title: miR-126 identifies a quiescent and chemo-resistant human B-ALL cell subset that correlates with minimal residual disease [BulkRNA 1]

first: Matteo

last: Barcella

summary: Complete elimination of B-cell acute lymphoblastic leukemia (B-ALL) by a risk-adapted primary treatment approach remains a clinical key objective, which fails in up to a third of patients. Recent evidence has implicated subpopulations of B-ALL cells with stem-like features in disease persistence. We hypothesized that microRNA-126, a core regulator of hematopoietic and leukemic stem cells, may resolve intra-tumor heterogeneity in B-ALL and uncover therapy-resistant subpopulations. We exploited patient-derived xenograft (PDX) models with B-ALL cells transduced with a miR-126 reporter allowing the prospective isolation of miR-126(high) cells for their functional and transcriptional characterization. Discrete miR-126(high) populations, often characterized by MIR126 locus de-methylation, were identified in 8/9 PDX models and showed increased repopulation potential, in vivo chemotherapy resistance and hallmarks of quiescence, inflammation and stress-response pathway activation. Cells with a miR-126(high) transcriptional profile were identified as distinct disease subpopulations by single cell RNA sequencing in diagnosis samples from adult and pediatric B-ALL. Expression of miR-126 and locus methylation were tested in several pediatric and adult B-ALL cohorts, which received standardized treatment. High microRNA-126 levels and locus de-methylation at diagnosis associate with sub-optimal response to induction chemotherapy (MRD > 0.05% at day +33 or MRD+ at day +78).

overall_design: To unravel the role of miR-126 in leukemia heterogeneity in unmanipulated blasts we performed an extensive transcriptomic analysis on 5 primary B-ALL (4 adults and 1 pediatric) by single cell RNAsequencing. The obtained data were interrogated with a miR-126 high signature and we were able to detect a miR-126 intra tumoral heterogeneity even on this un-manipulated primary samples

first: Carolina

last: Caserta

first: Silvia

last: Nucera

first: Bernhard

last: Gentner


title: miR-126 identifies a quiescent and chemo-resistant human B-ALL cell subset that correlates with minimal residual disease [scRNAseq]

status: Public on Aug 30 2023

city: Milano

address: Via Olgettina 60

country: Italy

institute: SR-TIGET

type: Expression profiling by high throughput sequencing

summary: Complete elimination of B-cell acute lymphoblastic leukemia (B-ALL) by a risk-adapted primary treatment approach remains a clinical key objective, which fails in up to a third of patients. We hypothesized that microRNA-126, a core regulator of hematopoietic and leukemic stem cells, may resolve intra-tumor heterogeneity in B-ALL and uncover therapy-resistant subpopulations. By means of a miR-126-high signature (obtained by transducing primary human-B-all cells with a miR-126 reporter vector) and single cell RNA sequencing we identified a miR-126 derived intra tumoral heterogeneity in unmanipulated primary human B-ALL blasts

overall_design: To unravel the role of miR-126 in leukemia heterogeneity in unmanipulated blasts we performed an extensive transcriptomic analysis on 5 primary B-ALL (4 adults and 1 pediatric) by single cell RNAsequencing. The obtained data were interrogated with a miR-126 high signature and we were able to detect a miR-126 intra tumoral heterogeneity even on this un-manipulated primary samples.


title: Inhibition of EB3 activates the Meis2-Pax6 regenerative program in wet AMD [RNA-seq]

city: Chicago

first: Mark

last: Maienschein-Cline

state: IL

address: 900 S Ashland Ave, Rm 1058, M/C 937

department: Research Resources Center

country: USA

institute: University of Illinois at Chicago

summary: Wet age-related macular degeneration (AMD), characterized by leaky neovessels emanating from the choroid, is a main cause of blindness. As current treatments for wet AMD require regular intravitreal injections of anti-VEGF biologics, there is a need for the novel development of less invasive treatments. Here, we developed a novel inhibitor of microtubule-associated End Binding 3 protein (EB3), herein termed EBIN, which blocked pathological Ca2+ signaling in activated endothelial cells and suppressed leakage of choroidal neovessels. Delivery of EBIN via eye drops in mouse and non-human primate (NHP) models of AMD prevented neovascular leakage and neovascularization as effectively as intravitreal injection of anti-VEGF therapy. EBIN activated Meis2-Pax6 regenerative pathways in metabolic-active endothelial cells comprising neovessels and promoted tissue regeneration. Furthermore, single nuclei assay for transposase-accessible chromatin sequencing (sn-ATAC-seq) analysis demonstrated that in metabolic-active endothelial cells, the RPE, and photoreceptors, EBIN induced global increases in chromatin accessibility, the biological process progressively inhibited in AMD patients. These results suggest the unique therapeutic mode of action of this novel drug candidate, which can potentially promote regeneration of eye tissue by reversing the degenerative processes underlying both the neovascular and atrophic forms of AMD.

overall_design: Human retinal microvascular endothelial cells were first treated with either EBIN or vehicle before been cultured under normoxic or acute hypoxic (0.1% O2) for 3 hours. Total RNA from the cells were then obtained by RNeasy Plus kit. The concentration and quality of the RNA were assessed before reverse transcription to cDNA. The resulting cDNAs were sent to University of Chicago for sequencing.

first: Quinn

last: Lee

first: Wan

middle: C

last: Chan

first: Xinyan

last: Qu

first: Ying

last: Sun

first: Hazem

last: Abdelkarim

first: Jonathan

last: Le

first: Uzma

last: Saqib

first: Mitchell

middle: Y

first: Kevin

last: Kruse

first: Avik

last: Banerjee

first: Ben

last: Hitchinson

first: Melissa

last: Geyer

first: Fei

last: Huang

first: Victor

last: Guaiquil

first: Amelia

middle: A

last: Mutso

first: Martin

last: Sanders

middle: I

last: Rosenblatt

first: Vadim

last: Gaponenko

first: Asrar

middle: B

last: Malik

first: Yulia

last: Komarova


title: Inhibition of EB3 activates the Meis2-Pax6 regenerative program in wet AMD [scRNA-seq]

first: Mark

last: Maienschein-Cline

overall_design: Choroidal neovascularization was induced on day 0 in African green monkeys’ eye by laser photocoagulation targeting the Bruch’s membrane. Monkeys then received either 30 µL topical instillation of Myr-EBIN or vehicle in each eye twice daily for 7 days and once a day for another 14 days. After 21 days, the eyes were dissected and separated by different sections of the eyes. Samples that undergo sequencing were flash frozen and been sent to UC San Diego Center for Epigenomics for sample preparation and sequencing.

first: Quinn

last: Lee

first: Wan

last: Chan

first: Xinyan

last: Qu


title: KSHV uses viral IL6 to expand infected immunosuppressive macrophages (scRNA-seq)

city: Sacramento

first: Clifford

middle: G.

last: Tepper

state: CA

address: 4645 2nd Avenue, Room 2300A

department: Biochemistry and Molecular Medicine

institute: UC Davis School of Medicine

summary: Kaposi’s sarcoma-associated herpesvirus (KSHV) is an oncogenic double-stranded DNA virus and the etiologic agent of Kaposi’s sarcoma and hyperinflammatory lymphoproliferative disorders. Understanding the mechanism by which KSHV increases infected cell populations is crucial for curing KSHV-associated diseases. Here, we demonstrate that KSHV preferentially infects CD14+ monocytes and sustains viral replication through the viral interleukin-6 (vIL-6)-mediated activation of STAT1 and 3. Using vIL6-sufficient and vIL6-deficient recombinant KSHV, we demonstrated that vIL6 plays a critical role in promoting the proliferation and differentiation of KSHV-infected monocytes into macrophages. The macrophages derived from vIL6-sufficient (wild type) KSHV infection showed a distinct transcriptional profile of elevated IFN-pathway activation with immune suppression and were compromised in T-cell stimulation function compared to those from vIL6-deficient KSHV infection or uninfected control. These results highlight a clever strategy, in which KSHV utilizes vIL6 to secure its initial viral pool by expanding infected dysfunctional macrophages. This mechanism also facilitates KSHV to escape from host immune surveillance and to establish a lifelong infection.

overall_design: The goal of this study was to define the specific peripheral blood mononuclear cell (PBMC) cell types or lineages that are susceptible to infection by KSHV. To this end, recombinant KSHV (rKSHV.291) virions were purified from the culture supernatant of the iSLK.291 producer cell line. PBMCs were infected with rKSHV.291 at a multiplicity of infection (MOI)=1, fixed at various time points (day 0, 1, 2, and 4) after infection, and subjected to scRNA-seq analysis. KSHV infection and lytic replication in individual single cells were then monitored by the expression of the KSHV K2 gene that encodes vIL6.

first: Yoshihiro

last: Izumiya

first: Michiko

last: Shimoda

middle: G


title: KSHV uses viral IL6 to expand infected immunosuppressive macrophages

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

overall_design: Refer to individual Series


title: Single cell transcriptome of cultured thymic cortical and medullary progenitor and stem cells and skin keratinocyte stem/progenitor cells

city: London

first: Paola

last: Bonfanti

address: 1 Midland Road

country: United Kingdom

institute: The Francis Crick Institute

summary:
Purpose: The goal of this study is to characterise expanding thymic progenitor and stem cells from whole epithelial as well as cortical, medullary thymus sub-compartments. As reference, we have sequenced skin keratinocyte progenitor and stem cells
Methods: Cells have been expanded for 4 passages on mouse feeder layer cells then loaded into the 10x Genomics Chromium Platform, and sequenced using Illumina HiSeq 4000.
Conclusions: Our study compared thymic progenitor and stem cells from different origin and dissected in vitro properties of thymic and skin progenitor and stem cells

overall_design: 6 cultivated epithelial progenitors and stem cell populations were used for single-cell RNA-sequencing (10xGenomics Chromium Platform), and sequenced using Illumina HiSeq 4000.

first: Roberta

last: Ragazzini

first: Stefan

last: Boeing


title: CRISPR-Screen Identifies Senescence-Related Genes in Human Dyskeratosis Congenita Cells

city: New York City

first: Alireza

last: Khodadadi-Jamayran

state: NY

address: 550 1st Ave, MSB 304

department: Division of Advanced Research Technologies (DART)

institute: New York University, NYU Langone Medical Center

summary: Aging is the consequence of intra- and extracellular events that promote cellular senescence. Dyskeratosis congenita (DC) is an example of a premature aging disorder caused by underlying telomere/telomerase-related mutations. Cells from these patients offer an opportunity to study telomere-related aging and senescence. Our previous work has found that telomere shortening stimulates DNA damage responses (DDR) and increases reactive oxygen species (ROS), thereby promoting entry into senescence. This work also found that telomere elongation via TERT expression, the catalytic component of the telomere-elongating enzyme telomerase, or p53 shRNA could decrease ROS by disrupting this telomere-DDR-ROS pathway. To further characterize this pathway, we performed a CRISPR/Cas9 knockout screen to identify genes that extend lifespan in DC cells. Of the cellular clones isolated due to increased lifespan, 34% had a guide RNA (gRNA) targeting CEBPB while gRNAs targeting WSB1, MED28 and p73 were observed multiple times. CEBPB is a transcription factor associated with activation of proinflammatory response genes suggesting that inflammation may be present in DC cells. The inflammatory response was investigated using RNA-Seq to compare DC and control cells. Expression of inflammatory genes were found to be significantly elevated (p<0.0001) in addition to a key subset of these inflammation-related genes (IL1B, IL6, IL8, IL12A, CXCL1 (GROa), CXCL2 (GROb), and CXCL5) which are regulated by CEBPB. Exogenous TERT expression led to downregulation of RNA/protein CEBPB expression and the inflammatory response genes suggesting a telomere-length dependent mechanism to regulate CEBPB. Furthermore, unlike exogenous TERT and p53 shRNA, CEBPB shRNA did not significantly decrease ROS suggesting that CEBPB’s contribution in DC cells’ senescence is ROS-independent. Our findings demonstrate a key role for CEBPB in engaging senescence by mobilizing an inflammatory response within DC cells.

