RNA-Seq Transcriptome Analysis Research Articles

Genetic variation in CCDC93 is associated with elevated central systolic blood pressure, impaired arterial relaxation, and mitochondrial dysfunction.

Genetic studies of blood pressure (BP) traits to date have been performed on conventional measures by brachial cuff sphygmomanometer for systolic BP (SBP) and diastolic BP, integrating several physiologic occurrences. Genetic associations with central SBP (cSBP) have not been well-studied. Genetic discovery studies of BP have been most often performed in European-ancestry samples. Here, we investigated genetic associations with cSBP in a Chinese population and functionally validated the impact of a novel associated coiled-coil domain containing 93 (CCDC93) gene on BP regulation. An exome-wide association study (EWAS) was performed using a mixed linear model of non-invasive cSBP and peripheral BP traits in a Han Chinese population (N = 5,954) from Beijing, China genotyped with a customized Illumina ExomeChip array. We identified four SNP-trait associations with three SNPs, including two novel associations (rs2165468-SBP and rs33975708-cSBP). rs33975708 is a coding variant in the CCDC93 gene, c.535C>T, p.Arg179Cys (MAF = 0.15%), and was associated with increased cSBP (β = 29.3 mmHg, P = 1.23x10-7). CRISPR/Cas9 genome editing was used to model the effect of Ccdc93 loss in mice. Homozygous Ccdc93 deletion was lethal prior to day 10.5 of embryonic development. Ccdc93+/- heterozygous mice were viable and morphologically normal, with 1.3-fold lower aortic Ccdc93 protein expression (P = 0.0041) and elevated SBP as compared to littermate Ccdc93+/+ controls (110±8 mmHg vs 125±10 mmHg, P = 0.016). Wire myography of Ccdc93+/- aortae showed impaired acetylcholine-induced relaxation and enhanced phenylephrine-induced contraction. RNA-Seq transcriptome analysis of Ccdc93+/- mouse thoracic aortae identified significantly enriched pathways altered in fatty acid metabolism and mitochondrial metabolism. Plasma free fatty acid levels were elevated in Ccdc93+/- mice (96±7mM vs 124±13mM, P = 0.0031) and aortic mitochondrial dysfunction was observed through aberrant Parkin and Nix protein expression. Together, our genetic and functional studies support a novel role of CCDC93 in the regulation of BP through its effects on vascular mitochondrial function and endothelial function.

Comprehensive analysis of splicing factor SRs-related gene characteristics: predicting osteosarcoma prognosis and immune regulation status.

To investigate the impact of SRs-related genes on the overall survival and prognosis of osteosarcoma patients through bulk and single-cell RNA-seq transcriptome analysis. In this study, we constructed a prognosis model based on serine/arginine-rich splicing factors (SRs) and predicted the survival of osteosarcoma patients. By analyzing single-cell RNA sequencing data and applying AUCell enrichment analysis, we revealed oncogenic pathways of SRs in osteosarcoma immune cells. Additionally, we described the regulatory role of SRSF7 in pan-cancer. Lasso regression analysis identified 6 key SRs-related genes, and a prognosis prediction model was established. The upregulation of these pathways revealed that SRs promote tumor cell proliferation and survival by regulating related signaling pathways and help tumor cells evade host immune surveillance. Additionally, by grouping single-cell data using AUCell, we found significant differences in T cell expression between high and low-risk groups. The analysis results indicated that the regulatory activity of SRs is closely related to T cell function, particularly in regulating immune responses and promoting immune evasion. Furthermore, SRSF7 regulates cell proliferation and apoptosis. SRs-related genes play a critical regulatory role in osteosarcoma. T cells are key in regulating immune responses and promoting immune evasion through SRs genes. SRSF7 is a significant gene influencing the occurrence and development of osteosarcoma.

Using RNA-Seq Analysis to Select Key Genes Related to Seed Dormancy in ALS-Inhibiting Resistant Descurainia sophia with Pro-197-Thr Mutation.

