title: Baricitinib with cyclosporine eliminates acute graft rejection in fully mismatched skin and heart transplant models

first: Li

last: Ding

address: 660 S. Euclid Avenue

institute: Washington University in St. Louis

summary: Solid organ transplant represents a potentially lifesaving procedure for patients suffering from end-stage heart, lung, liver, and kidney failure. However, rejection remains a significant source of morbidity and immunosuppressive medications have significant toxicities. Janus kinase (JAK) inhibitors are effective immunosuppressants in autoimmune diseases and graft versus host disease after allogeneic hematopoietic cell transplantation. Here we examine the role of JAK inhibition in preclinical fully major histocompatibility mismatched skin and heart allograft models. Baricitinib combined with cyclosporine A (CsA) preserved fully major histocompatibility mismatched skin grafts for the entirety of a 111-day experimental period. In baricitinib plus CsA treated mice, circulating CD4+T-bet+ T cells, CD8+T-bet+ T cells, and CD4+FOXP3+ regulatory T cells were reduced. Single cell RNA sequencing revealed a unique expression profile in immune cells in the skin of baricitinib plus CsA treated mice, including decreased inflammatory neutrophils and increased CCR2- macrophages. In a fully major histocompatibility mismatched mismatched heart allograft model, baricitinib plus CsA prevented graft rejection for the entire 28-day treatment period compared with 9 days in controls. Our findings establish that the combination of baricitinib and CsA prevents rejection in allogeneic skin and heart graft models and supports the study of JAK inhibitors in human solid organ transplantation.

overall_design: We performed single cell RNA (scRNA) sequencing on CD45+ cells harvested from skin grafts on POD 6.

first: Ramzi

last: Abboud

first: Jaebok

last: Choi

first: John

last: Dipersio


title: An in situ method for identification of transcriptome-wide protein-RNA interactions in cells [isstamp_lr]

status: Public on Jun 13 2024

city: La Jolla

first: Brian

last: Yee

state: CA

address: 9500 Gilman Dr

department: Health

country: USA

institute: UCSD

summary: RNA-binding proteins (RBPs) play important roles in RNA metabolism including splicing, stability, localization, and translation. RBP-RNA interaction profiles are indicative of many diseases. Existing methods for mapping RBP-RNA interactions transcriptome-wide have notable limitations: immunoprecipitation (IP)-based technologies require large quantities of input materials, and RNA shearing during processing prevents identification of RNA isoforms. Meanwhile, profiling methods using RNA-modifying enzymes require ectopic expression of fusion proteins in cells of interest, potentially distorting interaction profiles. Here we report in situ STAMP, an RBP-RNA profiling method that overcomes the limitations of existing methods. In situ STAMP utilizes a chimeric fusion of the cytosine deaminase APOBEC1 and an IgG-targeting single-domain antibody (nanobody). We demonstrate that this fusion protein can be specifically targeted to proteins of interest including the RBPs RBFOX2 and TDP-43 when combined with primary antibodies targeting these proteins, enabling identification of their binding sites in un-engineered HEK293T cells. The canonical binding motifs of both RBFOX2 (UGCAUG) and TDP43 (UGUGUG) could be identified by de novo motif analysis from in situ STAMP data, demonstrating the method’s high specificity. In situ STAMP preserves intact RNAs and is therefore compatible with direct cDNA PacBio long-read sequencing, enabling the method to distinguish between RNA isoforms. Importantly, in situ STAMP is compatible with multiple fixation methods including methanol and formaldehyde fixation, enabling its application to tissue samples collected in research or clinical settings. Thus, in situ STAMP enables the profiling of authentic RBP-RNA interactions using small quantities of primary cells or tissues, thereby bridging a critical gap in uncovering the roles of RBPs in RNA-related disease mechanisms in authentic biological contexts.

overall_design: INSCRIBE (C-to-U RNA editing) in fixed cells and mouse brain tissue

first: Qishan

last: Liang

first: Tao

last: Yu

first: Eric

last: Kofman

first: Pratibha

last: Jagannatha

first: Kevin

last: Rhine

middle: D

last: Corbett

first: Gene

middle: W

last: Yeo


title: Repeated intermittent clearance of p21-highly-expressing cells extends lifespan and confers sustained benefits to health and physical function

city: Farmington

first: William

middle: F

last: Flynn

state: CT

address: 10 Discovery Drive

institute: The Jackson Laboratory

type: Expression profiling by high throughput sequencing

summary: A key challenge in aging research is to extend lifespan in tandem with slowing down functional decline so that the life with good health (healthspan) can be extended. Here, we show that clearance of a small number of cells, which highly express p21Cip1 (p21high), starting from 20 months improves cardiac and metabolic function, and extends both median and maximum lifespan in mice. Importantly, by assessing health and physical function of these mice monthly until death, we show that clearance of p21high cells improves physical function at all remaining stages of life, suggesting morbidity compression and healthspan extension. Mechanistically, p21high cells encompass several cell types, with a conserved pro-inflammatory signature. Clearance of p21high cells reduces inflammation, and rejuvenates transcriptomic signatures of various tissues to younger states. These findings demonstrate the feasibility of morbidity compression in mice, and indicate p21high cells as a therapeutic target for healthy aging.

overall_design: p21-high cells were enriched by GFP using flow cytometry from stromal vascular fraction (SVF) of visceral fat from 5 25-30 months old p21-Cre transgenic mice. Cells from each donor were labelled with cell hashing antibodies (TotalSeq-A anti-mouse Hashtag, BioLegend) according to the manufacturer’s protocol. Cells were then washed, and resuspended in 0.04% BSA/PBS. Labelled cells from each donor were pooled at equal proportions, then immediately sorted on a BD FACSAria Fusion cell sorter (BD Biosciences) for viable GFPhigh and non-GFPhigh cell populations. Cells from wildtype mice were used as GFP background for gating and sorting.

first: Nie

last: Jia

first: Wang

last: Binsheng

first: Xu

last: Ming


title: Germline-specific RNA helicase DDX4 forms cytoplasmic granules in cancer cells and promotes tumor growth

city: Turku

first: Lin

last: Ma

address: Kiinamyllynkatu 10

country: Finland

institute: University of Turku

summary: Cancer cells undergo major epigenetic alterations and transcriptomic changes, including ectopic expression of many tissue- and cell type-specific genes. Here we show that the germline-specific RNA helicase DDX4 (DEAD-box helicase 4) forms germ granule-like cytoplasmic ribonucleoprotein (RNP) granules in various human tumors, but not in cultured cancer cells. These cancerous DDX4-complexes contain RNA-binding proteins and splicing regulators, including many known germ granule components. The deletion of DDX4 in cancer cells induces transcriptomic changes and affects the alternative splicing landscape of a large number of genes involved in cancer growth and invasiveness, leading to compromised capability of DDX4-null cancer cells to form xenograft tumors in immunocompromised mice. Importantly, occurrence of DDX4-granules is associated with poor survival in patients with head and neck squamous cell carcinoma, and higher histological grade of prostate cancer. Taken together, these results show that the germ granule-resembling cancerous DDX4-granules control gene expression and promote malignant and invasive properties of cancer cells.

overall_design: To understand the role of DDX4 in the regulation of cancer-promoting properties of PC3 cells, we carried out RNA-seq of the DDX4-null and WT PC3 cells cultured in monolayers and xenograft tumors.

first: Opeyemi

last: Olotu

first: Anna-Riina

last: Koskenniemi

first: Valeriy

last: Paramonov

first: Sini

last: Laasanen

first: Elina

last: Louramo

first: Matthieu

last: Bourgery

first: Tiina

last: Lehtiniemi

first: Samuli

first: Adolfo

last: Rivero-Müller

first: Eliisa

last: Löyttyniemi

first: Cecilia

last: Sahlgren

first: Jukka

last: Westermarck

first: Sami

last: Ventelä

first: Tapio

last: Visakorpi

first: Matti

last: Poutanen

first: Paula

last: Vainio

first: Juho-Antti

last: Mäkelä

first: Noora

last: Kotaja


title: Unveiling the regulatory network controlling natural transformation in lactococci

city: Louvain-la-Neuve

first: Frédéric

last: Toussaint

address: Place de l'Université 1

country: Belgium

institute: UCLouvain

summary: Lactococcus lactis is a lactic acid bacterium of major importance for food fermentation and biotechnological applications. The ability to manipulate its genome quickly and easily through competence for DNA transformation would accelerate its general use as a platform for a variety of applications. Natural transformation in this species requires the activation of the master regulator ComX. However, the growth conditions that lead to spontaneous transformation, as well as the regulators that control ComX production, are unknown. Here, we identified the carbon source, nitrogen supply, and pH as key factors controlling competence development in this species. Notably, we showed that these conditions are sensed by three global regulators (i.e., CcpA, CodY, and CovR), which repress comX transcription directly. Furthermore, our systematic inactivation of known signaling systems suggests that classical pheromone-sensing regulators are not involved. Finally, we discovered that the ComX-degrading MecA-ClpCP machinery plays a predominant role based on the identification of a single amino-acid substitution in the adaptor protein MecA of a highly transformable strain. Contrasting with closely-related streptococci, the master competence regulator in L. lactis is regulated both proximally by general sensors and distantly by the Clp degradation machinery. This study not only highlights the diversity of regulatory networks for competence control in Gram-positive bacteria, but it also paves the way for the use of natural transformation as a tool to manipulate this biotechnologically important bacterium.

overall_design: To investigate the effects of CodY and CovRS inactivation on the transcription of competence genes, codY- and covRS-deletion mutants were generated and cultured in their respective competence-activating medium. Cells were harvested during competence activation (glucose-maltose diauxic shift). Total RNA was extracted and sequenced. The transcriptomes of the mutants were compared to those of the wild-type strain grown in the same medium and conditions.To highlight the set of late competence genes, we also compared the  transcriptomes of a WT strain carrying an empty vector with a WT strain overexpressing the master comptence regulator ComX .

first: Pascal

last: Hols


title: An in situ method for identification of transcriptome-wide protein-RNA interactions in cells [in_situ_STAMP II]

first: Brian

last: Yee

overall_design: in situ STAMP (C-to-U RNA editing) in fixed cells

first: Qishan

last: Liang

first: Tao

last: Yu

first: Eric

last: Kofman

first: Pratibha

last: Jagannatha

first: Kevin

last: Rhine

last: Corbett

first: Gene

last: Yeo


title: A PRE loop at the dac locus acts as a topological chromatin structure that restricts and specifies enhancer promoter communication

city: Montpellier

first: Bernd

last: Schuettengruber

address: 141, rue de la cardonille

country: France

institute: Institute de Génétique Humaine

summary: 3D genome folding plays a fundamental role for the regulation of developmental genes by facilitating or constraining chromatin interactions between cis-regulatory elements (CREs). Polycomb response elements (PREs) are a specific kind of CREs involved in the memory of transcriptional states in Drosophila melanogaster. PREs act as nucleation sites for Polycomb group (PcG) proteins, which deposit the repressive histone mark H3K27me3, leading to the formation of a class of topologically associating domain (TADs) called Polycomb domains. PREs can establish looping contacts that stabilize gene repression of key developmental genes during development. However, the mechanism by which PRE loops fine tune gene expression is unknown. Using CRISPR/Cas9 genome engineering, we specifically perturbed PRE contacts or enhancer function and used complementary approaches including 4C-seq, Hi-C, and Hi-M to analyze how chromatin architecture perturbation affects gene expression. Our results suggest that the PRE loop at the dac gene locus acts as a constitutive 3D chromatin scaffold during Drosophila development that forms independently of gene expression states and has a versatile function: it restricts enhancer promoter communication and contributes to enhancer specificity.

