Downregulated Differentially Expressed Genes Research Articles

P180 RNA sequencing of regulatory T cells sorted from Rheumatoid Arthritis peripheral blood

Abstract Background/Aims In the presence of an inflammatory environment, regulatory T cells (Tregs) have the ability to adopt an effector T helper(Th)-like phenotype, enabling them to produce pro-inflammatory cytokines, particularly IFN-γ (resembling Th1-like Tregs) and IL-17(resembling Th17-like Tregs). This adaptability of Tregs may be driven by the increased activation of effector pathways. In order to unveil distinct gene expression patterns while simultaneously characterizing Tregs based on their phenotype, we did RNA sequencing of sorted Tregs from the peripheral blood(PB) of individuals with rheumatoid arthritis(RA), osteoarthritis(OA), and healthy controls(HC). Methods Phenotyping of Tregs, Tregs expressing CXCR3(Th1-like Treg), CCR6(Th17-like Treg) from PB and synovial fluid(SF) of RA(n = 20 each), OA(n = 20 each) and HC (n = 20) was done using flow cytometry. Cell frequencies between the groups were compared using Student t test or one-way ANOVA (Tukey's post hoc Test) as appropriate; p < 0.05 was considered as statistically significant. RNA was extracted from the sorted PB Tregs of RA, OA and HC(n = 3 each) and was sequenced using Illumina Novaseq 6000 platform. Sample clustering was done using principal component analysis generated using DeSeq2. Genes were categorized as upregulated or downregulated based on their log2 fold change cut off of 2. Significantly upregulated and downregulated differentially expressed genes (DEGs) were shortlisted using a false discovery rate(FDR) corrected p-value cut-off of ≤ 0.05. Heatmaps and volcano plots were generated using R package. Functional pathway enrichment analysis was done using GO and KEGG software. Results Proportion of Tregs in the PB of RA was significantly lower than OA and HC. This difference was not observed in the SF. Both RA PB and SF exhibited higher percentages of Th1 and Th17-like Tregs when compared to OA and HC (Table 1A).DEG analysis revealed altered gene regulation in RA Tregs compared to Tregs from HC and OA. Compared to HC, genes related to TCR signaling, cytokine signaling, and chemokine signaling pathways were enriched in RA. In contrast, compared to OA, an upregulation of pathway genes such as NR4A1(PI3K-Akt signaling), and TNFRSF1B(TNF signaling) was noted in RA. Furthermore, genes associated with Th1 differentiation and activation (IFNGR1, IL12RB1, STAT4) and genes associated with Th17 profile(RORC, STAT3, IL6R, IL17B) were upregulated in RA relative to HC and but not in OA compared to to HC.(Table 1B). Additionally, genes involved in glucose metabolism, purine metabolism, oxidative phosphorylation, and pyrimidine metabolism exhibited distinct patterns of regulation in RA. Conclusion Our Treg phenotyping data shows that there is higher conversion of Tregs into Th1 and Th17 like Tregs in RA and transcriptome data indicates cytokine(IFNγ, IL-6, IL-12 & IL-17) mediated transcriptional changes likely drove the conversion of Tregs towards Th1 and Th17 phenotype. Our data also suggests a possible metabolic re-programming of Tregs towards an inflammatory phenotype affecting its stability. Disclosure V. Kommoju: None. C. mariaselvam: None. S. Vembar: None. S. Bulusu: None. C. Kavadichanda: None. M. Thabah: None. V. Negi: None.

The phloem protein 2 (PP2) is positively regulated by ThNAC13 that enhances salt tolerance of Tamarix

NAC transcription factors play a significant role in regulating plant responses to biological processes and environmental stresses. Our previous study revealed 21 ThNAC proteins, and ThNAC13 is a nuclear protein that responds to saline stress in Tamarix hispida. To determine whether ThNAC13 regulates downstream genes, RNA sequencing (RNA-seq) and DNA affinity purification sequencing (DAP-seq) were used to identify the binding sites of ThNAC13 at the whole-genome level. In RNAi-silenced ThNAC13 transgenic Tamarix, a total of 4 downregulated differentially expressed genes (DEGs) and 114 peaks strongly associated with four different motifs were identified compared with those in empty vector transgenic Tamarix under salt stress. The c48149.graph_c0 gene contains the key domain of the phloem protein 2 (PP2) protein and is a candidate gene downstream of ThNAC13. Yeast one-hybrid (Y1H), chromatin immunoprecipitation (ChIP) and dual-luciferase reporter (LUC) assays were used to identify ThNAC13 as an upstream regulator of ThPP2. Transgenic Tamarix plants in which ThPP2 was transiently expressed or silenced via RNAi were used for functional gain and loss experiments. The ThPP2-overexpressing plants exhibited decreased hydrogen peroxide (H2O2), superoxide (O2•−), and electrolyte leakage (EL) and malondialdehyde (MDA) levels and increased superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) activity. In contrast, transient RNAi-mediated silencing of ThPP2 in Tamarix had the opposite effect. ThNAC13 binds to the promoter region of the ThPP2 gene and reduces the reactive oxygen species (ROS) accumulation and enhances antioxidant enzyme activity, positively regulating salt stress tolerance in Tamarix.

OsRH52A, a DEAD-Box protein, is required for embryo sac development by regulating functional megaspore specification in rice.

