title: Promoter mutagenesis and a massively parallel reporter screen of the MAPT locus identifies cis-regulatory elements and genetic variation effects [BAC MPRA]

city: Huntsville

first: Jesse

middle: Nicholas

last: Cochran

state: AL

address: 601 Genome Way

institute: HudsonAlpha Institute for Biotechnology

summary: Tau neurofibrillary tangles are a hallmark of several neurodegenerative diseases called tauopathies, including frontotemporal dementia and Alzheimer's Disease. Ongoing clinical trials for tauopathies seek to reduce Tau in the brain through immunotherapy, antisense oligonucleotides, and siRNA. MAPT codes for Tau, therefore understanding how the MAPT gene is regulated and the effect of genetic variation at its regulatory elements is likely to have high relevance for tauopathies. We screened a ~3 Mb region including the MAPT locus using 2 different massively parallel reporter assay (MPRA) strategies in KOLF2.1J h-NGN2 neurons and HEK293FT cells, identifying previously unannotated cis-regulatory elements (CREs). Using CRISPR interference (CRISPRi) in mixed neuron cultures, we identified a new CRE for MAPT, as well as 2 CREs for another nearby gene of interest, KANSL1. Known genetic variation from the Alzheimer's Disease sequencing project was tested in a separate MPRA at the top CREs near the MAPT gene, identifying variants with altered regulatory effects including those at previously identified CREs for MAPT. Using a saturation mutagenesis screen of a 2,000 bp region encompassing the MAPT promoter, we assessed regulatory effects of each possible single nucleotide variant in this region. We identified several neuron-specific regulatory variant effects at this region, including a high confidence binding site for the transcription factors EGR2, ZBTB14 and TCLF5 at a region of high MPRA activity and genetic conservation.

overall_design: LentiMPRA in KOLF2.1h-NGN2 Neurons (transduced on day 14, collected on day 18) and HEK293FT cells using sheared BACs as test sequences

first: Rebecca

last: Hauser

first: Nicholas


title: Promoter mutagenesis and a massively parallel reporter screen of the MAPT locus identifies cis-regulatory elements and genetic variation effects [Variation and Saturation Mutagenesis MPRA]

status: Public on Mar 18 2026

country: USA

overall_design: LentiMPRA in KOLF2.1h-NGN2 Neurons (transduced on day 14, collected on day 18) and HEK293FT cells testing known genetic variation at proximal CREs for MAPT as well as a saturation mutagenesis of the MAPT promoter

middle: M


title: Dendritic cell redundancy enables priming of anti-tumor CD4 T cells in pancreatic cancer

city: Boston

first: Chong

last: Zuo

address: 450 Brookline Ave

institute: Dana Farber Cancer Institute

type: Expression profiling by high throughput sequencing

summary: Pancreatic ductal adenocarcinoma (PDAC) is resistant to current immunotherapies and lacks effective CD8 T cell responses, which is potentially due to a paucity of cross-presenting dendritic cells (cDC1s). Here, we combine an innate immune adjuvant with anti-CTLA-4 and anti-PD-1 to achieve durable remissions and immunologic memory in multiple mouse models of poorly immunogenic PDAC. Tumor regression does not depend on CD8 T cells or tumor cell MHC class I expression but instead requires IFNγ-producing CD4 T cells (Th1s) that were primed by dendritic cells in lymph nodes. Combination immunotherapy induces an influx of activated cDC2s carrying tumor antigen into tumor-draining lymph nodes; cDC2s are required for orthotopic tumor clearance. Intratumoral CD4 T cells and cDC2s remain present in treatment-naïve and chemotherapy-exposed human PDAC. Even in chemotherapy-exposed patients’ blood, cDC2s outnumber cDC1s by ten-fold. Therefore, therapeutic targeting of the cDC2-CD4 T cell axis could be efficacious in pancreatic cancer.

overall_design:
Murine scRNAseq tissue preparation: Mice bearing ZsGreen-expressing 6694c2 tumors were treated as described above. 48 hours after treatment, tumors, spleens, and tumor-draining lymph nodes were harvested from mice. Spleens and lymph nodes were macerated. Tumors were macerated and digested in collagenase IV (Sigma #C5138) and soybean trypsin inhibitor (Life Technologies #17075029). Tumor chunks were removed via filtration with a 40 μM filter, and the resulting single cell suspension was washed with PBS. Part of the tumor, lymph node, and spleen sample was taken for flow cytometry analysis. The rest of the samples were hashed, sorted based on ZsGreen expression on a BD FACS Aria, and analyzed via single-cell sequencing analysis.
Bulk RNA sequencing: 6694c2 and B16F10 tumor cells were treated with IFNγ (100 ng/mL) (Peprotech #315-05), IFNβ (1000 U/mL) (BioLegend #581302), and STING agonist (225 ug/mL) in culture for 24 hrs. Cells were harvested and RNA isolated with a QIAGEN RNAeasy Plus Mini Kit (#74134). RNA was sent to Genewiz for paired-end RNA sequencing. In brief, FASTQ files were put through quality control (fastqc 0.11.9 & multiQC v1.5 for collating fastqc results), adaptor trimming (cutadapt v.1.14), genomic alignment (STAR v2.7.9a), and read count analyses (Rsubread) via the Harvard O2 cluster. Filtering for genes with less than 10 reads combined across samples was conducted, and analysis of differential gene expression was conducted with DESeq293. Plots were generated with ggplot2 in R.

first: Stephanie

last: Dougan

first: Courtney

last: Kureshi


title: CRISPR-Cas12a2

city: Salt Lake City

first: Emily

middle: Rose

last: Wilson

state: UT

address: 2000 Circle of Hope

department: Oncological Sciences

institute: Huntsman Cancer Institute

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

sample_organism:
Escherichia coli
Homo sapiens

platform_organism:
Escherichia coli
Homo sapiens

overall_design: Refer to individual Series


title: m6Am methyltransferase PCIF1 regulates osteogenesis via WNT signaling

city: Chengdu

first: Quan

last: Yuan

address: No.14, 3Rd Section of Ren Min Nan Road, Sichuan

country: China

institute: West China Hospital of Stomatology, Sichuan University

summary: Mesenchymal stem cells (MSCs) were isolated from Prrx1-Cre;Pcif1fl/fl mice and Pcif1fl/fl littermates and cultured in osteogenic medium for 7 days to investigate the role of Pcif1 in osteogenic differentiation. Total RNA was extracted using Trizol reagent and purified with poly-T oligo-attached magnetic beads. Sequencing libraries were prepared and sequenced on an Illumina PE150 platform. Raw reads were processed and mapped to the Mus musculus reference genome (mm10) using HISAT2. Differential gene expression analysis was performed with the DESeq2 R package using thresholds of |fold change| ≥ 2 and adjusted p-value ≤ 0.05. Gene Ontology (GO) and KEGG pathway enrichment analyses were conducted using the clusterProfiler R package to identify biological processes and pathways affected by Pcif1 deletion during osteogenic differentiation. This dataset provides insights into the transcriptional programs regulated by Pcif1 in MSC osteogenesis.

overall_design: Gene expression profiling of MSCs from Prrx1-Cre;Pcif1fl/fl and Pcif1fl/fl mice during osteogenic differentiation. Samples were collected in biological duplicates after 7 days of culture in osteogenic medium. RNA-seq libraries were prepared and sequenced using Illumina PE150.

first: Wei

last: Song

first: Qi

last: Yin


title: Identifying clones in myelodysplastic syndromes by using single-cell RNA sequencing

status: Public on Mar 21 2026

city: Be'er Sheva

first: Roy

last: Pollak

address: David Ben Gurion Blvd 1

department: Life sciences

country: Israel

institute: Ben Gurion University

summary: We employed single-cell RNA sequencing to sequence hematopoietic stem cells (HSC) derived from bone marrow aspirations of seven patients with myelodysplastic syndrome (MDS) and four control subjects. Based on gene expression and pathway-enrichment analyses, we inferred chromosomal Y deletion and characterized the transcriptome of chromosome Y-deleted HSC in comparison to chromosomally intact HSC. Our results support the premise that chromosome Y deletion is not a benign aging phenomenon and highlight the importance of upregulation of the anti-apoptotic pathway in the pathogenesis of MDS.

overall_design: Eleven subjects who were evaluated for MDS were recruited to the study. Fresh bone marrow aspirations were collected and sorted for HSC by using fluorescence-activated cell sorting (FACS). The Smart-Seq2 protocol was applied to sequence 1,150 HSC. Reads were mapped to the human genome, and gene expression levels were calculated. After application of quality control measures, the expression matrix included 1,101 cells and 26,199 genes.

first: Hadas

last: Ner-Gaon

first: Leonid

last: Olender

first: Itai

last: Levi

first: Roi

last: Gazit

first: Tal

last: Shay

first: Uri

last: Greenbaum


title: Developmental gene expression patterns driving species-specific cortical features

city: Geneva

first: Awais

last: Javed

address: Chemin des mines, 9

department: Fundamental Neurosciences

country: Switzerland

institute: University of Geneva

type: Genome binding/occupancy profiling by high throughput sequencing

summary: The cerebral cortex shows species-specific variations in size and organization, likely accounting for distinct behavioral abilities. These structural differences may reflect evolutionary changes in the developmental expression of shared genes. To investigate this possibility, we used machine vision to identify and compare cell-type-specific gene expression patterns in the developing mouse and human neocortex, and in human cortical organoids. This identified genes with evolutionary conserved/divergent transcriptional regulation, revealing species-specific cyto-temporal gene expression patterns. Among such genes, the transcription factor JUNB showed mutually exclusive expression in human progenitors and mouse neurons. Through cell-type-specific gain- and loss-of-function experiments in mice and human organoids, we show that JUNB bidirectionally controls human cortical features, including progenitor proliferation rates, neuronal production timing, and total neuronal output. We identify IRF1 as a human RG-specific regulator that, when expressed in mouse RG, activates JUNB and recruits human-like gene regulatory networks, demonstrating cross-species activation of poised developmental programs. Together, these findings reveal how cyto-temporal regulation of shared genes drives species-specific cortical features and provides a molecular framework to understand and manipulate these evolutionary programs.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: Single nucleus/cell RNA sequencing with or without TrackerSeq barcoding was performed on embryonic mouse cortex electroporated samples with control, hJUNB, hIRF1, hEGR1. CUT&RUN/Tag assays were performed on embryonic mouse cortex electroporated samples with control or hJUNB overexpression and iPSC-derived human cortical organoids.

