RNA-seq Technology Research Articles

IDENTIFICATION OF TARGET GENES INVOLVED IN RESPONSES OF Populus TO POLYCHLORINATED BIPHENYLS BASED ON RNA-SEQ

Steel slag and rice husk bokashi are the ameliorant that expected can decrease P-retention Phytoremediation represents an effective, low-cost, environmentally friendly alternative that improves soil properties, although there have been significant developments in the last decade. From the scientific point of life, the main challenge is to decipher the metabolic pathways involved in response to contaminants and to understand their regulation. This information is essential if we aspire to improve the natural capacities of some plant species to remediate contaminated soils. A comprehensive study of the molecular response to polychlorinated biphenyls (PCBs), a family of persistent organic pollutants of particular relevance on a global scale, was obtained. A transcriptomic approach based on RNA-seq technology has been used to identify the genes involved in the metabolism of in-plant compounds and to quantify their activation levels in different controlled situations. It is surprising that this response is virtually unknown at the molecular level, despite its great potential applied in the context of phytoremediation technology. We apply to hybrid poplar (Populus tremula x alba) crops with Aroclor 1221. And we took samples of RNA, thus generating a matrix of four elements with their corresponding controls. On the other hand, we perform functional analyzes with bioinformatic tools based on sequence comparisons and gene co-expression networks. The response of genes of particular interest was validated using qRT-PCR technology. This is the first comprehensive study of the response of a plant organism to the presence of PCBs. A considerable number of structural and regulatory genes have been found, defining new transcription factors whose expression is proportional to the concentration of pollutant in the medium or the time of exposure to it. Correlation analyzes allow us to state that the metabolic response to PCBs, including possible degrading pathways, is involved in at least fifteen transcription factors and approximately forty proteins or enzymes that are particularly induced.

Transcriptome profiling of Nile tilapia (Oreochromis niloticus) identifies candidate genes in response to riverine pollution

To delineate the response mechanism of Nile tilapia (Oreochromis niloticus) to the riverine pollution of river Ganga, a comparative transcriptomic analysis was performed utilizing fresh liver through RNA-Seq technology. A total of 51.39 million and 32.8 million reads were obtained after excluding low quality sequences from non-polluted (Barrackpore) and polluted (Kanpur) sites of Nile tilapia. About 81.4 % and 95.3% reads were perfectly mapped with the reference sequence of O. niloticus. Transcriptional analysis generated 363 differential expressed genes (DEGs) including 131 up-regulated and 232 down-regulated genes. Gene Ontology (GO) enrichment analysis revealed that significant DEGs were associated with ribosome biogenesis, alpha-amino acid metabolic process, translational initiation etc. as biological process (BP); unfolded protein binding, vitamin binding, carboxylic acid binding and etc. as molecular function (MF); ribosome, and ribosomal subunit as cellular component (CC). The KEGG analysis indicated that these DEGs were highly involved in ribosome, Lysine degradation and RNA transport pathways. Additionally, ten hub genes participated in Translation, Ribonucleoprotein complex biogenesis as BP, Ribosome, Eukaryotic translation initiation factor 3 complex as CC and Structural constituent of ribosome, RNA binding as MF were affected in riverine pollution. Overall, this transcriptome investigation provided an extensive overview of pollution triggered transcriptional mechanisms in Tilapia liver and would be highly significant for further exploration of the molecular processes in response to pollution.

Elucidation of mechanisms underlying active oxygen burst in Citrus sinensis after Diaporthe citri infection using transcriptome analysis.