The development of the embryo sac is an important factor affecting seed setting in rice. Numerous genes associated with embryo sac (ES) development have been identified in plants. However, the function of the DEAD-box RNA helicase family genes on ES is poorly known in rice. Here, we characterized a rice DEAD-box protein, OsRH52A, which was localized in the nucleus and cytoplasm and highly expressed in the floral organs in rice. The knockout mutant, rh52a, displayed partial ES sterility, including degenerated ES (21.0%) and the presence of double-female-gametophyte (DFG) structure (11.8%). The DFG developed from two functional megaspores (FM) near the chalazal end in one ovule, and 3.4% of DFG could fertilize via the sac near the micropylar pole in rh52a. OsRH52A was found to interact with OsMFS1 and ZIP4, both of which play a role in homologous recombination in rice meiosis. RNA-seq identified 234 down-regulated differentially expressed genes (DEGs) associated with reproductive development, including the two genes, OsMSP1 and HSA1b, required for female germline cell specification. Taken together, our study demonstrated that OsRH52A is essential for the development of the embryo sac and provided cytological evidence regarding the formation of DFG.

Deciphering Microbiome, Transcriptome, and Metabolic Interactions in the Presence of Probiotic Lactobacillus acidophilus against Salmonella Typhimurium in a Murine Model.

Salmonella enterica serovar Typhimurium (S. Typhimurium), a foodborne pathogen that poses significant public health risks to humans and animals, presents a formidable challenge due to its antibiotic resistance. This study explores the potential of Lactobacillus acidophilus (L. acidophilus 1.3251) probiotics as an alternative strategy to combat antibiotic resistance associated with S. Typhimurium infection. In this investigation, twenty-four BALB/c mice were assigned to four groups: a non-infected, non-treated group (CNG); an infected, non-treated group (CPG); a group fed with L. acidophilus but not infected (LAG); and a group fed with L. acidophilus and challenged with Salmonella (LAST). The results revealed a reduction in Salmonella levels in the feces of mice, along with restored weight and improved overall health in the LAST compared to the CPG. The feeding of L. acidophilus was found to downregulate pro-inflammatory cytokine mRNA induced by Salmonella while upregulating anti-inflammatory cytokines. Additionally, it influenced the expression of mRNA transcript, encoding tight junction protein, oxidative stress-induced enzymes, and apoptosis-related mRNA expression. Furthermore, the LEfSe analysis demonstrated a significant shift in the abundance of critical commensal genera in the LAST, essential for maintaining gut homeostasis, metabolic reactions, anti-inflammatory responses, and butyrate production. Transcriptomic analysis revealed 2173 upregulated and 506 downregulated differentially expressed genes (DEGs) in the LAST vs. the CPG. Functional analysis of these DEGs highlighted their involvement in immunity, metabolism, and cellular development. Kyoto Encyclopedia of Genes and Genome (KEGG) pathway analysis indicated their role in tumor necrosis factor (TNF), mitogen-activated protein kinase (MAPK), chemokine, Forkhead box O (FOXO), and transforming growth factor (TGF-β) signaling pathway. Moreover, the fecal metabolomic analysis identified 929 differential metabolites, with enrichment observed in valine, leucine, isoleucine, taurine, glycine, and other metabolites. These findings suggest that supplementation with L. acidophilus promotes the growth of beneficial commensal genera while mitigating Salmonella-induced intestinal disruption by modulating immunity, gut homeostasis, gut barrier integrity, and metabolism.

Differentially expressed genes involved in immune pathways following Vibro parahaemolyticus challenge: A study on red swamp crayfish Procambarus clarkii

This study conducted a comprehensive analysis of the damage mechanism of Vibrio parahaemolyticus (V. parahaemolyticus) on crayfish, utilizing pathological section observations, biochemical indicator detection, and transcriptomics analysis. Histopathological examination showed fragmentation of the gill lamellae, spotted pigment deposition on the branchial membrane, degeneration of epithelial cells, and disruption of the normal structure of gill tissues. Biochemical tests indicated a significant decrease in the activity of antioxidant-related enzymes in crayfish injected with V. parahaemolyticus compared with the phosphate-buffered saline group. Transcriptomics analysis identified 2307 upregulated and 1721 downregulated differentially expressed genes (DEGs). Gene ontology enrichment analysis highlighted that DEGs were primarily involved in the regulation of lipid metabolic processes, cellular nitrogen compound biosynthesis and metabolic processes, and the negative regulation of neuron death. Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed significant enrichment in N-glycan biosynthesis, fatty acid metabolism, and the mTOR signaling pathway. Gene set enrichment analysis indicated pathways related to immunity and oxidative stress, including oxidative phosphorylation and tyrosine metabolism. Quantitative reverse transcription–polymerase chain reaction results for selected genes involved in the oxidative phosphorylation pathway and immune-related genes corroborated the RNA-Seq trends, confirming the transcriptomics data's reliability. The findings suggest that infection with V. parahaemolyticus leads to excessive oxidative stress due to bacterial proliferation, overwhelming the crayfish's antioxidative defense. This causes a continuous increase in reactive oxygen species levels, leading to irreversible damage to the oxidative phosphorylation and innate immune systems. This study elucidated the mechanism of oxidative stress damage in crayfish following V. parahaemolyticus infection, providing valuable insights for reducing bacterial and viral diseases in aquaculture. A deeper understanding of pathogen affects the physiology and immune systems of aquatic organisms can help develop more effective prevention and control strategies, thereby improving the efficiency and sustainability of aquaculture.

