title: Gene expression in pre-cystic mouse kidneys after Pkd2 deletion.

city: Worcester

first: Gregory

last: Pazour

address: 373 Plantation St.

department: Program in Molecular Medicine

institute: UMass Chan School of Medicine

summary:
To identify early transcriptional changes downstream of Pkd2 mutations that promote cyst formation, we used tamoxifen-inducible CAG-Cre to induce Pkd2 deletion at postnatal day 2 and compared to wild-type mice at postnatal day 6 and 10.
We then performed gene expression profiling analysis using data obtained from RNA-seq of whole mouse kidneys.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for Pkd2 deleted mouse kidneys vs wild-type.

first: Abigail

middle: O

last: Smith

first: Julie

last: Jonassen


title: Small noncoding RNA expression in pre-cystic mouse kidneys after Pkd2 deletion.

status: Public on Nov 26 2024

state: MA

country: USA

type: Non-coding RNA profiling by high throughput sequencing

summary: To identify differentially expressed small noncoding RNAs downstream of Pkd2 mutations that may regulate early cyst growth, we used tamoxifen-inducible CAG-Cre to induce Pkd2 deletion at postnatal day 2 and compared to wild-type mice at postnatal day 6 and 10.

overall_design:
We then performed miRNA profiling analysis using data obtained from sequencing small RNA from whole mouse kidneys.
Comparative miRNA expression profiling analysis of small RNA-seq data for Pkd2 deleted mouse kidneys vs wild-type.

middle: J

middle: A

first: Yvonne

last: Edwards


title: Single cell RNA-seq of a laser treated alopecia model

city: Suita

first: Kouichi

last: Hasegawa

state: Osaka

address: 6F Techno Alliance Bldg. 2-8 Yamadaoka

department: StemRIM Institute of Regeneration-Inducing Medicine

country: Japan

institute: Osaka University

type: Expression profiling by high throughput sequencing

summary: Hair loss, including alopecia, is one of the most common dermatological issues worldwide. At present, the application of fractional CO2 laser in the treatment of alopecia has been few documented, however, the results are varied between the reports. These different results may come from limited knowledge of cellular action in the laser irradiated skin. The objective of this study was to investigate the molecular and cellular mechanism of laser treatment at the affective condition for hair cycle initiation.

overall_design: Fractional CO2 laser was applied and optimized to initiate their hair cycle in a mouse model of alopecia. The irradiated skin was analyzed by multiple cellular markers with immunofluorescent staining and flow cytometry. The cellular populations and their comprehensive gene expression was analyzed by single cell RNA sequencing and bioinformatics.

first: Takashi

last: Shimbo


title: Regulation and function of a polarly localized lignin barrier in the exodermis

city: Davis

first: Siobhan

last: Brady

state: CA

address: One Shields Avenue, GBSF Room 4409

department: Genome center

institute: UC Davis

summary: This experiment aimed to identify DEG in tomato hairy roots from the slscz and slexo1 mutant backgrounds.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for tomato hairy roots (wild-type, slscz and slexo1).

first: Concepcion

last: Manzano

middle: M


title: Control of Striatal Circuit Development by the  Chromatin Regulator Zswim6 [snRNA-Seq]

status: Public on Nov 25 2024

city: Philadelphia

first: Nathan

middle: Thomas

last: Henderson

state: PA

address: 415 Curie Blvd., Room 778

institute: University of Pennsylvania

summary: The pathophysiology of neurodevelopmental disorders involves vulnerable neural populations, including striatal circuitry, and convergent molecular nodes, including chromatin regulation and synapse function. Despite this, how epigenetic regulation regulates striatal development is understudied. Recurrent de novo mutations in Zswim6 are associated with intellectual disability and autism. We demonstrate that ZSWIM6 localizes to the nucleus where it associates with repressive chromatin regulators. Disruption of Zswim6 in ventral telencephalic progenitors leads to increased chromatin accessibility and transcriptional dysregulation. Ablating Zswim6 in either striatal direct or indirect pathway spiny projection neurons resulted in similar cell-autonomous changes in excitatory but not inhibitory synaptic transmission. Specifically, Zswim6 disruption altered the desensitization properties of AMPA receptors, leading to enhanced synaptic recruitment of SPNs, explaining SPN-subtype specific effects on activity and behavioral sub-structure. Finally, adult deletion of Zswim6 identified a continuing role in the maintenance of mature striatal synapses. Together, we describe a mechanistic role for Zswim6 in the epigenetic control of striatal synaptic development.

overall_design: Nuclei were isolated from striatal tissue of Control (Dlx5/6-Cre) and Zswim6 striatal neuron cKO (Dlx5/6-Cre; Zswim6c/c) P8 pups. snRNA-seq was then performed in collaboration with the Upenn NGS core.

first: Kyuhyun

last: Choi

middle: T

first: Emily

middle: R

last: Feireman

first: Sean

last: Louzon

first: Jamie

last: Galanaugh

first: Mariexcel

middle: F

last: Davatolhagh

first: Isha

last: Bandaru

first: David

last: Tischfield

first: Stewart

last: Anderson

first: Erica

last: Korb

first: Marc

middle: V

last: Fuccillo


title: NAC regulates metabolism and cell fate in intestinal stem cells

city: Amsterdam

first: William

last: Faller

address: Plesmanlaan 121

department: Oncogenomics

country: Netherlands

institute: Netherlands Cancer Institute

summary: mRNA translation is a highly dynamic process that has been shown to modulate cell adaptation to stress conditions. Upon ribosome impairment, ZAKα affects the metabolic profile of intestinal cells, however the mechanism by which this is promoted is still unclear. Here, we identified the nascent polypeptide-associated complex (NAC) as a central player in the metabolic regulation triggered by ribosome impairment. Furthermore, we show that NAC is essential for the survival of the highly oxidative phosphorylation (OXPHOS) reliant intestinal stem cells (ISCs), being translationally regulated itself. NAC interacts with the ribosome and promotes the transport of the translation machinery and newly-formed peptides to the proximity of the mitochondria, allowing the import of proteins essential for processes such as OXPHOS. Upon translation stress, NAC is inhibited through an interaction with ZAKα, leading to the downregulation of OXPHOS. Overall, our findings unveil  that NAC is a central player in translation-mediated metabolic regulation in intestinal cells.

overall_design: Ribo-seq and RNAseq based identification of translationally regulated genes in intestinal stem cells and their essentiality analysis with a CRISPR drop-out screen

first: Sofia

last: Ramalho

first: Ferhat

last: Alkan

first: Joana

last: Silva


title: Control of Striatal Circuit Development by the  Chromatin Regulator Zswim6 [ATAC-Seq]

type: Genome binding/occupancy profiling by high throughput sequencing

overall_design: Nuclei were isolated from striatal tissue of Control (Dlx5/6-Cre) and Zswim6 striatal neuron cKO (Dlx5/6-Cre; Zswim6c/c) P8 pups. ATAC-seq was then performed to compare chromatin accessibility between genotypes.


title: A single-nucleus RNA sequencing atlas of the postnatal retina of the shark Scyliorhinus canicula

city: Santiago de Compostela

first: Nicolás

last: Vidal-Vázquez

state: A Coruña

address: Av. Lope Gómez de Marzoa s/n

department: Departamento de Bioloxía Funcional

country: Spain

institute: Universidade de Santiago de Compostela

summary: The retina, whose basic cellular structure is highly conserved across vertebrates, constitutes an accessible system for studying the central nervous system. In recent years, single-cell RNA-sequencing studies have uncovered cellular diversity in the retina of a variety of species, providing new insights on retinal evolution and development. However, similar data in cartilaginous fishes, the sister group to all other extant jawed vertebrates, are still lacking. Here, we present a single-nucleus RNA-sequencing atlas of the postnatal retina of the catshark Scyliorhinus canicula, consisting of the expression profiles for 17,438 individual cells from three female, juvenile catshark specimens. Unsupervised clustering revealed 22 distinct cell types comprising all major retinal cell classes, as well as retinal progenitor cells (whose presence reflects the persistence of proliferative activity in postnatal stages in sharks) and oligodendrocytes. Thus, our dataset serves as a foundation for further studies on the development and function of the catshark retina. Moreover, integration of our atlas with data from other species will allow for a better understanding of vertebrate retinal evolution.

overall_design: Three retinas from three female, juvenile small-spotted catsharks (Scyliorhinus canicula) were analysed using snRNA-seq

first: Ismael

last: Hernández-Núñez

first: Pablo

last: Carballo-Pacoret

first: Sarah

last: Salisbury

first: Paula

last: Villamayor

first: Francisca

last: Hervas-Sotomayor

first: Xuefei

last: Yuan

first: Francesco

last: Lamanna

first: Céline

last: Schneider

first: Julia

last: Schmidt

first: Sylvie

last: Mazan

first: Henrik

last: Kaessmann

first: Fátima

last: Adrio

first: Diego

last: Robledo

first: Antón

last: Barreiro-Iglesias

first: Eva

last: Candal


title: AGS infected with H. pylori for 24 hours

city: Nanchang

first: Leyan

last: Li

state: Jiangxi

address: Donghu District

country: China

institute: The First Affiliated Hospital of Nanchang University

summary: Helicobacter pylori (H. pylori) is a type of pathogen in humans that has infected nearly half of the population worldwide. Infections with H. pylori are typically associated with chronic gastritis and may even lead to gastric and duodenal ulcers and stomach cancer. While the mechanisms behind persistent colonization by H. pylori and the development of gastritis associated with it remain unclear, it is generally believed that the gastric epithelial cells (GECs) modulated by H. pylori in the gastric mucosa play a crucial role. We  extensively studied the global gene expression patterns in H. pylori-infected AGS cells, a line of gastric epithelial cells, and identified genes that show increased expression upon infection with H. pylori.

overall_design: Gene expressions in H. pylori-infected and uninfected AGS cells (MOI=100, 24 h) were compared

first: Yanan

last: Zhou

first: Nianshuang

first: Yi

last: Hu

first: Yin

last: Zhu


title: Adolescent circadian rhythm disruption increases reward and risk-taking

city: Pittsburgh

first: Colleen

middle: A.

last: McClung

address: 450 Technology Drive

institute: University of Pittsburgh

summary: Circadian rhythm disturbances have long been associated with the development of psychiatric disorders, including mood and substance use disorders. Adolescence is a particularly vulnerable time for the onset of psychiatric disorders and for circadian rhythm and sleep disruptions. Preclinical studies have found that circadian rhythm disruption (CRD) impacts the brain and behavior, but this research is largely focused on adult disruptions. Here, we sought to determine the long-term behavioral and neurobiological effects of CRD during early adolescence by exposing mice to 12 h shifts in the light/dark cycle. We hypothesized that adolescent CRD would have a greater effect on psychiatric-related behaviors, relative to adult disruption. To identify possible mechanisms, we also measured gene expression in brain regions relevant to circadian rhythms, mood and reward. We found that disruption during early adolescence, but not adulthood, persistently increased exploratory drive (risk-taking behavior) and cocaine preference when tested later in life. Interestingly, we found sex differences when intravenous cocaine self-administration was tested. While female mice with a history of adolescent CRD had a greater propensity to self-administer cocaine, as well as increased motivation and cue-induced reinstatement, male adolescent CRD mice had reduced motivation and extinction responding. Overall, adolescent CRD in mice caused persistent increases in risky behavior, cocaine reward and cocaine self-administration, which suggests that CRD during adolescence may predispose individuals towards substance use disorders. Importantly, we found that many transcripts were affected by adolescent CRD and these were largely distinct across sex and brain region. Future research is required to elucidate how adolescent CRD affects behaviors relevant to mood- and substance use-related disorders across the 24-hour day, as well as to identify intervention strategies to alleviate disruption during adolescence and novel therapeutic approaches once symptoms have begun.

