title: Cyotoxicity in CD8+ T cells differs among adjuvant immune checkpoint blockade therapies

first: Ramin

last: Herati

state: New York

address: 430 E. 29th Street, 1st floor, room 135A-C

institute: NYU School of Medicine

summary: While immune checkpoint blockade therapies are commonly utilized during the course of metastatic cancer treatment, much remains unknown regarding the mechanisms underlying individual therapies. CD8+ T cells from 8 patients that had been treated with either adjuvant Ipilimumab or Nivolumab were compared to assess immunological differences between the therapies. CD8+ T cells from patients that had received Ipilimumab exhibited higher cytotoxic gene expression and pathway enrichment than those from patients that had received Nivolumab. Cytotoxic activity was especially driven by cluster 3 in both groups, with the proportion of CD8+ T cells in the cluster expanding significantly in Ipilimumab patients over the course of treatment, in contrast to Nivolumab patients where this pattern was not seen. This data suggests that, on the CD8+ T cell level, Ipilimumab and Nivolumab exhibit mechanistic differences that are driven by cytotoxic genes and pathways.

overall_design: Multimodal single-cell RNA sequencing was used to compare CD8+ T cells in 8 patients that had been treated with either adjuvant Ipilimumab or Nivolumab.

first: Elizabeth

last: Severa

first: Sabrina

last: Solis

middle: S

first: Jeff

last: Weber


title: CEBPβ/IL-1β/TNFα Feedback Loop Drives Drug Resistance to BCL2 and MDM2 Inhibitors in Monocytic Leukemia Cells

status: Public on Oct 17 2024

city: Portland

first: Peter

last: Ryabinin

state: OR

address: 3181 SW Sam Jackson Park Rd

country: USA

institute: Oregon Health and Science University

type: Expression profiling by high throughput sequencing

summary:
Ventoclax-based combinations are a new standard of care for patients with acute myeloid leukemia (AML) who are not eligible for intensive chemotherapy, but not all patients respond to these treatments, and those who do may relapse. MDM2 inhibitors are promising therapeutics for treating TP53 wild-type tumors, including most de novo AML cases, but clinical trials have shown modest and variable clinical activity. Functional genomic data suggest that venetoclax and the MDM2 inhibitor, idasanutlin, may be ineffective against monocytic leukemia (FAB M4/M5) or leukemia cells with a high immune signature. We hypothesize upregulated myeloid transcription factors and enrichment of certain environmental cues that confer intrinsic and extrinsic drug resistance to these cells.
We show that monocytic leukemia cells express a high level of CEBPB and CEBPB overexpression confers drug resistance to a broad range of BH3 mimetics, venetoclax combinations, and MDM2 inhibitors by downregulating CASP3, CASP6, BCL2, and p53 pathway targets, while upregulating MCL1, BCL2A1, and the NFKB/IL1/TNFA pathway. Moreover, leukemia monocytes can extrinsically protect leukemia blasts from venetoclax and MDM2 inhibition by secreting elevated IL1 and TNFA, which drive myelo/monocytic differentiation and upregulate inflammatory cytokines and receptors, including IL1/TNFA pathway in an autoregulatory loop. Remarkably, IL1A/IL1B and TNFA uniquely upregulate CEBPB expression in M4/M5 cells and protect them from apoptosis induced by venetoclax and MDM2 inhibitors. Conversely, TNFA treatment enhances extrinsic apoptosis in M0/M1 leukemia cells. Inhibitors of IRAK or MAPK14 (p38), which block IL1/TNFA signaling, showed synergistic cytotoxicity in M4/M5 AML when combined with venetoclax and idasanutlin.
In summary, we described a targetable, positive feedback loop between CEBPB and IL1/TNFA in monocytic leukemia that drives intrinsic and extrinsic drug resistance to BCL2 and MDM2 inhibitors, offering promising therapeutic strategies to enhance treatment efficacy for monocytic leukemia.

overall_design:
4 samples:
OCI-AML3 cell line: Plex CEBPB
OCI-AML3 cell line: Plex CEBPB
OCI-AML3 cell line: Plex Control
OCI-AML3 cell line: Plex Control

first: Basil

last: Allen

first: Anthony

last: Wang

first: Daniel

last: Bottomly

first: Shannon

last: McWeeney

first: Haijiao

last: Zhang


title: Extreme positive epistasis for fitness in monosomic yeast strains - part 2

status: Public on Oct 16 2024

city: Krakow

first: Hanna

last: Tutaj

address: Gronostajowa 7

country: Poland

institute: Jagiellonian University

summary: The loss of a single chromosome in a diploid organism halves the dosage of many genes and is usually accompanied by a substantial decrease in fitness. We asked whether this decrease simply reflects the joint damage caused by individual gene dosage deficiencies. We measured the fitness effects of single heterozygous gene deletions in yeast and combined them for each chromosome. This predicted a negative growth rate, i.e. lethality, for multiple monosomies. However, monosomic strains remained alive and grew as if much (often most) of the damage caused by single mutations had disappeared, revealing an exceptionally large and positive epistatic component of fitness. We looked for functional explanations by analyzing the transcriptomes. There was no evidence of increased (compensatory) gene expression on the monosomic chromosomes. Nor were there signs of the cellular stress response that would be expected if monosomy led to protein destabilization and thus cytotoxicity. Instead, all monosomic strains showed extensive upregulation of genes encoding ribosomal proteins, but in an indiscriminate manner that did not correspond to their altered dosage. This response did not restore the stoichiometry required for efficient biosynthesis, which probably became growth limiting, making all other mutation-induced metabolic defects much less important. In general, the modular structure of the cell leads to an effective fragmentation of the total mutational load. Defects outside the module(s) currently defining fitness lose at least some of their relevance, producing the epiphenomenon of positive interactions between individually negative effects.

overall_design: RNA-seq for 3 monosomic (experimental)  and 1 euploid (control) Saccharomyces cerevisiae strains. One sample per strain is provided.

first: Katarzyna

last: Tomala

first: Adrian

last: Pirog

first: Marzena

last: Marszałek

first: Ryszard

last: Korona


title: Interplay between nutrient stress and lysosomal signaling imprints immune memory in tissues [ATAC-seq]

city: Memphis

first: Hongbo

last: Chi

state: TN

address: 262 Danny Thomas Place

department: Immunology

institute: St Jude Children's Research Hospital

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Uncovered organelle signaling and metabolic processes shaping CD8+ tissue-resident memory (TRM) development

overall_design: We used ATAC seq to compare chromatin accessibility profiles of sgNTC or sgFlcn-transduced OT-1 cells purified from the spleen and siIEL from the same host on day 7.5 post-LM-OVA infection

first: Hao

last: Shi

first: Jana

middle: L

last: Raynor


title: A Molecular Switch from Tumor Suppressor to Oncogene in ER-Positive Breast Cancer: Role of Androgen Receptor, JAK-STAT, and Lineage Plasticity [ChIP-seq]

middle: Lee

last: Johnson

state: Tennessee

address: 71 S Manassas

department: Molecular Informatics Core

institute: UTHSC

summary: Cancers develop resistance to inhibitors of oncogenes mainly due to target-centric mechanisms such as mutations and splicing. While inhibitors or antagonists force targets to unnatural conformation contributing to protein instability and resistance, activating tumor suppressors may maintain the protein in an agonistic conformation to elicit sustainable tumor growth inhibition. Due to lack of tumor suppressor-agonists, this hypothesis and mechanisms underlying resistance are not understood. In estrogen receptor (ER)-positive breast cancer (BC), androgen receptor (AR) is a druggable tumor suppressor and offers a promising avenue for this investigation. Spatial genomics suggest that the molecular portrait of AR-expressing BC cells in tumor microenvironment corresponds to better overall patient survival, clinically confirming AR’s role as a tumor suppressor. Ligand-activation of AR-positive BC xenografts reprograms cistromes, inhibits oncogenic pathways, and promotes cellular elasticity towards a more differentiated state, while sustained treatment results in cistromes rearrangement towards transcription factor PROP1, transformation of AR into an oncogene, and activation of the JAK/STAT pathway, all culminating in lineage plasticity to an aggressive resistant subtype. While the molecular profile of AR agonist-sensitive tumors corresponds to better patient survival, the profile represented in the resistant phenotype corresponds to shorter survival. Inhibition of activated oncogenes in resistant tumors reduces growth and re-sensitizes them to AR agonists. Together, these findings indicate that persistent activation of a tumor suppressor is not fail-safe and leads to resistance through lineage plasticity-driven tumor metamorphosis. Our work provides a framework to explore the above phenomenon across multiple cancer types and underscores the importance of factoring the sensitization of tumor suppressor targets while developing agonist-like drugs

overall_design:
To determine the androgen receptor (AR), estrogen receptor (ER), and FOXA1 cistrome in breast cancer xenografts treated with AR agonists and those xenografts that have developed resistance to AR agonists.
G1 = Vehicle
G2 = DHT
G4 = Enobosarm
G4R = Enobosarm Resistant

first: Sarah

last: Asemota

first: Ramesh

last: Narayanan


title: Interplay between nutrient stress and lysosomal signaling imprints immune memory in tissues [scRNAseq]

overall_design: We used scRNAseq to compare gene transcriptome profiles of sgNTC or sgFlcn-transduced OT-1 cells purified from the siIEL on days 4.5 and 7.5 post-LM-OVA infection


title: Dissecting the temporal genetic networks programming soybean embryo development from embryonic morphogenesis to post-germination

city: Kannapolis

first: Tzung-Fu

last: Hsieh

state: NC

address: Tzung-Fu Hsieh, 600 Laureate Way

department: Plant and Microbial Biology

institute: North Carolina State University

summary: To understand the molecular events underlying seed desiccation and germination, we performed transcriptome analysis of soybean (Glycine max) embryos at three seed developmental stages (early, mid and late desiccation), mature dry seeds, and three germination stages (3, 16 and 27 hour-after-imbibition).

overall_design: RNA sequencing using embryos from seven stages was performed to examine global gene activity changes.

first: Jer-Young

last: Lin

first: Hsieh

last: Tzung-Fu


title: Extreme positive epistasis for fitness in monosomic yeast strains - part 1

overall_design: Examination of transcription levels of eight BY4743 Saccharomyces cerevisiae diploid strains lacking different single chromosomes from a pair (monosomics), three replicates each, control diploid strain BY4743 in three replicates included


title: A Molecular Switch from Tumor Suppressor to Oncogene in ER-Positive Breast Cancer: Role of Androgen Receptor, JAK-STAT, and Lineage Plasticity

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

overall_design: Refer to individual Series


title: Abortive infection of bat fibroblasts with SARS-CoV-2

status: Public on Oct 15 2024

city: Cambridge

first: Punam

last: Bisht

state: Massachusetts

address: 455 Main St

institute: Whitehead Institute

summary: Bats are tolerant to highly pathogenic viruses such as Marburg, Ebola, and Nipah, suggesting the presence of a unique immune tolerance toward viral infection. Here, we compared SARS-CoV-2 infection of human and bat (Rhinolophus ferrumequinum) pluripotent cells and fibroblasts. Since bat cells do not express an ACE2 receptor that allows virus infection, we transduced the human ACE2 receptor into the cells and found that transduced cells can be infected with SARS-CoV-2. Compared to human ESCs-hA, infected bat iPSCs-hA produced about a 100-fold lower level of infectious virus and displayed lower toxicity. In contrast, bat fibroblasts (BEF-hA) produced no infectious virus while being infectable and synthesizing viral RNA and proteins, suggesting abortive infection. Indeed, electron microscopy failed to detect virus-like particles in infected bat fibroblasts in contrast to bat iPSCs or human cells, consistent with the latter producing infectious viruses. This suggests that bat somatic but not pluripotent cells have an effective mechanism to control virus replication. Consistent with previous results by others, we find that bat cells have a constitutively activated innate immune system, which might limit SARS-CoV-2 infection compared to human cells.

