title: Native nucleosomes intrinsically encode genome organization principles

city: Baltimore

first: Taekjip

last: Ha

state: Maryland

address: 3400 N. Charles Street

institute: Johns Hopkins University

summary: The eukaryotic genome, first packed into nucleosomes of about 150 bp around the histone core, is organized into euchromatin and heterochromatin, corresponding to the A and B compartments, respectively. Here, we asked if individual nucleosomes in vivo know where to go. That is, do mono-nucleosomes by themselves contain A/B compartment information, associated with transcription activity, in their biophysical properties? We purified native mono-nucleosomes to high monodispersity and used physiological concentrations of biological polyamines to determine their condensability. The chromosomal regions known to partition into A compartments have low condensability and vice versa. In silico chromatin polymer simulations using condensability as the only input showed that biophysical information needed to form compartments is all contained in single native nucleosomes and no other factors are needed. Condensability is also strongly anticorrelated with gene expression, and especially so near the promoter region and in a cell type dependent manner. Therefore, individual nucleosomes in the promoter know whether the gene is on or off, and that information is contained in their biophysical properties. Comparison with genetic and epigenetic features suggest that nucleosome condensability is a very meaningful axis onto which to project the high dimensional cellular chromatin state. Analysis of condensability using various condensing agents including those that are protein-based suggests that genome organization principle encoded into individual nucleosomes is electrostatic in nature. Polyamine depletion in mouse T cells, by either knocking out ornithine decarboxylase (ODC) or inhibiting ODC, results in hyperpolarized condensability, suggesting that when cells cannot rely on polyamines to translate biophysical properties of nucleosomes to control gene expression and 3D genome organization, they accentuate condensability contrast, which may explain dysfunction known to occur with polyamine deficiency.

sample_organism:
Homo sapiens
Mus musculus
synthetic construct

overall_design: Native mono-nucleosomes are biochemically purified from human and mouse cells and followed by in vitro condensation using various condensing agents including polyamine. Using high-throughput sequencing of input and supernatant fractions after condensation, the condensabilities of mono-nucleosomes  were estimated on genome-wide in single-nuclesome resolution.

first: Sangwoo

last: Park

first: Raquel

last: Merino Urteaga

first: Violetta

last: Karwacki-Neisius

first: Gustavo

last: Ezequiel Carrizo

first: Advait

last: Athreya

first: Alberto

last: Marin-Gonzalez

first: Nils

last: Benning

first: Jonghan

first: Michelle

last: Mitchener

first: Natarajan

middle: V

last: Bhanu

first: Benjamin

last: Garcia

first: Bin

last: Zhang

first: Tom

middle: W

last: Muir

first: Erika

last: Pearce


title: Transcripts identified by microarray analysis that were deregulated in E16.5 Pax2-cre/+;Parg(fl/fl) (Pparg-cKO) ureters.

status: Public on Mar 10 2025

city: Hannover

first: Mark-Oliver

last: Trowe

address: Carl-Neuberg-Str. 1

department: Molekularbiologie

country: Germany

institute: Medizinische Hochschule Hannover

summary: Evaluation of the transcriptional changes in E16.5 ureters upon loss of Pparg in the ureteric epithelium.

overall_design: Total RNA from dissected ureters of E16.5 Pparg-cKO and control embryos was analyzed by microarray analysis.

first: Andreas

last: Kispert

first: Carsten

last: Rudat


title: G1 length dictates heterochromatin landscape [CUT&RUN]

city: Aurora

first: Srinivas

last: Ramachandran

state: CO

department: Biochemistry and Molecular Genetics

country: USA

institute: University of Colorado School of Medicine

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Stem cells have lower facultative heterochromatin as defined by trimethylation of histone H3 lysine 27 (H3K27me3) compared to differentiated cells. However, the mechanisms underlying these differential H3K27me3 levels remain elusive. Because H3K27me3 levels are diluted two-fold in every round of replication and then restored through the rest of the cell cycle, we reasoned that the cell cycle length could be a key regulator of total H3K27me3 levels. Here, we demonstrate that a fast cell cycle sets low levels of H3K27me3 in serum-grown murine embryonic stem cells (mESCs). Extending the G1 phase leads to an increase in global H3K27me3 in mESCs due to the formation of de novo Polycomb domains, and the length of the G1/S block correlates with the extent of gain in H3K27me3, arguing that levels of the modification depend on the time available between successive rounds of replication. Similarly, accelerating the G1 phase in HEK293 cells decreases H3K27me3 levels. Finally, we applied this principle in tumor cells harboring a dominant negative H3K27M allele that reduces H3K27me3 levels. In these cells, extending G1 increases H3K27me3 levels, pointing to an unexpected means to rescue the effect of oncohistones. Our results suggest cell cycle length as a universal mechanism to modulate heterochromatin formation and, thus, cellular identity.

overall_design: Cleavage Under Target and Release Under Nuclease (CUT&RUN) for H3K27 trimethylation (H3K27me3) after G1 block of mouse embryonic stem cells (mESCs) with thymidine.

first: Abby

last: Trouth

first: Kamesh

last: Ravichandran

first: Philip

middle: R

last: Gafken

first: Sara

last: Martire

first: Gabriel

middle: E

last: Boyle

first: Giovana

middle: M

last: Veronezi

first: Van

last: La

first: Stephanie

middle: J

last: Namciu

first: Laura

middle: A

last: Banaszynski

first: Jay

middle: F

last: Sarthy


title: PBMC single-cell transcriptome discriminates systemic immune changes associated with metastasis

status: Public on Mar 08 2025

city: Adelaide

first: Stefano

last: Mangiola

state: SA

address: North Terrace

department: SAiGENCI

country: Australia

institute: University of Adelaide

type: Expression profiling by high throughput sequencing

summary: Several single-cell studies have reported transcriptomic, genomic and epigenomic dysregulation in breast cancer tissue. However, the peripheral immune landscape of breast cancer patients remains poorly understood. We report the analysis of the single-cell transcriptomes of more than 110,000 peripheral blood mononuclear cells (PBMCs) of healthy and metastatic breast cancer patients. Integrated analysis revealed depleted memory B cells (BMEM) and T cell subpopulations, including transitional CD8 T, CD4 naïve, CD4 ribosome and MAIT cells in the circulating immune landscape of metastatic breast cancer patients. Nevertheless, metastatic breast cancer patients showed marked enrichment of pro-inflammatory NKG7 high monocytes, M1 macrophages, migratory myeloid, T memory stem cells (TSCM), CD4 T effector memory (TEM) and CD4 T central memory (TCM) cells. Further, interrogation of single-cell transcriptomes based on metastatic disease state, i.e. stable vs progressive, revealed distinct compositional and transcriptional changes in PBMCs correlated with the metastatic burden. The antigen inexperienced CD4 naïve T cells and cytotoxic MAIT cells are further depleted in the progressive metastatic stage. Surprisingly, the pro-inflammatory classical monocytes and CD4 T effector memory (TEM) cells are also depleted in the progressive metastatic state, indicating their potential role in stable metastatic disease. Lastly, the receptor-ligand relationships, such as cell-cell contact, ECM-receptor interactions, and cytokine transcription profiles, were tightly associated with metastatic burden. Collectively, our study provides unique molecular insight into the peripheral immune system operating in metastatic breast cancers and identified novel surrogate biomarkers of metastatic disease.

overall_design: single-cell transcriptomes of peripheral mononuclear blood cells of 5 oligometastatic and 5 progressive metastatic women were sequences with 10X technology

first: Rebecca

last: Brown

first: Chen

last: Zhan

first: Jean

last: Berthelet

first: Shalini

last: Guleria

first: Chamikara

last: Liyanage

first: Simone

last: Ostrouska

first: Jordan

last: Wilcox

first: Michal

last: Merdas

first: Paula

last: Fuge-Larsen

first: Caroline

last: Bell

first: Jan

last: Schroder

first: Lisa

last: Mielke

first: John

last: Mariadason

first: Simon

last: Chang-Hao Tsao

first: Yunshun

last: Chen

first: Vijay Kumar

last: Yadav

first: Sadanand

last: Vodala

first: Robin

middle: L

last: Anderson

first: Delphine

last: Merino

last: Behren

first: Belinda

last: Yeo

first: Anthony

middle: T

last: Papenfuss

first: Bhupinder

last: Pal


title: Gene expression analyses of HCT116 p53+/+ and HCT116 p53-/- cells knocked down for RPS19 or BYSL

status: Public on Mar 09 2025

city: Salamanca

first: L. Francisco

last: Lorenzo-Martín

state: Castilla y León

address: Campus Miguel de Unamuno

country: Spain

institute: University of Salamanca (USAL) – Centro de Investigación del Cáncer (CIC)

summary: A gene expression array was used to measure differential expresion of mRNAs upon knockdown of RPS19 or BYSL in HCT116 TP53+/+ and HCT116 TP53-/- cells. Two independent experiments were performed to analyze the eight conditions subject to analyses: HCT116 TP53+/+ and HCT116 TP53-/- cells harvested 24 h (si-ctrl and si-RPS19) and 48 h (si-ctrl and si-BYSL) after si-RNA transfection. Each experiment included three biological replicates of each condition. In silico gene set expression analyses (GSEA) revealed multiple gene sets that were differentially enriched in at least two of the RPS19 and/or BYSL knockdown conditions. Out of those, the upregulated gene sets showing the highest enrichment score in TP53+/+ cells included the signatures for p53-regulated genes and for oxidative phosphorylation. Our GSEAs also revealed that the siRNA-mediated depletion of either RPS19 or BYSL is associated with a marked downmodulation of gene signatures linked to the signaling of the mammalian target of rapamycin complex 1 (mTORC1) and the activation of the unfolded protein response (UPR). These two gene sets become downregulated at similar levels irrespectively of the p53 presence in the HCT116 cells, thus indicating that their expression must be under the control of other transcriptional regulators. Interestingly, these two gene signatures have not ever been pointed as relevant in DBA or any other ribosomopathy patients. We noticed that many of the genes commonly downregulated by the knockdowns of the RPS19 and BYSL transcripts are direct targets of the transcription factor ATF4. This led us to perform a GSEA analysis of a list of 41 genes (AmTOR signature) whose expression is commonly induced by ATF4 downstream of mTORC1 and phosphorylated IF2-alpha in normal and stressed cells, respectively. It was found that the mRNAs for ATF4 and most of the AmTOR signature genes display a similar downregulation in both TP53+/+ and TP53-/- HCT116 cells upon depletion of either the RPS19 or the BYSL transcript.

overall_design: A total of 48 samples were analyzed that correspond to eight different conditions: HCT116 TP53+/+ and HCT116 TP53-/- cells harvested 24 h (si-ctrl and si-RPS19) and 48 h (si-ctrl and si-BYSL) after si-RNA transfection. Two independent experiments were performed for each set of samples (batch 1 and batch 2). Each experiment included three biological replicates of each condition.

first: L

first: Javier

last: Robles-Valero

first: Rosa

last: Ramírez-Cota

first: Sonia

middle: G

last: Gaspar

first: Pedro

last: Fuentes

first: Antonio

last: Gentilella

first: Xosé

last: Bustelo

first: Mercedes

last: Dosil


title: DJ1 ablation enhances CMA activity to exacerbate BAT whitening in humanized mice

status: Public on Mar 07 2025

city: Shanghai

first: Kong

last: Xingxing

address: Songhu Road 2005

department: School of Life Sciences

country: China

institute: Fudan University

summary: Humans predominantly live under thermoneutral conditions, leading to brown adipose tissue (BAT) whitening, a reduction in mitochondrial content and thermogenesis. Here, the adaptations to thermal stress of BAT are examined using quantitative proteomics, finding that Parkinson’s disease (PD) pathways are sensitive to ambient temperature. Ablation of DJ-1, a PD gene, exaggerates BAT whitening, resulting in impaired insulin sensitivity and energy expenditure at thermoneutrality. We further identify that the activity of chaperon-mediated autophagy (CMA) contributes to the deletion of DJ-1-induced metabolic phenotypes. Importantly, DJ-1 interacts with HSC70 and subsequently decreases the activity of CMA, enhances the stability of thermogenic proteins. Moreover, inhibiting the activity of CMA by knockdown of LAMP2A counteracts the phenotypes induced by DJ-1 ablation. Additionally, while 4-PBA, an agonist of DJ-1, upregulates mitochondrial oxygen consumption; compound 23, an antagonist of DJ-1, exerts the opposite effects. Collectively, these data address a cell-autonomous route to regulate BAT whitening at thermoneutrality.

