title: Single-cell transcriptomic and spatial subcellular landscape of the articular disk synovium in mouse temporomandibular joint osteoarthritis

city: Sinagawa-ku

first: Kazuhiro

last: Shibusaka

state: Tokyo-to

address: 1-5-8, Hatanodai,1st building, 343

institute: showa university

summary: This study aimed to elucidate the molecular mechanisms underlying TMJ-OA by integrating subcellular spatial transcriptomics with single-cell RNA sequencing analysis in TMJ-OA mouse models.

overall_design: The TMJ-OA model mouse experiments were conducted using 9-week-old female C57Black/6J mice. The TMJ-OA models were divided into three groups: Control, Mechanical Stress (MS) model, and Articular Disc Displacement (ADD) model. Three weeks after establishing the TMJ models, the condyles and synovial tissues containing the articular disc were collected separately, RNA was extracted, and RNA-seq analysis was performed for each group (n=3)

first: Asuka

last: Terashima

first: Soichiro

last: Negishi

first: Masahiro

last: Hosonuma

first: Miki

last: Maemura

first: Hiroshi

last: Yoshida

first: Nobuhiro

first: YoungKwan

last: Kim

first: Yutaka

last: Suzuki

first: Hiroyuki

last: Okada

first: Fumiko

last: Yano


title: Mapping and Examination of a Quantitative Trait Locus Controlling Trichome Abundance in Brassica napus

status: Public on Mar 17 2026

city: Saskatoon

first: Dwayne

last: Hegedus

state: SK

address: 107 Science Place

country: Canada

institute: Agriculture and Agri-Food Canada

type: Expression profiling by high throughput sequencing

summary: Trichomes contribute to plant tolerance of abiotic stresses, such as heat, and biotic stresses, including insect herbivory. Leaves of Brassica napus are typically glabrous or near-glabrous; however, a small number of hirsute accessions were identified in international germplasm collections that produced up to 300 trichomes on the earliest leaves. To investigate the genetic basis of this trait, trichome density on the abaxial and adaxial leaf surfaces, leaf margins, and petioles of the 3rd and 6th leaves was assessed in a doubled haploid (DH) population derived from a cross between a glabrous line (NAM-0) and a hirsute line (DOS-2). Lines were genotyped using a Brassica 60K SNP BeadChip array. A major quantitative trait locus (QTL) controlling trichome density was identified on chromosome C01 that overlapped with regions previously mapped in the C-genome species B. oleracea and B. villosa. In B. napus, this QTL spanned 5.65 Mb and with a 1.7 Mb deletion in the hirsute DOS-2 parent. A gene encoding a novel, R3 single-repeat, MYB protein related to the trichome repressor TRIPTYCHON was identified within the shared region and designated TRIPTYCHON-LIKE (TRL). DOS-2 harbors a single BnTRL gene, whereas NAM-0 contains four tandemly-linked copies, although BnTRL transcript abundance did not correlate directly with trichome density. The expanded TRL gene family is unique to the Brassicaceae and BnTRL proteins physically interact with the GL1-binding domain of GL3, a core component of the GL1–GL3–TTG1 trichome initiation complex. Analysis of additional genes within the shared QTL region or absent in DOS-2 revealed regulators associated with chromatin modification (e.g. ADA2b, RBBP6-like), hormone signaling (ARR2, GH3, JAM3, HAT4), cytoskeletal dynamics (SPIRAL2, MAP65-2, MAP70-5, SPIKE1), and cell growth (SVH3, LIP5). Hormone application experiments demonstrated genotype- and tissue-specific responses on trichome production, particularly to jasmonate and gibberellin. Together, these results indicate that the CO1 locus controlling trichome abundance in B. napus is complicated and may affect regulation of chromatin state, hormonal pathways, cytoskeletal organization, and cell growth, in addition to the assembly of the trichome regulatory complex.

overall_design: Two near-isogenic doubled haploid lines were developed: DH6-47 (a hirsute DH line with ca. 600 trichomes/leaf) and DH6-110 (a glabrous DH line). Original parents were Quantum/LG3260 from the AAFC breeding program and CN113136 from the Plant Gene Resources of Canada.  Individual very early third leaves (two replicates from each line) were used for RNAseq analysis, to detect differentially expressed genes in early trichome cell determination and development.

first: Zohreh

last: Heydarian

first: Cathy

last: Coutu

first: Erin

last: Higgins

first: Sally

last: Vail

middle: D


title: APOBEC3B regulates HPV replication by inducing R-loop formation and genomic instability [RNA-Seq]

city: Chicago

first: Conor

last: Templeton

address: 300 E Superior Street

country: USA

institute: Northwestern University

summary: APOBECs are cytidine deaminases whose levels are increased in cells with high-risk HPV genomes and are responsible for most mutations in HPV associated cancers. APOBEC3B is a nuclear member of this family and is shown to be a positive regulator of HPV replication as well as expression. The proviral effects of A3B found in HPV positive cells contrast with its role as a restriction factor for many other viruses. Studies demonstrated that A3B can bind and regulate the formation of R-loops, which are trimeric nucleic acid structures consisting of an RNA paired with its complementary DNA strand, displacing one of the DNA strands. The present study demonstrates that A3B binds stably to both cellular and viral chromatin at sequences containing high R-loop levels, including the HPV URR and early polyadenylation sites. Importantly, A3B was found to play a critical role in the replication of HPV genomes and in regulating viral expression. Reduction of R-loop levels through overexpression of the R-loop specific RNase, RNase H1, impaired A3B binding to viral genomes as well as at multiple cellular sites. When A3B was depleted, total R-loop levels decreased by ~50%, leading to impaired viral transcription and an increase in the expression of immune genes, such as OASL, IL6, and IRF1. Mapping R-loop formation in A3B depleted cells revealed that A3B regulated a subset of R-loops that form on the transcriptional start (TSS) and termination sites (TTS) of cellular genes, including at the HPV URR.  Furthermore, A3B depletion resulted in over a 50% reduction of DNA breaks along with altered expression of DNA damage repair proteins. This study demonstrates that A3B is an inducer of R-loop formation and genomic instability in HPV positive cells, thereby regulating cellular and viral gene expression along with HPV replication.

overall_design: RNA-Seq profiling of shAPOBEC3B (shA3B01 is sample3 and sample4, and shA3B11 is sample5 and sample6) knockdowns of CIN612 cells compared to a shScramble control (sample1 and sample2).

first: Conor W.

first: Jasmine S.

last: Gulik

first: Laimonis A.

last: Laimins


title: A temporal factorial dissection of kidney organoid differentiation identifies the impact of WNT, FGF, BMP and GDF signaling on ‘off-target’ patterning

status: Public on Mar 18 2026

city: Parkville

first: Sean

last: Wilson

state: Victoria

address: 50 Flemington Road

country: Australia

institute: MCRI

summary: Human pluripotent stem cell-derived organoids are multicellular models of developing tissues proposed to recapitulate developmental stages of lineage commitment across time. Assessing how accurately protocols recapitulate development is challenged by the lack of accurate reference data sets for early human development, the presence of ‘off target’ states and the potential to form cellular states not presentin vivo. This study addresses these challenges with respect to differentiation of human pluripotent stem cells to kidney organoids. Based on a factorial single-cell transcriptomic analysis across a 27-day differentiation protocol, including 150,957 cells collected at five time points, we present a comprehensive classification of predicted identity, representing anin vitrotemporal transcriptional atlas. A significant component of all timepoints were neuromesodermal ‘off target’ populations which resulted in a variety of neural/neural crest and muscle endpoints. Transitional mesodermal populations with no clearin vivoequivalent were also present and varied with duration of initial mesodermal commitment. The analysis of cell trajectory across the timecourse identified distinct asynchrony of cell responses duringin vitrodifferentiation, with this driving ‘off target’ patterning.

overall_design: The iPSC line used in this study was CRL2429-MAFBmTagBFP2/GATA3mCherry (32), maintained and expanded at 37oC in 5% CO2 in Essential 8 medium (Thermo Fisher) on MatrigelTM-coated (Thermo Fisher) plates with daily media changes and passaged every 3-4 days with EDTA in 1x PBS buffer as described previously49. An initial seeding density of 7 x 104 cells into 6-well culture plates was then cultured with variations based on the Takasato protocol3 modified to use TeSR-E6 medium31 (STEMCELL technologies). Organoids were cultured from D0 in 1.5ml of E6 and 7µM CHIR for 3 (3c), 4 (4c) or 5 (5c) days. Following this, organoids were cultured in one of the following conditions; 2 (2F), 3 (3F) or 4 (4F and 4FG) days of 100ng/mL FGF9, 4 days of 100ng/mL FGF9 with one of 2.5µM AGN193189 (4FA), 1µM CHIR (4FC), 0.1µM LDN193109 (4FL), or 4 days of 200ng/mL FGF9 (4F200) or base media alone (4E). All conditions were then aggregated onto transwells by dissociating to single cell and transferring 1 x 104 cells as a 1ul cell/media droplet, as 3 sets of 2 technical replicates per transwell. Organoids were cultured in 1.1ml E6 with 5µM CHIR for 1 hour before replacing media with 1.1ml E6, 200ng/mL FGF9 and 1ug/mL heparin for 5 days. An additional set of 4F organoids were generated, with these receiving 50ng/mL GDF11 during these 4 days (4FG). After 5 days culture, all organoids were maintained in E6 media alone until day 27. Media was changed every 2 days for all organoids at all times, except when a culture change occurred in between. Samples were collected at the end of the four stages of differentiation as defined by changes in media composition, with the final organoids collected at day 27 of culture.