Identification of Down-Expressed CRNN Associated with Cancer Progression and Poor Prognosis in Laryngeal Squamous Cell Carcinoma.

The prevalence of laryngeal squamous cell carcinoma (LSCC) is increasing, and it poses a significant threat to human health; therefore, identifying specific targets for LSCC remains crucial. Bioinformatics analysis was used to compare the different expression genes expressed in LSCC. Immunohistochemical assay and western blotting were used to analysis protein expression. Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide)((4,5 Dimethyl thiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide)4,5 Dimethyl thiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide (MTT) and 5-ethynyl 2'-deoxyuridine (Edu) assay. Flow cytometry was used to measure the cell cycle. Cell migration was measured by wound healing assay and transwell assay. Our analysis revealed 36 upregulated and 65 downregulated differentially expressed genes (DEGs) when comparing LSCC tumors to adjacent tissues, with cornulin (CRNN) identified as a key hub gene connecting these DEGs. We observed a consistent downregulation of CRNN expression in LSCC cell lines and tissues and was associated with poor patient survival and the tumor microenvironment. CRNN overexpression was found to significantly inhibit cell growth, cell cycle progression, migration and invasion, while CRNN knockdown had the opposite effects. Additionally, in vivo experiments demonstrated that CRNN overexpression suppressed tumor growth in nude mice. CRNN functions as a potential tumor suppressor and regulates important aspects of LSCC, providing valuable insights into the role of CRNN in LSCC pathogenesis and potential for targeted therapeutic interventions.

Integrated analysis reveals the regulatory mechanism of the neddylation inhibitor MLN4924 on the metabolic dysregulation in rabbit granulosa cells

BackgroundNeddylation, an important post-translational modification (PTM) of proteins, plays a crucial role in follicular development. MLN4924 is a small-molecule inhibitor of the neddylation-activating enzyme (NAE) that regulates various biological processes. However, the regulatory mechanisms of neddylation in rabbit ovarian cells have not been emphasized. Here, the transcriptome and metabolome profiles in granulosa cells (GCs) treated with MLN4924 were utilized to identify differentially expressed genes, followed by pathway analysis to precisely define the altered metabolisms.ResultsThe results showed that 563 upregulated and 910 downregulated differentially expressed genes (DEGs) were mainly enriched in pathways related to cancer, cell cycle, PI3K-AKT, progesterone-mediated oocyte maturation, and PPAR signaling pathway. Furthermore, we characterized that MLN4924 inhibits PPAR-mediated lipid metabolism, and disrupts the cell cycle by promoting the apoptosis and proliferation of GCs. Importantly, we found the reduction of several metabolites in the MLN4924 treated GCs, including glycerophosphocholine, arachidic acid, and palmitic acid, which was consistent with the deregulation of PPAR signaling pathways. Furthermore, the increased metabolites included 6-Deoxy-6-sulfo-D-glucono-1,5-lactone and N-Acetyl-D-glucosaminyldiphosphodolichol. Combined with transcriptome data analyses, we identified genes that strongly correlate with metabolic dysregulation, particularly those related to glucose and lipid metabolism. Therefore, neddylation inhibition may disrupt the energy metabolism of GCs.ConclusionsThese results provide a foundation for in-depth research into the role and molecular mechanism of neddylation in ovary development.

Molecular insights into a non-lethal yellow bud mutant in Ilex × ‘Nellie R. Stevens’

While the mechanisms behind the yellow phenotype have been widely investigated in model plant seedlings, research on leaf mutations in woody species has been restricted. The purpose of this research is to characterize a holly bud mutant of Ilex × ‘Nellie R. Stevens’ with a non-lethal yellow phenotype. Through cytological, physiological, metabolomic, and transcriptomic analyses, we identified remarkable differences between the mutant and its wild-type. The mutant exhibited abnormal chloroplasts, reduced chlorophyll levels, and elevated carotenoid/chlorophyll ratios. Metabolomic and transcriptomic analyses identified differentially expressed genes (DEGs) associated with chlorophyll and heme biosynthesis disruption, contributing to the chlorophyll-deficient phenotype. Moreover, genes associated with carotenoid metabolism were significantly down-regulated, linking to decreased carotenoid content and impacting photoprotection. Down-regulated DEGs in photosynthesis-related underscored the mutant's diminished photosynthetic activity. Additionally, the study identified 382 DEGs encoding putative transcription factors such as bHLHs, NACs, and GLKs. The study suggests an intricate interplay between chlorophyll, heme and carotenoid metabolism as major regulators of leaf mutation in I. × ‘Nellie R. Stevens.’ These insights contribute to the understanding of leaf color variation, enabling targeted strategies for developing desirable ornamental cultivars.

