RNA-seq Technology Research Articles

Transcriptome sequencing analysis of overexpressed SikCDPK1 in tobacco reveals mechanisms of cold stress response

BackgroundCold is a significant limiting factor in productivity, particularly in northwestern and eastern China. Calcium-Dependent Protein Kinases (CDPKs), a primary calcium signaling sensor in plants, play an important role in their response to cold. Snow lotus (Sasussured involucrata Kar L) is a plant that thrives in harsh climates and grows in northwest China. However, there were no reports on the transcriptome of OE-SikCDPK1 transgenic tobacco in response to cold.ResultsWhen exposed to cold stress, OE-SikCDPK1 plants displayed a cold-tolerant phenotype compared to non-transgenic tobacco. Under cold conditions, relative water content reduced, relative conductivity increased, malondialdehyde levels rose, and cold-responsive gene expression increased. The OE-SikCDPK1 gene and non-transgenic tobacco were employed for research purposes. The transcriptome of leaves was sequenced using the HISAT2 sequencing platform, and the data were used to examine gene function annotation and differentially expressed genes (DEGs). 53,022 DEGs in tobacco leaves under cold treatment were obtained. The GO enrichment results revealed that it was enriched for biological-process, defense response and other processes under cold stress. The KEGG pathway enrichment analysis revealed that the metabolic pathways of significant enrichment of DEGs under cold stress mainly involved MAPK signaling pathway transduction. The transcription factor identification results showed that the transcription factors with the largest number of differential expressions under cold treatment were mainly from WRKY, AP2, MYB, bHLH, NAC and other transcription factor families.ConclusionThe cold tolerance mechanism of snow lotus SikCDPK1 was comprehensively analyzed at the transcriptional level for the first time using RNA-seq technology. This study demonstrates that SikCDPK1 can respond to cold by participating in the MAPK signaling pathway and regulating the expression levels of transcription factors, including WRKY, AP2, MYB, bHLH, and NAC. These results offer valuable insights for further exploration of the cold tolerance mechanism associated with SikCDPK1.

Identification and expression profiling of neuropeptides and neuropeptide receptor genes in a natural enemy, Coccinella septempunctata.

Neuropeptides and their receptors constitute diverse and abundant signal molecules in insects, primarily synthesized and released primarily from neurosecretory cells within the central nervous system Neuropeptides act as neurohormones and euromodulators, regulating insect behavior, lifecycle, and physiology by binding to receptors on cell surface. As a typical natural predator of agricultural pests, the lady beetle, Coccinella septempunctata, has been commercially mass-cultured and widely employed in pest management. Insect diapause is a physiological and ecological adaptative strategy acquired in adverse environments. In biological control programs, knowledge about diapause regulation in natural enemy insects provides important insight for improving long-term storage, transportation, and field adoption of these biological control agents. However, little is known about the function of neuropeptides and their receptors in controlling reproductive diapause of C. septempunctata. It is unclear which neuropeptides affect diapause of C. septempunctata. In this study, RNA-seq technology and bioinformatics were utilized to investigate genes encoding neuropeptides and their receptors in female adults of C. septempunctata. Quantitative real-time PCR (qRT-PCR) analysis was employed to examine gene expression across different development/diapause stages. A total of 17 neuropeptide precursor genes and 9 neuropeptide receptor genes were identified, implicated in regulating various behaviors such as feeding, reproduction, and diapause. Prediction of partial mature neuropeptides from precursor sequences was also performed using available information about these peptides from other species, conserved domains and motifs. During diapause induction, the mRNA abundance of AKH was notably higher on the 10th day compared to non-diapause females, but decreased by the 20th day. In contrast, GPHA showed lower expression levels on the 5th day of diapause induction compared to non-diapause females, but increased significantly by the 15th and 20th days. NPF was higher expressed in head and midgut while DH showed higher expression in the fat body and midgut. Additionally, NPF expression remained consistently lower throughout all stages of diapause induction compared to non-diapause conditions in females. This study represents the first sequencing, identification, and expression analysis of neuropeptides and neuropeptide receptor genes in C. septempunctata. Our results could provide a foundational framework for further investigations into the presence, functions, and potential targets of neuropeptides and their receptors, particularly in devising novel strategies for diapause regulation in C. septempunctata.

Transcriptomic insights into mycorrhizal interactions with tomato root: a comparative study of short- and long-term post-inoculation responses.

