title: Effect of Endogenous Stimuli-Responsive Separating Microneedles to Inhibit Hypertrophic Scar on Rabbit Ear

status: Public on Feb 04 2024

first: Zhuo-ran

last: Yang

address: No. 1037 Luoyu Road, Hongshan District

department: Hubei Engineering Research Center for Biomaterials and Medical Protective Materials, School of Chemistry and Chemical Engineering

institute: Huazhong University of Science and Technology (HUST)

summary: Hypertrophic scar (HS) considerably affects the appearance and causes tissue dysfunction in patients. Here we show a separating microneedle (MN) consisting of photo-crosslinked GelMA and 5-FuA-Pep-MA prodrug in response to high reactive oxygen species (ROS) levels and overexpression of matrix metalloproteinases (MMPs) in the HS pathological microenvironment. Bulk and single cell RNA sequencing analyses confirm that drug-loaded MNs could reverse skin fibrosis through down-regulation of BCL-2-associated death promoter (BAD), insulin-like growth factor 1 receptor (IGF1R) pathways, simultaneously regulate inflammatory response and keratinocyte differentiation via up-regulation of toll-like receptors (TOLL), interleukin-1 receptor (IL1R) and keratinocyte pathways, and promote the interactions between fibroblasts and keratinocytes via ligand-receptor pair of proteoglycans 2 (HSPG2)-dystroglycan 1(DAG1). This study reveals the potential therapeutic mechanism of drug-loaded MNs in HS treatment and presents a broad prospect for clinical application.

overall_design: To elucidate the RNA changes associated with drug-loaded MNs on HS, we performed single cell RNA sequencing (scRNA-seq) analysis using rabbit ear HS tissues and HS tissues with drug-loaded MNs. Comparative gene expression profiling analysis was performed.

first: Zhuoran

first: Huinan

last: Suo

first: Hao

last: Jiang

first: Juan

last: Tao


title: The global RNA-RNA interactome of Klebsiella pneumoniae unveils a small RNA regulator of cell division (RIL-Seq)

status: Public on Feb 05 2024

city: Jena

first: Eric

last: Ruhland

address: Winzerlaer Str. 2

country: Germany

institute: Friedrich Schiller University Jena

summary: The ubiquitous RNA chaperone Hfq is involved in the regulation of key biological processes in many species across the bacterial kingdom. In the opportunistic human pathogen Klebsiella pneumoniae, deletion of the hfq gene affects the global transcriptome, virulence, and stress-resistance, however, the ligands of the major RNA-binding protein in this species have remained elusive. In this study, we have combined transcriptomic, co-immunoprecipitation and global RNA interactome analyses to compile an inventory of conserved and species-specific RNAs bound by Hfq, and to monitor Hfq-mediated RNA-RNA interactions. In addition to dozens of previously unknown RNA-RNA pairs, our study revealed an Hfq-dependent sRNA, DinR, which is processed from the 3’ terminal portion of dinI mRNA. Transcription of dinI is controlled by the master regulator of the SOS response, LexA. As DinR accumulates in K. pneumoniae in response to DNA damage, the sRNA represses translation of the ftsZ transcript by occupation of the ribosome binding site. Ectopic overexpression of DinR causes depletion of ftsZ mRNA and inhibition of cell division, while deletion of dinR antagonizes cell elongation in the presence of DNA damage. Collectively, our work highlights the important role of RNA-based gene regulation in K. pneumoniae and uncovers the central role of DinR in LexA-controlled cell cycle inhibition during the SOS response.

overall_design: RNA-RNA interactions on HFQ were analyzed. For that, RIL-seq with Klebsiella pneumoniae MGH78578 wt or hfq-3XFLAG cells was performed.

first: Kathrin

middle: S

last: Fröhlich

first: Malte

last: Siemers


title: Distinct functions for the paralogous RBM41 and U11/U12-65K proteins in the minor spliceosome

city: Helsinki

first: Mikko

last: Frilander

address: Viikinkaari 5

department: Institute of Biotechnology

country: Finland

institute: University of Helsinki

type: Expression profiling by high throughput sequencing

summary: We identify RBM41 as a novel unique protein component of the minor spliceosome. RBM41 has no previously recognized cellular function but has been identified as a paralog of the U11/U12-65K protein, a known unique component of the minor spliceosome that functions during the early steps of minor intron recognition as a component of the U11/U12 di-snRNP. We show that both proteins use their highly similar C-terminal RRMs to bind to 3'-terminal stem-loops in U12 and U6atac snRNAs with comparable affinity. Our BioID data indicate that the unique N-terminal domain of RBM41 is necessary for its association with complexes containing DHX8, an RNA helicase, which in the major spliceosome drives the release of mature mRNA from the spliceosome. Consistently, we show that RBM41 associates with excised U12-type intron lariats, is present in the U12 mono-snRNP, and is enriched in Cajal bodies, together suggesting that RBM41 functions in the post-splicing steps of the minor spliceosome assembly/disassembly cycle. This contrasts with the U11/U12-65K protein, which uses the N-terminal region to interact with U11 snRNP during the intron recognition step. Finally, we show that while RBM41 knockout cells are viable, they show alterations in the splicing of U12-type introns, particularly differential U12-type 3' splice site usage. Together, our results highlight the role 3’-terminal stem-loop of U12 snRNA as a dynamic binding platform for the paralogous U11/U12-65K and RBM41 proteins, which function at distinct stages of minor spliceosome assembly/disassembly cycle.

overall_design: CRISPR/Cas9 knockout of the RBM41 locus in HEK293 cells followed by RNAseq analysis of the knockout and wt cell lines

first: Antto

middle: J

last: Norppa

first: Iftekhar

last: Chowdhury

first: Laura

middle: E

last: van Rooijen

first: Janne

last: Ravantti

first: Berend

last: Snel

first: Markku

last: Varjosalo


title: The global RNA-RNA interactome of Klebsiella pneumoniae unveils a small RNA regulator of cell division (dRNA-Seq)

overall_design: Transcriptional start sites in Klebsiella pneumoniae strain MGH78578 were analyzed. For that, dRNA-seq was performed with the wt strain under low (OD 0.25) and high (OD 2) cell density conditions and 50 % of samples were TEX-treated, the other half wasn't treated (ctrl).


title: Mtb HLA-E tetramer sorted CD8+ T-cells have a diverse TCR repertoire

city: Leiden

first: Linda

last: Voogd

state: South-Holland

address: Albinusdreef 2

department: Infectious Diseases

country: Netherlands

institute: Leiden University Medical Center

summary: HLA-E molecules can present self and pathogen-derived peptides to both NK-cells and T-cells. T-cells that recognize HLA-E peptides via their T-cell receptor (TCR) are termed donor-unrestricted T-cells due to restricted allelic variation of HLA-E. The composition and repertoire of HLA-E TCRs is not known so far. We performed TCR sequencing on CD8+ T-cells from 21 individuals recognizing HLA-E tetramers (TM) folded with 2 Mtb HLA-E restricted peptides. We sorted HLA-E Mtb TM+ and TMCD8+ T-cells directly ex vivo and performed bulk RNA-sequencing and single cell TCR sequencing. The identified TCR repertoire was diverse and showed no conservation between and within individuals. TCRs selected from our single cell TCR sequencing data could be activated upon HLA-E/peptide stimulation, although not robust, reflecting potentially weak interactions between HLA-E peptide complexes and TCRs. Thus, HLA-E Mtb specific T-cells have a highly diverse TCR repertoire.

overall_design: To determine RNA expression of HLA-E restricted Mtb specific CD8+ T-cells we stained PBMCs with HLA-E tetramers folded with two Mtb-derived peptides and sorted the tetramer positive CD8+ T-cells to perform RNAseq on these cells.

first: Anne

middle: M

last: Drittij

first: Calinda

middle: K

last: Dingenouts

first: Kees

middle: L

last: Franken

first: Vincent

last: Unen van

first: Krista

last: van Meijgaarden

first: Paula

last: Ruibal

first: Renate

last: Hagedoorn

first: Judith

middle: A

last: Leitner

first: Peter

last: Steinberg

first: Mirjam

middle: H

last: Heemskerk

first: Mark

last: Davis

first: Thomas

last: Scriba

first: Tom

last: Ottenhoff

first: Simone

last: Joosten


title: Source cell-type epigenetic memory persists in induced pluripotent cells but is lost in subsequently derived germline cells [RNA-seq]

status: Public on Feb 03 2024

city: San Antonio

first: Yu-Huey

last: Lin

state: Texas

address: One UTSA Circle,

department: The Department of Neuroscience, Developmental and Regenerative Biology

country: USA

institute: The University of Texas at San Antonio

summary: Retention of source cell-type epigenetic memory may mitigate the potential for induced pluripotent stem cells (iPSCs) to fully achieve transitions in cell fate in vitro. While this may not preclude the use of iPSC-derived somatic cell types for therapeutic applications, it becomes a major concern impacting the potential use of iPSC-derived germline cell types for reproductive applications. The transition from a source somatic cell type to iPSCs and then on to germ-cell like cells (GCLCs) recapitulates two major epigenetic reprogramming events that normally occur during development in vivo -embryonic reprogramming in the epiblast and germline reprogramming in primordial germ cells (PGCs). We examined the extent of epigenetic and transcriptomic memory persisting first during the transition from differentiated source cell types to iPSCs, and then during the transition from iPSCs to PGC-like cells (PGCLCs). We derived iPSCs from four differentiated mouse cell types including two somatic and two germ cell types and tested the extent to which each resulting iPSC line resembled a) a validated ES cell reference line, and b) their respective source cell types, on the basis of genome-wide gene expression and DNA methylation patterns. We then induced each iPSC line to form PGCLCs, and assessed epigenomic and transcriptomic memory in each compared to endogenous PGCs/M-prospermatogonia. In each iPSC line, we found residual gene expression and epigenetic programming patterns characteristic of the corresponding source differentiated cell type from which each was derived. However, upon deriving PGCLCs, we found very little evidence of lingering epigenetic or transcriptomic memory of the original source cell type. This result indicates that derivation of iPSCs and then GCLCs from differentiated source cell types in vitro recapitulates the two-phase epigenetic reprogramming that normally occurs in vivo, and that, to a significant extent, germline cell types derived in vitro from pluripotent cells accurately recapitulate epigenetic programming and gene expression patterns corresponding to equivalent endogenous germ cell types, suggesting that they have the potential to form the basis of in vitro gametogenesis as a useful therapeutic strategy for treatment of infertility.

overall_design: To investigate whether residual epigenetic programming from source cell types limits the extent to which complete epigenetic reprogramming can be achieved upon induction of transitions in cell fate in vitro.

