High-throughput Transcriptome Research Articles

Abstract PR017: Anti-metastatic immunotherapy discovery using ex vivo lung tissue cultures and high-throughput single-cell chemical transcriptomics

Abstract Introduction: Tumors systemically remodel the immune system during metastasis. Developing anti-metastatic immunotherapies that target these tumor-immune interactions to make the metastatic niche hostile colonization would represent a paradigm shift in cancer treatment. However, traditional high-throughput screening (HTS) platforms that use simple read-outs and contrived in vitro systems are ill-suited to identify such therapies. In contrast, HTS platforms that accurately model the metastatic niche and use high-dimensional read-outs such as multiplexed single-cell RNA-sequencing (scRNA-seq) can accurately profile nuanced perturbation responses in individual immune cell types and therefore have the potential to discover anti-metastatic immunotherapies. In this study, we describe the development of an ex vivo lung tissue culture HTS platform that preserves immune gene expression signatures observed in the in vivo metastatic niche and is amenable to single-cell chemical transcriptomic analysis using MULTI- seq (McGinnis et al., Nature Methods, 2019). Inspired by our recent description of myeloid cell TLR-NFκB inflammation during breast cancer lung metastasis (McGinnis et al., Cancer Cell, 2024), we then performed the first anti-metastatic immunotherapy drug screen and identified TLR-NFκB inhibitors that effectively operate on all desired myeloid cell types in the lung metastatic niche. Methods and Results: Building upon existing techniques for culturing precision-cut lung slices (Wu et al., Journal of Experimental Medicine, 2024) and patient- derived tumor fragments (Voabil et al., Nature Medicine, 2021), we established and optimized an ex vivo lung tissue culture system that enables 48-hour culturing of lung slices isolated from 4T1 tumor-bearing mice. scRNA-seq profiling revealed successful capture of all relevant immune cell types and retention of metastasis-associated gene expression programs (e.g., myeloid TLR-NFκB inflammation and neutrophil degranulation) following culture. We then scaled our platform to address how myeloid cells in the lung metastatic niche respond to perturbations targeting every component of the canonical TLR-NFκB signaling cascade. Cell-type-specific perturbation modeling and quantification of TLR-NFκB signaling inhibition identified compounds that optimally perturb all intended myeloid cell types (e.g., tissue-resident macrophages, neutrophils, and monocytes) and represent prioritized candidates for future in vivo validation studies to assess impact on metastatic disease progression. Conclusions: In this study, we describe the development and application of a novel HTS platform that couples ex vivo lung tissue cultures with single-cell chemical transcriptomics to identify anti-metastatic immunotherapy candidates. We demonstrate how our platform can identify TLR-NFκB signaling inhibitors that optimally operate in the lung metastatic niche, paving the way for future interrogation of additional metastasis-associated immune cell signaling pathways in different disease and tissue backgrounds. Citation Format: Chris McGinnis, Winnie Yao, Ansuman Satpathy. Anti-metastatic immunotherapy discovery using ex vivo lung tissue cultures and high-throughput single-cell chemical transcriptomics [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr PR017.

The first chicken oocyte nucleus whole transcriptomic profile defines the spectrum of maternal mRNA and non-coding RNA genes transcribed by the lampbrush chromosomes.

Lampbrush chromosomes, with their unusually high rate of nascent RNA synthesis, provide a valuable model for studying mechanisms of global transcriptome up-regulation. Here, we obtained a whole-genomic profile of transcription along the entire length of all lampbrush chromosomes in the chicken karyotype. With nuclear RNA-seq, we obtained information about a wider set of transcripts, including long non-coding RNAs retained in the nucleus and stable intronic sequence RNAs. For a number of protein-coding genes, we visualized their nascent transcripts on the lateral loops of lampbrush chromosomes by RNA-FISH. The set of genes transcribed on the lampbrush chromosomes is required for basic cellular processes and is characterized by a broad expression pattern. We also present the first high-throughput transcriptome characterization of miRNAs and piRNAs in chicken oocytes at the lampbrush chromosome stage. Major targets of predicted piRNAs include CR1 and long terminal repeat (LTR) containing retrotransposable elements. Transcription of tandem repeat arrays was demonstrated by alignment against the whole telomere-to-telomere chromosome assemblies. We show that transcription of telomere-derived RNAs is initiated at adjacent LTR elements. We conclude that hypertranscription on the lateral loops of giant lampbrush chromosomes is required for synthesizing large amounts of transferred to the embryo maternal RNA for thousands of genes.