overall_design: Brown adipose tissues(BAT) from BAT-specific DJ-1 knockout (BDJ1KO) mice and flox mice which were housed at room temperature or 29°C for 8 weeks extracted for RNA sequencing. The sequencing was conducted using Illumina BGIseq500 platform (BGI, China). The transcriptomics data processing was addressed as previous article described. The clean reads were mapped to the reference genome (mm10) using STAR (2.7.10a). Quantification of gene expression was calculated using RSEM (v1.2.28). Subsequently, differential expression analysis between groups was conducted using the DESeq2(1.3.40) with adjust P value < 0.05 and |FC|>1.5. GO enrichment analysis and KEGG pathway analysis was performed by clusterProfiler (4.2.2) with different expression genes.


title: Morphological and genetic screens reveal mechanisms of BiDAC-induced plasma membrane protein degradation

city: South San Francisco

first: Kirill

last: Bersuker

state: CA

address: 1130 Veterans Blvd.

institute: Calico Life Sciences

summary: The discovery of bifunctional degradation activating compounds (BiDACs) has led to the development of a new class of drugs that promote the clearance of their protein targets. BiDAC-induced ubiquitination is generally believed to direct cytosolic and nuclear proteins to proteolytic destruction by proteasomes. However, pathways that govern the degradation of other classes of BiDAC targets, such as integral membrane and intraorganellar proteins, have not been investigated in depth. In this study we use morphological profiling and CRISPR/Cas9 genetic screens to investigate the mechanisms by which BiDACs induce the degradation of plasma membrane receptor tyrosine kinases (RTKs) EGFR and Her2. We find that BiDAC-dependent ubiquitination triggers the trafficking of RTKs from the plasma membrane to lysosomes for degradation. Surprisingly, functional proteasomes are required for endocytosis of RTKs upstream of the lysosome. Additionally, our screen uncovers a non-canonical function of the lysosome-associated arginine/lysine transporter PQLC2 in EGFR degradation. Our data show that BiDACs can target proteins to proteolytic machinery other than the proteasome and motivate further investigation of mechanisms that govern the degradation of diverse classes of BiDAC targets.

overall_design: Cells expressing Her2-GFP or EGFR-RFP were infected with a full genome sgRNA library and treated with the EGFR/Her2 BiDAC Lap-VHL. Cells that failed to degrade Her2-GFP or EGFR-RFP were isolated by cell sorting, and genes enriched in the sorted versus bulk populations were identified by high throughout sequencing.


title: The TetR-like regulator SCO4385 and Crp-like regulator SCO3571 modulate heterologous production of antibiotics in Streptomyces coelicolor M512

status: Public on Mar 06 2025

city: Tübingen

first: Kay

last: Nieselt

state: DE

address: Sand 14

department: Department for Computer Science

institute: Institute for Bioinformatics and Medical Informatics

summary: Heterologous expression in well-studied model strains is a  routinely applied method to investigate biosynthetic pathways. Here, we pursue a comparative approach of large-scale DNA-affinity-capturing assays coupled with semi-quantitative mass spectrometry, to identify putative regulatory proteins from Streptomyces coelicolor M512, that bind to the heterologously expressed biosynthetic gene clusters of the liponucleoside antibiotics caprazamycin and liposidomycin. Both gene clusters share an almost identical genetic arrangement, including the location of promoter regions, as detected by RNA-sequencing. A total of 2214 proteins were trapped at the predicted promoter regions, with only three binding to corresponding promoters in both gene clusters. Among these, overexpression of a yet uncharacterized TetR-family regulator, SCO4385, increased caprazamycin but not liposidomycin production. Protein-DNA interaction experiments using biolayer-interferometry confirmed the binding of Sco4385 to Pcpz10 and PlpmH at different locations within both promoter regions, which might explain its functional variance. Sequence alignment allowed the determination of a consensus sequence present in both promoter regions,  to which Sco4385 was experimentally shown to bind. Furthermore, we show that overexpression of the Crp regulator Sco3571 leads to a three-fold increase in caprazamycin and liposidomycin production yields, possibly due to an increased expression of a precursor pathway.

overall_design: RNA-seq of the heterologously expressed caprazamycin and liposidomycin biosynthetic gene cluster in Streptomyces coelicolor M512 after 2 days and 4 days of growth.

first: Sarah

last: Wilcken

first: Theresa

last: Harbig

first: Bertolt

last: Gust


title: Microvascular immunity is organ-specific and remodeled after kidney injury

city: Münster

first: Carolin

last: Walter

address: Domagkstraße 9

department: Medical Faculty of the WWU Münster

summary: Blood tests are a common method for diagnosing and monitoring various health conditions. Nevertheless, the extent to which blood analysis can offer insights into immune and organ dysfunction remains uncertain. Here, we conducted a comprehensive analysis of blood-borne leukocytes in the vasculature of different mouse organs and compared it to peripheral blood and parenchymal samples. We observed that microvascular immune cells outnumber tissue-resident counterparts in the kidney, liver and lung. Here, classical monocytes and lymphocytes are diminished while nonclassical and SSC-high monocytes are enriched compared to blood. Utilizing single-cell sequencing, we identified specific cell populations up to 100-fold expanded in the kidney vasculature including macrophages, plasmacytoid dendritic cells, B cells, and innate lymphoid cells type 2. Microvascular enrichment can trigger a local phenotype switch as shown in B cells specifically dwelling in glomerular capillaries. Peritonitis and acute kidney injury (AKI) elicited a multifaceted and systemic response of microvascular leukocytes independent of tissue infiltration. It involves remote organ effects, such as a 16-fold increase of leukocytes in the splenic circulation or 64-fold increase of SSC-high monocytes in the liver circulation that is not detectable in peripheral blood. Following full recovery from AKI, persistent changes were observed particularly in the renal vasculature, while most leukocytes in the peripheral blood have already returned to baseline levels. Collectively, our findings suggest a paradigm of organ- and disease-specific microvascular immunity that largely eludes conventional blood and tissue analysis.

overall_design: We examined the single-cell transcriptomes of kidney tissue, renal microcirculation and peripheral blood from four untreated and four IRI-reg mice. For this purpose, single-cell suspensions were prepared from fresh tissue and peripheral blood material, labeled with sample tags (BD CTT Mouse Single Cell Multiplexing Kit) and were then subjected to microwell-based single-cell RNA sequencing (scRNA-seq) using BD Rhapsody Express system by BD Biosciences following manufacturer’s instructions. Samples were sequenced using the Illumina NovaSeq and analyzed using SevenBridges. Single-cell capture was performed using the BD Rhapsody Express following manufacturer’s instructions.

first: Konrad

last: Buscher

first: Rebecca

last: Rixen

first: Paula

last: Schütz


title: Sequential JAK inhibition boosts antitumor T cell responses to combined anti-PD-1 and anti-CTLA4

city: San Francisco

first: Mark

last: Bridge

address: 513 Parnassus Ave

institute: UCSF

summary: While immune Checkpoint Inhibition (CPI) has reshaped cancer treatment, the majority of cancer patients do not benefit from this treatment approach, and this treatment can lead to immune related adverse events. While induction of IFNγ responses are thought be necessary for anti-tumor immunity, growing evidence also implicates IFNγ as a tumor-intrinsic mediator of CPI resistance. CPI-induced IFNγ also mediates activation induced cell death in T cells as an immune-intrinsic mechanism of resistance. In this study, we show that transient block of IFNγ signaling through administration of the JAK1 inhibitor ABT-317 enhances anti-tumor T cell responses with CPI in pre-clinical models. Importantly sequential but not concomitant ABT-317 treatment, led to significantly reduced toxicity and improved tumor efficacy.  Sequential treatment reduced activation-induced T cell death and enhanced expansion of tumor-reactive T cell subsets with increased effector function in vivo and ex vivo. Only CPI in combination with ABT-317 was able to protect mice from tumor rechallenge. These results demonstrate that the JAK inhibition within a window following CPI can used to address an immune-intrinsic mechanism of therapeutic resistance.

overall_design: 8- to 10-week-old male WT C57BL/6J mice were implanted s.c. with 1 million TRAMP-C2 or MC38 cells and treated i.p. with Isotype or CPI (anti-CTLA4+antiPD-1) on days 3, 6 and 9, followed by vehicle (DPBS) or 20 mg/kg ABT-317 on day 12. On day 15 after implantation, tumor,  and TDLNs from mice were harvested and processed to single cell suspension as described. To enrich for CD45+ cells in tumors, total tumor lysates were stained for Live/Dead and CD45. Live CD45- or CD45+ (tumors)  were sorted with a FACS Aria into separate tubes and barcoded by with mouse TotalSeq-C03 hashtag antibodies 1-10 (155861, 155863, 155865, 155867, 155869, 155871, 155873, 155875, 155877, 155879, Biolegend) at a 1:200 dilution for 40 minutes at 4C to identify individual samples and treatment groups. Then, individual reactions of 6 x 104 cells each with 1:1 CD45+:CD45- (tumors) ratios (unsorted total cells for TDLNs) and equal hashtag representation for batch correction purposes were prepared at the Chromium Controller (10X Genomics) using the Chromium Next GEM Single Cell 5' Kit v2 (1000263, 10X Genomics) and the Chromium Single Cell Mouse TCR Amplification kit (1000254, 10X Genomics) and sequencing was performed on an Illumina NovaSeq 6000 with paired-end 100 base pair read lengths.

first: Marcel

last: Arias-Badia

first: PeiXi

last: Chen

first: Yee May

last: Lwin

first: Aahir

last: Srinath

first: Aram

last: Lyu

first: Zenghua

last: Fan

first: Serena

middle: S

last: Kwek

first: Diamond

last: Luong

first: Ali

last: Setayesh

first: Mason

last: Sakamoto

first: Matthew

last: Clark

first: Averey

last: Lea

first: Rachel

last: Wolters

first: Andrew

last: Goodearl

first: Fiona

last: Harding

first: Jacob

last: Gorman

first: Wendy

last: Ritacco

first: Lawrence

last: Fong


title: Tetracycline has no long-term effects on gut physiology and microbiome of the New World screwworm, Cochliomyia hominivorax, which has positive implications for transgenic male-only rearing systems

city: Kerrville

first: Mackenzie

last: Tietjen

state: TX

address: 2700 Fredericksburg Rd

department: Knipling-Bushland US Livestock Insects Research Laboratory

institute: USDA-ARS

summary: Insect control programs utilizing the sterile insect technique (SIT) are more efficient when all insects released are male. One approach to producing all males in a mass-rearing setting is development of tetracycline-repressible (Tet-Off) transgenic sexing systems. Tetracycline can have adverse impacts on insect health, therefore we reared screwworm, Cochliomyia hominivorax, like Tet-Off strains to investigate the impact on fitness, the midgut microbiota, and midgut transcriptomes. Overall, the impacts on the microbiota and transcriptome did not have adverse effects on the strain fitness, and changes caused by tetracycline exposure were not inherited by the offspring mimicking a male-only generation.

