Transcriptome Libraries Research Articles

CCKBR+ cancer cells contribute to the intratumor heterogeneity of gastric cancer and confer sensitivity to FOXO inhibition.

The existence of heterogeneity has plunged cancer treatment into a challenging dilemma. We profiled malignant epithelial cells from 5 gastric adenocarcinoma patients through single-cell sequencing (scRNA-seq) analysis, demonstrating the heterogeneity of gastric adenocarcinoma (GA), and identified the CCKBR+ stem cell-like cancer cells associated poorly differentiated and worse prognosis. We further conducted targeted analysis using single-cell transcriptome libraries, including 40 samples, to confirm these screening results. In addition, we revealed that FOXOs are involved in the progression and development of CCKBR+ gastric adenocarcinoma. Inhibited the expression of FOXOs and disrupting cancer cell stemness reduce the CCKBR+ GA organoid formation and impede tumor progression. Mechanically, CUT&Tag sequencing and Lectin pulldown revealed that FOXOs can activate ST3GAL3/4/5 as well as ST6GALNAC6, promoting elevated sialyation levels in CCKBR+ tumor cells. This FOXO-sialyltransferase axis contributes to the maintenance of homeostasis and the growth of CCKBR+ tumor cells. This insight provides novel perspectives for developing targeted therapeutic strategies aimed at the treating CCKBR associated gastric cancer.

Evaluation of reference genes and expression patterns of CONSTANS-LIKE genes in Tetrastigma hemsleyanum under different photoperiods.

CONSTANS-LIKE (COL ) genes are a key signalling molecule that regulates plant growth and development during the photoperiod. Our preliminary experiments showed that the photoperiod greatly influence the formation of Tetrastigma hemsleyanum root tubers. In this study, we examined the oscillation patterns and expression characteristics of COL genes in leaves of T. hemsleyanum under different photoperiod conditions. Six genes were selected as candidate reference genes for further analyses: (1) 18S ribosomal RNA (18S rRNA ); (2) α-tubulin (TUBA ); (3) 30S ribosomal RNA (30S rRNA ); (4) TATA binding protein (TBP ); (5) elongation factor 1α (EF-1α ); and (6) RNA polymerase II (RPII ). The geNorm, NormFinder, and BestKeeper software programs were used to evaluate expression stability. Two ThCOL genes were screened in the T. hemsleyanum transcriptome library, and their expression patterns under different photoperiod conditions were analysed using quantitative reverse transcription PCR. The genes EF-1α , TUBA , and 18S rRNA were used to analyse the expression profiles of CONSTANS genes (ThCOL4 and ThCOL5 ) under different photoperiods. The expression peaks of ThCOL4 and ThCOL5 appeared at different times, demonstrating that their oscillation patterns were influenced by the photoperiod. We speculate that these two ThCOL genes may be involved in different biological processes.

Screening and anti-angiogenesis activity of Chiloscyllium plagiosum anti-human VEGFR2 single-domain antibody.

Recently, the incidence of malignant tumors is on the rise and searching for new treatments on it has become the research priority. Blocking the vascular endothelial growth factor (VEGF) and its receptor (VEGFR) is one of the treatment strategies that used in the development of specific anti-angiogenic drugs. The deficiencies in tissue penetration and affinity maturation become the weakness of these drugs in anti-tumors applications. The single heavy chain antibody found in Chiloscyllium plagiosum, which has a low molecular weight and superior tissue penetration of variable region (variable new antigen receptor, VNARs), was considered to have the high antigen-binding activity and stability. This type of antibody has a simple structure that can be prokaryoticaly expressed, which makes it easily to produce new antiangiogenic target drugs. Specific anti-IgNAR rabbit multiple antibodies have been used to assess the level of VNARs in sharks and have shown a significant enrichment of IgNAR after triple immunization. An anti-VEGFR2 phage library was used for the targeted VNARs screening, and five candidate VNARs sequences were subsequently obtained by phage screening, followed by combined screening with the transcriptome library, and analysis of conserved regions along with 3D modelling matched the VNAR profile. ELISA and cell-based assays showed that two of the VNARs, VNAR-A6, and VNAR-E3, had a superior antigen affinity and anti-angiogenic activity thereby being able to inhibit human Umbilical Vein Endothelial Cells proliferation and migration. The anti-VEGFR2 VNARs derived from the immunized C. plagiosum and screened by phage library, which provide the new research ideas and specific approaches for the development of new drugs. The anti-VEGFR2 VNARs are capable for blocking the VEGF-VEGFR pathway, which of these may contribute to expanding the use of anti-angiogenic drugs.