Signature analysis of high-throughput transcriptomics screening data for mechanistic inference and chemical grouping.

High-throughput transcriptomics (HTTr) uses gene expression profiling to characterize the biological activity of chemicals in in vitro cell-based test systems. As an extension of a previous study testing 44 chemicals, HTTr was used to screen an additional 1,751 unique chemicals from the EPA's ToxCast collection in MCF7 cells using 8 concentrations and an exposure duration of 6 h. We hypothesized that concentration-response modeling of signature scores could be used to identify putative molecular targets and cluster chemicals with similar bioactivity. Clustering and enrichment analyses were conducted based on signature catalog annotations and ToxPrint chemotypes to facilitate molecular target prediction and grouping of chemicals with similar bioactivity profiles. Enrichment analysis based on signature catalog annotation identified known mechanisms of action (MeOAs) associated with well-studied chemicals and generated putative MeOAs for other active chemicals. Chemicals with predicted MeOAs included those targeting estrogen receptor (ER), glucocorticoid receptor (GR), retinoic acid receptor (RAR), the NRF2/KEAP/ARE pathway, AP-1 activation, and others. Using reference chemicals for ER modulation, the study demonstrated that HTTr in MCF7 cells was able to stratify chemicals in terms of agonist potency, distinguish ER agonists from antagonists, and cluster chemicals with similar activities as predicted by the ToxCast ER Pathway model. Uniform manifold approximation and projection (UMAP) embedding of signature-level results identified novel ER modulators with no ToxCast ER Pathway model predictions. Finally, UMAP combined with ToxPrint chemotype enrichment was used to explore the biological activity of structurally related chemicals. The study demonstrates that HTTr can be used to inform chemical risk assessment by determining in vitro points of departure, predicting chemicals' MeOA and grouping chemicals with similar bioactivity profiles.

Isolation and Identification of Alkaloid Genes from the Biomass of Fritillaria taipaiensis P.Y. Li

Background/Objectives: Fritillaria taipaiensis P.Y. Li is a valuable traditional Chinese medicinal herb that utilizes bulbs as medicine, which contain multiple alkaloids. Biomass, as a sustainable resource, has promising applications in energy, environmental, and biomedical fields. Recently, the biosynthesis and regulatory mechanisms of the main biomass components of biomass have become a prominent research topic. Methods: In this article, we explored the differences in the heterosteroidal alkaloid components of F. taipaiensis biomass using liquid chromatography–mass spectrometry and high-throughput transcriptome sequencing. Results: The experimental results demonstrated significant differences in the eight types of heterosteroidal alkaloid components among the biomass of F. taipaiensis, including peimisine, imperialine, peimine, peiminine, ebeinone, ebeiedine, ebeiedinone, and forticine. Transcriptomic analysis revealed substantial significant differences in gene expression patterns in the various samples. Three catalytic enzyme-coding genes, 3-hydroxy-3-methylglutaryl coenzyme A synthase (HMGS), 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), and terpene synthase (TPS), were speculated to contribute to the regulation of the differential accumulation of alkaloid synthesis in F. taipaiensis bulbs. A strong positive correlation was observed between the transcriptional level of the TPS gene and the alkaloid content of F. taipaiensis biomass, suggesting that TPS may be a key gene in the biosynthesis pathway of alkaloids. This finding can be used for subsequent gene function verification and molecular regulatory network analysis. Conclusions: This work provides fundamental data and novel insights for the subsequent research on alkaloid biosynthesis in F. taipaiensis.

Comparative Transcriptome Analysis of the Pest Galeruca daurica (Coleoptera: Chrysomelidae) Larvae in Response to Six Main Metabolites from Allium mongolicum (Liliaceae)

Plants are important ecological factors and food resources, which can significantly affect the occurrence and distribution of insects. The metabolites in host plants can affect the feeding, spawning, and avoidance behaviors of herbivorous insects. Galeruca daurica (Joannis) is a phytophagous pest that has seriously occurred in the desert steppe of Inner Mongolia in recent years, only infesting the leaves of Allium plants. In order to clarify the effects of plant metabolites on the gene expression in G. daurica larvae at the transcriptome level, we fed the larvae of G. daurica with Allium tuberosum leaves soaked in 10% DMSO solutions containing d-galactose, β-d-glucopyranose, l-rhamnose, isoquercitrin, isoflavone, and rutin, respectively, used the larvae fed on A. tuberosum leaves soaked in a 10% DMSO solution as the control, and screened out the differentially expressed genes (DEGs) by performing high-throughput transcriptome sequencing. The results showed that a total of 291 DEGs were identified compared to the solvent control (DMSO), including 130, 34, 29, 21, 72, and 97 in the isoquercitrin, isoflavone, rutin, d-galactose, β-d-glucopyranose, and l-rhamnose treatment groups, respectively. GO and KEGG enrichment analysis showed that most DEGs were enriched in various metabolic pathways, implying that these six main primary and secondary metabolites in Allium plants may affect various metabolic processes in the larvae of G. daurica.