overall_design:
Hi-C experiments of wilt type imaginal leg discs and pupae or imaginal leg discs carrying a gypsy insertion and/or enhancer deletion
4C-seq experiments of wilt type embryos, imaginal leg discs or pupae or embryos, imaginal leg discs or pupae carrying a gypsy insertion and/or enhancer deletion
Cut&Run experiments of wilt type imaginal leg discs or imaginal leg discs carrying a gypsy insertion and/or enhancer deletion

first: Sandrine

last: Denaud

first: Marion

last: Bardou

first: Giorgio-Lucio

last: Papadopoulos

first: Stefan

last: Grob

first: Marco

last: Di Stefano

first: Gonzalo

last: Sabaris

first: Marcelo

last: Nollmann

first: Giacomo

last: Cavalli


title: The Mohawk homeobox gene represents a marker and osteo-inhibitory factor in cranial suture stem cells

city: Baltimore

first: Aaron

last: James

state: Maryland

address: 720 Rutland Avenue, Ross Bldg., Room 529

department: Pathology

institute: Johns Hopkins University

summary: The regeneration of craniofacial bones of the mammalian skeleton necessitates the action of both intrinsic and extrinsic inductive factors from multiple cell types, which function in a hierarchical and temporal fashion to control the differentiation of osteogenic progenitors. Single-cell transcriptomics of developing mouse cranial suture recently identified a suture mesenchymal progenitor population with tendon- or ligament-associated gene expression profile previously uncharacterized. Here, we developed a Mohawk homeobox (MkxCG;R26RtdT) reporter mouse, finding that this teno-ligamentous gene identifies a cranial suture resident cell population within the adult mouse that gives rise to calvarial osteoblasts and osteocytes overtime during homeostatic conditions.  Single cell RNA-Sequencing (scRNA-Seq) demonstrated that Mkx+ suture cells demonstrate a stem-like phenotype with expression of teno-ligamentous genes.  Bone injury with Mkx+ cell ablation showed delayed bone healing.  Remarkably, Mkx gene played a critical role as an osteo-inhibitory factor in cranial suture cells, as knockdown or knockout resulted in increased osteogenic differentiation.  Furthermore, in vivo local deletion of Mkx in Mkx floxed mice resulted in robustly increased calvarial defect repair.  Finally, we observed that mechanical stretch dynamically regulates Mkx expression in turn regulating calvarial cell osteogenesis. Overall, we identify Mkx+ cells within the suture mesenchyme as a progenitor cell population for adult craniofacial bones required for bone repair and Mkx itself as mechanical stretch responsive gene which functions to prevent osteogenic differentiation within the stem cell niche.

overall_design: There are three samples that prepared from uninjured calvarial bone, day 7, and day 30 after calvarial defect  Uninjured is control.

first: Yiyun

last: Wang

first: Qizhi

last: Qin

first: Ziyi


title: Antisense oligonucleotide therapy for calmodulinopathy

city: Boston

first: William

middle: T.

last: Pu

state: Ma

address: 300 Longwood Ave

department: Cardiology

institute: Boston Children's Hospital

summary: Calmodulinopathies are rare inherited arrhythmia syndromes caused by dominant gain of function variants in one of three genes, CALM1, CALM2, and CALM3, which each encode the identical calmodulin (CaM) protein. We hypothesized that antisense oligonucleotide (ASO)-mediated depletion of an affected calmodulin gene would ameliorate disease manifestations, while the other two calmodulin genes would preserve CaM level and function. Here we tested this hypothesis using human induced pluripotent stem cell-derived cardiomyocyte (iPSC-CM) and mouse models of CALM1 pathogenic variants. Human CALM1F142L/+ iPSC-CMs exhibited prolonged action potentials, modeling congenital long QT syndrome. CALM1-depleting ASOs did not alter CaM protein level and normalized repolarization of CALM1F142L/+ iPSC-CMs. Similarly, an ASO targeting murine Calm1 depleted Calm1 transcript without affecting CaM protein level. This ASO alleviated drug-induced arrhythmia in CalmN98S/+ mice without causing observable toxicity. These results provide proof-of-concept that ASOs targeting individual calmodulin genes are potentially effective and safe therapies for calmodulinopathies.

overall_design: WT iPSC-CM were treated with ASOs (1,3, 4 and Control) or vehicle for 24hs. Total RNA were extracted 7 days later and the transcriptome were performed

first: Raul

middle: H

last: Bortolin

first: Farina

last: Nawar

first: Michael

middle: A

last: Trembley

first: Maksymilian

last: Prondzynski

first: Mason

middle: E

last: Sweat

first: Peizhe

first: Fujian

last: Lu

first: Carter

last: Liou

first: Paul

last: Berkson

first: Erin

middle: M

last: Keating

first: Park

last: Chaehyoung

first: Daisuke

last: Yoshinaga

first: Nikoleta

last: Pavlaki

first: Thomas

last: Samenuk

first: Peter

middle: T

last: Sage

first: Cecilia

middle: B

last: Cavazzoni

first: Robert

last: Whitehill

first: Dominic

middle: J

last: Abrams

first: Chrystalle

middle: K

last: Carreon

first: Juan

last: Putra

first: Sanda

last: Alexandrescu

first: Shuai

last: Guo

first: Wen-Chin

last: Tsai

last: Rubart

first: Dieter

last: Kubli

first: Adam

last: Mullick

first: Vassilios

last: Bezzerides


title: Multi-OMIC analysis of Huntington disease reveals a neuroprotective astrocyte state (bulk RNA-Seq)

city: New York

first: Osama

last: Al Dalahmah

state: NY

address: 622 West 168th Street

department: Pathology & Cell Biology

institute: Columbia University Irving Medical Center

summary: Huntington disease (HD) is an incurable neurodegenerative disease that is characterized by neuronal loss and astrogliosis. One hallmark of HD is the selective neuronal vulnerability of striatal medium spiny neurons and to date, the underlying mechanisms of this selective vulnerability have not been fully defined. Here, we employed a multi-omic approach including single nucleus RNAseq (snRNAseq), bulk RNAseq, lipidomics, CAG repeat length measurements, and multiplexed immunofluorescence on post-mortem brain tissue from multiple brain regions of HD and control donors. We defined a signature of genes that is driven by CAG repeat length and found it enriched in astrocytic and microglial genes. Moreover, weighted gene correlation network analysis showed loss of connectivity of astrocytic and microglial modules in HD and identified modules that correlated with CAG-repeat length which further implicated inflammatory pathways and metabolism. We performed lipidomic analysis of HD and control brains and identified several lipid species that correlate with HD grade, including ceramides and very long chain fatty acids. Integration of lipidomics and bulk transcriptomics identified a consensus gene signature that correlates with HD grade and HD lipidomic abnormalities and implicated the unfolded protein response pathway. Because astrocytes are critical for brain lipid metabolism and play important roles in regulating inflammation, we analyzed our snRNAseq dataset with an emphasis on astrocyte pathology. We found two main astrocyte types that spanned multiple brain regions; these types correspond to protoplasmic astrocytes, and fibrous-like, CD44-positive astrocytes. HD pathology was differentially associated with these cell types in a region-specific manner. One protoplasmic astrocyte cluster showed high expression of metallothionein genes, the depletion of which positively correlated with the depletion of vulnerable medium spiny neuronal clusters in the caudate nucleus. We confirmed that metallothioneins were increased in cingulate HD astrocytes but were unchanged or even decreased in caudate astrocytes. We combined existing genome-wide association studies (GWAS) with a GWA study conducted on HD patients from the original Venezuelan cohort and identified a single-nucleotide polymorphism in the metallothionein gene locus associated with delayed age of onset. Functional studies found that metallothionein overexpressing astrocytes are better able to buffer glutamate and were neuroprotective of induced pluripotent stem cells-derived HD MSNs as well as against rotenone-induced neuronal death in vitro. Finally, we found that metallothionein- overexpressing astrocytes increased the phagocytic activity of microglia in vitro and increased the expression of genes involved in fatty acid binding. Together, we identified an astrocytic phenotype that is regionally-enriched in less vulnerable brain regions that can be leveraged to protect neurons in HD.

overall_design: Analysis of human brain samples using a multi-omic approach merged with functional and imaging studies on astrocyte cells

first: Fahad

last: Paryani

first: Ji-Sun

last: Kwon

first: Chris

last: Ng

first: Nacoya

last: Madden

first: Kenneth

last: Ofori

first: Alice

last: Tang

first: Hong

last: Lu

first: Juncheng

last: Li

first: Aayushi

last: Mahajan

first: James

last: Goldman

first: Shawn

last: Davidson

first: Andrew

middle: S

last: Yoo

first: Vilas

last: Menon

last: Al-Dalahmah


title: An in situ method for identification of transcriptome-wide protein-RNA interactions in cells [in_situ_STAMP - Long-Read]"

first: Brian

last: Yee

overall_design: in situ STAMP (C-to-U RNA editing) in fixed cells and mouse brain tissue

first: Qishan

last: Liang

first: Tao

last: Yu

first: Eric

last: Kofman

first: Pratibha

last: Jagannatha

first: Kevin

last: Rhine

last: Corbett

first: Gene

last: Yeo


title: Single-Cell RNA Sequencing of Peripheral Blood Mononuclear Cells From Acute Myocardial Infarction

status: Public on Jun 12 2024

city: Shanghai

first: Xuebo

last: Liu

address: 389 Xincun Road,Putuo District

department: Department of Cardiology

country: China

institute: Tongji Hospital of Tongji University

summary:
Background: Acute myocardial infarction (AMI) can occur in patients with atherosclerotic disease, with or without plaque rupture. Previous studies have indicated a set of immune responses to plaque rupture. However, the specific circulating immune cell subsets that mediate inflammatory plaque rupture remain elusive.
Methods: Ten AMI patients were enrolled in our study (five with and five without plaque rupture; plaque characteristics were identified by optical coherence tomography). By single-cell RNA sequencing, we analyzed the transcriptomic profile of peripheral blood mononuclear cells.
Results: We identified 27 cell clusters among 82,550 cells, including monocytes, T cells, NK cells, B cells, megakaryocytes, and CD34+ cells. Classical and non-classical monocytes constitute the major inflammatory cell types, and pro-inflammatory genes such as CCL5, TLR7, and CX3CR1 were significantly upregulated in patients with plaque rupture, while the neutrophil activation and degranulation genes FPR2, MMP9, and CLEC4D were significantly expressed in the intermediate monocytes derived from patients without plaque rupture. We also found that CD4+ effector T cells may contribute to plaque rupture by producing a range of cytokines and inflammatory_x0002_related chemokines, while CD8+ effector T cells express more effector molecules in patients without plaque rupture, such as GZMB, GNLY, and PRF1, which may contribute to the progress of plaque erosion. Additionally, NK and B cells played a significant role in activating inflammatory cells and promoting chemokine production in the plaque rupture. Cell?cell communication elaborated characteristics in signaling pathways dominated by inflammatory activation of classical monocytes in patients with plaque rupture.
Conclusions: Our studies demonstrate that the circulating immune cells of patients with plaque rupture exhibit highly pro-inflammatory characteristics, while plaque erosion is mainly associated with intermediate monocyte amplification, neutrophil activation, and degranulation. These findings may provide novel targets for the precise treatment of patients with AMI.

