title: Challenges in the discovery of tumor-specific alternative splicing-derived cell-surface antigens in glioma

status: Public on Mar 17 2024

first: Hideho

last: Okada

department: Neurological Surgery

institute: University of California, San Francisco

summary: Background: Despite advancements in cancer immunotherapy, solid tumors remain formidable challenges. In glioma, profound inter- and intra-tumoral heterogeneity of antigen landscape hampers therapeutic development. Therefore, it is critical to consider alternative sources to expand the repertoire of targetable (neo-)antigens and improve therapeutic outcomes. Accumulating evidence suggests that tumor-specific alternative splicing (AS) could be an untapped reservoir of antigens.  Results: In this study, we investigated tumor-specific AS events in glioma, focusing on those predicted to generate major histocompatibility complex (MHC)-presentation-independent, cell-surface antigens that could be targeted by antibodies and chimeric antigen receptor (CAR)-T cells. We systematically analyzed bulk RNA-sequencing datasets comparing 429 tumor samples (from The Cancer Genome Atlas [TCGA]) and 9,166 normal tissue samples (from the Genotype-Tissue Expression project [GTEx]), and identified 13 AS events in 7 genes predicted to be expressed in more than 10% of the patients, including PTPRZ1 and BCAN, which were corroborated by an external RNA-sequencing dataset. Subsequently, we validated our predictions and elucidated the complexity of the isoforms using full-length transcript amplicon sequencing on patient-derived glioblastoma cells. However, analyses of the RNA-sequencing datasets of spatially mapped and longitudinally collected clinical tumor samples unveiled remarkable spatiotemporal heterogeneity of the candidate AS events. Furthermore, proteomics analysis did not reveal any peptide spectra matching the putative antigens.  Conclusions: Our investigation illustrated the diverse characteristics of the tumor-specific AS events and the challenges of antigen exploration due to their notable spatiotemporal heterogeneity and elusive nature at the protein levels. Redirecting future efforts toward intracellular, MHC-presented antigens could offer a more viable avenue.

overall_design: The PCR primer pair was designed to bind to the 5’- and 3’- untranslated regions (UTRs) of the PTPRZ1 gene, ensuring amplification of the entire coding sequence (CDS). Following PCR amplification and purification, we prepared the amplicon library, barcoded the samples, pooled them, and sequenced using a MINION sequencer. The sequencing data were acquired using the MinKNOW software (v4.0.20) with high-accuracy mode. The generated Fast5 data were analyzed, excluding the Fast5_fail data and only focusing solely on the Fast5_pass data. Base calling was performed using Guppy basecaller (4.2.2+effbaf8).

first: Takahide

last: Nejo

first: Lin

first: Kevin

middle: K

last: Leung

first: Albert

first: Senthilnath

last: Lakshmanachetty

first: Marco

last: Gallus

first: Darwin

last: Kwok

first: Chibo

last: Hong

first: Lee

last: Chen

first: Diego

last: Carrera

first: Michael

last: Zhang

first: Nicholas

middle: O

last: Stevers

first: Gabriella

middle: C

last: Maldonado

first: Akane

last: Yamamichi

first: Payal

last: Watchmaker

first: Akul

last: Naik

first: Anny

last: Shai

first: Joanna

last: Phillips

first: Susan

middle: M

last: Chang

first: Arun

last: Wiita

first: James

last: Wells

first: Joseph

last: Costello

first: Aaron

last: Diaz


title: Unraveling the molecular signature of BCR-ABLT3151 in the Drosophila melanogater CML model

status: Public on Mar 15 2024

city: Beirut

first: Margret

last: Shirinian

address: Bliss street

country: Lebanon

institute: American University of Beirut

type: Expression profiling by high throughput sequencing

summary: Chronic myeloid leukemia (CML) is a clonal hematopoietic stem cell disorder resulting from a balanced translocation namely the Philadelphia chromosome. It produces the BCR-ABL1 oncogene that encodes for the BCR-ABL1 fusion protein that demonstrates increased tyrosine kinase activity. In the present study we aim to investigate the transcriptomic and molecular profiles of the wild type BCR-ABLp210 and BCR-ABLT315I mutant in attempt to examine their pathogenesis and find potential targets in chronic myeloid leukemia (CML). The molecular profiles of the BCR-ABLp210 and BCR-ABLT315I mutant were highly similar, leading us to identify six genes that were consistently upregulated. Functional analysis of these genes revealed their ability to rescue the observed phenotypes associated with hemocytes when they were suppressed in flies expressing BCR-ABL1p210 and BCR-ABL1T315I. Additionally, we observed an increase in markers associated with lamellocyte differentiation in both BCR-ABL1p210 and BCR-ABL1T315I, with significant upregulation of genes involved in this process. Overall, our study provides a comprehensive analysis of gene signatures in BCR-ABL1p210 and BCR-ABL1T315I, which can serve as a foundation for further targeted investigations into the role of these genes in the pathogenesis of BCR-ABL1p210 and BCR-ABL1T315I in chronic myeloid leukemia (CML).

overall_design: For RNA sequencing, RNA was extracted from collected hemocytes form 3rd instar larval groups. The following three Drosophila Melanogaster groups were used: W1118 (control), BCR-ABLp210 and BCR-ABLT315I. Three different biological replicates (N=3) with 300 third-instar larvae for each replicate were used for RNA-seq (900 larvae total).

first: Amro

last: Baassiri

first: Ali

last: Ghais

first: Abdallah

last: Kurdi

first: Elias

last: Rahal

first: Rihab

last: Nasr


title: Identification of calcineurin-dependent gene expression changes in response to chronic CaCl2 stress

status: Public on Mar 19 2024

city: Worcester

first: Jennifer

last: Benanti

state: MA

address: 364 Plantation St., LRB 525

department: Molecular, Cell and Cancer Biology

country: USA

institute: University of Massachusetts Medical School

summary: Gene expression was quantified in S. cerevisae cells over a time course of three days of CaCl2 treatment to activate calcineurin. Wild type (WT) and  cnb1 mutant strains were compared to allow the identification of calcineurin-dependent gene expression changes.  We show that mitochondrial genes are downregulated, and ribosome biogenesis genes are upregulated, in both geneotypes over time.

overall_design: Gene expression was measured in wild type (WT) cells and cell lacking calcineurin (cnb1). For each strain samples were collected before treatment and 12, 48 and 72 hours following the addition of 200mM CaCl2. Triplicate time course experiments were performed.

first: Mackenzie

middle: J

last: Flynn

first: Rui

last: Li

first: Lihua

last: Zhu

middle: A


title: Identification of apolipoprotein B–reactive CDR3 motifs allows tracking of atherosclerosis-related memory CD4+T cells in multiple donors

city: La Jolla

first: Klaus

last: Ley

state: California

address: 9420 Athena Cir

institute: La Jolla Institute for Immunology

summary: Atherosclerosis is a major pathological condition that underlies many cardiovascular diseases (CVDs). Its etiology involves breach of tolerance to self, leading to clonal expansion of autoreactive apolipoprotein B (APOB)–reactive CD4+T cells that correlates with clinical CVD. The T-cell receptor (TCR) sequences that mediate activation of APOB-specific CD4+T cells are unknown. Methods: In a previous study, we had profiled the hypervariable complementarity determining region 3 (CDR3) of CD4+T cells that respond to six immunodominant APOB epitopes in most donors. Here, we comprehensively analyze this dataset of 149,065 APOB-reactive and 199,211 non-reactive control CDR3s from six human leukocyte antigen–typed donors. Results: We identified 672 highly expanded (frequency threshold > 1.39E-03) clones that were significantly enriched in the APOB-reactive group as compared to the controls (log10 odds ratio ≥1, Fisher’s test p < 0.01). Analysis of 114,755 naïve, 91,001 central memory (TCM) and 29,839 effector memory (TEM) CDR3 sequences from the same donors revealed that APOB+ clones can be traced to the complex repertoire of unenriched blood T cells. The fraction of APOB+ clones that overlapped with memory CDR3s ranged from 2.2% to 46% (average 16.4%). This was significantly higher than their overlap with the naïve pool, which ranged from 0.7% to 2% (average 1.36%). CDR3 motif analysis with the machine learning–based in-silico tool, GLIPHs (grouping of lymphocyte interactions by paratope hotspots), identified 532 APOB+ motifs. Analysis of naïve and memory CDR3 sequences with GLIPH revealed that ~40% (209 of 532) of these APOB+motifs were enriched in the memory pool. Network analysis with Cytoscape revealed extensive sharing of the memory-affiliated APOB+ motifs across multiple donors. We identified six motifs that were present in TCM and TEM CDR3 sequences from >80% of the donors and were highly enriched in the APOB-reactive TCR repertoire. Discussion: The identified APOB-reactive expanded CD4+T cell clones and conserved motifs can be used to annotate and track human atherosclerosis-related autoreactive CD4+T cells and measure their clonal expansion.

overall_design:
To identify APOB-reactive TCRs and track them within the antigen-experienced memory repertoire, we conducted high-throughput TCR sequencing of five CD4+T cell subsets from six donors - APOB-reactive (APOB_AIMpos), control non-APOB-reactive (APOB_AIMneg), naïve, central memory (TCM) and effector memory (TEM) T cells. The naive and memory T cell were sorted ex vivo at Day 0, while the AIM+ and AIM- subsets were isolated after 14 days of APOB-dependent expansion and restimulation.

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Adaptive Biotechnologies does not release raw data.
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first: Payel

last: Roy

first: Sujit

middle: S

last: Armstrong Suthahar

first: Jeffrey

last: Makings


title: Expression analysis of Plasmodium falciparum with MYST overexpression

status: Public on Mar 18 2024

city: Tampa

first: JUN

last: MIAO

state: Florida

address: 3720 Spectrum Boulevard, MDC84

department: Internal Medicine

institute: University of South Florida

summary:
Investigation of genome-wide gene expression changes in Plasmodium falciparum overexpressing PfMYST compared to the wild-type strain 3D7.
After introducing a single copy of the full-length PfMYST expression cassette into the parasite genome, parasites showed higher levels of H4-K5, -K8, and -K12 acetylation, a faster progression of intraerythrocytic developmental cycle (IDC), and shorter schizont development time (duration), which led to significantly fewer merozoites developed in mature schizonts than the control. The parasites with PfMYST overexpression substantially increased the resistance to DNA-damaging agents. These findings were published in Miao, Fan et al Mol Microbiol 2010 (PMID: 20807207). To understand the underlying mechanisms, we studied changes in the transcriptomes during the IDC by expression microarray.

overall_design: A 12 chip study using total RNA recovered from six time points (with 8h intervals from ring to schizont stage) of wild-type asexual cultures of Plasmodium falciparum 3D7 and six time points (with 8h intervals from ring to schizont stage) of asexual PfMYST-overexpression cultures.  Each chip measures the expression level of 5,367 genes with 45-60 mer probes with two replicates on the array of 71618 probes.

