Expression Of Early Response Genes Research Articles

MtTCP18 Regulates Plant Structure in Medicago truncatula.

Plant structure has a large influence on crop yield formation, with branching and plant height being the important factors that make it up. We identified a gene, MtTCP18, encoding a TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR (TCP) transcription factor highly conserved with Arabidopsis gene BRC1 (BRANCHED1) in Medicago truncatula. Sequence analysis revealed that MtTCP18 included a conserved basic helix-loop-helix (BHLH) motif and R domain. Expression analysis showed that MtTCP18 was expressed in all organs examined, with relatively higher expression in pods and axillary buds. Subcellular localization analysis showed that MtTCP18 was localized in the nucleus and exhibited transcriptional activation activity. These results supported its role as a transcription factor. Meanwhile, we identified a homozygous mutant line (NF14875) with a mutation caused by Tnt1 insertion into MtTCP18. Mutant analysis showed that the mutation of MtTCP18 altered plant structure, with increased plant height and branch number. Moreover, we found that the expression of auxin early response genes was modulated in the mutant. Therefore, MtTCP18 may be a promising candidate gene for breeders to optimize plant structure for crop improvement.

Modulation of early gene expression responses to water deprivation stress by the E3 ubiquitin ligase ATL80: implications for retrograde signaling interplay

BackgroundPrimary response genes play a pivotal role in translating short-lived stress signals into sustained adaptive responses. In this study, we investigated the involvement of ATL80, an E3 ubiquitin ligase, in the dynamics of gene expression following water deprivation stress. We observed that ATL80 is rapidly activated within minutes of water deprivation stress perception, reaching peak expression around 60 min before gradually declining. ATL80, despite its post-translational regulation role, emerged as a key player in modulating early gene expression responses to water deprivation stress.ResultsThe impact of ATL80 on gene expression was assessed using a time-course microarray analysis (0, 15, 30, 60, and 120 min), revealing a burst of differentially expressed genes, many of which were associated with various stress responses. In addition, the diversity of early modulation of gene expression in response to water deprivation stress was significantly abolished in the atl80 mutant compared to wild-type plants. A subset of 73 genes that exhibited a similar expression pattern to ATL80 was identified. Among them, several are linked to stress responses, including ERF/AP2 and WRKY transcription factors, calcium signaling genes, MAP kinases, and signaling peptides. Promoter analysis predicts enrichment of binding sites for CAMTA1 and CAMTA5, which are known regulators of rapid stress responses. Furthermore, we have identified a group of differentially expressed ERF/AP2 transcription factors, proteins associated with folding and refolding, as well as pinpointed core module genes which are known to play roles in retrograde signaling pathways that cross-referenced with the early ATL80 transcriptome.ConclusionsBased on these findings, we propose that ATL80 may target one or more components within the retrograde signaling pathways for degradation. In essence, ATL80 serves as a bridge connecting these signaling pathways and effectively functions as an alarm signal.

Transcriptomic profiling reveals key early response genes during GDF6-mediated differentiation of human adipose-derived stem cells to nucleus pulposus cells.

Stem cell-based therapies show promise as a means of repairing the degenerate intervertebral disc, with growth factors often used alongside cells to help direct differentiation toward a nucleus pulposus (NP)-like phenotype. We previously demonstrated adipose-derived stem cell (ASC) differentiation with GDF6 as optimal for generating NP-like cells through evaluating end-stage differentiation parameters. Here we conducted a time-resolved transcriptomic characterization of ASCs response to GDF6 stimulation to understand the early drivers of differentiation to NP-like cells. Human ASCs were treated with recombinant human GDF6 for 2, 6, and 12 h. RNA sequencing and detailed bioinformatic analysis were used to assess differential gene expression, gene ontology (GO), and transcription factor involvement during early differentiation. Quantitative polymerase chain reaction (qPCR) was used to validate RNA sequencing findings and inhibitors used to interrogate Smad and Erk signaling pathways, as well as identify primary and secondary response genes. The transcriptomic response of ASCs to GDF6 stimulation was time-resolved and highly structured, with "cell differentiation" "developmental processes," and "response to stimulus" identified as key biological process GO terms. The transcription factor ERG1 was identified as a key early response gene. Temporal cluster analysis of differentiation genes identified positive regulation NP cell differentiation, as well as inhibition of osteogenesis and adipogenesis. A role for Smad and Erk signaling in the regulation of GDF6-induced early gene expression response was observed and both primary and secondary response genes were identified. This study identifies a multifactorial early gene response that contributes to lineage commitment, with the identification of a number of potentially useful early markers of differentiation of ASCs to NP cells. This detailed insight into the molecular processes in response to GDF6 stimulation of ASCs is important for the development of an efficient and efficacious cell-based therapy for intervertebral disc degeneration-associated back pain.

