title: Homeostatic Preservation of Action Potential Waveform by Notch and Nerfin-1

city: San Francisco

last: Karmelic

state: CA

department: Biochemistry and Biophysics

institute: UCSF

summary: To understand the homeostatic mechanisms responsible for preservation of neuron type-specific firing we characterized the preservation of neuronal firing following the deletion of a potassium channel gene (Shal/Kv4). Patch-Seq experiments and subsequent genetic analyses reveal widespread Notch signaling-dependent transcriptional changes including a required function for Nerfin-1.

overall_design: Patch-seq analysis of identified motoneurons (MN1-Ib). Control is MN1>GFP (n=3), Shal RNAi is MN1> Shal RNAi (n=3), N RNAi is MN1> Notch RNAi (n=4), and N+Shal RNAi is MN1> Shal RNAi; N RNAi (n=3). Replication number is the number of independent reactions, with 4-8 patched cells from 1 or 2 larvae pooled into each sequencing reaction.

first: Yelena

last: Steinberg

first: Ryan

middle: T

last: Jones

first: Özgür

last: Genç

first: Graeme

middle: W

last: Davis


title: A human-specific enhancer modulates chromosomal interactions to promote neurogenesis [mouse RNA-seq]

status: Public on Feb 16 2026

city: Durham

first: Federica

last: Mosti

state: NC

address: 213 Research Dr

country: USA

institute: Duke University

type: Expression profiling by high throughput sequencing

summary: Changes to the human genome have driven the evolution of human features including a larger neocortex and enhanced cognition. Human Accelerated Regions (HARs) are highly-conserved loci containing human-specific variants, which can be transcriptional enhancers during neurodevelopment. However, the neurodevelopmental functions of HARs and their mechanism of gene regulation are still largely unknown. Here, we show that the HAR1984 promotes neural progenitor cell identity and neurogenesis by influencing species-specific transcription and chromatin interactions. Humanized HAR1984 knock-in of chimpanzee cortical organoids increased intermediate progenitors and neurons, whereas chimpanzee HAR1984 knock-in  in human cortical organoids produced the opposite phenotype. Moreover, humanized HAR1984 knock-in mouse brains showed increased progenitor proliferation and neuron number, resulting in a thicker cortex with cortical folding. Hi-C data revealed a chromatin loop of HAR1984 with its target genes, ETV5 and TRA2B, present in human fetal brains, but reduced in chimpanzee, macaque or mouse neural cells. We show that human-specific mutations in HAR1984 promote chromatin looping and that human-specific ETV5 binding auto-regulates enhancer activity. This work demonstrates new molecular mechanisms underlying human-specific neurodevelopment, linking HARs to chromatin architecture, cortical cell identity and evolutionary expansion of  human brains.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for Mm-HAR1984(Mm/Mm), Mm-HAR1984(Hs/Mm) and Mm-HAR1984(del/del) E16.5 mouse cortices.  3 biological replicated were analyzed for each genotype

first: Debra

last: Silver


title: STAT1 and STAT3 Signaling in Ovarian Cancer: Prognostic Biomarkers and Therapeutic Targets [RNA-seq]

city: Rochester

first: Esther

last: Rodman

state: Minnesota

address: 200 1st St SW

institute: Mayo Clinic

summary: Epithelial ovarian cancer is the 7th most common cancer in women worldwide and is the most fatal of all female reproductive cancers. JAK/STAT signaling is oncogenic in ovarian cancer and has been reported to be constitutively active in a proportion of chemotherapy resistant ovarian cancer cell lines and tumors. However, the precise molecular mechanisms by which STAT1 and STAT3 function in ovarian cancer are not well understood and the consequences of their specific inhibition have yet to be determined. Methods: To delineate the specific functions of STAT1 and STAT3 in high grade serous and endometrioid ovarian cancer cell lines, we treated OVSAHO and MDAH cells with IFNγ or IL6, canonical STAT1 and STAT3 activators respectively, with and without STAT1 or STAT3 specific siRNAs. We conducted RNA-sequencing and ChIP-sequencing following treatment to delineate direct and indirect target genes for STAT1 and STAT3. We generated a STAT1 and STAT3 specific gene signature and applied them to publicly available datasets to test their predictive and prognostic ability. Results: Using these global and unbiased datasets, we identified STAT1 and STAT3 specific pathways, developed STAT1 and STAT3 specific gene signatures, and applied these signatures to publicly available ovarian cancer patient data. We determined the STAT1 and STAT3 signature’s associations with tumor development, chemotherapy responsiveness and long-term patient outcomes. Conclusions: Our studies have further informed the precise mechanisms of STAT1 and STAT3 action in the two most common subtypes of ovarian cancer and have defined gene signatures that are both prognostic and predictive.

overall_design: MDAH and OVSAHO (OVS) cells treated with control, STAT1, or STAT3 siRNA for 72 hours followed by 2 hours of treatment with DMSO vehicle (Veh), 10ng/μl IFNγ, or 30ng/μl of IL6 in triplicate.

first: John

last: Hawse


title: Integrated single-cell and spatial analysis identifies context-dependent myeloid-T cell interactions in head and neck cancer immune checkpoint blockade response

city: Madison

first: Huy

last: Dinh

state: WI

address: 1111 Highland Avenue

institute: University of Wisconsin-Madison

summary:
Background Approximately 15-20% of head and neck cancer squamous cell carcinoma (HNSCC) patients respond favorably to immune checkpoint blockade (ICB). Previous single-cell RNA-Seq (scRNA-Seq) studies identified immune features, including macrophage subset ratios and T-cell subtypes, in HNSCC ICB response. However, the spatial features of HNSCC-infiltrated immune cells in response to ICB treatment need to be better characterized.
Methods Here, we perform a systematic evaluation of cell interactions between immune cell types within the tumor microenvironment using spatial omics data using complementary techniques from both 10X Visium spot-based spatial transcriptomics and Nanostring CosMx single-cell spatial omics with RNA gene panel including 435 ligands and receptors. In this study, we used integrated bioinformatics analyses to identify cellular neighborhoods of co-localizing cell types in single-cell spatial transcriptomics and proteomics data. In addition, we used both publicly available scRNA-Seq and in-house spatial RNA-Seq data to identify spatially constrained Ligand-Receptor interactions in Responder patients.
Results With 522,399 single cells profiled with both RNA and protein from 26 patients, in addition to spot-resolved spatial RNA-Seq from 8 patients treated with ICB together with bioinformatics analysis of publicly available single-cell and bulk RNA-Seq, we have identified a spatial and cell-type specific context-dependency of myeloid and T cell interaction difference between Responders and Non-Responders. We defined further cellular neighborhood and the sources of chemokine CXCL9/10-CXCR3 interactions in Responders, emerging targets in ICB, as well as CXCL16-CXCR6, CCL4/5-CCR5, and other underappreciated and potential markers and targets for ICB response in HNSCC. In addition, we have contributed a rich data resource of cell-cell Ligand Receptor interactions for the immunotherapy and HNSCC research community.
Discussion Our work provides a comprehensive single-cell and spatial atlas of immune cell interactions that correlate with response to ICB in HNSCC. We showcase how integrating multiple technologies and bioinformatics approaches can provide new insights into potential immune-based biomarkers of ICB response. Our results suggested refining future studies using preclinical animal models in a more context-specific manner to elucidate potential underlying mechanisms that lead to improved ICB responses.
What is already known on this topic Most cancer patients still do not experience clinical benefits from immune checkpoint blockade (ICB), necessitating the development of response biomarkers and new immunotherapeutic targets.
What this study adds Here, we use integrated high-dimensional omics and bioinformatics approaches to identify immune cell-cell interaction markers associated with ICB response in patients with Head and neck squamous cell carcinoma.
How this study might affect research, practice or policy We identified spatial and cell-type specificity of Ligand-Receptor interactions between myeloid and T cells in ICB Responder patients that may help inform further mechanistic studies and biomarker development

overall_design: Spatial transcriptomic profiling using 10X Visium was performed on 8 head and neck cancer patient samples for identification of cellular communities and communication that differentiates immunotherapy responders and non-responders

first: Athena

middle: E

last: Golfinos-Owens

first: Taja

last: Lozar

first: Parth

last: Khatri

first: Rong

last: Hu

first: Paul

middle: M

last: Harari

middle: F

last: Lambert

first: Megan

middle: B

last: Fitzpatrick

middle: Q


title: Schizophrenia-linked gene expression changes across cortical layers and cellular microenvironments in human prefrontal cortex [Xenium]

city: Baltimore

first: Keri

last: Martinowich

state: Maryland

address: 855 N Wolfe St Suite 300

institute: Lieber Institute for Brain Development

summary:
Molecular mechanisms underlying dorsolateral prefrontal cortex (dlPFC) dysfunction in schizophrenia (SCZ) are poorly understood. dlPFC cell types are spatially organized across the six layers into functional microcircuits, which regulate cognitive and emotional processes that are implicated in SCZ. While regional specificity across cortical layers and cell types has been demonstrated for some SCZ-linked genes, spatially-resolved transcriptomics (SRT) can more definitively map molecular associations of disease. We investigated spatial gene expression changes in the human dlPFC from neurotypical control (n=31) and SCZ (n=32) brain donors using the Visium platform with incorporation of immunostaining to label perineuronal nets, neurons and vasculature. SCZ-associated DEGs were then mapped across these labeled cellular microenvironments and cortical layers. Major transcriptional alterations were identified in synaptic and neuroimmune pathways, which we localized to neuropil and glia-enriched domains. Integrative analysis with bulk and single-cell RNA studies highlighted distinct roles for spatially-localized glial cell populations, and identified enrichment of novel DEGs in endothelial cells and microglia. These findings were supported by enrichment of SCZ genetic risk across similar domains, and association of laminae-specific transcription factors to SCZ risk variants. Cellular resolution SRT using Xenium extended laminar disease associations to spatially-organized cell types. The findings highlight unique advantages of SRT to identify novel SCZ-related biology, particularly for cellular populations and compartments that may be missed with alternative technologies. We provide data resources to enable sample-level visualization of the spatial transcriptomics data (Samui) and to explore SCZ-associated DEGs across spatial domains (iSEE-layer adjusted) and cellular microenvironments (iSEE-Neuropil, iSEE-Neuron, iSEE-vasculature, iSEE-PNNs).
The primary goal of the Xenium experiments was to cross-validate layer-adjusted schizophrenia-associated differentially expressed genes previously identified by Visium spatial transcriptomics at enhanced cellular resolution, along with a select set of previously reported SCZ-associated genes and risk factors.

overall_design: For Xenium experiments, 24 donors (neurotypical controls [NTC], 5 males and 7 females; schizophrenia [SCZ], 6 males and 6 females) were selected from the previously studied cohort of 63 donors analyzed with Visium, and the same tissue blocks were cryosectioned. For each of the 24 donors, one tissue section was obtained (n = 1 tissue section per donor; total 24 tissue sections). Xenium tissue slides were prepared and processed on the Xenium Analyzer (10x Genomics, PN-1000481) using the v1 workflow (10x Genomics, Pleasanton, CA). Slides were handled in pairs (each slide containing 3 sections; 6 sections per experimental run), with one slide comprising two NTC and one SCZ tissue sections, and the other comprising two SCZ and one NTC tissue sections. This design balanced the number of NTC and SCZ samples (3 each per experimental run) across four total experimental runs, minimizing potential batch effects. Procedures for cryosectioning and slide storage were carried out according to the protocol CG000579 rev C. A custom panel of 300 genes was designed to characterize spatial gene expression patterns at enhanced cellular resolution using the 10x Genomics Xenium platform. The 300-gene panel included four categories: (1) 168 layer-adjusted DEGs, (2) 85 cortical layer marker genes, (3) 18 SCZ-associated risk genes previously identified in GWAS and published literature, and (4) 35 broad brain cell-type markers, covering excitatory neurons, inhibitory neurons, astrocytes, oligodendrocytes, microglia, endothelial cells, and immune cells.