overall_design:
A total of 20 patients with AMI who underwent percutaneous coronary intervention (PCI) at the Shanghai Tongji Hospital from April to December 2020 were included in the study. AMI was classified as STEMI and non-ST segment elevation MI (NSTEMI). The STEMI diagnosis was based on typical chest pain and new ST-segment elevation of at least two contiguous leads >0.1 mV or new left bundle-branch block on the 12-lead electrocardiogram (ECG) and elevated cardiac markers (troponin T/I or creatine kinase-MB) (6). On the 12-lead ECG, NSTEMI was identified as persistent angina accompanied by elevated cardiac markers but without the ST-segment elevation. Exclusion criteria were a left ventricular ejection fraction <30%; severe infectious disease, sepsis, or autoimmune disease; or malignant tumor or life expectancy of <1 year. All patients gave written informed consent, and the ethics committee of the Shanghai Tongji Hospital approved the study protocol. This research was in line with the Declaration of Helsinki.
Ten AMI patients were enrolled in our study (five with and five without plaque rupture; plaque characteristics were identified by optical coherence tomography). By single-cell RNA sequencing, we analyzed the transcriptomic profile of peripheral blood mononuclear cells.

first: Jun

last: Qian

first: Yanhua

last: Gao

first: Yan

last: Lai

first: Zi

last: Ye

first: Yian

last: Yao

first: Keke

last: Ding

first: Jing

last: Tong

first: Hao

last: Lin

first: Guoqi

last: Zhu

first: Yunan

first: Haoran

first: Deqiang

last: Yuan

first: Fei

last: Chen


title: Affinity maturation broadens influenza vaccine-induced antibody responses in humans [gex]

city: New Haven

first: Steven

last: Kleinstein

state: Ct

address: 300 George St.

institute: Yale University

summary: Influenza virus vaccination remains the best strategy for combating virus infection, but vaccine efficacy is highly variable. An ideal influenza vaccine must have two attributes: one, it should be capable of inducing broadly cross-reactive antibodies that can neutralize diverse influenza virus strains; and two, it must induce long-lived antibody responses to maintain protective immunity for extended periods. Germinal center (GC) reactions are the major sites where diversification and affinity maturation of B cells occur. Whether a persistent GC response could expand the breadth of responding B cell clones following influenza vaccination in humans remains unknown. Here, we show that influenza virus vaccine-specific GC B cells persist for over nine weeks post vaccination in two out of seven individuals. These late vaccine-specific GC B cells exhibited increased somatic hypermutation (SHM) of their B cell receptors compared to early vaccine-specific GC B cells. After re-immunization with seasonal influenza virus vaccine, individuals with a persistent GC engaged vaccine-specific plasmablasts (PBs) with higher SHM frequency. Tracking the maturation of three clonally related GC B cell lineages over time revealed that late GC B cells had receptors that recognized and neutralized heterologous influenza virus strains. Thus, SHM induced by persistent GCs can broaden the antibody response to influenza virus vaccination. This indicates that seasonal influenza virus vaccination in humans can induce broadly cross-reactive antibodies that target diverse influenza virus strains.

overall_design: 119 samples from PBMC, bone marrow plasma cells and lymph node were taken at multiple time points pre- and post-vaccination from 3 subjects. The samples were examined with single-cell RNA-seq.

first: Katherine

last: McIntire

first: Julian

middle: Q

last: Zhou

first: Hailong

last: Meng

first: Ali

last: Ellebedy


title: An in situ method for identification of transcriptome-wide protein-RNA interactions in cells

first: Brian

last: Yee

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

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: Refer to individual Series


title: Multi-OMIC analysis of Huntington disease reveals a neuroprotective astrocyte state

first: Osama

last: Al Dalahmah


title: Affinity maturation broadens influenza vaccine-induced antibody responses in humans [bcr]

overall_design: 128 samples from PBMC and lymph node were taken at multiple time points pre- and post-vaccination from 3 subjects. The samples were examined with bulk & single-cell BCR sequencing


title: Increased PVR Expression on Bone Marrow Macrophages May Promote Resistance to TIGIT Blockade in Multiple Myeloma

city: Barcelona

first: Ester

last: Lozano

address: Diagonal Av. 643

department: Cell Biology, Physiology and Immunology

country: Spain

institute: University of Barcelona

summary: Gene expression analysis of CD8+ T cells isolated from PBMC from healthy donors was performed by using Nanostring Technology

overall_design: The aim was to assess changes in cytotoxic T cell gene expression caused by incubation onto PVR or nectin-2 for 24h

first: Laura

last: Rosiñol


title: Effect of CRY2 stabilizer and proteasome inhibitor on the activity of cis-regulatory elements in MDA-MB-231 cells [STARR-seq]

city: Los Angeles

first: Yuanzhong

last: Pan

state: California

address: 1002 Childs Way

institute: University of Southern California

summary: In this experiment we performed STARR-seq on MDA-MB-231 cells that are treated with different drugs to test the features of cis-regulatory elements that make their activity potentially repressed by these small molecules.

overall_design: The STARR-seq library comprises fragmented genomic DNA of MDA-MB-231. The experiment contains four treatment groups: DMSO (control), CRY stabilizer SHP1705, proteasome inhibitor MG132, and the combination of SHP1705 and MG132. Prior to library transfection, media containing different drugs were added to cultured cell, and transfection mix was distributed to all groups. Eight hours after transfection, RNA was extracted, processed and sequenced.

first: Evanthia

last: Roussos Torres

first: Steve

last: Kay


title: The response to influenza vaccination is associated with DNA methylation-driven regulation of T cell innate antiviral pathways

first: Matteo

last: Pellegrini

address: 610 Charles E. Young Drive South TLSB room 5146, Pellegrini Lab

department: Molecular, Cell, and Developmental Biology

institute: University of California, Los Angeles

type: Methylation profiling by high throughput sequencing

summary: The effect of vaccination on the epigenome remains poorly characterized. In previous research, we identified an association between seroprotection against influenza and DNA methylation at sites associated with the RIG-1 signaling pathway, which recognizes viral double-stranded RNA and leads to a type I interferon response. However, these studies did not fully account for confounding factors including age, gender, and BMI, along with changes in cell type composition. Here, we studied the influenza vaccine response in a longitudinal cohort vaccinated over two consecutive years (2019–2020 and 2020–2021), using peripheral blood mononuclear cells and a targeted DNA methylation approach. To address the effects of multiple factors on the epigenome, we designed a multivariate multiple regression model that included seroprotection levels as quantified by the hemagglutination-inhibition (HAI) assay test. Our findings indicate that 179 methylation sites can be combined as potential signatures to predict seroprotection. These sites were not only enriched for genes involved in the regulation of the RIG-I signaling pathway, as found previously, but also enriched for other genes associated with innate immunity to viruses and the transcription factor binding sites of BRD4, which is known to impact T cell memory. We propose a model to suggest that the RIG-I pathway and BRD4 could potentially be modulated to improve immunization strategies.

overall_design: As part of a longitudinal study led by the University of Georgia, Athens (UGA), a cohort of 690 individuals was recruited for five successive seasons from 2016 to 2020 (UGA 1-5) between the ages of 18 to 65 years old. Participants were administered the split-inactivated influenza vaccine, Fluzone, manufactured by Sanofi Pasteur. For this study, a subset comprising 42 participants, all of whom were enrolled in UGA 5 and had previously been part of the UGA4 season, were selected. DNA was isolated from each participant's blood samples using the standard phenol-chloroform extraction approach.

first: Hongxiang

last: Fu

first: Harry

last: Pickering

first: Liudmilla

last: Rubbi

first: Ted

last: Ross

first: Wanding

first: Elain

last: Reed


title: Effect of CRY2 stabilizer and proteasome inhibitor on the transcription of MDA-MB-231 cells [RNA-seq]

summary: In this study we tested if the transcriptional activity of circadian transcription factors, BMAL1 and CLOCK, can be repressed by small molecules in triple negative breast cancer cells. We validated that CRY inhibitors and proteasome inhibitors synergize and can recapitulate the phenotype of genetic knockdown of BMAL1 and CLOCK. To investigate the potential mechanism of the effect of this drug combination, we performed RNA sequencing analysis on cells treated with either single drug or drug combination at different time points.

overall_design: We tested the effect of two kinds of proteasome inhibitors, either as a single agent or in combination with CRY stabilizer SHP1705, on the transcription of triple negative breast cancer cell line MDA-MB-231. Three independent experiments are performed. Each experiment includes DMSO-treated (control), SHP1705-treated, proteasome inhibitor-treated, and the combination of two drugs-treated. For MG132 combination experiment, the cells were treated for eight hours. For carfilzomib (CFZ) combination experiment, the cells were treated for 8 or 24 hours.


title: mTORC1 signaling in brain endothelial progenitors contributes to CCM pathogenesis in a Pdcd10-deficient mouse model

city: West Haven

first: Francesc

last: Lopez

department: Department of Genetics

summary: Cerebral vascular malformations (CCM) are primarily found within brain, where they result in increased risk for stroke, seizures and focal neurological deficits. The unique feature of the brain vasculature is the blood-brain barrier (BBB) formed by the brain neurovascular unit, and recent studies suggest that loss of CCM genes causes disruptions of BBB integrity as the inciting event for the development of CCM. CCM lesion is proposed to be initially derived from a single clonal expansion of a subset of angiogenic venous capillary endothelial cells (ECs) and respective resident endothelial progenitor cells (EPCs). However, the critical signaling in the subclass of brain EC/EPC for the CCM lesion initiation and progression are unclear. Here we employed single-cell RNA-sequencing (scRNA-seq) analyses in early stages of the brain EC-specific Ccm3 deletion (Pdcd10BECKO) microvessels and identified a unique EPC cluster with high stem cell markers but low BBB-associated genes. mTORC1, but not mTORC2, is activated in mouse and human CCM lesions and EPCs. Mechanistic studies suggest that CCM3 suppresses Cav1/caveolae-mediated cellular trafficking and complex formation of the mTORC1 signaling proteins. Genetic deficiency of Raptor (mTORC1), but not of Rictor (mTORC2), prevents CCM lesion formation in the Pdcd10BECKO model. Importantly, mTORC1 pharmacological inhibitor Rapamycin ameliorate the CCM pathogenesis in the Pdcd10BECKO mice, by suppressing lesion-forming EPC expansion while enhancing BBB maturation. Our data suggest that CCM3 is critical for maintaining BBB integrity, and CCM3 loss-induced mTORC1 signaling in brain EPCs initiate the CCM pathogenesis.