overall_design: Recover fibroblast from CRISPR knockout screen that acquire an insertion/deletion mutation provding a rescue from early senescence

first: Erik

middle: R

last: Westin

first: Linh

middle: K

last: Pham

first: Moon

middle: L

last: Tung

first: Frederick

middle: D

last: Goldman


title: KSHV uses viral IL6 to expand infected immunosuppressive macrophages (RNA-seq)

overall_design: The goal of this study was to define the role of KSHV vIL6 in monocyte activation and differentiation by using de novo infection of PBMCs with vIL6-sufficient and vIL6-deficient recombinant KSHV followed by transcription profiling with RNA-seq. To this end, recombinant KSHV lacking vIL6 expression was prepared by inserting stop codons into the vIL6 gene (vIL6STOP; K2_neg) using the KSHV bacterial artificial chromosome (BAC) clone BAC16. A revertant KSHV with intact vIL6 expression (vIL6REV; K2_pos) was also generated by changing the sequence back to the original wild type sequence with BAC recombination. Duplicate PBMC cultures were each infected with vIL6REV, vIL6STOP, or mock-infected (PBS) and cells were harvested at 7 days post-infection for isolation of total RNA and RNA-seq analysis. Comparative gene expression profiling analysis of the RNA-seq data was then performed to identify the differentially expressed genes (DEGs) in monocytes infected with vIL6-sufficient or vIL6-deficient KSHV.


title: Single cell transcriptome of cultured thymic progenitor and stem cells

summary:
Purpose: The goal of this study is to characterise expanding thymic progenitor and stem cells in culture
Methods: FACS-isolation protocol to separate epithelial cells from mouse feeder layer cells then loaded into the 10x Genomics Chromium Platform, and sequenced using Illumina HiSeq 4000.
Conclusions: Our study dissected in vitro properties of thymic progenitor and stem cells

overall_design: 2 FACS sorted epithelial populations were used for single-cell RNA-sequencing (10xGenomics Chromium Platform), and sequenced using Illumina HiSeq 4000.


title: A single cell transcriptome atlas of Human thymic epithelial compartments

summary:
Purpose: The goal of this study is to characterise cortical (cTECs) and medullary (mTECs) epithelial cells of the human thymus.
Methods: FACS-isolation protocol to separate 4 different epithelial cells populations from total dissociation of human thymus that is negative for hCD45 and either positive for EPCAM or CD205, then loaded into the 10x Genomics Chromium Platform, and sequenced using Illumina HiSeq 4000.
Conclusions: Our study identified and characterised human thymic stem/progenitors cells, as well as identified new specialised cell clusters in each of the two compartments

overall_design: 4 FACS sorted epithelial populations were used for single-cell RNA-sequencing (10xGenomics Chromium Platform), and sequenced using Illumina HiSeq 4000.


title: Histone H3 K27M mediated Regulation of Cancer Cell Stemness and Differentiation in Diffuse Intrinsic Pontine Glioma (DIPG) [ChIP-seq]

city: Ann Arbor

first: Brian

last: Magnuson

state: MI

address: 1500 East Medical Center Drive

institute: University of Michigan

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Diffuse intrinsic pontine glioma (DIPG) is a rare pediatric brain tumor with a median survival of 10-15 months. Histone H3 is mutated in 80% of DIPGs, dictating tumor location, onset, and outcome. Therapeutic resistance remains a major obstacle to preventing tumor recurrence and is in part driven by cancer stem cells (CSCs), which exhibit self-renewal properties and tumorigenic potential. In previous studies, we have identified these proliferative and tumorigenic features in aldehyde dehydrogenase positive (ALDH+) CSCs in H3K27M mutant DIPG. We hypothesize that ALDH-mediated cancer stemness and resistance may in part be driven by H3K27M.  Histone H3 K27 acetylation was assayed by chromatin immunoprecipitation and sequencing (ChIP-seq).

overall_design: SU-DIPG-XIII cells (here also "DIPG13") were treated with PI3K/mTOR inhibitor GSK458 or DMSO (one sample each). ChIP DNA was obtained using anti-H3K27Ac antibodies, as well as IgG IP and input DNA for a total of 6 ChIP-seq libraries.

first: Monika

last: Sharma

first: Stefanie

last: Galban


title: To find target miRNA of Zeb1

status: Public on Sep 07 2023

city: Xiamen

first: Nengming

last: Xiao

state: 福建

address: Xiang an Campus Xiang an south Road Xiamen University, fengting 2 -2044

country: China

institute: Xiamen University

type: Non-coding RNA profiling by high throughput sequencing

summary:
To investigate potential target miRNA of Zeb1 that were involved in regulation of antigen cross-presentation.
We then performed gene expression profiling analysis using data obtained from miRNA-seq from WT and Zeb1-deficient BMDC after HKLM-OVA stimulating for 4 hours.

overall_design:
Comparative gene expression profiling analysis of miRNA-seq data from WT and Zeb1-deficient BMDC.
Nengming,Xiao

first: Yan

last: Wang

first: Quan

last: Zhang


title: A single cell atlas of CD19 chimeric antigen receptor T cells

status: Public on Aug 29 2023

city: Houston

first: Michael

last: Green

state: TX

address: 1515 Holcombe Blvd

department: Lymphoma/Myeloma

institute: MD Anderson Cancer Center

summary: We present a resource of scRNA-seq and scTCR-seq data from the infusion products of 59 patients with relapsed or refractory large B-cell lymphoma (rrLBCL) treated with standard-of-care axicabtagene ciloleucel (Axi-cel), that we have made publicly available to facilitate ongoing and future discovery efforts that will improve patient outcomes.

overall_design: Single cell gene expression profiles and TCR data for infusion products from 59 patients treated with axicabtagene ciloleucel CAR T cell therapies.


title: miR-126 identifies a quiescent and chemo-resistant human B-ALL cell subset that correlates with minimal residual disease [RNASeq 2]

first: Matteo

last: Barcella

overall_design: Chronic induction of miR-126 consist of 17 days of continuous miR-126 overexpression in primary human B-ALL PDXs previously transduced with a doxycycline-inducible miR-126-overexpressing lentiviral vector. This PDXs were exposed to doxycycline for 17 days and to Vincristine and Dexamethasone (or to PBS) for 2 weeks. At the end of the treatment  BM derived 126-OE-OFP+ fresh cells were sorted and sequenced by RNAsequencing. We compared blasts isolated from Vincristine and Dexamethasone (Vin+Dex) chemotherapy-treated (n=4 mice, 4 fractions 126-OE-OFP+, 4 fractions 126-OE-OFP-) vs. PBS-treated mice (n=3 mice, 3 fractions 126-OE-OFP+, 3 fractions 126-OE-OFP-)

first: Carolina

last: Caserta

first: Silvia

last: Nucera

first: Bernhard

last: Gentner


title: A mouse model for assessing mTOR kinase-independent role and off-target effect of mTOR kinase inhibitors [I]

city: Cincinnati

first: Yi

last: Zheng

state: OH

address: 3333 Burnet Ave MLC7013

department: Experimental Hematology and Cancer Biology

institute: CINCINNATI CHILDRENS HOSP MED CTR

summary: mTOR is a serine/threonine kinase with a crucial role in regulating fundamental cellular processes and is an attractive anticancer target. Multiple clinical trials of mTOR kinase inhibitors such as AZD2014 that suppress both mTORC1 and mTORC2 are ongoing, but their specificity and toxicity features remain a concern. We have developed an inducible kinase-dead D2338A mTOR mouse knock-in model and show that it is useful to study the kinase-independent function of mTOR and for assessing the off-target effects of mTOR kinase inhibitors. We found that while the mTOR KI mice in hematopoietic system displayed similar phenotypes to that of mTOR KO mice, they have significantly prolonged survival time than the KO mice. Whereas AZD2014 was mostly on-target in suppressing mTOR kinase activity, mimicking that seen in KI stem cells in transcriptome analysis, off-target genes in KI cells induced by AZD2014 including Ahnak2, Emp1, Lilrb4, Tpsb2, and Cma1 were detected. Our studies reveal kinase-independent function of mTOR in hematopoiesis and presents a mouse model for precisely assessing specificity of mTOR kinase inhibitors. Stringent test of drugs with such mouse models in the preclinical setting may provide useful information guiding the safety and efficacy of related clinical trials.

overall_design: WT Control, KO mutant, KI mutant.

first: Cuiqing

last: Fan


title: Single cell transcriptome of cultured skin and airways progenitor and stem cells

first: Paola

last: Bonfanti

summary:
Purpose: The goal of this study is to characterise expanding epithelial progenitor and stem cells in culture from different organs and compare them to thymic ones
Methods: FACS-isolation protocol to separate epithelial cells from mouse feeder layer cells then loaded into the 10x Genomics Chromium Platform, and sequenced using Illumina HiSeq 4000.
Conclusions: Our study dissected in vitro properties of airways and skin progenitor and stem cells and showed they differ from thymic ones

first: Roberta

last: Ragazzini

first: Stefan

last: Boeing


title: Generation Of Human Intestinal Organoids From Cronkhite Canada Syndrome Patients Reveals Serotonin As A Link To Intestinal Proliferation

first: Mary

last: Estes

address: 1 Baylor Plaza

department: Microbiology and Virology

institute: Baylor College of Medicine

summary: Cronkhite Canada Syndrome (CCS) is a rare, non-inherited polyposis syndrome affecting 1 in a million individuals. Despite over 50 years of CCS cases, the etiopathogenesis and optimal treatment for CCS remains unknown due to the rarity of the disease and lack of model systems. To investigate and better understand the etiology of CCS, we generated human intestinal organoids (HIOs) from intestinal stem cells isolated from two CCS patients. We discovered that CCS HIOs have unique growth characteristics and have increased serotonin (5-hydroxytryptamine, 5HT) positive cells. Further investigation revealed that 5HT can induce HIO proliferation, and inhibition of 5HT results in decreased proliferation in the CCS HIOs. Our findings indicate that dysregulation of 5HT may explain polyposis observed in CCS and provide new insight into regulation of the intestinal stem cell. This work illustrates the important contribution organoids can make to understanding disease etiology and identifying novel therapies.

overall_design: Biopsies from 2 CCS patients and controls were obtained and the stem cells were isolated to generate the organoids. Organoids were differentiated for 3 days prior to RNA extraction and sequencing.

first: Victoria

last: Poplaski

first: Carolyn

last: Bomidi

first: Amal

last: Kambal

first: Hoa

last: Nguyen-Phuc

first: Xi-Lei

last: Zeng

first: Linda

last: Feagins

first: Richa

last: Shukla

first: Sarah

middle: E

last: Blutt


title: Histone H3 K27M mediated Regulation of Cancer Cell Stemness and Differentiation in Diffuse Intrinsic Pontine Glioma (DIPG) [RNA-seq]

first: Brian

last: Magnuson

summary: Diffuse intrinsic pontine glioma (DIPG) is a rare pediatric brain tumor with a median survival of 10-15 months. Histone H3 is mutated in 80% of DIPGs, dictating tumor location, onset, and outcome. Therapeutic resistance remains a major obstacle to preventing tumor recurrence and is in part driven by cancer stem cells (CSCs), which exhibit self-renewal properties and tumorigenic potential. In previous studies, we have identified these proliferative and tumorigenic features in aldehyde dehydrogenase positive (ALDH+) CSCs in H3K27M mutant DIPG. We hypothesize that ALDH-mediated cancer stemness and resistance may in part be driven by H3K27M. ALDH1A3 expression was reduced in CRISPR-edited DIPG cells (SU-DIPG-XIII) where the H3K27M mutation had been removed (H3K27M-KO). Furthermore, these gained differentiation characteristics and lost their neurosphere-forming potential. The parental (H3K27M) and H3K27M-KO cells were also subjected to ionizing radiation to identify genes responsible for radioresistance in histone-mutated DIPG radioresistance.  Nascent transcriptomes were obtained from these cells using bromouridine labeling and capture followed by sequencing (Bru-seq).