first: Sang-Ho

last: Kwon

first: Boyi

last: Guo

first: Cindy

last: Fang

first: Madhavi

last: Tippani

first: Svitlana

middle: V

last: Bach

middle: A

last: Miller

first: Sarah

last: Maguire

first: Madeline

middle: R

last: Valentine

first: Seyun

last: Oh

first: Aarti

last: Jajoo

first: Artemis

last: Iatrou

first: Vijetha

last: Balakundi

first: Yufeng

last: Du

first: Annie

last: Nguyen

first: Ruth

last: Zhang

first: Uma

last: Kaipa

first: Heena

last: Divecha

first: Jashandeep

middle: S

last: Lobana

first: Joel

last: E Kleinman

first: Nikolaos

middle: P

last: Daskalaskis

first: Thomas

last: Hyde

first: Stephanie

middle: C

last: Page

first: Kristen

last: Maynard

last: Hicks


title: STAT1 and STAT3 Signaling in Ovarian Cancer: Prognostic Biomarkers and Therapeutic Targets

first: Esther

last: Rodman

type: Genome binding/occupancy profiling by high throughput sequencing

overall_design: MDAH and OVSAHO (OVS) cells treated with vehicle (Veh), 10ng/μl IFNγ, or 30ng/μl of IL6 in triplicate for 30 minutes. ChIP was performed with STAT1 (#SC417, Santa Cruz Biotechnology, Dallas, TX) and STAT3 (#CST9139, Cell Signaling, Danvers, MA).

first: John

last: Hawse


title: Phase separation of Heterochromatin protein 1 (HP1) controls virulence gene silencing in Plasmodium falciparum

city: Pune

first: Krishanpal

last: Karmodiya

state: Maharashtra

address: IISER PUNE, Dr. Homi Bhabha Road, Pashan

country: India

institute: IISER Pune

summary: Malaria caused by Plasmodium falciparum involves antigenic variation on the infected red blood cells through mutually exclusive expression of var genes. The repressed var genes have a distinct genome organization where they localize as a cluster near the nuclear periphery and are bound by PfHP1 (P. falciparum Heterochromatin Protein 1). However, the mode of regulation of var genes by PfHP1 remains unclear. In this study, we show that PfHP1 undergoes liquid-liquid phase separation (LLPS) in vitro in an RNA and DNA-dependent manner. Single-molecule DNA tethering experiments further revealed that AT-rich DNA sequences act as nucleation sites for the assembly and compaction of PfHP1-mediated heterochromatinization. We have also identified point mutations in the IDRs (intrinsically disordered regions) of PfHP1 that disrupt its phase separation as well as DNA compaction in vitro. An ectopic expression of PfHP1 phase separation and DNA compaction mutants led to dispersed nuclear localization, in contrast to the punctate appearance of the wild-type protein and altered chromatin binding at var genes. These results were corroborated with DiCre/loxP based conditional expression of the same PfHP1 mutants, which also led to the de-repression of multiple var genes (as many as 54 out of 60 var genes), mimicking the phenotype of PfHP1 depletion mutants. To assess the dynamic properties of PfHP1 condensates in vivo, we performed fluorescence recovery after photobleaching (FRAP) in live parasites, which revealed rapid fluorescence recovery, supporting their liquid-like behaviour. In conclusion, our study demonstrates the role of PfHP1 mediated LLPS in formation of heterochromatin and var gene silencing; unveiling a fundamental mechanism that drives antigenic variation in P. falciparum.

overall_design: ChIP-seq of PfHP1 and histone modifications in Plasmodium falciparum lines in ring stage. The mutant lines of PfHP1 are also subjected ChIP-seq.

first: D

last: Mamatharani


title: An epigenomic investigation of DNA methylation levels in matched left- and right- atrial fibrillation human cohort

city: London

first: Diego

last: Villar

address: 4 Newark Street, Blizard Institute

country: United Kingdom

institute: Queen Mary University of London

type: Methylation profiling by genome tiling array

summary: Atrial fibrillation (AF) is the most common form of cardiac arrhythmia, approximately affecting 2% of individuals worldwide with recent epidemiological reports expecting cases to double over the next two decades. Currently, there is a critical need to improve prevention and clinical management of AF patients. Recent genome-wide association studies have revealed over 150 genetic loci associated to AF susceptibility, the majority of which reside within non-coding portions of the human genome, suggesting they are involved in gene regulatory processes.   To investigate patient-specific epigenomic changes in AF, we employed epigenomic approaches across a set of patient atrial samples and sinus rhythm controls, comprising paired left and right atrial tissue. Here, we report profiling of DNA methylation levels, which we integrated with histone modification data and matched gene expression profiles. Interestingly, we identified AF-specific epigenomic regions associating with concordant gene expression and methylation patterns pointing to novel candidate disease loci. Additionally, we further validated candidate regions of interest in an independent cohort using quantitative PCR.

overall_design: The submission includes DNA methylation profiles on the EPIC array v2 Illumina platform, corresponding to left- and right- heart atrium biopsies across 7 atrial fibrillation and 4 synus rhythm controls (16 samples in total, 15 male and 1 female). Variables under investigation were anatomical side (right atrium or left atrium) and disease status (atrial fibrillation or synus rythm). Most of the samples were male and we therefore did not assess sex-specific changes.

first: Adrian

last: Rodriguez Gonzalez

last: Frost

first: Alison

last: Thomas

first: Andrew

last: Tinker


title: Functional genomics analysis of developing zebrafish and human endoderm reveals highly conserved cis-regulatory modules controlling vertebrate organogenesis [ATAC-seq]

city: Coventry

last: Nelson

address: Gibbet Hill Road

department: School of Life Sciences

institute: University of Warwick

summary: While vertebrate species are superficially diverse, they share key commonalities in terms of overall morphology, and organ configuration and function. Maintenance of these traits during evolution is partially explained by conservation of critical genes governing embryonic development. However, for conserved genes to deliver consistent developmental outcomes between species, similar gene regulatory programmes and gene expression patterns must also be maintained. The endoderm germ layer makes major contributions to the respiratory and gastrointestinal tracts, and associated organs including liver and pancreas. We used functional genomics approaches to identify highly conserved endodermal cis-regulatory modules (CRMs) functioning across the 400 million years of evolution separating zebrafish and humans. Our analyses suggest that there are few endoderm-specific CRMs, with many CRMs governing pancreas developmental also likely acting within the nervous system. Furthermore, these highly conserved CRMs are strongly enriched for binding sites of “neuro-pancreatic” transcription factors governing both pancreas and nervous system development, potentially suggesting function across these distinct organ systems. Additionally, we identify highly conserved CRMs likely participating endodermal patterning of adjacent craniofacial structures and sensory tissues. The highly conserved CRMs we identify are characterised by conserved patterns of transcription factor binding site co-occurrence. However, they are not characterised by rigid arrangement of binding sites, suggesting more complex or individual grammatical rules. Overall, our analyses provide key insights into critical gene regulatory control during vertebrate endoderm organogenesis, and define a compendium of highly conserved CRMs that should be prioritised for analysis of neuro-pancreatic gene transcriptional control, and anterior embryonic patterning.

overall_design: ATAC-seq analysis of cell populations isolated from zebrafish at 28 and 48 hours post-fertilisation using fluorescence activated cell sorting.  Samples are enriched based on sox17:GFP  reporter expression relative to endothelial and erythrocyte markers (kdrl:cherry and gata1a:dsRed respectively).  Sox17E is endoderm-enriched; sox17M is sox17+ hematovascular cells; sox17N is sox17-negative lineages.

first: Daniela

last: Figiel

first: Randa

last: Elsayed

first: Mark

middle: D

last: Walsh


title: Phase separation of Heterochromatin protein 1 (HP1) controls virulence gene silencing in Plasmodium falciparum [RNA-seq]

summary: Malaria caused by Plasmodium falciparum involves antigenic variation on the infected red blood cells through mutually exclusive expression of var genes. The repressed var genes have a distinct genome organization where they localize as a cluster near the nuclear periphery and are bound by PfHP1 (P. falciparum Heterochromatin Protein 1). However, the mode of regulation of var genes by PfHP1 remains unclear. In this study, we show that PfHP1 undergoes liquid-liquid phase separation (LLPS) in vitro in an RNA and DNA-dependent manner. Single-molecule DNA tethering experiments further revealed that AT-rich DNA sequences act as nucleation sites for the assembly and compaction of PfHP1-mediated heterochromatinization. We have also identified point mutations in the IDRs (intrinsically disordered regions) of PfHP1 that disrupt its phase separation as well as DNA compaction in vitro. An ectopic expression of PfHP1 phase separation and DNA compaction mutants led to dispersed nuclear localization, in contrast to the punctate appearance of the wild-type protein and altered chromatin binding at var genes. These results were corroborated with DiCre/loxP based conditional expression of the same PfHP1 mutants, which also led to the de-repression of multiple var genes (as many as 54 out of 60 var genes), mimicking the phenotype of PfHP1 depletion mutants. To assess the dynamic properties of PfHP1 condensates in vivo, we performed fluorescence recovery after photobleaching (FRAP) in live parasites, which revealed rapid fluorescence recovery, supporting their liquid-like behaviour. In conclusion, our study demonstrates the role of PfHP1 mediated LLPS in formation of heterochromatin and var gene silencing; unveiling a fundamental mechanism that drives antigenic variation in P. Falciparum.

overall_design: Studying the effect of PfHP1 mutation on virulence genes by RNA sequencing.

first: Mamatharani

last: D. V.


title: PAMD-Ch17, a Polymeric Analog of Plerixafor, Induces Mitochondrial Dysfunction in T-ALL Cells Independent of CXCR4

city: Omaha

first: Kate

last: Hyde

state: NE

address: 985802 Nebraska Medical Center

institute: University of Nebraska Medical Center

summary: PAMD-Ch17 is a polymer composed of the CXCR4 inhibitor AMD3100/Plerixafor with a cholesterol modification. In previous work, we showed that PAMD-Ch17, but not AMD3100, induces cell death and differentiation in mouse Acute Myeloid Leukemia cells. To investigate the mechanism of PAMD-Ch17’s novel anti-leukemic effects, we tested PAMD-Ch17 against a panel of human leukemia cell lines and found that PAMD-Ch17 is effective against a variety of acute leukemias, with T-ALL cell lines being highly sensitive. Surprisingly, CXCR4 knock out T-ALL cells were equally sensitive to PAMD-Ch17. Using a fluorescently tagged PAMD-Ch17, we found that the drug colocalized to the mitochondria. We also found that PAMD-Ch17 induced changes in expression of genes related to mitochondrial function, increased levels of mitochondrial superoxide, and decreased mitochondrial membrane potential. Using seahorse assays, we found that PAMD-Ch17 decreased baseline oxygen consumption, ATP production, and proton leakage. PAMD-Ch17 also decreased baseline extracellular acidification rate, indicating a decrease in overall metabolism. In mouse primary T-ALL but not healthy bone marrow cells, PAMD-Ch17 induced both mitochondrial superoxide and cell death. Using human bone marrow organoids, we found that PAMD-Ch17 induced mitochondrial superoxide and cell death in human primary T-ALL cells, but not in healthy stromal and hematopoietic cells. Collectively, our results indicate that PAMD-Ch17 has anti-leukemic effects against T-ALL cells but not healthy cells, likely mediated through a CXCR4 independent, mitochondrial based mechanism. These findings support further development of PAMDs as potential therapeutics for patients with T-ALL.