Flixweed (Descurainia sophia) is a weed that seriously affects wheat fields in China. Over the past 20 years, it has evolved resistance to the herbicide tribenuron-methyl. In the present study, a resistant D. sophia population with a Pro-197-Thr mutation of acetolactate synthetase (ALS) was found to have a resistance index of 457.37 for tribenuron-methyl. Under the same growth conditions, the seeds of resistant (R) and susceptible (S) populations exhibited similar vitality but the germination rates of R seeds were higher than those of S seeds. This result demonstrated that seed dormancy periods were shorter in the R seeds. RNA-Seq transcriptome analysis was then used to choose candidate genes that could regulate seed dormancy pathways in the R population. A total of 504,976,046 clean reads were selected from nine RNA-Seq libraries and assembled into 79,729 unigenes. Among these, 33,476 unigenes were assigned to 51 GO subgroups, and 26,117 unigenes were assigned to 20 KEGG secondary metabolic pathways. Next, 2473 differentially expressed genes (DEGs) were divided into three groups, as follows: G-24 h (germinating seeds) vs. D (dormant seeds); G-48 h (germinated seeds) vs. D; and G-48 h vs. G-24 h. From these 2473 DEGs, 8 were selected as candidate dormancy unigenes for the R population if their expression levels continuously decreased during the seed germination progress and their functional annotations were related to plant seed dormancy. One candidate unigene was annotated as CYP707A2; two unigenes were annotated as the transcription factors TGA4 and TGA2; one unigene was annotated as the cystathionine beta-synthase gene; and four unigenes could not be annotated as any gene listed in the six public databases. However, qRT-PCR-validated results showed that, during the germination of R seeds, the expression of the three candidate unigenes first decreased and then increased, indicating that they may have other growth-regulating functions in R populations. In brief, the dormancy function of the eight candidate dormancy unigenes needs to be further studied.

Western diet-induced cognitive and metabolic dysfunctions in aged mice are prevented by rosmarinic acid in a sex-dependent fashion

Unhealthy lifestyles, such as chronic consumption of a Western Diet (WD), have been associated with increased systemic inflammation and oxidative stress (OS), a condition that may favour cognitive dysfunctions during aging. Polyphenols, such as rosmarinic acid (RA) may buffer low-grade inflammation and OS, characterizing the aging brain that is sustained by WD, promoting healthspan. The aim of this study was to evaluate the ability of RA to prevent cognitive decline in a mouse model of WD-driven unhealthy aging and to gain knowledge on the specific molecular pathways modulated within the brain. Aged male and female C57Bl/6N mice were supplemented either with RA or vehicle for 6 weeks. Following 2 weeks on RA they started being administered either with WD or control diet (CD). Successively all mice were tested for cognitive abilities in the Morris water maze (MWM) and emotionality in the elevated plus maze (EPM). Glucose and lipid homeostasis were assessed in trunk blood while the hippocampus was dissected out for RNAseq transcriptomic analysis. RA prevented insulin resistance in males while protecting both males and females from WD-dependent memory impairment. In the hippocampus, RA modulated OS pathways in males and immune- and sex hormones-related signalling cascades (Lhb and Lhcgr genes) in females. Moreover, RA overall resulted in an upregulation of Glp1r, recently identified as a promising target to prevent metabolic derangements. In addition, we also found an RA-dependent enrichment in nuclear transcription factors, such as NF-κB, GR and STAT3, that have been recently suggested to promote healthspan and longevity by modulating inflammatory and cell survival pathways. Oral RA supplementation may promote brain and metabolic plasticity during aging through antioxidant and immune-modulating properties possibly affecting the post-reproductive hormonal milieu in a sex-dependent fashion. Thus, its supplementation should be considered in the context of precision medicine as a possible strategy to preserve cognitive functions and to counteract metabolic derangements.

Development of Mutated β-catenin Gene Signature to identify CTNNB1 mutations from whole and spatial transcriptomic data in patients with HCC

Background & AimsPatients with β-catenin (encoded by CTNNB1)-mutated hepatocellular carcinoma (HCC) demonstrate heterogenous responses to first-line immune checkpoint inhibitors (ICIs). Precision-medicine based treatments for this subclass are currently in clinical development. Here, we report derivation of Mutated β-catenin Gene Signature (MBGS) to predict CTNNB1-mutational status in patients for future personalized medicine. MethodsCo-expression of mutant-Nrf2 and hMet ± mutant-β-catenin in murine livers in mice led to HCC development. Using bulk RNA-seq and intersectional transcriptomic analysis of various β-catenin-mutated and non-mutated HCC models, MBGS was derived. Integrated RNA/whole-exome-sequencing and spatial transcriptomic data from multiple HCC patient cohorts was assessed to address ability of MBGS to detect CTNNB1 mutation, tumor immune microenvironment and/or predict therapeutic responses. ResultsBulk RNA-seq comparing HCCs in mutant β-catenin-Nrf2, β-catenin-Met and β-catenin-Nrf2-Met to Nrf2-Met HCC model yielded 95 common upregulated genes. In TCGA-LIHC, differential gene expression analysis with FDR=0.05 and log2FC>1.5 on the 95 common genes comparing CTNNB1-mutated vs wild-type patients narrowed the gene panel to 13-genes MBGS. MBGS predicted CTNNB1-mutations in TCGA (n=374) and French (n=398) patient cohorts with ROC AUC of 0.90 and 0.94, respectively. Additionally, increased MBGS expression score was associated with lack of significant overall survival or progression-free survival effects in the atezolizumab-bevacizumab arm versus sorafenib arm in IMbrave150 cohort. MBGS performed comparable or superior to other CTNNB1-mutant classifiers. MBGS overlapped with Hoshida S3, Boyault G5/G6, and Chiang CTNNB1 subclass tumors in TCGA and in HCC spatial transcriptomic datasets visually depicting these tumors to be situated in an immune excluded tumor microenvironment. ConclusionsMBGS will aid in patient stratification to guide precision medicine therapeutics for CTNNB1-mutated HCC subclass as a companion diagnostic, as anti-β-catenin therapies become available.

