title: Single cell RNA sequencing profile of 60 day old human iPSC-derived midbrain organoids from healthy individual, Parkinson’s disease patient (Miro1 p.R272Q mutation) and its isogenic control

city: Esch sur Alzette

first: Alise

last: Zagare

state: Belval

address: 7 Avenue des Hauts-Fourneaux

country: Luxembourg

institute: University of Luxembourg

summary: Use of patient specific models, such as midbrain organoids, have shown to be valuable tools to models Parkinson’s disease as well as to identify novel pathological cellular and molecular mechanisms. In here, we used single cell RNA sequencing analysis of 60 day old midbrain organoids obtained from a Parkinson’s disease patient carrying the p.R272Q Miro1 mutation, its isogenic control and a healthy control to explore the effect of RhoT1 gene in different cellular populations present within the midbrain organoids (e.g. dopaminergic neurons, astrocytes) and the subsequent effect on the PD pathology.

overall_design: Midbrain organoids generated from iPSC obtained from a Parkinson's disease patient carrying the Miro1 p.R272Q mutation, its respective isogenic control and from a healthy age and sex-matched donor were used. The midbrain organoids were culture for 60 days and then, dissociated into single cells and used for scRNA-seq analysis.

first: Cláudia

last: Saraiva

first: Jens

last: Schwamborn

first: Kyriaki

last: Barmpa

first: Rejko

last: Krueger


title: The significance of PAX5 in Merkel cell carcinoma (Methylation BeadChip)

status: Public on Jan 31 2025

city: Ulm

first: Emil

last: Chteinberg

address: Albert-Einstein-Allee 11

country: Germany

institute: Ulm University Medical Center

summary:
Merkel cell carcinoma (MCC) is a highly malignant skin cancer that expresses epithelial-, neuroendocrine-, and lymphoid-associated genes. Here, we focused on B-cell differentiation, which is characterised by the co-expression of PAX5 and TdT. PAX5 is the master regulator of B-cell commitment and is expressed in 65% of MCC cases. Yet, little is known about the underlying molecular biology of the frequently reported PAX5 expression in MCC.
Multi-omics analyses, including RNA next-generation sequencing, RT-qPCR, immunohistochemistry, and western blotting, were performed to assess PAX5 expression in MCC. Differential DNA methylation analysis at 61,043 PAX5 binding sites in enhancer and promoter elements was performed to detect differences between n=14 MCC tissues and n=91 various normal B-cell populations.
RNA analysis revealed full-length PAX5 expression in MCC at the transcriptional level using both PAX5 transcription start sites. PAX5 protein expression was found in 40 of 41 MCCs and six out of seven MCC cell lines. DNA methylation array analysis revealed 1,084 hypermethylated loci of enhancer and promoter elements located in PAX5 binding sites in MCC. Of these, 702 loci were associated with 257 genes which are not expressed. The PAX5-associated regulatory elements of these 257 genes were enriched for interferon regulatory factor 4 (IRF4) and SPi-proto-oncogene (SPI1) binding motifs. Neither IRF4 or SPI1 could be detected in MCC on a RNA or protein level. Thus, because of the absence of these transcription factors we conclude that full length PAX5 alone cannot induce B-cell differentiation.

overall_design: gene expression profiling analysis of RNA-seq data for Merkel cell carcinoma tissues, Merkel cell carcinoma cell lines and one pre-B-cell acute lymphoblastic leukemia cell line

first: Julia

last: Kolarova

last: Vogt

first: Amanda

last: Macamo

first: Felix

last: Bormann

first: Helene

last: Kretzmer

first: Ernst

middle: J

last: Speel

first: Joost

last: van den Oord

first: Christof

last: Schneider

first: Stephan

last: Stilgenbauer

first: Jürgen

middle: C

last: Becker

first: Véronique

last: Winnepenninckx

first: Erik

last: Biessen

first: Martin

last: Zenke

first: Anna

middle: K

last: Kurz

first: Reiner

last: Siebert

first: Axel

last: zur Hausen


title: Secreted neurofilament light chain (NfL) after neuronal damage induces myeloid cell activation and neuroinflammation [bulkRNA-Seq]

status: Public on Jan 27 2025

city: South San Francisco

first: Mitchell

last: Rezzonico

state: CA

address: 1 DNA Way

country: USA

institute: Genentech

type: Expression profiling by high throughput sequencing

summary: Neurofilament light chain (NfL) is a neuron-specific cytoskeletal protein that provides structural support for axons and is released into the extracellular space following neuronal injury. While NfL has been extensively studied as a disease biomarker, the underlying release mechanisms and role in neurodegeneration remain poorly understood. Here, we find that neurons secrete low baseline levels of NfL, while neuronal damage triggers calpain-driven proteolysis and release of fragmented NfL. Secreted NfL activates microglia cells, which can be blocked with anti-NfL antibodies. We utilize in vivo single-cell RNA sequencing to profile brain cells after injection of recombinant NfL into the mouse hippocampus and find robust macrophage and microglial responses. Consistently, NfL knock-out mice ameliorate microgliosis and delay symptom onset in the SOD1 mouse model of amyotrophic lateral sclerosis (ALS). Our results show that released NfL can activate myeloid cells in the brain and is, thus, a potential therapeutic target for neurodegenerative diseases.

overall_design: To examine the effect of exogenous NfL on microglia, primary murine microglia were treated with either a vehicle control (n=4), full length NfL (5 µg; n=4) or coiled-coil NfL (5 µg; n=4). After 24 hours, total RNA was isolated and sequencing libraries were prepared for further analysis.

first: Olga

middle: I

last: Kahn

first: Casper

last: Hoogenraad


title: The spatial transcriptomic atlas of human limbus and vital niche microenvironment regulating the fate of limbal epithelial stem cells

status: Public on Jan 30 2025

city: Shanghai

first: Shiding

last: Li

address: 639 Zhizaoju Rd, Shanghai, China, 200011

country: China

institute: Department of Ophthalmology, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine

summary: We profiled over 400 spots within each sample and spatially located major cell types through the integration with reference the single-cell RNA sequencing data. Based on Robust Cell Type Decomposition (RCTD) technology, limbal epithelial stem cells (LESCs) were mainly localized in the basement membrane, and limbal niche cells were predominantly situated within the stromal area. Following, the limbus was divided into 4 regions based on histological structure, and the differential expressed genes among the four regions were analyzed. Notably, our investigation revealed significantly higher levels of GPHB5 and PERP within the epithelium of the middle region, precisely where LESCs predominantly reside. Concurrently, the activation of critical pathways such as "Protein Binding" and "ECM-receptor interaction" were discovered in this same region, underscoring its pivotal role in maintaining the limbal niche. Subsequently, cell-cell communication analysis was conducted on essential cells inside the niche microenvironment. Remarkably, we observed that limbal mesenchymal stem cells exhibit the largest amounts of ligands associated with LESCs, with melanocytes closely trailing behind. The widespread activity of COL6A2/CD44 signaling among limbal mesenchymal stem cells, melanocytes, immune cells, and LESCs, indicate its essential role in mediating bidirectional communication via the Collagen pathway. Additionally, a myriad of ligand-receptor interactions and pathways regulatory crosstalk among diverse cell types were exhibited.

overall_design: The single-cell transcriptomic atlas of two human limbus was performed using 10X Genomics' Space Ranger software platform, followed by visualizing in two-dimensional space by UMAP and unbiased computational informatic analysis. Immunofluorescent staining assays were performed to further validate the analysis results.

first: Hao

last: Sun

first: Fei

last: Fang

first: Chunyi

last: Shao

first: Yao

last: Fu

first: Liangbo

last: Chen


title: Immune-aging is linked to clinical malignancy and vaccine responses in myeloma

city: Atlanta

first: Shivani

last: Kapadia

state: GA

address: 1365 Clifton Road NE

department: Hematology and Medical Oncology

institute: Emory University

summary: Several single-cell transcriptomic approaches have been applied to study immune system in MM, and previously implied “T-cell exhaustion” largely based on comparison with signatures derived in murine chronic viral infection models. However functional validation of such “signatures” has been lacking. In this paper, we have studied nearly 300+ racially diverse MM/MGUS patients/healthy controls. Initial functional studies did not support global T cell exhaustion, but instead illustrated paradoxically enhanced inflammatory cytokine production and proliferation in MM T cells, even in phenotypes previously thought to be “exhausted”. We therefore hypothesized that altered states of T cell differentiation (e.g. increased terminal effectors) may instead be related to immune aging. In order to quantify this, we first studied a cohort of 100+ healthy donors across 7 decades of life to quantify age-associated immune changes, and then applied findings to MM/MGUS cohorts. This led to the discovery that in chronologically age-matched cohorts, MM is associated with greater immune aging compared to MGUS. These findings were also supported by transcriptomic and phospho-proteomic analyses. Interestingly, MGUS patients with Black ancestry exhibited enhanced aging. These data therefore support potential contribution of immune aging in malignant transformation, as well as racial predisposition with earlier onset of MM in Blacks. Finally, to evaluate the impact of immune aging on immune function in vivo, we analyzed yet another cohort of patients undergoing SARSCov2 vaccination. Immune but not chronologic aging was associated with responses to vaccination.

overall_design: Peripheral blood and bone marrow specimens were obtained from patients with myeloma and MGUS following informed consent approved by Emory Institutional Review Board. BMMCs (n=23) were thawed, incubated at 37°C for 1 hour to rest. Cells were stained using a TotalSeq-C antibody cocktail  following the 10X Genomics protocol for Chromium Single-Cell Immune Profiling with Feature Barcoding Technology (ver. 1.0). Single cells were isolated using the Chromium Controller (10X Genomics). Gene expression and CITE-seq libraries were prepared using the following kits from 10× Genomics: Chromium Next GEM Single Cell 5’ Kit v2 (PN-1000263), Chromium 5’ Feature Barcode Kit (PN-1000541), Chromium Single-Cell 5′ Library Construction Kit (PN-1000190), Chromium Next GEM Chip K Single Cell Kit (PN-1000287), Dual Index Kit TT Set A (PN-1000215) and Dual Index Kit TN Set A (PN-1000250). Sequencing was conducted on an Illumina NovaSeq 6000 at a targeted depth of 50,000 reads/cell for GEX libraries and 5000 reads/cell for CITE-seq libraries.

