RNA-seq Technique Research Articles

Identification of potential target genes of breast cancer in response to Chidamide treatment.

Chidamide, a new chemically structured HDACi-like drug, has been shown to inhibit breast cancer, but its specific mechanism has not been fully elucidated. In this paper, we selected ER-positive breast cancer MCF-7 cells and used RNA-seq technique to analyze the gene expression differences of Chidamide-treated breast cancer cells to identify the drug targets of Chidamide's anti-breast cancer effect and to lay the foundation for the development of new drugs for breast cancer treatment. The results showed that the MCF-7 CHID group expressed 320 up-regulated genes and 222 down-regulated genes compared to the control group; Gene Ontology functional enrichment analysis showed that most genes were enriched to biological processes. Subsequently, 10 hub genes for Chidamide treatment of breast cancer were identified based on high scores using CytoHubba, a plug-in for Cytoscape: TP53, JUN, CAD, ACLY, IL-6, peroxisome proliferator-activated receptor gamma, THBS1, CXCL8, IMPDH2, and YARS. Finally, a combination of the Gene Expression Profiling Interactive Analysis database and Kaplan Meier mapper to compare the expression and survival analysis of these 10 hub genes, TP53, ACLY, PPARG, and JUN were found to be potential candidate genes significantly associated with Chidamide for breast cancer treatment. Among them, TP53 may be a potential target gene for Chidamide to overcome multi-drug resistance in breast cancer. Therefore, we identified four genes central to the treatment of breast cancer with Chidamide by bioinformatics analysis, and clarified that TP53 may be a potential target gene for Chidamide to overcome multi-drug resistance in breast cancer. This study lays a solid experimental and theoretical foundation for the treatment of breast cancer at the molecular level with Chidamide and for the combination of Chidamide.

Transcriptome analysis and prediction of the metabolic state of stress-induced viable but non-culturable Bacillus subtilis cells

Many bacteria adapt their physiology and enter the viable but non-culturable state to survive prolonged exposure to adverse environmental conditions. The VBNC cells maintain active metabolism, membrane integrity and gene transcription. However, they lose the ability to form colonies on a conventional culture media. Thus, standard colony counting methods cannot detect these alive but dormant cells. The Gram-positive bacterium Bacillus subtilis was found to enter the VBNC state when pre-exposed to osmotic stress and treated with a lethal dose of kanamycin. These cells reduced their metabolic activity, ceased growth and division and became kanamycin-tolerant. Interestingly, despite active metabolism, the majority of the kanamycin tolerant cells could not be revived on LB agar. In this study, we use a robust RNA-Seq technique to elucidate the differences in transcriptional profiles of B. subtilis VBNC cells. A comparative analysis of differently expressed genes and operons performed in this study indicates high similarities in transcriptional responses of VBNC and kanamycin-sensitive cells to antibiotic treatment. Moreover, this work reveals that VBNC cells strongly upregulate genes involved in proline uptake and catabolism, suggesting a putative role of proline as nutrient in VBNC cells.

Methods to study adult hippocampal neurogenesis in humans and across the phylogeny.

The hippocampus hosts the continuous addition of new neurons throughout life-a phenomenon named adult hippocampal neurogenesis (AHN). Here we revisit the occurrence of AHN in more than 110 mammalian species, including humans, and discuss the further validation of these data by single-cell RNAseq and other alternative techniques. In this regard, our recent studies have addressed the long-standing controversy in the field, namely whether cells positive for AHN markers are present in the adult human dentate gyrus (DG). Here we review how we developed a tightly controlled methodology, based on the use of high-quality brain samples (characterized by short postmortem delays and ≤24 h of fixation in freshly prepared 4% paraformaldehyde), to address human AHN. We review that the detection of AHN markers in samples fixed for 24 h required mild antigen retrieval and chemical elimination of autofluorescence. However, these steps were not necessary for samples subjected to shorter fixation periods. Moreover, the detection of labile epitopes (such as Nestin) in the human hippocampus required the use of mild detergents. The application of this strictly controlled methodology allowed reconstruction of the entire AHN process, thus revealing the presence of neural stem cells, proliferative progenitors, neuroblasts, and immature neurons at distinct stages of differentiation in the human DG. The data reviewed here demonstrate that methodology is of utmost importance when studying AHN by means of distinct techniques across the phylogenetic scale. In this regard, we summarize the major findings made by our group that emphasize that overlooking fundamental technical principles might have consequences for any given research field.

