title: CD32 captures committed haemogenic endothelial cells during human embryonic development.

city: Milan

first: Monah

last: Abou Alezz

address: Via Olgettina 60

country: Italy

institute: SR-TIGET

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

overall_design: Refer to individual Series


title: The Fc receptor CD32 identifies primed human hemogenic endothelial cells [RNA-seq]

status: Public on Feb 09 2024

type: Expression profiling by high throughput sequencing

summary: During embryonic development, blood cells emerge from a subset of specialized endothelial cells, named hemogenic endothelial cells (HECs), via a process known as endothelial-to-hematopoietic transition (EHT).  A thorough characterization of HECs and their EHT is essential to guide the efforts to derive this population from human pluripotent stem cells (hPSCs), a critical step to generate therapeutic blood products in vitro. However, current known markers used to isolate HECs are insufficient as they also enrich for arterial endothelial cells that are associated with HECs. To identify specific human HEC markers, we performed transcriptomic analysis of 28-32-day human embryos, a developmental stage characterized by active EHT. We observed that the expression of FCGR2B, encoding for the Fc receptor CD32 previously associated with other specialized endothelia, is highly enriched in the ACE+CD34+ endothelial cell population that contains HECs. Functional ex vivo analyses confirmed that multilineage hematopoietic potential is highly enriched in CD32+ endothelial cells isolated from the aorta-gonad-mesonephros region and yolk sac of human embryos. In addition, CD32 emerged as selective marker for hPSC-derived HECs across different hematopoietic programs. Remarkably, our analyses showed that CD32 expression identifies HECs that are irreversibly bound to undergo EHT. As such, CD32 expression enriches for cells with hemogenic potential with a higher specificity for hPSC-derived HECs than other known HEC markers. These findings provide a simple method for isolating HECs from human embryos and hPSC cultures, allowing the efficient generation of hematopoietic cells in vitro.

overall_design: H1 human embryonic stem cells were expanded in serum-free conditions and differentiated towards endothelial and hematopoietic fate as reported in Ditadi et al., 2015 (doi.org/10.1038/ncb3161); Ditadi et al, 2016 (doi.org/10.1016/j.ymeth.2015.10.001). At day8 of Wnt-dependent hematopoietic cultures, CD34+CD32+CD43-CD184-CD73-DLL4- (referred to as CD32+DLL4-) and CD34+CD73+CD184+DLL4+CD43- (referred to as CD184+DLL4+) cells were isolated from the same experimental sample by FACS sorting and used for RNA extraction, library preparation and RNA sequencing. 3 experiments were performed independently (identified as RS227, RS242, RS243).

first: Rebecca

last: Scarfò

middle: A

last: Alezz

first: Ivan

last: Merelli

first: Andrea

last: Ditadi


title: The Fc receptor CD32 identifies primed human hemogenic endothelial cells [scRNA-seq]

overall_design: H1 human embryonic stem cells were expanded in serum-free conditions and differentiated towards endothelial and hematopoietic fate as reported in Ditadi et al., 2015 (doi.org/10.1038/ncb3161); Ditadi et al, 2016 (doi.org/10.1016/j.ymeth.2015.10.001). At day8 of Wnt-dependent hematopoietic cultures, CD34+CD43-CXCR4-CD73- cells (referred to as HE) were isolated by FACS sorting and used for RNA extraction, library preparation and single cell RNA sequencing.

first: Manuela

last: Tavian

first: Stephanie

last: Luff

first: Christopher

middle: M

last: Sturgeon


title: Autofluorescence is a biomarker of neural stem cell activation state [Hippocampus]

city: Madison

first: Darcie

middle: L.

last: Moore

state: WI

address: 1111 Highland Ave

department: Neuroscience

country: USA

institute: University of Wisconsin-Madison

summary: Neural stem cells (NSCs) in the adult brain are primarily quiescent but can activate and enter the cell cycle to produce newborn neurons. NSC quiescence can be regulated by disease, injury, and age, however our understanding of NSC quiescence is limited by technical limitations imposed by the bias of markers used to isolate each population of NSCs and the lack of live-cell labeling strategies. Fluorescence lifetime imaging (FLIM) of autofluorescent metabolic cofactors has previously been used in other cell types to study shifts in cell states driven by metabolic remodeling that change the optical properties of these endogenous fluorophores. Here we asked whether autofluorescence could be used to discriminate NSC activation state. We found that quiescent NSCs (qNSCs) and activated NSCs (aNSCs) each have unique autofluorescence intensity and fluorescence lifetime profiles. Additionally, qNSCs specifically display an enrichment of a specific autofluorescent signal localizing to lysosomes that is highly predictive of cell state. These signals can be used as a graded marker of NSC quiescence to predict cell behavior and track the dynamics of quiescence exit at single cell resolution in vitro and in vivo. Through coupling autofluorescence imaging with single-cell RNA sequencing in vitro and in vivo, we provide a high-resolution resource revealing transcriptional features linked to rapid NSC activation and deep quiescence. Taken together, we describe a single-cell resolution, non-destructive, live-cell, label-free strategy for measuring NSC activation state in vitro and in vivo and use this tool to expand our understanding of adult neurogenesis.

overall_design: Cultured primary neural stem cells from male 6-week old hippocampus of C57BL6/J mice. Samples are activated, quiescent, 24 hours quiescence exit, and 48 hours quiescence exit.  47 cells collected per condition with equal numbers of cell collected from 4 gates made within the spread of punctate autofluorescence intensity.

middle: S

last: Morrow

first: Kelsey

last: Tweed

first: Zachary

middle: P

last: Arndt

first: Alex

middle: J

last: Walsh

first: Bo

last: Peng

first: Ryan

middle: D

last: Risgaard

first: Payton

middle: C

last: Klosa

first: Michelle

last: Chi

first: Eli

last: Wallace

first: Matthew

middle: V

last: Jones

first: Avtar

last: Roopra

first: Melissa

last: Skala

middle: L


title: RNA localization to stress granules

city: Aarhus

first: Ulf

middle: Andersson

last: Vang Ørom

address: Universitetsbyen 81

country: Denmark

institute: Aarhus University

summary: We determined the enrichment of mRNA in stress granules in WT and NAT10 KO cells to assess the impact of RNA acetylation on stress granule mRNA localization.

overall_design: We induced stress granules with arsenite treatment and isolated stress granules with G3BP-specific antibodies. Stress granule RNA was compared to total mRNA for calculation of enrichment.


title: Zeb2 drives the formation of CD11c+ atypical B cells to sustain germinal centers that control persistent infection [Bulk RNA-seq]

city: Canberra

first: Xin

last: Gao

state: ACT

address: 131 Garran Rd

department: Immunology

country: Australia

institute: The Australian National University

summary: CD11c+ atypical B cells (ABCs) are an alternative memory B cell lineage associated with immunization, infection, and autoimmunity. However, the factors that drive the transcriptional program of ABCs have not been identified, and the function of this population remains incompletely understood. Here we identified candidate transcription factors associated with the ABC population based on a human tonsillar B cell single cell dataset. We identified CD11c+ B cells in mice with a similar transcriptomic signature to human ABCs, and using an optimized CRISPR-Cas9 knockdown screen, we observed that loss of zinc finger E-box binding homeobox 2 (Zeb2) impaired ABC formation. Furthermore, ZEB2 haplo-insufficient Mowat Wilson syndrome (MWS) patients have decreased circulating ABCs in the blood. In Cd23Cre/+Zeb2fl/fl mice with impaired ABC formation, ABCs were dispensable for efficient humoral responses after Plasmodium sporozoite immunization but were required to control recrudescent blood-stage malaria. Immune phenotyping revealed that ABCs drive optimal T follicular helper (Tfh) cell formation and germinal center (GC) responses and they reside at the red/white pulp border likely permitting better access to pathogen antigens for presentation. Collectively, our study shows that ABC formation is dependent upon Zeb2, and these cells can limit recrudescent infection by sustaining GC reactions.

overall_design: In vitro cultured and in vivo generated CD11c+ atypical B cells and CD11c- classical memory B cells were sorted for bulk RNA-seq


title: Transcriptome profile of ACE-expressing embryonic endothelial cells

first: Monah

last: Abou Alezz

summary: During embryonic development, blood cells emerge from a subset of specialized endothelial cells, named hemogenic endothelial cells (HECs) via a process known as endothelial-to-hematopoietic transition (EHT). A more thorough characterization of HECs is essential to guide the efforts to derive this population from human pluripotent stem cells (hPSCs), a critical step to generate therapeutic blood products in vitro. To identify specific human HEC markers, we performed transcriptomic analysis of 28-32-day human embryos, a developmental stage characterized by active EHT.

overall_design: Analysis of gene expression profile of 4 (CD34+CD45-ACE+) samples vs (CD34+CD45-ACE-) in 4 embryos staged between day 28 and 32 from the aorta-gonad-mesonephros (AGM) region.

first: Rebecca

last: Scarfò

first: Sara

last: Valsoni

first: Ivan

last: Merelli

first: Manuela

last: Tavian

first: Andrea

last: Ditadi


title: Zeb2 drives the formation of CD11c+ atypical B cells to sustain germinal centers that control persistent infection [CITE-seq and scRNA-seq]

overall_design: Total, GC and memory B cells were sorted from human tonsils of two individuals, stained by CITE-seq antibodies and pooled for 10X single cell RNA-seq


title: Prebiotic proanthocyanidins inhibit bile reflux-induced esophageal adenocarcinoma through reshaping the gut microbiome

status: Public on Feb 08 2024

city: Ann Arbor

first: Laura

last: Kresty

state: MI

address: 1500 E Medical Center Dr

department: Surgery

institute: University of Michigan

summary: The gut and local esophageal microbiome progressively shift from healthy commensal bacteria to inflammatory-linked pathogenic bacteria in patients with gastroesophageal reflux disease, Barrett’s esophagus and esophageal adenocarcinoma (EAC). However, mechanisms by which microbial communities contribute to reflux-driven EAC remain incompletely understood and challenging to target. Herein, we utilized a rat reflux-induced EAC model to investigate targeting the gut microbiome-esophageal metabolome axis with cranberry proanthocyanidins (C-PAC) to inhibit EAC progression. Sprague Dawley rats, with or without reflux-induction received water or C-PAC ad libitum (700 µg/rat/day) for 25 or 40 weeks. C-PAC exerted prebiotic activity abrogating reflux-induced dysbiosis, and mitigating bile acid metabolism and transport, culminating in significant inhibition of EAC through TLR/NF-κB/TP53 signaling cascades. At the species level, C-PAC mitigated reflux-induced pathogenic bacteria (Streptococcus parasanguinis, Escherichia coli, and Proteus mirabilis). C-PAC specifically reversed reflux-induced bacterial, inflammatory and immune-implicated proteins and genes including Ccl4, Cd14, Crp, Cxcl1, Il6, Il1β, Lbp, Lcn2, Myd88, Nfkb1, Tlr2 and Tlr4 aligning with changes in human EAC progression, as confirmed through public databases. C-PAC is a safe promising dietary constituent that may be utilized alone or potentially as an adjuvant to current therapies to prevent EAC progression through ameliorating reflux-induced dysbiosis, inflammation and cellular damage.

overall_design: Sprague Dawley rats, with or without reflux-induction, received water or C-PAC ad libitum (700 µg/rat/day) for 40 weeks. Fecal pellets from animals were collected at 40 weeks and 16s rRNA sequencing was performing using DNA isolated from fecal pellets. Four groups were included in the 16s rRNA sequencing: reflux (n = 7), reflux + CPAC (n = 5), CPAC (n = 5), and water (n = 7).