Arbuscular mycorrhiza (AM) refers to a symbiotic association between plant roots and fungi that enhances the uptake of mineral nutrients from the soil and enables the plant to tolerate abiotic and biotic stresses. Although previously reported RNA-seq analyses have identified large numbers of AM-responsive genes in model plants, such as Solanum lycopersicum L., further studies are underway to comprehensively understand the complex interactions between plant roots and AM, especially in terms of the short- and long-term responses after inoculation. Herein, we used RNA-seq technology to obtain the transcriptomes of tomato roots inoculated with the fungus Rhizophagus irregularis at 7 and 30days post inoculation (dpi). Of the 1,019 differentially expressed genes (DEGs) in tomato roots, 635 genes showed differential expressions between mycorrhizal and non-mycorrhizal associations at the two time points. The number of upregulated DEGs far exceeded the number of downregulated ones at 7 dpi, and this difference decreased at 30dpi. Several notable genes were particularly involved in the plant defense, plant growth and development, ion transport, and biological processes, namely, GABAT, AGP, POD, NQO1, MT4, MTA, and AROGP3. In addition, the Kyoto encyclopedia of genes and genomes pathway enrichment analysis revealed that some of the genes were involved in different pathways, including those of ascorbic acid (AFRR, GME1, and APX), metabolism (CYP, GAPC2, and CAM2), and sterols (CYC1 and HMGR), as well as genes related to cell division and cell cycle (CDKB2 and PCNA). These findings provide valuable new data on AM-responsive genes in tomato roots at both short- and long-term postinoculation stages, enabling the deciphering of biological interactions between tomato roots and symbiotic fungi.

Recombinant adenoviruses expressing HPV16/18 E7 upregulate the HDAC6 and DNMT3B genes in C33A cells.

High-risk human papillomavirus (HPV) is a carcinogenic virus associated with nearly all cases of cervical cancer, as well as an increasing number of anal and oral cancers. The two carcinogenic proteins of HPV, E6 and E7, can immortalize keratinocytes and are essential for HPV-related cellular transformation. Currently, the global regulatory effects of these oncogenic proteins on the host proteome are not fully understood, and further exploration of the functions and carcinogenic mechanisms of E6 and E7 proteins is needed. We used a previously established platform in our laboratory for constructing recombinant adenoviral plasmids expressing the HPV16 E7 gene to further construct recombinant virus particles expressing HPV16/18 E6, E7, and both E6 and E7 genes. These recombinant viruses were used to infect C33A cells to achieve sustained expression of the HPV16/18 E6/E7 genes. Subsequently, total RNA was extracted and RNA-Seq technology was employed for transcriptome sequencing to identify differentially expressed genes associated with HPV infection in cervical cancer. RNA-Seq analysis revealed that overexpression of the HPV16/18 E6/E7 genes upregulated GP6, CD36, HDAC6, ESPL1, and DNMT3B among the differentially expressed genes (DEGs) associated with cervical cancer. Spearman correlation analysis revealed a statistically significant correlation between the HDAC6 and DNMT3B genes and key pathways, including DNA replication, tumor proliferation signature, G2M checkpoint, p53 pathways, and PI3K/AKT/mTOR signaling pathways. Further, qRT-PCR and Western blot analyses indicated that both HPV16/18 E7 can upregulate the expression of HDAC6 and DNMT3B, genes associated with HPV infection-related cervical cancer. The successful expression of HPV16/18 E6/E7 in cells indicates that the recombinant viruses retain the replication and infection capabilities of Ad4. Furthermore, the recombinant viruses expressing HPV16/18 E7 can upregulate the HDAC6 and DNMT3B genes involved in cervical cancer pathways, thereby influencing the cell cycle. Additionally, HDAC6 and DNMT3B are emerging as important therapeutic targets for cancer. This study lays the foundation for further exploration of the oncogenic mechanisms of HPV E6/E7 and may provide new directions for the treatment of HPV-related cancers.

Hemoglobin α-derived peptides VD-hemopressin (α) and RVD-hemopressin (α) are involved in electroacupuncture inhibition of chronic pain.