Systematic identification and functional analysis of root meristem growth factors (RGFs) reveals role of PgRGF1 in modulation of root development and ginsenoside production in Panax ginseng

Panax ginseng C.A. Mey., known for its medicinal and dietary supplement properties, primarily contains pharmacologically active ginsenosides. However, the regulatory mechanisms linking ginseng root development with ginsenoside biosynthesis are still unclear. Root meristem growth factors (RGFs) are crucial for regulating plant root growth. In our study, we identified five ginseng RGF peptide sequences from the ginseng genome and transcriptome libraries. We treated Arabidopsis and ginseng adventitious roots with exogenous Panax ginseng RGFs (PgRGFs) to assess their activities. Our results demonstrate that PgRGF1 influences gravitropic responses and reduces lateral root formation in Arabidopsis. PgRGF1 has been found to restrict the number and length of ginseng adventitious root branches in ginseng. Given the medicinal properties of ginseng, We determined the ginsenoside content and performed transcriptomic analysis of PgRGF1-treated ginseng adventitious roots. Specifically, the total ginsenoside content in ginseng adventitious roots decreased by 19.98 % and 63.71 % following treatments with 1 μM and 10 μM PgRGF1, respectively, compared to the control. The results revealed that PgRGF1 affects the accumulation of ginsenosides by regulating the expression of genes associated with auxin transportation and ginsenoside biosynthesis. These findings suggest that PgRGF1, as a peptide hormone regulator in ginseng, can modulate adventitious root growth and ginsenoside accumulation.

High-resolution transcriptome datasets during embryogenesis of plant-parasitic nematodes

Understanding the transcriptional regulatory characteristics throughout the embryogenesis of plant-parasitic nematodes is crucial for elucidating their developmental processes’ uniqueness. However, a challenge arises due to the lack of suitable technical methods for synchronizing the age of plant-parasitic nematodes embryo, it is difficult to collect detailed transcriptome data at each stage of embryonic development. Here, we recorded the 11 embryonic developmental time-points of endophytic nematode Meloidogyne incognita (isolated from Wuhan, China), Heterodera glycines (isolated from Wuhan, China), and Ditylenchus destructor (isolated from Jinan, China) species, and constructed transcriptome datasets of single embryos of these three species utilizing low-input smart-seq2 technology. The datasets encompassed 11 complete embryonic development stages, including Zygote, 2-cell, 4-cell, 8-cell, 24–44 cell, 64–78 cell, Comma, 1.5-fold, 2-fold, Moving, and L1, each stage generated four to five replicates, resulting in a total of 162 high-resolution transcriptome libraries. This high-resolution cross-species dataset serves as a crucial resource for comprehending the embryonic developmental properties of plant-parasitic nematodes and for identifying functional regulatory genes during embryogenesis.

Resolving candidate genes of duck ovarian tissue transplantation via RNA-Seq and expression network analyses