first: Jake

middle: D

last: Lehle

first: John

middle: R

last: McCarrey


title: The global RNA-RNA interactome of Klebsiella pneumoniae unveils a small RNA regulator of cell division

first: Eric

last: Ruhland

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

overall_design: Refer to individual Series


title: NR4a1/2 deletion promotes accumulation of TCF1+ stem-like precursors of exhausted CD8+ T cells in the tumor microenvironment [pd1tim3 scRNA-seq]

status: Public on Feb 02 2024

city: Shinjuku-ku

first: Taeko

last: Hayakawa

state: Tokyo

address: 35 Shinanomachi

department: Division of Tumor Immunology, Institute for Advanced Medical Research

country: Japan

institute: Keio University School of Medicine

summary: Cytotoxic T cells (CTLs) play a crucial role in the elimination of cancer cells. However, within the intra-tumor microenvironment, these cells eventually undergo cell death or enter a state of dysfunction known as "exhaustion". T cell exhaustion not only impairs the efficacy of tumor immunity but also contributes to resistance against immune-checkpoint inhibitors. Our previous investigations have demonstrated that the NR4a family of nuclear receptors, induced by chronic TCR stimulation, plays a pivotal role in driving exhaustion. These receptors enhance the transcription of inhibitory molecules, such as PD1, while suppressing the expression of functional molecules, including cytokines. However, it remains unclear at which stage of T cell differentiation NR4a exerts its influence and whether the loss of NR4a can "rejuvenate" T cell fate. In the present study, we confirmed that simultaneous deletion of NR4a1 and NR4a2 in CD8+ T cells led to a less exhausted phenotype characterized by reduced expression of PD1, Tim3, and Tox, as well as increased oxidative phosphorylation (OXPHOS) and glycolysis activities, resulting in potent suppression of tumor growth. Interestingly, the transfer of activated NR4a1-/-NR4a2-/-CD8+ T cells into tumor-bearing mice gave rise to TCF1+ (Tcf7+) early memory T cells. Wild-type PD1+TIM3+CD8+ tumor-infiltrating lymphocytes (TILs) diminished in the secondary transfer to tumor-bearing mice, while NR4a-deficient CD8+ TILs expanded even during the secondary transfer, indicating a strong stemness of memory T cells due to NR4a loss. We discovered that NR4a directly repressed the expression of Tcf7 by binding to the enhancer region of the Tcf7 gene. Collectively, these findings indicate that inhibiting NR4a in tumors represents a potent strategy for immuno-oncotherapy by enhancing stemness and reducing exhaustion of memory T cells.

overall_design: Rag2-/- mice were subcutaneously injected with 1×106 B16-OVA cells 10 days before adoptive transfer with CD8+ T cell isolated from Cd8CreNR4a1f/fNR4a2f/fOT-ITg and NR4a1f/fNR4a2f/fOT-ITg mice.

first: Tanakorn

last: Srirat

first: Akihiko

last: Yoshimura


title: NR4a1/2 deletion promotes accumulation of TCF1+ stem-like precursors of exhausted CD8+ T cells in the tumor microenvironmentg [scRNA-seq_TAM]

overall_design: CreERT2 expressing NR4a1flox/floxNR4a2flox/flox  CD8+T cells were generated by transducing them with a retrovirus obtained from transfection with pMX-Cre-IRES-GFP retroviral vector and pCL-Eco packaging vectors into Plate-E cells 293T HEK cells . Naïve CD8+ T cells were isolated from the spleen, inguinal, axillary, and mesenteric lymph nodes of NR4a1flox/floxNR4a2flox/flox, OT-I, tdTomato-reporter mice and were activated by plate-coated with 2 μgml-1 of anti-CD3 monoclonal antibody and 1 μg ml-1 of soluble anti-CD28 monoclonal antibody, supplemented with 10 ngml-1 of IL-2, for 36-48 hr. The harvested cells were adoptively transferred into Rag2-/- recipient mice. On 21 days after tumor inoculation, CD8+PD1+TIM3+ TILs were isolated using a FACS AriaIII and secondary transferred into Rag2-/- mice. After one day those mice were subcutaneously injected with B16-OVA cells.


title: Source cell-type epigenetic memory persists in induced pluripotent cells but is lost in subsequently derived germline cells [WGBS]

type: Methylation profiling by high throughput sequencing


title: NR4a1/2 deletion promotes accumulation of TCF1+ stem-like precursors of exhausted CD8+ T cells in the tumor microenvironment [scRNA-seq_2]

overall_design: Rag2-/- mice, were subcutaneously injected with 1×106 B16-OVA cells 10 days before adoptive transfer with CD8+ T cells from WT and Cd8Cre NR4a1flox/floxNR4a2flox/flox  OT-ITg/+ mice.


title: NR4a1/2 deletion promotes accumulation of TCF1+ stem-like precursors of exhausted CD8+ T cells in the tumor microenvironment [bulk_RNA-seq_vivo]


title: NR4a1/2 deletion promotes accumulation of TCF1+ stem-like precursors of exhausted CD8+ T cells in the tumor microenvironment [CD4cre scRNA-seq]

overall_design: Cd4CreNR4a1f/fNR4a2f/fandNR4a1f/fNR4a2f/fMice were subcutaneous injected with 1x106B16-melanoma cells.


title: Source cell-type epigenetic memory persists in induced pluripotent cells but is lost in subsequently derived germline cells


title: NR4a1/2 deletion promotes accumulation of TCF1+ stem-like precursors of exhausted CD8+ T cells in the tumor microenvironment[scRNA-seq_1]

overall_design: Fourteen days after B16 tumor (1x10^6 cells/mouse) transplantation, intratumor CD3⁺ cells were isolated using an autoMACS Pro Separator by positive selection after Percoll enrichment. To obtain scRNA-seq data, we used an BD Rhapsody system (BD Biosciences) and TAS-Seq. A single-cell suspension of ~20,000 cells was captured from all isolated cells, without selection, on an array of >200,000 microwells through a limited dilution approach. Beads with oligonucleotide barcodes were added to saturation so that one bead was paired with each cell in a microwell. After exposure to cell lysis buffer, polyadenylated RNA molecules hybridized to the beads. Beads were retrieved into a single tube for reverse transcription and Exonuclease treatment. cDNA amplification was performed by using TAS-Seq approach. Sequencing libraries were prepared by using NEBNext Ultra II FS library prep kit for Illumina (New England Biolabs). The sequencing analysis were performed by ImmunoGeneTeqs, Inc (Chiba, Japan) by a Novaseq 6000 sequencer (Illumina, San Diego, CA, USA) and a Novaseq S4 200 cycles v1.5 kit (Illumina).

first: Shigeyuki

last: Shichino

first: Satoshi

last: Ueha

first: Wu

last: Bin


title: Lentiviral expression of wildtype LAMA3A restores cell adhesion in airway basal cells from children with epidermolysis bullosa

status: Public on Feb 01 2024

city: London

first: Robert

middle: E.

last: Hynds

address: 20c Guilford Street

department: UCL GOS Institute of Child Health

country: United Kingdom

institute: University College London

summary: The hallmark of epidermolysis bullosa (EB) is the fragile attachment of epithelia to underlying tissues due to genetic variants in genes involved in cell adhesion. A variety of epithelial surfaces can be affected, and EB patients who develop airway involvement suffer from a high degree of morbidity and mortality from laryngotracheal stenosis. Here, we describe 15 EB patients that were treated in the Ear, Nose and Throat department of a tertiary pediatric hospital linked to the United Kingdom’s National EB unit between 1992 and 2023. Investigation of the underlying genotypic status revealed that variants in LAMA3 were common in EB patients managed with airway complications (9/14 patients where genotype was available). Since LAMA3 encodes a subunit of the laminin-332 heterotrimeric extracellular matrix protein complex and is expressed by airway basal epithelial stem cells, we investigated the possibility of correcting LAMA3 in EB patient basal cell cultures. Airway basal cell cultures initiated from EB patients with variants affecting LAMA3 demonstrated fragile adherence to culture flasks but were otherwise expanded similarly to non-EB patient cell cultures. For genetic modification, we generated a lentiviral vector containing LAMA3A under the control of a constitutively active promoter sequence. In vitro transduction of primary EB patient-derived basal cell cultures restored cell attachment to levels comparable to a healthy control donor culture. Transduced cells could be expanded extensively in cell culture, suggesting their potential as an airway cell therapy for EB. We anticipate that our proof-of-concept data will be a starting point for translational combined cell and gene therapy for this rare patient group.

overall_design: Non-transduced controls vs LAMA3-transduced cells. Replicates are independent transductions from a single EB patient.

first: Chun

last: Lau

first: Jessica

middle: C

last: Orr


title: Let-7 miRNAs repress HIC2 to regulate BCL11A transcription and hemoglobin switching

city: Guangzhou

first: Peng

last: Huang

state: Guangdong

address: Xinzao, Panyu District

department: GMU-GIBH Joint School of Life Sciences

country: China

institute: Guangzhou Medical University

type: Genome binding/occupancy profiling by high throughput sequencing


title: Glycerol-3-phosphate and Phosphoethanolamine homeostatic switch triggers senescence by rewiring lipid metabolism II

city: CRETEIL

first: José Américo

middle: Nabuco Leva Ferreira

last: Freitas

address: 8 rue du Général Sarrail

country: France

institute: Institut Mondor de Recherche Biomedicale

summary: Cellular senescence impacts many physiological and pathological processes. A durable cell cycle arrest, inflammatory secretory phenotype, and metabolic reprogramming characterize it. Identifying common and specific metabolic liabilities in senescence provide novel inroads to exploit senescence targeting for health benefits. Here, we use dynamic transcriptome and metabolome profiling in different senescence subtypes to reveal common and specific metabolic signatures. Specifically, we pinpoint the homeostatic switch of glycerol-3-phosphate (G3P) and phosphoethanolamine (PEtn) accumulation, intimately linking lipid metabolism to the senescence gene expression program. Mechanistically, p53-dependent glycerol kinase (GK) activation and post-translational inactivation of Phosphate Cytidylyltransferase 2- Ethanolamine (PCYT2) regulate this metabolic switch, which is senogenic. Conversely, G3P phosphatase (G3PP) and Ethanolamine-Phosphate Phospho-Lyase (ETNPPL)-based scavenging of G3P and PEtn is senomorphic.  Collectively, our study ties the G3P-PEtn homeostatic switch to controlling lipid droplet biogenesis and phospholipid flux in senescent cells, providing a potential, novel therapeutic avenue for senescence targeting in pathophysiology.

overall_design: We performed time-series gene expression profiling analysis in normal, human diploid fibroblasts (strain WI38) undergoing senescence after exposition to either DNA Damage (DDIS) or RAS-oncogene overexpression (RAS-OIS). We administered the mTOR inhibitor Rapamycin, an established senomorphic, to WI38 cells undergoing DDIS, and Dimethyloxalylglycine (DMOG), a hypoxia-mimetic and competitive alphaKG antagonist to cells undergoing RAS-OIS. Total RNA was collected at the time points indicated and global gene expression levels were determined using Affymetrix HTA2.0 microarrays.

first: Khaled

last: Tighanimine

first: José

last: Nabuco Leva Ferreira Freitas

first: Ivan

last: Nemazanyy

first: Alexia

last: Bankolé

first: Delphine

last: Benarroch-Popivker

first: Susanne

last: Brodesser

first: Gregory

last: Doré

first: Lucas

last: Robinson

first: Paule

last: Benit

first: Sophia

last: Ladraa

first: Yara

last: Bou Saada

first: Bertrand

last: Friguet

first: Philippe

last: Bertolino

first: David

last: Bernard

first: Guillaume

last: Canaud

first: Pierre

last: Rustin

last: Gilson

first: Oliver

last: Bischof

first: Stefano

last: Fumagalli

first: Mario

last: Pende


title: Glycerol-3-phosphate and Phosphoethanolamine homeostatic switch triggers senescence by rewiring lipid metabolism

first: José Américo

last: Freitas


title: Glycerol-3-phosphate and Phosphoethanolamine homeostatic switch triggers senescence by rewiring lipid metabolism I

overall_design:
We performed time-series gene expression profiling analysis in normal, human diploid fibroblasts (strain WI38) exposed to diverse forms of senescence-inducing stress including hyper-active RAS oncogene, replicative exhaustion (RS), and DNA damage (etoposide, i.e., DDIS). We confirmed these results in additional senescence models: SkMC human primary myoblasts undergoing RAS-OIS.

