title: Mapping the Cellular Biogeography of Human Bone Marrow Niches Using Single-Cell Transcriptomics

first: Kai

last: Tan

address: 3501 Civic Center Boulevard

department: Pediatrics

institute: Children's Hospital of Philadelphia

summary: Non-hematopoietic cells contribute essentially to hematopoiesis. However, heterogeneity and spatial organization of these cells in human bone marrow remain largely uncharacterized. We used single-cell RNA sequencing (scRNA-Seq) to profile 29,325 non-hematopoietic cells and discovered nine transcriptionally distinct subtypes. We simultaneously profiled 53,417 hematopoietic cells and predicted their interactions with non-hematopoietic subsets. We employed Co-Detection by Indexing (CODEX) to spatially profile over one million single cells with a novel 53-antibody panel. We integrated scRNA-Seq and CODEX data to link cellular signaling and spatial proximity. Spatial analysis also revealed a hyperoxygenated arterio-endosteal niche for early myelopoiesis, and an adipocytic, but not endosteal or perivascular, localization for early hematopoietic stem and progenitor cells. We used our CODEX atlas to automatically annotate cell types in new bone marrow images and uncovered MSC expansion and leukemic blast/MSC-enriched spatial neighborhoods in AML patient samples. This comprehensive, spatially-resolved multiomic atlas of human bone marrow serve as a reference for future investigation of cellular interactions that drive hematopoiesis.

overall_design: We simultaneously profiled enriched mesenchymal, HSPC, and unenriched RBC depleted cells from fresh, enzymatically digested femoral head human bone marrow from hematologically healthy patients undergoing total hip arthroplasties and subjected to paired-end single-cell RNA sequencing using 10X Genomics Single Cell 3’ v3.1 and libraries were sequenced using an Illumina Nova Seq 6000.

first: Shovik

last: Bandyopadhyay

first: Kyung

last: Ahn

first: Michael

last: Duffy

first: Ling


title: ChIP-seq analysis of gene expression regulated by LSD1 in CD8+ T cells

status: Public on Apr 24 2024

city: Hangzhou

first: Wanqiang

last: Sheng

state: Zhejiang

address: 866 Yuhangtang Road

department: Department of Immunology

country: China

institute: Zhejiang University School of Medicine

type: Genome binding/occupancy profiling by high throughput sequencing

summary: ChIP-seq assay was performed with GSK- or Vehicle-treated CD8+ T cells to interrogate the genome-wide distribution of LSD1, H3K4me1, H3K4me2, H3K4me3, H3K27ac, and Pol II, and their alterations in response to LSD1 inhibition.

overall_design: In vitro cultured CD8+ T cells were fixed in 1% formaldehyde for 10 min at RT on a shaker, followed by quenching in 125 mM glycine. Next, cells were washed twice with ice-cold PBS and lysed in sonication buffer, then sonicated to obtain DNA fragments mostly between 300-800 bp. Subsequent procedures were performed following the Epigenetics Protocols Database PROT-11 (https://www.epigenome-noe.net/researchtools/protocols.php.html). ChIP-seq libraries were prepared using the VAHTS Universal DNA Library Prep Kit (Vazyme, ND607-01) according to the manufacturer’s instructions. Library concentrations were quantified by Qubit (Invitrogen) and mixed equally for sequencing at Novaseq 6000 (Illumina) to generate 150 bp paired-end reads or at DNBSEQ-T7 (MGI Tech) to generate 100 bp paired-end reads.

first: Peishan

last: Jiang


title: Development and validation of AI/ML derived splice-switching oligonucleotides

status: Public on Apr 25 2024

city: Long Island City

first: Alyssa

last: Fronk

state: NY

address: 30-02 48th Avenue, Suite 140

country: USA

institute: Envisagenics

type: Expression profiling by high throughput sequencing

summary: Splice-switching oligonucleotides (SSOs) are antisense compounds that act directly on pre-mRNA to modulate alternative splicing (AS). This study demonstrates the value that artificial intelligence/machine learning (AI/ML) provides for the identification of functional, verifiable, and therapeutic SSOs. We trained XGboost tree models using splicing factor (SF) pre-mRNA binding profiles and spliceosome assembly information to identify modulatory SSO binding sites on pre-mRNA. Using Shapley and out-of-bag analyses we also predicted the identity of specific SFs whose binding to pre-mRNA is blocked by SSOs. This step adds considerable transparency to AI/ML-driven drug discovery and informs biological insights useful in further validation steps. We applied this approach to previously established functional SSOs to retrospectively identify the SFs likely to regulate those events. We then took a prospective validation approach using a novel target in triple negative breast cancer (TNBC), NEDD4L exon 13 (NEDD4Le13). Targeting NEDD4Le13 with an AI/ML-designed SSO decreased the proliferative and migratory behavior of TNBC cells via downregulation of the TGFβ pathway. Overall, this study illustrates the ability of AI/ML to extract actionable insights from RNA-seq data.

overall_design: RNA-seq analysis from breast cancer (and normal breast tissue) cell lines, including cell lines treated with splice-switching oligonucleotides

middle: D

first: Miguel

middle: A

last: Manzanares

first: Paulina

last: Zheng

first: Adam

last: Geier

first: Kendall

last: Anderson

first: Shaleigh

last: Stanton

first: Hasan

last: Zumrut

first: Sakshi

last: Gera

first: Robin

last: Munch

first: Vanessa

last: Frederick

first: Priyanka

last: Dhingra

first: Gayatri

last: Arun

first: Martin

last: Akerman


title: Cancer-stromal cell interactions in breast cancer brain metastases induce glycocalyx-mediated resistance to HER2-targeting therapies

city: Boston

first: Kornelia

last: Polyak

state: MA

address: 450 Brookline Ave

department: Medical Oncology

institute: Dana-Farber Cancer Institute

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

overall_design: Refer to individual Series


title: Regulation of muscle mass by Thrombospondin-1

status: Public on Apr 18 2024

city: Cincinnati

first: Jeffery

last: Molkentin

state: Ohio

address: 240 Albert Sabin Way MLC 7020

department: Molecular Cardiovascular Biology

institute: Cincinnati Children's Hospital Medical Center

summary: Loss of muscle mass is a pathological driver in both chronic illness and aging. We have discovered that skeletal muscle specific thrombospondin-1 (Thbs1) transgenic mice have profound muscle atrophy with age-dependent decreases in exercise capacity and premature lethality. Here, we compared the transcriptional profiles of skeletal muscle from control non-transgenic mice with those overexpressing Thbs1 in order to elucidate the transcriptional changes underlying the observed effects on skeletal muscle mass. The overall purpose was to identify new, effective targets to combat muscle atrophy and sarcopenia.

overall_design: Total skeletal muscle RNA was isolated and subjected to RNAseq analysis in order to identify the transcriptional effects of Thbs1 overexpression in skeletal muscle

first: Davy

last: Vanhoutte

first: Vikram

last: Prasad

first: Jeff


title: Gene expression profile at single cell level of hippocampal organoids

status: Public on Apr 20 2024

city: Shen Zhen

first: Yan

last: Wu

address: Tao Yuan

department: Department of biomedical engineering

institute: Southern University of Science and Technology

summary: Hippocampal organoids are derived from human induced pluripotent stem cells under the guidance of multiple growth factors, such as proteins, chemical inhibitors, and activators. We used single-cell RNA sequencing (scRNA-seq) to analyze the transcriptomic signature of hippocampal organoids so that we could characterize cell identities in them. We prepared two single-cell samples from day-70 and day-81 hippocampal organoids.

overall_design: We used 4 day-70 hippocampal organoids to prepare sample #1. Organoids were dissected into fragments and plated on flasks. We cultured them for 2-4 days, then isolated them with a Pierce Primary Neuron Isolation Kit with papain enzyme. We used 10 day-81 hippocampal organoids to prepare sample #2. Organoids were isolated directly with a Pierce Primary Neuron Isolation Kit with papain enzyme, broken up by pipetting up and down. The pre-sorting of live cells was completed using magnetic-activated cell sorting. The 3’ Gene expression Library was prepared with the quality-qualified cDNA based on Chromium Next GEM Single-cell 3’ GEM protocol. The quality of the library was assessed according to the standard protocol after fragmentation, adaptor ligation, and sample index PCR.

first: Jin

last: Wen


title: A conserved Pol II elongator SPT6L mediates Pol V transcription to regulate RNA-directed DNA methylation in Arabidopsis

status: Public on Apr 22 2024

city: Guangzhou

first: Chen

last: Chen

state: Guangdong

address: 723 Xinke Rd.

institute: South China Botanical Garden, Chinese Academy of Science

type: Methylation profiling by high throughput sequencing

type: Non-coding RNA profiling by high throughput sequencing

summary: In plants, the plant-specific RNA polymerase V (Pol V) transcripts non-coding RNA and provides a docking platform for the association of accessory proteins in the RNA-directed DNA methylation (RdDM) pathway. Various components were uncovered in the process of DNA methylation, but it is still not clear how the transcription of Pol V is regulated. Here, we found the conserved elongation factor, SPT6L, bound to thousands of intergenic regions in an RNA polymerase II (Pol II) independent manner. The intergenic enrichment of SPT6L, interestingly, co-occupied with the largest subunit of Pol V (NRPE1) and the mutation of SPT6L led to the reduction of DNA methylation but not Pol V enrichment. Furthermore, the association of SPT6L at Pol V loci was dependent on the Pol V associated factor, SPT5L, rather than the presence of Pol V, and the interaction between SPT6L and NRPE1 was compromised in spt5l. Finally, Pol V RIP-seq revealed that SPT6L is required to maintain the amount and length of Pol V transcripts. Our findings revealed the critical role of a Pol II conserved elongation factor in Pol V mediated DNA methylation and transcription, and shed light on the mutual regulation between Pol V and Pol II in plants.

overall_design: 10-day old seedlings were used for ChIP-seq, BS-seq, RNA-seq, and RIP-seq assays.

first: Yujuan

last: Liu

first: Jie

last: Shu

first: Zhi

last: Zhang

first: Ning

last: Ding

first: Jinyuan

first: Jun

first: Yuhai

last: Cui

first: Changhu

last: Wang


title: The Fur-like regulatory protein MAP3773c modulates key metabolic pathways in Mycobacterium avium subsp. paratuberculosis under in-vitro iron starvation

city: East Lansing

first: Srinand

last: Sreevatsan

state: MI

address: 784 WILSON RD

department: Pathobiology and Diagnostic investigation

institute: Michigan State University

summary: Mycobacterium avium subsp. paratuberculosis (MAP) carries MAP3773c, a putative metal regulator, on its genome which is absent in other mycobacteria. Homologues of this gene in enterobacteria have roles in global iron regulation and homeostasis. The role of MAP3773c in regulating intracellular iron in MAP is poorly understood. Functional analysis of DEGs in ΔMAP3773c revealed that pantothenate (Pan) biosynthesis, polysaccharide biosynthesis, sugar metabolism and N-acetyltransferase enhanced intracellular survival genes were downregulated at 30 minutes post-iron starvation whereas ATP-binding cassette (ABC) type metal transporters, putative siderophore biosynthesis, arginine and proline metabolism, PPE, PE family and mammalian cell entry (MCE) genes were upregulated at that same time point. Pathway analysis revealed that the MAP3773c knockout has an impairment in Pan and Coenzyme A (CoA) biosynthesis pathways at 30 minutes post-iron starvation suggesting that the absence of those pathways likely affect overall metabolic processes and cellular functions, which have consequences affecting MAP survival and pathogenesis

overall_design: A field isolate (K-10) and an in-frame MAP3773c deletion mutant (ΔMAP3773c) derived from K-10, were exposed to iron restricted conditions for 5, 30, 60, and 90 minutes in six replicates each. Total RNA was extracted at each time point, quality and integrity of the RNA was assessed. Two replicates from each strain and each time points were send for RNA-Sequencing. An iron replete baseline culture was also processed for mRNA extraction and sequencing. A comparison of transcriptional profiles between K-10 and ΔMAP3773c were performed.