Reactive oxygen species (ROS) generation is a common disease defense mechanism in plants. However, it is unclear whether Citrus host activates defense response against Diaporthe citri causing citrus melanose disease by producing ROS, and the underlying molecular mechanisms are unknown. DAB staining and RNA-Seq technology were used to compare the active oxygen burst and differential gene expression, respectively, in uninfected and infected Citrus sinensis leaves at different time points during D. citri infection in vivo. The functions of CsRBOH (a significant DEG) were confirmed in N. benthamiana through the Agrobacterium-mediated transient expression system. DAB staining indicated that C. sinensis initiated defense against D. citri infection within 24 h by generating ROS. Illumina sequencing revealed 25,557 expressed genes of C. sinensis. The most upregulated DEGs (n = 1,570) were identified 72 h after fungal inoculation (sample denoted as CD72). In the CD72 vs. Cs (samples at 0 h after fungal inoculation) comparison, the KEGG pathway category with the highest number of genes (n = 62) and most significant enrichment was Protein processing in endoplasmic reticulum, followed by Glutathione metabolism and MAPK signaling pathway-plant. GO analysis revealed that the DEGs of CD72 vs. Cs related to active oxygen burst and chitin recognition were significantly grouped into the regulation of biological processes and molecular functions, with GO terms including response to ROS, response to fungus, and oxidoreductase activity. Remarkably, CsRBOH was significantly enriched in the GO and KEGG analyses, and its expression pattern in qRT-PCR and DAB staining results were consistent. Among the 63 ROS-related DEGs, HSP genes and genes associated with the peroxidase family were highly significant as revealed by protein-protein interaction networks. Furthermore, ROS accumulation, cell death, and upregulation of defense-related genes were observed in N. benthamiana leaves with CsRBOH expressed through the Agrobacterium-mediated transient expression system. Our findings suggested that C. sinensis activates CsRBOH and ROS-related genes, leading to ROS accumulation to resist the invasion by D. citri. This study laid the foundation for future research on molecular mechanisms and breeding of C. sinensis cultivars resistant to citrus melanose.

Gubi Zhitong formula alleviates osteoarthritis in vitro and in vivo via regulating BNIP3L-mediated mitophagy

BackgroundOsteoarthritis (OA) is characterized by degeneration of articular cartilage, leading to joint pain and dysfunction. Gubi Zhitong formula (GBZTF), a traditional Chinese medicine formula, has been used in the clinical treatment of OA for decades, demonstrating definite efficacy. However, its mechanism of action remains unclear, hindering its further application. MethodsThe ingredients of GBZTF were analyzed and performed with liquid chromatography-mass spectrometry (LC-MS). 6 weeks old SD rats were underwent running exercise (25 m/min, 80 min, 0°) to construct OA model with cartilage wear and tear. It was estimated by Micro-CT, Gait Analysis, Histological Stain. RNA-seq technology was performed with OA Rats' cartilage, and primary chondrocytes induced by IL-1β (mimics OA chondrocytes) were utilized to evaluated and investigated the mechanism of how GBZTF protected OA cartilage from being damaged with some functional experiments. ResultsA total of 1006 compounds were identified under positive and negative ion modes by LC-MS. Then, we assessed the function of GBZTF through in vitro and vivo. It was found GBZTF could significantly up-regulate OA rats’ limb coordination and weight-bearing capacity, and reduce the surface and sub-chondral bone erosions of OA joints, and protect cartilage from being destroyed by inflammatory factors (iNOS, IL-6, IL-1β, TNF- α, MMP13, ADAMTS5), and promote OA chondrocytes proliferation and increase the S phage of cell cycle. In terms of mechanism, RNA-seq analysis of cartilage tissues revealed 1,778 and 3,824 differentially expressed genes (DEGs) in model vs control group and GBZTF vs model group, respectively. The mitophagy pathway was most significantly enriched in these DEGs. Further results of subunits of OA chondrocytes confirmed that GBZTF could alleviate OA-associated inflammation and cartilage damage through modulation BCL2 interacting protein 3-like (BNIP3L)-mediated mitophagy. ConclusionThe therapeutic effectiveness of GBZTF on OA were first time verified in vivo and vitro through functional experiments and RNA-seq, which provides convincing evidence to support the molecular mechanisms of GBZTF as a promising therapeutic decoction for OA.

Dynamics of alternative polyadenylation in single root cells of Arabidopsis thaliana.