first: Wilson

last: Sean

first: Little

last: Melissa


city: Shenzhen

first: Zhe

last: Dai

address: Xueyuan Avenue 1088

department: School of Medicine

country: China

institute: Southern University of Science and Technology

summary: Metabolic dysfunction-associated steatotic liver disease (MASLD) is characterized by profound remodeling of hepatic macrophages, including the emergence of lipid-associated macrophages (LAMs). However, the mechanisms through which LAMs promote fibrosis and their key molecular drivers remain elusive.

overall_design: Transcriptomic analysis of co-culture generated  lipid-associated macrophages (LAMs) and naive macrophages co-cultured with untreated hepatic cells (referred to as the Control, Ctrl group) by RNA sequencing (RNA-seq). There are six samples in the study which belonged to two groups, each groups contains three samples.

first: Yijin

last: Wang


title: Long-term efficacy of carbon dioxide laser in patients with Hailey-Hailey disease. Assessment of gene expression change. A clinical trial

city: Muenster

first: Marcos

middle: J

last: Araúzo-Bravo

address: Rontgenstr. 20

department: Cell and Developmental Biology

country: Germany

institute: Max Planck Institute for Molecular Biomedicine

summary: We conducted a randomized clinical trial to assess the efficacy and safety of CO2 laser therapy in 10 patients with HHD. We utilized the Ablative AcuPulse CO2 laser (10,600 nm) with the following parameters: Scanner F200, shape size 8 mm, depth 4, time 0.50 seconds, power 24 watts. Biopsies of healthy and affected skin were performed before treatment and 6 months post-treatment to evaluate changes in gene expression. RNA extraction followed by molecular analysis using microarray technology and Sanger method was conducted. Additionally, blood samples were collected for mutation analysis in peripheral blood. The validated application ImitioMeasure was used for measuring the reduction of the affected area. We used the Wilcoxon matched-pairs test to evaluate the intervention efficacy.

overall_design:
Study Population:  We conducted a randomized, controlled, intraindividual comparative clinical trial in a cohort of 10 patients with HHD refractory to conventional treatments. The study was performed at the Clínica Universidad de Navarra, Pamplona (Spain), between September 2021 and March 2024. The center's ethics committee approved the project with registration number: 2020.240. The trial was funded by the Agencia Estatal de Investigación (Spain) under the MINECO grants for research on rare diseases PID2020-114340RA-I00. All criteria of the Helsinki Declaration for research involving human subjects were met, as well as the CONSORT guidelines governing clinical trials. Informed consent was obtained from all patients in the sample. Sample Size Calculation: The total number of areas to evaluate improvement was 20, as two symmetrical areas per patient were included. These are paired samples (intraindividual), which significantly increases the power of the study. To determine the necessary sample size, the standard deviation was calculated using data from the affected area before and after treatment in patients previously treated at our center. Accepting an alpha risk of 0.05 and a beta risk of 0.2 in a two-sided test, 10 subjects were required to detect a difference equal to or greater than 2 units (assuming a standard deviation of 2.1). An estimated follow-up loss rate of 10% was accounted for. Sample Selection: Patients without histological confirmation of the disease, without bilateral involvement of the skin folds, or without disease activity in the last 3 months were excluded. Additionally, a washout period of 4 months was established for patients recently treated with photodynamic therapy or botulinum toxin, 1 month for those who received corticosteroids or other topical or oral immunosuppressants, and 2 weeks for antibiotics and/or antifungals. Recruitment took place during the COVID-19 pandemic and involved consecutive case sampling. Collected Variables: The following variables were collected for all patients at baseline and at 6, 12, and 24 months: age, sex, disease duration, affected locations, previous treatments, number of days with symptoms in the last month, quality of life (measured with the Skindex-29 questionnaire), area with active disease (cm²), presence of scars, hypopigmentation or hyperpigmentation, and iconographic control. The questionnaires detailing the variables to be collected, were administered to each patient at each visit independently for the right and left sides. Biopsy Collection: After confirming inclusion criteria and obtaining informed consent, the mentioned variables were collected separately for the left and right sides. At the initial visit, blood was drawn to perform ATP2C1 gene sequencing to confirm that they carry a mutation. Before performing laser treatment on the most affected location, randomization was done using OxMaR software to decide which side would be treated initially (treatment side), leaving the contralateral side (control side) untreated until 6 months later to compare results. Subsequently, on the treatment side, two 4-mm punch biopsies were performed in an area near but not affected by the disease (Biopsy 0), and two 4-mm punch biopsies were performed in the affected area where the laser would be applied (Biopsy 1). All four punch biopsies included the epidermis and dermis and were sutured under sterile conditions with a 4/0 silk stitch. For each patient, two of the biopsies were fixed in formalin and later embedded in paraffin for immunohistochemistry staining, and in the case of Biopsy 1, for histological confirmation of HHD. The other two biopsies were also collected under sterile conditions and stored in liquid nitrogen, kept at -80C for subsequent RNA extraction to study differential gene expression between an affected area and an adjacent healthy area. Six months after the baseline visit, before performing laser treatment on the contralateral untreated side (control side), another four 4-mm punch biopsies were taken from the treatment side, including two from the already treated area (Biopsy 2) and two from the untreated, unaffected area (Biopsy 3). Laser Treatment : The treatment was performed under anesthetic sedation, and in some cases where contraindications existed or at the patient's request, tumescent local anesthesia with mepivacaine and 0.9% saline solution was administered. We used the Lumenis AcuPulse CO2 ablative laser at 10,600 nm with the following parameters: F200 scanner, 8 mm spot size, 4 mm depth, 0.50-second duration, 24 watts power. Three passes were administered in each area until slight bleeding was achieved, indicating that the papillary dermis had been reached. Between each laser pass, the resulting scab from the epidermal ablation was removed with a saline-soaked gauze. Immediately afterward, medium potency topical corticosteroids (betamethasone) and a vaseline gauze were applied to the treated area. After 48 hours, the drressing was removed, and patients were instructed to perform daily washes with water and antiseptic soap and apply an alginate dressing until complete epithelialization, which occurred between 4 and 6 weeks. All patients were discharged with oral analgesics, and antibiotics were not prescribed unless an infection occurred during follow-up. Gene Expression Changes : After obtaining biopsies from all patients, those intended for gene expression change studies were selected, and RNA was extracted. The change in gene expression between different biopsies from the same patient was analyzed using arrays and bioinformatic analysis. Genes that varied their expression when comparing Biopsy 0 (unaffected skin, before treatment) and Biopsy 3 (unaffected skin, 6 months after treatment) were excluded from the study, as no laser was applied to the unaffected skin, and thus no gene expression changes were expected. Gene expression changes between Biopsies 1 and 2 (affected area before treatment and affected area after treatment) were interpreted as treatment-induced modifications. These changes were subsequently validated by quantitative polymerase chain reaction (qPCR). Finally, the functions of the genes whose expression had changed due to treatment were studied in the Ingenuity Pathway Analysis (IPA) database. Mutation Study : At the start of the clinical trial, EDTA-anticoagulated peripheral blood samples were collected from all patients, and genomic DNA was extracted using the Magna Pure 24 system (Roche Diagnostics). All coding exons and flanking regions of the ATP2C1 gene were analyzed using next-generation sequencing (NGS), with confirmation by Sanger sequencing. ATP2C1 gene sequencing was performed using SeqCap EZ probes and the DNBseq-G400 platform (MGI Genomics). Subsequently, a bioinformatic algorithm using Genosystem variant analysis software was applied. The algorithm included the following steps: initial sequence quality control, sequence filtering to remove terminators, adapters, and low-quality sequences, a second quality control that included frequency analysis and mapping to the reference genome Hg19, and finally, variant identification and copy number variations (CNVs) analysis, mapping coverage study, and variant annotation. This algorithm aimed to detect variants in exonic and splice regions (+/- 10 base pairs) with an allelic frequency >30%. CNV analysis was performed using Exomedepth software. The databases for variant classification were mainly ClinVar and ExAC. The criteria for variant classification followed the guidelines of the American College of Medical Genetics and Genomics (ACMG). For previously undescribed variants and VSIs, in silico prediction algorithms including REVEL, SIFT, PolyPhen-2, MutationTaster, and Prokcan were used. For splice variant prediction, Human Splicing Finder and AlamutTM Visual programs were employed. Statistical Analysis: The validated ImitioMeasure application was used to measure the affected area and calculate its reduction after laser treatment. Statistical analyses were performed using Stata version 14 (StataCorp LP, 2015, Stata Statistical Software: Release 14, College Station, Texas). Quantitative and qualitative variables were summarized using mean with standard deviation (SD) and percentages, respectively. The Wilcoxon matched-pairs test was used to assess the intervention's efficacy compared to the control. All statistical tests were two-sided. P-values less than 0.05 were considered statistically significant.
Samples:  The data 1G1-PrN-A (Gluteo,Pre-Treatment,Not-Treated,Affected), is performed with 1 replicate. The data 1G2-PoY-A (Gluteo,Post-Treatment,Treated,Affected), is performed with 1 replicate. The data 1G4-PoN-H (Gluteo,Post-Treatment,Not-Treated,Healthy), is performed with 1 replicate. The data 1G5-PoN-H (Gluteo,Post-Treatment,Not-Treated,Healthy), is performed with 1 replicate. The data 2S1-PrN-A (Undermammary fold,Pre-Treatment,Not-Treated,Affected), is performed with 1 replicate. The data 2S2-PoY-A (Undermammary fold,Post-Treatment,Treated,Affected), is performed with 1 replicate. The data 2S4-PoN-H (Undermammary fold,Post-Treatment,Not-Treated,Healthy), is performed with 1 replicate. The data 2S5-PoN-H (Undermammary fold,Post-Treatment,Not-Treated,Healthy), is performed with 1 replicate. The data 3I1-PrN-A (Groin,Pre-Treatment,Not-Treated,Affected), is performed with 1 replicate. The data 3I2-PoY-A (Groin,Post-Treatment,Treated,Affected), is performed with 1 replicate. The data 3I4-PoN-H (Groin,Post-Treatment,Not-Treated,Healthy), is performed with 1 replicate. The data 3I5-PoN-H (Groin,Post-Treatment,Not-Treated,Healthy), is performed with 1 replicate. The data 4A1-PrN-A (Armpit,Pre-Treatment,Not-Treated,Affected), is performed with 1 replicate. The data 4A2-PoY-A (Armpit,Post-Treatment,Treated,Affected), is performed with 1 replicate. The data 4A4-PoN-H (Armpit,Post-Treatment,Not-Treated,Healthy), is performed with 1 replicate. The data 4A5-PoN-H (Armpit,Post-Treatment,Not-Treated,Healthy), is performed with 1 replicate. The data 5I1-PrN-A (Groin,Pre-Treatment,Not-Treated,Affected), is performed with 1 replicate. The data 5I2-PoY-A (Groin,Post-Treatment,Treated,Affected), is performed with 1 replicate. The data 5I0-PrN-H (Groin,Pre-Treatment,Not-Treated,Healthy), is performed with 1 replicate. The data 5G4-PoN-H (Gluteo,Post-Treatment,Not-Treated,Healthy), is performed with 1 replicate. The data 5G5-PoN-H (Gluteo,Post-Treatment,Not-Treated,Healthy), is performed with 1 replicate. The data 5G5M-PoN-H (Gluteo,Post-Treatment,Not-Treated,Healthy), is performed with 1 replicate. The data 6I1-PrN-A (Groin,Pre-Treatment,Not-Treated,Affected), is performed with 1 replicate. The data 6I2-PoY-A (Groin,Post-Treatment,Treated,Affected), is performed with 1 replicate. The data 6I0-PrN-H (Groin,Pre-Treatment,Not-Treated,Healthy), is performed with 1 replicate. The data 6I3-PoN-H (Groin,Post-Treatment,Not-Treated,Healthy), is performed with 1 replicate. The data 7C1-PrN-A (Neck,Pre-Treatment,Not-Treated,Affected), is performed with 1 replicate. The data 7C2-PoY-A (Neck,Post-Treatment,Treated,Affected), is performed with 1 replicate. The data 7C0-PrN-H (Neck,Pre-Treatment,Not-Treated,Healthy), is performed with 1 replicate. The data 7C3-PoN-H (Neck,Post-Treatment,Not-Treated,Healthy), is performed with 1 replicate. The data 8I1-PrN-A (Groin,Pre-Treatment,Not-Treated,Affected), is performed with 1 replicate. The data 8I2-PoY-A (Groin,Post-Treatment,Treated,Affected), is performed with 1 replicate. The data 8I0-PrN-H (Groin,Pre-Treatment,Not-Treated,Healthy), is performed with 1 replicate. The data 8I3-PoN-H (Groin,Post-Treatment,Not-Treated,Healthy), is performed with 1 replicate. The data 9I1-PrN-A (Groin,Pre-Treatment,Not-Treated,Affected), is performed with 1 replicate. The data 9I2-PoY-A (Groin,Post-Treatment,Treated,Affected), is performed with 1 replicate. The data 9I0-PrN-H (Groin,Pre-Treatment,Not-Treated,Healthy), is performed with 1 replicate. The data 9I3-PoN-H (Groin,Post-Treatment,Not-Treated,Healthy), is performed with 1 replicate. The data 10I1-PrN-A (Groin,Pre-Treatment,Not-Treated,Affected), is performed with 1 replicate. The data 10I2-PoY-A (Groin,Post-Treatment,Treated,Affected), is performed with 1 replicate. The data 10I0-PrN-H (Groin,Pre-Treatment,Not-Treated,Healthy), is performed with 1 replicate. The data 10I3-PoN-H (Groin,Post-Treatment,Not-Treated,Healthy), is performed with 1 replicate.