title: Osteomacs promote maintenance of murine hematopoiesis through megakaryocyte-induced upregulation of Embigin and CD166

city: Boston

first: Safa

middle: F

last: Mohamad

state: MA

address: 125 Park Drive, Apt 15

institute: Boston Children's Hospital/ Harvard Medical School

summary: Maintenance of hematopoietic stem cell (HSC) function in the niche is an orchestrated event. Osteomacs (OM), are key cellular components of the niche. Previously, we documented that osteoblasts, OM, and megakaryocytes interact to promote hematopoiesis. Here, we further characterize OM and identify megakaryocyte-induced mediators that augment the role of OM in the niche. Single cell mRNAseq, mass spectrometry, and CyTOF examination of megakaryocyte-stimulated OM suggested that upregulation of CD166 and Embigin on OM augment their hematopoiesis maintenance function. CD166 knockout OM or shRNA-Embigin knockdown OM, confirmed that loss of these molecules significantly reduced OM ability to augment the osteoblast-mediated hematopoietic enhancing activity. Recombinant CD166 and Embigin partially substituted for OM function, characterizing both proteins as critical mediators of OM hematopoietic function. Our data identify Embigin and CD166 as OM-regulated critical components of HSC function in the niche and potential participants in various in vitro manipulations of stem cells.

overall_design: 1x105 neonatal calvarial cells (NCC) were cultured for 16hrs with or without 50,000 megakaryocytes were stained with CD45, F4/80, and CD41.  Sorted CD45+F4/80+CD41- cells were suspended at 3x105 cells/ml PBS then dispensed into medium sized IFC chips (Fluidigm C1 Single-Cell Auto Prep System)(Zhang et al., 2019). mRNA was isolated followed by cDNA synthesis (Clontech SMART-Se v4 Ultra Low Input RNA Kit). Up to 0.4ng cDNA was used for library preparation and indexing with Nextera XT DNA Library Prep Kit (Illumina, Inc., San Diego, CA). Pooled libraries (4nM, quality checked) were used for 150b paired-end sequencing (NextSeq 500). Cells that passed quality testing were analyzed.

first: Edward

last: Srour

first: Chi

last: Zhang


title: Autofluorescence is a biomarker of neural stem cell activation state [Subventricular zone]

first: Darcie

last: Moore

overall_design: Subventricular zone neural stem cells were dissected and purified from 8 male C57BL6/J mice from Envigo on 3 separate days of experiments, onto 3 separate sequencing plates. All cells were analyzed together.

first: Christopher

last: Morrow

first: Kelsey

last: Tweed

first: Zachary

last: Arndt

first: Alex

last: Walsh

first: Bo

last: Peng

first: Ryan

last: Risgaard

first: Payton

last: Klosa

first: Michelle

last: Chi


title: GLUT1-mediated glucose import in B cells is critical for anaplerotic balance and humoral immunity

city: Erlangen

first: Hans-Martin

last: Jäck

address: Glückstrasse 6

department: Molecular Immunology

country: Germany

institute: Universitätsklinikum Erlangen

summary: Glucose uptake increases during B cell activation and plasma cell differentiation. However, conflicting findings regarding the role of glucose at different stages of B cell activation prevents establishing a clear metabolic profile. To determine whether glucose is required for establishing humoral immunity, we deleted the glucose transporter type 1 (GLUT1)-encoding gene (Slc2a1fl/fl) in mature B cells by CD23-mediated Cre expression (GLUT1-cKO). While B cell development was normal, germinal center B cell and antibody-secreting cell numbers (ASCs) were severely reduced in GLUT1-cKO mice. In addition, these mice could hardly mount antigen-specific antibody titers after vaccination. Activation of GLUT1-deficient B cells in vitro was impaired and the remaining plasmablasts abolished glycolysis, relying on mitochondrial activity and fatty acid metabolism. Metabolomics combined with genome-wide transcriptome analysis revealed a dramatically altered anaplerotic balance in GLUT1-deficient ASCs. Although the cells attempt to compensate for glucose deprivation by increasing their mitochondrial mass and the expression of genes responsible for glycolysis, the tricarboxylic acid cycle and the hexosamine synthesis pathways, they lack both the necessary metabolites for energy production and functional mitochondrial respiration. Consequently, protein synthesis was limited in GLUT1-deficient ASCs. Thus, we identified GLUT1 as a critical metabolic player defining the germinal center response and humoral immunity.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for splenic plasma cells from CD23cre(+/-);Slc2a1(fl/fl) and CD23cre(+/-);Slc2a1(wt/wt) mice

first: Theresa

middle: E

last: Bierling

first: Amelie

last: Gumann

first: Shannon

middle: R

last: Ottmann

first: Sebastian

last: Schulz

first: Leonie

last: Weckwerth

first: Jana

last: Thomas

first: Arne

last: Gessner

first: Magdalena

last: Wichert

first: Frederic

last: Kuwert

first: Franziska

last: Rost

last: Hauke

first: Tatjana

last: Freudenreich

first: Dirk

last: Mielenz

first: Katharina

last: Pracht


title: Colitis associated with immune checkpoint inhibitors

city: Charlestown

first: Kamil

last: Slowikowski

state: Massachusetts

address: Mailstop CNY 149-8, 149 13th Street

institute: Massachusetts General Brigham

summary:
This SuperSeries is composed of 3 SubSeries listed below:
- blood_cells
- tissue_cells
- tissue_nuclei

overall_design:
In this study we set investigate four aspects of irColitis that are vital to the pathogenesis of most immunotherapy related adverse events:
(1) the interactions between CD8 T cells and other immune, epithelial, and mesenchymal cells that sustain organ specific inflammation;
(2) the contribution of circulating immune cells to disease;
(3) the epithelial and mesenchymal defects that define parenchymal organ injury and dysfunction;
(4) the transcriptional features of colitis associated with anti-PD-1 monotherapy versus anti-PD-1/CTLA-4 dual therapy.

We leveraged single-cell RNA-sequencing (scRNA-seq) and single-nuclei RNA-sequencing (snRNA-seq) of colon mucosal biopsies and paired blood specimens collected from a well-characterized cohort of irColitis cases and controls to define the cellular and molecular drivers of irColitis.


title: RNA-seq analysis of Pneumocystis murina in short term culture

city: Bethesda

first: Ousmane

middle: Hamadoun

last: Cisse

state: MD

address: 10 Center Dr,

institute: National Institutes of Health

summary: RNA-Seq analysis of Pneumocystis murina in culture at three time points post inoculation

overall_design: To investigate the transcriptional landscape of Pneumocystis murina in coculture with mammalian epithelial lung cells, we performed a gene expression analysis using data obtained RNA-seq of cells at three time points post inoculation

middle: H

first: Shelly

last: Curran

first: Joseph

last: Kovacs


title: DNA methylation analysis in Pneumocystis carinii using bisulfite sequencing

type: Methylation profiling by high throughput sequencing

summary: Pneumocystis carinii bisulfite_seq

overall_design: To determine whether DNA methylation plays a role in Pneumocystis centromeres, we performed bisulfite sequencing (5 methylcytosine) in P. carinii.

first: Liang

last: Ma


title: Lung epithelium releases growth differentiation factor 15 in response to pathogen-mediated injury

city: Pittsburgh

first: William

last: Bain

state: PA

address: 3459 Fifth Ave., MUH NW 628

department: Medicine

institute: University of Pittsburgh

summary: Rationale: Growth/differentiation factor 15 (GDF15) is a stress cytokine with numerous proposed roles including stress erythropoiesis. Higher circulating GDF15 levels are prognostic of mortality during acute respiratory distress syndrome, but the sources and downstream effects of GDF15 during pathogen-mediated injury remain unclear. Methods: We quantified GDF15 in plasma and lower respiratory tract (LRT) biospecimens from critically-ill humans with acute respiratory failure. Publicly available data from SARS-CoV-2 infection were re-analyzed. We utilized mouse models of acute lung injury mediated by P. aeruginosa exoproducts in wildtype mice and in mice genetically deficient for Gdf15 and its putative receptor, Gfral.Results: Plasma levels of GDF15 associated with worse outcomes and LRT GDF15 levels in critically-ill humans. Intra-tracheal P. aeruginosa Type 2 secretion system exoproducts (PA SN) were sufficient to induce airspace and plasma release of GDF15 in wildtype mice, which was attenuated during epithelial-specific Gdf15 deficiency. SARS-CoV-2 infection induced GDF15 expression in human lung epithelium. Mice with global Gdf15 deficiency had decreased airspace hemorrhage, an attenuated cytokine profile, and altered lung transcriptional profile during PA SN injury, which was not recapitulated in mice deficient for Gfral.  Airspace GDF15 reconstitution did not significantly modulate key lung cytokine levels but did increase circulating erythrocyte counts.Conclusions: Lung epithelium releases GDF15 during pathogen injury, which associates with plasma levels in both humans and mice and can increase erythrocyte counts in mice. Potential local and other extra-pulmonary roles for GDF15 remain undefined.

overall_design: Wildtype or Gdf15-/- mice were intra-tracheally inoculated with the supernatant of P. aeruginosa (PA SN), or vehicle, under direct visualization after isoflurane anesthesia. At the time of necropsy approximately 20 hours post-inoculation, mice received a terminal dose of ketamine/xylazine and the left lung was clamped, removed, and snap-frozen in liquid nitrogen prior to storage at -80°C. Snap frozen lungs were homogenized in Trizol (Life Sciences) and RNA was isolated per manufacturer instructions. RNA cleanup was performed using Qiagen RNeasy Mini Kit per manufacturer instructions. Quality control and mRNA library preparation of isolated lung RNA were performed by Novogene (Sacramento, CA) followed by paired-end whole genome sequencing using an Illumina NovaSeq platform.

first: Jonathan

middle: K

last: Alder


title: Polycomb repression of Hox genes involves spatial feedback but not domain compaction or phase-transition

city: Palo Alto

first: Sedona

middle: Eve

last: Murphy

state: California

address: 279 campus dr

institute: Stanford University

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Polycomb group proteins play a critical role in silencing transcription during development. It is commonly proposed that Polycomb dependent changes in genome folding, which compact chromatin, contribute directly to repression by blocking binding of activating complexes. Recently, it has also been argued that liquid-liquid demixing of Polycomb proteins facilitates this compaction and repression by phase-separating target genes into a membraneless compartment. To test these models, we utilized Optical Reconstruction of Chromatin Architecture (ORCA) to trace the Hoxa gene cluster, a canonical Polycomb target, in thousands of single cells. Across multiple cell types, we find that Polycomb-bound chromatin frequently explores decompact states and partial mixing with neighboring chromatin, while remaining uniformly repressed, challenging the repression-by-compaction or phase-separation models. Using polymer simulations, we show that these observed flexible ensembles can be explained by “spatial feedback”: transient contacts  that contribute to propagation of the epigenetic state, (epigenetic memory) without inducing a globular organization.

overall_design: CUT&RUN performed on untreated and RING1B + EED dTAG degron mouse embryonic stem cells. Polycomb was degraded for 8 hours. IgG negative input, H3k4me3 positive control and HA-Tagged RING1B were assayed.

first: Alistair

middle: N

last: Boettiger


title: Granulocytic myeloid-derived suppressor cell activity during biofilm infection is regulated by a glycolysis-HIF-1α axis

city: Omaha

first: Tammy

last: Kielian

state: NE

address: 985900 Nebraska Medical Center

institute: University of Nebraska Medical Center

summary: Staphylococcus aureus is a leading cause of biofilm-associated prosthetic joint infection (PJI). A primary contributor to infection chronicity is an expansion of granulocytic myeloid-derived suppressor cells (G-MDSCs) that are critical for orchestrating the anti-inflammatory biofilm milieu. Single-cell sequencing and bioinformatic metabolic algorithms were used to explore the link between G-MDSC metabolism and S. aureus PJI outcome. Glycolysis and the hypoxic response through hypoxia-inducible factor-1 alpha (HIF-1α) were significantly enriched in G-MDSCs. Interfering with both pathways in vivo, using a 2-deoxyglucose nanopreparation and granulocyte-targeted HIF-1α conditional knockout mice, respectively, attenuated G-MDSC-mediated immunosuppression and reduced bacterial burden in a mouse model of S. aureus PJI. In addition, scRNA-seq analysis of granulocytes from PJI patients also showed an enrichment in glycolysis and hypoxic response genes. These findings support the importance of a glycolysis/HIF-1α axis in promoting G-MDSC anti-inflammatory activity and biofilm persistence during PJI.