overall_design: The prefrontal cortex (PFC), nucleus accumbens (NAc), and suprachiasmatic nucleus (SCN) were punched and isolated from frozen brain tissue using a cryostat. Individual animals were each used as a sample (n=4-8 per condition). Tissue was homogenized and total RNA was isolated using the RNeasy Plus Micro Kit (Qiagen). RNA quantity and quality were assessed using fluorometry (Qubit RNA High Sensitivity Assay Kit and 322 Fluorometer; Invitrogen) and chromatography (Bioanalyzer and RNA 6000 Pico Kit; Agilent) respectively. Libraries were prepared with 10 ng of RNA from each sample using the Smartseq HT ultra-low input sample preparation kits (Illumina). Paired-end dual-indexed sequencing (75 bp) was performed using the NextSeq 500 platform (Illumina). A total of 30 million reads per sample was targeted. Sequencing was performed at the University of Pittsburgh Health Sciences Sequencing Core at UPMC Children’s Hospital of Pittsburgh.

first: Lauren

last: DePoy

first: Chelsea

last: Vadnie

first: Kaitlyn

last: Petersen

first: Madeline

last: Scott

first: Wei

last: Zong

first: RuoFei

last: Yin

first: Ross

middle: C

last: Matthaei

first: Fernanda

last: Anaya

first: Callie

middle: I

last: Kampe

first: George

last: Tseng


title: A comprehensive gene expression analysis to identify the key regulator suppressing the IFN-γ-inducible factors in tumor cells

city: Tokushima

first: atsushi

last: mitsuhashi

address: 3-18-15 Kuramoto-cho

department: Department of Respiratory Medicine and Rheumatology

institute: Tokushima university

summary: Immune checkpoint inhibitors (ICIs) provide clinical benefits for various advanced malignancies. However, the predictive factors that determine sensitivity to ICIs have not been fully elucidated. To identify the mechanisms underlying ICI resistance, we performed a microarray analysis to compare the IFN-γ-inducible genes between ICI-sensitive AB1-HA and ICI-resistant LLC in vitro.

overall_design: Total RNA was extracted from AB1-HA or LLC tumor cells incubated with or without IFN-γ for 24 h.

first: Yohei

last: Yabuki

first: Atsushi

last: Mitsuhashi

first: Yasuhiko

last: Nishioka


title: Co-isolation of human donor eye cells and development of oncogene-mutated melanocytes to study uveal melanoma

city: Montreal

first: Bioinformatics

last: Platform

state: Quebec

address: 1001 Decarie Blvd

country: Canada

institute: RI-MUHC

summary: Uveal melanoma (UM) is the most common intraocular tumor in adults, and its pathogenesis is only partly understood. UM can develop de novo from normal choroidal melanocytes (NCMs) or from pre-existing nevi that stem from NCMs and are thought to harbour UM-initiating mutations, most commonly in GNAQ or GNA11. However, there is a complete lack of commercially available NCM cell lines and no detailed protocol for developing oncogene-mutated CM line (MutCM) to study UM development. The purpose of this study was to establish and characterize premalignant CM models from human donor eyes to recapitulate the cell populations at the origin of UM.

overall_design: We isolated NCMs from human donor eyes and generated MutCMs harboring one of the most common UM driver mutations (GNAQQ209L) by lentiviral transduction. To understand potential molecular changes induced by GNAQQ209L mutation, transcriptomic analyses comparing between a pair of NCM and MutCM derived from the same donor and between NCM and a uveal melanoma cell line (92.1) were performed.

first: Julia

last: Burnier

first: Yunxi

last: Chen


title: Single cell RNA-sequencing of Lepr+ lung mesenchymal cells in normal and fibrotic mouse lungs

city: New York

first: Yinshan

last: Fang

state: New York

address: 650W 168th Street

institute: Columbia University Medical Center

summary: A hallmark of pulmonary fibrosis is aberrant activation of lung fibroblasts to become pathological fibroblasts producing excessive extracellular matrix (ECM). Here, we showed Lepr-CreERT2 lineage labels lung mesenchymal cells including the majority of alveolar fibroblasts, which signigicatly contribute to pathological fibroblasts in bleomycin induced lung fibrosis.

overall_design: We isolated CD45-EpCAM-CD31-tdtomato+ lung mesenchymal cells from Lepr-CreERT2;Rosa26-tdTomato mice treated with bleomycin or saline and then applied for single cell RNA-sequencing.

first: Jianwen

last: Que


title: CD4+ T cell-innate immune crosstalk is critical during Staphylococcus aureus biofilm infection [CD3+ scRNA-seq]

city: White/Caucasian

first: Tammy

last: Kielian

state: NE

address: 985900 Nebraska Medical Center

institute: UNMC

summary: Access to the brain for treating neurological sequalae requires a craniotomy, which can be complicated by infection. T cells preferentially home to the brain, but not other tissue sites, during Staphylococcus aureus (S. aureus) craniotomy infection; however, their functional importance is unknown. CD4+ T cells were critical for bacterial containment during craniotomy infection as Rag1-/- mice and WT animals treated with anti-CD4 or VLA-4 and LFA-1 antibodies exhibited elevated bacterial burdens. scRNA-seq revealed transcriptional heterogeneity in brain CD3+ infiltrates, with CD4+ cells most prominent and typified by both Th1 and Th17 signatures, and adoptive transfer of either subset in Rag1-/- mice prevented S. aureus outgrowth. scRNA-seq revealed a profound IFN-γ signature in innate immune cells from Rag1-/- mice during craniotomy infection, supporting extensive T cell-innate immune crosstalk that was validated by immunostaining in the brain parenchyma. A cooperative role for Th1 and Th17 driven responses was demonstrated by treatment of Ifng-/- mice with IL-17A/F neutralizing Ab that recapitulated phenotypes observed in Rag1-/- animals, which were not observed following the loss of either cytokine alone. Collectively, these results implicate a critical role for CD4+ T cells in S. aureus containment during craniotomy infection by shaping the innate immune landscape.

overall_design: To better understand the function of t cells during craniotomy infection, we collected immune cells from a mouse model of craniotomy infection to perform scRNA-seq. CD3+ cells were sorted from brain homogenates of wild-type (C57BL/6) mice subjected to craniotomy infection at days 3,7 and 14 post-craniotomy infections.

first: Gunjan

last: Kak

first: Zachary

last: van Roy

first: Rachel

last: Fallet

first: Lee

last: Korshoj


title: Heterochromatin-dependent gene silencing pathways control CD4 T cell susceptibility to regulatory T cell-mediated suppression [RNAtreg]

city: Toulouse

first: Olivier

last: Joffre

address: CHU Purpan

department: Infinity UMR1291

country: France

institute: Inserm

summary: The integrity of the organism depends on close and dynamic interactions between conventional and regulatory (Treg) T cells. The extracellular signals and signaling events that regulate this crosstalk have been extensively characterized. However, how conventional T cells translate Treg-dependent immunosuppressive signals at the chromatin level remains largely unknown. In differentiating CD4+ T helper (Th) cells, we and others previously reported that transcriptional specificity is largely controlled by heterochromatin-dependent gene silencing. We therefore hypothesized that Treg-mediated T cell suppression could also involve the repressive mark H3K9me3 and the histone-binding factor heterochromatin protein 1 (HP1). Using a bone marrow allograft model, in which graft rejection is coordinated by adoptively-transferred naive CD4+ T cells and can be inhibited by Treg, we show that Treg-dependent suppressive signals indeed mobilize HP1a to repress Th-cell effector genes. Unexpectedly, our screen also revealed that T cells deficient for HP1g or the lysine methyltransferase SUV39H1 are better tolerized than their wild-type counterparts. Mechanistically, our transcriptional and epigenetic profiling identified HP1g as a negative regulator of a network of genes functionally associated with T-cell anergy and exhaustion, including those encoding the transcription factor TOX and the inhibitory receptors PD-1, TIM-3 and LAG-3. In response to T cell receptor engagement, the expression of these immune checkpoints is deregulated in HP1g-deficient cells, thus promoting their better inhibition by Treg. Therefore, we demonstrate that H3K9me3-dependent epigenetic pathways critically regulate Th cell susceptibility to Treg-mediated suppression, and we identify HP1a and HP1g as new epigenetic players whose expression may be manipulated to restore protective immune responses or correct immunopathology, respectively.

overall_design: C57BL/6 mice were lethally irradiated and grafted with a mixture of C57BL/6 and B6D2F1 bone marrow. To investigate the role of HP1a and HP1g in CD4 T cells reponse to Treg-mediated immunosuppression, grafted-recipients were injected with WT, HP1a- or HP1g-deficient Tconv with or without Treg. Three weeks after bone marrow graft, Treg were isolated by Fluorescence-activated cell sorting (FACS) and analyzed by RNA-Seq.

first: Julie

last: Noguerol

first: Karl

last: Laviolette

first: Margot

last: Zahm

first: Adeline

last: Chaubet

first: Claire

last: Détraves

first: Véronique

last: Adoue

first: Florence

last: Cammas

first: Joost

middle: P

last: van Meerwijk


title: Heterochromatin-dependent gene silencing controls CD4 T cell response to Treg-mediated suppression [Tmem]


title: Heterochromatin-dependent gene silencing controls CD4 T cell response to Treg-mediated suppression.

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

overall_design: Refer to individual Series


title: The activity of the mammalian DNA transposon piggyBat from Myotis lucifugus is restricted by its own terminal inverted repeat

city: Bethesda

first: Hernan

last: Lorenzi

state: Maryland

address: 8 Center Dr, Bldg 8A

department: National Institutes of Diabetes and Digestive and Kidney Diseas

institute: National Institutes of Health

summary: The transposon piggyBat has already been used as a genomic tool for studies in human cells13.  Here, through a combination of in vitro DNase I footprinting experiments with cell culture-based transposition assays, we have been able to rationalize the organization of the piggyBat TIRs. Based on this, we solved the cryo-EM structure of the piggyBat pre-synaptic complex at 3.4Å resolution. By combining these results, we discovered that piggyBat's transposition activity in vivo is severely restricted by a transposase binding site on its Left End (LE).  Further modifications of the RE and the elimination of N-terminal phosphorylation sites of the transposase and the duplication of C-terminal site-specific DNA  domain increased transposition activity by approximately two orders of magnitude relative to wild type. Taken together, the results indicate a previously unobserved down-regulation of transposon activity by a elongated TIR. The TIR modifications described here lead to a transposition activity increase comparable to the most highly active reported piggyBac version with no detectable change in chromosomal integration fidelity

overall_design:
To determine the transposition profiles of WT and pBat-4StoA-2xCRD (mutant) transposases genomewide, we established HTC116 cells expressing WT or mutant transposases and carrying a Puromycin-resistance encoding self-reporting transposon cassette carrying a Puromycin-resistance gene.
We then performed selective RT-PCR amplification of transcripts carrying the left end (LE) of the pBAT transposon from Puromycin-resistant cells, followed by RNAseq analysis to characterize and compare the transposition insertion site profiles  generated by WT and mutant transposases across the HTC116 genome.

