title: Transcriptomic profile of recovered exogenous Tregs from injured bone, muscle, and skin of mice that were locally treated with Tregs, as well as heart, MLN and spleen from mice with MI that were systemically treated with Tregs.

summary:
Transcriptomic profile of recovered exogenous Tregs from injured bone, muscle, and skin of mice that were locally treated with Tregs, as well as heart, mediastinal lymph nodes (MLN) and spleen from mice with myocardial infarcts (MI) that were systemically treated with Tregs.

Mice with tissue injuries were treated with exogenous Tregs that were sorted from spleens of Foxp3(IRES-mRFP) mice - C57BL6/J mice with bone, muscle and skin injuries were treated via hydrogel-mediated local delivery of Tregs soon after injury, while mice with myocardial infarcts (MI) were treated with systemic (intravenous) delivery of Tregs one day post-MI. Three days after Treg-delivery, the injured bone, muscle, and skin tissues, as well as heart, mediastinal lymph nodes and spleens from mice with MI were harvested, and the exogenous (delivered) Tregs were recovered by FACS sorting. These sorted exogenous Tregs recovered at day 3 post-Treg delivery (Day 3 recovered Tregs) were then used for mini-bulk RNA sequencing along with spleen Tregs before delivery as a control (Day 0 Spleen Tregs).

overall_design:
Bone (cranial defect), muscle (volumetric muscle loss defect) and skin (full-thickness biopsy punch) injuries were induced in 10 week old male wild-type mice and treated with RFP+ Tregs sorted from Foxp3(IRES-mRFP) mice, locally delivered via a hydrogel directly polymerised at the site of injury. Control mice were treated with hydrogel alone. Heart injury (myocardial infarction, MI) was induced in 10 week old male wild-type mice by left coronary artery ligation. One day post-MI, the mice were administered with either RFP+Tregs, or PBS (control), via intravenous tail vein injection. In the heart injury model, the delivered Tregs homed to the heart, mediastinal lymph nodes and spleens of the mice with infarcts. These exogenous Tregs were recovered from all these 6 tissues of the mice, on day 3 post-Treg delivery, by cell sorting (n=3, each replicate represents one mouse). These sorted Day 3 exogenous Tregs, along with the Tregs before delivery, were used for mini-bulk RNA sequencing.

***Please note that the Series/Sample records have been updated on July 25, 2024****


title: The tumor intrinsic role of the m6A reader YTHDF2 in regulating immune evasion [RNA-seq]

status: Public on May 31 2024

city: Los Angeles

first: Sai

last: Xiao

state: California

address: 1500 E. Duarte Road

country: USA

institute: City of Hope

type: Expression profiling by high throughput sequencing

summary: Tumors evade attacks from the immune system through various mechanisms. Here, we identify a component of tumor immune evasion mediated by YTH domain-containing family protein 2 (YTHDF2), a reader protein that usually destabilizes m6A-modified mRNA. Loss of tumoral YTHDF2 inhibits tumor growth and prolongs survival in immunocompetent tumor models. Mechanistically, tumoral YTHDF2 deficiency promotes the recruitment of macrophages via CX3CL1 and enhances mitochondrial respiration of CD8+ T cells by impairing tumor glycolysis metabolism. Tumoral YTHDF2 deficiency promotes inflammatory macrophage polarization and antigen presentation in the presence of IFN-γ. In addition, IFN-γ induces autophagic degradation of tumoral YTHDF2, thereby sensitizing tumor cells to CD8+ T cell-mediated cytotoxicity. Last, we identified a small molecule compound that preferentially induces YTHDF2 degradation, which shows a potent antitumor effect alone but a better effect when combined with anti-PD-L1 or anti-PD-1 antibodies. Collectively, YTHDF2 appears to be a tumor-intrinsic regulator that orchestrates immune evasion, representing a promising target for enhancing cancer immunotherapy.

overall_design: Total RNA was isolated by TRIzol reagent from fifty million  WT and Ythdf2-KO MC38 tumor cells. m6A MeRIP sequencing was performed to detect the m6A modification alterations in two groups. YTHDF2 RNA immunoprecipitation sequencing was performed to map the target transcripts bound by YTHDF2 in MC38 tumor cells.

first: Jianhua

last: Yu


title: The tumor intrinsic role of the m6A reader YTHDF2 in regulating immune evasion

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

overall_design: Refer to individual Series


title: Transcriptomic profile of monocytes/macrophages from mice with bone, muscle, skin or heart injuries that were treated with systemic delivery of Tregs

city: Clayton

first: Mikaël

last: Martino

state: Victoria

address: 15 Innovation Walk

department: Australian Regenerative Medicine Institute

country: Australia

institute: Monash University

summary: Mice with either a cranial bone defect, skin biopsy punch injury, quadriceps volumetric muscle loss defect or myocardial infarction (MI) were treated with Tregs, either via local hydrogel-mediated delivery (for bone, muscle and skin), or with systemic (intravenous) delivery (for heart). Control mice were treated with hydrogel alone (for bone, muscle and skin) or PBS (for heart). On day 4 and 7 post-MI, the injured tissues were harvested and the endogenous monocytes/macrophages were sorted and used for mini-bulk RNA sequencing, to compare the transcriptomic profiles of injured tissue monocytes/macrophages in Treg-treated and untreated mice post-injury.

overall_design:
Bone (cranial defect), muscle (volumetric muscle loss defect) or skin (full-thickness biopsy punch) injuries were induced in 10 week old male wild-type mice and treated with Tregs locally delivered via a hydrogel directly polymerised at the site of injury. Control mice were treated with hydrogel alone.
Heart injury (myocardial infarction, MI) was induced in 10 week old male wild-type mice by left coronary artery ligation. One day post-MI, the mice were administered with either Tregs or PBS (control) via intravenous tail vein injection.
Only 1 injury per mouse was performed.
On day 4 and 7 post-injury (for all 4 tissues - bone, muscle, skin and heart), the injured tissues from Treg-treated and control mice were harvested, and the monocytes/macrophages accumulating in the injured tissues were sorted by fluorescence activated cell sorting (FACS), then used for mini-bulk RNA sequencing."

***Please note that the Series/Sample records have been updated on July 25, 2024***

first: Bhavana

last: Nayer

first: Yasmin

middle: K

last: Alshoubaki

middle: M


title: Bulk RNA Sequencing of SUM159PT cells grown in 2D or from tumors formed in NOD mice

city: Milano

first: Francesco

last: Nicassio

address: Via Adamello 16

department: Center for genomic science

country: Italy

institute: Istituto Italiano di Tecnologia

summary: Cancer is a heterogeneous disease, where multiple, phenotypically distinct subpopulations co-exist. Tumor evolution is a result of a complex interplay of genetic and epigenetic factors. To predict the molecular drivers of distinct cancer responses, we apply single-cell lineage tracing (scRNA-Seq of barcoded cells) on a triple-negative breast cancer model. We propose GALILEO, a framework providing lineage tracing, transcriptomic, and chromatin accessibility information simultaneously at single-cell resolution (Multiome ATAC + gene expression on barcoded cells). The combination of single-cell lineage tracing with phenotypic assays allows to link a cell state with its fate.

overall_design: SUM159PT cells were cultivated in 2D dishes (N=6) or transplanted (100k cells) in vivo in the mammary fat pad of NOD mice and collected when the tumors (N=5) reached 1.2 cm (with a typical latency of 30 days). Bulk RNA-seq was used to compare the transcriptome of SUM159 derived tumors compared to cells grown in 2D

first: Francesca

last: Nadalin

first: Matteo

last: Marzi

first: Patricio

last: Fuentes

first: Maria

last: Pirra Piscazzi

first: Simone

last: Procaccia

first: Montserrat

last: Climent

first: Paola

last: Bonetti

first: Irene

last: Papatheodorou

first: John

last: Marioni


title: Transcriptional noise, gene activation, and roles of SAGA and Mediator Tail  measured using nucleotide recoding single cell RNA-seq

status: Public on Jun 02 2024

city: Seattle

first: Jeremy

middle: Aspin

last: Schofield

state: Washington

address: 1100 Fairview Ave N

department: Basic Sciences Division

institute: Fred Hutchinson Cancer Center

summary: The populational variance of eukaryotic transcription differs by gene, and can be range from constitutive to bursty in nature. Exemplary bursty yeast genes tend to rely on SAGA and Mediator Tail (coactivator-redundant, CR) for transcription, and often contain TATA boxes in their promoters. To dissect gene-specific transcriptional bursting and the roles of coactivator complexes in regulating bursting in yeast, we performed genome-wide nascent single-cell RNA-seq. Examining the transcriptional variance-mean relationship (Fano factor) for thousands of yeast genes across cell populations, we found that CR-class genes generally display burstier transcription than TFIID-class genes, regardless of expression level. We also found that the cell cycle-dependent activation of yeast histone genes correlates with an increase in Fano. Finally, we assessed the consequences of rapid depletion of SAGA or Mediator Tail coactivator complex subunits. On a per-gene basis, we observed a strong relationship between bulk transcriptional fold change and the change in fraction of yeast cells actively transcribing (Fon), and a weak relationship between bulk transcriptional fold change and the change in mean expression per cell (MPC). We also observe gene-specific variation in Fon, MPC, and Fano following coactivator depletion. These results provide insight into gene-specific transcriptional kinetics and the roles of coactivator complexes in gene activation and transcriptional bursting.

overall_design: Time courses of transcriptional metabolic labeling with 4-thiouracil were performed in S. cerevisiae, followed by in-cell nucleotide recoding (NR) chemistry treatment to induce U-to-C mutations in nascent RNAs. NR-recoded yeast were then subjected to scRNA-seq, and nascent RNAs were identified by their T-to-C mutation content in cDNAs. The functions of SAGA and Mediator Tail were also assessed by performing rapid coactivator depletion (Spt3/7 or Med15) using the auxin-inducible degron approach immediately prior to metabolic labeling, NR chemistry treament and scRNA-seq.

middle: A

first: Steve

last: Hahn


title: Glycolysis inhibition by 2-Deoxy-D-Glucose sustains a distinct cell pluripotency state in human pluripotency stem cells

status: Public on Jun 01 2024

city: macau

first: Chengcheng

last: SONG

address: universtiy of macau

department: fhs

country: Macau

institute: university of macau

summary: To investigate the function glycolysis in the regulation of hPSC pluripotency, we processed glycolysis inhibition (2DG) treamtent in E8 medium for 5 cell passages in H1/H9 ESCs and NL1/NL4 hiPSCs.

overall_design: We then performed gene expression profiling analysis using data obtained from RNA-seq of 2DG treatment for 5 cell passages in H1/H9 ESCs and NL1/NL4 hiPSCs

last: Song

first: Guokai

last: Chen


title: SUM159PT

type: Genome binding/occupancy profiling by high throughput sequencing


title: Immunomodulation by the combination of statin and matrix-bound nanovesicle enhances optic nerve regeneration

status: Public on Jun 05 2024

city: Pittsburgh

first: Takaaki

last: Kuwajima

state: PA

address: 1622 Locus Street, 7.396

department: Ophthalmology

institute: University of Pittsburgh School of Medicine

summary: To identify a novel clinically-relevant therapy, fluvastatin and matrix bound nanovesicles (MBV) were focused in this study. We found that fluvastatin and MBV synergistically enhance optic nerve regeneration and RGC protection after optic nerve injury in mouse. To identify the molecular mechanisms underlying enhancement of RGC protection and axon regeneration after optic nerve injury, bulk RNA sequencing analysis of the whole retina treated with MBV, fluvastatin and the combination of MBV and fluvastatin or PBS as a control two days after optic nerve crush, was performed. Bulk RNA-seq analysis revealed that genes and cellular pathways related to inflammation, were robustly altered.