first: Lucia

last: Gomez

first: Veronica

last: Pravata

first: Moein

last: Sarhadi

first: Quentin

last: Lo Giudice

first: Timea

last: Szalai

first: Lea

last: Aubert

first: Theo

last: Ribierre

first: Laurent

last: Nguyen

first: Silvia

last: Capello

first: Denis

last: Jabaudon

first: Esther

last: Klingler


title: Light and excessive CO2 uptake inhibit growth of Synechococcus sp. PCC 7002 under very high CO2 levels

city: Helsingør

first: Niels-Ulrik

last: Frigaard

address: Strandpromenaden 5

department: Department of Biology

country: Denmark

institute: University of Copenhagen

summary: Cultivation of photosynthetic microbes under elevated CO2 is widely used in biotechnology to enhance growth. However, very high CO2 levels inhibit growth for reasons that are not always clear. We established a method that allowed cultivation under different CO2 concentrations and controlled pH to study the cyanobacterium Synechococcus sp. PCC 7002. Growth rates, cell composition, and transcriptomic profiles were investigated under limiting (0.04%), optimal (1-4%), and inhibitory high (15-30%) gas phase CO2 contents, while maintaining pH approx. 7.5. At intermediate light (200 µmol /m2 /s), the growth rate at 30% CO2 was approx. 45% of that under optimal conditions. Under low light (<100 µmol /m2 /s), growth was less inhibited by high CO2 than at higher light intensities. Pigmentation and photosynthesis likewise decreased under high CO2, except that carotenoids indicative of high-light stress increased. Transcriptome sequencing confirmed that the photosynthesis machinery was downregulated and stress responses were upregulated under high CO2. Genetic knockdown of CO2 uptake (ndhF3 and ndhF4) or overexpression of a heterologous Na+/H+ antiporter (nhaS3) improved tolerance to high CO2, while knockdown of the CcmR transcriptional regulator reduced it. We conclude that the inhibiting species under very high inorganic carbon conditions was CO2, and not HCO3-, and that reduced light intensity or genetically suppressing active CO2 uptake improved the tolerance of Synechococcus to very high CO2 conditions. This work provides a basis for cultivation under a very wide range of CO2 concentrations, thereby broadening the scope for metabolic manipulation and biotechnological applications of cyanobacteria. (Algal Research)

overall_design: We performed targeted genetic manipulation (CRISPR interference knockdown of CO2 uptake (ndhF3 and ndhF4) and overexpression of a heterologous Na+/H+ antiporter (nhaS3)) in Synechococcus sp. PCC 7002. Transcriptome sequencing was performed to understand the mechanisms of CO2 toxicity and improve cellular tolerance to very high CO2 concentrations.

first: Carrasquer-Alvarez

last: Elena

first: Sven Geissler

last: Adrian

first: Abraham

last: Joshua Papa

first: Gorodkin

last: Jan

first: Pfleger

last: Brian Frederick

first: Seemann

last: Stefan Ernst

first: Frigaard

last: Niels-Ulrik


title: NK cells promote cardiac cell death and regulate myelopoiesis in myocardial infarction

city: Paris

first: Jean-Sébastien

last: Hulot

address: Rue Leblanc

country: France

institute: Paris Centre de Recherche Cardiovasculaire

summary: Ischemic heart failure remains a major clinical challenge, underscoring the need to better understand post-infarction immune mechanisms and identify new therapeutic targets. Both innate and adaptive immunity contribute to adverse cardiac remodeling following myocardial infarction (MI), yet the role of cytotoxic cells such as natural killer (NK) cells remains poorly defined. Here, we show that after acute MI in mice, NK cells are recruited to the ischemic myocardium in a CCR2-dependent manner and become activated. Activated NK cells locally release granzyme B, promoting cardiomyocyte apoptosis, adverse ventricular remodeling, and impaired cardiac function. Genetic deletion or pharmacological depletion of NK cells reduces cardiomyocyte death, attenuates inflammation, limits myocardial injury, and improves cardiac function. In contrast, NK cell activation using an anti-NKG2A monoclonal antibody exacerbates ischemic heart failure. We further demonstrate that NK cells regulate bone marrow myelopoiesis through local GM-CSF production. Finally, we identify a distinct NK cellular and transcriptomic signature in human ischemic heart tissue at early stages. Together, these findings reveal a detrimental role for NK cells following acute MI and highlight NK cells as potential therapeutic targets to limit adverse cardiac remodeling.

overall_design: We performed transcriptomic analysis of human heart biopsies using the nCounter Human Organ Transplant Panel from Nanostring (770 genes) to compare early MI (< 14 days post-MI) and late MI (> 3 months port-MI) against healthy heart tissue.

first: Orianne

last: Domenge

first: Marine

last: Livrozet

first: Alexandre

last: Loupy

first: Marc

last: Diedisheim

first: Hafid

last: Ait-Oufella


title: RNA-seq of human articular chondrocytes after exposure to BMP7-derived peptide p[63-82].

city: maastricht

first: Marjolein

last: Caron

department: Orthopedic Surgery

country: Netherlands

institute: Maastricht University

summary: Osteoarthritis is a degenerative joint disease that can currently only be treated by alleviating pain or, ultimately, by a total joint replacement. The bone morphogenetic protein 7-derived peptide p[63-82] offers a potentially novel molecular treatment option for osteoarthritis. Mapping peptide-induced transcriptomic changes in human articular chondrocytes is expected to help reveal how p[63-82] confers its chondrocyte phenotype modulating bioactivity. To this end,  human articular chondrocytes were isolated from the articular cartilage of patients with late-stage osteoarthritis undergoing total knee arthroplasty (METC approved). Cells were stimulated with 100 nM p[63-82] peptide for 24 hours (or vehicle), after which RNA was isolated, and RNAseq was performed. We found a total of 1976 differentially expressed genes. The p[63-82] peptide induces profound transcriptomic changes in primary human articular chondrocytes. One of the dominant transcriptomic alterations was the differential expression of several collagen-related genes. Many of these collagen-related genes have established roles in cartilage homeostasis. The here-acquired transcriptomic insights provide clues into the therapeutic mechanisms of action of the p[63-82] peptide in human articular chondrocytes.

first: Jessica

last: Steijns

first: Tim

last: Welting


title: Genome-wide CRISPR sgRNA loss-of-function screening in PRCC-TFE3 inducible HK-2 cells

city: Kumamoto

first: Masaya

last: Baba

state: Kumamoto

address: 1-1-1 Honjo, Chuo-ku

department: Urology

country: Japan

institute: Kumamoto University

summary: PRCC-TFE3 is an oncogenic chimeric transcription factor that drives TFE3-rearranged renal cell carcinoma. Although PRCC-TFE3 can trigger either oncogene-induced senescence (OIS) or tumor progression depending on the cellular context, both phenotypes are fundamentally dependent on its transcriptional function. This study aims to identify genetic factors essential for the transcriptional activity and functional output of PRCC-TFE3. Using a doxycycline (Dox)-inducible PRCC-TFE3 HK-2 cell model where the fusion protein induces robust OIS, we performed a genome-wide CRISPR/Cas9 knockout screen. By identifying genes whose loss alleviates this OIS phenotype, we sought to uncover key regulators required for PRCC-TFE3-mediated transcriptional programs.

overall_design: A genome-wide CRISPR/Cas9 screen was conducted using the Brunello library in two independent experimental runs performed at different times. Each run consisted of a paired comparison between PRCC-TFE3 non-induced (Dox−) and induced (Dox+) conditions in HK-2 cells. Run 1: Pair of Doxy-_1 (control) and Doxy+_1 (PRCC-TFE3 induced). Run 2: Pair of Doxy-_2 (control) and Doxy+_2 (PRCC-TFE3 induced). Because PRCC-TFE3 induction in this cell model enforces OIS via its transcriptional activity, loss of genes required for PRCC-TFE3 function allows cells to bypass senescence and gain a growth advantage. sgRNA abundance was quantified by NGS, and differential enrichment between the paired populations was analyzed using the MAGeCK pipeline to identify essential mediators of PRCC-TFE3-mediated functional programs.

first: Hidekazu

last: Nishizawa

first: Shintaro

last: Funasaki

first: Shoichiro

last: Kuroda


title: RNA-seq analysis of PRCC-TFE3 dependent transcriptional programs and their modulation by MSC2530818 in HK-2 cells.

summary: PRCC-TFE3 is an oncogenic chimeric transcription factor identified in TFE3-rearranged renal cell carcinoma. To characterize PRCC-TFE3–dependent transcriptional programs and their regulation by the Mediator kinase module, we performed RNA sequencing in a doxycycline-inducible PRCC-TFE3 expression system. HK-2 cells expressing HA-tagged PRCC-TFE3 under doxycycline control were cultured under four conditions defined by the presence or absence of doxycycline and the CDK8/19 inhibitor MSC2530818. RNA-seq was conducted to assess global gene expression changes associated with PRCC-TFE3 induction and CDK8/19 inhibition. These data provide a resource for analyzing PRCC-TFE3–driven transcriptional reprogramming and its modulation by the Cyclin C–CDK8/19 Mediator kinase module.

overall_design: PRCC-TFE3 doxycycline (Dox)-inducible HK-2 cells were cultured for 48 h under four conditions defined by the presence or absence of Dox and the CDK8/19 inhibitor MSC2530818 (100 nM). Three independent biological replicates were analyzed for each condition.


title: A novel truncating variant c.1222dupC in RBM20 causes cardiomyopathy consistent with haploinsufficiency

city: Heidelberg

first: Maarten

last: van den Hoogenhof

address: Eppelheimer Str. 8,

country: Germany

institute: Institute of Experimental Cardiology

summary: RBM20 is a cardiac splicing factor responsible for splicing of several cardiac genes such as TTN, TRDN, RyR2, PDLIM1, and CAMK2D. Mutations in RBM20 are a major cause of familial dilated cardiomyopathy (DCM), and lead to missplicing of RBM20 target genes. Here, we describe a novel pathogenic truncating mutation, RBM20 c.1222dupC, identified in a patient with mitral valve prolapse and late onset familial DCM. This mutation introduces a premature termination codon and generates a truncated protein of ~55 kDa in vitro. Splicing assays demonstrated complete loss of activity and no dominant-negative effect on WT RBM20. Overexpression in NRCMs revealed that the truncated protein localized to both cytoplasm and nucleus, partially co-localizing with WT RBM20, despite lacking the RS and RRM domains. To model the patient’s condition, we generated heterozygous c.1222dupC iPSC-derived cardiomyocytes. Western blot analysis of endogenous RBM20 revealed a strong reduction in RBM20 protein level. RT-PCR revealed splicing defects in canonical RBM20 targets, and RNA-sequencing identified widespread splicing abnormalities, including in established RBM20 targets (TTN, RyR2, CAMK2D, and CACNA1G). Together, these findings establish RBM20 c.1222dupC as a pathogenic truncating variant that causes DCM primarily through haploinsufficiency.

overall_design: RNA sequencing of WT and DupC+/– hiPSC cardiomyocytes.


title: Comprehensive mass spectrometry screening-derived atlas of HDAC inhibitors reveals histone specific acetylation changes [ChIP-seq]

city: St. Louis

first: Rashmi

last: Karki

address: 660 S. Euclid Ave

institute: Washington University in St. Louis

summary: Histone deacetylases inhibitors (HDACi) have emerged as valuable therapeutics in cancer and other diseases, yet their effect on histone post-translational modification remains incompletely characterized. Here, we applied quantitative mass spectrometry and high throughput sequencing to systematically profile site-specific histone modification changes in response to a panel of HDAC inhibitors. This platform enabled mapping of histone modification changes across hundreds of sites including low abundant histone marks. Furthermore, integrative analysis of ChIP-seq and RNA-seq identified genome wide binding site for a low-abundant histone mark H2A.Z acetylation in HeLa cells and MDA-MB-231 breast cancer cells, highlighting the role of H2A.Z acetylation in regulating gene expression through various biological pathways and specific genes involved in tumor suppressor pathways. Our findings provide a functional resource for identification and quantification of epigenetic changes and transcriptional regulation of histone H2A.Z acetylation upon pharmacological perturbation

overall_design: ChIP-seq of histone H2A.Z K4+K7+K11 acetylation (H2A.Zac) in HeLa and MDA-MB-231 cells treated with Entinostat (1µM) for 24h. Controls were treated with DMSO. Two replicates were included for  each conditions.