This study aims to identify candidate genes related to ovarian development after ovarian tissue transplantation through transcriptome sequencing (RNA-seq) and expression network analyses, as well as to provide a reference for determining the molecular mechanism of improving ovarian development following ovarian tissue transplantation. We collected ovarian tissues from 15 thirty-day-old ducks and split each ovary into 4 equal portions of comparable sizes before orthotopically transplanting them into 2-day-old ducks. Samples were collected on days 0 (untransplanted), 3, 6, and 9. The samples were paraffin sectioned and then subjected to Hematoxylin-Eosin (HE) staining and follicular counting. We extracted RNA from ovarian samples via the Trizol method to construct a transcriptome library, which was then sequenced by the Illumina Novaseq 6000 sequencing platform. The sequencing results were examined for differentially expressed genes (DEG) through gene ontology (GO) function and the Kyoto encyclopedia of genes and genomes (KEGG) pathway analyses, gene set enrichment analysis (GSEA), weighted correlation network analysis (WGCNA), and protein-protein interaction (PPI) networks. Some of the candidate genes were selected for verification using real-time fluorescence quantitative PCR (qRT-PCR). Histological analysis revealed a significant reduction in the number of morphologically normal follicles at 3, 6, and 9 d after ovarian transplantation, along with significantly higher abnormality rates (P < 0.05). The transcriptome analysis results revealed 2,114, 2,224, and 2,257 upregulated DEGs and 2,647, 2,883, and 2,665 downregulated DEGs at 3, 6, and 9 d after ovarian transplantation, respectively. Enrichment analysis revealed the involvement multiple pathways in inflammatory signaling, signal transduction, and cellular processes. Furthermore, WGCNA yielded 13 modules, with 10, 4, and 6 candidate genes mined at 3, 6 and 9 d after ovarian transplantation, respectively. Transcription factor (TF) prediction showed that STAT1 was the most important TF. Finally, the qRT-PCR verification results revealed that 12 candidate genes exhibited an expression trend consistent with sequencing data. In summary, significant differences were observed in the number of follicles in duck ovaries following ovarian transplantation. Candidate genes involved in ovarian vascular remodeling and proliferation were screened using RNA-Seq and WGCNA.

Transcription factor PgNAC72 activates DAMMARENEDIOL SYNTHASE expression to promote ginseng saponin biosynthesis.

Ginsenosides, the primary bioactive constituents in ginseng (Panax ginseng), possess substantial pharmacological potential and are in high demand in the market. The plant hormone methyl jasmonate (MeJA) effectively elicits ginsenoside biosynthesis in P. ginseng, though the regulatory mechanism remains largely unexplored. NAC transcription factors are critical in intricate plant regulatory networks and participate in numerous plant physiological activities. In this study, we identified a MeJA-responsive NAC transcription factor gene, PgNAC72, from a transcriptome library produced from MeJA-treated P. ginseng callus. Predominantly expressed in P. ginseng flowers, PgNAC72 localizes to the nucleus. Overexpressing PgNAC72 (OE-PgNAC72) in P. ginseng callus notably elevated total saponin levels, particularly dammarane-type ginsenosides, by upregulating dammarenediol synthase (PgDDS), encoding a key enzyme in the ginsenoside biosynthesis pathway. Electrophoretic mobility shift assays and dual-luciferase assays confirmed that PgNAC72 binds to the NAC-binding elements in the PgDDS promoter, thereby activating its transcription. Further RNA-seq and terpenoid metabolomic data in the OE-PgNAC72 line confirmed that PgNAC72 enhances ginsenoside biosynthesis. These findings uncover a regulatory role of PgNAC72 in MeJA-mediated ginsenoside biosynthesis, providing insights into the ginsenoside regulatory network and presenting a valuable target gene for metabolic engineering.

Experimental and analytical pipeline for sub-genomic RNA landscape of coronavirus by Nanopore sequencer.

Coronaviruses (CoVs), including severe acute respiratory syndrome coronavirus 2, can infect a variety of mammalian and avian hosts with significant medical and economic consequences. During the life cycle of CoV, a coordinated series of subgenomic RNAs, including canonical subgenomic messenger RNA and non-canonical defective viral genomes (DVGs), are generated with different biological implications. Studies that adopted the Nanopore sequencer (ONT) to investigate the landscape and dynamics of viral RNA subgenomic transcriptomes applied arbitrary bioinformatics parameters without justification or experimental validation. The current study used bovine coronavirus (BCoV), which can be performed under biosafety level 2 for library construction and experimental validation using traditional colony polymerase chain reaction and Sanger sequencing. Four different ONT protocols, including RNA direct and cDNA direct sequencing with or without exonuclease treatment, were used to generate RNA transcriptomic libraries from BCoV-infected cell lysates. Through rigorously examining the k-mer, gap size, segment size, and bin size, the optimal cutoffs for the bioinformatic pipeline were determined to remove the sequence noise while keeping the informative DVG reads. The sensitivity and specificity of identifying DVG reads using the proposed pipeline can reach 82.6% and 99.6% under the k-mer size cutoff of 15. Exonuclease treatment reduced the abundance of RNA transcripts; however, it was not necessary for future library preparation. Additional recovery of clipped BCoV nucleotide sequences with experimental validation expands the landscape of the CoV discontinuous RNA transcriptome, whose biological function requires future investigation. The results of this study provide the benchmarks for library construction and bioinformatic parameters for studying the discontinuous CoV RNA transcriptome.IMPORTANCEFunctional defective viral genomic RNA, containing all the cis-acting elements required for translation or replication, may play different roles in triggering cell innate immune signaling, interfering with the canonical subgenomic messenger RNA transcription/translation or assisting in establishing persistence infection. This study does not only provide benchmarks for library construction and bioinformatic parameters for studying the discontinuous coronavirus RNA transcriptome but also reveals the complexity of the bovine coronavirus transcriptome, whose functional assays will be critical in future studies.