Single-cell RNA-seq (scRNA-seq) technologies have been widely used to reveal the diversity and complexity of cells, and pioneering studies on scRNA-seq in plants began to emerge since 2019. However, existing studies on plants utilized scRNA-seq focused only on the gene expression regulation. As an essential post-transcriptional mechanism for regulating gene expression, alternative polyadenylation (APA) generates diverse mRNA isoforms with distinct 3' ends through the selective use of different polyadenylation sites in a gene. APA plays important roles in regulating multiple developmental processes in plants, such as flowering time and stress response. In this study, we developed a pipeline to identify and integrate APA sites from different scRNA-seq data and analyze APA dynamics in single cells. First, high-confidence poly(A) sites in single root cells were identified and quantified. Second, three kinds of APA markers were identified for exploring APA dynamics in single cells, including differentially expressed poly(A) sites based on APA site expression, APA markers based on APA usages, and APA switching genes based on 3' UTR (untranslated region) length change. Moreover, cell type annotations of single root cells were refined by integrating both the APA information and the gene expression profile. We comprehensively compiled a single-cell APA atlas from five scRNA-seq studies, covering over 150,000 cells spanning four major tissue branches, twelve cell types, and three developmental stages. Moreover, we quantified the dynamic APA usages in single cells and identified APA markers across tissues and cell types. Further, we integrated complementary information of gene expression and APA profiles to annotate cell types and reveal subtle differences between cell types. This study reveals that APA provides an additional layer of information for determining cell identity and provides a landscape of APA dynamics during Arabidopsis root development.

Single cell proteomics analysis of drug response shows its potential as a drug discovery platform.

Single-cell analysis has clearly established itself in biology and biomedical fields as an invaluable tool that allows one to comprehensively understand the relationship between cells, including their types, states, transitions, trajectories, and spatial position. Scientific methods such as fluorescence labeling, nanoscale super-resolution microscopy, advances in single cell RNAseq and proteomics technologies, provide more detailed information about biological processes which were not evident with the analysis of bulk material. This new era of single-cell biology provides a better understanding of such complex biological systems as cancer, inflammation, immunity mechanism and aging processes, and opens the door into the field of drug response heterogeneity. The latest discoveries of cellular heterogeneity gives us a unique understanding of complex biological processes, such as disease mechanism, and will lead to new strategies for better and personalized treatment strategies. Recently, single-cell proteomics techniques that allow quantification of thousands of proteins from single mammalian cells have been introduced. Here we present an improved single-cell mass spectrometry-based proteomics platform called SCREEN (Single Cell pRotEomE aNalysis) for deep and high-throughput single-cell proteome coverage with high efficiency, less turnaround time and with an improved ability for protein quantitation across more cells than previously achieved. We applied this new platform to analyze the single-cell proteomic landscape under different drug treatment over time to uncover heterogeneity in cancer cell response, which for the first time, to our knowledge, has been achieved by mass spectrometry based analytical methods. We discuss challenges in single-cell proteomics, future improvements and general trends with the goal to encourage forthcoming technical developments.

Integration of full-length Iso-Seq, Illumina RNA-Seq, and flavor testing reveals potential differences in ripened fruits between two Passiflora edulis cultivars.

Passion fruit (Passiflora edulis) is loved for its delicious flavor and nutritious juice. Although studies have delved into the cultivation and enhancement of passion fruit varieties, the underlying factors contributing to the fruit's appealing aroma remain unclear. This study analyzed the full-length transcriptomes of two passion fruit cultivars with different flavor profiles: "Tainong 1" (TN1), known for its superior fruit flavor, and "Guihan 1" (GH1), noted for its strong environmental resilience but lackluster taste. Utilizing PacBio Iso-Seq and Illumina RNA-Seq technologies, we discovered terpene synthase (TPS) genes implicated in fruit ripening that may help explain the flavor disparities. We generated 15,913 isoforms, with N50 lengths of 1,500 and 1,648 bp, and mean lengths of 1,319 and 1,463 bp for TN1 and GH1, respectively. Transcript and isoform lengths ranged from a maximum of 7,779 bp to a minimum of 200 and 209 bp. We identified 14,822 putative coding DNA sequences (CDSs) averaging 1,063 bp, classified 1,007 transcription factors (TFs) into 84 families. Additionally, differential expression analysis of ripening fruit from both cultivars revealed 314 upregulated and 43 downregulated unigenes in TN1 compared to GH1. The top 10 significantly enriched Gene Ontology (GO) terms for the differentially expressed genes (DEGs) indicated that TN1's upregulated genes were primarily involved in nutrient transport, whereas GH1's up-regulated genes were associated with resistance mechanisms. Meanwhile, 17 PeTPS genes were identified in P. edulis and 13 of them were TPS-b members. A comparative analysis when compared PeTPS with AtTPS highlighted an expansion of the PeTPS-b subfamily in P. edulis, suggesting a role in its fruit flavor profile. Our findings explain that the formation of fruit flavor is attributed to the upregulation of essential genes in synthetic pathway, in particular the expansion of TPS-b subfamily involved in terpenoid synthesis. This finding will also provide a foundational genetic basis for understanding the nuanced flavor differences in this species.

