Specific Set Of Genes Research Articles

Metabolic adaptation to consume butyrate under prolonged resource exhaustion.

Bacteria must often survive following the exhaustion of their external growth resources. Fitting with this need, many bacterial species that cannot sporulate, can enter a state known as long term stationary phase (LTSP) in which they can persist for years within spent media. Several recent studies have revealed the dynamics of genetic adaptation of Escherichia coli under LTSP. Yet, the metabolic consequences of such genetic adaptation were not addressed. Here, we characterized the metabolic changes LTSP populations experience, over the first 32 days under LTSP. This allowed us to link genetic adaptations observed in a convergent manner across LTSP populations back to their metabolic adaptive effect. Specifically, we demonstrate that through the acquisition of mutations combinations in specific sets of metabolic genes, E. coli acquires the ability to consume the short chain fatty acid butyrate. Intriguingly, this fatty acid is not initially present within the rich media we used in this study. Instead, it is E. coli itself that produces butyrate during its initial growth within fresh rich media. The mutations that enable butyrate consumption allow E. coli to grow on butyrate. However, the clones carrying these mutations rapidly decrease in frequency, once the butyrate is consumed, likely reflecting an associated cost to fitness. Yet despite this, E. coli populations show a remarkable capability of maintaining these genotypes at low frequency, as standing variation. This in turn allows them to more rapidly re-adapt to consume butyrate, once it again becomes available to them.

HDAC1 regulates the chromatin landscape to control transcriptional dependencies in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is a quiescent B-cell malignancy that depends on transcriptional dysregulation for survival. The histone deacetylases are transcriptional regulators whose role within the regulatory chromatin and consequence on the CLL transcriptome is poorly characterized. Here, we profiled and integrated the genome-wide occupancy of HDAC1, BRD4, H3K27Ac, and H3K9Ac signals with chromatin accessibility, Pol2 occupancy, and target expression signatures in CLL cells. We identified that when HDAC1 was recruited within super-enhancers (SEs) marked by acetylated H3K27 and BRD4, it functioned as a transcriptional activator that drove the de novo expression of select genes to facilitate survival and progression in CLL. Targeting HDACs reduced BRD4 and Pol2 engagement to downregulate the transcript and proteins levels of specific oncogenic driver genes in CLL such as BLK, a key mediator of the B-cell receptor pathway, core transcription factors such as PAX5 and IKZF3, and the antiapoptotic gene, BCL2. Concurrently, HDAC1, when recruited in the absence of SEs, repressed target gene expression. HDAC inhibition reversed silencing of a defined set of protein-coding and noncoding RNA genes. We focused on a specific set of microRNA genes and showed that their upregulation was inversely correlated with the expression of CLL-specific survival, transcription factor, and signaling genes. Our findings identify that the transcriptional activator and repressor functions of HDACs cooperate within the same tumor to establish the transcriptional dependencies essential for survival in CLL.

Glucocorticoid Receptors Drive Breast Cancer Cell Migration and Metabolic Reprogramming via PDK4.

Corticosteroids act on the glucocorticoid receptor (GR; NR3C1) to resolve inflammation and are routinely prescribed to breast cancer patients undergoing chemotherapy treatment to alleviate side effects. Triple-negative breast cancers (TNBCs) account for 15% to 20% of diagnoses and lack expression of estrogen and progesterone receptors as well as amplified HER2, but they often express high GR levels. GR is a mediator of TNBC progression to advanced metastatic disease; however, the mechanisms underpinning this transition to more aggressive behavior remain elusive. We previously showed that tissue/cellular stress (hypoxia, chemotherapies) as well as factors in the tumor microenvironment (transforming growth factor β [TGF-β], hepatocyte growth factor [HGF]) activate p38 mitogen-activated protein kinase (MAPK), which phosphorylates GR on Ser134. In the absence of ligand, pSer134-GR further upregulates genes important for responses to cellular stress, including key components of the p38 MAPK pathway. Herein, we show that pSer134-GR is required for TNBC metastatic colonization to the lungs of female mice. To understand the mechanisms of pSer134-GR action in the presence of GR agonists, we examined glucocorticoid-driven transcriptomes in CRISPR knock-in models of TNBC cells expressing wild-type or phospho-mutant (S134A) GR. We identified dexamethasone- and pSer134-GR-dependent regulation of specific gene sets controlling TNBC migration (NEDD9, CSF1, RUNX3) and metabolic adaptation (PDK4, PGK1, PFKFB4). TNBC cells harboring S134A-GR displayed metabolic reprogramming that was phenocopied by pyruvate dehydrogenase kinase 4 (PDK4) knockdown. PDK4 knockdown or chemical inhibition also blocked cancer cell migration. Our results reveal a convergence of GR agonists (ie, host stress) with cellular stress signaling whereby pSer134-GR critically regulates TNBC metabolism, an exploitable target for the treatment of this deadly disease.