sample_organism:
Homo sapiens
Rhinolophus ferrumequinum

platform_organism:
Homo sapiens
Rhinolophus ferrumequinum

overall_design: RNA Sequencing

first: M

middle: I

last: Barrasa

first: Rudolf

last: Jaenisch


title: Predictive prioritization of functional enhancers in human pancreas cells [HiChIP]

city: bethesda

first: Efsun

last: Arda

state: Maryland

address: 41 medlars drive

institute: National Institutes of Health

summary: We mapped enhancer-promoter interactions genome-wide in purified human pancreas cell types

overall_design: We performed HiChIP using the H3K27ac antibody to identify enhancer-promoter interactions in pancreatic alpha, beta, delta, acinar and duct cells, isolated and purified from organ donors. We followed the protocol described in Mumbach et al (PMID: 27643841)

first: Li

last: Wang

first: Songjoon

last: Baek

first: H

middle: E


title: Rab8a restores diverse innate functions of CD11b+CD11c+ dendritic cells from aged mice

status: Public on Oct 14 2024

city: Mohali

first: Sharvan

last: Sehrawat

state: Punjab

address: SAS Nagar

department: Biological Sciences

country: India

institute: IISER Mohali

summary: Here, we demonstrate that Rab8a, a small GTPase, plays critical role in the functioning of dendritic cells (DCs) of aging host. Compared with the cells of young mice, CD11c+CD11b+ bone marrow derived dendritic cells (BMDCs) of aged mice generated a diminished type I IFN response when stimulated with Influenza A virus (IAV) and murine herpesvirus 68 (MHV68). In comparison to those of young mice, BMDCs of aged mice inefficiently presented exogenous antigens to CD8+ T cells in vitro and in vivo. While the Rab8a overexpressing BMDCs of aged animals regained functionality and generated a potent anti-viral IFN response, the Rab8a depleted cells of young animals were severely compromised in producing IFNs. BMDCs of young and aged animals were transcriptionally distinct but the Rab8a overexpressing cells of aged animals widely shared transcriptome with those of young animals. Ovalbumin fed Rab8a reconstituted BMDCs of aged animals upregulated Rab11 than did the control cells. Both Rab8a and Rab11 preferentially co-localised with the generated SIINFEKL peptide-H-2Kb complexes in the endosomal recycling compartment (ERC) to efficiently induce the surface display of such complexes. Compared with the control cells, the Ova fed Rab8a reconstituted BMDCs of aged animals expanded more of the TCR tg OT1 cells to better control IAV (WSN-SIINFEKL) infection. Therefore, targeting Rab8a might serve as a strategy to promote the functioning of DCs in the aging host to achieve favourable outcome of viral infection.

overall_design: Aged Bone marrow cells transduced with lentiviruses for Rab8a overexpression and untransduced Young and Aged Bone marrow cells were cultured in the presence of IL4 and GMCSF for 5 days. The CD11b+CD11c+ cells were then sorted for RNA isolation and sequencing.

first: Sudhakar

last: Singh

first: Azeez

last: Tehseen


title: Comprehensive identification of coronary artery disease-associated variants regulating vascular smooth muscle cell gene expression [lentiMPRA]

city: Charlottesville

first: Nicolas

last: Barbera

state: Virginia

address: 1335 Lee Street

department: Department of Genome Sciences

institute: University of Virginia

summary: Coronary artery disease (CAD) is a complex disorder with genetic and environmental influences. Genome-wide association studies (GWAS) have identified over 300 genomic loci associated with disease risk. However, identifying the functional variants within these loci has been limited in large part due to linkage disequilibrium. This represents a critical step in understanding the molecular mechanisms underlying disease risk. To identify and prioritize candidate causal CAD-associated variants, we performed lentivirus-based massively parallel reporter assays (lentiMPRAs) in primary vascular smooth muscle cells (SMCs), which play significant roles in atherosclerosis, the underlying cause of CAD. We tested 25892 CAD-associated variants for their allele-specific enhancer activity in quiescent and proliferative SMCs, modeling healthy and disease conditions. We identified 122 candidate variants showing significant enhancer activity and differences in reporter gene expression between risk and non-risk alleles. We also identified 23 variants showing condition-biased allelic imbalance and 41 variants showing sex-biased allelic imbalance. We further functionally characterized 25892 variants by performing CUT&RUN assays to identify variants in enhancer and promoter regions of SMCs. By integrating the results of these experiments, we identified a credible set of 49 CAD-associated variants in functionally relevant regions. Furthermore, by comparing these identified variants with our previously obtained expression quantitative trait loci (eQTL) data, 27 of these 49 variants were associated with SMC gene expression levels. Finally, we performed CRISPRi experiments on 8 variants comprising 9 variant-gene pairs, rs35976034 (MAP1S), rs4888409 (CFDP1), rs73193808 (MAP3K7CL), rs67631072 (INPP5B/FHL3), rs1651285 (SNHG18), rs17293632 (SMAD3), rs2238792 (ARVCF), rs4627080 (NRIP3), to confirm their regulatory potential of nearby gene expression. Taken together, our results comprehensively fine-map the causal variants that confer increased risk of CAD through their effects on vascular smooth muscle cells.

overall_design: lentiMPRA were performed on 6 vascular smooth muscle cell lines in two different culture conditions, testing 25892 CAD-associated variants for enhancer activity and allelic imbalance.

first: Alexia

last: Wallace

first: Noah

middle: R

last: Perry

first: Lily

last: Lei

first: Hester

middle: M

last: den Ruijter

first: Mete

last: Civelek


title: Effect of housing condition (group- or single-housing) on gene expression in African turquoise killifish

status: Public on Oct 13 2024

city: Kobe

first: Chika

last: Takahashi

address: 2-2-3 Minatojima-minamimachi,Chuo-ku

country: Japan

institute: RIKEN


title: Predictive prioritization of functional enhancers in human pancreas cells [ATAC-Seq]

summary: We mapped accessible chromatin regions genome-wide in purified human pancreas cell types

overall_design: We performed ATAC-seq on alpha, beta, delta, acinar and duct cell populations, isolated and purified from donor pancreas. We used the protocol described in Corces et al (PMID: 28846090).


title: Spatially Resolved Transcriptomic Signatures of Hippocampal Subregions and Arc-Expressing Ensembles in Active Place Avoidance Memory [Spatial Transcriptomics]

status: Public on Oct 11 2024

city: Brooklyn

first: Oleg

middle: V

last: Evgrafov

address: 450 Clarkson Ave

institute: SUNY Downstate Health Sciences University

summary: The rodent hippocampus is a spatially organized neuronal network that supports the formation of spatial and episodic memories. We conducted bulk RNA sequencing and spatial transcriptomics experiments to measure gene expression changes in the dorsal hippocampus following the recall of active place avoidance (APA) memory. Through bulk RNA sequencing, we examined the gene expression changes following memory recall across the functionally distinct subregions of the dorsal hippocampus. We found that recall induced differentially expressed genes (DEGs) in the CA1 and CA3 hippocampal subregions were enriched with genes involved in synaptic transmission and synaptic plasticity, while DEGs in the dentate gyrus (DG) were enriched with genes involved in energy balance and ribosomal function. Through spatial transcriptomics, we examined gene expression changes following memory recall across an array of spots encompassing putative memory-associated neuronal ensembles marked by the expression of the IEGs Arc, Egr1, and c-Jun. Within samples from both trained and untrained mice, the subpopulations of spatial transcriptomic spots marked by these IEGs were transcriptomically and spatially distinct from one another. DEGs detected between Arc+ and Arc- spots exclusively in the trained mouse were enriched in several memory-related gene ontology terms, including “regulation of synaptic plasticity” and “memory.” Our results suggest that APA memory recall is supported by regionalized transcriptomic profiles separating the CA1 and CA3 from the DG, transcriptionally and spatially distinct IEG expressing spatial transcriptomic spots, and biological processes related to synaptic plasticity as a defining the difference between Arc+ and Arc- spatial transcriptomic spots.

overall_design: To explore the spatial distribution molecular mechanisms following memory retention test, spatial transcriptomics was performed using coronal brain sections containing the dorsal hippocampus collected from mice trained in the active place avoidance task (Trained) or exposed to the rotating active place avoidance arena with no active shock zone (Untrained).

first: Isaac

last: Vingan

first: Shwetha

last: Phatarpekar

first: Victoria Sook Keng

last: Tung

first: A Iván

last: Hernandez

first: Juan Marcos

last: Alarcon


title: Effect of housing condition (group- or single-housing) on gene expression in African turquoise killifish I.

summary: To explore the effects of the housing condition (group- or single-housing) on killifish, we performed RNA-seq analysis for the whole bodies of male and female juveniles,  gonads of males and females from puberty to middle age, and livers of young and old males.

overall_design: In the GRZ strain, during the first three weeks after hatching, the fish were reared under the group- or single-housing condition. After three weeks, all fish were single-housed.

first: Takuya

last: Yamamoto

first: Eisuke

last: Nishida


title: Effect of housing condition (group- or single-housing) on gene expression in African turquoise killifish II.


title: P4HB maintains Wnt-dependent stemness in glioblastoma stem cells as a precision therapeutic target and serum marker

city: HENAN

first: ZHENG

last: YUAN

address: Jinming Campus, Henan University, Minglun Street, Longting District, Kaifeng, Henan, China

country: China

institute: HENAN University

summary:
Glioblastoma stem cells (GSCs) are pivotal in the recurrence and drug resistance of glioblastoma multiforme (GBM). However,
precision therapeutic and diagnostic markers for GSCs have not been fully established. Here, using bioinformatics and experimental
analysis, we identified P4HB, a protein disulfide isomerase, as a serum marker that maintains stemness in GSCs through the Wnt/β-
catenin signaling pathway. Transcriptional silencing of P4HB induces apoptosis and diminishes stem cell-like characteristics in GSCs.
Treatments with the chemical CCF624 or the China National Medical Products Administration (NMPA)-approved securinine significantly prolonged survival in patient-derived xenograft mouse models, underscoring P4HB’s potential as a therapeutic target and
presenting an expedited path to clinical application through drug repurposing. Additionally, elevated P4HB levels in patient serum were found to correlate with disease progression, underscoring its utility as a biomarker and its promise for precision medicine.

overall_design: To validate the downstream cytokines regulated by P4HB, we conducted an experiment involving X01 GSCs. These cells were transfected with a control shRNA lentivirus and two distinct shP4HB lentiviruses. Following the transfection, RNA sequencing was performed to analyze the expression profiles. This experimental design aims to elucidate the regulatory role of P4HB on cytokine expression in glioma stem cells.