Source code and additional data accessible at: https://zenodo.org/records/8199751

first: Khaled

last: Tighanimine

first: José

last: Nabuco Leva Ferreira Freitas

first: Ivan

last: Nemazanyy

first: Alexia

last: Bankolé

last: Benarroch-Popivker

first: Eric


title: Stem Leydig cells support macrophage immunological homeostasis through intercellular mitochondria transfer in a TRPM7-mediated manner in the testis

first: Ke

last: Song

address: No.1023,South Shatai Road, Baiyun District

institute: Southern Medical University

summary: As a unique population of testicular mesenchymal stromal cells, stem Leydig cells (SLCs) have shown great promise in the treatment of male hypogonadism. The therapeutic functions of MSCs are largely determined by their reciprocal regulation by immune responses. However, the immunoregulatory properties of SLCs have not been fully studied. Here, we found that transplanted SLCs restored testicular immunological homeostasis via damage signal-triggered transient receptor potential cation channel subfamily member 7 (TRPM7)-mediated mitochondrial transfer to macrophages. Specifically, the results showed that SLCs transplantation restored male fertility and testosterone production in an ischemia‒reperfusion injury-induced acute inflammation model. This was achieved mainly via a mechanism involving the prevention of inflammatory cascades but not the differentiation to Leydig cells (LCs), the main testosterone-producing cells. Furthermore, we identified that ROS released from activated macrophages, which are the primary testicular immunocytes, induced nanotube-mediated mitochondrial transfer from SLCs to macrophages via TRPM7. Notably, TRPM7 knockdown by interfering RNA in transplanted SLCs compromised the therapeutic outcomes in both testicular ischemia‒reperfusion and testicular aging mouse models. Collectively, SLC rescued testicular damage in testicular ischemia‒reperfusion mouse models, and the damage signal activated TRPM7 signaling and induced mitochondrial transfer from SLCs to macrophages, playing important roles in the repair of SLCs. These findings provide insight into the clinical translation of SLCs and suggest that strategies targeting mitochondrial transfer may be effective treatments for patients with male hypogonadism related to immune disorders.

overall_design: We analyzed the RNA profiles in mouse primary bone marrow mesenchymal stromal cells (BMSCs) and stem Leydig cells (SLCs)

first: Ani

last: Chi


title: Growth factor-induced activation of MSK2 leads to phosphorylation of H3K9me2S10 and corresponding changes in gene expression

city: Philadelphia

first: Karen

last: Wong

state: PA

address: 3400 Civic Center Blvd., SCTR 9-187

department: Cell and Developmental Biology

institute: University of Pennsylvania


title: NR4a1/2 deletion promotes accumulation of TCF1+ stem-like precursors of exhausted CD8+ T cells in the tumor microenvironment [bulk_RNA-seq_vitro]

first: Taeko

last: Hayakawa

overall_design: Naïve CD8+ T cells were isolated from Cd8Cre NR4a1flox/floxNR4a2flox/flox  mice and their wild type little mates, activated with 2 μgml-1 of anti-CD3 and 1μgml-1 of anti-CD28 supplements with 10 ngml-1 of  IL2 for 24 or 48 hr.

first: Tanakorn

last: Srirat

first: Akihiko

last: Yoshimura


title: ARID1A Orchestrates SWI/SNF-mediated Sequential Binding of Transcription Factors with ARID1A Loss Driving Pre-memory B Cell Fate and Lymphomagenesis

city: New York City

first: Christopher

middle: Russell

last: Chin

state: NY

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

institute: Weill Cornell

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus


title: Growth factor-induced activation of MSK2 leads to phosphorylation of H3K9me2S10 and corresponding changes in gene expression [CUT&RUN]

summary: Extracellular signals are transmitted through kinase cascades to modulate gene expression, but it remains unclear how epigenetic changes in chromatin architecture may regulate this response. Here we provide evidence supporting a mechanism through which growth factor-responsive genes located within H3K9me2-marked heterochromatin domains are made available for transcriptional activation via phosphorylation of the adjacent histone H3 serine 10 residue following growth factor signaling. Imaging and proteomic approaches show that Epidermal Growth Factor (EGF) stimulation results in H3K9me2S10 phosphorylation, which occurs in genomic regions enriched for regulatory enhancers of EGF-responsive genes. We also demonstrate that the EGF-induced increase in H3K9me2S10ph is dependent on the nuclear kinase MSK2, and a subset of EGF-induced genes is dependent on MSK2 for transcription. Taken together, our work indicates that growth factor-induced changes in chromatin state can mediate the activation of downstream genes.

overall_design: CUT&RUN sequencing for H3K9me2S10ph in human IMR90 lung fibroblast cells, serum starved for 24 hours and treated with either PBS or 750pM EGF for 30 minutes.

middle: G

first: Cheryl

last: Smith

first: Jonathan

last: Epstein


title: Growth factor-induced activation of MSK2 leads to phosphorylation of H3K9me2S10 and corresponding changes in gene expression [RNA-seq]

overall_design: RNA-sequencing from IMR90 lung fibroblast cells, serum starved for 24 hours and treated with: 1) either non-template control scramble siRNA or siRNA targeting MSK2; 2) PBS, 750pM EGF for 30 minutes, or 750pM EGF for 60 minutes.


title: Role of UPF1 in lncRNA-HEIH regulation for hepatocellular carcinoma therapy

city: Seoul

first: HyunHo

last: cha

address: 222, Wangsimni-ro, Seongdong-gu

department: Biomedical Science

country: South Korea

institute: hanyang university

summary: To investigate UPF1 can regulates lncRNA-HEIH transcripts and function of lncRNA-HEIH, we established UPF1 knock out HeLa cell using CRISPR/cas9 system and overexpressed lncRNA-HEIH using plasmids.

overall_design: We then performed gene expression profiling analysis using data obtained from RNA-seq of i) WT HeLa cells and UPF1 knock out clone 9 and 41. ii) control and lncRNA-HEIH overexpressed Huh7 cell

first: Hyunho

last: Cha

first: Minwoo

last: Kim

first: Seong Dong

last: Jeong

first: Narae

last: Ahn

first: Elizaveta

last: Ignatova

first: Sung Wook

first: Hyeon Ho

first: Jungwook

last: Hwang


title: Targeting DHX9 triggers interferon response and replication stress in Small Cell Lung Cancer [RNA-seq]

first: Israel

last: Canadas

address: 333 Cottman Ave.

department: Nuclear Dynamics and Cancer Program

institute: Fox Chase Cancer Center

summary: Activating innate immunity in cancer cells through cytoplasmic nucleic acid sensing pathways, a phenomenon known as “viral mimicry”, has emerged as an effective strategy to convert immunologically “cold” tumors into “hot”. Through a curated CRISPR-based screen of RNA Helicases, we identified DExD/H-box helicase 9 (DHX9) as a potent repressor of double-stranded RNA (dsRNA) in small cell lung cancers (SCLCs). Depletion of DHX9 induced accumulation of cytoplasmic dsRNA and triggered tumor-intrinsic innate immunity. Intriguingly, ablating DHX9 also induced aberrant accumulation of R-loops, which resulted in an increase of DNA damage-derived cytoplasmic DNA and replication stress in SCLCs. In vivo, DHX9 deletion promoted decrease in tumor growth while inducing a more immunogenic tumor microenvironment, invigorating responsiveness to immune checkpoint blockade. These findings suggest that DHX9 is a crucial repressor of tumor-intrinsic innate immunity and replication stress, representing a promising target for SCLC and other “cold” tumors where genomic instability contribute to pathology.

overall_design: To investigate the function of DHX9, we established H196, H82, H446 cell lines in which the target gene was depleted.

first: Takahiko

last: Murayama

first: Jun

last: Nakayama

last: Cañadas


title: Targeting DHX9 triggers interferon response and replication stress in Small Cell Lung Cancer [RIP-seq I]

overall_design: To investigate the function of DHX9, we established H446 cell lines in which the target gene was depleted. dsRNA was pulled down with J2 antibody.

first: Kenichi

last: Miyata


title: Differential gene expression analyses after noncanonical peptide overexpression

city: Chicago

first: Zhe

last: Ji

state: ILLINOIS

address: 303 E Superior St, Lurie 6-107

institute: Northwestern University

summary: Ribosome profiling analyses showed thousands of translated open reading frames in a cell encoding noncanonical peptides. To examine their functional roles, we stably overexpressed two noncanonical peptides (uSLC35A4 and iPGRMC1) in MCF-7 cells. And then we performed RNA-seq to examine differential gene expression upon gene overexpression.

overall_design: We perform RNA-seq of MCF-7 cells overexpression wide-type ORF sequences. The start codon mutated sequences were used as the controls.

first: Qianru

last: Li


title: Pericyte sGC signaling dictates tumor vessel formation and tumor microenvironment

city: Shanghai

first: junhao

last: hu

address: 100 Haike Road, Zhangjiang High tech Park, Pudong

institute: The Interdisciplinary Research Center on Biology and Chemistry

summary: Pericytes and endothelial cells (ECs) are building blocks of blood vessels. While the contributions of ECs to tumor angiogenesis and the tumor microenvironment are well established and multiple drugs that targeting ECs have been developed for clinic use to treat cancers, the underlying mechanisms of pericyte in supporting tumor vessel and in shaping tumor microenvironment remains largely unexplored and no pericyte targeting strategies have been approved yet. This study employs targeted deletion of the NO receptor sGC in pericytes and utilizes single-cell RNA sequencing to elucidate its impact on the tumor microenvironment. The results unveil a disruptive effect on EC-pericyte interactions, subsequently impeding Notch-mediated intercellular crosstalk and prompting extensive transcriptomic reprogramming in both cell types. This vascular alteration further relays to neighboring cancer-associated fibroblasts (CAFs) and tumor-associated macrophages (TAMs) through paracrine signaling, collectively suppressing tumor growth. Importantly, the inhibition of pericyte sGC has limited influence on quiescent vessels but significantly sensitizes angiogenic vessels to anti-angiogenic treatment. In conclusion, this study underscores the vital role of pericytes in governing tumor vessels and the tumor microenvironment, suggesting that targeting pericyte sGC holds promise for enhancing anti-angiogenic therapy.

overall_design: LLC tumor of both three Gucy1b1 flox/flox (CT/Ctr) and Cspg4-CreER^T2::Gucyc1b1 flox/flox (KO/Δpc) C57BL/6 mice were both sequenced using 10X chromium 3' scRNA-seq protocol separately to produce two libraries followed by further analysis.


title: Human cytomegalovirus infection triggers a paracrine senescence loop in renal epithelial cells.

city: Novara

first: Francesco

last: Favero

state: NO

address: Corso Trieste 15/A

department: Translational Medicine - CAAD (Center for Translational Research on Autoimmune and Allergic Diseases)

country: Italy

institute: University of Eastern Piedmont

summary: We evaluated the modulation of mRNA upon human cytomegalovirus (HCMV) infection in renal proximal tubule epithelial cell line (RPTEC).

