scholarly journals Calcium-Prolactin Secretion Coupling in Rat Pituitary Lactotrophs Is Controlled by PI4-Kinase Alpha

2021 ◽  
Vol 12 ◽  
Author(s):  
Marek Kučka ◽  
Arturo E. Gonzalez-Iglesias ◽  
Melanija Tomić ◽  
Rafael M. Prévide ◽  
Kosara Smiljanic ◽  
...  
Keyword(s):  
Calcium Signaling ◽  
Single Cell ◽  
Rna Sequencing ◽  
Intracellular Signaling ◽  
Specific Inhibitor ◽  
Pcr Analysis ◽  
Single Cell Rna Sequencing ◽  
Specific Inhibitors ◽  
Calcium Secretion

The role of calcium, but not of other intracellular signaling molecules, in the release of pituitary hormones by exocytosis is well established. Here, we analyzed the contribution of phosphatidylinositol kinases (PIKs) to calcium-driven prolactin (PRL) release in pituitary lactotrophs: PI4Ks - which control PI4P production, PIP5Ks - which synthesize PI(4, 5)P2 by phosphorylating the D-5 position of the inositol ring of PI4P, and PI3KCs – which phosphorylate PI(4, 5)P2 to generate PI(3, 4, 5)P3. We used common and PIK-specific inhibitors to evaluate the strength of calcium-secretion coupling in rat lactotrophs. Gene expression was analyzed by single-cell RNA sequencing and qRT-PCR analysis; intracellular and released hormones were assessed by radioimmunoassay and ELISA; and single-cell calcium signaling was recorded by Fura 2 imaging. Single-cell RNA sequencing revealed the expression of Pi4ka, Pi4kb, Pi4k2a, Pi4k2b, Pip5k1a, Pip5k1c, and Pik3ca, as well as Pikfyve and Pip4k2c, in lactotrophs. Wortmannin, a PI3K and PI4K inhibitor, but not LY294002, a PI3K inhibitor, blocked spontaneous action potential driven PRL release with a half-time of ~20 min when applied in 10 µM concentration, leading to accumulation of intracellular PRL content. Wortmannin also inhibited increase in PRL release by high potassium, the calcium channel agonist Bay K8644, and calcium mobilizing thyrotropin-releasing hormone without affecting accompanying calcium signaling. GSK-A1, a specific inhibitor of PI4KA, also inhibited calcium-driven PRL secretion without affecting calcium signaling and Prl expression. In contrast, PIK93, a specific inhibitor of PI4KB, and ISA2011B and UNC3230, specific inhibitors of PIP5K1A and PIP5K1C, respectively, did not affect PRL release. These experiments revealed a key role of PI4KA in calcium-secretion coupling in pituitary lactotrophs downstream of voltage-gated and PI(4, 5)P2-dependent calcium signaling.

Exploring the role of the skin microenvironment in cutaneous T-cell lymphoma using single cell RNA-sequencing

2021 ◽  
Vol 156 ◽  
pp. S3-S4
Author(s):  
Gabor Dobos ◽  
Andreea Calugareanu ◽  
Laurence Michel ◽  
Maxime Battistella ◽  
Caroline Ram-Wolff ◽  
...  
Keyword(s):  
T Cell ◽  
Single Cell ◽  
Rna Sequencing ◽  
Cell Lymphoma ◽  
T Cell Lymphoma ◽  
Cutaneous T Cell Lymphoma ◽  
Single Cell Rna Sequencing

scholarly journals EPCO-32. AN EPIGENETIC SINGLE-CELL ATLAS OF IDH-MUTANT GLIOMA REVEALS THE ROLE OF ATRX IN SHAPING TUMOR COMPOSITION

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii76-ii76
Author(s):  
Husam Babikir ◽  
Lin Wang ◽  
Karin Shamardani ◽  
Sweta Sudhir ◽  
Gary Kohanbash ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Single Cell ◽  
Rna Sequencing ◽  
Myeloid Cell ◽  
Cell Types ◽  
Open Chromatin ◽  
Helix Loop Helix ◽  
Local Immunosuppression ◽  
Single Cell Rna Sequencing