overall_design: RNA-seq profiling of midguts from production Cochliomyia hominivorax larval and adult flies before, during, and after rearing with tetracycline.

first: Alex

middle: P

last: Arp

first: Agustin

last: Sagel

first: Mario

last: Vasquez

first: Gladys

last: Quintero

first: Deanna

last: Bodine

first: Perot

last: Saelao

first: Paul

last: Hickner


title: Transcriptional landscapes underlying Notch-induced lineage conversion and reprogramming of mammary cells

city: Paris

first: Calvin

last: Rodrigues

address: 26 Rue d Ulm

department: CdR

country: France

institute: Institut Curie

summary: Ectopic activation of NOTCH1 (N1ICD) in adult unipotent basal cells induced a cell fate switch to luminal lineage. We investigated the transcriptomic evolution of the during this transition.

overall_design: In this study we used K5CreERT2/N1ICD and SMACreERT2/NICD mice induced at P21 and mammary glands were analysed 1,3,4 and 6 weeks later by SMARTSeq V2. We sequenced WT and N1ICD luminal, intermediate and basal cells afer cell sorting.

first: Candice

last: Merle

first: Sarah

last: Kinston

first: Bertie

last: Göttgens

first: Silvia

last: Fre


title: A versatile IRES toolbox to determine IRES-like activity exemplified by Hoxa mRNA expression and translation in mouse embryonic tissues

city: Singapore

first: Teodorus Theo

last: Susanto

address: 60 Biopolis St

country: Singapore

institute: Genome Institute of Singapore

summary: Widespread control of gene expression at the level of translation has emerged as a key point of protein expression regulation in space and time. Internal ribosomal entry sites (IRESes) are a prominent mechanism by which ribosomes can confer greater gene regulation. However, their rigorous functional characterization remains difficult. Here we present a versatile toolbox of technologies in embryos and cells including single-molecule mRNA isoform imaging, Pacbio long read sequencing, isoform-sensitive mRNA quantification along polysome profiles, and IRES-like translation of circular RNA (circRNA) reporters as a new guide to understanding IRES-mediated regulation. We investigate the disputed IRES-like RNA elements in embryonic Hoxa mRNAs. We show the relative expression, localization and translation of the IRES-like containing Hoxa9 mRNA isoform in specific embryonic tissues, and Hoxa IRES-like dependent translation in circRNAs. We thereby provide a new resource of technologies to elucidate the roles of IRES-like elements in gene regulation and embryonic development.

overall_design: somite and neural tube was microdissected from mouse E11.5 embryo. Iso-seq library was prepared using PacBio SMRTbell 3.0 kit, and then sequenced using Sequel II

first: Phillip

middle: B

last: Koch

first: Zijian

last: Zhang

first: Naomi

last: Genuth

first: Teodorus

first: Martin

last: Haimann

first: Alena

last: Khmelinskaia

first: WooByeon

last: Gun

first: Alina

last: Niedzwetzki

first: Maria

last: Barna

first: Kathrin

last: Leppek


title: Myrobalane fruit extracts modulate immunobiochemical pathways in vitro

city: Innsbruck

first: Pablo

last: Monfort-Lanzas

address: Innrain 80-82

department: Medical Biochemistry

country: Austria

institute: Medical University Innsbruck

summary: The study investigates the effects of myrobalane fruit extracts, essential in Asian traditional medicine and notably part of the Triphala formulation, on human cell models. The complexity of these botanical preparations suggests a multi-target mode of action, making it difficult to identify specific active ingredients. The in vitro study revealed that, beyond their antioxidant properties, myrobalane fruit extracts modulate tryptophan metabolism and affect immunobiochemical and cytoprotective signaling pathways in a dose-dependent manner. Integrated transcriptome analysis of treated cells showed impacts on immune response pathways, oxidative stress, and detoxification processes. Specifically, a synergistic activation of the endogenous antioxidant response was observed in liver epithelial cells treated with a combination of the three fruit extracts. These findings highlight the modulation of various signaling pathways and cellular processes, underpinning the complex multi-target effects of myrobalane fruit extracts. Despite being limited to in vitro data, this study enhances the understanding of the mode of action of these botanical mixtures.

overall_design: Triplicate cultures of THP-1 cells were treated with BB3 extract (25 µg/ml and 50 µg/ml) plus LPS [1 µg/ml] or vehicle control plus LPS for 24 hours. Following treatment, cells were harvested by centrifugation, and total RNA was extracted using the RNeasy Mini Kit (Qiagen, Germany) with an included DNase digest step. The RNA was quantified and assessed for quality via spectrophotometric measurement and evaluation of 18S/28S ribosomal RNA band intensities on an agarose gel. The RNA from triplicates was pooled and analyzed using Affymetrix Human Genome U133 Plus 2.0 microarrays (one array per treatment).

first: Stefanie

last: Hofer

first: Marcel

last: Jenny

first: Angela

last: Klein

last: Becker

first: Lucia

last: Parrakova

first: Florian

last: Überall

first: Markus

last: Ganzera

first: Dietmar

last: Fuchs

first: Hubert

last: Hackl

first: Johanna

last: Gostner


title: Radiation-induced amphiregulin drives tumor metastasis

status: Public on Mar 05 2025

city: Chicago

first: Andras

last: Piffko

state: IL

address: 900 E 57th St

institute: University of Chicago

summary: The anti-tumor effect of radiotherapy (RT) beyond the treatment field - the abscopal effect - has garnered much interest. By contrast, the potentially harmful impact of radiation in promoting metastasis is less well studied. Here, we show that RT induces the expression of epidermal growth factor receptor (EGFR) ligand amphiregulin (AREG) in tumor cells, which reprograms EGFR+ myeloid cells toward an immunosuppressive phenotype and reduces phagocytosis. This stimulates distant metastasis growth in both patients and pre-clinical murine tumor models. The inhibition of these tumor-promoting factors induced by RT may represent a novel therapeutic strategy to improve patient outcomes.

overall_design: RNA-seq profliing of two separate experiments. The first experiment contains WT and WT_IR samples. WT samples contain RNA isolated from LLC tumor cells expressing RFP (LLC-RFP) cultured in vitro. WT_IR samples contain RNA isolated from LLC-RFP tumor cells cultured in vitro and treated with 4Gy irradiation. RNA was collected 12 hours after irradiation - shown in Extended Data Fig. 4j-k of the manuscript - three technical repeats for each condition. The second experiment contains files labelled WT_mono_WT_tumor, WT_mono_AREG_KO_tumor, EGFR_KO_mono_WT_tumor and EGFR_KO_mono_AREG_KO_tumor.The samples contain RNA isolated from Ly6C+ mononuclear phagocytes (MNPs) generated from bone marrow culture of EGFR+ wildtype C57BL/6 mice or conditionally Egfr-deficient mice (LysMCre Egfr-/-) - also three technical repeats of each condition. Ly6C+ MNPs were collected after 24 co-culturing bone-marrow derived cells with LLC-RFP tumor cells (LLC cells expressing RFP, labelled LLCAR+ in the manuscript) or with LLC-AR- tumor cells (LLC cells expressing RFP in which Amphiregulin has been knocked out by CRISPR gene editing). After co-culture, only Ly6C+ MNPs were sorted and used for RNA isolation. The title of each sample contains the information of the co-culture setup, for example WT_mono_WT_tumor contain RNA from Ly6C+ MNPs generated from bone marrow of EGFR+ control mice which were co-cultured with WT LLC-RFP tumor cells, whereas WT_mono_AREG_KO_tumor samples contain RNA from Ly6C+ MNPs generated from bone marrow of EGFR+ control mice which were co-cultured with LLC-AREG-KO tumor cells and the same naming system applies to the Ly6C+ MNPs from EGFR- (LysmCre Egfr-/-) mice that were co-cultured either with LLC-RFP (WT) or AREG-KO LLC cells before sorting. (These data were used to generate Extended Data Fig. 8 f-i of the manuscript).

first: Kaiting

last: Yang

first: Arpit

last: Panda

first: Katarzyna

last: Zawieracz

first: Sean

last: Pitroda

first: Hua Laura

last: Liang

first: Ralph

last: Weichselbaum


title: T cell-specific RIPK1 ablation results in villus atrophy of the duodenum in mice

city: Ghent

first: Bruno

last: Verstraeten

state: Oost-Vlaanderen

address: Technologiepark 71

country: Belgium

institute: Ghent University

summary: RIPK1 is required in T cells with a history of CD4-expression to maintain intestinal homeostasis. We show that the pathology in the upper intestine is a result of conventional T cell apoptosis in absence of the RIPK1.

overall_design: Four samples in total: Two intraepithelial lymphocytes (IEL) samples: Ripk1FL/FL and Ripk1ΔCD4; Two lamina propria lymphocytes (LPL) samples: Ripk1FL/FL and Ripk1ΔCD4

first: Jelle

last: Huysentruyt

first: Andrew

last: Brown

first: Mario

last: Ruiz Perez

first: Kelly

last: Lemeire

first: Tatyana

last: Divert

first: Kayleigh

last: Flies

first: Nozomi

last: Takahashi

first: Marie

last: Joossens

first: Wim

last: Declercq

first: Tom

last: Vandenberghe

first: Jonathan

last: Maelfait

first: Peter

last: Vandenabeele

last: Tougaard

first: Wolf

last: Steels

first: Elke

last: De Bruyn

first: Bart

last: Lambrecht


title: Rickettsia rickettsii RoaM negatively regulates expression of a limited number of rickettsial genes

status: Public on Mar 04 2025

city: Hamilton

first: Ted

last: Hackstadt

state: MT

address: 903 S 4th St

department: Rocky Mountain Laboratories (National Institute of Allergy and Infectious Disease)

institute: National Institutes of Health

summary: The recently described rickettsial protein, RoaM, (Regulator of Actin-based Motility), negatively regulates the production of actin tails and its abrogation induces hyper-spreading behavior in many laboratory-adapted strains of Rickettsia rickettsii.  RoaM is not surface exposed thus its mechanism of regulating actin-based motility is unclear.  Using R. rickettsii strains derived from the virulent Sheila Smith strain that express varying levels of roaM, an RNA-seq experiment was performed.  We found that roaM-overexpressing strains downregulate expression of at least six genes which may link the regulatory effects of RoaM to the phenotypic effect on motility.  Genes regulated by RoaM were confirmed by RT-qPCR.  Among the genes regulated is the secreted effector RarP2 which disrupts the trans-Golgi network.  Two of the hypothetical proteins were shown to be secreted via fusion to a glycogen synthase kinase tag, which when phosphorylated reveals exposure to the host-cell cytosol. Taken together, these data support the hypothesis that RoaM affects transcription, downregulating rickettsial genes important for pathogenicity in the mammalian host but which are perhaps otherwise detrimental within the tick vector.  To determine how RoaM activity may itself be regulated, we investigated a role of temperature in roaM transcription.  RoaM expression itself is not temperature dependent but many other rickettsial genes are, including some also regulated by RoaM.  This suggests that rickettsiae utilize multiple mechanisms to control gene expression in response to environmental signals.

sample_organism: Rickettsia rickettsii str. 'Sheila Smith'

overall_design: This study includes two bulk RNA-seq experiments on Rickettsia rickettsii Sheila Smith. One experiment compares the expression of different genotypes to investigate the role of the gene RoaM. For this experiment, four genotypes of R. rickettsii Shelia Smith (wildtype, truncated RoaM, RoaM knockout, and RoaM overexpression) were grown in identical conditions. The other experiment compares the expression of R. rickettsia Shelia Smith wildtype and RoaM deletion strains at two temperatures, 34ºC (standard temperature) and 22ºC.

first: Nock

last: Adam M.

first: Clark

last: Tina R.

first: Anzick

last: Sarah L.