The effect of feeding on different hosts on the egg proteins in Haemaphysalis qinghaiensis tick.

The majority of ixodid ticks display host-specificity to varying extents. Feeding on different hosts affects their development and reproduction. Consequences can be analyzed at the level of the egg, as it is the initial stage of tick development. Tick egg proteins are abundant and diverse, providing nutrients for embryonic development. However, studies on tick egg profiles are scarce. In this study, we aimed to analyze whether feeding Haemaphysalis qinghaiensis ticks on the yaks (Bos grunniens) and domestic sheep (Ovis aries) has an impact on the variety and variability of the egg proteome. Detached engorged females were used to lay eggs, which were then collected, dewaxed, and subjected to protein extraction. The extracted egg proteins were enzymatically digested using Filter-Aided Sample Preparation (FASP), and the unique peptides were separated and detected by Liquid Chromatography-tandem Mass Spectrometry (LC-MS/MS). The MS data were searched against the previously constructed whole tick transcriptome library of H. qinghaiensis, and the UniProt database for the identification of tick-derived egg proteins. The analysis revealed 49 and 53 high-confidence proteins identified in eggs collected from B. grunniens (EggBg) and O. aries (EggOa), respectively. Of these, 46 high-confidence proteins were common to both egg types, while three were unique to EggBg and seven to EggOa. All the identified proteins mainly belonged to enzymes, enzyme inhibitors, transporters, and proteins with unknown functions. The differential abundance analysis showed that nine proteins were significantly more present in EggBg, while six were significantly more present in EggOa. Overall, enzymes were the most diverse group, while vitellogenin (Vg) was the most abundant. Blood meal uptake on different hosts has a certain effect on the egg proteome composition and the abundance of some proteins, but it may also lead to compensation of protein roles.

Abstract 4956: A fast and efficient bioinformatics analysis workflow for processing reads from single-cell multiomics assays captured on a microwell-based platform

Abstract Here, we present a significant update: version 2.0 of our sequencing analysis, it is a comprehensive primary analysis workflow that is up to 7X faster and consumes 2X less disk space than the previous release. We used it to process a library with 20,000 human PBMCs sequenced to a depth of 1.2 billion reads in 2.5 hours of wall-clock time. The pipeline can be used with reads from single-cell whole transcriptome, targeted mRNA, surface antigens (AbSeq), TCR/BCR and Sample Tag libraries captured on the BD Rhapsody™ System platform. The major processing steps include Quality Filtering, Cell Barcode Identification, Read Alignment, Feature Assignment, UMI Error Correction, Identification of Putative Cells, Sample De-Multiplexing, Dimensionality Reduction for Visualization and VDJ Contig Assembly and Annotation. Output files and metrics are available in easy-to-digest formats, including an html report with dynamic visualization. Integrated outputs in Seurat and Scanpy formats combine expression matrices and all cell annotation metadata (e.g., predicted cell type, sample assignment, TCR/BCR sequence and gene segments). These pre-generated files are ready to load into popular single-cell analysis tools. The pipeline uses CWL as the workflow manager and makes use of custom code written in C++ and Python along with various open-source packages for data processing. The pipeline can also process reads from non-human species and includes built-in support for specifying and building custom reference genome indices. In support of high-throughput multiomic discovery studies enabled by the BD Rhapsody™ HT Xpress System, this pipeline has been tested with datasets containing more than 800,000 putative cells and 14.5 billion reads. For Research Use Only. Not for use in diagnostic or therapeutic procedures. BD, the BD Logo and BD Rhapsody are trademarks of Becton, Dickinson and Company or its affiliates. © 2023 BD. All rights reserved. NPM-2535 (v1.0) 1023 Citation Format: Raghavendra Padmanabhan, Brent Weichel, Amie Radenbaugh, Yuefu Jiang, Charles Weeks, Thomas McCarthy, Youngsook Kim, Anthony Berno, Devon Jensen. A fast and efficient bioinformatics analysis workflow for processing reads from single-cell multiomics assays captured on a microwell-based platform [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 4956.