Knee osteoarthritis (KOA) is a chronic degenerative bone metabolic disease that primarily affects older adults, leading to chronic pain and disability that affect patients' daily activities. Electroacupuncture (EA) is a commonly used method for the treatment of chronic pain in clinical practice. Previous studies indicate that the endocannabinoid system is involved in EA analgesia, but whether endocannabinopeptide VD-hemopressin (α) and RVD-hemopressin (α) derived from hemoglobin chains are involved in EA analgesia is unclear. RNA-seq technology was used to screen which genes involved in EA analgesia. The expression of hemoglobin α chain and 26S proteasome were determined by Western blotting. The level of VD-hemopressin (α) and RVD-hemopressin (α) were measured by UPLC-MS/MS. Microinjection VD-Hemopressin (α), RVD-Hemopressin (α) and 26S proteasome inhibitor MG-132 into vlPAG, then observe mechanical and thermal pain thresholds. Therefore, we used RNA-seq to obtain differentially expressed genes Hba-a1 and Hba-a2 involved in EA analgesia in the periaqueductal gray (PAG), which were translated into the hemoglobin α chain. EA significantly increased the expression of the hemoglobin α chain and the level of hemopressin (α) and RVD-hemopressin (α). Microinjection of VD-hemopressin (α) and RVD-hemopressin (α) into the ventrolateral periaqueductal gray (vlPAG) mimicked the analgesic effect of EA, while CB1 receptor antagonist AM251 reversed this effect. EA significantly increased the expression of 26S proteasome in KOA mice. Microinjection of 26S proteasome inhibitor MG132 before EA prevented both the anti-allodynic effect and upregulation of the concentration of RVD-hemopressin (α) by EA treatment and upregulated the expression of the hemoglobin α chain. Our data suggest that EA upregulated the concentration of VD-hemopressin (α) and RVD-hemopressin (α) through enhancement of the hemoglobin α chain degradation by 26S proteasome in the PAG, then activated the CB1 receptor, thereby exerting inhibition of chronic pain in a mouse model of KOA. These results provide new insights into the EA analgesic mechanisms and reveal possible targets for EA treatment of chronic pain.

Exploring the potential mechanism of Radix Bupleuri in the treatment of sepsis: a study based on network pharmacology and molecular docking

AimTo explore, using network pharmacology and RNA-seq technologies, potential active targets and mechanisms underpinning Radix Bupleuri’s effectiveness during sepsis treatment.MethodsFollowing the Sepsis-3.0 criteria, the research cohort, comprising 23 sepsis patients and 10 healthy participants, was obtained from public databases. Peripheral blood samples were collected and subjected to RNA-seq analysis. Active ingredients and potential targets of Radix Bupleuri were identified using the Bioinformatics Analysis Tool for Molecular mechANism of Traditional Chinese Medicine 2.0 (BATMAN-TCM 2.0) database and TCMSP database. Subsequently, protein-protein interaction (PPI) network construction, Gene Ontology (GO) analysis, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were conducted to explore cross-targets between disease and drugs. Survival analysis of key targets was performed using the GSE65682 dataset, and single-cell RNA-seq was employed for cellular localization analysis of key genes. Finally, molecular docking and Molecular dynamics simulation of the core target was conducted.ResultsDifferential expression analysis revealed 4253 genes associated with sepsis. Seventy-six active components and 1030 potential targets of Radix Bupleuri were identified. PPI, GO, and pathway enrichment analyses indicated involvement in the regulation of transmembrane transport, monatomic ion transport, and MAPK signaling. Survival curve analysis identified PIK3CD, ARRB2, SUCLG1, and SPI1 as key targets associated with lower mortality in the high expression group, while higher mortality was observed in the high PNP and FURIN expression groups. Single-cell RNA sequencing unveiled the cellular localization of PIK3CD, PNP, SPI1, and FURIN within macrophages, while ARRB2 and SUCLG1 exhibited localization in both macrophages and T-cells. Subsequent molecular docking and Molecular dynamics simulation indicated a potential binding interaction for Carvone-PIK3CD, Encecalin-ARRB2, Lauric Acid-SUCLG1, Pulegone-FURIN, Nootkatone-SPI1, and Saikogenin F-PNP.ConclusionRadix Bupleuri could modulate immune function by affecting PIK3CD, ARRB2, SUCLG1, FURIN, SPI1, and PNP, thereby potentially improving the prognosis of sepsis.