overall_design: Two biological replicates each of SU-DIPG-XIII cells (here also stylized "DIPG13") with mutant histone (H3.3-K27M, here also "H3K27M") or CRISPR-mediated knockout of mutant histone (H3K27M-KO) were irradiated (5 Gy) or not, for a total of 8 samples.

first: Monika

last: Sharma

first: Stefanie

last: Galban


title: Kupffer cells coordinate anti-metastatic activity and immune resistance in the liver

status: Public on Sep 04 2023

city: Philadelphia

first: Max

middle: M

last: Wattenberg

state: PENNSYLVANIA

address: 3400 Civic Center Blvd

institute: University of Pennsylvania

summary: The goal of this study was to define the impact of beta-glucan on the liver

overall_design: This repository contains single cell RNA sequencing of mouse livers with intra-portal injection (8572-YFP mouse tumor cells) (n=3 samples/condition) untreated or 14 days post-treatment with beta-glucan x3. Tissues were digested in RPMI media containing collagenase/DNAse and passed through a 40 μm strainer. Subsequently, dead cells were removed using the Dead Cell Removal Kit (STEMCELL Technologies). CD45+ cells were isolated using the CD45+ isolation kit (STEMCELL Technologies). Kupffer cells were enriched by layering the flow through from the CD45+ selection step over Ficoll Paque Plus and cells from the buffy coat combined with CD45+ cells for sequencing. Single-cell cDNA libraries were prepared at the CHOP Single Cell Genomics Core at the University of Pennsylvania.

first: Stacy

last: Thomas

first: Shaanti

last: Choi-Bose

first: Mark

last: Uhlik

first: Ben

last: Harrison

first: Heather

last: Coho

first: Christopher

last: Cassella

first: Meredith

last: Stone

first: Dhruv

last: Patel

first: Kelly

last: Markowitz

first: Devora

last: Delman

first: Michael

last: Chisamore

first: Jeremy

last: Drees

first: Nandita

last: Bose

first: Gregory

last: Beatty


title: A mouse model for assessing mTOR kinase-independent role and off-target effect of mTOR kinase inhibitors [III]

first: Yi

last: Zheng

overall_design: Comparative gene expression profiling analysis of RNA-seq data for mTOR WT HSCs and 0.01% DMSO-treated mTOR WT HSCs.

first: Cuiqing

last: Fan


title: Hyperpolarization-Activated Cyclic Nucleotide-Gated Potassium Channel 3 Promotes HCC Development in a Female-biased Manner

city: East Lansing

first: Hua

last: Xiao

state: Michigan

address: 567 WILSON RD, RM 2201

department: Physiology

institute: Michigan State University

summary: Sex differences in hepatocellular carcinoma (HCC) development are regulated by sex and non-sex chromosomes, sex hormones, and environmental factors. We previously reported that Ncoa5+/- mice develop HCC in a male-biased manner. Here we show that NCOA5 expression was reduced in male patient HCCs while the expression of an NCOA5-interacting tumor suppressor, TIP30, was lower in female HCCs. Tip30 heterozygous deletion did not change HCC incidence in Ncoa5+/- male mice but dramatically increased HCC incidence in Ncoa5+/- female mice, accompanied by hepatic hyperpolarization-activated cyclic nucleotide-gated cation channel 3 (HCN3) overexpression. HCN3 overexpression cooperated with MYC to promote mouse HCC development, whereas Hcn3 knockout preferentially hindered HCC development in female mice. Furthermore, HCN3 amplification and overexpression occurred in human HCCs and correlated with a poorer prognosis of patients in a female-biased manner. Our results suggest that TIP30 and NCOA5 protect against female liver oncogenesis and that HCN3 is a female-biased HCC driver.

overall_design: mRNA profiles of liver from 5-month-old female mice with heterozygous deletion of Ncoa5, Tip30, both Ncoa5 and Tip30, and wild-type female mice. 4 mice in each group.

first: Yueqi

last: Zhang

first: Xinhui

last: Liu


title: Trained immunity induced by high salt diet impedes stroke recovery

city: Taiepi

first: Che-Feng

last: Chang

address: No. 1, Sec. 1, Jen-Ai Rd.

country: Taiwan

institute: National Taiwan University College of Medicine

summary: High salt diet-induced functional adaptions in bone marrow progenitor cells, which subsequently reduced monocyte-derived macrophage reparative phenotype and impeded stroke recovery

overall_design: We performed bulk RNA sequencing (RNA-seq) of hematopoietic stem and progenitor cells from 8-week normal diet and high salt diet mice with or without ICH induction.

first: Liang-Chuan

last: Lai

first: Kai-Chien

last: Yang


title: Lasting response by vertical inhibition with Cetuximab and Trametinib in KRAS mutated Colorectal Cancer Patient-derived Xenografts (PDX)

status: Public on Aug 28 2023

city: Bochum

first: Stephan

last: Hahn

address: Universitaetsstrasse 150

department: MGO

country: Germany

institute: Ruhr-University Bochum

summary: Global mRNA expression profiling of patient derived colon cancer xenograft models (N=13) were collected using Agilent human whole genome array (G4845A AMADID  026652, cRNA 4x44k V2) .  All xenograft models carried an activating. All Mice were either untreated (controls) or treated with  treated with cetuximab (25 mg/kg, twice weekly, i.p., Merck) and trametinib (0.5 mg/kg, five subsequent days per week, p.o., Hycultec)  to establish response characteristics. Tumors showing disease control  under the combination treatment were chronically treated to establish cetuximab secondary resistant tumors which was successful for BoC2 and BoC56 .Primary resistant PDX tumors (BoC51, 109, 117, and 122) and secondary resistant tumors (BoC2 and BoC56) were harvested at the end of treatment (days 29 to 59). For BoC2 and 56, untreated control Tumors (K) were harvested once tumors reached approx. 1000mm3 in volume.

overall_design: Three conditions, untreated control (K), 5 days cetuximab and trametinib treatment (5dTC) and cetuximab and trametinib resistant (CT) tumors were analyzed.


title: A mouse model for assessing mTOR kinase-independent role and off-target effect of mTOR kinase inhibitors

overall_design: Refer to individual Series


title: Temporal Analyses Reveal a Pivotal Role for Sense and Anti-sense Enhancer RNAs in Coordinate Immunoglobulin Lambda Locus Activation

city: Oxford

first: Alastair

last: Smith

state: England

address: The MRC Weatherall Institute of Molecular Medicine

institute: University of Oxford

summary: Transcription enhancers are essential activators of V(D)J recombination that orchestrate non-coding transcription through complementary, unrearranged gene segments. How transcription is coordinately increased at spatially distinct promoters, however, remains poorly understood. Using the murine immunoglobulin lambda (Igλ) locus as model, we find that three enhancer-like elements in the 3’ Igλ domain, Eλ3-1, HSCλ1 and HSE-1, show strikingly similar transcription factor binding dynamics and close spatial proximity, suggesting that they form an active enhancer hub. Temporal analyses show coordinate recruitment of complementary V and J gene segments to this hub, with comparable transcription factor binding dynamics to that at enhancers. We find further that E2A, p300, Mediator and Integrator bind to enhancers as early events, whereas YY1 recruitment and eRNA synthesis occur later, corresponding to transcription activation. Remarkably, the interplay between sense and anti-sense enhancer RNA is central to both active enhancer hub formation and coordinate Igλ transcription: Antisense Eλ3-1 eRNA represses Igλ activation whereas temporal analyses demonstrate that accumulating levels of sense eRNA boost YY1 recruitment to stabilise enhancer hub/promoter interactions and lead to coordinate transcription activation. These studies therefore demonstrate for the first time a critical role for threshold levels of sense versus antisense eRNA in locus activation.

overall_design: ATAC-seq performed on the PIPER-15 cell line

first: Zeqian

last: Gao

first: Joan

last: Boyes


title: Thymic mimetic cells function beyond self-tolerance [scATAC-Seq Multiome]

city: Rehovot

first: Jakub

last: Abramson

address: 234 Herzl st.

department: Department of Immunology

country: Israel

institute: Weizmann Institute of Science

summary: Deliniating thymic epithelial cell heterogeneity by combined single cell RNAseq and ATACseq

overall_design: 10x single cell multiome scATACseq profiles of thymic epithelial cells from four pooled EpCAM enriched stroma of 5 week old B6.Adig mice.

first: Yael

last: Goldfarb

first: Tal

last: Givony


title: Thymic mimetic cells function beyond self-tolerance [Spib]

summary: To examine the Microfold thymic epithelial cell transcriptome within the mTEC-III compartment we sorted this population from young Spib KO mice and performed bulk RNAseq

overall_design: mRNA profiles of mTEC-III from young (5 weeks old) Spib WT or Spib KO mice ,were generated by deep sequencing, using the MARSseq method.


title: Thymic mimetic cells function beyond self-tolerance [Insm1]

summary: To examine the Endocrine thymic epithelial cell transcriptome within the mTEC-III compartment we sorted this population from young Insm1 cKO mice and performed bulk RNAseq

overall_design: mRNA profiles of mTEC-III from young (4 weeks old) Insm1 wt or Insm1 cKO mice ,were generated by deep sequencing, using the MARSseq method.


title: A mouse model for assessing mTOR kinase-independent role and off-target effect of mTOR kinase inhibitors [II]

overall_design: WT Control, KI mutant, AZD.


title: ECM composition dictates the mode of cell competition and competence for tumor initiation

city: Bruxelles

first: Yura

last: Song

address: 808, route de Lennik

country: Belgium

institute: Université Libre de Bruxelles

summary: The skin epidermis is constantly renewed throughout life. Disruption of the balance between renewal and differentiation can lead to uncontrolled growth and tumor initiation. Basal cell carcinoma (BCC) is the most frequent cancer in human. Cell competition is a biological process that leads to the elimination of cells by winner cells, which have been proposed to be important for tumour initiation. Recent studies identified somatic mutations in cancer drivers in normal tissues that get colonized by clones carrying oncogenic mutations.  However, how the different oncogenic mutations impact the balance between renewal and differentiation and lead to clonal expansion, cell competition, tissue colonization and tumor development is currently unknown. Here, using multidisciplinary approaches combining in vivo clonal analysis using intra-vital microscopy, single cell analysis, and functional analysis, we defined at a single cell resolution in living animals how mutations associated with BCC development (SmoM2) affect clonal competition and tumor initiation in real time.  We found that SmoM2 expression in the ear epidermis induces a stereotypical pattern of cell competition with clonal expansion leading tumor initiation and invasion. In contrast, SmoM2 expression in the back skin epidermis led to a similar clonal expansion that induces lateral cell competition but without inducing dermal invasion and tumor formation. Single cell analysis showed that oncogene expression is associated with a cellular reprogramming of adult interfollicular cells into embryonic hair follicle progenitor (EHFP) state in the ear but not in the back skin. Comparison between ear and back skin epidermis revealed a very different composition of the dermis with increased stiffness and a denser collagen 1 network in the back skin. Decreasing Collagen 1 expression in the back skin overcame the natural resistance of these cells to undergo EHFP reprogramming and tumor initiation following SmoM2 expression. Altogether our study demonstrates that ECM composition controls the mode of clonal competition and competence to undergo oncogenic transformation upon oncogene expression.