Phenotypic divergence between broiler and layer chicken lines is regulated at the molecular level during development

BackgroundUnderstanding the molecular underpinnings of phenotypic variations is critical for enhancing poultry breeding programs. The Brazilian broiler (TT) and laying hen (CC) lines exhibit striking differences in body weight, growth potential, and muscle mass. Our work aimed to compare the global transcriptome of wing and pectoral tissues during the early development (days 2.5 to 3.5) of these chicken lines, unveiling disparities in gene expression and regulation.ResultsDifferent and bona-fide transcriptomic profiles were identified for the compared lines. A similar number of up- and downregulated differentially expressed genes (DEGs) were identified, considering the broiler line as a reference. Upregulated DEGs displayed an enrichment of protease-encoding genes, whereas downregulated DEGs exhibited a prevalence of receptors and ligands. Gene Ontology analysis revealed that upregulated DEGs were mainly associated with hormone response, mitotic cell cycle, and different metabolic and biosynthetic processes. In contrast, downregulated DEGs were primarily linked to communication, signal transduction, cell differentiation, and nervous system development. Regulatory networks were constructed for the mitotic cell cycle and cell differentiation biological processes, as their contrasting roles may impact the development of distinct postnatal traits. Within the mitotic cell cycle network, key upregulated DEGs included CCND1 and HSP90, with central regulators being NF-κB subunits (RELA and REL) and NFATC2. The cell differentiation network comprises numerous DEGs encoding transcription factors (e.g., HOX genes), receptors, ligands, and histones, while the main regulatory hubs are CREB, AR and epigenetic modifiers. Clustering analyses highlighted PIK3CD as a central player within the differentiation network.ConclusionsOur study revealed distinct developmental transcriptomes between Brazilian broiler and layer lines. The gene expression profile of broiler embryos seems to favour increased cell proliferation and delayed differentiation, which may contribute to the subsequent enlargement of pectoral tissues during foetal and postnatal development. Our findings pave the way for future functional studies and improvement of targeted traits of economic interest in poultry.

Integrated Transcriptomics and Metabolomics Analysis of Two Maize Hybrids (ZD309 and XY335) under Heat Stress at the Flowering Stage.

High temperature around flowering has a serious impact on the growth and development of maize. However, few maize genes related to flowering under heat stress have been confirmed, and the regulatory mechanism is unclear. To reveal the molecular mechanism of heat tolerance in maize, two maize hybrids, ZD309 and XY335, with different heat resistance, were selected to perform transcriptome and metabolomics analysis at the flowering stage under heat stress. In ZD309, 314 up-regulated and 463 down-regulated differentially expressed genes (DEGs) were detected, while 168 up-regulated and 119 down-regulated DEGs were identified in XY335. By comparing the differential gene expression patterns of ZD309 and XY335, we found the "frontloaded" genes which were less up-regulated in heat-tolerant maize during high temperature stress. They included heat tolerance genes, which may react faster at the protein level to provide resilience to instantaneous heat stress. A total of 1062 metabolites were identified via metabolomics analysis. Lipids, saccharides, and flavonoids were found to be differentially expressed under heat stress, indicating these metabolites' response to high temperature. Our study will contribute to the identification of heat tolerance genes in maize, therefore contributing to the breeding of heat-tolerant maize varieties.

Potential effect of MSR1 and C6 ceramide small molecules on nasopharyngeal carcinoma based on GSEA analysis

Purpose: To investigate some potential therapeutic agents and targets for nasopharyngeal carcinoma (NPC).
 Methods: Some potential therapeutic agents and target genes for NPC were identified by integrating bioinformatic analysis and in vitro experimental validation. Three datasets of NPC patients were gathered to reveal 26 upregulated and 344 downregulated differentially expressed genes (DGEs). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes pathway analysis (KEGG) of the DGEs were implemented. Separately, 316 drug and small molecule target genes were acquired from the SEA database, allowing for detection of C6-ceramide as a small molecule of high relevance to NPC. Then, the differentially upregulated genes were intersected with potential target genes of C6-ceramide small molecules to obtain macrophage scavenger receptor 1 (MSR1). Finally, the study validated the potential roles of MSR1 and C6-ceramide in NPC cell lines.
 Results: Knockdown of MSR1 expressions in NPC cells significantly decreased cell viability. Treatment with 10 μmol/L C6-ceramide also significantly reduced NPC cell viability (p < 0.0001). Furthermore, C6-ceramide attenuated the increase in MSR1 levels induced by MSR1 overexpression in NPC cells (p < 0.0001). Concurrently, MSR1 knockdown decreased expression of PI3K and AKT, while MSR1 overexpression upregulated AKT and PI3K levels.
 Conclusion: MSR1 modulates viability of NPC cells by regulating PI3K and AKT expression. Additionally, C6-ceramide exerts therapeutic effect on NPC by regulating MSR1 expression. These findings reveal new therapeutic targets and strategies for the clinical management of NPC. These results establish a rationale for further exploration of MSR1 and ceramides as novel targets in NPC.

Research on the targeted improvement of the yield of a new VB12-producing strain, Ensifer adhaerens S305, based on genomic and transcriptomic analysis

