title: Transcriptional analysis of developing Aspergillus fumigatus biofilms reveals metabolic shifts required for biofilm maintenance

city: Hanover

first: Charles

last: Puerner

address: 1 Rope Ferry Rd

institute: Geisel School of Medicine at Dartmouth College

summary: Aspergillus fumigatus is a filamentous fungus found in compost and soil that can cause invasive and/or chronic disease in a broad spectrum of individuals. Diagnosis and treatment of aspergillosis often occur during stages of infection when A. fumigatus has formed dense networks of hyphae within the lung. These dense hyphal networks are multicellular, encased in a layer of extracellular matrix, and have reduced susceptibility to contemporary antifungal drugs, characteristics which are defining features of a microbial biofilm. A mode of growth similar to these dense hyphal networks observed in vivo can be recapitulated in vitro using a static, submerged biofilm culture model. The mechanisms underlying filamentous fungal cell physiology at different stages of biofilm development remain to be defined. Here, we utilized an RNA sequencing approach to evaluate changes in transcript levels during A. fumigatus biofilm development. These analyses revealed an increase in transcripts associated with fermentation and a concomitant decrease in oxidative phosphorylation related transcripts. Further investigation revealed that ethanol and butanediol fermentation is important for mature biofilm biomass maintenance. Correspondingly, a gene (silG), a predicted transcription factor, was observed to also be required for mature biofilm biomass maintenance. Taken together, these data suggest temporal changes in A. fumigatus metabolism during biofilm development are required to maintain a fully mature biofilm.

overall_design: RNAseq profiling of A. fumigatus strain CEA10 submerged biofilm tissue harvested at 12-, 18-, 24-, and 30-hours of development in three biological replicates.

first: Kaesi

last: Morelli

first: Joshua

middle: D

last: Kerkaert

first: Jane

last: Jones

first: Katherine

middle: G

last: Quinn

first: Sandeep

last: Vellanki

first: Robert

last: Cramer


title: Small Extracellular Vesicles from Radioresistant H3K27M-Pediatric Diffuse Midline Glioma Cells Modulate Tumor Phenotypes and Radiation Response [bulk RNA-seq]

status: Public on Jun 02 2025

city: Lexington

first: Jessica

middle: S

last: Blackburn

state: Kentucky

address: 741 S. Limestone

country: USA

institute: University of Kentucky

type: Expression profiling by high throughput sequencing

summary: Pediatric diffuse midline gliomas harboring the Histone 3 lysine 27-to-methionine mutation (H3K27M-pDMG) represent a highly heterogenous group of brain tumors characterized by intrinsic resistance to radiation therapy, the current standard of care. Recent evidence suggests that intratumor small extracellular vesicle (sEV)-mediated signaling plays an oncogenic role among glioma stem cell populations. However, the dynamics and functional roles of H3K27M-pDMG derived sEVs in the context of radiation-induced stress remain unclear. In this study, we characterize sEV uptake dynamics between H3K27M tumor cells, identify potential sEV surface proteins involved in the process, and demonstrate that radioresistant (RR) H3K27M-pDMG-derived sEVs confer radioprotective effects to radiosensitive (RS) H3K27M-pDMG cells at the population level. RR-sEVs induce metabolic and gene expression changes in RS cell populations within the timescale of EV uptake and internalization, leading to improved DNA repair following radiation-induced stress. Furthermore, we identified the proteins, microRNAs (miRNAs), and metabolites present in RR-sEVs, shedding light on the molecular cargo that may be responsible for these effects. Our findings provide insights into the intrinsic radioresistant properties mediated by H3K27M-pDMG-derived sEVs and propose novel targets for disrupting radioresistant intratumoral communication in H3K27M-pDMGs.

overall_design: RNA-seq profiling of 3 bulk cell lines, SF7761, SF8628, and SF7761-Clone#1

first: Viral

last: Oza


title: Small Extracellular Vesicles from Radioresistant H3K27M-Pediatric Diffuse Midline Glioma Cells Modulate Tumor Phenotypes and Radiation Response [smRNA-Seq]

type: Non-coding RNA profiling by high throughput sequencing

overall_design: sEVs were isolated from 3 cells lines, SF7761, SF8628, and SF7761-Clone#1 using differential ultracentrifugation followed by 100kDa ultrafiltration. Small RNA was extracted from each sEV preparation and sequenced.


title: Overexpression of Mir-197 und Mir-769 in mouse iPSCs

status: Public on Jun 04 2025

city: bad nauheim

first: Stefan

last: Günther

address: ludwigtr. 43

country: Germany

institute: MPI for heart and lung research

summary: MicroRNAs (miRNAs) are post-transcriptional regulators that are involved in many essential biological processes. Recently, targeted searches for human miRNA orthologs revealed that 15 miRNA families are lost in the Eumuroidea lineage, including the model organisms mouse and rat, but not in other mammals. We here characterize this loss in more detail and investigate potential consequences for the Eumuroidean regulatory network. We confirm that the miRNA loss was not compensated by other miRNAs in rat or mouse, and we identify a significant overlap in the function of their target genes. We heterologously express two human miRNAs, Mir-197 and Mir-769, in mouse inducible pluripotent stem cells (iPSCs) and compare effects on the transcriptome to those of induced miRNA levels in human iPSCs. This reveals overlapping sets of down-regulated mRNAs in the two species, likely representing remnants of an ancient miRNA-dependent regulatory network that predates the diversification of the human and mouse lineages.  The consequent elimination of established miRNA regulatory connections on the Eumuroidean lineage is accompanied by the lineage-specific loss of 37 transcription factors. Our results indicate that mouse and rat have modified their regulatory network of gene expression on transcriptional and post-transcriptional level compared to other vertebrate model organisms. This regulatory shift likely adds noise to studies of gene expression that, for example, use murine cells to study human diseases.

overall_design: Differential gene expression analysis upon overexpression for 48h of either miRNA Precursor hsa-miR-197-3p or miRNA Precursor hsa-miR-769-5p in murine iPSCs compared to miRNA Precursor Negative Control

first: Felix

last: Langschied

first: Matthias

last: Leisegang

first: Fabian

last: Hahner

first: Ralf

middle: P

last: Brandes

first: Ingo

last: Ebersberger


title: Basal IFNλ2/3 expression regulates tight junction formation in human epithelial cells

city: Gainesville

first: Yagmur

last: Keser

state: Florida

address: 1200 Newell Dr.

department: Molecular Genetics and Microbiology (MGM)

institute: University of Florida

summary: Type III interferons (IFNλs) play crucial roles in antiviral defense and intestinal epithelial barrier integrity. While interferon expression has been primarily studied in response to pathogens, basal interferon expression occurs in pathogen-free environments. However, the mechanisms regulating basal IFNλ expression and its function have not yet been elucidated. Our findings reveal that basal IFNλ2/3 expression correlates with the development of an intact cellular epithelium characterized by formation of tight junctions and establishment of barrier function. We discovered that basal IFNλ2/3 expression depends on cGAS/STING-mediated mitochondrial DNA detection and is inhibited by the Hippo mechanotransduction pathway at low cellular densities. Notably, cells lacking basal IFNλ2/3 fail to develop proper tight junctions and barrier function. Mechanistically, we found that IFNλ2/3 negatively regulates claudin-2 expression, thereby promoting barrier formation as cells become confluent. These results demonstrate a previously unknown function of basal IFNλs in regulating epithelial cell junction formation and highlight their importance not only during pathogen challenges but also in maintaining epithelial cell function under steady-state conditions.

overall_design: To investigate  the interplay between cellular density and basal IFNλ signaling in human intestinal epithelial cells, we generated T84 IFNλ2/3 KO and IFNLR KO cells. T84 WT, IFNλ2/3 KO and IFNLR KO were seeded at high and low density. One day pos-seeding of low density and two-days post-seeding of high density, cells were harvested and sent for RNA sequencing.

first: Camila

last: Metz-Zumaran

first: Zina

middle: M

last: Uckeley

first: Megan

last: Stanifer

first: Steeve

last: Boulant


title: Germline-somatic liaison dictates cancer subtype via de novo steroid biosynthesis

status: Public on Jun 03 2025

city: Trento

first: Francesca

last: Demichelis

state: TN

address: Via Sommarive, 9

department: Department for Cellular, Computational and Integrative Biology - CIBIO

country: Italy

institute: University of Trento

summary: This study investigates the cooperation between germline genetic variants and somatic mutations in prostate cancer. Specifically, we dissected the interplay between a germline variant at the 7p14.3 locus (rs1376350, G>A) and early recurrent prostate cancer-specific mutation in the Speckle-Type POZ protein (SPOP) gene across human prostate adenocarcinomas. The transcriptomes of multiple edited models pointed to Gli3 and the Hedgehog signaling pathway in a genotype-specific manner.

overall_design: RNA-seq profiling of PC3 and LnCaP cellines with single-nucleotide, macrodeletion, microdeletion, and distruption of binging motifs at genomic site 7p14.3 (rs1376350)

first: Paola

last: Gasperini

first: Blerta

last: Stringa

first: Yari

last: Ciani

first: Orsetta

last: Quaini


title: Enhanced gliogenesis and delayed maturation underpin the neurodevelopmental defects in Lowe syndrome

city: Bangalore

first: Raghu

last: Padinjat

state: Karnataka

address: Kodigehalli

country: India

institute: National Centre for Biological Sciences (NCBS)

type: Genome binding/occupancy profiling by high throughput sequencing

summary: The activity of signaling pathways is required for coordinated cellular and physiological processes that lead to the normal development of brain structure and function. Mutations in OCRL, a phosphatidylinositol 4,5-bisphosphate (PIP₂) 5-phosphatase, lead to the neurodevelopmental disorder Lowe Syndrome (LS). However, the mechanism by which mutations in OCRL lead to the brain phenotypes observed in LS remains unclear.We found that LS patient-derived induced pluripotent stem cells (iPSCs), when differentiated into neural cultures, exhibited significantly reduced neuronal excitability along with increased expression of the astrocytic marker GFAP. Multiomic single-nucleus RNA and ATAC sequencing revealed an early fate switch of LS patient-derived neural stem cells (LSP-NSCs) toward gliogenesis. Pseudobulk analysis of snRNA-seq data showed increased levels of DLK1, a non-canonical Notch ligand, in LS patient NSCs. This was associated with elevated levels of cleaved Notch protein and increased expression of its transcriptional target HES5, indicating upregulated Notch signaling. Treatment of iPSC-derived brain organoids with an inhibitor of PIP5K1C, the lipid kinase responsible for synthesizing PIP₂, restored neuronal excitability and rescued Notch signaling defects in LS patient-derived organoid cultures. Overall, our results demonstrate a role for PIP₂-dependent regulation of Notch signaling, cell fate specification, and the development of neuronal excitability mediated by OCRL activity.

overall_design:
Multiomics (GEX+ATAC): Nuclei from WT1, LSP2 and LSP3 iPSC-derived neural stem cells (NSCs) were isolated using 10x Genomics protocol. Joint snRNA seq and snATAC libraries were prepared using the 10x genomics platform.