overall_design: We employed single-cell RNA-sequencing (scRNA-seq) analyses in early stages of the brain EC-specific Ccm3 deletion (Pdcd10BECKO) microvessels

first: Jenny

first: Lingfeng

last: Qin

first: Haifeng

last: Zhang

last: López-Giráldez

first: Yeaji

last: Kim

first: Katie

middle: N

last: Murray

first: Jaime

last: Grutzendler

first: Wang

last: Min


title: Ketogenicdiet versus standard high-carbohydrate nutrition in sepsis: a randomized controlled trial

city: Munich

first: David

last: Effinger

address: Marchioninistraße 15

department: Department of Anaesthesiology

country: Germany

institute: LMU University Hospital

summary: Sepsis patients suffer from severe metabolic and immunologic dysfunction that may be amplified by standard nutritional approaches relying primarily on carbohydrates. We here hypothesize that a ketogenic diet improves sepsis treatment. We conducted a monocentric open-labeled randomized controlled trial enrolling 40 adult sepsis patients. Patients were randomly assigned to either ketogenic diet (KD) or standard high-carbohydrate nutrition for 14 days and followed up until day 30. The primary outcome measure was β-hydroxybutyrate (BHB) serum concentration on day 14. We assessed feasibility and safety of KD, as well as diet-associated clinical and immunological changes. The respective nutrition protocols were successfully applied, dropouts did not occur. Regression analysis revealed a greater increase in BHB concentrations from baseline to day 14 in KD patients compared to controls. Metabolic side effects were not observed under ketogenic diet. Ventilation-free, vasopressor-free, dialysis-free and ICU-free days significantly increased in patients under ketogenic diet. Transcriptome profiling of both CD4+ and CD8+ T cells at day 14 revealed substantial differential regulation of gene expression in the KD vs. the control group indicating a reduced T-cell activation and a shift towards a more regulated immunity.

overall_design:
To investiagte the impact of an ketogenic diet on septic patients treated on intensive care unit we conducted a monocentric open-labeled randomized controlled trial enrolling 40 adult sepsis patients. Patients were randomly assigned to either ketogenic diet (KD) or standard high-carbohydrate nutrition for 14 days and followed up until day 30.
In order to analyse the influence of KD on the immune response in sepsis, we used Next-Generation Sequencing of mRNA from CD4+ and CD8+ T cells isolated before (T1) and after 14-day study phase (T14) for transcriptome profiling. CTRL=Control group, Keto = ketogenic group

first: Tim

last: Rahmel

first: Simone

last: Kreth


title: ssDRIP-seq: a high-throughput method for R-loop mapping and quantitative assessment

city: Shenzhen

first: Changbin

last: Sun

address: Dapeng

department: Agricultural Genomics Institute at Shenzhen

institute: Chinese Academy of Agricultural Sciences

summary: Current methods for R-loop mapping need to perform DNA:RNA immunoprecipitation for each sample individually, with consequent limitations in throughput. Here, we develop and validate mDRIP-seq, a multi-sample barcoding and pooling method for R-loop mapping. We show mDRIP-seq performs equivalently as conventional methods, but with the merits of high throughput and cost-efficiency. We also show the simplicity of mDRIP-seq for relative and absolute quantitation of genomic R-loop fractions for multiple samples. Together, mDRIP-seq is a high-throughput and cost-efficient method for R-loop mapping and quantitative assessment and can be widely applied to large-scale dynamic profiles of these important structures for diverse organisms.

sample_organism:
Escherichia coli
Homo sapiens
Mus musculus

overall_design: ssDRIP-seq for R-loop mapping

first: Wei

last: Xu


title: mDRIP-seq: a high-throughput method for R-loop mapping and quantitative assessment.

overall_design: mDRIP-seq development, and R-loop mapping in multiple samples


title: Global transcriptomic comparisons between fetal liver and bone marrow definitive erythropoiesis

status: Public on Jun 11 2024

first: Xiuli

last: An

address: 310 E 67th St

institute: New York Blood Center

summary: Erythropoiesis occurs first in the yolk sac as a transit “primitive” form, then is gradually re-placed by the “definitive” form in the fetal liver (FL) during fetal development and in the bone marrow (BM) postnatal. While it is well known that differences exist between primitive and de-finitive erythropoiesis, the similarities and differences between FL and BM definitive erythropoi-esis have not been studied. Here we performed comprehensive comparisons of erythroid progen-itors and precursors at all maturational stages sorted from E16.5 FL and adult BM. Transcriptome comparison revealed that genes with increased expression in FL BFU-E were en-riched in cell division. Interestingly, the expression levels of glucocorticoid receptor Nr3c1, Myc and Myc downstream target Ccna2, were significantly higher in FL BFU-E, indicating the role of Nr3c1-Myc-Ccna2 axis in the enhanced proliferation/cell division of FL BFU-E cells. At the CFU-E stage, the expression of genes associated with hemoglobin biosynthesis were much higher in FL CFU-E, indicating more hemoglobin production. During terminal erythropoiesis, overall temporal patterns in gene expression were conserved between FL and BM. While biological pro-cesses related to translation, TCA and hypoxia response were upregulated in FL erythroblasts, that related to antiviral signal pathway were upregulated in BM erythroblasts. Our findings un-covered previously unrecognized differences between FL and BM definitive erythropoiesis and provide novel insights into erythropoiesis.

overall_design: RNA was purified from the sorted erythroid cells at each progenitor and precursor stage from bone marrow and fetal liver, and then sequenced. We performed transcriptome comparisons of erythroid progenitors and precursors at all maturational stages sorted from E16.5 FL and adult BM. Adjacent-stage comparison and same-stage comparison of gene expression were done one BM and FL erythroid cells.

first: Huizhi

last: Zhao


title: B cells require DOCK8 to elicit and integrate T cell help when antigen is limiting

city: Oxford

first: Mukta

last: Deobagkar

address: Centre for Human Genetics, Roosevelt Drive

department: Nuffield Department of Medicine

country: United Kingdom

institute: University of Oxford

summary: Dedicator of cytokinesis 8 (DOCK8) immunodeficiency syndrome is characterized by a failure of the germinal center response, a process involving the proliferation and positive selection of antigen-specific B cells. While DOCK8-deficient B cells are recruited into germinal centers, we find that they are arrested at a light-zone stage. They are unable to respond to T cell–dependent survival and selection signals, and consequently differentiate into plasma cells or memory B cells. Although DOCK8-deficient B cells can acquire and present antigen to initiate activation of cognate T cells, integrin upregulation, B–T cell conjugate formation, and costimulation are insufficient for sustained activation of B and T cells when antigen availability is limited. Our findings provide an explanation for the failure of B cell-dependent humoral responses in DOCK8 immunodeficiency syndrome, and offer insights into how the level of available antigen modulates B–T cell interactions necessary for humoral immune responses and immune memory.

overall_design:
Hen egg lysozyme (HEL)-specific splenic germinal centre (GC) B cells were sorted from mouse splenocytes on day 8 after adoptive transfer of splenic MD4 B cell mixtures and immunization with SRBC-HEL.
CD95+ HEL+ GC B cells from 6 spleens (3 from WT group, 3 from DOCK8-deficient group) were hashtagged (HTO labeled), pooled and sorted by flow cytometry into CD45.1+ (WT) or CD45.2+ (WT or DOCK8-deficient) subsets. The CD45.1 and CD45.2 multiplexed samples were processed independently on the 10X scRNASeq platform.

last: Deobagkar-Lele

first: Greg

last: Crawford

first: Tanya

middle: L

last: Crockford

first: Jennifer

last: Back

first: Rose

last: Hodgson

first: Aneesha

last: Bhandari

first: Katherine

middle: R

last: Bull

first: Richard

last: Cornall


title: RNA-seq for HEK293T: a high-throughput method for R-loop mapping and quantitative assessment

overall_design: RNA-seq for HEK293T


title: USP44 overexpression drives a MYC-like gene expression program in neuroblastoma through epigenetic reprogramming

city: Stuttgart

first: Steven

last: Johnsen

address: Auerbachstrasse 112

institute: Robert Bosch Center for Tumor Diseases

summary: Neuroblastoma is an embryonic cancer that disproportionately contributes to death in young children. Sequencing data have uncovered few recurrently mutated genes in this cancer though epigenetic pathways have been implicated in disease pathogenesis. We performed a computational screen of deubiquitinating enzymes to identify potential new targets and identified the histone H2B deubiquitinating enzyme USP44 as significantly over-expressed in high-risk tumors. High levels of USP44 significantly correlate with metastatic disease, unfavorable histology, advanced patient age, and MYCN-amplification. The subset of patients with tumors expressing high levels of USP44 have a significantly worse survival, including those with tumors lacking MYCN-amplification. Depleting USP44 in neuroblastoma cell lines leads to diminished proliferation, migration, invasion, as well as impaired neuritic development in response to retinoic acid. Integrated analysis of RNA-seq and ChIP-seq demonstrates a distinct set of genes that is regulated by USP44, including those in Hallmark pathways critical to tumorigenesis – findings that were completely reversed by the re-introduction of USP44. We conclude that USP44 is a novel epigenetic regulator that restrains differentiation and promotes aggressive features. Our data further suggests that USP44 may be a novel target in this disease.

overall_design: mRNA-seq was performed in immortalized neuroblastoma cell line SH-SY5Y that were empty vector control or overexpressed USP44 to determine the contribution of USP44 on gene regulation.

first: Thomas

last: Ekstrom

first: Sajjad

last: Hussain

first: Paul

last: Galardy


title: Autotrophic adaptive laboratory evolution of the acetogen Clostridium autoethanogenum delivers the gas-fermenting strain LAbrini with superior growth, products, and robustness

city: Tartu

first: Kaspar

last: Valgepea

state: Tartumaa

address: Nooruse 1

department: Institute of Technology

country: Estonia

institute: University of Tartu

summary: Microbes able to convert gaseous one-carbon (C1) waste feedstocks are increasingly important to transition to the sustainable production of renewable chemicals and fuels. Acetogens are interesting biocatalysts since gas fermentation using Clostridium autoethanogenum has been commercialised. However, most acetogen strains need complex nutrients, display slow growth, and are not robust for bioreactor fermentations. In this work, we used three different and independent adaptive laboratory evolution (ALE) strategies to evolve the wild-type C. autoethanogenum to grow faster, without yeast extract and to be robust in operating continuous bioreactor cultures. Multiple evolved strains with improved phenotypes were isolated on minimal media with one strain, named “LAbrini”, exhibiting superior performance regarding the maximum specific growth rate, product profile, and robustness in continuous cultures. Whole-genome sequencing of the evolved strains identified 25 mutations. Of particular interest are two genes that acquired seven different mutations across the three ALE strategies, potentially as a result of convergent evolution. Reverse genetic engineering of mutations in potentially sporulation-related genes CLAU_3129 (spo0A) and CLAU_1957 recovered all three superior features of our ALE strains through triggering significant proteomic rearrangements. This work provides a robust C. autoethanogenum strain “LAbrini” to accelerate phenotyping and genetic engineering and to better understand acetogen metabolism.

overall_design: RNA sequencing of various strains of Clostridium autoethanogenum chemostat cultures grown on CO at various dilution rates; 15 samples in total with various number of biological replicate samples

first: Henri

last: Ingelman

first: James

last: Heffernan

first: Audrey

last: Harris

middle: D

last: Brown

first: Kurshedaktar

last: Shaikh

first: Asfand

last: Yar Saqib

first: Marina

last: Pinheiro

first: Lorena

last: de Lima

first: Karen

last: Rodriguez Martinez

first: Ricardo

last: Gonzalez-Garcia

first: Grant

last: Hawkins

first: Jim

last: Daleiden

first: Loan

last: Tran

first: Hunter

last: Zeleznik

first: Rasmus

middle: O

last: Jensen

first: Vinicio

last: Reynoso

first: Heidi

last: Schindel

first: Jürgen

last: Jänes

first: Séan

last: Simpson

first: Michael

last: Köpke

first: Esteban

last: Marcellin


title: snRNAseq analysis of mouse placenta during ZIKV infection

first: Scott

last: Handley

state: MO

address: 660 S Euclid Ave

institute: Washington University in St.Louis

summary: The aim of this study was to study placental immune responses to ZIKV infection at the cell level. WT or MAVS-deficient placentas from mock- or ZIKV- infected pregnant WT or mutant mice were collected, and placentas from male fetuses selected to facilitate the identification of maternal and fetal clusters, and doublets. Two placentas from the same genotype and condition were pooled and processed for isolation of nuclei. Approximately 10,000 nuclei per sample were subjected to gel bead-in-emulsion (GEM) generation, reverse transcription, and construction of libraries for sequencing according to the protocol provided in the 3' gene expression v3.1 kit manual (10X Genomics). We obtained 98,661 nuclei with a median of 3,077 genes detected per nucleus after data processing and quality control. Seurat integrated analysis and graph-based clustering identified cell types in maternal and fetal compartments based on analysis of canonical cellular markers. We performed differential expression test using Wilcoxon test from Seurat to identify the changes in expression across conditions. The DSGs were then used to align to the Hallmark mouse pathway database Msigdbr (Version 7.5.1) with package fgsea (Version 1.26.0) and pathways that with P-adjusted value < 0.05 were chosen as the significantly enriched pathways. We found cell-type specific antiviral pathways driven by MAVS, and MAVS-independent induction of interferon-stimulated and antiviral genes in other cell types.