first: Zheng

last: Yuan


title: Delivery of A Jagged1-PEG-MAL hydrogel with Pediatric Human Bone Cells Regenerates Critically-Sized Craniofacial Bone Defects

city: Atlanta

first: Steven

last: Goudy

state: GA

address: 2174 N Druid Hills Rd, 1st Floor

institute: Emory University

summary: Treatments for congenital and acquired craniofacial (CF) bone abnormalities are limited and expensive. Current reconstructive methods include surgical correction of injuries, short-term bone stabilization, and long-term use of bone grafting solutions, including implantation of (i) allografts which are prone to implant failure or infection, (ii) autografts which are limited in supply. Current bone regenerative approaches have consistently relied on BMP-2 application with or without addition of stem cells. BMP2 treatment can lead to severe bony overgrowth or uncontrolled inflammation, which can accelerate further bone loss. Bone marrow-derived mesenchymal stem cell-based treatments, which do not have the side effects of BMP2, are not currently FDA approved, and are time and resource intensive. There is a critical need for novel bone regenerative therapies to treat CF bone loss that have minimal side effects, are easily available, and are affordable. In this study we investigated novel bone regenerative therapies downstream of JAGGED1 (JAG1). We previously demonstrated that JAG1 induces murine cranial neural crest (CNC) cells towards osteoblast commitment via a NOTCH non-canonical pathway involving JAK2-STAT5 (1) and that JAG1 delivery with CNC cells elicits bone regeneration in vivo. In this study, we hypothesized that delivery of JAG1 and induction of its downstream NOTCH non-canonical signaling in pediatric human osteoblasts constitute an effective bone regenerative treatment in an in vivo murine bone loss model of a critically-sized cranial defect. Using this CF defect model in vivo, we delivered JAG1 with pediatric human bone-derived osteoblast-like (HBO) cells to demonstrate the osteo-inductive properties of JAG1 in human cells and in vitro we utilized the HBO cells to identify the downstream non-canonical JAG1 signaling intermediates as effective bone regenerative treatments. In vitro, we identified an important mechanism by which JAG1 induces pediatric osteoblast commitment and bone formation involving the phosphorylation of p70 S6K. This discovery enables potential new treatment avenues involving the delivery of tethered JAG1 and the downstream activators of p70 S6K as powerful bone regenerative therapies in pediatric CF bone loss.

overall_design: Pediatric human bone-derived osteoblast-like cell line treated with JAGGED1 ligand, DAPT (canonical NOTCH signaling inhibitor), both, or vehicle (n=3) for 24 hours.


title: IFN-ɣ derived from activated human CD4+ T cells inhibits the replication of SARS-CoV-2 depending on cell-type and viral strains.

status: Public on Oct 10 2024

city: Kyoto

first: Jun

last: Shimizu

state: Kyoto

address: Nishikyo-ku, 1-36 Goryo-ohara, KKVP 206

institute: MiCAN Technologies Inc.

summary: Severe acute respiratory syndrome coronavirus 2 (SARS‑CoV‑2) infection and vaccination induces T cell immunity as well as B cell responses in immunocompetent individuals. The mechanisms of anti-viral effect by CD4+ T cells as a part of T cell immunity are not fully understood. Here we used and analyzed the culture supernatant (SN) prepared from polyclonally stimulated human CD4+ T cells as a model of soluble mediator(s) from SARS-CoV-2-stimulated CD4+ T cells. Interestingly, this SN inhibited SARS-CoV-2 propagation in a viral strain- and host cell type-dependent manner. The most susceptible strain was original wild-type, and the insusceptible Delta strain in the case of human monocyte cell line. Antibody-dependent enhancement (ADE) of infection with original strain was also abolished in the presence of this SN. The following study revealed that the inhibitory effect against viral expansion by the SN was ascribed to interferon-ɣ (IFN-ɣ) included in SN, not to IFN-α, -β and -λ. These results highlight the important potential of IFN-ɣ as anti-SARS-CoV-2 mediator derived from CD4+ T cells and suggest that we need to understand the SARS-CoV-2 strain-dependent sensitivity against IFN-ɣ in controlling clinical manifestations. In addition, characterization of new SARS-CoV-2 variants in terms of IFN-ɣ-sensitivity will have important implications in selecting therapeutic strategy.

overall_design: To investigate the involvement of CD4 T cells in SARS-CoV-2 infection, we established the culture system of human monocyte cells (clone 35). We performed gene expression profiling analysis using data obtained from RNA-seq of 3 different conditions at two time points.  Comparative gene expression profiling analysis of RNA-seq data for clone 35 cells before and after treatment.

first: Guang

middle: H

last: Ong


title: RNA-seq Validation of MicroRNA Expression Signatures for Precision Melanoma Diagnosis and Prognostication

city: Balaclava

first: Ryan

last: Van Laar

state: VIC

address: PO Box 234

country: Australia

institute: Geneseq Biosciences

type: Non-coding RNA profiling by high throughput sequencing

summary: This study aimed to transition genomic signatures of invasive cutaneous melanoma (MEL38 for diagnosis and MEL12 for prognosis) from Nanostring to a high-throughput RNAseq platform using the gel-free NEXTflex Small RNA Library Prep Kit. We validated against 59 FFPE and 61 plasma samples from patients with melanoma and normal tissue . MEL38 accurately diagnosed invasive melanoma across different sample types and platforms, unaffected by disease stage or data origin. The MEL12 signature significantly stratified patients by survival rates in solid tissues, surpassing previous prognostic models based on DNA, mRNA, or protein analysis, with hazard ratios indicating high risk. In plasma, MEL12 also differentiated survival outcomes. Overall, the study confirms the reliability of MEL38 and MEL12 in predicting melanoma presence and prognosis, demonstrating their superiority and adaptability across specimen types and platforms, marking a substantial advancement in personalized melanoma management.

overall_design: miRNASEQ profiling of 61 plasma patient samples and 59 ffpe biopsy samples from individuals with invasive melanoma or related benign phenotypes. Plasma samples consisted of 49 invasive and 12 non-invasive/benign melanoma phenotypes. FFPE solid tissue biopsy samples consisted of 27 invasive and 32 non-invasive/benign melanoma phenotypes. Each sample was scored and assessed using MEL38 and MEL12 miRNA signatures.

first: Christopher

last: Love

first: Lauren

last: Coombs

last: van Laar


title: Cell type-specific weighting-factors to solve solid organs-specific limitations of single cell RNA-sequencing

status: Public on Oct 09 2024

city: Seika-cho, Soraku-gun

first: Thomas

middle: N

last: Sato

address: 2-2-2 Hikaridai

institute: Karydo TherapeutiX, Inc.

summary: While single-cell RNA-sequencing (scRNA-seq) is a popular method to analyze gene expression and cellular composition at single-cell resolution, it harbors shortcomings: The failure to account for cell-to-cell variations of transcriptome-size (i.e., the total number of transcripts per cell) and also cell dissociation/processing-induced cryptic gene expression. This is particularly a problem when analyzing highly heterogeneous solid tissues/organs, which requires cell dissociation for the analysis. As a result, there exists a discrepancy between bulk RNA-seq result and virtually reconstituted bulk RNA-seq result using its composite scRNA-seq data. To fix this problem, we propose a computationally calculated coefficient, “cell type-specific weighting-factor (cWF)”. Here, we introduce a concept and a method of its computation and report cWFs for 76 cell-types across 10 solid organs. Their fidelity is validated by more accurate reconstitution and deconvolution of bulk RNA-seq data of diverse solid organs using the scRNA-seq data and the cWFs of their composite cells. Furthermore, we also show that cWFs effectively predict aging-progression, implicating their diagnostic applications and also their association with aging mechanism. Our study provides an important method to solve critical limitations of scRNA-seq analysis of complex solid tissues/organs. Furthermore, our findings suggest a diagnostic utility and biological significance of cWFs.

overall_design: mRNA profiles of 11 weeks-old male wild-type mice were generated by QuantSeq 3' mRNA-sequencing.

first: Kengo

last: Tejima

first: Satoshi

last: Kozawa


title: Spatially Resolved Transcriptomic Signatures of Hippocampal Subregions and Arc-Expressing Ensembles in Active Place Avoidance Memory [RNA-seq]

first: Oleg

last: Evgrafov

overall_design: To explore the molecular mechanisms following memory retention test across the dorsal hippocampus, bulk RNA-sequencing was performed on the Dentate Gyrus (DG), CA3, and CA1 hippocampal subregions collected from mice trained in the active place avoidance task (Trained) or exposed to the rotating active place avoidance arena with no active shock zone (Untrained).

first: Vingan

last: Isaac

first: Phatarpekar

last: Shwetha

first: Tung

last: Victoria Sook Keng

first: Hernandez

last: A Iván

first: Evgrafov

last: Oleg

first: Alarcon

last: Juan Marcos


title: Reduced representation bisulfite sequencing (RRBS) of brown adipose tissue from postnatal day 12 mice perinatally exposed to vehicle or DDT

status: Public on Oct 05 2024

city: Davis

first: Michele

middle: Andrea

last: La Merrill

state: CA

address: One Shields Ave

department: Environmental Toxicology

institute: University of California Davis

type: Methylation profiling by high throughput sequencing

summary: The insecticide dichlorodiphenyltrichloroethane (DDT) and its persistent metabolite, dichlorodiphenyldichloroethylene (DDE), have been associated with increased adiposity and obesity in rodent and human studies. These lipophilic pollutants accumulate in adipose tissue and it is theorized that their exposure leads to decreased decrease energy expenditure through the impairment of thermogenesis in brown adipose tissue. We hypothesized that impaired thermogenesis is due to persistent epigenetic modifications of brown adipose tissue. To address this, we exposed C57BL/6J mice to DDT from gestational day 11.5 to postnatal day 5 and isolated brown adipose tissue for RRBS from one offspring per litter when mice reached postnatal day 12 (n=20 DDT litters and 22 vehicle litters). DNA methylation levels in brown adipose tissue of mice perinatally exposed to vehicle or DDT were determined using RRBS. Analyses of differential methylation and KEGG pathway enrichment were performed.

overall_design: RRBS was conducted on brown adipose tissue samples of postnatal day 12 mice perinatally exposed to DDT or vehicle (n=12 DDT, n=12 vehicle)

first: Juliann

middle: A

last: Jugan

first: Kyle

middle: B

last: Jackson

first: Sarah

last: Elmore


title: B cells in triple positive antiphospholipid syndrome

status: Public on Oct 08 2024

city: Paris

first: Mickael

middle: Mathieu

last: Menager

address: 24 Boulevard du Montparnasse

country: France

institute: Institut Imagine

summary: Peripheral B cell selection of autoreactive B cells is crucial for preventing autoimmunity. In this study, we identify that a significant proportion of antiphospholipid (aPL) B cells present in the naive compartment, exhibit polyreactivity and belong to the natural repertoire. We used scRNA Seq to follow B cells with aPL features throughout their development from naïve B cells to plasma cells in patients with PAPS.

overall_design: B cells from triple positive primary PAPS patients were magnetically sorted.