An inhibitor of BRD4, GNE987, inhibits the growth of glioblastoma cells by targeting C-Myc and S100A16

PurposeAmong children, glioblastomas (GBMs) are a relatively common type of brain tumor. BRD4 expression was elevated in GBM and negatively correlated with the prognosis of glioma. We investigated the anti-GBM effects of a novel BRD4 inhibitor GNE987.MethodsWe evaluated the anti-tumor effect of GNE987 in vitro and in vivo by Western blot, CCK8, flow cytometry detection, clone formation, the size of xenografts, and Ki67 immunohistochemical staining, and combined ChIP-seq with RNA-seq techniques to find its anti-tumor mechanism.ResultsIn vitro experiments showed that GNE987 significantly degraded BRD4, inhibited the proliferation of GBM cells, blocked the cell cycle, and induced apoptosis. Similarly, in vivo experiments, GNE987 also inhibited GBM growth as seen from the size of xenografts and Ki67 immunohistochemical staining. Based on Western blotting, GNE987 can significantly reduce the protein level of C-Myc; meanwhile, we combined ChIP-seq with RNA-seq techniques to confirm that GNE987 downregulated the transcription of S100A16 by disturbing H3K27Ac. Furthermore, we validated that S100A16 is indispensable in GBM growth.ConclusionGNE987 may be effective against GBM that targets C-Myc expression and influences S100A16 transcription through downregulation of BRD4.

KAT2A/E2F1 Promotes Cell Proliferation and Migration via Upregulating the Expression of UBE2C in Pan-Cancer.

Various studies have shown that lysine acetyltransferase 2A (KAT2A), E2F transcription factor 1 (E2F1), and ubiquitin conjugating enzyme E2 C (UBE2C) genes regulated the proliferation and migration of tumor cells through regulating the cell cycle. However, there is a lack of in-depth and systematic research on their mechanisms of action. This study analyzed The Cancer Genome Atlas (TCGA) to screen potential candidate genes and the regulation network of KAT2A and E2F1 complex in pan-cancer. Quantitative real-time PCR (qRT-PCR) and Western blotting (WB), cell phenotype detection, immunofluorescence co-localization, chromatin immunoprecipitation assay (ChIP), and RNA-Seq techniques were used to explore the functional of a candidate gene, UBE2C. We found that the expression of these three genes was significantly higher in more than 10 tumor types compared to normal tissue. Moreover, UBE2C was mainly expressed in tumor cells, which highlighted the impacts of UBE2C as a specific therapeutic strategy. Moreover, KAT2A and E2F1 could promote cell proliferation and the migration of cancer cells by enhancing the expression of UBE2C. Mechanically, KAT2A was found to cooperate with E2F1 and be recruited by E2F1 to the UBE2C promoter for elevating the expression of UBE2C by increasing the acetylation level of H3K9.

Dissection of the Inner Female and Male Cone Differentiation Mechanisms at Different Development Stages in Masson’s Pine (Pinus massonianaLamb.) by RNA-Seq Techniques

The differentiation of male and female cones is important for growth and reproduction for Masson’s pine (Pinus massonianaLamb.). The inner regulation mechanisms of the male and female cones differentiation will helpful for Masson’s pine breeding programs. In order to dissection molecular mechanisms ofP. massonianadifferentiation, male and female cones of early, middle and late development stages were used for comprehensive transcriptomic analysis and quantitative reverse transcriptase PCR (qRT-PCR) verifications. The total numbers of unigenes were 81,850, and mean length was 812 bp in transcriptome analysis. Gene function classification by GO analysis showed that main difference was found mainly in 3 processes, including biological process. KEGG classification showed that differential expressed genes (DEGs) were belong to metabolic pathways and transcriptional regulations. 10 DEGs expression, including flower-like protein (PmTFL), Floricaula (PmFL), APETALA2-related transcription factor2 (PmAP2) that appeared to have central roles in response to cones differentiation was verified via qRT-PCR. The transcriptomic analysis revealed difference of male and female cones differentiation were mainly related to biosynthesis of secondary metabolites, metabolic pathways genes and AP2 transcription factors. These results showed that inner mechanisms of male and female cones differentiations are related to transcriptional regulation of metabolic pathways and helpful for further selection and breeding programs ofPinus massonianaLamb.