overall_design: C57BL/6J mice received 2-DG-containing or empty nanoparticles at day 3 post-infection, whereupon live Ly6G+ cells were collected from the soft tissue adjacent to the knee joint 4 days later (i.e. day 7 post-infection) by FACS and RNA isolated for bulk RNA-sequencing.

first: Horn

last: Chris

first: Arumugam

last: Prabhakar

first: Van

last: Zachary

first: Heim

last: Cortney

first: Kielian

last: Tammy

first: Jagadesan

last: Sankarasubramanian

first: Xiao

last: Peng


title: Inhibition of ACSS2-mediated crotonylation alleviates kidney fibrosis via IL- 1β-dependent macrophage activation and tubular cell senescence

city: chengdu

first: Ting

last: Xiang

address: chengdu

country: China

institute: West China Hospital of Medicine: Sichuan Universit

summary: Chronic kidney disease (CKD) is characterized by sustained inflammation and progressive fibrosis, however therapies to slow CKD progression are limited. Histone lysine crotonylation (Kcr) as a novel post-translational modification is widespread as acetylation (Kac), but its roles in CKD are largely unknown. In the study, we firstly reported that the nuclear histone Kcr in tubular epithelial cells was significantly elevated in fibrotic kidney of patients and mice, which was positively correlated with the progression of disease. Interestingly, abnormal increase of histone 3 lysine 9 crotonylation (H3K9cr) in kidneys of unilateral ureteric obstruction (UUO) and folic acid nephropathy (FAN) was completely different to the stable expression of H3K9ac, indicating that H3K9cr may exert extraordinary functions in kidney fibrosis. By screening these crotonylated/acetylated factors, crotonyl-CoA-producing enzyme ACSS2 (acyl-CoA synthetase short chain family member 2) remarkably promoted H3K9cr without influencing H3K9ac to trigger proinflammatory cytokine IL-1β transcription. Furthermore, genetic and pharmacologic inhibition of ACSS2 both attenuated kidney injury and fibrosis, as well as suppressed H3K9cr-mediated IL-1β expression, which thus to alleviate IL-1β-dependent macrophage activation and tubular cell senescence. Collectively, our finding uncovers that H3K9cr plays a critical, previously unrecognized role in kidney fibrosis, where ACSS2 represents an attractive target for strategies that aim to slow fibrotic kidney disease progression

overall_design: To understand the functions of ACSS2 in kidney fibrosis, we established ACSS2 konckout mouse and perform rnaseq analysis

first: Lingzhi

last: Li


title: An organ-specific angiogenic control mechanism for endothelial tailoring

city: Stockholm

first: Michael

last: Vanlandewijck

address: Blickagången 6

department: Department of Medicine

country: Sweden

institute: Karolinska Institute

summary: Each organ of the human body requires locally-adapted blood vessels. The gain of such organotypic vessel specializations is often deemed molecularly unrelated to the process of organ vascularization. Opposing this model, we reveal a molecular mechanism for brain-specific angiogenesis, that operates under the control of Wnt7a/b ligands, well-known blood-brain barrier maturation signals. The control mechanism relies on Wnt7a/b-dependent expression of Mmp25 in brain endothelial cells. This hitherto poorly characterized GPI-anchored matrix metalloproteinase is selectively required in endothelial tip cells to enable their initial migration across the pial basement membrane lining the brain surface, which distinctive molecular composition is controlled by embryonic pial fibroblasts. Mechanistically, Mmp25 confers brain invasive competence by cleaving the pial basement membrane-enriched Col4a5/6 within a short non-collagenous region of the central helical part of the heterotrimer. Upon genetic interference with pial basement membrane composition, the Wnt/β-catenin-dependent organotypic control of brain angiogenesis is lost, resulting in properly patterned, yet blood-brain barrier-defective cerebrovasculatures. This work reveals an organ-specific angiogenesis mechanism, sheds light on tip cell mechanistic angiodiversity, and thereby illustrates how organs, by imposing local constraints on angiogenic tip cells, can select vessels matching their distinctive physiological requirements.

overall_design: The samples in this submission are single cells, sorted into single wells of a 96 or 384 well plates. The cells were lysed and transcriptomic sequencing data was obtained using Smart-seq2 chemistry

first: Giel

last: Schevenels

first: Pauline

last: Cabochette

first: Line

last: De Grande

first: Stefan

last: Guenther

first: Liqun

last: He

first: Marc

last: Dieu

first: Marjorie

last: Vermeersch

first: Raoul

last: Germano

first: Michelle

last: America

first: David

last: Perez-Morga

first: Patricia

last: Renard

first: Maud

last: Martin

first: Christer

last: Betsholtz

first: Benoit

last: Vanhollebeke


title: Joubert Syndrome-derived induced pluripotent stem cells show altered neuronal differentiation in vitro

first: Santo

last: Diprima

address: Via Olgettina 58

department: COSR

institute: San Raffaele Hospital

summary: Joubert syndrome (JS) is a recessively inherited congenital ataxia characterized by hypotonia, psychomotor delay, abnormal ocular movements, intellectual disability and a peculiar cerebellar and brainstem malformation, the ’molar tooth sign’. Over 40 causative genes have been reported, all encoding for proteins implicated in the structure or functioning of the primary cilium, a subcellular organelle widely present in embryonic and adult tissues. In this paper we developed an in vitro neuronal differentiation model using patient-derived induced pluripotent stem cells (iPSCs), to evaluate possible neurodevelopmental defects in JS. To this end, iPSCs from four JS patients harboring mutations in distinct JS genes (AHI1, CPLANE1, TMEM67 and CC2D2A) were differentiated alongside healthy control cells to obtain mid-hindbrain precursors and cerebellar granule cells. Differentiation was monitored over 31 days through the detection of lineage-specific marker expression by qRT-PCR, immunofluorescence, and transcriptomics analysis. All JS patient-derived iPSCs, regardless of the mutant gene, showed a similar impairment to differentiate into mid-hindbrain and cerebellar granule cells when compared to healthy controls. In addition, analysis of primary cilium count and morphology showed notable ciliary defects in all differentiating JS patient-derived iPSCs compared to controls. These results confirm that patient-derived iPSCs are an accessible and relevant in vitro model to analyze cellular phenotypes connected to the presence of JS gene mutations in a neuronal context.

overall_design: To evaluate possible neurodevelopmental defects in JS patients derived IPScells we performed RNAseq experiment on early (D8) and late (D24) differentiation time points during IPSc cerebellar granule cells differentiation and we compared the expression of cerebellar development genes in patients and control derived IPScells.

first: Roberta

last: De Mori


title: Transcriptome fine-mapping reveals FoxJ, a σE-dependent small RNA with unusual mRNA activation activity in Fusobacterium nucleatum

city: Würzburg

first: Falk

last: Ponath

address: Josef-Schneider-Straße 2

institute: Helmholtz Institute for RNA-based Infection Research (HIRI)

summary: The oral commensal Fusobacterium nucleatum can spread to extra-oral sites where it is associated with pathologies as diverse as pre-term birth or cancer. Due to the evolutionary distance of F. nucleatum to other model bacteria, we lack a deeper understanding of RNA regulatory networks that allow this bacterium to adapt to different environmental niches. As a first step in that direction, we recently showed that F. nucleatum harbors a global stress response governed by the extracytoplasmic function sigma factor σE, which displays a striking functional conservation with Proteobacteria and includes a noncoding arm in the form of a regulatory small RNA (sRNA), FoxI. To search for putative additional σE-dependent sRNAs, we comprehensively mapped 5’- and 3’-ends of transcripts in the model strain ATCC 23726. This enabled the discovery of FoxJ, a ~156 nucleotide sRNA previously misannotated as the 5’ UTR of ylmH. FoxJ is tightly controlled by σE and activated by the same stress conditions as FoxI. Both sRNAs act as mRNA repressors of the abundant porin FomA, but FoxJ also regulates genes that are distinct from the target suite of FoxI. Moreover, FoxJ differs from most other σE-dependent sRNAs in that it also positively regulates genes at the post-transcriptional level. We provide preliminary evidence for a new mode of sRNA-mediated mRNA activation, which involves the targeting of intra-operonic terminators. Overall, our study provides an important resource through the comprehensive annotation of 5’- and 3’ UTRs in F. nucleatum and expands our understanding of the σE-response in this evolutionarily distant bacterium.

sample_organism: Fusobacterium nucleatum subsp. nucleatum ATCC 23726

overall_design: Gene expression was evaluated for F. nucleatum during the mid-exponential phase. For this the FoxJ overexpression was compared to the control. dRNA-seq data were analyzed using ANNOgesic following manual curation to annotate TSS and 5' UTRs. Term-seq data were analyzed manually to annotate 3' UTRs.

first: Yan

last: Zhu

first: Jörg

last: Vogel


title: Characterizing Pneumocystis murina centromeres with ChIP-seq (pm_191010)

first: Ousmane

last: Cisse

summary: Pneumocystis is a relevant genetic system to study centromere formation in relation with host adaptation. How centromeres are formed and maintained in strongly host adapted fungal pathogens is poorly investigated. Centromeres are genomic regions that coordinate accurate chromosomal segregation during mitosis and meiosis. Yet, despite their essential function, centromeres evolve rapidly across eukaryotes. CENP-A, a variant of histone H3 is the epigenetic marker that define centromeres in most eukaryotes. Centromeres are often the sites of chromosomal breaks which contribute to genome shuffling and promote speciation by inhibiting gene flow. Genome shuffling allows genome reconfiguration suitable for survival in new environment such as pathogen adaptation to new hosts. Here, we study the evolution of centromeres in closely related species of mammalian specific pathogens of the fungal phylum of Ascomycota. Long term culture of Pneumocystis species is currently untenable. Using heterologous complementation, we show that Pneumocystis CENP-A ortholog is functionally equivalent to fission yeast Cnp1. Using a short-term in vitro culture, infected animal models and ChIP-seq, we identified centromeres in three Pneumocystis species that diverged ~100 Mya ago. Each species has 17 unique short regional centromeres (< 10kb) in 17 monocentric chromosomes. The centromeres are flanked by heterochromatin. They span active genes, lack conserved DNA sequence motifs, and repeats.These features suggest an epigenetic specification of centromere function.

overall_design: Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) for centromeric histone CENP-A and CENP-C in Pneumocystis murina. P. murina organisms were collected from an infected CD40 Ligand knock out mice (C1 seeder)

first: Shelly

last: Curran

first: Joseph

last: Kovacs


title: Characterizing Pneumocystis carinii centromeres with ChIP-seq (pc_210927)

overall_design: Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) for centromeric histone CENP-A and CENP-C in Pneumocystis carinii. P. carinii organisms were collected from infected lungs of corticoids-induced immunocompromised Sprague-Dawley male rats


title: Characterizing Pneumocystis carinii centromeres with ChIP-seq III

overall_design: Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) for centromeric histone CENP-A and CENP-C in Pneumocystis carinii. P. carinii organisms were collected from infected lungs of corticoids-induced immunocompromised Sprague-Dawley male rats. Organisms were cocultured with a suspension of A549 and LET1 cells for 7 days


title: Characterizing Pneumocystis carinii centromeres with ChIP-seq (pm_211220)


title: Profiling trancriptome changes that affect vein specification in maize (Zea mays) tml (TOO MANY LATERALS) mutants

status: Public on Feb 07 2024

city: Oxford

first: Maricris

last: Zaidem

state: Oxfordshire

address: South Parks Rd

department: Department of Biology

country: United Kingdom

institute: University of Oxford

summary: The biochemistry of C4 photosynthesis relies on a specific suite of leaf functional properties, often referred to as Kranz anatomy, where there is increased vein density and the proportion of bundle sheath cells surrounding the veins compared to C3 photosynthesizing species. From a suite of putative Kranz anatomy regulators, TML was identified.  Loss of function tml mutations lead to the development of large lateral veins in positions normally occupied by smaller intermediate veins in both C3 and C4 species tested. In order to elucidate the role of TML in vein patterning, we set out to profile transcriptional changes that happen in the leaf primordia upon tml knock-out in the C4 species Zea mays.

overall_design:
To investigate the effect of TML in vein patterning, we set out to profile transcriptional changes that happen in the leaf primordia, bulk plastochron 0 (P0) to plastochron 5 (P5) leaves including the shoot apical meristem (SAM) and the leaf sheath, of maize (Zea mays B104) upon tml1;tml2 knock-out. Loss of function alleles were generated by CRISPR/Cas9 mediated gene editing.
We then performed gene expression profiling analysis using leaf primordia samples (SAM, bulk P0 - P5) from 3 biological replicates of homozygous tml1;tml2 (segregating independently from the transgene) mutants and the respective segregating wildtypes.