Submandibular gland tissue RNAseq and spatial transcriptome analyses in IgG4-related disease.

To identify genes that could provide clues leading to the discovery of drugs to treat IgG4-related disease (IgG4-RD). Submandibular gland tissue bulk RNAseq analysis of 45 cases with a definite diagnosis of IgG4-RD was integrated with Visium spatial transcriptome analysis of 2 cases to identify pathogenic genes expressed in tertiary lymphoid tissues. Bulk RNAseq and pathway analyses showed upregulation of cell cycle and T cell-related signals in IgG4-RD. Spatial transcriptome analysis identified the cluster corresponding to germinal centers and the top 38 common genes that showed significant variations in expression compared with other clusters. The top 20 genes were extracted by comparing the bulk RNAseq data. Network analysis identified CDK1 as the ge most strongly associated of the top 20 genes. The CDK1 gene may be a regulator of the pathogenesis of IgG4-RD and provide clues for drug discovery.

Impact of age and sex on neuroinflammation following SARS-CoV-2 infection in a murine model.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the etiological agent of COVID-19, is known to infect people of all ages and both sexes. Senior populations have the greatest risk of severe COVID-19, and sexual dimorphism in clinical outcomes has been reported. Neurological symptoms are widely observed in COVID-19 patients, with many survivors exhibiting persistent neurological and cognitive impairment. The present study aims to investigate the impact of age and sex on the neuroinflammatory response to SARS-CoV-2 infection using a mouse model. Wild-type C57BL/6J mice were intranasally inoculated with SARS-CoV-2 lineage B.1.351, a variant known to infect mice. Older male mice exhibited a significantly greater weight loss and higher viral loads in the lung at 3 days post infection. Notably, no viral RNA was detected in the brains of infected mice. Nevertheless, expression of IL-6, TNF-α, and CCL-2 in the lung and brain increased with viral infection. RNA-seq transcriptomic analysis of brains showed that SARS-CoV-2 infection caused significant changes in gene expression profiles, implicating innate immunity, defense response to virus, and cerebrovascular and neuronal functions. These findings demonstrate that SARS-CoV-2 infection triggers a neuroinflammatory response, despite the lack of detectable virus in the brain. Aberrant activation of innate immune response, disruption of blood-brain barrier and endothelial cell integrity, and suppression of neuronal activity and axonogenesis underlie the impact of SARS-CoV-2 infection on the brain. Understanding the role of these affected pathways in SARS-CoV-2 pathogenesis helps identify appropriate points of therapeutic interventions to alleviate neurological dysfunction observed during COVID-19.

A gibberellin-assisted study of the transcriptional and hormonal changes occurring at floral transition in peach buds (Prunus persica L. Batsch)