BackgroundVitamin B12 (VB12) has a wide range of applications and high economic value. In this study, a new strain with high VB12 production potential, Ensifer adhaerens S305, was identified in sewage. Because E. adhaerens strains have become the main strains for VB12 production via fermentation in recent years, the directional modification of the S305 strain to obtain a strain suitable for the industrial production of VB12 has great potential and commercial value.Results16S rRNA and genome-wide phylogenetic tree analysis combined with average nucleotide identity (ANI) analysis showed that the high-yielding VB12 strain was a E. adhaerens strain and that its VB12 synthesis pathway genes were highly similar to related genes of strains of this and other species, including E. adhaerens Casida A, Pseudomonas denitrificans SC 510, and E. adhaerens Corn53. High-pressure liquid chromatography (HPLC) results indicated that the VB12 yields of the S305 strain were more than double those of the Casida A strain under different medium components. Multiple genes with significantly upregulated and downregulated transcription were identified by comparing the transcription intensity of different genes through transcriptome sequencing. KEGG enrichment analysis of the porphyrin metabolism pathway identified 9 significantly upregulated and downregulated differentially expressed genes (DEGs) in the VB12 synthesis pathway, including 7 transcriptionally upregulated genes (cobA, cobT, hemA, cobJ, cobN, cobR, and cobP) that were episomally overexpressed in the Casida A strain. The results showed that the VB12 yield of the overexpressed strain was higher than that of the wild-type strain. Notably, the strains overexpressing the cobA and cobT genes exhibited the most significant increases in VB12 yield, i.e., 31.4% and 24.8%, respectively. The VB12 yield of the S305 strain in shake-flask culture was improved from 176.6 ± 8.21 mg/L to 245.6 ± 4.36 mg/L by integrating the cobA and cobT genes into the strain.ConclusionPhylogenetic tree and ANI analysis showed that the Ensifer and Sinorhizobium strains were quite different at the genome level; the overexpression and integrated expression of significantly upregulated genes in the VB12 synthesis pathway could increase the yield of VB12, further improving the VB12 yield of the E. adhaerens S305 strain.

Transcriptomic analysis of large yellow croaker (Larimichthys crocea) reveals the suppression of the inflammatory response from Cryptocaryon irritans infection

Large yellow croaker (Larimichthys crocea) is the most productive marine fish in China. Cryptocaryon irritans is an extremely destructive parasite that causes great economic losses in large yellow croaker aquaculture industry. Therefore, it is very necessary to study the immune response of large yellow croaker in response to C. irritans infection. In this study, the transcriptomic profiles of large yellow croaker were sequenced and analyzed in the brain and head kidney at 72 h after C. irritans infection. Cytokines and chemokines related terms were significantly enriched based on the GO enrichment of down-regulated differentially expressed genes (DEGs) from the head kidney. Meanwhile, cytokine-cytokine receptor interaction was significantly enriched based on the KEGG enrichment of up-regulated DEGs from the brain and down-regulated DEGs from the head kidney, respectively. Moreover, the majority of inflammation-related DEGs were significantly up-regulated in the brain, but distinctly down-regulated in the head kidney. These results showed that the brain and head kidney might play different roles against C. irritans infection, and the inflammatory response of large yellow croaker may be restrained during C. irritans infection. Taken together, the transcriptomic analyses will be helpful to more comprehensively understand the immune mechanism of teleost against C. irritans infection, and provide a theoretical basis for the prevention and treatment of Cryptosporidiosis.

Clinical and Genetic Features By Next-Generation Sequencing and RNA Sequencing in Pre-Fibrotic Primary Myelofibrosis Patients

The 2016 WHO classification recognized pre-fibrotic primary myelofibrosis (pre-PMF) as a distinct entity. Such knowledge is essential to depict the landscape of pre-PMF and develop mechanism-based approaches to slow the progression of pre-PMF. Here, we conducted a retrospective study of 234 pre-PMF patients defined by 2016 WHO diagnostic criteria in 13 hematooncology centers between 01 January 2015 and 31 July 2023 (ChiCTR2200061208). We evaluated clinical characteristics, risk factors and genetic features of progression to overt-PMF in pre-PMF patients. Among 234 pre-PMF patients, 135 (57.7%) harbored JAK2V617F, 36 (15.4%) CALR and 3 (1.3%) MPL mutations; 60 (25.6%) patients were negative for all three mutations. JAK2V617F-mutated patients were older ( P=0.036), had more constitutional symptoms ( P=0.027) and cardiovascular risk factors ( P=0.045), higher white blood cell count (WBC) ( P<0.001), hemoglobin level (HB) ( P<0.001), neutrophile granulocyte (NEU) ( P<0.001), eosinophilic granulocyte (EOS) ( P<0.001), basophilic granulocyte (BAS) ( P<0.001), hematocrit (HCT) ( P=0.002) and red blood cell distribution width (RDW) ( P<0.001) (Figure A). CALR-mutated cases had higher platelet hematocrit (PCT) ( P=0.043) and lactic dehydrogenase (LDH) ( P=0.008). Besides, CALR group had a higher proportion of 25.0% to progress to overt-PMF than JAK2V617F group (9.6%) and TN group (6.7%) ( P=0.025) (Figure A). As for risk factors of progression to overt-PMF, univariable analysis found it was associated with mean platelet volume (MPV) ( P=0.019), HB ( P=0.027), WBC ( P=0.008), LDH ( P=0.004) and CALR mutations ( P=0.007). Multivariable analysis revealed that lower HB value ( P=0.041; HR 0.981, 95% CI 0.962-0.999) and CALR mutation ( P=0.001; HR 3.716, 95% CI 1.331-10.069) had negative impact on the progression to overt-PMF (Figure B). When divided by HB with cut-off point of 128 g/L, patients with HB value ≤128 g/L were more likely to progress to overt-PMF compared to >128 g/L groups ( P=0.007) (Figure C). And patients carried CALR mutation were also more likely to progress to overt-PMF ( P<0.001) (Figure D). We performed next-generation sequencing (NGS) in 18 pre-PMF patients, including 9 JAK2V617F, 3 CALR and 6 TN, 3 of which progressed to overt-PMF and 15 stayed in pre-PMF until the end of the follow-up. JAK2V617F was the most frequently mutated gene (9/38, 23.7%), followed by TET2 (7/38, 18.4%), RELN (4/38, 10.5%) and ASXL1 (3/38, 7.9%). JAK2V617F-mutated patients possessed more mutations related to activation signaling pathway ( P=0.025) and DNA methylation ( P=0.033) (Figure F). TN patients possessed more mutations related to transcription factor ( P=0.048) (Figure F). Patients who progressed to overt-PMF possessed more mutations associated with myeloid related transcription factor ( P=0.025) (Figure H). We further performed RNA-Seq analysis in 6 pre-PMF samples and 14 overt-PMF samples. Compared to pre-PMF, overt-PMF showed 49 up-regulated and 13 down-regulated differentially expressed genes (DEGs), of which CSCF3 and CXCL13 were significantly up-regulated (Figure I,J). Gene ontology (GO) functional enrichment analysis showed that DEGs were enriched in cell-cell signaling, cytokine activity, monocyte chemotaxis and chemokine activity (Figure K). KEGG biofunctional analysis showed that DEGs were significantly enriched in cytokine-cytokine receptor interaction, chemokine signaling pathway, IL-17 signaling pathway and NF-kappa B signaling pathway (Figure L). In conclusion, CALR-mutated pre-PMF patients with HB value ≤128 g/L were more likely to progress to overt-PMF. Genetic analysis found patients with high proportion of progress to overt-PMF tended to possess mutations associated with myeloid related transcription factor. And the upregulation of CSCF3 and CXCL13 may potentially drive the progression of overt-PMF in pre-PMF patients through inflammatory and cytokine related pathways. Acknowledgement: This research was funded by the Key R&D Program of Zhejiang, No. 2022C03137; Public Technology Application Research Program of Zhejiang, China, No. LGF21H080003; Zhejiang Medical Association Clinical Medical Research special fund project, No. 2022ZYC-D09. *Correspondence to: Jian Huang, M.D., Ph.D., Department of Hematology, The First Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou 310003, Zhejiang, China. E-mail: househuang@zju.edu.cn