Please note that the WTCHG_960488 and WTCHG_960489 represent two technical replicates of the same biological sample from different runs. The *_T1 data columns (in the processed data file) were generated from the WTCHG_960488* runs and *T2 data columns from the WTCHG_960489 runs.

first: Daniela

last: Vlad

first: Jane

last: Langdale


title: CDK12 maintains phosphorylation of RNA Polymerase II during epidermal differentiation

city: Guangzhou

first: Jingting

last: Li

address: No 1, Zhongshan 2 Road, 12th Floor

department: Institue of Precision Medicine

institute: The First Affiliated Hospital, Sun Yat-sen University

summary: CDK12 plays an important role in regulating epidermal differntaition through maintaining the phosphorylation of RNA polymerase II levels in differentiation associated genes.

overall_design: CDK12 knockdown RNA-Seq in calcium induced differentiated primary keratinocytes. CDK12 ChIP-Seq with CDK12 and RNA Pol II antibodies in calcium induced differentiated primary keratinocytes.

first: George

last: Sen


title: Characterizing Pneumocystis carinii centromeres with ChIP-seq (pc_210506)


title: A novel inhibitor of P. aeruginosa folate metabolism exploits metabolic differences for narrow-spectrum antibiotic targeting

city: Princeton

first: Connor

last: Chain

address: Lewis Thomas Lab

institute: Princeton University

summary: Pseudomonas aeruginosa is a leading cause of hospital acquired infections for which the development of new antibiotics is urgently needed. Unlike most enteric bacteria, P. aeruginosa lacks thymidine kinase and thymidine phosphorylase activity, and thus cannot scavenge exogenous thymine. An appealing strategy to selectively target P. aeruginosa while leaving the healthy microbiome largely intact would thus be to disrupt thymidine synthesis while providing exogenous thymine. However, this approach was previously intractable because known antibiotics that perturb thymidine synthesis are largely inactive against P. aeruginosa. Here, we characterize a novel dihydrofolate reductase inhibitor, fluorofolin, that exhibits significant activity against P. aeruginosa in culture and in a mouse thigh infection model. Fluorofolin is active against a wide range of clinical P. aeruginosa isolates resistant to known antibiotics, including critical antibiotic development priorities expressing the beta-lactamases KPC-5 and NDM-1. Importantly, in the presence of thymine supplementation, fluorofolin activity is selective for P. aeruginosa. Resistance to fluorofolin can emerge through overexpression of the efflux pumps MexCD-OprJ and MexEF-OprN. However, these mutants also decrease pathogenesis, in part due to increased export of quorum sensing precursors leading to decreased virulence factor production. Our findings thus demonstrate how understanding species-specific genetic differences and discovery of an antibiotic with a widely conserved target can enable selective targeting of important pathogens while revealing new tradeoffs between resistance and pathogenesis.

overall_design: To verify the phenotype of two flurofolin resistant mutants in P. Aeruginosa

last: Sheehan

first: Xincheng

last: Xu

first: Soodabeh

last: Ghaffari

first: Aneesh

last: Godbole

first: Hahn

last: Kim

first: Joel

last: Freundlich

first: Joshua

last: Rabinowitz

first: Zemer

last: Gitai


title: A Pre-Leukemic DNA Methylation Signature in Healthy Individuals Associated with Risk of Myeloid Malignancy

city: Singapore

first: Qiaoyang

last: Sun

address: 20 College Rd

department: Neurology

country: Singapore

institute: National Neuroscience Institute

summary: Methylation profiling by high throughput sequencing

overall_design: We performed global DNA methylation profiling in a case-control study nested within Singapore Chinese Health Study to evaluate if DNA methylation alterations were associated with AML/MDS development. Targeted deep sequencing and methylated DNA immunoprecipitation sequencing (MeDIP-seq) were performed on peripheral blood collected a median of 9.9 years prior to diagnosis of AML or MDS, together with age-matched still healthy individuals as controls.

first: Zhentang

last: Lao

first: Ling-Wen

last: Ding

first: Qiao-Yang

first: Henry

last: Yang


title: Slam-seq reveals that miR-430 regulates zygotic mRNA during zebrafish embryogenesis

city: Kansas City

first: Ariel

middle: Alejandro

last: Bazzini

state: Missouri

institute: Stowers Institute for Medical Research

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

overall_design: Refer to individual Series


city: bad nauheim

first: Stefan

last: Günther

address: ludwigtr. 43

institute: MPI for heart and lung research

summary: To address the role of endothelial Wnt/β-catenin signaling in CNS angiogenesis, we compared the bulk transcriptomes of WT and Wnt/β-catenin signaling-deficient PHBC endothelial cells, prior to CNS vascular invasion. To this end, we used three approaches to abrogate endothelial Wnt/β-catenin signaling: Morpholino-mediated knock-down of gpr124, reck or wnt7aa. We find that the expression of mmp25b is decreased in PHBC endothelial cells of all Wnt/β-catenin signaling deficient conditions as compared to the WT controls.

overall_design:
Tg(fli1:Gal4)ubs3;(UAS:Kaede)rk8 embryos were injected at the single cell stage with Morpholinos targeting gpr124, reck  or wnt7aa. Prior to CNS vascular invasion (30 hpf), PHBC endothelial cells expressing the Kaede fluorescent protein, were photoconverted (shifting green towards red fluorescence) by exposere to a 405 nm laser nanobeam, specifically at the level of the PHBC.
Subsequent embryo dissociation and FACS allowed for isolation of PHBC endothelial cells. RNA extraction and sequencing was performed on 4 samples per condition, each containing photoconverted PHBC endothelial cells of 80 embryos.

first: Pauline

last: Cabochette

first: Line

last: De grande

first: Giel

last: Schevenels

first: Benoit

last: Vanhollebeke


title: Deep-sequencing of endothelial-enriched RNAs from carotid arteries of C57 Bl6 mice subjected to partial carotid ligation (PCL) to determine the effect of disturbed blood flow and aging

city: Atlanta

first: Sandeep

last: Kumar

state: GA

address: 1760 Haygood Dr.

department: Biomedical Engineering

institute: Emory University

summary:
Purpose: To determine the effect of disturbed blood flow and aging on mouse carotid artery endothelial cells.
Methods: 8-week old  and 80-week-old male C57BL6 mice were subjected to partial carotid ligation surgery and two days post ligation surgery, the endothelial enriched RNAs were flushed out using Qiazol

first: Hanjoong

last: Jo


title: Effect of performing SLAM-seq chemistry in methanol fixed cells versus standard tube processing on quantification bias in nucleotide conversion RNA-seq data

status: Public on Feb 06 2024

first: Kevin

last: Berg

address: Versbacher Str. 7

institute: University of Würzburg

summary: To investigate the occuring 4sU dropout during library preparation we performed SLAM conversion either in intact, methanol fixed cells or in tubes

overall_design: SLAM conversion in intact, methanol fixed NIH-3T3 cells and in tubes in two replicates

first: Manivel

last: Lodha

first: Isabel

last: Delazer

first: Yiliam

last: Cruz Garcia

first: Thomas

last: Hennig

first: Elmar

last: Wolf

first: Alexandra

last: Lusser

first: Bhupesh

last: Prusty

first: Florian

last: Erhard


title: Lysine 36 of Drosophila histone H3.3 supports adult longevity

city: Chapel Hill

first: Gregory

last: Matera

state: North Carolina

address: Genome Science Building

institute: University of North Carolina at Chapel Hill

summary: Aging is a multifactorial process that disturbs homeostasis, increases disease susceptibility, and ultimately results in death. Although the definitive set of molecular mechanisms responsible for aging remain to be discovered, epigenetic change over time is proving to be a promising piece of the puzzle. Several posttranslational histone modifications (PTMs) have been linked to the maintenance of longevity. Here, we focus on lysine 36 of the replication independent histone protein, H3.3 (H3.3K36). To interrogate the role of this residue in Drosophila developmental gene regulation, we generated a lysine to arginine mutant that blocks the activity of its cognate modifying enzymes. We found that an H3.3BK36R mutation causes a significant reduction in adult lifespan, accompanied by dysregulation of the genomic and transcriptomic architecture. Transgenic co-expression of wild-type H3.3B completely rescues the longevity defect. Because H3.3 is known to accumulate in nondividing tissues, we carried out transcriptome profiling of young vs aged adult fly heads. The data show that loss of H3.3K36 results in age-dependent misexpression of NF-κB and other innate immune target genes, as well as defects in silencing of heterochromatin. We propose H3.3K36 maintains the postmitotic epigenomic landscape, supporting longevity by regulating both pericentric and telomeric retrotransposition and by suppressing aberrant immune signaling.

overall_design: RNA-Seq of Drosophila melanogaster heads. Three replicates of each condition (sex, age, and genotype). Sex: Male and Female. Age: Old and Young. Genotypes: H3.3Adelta and H3.3BK36R.

first: John

last: Brown

first: Benjamin

last: McMichael

first: Vasudha

last: Vandadi

first: Aadit

last: Mukherjee

first: Harmony

last: Salzler

first: A

middle: G


title: Human ASXL1-Mutant Hematopoiesis Is Driven by a Truncated Protein Associated with Aberrant Deubiquitination of H2AK119

city: Stanford

last: Koehnke

state: CA

address: 265 Campus Drive

summary: Mutations in additional sex combs like 1 (ASXL1) confer poor prognosis in myeloid malignancies and are also found in pre-malignant clonal hematopoiesis of indeterminate potential (CHIP). However, the mechanisms by which these mutations contribute to disease initiation remain unresolved and therapeutic targeting has remained elusive. Here, we demonstrate that ASXL1 mutations are frequently founding lesions in acute myeloid leukemia (AML) and lead to the expression of a functional, truncated protein. We developed a human disease model that faithfully recapitulates ASXL1-mutant CHIP with increased self-renewal and in vivo competitive advantage that can spontaneously progress to a lethal myeloid malignancy. We determined that truncated ASXL1 leads to global redistribution of the repressive chromatin mark H2AK119Ub, increased chromatin accessibility, and activation of both myeloid and stem cell gene expression programs. Finally, we identified dysregulation of H2AK119Ub as a therapeutic vulnerability and demonstrated that ASXL1-mutant cells are vulnerable to inhibition of the PRC1 complex.