MicroRNA-145-5p restricts cardiac scar resolution during cardiac regeneration

Abstract Background Human hearts have a very limited capacity to renew cardiomyocytes following injury. The lost myocardium is permanently replaced with noncontractile fibrotic tissue, consequently leading to heart failure. In contrast, zebrafish retain cardiac regenerative capacity during their lifetime. After injury, zebrafish hearts undergo transient fibrotic scarring that subsequently resolves and is replaced with new cardiomyocytes over time. Thus, understanding the molecular mechanisms governing cardiac scar resolution in zebrafish could offer insights into enhancing the regenerative capacities of human hearts. Objective We aimed to decipher the molecular pathways associated with zebrafish cardiac scar resolution. Methods We investigated the mRNA and miRNA responses to injury at 14 days post-injury by high-throughput transcriptome profiling of RNAs isolated from healthy or cryoinjured hearts. The prediction of miRNA-mRNA interactions was performed to identify the most differentially expressed miRNAs and their target genes. These miRNAs and genes were validated in zebrafish regenerating hearts and in the left ventricles of ischemic cardiomyopathy or dilated cardiomyopathy patients. The potential role of identified miRNAs in cardiac scar resolution was investigated in human cardiac fibroblasts. Results Principal component analyses identify two distinct clusters in both the mRNAome and miRNAome corresponding to healthy and injured hearts, indicating significant changes in transcriptome profiles during zebrafish cardiac regeneration. Gene set enrichment analyses indicate that molecular pathways involved in extracellular matrix organization and inflammatory response are markedly activated whereas genes associated with mitochondrial organization and oxidative phosphorylation are downregulated in injured hearts. We observed the downregulation of miR-145-5p and the upregulation of procollagen V in the injured hearts. In contrast to zebrafish, in the left ventricles of ischemic cardiomyopathy patients, miR-145-5p is significantly upregulated and procollagen V α1 is downregulated. Theoretical prediction of miRNA-mRNA interactions indicates procollagen V α1 is negatively correlated with miR-145-5p. Overexpression of miR-145-5p in human cardiac fibroblasts downregulates collagen V α1 and alpha smooth muscle actin, upregulates procollagen I and matrix metalloproteinase 9, and inhibits cell proliferation and migration. In contrast, inhibition of miR-145-5p upregulates collagen V α1 and alpha smooth muscle actin, downregulates procollagen I and matrix metalloproteinase 9, and stimulates cell proliferation and migration. Conclusion Our study suggests that miR-145-5p may play a role in the suppression of myofibroblast transdifferentiation via collagen V α1, which restricts scar resolution and cardiac repair.

Spatial transcriptomics analysis identifies therapeutic targets in diffuse high-grade gliomas.

Diffuse high-grade gliomas are the most common malignant adult neuroepithelial tumors in humans and a leading cause of cancer-related death worldwide. The advancement of high throughput transcriptome sequencing technology enables rapid and comprehensive acquisition of transcriptome data from target cells or tissues. This technology aids researchers in understanding and identifying critical therapeutic targets for the prognosis and treatment of diffuse high-grade glioma. Spatial transcriptomics was conducted on two cases of isocitrate dehydrogenase (IDH) wild-type diffuse high-grade glioma (Glio-IDH-wt) and two cases of IDH-mutant diffuse high-grade glioma (Glio-IDH-mut). Gene set enrichment analysis and clustering analysis were employed to pinpoint differentially expressed genes (DEGs) involved in the progression of diffuse high-grade gliomas. The spatial distribution of DEGs in the spatially defined regions of human glioma tissues was overlaid in the t-distributed stochastic neighbor embedding (t-SNE) plots. We identified a total of 10,693 DEGs, with 5,677 upregulated and 5,016 downregulated, in spatially defined regions of diffuse high-grade gliomas. Specifically, SPP1, IGFBP2, CALD1, and TMSB4X exhibited high expression in carcinoma regions of both Glio-IDH-wt and Glio-IDH-mut, and 3 upregulated DEGs (SMOC1, APOE, and HIPK2) and 4 upregulated DEGs (PPP1CB, UBA52, S100A6, and CTSB) were only identified in tumor regions of Glio-IDH-wt and Glio-IDH-mut, respectively. Moreover, Kyoto Encyclopedia of Genes and Genomes (KEGG) and gene ontology (GO) enrichment analyses revealed that upregulated DEGs were closely related to PI3K/Akt signaling pathway, virus infection, and cytokine-cytokine receptor interaction. Importantly, the expression of these DEGs was validated using GEPIA databases. Furthermore, the study identified spatial expression patterns of key regulatory genes, including those involved in protein post-translational modification and RNA binding protein-encoding genes, with spatially defined regions of diffuse high-grade glioma. Spatial transcriptome analysis is one of the breakthroughs in the field of medical biotechnology as this can map the analytes such as RNA information in their physical location in tissue sections. Our findings illuminate previously unexplored spatial expression profiles of key biomarkers in diffuse high-grade glioma, offering novel insight for the development of therapeutic strategies in glioma.