overall_design: To compare PAMD-Ch17 treated Jurkat cells (PAMD-Ch17 Treated Jurkat Cells 0.6uM) to wild type Jurkat cells (Wild Type Jurkat Cells Untreated), cells were treated with either 0.6uM of PAMD-Ch17 or vehicle for 24 hours. To compare CXCR4 Knock Out Jurkat cells (CXCR4 KO 2) to wild type Jurkat cells (Wild Type Jurkat Cells), cells were plated in fresh media for 24 hours. Triplicates were plated for each condition. Wild type Jurkat cells or untreated wild type Jurkat cells serve as the control for the experiments.

first: Calvin

last: Lam

first: Arjun

last: Dhir

first: Chinmay

last: Jogdeo

first: Sipra

last: Panda

first: Ekta

last: Kapoor

first: Siyuan

last: Tang

first: Victor

last: Rivero

first: Erin

last: McIntyre

first: Donald

last: Coulter

first: Peng

last: Xiao

last: Black

first: Kyle

last: Hewitt

first: Samantha

last: Swenson

first: Svetlana

last: Romanova

first: David

last: Oupicky

first: R

middle: K


title: NAND Hybrid Riboswtich Design by Deep Batch Bayesian Optimization

city: Darmstadt

first: Daniel

last: Kelvin

address: Merckstrasse 25

department: Centre for Synthetic Biology

country: Germany

institute: Technical University of Darmstadt

summary: The design of large genetic circuits requires genetic regulatory devices capable of performing complex logic operations. Hybrid riboswitches, synthetically enhanced compact RNA elements (<100 nucleotides) that form a tertiary structure with the ability to specifically bind two different target molecules, can be used to design genetic regulators that emulate Boolean logic. When inserted into the 5' UTR of an mRNA, these devices can regulate translation initiation upon specific binding of one or both ligands. The goal of this study is to design hybrid riboswitches that emulate Boolean NAND logic in yeast. We propose a novel machine learning-based design framework combining high-throughput in vivo screening and deep Bayesian optimization. Through an initial screening, we discovered a hybrid riboswitch with NAND behavior. Using batch Bayesian optimization with an ensemble neural network as surrogate, we further improve the NAND functionality of our hybrid riboswitch with respect to a performance score, thereby achieving near digital NAND behavior. With its focus on model-based and score-driven design, our proposed method can complement experiment driven approaches by allowing fine grained adaptation of functionality, including constructs sensitive to single nucleotide changes.

overall_design: During the first screening a communication module (connecting stem between the two binding pockets of the hybrid ribowitch) conferring NAND behavior was discovered using a massive parallel reporter assay. The fluorescence range of GFP regulated by the construct was divded into 8 gates using controls, with the areas occupied by the controls being gated as well. This sorting grid was used for every ligand condition of the hybrid riboswitch (no ligand(wo), tetracycline(Tc), neomycin(Neo), both(TcNeo)). The communication module sequence was randomized and a yeast culture containing the pool sequence upstream of a gfp gene was inoculated for each ligand condition. Each culture was separatly sorted using the same grid and the plasmid DNA from every subpopulation was extracted afterwards. Construct sequences were amplified from the plasmid DNA and sequenced using the AMPLICON-EZ service form Azenta/GENEWIZ. The NGS data of each subpopulation under every ligand condition was then used to approximate the switching pattern of each sequence contained within the sequence pool. The entire screening process was repeated once (replicate). For the second screening the base stem of a construct containing a NAND communication module discovered during the first screening was randomized and the sequence pool was sorted for low GFP fluorescence in the presence of both ligands and different levels of GFP fluorescence in the absence of both ligands (P7: 20-40%; P8: 40-60%; P9: 60-80%; P10: 80-100%). Plasmid DNA was again extracted from each subpopulation and the randomized area was amplified to be analyzed via AMPLICON-EZ by Azenta/GENEWIZ.

first: Erik

last: Kubaczka

first: Marianna

last: Karava

first: Heinz

last: Koeppl

first: Beatrix

last: Suess


title: Post-mitotic transcriptional activation and 3D regulatory interactions show locus- and differentiation-specific sensitivity to cohesin depletion [Micro-C]

city: New York

first: UkJin

last: Lee

state: NY

address: 413 E 69th St

department: Sanford I. Weill Department of Medicine

institute: Weill Cornell Medicine

summary: Structural loops are known to collapse in the absence of cohesin, whereas regulatory loops largely persist with only minor transcriptional changes, leaving unresolved questions about cohesin’s role in establishing regulatory interactions during cell state transitions. To investigate this, we degraded RAD21, a core cohesin subunit, in mouse embryonic stem cells (ESCs) transitioning from mitosis to G1 under self-renewal or differentiation conditions. We performed Micro-C to map changes in 3D chromatin architecture in ESCs upon RAD21 depletion and p300/CBP inhibition.

overall_design: Micro-C of ESCs in G1 treated with DMSO, dTAG, or A485.

first: Effie

last: Apostolou

first: Alejandra

last: Laguillo-Diego


title: Post-mitotic transcriptional activation and 3D regulatory interactions show locus- and differentiation-specific sensitivity to cohesin depletion [4C-seq]

summary: Structural loops are known to collapse in the absence of cohesin, whereas regulatory loops largely persist with only minor transcriptional changes, leaving unresolved questions about cohesin’s role in establishing regulatory interactions during cell state transitions. To investigate this, we degraded RAD21, a core cohesin subunit, in mouse embryonic stem cells (ESCs) transitioning from mitosis to G1 under self-renewal or differentiation conditions. We performed high-resolution in situ 4C-seq around the promoters of cohesin-sensitive genes (Klf4, Tbx3, Myc, and Fosl2) in ESCs and EpiLCs.

overall_design: 4C-seq for selected genes in ESCs and EpiLCs with or without RAD21 depletion


title: Post-mitotic transcriptional activation and 3D regulatory interactions show locus- and differentiation-specific sensitivity to cohesin depletion [ChIP-seq]

summary: Structural loops are known to collapse in the absence of cohesin, whereas regulatory loops largely persist with only minor transcriptional changes, leaving unresolved questions about cohesin’s role in establishing regulatory interactions during cell state transitions. To investigate this, we degraded RAD21, a core cohesin subunit, in mouse embryonic stem cells (ESCs) under self-renewal or differentiation conditions. We performed ChIP-seq for H3K4me3, H3K27ac, RAD21 and HA.

overall_design: Characterization of H3K4me3 and H3K27ac marked regions and RAD21 binding regions with ChIP-seq in ESCs and EpiLC


title: Phase separation of Heterochromatin protein 1 (HP1) controls virulence gene silencing in Plasmodium falciparum [HiC]

first: Krishanpal

last: Karmodiya

overall_design: We performed HiC-seq on PfHP1 mutants to study the effect of its phase separation on genome organization and var gene clustering.

first: Karmodiya

last: Krishanpal

first: D. V.

last: Mamatharani


title: Simultaneous profiling of native-state proteomes and transcriptomes of brain cell types using proximity labeling

city: Atlanta

first: Christina

last: Ramelow

address: 615 Michael St RM 375

institute: Emory University

type: Non-coding RNA profiling by high throughput sequencing

summary: Phenotyping cells at transcriptomic and proteomic levels is an essential step to understanding cellular contributions to development, aging, injury, and disease. Since proteome and transcrip-tome level abundances modestly correlate, complementary profiling of both is needed. We report a method called simultaneous protein and RNA -omics (SPARO) to capture the cell type-specific transcriptome and proteome simultaneously in vitro and in vivo. SPARO leverages TurboID to biotinylate RNA-interacting cytosolic proteins, enabling enrichment of proteins for proteomics and protein-associated RNA for transcriptomics. We validate SPARO first using well-controlled in vitro systems to verify that the proteomes and transcriptomes obtained reflect the global proteo-mes and transcriptomes. The effect of neuroinflammatory activation by lipopolysaccharide is al-so faithfully captured. We apply SPARO to obtain native-state proteomes and transcriptomes from astrocytes and neurons, thereby validating the approach in vivo. We interrogate mRNA-protein concordance and discordance, providing insights into molecular processes that exhibit uniform or cell type-specific patterns.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: Cell type-specific transcriptomes of BV2-microglia and HEK293 cells in vitro and Aldh1l1-expressing astrocytes and Camk2a-expressing neurons in vivo were obtained using the proximity labeling enzyme, TurboID, that biotinylates proteins that interact with RNA (e.g., ribosomal and RNA-binding proteins). Following streptavidin or A/G bead affinity purification, RNA was extracted and sent for RNA-sequencing. HEK293 cells expressing TurboID with and without a nuclear export sequence were contrasted. The TurboID-enriched and RiboTag-enriched astrocyte in vivo transcriptomes were also compared. MicroRNA-sequencing from cortical Aldh1l1-expressing astrocytes and the bulk cortex in vivo was also completed.

first: Srikant

last: Rangaraju

first: Steven

last: Sloan


title: Developmental reprogramming of the melanocortin neurons regulates energy homeostasis

city: Dallas

first: Li

last: Li

state: TX

address: 5323 Harry Hines Boulevard

institute: UT Southwestern Medical Center

summary: The central melanocortin neurons regulate satiety in mammalian species. Proopiomelanocortin (POMC) and agouti-related peptide (AgRP) neurons in the arcuate nucleus of the hypothalamus (ARH) play crucial and opposing roles in regulating food intake. POMC neurons promote satiety by releasing the anorexigenic peptide α-melanocyte-stimulating hormone (α-MSH), while AgRP neurons promote hunger through the release of the orexigenic peptides AgRP and neuropeptide Y (NPY). While distinct in function, these neurons arise, in part, from shared neuronal precursors during development. In mice, Pomc mRNA first appears in the developing hypothalamus at embryonic (E) day E10.5. The number of Pomc-expressing neurons gradually increases, reaching a peak around E13.5. However, Pomc expression in these neurons does not dictate terminal cell fate; instead, more than half of them lose Pomc expression as they adopt other peptidergic identities by the early postnatal period. Remarkably, some Pomc precursors begin expressing Npy, a marker for AgRP neurons, giving rise to a subset of adult AgRP neurons. However, the molecular mechanisms governing the fate switch between two antagonistic feeding neurons, and its physiological implications, have remained unknown.

overall_design: We conducted a single-cell multiomic analysis of Pomc-lineage neurons (hereafter referred to as PL neurons) in the adult mediobasal hypothalamus. To label PL neurons, we generated double transgenic mice (Pomc-Cre; R26LSL-Sun1-sfGFP), in which PL neurons were permanently marked by the expression of a nuclear GFP (Sun1-sfGFP). Under a stereomicroscope, we isolated the mediobasal hypothalamus from adult mice, extracted, and purified GFP+ nuclei via fluorescence-activated cell sorting (FACS).

first: Baijie

last: Xu

first: Chen

last: Liu


title: Post-mitotic transcriptional activation and 3D regulatory interactions show locus- and differentiation-specific sensitivity to cohesin depletion [ChIP-exo]

summary: Structural loops are known to collapse in the absence of cohesin, whereas regulatory loops largely persist with only minor transcriptional changes, leaving unresolved questions about cohesin’s role in establishing regulatory interactions during cell state transitions. To investigate this, we degraded RAD21, a core cohesin subunit, in mouse embryonic stem cells (ESCs) transitioning from mitosis to G1 under self-renewal or differentiation conditions. We performed ChIP-exo for H3K4me3 to map marked regions and for CTCF and RAD21 to map binding regions in ESCs.

overall_design: Characterization of H3K4me3-marked regions and CTCF and RAD21 binding regions with ChIP-exo in ESCs.