MicroRNAs associated with postoperative outcomes in patients with limited stage neuroendocrine carcinoma of the esophagus.

Esophageal neuroendocrine carcinoma (E-NEC) is an aggressive disease with a poor prognosis. The present study aimed to assess the role of surgery in the treatment of patients with resectable E-NEC, and identify a microRNA (miRNA/miR) signature in association with positive postoperative outcomes. Between February 2017 and August 2019, 36 patients with E-NEC who underwent curative surgery at the Japan Neuroendocrine Tumor Society partner hospitals were enrolled in the study. A total of 16 (44.4%) patients achieved disease-free survival (non-relapse group), whereas 20 (55.6%) patients developed tumor relapse (relapse group) during the median follow-up time of 36.5 months (range, 1-242) after surgery with a 5-year overall survival rate of 100 and 10.8%, respectively (P<0.01). No clinicopathological parameters, such as histological type or TNM staging, were associated with tumor relapse. Microarray analysis of 2,630 miRNAs in 11 patients with sufficient quality RNA revealed 12 miRNAs (miR-1260a, -1260b, -1246, -4284, -612, -1249-3p, -296-5p, -575, -6805-3p, -12136, -6822-5p and -4454) that were differentially expressed between the relapse (n=6) and non-relapse (n=5) groups. Furthermore, the top three miRNAs (miR-1246, -1260a and -1260b) were associated with overall survival (P<0.01). These results demonstrated that surgery-based multidisciplinary treatment is effective in a distinct subpopulation of limited stage E-NEC. A specific miRNA gene set is suggested to be associated with treatment outcome.

Transcriptional diversity in specific synaptic gene sets discriminates cortical neuronal identity

Synapse diversity has been described from different perspectives, ranging from the specific neurotransmitters released, to their diverse biophysical properties and proteome profiles. However, synapse diversity at the transcriptional level has not been systematically identified across all synapse populations in the brain. To quantify and identify specific synaptic features of neuronal cell types we combined the SynGO (Synaptic Gene Ontology) database with single-cell RNA sequencing data of the mouse neocortex. We show that cell types can be discriminated by synaptic genes alone with the same power as all genes. The cell type discriminatory power is not equally distributed across synaptic genes as we could identify functional categories and synaptic compartments with greater cell type specific expression. Synaptic genes, and specific SynGO categories, belonged to three different types of gene modules: gradient expression over all cell types, gradient expression in selected cell types and cell class- or type-specific profiles. This data provides a deeper understanding of synapse diversity in the neocortex and identifies potential markers to selectively identify synapses from specific neuronal populations.

Targeted transcriptome sequencing enables exponential scaling of combinatorial barcoding in leukemia samples.

Abstract Single-cell RNA-sequencing (scRNA-seq) has rapidly spread across multiple research fields, leading to new discoveries. Many applications of scRNA-seq are focused on cell type identification, gene regulatory networks, or biomarker discovery which require the interrogation of specific sets of well-characterized genes. In these cases, sequencing the entire transcriptome may be adding unnecessary project costs. To increase throughput and minimize sequencing costs, the development of a targeted gene enrichment method is required. Here, we extend our whole transcriptome (WT) split-pool combinatorial barcoding technology to enrich a subset of genes in 16 libraries representing hundreds of thousands of human bone marrow mononuclear cells (BMMCs) from three acute myeloid leukemia (AML) and one acute lymphocytic leukemia (ALL) donors. We used our 1,000 immune gene panel to enrich genes representing canonical immune cell markers and pathways. Our method increased the percent of reads on target from as low as 7% in the whole transcriptome libraries to 75% in the targeted libraries. Furthermore, despite a nearly ten-fold reduction in sequencing reads between unenriched and enriched libraries, the resulting clustering yielded high concordance of cell type identities and preserved leukemia-specific signatures such as FLT3, MKI67, and CD19. Overall, we demonstrate our modular enrichment strategy preserves biological structure and allows for deep characterization of gene signatures in health and disease. We envision our approach will enable researchers to simultaneously reduce sequencing costs while drastically scaling up the number of cells and samples across experiments.