BackgroundFlower load in peach is an important determinant of final fruit quality and is subjected to cost-effective agronomical practices, such as the thinning, to finely balance the sink-source relationships within the tree and drive the optimal amount of assimilates to the fruits. Floral transition in peach buds occurs as a result of the integration of specific environmental signals, such as light and temperature, into the endogenous pathways that induce the meristem to pass from vegetative to reproductive growth. The cross talk and integration of the different players, such as the genes and the hormones, are still partially unknown. In the present research, transcriptomics and hormone profiling were applied on bud samples at different developmental stages. A gibberellin treatment was used as a tool to identify the different phases of floral transition and characterize the bud sensitivity to gibberellins in terms of inhibition of floral transition.ResultsTreatments with gibberellins showed different efficacies and pointed out a timeframe of maximum inhibition of floral transition in peach buds. Contextually, APETALA1 gene expression was shown to be a reliable marker of gibberellin efficacy in controlling this process. RNA-Seq transcriptomic analyses allowed to identify specific genes dealing with ROS, cell cycle, T6P, floral induction control and other processes, which are correlated with the bud sensitivity to gibberellins and possibly involved in bud development during its transition to the reproductive stage. Transcriptomic data integrated with the quantification of the main bioactive hormones in the bud allowed to identify the main hormonal regulators of floral transition in peach, with a pivotal role played by endogenous gibberellins and cytokinins.ConclusionsThe peach bud undergoes different levels of receptivity to gibberellin inhibition. The stage with maximum responsiveness corresponded to a transcriptional and hormonal crossroad, involving both flowering inhibitors and inductors. Endogenous gibberellin levels increased only at the latest developmental stage, when floral transition was already partially achieved, and the bud was less sensitive to exogenous treatments. A physiological model summarizes the main findings and suggests new research ideas to improve our knowledge about floral transition in peach.

Unveiling major histocompatibility complex-mediated pan-cancer immune features by integrated single-cell and bulk RNA sequencing

Immune checkpoint inhibitors (ICIs) have transformed cancer therapy, yet persistent challenges such as low response rate and significant heterogeneity necessitate attention. The pivotal role of the major histocompatibility complex (MHC) in ICI efficacy, its intricate impacts and potentials as a prognostic marker, warrants comprehensive exploration. This study integrates single-cell RNA sequencing (scRNA-seq), bulk RNA-seq, and spatial transcriptomic analyses to unveil pan-cancer immune characteristics governed by the MHC transcriptional feature (MHC.sig). Developed through scRNA-seq analysis of 663,760 cells across diverse cohorts and validated in 30 solid cancer types, the MHC.sig demonstrates a robust correlation between immune-related genes and infiltrating immune cells, highlighting its potential as a universal pan-cancer marker for anti-tumor immunity. Screening the MHC.sig for therapeutic targets using CRISPR data identifies potential genes for immune therapy synergy and validates its predictive efficacy for ICIs responsiveness across diverse datasets and cancer types. Finally, analysis of cellular communication patterns reveals interactions between C1QC+macrophages and malignant cells, providing insights into potential therapeutic agents and their sensitivity characteristics. This comprehensive analysis positions the MHC.sig as a promising marker for predicting immune therapy outcomes and guiding combinatorial therapeutic strategies.

Employing Genomic Tools to Explore the Molecular Mechanisms behind the Enhancement of Plant Growth and Stress Resilience Facilitated by a Burkholderia Rhizobacterial Strain.

The rhizobacterial strain BJ3 showed 16S rDNA sequence similarity to species within the Burkholderia genus. Its complete genome sequence revealed a 97% match with Burkholderia contaminans and uncovered gene clusters essential for plant-growth-promoting traits (PGPTs). These clusters include genes responsible for producing indole acetic acid (IAA), osmolytes, non-ribosomal peptides (NRPS), volatile organic compounds (VOCs), siderophores, lipopolysaccharides, hydrolytic enzymes, and spermidine. Additionally, the genome contains genes for nitrogen fixation and phosphate solubilization, as well as a gene encoding 1-aminocyclopropane-1-carboxylate (ACC) deaminase. The treatment with BJ3 enhanced root architecture, boosted vegetative growth, and accelerated early flowering in Arabidopsis. Treated seedlings also showed increased lignin production and antioxidant capabilities, as well as notably increased tolerance to water deficit and high salinity. An RNA-seq transcriptome analysis indicated that BJ3 treatment significantly activated genes related to immunity induction, hormone signaling, and vegetative growth. It specifically activated genes involved in the production of auxin, ethylene, and salicylic acid (SA), as well as genes involved in the synthesis of defense compounds like glucosinolates, camalexin, and terpenoids. The expression of AP2/ERF transcription factors was markedly increased. These findings highlight BJ3's potential to produce various bioactive metabolites and its ability to activate auxin, ethylene, and SA signaling in Arabidopsis, positioning it as a new Burkholderia strain that could significantly improve plant growth, stress resilience, and immune function.