Transcriptome Analysis Reveals the Stress Tolerance Mechanisms of Cadmium in Zoysia japonica.

Cadmium (Cd) is a severe heavy metal pollutant globally. Zoysia japonica is an important perennial warm-season turf grass that potentially plays a role in phytoremediation in Cd-polluted soil areas; however, the molecular mechanisms underlying its Cd stress response are unknown. To further investigate the early gene response pattern in Z. japonica under Cd stress, plant leaves were harvested 0, 6, 12, and 24 h after Cd stress (400 μM CdCl2) treatment and used for a time-course RNA-sequencing analysis. Twelve cDNA libraries were constructed and sequenced, and high-quality data were obtained, whose mapped rates were all higher than 94%, and more than 601 million bp of sequence were generated. A total of 5321, 6526, and 4016 differentially expressed genes were identified 6, 12, and 24 h after Cd stress treatment, respectively. A total of 1660 genes were differentially expressed at the three time points, and their gene expression profiles over time were elucidated. Based on the analysis of these genes, the important mechanisms for the Cd stress response in Z. japonica were identified. Specific genes participating in glutathione metabolism, plant hormone signal and transduction, members of protein processing in the endoplasmic reticulum, transporter proteins, transcription factors, and carbohydrate metabolism pathways were further analyzed in detail. These genes may contribute to the improvement of Cd tolerance in Z. japonica. In addition, some candidate genes were highlighted for future studies on Cd stress resistance in Z. japonica and other plants. Our results illustrate the early gene expression response of Z. japonica leaves to Cd and provide some new understanding of the molecular mechanisms of Cd stress in Zosia and Gramineae species.

Differential expression of Dusp1 and immediate early response genes in the hippocampus of rats, subjected to forced swim test

The forced swim test (FST) is widely used to screen for potential antidepressant drugs and treatments. Despite this, the nature of stillness during FST and whether it resembles “depressive-like behavior” are widely debated issues. Furthermore, despite being widely used as a behavioral assay, the effects of the FST on the brain transcriptome are rarely investigated. Therefore, in this study we have investigated changes in the transcriptome of the rat hippocampus 20 min and 24 h after FST exposure. RNA-Seq is performed on the hippocampus tissues of rats 20 min and 24 h after an FST. Differentially expressed genes (DEGs) were identified using limma and used to construct gene interaction networks. Fourteen differentially expressed genes (DEGs) were identified only in the 20-m group. No DEGs were identified 24 h after the FST. These genes were used for Gene Ontology term enrichment and gene-network construction. Based on the constructed gene-interaction networks, we identified a group of DEGs (Dusp1, Fos, Klf2, Ccn1, and Zfp36) that appeared significant based on multiple methods of downstream analysis. Dusp1 appears especially important, as its role in the pathogenesis of depression has been demonstrated both in various animal models of depression and in patients with depressive disorders.

Expression of Salivary Immediate Early Response Gene X-1 as a Predictor of Malignancy in Epithelial Ovarian Tumor

Objective: This diagnostic study aims to determine the expression of salivary IEX-1 as a predictor of malignancy in epithelial ovarian tumors. 
 Methods: Samples were obtained from ovarian cancer patients who were scheduled for elective surgery. Patients' saliva was collected before surgery and used as the study's research material. Research subjects who met the inclusion and exclusion criteria were divided into two groups based on the post-operative histopathological results, benign and malignant epithelial ovarian tumors. The salivary IEX-1 expression was examined using the Real Time qPCR method. We compared and analyzed the salivary IEX-1 expression ​​in benign and malignant epithelial ovarian tumors. 
 Results: The results of this study were obtained from 47 epithelial ovarian tumors subjects, 22 malignant tumors and 27 benign tumors. The mean salivary IEX-1 expression in benign epithelial ovarian tumors was higher (1.976) than the malignant tumors (0.554) (p<0.001). The AUC value of IEX-1 expression was 0.949 (95% CI 0.894-1,000), cut off point of salivary IEX-1 is ≥ 0,9115 with sensitivity 84%, specificity 86,4%, positive predictive value 82.6% and negative predictive value 87.5%. There was a significant correlation between salivary IEX-1 expression and malignant epithelial ovarian tumors with an OR 5.031 (95% CI 2.039-12.4; p<0,001)
 Conclusion: Salivary IEX-1 expression declines in tandem with the development of malignant epithelial ovarian tumors, providing a very sensitive and specific indicator of the presence of these malignant tumors.
 Keywords: Expression of IEX-1, Malignant epithelial ovarian tumor, Salivary IEX-1, Tumor Marker.