Transforming Transcriptomics: The Impact of RNA Sequencing Technology

Aim: This study aimed to explore the advancements, methodologies, and applications of RNA sequencing (RNA-seq) technology, emphasizing its transformative impact on genomic research. Study Design: The research involved an in-depth review of RNA-seq technology, focusing on the steps of sample preparation, sequencing, and data analysis. Place and Duration of Study: Conducted through a detailed literature review over six months, at Hygia college of Pharmacy the study sourced information from various molecular biology and genomics publications. Methodology: Advanced computational techniques were utilized to map and quantify RNA sequences, facilitating a comprehensive analysis of gene expression patterns, alternative splicing, and novel transcript discovery. High-throughput sequencing platforms like Illumina and PacBio generated extensive datasets from diverse biological samples, including tissues, single cells, and environmental microbiomes. Results: The results highlighted RNA-seq's ability to provide a high-resolution view of the transcriptome, surpassing previous technologies in both sensitivity and accuracy. RNA-seq uncovered critical insights into differential gene expression, identifying significant pathways involved in development, disease, and environmental responses. The technology also discovered numerous previously unannotated transcripts and isoforms, contributing to the expansion of existing genomic databases. Conclusion: The study concludes that RNA-seq is a crucial advancement in molecular biology, offering unprecedented insights into gene regulation and expression. Its adoption has facilitated significant breakthroughs in understanding cellular processes, disease mechanisms, and therapeutic targets. As RNA-seq technology continues to evolve, it promises to drive further innovations and applications across diverse fields in the life sciences.

Unlocking the secrets of NPSLE: the role of dendritic cell-secreted CCL2 in blood-brain barrier disruption.

This study employed RNA-seq technology and meta-analysis to unveil the molecular mechanisms of neuropsychiatric systemic lupus erythematosus (NPSLE) within the central nervous system. Downloaded transcriptomic data on systemic lupus erythematosus (SLE) from the Gene Expression Omnibus (GEO) and analyzed differential genes in peripheral blood samples of NPSLE patients and healthy individuals. Employed WGCNA to identify key genes related to cognitive impairment and validated findings via RNA-seq. Conducted GO, KEGG, and GSEA analyses, and integrated PPI networks to explore gene regulatory mechanisms. Assessed gene impacts on dendritic cells and blood-brain barrier using RT-qPCR, ELISA, and in vitro models. Public databases and RNA-seq data have revealed a significant upregulation of CCL2 (C-C motif chemokine ligand 2) in the peripheral blood of both SLE and NPSLE patients, primarily secreted by mature dendritic cells. Furthermore, the secretion of CCL2 by mature dendritic cells may act through the RSAD2-ISG15 axis and is associated with the activation of the NLRs (Nod Like Receptor Signaling Pathway) signaling pathway in vascular endothelial cells. Subsequent in vitro cell experiments confirmed the high expression of CCL2 in peripheral blood dendritic cells of NPSLE patients, with its secretion being regulated by the RSAD2-ISG15 axis and inducing vascular endothelial cell pyroptosis through the activation of the NLRs signaling pathway. Clinical trial results ultimately confirmed that NPSLE patients exhibiting elevated CCL2 expression also experienced cognitive decline. The secretion of CCL2 by dendritic cells induces pyroptosis in vascular endothelial cells, thereby promoting blood-brain barrier damage and triggering cognitive impairment in patients with systemic lupus erythematosus.

RNA Sequencing of Sperm from Healthy Cattle and Horses Reveals the Presence of a Large Bacterial Population.

RNA molecules within ejaculated sperm can be characterized through whole-transcriptome sequencing, enabling the identification of pivotal transcripts that may influence reproductive success. However, the profiling of sperm transcriptomes through next-generation sequencing has several limitations impairing the identification of functional transcripts. In this study, we explored the nature of the RNA sequences present in the sperm transcriptome of two livestock species, cattle and horses, using RNA sequencing (RNA-seq) technology. Through processing of transcriptomic data derived from bovine and equine sperm cell preparations, low mapping rates to the reference genomes were observed, mainly attributed to the presence of ribosomal RNA and bacteria in sperm samples, which led to a reduced sequencing depth of RNAs of interest. To explore the presence of bacteria, we aligned the unmapped reads to a complete database of bacterial genomes and identified bacteria-associated transcripts which were characterized. This analysis examines the limitations associated with sperm transcriptome profiling by reporting the nature of the RNA sequences among which bacterial RNA was found. These findings can aid researchers in understanding spermatozoal RNA-seq data and pave the way for the identification of molecular markers of sperm performance.

ADAMTS12 promotes fibrosis by restructuring extracellular matrix to enable activation of injury-responsive fibroblasts.