***************************************************************
Raw files for human/patient samples were not submitted to GEO due to concerns about submitting personally identifiable sequence data for open access.
***************************************************************

first: Yojet

last: Sharma

first: Priyanka

last: Bhatia

first: Gagana

last: Rangappa

first: Sankhanil

last: Saha

first: Padinjat

last: Raghu


title: Neuroimmune gene signature of prurigo nodularis compared to psoriasis and atopic dermatitis

city: Baltimore

first: Shawn

last: Kwatra MD

state: MD

address: 1550 Orleans Street

department: Dermatology

institute: Johns Hopkins School of Medicine

summary: Prurigo nodularis (PN) is a debilitating neuroimmune skin disease characterized by pruritic, raised, nodular lesions. Psoriasis and AD are classically characterized by Th1/Th17 and Th2 polarization, respectively, but the neuroinflammatory profile of PN remains poorly defined. We characterized the neuroimmune phenotype of PN compared to AD, psoriasis, and healthy controls (HC), through transcriptomic analysis of lesional and nonlesional skin biopsies from 25 PN patients, 27 AD patients, 15 psoriasis patients, and 12 HC using a custom neuroinflammation-focused NanoString panel of 770 genes. Analysis of affected versus unaffected skin revealed differentially expressed genes (DEGs, fold change>1.5, p<0.05) associated with Th1, Th2, and Th17 activation in all three diseases. Upregulated DEGs in PN compared to AD and psoriasis were primarily associated with extracellular matrix remodeling or neural function. Expression of IL-31 was upregulated in PN compared to psoriasis but not AD. Downregulated DEGs in PN compared to AD were associated with Th2 activation and glutamate receptors; those compared to psoriasis were related to Th1 and Th17 activation.  PN has a distinct neuroinflammatory signature characterized by intermediate Th1/Th17 and Th2 immune axis activation compared to AD and psoriasis, with increased levels of ECM dysregulation, neuronal abnormalities, and IL-31 activity.

overall_design: Transcriptome analysis was performed on a total of 26 lesional and 26 non-lesional skin biopsies from 26 PN patients (mean age 52.5 years, 76% female, and 64% African American), 26 lesional and 15 nonlesional biopsies from 26 AD patients (mean age 48.5 years, 37% female, and 44% African American), and 15 lesional biopsies from 15 psoriasis patients (mean age 54.1 years, 40% female, and 47% African American) patients, as well as 12 matched controls.

first: Hannah

last: Cornman

first: Jaya

last: Manjunath

first: Shrey

last: Bhatt

first: Alexander

last: Kolhoff

first: Michael

middle: J

last: Diaz

first: Weiying

last: Lu

first: Selina

last: Yossef

first: Emily

middle: Z

last: Ma

first: Brenda

last: Umenita Imo

first: Jingyi

last: Zhang

first: Kevin

middle: K

last: Lee

first: Thomas

middle: W

last: Pritchard

last: Kwatra


title: The impact of high-cholesterol diet feeding on hepatic mRNA and small non-coding RNA profiles in mice

status: Public on Jul 02 2025

city: Riverside

first: Xiuchun

last: Li

address: 900 University Avenue

institute: UCR

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

overall_design: Refer to individual Series


title: Epithelial Transcription Factor Elf3 mediates host immune responses to microbiota and protects against aerocystitis in zebrafish

status: Public on Jun 01 2025

city: Durham

first: John

middle: F

last: Rawls

state: North Carolina

address: 213 Research Drive

institute: Duke University School of Medicine

summary: We identified Elf3 as a conserved microbially responsive transcription factor and generated an elf3 mutant  zebrafish allele to test whether it mediates host responses to microbes. We performed bulk RNA-sequencing of elf3 wildtype and mutant larvae reared without (germ-free) or with (conventionalized) the presence of microbes. We found that elf3 does mediate host-microbiota interactions in zebrafish and has an important role in the induction of many immune responsive genes.

overall_design: RNA-profiling of elf3 wildtype and mutant 6 days post-fertilization (dpf) zebrafish larvae reared in germ-free (GF) and conventionalized (CV) conditions

first: Briana

middle: R

last: Davis

first: Colin

last: Lickwar


title: Altered Cofactor Recruitment and Nucleosome Dynamics Underlie Bisphenol A's Impact on Estrogen Receptor-Mediated Transcriptional Bursting [CUT&Tag]

first: Christopher

last: Day

state: NC

address: 111 TW Alexander Dr.

department: ERBL

institute: NIH/NIEHS

summary: Gene regulation is complex, involving the coordination of hundreds of proteins to initiate transcription.  At individual loci, transcriptional initiation is stochastic, resulting in short periods of nascent RNA synthesis known as transcriptional bursts.  To understand how altered Estrogen Receptor α (ERα) function and cofactor recruitment regulates transcriptional bursting, we utilized single molecule imaging of estrogen responsive genes in Bisphenol A (BPA) treated cells. We observe that cells treated with BPA exhibited TFF1 burst initiation rates and sizes which were indistinguishable from cells induced with Estradiol (E2). However, we observed a 50% reduction in the number of active alleles in BPA treated cells.  This effect is gene specific, as GREB1 was unperturbed.  Although we observed no difference in chromatin accessibility, the TFF1 promoter exhibited an altered structure which coincided with reduced ERα and cofactor binding.  Lastly, deletion of the enhancer locus removed the BPA effect, indicating that enhancer function was perturbed.  Our results demonstrate gene specific effects of altered ERα recruitment and function which lead to a reduction of transcriptionally permissive states. Our work supports the model that the initial estrogen response occurs from alleles in primed transcriptionally permissive states with additional inactive alleles contributing to the cellular response over time.

overall_design: CUT&Tag, ATAC-seq and RNA-seq of MCF7 cells treated with E2 and BPA.

first: Pelin

last: Yesar

first: Gloria

last: Adedoyin

first: Brian

last: Bennett

first: Carson

middle: C

last: Chow

first: Joseph

last: Rodriguez


title: Small Extracellular Vesicles from Radioresistant H3K27M-Pediatric Diffuse Midline Glioma Cells Modulate Tumor Phenotypes and Radiation Response [EVtreat_RNA-Seq]

first: Jessica

last: Blackburn

overall_design: RNA-seq of SF7761 cells treated with 1) Control (RR-sEV 100 kDa flow through solution), 2) SF8628-sEV, or 3) SF7761-Clone #1-sEV for 18 hours

first: Viral

last: Oza


title: Development of a LRRC15-Targeted Radio-immunotheranostic Approach to Deplete Pro-tumorigenic Mechanisms and Immunotherapy Resistance

city: Los Angeles

first: Claire

last: Storey

state: CA

address: 10833 Le Conte Ave

institute: UCLA

summary: Leucine-rich repeat containing 15 (LRRC15) has emerged as an attractive biomarker and target for cancer therapy. We have developed a humanized monoclonal antibody (mAb), DUNP19, that specifically binds to a phylogenetically conserved LRRC15 epitope and is internalized by target-expressing cancer and stromal cells. In xenograft mouse models, Lutetium-177 labeled DUNP19 ([177Lu]-DUNP19) enables non-invasive imaging and precise radiotherapy to LRRC15-expressing cancer cells and murine cancer-associated fibroblasts (CAFs), halting tumor progression and prolonging survival with minimal toxicity. Transcriptomic analyses of [177Lu]-DUNP19-treated tumors reveal a loss of pro-tumorigenic mechanisms, including a transforming growth factor beta (TGFβ)-driven and LRRC15+ signature associated with immunotherapy resistance. Together, these results demonstrate that radio-theranostic targeting of LRRC15 with DUNP19 is a compelling precision medicine platform for image-guided diagnosis, eradication, and reprogramming of LRRC15+ tumor tissue that drives immuno-resistance and aggressive disease.

overall_design: Evaluation of transcriptomic changes induced by LRRC15-targeted radioimmunotherapy with 177Lu-DUNP19 in U118MG, HuO9, and HCC1954 xenograft models, compared to untreated (PBS-injected) controls

first: Mohamed

last: Altai

first: Katharina

last: Lückerath

first: Wahed

last: Zedan

first: Henan

last: Zhu

first: Marija

last: Trajkovic-Arsic

first: Julie

last: Park

first: Norbert

last: Peekhaus

first: Jens

last: Siveke

first: Diane

last: Abou

first: Haley

last: Marks

first: Enna

last: Ulmert

first: Hans

last: Lilja

first: Alexander

last: Ridley

first: Marcella

last: Safi

first: Constance

last: Yuen

first: Susanne

last: Geres

first: Liqun

last: Mao

first: Michael

last: Cheng

first: Johannes

last: Czernin

first: Ken

last: Herrmann

first: Laurent

last: Bentolila

first: Xia

last: Yang

last: Graeber

first: Robert

last: Damoiseaux

first: Daniel

last: Thorek

first: David


title: Sarcospan selectively interfaces with sarcoglycan subunits to stabilize the sarcolemma and prevent limb-girdle muscular dystrophy

last: Helzer

address: 610 Charles E. Young Drive East

institute: University of California, Los Angeles

summary: Gene duplication events provide redundancy to complex organisms, and homologous genes and their encoded proteins are potential replacements to treat loss-of-function genetic syndromes.  Skeletal muscle possesses redundant molecular mechanisms that partially or fully compensate for loss of gene function.  Within the dystrophin-glycoprotein complex (DGC), utrophin is known to substitute for dystrophin.  However, there has been little investigation of the orthologous relationships within the sarcoglycan subcomplex of the DGC.  In skeletal muscle, the sarcoglycan complex canonically consists of alpha-, beta-, gamma- and delta-subunits, with gamma- and delta-sarcoglycan showing the greatest homology but no functional redundancy.  We show that sarcospan, a transmembrane scaffolding protein, mediates assembly of a compensatory DGC with gamma-sarcoglycan replacement by zeta-sarcoglycan.  This alternative complex improved pathology ingamma-sarcoglycan muscular dystrophy, but not alpha- or beta-sarcoglycan muscular dystrophy where sarcospan was unable to form compensatory complexes.  Three-dimensional modeling of the compensatory DGC reveals that zeta-sarcoglycan maintains specific hydrophobic interactions with sarcospan and preserves overall quaternary arrangement.  This compensatory DGC protects the cell membrane from contraction-induced damage and all secondary consequences of disease.  These findings demonstrate a novel mechanism stabilizing the DGC by leveraging protein redundancy, with an important role for sarcospan in assembly and scaffolding of a compensatory DGC.

overall_design:
Quadriceps muscles form wildtype, Sgcg (gamma-sarcoglycan deficient), and SgcgTG (gamma-sarcoglycan deficient witih trangenic sarcospan expression) mice were scap frozen in liquid nitrogen (n = 1 female mouse per genotype). 7-AAD+ nuclei were isolated from snap frozen muscle by FACS and submitted for  RNA sequencing using the 10x genomics platform.