The role of C1473G polymorphism in triptophanhydroxylase-2 gene in the acute ethanol effects on <i>c-fos</i> gene expression and metabolism of biogenic amines in brain of mice

Tryptophan hydroxylase-2 is a key enzyme in the synthesis of the neurotransmitter serotonin, which plays an important role in the mechanisms of regulation of a wide range of physiological functions and behaviors. In this work, we studied the effect of acute ethanol administration on the expression of the c-fos early response gene and the metabolism of serotonin and catecholamines in the brain structures of B6-1473C and B6-1473G congenic mice, which differ in the C1473G single-nucleotide substitution in the Tph2 gene and enzyme activity. Acute alcoholization led to a significant increase in c-fos gene expression in the frontal cortex and striatum of B6-1473G mice and the hippocampus of B6-1473C mice. The introduction of ethanol caused a decrease in the index of serotonin metabolism in the nucleus accumbens in B6-1473C mice, as well as in the hippocampus and striatum of B6-1473G mice. Also, ethanol led to a decrease in the level of norepinephrine in the hypothalamus in B6-1473C mice. Thus, the C1473G polymorphism in the Tph2 gene has a significant effect on changes in the c-fos gene expression pattern and metabolism of biogenic amines in the brain induced by acute ethanol administration.

Phospho-mimetic cardiac troponin I Ser43/45 produces early changes in mitochondria.

Our work tests whether chronic protein kinase C (PKC)-mediated phosphorylation of cardiac troponin I (cTnI) Ser43/45 diminishes cardiac function and initiates progressive heart failure via early increases in oxidative stress and mitochondria. The current work focuses on early gene expression responses in mice after cardiac-specific phospho-mimetic cTnI S43/45D (SD) replacement of endogenous cTnI. Dose-dependent changes in cTnISD replacement do not significantly change function, when measured by echocardiography at 1 month (mos) of age in male or female mice. However, by 2 mos of age, the highest cTnISD replacement line develop significant dysfunction and 100% mortality by 16 weeks of age, with males (M) dying earlier than females (F; F = 14.6 ± 0.4 wks, n = 32; M = 9.3 ± 0.3 wks, n = 68). Based on gene expression measured by quantitative PCR (QPCR) analysis, 1 mos old hearts from this high replacement cTnISD line of mice develop significant reductions in mitochondrially-encoded cytochrome oxidase and cytochrome b in males, which are followed by similar changes at 2 mos of age in females. Reductions in gene expression of critical mitochondrial proteins PGC-1α and SOD2 also develop by 1 mos of age in males. In contrast, there are no detected changes in gene expression of the endoplasmic reticulum (ER) and nuclear stress markers, ATF6 and Nrf2, respectively for males and/or females by 2 mos of age in the high replacement cTnISD mice. Ongoing work is determining whether similar changes develop in the protein expression of these genes in young adult mice. The present results are consistent with the idea that chronic modification of the sarcomere produces a stress signal leading to early mitochondrial and oxidative stress responses, which are predicted to lay the foundation for the progressive deterioration in cardiac function observed in this model.

Djptpn11 is indispensable for planarian regeneration by affecting early wound response genes expression and the Wnt pathway

Planarian is an ideal model system of studying regeneration. Stem cell system and positional control genes (PCGs) are two important factors for perfect regeneration of planarians and they combine to promote their regeneration. Even so, how wounds regulate proliferation and neoblast fate is still important areas to address. Ptpn11 (Protein tyrosine phosphatase non-receptor type 11), one of PTP (Protein tyrosine phosphatase) family members, plays an important role in cellular processes including cell survival, proliferation, differentiation and apoptosis. Nevertheless, the role of ptpn11 in the planarian regeneration has not been fully studied. In this study, we identify the Djptpn11 gene to observe its function in planarian regeneration. The results reveal that the regeneration is severely inhibited and cause the disorder homeostasis in planarians. Furthermore, the stem cells proliferation and differentiation decreases while the apoptosis increases following Djptpn11 RNAi. At the same time, Djptpn11 affects the expression levels of early wound response genes (Djegr2, Dj1-jun, Djrunt1, Djwnt1 and Djnotum). Djwnt1 and Djnotum are two key Wnt signaling pathway genes and Djptpn11 affects the expression levels of Djwnt1 and Djnotum in the early and late stages of planarian regeneration. In general, Djptpn11 is indispensable for the homeostasis and regeneration of planarian by affecting the stem cells, early wound response genes and the Wnt pathway.