Fibrosis represents the uncontrolled replacement of parenchymal tissue with extracellular matrix (ECM) produced by myofibroblasts. While genetic fate-tracing and single-cell RNA-Seq technologies have helped elucidate fibroblast heterogeneity and ontogeny beyond fibroblast to myofibroblast differentiation, newly identified fibroblast populations remain ill defined, with respect to both the molecular cues driving their differentiation and their subsequent role in fibrosis. Using an unbiased approach, we identified the metalloprotease ADAMTS12 as a fibroblast-specific gene that is strongly upregulated during active fibrogenesis in humans and mice. Functional in vivo KO studies in mice confirmed that Adamts12 was critical during fibrogenesis in both heart and kidney. Mechanistically, using a combination of spatial transcriptomics and expression of catalytically active or inactive ADAMTS12, we demonstrated that the active protease of ADAMTS12 shaped ECM composition and cleaved hemicentin 1 (HMCN1) to enable the activation and migration of a distinct injury-responsive fibroblast subset defined by aberrant high JAK/STAT signaling.

Inhibition of Colorectal Cancer Metastasis by Total Flavones of Abelmoschus Manihot via Lncrna AL137782-mediated STAT3/EMT Pathway Regulation.

Colorectal cancer (CRC) ranks among the most lethal malignancies globally, particularly following metastasis which results in poor prognosis. In recent years, CRC incidence in China has persistently increased. Total flavonoids (TFA) from Abelmoschus manihot, a natural compound, are recognized for their anti-inflammatory, analgesic, and antioxidant properties. However, despite extensive research into the therapeutic potential of TFA, coverage of its role in cancer treatment is notably lacking. To address this research void, our study aims to unveil the role and potential mechanisms of TFA in treating CRC. We conducted a series of experiments to assess the impact of TFA on CRC cells. Two specific CRC cell lines, DLD-1 and HCT116, were employed in cell proliferation, colony formation, flow cytometry, and cell migration assays. Additionally, to test the in vivo effects of TFA, we developed a nude mouse xenograft tumor model to assess TFA's impact on tumor growth and liver metastasis. Furthermore, we meticulously analyzed the gene expression differences between CRC cells pretreated with TGF-β and those treated with TFA using RNA-seq technology. We also examined the molecular mechanisms of TFA and assessed the expression of proteins related to the STAT3/EMT signaling pathway through Western blotting and siRNA technology. Our research findings reveal for the first time the effect of TFA on CRC cells. Result shows that TFA could suppress cell proliferation, migration, and induce apoptosis. In vivo results showed that TFA inhibited tumor growth and liver metastasis. Molecular mechanism studies have shown that TFA exerts these effects by upregulating the expression of non-coding RNA AL137782, inhibiting the EMT/STAT3 signaling pathway. These results suggest that TFA is a potential candidate for mitigating CRC metastasis. However, further research is needed to comprehensively evaluate the efficacy and safety of TFA in animal models and clinical settings. These findings bring great hope for the development of innovative CRC treatment methods.

Transcriptomic analysis unveils alterations in the genetic expression profile of tree peony (Paeonia suffruticosa Andrews) infected by Alternaria alternata

BackgroundBlack spot disease in tree peony caused by the fungal necrotroph A. alternata, is a primary limiting factor in the production of the tree peony. The intricate molecular mechanisms underlying the tree peony resistance to A. alternata have not been thoroughly investigated.ResultsThe present study utilized high-throughput RNA sequencing (RNA-seq) technology to conduct global expression profiling, revealing an intricate network of genes implicated in the interaction between tree peony and A. alternata. RNA-Seq libraries were constructed from leaf samples and high-throughput sequenced using the BGISEQ-500 sequencing platform. Six distinct libraries were characterized. M1, M2 and M3 were derived from leaves that had undergone mock inoculation, while I1, I2 and I3 originated from leaves that had been inoculated with the pathogen. A range of 10.22–11.80 gigabases (Gb) of clean bases were generated, comprising 68,131,232 − 78,633,602 clean bases and 56,677 − 68,996 Unigenes. A grand total of 99,721 Unigenes were acquired, boasting a mean length of 1,266 base pairs. All these 99,721 Unigenes were annotated in various databases, including NR (Non-Redundant, 61.99%), NT (Nucleotide, 45.50%), SwissProt (46.32%), KEGG (Kyoto Encyclopedia of Genes and Genomes, 49.33%), KOG (clusters of euKaryotic Orthologous Groups, 50.18%), Pfam (Protein family, 47.16%), and GO (Gene Ontology, 34.86%). In total, 66,641 (66.83%) Unigenes had matches in at least one database. By conducting a comparative transcriptome analysis of the mock- and A. alternata-infected sample libraries, we found differentially expressed genes (DEGs) that are related to phytohormone signalling, pathogen recognition, active oxygen generation, and circadian rhythm regulation. Furthermore, multiple different kinds of transcription factors were identified. The expression levels of 10 selected genes were validated employing qRT-PCR (quantitative real-time PCR) to confirm RNA-Seq data.ConclusionsA multitude of transcriptome sequences have been generated, thus offering a valuable genetic repository for further scholarly exploration on the immune mechanisms underlying the tree peony infected by A. alternata. While the expression of most DEGs increased, a few DEGs showed decreased expression.