first: Meyer

last: Elisabeth A.

first: Miller

last: Rebecca

first: Holbrook

last: Myndi

first: Martens

last: Craig

first: Lack

last: Justin

first: Hackstadt

last: Ted


title: Long-lasting B cell convergence to distinct broadly reactive epitopes following vaccination with chimeric influenza virus hemagglutinins II

city: New York

first: Patrick

last: Wilson

state: NY

address: 413 E 69th St

department: Drukier Institute

institute: Weill Cornell Medicine

summary: In a phase I clinical trial, a chimeric hemagglutinin (cHA) immunogen induced antibody responses against the conserved HA stalk domain as designed. Here, we determined the specificity, function, and subsets of B cells induced by cHA vaccination by pairing single cell RNA-sequencing and B cell receptor repertoire sequencing. We have shown that the cHA inactivated vaccine with a squalene-based adjuvant induced a robust activated B cell and memory B cell (MBC) phenotype against two broadly neutralizing epitopes in the stalk domain. The overall specificities of the acute plasmablast (PB) and MBC  responses clonally overlapped, suggesting B cell convergence to these broadly protective epitopes. At one-year post-immunization, we identified that cHA vaccination reshaped the HA-specific MBC pool to enrich for stalk-binding B cells. Altogether, these data indicate the cHA vaccine induced robust and durable B cell responses against broadly protective epitopes of the HA stalk domain, in line with serological data.

overall_design: MBCs (CD19+IgD-Probe+) from cHA vaccine and seasonal influenza vaccine recipients were sort purified for analysis of transcriptome and V(D)J gene usage

first: Jenna

last: Guthmiller

first: Linda

middle: Y

last: Lan

first: Lei

last: Li

first: Sean

last: Nelson

first: Yanbin

last: Fu

first: Jordan

last: Chervin

first: Chloe

last: Troxell

middle: C


title: Shared and divergent transcriptomic regulation in nucleus accumbens D1 and D2 medium spiny neurons by morphine

first: Molly

last: Estill

address: 1 Gustave L. Levy Place

department: Neuroscience

institute: Mount Sinai School of Medicine

summary: Substance use disorders (SUDs) induce widespread molecular dysregulation in nucleus accumbens (NAc), a brain region pivotal for coordinating motivation and reward, which is linked to neural and behavioral disturbances promoting addiction. Despite the overlapping symptomatology of SUDs, different drug classes exert partly unique influences on neural circuits, cell types, physiology, and gene expression. To better understand common and divergent molecular mechanisms governing SUD pathology, we characterized the cell-type-specific restructuring of the NAc transcriptional landscape after psychostimulant or opioid exposure. We combined fluorescence-activated nuclei sorting and RNA sequencing to profile NAc D1 and D2 medium spiny neurons (MSNs) across morphine exposure paradigms, including initial exposure, prolonged withdrawal after repeated exposure, and re-exposure post-withdrawal. Our analyses reveal that D1 MSNs display many convergent transcriptional responses, whereas D2 MSNs manifest highly divergent responses, with morphine causing far more adaptations in this cell type.

overall_design:
Male double-transgenic mice expressing D1- or D2-specific nuclear-tagged GFP were generated by crossing LSL-eGFP::L10a (IMSR_JAX:022367) mice with either D1-Cre (MGI:3836633) or D2-Cre (MGI:3836635) mice. Morphine sulphate (from the National Institute on Drug Abuse) were dissolved in 0.9% saline and administered intraperitoneally at doses of 20 mg/kg or 10 mg/kg, respectively. These doses were chosen based on their roughly equivalent rewarding effects, as established in prior conditioned place preference experiments. In the cocaine cohort, mice received saline or morphine each day for 15 days prior to withdrawal. As depicted in Figure 1A, after 30 days of homecage withdrawal, mice received challenge injections of saline, or morphine and were sacrificed 1 h later.

Treatments:
SS = Saline exposure during acquisition, with Saline bolus during the challenge
SM = Saline exposure during acquisition, with Morphine bolus during the challenge
MS = Morphine exposure during acquisition, with Saline bolus during the challenge
MM = Morphine exposure during acquisition, with  Morphine bolus during the challenge

first: Caleb

last: Browne

first: Philipp

last: Mews

first: Xianxaio

last: Zhou

first: Leanne

last: Holt

first: Rita

last: Futamura

first: Li

last: Shen

first: Bin

last: Zhang

first: Eric

last: Nestler


title: Tebentafusp elicits on-target cutaneous immune responses driven by cytotoxic T cells in uveal melanoma patients

city: Schwerzenbach

first: Adhideb

last: Ghosh

address: Schorenstrasse 16

department: Health Sciences and Technology

country: Switzerland

institute: ETH Zurich

summary:
Background: Tebentafusp is the first T-cell receptor-based bispecific protein approved for clinical use in HLA-A*02:01+ adult patients with unresectable/metastatic uveal melanoma. It redirects T-cells toward gp100-expressing target cells, frequently inducing skin-related early adverse events.
Methods: This study investigated immunological and cellular responses using single-cell and spatial analysis of skin biopsies from patients with metastatic uveal melanoma treated with tebentafusp.
Results: 81.8% of patients developed acute cutaneous adverse events, which correlated with improved survival. Multimodal analysis revealed a brisk infiltration of CD4+ and CD8+ T-cells, while melanocyte numbers declined. Single-cell RNA-sequencing revealed T-cell activation, proliferation, and IFN-γ/cytotoxic gene upregulation. CD8+ T-cells co-localized with melanocytes and upregulated LAG3, suggesting potential for combination therapies with tebentafusp. Melanocytes upregulated antigen presentation and apoptotic pathways, while pigmentation gene expression decreased. However, gp100 remained stably expressed.
Conclusion: Sequential skin biopsies enable in vivo pharmacodynamic modeling of tebentafusp, offering insights into immune activation, toxicity, and treatment response. Examining the on-target effects of bispecifics in tissues amenable to longitudinal sampling enhances our understanding of toxicity and therapeutic escape mechanisms, guiding strategies for treatment optimization.

overall_design: Paired skin samples from patients at baseline and during treatment with tebentafusp.

first: Ramon

last: Staeger

first: Aizhan

last: Tastanova

first: Mitchell

last: Levesque

first: Reinhard

last: Dummer

first: Barbara

last: Meier-Schiesser


title: Dual DNA demethylation mechanisms implement epigenetic memory driven by the pioneer factor PAX7 [ATAC-seq]

city: Montréal

first: Juliette

middle: Emilie

last: Harris

state: Québec

address: 110 avenue des Pins O

country: Canada

institute: IRCM

summary: Pioneer transcription factors have the unique ability to open chromatin at enhancers to implement new cell fates. They also provide epigenetic memory through demethylation of enhancer DNA but the underlying mechanisms remain unclear. We now show that the pioneer PAX7 triggers DNA demethylation using two replication-dependent mechanisms, including direct PAX7 interaction with the UHRF1-DNMT1 complex that is responsible for DNA methylation maintenance. PAX7 binds to UHRF1 and prevents its interaction with DNMT1, thus blocking activation of its enzyme activity. The TET DNA demethylases also contribute to the replication-dependent loss of DNA methylation. Thus, PAX7 hijacks UHRF1 to block activation of DNMT1 after replication leading to loss of DNA methylation by dilution and the process is accelerated by the action of TET demethylases.

overall_design: ATAC-seq in mouse corticotrope AtT-20 cells with ER-Pax7 ectopic expression. Treated with tamoxifen (tam, ER-Pax7 activation).

first: Alexandre

last: Mayran

first: Arthur

last: Gouhier

first: Yves

last: Gauthier

first: Nawal

last: Hajj Sleiman

first: Samir

last: Merabet

first: Micheal

last: Dukatz

first: Pavel

last: Bashtrykov

first: Albert

last: Jeltsch

first: Haig

last: Djambazian

first: Shu-Huang

last: Chen

first: Aurelio

last: Balsalobre

first: Jacques

last: Drouin


title: Microglial mechanisms drive amyloid-β clearance in immunized patients with Alzheimer’s disease

status: Public on Mar 03 2025

first: David

last: Gate

address: 745 N Fairbanks Ct

department: Neurology

institute: Northwestern University

summary: Alzheimer’s disease (AD) therapies utilizing amyloid-β (Aβ) immunization have shown potential in clinical trials. Yet, the mechanisms driving Aβ clearance in the immunized AD brain remain unclear. Here, we use spatial transcriptomics to explore the effects of both active and passive Aβ immunization in the AD brain. We compare actively immunized AD patients with nonimmunized AD patients and neurologically healthy controls, identifying distinct microglial states associated with Aβ clearance. Using high-resolution spatial transcriptomics alongside single-cell RNA sequencing, we delve deeper into the transcriptional pathways involved in Aβ removal after lecanemab treatment. We uncover spatially distinct microglial responses that vary by brain region. Our analysis reveals upregulation of the triggering receptor expressed on myeloid cells 2 (TREM2) and apolipoprotein E (APOE) in microglia across immunization approaches, which correlate positively with antibody responses and Aβ removal. Furthermore, we show that complement signaling in brain myeloid cells contributes to Aβ clearance after immunization. These findings provide new insights into the transcriptional mechanisms orchestrating Aβ removal and shed light on the role of microglia in immune-mediated Aβ clearance. Importantly, our work uncovers potential molecular targets that could enhance Aβ-targeted immunotherapies, offering new avenues for developing more effective therapeutic strategies to combat AD.

overall_design: 3 samples: 2 AD Non-Immunized AD (nAD); 1 lecanemab immunized AD (LCMB)

first: Lynn

last: Van Olst


title: Microglial mechanisms drive amyloid-β clearance in immunized patients with Alzheimer’s disease [dataset2]

overall_design:
4 donors, 4 brain areas: Left Middle Frontal Gyrus, Left Superior Temporal Gyrus, Left Inferior Parietal Lobule, Posterior Hippocampus of 3 nAD donors and 1 lecanemab-immunized AD donor

Please note that Seurat object(s) were removed on March 17, 2025, as they were uploaded prior to revisions and contained outdated cell/spot annotations. Any Seurat objects downloaded before this date should not be used. For access to updated Seurat objects, please contact dgate@northwestern.edu.


title: Microglial mechanisms drive amyloid-β clearance in immunized patients with Alzheimer’s disease [dataset1]

overall_design:
25 samples: 6 Non-Neurological Diseased (NND); 6 AD Non-Immunized AD (nAD); 13 AN1792 Immunized AD (iAD)

Please note that Seurat object(s) were removed on March 17, 2025, as they were uploaded prior to revisions and contained outdated cell/spot annotations. Any Seurat objects downloaded before this date should not be used. For access to updated Seurat objects, please contact dgate@northwestern.edu.


title: Lifestyle-induced changes in alternative splicing patterns in mice [adipose]

city: Nuthetal

first: Heike

last: Vogel

state: BRB

address: Arthur-Scheunert-Allee 114-116

department: Department of Experimental Diabetology

institute: German Institute of Human Nutrition Potsdam-Rehbruecke

summary: Alternative splicing diversifies the cellular protein landscape but aberrant splicing is implicated in many diseases. We aimed to investigate to which extent mis-splicing contributes to insulin resistance as the causal defect of Type 2 Diabetes and whether diet- and exercise-based lifestyle interventions can reverse mis-splicing.