De novo transcriptome assembly of Dalbergia sissoo Roxb. (Fabaceae) under Botryodiplodia theobromae-induced dieback disease

Dalbergia sissoo Roxb. (Shisham) is a timber-producing species of economic, cultural, and medicinal importance in the Indian subcontinent. In the past few decades, Shisham's dieback disease caused by the fungus Botryodiplodia theobromae has become an evolving issue in the subcontinent endangering its survival. To gain insights into this issue, a standard transcriptome assembly was deployed to assess the response of D. sissoo at the transcriptomic level under the stress of B. theobromae infection. For RNA isolation, the control and infected leaf tissue samples were taken from 1-year-old greenhouse-grown D. sissoo plants after 20 days of stem-base spore inoculation. cDNA synthesis was performed from these freshly isolated RNA samples that were then sent for sequencing. About 18.14 Gb (Giga base) of data was generated using the BGISEQ-500 sequencing platform. In terms of Unigenes, 513,821 were identified after a combined assembly of all samples and then filtering the abundance. The total length of Unigenes, their average length, N50, and GC-content were 310,523,693 bp, 604 bp, 1,101 bp, and 39.95% respectively. The Unigenes were annotated using 7 functional databases i.e., 200,355 (NR: 38.99%), 164,973 (NT: 32.11%), 123,733 (Swissprot: 24.08%), 142,580 (KOG: 27.75%), 139,588 (KEGG: 27.17%), 99,752 (GO: 19.41%), and 137,281 (InterPro: 26.72%). Furthermore, the Transdecoder detected 115,762 CDS. In terms of SSR (Simple Sequence Repeat) markers, 62,863 of them were distributed on 51,508 Unigenes and on the predicted 4673 TF (Transcription Factor) coding Unigenes. A total of 16,018 up- and 19,530 down-regulated Differentially Expressed Genes (DEGs) were also identified. Moreover, the Plant Resistance Genes (PRGs) had a count of 9230. We are hopeful that in the future, these identified Unigenes, SSR markers, DEGs and PRGs will provide the prerequisites for managing Shisham dieback disease, its breeding, and in tree improvement programs.

Chromatin accessibility mediated transcriptome changes contribute to flavor substance alterations and jasmonic acid hyperaccumulation during oolong tea withering process.

During the oolong tea withering process, abiotic stresses induce significant changes in the content of various flavor substances and jasmonic acid (JA). However, the changes in chromatin accessibility during withering and their potential impact remain poorly understood. By integrating ATAC-seq, RNA-seq, metabolite, and hormone assays, we characterized the withering treatment-induced changes in chromatin accessibility, gene expression levels, important metabolite contents, and JA and JA-ILE contents. Additionally, we analyzed the effects of chromatin accessibility alterations on gene expression changes, content changes of important flavor substances, and JA hyperaccumulation. Our analysis identified a total of 3451 open- and 13 426 close-differentially accessible chromatin regions (DACRs) under withering treatment. Our findings indicate that close-DACRs-mediated down-regulated differentially expressed genes (DEGs) resulted in the reduced accumulation of multiple catechins during withering, whereas open-DACRs-mediated up-regulated DEGs contributed to the increased accumulation of important terpenoids, JA, JA-ILE and short-chain C5/C6 volatiles. We further highlighted important DACRs-mediated DEGs associated with the synthesis of catechins, terpenoids, JA and JA and short-chain C5/C6 volatiles and confirmed the broad effect of close-DACRs on catechin synthesis involving almost all enzymes in the pathway during withering. Importantly, we identified a novel MYB transcription factor (CsMYB83) regulating catechin synthesis and verified the binding of CsMYB83 in the promoter-DACRs regions of key catechin synthesis genes using DAP-seq. Overall, our results not only revealed a landscape of chromatin alters-mediated transcription, flavor substance and hormone changes under oolong tea withering, but also provided target genes for flavor improvement breeding in tea plant.