Defining the role of endoplasmic reticulum in the initiation of paligenosis

Reprogramming is crucial for cellular renewal in adult organs that lack dedicated stem cells for replacement of cell loss after injury. Because such cell plasticity is likely to be executed by a conserved cellular program, we have begun to identify the conserved cellular-molecular features of the process in which differentiated cells are recruited as progenitors. We have characterized three successive stages by which this occur with differentiated cells: 1) the cells downscale their organelle contents, 2) cells activate a progenitor-like gene network, and 3) cells reenter the cell cycle. We have given this conserved process the name paligenosis. Here, we investigate upstream triggers of paligenosis. Using a high-dose tamoxifen (HD-TAM) injury model to induce paligenosis in zymogenic chief cells of the murine stomach corpus, we observed ultrastructural changes in the endoplasmic reticulum (ER) during paligenosis initiation (e.g., swelling of ER lamellae, liberation of ribosomes from rough ER, and overall loss of ER). This led us to hypothesize that dynamic changes in ER were an upstream event in paligenosis. ER functioning is in part monitored by the Integrated Stress Response (ISR) with the paramount ER stress sensor being PERK, which then autophosphorylates and in turn phosphorylates the α subunit of the eukaryotic initiation factor 2 (eIF2α). Phospho-eIF2α halts global translation while upregulating a specific set of genes to restore homeostasis. Here, we go on to show that HD-TAM activates the ISR in paligenotic gastric chief cells, triggering global attenuation of protein synthesis. However, this “ER stress” ISR pathway must occur in a critical, temporally limited window early in paligenosis, as pharmaceutically-induced prolonged ER stress caused paligenosis to halt in early stages with cells failing to proliferate. Blocking the ISR also halted paligenosis in early stages. Ongoing experiments include the use of the glycosylation-inhibiting drug tunicamycin to determine if inducing the ISR via ER stress is sufficient to initiate paligenosis; and deleting PERK in chief cells during paligenosis using Perk flox/flox ; Mist1 CreERT2 mice. Our results provide initial insights on the dynamic functionality of the ER and its role and necessity during cellular regeneration. This work was supported by funding from the NIH through the National Institute of Diabetes and Digestive and Kidney Diseases and the National Cancer Institute. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Profiling spatiotemporal gene expression of the developing human spinal cord and implications for ependymoma origin

The spatiotemporal regulation of cell fate specification in the human developing spinal cord remains largely unknown. In this study, by performing integrated analysis of single-cell and spatial multi-omics data, we used 16 prenatal human samples to create a comprehensive developmental cell atlas of the spinal cord during post-conceptional weeks 5–12. This revealed how the cell fate commitment of neural progenitor cells and their spatial positioning are spatiotemporally regulated by specific gene sets. We identified unique events in human spinal cord development relative to rodents, including earlier quiescence of active neural stem cells, differential regulation of cell differentiation and distinct spatiotemporal genetic regulation of cell fate choices. In addition, by integrating our atlas with pediatric ependymomas data, we identified specific molecular signatures and lineage-specific genes of cancer stem cells during progression. Thus, we delineate spatiotemporal genetic regulation of human spinal cord development and leverage these data to gain disease insight.

Requirements for mammalian promoters to decode transcription factor dynamics.

In response to different stimuli many transcription factors (TFs) display different activation dynamics that trigger the expression of specific sets of target genes, suggesting that promoters have a way to decode dynamics. Here, we use optogenetics to directly manipulate the nuclear localization of a synthetic TF in mammalian cells without affecting other processes. We generate pulsatile or sustained TF dynamics and employ live cell microscopy and mathematical modelling to analyse the behaviour of a library of reporter constructs. We find decoding of TF dynamics occurs only when the coupling between TF binding and transcription pre-initiation complex formation is inefficient and that the ability of a promoter to decode TF dynamics gets amplified by inefficient translation initiation. Using the knowledge acquired, we build a synthetic circuit that allows obtaining two gene expression programs depending solely on TF dynamics. Finally, we show that some of the promoter features identified in our study can be used to distinguish natural promoters that have previously been experimentally characterized as responsive to either sustained or pulsatile p53 and NF-κB signals. These results help elucidate how gene expression is regulated in mammalian cells and open up the possibility to build complex synthetic circuits steered by TF dynamics.