first: Francisca

middle: N

last: Vitorino

first: Richard

last: Searfoss

first: Joanna

middle: K

last: Lempiäinen

first: Cheryl

last: Arrowsmith

first: Benjamin

middle: A

last: Garcia


title: Developmental systesms drift in dorsoventral patterning is linked to transitions to autonomous development in Annelida

city: London

first: Allan

last: Carrillo-Baltodano

address: Mile End

department: School of Biological and Behavioural Studies

country: United Kingdom

institute: Queen Mary University of London

summary: The Bone Morphogenetic Protein (BMP) pathway is the ancestral signalling defining the dorsoventral axis in bilaterally symmetrical animals. However, Spiralia, a large bilaterian clade including molluscs and annelids, uses the Fibroblast Growth Factor pathway and ERK1/2 as the ancestral cue to establish their posterodorsal side. How this profound change in axial patterning evolved and what it implied for BMP’s developmental role remains elusive. Here, we studied four annelid species and combined disruption of the BMP and Activin/Nodal pathways with transcriptomics and blastomere deletions to demonstrate that BMP is ancestrally downstream of ERK1/2 and promotes dorsoventral development in Spiralia. Importantly, this signalling hierarchy is lost in annelids that secondarily transitioned into a maternally controlled, autonomous development. While some, like Capitella teleta, use Activin/Nodal, the annelid Platynereis dumerilii relies on BMP to establish dorsoventral polarity only in the head. Unexpectedly, this divergence in upstream axial regulators implied extensive rewiring of downstream targets, as inferred by comparing C. teleta and Owenia fusiformis. Our data clarify the ancestral BMP’s axial role in Spiralia, unveiling a potential causal link between parallel shifts to autonomous cell-fate specification in early development and the emergence of developmental system drift, a pervasive yet poorly understood phenomenon in animal embryogenesis.

overall_design: We generated bulk RNA-seq in the annelid Owenia fusiformis grown in laboratory conditions,  treated with DMH1 and SB435421 (BMP and Nodal/Activin antagonists) and zBMP4 and Activin-A recombinant protein, and compared to DMSO treated control samples.

first: Allan M

first: Emmanuel

last: Hailot

first: Steffanie M

last: Meha

first: Imran

last: Luqman

first: Artenis

last: Pashaj

first: Yun-Ju

last: Lee

first: Tsai-Ming

last: Lu

first: David KE

last: Ferrier

first: Stephan Q

last: Schneider

first: Jose M

last: Martín-Durán

first: Jimena

last: Montagne


title: Exploring Mechanism of Action of Abemaciclib in Breast Cancer Through Circulating Chromatin Fragment

city: Grand Forks

first: Motoki

last: Takaku

state: ND

address: W221 Biomedical Sciences 1301 N. Columbia Road

institute: University of North Dakota

summary: Cell free DNA (cfDNA) analysis has a large potential for cancer patient diagnosis. Given the evidence that cfDNA partially preserves chromatin architecture of tumor cells, it potentially allows the detection of tumor responses after therapeutic treatment. In this study, we aimed to detect the responses of abemaciclib, a potent cyclin-dependent kinase 4 and 6 inhibitor (CDK4/6 inhibitor), in metastatic recurrent breast cancer patients through cfDNA analysis. Our results suggest that cfDNA can be used as a valuable marker to monitor cancer burden, cancer responses to drug treatment, and sensitivity against therapeutic reagents. cfDNA concentrations purified from serum are significantly higher in cancer patients compared to healthy donors, suggesting that a significant portion of the cfDNA originates from tumors in our samples. The cfDNA concentrations are also significantly decreased after abemaciclib treatment. The sequencing analysis of cfDNAs revealed a higher enrichment of cfDNAs at open chromatin regions and differentiated the cfDNA enrichment patterns between abemaciclib treated and non-treated samples. Altered cfDNA signals are frequently associated with the genes that are involved in apoptosis and CDK4/6 related pathways. Finally, the differentially enriched region analysis also detected the differences in abemaciclib cases that could be treated long-term and cases that had an exacerbation after short-term treatment. In conclusion, this research enhances our understanding of the biology of abemaciclib and provides new insights into CDK4/6 inhibitor’s action in metastatic breast cancer.

overall_design: Cell-free DNAs (cfDNAs) were isolated from metastatic luminal breast cancer patients and healthy donors. The study was designed to monitor the impact of abemaciclib using cDNAs. cfDNA enrichment patterns were analyzed at accessible chromatin regions defined by ATAC-seq. Differentially enriched regions were identified between abemaciclib-treated and non-treated samples.  Comparative analysis was performed to identify treatment-responsive genomic regions. Finally, gene ontology analyses provided insights into the biological processes affected by abemaciclib, revealing potential biomarkers for monitoring tumor response and therapeutic sensitivity.

first: Mamoru

last: Takada

first: Sakuntha

middle: D

last: Gunarathna

first: Regina

last: Nguyen

first: Muhan

last: Yu

first: Hideyuki

last: Yamada

first: Takeshi

last: Nagashima

first: Hiroshi

last: Fujimoto

first: Junta

last: Sakakibara

first: Hiroto

last: Yamamoto

first: Mayumi

last: Iida

first: Tsutomu

last: Kawaguchi

first: Otsuka

last: Masayuki


title: Single-cell sequencing reveals dynamic immune features of paraneoplastic pemphigus in a patient with follicular lymphoma

city: Shanghai

first: Yuxuan

last: Zheng

address: 825 Zhangheng Rd.

institute: Fudan University

summary: Background: Paraneoplastic pemphigus (PNP) is a highly fatal autoimmune blistering disease that commonly occurs in patients with underlying benign or malignant neoplasms. It poses significant challenges for diagnosis and treatment. To date, the cellular and molecular mechanisms underlying the pathogenesis of PNP remain largely unclear. Objective: This study aims to elucidate the cellular and molecular mechanisms of PNP, particularly when it occurs secondary to lymphoma, by analyzing the dynamic immune landscape throughout the course of treatment. Method: We performed single-cell transcriptome sequencing and single-cell T cell receptor (TCR) analysis on peripheral blood mononuclear cells (PBMCs) and bone marrow cells (BMCs) obtained from a patient with follicular lymphoma (FL) accompanied by PNP. Samples were collected at three critical time points: before treatment, during treatment, and after successful treatment. Result: Before treatment, we observed a specific enrichment of ITGAL+ T cells, abnormal γδT cell subtypes, and BCL2+ B cells in PBMCs. Notably, naïve T cells and an abnormal B cell subtype were also enriched in BMCs. The single-cell transcriptome results mapped the lymphoma-associated tumor microenvironment and revealed post-treatment immune reconstitution. This reconstitution involved activated DNA damage and T cell immune responses, which was further supported by the observation of a gradually expanded TCR clone as the treatment progressed. Conclusion: Our study delineates the dynamic immune landscape in a patient with FL-associated PNP throughout treatment, demonstrating that successful treatment correlates with immune reconstitution involving specific T cell responses and TCR clone expansion. These findings provide novel insights into the cellular and molecular mechanisms of PNP, especially in cases secondary to lymphoma.

overall_design:
To decipher the cellular and molecular characteristics and potential mechanisms of the PNP through this case, we performed single-cell multi-omics sequencing, including single-cell RNA-seq (scRNA-seq) and single T cell receptor repertoire sequencing (scTCR-seq), using PBMCs collected from four time points prior- and post-treatment (C0, C1, C2, and C4).

***************************************************************
The raw sequence data reported in this study have been deposited in the Genome Sequence Archive in National Genomics Data Center, China National Center for Bioinformation / Beijing Institute of Genomics, Chinese Academy of Sciences (GSA-Human: HRA016740) that are publicly accessible at https://ngdc.cncb.ac.cn/gsa-human.
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first: Rong

last: Wei

first: Bochao

last: Liu

first: Yun

first: Wenjing

last: Li

first: Zhiguo

last: Chen

first: Jin

last: Lu

first: Shenmiao

last: Yang


title: Intermittent Fasting Reprograms Chromatin Accessibility to Modulate Gene Expression in Brain and Muscle

city: Melbourne

first: Yibo

last: Fan

state: VIC

address: Science Dr, Bundoora VIC 3083

country: Australia

institute: La Trobe University

summary: Intermittent fasting (IF), a dietary regimen that mimics the natural feeding patterns observed across diverse organisms—from single-celled life to mammals—is thought to activate systemic survival responses and confer health benefits. However, the molecular mechanisms underlying these effects remain poorly understood. In this study, we utilized ATAC-Seq and RNA-Seq to investigate how IF influences chromatin accessibility and gene expression in brain and muscle tissues of mice, compared to ad libitum feeding. Our results reveal that IF induces significant changes in chromatin accessibility, modulating pathways related to metabolism, ribosome function, HIF-1 signaling, and glycolysis. Motif analysis identified tissue-specific transcription factors enriched in IF-regulated regions, including Sp1, Mef2a, NeuroD2, Banp, and NFIA in the brain, and SMAD4, TCF4, STAT5B, NKX3-1, and ZEB2 in muscle. Integrative analysis of ATAC-Seq and RNA-Seq data demonstrated that IF upregulates 50 genes and downregulates 15 in the cortex, while upregulating 31 genes and downregulating 10 in muscle. These gene expression changes are linked to pathways associated with neuroprotection and enhanced muscle function, offering mechanistic insights into the health benefits of IF. Our findings underscore the role of IF-induced chromatin remodeling in driving adaptive gene regulation.

overall_design: Male mice were divided into two groups: Ad Libitum (AL) and 16:8 fasting (IF) (n=8 per group). After 4 months on their respective dietary regimens, mice were sacrificed, and tissues from the cortex and muscle were harvested, prepared, and subjected to ATAC-seq and RNA-seq analyses

first: Xiangyuan

last: Peng

first: Thiruma

middle: V

last: Arumugam


title: Effect of AMP-dT on lymph node immune response in rhesus macaque

first: Martin

middle: P

last: Steinbuck

state: Massachusetts

address: 451D Street, Ste 501

department: Research

institute: Elicio Therapeutics

summary: Adjuvants are immuno-activators capable of shaping the magnitude and quality of antigen-specific immune responses induced by subunit immunization. Presently, there is an acute need for effective adjuvants that safely induce durable and balanced humoral and cellular responses. Here, we engineered a class of Amphiphile (AMP)-modified, immunostimulatory DNA-adjuvants designed for targeted delivery to lymph nodes and enhanced stimulation of TANK-binding kinase 1 (TBK1)-mediated danger-sensing pathways to generate strong adaptive immunity and long-term memory with potent recall potential. AMP-DNA adjuvants induced robust interferon type-I (IFN-I)-driven inflammatory environments in mouse and non-human primate (NHP) lymph nodes, leading to significantly enhanced cytokine secretion by polyfunctional CD8+ and CD4+ T cells in multiple tissues, as well as strongly elevated TH1-associated and neutralizing antibody responses, in the absence of systemic toxicity. These results demonstrate that AMP-modification enables lymph node-targeted DNA-adjuvants to potently activate IFN-I-signaling to generate substantial cellular and humoral responses crucial for vaccine efficacy.

overall_design: 1 group of two NHP (non-human primates) was injected with AMP-dT vaccine (SARS-CoV2-RBD with AMP-dT). Their immune responses within the draining iguinal lymph nodes at 24h and 48h post immunization were compared to baseline responses prior to immunization.