Abstract Recent single-cell RNA-sequencing studies have identified a hierarchy of cell types that is common to all isocitrate dehydrogenase (IDH) -mutant gliomas. This finding is somewhat paradoxical since the genetic differences between IDH-mutant astrocytomas and IDH-mutant oligodendrogliomas are prognostic, predictive of therapeutic response, and correlated with differences in immune infiltrates. To integrate these disparate findings, we constructed a single-cell atlas of 28 human IDH-mutant primary untreated grade-II/III gliomas. All specimens were profiled by single-cell assay for transposase-accessible chromatin, with additional cohorts profiled via single-cell RNA-sequencing and single-cell spatial proteomics. We determined the cell-type specific differences between IDH-mutant gliomas in transcription-factor utilization, associated targeting and cis-regulatory grammars. To elucidate the role of the chromatin remodeler ATRX (inactivated in over 86% of IDH-mutant astrocytomas) in shaping observed differences in open chromatin, we knocked out ATRX in an immunocompetent model of IDH-mutant glioma and subjected murine tumors to single-cell profiling. We found: 1. ATRX-deficient, IDH-mutant human and murine gliomas both upregulate an astrocytic regulatory program driven by Nuclear Factor I genes and downregulate an oligodendrocytic program driven by basic helix-loop-helix transcription factors. 2. Both human and mouse ATRX-deficient, IDH-mutant gliomas up-regulate genes that promote myeloid-cell chemotaxis and both have significantly higher percentages of myeloid-derived immune-suppressive cells than controls; 3. A transcription-factor program is conserved between human and murine ATRX-deficient tumors that shapes glial identity and promotes local immunosuppression. These studies elucidate how IDH-mutant gliomas from different subtypes can have distinct cellular morphologies and tumor micronenvironments despite a common lineage hierarchy.

scholarly journals TMIC-06. MYELOID POPULATIONS AND THE EFFECT OF NEOADJUVANT PD-1 INHIBITION IN THE GLIOBLASTOMA MICROENVIRONMENT: A SURFACEOMIC AND TRANSCRIPTOMIC DISSECTION AT THE SINGLE-CELL LEVEL

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi248-vi248
Author(s):  
Aaron Mochizuki ◽  
Alexander Lee ◽  
Joey Orpilla ◽  
Jenny Kienzler ◽  
Mildred Galvez ◽  
...  
Keyword(s):  
Brain Tumor ◽  
T Cell ◽  
Single Cell ◽  
Rna Sequencing ◽  
Myeloid Cells ◽  
Single Cell Level ◽  
Mass Cytometry ◽  
Cell Level ◽  
Single Cell Rna Sequencing

Abstract INTRODUCTION Glioblastoma (GBM) is the most common malignant brain tumor in adults and is associated with a dismal prognosis. Neoadjuvant anti-PD-1 blockade has demonstrated efficacy in melanoma, non-small cell lung cancer and recurrent GBM; however, responses vary. While T cells have garnered considerable attention in the context of immunotherapy, the role of myeloid cells in the GBM microenvironment remains controversial. METHODS We isolated CD45+ immune populations from patients who underwent brain tumor resection at UCLA. We hypothesized that myeloid cells in glioblastoma contribute to T cell dysfunction; however, this immune suppression can be mitigated by neoadjuvant PD-1 inhibition. To test this, we utilized mass cytometry and single-cell RNA sequencing to characterize these immune populations. RESULTS Mass cytometry profiling of tumor infiltrating lymphocytes from patients with GBM demonstrated a preponderance of CD11b+ myeloid populations (75% versus 25% CD3+). At the transcriptomic level, myeloid cells in newly diagnosed GBMs exhibited decreased expression of CCL4 (loge fold change -1.18, Bonferroni-adjusted P = 1.62x10-254) and its ligands compared to anaplastic astrocytoma. In ranked gene set enrichment analysis, patients who received neoadjuvant pembrolizumab demonstrated enrichment in TNFα-, NFκB- and lipid metabolism-related gene sets by bootstrapped Kolmogorov-Smirnov test (Benjamini-Hochberg adjusted P = 4.74x10-3, 1.45x10-2 and 2.48x10-3, respectively) in tumor-associated myeloid populations. Additionally, single-cell trajectory analysis demonstrated increased CCL4 and decreased ISG15 with neoadjuvant checkpoint inhibition. CONCLUSIONS Here, we utilize mass cytometry and single-cell RNA sequencing to demonstrate the predominance and transcriptomic features of myeloid populations in GBM. Myeloid cells in patients who receive neoadjuvant PD-1 blockade re-express increased levels NFκB, TNFα and CCL4, a cytokine crucial for the recruitment of dendritic cells to the tumor for antigen-specific T cell activation. By delving into the GBM microenvironment at the single-cell level, we hope to better delineate the role of myeloid populations in this uniformly fatal tumor.