overall_design: Comparative gene expression profiling analysis of bulk RNA-seq data for the whole retinae treated with PBS (control), fluvastatin, MBV, or a combination of MBV and fluvastatin two days after optic nerve crush in mouse.

first: Gregory

middle: P

last: Campbell

first: Dwarkesh

last: Amin

first: Kristin

last: Hsieh

first: George

middle: S

last: Hussey

first: Anthony

middle: J

last: St. Leger

first: Jeffrey

last: Gross

first: Stephen

middle: F

last: Badylak


title: Transcriptomic profile of endogenous Tregs in mouse injured bone, muscle, skin and heart tissues compared to healthy spleen Tregs

first: Mikaël

last: Martino

summary: We aimed to identify the changes in gene expression profile of Tregs isolated from the injured bone, muscle, skin and heart, compared to healthy (uninjured) spleen Tregs. The bone injury consisted of calvarial bone defects, the muscle injury involved volumetric muscle loss defect in the quadriceps and the skin injury involved full-thickness punch-biopsy wounds. The heart injury was performed using the left coronary artery ligation to induce myocardial infarction (MI). The Tregs accumulating within the injured tissues were isolated and purified by fluorescence activated cell sorting (FACS), at 7 days post-injury (which is the peak of Treg accumulation within these tissues post-injury).

overall_design:
Bone injury (cranial bone defect), muscle injury (quadriceps volumetric muscle loss), skin injury (full-thickness punch-biopsy wounds) or heart injury (myocardial infarction, MI by left coronary artery ligation) was induced in 10 week old male Foxp3(DTR/GFP) mice (1 injury per mouse) and the injured tissue was harvested 7 days after injury to sort Tregs by flow cytometry (n=3, each replicate containing cells pooled from 3 mice). As a control, healthy (uninjured) spleen Tregs from 10 week old male Foxp3(DTR/GFP) mice were also sorted by flow cytomtery (n=6). The sorted cells were then used for RNA extraction and bulk RNA sequencing.

***Please note that the Series annotations have been updated on July 25th, 2024, and additional sample records included on Aug 2, 2024****
***Submitters state that the records will be updated with two additional samples (Bone injury replicate 2, and Bone injury replicate 3)****

first: Bhavana

last: Nayer

first: Yasmin

last: Alshoubaki


title: RNA seq gene expression profile of SUM159PT cells sorted for TM4SF1 (high vs bulk).

first: Francesco

last: Nicassio

summary: Cancer is a heterogeneous disease, where multiple, phenotypically distinct subpopulations co-exist. Tumour evolution is a result of a complex interplay of genetic and epigenetic factors. To predict the molecular drivers of distinct cancer responses, we apply single-cell lineage tracing (scRNA-Seq of barcoded cells) on a triple-negative breast cancer model. We propose GALILEO, a framework providing lineage tracing, transcriptomic, and chromatin accessibility information simultaneously at single-cell resolution (Multiome ATAC + gene expression on barcoded cells). The combination of single-cell lineage tracing with phenotypic assays allows to link a cell state with its fate.

overall_design: SUM159PT cells were sorted according to the surface marker TM4SF1 (high or bulk) and then immediately processed by RNA-seq or cultivated for the indicated number of passages (P4, P19) and processed by RNA-seq.

first: Francesca

last: Nadalin

first: Matteo

last: Marzi

first: Patricio

last: Fuentes

first: Maria

last: Pirra Piscazzi

first: Simone

last: Procaccia

first: Montserrat

last: Climent

first: Paola

last: Bonetti

first: Irene

last: Papatheodorou

last: Marioni


title: Targeting PRMT3 Impairs Methylation and Oligomerization of HSP60 to Boost Anti-Tumor Immunity by Activating cGAS/STING Signaling

status: Public on May 29 2024

city: Guangzhou

first: Yunxing

last: Shi

address: Erheng Road, Member Village, Tianhe District

country: China

institute: sun-yat sen university

summary: Hepatocellular carcinoma (HCC) is a highly lethal cancer for which current available treatment options have limited efficacy. Immunotherapy has emerged as a promising therapeutic option for HCC, yet resistance to immunotherapy is a major challenge. Here, we uncover protein arginine methyltransferase 3 (PRMT3) as a novel driver of immunotherapy resistance in HCC. We show that PRMT3 expression is induced by activated T cells in response to immune checkpoint blockade (ICB) via an interferon-gamma (IFN)STAT1 signaling pathway, and that high PRMT3 expression inversely correlates with tumor-infiltrating CD8+ T cells and predicts poor response to ICB in HCC patients. We demonstrate that genetic depletion and pharmacological inhibition of PRMT3 induce a profound influx of T cells into tumors and activate anti-tumor immunity to suppress HCC progression. Mechanistically, we demonstrate that PRMT3 methylates HSP60, a mitochondrial chaperone protein, at R446, and that this novel post-translational modification is required for HSP60 oligomerization and maintaining mitochondrial homeostasis. We reveal that targeting PRMT3-dependent HSP60-R446 methylation boosts anti-tumor immunity by disrupting mitochondrial function, increasing mitochondrial DNA (mtDNA) leakage, and activating the cGAS/STING pathway, a key innate immune sensor of cytosolic DNA. Importantly, we provide genetic and pharmacologic evidence that targeting PRMT3 enhances anti-PD1 efficacy and anti-tumor immunity in HCC mouse models. Our study identifies PRMT3 as a potential biomarker and therapeutic target to overcome immunotherapy resistance in HCC.

overall_design: We sorted CD45+ cells from Prmt3-KO and Prmt3-WT Hepa1-6 subcutaneous (subQ) tumors, which were subjected to scRNA-seq using the 10X Genomics platform.

first: Zongfeng

last: Wu

first: Shaoru

last: Liu


title: Citrullination modulation maintains HIF-1A stabilization to drive tumor progression

status: Public on May 28 2024

city: guangzhou

first: shengqi

last: shen

address: zhongshan road #104

institute: GDPH

summary:
HIF-1 (Hypoxia-inducible factor 1) is the master regulator responding to hypoxic conditions. Here, we found that HIF-1A is citrullinated by peptidyl arginine deiminase 4 (PADI4) at arginine 698, promoting HIF-1A stabilization and thus HIF-1-driven tumor growth. The knockdown of PADI4 could dramatically decrease HIF-1 protein expression without affecting the mRNA level, and this could be rescued by the proteasome inhibitor MG132 treatment under hypoxia. And PADI4-HIF-1A interaction is critical for HIF-1A stability and tumor progression, depending on the enzymatic activity of PADI4 and the pocket structure of PADI4.  DHE, an FDA-approved agent for the treatment of migraine, was found serve as a potential antitumor agent throgh disrupting PADI4-HIF-1A interaction and suppressing HIF-1A stability. Taken together, we found the anti-tumor effect of DHE due to its effect on blocking PADI4-HIF-1A interaction and downregulating HIF-1A pathway in cancer cells.
To figure out the influence of DHE on liver cancer cell under hypoxia, we treated Hep3B cell lines with two does DHE in 6 hours under Hypoxia and extracted mRNA for furthur RNA seq.

first: Rui

last: Chen

first: Zhiyuan

last: Lin

first: Shengqi

last: Shen


title: Targeted DNA sequencing of barcoded SUM159PT cells

last: Nicassio

overall_design: Analysis of barcoded SUM159PT by targeted DNA-seq. Samples were collected before or after treatment with Paclitaxel (PTX), either in vitro or in vivo (transplantation into the mammary fat pad). Reference samples are called "D0". Pre-treated = cells received a first round of paclitaxel treatment before being barcoded and treated for a second round.

first: John


title: Expression changes in respiratory epithelial cells when treated with TGF-β1

city: Ilsandong-gu, Goyang-si

first: Dong-Hee

last: Han

state: Gyeonggi-do

address: 32, Dongguk-ro

department: Life science

country: South Korea

institute: Dongguk University

summary: TGF-β1 is a key regulator of diverse biological processes. It can be overexpressed by noxious stimuli, and activation of downstream signals leads to excessive accumulation of extracellular matrix. Therefore, in order to study diseases related to these symptoms, we sought to compare changes in expression of genes in cells. Our study investigated differentially expressed genes in two types of respiratory cells following TGF-β1 exposure and identified the molecular mechanism through biological analysis.

overall_design: To investigate changes in expression of two respiratory epithelial cells according to TGF-β1 exposure, A549 and BEAS-2B were treated with 1 and 5 ng/mL of TGF-β1.

first: Min

last: Kim


title: Uracil phosphoribosyltransferase is required to establish a functional cytochrome b6/f complex

city: Kaiserslautern

first: Torsten

last: Möhlmann

address: Paul-Ehrlich-Str

department: Biology

country: Germany

institute: University of Kaiserslautern

summary: Arabidopsis uracil phosphoribosyltransferase (UPP) is an essential enzyme and plants lacking this enzyme are strongly compromised in chloroplast function. Our analysis of UPP amiRNA mutants has confirmed that this vital function is crucial to establish a fully functional photosynthesis as the RIESKE iron sulfur protein (PetC) is almost absent, leading to a block in photosynthetic electron transport. Interestingly, this function appears to be unrelated to nucleotide homeostasis since nucleotide levels were not altered in the studied mutants. Transcriptomics and proteomic analysis showed that protein homeostasis but not gene expression is most likely responsible for this observation and high light provoked an upregulation of protease levels, including thylakoid filamentation temperature-sensitive 1, 5 (FtsH), caseinolytic protease proteolytic subunit 1 (ClpP1), and processing peptidases, as well as components of the chloroplast protein import machinery in UPP amiRNA lines. Strongly reduced PetC amounts were not only detected by immunoblot from mature plants but in addition in a de-etiolation experiment with young seedlings and are causing reduced high light-induced non-photochemical quenching Φ(NPQ) but increased unregulated energy dissipation Φ(NO). This impaired photosynthesis results in an inability to induce flavonoid biosynthesis. In addition, the levels of the osmoprotectants raffinose, proline, and fumarate were found to be reduced. In sum, our work suggests that UPP assists in stabilization PetC during import, processing or targeting to the thylakoid membrane, or protects it against proteolytic degradation.

overall_design: To analyze the effect of UPP in the establishment of photosynthesis, Col-0 (wild-type control) plants were compared to ami-UPP knock down (R4) plants. Plants were grown on soil at 120 µmol photons m-2 s-1 (ML), and samples were harvested either after 3 hours of light (ML, control) or after three hours of high light (HL) at 1000μmol in three biological replicates.

first: Scherer

last: Vanessa

first: Bellin

last: Leo

first: Möhlmann

last: Torsten


title: Strain engineering for de novo synthesis of sedoheptulose from glucose

city: Tianjin

first: Jianggang

last: Yang

address: 32 West 7th Avenue, Tianjin Airport Economic Area, Tianjin 300308, China

institute: Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences

summary: We propose a strain engineering approach for synthesizing sedoheptulose using glucose as sole feedstock. The gene pfkA encoding 6-phosphofructokinase in Corynebacterium glutamicum was inactivated to direct the carbon flux towards pentose phosphate pathway in the cellular metabolic network. This genetic modification successfully enabled the synthesis of sedoheptulose from glucose. Additionally, we identified key enzymes responsible for product formation through transcriptome analysis, and their corresponding genes were overexpressed, resulting in a further 20% increase in sedoheptulose production.