overall_design: RNA-Seq, ATAC-Seq and CUT&RUN Sequencing of H2AK119Ub in cord-blood derived human HSPCs

middle: A

last: Nuno

first: Ravindra

last: Majeti


title: Comprehensive Transcriptomic Analysis of EWSR1::WT1 Targets Identifies CDK4/6 Inhibitors as a Novel Therapeutic for Desmoplastic Small Round Cell Tumor

city: New Orleans

first: Sean

middle: Bong

last: Lee

state: LA

address: 1700 Tulane Ave

department: Pathology

institute: Tulane University School of Medicine

summary: Desmoplastic small round cell tumor (DSRCT) is an aggressive, pediatric tumor characterized by the EWSR1::WT1 oncogene. Targeted therapies have not been developed and multimodal therapy is insufficient, leading to a 5-year survival rate of only 15-25%. Here, we deplete EWSR1::WT1 in DSRCT and for the first time establish its essentiality in vivo. Through transcriptomic analysis, we discover novel mechanistic insights into EWSR1::WT1 functionality including the uniqueness of its transcriptional alterations, the direct role of EWSR1::WT1 binding in gene upregulation, and the dominant role of the one of its two isoforms in transcription. We show that the dominant isoform binds to the CCND1 promoter and stimulates DSRCT growth through the Cyclin D-CDK4/6-RB axis. Treatment with the CDK4/6 inhibitor palbociclib successfully reduced growth in two DSRCT xenograft models. Given palbociclib’s previous approval by the FDA for the treatment of breast cancer, we advance palbociclib as an exciting DSRCT therapy that warrants urgent clinical investigation.

overall_design: DSRCT cell lines with doxycycline-indicuble shRNA against the EWSR1::WT1 fusion gene were cultured for 4-days  with or without doxycycline addition and harvested for analysis by RNA-seq.

middle: B

first: Justin

middle: W

last: Magrath


title: The RNA sequencing results revealed the expression of different genes and signaling pathways during chemotherapy resistance in peripheral T-cell lymphoma

city: Kunming

first: Yunyi

last: Lan

state: Yunnan

address: NO.519,Kunzhou Road,Xishan District

department: Molecular Diagnostic Center

institute: The Third Affiliated Hospital of Kunming Medical University

summary: Background: NK/T cell lymphoma (NKTCL) is a type of non-Hodgkin's lymphoma that occurs primarily extra nodal. The main treatment methods are radiotherapy and chemotherapy. The 5-year recurrence rate is still high, and there is currently no standard treatment for relapse-resistant patients. Compared with other lymphoid tumors, its malignancy is relatively strong, and the prognosis is not very good. Here, we try to identify the key genes and signaling pathways related to chemotherapy resistance in extra nodal NK/T cell lymphoma.  Methods: Firstly, we collect white blood cells from 5 healthy controls and 7 different clinical patients,4 patients were chemotherapy sensitive while 3 patients were chemotherapy resistant. Secondly, after total RNA extraction from those white blood cells, RNA sequence was performed on the Illumina HiSeq platform. Finally, we used qRT-PCR to confirm the expression patterns of different expression genes(DEGs) from the mostly enriched signaling pathways, especially the cancer related genes on the NK/T cell lymphoma clinical individuals. Results: The RNA sequence results displayed 4063 DEGs, including 1128 up regulated and 2935 down regulated genes in the chemotherapy resistance cases of extra nodal NK/T cell lymphoma specimens. Further quantitative gene expression analysis was performed to identify the differential expression pattern in all libraries. And 9 down regulated genes and 10 up regulated genes were confirmed by quantitative real time PCR comparison of chemotherapy resistance in extra nodal NK/T cell lymphoma from 6 clinical specimens. KEGG analysis revealed significant pathway changes, including 6 down regulated and 9 up regulated gene enrichment pathways. The TNF signaling pathway, one of the most extensively regulated, was among the pathways. DEGs and the TNF signaling pathway may be essential in the resistance to chemotherapy in extra nodal NK/T cell lymphoma. Conclusion:Our finding indicated that different genes expression may be important for chemotherapy resistance in extra nodal NK/T cell lymphoma, particularly TNFRSF1B, TRADD2 and MAP3K7.The TNF signaling pathway, a critical signaling pathway, was pinpointed as a potential factor in the emergence of chemotherapy resistance in extra nodal NK/T cell lymphoma.

first: Wei

last: Tao

first: Luyao

last: Ma

first: Xiaoxiong

last: Wang

first: Hongsheng

last: Li

first: Yaxi

last: Du

first: Ruijiao

last: Yang

first: Shunxian

last: Wu

first: Yingxin

last: Ou

first: Xin

last: Liu

first: Yunchao

last: Huang

first: Yongchun

last: Zhou


title: The ALOG domain defines a new family of plant-specific Transcription Factors acting during Arabidopsis flower development.

city: Cassina Rizzardi

first: Veronica

middle: Maria

last: Beretta

state: Co

address: Via A. Vivaldi 36

institute: Università degli Studi di Milano

summary: Analysis of the transcriptome of the reproductive meristems of Arabidopsis thaliana Col-0. We compared the transcriptome obtained from lsh1 lsh3 lsh4 triple mutant to a wild type control

overall_design: sampling reproductive meristems enriched in IM, FM and first stages of flower development until stage 8 from lsh1 lsh3 lsh4 and wild type background. Four independent replicates for each genotype were performed

middle: M

first: Francesca

last: Caselli

first: Chiara

last: Paleni

last: Gregis

first: Martin

last: Kater


title: Effect of TUC-seq protocol versus SLAM-seq on quantification bias in nucleotide conversion RNA-seq data

first: Kevin

last: Berg

summary: To investigate the effect of TUC-seq versus SLAM-seq on quantification bias in nucleotide conversion RNA-seq, we performed SLAM-seq and TUC-seq on NIH cells. We then measured 4sU dropout in 4sU samples compared to 4sU naive samples.

overall_design: Control sample, Uridine treatment (no conversion), 4sU treatment (no conversion), SLAM-seq and TUC-seq on NIH cells in two replicates.

first: Manivel

last: Lodha

first: Isabel

last: Delazer

first: Yiliam

middle: C

last: Garcia

first: Thomas

last: Hennig

first: Elmar

last: Wolf

first: Alexandra

last: Lusser

first: Bhupesh

last: Prusty

first: Florian

last: Erhard


title: Size fractionated NET-Seq reveals a conserved architecture around yeast genes

first: Jane

last: Mellor

address: South Parks Road

department: Biochemistry

summary: Genomes from yeast to human are subject to pervasive transcription. A single round of pervasive transcription is sufficient to alter local chromatin conformation, nucleosome dynamics and gene expression, but is hard to distinguish from background signals. Size fractionated native elongating transcript sequencing (sfNET-Seq) was developed to precisely map nascent transcripts independent of expression levels. RNAPII-associated nascent transcripts are fractionation into different size ranges before library construction. When anchored to the transcription start sites (TSS) of annotated genes, the combined pattern of the output metagenes gives the expected reference pattern. Bioinformatic pattern matching to the reference pattern identified 9542 transcription units in Saccharomyces cerevisiae, of which 47% are coding and 53% are non-coding. 3113 (33%) are unannotated non-coding transcription units. Anchoring all transcription units to the TSS or polyadenylation site (PAS) of annotated genes reveals distinctive architectures of linked pairs of divergent transcripts approximately 200nt apart. The Reb1 transcription factor is enriched 30nt downstream of the PAS only when an upstream (TSS -60nt with respect to PAS) non-coding transcription unit co-occurs with a downstream (TSS+150nt) coding transcription unit and acts to limit levels of upstream antisense transcripts. The potential for extensive transcriptional interference is evident from low abundance unannotated transcription units with variable TSS (median -240nt) initiating within a 500nt window upstream of, and transcribing over, the promoters of protein coding genes. This study confirms a highly interleaved yeast genome with different types of transcription units altering the chromatin landscape in distinctive ways, with the potential to exert extensive regulatory control.

overall_design: Size fractionated NET-Seq was developed to map new transcription units and their putative transcription start sites (TSS) on the S.cerevisiae genome.

first: Shidong

last: Xi

first: Philipp

last: Lorenz

first: Struan

last: Murray

first: Tania

last: Nguyen


title: Effect of 4sU labeling durations on quantification bias in nucleotide conversion RNA-seq data

summary: To investigate the effect of labeling durations on quantification bias in nucleotide conversion RNA-seq, we labeled NIH cells with 4sU. We then measured 4sU dropout in 4sU samples compared to 4sU naive samples.

overall_design: Increasing labeling durations in NIH cells for up to 2 hours in 2 replicates.

last: Cruz Garcia


title: Effect of 4sU labeling on quantification bias in nucleotide conversion RNA-seq data

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus


title: Effect of 4sU labeling concentrations on quantification bias in nucleotide conversion RNA-seq data

summary: To investigate the effect of increasing 4sU labeling concentrations on quantification bias in nucleotide conversion RNA-seq, we labeled 3 different cell lines with increasing concentrations of 4sU. We then measured 4sU dropout in 4sU samples compared to 4sU naive samples.

overall_design: Increasing labeling concentrations (up to 800 µM) in U2OS, HCT116 and HFF-TerT cells for up to 2 hours in 2 replicates.


title: microRNA-33 controls feeding behavior by activating hypothalamic AgRP neurons

city: New Haven

first: PABLO

last: FERNANDEZ-TUSSY

state: CT

address: 10 amistad street, 420C

department: Comparative Medicine

institute: Yale University

summary: miRNA-33a/b provides a critical link between the regulation of cholesterol and fatty acid biosynthesis by SREBPs, and cholesterol efflux, high-density lipoprotein (HDL) biogenesis and fatty acid oxidation pathways. Notably, pharmacological inhibition of miR-33 elevates hepatic ABCA1 expression, thereby increasing circulating HDL-C and attenuating the progression of atherosclerosis, highlighting the therapeutic potential of miR-33 inhibitors for the treatment of cardiovascular disease. However, work with genetic models of miR-33 deficiency has clearly demonstrated that global loss of miR-33 promotes the development of obesity and metabolic dysfunction. We sought to determine if miR-33 is directly involved in regulating the activity of the AgRP and POMC neurons that promote signals of hunger and satiety, respectively. We have generated AgRP conditional KO mouse to analyze the effect of removing miR-33 in these neurons. After miR-33 removal, mice were fed a HFD and then the expression of different markers related to activiation or inhibition of AgRP neurons was analyzed by scRNAseq.

overall_design: Hypothalamus from WT and miR-33 AgRP-CRE mice were dissected after 1 month of 60% HFD. Single cell suspension was prepared and cells were submitted for sc-RNAseq

first: Pablo

last: Fernández-Tussy

first: Carlos

last: Fernández-Hernando


title: Transcriptomics of in vitro brain endothelial cells

city: Rancagua

first: Nicolas

last: Santander

address: Libertador Bernardo O'Higgins 611

country: Chile

institute: Universidad de O´Higgins

summary: Brain endothelial cells have specific characteristics that differentiate them from other vascular beds. We used transcriptomics of mouse and human brain endothelial cells to explore this differences.

overall_design: We obtained total RNA from bEnd3.1 cells as a model of mouse brain endothelium and HBEC cells for human primary brain endothelial cells.