first: Sayalee

middle: V

last: Potdar

first: Maryam

last: Azeem

first: Roman

last: Radzievski

first: Kiran

last: Lakhani

first: Nisha

middle: S

last: Joseph

first: Jonathan

middle: L

last: Kaufman

first: Craig

last: Hofmeister

first: Vikas

middle: A

last: Gupta

first: Sagar

last: Lonial

first: Ajay

last: Nooka

first: Kavita

middle: M

last: Dhodapkar

first: Madhav


title: Thymic B lineage cell landscape in Myasthenia gravis

status: Public on Jan 28 2025

city: Muenster

first: I-Na

last: Lu

state: NRW

address: Domagkstraße 20

department: Department of Pediatric Hematology and Oncology

institute: University Children's Hospital Münster

summary: Here, we provide a comprehensive and unbiased atlas of hematopoietic cell colonization in thymi derived from 12 immunotherapy-naïve patients with early-onset AChR-Ab+ MG (EOMG) using single-cell RNA sequencing (scRNA‑seq).

overall_design: We performed scRNA‑seq in thymic cell suspensions derived from 10 immunotherapy-naïve female patients and two immunotherapy-naïve male patients with AChR-Ab+ EOMG following enrichment for CD45+ hematopoietic cells.

first: Paula

last: Terroba Navajas

first: Gerd

last: Meyer zu Hörste

first: Jan

middle: D

last: Lünemann


title: Loss of the APP regulator RHBDL4 preserves memory in an Alzheimer’s disease mouse model.

status: Public on Jan 29 2025

city: Montreal

first: Lisa

last: Munter

state: QC

address: 3649 Sir-William-Osler Promenade

department: Pharmacology and Therapeutics

country: Canada

institute: McGill University

summary: Characteristic cerebral pathological changes of Alzheimer’s disease (AD) such as glucose hypometabolism or the accumulation of cleavage products of the amyloid precursor protein (APP), known as Aβ peptides, lead to sustained endoplasmic reticulum (ER) stress and neurodegeneration. To preserve ER homeostasis, cells activate their unfolded protein response (UPR). The rhomboid-like-protease 4 (RHBDL4) is an enzyme that participates in the UPR by targeting proteins for proteasomal degradation. We demonstrated previously that RHBLD4 cleaves APP in HEK293T cells, leading to decreased total APP and Aβ. More recently, we showed that RHBDL4 processes APP in mouse primary mixed cortical cultures as well. Here, we aim to examine the physiological relevance of RHBDL4 in the brain. We first found that brain samples from AD patients and an AD mouse model (APPtg) showed increased RHBDL4 mRNA and protein expression. To determine the effects of RHBDL4’s absence on APP physiology in vivo, we crossed APPtg mice to a RHBDL4 knockout (R4-/-) model. RHBDL4 deficiency in APPtg mice led to increased total cerebral APP and amyloidogenic processing when compared to APPtg controls. Contrary to expectations, as assessed by cognitive tests, RHBDL4 absence rescued cognition in 5-month-old female APPtg mice. Informed by unbiased RNAseq data, we demonstrated in vitro and in vivo that RHBDL4 absence leads to greater levels of active β-catenin due to decreased proteasomal clearance. Decreased β-catenin activity is known to underlie cognitive defects in APPtg mice and AD. Our work suggests that RHBDL4’s increased expression in AD, in addition to regulating APP levels, leads to aberrant degradation of β-catenin, contributing to cognitive impairment.

overall_design: RNAseq profiling of wild-type and RHBDL4-knockout mouse ebryonic fibroblasts at standard culturing conditions after 48 h of seeding.

first: Ylauna

last: Penalva

first: Sandra

last: Paschkowsky

first: Jingyun

last: Yang

first: Sherilyn

last: Recinto

first: Jessica

last: Cinkornpumin

first: Marina

last: Ruelas

first: Bin

last: Xiao

first: Albert

last: Nitu

first: Helen

middle: Y

last: Wu

first: Hans

first: Bernadeta

last: Michalski

first: Margaret

last: Fahnestock

first: William

last: Pastor

first: David

last: Bennett


title: Functional inhibition of core spliceosomal machinery activates intronic premature cleavage and polyadenylation of pre-mRNAs

city: guangzhou

first: chengguo

last: yao

address: No. 74 zhongshan 2rd road

department: school of medicine

institute: sysu

summary: To understand if U1/U6/U4/U2 snRNPs could regulate 3'-UTR APA, we treated NIH3T3 cells with different concentration of U1/U6/U4/U2 AMOs

overall_design: Total RNAs were harvested at 12 hour time points upon U1/U6/U4/U2 AMOs transfection,mRNA 3'-seq library was constructed using lexogen kit (REV. 016.24). Bioinformatics analysis were subsequently carried out using our published pipeline QuantifyPoly(A) (https://pubmed.ncbi.nlm.nih.gov/34255024/)

first: Qiuming

last: Feng

first: Zejin

last: Lin

first: Danhui

last: Zhao

first: Congting

last: Ye

first: Chengguo

last: Yao


title: DNA methylation entropy is a biomarker for aging

city: Los Angeles

first: Jonathan

last: Chan

address: 610 Charles E. Young Drive South

department: Molecular, Cellular, Developmental Biology

institute: University of California, Los Angeles

type: Methylation profiling by high throughput sequencing

summary: The dynamic nature of epigenetic modifications has been leveraged to construct epigenetic clocks that accurately predict an individual’s age based on DNA methylation levels. Here we explore whether the accumulation of epimutations, which can be quantified by Shannon’s entropy, changes reproducibly with age. Using targeted bisulfite sequencing, we analyzed the associations between age, entropy, and methylation levels in human buccal swab samples. We find that epigenetic clocks based on the entropy of methylation states predict chronological age with similar accuracy as common approaches that are based on methylation levels of individual cytosines. Our approach suggests that across many genomic loci, methylation entropy changes reproducibly with age.

overall_design: Targeted bisulfite sequencing with 3000 regions that covered age associated CpGs identified in multiple epigenetic clocks. Data was collected from the buccal swab of 100 human participants.

first: Liudmilla

last: Rubi

first: Matteo

last: Pellegrini


title: Metabolic dysfunction-associated steatotic liver disease may increase intrahepatic interferon gene signatures in patients with chronic hepatitis B

city: Chongqing

first: Zhiwei

last: Chen

address: The Second Affiliated Hospital of Chongqing Medical University, 74  Linjiang Road, Yuzhong District, Chongqing

summary: As metabolic dysfunction-associated steatotic liver disease (MASLD) frequently co_x0002_occurs in patients with chronic hepatitis B (CHB), the interplay between these two common liver conditions remains largely unexplored. A recent  study suggest that MASH comorbidity can reduce intrahepatic interferon pathway activity and macrophage gene signatures in HBeAg_x0002_negative chronic HBV (ENEG) patients, potentially contributing to persistent infection and fibrosis. However, it remains unclear whether this phenomenon also occurs in MASLD with CHB patients.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: Firstly, we evaluated the results in our cohort of CHB patients. Liver samples from seven ENEG patients and eleven ENEG patients with MASLD were analyzed using RNA-seq. Further, we validated our findings through an animal study. rAAV8-1.3 HBV was administered intravenously to C57BL/6 mice to mimic chronic HBV infection. A high-fat diet was maintained for 16 weeks to establish the MASLD model ,We collected livers from each group of mice and sent the livers for RNA-seq analysis.

first: Yan

last: Han

first: Jing

last: Tang

first: Peng

last: Hu

first: Hong

last: Ren


title: Antifungal activity and potential inhibitory mechanism of Huangqin decoction on Trichophyton mentagrophytes

city: Nanchang

first: Suqing

last: Yang

address: Nanchang

institute: Jiangxi Provincial People's Hospital (the First Affiliated Hospital of Nanchang Medical College)

summary: Trichophyton mentagrophytes (T. mentagrophytes) is a prevalent pathogen that causes human and animal dermatophytosis. The clinical treatment of these infections is challenging due to the prolonged treatment duration, limited efficacy, antifungal resistance and side effects of existing drugs. It has been reported that the classic Traditional Chinese medicine (TCM) prescription Huangqin decoction (HQD) along with its principal ingredients could exhibit antifungal properties. Given the valued advantages of TCM such as broad-spectrum antifungal activity, low incidence of drug resistance and low toxicity, this study investigated the antifungal activity of HQD against T. mentagrophytes and explored the potential inhibitory mechanism, aimed to provide new clues for the treatment of dermatophytosis. By detecting minimal inhibitory concentration (MIC) using the broth microdilution method, the result showed that HQD could significantly inhibit the growth of T. mentagrophytes, with a MIC of 3.13 mg/mL. The transcriptome sequencing and quantitative real-time PCR (qRT-PCR) technology were combined to shed light on the complicated adaptive responses of T. mentagrophytes upon HQD. The results demonstrated that a total of 730 differentially expressed genes (DEGs) were detected in T. mentagrophytes after HQD exposure, of which 547 were up-regulated and 183 were down-regulated. These DEGs were abundant in “single-organism metabolic process”, “catalytic activity” and “oxidoreductase activity”, and were significantly enriched in seven signaling pathways including glutathione metabolism, DNA replication, glyoxylate and dicarboxylate metabolism, taurine and hypotaurine metabolism, carotenoid biosynthesis, ubiquitin mediated proteolysis, and cyanoamino acid metabolism. The results of transcriptome profiling were verified using qRT-PCR for a subset of 10 DEGs. The overall evidence indicated that HQD had a significant anti-dermatophyte activity through the modulation of glutathione metabolism by regulating GST, γ-GT genes, the inhibition of DNA replication pathway by downgrading MCM3, MCM5, RNaseH1 genes, and the repressed expression of PSD gene related to phospholipid synthesis in T. mentagrophytes.