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Submitter states that missing raw files are due to file loss.
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title: Altered Cofactor Recruitment and Nucleosome Dynamics Underlie Bisphenol A's Impact on Estrogen Receptor-Mediated Transcriptional Bursting [RNA-seq]

first: Christopher

last: Day

first: Pelin

last: Yesar

first: Gloria

last: Adedoyin

first: Brian

last: Bennett

first: Carson

last: Chow

first: Joseph

last: Rodriguez


title: Somatic mtDNA mutations at intermediate levels of heteroplasmy are a source of functional heterogeneity among primary leukemic cells

city: Memphis

first: Mondira

last: Kundu

address: 262 Danny Thomas Place

institute: St. Jude Children's Hospital

summary: Purpose: Metabolic reprogramming is a key feature of cellular transformation. Although the mitochondrial genome encodes components of the electron transport chain that produce tumor-modifying metabolic signals, the contribution of somatic mtDNA mutations in cancer pathophysiology remains unclear. Here, we employed scRNA-seq to capture cell state changes associated with pathogenic, tumor-enriched mtDNA variants in primary leukemic blasts using single-cell RNA sequencing (scRNA-seq)

overall_design: Single-cell RNA sequencing (Chromium, 10X Genomics) of three B-ALL tumor samples that were positive for the t(1;19)(q23;p13) translocation (TCF3-PBX1+/E2A-PBX1+)

first: Kelly

last: McCastlain

first: Wenan

last: Chen

first: Ti-Cheng

last: Chang

first: Gang

last: Wu


title: Altered cofactor recruitment and nucleosome dynamics underlie bisphenol A's impact on ERα-mediated transcriptional bursting


title: GmMEKK2 enhanced mosaic virus resistance in soybean (Glycine max L.)

city: Hangzhou

first: Xuanbo

last: Zhong

address: 866 Yuhangtang Road

country: China

institute: Zhejiang university

summary: Mitogen-activated protein kinase kinase kinase (MAPKKK) assume a pivotal position within the MAPK cascade signaling pathway that converts external stimuli into intracellular responses, and plays a central role in adaptation and resistance to biotic and abiotic stress. Only a limited number of researches have reported that MAPKKK conducted on regulating the resistance to soybeans mosaic viruses (SMV). Here, we identified a MAPKKK 2-like gene named as GmMEKK2 from SMV resistance cultivar. Overexpression of GmMEKK2 in soybean not only reduced SMV content but also decreased the disease index of virus. Importantly, overexpression of GmMEKK2 mitigated the yield loss after SMV inoculation and improved the yield-related traits of soybean. These demonstrated that GmMEKK2 had a favorable role in SMV resistance. This study explored the functions of GmMEKK2 in soybean and provided an assertive solution for effectively improving SMV resistance.

overall_design: The top trifoliate leaves of wildtype and the GmMEKK2 overexpression line at 7 dpi and 14 dpi SMV were sampled, and the leaves before inoculation were used as controls.

first: Guixiang

last: Tang


title: The histone modifier KDM5 functions as an insulator [ATAC-seq]

city: Bronx

first: Matanel

last: Yheskel

state: NY

address: 1300 Morris Park

department: Genetics

institute: Albert Einstein College of Medicine

summary: KDM5 family proteins are best known for their demethylation of the promoter proximal chromatin mark H3K4me3. KDM5-regulated transcription is critical in the brain, with variants in the X-linked paralog KDM5C causing the intellectual disability (ID) disorder, Claes-Jensen syndrome. Although the demethylase activity of KDM5C is known to be important for neuronal function, the contribution of non-enzymatic activities remains less characterized. We therefore used Drosophila to model the ID variant Kdm5L854F that disrupts a C5HC2 zinc finger adjacent to the enzymatic JmjC domain. Kdm5L854F causes similar transcriptional changes in the brain to a demethylase dead strain, Kdm5JmjC*, despite having little effect on enzymatic activity. KDM5L854F is also distinct from KDM5JmjC* in its reduced interactions with insulator proteins and enhancement of position effect variegation. Instead, the common transcriptional deficits likely result from both the JmjC and C5HC2 domains driving proper genomic organization through their activity in promoting proper loop architecture.

overall_design: ATAC-Seq in KDM5 WT, JmjC, and L854F brains

first: Melissa

last: Castiglione

first: Richard

last: Kelly

first: Julie

last: Secombe


title: The histone modifier KDM5 functions as an insulator [Targeted DamID]

overall_design: Targeted DamID in KDM5 WT, JmjC, and L854F brains


title: Temporally resolved single-cell RNA sequencing reveals protective and pathological responses during herpes simplex virus 1 CNS infection

city: Aarhus

first: Soren

last: Paludan

address: CF Møllers Alle 6, Building 1115

department: Biomedicine

country: Denmark

institute: Aarhus university

summary: We profile the transcriptomic changes in the infected brain stem at the single-cell level with temporal resolution, supported by GeoMx spatial transcriptomic data. Microglia proportions increased during infection, while blood-brain barrier cells declined. Transcriptional analysis revealed strong antiviral and inflammatory responses, and impairment of physiological brain activities. This study highlights the complex immune response in virus-infected brains, balancing infection elimination with immunopathology.

overall_design: We allowed the infection of HSV-1 to progress for days 4, 6, and 8. Brainstem of HSV1 infected mice and control mice were collected for scRNA sequencing


title: Determining the transcriptional regulation of CDK4/6 inhibitor resistance in estrogen receptor-positive breast cancer

city: Boston

first: Renyan

last: Liu

state: MA

address: 450 Brookline Avenue

institute: Dana-Farber Cancer Institute

summary: CDK4/6 inhibitor (CDK4/6i) resistance is a pressing clinical problem for patients with ER+ breast cancer.  Previous work identified CDK6 overexpression as one critical determinant of acquired resistance to CDK4/6 inhibition. This dataset interrogates CDK6 interactors/recruiters to determine their role in transcriptional regulation of CDK4/6i resistance mechanisms and will provide novel therapeutic strategies to overcome resistance.

overall_design: RNA was extracted from cultured cells treated with vehicle (Palcociclib-Resitant 100nM) or the addition of JQ1 (300nM - 24hr) using the RNeasy Mini Kit (Qiagen). Prior to RNA sequencing, RNA was treated with DNase using RNase-Free DNase Set (Qiagen). rRNA depletion was performed from 100ng of purified RNA using QIAseq FastSelect rRNA HMR reagents according to manufacturer’s protocol. Libraries were prepared using Roche Kapa Biosystems RNA HyperPrep sample preparation reagents on a Beckman Coulter Biomek i7. Finished dsDNA libraries were quantified by Qubit fluorometer and Agilent TapeStation 4200. Uniquely dual indexed libraries were pooled in an equimolar ratio and shallowly sequenced on an Illumina MiSeq to further evaluate library quality and pool balance. The final pool was sequenced with paired-end 150bp reads on an Illumina NovaSeq 6000at the Dana-Farber Cancer Institute Molecular Biology Core Facilities. Sequenced reads were aligned to the UCSC hg38 reference genome assembly and gene counts were quantified using STAR (v2.7.3a).

first: Geoffrey

last: Shapiro


title: Implant renal injury-responsive cells to supplement erythropoietin and protect kidney [Spatial Transcriptomics]

city: Shanghai

first: Wei

last: Zuo

state: Shanghai

address: No. 500 Zhennan Road, Putuo District, Shanghai

institute: University

summary: Anemia poses a life-threatening risk to individuals with chronic kidney diseases (CKDs). Insufficient production of erythropoietin (EPO) from injured kidney is the major reason for anemia, while lack of EPO would further aggravate the kidney injury and make a “vicious cycle”. In this study, we dissected the change of endogenous EPO signaling in injured kidney by spatial transcriptomic analysis and validated the dual beneficial effects of local recombinant EPO administration on both anemia correction and kidney protection. Next, we constructed an injury-responsive EPO producing (iREP) element to sense the kidney damage signal and drive the synthesis and secretion of native EPO. After intra-renal implantation of iREP engineered HEK-293T embryonic kidney cells, the mouse kidney would automatically produce more EPO when sensing injury signal, which in turn enhanced red blood cell mass and protected kidney from further injury. Moreover, aided with automated equipment, we cloned urine-derived SOX9+ epithelial cells (USCs) from both healthy donors or CKD patients. The USCs could be transplanted into mouse kidney and form tubule-like structures, which makes them an alternative candidate cell for iREP engineering. Altogether, the current work proposed a potential approach based on engineered “smart” cell to treat CKDs.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: 6.5*6.5mm fresh tissues were concurrently frozen and embedded in optical cutting tissue (OCT) compound in liquid nitrogen and delivered in dry ice for spatial transcriptome sequencing.

first: Hao

last: Nie

first: Chen

last: Liang

first: Zixian

last: Zhao

first: Yingxin

last: He


title: Gene expression during PMA-induced neutrophil NETs formation with or without knockout of neutrophils of Src kinase

city: Yangzhou

first: Guotao

last: Lu

address: 368 Hanjiang Middle Road, Yangzhou

institute: The Affiliated Hospital of Yangzhou University, Yangzhou University

summary: Src kinase has functions in regulating reactive oxygen species production, degranulation, NETs formation, integrin activation, and migration to human infection sites in neutrophils. RNA sequencing was performed by extracting mRNA from PMA-induced mouse neutrophils with or without Src kinase knockout(Src fl/fl S100a8 Cre or Src fl/fl  mice). Gene enrichment analysis by Genomes (Reactome) identified RAF/MAP kinase cascade in the top ten significantly changed pathways.

overall_design: Fresh primary  neutrophils were quickly isolated from neutrophil Src kinase knockout or non knockout mice(Src fl/fl S100a8 Cre or Src fl/fl  ) using  the mouse bone marrow neutrophil isolation kit. NETs formation was induced by adding 100 nM Phorbol 12-myristate 13-acetate (PMA) for 4 h.

first: Fei

last: Han

first: Yaodong

last: Wang

first: Chenchen

last: Yuan

first: Lianghao

last: Hu

first: Weijuan

last: Gong


title: Altered Cofactor Recruitment and Nucleosome Dynamics Underlie Bisphenol A's Impact on Estrogen Receptor-Mediated Transcriptional Bursting [ATAC-seq]

first: Christopher

last: Day

first: Pelin

last: Yesar

first: Gloria

last: Adedoyin

first: Brian

last: Bennett

first: Carson

last: Chow

first: Joseph

last: Rodriguez


title: Dissection of the T-cell infiltrate in mouse pancreatic tumors reveals an extensive and diverse tumor-reactive T-cell repertoire

city: Heidelberg

first: Rienk

last: Offringa

state: Baden-Wuerttemberg

address: Im Neuenheimer Feld 280

department: Division of Molecular Oncology of Gastrointestinal Tumors

institute: DKFZ Heidelberg

summary:
Background: Pancreatic ductal adenocarcinoma (PDAC) is refractory to cytostatic and immunotherapeutic treatment regimens, including immune checkpoint inhibition (ICI). The latter is due to the limited mutational load of most of these tumors, in combination with the immunosuppressive hurdles imposed by the tumor microenvironment. Here we present an in-depth analysis of the T-cell response at the single-cell level in an orthotopic PDAC mouse model that recapitulates the therapy resistance of the human disease. In parallel, we investigated an ICI-sensitive variant of this model expressing an immunogenic neo-antigen.

Methods: Single-cell RNA and T-cell receptor (TCR) from tumor-infiltrating lymphocytes (TILs) of 14 tumor-bearing mice were combined with functional in vitro screening of TCR specificity against tumor. Transcriptomic differences between tumor-reactive (TR) and non-tumor-reactive (NTR) T-cells were harnessed to define gene signatures for the purpose of predicting the specificity of additional TCRs.

Methods: We used scRNA and scTCR sequencing to analyze tumor-infiltrating T-cells in a model of murine PDAC for tumor-reactivity. Testing of T-cell receptors in vitro against tumor cells revealed specific anti-tumor activity of the majority of T-cells in the PDA-OVA model, and to a lesser but significant extent in the less immunogenic PDA30364 model. Localization of tested T-cells on the transcriptome revealed distinct profiles of tumor-specific T-cells, dominated by an exhausted phenotype, and of bystander T-cells, which displayed a T effector memory phenotype. Underlying differences in gene expression were delineated for generation of gene signatures that confidently discriminated tumor-reactive and bystander T-cells in this PDAC model, as well as in the CRC model MC38.