first: Sajani

last: Thapa

first: Govardhan

last: Rathnaiah

first: Denise

middle: K

last: Zinniel

first: Raul

middle: G

last: Barletta

first: John

middle: P

last: Bannantine

first: Marianne

last: Huebner


title: plasma exosome from mice bearing Ago2wt and Ago2dm tumor

city: Miaoli County

first: LU

middle: LI

last: YU

address: 35, Keyan Road, Zhunan Town,

country: Taiwan

institute: ational Health Research Institutes

summary: Exosomes are small membrane bound cell-derived vesicles that are present in biological fluids include blood and cell culture medium. Exosomes contain various functional proteins, mRNAs and microRNAs (miRNAs). We used miRNA microarrays to detail the miRNA content in plasma exosomes of mice bearing A549Ago2-KO/HA-Ago2Wt and A549Ago2-KO/HA-Ago2Δ (Dm) tumors.

overall_design: Plasma exosomes of mice bearing A549Ago2-KO/HA-Ago2Wt and A549Ago2-KO/HA-Ago2Δ (Dm) tumors were collected for miRNA extraction and hybridization on Affymetrix GeneChip® miRNA 4.0 array.

first: Hua-Jung

last: Li

first: Li-yu

last: Lu


title: miRNA differentially expressed in P2-peptide and P2S-peptide treated A549 cancer cells.

city: Miaoli

first: meng chieh

last: lin

address: 35 Keyan Road

institute: National Health Research Institutes

summary: To determine the role of Ago2/Cav1 interaction in Ago2 function, we used   miRNA arrays to examine the levels of miRNA in cancer cells whose Ago2/Cav1 interaction was inhibited by P2 or P2S peptides. Disruption of Ago2/Cav1 interaction by P2 peptides resulted in fluctuations in different  miRNAs  in  cancer  cells

overall_design: After P2-peptide and P2S-peptide treated  A549 cancer cells, toatl cells were collected from for miRNA extraction and hybridization on Affymetrix GeneChip® miRNA 4.0 array.

first: Meng-chieh

last: Lin


title: Uncovering shared Molecular and Histopathological features of accepted versus rejected donor livers for transplantation

city: Albuquerque

first: Rajanikanth

last: Vadigepalli

state: PA

address: 1 University of New Mexico

institute: University of New Mexico

summary: Liver transplantation is the solution for acute or chronic liver failure. There is an unmet demand for liver transplantation, as not all donated organs are transplanted. The clinical selection criteria for donated livers depends on histopathological evaluation and liver function tests. A fraction of the donor livers are rejected for transplantation due to the variability in applying the selection criteria. We applied an integrated transcriptomics and histopathological approach to characterize the donor livers evaluated for transplantation. We obtained liver biopsies from deceased donors during organ collection (n=10 accepted and n= 21 rejected for transplantation). We performed RNA sequencing to characterize the global gene expression profiles. We assessed steatosis, fibrosis, and necrosis via manual histopathological evaluation and a custom artificial intelligence-based image analysis. We identified two transcriptomically distinct subsets in the rejected donor liver group (termed rejected-1 and rejected-2), where the rejected-2 subset had a near complete transcriptomic overlap with the accepted livers, suggesting the potential for acceptability from a molecular standpoint. The liver metabolic functional genes were similarly upregulated in the accepted and rejected-2 groups compared to the rejected-1 subset. Expression levels of several extracellular matrix genes were similarly low in the accepted and rejected-2 groups compared to the rejected-1 subset. Filtering the rejected-2 subset based on histopathological evaluation identified the cases with borderline scores and extensive molecular overlap with the accepted group. Our integrated molecular and histopathological approach identified a subset of rejected donor livers that may be suitable for transplantation, thereby expanding the pool of transplantable livers.

overall_design: Transcriptomic profiling of the deceased donor liver biopsy samples either accepted (n=10) or rejected (n=22) for transplantation.

first: Ankita

last: Srivastava

first: Alexandra

last: Manchel

first: Benjamin

last: Barnhart

first: Jan

middle: B

last: Hoek


title: Gene expression in kidney cells after unilateral ischemia-reperfusion

status: Public on Apr 17 2024

city: Durham

first: Jamie

last: Privratsky

state: NC

address: 303 Research Dr., Sands 101

department: Anesthesiology

institute: Duke University

summary: To determine changes in gene expression due to ischemia-reperfusion AKI in different intra-renal cell populations

overall_design: Male wildtype mouse subjected to left-sided unilateral ischemia (27 minutes) followed by reperfusion. Ischemic kidney (IR) along with contralateral, uninjured kidney (control) harvested at 24 hrs and single cell RNA sequencing performed.

middle: R

first: Yanting

last: Chen


title: Frataxin Deficiency Drives a Shift from Mitochondrial Metabolism to Glucose Catabolism, Triggering an Inflammatory Phenotype in Microglia (scRNA-Seq)

status: Public on Apr 16 2024

city: Roma

first: Andrea

last: Ninni

state: RM

address: Via della Ricerca Scientifica

department: Biology

country: Italy

institute: Tor Vergata University

summary: Immunometabolism investigates the complex interplay between the immune system and cellular metabolism. This study highlights the effects of mitochondrial frataxin (FXN) depletion, which causes Friedreich's ataxia (FRDA), a neurodegenerative condition characterized by coordination and muscle control deficiencies. Using single-cell RNA sequencing, we identified specific cell groups in the cerebellum of a FRDA mouse model, emphasizing a notable inflammatory microglial response. These FXN-deficient microglia cells exhibited enhanced inflammatory reactions. Furthermore, our metabolomic analyses revealed increased glycolysis and itaconate production in these cells, possibly driving the inflammation. Remarkably, butyrate treatment counteracted these immunometabolic changes, triggered an antioxidant response via the itaconate-Nrf2-GSH pathways, and dampened inflammation. The study also pinpointed Hcar2 (GPR109A) as a potential agent for butyrate anti-inflammatory impact on microglia. Tests on FRDA mice highlighted the neuroprotective attributes of butyrate intake, bolstering neuromotor performance. In essence, our findings shed light on how cerebellar microglia activation contributes to FRDA and highlight butyrate potential to alleviate neuroinflammation, rectify metabolic imbalances, and boost neuromotor capabilities in FRDA and similar conditions.

overall_design: The transcriptional profile of the cerebellum in frataxin-deficient (KIKO) mice was compared to that of wild-type (WT) mice at the single-cell level.

first: Daniele

last: Lettieri Barbato

first: Francesca

last: Sciarretta

first: Fabio

last: Zaccaria


title: CEBPA restricts alveolar type 2 cell plasticity during development and injury-repair [scATAC-seq]

city: Houston

first: Dalia

last: Hassan

state: TX

department: Pulmonary Medicine

institute: MD Anderson Cancer Center

summary: Cell plasticity theoretically extends to all possible cell types, but naturally decreases as cells differentiate, whereas injury-repair re-engages the developmental plasticity. Here we show that the lung alveolar type 2 (AT2)-specific transcription factor (TF), CEBPA, restricts AT2 cell plasticity in the mouse lung. AT2 cells undergo transcriptional and epigenetic maturation postnatally. Without CEBPA, both neonatal and mature AT2 cells reduce the AT2 program, but only the former reactivate the SOX9 progenitor program. Sendai virus infection bestows mature AT2 cells with neonatal plasticity where Cebpa mutant, but not wild type, AT2 cells express SOX9, as well as more readily proliferate and form KRT8/CLDN4+ transitional cells. CEBPA promotes the AT2 program by recruiting the lung lineage TF NKX2-1. The temporal change in CEBPA-dependent plasticity reflects AT2 cell developmental history. The ontogeny of AT2 cell plasticity and its transcriptional and epigenetic mechanisms have implications in lung regeneration and cancer.

overall_design: FACS-purified lung epithelial, immune, endothelial, and mesenchymal cells were processed using the 3′ Library and Gel Bead Kit following the manufacturer’s users guide (v2 rev D) on the Chromium Single-Cell Gene Expression Solution Platform (10X Genomics).

first: Jichao


title: Targeting Set7 in an experimental model of diabetic nephropathy

city: Melbourne

first: Assam

last: El-Osta

address: 75 Commercial Road

country: Australia

institute: Baker Heart and Diabetes Institute

summary: Set7 knockout (Set7KO) improved glomerular structure and albuminuria in a mouse model of diabetes. Analysis of mouse single-cell RNA-sequencing data showed dynamic transcriptional changes in diabetic renal cells. Set7KO controls phenotype switching of GEN cell populations through transcriptional regulation of the insulin growth factor binding protein 5 (IGFBP5). Chromatin immunoprecipitation assays confirmed that the expression of the IGFBP5 gene was associated with mono- and dimethylation of histone H3 lysine 4 (H3K4me1/2). This generalizability was investigated in human renal and circulating hyperglycemic cells exposed to TGFb1. We showed that the highly selective Set7 inhibitor PFI-2 attenuated indices associated with renal cell damage and mesenchymal transition, specifically (1) reactive oxygen species production, (2) IGFBP5 gene regulation, and (3) expression of mesenchymal markers. Furthermore, renal benefit observed in Set7KO diabetic mice closely corresponds in human glomerular endothelial cells with PFI-2 inhibition or Set7 shRNA silencing.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: Single cell RNA analysis (Drop-seq) of diabetic kidney tissue from streptozotocin-induced Setd7 deficient (Setd7-/-) mice. Mouse models included: Diabetic (Setd7-/- ApoE-/-); Control (Setd7-/- ApoE-/-); Diabetic (Setd7+/+ ApoE-/-); Control (Setd7+/+ ApoE-/-). For target validation experiments, human immortalized glomerular endothelial (GEN) cells were cultured with or without 15 mM (R)-PFI-2 (PFI-2; Cayman Chemicals) dissolved in DMSO for 24 hours before exposing them to 5.5 or 25 mM D-glucose in the presence or absence of 5 ng/ml TGF-b1 (R&D Systems) for 48 hours at 37°ockdown of Set7 was performed in GEN cells using MISSION shRNA expressing lentivirus vector (Sigma). Cells transduced with MISSION Non-target shRNA control vector (Sigma) were used as a control. GEN treatment groups included: GEC_NG (5.5 mM D-glucose); GEC_PFI (25 mM D-glucose + 15 mM (R)-PFI-2 + 5 ng/ml TGF-b1); GEC_TGF (25 mM D-glucose + 5 ng/ml TGF-b1); KD_TGF (25 mM D-glucose + Set7 shRNA + 5 ng/ml TGF-b1); NT_TGF (25 mM D-glucose + MISSION Non-target shRNA + 5 ng/ml TGF-b1). Single-cell transcriptomics was used to investigate Set7 regulation in a mouse model of DKD, followed by validation of findings using pharmacological and shRNA inhibition of Set7.

first: Jun

last: Okabe

first: Scott

last: Maxwell


title: MNase-Seq analysis of wild-type Candida glabrata strain in RPMI-growth and macrophage-internalized conditions at 2 h and 10 h post-infection.

city: Hyderabad

first: Rupinder

last: Kaur

state: Telangana

address: Inner ring road, Uppal

country: India

institute: Centre for DNA Fingerprinting and Diagnostics

summary: MNase-sequencing analysis led to identification of 56,637 to 71,823 nucleosomes under RPMI-growth or macrophage-internalized conditions. Mapping of dynamic nucleosomes revealed that 12 to 24% of total nucleosomes were altered in their position, occupancy or fuzziness. Notably, while the nucleosome position shift was the most common dynamic event, the nucleosome fuzziness was the least observed change. Promoter regions were found to be nucleosome-depleted.