Regulation of renal ischemia-reperfusion injury and tubular epithelial cell ferroptosis by pparγ m6a methylation: mechanisms and therapeutic implications

This study aimed to elucidate the role and underlying mechanisms of Peroxisome proliferator-activated receptor gamma (PPARγ) and its m6A methylation in renal ischemia-reperfusion (I/R) injury and ferroptosis of tubular epithelial cells (TECs). High-throughput transcriptome sequencing was performed on renal tissue samples from I/R injury models and sham-operated mice, complemented by in vivo and in vitro experiments focusing on the PPARγ activator Rosiglitazone and the manipulation of METTL14 and IGF2BP2 expression. Key evaluations included renal injury assessment, ferroptosis indicator measurement, and m6A methylation analysis of PPARγ. Our findings highlight the critical role of the PPARγ pathway and ferroptosis in renal I/R injury, with Rosiglitazone ameliorating renal damage and TEC ferroptosis. METTL14-mediated m6A methylation of PPARγ, dependent on IGF2BP2, emerged as a pivotal regulator of PPARγ expression, renal injury, and ferroptosis. This study reveals that PPARγ m6A methylation, orchestrated by METTL14 through an IGF2BP2-dependent mechanism, plays a crucial role in mitigating renal I/R injury and TEC ferroptosis. These insights offer promising avenues for therapeutic strategies targeting acute kidney injury.

Transcriptome analysis reveals a role of FOXO3 in antileukemia/lymphoma properties of panduratin A

Boesenbergia rotunda, commonly known as fingerroot, is a medicinal and culinary plant native to the Indochina Peninsula. We found that panduratin A (Pan-A), one of the compounds present in the rhizome extract of fingerroot, inhibited cell proliferation, induced apoptosis, and promoted cell cycle arrest at G0/G1 phase in multiple hematologic malignant cell lines including leukemia and lymphoma lines. Pan-A inhibited these activities in leukemia and lymphoma cells in a concentration-dependent manner. High-throughput transcriptome analysis indicated that Pan-A is involved in the cell cycle, cellular senescence, apoptosis, and multiple canonical signaling pathways in lymphoma cells. The Forkhead box O (FOXO) transcription factor family was identified as a potential target of Pan-A. Western blot showed elevated caspase 7 and cPARP/PARP in the B-cell lymphoma cells after treatment with Pan-A. The inhibitory effects were accompanied by stimulation of Akt signaling and phosphorylation of FOXO3. Immunohistochemistry of tissues from patients with B-cell lymphoma revealed detectable levels of FOXO3 protein specifically in neoplastic B cells. Overall, our results highlight FOXO3 as a player underlying antileukemia/lymphoma effects of Pan-A.

Mode of action exploration for prostate epithelial cell injury caused by bisphenol A