title: Immune response of human lung tissue after infection with human parainfluenza virus-3 – canonical pathways and treatment

city: Hannover

first: Maximilian

last: Fuchs

address: Carl-Neuberg-Str. 1

department: IMTTS

institute: Hanover Medical School

summary: Ex vivo human precision-cut lung slices (PCLS) were infected with human parainfluenza virus 3 (hPIV-3). We used microarry to explore the changes in gene expression between infected and non-infected, same-donor PCLS. We particularly analyzed changes in mRNA and miRNA expression levels linked to inflammation and antiviral immunity.

overall_design: Human PCLS were infected with hPIV-3 and RNA isolation was performed 72 hours post infection to see the effect of the viral infection upon gene expression

first: Danov

last: O.

first: Fuchs

last: M.

first: Obernolte

last: H.

first: Röpken

last: F.

first: Vollmer Barbosa

last: P.

first: Sewald

last: K.


title: Global and region-specific rules of airway epithelial development [Scgb1a1_FGFR]

city: Stockholm

first: Andreas

last: Liontos

state: Stockholm

address: Svante Arrheniusväg 20C

department: Molecular Biosciences-The Wenner Gren Institute

country: Sweden

institute: Stockholm University

summary: Evaluate the role of FGFR activity in the differentiation and maturation of bronchiolar secretory cells, postanatally.

overall_design:
Airways epithelial cells from Scgb1a1CREERT2 -/- ; FGFR2 flox/flox ; Rosa26R-Ai14/Ai9 and Scgb1a1CREERT2 +/- ; FGFR2 flox/flox ; Rosa26R-Ai14/Ai9 mice were isolated by Fluorescence-activated cell sorting (FACS) according to the presence of Epcam.
Epcam positive cells from Scgb1a1CREERT2 +/- ; FGFR2 flox/flox ; Rosa26R-Ai14/Ai9 mice were separated further by FACS based on the presence or absence of tdTomato signal. All isolated cells were analyzed using scRNAseq.

first: Alexandros

last: Sountoulidis

first: Alexandra

last: Firsova

first: Athanasios

last: Fysikopoulos

first: Janine

last: Koepke

first: Jonas

last: Theelke

first: Åsa

last: Björklund

first: Konstantin

last: Gaengel

first: Christer

last: Betsholtz

first: Werner

last: Seeger

first: Christos

last: Samakovlis


title: H3K27ac and H3K4me3 ChIP-Seq profiling of a human paired left and right atrial cohort of atrial fibrillation and healthy sinus rhythm individuals

city: LONDON

first: Adrian

last: Rodriguez

state: London

address: 4 Newark St

department: Blizard Institute - Genomics and Child Health

summary: H3K27ac and H3K4me3 ChIP-Seq profiling was performed on atrial appendages samples from 12 individuals aged over 60 years, either classified as persistent atrial fibrillation (AF) or normal sinus rhythm (SR). For all individuals, careful examination of medical history displayed no indications of hypertension, hypothyroidism or diabetes.

overall_design: Chromatin immunoprecipitation sequencing (ChIP-seq) for histone postranslational modifications H3K27ac and H3K4me3 in paired left and right atrium from 12 individuals (either classified as sinus rhythm controls or persistent AF). Average age: 71 ± 10.63; Gender (males, females): 11, 1.

first: Rodriguez

last: Adrian

first: Diego

last: Villar


title: Global and region-specific rules of airway epithelial development [Scgb1a1_Tom_Sftpc_GFP]

summary: Evaluate heterogeneity of the airway secretory cells, including club secretory, Scgb1a1/Sftpc double positive cells and alveolar secretory cells

overall_design: Airways epithelial cells from a double transgenic reporter mouse, Scgb1a1CREERT2/+; Rosa26RAi14/+; Sfptc;GFP/+ were isolated. Labelled cells were isolated using Fluorescence-activated cell sorting (FACS) according to the presence or absence of tdTomato and GFP. We isolated Tomato+ cells (club secretory cells), Tomato/GFP double positive cells (Scgb1a1/Sftpc double positive cells) and GFP positive cells (alveolar secretory cells). Tomato+ cells (club secretory cells) were further separated by FACS, based on podoplanin (Pdpn) expression.


title: Post-mitotic transcriptional activation and 3D regulatory interactions show locus- and differentiation-specific sensitivity to cohesin depletion.

first: UkJin

last: Lee

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

overall_design: Refer to individual Series


title: A pH-Responsive CS-HA-DAP Nanodelivery System Induces Mitotic Catastrophe and Mixed-Type Cell Death via Dual-Targeting of TOP2A/CHK1 for Triple Negative Breast Cancer Therapy

status: Public on Feb 15 2026

city: Changchun

first: Boyin

last: Jia

state: Jilin

address: 2888 Xincheng Street

department: College of Animal Science and Technology

country: China

institute: Jilin Agricultural University

summary: Triple-negative breast cancer (TNBC) is characterized by a highly aggressive nature and poor clinical prognosis, largely due to the absence of effective therapeutic targets. Although natural bioactive peptides hold tremendous promise for anti-tumor therapy, their clinical application is restricted by short in vivo half-lives and insufficient targeting specificity. Herein, we aimed to develop a smart nanodelivery system to deliver deer antler polypeptide (DAP) for the precise treatment of TNBC.We constructed a pH-responsive nanoparticle (CS-HA-DAP) comprising chitosan (CS)-coated hyaluronic acid (HA)-DAP conjugates. Tumor accumulation of the drug was enhanced via HA-mediated active targeting of CD44 receptors and the enhanced permeability and retention (EPR) effect of the nanocarrier. Through RNA sequencing (RNA-seq), molecular docking, and multi-dimensional molecular biology experiments, we systematically elucidated the anti-tumor targets and lethal mechanism of DAP. Furthermore, its biodistribution, therapeutic efficacy, and safety were evaluated in a 4T1 tumor-bearing mouse model. CS-HA-DAP exhibited excellent physicochemical stability and tumor-targeting capability. Mechanistic studies revealed, for the first time, that DAP functions as a topoisomerase II alpha (TOP2A) inhibitor to induce extensive DNA double-strand breaks; concurrently, it inhibits checkpoint kinase 1 (CHK1), leading to the functional loss of the S/G2 cell cycle checkpoint. This synergistic effect of DNA damage and repair blockade forces cancer cells harboring severe genomic injury to undergo mitotic slippage, ultimately driving them into irreversible mitotic catastrophe. Subsequent signaling cascades triggered a mixed-type cell death program. In vivo experiments confirmed that CS-HA-DAP significantly suppressed tumor growth without inducing notable systemic toxicity. In summary, we successfully constructed the CS-HA-DAP nanosystem and validated a novel anti-tumor mechanism driven by TOP2A/CHK1 dual-targeting-induced mitotic catastrophe and mixed-type death. This strategy not only overcomes the barriers to peptide druggability but also provides a promising, innovative solution for the treatment of refractory TNBC.

overall_design: 4T1 cells were collected from C_HA_DAP group and the control group. Total RNA pooled was collected from 4T1 cells of three individuals to construct a cDNA library. 4T1 cells transcriptomics were analyzed by Illumina NovaSeq 6000 for high-throughput deep sequencing.

first: Wei

last: Liu

first: Rui

last: Han

first: Chang

first: Xintong


title: Post-mitotic transcriptional activation and 3D regulatory interactions show locus- and differentiation-specific sensitivity to cohesin depletion [RNA-seq]

summary: Structural loops are known to collapse in the absence of cohesin, whereas regulatory loops largely persist with only minor transcriptional changes, leaving unresolved questions about cohesin’s role in establishing regulatory interactions during cell state transitions. To investigate this, we degraded RAD21, a core cohesin subunit, in mouse embryonic stem cells (ESCs) transitioning from mitosis to G1 under self-renewal or differentiation conditions. We performed RNA-seq to profile gene expression in ESCs and EpiLCs.

overall_design: RNA-seq to identify transcriptional changes upon RAD21 depletion or CBP/p300 inhibition in ESCs and EpiLCs.

first: Effie

last: Apostolou

first: Alejandra

last: Laguillo-Diego


title: Transcriptomic atlas of POSA mice versus normoxic controls

city: Houston

first: Akdes

last: Serin Harmanci

address: One Baylor Plaza

institute: Baylor College of Medicine

summary: We exposed p14 mice to 56 day of forced intermittent hypoxia as a proxy stimlulus for pediatric OSA. After this exposure, we performed sn-RNA seq of the hippocampus of both normoxic controls and IH exposed mice to study potentially DEGs.

overall_design: sn RNA sequencing of Pediatric OSA hippocampus

first: Williamson

last: Michael

first: Chandrakantan

last: Arvind

first: Serin Harmanci

last: Akdes


title: RNA-seq analysis of Tabanus nipponicus Salivary Glands

city: Obihiro

first: Noboru

last: Inoue

state: Hokkaido

address: 11 Nishi 2-sen, Inada-cho

department: National Research Center for Protozoan Diseases

country: Japan

institute: Obihiro University of Agriculture and Veterinary Medicine

summary: Tabanus nipponicus is a hematophagous insect species with high activity in summer, and its salivary gland secretions play a critical role in mediating successful blood-feeding. In this study, pooled salivary gland samples from three T. nipponicus individuals collected during peak summer activity were used for RNA-seq analysis. Total RNA was extracted under sterile and low-temperature conditions prior to sequencing. Paired-end sequencing generated 45,310,280 raw reads, with 88.75% of reads successfully mapped. De novo assembly yielded 35,175 unigenes, which were aligned to the Swiss-Prot database to obtain UniProt IDs. These IDs were mapped to the GO database, and classified into three GO categories; functional annotation revealed enrichment in metabolic, membrane-related biological processes, and binding/catalytic molecular activities, which underpin the synthesis and secretion of anticoagulant saliva components essential for blood-feeding adaptation and microbial stress resistance. Alignment with the KEGG database identified KEGG IDs, with unique KOs following mapping, and subsequent KEGG enrichment analysis was performed. This high-quality transcriptomic dataset represents the first gene expression profile for T. nipponicus, and builds on previous experimental foundations to provide valuable insights into key physiological processes in tabanids, including anticoagulant, blood meal digestion, antioxidant defense, mammalian host immune interaction, and microbial resistance.

overall_design: RNA-seq data from three pooled samples of salivary gland tissue from female Tabanus japonica

first: Keisuke

last: Suganuma

first: Ercha

last: Hu


title: HER2d16 promotes breast cancer metastasis through integrin-driven signaling

city: Montreal

first: Philippe

last: Roux

state: Quebec

address: 2950 Polytechnic Rd

country: Canada

institute: Université de Montréal

summary: The HER2d16 splice variant, generated by skipping exon 16 of the ERBB2 transcript, is frequently detected in aggressive HER2-positive breast tumors and has been associated with enhanced tumorigenicity. However, the mechanisms by which HER2d16 promotes metastasis remain poorly understood. Here, we demonstrate that HER2d16 expression in mammary epithelial cells drives a pro-migratory and pro-invasive phenotype both in vitro and in vivo. Through integrative cell surface proteomics and transcriptomic profiling, we identify a distinct upregulation of RGD-binding integrins, including integrin β3, in HER2d16-expressing cells. Functional analyses demonstrate that integrin β3 signaling is required for HER2d16-driven invasion, and that pharmacological inhibition of FAK downstream of integrin engagement significantly reduces metastatic dissemination in vivo. Together, these findings reveal a critical link between HER2d16 expression and integrin-FAK signaling programs that drive metastatic progression, highlighting integrin-FAK signaling as a therapeutically actionable vulnerability in HER2-driven breast cancer.