overall_design: The study was designed to delineate the effect of the macrophage internal milieu on the chromatin dynamics in C. glabrata. C. glabrata wild-type cells were grown either in RPMI medium or infected to human THP-1 macrophages. After 2 h and 10 h, C. glabrata cells were collected, and chromatin structure was analysed via MNase-Sequencing approach. The experiment was performed with two biological replicates.

first: Kundan

last: Kumar

first: Aditi

last: Pareek


title: Transposable elements regulate T cell maturation and education in the thymus

city: Montréal

first: Claude

last: Perreault

state: Québec

address: 2950, Chemin de Polytechnique

country: Canada

institute: Institute for Research in Immunology and Cancer

summary: Antigen presentation by cortical and medullary thymic epithelial cells (cTEC and mTEC) ensures the formation of a self-restricted and self-tolerant T cell repertoire, respectively. As such, a broad diversity of self-antigens needs to be presented by mTEC to induce T cell\u2019s self-tolerance. Even though the expression and antigen presentation of protein coding genes in mTEC has been abundantly described, little is known of the implication of allegedly noncoding regions of the genome to tolerance induction. In this study, we focused on transposable elements (TE), which have been shown to be highly expressed by mTEC.

overall_design: Transcriptomic data from murine cTEC and mTEC were used to build personnalized databases for the identification of MHC I-associated peptides in mass spectrometry

first: Jean-David

last: Larouche


title: Murine Model of Long- COVID19 following SARS-CoV-2 Variant Infection

status: Public on Apr 15 2024

city: Galveston

first: Haiping

last: Hao

state: Texas

address: 301 University Blvd

institute: UTMB NGS Core

summary: Viral variant is one known risk factor associated with post-acute sequelae of COVID-19 (PASC), yet the pathogenesis is largely unknown.  We studied SARS-CoV-2 Delta variant-induced PASC in K18-hACE2 mice. The virus replicated productively, induced robust inflammatory responses in lung and brain tissues, and caused weight loss and mortality during the acute infection.  Longitudinal behavior studies, in surviving mice up to 4 months post-acute infection, revealed persistent abnormalities in neuropsychiatric state and motor behaviors, while reflex and sensory functions recovered over time. Surviving mice showed no detectable viral RNA in the brain and minimal neuroinflammation post-acute infection.  Transcriptome analysis revealed persistent activation of immune pathways, including humoral responses, complement, and phagocytosis, and reduced levels of genes associated with ataxia telangiectasia, impaired cognitive function and memory recall, and neuronal dysfunction and degeneration.  Furthermore, surviving mice maintained potent T helper 1 prone cellular immune responses and high neutralizing antibodies in the periphery for months post-acute infection. Overall, infection in K18-hACE2 mice recapitulates the persistent clinical symptoms reported in long COVID patients.

overall_design: 6- to 8-week-old K18-hACE2 mice were infected intranasally with 600PFU of SARS-CoV-2 Delta strain. At 1-month (n =4), and 4-months (n=3) post infection, brain samples were collected for RNA extraction.  PBS-infected mice (n=4) were collected at 4-month as mock-samples for comparison.

first: Ankita

last: Singh

first: Tian

last: Wang

first: Awadalkareem

last: Adam


title: Frataxin Deficiency Drives a Shift from Mitochondrial Metabolism to Glucose Catabolism, Triggering an Inflammatory Phenotype in Microglia

first: Andrea

last: Ninni

overall_design: The transcriptional profile of the cerebellum in frataxin-deficient (KIKO) mice was compared to that of wild-type (WT) mice at the bulk and single-cell level.


title: Convergent Epigenetic Evolution Drives Relapse in Acute Myeloid Leukemia

city: Stanford

first: Ravindra

last: Majeti

state: CA

address: 875 Blake Wilbur Dr Clinic F

institute: Stanford University

summary: Relapse of acute myeloid leukemia (AML) is highly aggressive and often treatment refractory. We analyzed previously published AML relapse cohorts and found that 40% of relapses occur without changes in driver mutations, suggesting that non-genetic mechanisms drive relapse in a large proportion of cases. We therefore characterized epigenetic patterns of AML relapse using 26 matched diagnosis-relapse samples with ATAC-seq. This analysis identified a relapse-specific chromatin accessibility signature for mutationally stable AML, suggesting that AML undergoes epigenetic evolution at relapse independent of mutational changes. Analysis of leukemia stem cell (LSC) chromatin changes at relapse indicated that this leukemic compartment underwent significantly less epigenetic evolution than non-LSCs, while epigenetic changes in non-LSCs reflected overall evolution of the bulk leukemia. Finally, we used single-cell ATAC-seq paired with mitochondrial sequencing (mtscATAC) to map clones from diagnosis into relapse along with their epigenetic features. We found that distinct mitochondrially-defined clones exhibit more similar chromatin accessibility at relapse relative to diagnosis, demonstrating convergent epigenetic evolution in relapsed AML. These results demonstrate that epigenetic evolution is a feature of relapsed AML and that convergent epigenetic evolution can occur following treatment with induction chemotherapy.

overall_design: Bulk ATAC sequencing of bulk AML cells, AML blasts, and AML LSCs at diagnosis and relapse post-chemotherapy. Single cell ATAC sequencing of diagnosis and relapse bulk AML cells.

first: Kevin

last: Nuno

first: Armon

last: Azizi

first: Thomas

last: Köhnke

first: Caleb

last: Lareau

first: Asiri

last: Ediwirickrema

first: M

last: Corces

first: Ansuman

middle: T

last: Satpathy


title: High-altitude hypoxia exposure inhibits erythrophagocytosis by inducing macrophage ferroptosis in the spleen

city: Nantong

first: Qianqian

last: Luo

state: Jiangsu

address: No.9, Seyuan Road, Chongchuan district, Nantong, Jiangsu Province, China

institute: Nantong University

summary: High-altitude polycythemia (HAPC) is a chronic condition prevalent in individuals residing at high altitudes (HA), characterized by an augmented production of red blood cells (RBCs) due to hypoxic conditions. Despite its prevalence, the pathophysiological basis and molecular mechanisms driving HAPC remain largely unelucidated. In our study, we utilized a mouse model exposed to hypobaric hypoxia (HH), replicating the environmental conditions experienced at 6000 m above sea level, coupled with in vitro analysis of primary splenic macrophages under 1% O2 to investigate these mechanisms. Our findings revealed that HH exposure significantly stimulates erythropoiesis, leading to erythrocytosis. This was accompanied by a notable sequence of splenic changes, initially characterized by splenic contraction, which subsequently progressed to splenomegaly over 14 days. A key observation was the impairment on the capacity of spleen to process RBCs, primarily attributed to a reduction in splenic macrophages located in the red pulp. Extended HH treatment over 7 and 14 days precipitated increased iron mobilization and the onset of ferroptosis within the spleen. This was evidenced by altered expression levels of iron metabolism and ferroptosis-related proteins, paralleling gene expression patterns observed in human peripheral blood mononuclear cells. Single-cell sequencing of splenic tissue post-HH exposure demonstrated a marked decrease in macrophage populations after 7 days. Our study further disclosed a significant increase in RBCs retention in the spleen following HH exposure, likely a consequence of diminished red pulp macrophages (RPMs) and erythrophagocytosis. This hypothesis was corroborated through comprehensive analyses involving flow cytometry, histological staining, and immunostaining, which collectively provided a detailed understanding of RBCs dynamics under HH conditions. In vitro analyses substantiated the decline in primary splenic macrophages and induction of ferroptosis under hypoxic treatment, effects that were relieved by pre-treatment with the ferroptosis inhibitor ferrostatin-1. Collectively, our data suggest that HH exposure initiates splenic ferroptosis, leading primarily to a decrease in RPMs. This decrease potentially impacts erythrophagocytosis, contributing to increased RBCs retention and subsequent splenomegaly. Such changes could potentially foster continuous RBCs production and accelerate the progress of HAPC. In conclusion, our study highlights the important role of the spleen and splenic macrophages in the pathogenesis of HAPC, providing crucial insights into the disease progression and potential therapeutic targets.

overall_design: C57BL/6J mice were exposed to a hypobaric hypoxia (HH) chamber for 7 days to establish an animal model simulating high altitude exposure at 6000 meters, while the control group animals were kept in a similar chamber under normobaric normoxia (NN) conditions for 7 days. Spleen tissues were surgically extracted and preserved in MACS Tissue Storage Solution (Miltenyi Biotec, Bergisch Gladbach, Germany) until further processing. Single-cell dissociation was performed by researchers at the GENECHEM laboratory (Shanghai, China). Dissociated single cells were then stained with Calcein-AM (BD Biosciences, USA) and Draq7 (BD Biosciences) for viability assessment. The BD Rhapsody system was utilized to capture transcriptomic information from individual cells. Library quantification was conducted using a High Sensitivity D1000 ScreenTape and High Sensitivity D1000 Reagents on a 4150 TapeStation System (Agilent, Palo Alto, CA, USA), as well as the Qubit High Sensitivity DNA assay (Thermo Fisher Scientific). Sequencing of libraries was carried out on an Illumina sequencer (Illumina Nova Seq 6000, San Diego, CA) through a paired-end run of 150 bp.

first: Wan-ping

last: Yang

first: Mei-qi

last: Li

first: Jie

last: Ding

first: Jia-yan

first: Guo-hua

first: Qian-qian


title: CEBPA restricts alveolar type 2 cell plasticity during development and injury-repair

first: Dalia

last: Hassan

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

overall_design: Refer to individual Series


title: The Nucleic Acid Binding Protein SFPQ Represses EBV Lytic Reactivation by Promoting Histone H1 Expression

city: Boston

first: Benjamin

last: Gewurz

address: 181 Longwood Avenue, Channing Laboratory, 8th Floor

institute: Brigham and Women's Hospital

summary: Epstein-Barr virus (EBV) uses a biphasic lifecycle of latency and lytic reactivation to infect >95% of adults worldwide. Despite its central role in EBV persistence and oncogenesis, much remains unknown about how EBV latency is maintained. We used a human genome-wide CRISPR/Cas9 screen to identify that the nuclear protein SFPQ was critical for latency. SFPQ supported expression of linker histone H1, which stabilizes nucleosomes and regulates nuclear architecture, but has not been previously implicated in EBV gene regulation. H1 occupied latent EBV genomes, including the immediate early gene BZLF1 promoter. Upon reactivation, SFPQ was sequestered into sub-nuclear puncta, and EBV genomic H1 occupancy diminished. Enforced H1 expression blocked EBV reactivation upon SFPQ knockout, confirming it as necessary downstream of SFPQ. SFPQ knockout triggered reactivation of EBV in B and epithelial cells as well as in Kaposi’s Sarcoma Associated Herpesvirus, suggesting a conserved gamma-herpesvirus role. These findings highlight SFPQ as a major regulator of H1 expression and EBV latency.

overall_design: SFPQ sgRNA or a control sgRNA transduced into Cas9 expressing cells. SFPQ was knocked out using CRISPR-Cas9. Cells were collected, and rRNA-depletion was performed prior to library preparation and sequencing.

first: Laura

last: Murray-Nerger

first: Clarisel

last: Lozano

first: Eric

middle: M

last: Burton

first: Yifei

last: Liao

first: Nathan

last: Ungerleider

first: Rui

last: Guo

middle: E


title: Single-cell gene expression profiling of interstitial macrophages exposed to hypoxia

city: Aurora

first: Sushil

last: Kumar

institute: University of Colorado Anschutz medical campus

summary: Employing single-cell transcriptomic analyses, we investigated the repertoire and functional profiles of pulmonary interstitial macrophages in response to exposure at 0, 1, 3, 7, and 21 days of hypobaric hypoxia (at an altitude of 5,486 meters). The repertoire and functional profiles of interstitial macrophages (IMs) undergo dynamic changes based on the duration of hypoxia exposure. Acute hypoxia leads to type 1 acute inflammatory responses in most IM populations, which largely subside by Day 7. The resolution of the inflammatory phase is succeeded by the accumulation of dysregulated IMs, contributing to abnormal pulmonary vascular repair and remodeling. The classical and alternative complement pathways may play distinct roles in regulating early inflammation and later vascular remodeling phases in pulmonary hypertension (PH) pathogenesis. These findings highlight the dynamic nature of IMs, emphasizing that understanding the timing and mechanisms through which they influence PH pathogenesis will contribute to the development of IM-specific therapeutic targets.