Survey of clinical and commensal Escherichia coli from commercial broilers and turkeys, with emphasis on high-risk clones using APECTyper

Molecular characterization of avian pathogenic Escherichia coli (APEC) is challenging due to the complex nature of its associated disease, colibacillosis, in poultry. Numerous efforts have been made toward defining APEC, and it is becoming clear that certain clonal backgrounds are predictive of an avian E. coli isolate's virulence potential. Thus, APEC can be further differentiated as high-risk APEC based upon their clonal background's virulence potential. However, less clear is the degree of overlap between clinical isolates of differing bird type, and between clinical and gastrointestinal isolates. This study aimed to determine genomic similarities and differences between such populations, comparing commercial broiler vs. turkey isolates, and clinical vs. gastrointestinal isolates. Differences were observed in Clermont phylogenetic groups between isolate populations, with B2 as the dominant group in turkey clinical isolates and G as the dominant group in broiler clinical isolates. Nearly all clinical isolates were classified as APEC using a traditional gene-based typing scheme, whereas 53.4% and 44.1% of broiler and turkey gastrointestinal isolates were classified as APEC, respectively. High-risk APEC were identified among 31.0% and 46.9% of broiler and turkey clinical isolates, compared with 5.7% and 2.9% of broiler and turkey gastrointestinal isolates. As found in previous studies, no specific known virulence or fitness gene sets were identified which universally differentiate between clinical and gastrointestinal isolates. This study further demonstrates the utility of a hybrid APEC typing approach, considering both plasmid content and clonal background, for the identification of dominant and highly virulent APEC clones in poultry production.

Mechanism of Action of Lactic Acid on Histones in Cancer.

Significance: Metabolic end products and intermediates can exert signaling functions as chemical sources for histone posttranslational modifications, which remodel chromatin and affect gene expression. Among them, lactic acid is responsible for histone lactylation, a recently discovered histone mark that occurs in high lactate conditions, such as those resulting from the Warburg effect in cancer cells. Recent Advances: Late-breaking studies have advanced the knowledge on the mechanisms involved in histone lactylation, requiring independent nonenzyme and enzyme-dependent reactions, which is emerging as an important hallmark of cancer cells linking metabolic changes to gene expression reprogramming. Critical Issues: In this study, we give an overview about this new epigenetic modification, focusing on its mechanism of action in tumors and tumor microenvironment. Future Directions: Further investigation on the competition mechanism between lactylation and acetylation, as well as on the mechanisms by which lactate fluctuation can control a specific gene set in a given tissue, is needed in the coming years to exploit new anticancer therapeutic approaches. Antioxid. Redox Signal. 40, 236-249.

Detect Cytochrome C Oxidase- and Glutathione-S-Transferase-Mediated Detoxification in a Permethrin-Resistant Population of Lygus lineolaris.

Frequent sprays on cotton prompted resistance development in the tarnished plant bug (TPB). Knowledge of global gene regulation is highly desirable to better understand resistance mechanisms and develop molecular tools for monitoring and managing resistance. Novel microarray expressions of 6688 genes showed 3080 significantly up- or down-regulated genes in permethrin-treated TPBs. Among the 1543 up-regulated genes, 255 code for 39 different enzymes, and 15 of these participate in important pathways and metabolic detoxification. Oxidase is the most abundant and over-expressed enzyme. Others included dehydrogenases, synthases, reductases, and transferases. Pathway analysis revealed several oxidative phosphorylations associated with 37 oxidases and 23 reductases. One glutathione-S-transferase (GST LL_2285) participated in three pathways, including drug and xenobiotics metabolisms and pesticide detoxification. Therefore, a novel resistance mechanism of over-expressions of oxidases, along with a GST gene, was revealed in permethrin-treated TPB. Reductases, dehydrogenases, and others may also indirectly contribute to permethrin detoxification, while two common detoxification enzymes, P450 and esterase, played less role in the degradation of permethrin since none was associated with the detoxification pathway. Another potential novel finding from this study and our previous studies confirmed multiple/cross resistances in the same TPB population with a particular set of genes for different insecticide classes.