overall_design: FACS isolated keratinocyte CD34- YFP+ cells from K14CreER/Rosa-SmoM2-YFP mouse; FACS isolated keratinocyte CD34- Alpha6+ cells from CD1 mouse (Epidermis) / Separation of dermis by mechanical process from epidermis(Dermis)

first: Cédric

last: Blanpain

first: Nordin

last: Bansaccal

first: Rahul

last: Sarate


title: Thymic mimetic cells function beyond self-tolerance [IgM and Ccr6]

summary: To examine the microfold thymic epithelial cell transcriptome within the mTEC-III compartment we sorted this population from young IgM or Ccr6 KO mice and performed bulk RNAseq

overall_design: mRNA profiles of mTEC-III from young (5 weeks old)  IgM or Ccr6 KO mice ,were generated by deep sequencing, using the MARSseq method.


title: Transcriptional Regulation of Sunflower Heliotropism and Phototropism

city: Davis

first: Stacey

last: Harmer

state: California

address: One Shields Ave

department: Plant Biology

institute: University of California Davis

summary: Sunflowers are famous for their ability to track the sun throughout the day, and then reorient at night to face east the following morning. This process, termed heliotropism, is generally believed to be a specialized form of phototropism; however, the underlying mechanism is unknown. To better understand heliotropism we used RNA sequencing to compare the transcriptional profiles of sunflower stems during heliotropism, and blue light-induced phototropism.

overall_design: Comparative gene expression profiling analysis of RNA-seq data from stem tissue of sunflowers undergoing either phototropism or heliotropism.

first: Christopher

middle: J

last: Brooks

first: Hagop

middle: S

last: Atamian


title: Thymic mimetic cells function beyond self-tolerance [scRNA-Seq Spib chimeras]

summary: Deliniating the antigen-presenting compartment of thymic  Spib–/– and Spib+/– chimeric mice

overall_design: 10x single cell RNAseq profiles of thymic CD45.1+ CD3– (CD4/CD8DP)–MHCIImid-high cells from perfused Spib–/– and Spib+/– chimeric mice


title: Thymic mimetic cells function beyond self-tolerance [mimetics]

summary: To examine the transcriptome of various mimetic thymic epithelial cell populations (CilTEC, MyoTEC, Hetero-post TEC, microfold TEC, endocrine TEC and corneoTEC) we sorted these populations from young WT mice and performed bulk RNAseq

overall_design: mRNA profiles of various thymic epithelial cells (TEC) (e.g. CilTEC, MyoTEC, Hetero-post TEC, microfold TEC, endocrine TEC and corneoTEC) from young (5 weeks old)  C56Bl/6 mice ,were generated by deep sequencing, using the MARSseq method.


title: Chromatin remodeling of histone H3 variants by DDM1 underlies epigenetic inheritance of DNA methylation.

city: Cold Spring Harbor

first: Jonathan

last: Cahn

address: 1 Bungtown Rd

institute: CSHL

type: Methylation profiling by high throughput sequencing

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


title: Myo-differentiation reporter screen reveals NF-Y as an activator of PAX3-FOXO1 in rhabdomyosarcoma [4C-seq]

last: Vakoc

state: New York

institute: Cold Spring Harbor Laboratory

summary: Recurrent chromosomal rearrangements found in rhabdomyosarcoma (RMS) produce the PAX3-FOXO1 fusion protein, which is an oncogenic driver and a dependency in this disease. One important function of PAX3-FOXO1 is to arrest myogenic differentiation, which is linked to the ability of RMS cells to gain an unlimited proliferation potential. Here, we developed a phenotypic screening strategy for identifying factors that collaborate with PAX3-FOXO1 to block myo-differentiation in RMS. Unlike most genes evaluated in our screen, we found that loss of any of the three subunits of the Nuclear Factor Y (NF-Y) complex leads to a myo-differentiation phenotype that resembles the effect of inactivating PAX3-FOXO1. While the transcriptomes of NF-Y- and PAX3-FOXO1-deficient RMS cells bear remarkable similarity to one another, we found that these two transcription factors occupy non-overlapping sites along the genome: NF-Y preferentially occupies promoters, whereas PAX3-FOXO1 primarily binds to distal enhancers. By integrating multiple functional approaches, we map the PAX3 promoter as the point of intersection between these two regulators. We show that NF-Y occupies CCAAT motifs present upstream of PAX3 to function as a transcriptional activator of PAX3-FOXO1 expression in RMS. These findings reveal a critical upstream role of NF-Y in the oncogenic PAX3-FOXO1 pathway, highlighting how a broadly essential transcription factor can perform tumor-specific roles in governing cellular state.

overall_design: 4C-seq validation of interactions between NF-Y bound promoters and PAX3-FOXO1-bound distant enhancers at 4 different loci (ERRFI1, SIX1, PEG3, JUN) in RH4 rhabdomyosarcoma cells. Performed in 2 biological replicates.

first: Martyna

middle: W

last: Sroka

first: Marit

last: Vermunt

first: Gerd

last: Blobel


title: Myo-differentiation reporter screen reveals NF-Y as an activator of PAX3-FOXO1 in rhabdomyosarcoma


title: Chromatin remodeling of histone H3 variants by DDM1 underlies epigenetic inheritance of DNA methylation [ChIP-seq]

summary: Epigenetic inheritance refers to the faithful replication of DNA and histone modification independent of DNA sequence. Nucleosomes block access to DNA methyltransferase during S phase, unless they are remodeled by Decrease in DNA methylation 1 (DDM1[Lsh/HELLS]), a Snf2-like master regulator of epigenetic inheritance. We show that DDM1 activity results in replacement of the transcriptional histone variant H3.3 for the replicative variant H3.1. Inddm1mutants, DNA methylation can be restored by loss of the H3.3 chaperone HIRA, while the H3.1 chaperone CAF-1 becomes essential. The single-particle cryo-EM structure at 3.2 Å of DDM1 with a variant nucleosome reveals direct engagement at SHL2 with histone H3.3 at or near variant residues required for assembly, as well as with the deacetylated H4 tail. An N-terminal autoinhibitory domain binds H2A variants to allow remodeling, while a di-sulphide bond in the helicase domain is essential for activity in vivo and in vitro. Differential remodeling of H3 and H2A variants in vitro reflects preferential deposition in vivo. DDM1 co-localizes with H3.1 and H3.3 during the cell cycle, and with the DNA methyltransferase MET1[Dnmt1]. DDM1 localization to the chromosome is blocked by H4K16 acetylation, which accumulates at DDM1 targets in ddm1 mutants, as does the sperm cell specific H3.3 variant MGH3 in pollen, which acts as a placeholder nucleosome and contributes to epigenetic inheritance.

overall_design:
Chromatin Immunoprecipitation sequencing was performed on wild-type and ddm1 mutants for DDM1 (vs Input), H3.3 (vs H3), H3K27me1 (vs H3), H4K16ac (vs H4) and MGH3 (vs Input)
Please note that processed data generated from both ChIP and Input/control sample is linked to the corresponding ChIP sample records.

first: Seung

last: Lee

first: Robert

last: Martienssen


title: Myo-differentiation reporter screen reveals NF-Y as an activator of PAX3-FOXO1 in rhabdomyosarcoma [scRNA-Seq]

overall_design: scRNA-seq of RH4 human rhabdomyosarcoma cells transduced with sgRNAs targeting NFYC, PAX3-FOXO1 or ROSA26 control. Control/reference sample: CRISPR-mediated targeting of ROSA26; n=1 biological replicate.

first: Sroka

last: Martyna

first: Preall

last: Jonathan

first: Vakoc

last: Christopher


title: Elongation rate of RNA polymerase II affects pausing patterns across the 3’ UTR

city: Chapel Hill

first: Alexandra

last: Khitun

state: North Carolina

address: 250 Bell Tower Drive, 3244Genome Sciences Building

department: Chemistry

institute: UNC Chapel Hill

summary: Yeast mRNAs are polyadenylated at multiple sites in their 3’ untranslated regions (3’UTRs), and poly(A) site usage is regulated by the rate of transcriptional elongation by RNA polymerase II (Pol II).  Slow Pol II derivatives favor upstream poly(A) sites and fast Pol II derivatives favor downstream poly(A) sites.  Transcriptional elongation and polyadenylation are linked at the nucleotide level, presumably reflecting Pol II dwell time at each residue that influences the level of polyadenylation.  Here, we investigate the relationship between Pol II pause sites and poly(A) sites within 3’UTRs.

overall_design:
Elongation behavior of RPO21 variants bearing point mutations that affect RNA polymerase II elongation speed were assayed by NET-seq in biological duplicate.
Polyadenylation patterns in ubp8Δ and rpb4Δ deletion strains were profiled using 3'READS in biological duplicate.

first: Kevin

last: Struhl


title: Myo-differentiation reporter screen reveals NF-Y as an activator of PAX3-FOXO1 in rhabdomyosarcoma [CRISPR screens]

overall_design: Comparative analysis of sgRNA abundance in RH4 rhabdomyosarcoma cells following negative selection or myo-differentiation screening.

first: Skopelitis

last: Damianos


title: Mycobacterium smegmatis secreting methionine sulfoxide reductase A (MsrA) modulates cellular processes in mouse macrophages

city: El Paso

first: Enrique

middle: Ivan

last: Ramos

state: Texas

address: 5001 El Paso Dr.

department: Molecular and Translational Medicine

institute: Texas Tech University Health Sciences Center El Paso

summary: Methionine sulfoxide reductase A (MsrA) is an antioxidant repair enzyme that reduces the oxidized methionine (Met-O) in proteins to methionine (Met). It plays a pivotal role in the cellular processes has been well established by overexpressing, silencing and knocking down of MsrA or by deleting the gene encoding MsrA in several species. We are specifically interested in understanding the role of secreted MsrA of bacterial pathogens. To elucidate this, we infected mouse bone marrow derived macrophages (BMDMs) with recombinant Mycobacterium smegmatis strain (MSM) secreting a bacterial MsrA or M. smegmatis strain (MSC) carrying only the control vector. BMDMs infected with MSM induced higher levels of ROS and TNF-α than BMDMs infected with MSC. The increased ROS and TNF-α in BMDMs infected with MSM correlated with elevated necrotic cell death.  Further, RNA-Seq transcriptome analysis of BMDMs infected with MSC and MSM revealed differential expression of protein and RNA coding genes, suggesting that bacterial delivered MsrA could modulate the host cellular processes. Finally, KEGG pathway enrichment analysis identified down-regulation of cancer related signaling genes in MSM infected cells, indicating that MsrA has the potential to regulate the development and progression of cancer.

overall_design: BMDMs (1 x10^6 cells/well) in each well of a 6-well culture plate were infected with MSC or MSM at an MOI of 1:5 or uninfected (control cells) and incubated for 2 h at 37°C in a CO2 incubator. After the incubation, the cells were washed with 1xPBS to remove the extracellular bacteria. Then the cells were lifted using cell scraper and collected via centrifugation at 200xg for 10 minutes. Total RNA was isolated from the infected BMDMs using EZ-10 DNAaway RNA Mini-Preps Kit (Bio Basic, Canada). TapeStation (Agilent Technologies 4200) was used to analyze RNA integrity of the preparation, and RNA quantification was done using Nanodrop (Thermo Scientific, USA). 4 ug of total RNA with >9 RIN (RNA Integrity Number) was used for RNA-Seq library preparation.