Abstract 1778: NR2F2 mediates cell growth and endocrine resistance in NF1 loss, ER+ breast cancer

Abstract Genomic loss of in Neurofibromin 1 (NF1) is a recurrent mechanism driving metastatic progression and resistance to endocrine therapy in ER-positive breast cancer, via activation of the MAPK pathway, induction of cyclin D1 expression and modulation of estrogen receptor signaling. To identify specific mediators of tumor growth in NF1 mutant cancers, we performed a genetic knockout (KO) screen in isogenic WT and NF1 KO breast cancer cells. After employing stringent cutoff criteria, we found 28 genes that are specifically essential for NF1 KO cells but not WT cells. Among these genes, NR2F2, encoding an orphan nuclear receptor, was one of the top candidates. We validated that its loss significantly impaired the growth of multiple models of NF1 null, ER+ breast cancer. Loss of NF1 leads to the hyperactivation of Ras which sequentially activates multiple effector pathways, including the MAPK pathway and the PI3K-AKT pathway. We found that MAPK activation upon NF1 loss led to elevated NR2F2 expression levels. A negative correlation between NF1 and NR2F2 expression was further confirmed in public gene expression databases. These data suggested that induction of NR2F2 expression might be one mechanism that mediates endocrine resistance caused by NF1 loss. Consistently, we found that exogenous expression of NR2F2 could confer endocrine resistance while its knockout hypersensitized ER+ breast cancer cells to endocrine therapies and significantly attenuated NF1 loss induced resistance. RNA-seq analysis revealed that estrogen response gene sets were among the most enriched signatures upon NF1 KO, NR2F2 over-expression (OE) or NR2F2 KO. NF1 KO or NR2F2 OE decreased the expression of early estrogen response genes and impaired their E2-induced expression, most of which could be rescued by NR2F2 KO, suggesting a regulatory effect of NR2F2 on ER transcriptional activity. To further define these mechanisms, ongoing integrative analyses of NR2F2 qPlex-RIME, ER and NR2F2 ChIP-Sequencing and ATAC-Sequencing will be presented. Taken together, our studies identify NR2F2 as a MAPK driven transcript whose induction mediates endocrine resistance, and may serve as a novel therapeutic target to overcome anti-estrogen resistant breast cancer growth. Citation Format: Yanyan Cai, Peihua Zhao, Fan Wu, Hong Shao, Pedram Razavi, Guotai Xu, Maurizio Scaltriti, Sarat Chandarlapaty. NR2F2 mediates cell growth and endocrine resistance in NF1 loss, ER+ breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1778.

The dual role of transforming growth factor-beta signatures in human B viral multistep hepatocarcinogenesis: early and late responsive genes.

Transforming growth factor-beta (TGF-β) has a dichotomous role, functioning as a tumor suppressor and tumor promoter. TGF-β signatures, explored in mouse hepatocytes, have been reported to predict the clinical outcomes of hepatocellular carcinoma (HCC) patients; HCCs exhibiting early TGF-β signatures showed a better prognosis than those with late TGF-β signatures. The expression status of early and late TGF-β signatures remains unclear in defined lesions of human B-viral multistep hepatocarcinogenesis. The expression of TGF-β signatures, early and late responsive signatures of TGF-β were investigated and analyzed for their correlation in cirrhosis, low-grade dysplastic nodules (DNs), high-grade DNs, early HCCs and progressed HCCs (pHCCs) by real-time PCR and immunohistochemistry. The expression levels of TGF-β signaling genes (TGFB1, TGFBR1, TGFBR2 and SMAD4) gradually increased with the progression of hepatocarcinogenesis, peaking in pHCCs. The expression of early responsive genes of TGF-β (GADD45B, FBP1, CYP1A2 and CYP3A4) gradually decreased, and that of the late TGF-β signatures (TWIST and SNAI1) significantly increased according to the progression of multistep hepatocarcinogenesis. Furthermore, mRNA levels of TWIST and SNAI1 were well correlated with those of stemness markers, with upregulation of TGF-β signaling, whereas FBP1 expression was inversely correlated with that of stemness markers. The enrichment of the late responsive signatures of TGF-β with induction of stemness is considered to be involved in the progression of the late stage of multistep hepatocarcinogenesis, whereas the early responsive signatures of TGF-β are suggested to have tumor-suppressive roles in precancerous lesions of the early stage of multistep hepatocarcinogenesis.

P. aeruginosa type III and type VI secretion systems modulate early response gene expression in type II pneumocytes in vitro