Integrative Proteomics and N-Glycoproteomics Analyses of Rheumatoid Arthritis Synovium Reveal Immune-Associated Glycopeptides

Rheumatoid arthritis (RA) is a typical autoimmune disease characterized by synovial inflammation, synovial tissue hyperplasia, and destruction of bone and cartilage. Protein glycosylation plays key roles in the pathogenesis of RA but in-depth glycoproteomics analysis of synovial tissues is still lacking. Here, by using a strategy to quantify intact N-glycopeptides, we identified 1260 intact N-glycopeptides from 481 N-glycosites on 334 glycoproteins in RA synovium. Bioinformatics analysis revealed that the hyper-glycosylated proteins in RA were closely linked to immune responses. By using DNASTAR software, we identified 20 N-glycopeptides whose prototype peptides were highly immunogenic. We next calculated the enrichment scores of nine types of immune cells using specific gene sets from public single-cell transcriptomics data of RA and revealed that the N-glycosylation levels at some sites, such as IGSF10_N2147, MOXD2P_N404, and PTCH2_N812, were significantly correlated with the enrichment scores of certain immune cell types. Furthermore, we showed that aberrant N-glycosylation in the RA synovium was related to increased expression of glycosylation enzymes. Collectively, this work presents, for the first time, the N-glycoproteome of RA synovium and describes immune-associated glycosylation, providing novel insights into RA pathogenesis.

Abstract 4281: Data driven refinement of gene signatures for enrichment analysis and cell state characterization

Abstract The use of gene expression data has been crucial to the functional characterization of changes in molecular pathway activity and for identifying targets for novel treatments. However, the interpretation of this data is complicated by its high dimensionality and the difficulty of identifying biological signals within a list of differentially expressed genes. Gene Set Enrichment Analysis (GSEA) is a standard method for identifying pathway enrichment in gene expression data by testing whether a set of genes whose expression would indicate the activity of a specific process or phenotype are coordinately up- or downregulated more than would be expected by chance. As GSEA relies on high quality gene sets with coordinately regulated member genes, we maintain the Molecular Signatures Database (MSigDB) which contains 9 collections of curated gene sets representing different biological pathways and processes. Over time, we have observed that some of the MSigDB gene sets, especially those that are manually curated or defined in a very specific biological context, may not provide a sensitive and specific enough co-regulation signature. In response, we have created a data-driven, matrix-factorization-based refinement method to build more sensitive and specific gene sets. This method incorporates large-scale datasets from multiple sources such as the Cancer Dependency Map as well as curated protein-protein interaction networks. We will present the initial results of this refinement method and our ongoing work which will yield a new collection of refined gene sets that will be made freely available in MSigDB for use with GSEA and many other applications. Citation Format: Alexander T. Wenzel, Pablo Tamayo, Jill P. Mesirov. Data driven refinement of gene signatures for enrichment analysis and cell state characterization. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4281.

Abstract 3563: SWI/SNF-associated DPF1 is a unique transcriptional regulator of malignant peripheral nerve sheath tumors