overall_design: RNA-seq was performed in Trichophyton mentagrophytes (XM109) treated with or without HQD to detect the differential genes that HQD may affect.

first: Su-Qing

first: You-Jin

last: Ge

first: Chen-Ying

last: Shen


title: Clonal cell states link gastroesophageal junction tissues, metaplasia, and cancer [MAESTER]

city: Philadelphia

first: Sydney

last: Shaffer

state: PA

address: 421 Curie Boulevard

department: Penn Medicine

institute: University of Pennsylvania

summary: Barrett's esophagus is a common type of metaplasia and a precursor of esophageal adenocarcinoma. However, the cell states and lineage connections underlying the origin, maintenance, and progression of Barrett’s esophagus have not been resolved in humans. Here, we performed single-cell lineage tracing and transcriptional profiling of patient cells isolated from metaplastic and healthy tissue. Our analysis unexpectedly revealed that the squamous esophagus and gastric cardia contained cells belonging to common lineages that also included transitional basal progenitor cells; both esophageal and gastric tissues were also related to Barrett's esophagus. Barrett’s esophagus biopsies consisted of multiple clones, with lineages that contained all progenitor and differentiated cell types. In contrast, precancerous dysplastic lesions were initiated by the expansion of a single molecularly aberrant Barrett’s esophagus clone. Together, these findings provide a comprehensive view of the cell dynamics of Barrett's esophagus, linking cell states along the disease trajectory, from its origin to cancer.

overall_design: Performed single-cell lineage tracing and transcriptional profiling of patient cells isolated from metaplastic and healthy tissue.

first: Rodrigo

last: Gier

first: Raúl

last: Reyes Hueros

first: Jiazhen

last: Rong

first: Maureen

last: DeMarshall

last: Bracht

first: Tatiana

last: Karakasheva

first: Amanda

middle: B

last: Muir

first: Gary

middle: W

last: Falk

first: Nancy

middle: R

last: Zhang


title: Ribosome Profiling Reveals Translational Reprogramming via mTOR Activation in Omacetaxine Resistant Multiple Myeloma

city: Aurora

first: Neelanjan

last: Mukherjee

state: Colorado

address: Research Complex 1 South, 12801 E 17th Ave

department: Biochemistry and Molecular Genetics

institute: University of Colorado School of Medicine

summary: Protein homeostasis is critical to the survival of multiple myeloma (MM) cells. While this is targeted with proteasome inhibitors, mRNA translation inhibition has not entered trials. Recent work illustrates broad sensitivity MM cells to translation inhibitor omacetaxine. We hypothesized that understanding how MM cells become omacetaxine resistant will lead to the development of drug combinations to prevent or delay relapse. We generated omacetaxine resistance in H929 and MM1S MM cell lines and compared them to their parental lines. Resistant lines displayed decreased sensitivity to omacetaxine, with EC50 > 100 nM, compared to parental line sensitivity of 24-54 nM. To adapt to omacetaxine, H929 and MM1S exhibited an increased percentage of multi-nucleated polyaneuploid cells that led to distinct molecular mechanisms of resistance Interestingly, both resistant lines showed a defect in oncologic potential via extended survival in a MM xenograft model. Since omacetaxine inhibits protein synthesis, we performed both RNA-sequencing and ribosome profiling (Ribo-seq) to identify shared and unique regulatory strategies of resistance. Transcripts encoding translation factors and containing TOP motifs in their 5’ UTR were translationally upregulated in both resistant cell lines. The mTOR pathway promotes the translation of TOP motif containing mRNAs. Indeed, mTOR inhibition restored partial sensitivity to omacetaxine in both MM1S and H929 cells. Primary MM cells from patient samples were sensitive to combinations of omacetaxine and mTOR inhibitors rapamycin and Torin 1. These results provide a rational approach for omacetaxine-based combination in patients with multiple myeloma, which have historically shown better responses to multi-agent regimens.

overall_design: We generated omacetaxine resistance in H929 and MM1S MM cell lines and compared them to their parental lines using ribo-seq and RNA-seq

first: Zachary

last: Walker

first: Katherine

last: Vaeth

first: Amber

last: Baldwin

first: Denis

last: Ohlstrom

first: Lauren

middle: T

last: Reiman

first: Beau

last: Idler

first: Brett

last: Stevens

first: Daniel

last: Sherbenou


first: chengguo

last: yao

summary: To investigate the extent to which U6 inhibits premature cleavage and polyadenylation at the transcriptome level.  we performed 3’-seq in control, U1 and U6-AMO treated sample using lexogen 3’-seq kit (Cat. 016.24) that could accurately quantify the global PAS usage. Indeed, we found both U1 and U6 could globally suppress intronic PCPA.

overall_design: we performed 3’-seq in control, U1 and U6-AMO treated sample using lexogen 3’-seq kit (Cat. 016.24). Five cell lines were used in our study, namely, HeLa cells, HEK-293 cells, SW480, NIH3T3, Neuro-2A

first: Qiumin

last: Feng

first: Zejin

last: Lin

first: Danhui

last: Zhao

first: Congting

last: Ye

first: Chengguo

last: Yao


title: Bulk RNA-seq analysis of wildtype and MYF5/MYOD/MYF6 knockdown porcine embryo

city: Minneapolis

last: Garry

state: Minnesota

address: 2231 6th Street SE, Suite 4-156

department: Lillehei Heart Institute

institute: University of Minnesota

summary: The pig is an important animal model that is increasingly used for biomedical research.  For example, pigs produced using somatic cell nuclear transfer (SCNT) technology are relevant for transplantation strategies either involving xenotransplantation or the development of human organs in pig for exotransplantation. Pigs, however, are less well characterized than other animal models such as rodents. In previous studies, we produced pigs that lacked skeletal muscle as this was the first step in humanizing this lineage, and we characterized these MYF5/MYOD/MYF6 knockdown embryos to E35. In the current studies, we have evaluated the development and myogenesis in mid-stage porcine embryos produced by SCNT (E41-E90). We comprehensively examined mid-stage MYF5/MYOD/MYF6 knockdown pigs produced by deleting MYF5/MYOD/MYF6 from porcine fibroblasts, cloning these fibroblasts, transferring the early embryos to surrogate gilts, and harvesting the resulting fetal pigs at defined gestational ages. Here, we observed that these genetic deletions resulted in a complete absence of skeletal muscle in these embryos and fetal pigs, an absence of ribs, vertebral abnormalities and an absence of a sternum. Importantly, we observed that these embryos failed to progress beyond E62 indicating embryonic lethality. In addition, we examined porcine myogenesis by evaluating wildtype comparators using gross anatomical, morphological, histological, molecular biological analyzes as well as magnetic resonance imaging of both WT and knockdown embryos and fetal pigs between the ages of E41-E90. We have determined that the knockdown phenotype in pigs is more severe than that observed in rodents as mice with this multiplexed deletion were lethal in the immediate postnatal period. These studies provide an important platform for engineering humanized muscle in gene edited pigs.

overall_design: Limb muscle tissue form wild-type and MYF5/MYOD/MYF6 knockout E41 and E62 pig embryonic stages.

first: Xiao

last: Ma

first: Satyabrata

last: Das


title: KLF5 promotes a mixed basal, club, and hillock epithelial cell identity in castration-resistant prostate cancer

first: Scott

last: Dehm

address: Mayo Mail Code 806, 420 Delaware St SE

institute: Masonic Cancer Center, University of Minnesota

summary: Inhibiting the androgen receptor (AR) is effective for advanced prostate cancers because of their AR-dependent luminal epithelial cell identity. Tumors progress during therapy to castration resistant prostate cancer (CRPC) by restoring AR signaling and maintaining luminal identity or by converting through lineage plasticity to a neuroendocrine identity (NEPC) or double negative CRPC lacking luminal and neuroendocrine identity (DNPC). Here, we show that DNPC cells express genes defining basal, club, and hillock epithelial cells from benign prostate. We identified KLF5 as a regulator of DNPC growth and expression of genes defining this mixed basal, club, and hillock cell identity. KLF5-mediated upregulation of RARG exposed a DNPC growth sensitivity to retinoic acid receptor agonists, which down-regulated KLF5 and up-regulated AR. These findings offer new CRPC classifications based on prostate epithelial cell identities, and nominate KLF5 and RARG as therapeutic targets for CRPC displaying a mixed basal, club, and hillock identity.

overall_design: To investigate the effects of knockdown of KLF5 on gene expression in LTL331CL, we knocked down KLF5 with 2 independent siRNAs or control siRNA. Cells were maintained in LTL331CL-specific media for 72 hours before collection. Three biological replicates were performed for each siRNA (siC, siKLF5-1, siKLF5-2 for n=9 total samples sequenced).

first: Samuel

middle: P

last: Pitzen

first: Sarah

last: Munro


type: Genome binding/occupancy profiling by high throughput sequencing

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

overall_design: Refer to individual Series


title: A single-cell atlas of immune heterogeneity  in anti-PD1-treated non-small cell lung cancer

city: Beijing

first: Wenjie

last: Zhang

state: Not applicable

address: No. 5, Summer Palace Road, Haidian District, Beijing

department: BIOPIC

institute: Peking University

summary: Anti-PD(L)1 with chemotherapy is a standard of care for non-small cell lung cancer (NSCLC), but a varying degree of response to the same regimen is observed in patients. The tumor immune microenvironment (TIME) plays a key role in response to immunotherapy, and the TIME heterogeneity in association with therapeutic outcome is incompletely understood. Here we prospectively applied single-cell RNA and TCR sequencing to characterize post-neoadjuvant chemo-immunotherapy treatment tumor samples of 234 NSCLC patients. Our study provides a fine-grained dissection of the TIME heterogeneity underlying response to chemo-immunotherapy in NSCLC, thus representing a valuable resource for improved management of NSCLC.