Single-cell RNA sequencing reveals Immune Education promotes T cell survival in mice subjected to the cecal ligation and puncture sepsis model.

Individual T cell responses vary significantly based on the microenvironment present at the time of immune response and on prior induced T cell memory. While the cecal ligation and puncture (CLP) model is the most commonly used murine sepsis model, the contribution of diverse T cell responses has not been explored. We defined T cell subset responses to CLP using single-cell RNA sequencing and examined the effects of prior induced T cell memory (Immune Education) on these responses. We hypothesized that Immune Education prior to CLP would alter T cell responses at the single cell level at a single, early post-CLP time point. Splenic T cells were isolated from C57BL/6 mice. Four cohorts were studied: Control, Immune-Educated, CLP, and Immune-Educated CLP. At age 8 weeks, Immune-Educated and Immune-Educated CLP mice received anti-CD3ϵ antibody; Control and CLP mice were administered an isotype control. CLP (two punctures with a 22-gauge needle) was performed at 12-13 weeks of life. Mice were sacrificed at baseline or 24-hours post-CLP. Unsupervised clustering of the transcriptome library identified six distinct T cell subsets: quiescent naïve CD4+, primed naïve CD4+, memory CD4+, naïve CD8+, activated CD8+, and CD8+ cytotoxic T cell subsets. T cell subset specific gene set enrichment analysis and Hurdle analysis for differentially expressed genes (DEGs) were performed. T cell responses to CLP were not uniform - subsets of activated and suppressed T cells were identified. Immune Education augmented specific T cell subsets and led to genomic signatures favoring T cell survival in unoperated and CLP mice. Additionally, the combination of Immune Education and CLP effected the expression of genes related to T cell activity in ways that differed from CLP alone. Validating our finding that IL7R pathway markers were upregulated in Immune-Educated CLP mice, we found that Immune Education increased T cell surface IL7R expression in post-CLP mice. Immune Education enhanced the expression of genes associated with T cell survival in unoperated and CLP mice. Induction of memory T cell compartments via Immune Education combined with CLP may increase the model's concordance to human sepsis.

Comparative Transcriptomics Analysis Reveals Rusty Grain Beetle's Aggregation Pheromone Biosynthesis Mechanism in Response to Starvation.

Pheromones are the basis of insect aggregation, mating, and other behaviors. Cucujoid grain beetles produce macrocyclic lactones as aggregation pheromones, yet research on their biosynthesis at the molecular level remains limited. The rusty grain beetle, C. ferrugineus, is an important economic species in China. Although two aggregation pheromone components have been identified, their suspected biosynthesis via the MVA pathway and the FAS pathway lacks molecular elucidation. Previous evidence supports that starvation affects the production of aggregation pheromones. Therefore, we constructed comparative transcriptome libraries of pheromone production sites in C. ferrugineus under starvation stress and identified genes related to pheromone biosynthesis and hormone regulation. A total of 2665 genes were significantly differentially expressed, of which 2029 genes were down-regulated in starved beetles. Putative C. ferrugineus genes directly involved in pheromone biosynthesis were identified, as well as some genes related to the juvenile hormone (JH) pathway and the insulin pathway, both of which were depressed in the starved beetles, suggesting possible functions in pheromone biosynthesis and regulation. The identification of genes involved in macrolide lactone biosynthesis in vivo holds great significance, aiding in the elucidation of the synthesis and regulatory mechanisms of cucujoid grain beetle pheromones.