first: Laurie

last: Lannes

first: Christopher

last: Furman

first: Christina

last: Hong

first: Lidiya

last: Franklin

first: Rodolfo

last: Ghirlando

first: Arpita

last: Ghosh

first: Anne

last: Grove

first: Wentian

last: Luo

first: Matthew

middle: H

last: Wilson

first: Parthena

last: Konstantinidou

first: Astrid

last: Haase

first: Alison

middle: B

last: Hickman

first: Fred

last: Dyda


title: Single cell RNA-sequencing of Lepr+ cells in normal and fibrotic mouse lungs

first: Yinshan

last: Fang

summary: A hallmark of pulmonary fibrosis is aberrant activation of lung fibroblasts to become pathological fibroblasts producing excessive extracellular matrix (ECM). Here, we showed Lepr-Cre lineage labels multiple cells types including the majority of alveolar fibroblasts, which signigicatly contribute to pathological fibroblasts in bleomycin induced lung fibrosis.

overall_design: We isolated tdtomato+ cells from Lepr-Cre;Rosa26-tdTomato mice treated with bleomycin or saline and then applied for single cell RNA-sequencing.

first: Jianwen

last: Que


title: U937 cells with CRISPR interference at regulatory genetic variants [4C-seq]

status: Public on Nov 24 2024

city: London

first: Mikhail

last: Spivakov

address: Du Cane Road

country: United Kingdom

institute: MRC London Institute of Medical Sciences

summary: Gene enhancers often form long-range contacts with promoters, but it remains unclear if enhancer activity and their chromosomal contacts are mediated by the same DNA sequences and recruited factors. Here, we study the effects of expression quantitative trait loci (eQTLs) on enhancer activity and promoter contacts in primary monocytes isolated from 34 individuals. Using eQTL-Capture Hi-C and a Bayesian approach considering both intra- and inter-individual variation, we detect 19 eQTLs associated with enhancer-eGene promoter contacts, most of which also associated with enhancer accessibility and activity. Capitalising on these shared effects, we devise a multi-modality Bayesian strategy, which identifies 629 “trimodal QTLs” jointly associated with enhancer accessibility, eGene promoter contact, and gene expression. Causal mediation analysis and CRISPR interference reveal causal relationships between these three modalities. Many detected QTLs overlap disease susceptibility loci and influence the predicted binding of myeloid transcription factors, including SPI1, GABPB and STAT3. Additionally, a variant associated with PCK2 promoter contact directly disrupts a CTCF binding motif and impacts promoter insulation from downstream enhancers. Jointly, our findings suggest an inherent genetic coupling between enhancer activity and connectivity with relevance for human disease, and highlight the role of genetically-determined chromatin boundaries in gene control.

overall_design: This study focused on the effects of regulatory genetic variants (cQTLs, as described in the publication) on enhancer function. The data in this repository comprise a CRISPRi experiment where dCas9 KRAB was directed to cQTLs, associated with expression of THBS1, in U937 monocyte-like cells. The sequencing outputs were 1) ATAC-seq, as a proxy of enhancer/promoter activity, and 2) 4C-seq, to determine the effect on 3D promity with the THBS1 promoter.

first: Ray-Jones

last: Helen

first: Malysheva

last: Valeriya

first: Bottolo

last: Leonardo

first: Vigorito

last: Elena

first: Spivakov

last: Mikhail


title: Heterochromatin-dependent gene silencing pathways control CD4 T cell susceptibility to regulatory T cell-mediated suppression [RNAnaivetconv]

first: Olivier

last: Joffre

overall_design: C57BL/6 mice were lethally irradiated and grafted with a mixture of C57BL/6 and B6D2F1 bone marrow. To investigate the role of HP1a and HP1g in CD4 T cells reponse to Treg-mediated immunosuppression, grafted-recipients were injected with WT, HP1a- or HP1g-deficient Tconv with or without Treg. Three weeks after bone marrow graft, TCR Vb6 positive allospecific Tconv were isolated by Fluorescence-activated cell sorting (FACS) and analyzed by RNA-Seq.

first: Julie

last: Noguerol

first: Karl

last: Laviolette

first: Margot

last: Zahm

first: Adeline

last: Chaubet

first: Claire

last: Détraves

first: Véronique

last: Adoue

first: Florence

last: Cammas

first: Joost

last: van Meerwijk


title: U937 cells with CRISPR interference at regulatory genetic variants [ATAC-seq]


title: An atlas of pancreatic function for ruminating goats mapped by single cell transcriptomics

city: changsha

first: yan

last: cheng

state: Hunan

address: Changsha, Hunan, China

institute: Institute of Subtropical Agriculture, Chinese Academy of Sciences

summary: The objectives of this study were to elucidate the development of pancreatic function from the non-ruminating to ruminating stage, including characterizing the inventory of cell types and their corresponding marker genes in the pancreas, using single-cell transcriptomic techniques.Pancreas of adult twin female goats at ruminating stage were collected  after harvesting and the pancreas of neonatal  twin female goats at non-ruminating stage were used as control.

overall_design: Four Liuyang black goats aged of 10 days old (neonatal goats at non-ruminating stage) and 6 months old (adult goats at ruminating stage) were selected from commercial goat farms and divided into XY group (10-days old, n=2, control) and DY group (6-months old, n=2), respectively. Goats were humanely euthanized, and the pancreas was freshly sampled.

first: Yan

last: Cheng

first: Tianxi

last: Zhang

first: Chao

last: Yang

first: Kefyalew

last: Gebeyew

first: Chengyu

last: Ye

first: Xinxin

last: Zhou

first: Zhixiong

last: He

first: Zhiliang

last: Tan


title: Heterochromatin-dependent gene silencing controls CD4 T cell response to Treg-mediated suppression [ATAC-seq]

first: Olivier

last: Joffre

overall_design: C57BL/6 mice were lethally irradiated and grafted with a mixture of C57BL/6 and B6D2F1 bone marrow. To investigate the role of HP1a and HP1g in CD4 T cells reponse to Treg-mediated immunosuppression, grafted-recipients were injected with WT, HP1a- or HP1g-deficient Tconv with or without Treg. Three weeks after bone marrow graft, TCR Vb6 positive allospecific Tconv were isolated by Fluorescence-activated cell sorting (FACS) and analyzed by ATAC-Seq.

first: Julie

last: Noguerol


title: Local ablation disrupts immune evasion in pancreatic cancer

city: Verona

first: Francesco

last: De Sanctis

address: Piazzale L.A. Scuro, 10

department: Department of Medicine

country: Italy

institute: University and Hospital Trust of Verona


title: DUX4 induced by HSV-1 infection promotes viral replication

city: Wuhan

first: Junjie

state: Hubei

address: Donghu Road, No. 115

department: Medical Research Institute

institute: Wuhan University

summary: RNAseq analysis of THP-1 cells mock-infected or infected with HSV-1 without or with DUX4 knockdown

overall_design: THP-1 cells (2 × 106, three replicates per sample) were mock-infected or infected with HSV-1 (MOI=1) for 12 h. RNA was extracted and RNA-seq was performed. Three replicates are included for each samples.

first: Ming

last: Gao


title: Melanoma bone metastasis-induced osteocyte ferroptosis via the HIF1α-HMOX1 axis II

city: Erlangen

first: Yewei

last: Jia

address: Werner-von-Siemens-Straße 7

department: Department of Internal Medicine 3

country: Germany

institute: Friedrich-Alexander-University Erlangen-Nürnberg (FAU) and Universitätsklinikum Erlangen

summary: Osteocytes are the main cells in mineralized bone tissue. Elevated osteocyte apoptosis has been observed in lytic bone lesions of patients with multiple myeloma. However, their precise contribution to bone metastasis remains unclear.Here, we investigated the pathogenic mechanisms driving melanoma-induced osteocyte death. Both in vivo models and in vitro assays were combined with untargeted RNA sequencing approaches to explore the pathways governing melanoma-induced osteocyte death. We could show that ferroptosis is the primary mechanism behind osteocyte death in the context of melanoma bone metastasis. HMOX1 was identified as a crucial regulatory factor in this process, directly involved in inducing ferroptosis and affecting osteocyte viability. We uncover a non-canonical pathway that involves excessive autophagy-mediated ferritin degradation, highlighting the complex relationship between autophagy and ferroptosis in melanoma-induced osteocyte death. In addition, HIF1α pathway was shown as an upstream regulator, providing a potential target for modulating HMOX1 expression and influencing autophagy-dependent ferroptosis. In conclusion, our study provides insight into the pathogenic mechanisms of osteocyte death induced by melanoma bone metastasis, with a specific focus on ferroptosis and its regulation. This would enhance our comprehension of melanoma-induced osteocyte death.

overall_design: To investigate the alternation of osteocyte involved in melanoma bone metastasis, osteocyte cell line MLO-Y4 was exposed to 50% melanoma cell line B16F10 derived conditioned medium for 48h.

first: Aline

last: Bozec


title: CD133+PD-L1+ cancer cells confer resistance to adoptively transferred engineered macrophage-based therapy in melanoma

city: 上海

first: Xu

last: Jiaojiao

state: 上海Shanghai

address: Zhangheng Road

institute: Fudan university

summary: Adoptive transfer of genetically or nanoparticle-engineered macrophages represents a promising cell therapy modality for treatment of solid tumor. However, the therapeutic efficacy is suboptimal without achieving a complete tumor regression, and the underlying mechanism remains elusive. Here, we discover a subpopulation of cancer cells with upregulated CD133 and programmed death-ligand 1 in mouse melanoma, resistant to the phagocytosis by the transferred macrophages. Compared to the CD133-PD-L1- cancer cells, the CD133+PD-L1+ cancer cells express higher transforming growth factor-β signaling molecules to foster a resistant tumor niche, that restricts the trafficking of the transferred macrophages by stiffened extracellular matrix, and inhibits their cell-killing capability by immunosuppressive factors. The CD133+PD-L1+ cancer cells exhibit tumorigenic potential. The CD133+PD-L1+ cells are further identified in the clinically metastatic melanoma. Hyperthermia reverses the resistance of CD133+PD-L1+ cancer cells through upregulating the ‘eat me’ signal calreticulin, significantly improving the efficacy of adoptive macrophage therapy. Our findings demonstrate the mechanism of resistance to adoptive macrophage therapy, and provide a de novo strategy to counteract the resistance.

overall_design: Human A375 melanoma tissue from relapsed tumors and the corresponding sorted melanoma cells were collected for scRNA-seq

first: Jiaojiao

last: Xu

first: Zhe

last: Li

first: Qinli

last: Tong

first: Sihang

first: Jianchen

last: Fang

first: Aihua

last: Wu

first: Guoguang

last: Wei

first: Chen

first: Sheng

last: Yu

first: Binbin

last: Zheng

first: Hongzheng

last: Lin

first: Xueling

last: Liao

first: Zeyu

last: Xiao

first: Wei

last: Lu


title: Melanoma bone metastasis-induced osteocyte ferroptosis via the HIF1α-HMOX1 axis.

first: Yewei

overall_design:
The injection of B16F10 melanoma cells was performed intracardially by administering 1x10^5 cells in a 100 μl volume after inducing anesthesia with isoflurane (Abbott; IsoFlo®, Cat. #05260-05), following established protocols. Mice without tumors received PBS injections as controls and were continuously monitored for 24 hours.
The treatment group received B16F10 melanoma cells injected as previously described. Subsequently, daily intraperitoneal injections of Zinc Protoporphyrin (Znpp, 10 mg/kg) (MedChemExpress, Cat. # HY-101193), and Roxadustat (10 mg/kg) (Invivochem, Cat.# V0293-500mg) were administered for 13 consecutive days. The control group received an equal volume of DMSO in PBS.