Abstract Background: Malignant peripheral nerve sheath tumors (MPNST) are rare soft tissue sarcomas that are typically therapy resistant and associated with a poor prognosis. Genetic aberrations of the polycomb repressive complex 2 (PRC2) occur in up to 75% of MPNST cases. PRC2 functions antagonistically to SWI/SNF protein complexes, regulating expression of target loci in a dynamic relationship that is critical to normal cellular development and maintenance of cell identity. The consequences of PRC2 loss on chromatin state maintained by SWI/SNF in MPNST is unexplored. Therefore, this study used functional genomics to elucidate the role of SWI/SNF in MPNST. Methods: To define the transcriptional regulatory role of SWI/SNF complexes in MPNST, SWI/SNF components were targeted via a CRISPR knock-out (KO) screen combined with a single cell RNA sequencing (RNAseq) readout. Genes of interest highlighted by this screen were further investigated using bulk CRISPR RNAseq. Phenotypic effects of the expression of these genes were studied using loss-of-function assays coupled with colony formation assays. SWI/SNF complex heterogeneity was characterized using glycerol gradient sedimentation, co-immunoprecipitation (co-IP), and mass spectrometry experiments. Results: The investigation of SWI/SNF transcriptional regulation in MPNST highlighted the Double PHD Finger family proteins (DPF1,2,3) as regulating distinct downstream targets. Notably, DPF1 had a unique transcriptional profile compared to other SWI/SNF components. Bulk CRISPR KO RNAseq confirmed these findings and highlighted a specific set of DPF1 target genes including many long non-coding RNAs. The phenotypic role of DPF1 in MPNST was investigated using in vitro siRNA and CRISPR experiments, where DPF1 KO reduced proliferation and viability of MPNST cells in both 2D and 3D cell culture assays. Further, DPF1 was found contribute to anchorage independent cell growth of MPNST cells using soft agar assays. Glycerol gradient sedimentation assays demonstrated that DPF1 co-migrated with core components of a SWI/SNF complex known as canonical BAF (cBAF) and not the alternate GBAF and PBAF SWI/SNF complexes. These findings were further validated using co-immunoprecipitation assays where core the SWI/SNF ATPase, SMARCA4, pulled down DPF1, while components unique to the GBAF and PBAF complexes did not. An ATPase directed proteolysis targeting chimers (PROTAC) was used to therapeutically target cBAF in MPNST cells, reducing their growth and viability. Combination treatment with standard of care chemotherapy synergistically increased the efficacy of this drug. Conclusions: DPF1 was identified as a unique transcriptional regulator of MPNST cells and acts as a member of the cBAF SWI/SNF complex to play important phenotypic roles in this cancer type. Citation Format: Bega Murray, Xiyuan Zhang, Shahroze Abbas, Haiyan Lei, Hilda Jafarah, Jack F. Shern. SWI/SNF-associated DPF1 is a unique transcriptional regulator of malignant peripheral nerve sheath tumors. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3563.

Abstract 226: Targeted transcriptome sequencing enables exponential scaling of combinatorial barcoding in AML samples

Abstract In the thirteen years since its inception, single-cell RNA-sequencing (scRNA-seq) has rapidly spread across multiple fields of research, leading to many new discoveries. As technologies have matured, the number of cells that can be processed in a single experiment has seen exponential growth with workflows now assaying up to one million cells in an individual experiment. While high throughput sequencing methods have facilitated the discovery and characterization of various cell types, sequencing costs can be prohibitively high for routine use. Many applications of scRNA-seq are focused on cell type identification, gene regulatory networks, or biomarker discovery. These applications often do not require surveying the entire transcriptome, but rather require the interrogation of specific sets of well-characterized genes. In these cases, sequencing the entire transcriptome may be adding unnecessary project costs. To increase throughput and minimize sequencing costs, the development of a targeted gene enrichment method is required. Here we extend our whole transcriptome split-pool combinatorial barcoding technology, to enable enrichment of a subset of genes in the final single cell sequencing libraries. To illustrate the power of our technology, we enriched a whole transcriptome library of human bone marrow mononuclear cells (BMMCs) from four acute myeloid leukemia (AML) donors and four control donors. From a library of 27,000 cells, we used our immune gene panel to enrich 1,000 genes representing canonical immune cell markers and pathways. Our method increased the percent of reads on target from as low as 1.6% in the whole transcriptome libraries to 75% in the targeted libraries. Furthermore, despite a nearly ten-fold reduction in sequencing reads between unenriched and enriched libraries, the resulting clustering yielded very high concordance of cell type identities and preserved AML-specific signatures such as FLT3, MKI67, and CD19. Overall, we demonstrate our modular enrichment strategy preserves biological structure of the data and allows for deep characterization of gene signatures in health and disease. We envision our approach will enable researchers to simultaneously reduce sequencing costs while drastically scaling up the number of cells and samples across experiments. Citation Format: Grace Kim, Efthymia Papalexi, Peter Matulich, Ryan Koehler, Sarah Schroeder, Vuong Tran, Charles Roco, Alexander Rosenberg. Targeted transcriptome sequencing enables exponential scaling of combinatorial barcoding in AML samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 226.