overall_design: Alternative splicing analysis was conducted with Whippet and rMATS using RNA sequencing data from skeletal muscle and adipose tissue of diabetes susceptible NZO mice fed ad libitum, with intermittent fasting or caloric restriction and from healthy C57BL/6J mice subjected to exercise

first: Annette

last: Schürmann

first: Jasmin

last: Gaugel

first: Markus

last: Jähnert

first: Alexander

last: Neumann


title: Methylation Fraxinus excelsior 2451S ONT genome

city: Heslington, York

first: Sara

last: Franco Ortega

address: Wentworth Way

department: Department of Biology

country: United Kingdom

institute: Centre for Novel Agricultural Products, University of York

type: Methylation profiling by high throughput sequencing

summary: A new genome of Fraxinus excelsior was assembled using a hybrid approach combining Nanopore and Illumina data (BioProject PRJNA865134, SAMN30100368, genome JANJPF000000000 ). Methylation was also assessed in the genome. Manuscript title: Fraxinus excelsior updated long-read genome reveals the importance of MADS-box genes in tolerance mechanisms against ash dieback, G3:Genes|Genomes|Genetics

overall_design: Ash leaf samples taken from a graft of the F.excelsior 2451S tree growing in Paradise Wood. DNA was extacted following the High molecular weight gDNA extraction from plant leaves protocol, Oxford Nanopore Technologies [ONT]), which utilised Carlson buffer and further purification using QIAGEN genomic-tip, see preprint Vaillancourt and Buell (2019) for details.The libraries were prepared using the SQK-LSK109 ligation sequencing kit and run with PromethION R9.4.1 (FLO-PRO002) flow cells. Base calling was obtained onboard with Guppy. Nanopolish (v 0.13.2) (Simpson et al., 2017) was used to identify the methylated regions as well as the frequency of methylation (using the script calculate_methylation_frequency.py) in the whole genome by using the new genome as reference.

first: Andrea

last: Harper

last: Ortega

first: Sally

last: James

first: Katherine

last: Newling


title: Methylation comparison between ash tolerant and susceptible trees to ash dieback [methylation]

summary: DNA was extracted from two ash tree samples, one tolerant and one susceptible to ash dieback. The DNA was sequenced using Nanopore technologies and the methylation was called against the new genome (BioProject PRJNA865134, SAMN30100368, genome JANJPF000000000 ) to identify differentially methylated regions between both samples. Manuscript title: Fraxinus excelsior updated long-read genome reveals the importance of MADS-box genes in tolerance mechanisms against ash dieback, G3:Genes|Genomes|Genetics

overall_design: DNA was extracted using the method of Workman et al., (Workman et al., 2018) from a pair of Danish trees previously described in Harper et al. (Harper et al., 2016): association panel tree Ash66, and prediction panel tree DNASH35, which had ash dieback disease damage scores of 0 and 75% respectively. The libraries were prepared using the SQK-LSK109 ligation sequencing kit and run with PromethION R9.4.1 (FLO-PRO002) flow cells, base calling was generated automatically. Nanopolish was used to call the methylation profile of each sample.


title: Fraxinus excelsior transcriptome and methylome

summary: This SuperSeries is composed of the SubSeries listed below. Manuscript title: Fraxinus excelsior updated long-read genome reveals the importance of MADS-box genes in tolerance mechanisms against ash dieback, G3:Genes|Genomes|Genetics

overall_design: Refer to individual Series


title: Gene expression of the 182 Danish ash accessions (Harper et al., 2016, ENA PRJEB10202) using the new long-read F.excelsior genome (PRJNA865134, SAMN30100368)

summary: A new genome of Fraxinus excelsior (PRJNA865134) was assembled using a hybrid approach combining Nanopore and Illumina data. The gene expression of a 182 Danish tree panel (Harper et al. 2016) was assessed using the new genome as reference (BioProject PRJNA865134, SAMN30100368, genome JANJPF000000000 ).Manuscript title: Fraxinus excelsior updated long-read genome reveals the importance of MADS-box genes in tolerance mechanisms against ash dieback, G3:Genes|Genomes|Genetics

overall_design:
Sequence reads for the 182 Danish ash accessions (Harper et al., 2016, European Nucleotide Archive accession number PRJEB10202, Please check Harper et al., 2016, https://doi.org/10.1038/srep19335) were trimmed with trimmomatic, mapped against the CDS models of the new genome using salmon (version 0.8) (Patro et al., 2017). Salmon counts were inputted into R-studio with tximport to obtain TPMs table.
Please note that PRJNA865134 is still being processed by GenBank and it is not publicly available yet.


title: Characterization of chicken peripheral blood mononuclear cells (PBMCs) during prime vaccinations at single-cell resolution

city: Xi'an

first: Hai

last: Li

address: No.76 Yanta West Road

department: Department of Pathogenic Microbiology and Immunology

institute: Xi'an Jiaotong University

summary: We performed scRNA-seq for PBMCs isolated from 35-day-old healthy male SPF chicken at the onset or upon vaccination with live-attenuated or inactivated IBV vaccines.

overall_design: PBMC samples freshly isolated from 35-day-old healthy male SPF chickens were subjected to scRNA-seq using the 10x Chromium platform.


title: Transcription-coupled AID Deamination Damage Depends on ELOF1-associated RNA Polymerase II [ChIP-seq]

status: Public on Mar 01 2025

city: Shanghai

first: Fei-Long

last: Meng

state: Shanghai

address: 320 Yue Yang Road

institute: Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences

summary: In adaptive immunity, transcription-coupled damage is introduced into antibody genes by activation-induced cytidine deaminase (AID) to diversify antibody repertoire. However, the coordination between transcription and DNA damage/repair remains elusive. Here, we find that transcription elongation factor 1 (ELOF1) stabilizes paused RNA polymerase II (RNAPII) at transcription barriers, providing a platform for transcription-coupled DNA damage/repair. Using a genetic screen, we discover that ELOF1 is required for AID targeting and that ELOF1 deficiency results in defective antibody class switch recombination and somatic hypermutation in mice. While downstream transcription-coupled repair factors are dispensable for AID-damage, ELOF1 mechanistically facilitates both transcription-coupled damage and repair by stabilizing chromatin-bound RNAPII. In ELOF1-deficient cells, paused RNAPII tends to detach from chromatin and fails to recruit factors to induce or repair DNA damage. Our study places ELOF1 at the center of transcription-coupled DNA metabolism processes and suggests a transition of RNAPII from elongation to a DNA damage/repair scaffold.

overall_design: For ChIP-seq, 15 million CH12F3 cells were fixed and mixed with 5 million k562 cells before sonication. For damage-seq, CH12F3 cells were treated with 20J/m^2 of 254 nm UV. Cells were harvested immediately or cultured for an additional 8 hours. For PADD-seq, NVP-2 was added to the cell culture medium to a final concentration of 250 nM. About 5x10^7 CH12F3 cells were irradiated with 20 J/m^2 of 254 nm UV. Cells were further incubated for 30 or 60 minutes and subjected to PADD-seq.

first: Pengfei

last: Dai

first: Yuanqing

last: Tan


title: Transcription-coupled AID Deamination Damage Depends on ELOF1-associated RNA Polymerase II [RNA-seq]

overall_design: RNA-seq of ELOF1 proficient and deficient CH12F3 cells and splenic primary B cells. CH12F3 cells were stimulated with α-CD40, IL4, TGF-β for 1.5, 3 or 7 days before experiments. Splenic primary B cells were stimulated with IL4 and LPS for 2.5 or 3.5 days. For each sample, polyA RNA from Drosophia S2 cells were added at a ratio of 76ng/million cells as spike-in control. The library were prepared with NEB kits (E7770S, E7400L) using rRNA depletion method.

first: Yifeng

last: Luo


title: Long-lasting B cell convergence to distinct broadly reactive epitopes following vaccination with chimeric influenza virus hemagglutinins

first: Patrick

last: Wilson

first: Jenna

last: Guthmiller

first: Linda

last: Lan

first: Lei

first: Sean

last: Nelson

first: Yanbin

last: Fu

first: Jordan

last: Chervin

first: Chloe

last: Troxell


title: TFEB Orchestrates Stress Recovery and Paves the Way for Senescence Induction in Human Dermal Fibroblasts

status: Public on Mar 02 2025

first: Pablo

last: Monfort-Lanzas

summary: Cells experience oxidative stress and widespread cellular damage during stress-induced premature senescence (SIPS). Senescent cells show an increase in lysosomal content, which may contribute to mitigating cellular damage by promoting autophagy. This study investigates the dynamics of lysosomal quality control in human dermal fibroblasts (HDF), specifically examining lysosomal signaling pathways during oxidative stress-induced SIPS. Our results reveal distinct signaling responses between the initial stress phase and the ensuing senescent phenotype. During the stress phase, treatment with tBHP, which undermines the antioxidant response, leads to elevated reactive oxygen species (ROS) and lysosomal damage. ROS accumulation activates AMP-activated protein kinase (AMPK) and inhibits Akt, which correlates with the suppression of mammalian target of rapamycin (mTOR). Inactivation of mTOR during this phase aligns with the activation of transcription factor EB (TFEB), a key regulator of autophagy and lysosomal biogenesis. TFEB knockdown under stress increased apoptosis, highlighting the protective role of TFEB in the stress response. As cells transition to senescence, TFEB activity, required for the autophagic damage repair, becomes less critical. The decrease in ROS levels leads to the normalization of AMPK and Akt signaling, accompanied by the reactivation of mTOR. This reactivation of mTOR, which is critical for establishing the senescent state, is observed alongside the inactivation of TFEB. Consequently, as damage decreases, TFEB activity decreases. Our results suggest a dynamic interplay between TFEB and mTOR, highlighting a critical role of TFEB in ensuring cellular survival during SIPS induction but becoming dispensable once senescence is established.

overall_design: Bulk RNA-seq was performed on HFF-2 human dermal fibroblasts with TFEB knockdown and control samples under oxidative stress (tBHP treatment for 4 days). RNA extraction was done using the Monarch Total RNA Miniprep kit, and sequencing was performed using Illumina NovaSeq 6000 with paired-end 150bp reads. STAR was used for alignment, Salmon for quantification, and DESeq2 for differential expression analysis.

first: Lena

last: Guerrero-Navarro

first: Vinzenz

last: Krichbaumer

first: Mariana

last: De Araújo

first: Jlenia

last: Monfregola

first: Lukas

last: Huber

last: Ballabio

first: Pidder

last: Jansen-Dürr

first: Maria

last: Cavinato


title: Transcriptomic signature of L5 pyramidal neuron subpopulations during early steps of inhibitory synapse formation

city: London

first: Julie

last: Jezequel

address: Newcomen Street

department: Centre for Developmental Neurobiology

institute: King's College London

summary: The aim of our project is to identify L5 pyramidal neuron-subtype specific molecules involved in shaping interneurons connectivity.

overall_design:
We used a retrograde tracing approach (retrobreads) to specifically target L5 IT and L5 ET
We then isolated retrobeads-positive cells via Fluorescence Activated Sorting (FACS) at P10, and performed differential gene expression analysis between these 2 L5 pyramidal neuron subpopulations

last: Jézéquel

first: Fursham

last: Hamid

first: Beatriz

last: Rico


title: LAG-3 sustains TOX and regulates the CD94/NKG2-Qa-1b axis to govern exhausted CD8 T cell NK receptor expression and cytotoxicity

city: Philadelphia

first: E

middle: JOHN

last: WHERRY

state: PA

address: 421 Curie Blvd,354 BRB II/III

department: Systems Pharmacology       and Translational Therapeutics,Institute for Immunology

institute: University of Pennsylvania

summary: Exhausted CD8 T cells (Tex) found in chronic viral infection and cancer are defined by sustained co-expression of inhibitory receptors (IRs). Tex can be reinvigorated by blocking IRs, such as PD-1, but synergistic reinvigoration of Tex and enhanced disease control can be achieved by co-targeting multiple IRs including co-blockade of PD-1 and LAG-3, a treatment recently FDA-approved for melanoma. Despite the promise of anti-PD-1/anti-LAG-3 therapy, the cellular and molecular changes intrinsic to CD8 T cells when one or more of these IR pathways are disrupted remain poorly understood. To dissect these IR pathways and their interactions with each other, we used a quadruple co-transfer model to investigate the impact of singular or combined loss of PD-1 and LAG-3 on the differentiation of Tex during chronic viral infection. These analyses revealed distinct roles of PD-1 and LAG-3 in regulating Tex proliferation and cytokine/cytotoxic functions, respectively. Moreover, these studies identified key roles for LAG-3 that complement PD-1 biology including an essential role for LAG-3 in sustaining TOX expression and associated Tex durability. Finally, these studies uncovered a LAG-3-dependent circuit for the generation of an effector-like CD94/NKG2+ subset of Tex capable of enhanced cytotoxicity mediated by recognition of the stress-induced ligand Qa-1b with similar regulation of CD94/NKG2 family members in humans with combination anti-PD-1/anti-LAG-3. These analyses identify a role for LAG-3 in governing CD8 T cell Qa-1b-restricted immune sensing and surveillance. Together, these results disentangled the underlying non-redundant mechanisms of PD-1 and LAG-3 and their synergy in regulating Tex revealing additional immunotherapeutic opportunities based on these pathways.