Results: Our efforts revealed extensive tumor-reactive and bystander CD8+ and CD4+ T-cell repertoires, not only in the immunogenic but also in the ICI-refractory tumors. By integrating the functional screening and single-cell transcriptome data sets, we delineated gene signatures that distinguish between tumor-reactive and bystander T-cells in the mouse model as well as in human cancers. The TCRs in the murine models detect a range of tumor-associated antigens beyond mutanome-encoded neoepitopes and retain functional anti-tumor activity in vitro.

overall_design: Transplantable tumor cell lines PDA30364 and PDA-OVA, which are derived from a genetically engineered mouse model (Elas-tTA/TetO-Cre Kras+/LSL-G12D Trp53+/LSL-R172H), were implanted orthotopically into the pancreas of immune-competent C57BL/6 mice. Mice were subjected to control treatment or treatment with MEK inhibitor GDC-0623 in combination with agonist anti-CD40 antibody treatment. After two cycles of treatment, tumors were harvested from mice, dissociated into single-cells for the sorting of tumor-infiltrating T-cells. T-cells were analyzed for single-cell RNA and VDJ expression using 10xGenomics for discrimination of tumor-reactive T-cells from bystander T-cells. Transplantable tumor cell line MC38 was implanted subcutaneously in immune-competent C57BL/6 mice. Tumors were harvested from untreated mice, dissociated into single-cells for the sorting of tumor-infiltrating T-cells. T-cells were analyzed for single-cell RNA and VDJ expression using 10xGenomics for discrimination of tumor-reactive T-cells from bystander T-cells based on the gene expression signatures derived from the PDAC tumor-infiltrating T-cells.

first: Hannes

last: Kehm

first: Stefan

last: Zens


title: The histone modifier KDM5 functions as an insulator [CUT&RUN]

first: Matanel

last: Yheskel

overall_design: CUT & RUN for H3K4me3 in KDM5 WT, JmjC, and L854F brains

first: Melissa

last: Castiglione

first: Richard

last: Kelly

first: Julie

last: Secombe


title: RNA proximity labeling in log phase Caulobacter crescentus NA1000

status: Public on May 31 2025

city: Bloomington

first: Jared

middle: Michael

last: Schrader

state: IN

address: 212 S Hawthorne Dr

department: Biology

institute: Indiana University

summary: APEX2 RNA proximity labeling is a powerful method for determining localized RNAs in vivo.  APEX2 RNA proximity labeling was adapted to bacterial cells, using an APEX2 fusion to the core scaffold of BR-bodies, RNase E.  As a control, APEX2 was also fused to a variant of RNase E that lacks its C-terminal IDR and is unable to form BR-bodies, RNase E delta CTD.  RNA proximity labeling was performed, and we observed a similar pattern of enriched RNAs to density centrifugation isolated BR-bodies (Al-Husini et al. Mol Cell 2020).

overall_design: Duplicate cultures of RNase E-APEX2 and RNase E(delta CTD)-APEX2 strains were growth to mid-log phase, and incubated with alkyne phenol for 30 minutes.  The cells were incubated with H2O2 for 1 minute, and then harvested for RNA extraction.  An aliquot of the lysate of each biological replicate was saved for normalization, and the proximity labled RNA was isolated by attaching a biotin-azide via click chemistry followed by purificaiton on streptavidin resin.  Lysate RNA and proximity labeled RNA samples were then prepared for RNA-seq by the IU CGM core facility.

first: Hadi

last: Yassine


title: Midline Assembloids Reveal Regulators of Human Axon Guidance

city: Stanford

first: Sergiu

last: Pasca

state: California

address: 318 Campus Drive

department: Psychiatry

institute: Stanford University

summary: Organizers are specialized cell populations that orchestrate cell patterning and axon guidance in the developing nervous system. Although non-human models have led to fundamental discoveries about the organization of the nervous system midline by the floor plate, an experimental model of human floor plate would enable broader insights into regulation of human neurodevelopment and midline connectivity. Here, we have developed stem cell-derived organoids resembling human floor plate (hFpO) and assembled them with spinal cord organoids (hSpO) to generate midline assembloids (hMA). We demonstrate that hFpO promote Sonic hedgehog-dependent ventral patterning of human spinal progenitors and Netrin-dependent guidance of human commissural axons, paralleling non-human models. To investigate evolutionary-divergent midline regulators, we profiled the hFpO secretome and identified 27 evolutionarily divergent genes between human and mouse. Utilizing the hMA platform, we targeted these candidates in an arrayed CRISPR knockout screen and reveal that GALNT2, a gene involved in O-linked glycosylation, impairs floor plate-mediated guidance of commissural axons in humans. This novel platform extends prior axon guidance discoveries into human-specific neurobiology with implications for mechanisms of nervous system evolution and neurodevelopmental disorders.

overall_design: single cell RNA-sequencing of day 8 floor plate organoids (3 replicates) , day 11 spinal organoids (4 replicates), day 11 floor plate organoid-assembled spinal organoids (4 replicates), and day 35 spinal organoids (3 replicates)

first: Massimo

last: Onesto

first: Neal

last: Amin


title: Transcriptional profiling of yeast to hyphae and hyphae to yeast transitions for Histoplasma strains differing in chromosome copy number.

status: Public on May 30 2025

city: San Francisco

first: Mark

last: Voorhies

address: 513 Parnassus, S472

department: Microbiology and Immunology

institute: University of California

summary: Heritable phenotypic switches are fundamental to the ability of cells to respond to specific conditions. Such switches are key to the success of environmental pathogens, which encounter disparate conditions as they transition between the environment and host. We determine that the copy number of chromosome seven in the thermally dimorphic fungus Histoplasma dramatically affects the rate of transition. Though Histoplasma is haploid, a second copy of this chromosome is present in natural isolates of multiple Histoplasma species and is gained and lost at a high rate. Cells carrying two copies of this chromosome exhibit aspects of the environmental transcriptome even under host-like conditions and have a competitive advantage in the transition to the environmental form. Conversely, these cells are considerably less virulent than euploid cells and have a competitive disadvantage in the mouse model of infection. Chromosome seven contains a previously unstudied transcription factor that, when overexpressed in euploid Histoplasma, is sufficient to promote some of the key phenotypes of aneuploidy. We hypothesize that rapid gain and loss of this chromosome benefits Histoplasma by increasing phenotypic variation, thus helping populations of cells survive abrupt transitions between environment and host.  This series contains expression profiling of yeast to hyphae and hyphae to yeast transitions for strains differing in chromosome seven copy number.

overall_design: Three time points were collected for three replicates of two genotypes for transitions in two directions for a total of 36 samples.

first: Sarah

last: Heater

first: Anita

last: Sil


title: Macrophages expressing Macrophage Receptor with Collagen Structure Attenuate Liver Fibrosis Through a Tissue Restoration Phenotype [macrophage_liverFibrosis_scRNA-seq]

city: ROCHESTER

first: William

last: Sherman

state: MN

address: 200 1st St SW

department: Quantitative Health Sciences

institute: Mayo Clinic

summary: Liver macrophages are central in maintaining hepatic homeostasis and mediating immune responses during liver injury, including fibrosis. Macrophages may have proinflammatory or anti-inflammatory properties, but which properties influence fibrosis remains unclear. To explore the role of macrophages in liver fibrosis, we performed single-cell RNA sequencing in a mouse model of liver injury and found that macrophage diversity was increased. A subset of macrophages expressing high levels of Marco gene was the most significantly upregulated after injury but was spatially segregated to non-fibrotic areas. The macrophage receptor with collagenous structure (MARCO) protein is a scavenger receptor expressed by specific subsets of macrophages, and its role in liver fibrosis is unclear. In vitro induction of Marco in bone marrow-derived macrophages decreased proinflammatory gene expression, increased anti-inflammatory and antifibrotic gene expression, and enhanced phagocytosis, indicating a restorative phenotype. Adoptive transfer of MARCO-positive macrophages in a mouse model of liver fibrosis reduced the expression of extracellular matrix (ECM)-associated genes in hepatic stellate cells (HSCs) and reduced collagen deposition, which did not occur with the transfer of MARCO-negative macrophages. Therefore, MARCO-positive macrophages have a tissue restorative role in the liver and attenuate fibrogenesis through interaction with HSCs, thereby providing a novel therapeutic pathway for liver fibrosis.

overall_design:
Isolated primary cells from mice livers that underwent 6 weeks of Carbon tetrachloride administration to induce fibrosis were subjected to Single Cell RNA sequencing.

C57B6 mice underwent 6 weeks of Carbon tetrachloride administration to induce liver fibrosis. Intrahepatic leukocytes were isolated from perfused livers and subjected to scRNAseq via the 10X Genomics GEM-X FLEX assay.

first: Sofia

last: Jerez

first: Shawna

last: Cooper

first: Usman

last: Yaqoob

first: Bushra

last: Arif

first: Maleeha

last: Kalaiger

first: Abid

last: Anwar

first: Mandy

last: Wong

first: Nidhi

last: Jalan-Sakrikar

first: Ying

last: Li

first: Moira

middle: B

last: Hilscher

first: Enis

last: Kostallari

first: Sheng

last: Cao

first: Vijay

last: Shah


title: Transcriptional profiling of HYF1 and HYF2 increased copy number strains.

first: Mark

last: Voorhies

summary: Heritable phenotypic switches are fundamental to the ability of cells to respond to specific conditions. Such switches are key to the success of environmental pathogens, which encounter disparate conditions as they transition between the environment and host. We determine that the copy number of chromosome seven in the thermally dimorphic fungus Histoplasma dramatically affects the rate of transition. Though Histoplasma is haploid, a second copy of this chromosome is present in natural isolates of multiple Histoplasma species and is gained and lost at a high rate. Cells carrying two copies of this chromosome exhibit aspects of the environmental transcriptome even under host-like conditions and have a competitive advantage in the transition to the environmental form. Conversely, these cells are considerably less virulent than euploid cells and have a competitive disadvantage in the mouse model of infection. Chromosome seven contains a previously unstudied transcription factor that, when overexpressed in euploid Histoplasma, is sufficient to promote some of the key phenotypes of aneuploidy. We hypothesize that rapid gain and loss of this chromosome benefits Histoplasma by increasing phenotypic variation, thus helping populations of cells survive abrupt transitions between environment and host.  This series contains expression profiling of increased copy number constructs for the HYF1 and HYF2 transcription factors.

overall_design: Three yeast culture replicates for each of three genotypes for a total of 9 samples.


title: Identifying downstream targets of ankrd1a in skeletal muscle injury repair in adult zebrafish

status: Public on May 29 2025

city: Belgrade

first: Mirjana

last: Novkovic

address: Vojvode Stepe 444a

country: Serbia

institute: Institute of Molecular Genetic and Genetic Engineering

summary: The ANKRD1 gene is responsive to different forms of mechanical stress, including injury, stretching, resistance exercise, and eccentric contractions. We showed that ankrd1a, zebrafish homologue of mammalian ANKRD1, gets activated in the heart, after cryoinjury of the ventricle and in skeletal muscle after stab wound, suggesting its role in muscle healing processes. To identify targets of ankrd1a involved in skeletal muscle repair we performed RNA-seq of injured adult ankrd1a mutant and wt zebrafish muscle at 5 days post injury. Non-injured skeletal muscle of both genotypes were used as controls. The loss of ankrd1a function affected the expression of genes involved in muscle contraction, muscle cell differentiation, MAPK and integrin-mediated signaling pathways, and cell-substrate adhesion. Our findings offer novel insights into the ankrd1a function and skeletal muscle repair in adult zebrafish.

overall_design: The ankrd1abns502 mutant zebrafish line was generated by CRISPR/Cas9 genome editing. Muscle injury was performed by needle stab into the dorsal myotome of the tail. After 5 days of recovery, RNA was isolated from the muscle tissue around the injury, anterior and posterior to the needle lesion or corresponding non-injured region in control fish. Four experimental groups (injured wt, non-injured wt, injured mutant, and non-injured mutant) were subjected to RNA-seq. Each experimental group comprised four biological samples and one sample contained RNA isolated from the skeletal muscle of one fish.