Biochemical and molecular responses to long-term salinity challenges in northern quahogs Mercenaria mercenaria

Salinity is a key environmental factor for aquatic organisms for survival, development, distribution, and physiological performance. Salinity fluctuation occurs often in estuary and coastal zones due to weather, tide, and freshwater inflow and thus heavily affects coastal marine aquaculture. The northern quahog Mercenaria mercenaria is an important aquaculture species along the Atlantic coast in the US, but information on the effects of salinity stress on physiological, immunological, and molecular responses is still scarce. The goal of this study was to investigate cellular and molecular responses through challenges of long-term hypo- and hyper-salinities in northern quahogs. The objectives were to: 1) measure the survival of market-sized quahogs under a three-month salinity challenge at 15 (hyposalinity), 25 (control), and 35 ppt (hypersalinity); 2) determine cellular changes of hemocytes through analysis of immune functions; 3) determine changes of the total free amino acid concentration in gills, and 4) evaluate the molecular responses in gills using RNAseq technology with qPCR verification. After a three-month salinity challenge, no mortality was observed, and increases in body weight were identified with a significantly higher increase in the hypersalinity group. Northern quahogs equilibrated their hemolymph osmolality with the ambient seawater and were verified to be osmoconformers. Significant differences were observed in total hemocyte concentration, lysosomal presence, ROS production, and phagocytic rate, but no differences were found in cell viability. The total free amino acid concentration within gills was positively correlated to water salinity, indicating amino acids were critical organic osmolytes. The transcriptome of gills using RNAseq revealed differential expression genes (DEG) encoding amino acid transporters (SLC6A3, SLC6A6, SLC6A13, SLC25A38), ion channel proteins (T38B1, GluCl, ATP2C1), and water channel protein (AQP8) in hyposalinity or/and hypersalinity groups, indicating these genes play critical roles in intracellular isosmotic regulation. Overall, the findings in this study provided new insights into osmoregulation in northern quahogs.

RNA Sequencing Reveals the Involvement of Serum Exosomal miRNAs in Early Pregnancy in Cattle.

Low fertility is the main cause of the low productivity in beef cattle and is mainly associated with a lack of conception after fertilization. The establishment of early pregnancy in cattle is a complex physiological process, and embryo implantation is crucial for the successful establishment of pregnancy. Exosomal miRNAs play an important role in regulating mammalian embryo implantation and development. This study used synchronous estrus technology to extract exosomes from bovine serum at 0, 14, and 21 days of early pregnancy and analyzed the expression profile of exosomal miRNAs through RNA-seq technology. We identified 472 miRNA precursor sequences and 367 mature miRNA sequences in the three sample groups, with the majority of the miRNAs having high abundance. Differentially expressed miRNAs (DEmiRNAs) were screened, and 20 DEmiRNAs were obtained. The differential expression analysis results show that compared to day 0, there were 15 DEmiRNAs in the serum on day 14 and 5 on day 21 of pregnancy. Compared to the 14th day of pregnancy, there were eight DEmiRNAs in the serum on the 21st day of pregnancy. Bioinformatics analysis shows that the target genes of DEmiRNAs regulated the signaling pathways closely related to early pregnancy, including the VEGF, NF-κB, and MAPK signaling pathways. In addition, the newly discovered miRNAs were bta-miR-3604, bta-miR-2889, bta-miR-3432a, and bta-miR-409b. These results provide a theoretical reference for screening the molecular markers for early pregnancy establishment and maternal recognition of pregnancy (MRP) in cattle and new ideas for shortening the calving interval in cows.

Reduced B cell frequencies in cord blood of HIV-exposed uninfected infants: an immunological and transcriptomic analysis.