overall_design:
We used a quadruple (Quad) adoptive transfer strategy with separately congenically marked antigen-specific WT, PD-1 KO, LAG-3 KO, and PD-1 + LAG-3 KO (Double KO; DKO) TCR transgenic CD8 T cells co-adoptively transferred into the same recipient mice infected with LCMV clone 13 resulting in a chronic infection
RNA sequencing (RNA-seq) was done on four replicates of P14 cells of each genotype on d8 post LCMV clone 13 infection in the Quad adoptive transfer setting described above

first: Shin

last: Ngiow

last: Wherry

first: Sasikanth

last: Manne


title: Microglial mechanisms drive amyloid-β clearance in immunized patients with Alzheimer’s disease [dataset3]

first: David

last: Gate

first: Lynn

last: Van Olst


title: Spatial Transcriptomic Profiling of the Human Fallopian Tube Epithelium Reveals Region-specific Gene Expression Patterns

status: Public on Feb 28 2025

city: Kansas City

first: Andrew

last: Godwin

state: Kansas

address: 3901 Rainbow Blvd

department: Pathology

institute: Kansas University Medical Center

summary: The fallopian tube (FT) plays a crucial role in fertility, gynecological health, and high grade serous ovarian cancer (HGSOC) development. Despite its importance, the spatial transcriptome of the FT’s distinct anatomical regions (fimbria, infundibulum, ampulla, and isthmus) remains underexplored. Using the GeoMx Digital Spatial Profiler (DSP) and a targeted ~1,800 gene panel, we analyze premenopausal FT epithelium, identifying region-specific gene expression patterns. Our analysis reveals upregulation of mature ciliated cell markers (FOXJ1, MLF1, SPA17, and CTSS) approaching the fimbria, elevated ROS and apoptosis-related transcripts (TXNIP, PRDX5, BAD, GAS1) in the distal FT, and a switch in cell-cell adhesion transcripts (CDH1, CDH3) along the distal-to-proximal axis. We also provide evidence that MHC-II transcripts in the FT are differentially regulated throughout the menstrual cycle, with lower expression in follicular phase.   These results suggest spatially regulated expression of FT transcripts with implications for fertilization and early neoplastic changes contributing to HGSOC.

overall_design:
To profile the fallopian tube along the proximal to distal axis, we collected seven tissue specimens from premenopausal patients (age 25-48, mean 39) undergoing salpingectomy for non-cancer indications unrelated to fallopian tube health. Samples were divided into four anatomical regions (isthmus, ampulla, infundibulum, and fimbria). We used immunofluorescence to distinguish secretory (PAX8) and ciliated (FOXJ1) epithelial cells and selected representative segments in each anatomical region. Transcriptomes were profiled using the GeoMx Cancer Transcriptome Atlas, covering ~1,800 cancer-related and cell marker transcripts.
Please note that "DSP-1001660037560-B-A07" is mentioned in the annotations, but there are no associated .dcc or fastq files (due to an error with sequencing for well A07)

first: Jared

last: Sipes

first: Sagar

last: Rayamajhi

first: Leonidas

last: Bantis

first: Rashna

last: Madan

first: Amrita

last: Mitra

first: Rajni

middle: V

last: Puri

first: Mohammod Mahmudur

last: Rahman

first: Foyez

last: Ahmmed

first: Harsh

last: Pathak

middle: K


title: A Satellite Cell-Dependent Epigenetic Signature Of Adult Skeletal Muscle Identity After Lifelong Physical Activity

city: Lexington

first: Yuan

last: Wen

state: Kentucky

address: 760 Press Ave

institute: University of Kentucky

summary: Satellite cells comprise only 2-3% of cells in adult skeletal muscle yet play a critical role in muscle plasticity, particularly during prolonged exercise training adaptation. The mechanisms by which these mononuclear cells mediate muscle fiber hypertrophy or attenuate atrophy with physical activity throughout the lifespan are still being elucidated, but epigenetic regulation may play a role. To investigate this possibility, we analyzed DNA methylation patterns in muscle tissue from female mice that engaged in lifelong voluntary unweighted wheel running with or without satellite cells; satellite cells were ablated in adulthood using the tamoxifen-inducible Pax7-DTA model. Wheel running for 13 months induced DNA methylation changes in the promoter regions of numerous muscle fiber-enriched genes - Dnm2, Mlip, Myl1, Myom2, Mstn, Tnnc1, Tnni2, Tpm1, and Ttn - only when satellite cells were present. These genes are fundamental to muscle fiber identity, cytoarchitecture, size, and function; the latter two we have shown to be compromised by the absence of satellite cells in these mice. Musk promoter methylation was altered only in the absence of satellite cells with lifelong running, which may relate to how satellite cells can influence muscle innervation status. We reveal novel epigenetic contributions from satellite cells to the myogenic identity of muscle fibers during lifelong physical activity, providing new directions for how these rare stem cells can promote muscle adaptation and function throughout the lifespan.

overall_design: At five months of age, mice were injected intraperitoneally with vehicle (15% ethanol in sunflower seed oil) or tamoxifen (2 mg/day, suspended in ethanol and sunflower seed oil) for five days, followed by a two-month washout period, then assignment to sedentary or wheel running groups – satellite cell replete (vehicle) or depleted (tamoxifen). Mice ran until ~20 months of age. Gastrocnemius muscles from subsets of mice (n=6/group) were used for methylation array analysis.

first: Kevin

last: Murach

first: Hunter

last: Porter


title: Dominant interfering CARD11 variants disrupt JNK signaling to promote GATA3 expression in T cells

city: Bethesda

first: Bradly

last: Bauman

state: MD

address: 4301 Jones Bridge Rd. C2033

institute: Uniformed Services University of the Health Sciences

summary: Several “primary atopic disorders” are linked to monogenic defects that attenuate TCR signaling, favoring T helper 2 (TH2) cell differentiation. Patients with CARD11-associated atopy with dominant interference of NF-κB signaling (CADINS) disease suffer from severe atopy, caused by germline loss-of-function/dominant interfering (LOF/DI) CARD11 variants. The CARD11 scaffold enables TCR-induced activation of NF-κB, mTORC1, and JNK signaling, yet the function of CARD11-dependent JNK signaling in T cells remains nebulous. Here we show that CARD11 is critical for TCR-induced activation of JNK1 and JNK2, as well as canonical JUN/FOS AP-1 family members. Patient-derived CARD11 DI variants attenuated wild-type CARD11 JNK signaling, mirroring effects on NF-κB. Transcriptome profiling revealed JNK inhibition upregulated TCR-induced expression of GATA3 and NFATC1, key transcription factors for TH2 cell development. Further, impaired CARD11-JNK signaling was linked to enhanced GATA3 expression in CADINS patient T cells. Our findings reveal a novel intrinsic mechanism connecting impaired CARD11-dependent JNK signaling to enhanced GATA3/NFAT2 induction and TH2 cell differentiation in CADINS patients.

overall_design: RNA-seq profiling of wild-type and CARD11 KO Jurkat T cells stimulated for 2 hours with PMA and ionomycin in the absence or presence of inhibitors of JNK signaling (JNK inhibitor IX, JNK2 (MAPK9)-specific inhibitor; JNK-IN-8, JNK1/2 (MAPK8/9) inhibitor).

first: Jeffrey

middle: R

last: Stinson

first: Melissa

last: Kallarakal

first: Lei

middle: H

last: Huang

last: Frank

first: Gauthaman

last: Sukumar

first: Nermina

last: Saucier

first: Clifton

last: Dalgard

first: Alice

last: Chan

first: Joshua

middle: D

last: Milner

first: Megan

last: Cooper

last: Snow


title: Enforcement of stem-cell dormancy by NPMc+ is a critical determinant of unrestricted self-renewal during myeloid leukemogenesis

city: Milano

first: Pier Giuseppe

last: Pelicci

address: Via Adamello, 16

department: Department of Experimental Oncology

country: Italy

institute: European Institute of Oncology

summary: Analysis of the transcriptional profile imposed by NPMc+ and Flt3-ITD oncogenes, both alone and in cooperation, on HSCs at single cell level

overall_design: 4 LT-HSC samples have been single cell sequenced (Mx, NPMc+,  Flt3-ITD, NPMc+/Flt3-ITD). Mx samples should be considered the reference control.

first: Maria

middle: E

last: Boggio Merlo

last: Mallardo

first: Lucilla

last: Luzi

first: Pier

first: Emanuela

last: Colombo


title: Role of KLF5 in Ischemia Reperfusion Injury

city: Cincinnati

first: Georgios

last: Siokatas

state: OH

address: 231 ALBERT SABIN WAY

department: Pharmacology and Systems Physiology

institute: University of Cincinnati

summary: Acute myocardial infarction remains a leading cause of morbidity and mortality worldwide. Our previous studies have investigated the role of the transcription factor Kruppel-like factor 5 (KLF5) in various cardiac disease models, including diabetic cardiomyopathy and ischemic heart failure, and have linked it to the regulation of cellular stress responses, particularly those associated with oxidative stress and metabolic dysregulation. This study examines the role of KLF5 in exacerbating myocardial ischemia/reperfusion (I/R) injury and explores the potential cardioprotective benefit of KLF5 inhibition. Using the surgical model of myocardial I/R injury in both pigs and mice, we observed that KLF5 expression is upregulated in cardiomyocytes during early reperfusion. This increase in KLF5 expression is accompanied by enhanced oxidative stress, apoptosis, fibrosis, and adverse cardiac remodeling. Pharmacologic and cardiomyocyte-specific genetic inhibition of KLF5 significantly reduced infarct size, oxidative stress, and cell apoptosis markers, while preserving cardiac function over both acute and long-term reperfusion periods.

overall_design: To explore the molecular pathways regulated by KLF5 in Ischemia Reperfusion, bulk RNA sequencing was performed on myocardial tissue obtained from three groups of mice: sham-operated, I/R (30' Ischemia/2hr reperfusion) operated and I/R operated with pharmacological inhibition of KLF5 (ML264) treatment.