first: Lisa

last: McNeil

first: Peter

middle: C

last: DeMuth

first: Lochana

last: Seenappa


title: Epigenetic control of telomeric RNA maintains heterochromatin in telomerase-driven cancers [ChIP-Seq]

city: Saskatoon

first: Franco

middle: J

last: Vizeacoumar

state: Saskatchewan

address: 4D40 Health Sciences Building, 107 Wiggins Road

department: Oncology

country: Canada

institute: University of Saskatchewan

summary: Tumor heterogeneity presents a major clinical challenge. Reactivation of telomerase (hTERT) is a near-universal hallmark of cancer, yet direct inhibition of hTERT has shown limited therapeutic benefit. To uncover tractable telomerase-related vulnerabilities, we applied a synthetic dosage lethality (SDL) strategy to identify genes required only when hTERT is overexpressed. We performed genome-wide CRISPR/Cas9 and shRNA screens across multiple isogenic cell line pairs differing in hTERT expression. From these datasets, 100 high-confidence candidates were prioritized and validated using an arrayed in vitro CRISPR screen and a pooled in vivo CRISPR screen across diverse cancer models, non-malignant cells, and patient-derived organoids. Through this pipeline, we identified FTSJ3, an RNA 2′-O-methyltransferase, as a top SDL target of hTERT. Depletion of FTSJ3 selectively impaired the viability of hTERT-positive cancer cells while sparing normal cells. Mechanistically, FTSJ3 installs 2′-O-methylation on the telomeric RNA TERRA, a modification essential for TERRA stability and function. Loss of FTSJ3 destabilizes TERRA, disrupts recruitment of the histone methyltransferase SUV39H1, and diminishes H3K9 trimethylation and HP1 assembly at sub-telomeric regions. This breakdown of repressive telomeric chromatin leads to genome instability and apoptosis specifically in hTERT-positive cells. These findings highlight SDL as a powerful strategy for uncovering telomerase-dependent liabilities and establish FTSJ3 as a central regulator of telomere stability in telomerase-active cancers. Targeting FTSJ3 enzymatic activity offers a promising therapeutic entry point, acting upstream of TERRA to eliminate telomerase-driven malignancies selectively.

overall_design: H3K9Me3 and H3K27ac (two replicates) analysis of MIA PaCa-2 and U-2 OS cells with shFTSJ3 knockdown. One experiment, here titles H3K9Me3-1, was performed in a different run than the others, and thus uses a different input.

first: Jared

last: Price

first: Frederick

middle: S

first: Connor

last: Denomy

first: Judy

last: Wong

first: Andrew

last: Freywald


title: Effect of AMP-dT on lymph node immune response in mouse

overall_design: 3 groups of mice were injected with the following vaccines: mock control (SARS-CoV2-RBD without adjuvant), SOL dT (SARS-CoV2-RBD with dT), AMP-dT (SARS-CoV2-RBD with AMP-dT). 4 time points (2h, 6h, 24h, 72h) with 2 mice per time point.

first: Erica

last: Palmer


title: Atypical memory B cells form a pre-plasmacellular reservoir for steady-state IgD responses to common nasopharyngeal antigens

city: Barcelona

first: Júlia

last: Perera-Bel

address: Doctor Aiguader, 88

department: MARData-BU

country: Spain

institute: Hospital del Mar Research Institute

summary: The human nasopharyngeal mucosa includes organized lymphoepithelial structures continually engaged in frontline immune responses to aerodigestive antigens. Advancing our understanding of these responses might lead to the development of new strategies for the prevention and treatment of common immune disorders such as allergies. Here we identified a hitherto elusive tonsillar subset of atypical IgD class-switched IgD+IgM- memory (IgD-ME) B cells that were clonally related to IgD+IgM− germinal center (IgD-GC) B cells and IgD-secreting IgD+IgM− plasma cells (IgD-PCs) but not anergic IgD+IgM− B cells. Consistent with their pre-plasmacellular properties, IgD-ME B cells served as preferential precursors of IgD-PCs over IgD-GC B cells. IgD antibodies from IgD+IgM− cells acquired reactivity to multiple oral, airborne and commensal antigens through a mutation-dependent pathway involving both innate and adaptive signals. Thus, IgD-ME B cells may form a ready-to-use pre-plasmacellular reservoir for steady-state IgD responses likely aimed at enhancing nasopharyngeal homeostasis.

overall_design: The primary aim of this study was to identify tonsillar IgD-ME B cells and characterize their phenotype, clonal architecture, transcriptional signatures, and reactivity properties. To address these questions and better understand the ontogeny and regulation of IgD responses, we combined standard and high-throughput approaches with sorting of 13 tonsillar B cell subsets, immunoglobulin gene repertoire analysis, cloning and expression of recombinant IgD mAbs, and reactivity analysis of these antibodies as well as native IgD pAbs from both healthy controls and patients with rare inborn errors of immunity. We first defined the topography, phenotype, transcriptional signatures, ultrastructural properties, and fundamental functions of tonsillar IgD-ME B cells as well as canonical IgG-ME and IgA-ME B cells by combining standard approaches with high-dimensional flow cytometry, RNA sequencing, and transmission electron microscopy. To further investigate the clonal architecture of IgD-ME B cells and their relationship with other tonsillar IgD class-switched B cell subsets, we performed high-throughput Ig gene sequencing, followed by clonal lineage and phylogeny analyses. Next, Ig cloning-expression technology, ELISA and bacterial flow cytometry were applied to assess the reactivity of secreted IgD to common nasopharyngeal antigens. Finally, total serum IgD from patients with rare inborn errors of immunity provided insights into the in vivo regulation of IgD responses. Experiments were performed 2-6 times at least in triplicate (where applicable) and showed excellent reproducibility.

first: Andrea

last: Cerutti

first: Martyna

last: Filipska


title: Epigenetic control of telomeric RNA maintains heterochromatin in telomerase-driven cancers [Invivo]

overall_design: pooled subset of CRISPR knock-out screen for synthetic dosage lethal interactions of telomerase overexpressing cells. There are two model cells used and for each model there are 6 samples done in duplicates. Total of 24 samples

first: Vincent

last: Maranda

first: Yup

last: Zhang


title: Epigenetic control of telomeric RNA maintains heterochromatin in telomerase-driven cancers

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

overall_design: Refer to individual Series


title: Adipocyte small extracellular vesicle-derived microRNA-30a-3p exacerbates hepatic steatosis in high fat diet-fed male mice

status: Public on Mar 17 2026

city: Anshun

first: Longkun

last: Hu

state: Guizhou

address: Machang Town, Yunmanhu Campus of Guizhou Medical University

institute: Guizhou Medical University

type: Non-coding RNA profiling by high throughput sequencing

summary: Adipose tissue dysfunction drives hepatic lipid overload in metabolic dysfunction-associated steatotic liver disease (MASLD), yet the involvement of adipose tissue-derived small extracellular vesicles (sEVs) remains unclear. Herein, we showed that transplanting adipose tissue from high‑fat diet-fed male mice exacerbated insulin resistance and hepatic steatosis in lean recipients. Adipose‑specific Sirt3 overexpression (Sirt3AKI) alleviated insulin resistance and liver steatosis in high fat diet-fed male mice, whereas adipose‑specific Sirt3 knockdown aggravated these phenotypes. Moreover, adipose‑derived sEVs play key roles in hepatic lipid metabolism by delivering miRNAs in Sirt3AKI male mice. MicroRNA sequencing identified miR-30a-3p was increased in the circulating sEVs from high fat diet-fed male mice, while decreased in sEVs from Sirt3OE adipocytes and Sirt3AKI male mice. Mechanistically, miR‑30a‑3p promoted hepatic steatosis by targeting Becn1; this process was suppressed when Sirt3 downregulated miR‑30a‑3p transcription via deacetylation of H3K56. These findings highlight the critical role of adipose sEVs microRNAs in driving hepatocyte lipotoxicity, and suggest miR-30a-3p inhibition could be a therapeutic intervention for MASLD.

overall_design: Small RNA sequencing was performed on serum-derived small extracellular vesicles (sEVs) vesicles of chow-fed WT mice, HFD-fed WT Mice, and HFD-fed Sirt3AKI mice.

first: Tian

first: Diao

last: Chen

first: Yongxin

first: Fei

last: Zhou

first: Ruohan

last: lou

first: Yuxia


title: Epigenetic control of telomeric RNA maintains heterochromatin in telomerase-driven cancers [sequencing]

first: Franco

overall_design: Genome-wide CRISPR knock-out screen for synthetic dosage lethal interactions of telomerase-overexpressing cells. There are two model cells used and for each model there are 6 samples done in duplicates. Total of 24 samples.

first: Jared

first: Frederick

first: Omar

last: Abuhussein


title: H3K27me3 ChIP-seq in Ranbp2 Wild-type and knockout 10T1/2 cells

city: Singapore

first: Roger

last: Foo

address: 14 Medical Drive, MD6

country: Singapore

institute: National University of Singapore

summary: Mitochondria regulate cellular processes through direct and indirect interactions with other organelles. A well-studied example is contact with the endoplasmic reticulum at mitochondrial-associated ER membranes (MAMs), which control pathways including redox and calcium homeostasis. Recent studies also reported direct mitochondria–nuclear membrane contacts in cancer cells and yeast that promote pro-survival signaling, but how exchange occurs across two largely impermeable membranes remains unclear. Here, we describe the existence of direct interactions between the nuclear pore and the mitochondria.  In two unbiased proteomic screens, GST pulldown and BioID, VDAC1 was the top mitochondrial candidate that interacts with the nuclear pore protein RANBP2. In vitro RANBP2 CRISPR knock-out, RANBP2 truncation, or site directed mutagenesis of RANBP2-interacting amino acids in VDAC1 resulted in reduced mitochondria-nucleus proximity and decreased nuclear ATP and phosphocreatine levels. This was accompanied by a decline in levels of the nuclear phosphoproteome, and downregulation of pathways involved in histone modification, cellular differentiation and transcriptional regulation in vitro. We show differential H3K27me3 histone epigenetic landscape in Ranbp2 Wildtype and knockout cells in 10T1/2. Moreover, deletion of RANBP2 C-terminal domain in vivo in mice resulted in embryonic lethality due to cardiac and neural crest differentiation defects.  Collectively, these results describe a mechanism by which mitochondria directly interact with the NPC, a phenomenon which is critical for regulation nuclear energetics and cellular differentiation during embryonic development.

overall_design: Generation of RANBP2 KO 10T1/2 cells: CRISPR-Cas9 guides for mouse Ranbp2  were designed using ChopChop (https://chopchop.cbu.uib.no/) and cloned into plasmid pX458 (48138, Addgene, MA, USA) using BbsI site.  Efficiency of the guides was analyzed using T7-endonuclease 1 assay (M0302, NEB, MA, USA) following manufacturer’s protocol. Each candidate plasmid containing a single guide was transfected into 10T1/2 cells using Lipofectamine 3000 (L3000015, ThermoFisher, MA, USA) following manufacturer’s protocol. 36 hours after transfection, single cells were sorted in base of their GFP fluorescence using a FACSAria cell sorter (BD, NJ, USA) into p96 wells, expanded and analyzed for efficient deletion of the target gene by qPCR, western-blot and immunostaining. Final clone selected for the studies was generated using the guide sequence: 5’-GCTTACCTCTCGGGGCGACG-3’ for Ranbp2. H3K27me3 ChIP-seq was performed on Ranbp2 WT and KO cells respectively.

first: Ivan

last: Menendez-Montes

first: Chang Jie Mick

last: Lee

first: Chukwuemeka George

last: Anene-Nzelu

first: Hesham A.

last: Sadek

first: Roger Sik-Yin


title: ATAC-seq in Ranbp2 Wild-type and knockout 10T1/2 cells

summary: Mitochondria regulate cellular processes through direct and indirect interactions with other organelles. A well-studied example is contact with the endoplasmic reticulum at mitochondrial-associated ER membranes (MAMs), which control pathways including redox and calcium homeostasis. Recent studies also reported direct mitochondria–nuclear membrane contacts in cancer cells and yeast that promote pro-survival signaling, but how exchange occurs across two largely impermeable membranes remains unclear. Here, we describe the existence of direct interactions between the nuclear pore and the mitochondria.  In two unbiased proteomic screens, GST pulldown and BioID, VDAC1 was the top mitochondrial candidate that interacts with the nuclear pore protein RANBP2. In vitro RANBP2 CRISPR knock-out, RANBP2 truncation, or site directed mutagenesis of RANBP2-interacting amino acids in VDAC1 resulted in reduced mitochondria-nucleus proximity and decreased nuclear ATP and phosphocreatine levels. This was accompanied by a decline in levels of the nuclear phosphoproteome, and downregulation of pathways involved in histone modification, cellular differentiation and transcriptional regulation in vitro. We show differential ATAC peaks in Ranbp2 Wildtype and knockout cells in 10T1/2. Moreover, deletion of RANBP2 C-terminal domain in vivo in mice resulted in embryonic lethality due to cardiac and neural crest differentiation defects.  Collectively, these results describe a mechanism by which mitochondria directly interact with the NPC, a phenomenon which is critical for regulation nuclear energetics and cellular differentiation during embryonic development.