BackgroundLung airway epithelial cells are part of innate immunity and the frontline of defense against bacterial infections. During infection, airway epithelial cells secrete proinflammatory mediators that participate in the recruitment of immune cells. Virulence factors expressed by bacterial pathogens can alter epithelial cell gene expression and modulate this response. Pseudomonas aeruginosa, a Gram-negative opportunistic pathogen, expresses numerous virulence factors to facilitate establishment of infection and evade the host immune response. This study focused on identifying the role of two major P. aeruginosa virulence factors, type III (T3SS) and type VI (T6SS) secretion systems, on the early transcriptome response of airway epithelial cells in vitro.ResultsWe performed RNA-seq analysis of the transcriptome response of type II pneumocytes during infection with P. aeruginosa in vitro. We observed that P. aeruginosa differentially upregulates immediate-early response genes and transcription factors that induce proinflammatory responses in type II pneumocytes. P. aeruginosa infection of type II pneumocytes was characterized by up-regulation of proinflammatory networks, including MAPK, TNF, and IL-17 signaling pathways. We also identified early response genes and proinflammatory signaling pathways whose expression change in response to infection with P. aeruginosa T3SS and T6SS mutants in type II pneumocytes. We determined that T3SS and T6SS modulate the expression of EGR1, FOS, and numerous genes that are involved in proinflammatory responses in epithelial cells during infection. T3SS and T6SS were associated with two distinct transcriptomic signatures related to the activation of transcription factors such as AP1, STAT1, and SP1, and the secretion of pro-inflammatory cytokines such as IL-6 and IL-8.ConclusionsTaken together, transcriptomic analysis of epithelial cells indicates that the expression of immediate-early response genes quickly changes upon infection with P. aeruginosa and this response varies depending on bacterial viability and injectosomes. These data shed light on how P. aeruginosa modulates host epithelial transcriptome response during infection using T3SS and T6SS.

Early gene expression response of barley root tip to toxic concentrations of cadmium

Already a short-term Cd treatment induces changes in gene expression in barley root tips via IAA and ROS signaling during mild and severe Cd stress, respectively. Even a short, 30min, Cd treatment of roots induced a considerable alteration in gene expression in the barley root tips within an hour after the treatments. The very early activation of MYB1 transcription factor expression is partially regulated by auxin signaling in mildly stressed seedlings. An increase in allene oxide cyclase and NADPH oxidase expression was a distinguishing feature of root tips response to mild Cd stress and their expression is activated via IAA signaling. Meanwhile, early changes in the level of dehydrin transcripts were detected in moderately and severely stressed root tips, and their induction is related to altered ROS homeostasis in cells. The early activation of glutathione peroxidase expression by mild Cd stress indicates the involvement of IAA in the signaling process. In contrast, early ascorbate peroxidase expression was induced only with Cd treatment causing severe stress and ROS play central roles in its induction. The expression of cysteine protease was activated similarly in both mildly and severely Cd-stressed roots; consequently, both increased IAA and ROS levels take part in the regulation of cysteine protease expression. The Cd-evoked accumulation of BAX Inhibitor-1 mRNA was characteristic for moderately and severely stressed roots. Whereas decreased IAA level did not affect its expression, rotenone-mediated ROS depletion markedly reduced the Cd-induced expression of BAX Inhibitor-1. An early increase of alternative oxidase levels in the root tip cells indicated that the reduction of mitochondrial superoxide generation is an important component of barley root response to severe Cd stress.

CARD19, a Novel Negative Regulator of B-Cell Tolerance

The CARD11-Bcl10-Malt1 (CBM) signalosome controls TAK1 activation to regulate B-cell receptor (BCR)-induced NF-κB activation and B cell biology. The biological function of caspase recruitment domain family member 19 (CARD19), originally identified as a BCL10-interacting CARD protein (BinCARD), is not known. Here we found CARD19 strongly interacted with TAK1 but not BCL10 or other CBM components and prevented TAK1's association with TAB2, thereby inhibiting TAB2-mediated TAK1 ubiquitination and activation and subsequent NF-κB activation. CARD19 was ubiquitously expressed in hematopoietic lineages but its deficiency in mice had no effect on hematopoiesis, including B cell development and humoral immune response. CARD19 deficiency enhanced clonal deletion, receptor editing and anergy of self-reactive B cells, thus reducing autoantibody production in vivo. Mechanistically, CARD19 deficiency led to an increase of BCR/TAK1-mediated NF-κB activation. Activation of NF-κB, such as c-Rel, was responsible for the up-regulation of BCR-induced expression of the transcription factor early growth response genes 2 and 3 (Egr2, Egr3) and the E3 ubiquitin ligases, c-Cbl and Cbl-b, the important inducers of B-cell tolerance in B cells. Further, high-throughput RNA sequencing analysis revealed that CARD19 deficiency did not affect the overall antigen-induced gene expression in naïve B cells but suppressed BCR signaling to increase hyporesponsiveness of self-reactive B cells. Consequently, CARD19 deficiency prevented Bm12-induced experimental systemic lupus erythematosus (SLE) and autoimmunity in a B cell-intrinsic manner. Taken together, CARD19 negatively regulates BCR-induced NF-κB activation via blocking TAK1/TAB2 interaction and its deficiency leads to NF-κB-induced expression of Egr2/3 and c-Cbl/Cbl-b in self-reactive B cells, which enhances B-cell tolerance and thus prevents autoimmunity. DisclosuresNo relevant conflicts of interest to declare.