first: Emilija

last: Milosevic

first: Jovana

last: Jasnic

first: Andjela

last: Milicevic

first: Snezana

last: Kojic


title: Combined single-cell RNA-sequencing and single-cell bisulfite-sequencing reveal key regulatory mechanisms associated with oocyte in vitro maturation in pig [scBS-seq]

city: Murcia

first: Francisco

last: Huertas

address: C/ Campus Universitario de Espinardo Nº7 (Edificio CEEIM)

country: Spain

institute: Marbyt - Smart Solutions for Biotechnology

type: Methylation profiling by high throughput sequencing

summary: In vitro maturation (IVM) is a critical step in current animal in vitro embryo production protocols. However, in vitro matured oocytes often exhibit lower developmental competence than their in vivo counterparts, and the molecular mechanisms underlying successful maturation remain unclear. In this study, we compared the developmental competence up to the blastocyst stage of IVM and in vivo matured porcine oocytes (N=397) and also analysed simultaneously their gene expression and DNA methylation profiles at single cell resolution (N=40) using RNA sequencing and bisulfite sequencing. Our final aim was to find the associations between the developmental and molecular differences between the two groups of oocytes. All the oocytes were obtained by aspiration of either ovarian follicles between 3-6 mm diameter, and the subsequent IVM, or ovarian follicles from 8-10 mm diameter, with no need of maturation (in vivo matured oocytes). Significant differences were found at day 2 for cleavage rates (58.2 ± 3.0 and 45.7 ± 4.4, P = 0.010) and at day 7 for the blastocyst rates (31.4 ± 3.7 and 47.5 ± 6.6, P = 0.014) for IVM and in vivo groups, respectively, but not for the total cell number per blastocyst (48.1 ± 1.9 and 48.2 ± 2.7). Using the in vivo group as a reference, 1297 and 476 differentially expressed genes (DEGs) were down-regulated and up-regulated, respectively, in the IVM group. The up-regulated DEGs in IVM oocytes were mainly associated with organelle organization, DNA methylation, and cell cycle processes; down-regulated genes were mainly enriched in ribosomal RNA processing, protein synthesis, oxidative phosphorylation and metabolomic processes such as glycosyl and aldehyde compound pathways. The global percentage of methylation was similar between the IVM and in vivo groups, but 321, 344, and 843 differentially methylated regions (DMRs) were detected (P < 0.05) in CpG islands, promoters and coding regions, respectively. Integrative analysis of RNA-sequencing and DNA methylation data identified the main methylated regions and genes that define each group. A total of 236 loci and 296 genes defined the IVM group, while 856 loci and 688 genes were related to the in vivo group. In addition, in the IVM group, we found a higher number of negative correlations between gene expression and DNA methylation, while the in vivo group showed higher number and stronger positive correlations. Taken together, these results indicate a discrete effect of IVM on the DNA methylation landscape in mature porcine oocytes, but these changes seem to have a greater impact on gene expression regulation.

overall_design: Ovaries from Landrace-Large-White gilts were collected at the slaughterhouse and transported to the laboratory (within 60 minutes) in saline solution containing 100 µg/mL kanamycin sulphate at 38.5°C, washed once in 0.04% cetrimide solution (w/v) and then twice in saline. Then, the IVM protocol was the same as in Romero-Aguirregomezcorta et al. (2024). As for the in vivo matured oocytes, the contents of follicles of 8-10 mm diameter were aspirated and washed twice in Dulbecco's PBS supplemented with 1 mg mL-1 PVA. All IVM and in vivo matured COCs were identified under a stereomicroscope and mechanically detached from the cumulus cells using an automatic pipette set to a volume of 200 microlitres. To do this, the COCs were gently aspirated and repeatedly ejected into the Petri dish until the zona pellucida was visualised free of cumulus cells at 50x magnification. Only oocytes with a visible polar body under the stereomicroscope were selected and snap frozen in LN2 and stored at -80°C until use (n = 20 IVM and 20 in vivo matured oocytes).

first: Pilar

last: Fuster Coy

first: Laura

last: Abril-Parreño

first: Gavin

last: Kelsey

first: Antonio

last: Galvao

last: Huertas-López

first: Emilio

last: Serrano-López

first: David

last: Martínez-Moreno


title: An alternative transcription factor isoform controls AML chemoresistance [LongRead_mRNA]

city: Farmington

first: Harshpreet

last: Chandok

state: CT

address: 10 Discovery Dr

institute: The Jackson Laboratory

summary: Aberrant splicing is a hallmark of many cancers, including Acute Myeloid Leukemia (AML), but its role in post-treatment relapse remains poorly understood. Here, we analyzed the alternative splicing landscape of 19 AML patients during chemotherapy treatment. We found an upregulation of the long isoform of the transcription factor RUNX1 (also known as RUNX1C) in relapsed cohorts, which conferred chemotherapy resistance. Notably, these patients showed elevanted intragenic DNA methylation at the RUNX1 proximal promoter, thereby activating RUNX1C transcript through its alternative distal promoter. Mechanistically the N-terminus region of RUNX1C is required to promote chemoresistance by regulating a distinct RUNX1 transciptional program. Among these target genes, B-cell translocation gene 2 (BTG2) mediates a quiescent-like phenotype by deadenylating ribosomal RNAs to reduce global protein synthesis. Lastly, we harnessed orthogonal RNA-targeting strategies to target RUNX1C isoform, which led to augmented chemotherapy efficacy. Collectively, our finidngs delineate a transcriptional circuity orchestrated by a transcription factor isoform that underpins chemotherapy responsiveness and a potential therapeutic strategy to mitigate disease recurrence.

overall_design: To determine the role of BTG2 on mRNA stability, MOLM13 cells were transcuded with a cDNA to overexpress BTG2 or with an empty vector control, and mRNA was isolated for long-read sequencing.

first: Cuijuan

last: Han

first: Eric

last: Wang


title: Amygdala-liver signaling orchestrates rapid glycemic responses to stress and drives stress-induced metabolic dysfunction

city: New York

first: Sarah

last: Stanley

address: 1230 York Avenue

department: Dept of Molecular Genetics

institute: Rockefeller University

summary: Behavioral adaptations to environmental threats are crucial for survival and necessitate rapid deployment of energy reserves. The amygdala coordinates behavioral adaptations to threats, but little is known about its involvement in underpinning metabolic adaptations. Here, we show that acute stress activates medial amygdala (MeA) neurons that innervate the ventromedial hypothalamus (MeAVMH neurons), which precipitates hyperglycemia and hypophagia. The glycemic actions of MeAVMH neurons occur independent of adrenal or pancreatic glucoregulatory hormones. Instead, using whole-body virus tracing, we identify a polysynaptic connection from MeA to the liver, which promotes the rapid synthesis of glucose by hepatic gluconeogenesis. Repeated stress exposure disrupts MeA control of blood glucose, resulting in diabetes-like dysregulation of glucose homeostasis. Our findings reveal a novel amygdala-liver axis that regulates rapid glycemic adaptations to stress and links recurrent stress to metabolic dysfunction.

overall_design: 12 sections (2 sections per mouse) of formalin-fixed & paraffin-embedded (FFPE) mouse brains with AAV hSyn-mCherry injection into the ventromedial hypothalamus.

first: Jamie

last: Carty

middle: A


title: An alternative transcription factor isoform controls AML chemoresistance [Cut&Run]

overall_design: To determine any unique binding sites for different RUNX1 isoforms, MOLM-13 cells were infected with lentiviral constructs expressing each isoform individually. This construct contained a GFP reporter and 1x10^6 cells per replicate were sorted on a BD FACS Aria Fusion. After sorting, cells were subjected to CUT&RUN using the CUTANA™ kit from Epicypher. Libraries were generated using Epicypher's CUTANA™ CUT&RUn LIbrary Prep Kit. All procedures followed the manufacturer's protocol.

first: Han

last: Cuijuan

first: Wang

last: Eric


title: An alternative transcription factor isoform controls AML chemoresistance

overall_design: To investigate the functions of unique RUNX1 isoforms, MOLM-13 cells were infected with lentiviral constructs expressing each isoform individually. Three days after transduction, cells were collected and RNA isolated and polyA mRNAs were enriched and used to generate cDNA libraries. Barcoded libraries were then sequenced on a NovaSeq. Gene expression analysis was then performed on cells expressing RUNX1 Isoforms, compared to an empty vector control.


title: Synthetic lethality from the combination of a histone methyltransferase, SUV39H2 inhibitor, and a poly (ADP-ribose) polymerase inhibitor for uterine leiomyosarcoma [RNA-Seq]

status: Public on May 28 2025

city: Tokyo

first: Kohei

last: Kumegawa

address: 3-8-31 Ariake, Koto-ku

country: Japan

institute: Japanese Foundation for Cancer Research

summary: Background: Uterine leiomyosarcoma (uLMS) has a poor prognosis owing to its resistance to chemotherapy. Therefore, novel therapeutic targets for uLMS should be identified. Suppressor license of variegation 3-9 homolog 2 (SUV39H2) is a histone methyltransferase that promotes the repair of double-stranded DNA breaks by recruiting phosphorylated H2AX (γH2AX). In this study, we investigated the potential therapeutic targets of SUV39H2 in uLMS and the mechanism of synthetic lethality between PARP inhibitors and the SUV39H2 inhibitor OTS186935. Methods: First, we analyzed the mRNA and protein expression of SUV39H2 in the clinical tissues of uLMS, normal myometrium, and leiomyomas using real-time polymerase chain reaction and immunohistochemistry, respectively. Next, we conducted drug sensitivity assays for OTS186935 alone and in combination with olaparib, a poly (ADP-ribose) polymerase inhibitor, using the uLMS cell lines SK-LMS-1 and SK-UT-1. We performed Western blotting, immunofluorescence, and chromatin immunoprecipitation sequencing (ChIP-seq) to investigate γH2AX following OTS186935 treatment in addition to in vivo experiments using nude mice with subcutaneously implanted uLMS. Results: SUV39H2 expression in uLMS was significantly higher than that in the normal myometrium and leiomyomas. OTS186935 decreased the viability of both cell lines, and its combination with olaparib resulted in synthetic lethality in SK-UT-1 cells (combination index = 0.88). After treatment with OTS186935, γH2AX accumulation decreased. ChIP-seq also showed downregulation of γH2AX following OTS186935 treatment. Notably, the combination of OTS186935 and a PARP inhibitor was significantly more effective in vivo. Conclusion: OTS186935 inhibited double-stranded DNA break repair, as evidenced by γH2AX downregulation by ChIP-seq and other assays. OTS186935 combined with olaparib induces synthetic lethality in patients with uLMS.

overall_design: RNA-seq was conducted for SK-LMS-1 and SK-UT-1 cell lines treated with DMSO

first: Yusuke

last: Toyohara

first: Kenbun

last: Sone

first: Reo

last: Maruyama


title: Human pancreatic beta cell transcriptomes from donors with and without T1D

city: Worcester

first: Mason

last: Tarpley

address: 368 Plantation Street Albert Sherman Center, AS7.2049

department: Diabetes Center of Excellence

institute: UMass Chan Medical School

summary: We compared the transcriptome of bulk sorted T1D donor beta cells to those from non-diabetic donors.

overall_design: Isolated human donor islets were fiexed, permeabilized, and atained for hormones.  Beta cells were enriched by sorting for Insulin-positve cells and lysed for RNA purification. mRNA llibrarries prepared with either Nugen Ovation or Clotech SMARTER Pico kits were sequenced using Illumna NextSeq.  Sequnce data was process through the DolphinNext RNA-Seq pipeline and Harman was used normalize based on library kit.  DEBrowser was used to create differential gene expression lists.