first: Siokatas

last: Georgios

first: Drosatos

last: Konstantinos

first: Mylonas

last: Nikolaos


title: Multi-Omics Analysis of the Immunological State at Baseline and Following SARS-CoV-2 mRNA Vaccination in Lung Transplant Recipients [RNA-seq]

city: Palo Alto

first: Dmitri

middle: A

last: Kazmin

address: 240 Pasteur Dr

department: Institute for Immunity, Transplantation and Infection

institute: Stanford University

summary: Despite their remarkable success, the effectiveness of COVID-19 mRNA vaccines is notably diminished in organ transplant patients. Here, we employed a multi-omics approach to elucidate the immunological state at baseline and following SARS-CoV-2 mRNA vaccination of lung transplant (LTX) patients compared to healthy controls (HC). Our analysis revealed a baseline immune profile in LTX patients that mirrors the immune alterations observed in severe COVID-19 cases and sepsis. This profile is characterized by: (i) elevated pro-inflammatory cytokines in plasma, notably high levels of EN-RAGE and IL-6; (ii) reduced expression of HLA-DR on blood monocytes and dendritic cells (DCs); (iii) diminished cytokine production by blood monocytes and DCs in response to toll-like receptor (TLR) activation; (iv) increased plasma concentrations of microbial products. In addition to these alterations, single-cell RNA-seq analysis revealed 4 transcriptionally distinct clusters of classical monocytes in HC, with a striking enrichment of a unique classical monocyte cluster in LTX patients, differing from the predominant classical monocytes in HC, marked by heightened expression of the S100A family and reduced expression of genes related to cytokine production and antigen presentation. Following vaccination, LTX patients displayed a reduced magnitude and breadth of binding and neutralizing antibody responses, and impaired memory B and T cell responses. Furthermore, there was a blunted innate immune response to vaccination, evidenced by decreased transcriptional signatures linked to antigen presentation, dendritic cell activation, interferon and monocytes at the single-cell level. Integrative multiscale, multiresponse network (MMRN) analysis revealed an inverse correlation between the distinctive baseline immunological state observed in LTX patients and the adaptive immune responses to vaccines. These findings underscore the distinct immunological profile of lung transplant recipients, providing insights into their immunosuppressed condition and reduced immune responses to vaccines.

overall_design: Paxgene tubes collected blood samples from Baseline, day 1 and day 7 timepoints following the vaccination from each cohort, totaling 106 samples from 19 lung transplant patients and 21 healthy individuals.

first: Mengyun

last: Hu

first: Ana Paula

last: Oliveira

first: Thomas

last: Hagan

first: Bali

last: Pulendran


title: Gene Expression Patterns in the Preoptic Area of Mice with Defective FGF Signaling in GnRH Neurons

city: Boulder

first: Pei-San

last: Tsai

state: CO

address: 354 UCB

department: Integrative Physiology

institute: University of Colorado Boulder

summary: Sexual interactions have previously been shown to improve reproductive health through unknown mechanisms. In this study, we used RNA-Seq to examine sex-induced gene expression changes in the preoptic area (POA), a critical reproductive brain region. Using a mouse model defective in fibroblast growth factor signaling (dnFGFR mouse), previously shown to disrupt the gonadotropin-releasing hormone (GnRH) system, we examined the impact of opposite sex (OS) housing on gene expression in the POA of a reproductively compromised animal. Bulk RNA-Seq followed by gene set enrichment analysis (GSEA) were used to analyze changes in gene expression and biological processes in control and dnFGFR mice after 300 days of cohabitation with a same sex or OS partner. OS housing of dnFGFR mice, but not control mice, significantly improved reproductive anatomy and gonadotropins in dnFGFR mice. These changes occurred concomitantly with novel biological processes related to estradiol metabolism and neuron excitation. Our results suggest a new role of neuron- or astrocyte-derived estradiol in the plasticity of the GnRH neuron population and offer a promising new direction for the treatment of reproductive disorders stemming from GnRH deficiency.

overall_design: RNA-Seq profiling of the preoptic area in male control or dnFGFR mice that were housed with a same or opposite sex partner until PND320.

first: Tyler

middle: N

last: Akonom

first: Mary

last: Allen


title: DYRK1A Inhibition via Harmine or via Conditional Knockout

city: Seattle,

first: Stephanie

last: Osmond

state: WA

address: 1201 9th Ave

institute: Benaroya Research Institute

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


title: Elicitor specific mechanisms of defense priming in oak seedlings against powdery mildew

city: Birmingham

first: Marco

last: Catoni

address: Edgbaston

department: School of Biosciences

institute: University of Birmingham

summary: Defense priming sensitises plant defenses to enable a faster and stronger response to subsequent stress. Various chemicals can trigger priming, however the response remains unexplored in oak. Following treatment with salicylic acid (SA), jasmonic acid (JA), or β-aminobutyric acid (BABA), oak (Quercus robur) seedlings were infected with oak powdery mildew (Erysiphe alphitoides, PM). Whilst JA increased susceptibility to PM, BABA and SA enhanced resistance by priming callose deposition and SA-dependent gene expression, respectively. All three treatments had no impact on growth. To characterise molecular markers of priming, untargeted transcriptome and metabolome analyses were performed using RNAseq and LC-MS/MS. Differential gene expression analysis revealed around 2900, 1600, and 900 genes uniquely primed by each treatment BABA, SA, and JA, respectively. A limited number of enriched GO terms differentiated the three treatments. Meanwhile, metabolome analysis found roughly 340, 220, and 40 accumulated masses uniquely primed by BABA, SA, and JA, respectively. Pathway enrichment analysis linked BABA priming to alkaloids biosynthesis, whereas no specific pathways were identified for SA and JA priming.  Our results confirm the existence of chemical-induced priming in oak and putatively identify associated molecular markers.

overall_design: Examination of the trascriptome  of leaves of Quercus robur (Forestart), from plants exposed to elicitors, including β-aminobutyric (BABA), Salicylic acid (SA) and Jasmonic acid (JA), and then infected with the  powdery mildew  causal agent, Erysiphe alphitoides; along with water-treated and mock inoculated controls.

first: Estrella

last: Luna-Diez

first: Rosa

last: Sanchez Lucas


title: Role of FGF21 in cardiac hypertrophy

summary: Fibroblast Growth Factor (FGF) 21 is a hormone produced mainly by the liver but also other organs, including the heart. FGF21 analogs are used for treating obesity and metabolic syndrome in humans. In this study, we explored involvement of FGF21 in cardiac hypertrophy. Our mouse studies in wild type mice as well as in mice with either hepatocyte-specific or cardiomyocyte-specific FGF21 ablation demonstrated an interorgan liver-heart crosstalk mechanism, which is controlled by initial hepatic FGF21 release followed by induction of cardiomyocyte FGF21 expression. Conclusively, a hepato-cardiac FGF21-based signaling axis governs cardiac hypertrophy.

overall_design: To assess the gene expression changes that FGF21 stimulates in the heart over the course of cardiac hypertrophy progression, transcriptomic analysis was conducted on mouse hearts at 3 days and 2 weeks post TAC.

first: Mia

last: Sobuj


title: Single-cell RNA sequencing of digested whole lungs of mb1Cre-/-ChATfl/fl and mb1Cre+/-Chatfl/fl mice taken before and at 24h after infection with a sublethal dose of Influenza A/Puerto Rico/8/24

city: Baltimore

first: Antonio

last: Cembellin-Prieto

address: 1742 Fleet St, Apt. 102

institute: Johns Hopkins University

summary: Respiratory viral infections can lead to dysregulated inflammatory responses in the lungs. Acetylcholine (ACh), synthesized by the enzyme choline acetyltransferase (ChAT), is a well-known neurotransmitter but has also been proposed as an immunomodulator and a potential mediator of inflammation. Our findings demonstrate that ACh treatment inhibits lung macrophage activation in vitro and intranasal ACh administration 12h prior to infection with influenza A/Puerto Rico/8/34 reduces lung inflammation in vivo in mice 24h following influenza virus infection. Furthermore, we identified B cells as the primary source of ChAT in mice, both under steady-state conditions and during the first 7 days post influenza infection. Based on these findings, we hypothesized that B cell-derived ACh regulates lung innate immunity.  To investigate this, we performed single-cell RNA sequencing on 4 samples using the Standard 3' 10X Chromium platform on >85% viable lung single-cell suspensions from 1 sample each of control (mb1Cre-/-Chatfl/fl) and ChATBKO (mb1Cre+/-Chatfl/fl) mice, collected before infection and 24 hours post-infection. Each sample (n=4) contained cells pooled from 2 mouse lungs.  Our analysis identified 15 distinct cell clusters of lung cells. Mice lacking B cell-derived ACh showed transcriptional changes among lung interstitial macrophages but not alveolar macrophages, specifically affecting TNF-α signaling via NF-κB and apoptosis pathways.

overall_design: Prior to and following intranasal influenza A/PR8 infection, live lung cell suspensions were isolated using fluorescently activated cell sorting (FACS) (>85%) with propidium iodide to distinguish live and dead cells. The isolated cells were then submitted for library preparation and Standard 3' Chromium single-cell RNA sequencing technology.  Four different samples were compared:  S1 WT (mb1Cre-/-Chatfl/fl) non-infected S3 ChATBKO (mb1Cre+/-Chatfl/fl) non-infected S5 WT (mb1Cre-/-Chatfl/fl) 24h post-infection S7 ChATBKO (mb1Cre+/-Chatfl/fl) 24h post-infection  Single cell suspension of lung parenchyma from 2 female,12 wk-old mice were pooled together for each sample

first: Zheng

last: Luo

first: Nicole

last: Baumgarth


title: Transcription-coupled AID Deamination Damage Depends on ELOF1-associated RNA Polymerase II [PRO-seq]

first: Fei-Long

last: Meng

overall_design: CH12F3 cells were stimultated with CIT for 1.5 days, treat with/w.o. NVP-2, or irradiated with UV, then subjected to PRO-seq

first: Pengfei

last: Dai

first: Yifeng


title: Continuous expression of TOX safeguards exhausted CD8 T cell epigenetic fate [coassay_TOXKO_LCMV_Arm_rechalle]

first: E

last: WHERRY

summary: CD8 T cell exhaustion is a major barrier limiting anti-tumor therapy. Though checkpoint blockade temporarily improves exhausted CD8 T cell (Tex) function, the underlying epigenetic landscape of Tex remains largely unchanged, preventing their durable “reinvigoration.” Whereas the HMG-box transcription factor TOX has been identified as a critical initiator of Tex epigenetic programming, it remains unclear whether TOX plays an ongoing role in preserving Tex biology after cells commit to exhaustion. Here, we decoupled the role of TOX in the initiation versus maintenance of CD8 T cell exhaustion by temporally deleting TOX in established Tex cells. Induced TOX ablation in committed Tex resulted in apoptotic-driven loss of Tex, reduced expression of inhibitory receptors including PD-1, and a pronounced decrease in terminally differentiated subsets of Tex cells. Simultaneous gene expression and epigenetic profiling revealed a critical role for TOX in ensuring ongoing chromatin accessibility and transcriptional patterns for key Tex gene modules in committed Tex cells. Moreover, when exposed to strong effector-differentiation signals, established Tex in which TOX had been deleted acquired an altered chromatin landscape with increased accessibility at cytotoxic genes typically accessible in Teff cells, undergoing partial reprogramming into a more functional state. Together, these findings suggest that continuous TOX expression in established Tex functions as a durable epigenetic barrier to reinforce the Tex developmental fate by simultaneously maintaining Tex epigenetic commitment while restraining effector reprogramming. Manipulation of TOX even after Tex establishment could therefore provide a therapeutic opportunity to rewire Tex biology in settings of chronic infection or cancer

overall_design: After inducing TOX ko in T exhausted cells,after 11days adoptively transferred WT and TOX iKO Tex into new recipient mice. We then rechallenged these mice with LCMV Armstrong (Arm).In parallel,  adoptively transferred naïve P14 cells as a benchmark. After LCMV Armstrong infection rechallenge (8 days post infection), similar proportions of WT and TOX iKO T exhausted cells were used for scRNA and scATAC coassay.