first: Raja

last: Veerapandian

first: Mahalakshmi

last: Vijayaraghavan

first: Melina

last: Sedano

first: Areanna

last: Carmona

first: Kishore

last: Das

first: Jessica

last: Chacon

first: Shrikanth

last: Gadad

first: Subramanian

last: Dhandayuthapani


title: Oxidized LDL accumulation suppresses glycolysis and attenuates the macrophage inflammatory response by diverting transcription from the HIF-1α to the Nrf2 pathway

city: Toronto

first: Myron

last: Cybulsky

state: Ontario

address: 101 College Street

department: Toronto General Hospital Research Institute

country: Canada

institute: University Health Network

summary: Lipid accumulation in macrophages (Mφs) is a hallmark of atherosclerosis, yet how lipid accumulation affects inflammatory responses through rewiring of Mφ metabolism is poorly understood. We modeled lipid accumulation in cultured wild type mouse thioglycolate-elicited peritoneal Mφs and bone marrow-derived Mφs with conditional (Lys2-Cre) or complete genetic deficiency of Vhl, Hif1a, Nos2 and Nfe2l2. Transfection studies employed RAW264.7 cells. Mφs were cultured for 24 hours with oxidized LDL (oxLDL) or cholesterol and then were stimulated with LPS. Transcriptomics revealed that oxLDL accumulation in Mφs down-regulated inflammatory, hypoxia and cholesterol metabolism pathways, while antioxidant pathway, fatty acid oxidation and ABC family proteins were up-regulated. Metabolomics and extracellular metabolic flux assays showed that oxLDL accumulation suppressed LPS-induced glycolysis. Intracellular lipid accumulation in Mφs impaired LPS-induced inflammation by reducing both HIF-1α stability and transactivation capacity; thus, the phenotype was not rescued in Vhl-/- Mφs. Intracellular lipid accumulation in Mφs also enhanced LPS-induced Nrf2-mediated antioxidative defense that destabilizes HIF-1α, and Nrf2-deficient Mφs resisted the inhibitory effects of lipid accumulation on glycolysis and inflammatory gene expression. Furthermore, oxLDL shifted NADPH consumption from HIF-1α- to Nrf2-regulated apoenzymes. Thus, we postulate that repurposing NADPH consumption from HIF-1α to Nrf2 transcriptional pathways is critical in modulating inflammatory responses in Mφs with accumulated intracellular lipid. The relevance of our in vitro models was established by comparative transcriptomic analyses, which revealed that Mφs cultured with oxLDL and stimulated with LPS shared similar inflammatory and metabolic profiles with foamy Mφs derived from the atherosclerotic mouse and human aorta.

overall_design: To investigate the effects of oxLDL loading on the transcriptome of mouse peritoneal macrophages before and after LPS stimulation.   Control and OxLDL-loaded peritoneal macrophages before and after stimulation with LPS; 3 replicates per sample.

first: Kenneth

last: Ting

first: Pei

last: Yu

first: Sharon

last: Hyduk


title: Variation in transcription regulator expression underlies differences in white-opaque switching between the SC5314 reference strain and the majority of Candida albicans clinical isolates.

city: San Francisco

first: Matthew

last: Lohse

address: 44 Vicksburg Street

institute: BioSynesis, Inc.

summary: Expression of 68 genes was tracked across different C. albicans strains and enviornmenal conditions using a custom 72 probe Nanostring library

overall_design: In the study presented here, we used a custom 72-probe Nanostring library to examine expression of genes of interest from 37 distinct strain-condition pairings, one with four biological replicates and 36 with 2 biological replicates each.

first: Naomi

last: Ziv

first: Alexander

last: Johnson


title: Thymic mimetic cells function beyond self-tolerance

status: Public on Aug 27 2023

first: Jakub

last: Abramson

overall_design: Refer to individual Series


title: Gene expression of HUVECS treated with either siRNA to CTRL or PDHA1 and treated with TNF 16 hours

status: Public on Aug 26 2023

city: New Haven

first: Nabil

last: Boutagy

state: CT

address: 10 Amistad St

department: Pharmacology

institute: Yale University

summary: The endothelium is a major target of the proinflammatory cytokine, tumor necrosis factor alpha (TNFα). Exposure of endothelial cells (EC) to proinflammatory stimulileads to an increase in mitochondrial metabolism, however, the function and regulation of elevated mitochondrial metabolism in EC in response to pro-inflammatory cytokines remains unclear. Using high-resolution metabolomics and13C-glucose labeled flux techniques, we demonstrate thatpyruvate dehydrogenase activity (PDH) and tricarboxylic acid cycle flux is elevated in human umbilical vein ECs (HUVECs) in response to overnight (16 hrs) treatment withTNFα (10 ng/mL).Mechanistic studies indicate thatTNFα mediates these metabolic changes via mitochondrial specific protein degradation of pyruvate dehydrogenase kinase 4 (PDK4, inhibitor of PDH) by the Lon protease via an NF-κB dependent mechanism. Using RNA sequencing following siRNA mediated knockdown of thecatalytically active subunit of PDH, PDHE1a(PDHA1 gene), we show that PDH fluxcontrols the transcription of approximately one-third of the genes that are upregulated by TNFastimulation. Notably, TNFainduced PDHflux regulates a unique signature of proinflammatory mediators (cytokines and chemokines) but not inducible adhesion molecules.Using metabolomics and ChIP sequencing (H3AcK27), we demonstrate that TNFα induced PDH flux promotes histone acetylation of specific gene sets via citrate accumulation and ATP-citrate lyase activity.Together, these resultsindicate that targeting endothelial glucose oxidation and/or acetyl CoA generation may offer a novel therapeutic strategy in the treatment of vascular inflammation.

overall_design: Comparative gene expression and acetylation of early passage HUVECs treated with either siRNA to CTRL or PDHA1 and treated with TNF 16 hours.

first: Joseph

last: Fowler

first: Kariona

last: Grabinska

first: Rebecca

last: Cardone

first: Qiushi

last: Sun

first: Kyla

last: Vazquez

first: Michael

last: Whalen

first: Xiaolong

last: Zhu

last: Chakraborty

first: Kathleen

last: Martin

first: Casey

last: Romanoski

last: Simons

first: Richard

last: Kibbey

first: William

middle: C

last: Sessa


title: Nucleosome repositioning in chronic lymphocytic leukemia

city: Colchester

first: Vladimir

last: Teif

address: Wivenhoe Park

department: School of Life Sciences

institute: University of Essex

summary: The location of nucleosomes in the human genome determines the primary chromatin structure and regulates access to regulatory regions. However, genome-wide information on deregulated nucleosome occupancy and its implications in primary cancer cells is scarce. Here, we conducted a genome-wide comparison of high-resolution nucleosome maps in peripheral-blood B cells from patients with chronic lymphocytic leukaemia (CLL) and healthy individuals at single base pair resolution. Our investigation uncovered significant changes of nucleosome positioning in CLL. Globally, the spacing between nucleosomes – the nucleosome repeat length (NRL) – was shortened in CLL. This effect was stronger in the more aggressive IGHV-unmutated than in the IGHV-mutated CLL subtype. Changes in nucleosome occupancy at specific sites were linked to active chromatin remodelling and reduced DNA methylation. Nucleosomes lost or gained in CLL marked differential binding of 3D chromatin organisers such as CTCF as well as immune response-related transcription factors and delineated mechanisms of epigenetic deregulation. The principal component analysis of nucleosome occupancy in cancer-specific regions allowed classification of samples between cancer subtypes and normal controls. Furthermore, patients could be better assigned to CLL subtypes according to differential nucleosome occupancy than based on DNA methylation or gene expression. Thus, nucleosome positioning constitutes a novel readout to dissect molecular mechanisms of disease progression and to stratify patients. Furthermore, we anticipate that the global nucleosome repositioning detected in our study, such as changes in the NRL, can be exploited for liquid biopsy applications based on cell-free DNA to monitor disease progression.

overall_design:
We have performed genome-wide comparison of nucleosome positioning in B-cells from peripheral blood of CLL patients and healthy donors at a sequencing depth of more than 4 billion DNA reads. The median age of the CLL patients was 60 years (range, 47 to 75). Non-malignant control samples were enriched from peripheral blood of in total 36 age-matched donors (median, 58 years, range, 51 to 70) that were collected in 9 sample pools. All samples, from CLL patients and from non-malignant donors, were enriched for CD19-positive B cells. The purity of the CD19-positive fraction was 98 ± 2% in non-malignant donor samples and 99 ± 1% in CLL patient samples. 79% of CLL patients had favorable genetic aberrations [del(13q) with deletions of SF3B1 and ATM] as sole abnormality associated with increased survival times, 21% had normal karyotype. 71% of the patients were diagnosed with hypermutated IGHV associated with good prognosis. Due to the high numbers of B-cells required for multiple sequencing, we selected asymptomatic CLL patients who had been approached with the watch-and-wait strategy. None of the patients analyzed had previous treatment of the CLL disease, thereby high numbers of CLL cells unaffected by the potential impact of prior therapy had accumulated in the peripheral blood with a median leukocyte count of 105 x 109/L (range, 82 to 187 x 109/L). Patient derived cells were crosslinked with 1 % methanol-free formaldehyde for 10 min. After quenching with glycine, cells were washed three times with PBS and the cell pellet was frozen in liquid nitrogen. For analysis, the cell pellet was thawn and treated with 4U MNase per 1x106 cells for 15 min. MNase was stopped with 10x covaris buffer and the chromatin was sheared for an additional 15 min with the S2 covaris device and the soluble chromatin was then recovered and subjected to a background-minimizing pre-clearing step with an unspecific IgG antibody. For each ChIP-assay an equivalent of 3x106 cells were used with the following antibody: H3 (Abcam, ab1791). After the IP, chromatin was digested with RNaseA and proteinase K. The purified DNA was cloned into illumina sequencing libraries with the NEBNext Ultra library preparation kit (NEB).

Original sequencing data for all samples are available at the European Genome-phenome Archive (http://www.ebi.ac.uk/ega/), hosted by the European Bioinformatics Institute under the accession number EGAS00001002518.

first: Kristan

middle: V

last: Piroeva

first: Charlotte

last: McDonald

first: Babis

last: Xanthopoulos

first: Chelsea

last: Fox

first: Christopher

middle: T

last: Clarkson

first: Jan-Philipp

last: Mallm

first: Yevhen

last: Vainshtein

first: Luminita

last: Ruje

first: Lara

last: Klett

first: Stephan

last: Stilgenbauer

first: Daniel

last: Mertens

first: Efterpi

last: Kostareli

first: Karsten

last: Rippe


title: Myo-differentiation reporter screen reveals NF-Y as an activator of PAX3-FOXO1 in rhabdomyosarcoma [Cut & Run]

last: Vakoc

overall_design: Chromatin profiling of different NF-Y subunits in RH4 and RH30 rhabdomyosarcoma cell lines using CUT&RUN. In RH30 cells, CCAAT NF-Y binding motif within the endogenous PAX3 promoter was mutated via base editing.

first: Martyna

last: Sroka

first: Gryder

last: Berkley


title: Intestinal Paneth cell differentiation relies on asymmetric regulation of Wnt 2 signaling by Daam1/2

status: Public on Aug 25 2023

city: Daejeon

first: Bon-Kyoung

last: Koo

state: Yuseong-gu

address: Expo-ro 55

department: Center for genome engineering

country: South Korea

institute: Institute for Basic Science

summary: The mammalian intestine is one of the most rapidly self-renewing tissues, driven by actively cycling stem cells residing at the crypt bottom . Together with stromal cells, Paneth cells form a major element of the niche microenvironment that provides various growth factors to orchestrate intestinal stem cell homeostasis, such as Wnt3. With 19 family members, different Wnt ligands can selectively activate βcatenin dependent (canonical) or independent (non-canonical) signaling. Here, we report that Dishevelled-associated activator of morphogenesis 1 (Daam1) and its paralogue Daam2 asymmetrically regulate canonical and non-canonical Wnt (Wnt/PCP) signaling, and their function is required for Paneth cell progenitor differentiation. We found that Daam1/2 interacts with the Wnt antagonist Rnf43, and Daam1/2 double knockout stimulates canonical Wnt signaling by preventing Rnf43-dependent endo-lysosomal degradation of the ubiquitinated Wnt receptor, Frizzled (Fzd). Moreover, single-cell RNA sequencing analysis revealed that Paneth cell differentiation is impaired by Daam1/2 depletion, as a result of defective Wnt/PCP signaling. Taken together, we identified Daam1/2 as an unexpected hub molecule coordinating both canonical and non-canonical Wnt signaling, the regulation of which is fundamental for specifying an adequate number of Paneth cells while maintaining intestinal stem cell homeostasis.