last: Harlan

first: Sambra

last: Redick

first: Sally

last: Kent

last: Blodgett


title: Partial restoration of the mitochondrial dysfunction by AAV-Ant1 (Slc25a4) transduction protects against dilated cardiomyopathy in Slc25a4-/- plus mtDNA mutant mice

city: Philadelphia

first: Joseph

last: Guarnieri

state: PA

address: 3501 Civic Center Blvd

department: Center for Mitochondrial and Epigenomic Medicine

institute: Children's Hosptial of Philadelphia

summary: Primary mitochondrial disease (PMD) patients manifesting cardiomyopathy are twice as likely to die as other PMD patients. One PMD with cardiomyopathy is caused by null mutations in the heart-muscle isoform of the adenine nucleotide translocator (SLC25A4, ANT1) gene, the severity of the cardiomyopathy being mediated by the mitochondrial DNA (mtDNA). To optimize strategies for addressing mitochondrial cardiomyopathy, we generated an Ant1 null mouse and combined it with the ND6P25L mtDNA mutation to mimic the hypertrophic versus dilated cardiomyopathies observed in patients. Here, we transduce the neonatal Ant1-/- and Ant1-/-+ND6P25L mouse hearts with an AAV2/9-pDes-Gfp-mAnt1 cDNA vector. We show that restoration of just 10% of Ant1 gene expression was sufficient to ameliorate the cardiomyopathies in these mice. Proteomics and single-nucleus RNA sequencing reveal the reversal of dysregulated mitochondrial metabolic genes, including PGC1α, as well as cardiac contractile and extracellular matrix proteins. Hence, a modest increase in cardiac mitochondrial energetics can have profound benefits on cardiac function and is effective in treating mitochondrial cardiomyopathy.

overall_design: Bulk RNA sequencing and single nucleus RNA Sequencing (snRNA-seq) analysis on C57BL/6JEiJ, C57BL/6JEiJ Ant1-/-, and C57BL/6JEiJ Ant1-/- ND6P25L mice (WT, ANT1, ANT1 ND6), pericardially injected with 2.5x10^11 vg AAV2/9Des-Gfp-mAnt1 or 2.5x10^11 vg AAV2/9Des-Gfp . 1, 3, 6, 9, and 12 months post-infection RNA was collected using TRIzol™ Reagent (Invitrogen, 15596026) from heart samples. The RNA was assessed by NanoDrop spectrophotometer (ThermoFisher Scientific) and quantified by Qubit Flex (Thermofisher Scientific) using the Qubit RNA HS Assay Kit (Thermofisher Scientific, Q32855). The RNA was diluted to 1.43 ng/ul and reverse transcribed into cDNA using the Ion Torrent™ NGS Reverse Transcription Kit (Cat. No. A45003). The RNA library was prepared using the Ion Torrent S5 Chef and Ion AmpliSeq Kit for Chef DL8 (Thermofisher Scientific, A29024) and sequenced using the Ion Torrent S5 Sequencer and Ion 540 Kit (Thermofisher Scientific, A30011). For snRNA-seq, isolated nuclei were processed using the Chromium Single Cell 3ʹ Reagent V3.1 kit from 10X Genomics. A total of 8,000 nuclei per sample were loaded into a Chip G. Barcoding, cDNA amplification, and library construction were performed according to the Chromium 3ʹ V3.1 protocol. Sequencing was performed on a DNBSEQ-T7 platform targeting 20,000 reads per nucleus.

first: Alessia

last: Angelin

first: Kierstin

last: Keller

first: Peiran

last: Li

first: Joeseph

first: Gabrielle

last: Widjaja

first: Rasheed

last: Sule

first: Kevin

last: Janssen

first: Arnold

last: Olali

first: Valeria

last: Carosi

first: Maina

last: Beauplan

first: Deborah

last: Murdock

first: Prasanth

last: Potluri

first: Zimu

last: Cen


title: Effect of depletion of hepcidin-1 on gene expression  of mouse hepatic Kupffer cells

city: Hangzhou

first: yihang

last: Pan

state: Zhejiang

address: erkang road

institute: Zhejiang University

summary: To investigate the effect of hepcidin-1 deficiency on gene expression of mouse hepatic Kupffer cells, we isolated hepatic non-parenchymal cells from hepcidin-1 knockout mice (KO) and wild type (WT) mice with C57BL/6 genetic background, and then obtained fluorescence-activated cell sorting (FACS) sorted-Kupffer cells (gated on live CD45+ F4/80+ CD11bint hepatic nonparenchymal cells). We then performed switching mechanism at the 5′ end of the RNA transcript (SMART-seq) analysis of isolated Kufpffer cells.

overall_design: Comparative gene expression profiling analysis of SMART-seq data for Kupffer cells isolated from  hepcidin-1 knockout mice (KO) and wild type (WT) mice.

first: Yihang

first: Qixing

last: Chen


title: Early IKKb-dependent anabolic and fibrotic signature governs vascular smooth muscle cells fate and abdominal aortic aneurysm development

city: Montreal

first: Simon-Pierre

last: Gravel

state: QC

address: 2940 ch. de la polytechnique

department: Faculty of pharmacy

country: Canada

institute: University of Montreal

summary: Abdominal aortic aneurysm (AAA) is a serious disease with no effective pharmacological therapy. Although inflammation is recognized as a key regulator of AAA, targeting inflammatory pathways once the disease is established does not improve outcomes. Understanding the earliest molecular indicators could clarify precise biological targets and prognostic markers for AAA. Using ApoE-deficient mice, we performed RNA-Seq on suprarenal abdominal aortas (SRAs) from Ang II- and saline-treated mice 24 h after infusion. We further developed a unique model of hyperlipidemic mice in which the expression of the inhibitor of nuclear factor kappa B kinase subunit beta (IKKβ) can be conditionally suppressed in vascular smooth muscle cells (VSMCs). RNA-Seq data revealed early IKKβ-dependent cellular anabolic processes in SRAs, including activation of the mammalian target of rapamycin complex 1 (mTORC1) pathway. Furthermore, deletion of the Ikbkb gene in VSMCs significantly reduced the rate of aneurysm rupture in mice exposed to Ang II. In situ analysis further confirmed that the absence of IKKβ in VSMCs is associated with a reduced inflammatory response and the preservation of their contractile phenotypes. Our results reinforce the crucial role of VSMCs in rapid adaptation, leading to deleterious inflammation-dependent remodeling of the vascular wall, and define a previously unrecognized anabolic role of IKKβ in AAA pathogenesis.

overall_design: We used a transgenic homozygous mouse model referred to as SMA-CreERT2IkkbFlox/FloxApoE-/- in which IKKb expression can be temporally and selectively prevented in ApoE-/- vascular SMCs on a C57BL6 background (referred as KO). Wild-type and KO mice were treated with saline or angiotensin II (Ang II) (500 to 1000 ng/min/kg) s.c. with constant infusion using osmotic pumps for 24 h. Total RNA from the suprarenal aorta was extracted using the RNeasy Mini Kit (No. 74106, Qiagen).

first: Priscilla

last: Doyon

first: Ozge

last: Kizilay Mancini

first: Florence

last: Dô

first: David

middle: H

last: Huynh

first: Gaétan

last: Mayer

first: Huy

last: Ong

first: Maëlle

last: Batardière

first: Vincent

middle: Q

last: Trinh

first: Sylvie

last: Marleau

first: Pierre

last: Chambon

first: Marc

last: Servant


title: Cold-induced suppression of myogenesis in skeletal muscle stem cells contributes to delayed muscle regeneration during hibernation

city: Hiroshima

first: Mitsunori

last: Miyazaki

state: Hiroshima

address: 1-2-3 Kasumi, Minami-Ku

department: Graduate School of Biomedical and Health Sciences

institute: Hiroshima University

summary: Mammalian hibernators experience profound cold stress and prolonged physical inactivity during torpor periods; however, it is unclear how skeletal muscle stem cells (satellite cells; SCs) respond to these challenges. In this study, we demonstrate that SCs from a mammalian hibernator, Syrian hamster exhibit remarkable resistance to cold-induced cell death, which is associated with intrinsically higher expression of the antioxidant enzyme GPX4, which likely contributes to the suppression of ferroptosis. RNA-seq analysis revealed widespread downregulation of myogenesis-related genes following cold exposure, which suggests suppression of the myogenic program. Consistently, SCs exposed to cold stress exhibited reduced activation and differentiation capacities upon subsequent rewarming, with an increased number of quiescent Pax7-positive/MyoD-negative cells. Muscle regeneration was markedly delayed during hibernation, accompanied by decreased SC activation and macrophage infiltration, suggesting that cold-induced suppression of SC function underlies limited regenerative capacity in hibernating hamsters. Our results provide insight into the unique physiology of mammalian hibernators: SC viability is preserved, whereas regenerative activity is selectively suppressed during hibernation.

overall_design: Satellite cells (SCs) isolated from Syrian hamsters were cultured at 37°C for 48 hours after plating, followed by 24-hour incubation in GM-HEPES at 37°C, and then exposed to 4°C for 24 hours. Total RNA was extracted before and after cold exposure for RNA sequencing.

first: Tatsuya

last: Miyaji


title: Synthetic lethality from the combination of a histone methyltransferase, SUV39H2 inhibitor, and a poly (ADP-ribose) polymerase inhibitor for uterine leiomyosarcoma [ChIP-Seq]

first: Kohei

last: Kumegawa

overall_design: ChIP-seq was conducted for SK-UT-1 cell line under four conditions: 0.1% DMSO (negative control), 1 µM OTS186935, 0.5 µM doxorubicin, and a combination of 1 µM OTS186935 with 0.5 µM doxorubicin.

first: Yusuke

last: Toyohara

first: Kenbun

last: Sone

first: Reo

last: Maruyama


title: Nuclear functions of CFAP20 in transcription and replication [ChIP-seq TY-CFAP20]

city: Leiden

first: Martijn

middle: S.

last: Luijsterburg

state: zuid-holland

address: Einthovenweg 20

department: Human Genetics

country: Netherlands

institute: LUMC

summary: Fine-tuning DNA replication and transcription is crucial to prevent collisions between their machineries. This is particularly important near promoters, where RNA polymerase II (RNAPII) initiates transcription and frequently arrests, forming R-loops. Arrested RNAPII can obstruct DNA replication, which often initiates near promoters. The mechanisms that rescue arrested RNAPII during elongation to avoid conflicts with co-directional replisomes remain unclear. Here, using genome-wide approaches and genetic screens, we identify CFAP20 as part of a protective pathway that salvages arrested RNAPII in promoter-proximal regions, diverting it from the path of co-directional replisomes. CFAP20-deficient cells accumulate R-loops near promoters, which leads to defects in replication timing and dynamics. These defects stem from accelerated replication-fork speeds that cause a secondary reduction in origin activity. Co-depletion of the Mediator complex or removal of R-loop-engaged RNAPII restores normal replication. Our findings suggest that transcription-dependent fork stalling in cis induces accelerated fork progression in trans, generating single-stranded DNA gaps. We propose that CFAP20 facilitates RNAPII elongation under high levels of Mediator-driven transcription, thereby preventing replisome collisions. This study provides a transcription-centred view of transcription-replication encounters, revealing how locally arrested transcription complexes propagate genome-wide replication phenotypes and defining CFAP20 as a key factor that safeguards genome stability.

overall_design: To explore spatial connections between transcription and replication at the genome-wide level, as well as the role of CFAP20 in these processes, we mapped RNA polymerase II (RNAPII) and CFAP20 occupancy using chromatin immunoprecipitation (ChIP)-seq, nascent transcription by bromouridine (BrU)-seq and TTchem-seq, and R-loops by DNA-RNA hybrid immunoprecipitation (DRIP)-seq in RPE1 cells being either wild type (WT), CFAP20-KO, CFAP20-KO rescued with CFAP20-GFP, or CFAP20/CCNC-dKO.