first: Javier

last: Antoñanzas Pérez

first: Rafael

last: Salido-Vallejo

first: Daniela

last: Gerovska

first: Ana

last: Gorostidi

first: Agustín

last: España

first: Leyre

last: Aguado-Gil


title: Parity-conditioned Hedgehog/VDR-linked NanoString profiling of histologically normal human breast tissue (BC360)

city: Jacksonville

first: Derek

middle: Charles

last: Radisky

state: Florida

address: 4500 San Pablo Road

department: Cancer Biology

institute: Mayo Clinic

summary:
Histologically normal human breast FFPE specimens were profiled by NanoString nCounter to study parity-conditioned basal/progenitor, Hedgehog, and vitamin D receptor-associated biology in normal breast tissue.
This workbook contains the Breast Cancer 360 (BC360) arm of the study and is paired with a companion workbook for the other NanoString panel. Each of the 74 analyzed specimens contributes one GEO sample to this platform-specific workbook and one GEO sample to the companion panel workbook.

overall_design: Seventy-four histologically normal breast FFPE specimens were analyzed on both NanoString panels. This BC360 workbook contains the selected raw RCC files and a complete panel-specific raw-count matrix for the 74 paired specimens. Raw RCC files were matched to specimens by exact specimen ID; when multiple candidate RCC files existed for a specimen and panel, the latest RCC date was selected. One special mapping was applied per submitter instruction: the processed workbook sample ORG-0098 was matched to raw IO360 specimen ORG-0098 R.

first: Nicole

last: Cruz-Reyes

first: Joshua

last: Ogony

first: Pria J

last: Westerman

first: Melody

last: Stallings-Mann

first: Tanya

last: Hoskin

first: Bryan M

last: McCauley

first: Robert

middle: A

last: Vierkant

first: Laura

last: Pacheco-Spann

first: Jessica

middle: L

last: Fischer

first: Denice L

last: Gehling

first: Lisa

middle: R

last: Seymour

first: Mark

middle: E

last: Sherman

first: Amy

middle: C

last: Degnim

first: Stacey

last: Winham


title: Parity-conditioned Hedgehog/VDR-linked NanoString profiling of histologically normal human breast tissue

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

overall_design: Refer to individual Series


title: Feeder-Free Generation of Lymphatic Endothelial Cells from Human Induced Pluripotent Stem Cells

city: New York

first: Bruce

last: Gelb

state: New York

address: 1470 Madison Ave

department: Pediatrics and Genetics and Genomic Sciences

institute: Icahn School of Medicine at Mount Sinai

summary: The lymphatic system is vital for fluid balance, lipid transport, and immune function, but studying human lymphatic vasculature has been limited by poor access to primary LECs. Here, we present an efficient, scalable, feeder-free protocol to differentiate human iPSCs into iLECs that closely resemble primary fetal LECs transcriptionally and phenotypically, enabling disease-relevant studies and precise genetic perturbations supported by marker validation and bulk RNA-seq comparisons to human fetal endothelial datasets.