Bisphenol A (BPA) is a typical food chemical contaminant with various detrimental effects, especially on reproductive system. Male prostate damage is also one of its major adverse health effects, of which mode of action (MOA) remains unclear. This study aims to explore the MOA for prostate toxicity of BPA using human normal prostate epithelial cell RWPE-1 for 28-day human-relevant-level exposure. A physiological based pharmacokinetic model was used to determine the concentration of BPA based on the actual oral exposure in China. The possible key events were identified by high-throughput transcriptome sequencing and validated by qPCR, Western blot and cell cycle assay, and the benchmark concentration analysis were conducted. The enriched KEGG pathways include the endocytosis, cell cycle, cellular senescence, MAPK and TNF signaling pathways. With increasing BPA concentrations, the increased mRNA and/or protein expressions of MAPKAPK2, c-JUN and c-fos in the MAPK signaling pathway, the increased mRNA expressions of CCND1 and CDKN1A, the decreased mRNA expression of CDC25C, the increased proportion of G0/G1 phase and S phase, as well as the decreased proportion of G2/M phase, were observed. The lowest value of benchmark concentration lower confidence limit (BMCL) was retrieved from G2/M phase ratio, with 110.580 and 175.862 nM for BMCL5 and BMCL10, respectively, much higher than the male gonad maximum concentration of 0.019 nM of BPA at the current exposure level of adult Chinese males. In conclusion, the MOA of BPA induced male prostatic toxicity at human-relevant levels may include: key event (KE)1-MAPK signaling pathway activation, KE2-disorder of cell cycle regulatory gene expression (increased expression of CCND1 and CDKN1A, decreased expression of CDC25C), and KE3-disturbance of cell cycle (increased proportion of G0/G1 and S phases, decreased proportion of G2/M phases). However, more studies are needed to validate and complete the MOA.

Infection mechanism of Botryosphaeria dothidea and the disease resistance strategies of Chinese hickory (Carya cathayensis)

Botryosphaeria dothidea is the main fungal pathogen responsible for causing Chinese hickory (Carya cathayensis) dry rot disease, posing a serious threat to the Chinese hickory industry. Understanding the molecular basis of B. dothidea infection and the host’s resistance mechanisms is crucial for controlling and managing the ecological impact of Chinese hickory dry rot disease. This study utilized ultrastructural observations to reveal the process of B. dothidea infection and colonization in Chinese hickory, and investigated the impact of B. dothidea infection on Chinese hickory biochemical indicators and plant hormone levels. Through high-throughput transcriptome sequencing, the gene expression profiles associated with different stages of B. dothidea infection in Chinese hickory and their corresponding defense responses were described. Additionally, a series of key genes closely related to non-structural carbohydrate metabolism, hormone metabolism, and plant-pathogen interactions during B. dothidea infection in Chinese hickory were identified, including genes encoding DUF, Myb_DNA-binding, and ABC transporter proteins. These findings provide important insights into elucidating the pathogenic mechanisms of B. dothidea and the resistance genes in Chinese hickory.

Toe1 promotes proliferation and differentiation of neural progenitor cells

Toe1 (target of EGR1, member 1) is a 3′- exonuclease in the deadenylase family, essential for the maturation of small nuclear RNAs. Mutations in Toe1 are linked to pontocerebellar hypoplasia 7 (PCH7), a severe neurodegenerative syndrome affecting infants, characterized by progressive neurodegeneration, developmental delay, and genital abnormalities. The pathogenic mechanisms of PCH7 are unclear but are thought to involve abnormal neural stem cell (NSC) development during embryogenesis. This study investigates Toe1's role in NSC development using the C17.2 NSC line. Colony formation, EdU incorporation, and CFSE staining assays showed that Toe1 knockout inhibited C17.2 cell proliferation. Upon inducing differentiation, Toe1 knockout significantly reduced cell dendrites. Immunofluorescence, qPCR, and Western blot analyses indicated that Toe1 knockout suppressed the expression of neuronal marker βIII-tubulin and glial cell marker Gfap, thereby inhibiting C17.2 cell differentiation. Additionally, Toe1 knockout reduced the expression of Dll1 and Jag1, suggesting an inhibition of Notch signaling. High-throughput transcriptome sequencing revealed that Toe1 influenced calcium ion binding, ECM, and amino acid catabolism in undifferentiated C17.2 cells, and peptidase activity, chemotactic factors, ECM, and TNF signaling in differentiated cells. These findings underscore Toe1's critical regulatory role in NSC proliferation and differentiation, with significant implications for developing therapeutic targets for neurodegenerative diseases such as PCH7.

Progress in Research on Terpenoid Biosynthesis and Terpene Synthases of Lauraceae Species