title: Brd4 knockout colonic epithelial organoids are partially resistant to IFNg-induced cell death.

city: Chuo-ku

first: Haruna

last: Takeda

state: Tokyo

address: 5-1-1 Tsukiji,

institute: National Cancer Center Japan

summary: To identify the role of Brd4 in colorectal cancer (CRC) development , we performed Sleeping Beauty (SB) transposon mutagenesis in mice. We identified several candidate genes including Brd4.  To provide functional validation, we knocked out Brd4 in mouse tumor organoids carrying an activating KrasG12D, and an inactivating ApcD716 allele, and performed RNAseq. Brd4 knockout colonic organoids showed deregulation of genes in IFNg signaling. We treated Brd4 knockout organoids with IFNg and found that Brd4 konckout organoids showed partial resistance  to IFNg-induce cell death. The mechanism may explain the function of Brd4 in colon cancer development.

overall_design: Brd4 was knocked out by CRISPR-Cas9 in mouse colon tumor organoids carrying an activating KrasG12D, and an inactivating ApcD716 allele. RNA was collected from control (4 replicates) and Brd4 KO (3 replicates). Also, IFNg was treated with control organoids  and Brd4 KO organoids, and RNA was collected from IFNg-treated conrol organoids (3 replicates) and IFNg-treated Brd4 KO organoids to perform RNA-seq.

first: Akimi

last: Yonezawa

first: Yukari

last: Muranaka

first: Yujiro

last: Takegami


title: Nociceptive neurons interact with gastric cancer cells via a CGRP/Ramp1 axis to promote tumor progression

first: Timothy

middle: Cragin

last: Wang

address: 1130 St Nicholas Ave

department: Dept Med Digestive & Liver Dis

institute: Columbia University

summary: To determine how DRG affects mouse gastric cancer cells, we cocultured GC cells with DRG

overall_design: Comparative gene expression profiling analysis of RNA-seq data for mouse gastric cancer cells with or without DRG coculture

first: Xiaofei

last: Zhi

first: Feijing

first: Jin

last: Qian

first: Yosuke

last: Ochiai

first: Guodong

last: Lian

first: Ermanno

last: Malagola

first: Duan

last: Chen

first: Sandra

middle: W

last: Ryeom


title: Transcriptomic Changes in Adult Hypothalamic Neurons Following Leptin Sensitivity Modulation

status: Public on Nov 22 2024

city: Kraków

first: Artur

last: Gurgul

address: Rędzina 1C

department: Center for Experimental and Innovative Medicine

country: Poland

institute: University of Agriculture in Krakow

summary: Modulating leptin sensitivity in hypothalamic neurons is critical for metabolic regulation and plays a key role in the development of obesity. This study examines the effects of three distinct approaches—leptin exposure (LEPA), leptin antagonist administration (LANTA), and palmitate treatment (PA)—on adult-derived mHypoA-2/12 neurons. Transcriptomic analysis using 3' mRNA-Seq revealed significant downregulation of several pathways, including the NOD-like receptor signaling pathway, C-type lectin receptor signaling pathway, NF-κB signaling pathway, and IL-17 signaling pathway. These pathways are closely linked to neuroinflammatory processes, suggesting that modulating leptin sensitivity may influence inflammation in the hypothalamus. The observed downregulation indicates a complex interaction between leptin signaling and cellular stress responses, which may represent an adaptive mechanism to prolonged exposure to leptin or fatty acids. Understanding these dynamics is crucial for gaining insight into how neuroinflammation contributes to the development of leptin resistance and related metabolic disorders.

overall_design: The adult mouse hypothalamic cell line mHypoA-2/12 (Clu177, Cedarlane, Canada) was cultured in Dulbecco's Modified Eagle's Medium with GlutaMAX (DMEM, 4500 mg/L, Gibco, Thermo Fisher Scientific, USA), supplemented with 10% heat-inactivated fetal bovine serum (FBS, Gibco) and 1% Antibiotic-Antimycotic solution (containing 10,000 units/mL penicillin, 10,000 µg/mL streptomycin, and 25 µg/mL amphotericin B, Gibco). Cells were seeded at a density of 1.5x10⁵ cells per well in 24-well plates and incubated at 37°C with 5% CO₂. For treatments, cells were exposed to 200 nM leptin (LEPA, PLR, Israel), 30 µM leptin antagonist (LANTA, PLR, Israel), or 5 mM palmitic acid (PA, Merck, USA), which was conjugated with fatty acid-free bovine serum albumin (NEFA-free BSA) and dissolved in serum-free DMEM, based on the method of Mayer and Belsham (2010). Vehicle controls were prepared with serum-free DMEM and 5% NEFA-free BSA (CTR). After treatment, cells were harvested for analysis using the RealTime-Glo™ MT Cell Viability Assay for viability, the Caspase-Glo® 3/7 Assay for apoptosis, and the Fluo-4 NW Calcium Assay Kit for calcium measurement.

first: Ewa

last: Oclon

first: Tomasz

last: Szmatola

first: Igor

last: Jasielczuk

first: Miroslaw

last: Kucharski

first: Joanna

last: Zubel-Łojek

first: Dorota

last: Zieba-Przybylska


title: PRMT5 promotes growth of pediatric high grade glioma [bulk RNA-seq]

city: Aurora

first: John

last: DeSisto

state: CO

department: Pediatrics

institute: University of Colorado

summary: Background. Pediatric high-grade gliomas (PHGG) are aggressive, undifferentiated CNS tumors with poor outcomes, for which no standard-of-care drug therapy currently exists. Through a knockdown screen for epigenetic regulators, we identified PRMT5 as essential for PHGG cell growth. We hypothesized that, similar to its effect in normal cells, PRMT5 promotes self-renewal of stem-like PHGG tumor initiating cells (TICs) essential for tumor growth. Methods. We conducted in vitro analyses, including limiting dilution studies of self-renewal, to determine the phenotypic effects of PRMT5 KD. We performed ChIP-Seq to identify  PRMT5-mediated epigenetic changes and gene set enrichment analysis to identify pathways that PRMT5 regulates. Using an orthotopic xenograft model of PHGG, we tracked survival and histological characteristics resulting from PRMT5 KD or administration of a PRMT5 inhibitor ± radiation therapy (RT). Results. In vitro, PRMT5 KD slowed cell cycle progression, tumor growth and self-renewal.  PRMT5 KD reduced H3K4me3 occupancy at genes associated with self-renewal, tumor formation and growth. In vivo, PRMT5 KD increased survival and reduced tumor aggressiveness; however, pharmacological inhibition of PRMT5 with or without RT did not improve survival. Conclusion. PRMT5 KD epigenetically reduced TIC self-renewal, leading to increased survival in preclinical models. Pharmacological inhibition of PRMT5 enzymatic activity may have failed in vivo due to insufficient reduction of PRMT5 activity by chemical inhibition, or this failure may suggest that non-enzymatic activities of PRMT5 are more relevant.

overall_design: Bulk RNA-Seq data comparing gene expression in pediatric high grade glioma (DMG or cortical PHGG) cells with PRMT5 knockdown (KD) by lentiviral transduction with cells containing empty vector control shRNA.  There are 5 cell lines:  BT245, SU-DIPG-IV, HSJD-DIPG-7, SU-DIPG-XIII* and HSJD-GBM-001. For each cell line, there is one control (empty vector) shRNA and three PRMT5 KD shRNAs used, for a total of four samples per cell line. The shRNA vectors are:  Control - SHC002; PRMT5 targeting shRNAs:  TRCN0000379612, TRCN0000303446, and TRCN0000381130.

first: Adam

last: Green


title: PRMT5 promotes growth of pediatric high grade glioma.


title: Motile cilia modulate neuronal and astroglial activity in the zebrafish larval brain

city: Trondheim

first: Nathalie

last: Jurisch-Yaksi

address: Erling Skjalgsons gate 1

country: Norway

institute: NTNU

summary: The brain uses a specialized system to transport cerebrospinal fluid (CSF), consisting of interconnected ventricles lined by motile ciliated ependymal cells. These cells act jointly with CSF secretion and cardiac pressure gradients to regulate CSF dynamics. To date, the link between cilia-mediated CSF flow and brain function is poorly understood. Using zebrafish larvae as a model system, we identify that loss of ciliary motility does not alter progenitor proliferation, brain morphology, or spontaneous neural activity despite leading to an enlarged telencephalic ventricle. We observe altered neuronal responses to photic stimulations in the optic tectum and hindbrain and brain asymmetry defects in the habenula. Finally, we investigate astroglia since they contact CSF and regulate neuronal activity. Our analyses reveal a reduction in astroglial calcium signals during both spontaneous and light-evoked activity. Our findings highlight a role of motile cilia in regulating brain physiology through the modulation of neural and astroglial networks.

overall_design: To analyze the impact of cilia paralysis on gene expression, we performed RNA sequencing analysis of 4 days old smh mutant and sibling control zebrafish larvae. We extracted total RNA from mutant (selected based on their curly tail down phenotype) and sibling controls (mixture of heterozygous and wild-type). RNAsequencing was performed on 4 different clutches.

first: Percival

last: D'Gama


title: PRMT5 promotes growth of pediatric high grade glioma [ChIP-seq]

overall_design: ChIP-Seq data comparing peak height in pediatric high grade glioma (DMG or cortical PHGG) cells with PRMT5 knockdown (KD) by lentiviral transduction with cells containing empty vector control shRNA. Two cells were used for ChIP-Seq, BT245 and HSJD-GBM-001. For each line, one control (empty vector) and two PRMT5 KD shRNAs were used. The targets were H3K4me3, H3K27me3 and H3K27ac.  The control shRNA for both cell lines is SHC202. The PRMT5 targeting shRNAs for BT245 are: TRCN0000303446, and TRCN0000381130, and for GBM1 are: TRCN0000379612 and TRCN0000303446.


title: Optimized Methods for scRNA-seq and snRNA-seq of Skeletal Muscle Stored in Nucleic Acid Stabilizing Preservative

status: Public on Nov 21 2024

first: Elisabeth

last: Heuston

state: MD

address: 50 South Drive, Bldg 50, Rm 5531

department: National Human Genome Research Institute

institute: NIH/NHGRI

summary: Single cell studies have transformed our understanding of cellular heterogeneity in disease but the need for fresh starting material can be an obstacle, especially in the context of international multicenter studies and archived tissue. We developed a protocol to obtain high-quality cells and nuclei from dissected human skeletal muscle archived in the preservative Allprotect® Tissue Reagent. After fluorescent imaging microscopy confirmed intact nuclei, we performed four protocol variations that compared sequencing metrics between cells and nuclei enriched by either filtering or flow cytometry sorting. Cells and nuclei (either sorted or filtered) produced statistically identical transcriptional profiles and recapitulated 8 cell types present in skeletal muscle. Flow cytometry sorting successfully enriched for higher-quality cells and nuclei but resulted in an overall decrease in input material. Our protocol provides an important resource for obtaining high-quality single cell genomic material from archived tissue and to streamline global collaborative efforts.

overall_design: Primary human skeletal muscle tissue from multiple donors was pooled, homogenized, filtered to create a single cell suspension, and divided into 4 aliquots. Aliquot1: unaltered. Aliquot2: nuclei extracted; Aliquot3: stained with Nuclear Pore Complex antibody (BioLegend #682202), and positively selected by fluorescence-activated cell sorting (FACS); Aliquot4: stained with Nuclear Pore Complex antibody, positively selected by FACS, and nuclei extracted.

