterion was met by ATM, MYD88, NOTCH1, SF3B1, and TP53. We observed high concordance between routine and WGS data ( ATM 99.4%, MYD88 100%, NOTCH1 97.8%, SF3B1 100%, TP53 99.1%). Five mutations were detected in routine only and three in WGS only (routine only/WGS only: ATM 1/0, MYD88 0/0, NOTCH1 2/2, SF3B1 0/0, TP53 2/1) and found to be caused by low variant allele frequency (5 miscalls, VAF &amp;lt; 5%) and/or filtering out of low quality variants (5 cases). IGHV rearrangements analyzed by Sanger (248 patients), WGS (242/248 patients), and WTS (236/248 patients) data were compared for matches in the rearranged IGHV. IGHVms calling employed 98% of sequence identity as cutoff. Calling the rearranged IGHV was concordant in 93%, 95%, and 92% when comparing Sanger/MiXCR, Sanger/IgCaller, and MiXCR/IgCaller calls, respectively. For IGHVms, the corresponding percentages of concordance were 91%, 86%, and 83%. Mismatches in calling the IGHVms were found in borderline cases. Detection of stereotyped IGHV was fully concordant. In addition to reliably replicating routine results, we find that WGS/WTS data can also outperform routine procedures. In our hands, the highest hazard ratio (4.94) in survival analysis distinguishing mutated CLL (mCLL) from unmutated CLL (uCLL) is observed using WTS for IGHVms determination and 97% sequence identity as cutoff (208 patients, up to 30 years follow up). Survival for borderline cases (97.00-97.99% sequence identity) was statistically indistinguishable from uCLL in routine diagnostics as well as WGS/WTS. Furthermore, using WGS, CN-LOH regions and deletions below the detection limit of CBA (10 MB) can be detected. We observed such regions in samples with mutated TP53 (5 CN-LOH), ATM (2 CN-LOH, 4 deletions &amp;lt; 10MB), and deletions of minimally overlapping region chr8:21,700,000-23,600,000, whose loss was associated with poor survival (3/29 deletions &amp;lt; 3.1 MB). Conclusions: WGS/WTS data reliably replicate results obtained by standard procedures in a diagnostic setting. Sequencing offers advantages such as a unified workflow, better sensitivity for small deletions, CN-LOH detection, genome-wide analyses of coding and non-coding mutations, among others. Challenges of short read based sequencing regard the detection limit for point mutations and specificity/sensitivity in detecting chromosomal aberrations.

Heterogeneous Gene Fusion Transcripts Found in t(7;12)(q36;p13) Acute Myeloid Leukemia but with Similar Gene Expression Profile

Cytogenetic aberrations are often involved in acute myeloid leukemia (AML) and can serve as diagnostic markers, prognosis predictors and impact the choice of therapy. A translocation t(7;12)(q36;p13) is reported in up to 20-30% of AML patients typically diagnosed before 24 months of age (Beverloo, Panagopoulos et al. 2001, Cancer Res 61(14): 5374-5377). However, in a retrospective survey of the Nordic Organization of Pediatric Hematology and Oncology (NOPHO) registry, only 4% of reported AML diagnosed before 24 months of age had t(7;12) (Espersen, Noren-Nystrom et al. 2018, Genes Chromosomes Cancer 57(7): 359-365). The t(7;12) is difficult to identify with conventional karyotyping, why the true frequency of t(7;12) may be underestimated. Similarly, the reported prognosis for t(7;12) patients has varied ranging from good (Espersen, Noren-Nystrom et al. 2018) to dismal (von Bergh, van Drunen et al. 2006, Genes Chromosomes Cancer 45(8): 731-739). The translocation has been reported to give rise to an in-frame fusion transcript, MNX1::ETV6. However, detection of this fusion is reported only in 50% of cases in contrast to high expression of MNX1 in 100% (von Bergh, van Drunen et al. 2006). The aim of this study was to determine the frequency of the t(7;12) in pediatric AML patients in the NOPHO AML-2004/2012 protocols, their event-free and overall survival and to investigate the presence of additional genetic alterations. Patients in Sweden, Denmark, or Iceland between 2004 to 2020 diagnosed with AML before 2 years of age that had not been reported with a recurrent genetic aberration plus all patients diagnosed with t(7;12) AML were identified. Bone marrow, or peripheral blood samples from patients were retrieved from the NOPHO biobank. This cohort constituted 86% (31 out of 36) of all patients that fulfilled these criteria. In total 89 AML patients were diagnosed before 2 years of age. The t(7;12) AML cases were identified by screening for high expression of MNX1 and presence of fusion transcripts using whole transcriptome sequencing (WTS). Whole genome sequencing (WGS) was done to identify chromosomal rearrangements, and translocation breakpoints. At diagnosis, four patients were diagnosed with t(7;12). A central karyotype review of all patients reported two additional cases. No further cases were identified using WTS. Thus, the frequency of t(7;12) AML was 7% (6 out of 89) and for patients diagnosed before 12 months of age 11% (5 out of 47). In total 12 cases with t(7;12) were identified in the complete NOPHO registry but only 11 patients received treatment according to NOPHO protocol. The relapse rate was 54% (6 out of 11). All relapse cases underwent allogeneic hematopoietic stem cell (HSC) transplantation in complete remission two (CR2), and their overall survival (OS) was 83% (5 out of 6). Hence OS for all t(7;12) patients treated according to the NOPHO2004 and NOPHO2012 protocols was 91% (10 out of 11). Analysis of WTS data showed similar gene expression profile between our t(7;12) cases and t(7;12) cases retrieved from the TARGET database. Genes that were typically overexpressed in all t(7;12) AML were, MNX1, MNX1-AS1, MNX1-AS2, LIN28B, BAMBI, MAF, CRISP3, EDIL3, CTTNBP2, KRT72, and AGR2, where MNX1 seems to be uniquely expressed in this type of leukemia (Nilsson et al. 2022, Int J Cancer 151(5): 770-782). Analysis of fusion transcripts showed presence of MNX1::ETV6 in just one case. The remaining cases instead showed presence of other fusion transcripts involving ETV6, NOM1::ETV6, ETV6::NOM1, ETV6::LMBR1 and ETV6::BMAL2. WGS verified a fusion between NOM1 and ETV6 in 3 cases. The patient with ETV6::BMAL2 showed that 16 Mb of chromosome 12 was inserted into chromosome 7 placing a large part of ETV6 near MNX1. The fusion transcript ETV6::BMAL2 is the product of what remains on chromosome 12. In conclusion, frequency of t(7;12) AML in the NOPHO treatment cohort was approximately 7%, with 54% relapse rate, but most patients were salvaged by HSCT in CR2. Heterogeneous gene fusion transcripts were identified in this subgroup of AML where ETV6::NOM1 was most frequent. However, all cases showed similar gene expression signatures which underlines that the leukemia driving event is ectopic expression of MNX1 (Waraky, A., 2023, Haematologica, DOI: 10.3324/haematol.2022.282255) and should therefore be the defining classifying criteria of this type of AML.