overall_design: Nuclei were obtained from placentas belonging to male WT or MAVS-/- fetuses from mock- or ZIKV-infected mouse fetuses at different gestational stages

first: Yael

last: Alippe

first: Leran

last: Wang

first: Reyan

last: Coskun

first: Stefanie

last: Muraro

first: Fang

last: Zhao

first: Michelle

last: Elam-Noll

last: White

first: Daiana

last: Vota

first: Vanesa

last: Hauk

first: Jeffrey

middle: I

last: Gordon

last: Diamond


title: Multi-OMIC analysis of Huntington disease reveals a neuroprotective astrocyte state (snRNA-Seq)

first: Osama

last: Al Dalahmah

overall_design: Analysis of human brain samples using genomic and lipidomic approaches merged with functional and imaging studies on astrocyte cells

first: Fahad

last: Paryani

first: Ji-Sun

last: Kwon

first: Chris

last: Ng

first: Nacoya

last: Madden

first: Kenneth

last: Ofori

first: Alice

last: Tang

first: Hong

last: Lu

first: Juncheng

last: Li

first: Aayushi

last: Mahajan

first: James

last: Goldman

first: Shawn

last: Davidson

first: Andrew

last: Yoo

first: Vilas

last: Menon

last: Al-Dalahmah


title: Defective mitochondrial COX1 translation due to loss of COX14 function triggers ROS-induced inflammation in mouse liver

city: Gottingen

first: Andre

last: Fischer

state: NS

address: Von Sieboldstr 3a

department: Epigenetic Mechanisms in Dementia

institute: German Center for Neurodegenerative Diseases (DZNE)

summary: Mitochondrial oxidative phosphorylation (OXPHOS) fuels cellular ATP demands. OXPHOS defects lead to severe human disorders with unexplained tissue specific pathologies. Mitochondrial gene expression is essential for OXPHOS biogenesis since core subunits of the complexes are mitochondrial-encoded. COX14 is required for translation of COX1, the central mitochondrial-encoded subunit of complex IV. Here we describe a COX14 mutant mouse corresponding to a patient with complex IV deficiency. COX14M19I mice display broad tissue-specific pathologies. A hallmark phenotype is severe liver inflammation linked to release of mitochondrial RNA into the cytosol sensed by RIG-1 pathway. We find that mitochondrial RNA release is triggered by increased reactive oxygen species production in the deficiency of complex IV. Additionally, we describe a COA3Y72C mouse, affected in an assembly factor that cooperates with COX14 in early COX1 biogenesis, which displays a similar yet milder inflammatory phenotype. Our study provides insight into a link between defective mitochondrial gene expression and tissue-specific inflammation.

overall_design: To compare differences in gene expression between WT and COX14M19I mouse liver RNA samples

first: Abhishek

last: Aich

first: Angela

last: Boshnakovska

first: Steffen

last: Witte

first: Tanja

last: Gall

first: Kerstin

last: Unthan-Fechner

first: Roya

last: Yousefi

first: Arpita

last: Chowdhury

first: Drishan

last: Dahal

first: Aditi

last: Methi

first: Svenja

last: Kaufmann

first: Ivan

last: Silbern

first: Jan

last: Prochazka

first: Zuzana

last: Nichtova

first: Marcela

last: Palkova

first: Miles

last: Raishbrook

first: Gizela

last: Koubkova

first: Radislav

last: Sedlacek

first: Simon

last: Tröder

first: Branko

last: Zevnik

first: Dietmar

last: Riedel

first: Susann

last: Michanski

first: Wiebke

last: Möbius

first: Philipp

last: Ströbel

first: Christian

last: Lüchtenborg

first: Patrick

last: Giavalisco

first: Henning

last: Urlaub

first: Britta

last: Brügger

first: Stefan

last: Jakobs

first: Peter

last: Rehling


title: Investigating the noncanonical Nfkb2 pathway regulation of gene expression in modulating dendritic cell function.

city: New Delhi

first: Soumen

last: Basak

state: Delhi

address: Aruna Asaf Ali Marg

department: Infection and immunity

country: India

institute: National Institute of Immunology

summary: Dendritic cells (DCs) orchestrate intestinal inflammation in health and diseases. We found that human IBD was associated with heightened non-canonical NF-κB signaling in intestinal DCs. The non-canonical NF-κB pathway, which induces RelB:p52-mediated immune gene expressions, has been implicated in DC functions and that genetic inactivation of RelB:p52 in DCs alleviated experimental colitis in mice. Here, we aim to investigate the regulation of gene expression by noncanonical Nfkb2 pathway in modulating DC function.

overall_design: The dendritic cell were cultured from the bone marrow of C57BL/6 WT mice and the Nfkb2-/- mice using a GMCSF and IL4 cocktail. The total RNA was isolated from these BMDCs and subjected to RNASeq analysis.

first: Alvina

last: Deka

first: Naveen

last: Kumar


title: Deleterious variants in RNF111 impair female fertility and induce premature ovarian insufficiency in humans and mice

first: Chengcheng

last: Song

state: Shanghai

address: 220 Handan Rd.

institute: Fudan University

summary: Premature ovarian insufficiency (POI) is a heterogeneous female disorder characterized by the loss of ovarian function before the age of 40. It represents a significant detriment to female fertility. However, the known POI-causative genes currently account for only a fraction of cases. To elucidate the genetic factors underlying POI, we conducted whole-exome sequencing on a family with three fertile POI patients and identified a deleterious missense variant in RNF111. In a subsequent replication study involving 1030 POI patients, this variant was not only confirmed but also accompanied by the discovery of three additional predicted deleterious RNF111 variants. These variants collectively account for eight cases, representing 0.78% of the study cohort. A further study involving 500 patients with diminished ovarian reserves also identified two additional RNF111 variants. Notably, RNF111 encodes an E3-ubiquitin ligase with a regulatory role in the TGF-β/BMP signaling pathway. Our analysis revealed that RNF111/RNF111 is predominantly expressed in the oocytes of mice, monkeys, and humans. To further investigate the functional implications of RNF111 variants, we generated two mouse models: one with a heterozygous missense mutation (Rnf111+/M) and another with a heterozygous null mutation (Rnf111+/−). Both mouse models exhibited impaired female fertility, characterized by reduced litter sizes and small ovarian reserve. Additionally, RNA-seq and quantitative proteomics analysis unveiled that Rnf111 haploinsufficiency led to dysregulation in female gonad development and negative regulation of the BMP signaling pathway within mouse ovaries. In conclusion, our findings strongly suggest that monoallelic deleterious variants in RNF111 can impair female fertility and induce POI in both humans and mice.

overall_design: To investigate the functional implication of monoallelic deleterious variants in RNF111 to premature ovarian insufficiency, we generated two mouse models using CRISPR-Cas9. To further investigate the functional alterations within the mutant mouse ovaries, We then performed bulk RNA-seq analyses on ovaries from wild-type and Rnf111+/- mice.

first: Yingying

last: Qin

first: Yan

last: Li

first: Bingyi

last: Yang

first: Ting

last: Guo

first: Wenqing

last: Ma

first: Dian

last: Xu

first: Keyan

first: Fangfang

last: Fu

first: Li

last: Jin

first: Yanhua

last: Wu

first: Shuyan

last: Tang

first: Xiaojun

last: Chen

first: Feng

last: Zhang


title: Transcriptome profile of RCS WT and TFEB/3 DKO cells transfected with mutant COL2R789C compared to control

city: Pozzuoli

first: Rossella

last: De Cegli

address: Via Campi Flegrei 34

country: Italy

institute: Tigem

summary: To analyse gene expression differences between RCS COL2R789C transfected cells WT and TFEB/3 DKO compared to their relative scramble controls

overall_design: Total RNA was extracted from n=3, for each conditions.

first: Chiara

last: De Leonibus

first: Marianna

last: Maddaluno


title: HeLa COL1G610C transfected cells; RCS WT and TFEB/3 DKO cells transfected with mutant COL2R789C

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

sample_organism:
Homo sapiens
Rattus norvegicus

platform_organism:
Homo sapiens
Rattus norvegicus

overall_design: Refer to individual Series


title: Transcriptome profile of HeLa COL1G610C transfected cells compared to control cells

summary: To analyse gene expression differences between HeLa tranfected cells with mutant COL1 (COL1G610C) compared to their relative scramble controls

overall_design: Total RNA was extracted from n=5 for each conditions.


title: scRNAseq of RPE-hTert cells expressing different levels of predictive reporter construct

city: Cambridge

first: Adelyne

middle: SL

last: Chan

address: Li Ka Shing Way

department: Cancer Research UK

institute: University of Cambridge

summary: Single cell sequencing of RPE1-hTert cells transduced with the NLS-mVenus-P2A-ER:HRAS^G12V construct, and then flow-sorted into subpopulations based on increasing mVenus intensity, corresponding to different levels of oncogenic RAS stabilised upon induction with 4OHT.

overall_design: RPE1-hTert cells were infected with the predictive reporter (NLS-mVenus-P2A-ER:HRAS^G12V) construct then flow sorted into subpopulations based on increasing mVenus intensity ('S' < 'M' < 'L' < 'XL'). Cells were grown either in monoculture i.e. each subpopulation separately, or in coculture i.e. subpopulations mixed together in equal proportions, and induced with 4OHT. A total of 6 samples (uninduced coculture, induced coculture, induced 'S', induced 'M', induced 'L' and induced 'XL') were hashtagged with TotalSeq-A hashtag antibodies and pooled for encapsulation and single-cell sequencing using the 10x Genomics platform. Induced samples were harvested on Day 6 post-induction.

first: Masashi

last: Narita


title: A PIKfyve Modulator Combined with an Integrated Stress Response Inhibitor to treat Lysosomal Storage Diseases

city: La Jolla

first: Jeffery

last: Kelly

address: 10550 North Torrey Pines Road

department: Chemistry

institute: The Scripps Research Institute

summary: Lysosomal degradation pathways coordinate the clearance of superfluous and damaged cellular components. Compromised lysosomal degradation is a hallmark of many degenerative diseases, including lysosomal storage diseases, which are caused by loss-of-function mutations within both alleles of a lysosomal hydrolase, leading to lysosomal substrate accumulation. Gaucher’s disease, characterized by <15% of normal glucocerebrosidase function, is the most common lysosomal storage disease and is a prominent risk factor for developing Parkinson’s disease. Here, we show that either of two structurally distinct small molecules that modulate PIKfyve activity, discovered from a high-throughput cellular lipid droplet clearance screen, can improve glucocerebrosidase function in Gaucher patient–derived fibroblasts through an MiT/TFE transcription factor that promotes lysosomal gene translation. An ISR antagonist used in combination with a PIKfyve modulator further improves cellular glucocerebrosidase activity, likely because integrated stress response (ISR) signaling appears to also be slightly activated by treatment by either small molecule at the higher doses employed, This strategy of combining a PIKfyve modulator with an integrated stress response inhibitor improves mutant lysosomal hydrolase function in cellular models of additional lysosomal storage diseases.