overall_design:
Single-cell RNA-seq and single-cell TCR-seq of post-treatment surgical samples(n=234) from 234 non-small-cell lung cancer patients with newly-diagnosed lung squamous carcinoma (LUSC) or lung adenocarcinoma (LUAD) without EGFR mutation and ALK rearrangement undergoing treatment of neoadjuvant anti-PD1 agents combined with platinum-based chemotherapy doublets or chemotherapy alone before successful surgery.
**RAW data not provided** According to the requirements of the Chinese Genetic Information Data Management, we have uploaded the raw sequencing data to the website: https://ngdc.cncb.ac.cn/ (National Genomics Data Center)

first: Zemin

first: Zedao

last: Liu

first: Yuxuan

last: Sun


title: Longitudinal single-cell multiomics data for different severity of COVID-19

city: Seoul

first: Hang-Rae

last: Kim

address: 103 Daehak-ro, Jongno-gu

department: Anatomy and Cell Biology

country: South Korea

institute: Seoul National University College of Medicine

summary: To trace immune responses in COVID-19 patients with severity, we performed in-depth, longitudinal single-cell multiomics involving T-cell receptor (TCR)/B-cell receptor (BCR) sequencing, feature barcoded antibody (Ab) panel detection (i.e., cellular indexing of transcriptomes and epitopes by sequencing, CITE-seq) followed by RNA sequencing in a single-cell resolution.

overall_design: We collected PBMCs from 25 samples in 9 patients with different severity of COVID-19, consisting of two asymptomatic, two mild, five severe patients and two healthy individual.

first: Seung

last: Lee

first: Ik


title: Human pluripotent stem cell (hPSC)-derived progenies (RNA-Seq)

city: Carpi

first: Emiliano

last: Bolesani

state: Modena

address: Via Giuliano Benassi, 7

department: Process Development & Cell Therapy

country: Italy

institute: Evotec SE

summary: The in vitro culture of human cardiac progenitor cells remains a major challenge in biomedicine. The molecular identity of hPSC-derived cardiac progenies and mechanisms controlling their proliferation and differentiation remain unclear. Here, we show that chromatin remodeling and WNT pathway modulation by chemical inhibitors (IQ-1 and CHIR, respectively) synergistically enables stabilization of human cardiac progenitors (SCPs), in a process of transcriptional uncoupling. SCPs are characterized by ISL1pos/KI-67pos/NKX2-5neg expression and are maintained in a quiescent state in the presence of the inhibitors. Upon compound removal, cell autonomous NKX2-5 upregulation hallmarks the recoupling to the cardiomyogenic program, whilst directed differentiation generates endothelial and smooth muscle cells. Chip-sequencing revealed a narrowly defined open chromatin state in SCPs and, when combined with single cell transcriptome analyses, showed a yet unreported Cardiac Neural Crest Cells (CNCCs) footprint. Enforced expression of the oncogene c-MYC could notably not overcome SCPs proliferative quiescence but disrupted the cell autonomous cardiomyogenic potential instead. In contrast, Retinoic Acid (RA) stimulates SCPs proliferation and further supports cells’ phenotypic homogeneity. We consequently show that our in vitro established treatment with IQ1 can also retain ISL1pos cardiac progenies in vivo in a dose and stage specific manner during zebrafish heart development. Due to its chemically defined and reversible nature, our approach provides an unprecedented opportunity to dissect the key mechanisms in cardiac progenitor cell biology, providing a new tool for the advancement of human heart regeneration.

first: Robert

last: Zweigerdt

first: Lavanya

last: Iyer

first: Laura

last: Zelarayan


title: Human pluripotent stem cell (hPSC)-derived progenies


title: Smoking aggravates neovascular age-related macular degeneration via Sema4D-PlexinB1 axis-mediated activation of pericytes [scRNA-seq]

status: Public on Jan 26 2025

city: Tianjin

first: 钰云

last: 王

address: Tianjin

institute: 天津医科大学

summary: Age-related macular degeneration (AMD) is a prevalent neuroinflammation condition and the leading cause of irreversible blindness among the elderly population. Smoking significantly increases AMD risk, yet the mechanisms remain unclear. Here, we investigated the role of Sema4D-PlexinB1 axis in the progression of AMD, in which Sema4D-PlexinB1 is highly activated by smoking. Using patient-derived samples and mouse models, we discovered that smoking increased the presence of Sema4D on the surface of CD8+ T cells that migrated into the choroidal neovascularization (CNV) lesion via CXCL12-CXCR4 axis and interacted with its receptor PlexinB1 on choroidal pericytes. This led to ROR2-mediated PlexinB1 phosphorylation and pericytes activation, hence disrupted vascular homeostasis and promoted neovascularization. Inhibition of Sema4D reduced CNV and improved the benefit of anti-VEGF treatment. In conclusion, this study unveils the molecular mechanisms through which smoking exacerbates AMD pathology, and presents a potential therapeutic strategy by targeting Sema4D to augment current AMD treatments.

overall_design: To investigate the effects of smoking on neovascular age-related macular degeneration (nvAMD) in mice, we performed single cell  transcriptome sequencing on the retinal-choroid complex of C57BL/6J mice. The mice were exposed to cigarette smoke, with or without laser-induced choroidal neovascularization (CNV) modeling. Transcriptome sequencing was conducted on the retinal-choroid complex three days after modeling, with four groups: Smoke group and Smoke+CNV group.

first: Kai

last: He

first: Xue

last: Dong

first: Tianjing

last: Yang


title: Astrocytic heterogeneous nuclear ribonucleoprotein U facilitates scar formation after spinal cord injury

city: Chiba

first: Ichiro

last: Manabe

address: 1-8-1 Inohana, Chuo

country: Japan

institute: Chiba University

summary: In this study, by a genome-wide RNA interference screen, we identified heterogeneous nuclear ribonucleoprotein U (Hnrnpu) as an endogenous key molecule in astrocyte activation. Inhibition of Hnrnpu in astrocytes impairs the formation of astrocytic glial scar, motor function recovery, and neuronal regeneration after spinal cord injury (SCI) in mice. Therefore, we then performed gene expression profiling analysis to evalute the function of HNRNPU in human astrocytes. Our findings uncover that modulation of endogenous astrocytic function through Hnrnpu would be a promising therapeutic avenue to restore neurological function after the injury.

overall_design: mRNA profiles of human astrocytes after transfected with either control or HNRNPU siRNA for 2 days.

first: Lili

last: Quan

first: Rieko

last: Muramatsu


title: Ethanol treatment enhances drought stress avoidance in cassava (Manihot esculenta Crantz)

city: Yokohama

first: motoaki

last: seki

state: Kanagawa

address: Suehiro-cho 1-7-22, Tsurumi-ku

department: CSRS

institute: RIKEN

summary: The external application of ethanol has been reported to enhance salinity, drought and heat stress tolerance in various plant species (Nguyen et al. 2017 ; Bashir et al. in prep.; Matsui et al. in prep). However, the effects of ethanol application on increased drought tolerance in cassava, an important tropical starch crop used for food security and industrial application by a billion people world-wide, remain unknown. In the present study, morphological, physiological, and molecular responses to drought were analyzed in cassava after the treatment with ethanol. The present study showed that the ethanol treatment increased drought avoidance in cassava than the water treatment. Ethanol treatment induced stomatal closure, resulting in the increase of leaf temperature. The decrease in water content of the leaves during drought stress treatment was reduced in ethanol-treated plants compared with control plants. Transcriptome analysis revealed that the expression of drought stress response-related genes and ABA response-related genes was lower in ethanol-treated plants compared with control plants on 12 days after drought treatment.

overall_design: The first to third leaves from the top of stems from 1.0% ethanol-treated or water treated plant for 5 days, drought-treated plant treated drought stress for 6 days after 1.0% ethanol or water pretreatment and drought-treated plant treated drought stress for 12 days after 1.0% ethanol or water pretreatment were collected for transcriptome analysis.

first: Vu

last: Thu

first: Yoshinori

last: Utsumi

first: Maho

last: Tanaka

first: Chikako

first: Satoshi

last: Takahashi

first: Daisuke

last: Todaka

first: Yuri

last: Kanno

first: Mitsunori

last: Seo

first: Eigo

last: Ando

first: Kaori

last: Sako

first: Khurran

last: Bashir

first: Toshinori

last: Kinoshita

first: Pham

middle: X

last: Hoi

first: Motoaki

last: Seki


title: Secreted neurofilament light chain (NfL) after neuronal damage induces myeloid cell activation and neuroinflammation [scRNA-Seq]

city: South San Francisco

first: Mitchell

last: Rezzonico

address: 1 DNA Way

institute: Genentech

summary: Neurofilament light chain (NfL) is a neuron-specific cytoskeletal protein that provides structural support for axons and is released into the extracellular space following neuronal injury. While NfL has been extensively studied as a disease biomarker, the underlying release mechanisms and role in neurodegeneration remain poorly understood. Here, we find that neurons secrete low baseline levels of NfL, while neuronal damage triggers calpain-driven proteolysis and release of fragmented NfL. Secreted NfL activates microglia cells, which can be blocked with anti-NfL antibodies. We utilize in vivo single-cell RNA sequencing to profile brain cells after injection of recombinant NfL into the mouse hippocampus and find robust macrophage and microglial responses. Consistently, NfL knock-out mice ameliorate microgliosis and delay symptom onset in the SOD1 mouse model of amyotrophic lateral sclerosis (ALS). Our results show that released NfL can activate myeloid cells in the brain and is, thus, a potential therapeutic target for neurodegenerative diseases.

overall_design: To examine the effect of exogenous NfL on brain cells in vivo, 11-week-old male mice were bilaterally injected in the hippocampus with either vehicle (n=4), full length NfL (1 mg/ml; n=3) or coiled-coil NfL (1 mg/ml; n=3). After three days, the hippocampi were collected, the cells were dissociated, and live cells were sorted. Single-cell libraries were prepared for further analysis.