overall_design: Gene expression profile study of RPTEC 2 days post hCMV infection

first: Marco

last: De Andrea

first: Marisa

last: Gariglio

first: Davide

last: Corà

first: Stefano

last: Raviola

first: Gloria

last: Griffante


title: Nuclear Morphology is Instructed by the Loop Extrusion Program [scRNA-seq]

status: Public on Feb 11 2024

city: SAN DIEGO

first: Indumathi

last: Patta

state: CA

address: 9500 Gilman Drive, NSB5114A, LaJolla

department: Molecular Biology

institute: UCSD

summary: More than a century ago Metchnikoff defined neutrophils as small amoeboid-like cells that harbored peculiar polymorphonuclear structures. While since these early studies much has been learned about neutrophil physiology, mechanistic insight into neutrophil polymorphonuclear assembly remains rudimentary. Here we found that depletion of factors, that initiate the loop extrusion program, including NIPBL and MAU2, greatly accelerated the conversion of mononuclear to polymorphonuclear cells and orchestrated the induction of a neutrophil specific transcription signature. Remarkably, mere ablation of either NIPBL or MAU2 expression under conditions that normally prevent neutrophil differentiation, swiftly converted mono-nuclear into polymorphonuclear cells that expressed a neutrophil specific gene program.  Depletion of NIPBL and MAU2 in neutrophil progenitors activated an armamentarium of neutrophil-specific enhancers to establish a neutrophil genome depleted for loop extrusion-induced looping. These observations indicate that extinction of the loop extrusion program converts mononuclear progenitor cells into polymorphonuclear cells and suffices to induce a neutrophil specific program of gene expression.

overall_design: Mouse bone marrow progenitros including precursor neutrophils were isolated and electroporated with Cas9-sgRNA RNP complexes against NIPBL.  We performed single cell RNA-Seq.

first: Kathleen

last: Fish

first: Cornelis

last: Murre


title: Targeting DHX9 triggers interferon response and replication stress in Small Cell Lung Cancer [RIP-Seq II]

last: Canadas

overall_design: To investigate the function of DHX9, we sequenced MDA5-bound and RIG-I-bound RNAs in H446 cell lines in which the target gene was depleted. We also sequenced DHX9-bound RNAs by pulling down 3xFlag-DHX9-K417R.


title: Comparative Analysis of Paraspinal Muscle Imbalance Between Idiopathic Scoliosis and Congenital Scoliosis from the Transcriptome Aspect

status: Public on Jan 31 2024

city: Beijing

first: Zhen

last: Wang

address: No.1 Shuaifuyuan

institute: Peking uinon medical college

summary: Comparison of paraspinal muscle imbalance between idiopathic scoliosis and congenital scoliosis  may shed some light on the causality of paraspinal muscle imbalance and idiopathic scoliosis. This study aims to compare the transcriptomic profiles of paraspinal muscle imbalance between idiopathic scoliosis and congenital scoliosis.

overall_design:
Bilateral paraspinal muscles of idiopathic scoliosis and congenital scoliosis patients were collected for transcriptome sequencing. Differentially expressed genes in paraspinal muscles of IS and CS between the convexity and the concavity were screened out.

Please note that 'A' samples indicate paraspinal muscles on the concavity from idiopathic or congenital scoliosis patients, whereas 'T' samples are muscles on the convexity in idiopathic or congenital scoliosis.

first: Junduo

last: Zhao

first: Haining

last: Tan

first: Yang

last: Jiao

first: Xin

last: Chen

first: Jianxiong

last: Shen


title: T cell Immune Escape in Blast Crisis Transformation of Chronic Myeloid Leukemia

city: Duarte

first: Hsun

middle: Teresa

last: Ku

address: 1500 E. Duarte Road

department: Translational Research and Cellular Therapeutics

institute: City of Hope

summary: Chronic myeloid leukemia (CML) may transform from a chronic phase (CP) into blast crisis (BC), which is often incurable. Herein, we report that miR-142 deficit acquired by T lymphocytes contributes to BC transformation by promoting immune escape. We observe that T cells of BC patients lack miR-142 and are fewer and exhausted compared with CP patients. Similarly, BC miR-142−/−/BCR/ABL+/+ mouse presents with T lymphopenia compared with the CP miR-142+/+/BCR/ABL+/+ mouse. In the BC mouse, miR-142 deficit impairs thymic differentiation of lymphoid-primed multipotent progenitors (LMPP) into mature T cells and redirects them toward myeloid lineage. The fewer mature T cells in the BC mouse are enriched with exhausted T effectors. MiR-142 deficit, driven by leukemic blasts-secreted inflammatory cytokines (i.e., IL-6), induces T cell hypofunction by preventing the metabolic reprogramming that allows activated T cells to thrive and expand. This ultimately results in an increase in T cell spontaneous apoptosis and BC immune escape. In fact, NSG mice transplanted with BC CML LSKs and miR-142 KO T cells had shorter survival than mice transplanted with BC CML LSKs and miR-142 wild-typewt T cells. Conversely, BC patient-derived xenograft (PDX) mice receiving autologous T cells and synthetic miR-142 lived longer than those receiving autologous T cells and scramble RNA. Combination of tyrosine kinase inhibitors (TKI) plus synthetic miR-142 and/or PD-1 antibody induced a prolonged survival compared to TKI alone, suggesting that harnessing the host immune system with synthetic miR-142 and immunocheckpoint inhibitors along with BCR/ABL inhibition may provide novel therapeutic opportunities.

overall_design: Bone marrow (BM) LMPPs from miR-142+/+, miR-142−/−, miR-142+/+BCR-ABL (CP CML), and miR-142−/−BCR-ABL (BC CML) mice (BCR-ABL was induced for two week by tet-off) were sorted for scRNA-seq.

first: Bin

last: Zhang

first: Min-Hsuan


title: Idd5 favours robust neonatal development of highly autoreactive regulatory T cells in the NOD mouse

city: Toulouse

first: Joost

last: van Meerwijk

state: HG

address: CHU Purpan – BP 3028

institute: INSERM

summary: Regulatory T lymphocytes expressing the transcription factor Foxp3 (Treg) play an important role in the prevention of autoimmune diseases and other immunopathologies. Aberrations in Treg-mediated immunosuppression are therefore thought to be involved in the development of autoimmune pathologies but few have been documented. Recent reports indicated a central role for Treg developing during the neonatal period in the prevention of autoimmune pathology. We therefore investigated the development of Treg in neonatal NOD mice, an important animal model for autoimmune type 1 diabetes. Surprisingly, we found that, as compared to seven other commonly studied inbred mouse-strains, in neonatal NOD mice exceptionally large proportions of developing Treg express high levels of GITR and PD-1. The latter phenotype was previously associated with high Treg-autoreactivity in C57BL/6 mice, which we here confirm for NOD animals. Proportions of newly developing GITRhighPD-1+ Treg rapidly drop during the first week of age. A genome-wide genetic screen indicated involvement of several diabetes susceptibility loci in this trait. Analysis of a congenic mouse-strain confirmed that Idd5 contributes to the genetic control of GITRhighPD-1+ Treg-development in neonates. Our data thus demonstrate an intriguing and paradoxical correlation between an idiosyncrasy in Treg development in NOD mice and their susceptibility to type 1 diabetes.

first: Jeremy

last: Santamaria

first: Sylvia

last: Vuillier

first: Ariel

last: Galindo-Albarrán

first: Sarah

last: Castan

first: Claire

last: Detraves

first: Olivier

middle: P

last: Joffre

first: Paola

last: Romagnoli

last: an Meerwijk


title: Single cell atlas of human gastric muscle immune cells and macrophage-driven changes in idiopathic gastroparesis

city: Rochester

first: Madhusudan

last: Grover

state: MN

address: 200 First St SW

institute: Mayo Clinic

summary: Gastrointestinal immune cells, particularly muscularis macrophages (MM) interact with enteric nervous system and influence gastrointestinal motility. Here we determine the human gastric muscle immunome and its changes in patients with idiopathic gastroparesis (IG). Single cell sequencing was performed on 26,000 CD45+ cells obtained from gastric tissue of 20 subjects (13 controls, 7 IG). Canonical markers, differential expression, and signalling were determined. We demonstrate 11 immune cell clusters with T cells being most abundant followed by myeloid cells. The proportions of cells belonging to the 11 clusters were similar between IG and controls. However, 9/11 clusters showed 578-11,429 differentially expressed genes (FDR<0.05). In IG, MM had decreased expression of tissue-protective and microglial genes and increased expression of monocyte trafficking and stromal activating genes. Furthermore, in IG, IL12 mediated JAK-STAT signaling involved in activation of tissue-resident macrophages and Eph-ephrin signaling involved in monocyte chemotaxis to inflammatory sites were upregulated. In summary, human gastric muscle has a rich representation of immune cells; and IG patients demonstrate a higher expression of pro-inflammatory genes and signaling in MM. These data further link immune dysregulation to pathophysiology of gastroparesis.

overall_design:
Our aim in this study was to characterize the immune cell populations in human gastric muscle and determine changes in patients with idiopathic gastroparesis.
[contributor] NIDDK Gastroparesis Clinical Research Consortium

first: Lakshmikanth

last: Chikkamenahalli

first: Erik

last: Jessen

first: Cheryl

last: Bernard

first: W

last: Ip

first: Margaret

last: Breen-Lyles

first: Gianluca

last: Cipriani

first: Suraj

last: Pullapantula

first: Ying

last: Li

first: Laura

last: Wilson

first: Kenneth

last: Koch

first: Braden

last: Kuo

first: Robert

last: Shulman

first: Bruno

last: Chumpitazi

first: Travis

last: McKenzie

first: Todd

last: Kellogg

first: James

last: Tonascia

first: Frank

last: Hamilton

first: Irene

last: Sarosiek

first: Richard

last: McCallum

first: Henry

last: Parkman

first: Pankaj

last: Pasricha

first: Thomas

last: Abell

first: Gianrico

last: Farrugia

first: Surendra

last: Dasari


title: A mutational atlas for Parkin

city: Copenhagen

first: Kristoffer

last: Johansson

address: Ole Maaløes Vej 5

department: Biology

country: Denmark

institute: University of Copenhagen

summary: More than half of disease-causing missense variants are thought to lead to protein degradation, but the molecular mechanism of how these variants are recognized by the cell remains enigmatic. To approach this issue we have applied deep mutational scanning experiments to test the degradation of thousands of missense protein variants in large multiplexed experiments in cultured human cells. As a model protein we selected the ubiquitin-protein ligase Parkin, where known missense variants result in an autosomal recessive early onset Parkinsonism. The resulting mutational map comprises 9219 out of the 9300 (>99%) possible single-amino-acid substitution and nonsense Parkin variants. With a few notable exceptions, the majority of the destabilizing mutations are located within the structured domains of the protein, while the flexible linker regions are more tolerant to mutations. The cellular abundance data correlate with Parkin structural stability, evolutionary conservation, and separates known disease-linked variants from benign variants. Systematic mapping of degradation signals (degrons) shows that inherent primary degrons in Parkin largely overlap with regions that are highly sensitive to mutations. We identify a degron region proximal to the ACT element, which is enhanced by substitutions to hydrophobic residues. The vast majority of unstable Parkin variants are degraded through the ubiquitin-proteasome system and are stabilized at lowered temperatures. In conclusion, in addition to providing a diagnostic tool for rare genetic disorders, deep mutational scanning technologies have the potential to reveal both protein specific and general information on the specificity of the protein quality control network and the ubiquitin-proteasome system.

overall_design: To investigate cellular abundance levels of parkin single amino acid variants, a stie saturation library was GFP labeled and FACS sorted in 3 replicates and 4 biological replicates

first: Lene

last: Clausen

first: Vasileios

last: Voutsinos

first: Rasmus

last: Hartmann-Petersen


title: Targeting DHX9 triggers interferon response and replication stress in Small Cell Lung Cancer

first: Israel

last: Canadas

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

overall_design: Refer to individual Series


title: One-step generation of auxin-inducible degron cells with high-efficiency homozygous tagging

city: Cardiff

first: Van Dien

last: Nguyen

address: Heath Park

department: School of Medicine

institute: Cardiff University

summary: Auxin-inducible degron (AID) technology is powerful for chemogenetic control of proteolysis. However, generation of human cell lines to deplete endogenous proteins with AID remains challenging. Typically, homozygous degron-tagging efficiency is low and overexpression of an auxin receptor requires additional engineering steps. Here, we establish a one-step genome editing procedure with high-efficiency homozygous tagging and auxin receptor expression. We demonstrate its application in 5 human cell lines, including embryonic stem (ES) cells. The method allowed isolation of AID single-cell clones in 10 days for 11 target proteins with >80% average homozygous degron-tagging efficiency in A431 cells, and >50% efficiency for 5 targets in H9 ES cells. The tagged endogenous proteins were inducibly degraded in all cell lines, including ES cells and ES-cell derived neurons, with robust expected functional readouts. This method facilitates the application of AID for studying endogenous protein functions in human cells, especially in stem cells.