first: Sidrit

last: Uruci

first: Daphne

middle: E

last: Boer

first: Paul

last: Chrystal

first: Maxime

last: Lalonde

first: Andreas

last: Panagopoulos

first: George

last: Yakoub

first: Idil

last: Kirdök

first: Klaas

last: de Lint

first: Melanie

last: van der Woude

first: Tiemen

last: Wendel

first: Sem

last: Brussee

first: Annelotte

last: Wondergem

first: Nila

last: van Overbeek

first: Nini

last: Schotman

first: Jolanthe

last: Lingeman

first: Mats

last: Ljungman

first: Alexander

last: van Oudenaarden

first: Haico

last: van Attikum

first: Alfred

last: Vertegaal

last: Noordermeer

first: Rob

last: Wolthuis

first: Matthias

last: Altmeyer

first: Stephan

last: Hamperl

last: Tropepe

first: Jeroen

last: van den Berg

first: Diana

last: van den Heuvel


title: Rapid gain and loss of a chromosome drives key morphology and virulence phenotypes in Histoplasma, a fungal pathogen of humans

first: Mark

last: Voorhies

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

overall_design: Refer to individual Series


title: Tributyltin chloride alters the structural, genomic, and epigenomic integrity of postejaculatory mammalian sperm

status: Public on May 27 2025

city: Gainesville

first: Muhammad

middle: Sohail

last: Siddique

state: Florida

address: 1911 SW 40th Terrace Apt E

institute: Department of Animal Sciences

summary:
A global top priority for ameliorating male factor infertility includes identification of environmental factors and mechanisms that impact sperm function. Detection of endocrine disruptors (EDs) in seminal plasma and within the female reproductive tract has created an urgent need to understand how environmental stressors alter postejaculatory sperm function. Tributyltin chloride (TBT) is a model organotin, ED and epigenetic modifier that causes reproductive disorders in testicular sperm; however, consequences of TBT exposure on postejaculatory sperm prior to fertilization remain unknown. Our previous data indicate that TBT-exposed sperm affects initial cleavage and lowers embryo development potential. However, identifying reasons for idiopathic male factor subfertility is challenged by lack of routine clinical assessment for subtle phenotypes that compromise sperm function and therefore remain undetected with current diagnostic tools.
Results
The present study was aimed at identifying structural, genomic, and epigenomic consequences of TBT exposure to postejaculatory sperm in a temporal manner reflective of sperm transport prior to fertilization within the female reproductive tract. Bovine sperm were exposed to TBT (0 ,1, 10, 100nM) for 24 h under non-capacitating conditions at 25°C followed by quantification of sperm kinematics at 37°C, DNA integrity, and methylation status. No differences in sperm kinematics or capacitation status were detected after 24 h. However, acrosome integrity was compromised at both 0 and 24 h (P ≤ 0.05). Sperm DNA integrity was also negatively affected after 24 h. The methylation levels of select loci were quantified by PacBio single-molecule real-time (SMRT) long-read high-fidelity sequencing. Whole genome methyl-seq revealed 750 differentially methylated regions (DMRs) associated with exposure to TBT for 24 h. IPA and GO analyses identified embryo development, cell signaling, and transcriptional regulation as the most relevant bio-functions of TBT altered DMRs.
Conclusions
Postejaculatory mammalian sperm exposure to TBT negatively affects parameters important for sperm function while altering DNA integrity and the methylation profile of promotor regions. Consequences of sperm exposure to TBT include cellular and molecular mechanisms that are important for sperm function but remain undetected by routine clinical analyses. These findings provided new insight towards environmental impacts on postejaculatory sperm function.

overall_design: Frozen-thawed sperm from two different commercial bulls were used for all experiments except where stated otherwise. Four exposure level of TBT (100nm, 10nm, 1nM and 0nM) were used in all experimnets unless stated otherwise.In each replicate,sperm were isolated from four frozen straws on a discontinuous Percoll gradient (90:45%).DNA was isolated from TBT exposed sperm following TBT exposure at 24 h followed by enzymatic Methyl seq Conversion. The enzymatic methyl seq converted DNA from sperm were PCR amplified for three hypermethylated loci (PTK2B, HDAC11, PAK1) and three imprinted loci(SNRPN, H19, KCNQ1) and sequenced for loci specific PacBio single molecule real time long-read high-fidelity sequencing (SMRT).In SMRT sequencing, only two treatments (VC and TBT 10nM) were used (n = 2 bulls, 4 pooled independent reps/ bull/trt). For whole genome methyl-seq analysis, three treatments were used (VC, TBT 100nM and TBT 10nM) n = 1 bull, 4 pooled independent reps/bull/trt).Data were analyzed by logistic regression using a generalized linear mixed effect model. Treatment and time were considered fixed effects while bull and replicate were assigned as random effects.

middle: S

first: Santosh

last: Anand

first: Marie-Pierre

middle: L

last: Gauthier

first: Brant

middle: O

last: Jason

first: Michael

last: Kladde

first: Ramji

last: Bhandari

first: Bradford

last: Daigneault


title: Human pancreatic islet responses to cytokine treatment

first: Mason

last: Tarpley

summary: We performed CITE-Seq on isolated human donor islets cultured 48hrs in the presence or absence of a cytokine cocktail. Major cell types were identified based on gene expression and changes in gene expression in beta cells were examined.

overall_design: CITE antibody labeling, cell partitioning and library construction using antibodies from BioLegned and 10x Chrmium 3' technology.

first: David

middle: M

last: Harlan

first: Sambra

middle: D

last: Redick

first: Sally

last: Kent


title: Comprehensive transcription factor perturbation responses for Cryptococcus neoformans

city: St Louis

first: Chase

last: Mateusiak

state: MO

address: 4515 McKinley Ave

institute: Washington University, St Louis

summary: Cryptococcus neoformans is a deadly fungal pathogen. Upon entering a mammalian host, it deploys a voluminous polysaccharide capsule that is necessary for it to survive host defenses and maintain an infection. Capsule expansion is regulated transcriptionally, as deletion of many transcription factors alters capsule. Thus, we set out to map the transcriptional regulatory network of C. neoformans – that is, to identify the TFs that directly regulate each gene in the genome. First, we carried out RNA-seq of 120 single-TF-deletion strains, together with wild-type controls. We then applied NetProphet3, a TF network mapping algorithm, to predict the direct functional targets of each TF. Unexpectedly, analysis of this network indicated that there are no TFs that primarily regulate genes involved in capsule formation. Rather, the TFs that play a role in deploying capsule also regulate many other genes and processes. Comparison to a TF network map we built for Saccharomyces cerevisiae, a distantly related model yeast, identified pairs of TFs that are functionally orthologous – that is, their targets are enriched for orthologous genes. In many cases, these pairs are different from the ones identified by sequence homology alone. We suggest that network analyses should be used to complement sequence comparison when searching for functionally orthologous transcription factors. Our network map can be searched and visualized at http://cryptococcus.net.

overall_design: After growth in YPD at 30C, WT cells and deletion cells were washed in PBS and transferred to DMEM medium at 37C. Samples were taken for RNA-Seq at 90 minutes after transfer. We aimed for at least four replicates of each concentration at each time point. Replicates were grown and library-prepped on separate days to account for day-to-day variability.

last: Brent

first: Tamara

last: Doering


title: Evaluating the practical aspects and performance of commercial single-cell RNA sequencing technologies

city: Seattle

first: Anna

last: Elz

state: WA

address: 1201 Eastlake Ave E

institute: Fred Hutchinson Cancer Center

summary: The rapid development of updated and new commercially available single-cell transcriptomics platforms is providing a range of experimental options.  Cost, sensitivity, throughput, flexibility, and ease of use influence that selection.  We performed a comprehensive comparison of single-cell transcriptomic approaches using multiple standardized PBMCs. In evaluating seven recently available kits that interrogate whole transcriptome mRNA and two including TCR sequencing, we report on standard single-cell sensitivity metrics including gene saturation, UMI detection, cell recovery, sequencing efficiency, and cell subset resolution.  We also discuss workflow throughput, sample requirements, timing, cost, and labor as critical factors to consider based on project goals. In addition to variable experimental constraints imposed by each platform, our findings highlight differences in cell recovery and sensitivity, which we found to significantly influence cell subset resolution. This work provides a basis by which users can balance performance and practical considerations when selecting an RNA single-cell sequencing platform.

overall_design: For our first experiment,  Benchmark 1 (BM1), we used two healthy PBMC donor samples in replicate, to compare seven platforms: 10x Genomics’ Chromium NextGEM 3′ v3.1 (labeled “NextGEM 3’” in figures and results),  GEM-X 3’ v4 (GEM-X 3’), and Fixed RNA Profiling (Flex), Parse’s Evercode WT v.3 (Parse v3), Scale Biosciences Single Cell RNA kit (Scale), and Fluent Biosciences PIP Seq T20 3′ v4 (Fluent v4) and PIPseq V T20 3’ (Fluent V).  Each of these kits target 3′ and whole transcriptome RNA from single cells.  All platforms were run in parallel on the same day except for Scale and Fluent PIPseq V assays, which were processed separately using aliquots from the same donors’ leukapheresis samples (Figure 1a). For our second experiment, Benchmark 2 (BM2), we compared platforms that generate TCR repertoire profiling (Supplemental Figure 1a).  For this, we sorted T-cells based on CD3 expression from 4 different healthy donor PBMCs, with overlap of two BM1 donors, to run on 10x Genomic’s Chromium NextGEM 5′ v2 (NextGEM 5′), GEM-X 5′ v3 (GEM-X 5’) and Parse Evercode v.2 TCR (Parse v2).

first: Derrik

last: Gratz

first: Annalyssa

last: Long

last: Sowerby

first: Azi

last: Hadadianpour

first: Evan

last: Newell


title: RNA sequencing of (1) isolated retinal endothelial cells from wildtype and Zmiz1-KO endothelial cells and (2) control and Zmiz1 shRNA treated MS1 cells

city: new orleans

first: Rajan

last: K C

state: LA

address: 6602 Freret St

department: Cell and Molecular Biology

institute: Tulane University

summary:
Purpose: Zinc Finger MIZ-Type Containing 1 (Zmiz1) is a member of the PIAS family of protein and function as a transcriptional coactivator of Notch, Androgen Receptor (AR), p53, Estrogen Receptor (ER), and Smad3/4 . Despite Zmiz1 association with vascular system, its role in physiological and pathological angiogenesis is significantly unknown. Here, we use (1) MS1 cell line treated with control shRNA and Zmiz1 shRNA and (2) murine model to knockout Zmiz1 in endothelial cells using Cdh5CreERT2 and isolate retinal endothelial cells (iRECs) then performed RNA sequencing to profile transcriptional changes upon Zmiz1 deletion.
Retinal ECs were isolated from P7 pups as previously described (Patel et al., 2022. JCI Insight). Briefly, isolation of ECs from the retinas was performed using the Miltenyi Neural tissue Dissociation Kit (P) (Miltenyi, 130-092-628). For each sample, 8-10 retinas were pooled and digested to obtain a single cell suspension. CD31+ cells were separated from the cell mixture via magnetic activated cell sorting. MS1 cells were transfected with 20μM of either control-shRNA or Zmiz1-shRNA using Lipofectamine3000 (Thermofisher, L3000015).  Next, RNA was isolated from the cells and used for downstream RNA-sequencing.  RNA concentration and RNA integrity number were determined. RNA library was prepared, quantified, and verified using TruSeq RNA Library Prep Kit v2, Qubit dsDNA High Sensitivity Assay kit and Bioanalyzer DNA1000 assay kit respectively. Verified samples were sequenced on a Nextseq 500/550 platform. RNA-seq data analysis was performed using illumina BaseSpace Sequence Hub.
Results: For iRECs , we found 2,364 differentially expressed genes of which 834 genes were upregulated while 1530 genes were downregulated. Downregulated genes were enriched in biological processes such as angiogenesis, vascular development, and cell growth. For MS1 cells,  we found 1,711 differentially expressed genes of which 718 genes were upregulated while 993 genes were downregulated. Downregulated genes were enriched in biological processes such as regulation of angiogenesis.
Conclusions: We assessed changes in transcriptional landscape in MS1 and iRECs following Zmiz1 deletion.