overall_design: Cx3cr1+/GFP reporter mice were crossbred with C57BL/6J and raised. To eliminate potential confounding factors associated with adaptation to reduced oxygen levels at Denver altitude, Cx3cr1+/GFP animals were placed in a sea-level chamber (~21% fraction of inspired oxygen (FiO2)) at 5 weeks of age for 4 weeks to acclimate to sea-level conditions. Subsequently, animals were kept at sea level (Day 0) or exposed to hypoxic conditions (simulated altitude of 5,486 meters; ~10% FiO2) for 1, 3, 7, or 21 days. At each time point, the lungs of a pair of age- and sex-matched mice (1 male and 1 female) were harvested for subsequent analyses. Pulmonary interstitial macrophages were sorted by flow cytometry as previously described [PMID:]. Single cells were captured using a Chromium Box (10X Genomics, Pleasanton, CA). Libraries were sequenced using the Illumina NovaSEQ 6000 sequencer (Illumina, Inc., San Diego, CA). Subsequently, RNA sequence reads were aligned to the Mus musculus GRCm39 reference genome using Cell Ranger v6 (10x Genomics). Downstream analysis was performed using the Seurat package in R.

first: Claudia

last: Mickael

first: Rahul

first: Ram

last: Prasad

first: Nzali

middle: V

last: Campbell

first: Hui

last: Zhang

first: Min

first: Brittany

last: McKeon

first: Thaddeus

last: Allen

first: Brian

last: Graham

first: A

middle: Y

last: Yu

first: Kurt

last: Stenmark


title: Prophage-encoded methyltransferase upregulates virulence to drive adaptation in an outbreak of community-acquired methicillin-resistant Staphylococcus aureus

city: New York

first: Alejandro

last: Pironti

address: 430 East 29th Street

department: Microbiology

institute: NYU Langone Health

summary: To determine the effects of phage adenine methyltransferase (pamA) on gene expression in WT USA300 (LAC*)

overall_design: Comparative gene expression analysis of WT::pamA (USA300 LAC* containing a single copy of constituatively expressed pamA) and WT::EV control.

first: Robert

middle: J

last: Ulrich

first: Bo

last: Shopsin


title: Disulfiram treatment suppresses antibody-producing reactions by inhibiting macrophage activation and B cell pyrimidine metabolism

city: Bunkyo-ku

first: Etsuko

last: Toda

state: Tokyo

country: Japan

institute: Nippon Medical School

summary: Antibody responses, involving B cells, CD4+ T cells, and macrophages, are implicated in autoimmune diseases and organ transplant rejection. We have previously shown that inhibiting FROUNT with disulfiram (DSF) suppresses macrophage activation and migration, effectively treating inflammatory diseases. In this study, we investigated the effectiveness of DSF in antibody-producing reactions. Using a heart transplantation mouse model with antibody-mediated rejection, we administered anti-CD8 antibody to exclude cellular rejection. DSF directly inhibited B cell responses in vitro and significantly reduced plasma donor-specific antibodies and graft antibody deposition in vivo, resulting in prolonged survival of the heart graft. DSF also mediated various effects, including decreased macrophage infiltration and increased Foxp3+ regulatory T-cells in the grafts. Additionally, DSF inhibited pyrimidine metabolism-related gene expression induced by B-cell stimulation. These findings demonstrate that DSF modulates antibody production in the immune response complexity by regulating B-cell and macrophage responses.

overall_design: To investigate the effect of disulfiram on B cell gene expression upon stimulation, mouse splenic B cells were treated with disulfiram and then stimulated with LPS or IL-4+CD40 antibody for 3 hours. Gene expression analysis was conducted using RNA-seq data from 4 replicates in experiment 1 and a single replicate in experiment 2. In experiment 2, for comparison, cells were also treated with ibrutinib instead of disulfiram.

first: Weili

last: Chen

first: Yurika

last: Sawa

first: Naomi

last: Kuwahara

first: Yuya

last: Terashima

first: Akira

last: Shimizu


title: RNA-Seq analysis of Candida glabrata wild-type and Cgsnf2Δ strains in RPMI-grown or macrophage-internalized conditions at 2 h and 10 h post-infection.

first: Rupinder

last: Kaur

summary: The goal of the current study was to identify differentially-expressed genes upon CgSNF2 deletion, as well as, upon macrophage internalization in C. glabrata wild-type (wt) and Cgsnf2Δ strains. For this study, RNA samples were collected from RPMI-grown and macrophage-internalized cells of C. glabrata wild-type and Cgsnf2Δ strains at 2 h and 10 h post-infection. Comparative transcriptome analysis shows the differential regulation of 1419 genes in C. glabrata wild-type cells upon macrophage internalization, whereas the CgSNF2 deletion leads to altered regulation of 935 genes in C. glabrata wild-type cells.

overall_design: The study was designed to profile the transcriptomes of C. glabrata wild-type and Cgsnf2Δ cells grown either in RPMI medium or internalized by macrophages, via RNA-Sequencing approach, at two time points viz., 2 h and 10 h. The experiment was performed with two biological replicates.

first: Kundan

last: Kumar

first: Aditi

last: Pareek


title: Gene expression profile at single cell level of p16INK4a+ fibroblasts from fibrotic mouse lung

city: San Francisco

first: Jinyoung

last: Lee

address: 513 Parnassus Avenue

institute: UCSF

summary: We studied the role of p16INK4a+ fibroblasts in lung fibrosis. We used single cell RNA seq (scRNA-seq) to characterize p16INK4a+ fibroblasts in fibrotic lung.

overall_design: The p16INK4a+ lung fibroblasts of INKBRITE mice were isolated by Fluorescence-activated cell sorting (FACS) according to the presence of eGFP signal and analyzed using scRNAseq.

first: Tien

last: Peng


title: Prophage-encoded methyltransferase upregulates virulence to drive adaptation in an outbreak of community-acquired methicillin-resistant Staphylococcus aureus [Harm analysis]

summary: To determine the effects of phage m⏀11 on gene expression in  USA300 (LAC*)

overall_design: Comparative gene expression analysis of USA300 LAC* with and without m⏀11, using two biological replicates grown to late exponential phase


title: SWI/SNF complex-mediated chromatin remodelling in Candida glabrata is vital for immune evasion


title: Proteostatic reactivation of the developmental transcription factor TBX3 drives BRAF/MAPK-mediated tumorigenesis.

city: Tianjin

first: Zhenlei

last: Zhang

address: Qixiangtai Road 22

institute: Tianjin Medical University

summary: MAPK pathway-driven tumorigenesis, often induced by BRAFV600E, relies on epithelial dedifferentiation. However, how lineage differentiation events are reprogrammed remains unexplored. Here, we demonstrate that proteostatic reactivation of developmental factor, TBX3, accounts for BRAF/MAPK-mediated dedifferentiation and tumorigenesis. During embryonic development, BRAF/MAPK upregulates USP15 to stabilize TBX3, which orchestrates organogenesis by restraining differentiation. The USP15-TBX3 axis is reactivated during tumorigenesis, and Usp15 knockout prohibits BRAFV600E-driven tumor development in a Tbx3-dependent manner. Deleting Tbx3 or Usp15 leads to tumor redifferentiation, which parallels their overdifferentiation tendency during development, exemplified by disrupted thyroid folliculogenesis and elevated differentiation factors such as Tpo, Nis, Tg. The clinical relevance is highlighted in that both USP15 and TBX3 highly correlates with BRAFV600E signature and poor tumor prognosis. Thus, USP15 stabilized TBX3 represents a critical proteostatic mechanism downstream of BRAF/MAPK-directed developmental homeostasis and pathological transformation, supporting that tumorigenesis largely relies on epithelial dedifferentiation achieved via embryonic regulatory program reinitiation.

overall_design: mRNA profiles of mouse thyroidcontrol and Tbx3 knockout were generated by deep sequencing, in duplicate, using Illumina novaseq6000.

first: Li

last: Zhao

first: Yufan

last: Wu


title: Role of MIR9-2 in testicular germ cell tumours

city: Nottingham

first: Cinzia

last: Allegrucci

state: Notts

address: Science Road

department: Biodiscovery Institute

country: United Kingdom

institute: University of Nottingham

summary: The testicular germ cell tumour cell lines  were transfected with MIR9-2-5p mimic to study the effect on gene expression by RNA sequencing.

overall_design: NTERA-2 and TCam-2 cells lines were transfected with MIR9-2-5p and scrambled mimics and gene expression compared after 3 days treatment.

first: Ryan

last: Cardenas

first: Ahmad

last: Zyoud

first: Nigel

last: Mongan


title: FOXC1 regulates endothelial CD98 (LAT1/4F2hc) expression in retinal angiogenesis and blood-retina barrier formation

city: Chicago

first: Teena

last: Bhakuni

state: Illinois

address: 303 E Superior St

institute: Northwestern University

summary: Angiogenesis, the growth of new blood vessels from pre-existing vasculature, is essential for the development of new organ systems, but transcriptional control of angiogenesis remains incompletely understood. Here we report that FOXC1 is essential for retinal angiogenesis. Endothelial cell (EC)-specific loss of Foxc1 impairs retinal vascular growth and expression of Slc3a2 and Slc7a5, which encode the heterodimeric CD98 (LAT1/4F2hc) amino acid transporter and regulate the intracellular transport of essential amino acids and activation of the mammalian target of rapamycin (mTOR). EC-Foxc1 deficiency diminishes mTOR activity, while administration of the mTOR agonist MHY-1485 rescues perturbed retinal angiogenesis. EC-Foxc1 expression is required for retinal revascularization and resolution of neovascular tufts in a model of oxygen-induced retinopathy. Foxc1 is also indispensable for pericytes, a critical component of the blood-retina barrier during retinal angiogenesis. Our findings establish FOXC1 as a crucial regulator of retinal vessels and identify therapeutic targets for treating retinal vascular disease.

overall_design: To investigate transcriptional regulation of retinal angiogenesis by transcription factor FOXC1, we administered tamoxifen to endothelial-specifc FOXC1 knockout (EC-Foxc1-KO) mice and littermate WT controls during early postnatal retinal angiogenesis. Retinal endothelial cells were isolated from pooled retinas of WT control or littermate EC-Foxc1-KO mice using CD31 pre-conjugated magnetic beads and processed for RNA extraction and transcriptomics analysis.

first: Pieter

last: Norden

first: Naoto

last: Ujiie

first: Can

last: Tan

first: Sun

first: Thomas

last: Tedeschi

first: Yi-Wen

last: Hsieh

first: Ying

last: Wang

first: Ting

last: Liu

first: Amani

last: Fawzi

first: Tsutomu

last: Kume


title: Frataxin Deficiency Drives a Shift from Mitochondrial Metabolism to Glucose Catabolism, Triggering an Inflammatory Phenotype in Microglia (bulk RNA-Seq)

first: Andrea

last: Ninni

summary: Immunometabolism investigates the complex interplay between the immune system and cellular metabolism. This study highlights the effects of mitochondrial frataxin (FXN) depletion, which causes Friedreich's ataxia (FRDA), a neurodegenerative condition characterized by coordination and muscle control deficiencies. Using single-cell RNA sequencing, we identified specific cell groups in the cerebellum of a FRDA mouse model, emphasizing a notable inflammatory microglial response. These FXN-deficient microglia cells exhibited enhanced inflammatory reactions. Furthermore, our metabolomic analyses revealed increased glycolysis and itaconate production in these cells, possibly driving the inflammation. Remarkably, butyrate treatment counteracted these immunometabolic changes, triggered an antioxidant response via the itaconate-Nrf2-GSH pathways, and dampened inflammation. The study also pinpointed Hcar2 (GPR109A) as a potential agent for butyrate anti-inflammatory impact on microglia. Tests on FRDA mice highlighted the neuroprotective attributes of butyrate intake, bolstering neuromotor performance. In essence, our findings shed light on how cerebellar microglia activation contributes to FRDA and highlight butyrate potential to alleviate neuroinflammation, rectify metabolic imbalances, and boost neuromotor capabilities in FRDA and similar conditions.