overall_design: Generation of RANBP2 KO 10T1/2 cells: CRISPR-Cas9 guides for mouse Ranbp2  were designed using ChopChop (https://chopchop.cbu.uib.no/) and cloned into plasmid pX458 (48138, Addgene, MA, USA) using BbsI site.  Efficiency of the guides was analyzed using T7-endonuclease 1 assay (M0302, NEB, MA, USA) following manufacturer’s protocol. Each candidate plasmid containing a single guide was transfected into 10T1/2 cells using Lipofectamine 3000 (L3000015, ThermoFisher, MA, USA) following manufacturer’s protocol. 36 hours after transfection, single cells were sorted in base of their GFP fluorescence using a FACSAria cell sorter (BD, NJ, USA) into p96 wells, expanded and analyzed for efficient deletion of the target gene by qPCR, western-blot and immunostaining. Final clone selected for the studies was generated using the guide sequence: 5’-GCTTACCTCTCGGGGCGACG-3’ for Ranbp2. Chromatin Accessibility was assessed on Ranbp2 WT and KO cells respectively.


title: RNA-sequencing to analyze global changes in transcriptome during local infection and systemic acquired resistance of Col-0 and rpt2a-2 to Pseudomonas syringae pv. maculicola ES4362

city: Wien

first: Gautier

last: Langin

address: Muthgasse 18

country: Austria

institute: BOKU

summary: The ubiquitin-proteasome-system (UPS) is a cellular cascade involving three enzymatic steps for protein ubiquitination to target them to the 26S proteasome for proteolytic degradation. Several components of the UPS have been shown to be central for regulation of defense responses during infections with phytopathogenic bacteria. Upon recognition of the pathogen, local defense is induced which also primes the plant to acquire systemic resistance (SAR) for enhanced immune responses upon challenging infections. In this experiment we analyzed the immune response of Col-0 and rpt2a-2 mutant to the pathogenic bacteria Pseudomonas syringae pv. maculicola ES4362 at the site of infection or during SAR.

overall_design: 5 weeks old Arabidopsis thaliana plant lower leaves were infiltrated with Pseudomonas syringae pv. maculicola ES4362 solution in 10mM MgCl2 OD600=0.005 using a needleless syringe. For control 10mM MgCl2 solution was used. For SAR experiment, upper leaves were infiltrated 38h after the first infiltration. Samples were processed 48h post 1st-infiltration.

first: Suayb

last: Üstün

first: Daniela

last: Spinti

first: Frederik

last: Börnke


title: RNA-sequencing to analyze immune response to Pseudomonas syringeae pv tomato DC3000 in Col-0 or nac53-1 78-1

summary: Proteotoxic stress, characterized by the accumulation of damaged proteins, poses a significant challenge to cellular homeostasis. To mitigate proteotoxicity, eukaryotes rely on the proteasome that is regulated by proteasome transcriptional activators. As proteotoxicity can originate in different compartments, cells must integrate signals from multiple locations, yet the mechanisms coordinating this response remain unclear. In this experiement we analyzed the importance of the plant proteasome transcriptional activators NAC53 and NAC78 in response to Pseudomonas syringeae pv tomato DC3000 (Pst) infection, a context known to trigger proteotoxic stress through several subcellular compartments.

overall_design: 5 weeks old Arabidopsis thaliana leaves of Col-0 or nac53-1 78-1 were infiltrated with Pst solution in MgCl2 10mM at OD600=0.2 or control soluzion MgCl2 10mM. After 24h of infection samples were processed for subsequent RNA exctraction.


title: Functional and genomic characterization of LCN2-deficient PC-3 cells reveals insights into prostate cancer progression

city: Aachen

first: Ralf

last: Weiskirchen

address: Pauwelsstr. 30

department: IFMPEGKC

institute: RWTH University Hospital Aachen

summary: Prostate Cancer-3 (PC-3) cells, commonly used as a model for aggressive, androgen-independent prostate cancer, display numerous genetic alterations that contribute to advanced disease, including the loss of tumor suppressors and dysregulated inflammatory signaling. Recent evidence has highlighted the pleiotropic roles of lipocalin 2 (LCN2) in promoting tumor cell proliferation, adhesion, and stress resistance. This study aimed to investigate the functional and molecular effects of LCN2 depletion in PC-3 cells.

overall_design: We conducted a genetic analysis of both the parental PC-3 cell line and a newly created LCN2-deficient PC-3 clone #1 (PC-3 LCN2-KO#1), developed using CRISPR/Cas9 technology. Short tandem repeat (STR) analyses confirmed the authenticity and lineage of each cell line, while next-generation sequencing (NGS) coupled with RT-qPCR validation was used to identify differentially expressed genes and any potential genomic changes resulting from the CRISPR/Cas9 editing process. Our analysis aligned with our previous findings showing that LCN2 is involved in inflammation, endoplasmic reticulum stress responses, and cytoskeletal organization. Previously we have shown that LCN2-deficient cells exhibited decreased invasiveness, disrupted F-actin dynamics, and increased sensitivity to stress-inducing conditions. Consistent with these observations, spectral karyotyping (SKY) and analysis of spontaneously occurring micronuclei revealed an elevated level of chromosomal aberrations in the LCN2-deficient cell line. These results emphasize the significance of LCN2 in driving prostate cancer aggressiveness and provide a foundation for exploring targeted interventions that disrupt LCN2-mediated pathways in advanced disease.


title: Pervasive Cas9 expression driven by mammalian promoter activity in E. coli affects representation of CRISPR sgRNA libraries [II]

city: Vancouver

first: Jana

last: Jajarmi

state: BC

institute: University of British Columbia

summary: CRISPR–Cas9 screening relies on uniform representation of single-guide RNA (sgRNA) libraries to enable accurate gene discovery. However, technical biases during library preparation can compromise screen performance. Here we show that commonly used “all-in-one” CRISPR vectors expressing both Cas9 and sgRNAs drive unintended Cas9 protein expression in Escherichia coli during plasmid amplification. This bacterial Cas9 expression causes guide-specific toxicity, leading to selective loss of sgRNAs and highly skewed library representation. We demonstrate that this effect occurs across multiple bacterial strains and affects both targeted and genome-wide libraries, including widely used human CRISPR libraries. Mechanistically, toxicity is driven by Cas9 expression rather than plasmid size and is only partially alleviated by catalytically inactive Cas9. Importantly, replacing the EF-1α promoter with a mouse phosphoglycerate kinase promoter suppresses Cas9 expression in bacteria while preserving genome editing efficiency in mammalian cells and restoring sgRNA uniformity. These findings identify a previously unrecognized source of bias in CRISPR library preparation and provide a practical solution to improve screening fidelity.

overall_design: All other sequencing data included dCas9 library (Fig4), resequence of original LcV2 library and PGK library (Fig6).


title: Prolactin, cortisol, and extracellular osmolality regulate cftr, ostf1, and sgk1 in tilapia ionocytes

city: Saratoga Springs

first: Jason

last: Breves

state: New York

address: 815 N. Broadway

department: Biology

institute: Skidmore College

summary: Objective: Euryhaline fishes can inhabit salinities ranging from fresh water (FW) to seawater (SW), in part because their endocrine system aligns the ion-transporting capacities of branchial ionocytes with the external environment. Ionocytes also directly sense extracellular osmolality and adjust their functions accordingly; therefore, this study investigated the interplay between osmotic conditions and osmoregulatory hormones in Mozambique tilapia (Oreochromis mossambicus) to further elucidate the basis of euryhalinity. We sought to determine whether prolactin (Prl) supports FW acclimation by counteracting osmotic and hormonal signals that initiate branchial responses to SW environments. Methods: We first combined hypophysectomy, hormone replacement, RNA-Seq, and qPCR to identify cystic fibrosis transmembrane conductance regulator (cftr), osmotic stress transcription factor 1 (ostf1), and serum- and glucocorticoid-inducible kinase 1 (sgk1) as Prl-repressed genes. We then leveraged a series of in vivo and in vitro experimental paradigms to characterize their regulation by environmental salinity, Prl, cortisol, and extracellular osmolality. Results: Our findings indicate that 1) Prl’s capacity to antagonize cortisol-stimulated cftr expression depends on extracellular osmotic conditions; 2) Prl and cortisol play opposing roles in regulating branchial ostf1 expression; 3) Sgk1 is expressed in ‘SW-type’ ionocytes, where interactions among Prl, cortisol, and osmotic conditions influence its expression; and 4) Prl promotes the expression of Na+/Cl- cotransporter 2 and Clc family Cl− channel 2c, as shown previously. Conclusions: The combined actions of osmotic stimuli, Prl, and cortisol shape the branchial expression of cftr, ostf1, and sgk1. While hyperosmotic extracellular conditions and cortisol promote the activation of these genes during SW acclimation, Prl supports FW adaptation by simultaneously suppressing these genes and promoting processes underlying active ion uptake. Thus, euryhaline tilapia adjust the ion-transporting activity of their branchial ionocytes to meet environmental demands by integrating multiple regulatory cues.

overall_design: RNA-Seq analysis of gill filaments collected from hypophysectomized male tilapia (80-110 g) following IP-injection with ovine prolactin (5 ug/g body weight) or saline vehicle. Hypophysectomy was performed by the transorbital technique (see Nishioka, 1994 and Breves et al., 2010).

first: Ming-Wen

first: Mayu

last: Inokuchi

first: Lucia

last: Seale

first: Andre


title: Pervasive Cas9 expression driven by mammalian promoter activity in E. coli affects representation of CRISPR sgRNA libraries

overall_design: Original plasmid library preparations for the Lockwood23 sgRNA library (Figure 1).


title: Spatial transcriptomics of squamous cell carcinoma from different tissues

city: Manchester

first: Amaya

last: Viros

address: Wilmslow Road

department: Skin Cancer Ageing

institute: Cancer Research UK

summary: We performed spatial transcriptomics on primary squamous cell carcinomas (SCC) from the head and neck, lung and skin to characterise tissue-specific stromal and epithelial cell interactions

overall_design: Two primary cutaneous SCC, two head and neck SCC, and one lung SCC FFPE samples were analysed using spatial transcriptomics

last: Virós

first: Kevin

last: Harrington

first: Malin

last: Pederson

first: Ben

last: O'Leary

first: Antonio

last: Rullan


title: Tissue-specific gene expression patterns in fibroblast cell lines

summary: Fibroblast cell lines from the dermis of the skin, the oral cavity, and the lungs were cultured in serum free DMEM and gene expression patterns were examined by RNA sequencing to determine tissue specific gene expression and programs expressed in fibroblasts in different tissues.

overall_design: RNA sequencing of fibroblast cell lines from the skin (dermis), oral cavity and lungs cultured in serum free DMEM

first: Timothy

last: Budden


title: Effect of tissue specific fibroblast secretome on squamous cell carcinomas

summary: Fibroblasts impact epithelial cell development and function and drive the progression of squamous cell carcinoma. We examined how fibroblasts from different tissues impact SCC behaviour and biology in tissue specific manner. Here we report tissue specific interactions between fibroblasts and SCC cell lines that drive invasion and agressive phenotypes in specific tissues but not others.

overall_design: RNA sequencing of cutaneous, head and neck, and lung squamous cell carcinoma cell lines treated with conditioned media from dermal, oral, or lung fibroblasts.


