Expression Of Delta Research Articles

Hinokiol regulates the cell cycle and apoptosis of nasopharyngeal carcinoma CNE1 cells via Hippo-YAP signaling pathway

Objective: To explore the effects of hinokiol on the cell cyle and apoptosis of CNE1 nasopharyngeal carcinoma cells and the relevant molecular mechanism. Methods: The CNE1 cells were cultured in vitro and incubated with different concentrations of honokiol, and the cells were divided into blank control group, 10 μmol/L, 20 μmol/L and 40 μmol/L hinokiol treatment groups, and 10 μg/ml cisplatin group. Cell viability was determined by methylthiazolyldiphenyl- tetrazolium bromide (MTT) method, the cell cycle distribution was detected by flow cytometry, mitochondrial membrane potential was detected by mitochondrial membrane potential test kit, apoptosis was detected by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) method, and the proteins expression of proliferating cell nuclear antigen (PCNA) and G1/S specific cyclin D1 (cyclin D1) were detected by immunoblotting. RNA-Seq was conducted in the hinokiol-treated cells. The mRNA expression of yes-associated protein delta (YAP) was detected by quantitative reverse transcription polymerase chain reaction (RT-qPCR). The proteins expression of phosphor-YAP (p-YAP) and nuclear YAP were detected by immunoblotting, the nuclear distribution of YAP protein was detected by immunofluorescence in the cells with or without treated with the mammalian STE20-like kinase 1/2 (MST1/2) inhibitor (XMU-MP-1), hinokiol, and XMU-MP-1+hinokiol. Statistical analysis of the data was conducted using GraphPad Prism 8.0 software. Resluts Compared with the control group, the cell viablity of CNE1 cells, the levels of mitochondrial membrane potential, the proteins expression of PCNA and cyclin D1 in hinokiol treatment groups were markedly decreased (all P values<0.05), while the proportion of G0/G1 phase cells and the ratio of TUNEL-positive cells were significantly increased (both P values<0.05). Transcriptome analysis showed that differential genes were mainly enriched in Wnt signaling pathway, tumor necrosis factor pathway, and Hippo signaling pathway. The mRNA level of YAP and the protein expression of YAP in the nucleus were decreased and the level of p-YAP protein was increased in cells treated with hinokiol, which were significantly different from control group (all P values<0.05). Compared with the hinokiol group, XMU-MP-1+hinokiol groups showed the decrease of p-YAP protein expression (1.157±0.076 vs 0.479±0.038, t=37.120, P<0.05), the increase of YAP protein expression in the nucleus (0.143±0.012 vs 0.425±0.031, t=29.181, P<0.05), the reduced proportion of cells in G0/G1 phase [(72.494±3.309)% vs (58.747±2.865)%, t=17.265, P<0.05], and the decrease of apoptosis ratio [(53.158±3.376)% vs (29.621±2.713)%, t=28.584, P<0.05]. Conclusion: Hinokiol can arrest the cell cycle and induce the cell apoptosis of CNE1 cells via Hippo/YAP signaling pathway.

Intranasal delivery of small extracellular vesicles from specific subpopulation of mesenchymal stem cells mitigates traumatic spinal cord injury

Vascular injury following spinal cord injury (SCI) can significantly exacerbate secondary SCI and result in neurological dysfunction. Strategies targeting angiogenesis have demonstrated potential in enhancing functional recovery post-SCI. In the context of angiogenesis, the CD146+ and CD271+ subpopulations of mesenchymal stem cells (MSCs) have been recognized for their angiogenic capabilities in tissue repair. Small extracellular vesicles (sEVs) derived from MSCs are nanoscale vesicles containing rich bioactive components that play a crucial role in tissue regeneration. However, the precise role of sEVs derived from CD146+CD271+ UCMSCs (CD146+CD271+ UCMSC-sEVs) in SCI remain unclear. In this study, CD146+CD271+ UCMSC-sEVs were non-invasively administered via intranasal delivery, demonstrating a significant capacity to stimulate angiogenesis and improve functional recovery in mice following SCI. Furthermore, in vitro assessments revealed the effective enhancement of migration and tube formation capabilities of the murine brain microvascular endothelial cell line (bEnd.3) by CD146+CD271+UCMSC-sEVs. MicroRNA array analysis confirmed significant enrichment of multiple microRNAs within CD146+CD271+ UCMSC-sEVs. Subsequent in vivo and in vitro experiments demonstrated that CD146+CD271+ UCMSC-sEVs promote enhanced angiogenesis and improved functional recovery mediated by miR-27a-3p. Further mechanistic studies revealed that miR-27a-3p sourced from CD146+CD271+ UCMSC-sEVs enhances migration and tube formation of bEnd.3 cells in vitro by suppressing the expression of Delta Like Canonical Notch Ligand 4 (DLL4), thereby promoting angiogenesis in vivo. Collectively, our results demonstrate that a crucial role of CD146+CD271+ UCMSC-sEVs in inhibiting DLL4 through the transfer of miR-27a-3p, which leads to the promotion of angiogenesis and improved functional recovery after SCI.