first: Ayo

last: Doumatey

first: Faiza

last: Naz

first: Shamima

last: Islam

first: Stacie

last: Anderson

first: Martha

last: Kirby

first: Stephen

last: Wincovitch

first: Stefania

last: Dell’Orso

first: Adebowale

last: Adeyemo

first: Charles

middle: N

last: Rotimi


title: Identifying bone marrow microenvironmental alterations in multiple myeloma and its precursor conditions using whole bone marrow biopsies

city: Little Rock

first: Yan

last: Cheng

state: Arkansas

address: 4301 W. Markham St.

department: Internal Medicine

institute: University of Arkansas for Medical Sciences

summary:
Tumor immune microenvironmental alterations occur early in multiple myeloma (MM) development. In this study, we aim to systematically characterize the tumor immune microenvironment (TME) and the tumor-immune interactions from precursor stages, i.e., monoclonal gammopathy of undetermined significance (MGUS) and smoldering MM (SMM), to newly diagnosed MM, comparing these to healthy donors. CIBERSORT analysis revealed proportions of 27 cell types, including 10 innate immune cells (monocytes, M0/M1/M2 macrophages, resting/activated dendritic cells, resting/activated mast cells, eosinophils, and neutrophils), 7 T cell subsets (CD8+ T cells, naive CD4+ T cells, resting/activated memory CD4+ T cells, follicular helper T cells, regulatory T cells, and gamma delta T cells), resting/activated NK cells, naive B cells, memory B cells, plasma cells, myeloma plasma cells, memory plasma cells, osteoblasts, osteoclasts and adipocytes. We removed plasma cells (PC) from the 100% total cell proportions to avoid bias resulting from different tumor burdens.
We found that the proportions of neutrophils, mast cells, and monocytes account for the majority of innate immune cells and were decreased in NDMM and its precursor stages when compared with NBM. We noticed a significant abundance of M2 macrophages in NDMM, but not in the precursor stages. The proportion of CD8+ T cells was increased at the SMM and MM stages, and the proportion of activated memory CD4+ T cells continued to decrease from NBM to MGUS, SMM, and MM. We performed correlation analysis of immune cell proportions with the time of progression of MGUS and SMM patients. We observed neutrophil proportions were negatively correlated with the time to progression in SMM patients, indicating more neutrophils predict inferior outcomes for SMM patients. However, in NDMM patients, we observed that the neutrophil percentage was decreased in patients with high-risk status based on the 70-gene Prognostic Risk Score (GEP-70) or at stage III of International Staging System (ISS), and patients with high proportions of neutrophils had a significantly superior overall survival (OS) and event-free survival (EFS). For T cell populations, we observed that the proportions of CD8+ T cells were negatively correlated with the time of progression in SMM patients and the γδ T cells were positively correlated. We observed the proportions of γδ T cells were also positively correlated with the time of progression in MGUS patients. Consistent with the time-to-progression data, high-risk SMM patients showed higher proportions of CD8+ T cells and lower proportions of naïve CD4+ T cells and γδ T cells.

overall_design: Bone marrow biopsies were processed for RNA extraction and hybridization on Affymetrix microarrays. CIBERSORT-based analysis was used to deconvolution the bone marrow cell types. Specifically, the 27-myeloma genome signature matrix (MGSM27), a gene-expression–based computational technique, was implemented.

middle: D

last: Shaughnessy

first: Fenghuang

last: Zhan


title: Multivalent S2-subunit Vaccines Provide Broad Protection Against Clade 1 Sarbecoviruses

first: Patrick

last: Wilson

state: NY

address: 413 E 69th St

department: Drukier Institute

institute: Weill Cornell Medicine

summary: The continuing emergence of immune evasive SARS-CoV-2 variants and the previous SARS-CoV-1 outbreak have accentuated the need for broadly protective sarbecovirus vaccines. Targeting the conserved S2-subunit of SARS-CoV-2 is a particularly promising approach to elicit broad protection. Here, expanding on our previous work with S2-based vaccines, we developed a nanoparticle vaccine displaying multiple copies of the SARS-CoV-1 S2 subunit. This vaccine alone, or as a cocktail with a SARS-CoV-2 S2 subunit vaccine, protected female transgenic K18-hACE2 mice from challenges with Omicron subvariant XBB as well as several sarbecoviruses identified as having pandemic potential including the bat sarbecovirus WIV1, BANAL-236, and a pangolin sarbecovirus.  Challenge studies in female Fc- receptor knockout mice revealed that antibody-based cellular effector mechanisms played a role in protection elicited by these vaccines. These results demonstrate that our S2-based vaccines provide broad protection against clade 1 sarbecoviruses and offer insight into the mechanistic basis for protection.

overall_design: Specific B cells against SARS-CoV-2 Spike were isolated from mice immunized with S2 subunit vaccine and analyzed by 10x Genomics 5' scRNA-seq. Includes 5' GEX library and V(D)J library.

first: Yasuhara

last: Atsuhiro

first: Wilson

last: Patrick C.

first: Chervin

last: Jordan

first: Troxell

last: Chloe


title: Autocrine TGF-β1 drives tissue-specific differentiation and function of resident NK cells

city: Zürich

first: Colin

last: Sparano

address: Winterthurerstrasse 190

country: Switzerland

institute: University of Zürich

summary: Group 1 innate lymphoid cells (ILCs) encompass NK cells and ILC1s which have non-redundant roles in host protection against pathogens and cancer. Despite their circulating nature, NK cells can establish residency in selected tissues during ontogeny, forming a distinct functional subset. The mechanisms that initiate, maintain, and regulate the conversion of NK cells into tissue-resident NK (trNK) cells are currently not well understood. Here, we identify autocrine transforming growth factor-b (TGF-b) as a cell-autonomous driver for NK cell tissue-residency across multiple glandular tissues during development. Cell-intrinsic production of TGF-β was continuously required for the maintenance of trNK cells and synergized with Hobit to enhance cytotoxic function. Whereas autocrine TGF-β was redundant in tumors, our study revealed that NK cell-derived TGF-β allowed expansion of cytotoxic trNK cells during local infection with murine cytomegalovirus (MCMV) and contributed to viral control in the salivary gland. Collectively, our findings reveal tissue-specific regulation of trNK cell differentiation and function by autocrine TGF-β1, which is relevant for anti-viral immunity.

overall_design: Salivary glands were dissociated and CD45+ cells sorted before scRNA-seq using 10x Genomics platform.

first: Dario

last: Solis-Sayago

first: Frederike

last: Westermann


title: Assembly of Genetically Engineered Ionizable Protein Nanocage-based Nanozymes for Intracellular Superoxide Scavenging

city: tinajin

first: qiqi

last: liu

state: other

address: nankai university

institute: nankai university

summary: Nanozymes play a pivotal role in mitigating excessive oxidative stress, however, determining their specific enzyme-mimicking activities for intracellular free radical scavenging is challenging due to endo-lysosomal entrapment. In this study, we employed a genetic engineering strategy to generate ionizable ferritin nanocages (iFTn), enabling their escape from endo-lysosomes and entry into the cytoplasm. Specifically, ionizable repeated Histidine-Histidine-Glutamic acid (9H2E) sequences were genetically incorporated into the outer surface of human heavy chain FTn, followed by the assembly of various chain-like nanostructures via a two-armed polyethylene glycol (PEG). Utilizing endosome-escaping ability, we designed iFTn-based tetrameric cascade nanozymes with high superoxide dismutase- and catalase-mimicking activities. The in vivo protective effects of these ionizable cascade nanozymes against cardiac oxidative injury were demonstrated in mouse models of cardiac ischemia-reperfusion (IR). RNA-sequencing analysis highlighted the crucial role of these nanozymes in modulating superoxide anions-, hydrogen peroxide- and mitochondrial functions-relevant genes in IR injured cardiac tissue. These genetically engineered ionizable protein nanocarriers provide opportunities for developing ionizable drug delivery systems.

overall_design: Mouse ischemia-reperfusion (IR) models were established following treatment with PBS, iFTn-M4, iFTn-R4 and iFTn-MR4 for two doses (equivalent protein concentration of 2.5 mg/kg) given every other day. After three days, RNA was extracted from the isolated cardiac IR tissue.

first: Qiqi

last: Liu

first: Zhanxia

last: Gao

first: Xiangyun

last: Zhang

first: Qiannan

last: Duan

first: Yue

first: Adam

last: Midgley

first: Li

last: Jiao

first: Ruming

first: Mingsheng

last: Zhu

first: Deling

last: Kong

first: Jie

last: Zhuang

first: Xinglu

last: Huang


title: The spatial zonation of the placental vasculature is specified by epigenetic mechanisms [WGBS]

city: Heidelberg

first: Stephanie

last: Gehrs

address: Im Neuenheimer Feld

institute: DKFZ

type: Methylation profiling by high throughput sequencing

summary: The labyrinthian fetoplacental capillary network is of vital importance for proper nourishment of the developing embryo. An inadequate function of the maternal-fetal circulation has emerged as one of the primary causes of placental insufficiency. Here, we show that the spatial zonation of the placental labyrinth vasculature is controlled by flow-regulated epigenetic mechanisms. The spatial and temporal analysis of gene transcription revealed a gradual change in the expression of epigenetic enzymes with the de novo DNA methyltransferase 3a (DNMT3A) as primary enzyme introducing DNA methylation in cells of the vascular system. Loss of Dnmt3a resulted in DNA hypomethylation and disturbance of the spatial-zonated placental gene expression. The resulting global DNA hypomethylation impaired the angiogenic capacity of endothelial cells. Global as well as endothelium-predominant deletion of Dnmt3a resulted in impaired placental vascularization and fetal growth retardation. A meta-analysis of human placental endothelial cell gene expression identified an association between preeclampsia and reduction of DNMT3A. Collectively, our study identified DMNT3A as critical methylome-regulator of placental endothelial cell gene expression and function with clinical implications for the pathogenesis of placental dysfunction, as it occurs during preeclampsia or fetal growth retardation.

overall_design: WGBS was performed from FACS-sorted placenta EC from litter-matched Dnmt3aflox/flox xCdh5-Cre and Dnmt3aflox/flox embryos in triplicates.

first: Hellmut

middle: G

last: Augustin


title: The spatial zonation of the placental vasculature is specified by epigenetic mechanisms

overall_design: We performed single-cell RNA-seq on labyrinth endothelial cells (EC) dissected from four litter-matched E16.5 wildtype placentas.


title: The spatial zonation of the placental vasculature is specified by epigenetic mechanisms [bulk RNA-seq]

overall_design: For bulk RNA-seq, RNA was isolated from FACS-sorted labyrinth placenta EC from Dnmt3aflox/flox xCdh5-Cre and Dnmt3aflox/flox embryos in triplicates.


title: Gene expression profile at single vell level of growth plate chondrocytes [scRNA-seq]

city: Suita

first: Kenji

last: Hata

state: Osaka

address: 1-8 Yamadaoka

department: Biochemistry

institute: Osaka University graduate school of dentistry

summary: Gowth plate chondrocytes include heterogenous cell population and cellular diversity of these chondrocytes remains to be elucidated. We used single cell RNA sequencing to uncover the diverssity of growth plate chondrocytes and identify noel marker genes selectively expressed in pre-hypertrophic chondrocytes.