Ephrin type-A receptor 2-antisense RNA1/2 promote proliferation and migration of MDA-MB-231 cells through EPHA2-dependent Ras signaling pathway mediated by MAPK8/JNK1, MAPK9/JNK2-NFATC2/NFAT1 and JUND

Ephrin type-A receptor 2 (EPHA2) is a receptor tyrosine kinase that is overexpressed in a variety of cancers, including breast cancer. EPHA2 expression may be causally related to tumorigenesis; therefore, it is important to understand how EPHA2 expression is regulated. We previously reported that EPHA2 antisense RNA (EPHA2-AS), a natural antisense transcript, is an important modulator of EPHA2 mRNA levels and hence production of EPHA2 protein. EPHA2-AS encodes two splice variants, EPHA2-AS1 and EPHA2-AS2. The two variants are constitutively expressed in a concordant manner with EPHA2 mRNA in human breast adenocarcinoma cell lines and in patient samples, with the highest levels detected in the basal-like/triple-negative molecular subtype of breast cancer cells. In this study, we investigated the mechanism of EPHA2-AS1/2 in triple-negative breast cancer using MDA-MB-231 cells. We performed RNA-seq transcriptome analyses of MDA-MB-231 cells treated with AHCC®, which suppressed expression of EPHA2-AS1/2 and EPHA2 mRNA, and EPHA2-AS1/2-silenced MDA-MB-231 cells. Bioinformatics analyses identified 545 overlapping differentially expressed genes that were significantly up- or down-regulated by these treatments. Subsequent functional enrichment analyses of the overlapping genes in combination with in vitro assays indicated that EPHA2-AS1/2 may promote the proliferation and migration of MDA-MB-231 cells through the EPHA2-dependent Ras signaling pathways mediated by MAPK8/JNK1, MAPK9/JNK2-NFATC2/NFAT1 (proliferation and migration) and JUND (migration). These results thus suggest that EPHA2-AS1/2 may represent a potential molecular target for triple-negative breast cancer treatment.

Distinct actions of testicular endocrine and lumicrine signaling on the proximal epididymal transcriptome.

The epididymal function and gene expression in mammals are under the control of the testis. Sex steroids are secreted from the testis and act on the epididymis in an endocrine manner. There is another, non-sex steroidal secreted signaling, named lumicrine signaling, in which testis-derived secreted proteins go through the male reproductive tract and act on the epididymis. The effects of such multiple regulations on the epididymis by the testis have been investigated for many genes. The recent development of high-throughput next-generation sequencing now enables us a further comparative survey of endocrine and lumicrine action-dependent gene expression. In the present study, testis-derived endocrine and lumicrine actions on epididymal gene expression were comparatively investigated by RNA-seq transcriptomic analyses. This investigation utilized experimental animal models in which testis-derived endocrine and/or lumicrine actions were interfered with, such as unilateral or bilateral orchidectomy. By bilateral orchidectomy, which interferes with both endocrine and lumicrine actions, 431 genes were downregulated. By unilateral orchidectomy, which also interferes with endocrine and lumicrine actions by the unilateral testis, but the endocrine action was compensated by the contralateral testis, 283 genes were downregulated. The content of such genes downregulated by unilateral orchidectomy was like those of lumicrine action-interfered efferent duct-ligation, W/Wv, and Nell2-/- mice. When genes affected by unilateral and bilateral orchidectomy were compared, 154 genes were commonly downregulated, whereas 217 genes were specifically downregulated only by bilateral orchidectomy, indicating the distinction between endocrine and lumicrine actions on the proximal epididymal transcriptome. Comparative transcriptome analyses also showed that the expressions of genes emerging since Amniota were notably impacted by bilateral orchidectomy, unilateral orchidectomy, and lumicrine action-interfering treatments; the degree of influence from these treatments varied based on the evolutionary stage beyond Amniota. These findings unveil an evolutional transition of regulated gene expression in the proximal epididymis by two different testis-derived signaling mechanisms.

Nontarget site-based resistance to nicosulfuron and identification of candidate genes in Cucumis melo L. var. agrestis Naud. via RNA-Seq transcriptome analysis