overall_design: Total RNAsequencing of L444P-GBA1 fibroblasts following treatment with A16 (500 nM), A18 (1 uM), Apilimod (50 nM), and ISRIB (200 nM) for 24 h. RNA was submitted to BGI Genomics Co., Ltd. for strand-specific mRNA sequencing on their DNBSEQ platform with three biological replicates per condition.

first: William

middle: C

last: Hou

first: Lynée

last: Massey

middle: W


title: RNA-seq analysis of Mpwrky10 in Marchantia under nitrate and phosphate starvation, and comparative analysis with Lactuca sativa var. Redfire

city: Singapore

first: Shalini

last: Ravi

address: 1 Research link

country: Singapore

institute: Temasek lifesciences laboratory

summary: To understand the role of WRKY10 transcription factor in foliar pigmentation patterns in Marchantia (liverwort) and assess the conservation of its regulatory network in Lactuca sativa var. Redfire(angiosperm).

sample_organism:
Marchantia polymorpha
Lactuca sativa

platform_organism:
Marchantia polymorpha
Lactuca sativa

overall_design:
Marchantia Polymorpha; Lactuca sativa var Redfire
7-day old gemmalings of wildtype Marchantia Tak-1 and Mpwrky10 were treated with control (2 mM phosphate and 10 mM nitrate), phosphate deficiency (0 mM phosphate) and nitrate deficiency (0 mM nitrate) for 5 days.
4-day old seedlings of Lettuce were treated with control (2 mM phosphate and 10 mM nitrate), phosphate deficiency (0 mM phosphate) and nitrate deficiency (0 mM nitrate) for 7 days.

last: Krishnamoorthi

first: Daisuke

last: Urano


title: Myb is regulated by -81 enhancer eRNA transcript Myrlin through recruitment of MLL1 complex

city: Bethesda

first: Ann

last: Dean

state: MD

address: 50 South Drive, Bldg 50

department: NIDDK

institute: NIH

type: Genome binding/occupancy profiling by high throughput sequencing

summary: The Myb proto-oncogene encodes the transcription factor c-MYB, which is critical for the proliferation and differentiation of hematopoietic stem and progenitor cells. Distant enhancers of Myb expression have been characterized but the regulation of Myb during hematopoiesis is still incompletely understood. Here we identified a novel nuclear Myb enhancer long intergenic non-coding RNA (Myrlin) that originates from the -81 kb murine Myb enhancer within the Myb ─ Hbs1l intergenic region. Myrlin and Myb are coordinately regulated in a developmental stage-specific fashion during maturation of erythroid progenitors and upon differentiation of MEL cells. CRISPR/Cas9 genome editing of the Myrlin transcription start site at the -81kb enhancer reduced both Myrlin and Myb expression. The deletion of Myrlin TSS reduces the occupancy of LDB1, which mediates chromatin looping, and compromises long-range contacts between the Myb promoter and enhancer and RNA Pol II occupancy decreases across the Myb locus. In contrast, silencing of Myrlin using CRISPRi similarly reduced both Myrlin and Myb expression but left the Myb enhancer hub undisturbed, separating chromatin looping from transcription activation of Myb. In unedited cells, we found that Myrlin interacts with MLL1 complex, a transcriptional coactivator that plays an essential role in regulating gene expression during hematopoiesis. Myrlin CRISPRi compromised MLL1 occupancy in the Myb locus and decreased CDK9 and RNA Pol II binding. Myrlin CRISPRi further resulted in pausing of RNA Pol II in the Myb first exon/intron. These data suggest that Myrlin directly participates in activating Myb transcription by recruiting MLL1.

overall_design: CUT&TAG sequencing for Phospho-Rpb1 CTD Ser5 and Ser2 in dCAS_control and dCAS_Myrlin MEL cells.

first: Juhyun

last: Kim

first: Lecong

last: Zhou


first: Steven

middle: A

last: Johnsen

overall_design: mRNA-seq was performed in immortalized mouse embryonic fibroblasts (MEF) that were USP44 null or were rescued with re-introduction of USP44 to determine the contribution of USP44 on gene regulation.

first: Thomas

last: Ekstrom

first: Sajjad

last: Hussain

first: Paul

last: Galardy


title: Single cell RNAseq of control vs. OIS hepatocytes

summary: Single cell sequencing of hepatocytes isolated from mice injected via HDTV with transposons containing mVenus and either active (^G12V) or inactive (^D38A/G12V) oncogenic NRAS

overall_design: Multiplexed single-cell RNAseq experiment: Three mice were used in this experiment, all having received hydrodynamic tail vein (HDTV) injection of either mVenus-NRAS^G12V-containing or mVenus-NRAS^G12V/D38A-containing  transposons. At Day 6 post-injection, livers were harvested and dissociated to single cells. Mvenus-expressing hepatocytes were flow sorted from the dissociated livers and labelled with Hashtag oligos before multiplexing and single cell RNA sequencing.


title: Gene expression of glucose homeostasis in a pancreas-liver microphysiological system

status: Public on Jun 10 2024

city: Molndal

first: Fredrik

last: Karlsson

address: Pepparedsleden 1

country: Sweden

institute: AstraZeneca

summary: Current research on metabolic disorders and diabetes relies on animal models because multi-organ diseases cannot be well studied with the standard in vitro assays. Here, we connect models of key metabolic organs, pancreas and liver, on a microfluidic chip to enable diabetes research in a human-based preclinical system. Aided by mechanistic mathematical modelling, we show that hyperglycemia and high cortisone induce glucose dysregulation in the pancreas-liver microphysiological system (MPS) mimicking a diabetic phenotype seen in patients with glucocorticoid-induced diabetes. In this diseased condition, pancreas-liver MPS displays beta-cell dysfunction, steatosis, elevated ketone-body secretion, increased glycogen storage, and upregulated gluconeogenic gene expression. In turn, a physiological culture condition rescues the glucose tolerance and beta cell function. This method was evaluated for repeatability in two laboratories and was effective in multiple pancreatic islet donors. The model also provides a platform to identify new therapeutic targets as demonstrated with a combined transcriptome and proteome analysis.

overall_design: We performed an exploratory transcriptome and proteome analyses of liver lysates and supernatants from hyperglycemic and normoglycemic co-culture conditions at the end of the chip co-culture.

first: Maryam

last: Clausen

first: Liisa

last: Vilén


title: Titration of RAS alters senescent state and influences tumour initiation

first: Adelyne

last: Chan

last: Narita


title: Single cell RNAseq of hepatocytes isolated from UBC-injected mice at different timepoints post-HDTV

summary: Single-cell sequencing of mVenus-expressing hepatocytes from mouse livers following introduction of either control or oncogenic NRAS-containing transposons

overall_design: Multiplexed single-cell RNAseq experiment: Ten mice were used in this experiment, all having received hydrodynamic tail vein (HDTV) injection of either mVenus-NRAS^G12V-containing or control mVenus only transposons. At different timepoints post-injection (Day 12, Day 30 and Day 218 – after formation of liver tumours detectable by ultrasound imaging), livers were harvested and dissociated to single cells. Mvenus-expressing hepatocytes were flow sorted from the dissociated livers and labelled with Hashtag oligos before multiplexing and single cell RNA sequencing.

first: Masashi


title: RNAseq of RPE-hTert cells expressing different levels of predictive reporter construct

summary: RNAseq of different subpopulations of RPE1 cells expressing predictive reporter construct, pre- and on Day 6 post-induction with 4OHT

overall_design: Parent ('N') RPE1-hTert cells were infected with the predictive reporter (NLS-mVenus-P2A-ER:HRAS^G12V) construct then flow sorted into subpopulations based on increasing mVenus intensity ('S' < 'M' < 'L' < 'XL'). Cells were harvested for RNA extraction pre- and on Day 6 post-induction with 4OHT. Five biological replicates of each sample were sequenced.


title: USP44 overexpression drives a MYC-like gene expression program in neuroblastoma through epigenetic reprogramming

summary: Neuroblastoma is an embryonic cancer that disproportionately contributes to death in young children. Sequencing data have uncovered few recurrently mutated genes in this cancer though epigenetic pathways have been implicated in disease pathogenesis. We performed a computational screen of deubiquitinating enzymes to identify potential new targets and identified the histone H2B deubiquitinating enzyme USP44 as significantly over-expressed in high-risk tumors. High levels of USP44 significantly correlate with metastatic disease, unfavorable histology, advanced patient age, and MYCN-amplification. The subset of patients with tumors expressing high levels of USP44 have a significantly worse survival, including those with tumors lacking MYCN-amplification. Depleting USP44 in neuroblastoma cell lines leads to diminished proliferation, migration, invasion, as well as impaired neuritic development in response to retinoic acid. Integrated analysis of RNA-seq and ChIP-seq demonstrates a distinct set of genes that is regulated by USP44, including those in Hallmark pathways critical to tumorigenesis – findings that were completely reversed by the re-introduction of USP44. We conclude that USP44 is a novel epigenetic regulator that restrains differentiation and promotes aggressive features. Our data further suggests that USP44 may be a novel target in this disease.

overall_design: ChIP-seq for UbH2B, H3K4Me3, H3K27Ac, and H3K27Me3 was performed in immortalized mouse embryonic fibroblasts (MEF) that were USP44 null or were rescued with re-introduction of USP44 to determine the contribution of USP44 on epigenetic regulation.


title: CRISPRi screen identifies core regions in IGH enhancers essential for non-Hodgkin lymphoma cells survival

city: Poznan

first: Agnieszka

last: Dzikiewicz-Krawczyk

address: Strzeszynska 32

country: Poland

institute: Institute of Human Genetics, Polish Academy of Sciences

summary: Chromosomal translocations in non-Hodgkin lymphoma (NHL) are known to bring oncogenes (e.g. MYC, BCL2) under the regulation of immunoglobulin heavy chain (IGH) super-enhancers, composed of the intronic Eµ enhancer and 3’ regulatory regions (3’RR1, 3’RR2). Deregulation of translocated oncogenes by enhancer activity can contribute to lymphomagenesis.  The role of IGH enhancers in normal B-cell development is well established, but the knowledge regarding precise mechanisms of their involvement in control of the translocated oncogenes is limited. We previously identified exact regions within IGH regulatory elements, one in the Eµ and two in each of the 3'RRs, whose targeting profoundly inhibited NHL cells growth. The goal of this project was to reveal enhancer RNAs (eRNA) transcribed from Eµ and 3'RRs and their essential regions. With the use of chromatin-enriched RNA-Seq we confirmed bi-directional transcription from those core enhancer regions and further validated eRNAs expression in a large panel of NHL cell lines and in patient-derived samples. We showed in several NHL cell lines that CRISPR/dCas9-KRAB-mediated inhibition of essential IGH enhancer regions decreased expression of eRNAs and translocated oncogenes in the fashion likely related to their breakpoints pattern within IGH locus. Our results provide new insights into the current understanding of IGH enhancers role in B-cell NHLs.