Efficient and Powerful Integration of Targeted Metabolomics and Transcriptomics for Analyzing the Metabolism Behind Desirable Traits in Plants.

Through current mass spectrometry methods and multiple RNA-Seq technologies, large metabolomics and transcriptomics datasets are readily obtainable, which provide a powerful and global perspective on metabolism. Indeed, one "omics" method is often not enough to draw strong conclusions about metabolism. Combining and interpreting multiple "omics" datasets remains a challenging task that requires careful statistical considerations and pre-planning. Here we describe a protocol for obtaining high-quality metabolomics and transcriptomics datasets in developing plant embryos followed by a robust approach to integration of the two. This protocol is readily adjustable and scalable to any other metabolically active organ or tissue.

Omics-Based Investigation on Mechanisms Controlling Cellular Internalization of Keratin Monomers during Biodegradation by Stenotrophomonas maltophilia DHHJ.

The global poultry industry produces millions of tons of waste feathers every year, which can be bio-degraded to make feed, fertilizer, and daily chemicals. However, feather bio-degradation is a complex process that is not yet fully understood. This results in low degradation efficiency and difficulty in industrial applications. Omics-driven system biology research offers an effective solution to quickly and comprehensively understand the molecularmechanisms involved in a metabolic pathway. In the early stage of this process, feathers are hydrolyzed into water-soluble keratin monomers. In this study, we used high-throughput RNA-seq technology to analyze the genes involved in the internalization and degradation of keratin monomers in Stenotrophomonas maltophilia DHHJ strain cells. Moreover, we used Co-IP with LC-MS/MS technology to search for proteins that interact with recombinant keratin monomers. We discovered TonB transports and molecular chaperones associating with the keratin monomer, which may play a crucial role in the transmembrane transport of keratin. Meanwhile, multiple proteases belonging to distinct families were identified as binding partners of keratin monomers, among which ATPases associated with diverse cellular activity (AAA+) family proteases are overrepresented. Four genes, including JJL50_15620, JJL50_17955 (TonB-dependent receptors), JJL50_03260 (ABC transporter ATP-binding protein), and JJL50_20035 (ABC transporter substrate-binding protein), were selected as representatives for determining their expressions under different culture conditions using qRT-PCR, and they were found to be upregulated in response to keratin degradation consistent with the data from RNA-seq and Co-IP. This study highlights the complexity of keratin biodegradation in S. maltophilia DHHJ, in which multiple pathways are involved such as protein folding, protein transport, and several protease systems. Our findings provide new insights into the mechanism of feather degradation.

Dual RNA-seq Analysis of Patients' Cells and Viral Genome After Measles Infection.

During the infection of a host cell by an infectious agent, a series of gene expression changes occurs as a consequence of host-pathogen interactions. Unraveling this complex interplay is the key for understanding of microbial virulence and host response pathways, thus providing the basis for new molecular insights into the mechanisms of pathogenesis and the corresponding immune response. Dual RNA sequencing (dual RNA-seq) has been developed to simultaneously determine pathogen and host transcriptomes enabling both differential and coexpression analyses between the two partners as well as genome characterization in the case of RNA viruses. Here, we provide a detailed laboratory protocol and bioinformatics analysis guidelines for dual RNA-seq experiments focusing on - but not restricted to - measles virus (MeV) as a pathogen of interest. The application of dual RNA-seq technologies in MeV-infected patients can potentially provide valuable information on the structure of the viral RNA genome and on cellular innate immune responses and drive the discovery of new targets for antiviral therapy.