Transcriptome Analysis and QTL Mapping Identify Candidate Genes and Regulatory Mechanisms Related to Low-Temperature Germination Ability in Maize.

Low-temperature germination ability (LTGA) is an important characteristic for spring sowing maize. However, few maize genes related to LTGA were confirmed, and the regulatory mechanism is less clear. Here, maize-inbred lines Ye478 and Q1 with different LTGA were used to perform transcriptome analysis at multiple low-temperature germination stages, and a co-expression network was constructed by weighted gene co-expression network analysis (WGCNA). Data analysis showed that 7964 up- and 5010 down-regulated differentially expressed genes (DEGs) of Ye478 were identified at low-temperature germination stages, while 6060 up- and 2653 down-regulated DEGs of Q1 were identified. Gene ontology (GO) enrichment analysis revealed that ribosome synthesis and hydrogen peroxide metabolism were enhanced and mRNA metabolism was weakened under low-temperature stress for Ye478, while hydrogen peroxide metabolism was enhanced and mRNA metabolism was weakened for Q1. DEGs pairwise comparisons between the two genotypes found that Ye478 performed more ribosome synthesis at low temperatures compared with Q1. WGCNA analysis based on 24 transcriptomes identified 16 co-expressed modules. Of these, the MEbrown module was highly correlated with Ye478 at low-temperature stages and catalase and superoxide dismutase activity, and the MEred, MEgreen, and MEblack modules were highly correlated with Ye478 across low-temperature stages, which revealed a significant association between LTGA and these modules. GO enrichment analysis showed the MEbrown and MEred modules mainly functioned in ribosome synthesis and cell cycle, respectively. In addition, we conducted quantitative trait loci (QTL) analysis based on a doubled haploid (DH) population constructed by Ye478 and Q1 and identified a major QTL explanting 20.6% of phenotype variance on chromosome 1. In this QTL interval, we found three, four, and three hub genes in the MEbrown, MEred, and MEgreen modules, of which two hub genes (Zm00001d031951, Zm00001d031953) related to glutathione metabolism and one hub gene (Zm00001d031617) related to oxidoreductase activity could be the candidate genes for LTGA. These biological functions and candidate genes will be helpful in understanding the regulatory mechanism of LTGA and the directional improvement of maize varieties for LTGA.

OR11-01 A New Model for Estrogen Receptor-alpha Antagonist Mechanism of Action

Abstract Disclosure: J.C. Nwachukwu: None. J.W. Njeri: None. T. Venables: None. C. Seath: None. M.E. Pipkin: None. Y. Hou: None. B.S. Katzenellenbogen: None. J.A. Katzenellenbogen: None. K.W. Nettles: None. We recently developed Dual Mechanism Estrogen Receptor-α (ERα) Inhibitors (DMERI) with more antagonist efficacy than selective ERα modulators (SERMs) such as tamoxifen, and selective ERα degraders (SERDs) such as fulvestrant in certain breast cancer models. Current models of anti-estrogen action are largely based on genomics studies in growth arrested cells, where the effects primarily reversed estradiol-mediated gene expression. In that context, most ER antagonists, especially the SERDs, show very little gene activation. We were interested in understanding antagonist mechanism of action in asynchronous cells that were growing in intact medium that had not been charcoal-stripped to remove estradiol and growth factors. RNA-seq of asynchronous MCF-7 cells treated with 4-hydroxytamoxifen, fulvestrant, a SERM-like DMERI, and a SERD-like DMERI showed that all four classes of ligands equally up-regulated and down-regulated differentially expressed genes (DEGs), which included a core set of regulatory networks, as well as ligand-selective gene networks. The number of DEGs predicted efficacy, with tamoxifen regulating few genes, and the SERD-like DMERI regulating the most genes. The SERD-like DMERI also inhibited expression of many more genes that encode cell cycle regulators and key cancer drivers, such as ERα, AKT1, HRAS, and several growth factor receptors. Landscape in silico analysis based on chromatin immunoprecipitation (ChIP)-seq data suggests that recruitment of the ERα MegaTrans complex in collaboration with the oxysterol receptor, LXRα, predicts up-regulation, while cooperation with the retinoblastoma-associated proteins, RB1 and RBL1, predicts down-regulation of target genes. APEX2-mediated proximity labeling in asynchronous cells revealed that fulvestrant and the SERD-like DMERI labeled both coactivators and corepressors. Both compounds also increase labeling of STAG1 and other cohesin subunits that coordinate chromatin looping, while also associating with the nuclear matrix protein Lamin B1 and heterochromatin-associated proteins. These data suggest that antagonists have dual roles in genome organization. An shRNAmiR screen targeting over 300 chromatin regulatory factors revealed networks of shared and exclusive coregulators used by antiestrogens, including the heterochromatin protein, CBX1, which was exclusively required for growth inhibition by fulvestrant, and subunits of histone acetylase and methylase complexes such as KMT2A (the catalytic subunit of the MLL1 complex), which were broadly required. Thus, antiestrogen-driven growth suppression involves both common and ligand-selective transcriptional regulatory networks through utilization of both coactivators and corepressors. Supported by NIH R01CA220284 and BCRF-083 and BCRF-084 grants. Presentation Date: Friday, June 16, 2023