Lauraceae, an important family of Angiospermae, comprises over 2500 species widely distributed in tropical and subtropical evergreen broad-leaved forests. This family is renowned for its rich resource of terpenoids, particularly monoterpenes, sesquiterpenes, and diterpenes. These compounds not only impart specific scents to Lauraceae species but also play crucial roles in plant growth, development, and environmental adaptation. These compounds also possess extensive bioactivities, such as antioxidant, antibacterial, anti-inflammatory, and neuroprotective effects, making them valuable in the fields of perfumery, cosmetics, food, and medicine, and thus holding significant economic value. Recent advancements in high-throughput technologies, especially genomics, transcriptomics, and metabolomics, have significantly advanced our knowledge of the chemical constituents and biosynthetic pathways of terpenoids in Lauraceae species. Such progress has also shed light on the diversity and functionality of the terpene synthases (TPSs) gene family, a key enzyme involved in terpenoid biosynthesis. This paper reviews the latest research findings on the biosynthetic pathways of terpenoids and their key enzyme-encoding gene families in Lauraceae plants. We also analyze the evolutionary patterns of TPS gene family members of four Lauraceae species at the whole-genome level and summarize their mechanisms of action in secondary metabolite synthesis. Furthermore, this paper highlights the current research challenges and proposes prospects, such as the complexity of gene families, the uncertainties in functional predictions, and unclear regulatory mechanisms. Our objective is to provide scientific foundations for the in-depth analysis of terpenoid biosynthesis mechanisms and the development and utilization of natural products in Lauraceae plants.

Bulk RNA Barcode Sequencing Reveals Role of RNA Splicing in Aging Dermal Stem Cell Modulation by a Botanical Extract

Skin aging is a complex, multifaceted process influenced by both intrinsic and extrinsic factors. Understanding the molecular mechanisms underlying skin aging is crucial for developing effective anti-aging strategies. Dermal stem cells play a pivotal role in maintaining skin homeostasis, but their functionality is compromised with aging. This study investigated the impact of aging on dermal stem cells and explored the potential of natural extracts in modulating their biological characteristics. Using bulk RNA barcoding and sequencing (BRB-seq), we identified differentially expressed genes (DEGs) between young and aged dermal stem cells, revealing alterations in cellular processes, including cell proliferation, ECM synthesis, and RNA splicing. We also demonstrated that a natural extract, comprising callus cells and Alpine rose leaf extracts, influenced RNA splicing in aged dermal stem cells, leading to improved dermal structure and integrity in vitro. Our findings suggest that natural extracts may exert their effects through senolytic activity and the modulation of RNA splicing, a process crucial to gene expression and cellular function. This study underscores the potential of integrating high-throughput transcriptomics in understanding skin aging, presenting new avenues for the development of innovative, sustainable, and effective anti-aging strategies.

A novel spherical GelMA-HAMA hydrogel encapsulating APET×2 polypeptide and CFIm25-targeting sgRNA for immune microenvironment modulation and nucleus pulposus regeneration in intervertebral discs

MethodsSingle-cell transcriptomics and high-throughput transcriptomics were used to screen factors significantly correlated with intervertebral disc degeneration (IDD). Expression changes of CFIm25 were determined via RT-qPCR and Western blot. NP cells were isolated from mouse intervertebral discs and induced to degrade with TNF-α and IL-1β. CFIm25 was knocked out using CRISPR-Cas9, and CFIm25 knockout and overexpressing nucleus pulposus (NP) cell lines were generated through lentiviral transfection. Proteoglycan expression, protein expression, inflammatory factor expression, cell viability, proliferation, migration, gene expression, and protein expression were analyzed using various assays (alcian blue staining, immunofluorescence, ELISA, CCK-8, EDU labeling, transwell migration, scratch assay, RT-qPCR, Western blot). The GelMA-HAMA hydrogel loaded with APET×2 polypeptide and sgRNA was designed, and its effects on NP regeneration were assessed through in vitro and mouse model experiments. The progression of IDD in mice was evaluated using X-ray, H&E staining, and Safranin O-Fast Green staining. Immunohistochemistry was performed to determine protein expression in NP tissue. Proteomic analysis combined with in vitro and in vivo experiments was conducted to elucidate the mechanisms of hydrogel action.ResultsCFIm25 was upregulated in IDD NP tissue and significantly correlated with disease progression. Inhibition of CFIm25 improved NP cell degeneration, enhanced cell proliferation, and migration. The hydrogel effectively knocked down CFIm25 expression, improved NP cell degeneration, promoted cell proliferation and migration, and mitigated IDD progression in a mouse model. The hydrogel inhibited inflammatory factor expression (IL-6, iNOS, IL-1β, TNF-α) by targeting the p38/NF-κB signaling pathway, increased collagen COLII and proteoglycan Aggrecan expression, and suppressed NP degeneration-related factors (COX-2, MMP-3).ConclusionThe study highlighted the crucial role of CFIm25 in IDD and introduced a promising therapeutic strategy using a porous spherical GelMA-HAMA hydrogel loaded with APET×2 polypeptide and sgRNA. This innovative approach offers new possibilities for treating degenerated intervertebral discs.Graphical Molecular mechanism of GCA targeting CFIm25 in NP cells to promote regeneration of degenerated intervertebral disc NP