Effects of Organic Selenium and Nanoselenium on Drought Stress of Pak Choi (Brassica chinensis var. pekinensis. cv. ‘Suzhouqing’) and Its Transcriptomic Analysis

In order to investigate physiological and molecular mechanisms underlying the impacts of organic selenium and nanoselenium on the drought stress of pak choi (Brassica chinensis var. pekinensis. cv. ‘Suzhouqing’), we sprayed 20 mL of 0, 5, 10, 20 mg L−1 concentrations of organic selenium (DCK, DO5, DO10, DO20) and nanoselenium (DN5, DN10, DN20). We measured photosynthetic pigment, antioxidant and nutritional quality, performed transcriptome sequencing of leaves using RNA-seq technology, obtained 297,176,114 clean sequences, and identified differential expressed genes. Organic selenium and nanoselenium increased the photosynthetic capacity by upregulating light-trapping pigment proteins, regulated reactive oxygen species homeostasis by activating the antioxidant system through the upregulation of glutathione S-transferase, and maintained water homeostasis through glutathione oxidase activity, thus enhancing weight. They contributed to the elevated total selenium content by increasing expression levels of ABC transporter, adenylate kinase, and cysteine desulphurase. Additionally, hub genes related to these metabolic pathways were identified using a weighted gene coexpression network analysis. The consistent results with the upregulated genes identified in the Kyoto Encyclopedia of Genes and Genomes pathway confirmed the accuracy and reliability of the transcriptome sequencing data. This study provides scientific reference to further in-depth research on the molecular effect of selenium on crops under drought stress conditions.

A Physiogenomic Study of the Tolerance of Saccharomyces cerevisiae to Isoamyl Alcohol

Isoamyl alcohol is a clear, unpleasantly odorous, colorless liquid of higher alcohol that emits a fruity aroma when heavily diluted. It has received much attention in recent years as a new fuel with a high energy density. Isoamyl alcohol can be produced industrially by microbial fermentation. Still, its toxicity to host cells has limited its potential for industrial production, and the molecular mechanism of its toxic effects has not yet been elucidated. In this study, RNA-Seq technology was used to analyze the transcripts of Saccharomyces cerevisiae under normal conditions and in the presence of isoamyl alcohol (0.5 g/L and 2.5 g/L). The results showed that the expression of the cell wall (CCW12, BGL2, NCW2 and SUN4), cell membrane (ELO1, ERG2, FAA1, and OPI3), translation and other structural genes were significantly down-regulated. The expression of genes related to ATP biosynthesis, NADPH biosynthesis (ZWF1), and metal ion transport (PMC1) proteins were up-regulated. Strains with key genes knocked out were cultured without isoamyl alcohol. Combined results suggested that isoamyl alcohol may affect cell wall stability and cell membrane fluidity, and the expression of genes related to ion homeostasis and energy production may play a protective role against isoamyl alcohol stress. By maintaining cell wall stability/membrane fluidity under isoamyl alcohol pressure, improving certain ion homeostasis, and generating energy/NADPH, it is possible to overcome the toxicity of isoamyl alcohol in industrial fermentation processes to a certain extent.

Analysis of transcriptome differences between subcutaneous and intramuscular adipose tissue of Ningxiang pigs.