first: Miao

last: Jun


title: Early life phenobarbital exposure dysregulates the hippocampal transcriptome

city: Washington

first: Patrick

middle: Alexander

last: Forcelli

state: DC

address: 3970 Reservoir Road NW NRB W209B

department: Pharmacology & Physiology

institute: Georgetown University

summary:
Introduction: Phenobarbital (PB) and levetiracetam (LEV) are the first-line therapies for neonates with diagnosed seizures, however, a growing body of evidence shows that these drugs given during critical developmental windows trigger lasting molecular changes in the brain. While the targets and mechanism of action of these drugs are well understood-what is not known is how these drugs alter the transcriptomic landscape, and therefore molecular profile/gene expression during these critical windows of neurodevelopment. PB is associated with a range of neurotoxic effects in developing animals, from cell death to altered synaptic development to lasting behavioral impairment. LEV does not produce these effects.
Methods: Here we evaluated the effects of PB and Lev on the hippocampal transcriptome by RNA sequencing. Neonatal rat pups were given a single dose of PB, Lev or vehicle and sacrificed 72 h later-at time at which drug is expected to be cleared.
Results: We found PB induces broad changes in the transcriptomic profile (124 differentially expressed transcripts), as compared to relatively small changes in LEV-treated animals (15 transcripts). PB exposure decreased GABAergic and oligodendrocyte markers pvalb and opalin, and increased the marker of activated microglia, cd68 and the astrocyte- associated gene vegfa. These data are consistent with the existing literature showing developmental neurotoxicity associated with PB, but not LEV.
Discussion: The widespread change in gene expression after PB, which affected transcripts reflective of multiple cell types, may provide a link between acute drug administration and lasting drug toxicity.

overall_design: Wild type Sprague Dawley pups were treated with phenobarbital (75 mg/kg), levetiracetam (200 mg/kg) or vehicle on postnatal day 7. 72 hours later, pups were euthanized, hippocampi microdissected and processed for RNA sequencing. We peformed gene expression profiling analysis comparing PB to VEH and LEV to VEH.

first: Sean

last: Quinlan


title: Single-cell RNA sequencing unveils the hidden powers of zebrafish kidney for generating both hematopoiesis and adaptive antiviral immunity

city: Hangzhou

first: Chongbin

last: Hu

state: Zhejiang Province

address: 866 Yuhangtang Rd

department: College of Life Sciences

country: China

institute: Zhejiang University

summary: The vertebrate kidneys play two evolutionary conserved roles in waste excretion and osmoregulation. Besides, the kidney of fish is considered as a functional ortholog of mammalian bone marrow that serves as a hematopoietic hub for generating blood cell lineages and immunological responses. However, knowledge about the properties of kidney hematopoietic cells, and the functionality of kidney in fish immune systems remain to be elucidated. To this end, our present study generated a comprehensive atlas with 59 hematopoietic stem/progenitor cell (HSPC) and immune-cell types from zebrafish kidney via single-cell transcriptome profiling analysis. These populations included almost all known cells associated with innate and adaptive immunity, and displayed differential responses to viral infection, indicating their diverse functional roles in antiviral immunity. Remarkably, HSPCs were found to have extensive reactivities to viral infection, and the trained immunity can be effectively induced in certain HSPCs. In addition, the antigen-stimulated adaptive immunity can be fully generated in kidney, suggesting kidney acting as a secondary lymphoid organ. These results indicated that fish kidney is a dual-functional entity with functionalities of both primary and secondary lymphoid organs. Our findings illustrated the unique features of fish immune system, and highlighted the multifaced biology of kidney in ancient vertebrates.

overall_design: Kidney leukocyte (PBS-administered (control), SVCV-infected (infected) and SVCV-vaccinated plus SVCV-infected (vaccinated+infected) groups) of the AB zebrafish were isolated by Ficoll-Hypaque (1.080 g/mL) density-gradient centrifugation and analyzed using scRNAseq.

last: Zhang

first: Yun

last: Hong

first: Ruxiu

last: Tie

first: Dongdong

last: Fan

first: Aifu

last: Lin

first: Ye

last: Chen

first: Lixin

last: Xiang

first: Jianzhong

last: Shao


title: SIX2 promotes cell plasticity via Wnt/beta-catenin signalling in androgen receptor independent prostate cancer (ATAC-seq)

city: Kuopio

first: Ville

last: Paakinaho

address: Yliopistonranta 8

department: School of Medicine

country: Finland

institute: University of Eastern Finland

type: Genome binding/occupancy profiling by high throughput sequencing

summary: The use of androgen receptor (AR) inhibitors in prostate cancer gives rise to increased cellular lineage plasticity resulting resistance to AR-targeted therapies. By examining the chromatin landscape of AR positive prostate cancer cells following exposure to the AR inhibitor enzalutamide, we have identified a novel regulator of cell plasticity, homeobox transcription factor SIX2, whose motif is enriched in accessible regions post-treatment. Our investigation demonstrates that depletion of SIX2 in androgen-independent PC-3 prostate cancer cells is sufficient to induce a switch from a stem-like to an epithelial state, leading to the reduction of key cancer-related properties such as proliferation, colony formation, and metastasis both in vitro and in vivo. These effects are mediated through downregulation of Wnt/β-catenin signalling pathway and subsequent reduced nuclear localization of β-catenin. Collectively, our findings provide compelling evidence that depletion of SIX2 may represent a promising strategy for overcoming cell plasticity mechanisms driving AR resistance in prostate cancer.

overall_design: Examination of chromatin accessibility by ATAC-seq from LNCaP prostate cancer cells grown in the presence or absence of ENZ. All sequencing was done with Illumina NextSeq 500.

first: Noora

last: Leppänen

first: Kirsi

last: Ketola


title: Case Report: Molecular Profiling Facilitates the Diagnosis of a Challenging Case of Lung Cancer with Choriocarcinoma Features

status: Public on Mar 14 2024

city: Irving

first: Phoenix

last: Holmes

state: Texas

address: 750 W John Carpenter Fwy Suite 800

institute: Caris Life Sciences

summary: Accurate diagnoses are crucial in determining the most effective treatment across different cancers. In challenging cases, morphology-based traditional pathology methods have important limitations, while molecular profiling can provide valuable information to guide clinical decisions. We present a 35-year female with lung cancer with choriocarcinoma features. Her disease involved the right lower lung, brain, and thoracic lymph nodes. The pathology from brain metastasis was reported as “metastatic choriocarcinoma” (a germ cell tumor) by local pathologists. She initiated carboplatin and etoposide, a regimen for choriocarcinoma. Subsequently, her case was assessed by pathologists from an academic cancer center, who gave the diagnosis of “adenocarcinoma with aberrant expression of β-hCG” and finally pathologists at our hospital, who gave the diagnosis of “poorly differentiated carcinoma with choriocarcinoma features”. Genomic profiling detected a KRAS G13R mutation and transcriptomics profiling was suggestive of lung origin. The patient was treated with carboplatin/paclitaxel/ipilimumab/nivolumab followed by consolidation radiation therapy. She had no evidence of progression to date, 13 months after the initial presentation. The molecular profiling could facilitate diagnosing of challenging cancer cases. In addition, chemoimmunotherapy and local consolidation radiation therapy may provide promising therapeutic options for patients with lung cancer exhibiting choriocarcinoma features.

overall_design: The lung tumor biopsy samples were sent to Caris Life Sciences to investigate genetic mutations that could offer more information about the tumor's origin. This subtype of lung cancer is rare, and identifying diverse driver mutations could also guide clinical treatment decisions. Caris Life Science performed the whole transcriptome sequencing (WTS) and whole exome sequencing (WES), and interpreted the findings.

first: Hui

last: Li

first: Xin

first: Mathew

last: Ning

first: Gregory

middle: N

last: Fuller

first: John

last: Stewart

first: Jared

last: Gilliam

first: Jia

last: Wu

first: Xiuning

last: Le

first: Ara

last: Vaporciyan

first: J

last: Lee

first: Don

middle: L

last: Gibbons

middle: V

last: Heymach

first: Andrew

last: Futreal

first: Jianjun


title: EP300/CREBBP acetyltransferase inhibition limits steroid receptor and FOXA1 signaling in prostate cancer cells

first: Ville

summary: This SuperSeries is composed of the SubSeries listed below.

overall_design: Refer to individual Series


title: EP300/CREBBP acetyltransferase inhibition limits steroid receptor and FOXA1 signaling in prostate cancer cells (ATAC-seq)

summary: The androgen receptor (AR) serves as the primary target for therapeutic intervention in prostate cancer (PCa), and AR-targeted treatments constitute the cornerstone of clinical management for this malignancy. Nevertheless, their effectiveness is often compromised by the emergence of resistance mechanisms. These resistant forms of PCa persist in activating AR signaling, highlighting the urgent need for new therapeutic strategies to combat therapy-resistant PCa. Resistance to AR-targeted therapies can manifest through a multitude of mechanisms, including the overexpression of AR splice variants and altered activities of other transcription factors, such as the glucocorticoid receptor (GR) and FOXA1. A shared characteristic among these transcription factors is their dependence on a common set of coregulators, with EP300/CREBBP being a prominent example. This suggests a compelling rationale for employing coregulatory-targeted therapies in the management of treatment-resistant PCa. Here, we aimed to explore the impact of EP300/CREBBP acetyltransferase inhibition on steroid receptor and FOXA1 signaling in PCa cells, employing genome-wide techniques. Our findings illuminate that EP300/CREBBP inhibition exerts a substantial disruptive effect on the AR-regulated transcriptome and receptor chromatin binding, primarily by downregulating the AR-gene. Similarly, the transcriptome regulated by GR and the chromatin binding of the receptor were also hindered, although this was not associated with decreased GR expression levels. Instead, EP300/CREBBP acetyltransferase inhibition leads to a significant reduction in FOXA1 chromatin binding, consequently constraining GR signaling. In summary, our results emphasize how EP300/CREBBP acetyltransferase inhibition distinctly curtails the signaling activities of oncogenic transcription factors, underscoring the potential effectiveness of coregulatory-targeted therapies in PCa.

overall_design: Examination of chromatin accessibility by ATAC-seq from 22Rv1 and VCaP prostate cancer cells treated with or without A485 and grown in the presence or absence of ENZ. Sequencing was done with Illumina NextSeq 500 or Illumina NextSeq 2000.

first: Jasmin

last: Huttunen


title: EP300/CREBBP acetyltransferase inhibition limits steroid receptor and FOXA1 signaling in prostate cancer cells (RNA-seq)

last: Paakinaho

overall_design: Gene expression profiling by RNA-seq from 22Rv1 and VCaP cells treated with or without A485 and grown in the presence or absence of ENZ. Sequencing was done with Illumina NextSeq 500 or Illumina NextSeq 2000.


title: Total and dendritic ribosome profiling from primary mouse cortical neurons

city: New York

first: Caryn

middle: R

last: Hale

state: New York

address: 1230 York Avenue

institute: Rockefeller University

summary: Activity-dependent protein synthesis is critical for determining changes in dendritic proteomes underlying brain function, yet the mechanisms governing these changes are lacking. Here, we combined proximity-based labeling of dendritic transcriptome, translatome, and proteome to study the dynamics of RNA regulation in activated synapses. We discovered that depolarization leads to a switch from RNAs translated under basal conditions to new translation of previously unrecognized subsets of depolarization-dependent transcripts. Dynamically regulated RNAs bound by specific regulatory proteins, including NOVA1, FMRP, and ELAVLs, rapidly generate proteins with roles in mitochondrial regulation, RNA metabolism, translational control, and synaptic signaling in dendrites. Knockdowns of activity-induced dendritic RNAs altered neuronal physiology, underscoring how dynamic switches in the regulation of RNAs encoding coordinated sets of proteins underlie synaptic plasticity.