overall_design: A431 wild-type cells were treated with different inducers: Pico_cvxIAA (0.5 μM), cvxIAA (5 μM) and IAA (500 μM); for 24 h compared to control.

first: Shiqian

last: Li

first: You

last: Zhou

first: VanDien


title: Effect of A1 on the transcriptome of mesenchymal stem cells

city: Montreal

first: Moutih

last: Rafei

address: Montreal

country: Canada

institute: Universite de Montreal

summary: To investigate whether pharmacological stimulation of mesenchymal stromal cells (MSCs) could induce cross-presentation abilities, we treated MSCs with A1. We performed gene expression profiling analysis using data obtained from RNA-seq of untreated MSCs, MSCs treated only with the molecule A1, and MSCs treated with the molecule A1 and the model antigen ovalbumin. A1 is an Accum™ variant, a molecule design to enhance intracellular accumulation of associated molecules through endosomal disruption. Endosomal break was targeted for its crucial role in antigen export to the cytosol and cross-presentation of endocytosed antigen.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for untreated mesenchymal stromal cells and A1-treated cells, co-treated with the antigen ovalbumin, or not.

first: Marina

last: Pereira Gonçalves

first: Nehme

last: El-Hachem


title: Quiescence Enables Unrestricted Cell Fate in Naive Embryonic Stem Cells

city: Los Angeles

first: Wentao

last: Yang

state: California

address: 1441 Eastlake Ave

institute: university of southern california

summary: This project will include 42 datasets (24 RNA-seq datasets, 8 CUT&RUN datasets, 4 ATAC-seq datasets and 6 CRISPR target bisulfite-seq datasets), generated from illumine Hi-seq sequencer to investigate the stem cell quiescence in mouse embryonic stem cells.

overall_design: Expression profiling by high throughput sequencing, Genome binding/occupancy profiling by high throughput sequencing, Elf5 promoter methylation profiling by CRISPR sequencing

first: Yali

last: Dou

first: Le

last: Khoa


title: Effect of Bmal1 depletion on gene expression in poly(I:C)-stimulated myeloid peritoneal cells.

first: Do-Wan

last: Shim

address: 50-1, Yonsei-ro, Seodaemun-gu

department: Microbiology

institute: Yonsei university

summary: To investigate the effect of Bmal1 on poly(I:C) response, we isolate mRNA from PBS or poly(I:C)-injected WT and myeloid Bmal1 KO mice. We then compare the poly(I:C)-induced transcriptomic change between WT and Bmal1 KO peritoneal myeloid cells.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for WT and Bmal1 KO peritoneal myeloid cells.

first: Je-Wook

last: Yu


title: Cold exposure impacts DNA methylation patterns in cattle sperm

city: Pullman

first: Min

last: Du

state: WA

address: 1815 Ferdinand's Ln

department: Animal Science

institute: Washington State University

summary: Epigenetic markers, such as DNA methylation, in sperm plays crucial roles in spermatogenesis and fertilization, and a portion of methylation escape the early embryonic genome-wide DNA demethylation and affect later growth and development of offspring. DNA methylation is influenced by various exogenous factors such as nutrition, temperature, toxicants, and stress. The effects of this cold exposure on the methylation dynamics of bovine sperm remain unexamined. Methylation profiles of sperm, collected from bulls during summer and winter seasons were analyzed by Whole Genome Bisulfite Sequencing (WGBS). Cold exposure during winter does not have a significant effect on the global methylation level, it altered the methylation levels at certain genomic loci and genes that may impact the maintenance of methylation status of imprinted genes and other loci, which may impact the development and growth of offspring.

overall_design: Semen samples collect from each of the three bulls during Late Spring (summer) and winter, Total 6 samples

first: Md Nazmul

last: Hossain

first: Yao

last: Gao

first: Jeanene

last: M. de Avila

first: Michael

last: J. Hatfield

first: Matthew

last: C. McClure


title: Transcriptome Sequencing of mice eosinophils upon Listeria monocytogenes (L.m.) infection

city: Hangzhou

first: Jun

address: 866 Yuhangtang Road

department: Institute of Immunology

institute: Zhejiang University School of Medicine

summary:
Mature eosinophils were differentiated from mouse bone marrow progenitors
We performed transcriptome sequencing on Listeria monocytogenes infected eosinophils and resting eosinophils to shed light on the transcriptional changes of cytokines and  chemokines.

overall_design: 3×10^6 Eosinophils were infected with Listeria monocytogenes for 8 hours. Resting and infected eosinophils were used for RNA extraction, and sequencing. Total RNA was isolated using RNeasy mini kit (Qiagen, Germany). Strand-specific libraries were prepared using the TruSeq® Stranded Total RNA Sample Preparation kit (Illumina, USA) following the manufacturer’s instructions.Ribosomal RNA was removed from total RNA using Ribo-Zero rRNA removal beads. Following purification, the mRNA is fragmented into small pieces using divalent cations under 94℃ for 8 min. The cleaved RNA fragments are copied into first strand cDNA using reverse transcriptase and random primers. This is followed by second strand cDNA synthesis using DNA Polymerase I and RNase H. These cDNA fragments then go through an end repair process, the addition of a single ‘A’ base, and then ligation of the adapters. The products are then purified and enriched with PCR to create the final cDNA library. Purified libraries were quantified by Qubit® 2.0 Fluorometer (Life Technologies, USA) and validated by Agilent 2100 bioanalyzer (Agilent Technologies, USA) to confirm the insert size and calculate the mole concentration. Cluster was generated by cBot with the library diluted to 10 pM and then were sequenced on the Illumina NovaSeq 6000 (Illumina, USA).


summary: This project will include 42 datasets (24 RNA-seq datasets, 8 CUT&RUN datasets, 4 ATAC-seq datasets, and 6 CRISPR target bisulfite-seq datasets), generated from illumine Hi-seq sequencer to investigate the stem cell quiescence in mouse embryonic stem cells.

middle: T

last: Phuc Khoa


overall_design: Expression profiling by high throughput sequencing, Genome binding/occupancy profiling by high throughput sequencing, Elf5 promoter methylation profiling by CRISPR sequencing.


title: Induced pluripotent stem cell derived pericytes respond to endogenous mediators of proliferation and contractility

status: Public on Jan 30 2024

city: HOBART

first: Brad

middle: A.

last: Sutherland

state: TAS

address: Medical Sciences Precinct, 17 Liverpool Street

country: Australia

institute: University of Tasmania

summary: Pericytes are multifunctional contractile cells that reside on capillaries. Pericytes are critical regulators of cerebral blood flow and blood-brain barrier function, and pericyte dysfunction may contribute to the pathophysiology of human neurological diseases including Alzheimer’s disease, multiple sclerosis, and stroke. Induced pluripotent stem cell (iPSC)-derived pericytes (iPericytes) are a promising tool for vascular research. However, it is unclear how iPericytes functionally compare to primary human brain vascular pericytes (HBVPs). We differentiated iPSCs into iPericytes of either the mesoderm or neural crest lineage using established protocols. We compared iPericyte and HBVP morphologies, quantified gene expression by qPCR and bulk RNA sequencing, and visualised pericyte protein markers by immunocytochemistry. To determine whether the gene expression of neural crest iPericytes, mesoderm iPericytes or HBVPs correlated with their functional characteristics in vitro, we quantified EdU incorporation following exposure to the key pericyte mitogen, platelet derived growth factor (PDGF)-BB and, contraction and relaxation in response to the vasoconstrictor endothelin-1 or vasodilator adenosine, respectively. iPericytes were morphologically similar to HBVPs and expressed canonical pericyte markers. Consistent with the ability of HBVPs to respond to PDGF-BB signalling, PDGF-BB enhanced and PDGF receptor (PDGFR)β inhibitors impaired iPericyte proliferation. Administration of endothelin-1 led to iPericyte contraction and adenosine led to iPericyte relaxation, of a magnitude similar to the response evoked in HBVPs. We determined that neural crest iPericytes were less susceptible to PDGFRβ inhibition, but responded most robustly to vasoactive meditators.  iPericytes express pericyte-associated genes and proteins and, exhibit an appropriate physiological response upon exposure to a key endogenous mitogen or vasoactive mediators. Therefore, this protocol to generate functional iPericytes would be suitable for use in future investigations exploring pericyte function or dysfunction in neurological diseases.

overall_design: To investigate the suitability of iPSC-derived pericytes for investigations into neurological diseases we differentiated iPSCs into iPericytes via either mesoderm or neural crest lineage and compared these to each other and to primary human brain vascular pericytes.

first: Natalie

last: King

first: Jo-Maree

last: Courtney

first: Lachlan

middle: S

last: Brown

first: Alastair

middle: J

last: Fortune

first: Nicholas

middle: B

last: Blackburn

first: Jessica

last: Fletcher

first: Jake

last: Cashion

first: J

last: Talbot

first: A

last: Pebay

first: Alex

middle: W

last: Hewitt

first: Gary

last: Morris

first: Kaylene

last: Young

first: Anthony

last: Cook


first: Wentao

last: Yang


title: Effect of Endogenous Stimuli-Responsive Separating Microneedles to Inhibit Hypertrophic Scar on Rabbit Ear

city: Wuhan

first: Huinan

last: Suo

state: Hubei Province

address: 1277 Jiefang Avenue, Wuhan, Hubei Province, 430022, China

department: Dermatology

institute: Huazhong University of Science and Technology

summary: Hypertrophic scar (HS) considerably affects the appearance and causes tissue dysfunction in patients. Here we show a separating microneedle (MN) consisting of photo-crosslinked GelMA and 5-FuA-Pep-MA prodrug in response to high reactive oxygen species (ROS) levels and overexpression of matrix metalloproteinases (MMPs) in the HS pathological microenvironment. RNA sequencing analyses confirm that drug-loaded MNs could reverse skin fibrosis through down-regulation of BCL-2-associated death promoter (BAD), insulin-like growth factor 1 receptor (IGF1R) pathways, simultaneously regulate inflammatory response and keratinocyte differentiation via up-regulation of toll-like receptors (TOLL), interleukin-1 receptor (IL1R) and keratinocyte pathways.

overall_design: To elucidate the RNA changes associated with drug-loaded MNs on HS, we performed RNA sequencing (RNA-seq) analysis using rabbit ear normal skin (NS), rabbit ear HS tissues, HS tissues with drug-loaded MNs, and HS tissues with 5-Fu injection treatment. Comparative gene expression profiling analysis of RNA-seq data for HS versus NS, HS with drug-loaded MNs versus HS, and HS with 5-Fu injection versus HS.