overall_design: Mouse Small intestine oragnoids from Vil-CreERT2-Rnf43fl/fl-Znrf3fl/fl mice (RZ sample) and Vil-CreERT2-Daam1fl/fl-Daam2fl/fl mice (D1/2 sample). For the samll intestinal organoid lines, 1 μg/ml 4-hydroxytamoxifen (4-OHT) was added overnight in WENR+Nic (Wnt, Egf, Noggin, R-spondin1 + nicotinamide) organoid culture medium. After recombination, D1/2 and RZ mutant organoids were cultured in WEN+Nic medium for selection purposes, as only successfully recombined mutant cells survive in the absence of Rspo1. WT, D1/2 cDKO and RZ cDKO organoids were maintained in regular WENR+Nic (WT) or WEN+Nic (D1/2 and RZ cDKO) culture medium for 10 days prior to single-cell analysis.

first: Gabriele

last: Colozza

first: Heetak

last: Lee

first: Alessandra

last: Merenda

first: Szu-Hsien

last: Wu

first: Andrea

last: Català-Bordes

first: Tomasz

last: Radaszkiewicz

first: Ingrid

last: Jordens

first: Ji-Hyun

first: Aileen-Diane

last: Bamford

first: Fiona

last: Farnhammer

first: Teck

middle: Y

last: Low

first: Madelon

last: Maurice

first: Vítězslav

last: Bryja

first: Jihoon

last: Kim


title: Myo-differentiation reporter screen reveals NF-Y as an activator of PAX3-FOXO1 in rhabdomyosarcoma [RNA-Seq]

first: Christopher

last: Vakoc

overall_design: Comparative gene expression profiling using RNA-seq. Performed on human rhabdomyosarcoma cell lines transduced with sgRNAs: sgRosa (reference), sgPAX3-FOXO1, or sgNFYC. Analyzed at day 7 post-transduction. Experiment performed in 2 biological replicates, with the exception of RH30 and Dbt-P3F1, where 1 biological repeat was performed.

first: Martyna


title: Cell-type-specific tumour sensitivity identified with a bromodomain targeting PROTAC in adenoid cystic carcinoma.

city: London

first: Amanda

last: Swain

state: London

address: 237 Fulham Road, 3n17

institute: The Institute of Cancer Research

summary: Salivary gland adenoid cystic carcinoma (ACC) is a rare malignancy with limited treatment options. The development of novel therapies is hindered by a lack of preclinical models. We have generated several ACC patient-derived xenograft (PDX) lines that retain the physical and genetic properties of the original tumours, including the presence of the common MYB/MYBL1-NFIB translocation. Using these PDXs, we have developed the conditions for the generation of 2D and 3D ACC models that retain MYB expression and can be used for drug studies. Using these models, we show in vitro and in vivo sensitivity of ACC cells to the bromodomain degrader, dBET6. Molecular studies show a decrease in BRD4 and MYB protein levels and target gene expression with treatment. The most prominent effect of dBET6 on tumours in vivo was a change in the relative composition of ACC cell types expressing either myoepithelial or ductal markers. We show that dBET6 inhibits the progenitor function of ACC cells, particularly in the myoepithelial marker-expressing population, revealing a cell-type-specific sensitivity. These studies reveal a novel mechanistic impact of bromodomain inhibitors on tumours and highlight the need to impact both cell-type populations for more effective treatments in ACC patients.

overall_design: Single cells from a PDX line were injected into the flanks of NSG mice. Once tumours developed, mice were allocated to either receive vehicle or dBET6 via IP injection twice daily. Following short term (4 day) treatment with 5mg/kg of dBET6 or vehicle, tumours were harvested and snap frozen. RNA was extracted from these samples and sequenced.

first: Alexandra

last: Rose

first: Mercedes

last: Fleming

first: Jeffrey

last: Francis

first: Jian

last: Ning

first: Anton

last: Patrikeev

first: Ritika

last: Chauhan

first: Kevin

last: Harrington


title: A cell circuit approach to dissect fibroblast-macrophage interactions in the tumor microenvironment [organs]

first: Shimrit

last: Mayer

state: Central

address: Herzl St 234

department: Bimolecular sciences

institute: Weizmann Institute

summary: We preformed RNA seq on invitro macrophages in mono-cultured or co-cultured with from fibroblasts different organs. In order to understand the differences in macrophages-fibroblasts interactions between organs

overall_design: We performed RNA-sequencing of the co-cultures at the ON state. As control, we analyzed mono-cultured fibroblasts and macrophages. Fibroblasts from different organs and BMDMs were seeded at 3× 10^5 into a pre-coated 6 well plate. The co-cultures and the mono-cultures were grown in DMEM for 7 days . The mono-cultured macrophages were collected at day 0 since they cannot maintain themselves in control medium. 10^4 cells of macrophages were sorted using the FACSMelody instrument (BD-biosciences). Dead cells were excluded using PI, the macrophages were postive stained for anti-F4/80-APC cy7 (Biolegend. cat.123117), anti-CD11b-Pacific blue antibody (Miltenyi, 130-110-802) . The cells were collected directly into lysis/binding buffer (Life Technologies) and mRNA was isolated using Dynabeads oligo (dT) (Life Technologies). RNA-seq was performed as previously described (“Massively Parallel Single-Cell RNA-Seq for Marker-Free Decomposition of Tissues into Cell Types” n.d.).

first: Tomer

last: Milo

first: Uri

last: Alon

first: Ruth

last: Scherz Shouval


title: A GPI-anchored protein Lypd1 regulates odontoblast differentiation in tooth development

city: Sendai

first: Yuta

last: Chiba

state: Miyagi

address: 4-1 Seiryo-cho, Aoba-ku

country: Japan

institute: Tohoku University

summary: Lipid rafts are membrane microdomains rich in cholesterol, sphingolipids, glycosylphosphatidylinositol-anchored proteins (GPI-APs), and receptors. They are localized at the plasma membrane and are essential for signal transmission and organogenesis. However, few reports on the specific effects of lipid rafts on organogenesis have been reported. Here, we focused on the odontoblast differentiation that secretes the dentin matrix and generates dentin. We found the GPI-AP, lymphocyte antigen-6 (Ly6)/Plaur domain-containing1 (Lypd1), specifically expressed in the dental papilla, especially preodontoblasts, using single-cell RNA sequence (scRNA-seq). Depletion experiments of lipid rafts and Lypd1 using ex vivo culture and cell cultures showed that differentiation of tooth germ and dental mesenchymal cells was inhibited. Furthermore, the C-terminal structural domain containing the omega site, which is necessary to localize the plasma membrane, of Lypd1 is necessary for odontoblast differentiation and the morphological change like odontoblasts. Stimulating downstream Bone morphogenetic protein (Bmp) signaling by BMP2 caused it to activate the phosphorylation of Smad1/5/8 and promote the expression of odontoblast differentiation markers such as Pannexin 3 (Panx3), Alkaline phosphatase (Alp), and Dentin sialophosphoprotein (Dspp). In contrast, suppression of Lypd1 inhibited the phosphorylation of Smad1/5/8. In addition, the expression of Lypd1 predates any other marker of odontoblast differentiation previously described. That implies that Lypd1 could be employed as a novel earlier differentiation marker of odontoblasts. These results suggest that Lypd1 as GPI-AP of lipid rafts is important for tooth organogenesis and plays a pivotal role in odontoblast differentiation by regulating phosphorylation of Smad1/5/8.

overall_design: Examination of epithelial and mesenchymal cells in mouse embryonic day 16 molars.

first: Yao

last: Fu

first: Kanako

last: Miyazaki

first: Jumpei

last: Kawahara

first: Keita

last: Funada

first: Tomomi

last: Yuta

first: Tian

last: Tian

first: Kanji

last: Mizuta

first: Daniel

last: Martin

first: Ichiro

last: Takahashi

first: Satoshi

last: Fukumoto

first: Keigo

last: Yoshizaki


title: Blastoidverse

city: Dallas

first: Gary

last: Hon

address: 5323 Harry Hines Blvd.

department: Department of Obstetrics and Gynecology

institute: UT Southwestern


title: Oncogene-induced MALT1 protease activity drives post-transcriptional gene expression in malignant lymphomas

city: Parkville

first: Göksu

last: Avar

state: Victoria

address: 30 Flemington Road

department: Biochemistry and Pharmacology

country: Australia

institute: University of Melbourne

summary: Constitutive MALT1 activity drives survival of malignant lymphomas addicted to chronic B-cell receptor (BCR) signaling, oncogenic CARD11, or the API2-MALT1 fusion oncoprotein. While MALT1 scaffolding induces NF-kB-dependent survival signaling, MALT1 protease function is thought to augment NF-kB activation by cleaving signaling mediators and transcriptional regulators in B-cell lymphomas. However, the pathological role of MALT1 protease function in lymphomagenesis is not well understood. Here, we show that TRAF6 controls MALT1-dependent activation of NF-kB transcriptional responses, but is dispensable for MALT1 protease activation driven by oncogenic CARD11. To uncouple enzymatic and non-enzymatic functions of MALT1, we analyzed TRAF6-dependent and -independent as well as MALT1 protease-dependent gene expression profiles downstream of oncogenic CARD11 and API2-MALT1. By cleaving and inactivating the RNA binding proteins Regnase-1 and Roquin-1/2, MALT1 protease induces post-transcriptional upregulation of genes like NFKBIZ/IkBz, NFKBID/IkBNS and ZC3H12A/Regnase-1 in activated B-cell-like diffuse large B-cell lymphoma (ABC DLBCL). We demonstrate that oncogene-driven MALT1 activity in ABC DLBCL cells regulates NFKBIZ and NFKBID induction on mRNA level via releasing a brake imposed by Regnase-1 and Roquin-1/2. Furthermore, MALT1 protease drives post-transcriptional gene induction in the context of the API2-MALT1 fusion created by the recurrent t(11;18)(q21;q21) translocation in mucosa-associated lymphoid tissue (MALT) lymphoma. Thus, MALT1 paracaspase acts as a bifurcation point for enhancing transcriptional and post-transcriptional gene expression in malignant lymphomas. Moreover, the identification of MALT1 protease selective target genes will provides specific biomarkers for the clinical evaluation of MALT1 inhibitors.

overall_design: 1)Bulk RNA-Seq profiling of CARD11 L232LI mutant and/or TRAF6 knockout BJAB cells. Additionally, profiling of MLT-748 treatment for TRAF6 KO CARD11 mut cells. 2)Bulk RNA-Seq profiling of (CARD11 L232LI mutant) + (MALT1 C453A protease and/or E795A TRAF6 binding mutant) BJAB cells. 3)Bulk RNA-Seq profiling of API2:MALT1 fusion protein expressing BJAB cells, where MALT1 carries the mutations described in the second bullet point.

first: Nicole

last: Wimberger

first: Franziska

last: Ober

first: Michael

last: Grau

first: Wendan

last: Xu

first: Georg

last: Lenz

middle: P

last: Menden

last: Krappmann


title: Determinants of Motor Neuron Functional Subtypes Important for Locomotor Speed

status: Public on Aug 24 2023

city: New York

first: Jeremy

last: Dasen

state: NY

address: 435 E 30th St

department: Neuroscience Institute

institute: New York University Grossman School of Medicine

summary: Locomotion requires precise control of the strength and speed of muscle contraction and is achieved by recruiting functionally-distinct subtypes of motor neurons (MNs). MNs are essential to movement and differentially susceptible in disease, but little is known about how MNs acquire functional subtype-specific features during development. Using single-cell RNA profiling in embryonic and larval zebrafish, we identify novel and conserved molecular signatures for MN functional subtypes, and identify genes expressed in both early post-mitotic and mature MNs. Assessing MN development in genetic mutants, we define a molecular program essential for MN functional subtype specification. Two evolutionarily-conserved transcription factors, Prdm16 and Mecom, are both functional subtype-specific determinants integral for fast MN development. Loss of prdm16 or mecom causes fast MNs to develop transcriptional profiles and innervation similar to slow MNs. These results reveal the molecular diversity of vertebrate axial MNs and demonstrate that functional subtypes are specified through intrinsic transcriptional codes.