P-065: CyTOF and single cell RNA sequencing reveal altered T cell phenotypes in Multiple Myeloma patients: implications for immunotherapy

<h3>Background</h3> Immune therapies have had a major impact in multiple myeloma (MM). Responses induced in relapsed and/or refractory MM (RRMM) patients (pts) are unprecedented; however the key challenge is their durability. Most immunotherapeutic approaches rely on the fitness of pts' T cells but exposure to multiple therapies may alter immune cell numbers and function. Here we analyzed and compared the transcriptome and phenotype of T cells from healthy donors (HD), newly diagnosed MM (NDMM) and RRMM pts. <h3>Methods</h3> Peripheral Blood Mononuclear Cells (PBMCs) from age-matched HD, NDMM and RRMM pts were collected before the start of a new line of treatment. Single cell RNA sequencing (scRNAseq) was performed and proportion of cell types were inferred using a reference PBMC dataset with Seurat. Cell type proportions were also estimated with CyTOF and conventional flow cytometry <h3>Results</h3> PBMCs from 10 HDs (median age 69, range 53-79), 20 NDMM pts (median age 71, range 54-88), 20 RRMM pts (median age 71, range 54-80) were analyzed. RRMM pts were exposed to 1 (n=8), 2 (n=6) or 3 (n=6) lines of treatment. All RRMM pts were exposed to a proteasome inhibitor, 75% were exposed to an immunomodulatory drug, and 65% received autologous stem cell transplantation. 48 out of 50 samples were processed by scRNAseq, 45 by CyTOF and 33 by flow cytometry. NDMM pts demonstrated a trend towards lower frequency of CD4 T cells compared to HD. This decreased number was accompanied by a decrease in the expression of genes involved in mitochondrial oxidative phosphorylation and an increase in expression of immediate early response genes (e.g. FOS, JUN, JUNB). In the CD8 compartment, CD8 naive cells were lower in NDMM compared to HD. No differences were found on CD8 central memory, effector memory and/or terminal effector populations In RRMM, the proportion of naive CD4 cells was significantly lower compared to NDMM (p < 0.05 by scRNAseq and CyTOF). This was confirmed by conventional flow cytometry (p < 0.05). No differences in CD4 central memory, effector memory and/or terminal effector proportions were observed. Decreases in the proportion of naive CD4 cells were observed as early as after 1 line of treatment and did not correlate with age, absolute lymphocyte count, previous exposure to lenalidomide or cyclophosphamide cumulative dose. Exposure to higher melphalan doses corelated with lower proportion of CD4 naive T cells. Compared with T cells from NDMM, T cells harvested from RRMM pts expressed higher levels of activation/exhaustion markers such as PD1 (p < 0.05) and HLA-DR (p < 0.05). <h3>Conclusion</h3> T cell phenotypes in MM pts are altered, especially after treatment. These findings may provide the rationale for the investigation of immunotherapy in NDMM and less heavily pretreated MM. Ongoing functional studies will demonstrate whether these phenotypic and transcriptomic changes lead to less fit T cells with consequent impact on effectiveness of T cell directed therapies.

Abstract P140: Molecular Signatures Of The Delayed Healing Response To Arterial Injury In Diabetic Rats

Aim: Atherosclerosis is a major complication of diabetes mellitus and a leading cause of mortality in diabetic patients. Vascular interventions in diabetic patients can lead to complications attributed to defective vascular remodeling and impaired healing response. In this study, we aim to elucidate the physiological and molecular differences in the vascular healing response over time using a rat model of arterial injury applied in healthy and diabetic conditions. Methods: Wistar (healthy) and Goto-Kakizaki (GK, diabetic) rats (n = 40 per strain) were subjected to left common carotid artery (CCA) balloon injury and euthanized at different timepoints: 0 and 24 hours, 5 days, 2, 4 and 6 weeks. Non-invasive morphological and physiological assessment, and microarray profiling of the CCA was performed for each timepoint. Bioinformatic analyses were conducted using R software, DAVID bioinformatic tool, online STRING database and Cytoscape software. Results: Significant increase in the neointimal thickness (p&lt;0.01; 2-way ANOVA) was observed after 2 weeks of injury in diabetic compared to heathy rats, which was confirmed by histological analyses. Moreover, a decrease in the reendothelialization rate was also detected in the diabetic rats at 4 and 6 weeks after injury. Bioinformatic analyses showed that expression of early response genes related to inflammation and proliferation were delayed in diabetic rats, coupled with the dysregulation of key pathways related to vascular healing. Defective expression patterns were observed for endothelial, macrophage and lymphocyte markers indicating potential faults on cell regulation level. TF-PPI analysis provided mechanistic evidence wherein an array of transcription factors was dysregulated in diabetic rats specifically from 2 weeks after injury. Conclusions: In this study, we investigated the effect of diabetes on vessel wall healing. We demonstrate that diabetic rats exhibit impaired arterial remodelling characterised by a delayed healing response. These results further corroborate the higher prevalence of restenosis in diabetic patients and provide vital molecular insights into the mechanisms contributing to the impaired arterial healing response in diabetes.