Analysis of the miRNA expression profile of laboratory red crucian carp under low-dose caesium-137 irradiation.

Radiation can cause the differential expression of biological miRNA molecules. This research was based on the development of the laboratory red crucian carp (LRCC) to explore the feasibility of its application in the detection of low-dose ionizing radiation-induced biological damage in aquatic environments and the development of related molecular markers. Adult LRCC were irradiated with caesium-137 at 0.3 Gy, while RNA-seq and bioinformatics techniques were used to identify miRNAs that were differentially expressed relative to their levels in the nonirradiation group. Analysis of liver sections showed that liver cells in the radiation group showed nuclear pyknosis. In this study, 34 miRNAs differentially expressed in the liver of LRCC after irradiation were identified, among which seven were new crucian carp miRNAs; a total of 632 target genes were predicted in the prediction analysis. The results of comprehensive GO enrichment and KEGG pathway analyses showed that these target genes were mainly involved in energy transfer and material catabolism, especially malonyl-CoA biosynthesis, acetyl-CoA carboxylase activity, fatty acid biosynthesis and metabolism, and pyruvate metabolism; in addition, the AMPK signalling pathway was the most active pathway. This study shows that the LRCC is sensitive to radiation, or can be used as a candidate experimental animal to study the biological effects of radiation, and the screened miRNA can be used as a pre-selected biomarker for radiation damage detection and radiation biological environmental monitoring. CLINICAL TRIALS REGISTRATION: None.

P421 Whole-transcriptome analysis of Sporothrix brasiliensis grown in mold- and yeast-inducing conditions

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PM Objectives Sporothrix brasiliensis is an emerging Sporothrix species limited to Brazil capable of causing sporotrichosis in humans and animals, especially in cats. Like other pathogenic Sporothrix species, S. brasiliensis exhibits a temperature-dependent dimorphic switch and is therefore, able to undergo a reversible morphological transition (mold and yeast), in response to environmental thermal stimuli.While dimorphism appears to be essential for virulence in Sporothrix spp, the molecular mechanisms involved in this phenomenon have not yet been fully elucidated.In this study, we used the strand-specific RNA-Seq technique and bioinformatics analysis to investigate the transcriptomic signatures associated with mold and yeast phases of S. brasiliensis. Furthermore, we generated an accurate version of the S. brasiliensis genome annotation in order to perform high-quality gene expression analysis and other functional or structural genomic studies.MethodsThe whole transcriptome of S. brasiliensis ATCC-MYA-4823, grown in both yeast-inducing (YPD medium at 37°C) and mold-inducing (YPD medium at 25°C) conditions, was sequenced in this study. High-quality RNA was used to prepare Illumina TruSeq Stranded mRNA-paired-end sequencing libraries (2 × 150 bp) that were sequenced using the HiSeq-2500 platform. A total of three biological replicates were sequenced for each condition.Before transcriptome assembly, adapters and low-quality reads (Phred-score <25) were removed. The StringTie software was used to assemble the transcriptomes imported into the Apollo webtool to manually curate the genome annotation. Transcripts were investigated using TransDecoder and CPC2 programs to determine whether a gene was potentially protein-coding or non-coding. Finally, differential gene expression analysis between yeast and mold forms of S. brasiliensis was conducted using the edgeR package.ResultsIllumina sequencing resulted in a total of ∼217 million raw reads. After quality filtering and trimming, ∼99.5% of reads were used for downstream bioinformatics analysis. The updated S. brasiliensis genome annotation consisted of a total of 14 664 genes of which 10 243 protein-coding genes, 4259 lncRNAs, 140 tRNAs, and 22 rRNAs.Gene expression analysis revealed a total of 13 838 and 13 938 transcripts expressed in mold- and yeast-form, respectively. Of these, 192 and 292 were expressed exclusively in the mold and yeast-phase, respectively. Moreover, a total of 6802 genes (FDR <0.05) were differentially expressed between the two examined conditions. In particular, 3420 of these genes were up-regulated in the yeast-form (2450 coding, 970 non-coding), and 3382 genes in the filamentous form (2507 coding, 875 non-coding). The raw reads have been deposited into the SRA database and are available under BioProjectID: PRJNA646214.ConclusionsThe characterization of the whole-transcriptome of S. brasiliensis mycelial and yeast-like forms represents an essential starting point for investigating the molecular pathways and regulatory frameworks associated with these two morphological stages. Our results provide new insight into global gene expression profiles of S. brasiliensis, emphasizing the role of non-coding RNAs in its complex transcriptional network.All transcriptomic data have also been integrated into the ‘Sporothrix Genome DataBase’ (www. sporothrixgenomedatabase.unime.it) in order to expand the current knowledge of Sporothrix genomics and to allow a more in-depth structural exploration of S. brasiliensis gene models, including gene expression patterns related to its saprophytic and pathogenic lifestyle.