overall_design: Four 150 mm cell culture dishes for resting and depolarized primary cortical neurons were combined per sample per replicate, 1/4th of which was spared for proteomics and rest for the ribosome profiling experiments. After a two-minute cycloheximide (Chx) treatment followed by a 30-minute biotin pulse, cells were quickly rinsed in ice-cold polysome gradient buffer (20 mM Tris pH7.5, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, and 100 μg/ml CHX), then scraped and lysed on plates on ice in 1 mL of ice-cold polysome lysis buffer (20 mM Tris pH7.5, 150 mM NaCl, 5 mM MgCl2 supplemented with 20 U/mL SUPERase-In RNase inhibitor, 24 U/mL Turbo DNase, 1 mM DTT, and 100 μg/ml CHX, 1x EDTA-free protease inhibitor. Lysates were clarified by centrifugation at 20,000 g for 2 minutes at 4°C, after which the supernatant was immediately loaded on to the cold 5 mL 7 kDa MWCO Zeba desalting column that was previously equilibrated with the ice-cold polysome gradient buffer. Lysates were subsequently flash-frozen or used for monosome fractionation. For monosome fractionation, lysates were added 5 mM CaCl2, incubated with micrococcal nuclease for 45 minutes at room temperature, quenched with 6.25 mM EGTA, and loaded on sucrose gradients. Gradients were then spun for 2 hours at 41K rpm in an SW-41 rotor and fractionated using BioComp Gilson fraction collection. 10% of the monosome fraction was kept for input. Triton X-100 was added to the remaining monosomes to a final concentration of 0.05%. The biotinylated monosomes were isolated using 50 µl of MyOne streptavidin C1 magnetic Dynabeads that were prepared according to the manufacturer’s guidelines and washed twice with Buffer A, twice with Buffer B, and once with polysome gradient buffer (with DTT and CHX). The monosome and bead mix was nutated at 4°C overnight. The supernatant was removed the next day, and the beads were moved into a new tube with low salt wash buffer (20 mM Tris pH8, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, and 100 μg/ml CHX, and 0.1% Triton X-100), and consequently washed three times for 10 minutes at 4°C with high salt wash buffer (20 mM Tris pH8, 500 mM NaCl, 5 mM MgCl2, 1 mM DTT, 100 μg/ml CHX, and 0.1% Triton X-100).

first: Ezgi

last: Hacisuleyman

first: Ji-Dung

last: Luo

first: Thomas

last: Carroll

first: Robert

middle: B

last: Darnell


title: Total and dendritic RNA-sequencing from primary mouse cortical neurons

overall_design: For the UV-crosslinking experiments, resting and depolarized neurons were treated with cycloheximide (Chx) and biotin and then were washed twice with 1x PBS supplemented with 100 μg/ml CHX and crosslinked on 150 mm plates on ice in 1x PBS with CHX with one pulse of 400 mJ/cm2 and one pulse of 200 mJ/cm2. Cells were then immediately scraped in fresh 1x ice-cold PBS with CHX and centrifuged at 7000 rpm for 5 minutes at 4°C. The pellets were either flash frozen or handled for the downstream steps as described previously by Kaewsapsak et al. (eLife, 2017) and Hendrickson et al. (Genome Biol., 2016). The pellets were lysed in 0.5 ml of ice-cold RIPA buffer (50 mM Tris (pH8), 150 mM KCl, 0.1% SDS, 1% Triton-X, 5 mM EDTA, 0.5% sodium deoxycholate, 0.5 mM dithiothreitol (DTT),  1x EDTA-free protease inhibitor cocktail, and 100 U/ml RNaseOUT for 10 minutes on ice. The lysates were then briefly sonicated on the Branson® digital sonifier using 10% amplitude for 3 seconds, during which they were held in cold metal blocks, and clarified by centrifugation at 15,000 g for 10 minutes at 4°C. Samples were diluted by adding 0.5 ml of Native Lysis Buffer (NLB) (150 mM KCl, 25 mM Tris pH7.5, 5 mM EDTA, 0.5 % NP-40, 0.5 mM DTT, 1x protease inhibitor, 100 U/mL RNaseOUT). 15% of the lysate was kept as input RNA. The remaining lysate was added 50 µl of RIPA:NLB equilibrated C1 magnetic beads. The pulldown was allowed to proceed overnight at 4°C. Beads were then washed briefly at 4°C with ice-cold: 1) RIPA buffer twice, 2) high salt buffer (1 M KCl, 50 mM Tris, pH8, 5 mM EDTA), 3) urea buffer (2 M Urea, 50 mM Tris, pH8, 5 mM EDTA), 4) RIPA Buffer, 5) 1:1 RIPA: NLB, 6) NLB, and 7) TE (10 mM Tris, pH7.5, 1 mM EDTA). The inputs and beads were then treated with sarkosyl and Proteinase K (2% N-lauryl sarkosyl, 10 mM EDTA, 5 mM DTT, in 1x PBS, supplemented with 200 µg of Proteinase K (Roche) and 4 U of RNaseOUT) at 42°C for 1 hour followed by 55°C for 1 h prior to RNA isolation to digest the biotinylated proteins and free the bound RNAs.


title: eIF4G2 cross-linking immunoprecipitation (CLIP) in primary cortical neurons

summary: Learning and memory require activity-induced changes in mRNA translation within dendrites, but which mRNAs are involved and how they are regulated remain unclear. We combined proximity labeling with ribosome profiling and CLIP to monitor how depolarization impacts dendritic translation. For a functionally coherent set of transcripts highly enriched in mitochondrial genes, depolarization leads to enhanced uORF translation, eIF4G2 binding, and increased translation. Engineered reporters demonstrate that activity-dependent translational control is conferred by the 5’UTRs and that dendritic localization, eIF4G2 binding, and uORF translation are necessary and sufficient to mediate this regulation. Downstream, this drives activity-dependent changes in dendritic mitochondrial function. Our studies uncover an unanticipated mechanism by which activity-dependent uORF translational control by eIF4G2 enables the coupling of synaptic activity to local remodeling of dendrites.

overall_design: Neurons were washed twice with 1x PBS with 100 μg/ml cycloheximide (CHX) and UV-crosslinked on ice in the same wash buffer with one pulse of 400 mJ/cm2 and one pulse of 200 mJ/cm2. Cells were then immediately scraped in fresh 1x ice-cold PBS with CHX and centrifuged at 7500 rpm for 5 minutes at 4°C. The pellets were flash frozen or processed as described previously for established CLIP protocols with modifications. Three biological replicates were used, each replicate prepared from three 15-cm cell culture dishes. Pellets were resuspended in 0.5 m lysis buffer, and immunoprecipitations (IP) were performed overnight at 4°C using the rabbit monoclonal antibody against eIF4G2.


title: ORFRATER analysis of ribosome profiling data from primary mouse cortical neurons

overall_design: Three 150 mm cell culture dishes were combined per sample. Neurons were either treated with nothing (WT), cycloheximide for 2 minutes (Chx), or harringtonine for 2 minutes followed by a Chx pulse. Cells were quickly rinsed in ice-cold polysome gradient buffer (20 mM Tris pH7.5, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, and 100 μg/ml CHX), then scraped and lysed on plates on ice in 1 mL of ice-cold polysome lysis buffer (20 mM Tris pH7.5, 150 mM NaCl, 5 mM MgCl2 supplemented with 20 U/mL SUPERase-In RNase inhibitor, 24 U/mL Turbo DNase, 1 mM DTT, and 100 μg/ml CHX, 1x EDTA-free protease inhibitor. Lysates were clarified by centrifugation at 20,000 g for 2 minutes at 4°C and subsequently flash-frozen or used for monosome fractionation. For monosome fractionation, lysates were added 5 mM CaCl2, incubated with micrococcal nuclease for 45 minutes at room temperature, quenched with 6.25 mM EGTA, and loaded on sucrose gradients. Gradients were then centrifuged for 2 hours at 41K rpm in an SW-41 rotor, and monosomes were collected using the BioComp Gilson fraction collection.


title: Lansoprazole interferes with fungal respiration and acts synergistically with amphotericin B against multidrug-resistant Candida auris

status: Public on Mar 13 2024

city: West Lafayette

first: Tony

last: Hazbun

state: Indiana

address: 201 South University Street

department: Borch Medicinal Chemistry and Molecular Pharmacology

institute: Purdue University

summary: Candida auris has emerged as a problematic fungal pathogen associated with high morbidity and mortality. Amphotericin B (AmB) is the most effective antifungal used to treat invasive fungal candidiasis, with resistance rarely observed among clinical isolates. However, C. auris possesses extraordinary resistant profiles against all available antifungal drugs, including AmB. In our pursuit of potential solutions, we conducted a screening of a panel of 727 FDA-approved drugs and identified the proton pump inhibitor lansoprazole (LNP) as a potent enhancer of AmB’s activity against C. auris. LNP also potentiates the antifungal activity of AmB against other medically important species of Candida and Cryptococcus. Our investigations into the mechanism of action unveiled that LNP metabolite(s) interact with a crucial target in the mitochondrial respiratory chain (complex III, known as cytochrome bc1). This interaction increases oxidative stress within fungal cells. Our results demonstrated the critical role of an active respiratory function in the antifungal activity of LNP. Most importantly, LNP restored the efficacy of AmB in an immunocompromised mouse model, resulting in a 1.7-log (~98%) CFU reduction in the burden of C. auris in the kidneys. Our findings strongly advocate for a thorough and comprehensive evaluation of LNP as a cytochrome bc1 inhibitor for combating drug-resistant C. auris infections.

overall_design: Exponential C. auris AR0390 in RPMI 1640 medium were treated with DMSO (control), AmB (0.5 µg/ml), LNP (32 µg/ml), or the combination of AmB/LNP in triplicates for 3 hr at 35 °C.

first: Ehab

last: Salama

first: Yehia

last: Elgammal

first: Aruna

last: Wijeratne

first: Nadia

last: Lanman

first: S

last: Utturkar

first: Atena

last: Farhangian

first: Jianing

last: Lie

first: Brigitte

last: Meunier

first: Mohamed

last: Seleem


title: A personalized medicine approach identifies enasidenib as an efficient treatment for IDH2 mutant chondrosarcoma [RNAseq_Enasidenib_L2975_SW1353]

city: Oviedo

first: Rene

last: Rodriguez

state: Asturias

address: Av. Hospital Universitario s/n

country: Spain

institute: Health Research Institute of Asturias (ISPA)

summary:
The heterogenous genomic nature of most sarcoma subtypes makes them especially indicated for personalized treatment approaches. Here, we developed a personalized medicine strategy based in the use of patient-derived cell lines as a drug-testing platform. Targeted sequencing of a panel of cancer-related genes in these models revealed the presence of IDH1 and IDH2 mutations in two chondrosarcomas. Mutant IDH (mIDH) enzymes produce the oncometabolite 2-HG which contributes to driving tumor growth. Thus, we treated several chondrosarcoma models with specific mIDH1/2 inhibitors. Among these treatments, only the mIDH2 inhibitor enasidenib was able to decrease 2-HG levels and to efficiently reduce the viability of mIDH2 chondrosarcoma cells.
Importantly, oral administration of enasidenib in xenografted mice resulted in a complete abrogation of tumor growth. Enasidenib induced a profound remodeling of the transcriptomic landscape not associated to changes in the 5mC methylation levels and its anti-tumor effects were associated to the repression of proliferative pathways such as those controlled by E2F factors.
Overall, this work provides the first preclinical evidence for the use of enasidenib to treat mIDH2 chondrosarcomas.