overall_design: mRNA profiles of cells of  Corynebacterium glutamicum Wildtpye and  ΔpfkA mutant were analysized.

first: Jiangang


title: Transcriptomic analysis of HIV-associated nephropathy (Tg26) mouse kidney

status: Public on May 27 2024

city: Bethesda

first: Teruhiko

last: Yoshida

state: Maryland

address: 10 Center Drive, 9N208

department: NIDDK - KDB

institute: National Institutes of Health


title: Microglia are not necessary for maintenance of blood-brain barrier properties in health, but PLX5622 alters brain endothelial cholesterol metabolism (Liver Seq)

status: Public on May 22 2024

city: La Jolla

first: Caterina

last: Profaci

address: 9500 Gilman Drive

institute: University of California, San Diego

summary: Microglia are resident immune cells of the central nervous system, yet their functions far exceed those related to immunology. From pruning neural synapses during development to preventing excessive neural activity throughout life, microglia are intimately involved in the brain’s most basic processes. Studies have reported a close interaction between microglia and endothelial cells, as well as both helpful and harmful roles for microglia at the blood-brain barrier (BBB) in the context of disease. However, much less work has been done to understand microglia-endothelial cell interactions in the healthy brain. Here, we aim to determine the role of microglia at the healthy BBB. We used the colony-stimulating factor 1 receptor (CSF1R) inhibitor PLX5622 to deplete microglia and analyzed BBB ultrastructure, permeability, and transcriptome. Interestingly, we found that, despite their direct contact with endothelial cells, microglia are not necessary for maintenance of BBB structure, function, or gene expression in the healthy brain. However, we found that PLX5622 treatment alters brain endothelial cholesterol metabolism, and this effect was independent from microglial depletion, suggesting PLX5622 has off-target effects on brain vasculature.

overall_design: We aimed to determine whether PLX5622 affects cholesterol metabolism in peripheral endothelial cells (in addition to central nervous system endothelial cells). We put mice on control or PLX5622 diet for one month and then acutely isolated liver endothelial cells for RNA sequencing.

first: Sean

last: Harvey

first: Richard

last: Daneman


title: Microglia are not necessary for maintenance of blood-brain barrier properties in health, but PLX5622 alters brain endothelial cholesterol metabolism (Brain Endo Bulk Seq)

overall_design: To determine how microglial depletion affects brain vasculature, we put C57BL6 mice on PLX5622 diet or control diet for one month. PLX5622 is a CSF1R inhibitor that eliminates ~95% of microglia. We then acutely isolated brain endothelial cells for gene expression analysis.


title: Whole blood transcriptome in long-COVID patients reveals association with lung function and immune response

city: Amsterdam

first: Jelle

middle: Maarten

last: Blankestijn

address: meibergdreef 9

country: Netherlands

institute: Amsterdam UMC

summary: It is estimated that 10% of COVID survivor still experience from complaints months after infection. Here we collected blood from 95 patients in the P4O2 COVID-19 study and performed RNA-seq at 3-6 months and 12-15 after infection. Our aim was to discover biomarkers related to long COVID and perform unsupervised clustering to potentially reveal pathological underlying mechanism for long COVID

overall_design: Blood was collected and sequenced at 3-6 months and 12-15 months after infection in this observational study. No experimental treatments were performed. Additional clinical variables can be requested through the P4O2 data committee (via p4o2@amsterdamumc.nl). For more info see https://pmc.ncbi.nlm.nih.gov/articles/PMC10381397/#notes4


title: Enhanced Phase Separation Mediated Oncogenicity

status: Public on May 24 2024

city: hangzhou

first: kuang

last: junqi

address: No.18 Shilongshan Road

institute: westlake University


title: Microglia are not necessary for maintenance of blood-brain barrier properties in health, but PLX5622 alters brain endothelial cholesterol metabolism (Astro Seq)

overall_design: To determine whether PLX5622 alters cholesterol metabolism in astrocytes (the main cholesterol-producing cells in the adult brain), we put GFAP-Cre; RiboTag mice on PLX5622 diet or control diet for one month. We then attempted to isolate astrocytes using RiboTag methods. Ultimately, the sequenced population also included some neurons and oligodendrocytes, the other two cell types in the brain that produce cholesterol. Thus, we were able to assess whether PLX5622 alters cholesterol metabolism across multiple neuroglial cell types.


title: Microglia are not necessary for maintenance of blood-brain barrier properties in health, but PLX5622 alters brain endothelial cholesterol metabolism (Brain Endo SC Seq)

overall_design: To determine how microglial depletion affects brain vasculature, we put C57BL6 mice on PLX5622 diet or control diet for one month. PLX5622 is a CSF1R inhibitor that eliminates ~95% of microglia. We then acutely isolated brain endothelial cells for single-cell RNA sequencing


title: An age specific platelet differentiation path from hematopoietic stem cells causes thrombocytosis and exacerbated thrombosis

status: Public on May 21 2024

city: Santa Cruz

first: Camilla

last: Forsberg

address: 1156 High Street

institute: Institute for the Biology of Stem Cells, University of California Santa Cruz

summary: Here we reveal a direct differentiation pathway from hematopoietic stem cells into platelets that is unique to aging. We used single cell RNA sequencing (scRNA-seq) to analyze age specific platelet pathway at the clonal level.

overall_design:
Cell preparation and sorting: Cells were harvested and c-Kit-enriched from the long bones and hips of the hind legs of young and old mice as described above. Cc-Kit-enriched cells were then stained for lineage markers via a purified rat-anti-mouse antibody cocktail (CD3, CD4, CD5, CD8, CD11b, B220, Gr-1, and Ter119) on ice for 30 minutes. Cells were washed three times followed by incubation with goat-anti-rat Alexa Fluor 680 secondary on ice in the dark for 20 minutes. Cells were washed three times then resuspended in blocking solution (10% bovine calf serum (BCS), 10% normal mouse serum (Invitrogen), and 10% normal rat serum (Invitrogen) in 1X PBS (Cytiva). Cells were blocked for 10 minutes on ice in the dark. Following block, directly conjugated antibodies were directly added to the cells (CD117/cKit and CD150) and incubated on ice in the dark for 45 minutes, gently mixing the cells at the 30-minute mark. A small aliquot of cells was also used to perform a CD150 fluorescence minus one (FMO) control. Cells were washed three times, resuspended in 10% BCS in 1X PBS (wash buffer), with three drops of propidium iodide (diluted 1:5000 in wash buffer) added. Live, lineage-c-Kit+CD150- and live, lineage-c-Kit+CD150+ young and old cells were sorted on a BD FACSAria IIu (BD Biosciences) (Figure S6). The CD150- and CD150+ gates were touching to ensure a continuum of cells across CD150 expression were sorted. Cells were sorted into chilled 1X PBS (without Mg2+) containing 20% BCS and checked for sort purity. Following sorting, cells were washed and each of the four sorted populations (young CD150-, young CD150+, old CD150-, and old CD150+) were split into three aliquots for Cell Multiplexing Oligo (CMO) labeling (10X Genomics) following manufacturer’s instructions, with each individual group of cells receiving a single unique CMO tag. Cells were washed three times with wash buffer and manually counted via a hemocytometer. When possible, equal numbers of cells from each of the 12 CMO labeled aliquots were combined to artificially inflate the numbers of rare CD150+ cells and to equalize numbers between young and old mice. The pooled cells were then split into two replicates for further processing.
Sample sequencing and preparation: Both pooled replicates were used in parallel in the Chromium Next GEM single Cell 3’ v3.1 protocol (10X Genomics) following manufacturer’s directions. GEM generation and barcoding was performed immediately post-sorting, with resulting samples stored at -20ºC per manufacturer’s instructions. All other steps were performed simultaneously for all samples from each of the two independent experiments. Following library construction, samples were assessed via an Agilent Bioanalyzer with a High Sensitivity chip (Agilent Technologies). Final sample libraries were sequenced on a Novaseq P100 at the University of California Davis DNA Technologies and Expression Analysis Core, targeting 50,000 read pairs per cell for the gene expression libraries and 5,000 reads per cell for the CMO libraries.
Data analysis – quality control and data processing. Sequencing data was initially processed via the 10X Genomics Cell Ranger Cloud pipeline, using the mouse genome (mm10) modified to allow for detection of reporter transgene RNA expression. For the downstream analysis, cells with expression of fewer than 200 genes were excluded, and genes detected in fewer than three cells were removed from subsequent analysis steps. Mitochondrial and ribosomal gene transcripts, as well as Malat1 transcripts, were removed to reduce technical bias. This resulted in a dataset of 22,790 cells and 22,870 genes. Additionally, 13 cells with absent Kit RNA were filtered out. Doublets identified by DoubletDetection 2.4.0 (p_thresh=1e-16, voter_thresh=0.5) removed 157 cells in total (Gayoso et al. 2018).  The UMI counts for each cell were normalized by the total counts across all genes (target_sum=1e4), and log-transformed with an added pseudo count of 1.

first: Donna

last: Poscablo

first: Paloma

last: Medina


title: A systematic screen identifies Saf5 as a link between splicing and transcription in fission yeast

status: Public on May 20 2024

city: Barcelona

first: Montserrat

last: Vega

state: Barcelona

address: C/ Dr. Aiguader 88, lab 383

department: MELIS

country: Spain

institute: Universitat Pompeu Fabra

summary: Splicing is an important step of gene expression regulation in eukaryotes, as there are many mRNA precursors that can be alternatively spliced in different tissues, at different cell cycle phases or under different external stimuli. We have developed several life constructs that allow the quantification of fission yeast splicing in vivo on intact cells, and we have compared their splicing efficiency in a wild type strain and in a prp2-1 (U2AF65) genetic background, showing a clear dependency between Prp2 and a consensus signal at 5’ splicing site (5’SS). To isolate novel genes involved in regulated splicing, we have crossed the reporter containing the weakest splicing rate with the Schizosaccharomyces pombe knock out collection.  Among the candidate genes involved in the regulation of splicing, we have detected strong splicing defects in two of the mutants (Δcwf12 and Δsaf5), both of them related to the NineTeen Complex (NTC). We have identified that strains with mutations in cwf12 have inefficient splicing, mainly when the 5’SS differs from the consensus. However, although Δsaf5 cells also have some dependency on 5’SS sequence, we noticed that when one intron of a given pre-mRNA was affected, the rest of the introns of the same pre-mRNA had high probabilities of being also affected. This observation points Saf5 as a link between transcription rate and splicing.