Abstract A003: ASAP1 regulates myogenic differentiation in rhabdomyosarcoma by modulating YAP localization

Abstract Despite aggressive, multimodal therapies, the prognosis of patients with refractory or recurrent rhabdomyosarcoma (RMS) has not improved in four decades. RMS is thought to arise due to defective differentiation in skeletal muscle precursor cells. Differentiation-inducing therapy may improve outcomes for RMS patients with advanced disease. In RAS-mutant PAX fusion-negative RMS (FN-FMS), targeting ERK1/2 activation through MEK1/2 inhibition (MEKi) induces differentiation, slows tumor growth, and extends survival in preclinical studies. However, the duration of therapeutic response is short lived. Identifying additional targets for differentiation therapy is necessary. We propose that ASAP1, an Arf GTPase-activating protein (Arf GAP) highly expressed in FN-RMS and implicated in breast and colorectal cancer progression as well as osteogenic and retinal endothelium differentiation, contributes to FN-RMS differentiation. We find that knockdown (KD) of ASAP1 inhibits differentiation in myoblasts and FN-RMS cells, and its overexpression enhances differentiation. Moreover, myogenic differentiation-associated genes are not enriched upon loss of ASAP1. We discover that KD of ASAP1 homologs, ASAP2 and ASAP3, also blocks differentiation. However, loss of a paralogous Arf GAP, ARAP1, does not, indicating that ASAP regulates differentiation through a mechanism not explained by GAP activity alone. Interestingly, KD of Arf1 or Arf5, small GTPases inactivated by ASAP, also blocks differentiation of FN-RMS, suggesting a novel relationship between Arf and ASAP. Using RNAseq, qPCR, and immunoblotting techniques, we show that loss of ASAP blocks myogenic transcription factor expression. To determine the mechanism by which ASAP1 regulates myogenic transcription factor expression, we investigate the PI3K/AKT, MAPK, and Hippo pathways, which are known to be regulated by ASAP1. While the PI3K/AKT and MAPK pathways are unaffected, downstream components of the Hippo pathway are modulated by ASAP1 KD in FN-RMS cells treated with MEKi. YAP and TAZ are transcriptional coactivators that promote proliferation. Upon Hippo pathway activation, YAP/TAZ activity is blocked by phosphorylation at a nuclear exclusion site. Using cell fractionation and immunoblotting techniques, we find that induction of differentiation by MEKi increases TAZ phosphorylation, excluding it from the nucleus and blocking pro-proliferative transcription. Further, upon ASAP1 KD, TAZ phosphorylation is blocked, restoring nuclear localization, and inhibiting MEKi-induced differentiation. In conclusion, we discover that ASAP1 regulates MEKi-induced differentiation of FN-RMS cells by modulating TAZ localization and supports targeting the YAP pathway as a strategy for FN-RMS differentiation therapy. Our work also identifies ASAP1 as a potential effector of Arf1 activity, a novel interaction of these two proteins. The results described herein provide a deeper understanding of differentiation in FN-RMS and establish the groundwork for advancing differentiation therapy in FN-RMS. Citation Format: Katie E. Hebron, Olivia Feehan-Nelson, Angela Kim, Xiaoying Jian, Sofia A Girald, Paul Randazzo, Marielle E. Yohe. ASAP1 regulates myogenic differentiation in rhabdomyosarcoma by modulating YAP localization [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr A003.