overall_design: To investigate the effect of enasidenib in the transcriptomic landscape, we treated IDH2 mutated chondrosarcoma primary cell lines (SW1353 and L2975) for 48 h with the inhibitor enasidenib (20 μM) and with the vehicle DMSO (control)

first: V

last: Rey

first: J

last: Tornin

last: Alba-Linares

first: C

last: Robledo

first: D

last: Murillo

first: A

first: B

last: Galego

last: Huergo

last: Braña

last: Astudillo

last: Heymann

first: K

last: Szuhai

last: Bovée

last: Fernandez

first: M

last: Fraga

last: Alonso

first: R


title: EP300/CREBBP acetyltransferase inhibition limits steroid receptor and FOXA1 signaling in prostate cancer cells (ChIP-seq)

first: Ville

last: Paakinaho

overall_design: Examination of AR, GR, H3K27ac and FOXA1 chromatin binding by ChIP-seq from 22Rv1 prostate cancer cells treated with or without A485 and grown in the presence or absence of ENZ. Examination of AR and GR chromatin binding by ChIP-seq from VCaP prostate cancer cells treated with or without A485 and grown in the presence or absence of ENZ. Examination of EP300 chromatin binding by ChIP-seq from 22Rv1 and VCaP prostate cancer cells grown in the presence or absence of ENZ. Sequencing was done with Illumina NextSeq 500 or Illumina NextSeq 2000.

first: Jasmin

last: Huttunen


title: Changes in cellular transcriptome in response to inhibitors of Epstein-Barr virus

city: La Crosse

first: Kelly

last: Gorres

state: WI

address: 1725 State St, Cowley 4015

department: Chemistry & Biochemistry

institute: University of Wisconsin-La Crosse

summary: Burkitt lymphoma cells can be latently infected with Epstein-Barr virus (EBV). The virus may be activated into its lytic cycle by small molecules, such as sodium butyrate. Other molecules, such as valproate and valpromide, block viral lytic reactivation. These pharmacological agents alter the cellular physiology that controls viral lytic gene expression. Changes in the cellular transcription were measured in response to one activator and two inhibitors of the Epstein-Barr virus lytic cycle in order to identify cellular genes that are potential regulators of the viral life cycle.

overall_design: RNA-seq transcriptome analysis of EBV-positive Burkitt lymphoma cells treated for 6 hours with DMSO (control), butyrate (3 mM), valproate (10 mM), valpromide (10 mM), valproate plus butyrate, or valpromide plus butyrate. Each condition was performed in duplicate.

first: David

last: Reineke

first: George

last: Miller


title: Single cell gene expression profiling of CARD9 WT and R101C murine skin infected with Trichophyton rubrum

status: Public on Mar 12 2024

city: Cambridge

first: Ramnik

last: Xavier

address: 415 Main Street

institute: Broad Institute of MIT and Harvard

summary: We used scRNA-seq to understand how CARD9 R101C remodels skin immunity and cell state in response to fungal infection in a mouse model of dermatophytosis.

overall_design: We performed single cell RNA sequencing (scRNAseq) of skin from WT and CARD9 R101C mice infected with T. rubrum at day two (D2) and day nine (D9) post-infection (3 replicates of each WT and CARD9 R101C at each timepoint)

first: Marta

last: Brandt

first: Chirag

last: Krishna


title: A personalized medicine approach identifies enasidenib as an efficient treatment for IDH2 mutant chondrosarcoma [MethArray_5days]

first: Rene

last: Rodriguez

type: Methylation profiling by genome tiling array

overall_design: To investigate the effect of enasidenib in the methylation, we treated IDH2 mutated chondrosarcoma primary cell line for 120 h with the inhibitor (20 μM) and with the vehicle DMSO (control). The EZ-96 DNA Methylation Kit (Zymo Research) was used to perform the bisulphite conversion. The processed DNA samples were then hybridised to Infinium MethylationEPIC v2.0 BeadChip following the Illumina Infinium HD Methylation Assay protocol.

first: V

last: Rey

last: Tornin

last: Alba-Linares

last: Robledo

last: Murillo

last: Galego

last: Huergo


title: Early-life exercise induces immunometabolic epigenetic modification enhancing anti-inflammatory immunity in middle-aged male mice

city: xi'an

first: Xinpei

last: Wang

address: 169 Changle West Road

institute: Fourth Military Medical University

summary: Exercise is usually regarded to have short-term beneficial effects on immune health. Here we show that early-life regular exercise exerts long-term beneficial effects on inflammatory immunity. Swimming training for 3 months in male mice starting from 1-month-old curbed cytokine response and mitigated sepsis when exposed to lipopolysaccharide (LPS) challenge, even after 11-month interval of detraining. Metabolomics analysis of serum and liver identified pipecolic acid (a non-encoded amino acid) as a pivotal metabolite responding to early-life regular exercise. We then explored histone epigenetic modifications and observed a significant increase of H3K4me3 expression in the liver of 15-month-old mice exposed to early-life exercise. To further unravel the prolonged increased pipecplic acid production raised by early-life exercise, we conducted ChIP-seq analysis and found H3K4me3 occupancy at Crym (a key enzyme responsible for catalyzing pipecolic acid production) promoter has a significant increase in hepatocytes of early-life exercised mice. Our findings demonstrate that early-life regular exercise enhances anti-inflammatory immunity during middle-aged phase in male mice via epigenetic immunometabolic modulation, in which hepatic pipecolic acid production plays a pivotal role.

overall_design: Chromatin  immunoprecipitation DNA-sequencing (ChIP-seq) for H3K4me3 in primary hepatocytes isolated from 15-month-old early-life exercised or sedentary mice

first: Nini

last: Zhang

first: Jia

last: Li

first: Feng

last: Gao


title: Molecular patterns of resistance to immune checkpoint blockade in melanoma [NanoString GeoMx]

city: Lund

first: Martin

last: Lauss

address: Barngatan 2B

department: Dept of Oncology

country: Sweden

institute: Lund University

summary: Tumor cell-containing Regions of Interest from 17 patients with melanoma after progression to Immune Checkpoint Blockade were profiled at the transcriptomic level.

overall_design: Spatial digital profiling of 33 Regions of Interest from 17 patients with melanoma, using NanoString GeoMx platform for 1825 gene identifiers from the Cancer Transcriptome Atlas.

first: Göran

last: Jönsson


title: Profiling data from primary mouse cortical neurons

first: Caryn

last: Hale


title: Decoding Spatiotemporal Transcriptional Dynamics and Epithelial Fibroblast Crosstalk during Gastroesophageal Junction Development through Single Cell Analysis (Organoids)

city: Würzburg

first: Cindrilla

last: Chumduri

address: Am Hubland

department: Chair of Microbiology, Biozentrum

country: Germany

institute: Julius-Maximilians-Universität Würzburg

summary: The gastroesophageal squamocolumnar junction (GE-SCJ) is a critical tissue interface between the esophagus and stomach, with significant relevance in the pathophysiology of gastrointestinal diseases. Despite this, the molecular mechanisms underlying GE-SCJ development remain unclear. Using single-cell transcriptomics, organoids, and spatial analysis, we examine the cellular heterogeneity and spatiotemporal dynamics of GE-SCJ development from embryonic to adult mice. We identify distinct transcriptional states and signaling pathways in the epithelial and mesenchymal compartments of the esophagus and stomach during development. Fibroblast-epithelial interactions are mediated by various signaling pathways, including WNT, BMP, TGF-β, FGF, EGF, and PDGF. Our results suggest that fibroblasts predominantly send FGF and TGF-β signals to the epithelia, while epithelial cells mainly send PDGF and EGF signals to fibroblasts. We observe differences in the ligands and receptors involved in cell-cell communication between the esophagus and stomach. Our findings provide insights into the molecular mechanisms underlying GE-SCJ development and fibroblast-epithelial crosstalk involved, paving the way to elucidate mechanisms during adaptive metaplasia development and carcinogenesis.

overall_design: Mouse epithelial stem cells derived from esophagus and stomach tissues were grown into 3D organoids using a unique cocktail of growth factors were harvested separately and dissociated to single-cell suspension for single-cell RNA sequencing as described in the manuscript.

first: Naveen

last: Kumar

first: Rajendra

last: Kumar Gurumurthy

first: Pon

last: Ganish Prakash

first: Tobias

last: Krammer

first: Christophe

last: Toussaint

first: Antoine-Emmanuel

last: Saliba


title: Decoding Spatiotemporal Transcriptional Dynamics and Epithelial Fibroblast Crosstalk during Gastroesophageal Junction Development through Single Cell Analysis (Tissue-scRNAseq)

institute: University of Wuerzburg

overall_design: Single cells from esophagus, gastro-esophageal junction and stomach were isolated from embryonic day E15 (n=6), E19 (n=2), Newborn Day-2 (Pup, n=2) and 8-week-old mice (Adult, n=3). Replicates were pooled and cell multiplexed using  Cell Multiplexing Oligos (CMOs) based on their tissue region and time points using 10x Genomics 3' CellPlex and Single-Cell 3′ v3.1 RNA-seq kits.

first: Pon Ganish

last: Prakash

first: Rajendra Kumar

last: Gurumurthy


title: Gene expression profiles of mouse derived esophagus and stomach epithelial stem cells 3D organoids

overall_design: Mouse-derived esophagus and stomach epithelial stem cells were isolated and cultured as 3D organoids as described in the accompanying manuscript (Materials and Methods). Total RNA was extracted from samples and hybridized on Agilent-8x60k Mouse custom arrays as single-color hybridizations.

last: Kumar Nirchal

first: Christian

last: Wentland

first: Hans-Joachim

last: Mollenkopf


title: Z-DNA underlies the target choice of Aire by facilitating promoter poising [scRNA-Seq]

status: Public on Mar 11 2024

city: Boston

first: CBDM

last: Lab

address: 77 Avenue Louis Pasteur

department: Microbiology and Immunobiology

institute: Harvard Medical School

summary: Aire is an unconventional transcription factor (TF) that enhances the expression of thousands of genes in medullary thymic epithelial cells (mTECs), which promotes clonal deletion or phenotypic diversion of self-reactive T cells. The biological logic of Aire’s target specificity remains largely unknown as, unlike many TFs, it does not bind to a particular DNA-sequence motif. We implemented two orthogonal approaches to investigate Aire’s cis-regulatory mechanisms: construction of a convolutional neural network and leveraging of natural genetic variation via analysis of F1-hybrid mice. Both approaches nominated Z-DNA and Nfe2•Maf as putative positive influences on Aire’s target choices. Genome-wide mapping studies revealed that Z-DNA-forming and Nfe2l2-binding motifs were positively associated with the inherent ability of a gene’s promoter to generate DNA double-strand breaks (DSBs), and promoters showing strong DSB generation were more likely to enter a poised state with accessible chromatin and already-assembled transcriptional machinery. Consequently, Aire preferentially targeted genes with poised promoters. We propose a model whereby Z-DNA anchors the Aire-mediated transcriptional program by enhancing DSB generation and promoter poising. Beyond resolving a long-standing mechanistic conundrum, these findings suggest previously unexplored routes of manipulating T-cell tolerance.

overall_design: We studied how spermidine injection influences the transcriptome of mTECs. To do so, 4-week-old female B6 WT mice were ip-injected with 15mg/kg spermidine (Sigma-Aldrich) in PBS, or just PBS as control for seven consecutive days. mTEChi and post-Aire mTEClo were then sorted. Samples were hashed using the TotalSeq-A anti-mouse (anti-CD45/MHC class I) hashtags (BioLegend). Cells were then submitted to the Broad Institute Genomics Platform for encapsulation and library preparation following 10X Genomics protocols. Two replicates were prepared for each condition.