overall_design: Gene expression profiling using RNA-seq obtained from a wild-type, and saf5 and cwf12 deleted strains

first: Borao

last: Sonia

first: Vega

last: Montserrat

first: Boronat

last: Susanna

first: Hidalgo

last: Elena

first: Hummer

last: Stefan

first: Ayte

last: Jose


title: Regulation of germline proteostasis by insulin/IGF-1 signaling

city: Valhalla

first: Jian

last: Li

state: NY

address: 15 Dana Road

institute: New York Medical College

summary: Gametogenesis involves active protein synthesis and heavily relies on proteostasis. How animals regulate germline proteostasis at the organismal level is poorly understood. Our recent work in C. elegans indicates that germline development requires coordinated activities between insulin/IGF-1 signaling and HSF-1, the transcriptional activator of many molecular chaperones in stress and physiological conditions. In this study, we show that HSF-1 is important for germline proteostasis at ambient temperature. Depletion of HSF-1 from germ cells impairs chaperone gene expression, causing protein degradation and aggregation and, consequently, declines in fecundity and gamete quality. Reduced insulin/IGF-1 signaling confers germ cells' tolerance to limited protein folding capacity and proteotoxic stress by lowering ribosome biogenesis and translation. Interestingly, regulation of germline proteostasis by insulin/IGF-1 signaling occurs non-cell-autonomously. Our data suggest that insulin/IGF-1 signaling controls the expression of the evolutionarily conserved intestinal peptide transporter PEPT-1 via its downstream transcription factor FOXO/DAF-16, therefore allowing dietary proteins to be incorporated into an amino acid pool that fuels ribosomal biogenesis and translation in the germline. We propose that this pathway plays a critical role in regulating germline protein synthesis, which must be at balance with HSF-1-dependent protein folding to achieve proteostasis in gametogenesis.

overall_design: To understand the role of IIS in regulating germline proteostasis, we performed two series of transcriptomic analysis using a reduction-of-function mutant of IGF-1R/DAF-2. First, we depleted HSF-1 from the germline in the daf-2(e1370) mutant worms either starting from egg-lay for 88 hours or starting from the young adult stage for 16 hours till the worms grow to gravid adults. These experiments were to check if there are chaperone expression change and proteotoxic stress responses associated with loss of HSF-1. Auxin-inducible degron system was used for HSF-1 depletion upon auxin treatment. Mock-treatment with ethanol (EtOH) was used as the negative control.  Second, we isolated and enriched germline nuclei from both the wild-type control and the daf-2(e1370) mutant at the young adult stage, and examine the expression of proteostasis network players in the germline upon reduction of IIS.


title: RDD reveals the landscape of RNA-associated chromatin DNA-DNA interactome and gene regulation

city: Shenzhen

first: Tian

last: (Simon) Zhongyuan

state: Guangdong

address: 1088 Xueyuan Avenue

department: School of Life Sciences

institute: Southern University of Science and Technology (SUSTech)

summary: Although non-coding RNAs (ncRNAs) have been proposed to play important roles in nuclear organization with three-dimensional (3D) conformation and gene transcription regulation, exploring the targeting and diffusion patterns of ncRNAs on chromatin has been challenging because it requires a technique that can simultaneously detect high-affinity sites and their higher-order contacts in 3D structures. Here we developed a new method, RNA-associated chromatin DNA-DNA interactions (RDD) in solution, which enables genome-wide detection of genomic binding sites and high-resolution chromatin interactions for a specific ncRNA. Using RDD to investigate the well-known ncRNA roX2 that involved in Drosophila dosage compensation, we not only recapitulate roX2 targeting chromatin sites but also reveal the landscape of chromatin DNA-DNA contacts at various resolutions from loops, domains to territories. We further show that roX2 anchors at the target gene TES and spreads around as a 'boot-like' structure for maintaining gene activity, where tethered daisy-chain chromatin loops, bridging to TSS to dock RNAPII-related contact hubs to achieve a precise twofold expression of male X-linked genes, further crossing active domains to form distinct hubs to exhibit the condensed properties of roX2 in high-throughput sequencing. These results provide a genome-wide 3D model for explaining the mechanism of ncRNA roX2 involved dosage compensation in Drosophila. Together, we demonstrate the high specificity and robustness of the RDD method to capture RNA-associated chromatin DNA-DNA interactome, which provides topological insights into the mechanism of gene transcription regulation by ncRNAs.

first: Simon Zhongyuan

last: Tian

first: Meizhen

last: Zheng


title: Analysis of Gene Expression in the Recovered Mouse Brain following Zika Virus Infection

city: Seoul

first: Nahyun

last: Kim

address: 120 Neungdong-ro, Gwangjin-gu, Seoul

institute: Konkuk university

summary: Zika virus (ZIKV) is a member of the Flaviviridae family, transmitted by Aedes mosquitoes. ZIKV can be transmitted from pregnant woman to fetus and causes microcephaly and neuronal defects. Patients infected with ZIKV showed increased levels of proinflammatory cytokines compared to healthy individuals and neurological changes even after recovery. However, the detailed mechanism of how neuronal defects occurs after recovery from ZIKV infection has never been investigated in vivo. Here, we found that viral RNA loads were highest in mouse brain tissue 7 days after ZIKV infection and were equivalent to uninfected controls after 21 days, suggesting that mice recovered after 21 days of ZIKV infection. To explore the pathological mechanisms of neuronal defects after recovery from ZIKV infection, we performed RNA-sequencing in mouse cerebral cortex obtained 7 days and 21 days after ZIKV infection.

overall_design: Gene expression profiling analysis of RNA-seq data from ZIKV-infected mouse brains at 7 and 21 days post-infection


title: Integrated analyses highlight interactions between the 3D-genome and DNA, RNA, and epigenomic alterations in metastatic prostate cancer

status: Public on May 16 2024

city: San Fransisco

first: Felix

last: Feng

state: CA

address: 1450 3rd Street

institute: University of California

summary: Advances in next generation sequencing have expanded our understanding of the molecular drivers of prostate cancer progression from curable androgen-sensitive disease to metastatic castration-resistant prostate cancer (mCRPC). The focus to date has been on the linear genome, but the impact of variations in the 3D structure of the genome on tumor development and progression has been increasingly recognized. However, studies of human cancer tissues, and especially solid tumors, are extremely limited. Herein, we performed the first integrated low-input deep Hi-C sequencing with matched whole-genome sequencing, whole-genome bisulfite sequencing, 5hmC-seq, and RNA-seq across a cohort of 80 mCRPC biopsy samples. We identify dramatic differences in gene expression, 5mC and 5hmC methylation, and mutation rate between A/B (open/closed) chromatin compartments, as well as a novel association between structural variation and A compartments. By probing the 3D genome, we identify samples with depleted regional chromatin contacts at the Androgen Receptor locus linked to extra-chromosomal circular DNA (ecDNA) and worse response to Androgen Signaling Inhibitors (ASIs). We also identify two never-before described topological subtypes with broad vs. narrow Topologically Associated Domain (TAD) structures, which are associated with stark differences in methylation structure, gene expression, and prognosis. Structural Variants (SVs) can be identified using Hi-C, but in the first such observation in clinical tumor samples, we demonstrate that DNA interactions may contribute and predispose to SV formation, exemplified by the recurrent TMPRSS2-ERG fusion. This large and comprehensive effort provides unprecedented insights into how the 3D genome interacts with the genomic and epigenomic drivers of cancer.

overall_design:
Patient biopsies with pre-existing multi-omic profiling were sequenced using HiC-seq to determine the relationship of the 3D genome with classical genomic landscape measures, within metastatic prostate cancer
--------------------------------
Authors state "Raw data files have been uploaded to EGA. Sample-specific accession IDs are included in the metadata table."


title: Single-cell RNAseq of mouse ventral prostate cells in steroid hormone imbalance

status: Public on May 15 2024

city: Norfolk

first: Petra

last: Popovics

state: Virginia

address: 651 Colley Avenue Room 422

institute: Eastern Virginia Medical School

summary: We used scRNA-seq to study prostate cellular heterogeneity in mice impanted with testosterone and estradiol pellets.

overall_design: Mice were implanted with hormone pellets and the ventral prostates were harvested two weeks later for cell dissociation.


title: Estradiol-dependent 3D chromatin structure changes in MCF7 cells assessed by Capture-C, covering GREB1 and NRIP1 gene and enhancer regions

status: Public on May 13 2024

city: Edinburgh

first: Luciana

middle: Ines

last: Gomez Acuna

address: Crewe Road

country: United Kingdom

institute: MRC-University of Edinburgh

summary: Estrogen-receptor alpha (ERα)-dependent enhancers in the human breast cancer cell line MCF7 are a well-studied model system that allows high temporal resolution tracking of the different molecular events after hormone treatment and in which the enhancer and their target genes, as well as many of the molecular mechanisms operating in enhancer activation, are well defined. We use Capture-C to look at enhancer-promoter contact frequencies of GREB1 and NRIP1 genes across a time course (5, 30 and 60 min) after E2 addition to MCF7 cells which had been extensively starved of hormone. Additionally, ee look at the E2-dependent enhancer-promoter contact frequencies at these two loci in different contexts: in cells where the enhancer regions have been deleted; in cells treated with tamoxifen as well as with estradiol to assess the effect of different ERα ligands; and in cells pre-treated with the transcription inhibitors Flavopiridol and Triptolide to assess the role of active transcription.

overall_design:
Capture-C of MCF7 cells, hormone starved for 4 days and treated with vehicle or E2 for 5, 30 or 60 minutes. Two biological replicates were done.
Capture-C of  WT, GREB1 enhancer deleted or NRIP1 enhancer deleted MCF7 cells, hormone starved for 4 days and treated with vehicle or E2 for 30 min. Two clones per deletion were analyzed; two biological replicates were done.
Capture-C of MCF7 cells, hormone starved for 4 days and treated with vehicle, E2 or Tamoxifen 30 minutes. Two biological replicates were done.
Capture-C of MCF7 cells, hormone starved for 4 days and treated with DMSO, Flavopiridol or Triptolide for 5 min and then with Vehicle or E2 for 30 additional minutes. Two biological replicates were done.

middle: I

last: Gomez Acuña

first: Wendy

last: Bickmore


title: Effect of Wolbachia (wMel strain) on gene expression in Dirofilaria immitis infected-Aedes aegypti

city: Toyoake

first: Takahiro

last: Shirozu

state: Aichi

address: 1-98 Dengakugakubo, Kutsukake-cho

country: Japan

institute: Fujita Health University

summary: Wolbachia pipientis is an intracellular symbiotic bacterium found in insects and arthropods. Wolbachia can decrease the vectorial capacity for various pathogens, such as the dengue virus, in Aedes aegypti. The purpose of this study was to determine the effect of Wolbachia (wMel strain) on the vectorial capacity of Ae. aegypti for Dirofilaria immitis. We analyzed gene expression patterns by RNA-seq in addition to the D. immitis infection phenotype in Ae. aegypti infected with and without wMel. Four Ae. aegypti strains, MGYP2.tet, MGYP2, Liverpol (LVP)-Obihiro (OB), and LVP-OB-wMel (OB-wMel) were analyzed for transcriptome comparison in Malpighian tubule at 2 days post infection. The correlation between Wolbachia infection, D. immitis infection phenotype and immune-related genes expression in Ae. aegypti was investigated.

overall_design: MGYP2 were artificially infected with Wolbachia wMel. MGYP2.tet was maintained free of wMel infection by tetracycline treatment. OB is an original D. immitis-susceptible strain in our laboratory. OB-wMel was established by eight repeated backcrossing female progenies of OB males and MGYP2 females with OB males to obtain a D. immitis-susceptible strain infected with wMel. The differentially expressed genes between MGYP2.tet and MGYP2, OB and OB-wMel, MGYP2.tet and OB, and MGYP2 and OB-wMel were analyzed to determine the effect of Wolbachia and strain differences on gene expression in Malpighian tubules of Ae. aegypti at 2 days post infection of D. immitis.