Effects of therapeutic hypercapnia on the expression and function of γδT cells in transplanted lungs in rats.

To investigate the effect of therapeutic hypercapnia on the expression and function of gamma delta T (γδ T) cells during ischemia-reperfusion injury (IRI) after lung transplantation. We randomly divided male Wistar rats into three groups (n = 6 in each group), the control group (group N), the IRI group (group I), and the therapeutic hypercapnia group (group H). We then assessed pulmonary edema, neutrophil infiltration, wet-to-dry (W/D) weight ratio, and microscopic histopathology and separately measured the levels of γδT cell surface antigen (TCR) and Interleukin-17 (IL-17) using flow cytometry and enzyme-linked immunosorbent assays (ELISAs). The infiltration of neutrophils and the expression of TCR and IL-17 were significantly increased in the I group compared to the control, and the biopsy edema in group I was more severe. Arterial partial pressure of oxygen (PaO2) was decreased after reperfusion in group I compared with the control group. W/D weight ratio, neutrophil infiltration, and the expression of TCR and IL-17 decreased drastically in the H group compared to the I group. Our findings suggest that γδ T lymphocytes were directly involved in lung injury. In addition, therapeutic hypercapnia effectively reduced the expression of γδ T cells and IL-17, and this has the potential to become a treatment strategy for IRI and an intervention to improve lung function.

Stem cell mTOR signaling directs region-specific cell fate decisions during intestinal nutrient adaptation.

The adult intestine is a regionalized organ, whose size and cellular composition are adjusted in response to nutrient status. This involves dynamic regulation of intestinal stem cell (ISC) proliferation and differentiation. How nutrient signaling controls cell fate decisions to drive regional changes in cell-type composition remains unclear. Here, we show that intestinal nutrient adaptation involves region-specific control of cell size, cell number, and differentiation. We uncovered that activation of mTOR complex 1 (mTORC1) increases ISC size in a region-specific manner. mTORC1 activity promotes Delta expression to direct cell fate toward the absorptive enteroblast lineage while inhibiting secretory enteroendocrine cell differentiation. In aged flies, the ISC mTORC1 signaling is deregulated, being constitutively high and unresponsive to diet, which can be mitigated through lifelong intermittent fasting. In conclusion, mTORC1 signaling contributes to the ISC fate decision, enabling regional control of intestinal cell differentiation in response to nutrition.

Notch signaling and Bsh homeodomain activity are integrated to diversify Drosophila lamina neuron types

Notch signaling is an evolutionarily conserved pathway for specifying binary neuronal fates, yet how it specifies different fates in different contexts remains elusive. In our accompanying paper, using the Drosophila lamina neuron types (L1-L5) as a model, we show that the primary homeodomain transcription factor (HDTF) Bsh activates secondary HDTFs Ap (L4) and Pdm3 (L5) and specifies L4/L5 neuronal fates. Here we test the hypothesis that Notch signaling enables Bsh to differentially specify L4 and L5 fates. We show asymmetric Notch signaling between newborn L4 and L5 neurons, but they are not siblings; rather, Notch signaling in L4 is due to Delta expression in adjacent L1 neurons. While Notch signaling and Bsh expression are mutually independent, Notch is necessary and sufficient for Bsh to specify L4 fate over L5. The NotchON L4, compared to NotchOFF L5, has a distinct open chromatin landscape which allows Bsh to bind distinct genomic loci, leading to L4-specific identity gene transcription. We propose a novel model in which Notch signaling is integrated with the primary HDTF activity to diversify neuron types by directly or indirectly generating a distinct open chromatin landscape that constrains the pool of genes that a primary HDTF can activate.

Notch signaling and Bsh homeodomain activity are integrated to diversify Drosophila lamina neuron types.