first: Yuan

last: Fang

last: Benoist

first: Diane

last: Mathis


title: Targeting Glycomes Sensitizes Ovarian Tumors to Immune Checkpoint Blockade

city: Philadelphia

first: Priyankara

last: Wickramasinghe

state: PA

address: 3601 Spruce Street

department: Bioinformatics

institute: The Wistar Institute

summary: Aberrant glycosylation is a crucial strategy employed by cancer cells to evade cellular immunity. However, homologous recombination (HR) status-dependent glycosylation has never been explored therapeutically. Here, we show that the inhibition of branched N-glycans sensitizes HR-proficient, but not HR-deficient, epithelial ovarian cancers (EOCs) to immune checkpoint blockade (ICB). In contrast to fucosylation whose inhibition sensitizes EOCs to anti-PD-L1 immunotherapy regardless of HR-status, we observe a unique enrichment of branched N-glycans on HR-proficient compared to HR-deficient EOCs. Mechanistically, BRCA1/2 transcriptionally promotes the expression of MGAT5, the enzyme responsible for catalyzing branched N-glycans. The branched N-glycans on HR-proficient tumors augment their resistance to anti-PD-L1 by enhancing its binding with PD-1 on CD8_ T cells. In orthotopic syngeneic EOC mouse models, inhibiting branched N-glycans, using 2-Deoxy-D-glucose, sensitizes HR-proficient, but not HR-deficient EOCs, to anti-PD-L1. These findings indicate branched N-glycans as promising therapeutic targets whose inhibition sensitizes HR-proficient EOCs to ICB by overcoming immune evasion

overall_design: single cell RNA-seq in young and aged treated and naïve mice

first: Hao

last: Nie

first: Rugang

last: Zhang


title: Obesity-related T cell dysfunction impairs immunosurveilance and increases cancer risk

city: St Louis

first: Ryan

last: Teague

state: Missouri

address: 1100 S Grand Blvd

department: Molecular Microbiology and Immunology

institute: Saint Louis University School of Medicine

summary: The current study used a mouse model of diet-induced obesity to investigate tumor-infiltrating CD8+ T cell responses.

overall_design: Tumors from lean or obese C57Bl/6J mice were harvested and CD8 T cells were isolated by CD8 (TIL) mouse magnetic Microbeads (Miltenyi) and Flurescence-activated cell sorting.

first: Alexander

last: Piening

first: Emily

last: Ebert


title: RAD51AP1 associated with RNA in HEK293T cells

first: Xiaochun

last: Yu

state: Zhejiang

address: 18 Shilongshan Street

institute: Westlake University

summary: RNAs have versatile roles in DNA damage repair. We here report RAD51AP1 associated with RNAs by PAR-CLIP.

overall_design: To investigate the function of RNAs which are associated with RAD51AP1 in DNA damage repair, we established RAD51AP1 stable cell line. We then performed PAR-CLIP and analysied RAD51AP1 associated RNAs.

first: Linlin

last: Chen

first: Qilin

last: Li


title: A personalized medicine approach identifies enasidenib as an efficient treatment for IDH2 mutant chondrosarcoma [MethArray_2days]

first: Rene

last: Rodriguez

summary:
The heterogenous genomic nature of most sarcoma subtypes makes them especially indicated for personalized treatment approaches. Here, we developed a personalized medicine strategy based in the use of patient-derived cell lines as a drug-testing platform. Targeted sequencing of a panel of cancer-related genes in these models revealed the presence of IDH1 and IDH2 mutations in two chondrosarcomas. Mutant IDH (mIDH) enzymes produce the oncometabolite 2-HG which contributes to driving tumor growth. Thus, we treated several chondrosarcoma models with specific mIDH1/2 inhibitors. Among these treatments, only the mIDH2 inhibitor enasidenib was able to decrease 2-HG levels and to efficiently reduce the viability of mIDH2 chondrosarcoma cells.  Importantly, oral administration of enasidenib in xenografted mice resulted in a complete abrogation of tumor growth. Enasidenib induced a profound remodeling of the transcriptomic landscape not associated to changes in the 5mC methylation levels and its anti-tumor effects were associated to the repression of proliferative pathways such as those controlled by E2F factors.
Overall, this work provides the first preclinical evidence for the use of enasidenib to treat mIDH2 chondrosarcomas.

overall_design: To investigate the effect of enasidenib in the methylation, we treated IDH2 mutated chondrosarcoma primary cell line for 48 h with the inhibitor (20 μM) and with the vehicle DMSO (control). The EZ-96 DNA Methylation Kit (Zymo Research) was used to perform the bisulphite conversion. The processed DNA samples were then hybridised to Infinium MethylationEPIC BeadChips following the Illumina Infinium HD Methylation Assay protocol.

first: V

last: Rey

first: J

last: Tornin

last: Alba-Linares

first: C

last: Robledo

first: D

last: Murillo

first: A

first: B

last: Galego

last: Huergo

last: Braña

last: Astudillo

last: Heymann

first: K

last: Szuhai

last: Bovée

last: Fernandez

first: M

last: Fraga

last: Alonso

first: R


title: A personalized medicine approach identifies enasidenib as an efficient treatment for IDH2 mutant chondrosarcoma [RNAseq_Enasidenib_T-CDS17#1]

summary: The heterogenous genomic nature of most sarcoma subtypes makes them especially indicated for personalized treatment approaches. Here, we developed a personalized medicine strategy based in the use of patient-derived cell lines as a drug-testing platform. Targeted sequencing of a panel of cancer-related genes in these models revealed the presence of IDH1 and IDH2 mutations in two chondrosarcomas. Mutant IDH (mIDH) enzymes produce the oncometabolite 2-HG which contributes to driving tumor growth. Thus, we treated several chondrosarcoma models with specific mIDH1/2 inhibitors. Among these treatments, only the mIDH2 inhibitor enasidenib was able to decrease 2-HG levels and to efficiently reduce the viability of mIDH2 chondrosarcoma cells. Importantly, oral administration of enasidenib in xenografted mice resulted in a complete abrogation of tumor growth. Enasidenib induced a profound remodeling of the transcriptomic landscape not associated to changes in the 5mC methylation levels and its anti-tumor effects were associated to the repression of proliferative pathways such as those controlled by E2F factors. Overall, this work provides the first preclinical evidence for the use of enasidenib to treat mIDH2 chondrosarcomas.

overall_design: To investigate the effect of enasidenib in the transcriptomic landscape, we treated (in triplicates) IDH2 mutated chondrosarcoma primary cell line (T-CDS17#1) for 48 h with the inhibitor enasidenib (20 μM) and with the vehicle DMSO (control)


title: FixNCut: Single-cell genomics through reversible tissue fixation and dissociation

city: Barcelona

first: Laura

last: Jiménez-Gracia

address: c/ Baldiri Reixac, 4  Barcelona Science Park - Tower I

institute: Centre Nacional d'Anàlisi Genòmic (CNAG)

summary: The use of single-cell technologies for clinical applications requires disconnecting sampling from downstream processing steps. Early sample preservation can further increase robustness and reproducibility by avoiding artifacts introduced during specimen handling. We present FixNCut, a methodology for the reversible fixation of tissue followed by dissociation that overcomes current limitations. We applied FixNCut to human and mouse tissues to demonstrate the preservation of RNA integrity, sequencing library complexity, and cellular composition, while diminishing stress-related artifacts. Besides single-cell RNA sequencing, FixNCut is compatible with multiple single-cell and spatial technologies, making it a versatile tool for robust and flexible study designs.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: 12 samples

first: Holger

last: Heyn

first: Luciano G.

last: Martelotto


title: Compensatory growth and recovery of tissue cytoarchitecture after transient cartilage-specific cell death in foetal mouse limbs

first: Alberto

last: Rosello-Diez

address: Physiology building. Downing St.

department: Physiology, Development and Neuroscience

country: United Kingdom

institute: University of Cambridge

summary: A major question in developmental and regenerative biology is how organ size is controlled by progenitor cells. For example, while limb bones exhibit catch-up growth (recovery of a normal growth trajectory after transient developmental perturbation), it is unclear how this emerges from the behaviour of chondroprogenitors, the cells sustaining the cartilage anlagen that are progressively replaced by bone. Here we show that transient sparse cell death in the mouse foetal cartilage was repaired postnatally, via a two-step process. During injury, progression of chondroprogenitors towards more differentiated states was delayed, leading to altered cartilage cytoarchitecture and impaired bone growth. Then, once cell death was over, chondroprogenitor differentiation was accelerated and cartilage structure recovered, including partial rescue of bone growth. At the molecular level, ectopic activation of mTORC1 correlated with, and was necessary for, part of the recovery, revealing a specific candidate to be explored during normal growth and in future therapies.

overall_design: The use of animals in this study was approved by the Monash Animal Research Platform animal ethics committee at Monash University. The Pitx2-ASE-Cre (aka Pitx2-Cre) mouse line was crossed with the Col2a1-rtTA mouse line and then inter-crossed to generate Pitx2-Cre/Cre; Col2a1-rtTA mice. Experimental and control animals were generated by crossing Pitx2-Cre/Cre; Col2a1-rtTA/+ females with TigreDragon-DTA/DTA males (i.e. homozygous for the conditional misexpression allele). The separation of control and experimental animals was based on the rtTA genotype. Pregnant females were administered doxycycline hyclate (Sigma, 1mg/ml in drinking water, with 0.5% sucrose for palatability) from E12.25 to E13.75. The day of vaginal plug detection was designated as E0.5, and E19.5 was referred to as P0. Mouse embryos were euthanized using hypothermia in cold PBS, while mouse pups were euthanized by decapitation. Control and experimental samples (2-3 biological replicates each) were collected for 4 different stages: E15.5, E17.5, P0 and P3. Left and right cartilage samples (each containing proximal and distal tibia and femur) were kept separated for each sample, and flash-frozen upon dissection. Total RNA was extracted using the Monarch Total RNA Miniprep Kit (New England Biolabs), following manufacturer instructions. From this, RNA-Seq of messenger RNAs (mRNAs) was performed using a custom in-house multiplex method, which allowed us to sequence up to 24 different samples in the same sequencing lane. Briefly, samples were given a unique i7 index (together with UMI) during individual polyA-priming and first-strand synthesis, which also added a template-switch sequence to the 5’ end. Samples were then pooled and amplified using P7 and an oligo which binds the template-switch sequence. Final library construction was completed by tagmentation and addition of P5 (with i5 index) by PCR. Sequencing was performed on an Illumina NSQ2k run with up to 101nt SR (cDNA). An 18-nt i7 read contains the 8-nt index and 10-nt UMI and, where required, an 8-nt i5 index read is also generated. We obtained a read depth of at least 15 million reads per library.

first: Chee

middle: H

last: H’ng

first: Shanika

last: Amarasinghe

first: Boya

last: Zhang

first: Hojin

last: Chang

first: Xinli

last: Qu

first: David

last: Powell


title: A personalized medicine approach identifies enasidenib as an efficient treatment for IDH2 mutant chondrosarcoma

first: Rene

last: Rodriguez

summary: This SuperSeries is composed of the SubSeries listed below.

overall_design: Refer to individual Series


title: Dynamic changes of lung homeostasis from development-to-aging single-cell atlas

status: Public on Mar 10 2024

city: Beijing

first: Jiangping

last: Song

address: 167A Beilishi Road, Xi Cheng District

institute: Fuwai Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College

summary: Aging is a global problem, in which lung aging is accompanied by functional decline and structural disorders, disturbing the health of the elderly population. To explore anti-aging methods, we constructed a dynamic atlas of 45 cell types, from embryonic development to aging on human lung samples, and we hoped to use the discoveries of development to solve the problems of aging. During development and aging, epithelial and immune cells underwent a process of acquisition and loss of obligatory function. During aging, we identified cellular phenotypic changes that result in decreased pulmonary compliance and immune homeostasis. Furthermore, we found a characteristic expression pattern for the ferritin light chain (FTL) gene, which was regulated upward during development and downward during aging in multiple component cells of the lung.