first: Shinya

last: Fukumoto


title: Rapid enhancer-dependent gene activation in MCF7 cells in response to estradiol.

summary: Estrogen-receptor alpha (ERα)-dependent enhancers in the human breast cancer cell line MCF-7 are a well-studied model system that allows high temporal resolution tracking of the different molecular events after hormone treatment and in which the enhancer and their target genes, as well as many of the molecular mechanisms operating in enhancer activation, are well defined. We use nascent RNA-seq (TT-seq) across a time course (5, 30 and 60 min) after E2 addition to MCF-7 cells which had been extensively starved of hormone to assess the transcription dynamics of E2-responsive genes and enhancers and to link this with other aspects of rapid gene activation under study, i.e. enhancer-promoter communication.

overall_design: TT-seq of MCF7 cells, hormone starved for 4 days and treated with vehicle or E2 for 5, 30 or 60 minutes and a 4Su pulse of 5 minutes. Two biological replicates per time point  were done.

last: Acuña


title: Expansion of profibrotic monocyte-derived alveolar macrophages in patients with persistent respiratory symptoms and radiographic abnormalities after COVID-19 [PASC]

status: Public on May 12 2024

city: Chicago

first: Alexander

middle: V.

last: Misharin

state: IL

address: 303 E Superior

institute: Northwestern University

summary: As many as 10–30% of the nearly 750 million survivors of COVID-19 develop persistent symptoms that define the post-acute sequelae of COVID-19 (PASC) syndrome. To understand the molecular basis of this syndrome, we combined a machine learning based analysis of lung computed tomography (CT) with flow cytometry and single cell RNA-sequencing analysis of bronchoalveolar lavage fluid and nasal curettage samples in a cohort of thirty-five patients with respiratory symptoms and radiographic abnormalities more than 90 days after infection with COVID-19. CT images from patients with PASC revealed primarily fibrotic abnormalities involving 73 +/- 28% of the lung, most of which improved on subsequent imaging. The presence of profibrotic monocyte-derived alveolar macrophages in BAL fluid was significantly associated with increased levels of alveolar cytokines and fibrotic abnormalities on CT. Persistent infection with SARS-CoV-2 was identified in 6 patients and secondary bacterial or viral infections in two others. These findings suggest persistent alveolar inflammation characterized by the ongoing recruitment of monocyte derived alveolar macrophages is associated with fibrotic CT changes in some COVID-19 survivors with implications for diagnosis and therapy.

overall_design: Bronchoalveolar lavage (BAL) fluid from 25 patients with respiratory PASC was obtained at Northwestern Medicine Comprehensive COVID-19 Center. BAL fluid was flow-sorted to enrich for live CD45+CD15- cells and single cell RNA-seq libraries were prepared with 10x 5' gene expression kit. Nasal epithelial samples were collected from 5 patients with respiratory PASC was obtained at Northwestern Medicine Comprehensive COVID-19 Center. Briefly, patients were seated and asked to extend their neck. A nasal curette (Rhino-Pro; VWR) was inserted into either nare and gently slid in the direction of posterior to anterior ~1 cm along the lateral inferior turbinate. Five curettes were obtained per participant. The curette tip was then cut and placed in 2 ml hypothermosol and stored at 4 C until processing. A single-cell suspension was generated using the cold-active dispase protocol reported by Deprez et al. AJRCCM, 2020 with slight modification. Specifically, ethylenediaminetetraacetic acid (EDTA) was omitted and cells were dispersed by pipetting 20 times every 5 min using a 1 ml tip instead of tritration using a 21/23 G needle. The final concentration of protease from Bacillus licheniformis was 10 mg ml−1. The total digestion time was 30 min. Single cell RNA-seq libraries were prepared with 10x 5' gene expression kit. Libraries were sequenced on Illumina NovaSeq 6000. Gene expression matrices were generated with 10x Cell Ranger v7.0.0 (exon-only mode) software and reads were aligned to a custom hybrid genome containing GRCh38.98 and SARS-CoV-2 (NC_045512.2).

first: Joseph

last: Bailey

first: Connor

middle: H

last: Puritz

first: Karolina

last: Senkow

first: Nikolay

last: Markov

first: Ziyan

last: Lu

first: Robert

last: Tighe

middle: V

first: Marc

last: Sala


title: Expansion of profibrotic monocyte-derived alveolar macrophages in patients with persistent respiratory symptoms and radiographic abnormalities after COVID-19 [BAL]

overall_design: Bronchoalveolar lavage (BAL) fluid from 6 heathy volunteers was obtained the Duke University School of Medicine. BAL fluid was flow-sorted to enrich for live CD45+CD15- cells and single cell RNA-seq libraries were prepared with 10x 5' gene expression kit. Libraries were sequenced on Illumina NovaSeq 6000. Gene expression matrices were generated with 10x Cell Ranger v7.0.0 (exon-only mode) software and reads were aligned to a custom hybrid genome containing GRCh38.98 and SARS-CoV-2 (NC_045512.2).


title: Transcriptomic analysis of HIV-associated nephropathy (Tg26) mouse kidney [single-nuclear RNA-seq]

first: Teruhiko

last: Yoshida

summary: We analyzed the transcriptomic profiles of Tg26 mouse kidney by single-nuclear RNA-seq with or without treatment by PKR inhibitor (C16).

overall_design: Transcriptomic profiles of Tg26 mouse kidney by single-nuclear RNA-seq

first: Khun

middle: Z

last: Latt

first: Avi

last: Rosenberg

first: Yongmei

last: Zhao

first: Shashi

last: Shrivastav

first: Koji

last: Okamoto

first: Cheryl

last: Winkler

first: Jeffrey

middle: B

last: Kopp


title: Expansion of profibrotic monocyte-derived alveolar macrophages in patients with persistent respiratory symptoms and radiographic abnormalities after COVID-19 [NEP]

overall_design: Nasal epithelial samples were collected from healthy volunteers who provided informed consent at Northwestern Medicine in Chicago. The protocol was approved by the Northwestern University IRB (STU00214826). Briefly, donors were seated and asked to extend their neck. A nasal curette (Rhino-Pro; VWR) was inserted into either nare and gently slid in the direction of posterior to anterior ~1 cm along the lateral inferior turbinate. Five curettes were obtained per participant. The curette tip was then cut and placed in 2 ml hypothermosol and stored at 4 C until processing. A single-cell suspension was generated using the cold-active dispase protocol reported by Deprez et al. AJRCCM, 2020 with slight modification. Specifically, ethylenediaminetetraacetic acid (EDTA) was omitted and cells were dispersed by pipetting 20 times every 5 min using a 1 ml tip instead of tritration using a 21/23 G needle. The final concentration of protease from Bacillus licheniformis was 10 mg ml−1. The total digestion time was 30 min. Single cell RNA-seq libraries were prepared with 10x 5' gene expression kit. Libraries were sequenced on Illumina NovaSeq 6000. Gene expression matrices were generated with 10x Cell Ranger v7.0.0 (exon-only mode) software and reads were aligned to a custom hybrid genome containing GRCh38.98 and SARS-CoV-2 (NC_045512.2).


title: Epigenomic signatures of sarcomatoid differentiation to guide the treatment of renal cell carcinoma

status: Public on May 23 2024

city: Boston

first: Karl

last: Semaan

state: MA

address: Brookline Ave 450

institute: Dana-Farber Cancer Institute

type: Methylation profiling by high throughput sequencing

summary: Renal cell carcinoma with sarcomatoid differentiation is associated with poor survival and heightened response to immune checkpoint inhibitors. Two major barriers to improving outcomes for sRCC are the limited understanding of its gene regulatory programs and the low diagnostic yield of tumor biopsies due to spatial heterogeneity.

overall_design: Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) for histone modifications H3K27ac, H3K4me2 on tissue samples. Methylated immunoprecipitation and high-throughput sequencing (MeDIP-seq)  on plasma and tissue samples.

first: T

last: El Zarif

first: K

last: Choueiri

first: M

middle: L

last: Freedman

first: S

middle: C

last: Baca


title: Identifying the transcriptomic effects of tRNA-induced proline-to-serine mistranslation in Drosophila melanogaster

status: Public on May 11 2024

city: London

first: Amanda

middle: Jean

last: Moehring

state: Ontario

address: 1151 Richmond Street, Western Science Centre 307

country: Canada

institute: Western University

summary: Mistranslation describes the misincorporation of an amino acid into a nascent polypeptide. Mistranslation has diverse effects on multicellular eukaryotes and is implicated in several human diseases. We introduced a mistranslating serine transfer RNA (tRNA) into Drosophila melanogaster that misincorporates serine at proline codons and found that it affected male and female flies differently. Here, we compare the transcriptomic response of male and female flies to mistranslation to identify cellular pathways that underlie this sex-specific response. Both males and females downregulated genes associated with metabolism in response to proline-to-serine mistranslation. Only males downregulated genes associated with developmental processes and response to negative stimuli such as infection, whereas only females downregulated aerobic respiration and ATP synthesis genes. Both sexes upregulated genes associated with gametogenesis but females upregulated cell cycle and DNA maintenance genes, suggesting that mistranslation may compromise genome integrity in females. This transcriptomic analysis advances our understanding of how males and females respond to mistranslation and has important implications for future studies that examine the influence of mistranslation on disease.

overall_design: RNA sequencing performed on 10 male or female flies containing an additional wildtype serine tRNA with a UGA anticodon (FlyBase ID: FBgn0050201) or an additional serine tRNA modified to contain a UGG anticodon and a G26A mutation causing misincorporation of serine at proline codons

first: Joshua

middle: R

last: Isaacson

first: Matthew

middle: D

last: Berg

first: William

last: Yeung

first: Christopher

last: Brandl


title: Analysis of gene expression in Mel Z cells grown on Matrigel in the presence of inhibitor(MMI).

status: Public on May 10 2024

city: Moscow

first: Nickolai

last: Tchurikov

address: 32, Vavilova str.

department: Department of Epigenetic Mechanisms of Gene Expression Regulation

country: Russia

institute: Engelhardt Institute of Molecular Biology

summary: RNA-Seq procedure was used for analysis of expression in melanoma cells. Cells were separately lysed with Trizol reagent (Invotrogen). Total RNA was extracted from cell lysed with Trizol reagent and PureLink RNA Micro Kit (Invitrogen) according to manufacture instruction. RNA quality was checked with  BioAnalyser and  RNA 6000 Nano Kit (Agilent). Poly(A)+ RNA was purified with Dynabeads® mRNA Purification Kit (Ambion). Illumina library was prepared from poly(A)+RNA with NEBNextNEBNext® Ultra™II RNA Library Prep Kit for Illumina® (NEB) according to manual. Sequencing was performed on HiSeq1500 with 50 bp read length. At least 14.8 millions of reads were generated for each sample.

overall_design: Analysis of gene expression in Mel Z cells grown on plastic (MP), Matrigel (MM), and on Matrigel in the presence of inhibitor(MMI).