first: Dibyanti

last: Mukherjee

last: Arnold


title: Unraveling the Genetic Landscape of Eye Migration and Visual Adaptation during Flatfish Metamorphosis

city: Vigo

first: Laura

middle: Guerrero

last: Peña

state: Pontevedra

address: Eduardo Cabello, 8

department: Aquaculture and Biotecnology

country: Spain

institute: CSIC

summary: Flatfish have evolved their visual system to meet the demands of their environment through a fascinating process of metamorphosis. This morphological transformation involves a significant change in lifestyle from pelagic to benthic, accompanied by changes in body structure to achieve an asymmetrical flat shape. However, the fundamental change lies in the migration of one eye from its original position to the opposite location on the body. Although the role of thyroid hormone in triggering flatfish metamorphosis is well established, the specific gene regulatory network responsible for this extraordinary eye migration is still unknown. Here we show a global view through the creation of a general map of gene expression during the metamorphic process, encompassing both migrant and non-migrant eyes. Our research uncovers significant differences between migrant and non-migrant eyes of turbot in the pre-metamorphic and climax phases, emphasizing genetic disparities crucial for benthic adaptation.

overall_design:
To investigate differences in gene expression during flatfish metamorphosis we selected three key stages in turbot development: pre-metamorphic stage (15 dpf); climax of metamorphosis (30dpf); and post-metamorphic stage (57dpf). These fish were reared under standard production conditions at 18ºC.
The migrant and non-migrant eyes and their respective surrounding tissue were directed for each stage and RNA were extracted. Libraries were sequenced on a BGISEQ-500 platform and single-end reads of 50 base pairs length were generated per sample.

last: Guerrero-Peña

first: Paula

last: Suarez-Bregua

first: Lucía

last: Sánchez-Ruiloba

first: Luis

last: Méndez-Martínez

last: García-Fernández

first: Ricardo

last: Tur

first: Juan

middle: J

last: Tena

first: Josep

last: Rotllant


title: Transgenic tools targeting striatal and pallidal subpopulations reveal both evolutionary conservation and specialization of cortico-basal ganglia circuits in zebrafish

status: Public on Feb 05 2024

city: Wako

first: Yuki

last: Tanimoto

state: Saitama

address: 2-1 Hirosawa

department: CBS

country: Japan

institute: RIKEN

summary: The cortico-basal ganglia circuit mediates decision-making. Here, we generated transgenic tools for adult zebrafish targeting specific subpopulations of the components of this circuit and utilized them to identify evolutionary homologs of the mammalian direct- and indirect-pathway striatal neurons which respectively project to the homologs of the internal and external segment of the globus pallidus (dEN and Vl) as in mammals. Unlike in mammals, the Vl mainly projected to the dEN directly, not by way of the subthalamic nucleus. Further single-cell RNA sequencing analysis revealed two pallidal output pathways: a major shortcut pathway directly connecting the dEN with the pallium and the evolutionarily conserved closed loop by way of the thalamus. Our resources and circuit map provide the common basis for the functional study of the basal ganglia in a small and optically tractable zebrafish brain for the comprehensive mechanistic understanding of the cortico-basal ganglia circuit.

overall_design: To identify genetic markers of the npy-negative thalamus-projecting neurons in the dEN, we conducted single-cell RNA sequencing analysis. We dissected the dEN and its surrounding regions from six adult individuals of TgBAC(npy:GAL4VP16);Tg(UAS:GFP). We then dissociated the dissected tissue and used the droplet-based 3’end scRNAseq system Chromium (10x Genomics). We obtained transcriptomic data from 3,381 cells and performed unbiased clustering by Seurat.

first: Hisaya

last: Kakinuma

first: Hitoshi

last: Okamoto


title: Smarca4 (Brg1) is a haploinsufficient B-cell tumor suppressor that fine-tunes centrocyte cell fate decisions [raji_R1192C_atac]

city: Houston

first: Michael

last: Green

state: Texas

address: 7455 Fannin Street

department: Department of Lymphoma/Myeloma

institute: The University of Texas MD Anderson Cancer Center

summary: SMARCA4 encodes one of two mutually-exclusive ATPase subunits in the Brg/Brm associated factor (BAF) complex that is recruited by transcription factors to drive chromatin accessibility and transcriptional activation. SMARCA4 is among the most recurrently mutated genes in human cancer, including ~30% of germinal center (GC)-derived Burkitt lymphomas. In mice, GC-specific Smarca4 haploinsufficiency cooperates with Myc over-expression to drive lymphomagenesis. Furthermore, monoallelic Smarca4 deletion drove GC hyperplasia with centroblast polarization via significantly increased rates of centrocyte recycling to the dark zone. Mechanistically, Smarca4 loss reduced the activity of transcription factors that are activated in centrocytes to drive GC-exit, including SPI1 (PU.1), IRF family and NFκB. Loss of activity for these factors phenocopied aberrant Bcl6 activity within murine centrocytes and human BL cells. Smarca4 therefore facilitates chromatin accessibility for transcription factors that shape centrocyte trajectories, and loss of fine-control of these programs biases towards centroblast cell-fate and GC hyperplasia.

overall_design: Chromatin accessibility analysis by comparing SMARCA4 WT or R1192C mutant in a human BL cell line


title: Smarca4 (Brg1) is a haploinsufficient B-cell tumor suppressor that fine-tunes centrocyte cell fate decisions [smarca4_atac]

overall_design: Dynamics of genome-wide chromatin accessibility in Smarca4 knockout and WT mouse GC B-cells


title: Smarca4 (Brg1) is a haploinsufficient B-cell tumor suppressor that fine-tunes centrocyte cell fate decisions [rajiAUD_cutrun]

overall_design: Chromatin oocupancy analysis of PU.1 upon SMARCA4 degradation in an established auxin-inducible degron system


title: Smarca4 (Brg1) is a haploinsufficient B-cell tumor suppressor that fine-tunes centrocyte cell fate decisions [pbrm1_atac]

overall_design: Map genome-wide chromatin accessibility for PBAF-specific subunit Pbrm1 knockout in mouse GC B-cell


title: Differentiation Shifts from a Reversible to an Irreversible Heterochromatin State at the DM1 locus

city: jerusalem

first: Rachel

last: Eiges

address: Shmu'el Bait St 12, Jerusalem, 9103102

department: Genetics

country: Israel

institute: Shaare Zedek medical center

summary: Epigenetic defects caused by hereditary or de novo mutations are implicated in various human diseases. It remains uncertain whether correcting the underlying mutation can reverse these defects in patient cells. Focusing on myotonic dystrophy type 1 (DM1), we discovered a fundamental difference between undifferentiated and differentiated cells. While in mutant human embryonic stem cells (hESCs), DNA methylation and H3K9me3 enrichments are completely abolished by repeat excision (2000CTG), in patients' myoblasts (CTG2600 expansion) repeat deletion fails to do so. This distinction stems from cell differentiation, and can be set back by reprogramming gene-edited myoblasts. We demonstrate that abnormal methylation in DM1 is distinctively maintained in the undifferentiated state by the activity of the de novo DNMTs (DNMT3b and/or DNMT3a). Overall, these findings highlight a crucial difference in heterochromatin maintenance between undifferentiated (sequence-dependent) and differentiated (sequence-independent) cells, underscoring the role of differentiation as a locking mechanism for repressive epigenetic modifications at the DM1 locus.

overall_design:
Total RNA was extracted using NucleoSpin RNA Plus kit (Macherey-Nagel, Cat # 740984.50) following the manufacturer's instructions. RNA was quantitated by NanoDrop 200 (ThermoFisher). RNA libraries were prepared for sequencing using standard Illumina protocols.
Genomic DNA (2 µg) was modified by bisulfite treatment (EZ DNA methylation kit, Zymo Research) and amplified by FastStart DNA polymerase (Roche). PCR was carried out using 5? primer: 5?-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGTGGTTGTGGGTTAGTGTT-3?, and 3? primer: 5?-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCCCAACAACCTACAACTATTAT-3? (DMPK-specific sequences are underlined; adaptors for library preparation are at the 5? end of each primer). Amplified products were quality controlled for size using the D1000 ScreenTape kit; Agilent Technologies) and for concentration using the Qubit® DNA HS Assay kit (catalog #32854; Invitrogen). Subsequently, the PCR products were bead purified with Agencourt Ampure XP beads (Beckman Coulter) according to the manufacturer?s protocol. Next, amplicons were subjected to a second PrimMax Takara PCR reaction (1st purified PCR DNA (7.5 ?l), 2.5 ?l N7XX primer (nextera barcode 1), 2.5 ul S5XX primer (nextera barcode 2), 12.5 ?l 2x Primstar ReadyMix). PCR program: 98°C for 1 minute followed by 8 cycles of 98°C for 10 seconds, 55°C for 10 seconds, 72°C for 30 seconds, then 72°C for 5 minutes and finally held at 10°C. The 2nd PCR was bead purified, quality controlled using the D1000 ScreenTape kit and the Qubit® DNA HS Assay kit, and all resultant libraries were normalized and pooled at a 10 nM concentration. Denaturation and dilution of the sequencing pool were performed according to the standard Illumina protocol. Ultimately, 1.5 pM of pool (combined with 40% spiked?in) was loaded onto a NextSeq 500 Mid?Output Kit (150 cycles) cartridge (catalog #FC?102?1001; Illumina, San Diego, CA) for high throughput sequencing on a NextSeq 500 instrument (Illumina), with 150 cycle, single?read run conditions.


title: Smarca4 (Brg1) is a haploinsufficient B-cell tumor suppressor that fine-tunes centrocyte cell fate decisions [ca46_atac]


title: Smarca4 (Brg1) is a haploinsufficient B-cell tumor suppressor that fine-tunes centrocyte cell fate decisions [raji_R1192C_rna]

overall_design: Gene expression profiling analysis by comparing SMARCA4 WT or R1192C mutant in a human BL cell line


title: Smarca4 (Brg1) is a haploinsufficient B-cell tumor suppressor that fine-tunes centrocyte cell fate decisions [smarca4_rna]

overall_design: Compararive gene expression profiling analysis using WT or Smarca4 HET knockout in mouse GC B-cell


title: Smarca4 (Brg1) is a haploinsufficient B-cell tumor suppressor that fine-tunes centrocyte cell fate decisions [rajiAUD_rna]

overall_design: Gene expression analysis of SMARCA4 protein degradation in an established auxin-inducible degron system


title: Smarca4 (Brg1) is a haploinsufficient B-cell tumor suppressor that fine-tunes centrocyte cell fate decisions [rajiAUD_atac]

overall_design: Dynamic chromatin accessibility analysis of SMARCA4 protein degradation in an established auxin-inducible degron system


title: Smarca4 (Brg1) is a haploinsufficient B-cell tumor suppressor that fine-tunes centrocyte cell fate decisions [rajiAUD_FX1_rna]

overall_design: Gene expression profiling analysis by comparing FX1 treatment vs non-treatment upon SMARCA4 protein degradation in an established auxin-inducible degron system.


title: Smarca4 (Brg1) is a haploinsufficient B-cell tumor suppressor that fine-tunes centrocyte cell fate decisions

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

overall_design: Refer to individual Series


title: Smarca4 (Brg1) is a haploinsufficient B-cell tumor suppressor that fine-tunes centrocyte cell fate decisions [pbrm1_rna]

overall_design: Compararive gene expression profiling analysis using WT or Pbrm1 knockout in mouse GC B-cell


title: Smarca4 (Brg1) is a haploinsufficient B-cell tumor suppressor that fine-tunes centrocyte cell fate decisions [ca46_rna]


title: Serine/arginine-rich splicing factor 7 (SRSF7) promotes the type I interferon response by activating Irf7 transcription

city: Bryan

first: Kristin

middle: L

last: Patrick

address: 8447 Riverside Pkwy, MREB 3102

department: Microbial Pathogenesis and Immunology

institute: Texas A&M University, College of Medicine

summary: Tight regulation of macrophage immune gene expression is required to fight infection without risking harmful inflammation. The contribution of RNA binding proteins (RBPs) to shaping the macrophage response to pathogens remains poorly understood. Transcriptomic analysis revealed that a member of the serine/arginine-rich (SR) family of mRNA processing factors, SRSF7, is required for optimal expression of a cohort of interferon stimulated genes (ISGs) in macrophages. Using genetic and biochemical assays, we discovered that in addition to its canonical role in regulating alternative splicing, SRSF7 drives transcription of interferon regulatory transcription factor 7 (IRF7) to promote antiviral immunity. At the Irf7 promoter, SRSF7 maximizes STAT1 transcription factor binding and RNA polymerase II elongation via cooperation with the H4K20me1 histone methyltransferase KMT5a (SET8). These studies define an unorthodox role for an SR protein in activating transcription and reveal an unappreciated RNA binding protein-chromatin network that orchestrates macrophage antiviral gene expression.