PD02-04 SINGLE CELL TRANSCRIPTIONAL PROFILES OF BENIGN PROSTATIC HYPERPLASIA

You have accessJournal of UrologyCME1 Apr 2023PD02-04 SINGLE CELL TRANSCRIPTIONAL PROFILES OF BENIGN PROSTATIC HYPERPLASIA Rei Unno, Jon Akutagawa, Hanging Song, Heiko Yang, Thomas Chi, and Franklin W. Huang Rei UnnoRei Unno More articles by this author , Jon AkutagawaJon Akutagawa More articles by this author , Hanging SongHanging Song More articles by this author , Heiko YangHeiko Yang More articles by this author , Thomas ChiThomas Chi More articles by this author , and Franklin W. HuangFranklin W. Huang More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003219.04AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Benign prostatic hyperplasia (BPH) affects the majority of aging men and leads to lower urinary tract symptoms. Based on histological characteristics and bulk sequencing data, the pathophysiology and underlying mechanisms of BPH are poorly understood. The aim of this study was to investigate the transcriptional profile of BPH at single cell resolution to improve our understanding of the molecular features of BPH. METHODS: Fifteen BPH specimens were collected from patients undergoing holmium laser enucleation of the prostate. Cells from each specimen were isolated, captured, and sequenced using a bead-based single-cell RNA sequencing platform (Seq-well). Transcriptomic analysis of the gene expression matrices was performed using software including Scanpy and cellxgene. The absence of malignant pathologic diagnoses was independently confirmed. RESULTS: 16,234 cells from 15 BPH specimens were analyzed. By canonical cell type markers, we annotated three major cell types (epithelial, stromal, and immune cells). Using significantly upregulated genes in BPH compared to normal prostates previously derived from a bulk RNA sequencing comparison, the signature score of the bulk sequencing data was high only in the fibroblast populations. After performing a sub-clustering analysis of the 6249 epithelial cells, we identified four luminal cell subclusters showing similar levels of luminal epithelial markers KLK3, KLK2, and NKX3-1, as well as one distinct basal and one club cell population. Analysis of the differentially expressed genes among the four luminal subclusters revealed that luminal subcluster 4 was enriched in a specific signature gene set, including ribosomal genes and KLK4, which has been shown to be abundant in BPH, compared to the other subclusters. Gene ontology analysis using this gene set demonstrated that luminal subcluster 4 was associated with pathways such as oxidative phosphorylation and adipogenesis. Analyses of the BPH immune microenvironment showed a larger proportion of pro-inflammatory cells, such as M1 macrophages, compared to anti-inflammatory cells within the myeloid cell population. CONCLUSIONS: Single cell profiling of BPH identified epithelial cells, stromal cells, and immune cells. We confirmed the upregulation of previously derived BPH-associated genes in the fibroblast population and the enrichment of pro-inflammatory myeloid cells in the immune microenvironment. Within the epithelial cell population, we identified a luminal epithelial subcluster that may represent a cell state associated with BPH. Source of Funding: none © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e70 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Rei Unno More articles by this author Jon Akutagawa More articles by this author Hanging Song More articles by this author Heiko Yang More articles by this author Thomas Chi More articles by this author Franklin W. Huang More articles by this author Expand All Advertisement PDF downloadLoading ...

Role of human milk oligosaccharide metabolizing bacteria in the development of atopic dermatitis/eczema.

Despite affecting up to 20% of infants in the United States, there is no cure for atopic dermatitis (AD), also known as eczema. Atopy usually manifests during the first six months of an infant's life and is one predictor of later allergic health problems. A diet of human milk may offer protection against developing atopic dermatitis. One milk component, human milk oligosaccharides (HMOs), plays an important role as a prebiotic in establishing the infant gut microbiome and has immunomodulatory effects on the infant immune system. The purpose of this review is to summarize the available information about bacterial members of the intestinal microbiota capable of metabolizing HMOs, the bacterial genes or metabolic products present in the intestinal tract during early life, and the relationship of these genes and metabolic products to the development of AD/eczema in infants. We find that specific HMO metabolism gene sets and the metabolites produced by HMO metabolizing bacteria may enable the protective role of human milk against the development of atopy because of interactions with the immune system. We also identify areas for additional research to further elucidate the relationship between the human milk metabolizing bacteria and atopy. Detailed metagenomic studies of the infant gut microbiota and its associated metabolomes are essential for characterizing the potential impact of human milk-feeding on the development of atopic dermatitis.

Genetic Networks of Alzheimer's Disease, Aging, and Longevity in Humans.