overall_design: To reveal enhancer RNAs transcription from IGH enhancer regions cellular fractionation of wild type B-cell lymphoma and control cell lines was performed and chromatin-associated RNA was subjected to RNA-Sequencing.

first: Marta

middle: E

last: Kasprzyk

first: Weronika

last: Sura

last: Podralska

last: Kazimierska

first: Annika

last: Seitz

first: Wojciech

last: Łosiewski

first: Tomasz

last: Woźniak

first: Jeroen

last: Guikema

first: Joost

last: Kluiver

first: Anke

last: van den Berg

first: Natalia

last: Rozwadowska


title: Immunotherapy-activated T cells install a subset of late-stage activated M1-like macrophages that are critical for therapeutic efficacy

city: Leiden

first: Sjoerd

middle: H

last: van der Burg

address: Albinusdreef 2

department: Medical Oncology

country: Netherlands

institute: LUMC

summary: Total tumor clearance through immunotherapy is associated with a fully coordinated innate and adaptive immune response, but knowledge on the exact contribution of each immune cell subset is limited. We show that therapy-induced intratumoral CD8 T cells recruited and installed late-stage activated M1-like macrophages, which were critical for effective tumor control in two different murine models of cancer immunotherapy. The activated CD8 T cells summon these macrophages into the tumor and their close vicinity via CCR5-signaling. Exposure of non-polarized macrophages to activated T-cell supernatant and tumor lysate recapitulates the late-stage activated and tumoricidal phenotype in vitro.The transcriptomic signature of these macrophages is also detected in a similar macrophage population present in human tumors and coincides with clinical response to immune checkpoint inhibitors. The requirement of a functional co-operation between CD8 T cells and effector macrophages for effective immunotherapy gives warning to combinations with broad macrophage targeting strategies.

overall_design: To gain an understanding of different subsets of intratumoral M1-like macrophages, we performed single cell RNA-sequencing (scRNAseq) on MHCII+ and MHCII- macrophage populations (CD11b+F4/80+) isolated from RMA-Qa1b-/- tumors that responded to therapeutic vaccination.

first: Marit

last: van Elsas

first: Jim

last: Middelburg

first: Camilla

last: Labrie

first: Jessica

last: Roelands

first: Gaby

last: Schaap

first: Marjolein

last: Sluijter

first: Ruxandra

last: Tonea

first: Vitalijs

last: Ovcinnikovs

first: Katy

last: Lloyd

first: Janine

last: Schuurman

first: Samantha

last: Riesenfeld

first: Thomas

middle: F

last: Gajewksi

first: Noel

last: de Miranda

first: Thorbald

last: van Hall


title: Gene expression profile of HSC/Ps and mature myeloid cells in HMGA2-OE MDS mutant clone

city: Hachioji

first: Yoshihiro

last: Hayashi

state: Tokyo

address: 1432-1 Horinouchi

country: Japan

institute: Tokyo University of Pharmacy and Life Sciences

summary: Although HMGA2 overexpression (OE) has been reported in several myeloid malignancies, its role in MDS remains unclear. To determine the role of HMGA2 in MDS pathogenesis, we generated MDS mice using patient-derived RUNX1 mutant, and overexpressed HMGA2 in this model. HMGA2-OE MDS mice developed non-infectious pneumonia. Prominent infiltration of neutrophils was observed in the lung tissue. RNA sequencing of HSC/Ps and neutrophils revealed that genes related to platelet activation are significantly activated in HMGA2-OE MDS mice. Flow cytometric analysis showed that P-selectin+ activated platelets tightly interact with neutrophils in the HMGA2-OE MDS mice. Genetic inhibition of P-selectin attenuated neutrophil activation and pneumonia development in the MDS mice.  These findings provide new insights into mechanistic basis of systemic complications of MDS.

overall_design: Expression profiles of mRNA in HSC/Ps and Ly6G+ myeloid cells from RUNX1 mutant mice with or without HMGA2-OE and their control mice.

first: Hironori

last: Harada


title: Single cell transcriptome analysis of primary ANKL cells derived from ANKL-PDX treated with PPMX-T003

city: Saga

first: Ryo

last: Yanagiya

state: Saga

address: 5-1-1 Nabeshima

department: Department of Internal Medicine

institute: Saga University

summary: PPMX-T003, an humanized anti-transferrin receptor 1 inhibitory antibody, has anti-neoplastic potential against ANKL. However, phenotype of refractory subclone of ANKL against PPMX-T003 remains unclear. To investigate, we performed single cell transcriptome analysis of ANKL1, a primary ANKL cells derived from ANKL-patient-derived xenograft mouse model (ANKL-PDX) with/without treatment of PPMX-T003 injection.

overall_design: Total of six ANKL1-PDX were established. Three mices were sacrificed and ANKL cells were harvested from liver and spleen 14 days after establishment, and others were treated with intravenously inoculation of 10 mg/kg PPMX-T003 on 5, 8, 12, 15 days after establishment, and sacrificed 3 days after last treatment, followed by collection of ANKL cells from spleen.

first: Natsumi

last: Watanabe

first: Ai

last: Kotani


title: Mapping cellular plasticity through the lens of Fra1 during KrasG12D mediated pancreatic acinar to ductal metaplasia [CUT&RUN]

status: Public on Jun 09 2024

city: New York

first: Anil

middle: K.

last: Rustgi

state: New York

address: 1130 St. Nicholas Avenue

institute: Columbia University

summary: Acinar cells have been proposed as a cell-of-origin for pancreatic intraepithelial neoplasia (PanIN) after undergoing a highly regulated acinar to ductal metaplasia (ADM) process. ADM can be triggered by pancreatitis causing acinar cells de-differentiate to a ductal-like state. We identify Fra1 (gene name Fosl1) as the most enriched transcription factor during KrasG12D acute pancreatitis mediated injury. We have elucidated the functional role of Fra1 by generating an acinar-specific Fosl1 knockout mouse expressing KrasG12D (Ptf1aCreERT;KrasG12D;Fosl1fl/fl;YFP) . Using single nuclei ATAC-seq and bulk-RNA seq, we used pseudotime analysis and developed a gene-regulatory network governing de-differentiation to demonstrate that Fosl1 knockout mice are delayed in the onset of ADM and accompanying recovery.  Fosl1 depletion prevents the pro-inflammatory effects of G-CSF, an ADM-promoting cytokine, suggesting that the G-CSF/Fra1 signaling axis can modulate ADM. Overall, our studies mark the first time a discrete transcriptional factor has been linked to the temporal regulation of ADM.

overall_design: CUT&RUN H3K27ac data and background IgG of Ptf1aCreERT;KrasG12D;YFP mice on Day 5 after acute pancreatitis

first: Alina

last: Li

first: Kensuke

last: Suguira

last: Suzuki

first: Dorsay

last: Sadeghian

first: Jun

last: Zhao

first: Anirban

last: Maitra

first: David

last: Falvo

first: Rohit

last: Chandwani

first: Jason

last: Pitaressi

first: Peter

last: Sims


title: in vivo CRISPR screening of ANKL-PDX targeting to iron-requirement molecules

summary: ANKL cell proliferates predominantly in liver sinusoid depending on transferrin. However, little is known about molecular mechanism of iron-dependency of ANKL. To investigate, we performed in vivo CRISPR screening of liver resident ANKL cells targeting to genes encoding 482 iron-requirement molecules.

overall_design: ANKL1 cells, an primary ANKL cells harvested from ANKL-patient-derived xenograft mouse model (ANKL-PDX) previously reported, were transduced with Cas9- and small guide RNA (sgRNA) library-expressive plasmids via lentivirus vector, and PDX were established with them. Fourteen days after, ANKL cells were re-harvested from liver of established PDX, followed by genomic DNA extraction and integrated sgRNA amplification. After deep sequencing, dropped-out guide sgRNAs were extracted using MAGeCK program.

first: Masaya

last: Ueno

first: Atsushi

last: Hirao


title: Mapping cellular plasticity through the lens of Fra1 during KrasG12D mediated pancreatic acinar to ductal metaplasia [snATAC]

overall_design: single-nuclei ATAC sequencing data of Ptf1aCreERT;KrasG12D;Fosl1+/+;YFP or Ptf1aCreERT;KrasG12D;Fosl1fl/fl;YFP mice on Day 5 after acute pancreatitis.

middle: K


title: Mapping cellular plasticity through the lens of Fra1 during KrasG12D mediated pancreatic acinar to ductal metaplasia [RNA-seq]

overall_design: Stranded RNA-sequencing data of Ptf1aCreERT;KrasG12D;YFP or Ptf1aCreERT;KrasG12D;Fosl1fl/fl;YFP mice on Day 5 after acute pancreatitis.


title: Endogenous p53 Inhibitor TIRR Dissociates Systemic Metabolic Health from Oncogenic Activity - RNAseq data

city: Łódź

first: Jędrzej

last: Chrzanowski

state: łódzkie

address: Mazowiecka 15

department: Department of Biostatistics and Translational Medicine

institute: Medical University of Lodz

summary: It is unclear whether metabolic health corresponds to reduced oncogenesis or vice versa. We study Tudor-interacting repair regulator (TIRR), an inhibitor of p53 binding protein 1 (53BP1)-mediated p53 activation, and the physiological consequences of enhancing tumor suppressor activity. Deleting TIRR selectively activates p53, significantly protecting against cancer but leading to a systemic metabolic imbalance in mice. TIRR-deficient mice are overweight and insulin resistant, even under normal chow diet. Similarly, reduced TIRR expression in human adipose tissue correlates with higher BMI and insulin resistance. Despite the metabolic challenges, TIRR loss improves p53 heterozygous (p53HET) mouse survival and correlates with enhanced progression-free survival in patients with various p53HET carcinomas. Finally, TIRR’s oncoprotective and metabolic effects are dependent on p53 and lost upon p53 deletion in TIRR-deficient mice, with glucose homeostasis and orexigenesis being primarily regulated by TIRR expression in the adipose tissue and the CNS, respectively, as evidenced by tissue-specific models. In summary, TIRR deletion provides a paradigm of metabolic deregulation accompanied by reduced oncogenesis.

overall_design: Comparative gene expression profiling analysis of RNA-seq data from mouse (C57Bl6 background) for: (1) perigonadal adipose tissue and brown adipose tissue models of Nudt16l1 knock-out (whole organism and adipose-specific) and Tp53 (whole organism) knock-out and their combinations; (2) liver, hypothalamus and perigonadal adipose tissue (2nd cohort) models of Nudt16l1 knock-out (whole organism); (3) mouse embryonic fibroblasts models of Nudt16l1 knock-out (whole organism) and Tp53 (whole organism) knock-out and their combinations.

first: Eva

last: Tsaousidou

first: Jedrzej

first: Pascal

last: Drané

first: Yizhou

last: He

first: Mateusz

last: Kaminski

first: Dominika

last: Michalek

first: Grace

middle: Y

last: Lee

first: Ekin

last: Güney

first: Kalindi

last: Parmar

first: Wojciech

last: Fendler

first: Dipanjan

last: Chowdhury

first: Gökhan

middle: S

last: Hotamışlıgil


title: Transcriptome analysis of NK92 teated with PPMX-T003 for connectivity scoring analysis

first: Ryo

last: Yanagiya

summary: PPMX-T003, an humanized anti-transferrin receptor 1 inhibitory antibody, has anti-neoplastic potential against mature NK cell neoplasms. However, its molecular mechanism is not elucidated. To investigate, we performed RNAseq of NK92 cells treated with PPMX-T003 to obtain defferentially expressed gene signature and compared it to those of well-known anti-neoplastic agents for prediction of similarity in transcriptomic change.