middle: A

first: Yuri

last: Kravatsky

first: Amalia

last: Vartanian


title: Cryptic splicing mediates genetic and therapeutic perturbation of human gene expression levels [naRNA_LCLs]

first: Carlos F

last: Buen Abad Najar

state: Illinois

address: 920 E 58th St

department: Medicine

institute: University of Chicago

summary: Alternative splicing (AS) in human genes is widely viewed as a mechanism for enhancing proteomic diversity. AS can also impact gene expression levels without increasing protein diversity by producing “unproductive” transcripts that are targeted for rapid degradation by nonsense-mediated decay (NMD). However, the relative importance of this regulatory mechanism remains underexplored. To better understand the impact of AS-NMD relative to other regulatory mechanisms, we analyzed population-scale genomic data across eight molecular assays, covering various stages from transcription to cytoplasmic decay. We report threefold more unproductive splicing compared to prior estimates using steady-state RNA. This unproductive splicing compounds across multi-intronic genes, resulting in 15% of all transcript molecules from protein-coding genes being unproductive. Leveraging genetic variation across cell lines, we find that GWAS trait-associated loci explained by AS are as often associated with NMD-induced expression level differences as with differences in protein isoform usage. Employing the splice-switching drug risdiplam to manipulate AS at hundreds of genes, we find that ~3/4 of drug-induced isoforms are targeted by NMD, suggesting that most aberrant splicing influences expression levels. Our findings suggest much of the impact of AS is mediated by NMD-induced changes in gene expression rather than diversification of the proteome.

overall_design: Nuclei were isolated through a sucrose cushion followed by high-salt washes to dissociate nucleoplasm and weakly bound RNAs and proteins from chromatin, following the protocol in PMID: 19656867. RNA yield was 5-20ug naRNA per sample.

first: Benjamin

last: Fair

first: Carlos

first: Yang

last: Li


title: Cryptic splicing mediates genetic and therapeutic perturbation of human gene expression levels [Cut&Tag]

overall_design: CUT&Tag was performed as described in protocol DOI: dx.doi.org/10.17504/protocols.io.z6hf9b6 using 14 cycles instead of 13 to ensure we had enough DNA material to sequence.


title: Phase separation of phospho-HDAC6 drives aberrant chromatin architecture in triple-negative breast cancer [ChIP-seq, Cut&Tag, ATAC-seq]

status: Public on May 09 2024

city: Guangzhou City

first: Ru

last: Qiu

state: Guangdong Province

address: No. 74, 2nd Yat-sen Road, Yuexiu District

institute: Sun Yat-sen University

summary: To further elucidate the role of phospho-HDAC6 LLPS in transcriptional regulation, we performed RNA-seq experiments in TNBC cells treated with Nexturastat A or HDAC6 knockdown

overall_design: Comparative gene expression profiling analysis of ChIP-seq data (HDAC6, Pan-acetyl, polII) and ATAC-seq for MDA-MB231 cells with and without HDAC6 inhibitor Nexturastat A treatment. Comparative gene expression profiling analysis of ChIP-seq data (HDAC6, Pan-acetyl, polII) and ATAC-seq  for MDA-MB231 cells  and its HDAC6 KD derivatives (shHDAC6).

first: Wang

first: Qiu

last: Ru


title: Compare transcriptome profile of reference strain and mutant of HapX during A.niger grown on maltose

status: Public on May 17 2024

city: Utrecht

first: Ronald

last: de vries

state: Utrecht

address: Uppsalalaan 8

institute: westerdijkinstitute

summary: Transcriptome analysis was performed to identify the differentially expressed genes between hapX mutant and reference strains during their growth on maltose

overall_design: The strains were pre-grown in complete medium containing 2% (w/v) fructose for 16h. The mycelium was harvested and washed with minimal medium (MM) , and then was transferred to 50 ml MM (pH 6) with 25 mM maltose. After 2h of incubation, the mycelium was harvested and frozen in liquid nitrogen. RNA was extracted from grinded mycelia and were single-end sequenced using Illumina HiSeqTM 2000 platform . The reads were mapped to the genome of Aspergillus niger NRRL3 (https://mycocosm.jgi.doe.gov/Aspni_NRRL3_1),

first: Joanna

middle: E

last: Kowalczyk

first: Mao

last: Peng

middle: P

last: de Vries

first: Astrid

last: Mueller


title: Global impact of unproductive splicing on human gene expression.

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

relation:
SuperSeries of: GSE252003
SuperSeries of: GSE252005

overall_design: Refer to individual Series


title: O-GlcNAcylation regulates c-Myc/PDK2 axis to rewire TCA cycle and promote colorectal tumor growth

city: 浙江省

first: Huijuan

last: Wang

address: Yuhangtang Road No.866

institute: 浙大

summary: To investigate the function OGT in the regulation of colorectal tumor growth, we established HT-29 cell lines in which OGT has been knocked down by shRNA.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for HT-29 cells and its knock down OGT (shOGT).

first: Wen

last: Yi


title: Characterization of Transcriptome and Chromatin Interactome in Accessible Regions at Single-cell Resolution by scAIR

first: Tian

last: (Simon) Zhongyuan

summary: The increasing prevalence of single-cell technologies has brought significant advancements in the study of cellular heterogeneity. However, there is a lack of techniques that can simultaneously capture both the transcriptome and chromatin interactions. In this study, we introduce a novel approach called scAIR, which utilizes the 10x Genomics platform to perform single-cell RNA-seq and ATAC-seq analysis on pre-proximity-ligated nuclei. By leveraging this technology, we were able to obtain comprehensive data that offers a deeper understanding of transcriptional regulation and chromatin dynamics at the single-cell level. This innovative methodology holds great promise in unraveling the complex intricacies of cellular processes and their underlying regulatory mechanisms.

overall_design: To perform scAIR (single-cell Assay for Integration of scATAC-seq, chromatin interactome, and RNA-seq data) on Drosophila embryo nuclei, we first targeted chromatin using a restriction enzyme. This allowed us to label specific chromatin regions of interest. Next, we performed proximity ligation to capture the interactions between these targeted chromatin regions. The proximity-ligated nuclei were then loaded onto the 10x Genomics platform, enabling us to perform scRNA-seq and scATAC-seq simultaneously. After library construction, the samples were sequenced using Illumina Nova-seq6000 with a paired-end sequencing approach. By integrating the scRNA-seq and ATAC-seq data, we were able to identify the accessible regions of the chromatin and the interactions within, providing valuable insights into the cell-specific transcriptome and chromatin landscape on Drosophila embryo nuclei.

first: Meizhen

last: Zheng


title: Mucosal immune response against equine herpesvirus type 1

status: Public on May 08 2024

city: Ithaca

first: Bettina

last: Wagner

address: 240 Farrier Drive

department: Population Medicine and Diagnostic Sciences

institute: Cornell University

summary:
To investigate pre-existing mucosal immunity in equine herpesvirus type 1 (EHV-1) immune and non-immune horses during experimental viral challenge.
RNAseq was performed on nasal mucosal swab samples from immune and non-immune horses from  pre (d-2), early (d1pi and d3pi), mid (d8pi and d10pi), and late (d18pi) infection.

overall_design: Comparative gene expression analysis of immune and non-immune horses throughout viral challenge.

first: Camille

middle: M

last: Holmes

first: Susanna

last: Babasyan

first: Naya

last: Eady

first: Christiane

last: Schnabel


title: Two distinct epithelial-to-mesenchymal transition programs control invasion and inflammation in segregated tumor cell populations

city: San Juan de Alicante

first: Angela

last: Nieto

state: Alicante

address: Av. Ramón y Cajal s/n

department: Developmental Neurobiology

institute: Instituto de Neurociencias (CSIC-UMH)

summary: The goal of this study is to analyze mRNA transcriptional changes associated with partial and full EMTs undergone by kidney epithelial dog MDCK cells upon treatment with TGFb.

overall_design: We performed mRNA-seq on 24 samples from two MDCK sublines: MDCK-II and MDCK-NBL2

first: Khalil

middle: K

last: Youssef

first: Aida

last: Arcas

first: M


title: Next-Generation Sequencing to compare RNA transcripts between Adam19 wildtype and knockout mice

city: RTP

first: Huiling

last: Li

state: NC

address: 111 T.W. Alexander Dr

institute: NIEHS

summary: ADAM19 has been associated with pulmonary function traits in a number of genome wide association analyses by our group and others. We want to use a mouse model to examine the role Adam19 in lung function to clarify whether ADAM19 is truly the causal gene at this locus. We created a whole body Adam19 knockout mouse model targeting exon6 and 7 to disrupt the transcript expression from exon 8 through the end of gene sequences. In previous publications, mice lacking Adam19 exon 11, which harbor a catalytic site, were not postnatal viable. Contradictory to the previous publications, our Adam19 knockout survived postnatally. Our objectives were to determine if this novel mouse model is truly Adam19-deficient rather than a hypomorph or neomorph rescuing the previously published lethal phenotype of Adam19-deficient mice.

overall_design: mRNA profiles of the heart and lung of wildtype and Adam19 knockout generated by deep sequencing, in triplicate, using Illumina NextSeq-High.

first: James

last: Ward

first: Stephanie

last: London

first: Artiom

last: Gruzdev


title: Targeting senescence induced by age or chemotherapy with a polyphenol-rich natural extract improves longevity and healthspan in mice

status: Public on May 01 2024

city: Bellinzona

first: Marco

last: Bolis

address: Via Francesco Chiesa

country: Switzerland

institute: IOR Institute of Oncology Research

summary: The accumulation of senescent cells within tissues contributes to age-related diseases through the senescence-associated secretory phenotype (SASP). To fight senescence and maintain a healthy tissue microenvironment, the quest for longevity-promoting compounds has resulted in promising discoveries from botanical sources. Here we show that daily administration with a low dose of Haenkenium (HK), a standardized extract of Salvia haenkei, extended the lifespan and healthspan of aged C57BL/6 mice by delaying senescence and consequently ameliorating systemic inflammation and fibrosis markers in multiple tissues including skin, muscle, cartilage, and kidney. Additionally, treated aged mice displayed increased muscle strength, vitality, and thicker fur coverage than age-matched controls. Notably, HK treatment also effectively reduced doxorubicin (Doxo)-induced senescence in p16LUC reporter mice and protected against Doxo-induced cardiotoxicity. The composition of HK was analyzed by UPLC-QTOF-MS, which revealed multiple polyphenols that were then screened for their putative anti-senescence activity in vitro. Among these, luteolin, luteolin- 7-O-glucuronide and 3,4-dicaffeoylquinic were identified as HK-derived constituents with comparable activity to HK. Mechanistically, we report that luteolin could disrupt the p16-CDK6 interaction, which was validated by surface plasmon resonance (SPR) and by in situ proximity ligation assay in vitro. Our findings suggest that phytoconstituents such as luteolin can alter the senescence phenotype by disrupting the activity of p16 which subsequently is associated with improved longevity in mice.

overall_design: Gastrocnemius muscle of untreated (n=10) and Haenkenium (HK) treated (n=10) mice were subjected to bulk RNASeq. Each of the 2 experimental groups consisted of 5 male and 5 female mice.

first: Andrea

last: Alimonti


title: Dynamic Chromatin Alteration Induces Oncogenic Hijacking by Essential Transcriptional Factors during Medulloblastoma Tumorigenesis [Multiome]

status: Public on May 03 2024

city: Tokyo

first: Kohei

last: Kumegawa

address: 3-8-31 Ariake, Koto-ku

institute: Japanese Foundation for Cancer Research

summary: Using ATAC-seq analysis from GNPs to MBSHH we discovered a massive modification in chromatin accessibility during MBSHH formation. Next, using integrative bioinformatics, we identified genes of the nuclear factor I (NFI) family that function as oncogenes on the MBSHH epigenome. We demonstrate not only that these genes are essential in the early stages of murine MBSHHs tumorigenesis, but also that their genetic silencing inhibits tumor growth in murine and human MBSHHs in vitro and in vivo. Finally, we discovered that NFIA/B are post-translationally modified by an activating methylation in MBSHH, making it possibility to target this pathway. Further studies revealed that pharmacological inhibition of this methylated NFIA/B hindered tumor proliferation, demonstrating the requirement of NFIA/B for tumor progression. Collectively our data shed light on the mechanism underlying the epigenome during the formation of the most malignant pediatric brain tumor and emphasizes the importance of deciphering cancer-specific epigenome for identification of new therapeutic avenues.