title: Targeted Cellular Micropharmacies Deliver Therapeutic Agents to the Brain

city: New York

first: David

last: Scheinberg

state: NY

address: 417 E 68th St

department: Pharmacology

institute: Memorial Sloan Kettering Cancer Center

summary: The systemic administration of therapeutic agents, particularly large, charged molecules such as antibodies, has limited efficacy in treating central nervous system (CNS) disorders. In addition, the slow progression of neurodegenerative diseases makes repeated intrathecal injections unfeasible. Alzheimer’s disease is characterized by the accumulation of Aβ amyloid plaques. Microglia contribute to the clearance of Aβ, but are inhibited by the expression of CD33. Therefore, antibody blocking of CD33 may enhance the phagocytosis of Aβ by microglial cells, slowing AD progression. Here, we use cells as “targeted cellular micropharmacies” that are retained in the CNS to deliver therapeutic proteins directly into the brain. To achieve this, we genetically engineered CD4 T-cells to express: (1) a chimeric antigen receptor against GD2 to retain the cells into the brain, (2) ectopic FoxP3 to reduce inflammation, (3) secreted IL-2 to promote cell longevity, and (4) secreted anti-CD33 scFv antibody. Our proof-of-concept demonstrates that therapeutic antibodies can be delivered to the brain for at least 8 weeks to treat neurological disorders. Other agents could be similarly delivered into the brain by this platform.

overall_design: RNA-seq profiling of GD2 CAR-FOXP3 CD4 T cells vs. GD2 CAR CD4 T cells. 1 million  cells per sample were sent to the MSKCC IGO facility for sequencing after 3 days in culture following the sorting of CAR-positive CD4 T cells.

first: Manish

last: Malviya


title: Xist RNA Dependent and Independent Mechanisms Regulate Dynamic X Chromosome Inactivation in B Lymphocytes

city: Philadelphia

first: Montserrat

last: Anguera

state: PA

address: 3800 Spruce Street, Rosenthal Room 390EB

department: Biomedical Sciences

institute: University of Pennsylvania School of Veterinary Medicine

type: Methylation profiling by high throughput sequencing


title: Xist RNA Dependent and Independent Mechanisms Regulate Dynamic X Chromosome Inactivation in B Lymphocytes [CUTandRUN]

summary: X-chromosome inactivation (XCI) maintenance in female B cells is dynamic (dXCI), where naïve cells lack cytological enrichment of Xist RNA and heterochromatic marks on the inactive X-chromosome (Xi), and these marks re-localize after stimulation. Here, we report the epigenetic profiling of silencing (H3K27me3, H2AK119Ub, H3K9me3, DNA methylation (DNAm)) and activating (H3K27ac) marks on the Xi during dXCI and investigate the requirement for Xist RNA in maintaining the Xi epigenetic landscape. We find that the Xi in naïve B cells is depleted for H2AK119Ub and H3K9me3 but enriched for DNAm and H3K27me3 which maintain an epigenetic memory of transcriptional silencing dependent on Xist RNA. Upon stimulation, Xist-independent H3K27me3 and Xist-dependent H2AK119Ub modifications accumulate across the Xi. Thus, naïve B cells maintain an epigenetic memory of silencing in the absence of Xist RNA localization via retention of DNAm and H3K27me3, and both H3K27me3 and H2AK119Ub accumulate on the Xi post-stimulation in an Xist-dependent and -independent manner. These data have critical implications for understanding molecular mechanisms underlying female-biased immune responses.

overall_design: CUT&RUN analysis for the histone modifications H3K27me3 and H2AK119Ub in CD23+ B cells with an ex vivo deletion of Xist RNA and stimulated in the presence of CpG and PRC2 or DNMT1 inhibitors. Experiments use B cells from female WT and Xist flox/flox mice that were unsitmulated or treated ex vivo with TAT-Cre recombinant protein then stimulated with CpG in combination with 6µM UNC1999 (Cat # ab146152, Abcam), 0.75µM 6-Thioguanine (Cat # A4882-100MG, Millipore Sigma), or 0.0006% DMSO for 24 hours (n=3 per condition).

first: Natalie

middle: E

last: Toothacre


title: Excitatory cortical neurons from CDKL5 deficiency disorder patient-derived organoids show early hyperexcitability not identified in neurogenin2 induced neurons [Ampliseq]

city: Boston

first: Madison

last: Glass

state: MA

address: 3 Blackfan St

department: Neurobiology

institute: Boston Children's Hospital

summary: CDKL5 deficiency disorder (CDD) is a rare developmental and epileptic encephalopathy resulting from variants in cyclin-dependent kinase-like 5 (CDKL5) that lead to impaired kinase activity or loss of function. CDD is one of the most common genetic etiologies identified in epilepsy cohorts. To study how CDKL5 variants impact human neuronal activity, gene expression and morphology, CDD patient-derived induced pluripotent stem cells and their isogenic controls were differentiated into excitatory neurons using either an NGN2 induction protocol or a guided cortical organoid differentiation. Patient-derived neurons from both differentiation paradigms had decreased phosphorylated EB2, a known molecular target of CDKL5. Induced neurons showed no detectable differences between cases and isogenic controls in network activity using a multielectrode array, or in MAP2+ neurite length, and only two genes were differentially expressed. However, patient-derived neurons from the organoid differentiation showed increased synchrony and weighted mean firing rate on the multielectrode array within the first month of network maturation. CDD patient-derived cortical neurons had lower expression of CDKL5 and HS3ST1, which may change the extracellular matrix around the synapse and contribute to hyperexcitability. Similar to the induced neurons, there were no differences in neurite length across or within patient-control cell lines. Induced neurons have poor cortical specification while the organoid derived neurons expressed cortical markers, suggesting that the changes in neuronal excitability and gene expression are specific to cortical excitatory neurons. Examining molecular mechanisms of early hyperexcitability in cortical neurons is a promising avenue for identification of CDD therapeutics.

first: Madison R

first: Dosh

last: Whye

first: Nickesha C

last: Anderson

first: Delaney

last: Wood

first: Nina R

last: Makhortova

first: Taryn

last: Polanco

first: Kristina H

last: Kim

first: Kathleen E

last: Donovan

first: Gayathri Rajaram

last: Srinivasan

first: Lorenzo

last: Vaccaro

first: Ashish

last: Jain

first: Davide

last: Cacchiarelli

first: Liang

last: Sun

first: Elizabeth D

last: Buttermore

first: Mustafa

last: Sahin


title: Effect of DHX29 depletion on gene expression in U937 cells

status: Public on Mar 19 2026

city: Kyoto

first: Masanori

last: Yoshinaga

state: Kyoto

address: Yoshida-konoe-cho, Sakyo-ku

department: Graduate School of Medicine

institute: Kyoto University

summary: Synonymous codon usage controls global gene expression in both prokaryotic and eukaryotic species. Non-optimal codons are known to induce mRNA decay; however, the underlying molecular mechanism remains poorly understood in human cells. Through genome-wide CRISPR screening, we identified the RNA-binding protein DHX29 as a critical regulator of codon-dependent gene expression. Cryogenic electron microscopy and selective ribosome profiling demonstrated that DHX29 directly interacts with the A-site entrance of the translating 80S ribosome, the binding site for the eEF1A•GTP•aminoacyl-tRNA ternary complex, suggesting a role in monitoring aminoacyl-tRNA sampling. Proteomic analysis further revealed that DHX29 recruits the GIGYF2•4EHP complex to mediate global suppression of non-optimal mRNAs. These findings establish a mechanistic link between synonymous codon usage and the regulation of gene expression.

overall_design: Control and DHX29-deficient U937 cells were generated by transducing Cas9-expressing cells with vectors expressing control and DHX29-targeting gRNAs, and subjected to RNA-seq analysis.

first: Fabian

last: Hia

first: Ting

last: Cai

first: Osamu

last: Takeuchi


title: Spatial and Bulk Transcriptomics Analyses Reveal Distinct Gene Expression Profiles in Archival Skin Kaposi Sarcoma Lesions Based on Disease Characteristics (NCounter Data)

city: Bethesda

first: Bahman

last: Afsari

state: MD

address: Building 10, 9000 Rockville Pike

institute: NIH

summary:
Targeted transcriptomics assay of immuno-oncology genes using NanoString PanCancer IO 360 panel (770 gene) was carried out with archival skin Kaposi sarcoma samples.
42 participants with HIV-associated KS with and without other KSHV-associated diseases (KICS, PEL and/or MCD) included

overall_design: NanoString PanCancer IO 360 panel is supplemented with KSHV custom probe - 42 FFPE samples were included in the analyses

first: Ramya

last: Ramaswami

first: Edmund

last: Culley

first: Baman

first: Quashawn

last: Chadwick

first: Xiaolin

last: Wu


title: 5ɑ-Dihydrotestosterone Upregulates Interferon Signaling in Osteoclast Differentiation During Inflammatory State

city: Rochester

first: Kiana

middle: Lee

last: Chen

address: 601 Elmwood Avenue

institute: University of Rochester Medical Center

summary: Rheumatoid arthritis (RA) is characterized by chronic inflammatory erosions and a more prevalent disease in females. In RA, the differentiation of osteoclast precursor cells (OCPs) into bone-resorbing osteoclasts is stimulated by the inflammatory environment, causing significant erosive disease. Notably, androgen, the dominant sex hormone in males, is protective against this inflammation-driven bone loss. To investigate how androgen regulates osteoclastogenesis in an inflammatory state, bone marrow-derived cells were treated with or without androgen in a TNF-mediated osteoclast culture. 5ɑ-dihydrotestosterone (DHT)-treated and vehicle-treated cells were collected, analyzed, and compared with single-cell RNA-sequencing which determined differences in cell populations, gene expression, functional gene ontology, and differentiation.

overall_design: Bone marrow derived cells from a C57BL/6 male mouse were treated with either DHT or vehicle control and grown in an inflammatory osteoclast cell culture system. Cells were collected after 24 hours with RANKL and hTNFɑ stimulation and analyzed with single-cell RNA-sequencing.

middle: L

first: Michael

last: Christof

first: Gulzada

last: Kulzhanova

first: Chia-Lung

first: Homaira

last: Rahimi


title: Lcn2 deficiency leads to social impairments independent of maternal immune activation

city: Warsaw

first: Laboratory

last: of Sequencing

state: mazowieckie

address: Pasteura 3

country: Poland

institute: Nencki Institute of Experimental Biology PAS

summary: Maternal infection during pregnancy is a well-established risk factor for neurodevelopmental disorders (NDDs), yet the underlying molecular mechanisms remain poorly understood. Lipocalin-2 (Lcn2), an innate immune protein that is highly upregulated during infection, also affects neuronal and glial function. This study investigates the role of Lcn2 in shaping brain development, particularly after maternal immune activation (MIA). To mimic maternal infection, pregnant mice received intraperitoneal injections of either lipopolysaccharide (LPS) or saline on embryonic days 16 to 18 to model infection during the second trimester of pregnancy in humans. We first showed that Lcn2 mRNA is expressed in the fetal brain and that MIA significantly upregulates Lcn2 mRNA and protein in the hippocampus and neocortex of both sexes. To assess functional relevance, we employed Lcn2 heterozygous females to generate wild-type and Lcn2 KO offspring from the MIA and control groups. Both female and male offspring underwent a battery of behavioral assays. Lcn2 deletion and MIA independently induced deficits in social behavior and increased repetitive behavior phenotypes relevant to NDDs. However, their combination did not exacerbate these effects, suggesting an occlusion effect. Importantly, the impact of Lcn2 deletion and MIA appeared to be specific to social and repetitive behavior, as no deficits were observed in the learning and memory task.  To investigate potential shared molecular mechanisms, we performed RNA sequencing of the fetal forebrain 4 hours after the final LPS injection. This analysis revealed an overlapping group of differentially expressed genes in the Lcn2 KO and MIA groups, indicating convergence on similar transcriptional pathways that may underlie the observed behavioral phenotypes. These results suggest that while Lcn2 may not mediate the pathological effects of prenatal immune challenge, it plays a critical role in normal brain development.