Herbicide resistance is a worldwide concern for weed control. Cucumis melo L. var. agrestis Naud. (C. melo) is an annual trailing vine weed that is commonly controlled by nicosulfuron, acetolactate synthase (ALS)-inhibiting herbicides. However, long-term use of this herbicide has led to the emergence of resistance and several nicosulfuron resistant populations of C. melo have been found. Here we identified a resistant (R) C. melo population exhibiting 7.31-fold resistance to nicosulfuron compared with a reference sensitive (S) population. ALS gene sequencing of the target site revealed no amino acid substitution in R plants, and no difference in enzyme activity, as shown by ALS activity assays in vitro. ALS gene expression was not significantly different before and after the application of nicosulfuron. Pretreatment with the cytochrome P450 monooxygenase (P450) inhibitor malathion reduced nicosulfuron resistance in the R population. RNA-Seq transcriptome analysis was used to identify candidate genes that may confer metabolic resistance to nicosulfuron. We selected genes with annotations related to detoxification functions. A total of 20 candidate genes (7 P450 genes, 1 glutathione S-transferase (GST) gene, 2 ATP-binding cassette (ABC) transporters, and 10 glycosyltransferase (GT)) were identified; 12 of them (7 P450s, 1 GST, 2 ABC transporters, and 2 GTs) were demonstrated significantly differential expression between R and S by quantitative real-time RT-PCR (qRT–PCR). Our findings revealed that the resistance mechanism in C. melo was nontarget-site based. Our results also provide a valuable resource for studying the molecular mechanisms of weed resistance.

Pectolinarigenin ameliorates osteoporosis via enhancing Wnt signaling cascade in PPARβ-dependent manner

BackgroundOsteoporosis is a prevalent metabolic bone disease in older adults. Peroxisome proliferator-activated receptor β (PPARβ), the most abundant PPAR isotype expressed in bone tissues, plays a critical role in regulating the energy metabolism of osteoblasts. However, the botanical compounds targeting PPARβ for the treatment of osteoporosis remain largely unexplored. PurposeTo discover a potent PPARβ agonist from botanical compounds, as well as to investigate the anti-osteoporosis effects and to elucidate the underlying mechanisms of the newly identified PPARβ agonist. MethodsThe PPARβ agonist effects of botanical compounds were screened by an in vitro luciferase reporter gene assay. The PPARβ agonist effects of pectolinarigenin (PEC) in bone marrow mesenchymal stromal cells (BMSCs) were validated by Western blotting. RNA-seq transcriptome analyses were conducted to reveal the underlying osteoporosis mechanisms of PEC in BMSCs. The PPARβ antagonist (GSK0660) and Wnt signaling inhibitor (XAV969) were used to explore the role of the PPARβ and Wnt signaling cascade in the anti-osteoporosis effects of PEC. PEC or the PEG-PLGA nanoparticles of PEC (PEC–NP) were intraperitoneally administrated in both wild-type mice and ovariectomy-induced osteoporosis mice to examine its anti-osteoporotic effects in vivo. ResultsPEC, a newly identified naturally occurring PPARβ agonist, significantly promotes osteogenic differentiation and up-regulates the osteogenic differentiation-related genes (Runx2, Osterix, and Bmp2) in BMSCs. RNA sequencing and functional gene enrichment analysis suggested that PEC could activate osteogenic-related signaling pathways, including Wnt and PPAR signaling pathways. Further investigations suggested that PEC could enhance Wnt/β-catenin signaling in a PPARβ-dependent manner in BMSCs. Animal tests showed that PEC–NP promoted bone mass and density, increased the bone cell matrix protein, and accelerated bone formation in wild-type mice, while PEC–NP also played a preventive role in ovariectomy-induced osteoporosis mice via maintaining the expression level of bone cell matrix protein, balancing the rate of bone formation, and slowing down bone loss. Additionally, PEC–NP did not cause any organ injury and body weight loss after long-term use (11 weeks). ConclusionPEC significantly promotes bone formation and reduces bone loss in both BMSCs and ovariectomy-induced osteoporosis mice via enhancing the Wnt signaling cascade in a PPARβ-dependent manner, providing a new alternative therapy for preventing estrogen deficiency-induced osteoporotic diseases.

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.

Abstract 6552: Intratumoral (ITu) delivery of mRNA-2752 encoding human OX40L/IL-23/IL-36γ in combination with durvalumab induces an immunostimulatory effect within the tumor microenvironment (TME) of patients with advanced solid tumors