To compare and analyze the molecular mechanisms of adipose deposition in subcutaneous fat (SAF)and intramuscular fat (IMF) tissues in Ningxiang pigs, differential gene expression profiles in SAF and IMF tissues of Ningxiang pigs were identified and analysed using RNA-seq technology. Six healthy 250-day-old male Ningxiang pigs with similar body weights (approximately 85 kg) of intraspecific individuals were selected as experimental material and samples of SAF and IMF tissues were collected. Differential genes associated with fat deposition and lipid metabolism were obtained by sequencing two adipose tissue transcriptomes and performing GO (Gene Ontology) functional annotation and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analysis. To verify the reliability of the sequencing results, six differential genes were randomly selected to validate using qRT-PCR. The results showed that we identified 2406 DEGs, with 1422 up-regulated and 984 down-regulated genes in two tissues. GO functional annotation analysis revealed that the differentially expressed genes were mainly involved in lipid metabolism related pathways, such as steroid biosynthesis, unsaturated fatty acid biosynthesis, glycerophospholipid metabolism and autophagy pathway. KEGG pathway enrichment showed that the differentially expressed genes were mainly enriched in the biological processes related to lipid binding, fatty acid metabolism, glycol ester metabolism, lipid biosynthesis and other biological processes related to lipid metabolism. Genes related to lipid metabolism, such as TCAP, NR4A1, ACACA, LPL, ELOVL6, DGAT1, PRKAA1, ATG101, TP53INP2, FDFT1, ACOX1 and SCD were identified by bioinformatic analyses and verified by qRT-PCR. Our results indicated that these genes may play important roles in the regulation of fat deposition and metabolism in the SAF and IMF tissue, providing the further mechanistic investigation of fat deposition in Ningxiang pigs.

Transcriptome studies of congenital heart diseases: identifying current gaps and therapeutic frontiers.

Congenital heart disease (CHD) are genetically complex and comprise a wide range of structural defects that often predispose to - early heart failure, a common cause of neonatal morbidity and mortality. Transcriptome studies of CHD in human pediatric patients indicated a broad spectrum of diverse molecular signatures across various types of CHD. In order to advance research on congenital heart diseases (CHDs), we conducted a detailed review of transcriptome studies on this topic. Our analysis identified gaps in the literature, with a particular focus on the cardiac transcriptome signatures found in various biological specimens across different types of CHDs. In addition to translational studies involving human subjects, we also examined transcriptomic analyses of CHDs in a range of model systems, including iPSCs and animal models. We concluded that RNA-seq technology has revolutionized medical research and many of the discoveries from CHD transcriptome studies draw attention to biological pathways that concurrently open the door to a better understanding of cardiac development and related therapeutic avenue. While some crucial impediments to perfectly studying CHDs in this context remain obtaining pediatric cardiac tissue samples, phenotypic variation, and the lack of anatomical/spatial context with model systems. Combining model systems, RNA-seq technology, and integrating algorithms for analyzing transcriptomic data at both single-cell and high throughput spatial resolution is expected to continue uncovering unique biological pathways that are perturbed in CHDs, thus facilitating the development of novel therapy for congenital heart disease.

Unraveling C-to-U RNA editing events from direct RNA sequencing

ABSTRACT In mammals, RNA editing events involve the conversion of adenosine (A) in inosine (I) by ADAR enzymes or the hydrolytic deamination of cytosine (C) in uracil (U) by the APOBEC family of enzymes, mostly APOBEC1. RNA editing has a plethora of biological functions, and its deregulation has been associated with various human disorders. While the large-scale detection of A-to-I is quite straightforward using the Illumina RNAseq technology, the identification of C-to-U events is a non-trivial task. This difficulty arises from the rarity of such events in eukaryotic genomes and the challenge of distinguishing them from background noise. Direct RNA sequencing by Oxford Nanopore Technology (ONT) permits the direct detection of Us on sequenced RNA reads. Surprisingly, using ONT reads from wild-type (WT) and APOBEC1-knock-out (KO) murine cell lines as well as in vitro synthesized RNA without any modification, we identified a systematic error affecting the accuracy of the Cs call, thereby leading to incorrect identifications of C-to-U events. To overcome this issue in direct RNA reads, here we introduce a novel machine learning strategy based on the isolation Forest (iForest) algorithm in which C-to-U editing events are considered as sequencing anomalies. Using in vitro synthesized and human ONT reads, our model optimizes the signal-to-noise ratio improving the detection of C-to-U editing sites with high accuracy, over 90% in all samples tested. Our results suggest that iForest, known for its rapid implementation and minimal memory requirements, is a promising tool to denoise ONT reads and reliably identify RNA modifications.