In the course of immune development, HIV-exposed uninfected (HEU) infants exhibit abnormal immune function and increased infectious morbidity compared to HIV-unexposed uninfected (HUU) infants. Yet the specific functional phenotypes and regulatory mechanisms associated with in-utero HIV and/or ART exposure remain largely obscure. We utilized flow cytometry and RNA-seq technologies to conduct the immunological and transcriptomic profiling in cord blood from 9 HEU mother-infant pairs and 24 HUU pairs. On top of that, we compared the cord blood dataset with the maternal venous blood dataset to characterize unique effects induced by in-utero HIV and/or ART exposure. Flow cytometry immunophenotyping revealed that the level of B lymphocyte subsets was significantly decreased in HEU cord blood as compared to HUU (P < 0.001). Expression profiling-based cell abundance assessment, includes CIBERSORT and ssGSEA algorithm, showed a significantly reduced abundance of naive B cells in HEU cord blood (both P < 0.05), supporting the altered composition of B lymphocyte subsets in HEU. Functional enrichment analysis demonstrated suppressed innate immune responses and impaired immune regulatory function of B cells in HEU cord blood. Furthermore, through differential expression analysis, co-expression network analysis using WGCNA, and feature selection analysis using LASSO, we identified a 4-gene signature associated with HEU status. This signature effectively assesses B cell levels in cord blood, enabling discrimination between HEU and HUU infants. Our study provides the first comprehensive immunological and transcriptomic characterization of HEU cord blood. Additionally, we establish a 4-gene-based classifier that holds potential for predict immunological abnormalities in HEU infants.

CTGF Inhibits the Differentiation of Chicken Preadipocytes via the TGFβ/Smad3 Signaling Pathway or by Inducing the Expression of ACTG2.

Chicken is the main source of protein for humans in most parts of the world. However, excessive fat deposition in chickens has become a serious problem. This adversely affects the growth of chickens and causes economic losses. Fat formation mainly occurs through preadipocyte differentiation, and excessive fat deposition results from the accumulation of preadipocytes after differentiation. Our previous studies have found that the connective tissue growth factor (CTGF) may be an important candidate gene for fat deposition. However, its function and mechanism in preadipocyte differentiation are still unclear. In this study, the RT-qPCR and Western blot results showed that the expression of CTGF mRNA and protein in the abdominal adipose of lean chickens was significantly higher than that of fat chickens. Therefore, we studied the function and mechanism of the CTGF in the differentiation of chicken preadipocytes. Functionally, the CTGF inhibited the differentiation of chicken preadipocytes. Mechanistically, the CTGF mediated the TGFβ1/Smad3 signaling pathway, thereby inhibiting the differentiation of chicken preadipocytes. In addition, we used the unique molecular identifier (UMI) RNA-Seq technology to detect genes that can be regulated by the CTGF in the whole genome. Through transcriptome data analysis, we selected actin gamma 2 (ACTG2) as a candidate gene. Regarding the function of the ACTG2 gene, we found that it inhibited the differentiation of chicken preadipocytes. Furthermore, we found that the CTGF can inhibit the differentiation of preadipocytes through the ACTG2 gene. In summary, this study found the CTGF as a new negative regulator of chicken preadipocyte differentiation. The results of this study help improve the understanding of the molecular genetic mechanism of chicken adipose tissue growth and development and also have reference significance for the study of human obesity.

Unraveling flavivirus pathogenesis: from bulk to single-cell RNA-sequencing strategies.

The global spread of flaviviruses has triggered major outbreaks worldwide, significantly impacting public health, society, and economies. This has intensified research efforts to understand how flaviviruses interact with their hosts and manipulate the immune system, underscoring the need for advanced research tools. RNA-sequencing (RNA-seq) technologies have revolutionized our understanding of flavivirus infections by offering transcriptome analysis to dissect the intricate dynamics of virus-host interactions. Bulk RNA-seq provides a macroscopic overview of gene expression changes in virus-infected cells, offering insights into infection mechanisms and host responses at the molecular level. Single-cell RNA sequencing (scRNAseq) provides unprecedented resolution by analyzing individual infected cells, revealing remarkable cellular heterogeneity within the host response. A particularly innovative advancement, virus-inclusive single-cell RNA sequencing (viscRNA-seq), addresses the challenges posed by non-polyadenylated flavivirus genomes, unveiling intricate details of virus-host interactions. In this review, we discuss the contributions of bulk RNA-seq, scRNA-seq, and viscRNA-seq to the field, exploring their implications in cell line experiments and studies on patients infected with various flavivirus species. Comprehensive transcriptome analyses from RNA-seq technologies are pivotal in accelerating the development of effective diagnostics and therapeutics, paving the way for innovative treatments and enhancing our preparedness for future outbreaks.