Transcriptional sequencing analysis reveals the potential use of deer antler for “tonifying the kidney and strengthening bone”

BackgroundIt is recorded in the Chinese Pharmacopoeia that deer antlers can be used to tonify the kidney and strengthen bone. Although numerous studies have demonstrated that deer antler has protective effects on the kidney and bone, its molecular mechanisms remain to be elucidated. The aim of this study was to explore the molecular mechanism underlying its effects on the bone and kidney.MethodsWater extract of pilose antler was prepared and then filtered through a 0.45 μm Hollow Fiber Cartridge (GE Healthcare, USA). The filtrate was freeze-dried by a Heto PowerDry LL3000 Freeze Dryer (Thermo, USA) and stored at − 80 °C. Rats were treated with deer antler extract (DAE) prepared in advance, and gene regulatory network in the kidney and bone was detected by RNA-Seq technique. Micro-CT was used to detect bone trabecular formation, bone mineral density (BMD) and bone volume fraction (BV/TV).ResultsThe results demonstrate that DAE could jointly heighten renal function by maintaining renal homeostasis, combating renal fibrosis, and reducing renal inflammation by regulating ion transport. Furthermore, DAE can strengthen the bone system by stimulating osteoblast differentiation and regulating bone regeneration and the bone marrow microenvironment. Micro-CT results confirmed that DAE can promote bone trabecular formation and increase BMD and BV/TV. We also identified many genes that can regulate both the kidney and bone simultaneously, which explained the theory of “kidney governing bone” at the molecular level and provided possible strategies for further application of this theory to treat diseases.ConclusionsDAE enhances renal function, maintains renal homeostasis, positively regulates skeletal system development, and increases bone mineral density. The underlying mechanism involves improving the expression levels of functional genes involved in renal function and regulation and repair, as well as genes that positively regulate skeletal system development.

Transcription analysis of chicken embryo fibroblast cells infected with the recombinant avian leukosis virus isolate GX14FF03.

Infection with recombinant avian leukosis virus (ALV) has previously been linked to malignancies and immunosuppression. However, the processes behind the unique pathophysiology of recombinant ALV are poorly understood. In this study, we analyzed gene expression patterns in chicken fibroblast cells (CEFs) infected with the recombinant ALV isolate GX14FF03 and used the RNA-seq technique to perform a complete analysis of the transcribed mRNAs. A total of 907 significant differentially expressed genes (SDEGs) were identified. Among these SDEGs, the most significantly upregulated gene was interleukin 8-like 1 (IL8L1), while the most significantly downregulated gene was fibroblast growth factor 16 (FGF16). The 907 SDGEs were highly enriched (p < 0.05) for 252 Gene Ontology (GO) terms, including 197 BP, 3 CC, and 52 MF. According to KEGG data analysis, SDEGs are implicated in eight significant pathways (p < 0.05). Furthermore, protein-protein interaction (PPI) network analysis revealed that IL8L1 interacts with 17 genes. These findings shed light on the molecular mechanisms involved in recombinant ALV infection by showing the mRNA expression profile in CEFs infected with GX14FF03 virus.

Investigation of the mechanisms leading to human sperm DNA damage based on transcriptome analysis by RNA-seq techniques

Research questionWhat are the molecular mechanisms leading to human sperm DNA damage? DesignSemen samples were collected and the sperm DNA fragmentation index (DFI) was assessed. Differentially expressed RNA in spermatozoa with a high (DFI ≥30%, experimental group) or normal (DFI <30%, control group) DFI were identified by RNA-sequencing (RNA-seq) technology, and Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis was performed. Three differentially expressed RNA related to sperm DNA damage and repair, namely PMS1, TP53BP1 and TLK2, were validated using real-time quantitative (RT-qPCR). ResultsA total of 19,970 expressed RNA were detected in the two groups. Compared with the control group, the expression levels of 189 RNA in the experimental group were significantly increased and those of 163 genes decreased. Gene Ontology enrichment analysis showed that these RNA were mainly concentrated in the ATPase-dependent transmembrane transport complex, extracellular exosome, somatic cell DNA recombination, protein binding, cytoplasm and regulation of localization. KEGG pathway analysis showed that these RNA were mainly related to the PI3K-Akt signalling pathway, endocytosis, p53 signalling pathway and cGMP-PKG signalling pathway. The RT-qPCR results showed that the expression levels of PMS1, TP53BP1 and TLK2 in the experimental group were significantly lower than in the control group (P = 0.01, 0.015 and 0.004, respectively), which was identical to the results of RNA sequencing. ConclusionsDifferentially expressed RNA related to sperm DNA damage and repair may be identified by RNA-seq technology, which provides new insights into the understanding of sperm DNA damage and repair, and will help to discover new biomarkers related to sperm DNA damage.