Notch signaling is an evolutionarily conserved pathway for specifying binary neuronal fates, yet how it specifies different fates in different contexts remains elusive. In our accompanying paper, using the Drosophila lamina neuron types (L1-L5) as a model, we show that the primary homeodomain transcription factor (HDTF) Bsh activates secondary HDTFs Ap (L4) and Pdm3 (L5) and specifies L4/L5 neuronal fates. Here we test the hypothesis that Notch signaling enables Bsh to differentially specify L4 and L5 fates. We show asymmetric Notch signaling between newborn L4 and L5 neurons, but they are not siblings; rather, Notch signaling in L4 is due to Delta expression in adjacent L1 neurons. While Notch signaling and Bsh expression are mutually independent, Notch is necessary and sufficient for Bsh to specify L4 fate over L5. The NotchON L4, compared to NotchOFF L5, has a distinct open chromatin landscape which allows Bsh to bind distinct genomic loci, leading to L4-specific identity gene transcription. We propose a novel model in which Notch signaling is integrated with the primary HDTF activity to diversify neuron types by directly or indirectly generating a distinct open chromatin landscape that constrains the pool of genes that a primary HDTF can activate.

Delta and Theta Operator Expansions

Abstract We give an elementary symmetric function expansion for the expressions $M\Delta _{m_\gamma e_1}\Pi e_\lambda ^{\ast }$ and $M\Delta _{m_\gamma e_1}\Pi s_\lambda ^{\ast }$ when $t=1$ in terms of what we call $\gamma $ -parking functions and lattice $\gamma $ -parking functions. Here, $\Delta _F$ and $\Pi $ are certain eigenoperators of the modified Macdonald basis and $M=(1-q)(1-t)$ . Our main results, in turn, give an elementary basis expansion at $t=1$ for symmetric functions of the form $M \Delta _{Fe_1} \Theta _{G} J$ whenever F is expanded in terms of monomials, G is expanded in terms of the elementary basis, and J is expanded in terms of the modified elementary basis $\{\Pi e_\lambda ^\ast \}_\lambda $ . Even the most special cases of this general Delta and Theta operator expression are significant; we highlight a few of these special cases. We end by giving an e-positivity conjecture for when t is not specialized, proposing that our objects can also give the elementary basis expansion in the unspecialized symmetric function.

Abstract P3195: Mitochondrial CaMKII Drives Cardiometabolic Dysfunction In Heart Failure

Background: Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) is an established negative regulator of cardiac injury. Both the expression and activity levels of CaMKII delta, the primary cardiac isoform of this kinase, are elevated in models of heart failure (HF) such as ischemia-reperfusion (I/R) injury and following myocardial infarction (MI). This upregulation is due in part to CaMKII’s role in the regulation of excitation-contraction coupling, apoptosis, activation of hypertrophic programming, arrhythmias and pro-inflammatory signaling. Recently, a pool of mitochondrial CaMKII (mtCaMKII) has been identified; we have demonstrated that there is an observed increase in mitochondrial CaMKII activation with left ventricular dilation acutely following injury in a mouse model of MI. This deleterious post-MI phenotype is rescued with genetic mitochondrial CaMKII inhibition; conversely, mice with myocardial mitochondrial CaMKII overexpression present with a severe dilated cardiomyopathy phenotype, alterations in electron transport chain (ETC) activity, and decreased ATP production. Objective: To date, the molecular mechanisms by which CaMKII regulates mitochondrial energetics are unknown and present a novel therapeutic target for HF which we are currently studying. Methods: As we have identified changes in the activity of enzymes, particularly complexes I and II, in the mitochondrial ETC as well as TCA cycle in response to increased CaMKII levels or activity, we are in the process of assessing changes in the mitochondrial CaMKII interactome between uninjured and failing hearts. Utilizing liquid chromatography-mass spectrometry (LCMS) and proteomics analysis of both scaffolding interactions alongside a novel ATP analogue labeling technique, we have mapped novel mitochondrial proteins which interact with mtCaMKII. Significance: The identification of previously unknown mitochondrial pathways involving CaMKII may uncover promising pharmacological targets for cardiovascular therapeutics, as this kinase is a critical player in the progression of HF.

Revealing distinct immune landscape in EGFR mutant and EGFR wild type lung adenocarcinoma by using single-cell RNA sequencing.