FGF7 Supports Hematopoietic Stem and Progenitor Cells and Niche-Dependent Myeloblastoma Cells Via Autocrine Action On Bone Marrow Stromal Cells In Vitro

Some fibroblast growth factor (FGF)/FGF receptor (FGFR) systems, namely FGF1 and FGF2 which are produced mainly by megakaryocytes, and their cognate receptors expressed on either hematopoietic stem or progenitor cells (HSPCs) or bone marrow (BM) stromal cells, are reported to support HSPCs under stressed conditions. In this study, we show that mesenchymal cell-derived FGF7 (also called keratinocyte growth factor or KGF) may be a novel niche factor for HSPC support and niche-dependent leukemia-initiating cell growth. We previously reported that mouse embryonic fibroblasts (MEFs) have characteristics that mimic BM stromal cells and possess an ability of HSPCs support in vitro, and that this ability is profoundly attenuated in MEFs deficient for the MED1 subunit of the Mediator transcriptional master coregulator complex, subcomplex of RNA polymerase II holoenzyme. We found through the microarray analyses that the FGF7 expression (both at mRNA and protein levels) was specifically and profoundly attenuated in Med1-/- MEFs compared with Med1+/+ MEFs. When normal mouse BM cells were cocultured with mitomycin C-treated Med1+/+ MEFs or BM stromal cells (OP-9 or MS-5) in the presence of anti-FGF7 antibody, the DNA synthesis and resultant growth of BM cells, and the number of long-term culture-initiating cells (LTC-ICs), were significantly suppressed. However, the addition of anti-FGF7 antibody did not enhance cell death in BM cells. In contrast, the addition of recombinant FGF7 to the coculture of BM cells with mitomycin C-treated Med1-/- MEFs robustly upregulated the DNA synthesis and growth of BM cells, and the number of LTC-ICs. The Ph+ myeloblastoma cells MB-1, originally isolated from a patient with blast crisis chronic myeloid leukemia, is niche-dependent myeloblastic cells that require mesenchymal stromal cells for survival and growth. During coculture typically with OP-9 BM stromal cells, these MB-1 cells form cobblestone areas, which most probably reconstitute a stochastic model of leukemic stem or initiating cells in vitro. We next analyzed the role of FGF7 in support of MB-1 cells for growth and survival during coculture. Indeed, the addition of anti-FGF7 antibody to the coculture with mitomycin C-treated BM stromal cells (either OP-9 or MS-5) substantially attenuated DNA synthesis and resultant proliferation of stromal cell-dependent myeloblastoma cells MB1. Notably, cobblestone formation (i.e., signature of stem cell phenotype) of MB-1 cells was significantly attenuated when anti-FGF7 antibody was added to the coculture with OP-9 cells. However, the addition of anti-FGF7 antibody did not elicit cell death of MB-1 cells. Thus, FGF7 appears to promote both proliferation and maintenance of leukemia stem or initiating cell feature of MB-1 cells. While neither FGF7 nor its cognate receptor FGFR2IIIb mRNAs were detectable in BM cells, the mesenchymal cells including Med1+/+ MEFs and (OP-9 and MS-5) BM stromal cells expressed them. The expression levels of FGFR2IIIb were comparable between Med1+/+ and Med1-/- MEFs, and BM stromal cells. Therefore, we hypothesized that FGFR2IIIb on these mesenchymal stromal cells might be functional and possibly elicit secretion of niche signal(s) secondarily in response to the downstream target gene(s) of FGFR2IIIb, followed by subsequent support of HSPCs and leukemic cells. To prove that FGF7 is functional in these cells, we examined response of the downstream targets of FGFR2IIIb in these cells, including phosphorylation of FRS2 (the immediate downstream target of FGFR2IIIb tyrosine kinase), phosphorylation of MAP kinases (the intermediate hub of various intracellular signals), and mRNA expression of immediate early response genes including c-Jun, c-Fos and c-Myc. Indeed, exogenously added recombinant FGF7 elicited phosphorylation of both FRS2 and MAP kinases within 10 min, and full induction of these immediate early response genes within 30 min, similarly in both Med1+/+ and Med1-/- MEFs. The OP-9 and MS-5 BM stromal cells also exhibited similar responses to exogenously added FGF7. These results indicate that FGFR2IIIb expressed on mesenchymal stromal cells are functional, and that FGF7 and FGFR2IIIb produced by these cells constitute an autocrine (or paracrine) loop. Taken together, we propose that FGF7 produced by BM stromal cells supports HSPCs and leukemic progenitor cells indirectly via FGFR2IIIb expressed on the BM stromal cells. Disclosures:No relevant conflicts of interest to declare.

1α,25-Dihydroxyvitamin D3 Encapsulated in Nanoparticles Prevents Venous Neointimal Hyperplasia and Stenosis in Porcine Arteriovenous Fistulas.