first: Yannick

last: Dieudonné

first: Francesco

last: Carbone

first: Vincent

last: Gies


title: DHX36 binding induces RNA structurome remodeling and regulates RNA abundance via m6A/YTHDF1

status: Public on Oct 03 2024

city: Hong Kong

first: Yuwei

last: ZHANG

state: Hong Kong

address: Rm503, Li Ka Shing Medical Sciences Building, Prince of Wales Hospital, Shatin, NT, HK

department: Chemical Pathology

country: Hong Kong

institute: The Chinese University of Hong Kong

summary: RNA structure constitutes a new layer of gene regulatory mechanisms. RNA binding proteins can modulate RNA secondary structures, thus participating in post-transcriptional regulation. The DEAH-box helicase 36 (DHX36) is known to bind and unwind RNA G-quadruplex (rG4) structure but the transcriptome-wide RNA structure remodeling induced by DHX36 binding and the impact on RNA fate remain poorly understood. Here, we investigate the RNA structurome alteration induced by DHX36 depletion. Our findings reveal that DHX36 binding induces structure remodeling not only at the localized binding sites but also on the entire mRNA transcript most pronounced in3’UTR regions. DHX36 binding increase structural accessibility at 3’UTRs which is correlated with decreased post-transcriptional mRNA abundance. Further analyses and experiments uncover that DHX36 binding sites are enriched for N6-methyladenosine (m6A) modification and YTHDF1 binding; and DHX36 induced structural change may facilitate YTHDF1 binding to m6A sites leading to RNA degradation. Altogether, our findings uncover the structural remodeling effect of DHX36 binding and its impact on RNA abundance through regulating m6A dependent YTHDF1 binding.

overall_design: To investigate the effect of m6A levels on RNA structure changes,  we conducted RNA Structure-seq in STM2457-treated and control HEK293T cells.

last: Zhang

first: Jieyu

last: Zhao

first: Chun

middle: K

last: Kwok

first: Huating

last: Wang


title: Germ cell progression through zebrafish spermatogenesis declines with age

status: Public on Oct 04 2024

city: Salt Lake City

first: Andrea

middle: Lynn

last: Sposato

state: UT

address: 1390 President's Circle

department: Biology

institute: University of Utah

summary: Vertebrate spermatogonial stem cells maintain sperm production over the lifetime of an animal but fertility declines with age. While morphological studies have informed our understanding of typical spermatogenesis, the molecular and cellular mechanisms underlying the maintenance and decline of spermatogenesis are not yet understood. We used single-cell RNA sequencing to generate a developmental atlas of the aging zebrafish testis. All testes contained spermatogonia, but we observed a progressive decline in spermatogenesis that correlates with age. Testes from some older males only contained spermatogonia and a reduced population of spermatocytes. Spermatogonia in older males are transcriptionally distinct from spermatogonia in testes capable of robust spermatogenesis. Immune cells including macrophages and lymphocytes drastically increase in abundance in testes that cannot complete spermatogenesis. Our developmental atlas reveals the cellular changes as the testis ages and defines a molecular roadmap for the regulation of spermatogenesis.

overall_design: scRNA-seq of dissected zebrafish testes from 5, 12, 20, 22, and 27 month individuals

first: Hailey

last: Hollins

first: Darren

last: Llewellyn

first: Jenna

last: Weber

first: Madison

last: Schrock

first: Jeffrey

last: Farrell

first: James

last: Gagnon


title: Smarcd1 is a “Goldilocks” metastasis modifier gene [RNA-seq_on_plastic]

city: Bethesda

first: Kent

last: Hunter

state: MD

address: 37/4048 37 Convent Drive

department: Center for Cancer Research

institute: National Cancer Institute

summary: Breast cancer is the most highly diagnosed cancer worldwide, making up around 12% of all diagnosed cancers in 2020. Mortality caused by breast cancer is largely due to metastasis to distant essential organs, and a lack of metastasis-targeted therapies perpetuates incredibly poor outcomes for late-stage patients. Here we have identified a new class of “Goldilocks” genes for metastasis-targeted therapeutic development, through our focus on meiotic genetics and inherited transcriptional network regulation. Building on previous work identifying the CCR4-NOT RNA deadenylase complex in metastasis, we further demonstrate that RNA binding proteins NANOS1, PUM2, and CPSF4 also regulate metastatic potential. Using cell lines, 3D culture, mouse models, and clinical data, we have identified Smarcd1 mRNA, a key target of all three factors, for which high and low expression is associated with positive clinical outcome, but medium expression significantly reduces probability of survival. Applying the theory of “essential genes” from evolution, we have identified an additional 50 genes that span several cellular processes and must be maintained within a discrete window of expression for metastasis to occur. In the case of Smarcd1, small perturbations in expression level significantly reduced metastasis in laboratory mouse models and altered splicing programs of particular relevance to the ER+HER2 enriched patient subtype. Identification of subtype-specific “Golidlocks” metastasis modifier genes creates a new class of genes and catalogue of potential novel targets that when therapeutically “nudged” in either direction may significantly improve late-stage patient outcomes.

overall_design: To investigate the affect of Smarcd1 depletion on mammary tumor transcription shRNA knockdown or overexpression cells were generated and RNA sequencing performed from control and experimental cells grown on standard tissue culture conditions

first: Christina

last: Ross

first: Li-Yun

last: Gong

first: Lisa

last: Jenkins

first: Ngoc-Han

last: Ha

first: Megan

last: Majocha


title: Smarcd1 is a “Goldilocks” metastasis modifier gene

overall_design: To assess the affect of Smarcd1 expression on accessible chromatin, knockdown or overexpression cells grown as spheroids were assayed by ATAC-seq


title: Root-specific secondary metabolism at the single-cell level: a case study of theanine metabolism and regulation in the roots of tea plants (Camellia sinensis)

status: Public on Oct 07 2024

city: Hefei

first: Shijia

last: Lin

state: Anhui

address: No. 130, Changjiang West Road

institute: Anhui Agricultral University

summary: Root-synthesized secondary metabolites are critical quality-conferring compounds of foods, plant-derived medicines, and beverages. However, information at a single-cell level on root-specific secondary metabolism remains largely unexplored. L-theanine, an important quality component of tea, is primarily synthesized in roots, from which it is then transported to new tea shoots. In this study, we present a single-cell RNA sequencing (scRNA-seq)-derived map for the tea plant root, which enabled cell-type-specific analysis of glutamate and ethylamine (two precursors of theanine biosynthesis) metabolism, and theanine biosynthesis, storage, and transport. Our findings support a model in which the theanine biosynthesis pathway occurs via multicellular compartmentation and does not require high co-expression levels of transcription factors and their target genes within the same cell cluster. This study provides novel insights into theanine metabolism and regulation, at the single-cell level, and offers an example for studying root-specific secondary metabolism in other plant systems.

overall_design: In this study, we performed a scRNA-seq-based study of tea plant roots, as an example, to elucidate the biosynthesis and regulatory processes for theanine, a root-specific secondary metabolite.

first: Yiwen

last: Zhang

first: Shupei

first: Yijie

last: Wei

first: Mengxue

last: Han

first: Yamei

last: Deng

first: Jiayi

last: Guo

first: Biying

last: Zhu

first: Tianyuan

last: Yang

first: Enhua

last: Xia

first: Xiaochun

last: Wan

first: William

middle: J

last: Lucas

first: Zhaoliang


title: Smarcd1 is a “Goldilocks” metastasis modifier gene [rnaseq_spheroids]

first: Kent

last: Hunter

overall_design: To investigate the affect of Smarcd1 depletion on mammary tumor transcription shRNA knockdown or overexpression cells were generated and RNA sequencing performed from control and experimental cells grown in 3D as tumor spheroids

first: Christina

first: Li-Yun


title: Transcriptomic  RNA structure mapping unveils the structure dynamic induced by DHX36 binding

first: Yuwei

last: ZHANG

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

sample_organism:
Homo sapiens
Mus musculus
synthetic construct

overall_design: Refer to individual Series


title: Doxorubicin resistance involves modulation of interferon signaling, transcriptional bursting, and gene co-expression patterns of U-ISGF3-related genes

first: Seolkyoung

last: Jung

address: 10 Center Dr

department: NIAMS

institute: NIH

summary: we investigate the transcriptomic alterations induced by doxorubicin (DOX), a commonly used chemotherapeutic agent, in human colon cancer cells. Using single-cell RNA sequencing, we identified distinct cell populations and their transcriptional profiles following subtoxic dose DOX treatment, revealing cell clusters characterized by differential expression of genes involved in cell cycle regulation and interferon (IFN) signaling. We observed that expression of JAK/STAT pathway genes, activated by IFNs, did not correlate with expression of senescence markers. In contrast, DOX-persisting proliferating cells exhibited upregulation of genes induced by the unphosphorylated form of ISGF3 (U‐ISGF3) transcription factor. Further analysis revealed that U‐ISGF3 drives the expression of target genes by modulating the number of mRNA produced per transcriptional burst, potentially contributing to chemoresistance. Importantly, we found high upregulation of HSH2D, a poor prognostic marker, in doxorubicin-resistant U-ISGF3-dependent cells. Moreover, analysis of gene co-expression networks demonstrated that the altered network in DOX-treated cells, rather than individual gene expression levels, may dictate cancer cell fate after chemotherapy.

overall_design: scRNAseq. mRNAseq to define bursting kinetics and expression difference on DOX treatment

first: Pawel

last: Trzaskoma

first: Yuka

last: Kanno

first: John

last: O'Shea

first: Carson

middle: C

last: Chow


title: Paraventricular hypothalamic RUVBL2 neurons suppress appetite by enhancing excitatory synaptic transmission in distinct neurocircuits [RNA-seq]

status: Public on Oct 02 2024

city: tianjin

first: mingming

last: xing

address: Huanrui Road，Beichen District

institute: Tianjin Medical University

summary: Synaptic plasticity has been shown to participate in control of energy homeostasis.The paraventricular hypothalamus (PVH) is crucial for satiety control, though the underlying synaptic mechanisms remain unclear. Here, we show that RUVBL2 in the PVH is significantly reduced during energy deficit, and knockout (KO) of PVH RUVBL2 leads to hyperphagic obesity. We further show that KO of PVH RUVBL2 impairs the excitatory synaptic transmission by reducing presynaptic boutons and altering synaptic vesicle distribution.  Chromatin immunoprecipitation-sequencing (ChlP-seq) data show that RUVBL2 binds to the promoters of numerous genes related to synaptic plasticity, in particular synaptic vesicle and neuron axonogenesis. Meanwhile, RNA-seq data show significant changes in the expression of some genes in these two pathways. Together, this study demonstrates an essential role for PVH RUVBL2 in food intake control, and suggests that modulating synaptic plasticity could be an effective way to curb appetite and obesity.

overall_design: To investigate RUVBL2 in the regultion of synaptic plasticity, we used AAV-Cre to knock out RUVBL2 in the PVH of Ruvbl2fl/fl mice. We then performed gene expression profiling using data obtained from RNA-seq of mouse PVH.


title: Transcriptomic RNA structure mapping unveils the structure dynamic induced by DHX36 binding (RBNS)

summary: RNA binding proteins can modulate RNA secondary structures, thus participating in post-transcriptional regulation. The DEAH-box helicase 36 (DHX36) has a remarkable ability to bind and unwind RNA G-quadruplex (rG4) and duplex. However, the transcriptome-wide RNA structure dynamic induced by DHX36 and how structure change subsequently influences RNA fate remain poorly understood. Here, we first identify the endogenous binding sites and specificity of DHX36 based on binding profiles. Next, we capture in vivo RNA structuromes to investigate the structure change of DHX36-bound mRNAs following DHX36 knockout. DHX36 induces structure remodeling on not only the localized binding sites but also the other sites across the entire mRNA especially in 3’UTR. DHX36-induced more accessible structures of 3’UTR are revealed to correlate with post-transcriptional mRNA decrease. Furthermore, we demonstrate that DHX36 binding sites are enriched for N6-methyladenosine (m6A) modification and YTHDF1 binding. Finally, we experimentally validate that YTHDF1 binding is repelled to DHX36 loss-induced structure inaccessibility and YTHDF1 loss-induced mRNA stabilization could be a source of DHX36 loss-induced mRNA increase. Altogether, our findings uncover the effect of DHX36 binding on in vivo mRNA structure and propose a plausible mechanism of how RNA secondary structure change involves in post-transcriptional regulation through orchestrating YTHDF1 binding.