Unveiling CRESS DNA Virus Diversity in Oysters by Virome.

Oysters that filter feed can accumulate numerous pathogens, including viruses, which can serve as a valuable viral repository. As oyster farming becomes more prevalent, concerns are mounting about diseases that can harm both cultivated and wild oysters. Unfortunately, there is a lack of research on the viruses and other factors that can cause illness in shellfish. This means that it is harder to find ways to prevent these diseases and protect the oysters. This is part of a previously started project, the Dataset of Oyster Virome, in which we further study 30 almost complete genomes of oyster-associated CRESS DNA viruses. The replication-associated proteins and capsid proteins found in CRESS DNA viruses display varying evolutionary rates and frequently undergo recombination. Additionally, some CRESS DNA viruses have the capability for cross-species transmission. A plethora of unclassified CRESS DNA viruses are detectable in transcriptome libraries, exhibiting higher levels of transcriptional activity than those found in metagenome libraries. The study significantly enhances our understanding of the diversity of oyster-associated CRESS DNA viruses, emphasizing the widespread presence of CRESS DNA viruses in the natural environment and the substantial portion of CRESS DNA viruses that remain unidentified. This study's findings provide a basis for further research on the biological and ecological roles of viruses in oysters and their environment.

Identification of Differentially Expressed mRNAs and miRNAs and Related Regulatory Networks in Cumulus Oophorus Complexes Associated with Fertilization.

Cumulus oophorus complexes (COCs) are the first extracellular barriers that sperm must pass through to fuse with oocytes, which have an important role in oocyte maturation and fertilization. However, little is known about the molecular mechanisms of COCs involved in fertilization. In this study, COCs were collected and then randomly divided into a test group that interacted with sperm and a control group that did not interact with sperm. Then, the total RNA was extracted; RNA transcriptome and small RNA libraries were prepared, sequenced, and analyzed. The results showed that 1283 differentially expressed genes (DEGs), including 560 upregulated and 723 downregulated genes. In addition, 57 differentially expressed miRNAs (DEMIs) with 35 upregulated and 22 downregulated were also detected. After the RNA-seq results were verified by RT-qPCR, 86 effective DEGs and 40 DEMIs were finally screened and a DEMI-DEG regulatory network was constructed. From this, the top ten hub target genes were HNF4A, SPN, WSCD1, TMEM239, SLC2A4, E2F2, SIAH3, ADORA3, PIK3R2, and GDNF, and they were all downregulated. The top ten hub DEMIs were miR-6876-5p, miR-877-3p, miR-6818-5p, miR-4690-3p, miR-6789-3p, miR-6837-5p, miR-6861-5p, miR-4421, miR-6501-5p, and miR-6875-3p, all of which were upregulated. The KEGG signaling pathway enrichment analysis showed that the effective DEGs were significantly enriched in the calcium, AMPK, and phospholipase D signaling pathways. Our study identified several DEGs and DEMIs and potential miRNA-mRNA regulatory pathways in COCs and these may contribute to fertilization. This study may provide novel insights into potential biomarkers for fertilization failure.

Identification of genes regulated by 20-Hydroxyecdysone in Macrobrachium nipponense using comparative transcriptomic analysis

BackgroundMacrobrachium nipponense is a freshwater prawn of economic importance in China. Its reproductive molt is crucial for seedling rearing and directly impacts the industry’s economic efficiency. 20-hydroxyecdysone (20E) controls various physiological behaviors in crustaceans, among which is the initiation of molt. Previous studies have shown that 20E plays a vital role in regulating molt and oviposition in M. nipponense. However, research on the molecular mechanisms underlying the reproductive molt and role of 20E in M. nipponense is still limited.ResultsA total of 240.24 Gb of data was obtained from 18 tissue samples by transcriptome sequencing, with > 6 Gb of clean reads per sample. The efficiency of comparison with the reference transcriptome ranged from 87.05 to 92.48%. A total of 2532 differentially expressed genes (DEGs) were identified. Eighty-seven DEGs associated with molt or 20E were screened in the transcriptomes of the different tissues sampled in both the experimental and control groups. The reliability of the RNA sequencing data was confirmed using Quantitative Real-Time PCR. The expression levels of the eight strong candidate genes showed significant variation at the different stages of molt.ConclusionThis study established the first transcriptome library for the different tissues of M. nipponense in response to 20E and demonstrated the dominant role of 20E in the molting process of this species. The discovery of a large number of 20E-regulated strong candidate DEGs further confirms the extensive regulatory role of 20E and provides a foundation for the deeper understanding of its molecular regulatory mechanisms.