e21008 Background: Non-small cell lung cancer (NSCLC) with EGFR mutations displays heterogeneity in the extent of intratumoral immunocytes infiltration compared to those with EGFR wild types, which may explain their variable responsiveness to PD-1 blockade. Here, we performed single-cell RNA sequencing (scRNA-seq) to unveil the immunescape and heterogeneity of lung adenocarcinoma (LUAD) between EGFR mutant (EGFR-mut) patients and wild type (EGFR-wt) patients. Methods: Eleven EGFR-mut and 7 EGFR-wt NSCLC tumor samples were analyzed. Immune cells (CD45+) were enriched through fluorescence-activated cell sorting (FACS) and subjected to single-cell RNA sequencing (scRNA-seq) using the 10X Genomics platform. Sequencing reads were normalized and analyzed using the R/Seurat package. Cellular components of each sample were determined based on known marker genes. Wilcox test was used to compare promotion by subtype composition and immune cell infiltrates. Results: Different immune cell compositions were observed between EGFR-mut and EGFR-wt LUAD tumor samples. We found EGFR-wt tumors are marked by an enrichment of CCL15+ tumor-associated macrophages(TAMs), which display pro-inflammatory properties and high expressions of TNF-a-signaling genes. In contrast, EGFR-mut tumors exhibit enrichment of SPARCL1+TAMs, which are involved in epithelial-mesenchymal transition (EMT) and angiogenesis. EGFR-wt tumors exhibited enrichment of CD8-Teff-MX1 and Gamma delta T cells, both of which displayed higher cytotoxic and naive features. Moreover, the high expression of Gamma delta T corresponds with a better clinical prognosis. In contrast, CD8-Teff-XCL1 and NKT-FGFBP2 were found enriched in EGFR-mut tumors, both of which contributed to higher exhaustion and decreased cytotoxic function. Additionally, NK-IL32 was found enriched in EGFR-mut tumors, showing a downregulated T cell receptor signaling and stronger exhausted features. The high expression of this subcluster correlated with poor outcomes in LUAD patients harboring EGFR mutation. Conclusions: Our study provided a deeper understanding of the immune landscape of EGFR-mut and EGFR-wt LUAD and gained better insights into the immunological mechanisms involved in the response to ICIs. The results of our study could be instructive for the development of future immunotherapy strategies for NSCLC patients harboring EGFR mutations.

Effects of dietary n-6 polyunsaturated fatty acids (PUFA) composition on growth performances and non-specific immunity in pacific white shrimp (Litopenaeus vannamei)

This study was aimed to investigate the effects of dietary arachidonic acid (ARA) and linoleic acid (LA) on growth, fatty acid composition, antioxidant capacity and immunity of Litopenaeus vannamei. Eight diets were prepared with different LA or ARA content and approximately equal amounts of α-linolenic acid (ALA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), including a control diet (CK0) without ARA and LA supplementation. 720 shrimp were fed above diets in triplicate for 56 days. The results showed that, the specific growth rate (SGR) and weight gain (WG) of shrimp fed the experimental diets were significantly enhanced compared to the control group. In general, the content of LA or ARA in the experimental shrimp tissues closely mirrored their respective dietary fatty acid levels. The expressions of delta 6 fatty acid desaturase (Δ6FAD) and fatty acid elongase 5 (Elovl5) genes in the intestine and hepatopancreas increased significantly with increasing dietary LA from 5.3 to 9.5, or ARA from 0.2 to 11.1 g kg−1. The activities of superoxide dismutase (SOD) and catalase (CAT), and content of malondialdehyde (MDA) decreased with increasing dietary LA from 5.3 to 9.5, or ARA from 0.2 to 3.8 g kg−1, then increased as dietary LA increased to 16.2, or ARA increased to 11.1 g kg−1. The activities of lysozyme (LZM), acid phosphatase (ACP) and alkine phosphatase (AKP) increased accordingly with the increase of dietary LA from 5.3 to 9.5 or ARA from 0.2 to 1.9 g kg−1. SOD and CAT gene expressions were opposite to the enzyme activity in ARA groups. The results suggested that appropriate dietary LA (9.5 g kg−1) or ARA (3.8 g kg−1) had positively effects on growth, EPA biosynthesis, antioxidant ability and immunity of L. vannamei.

Epigenetic Modulation of Opioid Receptors by Drugs of Abuse.

Chronic exposure to drugs of abuse produces profound changes in gene expression and neural activity associated with drug-seeking and taking behavior. Dysregulation of opioid receptor gene expression is commonly observed across a variety of abused substances including opioids, cocaine, and alcohol. Early studies in cultured cells showed that the spatial and temporal gene expression of opioid receptors are regulated by epigenetic mechanisms including DNA and histone modifications and non-coding RNAs. Accumulating evidence indicate that drugs of abuse can modulate opioid receptor gene expression by targeting various epigenetic regulatory networks. Based on current cellular and animal models of substance use disorder and clinical evidence, this review summarizes how chronic drug exposure alters the gene expression of mu, delta, kappa, and nociceptin receptors via DNA and histone modifications. The influence of drugs of abuse on epigenetic modulators, such as non-coding RNAs and transcription factors, is also presented. Finally, the therapeutic potential of manipulating epigenetic processes as an avenue to treat substance use disorder is discussed.