overall_design: The transcriptional profile of the cerebellum in frataxin-deficient (KIKO) mice was compared to that of wild-type (WT) mice at the single-cell level.

first: Daniele

last: Lettieri Barbato

first: Francesca

last: Sciarretta

first: Fabio

last: Zaccaria


title: Vitamin D opposes multilineage cell differentiation induced by Notch inhibition and BMP pathway activation in human colon organoids

city: Madrid

first: Jaime

last: Martinez de Villarreal

state: Madrid

address: Melchor Fernández Almagro, 3

department: Molecular Oncology Programme

country: Spain

institute: CNIO

summary: Understanding the mechanisms involved in colonic epithelial differentiation is key to unraveling the alterations causing inflammatory conditions and cancer. Organoid cultures provide a unique tool to address these questions but studies are scarce. We report a differentiation system toward enterocytes and goblet cells, the two major colonic epithelial cell lineages, using organoids from human healthy tissue. Culture of normal patient-derived colon organoids in medium lacking stemness agents resulted in a modest ultrastructural differentiation phenotype with low-level expression of enterocyte (KLF4, KRT20, CA1) and goblet cell (TFF2, TFF3, AGR2) lineage markers. BMP pathway activation through depletion of Noggin and addition of BMP4 resulted in enterocyte-biased differentiation. Contrarily, blockade of the Notch pathway using the g-secretase inhibitor dibenzazepine (DBZ) favored goblet cell differentiation. Combination treatment with BMP4 and DBZ caused a balanced strong induction of both lineages. In contrast, colon tumor organoids responded poorly to BMP4 and DBZ showing only weak signals of cell differentiation. We also investigated the effects of 1α,25-dihydroxyvitamin D3 (calcitriol) on differentiation. Calcitriol attenuated the effects of the BMP4 and DBZ on normal colon organoids, with reduced expression of differentiation genes and phenotype. Consistently, in normal organoids, calcitriol inhibited early signaling by BMP4 as assessed by reduction of the level of phospho-SMAD1/5/8. Our results show that BMP and Notch signaling play key roles in human colon stem cell differentiation to the enterocytic and goblet cell lineages and that calcitriol modulates these processes favoring stemness features.

overall_design: We have performed global transcriptomic analyses (RNA-seq) in colon normal organoids derived from 6 patients that were incubated in proliferation medium (PROL) or differentiation medium (B+D) containing Bone Morphogenetic Protein 4 (BMP4, 20 ng/mL) and the Notch inhibitor dibenzazepine (DBZ, 2.5 µM) for 48 h. Additionally, organoids cultured in B+D medium were treated with calcitriol (1,25(OH)2D3, 100 nM) or vehicle (ethanol) to investigate the effect of vitamin D on the differentiation of colon normal organoids.

first: Pilar

last: Bustamante-Madrid

first: Antonio

last: Barbáchano

first: David

last: Albandea-Rodríguez

first: Javier

last: Rodríguez-Cobos

first: Nuria

last: Rodríguez-Salas

first: Isabel

last: Prieto

first: Aurora

last: Burgos

last: Martínez de Villarreal

first: Francisco

middle: X

last: Real

first: José

last: González-Sancho

first: Maria

last: Larriba

first: Miguel

last: Lafarga

first: Alberto

last: Muñoz

first: Asunción

last: Fernández-Barral


title: Intracrine Vascular Endothelial Growth Factor Maintains Hippocampal Neural Stem Cell Quiescence

city: Columbus

first: Tyler

middle: Joseph

last: Dause

address: 1835 Neil Avenue

department: Psychology

institute: The Ohio State University

summary: In the dentate gyrus (DG) of the adult mouse hippocampus, neural stem cells (NSCs) balance self-renewal and differentiation to produce neurons that support hippocampal function. Vascular endothelial growth factor (VEGF) is a well-known supporting factor for adult neurogenesis, but conflicting studies have left it uncertain how VEGF signals to NSCs. Here, we identified a VEGF-VEGFR2 intracrine signaling mechanism within adult DG NSCs that prevents their exhaustion. We show both in vitro and in vivo that NSC-VEGF loss caused cell-autonomous exhaustion of adult DG NSCs. In contrast, extracellular VEGF was neither necessary nor sufficient to maintain NSC quiescence or to stimulate VEGFR2 signaling, most likely due to sheddase-mediated cleavage of extracellular VEGFR2 ligand binding domains. Our findings support an exclusively intracellular mechanism for VEGF signaling in adult DG NSCs, thereby providing resolution to previously conflicting studies and suggesting cellular source can dictate the functional impact of soluble ligands in DG NSCs.

overall_design: An in vivo model of conditional VEGF knockdown was used where adult NestinCreERT2;VEGFfloxed;ROSA-STOP-EYFP mice and VEGFWT controls were submitted to TAM administration to knockdown NSC-specific VEGF expression. 21 days after TAM administration DGs from 5 WT and 5 KO mice were harvested and submitted to fluorescently activated cell sorting for EYFP+ cells and then single cell sequencing on the 10x Genomics platform.

first: Jiyeon

last: Denninger

first: Joshua

last: Rieskamp

first: Akela

last: Kuwahara

first: Elizabeth

last: Kirby


title: Single-cell 5' RNA sequencing of Bactrian camel PBMCs

city: Shanghai

first: Zhen

last: Wang

address: 320 Yueyang Road

institute: Shanghai Institute of Nutrition and Health

summary: Camelids are capable of producing both conventional tetrameric antibodies (Abs) and dimeric heavy-chain antibodies (HCAbs). While B cells generating these two types of Abs exhibit distinct B-cell receptors (BCRs), it remains unclear whether these two B cell populations differ in their phenotypes and developmental processes. Here, we collected eight PBMC samples before and after immunization from four Bactrian camels and conducted single-cell 5’ RNA sequencing. We characterized the functional subtypes and differentiation trajectories of circulating B cells in camels, including native B cells, memory B cells, intermediate B cells, atypical B cells, and plasma cells. Additionally, we reconstructed single-cell BCR sequences and revealed the IGHV and IGHC gene types. We found that B cells with variable genes of HACbs (VHH) were widely present in various functional subtypes and showed highly overlapping differentiation trajectories to B cells with variable genes of conventional Abs (VH). After immunization, the transcriptional changes in VHH+ and VH+ B cells were also largely consistent. Our study not only elucidates the cellular context of HCAb production in camels, but also lays the foundation for the development of single B cell-based nanobody screening.

overall_design: We conducted single-cell 5' RNA sequencing on PBMCs from four adult healthy camels (C1-C4) from Inner Mongolia. Additionally, two camels (C3, C4) were immunized with BSA as an antigen, and PBMCs were collected at two time points after four immunizations (42 days and 56 days) for single-cell 5' RNA sequencing. We identified cell types in camel PBMCs, focusing on B cell subtypes and differentiation trajectories. Furthermore, we reconstructed single-cell BCR sequences and investigated the associations between B cell phenotypes and BCR types.

first: Li

last: Yi

first: Xin

last: Guo

first: Yuexing

last: Liu

first: J

last: Jirimutu


title: The effect of T/B cell maturation deficiency on the pulmonary pathology of acute SARS-CoV-2 infection

status: Public on Apr 14 2024

city: New Orleans

first: Jay

last: Kolls

state: Louisiana

address: 333 S. Liberty St

department: Medicine

institute: Tulane University

summary:
To investigate the function T and B cells in SARS-CoV-2 infection, we utilized Rag2-/- mice and infected them with a mouse adapted strain of SARS-CoV-2 virus (MA30)
We then performed gene expression analysis using data obtained from RNA-seq of the lungs of 3 Rag2+/+ and 3 Rag2-/- mice at 3 days post-infection (DPI) with MA30.

overall_design: Comparative gene expression analysis of RNA-seq data for 3 Rag2+/+ (WT) and 3 Rag2-/- (KO) mice at 3 DPI using MA30 1×104 TCID50

first: Calder

last: Ellsworth

first: Haoran

last: Yang


title: Single-Cell Transcriptomics Links Loss of Human Pancreatic β-Cell Identity to ER Stress

status: Public on Apr 13 2024

city: Leiden

first: Françoise

last: Carlotti

address: Albinusdreef 2

country: Netherlands

institute: Leiden University Medical Center

summary: The maintenance of pancreatic islet architecture is crucial for proper β-cell function. We previously reported that disruption of human islet integrity could result in altered β-cell identity. Here we combine β-cell lineage tracing and single-cell transcriptomics to investigate the mechanisms underlying this process in primary human islet cells. Using drug-induced ER stress and cytoskeleton modification models, we demonstrate that altering the islet structure triggers an unfolding protein response that causes the downregulation of β-cell maturity genes. Collectively, our findings illustrate the close relationship between endoplasmic reticulum homeostasis and β-cell phenotype, and strengthen the concept of altered β-cell identity as a mechanism underlying the loss of functional β-cell mass.

overall_design: Islets were dispersed into single cells by incubation with 0.025% trypsin (Gibco) supplemented with 10 mg/mL DNase (Pulmozyme, Genentech, San Francisco, CA, USA) for 6–7 min and filtered through a 70 μm cell-strainer. For β-cell lineage tracing, the two lentiviral vectors pTrip-RIP405Cre-ERT2-ΔU3 (RIP-CreERT2) and pTrip–loxP-NEO-STOP-loxP-eGFP-ΔU3 (CMVstopGFP) were used, kindly provided by P. Ravassard. Dispersed cells were transduced overnight with a 1:1 mixture of the two lentiviruses in regular CMRL medium containing 8 μg/mL polybrene. In the morning the medium was refreshed, and 4-hydroxy-tamoxifen (Sigma-Aldrich, St. Louis, MO, USA) was added to a final concentration of 1 μmol/L in the evening to activate RIP-CreERT2 and induce GFP expression in β-cells specifically. After overnight incubation, the medium was refreshed and all cells were seeded onto 2% w/v agarose microwell chips containing 2865 microwells/chip with a diameter of 200 μm/microwell, resulting in cellular aggregates of ~1000 islet cells.

first: Amadeo

last: Muñoz Garcia

first: Nathalie

last: Groen

first: Eelco

last: de Koning


title: SUMOylation Regulates Super-Enhancers Through Transcription Factor TFAP2C Binding on Chromatin [RNA-seq]

city: Duarte

first: Xiwei

last: Wu

address: 1500 E. Duarte Rd.

department: Computational and Quantitative Medicine

institute: City of Hope National Medical Center

summary: Genome wide localization of SUMO1 and SUMO2/3 proteins revealed an asscociation of SUMO proetins with active chromatin. SUMO proteins are enriched at super enhancers and enhancers and SUMOylation regulates a subset of these super enhancers. Super enhancers regulated by SUMOylation were enriched for transcription factor TFAP2C and SUMOylation negatively regulates TFAP2C localization to enhancers and super enhancers. Proteomics and ChIP-PCR at MYC SE suggests that chromatin bound TFAP2C recruits histone deacetylation complexes that increases upon SAE2 knockdown. Conversely, SUMOylation promoted TFAP2C asscociation with pre-mRNA splicing machinery components. Taken together, our study revealed a critical role of SUMOylation in chromatin modification through an AP-2 family of transcription factor, TFAP2C  and a potential role of TFAP2C in pre-mRNA splicing.