overall_design: To investigate the binding specificity of DHX36 and the effect of DHX36 on target RNA structures, we conducted RNA Bind-n-Seq using RHAU53 peptide and conducted RNA Structure-seq in WT and DHX36-KO HEK293T and C2C12 cells.

last: Zhang

first: Jieyu

last: Zhao

first: Chun

last: Kwok

first: Huating

last: Wang


title: Paraventricular hypothalamic RUVBL2 neurons suppress appetite by enhancing excitatory synaptic transmission in distinct neurocircuits

summary: The paraventricular hypothalamus (PVH) is crucial for food intake control, yet the presynaptic mechanisms underlying PVH neurons remain unclear. Here, we show that RUVBL2 in the PVH is significantly reduced during energy deficit, and knockout (KO) of PVH RUVBL2 results in hyperphagic obesity in mice. RUVBL2-expressing neurons in the PVH (PVHRUVBL2) exert the anorexigenic effect by projecting to the arcuate hypothalamus, the dorsomedial hypothalamus, and the parabrachial complex. We further demonstrate that PVHRUVBL2 neurons form the synaptic connections with POMC and AgRP neurons in the ARC. PVH RUVBL2 KO impairs the excitatory synaptic transmission by reducing presynaptic boutons and synaptic vesicles near active zone. Finally, RUVBL2 overexpression in the PVH suppresses food intake and protects against diet induced obesity. Together, this study demonstrates an essential role for PVH RUVBL2 in food intake control, and suggests that modulation of synaptic plasticity could be an effective way to curb appetite and obesity.

overall_design: Chromatin immunoprecipitation DNA-sequencing (ChIP-Seq) for RUVBL2 in hypothalamus of wild-type mice


title: Transcriptomic  RNA structure mapping unveils the structure dynamic induced by DHX36 binding (Structure-seq)

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus


title: Histone Methylation Patterns in Acute Leukemias are Defined by Genotype Independent of the Disease Phenotype

status: Public on Nov 01 2024

city: Burlington

first: Jonathan

middle: AR

last: Gordon

state: VT

address: 89 Beaumont Ave Given E209

department: Biochemistry

institute: University of Vermont

summary: Epigenetic mechanisms, including acetylation, phosphorylation, and methylation of nucleosomal histones, contribute greatly to physiological control of gene expression required for hematopoietic cell differentiation. Perturbations in histone methylation patterns and mutations in histone methyltransferases and demethylases are frequently identified in acute lymphoid and myeloid leukemia. Histone H3 methylation patterns on lysine 4 and 27 residues define bivalent, or poised, chromatin in the embryonic and hematopoietic stem cells. We examined how the bivalent histone methylation patterns change through differentiation from an embryonic stem cell to a hematopoietic stem cell, and followed these changes through malignant lymphoid and myeloid hematopoiesis. Using an integrative bioinformatics analysis of genome-wide histone methylation patterns, we find that a common bivalent histone methylation pattern does not exist for lymphoid or myeloid leukemia. Instead, a select subset of key hematopoietic genes retains the activating H3K4 trimethyl mark in leukemia cells when compared with hematopoietic stem cells. A close examination of genes marked for active transcription suggests that the malignant transcriptional program is dictated primarily by the cytogenetics and the mutations present in leukemia subtypes. Our findings have implications in determining the treatment for leukemia using epigenetic inhibitors, particularly for those malignancies with a mixed immunophenotype.

overall_design: ChIP-seq, 2 biological pulldown replicates and 1 input control for 2 cell lines.  Re-processed data for hESC cell lins, CD34+ donor samples, Kasumi1, and DOHH2 were downloaded from sources detailed in public_data_sources.csv.

first: Jessica

middle: L

last: Heath

first: Joseph

last: Boyd


title: Smarcd1 is a “Goldilocks” metastasis modifier gene [ATAC_on_plastic]

overall_design: To assess the affect of Smarcd1 expression on accessible chromatin, knockdown or overexpression cells grown on standard tissue culture conditions were assayed by ATAC-seq

last: Ross

last: Gong

first: Lisa

last: Jenkins

first: Ngoc-Han

last: Ha

first: Megan

last: Majocha


title: Dynamic chromatin accessibility and transcriptome changes following PDGF-BB treatment of bone-marrow derived mesenchymal stem cells

status: Public on Oct 01 2024

city: Indianapolis

first: Yue

address: 975 W. Walnut street

department: Medical Genetics

institute: Indiana University

summary: Mesenchymal stem cells (MSCs) are multipotent stem cells that are under investigation for use in clinical trials because they are capable of self-renewal and differentiating into different cell types under defined conditions. Nonetheless, the therapeutic effects of MSCs have been constrained by low engraftment rates, cell fusion, and cell survival. Various strategies have been explored to improve the therapeutic efficacy of MSCs, with platelet-derived growth factor (PDGF)-BB emerging as a promising candidate. To enhance our comprehension of the impact of PDGF-BB on the gene expression profile and chromosomal accessibility of MSCs, RNA-sequencing and analysis of chromatin accessibility profiles were conducted on three human primary MSCs in culture, both with and without stimulation by PDGF-BB.  Integrative analysis of gene expression and chromatin accessibility demonstrated that PDGF-BB treatment modified the chromatin accessibility landscape, marking regions for activation or repression through the AP-1 family transcription factors TEAD, CEBP, and RUNX2. These changes in AP1 transcription factor expression, in turn, led to cell proliferation and differentiation potential towards osteoblasts, adipocytes, or chondrocytes. The degree of MSC differentiation varies among cells isolated from different donors. The presence of an enrichment of exosome-related genes is also noted among all the differentially expressed genes.  In conclusion, the observed changes in AP1 transcription factor expression not only induced cellular proliferation and differentiation, but also revealed variations in the degree of MSC differentiation based on donor-specific differences. Moreover, the enrichment of exosome-related genes among differentially expressed genes suggests a potential significant role for PDGF-BB in facilitating intercellular communication.

overall_design: MSCs: PoieticsTM Normal Human Bone Marrow Derived Mesenchymal Stem Cells (hMSC) are isolated from normal (non-diabetic) adult human bone marrow withdrawn from bilateral punctures of the posterior iliac crests of normal volunteers. Three PoieticsTM hMSC cells, namely 36015, 36461, and 36550, sourced from three distinct donors, were acquired from Lonza. These cells were designated as follows: 1 (batch 18TL 169252 from donor 36015), 2 (batch 18TL 241909 from donor 36461), and 3 (batch 18TL 262066 from donor 36550). The MSC cells were grown in a humidified chamber maintained at 37°C and 5% CO2, utilizing MSCBMTM Basal Media (PT-3238) and MSCGMTM SingleQuots Supplement Kit (PT-4105) obtained from Lonza. Media was changed every three days.  MSCs were cultured in media supplemented with 10 ng/ml PDGF-BB (Invitrogen) for 48 hours as the treated sample, while the control sample was cultivated without PDGF-BB. Control and PDGF-BB treated MSCs were concurrently collected from distinct plates for RNAseq analyses separately.


title: snPATHO-seq: unlocking the FFPE archives for single nucleus RNA profiling [Visium]

city: Darlinghurst

first: Taopeng

state: New South Wales

address: 384 Victoria St

department: Cancer ecosystem

institute: Garvan institute of medical research

summary: FFPE tissue samples have long been considered challenging for sc-/sn-RNA sequencing assays. In this study, we developed a novel snRNA-seq workflow, snPATHO-seq, for FFPE tissue samples utilising isolated high-quality nuclei from FFPE samples and 10X Genomics FLEX chemistry. The snPATHO-seq demonstrated consistent performance with the established 10X Genomics 3' assay. In addition, it allows more accurate single-nucleus and spatial transcriptomics integration with the FFPE Visium dataset generated from the same FFPE block. We envision this new protocol can facilitate the transcriptomic interpretation of millions of FFPE archives generated and simplify the sample collection process and data generation.

overall_design: 5um thick tissue sections were obtained from 3 breast cancer tissue FFPE blocks and processed for FFPE Visium V2 chemistry using the CytAssist machine for processing.

first: Alexander

last: Swarbrick

first: Luciano

last: Martelotto


title: Coordination between the eIF2 kinase GCN2 and p53 signaling supports purine metabolism and the progression of prostate cancer

first: Ricardo

last: Cordova

state: IN

address: 635 Barnhill Drive, MS4067

institute: Indiana University School of Medicine

summary: Cancers invoke various pathways to mitigate external and internal stresses to continue their growth and progression. We previously reported that the eIF2 kinase GCN2 and the integrated stress response are constitutively active in prostate cancer (PCa) and are required to maintain amino acid homeostasis needed to fuel tumor growth. However, although loss of GCN2 function reduces intracellular amino acid availability and PCa growth, there is no appreciable cell death. Here, we discovered that the loss of GCN2 in PCa induces prosenescent p53 signaling. This p53 activation occurred through GCN2 inhibition-dependent reductions in purine nucleotides that impaired ribosome biogenesis and, consequently, induced the impaired ribosome biogenesis checkpoint. p53 signaling induced cell cycle arrest and senescence that promoted the survival of GCN2-deficient PCa cells. Depletion of GCN2 combined with loss of p53 or pharmacological inhibition of de novo purine biosynthesis reduced proliferation and enhanced cell death in PCa cell lines, organoids, and xenograft models. Our findings highlight the coordinated interplay between GCN2 and p53 regulation during nutrient stress and provide insight into how they could be targeted in developing new therapeutic strategies for PCa.

overall_design: To investigate how loss of p53 in combination with GCN2 inhibition affects gene expression in the human prostate cancer cell line LNCaP. We performed RNA-seq on wild-type (WT) and p53 KO LNCaP cells treated with the GCN2 inhibitor GCN2iB.

first: Noah

last: Sommers

first: Andrew

middle: S

last: Law

first: Angela

last: Klunk

first: Katherine

last: Brady

first: David

middle: W

last: Goodrich

first: Tracy

middle: G

last: Anthony

first: Jeffrey

last: Brault

first: Roberto

last: Pili

first: Ronald

last: Wek

first: Kirk

last: Staschke


title: NG2+ and Tbx18+ pericyte subtypes exhibit distinct reprogramming potency following ischemic stroke

city: Ottawa

first: Jing

last: Wang

state: Ontario

address: 501 smyth road

country: Canada

institute: University of Ottawa

summary: Increasing evidence shows that brain pericytes can acquire multipotency to produce multi-lineage cells following injury. However, pericytes are a heterogenous population and it remains unknown whether there are different potencies from different subsets of pericytes in response to injury. Here, using single-cell RNA-sequencing (scRNA-seq) and immunohistochemistry analysis following ischemic stroke, we showed that NG2+ pericyte subset expressed very strong neural reprogramming potential to produce newborn neurons, while Tbx18+ pericytes displayed strong multipotency to produce endothelial cells, fibroblasts, and microglia but with minimal neural reprogramming potency. In addition, we mimicked ischemic stroke in vitro and developed an NG2+ pericyte neural reprogramming culture model. Here we discovered that AMP-dependent kinase (AMPK) modulators facilitated pericyte to neuron conversion by modulating Ser436 phosphorylation status of CREB-binding protein (Cbp), a histone acetyltransferase, which coordinated an acetylation shift between the non-histone substrate (Sox2) and the histone substrate (H2B) and modulated Sox2 nuclear-cytoplasmic trafficking during reprogramming/differentiation process. Finally, we identified that sequential treatment of compound C (CpdC) and metformin, AMPK inhibitor and activator respectively through reprogramming/differentiation process, robustly facilitated the conversion of human pericyte into functional neurons via transient pericyte-derived neural stem cell population.