first: Zhuoran

first: Hao

last: Jiang

first: Juan

last: Tao

first: Jintao

last: Zhu


title: GFP and mCherry gene expression at single cell level of all liver cell types from B6J mice

city: Dallas

first: Simeng

last: Wang

state: TX

address: 5323 Harry Hines Blvd

institute: UT Southwestern Medical Center

summary: Diacylglycerol acyltransferase (DGAT)-2 catalyzes the final step of triglyceride (TG) synthesis. DGAT2 deletion in mice lowers liver TGs and DGAT2 overexpression might have the opposite effect. Mouse DGAT2 was overexpressed in C57Bl/6J mice using adeno-associated virus 8 (AAV8 and AAV-DJ). The tissue specificity of AAV8 and AAV-DJ was first evaluated.  To confirm that AAV8 and AAV-DJ only infected hepatocytes and not other cells in liver, we carried out single cell sequencing of liver cells isolated from mice infected with AAV8-mCherry and AAV-DJ-GFP. The mCherry and GFP signals were highly expressed in hepatocytes and not in stellate, endothelial, or Kupffer cells.

overall_design: An 8-week-old C57BL/6J male mouse was injected retro-orbitally with an AAV-DJ virus that was engineered to express GFP and AAV-8 engineered to express mCherry. The mouse was fed a chow diet ad lib and sacrificed in 3 weeks after virus injection. Hepatocytes and non-parenchymal liver cells (NPCs) were isolated for the sc-RNA seq. New custom mice gene reference genomes were created by adding both GFP and mCherry into the existing mouse gene reference genomes. GFP and mCherry gene expression were assessed in both hepatocytes and NPCs.

first: Jay

last: Horton


first: Le

last: Phuc Khoa

first: Yali

last: Dou


title: Arabidopsis AGO1 N-terminal Poly-Q domain promotes phase separation and association with stress granules during heat stress [miRNA-seq]

city: Davis

first: Patricia

last: Baldrich Gonzalez

address: 451 Health Sciences Dr., Davis, CA 95616

department: Genome Center

institute: University of California - Davis

type: Non-coding RNA profiling by high throughput sequencing

summary: In Arabidopsis thaliana, ARGONAUTE1 (AGO1) plays a central role in microRNA (miRNA) and small interfering RNA (siRNA)-mediated silencing. Nuclear AGO1 is loaded with miRNAs and exported to the cytosol where it associates to the rough ER to conduct miRNA-mediated translational repression, mRNA cleavage and biogenesis of phased siRNAs. These latter, as well as other cytosolic siRNAs, are loaded into cytosolic AGO1, but in which compartment this happens is not known. Moreover, the effect of stress on AGO1 localization is still unclear. Here, we show that heat stress (HS) promotes AGO1 protein accumulation, which co-localizes with components of the siRNA bodies and of stress granules (SGs). AGO1 does not need SGS3, a key component of siRNA bodies, to efficiently form condensates during HS. Instead, we found that the still poorly characterized N-terminal Poly-Q domain of AGO1, which contains a prion-like domain, is sufficient to undergo phase separation. Moreover, an exposure of 1 hour to HS only moderately affected AGO1 loading by miRNAs and target cleavage, suggesting that its localization in condensates protects AGO1 rather than promoting its activity in reprograming gene expression during stress. Collectively, our work shed new light on the impact of high temperatures on the main effector of RNA silencing in plants.

overall_design: small RNAseq from Arabidopsis seedlings under control, heat stress, and recovery conditions. Three biological replicates each.

last: Baldrich

first: Aleksandar

last: Blagojevic

first: Esther

last: Lechner

first: Blake

last: Meyers

first: Pascal

last: Genschik


title: Arabidopsis AGO1 N-terminal Poly-Q domain promotes phase separation and association with stress granules during heat stress [nanoPARE]

overall_design: RNAseq from Arabidopsis seedlings under control, heat stress, and recovery conditions. Three biological replicates each.


title: Arabidopsis AGO1 N-terminal Poly-Q domain promotes phase separation and association with stress granules during heat stress


title: GPATCH4 regulates rRNA and snRNA 2’-O-methylation in both DHX15-dependent and -independent manners [RiboMeth-seq]

city: Göttingen

first: Katherine

last: Bohnsack

address: Humboldtallee 23

institute: University Medical Centre Göttingen

summary: Strict regulation of RNA helicase activity, often accomplished by protein cofactors, is essential to ensure target specify within the complex cellular environment. The largest family of RNA helicase cofactors are the G-patch proteins, but the cognate RNA helicases and cellular functions of numerous human G-patch proteins remain elusive. Here, we discover that GPATCH4 is a stimulatory cofactor of DHX15 that interacts with the helicase in the nucleolus via residues in its G-patch domain. We reveal that GPATCH4 associates with pre-ribosomal particles, and crosslinks to the transcribed ribosomal DNA locus and precursor ribosomal RNAs as well as binding to small nucleolar- and small Cajal body-associated- RNAs that guide rRNA and snRNA modifications. Global analysis of rRNA and snRNA 2’-O-methylation revealed that lack of GPATCH4 impairs methylation at various sites. We further demonstrated that the regulation of 2’-O-methylation by GPATCH4 is both dependent on, and independent of, its interaction with DHX15. Intriguingly, the ATPase activity of DHX15 is necessary for efficient methylation of DHX15-dependent sites, suggesting a function of DHX15 in regulating snoRNA-guided 2’-O-methylation of rRNA that requires activation by GPATCH4. Overall, our findings extend knowledge on RNA helicase regulation by G-patch proteins and also provide important new insights into the mechanisms regulating installation of rRNA and snRNA modifications, which are essential for ribosome function and pre-mRNA splicing.

overall_design: WT and GPATCH4 KO Flp-In T-Rex HEK293 cell lines or WT Flp-In T-Rex HEK293 cells treated with non-target siRNAs or those targeting DHX15 were  analysed by RiboMeth-seq.

first: Nidhi

last: Kanwal

first: Nicolai

last: Krogh

first: Indira

last: Memet

first: Nicolas

last: Lemus-Diaz

first: Chairini

last: Thome

first: Luisa

last: Welp

first: Athanasia

last: Mizi

first: Philipp

last: Hackert

first: Argyris

last: Papantonis

first: Henning

last: Urlaub

first: Henrik

last: Nielsen

first: Markus


title: GPATCH4 regulates rRNA and snRNA 2’-O-methylation in both DHX15-dependent and -independent manners [ChIP-Seq]

summary: RNA helicases are critical regulators of gene expression and therefore strict regulation of their activities is essential to limit promiscuity and ensure target specify. While some RNA helicases appear dedicated for particular substrates others, such as DHX15, are multifunctional, contributing to more than one gene expression process. Protein cofactors play key roles both in modulating RNA helicase activities and also directing them to appropriate substrate RNAs. The largest family of RNA helicase cofactors, with more than 20 members expressed in human cells, are the G-patch proteins, characterised by a glycine-rich domain via which they associate with specific DEAH/RHA helicases. However, the cognate RNA helicases and cellular functions of numerous human G-patch proteins still remain elusive.  Here, we reveal that the G-patch protein GPATCH4 is a cofactor of DHX15 that stimulates its ATPase activity. We show that GPATCH4 interacts with DHX15 in the nucleolus via residues in its G-patch domain and co-migrates with pre-ribosomal particles, wherein loss of DHX15 reduces pre-ribosomal co-migration of GPATCH4. Using a combination of high-throughput sequencing approaches (ChIP, CRAC and small RNA-seq), we discovered that GPATCH4 crosslinks to the ribosomal DNA locus and precursor ribosomal RNAs as well as binding to small nucleolar- and small Cajal body-associated- RNAs that guide rRNA and snRNA modifications. In line with this, using RiboMeth-seq analysis to globally analyse rRNA and snRNA 2’-O-methylation revealed that knockout of GPATCH4 impairs methylation at various sites. Utilizing targeted 2’-O-methylation-sensitive RNase H-based cleavage assays, we demonstrated that the regulation of 2’-O-methylation by GPATCH4 is both dependent on, and independent of, its interaction with DHX15. Intriguingly, the ATPase activity of DHX15 is necessary for efficient methylation of DHX15-dependent sites, suggesting a function of DHX15 in regulating snoRNA-guided 2’-O-methylation of rRNA that requires activation by GPATCH4. Overall, our findings not only extend knowledge on RNA helicase regulation by G-patch proteins but also provide important new insights into how the installation of rRNA and snRNA modifications, which are essential for ribosome assembly/function and pre-mRNA splicing, is regulated.

overall_design: Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) was performed on stably transfected HEK293 Flp-In cell lines expressing  GPATCH4-His-FLAG or the His-FLAG tag.

first: Kanwal

last: Nidhi

first: Krogh

last: Nicolai

first: Memet

last: Indira

first: Lemus-Diaz

last: Nicolas

first: Thome

last: Chairini

first: Welp

last: Luisa

first: Mizi

last: Athanasia

first: Hackert

last: Philipp

first: Papantonis

last: Argyris

first: Urlaub

last: Henning

first: Nielsen

last: Henrik

first: Bohnsack

last: Katherine

last: Markus


title: ISR inhibition reverses pancreatic β-cell failure in Wolfram syndrome models

city: Shanghai

first: Xiangyi

last: Chen

address: No. 1239, Siping Road, Shanghai, China

institute: Weida Li

summary: Pancreatic β-cell failure induced by WFS1 deficiency is manifested in wolfram syndrome (WS). The lack of a suitable human model in WS has hampered the progress in developing new treatments. Here, human pluripotent stem cell derived pancreatic β cells (SC-β cells) harboring WFS1-deficiency and mouse model of β cell-specific Wfs1 knockout were applied to model β-cell failure in WS. Single-cell RNA sequencing of WFS1-deficient SC-β cells revealed two cell fates along pseudotime trajectory including maturation and stress branch. WFS1 deficiency blocked β-cell fate trajectory to maturation but pushed it towards stress trajectory leading to β-cell failure.

overall_design: We generated hESCs reporter cell line (MEL1 Nkx6.1:linker2a:mCherry; INSGFP/w) that enable to precisely and dynamically trace SC-β cells during differentiated stages. Meanwhile, WFS1 knockout hESCs reporter cell line (WFS1-/-) was established by using a CRISPR/Cas9 knock-out strategy to mimic severe mutations identified from WS patients. We performed single-cell RNA sequencing (scRNA-Seq) for WT and WFS1-/- SC-islets by sorting out GFP and mCherry double-positive SC-β cells with high INS and NKX6.1 expression

first: Rui

last: Hu

first: Qiang

last: Su

first: Zhaoyue

last: Wang

first: Xushu

first: Minglu

last: Xu

first: Zhenning

last: Zhang

first: Li

last: Shao

first: Weida

last: Li


title: Arabidopsis AGO1 N-terminal Poly-Q domain promotes phase separation and association with stress granules during heat stress [RNA-seq]

first: Patricia

last: Baldrich Gonzalez

last: Baldrich

first: Aleksandar

last: Blagojevic

first: Esther

last: Lechner

first: Blake

last: Meyers

first: Pascal

last: Genschik


title: GPATCH4 regulates rRNA and snRNA 2’-O-methylation in both DHX15-dependent and -independent manners [CRAC]

first: Katherine

last: Bohnsack

overall_design: Flp-In T-Rex HEK293 cell lines expressing GPATCH4-His6-2xFLAG (FLAG) or the FLAG tag alone were crosslinked using UV and analysed by CRAC.

first: Nidhi

last: Kanwal

first: Nicolai

last: Krogh

first: Indira

last: Memet

first: Nicolas

last: Lemus-Diaz

first: Chairini

last: Thome

first: Luisa

last: Welp

first: Athanasia

last: Mizi

first: Philipp

last: Hackert

first: Argyris

last: Papantonis

first: Henning

last: Urlaub

first: Henrik

last: Nielsen

first: Markus


title: GPATCH4 regulates rRNA and snRNA 2’-O-methylation in both DHX15-dependent and -independent manners [small RNA-seq]

overall_design: Small RNA-seq was performed on RNAs immunoprecipitated with GPATCH4-His-FLAG or the His-FLAG tag.