221 Putative Gene Regulators in the Equine Endometrium, Corpus Luteum and Embryo During Early Pregnancy in the Horse

Abstract It is estimated that 17.3% of all pregnancies in the horse are lost from the time of detection to day 40, which has significant economic impact. But still, equine reproductive research has limited funding opportunities. Understanding the dynamics between the embryo, endometrium, and corpus luteum (CL) is essential to ameliorate those losses. Pig embryonic interferon (IFN) gamma is essential for the establishment of pregnancy. In ruminants, IFN tau is the maternal recognition signal. The equine embryo seems to express IFN alpha, gamma, delta, and epsilon but higher expression was only identified on day(D) 16 embryos, after the maternal recognition of pregnancy (MRP), so the IFN role during pregnancy is unknown. Oxytocin and prostaglandins have been demonstrated to have a role during luteolysis and luteostasis in the horse, and therefore investigation the embryonic, endometrium, and CL putative gene regulators during the equine MRP is warranted. Oxytocinase (LNPEP), an aminopeptidase that metabolizes oxytocin (OXT) has been recently investigated with other putative gene regulators in the horse during the expected time of MRP. OXT and LNPEPmRNA were identified in the equine embryo, endometrium, and CL, but OXT/neurophysin-1 protein was not detected in the embryo, and present in the CL. Most of the endometrial and luteal mRNA differences were identified around D12 post-ovulation, suggesting being a critical time for MRP. Luteal OXT was not different between pregnant and non-pregnant mares. LNPEP mRNA was upregulated in pregnant and non-pregnant mares D10 and 12 luteal tissues along with prostaglandin E synthase (PTGES). Embryonic trophoblast LNPEP and PTGES have higher relative abundance on D15 conceptus compared earlier embryos, suggesting a potential role during embryonic fixation. The increased expression of LNPEP is suggested to have a role in decreasing oxytocin availability and shifting the ratio PGE:PGF to a PGE that will support luteal function during early pregnancy.

Therapeutic targets and functions of curcumol against COVID-19 and colon adenocarcinoma.

Since 2019, the coronavirus disease (COVID-19) has caused 6,319,395 deaths worldwide. Although the COVID-19 vaccine is currently available, the latest variant of the virus, Omicron, spreads more easily than earlier strains, and its mortality rate is still high in patients with chronic diseases, especially cancer patients. So, identifying a novel compound for COVID-19 treatment could help reduce the lethal rate of the viral infection in patients with cancer. This study applied network pharmacology and systematic bioinformatics analysis to determine the possible use of curcumol for treating colon adenocarcinoma (COAD) in patients infected with COVID-19. Our results showed that COVID-19 and COAD in patients shared a cluster of genes commonly deregulated by curcumol. The clinical pathological analyses demonstrated that the expression of gamma-aminobutyric acid receptor subunit delta (GABRD) was associated with the patients' hazard ratio. More importantly, the high expression of GABRD was associated with poor survival rates and the late stages of COAD in patients. The network pharmacology result identified seven-core targets, including solute carrier family 6 member 3, gamma-aminobutyric acid receptor subunit pi, butyrylcholinesterase, cytochrome P450 3A4, 17-beta-hydroxysteroid dehydrogenase type 2, progesterone receptor, and GABRD of curcumol for treating patients with COVID-19 and COAD. The bioinformatic analysis further highlighted their importance in the biological processes and molecular functions in gland development, inflammation, retinol, and steroid metabolism. The findings of this study suggest that curcumol could be an alternative compound for treating patients with COVID-19 and COAD.

Protein kinase c delta expression in primary central nervous system lymphomas

Protein kinase c delta expression in primary central nervous system lymphomas

Integrative Analysis of Biomarkers and Mechanisms in Adamantinomatous Craniopharyngioma.