overall_design: Growth plate chondrocytes of the Col11a2-enh-ZsGreen mice were isolated by FACS and analyzed using scRNAseq


title: The regulatory mechanism of Serinc5 expression in chondrocytes [ATAC-seq]

summary: Chondrocytes undergo a series of sequential differentiation steps which result in the formation of a unique hierarchical columnar structure including proliferating, pre-hypertrophic and hypertrophic chondrocytes. Pre-hypertrophic chondrocytes, which exist in the transitional stage between proliferating and hypertrophic chondrocytes, are a rare cell population in the growth plate but critical for endochondral bone development. However, the molecular basis of pre-hypertrophic chondrocytes largely remains elusive. The purpose of this study is to identify novel marker gene and uncover regulatory mechanism of marker gene expression.

overall_design: ATAC-seq in Bone Marrow mesenchymal Stem cells


title: spatialGE is a User-Friendly Web Application that Facilitates Spatial Transcriptomics Data Analysis

city: St. Petersburg

first: Oscar

middle: E

last: Ospina

state: FL

address: 600 5th Street South

department: Health Informatics

institute: Johns Hopkins All Children's Hospital

summary: Spatial transcriptomics (ST) is a powerful tool for understanding tissue biology and disease mechanisms. However, the advanced data analysis and programming skills required can hinder researchers from realizing the full potential of ST. To address this, we developed spatialGE, a web application that simplifies the analysis of ST data. The application spatialGE provided a user-friendly interface that guides users without programming expertise through various analysis pipelines, including quality control, normalization, domain detection, phenotyping, and multiple spatial analyses. It also enabled comparative analysis among samples and supported various ST technologies. The utility of spatialGE was demonstrated through its application in studying the tumor microenvironment of two data sets: 10× Visium samples from a cohort of melanoma metastasis and NanoString CosMx fields of vision from a cohort of Merkel cell carcinoma samples. These results support the ability of spatialGE to identify spatial gene expression patterns that provide valuable insights into the tumor microenvironment and highlight its utility in democratizing ST data analysis for the wider scientific community.

overall_design: This study presents three brain melanoma metastasis samples from three patients profiled with Visium.

first: Inna

last: Smalley


title: The effect of ORF57 knockout (KO) on host expression in iSLK/Bac16 cells.

city: Frederick

first: Zhi-Ming

last: Zheng

department: Center for Cancer Research

institute: National Cancer Institute

summary: Kaposi’s sarcoma-associated herpesvirus (KSHV) is a human oncogenic virus associated with various malignancies, including Kaposi’s sarcoma, primary effusion lymphoma, and multicentric Castleman’s disease. The expression of viral products is essential for initiating and sustaining KSHV-induced tumors. KSHV ORF57, a viral RNA-binding protein, plays a crucial role in regulating viral gene expression at the posttranscriptional level by promoting viral RNA stability, splicing, and translation. In addition, ORF57 dysregulates host gene expression to promote viral replications.

overall_design: The iSLK/Bac16 cells were generated by stable transfection of the KSHV genome cloned into bacterial artificial chromosome (Brulois et al. 2012; PMID: 22740391). The ORF57 knockout (57KO) was generated using Cas9/CRISPR by expressing two gRNA flanking ORF57 locus in the KSHV genome (BeltCappellino et al., 2019; PMID: 31413125). The cells expressing Cas9 but no gRNA were used as control wild-type samples.

first: Vladimir

last: Majerciak

first: Alexei

last: Lobanov

first: Maggie

last: Cam


title: Lymphatic-derived 25-hydroxycholesterol promotes immunity in melanoma by inhibiting PPAR-γ in tumor associated macrophages and monocytes

city: Lausanne

first: Nadine

last: Fournier

state: Vaud

address: Agora Cancer Research Center, Bugon 25A

institute: Agora Cancer Research Center & Swiss Institute of Bioinformatics

summary: In melanoma, lymphangiogenesis correlates with metastasis and poor prognosis and promotes immunosuppression. However, it also potentiates immunotherapy by supporting trafficking of immune cells. Lymphatic endothelial cells (LECs) are highly plastic cells that shape their phenotype according to distinct microenvironments, which likely define their functional properties, such as immunomodulation. Here, we show in a lymphangiogenic murine melanoma model that LECs upregulate the enzyme Ch25h, which catalyzes the formation of 25-hydroxycholesterol (25-HC) from cholesterol and plays important roles in regulating lipid metabolism, gene expression, and immune activation. LECs represent the main source of extracellular 25-HC in tumors, leading to the inhibition of PPAR- in intra-tumoral macrophages and monocytes. This prevents their immunosuppressive phenotype and instead promotes their conversion into proinflammatory myeloid cells that support effector T cell functions. We identify here in mechanistic detail a novel LEC function that supports anti-tumor immunity, which can be therapeutically exploited in combination with immunotherapy.

overall_design: Ch25h lymphatic-specific knock-out mice and their littermate control were transplanted with lymphangiogenic mouse melanoma cell line B16-OVA-VEGFC . Macrophages and monocytes are sorted from tumor and extracted mRNA for sequencing.

first: Mengzhu

last: Sun

first: Dale

last: Brighouse

first: Natacha

last: Civic

first: Stéphanie

last: Hugues


title: SnoRNA-facilitated protein secretion revealed by transcriptome-wide snoRNA target identification

city: Chicago

first: Bei

last: Liu

state: IL

address: 929 EAST 57TH STREET

department: Chemistry

institute: The University of Chicago

summary: Small nucleolar RNAs (snoRNA) are non-coding RNAs known for guiding RNA modifications including 2ʹ-O-methylation (Nm) and pseudouridine (Ψ). While snoRNAs may also interact with other RNAs such as mRNA, the full repertoire of RNAs targeted by snoRNA remains elusive due to the lack of effective technologies that identify snoRNA targets transcriptome-wide. Here we develop a chemical crosslinking-based approach that comprehensively detects cellular RNA targets of snoRNAs, yielding thousands of previously unrecognized snoRNA-mRNA interactions in human cells and mouse brain tissues. Many interactions occur outside of snoRNA-guided rRNA modification sites, hinting at non-canonical functions beyond RNA modification. We find that one of these snoRNAs, SNORA73, targets mRNAs that encode secretory proteins and membrane proteins. SNORA73 also interacts with 7SL RNA, part of the signal recognition particle (SRP) required for protein secretion. The mRNA-SNORA73-7SL RNA interactions enhance the association of the SNORA73-target mRNAs with SRP, thereby facilitating secretion of the encoded proteins.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design:
Sample 1-34 are duplicates for snoKARR-seq experiments detecting RNA-RNA interactions. Homogenized tissue or live cells are labeled with N3-kethoxal, which reacts with cellular RNA close to saturation, followed by formaldehyde-assisted fixing. Physically proximal RNAs labeled by N3-kethoxal are crosslinked by a chemically modified dendrimer bearing an activated alkyne group through the  click  chemistry. Subsequently, proteins bound to RNAs are digested by proteinase K. Cross-linked RNAs are fragmented and enriched using streptavidin beads. Enriched RNAs undergo proximity ligation to generate chimeric RNAs, after which the kethoxal labeling is reversed. The resulting RNAs are subjected to RT to generate cDNAs. 10% of the cDNA was saved as input samples. Next, a pool of biotinylated ASOs targeting a group of specific snoRNAs is used to enrich cDNAs derived from snoRNAs of interest. Both the input sample and the enriched cDNAs are amplified for high-throughput sequencing using the SMARTer Stranded Total RNA-seq kit v2   pico input mammalian. Sample 35-40 are duplicates for small RNA-seq experiments from cellular small RNA fractions

Dot-Bracket Notation is described here: https://wiki.eternagame.org/wiki/Dot-Bracket_Notation

first: Chuan

last: He


title: Anti-EGFR aptamer exhibits direct anti-cancer effects in NSCLC cells harboring EGFR L858R mutations

city: Columbia

first: Donald

last: Burke-Agüero

state: Missouri

address: 1201 Rollins St

department: molecular microbiology and immunology

institute: University of Missouri

summary: Non-small cell lung cancer (NSCLC) lung adenocarcinoma (LUAD) is a leading cause of death worldwide. Activating mutations in the tyrosine kinase domain of the oncogene epidermal growth factor receptor (EGFR) are responsible for ~10-50% of all LUAD cases. Although EGFR tyrosine kinase inhibitors (TKIs) have been effective in prolonging NSCLC patient survival and quality of life, acquired resistance mechanisms and disease progression are inevitable. Contemporary second- and third-line treatments, such as immunotherapy, remain ineffective for these patients, presenting a clear and unmet need for alternative or adjuvant therapeutics for the treatment of mutant EGFR positive NSCLC. Here we show that an anti-EGFR aptamer (EGFRapt) decreases viability of NSCLC cell lines harboring the L858R ± T790M mutation in EGFR but not cell lines harboring wild-type or exon 19 deletions. In a humanized xenograft mouse model, EGFRapt decreased tumor burden compared to controls when delivered intratumorally over multiple doses. To elucidate the mechanism by which EGFRapt exerts these effects, we examined kinase-dependent and kinase-independent mechanisms and found that it is cell line dependent, either inhibiting cellular proliferation or inducing cell death. Post hoc transcriptomics analysis comparing EGFRapt to control aptamer or vehicle validated these findings and provided additional mechanistic insights. Overall, these data establish that EGFRapt has direct anti-cancer activity in mutant EGFR positive NSCLC via targetable mechanisms that are independent of existing approaches and provide a foundation for further development of nucleic acid-based therapies that target EGFR.

overall_design: H3255, H1975, H820, and A549 cells were seeded at ~10,000 cells/well in a 48-well flat-bottom tissue culture plate 24 h prior to treatment. Cells were treated with 3 µM EGFRapt (MinE07), control apt (extended version of C36), or vehicle (DPBS and Mg2+) for 24 h, after which the cells were washed twice with DPBS and total RNA was extracted using the Monarch® Total RNA Miniprep Kit (NEB, Ipswich, MA). Total RNA was stored at -80°C until sequencing. Illumina NGS was performed on total RNA from three biologically independent experiments by the University of Missouri DNA Genomics Technology Core using NovaSeq technology to provide 100 base paired end (PE) reads.

first: Brian

middle: J

last: Thomas

first: Sania

middle: Z

last: Awan

first: Trupti

last: Joshi

first: Mark

middle: A

last: Daniels

first: David

last: Porciani

last: Burke


title: Deciphering head and neck cancer microenvironment: Single‐cell and spatial transcriptomics reveals human papillomavirus‐associated differences

status: Public on Nov 20 2024

city: Yangsan

first: Hansong

last: Lee

address: 49 Busandaehak-ro

country: South Korea

institute: Pusan National University

summary: Human papillomavirus (HPV) is a major causative factor of head and neck squamous cell carcinoma (HNSCC), and the incidence of HPV-associated HNSCC is increasing. The role of tumor microenvironment (TME) in viral infection and metastasis needs to be explored further. Thus we studied the molecular characteristics of primary tumors (PTs) and lymph node metastatic tumors (LNMTs) by stratifying them based on their HPV status using spatial transcriptomics.

overall_design: Six samples for spatial transcriptomics (ST), composed of matched primary tumors (PT) and lymph node metastases (LNMT), were collected from both HPV-negative (HPV−) and HPV-positive (HPV+) patients using the 10x Genomics Visium platform.