first: Yinghui

middle: J

last: Huang

last: Wherry


first: Pier Giuseppe

last: Pelicci

overall_design: Refer to individual Series

first: Maria

last: Boggio Merlo

last: Mallardo

first: Lucilla

last: Luzi

first: Pier

middle: G

first: Emanuela

last: Colombo


title: Proline metabolic reprogramming modulates cardiac remodeling induced by pressure overload in the heart

city: Hangzhou

first: Qingbo

last: Lv

state: Zhejiang

address: 3 East Qingchun Road

institute: Sir Run Run Hospital, School of Medicine, Zhejiang University

summary: Metabolic reprogramming is critical in the onset of pressure overload-induced cardiac remodeling. Our study reveals that proline dehydrogenase (PRODH), the key enzyme in proline metabolism, reprograms cardiomyocyte metabolism to protect against cardiac remodeling. We induced cardiac remodeling using transverse aortic constriction (TAC) in  both cardiac-specific PRODH knockout and overexpression mice. Our results indicate that PRODH expression is suppressed post-TAC. Cardiac-specific PRODH knockout mice exhibited worsened cardiac dysfunction, while mice with PRODH overexpression demonstrated a protective effect. Additionally, we simulated cardiomyocyte hypertrophy in vitro using neonatal rat ventricular myocytes treated with phenylephrine. Through RNA sequencing, metabolomics, and metabolic flux analysis, we elucidated that PRODH overexpression in cardiomyocytes redirects proline catabolism to replenish tricarboxylic acid (TCA) cycle intermediates, enhance energy production, and restore glutathione redox balance. In summary, our findings suggest PRODH as a modulator of cardiac bioenergetics and redox homeostasis duing cardiac remodeling induced by pressure overload. This highlights the potential of PRODH as a therapeutic target for cardiac remodeling.

overall_design:
To investigate the role of PRODH in the regulation of pressure overload-induced cardiac remodeling, cardiac-specific overexpression of PRODH was achieved through AAV9-mediated delivery system.
Two types of viruses, AAV9-Vector and AAV9-PRODH, were injected into mice, and transverse aortic constriction (TAC) surgery was performed. Four weeks post-surgery, cardiac tissues were collected for RNA-seq analysis.
The primary aim of our study was to compare the changes between the TAC group and the Sham group in AAV9-Vector mice, as well as to assess the transcriptomic differences between the AAV9-Vector and AAV9-PRODH groups in mice following TAC surgery.

first: Wenbin

last: Zhang


title: Transcription-Replication conflicts are linked to histone H3K79 methylation and R-loop dependent nucleosome eviction

city: Munich

first: Stephan

last: Hamperl

state: Bayern

address: Feodor-Lynen-Strasse 21

department: Institute of Epigenetics and Stem Cells

institute: Helmholtz-Zentrum Munich


title: Clonal expansion of T regulatory cells in rejecting versus tolerant mouse lung grafts

city: Saint Louis

first: Daniel

last: Kreisel

state: Missouri

address: 660 S Euclid Ave

department: Surgery

institute: Washington University

summary: It has been previously shown that T regulatory cells play a key role in regulating local immune responses within lung grafts after transplantation to promote tolerance. However, it remains unknown whether T regulatory cells within the graft undergo clonal expansion after transplantation. In this study, we examine the T cell receptor repertoire of T regulatory cells within the local graft and also within the recipient spleen.

overall_design: Transplanted left lungs and recipient spleens from four mice were collected 7 days following transplantation. Two samples were collected at a time and pooled for further processing. Lung tissue was digested, and single-cell suspensions were prepared as previously described (PMID: 28615357). Cells were stained using DAPI (BD Biosciences, #564907) and were sorted by flow cytometry for CD45+CD4+Foxp3+ Tregs into 250 μL cell resuspension buffer (0.04% BSA in PBS). Collected cells were centrifuged (300 rcf for 5 min at 4 °C) and resuspended in collection buffer to a target concentration of 1,000 cells/μL. Cells were counted on a hemocytometer before proceeding. Single-cell suspensions of sorted lung and spleen Tregs were submitted to the Genome Technology Access Center core facility (Washington University in St. Louis) for single-cell GEM construction and cDNA synthesis and library construction. Two sets of hashtagged samples were submitted as follows: tolerant lung CD4+, tolerant lung Treg, tolerant spleen Treg, rejecting lung Treg. Sorted cell types were labeled using a TotalSeq anti-mouse hashtag antibody (Biolegend, #155861, #155863, #155865, #155807) prior to pooling for sample submission. Samples were processed using the Chromium Single Cell 3′ Library & Gel Bead Kit (10X Genomics, v3) and Chromium Single Cell V(D)J Reagent Kits (10x Genomics) following manufacturer’s protocols. The libraries were sequenced on NovaSeq S4 (Illumina) targeting 50,000 reads per cell and 500 million read pairs per library. Cells were aligned to the mouse mm10-2020-A transcriptome using CellRanger (10× Genomics, v6.1.1) to generate feature-barcoded count matrices.

first: Hailey

last: Shepherd

first: Wenjun

last: Li

first: Yun

middle: Z

last: Bai


summary:
Purpose: Analysis of downstream pathways activated by NPMc+ and Flt3-ITD oncogenes in preleukemic hematopoietic stem cells (HSCs).
Methods: RNA extracted and processed from MxCRE, Flt3-ITD and NPMc+/Flt3-ITD FACS sorted HSCs
Results: These data demonstrate that NPMc+ expression in Flt3-ITD HSCs promote a transcriptional program that supports both self-renewal and quiescence.

overall_design: 3 LT-HSC samples have been sequenced (Mx, Flt3-ITD, NPMc+/Flt3-ITD). Biological replicates is provided. Mx samples should be considered the reference control.


title: Reduced function of the adaptor SH2B3 promotes T1D via altered gc cytokine-regulated, T cell intrinsic immune tolerance

status: Public on Feb 27 2025

city: Seattle

first: Kimberly

last: Dill-McFarland

address: 750 Republican St

department: Allergy and Infectious Diseases

institute: University of Washington

summary: Genome-wide assocation studie have identified SH2B3 as an important non-MHC gene for islet autoimmunity and type 1 diabetes (T1D). In this study, we found a single haplotype strongly associated with risk for human T1D that includes the sinlge nucleotide variant rs3184505*T in SH2B3. To better characterize the role of SH2B3 in T1D, we used moouse modeling and found a T cell-intrisic role for SH2B3 regulating peripheral tolerance. SH2B3 deficiency had minimal effect on TCR signaling or. proliferation across antigen doses, yet enhance cell survival and cytokine signaling including common gamma chain-dependent and interferon-gamma receptor signaling. SH2B3 deficient CD8+T cells showed augmented STAT5-Myc and effector function gene expression partially reversed when blocking autocrine IL-2 in culture. Using the RIP-mOVA model, we found T cells lacking SH2B3 promoted early islet destruction and diasbetes without requiring CD4+ T cell help. SH2B3-deficient cells demontrated increased survival post-transfer compared to control cells despite a similar proliferation profile in the same host. Next we created a spontatneous NOD.Sh2b3-/- mouse model and found markedly increased incidence and accelerated T1D across genders. Collectively, these studies identify SH2B3 as a critical mediator of peripheral T cell tolerance lmiting the T cell response to self-antigens.

overall_design: mRNA transcripts from resting naive OT-I transgenic T cells from Sh2b3+/+ versus Sh2b3-/- mice at early time points (0, 2 and 6 hours post-TCR stimulation) or late time points ( 24-hours) with or without blocking IL-2 antibody.

first: Taylor

last: Watson

first: Aaron

last: Rosen

first: Travis

last: Drow

first: Jacbo

last: Medjo

first: Matthew

last: MacQuivey

first: Yan

last: Ge

first: H

last: Liggett

first: Dane

last: Grosvenor

last: Altman

first: Patrick

last: Concannon

first: Jane

last: Buckner

first: David

last: Rawling

first: Eric

last: Allenspach


title: An arrayed CRISPR screen identifies knockout combinations improving antibody productivity in HEK293 cells

city: South San Francisco

first: Ittai

middle: Ethan

last: Eres

state: CA

address: 750 Gateway Boulevard

department: CRADI

institute: Amgen

summary: Mammalian cells are used to express complex biologics such as multispecific antibodies. While multispecifics enable promising new strategies for treating human disease, their production at high expression titer and purity can be challenging. To understand how cells respond to antibody and multispecific expression, five molecules were selected for bulk RNA-seq early after transfection of a HEK293 host. All five molecules shared a differential expression signature of secretory and protein folding stress, but this signature was stronger for molecules with low titer. We then designed an arrayed CRISPR knockout screen of 206 differentially-expressed target genes and 223 literature-motivated targets to identify knockouts that impact antibody productivity. Eight novel knockout targets were identified that increased expression titers by 20 to 80%. Notably, seven of these top eight hits were from the differentially- expressed set of candidate-gene knockouts. The top knockout target, HIST2H3C, showed evidence for additivity with five other hits, including a knockout combination that increased the titer of a difficult-to-express antibody by up to 100%. Findings for both HIST2H3C and INHBE knockout targets generalized to an alternate HEK293 host expressing an additional antibody and a multispecific with no meaningful impact on product purity. Thus, we propose HIST2H3C and INHBE disruption to be a promising and novel strategy for host-cell engineering to improve antibody and multispecific productivity.

overall_design: Five different molecules were selected for transfection into HEK293-6E cells, with 4 replicats of each treatment molecule condition. Bulk RNA was extracted two days after transfection to investigate RNA expression changes relative to empty vector control.

first: Eric Edward

last: Bryant

first: Danyang

last: Gong

first: Cai

last: Guo

first: Fernando

last: Garces

first: René

last: Hubert

first: Irwin

last: Chen


title: Systemic 4-1BB stimulation augments extrafollicular memory B cell formation and recall responses during Plasmodium infection [scATAC-seq]

city: NORTH CHICAGO

first: Rahul

last: Vijay

state: IL

address: 3333 GreenBay Road, BSB 2.450

department: Microbiology and Immunology

institute: Rosalind Franklin University

summary: T-dependent germinal center (GC) output, comprising plasma cells (PC) and memory B cells (MBC), is crucial for the clearance of Plasmodium infection and protection against reinfection. In this study, we examined the effect of an agonistic antibody targeting 4-1BB (CD137), a member of the Tumor Necrosis Factor Receptor Superfamily (TNFRSF), during experimental malaria. We found that exogenous 4-1BB stimulation dramatically enhanced humoral immune memory and protection from reinfection, despite delaying the effector GC response. Although fewer in number, single cell RNA and ATAC sequencing of MBCs from mice that received 4-1BB stimulation revealed clusters with a transcriptional and epigenetic signature indicative of superior recall and proliferative potential. Importantly, our results indicate that these effects are independent of parasite load or the inflammatory milieu but are dependent on IL-9R signaling in B cells. Our study proposes an immunomodulatory approach to enhance the quality of the MBC pool, providing superior protection during infection and vaccination, particularly in the context of malaria.

overall_design: C57BL/6 mice were infected with Plasmodium yoelii and treated with 3H3 (4-1BB agonist antibody) or isotype control on days 5 and 7 post infection. On 38 days post infection, the spleens were harvested and MSP-1 specific (B220+IgD-GL7-CD38+Decoy-MSP1+) and polyclonal (B220+IgD-GL7-CD38+) memory B cells were flow sorted. Nuclei were isolated and transposition, barcoding on the 10X Chromium (10X Genomics) and library construction were performed according to the manufacturer’s protocol for Chromium Next GEM Single Cell ATAC Reagents Kits v2 (10X Genomics).

first: Carolina

last: Caloba

first: Alexandria

last: Sturtz

first: Taylor

last: Lyons

first: Lijo

last: John

first: Akshaya

last: Ramachandran

first: Allen

middle: M

last: Minns

first: Anthony

last: Cannon

first: Justin

middle: P

last: Whalley

first: Tania

last: Watts

first: Mark

last: Kaplan

first: Scott

last: Lindner