overall_design: Chromium Single-Cell Multiome ATAC + Gene Expression for mouse medulloblastoma GEMM model

first: Ryo

last: Shiraishi

first: Gabriele

last: Cancila

first: Reo

last: Maruyama

first: Olivier

last: Ayrault

first: Daisuke

last: Kawauchi


title: Transcriptome of DLD-1 co-cultured with or without extracellular vesicels derived from Fusobacterium nucleatum

city: Chengdu

first: Xin

last: Zheng

state: Sichuan

address: No. 14, Section 3, Renmin South Road

institute: Sichuan University

summary: To explore the impact of FnEV on transcriptome of colorectal cancer cell line DLD-1

overall_design: The cells cultured in 10% FBS DMEM were grown to ~90% confluency, followed by treatemnt of 5 μg/mL FnEVs or PBS for 2 hr at 37°C in a humidified 5% CO2 atmosphere.

first: Tao

last: Gong

first: Wanyi

last: Luo

first: Jiaxin

last: Liu


title: STC1 promotes temozolomide resistance of glioblastoma through STAT3-mediated regulation of MGMT.

status: Public on Apr 30 2024

city: Wuhan

first: Bincan

last: He

address: 1 Shizishan Rd

institute: Huazhong Agricultural University

summary: Resistance to temozolomide (TMZ) is one of the major challenges for glioblastoma (GBM) therapy while the underlying mechanisms demand further exploration. Tumor-repopulating cells (TRCs) have been proven to be involved in chemotherapy resistentce. We first enriched GBM TRCs by culturing DBTRG cells in 3D soft fibrin gels and performed RNA-seq. By anlyzing the differentially expressed genes (DEGs) between TRCs and 2D conventionally cultured DBTRG cells, we identified the glycoprotein gene Stanniocalcin-1 (STC1), which is highly expressed in TRCs. Our analyses using patient data from CGGA disclosed that high STC1 expression was associated with poor prognosis, high glioma grade and TMZ therapy resistance. Both our in vitro and in vivo expreriments showed that overexpression of STC1 promoted while knockdown of STC1 inhibited GBM cell proliferation and TMZ resistance. Our mechanistic study revealed that STC1 elevated the phosphoralation of STAT3 to increase MGMT expression, which inhibited the TMZ-induced DNA damage and apoptosis. Our study provides rationale for targeting STC1 to overcome TMZ resistance.

overall_design: We cultured DBTRG cells with rigid dish or soft gel, cells were harvested cells separately for RNA-seq analysis after being cultured for 5 days.

first: Chao

last: Duan

first: Zhiqiang

last: Dong


title: ESR1 fusions invoke breast cancer subtype-dependent enrichment of ligand independent oncogenic signatures and phenotypes [ChIP-seq]

status: Public on May 02 2024

city: Pittsburgh

first: Megan

middle: E.

last: Yates

state: PA

address: 5051 Centre Ave

institute: University of Pittsburgh

summary: Breast cancer is a leading cause of female mortality and despite advancements in personalized therapeutics, metastatic disease largely remains incurable due to drug resistance. The estrogen receptor (ER, ESR1) is expressed in two-thirds of all breast cancer, and under endocrine stress, somatic ESR1 mutations arise in ~30% of cases that result in endocrine resistance. We and others reported ESR1 fusions as a mechanism of ER mediated endocrine resistance. ER fusions, which retain the AF1 and DNA binding domains, harbor ESR1 exons 1-6 fused to an in-frame gene partner resulting in loss of the ER ligand binding domain (LBD). We demonstrate that in a no-special type (invasive ductal carcinoma (IDC)- NST) and an invasive lobular carcinoma (ILC) cell line, ER fusions exhibit robust hyperactivation of canonical ER signaling pathways independent of estradiol or anti-endocrine therapies. We employ cell line models stably overexpressing ER fusions with concurrent endogenous ER knockdown to minimize endogenous ER influence. Cell lines exhibited shared transcriptomic enrichment in pathways known to be drivers of metastatic disease, notably MYC signaling. Cells expressing the 3’ fusion partners SOX9 and YAP1 consistently demonstrated enhanced growth and cell survival. ILC cells expressing the DAB2 fusion led to enhanced growth, survival, and migration; phenotypes not appreciated in the IDC- NST DAB2 model. Herein, we report that cell line activity is subtype-, fusion-, and assay-specific suggesting that LBD loss, the fusion partner, and the cellular landscape all influence fusion activities. Therefore, it will be critical to assess fusion frequency, in the context of the tumor clinicopathology.

overall_design: Chromatin immunoprecipitation DNA sequencing (ChIP-seq) of six transgenic breast cancer cell lines derived from the T47D parental cell line. Cells stably express HA tagged wildtype ER, HA tagged truncated ER, or a HA tagged ER fusion protein (ESR1-DAB2, ESR1-LPP, ESR1-SOX9, ESR1-YAP1), with concurrent endogenous ER knockdown. Each cell type is treated with vehicle for 45 minutes prior to cell harvest, HA immunoprecipitation, and sequencing. An additional wildtype ER sample was treated with 1nM estradiol for 45 minutes to serve as a positive control.

first: Hunter

last: Waltermire

first: Kanako

last: Mori

first: Zheqi

last: Li

first: Yiting

first: Hannah

last: Guzolik

first: Xiaosong

last: Wang

first: Tiantong

first: Jennifer

last: Atkinson

first: Jagmohan

last: Hooda

first: Adrian

last: Lee

first: Steffi

last: Oesterreich


title: ESR1 fusions invoke breast cancer subtype-dependent enrichment of ligand independent oncogenic signatures and phenotypes


title: Single-cell transcriptomic analysis of human skin organoids during folliculogenesis

first: Karl

middle: Russell

last: Koehler

address: 3 Blackfan Circle

department: Otolaryngology-Head and Neck Surgery

institute: Boston Children's Hospital-Harvard Medical School

summary: In this study, we defined a differentiation approach for guiding human pluripotent stem cells in to complex hair-bearing skin tissue, known as skin organoids. The primary goal of this single-cell RNA-sequencing analysis was to define the cellular composition of skin organoids before, during, and after the process of hair folliculogenesis. The secondary goal was to determine whether skin organoids generated using different cell lines (WA25 hESCs and DSP-GFP hiPSCs) contain similar cell types and accurately reflect features of developing fetal skin tissue.

overall_design: Single-cell RNA-sequencing data from three different timepoints using two different stem cell lines.

first: Jiyoon


title: Transcriptomic signature and pro-osteoclastic secreted factors of abnormal bone marrow stromal cells in fibrous dysplasia (Mouse)

status: Public on Apr 29 2024

city: Bethesda

first: Luis

last: Fernandez de Castro Diaz

state: Maryland

address: 30 Convent Drive

department: SDMHS

institute: NIDCR/NIH

summary: Fibrous dysplasia (FD) is a mosaic skeletal disorder caused by somatic activating variants in GNAS, encoding for Gαs, which leads to excessive cAMP signaling in bone marrow stromal cells (BMSCs). Despite advancements in our understanding of FD pathophysiology, the effect of Gαs activation in the BMSC transcriptome remains unclear, as well as how this translates into their local influence in the lesional microenvironment. In this study, we analyzed changes induced by Gαs activation in BMSC transcriptome and performed a comprehensive analysis of their production of cytokines and other secreted factors. We performed RNAseq of cultured BMSCs from patients with FD and healthy volunteers, and from an inducible mouse model of FD, and combined their transcriptomic profiles to build a robust FD BMSC genetic signature. Pathways related to Gαs activation, cytokine signaling, and extracellular matrix deposition were identified. In addition, a comprehensive profile of their secreted cytokines and other factors was performed to identify modulation of several key factors we hypothesized to be involved in FD pathogenesis. We also screened circulating cytokines in a collection of plasma samples from patients with FD, finding positive correlations of several cytokines to their disease burden score, as well as to one another and bone turnover markers. Overall, these data support a pro-inflammatory, pro-osteoclastic behavior of BMSCs bearing hyperactive Gαs variants, and point to several cytokines and other secreted factors as possible therapeutic targets and/or circulating biomarkers for FD.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for bone marrow stromal cells derived from a doxycycline-inducible mouse model of fibrous dysplasia

first: Zachary

last: Michel

first: Layne

middle: N

last: Raborn

first: Tiahna

last: Spencer

first: Kristen

last: Pan

first: Daniel

last: Martin

first: Kelly

last: Roszko

first: Yan

first: Pamela

middle: G

last: Robey

first: Michael

middle: T

last: Collins

first: Alison

last: Boyce

middle: F

last: de Castro


title: Phase separation of phospho-HDAC6 drives aberrant chromatin architecture in triple-negative breast cancer [RNA-seq]

first: Ru

last: Qiu

overall_design: Comparative gene expression profiling analysis of RNA-seq data for MDA-MB231 cells with and without HDAC6 inhibitor Nexturastat A treatment. Comparative gene expression profiling analysis of RNA-seq data for MDA-MB231 cells  and its HDAC6 KD derivatives (shHDAC6).

first: Qiu

last: Ru

first: Wang

last: Li


title: Phase separation of phospho-HDAC6 drives aberrant chromatin architecture in triple-negative breast cancer [Hi-C]

summary: To investigate the Nexturastat A effects on chromatin structure which disrupt phospho-HDAC6 LLPS in triple-negative breast cancer (TNBC), we conducted the analysis of Hi-C in TNBC cells treated with or without Nexturastat A.

overall_design: control group:untreat BT-549. treatment group: 5uM Nexturastat A treated BT-549 for 2 days

first: Lu

last: Bing

first: Li

last: Mingsen


title: aChIP: efficient, sensitive, robust ChIP-seq for economically important plant organs

status: Public on Apr 28 2024

first: Wenying

last: Zhong

address: Lion Rock Street

summary: Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is crucial for profiling histone modifications and transcription factor binding throughout the genome. However, its application in economically important plant organs (EIPOs) such as seeds, fruits and flowers is challenging due to their sturdy cell walls and complex constituents. Here we present advanced ChIP (aChIP), an optimized method that efficiently isolates chromatin from plant tissues while simultaneously removing cell walls and cellular constituents. aChIP precisely profiles histone modifications in all 14 tested EIPOs and identifies transcription factor and chromatin-modifying enzyme binding sites. In addition, aChIP enhances ChIP efficiency, revealing numerous novel modified sites compared with previous methods in vegetative tissues. aChIP reveals the histone modification landscape for rapeseed dry seeds, highlighting the intricate roles of chromatin dynamics during seed dormancy and germination. Altogether, aChIP is a powerful, efficient and sensitive approach for comprehensive chromatin profiling in virtually all plant tissues, especially in EIPOs.