overall_design: We use CHIR, bFGF, BMP4, VEGF-A, EGF, and VEGF-C to differentiate iPSCs into LECs

first: Aditi

last: Prasad

first: Shrey

last: Patel

first: Simon

last: Ng

first: Clifford

last: Liu


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

first: Franco

last: Vizeacoumar

state: Saskatchewan

address: 4D40 Health Sciences Building, 107 Wiggins Road

department: Oncology

institute: University of Saskatchewan

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

overall_design: Samples were collected at different time points after infection with the 90k shRNA libraries and probes were prepared to hybridize to microarray chips to identify SDL partners of TERT gene.

first: Jared

last: Price

first: Frederick

middle: S

first: Omar

last: Abuhussein

first: Judy

middle: M

last: Wong

first: Andrew

last: Freywald


title: SLC1A5 prevents aortic aneurysm and dissection by glutaminolytic-epigenetic orchestration of vascular smooth muscle cell homeostasis [CUT&Tag]

city: Guangzhou

first: Zhiping

state: Guangdong

address: No. 855, East Xingye Avenue

institute: Jinan University

type: Genome binding/occupancy profiling by high throughput sequencing

summary: In this study, we found that H3K9ac level were reduced both in vitro (Slc1a5 knockdown VSMCs) and in vivo (Slc1a5 knockout mice). To further investigate the potential functional significance that SLC1A5-mediated H3K9ac contributed to the pathogenesis of aortic aneurysm and dissection (AAD), we performed genome-wide cleavage under targets and tagmentation (CUT&Tag) analysis to identify candidate genes regulated by H3K9ac (CST, 9649S) in Slc1a5 knockdown mouse VSMCs. Following CUT&Tag, H3K9ac associated DNAs were amplified using non-biased conditions, labeled, and sequenced with Illumina novaseq Xplus.

overall_design: The CUT&Tag assay was performed on Slc1a5 knockdown mouse vascular smooth muscle cells to specifically capture and sequence the DNA fragments associated with H3K9ac binding sites regulated by SLC1A5.

first: Pinglian

last: Yang

first: Zhechang

last: Gao

first: Weile

last: Ye

first: Ke

last: He

first: Suowen

last: Xu

first: Jiaojiao

last: Wang


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

city: Singapore

first: Roger

last: Foo

address: 14 Medical Drive, MD6

country: Singapore

institute: National University of Singapore

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

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

first: Ivan

last: Menendez-Montes

first: Chang Jie Mick

last: Lee

first: Chukwuemeka George

last: Anene-Nzelu

first: Hesham A.

last: Sadek

first: Roger Sik-Yin


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

first: Franco

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

first: Jared

first: Frederick

first: Vincent

last: Maranda

first: Yup

last: Zhang


title: Gammaherpesviruses reprogram long-term myelopoiesis through CD8⁺ T cell–monocyte dialogue within the bone marrow niche

city: Liège

first: Bénédicte

last: Machiels

state: Liège

address: Avenue de Cureghem 7b, B43b

department: DMI

country: Belgium

institute: Uliège


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

last: Vizeacoumar

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

last: Price

first: Connor

last: Denomy

first: Judy

first: Andrew


title: Gammaherpesviruses reprogram long-term myelopoiesis through CD8⁺ T cell–monocyte dialogue within the bone marrow niche [scATAC-Seq]

summary: The immune system is continuously shaped by microorganisms, with viruses emerging as key drivers of immune variability. Gammaherpesviruses (γHVs), including Epstein–Barr virus, have coevolved with their hosts to establish lifelong infections, and may imprint durable innate immune memory. Using Murid herpesvirus 4 in wild-type mice and EBV in humanized models, we show that γHVs remodel monocytes and hematopoietic stem cells at phenotypic, transcriptomic, and epigenetic levels, resulting in altered responses to heterologous challenge. Viral persistence is required for immune priming, and IFN-γ signaling directly reprograms myelopoiesis. During latency, virus-specific resident CD8⁺ T cells accumulate in the bone marrow, and remain poised for rapid IFN-γ release. Upon low-dose LPS stimulation, monocyte-derived IL-27, IL-18, and IL-12 promote bystander IFN-γ secretion from these CD8+ T cells, amplifying myelopoietic rewiring. Thus, γHV latency establishes a cytokine circuit that shapes innate immune training and defines a new equilibrium between host and persistent viruses.

overall_design: C57BL/6 mice were treatead or not with MuHV4 WT or MuHV4-Del73 (deleted) virus. Bone marrow LSK cells and blood monocytes were isolated by Fluorescence-activated cell sorting (FACS) according to the presence or absence of surface markers at day 8, 30 and 120 post-infection.

first: Arthur

last: Poncelet

first: Olivier

last: Botman

first: Abdulkader

last: Azouz

first: Aurélie

last: Detavernier

last: Martens

first: Muriel

last: Nguyen

first: Séverine

last: Thomas

first: Valérie

last: Acolty

first: Adeline

last: Rosu

first: Céline

last: Maquet

first: Lucia

last: Rodriguez-Rodriguez

first: Rémy

last: Sandor

first: Justine

last: Javaux

first: Malyvanh

last: Pathammavong

first: Christian

last: Münz

first: Anthony

last: Rongvaux

first: Laurent

last: Gillet

first: Stanislas

last: Goriely


title: Hierarchical microtopology and phase-specific delivery functionally restore ultra-long nerve continuity across species

city: Jinhua

first: QI

last: TU

address: 365 Renmin East Road, Wucheng District

institute: Zhejiang University School of Medicine

summary: Repairing ultra-long peripheral nerve defects remains a major clinical challenge, primarily due to the requirement for regenerative platforms to be capable of integrating spatial guidance with temporally resolved biochemical cues. To address this, we developed a clinically translatable, fully synthetic nerve conduit that aligns with native regenerative principles through hierarchical microtopological engineering and phase-specific molecular delivery. This design creates a pro regenerative microenvironment by synchronizing structural cues with repair cascades. The neuroanatomically inspired core mimics the endoneurium and perineurium, providing extensive cell scale contact guidance for axonal alignment and fasciculation. A dual-layered sheath ensures mechanical integrity, metabolic permeability, and selective exclusion of fibrotic tissue. Tailored to align with distinct phases of nerve repair, the conduit enables sequential release of spermidine and ascorbic acid. Spermidine resolves early inflammation, priming the niche for ascorbic acid-mediated debris clearance, axonal elongation, and remyelination. In both rat (2 cm) and beagle (5 cm) models of critical-sized sciatic nerve defects, the conduit supports structural and functional regeneration comparable to autografts, while yielding superior outcomes in motor coordination and suppression of autotomy. This strategy offers a scalable and mechanistically informed solution for repairing ultra long nerve injuries with high translational promise.

overall_design:
In this study, we developed a clinically translatable, fully synthetic nerve conduit that aligns with native regenerative principles through hierarchical microtopological engineering and phase-specific molecular delivery. This design creates a pro regenerative microenvironment by synchronizing structural cues with repair cascades. The neuroanatomically inspired core mimics the endoneurium and perineurium, providing extensive cell scale contact guidance for axonal alignment and fasciculation. A dual-layered sheath ensures mechanical integrity, metabolic permeability, and selective exclusion of fibrotic tissue. Tailored to align with distinct phases of nerve repair, the conduit enables sequential release of spermidine and ascorbic acid. Spermidine resolves early inflammation, priming the niche for ascorbic acid-mediated debris clearance, axonal elongation, and remyelination.
Experimental model and sample source:
The transcriptomic data were obtained from regenerated rat sciatic nerve tissues. A 2-cm long-gap sciatic nerve defect model was established in rats, and the nerve gap was bridged using three different types of nerve conduits.
Fourteen days after implantation, regenerated tissues within the conduits were collected. Total RNA was extracted from these regenerated nerve tissues for transcriptomic sequencing, with the aim of investigating the influence of conduit structural guidance and bioactive molecule delivery on early-stage nerve regeneration.
Three experimental groups were included in this dataset:
Hollow group:
A hollow nerve conduit without any internal guidance structures.
AFMC group:
A biomimetic nerve conduit containing internal guidance structures. The lumen of the conduit is filled with aligned fibers (AF) and multiple longitudinal microchannels (MC), which provide physical guidance cues for axonal regeneration. Because the conduit integrates both aligned fibers and microchannels, it is referred to as the AFMC conduit.
AFMC-AA-SP group:
Based on the AFMC conduit, bioactive molecules were incorporated with spatially differentiated loading. Spermidine (SP) was adsorbed onto the surface of the aligned fibers, while L-ascorbic acid (AA) was loaded inside the fibers. This design enables sequential release of the two bioactive molecules. In the early stage after implantation, SP is rapidly released from the fiber surface to modulate the inflammatory microenvironment. During later stages of regeneration, AA is gradually released from the fiber interior, contributing to neurotrophic support and promoting myelin remodeling.
This dataset therefore allows the evaluation of:
The effect of multiscale structural guidance on nerve regeneration (AFMC vs. Hollow)
The additional effect of bioactive molecule delivery on tissue regeneration (AFMC-AA-SP vs. AFMC)

first: Shuxin

last: Dai


title: Loss of BAP1 cooperates with TP53 deficiency to transform a common myelo-erythroid progenitor (scRNA-seq - mus musculus)

city: Washington

first: Alexandros

last: Tzatsos

state: DC

address: 800 22nd St NW SEH 8850

department: GW Cancer Center

institute: George Washington University

summary: We performed scRNA in sorted LinnegcKit+ bone marrow cells from single and double Bap1 and Trp53 knockout mice to identify transcirptional programs driving erythroleukemia.