overall_design: mRNA profiles of control and SAE2 kd (SUMOylation depleted) cells  and mRNA profiles of control and TFAP2C kd cells

first: Yuan

last: Chen


title: SUMOylation Regulates Super-Enhancers Through Transcription Factor TFAP2C Binding on Chromatin

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

overall_design: Refer to individual Series


title: Wnt signaling regulates chemokine production and cell migration of circulating human monocytes

city: Tel Aviv

first: Rina

last: Rosin-Arbesfeld

address: Haim Levanon

department: Clinical Microbiology and Immunology

country: Israel

institute: Tel Aviv University

summary:
The β-catenin dependent canonical Wnt signaling pathway plays a crucial role in maintaining normal homeostasis. However, when dysregulated, Wnt signaling is closely associated with various pathological conditions, including inflammation and different types of cancer.
Here, we show a new connection between the leukocyte inflammatory response and the Wnt signaling pathway. Specifically, we demonstrate that circulating human primary monocytes express distinct Wnt signaling components and are susceptible to stimulation by the classical Wnt ligand - Wnt-3a. Although this stimulation increased the levels of β-catenin protein, the expression of the classical Wnt-target genes was not affected. Intriguingly, treating circulating human monocytes with Wnt-3a induces the secretion of cytokines and chemokines, enhancing monocyte migration. Mechanistically, the enhanced monocyte migration in response to Wnt stimuli is mediated through CCL2, a strong monocyte-chemoattractant.
To further explore the physiological relevance of these findings, we conducted ex-vivo experiments using blood samples of patients with rheumatic joint diseases (RJD) – conditions where monocytes are known to be dysfunctional. Wnt-3a generated a unique cytokine expression profile, which was significantly distinct from that observed in monocytes obtained from healthy donors.
Thus, our results provide the first evidence that Wnt-3a may serve as a potent stimulator of monocyte-driven immune processes. These findings contribute to our understanding of inflammatory diseases and, more importantly, shed light on the role of a core signaling pathway in the circulation.

overall_design: Freshly isolated primary human classical monocytes (CD14+CD16-) from two donors were cultured for 8 hours in triplicates in cell repellent plates in the presence of control or Wnt-3a conditioned media.   Media were comprised of 80% L- or L-Wnt-3a- CM and 20% RPMI-1640 (both containing 10% FBS and 1% Penicillin/Streptomycin) at 37°C in 5% CO2. L cells and L-Wnt-3a cells were propagated according to the ATCC guidelines and CM were produced according to the ATCC protocols.  Total RNA was extracted using the Direct-zol RNA MiniPrep kit. Replicates of high RNA integrity (RIN ≥ 7.8) were processed. Libraries were prepared using the NEBNext Ultra II RNA Library Prep kit with the NEBNext Poly(A) mRNA Magnetic Isolation Module, starting with ~ 500 ng of total RNA. Ten PCR cycles were performed during library amplification. Libraries were quantified by Qubit and TapeStation and sequenced on a NextSeq 500 instrument using a NextSeq 500/550 High Output Kit v2.5 (75 Cycles) kit.  Twelve FastQ files were uploaded to Partek Flow (Build version 10.0.21.1116; https://www.partek.com) for processing. Poly-A/T stretches and Illumina adapters were trimmed from the reads. Resulting reads with Phred score < 20 were filtered. Reads were mapped to the human GRCh38.p13 reference genome using STAR 2.7.8a with default parameters to reveal ~ 20 million reads per sample. Quantification to annotation model was performed using Partek Expectation/Maximization (E/M) algorithm  obtaining 33,331 genes.

first: Natalie

last: Zelikson

first: Shaina

last: Ben

first: Michal

last: Caspi

first: Raneen

last: Tarabe

first: Yonatan

last: Shaleve

first: Yael

last: Peri-Paz Basson

first: Oshrat

last: Tayer-Shifman

first: Elad

last: Goldberg

first: Shaye

last: Kivity


title: The mRNA-binding protein KSRP limits the inflammatory response of macrophages

status: Public on Apr 12 2024

city: Mainz

first: Matthias

last: Bros

address: Langenbeckstrasse 1

department: Dermatology

country: Germany

institute: University Medical Center Mainz

summary: KH-type splicing regulatory protein (KSRP) is a single-stranded nucleic acid-binding protein with multiple functions. It is known to bind AU-rich motifs within the 3´-untranslated region of mRNA species, which in many cases encode dynamically regulated proteins like cytokines. In the present study, we investigated the role of KSRP for the immunophenotype of macrophages using bone marrow-derived macrophages (BMDM) from wild type (WT) and KSRP-/- mice. RNA sequencing revealed that KSRP-/- BMDM displayed significantly higher mRNA expression levels of genes in-volved in inflammatory and immune responses, particularly type I interferon responses, following LPS stimulation. In line, time kinetics studies revealed increased levels of interferon-γ (IFN-γ), in-terleukin (IL)-1β and IL-6 mRNA in KSRP-/- macrophages after 6 h subsequent to LPS stimulation as compared to WT cultures. On protein level KSRP-/- BMDM displayed higher levels of these cy-tokines after overnight stimulation. Matching results were observed for primary peritoneal mac-rophages of KSRP−/− mice. These showed higher IL-6, Tumor necrosis factor-α (TNF-α), C-X-C mo-tif chemokine 1 (CXCL1) and CC-chemokine ligand 5 (CCL5) protein levels in response to LPS stimulation than the WT controls. As macrophages play a key role in sepsis, the in vivo relevance of KSRP deficiency for cytokine/chemokine production was analyzed in an acute inflammation model. In agreement with our in vitro findings, KSRP-deficient animals showed higher cytokine production upon LPS administration in comparison to WT mice. Taken together, these findings demonstrate that KSRP constitutes as an important negative regulator of cytokine expression in macrophages

overall_design: We isolated bone marrow cells of WT and KSRP-KO mice and cultured them for 7 days in M-CSF containing medium to induce differentiation to macrophages (BMDM). BMDM were either treated with 1 µg/µl LPS for 6 h or left untreated. 5x10^4 cells of each samples were lysed and total RNA was purified for sequencing.

first: Vanessa

last: Bolduan


title: SUMOylation Regulates Super-Enhancers Through Transcription Factor TFAP2C Binding on Chromatin [ChIP-seq]

overall_design: Genomic distribution of SUMO 1 and SUMO2/3 proteins and their association with chromatin marks H3K72Ac, H4K4me3, H3K27me3  and coactivator Med1 were probed. Chages to super enhancer components H3K27Ac and Med1 were analysed upon SAE2 kd (absence of SUMOylation). Genomic distrubution of TFAP2C in control and SUMOylation depleted cells were measured.


title: Nuclear actin structure regulates chromatin accessibility [ATAC-seq]

city: Chapel Hill

first: Terry

last: Furey

address: 120 Mason Farm Road

department: Genetics

institute: University of North Carolina, Chapel Hill

summary: Polymerized B-actin may provide a structural basis for chromatin accessibility and actin transport into the nucleus can guide mesenchymal stem cell (MSC) differentiation. Using MSC, we show that using CK666 to inhibit Arp2/3 directed secondary actin branching results in decreased nuclear actin structure, and significantly alters chromatin access measured with ATACseq at 24 h. The ATAC-seq results due to CK666 are distinct from those caused by cytochalasin D (CytoD), which enhances nuclear actin structure. In addition, nuclear visualization shows Arp2/3 inhibition decreases pericentric H3K9me3 marks. CytoD, alternatively, induces redistribution of H3K27me3 marks centrally. Such alterations in chromatin landscape are consistent with differential gene expression associated with distinctive differentiation patterns. Further, knockdown of the non-enzymatic monomeric actin binding protein, Arp4, leads to extensive chromatin unpacking, but only a modest increase in transcription, indicating an active role for actin-Arp4 in transcription. These data indicate that dynamic actin remodeling can regulate chromatin interactions.

overall_design: Mouse marrow-derived mesenchymal stem cells (MSCs) were harvested from C57BL/6J murine. In the first set of experiments, MSCs were treated with either CK666, an Arp2/3 inhibitor, or cytochalasin D (CytoD) for 24 hours, and ATAC-seq and RNA-seq were performed on three replicates each of control and stimulated cells. In the second set of experiments, Arp4 was knocked down using an siRNA, and ATAC-seq and RNA-seq were performed on siRNA mock transfected control MSCs, siRNA Arp4 transfected MSCs, and siRNA Arp4 transfected MSCs also stimulated with CK666 for 24 hours. In a third set of experiments, MSCs and NIH3T3 cells were stimulated with CK666 for 4 hours, and ATAC-se qwas performed on three replicates each of stimulated and control cells for both cell types.

first: Janet

last: Rubin

first: Buer

last: Sen


title: Histone H3K27 methyltransferase EZH2 interacts with MEG3-lncRNA to directly regulate integrin signaling and endothelial cell function

city: Edinburgh

first: Hywel

last: Dunn-Davies

address: Max Born Crescent

department: Wellcome Trust Centre for Cell Biology

institute: University of Edinburgh

summary: We profiled EZH2-RNA interactions using formaldehyde/UV assisted cross-linking ligation and sequencing of hybrids (FLASH-seq) in primary human ECs. Transcriptome-wide EZH2-associated ncRNAs and RNA–RNA interactome were obtained.

overall_design: Nine different FLASH experiments were carried out on human HUVEC cells, with EZH2 or an IgG control used as bait proteins.

first: T

last: Dudnakova

first: H

first: A

last: Nogara

first: J

last: Rodor

last: Thomas

first: E

last: Parish

first: P

last: Gautier

last: Meynert

last: Madeddu

last: Caporali

last: Baker

first: D

last: Tollervey

last: Mitic


title: Nuclear actin structure regulates chromatin accessibility.


title: Long Non-Coding RNA MEG3 Promotes Angiogenesis by directly regulating integrin signaling impairing endothelial cell function [ChIRP-seq]

summary: We profiled MEG-DNA interactions using gluteraldehyde assisted chromatin immunoprecipitation by RNA purification (ChIRP-seq) in primary human ECs. Genome-wide MEG3-associated loci were obtained.

overall_design: Eight different ChIRP experiments were carried out using human HUVEC cells, with MEG3 or LacZ control biotynilated probes used as bait against MEG3-lncRNA.


title: Modelling Myeloma Dissemination in vitro with hMSC-Interacting Subpopulations of INA-6 Cells and their Aggregation/Detachment Dynamics

status: Public on Apr 11 2024

city: Würzburg

first: Regina

last: Ebert

address: Friedrich-Bergius-Ring 15

department: Department of Musculoskeletal Tissue Regeneration

institute: Universität Würzburg

summary:
Multiple myeloma involves early dissemination of malignant plasma cells across the bone marrow; however, the initial steps of dissemination remain unclear. Human bone marrow-derived mesenchymal stromal cells (hMSCs) stimulate myeloma cell expansion (e.g., IL-6) and simultaneously retain myeloma cells via chemokines (e.g., CXCL12) and adhesion factors. Hence, we hypothesized that the imbalance between cell division and retention drives dissemination.
We present an in vitro model using primary hMSCs co-cultured with INA-6 myeloma cells. Time-lapse microscopy revealed proliferation and attachment/detachment dynamics. Separation techniques (V-well adhesion assay and well plate sandwich centrifugation) were established to isolate MSC-interacting myeloma subpopulations that were characterized by RNAseq, cell viability and apoptosis. Results were correlated with gene expression data (n=837) and survival of myeloma patients (n=536).
On dispersed hMSCs, INA-6 saturate hMSC-surface before proliferating into large homotypic aggregates, from which single cells detached completely. On confluent hMSCs, aggregates were replaced by strong heterotypic hMSC-INA-6 interactions, which modulated apoptosis time-dependently. Only INA-6 daughter cells (nMA-INA6) detached from hMSCs by cell division but sustained adherence to hMSC-adhering mother cells (MA-INA6).
Isolated nMA-INA6 indicated hMSC-autonomy through superior viability after IL6 withdrawal and upregulation of proliferation-related genes. MA-INA6 upregulated adhesion and retention factors (CXCL12), that, intriguingly, were highly expressed in myeloma samples from patients with longer overall and progression-free survival, but their expression decreased in relapsed myeloma samples.
Altogether, in vitro dissemination of INA-6 is driven by detaching daughter cells after a cycle of hMSC-(re)attachment and proliferation, involving adhesion factors that represent a bone marrow-retentive phenotype with potential clinical relevance.