Structural Characterization and Anti-Inflammatory Activity of Polysaccharides from Tremella fuciformis on Monosodium Urate-Stimulated RAW264.7 Macrophages.

The structural characteristics and anti-inflammatory activity of Tremella fuciformis polysaccharides (TFPs) were investigated. The study showed that TFPs were mainly composed of mannose, rhamnose, glucuronic acid, glucose, galactose, xylose, and fucose. TFPs significantly inhibited monosodium urate (MSU)-induced inflammation of RAW264.7 cells, as well as the secretion levels of TNF-α, IL-1β, and IL-18 cytokines. The concentrations of malondialdehyde and reactive oxygen species in RAW264.7 macrophages were reduced, but superoxide dismutase activity was increased. RNA-Seq technology was applied to explore the mechanisms of TFPs ameliorating MSU-induced inflammation of RAW264.7 macrophages. Results revealed that TFPs significantly reduce MSU-stimulated inflammatory damage in RAW 264.7 cells by inhibiting signaling pathways like the hypoxia inducible factor-1 (HIF-1) signaling pathway and erythroblastic oncogene B (ErbB) signaling pathway. This study provides a foundation for TFPs to be developed as novel anti-inflammatory drugs.

Identification of HpMYB1 inducing anthocyanin accumulation in Hippeastrum Hybridum tepals by RNA-seq

BackgroundCultivated Hippeastrum × hybridum is a popular ornamental plant with large and colorful flowers, long flowering duration, and high commercial value. As its main ornamental feature, its flower color is related to the anthocyanin content in the tepals. However, the molecular regulatory mechanisms of anthocyanin biosynthesis in H. × hybridum have not yet been elucidated.ResultsIn the present study, 12 cDNA libraries of four stages of H.× hybridum ‘Royal Velvet’ tepal development were used for RNA-seq, obtaining 79.83 gigabases (GB) of clean data. The data were assembled into 148,453 unigenes, and 11,262 differentially expressed genes were identified. Forty key enzymes participating in anthocyanin biosynthesis were investigated, and the results showed that most of the anthocyanin structural genes were expressed at low levels in S1 and were markedly upregulated in S2 and S3. The expression profiles of 12 selected genes were verified by qRT-PCR. Furthermore, the R2R3-MYB transcription factor (TF), HpMYB1, involved in the regulation of anthocyanin biosynthesis was identified by sequence, expression pattern, and subcellular localization analyses. Its overexpression in tobacco significantly increased the anthocyanin levels in various tissues and activated anthocyanin-related genes.ConclusionsUsing RNA-seq technology, we successfully identified a potential R2R3-MYB gene, HpMYB1, that regulates anthocyanin biosynthesis in H.× hybridum ‘Royal Velvet’. Our findings provide basic transcript information and valuable transcriptome data for further identification of key genes involved in anthocyanin biosynthesis and can be applied in the artificial breeding of new H. × hybridum cultivars with enhanced ornamental value.

Identifying low salinity adaptation gene expression in the anterior and posterior gills of the mud crab (Scylla paramamosain) by transcriptomic analysis

In the present study, BGISEQ-500 RNA-Seq technology was adopted to investigate how Scylla paramamosain adapts to salinity tolerance at the molecular level and explores changes in gene expression linked to salinity adaptation following exposure to both low salinity (5 ‰) and standard salinity (23 ‰) conditions. A total of 1100 and 520 differentially expressed genes (DEGs) were identified in the anterior and posterior gills, respectively, and their corresponding expression patterns were visualized in volcano plots and a heatmap. Further analysis highlighted significant enrichment of well-established gene functional categories and signaling pathways, including those what associated with cellular stress response, ion transport, energy metabolism, amino acid metabolism, H2O transport, and physiological stress compensation. We also selected key DEGs within the anterior and posterior gills that encode pivotal stress adaptation and tolerance modulators, including AQP, ABCA1, HSP 10, A35, CAg, NKA, VPA, CAc, and SPS. Interestingly, A35 in the gills might regulate osmolality by binding CHH in response to low salinity stress or serve as a mechanism for energy compensation. Taken together, our findings elucidated the intricate molecular mechanism employed by S. paramamosain for salinity adaptation, which involved distinct gene expression patterns in the anterior and posterior gills. These findings provide the foothold for subsequent investigations into salinity-responsive candidate genes and contribute to a deeper understanding of S. paramamosain's adaptation mechanisms in low-salinity surroundings, which is crucial for the development of low-salinity species cultivation and the establishment of a robust culture model.