overall_design: The goal of the experiment was to determine the contribution of IRF7 to basal and induced type I IFN expression in RAW 264.7 macrophages. RAW 264.7 macrophages expressing Cas9 were transduced with lentivirus expressing either a GFP gRNA (untargeted, control) or a gRNA targeted to exon 3 of Irf7 (KO1 and KO2). Knockout of IRF7 was confirmed by immunoblot. RNA was isolated from GFP gRNA and Irf7 KO cell lines at baseline (UN) and after 4h LPS (100ng/ml) using the Zymo Direct-Zol RNA purification kit and eluted in nuclease-free water.

first: Haley

last: Scott

first: Mackenzie

last: Smith

first: Aja

last: Coleman

first: Kaitlyn

last: Armijo

first: Morgan

last: Chapman

first: Summer

last: Apostalo

first: Allison

last: Wagner

first: Robert

last: Watson


title: Smarca4 (Brg1) is a haploinsufficient B-cell tumor suppressor that fine-tunes centrocyte cell fate decisions [scMultiome]

summary: SMARCA4 encodes one of two mutually-exclusive ATPase subunits in the Brg/Brm associated factor (BAF) complex that is recruited by transcription factors to drive chromatin accessibility and transcriptional activation. SMARCA4 is among the most recurrently mutated genes in human cancer, including ~30% of germinal center (GC)-derived Burkitt lymphomas. In mice, GC-specific Smarca4 haploinsufficiency is able to cooperate with Myc over-expression to drive lymphomagenesis. Furthermore, monoallelic Smarca4 deletion drove GC hyperplasia with centroblast polarization via significantly increased rates of centrocyte recycling to the dark zone. Mechanistically, Smarca4 loss reduced the activity of transcription factors that are activated in centrocytes to drive GC-exit, including SPI1, IRF4 and NFκB. Loss of activity for these factors drove aberrant Bcl6 activity within murine centrocytes and human BL cells. Smarca4 therefore facilitates chromatin accessibility for transcription factors that shape centrocyte trajectories, and loss of fine-control of these programs biases towards centroblast cell-fate and GC hyperplasia.

overall_design: GC B cells were isolated from mice spleens at day 10 post-immunization using the Germinal Center B Cell (PNA) MicroBead Kit (Miltenyi Biotec,130-110-479), and sorted by FACS, followed by single-cell multiome analysis


title: Sildenafil as a candidate drug for Alzheimer’s disease: Real-world patient data observation and mechanistic observations from patient-induced pluripotent stem cell-derived neurons.

city: CLEVELAND

first: Feixiong

last: Cheng

state: OH

address: 9620 Carnegie Ave.

department: Genome center

institute: The Cleveland Clinic Lerner Research Institute

summary: Alzheimer’s disease (AD) is a chronic neurodegenerative disease needing effective therapeutics urgently. Sildenafil, one of the approved phosphodiesterase-5 inhibitors, has been implicated as having potential beneficial effect in AD. We showed that sildenafil usage is associated with reduced likelihood of AD across four new drug compactor cohorts, including bumetanide, furosemide, spironolactone, and nifedipine. For instance, sildenafil usage is associated with a 54% reduced prevalence of AD in MarketScan® (hazard ratio [HR] = 0.46, 95% CI 0.32-0.66) and a 30% reduced prevalence of AD in Clinformatics® (HR = 0.70, 95% CI 0.49-1.00) compared to spironolactone. We found that sildenafil treatment significantly reduced tau hyper-phosphorylation (pTau181, pTau205) in a dose-dependent manner in both familial and sporadic AD patient derived neurons. Further RNA-seq data analysis of sildenafil-treated AD patient iPSC-derived neurons revealed that sildenafil specifically targeting AD related genes and molecular pathways involved in axon guidance, AD-presenilin, neurogenesis, neurodegeneration, synaptic dysregulation, vascular smooth muscle contraction (VSMC) and cyclic guanosine monophosphate (cGMP)-protein kinase G (PKG) signaling pathway, mechanistically supporting the potential beneficial effect of sildenafil in AD. These real-world patient data validation and mechanistic observations from patient iPSC-derived neurons further suggested that sildenafil is a potential repurposable drug for AD. However, randomized clinical trials are required to validate sildenafil as a potential treatment of AD.

overall_design:
To investigate the potential therapeutic benefit of sildenafil on AD outcome, we used both familial and sporadic AD patient induced pluripotent stem cells (iPSC) derived neurons to evaluate the sildenafil’s mechanism-of-action using ELISA, Western blotting, and RNA-sequencing analysis.
We performed comparative gene expression profiling analysis of RNA-seq data for a male FAD patient derived iPSCs treated with and without sildenafil (100 µM) in replicates.

first: Dhruv

last: Gohel

first: Amit

last: Gupta


title: WWP2 MEDIATES THE METABOLIC REPROGRAMMING OF RENAL MYOFIBROBLASTS TO PROMOTE KIDNEY FIBROSIS [bulk RNA-seq]

city: Singapore

first: Jing

last: Guo

state: State...

address: 8 Collage Road, DUKE-NUS Medical School, level 5

department: Center for Computational Biology

institute: Duke-NUS Medical School

summary: Renal fibrosis is a common pathological endpoint that is challenging to reverse in chronic kidney disease (CKD) independently of the underlying causes. Although myofibroblasts are mainly responsible for the accumulation of a fibrillar collagen-rich extracellular matrix (ECM), recent reports revealed their heterogeneity in proliferative and fibrotic activities, mirroring specific metabolic states that drive fibrosis. Here, we investigate the role of E3 ubiquitin-protein ligase WWP2 in the metabolic reprogramming of renal myofibroblasts in fibrosis. The tubulointerstitial expression of WWP2 contributes to the progression of fibrosis in CKD patients and in pre-clinical models of CKD. WWP2 deficiency leads to increased fatty acid oxidation, boosting mitochondrial respiration, promoting myofibroblast proliferation and arresting pro-fibrotic activation, thus ameliorating kidney fibrosis. Specifically, WWP2 suppresses the transcription of PGC-1α, which mediates the metabolic and proliferative changes in fibrotic myofibroblasts. Pharmacological intervention targeting PGC-1α reverses the pro-fibrotic effect of WWP2. These findings reveal a previously unappreciated WWP2-PGC-1α axis underlying the metabolic reprogramming of myofibroblasts during renal fibrosis, which could provide a new target for therapeutic intervention in CKD.

overall_design: RNA-seq analysis of fibroblast derived from wildtype and WWP2-/- mouse kidney with and without TGFβ1 treatment.

first: Huimei

last: Chen

first: Gabriel

last: Chow

first: Enrico

last: Petretto


title: WWP2 MEDIATES THE METABOLIC REPROGRAMMING OF RENAL MYOFIBROBLASTS TO PROMOTE KIDNEY FIBROSIS [scRNA-seq]

overall_design: Kidneys from wwp2 knockout and wildtype mice were collected and processed according to 10X Genomics Chromium protocol for single-cell RNAseq.

last: G Petretto


title: WWP2 MEDIATES THE METABOLIC REPROGRAMMING OF RENAL MYOFIBROBLASTS TO PROMOTE KIDNEY FIBROSIS


title: Effect of Morc1 knock out on gonocyte chromatin and transcriptome [RNA-Seq 2]

city: Bunkyo-ku

first: Soichiro

last: Yamanaka

institute: The University of Tokyo

summary: To investigate the function of Morc1 to chromatin state during gonocyte stage when around 10,000 copies of transposons are deregulated.

overall_design: We performed ATAC-seq, RNA-seq, ChIP-seq (for H3K9me3) using gonocyte at four developmental timepoints (E13.5, E16.5, P0, P3) during gonocyte stage

first: Yuta

last: Uneme


title: The CoREST Repressor Complex Mediates Phenotype Switching and Therapy Resistance in Melanoma

city: Boston

last: Fisher

state: MA

address: 609 Albany St

department: Dermatology

institute: Boston University


title: Effect of Morc1 knock out on gonocyte chromatin and transcriptome [RNA-Seq 1]


title: Effect of Morc1 knock out on gonocyte chromatin and transcriptome [ChIP-Seq]


title: Effect of Morc1 knock out on gonocyte chromatin and transcriptome


title: Effect of Morc1 knock out on gonocyte chromatin and transcriptome [ATAC-Seq]


title: Let-7 miRNAs repress HIC2 to regulate BCL11A transcription and hemoglobin switching [ChIP-Seq]

city: Guangzhou

first: Peng

last: Huang

state: Guangdong

address: Xinzao, Panyu District

department: GMU-GIBH Joint School of Life Sciences

institute: Guangzhou Medical University

summary: The fetal hemoglobin (HBG) regulator BCL11A is repressed by HIC2 to regulate hemoglobin switching. HIC2 itself is under developmental control, highly expressed in fetal tissues and decreased in adult cells, but the mechanism of this developmental regulation is unclear. Here, we demonstrate that HIC2 is developmentally controlled by miRNAs, and loss of global miRNA biogenesis through DICER1 depletion leads to upregulation of HIC2 and HBG. We subsequently identify the adult-expressed let-7 miRNA family as a repressor of HIC2 expression. Ectopic expression of let-7 in fetal-type cells repressed HIC2 levels, while let-7 inhibition in adult erythroblasts upregulated HIC2 levels. Let-7 regulates BCL11A and HBG through induction of HIC2 and reduction of GATA1 binding at the BCL11A erythroid enhancer. Finally, HIC2 depletion rescues BCL11A-mediated repression of fetal hemoglobin in let-7 inhibited cells. Together these data demonstrate a key miRNA-mediated mechanism for developmental regulation of BCL11A expression and fetal hemoglobin repression in adult erythroid cells.

overall_design: Experiments were performed with 2 replicates from independent donors. Scramble samples were used as controls.


title: Effect of USB1 depletion on global miRNome in human embryonic stem cells and K562

city: boulder

first: Roy

last: parker

address: colorado ave

institute: university of colorado boulder

type: Non-coding RNA profiling by high throughput sequencing

summary: USB1 mutations lead to poikiloderma with neutropenia. We investigated the effect of inactivating USB1 on the global miRNome in undifferentiated hESCs and K562

overall_design: WT/USB1 mutant hESCs and WT/USB1 KO K562 cells were harvested and total RNA was used for miRNome sequencing

first: Ho-Chang

last: Jeong

first: Siddharth

last: Shukla

first: Thao

last: Huynh


title: Let-7 miRNAs repress HIC2 to regulate BCL11A transcription and hemoglobin switching [RNA-Seq]


title: Enhanced genome-wide knockout screens in bacteria with CRISPR base editors

city: Wuerzburg

first: Chase

last: Beisel

address: Josef-Schneider-Str. 2 / Bau D15

institute: Helmholtz Institute for RNA-based Infection Research

summary: Genome-wide knockout or knockdown screens have become powerful tools for the investigation of genotype-to-phenotype relationships. In bacteria, these screens commonly rely on transcriptional repression by dCas9, gene knockouts through Cas9 editing or random transposon mutagenesis, but depending on the technique, suffer from incomplete gene silencing, low editing efficiencies or they require massive library sizes. Here, we take a distinct approach with base editing to introduce premature stop codons or mutate start codons in Escherichia coli using a ScCas9 nickase derived base editor (ScBE3) that exhibits flexible PAM recognition. We then derive guide design rules by applying machine learning to a gene essentiality screen conducted in E. coli. For further improvement, we combined base-editing with Cas9-induced cleavage of the unedited cell fraction. The efficiency of this dual system was validated through a screen of conditionally essential E. coli genes. This improved setup that decouples the gene editing from the screening leads to more efficient guide depletion and confirmed previously published conditionally essential genes. Overall, base editing represents a useful tool for genome-wide knockout screens in bacteria and will eventually enable genome-wide knockout screens in a broader range of bacterial species to study their diverse genetics.