overall_design: We performed scRNA-seq using ET-1/L-NAME stroke-injured cerebral cortical tissues isolated from two pericyte lineage tracing mice (NG2-Cre-ERT2/Ai14-flx and Tbx18-CreERT2/Ai14-flx) that received intracerebral injections of ET-1/L-NAME (or saline) 3 days prior to scRNA-seq. Both transgenic mice were also treated with tamoxifen 7 days before injury to enable lineage tracing of Tbx18-Ai14+ and NG2-Ai14+ cells. Three groups of Tbx18-Ai14+ and NG2-Ai14+ cells from 1) no injury (naïve), 2) physical injury (saline), and 3) ischemic injury (ET-1/L-NAME) were Fluorescence-activated Cell (FAC) sorted for tdT (Ai14)+/DAPI- live cells to perform scRNA-seq.

first: Allison

last: Loan

first: Nidaa

last: Awaja

first: Margarita

last: Lui

first: Edward

last: Sun

first: Sailendra

last: Sarma

first: Ragav

last: Chona

first: Billy

last: Johnston

first: John

last: Albayay

first: Anabella

last: Nakhlé

first: Kaela

last: O’Connor

first: Jacob

last: Thomas

first: Joseph

last: Leung

first: Matthew

last: Seegobin

first: Ling

last: He

first: Fredric

middle: E

last: Wondisford

last: Picketts

first: Eve

last: Tsai

first: Laurie

last: Chan


title: Transcriptomic analysis of post-traumatic osteoarthritis mouse models identifies miR-199a-5p as a regulator of abnormal mechanical loading in articular cartilage (miRNA-Seq)

city: NEWCASTLE UPON TYNE

first: David

last: Young

address: Centre For Life

country: United Kingdom

institute: Newcastle University

summary: Objective  Animal models of post-traumatic osteoarthritis (PTOA) recapitulate the pathological changes observed in human PTOA. Here we aimed to compare the cartilage transcriptome responses of a non-surgical, mechanically induced rupture of the anterior cruciate ligament (ACL) model and the surgical destabilisation of the medial meniscus (DMM) model.   Methods  Skeletally mature male C57Bl6 mice were subjected to either the non-surgical, mechanical ACL rupture or surgical DMM models and transcriptome profiling performed on micro-dissected cartilage at day 7 and 42 post-procedure, respectively; in general, naïve animals served as controls. MicroRNA profiling was also performed on the ACL rupture model. Expression levels of a miRNA of interest, miR-199-5p, were inhibited in primary human articular chondrocytes (HAC) with RNA-seq and 3’UTR assays used to identify and valid potential target genes.    Results  The number of differentially expressed genes between the two models were comparable and highly correlative (Spearman R =0.8, P<2.2E-16).  Gene ontology enrichment analysis identified similarly enriched pathways, containing anabolic terms including ‘extracellular matrix organisation’ enriched for the upregulated genes. Within the ACL rupture miRNA transcriptome, miR-199-5p family members were amongst the most abundantly, and differentially expressed, which was replicated in the DMM cartilage by qRT-PCR. Inhibition of miR-199-5p in HAC led to a comparable transcriptome response to that observed in both human OA damaged vs intact cartilage and murine DMM cartilage datasets. Several genes, including GIT1, NCEH1, SOS2 and ECE1 were all experimentally verified as targets.  Conclusion  For the first time, we have characterised both the mRNA and miRNA articular cartilage signature in the ACL rupture model and demonstrated highly correlative responses with the DMM PTOA model. These data support the use of the ACL rupture model as a non-invasive alternative to DMM.

overall_design: Wild-type C57bl6 mice were used.  On the injured mice the the anterior cruciate ligament (ACL) rupture model was performed on the  right leg. The contralateral left leg acts as a control. mice were sacrifised at 1 or 7 day post-injury. Mice naive to treatment (day_7_naive) also acted as a further control. Medial knee cartilage caps were dissected, washed with PBS and frozen in liquid nitrogen and RNA isolated using TRIzol® reagent according to manufacturer's protocol (ThermoFisher Scientific). Total RNA and miRNA were purified from the TRIzol®-cartilage mixture using a mirVana™ miR Isolation Kit followed by DNAse treatment (Ambion, Fisher Scientific, Loughborough, UK) following the manufacturer's protocol   For each sample cartilage is pooled from 4 mice/treatment.  Breifly. for the ACL rupture, mice were anaesthetized with isoflurane and custom‐built cups used to hold the right ankle and knee in flexion with a 30o offset prior to the application of a 0.5 N pre‐load (ElectroForce3200; TA Instruments, Elstree, UK). A single 12 N load at a velocity of 1.4 mm s−1 was then applied resulting in anterior cruciate ligament (ACL) rupture; mechanical loading was always conducted in the morning. Buprenorphine (0.05 mg kg−1) was administered subcutaneously to mice at the start of the experiment; animals were able to move freely and were monitored for welfare and lameness until termination of the experiment by cervical dislocation.

first: Emma

last: Blain

first: Sophie

last: Gilbert

first: Matt

last: Barter

first: Jamie

last: Soul


title: Transcriptomic analysis of post-traumatic osteoarthritis mouse models identifies miR-199a-5p as a regulator of abnormal mechanical loading in articular cartilage (RNA-Seq)


title: Analysis of gene expression in SLMAP3 knockout e11.5 embryos

first: Balwant

last: Tuana

address: 451 Smyth Road

department: Cellular and Molecular Medicine

summary: Global loss of SLMAP3 resulted in developmental defects, therefore we wanted to analyze developmental gene expression in SLMAP3-knockout embryos when compared to wildtype embryos.

overall_design: 4 wildtype and 3 SLMAP3-knockout whole e11.5 embryos messenger RNA was quantified using RNA-Seq to measure differential gene expression.

first: Taha

last: Rehmani

first: Ana

middle: P

last: Dias


title: Transcriptomic analysis of post-traumatic osteoarthritis mouse models identifies miR-199a-5p as a regulator of abnormal mechanical loading in articular cartilage

overall_design: Wild-type C57bl6 mice were used (n=4 per group). Mice were either naive to surgery (D0) or were subjected to destabilisation of the medial meniscus (DMM) surgery (D42 group) and left for disease to develop for 42 days (6 weeks).  For the naive group (D0) the mice were 11 weeks of age, for the surgery group (D42)the mice were 17 weeks of age (e.g. 11 weeks plus 42 days). For all mice, Medial knee cartilage caps were dissected, washed with PBS and frozen in liquid nitrogen. Tissue was ground by being placed in an autoclaved chamber with a ball (Retsch) and 250µl QIAzol lysis reagent (QIAGEN). The chambers were transferred to Retsch MM200 mixer mill and tissue ground at vibration frequency of 25 Hz for 90 seconds. To this was added an additional 250µl QIAzol lysis reagent and the mixture transferred to an RNase free tube and incubated at room temperature for 5 minutes. 100µl of chloroform was added and the sample vortexed for 15 s, incubated at room temperature for 10 minutes then centrifuged at 12,500 x g at 4oC for 5 minutes. The upper RNA containing aqueous phase was transferred into a new Eppendorf and RNA and miRNA were purified using a mirVana™ miR Isolation Kit (Ambion, Fisher Scientific, Loughborough, UK) followed by DNAse treatment (DNA-free™ DNA Removal Kit, Invitrogen) following the manufacturer's protocol.

first: Yao

last: Hao

first: Hua

last: Lin


status: Public on Sep 30 2024

city: Changchun

first: Xiaorui

last: Yang

address: Xincheng Street

institute: Jilin Agricultural University

summary: Exploring the effect of m6A methylation modification on the progression of bovine pituitary tissue before and after the onset of puberty

first: Ziming

last: Wang

first: Wenfa

last: Lu


title: Effects of Smoldering Eucalyptus Wood Smoke Extract on Primary Human Brain Microvascular Endothelial Cells (HBMEC) and Immortalized Human Cerebral Microvascular Endothelial Cell Line (hCMEC/D3)

city: Livermore

first: Aimy

last: Sebastian

address: 7000 East Ave

department: BBTD

institute: LLNL

summary: Increase in frequency and size of wildfire raises a significant public health concern due to rapid climate change. Epidemiological studies have shown that wildfire smoke exposure can increase the risk of neurological disorders. Our study aims to understand how wildfire smoke particles affect brain endothelial cells using both primary human brain microvascular endothelial cells (HBMEC) and an immortalized human cerebral microvascular endothelial cell line (hCMEC/D3). HBMEC and hCMEC/D3 were exposed to different levels of smoldering eucalyptus smoke particles (10, 30, 50 ug/mL) for 24 hr. Supernatants were collected for LDH and ELISA array, cell lysates were collected for RNAseq, and cells were fixed and stained for tight junction marker (Zonula Occludens-1, ZO-1) via ICC. Our study found that treatment to smoldering eucalyptus particles can increase inflammatory cytokine production dose-dependently in HBMECs and hCMEC/D3. Moreover, RNAseq analyses resulted in dose-dependent changes in HBMEC and hCMEC/D3 from wood smoke treatment that could be mediated via AhR and Nrf2 pathways.

overall_design: Both HBMEC and hCMEC/D3 were plated in 96 well plates at 5x103 cells/well. Cells were cultured in coated 96 well plates to confluency before the treatment with eucalyptus wood smoke extract (10,30,50 mg/mL or 20,70, 100 mg/mL), lipopolysaccharide (LPS) (1 mg/mL), or LiCl (10 mM) for 24 hours. At the end of treatment, cells were fixed with 4% paraformaldehyde (PFA) for ICC, lysed to collect mRNA for bulk-RNA sequencing, or supernatants were collected for ELISA and cytotoxicity assays. All experiments were repeated with at least two different seedings with n=2 or 3.

first: Dorothy

last: You

first: Bruce

last: Buchholz

first: Heather

last: Enright


title: miR-324 regulates both osteoblast and osteoclast differentiation and function to control bone homeostasis

first: David

last: Young

summary: microRNAs are non-coding RNAs which modulate the expression of other RNA molecules. One microRNA can target many transcripts, allowing each microRNA to play key roles in many biological pathways. miR-324 is a microRNA previously implicated in bone and cartilage maintenance, defects of which result in common age-related diseases, such as osteoporosis or osteoarthritis (OA).  Cartilage damage was increased in both surgically and ageing-induced OA however changes in the cartilage transcriptome were minimal, with few miR-324 predicted targets dysregulated. However, in vivo micro-computed tomography and histology demonstrated that global miR-324-null mice had an increase in bone mineral density, trabecular thickness and cortical thickness, with many parameters increasing with age. The bones of the miR-null mice also had decreased osteocytes numbers and lipid droplets. In vivo TRAP staining revealed a decrease in osteoclasts with histomorphometry demonstrating an increased rate of bone formation in miR-324-null mice.  Ex vivo assays revealed that the high bone mass phenotype of the miR-324-null mice resulted from increased osteoblast activity and decreased osteoclastogenesis. RNA-seq and qRT-PCR followed by miR-324 target prediction and validation in osteoblasts, bone marrow macrophages and osteocytes, revealed that the osteoclast fusion regulator Pin1 was a miR-324 target in the osteoclast lineage, Hoxa9 and Samd5 were osteocyte target genes and the master osteogenic regulator Runx2 was a target of miR-324-5p in osteoblasts, the in vitro overexpression of which recapitulated the increased osteogenesis and decreased adipogenesis phenotype observed in vivo.  These data point to important roles of miR-324 in skeletal biology. Elucidation of pathways regulated by miR-324 offers promise for the treatment of bone diseases such as osteoporosis.