overall_design: Transcriptional assessment on Zmiz1 depleted MS1 cells and deleted iRECs by RNA sequencing

first: Nehal

last: Patel

first: K C

last: Rajan

first: Stryder

last: Meadows


title: NF1-depleted ER+ breast cancers are differentially sensitive to CDK4/6 inhibitors

city: Houston

first: Jonathan

middle: Thomas

last: Lei

state: TX

address: 1 Baylor Plaza

department: Medicine - Lester and Sue Smith Breast Center

institute: Baylor College of Medicine

summary: Neurofibromin/NF1-depletion (NF1low) is associated with endocrine therapy resistance in approximately 20% of ER+/HER2– early-stage breast cancer but specific treatments for NF1low tumors are not established.  Proteogenomic analyses on ER+/HER2– breast cancer demonstrated that NF1low tumors exhibit elevated CDK4/6 activity.  NF1-silencing in cell lines promoted ER recruitment to CCND1, thus increasing cyclin-D1 levels.  Additionally, RAF is demonstrated to directly phosphorylate the CDK4 activation-loop (pT172) thus providing a direct connection between NF1 loss and an increase in CDK4 activity.  Consequently, NF1low cancer cells are differentially sensitive to a fulvestrant plus CDK4/6 inhibitor (CDK4/6i) combination, with cell-death induction in vitro, and durable tumor regressions in ER+ NF1low PDX models in vivo. Furthermore, NF1low ER+/HER2– tumors treated neoadjuvantly with 4 weeks of anastrozole exhibited a minimal reduction of a multigene mRNA proliferation score vs NF1high tumors but exhibited marked sensitivity to the subsequent addition of palbociclib. In conclusion, dual ER and downstream RAF activation following NF1-loss drives endocrine therapy resistance and CDK4/6i sensitivity in NF1low ER+ breast cancer.  NF1 status should therefore be prioritized for biomarker investigations in adjuvant CDK4/6i trials to determine whether the NF1low status identifies a particularly sensitive subset where CDK4/6i therapy is critical.

overall_design: ER-ChIP-seq of doxycycline inducible knockdown of NF1 in hormone-deprived MCF7 breast cancer cell lines  -/+ E2.

first: Eric

last: Chang

first: Ze-Yi

last: Zheng

middle: T

first: Meenakshi

last: Anurag


title: Spatiotemporal regulation of target mRNA cleavage by 21-nt phasiRNAs in maize anthers [sRNA-seq]

status: Public on May 26 2025

city: Beiing

first: Yijun

last: Qi

address: NO.1 Qinghuayuan

department: School of Life Sciences

institute: Tsinghua University

summary: In grasses, 21- and 24-nt phasiRNAs are abundantly expressed in anthers and play crucial roles in anther development and male reproduction. 21-nt phasiRNAs can act in cis to direct the cleavage of their own precursor transcripts and is thought to lack trans cleavage targets in maize. Here we show that 21-nt phasiRNAs direct the cleavage of hundreds of target mRNAs in maize, with the cleavage activity being enriched in male germ cells. Interestingly, whereas 21-nt phasiRNAs in anthers (somatic anther wall cells) do not have obvious preference for a particular nucleotide at the 5’ end and target genes implicated in stress response and cellular homeostasis, 21-nt phasiRNAs in male germ cells preferentially initiate with 5’ C and associate with ZmAGO5c/MAGO2/MS28 to target genes related to meiotic processes. Our results indicate that mRNA cleavage is a conserved mechanism used by 21-nt phasiRNAs for gene regulation. However, the sequences of 21-nt phasiRNAs and their targets show low conservation in maize and rice, suggesting that 21PHAS loci and 21-nt phasiRNA targets have undergone fast divergence in monocots.

overall_design: To explore potential targets of 21-nt phasiRNA  and their regulatory roles in their targets in  maize，we collected maize anthers,male germ cells across various these developmental stages. and embryos from seeds  .We then using these samples performed low-input sRNA-seq, RNA-seq and high sensitive degradome-seq

first: Bi

last: Lian

first: Tianxiao

last: Huang

first: Wei

first: Pengfei

last: Jiang

first: Weiwei

last: Jin


title: Spatiotemporal regulation of target mRNA cleavage by 21-nt phasiRNAs in maize anthers [RNA-seq]


title: NR4A1 Expression Aberrations Contribute to Radiotherapy Resistance in Gastric Cancer

status: Public on May 24 2025

city: Xuzhou

first: Peidong

last: Ni

address: 199 Jiefang South Road

department: General Surgery

institute: xuzhou central hospital

summary: The expression of NR4A1 was significantly reduced in radioresistant cells, namely MKN45-R and AGS-R, as opposed to their parental counterparts. Overexpressing NR4A1 in MKN45-R and AGS-R cells resulted in heightened sensitivity to radiation. Additionally, NR4A1 overexpression exerted inhibitory effects on the growth and metastasis of GC tissues in animal models following irradiation (IR). Our observations indicate that NR4A1 plays a crucial role in enhancing the radiation sensitivity of gastric cancer cells, underscoring the therapeutic potential of NR4A1 in GC treatment.

overall_design: RNA-seq profiling of radioresistant cells MKN45-R and their parental MKN45 cells.

first: Chuming

last: Zhu


title: 6K-plex RNA  CosMx data from DLBCL sections.

first: Michael

middle: R

last: Green

address: 7455 Fannin St

institute: MD Anderson Cancer Center

summary: Diffuse large B-cell lymphomas (DLBCLs) are a common and heterogeneous group of mature B-cell malignancies that typically arise in lymph nodes; sites in which stromal and endothelial cells, antigen-presenting cells and other myeloid cells facilitate adaptive immune responses by T-cells and B-cells against pathogens. In this study, we have leveraged the spatial transcriptomic platform of CosMx together with CODEX imaging technology to investigate the spatial immunology and pathobiology of DLBCL.

overall_design: CosMxTM technology [CosMxTM Human Universal Cell Characterization RNA Panel (1000-plex) + 20 custom genes; Bruker Spatial, USA] was applied to  6 tissue microarrays (TMAs i.e, TMA DLBCL 1, TMA DLBCL 2, TMA DLBCL 3, TMA DLBCL 4, TMA DLBCL 5, TMA DLBCL 6  ) from excisional biopsies of 78 large B-cell lymphomas and 5 controls (4 tonsil, 1 lymph node). Large B-cell lymphomas included 66 DLBCL not otherwise specified (DLBCL NOS); 5 EBV+ DLBCL NOS; 4 T-cell/histiocyte-rich large B-cell lymphoma (TCHRBCL); 2 primary mediastinal large B-cell lymphoma (PMBCL); and 1 post-transplant lymphoproliferative disease (PTLD). Note: The raw data is common for all the samples in the corresponding TMA and the corresponding FOV for each sample has been described in the Supplementary File or can be requested from the contributor.

first: Atish

last: Kizhakeyil

first: Dai

last: Yibo


title: Analysis of iECM Immunomodulatory Capacities in an Animal Model of Systemic Inflammation

status: Public on May 23 2025

city: La Jolla

first: Maria

last: Karkanitsa

state: CA

address: 9100 Regents Rd, Apt G

department: Shu Chien Gene Lay Department of Bioengineering

institute: University of California San Diego

summary: Immunomodulatory effects of an infusible extracellular matrix (iECM) material were evaluated by a 547 mouse immunology panel using the nanostring nCounter mouse immunology panel (n=547 genes)

overall_design: Female C57 bl/6 were treated with a dual dose of lipopolysaccharide (LPS) to induce severe systemic inflammation leading to a Multiple Organ Dysfunction Syndrome (MODS) phenotype over a 30 hour period. Animals were treated with an infusible extracellular matrix (iECM) material or saline 10 hours post initial LPS dosage. Animals were sacrificed 30 hours post initial LPS dosage and RNA from blood, heart, lung, kidney, and brain tissue were isolated and transcriptional differences due to iECM treatment  were analyzed using nanostring nCounter analysis.

first: Anne

last: Lyons

first: Raymond

last: Wang


title: Spatiotemporal regulation of target mRNA cleavage by 21-nt phasiRNAs in maize anthers [degradome-seq]


title: Single cell RNAseq of human macrophage polarization states

status: Public on May 22 2025

city: Los Angeles

first: Alexander

last: Hoffmann

address: 570 Boyer Hall

department: MIMG

institute: University of California, Los Angeles

summary: We employed the BD Rhapsody scRNAseq platform to identify human macrophage polarization states and their response to various stimulaiton conditions. Using a select panel of genes (BD Immune Response Panel Hs) we were able to identify unique transcriptomes for naive, IFNy, IFNb, IL-4, IL-10, and IL-13 polarized macrophages under unstimulated conditions and in response to 3 hours of acute stimulation with LPS, polyIC and TNF.

overall_design: Human blood was obtained from a deidentified donor from the UCLA CFAR Centralized Laboratory Support Core in accordance with UCLA IRB 11-000443. Peripheral blood mononuclear cells were isolated from blood by Ficoll (Cytiva, 17-1440-03) density centrifugation. Monocytes were isolated using CD14+ bead selection (Milltenyi, 130-050-201), plated on 6-well tissue culture plates at a density of 5x105 cells/well, and cultured in RPMI (Gibco, 11875-093) supplemented with 10% ES fetal bovine serum (Gibco, 10439-024), penicillin-streptomycin (Corning, 30-002-Cl), L-glutamine (2mM; Corning, 25-005-CI) and 20ng/ml human macrophage colony-stimulating factor (M-CSF) (R&D systems, 216-MC-100). Media was replaced at day 4, and on 6 days from isolation, the cells were polarized for 24 hours with 100 U/mL IFN-β (PBL Assay Science, 11415-1), 20 ng/mL IFN-γ (Peprotech, 300-02), 50 ng/mL IL-10 (R&D, 217-IL), 40 ng/mL IL- 13 (R&D, 213-ILB), 20 ng/mL IL-4 (R&D, 204-IL), or left naïve. After 24 hours of polarization the cells were either left unstimulated or stimulated for 3 hours with LPS 1ng/ml (Sigma-Aldrich, L6529), TNF 10ng/ml (R&D, 210-TA-020), or polyIC 50ug/ml (Invivogen-TLRL-PIC). To collect the macrophages, the plate was washed with PBS, then incubated with 1mL warm 1mM EDTA for 15 minutes. Cells were lifted with a cell scraper, multiplexed using the BD Human Single-Cell Multiplexing Kit (BD, 633781), and loaded onto the cartridge following manufacturer’s instructions (BD Rhapsody #210967). The LPS and TNF sitmulated samples were loaded on Cartridge 1 and the polyIC and unstimulated on Cartridge 2. Targeted mRNA amplification was performed utilizing the Immune Response Panel Hs primer panel (BD, 633750), with subsequent library preparation. The pooled library was sequenced with paired-end 100bp reads on an Illumina NovaSeq X Plus. After sequencing, the FASTQ files were aligned to the Immune Response Panel reference file, generating raw read counts per gene and per cell, which were then linked to cell samples of origin, with the final output being a cell by molecular matrix.

first: Sarina

last: Lowe

first: Noah

last: Yan

first: Madelaine

last: Leitman