overall_design: RNA-seq profiling of MCF10A cells stably expressing HER2 WT, HER2Δ16, or an empty vector.

first: Guillaume

last: de Lhoneux

first: Jonathan

last: Boucher


title: NIK-Driven IL-23 Production by Myeloid Cells is a Key Factor in the Development of Autoimmune Inflammation [scRNA-seq]

city: Mainz

first: Matthias

last: Klein

address: Langenbeckstrasse 1

department: Inst. for Immunology

institute: University Medical Center Mainz

summary: We have employed a single cell sequencing approach with BD Rhapsody to study the differences within myeloid cells in the draining lymph nodes four days after MOG immunisation between NIKfl/fl control mice and NIK^CX3CR1 mice

overall_design: Following MOG immunisation cells were isolated from the inguinal lymph nodes at 5dpi. MACS sorting for CD90 and CD19 positive cells was used to remove T and B cells. enriched myeloid cells were analysed with scRNA-seq

first: Nishada

last: Ramphal

first: Ari

last: Waisman


title: Anaerobic riboflavin degradation by human gut Lachnospiraceae

city: Columbus

first: Justin

last: North

address: 484 W 12th Ave

department: Department of Microbiology

institute: Ohio State University

summary: Vitamins mediate a web of cross-feeding interactions in the human gut. Many Gram-positive gut microbes, in particular, are predicted to be vitamin auxotrophs. Previous studies of these microbes, however, have tended to use rich media, precluding controlled perturbations of low abundance nutrients. We tested the ability of diverse Lachnospiraceae, the most common Gram-positives in the gut, to grow on a chemically defined medium. Even though this medium contained riboflavin, we found that predicted riboflavin auxotrophs grew poorly, including the bile metabolizer Clostridium scindens. Riboflavin supplementation increased C. scindens final density in a concentration-dependent manner up to at least 200μM. Surprisingly, despite being an auxotroph, C. scindens also catabolized riboflavin into lumichrome. The only previously described catabolic pathway for riboflavin requires oxygen and has no homologs in C. scindens. In 200µM riboflavin, a single gene neighborhood with an aldolase, oxidoreductases, and a riboflavin kinase/adenylyltransferase was upregulated, suggesting an alternative anaerobic degradation or overflow pathway. Similar neighborhoods were detected in several other Lachnospiraceae, including Faecalicatena fissicatena, the only other anaerobe reported to degrade riboflavin. Reanalysis of published metabolomic data showed that in vivo, both riboflavin and lumichrome were more abundant in colonized (vs. germ-free) mouse ceca, and that in vitro, Lachnospiraceae isolates depleted riboflavin while certain Gram-negative isolates overproduced it. These results demonstrate that a member of the Lachnospiraceae can anaerobically convert an essential B vitamin into lumichrome, a molecule recently shown to have anti-inflammatory properties. Vitamin catabolism may both structure cross-feeding interactions in the gut and affect host health.

overall_design: Cultures of Clostridium scindens were grown anaerobically in defined media or defined media supplemented with riboflavin. The cultures were incubated anaerobically at 37C and samples were taken at 11, 12, 14, and 24 hours.

first: Christian

middle: J

last: Quiles-Pérez

first: Ami

last: Fofana

first: Kamal

last: Deep

first: Calissa

last: Carlisle

first: Patrick

middle: H

last: Bradley


title: Vascular STING activation facilitates NK cell anti-tumor immunity in small cell lung cancer [Spatial_Transcriptome]

city: Boston

first: Zhaorong

last: Li

address: 60 Fenwood Road

institute: Dana Farber Cancer Institute

summary: Small cell lung cancer (SCLC) typically displays a "cold" tumor microenvironment with a paucity of immune infiltrate. Neuroendocrine SCLC cells also profoundly repress MHC-I expression, rendering them vulnerable to NK cell-mediated cytotoxicity. Here, we confirm that neuroendocrine SCLC cells are sensitive to NK cell mediated attack, yet quantitative spatial profiling of the SCLC immune microenvironment in patient samples reveals that effector immune cells, including NK cells, are excluded from MHC-Ilow/neg SCLC regions. To study this biology, we develop dynamic single cell RNA sequencing of microphysiological immune tumor environments (DynaMITE-seq) and integrate findings with spatial transcriptomics in patient tissue, unveiling the microvasculature as a major checkpoint restricting NK cell extravasation/recruitment. We demonstrate that activation of vascular Stimulator of Interferon Genes (STING) signaling restores NK cell infiltration and killing of neuroendocrine SCLC, suggesting a strategy to overcome this key SCLC immunologic barrier and prime therapeutic response to DLL3-targeted CAR-NK cell therapy.

overall_design: Spatial transcriptomics of SCLC patient samples  from FFPE tissues (Visium platform)

first: Marco

last: Campisi

first: Ian

last: Dryg

first: Jason

last: Weirather

first: David

last: Barbie

first: Navin

last: Mahadevan


title: Vascular STING activation facilitates NK cell anti-tumor immunity in small cell lung cancer


title: Vascular STING activation facilitates NK cell anti-tumor immunity in small cell lung cancer [CHIP-seq RNA-seq]

overall_design:
CHIP-seq of the histone modification H3K27ac in SCLC lines H69EZ-G, H69EZ-GV (HLA negative vs HLA positive) compared to H69
RNA-seq of the histone modification H3K27ac in SCLC lines  H69EZ-GV (HLA negative vs HLA positive)

first: Sung Rye

last: Park

first: Caroline

middle: G

last: Fahey

first: Michael

middle: Y

last: Tolstorukov


title: Cohesin prevents local mixing of condensed euchromatic domains in living human cells

city: Cleveland

first: Le

last: Xiong

address: 9620 Carnegie Ave N Bldg

institute: Cleveland Clinic

summary: The human genome is folded into chromatin loops by the cohesin complex, forming functional chromatin domains that underlie transcription and DNA replication/repair. However, the physical properties of these domains in living cells, especially in active euchromatin, and how they are regulated by cohesin remain elusive. To address these questions, we combined singlenucleosome imaging/tracking and super-resolution 3D-structured illumination microscopy (3DSIM) with euchromatin-specific labeling of histone H3.3. Using this nanoscopic approach, we revealed that euchromatin is organized into condensed domains constrained by cohesin-mediated loops, challenging the established textbook view of euchromatin as largely open. Transcription machinery appear to be located near the condensed domain surfaces. Cohesin loss increased nucleosome fluidity of these domains without altering their compaction, leading to local mixing of domains and compromising transcriptional insulation. These findings uncover an unexpected physical role of cohesin in maintaining the integrity of condensed euchromatic domains and ensuring proper higher-order regulation of gene expression.

overall_design: ATAC-seq of human colorectal cancer cell line HCT116 with and without 5-Ph IAA treatment

first: Liangqi

last: Xie


title: Global and region-specific rules of airway epithelial development

first: Andreas

last: Liontos

summary: Lineage tracing experiment using a airway secretory specific Cre driver mouse. Airway secretory (club) cells were labelled from the beginning of the expression of the pansecretory marker Scgb1a1, at embryonic day 16.5 (E16.5) and samples were collected in four developmental stages (E19.5, P2, P21 and P60). Samples were collected right before activation of the Cre reporter, at E16.5, and served as control. We were interested to characterize the transcriptomic changes and uncover the molecular mechanisms that control the differentiation and maturation of the secretory cell lineage.

overall_design: Airways epithelial secretory cells from Scgb1a1CREERT2;Rosa26R-fGFP mice were isolated by Fluorescence-activated cell sorting (FACS) according to the presence of GFP, at E19.5, P2, P21 and P60 stages. E16.5 stage cell samples were isolated by FACS based on Epcam and CD45 (EpCam positive and CD45 negative cells were selected) expression.

first: Alexandros

last: Sountoulidis

first: Alexandra

last: Firsova

first: Athanasios

last: Fysikopoulos

first: Janine

last: Koepke

first: Jonas

last: Theelke

first: Åsa

last: Björklund

first: Konstantin

last: Gaengel

first: Christer

last: Betsholtz

first: Werner

last: Seeger

first: Christos

last: Samakovlis


title: A pioneering single-nucleus multi-omic atlas of Dermatophagoides farinae defines allergen origins and transcriptional regulators

city: Wuxi

first: YUBAO

last: CUI

state: Jiangsu

address: No. 299 at Qingyang Road

institute: The Affiliated Wuxi People's Hospital of Nanjing Medical University

summary: The cellular origins and transcriptional regulation of allergens from house dust mites (HDMs), a predominant source of indoor aeroallergens, remain poorly understood. To address this, we constructed the first single-nucleus multi-omic atlas of Dermatophagoides farinae to delineate its cellular composition, map allergen expression, and identify transcriptional regulators. We performed single-nucleus RNA-seq (snRNA-seq) and ATAC-seq (snATAC-seq) on whole mites, using clustering, functional enrichment, and integrative analyses to define cell types and chromatin landscapes. Focusing on the allergen Der f 9, candidate regulatory transcription factors (TFs) were validated by qPCR and dual-luciferase assays. snRNA-seq of 16,335 high-quality nuclei resolved six major cell types: Stromal_cell, Proliferative, Neuron1, Neuron2, Metabolism1, and Metabolism_lipid. Stromal_cells were identified as the dominant allergen source, with 28 of 40 allergen groups enriched, including major (Der f 1, Der f 2) and mid-tier (Der f 5, Der f 7, Der f 21) allergens. Integrated snATAC-seq confirmed distinct epigenomic states for each cell type and revealed a highly accessible promoter region for Der f 9. Motif analysis nominated Br-family TFs as putative regulators. Among six candidate br homologs identified, qPCR confirmed their expression in D. farinae. Dual-luciferase assays demonstrated that Def04G00825 and Def10G00316 significantly transactivated the Der f 9 promoter. In conclusion, this first single-nucleus atlas of D. farinae defines cellular heterogeneity, identifies Stromal_cells as the primary allergen reservoir, and validates TFs regulating Der f 9, thereby providing a foundational resource for understanding allergen biology and advancing targeted diagnostic and therapeutic strategies.

overall_design: snRNA-seq:We performed single-nucleus RNA-seq (16,335 nuclei) and ATAC-seq (7,150 nuclei) on whole Dermatophagoides farinae mites to construct a cellular atlas. Clustering and annotation defined six major cell types. Allergen expression was mapped via gene-set scoring, and chromatin accessibility analysis identified regulatory regions. Candidate transcription factors for Der f 9 were validated by qPCR and dual-luciferase assays. Three biological replicates were used throughout.

first: Yubao

last: Cui

first: Ying

last: Zhou

first: Ximeng

last: Ma

first: Xinhui

last: Gong

first: Cunyin

last: Yuan

first: Pavel

last: B. Klimov

first: Jing

last: Du

first: Dongmei


title: Vascular STING activation facilitates NK cell anti-tumor immunity in small cell lung cancer [scRNA-seq]

first: Zhaorong

last: Li

overall_design: A 3D microphysiological immune tumor environment (MITE) system comprises a microvascular network (MVN) composed of human umbilical vein endothelial cells (HUVEC) and human lung fibroblasts (HLF), surrounding MHC-Ilow/neg CORL47 SCLC (for 6 days). Human NK cells were perfused through the vasculature either without or with human monocytes, without or with the endogenous STING ligand, 2’3’-cGAMP (day 7, endpoint). Next, we developed a dynamic scRNAseq of the MITE (DynaMITE-seq) to investigate the phenotypes of the individual components of the MITE, effectively liberating and recovering cells from the extracellular matrix for scRNAseq at 7 days endpoint.  Experimental conditions (control; cGAMP; control + monocyte; and cGAMP + monocyte).