Whole transcriptome sequencing for revealing the pathogenesis of sporotrichosis caused by Sporothrix globosa

This study aimed to investigate the molecular mechanism of sporotrichosis and identify possible novel therapeutic targets. Total RNA was extracted from skin lesion samples from sporotrichosis patients and used to construct a long-chain RNA transcriptome library and miRNA transcriptome library for whole transcriptome sequencing. The differentially expressed genes (DEGs) between the groups were identified, and then Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, and Gene Set Enrichment Analysis enrichment analyses were performed based on the DEGs. An lncRNA-miRNA-mRNA ceRNA network was constructed. The expressions of JAK/STAT pathway-related proteins were detected in the patient and control tissues using RT-qPCR and Western blot analysis. Enrichment analysis showed that the DEGs were mainly enriched in various infectious diseases and immune response-related signaling pathways. Competing endogenous RNA network analysis was performed and identified the hub lncRNAs, miRNAs, and mRNAs. Compared with the control group, the mRNA expressions of SOCS3, IL-6, and JAK3 were significantly upregulated, while the expression of STAT3 did not change significantly. Also, the protein expressions of SOCS3, IL-6, JAK3, and STAT3, as well as phosphorylated JAK3 and STAT3, were significantly upregulated. We identified 671 lncRNA DEGs, 3281 mRNA DEGs, and 214 miRNA DEGs to be involved in Sporothrix globosa infection. The study findings suggest that the JAK/STAT pathway may be a therapeutic target for sporotrichosis.

Transcriptome blood profile of the Yili horse before and after training

Proper training improves the horse’s performance. In the present study, blood transcriptome of the Yili horse was analysed before and after training. Overall, six Yili horses were trained for 3 months; blood samples at rest were collected before and after the training period. Transcriptome libraries were created from the blood samples using high-throughput sequencing; Gene Ontology functional enrichment and Kyoto Encyclopaedia of Genes and Genomes pathway analyses were performed for differentially expressed genes to identify the pathways and candidate genes associated with training and locomotor performance. In total, transcriptome analysis screened 33 significantly differentially expressed genes; of these, 23 and 10 were up-regulated and down-regulated after training, respectively. These differentially expressed genes were significantly enriched in metabolic pathways associated with exercise physiology, including sphingolipid metabolism, tryptophan metabolism, and the cGMP-PKG signalling pathway. The results of this study provide a theoretical basis for exploring the molecular mechanisms underlying locomotor performance of the Yili horse as well as a novel insight into the kinematics of this breed.

Mechanistic analysis of the hardening process of the thorns on stems of Bougainvillea glabra "Elizabeth Angus".

Introduction: Bougainvillea glabra "Elizabeth Angus" is a thorny woody vine or shrub. However, the hard thorns are considered a deficiency in its ornamental value. Methods: To find the genes and pathways related to the hardening process of the thorns on the stems of B. glabra, the eukaryotic unreferenced transcriptome sequencing analysis was conducted to explore the 3 stages of the thorn-hardening process. Total RNA was extracted from thorns and stems, and transcriptome libraries were constructed and sequenced using unreferenced Illumina sequencing. Results: Gene function annotation was performed using various databases, resulting in 8937 co-annotated genes. The density distribution of Fragments Per Kilobase of transcript per Million mapped reads (FPKM) depicted the overall gene expression patterns. The study found that stage 2 as the period of highest gene expression activity during the thorns hardening process in B. glabra. Differential expression analysis revealed that during thorn-hardening, 1045 genes up-regulated and 391 genes down-regulated significantly in thorns at stage 2 compared to stage 1 (early stage of thorns formation). Meanwhile, 938 genes up-regulated and 784 genes down-regulated significantly in stems. At stage 3, as thorns became harder, 63 genes exhibited notable expression increase and 98 genes' expression decreased obviously within thorns, and 46 genes up-regulated and 29 genes down-regulated in stems, compared to stage 2. Phenylpropanoid biosynthesis was the key step in the hardening process of the thorns of B. glabra. The formation and hardening of thorns on the stem of B. glabra was a process in which lignin gradually accumulated in the thorns, and several genes were involved in this process. They include PAL (EC:4.3.1.24), CYP73A (EC:1.14.14.91), 4CL (EC:6.2.1.12), CCR (EC:1.2.1.44), CAD (EC:1.1.1.195) and POX (EC:1.11.1.7). Discussion: This transcriptome analysis offers insights into the molecular mechanisms underlying thorns development in this plant species.