Human genomic analysis and genome-wide association studies (GWAS) have identified genes that are risk factors for early and late-onset Alzheimer's disease (AD genes). Although the genetics of aging and longevity have been extensively studied, previous studies have focused on a specific set of genes that have been shown to contribute to or are a risk factor for AD. Thus, the connections among the genes involved in AD, aging, and longevity are not well understood. Here, we identified the genetic interaction networks (referred to as pathways) of aging and longevity within the context of AD by using a gene set enrichment analysis by Reactome that cross-references more than 100 bioinformatic databases to allow interpretation of the biological functions of gene sets through a wide variety of gene networks. We validated the pathways with a threshold of p-value < 1.00 × 10-5 using the databases to extract lists of 356 AD genes, 307 aging-related (AR) genes, and 357 longevity genes. There was a broad range of biological pathways involved in AR and longevity genes shared with AD genes. AR genes identified 261 pathways within the threshold of p < 1.00 × 10-5, of which 26 pathways (10% of AR gene pathways) were further identified by overlapping genes among AD and AR genes. The overlapped pathways included gene expression (p = 4.05 × 10-11) including ApoE, SOD2, TP53, and TGFB1 (p = 2.84 × 10-10); protein metabolism and SUMOylation, including E3 ligases and target proteins (p = 1.08 × 10-7); ERBB4 signal transduction (p = 2.69 × 10-6); the immune system, including IL-3 and IL-13 (p = 3.83 × 10-6); programmed cell death (p = 4.36 × 10-6); and platelet degranulation (p = 8.16 × 10-6), among others. Longevity genes identified 49 pathways within the threshold, of which 12 pathways (24% of longevity gene pathways) were further identified by overlapping genes among AD and longevity genes. They include the immune system, including IL-3 and IL-13 (p = 7.64 × 10-8), plasma lipoprotein assembly, remodeling and clearance (p < 4.02 × 10-6), and the metabolism of fat-soluble vitamins (p = 1.96 × 10-5). Thus, this study provides shared genetic hallmarks of aging, longevity, and AD backed up by statistical significance. We discuss the significant genes involved in these pathways, including TP53, FOXO, SUMOylation, IL4, IL6, APOE, and CEPT, and suggest that mapping the gene network pathways provide a useful basis for further medical research on AD and healthy aging.

SpHsfA4c from Sedum plumbizincicola Enhances Cd Tolerance by the AsA–GSH Pathway in Transgenic Populus × canescens

The ascorbate (AsA)–glutathione (GSH) metabolism pathway is an important antioxidant system in cadmium (Cd) detoxification; the AsA–GSHpathway is generally regulated by a specific set of functional genes. However, transcription factors involved in AsA–GSH pathway have yet to be identified. Herein, we transformed a heat shock transcription factor SpHsfA4c from Sedum plumbizincicola into Populus. × canescens. Under 100 μM CdCl2 stress for 30 d, the leaf chlorosis of wild-type poplars (WT) is more serious than that in transgenic poplars. The root biomass, shoot biomass and tolerance index (TIs) of transgenic poplars were higher than those in WT. In addition, transgenic poplars have higher Cd2+ uptake and Cd content. Compared with WT, the contents of hydrogen peroxide (H2O2) and superoxide anion (O2•−) in transgenic poplars were significantly reduced in leaves under Cd treatment. The expression levels of five enzymes (ascorbate peroxidase (APX), catalases (CAT), superoxide dismutase (SOD), peroxidase (POD) and glutathione S-transferase (GST)) were higher in transgenic poplars than those in WT. Transgenic poplars contained higher concentrations of intermediate metabolites, including GSH, AsA and phytochelatins (PCs), and a higher GSH/GSSG ratio in the AsA–GSH metabolism pathway. In Fourier transform infrared (FTIR) spectra, the characteristic peaks indicated that the contents of cysteine, GSH and AsA in transgenic poplars were exceeded compared to those in WT. These results suggested that SpHsfA4c can activate the AsA–GSH metabolism pathway to reduce Cd-associated oxidative stress. Therefore, overexpressing SpHsfA4c in P. × canescens can give rise to a superior Cd tolerance. Our results provide a theoretical significance for breeding potential new germplasm resources with high biomass and high Cd tolerance for remediation of soil heavy metal pollution.