title: BHLHE40/41 regulate microglia and peripheral macrophage responses associated with Alzheimer’s disease and other disorders of lipid-rich tissues

status: Public on Jan 29 2024

city: New York

first: Anna

last: Podlesny-Drabiniok

address: 1425 Madison Avenue

institute: Icahn School of Medicine at Mount Sinai

summary: Genetic and experimental evidence suggests that Alzheimer’s disease (AD) risk alleles and genes may influence disease susceptibility by altering the transcriptional and cellular responses of macrophages, including microglia, to damage of lipid-rich tissues like the brain. Recently, sc/nRNA sequencing studies identified similar transcriptional activation states in subpopulations of macrophages in aging and degenerating brains and in other diseased lipid-rich tissues. We collectively refer to these subpopulations of microglia and peripheral macrophages as DLAMs. Using macrophage sc/nRNA-seq data from healthy and diseased human and mouse lipid-rich tissues, we reconstructed gene regulatory networks and identified 11 strong candidate transcriptional regulators of the DLAM response across species. Loss or reduction of two of these transcription factors, BHLHE40/41, in iPSC-derived microglia and human THP-1 macrophages as well as loss of Bhlhe40/41 in mouse microglia, resulted in increased expression of DLAM genes involved in cholesterol clearance and lysosomal processing, increased cholesterol efflux and storage, and increased lysosomal mass and degradative capacity. These findings provide novel targets for therapeutic modulation of macrophage/microglial function in AD and other disorders affecting lipid-rich tissues

overall_design: We performed bulk RNA-seq of FACS sorted microglia from the mouse brain of wild type (WT) and Bhlhe40/41 double knockouts. We used following gating strategy: viability dye-Ly6C-Ly6G-MHCII-CD38-CD45.2+CD11b+.

first: Yiyuan

last: Liu

first: Josefine

last: Dunst

first: Taras

last: Kreslavsky

first: Edoardo

last: Marcora

first: Alison

last: M Goate


city: Austin

last: Warden

address: 2500 Speedway

institute: University of Texas at Austin

summary: Friedreich ataxia (FRDA) is a multisystemic, autosomal recessive disorder caused by a homozygous GAA expansion mutation in the first intron of frataxin (FXN) gene. FXN is a mitochondrial protein critical for iron-sulfur cluster biosynthesis and deficiency impairs mitochondrial electron transport chain functions and iron homeostasis within the organelle. Currently, there is no effective treatment for FRDA. We have previously demonstrated that a single infusion of wild-type hematopoietic stem and progenitor cells (HSPCs) resulted in the prevention of the neurologic and cardiac complications of FRDA in YG8R mice, and the rescue was mediated by FXN transfer from tissue engrafted HSPC-derived microglia/macrophages to diseased neurons/myocytes. For a future clinical translation of this approach, we developed an autologous stem cell transplantation approach using CRISPR/Cas9 for the excision of the GAA repeats in FRDA patients’ CD34+ HSPCs; this strategy leading to increased FXN expression and improved mitochondrial functions in the cells. The aim of the current study is to validate the efficiency and safety of our gene editing approach in a disease-relevant model. To this end, we generated a cohort of FRDA patient-derived iPSCs and isogenic lines that were gene edited with our CRISPR/Cas9 approach. FRDA neurons generated from these iPSCs displayed characteristic apoptotic and mitochondrial phenotype of the disease, such as non-homogenous microtubule staining in neurites, increased caspase-3 expression, mitochondrial superoxide levels, mitochondrial fragmentation, and partial degradation of the cristae compared to healthy controls. These defects were fully prevented in the gene edited neurons. RNASeq analysis of FRDA and gene edited neurons demonstrated striking improvement in gene clusters associated with endoplasmic reticulum (ER) stress in the isogenic lines. These results were validated by molecular and functional studies, and gene edited neurons demonstrated improved ER-calcium release, normalization of ER stress response gene, XBP-1, and significantly increased ER-mitochondrial contacts that are critical for functional homeostasis of both organelles, as compared to FRDA neurons. Ultrastructural analysis for these contact sites displayed severe structural damage to the ER in FRDA neurons, that was undetected in gene edited neurons. Taken together, these results represent a novel finding for disease pathogenesis showing dramatic ER structural damage and function in FRDA. In addition, these results validate the efficacy profile of our FXN gene editing approach, in a disease relevant model improving mitochondrial and cellular, and supporting our approach as an effective strategy for therapeutic intervention for Friedreich’s ataxia.

overall_design:
To investigate the normalization effect of the excision of the GAA repeats in FRDA patient neurons, we generated a cohort of FRDA patient-derived iPSCs and isogenic lines that were gene edited with our CRISPR/Cas9 approach.
We then performed gene expression profiling analysis using data obtained from RNA-seq of 5 cell lines for both edited and unedited GAA repeats in FRDA patient iPSC-derived neurons at 2 weeks of age.
Comparative gene expression profiling analysis of RNA-seq data for edited and unedited GAA repeats in FRDA patient iPSC-derived neurons  was performed.

first: Priyanka

last: Mishra

first: Anusha

last: Sivakumar

first: Avalon

last: Johnson

first: Carla

last: Pernaci

first: Lilas

last: El-Hachem

first: Emily

last: Hansen

first: Rafael

middle: A

last: Badel-Grau

first: Veenita

last: Khare

first: Gabriela

last: Ramirez

first: Sydney

last: Gillette

first: Peng

last: Guo

first: Nicole

last: Coufal

first: Stephanie

last: Cherqui


title: Nitrogen nutrition of E. coli in the mammalian intestine

city: STILLWATER

first: Tyrrell

last: Conway

state: OKLAHOMA

address: 307 Life sciences east, OKLAHOMA STATE UNIVERSITY

department: Microbiology and Molecular Genetics

institute: Oklahoma state university

summary: Our focus of this study is to determine the differential expression of genes related to uptake and degradation of nitrogenous compounds in E. coli colonizing the mouse intestine as compared to E. coli grown in minimal medium in vitro. We colonized CD-1 male mice with E. coli MG1655 StrR wild type and extracted the total RNA from mouse cecal mucus (GSE217743). We also colonized CD-1 male mice with E. coli MG1655 ∆glnG StrR CamR and extracted the total RNA from mouse cecal mucus. We grew E. coli MG1655 StrR wild type in MOPS minimal medium containing high nitrogen (10 mM NH4Cl) and low nitrogen (3 mM NH4Cl) and extracted the total RNA from exponential phase and stationary phase culture. We also grew E. coli MG1655 ∆glnG StrR CamR in MOPS minimal medium containing low nitrogen (3 mM NH4Cl) and extracted the total RNA from exponential phase culture. The extracted RNA samples were sequenced, and differential expression analysis was done to determine the genes related to uptake and degradation of nitrogenous compounds that are important for E. coli colonization. Our results indication nitrogen is not limiting for E. coli in the intestine and genes related to uptake and degradation of several aminoacids, di- and tripeptides, aminosugars, ethanolamine, urea among others are upregualated in the mouse intestine as compared to invitro culture grown in high nitrogen conditions.

sample_organism: Escherichia coli str. K-12 substr. MG1655

overall_design: E. coli MG1655 StrR and E. coli MG1655 ∆glnG StrR CamR were fed separately to CD-1 male mice and on the 9th day, the mice were euthanized, mucus from the cecum was obtained and total RNA was extracted and sequenced. E. coli MG1655 StrR and E. coli MG1655 ∆glnG StrR CamR were cultured in MOPS minimal medium containing high or low nitrogen, and the total transcriptome of exponential phase culture and/or stationary phase culture was sequenced.

first: Sudhir

last: Doranga


title: Single cell profiling in COVID-19 associated acute kidney injury reveals patterns of tubule injury and repair in human

city: Lugano

first: Anna

last: Rinaldi

state: Ticino

address: Tesserete

department: Nephrology

country: Switzerland

institute: Ente Ospedaliero Cantonale

summary: The cellular mechanisms of kidney tubule repair are poorly characterized in human. Here, we applied single-cell RNA sequencing to analyze the kidney in the first days after acute injury in 5 patients with severe COVID19. We found that tubule repair follows two converging patterns involving the plasticity of mature tubule cells and the expansion and differentiation of progenitor-like cells. Tubule repair by cell plasticity displayed substantial similarities between mouse and man and determined the transient expansion of undifferentiated tubule cells with altered functional and metabolic properties. Progenitor-like cells marked by PROM1 proliferated in response to injury and followed a differentiation process characterized by the sequential activation of the WNT, NOTCH and HIPPO signaling pathways. Taken together, our analyses reveal cell state transitions and fundamental cellular hierarchies underlying kidney injury and repair in patients.

overall_design: We characterized by single nucleus RNA sequencing (snRNAseq) kidney biopsies obtained from 5 critically ill patients with COVID-19 before planned withdrawal of resuscitation measures. snRNAseq was carried out by using 10X Genomics Chromium Drop-Seq technology.  Illumina Next-Seq sequencing of barcoded single nuclei libraries was performed and aligned to a pre-mrna genome(GRCh38-2020) with STAR/CellRanger. Seurat v4 in R v4 was used for downstream analyses, including normalization, scaling, and clustering of nuclei. The snRNAseq data were integrated with 3 controls from available datasets generated with similar tissue processing and single cell technology for data control and validation .

first: David

last: Legouis

first: Gregoire

last: Arnoux

first: Thomas

last: Verissimo

first: Jennifer

last: Scotti-Gerber

last: Faivre

first: Manuel

last: Schibler

first: Andrea

first: Maarten

last: Naesens

first: Kari

last: Koppitch

first: Jerome

last: Pugin

first: Solange

last: Moll

first: Andrew

last: McMahon

first: Sophie

last: de Seigneux

first: Pietro

middle: E

last: Cippà


title: Antibody-drug conjugate αEGFR-E-P125A reduces triple-negative breast cancer vasculogenic mimicry, motility, and metastasis through inhibition of EGFR, integrin, and FAK/STAT3 signaling

city: Miami

first: Ankita

middle: Padma

last: Sankar

state: Florida

address: 1580 NW 10th Ave, Batchelor Children's Research Institute

institute: University of Miami

summary: Primary tumor growth and metastasis in triple-negative breast cancer (TNBC) require supporting vasculature, which develop through a combination of endothelial angiogenesis and vasculogenic mimicry (VM), a process associated with aggressive metastatic behavior in which vascular-like structures are lined by tumor cells. We developed αEGFR-E-P125A, an antibody-endostatin fusion protein that delivers a dimeric, mutant endostatin (E-P125A) payload that inhibits TNBC angiogenesis and VM in vitro and in vivo. To characterize the mechanisms associated with induction and inhibition of VM, RNA-seq of MDA-MB-231-4175 TNBC cells grown in a monolayer (2D) was compared to cells plated on Matrigel undergoing VM (3D). We then compared RNA-seq between TNBC cells in 3D and cells in 3D with VM inhibited by αEGFR-E-P125A (αEGFR-E-P125A). Gene set enrichment analysis (GSEA) demonstrated that VM induction activated the IL6-JAK-STAT3 and angiogenesis pathways, which were downregulated by αEGFR-E-P125A treatment. Correlative analysis of the phospho-proteome demonstrated decreased EGFR phosphorylation at Y1069, along with decreased phosphorylation of focal adhesion kinase (FAK) Y397 and STAT3 Y705 sites downstream of α5β1 integrin. Suppression of phosphorylation events downstream of EGFR and α5β1 integrin demonstrated that αEGFR-E-P125A interferes with ligand-receptor activation, inhibits VM, and overcomes oncogenic signaling associated with EGFR and α5β1 integrin crosstalk. In vivo, αEGFR-E-P125A treatment decreased primary tumor growth and VM, reduced lung metastasis, and confirmed the inhibition of signaling events observed in vitro. Simultaneous inhibition of EGFR and α5β1 integrin signaling by αEGFR-E-P125A is a promising strategy for the inhibition of VM, tumor growth, motility, and metastasis in TNBC and other EGFR-overexpressing tumors.