Lifting the veil on macrophage diversity in tissue regeneration and fibrosis

2019 ◽  
Vol 4 (40) ◽  
pp. eaaz0749 ◽  
Author(s):  
Catia T. Perciani ◽  
Sonya A. MacParland
Keyword(s):  
Single Cell ◽  
Rna Sequencing ◽  
Tissue Regeneration ◽  
Tissue Repair ◽  
Single Cell Rna Sequencing ◽  
The Veil

Sommerfeld et al. have used single-cell RNA sequencing to unravel the role of macrophages in driving tissue repair and fibrosis.

Role of FOS transcription factor in diabetes associated atherosclerosis; insight from single cell RNA sequencing

2021 ◽  
Vol 331 ◽  
pp. e69-e70
Author(s):  
A.W. Khan ◽  
M.S.K. Lee ◽  
A.M. Watson ◽  
S. Maxwell ◽  
M.E. Cooper ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Single Cell ◽  
Rna Sequencing ◽  
Single Cell Rna Sequencing

scholarly journals Single-cell transcriptomic profile reveals macrophage heterogeneity in medulloblastoma and their treatment-dependent recruitment

2020 ◽  
Author(s):  
Mai T. Dang ◽  
Michael Gonzalez ◽  
Krutika S. Gaonkar ◽  
Komal S. Rathi ◽  
Patricia Young ◽  
...  
Keyword(s):  
Radiation Therapy ◽  
Single Cell ◽  
Rna Sequencing ◽  
Sonic Hedgehog ◽  
Radiation Treatment ◽  
Pediatric Brain Tumor ◽  
Single Cell Rna Sequencing ◽  
Human Medulloblastoma ◽  
Pediatric Brain

AbstractThe role of macrophages in medulloblastoma, the most common malignant pediatric brain tumor, is unclear. Using single-cell RNA sequencing in a mouse model of sonic hedgehog medulloblastoma and analysis of bulk RNA sequencing of human medulloblastoma, we investigated macrophage heterogeneity. Our findings reveal differential recruitment of macrophages with molecular-targeted versus radiation therapy and identify an immunosuppressive monocyte-derived macrophages following radiation treatment of mouse medulloblastoma, uncovering potential strategies for immunomodulation as adjunctive therapy.

Decoding the role of macrophages in periodontitis and type 2 diabetes using single‐cell RNA‐sequencing

2022 ◽  
Vol 36 (2) ◽  
Author(s):  
Panagiota Agrafioti ◽  
Joshua Morin‐Baxter ◽  
Kranthi K. K. Tanagala ◽  
Sunil Dubey ◽  
Peter Sims ◽  
...  
Keyword(s):  
Type 2 Diabetes ◽  
Single Cell ◽  
Rna Sequencing ◽  
Single Cell Rna Sequencing

scholarly journals Genetic Heterogeneity Profiling by Single Cell RNA Sequencing

2018 ◽  
Author(s):  
Zilu Zhou ◽  
Bihui Xu ◽  
Andy Minn ◽  
Nancy R Zhang
Keyword(s):  
Single Cell ◽  
Rna Sequencing ◽  
Evolutionary Dynamics ◽  
Single Nucleotide ◽  
Dna Mutations ◽  
Single Cell Rna Sequencing ◽  
Mouse Melanoma Model ◽  
Melanoma Model ◽  
The Relationship

AbstractDetection of genetically distinct subclones and profiling the transcriptomic differences between them is important for studying the evolutionary dynamics of tumors, as well as for accurate prognosis and effective treatment of cancer in the clinic. For the profiling of intra-tumor transcriptional heterogeneity, single cell RNA-sequencing (scRNA-seq) is now ubiquitously adopted in ongoing and planned cancer studies. Detection of somatic DNA mutations and inference of clonal membership from scRNA-seq, however, is currently unreliable. We propose DENDRO, an analysis method for scRNA-seq data that detects genetically distinct subclones, assigns each single cell to a subclone, and reconstructs the phylogenetic tree describing the tumor’s evolutionary history. DENDRO utilizes information from single nucleotide mutations in transcribed regions and accounts for technical noise and expression stochasticity at the single cell level. The accuracy of DENDRO was benchmarked on spike-in datasets and on scRNA-seq data with known subpopulation structure. We applied DENDRO to delineate subclonal expansion in a mouse melanoma model in response to immunotherapy, highlighting the role of neoantigens in treatment response. We also applied DENDRO to primary and lymph-node metastasis samples in breast cancer, where the new approach allowed us to better understand the relationship between genetic and transcriptomic intratumor variation.

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