overall_design: The use of human lung tissue in this study was approved by the Human Ethics Committee of Fuwai Hospital, Chinese Academy of Medical Sciences. Adult lung specimens were collected with the informed consent of the patients, and aborted embryo specimens were obtained with the informed consent of the pregnant women. Embryonic lung tissue samples were collected from aborted embryos at 10, 12, 17, 20, 25, and 40 weeks of gestation. Adult lung tissue samples were collected from adults 47, 54, 67, and 74 years of age who underwent lung surgery. These adult patients underwent surgery for lung nodules (less than 30mm in diameter), and the samples were obtained from the normal tissue far away from the nodules that had been surgically removed. Pulmonary nodules were reported as non-cancerous after surgery.

first: Hao

last: Jia

first: Yuan

first: Yulin

last: Chen

first: Xiao

first: Hang

first: Xiumeng

last: Hua

first: Mengda

last: Xu

first: Yixuan

last: Sheng

first: Ningning

last: Cui

first: Lei

last: Han

first: Jian

first: Xiaodong

last: Fu


title: Z-DNA underlies the target choice of Aire by facilitating promoter poising [CUT&Tag]

first: CBDM

last: Lab

summary: We profiled Pol II-pS5 binding in medullary thymic epithelial cells using CUT&Tag to investigate the logic of Aire's target choices

overall_design: CUT&Tag of mTECs

last: Fang

first: Diane

last: Mathis

first: Christophe

last: Benoist


title: Z-DNA underlies the target choice of Aire by facilitating promoter poising [RNA-seq]

summary: We profiled transcriptome of medullary thymic epithelial cells from mice of various treatments and genotypes to investigate the logic of Aire's target choices

overall_design: RNA-seq of mTECs


title: Z-DNA underlies the target choice of Aire by facilitating promoter poising [ChIP-seq]

summary: We profiled Pol II binding in medullary thymic epithelial cells using ChIP-seq to investigate the logic of Aire's target choices

overall_design: ChIP-seq of mTECs

first: Kushagra

last: Bansal


title: Z-DNA underlies the target choice of Aire by facilitating promoter poising


title: Z-DNA underlies the target choice of Aire by facilitating promoter poising [ChIPmentation]

summary: We profiled NELF and Aire binding in medullary thymic epithelial cells using ChIPmentation to investigate the logic of Aire's target choices

overall_design: ChIPmentation of mTECs


title: Z-DNA underlies the target choice of Aire by facilitating promoter poising [BLISS]

summary: We profiled DNA double-strand break (DSB) formation genome-wide in medullary thymic epithelial cells to investigate how DSBs regulate Aire's target choices

overall_design: BLISS of mTECs


title: Z-DNA underlies the target choice of Aire by facilitating promoter poising [ATAC-seq]

summary: We profiled chromatin accessibility of medullary thymic epithelial cells to investigate the logic of Aire's target choices

overall_design: ATAC-seq of mTECs


title: An Intricate Network Involving the Argonaute ALG-1 Modulates Organismal Resistance to Oxidative Stress miRNAs in a longevity model and an alg-1 overexpression model.

status: Public on Mar 09 2024

city: Campinas

first: SILAS

middle: PINTO

last: DA SILVA

state: SP

address: Rua Monteiro Lobato, 255

country: Brazil

institute: UNICAMP

type: Non-coding RNA profiling by high throughput sequencing

summary: Cellular response to redox imbalance is crucial for organismal health. microRNAs are implicated in stress responses. ALG-1, the C. elegans ortholog of human AGO2, plays an essential role in microRNA processing and function. Here we investigated the mechanisms governing ALG-1 expression in C. elegans and the players controlling lifespan and stress resistance downstream of ALG-1. We found that upregulated ALG-1 is a shared feature in conditions linked to increased longevity (e.g., germline-deficient glp-1 mutants). ALG-1 knockdown reduces lifespan and oxidative stress resistance, while overexpression enhances survival against pro-oxidant agents but not heat or reductive stress. R02D3.7 represses alg-1 expression, impacting oxidative stress resistance at least in part via ALG-1. microRNAs upregulated in glp-1 mutants (miR-87-3p, miR-230-3p, and miR-235-3p) can target genes in the protein disulfide isomerase pathway and protect against oxidative stress. This study unveils a tightly regulated network involving transcription factors and microRNAs which controls organisms’ ability to withstand oxidative stress.

overall_design: To investigate the role of specific miRNAs in oxidative stress resistance in long-lived worms, we examined the miRNA expression profiles in C. elegans strains with either alg-1 overexpression (ALG-1 O/E) or the glp-1(e2144) mutation. We conducted gene expression profiling analysis using data obtained from small RNA-seq, which was enriched for miRNAs, from four different strains. The comparative gene expression profiling analysis included the wild type (WT) strain and the germline-less longevity (glp-1 mutant) strain, as well as animals with alg-1 overexpression (ALG-1 O/E) and its control (CT20) strain.

first: Silas

last: Pinto

first: Raíssa

middle: P

last: Moro

first: Marcelo

last: Mori


title: PRMT1 promotes pancreatic cancer development and resistance to chemotherapy

city: Daejeon

first: Bomin

last: Ku

address: 335 Gwahak-ro

department: Biological Sciences

country: South Korea

institute: Korea advanced institute of science and technology (KAIST)

summary: Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal types of cancer, and novel treatment regimens are direly needed. Epigenetic regulation contributes to the development of various cancer types, but its role in the development of, and potential as a therapeutic target for, PDAC remains underexplored. Here, we show that PRMT1 is highly expressed in murine and human pancreatic cancer and is essential for cancer cell proliferation and tumorigenesis. Deletion of PRMT1 delays pancreatic cancer development in a KRAS-dependent mouse model, and multi-omics analyses reveal that the PRMT1 depletion leads to global changes in chromatin accessibility and transcription, resulting in reduced glycolysis and a decrease in tumorigenic capacity. Pharmacological inhibition of PRMT1 in combination with gemcitabine has a synergistic effect on pancreatic tumor growth in vitro and in vivo. Collectively, our findings implicate PRMT1 as a key regulator of pancreatic cancer development and a promising target for combination therapy.

overall_design: RNA-seq data of PRMT1 knockdown from MiaPaca-2 cells

first: David

last: Eisenbarth

first: Dae-Sik

last: Lim


overall_design: H3K27ac ChIP-seq data of PRMT1 knockdown from MiaPaca-2 cells


title: Effects of overexpressing HNF4A on 3D cultured mouse and human colon organoid

status: Public on Mar 08 2024

first: Sanlan

last: Li

state: NY

address: 1300 York Avenue

institute: Weill Cornell Medicine

summary: To test the hypothesis that HNF4A should engage small intestine enhancers in colon and activate transcription, we over-expressed HNF4A in 2 independent mouse colonic orgnaoid lines and 5 independent human colonic organoid lines and performed RNA-seq studies. Both RNA-seq data revealed that small intestine functional pathways including fat, protein and carbohydrate digestion and absorption were activated after HNF4A OE. This gain-of-function study of HNF4A in mouse and human colon organoid supports HNF4A as a central mediator of small intestine gene activation in colonic plasticity, conserved across species.

overall_design: To investigate the effects of HNF4A in mouse and human colon, we established 2 independent mouse colon organoid lines and 5 independent human colon organoid lines and over-expressed HNF4A protein by lentivirus infection of the cultured organoids. We also included 2 control and 5 control groups for mouse and human respectively where the same colonic organoids were infected with control virus without HNF4A overexpression. We havested the cells, extracted RNA and used for RNA-profiling.

first: Wei

last: Gu

first: Xiaofeng

last: Huang

first: Joe

last: Zhou

first: Ying

last: Lan


title: Hepatocyte CCAAT/enhancer-binding protein alpha restricts liver fibrosis by inhibiting secreted phosphoprotein 1 expression

city: Bethesda

first: Tingting

last: Yan

state: Maryland

address: Building 37, Room 3106, Mail Stop 4255

institute: National Institutes of Health


title: Restored glyoxylate metabolism in induced-hepatocytes after CRISPR/Cas-mediated AGXT gene correction and direct cell reprogramming of Primary Hyperoxaluria Type 1 fibroblasts

status: Public on Mar 27 2024

city: Madrid

first: Ramon

last: Garcia-Escudero

state: Madrid

address: Avda Complutense 40, Ed 70A

department: Biomedical Innovation

institute: CIEMAT

summary: Primary Hyperoxaluria Type 1 (PH1) is a rare inherited metabolic disorder characterized by oxalate overproduction in the liver, resulting in renal damage. It is caused by mutations in the AGXT gene. Combined liver and kidney transplantation is currently the only permanent curative treatment. We combined locus-specific gene correction and hepatic direct cell reprogramming to generate autologous healthy induced hepatocytes (iHeps) from PH1 patient-derived fibroblasts. First, site-specific AGXT corrected cells were obtained by homology directed repair (HDR) assisted by CRISPR/Cas9, following two different strategies: accurate point mutation (c.853T>C) correction or knock-in of an enhanced version of AGXT cDNA. Then, iHeps were generated, by overexpression of hepatic transcription factors. Generated AGXT-corrected iHeps showed hepatic gene expression profile and exhibited in vitro reversion of oxalate accumulation compared to non-edited PH1-derived iHeps. This strategy set up a potential alternative cellular source for liver cell replacement therapy and a personalized PH1 in vitro disease model.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for healthy donor, PH1 patient-derived and AGXT-corrected immortalized human fibroblasts and the generated induced-hepatocytes.

first: Virginia

last: Nieto-Romero

first: Aida

last: García-Torralba

first: Andrea

last: Molinos-Vicente

first: Ramón

last: García-Escudero

first: Eduardo

last: Salido

first: Jose

last: Segovia

first: María

last: García-Bravo


title: Hepatocyte CCAAT/enhancer-binding protein alpha restricts liver fibrosis  by inhibiting secreted phosphoprotein 1 expression (I)

summary: Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease. Persistent NAFLD can progress to nonalcoholic steatohepatitis (NASH) with inflammation and fibrosis, which predisposes patients to cirrhosis and hepatocellular carcinoma. CCAAT/enhancer-binding protein alpha (CEBPA) is a transcription factor that modulates glycolipid homeostasis, cell differentiation and tumor progression. MTL-CEBPA, a first-in-human small activating RNA therapeutic, has been used to overexpress CEBPA and treat hepatocellular carcinoma by targeting myeloid cells in clinical trials. However, whether and how hepatocyte-specific CEBPA modulates NASH are unknown. Here we found that CEBPA expression was inhibited in the livers of high-fat, high-cholesterol, and high-fructose diet (HFCFD)-fed mice, NASH patients and palmitic/oleic acid-treated primary hepatocytes. Liver-specific Cebpa knockout mice (Cebpa-dLiv) and liver-specific tamoxifen-inducible ERT2-Cre mice (Cebpa-dLiv-ERT2) were then generated. Both Cebpa-dLiv and Cebpa-dLiv-ERT2 mice (treated with tamoxifen) developed enhanced NASH and fibrosis compared to control mice. Global transcriptome analyses demonstrated hepatic secreted phosphoprotein 1 (Spp1) encoding the fibrosis-promoting factor SPP1, was markedly induced by hepatocyte CEBPA knockout in HFCFD-fed mice. Consistently, hepatic SPP1 was upregulated by NASH and negatively correlated with CEBPA expression in humans. Hepatocyte loss of CEBPA enhanced hepatic SPP1 expression and serum SPP1 levels both at the early and late stage of NASH in mice. CEBPA-knockout primary hepatocytes released more SPP1 to the culture medium than wild-type mouse hepatocytes, resulting in enhanced fibrogenesis of hepatic stellate cells, a phenotype that was rescued by the Spp1 shRNA. Hepatocyte-directed AAV8-deliverey of Spp1 shRNA rescued the fibrosis in HFCFD-fed Cebpa-dLiv mice. Mechanistically, CEBPA overexpression reduced, while CEBPA knockout enhanced, SPP1 expression in primary hepatocytes in vitro. The Spp1 promoter had functional CEBPA response elements, while CEBPA modulated histone acetylation to restrict SPP1 expression in a novel feedback loop. Furthermore, overexpression of CEBPA in hepatocytes with AAV8-TBG-CEBPA, inhibited hepatic SPP1 expression and reduced fibrosis in HFCFD-fed wild-type mice. This study demonstrates a novel hepatocyte CEBPA-SPP1 axis involved in modulating NASH fibrosis that supports hepatocyte CEBPA as an anti-NASH target.