Transcriptomic Profiling Reveals That the Differentially Expressed PtNAC9 Transcription Factor Stimulates the Salicylic Acid Pathway to Enhance the Defense Response against Bursaphelenchus xylophilus in Pines

Pinus, a conifer, dominates the world’s forest ecosystems. But it is seriously infected with pine wood nematode (PWN). Transcription factors (TFs) are key regulators in regulating plant resistance. However, the molecular mechanism of TFs remains thus far unresolved in P. thunbergii inoculated with Bursaphelenchus xylophilus. Here, we used RNA-seq technology to identify differentially expressed TFs in resistant and susceptible pines. The results show that a total of 186 differentially expressed transcription factors (DETFs), including 99 upregulated and 87 downregulated genes were identified. Gene ontology (GO) enrichment showed that the highly enriched differentially expressed TFs were responsible for secondary biosynthetic processes. According to KEGG pathway analysis, the differentially expressed TFs were related to chaperones and folding catalysts, phenylpropanoid biosynthesis, and protein processing in the endoplasmic reticulum. Many TFs such as NAC, LBD, MYB, bHLH, and WRKY were determined to be quite abundant in the DETFs. Moreover, the NAC transcription factor PtNAC9 was upregulated in PWN-resistant and susceptible P. thunbergii and especially distinctly upregulated in resistant pines. By purifying recombinant PtNAC9 protein in vitro, we found that overexpression of PtNAC9 at the early stage of B. xylophilus infection could reduce the degree of disease. We also demonstrated the content of salicylic acid (SA) and the related genes were increased in the PtNAC9 protein-treated plants. These results could be helpful in enhancing our understanding of the resistance mechanism underlying different resistant pine.

Comparative transcriptome and coexpression network analysis revealed the regulatory mechanism of Astragalus cicer L. in response to salt stress

BackgroundAstragalus cicer L. is a perennial rhizomatous legume forage known for its quality, high biomass yield, and strong tolerance to saline-alkaline soils. Soil salinization is a widespread environmental pressure. To use A. cicer L. more scientifically and environmentally in agriculture and ecosystems, it is highly important to study the molecular response mechanism of A. cicer L. to salt stress.ResultsIn this study, we used RNA-seq technology and weighted gene coexpression network analysis (WGCNA) were performed. The results showed 4 key modules were closely related to the physiological response of A. cicer. L. to salt stress. The differentially expressed genes (DEGs) of key modules were mapped into the KEGG database, and found that the most abundant pathways were the plant hormone signal transduction pathway and carbon metabolism pathway. The potential regulatory networks of the cytokinin signal transduction pathway, the ethylene signal transduction pathway, and carbon metabolism related pathways were constructed according to the expression pathways of the DEGs. Seven hub genes in the key modules were selected and distributed among these pathways. They may involved in the positive regulation of cytokinin signaling and carbon metabolism in plant leaves, but limited the positive expression of ethylene signaling. Thus endowing the plant with salt tolerance in the early stage of salt stress.ConclusionsBased on the phenotypic and physiological responses of A. cicer L. to salt stress, this study constructed the gene coexpression network of potential regulation to salt stress in key modules, which provided a new reference for exploring the response mechanism of legumes to abiotic stress.

Improvement of olfactory function in AD mice mediated by immune responses under 40 Hz light flickering

Background40 Hz light flickering has shown promise as a non-invasive therapeutic approach for alleviating both pathological features and cognitive impairments in Alzheimer’s disease (AD) model mice and AD patients. Additionally, vision may influence olfactory function through cross-modal sensory interactions. ObjectiveTo investigate the impact of 40 Hz light flickering on olfactory behavior in AD model mice and to explore the underlying mechanisms of this intervention. MethodsWe used immunofluorescence techniques to observe the activation of the olfactory bulb (OB) in C57BL/6J mice under 40 Hz light flickering. A buried food test was conducted to evaluate olfactory behavior in AD mice. Additionally, RNA sequencing technology was employed to detect transcriptional alterations in the OBs of AD mice following light stimulation. Results40 Hz light flickering was found to effectively activate the OB. This stimulation led to enhanced olfactory behavior and did not alter P-tau protein mRNA levels within the OBs of AD mice. RNA sequencing revealed significant transcriptional changes in the OBs under flickering, particularly related to immune responses. ConclusionVision can influence olfactory function through cross-modal sensory interactions in rodent models. 40 Hz light stimulation improved olfactory performance in AD mice. However, the improvement in olfaction in AD mice is not related to changes in P-tau mRNA levels. Instead, it may be associated with an altered immune response, providing a scientific basis for the clinical treatment of olfactory disorders in Alzheimer’s disease.