sample_organism:
Citrus sinensis
Dianthus caryophyllus
Populus
Arabidopsis thaliana
Brassica napus
Glycine max
Solanum lycopersicum
Solanum tuberosum
Oryza sativa
Saccharum officinarum
Zea mays

overall_design: We demonstrate the widespread utility of advanced ChIP-seq (aChIP) by profiling diverse chromatin proteins in 14 representative EIPOs including the extremely challenging dry seeds. aChIP shows excellent efficiency and sensitivity in vegetative tissues compared with previous methods. Finally, we profiled the landscape of histone modifications and unveiled their potential functions in rapeseed dry seeds and germinated seeds. Collectively, the data illustrate the great potential of using aChIP to unveil the intricacies of chromatin biology and shed light on their biological functions in plants, particularly within EIPOs.


title: Fetal-derived activated CD83+ γδ T cells expand in preterm infants with sepsis.

city: Hannover

first: Ximena

last: Leon Lara

state: Please select a region.

address: Carl-Neuberg-Strasse 1

institute: Hannover Medical School

summary: Here, we investigated γδ T cells in a longitudinal cohort of preterm neonates during the first year of life. Our data reveal dynamic postnatal maturation patterns of γδ T cell subsets, which are largely age-dependent. We report on the expansion of fetal-derived γδ T cells in preterm neonates with sepsis. Single cell transcriptome analyses identified HLA-DRhigh and CD83+ γδ T cells in neonatal sepsis.

overall_design: Combined single-cell transcriptome (scRNA-seq) and single-cell γδTCR-seq (scTCR-seq) analysis was conducted from peripheral blood.

first: X

last: Leon-Lara

first: T

last: Yang

first: S

last: Ravens


title: Transcriptomic signature and pro-osteoclastic secreted factors of abnormal bone marrow stromal cells in fibrous dysplasia (Human)

first: Luis

last: Fernandez de Castro Diaz

overall_design: Comparative gene expression profiling analysis of RNA-seq data for bone marrow stromal cells derived from patients with fibrous dysplasia and healthy donors

first: Zachary

last: Michel

first: Layne

last: Raborn

first: Tiahna

last: Spencer

first: Kristen

last: Pan

last: Wang


title: Functional genomics screens reveal a role for TBC1D24 and SV2B in antibody-dependent enhancement of dengue virus infection.

status: Public on Apr 27 2024

city: Seattle

first: Laura

last: Belmont

state: Washington

address: 1100 Fairview Ave N

institute: Fred Hutchinson Cancer Research Center

summary: Dengue virus (DENV) can hijack non-neutralizing IgG antibodies to facilitate its uptake into target cells expressing Fc gamma receptors (FcgR) - a process known as antibody-dependent enhancement (ADE) of infection. Beyond a requirement for FcgR, host dependency factors for this non-canonical infection route remain unknown. To identify cellular factors exclusively required for ADE, here, we performed CRISPR knockout screens in an in vitro system permissive to infection only in the presence of IgG antibodies. Validating our approach, a top hit was FcgRIIa, which facilitates binding and internalization of IgG-bound DENV but is not required for canonical infection. Additionally, we identified host factors with no previously described role in DENV infection, including TBC1D24 and SV2B, both of which have known functions in regulated secretion. Using genetic knockout and trans-complemented cells, we validated a functional requirement for these host factors in ADE assays performed with monoclonal antibodies and polyclonal sera in multiple cell lines and using all four DENV serotypes. We show that TBC1D24 and SV2B promote binding of IgG-DENV complexes to cells without affecting FcgRIIa expression levels. Thus, we identify cellular factors beyond FcgR that are required for ADE of DENV infection. Our findings represent a first step towards advancing fundamental knowledge behind the biology of ADE that can ultimately be exploited to inform vaccination and therapeutic approaches.

overall_design: Genome-wide and follow-up targeted CRISPR screens were performed on K562 cells (Cat #CCL-243, ATCC) mutagenized with either the human GeCKO v2 library (#1000000048 and #1000000049, Addgene) or a custom sub-library targeting the 500 highest-ranking candidates from the genome-wide screen, respectively. For the latter, we designed six guides per gene plus 200 non-targeting controls (NTC) using CHOPCHOP and Guides [PMIDs 31106371, 28858339]. The genome-wide screen was performed once in two technical replicates (one for each GeCKO v2 library A or B) and targeted screens were performed in biological triplicate using three independently mutagenized libraries. Following each screen, genomic DNA was isolated using the QIAamp DNA Blood Mini Kit (Cat #51185, Qiagen), and sgRNA sequences were amplified and prepared for next-generation sequencing (Illumina). The enrichment of each sgRNA in the selected cells relative to the unselected libraries cultured and harvested in parallel was calculated using MAGeCK [PMID: 25476604].

first: Maya

last: Contreras

first: Catiana

middle: H

last: Cartwright-Acar

first: Caleb

last: Marceau

first: Aditi

last: Agrawal

first: Lisa

last: Levoir

first: Jay

last: Lubow

first: Leslie

last: Goo


title: Molecular and functional profiling of cell diversity and identity in the lateral superior olive, an auditory brainstem center with ascending and descending projections

status: Public on Apr 24 2024

city: Saarbrücken

first: Kathrin

last: Kattler-Lackes

state: non-US Resident

address: Campus A2.4 Raum 0.13

department: Epigenetics

institute: Saarland University

summary: The lateral superior olive (LSO), a conspicuous integration center in the auditory brainstem, contains a remarkably heterogeneous neuron population. Ascending neurons, predominantly principal neurons (pLSOs), process interaural level differences for sound localization. Descending neurons (lateral olivocochlear neurons, LOCs) provide feedback into the cochlea and likely protect from acoustic overexposure. The molecular determinants of the neuronal diversity in the LSO are largely unknown. Here, we employed patch-seq analysis in juvenile mice to classify LSO neurons by their functional and molecular profiles, including developmental aspects. Across the sample (n=86), genes involved in ATPsynthesis were particularly highly expressed, confirming the energy expenditure of auditory neurons. Two clusters were identified, pLSOs and LOCs. They were distinguishable by 353 differentially expressed genes (DEGs), most being novel for the LSO. Electrophysiological analysis corroborated the transcriptomic clustering. We focused on genes impacting neuronal input-output properties and validated some by immunohistochemistry, electrophysiology, and pharmacology. These genes encode proteins like osteopontin, Kv11.3, and Kvb3 (pLSO-specific), calcitonin-gene-related peptide (LOC-specific), or Kv7.2 and Kv7.3 (no DEGs). We identified 12 ‘Super DEGs’ and 12 genes demonstrating ‘Cluster similarity’. Collectively, we provide fundamental and comprehensive insights into the molecular composition of individual ascending and descending neurons in the juvenile auditory brainstem and how this may relate to their specific functions, including developmental aspects.

overall_design: Patch-seq on mouse neurons located in the lateral superior olive of the auditory brainstem

first: Ayse

last: Maraslioglu

first: Erika

last: Pizzi

first: Jonas

last: Fisch

last: Kattler

first: Tamara

last: Ritter

first: Eckhard

last: Friauf


title: PML restrains p53 activity and cellular senescence in clear cell renal cell carcinoma

status: Public on Apr 23 2024

city: Milan

first: Rosa

last: Bernardi

address: via Olgettina 60

institute: San Raffaele Scientific Institute

summary: Clear cell renal cell carcinoma (ccRCC), the major subtype of RCC, is frequently diagnosed at late/metastatic stage with 13% 5-year disease-free survival. Functional inactivation of the wild type p53 protein is implicated in ccRCC therapy resistance, but the detailed mechanisms of p53 malfunction are still poorly characterized. Thus, a better understanding of mechanisms of disease progression and therapy resistance is required. Here, we report a novel ccRCC dependence on the promyelocytic leukemia PML protein. We show that PML is overexpressed in ccRCC and that PML depletion inhibits cell proliferation and relieves pathologic features of anaplastic disease in vivo. Mechanistically, PML loss unleashed p53-dependent cellular senescence thus depicting a novel regulatory axis to limit p53 activity and senescence in ccRCC. Treatment with the FDA-approved PML inhibitor arsenic trioxide induced PML degradation and p53 accumulation, and inhibited ccRCC expansion in vitro and in vivo. Therefore, by defining non-oncogene addiction to the PML gene, our work uncovers a novel ccRCC vulnerability and lays the foundation for repurposing an available pharmacological intervention to restore p53 function and chemosensitivity.

overall_design: RNA-sequencing (RNA-seq) in control and PML silenced RCC4 cells.

first: Matilde

last: Simoni

first: Chiara

last: Menegazzi

first: Cristina

last: Fracassi

first: Claudia

last: Biffi

first: Francesca

last: Genova

first: Nazario

last: Tenace

first: Roberta

last: Lucianò

first: Andrea

last: Raimondi

first: Carlo

last: Tacchetti

first: James

last: Brugarolas

first: Davide

last: Mazza


title: Effect of CSF1R targeting on human macrophages in normoxia and hypoxia

status: Public on Apr 18 2024

city: La Tronche

first: arnaud

last: MILLET

address: Allée des Alpes

country: France

institute: Institute for Advanced Studies

summary: CSF1R is a receptor involved in differentiation and survival of macrophages. We designed this study to explore the consequences of CSF1R targeting on human macrophage transcriptome. We also seek to explore the influence of oxygen environnment on the consequences of CSF1R blocakde . Our data provide an extensive characterization of the various pathways connected to CSF1/CSF1R pathway in human macrophages.

overall_design: To investigate the effect of a tyrosine kinase inhibitor edicotinib on CSF1R in human macrophages. We designed an experiment with macrophages treated for 48h with edicotinib 10µM or DMSO in normoxia (oxygen at 140 mmHg) or hypoxia (25 mmHg). We then performed gene profile expression analysis using data from RNAseq.

first: Arnaud

last: Millet

first: Khaldoun

last: Gharzeddine


title: RNA Sequencing Gene Expression of Endothelial differentiation in DMP1 treated DPSCs.

status: Public on May 07 2024

city: Chicago

first: Anne

last: George

address: 801 S Paulina St

institute: University of Illinois Chicago College of Dentistry

summary: Regenerative vascular medicine has received considerable attention due to recent advancements suggesting that the conversion of somatic cells into endothelial cells offers a promising approach to address diseases related to abnormal vessel growth and endothelial cell dysfunction. The therapeutic potential of this method lies in the ability of DPSCs, upon exposure to DMP1, to differentiate into endothelial cells and support the formation of new blood vessels. Despite this progress, there is a need for a comprehensive understanding of the expression profile during endothelial differentiation. Gene ontology and genome functional enrichment analyses have provided insight into the differentiation of ECs from DMP1-treated DPSCs in conjunction with HUVEC-ECM, as compared to positive HUVECs. Our RNA sequence data underscores the striking similarity in gene expression patterns between the sorted DPSCs treated with DMP1 and the positive control HUVECs. These findings could contribute to a comprehensive understanding of the molecular mechanisms underlying the impact of DMP1 treatment on DPSCs, particularly in the context of guiding endothelial cells.

overall_design:
We analyzed gene expression to examine endothelial-specific gene expression, cell morphogenesis, and tube development in sequencing data for sorted DPSCs treated with DMP1 and HUVECs.
After 7 days culture, the cells were collected, rinsed with PBS, and then incubated with an endothelial specific antibodies CD31 and C144 under dark at ambient conditions for 40 minutes followed by flow cytometry sorting using a Moflo Astrios EQ device (UIC RRC facility). The pre-sorted and post-sorted DPSCs were used immediately for RNA Seq-Analysis.