overall_design: Examination of 3 different batches of hepatocyte-specific CEBPA knockout mice fed a NASH-promoting 40% HFCFD

first: Frank

last: Gonzalez

first: Vorthon

last: Sawaswong


title: Symmetrically fated progenitors dynamically accommodate tissue maintenance in the esophagus [scRNA-seq]

status: Public on Mar 07 2024

city: Stockholm

first: David

last: Grommisch

address: Solnavägen 9

institute: Karolinska Institutet

summary: Progenitor cells adapt their behavior in response to tissue demands. However, the molecular mechanisms controlling esophageal progenitor decisions remain largely unknown. Here we demonstrate the presence of a Troy(Tnfrsf19)-expressing progenitor subpopulation localized to defined regions along the esophageal axis. Lineage tracing and mathematical modelling demonstrate that Troy-positive progenitor cells are prone to undergoing symmetrical fate choices and contribute to esophageal tissue homeostasis long-term. Functionally, TROY inhibits progenitor proliferation and enables commitment to differentiation without affecting fate symmetry. Whereas Troy expression is stable during esophageal homeostasis, progenitor cells downregulate Troy in response to tissue stress, enabling proliferative expansion of basal cells refractory to differentiation and reestablishment of tissue homeostasis. Our results demonstrate functional, spatially restricted, progenitor heterogeneity in the esophageal epithelium and unveil how dynamic regulation of Troy coordinates tissue generation.

overall_design: To investigate esophageal epithelial progenitor cells, CD104/CD49f double-positive epithelial cells of TroyWT/WT mice were FACS isolated and subjected to 10X Chromium single-cell sequencing.

first: Menghan

last: Wang

first: Maria

last: Genander


title: Effect of inhibition of Phospholipase D on gene expression during spinal cord injury

status: Public on Mar 06 2024

first: Zihan

last: Li

address: No. 10, Jiaomen North Road, Fengtai District, Beijing

institute: China Rehabilitation Science Institute

summary: Phospholipase D (PLD), a critical lipid signaling molecule, is known to be involved in various physiological processes. Despite this knowledge, the specific role of PLD in spinal cord injury remains unclear. In this study, we constructed mouse models of SCI and administered PLD inhibitor(FIPI) treatment to investigate the efficacy of PLD. Transcriptome sequencing analysis implicated inhibition of PLD altered gene expression in the inflammation regulation.

overall_design: To investigate the function of PLD in the regulation of spinal cord injury, we constructed mouse models of SCI and administered PLD inhibitor (FIPI) treatment. We then performed gene expression profiling analysis using data obtained from RNA-seq of 3 groups at 7 days after injury. Then comparative gene expression profiling analysis of RNA-seq data.


title: Hepatocyte CCAAT/enhancer-binding protein alpha restricts liver fibrosis by inhibiting secreted phosphoprotein 1 expression (II)

overall_design: Examination of the gene profiles changed by hepatocyte CEBPA knockout or overexpression in primary hepatocytes


title: Effect of Mip inhibition on J774A.1 macrophage immune response during Burkholderia pseudomallei infection

city: Crawley

first: Jua

last: Iwasaki

address: 35 Stirling Hwy

country: Australia

institute: University of Western Australia

summary:
Melioidosis, caused by the Gram-negative bacterium Burkholderia pseudomallei, is a disease endemic to South-East Asia and Northern Australia. Clinical presentation is highly variable, ranging from asymptomatic to fatal septicaemia, and thus the outcome of infection can depend on the host immune responses. The aim of this study was to characterise the macrophage immune response to B. pseudomallei in the presence of novel inhibitors targeting the virulence factor, Macrophage Infectivity Potentiator (Mip) protein.  To do this. murine macrophage J774A.1 cells were infected with B. pseudomallei K96243 in the presence and absence of two small-molecule inhibitors designed to target the Mip protein. Global transcriptional profiling of macrophages infected with B. pseudomallei was analysed by RNA-Seq four hours post-infection. In the presence of Mip inhibitors, we found a significant reduction in the expression of pro-inflammatory cytokines highlighting the potential to utilize Mip inhibitors to dampen potentially harmful pro-inflammatory responses resulting from B. pseudomallei infection in macrophages.
We then performed gene expression profiling analysis using data obtained from RNA-seq of J774A.1 macrophages infected with Burkholderia pseudomallei in the presence of two Mip inhibitors or vehicle control 4 hours post-infection

overall_design: Comparative gene expression profiling analysis of RNA-seq data for J774A.1 macrophage cells in the presence of two Mip inhibitors SF235 or AN_CH_37, and the vehicle control, following infection with Burkholderia pseudomallei

first: Mitali

last: Sarkar-Tyson


title: A Single Laccase Acts as a Key Component of Environmental Sensing in a Broad Host Range Fungal Pathogen

city: Madison

first: Mehdi

last: Kabbage

state: Wisconsin

address: 583 Russell Labs, 1630 Linden Dr.

department: Plant Pathology

institute: University of Wisconsin - Madison

summary: Secreted laccases are important enzymes on an ecological scale for their role in mediating plant-fungal interactions, but their function in fungal pathogenesis has yet to be elucidated. Ascomycete laccases have been primarily associagted with cell wall melanin deposition, and laccase mutants in ascomycete species often demonstrate reduced pigmentation. In this study, a putatively secreted laccase, Sslac2, was characterized from the broad host-range plant pathogen Sclerotinia sclerotiorum, which is largely unpigmented and is not dependent on melanogenesis for plant infection. Of the seven putative laccases in the S. sclerotiorum genome, Sslac2 was the only one found to be highly upregulated during pathogenesis of soybeans and was additionally found to be induced during growth on solid surfaces. Gene knockoues ot Sslac2 demonstrate wide ranging developmental defects, including abolished sclerotial formation, and are functionally non-pathogenic on unwounded tissue. While these mutants demonstrated a clear radial growth defect, enhanced growth was observed in liquid culture, likely due to altered hydrophobicity and thigmotropic responsiveness. Sslac2 mutants were also unable to respond to a host environment, and accordingly unable to differentiate penetration structures, respond appropriately to chemical stress, or produce the key virulence factor oxalic acid. Transmission and scanning electron microscopy of WT and mutant strains show apparent differences in extracellular matrix structure that may explain the inability of the mutant to respond to environmental cues. Targeting Sslac2 using host-induced gene silencing significantly improved resistance to S. sclerotiorum, suggesting that fungal laccases could be a valuable target of disease control. Collectively, we identified a laccase critical to the development and virulence of the broad host-range pathogen S. sclerotiorum and propose a potentially novel role for funal laccases in modulating environmental sensing.

overall_design: To investigate the function of Sslac2, the gene was knocked out in S. sclerotiorum (ΔSslac2-1). We then conducted gene expression profiling analysis using data obtained from RNA-seq of ΔSslac2-1 and the wild-type (1980) strain grown in both defined glucose minimal media (GMM) and GMM with the addition of soybean green stem extract to simulate the presence of plant material.

first: Nathaniel

last: Westrick

first: Eddie

middle: G

last: Dominguez

first: Madeline

middle: E

last: Bondy

first: Christina

last: Hull

first: Damon

last: Smith


title: Overexpression of SLIM1 transcription factor accelerates vegetative development in Arabidopsis thaliana

first: Sarah

last: Whitcomb

address: 502 Walnut St

department: Cereal Crops Research Unit

institute: USDA-ARS

summary: SLIM1 has a well established role in regulating transcriptional responses to sulfur deficiency in Arabidopsis thaliana. In order to investigate the impact of SLIM1 expression under sufficient nutrient conditions, we generated 35S::SLIM1 over-expression lines. SLIM1OX plants were found to have larger rosette area, bolt earlier, and enter developmental senescence earlier than Col-0 and slim1KO (slim1-cr) plants. RNA-seq followed by differential expression analysis was performed on rosette tissue at three timepoints.

overall_design: Gene expression profiling analysis was performed with RNA-seq data from Arabidopsis thaliana rosettes to investigate the function of SLIM1/EIL3 (AT1G73730) in sufficient nutrient conditions. 5 geneotypes were included in the study: Col-0, slim1KO (slim1-cr), pH7WG (EV), and two independent 35S::SLIM1 OX lines. Individual rosettes of each genotype were harvested at three timepoints: 30 days after sowing, 37 days after sowing, and 44 days after sowing.

first: Anastasia

last: Apodiakou

first: Rainer

last: Hoefgen


title: Excitatory Neurons Derived from Human-Induced Pluripotent Stem Cells Show Transcriptomic Differences in Alzheimer’s Patients from Controls

city: Baltimore

first: Dimitrios

last: Avramopoulos

address: 733 North Broadway

department: Genetic Medicine

institute: Johns Hopkins University

summary: The recent advances in creating pluripotent stem cells from somatic cells and differentiating them into a variety of cell types is allowing us to study them without the caveats associated with disease-related changes. We generated induced Pluripotent Stem Cells (iPSCs) and used lentiviral delivery to differentiate them into excitatory glutamatergic neurons, on which we performed RNA sequencing. We  compared transcriptomes of cells derived from 7 Alzheimer’s disease (AD) and 6 control patients. We found that 621 genes show differences in expression levels at adjusted p < 0.05 between the case and control derived neurons. These genes show significant overlap and directional concordance with genes reported from a single-cell transcriptome study of AD patients; they include five genes implicated in AD from genome-wide association studies and they appear to be part of a larger functional network as indicated by an excess of interactions between them observed in the protein–protein interaction database STRING. Exploratory analysis with Uniform Manifold Approximation and Projection (UMAP) suggests distinct clusters of patients, based on gene expression, who may be clinically different. Our research outcomes will enable the precise identification of distinct biological subtypes among individuals with Alzheimer’s disease, facilitating the implementation of tailored precision medicine strategies.