Abstract Background mRNA-2752 is a first-in-class ITu therapy composed of mRNA encoding human OX40L/IL-23/IL-36γ in a lipid nanoparticle. The combination of T cell costimulation and proinflammatory cytokines were developed to modulate the TME and allow a more robust immune response. Here we show evidence of the pharmacodynamic effect of ITu administration of mRNA-2752 in combination with PD-L1 antibody. Methods In this ongoing phase 1 study (NCT03739931), patients (pts) with advanced solid tumors received 0.25-8 mg mRNA-2752 alone (Arm A) or in combination with 1500 mg IV durvalumab (Arm B)1,2. Pre and on-treatment peripheral and tumor samples were collected to monitor changes in cytokines by ligand binding assays, gene signatures by RNA sequencing, and T cell infiltration by immunohistochemistry. Tumor biopsies were taken at baseline, 24h, 15 and 28 days post treatment. Results mRNA-2752 administration in combination with durvalumab revealed ≥2-fold plasma level increases of IL-23 (34/40, 85%) and IFN-γ (50/65, 77%) at 24h after first injection; exposure was induced in a dose-dependent manner. We observed induction of CD8 T cell abundance (20/49, 41%) and proliferation (23/49, 47%), and OX40L protein expression (28/49, 57%) in pts with evaluable post-treatment injected lesion biopsies. CD8 T cell abundance increased in 2 pts with evaluable post-treatment un-injected lesion biopsies. A positive correlation was observed between T cell abundance and T cell effector genes. All three mRNA (IL-23, IL-36γ, and OX40L) encoded genes were detected post treatment in tumor samples from 43 evaluable patients. In 18 pts with injected lesion biopsy samples at baseline and day 28 samples, the genes related to cytotoxic function (IFNG, GZMB, PRF1), T cell migration (CCL21, CCR7, KLF2), T cell activation/memory phenotype (TCF7, CD69, ITGAE), and conventional type 1 dendritic cells (CLEC9A, XCR1) were induced 28 days post-treatment in pts with complete/partial response or stable disease (CR/PR/SD, n=7) compared to progressive disease (PD, n=11) pts. RNA-Seq transcriptome analysis showed increases in the immune inflamed gene signatures related to antitumor activity in 24/43 (56%) pts post-treatment, and the pre-existing immune inflamed phenotype within the TME was associated with clinical benefit. Conclusions mRNA-2752 ITu injection induced a systemic immunomodulatory effect by elevating effector cytokines. This was associated with pharmacodynamic changes within the TME, including increases in T cell abundance and proliferation, and upregulation of immune gene signatures that correlate with antitumor response.

Deletion of Slc9a1 in Cx3cr1+ cells stimulated microglial subcluster CREB1 signaling and microglia-oligodendrocyte crosstalk

Microglial Na/H exchanger-1 (NHE1) protein, encoded by Slc9a1, plays a role in white matter demyelination of ischemic stroke brains. To explore underlying mechanisms, we conducted single cell RNA-seq transcriptome analysis in conditional Slc9a1 knockout (cKO) and wild-type (WT) mouse white matter tissues at 3 days post-stroke. Compared to WT, Nhe1 cKO brains expanded a microglial subgroup with elevated transcription of white matter myelination genes including Spp1, Lgals3, Gpnmb, and Fabp5. This subgroup also exhibited more acidic pHi and significantly upregulated CREB signaling detected by ingenuity pathway analysis and flow cytometry. Moreover, the Nhe1 cKO white matter tissues showed enrichment of a corresponding oligodendrocyte subgroup, with pro-phagocytosis and lactate shuffling gene expression, where activated CREB signaling is a likely upstream regulator. These findings demonstrate that attenuation of NHE1-mediated H+ extrusion acidifies microglia/macrophage and may underlie the stimulation of CREB1 signaling, giving rise to restorative microglia-oligodendrocyte interactions for remyelination.

RNA-seq transcriptome and pathway analysis of the medicinal mushroom Lignosus tigris (Polyporaceae) offer insights into its bioactive compounds with anticancer and antioxidant potential