overall_design: LinnegcKit+ cells were sorted and approximately 8,000-10,000 cells per mouse were partitioned with the help of the Chromium Controller (10x Genomics) into droplets with barcoded gel beads. Library preparations took place using Single Cell 3' v3.1 chemistry (catalog number 1000268; 10x Genomics) and sequenced in a NovaSeq 6000 instrument. Demultiplexing, barcode processing, gene counting, and aggregation performed with Cell Ranger (v7.1.0) using the ‘intron mode’ and the ‘mm10-2020-A’ reference genome.


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

city: Anshun

first: Longkun

last: Hu

state: Guizhou

address: Machang Town, Yunmanhu Campus of Guizhou Medical University

institute: Guizhou Medical University

type: Non-coding RNA profiling by high throughput sequencing

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

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

first: Tian

last: Zhang

first: Diao

last: Chen

first: Yongxin

first: Fei

last: Zhou

first: Ruohan

last: lou

first: Yuxia


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

first: Roger

last: Foo

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

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

first: Ivan

last: Menendez-Montes

first: Chang Jie Mick

last: Lee

first: Chukwuemeka George

last: Anene-Nzelu

first: Hesham A.

last: Sadek

first: Roger Sik-Yin


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

city: Saratoga Springs

first: Jason

last: Breves

address: 815 N. Broadway

department: Biology

institute: Skidmore College

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

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

middle: P

first: Ming-Wen

first: Mayu

last: Inokuchi

first: Lucia

last: Seale

first: Andre


title: Xenium single cell spatial transcriptomics from patient-derived glioblastoma organoid cultured on brain slice and a primary glioblastoma sample

city: Bonn

first: Michael

last: Holzel

address: Sigmund-Freud Str. 25

department: Institute of Clinical Chemistry and Clinical Pharmacology

institute: University Hospital Bonn

summary: Glioblastoma shows marked spatial heterogeneity and hypoxia-associated signaling. Here, we performed probe-based Xenium single-cell spatial transcriptomics on a primary human glioblastoma sample and on a patient-derived glioblastoma organoid cultured on a brain slice. The aim was to assess the in situ single-cell expression of hypoxia-related genes, with a particular focus on VEGFA and CXCL12. These datasets provide spatially resolved transcriptomic information to study hypoxic niches and tumor microenvironmental interactions in glioblastoma.

overall_design: A primary glioblastoma tumor specimen obtained at surgical resection was analyzed as an FFPE sample. In addition, a patient-derived glioblastoma organoid was established from a different primary glioblastoma specimen and cultured on a brain slice prior to probe-based Xenium spatial transcriptomic analysis. The study was designed to assess hypoxia-associated gene expression in situ at single-cell resolution, with particular focus on VEGFA and CXCL12.

last: Hölzel

first: Frank

last: Giordano

first: Julian

last: Layer

first: Matthias

last: Schneider

first: Anna-Laura

last: Potthoff

first: Barbara

last: Pregler


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

city: Vancouver

first: Jana

last: Jajarmi

state: BC

institute: University of British Columbia

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

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


city: Kawasaki

first: Yuko

last: Ogata

state: Kanagawa

address: 6-21-1 Nagao, Tama-Ku

department: Division of Chemical Information, Research Center for Chemical Information and Management

country: Japan

institute: National Institute of Occupational Safety and Health, Japan

summary: Gene expression analysis of human lymphoblastoid TK6 cells exposed to the carcinogen 3-methylcholanthrene (3-MCA) was conducted. The information entropy of the transcriptome was measured to assess whether a change with biological significance occurred in TK6 cells exposed to 3-MCA. Pathway and literature analyses for DEGs were also performed.

overall_design: RNA-Seq was performed. TK6 cells obtained from JCRB Cell Bank were incubated with 0, 0.0004, 0.04 and 4 micrograms per milliliter 3-methylcholanthrene for 24 hours.


title: Hippo signaling differentially regulates distal progenitor subpopulations and their transitional states to construct the mammalian lungs

city: San Francisco

first: Erica

last: Yao

state: CA

address: 555 Mission Bay Blvd. South, Box 3118

institute: University of California, San Francisco

summary: Lung cells from Lats1f/f; Lats2f/f; SftpcCre/+ and littermate control mice were collected at embryonic day 14.5 (E14.5) and 17.5 (E17.5) for multiomics.

overall_design: Lungs were isolated from E14.5 and E17.5 Lats1f/f; Lats2f/f; SftpcCre/+ and littermate control embryos and dissociated into single-cell suspensions, washed with PBS, and frozen. On the day of scRNA-seq, cells were thawed, then incubated with antibodies against CD45 and TER119 to label immune cells and blood cells, respectively. The samples were sorted by FACS, and CD45- TER119- live cells were collected. Nuclei were isolated from the collected cells and used for snATAC-seq/snRNA-seq. Libraries were prepared using the 10x Genomics Chromium Next GEM Single Cell Multiome ATAC + Gene Expression Reagent Kit.

first: Kuan

last: Zhang

first: Madhuri

last: Basak

first: Youssef

last: Zaher

first: Pao-Tien

last: Chuang


title: Gammaherpesviruses reprogram long-term myelopoiesis through CD8⁺ T cell–monocyte dialogue within the bone marrow niche [bulk ATAC-Seq]

first: Bénédicte

last: Machiels

first: Arthur

last: Poncelet

first: Olivier

last: Botman

first: Abdulkader

last: Azouz

first: Aurélie

last: Detavernier

first: Vincent

last: Martens

first: Muriel

last: Nguyen

first: Séverine

last: Thomas

first: Valérie

last: Acolty

first: Adeline

last: Rosu

first: Céline

last: Maquet

last: Rodriguez-Rodriguez

first: Rémy

last: Sandor

first: Justine

last: Javaux

first: Malyvanh

last: Pathammavong

first: Christian

last: Münz

first: Anthony

last: Rongvaux

first: Laurent

last: Gillet

first: Stanislas

last: Goriely


title: Gammaherpesviruses reprogram long-term myelopoiesis through CD8⁺ T cell–monocyte dialogue within the bone marrow niche [scMultiome]

first: Bénédicte

last: Machiels

overall_design: CITE-seq: C57BL/6 mice were treatead or not with MuHV4 WT or MuHV4-Del73 (deleted) virus. Bone marrow LSK cells and blood monocytes were isolated by Fluorescence-activated cell sorting (FACS) according to the presence or absence of surface markers at day 8, 30 and 120 post-infection.

first: Arthur

last: Poncelet

last: Botman

first: Lucia


title: Parity-conditioned Hedgehog/VDR-linked NanoString profiling of histologically normal human breast tissue (IO360)

first: Derek

last: Radisky

summary:
Histologically normal human breast FFPE specimens were profiled by NanoString nCounter to study parity-conditioned basal/progenitor, Hedgehog, and vitamin D receptor-associated biology in normal breast tissue.
This workbook contains the PanCancer IO 360 (IO360) arm of the study and is paired with a companion workbook for the other NanoString panel. Each of the 74 analyzed specimens contributes one GEO sample to this platform-specific workbook and one GEO sample to the companion panel workbook.

overall_design: Seventy-four histologically normal breast FFPE specimens were analyzed on both NanoString panels. This IO360 workbook contains the selected raw RCC files and a complete panel-specific raw-count matrix for the 74 paired specimens. Raw RCC files were matched to specimens by exact specimen ID; when multiple candidate RCC files existed for a specimen and panel, the latest RCC date was selected. One special mapping was applied per submitter instruction: the processed workbook sample ORG-0098 was matched to raw IO360 specimen ORG-0098 R.