overall_design: cells from 7 Alzheimers patients and 6 controls were used to create hiPSCs and then differentiated to excitatory neurons. RNA was extracted and subjected to RNA sequencing, after which the transcriptomes of patient and control derived cells were compared. Details in our publication Cells 2023, 12(15), 1990; https://doi.org/10.3390/cells12151990

first: R

last: Sagar

first: I

last: Azoidis

first: C

last: Zivko

first: A

last: Xydia

first: E

last: Oh

first: P

last: Rosenberg

last: Lyketsos

first: V

last: Mahairaki

first: D


title: Cooperative super-enhancer inactivation caused by heterozygous loss of CREBBP and KMT2D skews B-cell fate decisions and accelerates development of T-cell depleted lymphomas [Mint-ChIP]

status: Public on Mar 05 2024

city: New York City

first: Christopher

middle: Russell

last: Chin

address: 413 E 69th Street, Belfer Building, BB-1462

institute: Weill Cornell

summary: Mutations in chromatin modifiers are a hallmark of many tumors, especially lymphomas arising from germinal center (GC) B cells. Given that these lymphoma mutations all induce aberrant gene repression, it is surprising that they often co-occur in individual patients. The most common pairing are mutations affecting CREBBP and KMT2D. Both impair enhancer activity in overlapping pathways to facilitate lymphomagenesis, hence their co-occurrence is especially puzzling. Herein, we report that combined haploinsufficiency of CREBBP and KMT2D (C+K) do indeed accelerate lymphomagenesis (vs either allele alone) and confer a more malignant phenotype. Single cell RNA-seq analysis of GC reaction showed that C+K haploinsufficiency induced aberrant GC hyperplasia by altering cell fate decisions, skewing B cells away from memory B and plasma cell differentiation and favored instead expansion of centroblasts. Integrative epigenomic studies in murine and human B cells showed that C+K deficiency particularly impairs enhancer activation for immune synapse genes involved in exiting the GC reaction. This effect was especially severe at super-enhancers for genes governing cell fate decisions induced by T cell help, pointing to a particular dependency for both co-activators at these specialized regulatory elements. Mechanistically, CREBBP and KMT2D formed a complex, were highly co-localized on chromatin, and were required for each-other’s stable recruitment to enhancers. Given the impaired expression of immune synapse genes, it was notable that C+K lymphomas in mice and humans manifested significantly reduced CD8+ T cell abundance. This suggests that deficiency of the two chromatin modifiers cooperatively induced an immune evasive phenotype due to failure to activate key immune synapse super-enhancers, associated with altered immune cell fate decisions. These findings point to the potential need for epigenetic adjuvant therapy to augment reactivity with immunotherapy approaches in patients with C+K deficiency.

overall_design: Mint-ChIP sequencing was performed with 3 replicates of wild-type germinal center (GC) cells using H3K4me1 (Abcam ab8895, RRID:AB_306847) and H3K4me3 (Abcam ab8580, RRID:AB_306649) antibodies following the protocol from the Berstein lab.

first: Jie

last: Li

first: Cem

last: Meydan

first: Martin

last: Rivas

first: Wendy

last: Béguelin

first: Ari

last: Melnick


title: Cooperative super-enhancer inactivation caused by heterozygous loss of CREBBP and KMT2D skews B-cell fate decisions and accelerates development of T-cell depleted lymphomas [WES]

overall_design: Whole exome sequencing for splenic germinal center B cells sorted from lymphoma-bearing mice at day 235 post bone marrow transplant. We sequenced 4 mice replicates for each genotype (i.e., BCL2, BCL2+C, BCL2+K, BCL2+CK)


first: Bomin

last: Ku

overall_design: Refer to individual Series


title: Single cell analysis of CD45+ immune cells from the islets of non-obese diabetic (NOD) mice

city: Fitzroy

first: Balasubramanian

last: Krishnamurthy

state: VIC

address: 41 Victoria Parade

department: Department of Endocrinology and Diabetes

institute: St. Vincent’s Hospital Melbourne

summary: Persistent antigen exposure results in the differentiation of functionally impaired, also termed exhausted, T cells which are maintained by a distinct population of precursors of exhausted T (TPEX) cells. T cell exhaustion is well studied in the context of chronic viral infections and cancer, but it is unclear if and how antigen-driven T cell exhaustion controls progression of autoimmune diabetes and whether this process can be harnessed to prevent diabetes. Using non-obese diabetic (NOD) mice, we show that some CD8+ T cells specific for the islet antigen, islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) displayed terminal exhaustion characteristics within pancreatic islets but were maintained in the TPEX cell state in peripheral lymphoid organs. More IGRP-specific T cells resided in the peripheral lymphoid organs than in islets. To examine the impact of extra-islet antigen exposure on T cell exhaustion in diabetes, we generated transgenic NOD mice with inducible IGRP expression in peripheral antigen presenting cells. Antigen exposure in the extra-islet environment induced severely exhausted IGRP-specific T cells with reduced ability to produce IFNγ, which protected these mice from diabetes. Our data demonstrate that T cell exhaustion induced by delivery of antigen can be harnessed to prevent autoimmune diabetes.

overall_design: Single cell suspensions prepared from dispersed mouse pancreatic islets from non-obese diabetic (NOD) mice were stained with immune cell markers and sorted by flow cytometry based on the pan-immune cell marker, CD45, and processed for sc-RNA seq analysis.

first: David

last: De George


title: MERTK inhibition for the treatment of tissue fibrosis

city: Westmead

first: Ziyan

last: Pan

state: NSW

address: 176 Hawkesbury Rd

department: Storr Liver Centre

institute: Westmead Institute for Medical Resea

summary: This SuperSeries is composed of the SubSeries listed below.


title: SARS-CoV-2 infection causes heightened disease severity and mortality in a mouse model of Down syndrome

status: Public on Mar 04 2024

city: LITTLE ROCK

first: Zijing

last: Zhang

address: 4301 W Markham St

institute: University of Arkansas for Medical Sciences

summary: Recent epidemiological studies suggest that individuals with Down syndrome are more susceptible to SARS-CoV-2 infection and have higher rates of hospitalization and mortality than the general population. However, the main drivers behind these disparate health outcomes remain unknown. Herein we performed experimental infections with SARS-CoV-2 in a well-established mouse model of Down syndrome, Dp(16)1Yey/+;Tg(K18-ACE2)2Prlmn/+ (abbreviated as Dp16;ACE2). We observed similar SARS-CoV-2 replication kinetics and dissemination in the primary and secondary organs between mice with and without Down syndrome (i.e., Dp16;ACE2 and ACE2), suggesting that both groups have similar susceptibilities to SARS-CoV-2 infection. However, Down syndrome mice exhibited more severe disease as defined by clinical features including symptoms, weight loss, pulmonary function, and survival of mice. We found that increased disease severity in Down syndrome mice could not be attributed solely to increased infectivity or a more dramatic pro-inflammatory response to infection. Rather, results from RNA sequencing suggested that differences in expression of genes from other physiological pathways such as deficient oxidative phosphorylation, cardiopulmonary dysfunction and deficient mucociliary clearance in the lungs may also contribute to heightened disease severity and mortality in Down syndrome mice following SARS-CoV-2 infection.

overall_design:
To investigate whether individuals with Down syndrome are more susceptible to SARS-CoV-2 infection and have more severe COVID-19 in a mouse model of Down syndrome, we made a mutant mouse strain (Dp16;ACE2 or DS) by crossing Dp16 mouse, which has Down syndrome features, and ACE2 mouse (WT), which expresses human ACE2 gene and is susceptible to SARS-CoV-2 infection.
We then performed gene expression profiling analysis using data obtained from RNA-seq of the two mouse mutants either mock-infected or infected with SARS-CoV-2 at 2 different time points.

first: Roger

middle: D

last: Pechous

first: Priyangi

last: Malaviarachchi

first: Zhuo

last: Xing

first: Avrium

last: Douglas

first: Samantha

last: Crane

first: Hayley

last: Theriot

first: Alireza

last: Ghaffrieh

first: Lu

last: Huang

first: Yuejijng

last: Yu

first: Xuming


title: MERTK inhibition for the treatment of tissue fibrosis [scMultiome]

summary: Fibrosis is a core pathway that drives the progression of multiple chronic diseases for which there is a paucity of safe and effective treatments. In these diseases, transforming growth factor–β (TGF-β)–driven scarring propels disease progression. However, targeting this ubiquitously expressed cytokine is unlikely to yield a viable and safe antifibrotic therapy; thus, identification of alternative mechanisms to inhibit TGF-β signalling is required. We identified Mer tyrosine kinase (MERTK) as a TGF-β–inducible nodal effector of fibrosis that is up-regulated with fibrosis in multiple organs in both mice and humans. MERTK also induces TGF-β expression and promotes it’s signalling resulting in a positive feedback loop that promotes fibrosis. Mechanistically, MERTK regulates both canonical and non-canonical TGFβ signalling. Further downstream, MERTK modulates the fibrotic regulatory gene transcription network by regulating chromatin accessibility, RNA polymerase II (pol II) pausing and reprograming the enhancer landscape. Using mouse models of kidney, lung, and liver fibrosis, we demonstrate that this fibrosis-promoting signalling loop can be interrupted by loss of MERTK expression, leading to marked attenuation of fibrosis. Pharmacologic MERTK inhibition reduced fibrosis either when initiated immediately after injury or when initiated after fibrosis is established. Together, this data suggests that MERTK plays a critical role in modulating organ fibrosis, while small-molecule MERTK inhibitors are an attractive target for the treatment of diseases characterized by fibrosis.

overall_design: Compare the gene expression and chromatin accessibility between WT and Mertk knockout mice liver samples in the liver fibrosis model using scMultiome.

first: Rasha

last: El Sharkway

first: Ali

last: Bayoumi

first: Mayada

last: Metwally

first: Brian

last: Gloss

first: Robert

last: Brink

last: Lu

first: Christopher

last: Liddle

first: Jun

first: Philip

last: O'Connell

first: Jacob

last: George

first: Mohammed

last: Eslam


title: DNA methylation profiles of lymph node tissue of head and neck cancer of unknown primary

city: Munich

first: Christof

last: Winter

address: Ismaninger Str. 22

institute: Technical University of Munich

summary: Background. The unknown tissue of origin in head and neck cancer of unknown primary (hnCUP) leads to invasive diagnostic procedures and unspecific and potentially inefficient treatment options for patients. The most common histological subtype, squamous cell carcinoma, can stem from various tumor primary sites, including the oral cavity, oropharynx, larynx, head and neck skin, lungs, and esophagus. DNA methylation profiles are highly tissue-specific and have been successfully used to classify tissue origin. We therefore developed a support vector machine (SVM) classifier trained with publicly available DNA methylation profiles of commonly cervically metastasizing squamous cell carcinomas (n = 1,103) in order to identify the primary tissue of origin of our own cohort of squamous cell hnCUP patient’s samples (n = 28). Methylation analysis was performed with Infinium MethylationEPIC v1.0 BeadChip by Illumina.   Results. The SVM algorithm achieved the highest overall accuracy of tested classifiers, with 87%. Squamous cell hnCUP samples on DNA methylation level resembled squamous cell carcinomas commonly metastasizing into cervical lymph nodes. The most frequently predicted cancer localization was the oral cavity in 11 cases (39%), followed by the oropharynx and larynx (both 7, 25%), skin (2, 7%), and esophagus (1, 4%). These frequencies concord with the expected distribution of lymph node metastases in epidemiological studies.   Conclusions. On DNA methylation level, hnCUP is comparable to primary tumor tissue cancer types that commonly metastasize to cervical lymph nodes. Our SVM-based classifier can accurately predict these cancers’ tissues of origin and could significantly reduce the invasiveness of hnCUP diagnostics and enable a more precise therapy after clinical validation.

overall_design: Bisulphite treated genomic DNA from 28 lymph nodes (FFPE) and 28 patients, one lymph node per patient, were hybridised to an Illumina Infinium MethylationEPIC v1.0 Array. One array per sample, no replicates, no control samples.

first: Leo

last: Stark

first: Markus

last: Wirth