overall_design: RNA-seq profiling of RNA isolated from the forebrain of WT or Lcn2KO mouse fetuses at E18, four hours after the final of three injections of LPS (40 μg/kg of body weight) or saline administered to Lcn2Het dams.

first: Martyna

last: Pekala

first: Sylwia

last: Zawiślak

first: Sandra

last: Romanis

first: Karolina

last: Nader

first: Aleksandra

last: Cabaj

first: Anna

last: Madecka

first: Alicja

last: Puścian

first: Ewelina

last: Knapska

first: Robert

last: Pawlak

first: Leszek

last: Kaczmarek

first: Katarzyna

last: Kalita


title: Imidacloprid susceptibility is linked to life history regulation in honey bees (Apis mellifera)

city: Edmonton

first: Olav

last: Rueppell

state: Alberta

address: 11355 - Saskatchewan Drive

department: Biological Sciences

institute: University of Alberta

summary: The western honey bee (Apis mellifera) often encounters chemical stressors in the form of phytochemicals, or pesticide residues. The first line of defense against these agrochemicals is constitutively expressed detoxification enzymes such as cytochrome P450s.Honey bees with lower levels of constitutively expressed enzymes experience greater immediate stress, which can lead to death, sub-lethal effects, and precocious foraging, if nursing. Precocious foragers are young, less developed honey bees that are recruited into the foraging population, whereas normal-aged foragers are much older. These precocious foragers are less capable flyers, and are more likely to die during their flights, which can in turn further stress the colony due to reduced food stores. In our study, we hypothesize that chemical stress sensitivity is directly linked to the onset of precocious foraging. Additionally, we believe that precocious foragers are less suited for foraging due to their rushed development, and are likely more transcriptionally similar to nurses instead of older foragers. We measured the survival of honey bees taken from 26 different colonies when exposed to a chemical stressor over 48 hours. Honey bees from each of these colonies were also all added to the same colony, and we measured the age and number of foragers from each colony over a 2-week period. Lastly, we performed transcriptomics of young and old nurses and foragers. Our results suggest that chemical stress tolerance was linked to the honey bee’s origin colony. Moreover, we found no significant correlation of the honey bees’ chemical tolerance to the age at which they began foraging. Rather, we found that bees from more tolerant colonies exhibited lower volumes of foraging by two weeks. Furthermore, transcriptomic analysis showed that precocious foragers exhibited global gene expression patterns more closely aligned with nurses than with older foragers, reinforcing age as the primary determinant of gene expression profiles. Susceptible bees displayed distinct expression profiles compared to more tolerant ones across different life stages, with notable overlap in genes encoding cytochrome P450e and a vitellogenin receptor, highlighting their potential roles in stress response and behavioral maturation.

overall_design: RNA-seq and chemical mortality assay of nurse and forager bees of different origin colonies that have been concatenated into 3, equally dispersed replicate nuclear hives.

first: Gursimran

last: Toor

last: Lu


title: One-step knock-in CAR constructs in human NK cells enable scalable, TGFβ1-resistant immunotherapy for solid tumors

city: Seoul

first: Mihue

last: Jang

address: 5, Hwarang-ro 14-gil, Seongbuk-gu

department: Medicinal Materials Research Center, Biomedical Research Division

country: South Korea

institute: Korea Institute of Science and Technology

summary: Chimeric antigen receptor (CAR)-engineered natural killer (NK) cells offer a promising immunotherapy for various malignancies. However, their efficacy in treating solid tumors is often compromised by the immunosuppressive tumor microenvironment (TME), where TGF-β1 plays a key role in inhibiting NK cell function. To address this challenge, our study focused on developing a simple, one-step process for generating CAR-NK cells, termed “one-step CAR-NK”, by simultaneously knocking out TGFβR2 and knocking in a CAR gene into human primary NK cells. We first demonstrated that knocking out TGFβR2 using CRISPR-Cas9 enhanced NK-mediated cancer killing abilities with upregulated CD107a expression when co-cultured with AsPC-1 cells. Additionally, we optimized the donor DNA by inserting a binding motif for dexamethasone, which encodes a CAR targeting mesothelin (MSLN) as a tumor antigen, to integrate into the TGFβR2 locus. Using electroporation, the Cas9 protein/gRNA complex combined with CAR donor DNA significantly increased the knock-in efficacy, resulting in potent cancer killing against solid tumors. Furthermore, we explored a two-step CAR-NK manufacturing process, using AAV to deliver DNA instead of naked DNA. This method also showed high KI efficacy and robust cancer-killing capabilities. As with the one-step process, the supplement of dexamethasone further improved NK functions in two-step CAR-NK cells. Thus, our one-step CAR-NK platform, particularly in the presence of dexamethasone, holds promise as off-the-shelf therapeutics with a simple and scalable manufacturing process for treating solid tumors.

overall_design: RNA-seq profiling of wild type human primary NK cells and human primary NK cell-derived CAR-NK cells treated with or without dexamethasone.


overall_design: ATAC-seq profiling of wild type human primary NK cells treated with or without dexamethasone.


title: Steroid hormone antagonism reduces the rate of aortic rupture and improves survival in a mouse model of Vascular Ehlers-Danlos Syndrome

city: Baltimore

first: Elena

last: MacFarlane

state: Maryland

address: 733 N Broadway St

department: Department of Genetic Medicine

country: USA

institute: Johns Hopkins

summary: Vascular Ehlers-Danlos syndrome (VEDS) carries a high risk of vascular rupture. In the Col3a1G938D/+ mouse model, the rate of aortic rupture accelerates postnatally, especially in males, concurrently with the rise in androgens. Here we show that this vulnerability is associated with the postnatal transition of the aorta to a non-synthetic state, and that androgen receptor (AR ) antagonism partially reversed this shift, resulting in a non-physiological increase in the expression of extracellular matrix components. Both pharmacological and genetic AR antagonism improved survival in males, while dual AR and mineralocorticoid (MR) inhibition, or selective MR antagonism, reduced the risk of aortic rupture in both sexes. In all major aortic cell types, there was substantial overlap in the transcriptional alterations imposed by AR, MR and dual AR/MR inhibition, suggesting that both AR/MR antagonism and selective targeting of the MR are potential therapeutic strategies in this condition.

overall_design: RNA-seq of wildtype and Vascular Ehlers-Danlos Syndrome (vEDS) model mice. Wildtype and vEDS mice include untreated control mice, mice treated with hydralazine, mice treated with a combination of bicalutamide and hydralazine, mice treated with a combination of spironolactone and hydralazine

first: Emily

last: Juzwiak

first: Caitlin

last: Bowen

first: Rhiannon

last: Edwards

first: Maya

last: Black

first: Anthony

last: Zeng

last: Bramel

first: Zachary

last: Burger

first: Harry

last: Dietz

last: MacFarlane Gallo


title: Selective clonal attrition of high-affinity B cells in chronic viral infection

city: Basel

first: DBM Bioinformatics

last: Core Facility

state: BS

address: Hebelstrasse 20

department: Departement of Biomedicine

institute: University of Basel

summary: Perturbed B cell responses and late formation of neutralizing antibodies are hallmarks of chronic infections but the mechanisms underlying the dysfunctional humoral responses remain poorly defined. We used adoptive transfer of monoclonal anti-viral B cells (KL25) in the context of chronic lymphocytic choriomeningitis virus (LCMV) infection in mice and we observed late clonal deletion of the KL25 cells. This process was governed by the binding affinity of the KL25 B cell receptor (BCR) to its target, the viral glycoprotein (GP), and the persistence of the antigen. We found that the loss of the KL25 B cells was independent of IFN-I inflammation, and it was the result of Blimp-1-dependent differentiation. The late deletion of high-affinity B cells in chronic infection may explain the delayed neutralizing antibody response to persistent pathogens. Strategies to counter B cell clonal deletion should help us improve humoral immunity against chronic infectious diseases

overall_design: On day -6,  4 mice were infected with high-affinity DOCILE infection (DOC) (S1-S4), and 4 mice were infected wtih low-affinity antigen (DOC-LAV) (S5-S8). Low numbers of LCMV-specific KL25 B cells were adoptively tranferred at day 0 and isolated from chronically infected recipients 14 days after adoptive transfer using FAC sorting.

first: Mirela

last: Dimitrova

first: Julien

last: Roux

first: Daniel

last: Pinschewer


title: Deep sequencing data from PFS screen of GeCas12a2 CRISPR-Cas nuclease

city: Salt Lake City

last: Wilson

state: UT

address: 2000 Circle of Hope

department: Oncological Sciences

institute: Huntsman Cancer Institute

summary: In this work we analyzed possible protospacer flanking sequences recognized by GeCas12a2 Type V CRISPR-Cas nuclease.

overall_design: GeCas12a2 was challenged with a library of targets containing randomized protospacer flanking sequences. Sequences which result in GeCas12a2 activation are consequently depleted from the library. Conditions include a library prepared with GeCas12a2 under non-targeting conditions (NT) and a library with GeCas12a2 under targeting conditions (T). Sequences were extracted from the Illumina read data and their corresponding depletion scores calculated.

first: Jared

last: Thompson

first: Oleg

last: Dmytrenko

middle: R

first: Friso

middle: T

last: Schut

first: Xiaoyang

last: Zhang

first: Chase

last: Beisel

first: Yang

last: Liu


title: Targeting branched-chain amino acids alleviates pulmonary fibrosis [RNA-seq]

city: wuhan

first: Jie

last: Yao

state: 湖北

address: wuhan hubei province

country: China

institute: wuhan university

summary:
Purpose: Previous studies demonstrated that BCAA metabolic reprogramming induces idiopathic pulmonary fibrosis (IPF). This study investigates BCAA-mediated gene expression regulation in primary mouse lung fibroblasts (MLFs).
Methods: In vitro: MLFs were cultured in three media conditions: complete medium (Complete), BCAA-free medium (Free), or BCAA-free medium supplemented with BCAAs (Free+BCAA) or BCKA (Free+BCKA) for 24 hours prior to TGF-β1 (20 ng/mL) stimulation for 48 hours. Triplicate RNA-seq analyses were conducted on treated MLFs.
In vivo: Comparative RNA-seq profiling was performed on lung tissues from three experimental groups: PBS-treated controls, bleomycin (BLM)-exposed mice, and BLM-treated mice receiving BCAA supplementation (3 mmol/L in drinking water) or BCAA-free diets or BT2 treatment.
Conclusions: BCAA metabolism is essential for myofibroblast activation and mechanistically promotes lung fibrogenesis in both in vitro and in vivo models.

overall_design: In vitro: Primary mouse lung fibroblasts (MLFs) were isolated from lungs of 6–8-week-old mice using a combination of collagenase I digestion and differential adhesion and cultured in DMEM/F12 medium. Triplicate RNA-seq analyses were conducted on treated MLFs. In vivo: 10–12-week-old male C57BL/6 mice were randomly allocated into three batchs. Batch 1: ① Normal diet throughout (PBS); ② Normal diet for 7 days followed by intratracheal administration of 1.25 mg/kg BLM (BLM); ③ BCAA-supplemented drinking water (3 mM) for 7 days, followed by 1.25 mg/kg BLM (BLM+BCAA). Lung tissues were collected 14 days post-BLM treatment. Batch 2: ① Normal diet throughout (PBS); ② BLM administration (1.25 mg/kg) with normal diet (BLM); ③ BLM administration (1.25 mg/kg) for 7 days, followed by BCAA-free diet for 14 days (BLM+BCAA free). Lung tissues were collected 21 days post-BLM treatment. Batch 3:  ① Normal diet throughout (PBS); ② BLM administration (3.5 mg/kg) for 3 days, followed by daily intraperitoneal injection of 10% DMSO for 11 days (BLM); ③ BLM administration (3.5 mg/kg) for 3 days, followed by daily intraperitoneal injection of 20 mg/kg BT2 for 11 days (BLM+BT2). Lung tissues were collected 14 days post-BLM treatment.