Few therapies prevent venous neointimal hyperplasia (VNH) and venous stenosis (VS) formation in arteriovenous fistulas (AVF). Expression of the immediate early response gene X-1 (Iex-1), also known as Ier3, is associated with VNH and stenosis in murine AVFs. The study aimed to determine if local release of Ier3 long-acting inhibitor 1α,25(OH)2D3 from poly(lactic-co-glycolic acid) (PLGA) nanoparticles embedded in a thermosensitive Pluronic F127 hydrogel (1,25 NP) could affect VNH/VS formation in a large animal model. Immediately after AVF creation in a porcine model of renal failure, 1,25 NP or vehicle control was injected into the adventitia space of AVF outflow veins. Scanning electron microscopy and dynamic light scattering characterized drug and control nanoparticles. Animals were sacrificed 3 and 28 days later for gene expression, immunohistologic, magnetic resonance imaging and angiography, and ultrasound analyses. Whole transcriptome RNA sequencing with differential gene expression analysis was performed on outflow veins of AVF. Encapsulation of 1α,25(OH)2D3 in PLGA nanoparticles formed nanoparticles of uniform size that were similar to nanoparticles without 1α,25(OH)2D3. The 1,25 NP-treated AVFs exhibited lower VNH/VS, Ier3 gene expression, and IER-3, MCP-1, CD68, HIF-1α, and VEGF-A immunostaining, fibrosis, and proliferation. Blood flow and lumen area increased significantly, whereas peak systolic velocity and wall shear stress decreased. Treatment increased Young's modulus and correlated with histologic assessment of fibrosis and with no evidence of vascular calcification. RNA sequencing analysis showed changes in the expression of genes associated with inflammatory, TGFβ1, and apoptotic pathways. Local release of 1,25 NP improves AVF flow and hemodynamics, and reduces stenosis in association with reduction in inflammation, apoptosis, and fibrosis in a porcine model of arteriovenous fistula.

The adverse effects of acrylamide exposure on the early development of marine medaka (Oryzias melastigma) and its mechanisms

Acrylamide (AA) can have deleterious effects on freshwater fish. However, its adverse effects on euryhaline fish are still unknown. In this study, embryos of Oryzias melastigma were exposed to different concentrations of AA to investigate its effect on early developmental disorders. After 21 days of exposure, AA significantly inhibited the hatching rate and delayed the hatching time of embryos, and led to developmental delay, teratogenesis, and locomotion impairments in larvae. RNA-sequencing data of larvae indicated that AA upregulated the expression of hemoglobin and myoglobin involved in oxygen transport and angiopoietin 1, integrin, and matrix metallopeptidases related to angiogenesis and downregulated the expression of early growth response genes and synaptotagmin-2 related to neural plasticity and neurotransmitter release. Overall, our study showed that AA caused deleterious effects on the early development of euryhaline fish through hypoxic stress and neurotoxicity, providing a scientific basis for the environmental risk assessment of marine AA.

Temporal unsnarling of brain\u2019s acute neuroinflammatory transcriptional profiles reveals panendothelitis as the earliest event preceding microgliosis

Sepsis-associated encephalopathy (SAE) is an acutely progressing brain dysfunction induced by systemic inflammation. The mechanism of initiation of neuroinflammation during SAE, which ultimately leads to delirium and cognitive dysfunction, remains elusive. We aimed to study the molecular events of SAE to capture its onset and progression into the central nervous system (CNS), and further identify the cellular players involved in mediating acute inflammatory signaling. Gene expression profiling on the cerebral vessels isolated from the brains of the mice treated with peripheral lipopolysaccharide (LPS) revealed that the cerebral vasculature responds within minutes to acute systemic inflammation by upregulating the expression of immediate early response genes, followed by activation of the nuclear factor-κB pathway. To identify the earliest responding cell type, we used fluorescence-activated cell sorting (FACS) to sort the glial and vascular cells from the brains of the mice treated with LPS at different time points, and RNA-seq was performed on microglia and cerebral endothelial cells (CECs). Bioinformatic analysis followed by further validation in all the cell types revealed that panendothelitis. i.e., the activation of CECs is the earliest event in the CNS during the inception of acute neuroinflammation. Microglial activation occurs later than that of CECs, suggesting that CECs are the most likely initial source of proinflammatory mediators, which could further initiate glial cell activation. This is then followed by the activation of apoptotic signaling in the CECs, which is known to lead to the blood–brain barrier disruption and allow peripheral cytokines to leak into the CNS, exacerbate the gliosis, and result in the vicious neuroinflammatory cascade. Together, our results model the earliest sequential events during the advancement of systemic inflammation into the CNS and facilitate to understand the interplay between the vascular and glial cells in initiating and driving acute neuroinflammation during SAE.