first: Marco

last: Campisi

first: Tatsuya

last: Osaki

first: David

last: Barbie

first: Navin

last: Mahadevan


title: RNA sequencing of slow-and fast-cycling epidermal stem cells vs hair follicle stem cells in murine back skin, tail skin vs oral epithelium

city: Kumamoto City

first: Yen

middle: Xuan

last: Ngo

state: Kumamoto

address: Honjo 2-1-1, Chuo-ku

department: International Research Center for Medical Sciences

institute: Kumamoto University

summary: This study was performed to compare transcriptomic changes in the heterogeneous mouse skin epidermal stem cells and mouse oral epithelial stem cells during homeostasis at young age. Slow-cycling stem cells (label retaining cells, LRCs), fast-cycling stem cells (non-label retaining cells, nLRCs) express unique gene signatures in both skin and oral tissues and have independent stem cell identities. Our data indicates that there are similarities and differences in gene expression patterns between LRC and non-LRC populations in different epithelial tissues, suggesting stem cell autonomous heterogeneity and environmental differences.

overall_design: mRNA expression analysis from slow-and fast-cycling epidermal stem cells in mouse back skin, tail skin, tongue and palate at 2 months old.

middle: X

first: Gopakumar

last: Changarathil

first: Hiromi

last: Yanagisawa

first: Aiko

last: Sada


title: Intraperitoneal BSN-37 activates peritoneal macrophages in Balb/c mice and reshapes the circRNA–mRNA transcriptome

city: xinxing

first: xiaojing

last: xia

address: hualandadao

institute: HENAN INSTITUTE OF SCIENCE AND TECHNOLOGY

summary: Our preliminary work indicates that the antimicrobial peptide BSN-37 exhibits immunostimulatory activity; however, its in vivo molecular mechanisms remain insufficiently defined. Here, using male Balb/c mice, we profiled mRNA and circRNA expression in peritoneal macrophages under optimized dosing and timing conditions. By integrating these in vivo datasets with our in vitro RAW264.7 results, we aimed to identify key circRNAs involved in BSN-37–mediated immune enhancement and to construct putative circRNA–miRNA–mRNA regulatory networks.Male Balb/c mice (6–8 weeks old) were randomly assigned to a control group or a BSN-37–treated group. To enrich peritoneal macrophages, mice received an intraperitoneal injection of 3% thioglycollate broth three days prior to sampling. The control group was then injected intraperitoneally with PBS, whereas the treatment group received BSN-37 (48 μg/mL, 0.2 mL per mouse). Twelve hours later, peritoneal lavage fluid was collected for macrophage isolation. Total RNA was extracted and subjected to quality control. CircRNA and mRNA transcriptomes were obtained using the Mouse circular RNA Array V2.0 and the Arraystar Mouse LncRNA Expression Microarray V3.0, respectively. Differentially expressed molecules were identified using the criteria of P < 0.05 and fold change > 2, followed by hierarchical clustering, GO/KEGG enrichment analyses, and ceRNA network construction. To reduce false positives, multiple prediction algorithms were applied and only intersecting targets were retained. Network visualization was performed using Cytoscape. Compared with the control group, a total of 149 differentially expressed mRNAs were identified in peritoneal macrophages from the BSN-37–treated group (104 upregulated and 45 downregulated), together with 20 differentially expressed circRNAs (6 upregulated and 14 downregulated). Volcano plots and hierarchical clustering heatmaps demonstrated a clear separation between the two groups at the transcriptomic level. Functional enrichment analyses indicated that these differentially expressed molecules were involved in multiple biological processes and signaling pathways related to immune regulation. By further integrating in vivo and in vitro datasets, we constructed a circRNA–miRNA–mRNA tripartite regulatory network comprising 5 circRNAs, 6 miRNAs, and 50 mRNAs, providing an omics-based foundation for prioritizing key candidate circRNAs and subsequent mechanistic validation. BSN-37 markedly reshapes the mRNA/circRNA expression landscape of peritoneal macrophages in vivo and establishes immune regulation–associated molecular networks. The candidate circRNAs and ceRNA network identified here offer important clues for elucidating the immunostimulatory mechanisms of BSN-37 and for screening critical regulatory targets.

overall_design: Experimental design 1) Animals and grouping Animals: 36 healthy male Balb/c mice (6–8 weeks old; 25 ± 2 g). Grouping: Randomly assigned to two groups: Control (PBS) and BSN-37; 18 mice per group. Biological replicates: Every 6 mice were pooled as one biological replicate; 3 biological replicates per group (n = 3/group). Housing conditions: One-week acclimation; 12 h light/12 h dark cycle, with ad libitum access to food and water. 2) Peritoneal macrophage collection and treatment workflow Macrophage elicitation: Three days before sampling, mice were intraperitoneally injected with 2 mL of 3% thioglycollate broth at a fixed time each day (injections may be split to reduce irritation). In vivo treatment: Control group: Intraperitoneal injection of 0.2 mL PBS per mouse. BSN-37 group: Intraperitoneal injection of BSN-37 solution (48 μg/mL), 0.2 mL per mouse. Sampling time point: Mice were euthanized 12 h post-injection. Peritoneal lavage and cell collection: The peritoneal cavity was lavaged in multiple rounds with ice-cold PBS and lavage fluid was collected. Cells were pelleted by centrifugation at 4℃, 1,000 rpm for 10 min, followed by three PBS washes. Adherence purification: Cells were resuspended in RPMI-1640 containing 10% FBS, plated in six-well plates, and allowed to adhere for 4 h. Medium was then replaced to further enrich macrophages. RNA sample preparation: After PBS washing, cells were lysed with TRIzol (500 μL–1 mL per well). Lysates were collected into RNase-free tubes and stored at −80℃. Key control points: During intraperitoneal injection and lavage, avoid puncturing the intestine or organs to prevent contamination; if substantial erythrocyte contamination is present, apply red blood cell lysis buffer as needed. 3) RNA extraction and microarray profiling RNA extraction: Total RNA was extracted using the TRIzol method. Quality control: RNA concentration and purity were assessed (e.g., A260/280). Only qualified samples proceeded to microarray analysis. Platforms: circRNA: Mouse circular RNA Array V2.0 mRNA: Arraystar Mouse LncRNA Expression Microarray V3.0 (used to obtain the mRNA expression profile) Differential-expression threshold: P < 0.05 and mean fold change > 2. 4) Bioinformatics and network analysis Visualization: Volcano plots for differentially expressed mRNAs and circRNAs; hierarchical clustering and heatmap visualization of the two groups. Functional enrichment: GO and KEGG enrichment analyses were performed separately for host genes of differential circRNAs and for differential mRNAs. Integrated in vivo–in vitro screening: In vivo transcriptomic data from peritoneal macrophages were integrated with in vitro datasets from RAW264.7 cells to identify candidates showing overlap or consistent directional changes. ceRNA network construction: Multiple algorithms were used to predict circRNA–miRNA interactions and miRNA–mRNA targeting. Only intersecting predictions were retained to reduce false positives. A circRNA–miRNA–mRNA tripartite network was constructed and visualized in Cytoscape (example output: 5 circRNAs, 6 miRNAs, and 50 mRNAs). 5) Statistical analysis Appropriate software was used for differential-expression and enrichment analyses. Statistical significance was set at P < 0.05. The specific statistical tests and multiple-testing correction procedures followed standard microarray analysis workflows.

first: Xiaojing

last: Xia

first: Xujie

last: Zhao

first: Yanhe

last: Lv

first: Bingze

last: Jiao

first: Haipeng

first: Ke

last: Ding

first: Jianhe

last: Hu


title: Discovery of a Linked Constellation of Gene Expression Revealed by Local Editing of Fibroblasts in Tumors

city: San Francisco

first: Nicholas

middle: F

last: Kuhn

address: 513 Parnassus Ave, HSW518

department: Pathology

institute: University of California, San Francisco

summary: Fibroblasts play critical roles in regulating cellular relationships during tissue homeostasis, immunity, and tumor biology at multiple sites. However, tools to perturb fibroblasts at just one site in vivo are limited, restricting our understanding of how these cellular relationships develop on a local level. We optimized local gene editing of fibroblasts in multiple mouse tumor models to investigate how locally restricted fibroblast perturbations affect the cellular tumor microenvironment (TME). By knocking out surface receptors Osmr, Tgfbr2, or Il1r1 on cancer-associated fibroblasts (CAFs), we uncover that TGFBR2 signaling loss uniquely induces the emergence of a Col18a1hi CAF cell state that is distinct from previously described fibroblast states and is associated with worse survival in human PDAC patients. Further application of a local as well as combinatorial gene knockout technology in CAFs reveals a circuit in which these Col18a1hi CAFs reshape the TME by recruiting Siglec-Fhi neutrophils via Cxcl5 expression; and that the Col18a1hi CAF cell state is further dependent on fibroblast TNFR1 and canonical Wnt signaling. Together, a fast, affordable, and modular engineering method is demonstrated, allowing discovery of a modified fibroblast identify, and through this the network details of a local inter-cellular circuitry in a tumor.

overall_design: live cells from s.c. grown pancreatic tumors were sorted by FACS after AAV-mediated knockout in cancer-associated fibroblasts and then analyzed using scRNA-seq (10x Genomics FLEX)

first: Matthew

last: Krummel


title: Regulation of cell identity by 3D super-enhancers [WGS]

city: Memphis

first: Brian

last: Abraham

state: TN

address: 262 Danny Thomas Blvd

department: Computational Biology

institute: St. Jude Children's Research Hospital

summary: Gene regulation and genome structure are intimately linked, but accurate predictions of the elements that form topological regulatory assemblies in mammalian genomes remain elusive.  To address this challenge, we developed BOUQUET, a graph theory-derived algorithm that uses multiple facets of genome topology to discern and interrogate communities of cis-regulatory elements (CREs). BOUQUET’s unbiased approach reveals otherwise undetectable CRE communities, including those that cross presumed insulating boundaries and comprise different categories of CRE. Critically, some communities, which we term “3D super-enhancers (3D-SEs)”, accrue exceptional amounts of regulatory apparatus. 3D-SEs are associated with cell identity-defining genes across mammalian tissues. Microscopy analyses show co-localization and co-expression of genes from the same 3D-SE community within transcriptional co-factor puncta, resembling microcompartments. Gene pairs within 3D-SE communities are co-expressed, and CREs within 3D-SEs are co-accessible. Overall, our model more accurately represents the architectural complexity of CRE interactions, the coordination between chromatin conformation and gene regulation, and the connection between super-enhancers and cell identity.

overall_design: genome-wide gene expression profiling of wild type and Mir290 enhancer-deleted mESC v6.5 cell line is conducted by performing RNA-seq

first: Jie

first: Kelsey

last: Maher

first: Alaguraj

last: Veluchamy

first: Yuna

last: Kim

first: Li

last: Dong

first: Bensheng

last: Ju

first: Virginia

last: Valentine

first: Marc

first: Steven

last: Burden

first: John

last: Easton

first: Stanley

middle: B

last: Pounds


title: Aberrant medial ganglionic eminence (MGE) GABAergic neurogenesis contributes to Huntington’s disease pathogenesis

city: Bronx

first: Aldrin

middle: Enrique

last: Molero

state: US-NY

address: 1410 Pelham Parkway S

department: Neurology

institute: Albert Einstein College of Medicine

summary: This study used single-cell RNA sequencing to characterize early transcriptional alterations in medial ganglionic eminence (MGE) development caused by mutant huntingtin expression in a Huntington’s disease mouse model. Single-cell transcriptomes were generated from fluorescence-activated cell sorted (FACS) Dlx1-eGFP–positive MGE cells isolated from embryonic day 12.5 (E12.5) BACHD and wild-type control embryos. To control for biological variability, MGE tissue from eight embryos per genotype was collected and pooled into two independent biological replicates per condition, with each replicate consisting of four paired MGEs. Following dissociation and sorting, each replicate was processed independently for single-cell library preparation using the Chromium Next GEM Single Cell 3′ v3.1 platform (10x Genomics). Libraries were sequenced on an Illumina NextSeq 2000, yielding an average depth of approximately 26,000 reads per cell. Single-cell transcriptomic profiling was used to (i) resolve the cellular composition of the E12.5 MGE, (ii) define major progenitor and precursor populations along the MGE neurogenic trajectory, and (iii) assess genotype-dependent transcriptional changes using pseudobulk differential expression approaches. Particular emphasis was placed on basal GABAergic progenitors and postmitotic precursor cells. Together, this scRNA-seq dataset provides a high-resolution framework for interrogating how mutant huntingtin perturbs early progenitor states and downstream lineage programs during ventral telencephalic development, particularly the MGE.