first: Miao

last: Lei

last: He

first: Su

last: Fang

first: Wanli

last: Peng

first: Wenxiang

last: Hu


title: Targeting branched-chain amino acids alleviates pulmonary fibrosis [scRNA-seq]

summary: We previously confirmed that BCAT2 was reduced in TGF-β1-treated mouse and human lung fibroblasts. Knockdown of BCAT2 significantly promoted COL1A1 expression in response to TGF-β1 treatment. However, the roles of BCAT2 on other cell types, changes in cellular states, and cellular interactions are currently unknown. Here, we used single-cell RNA sequencing to analysis healthy mouse lungs (WT_PBS), mouse lungs with bleomycin-induced fibrosis (WT_BLM), and heterozygous BCAT2+/− (BCAT2 Het) mouse lungs treated with BLM (Het_BLM). Notably, we observed a reduced number of epithelial cells in the BCAT2 Het mice (Figure 3M and S3L). In contrast, the fibroblast and macrophage populations were significantly increased in the BCAT2 Het mice compared to WT mice after bleomycin-induced lung injury.

overall_design: 10–12-week-old male C57BL/6 mice (WT and BCAT2 Het) were injected intratracheally with bleomycin (1.25 mg/kg) for 14 days. Lung tissues were collected to perform scRNA-seq.


title: Targeting branched-chain amino acids alleviates pulmonary fibrosis [CUT&Tag]

summary:
Purpose: We previously observed significant dysregulation in the expression of key BCAA metabolic genes during idiopathic pulmonary fibrosis (IPF). This study aims to elucidate the molecular mechanisms underlying BCAA metabolic genes dysregulation during IPF progression.
Methods: ATF4 CUT&Tag were performed in MLFs and PPARγ CUT&Tag were performed in MLFs treated with TGF-β1 (20 ng/mL) for 48 h.
Conclusions: ATF4 and PPARγ respectively regulate the expression of Slc7a5 and BCAA catabolic genes in primary mouse lung fibroblasts (MLFs)


title: Targeting branched-chain amino acids alleviates pulmonary fibrosis [ATAC-seq]

summary:
Purpose: Previous studies demonstrated that BCAA metabolic reprogramming induces idiopathic pulmonary fibrosis (IPF). This study investigates BCAA-mediated gene expression regulation in primary mouse lung fibroblasts (MLFs).
Methods: MLFs were cultured in three media conditions: complete medium (Complete), BCAA-free medium (Free), or BCAA-free medium supplemented with BCAAs (Free+BCAA) for 24 hours prior to TGF-β1 (20 ng/mL) stimulation for 48 hours. ATAC-seq analyses were conducted on treated MLFs.
Conclusions: BCAA plays a crucial role in modulating the chromatin state of key fibrotic genes.

overall_design: Primary mouse lung fibroblasts (MLFs) were isolated from lungs of 6–8-week-old mice using a combination of collagenase I digestion and differential adhesion and cultured in DMEM/F12 medium. ATAC-seq analyses were conducted on treated MLFs.


title: Targeting branched-chain amino acids alleviates pulmonary fibrosis [CUT&Tag, histone_modification]

summary:
Purpose: Previous studies demonstrated that BCAA metabolic reprogramming induces idiopathic pulmonary fibrosis (IPF). This study investigates BCAA-mediated gene expression regulation in primary mouse lung fibroblasts (MLFs).
Methods: MLFs were cultured in two media conditions: complete medium (Complete) or BCAA-free medium (Free) for 24 hours prior to TGF-β1 (20 ng/mL) stimulation for 48 hours. H3K4me3, H3K27ac, H3K27me3, H3K36me3, and H3K9me3 CUT&Tag analyses were conducted on treated MLFs.
Conclusions: BCAA plays a crucial role in modulating H3K36me3 of key fibrotic genes.

overall_design: Primary mouse lung fibroblasts (MLFs) were isolated from lungs of 6–8-week-old mice using a combination of collagenase I digestion and differential adhesion and cultured in DMEM/F12 medium. CUT&Tag analyses were conducted on treated MLFs.


title: Weak influence of light and time of day on transcriptional response to SAV3 infection in Atlantic salmon (Salmo salar) smolts

city: Tromsø

first: Alexander

middle: Christopher

last: West

state: Troms

address: Framstredet 39, Tromsø

department: Arctic Chronobiology and Physiology

country: Norway

institute: Universitetet i Tromsø

summary: Seventeen percent of aquaculture Atlantic salmon in Norway die after transfer from freshwater to seawater facilities, many from infectious disease. The degree to which these deaths are attributable to unseen weakness in smolt preparation is therefore an important concern. Strong evidence in mammals and supporting evidence in other teleosts show that the light environment shapes the immune response via entrainment of the circadian clock. These data are alarming as most smoltification protocols use constant light, which does not entrain the circadian clock and is associated with negative survival outcomes to disease challenge in other species. The goal of our study was to establish how different light environments affected the anti-viral immunity of Atlantic salmon smolts. To achieve this goal, we measured the transcriptional response of select type I and type II interferons and major histocompatibility complexes in the heart and head kidney following an intraperitoneal SAV3 viral challenge delivered under different lighting conditions. We began with two hypotheses. First, that compared with a regular light-dark cycle (long photoperiod; LP), constant light (LL) would have a weaker resistance to viral challenge in Atlantic salmon smolts. Second, that Atlantic salmon smolts kept in a regular light-dark cycle would have altered immune sensitivity between the light and dark phases. To test our first hypothesis, we photostimulated smoltification in parr through exposure to either LP or LL conditions for 6 weeks then challenged them by SAV3 infection. LP and LL groups showed indistinguishable measurements in classical smolt characteristics and comparable levels of SAV3 mRNA in the heart after 8 and 14 days post infection. They also showed comparable transcriptional responses of select type I interferons (IFNa, IFNb, IFNc) and both class I and class II major histocompatibility complexes (MHC I and MHC II) in both heart and head kidney tissue. IFNγ induction in the heart, however, was marked higher in the LL compared to the LP group. While this data suggests a heightened sensitivity of the LL group to SAV3 viral challenge, the consistent induction of MHC II, a key target gene of IFNγ signaling, to SAV3 infection in both LL and LP groups indicates that photoperiod may only have a minor impact on interferon regulation, rather than a more general influence on the viral immune response. To test our second hypothesis, we compared the transcriptomic response in the heart to SAV3 infection delivered either at the mid-light or mid-dark phase in the LP group. Both mid-light and mid-dark groups showed an upregulation of immune related genes and down-regulation of structural genes of mitochondria, but an analysis of the interaction between infection and time of day identified no differentially regulated genes. This second experiment suggests that, unlike in mammals, daily timing of viral infection may not play a major role in the immune response of Atlantic salmon.

overall_design: RNAseq profiling of hearts from Salmo salar. Parr were exposed to 6 weeks short photoperiod, then 6 weeks long photoperiod before intraperitoneal viral challenge with SAV3 or PBS at the mid-light or mid-dark timepoint. Hearts were collected 14 days after infection and snap frozen on dry ice before RNA processing.

first: Therese

last: Solberg

first: Chandra

last: Ravuri

first: Mattis

last: Jayme van Dalum

first: Jo

last: Espen Tau Strand

first: David

last: Hazlerigg

first: Eva

last: Stina Edholm


title: Proton-activated chloride channel 1 is essential for innate host defense against bacterial sepsis

first: Markus

last: Bosmann

address: 72 E Concord Street

department: Pulmonary Centre, Department of Medicine

institute: Boston University

summary: Bacterial sepsis remains a devastating clinical problem. Here, we describe a protective role for the recently discovered acid-sensitive, proton-activated chloride channel, PACC1 (PAC/ASOR/TMEM206), during sepsis. Initially, we found PACC1 was enriched in healthy human and mouse mononuclear phagocytes, particularly macrophages, and differentially regulated by inflammatory stimuli, suggesting PACC1 involvement in innate immunity. To further investigate, we generated de novo Pacc1 knockout (−/−) mice, which presented without major immunologic abnormalities at baseline. Compared to wild type (WT), Pacc1−/− myeloid cells showed normal phagocytic uptake of acid-insensitive Escherichia coli BioParticles, but impaired development of the acidifying phagolysosome using acid-sensitive E. coli BioParticles. Transcriptomic profiling of Pacc1−/− macrophages revealed dysregulated phagolysosomal and cytokine networks (e.g., interferons). Because phagolysosomal bacterial clearance is essential to resolve infection, we challenged Pacc1−/− mice with intraperitoneal gram-negative E. coli sepsis. Pacc1−/− mice displayed increased bacterial burden, immune cell infiltration, inflammation, and lethality. In contrast, phagocytosis-independent E. coli lipopolysaccharide (LPS)-induced endotoxemia yielded comparable WT and Pacc1−/− survival, as well as similar inflammatory responses. Finally, we engineered Pacc1-floxed (fl/fl) mice crossed with a myeloid lineage Cre-deleter strain to interrogate myeloid cell–intrinsic PACC1 in vivo. Consistent with a predominate role for PACC1 during phagocytosis and bacterial clearance in these cells, LysM-Cre/Pacc1fl/fl mice exhibited impaired E. coli sepsis survival but indifferent endotoxemia phenotypes. In conclusion, PACC1 links sterilizing phagolysosomal activity with immune networks in sepsis pathobiology.

overall_design: Bone marrow-derived macrophages (BMDMs; 2x10^6) from wild type and Pacc1-/- (C57BL/6J-Pacc1em1Bosm/Bosm, MGI: 6283429) mice were plated in 6-well tissue-culture treated plates in macrophage media and allowed to attach for 2 hours. BMDMs were then either left unstimulated for 12h or stimulated with inactivated E. coli pHrodo Bioparticles at 100ug/mL for 12h (n=6 mice/group). RNA-seq was performed from these BMDMs.

first: Lucien

last: Garo

first: Archana

last: Jayaraman


title: T cell development from expanded hematopoietic progenitors reveals initiation control by Lmo2 and Flt3L priming

city: Pasadena

first: Brendan

middle: W

last: MacNabb

state: CA

address: 1200 E California Blvd

institute: California Institute of Technology

summary: To access the earliest stages of murine T cell development, we adapted a serum-free expansion system for hematopoietic stem and progenitor-like cells. These cells undergo normal T cell differentiation in vivo and in vitro, verified by gene expression trajectories from single-cell RNA sequencing. Their absolute differentiation speed is slower than that of fresh progenitors but is modulated with Flt3L priming. Leveraging this system, the earliest T cell lineage-initiating events in response to Notch signaling included chromatin opening and transcriptional activation of the TCR-Cβ locus. Acute CRISPR knockouts revealed that some transcription factors normally inherited from bone marrow progenitors impede early T cell development, with differing effects on proliferation. Among 23 factors tested, Lmo2 knockout greatly accelerated the onset of germline TCR-Cβ locus transcription and expression of Tcf7, Gata3, and Runx1 and their targets. Thus, normal endogenous expression of this progenitor- and leukemia-associated factor markedly restrains T cell program initiation.

overall_design: ATAC-seq of in vivo DN thymocyte populations:  Thymi were pooled from 8-10 sex-matched mice (4.5 - 7 weeks old) per replicate. Each population was purified by flow cytometry cell sorting.

first: Boyoung

last: Shin

first: Samantha

last: Chang

first: Ellen

last: Rothenberg