overall_design:
To explore differences within MSC-adherent subpopluations of multiple myeloma cells, the myeloma cell line INA-6 was co-cultured on primary human mesenchymal stromal cells (hMSCs) for 24h and separated into non-MSC-adhering (nMA-INA6) and MSC-adhering (MA-INA6) subpopulations using well plate sandwich centrifugation (WPSC). As a control, INA-6 were incubated in hMSC conditioned medium.
All myeloma samples were purified by anti-CD45 magnetic-assisted cell sorting.
Samples:
F1 = INA-6 incubated in hMSC-conditioned Medium (CM-INA6)
F2 = INA-6 incubated on hMSCs removed by centrifugation (nMA-INA6)
F3 = INA-6 incubated on hMSCs remaining after centrifugation (nMA-INA6)
Each sample was measured from 5 independent replicates or co-cultures with unique hMSC donors.
We performed RNAseq and differential gene expression analysis.

first: Martin

last: Kuric

first: Susanne

last: Beck

first: Doris

last: Schneider

first: Wyonna

last: Rindt

first: Marietheres

last: Evers

first: Jutta

last: Meißner-Weigl

first: Sabine

last: Zeck

first: Melanie

last: Krug

first: Marietta

last: Herrmann

first: Tanja

middle: N

last: Hartmann

first: Ellen

last: Leich

first: Maximilian

last: Rudert

first: Denitsa

last: Docheva

first: Anja

last: Seckinger

first: Dirk

last: Hose

first: Franziska

last: Jundt


title: PP2A inhibition instructs spliceosome phosphorylation patterns to create splicing vulnerability in colon adenocarcinoma

city: Warsaw

first: Michal

last: Krzyszton

address: Pawinskiego 5a

country: Poland

institute: Institute of Biochemistry and Biophysics, Polish Academy of Sciences

summary: Protein phosphatase 2A (PP2A) is a promiscuous enzyme affecting cellular physiology. It is unclear however, which of the cellular processes are most perturbed upon PP2A inhibition and how such perturbations could be exploited therapeutically. Here, we report an unanticipated sensitivity of the splicing machinery to phosphorylation changes in response to PP2A inhibition by LB-100 in colorectal adenocarcinoma. We observe enrichment for differentially phosphorylated sites within cancer-critical splicing nodes of U2 snRNP, SRSF and hnRNP proteins. Changed phosphorylation endows LB-100 treated colorectal adenocarcinoma cells with differential splicing patterns. Over 1000 exon skipping and intron retention events in PP2A-inhibited cells affect predominantly regulators of genomic integrity. Finally, LB-100-evoked alternative splicing is predicted to be a source of neoantigens that can improve cancer treatment responses to immune modulators. Our findings provide a potential explanation for the pre-clinical and clinical observations that PP2A inhibition sensitizes cancer cells to immune checkpoint blockade and genotoxic agents.

overall_design: Experiment was performed with two cell lines of human colorectal adenocarcinoma, SW-480 (harboring Kras mutation) and HT-29 (Braf mutated), treated for 12 hrs with PP2A inhibitor, LB-100 in 4 uM concentration.

first: Matheus

middle: H

last: Dias

first: Vladyslava

last: Liudkovska

first: Onno

last: Bleijerveld

first: Arno

last: Velds

first: Rene

last: Bernards

first: Maciej

last: Cieśla


title: Gene expression data of the adipogenesis process of mesenchymal stem cells derived from two different types of synovial membranes of German Landrace (DL) and Angeln Saddleback (AS) pigs.

city: Dummerstorf

first: Siriluck

last: Ponsuksili

address: Wilhelm-stahl-allee 2

institute: FBN

summary: 47,845 probe-sets were screened, This study compared expression profiles of mesenchymal stem cells derived from the two breed DL and AS and then analyzed the expression profile genes between two different sorts of synovial membrane in each breed.

overall_design:
RNAs of mesenchymal stem cells derived from two different sorts of synovial membrane of DL and AS were hybridized to Affymetrix Porcine Snowball Array
ANOVA


title: Functional investigation of inherited noncoding genetic variation impacting the pharmacogenomics of childhood acute lymphoblastic leukemia treatment [HiChIP]

status: Public on Apr 10 2024

city: Memphis

first: Daniel

last: Savic

state: TN

address: 262 Danny Thomas pl.

department: Pharmaceutical Sciences

institute: St. Jude Children's Research Hospital

summary: Although acute lymphoblastic leukemia (ALL) is the most prevalent childhood cancer, there is limited understanding of the contribution of inherited genetic variation on inter-individual differences in chemotherapy response, and defining genetic factors impacting therapy failure can help better predict response and identify drug resistance mechanisms. Using inherited noncoding variants associated with chemotherapeutic drug resistance and/or treatment outcome, we mapped these variants to ALL cis-regulatory elements and investigated their gene regulatory potential and genomic connectivity using massively parallel reporter assays and promoter capture Hi-C, respectively. We identified 53 variants with reproducible allele-specific effects on transcription and high-confidence gene targets. Subsequent functional interrogation of the top variant (rs1247117) determined that it disrupted a PU.1 consensus motif and PU.1 binding affinity. Importantly, deletion of the genomic interval containing rs1247117 sensitized ALL cells to vincristine. Together, these data demonstrate that noncoding regulatory variation associated with diverse pharmacological traits harbor significant effects on allele-specific transcriptional activity and impact sensitivity to chemotherapeutic agents in ALL.

overall_design: H3K27Ac HiChIP in 8 ALL cell lines

first: Kashi

last: Raj Bhattarai

first: Robert

last: Mobley

first: Kelly

last: Barnett

middle: C

last: Ferguson

first: Jonathan

middle: D

last: Diedrich

first: Brennan

last: Bergeron

first: Satoshi

last: Yoshimura

first: Wenjian

last: Yang

first: Kristine

last: Crews

first: Elias

last: Jabbour

first: Christopher

middle: S

last: Manring

first: Elisabeth

last: Paietta

first: Steven

last: Kornblau

first: Wendy

last: Stock

first: Hiroto

last: Inaba

first: Sima

last: Jeha

first: Ching-Hon

last: Pui

first: Cheng

last: Cheng

first: Mary

last: Relling

first: William

last: Evans

first: Jun


title: Apolipoprotein L1 is a tumor suppressor in clear cell renal cell carcinoma metastasis

city: Cheonan-si

first: Seongho

last: Ryu

address: 25 Bongjeong-ro Dongnam-gu

country: South Korea

institute: Soonchunhyang University


title: Nuclear actin structure regulates chromatin accessibility [RNA-seq]

first: Terry

last: Furey

summary: Polymerized b-actin may provide a structural basis for chromatin accessibility. Nuclear actin transport into the nucleus can determine mesenchymal stem cell (MSC) differentiative outcomes through regulated control of gene expression. Using MSC, we show that inhibiting Arp2/3 directed secondary actin branching with CK666, which results in decreased nuclear actin structure, significantly alters chromatin access measured with ATAC-seq at 24 h. The ATAC-seq results due to CK666 are distinct from those caused by cytochalasin D (CytoD), which increases nuclear actin structure. Nuclear visualization shows Arp2/3 inhibition is associated with decreased pericentric H3K9me3 marks. CytoD, in contrast, induces relocation of H3K27me3 marks away from the inner membrane. Treatment induced alterations in the chromatin landscape at 24 hours prompts differential gene expression associated with decreased proliferation and increased differentiation. Further, knockdown of the non-enzymatic monomeric actin binding protein, Arp4, leads to extensive chromatin unpacking, but only a modest change in transcription, indicating an active role for actin-Arp4 in transcription. These data indicate that dynamic actin remodeling can regulate chromatin interactions.

overall_design: Mouse marrow-derived mesenchymal stem cells (MSCs) were harvested from C57BL/6J murine. In the first set of experiments, MSCs were treated with either CK666, an Arp2/3 inhibitor, or cytochalasin D (CytoD), and ATAC-seq and RNA-seq were performed on three replicates each of control and stimulated cells. In the second set of experiments, Arp4 was knocked down using an siRNA, and ATAC-seq and RNA-seq were performed on siRNA mock transfected control MSCs, siRNA Arp4 transfected MSCs, and siRNA Arp4 transfected MSCs also stimulated with CK666. bone marrow

first: Janet

last: Rubin

first: Buer

last: Sen


title: Dysregulation of Neuroprotective Lipoxin Pathway in Astrocytes in Response to Cytokines and Ocular Hypertension

city: Berkeley

first: Shubham

last: Maurya

address: Rm 596, Minor Hall

institute: University of California Berkeley

summary: Glaucoma leads to vision loss due to retinal ganglion cell death. Astrocyte reactivity contributes to neurodegeneration. Our recent study found that lipoxin B4 (LXB4), produced by retinal astrocytes, has direct neuroprotective actions on retinal ganglion cells. In this study, we aimed to investigate how the autacoid LXB4 influences astrocyte reactivity in the retina under inflammatory cytokine-induced activation and ocular hypertension conditions. The protective activity of LXB4 was investigatedin vivousing the mouse silicone-oil model of chronic ocular hypertension (n=40). By employing a range of analytical techniques, including bulk RNA-seq, RNAscope in-situhybridization, qPCR, and lipidomic analyses, we discovered the formation of neuroprotective lipoxins in rodents (including the retina and optic nerve), primates (optic nerve), and human brain astrocytes, indicating their presence across various species. Our findings in the mouse retina demonstrated significant dysregulation of the lipoxin pathway in response to chronic ocular hypertension, leading to an increase in 5-lipoxygenase (5-LOX) activity and a decrease in 15-lipoxygenase activity. This dysregulation was coincident with a marked upregulation of astrocyte reactivity. Reactive human brain astrocytes also showed a significant increase in 5-LOX. Administration of LXB4 regulated the lipoxin pathway, restored and amplified LXA4 generation (another lipoxin with distinct bioactions), and mitigated astrocyte reactivity in mouse retinas and human brain astrocytes. In conclusion, the lipoxin pathway is functionally expressed in rodents, primates, and human astrocytes, and is a resident neuroprotective pathway that is downregulated in reactive astrocytes. Novel cellular targets for LXB4’s neuroprotective action are inhibition of astrocyte reactivity and restoration of lipoxin generation. Amplifying the lipoxin pathway is a potential target to disrupt or prevent astrocyte reactivity in neurodegenerative diseases.

overall_design: Optic nerves from two different macaques were analyzed.

first: Shruthi

last: Karnam

first: Elainna

last: Ng

first: Amodini

last: Choudhary

first: Arzin

last: Thobani

first: John

middle: G

last: Flanagan

first: Karsten

last: Gronert


title: Mitochondrial ACSS1-K-635 acetylation mimic knock-in mice exhibit altered lipid metabolism, hepatic cell senescence, and acute non-alcoholic fatty liver [2ndSet_analysis]

city: San Antonio

first: Yidong

last: Chen

address: 8403 Floyd Curl Drive, MSC 7784

department: Population Health Sciences

institute: UT Health Science Center at San Antonio

summary: We generated an ACSS1-acetylation (Ac) mimic mouse, where lysine 635 was mutated to glutamine (K635Q). Male Acss1K635Q/K635Q mice were smaller with higher metabolic rate and blood acetate, and decreased liver/serum ATP and lactate levels. After a 48-hour fast, Acss1K635Q/K635Q mice presented hypothermia and liver aberrations, including enlargement, discoloration, lipid droplet accumulation, and microsteatosis – consistent with nonalcoholic fatty liver disease (NAFLD). RNAseq analysis suggested dysregulation of fatty acid metabolism, cellular senescence, and hepatic steatosis networks, consistent with NAFLD

overall_design: Fasted and fed C57BL/6J WT and ACSS1K635Q mice were studied in the context of non-alcoholic fatty liver disease. The liver tissue was collected and prepared for RNAseq as well as for Western blotting and qPCR analysis.