first: Sohee

last: Park

first: Ju Hyun

last: Yun

first: Yun Hak

last: Kim

first: Jeon Yeob

last: Jang


title: Aurora-A phosphorylates splicing factors and regulates alternative splicing

city: Barcelona

first: Estefania

last: Mancini

state: Barcelona

address: C/ Dr. Aiguader, 88

department: GENE REGULATION, STEM CELLS AND CANCER

institute: Centre for Genomic Regulation

summary: The goal of this study is to identify the effect of inhibition of Aurora-A kinase activity on gene expression and RNA splicing. The perturbation of Aurora-A is well known to affect cell cycle distribution. Therefore, we coupled the inhibition of Aurora-A with cell synchronization procedure in order to avoid the indirect effect of cell cycle perturbation on splicing changes. The mRNA -seq libraries were prepared and subjected to paired-end sequencing on Illumina HiSeq 2500 lanes. Differential gene expression and splicing analysis were carried using the edgeR tool and VAST-tools respectively. The RNA seq analysis identified that pharmacological inhibition of Aurora-A affects alternative splicing of 505 genes while having a marginal effect on gene expression. Overall our work identified Aurora-A as a novel splicing kinase and for the first time, describes a broad role of Aurora-A in regulating alternative splicing.

overall_design: We compared differential gene expression and alternative splicing profiles between DMSO and MLN8237 (Aurora-A kinase inhibitor) conditions in G1, G2 and mitotic phases of the cell cycle.

first: Arun

last: Damodaran

first: Malgorzata

last: Rogalska

first: Juan

last: Valcárcel

first: Erwan

last: Watrin

first: Claude

last: Prigent


title: The respective roles of TMPRSS2 and cathepsins for SARS-CoV-2 infection in human respiratory organoids

city: Kyoto

first: Kazuo

last: Takayama

state: Kyoto

address: Shogoin Kawaharacho 53, Sakyo-ku

department: Center for iPS Cell Research and Application

institute: Kyoto University

summary: A critical aspect of the mechanism of SARS-CoV-2 infection is the protease-mediated activation of the viral spike (S) protein. The type II transmembrane serine protease TMPRSS2 is crucial for SARS-CoV-2 infection in lung epithelial Calu-3 cells and murine airways. However, the importance of TMPRSS2 needs to be re-examined because the ability to utilize TMPRSS2 is significantly reduced in the Omicron variants that spread globally. For this purpose, replication profiles of SARS-CoV-2 were analyzed in human respiratory organoids. All tested viruses, including Omicron variants, replicated efficiently in these organoids. Notably, all SARS-CoV-2 strains retained replication ability in TMPRSS2-gene knockout (KO) respiratory organoids, suggesting that TMPRSS2 is not essential for SARS-CoV-2 infection in human respiratory tissues. However, TMPRSS2-gene knockout significantly reduces the inhibitory effect of nafamostat, suggesting the advantage of TMPRSS2-utilizing ability for the SARS-CoV-2 infection in these organoids. Interestingly, Omicron variants regained the TMPRSS2-utilizing ability in recent subvariants. The basal infectivity would be supported mainly by cathepsins because the cathepsin inhibitor, EST, showed a significant inhibitory effect on infection with any SARS-CoV-2 strains, mainly when used with nafamostat. A supplementary contribution of other serine proteases was also suggested because the infection of the Delta variant was still inhibited partially by nafamostat in TMPRSS2 KO organoids. Thus, various proteases, including TMPRSS2, other serine proteases, and cathepsins, co-operatively contribute to SARS-CoV-2 infection significantly in the respiratory organoids. Thus, SARS-CoV-2 infection in the human respiratory tissues would be more complex than observed in cell lines or mice.

overall_design: The human respiratory organoids were recovered from the culture plates, and then total RNA was collected and RNA-seq was performed.

first: Makoto

last: Takeda


title: Dissecting AP-1 complexity  in hepatic health and disease using Genetically Modified Mice

city: Vienna

first: Latifa

last: Bakiri

address: Lazarettgasse 14, BT25/2, 6th floor Lab 2

department: Laboratory Medicine

country: Austria

institute: Medical University of Vienna

summary: Hepatocellular carcinoma (HCC) is a leading cause of cancer-related death with limited, while treatment options. Thus, a better understanding of the molecular pathways involved in HCC development has become a priority to guide future therapies. While previous studies implicated the AP-1 (Fos/Jun) transcription factor family members c-Fos and c-Jun in HCC formation, the contribution of Fos-related antigens 1 and 2 (Fra-1/2) is unknown. Here we analyse the transcriptional consequences of hepatocyte-restricted expression of Fra-1 and Fra-2 proteins in the liver of young adult mice during 2 months by bulk RNA-seq.

overall_design:
RNA from murine (adult males, C57BL/6) liver samples was sequenced 2 months after transgene induction from two closely related strains Fra1hep (in 3 controls and 3 mutants, littermates) and Fra-2hep (in 3 controls and 3 mutants, littermates).   Transgene expression in the mutants was always started at weaning (3 weeks of age) by doxyciclin removal. Controls were littermates kept in the same cage and that received the same treatment. Comparative expression profiling was conducted across genotype (mutants compared to controls) and also compared to control and mutants from an already published strain that were kept in identical conditions (GSE261005).

Grant ID: 741888 (ERC AdG 2016)
Grant title: CSI-Fun
Funding source: European Research Council
Grantee name: Erwin F.Wagner

Grant ID: 859860 (ITN 2019)
Grant title: CANCERPREV
Funding source: H2020 - MSCA
Grantee name: Erwin F. Wagner


title: IL-2-inducible T cell kinase deficiency sustains chimeric antigen receptor T cell therapy against tumor cells

city: Changsha

first: Zineng

last: Huang

address: No.139 of Renmin middle Road

department: Department of Hematology

institute: The Second Xiangya Hospital

summary: Despite the revolutionary achievements of chimeric antigen receptor (CAR) T cell therapy in treating cancers, especially leukemia, several significant challenges still limit its therapeutic efficacy. Of particular relevance is the relapse of cancer in large part, as a result of exhaustion and short persistence of CAR-T cells in vivo. IL-2-inducible T cell kinase (ITK) is a critical modulator of the strength of TCR signaling, while its role in CAR signaling is unknown. By electroporation of Cas9 ribonucleoprotein (RNP) complex into CAR-T cells, we successfully deleted ITK in CD19-CAR-T cells with high efficiency. Bulk and single-cell RNA sequencing (scRNA-seq) analyses revealed down-regulation of exhaustion and up-regulation of memory gene signatures in ITK-deficient CD19-CAR-T cells. Our results further demonstrated a significant reduction of T cell exhaustion and enhancement of T cell memory, with significant improvement of CAR-T cell expansion and persistence both in vitro and in vivo. Moreover, ITK-deficient CD19-CAR-T cells showed better control of tumor relapse. Our work provides a promising strategy of targeting ITK to develop sustainable CAR-T products for clinical use.

overall_design: To investigate the transcriptomic regulation by ITK in CD19-CAR-T cells, we generated ITK-KO CD19-CAR-T cells in which ITK gene has been deleted by Clustered regularly interspaced short palindromic repeats (CRISPR)-mediated gene editing technology.


title: Chimeric antigen receptor macrophages (CAR-M) sensitize HER2+ solid tumors to PD1 blockade

city: Philadelphia

first: Michael

last: Klichinsky

state: Pennsylvania

address: 3675 Market Street Ste. 401

institute: Carisma Therapeutics

summary: We previously developed human CAR macrophages (CAR-M) and demonstrated redirection of macrophage anti-tumor function leading to tumor control in immunodeficient xenograft models. Here, we developed clinically relevant fully immunocompetent syngeneic models to evaluate the potential for CAR-M to remodel the tumor microenvironment (TME), induce T cell anti-tumor immunity, and sensitize solid tumors to PD1/PDL1 checkpoint inhibition. In vivo, anti-HER2 CAR-M significantly reduced tumor burden, prolonged survival, remodeled the TME, increased intratumoral T cell and natural killer (NK) cell infiltration, and induced epitope spreading. CAR-M therapy protected against antigen-negative relapse in a T cell dependent fashion, confirming long-term anti-tumor immunity. In HER2+ solid tumors resistant to anti-PD1(aPD1) monotherapy, the combination of CAR-M and aPD1 significantly improved tumor growth control, survival, and remodeling of the TME. These results demonstrate synergy between CAR-M and T cell checkpoint blockade and provide a strategy to enhance response to aPD1 therapy for patients with non-responsive tumors.

overall_design: HSCs and monocytes were differentiated into macrophages by treatment with either M-CSF or GM-CSF. Cells were either untransduced or Adf5f35 virally transduced HER2 Chimeric Antigen Receptor (CAR) macrophages. Untreated untransduced monocytes and hematopoietic stem cells were used as controls.

first: Stefano

last: Pierini

first: Rashid

last: Gabbasov

first: Maria

last: Oliveira-Nunes

first: Rehman

last: Qureshi

first: Alison

last: Worth

first: Shuo

first: Karan

last: Nagar

first: Crystal

last: Griffin

first: Lurong

last: Lian

first: Yumi

last: Ohtani

first: Kayleigh

last: Ross

first: Christopher

last: Sloas

last: Ball

first: Benjamin

last: Schott

first: Poonam

last: Sonawane

first: Linara

last: Cornell

first: Daniel

last: Blumenthal

first: Sotheavy

last: Chhum

first: Nicholas

last: Minutolo

first: Kerri

last: Ciccaglione

first: Lauren

last: Shaw

first: Isaac

last: Zentner

first: Hyam

last: Levitsky

first: Olga

last: Shestova

first: Saar

last: Gill

first: Bindu

last: Vargese

last: Cushing

first: Sabrina

last: DeLong

first: Sascha

last: Abramson

first: Thomas

last: Condamine


title: Distinct effects of CDK8 module subunits on cellular growth and proliferation in Drosophila

city: New Orleans

first: Jun-yuan

last: Ji

state: LA

address: 1700 Tulane Avenue

department: Department of Biochemistry and Molecular Biology

institute: Tulane University

summary: The Mediator complex plays a pivotal role in facilitating RNA polymerase II-dependent transcription in eukaryotes. Within this complex, the CDK8 kinase module (CKM), comprising CDK8, Cyclin C (CycC), Med12, and Med13, serves as a dissociable subcomplex that modulates the activity of the small Mediator complex. Genetic studies in Drosophila have revealed distinct phenotypes associated with mutations in CKM subunits, yet the underlying mechanism has remained unknown. Using Drosophila as a model, we generated transgenic strains to individually or simultaneously deplete the four CKM subunits in all possible combinations, uncovering unique phenotypes in the eyes and wings. Depletion of CDK8-CycC enhanced E2F1 target gene expression and promoted cell-cycle progression, whereas Med12-Med13 depletion had no significant impact on these processes. Conversely, depleting Med12-Med13 altered the expression of ribosomal protein genes and fibrillarin, reduced nascent protein synthesis, indicating a severe reduction in ribosome biogenesis and cellular growth compared to the loss of CDK8-CycC. These findings reveal distinct in vivo roles for CKM subunits, with Med12-Med13 disruption having a more pronounced effect on ribosome biogenesis and protein synthesis .

overall_design: To investigate how different CKM subunits affect cell proliferation and tissue size in the same tissue and developmental stage, we performed single-cell RNA sequencing (scRNA-seq) on wing discs with depleted CKM subunits (CDK8-CycC, Med12, or CDK8-CycC-Med12) using the 10x Genomics platform. Control samples were ap-Gal4>GFP heterozygous wing discs.