overall_design:
To investigate the mechanisms associated with the induction of VM, RNA-seq of MDA-MB-231-4175 TNBC cells grown in a monolayer (2D) was compared to cells plated on Matrigel and undergoing VM (3D). To investigate the mechanisms associated with the inhibition of VM by αEGFR-E-P125A, RNA-seq of TNBC cells in 3D was compared to cells in 3D with VM inhibited by αEGFR-E-P125A.
We then performed differential gene expression analysis and gene set enrichment analysis using RNA-seq data to identify genes and pathways which were inversely dysregulated upon the induction of VM from 2D to 3D and the inhibition of VM from 3D to αEGFR-E-P125A treatment.
RNA was isolated from 2D, 3D, and αEGFR-E-P125A-treatment conditions in four biological replicates per condition.

first: Joseph

middle: D

last: Rosenblatt

first: Hava

last: Gil-Henn


title: Expression signatures of long non-coding RNAs and mRNAs in lung tissues in obesity and obesity-related acute lung injury

city: Chongqing

first: Wenjing

last: Tang

institute: The First Affiliated Hospital of Chongqing Medical University

type:
Expression profiling by array
Non-coding RNA profiling by array

summary: This result is expected to identify several lncRNAs and mRNAs that are dysregulated in the lung due to obesity, as well as obesity-associated lncRNAs and mRNAs that are further altered in acute lung injury, as biomarkers specific to obesity-associated acute lung injury.  To provide useful information for elucidating the development of acute lung injury exacerbated by obesity and exploring potential therapeutic targets

overall_design: Normal weight mice and obese mice were obtained by feeding a standard diet and a high fat diet of 60%fat for 12 weeks. Then, lipopolysaccharide induced acute lung injury in the obese group of mice. Lung tissue samples were taken from three groups of mice, three from each group. The expression of lncRNA and mRNA was compared between groups in an attempt to reveal the association of lncRNA with inflammatory aggravation of obesity-related acute lung injury.

first: Ke

last: Wei

first: Ling

last: Liu

first: Siqi

last: Wu

first: Yin


title: The chromatin factors SET-26 and HCF-1 oppose the histone deacetylase HDA-1 in longevity and gene regulation in C. elegans [RNA-Seq]

city: Ithaca

first: Siu

middle: Sylvia

last: Lee

address: 520 Campus road

institute: Cornell University

summary: SET-26, HCF-1, and HDA-1 are highly conserved chromatin factors with key roles in development and aging. Here we present mechanistic insights into how these factors regulate gene expression and modulate longevity in C. elegans. We show that SET-26 and HCF-1 cooperate to regulate a common set of genes, and both antagonize the histone deacetylase HDA-1 to limit longevity. We propose a model in which SET-26 recruits HCF-1 to chromatin in somatic cells, where they stabilize each other at the promoters of a subset of genes, particularly mitochondrial function genes, and regulate their expression. HDA-1 opposes SET-26 and HCF-1 on the regulation of a subset of their common target genes and in longevity. Our findings suggest that SET-26, HCF-1, and HDA-1 comprise a mechanism to fine-tune gene expression and longevity and likely have important implications for the mechanistic understanding of how these factors function in diverse organisms, particularly in aging biology.

overall_design: Gene expression profiling through 3' mRNA sequencing of germline-less controls, set-26(tm2467), and hcf-1(pk924) mutants at day 1, day 3, and day 12 of adulthood on empty vector control bacteria or hda-1 RNAi initiated at day 1 of adulthood. Two biological replicates are included for each condition.

first: Felicity

last: Emerson


title: ApoE maintains neuronal integrity via miRNA and H3K27me3-mediated repression [miRNA-seq]

city: Singapore

first: Chin Tong

last: Ong

address: 1 Research Link

country: Singapore

institute: Temasek Life Sciences Laboratory

summary: To better understand the regulatory roles of ApoE in neurogenesis, we differentiated isogenic control and ApoE-/- human neural stem cells (NSC) into mature cortical neurons and cultured them for additional three weeks. In this study, we found that ApoE is only expressed in control NSC and neural progenitor cell (NPC) stages, but not in mature neurons, suggesting that ApoE may epigenetically regulate the integrity and maintenance of the mature neurons. Mechanistic studies revealed that absence of ApoE in NSC led to upregulation of certain microRNAs, including miR-199a-5p. These microRNAs targeted EZH1 to reduce its protein expression, resulting in the lower level of repressive H3K27me3 marks in ApoE-/- NPC and mature neurons. Chromatin immunoprecipitation experiments suggests that the lower level of H3K27me3 at various extracellular matrix organization and angiogenesis genes is responsible for their higher expression and aberrant phenotypes observed in ApoE-/- neurons. Taken together, our results indicate that ApoE maintains pure population of mature neurons via H3K27me3-mediated repression of genes that drive non-neuronal cell proliferation in NPC stage.

overall_design: RNA-seq of poly-A and small RNA in neural stem cells and neurons

first: Jiazi

last: Tan

first: Zhen-Kai

last: Ngian

first: Vinay-Kumar

last: Rao

first: Xianmin

last: Zeng

first: Chin-Tong


title: Influence of Sox4 on chromatin accessibility of adult hepatocytes II

status: Public on Jan 27 2024

city: Philadelphia

first: Takeshi

last: Katsuda

address: 421 Curie Blvd, BRB II/III Rm531

department: Perelman School of Medicine

institute: University of Pennsylvania

summary: Recent studies have demonstrated that hepatocytes can be reprogrammed into biliary epithelial cell-like cells. An earlier work in our laboratory nominated Sox4 as an initiation factor of this reprogramming event To investigate the effect of Sox4 on the change of chromatin landscape of hepatocytes, we performed ATAC-seq of exogenously Sox4-expressed hepatocytes.

overall_design: Mice were injected with adeno-associated viruses (AAVs) packaged with mouse Sox4 (AAV8-TBG-HA-Sox4-P2A-Cre) or empty virus (AAV8-TBG-Cre), and hepatocytes were harvested 4 days post injection by a standard 2-step liberase digestion method followed by a Percoll enrichment step.

first: Jonathan

last: Sussman

first: Ben

middle: Z

last: Stanger


title: ApoE maintains neuronal integrity via miRNA and H3K27me3-mediated repression

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

overall_design: Refer to individual Series


title: The chromatin factors SET-26 and HCF-1 oppose the histone deacetylase HDA-1 in longevity and gene regulation in C. elegans


title: GPATCH4 regulates rRNA and snRNA 2'-O-methylation in both DHX15-dependent and -independent manners

first: Katherine

last: Bohnsack


title: Factors governing the transcriptome changes and chronological lifespan of fission yeast during phosphate starvation II

first: Ana

middle: M

last: Sanchez

address: 430 E 67th Street, RRL-865

institute: Memorial Sloan Kettering Cancer Center

summary: This RNA-Seq analysis compares gene expression of the wild-type fission yeast (Schizosaccharomyces pombe) strain at various times after transfer to defined phosphate-replete ePMGT(+PO4) medium (2, 4, 8 HR) contrasting to the WT fission yeast S. pombe cells grown in phosphate-replete YES medium (0HR).

overall_design: 3 test samples and 1 control sample was processed (each sample has 3 replicate experiments)

first: Angad

last: Garg

first: Beate

last: Schwer

first: Stewart

last: Shuman


title: ApoE maintains neuronal integrity via miRNA and H3K27me3-mediated repression [RNA-seq]


title: Targeting PHGDH reverses the immunosuppressive function in macrophages and enhances cancer immunotherapy

city: Vienna

first: Wolfram

last: Weckwerth

address: Djerassiplatz 1

country: Austria

institute: University of Vienna

summary: Phosphoglycerate dehydrogenase (PHGDH) has emerged as a crucial factor in facilitating macromolecule synthesis, neutralizing oxidative stress, and regulating methylation reactions in cancer cells, lymphocytes, and endothelial cells. However, the role of PHGDH in tumor-associated macrophages (TAMs) remains poorly understood. Here, we find that T helper 2 (Th2) cytokine interleukin-4 and the tumor-conditioned media increase the expression of PHGDH in macrophages and promote immunosuppressive M2 activation and proliferation. Loss of PHGDH disrupts cellular metabolism and mitochondrial respiration essential for immunosuppressive macrophages. Mechanistically, PHGDH-mediated serine biosynthesis promotes α-ketoglutarate production, which activates mTORC1 signaling and contributes to the maintenance of a M2-like macrophage phenotype in the tumor microenvironment. Genetically ablating PHGDH in macrophages of tumor-bearing mice results in attenuated tumor growth, reduced TAM infiltration, a phenotypic shift of M2-like TAMs towards an M1-like phenotype and enhanced anti-tumor T cell immunity. Our study provides a strong basis for further exploration of PHGDH as a potential target to counteract TAM-mediated immunosuppression and hinder tumor progression.

overall_design: Gene expression profiling analysis of RNA-seq data obtained from Phgdhfl/fl Cx3cr1-Cre (macrophage-specific Phgdh deletion) bone marrow-derived macrophages (BMDMs) and Phgdhfl/fl BMDMs that were stimulated with AE17-tumor-conditioned medium for 24 hours. Each condition contains 4 biological replicates.

first: Zhengnan

last: Cai


title: Factors governing the transcriptome changes and chronological lifespan of fission yeast during phosphate starvation I

summary: RNA-seq analysis compares gene expression of the fission yeast (Schizossacharomyces pombe) ecl123∆ mutant at various times after phosphate starvation (4, 8, 12, 24, 36 and 48h) to the ecl123∆ mutant fission yeast strain grown in phosphate replete conditions (0h, prior to phosphate starvation).

overall_design: 6 test samples and 1 control sample were processed (each sample has 3 replicate experiments).


title: Oncogenic c-Myc induces replication stress by increasing chromatin occupancy of cohesins at CTCF sites

status: Public on Jan 26 2024

city: London

first: Chiara

last: Perrod

state: England

address: Gower St

department: LMCB

institute: UCL

summary: Oncogenic c-Myc induces replication stress via increasing the amount of cohesins bound to chromatin accumulating at CTCF sites

overall_design: Upon MYC induction, and upon CTCF downregulation, cohesin binding was assessed quantitatively by qChIP-seq.

first: Robertus

last: de Bruin


title: Gene expression analysis in the zebrafish fractured scale exposed to ELF-EMFs

city: Kanazawa

first: Isao

last: Kobayashi

state: Ishikawa

address: Kakumamachi

institute: Kanazawa University

summary: The zebrafish scale is a thin membranous bone embedded in the skin and consists of osteoblasts, osteoclasts, and bone matrix, providing an elegant model to understand bone metabolisms. we developed an in vivo model system using zebrafish scales to investigate the effect of extremely low-frequency-electromagnetic fields (ELF-EMFs) on fracture healing. In this study, we have performed RNA-seq analysis on intact scales, fractured scales not exposed to ELF-EMFs, and fractured scales exposed to 10 militesra (mT) of ELF-EMFs.

overall_design: mRNA profiles of zebrafish scales were generated by Lasy-seq using Illumina NovaSeq X.

first: Jingjing

last: Kobayashi-Sun