Combining a lung microfluidic chip exposure model with transcriptomic analysis to evaluate the inflammation in BEAS-2B cells exposed to cigarette smoke

BackgroundTypically, in vitro studies on the exposure of complex gaseous substances are performed in multi-well plate experiments by trapping and redissolving them, which could introduce potential bias into the results due to the use of inadequate trapping methods. Therefore, a more effective method is to expose complex gaseous substances in gaseous form online, such as using microfluidic chips in experiments. To address these challenges, we introduce a methodology that integrates a self-designed bionic-lung chip with transcriptome analysis to assess the impact of cigarette smoke (CS) exposure on changes in BEAS-2B cells cultured on-chip. ResultsAfter the microfluidic chip underwent online gas exposure, total RNA was extracted via in situ cell lysis, and RNA-Seq transcriptome analysis was conducted. And the RNA-Seq findings revealed the significant involvement of the MAPK signaling pathway associated with the inflammatory response in the cellular effects induced by CS exposure. Moreover, the validation of inflammatory response-related biomarkers through in situ fluorescence corroborated the outcomes of the transcriptome analysis. Besides, the experiment involving the inhibition of inflammation by DEX on the microfluidic chip provided additional confirmation of the previous experimental findings. SignificantIn this study, we present an analytical strategy that combines microfluidic-based CS in situ exposure method with RNA-Seq technology. This strategy offers an experimental scheme for in situ exposure to complex gases, transcriptome analysis, and in situ fluorescence detection. Through the integration of the comprehensiveness of transcriptome analysis with the chip's direct and intuitive in situ fluorescence detection with the stability and reliability of RT-PCR and Western blot experiments, we have successfully addressed the challenges associated with in vitro risk assessment for online exposure to complex gaseous substances.

Time-Course Transcriptome Analysis of Aquilegia vulgaris Root Reveals the Cell Wall's Roles in Salinity Tolerance.

Salt stress has a considerable impact on the development and growth of plants. The soil is currently affected by salinisation, a problem that is becoming worse every year. This means that a significant amount of salt-tolerant plant material needs to be added. Aquilegia vulgaris has aesthetically pleasing leaves, unique flowers, and a remarkable tolerance to salt. In this study, RNA-seq technology was used to sequence and analyse the transcriptome of the root of Aquilegia vulgaris seedlings subjected to 200 mM NaCl treatment for 12, 24, and 48 h. In total, 12 Aquilegia vulgaris seedling root transcriptome libraries were constructed. At the three time points of salt treatment compared with the control, 3888, 1907, and 1479 differentially expressed genes (DEGs) were identified, respectively. Various families of transcription factors (TFs), mainly AP2, MYB, and bHLH, were identified and might be linked to salt tolerance. Gene Ontology (GO) analysis of DEGs revealed that the structure and composition of the cell wall and cytoskeleton may be crucial in the response to salt stress. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of the DEGs showed a significant enrichment of the pentose and glucuronate interconversion pathway, which is associated with cell wall metabolism after 24 and 48 h of salt treatment. Based on GO and KEGG analyses of DEGs, the pentose and glucuronate interconversion pathway was selected for further investigation. AP2, MYB, and bHLH were found to be correlated with the functional genes in this pathway based on a correlation network. This study provides the groundwork for understanding the key pathways and gene networks in response to salt stress, thereby providing a theoretical basis for improving salt tolerance in Aquilegia vulgaris.