Craniopharyngioma is a benign tumor, and the predominant treatment methods are surgical resection and radiotherapy. However, both treatments may lead to complex complications, seriously affecting patients’ survival rate and quality of life. Adamantinomatous craniopharyngioma (ACP), as one of the histological subtypes of craniopharyngioma, is associated with a high incidence and poor prognosis, and there is a gap in the targeted therapy of immune-related genes for ACP. In this study, two gene expression profiles of ACP, namely GSE68015 and GSE94349, were downloaded from the Gene Expression Omnibus (GEO) database. The differentially expressed genes (DEGs) were identified by the Limma package, and 271 differentially expressed immune-related genes (DEIRGs) were obtained from the Immport database. The gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Gene Set Enrichment Analysis (GSEA) were performed for annotation, visualization, and integrated discovery. Five hub genes, including CXCL6, CXCL10, CXCL11, CXCL13, and SAA1, were screened out through protein-protein interaction (PPI) network interaction construction. Two diagnostic markers, namely S100A2 and SDC1 (both of which have the Area Under Curve value of 1), were screened by the machine learning algorithm. CIBERSORT analysis showed that M2 macrophages, activated NK cells, and gamma delta T cells had higher abundance in ACP infiltration, while CD8+ T cells, regulatory T cells, and Neutrophils had less abundance in ACP infiltration. The expression of gamma delta T cells was positively correlated with CXCL6, S100A2, SDC1, and SAA1, while CD8+ T cells expression was negatively correlated with CXCL6, S100A2, SDC1, and CXCL10. ACP with high CXCL6 showed remarkable drug sensitivity to Pentostatin and Wortmannin via CellMiner database analysis. Our results deepened the understanding of the molecular immune mechanism in ACP and provided potential biomarkers for the precisely targeted therapy for ACP.

Helicid Reverses the Effect of Overexpressing NCALD, Which Blocks the sGC/cGMP/PKG Signaling Pathway in the CUMS-Induced Rat Model.

Background Increasing evidence has shown that apoptosis in the hippocampus is closely related to depressive-like behavior. We previously reported that helicid had good antidepressant activities, which manifested as the alleviation of depression-like behaviors and the reversal of the high expression of neurocalcin delta (NCALD) in chronic unpredictable mild stress (CUMS) rats. The aim of this study was, therefore, to characterize the antidepressant-like effects and underlying mechanism of helicid on CUMS rats by silencing NCALD and using rescue experiments. Methods We developed the CUMS rat model using CUMS stimulation from week 0 to week 6. The rats were treated with helicid, or NCALD silenced, then we overexpressed NCALD using adeno-associated virus. We also measured the protein levels of sGCα1, sGCβ1, PKG1/2, and cleaved caspase-3 in hippocampal tissues using western blotting and measured cGMP using an ELISA. Results Treating CUMS rats by silencing NCALD or by the administration of helicid improved the depressive-like behavior. The levels of proteins, including sGC, PKG, cleaved caspase-3, and cGMP, in hippocampus all decreased. NCALD overexpression reversed these decreases and reversed the alleviation of depression-like behaviors in CUMS rats. Limitation. We only detected the antidepressant effects of helicid in the hippocampus; therefore, other parts of brain should also be studied. Conclusions Inhibition of NCALD, as well as helicid administration, alleviated antidepressant-like behavior by regulating the expressions of apoptotic cytokines and the sGC/cGMP/PKG signaling pathway. Overexpressing NCALD reversed the amelioration effects of silenced NCALD and helicid administration.

Single-Cell Transcriptional Profiling of Zebrafish Hematopoiesis Offers Insight into Early Lymphocyte Development and Reveals Novel Immune Cell Populations

The advent of single-cell RNA sequencing (scRNA-seq) has greatly expanded our appreciation for cell state diversity beyond classical developmental hierarchies and simple population subsets. In particular, rich transcriptional heterogeneity has been observed within immune cell populations leading to the identification of novel cell types. Except for a few notable exceptions, intensive work in this area has largely been confined to mammals. Much of the transcriptional profiling of blood cell development in zebrafish has failed to capture early stages of lymphocyte development as the majority of research has not included thymus datasets or captured sufficient marrow B cells to explore developmental trajectories. To gain insight into T and B cell development in the zebrafish and immune cell diversity more broadly, we performed scRNA-seq using 10x Genomics Next GEM technology on adult zebrafish kidney marrows (n = 5 biological replicates) in addition to whole juvenile thymi at 4 weeks post-fertilization (wpf) (n = 4 technical replicates) and whole adult thymi at 3-4 months post-fertilization (n = 4 biological replicates). After filtering out low quality cellular barcodes, 34,492 kidney marrow cells and 35,268 thymus cells remained for analysis. With respect to T cell development, we identified putative early thymic progenitors from their clustering with hematopoietic stem and progenitor cells and shared transcriptional signatures, including the enrichment of CD34, CSF1R, FLI1, and DNMT3B human orthologs. Multiple subsets of thymic and marrow T cells were characterized, including a subset of gamma delta T cells readily identified by their expression of T-cell receptor gamma and delta chain components and expression of a SOX13 ortholog in addition to a Th2-like population expressing IL4, IL13, and GATA3 orthologs. Among other immune cell populations, rich transcriptional diversity was present. Two distinct populations of B cells, largely mutually exclusive for ighd and ighz expression (dual detection <1%), were present across all datasets, including the 4 wpf thymi, a surprisingly early time point in zebrafish B cell ontogeny. A clustering of the adult datasets demonstrated ighz predominance in the thymus (63% of thymic B cells), whereas the majority of marrow B cells (85%) fell within the ighd cluster. Stages of B cell development were also clearly evident, with the earliest B cell progenitors expressing orthologs of human PAX5, DNTT, RAG1, and RAG2, in addition to sid1, an understudied gene proposed to be orthologous to VPREB1. The expression of dntt was notably absent from more mature subsets of rag1 and rag2+ B cell progenitors, analogous to its expression in mammals. Transcriptional signatures unique but reminiscent of mammalian dendritic cell subsets were identified: Plasmacytoid-like dendritic cells characterized by high expression of TLR7, TLR9, and IRF8 orthologs and conventional-like dendritic cells characterized by high expression of CKB, BATF3, and ZNF366 were present in both marrow and thymus datasets, suggestive of greater dendritic-like cell diversity in the zebrafish than previously appreciated. These findings illustrate the power of single-cell transcriptional profiling for illuminating immune cell development and heterogeneity in the zebrafish, demonstrating increasing parallels to the mammalian system. DisclosuresZon: Celularity: Consultancy; Branch Biosciences: Current holder of individual stocks in a privately-held company, Other: Founder; Scholar Rock: Current equity holder in publicly-traded company, Other: Founder; Amagma Therapeutics: Current holder of individual stocks in a privately-held company, Other: Founder; CAMP4 Therapeutics: Current holder of individual stocks in a privately-held company, Other: Founder; Fate Therapeutics: Current equity holder in publicly-traded company, Other: Founder; Cellarity: Consultancy.