first: Olga

last: Kahn

first: Casper

last: Hoogenraad


title: A 3D Self-Assembly Platform Integrating Decellularized Matrix Recapitulates In Vivo Tumor Phenotypes and Heterogeneity

status: Public on Jan 25 2025

city: Frederick

first: Liping

last: Yang

state: MD

address: 1050 Boyles St

institute: NIH-NCI

summary: The tumor microenvironment consists of resident tumor cells organized within a compositionally diverse, three-dimensional (3D) extracellular matrix (ECM) network that cannot be replicated in vitro using bottom-up synthesis. We report a system to engineer ECM-rich 3D MatriSpheres wherein tumor cells actively organize and assemble microgram quantities of decellularized ECM which in turn modulate cancer phenotype. 3D colorectal cancer (CRC) MatriSpheres were created using decellularized small intestine submucosa (SIS) as an orthotopic ECM source that had greater proteomic homology to CRC tumor ECM than traditional ECM formulations such as Matrigel. SIS ECM was rapidly concentrated from its environment and assembled into ECM-rich 3D stroma-like regions by colorectal cancer cell lines within 4-5 days via a mechanism that was rheologically distinct from bulk hydrogel formation. Both ECM organization and transcriptional regulation by 3D ECM cues were cell line specific, affecting programs of malignancy, lipid metabolism, and immunoregulation that corresponded to an in vivo tumor cell subpopulation identified via single cell RNA sequencing. This 3D modeling approach stimulates tumor specific tissue morphogenesis that incorporates the complexities of both cancer cell and ECM compartments in a scalable, spontaneous assembly process that may further facilitate precision medicine.

overall_design: MatriSpheres (SIS ECM) and traditional cells alone spheroids (CA) (n = 5-6/group) generated from mouse (MC38, CT26) and human (HT-29) colorectal cell lines and cultured for 7 days. RNA was isolated at day 7 and sent for Bulk RNA Sequencing. The intercellular role of the ECM biomaterial (decellularized SIS ECM) on cancer cells within the 3D MatriSpheres were evaluated based upon their transcriptome profiles when compared to traditional cells alone spheroids. To validate the MatriSphere model, in vivo MC38 tumors (N = 2 with two replicates per sample) were isolated for scRNA Seq and compared to in vitro Bulk Seq data. A Scissor analysis was used to identify correlation between the in vitro and in vivo transcriptome profiles.

first: Michael

middle: J

last: Buckenmeyer

first: Elizabeth

last: Brooks

first: Madison

last: Taylor

first: Ronald

last: Holewinski

first: Thomas

last: Meyer

first: Melissa

last: Galloux

first: Marcial

last: Garmendia-Cedillos

last: Pohida

first: Thorkell

last: Andresson

first: Brad

last: St. Croix

first: Matthew

last: Wolf


title: OGT prevents DNA demethylation and suppresses the expression of transposable elements in heterochromatin by restraining TET activity genome-wide

city: Saffron Walden

first: Robert

last: Crawford

address: The Trinity Building

country: United Kingdom

institute: biomodal

summary: The O-GlcNAc transferase OGT interacts robustly with all three mammalian TET methylcytosine dioxygenases. We show here that deletion of the Ogt gene in mouse embryonic stem cells (mESC) results in a widespread increase in the TET product 5-hydroxymethylcytosine (5hmC) in both euchromatic and heterochromatic compartments, with concomitant reduction of the TET substrate 5-methylcytosine (5mC) at the same genomic regions. mESC treated with an OGT inhibitor displayed increased 5hmC, and attenuating the TET1-OGT interaction in mESC resulted in a genome-wide decrease of 5mC, indicating that OGT restrains TET activity and limits untoward DNA demethylation in a manner that requires the TET-OGT interaction and the catalytic activity of OGT. DNA hypomethylation in OGT-deficient cells was accompanied by de-repression of transposable elements (TEs) predominantly located in heterochromatin. We suggest that OGT protects the genome against TET-mediated DNA demethylation and loss of heterochromatin integrity, preventing the aberrant increase in TE expression noted in cancer, autoimmune-inflammatory diseases, cellular senescence and ageing.

overall_design: To investigate the effect of knock out of the OGT gene on genome-wide distribution of methylated and hydroxymethylated cytostine bases at base resolution using 6-base sequencing

first: Fabio

last: Puddu

first: Jamie

last: Scotcher

first: Páidí

last: Creed

first: Hugo

last: Sepulveda

first: Anjana

last: Rao

first: Aurélie

last: Modat


title: Human pluripotent stem cell (hPSC)-derived progenies (ChIP-Seq)

first: Emiliano

last: Bolesani

overall_design: Examination of H3K27ac regions in human pluripotent stem cell (hPSC)-derived progenies, in biphasic WNT pathway differentiation protocol. Comparison between Mesodermal Progenies (differentiation day 4 - dd4), Cardiomyocytes (dd10), SCPs (IQ-1/CHIR dd10), and respective CBP inhibition (ICG-001 dd10).

last: Zweigerdt

first: Lavanya

last: Iyer

first: Laura

middle: C

last: Zelarayan


title: Engineered biomaterial immunological niches as a surrogate for diseased tissue in chronic progressive EAE

status: Public on Jan 24 2025

city: Ann Arbor

first: Lonnie

last: Shea

state: MI

address: 1600 Huron Parkway

department: Biomedical Engineering

institute: University of Michigan

summary: We use a subcutaneous biomaterial implant as a surrogate for sampling immune cells phenotypes in murine chronic EAE. These implants are microporous polycaprolactone scaffolds that form immunological niches that get vascularized and capture both innate and adaptive immune cells. These niches can be minimally invasively biopsied, allowing for multiple samples to be collected from the same mouse at various timepoints in disease: presymptomatic (Day 7) and early state disease (Day 9). This study investigates immune dynamics over time in subclinical and early stage chronic progressive EAE at a distal biomaterial-based immunological niche.

overall_design: Following subcutaneous implant of PCL microporous scaffolds, C57/B6J mice were given an adoptive transfer (AT) of MOG-reactive cells to induce chronic EAE or OVA-reactive cells as a healthy control. Scaffolds were explanted 7 (pre-symptomatic) and 9 days (early-stage disease; symptomatic) after AT and analyzed using scRNAseq.

first: Aaron

middle: H

last: Morris

first: Laila

last: Rad


title: Identification of a new type of endocycle during multiciliated cell differentiation

city: Paris

first: Romain

last: Koszul

address: 25 Rue du Dr Roux

department: Genetics and Genomics

country: France

institute: Institut Pasteur

summary: Centriole duplication and DNA replication are two tightly synchronized events of the cell cycle. Perturbations of this coordination lead to genomic instabilities and increased cancer pathologies. Yet, uncoupling of these two molecular events can occur in physiological contexts, such as in post-mitotic multiciliated cell (MCCs) differentiation, during which the activity of the mitotic oscillator is finely tuned to allow centriole amplification while preventing nuclear and cell division. Using single-cell RNA sequencing of MCC-containing tissues, we uncovered that up to 70% of the cell cycle genes are actually co-opted in differentiating MCC progenitors. These progenitors progress through differentiation following a circular transcriptional trajectory characterized by consecutives cell cycle-like phases and successive waves of cyclin expression, mimicking the canonical cell cycle. However, non-canonical cyclins replace cyclins involved in DNA synthesis and mitosis onset, potentially explaining the absence of DNA replication and cell division. Altogether, our results suggest that post-mitotic multiciliated cells have evolved across tissues and mammals by reusing the cell cycle protein toolbox but also the optimality principles of gene expression regulation of the cell cycle, to orchestrate the elaborated stepwise centriole amplification dynamics. This prompts us to propose the existence of a new type of endocycle, where multiciliating cell achieves cytoplasmic organelle amplification rather than ploidy amplification, while still escaping cell division, as in other endocycles.

overall_design: scRNAseq was performed in duplicates for primary cultures of dissected brain lateral ventricular walls from newborn mice, to profile the multiciliated cell differentiation lineage.

first: Jacques

last: Serizay

first: Alice

last: Meunier


status: Public on Jan 23 2025

city: Wien

first: Jörg

last: Menche

state: Wien

address: Augasse 2-6,

country: Austria

institute: Ludwig Boltzmann Institute for Network Medicine

summary: Microbial exposure at barrier interfaces drives development and balance of the immune system but the consequences of local infections for systemic immunity and secondary inflammation are unclear. Here, we show that epicutaneous exposure to the bacterium Staphylococcus aureus persistently shapes the immune system of mice with specific impact on progenitor and mature bone marrow neutrophil and eosinophil populations. The infection-imposed changes in eosinophils were long-lasting and associated with functional as well as imprinted epigenetic and metabolic changes. Bacterial exposure enhanced skin allergic sensitization and resulted in exacerbated allergen-induced lung inflammation. S. aureus-mediated functional bone marrow eosinophil reprogramming and pulmonary allergen responses were driven by the alarmin interleukin 33 and the complement cleavage fragment C5a. Our study highlights the systemic impact of skin inflammation and reveals eosinophil progenitor training and organ-crosstalk mechanisms that modulate systemic responses to allergens.

overall_design:
To investigate the systemic effects of local Staphylococcus aureus (SA) skin infection or PBS, we performed single cell RNA-seq on bone marrow immune mature and progenitor cells at day 7 post infection in 9-week old C57BL/6J mice. We performed 2 replicate experiments with 2 treatment groups PBS and SA (n=3-4). These experiments resulted in the sequencing of 2 libraries (MR_30_10X_SA_PBS_BM_22112021_Transcriptome, MR_33_10X_SA_PBS_BM_Transcriptome).
This dataset is associated with the publication "Bacterial skin infection imprints bone marrow eosinophils to promote allergic skin-lung crosstalk", Radhouani et al., 2025 in Science Immunology, and was generated as part of a collaborative effort with the co-authors of the manuscript. For the full list of contributors, please refer to the publication.