overall_design: Osteoblasts and haematopoietic stromal cells were isolated from leg bone chips and marrow respectively, dissected from the long bones of 20-week-old mice. The epiphyses were removed to expose the marrow, which was flushed from the bones by centrifugation and cultured directly with α-MEM, supplemented with 100 ng/mL M-CSF, for 3 days to obtain M-CSF-dependent bone marrow macrophages (BMMs). The remaining bone was cut into small chips of approximately 2 mm3 and collagenase digested for 1 hour. Subsequently, the bone chips were washed with PBS and cultured in supplemented α-MEM and cultured for approximately 6 weeks until semi confluent layers of osteoblasts were present. Osteoblasts were subsequently plated in osteogenic growth media (complete α-MEM supplemented with 2 mM β-glycerophosphate (BGP) and 50 µg/mL L-ascorbic acid (both Sigma-Aldrich)) for 18 days, before RNA was extracted. BMMs were plated in complete α-MEM, supplemented either with 30 ng/mL M-CSF and 50 ng/mL RANK-L to stimulate osteoclastogenesis (samples labelled osteoclasts) or 30 ng/mL M-CSF only (samples labelled BMMs) and stimulated for 5 days, before RNA was extracted. Total RNA was extracted from BMMs, osteoclasts and osteoblasts using the mirVana miRNA Isolation Kit (Thermo Fisher Scientific), following the manufacturer’s protocols, before all samples were purified using the DNA-free™ DNA Removal Kit (ThermoFisher Scientific) to remove any DNA contamination.

first: Dan

last: Hayman


title: Liver dysfunction in obesity is associated with cerebrovascular health independently of diabetes and hypertension.

city: Leiden

first: Robert

last: Kleemann

address: Sylviusweg 71

country: Netherlands

institute: The Netherlands Organisation for Applied Scientific Research

summary: It is unclear whether liver dysfunction is implicated in obesity-related detrimental changes in brain structure and function. This study examineed associations between liver dysfunction and brain health in middle-aged participants with obesity and early-stage metabolic dysfunction-associated steatotic liver disease (MASLD). Using brain magnetic resonance imaging, neuropsychological tests, histological and biochemical characterization of liver biopsies, we showed that specific hallmarks of liver dysfunction (e.g. free cholesterol accumulation, early fibrosis) are associated with increased white matter hyperintensities and larger variations in cerebral blood flow indicating poorer cerebrovascular function. These associations were independent of age, sex, body mass index, diabetes and hypertension and corroborated by independent liver RNA-seq and pathway analysis highlighting the role of liver inflammation and cellular stress. Further associations with circulating IL-6 suggest systemic low-grade inflammation as potential mediator between liver and brain. Hence, in obesity at midlife, cerebrovascular health is independently associated with the pathological state of the liver.

overall_design: This study examined associations between liver dysfunction and brain health in middle-aged participants with obesity and early-stage metabolic dysfunction-associated steatotic liver disease (MASLD) by measuring brain magnetic resonance imaging (and quatification), neuropsychological tests and characterization of liver biopsies (histological, biochemical and liver RNAseq gene-expression).

first: Florine

last: Seidel

first: Debby

last: Vreeken

first: Emma

last: Custers

first: Maximilian

last: Wiesmann

first: Serdar

last: Özsezen

first: Wim

last: van Duyvenvoorde

first: Martien

last: Caspers

first: Aswin

last: Menke

first: Martine

last: Morrison

first: Lars

last: Verschuren

first: Marco

last: Duering

first: Eric

last: Hazebroek

first: Amanda

last: Kiliaan


title: Deciphering engraftment of bone marrow human single cells after allogeneic stem cell transplantation

first: Jennifer

last: Bordenave

address: 1 avenue Claude Vellefaux

institute: INSERM

summary: Engraftment of donor cells is a prerequisite to successful allogeneic hematopoietic stem cell transplantation. However, it has been poorly characterized in detail both in experimental models and in human. Bone marrow examination by mass cytometry in 26 patients with myeloid malignancies and 20 healthy donors provides evidence that the proportions of myeloid and B-cell progenitors profoundly differed as compared to healthy controls with a shift toward terminal myeloid differentiation and sharp decrease of myeloid and B-cell progenitors. Principal component analysis clearly separated in 2 recipient groups (R1 and R2) driven by previous GVHD (in R2 patients). We then used single cell CITEseq to decipher donor cell engraftment in 10/26 patients (compared with 4 healthy donors). Forty-one clusters were resolved encompassing all bone marrow cellular components. Projection of patients and R1 and R2 showed drastic segregation of the 2 patient groups which were highly significant different repartition in a compositional analysis. In R2 patients sustained engraftment relied on only 10 clusters that included only committed myeloid progenitors, one stem-cell memory T-cell cluster and activated NK-cell clusters. Gene expression hallmarks disclosed repeatedly similar pathway in transplant recipients, namely, TNFα signaling via NFĸ, interferonƔ response, and allograft rejection. Thus, the cellular composition of the engrafted bone marrow in human is strongly influenced by T-cell mediated processes and inflammation leading to an emergency hematopoiesis.

overall_design: Bone marrow mononuclear cells (BMMNC) from patients and healthy donors were isolated by density gradient centrifugation (Ficoll-Paque) within 3h after bone marrow sampling and cryopreserved in heat-inactivated fetal calf serum (FCS) with 10% dimethyl sulfoxide (DMSO). The cell were stained with CITE-seq antibodies (ADT)

first: Dorota

last: Desaulle

last: Michonneau

first: Nicolas

last: Vallet

first: Mathieu

last: Chevalier

first: Emmanuelle

last: Clappier

first: Pierre

last: Lemaire

first: Stephanie

last: Mathis

first: Marie

last: Robin

first: Alienor

last: Xhaard

first: Flore

last: Sicre de Fontbrune

first: Sophie

last: Caillat-Zucamn

first: Regis

last: Peffault de Latour

first: Emmanuel

last: Curis

first: Gerard

last: Socie


title: Dynamic chromatin accessibility and transcriptome changes following PDGF-BB treatment of bone-marrow derived mesenchymal stem cells [ATAC-seq]

first: Yue

last: Wang

overall_design: MSCs: PoieticsTM Normal Human Bone Marrow Derived Mesenchymal Stem Cells (hMSC) are isolated from normal (non-diabetic) adult human bone marrow withdrawn from bilateral punctures of the posterior iliac crests of normal volunteers. Three PoieticsTM hMSC cells, namely 36015, 36461, and 36550, sourced from three distinct donors, were acquired from Lonza. These cells were designated as follows: 1 (batch 18TL 169252 from donor 36015), 2 (batch 18TL 241909 from donor 36461), and 3 (batch 18TL 262066 from donor 36550). The MSC cells were grown in a humidified chamber maintained at 37°C and 5% CO2, utilizing MSCBMTM Basal Media (PT-3238) and MSCGMTM SingleQuots Supplement Kit (PT-4105) obtained from Lonza. Media was changed every three days.  MSCs were cultured in media supplemented with 10 ng/ml PDGF-BB (Invitrogen) for 48 hours as the treated sample, while the control sample was cultivated without PDGF-BB. Control and PDGF-BB treated MSCs were concurrently collected from distinct plates for ATACseq analyses.


title: Silencing of maternally expressed RNAs in Dlk1-Dio3 domain is critical for embryonic development

city: Harbin

first: Haoran

last: Yu

address: 92 West Dazhi Street

department: School of Life Science and Technology

institute: Harbin Institute of Technology

summary: The mammalian imprinted Dlk1-Dio3 domain contains multiple lncRNAs, mRNAs, the largest miRNA cluster in the genome and four differentially methylated regions (DMRs), and deletion of maternal RNA within this locus results in embryonic lethality, but the mechanism by which this occurs is not clear. Here, we optimized the model of maternally expressed RNAs transcription termination in the domain and found that the cause of embryonic death was apoptosis in the embryo, particularly in the liver. We generated a mouse model of maternally expressed RNAs silencing in the Dlk1-Dio3 domain by inserting a 3×polyA termination sequence in Gtl2 locus. By analyzing mouse embryos RNA-Seq data combined with histological analysis, we found that silence of maternally expressed RNAs in the domain activated apoptosis, causing vascular rupture of fetal liver, resulting hemorrhage and injury. Mechanistically, termination of Gtl2 transcription results in the silencing of the maternally expressed RNAs and activation of the paternally expressed genes in the interval, and it is the gene itself rather than the IG-DMR and Gtl2-DMR that causes the above phenotypes. In conclusion, these findings illuminate a novel mechanism by which silencing of the maternally expressed RNAs within Dlk1-Dio3 domain leads to hepatic hemorrhage and embryonic death through activation of the apoptosis.

overall_design: We used Easi-CRISPR technology to insert three polyadenylation cassettes into the promoter of Gtl2 in the Dlk1-Dio3 imprinted domain to generate Gtl2 polyA knock-in mouse model.  At embryonic day 12.5 (E12.5), the embryos of paternally Gtl2 polyA knock-in (PKI), maternally Gtl2 polyA knock-in (MKI), homozygous (HOMO) and wild-type (WT) mice were selected to do the RNA-sequencing.

first: Haroan

first: Qiong

last: Wu


title: snPATHO-seq: unlocking the FFPE archives for single nucleus RNA profiling [snRNA-seq]

first: Taopeng

overall_design: Snap frozen and FFPE samples from a wide range of tissue types were dissociated for nuclei isolation and characterised using 10X Genomics 3' v3.1 or FLEX chemistry for direct workflow comparison.

first: Alexander

last: Swarbrick

first: Luciano

last: Martelotto


title: Regulation of heterochromatin formation and tumor suppression in leukemia by IKAROS, HDAC1 and EZH2 [ChIP-seq]

city: Hershey

first: Bing

last: He

state: PA

address: 500 University Dr

institute: Penn State University

summary: The IKZF1 gene encodes IKAROS – a DNA binding protein that acts as a tumor suppressor in  T-cell acute lymphoblastic leukemia (T-ALL). IKAROS can act as a transcriptional repressor via chromatin remodeling, however the mechanisms through which Ikaros exerts its tumor suppressor function via heterochromatin in T-ALL are largely unknown. We studied human and mouse T-ALL using loss-of-function and Ikzf1 re-expression approach, along with Ikzf1-wildtype primary human and mouse T-ALL and thymocytes to establish the role of  Ikaros and Ikaros-associated protein histone deacetylase 1 (HDAC1)  in global regulation of facultative heterochromatin and transcriptional repression in T-ALL.   Results identified novel Ikaros and HDAC1 functions in T-ALL: Ikaros and HDAC1 are essential for EZH2 histone methyltransferase activity, and formation of facultative heterochromatin; Recruitment of HDAC1 by Ikaros is critical for establishment of H3K27me3 and repression of active enhancers; and Ikaros-HDAC1 complexes promote formation and expansion of H3K27me3 large organized chromatin lysine domains (LOCKs) and broad genic repression domains (BGRDs) in T-ALL.  Our results establish that Ikaros’ tumor suppressor function in T-ALL occurs via activation of EZH2 and HDAC1 function, global regulation of the facultative heterochromatin landscape and silencing of active enhancers that regulate oncogene expression.