Macrophages induce cardiomyocyte ferroptosis via mitochondrial transfer.

Mitochondrial transfer is a new cell-to-cell communication manner. Whether the mitochondrial transfer is also involved in the macrophage infiltration-induced cardiac injury is unclear. This study aimed to determine whether macrophage mitochondria can be transferred to cardiomyocytes, and to investigate its possible role and mechanism. Mitochondrial transfer between macrophages and cardiomyocytes was detected using immunofluorescence staining and flow cytometry. Cellular metabolites were analyzed using LC-MS technique. Differentially expressed mRNAs were identified using RNA-seq technique. (1) After cardiomyocytes were cultured with macrophage-conditioned medium (COND+group), macrophage-derived mitochondria have been found in cardiomyocytes, which could be blocked by dynasore (an inhibitor of clathrin-mediated endocytosis). (2) Compared with control (CM) group, there were 545 altered metabolites found in COND+group, most of which were lipids and lipid-like molecules. The altered metabolites were mainly enriched in the β-oxidation of fatty acids and glutathione metabolism. And there were 4824 differentially expressed mRNAs, which were highly enriched in processes like lipid metabolism-associated pathway. (3) Both RNA-seq and qRT-PCR results found that ferroptosis-related mRNAs such as Ptgs2 and Acsl4 increased, and Gpx4 mRNA decreased in COND+group (P<0.05 vs CM group). (4) The levels of cellular free Fe2+ and mitochondrial lipid peroxidation were increased; while GSH/GSSG ratio, mitochondrial aspect ratio, mitochondrial membrane potential, and ATP production were decreased in cardiomyocytes of COND+group (P<0.05 vs CM group). All the above phenomena could be blocked by a ferroptosis inhibitor ferrostatin-1 (P<0.05). Macrophages could transfer mitochondria to cardiomyocytes. Macrophage-derived mitochondria were internalized into cardiomyocytes through clathrin- and/or lipid raft-mediated endocytosis. Uptake of exogenous macrophage mitochondria induced cardiomyocyte injury via triggering ferroptosis.

Most reactions leading to neuropathic pain at dorsal root ganglion of rats with spinal nerve ligation have occurred in the early phase

In order to study the mechanism of early and late neuropathic pain, Ribonucleic acid sequencing (RNA-seq) technique was used to analyze the dorsal root ganglion (DRG) transcriptome of rat induced by spinal nerve ligation (SNL). It was found that most of the reactions leading to neuropathic pain at dorsal root ganglion, such as pro-inflammatory response and immunity, occurred in the early phase.378 genes are up-regulated and 42 genes are down-regulated in the early phase, while in the late phase, there are 27 up-regulated genes and 23 down-regulated genes. Four new pain related genes are found: Cd8a, Bub1b, Ccna2 and Cdk1. From the analysis of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and Gene Ontology (GO) functions enriched by differentially expressed genes (DEGs), dorsal root ganglion should carry out immunities, pro-inflammations and related compensatory regulation in response to peripheral nerve injury in the early phase. The late phase is characterized by maintaining long-term or chronic inflammation.

Comparative transcriptome and proteome profiles reveal the regulation mechanism of low temperature on garlic greening

Garlic stored at low temperature (0–13 ℃) for some times and subsequently crushed and placed at room temperature would turn green, while the one stored at high temperature (30 ℃) would not. In order to elucidate the regulatory mechanism of low temperature on garlic greening, transcriptome and proteome profiles of garlic stored at 4 ℃ and 30 ℃ were explored by RNA-seq and iTRAQ techniques. Principal component analysis showed that garlic at different storage temperatures were of significant differences on both gene and protein levels. 14,381 and 861 differential expression genes (DEGs) and proteins (DEPs) were identified respectively, in which 268 factors were shared according to their joint analysis, including 186 (144) up-regulated genes (proteins) and 82 (124) down-regulated genes (proteins) in comparing garlic stored at 4 ℃ with ones at 30 ℃. These 268 factors were mainly attributed to biological process (metabolic process) and molecular function (catalytic activity, binding) categories by Gene Ontology classification. The KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways enrichment of DEGs and DEPs revealed that GSSG production, GSH degradation, amino acid biosynthesis (cysteine and methionine) and energy metabolism (TCA and HMP cycles) were promoted by low-temperature storage to responding to oxidative stress and prepared for pigment synthesis in garlic. These results provide valuable information for the regulation of garlic greening during processing.