Unbiased multitissue transcriptomic analysis reveals complex neuroendocrine regulatory networks mediated by spinal cord injury-induced immunodeficiency

BackgroundSpinal cord injury (SCI), which causes loss of sensory and motor function in the body below the level of injury, is a devastating disease of the central nervous system. SCI leads to severe secondary immunosuppression, called SCI-induced immunodeficiency syndrome (SCI-IDS), which is characterized by increased susceptibility to infection and further exacerbates neurological dysfunction. Several studies have suggested that SCI-IDS is an independent risk factor for poor neurological prognosis. SCI-IDS predominantly occurs following injury above the T5 levels and eventually leads to systemic immune failure, possibly via the sympathetic–adrenal medullary axis and the hypothalamic‒pituitary‒adrenal (HPA) axis. However, the mechanism remains unclear.Methods and objectivesThe concentrations of adrenocorticotropic hormone and cortisol in plasma, as well as changes in sympathetic activity (blood pressure and catecholamine levels in plasma), were assessed in rats in the high-level (T3) spinal cord injury (T3-SCI) group and the low-level (T10) spinal cord injury (T10-SCI) group. Second, the differential regulation of the gene network between the sympathetic–adrenal medullary axis and the HPA axis was explored by histology and multitissue transcriptomics, and the neuroendocrine–immune network associated with SCI-IDS was further elucidated.ResultsThe spleen and thymus gland, which are secondary immune organs, were significantly atrophied in rats in the T3-SCI group, and the white pulp of the spleen was significantly atrophied. The level of cortisol, which is mediated by the adrenal glands, was markedly elevated, but norepinephrine levels were markedly decreased. There was no difference in adrenocorticotropic hormone expression between any of the groups. The transcriptome analysis results showed that the downregulated differentially expressed genes (DEGs) in the T3-SCI group were enriched in the GO term immunoregulation, indicating that splenic immune function was markedly impaired after high-level SCI. The upregulated DEGs in the hypothalamus (hub genes: Nod2, Serpine1, Cebpb, Nfkbil1, Ripk2, Zfp36, Traf6, Akap8, Gfer, Cxcl10, Tnfaip3, Icam1, Fcgr2b, Ager, Dusp10, and Mapkapk2) were significantly enriched in inflammatory pathways, and the downregulated genes (hub genes: Grm4, Nmu, P2ry12, rt1-bb1, Oprm1, Zfhx2, Gpr83, and Chrm2) were enriched in pathways related to inhibitory Gi-mediated G protein-coupled receptor (Gi-GPCR) neurons and neuropeptide changes. The upregulated genes in the adrenal glands (hub genes: Ciart, per2, per3, cry1, and cry2) were enriched in cortisol secretion and circadian rhythm changes, and the downregulated genes (hub genes: IL7r, rt1-bb, rt1-bb1, rt1-da, rt1-ba, cd74, cxcr3, vcam1, ccl5, bin1, and IL8) were significantly enriched in MHC-mediated immune responses.ConclusionsTo explore the possible mechanism underlying SCI-IDS, this study assessed the differential regulation of the gene network associated with neuroendocrine immunity after SCI. Progressive neuroinflammation spreads after injury, and neurotransmission through Gi-mediated G protein-coupled receptors in the HPA axis and neuropeptide production by the hypothalamus are inhibited. Disruption of the connection between the hypothalamus and the adrenal glands causes autonomous regulation of the adrenal glands, disturbance of circadian rhythm and finally hypercortisolemia, leading to general suppression of peripheral adaptive immunity. Neuraxial nerve inflammation caused by SCI persists indefinitely, blocking nerve repair; persistent system-wide immunosuppression in the periphery results in increased susceptibility to infection, leading to poor neurological prognosis.

Methamphetamine exposure drives cell cycle exit and aberrant differentiation in rat hippocampal-derived neurospheres.

Introduction: Methamphetamine (METH) abuse by pregnant drug addicts causes toxic effects on fetal neurodevelopment; however, the mechanism underlying such effect of METH is poorly understood. Methods: In the present study, we applied three-dimensional (3D) neurospheres derived from the embryonic rat hippocampal tissue to investigate the effect of METH on neurodevelopment. Through the combination of whole genome transcriptional analyses, the involved cell signalings were identified and investigated. Results: We found that METH treatment for 24h significantly and concentration-dependently reduced the size of neurospheres. Analyses of genome-wide transcriptomic profiles found that those down-regulated differentially expressed genes (DEGs) upon METH exposure were remarkably enriched in the cell cycle progression. By measuring the cell cycle and the expression of cell cycle-related checkpoint proteins, we found that METH exposure significantly elevated the percentage of G0/G1 phase and decreased the levels of the proteins involved in the G1/S transition, indicating G0/G1 cell cycle arrest. Furthermore, during the early neurodevelopment stage of neurospheres, METH caused aberrant cell differentiation both in the neurons and astrocytes, and attenuated migration ability of neurospheres accompanied by increased oxidative stress and apoptosis. Conclusion: Our findings reveal that METH induces an aberrant cell cycle arrest and neuronal differentiation, impairing the coordination of migration and differentiation of neurospheres.