first: Mariem

last: Radhouani

first: Anna

last: Hakobyan

first: Philipp

last: Starkl

first: Sylvia

last: Knapp


title: Bacterial skin infection imprints bone marrow eosinophils to promote allergic skin-lung crosstalk [Quant_seq]

overall_design: To investigate the impact of local Staphylococcus aureus (SA) skin infection or PBS on bone marrow eosinophils and eosinophil progenitors, we performed bulk RNA-seq on those two sorted cell populations at day 7 post infection in 9-week old C57BL/6J mice (n=6).


title: The reparative immunologic consequences of stem cell transplantation as a cellular therapy for refractory Crohn’s disease

city: New York

first: Louis

last: Cohen

state: NY

address: 1470 Madison Avenue, 8th Fl.

department: Gastroenterology

institute: Icahn School of Medicine at Mount Sinai

summary: Background Treatment strategies for Crohn’s disease (CD) suppress diverse inflammatory pathways but many patients remain refractory to treatment. Autologous hematopoietic stem cell transplantation (SCT) is an emerging therapy for medically-refractory CD though the mechanisms through which it circumvents refractory pathophysiology are unknown.  Objective The objective of this study is to understand how the immune system reconstitutes post-SCT and whether SCT may function as a cellular therapy restoring appropriately responsive immune cell populations from hematopoietic stem cells (HSCs).  Design Adults with CD with active clinical and endoscopic disease who failed available medical therapies were enrolled in a Phase II study of SCT for refractory CD (n=19).  Blood and intestinal samples were collected longitudinally and analyzed using CyTOF, scRNA-seq and TCRβ-seq. Stem cell autografts were functionally assayed in mouse xenograft models.  Results  scRNA-seq and CyTOF analyses reveal that SCT predominantly affected the intestinal myeloid lineage with loss of inflammatory populations and return of macrophages capable of supporting mucosal healing. Xenograft models using patient HSCs suggested HSCs support the early reconstitution of the myeloid lineage and reveal an impairment of short and long-term HSC engraftment that may determine SCT outcomes.   Conclusions  This study suggests SCT functions as a myeloid-directed cellular therapy reinforcing the critical role of macrophages in refractory CD pathophysiology and as a target for cellular therapies. Furthermore, we report an unrecognized functional heterogeneity among HSC subpopulations in CD that may be relevant to our understanding of CD treatment and pathophysiology.

overall_design: Samples of tissue from endoscopic biopsies, peripheral blood and peripheral mobilized stem cells were collected from subjects with CD enrolled for SCT. Blood and tissue samples were processed for 10x Genomics single cell RNA sequencing (scRNA-seq) and bulk T cell receptor beta (TCRβ) sequencing through Adaptive technologies.

first: Daniela

last: Guisado

first: Sayali

last: Talware

first: Xiaoli

last: Wang

first: Andrew

last: Davis

first: Elbek

last: Fozilov

last: Etra

first: Jean-Frederic

last: Colombel

first: Christoph

last: Schaniel

first: Christopher

last: Tasad

first: John

middle: E

last: Levine

first: James

last: Ferrara

first: Ling-Shiang

last: Chuang

first: Ksenija

last: Sabic

first: Shishir

last: Singh

first: Bridget

middle: K

last: Marcellino

first: Ronald

last: Hoffman

first: Judy

last: Cho


title: Loss of Ing4 enhances hematopoietic regeneration in multipotent progenitor cells

city: Columbia

first: Katie

last: Kathrein

state: SC

address: 715 Sumter St CLS 304

institute: University of South Carolina

summary: Gene expression of ING4-/- LT-HSCs, ST-HSCs, and MPPs were analyzed for gene expression at steady-state.

overall_design: Bone marrow from 8-10 mice was harvested and pooled, and lineage depleted. 1,000-10,000 LT- HSCs, ST-HSCs, and MPPs were sorted separately into Buffer RLT Plus (Qiagen) for both WT or Ing4-/- mice. Total RNA was isolated with the RNeasy Micro column (QIAGEN). Library construction, sequencing by Illumina HiSeq, and initial bioinformatics analyses were conducted by GENEWIZ. Counts obtained were used to perform Gene Set Enrichment Analysis. Ing4-/- LT- and ST-HSCs were compared together against WT LT- and ST-HSCs or Ing4-/- LT-, ST-HSC and MPPs were compared together against WT.

first: Georgina

last: Anderson

first: Zanshé

last: Thompson

first: Marco

last: Hernadez

first: Carlos

last: Alfaro Quinde

first: Vera

last: Binder-Blaser

first: Alison

last: Taylor


title: A unified molecular approach for spatial epigenome, transcriptome, and cell lineages

city: Stanford

first: Julia

last: Belk

state: California

address: 450 Serra Mall

institute: Stanford University

summary: Spatial epigenomics and multiomics can provide fine-grained insights into cell state but widespread adoption is limited by the requirement for bespoke slides and capture chemistries for each data modality. Here, we develop an approach (SPACE-seq) that uses a transposome with polyadenine adaptors to generate polyA-tailed epigenomic libraries, enabling facile spatial epigenomics and multiomics using standard whole transcriptome reagents. Application of SPACE-seq to a human glioblastoma specimen defines the spatial localization and accessible chromatin of distinct sub-populations within the tumor, and putative mitochondrial DNA variants.

overall_design: Tissue sections from mouse brain were used to develop SPACE-seq, a genomics method that adapts the commercially available 10X Visium CytAssist v2 workflow to capture epigenomic data (spatial ATAC-seq) alongside spatial transcriptomics. SPACE-seq was then applied to a clinical human glioblastoma sample.

first: Yung-Hsin

last: Huang

first: Howard

last: Chang


title: Condensin IDC, H4K20me1, and perinuclear tethering maintain X chromosome repression in C. elegans

first: Gyorgyi

last: Csankovszki

address: 1105 N University Ave

department: MCDB

summary: Dosage compensation in Caenorhabditis elegans equalizes X-linked gene expression between XX hermaphrodites and XO males. The process depends on a condensin-containing dosage compensation complex (DCC), which binds the X chromosomes in hermaphrodites to repress gene expression. Condensin IDC and an additional five DCC components must be present on the X during early embryogenesis in hermaphrodites to establish dosage compensation. However, whether the DCC's continued presence is required to maintain the repressed state once established is unknown. Beyond the role of condensin IDC in X chromosome compaction, additional mechanisms contribute to X-linked gene repression. DPY-21, a non-condensin IDC DCC component, is an H4K20me2/3 demethylase whose activity enriches the repressive histone mark, H4 lysine 20 monomethylation on the X chromosomes. In addition, CEC-4 tethers H3K9me3-rich chromosomal regions to the nuclear lamina, which also contributes to X-linked gene repression. To investigate the necessity of condensin IDC during the larval and adult stages of hermaphrodites, we used the auxin-inducible degradation system to deplete the condensin IDC subunit DPY-27. While DPY-27 depletion in the embryonic stages resulted in lethality, DPY-27 depleted larvae and adults survive. In these DPY-27 depleted strains, condensin IDC was no longer associated with the X chromosome, the X became decondensed, and the H4K20me1 mark was gradually lost, leading to X-linked gene derepression. These results suggest that the stable maintenance of dosage compensation requires the continued presence of condensin IDC. A loss-of-function mutation in cec-4, in addition to the depletion of DPY-27 or the genetic mutation of dpy-21, led to even more significant increases in X-linked gene expression, suggesting that tethering heterochromatic regions to the nuclear lamina helps stabilize repression mediated by condensin IDC and H4K20me1.

overall_design: RNA-seq profiles of L3 larval stage C. elegans samples. Synced L1s were placed on HGM plates with or without auxin, and NA22. 24-hours later, L3 larvae were collected for RNA extraction. Genotype and treatment for each sample are listed separately.

first: Jessica

last: Trombley

first: Audry

middle: I

last: Rakozy

first: Christian

last: McClear

first: Eshna

last: Jash

first: Györgyi


title: To investigate the role of WDR4 in sorting miRNAs into exosomes

city: Taipei

first: Nai Yang

last: Yeat

address: 128, Academia Road Sec. 2, Nankang

country: Taiwan

institute: Academia Sinica

type: Non-coding RNA profiling by high throughput sequencing

summary: Exosomes play pleiotropic tumor-promoting functions and are secreted by fusion of multivesicular bodies (MVBs) with the plasma membrane. We identified that WDR4-derived exosomes preferentially sorted pro-metastatic proteins to promote cancer metastasis and tumor progression. As miRNAs were also encapsulated by exosomes to perform certain tumor promoting effects, we wondered if WDR4 can also alter the sorting of miRNAs in exosomes.

overall_design: Exosomes were isolated from H1299 cells stably expressing control or WDR4 shRNA. miRNAs were extracted from exosomes and sent for small RNA sequencing. Differential expression analysis of high throughput miRNA-seq data was obtained from 3 independent biological repeats.

first: Nai-Yang

first: Ruey-Hwa

last: Chen


title: A single-cell atlas of normal and KRASG12D-malformed lymphatic vessels

city: Dallas

first: Michael

last: Dellinger

state: Texas

address: 5323 Harry Hines Blvd

institute: University of Texas Southwestern Medical Centre

summary: Somatic activating mutations in KRAS can cause complex lymphatic anomalies (CLAs). However, the specific cellular and molecular processes that drive KRAS-mediated CLAs have yet to be fully elucidated. Here, we use single-cell RNA sequencing to construct an atlas of normal and KrasG12D-malformed lymphatic vessels. We show that adult wild-type mice have six subtypes of lymphatic endothelial cells (LECs) in their lungs (Ptx3, capillary, collecting, valve, mixed, and proliferating). To determine when the LEC subtypes are specified during development, we integrated our data with data from four stages of development. We show that proliferating and Ptx3 LECs are prevalent during early lymphatic development and that collecting and valve LECs emerge later in development. Additionally, we demonstrate that the proportion of Ptx3 LECs decreases as the lymphatic network matures but remains high in KrasG12D mice. We also show that KrasG12D mice have fewer collecting and valve LECs than wild-type mice. Last, we demonstrate that immature lymphatic vessels in young mice are more sensitive to the pathologic effects of KrasG12D than mature lymphatic vessels in older mice. Together, our results expand the current model for the development of the lymphatic system and suggest that KRAS mutations impair the maturation of lymphatic vessels.