first: Nicole

last: Cruz-Reyes

first: Joshua

last: Ogony

first: Pria J

last: Westerman

first: Melody

last: Stallings-Mann

first: Tanya

last: Hoskin

first: Bryan M

last: McCauley

first: Robert

last: Vierkant

first: Laura

last: Pacheco-Spann

first: Jessica

last: Fischer

first: Denice L

last: Gehling

first: Lisa

last: Seymour

first: Mark

last: Sherman

first: Amy

last: Degnim

first: Stacey

last: Winham


title: APOBEC3B regulates HPV replication by inducing R-loop formation and genomic instability [ChIP-Seq]

city: Chicago

first: Conor

last: Templeton

address: 300 E Superior Street

institute: Northwestern University

summary: APOBECs are cytidine deaminases whose levels are increased in cells with high-risk HPV genomes and are responsible for most mutations in HPV associated cancers. APOBEC3B is a nuclear member of this family and is shown to be a positive regulator of HPV replication as well as expression. The proviral effects of A3B found in HPV positive cells contrast with its role as a restriction factor for many other viruses. Studies demonstrated that A3B can bind and regulate the formation of R-loops, which are trimeric nucleic acid structures consisting of an RNA paired with its complementary DNA strand, displacing one of the DNA strands. The present study demonstrates that A3B binds stably to both cellular and viral chromatin at sequences containing high R-loop levels, including the HPV URR and early polyadenylation sites. Importantly, A3B was found to play a critical role in the replication of HPV genomes and in regulating viral expression. Reduction of R-loop levels through overexpression of the R-loop specific RNase, RNase H1, impaired A3B binding to viral genomes as well as at multiple cellular sites. When A3B was depleted, total R-loop levels decreased by ~50%, leading to impaired viral transcription and an increase in the expression of immune genes, such as OASL, IL6, and IRF1. Mapping R-loop formation in A3B depleted cells revealed that A3B regulated a subset of R-loops that form on the transcriptional start (TSS) and termination sites (TTS) of cellular genes, including at the HPV URR.  Furthermore, A3B depletion resulted in over a 50% reduction of DNA breaks along with altered expression of DNA damage repair proteins. This study demonstrates that A3B is an inducer of R-loop formation and genomic instability in HPV positive cells, thereby regulating cellular and viral gene expression along with HPV replication.

overall_design: Enrichment of R-loop formation (S9.6 binding) was compared between Scramble control (Samples 2 and 3) and A3B-depleted CIN 612 cells (Samples 5 and 6). Sample 1 is an input control from the Scramble control CIN 612 cells, and Sample 4 is an input control from A3B-depleted CIN 612 cells.

first: Templeton

last: Conor

first: Gulik

last: Jasmine

first: Laimins

last: Laimonis


title: Gammaherpesviruses reprogram long-term myelopoiesis through CD8⁺ T cell–monocyte dialogue within the bone marrow niche [scRNA-Seq]

first: Bénédicte

last: Machiels

first: Arthur

last: Poncelet

first: Olivier

last: Botman

first: Abdulkader

last: Azouz

first: Aurélie

last: Detavernier

first: Vincent

last: Martens

first: Muriel

last: Nguyen

first: Séverine

last: Thomas

first: Valérie

last: Acolty

first: Adeline

last: Rosu

first: Céline

last: Maquet

first: Lucia

last: Rodriguez-Rodriguez

first: Rémy

last: Sandor

first: Justine

last: Javaux

first: Malyvanh

last: Pathammavong

first: Christian

last: Münz

first: Anthony

last: Rongvaux

first: Laurent

last: Gillet

first: Stanislas

last: Goriely


title: Integrated spatial transcriptomics (Stereo-seq) and bulk RNA-seq of cardiac sonogenetics in rodent heart tissue across time points [RNA-Seq]

city: Los Angeles

first: Jun

last: Chen

address: 420 Westwood Plaza 5121 Engineering V

department: Bioengineering

institute: University of California, Los Angeles

summary: This GEO Series contains two complementary transcriptomic datasets generated to study cardiac sonogenetics in rodent heart tissue. First, spatial transcriptomics was performed using Stereo-seq (SAW v8.1.3) to generate spatially resolved gene-expression maps from heart sections, providing GEF outputs, tissue images, and associated QC files. Second, bulk RNA-seq was performed on matched heart tissue samples from wild-type (WT) and sonogenetic (MscL-G22S) groups across multiple time points to quantify gene-level expression changes at the whole-tissue level. Raw sequencing files (FASTQ) and processed expression matrices (counts/TPM tables) are provided for both assays. Together, these datasets enable cross-validation of spatially localized transcriptional patterns with bulk gene-expression measurements.

overall_design: The study combines spatial transcriptomics and bulk RNA-seq to profile heart tissue from control (WT) and sonogenetic (AAV2/9-cTnT-MscL-G22S-mCherry) groups across longitudinal time points. Spatial transcriptomics (Stereo-seq) provides tissue section–level spatial gene-expression maps, while bulk RNA-seq provides gene-level expression matrices from whole heart tissue. Each assay is submitted as GEO Samples (GSMs) within this Series, with assay-specific protocols and data-processing details described in their respective metadata templates.

first: Gong

first: Ziyuan

last: Che


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

city: Manchester

first: Amaya

last: Viros

address: Wilmslow Road

department: Skin Cancer Ageing

country: United Kingdom

institute: Cancer Research UK

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

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

last: Virós

first: Timothy

last: Budden


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

city: Wien

first: Gautier

last: Langin

address: Muthgasse 18

country: Austria

institute: BOKU

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

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

first: Suayb

last: Üstün


title: Targeted Cellular Micropharmacies Deliver Therapeutic Agents to the Brain

first: David

last: Scheinberg

state: NY

address: 417 E 68th St

department: Pharmacology

institute: Memorial Sloan Kettering Cancer Center

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

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

first: Manish

last: Malviya


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

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

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

first: Daniela

last: Spinti

first: Frederik

last: Börnke


title: Genetic and heat-stress related environmental influences on pig whole-blood gene expression levels

city: CASTANET-TOLOSAN cedex

first: Laure

last: GRESS

address: 24 chemin de Borde Rouge CS52627

department: Occitanie-Toulouse

country: France

institute: INRAE

summary:
This study aims to use whole blood omics data to understand environmental and genetics influences between back-crossed pigs from tropical and temperate facilities or during an experimental heat stress.
Gene expression levels are affected by genetics and environmental effects. However, quantification of the influence of genetics and environmental effects on gene expression remains limited, especially in farm animals. Here, the relative influence of genetic and heat-related environmental variations on gene expression levels was investigated in pigs, using a backcross herd of diverse heat adaptation levels. Backcross animals were raised in either a tropical or temperate environment. Animals raised in temperate environment were subjected to an experimental heat stress at the end of their growth.  We identified 1,967 differentially expressed genes (DEGs) between pigs raised in the tropical (n = 181) and temperate (n = 180) facilities, and 472 DEGs throughout a 3 weeks experimental heat stress. Transcriptome-wide association (TWAS) study identified 139 associations between gene expression levels and thermoregulation/production traits. We detected 6,014 expression quantitative trait loci (eQTLs) associated with the expression level of 3,297 genes. Genetic variance was estimated to explain 36.3% of gene expression variance on average, and was the main source of variance for 27.7% of transcripts. Most eQTLs found are located in proximal regions (cis-eQTLs) and few within distal regions (trans-eQTLs) to their assigned genes. A trans-eQTL hotspot highlighted a hematopoietic mechanism driven by GPATCH8. An integration of GWAS and TWAS pointed to TMCO1 and ZNF184 as candidate genes for backfat thickness.  This study provides a better understanding of the impact of climate, heat stress and genetic influences on the pig whole blood transcriptome.

overall_design: Blood samples were collected at 23 weeks (d0) of age in each environment (temperate (N = 180) or tropical (N = 181) experimental facility). Pigs from the temperate facility were also blood sampled at two additional times during the heat stress challenge:three days (d3, N = 180) and eighteen days (d18, N = 177).

last: Gress

first: Katia

last: Fève

first: Arthur

last: Durante

first: Claire

last: Naylies

first: Yann

last: Labrune

first: Yannick

last: Lippi

first: Patrice

last: Dehais

first: Yvon

last: Billon

first: Mario

last: Giorgi

first: Denis

last: Milan

first: Jean-Luc

last: Gourdine

first: Hélène

last: Gilbert

last: Renaudeau

first: Juliette

last: Riquet

first: Guillaume

last: Devailly


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

first: Jana

last: Jajarmi

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


title: Spatially Resolved Panoramic in vivo CRISPR Screen via Perturb-DBiT [Spatial Transcriptomics]

city: New Haven

first: Rong

last: Fan

state: CT

address: 55 prospect street

institute: Yale University

summary: Spatially resolved in vivo CRISPR screening integrates gene editing with spatial transcriptomics to examine how genetic perturbations alter gene expression within native tissue environments. However, current methods are mostly for small perturbation panels and the detection of a  subset of protein-coding RNAs. We present Perturb-DBiT, a versatile approach for the simultaneous co-sequencing of spatial total RNA whole-transcriptome and single-guide RNAs (sgRNAs), base-by-base, on the same tissue section. This method enables unbiased discovery of how genetic perturbations influence RNA regulation, cellular dynamics, and tissue architecture in situ.  Applying Perturb-DBiT to a human cancer metastatic colonization model, we mapped large panels of sgRNAs across tumor colonies in multiple consecutive tissue sections alongside their corresponding total RNA transcriptomes, providing insights into how perturbations affect long non-coding RNA (lncRNA) co-variation, microRNA–mRNA interactions, and distinct amino acid-specific tRNA alterations linked to tumor migration and growth. By integrating transcriptional pseudotime trajectories, we further observed the impact of perturbations on clonal dynamics and cooperation. In an immune-competent syngeneic mouse model, Perturb-DBiT enabled investigation of genetic perturbations within the tumor immune microenvironment, potentially revealing distinct and synergistic effects on immune infiltration and suppression. Perturb-DBiT provides a spatially resolved comprehensive view of how genetic knockouts influence diverse molecular and cellular responses including small and large RNA regulation, tumor proliferation, migration, metastasis, and immune interactions, offering a panoramic perspective on perturbation responses in complex tissues.