first: David

last: Gius

first: Guogang

last: Xu

first: Joseph

last: Schell

first: Nobuo

last: Horikoshi

first: Zhao

last: Lai


title: Apolipoprotein L1 is a tumor suppressor in clear cell renal cell carcinoma metastasis [miRNA-Seq]

first: Seongho

last: Ryu

summary: The 5-year survival rate of kidney cancer drops dramatically from 93% to 15% when it is metastatic. Metastasis constitutes for 30% of kidney cancer cases, in which clear cell renal cell carcinoma (ccRCC) is the most prominent subtype. By sequencing mRNA of ccRCC patient samples, we found that apolipoprotein L1 (APOL1) was highly expressed in tumors compared to their adjacent normal tissues. This gene has been previously identified in a large body of kidney disease research and was reported as a potential prognosis marker in many types of cancers. However, the molecular function of APOL1 in ccRCC, especially in metastasis, remained unknown. In this study, we modulated the expression of APOL1 in various renal cancer cell lines and analyzed their proliferative, migratory, and invasive properties. Strikingly, APOL1 overexpression suppressed ccRCC metastasis both in vitro and in vivo. We then explored the mechanism by which APOL1 alleviated ccRCC malignant progression by investigating its downstream pathways. APOL1 overexpression diminished the activity of focal adhesive molecules, Akt signaling pathways, and EMT processes. Furthermore, in the upstream, we discovered that miR-30a-3p could inhibit APOL1 expression. In conclusion, our study revealed that APOL1 play a role as a tumor suppressor in ccRCC and inhibit metastasis, which may provide novel potential therapeutic approaches for ccRCC patients.

overall_design: To further investigate the microRNA that could regulate APOL1 expression and regulate ccRCC metastasis, we analyzed small RNA sequencing data of 9 patient samples.

first: Linh

last: Le

first: Cheolwon

last: Choi

first: Jae-A

last: Han

first: Yong-June

last: Kim


title: Apolipoprotein L1 is a tumor suppressor in clear cell renal cell carcinoma metastasis [RNA-Seq]

overall_design: To characterize the molecular mechanisms related to malignant properties of ccRCC, we first performed RNA sequencing on 9 pairs of tumors and adjacent normal tissues.


title: Long non-coding RNA MEG3 promotes angiogenesis by directly regulating integrin signaling impairing endothelial cell function

first: Hywel

last: Dunn-Davies

summary: We depleted MEG3-lncRNA using GapmeRs in primary human ECs. Total MEG3-regulated targets were obtained.

overall_design: Four different siRNA experiments were carried out using human HUVEC cells, with MEG3 or SCR control to deplete MEG3-lncRNA.

first: H

first: T

last: Dudnakova

first: A

last: Nogara

first: J

last: Rodor

last: Thomas

first: E

last: Parish

first: P

last: Gautier

last: Meynert

first: I

last: Ulitsky

last: Madeddu

last: Caporali

last: Baker

first: D

last: Tollervey

last: Mitic


title: Intravenous immunoglobulin (IVIg) ameliorates disease in a murine model of anti-laminin 332 mucous membrane pemphigoid

city: Lübeck

first: Axel

last: Künstner

address: Ratzeburger Allee 160

department: LIED

institute: University of Lübeck

summary: Intravenous immunoglobulin (IVIg) is used to treat mucous membrane pemphigoid (MMP), although its therapeutic effectivity is not sufficiently supported by randomized controlled clinical trials and its mode of action is only insufficiently understood. We have examined the effect of IVIg in a mouse model of anti-laminin 332 MMP and found that IVIg ameliorates both cutaneous and mucosal inflammatory lesions. Our investigation into the modes of action of IVIg in MMP indicated effective antiinflammatory mechanisms beyond the enhanced degradation of IgG mediated through inhibition of the neonatal Fc receptor. Our results suggest that IVIg curbs the activation of neutrophils at several levels. This includes a direct, immediate inhibitory effect on neutrophil activation by immune complexes but not C5a which blunts the release of reactive oxygen species and leukotriene B4 from neutrophils. IVIg also suppresses the formation of neutrophil extracellular traps in response to Ca2+ ionophore. In vivo treatment with IVIg altered the transcriptome of blood leukocytes and bone marrow neutrophils towards less proinflammatory phenotypes. Collectively, our results support the effectivity of IVIg in the treatment of MMP and indicate that effects on neutrophils at multiple levels may significantly contribute to its therapeutic effects.

overall_design: The effect of IVIg was investigated using a mouse model. Sampling was performed using blood, neutrophils and monocytes as well all skin samples.

first: Christian

last: Sadik

first: Hauke

last: Busch


title: Functional investigation of inherited noncoding genetic variation impacting the pharmacogenomics of childhood acute lymphoblastic leukemia treatment [ChIP-Seq]

first: Daniel

last: Savic

overall_design: ChIP seq

first: Kashi

last: Bhattarai

first: Robert

last: Mobley

first: Kelly

last: Barnett

last: Ferguson

first: Jonathan

last: Diedrich

first: Brennan

middle: P

last: Bergeron

first: Wenjian

last: Yang

first: Kristine

last: Crews

first: Elias

last: Jabbour

first: Christopher

last: Manring

first: Elisabeth

last: Paietta

first: Steven

last: Kornblau

first: Wendy

last: Stock

first: Hiroto

last: Inaba

first: Sima

last: Jeha

first: Ching-Hon

last: Pui

first: Cheng

last: Cheng

first: Mary

last: Relling

first: William

last: Evans

first: Jun


title: Ketogenic diet induces p53-dependent cell senescence in multiple organs

first: Yidong

last: Chen

summary: Mice on two different ketogenic diets induce p53 and cellular senescence in multiple organs, including heart and kidney. This is mediated through inactivation of MDM2 by caspase-2 cleavage, leading to p53 accumulation and induction of p21. Ketogenic diet also induced pAMPK, suggesting that persistent activation leads to p53-dependent senescence.

overall_design: Mice were placed on ketogenic diet for three weeks. Their tissues (heart, kidney and liver) were collected (3 replicates each) and analyzed via immunoblotting and RNA-seq as compared to mice on a standard chow diet.

first: Nobuo

last: Horikoshi

first: David

last: Gius

first: Mahboubeh

last: Varmazyad

first: Joseph

last: Schell

first: Sung-Jen

last: Wei

first: Erin

last: Munkacsy

first: Zhao

last: Lai


title: Functional investigation of inherited noncoding genetic variation impacting the pharmacogenomics of childhood acute lymphoblastic leukemia treatment [MPRA]

first: Daniel

last: Savic

sample_organism:
Homo sapiens
synthetic construct

platform_organism:
Homo sapiens
synthetic construct

overall_design: MPRA in 10 ALL cell lines

first: Kashi

last: Raj Bhattarai

first: Robert

last: Mobley

first: Kelly

last: Barnett

last: Ferguson

first: Jonathan

last: Diedrich

first: Brennan

last: Bergeron

first: Wenjian

last: Yang

first: Kristine

last: Crews

first: Elias

last: Jabbour

first: Christopher

last: Manring

first: Elisabeth

last: Paietta

first: Steven


title: Long Non-Coding RNA MEG3 Promotes Angiogenesis by directly regulating integrin signaling impairing endothelial cell function [ChIP-seq]

first: Hywel

last: Dunn-Davies

summary: We immunoprecipitated EZH2, together with associated chromatin isolated from the HUVECs (2 x 10^6) that were transfected with MEG3 GapmeRs (10 nM, 48 h) or a scrambled control GapmeRs (Ctr). For transfection, we used 10 nM scrambled LNA (locked nucleic acids) GapmeR control (Cat. No. 339515) or phosphorothioate antisense standard GapmeRs MEG3-lncRNA (Cat No. 339511, Qiagen). On beads crosslinked chromatin complexes were reversed, and DNA purified using QIAquick® PCR Purification Kit and quantified by Qubit HS assay (Q33230)

first: T

last: Dudnakova

first: H

first: A

last: Nogara

first: J

last: Rodor

last: Thomas

first: E

last: Parish

first: P

last: Gautier

last: Meynert

last: Madeddu

last: Caporali

last: Baker

first: D

last: Tollervey

last: Mitic


title: A heterogenous pharmaco-transcriptomic landscape induced by targeting a single oncogenic kinase

status: Public on Apr 09 2024

city: New York

first: José

middle: L

last: McFaline-Figueroa

email: jm5200@columbia.edu, joselmc@uw.edu

state: NY

department: Biomedical Engineering

institute: Columbia University

summary: Over-activation of the epidermal growth factor receptor (EGFR) is a hallmark of glioblastoma. However, EGFR-targeted therapies have led to minimal clinical response. While delivery of EGFR inhibitors (EGFRis) to the brain constitutes a major challenge, how additional drug-specific features alter efficacy remains poorly understood. We apply highly multiplex single-cell chemical genomics to define the molecular response of glioblastoma to EGFRis. Using a deep generative framework, we identify shared and drug-specific transcriptional programs that group EGFRis into distinct molecular classes. We identify programs that differ by the chemical properties of EGFRis, including induction of adaptive transcription and modulation of immunogenic gene expression. Finally, we demonstrate that pro-immunogenic expression changes associated with a subset of tyrphostin family EGFRis increase the ability of T-cells to target glioblastoma cells.

overall_design: Single-cell RNA-seq libraries were generated using three-level single-cell combinatorial indexing RNA sequencing (sci-RNA-seq) of untreated or small molecule inhibitor exposed A172, T98G, U87MG, BT112, BT228 and BT333 glioblastoma brain cancer cells. Different cells and different treatments were hashed and pooled prior to sci-RNA-seq using the sci-Plex nuclear barcoding strategy. This nuclear barcoding strategy relies on fixation of barcode containing well-specific oligos that are specific to a given cell type, replicate, or treatment condition.

first: R

last: Giglio

first: N

last: Hou

last: Wyatt


title: Functional investigation of inherited noncoding genetic variation impacting the pharmacogenomics of childhood acute lymphoblastic leukemia treatment

first: Daniel

last: Savic

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

overall_design: Refer to individual Series


title: Functional investigation of inherited noncoding genetic variation impacting the pharmacogenomics of childhood acute lymphoblastic leukemia treatment [HiC]

overall_design: HiC

first: Kashi

middle: R

last: Bhattarai

first: Robert

last: Mobley

first: Kelly

last: Barnett

last: Ferguson

first: Jonathan

last: Diedrich

first: Brennan

last: Bergeron

first: Wenjian

last: Yang

first: Kristine

last: Crews

last: Jabbour

last: Manring

last: Paietta

last: Kornblau

first: Wendy

last: Stock

first: Hiroto

last: Inaba

first: Sima

last: Jeha

first: Ching-Hon

last: Pui

first: Cheng

last: Cheng

first: Mary

last: Relling

first: William

last: Evans

first: Jun


title: Long Non-Coding RNA MEG3 Promotes Angiogenesis by directly regulating integrin signaling impairing endothelial cell function.

first: Hywel

last: Dunn-Davies


title: Functional investigation of inherited noncoding genetic variation impacting the pharmacogenomics of childhood acute lymphoblastic leukemia treatment [ATAC-seq]

first: Daniel

last: Savic

overall_design:
ATAC-seq

ATAC-seq on ALL patient derived xenografts (PDX_ATAC) and ALL cell models

last: Bhattarai

last: Mobley

last: Barnett

last: Ferguson

first: Elias

first: Christopher

first: Elisabeth

first: Steven