Engineering exosomes derived from TNF-α preconditioned IPFP-MSCs enhance both yield and therapeutic efficacy for osteoarthritis

BackgroundThe pathogenesis of osteoarthritis (OA) involves the progressive degradation of articular cartilage. Exosomes derived from mesenchymal stem cells (MSC-EXOs) have been shown to mitigate joint pathological injury by attenuating cartilage destruction. Optimization the yield and therapeutic efficacy of exosomes derived from MSCs is crucial for promoting their clinical translation. The preconditioning of MSCs enhances the therapeutic potential of engineered exosomes, offering promising prospects for application by enabling controlled and quantifiable external stimulation. This study aims to address these issues by employing pro-inflammatory preconditioning of MSCs to enhance exosome production and augment their therapeutic efficacy for OA.MethodsThe exosomes were isolated from the supernatant of infrapatellar fat pad (IPFP)-MSCs preconditioned with a pro-inflammatory factor, TNF-α, and their production was subsequently quantified. The exosome secretion-related pathways in IPFP-MSCs were evaluated through high-throughput transcriptome sequencing analysis, q-PCR and western blot analysis before and after TNF-α preconditioning. Furthermore, exosomes derived from TNF-α preconditioned IPFP-MSCs (IPFP-MSC-EXOsTNF−α) were administered intra-articularly in an OA mouse model, and subsequent evaluations were conducted to assess joint pathology and gait alterations. The expression of proteins involved in the maintenance of cartilage homeostasis within the exosomes was determined through proteomic analysis.ResultsThe preconditioning with TNF-α significantly enhanced the exosome secretion of IPFP-MSCs compared to unpreconditioned MSCs. The potential mechanism involved the activation of the PI3K/AKT signaling pathway in IPFP-MSCs by TNF-α precondition, leading to an up-regulation of autophagy-related protein 16 like 1(ATG16L1) levels, which subsequently facilitated exosome secretion. The intra-articular administration of IPFP-MSC-EXOsTNF−α demonstrated superior efficacy in ameliorating pathological changes in the joints of OA mice. The preconditioning of TNF-α enhanced the up-regulation of low-density lipoprotein receptor-related protein 1 (LRP1) levels in IPFP-MSC-EXOsTNF−α, thereby exerting chondroprotective effects.ConclusionTNF-α preconditioning constitutes an effective and promising method for optimizing the therapeutic effects of IPFP-MSCs derived exosomes in the treatment of OA.Graphical

Unveiling osmoregulation and immunological adaptations in Eleutheronema tetradactylum gills through high-throughput single-cell transcriptome sequencing

The fourfinger threadfin fish (Eleutheronema tetradactylum) is an economically significant species renowned for its ability to adapt to varying salinity environments, with gills serving as their primary organs for osmoregulation and immune defense. Previous studies focused on tissue and morphological levels, whereas ignored the cellular heterogeneity and the crucial gene information related to core cell subsets within E. tetradactylum gills. In this study, we utilized high-throughput single-cell RNA sequencing (scRNA-seq) to analyze the gills of E. tetradactylum, characterizing 16 distinct cell types and identifying unique gene markers and enriched functions associated within each cell type. Additionally, we subdivided ionocyte cells into four distinct subpopulations for the first time in E. tetradactylum gills. By employing weighted gene co-expression network analysis (WGCNA), we further investigated the cellular heterogeneity and specific response mechanisms to salinity fluctuant. Our findings revealed the intricate osmoregulation and immune functions of gill cells, highlighting their crucial roles in maintaining homeostasis and adapting to fluctuating salinity levels. This comprehensive cell-type atlas provides valuable insights into the species adaptive strategies, contributing to the conservation and management of this commercially significant fish as well as other euryhaline species.

ToxDAR: A Workflow Software for Analyzing Toxicologically Relevant Proteomic and Transcriptomic Data, from Data Preparation to Toxicological Mechanism Elucidation.