The Gene Expression Profile Differs in Growth Phases of the Bifidobacterium Longum Culture.

To date, transcriptomics have been widely and successfully employed to study gene expression in different cell growth phases of bacteria. Since bifidobacteria represent a major component of the gut microbiota of a healthy human that is associated with numerous health benefits for the host, it is important to study them using transcriptomics. In this study, we applied the RNA-Seq technique to study global gene expression of B. longum at different growth phases in order to better understand the response of bifidobacterial cells to the specific conditions of the human gut. We have shown that in the lag phase, ABC transporters, whose function may be linked to active substrate utilization, are increasingly expressed due to preparation for cell division. In the exponential phase, the functions of activated genes include synthesis of amino acids (alanine and arginine), energy metabolism (glycolysis/gluconeogenesis and nitrogen metabolism), and translation, all of which promote active cell division, leading to exponential growth of the culture. In the stationary phase, we observed a decrease in the expression of genes involved in the control of the rate of cell division and an increase in the expression of genes involved in defense-related metabolic pathways. We surmise that the latter ensures cell survival in the nutrient-deprived conditions of the stationary growth phase.

Integration of RNA-seq and ATAC-seq identifies muscle-regulated hub genes in cattle.

As the main product of livestock, muscle itself plays an irreplaceable role in maintaining animal body movement and regulating metabolism. Therefore, it is of great significance to explore its growth, development and regeneration to improve the meat yield and quality of livestock. In this study, we attempted to use RNA-seq and ATAC-seq techniques to identify differentially expressed genes (DEGs) specifically expressed in bovine skeletal muscle as potential candidates for studying the regulatory mechanisms of muscle development. Microarray data from 8 tissue samples were selected from the GEO database for analysis. First, we obtained gene modules related to each tissue through WGCNA analysis. Through Gene Ontology (GO) functional annotation, the module of lightyellow (MElightyellow) was closely related to muscle development, and 213 hub genes were screened as follow-up research targets. Further, the difference analysis showed that, except for PREB, all other candidate hub genes were up-regulated (muscle group vs. other-group). ATAC-seq analysis showed that muscle-specific accessible chromatin regions were mainly located in promoter of genes related to muscle structure development (GO:0061061), muscle cell development (GO:0055001) and muscle system process (GO:0003012), which were involved in cAMP, CGMP-PKG, MAPK, and other signaling pathways. Next, we integrated the results of RNA-seq and ATAC-seq analysis, and 54 of the 212 candidate hub genes were identified as key regulatory genes in skeletal muscle development. Finally, through motif analysis, 22 of the 54 key genes were found to be potential target genes of transcription factor MEF2C. Including CAPN3, ACTN2, MB, MYOM3, SRL, CKM, ALPK3, MAP3K20, UBE2G1, NEURL2, CAND2, DOT1L, HRC, MAMSTR, FSD2, LRRC2, LSMEM1, SLC29A2, FHL3, KLHL41, ATXN7L2, and PDRG1. This provides a potential reference for studying the molecular mechanism of skeletal muscle development in mammals.

Upregulation of Netrin-1 in the hippocampus mediates the formation of visceral hypersensitivity induced by maternal separation