overall_design: DN3 cells were transduced with retrovirus that contains HA-tagged wild-type murine Ikaros for 1 day, 2 days and 3 days. ChIP-seqs (Ikaros, HDAC1, H3K27me3 and EZH2) were performed for cells from these four time points.

first: Sinisa

last: Dovat

first: Yali

last: Ding


title: Characterization of human monocytic cell line THP-1 associated with human 3D skin models under UVA exposure

city: Bangkok

first: Tanapat

last: Palaga

address: Payathai Road, Pathumwan

department: Microbiology

country: Thailand

institute: Chulalongkorn University

summary: Tissue resident macrophages play important roles in tissue homeostasis and acute response to external stimuli. Human skin equivalents (HSEs) incorporating human monocytic cell line THP-1, were fabricated to generate immunocompetent human skin models. These HSEs were used to investigate the influence of the skin microenvironment and UVA exposure on the phenotypes of macrophages. THP-1 in HSEs exhibited mixed M1 and M2 macrophage phenotypes. Transcriptomic analysis demonstrated that THP-1 in HSEs enriched extracellular matrix interaction while downregulated a DNA replication hallmark. Upon UVA exposure, immunocompetent HSEs exhibited epidermal distortion and increased DNA double-strand breaks (DSB). THP-1 isolated from UVA-exposed HSEs revealed significant upregulation of genes associated with oxidative stress, antioxidant regulation, inflammatory and UV response. A photoprotective agent, mycosporine-2-glycine (M2G), derived from cyanobacteria was applied on to immunocompetent HSEs and the responses of THP-1 cell line was investigated after UVA exposure. The result showed that UVA-induced DSB was significantly lower in M2G-treated HSEs. In addition, the inflammatory and UV response hallmarks were downregulated while the oxidative phosphorylation hallmark was upregulated in THP-1 from M2G-treated UVA exposed HSEs. Taken together, this study provides an immunocompetent 3D skin model that can be used to interrogate responses of skin resident innate immune cells to microenvironment and external stimuli such as UVA irradiation.

overall_design:
Human skin equivalents (HSEs) fabrication: NHDFs were mixed with fibrinogen and thrombin, and seeded immediately into the transwell insert treated with plasma ion generated from plasma generator device for 2 mins. For immunocompetent HSEs, NHDFs and THP-1 cells were co-cultured in fibrin hydrogel. After gelation, the dermal layer was submerged in DMEM for 24 h. NHEKs were seeded on the dermal layer coated with collagen type IV, then submerged in the mixed medium and incubated for 24 h. Epidermal differentiation was initiated by replacing the medium with L-ascorbic acid, followed by air-liquid interface cultivation for 7 days.
UVA irradiation and procedure: HSEs were exposed to UVA irradiation by using a BlueWave® MX-250 LED lamp, RediCure™ at a single dose of 80 J/cm2. The UVA lamp was mounted on a stable support allowing an exposure distance of 6 cm from the sample surface. Before irradiation, the culture medium was replaced with 1ΧPBS to prevent phototoxic effects. PBS solution was removed, and a fresh differentiated medium was added after UVA exposure. The UVA‐induced damage was evaluated 24 h post-UVA exposure.
Applying Mycosporine-2-Glycine (M2G) on HSEs: M2G was diluted in Milli-Q water was applied for 2 h before, during, and 30 min after UVA irradiation. Afterward, M2G was removed, and HSEs were subsequently incubated. Harvesting and tissue histology, along with gene expression analysis, were performed 24 h post-UVA exposure.
CD45+ cell isolation, RNA extraction, and Library preparation: HSEs were digested with 0.05% trypsin/ 0.01% EDTA in 1ΧPBS. Labeled immune cells with Alexa Fluor® 488 anti-human CD45 Antibody were isolated using SH800S Cell Sorter. Harvested CD45+ cells underwent total RNA extraction with PicoPureTM RNA Isolation Kit. Total RNA was subjected to RNA-sequencing.

first: Suphanun

last: Phuphanitcharoenkun

first: Fiona

last: Louis

first: Misa

last: Katsuyama

first: Kentaro

last: Uchida

first: Rungaroon

last: Waditee-Sirisattha

first: Hakuto

last: Kageyama

first: Michiya

last: Matsusaki


title: Characterization of the enzyme for 5-hydroxymethyluridine production and its role in silencing transposable elements in dinoflagellates

status: Public on Sep 29 2024

city: Shenzhen

first: Hao

last: Chen

address: 1088 Xueyuan Avenue

institute: Southern University of Science and Technology (SUSTech)

summary: Dinoflagellate chromosomes are extraordinary, as their organization is independent of architectural nucleosomes unlike typical eukaryotes and shows a cholesteric liquid crystal state. 5-hydroxymethyluridine (5hmU) is present at unusually high levels and its function remains an enigma in dinoflagellates chromosomal DNA for several decades. Here, we demonstrate that 5hmU contents vary among different dinoflagellates and are generated through thymidine hydroxylation. Importantly, we identified the enzyme, which is a putative dinoflagellate TET/JBP homologue, catalyzing 5hmU production using both in vivo and in vitro biochemical assays. Based on the near-chromosomal level genome assembly of dinoflagellate Amphidinium carterae, we depicted a comprehensive 5hmU landscape and found that 5hmU loci are significantly enriched in repeat elements. Moreover, inhibition of 5hmU via dioxygenase inhibitor leads to transcriptional activation of 5hmU-marked transposable elements (TEs), implying that 5hmU appears to serve as an epigenetic mark for silencing transposon. Together, our results revealed the biogenesis, genome-wide landscape and molecular function of dinoflagellate 5hmU, providing mechanistic insight into the function of this enigmatic DNA mark.

overall_design: In our study, we successfully obtained the near chromosome-scale genome assembly of A. carterae based on a combination of genome survey approach, long-read sequencing through Pacbio sequel II platform with the circular consensus sequencing (CCS) mode, and Hi-C sequencing data. For A. carterae genome annotation, we also performed transcriptome sequencing using both short-read RNA-seq (DNBSEQ PE150) and long-read sequencing (PacBio Sequel platform). To map the genome-wide distribution of 5hmU sites in A. carterae, we utilized an anti-5hmU antibody-based DNA immunoprecipitation method. To examine the effects of altered 5hmU levels on repeat elements, we treated A. carterae cells with and without 5hmU synthase inhibitor, 2,4-pyridinedicarboxylic acid (2-4P), followed by directional RNA sequencing to reveal their transcriptional change. Additionally, we conducted Whole Genome Bisulfite Sequencing (WGBS) to investigate the correlation between 5hmU and genome-wide 5mC loci in A. carterae. All treatments in this study have at least two biological replicates.

first: Chongping

last: Li

first: Ying

first: Yuci


title: SATB1 prevents immune cell infiltration by regulating chromatin organization and gene expression of a chemokine gene cluster in T cells [RNA-seq]

status: Public on Sep 27 2024

city: shanghai

first: bao

last: wang

address: jin zun road

institute: shanghai jiao tong university school of medicine

summary: The precise regulation of T lymphocyte development and functions is crucial for preventing the initiation and progression of autoimmunity. SATB1, a key regulator of T cell development, governs lineage-specific transcriptional programs. However, its role in autoimmunity remains unclear. Here we show that conditional knockout of Satb1 in CD4+ T cells in mice led to T cell hyperactivation and inflammatory cell infiltration in the lungs and stomach. By performing transcriptome profiling, we show that the loss of SATB1 led to the aberrant upregulation of chemokines, particularly the ones belonging to the CC subfamily. Treating Satb1 conditional knockout mice with the inhibitor of the CC chemokine receptor alleviated inflammatory cell infiltration and autoimmune phenotypes. Intriguingly, SATB1's transcriptional regulation of chemokine genes could not be attributed to its direct binding to chemokine promoters or modulation of H3K27Ac. Instead, SATB1 exerted its regulatory effects through the control of higher-order chromatin organization at the CC chemokine locus. The loss of SATB1 led to the emergence of a new chromatin domain encompassing the Ccl3, Ccl4, Ccl5, Ccl6, and Ccl9 genes and a distal enhancer, resulting in increased contacts between the enhancer and all five chemokine genes, thus inducing their upregulation. Collectively, these results demonstrate that SATB1 safeguards organisms against autoimmunity by ensuring proper chemokine expression and preventing inflammatory cell infiltration, providing valuable insights into the understanding of autoimmune diseases.

overall_design: Naive CD4+ T cells were isolated from spleens of control and Satb1 flox/flox CD4-Cre mice and then activated using CD3/CD28 beads for 24 hours followed by RNA-seq experiments.

first: Bao

last: Wang

first: Qian

last: Bian


title: SATB1 prevents immune cell infiltration by regulating chromatin organization and gene expression of a chemokine gene cluster in T cells [CUT&Tag]

summary: Autoimmune diseases are systemic diseases caused by abnormalities of the immune system. Satb1 is a nuclear protein highly expressed in T cells and plays an important role in regulating the expression of T cell functional genes. However, its role in autoimmune diseases remains unclear. Our study found that conditional knockout of Satb1 in CD4+ T cells led to T cell hyperactivation and inflammatory cell infiltration in the lungs and stomach. By performing transcriptome profiling, we showed that the loss of SATB1 led to the aberrant upregulation of chemokines, particularly the ones belonging to the CC subfamily. We further found that SATB1 could bind to the promoters of CC chemokines and reduce the levels of H3K27ac, thereby repressing the expression of these genes. Such a transcriptional repressive function is likely attributed to the ability of SATB1 to recruit HDAC1. Treating Satb1 conditional knockout mice with the inhibitor of the CC chemokine receptor alleviated inflammatory cell infiltration and autoimmune phenotypes. Collectively, these results demonstrate that Satb1 plays a critical role in regulating chemokine expression and preventing inflammatory cell infiltration, providing new perspectives on the understanding and clinical treatment of autoimmune diseases.

overall_design: Naive CD4+ T cells were isolated from spleens of control (CON) and Satb1 flox/flox CD4-Cre (CKO) mice. Cells were activated and expanded using CD3/CD28 antibody followed by H3K27ac CUT&Tag experiments.


title: Transcriptome analysis of lung neutrophils from mice infected with Mycobacterium tuberculosis

city: Albany

first: Ramon

last: Bossardi Ramos

state: Ny

address: 43 New Scotland Avenue

department: Department of Molecular and Cellular Physiology

institute: Albany Medical College

summary: Neutrophils are accumulated in mouse lungs following Mycobacterium tuberculosis infections. Increased influx of neutrophils is associated with increased susceptibility to disease, but the underlying mechanisms of neutrophil mediated Tuberculosis disease pathogenesis is poorly understood. To understand the transcriptomic differences between resistant (C57 BL/6) and susceptible (C3HeB/FeJ) mouse lung neutrophils, we sorted them at Day 29 post infection and analyzed them by bulk mRNA sequencing.

overall_design: Bulk mRNA sequencing to study transcriptomic differences in neutrophils at Day 29 post infection with Mycobacterium tuberculosis HN878 from C57 BL/6 and C3HeB/FeJ mouse lungs.

first: Poornima

last: Sankar

first: Bibhuti

last: Mishra