overall_design: Perturb-DBiT is performed on tissue sections utilizing microfluidic deterministic barcoding to simultaneously co-sequence sgRNAs and the total RNA transcriptome. This enables spatially resolved, base-by-based detection of genetic perturbations and their corresponding trancsriptional phenotypes in situ.

first: Alev

last: Baysoy

first: Xiaolong

last: Tian

first: Paul

last: Renauer

first: Feifei

last: Zhang

first: Sidi

last: Chen


title: Spatially Resolved Panoramic in vivo CRISPR Screen via Perturb-DBiT

summary: Spatially resolved in vivo CRISPR screening integrates gene editing with spatial transcriptomics to examine how genetic perturbations alter gene expression within native tissue environments. However, current methods are mostly for small perturbation panels and the detection of a  subset of protein-coding RNAs. We present Perturb-DBiT, a versatile approach for the simultaneous co-sequencing of spatial total RNA whole-transcriptome and single-guide RNAs (sgRNAs), base-by-base, on the same tissue section. This method enables unbiased discovery of how genetic perturbations influence RNA regulation, cellular dynamics, and tissue architecture in situ.  Applying Perturb-DBiT to a human cancer metastatic colonization model, we mapped large panels of sgRNAs across tumor colonies in multiple consecutive tissue sections alongside their corresponding total RNA transcriptomes, providing insights into how perturbations affect long non-coding RNA (lncRNA) co-variation, microRNA–mRNA interactions, and distinct amino acid-specific tRNA alterations linked to tumor migration and growth. By integrating transcriptional pseudotime trajectories, we further observed the impact of perturbations on clonal dynamics and cooperation. In an immune-competent syngeneic mouse model, Perturb-DBiT enabled investigation of genetic perturbations within the tumor immune microenvironment, potentially revealing distinct and synergistic effects on immune infiltration and suppression. Perturb-DBiT provides a spatially resolved comprehensive view of how genetic knockouts influence diverse molecular and cellular responses including small and large RNA regulation, tumor proliferation, migration, metastasis, and immune interactions, offering a panoramic perspective on perturbation responses in complex tissues.

overall_design: Xenium In Situ analysis was performed on FFPE tissue sections from the HT29 lung metastatic colonization model to validate sgRNA expression. The experiment utilized a combinatorial probe strategy, merging the Xenium Prime 5K human pan-tissue and pathway panel with a custom panel designed to directly target the top 27 sgRNA species identified via Perturb-DBiT. This approach enables the high-resolution, spatially resolved detection of specific genetic perturbations alongside a broad transcriptomic profile in situ.


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

city: Philadelphia

first: Montserrat

last: Anguera

state: PA

address: 3800 Spruce Street, Rosenthal Room 390EB

department: Biomedical Sciences

institute: University of Pennsylvania School of Veterinary Medicine

type: Methylation profiling by high throughput sequencing

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

overall_design: Refer to individual Series


title: Integrated spatial transcriptomics (Stereo-seq) and bulk RNA-seq of cardiac sonogenetics in rodent heart tissue across time points [Stereo-seq]

first: Jun

first: Gong

first: Ziyuan

last: Che


title: Unnatural cytosine analogs potentiate a customizable, enzymatic method for integrated epigenetic and four-base genetic sequencing

first: Rahul

middle: M.

last: Kohli

state: Pennsylvania

address: 3610 Hamilton Walk, 502B Johnson Pavilion

department: Department of Medicine

institute: University of Pennsylvania

summary: The interplay of genetic and epigenetic information shapes cell identity, development, and disease. However, standard methods for profiling DNA modifications (e.g., bisulfite-sequencing) rely on selective C-to-T conversions, hindering the simultaneous examination of both genetic and epigenetic information. Here, we introduce Integrated-Sequencing, which provides high-fidelity mapping of DNA modifications while preserving the native four-base genetic code in single molecules. Integrated-Seq leverages the synthesis of a tethered copy strand with unnatural cytosine analogs that resist enzymatic conversion, combined with a novel DNA deaminase-helicase fusion that drives selective C-to-T conversion of natural cytosines in the original template strand. We demonstrate that Integrated-Seq is compatible with customizable enzymatic readouts to parse 5-methylcytosine and 5-hydroxymethylcytosine, and that preservation of the original four-base genetic code markedly improved enrichment, facilitating analysis of targeted genomic regions. Integrated-Seq thus provides a platform for simultaneous genetic and epigenetic analyses, paving the way for deep insights into fundamental biology and next-generation diagnostics.

overall_design: Integrated-ACE-Seq, Integrated-EM-Seq, ACE-Seq, and EM-Seq were performed in triplicate on gDNA isolated from various mouse tissues to quantify levels of 5hmC and 5modC for the purposes of benchmarking, validation, and application. Samples consisted of either mouse Cerebellum, Heart, Kidney, or Blood with unmodified lambda phage gDNA and either 5mCpG-methylated M.MpeI plasmid (ACE) or 5mCpG-methylated pUC19 plasmid (EM) spiked in as controls. Cerebellar ACE-Seq and Integrated-ACE-Seq samples were also prepared for deep sequencing. The cerebellar Integrated-ACE-Seq sample was also subjected to enrichment with a commercial four-base probe set.

first: Christian

last: Loo

first: Jared

middle: B

last: Parker

first: Aleksia

last: Barka

first: Ruiyao

last: Zhu

first: Matthew

last: Schnell

first: Rosella

middle: G

last: Cuomo

first: Heqiao

first: Laura

last: Liu

first: Ashley

first: Jasleen

last: Gill

first: Wanding

last: Zhou


title: Spatial transcriptomics of squamous cell carcinoma from different tissues

first: Amaya

last: Viros

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

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

last: Virós

first: Kevin

last: Harrington

first: Malin

last: Pederson

first: Ben

last: O'Leary

first: Antonio

last: Rullan


title: Metabolites from plasma-like medium fuel nitrogen metabolism and influence proliferation in Leptospira interrogans

city: St. Louis

middle: Harrison

last: Ward

state: Missouri

address: 1 Brookings Dr.

department: Chemistry

institute: Washington University in St. Louis

summary: The adaptation and survival of bacterial pathogens in a host involves a complex series of processes, with bacterial metabolism as a critical checkpoint and facilitator of infection. The pathogen Leptospira interrogans expands from a small inoculum to reach high bloodstream titers before colonizing host kidneys; however the supporting metabolic programs and the role of the host environment are largely unknown, and tools to investigate these host-pathogen metabolic interactions in a controlled in vitro setting are in their infancy. Here, we built upon the concept of human plasma-like medium (HPLM) to develop a more physiological system for L. interrogans culture. This supplemented HPLM (sHPLM) supported robust growth, and we used RNA-sequencing analyses to demonstrate the fidelity of the transcriptome of sHPLM cultures to in vivo models. Through the use of sHPLM, we identified the amino acid glutamine as a major nitrogen source supplying the biosynthetic activities of L. interrogans. Further study of the role of glutamine revealed that exposure altered the proliferative behavior and biofilm formation of L. interrogans, although RNA-sequencing revealed few strong transcriptional consequences of this environmental signal. This work adds to our understanding of the metabolic mechanisms supporting L. interrogans infection which could be targeted for new anti-leptospiral therapies. We propose a dual, pathogen-supporting role for glutamine: as a metabolic fuel for continued biosynthesis and replication, and as a metabolite signal to inform pathogen adaptation in the process of host infection.

overall_design: Leptospira interrogans grown in standard EMJH medium at 30 degrees Celsius was cultured for four hours at late exponential phase density in either fresh EMJH, sHPLM at 37 degrees Celsius, or EMJH with 550uM L-glutamine.