Single-nuclei transcriptome analysis of channel catfish spleen provides insight into the immunome of an aquaculture-relevant species.

The catfish industry is the largest sector of U.S. aquaculture production. Given its role in food production, the catfish immune response to industry-relevant pathogens has been extensively studied and has provided crucial information on innate and adaptive immune function during disease progression. To further examine the channel catfish immune system, we performed single-cell RNA sequencing on nuclei isolated from whole spleens, a major lymphoid organ in teleost fish. Libraries were prepared using the 10X Genomics Chromium X with the Next GEM Single Cell 3' reagents and sequenced on an Illumina sequencer. Each demultiplexed sample was aligned to the Coco_2.0 channel catfish reference assembly, filtered, and counted to generate feature-barcode matrices. From whole spleen samples, outputs were analyzed both individually and as an integrated dataset. The three splenic transcriptome libraries generated an average of 278,717,872 reads from a mean 8,157 cells. The integrated data included 19,613 cells, counts for 20,121 genes, with a median 665 genes/cell. Cluster analysis of all cells identified 17 clusters which were classified as erythroid, hematopoietic stem cells, B cells, T cells, myeloid cells, and endothelial cells. Subcluster analysis was carried out on the immune cell populations. Here, distinct subclusters such as immature B cells, mature B cells, plasma cells, γδ T cells, dendritic cells, and macrophages were further identified. Differential gene expression analyses allowed for the identification of the most highly expressed genes for each cluster and subcluster. This dataset is a rich cellular gene expression resource for investigation of the channel catfish and teleost splenic immunome.

Identification of a receptor for the sex pheromone of the vine mealybug, Planococcus ficus

The vine mealybug, Planococcus ficus, is a significant pest of vineyards in all major grape growing regions of the world. This pest causes significant aesthetic damage to berry clusters through its feeding behavior and secretion of "honeydew", which leads to significant decreases in crop marketability. More importantly, the vine mealybug is a vector of several grapevine viruses which are the causal agent of grapevine leafroll disease, one of the most destructive and economically devastating diseases of the grape industry worldwide. As there is no cure for grapevine leafroll disease, the only control measures available to reduce its spread are to remove infected vines whilst simultaneously controlling mealybug populations. Using transcriptomic libraries prepared from male and female mealybugs and a draft genome, we identified and evaluated expression levels of members of the odorant receptor gene family. Interestingly, of the 50 odorant receptors identified from these P. ficus genetic resources, only 23 were found to be expressed in females, suggesting this flightless life stage has a decreased reliance on the olfactory system. In contrast, 46 odorant receptors were found to be expressed in the alate male life stage. Heterologous expression of eight of these receptors, along with the obligate co-receptor, Orco, in HEK293 cells allowed for the identification of two receptors that respond to lavandulyl senecioate, the sole constituent of the sex pheromone used by this species. Interestingly, one of these receptors, PficOR8, also responded to the sex pheromone used by the Japanese mealybug, Planococcus kraunhiae. The data presented here represent the first report of odorant receptor gene family expression levels, as well as the identification of the first sex pheromone receptor, in soft-scale insects. The identification of a receptor for the vine mealybug sex pheromone will allow for the development of novel, species-specific pest control tools and monitoring devices.