Effects of dezocine on morphine tolerance and opioid receptor expression in a rat model of bone cancer pain

BackgroundClinically, the coadministration of opioids to enhance antinociception and decrease tolerance has attracted increasing research attention. We investigated the effects of dezocine, a mu- and kappa-opioid receptor agonist/antagonist, on morphine tolerance and explored the involvement of opioid receptor expression in a rat model of bone cancer pain.MethodsThermal nociceptive thresholds were measured after the subcutaneous injection of morphine (10 mg/kg) alone or combined with dezocine (10 or 1 mg/kg) for 7 consecutive days. Real-time PCR and western blot analysis were used to examine opioid receptor expression in the periaqueductal gray (PAG) and spinal cord.ResultsThe analgesic effect was significantly decreased after 4 days of morphine administration. We observed that low-dose dezocine significantly attenuated morphine tolerance without reducing the analgesic effect of morphine. Low-dose dezocine coadministration significantly reversed the downregulated expression of mu (MOR) and delta (DOR) opioid receptors in the PAG and the upregulated expression of kappa (KOR) and DOR in the spinal cord induced by morphine. Moreover, low-dose dezocine coadministered with morphine significantly inhibited KOR expression in both the PAG and spinal cord.ConclusionsThe combination of low-dose dezocine with morphine may prevent or delay the development of morphine tolerance in a rat model of bone cancer pain. The regulation of opioid receptor expression in the PAG and spinal cord may be part of the mechanism.

Abstract 610: Paired alterations of 35-gene expressing profiles before and after neoadjuvant chemoradiation in esophageal squamous cell carcinoma patients impact clinical prognosis

Abstract Purpose: Neoadjuvant concurrent chemoradiotherapy (CCRT) followed by surgery is widely used for treating locally advanced esophageal cancer in the thorax. The aim of this study was to evaluate alterations of the 35-gene expression profiles in irradiated esophageal tissues before and after chemoradiation. Materials and Methods: A total number of 50 patients with Esophageal squamous cell carcinoma (ESCC) received neoadjuvant CCRT were retrospectively reviewed. The prescribed radiation dose for gross tumor and metastatic lymph nodes or subclinical mucosal/submucosal disease and regional lymphatic basin were 48 Gy and 43.2 Gy, respectively. All patients underwent CCRT using the intensity-modulated radiation therapy in 24 fractions and received concurrent chemotherapy with cisplatin. A 35-gene expression profile derived from reported ESCC-specific next-generation sequencing was performed. Paired 35-gene expression profiles were analyzed using pre-CCRT biopsy and post-CCRT esophagectomy specimens. In vitro validation of differential expression genes by using human ESCC cell lines 81T and TE2 was performed using the same 35-gene panel. Results: The alterations in mutation of genes between pre-CCRT biopsy and post-CCRT surgical specimens (delta expression profiles) correlated with pathological complete remission, overall survival and progression free survival. Among the delta expression profiles, the most significant alterations with prognostic impact were noted in MYH4 (p.Gln1210fs) and TTN (p.Asp7145His). Mutation of MYH4 (p.Gln1210fs) and TTN (p.Asp7145His) were demonstrated with favorable and unfavorable impact on rate of pathological complete remission, respectively. The mutation of MYH4 was also noted in human ESCC cell lines 81T and TE2 cells. Conclusion: By using the 35-gene expression profiles, specific mutations correlated to prognosis in esophageal squamous cell carcinoma patients receiving neoadjuvant chemoradiation and esophagectomy were found. The role of MYH4 mutation in treatment outcome and development as therapeutic target of esophageal squamous cell carcinoma remains to be clarified. Citation Format: Yu-Jen Chen, Prerana Bhan, Terry Wu, Hang Chang, Chi-Jung Lee. Paired alterations of 35-gene expressing profiles before and after neoadjuvant chemoradiation in esophageal squamous cell carcinoma patients impact clinical prognosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 610.