overall_design: This study was designed to define how mutant huntingtin (mHTT) alters early medial ganglionic eminence (MGE) neurogenesis at single-cell resolution, with particular emphasis on progenitor cell states, lineage trajectories, and cell-cycle–associated transcriptional regulation. Single-cell RNA sequencing (scRNA-seq) was performed on fluorescence-activated cell sorted (FACS) Dlx1-eGFP–positive cells isolated from the MGEs of embryonic day 12.5 (E12.5) mouse embryos. Two experimental conditions were analyzed: (i) BACHD embryos expressing full-length human mutant huntingtin and (ii) wild-type (WT) littermate controls. The Dlx1-eGFP reporter was used in both genotypes to enrich for MGE-derived GABAergic progenitors and early postmitotic precursors, enabling selective interrogation of ventral telencephalic lineages. To generate biological replication while minimizing litter-specific bias, eight embryos per genotype were harvested by cesarean section and pooled into two independent biological replicates per condition, each consisting of four pairs of MGEs. Thus, the experimental design comprised four scRNA-seq libraries in total (two BACHD pools and two WT pools), with each pool representing an independent biological replicate derived from multiple embryos. Following enzymatic dissociation, Dlx1-eGFP⁺ cells were isolated by high-speed FACS and processed independently for scRNA-seq library preparation using the 10x Genomics Chromium Next GEM Single Cell 3′ v3.1 platform. Libraries were sequenced to comparable depth across samples, yielding between ~8,000 and ~13,000 cells per library. Raw sequencing data were generated as multiplexed FASTQ files and processed uniformly across all samples. The primary experimental variable under investigation was genotype (BACHD vs WT). Secondary analytical variables included cell type, developmental lineage, and cell-cycle state, inferred computationally from gene-expression profiles. After quality control, ambient RNA correction, and doublet removal, datasets were integrated to mitigate batch effects while preserving biological differences between genotypes. Unsupervised clustering resolved transcriptionally distinct cell populations spanning apical radial glia, basal intermediate progenitors, newly postmitotic progenitors, interneuron precursors, projection neuron precursors, and minor non-MGE populations. Differential gene-expression analyses were performed using a pseudobulk strategy, aggregating cells by cluster, biological replicate, genotype, and—where indicated—cell-cycle phase. Cell-cycle state was inferred using Seurat’s CellCycleScoring method, generating continuous S-phase and G2/M-phase transcriptional scores and assigning cells to G1-like, S, or G2/M categories. This enabled explicit testing of whether genotype-associated transcriptional changes reflected altered cell-cycle composition or phase-intrinsic dysregulation. Phase-restricted pseudobulk analyses were performed to assess genotype effects within matched cell-cycle states. Overall, this experimental design combines controlled genetic comparison, biological replication, lineage-restricted cell selection, and cell-cycle–resolved single-cell transcriptomics to interrogate early developmental mechanisms by which mutant huntingtin perturbs MGE progenitor biology

middle: E

first: Gnanapackiam

last: Devakanmalai

first: Yagiz

middle: M

last: Altum

first: Teresa

last: Jover-Mengual

first: Junya

last: Zhang

first: Nusrat

last: Khan

first: Mark

last: Mehler


title: Improved co-transcriptional splicing of readthrough transcripts during chemotherapy treatment [CML_RNA-seq]

city: New Haven

first: Paulina

last: Podszywalow-Bartnicka

state: CT

address: 333 Cedar Street

department: Department of Molecular Biophysics and Biochemistry

institute: Yale University

summary: The cellular stress response is an important component of cancer cell response to chemotherapy that can determine cell survival. Herein, we study how the coupling of the RNA processes in leukemia cells is affected in response to stress and therapeutic agents. We used short- and long-read sequencing to follow RNA processing upon treatment.

overall_design: To determine level of gene expression as well as determine changes in alternative splicing we used short-read RNA-seq. We analyzed  polyA selected RNA isolated from whole cells (mRNA) that were CD34+ cell subpopulation enriched from CML patients' samples. Before isolation of total RNA the cells were treated with 1uM Imatinib (im) for 1h  and the cells were collected in paralell with the untreated control cells (untr). Samples were analyzed in a single replicate.

first: Morgan

last: Shine

first: Jing

last: Lin

first: Karla

last: Neugebauer


title: A covalent fragment screen identifies ferroptosis induced by GPX4 inhibition as a vulnerability of senescent cells [PC3]

city: London

first: LMS

last: Bioinformatics Core

address: Hammersmith Hospital Campus, Du Cane Road

department: Bioinformatics Core

institute: MRC London Institute of Medical Sciences

summary: NOT PROVIDED; REQUESTED

overall_design: These samples come from an in vivo experiment performed with PC3 xenografts prostate tumor model. We have 4 different experimental groups: vehicle (V), RSL3 (R), Palbociclib (P) and palvociclib+RSL3 (P+R).

first: Mariantonietta

last: D'Ambrosio

first: Jesus

last: Gil

first: Sanjay

last: Khadayate


title: A covalent fragment screen identifies ferroptosis induced by GPX4 inhibition as a vulnerability of senescent cells

summary: We report the analysis of RNA sequencing aimed at understanding senescence induction in SK-MEL103 cells.

overall_design: SK-MEL103 cells were treated for 6 days with DMSO, Etopodise and Tozasertib.


title: Time‑resolved multi-omic analysis of paclitaxel exposure in human iPSC‑derived sensory neurons unveils mechanisms of chemotherapy‑induced peripheral neuropathy

city: Berlin

first: Christian

last: Schinke

address: Charitéplatz 1

institute: Charité Universitätsmedizin Berlin

summary: The microtubule-stabilizing drug paclitaxel remains standard of care for various solid malignancies but frequently leads to chemotherapy-induced peripheral neuropathy (CIPN). CIPN is a leading cause for premature treatment termination and a significantly reduced quality of life in long-term cancer survivors. The molecular mechanisms of neuro-axonal degeneration, neuroinflammation and pain in patients treated with paclitaxel remain incompletely understood, and there are currently no predictive biomarkers or preventive treatments. We used human iPSC-derived sensory neurons exposed to paclitaxel to comprehensively model the pathophysiology of CIPN. Neurotoxicity was assessed over time using viability assays and sequential RNA sequencing as well as deep proteome and lipidomic analyses. We observed a time and dose-dependent decline of cell viability at clinically relevant paclitaxel doses. Sequential RNA sequencing defined JUN as an early immediate gene, followed by the overexpression of genes of the neuronal stress response (e.g., ARID5A, WEE1, DUSP16, GADD45A), neuronal injury and apoptotic pathways (e.g., ATF3, HRK, BBC3 [PUMA], BCL2L11 [BIM], CASP3), neuroinflammation and nociception (CALCB, MMP10, IL31RA, CYSLTR2, C3AR1, TNFRSF12A) and neuronal transduction (e.g., CAMK2A, STOML3, PIRT), while key enzymes of lipid biosynthesis were markedly downregulated (e.g., LSS, HMGCS1, HMGCR, DHCR24). Deep proteome analyses following 48 hours of exposure to 100nM paclitaxel revealed a strong correlation of differentially expressed RNA with proteins, and a marked degradation of essential axonal transport proteins such as kinesins, stathmins and scaffold proteins. Consistent with the downregulation of rate-limiting enzymes of lipid biosynthesis, lipidome analysis confirmed deregulation of neuronal lipid homeostasis. In summary, paclitaxel induces transcriptomic and proteomic signatures of the neuronal stress response, neuroinflammation, nociception and disturbed metabolism. These may explain, in part, the clinical phenotype of sensory loss, hypersensitivity and neuropathic pain frequently observed in patients suffering from CIPN, but constitute pharmacologically addressable targets.

overall_design: 72 samples were included in RNA sequencing analyses from 12 different experimental units (plates): Seven time points from BIHi264-A (7 × 6-well plates) and 48-h samples from five cell lines (5 × 6-well plates). iPSC-DSN were maintained in geltrex coated 6-well plates at 10^6 cells/well, as described in the CDDis manuscript entitled above. After maturation for >60d, three wells were either treated with DMSO at 1/60.000 or paclitaxel at 100 nM, respectively. Investigated time points were 48h of paclitaxel incubation for all five cell lines (30 samples), and for BIHi264-A, the additional time points of 2h, 6h, 12h, 24h and 24h or 120h following removal of the drug after 48h incubation (s.c. 72h and 168h timepoints; in total, 42 samples). RNA was harvested with the Aurum™ Total RNA Mini Kit according to manufacturers’ instructions. RNA sequencing was performed by Brooks Life Sciences Genewiz® with PolyA selection for RNA removal, 2x150 bp sequencing configuration and 20-30 million reads per sample. RNA Library Preparation and NovaSeq Sequencing by AzentaLifeSciences Genewiz: RNA samples were quantified using Qubit 4.0 Fluorometer (Life Technologies) and RNA integrity was checked with an RNA Kit on Agilent 5300 Fragment Analyzer (Agilent Technologies). RNA sequencing libraries were prepared using the NEBNext Ultra RNA Library Prep Kit for Illumina following the manufacturer’s instructions (NEB, Ipswich). Briefly, mRNAs were first enriched with Oligo(dT) beads. Enriched mRNAs were fragmented for 15 minutes at 94 °C. First-strand and second strand cDNAs were subsequently synthesized. cDNA fragments were end repaired and adenylated at 3’ends, and universal adapters were ligated to cDNA fragments, followed by index addition and library enrichment by limited-cycle PCR. Sequencing libraries were validated using NGS Kit on the Agilent 5300 Fragment Analyzer (Agilent Technologies), and quantified by using Qubit 4.0 Fluorometer (Invitrogen). The sequencing libraries were multiplexed and loaded on the flowcell on the Illumina NovaSeq 6000 instrument according to manufacturer’s instructions. The samples were sequenced using a 2x150 Pair-End (PE) configuration v1.5. Image analysis and basecalling were conducted by the NovaSeq Control Software v1.7 on the NovaSeq instrument. Raw sequence data (.bcl files) generated from Illumina NovaSeq was converted into fastq files and de-multiplexed using Illumina bcl2fastq program version 2.20. One mismatch was tolerated for index sequence identification. Computational Methods. RNA Seq reads were mapped to human genome (GRCh38.p7) with STAR-version 2.7.3a [100] using the default parameters. Reads were assigned to genes with featureCounts version 2.0.0 [101] with the following parameters: -t exon -g gene_id -s 0 -p, gene annotation - Gencode GRCh38/v25. The differential expression analysis was carried out with DESeq2-version 1.32.0 [102] using the default parameters. We kept genes that have at least 5 reads in at least 3 samples. Gene set enrichment analysis was carried out with R/tmod package version 0.50.07 using MSigDB gene sets [103]. For Reference Numbers: Please refer to CDDis manuscript CDDis-25-4526.