Ethnopharmacological relevanceMedicinal mushrooms belonging to the Lignosus spp., colloquially known as Tiger Milk mushrooms (TMMs), are used as traditional medicine by communities across various regions of China and Southeast Asia to enhance immunity and to treat various diseases. At present, three Lignosus species have been identified in Malaysia: L. rhinocerus, L. tigris, and L. cameronensis. Similarities in their macroscopic morphologies and the nearly indistinguishable appearance of their sclerotia often lead to interchangeability between them. Hence, substantiation of their traditional applications via identification of their individual bioactive properties is imperative in ensuring that they are safe for consumption. L. tigris was first identified in 2013. Thus far, studies on L. tigris cultivar sclerotia (Ligno TG-K) have shown that it possesses significant antioxidant activities and has greater antiproliferative action against selected cancer cells in vitro compared to its sister species, L. rhinocerus TM02®. Our previous genomics study also revealed significant genetic dissimilarities between them. Further omics investigations on Ligno TG-K hold immense potential in facilitating the identification of its bioactive compounds and their associated bioactivities. Aim of studyThe overall aim of this study was to investigate the gene expression profile of Ligno TG-K via de novo RNA-seq and pathway analysis. We also aimed to identify highly expressed genes encoding compounds that contribute to its cytotoxic and antioxidant properties, as well as perform a comparative transcriptomics analysis between Ligno TG-K and its sister species, L. rhinocerus TM02®. Materials and methodsTotal RNA from fresh 3-month-old cultivated L. tigris sclerotia (Ligno TG-K) was extracted and analyzed via de novo RNA sequencing. Expressed genes were analyzed using InterPro and NCBI-Nr databases for domain identification and homology search. Functional categorization based on gene functions and pathways was performed using Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Clusters of Orthologous Genes (COG) databases. Selected genes were subsequently subjected to phylogenetic analysis. ResultsOur transcriptomics analysis of Ligno TG-K revealed that 68.06% of its genes are expressed in the sclerotium; 80.38% of these were coding transcripts. Our analysis identified highly expressed transcripts encoding proteins with prospective medicinal properties. These included serine proteases (FPKM = 7356.68), deoxyribonucleases (FPKM = 3777.98), lectins (FPKM = 3690.87), and fungal immunomodulatory proteins (FPKM = 2337.84), all of which have known associations with anticancer activities. Transcripts linked to proteins with antioxidant activities, such as superoxide dismutase (FPKM = 1161.69) and catalase (FPKM = 1905.83), were also highly expressed. Results of our sequence alignments revealed that these genes and their orthologs can be found in other mushrooms. They exhibit significant sequence similarities, suggesting possible parallels in their anticancer and antioxidant bioactivities. ConclusionThis study is the first to provide a reference transcriptome profile of genes expressed in the sclerotia of L. tigris. The current study also presents distinct COG profiles of highly expressed genes in Ligno TG-K and L. rhinocerus TM02®, highlighting that any distinctions uncovered may be attributed to their interspecies variations and inherent characteristics that are unique to each species. Our findings suggest that Ligno TG-K contains bioactive compounds with prospective medicinal properties that warrant further investigations. ClassificationSystems biology and omics

Developmental Dopaminergic Signaling Modulates Neural Circuit Formation and Contributes to Autism Spectrum Disorder–Related Phenotypes

Autism spectrum disorder (ASD) is a prevalent neurodevelopmental disorder with a complex etiology. Recent evidence suggests that dopamine plays a crucial role in neural development. However, it remains unclear whether and how disrupted dopaminergic signaling during development contributes to ASD. In this study, human brain RNA-seq transcriptome analysis revealed a significant correlation between changes in dopaminergic signaling pathways and neural developmental signaling in ASD patients. In the zebrafish model, disrupted developmental dopaminergic signaling led to neural circuit abnormalities and behavior reminiscent of autism. Dopaminergic signaling may impact neuronal specification by potentially modulating integrins. These findings shed light on the mechanisms underlying the link between disrupted developmental dopamine signaling and ASD, and they point to the possibility of targeting dopaminergic signaling in early development for ASD treatment.

Genome-wide identification and expression analysis of Autophagy-Related Genes in eggplant (Solanum melongena L.)

Autophagy, a highly conserved cellular process in plants, plays a vital role in regulating abiotic stress responses and senescence. Eggplant is a widely cultivated vegetable crop, its fruit is susceptible to cold damage, and its molecular mechanisms underlying stress tolerance, fruit ripening, and senescence remain largely uncharacterized. In this study, we systematically identified 41 Autophagy-Related Genes (ATGs) in eggplant and through bioinformatics methods, we analyzed their gene structures, evolutionary features, chromosomal locations, and promoter elements. Additionally, we conducted RNA-seq transcriptome analysis of various eggplant organs as well as under different stress conditions, and assessed the expression patterns of the SmATG8 under cold stress in eggplant fruit. Our analysis results show that the SmATGs promoters have diverse cis-regulatory elements, with most SmATGs exhibiting low and stable expression levels in different tissues. Some genes displayed tissue-specific expression patterns. Under 4 °C low-temperature stress, the expression of the SmATG8 family was continuously upregulated, indicating that autophagy plays a crucial role in defending against cold damage in eggplant. Furthermore, we found that ATGs in dicots and monocots exhibit distinct homology patterns. Within the dicots, eggplant and pepper, both being Solanaceae plants, their ATGs exhibit high homology, suggesting that SmATGs in Solanaceae plants have unique homologous relationships. The analysis of SmATGs unveiled their potential contributions to plant stress responses, fruit ripening, and senescence, thus offering a theoretical foundation for further exploration of stress resistance and senescence mechanisms in eggplant.