overall_design: Illumina sequencing reads from the genome-wide essentiality screen in E. coli; samples include the extracted library DNA from experiments with two base editor variants (sample label “s” and “e”) and 4 time points each (t1-4). The experiment was conducted in duplicate (e.g. e1 and e2). Illumina sequencing reads from the dual-sgRNA screen in E. coli; sample include the initial library, the extracted library after culturing in LB medium, after the base editing step, after the killing step and after screening in MOPS and M9 minimal media. Except for the initial library reads, all sample reads are provided in duplicates.

first: Sandra

last: Gawlitt

first: Scott

middle: P

last: Collins

first: Yanying

last: Yu

first: Sam

last: Blackman

first: Lars

last: Barquist


title: Improving prime editing with an endogenous small RNA-binding protein

status: Public on Feb 04 2024

city: San Francisco

first: Carl

last: Ward

address: 1650 Owens Street

department: Gladstone-UCSF Institute of Genomic Immunology

institute: Gladstone Institutes

summary: Prime editing allows precise modification of genomes. To identify cellular determinants of prime editing, we developed scalable prime editing reporters and performed genome-scale CRISPR-interference screens. From these screens, a single factor emerged as the strongest mediator of prime editing: the small RNA-binding exonuclease protection factor La (SSB). Further investigation revealed that La promotes prime editing across approaches (PE2, PE3, PE4, PE5), edit types (substitutions, insertions, deletions), guide RNA designs (pegRNAs, epegRNAs), endogenous loci, and cell types, but has no consistent effect on genome editing approaches that rely on standard, unextended guide RNAs. La binds polyuridine tracts at the 3' ends of RNA polymerase III transcripts and protects those transcripts from cellular exonucleases. We found that La stabilizes (e)pegRNAs, with particular effect on their 3' extensions, where edits are encoded. Guided by these insights, we developed a prime editing protein (PE7) fused to the RNA-binding, N-terminal domain of La. This editor improved prime editing with expressed (e)pegRNAs and synthetic pegRNAs optimized for La binding. Our results provide key insights into how prime editing components interact with the cellular environment and suggest general strategies for stabilizing exogenous small RNAs therein.

overall_design:
Primary human T cells were electroporated with PE7 or PEmax mRNA and synthetic pegRNAs with different 3' end configurations.
Related to GSE253424.

first: Jun

last: Yan

first: Paul

last: Oyler-Castrillo

first: Purnima

last: Ravisankar

first: Sébastien

last: Levesque

first: Yangwode

last: Jing

first: Danny

last: Simpson

first: Anqi

last: Zhao

first: Hui

last: Li

first: Weihao

first: Laine

last: Goudy

first: Ralf

last: Schmidt

first: Sabrina

last: Solley

first: Luke

last: Gilbert

first: Michelle

last: Chan

first: Daniel

middle: E

last: Bauer

first: Alexander

last: Marson

first: Lance

middle: R

last: Parsons

first: Britt

last: Adamson


title: Let-7 miRNAs repress HIC2 to regulate BCL11A transcription and hemoglobin switching [ATAC-Seq]

first: Peng

last: Huang


title: Let-7 miRNAs repress HIC2 to regulate BCL11A transcription and hemoglobin switching [Capture-C]

overall_design: Experiments were performed with 4 replicates. Scramble samples were used as controls.


title: Ciliopathy patient variants reveal organelle-specific functions for TUBB4B in axonemal microtubules

city: Edinburgh

middle: Oliver

last: Dodd

address: Western General Hospital  Crewe Road South

department: Institute of Genetics and Cancer

institute: University of Edinburgh

summary: Tubulin, one of the most abundant cytoskeletal building blocks, exhibits extensive isotype diversity in humans. Displaying high similarity, whether these distinct isotypes form cell-type and context specific microtubule structures is poorly understood. Studying a cohort of 11 patients with the motile ciliopathy primary ciliary dyskinesia as well as mouse mutants, we report mutations in the TUBB4B isotype specifically perturb centriole and cilium biogenesis. We demonstrate that distinct TUBB4B mutations differentially affect microtubule dynamics and cilia formation in a dominant negative manner. Finally, structure-function studies reveal that different TUBB4B mutations disrupt distinct tubulin interfaces allowing clear stratification of patients into three classes of ciliopathic disease. These findings illustrate that specific tubulin isotypes have unique and non-redundant subcellular functions and establishes the missing link between human tubulinopathies and ciliopathies.

overall_design: To investigate whether the loss of TUBB4B protein was due to an effect at the RNA level and to see what effect this loss had on the overall trancriptional landscape of multi-ciliated cells, especially on other beat tubulin isotypes which could theoretically compensate for the loss we sequenced the whole transcriptomes of mouse tracheas from  5 WT, 4 HET and 3 Hom mice.


title: Investigating the impact of antibiotic-induced dysbiosis on protection from C. difficile colitis by mouse colonic Innate Lymphoid Cells

city: CHARLOTTESVILLE

first: Md Jashim

last: Uddin

state: VA

address: 257 COLONNADE DR APT 12

institute: University of Virginia

summary: Innate lymphoid cells (ILCs) play a critical role in maintaining intestinal health in homeostatic and diseased conditions. During C. difficile infection (CDI), IL-33 activates ILC2 to protect from colonic damage and mortality. The function of IL-33 and ILC is tightly regulated by the intestinal microbiota. We set out to determine the impact of antibiotic-induced disruption of the microbiome on ILC function. Our goal was to understand antibiotic-induced changes in ILC function on susceptibility to C. difficile colitis in a mouse model. We utilized high-throughput single-cell RNAseq to investigate the phenotypic features of colonic ILC at baseline, after antibiotic administration with or without IL-33 treatment. We identified a heterogeneous landscape of colonic ILCs with gene signatures of inflammatory, anti-inflammatory, migratory, progenitor, plastic, and antigen-presenting ILC. Antibiotic treatment decreased ILC2 while coordinately increasing ILC1 and ILC3 phenotypes. Notably, Ifng+, Ccl5+, and Il23r+ ILC increased after antibiotics. IL-33 treatment counteracted the antibiotic effect by downregulating ILC1 and ILC3 and activating ILC2. In addition, IL-33 treatment markedly induced the expression of type 2 genes, including Areg and Il5. Finally, we identified amphiregulin, produced by ILC2, as protective duringC. difficileinfection. Together, our data expand our understanding of how antibiotics induce susceptibility to C. difficile colitis through their impact on ILC subsets and function.

overall_design: Here, samples were mouse colon ILCs. We flow-sorted colon ILCs at three different conditions. Conditions were -No Abx (Baseline), Abx (mice were administered an antibiotic cocktail -45 mg/L Vancomycin, 35 mg/L Colistin, 35 mg/L Gentamycin, and 215 mg/L Metronidazole, in drinking water for three days and followed by ip injection of Clindamycin, 0.016mg/g of body weight), Abx+IL-33. We pooled samples from 10 mice in each condition. The whole colon from mice was dissected and opened longitudinally, followed by washing with PBS. Tissues were then left in buffer 1 (HBSS with 25 mM HEPES and 5% FBS) until harvesting all the mice. Colon tissues were incubated at 37°C temperature with gentle shaking for 40 min to remove the epithelial layer into 40 ml Buffer 2 (HBSS with 15 mM HEPES, 5 mM EDTA, 10% FBS, and 1 mM DTT). Tissue sections were cut into small pieces with a fine scissor and incubated into 5 ml digestion buffer (RPMI 1640 containing 0.17 mg/ml liberase, 30 mg/ml DNase) for 30-35 minutes at 37°C temperature with gentle shaking. Solutions were then passed through 100-micron and 40-micron cell strainers to collect single-cell suspensions. Suspensions were centrifuged at 500g for 5 minutes to pellet cells, followed by resuspension into 300 ml FACS buffer (2% FBS in PBS). From the cell suspensions, ILCs were enriched using a mouse Lineage cell depletion kit (Milteny Biotec) following the manufacturer's protocol

first: Md

first: Jhansi

last: Leslie

first: Brandon

last: Thompson

first: Casey

last: Fishman

first: Katia

last: Sol-church

first: Pamkaj

last: Kumar

first: William

last: Petri Jr.


title: Identification of TREM1+CD163+ myeloid cells as a deleterious immune subset in HCC [Spatial Transcriptomics]

city: Laval

first: Maya

last: Saleh

state: Qc

address: 531 Boulevard des Prairies

department: Armand Frappier Santé Biotechnologie

country: Canada

institute: INRS

summary: Hepatocellular carcinoma (HCC) is an inflammation-associated cancer arising from viral and non-viral etiologies. Expansion of suppressive myeloid cells is a hallmark of chronic inflammation and cancer, but their heterogeneity in HCC is not fully resolved and might underlie immunotherapy resistance in the steatohepatitis setting. Here, we present a high resolution atlas of hepatic innate immune cells from patients with HCC that unravels a steatohepatitis contexture characterized by influx of inflammatory and immunosuppressive myeloid cells. A discrete myeloid cell population identified by selective expression of TREM1 and CD163 expands in steatohepatitis-HCC. We refer to this population as TREM1+ regulatory myeloid cells (Mreg), as it potently suppresses T cell effector functions, highly expresses TGFB1 and IL13RA1 and localizes to HCC fibrotic lesions.

overall_design: Single-cell RNA-seq (10x Genomics) on FACS-sorted CD45 + panTCRαβ - CD19 - innate immune cells freshly isolated from tumors (HCC) and adjacent non-tumoral (NT) liver sections were realized for 10 patients (10 HCC/CHC samples and 10 NT/TS samples). In parallel, we applied spatial transcriptomics (ST; Visium ® , 10x Genomics) on frozen tissue sections from two of the previous patients (2 CHC visium samples). Furthermore, bulk RNA-seq sequencing were performed on innate immunity cells that were isolated using the same sorting strategy designed for scRNAseq experiments, for six of the previous patients. Bulk RNA-seq sequencing were also realized on FACS-sorted cell population, namely THBS1_M (c0, c3 and c4) populations that were isolated according to specific sorting strategy. In summary, available data concerns 20 scRNA-seq samples, 2 visium spatial transcriptomics and 9 bulk RNA-seq.

first: Julie

last: Giraud

first: Domitille

last: Chalopin

first: Eloïse

last: Ramel

first: Thomas

last: Boyer

first: Atika

last: Zouine

first: Marie-Alix

last: Derieppe

first: Nicolas

last: Larmonier

first: Olivier

last: Adotevi

first: Brigitte

last: Le Bail

first: Jean-Frédéric

last: Blanc

first: Christophe

last: Laurent

first: Laurence

last: Chiche

first: Marc

last: Derive

first: Macha

last: Nikolski


title: Identification of TREM1+CD163+ myeloid cells as a deleterious immune subset in HCC