Early adverse life events (EALs), such as maternal separation (MS), can cause visceral hypersensitivity, which is thought to be a key pathophysiological mechanism of irritable bowel syndrome (IBS). Previous studies mainly focused on EALs-induced visceral hypersensitivity in adulthood but did not consider that it may have occurred in the preadult period. We previously found that rats who experienced MS suffered from visceral hypersensitivity starting from the post-weaning period. Moreover, the hippocampus is considered to be critical in regulating the formation of visceral hypersensitivity induced by MS. But the underlying mechanisms throughout different life periods are unclear. In this study, behavioral tests, RNA-seq, lentiviral interference, and molecular biology techniques were applied to investigate the molecular mechanism in the hippocampus underlying MS-induced long-lasting visceral hypersensitivity. It was found that both visceral sensitivity and anxiety-like behaviors were significantly increased in MS rats in post-weaning, prepubertal, and adult periods, especially in the prepubertal period. Subsequently, RNA-seq targeting the hippocampus identified that the expression level of Netrin-1 was significantly increased in all periods, which was further confirmed by quantitative real-time PCR and Western blot. Knocking-down hippocampal Netrin-1 in the post-weaning period by lentivirus interference alleviated visceral hypersensitivity and anxiety-like behaviors of MS rats in the later phase of life. In addition, deleted in colorectal cancer (DCC), instead of neogenin-1(Neo-1) or uncoordinated (UNC5), was proved to be the specific functional receptor of Netrin-1 in regulating visceral hypersensitivity, whose upregulation may result in the most severe symptoms in the prepubertal period. Furthermore, the activation of the Netrin-1/DCC pathway could enhance long-term potentiation (LTP) in the hippocampus, probably via recruitment of the AMPA receptor subunit GluA1, which finally resulted in the formation of visceral hypersensitivity. These novel findings suggest that long-lasting over-expression of Netrin-1 can mediate visceral hypersensitivity and anxiety disorder from the post-weaning period to adulthood by activating DCC/GluA1 pathway in the hippocampus. Moreover, early intervention of Netrin-1 in the post-weaning period could lead to significant symptom relief afterward, which provides evidence that the Netrin-1/DCC/GluA1 signaling pathway may be a potential therapeutic target for the treatment of visceral hypersensitivity in clinics.

Poly(a) selection introduces bias and undue noise in direct RNA-sequencing

BackgroundGenome-wide RNA-sequencing technologies are increasingly critical to a wide variety of diagnostic and research applications. RNA-seq users often first enrich for mRNA, with the most popular enrichment method being poly(A) selection. In many applications it is well-known that poly(A) selection biases the view of the transcriptome by selecting for longer tailed mRNA species.ResultsHere, we show that poly(A) selection biases Oxford Nanopore direct RNA sequencing. As expected, poly(A) selection skews sequenced mRNAs toward longer poly(A) tail lengths. Interestingly, we identify a population of mRNAs (> 10% of genes’ mRNAs) that are inconsistently captured by poly(A) selection due to highly variable poly(A) tails, and demonstrate this phenomenon in our hands and in published data. Importantly, we show poly(A) selection is dispensable for Oxford Nanopore’s direct RNA-seq technique, and demonstrate successful library construction without poly(A) selection, with decreased input, and without loss of quality.ConclusionsOur work expands the utility of direct RNA-seq by validating the use of total RNA as input, and demonstrates important technical artifacts from poly(A) selection that inconsistently skew mRNA expression and poly(A) tail length measurements.

Skin immune response to Aeromonas hydrophila infection in crucian carp Carassius auratus revealed by multi-omics analysis

Fish skin is an essential protective barrier and functions as the first line of immune defense against pathogens. However, the molecular mechanism at the proteome-level remains unclear in the skin of fish. In this study, the comparative proteomics of skin immune responses of crucian carp Carassius auratus infected with Aeromonas hydrophila was investigated by isobaric tags for relative and absolute quantification (iTRAQ), two-dimensional gel electrophoresis combined with mass spectrometry (2-DE/MS) as well as high-throughput transcriptome (RNA-seq) techniques. A total of 241 and 178 differentially expressed proteins (DEPs) at 6 and 12 h post-infection (hpi) were respectively identified by iTRAQ, and key-DEPs were furtherly verified with 2-DE/MS analysis. GO and KEGG analysis showed that these DEPs were mostly related to metabolism, regulation of the cytoskeleton, stress and immune responses. Co-association results of proteome and transcriptome revealed the lysozyme (LYZ), complement C3, DnaJ (Hsp40) homolog subfamily C member 8 (DNAJC8) and allograft inflammatory factor 1-like (AIF1L) play important roles in skin immune responses of crucian carp. The significantly up-regulated expression of detected immune-related genes (c3, mapk3, f5, nlr, hsp90, itgb2, fnl, flnca, p47, mhc and pros1) were validated by qRT-PCR analysis. To our knowledge, this is first report on multi-omics analysis of the differential proteomics for the skin immune response of C. auratus against A.hydrophila infection, which contribute to the understanding the mechanisms of skin mucosal immunity in cyprinid fish.