first: Smilla

last: Maierhof

first: Lois

last: Hew

first: Valeria

last: Fernandez Vallone

first: Silke

last: Frahm

first: Narasimha

last: Swamy Telugu

first: Sebastian

last: Diecke

first: Andranik

last: Ivanov

first: Richard

last: Kovács

first: Dieter

last: Beule

first: Marieluise

last: Kirchner

first: Philipp

last: Mertins

first: Ulrike

last: Brüning

first: Jennifer

last: Kirwan

first: Harald

last: Stachelscheid

first: Matthias

last: Endres

first: Petra

last: Huehnchen

first: Wolfgang

last: Boehmerle


title: Bulk RNA-seq analysis of WT C57BL/6 mise treated with Control, Choleste-help normal lot, toxic lot, and Puberulic acid

city: Bunkyoku

first: Yuta

last: Sekiguchi

state: Tokyo

address: 1-5-45, Yushima

institute: Institute of Science Tokyo

summary: Puberulic acid has been suggested as the causative agent in the toxic lot of Choleste-Help, but no detailed toxicological data have been published to date. In this study, we demonstrated a striking similarity in the renal transcriptomic profiles induced by puberulic acid and the toxic lot of Choleste-Help, supporting the conclusion that puberulic acid is the likely causative compound. Furthermore, both treatments led to a significant downregulation of mitochondria-related transcripts, suggesting that the kidney injury involves mitochondrial dysfunction.

overall_design: Male C57BL/6 mice were orally administered one of the following for 6 consecutive days: vehicle control, a normal lot of Choleste-Help, a toxic lot of Choleste-Help, or puberulic acid. On day 7 (after one drug-free day), whole kidney tissues were harvested for transcriptome analysis.

first: Makiko

last: Mori

first: Hiroaki

last: Kikuchi

first: Yutaro


title: Epas1 expression is controlled by the progesterone receptor and regulates hCG-induced gene expression in Granulosa Cells during ovulation in mice.

city: Adelaide

first: Alison

middle: Elizabeth

last: Roennfeldt

state: SA

address: Adelaide Health and Medical Sciences Building

country: Australia

institute: The University of Adelaide

summary: Ovulation is induced via a surge in gonadotrophin hormones, which increases the expression of the essential ovulatory transcription factor progesterone receptor (PGR) and its target genes. The importance of PGR in ovulation is well defined; however, the role of its many downstream genes largely remains unknown. Using mouse models of ovulation, we show that the Epas1 gene, which encodes the hypoxia inducible transcription factor 2α (HIF-2α), is expressed in a PGR-dependent manner during ovulation. Numerous HIF target genes increase in expression upon gonadotrophin stimulation in mouse granulosa cells, with expression of Epas1, but not its related isoform, Hif1a, increasing in a PGR-dependent manner. PGR directly binds introns of the Epas1 locus to enhance chromatin accessibility in ovarian granulosa cells in vivo, yet no evidence of PGR-dependent Epas1 expression was observed in PGR-expressing breast cancer cell lines, suggesting ovary-specific mechanisms of PGR-dependent Epas1 regulation. PGR activation in response to hormonal stimulation induced expression of a HIF reporter system in primary human granulosa cells, with HIF-2 inhibition with the small molecule PT-2385 confirming a HIF-2 contribution to this response. Upon HIF-2 inhibition with PT-2385 in mice, no change in ovulation counts were observed. However, gonadotrophin-induced ovary gene expression was significantly disrupted, supporting a model where HIF-2α contributes to the control of periovulatory gene expression downstream of PGR. In particular, inflammatory gene expression was dysregulated and a cohort of gonadotrophin-dependent genes, including Pgr, were elevated, suggesting impaired downregulation post-ovulation. These findings provide an important insight into regulation of the hypoxia inducible transcription factors during ovulation and how targeting HIF-2α may be of benefit in future fertility treatments.

overall_design: RNA-seq analysis of ovaries and kidney collected from 21-day old CBAF1 female mice treated with PT-2385 or DMSO vehicle control 12-hourly for 4 days, with ovulation induced with PMSG (48h) followed by hCG. Ovary and kidney collected at the post-ovulation 13-hour hCG timepoint.

first: Doan

middle: T

last: Dinh

first: Darryl

middle: L

last: Russell


title: Transcriptomic Changes in THP-1 Macrophages Infected with Candida albicans SC5314 in the Presence or Absence of Cinnamaldehyde

city: Hefei

first: Wenqian

last: Li

address: Longzihu Road

institute: Anhui University of Chinese Medicine

summary: This study investigated the transcriptomic changes in human THP-1 macrophages in response to Candida albicans SC5314 infection, with or without cinnamaldehyde treatment. RNA-seq revealed 1,996 differentially expressed genes (DEGs) (|log2FC |≥ 1, adjusted p-value < 0.05), comprising 691 upregulated and 1,305 downregulated genes. Additionally, we provide RNA sequencing data for macrophages uninfected with C. albicans. Between macrophages infected with C. albicans and uninfected macrophages, a total of 18 DEGs were identified, with 15 upregulated and 3 downregulated.

overall_design: Plate THP-1 cells at a density of 8 × 10⁵ cells/mL in a 6-well plate. Induce differentiation with 100 ng/mL PMA for 24 h. After cells adhere, add Candida albicans (final concentration: 2.4 × 10⁶ CFU/mL) and cinnamaldehyde (final concentration: 62.5 μg/mL), then incubate for 2 h. Following incubation, aspirate the medium. Wash cells by slowly adding 1 mL of ice-cold PBS down the side of each well, gently rocking the plate for a few seconds, and aspirating the PBS. Add 500 μL of TRIzol reagent to each well, pipette to homogenize, and transfer the lysate to pre-chilled RNase-free cryovials.

first: Zhaoling

last: Shi

first: Kelong

last: Ma


title: Differential gene expression analysis with or without IL-6 in multiple myeloma cells

city: Kumamoto

first: Hiroto

last: Ohguchi

address: 2-2-1 Honjo, Chuo-ku

department: IRDA

summary: Multiple myeloma (MM) is a plasma cell neoplasm that depends on the bone marrow (BM) microenvironment; however, the underlying mechanisms of epigenetic contribution to the pathogenesis of MM are incompletely understood. Here, we delineate the epigenetic-driven transcriptional and splicing program crucial for MM. We recharacterized transcriptional program induced by IL-6/JAK/STAT3 pathway by ChIP-seq and RNA-seq analyses combined with CRIPSR knockout screening database, and identified B cell lineage factors, POU2AF1 and ELL2, as crucial IL-6/JAK/STAT3 targets that are essential for MM cell growth and survival. Genetic depletion of these factors suppressed MM cell growth in vitro and in the xenograft model of IL-6 humanized mice. Mechanistically, POU2AF1 and ELL2 govern MM-distinct transcriptional program representing immaturity, and IL-6/JAK/STAT3 pathway augments this program through upregulating and recruiting these factors to the MM-signature genes. Furthermore, we showed that IL-6 induces alternative splicing that is partly mediated by POU2AF1. Proteomic study identified novel POU2AF1-associated factors that converge in regulation of RNA, including RNA splicing. Indeed, POU2AF1 is adjacent to multiple splicing factors required for MM cell survival and is involved in the organization of nuclear speckles. Finally, we showed that gapmer antisense oligonucleotides (ASOs) targeting POU2AF1 inhibit its expression and MM cell growth in the presence of soluble BM stromal cell factors, including IL-6. Our data demonstrate that IL-6-driven B cell-lineage factors are the vulnerability of MM cells and may represent novel therapeutic targets for this incurable tumor.

overall_design: Multiple myeloma cells (MM.1S, INA-6, and primary patient sample) were treated with or without IL-6 (control), and subjected to RNA-seq analysis. MM.1S and INA-6 cells expressing Cas9 were transduced with either sgRNA against POU2AF1, ELL2 or EGFP (control), treated with or without IL-6, and subjected to RNA-seq analysis.

first: Yasuyo

first: Shingo

last: Usuki


title: Alterations of splicing and gene expression in DMSO or 4OHT treated-IMR90 ER:HRAS G12V cells upon knockdown of SF3B1 and RBM39

first: LMS

last: Bioinformatics Core

summary: The goal of this study was to analyse changes of splicing and gene expression induced in IMR90 Control and IMR90 RAS (HRASG12V) cells upon knockdown of splicing factors SF3B1 and RBM39

overall_design: Comparative analysis of splicing and gene expression changes in DMSO-treated IMR90 ER:RAS (control) and 4OHT-induced IMR90 ER:RAS cells following the knockdown of SF3B1 and RBM39 by siRNA (siSF3B1 and siRBM39) compared to non-targeting siRNA (siNT).

first: Laura

last: Bousset

first: Sanjay

last: Khadayate

first: Marian

last: Dore

first: Ivan

last: Andrew

first: Ka Lok

last: Choi

first: Laurence

last: Game

first: Alexandre

last: da Silva Alfonso

first: Rita

last: Borges da Silva

first: Nuno

last: Barbosa Morais

first: Jesus

last: Gil


title: mRNA vaccines engage unconventional pathways in CD8 T cell priming

city: SAINT LOUIS

first: Suin

last: Jo

state: MO

address: 4939 Childrens PI

department: Division of Biology & Biomedical Sciences

institute: Washington University in St. Louis

summary: Vaccines comprised of mRNA and lipid nanoparticles (LNP) activate B and T cells by inducing in vivo production of specific protein antigens. While B cells can be activated directly by antigens, activation of T cells requires antigen processing and presentation by MHC molecules on specialized antigen presenting cells (APCs). In response to viral infections, tumors and current vaccines, antigen presentation to CD8 T cells is particularly dependent on type 1 conventional dendritic cells (cDC1s) which are specialized for efficient antigen cross-presentation of exogenous antigens. However, whether similar mechanisms are employed by mRNA-LNP vaccination is currently unknown. Here, we show that mRNA LNP vaccines do not depend on cDC1 to induce CD8 T cell responses but engage cDC1 and cDC2 redundantly. In addition, these responses do not involve the normal WDFY4-dependent cross-presentation pathway. Rather, cDC2 are a prominent component driving CD8 T cells response against mRNA-LNP vaccination and engage cross-dressing of peptide-MHC-I complexes derived from non-hematopoietic cells. Notably, multimodal single cells analysis revealed that CD8 T cells primed either by cDC1 or cDC2 exhibit phenotypical differences. These results suggest that mRNA-LNP vaccination should be further evaluated for its ability to sustain normal functional memory that emerges from natural infections or vaccines that engage cDC1 for CD8 T cell priming.

overall_design: WT, Irf8 +32-/- (d32), or Zeb2 -165(d1+2+3) mice were immunized with noncoding (dead) mRNA- or OVA mRNA-LNP vaccine on day 0 and 7. On day 11, spleens were harvested, stained with HTO antibodies, and Kb-SIINFEKL tetramer+ or tetramer- CD8 T cells were isolated by Florescence-activated cell sorting (FACS).

first: Chandrani

last: Thakur

first: Naresha

last: Saligrama

first: Kenneth

last: Murphy


title: Differential RNA splicing analysis with or without IL-6 in multiple myeloma cells

first: Hiroto

last: Ohguchi

overall_design: Multiple myeloma cells (MM.1S and INA-6) were treated with or without IL-6 (control), and subjected to alternative splicing analysis. MM.1S and INA-6 cells expressing Cas9 were transduced with either sgRNA against POU2AF1, ELL2, SRSF1, HNRNPL, or EGFP (control), treated with IL-6, and subjected to alternative splicing analysis.

first: Yasuyo

first: Mashahiko

last: Ajiro

first: Akihide

last: Yoshimi