overall_design: To evaluate the influence of hyperactive KRAS signaling on the cellular and transcriptional landscape of lymphatic vessels, we bred Prox1-CreERT2, mT/mG, and LSL-KrasG12D mice together to generate Prox1-CreERT2;mT/mG (control) and Prox1-CreERT2;mT/mG;LSL-KrasG12D (KrasG12D) mice. Prox1-CreERT2 mice express a tamoxifen-inducible form of Cre recombinase in lymphatic endothelial cells (LECs), and LSL-KrasG12D mice express an active form of Kras (KRAS.pG12D) from the endogenous Kras locus following Cre-mediated recombination. We included the mT/mG reporter strain in our experiment to label cells that had undergone Cre-mediated recombination with green fluorescence protein (GFP). Newborn mice were fed tamoxifen from postnatal day (P)0 to P2 to induce Cre-mediated recombination in Prox1-expressing cells (Figure 1B). Following tamoxifen administration, lungs from three-week-old control (n= 5 mice) and KrasG12D mice (n= 4 mice) were dissociated into single-cell suspensions, and live GFP-positive cells were sorted by FACS. The cells were then subjected to scRNA-Seq using 10X genomics 3’ chemistry.

first: L

last: Fernandes

first: M


title: Human pluripotent stem cell (hPSC)-derived progenies (scRNA-Seq)

first: Emiliano

last: Bolesani

first: Robert

last: Zweigerdt

first: Lavanya

last: Iyer

first: Laura

last: Zelarayan


title: Functional optic tract rewiring via subtype- and target-specific axonal regeneration and presynaptic activity enhancement

city: Hong Kong

first: Kai

last: Liu

address: Clear water bay

department: LIFS

institute: HKUST

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

overall_design: Refer to individual Series


title: Single cell RNA sequencing of urine sediment from early and advanced Diabetic Kidney Disease (DKD) patients

city: Guangzhou

first: Dongzhao

last: Ma

address: No.58 Zhongshan Second Road

institute: First Affiliated Hospital of Sun Yat-sen University

summary: Urine sediment collected from patients diagnosed with early and advanced DKD are subject to single cell RNA sequencing. The resulting data are used to define early tubule injury/repairing signatures.

overall_design: Three female and five male hospitalized patients who diagnosed as DKD are enrroled in the study, with age range of 49–78 years old. Based on their eGFR, 8 patients are assigned into early DKD (eGFR ≥ 30 ml/min/1.73 m²) and advanced DKD (eGFR<30 ml/min/1.73 m²) group respectively. We used morning urine samples for the urine cell scRNA-sequencing. Due to the limited cell number in the urine samples, the 8 samples were combined for two batches, each batch contains 2 early and 2 advanced subjects.

first: Dan

last: Wang

first: Jianwen

last: Yu

first: Naya

first: Yue

last: Yang

first: Minghui

last: Xu

first: Jianbo

last: Li

first: Yagui

last: Qiu

first: Jinjin

last: Fan

first: Zhijian

first: Wei

first: Qin

last: Zhou


title: Functional Optic Tract Rewiring via Subtype- and Target-specific Axonal Regeneration and Presynaptic Activity Enhancement (scRNA-Seq)

summary: Mechanisms underlying functional axonal rewiring after adult mammalian central nervous system (CNS) injuries remain unclear partially due to limited models. Using a mouse intracranial pre–olivary pretectal nucleus (OPN) optic tract injury model, we demonstrate that Pten/Socs3 knockout and CNTF expression in retinal ganglion cells (RGCs) promotes optic tract regeneration and reinnervation into OPN. Revealed by transmission electron microscopy, trans-synaptic labeling and electrophysiology, functional synapses are formed in OPN mainly by intrinsically photosensitive RGCs, thereby partially restoring the pupillary light reflex (PLR). Furthermore, combining with Lipin-1 knockdown accelerates the recovery and achieves functional reconnection in the chronic injury paradigm. PLR can be further boosted by increasing either RGC photosensitivity with melanopsin or optic tract presynaptic release with a voltage-gated calcium channel modulator, the latter of which also improves corticospinal-tract-sprouting-induced functional recovery after stroke. These findings highlight the importance of neuronal types and presynaptic activity for functional reconnection after CNS injuries.

overall_design: We showed that with genetic manipulation as Pten/Socs3 knockout (KO), CNTF overexpression and Lipin1 knockdown (PSCL), RGCs could regenerate and reinnervate into OPN nuclei after pre-OPN optic tract injury. To probe the regenerative transcriptome dynamics, we isolated RGCs labelled with CTB-555 from PSCL regeneration and injury control mice at various time points, conducting single-cell RNA sequencing mainly targeting on OPN-projecting RGCs. We had total 8 groups: GFP-intact, GFP-5dpi (5 days post injury), GFP-1.5mpi (1.5 months post injury), GFP-3mpi, PSCL-intact, PSCL-5dpi, PSCL-1.5mpi, PSCL-3mpi.

first: Chengle

last: Zhang

first: Xin

first: Chao


title: Spatial hepatocyte plasticity of gluconeogenic gene expression and gluconeogenic activity during the metabolic transitions between fed, fasted and starvation states [scRNA-Seq]

city: Bronx

first: Jeffrey

last: Pessin

address: 1301 Morris Park Avenue

department: Medicine and Molecular Pharmacology

institute: Albert Einstein College of Medicine

summary: Single cell analyses of hepatocytes across the liver lobule demonstrated that gluconeogenic gene expression (Pck1 and G6pc) is relatively low in the fed state and gradually increase first in the periportal hepatocytes during the initial fasting period. As the time of fasting progresses, pericentral hepatocyte gluconeogenic gene expression increases and following entry into the starvation state the pericentral hepatocytes are not significantly different than the periportal hepatocytes. Similarly, pyruvate-dependent gluconeogenic activity is approximately 10-fold higher in the periportal hepatocytes during the initial fasting states but with only 1.5-fold different between the pericentral and periportal hepatocytes in the starvation state. In parallel, starvation induced a reduction of canonical beta-catenin signaling and redistribution of pericentral and periportal glutamine synthetase and glutaminase resulting in an enhanced pericentral glutamine-dependent gluconeogenesis. These data demonstrate that hepatocyte gluconeogenic gene expression and gluconeogenic activity are highly spatially and temporally plastic across the liver lobule and underscore the critical importance of using well-defined feeding and fasting times to define the basis of hepatic insulin resistance and glucose production.

overall_design: Mice were trained for 3 days by removing food at 5PM and feeding at 9:00 the next day (16h fasting overnight and 8h feeding daytime). On the day of experiment, mice were provided food with drinking water that contained 20% of sucrose. The fed (0h) mice were sacrificed at 13:00 equal to 4h of feeding (0h). For fasted mice, the food and sucrose water were taken out at 13:00, then sacrificed at 17:00 for fast 4h (4h), 21:00 for fast 8h (8h), 5:00 the next day for fast16h (16h), and 13:00 the next day for fast 24h (24h).

first: Junichi

last: Okada

first: Li

first: Kosaku

last: Shinoda


title: Spatial hepatocyte plasticity of gluconeogenic gene expression and gluconeogenic activity during the metabolic transitions between fed, fasted and starvation states [RNA-Seq]

overall_design: Mice were trained for 3 days by removing food at 5PM and feeding at 9:00 the next day (16h fasting overnight and 8h feeding daytime). On the day of experiment, mice were provided food with drinking water that contained 20% of sucrose. The fed (0h) mice were sacrificed at 13:00 equal to 4h of feeding (0h). For fasted mice, the food and sucrose water were taken out at 13:00, then sacrificed at 21:00 for fast 8h (8h), and 13:00 the next day for fast 24h (24h).


title: Smoking aggravates neovascular age-related macular degeneration via Sema4D-PlexinB1 axis-mediated activation of pericytes [bulk RNA-seq]

city: Tianjin

first: 钰云

last: 王

address: Tianjin

institute: 天津医科大学

summary: Age-related macular degeneration (AMD) is a prevalent neuroinflammation condition and the leading cause of irreversible blindness among the elderly population. Smoking significantly increases AMD risk, yet the mechanisms remain unclear. Here, we investigated the role of Sema4D-PlexinB1 axis in the progression of AMD, in which Sema4D-PlexinB1 is highly activated by smoking. Using patient-derived samples and mouse models, we discovered that smoking increased the presence of Sema4D on the surface of CD8+ T cells that migrated into the choroidal neovascularization (CNV) lesion via CXCL12-CXCR4 axis and interacted with its receptor PlexinB1 on choroidal pericytes. This led to ROR2-mediated PlexinB1 phosphorylation and pericytes activation, hence disrupted vascular homeostasis and promoted neovascularization. Inhibition of Sema4D reduced CNV and improved the benefit of anti-VEGF treatment. In conclusion, this study unveils the molecular mechanisms through which smoking exacerbates AMD pathology, and presents a potential therapeutic strategy by targeting Sema4D to augment current AMD treatments.

overall_design: To investigate the effects of smoking on neovascular age-related macular degeneration (nvAMD) in mice, we performed transcriptome sequencing on the retinal-choroid complex of C57BL/6J mice. The mice were exposed to cigarette smoke or air, with or without laser-induced choroidal neovascularization (CNV) modeling. Transcriptome sequencing was conducted on the retinal-choroid complex three days after modeling, with four groups: Ctrl group, Smoke group, CNV group, and Smoke+CNV group.