middle: H

first: Nathan

last: Scherer

first: Leah

last: Shriver

first: Gary

last: Patti


title: Hippo signaling differentially regulates distal progenitor subpopulations and their transitional states to construct the mammalian lungs

first: Erica

last: Yao

summary: Lung cells from Lats1f/f; Lats2f/f; SftpcCre/+ and littermate control mice were collected at embryonic day 14.5 (E14.5) and 17.5 (E17.5) for scRNA-seq.

overall_design: Lungs were isolated from E14.5 and E17.5 Lats1f/f; Lats2f/f; SftpcCre/+ and littermate control embryos and dissociated into single-cell suspensions, washed with PBS, and frozen. On the day of scRNA-seq, cells were thawed, then incubated with antibodies against CD45 and TER119 to label immune cells and blood cells, respectively. The samples were sorted by FACS, and CD45- TER119- live cells were collected and used for scRNA-seq. Libraries were prepared using 10x Genomics Chromium GEM-X Universal 3' Gene Expression Kit (v4).

first: Kuan

last: Zhang

first: Madhuri

last: Basak

first: Youssef

last: Zaher

first: Pao-Tien

last: Chuang


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

city: Aachen

first: Ralf

last: Weiskirchen

address: Pauwelsstr. 30

department: IFMPEGKC

institute: RWTH University Hospital Aachen

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

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


title: BIN2 phosphorylation of GRF5 suppresses low nitrate root foraging by inhibiting transcriptional activity and UBP12/13-mediated deubiquitination

city: Pingdingshan

first: Dongxiao

last: Liu

address: Longxiang Avenue & Yuying Road Intersection, New Urban Area

institute: Henan University of Urban Construction

summary: Root developmental plasticity enables plants to adapt to nutrient-deficient conditions. Under low nitrate conditions, enhanced exploratory root growth—characterized by increased primary and lateral root elongation—facilitates nutrient foraging. Although nitrate-hormone crosstalk regulates this process, the underlying molecular mechanisms remain poorly understood. Here, we identify a BIN2-GRF5-UBP12/13 module governing root foraging responses to low nitrate in Arabidopsis. We demonstrate that BIN2 phosphorylates GRF5 at Ser-205, reducing its stability and transcriptional activity. Integrative DAP-seq and transcriptome analyses revealed that GRF5 directly regulates key nitrate-responsive genes, including the dual-affinity transporter NRT1.1 and the high-affinity uptake gene NRT2.1. Furthermore, dephosphorylated GRF5 preferentially interacts with UBIQUITIN-SPECIFIC PROTEASES 12 and 13 (UBP12/13), which stabilize GRF5 to promote lateral root elongation under low nitrate conditions. Our work delineates a phosphorylation-dependent regulatory circuit that fine-tunes root foraging adaptation, advancing the mechanistic understanding of nitrate sensing in plants.

overall_design: Arabidopsis thaliana seedlings were subjected to low nitrate (0.2 mM NO₃⁻) treatment for 3 days to induce root foraging responses, with normal nitrate (5 mM NO₃⁻) treatment as a control. 2. ChIP-seq was performed on root tissues of low nitrate-treated seedlings to profile the chromatin-binding sites of GRF5, including two biological replicates (GRF5_1, GRF5_2) and an input control (GRF5_Input) to eliminate non-specific binding signals. 3. DAP-seq (DNA Affinity Purification Sequencing) was conducted to identify direct DNA targets of GRF5, complemented by transcriptome analysis (RNA-seq) of wild-type and GRF5-modified lines (overexpression/knockdown) under low nitrate conditions to reveal GRF5-regulated genes. 4. Integrative analysis of ChIP-seq, DAP-seq, and transcriptome data was performed to validate key nitrate-responsive target genes (e.g., NRT1.1, NRT2.1) of GRF5. 5. Functional validation experiments (e.g., phosphorylation assay, protein-protein interaction assay, root growth phenotype analysis) were carried out to verify the regulatory role of the BIN2-GRF5-UBP12/13 module in root foraging under low nitrate stress.

first: Taotao

last: Li

first: Xiuhong

first: Xinke

last: Kang


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

first: Amaya

last: Viros

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

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

last: Virós

first: Timothy

last: Budden


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

city: Boston

first: Madison

middle: Rose

last: Glass

state: MA

address: 3 Blackfan St

department: Neurobiology

institute: Boston Children's Hospital

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

first: Madison R

first: Dosh

last: Whye

first: Nickesha C

last: Anderson

first: Delaney

last: Wood

first: Nina R

last: Makhortova

first: Taryn

last: Polanco

first: Kristina H

last: Kim

first: Kathleen E

last: Donovan

first: Gayathri Rajaram

last: Srinivasan

first: Lorenzo

last: Vaccaro

first: Ashish

last: Jain

first: Davide

last: Cacchiarelli

first: Liang

last: Sun

first: Elizabeth D

last: Buttermore

first: Mustafa

last: Sahin


title: The post-reproductive ovary shifts from a reproductive to an immune organ

first: Aubrey

last: Converse

state: IL

address: 303 E superior

institute: Northwestern University Feinberg School of Medicine

summary: Reproductive aging in females is characterized by the irreversible depletion of ovarian follicles, yet the structure and function of the post-reproductive ovary remain poorly defined. Using paired histological and bulk transcriptomic analyses of ovaries from reproductively young (2 m), reproductively old (18 m), and post-reproductive (24 m) mice, we mapped how ovarian identity evolves beyond follicle exhaustion. As expected, follicle loss, stromal remodeling, and increased collagen deposition were observed in the reproductively old and post-reproductive cohorts. Transcriptomic analyses revealed a shift from reproductive functionality to an immune-dominant signature with age. Correspondingly, post-reproductive ovaries exhibited increased infiltration of T cells, macrophages, and multinucleated giant cells. Although old and post-reproductive ovaries diverged substantially from young ovaries, they also showed discrete transcriptomic differences, indicating that the ovary continues to undergo molecular changes after reproductive senescence. Lastly, age-dependent changes in ovarian factors that are predicted to be secreted suggest that the post-reproductive ovary may be a source of pro-inflammatory signaling mediators with the potential to modulate extra-ovarian tissues. These findings challenge the assumption that the post-reproductive ovary is inert, instead indicating that it acquires an immune identity with potential endocrine and paracrine influence on whole-body aging.

overall_design: Transcriptomic profiling of mouse ovaries from reproductively young, reproductively old, and post-reproductive ovaries

first: Ian

last: Schowe

first: Shweta

last: Dipali

first: Shivani

last: Jambunathan

first: Emmett

last: Kelly

first: Sarah

last: Ocanas

first: Michael

last: Stout

first: Michele

middle: T

last: Pritchard

first: Francesca

last: Duncan


title: Versatile SMAD2 and SMAD3 epitope-tagged mouse models for genome-wide profiling of TGFβ superfamily signaling: uncovering novel GDF9-SMAD2/3 target genes

city: Houston

first: Martin

last: Matzuk

state: TX

address: One Baylor Plaza

department: Pathology and Immunolodgy

institute: Baylor College of Medicine

summary: Transforming growth factor β (TGFβ) signaling pathways are integral for a plethora of biological processes. SMAD2 and SMAD3 are the principal transcriptional effectors of TGFβ superfamily ligands, yet quantitative, genome-wide mapping of their DNA-associated complexes under physiological contexts has remained limited due to the lack of specific, robust models. Here, we generated two versatile epitope-tagged mouse models in which endogenous SMAD2 and SMAD3 proteins are globally tagged with HA and PA, respectively. To demonstrate the broad application of our models, we evaluated our lines in ovarian biology. By integrating genomic and transcriptomic analyses, we identified direct genes induced by the GDF9-SMAD2/3 axis and discovered gene sets suppressed by this signaling cascade, highlighting a previously underappreciated role of GDF9 in attenuating competing pathways to ensure proper ovarian granulosa cell fate transitions. Together, these epitope-tagged SMAD mouse models provide extensive, applicable in vivo genetic toolkits for tissue-specific dissection of TGFβ family signaling.

overall_design: RNA-seq profiles of mouse granulosa cells stimulated with Veh and mouse GDF9; CUT&RUN profiles of SMAD2/3/4 and H3K27ac in the mouse granulosa cells stimulated with Veh and mouse GDF9.

first: Zian

last: Liao

first: Qian

last: Zhang

first: Keisuke

last: Shimada

first: Kaori

last: Nozawa

first: Suni

last: Tang

first: Masahito

last: Ikawa

first: Diana

last: Monsivais


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

city: Bethesda

first: Laurie

last: Krug

state: MD

address: Building 10, 9000 Rockville Pike

institute: NIH

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

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

first: Ramya

last: Ramaswami

first: Edmund

last: Culley

first: Baman

last: Afsari

first: Quashawn

last: Chadwick

first: Xiaolin

last: Wu


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

first: Montserrat

last: Anguera

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

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

first: Natalie

last: Toothacre