overall_design: Spinal cord MNs of WT, prdm16-/-, and mecom -/- zebrafish embryos at two developmental timepoints (2dpf and 5dpf) were isolated by fluorescence activated cell sorting (FACS) according to the presence of dsRed and GFP signal, then analyzed using scRNAseq.

first: Kristen

last: D'Elia

first: Hanna

last: Hameedy

first: Dena

last: Goldblatt

first: Paul

last: Frazel

first: Mercer

last: Kriese

first: Yunlu

last: Zhu

first: Kyla

last: Hamling

first: Shane

last: Liddelow

first: Koichi

last: Kawakami

first: David

last: Schoppik


title: RNA molecular recording with an engineered RNA deaminase

first: Yizhu

last: Lin

state: CA

address: 513 Parnassus

department: Cell and Tissue Biology

institute: University of California, San Francisco


title: Dip2b knockout affects neural differentiation of mESCs by depressing axonogenesis and axon guidance and facilitating ECM-receptor interaction

city: Taiyuan city

first: yuanqing

last: Pan

address: No. 92 Wucheng road, Xiaodian district, Taiyuan city, Shanxi Province, China

institute: Shanxi university

summary: To investigate the biological function of Dip2b during the neural differentiation process of mESCs, we established a Dip2b homozygous knockout 46C ES cell line using the CRISPR/Cas9 genome editing technology

overall_design: Comparative gene expression profiling analysis of RNA-seq data for Dip2b knockout cells and WT cells during the neural differentiation at different time points

first: Yuanqing

first: Mingze

last: Yao


title: Development of nanoRibo-seq enables study of regulated translation by cortical neuron subtypes, showing uORF translation in synaptic-axonal genes

city: Cambridge

first: John

middle: Edward

last: Froberg

state: MA

address: 7 Divinity Avenue

department: Stem Cell & Regenerative Biology

institute: Harvard University

summary: Investigation of translation in rare cell types or subcellular contexts is challenging due to large input requirements for standard approaches. Here, we present “nanoRibo-seq” an optimized approach using 102- to 103-fold less input material than bulk approaches. nanoRibo-seq exhibits rigorous quality control features consistent with quantification of ribosome protected fragments with as few as 1,000 cells. We compare translatomes of two closely related cortical neuron subtypes, callosal projection neurons (CPN) and subcerebral projection neurons (SCPN), during their early postnatal development. We find that, while translational efficiency is highly correlated between CPN and SCPN, several dozen mRNAs are differentially translated. We further examine upstream open reading frame (uORF) translation and identify that mRNAs involved in synapse organization and axon development are highly enriched for uORF translation in both subtypes. nanoRibo-seq enables investigation of translational regulation of rare cell types in vivo and offers a flexible approach for globally quantifying translation from limited input material.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: nanoRibo-seq: low input ribosome profiling


title: Epigenetic profiling of human macrophages following adjuvant stimulation

first: Boris

last: Novakovic

address: Murdoch Children's Research Institute, Royal Children's Hospital, Flemington Road

institute: Murdoch Children's Research Institute

summary: Trained immunity is the phenomenon whereby innate immune cells such as monocytes or macrophages undergo functional reprogramming after exposure to certain microbial components, altering their responses to future exposures. Since trained immunity was first discovered, and has been commonly studied with the BCG vaccine, studies confirming trained immunity have often focused on human PBMCs. Thus, to study the mechanism of the vaccine in a well-established trained immunity model, we used human macrophages differentiated from monocytes isolated from PBMCs.  The macrophages were expose to A + M + MA for 24 h, and rested two or five days prior to exposure to bacteria, as an ex vivo model with a five-day rest period has been used in numerous studies to confirm trained immunity.

overall_design: PBMCs were obtained from healthy donors, and stimulated with the vaccine for 24 hours and rested for 2 or 5 days. Expeirment was repeated 3 times to obtain three biological replicates.

first: Jun

last: Yan

first: Travis

middle: B

last: Nielsen

first: Peggy

last: Lu

first: Yuli

last: Talyansky

first: Matt

last: Slarve

first: Hernan

last: Reza

first: Mihai

last: Netea

first: Ashley

last: Keller

first: Troy

last: Warren

first: Antonio

last: DiGiandomenico

first: Bret

last: Sellman

first: Brian

last: Luna

first: Brad

last: Spellberg


title: Human blastoid

first: Gary

last: Hon

summary: Based on our previous works, we established an optimized protocol that enables the generation of blastocyst-like structures from human pluripotent stem cells.

overall_design: To model human pre-/peri-implantation development, we reported a optimized protocol to generate blastocyst-like structures from human pluripotent stem cells (hPSCs) in vitro, which resemble blastocysts in morphology and cell-lineage allocation. Human blastoids provide an accessible, scalable, versatile, and manipulable in vitro model to study the early stages of human development and implantation.

first: Jialei

last: Duan


title: Transcriptomic profiling of human macrophages following adjuvant stimulation

middle: R


title: Human PSC-derived articular cartilage chondrocytes scRNA-seq

city: Boston

first: April

last: Craft

address: 300 Longwood Ave

institute: Boston Children's Hospital

summary: Objectives Traditional approaches to study Progressive Pseudorheumatoid Arthropathy of Childhood (PPAC) failed to uncover how loss-of-function mutations in Wnt inducible secreted protein 3 (WISP3) cause premature cartilage failure in children. We developed a human induced pluripotent stem cell (hiPSC)-based model of cartilage development to elucidate pathological changes in WISP3-deficient articular cartilage tissues compared to WISP3-sufficient isogenic cartilage tissues.    Methods We generated iPSCs from 2 patients with PPAC, and we corrected the disease-causing mutation in one of these lines using CRISPR/Cas9. We also created a WISP3-knockout human embryonic stem cell line. We generated articular cartilage tissues from these hPSCs by established directed differentiation methods. Bioinformatic analyses were used to identify differentially expressed genes (DEGs) and cell type abundances, which were validated by quantitative RT-qPCR and in situ hybridization.  Results WISP3-deficient articular cartilage tissues exhibited significantly different transcriptomic profiles and cellular composition compared with their isogenic controls. WISP3-deficient cartilage transcriptomes exhibited enriched biological processes such as TGFb signaling and epithelial to mesenchymal transition. We validated increased expression of TGFb-related genes in WISP3-deficient cartilage, and determined that the abundance of chondrocyte subtypes was altered compared with isogenic controls.     Conclusions We developed a robust and reproducible model of iPSC-derived cartilage development to better understand the role of WISP3 in cartilage biology, and the pathology of cartilage failure associated with loss of WISP3 in patients with PPAC. We identified several altered biological processes and signaling pathways in WISP3-deficient cartilage that can be validated in the future with additional patient-derived cell lines, or large animal models.

overall_design:
comparative gene expression profiling of in vitro derived cartilage chrondrocytes from human PSCs

Please note: Raw data are not available for the PPAC iPSC samples due to patient privacy concerns.

first: Chaochang

last: Li

first: Mireia

last: Alemany Ribes

first: Rosanne

last: Raftery

first: Uzochi

last: Nwoko

first: Matthew

last: Warman


title: Generation of Locus Coeruleus Norepinephrine Neurons from Human Pluripotent Stem Cells [snRNA-seq]

city: Nanjing

first: Yunlong

last: Tao

state: Jiangsu

address: 163 Xianlin Road

department: School of life sciences

institute: Nanjing University

summary: Central norepinephrine (NE) neurons, mainly located in the Locus coeruleus (LC), play roles in a wide range of behavioral and physiological processes. How the human LC-NE neurons develop and what roles they play in the pathophysiology of human diseases is poorly understood, partly due to the unavailability of functional human LC-NE neurons. Here we established a technology for efficient generation of LC-NE neurons from human pluripotent stem cells by identifying a novel role of ACTIVIN A in regulating the LC-NE transcription factors in the dorsal rhombomere 1 (r1) progenitors. The in vitro generated human LC-NE neurons not only display extensive axonal arborization and release/uptake NE, but also exhibit the pacemaker activity, calcium oscillation, and in particular chemoreceptor activity in response to CO2. Multiple timepoint single nucleus RNA-Seq (snRNA-Seq) analysis captured the dynamic NE differentiation process, confirmed the NE cell population and revealed the differentiation trajectory from hindbrain progenitors to NE neurons via ASCL1 expressing precursor stage.   The LC-NE neurons engineered with a NE sensor reliably reported the extracellular NE level. The availability of functional human LC-NE neurons enables investigation of their roles in the pathogenesis of and development of therapeutics for neural psychiatric and degenerative diseases.

overall_design: we performed single nucleus RNA-Seq (snRNA-Seq) to systematically investigate the NE differentiation process unbiasedly. Given the dynamic changes of NE related genes during this process, we collected the differentiation cells at day 6, day 11 and day 14 to capture the key statuses during NE differentiation

first: Su-chun

last: Zhang


title: Combinatorial treatment with PARP and MAPK inhibitors overcomes phenotype switch-driven drug resistance in advanced melanoma

city: Parma

first: Roberto

last: Ferrari

state: Parma

address: Parco Area delle Scienze 23/A

department: Department of Chemistry, Life Sciences and Environmental Sustainability

institute: University of Parma

summary: Metastatic melanoma is either intrinsically resistant or rapidly acquires resistance to targeted therapy treatments, such as MAPK inhibitors. A leading cause of resistance to targeted therapy is a dynamic transition of melanoma cells from a proliferative to a highly invasive state, a phenomenon called phenotype switching. Mechanisms regulating phenotype switching represent potential targets for improving treatment of melanoma patients. Using a drug screen targeting chromatin regulators in patient-derived 3D MAPK inhibitor-resistant melanoma cell cultures, we discovered that PARP inhibitors restore sensitivity to MAPK inhibitors, independent of DNA damage repair pathways. Integrated transcriptomic, proteomic, and epigenomic analyses demonstrated that PARP inhibitors induce lysosomal autophagic cell death, accompanied by enhanced mitochondrial lipid metabolism that ultimately increases antigen presentation and sensitivity to T-cell cytotoxicity. Moreover, transcriptomic and epigenetic rearrangements induced by PARP inhibition reversed EMT-like phenotype switching, which redirected melanoma cells toward a proliferative and MAPK inhibitor-sensitive state. The combination of PARP and MAPK inhibitors synergistically induced cancer cell death both in vitro and in vivo in patient-derived xenograft models. Therefore, this study provides a scientific rationale for treating melanoma patients with PARP inhibitors in combination with MAPK inhibitors to abrogate acquired therapy resistance.

overall_design: ChIP-seq of H3K27ac of M121224 cells and M121224 treated with TAL + LGX818

first: Lorenza

last: Ferretti

first: Flurina

last: Böhi

first: Deena

last: Leslie Pedrioli

first: Phil

last: Cheng

first: Elena

first: Petra

last: Baumgartner

first: Abdiel

last: Alvarado-Diaz

first: Federica

last: Sella-Diaz

first: Alessandra

last: Cereghetti

first: Patrick

last: Turko

first: Roni

middle: H

last: Wright

first: Katrien

last: De Bock

first: Daniel

last: De Speiser

first: Mitchell

last: Levesque

first: Michael

middle: O

last: Hottiger


title: Epigenetic profiles of mouse splenic macrophages responding to a Protein-Free Vaccine that Induces Trained Immunity and Protects against Nosocomial Pathogens

first: Boris

last: Novakovic

summary: Traditional vaccines are difficult to deploy against the diverse antibiotic-resistant, nosocomial pathogens that cause Hospital Acquired Infections (HAIs).  We developed a unique, protein-free vaccine to present antibiotic-resistant HAIs.  This vaccine protected mice from invasive infections caused by methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus faecalis, multidrug resistant Acinetobacter baumannii, Klebsiella pneumoniae, Pseudomonas aeruginosa, Rhizopus delemar, and Candida albicans.  Protection persisted even in neutropenic mice infected with A. baumannii or R. delemar.  Protection was already apparent after 24 hours and lasted for up to 21 days after a single dose, with a second dose restoring efficacy.  Protection persisted without lymphocytes but was abrogated with macrophages depletion. This vaccine induced trained immunity by altering the macrophage epigenetic landscape and the inflammatory response to infection.