Exploration of toxicological mechanisms is imperative for the assessment of potential adverse reactions to chemicals and pharmaceutical agents, the engineering of safer compounds, and the preservation of public health. It forms the foundation of drug development and disease treatment. High-throughput proteomics and transcriptomics can accurately capture the body's response to toxins and have become key tools for revealing complex toxicological mechanisms. Recently, a vast amount of omics data related to toxicological mechanisms have been accumulated. However, analyzing and utilizing these data remains a major challenge for researchers, especially as there is a lack of a knowledge-based analysis system to identify relevant biological pathways associated with toxicity from the data and to establish connections between omics data and existing toxicological knowledge. To address this, we have developed ToxDAR, a workflow-oriented R package for preprocessing and analyzing toxicological multi-omics data. ToxDAR integrates packages like NormExpression, DESeq2, and igraph, and utilizes R functions such as prcomp and phyper. It supports data preparation, quality control, differential expression analysis, functional analysis, and network analysis. ToxDAR's architecture also includes a knowledge graph with five major categories of mechanism-related biological entities and details fifteen types of interactions among them, providing comprehensive knowledge annotation for omics data analysis results. As a case study, we used ToxDAR to analyze a transcriptomic dataset on the toxicology of triphenyl phosphate (TPP). The results indicate that TPP may impair thyroid function by activating thyroid hormone receptor β (THRB), impacting pathways related to programmed cell death and inflammation. As a workflow-oriented data analysis tool, ToxDAR is expected to be crucial for understanding toxic mechanisms from omics data, discovering new therapeutic targets, and evaluating chemical safety.

Silver Nanoparticles Exposure Impairs Cardiac Development by Suppressing the Focal Adhesion Pathway in Zebrafish.

The potential toxic effects of wastewater discharges containing silver nanoparticles (AgNPs) and their release into aquatic ecosystems on aquatic organisms are becoming a major concern for environmental and human health. However, the potential risks of AgNPs to aquatic organisms, especially for cardiac development by Focal adhesion pathway, are still poorly understood. The cardiac development of various concentrations of AgNPs in zebrafish were examined using stereoscopic microscope. The expression levels of cardiac development-related genes were analyzed by qRT-PCR and Whole-mount in situ hybridization (WISH). In addition, Illumina high-throughput global transcriptome analysis was performed to explore the potential signaling pathway involved in the treatment of zebrafish embryos by AgNPs after 72 h. We systematically investigated the cardiac developing toxicity of AgNPs on the embryos of zebrafish. The results demonstrated that 2 or 4 mg/L AgNPs exposure induces cardiac developmental malformations, such as the appearance of pericardial edema phenotype. In addition, after 72 h of exposure, the mRNA levels of cardiac development-related genes, such as myh7, myh6, tpm1, nppa, tbx5, tbx20, myl7 and cmlc1, were significantly lower in AgNPs-treated zebrafish embryos than in control zebrafish embryos. Moreover, RNA sequencing, KEGG (Kyoto Encyclopedia of Genes) and Genomes and GSEA (gene set enrichment analysis) of the DEGs (differentially expressed genes) between the AgNPs-exposed and control groups indicated that the downregulated DEGs were mainly enriched in focal adhesion pathways. Further investigations demonstrated that the mRNA levels of focal adhesion pathway-related genes, such as igf1ra, shc3, grb2b, ptk2aa, akt1, itga4, parvaa, akt3b and vcla, were significantly decreased after AgNPs treatment in zebrafish. Thus, our findings illustrated that AgNPs could impair cardiac development by regulating the focal adhesion pathway in zebrafish.

Long Non-coding RNAs Influence Aging Process of Sciatic Nerves in SD Rats.

To investigate the long non-coding RNAs (lncRNAs) changes in the sciatic nerve (SN) in Sprague Dawley (SD) rats during aging. Eighteen healthy SD rats were selected at the age of 1 month (1M) and 24 months (24M) and SNs were collected. High-throughput transcriptome sequencing and bioinformatics analysis were performed. Protein-protein interaction (PPI) networks and competing endogenous RNA (ceRNA) networks were established according to differentially expressed genes (DEGs). As the length of lncRNAs increased, its proportion to the total number of lncRNAs decreased. A total of 4079 DElncRNAs were identified in Con vs. 24M. GO analysis was primarily clustered in nerve and lipid metabolism, extracellular matrix, and vascularization-related fields. There were 17 nodes in the PPI network of the target genes of up-regulating genes including Itgb2, Lox, Col11a1, Wnt5a, Kras, etc. Using quantitative RT-PCR, microarray sequencing accuracy was validated. There were 169 nodes constructing the PPI network of down-regulated target genes, mainly including Col1a1, Hmgcs1, Hmgcr. CeRNA interaction networks were constructed. Lipid metabolism, angiogenesis, and ECM fields might play an important role in the senescence process in SNs. Col3a1, Serpinh1, Hmgcr, and Fdps could be candidates for nerve aging research.