overall_design: NK92 cells were in vitro cultured for 18 hours, then equally splitted into two plates, followed by additional 6 hours cultivation with/without 10 µg/mL PPMX-T003.

first: Sachiko

last: Okabe

first: Tetsuo

last: Mashima

first: Ai

last: Kotani


title: Single-cell-resolved interspecies comparison shows a shared inflammatory axis and a dominant neutrophil-endothelial program in severe COVID-19

city: Heidelberg

first: Stefan

last: Peidli

state: BW

address: Meyerhofstraße 1

department: Genome Biology

institute: European Molecular Biology Laboratory

summary: Key issues for research of COVID-19 pathogenesis are the lack of biopsies from patients and of samples at the onset of infection. To overcome these hurdles, hamsters were shown to be useful models for studying this disease. Here, we further leveraged the model to molecularly survey the disease progression from time-resolved single-cell RNA-sequencing data collected from healthy and SARS-CoV-2-infected Syrian and Roborovski hamster lungs. This series contains scRNA-seq data from Roborovski hamster lungs.

overall_design: Roborovski hamsters (Phodopus roborovskii) were infected with either 1x10^4 plaque-forming units (pfu) or 1x10^5 pfu SARS-CoV-2 Variant B.1. scRNA-seq data from hamster lungs collected at 0 (naive), 2, and 3 days post infection (dpi) were generated using the 10x Genomics platform: (1 naive + 3 timepoints x 2 doses) x 3 replicates = 15 samples in total.

first: Geraldine

last: Nouailles

first: Emanuel

last: Wyler

first: Julia

middle: M

last: Adler

first: Sandra

last: Kunder

first: Anne

last: Voß

last: Kazmierski

first: Fabian

last: Pott

first: Peter

last: Pennitz

first: Dylan

last: Postmus

first: Luiz

middle: G

last: Teixeira Alves

first: Christine

last: Goffinet

first: Achim

last: Gruber

first: Nils

last: Blüthgen

first: Martin

last: Witzenrath

first: Jakob

last: Trimpert

first: Markus

last: Landthaler

first: Samantha

last: Praktiknjo


title: An immunoreactive signature of monocyte-derived dendritic cells from patients with Down syndrome

city: Fukuoka

first: Yasunari

last: Sakai

address: 3-1-1 Maidashi, Higashi-ku

department: Department of Pediatrics

institute: Kyushu University

summary:
Monocyte-derived dendritic cells (MoDCs) were obtained from two controls and two Down syndrome patients.
A microarray-based transcriptomic analysis was performed on the Clariom D Assay, Human platform (ThermoFisher Scientific).

overall_design: Comparative analysis of differentially expressed genes for LPS stimulate between two control samples and two samples from patients with Down syndrome.

first: Kentaro

last: Nakashima


title: Identification of immune-related genes and small-molecule drugs in hypertension-induced left ventricular hypertrophy based on machine learning algorithms and molecular docking [2]

status: Public on Jun 08 2024

city: Beijing

first: Mingxuan

last: Zhou

state: China

address: No. 2 Nanwei Road, Tianqiao Street

institute: Institute of Materia Medica, Chinese Academy of Medical Sciences

summary: Left ventricular hypertrophy (LVH) is a common consequence of hypertension and leads to heart failure. Immune response plays an important role in hypertensive LVH, whereas there is no comprehensive method to investigate the mechanistic relationships between immune response and hypertensive LVH or to find novel therapeutic targets. This study aimed to screen hub immune-related genes involved in hypertensive LVH as well as to explore immune target-based therapeutic substance. A total of 1215 differentially expressed genes (DEGs) were obtained, most of which were significantly enriched in immunoregulation and collagen synthesis. WGCNA and multiple machine learning strategies discovered six hub immune-related genes (Ankrd1, Birc5, Nuf2, C1qtnf6, Fcgr3, and Cdca3) that may accurately predict hypertensive LVH diagnosis. Immune analysis revealed that fibroblasts and macrophages were closely correlated with hypertensive LVH, and hub gene expression was significantly associated with these immune cells. A regulatory network of transcription factor-mRNA and a ceRNA network of miRNA-lncRNA was established. Notably, six hub immune-related genes were significantly increased in the hypertensive LVH model, which were positively linked to the left ventricle wall thickness. Finally, twelve small molecule compounds with the potential to reverse the high expression of hub genes were ruled out as potential therapeutic agents for hypertensive LVH.This study identified and validated six hub immune-related genes that may play essential roles in hypertensive LVH, providing new insights into the potential pathogenesis of cardiac remodeling and novel targets for medical interventions.

overall_design: We collected RNA-seq data from the control group and the ANG II-induced hypertensive-LVH group.

first: Min

last: Yang

first: Tiegang

last: Li

first: Silin

last: Lv

first: Wenqiang

last: Gan

first: Fang

last: Zhang

first: Yuexia

last: Che

first: Liu

first: Yufang

last: Hou

first: Zheng

last: Yan

first: Zifan

last: Zeng

first: Wenyi

last: Zhao


title: Identification of immune-related genes and small-molecule drugs in hypertension-induced left ventricular hypertrophy based on machine learning algorithms and molecular docking


title: IFN𝛾-induced Immunosuppression in Lung Carcinoma is Mediated by an Environmental Chemical Receptor (AhR) through PD-L1 and IDO Control (cmt167 scRNA)

city: Boston

first: David

last: Sherr

state: MA

address: 72 East Concord St

department: Environmental Health

institute: Boston University

summary: While immunotherapy has shown efficacy in non-small cell lung cancer (NSCLC) patients, many respond only partially or not at all. One limitation in improving outcomes is the lack of a complete understanding of factors regulating immune checkpoint targets. Here, we sought to address a possible link between an environmental chemical receptor implicated in NSCLC and immune regulation, the aryl hydrocarbon receptor (AhR), and a known but counterintuitive mediator of immunosuppression, IFN𝛾, in regulation of two immune checkpoints, PD-L1 and the IDO1, in lung adenocarcinoma (LUAD). To this end we used AhR, PD-L1, and IFN𝛾R gene-edited LUAD cell lines, a syngeneic LUAD mouse model, bulk- and single cell-RNA sequencing of LUADs and tumor-infiltrating leukocytes, and existing human transcriptomic databases. The data demonstrate that: 1) the AhR regulates both PD-L1 and IDO1 in murine and human LUAD cells, 2) AhR-driven IDO1 results in production of Kyn which likely mediates an AhR→IDO1→Kyn→AhR amplification loop, 3) the previously characterized induction by IFN𝛾of PD-L1 and IDO is mediated by the AhR, 4) transplantation of LUAD cells in which the AhR is deleted results in long-term tumor immunity in approximately half of the mice; slow growing tumors in the other half exhibit significantly higher densities of CD4+ and CD8+ T cells expressing immunocompetence markers, and 5) deletion of either IFN𝛾R1 or PD-L1 does not provide the same level of immune protection as AhR deletion. The data definitively link IFN𝛾- mediated immunosuppression to the AhR and support the targeting of the AhR in the context of LUAD.

overall_design: To investigate the presence or absence of Ahr in malignant cells of the lung in regards to the functions related to tumorogenicity and immune signalling using an average of 5,792 viable cells from wildtype tumors from two mice and an average of 3,173 viable cells from AhR-KO tumors

first: Megan

last: Snyder

first: Zhongyan

last: Wang

first: Brian

last: Lara

first: Jocelyn

last: Fimbres

first: Muzamil

last: Mohammed

first: Stefano

last: Monti


title: IFN𝛾-induced Immunosuppression in Lung Carcinoma is Mediated by an Environmental Chemical Receptor (AhR) through PD-L1 and IDO Control

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

overall_design: Refer to individual Series


title: IFN𝛾-induced Immunosuppression in Lung Carcinoma is Mediated by an Environmental Chemical Receptor (AhR) through PD-L1 and IDO Control (cm167 bulk RNA)

overall_design: The experiment is comprised of 6 samples, profiling RNA extracted from mouse CMT 167 (C57 mouse tumor; referred to herein as CMT) cell lines. Each cell line was either unmodified (naive, or "wild-type"), subjected to CRISPR with a non-targeting control sgRNA, or subjected to CRISPR with an sgRNA designed to knock out the aryl hydrocarbon receptor (Ahr) gene. Each experimental group was represented in triplicate.


title: Inferring pattern-driving intercellular flows from single-cell transcriptomics of young human cortical organoids

city: Irvine

first: Yuan-Chen

last: Tsai

state: CA

address: 845 Health Sciences Road

department: Anatomy and Neurobiology

institute: University of California, Irvine

summary: Intercellular communication drives directed flows of information that are mediated by intracellular gene modules, in turn triggering outflows of other signals. Methodologies to describe such intercellular flows are severely lacking. We present FlowSig, a method that infers communication-driven intercellular flows from scRNA-seq using graphical causal modeling and conditional independence. We used human cortical organoid samples collected at D18 and D35 to demonstrate the functions of FlowSig.

overall_design: Organoids were self-assembled using 9,000 stem cells per organoid and cultured in brain cortical region-inducing media. 160 cortical organoids were pooled for a D18 sample, and 25 cortical organoids were pooled for a D35 sample. There were two biological repeats per time point. The samples were subjected to scRNA-seq to identify key signaling pathways from D18 to D35 using FlowSig.

first: Axel

last: Almet

first: Qing

last: Nie

first: Momoko

last: Watanabe


title: PHF6 cooperates with SWI/SNF complexes to facilitate transcriptional progression[CUT&RUN]

status: Public on Jun 07 2024

city: Memphis

first: Jacquelyn

last: Myers

state: TN

address: 262 Danny Thomas Pl

institute: St. Jude Children’s Research Hospital

summary: Genes encoding subunits of SWI/SNF chromatin remodeling complexes are mutated in nearly 25% of cancers.  The SWI/SNF subunit SMARCB1 is the driving mutation in nearly all cases of rhabdoid tumor (RT), highly malignant cancers of childhood.  While loss of SMARCB1 drives cancer, its absence also creates unique dependencies, which can illuminate mechanism and reveal potential therapeutic vulnerabilities.  To identify such dependencies we undertook a genome-scale CRISPR screen comparing RT cell lines to 800 other cancer lines. We identified PHF6 as specifically essential for RT cell survival.  Germline mutations in both SMARCB1 and PHF6  cause Coffin-Siris syndrome, and somatic mutations in PHF6 or SWI/SNF occur in T-ALL, suggesting a mechanistic link.  We establish that PHF6 co-localizes with residual SWI/SNF complexes at active promoters and enhancers.  We find that PHF6 is specifically required for H3K14ac accumulation immediately downstream of active promoters and for release of paused Pol II.  Collectively, our work identifies novel integrated roles for PHF6 in chromatin and Pol II regulation, establishes a mechanistic basis for dependence upon PHF6 in RT, and reveals a novel potential therapeutic vulnerability in RT.

overall_design: ChIP-Seq was performed in biological triplicate in either shPHF6 or non-targeting control for PHF6 antibody

first: Priya

last: Mittal

first: Charles

last: Roberts