Protein Kinase C Delta in Diffuse Large B-Cell Lymphoma Post-Rituximab Era: Potential Prognostic Marker in Germinal Center B-Cell Lymphoma

Background: Diffuse Large B-cell lymphoma (DLBCL) is the most common subtype of lymphoma. Acquirement of resistance in DLBCL is a major clinical problem. The identification of biomarkers of clinical behavior may aid in the implementation of novel therapeutic strategies. Protein Kinase C (PKC) delta is a class serine/threonine kinase among the PKC family and contributes to cell proliferation, survival and apoptosis. The role of PKC delta in apoptosis is still controversial. Previously studies reported that overexpressed PKC delta promoted apoptosis. However, recently data showed inhibition of PKC delta blocked proliferation and survival of cancer stem cells in vitro and in vivo . The value of PKC delta expression in lymphoma patients remains unclear. We conducted a retrospective study to investigate the impact of PKC delta expression in DLBCL patients. Methods: Histologically confirmed DLBCL patients (N=98) treated at Fudan University Shanghai Cancer Center with available diagnostic biopsy material were identified. Forty-three patients were treated with CHOP and fifty-nine patients with R+CHOP. PKC delta expression was determined and quantified by immunohistochemistry (IHC) with staining intensity or percentage of positive cells. Tumors were considered positive when at least IRS>9 of tumor cells expressed PKC delta. Kaplan-Meier method was used to calculate progression free survival (PFS) and overall survival (OS) and curves were compared with the long-rank test in both groups. Correlation between the PKC delta expression and clinical variables were tested by Pearson Chi Square test and a two-sided P value of <0.05 was considered to be statistically significant. Additionally, PKC delta was inhibited using a PKC delta inhibitor (Rotterin) in DBCL cell lines. Changes in cell viability following in vitro exposure to rotterin were determined by Presto Blue Assay. Result: In CHOP treatment DLBCL, PKC delta expression was detected in 10/41 (24.4%) DLBCL patients. PKC delta expression was associated with a worse progression free survival (PFS) compared to PKC delta negative patients (50% vs 77%, p=0.036) treated with CHOP. The overall survival (OS) of PKC delta positive patients was also worse compared to the negative group (50% vs. 80%, p=0.023). In rituximab-CHOP (R-CHOP) treated patients, PKC delta was present in 17/57 (29.8%) patients. Surprisingly, no differences between the PKC delta positive group and negative R-CHOP treated groups were observed in terms of PFS or OS. Of interest, a subset analysis of R-CHOP treated DLBCL patients base on the cell of origin (COO) demonstrated that PKC delta expression in germinal center B-cell lymphoma (GCB) DLBCL was associated with an inferior OS outcome. The OS was 67% for GCB-DLBCL expressing PKC delta vs 83% for GCB-DLBCL patients without PKC delta expression (p=0.01). To identify the contribution of PKC delta to the survival of B-cell lymphoma, we performed the in vitro viability assay in different subtype of lymphoma cell lines using Rotterin, a PKC delta inhibitor. We found that in vitro exposure of cell lines to rotterin resulted in cell death in vitro suggesting that PKC delta can serve as a targetable biomarker in DLBCL. Additional pre-clinical studies are underway. Conclusion: This is the first report demonstrating that, PKC delta expression is a negative predictor of clinical outcome. PKC delta is a potential novel target to treat lymphoma and further investigation of PKC delta need to be done in the near future. (Research supported by Shanghai Municipal Commission of Health and Family Planning Project (no.20134030), National Natural Science Foundation of China (no.81670177, no.81001048), Roswell Park Cancer Institute Alliance Grant) [Display omitted] DisclosuresNo relevant conflicts of interest to declare.