overall_design: Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) for histone modification H3K27ac from mouse splenic f4/80+ macrophages extracted from Naïve (non vaccinated) mice, and vaccinated mice at 3 days and 21 days following vaccination.

first: Jun

last: Yan

first: Travis

last: Nielsen

first: Peggy

last: Lu

first: Yuli

last: Talyansky

first: Matt

last: Slarve

first: Hernan

last: Reza

first: Mihai

last: Netea

first: Brian

last: Luna

first: Brad

last: Spellberg


title: Lowering dietary BCAA reduces fibrosis, enhances mitochondrial respiration, and curbs cardiac hypertrophy, via regulating histone H3K23-propionylation

city: Newark

first: Maha

last: Abdellatif

state: NJ

address: 185 S. Orange Ave., MSB

department: Cell Biology and Molecular Medicine

institute: Rutgers University

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

overall_design: Refer to individual Series


title: SnapHiC-D: a computational pipeline to identify differential chromatin contacts from single cell Hi-C data

city: Cleveland

first: Ming

last: Hu

state: OH

address: 9500 Euclid Avenue

institute: Cleveland Clinic

summary: Single cell Hi-C (scHi-C) has been used to map genome organization in complex tissues. However, computational tools to detect dynamic chromatin contacts from scHi-C datasets in development and through disease pathogenesis are still lacking. Here, we present SnapHiC-D, a computational pipeline to identify differential chromatin contacts (DCCs) between two scHi-C datasets. Compared to methods designed for bulk Hi-C data, SnapHiC-D detects DCCs with high sensitivity and accuracy. We used SnapHiC-D to identify cell-type-specific chromatin contacts at 10 kilobase resolution in mouse hippocampal and human prefrontal cortical tissues, and demonstrated that DCCs detected in the cortical and hippocampal cell types are generally correlated with cell-type-specific gene expression patterns and epigenomic features. SnapHiC-D is freely available at https://github.com/HuMingLab/SnapHiC-D.

overall_design: We performed single-nucleus Hi-C on 94 mouse embryonic stem cells (ESCs)  and 188 mouse neuron progenitor cells (NPCs).

first: Lindsay

last: Lee

first: Miao

last: Yu

first: Xiaoqi

last: Li

first: Chenxu

last: Zhu

first: Yanxiao

last: Zhang

first: Hongyu

first: Bing

last: Ren

first: Yun


title: Transcriptomic profiling of mouse spleen mascrophages following adjuvant vaccination

first: Boris

last: Novakovic

summary: Trained immunity is the phenomenon whereby innate immune cells such as monocytes or macrophages undergo functional reprogramming after exposure to certain microbial components, altering their responses to future exposures. Intravesical instillation with Bacillus Calmette-Guérin (BCG) is the most effective adjuvant therapy in patients with high-risk non-muscle invasive bladder cancer (HR-NMIBC). HR-NMIBC is associated with a high risk of tumor recurrence and progression to muscle-invasive bladder cancer. The precise immunological mechanisms through which BCG mediates anti-tumor immunity are still unclear. The aim of our study was to investigate the (long-term) induction of trained immunity by repeated BCG instillations in NMIBC patients and elucidate the immunological and epigenetic mechanisms that are involved. Another aim was to assess the relationship between trained immunity response and clinical response of NMIBC patients, in terms of recurrence free survival and progression free survival. To determine whether BCG instillations induce trained immunity in peripheral blood mononuclear cells (PBMCs) we performed a prospective cohort study (‘Tribute’) and isolated PBMCs collected before BCG therapy and at 8 time points during BCG therapy. A total of 17 BCG-naïve HR-NMIBC patients were included. After isolation of PBMCs, monocytes were further purified using an isolation procotol with a percoll gradient. RNA was isolated from these purified monocytes were and used as input for RNA-seq analysis.

overall_design: Blood was collected using venapuncture and PBMCs were isolated at three time points during the BCG induction cycle: pre-BCG1, BCG2 and BCG6; and two time points during each subsequent BCG maintenance cycle: pre-BCG7, BCG9, pre-BCG10, BCG12, pre-BCG13 and BCG15. For each donor one replicate for each time point was included. RNA from purified monocytes was isolated for the time points pre-BCG1, BCG6, and pre-BCG7 and the RNA-seq data was analysed. For some patients RNA-seq data for the pre-BCG10 time point was also available, but this time point was not included in the analysis because not every patient had data available for this time point. Comparisons for differentially expressed genes were made using the pre-BCG1 time point as a reference.

first: Jun

last: Yan

first: Travis

first: Peggy


title: The Hippo and Canonical WNT Pathways Are Suppressed Prior to and in the Absence of Cardiac Dysfunction in Human Arrhythmogenic Cardiomyopathy Caused by Desmoplakin Truncations

city: Houaton

first: Ali

last: Marian

address: 6770 Bertner Ave DAC C900a

department: Centre for Cardiovascular Genetics

institute: UTHSC, Houston

summary: The RNA-Seq studies performed in the endomyocardial biopsy samples from human patients with defined truncating mutations in the DSP gene and normal right ventricular size and function, identified dysregulation of over two-dozen TRs, including the Hippo/cWNT pathways, early in the course of ACM. The findings of the RNA-Seq were corroborated by immunoblotting and immunofluorescence studies showing reduced protein levels of the TRs of the Hippo/cWNT pathways in the ACM hearts. Dysregulation of these pathways early and prior to the onset of cardiac dysfunction suggest their pathogenic role in ACM.

overall_design: Right ventricular endomyocardial biopsy samples from three independent individuals with familial ACM were used for RNA-sequencing (RNA-Seq) along with right ventricular tissues from 5 controls.

first: Siyang

last: Fan

first: Sirisha

last: Cheedipudi

first: Leila

last: Rouhi

first: Aitana

last: Braza-Boïls

first: Maria

last: Molina

first: Yan

last: Yao

first: Matthew

last: Robertson

first: Cristian

last: Coarfa

first: Juan

last: Gimeno

first: Esther

last: Zorio

first: Priyatansh

last: Gurha

first: A


title: Gene expression at single cell level of untreated and 10 day palbociclib treated LS8817 cells

status: Public on Aug 23 2023

first: Andrew

last: Koff

address: 1275 York Avenue

institute: Memorial Sloan Kettering Cancer Center

summary: Orthoganally complementing our RNA-seq (GSE241031) efforts to define transcriptional changes over time in LS8817(TetOnFMDM2) cells as they progressed from quiesence to senescence, we chose to look at gene expression in unperturbed parental and 10 day palbociclib treated LS8817 parental cells

overall_design: Asynchronously growing LS8817 cells were treated with 1 uM palbociclib and cultured for 10 days with media changes every three days.  Treated cells and untreated asynchronouysly growing cells were collected at that time and subjected to single cell RNA seq

first: Rodrigo

last: Gularte-Merida

first: Samuel

last: Singer


title: Immune surveillance of acute myeloid leukemia is mediated by HLA-presented antigens on leukemia progenitor cells

city: Freiburg im Breisgau

first: Andreas

last: Hecht

address: Stefan-Meier-Str. 17

department: Institute of Molecular Medicine and Cell Research

institute: Albert Ludwig University of Freiburg

summary: Persistent therapy-resistant leukemia progenitor cells (LPC) are a main cause of disease relapse and recurrence in acute myeloid leukemia (AML). Specific LPC-targeting therapies may thus improve treatment outcome of AML patients. We demonstrated that LPCs present human leukocyte antigen (HLA)-restricted cancer antigens that induce T cell responses allowing for immune surveillance of AML. Using a mass spectrometry-based immunopeptidomics approach, we characterized the antigenic landscape of patient LPCs and identified AML/LPC-associated HLA-presented antigens including mutation-derived and cryptic neoepitopes as prime targets for development of T cell-based immunotherapeutic approaches. We observed frequent spontaneous memory T cells targeting these AML/LPC-associated antigens in AML patients and showed that antigen-specific T cell recognition and HLA class II immunopeptidome diversity impacts clinical outcome. Our results pave the way for implementation of AML/LPC-associated antigens for T cell-based immunotherapeutic approaches to specifically target and eliminate residual LPCs in AML patients.

overall_design: In this study, we analyzed the expressional profile and T cell receptor (TCR) repertoire of antigen-specific CD4+ T cells from acute myeloid leukemia (AML) patients (n = 3). Therefore, PBMCs of AML patients were peptide-stimulated and sorted based on their IFN-g secretion. Single cell RNA sequencing (scRNA-seq) and single cell TCR profiling were performed using 10x Genomics single cell immune profiling.

first: Annika

last: Nelde

first: Heiko

last: Schuster

first: Jonas

last: Heitmann

first: Jens

last: Bauer

first: Yacine

last: Maringer

first: Melissa

last: Zwick

first: Jens-Peter

last: Volkmer

first: James

last: Chen

first: Anna

last: Paczulla Stanger

first: Ariane

last: Lehmann

first: Bismark

last: Appiah

first: Melanie

last: Märklin

first: Elke

last: Rücker-Braun

first: Helmut

last: Salih

first: Malte

last: Roerden

first: Sarah

last: Schroeder

first: Max-Felix

last: Häring

last: Schlosser

first: Johannes

last: Schetelig

first: Marc

last: Schmitz

last: Boerries

first: Natalie

last: Köhler

first: Claudia

last: Lengerke

first: Ravindra

last: Majeti

first: Irving

middle: L

last: Weissmann

first: Hans-Georg

last: Rammensee

first: Juliane

last: Walz


title: Directed protein evolution of an RNA editor for single molecule RNA-binding protein co-occupancy (II)

city: San Francisco

first: Yizhu

last: Lin

summary: RNA base editors have become powerful tools for both therapeutic and research purposes. However, currently available RNA editors such as ADAR or APOBEC family proteins have limitations due to RNA structure and sequence dependence. Here, we designed a protein engineering platform for putative RNA editor screening and directed enzyme evolution. We engineered an RNA A-to-I base editor (rABE) that has high activity, low local sequence or structure bias, and low background. Utilizing rABE, we present REMORA (RNA Encoded Molecular Recording in Adenosines), a new strategy to measure RNA-binding events on single RNA molecules in cells. In REMORA, adenosine deamination serves as a molecular record of RNA-protein interactions that are identified by mutations by sequencing. By combining our improved A-to-I RNA deaminase with the C-to-U deaminase APOBEC1 and long-read RNA sequencing, our approach enables simultaneous recording of the locations two RNA binding proteins on single mRNA molecules. Orthogonal RNA molecular recording of two Pumilio family proteins, PUM1 and PUM2, reveals that PUM1 competes with PUM2 for some but not all Pumilio binding sites in cells, despite having the same in vitro binding preferences. Our work thus measures competition between RNA-binding proteins for RNA sites in cells, and our genetically encodable RNA deaminase enables single-molecule identification of RNA-protein interactions with cell type specificity.

overall_design: Stable HEK293T cell lines expressing RBP-rABE, RBP-APOBEC1 or the deaminase only controls were generated with lentivral infection. Expression was driven by doxycycline promoters and cells were treated with 1000 ng/ml doxycycline for 48 hours.

first: Samentha

last: Kwok

first: Stephen

middle: N

last: Floor