first: Xiao

last: Li

first: Mengmeng

last: Liu

first: Yue

last: Xing

first: Ye

last: Niu

first: Tzu-Hao

first: Jasmine

middle: L

last: Sun

first: Yanwu

first: Rajitha-Udakara-Sampath

last: Hemba-Waduge

first: Jun-Yuan


title: Transcriptome and BCR profile at single cell level of influenza neuraminidase (NA) specific pre-exisiting memory B cells in health adults

city: Hang Zhou

first: Zeli

last: Zhang

address: ZhuanTang Jie Dao Shi long Shan Lu Nr. 17

institute: Westlake University

summary: Influenza virus NA is important for viral replication. The antibody targeting NA can block viral replication. We used single B cell RNA and BCR sequencing to analysis the heterogeneity of NA specific pre-existing memory B cells in healthy adults.

overall_design: NA-specific memory B cells in human PBMCs were sorted by FACS with NA-probes, and subjected to single B cell RNA and BCR sequencing.

first: Xudong

last: Wu

first: Xin

last: Wang


title: Genipin rescues developmental and degenerative defects in familial dysautonomia models and accelerates axon regeneration

city: Athens

first: Nadja

last: Zeltner

state: GA

address: 325 Riverbend Road

institute: University of Georgia

summary: The peripheral nervous system (PNS) is essential for proper body function. A high percentage of the world’s population suffers nerve degeneration or peripheral nerve damage. Despite this, there are major gaps in the knowledge of human PNS development and degeneration and therefore, there are no available treatments. Familial Dysautonomia (FD) is a devastating disorder caused by a homozygous point mutation in the gene ELP1. FD specifically affects the development and causes degeneration of the PNS. We previously employed patient-derived induced pluripotent stem cells (iPSCs) to show that peripheral sensory neurons (SNs) recapitulate the developmental and neurodegenerative defects observed in FD. Here, we conducted a chemical screen to identify compounds that rescue the SN differentiation inefficiency in FD. We identified that genipin restores neural crest and SN development in patient derived iPSCs and in two mouse models of FD. Additionally, genipin prevented FD degeneration in SNs derived from patients with FD, suggesting that it could be used to ameliorate neurodegeneration. Moreover, genipin crosslinked the extracellular matrix (ECM), increased the stiffness of the ECM, reorganized the actin cytoskeleton, and promoted transcription of YAP-dependent genes. Finally, genipin enhanced axon regeneration in healthy sensory and sympathetic neurons (part of the PNS), and in prefrontal cortical neurons (part of the central nervous system), in in vitro axotomy models. Our results suggest that genipin has the potential to treat FD-related neurodevelopmental and neurodegenerative phenotypes, and to enhance neuronal regeneration of healthy neurons after injury. Moreover, this suggests that the ECM can be targeted to treat FD.

overall_design: To study the effects of genipin in FD, we differentiated iPSC-FD-S3 cells into SNs with or without genipin. Genipin (10 μM) was added to the media of the treated cells throughtout the differentation. To characterize their transcriptome, we collected RNA on day 20. We also collected RNA from untreated healthy SNs (day 20) differentiated from hPSC-ctr-H9 cells. We performed RNAseq of each sample.

first: Kenyi

last: Saito-Diaz


title: Identify the interplay between N6-methyladenine and gene regulation under hypoxia by randomized empirical model (ATAC-seq)

city: Taoyuang

first: Kou-Juey

state: Taiwan

address: No.15, Wenhua 1st Road, Gueishan Dist

department: Cancer Genome Research Center

country: Taiwan

institute: Chang Gung Memorial Hospital at Linkou

summary: In this study, we employed ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing) to investigate the role of N6-methyladenine (6mA) in chromatin accessibility under hypoxic conditions.

overall_design: FaDu, a head and neck cancer cell line, was purchased from Bioresource Collection and Research Center (BCRC, Hsinchu, Taiwan). The cell line was cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS, 1 mM sodium pyruvate, and 1× Antibiotic-Antimycotic (Gibco), under the conditions of 5% CO2 at 37°C. The mycoplasma absence was tested by Mycoplasma PCR Detection Kit (abm) before the experiments. Knockdown of METTL4 was achieved using a previously established method with lenti-virus infection. For hypoxic conditions, cells were incubated in an environment of 1% O2, 5% CO2, and 94% N2 at 37°C for 18 hours.

first: Kai-Wen

last: Hsu

first: Joseph

last: Lai


title: Etoposide promotes tumor cells pyroptosis via down-regulation of O-GlcNAcylation to regulating p53 stability

city: Chengdu City

first: xiangyu

last: jin

address: No. 24, South Section 1, First Ring Road

institute: sichuan university

summary: As a new type of cell death mediated by Gasdermin (GSDM) family, pyroptosis has increasingly become a research hotspot in the fields of biology and medicine. Studies have found that a variety of drugs (such as etoposide and traditional Chinese medicine extracts) can effectively inhibit the occurrence and development of tumors by activating caspase family and GSDM protein family to induce pyroptosis. Promoting pyroptosis has become an effective method for the treatment of tumors, but the research on its regulatory mechanism is still lacking. In order to explore the key molecular pathways of etoposide-induced pyroptosis, transcriptome sequencing analysis was performed

overall_design: To explore the key molecular pathways involved in etoposide-induced pyroptosis, SH-SY5Y cells were treated with etoposide, and then the control and treatment groups were examined by RNA-seq in triplicate

first: Jing

first: Yida

last: wang

first: Xiangyu

last: Jin


title: Single-nucleus and spatial landscape of the sub-ventricular zone in human glioblastoma

city: Charleston

first: Stefano

last: Berto

state: SC

address: 68 President Street

department: Neuroscience

institute: Medical University of South Carolina

summary: Glioblastoma (GBM) clinical management is challenging due to its heterogeneous nature, invasive potential, and poor response to radio- and chemo-therapy. As a result, GBM inevitably recurs and only a minority of patients survive 5 years post-diagnosis. We were the first to show that in the majority of GBM patients, the sub-ventricular zone (SVZ) of the lateral ventricles is a reservoir of cancer stem-like cells (CSCs) that show distinct patterns of treatment resistance when compared to matched CSCs from the tumor mass and contribute to the seeding of the recurrent tumor. However, the sampling and the characterization of the SVZ in GBM patients pose several challenges, as this area is extremely small and needs to be objectively identified during tumor surgical resection.

overall_design: To overcome this challenge, by using our fluorescence-guided multiple sampling scheme, we built a single-nucleus RNA-sequencing-based microenvironment landscape of the SVZ in GBM patients (T_SVZ). By performing a systematic comparison with tumor mass (T_Mass) samples isolated from the same patients and using two histologically normal SVZ (N_SVZ) samples through a number of computational tools and experimental methods, we identified novel therapeutic vulnerabilities in the T_SVZ of GBM patients.

first: Suganya

last: Subramanian

first: Bryan

last: Granger

first: Yamhilette

last: Licón-Muñoz

first: Vanessa

last: Avalos

first: Sara

last: Piccirillo


title: Transcriptomic DN3 clock neuron subtypes regulate Drosophila sleep

city: Waltham

first: Michael

last: Rosbash

address: 415 South street

institute: HHMI/Brandeis University

summary: Circadian neurons within animal brains orchestrate myriad physiological processes and behaviors, but the contribution of these neurons to the regulation of sleep is not well understood. To address this deficiency, we leveraged single-cell RNA sequencing to generate a new and now comprehensive census of transcriptomic cell types of Drosophila clock neurons. We focused principally on the enigmatic DN3s, which constitute most fly brain clock neurons and were previously almost completely uncharacterized. These DN3s are organized into 12 clusters with unusual gene expression features compared to the more well-studied clock neurons. We further show that previously uncharacterized DN3 subtypes promote sleep through a G protein–coupled receptor, TrissinR. Our findings indicate an intricate regulation of sleep behavior by clock neurons and highlight their remarkable diversity in gene expression and functional properties.

overall_design: We developed a novel split GAL4 driver line that is expressed in almost all of the DN3 neurons. To learn more about their gene expression profiles, we utilized a droplet-based single cell RNA sequencing method and generated six time points of single cell data around the clock.

first: Dingbang

last: Ma


title: Dual nuclease single-cell lineage tracing by Cas9 and Cas12a

city: Guangzhou

first: Cheng

last: Chen

state: Guangdong

address: Kaiyuan Ave 190

institute: Guangzhou Institute of Biomedicine and Health, Chinese Academy of Science

summary: Single-cell lineage tracing based on CRISPR/Cas9 gene editing enables the simultaneous linkage of cell states and lineage history at a high resolution. Despite its immense potential in resolving the cell fate determination and genealogy within an organism, existing implementations of this technology suffers from the limitations in recording capabilities and considerable information dropout. Here, we introduce a versatile tool, DuTracer, which utilizes two orthogonal gene editing systems to record deep cell lineage history at single-cell resolution in an inducible manner. DuTracer shows the ability of enhanced lineage recording with minimized target dropouts and deepened tree depth. Application of DuTracer in mouse embryoid bodies and neuromesodermal organoids illustrates the transition pattern of the lineage relationship of different cell types and proposes potential lineage-biased molecular drivers. Moreover, we have developed an entropy-based approach to quantify the lineage recording ability of DuTracer in cell differentiation models. Together, DuTracer facilitates the precise and regulatory interrogation of cellular lineages of complex biological processes.

overall_design: All samples have undergone DuTracer-based lineage tracing. For neuromesodermal organoids (NMO), two biological batches of samples were collected. Batch 1 only contained NMO D30, while batch 2 included both neuromesodermal progenitor D3 and NMO D15. To increase the harvest rate, NMO D15 were sampled for three times from the same organoid. To generate 10 neuromesodermal organoid libraries, the samples were prepared halfway through the DNBelab C4 protocol and the cDNA pool was split. One quarter the pool is used to generate the RNA-seq libraries and one quarter is used to make amplicon libraries (Note: target site amplicon samples). For mouse embryonic stem cells and HEK293T samples, cells were collected every two days from D0-D16. Genomic DNAs were extracted and subsequently subjected to amplicon sequencing.

first: Yuanxin

last: Liao


title: The widely used Ucp1-CreEvdr transgene elicits complex developmental and metabolic phenotypes.

city: Cincinnati

first: Joan

last: Sanchez-Gurmaches

address: 3333 Burnet Ave

institute: CIncinnati Children's Hospital Medical Center

summary: Bacterial artificial chromosome transgenic models, including most Cre-recombinases, enable potent interrogation of gene function in vivo but require rigorous validation as limitations emerge. Due to its high relevance to metabolic studies, we performed comprehensive analysis of the Ucp1-CreEvdr line which is widely used for brown fat research. Hemizygotes exhibited major brown and white fat transcriptomic dysregulation, indicating potential altered tissue function. Ucp1-CreEvdr homozygotes also show high mortality, tissue specific growth defects, and craniofacial abnormalities. Mapping the transgene insertion site revealed insertion in chromosome 1 accompanied by large genomic alterations disrupting several genes expressed in a range of tissues. Notably, Ucp1-CreEvdr transgene retains an extra Ucp1 gene copy that may be highly expressed under high thermogenic burden. Our multi-faceted analysis highlights a complex phenotype arising from the presence of the Ucp1-CreEvdr transgene independently of intended genetic manipulations.

overall_design: Experimental mice were generated by breeding UCP1-CreEvdr heterozygous mice. Mice were sacrified and intersacpular brow and posterior subcutaneous adipose tissues obtained at 6 weeks of age without farther manipulation. RNA was isolated using Trizol and RNAeasy colums.

first: Manasi

middle: S

last: Halurkar

first: Christopher

last: Ahn

first: Hee-Woong

last: Lim