first: Kai

last: He

first: Xue

last: Dong

first: Tianjing


title: Functional Optic Tract Rewiring via Subtype- and Target-specific Axonal Regeneration and Presynaptic Activity Enhancement (bulk RNA-Seq)

last: Liu

overall_design: To compare the transcriptomic responses of retinal ganglion cells (RGCs) under two distinct injury models, optic nerve crush (ONC) and optic tract injury (OTI), a bulk RNA sequencing approach was employed. Specifically, RGCs were labelled by injecting AAV-DIO-GFP into the vitreous body of vGlut2-cre mice one month prior to any injury was applied to the animals. Subsequently, RGCs were collected for bulk sequencing from various treatment groups, including intact (non-injured) samples, ONC 1D (1 day post-injury), ONC 3D (3 days post-injury), ONC 7D (7 days post-injury), OTI 1D, OTI 3D, and OTI 7D. Fluorescence-activated cell sorting (FACS) was employed for the isolation of RGCs from each treatment group. Principal component analysis (PCA) and differential expression analysis were then performed to elucidate the distinctions between the two injury models.


title: Sphingolipid metabolism orchestrates the establishment of the adult hair follicle stem cell niche to control skin homeostasis

city: Münster

first: Sara

last: Wickström

address: Röntgenstraße 20

department: Cell & Tissue Dynamics

institute: Max Planck Institute for Molecular Biomedicine

summary: Bioactive sphingolipids serve as an essential building block of membranes, forming a selective barrier ensuring subcellular compartmentalization and facilitating cell type-specific intercellular communication through regulation of the plasma membrane receptor repertoire. How the cell type-specific lipid compositions are achieved and what is their functional significance in tissue morphogenesis and maintenance has remained unclear. Here, we  identify a stem-cell specific role for ceramide synthase 4 (CerS4) in orchestrating fate decisions in the skin epidermis. Deletion of CerS4 in the epidermis prevents the effective establishment of the adult hair follicle bulge stem cell (HFSCs) niche due to altered differentiation trajectories of HFSC precursors towards upper hair follicle and inner bulge fates. Mechanistically, the HFSC differentiation defects arise from a stem cell intrinsic imbalance of key ceramides and sphingolipids, and associated hyperactivity of canonical Wnt signaling. The lack of HFSCs leads to disruption of hair follicle architecture and hair follicle barrier function, ultimately triggering a Th2-dominated immune infiltration closely resembling human atopic dermatitis. This work uncovers a fundamental role for a cell state-specific sphingolipid profile in epidermal stem cell homeostasis and the role of an intact stem cell niche in maintaining an intact skin barrier.

overall_design: C57BL/6N mice with floxed CerS4 alleles were generated in cooperation with Taconic Artemis (Cologne, Germany). CerS4 complete knockout mice were generated as described before (Peters et al, 2015). To achieve epidermis-specific deletion, CerS4fl/fl mice were crossed to K14-Cre mice (Hafner et al, 2004), as described before (Peters et al, 2015). Experiments were performed with CerS4fl/flK14Cre+ (termed: CerS4epi-/-) and CerS4fl/flK14Cre- (termed: ctr) male and female littermates at the indicated postnatal days (P)

first: Franziska

last: Peters

last: Kruse


title: Spatial hepatocyte plasticity of gluconeogenic gene expression and gluconeogenic activity during the metabolic transitions between fed, fasted and starvation states [targeted scRNA-seq]

overall_design: Mice were trained for 3 days by removing food at 5PM and feeding at 9:00 the next day (16h fasting overnight and 8h feeding daytime). On the day of experiment, mice were provided food with drinking water that contained 20% of sucrose. The fed (0h) mice were sacrificed at 13:00 equal to 4h of feeding (0h). For fasted mice, the food and sucrose water were taken out at 13:00, then sacrificed at 21:00 for fast 8h (8h), 5:00 the next day for fast16h (16h), 13:00 the next day for fast 24h (24h), and 19:00 the next day for fast 30h (30h).


title: Genetic background and aging-driven neuroimmune and disc changes underscore herniation susceptibility and pain-associated behaviors in SM/J mice

city: Philadelphia

first: Emanuel

middle: José

last: Novais

address: 1025 Walnut St., Suite 511 College Bldg.

department: Department of Orthopaedic Surgery

institute: Thomas Jefferson

summary: There are no appropriate mouse models to study the pathophysiology of spontaneous disc herniations in a wild-type setting. We demonstrate that SM/J mice show a high incidence of age-associated lumbar disc herniations with neurovascular innervations. Transcriptomic comparisons of the SM/J annulus fibrosus with human tissues showed shared pathways related to immune cell activation and inflammation. Notably, aged SM/J mice showed increased pain sensitization and neuroinflammation with altered ECM regulation in the DRG and spinal cord. There were increased T cells in the vertebral marrow, and CyTOF analysis showed increased splenic CD8+ T cells, nonspecific activation of CD8+ memory T cells, and enhanced IFN-γ production in the myeloid compartment. ScRNA-seq of PBMCs showed more B cells, with lower proportions of T cells, monocytes, and granulocytes. This study highlights the contribution of genetic background and aging to increased susceptibility of spontaneous disc herniations and clinically relevant pain phenotypes in SM/J mice.

overall_design: DRG and AF tissue were dissected from lumbar discs from BL6 and SM/J mice (n=4-6 per group). RNA extraction and hybridization on Affymetrix microarrays.


title: Zmiz1-dependent Native Stem Cell Transcriptional Circuits in Normal and Malignant Early-stage T cells [ChIP-seq]

first: Mark

middle: Yat-fung

last: Chiang

address: 109 Zina Pitcher Place

department: Internal Medicine/Heme-onc

summary: The discovery of early T-cell precursor acute lymphoblastic leukemia (ETP-ALL) was based on expression of stem cell genes that are characteristic of mouse ETP cells, the most primitive multipotent cells in the thymus. Using complementary mouse and human genetic models and genome-wide expression and chromatin profiling integrated with 3D chromatin mapping, we show that the PIAS-like coactivator ZMIZ1 promotes immature T-ALL proliferation by recruiting the transcription factor MYB in feedforward circuits to cooperatively induce MYCN, MEF2C, and BCL2, which were recently associated with the high-risk bone marrow progenitor (BMP-like) subset.

overall_design: ChIP-seq analysis of LOUCY cells, a human immature T-ALL cell line.

first: Ashley

last: Melnick

first: Russell

last: Ryan

first: Qing

last: Wang


title: Zmiz1-dependent Native Stem Cell Transcriptional Circuits in Normal and Malignant Early-stage T cells [ATAC-seq]

overall_design: ATAC-seq analysis of LOUCY and THP-6 cells, a near ETP-ALL cell line.


title: Cholesterol-Associated Locus EHBP1 Protects Against MASH Fibrosis

city: Los Angeles

first: Bishuang

last: Cai

state: CA

address: 675 Charles E. Young Dr South, Rm 1220

department: Medicine

institute: University of California, Los Angeles

summary: Excess cholesterol accumulation contributes to fibrogenesis in metabolic dysfunction-associated steatohepatitis (MASH, formerly known as nonalcoholic steatohepatitis [NASH]), but how hepatic cholesterol metabolism becomes dysregulated in MASH is not completely understood. We show here that human fibrotic MASH livers have decreased EHBP1, a novel GWAS locus associated with LDL cholesterol, and that the EHBP1 rs10496099 T>C variant in MASH patients is associated with decreased hepatic EHBP1 expression and with augmented MASH fibrosis. Congruent with the human data, EHBP1 loss- and gain-of-function increases and decreases MASH fibrosis in mice, respectively. Mechanistic studies reveal that EHBP1 promotes sortilin (SORT1)-mediated PCSK9 secretion, leading to LDLR degradation, decreased LDL uptake, and reduced TAZ, a fibrogenic effector. At a cellular level, EHBP1 deficiency affects intracellular localization of retromer, a protein complex required for sortilin stabilization. Our novel therapeutic approach to stabilizing retromer is effective in mitigating MASH fibrosis. Moreover, we show that the TNFα/PPARα pathway suppresses EHBP1 in MASH. These data not only provide new mechanistic insight into the role of EHBP1 in cholesterol metabolism and MASH fibrosis by uncovering the interaction between EHBP1 and other cholesterol-related loci, including SORT1, PCSK9, and LDLR, but also elucidate a novel interplay between inflammation and EHBP1-mediated cholesterol metabolism.

overall_design: Total liver RNA from Chow, MASH, and shEhbp1 MASH mice was extracted for RNA-sequencing

first: Xin

last: Huang


title: Zmiz1-dependent Native Stem Cell Transcriptional Circuits in Normal and Malignant Immature T cells


title: Simvastatin modulates osteogenic differentiation in stem cells isolated from apical papilla

status: Public on Jan 22 2025

city: Bangkok

first: Paak

last: Rewthamrongsris

state: Patumwan

address: 34 Henri-Dunant Road, Wangmai

department: Department of Anatomy

country: Thailand

institute: Faculty of Dentistry, Chulalongkorn University

summary:
Background Simvastatin modulates numerous stem cell functions, including stemness maintenance and differentiation. The present study aimed to explore the effect of simvastatin on the osteogenic differentiation of SCAPs in vitro.
Methods Cells were isolated from apical papilla, and mesenchymal stem cell features were characterised. Cells were treated with various concentrations of simvastatin (100-1,000 nM). The mRNA expression profile of simvastatin-treated SCAPs was examined using RNA sequencing technique. The osteogenic differentiation abilities were assessed. Alkaline phosphatase activity was determined. The mineralisation was visualised using Alizarin Red S and Von Kossa staining. The osteogenic marker gene expression was determined using a quantitative polymerase chain reaction.
Results RNA sequencing data demonstrated that simvastatin upregulated genes enriched in those pathways involving osteogenic differentiation, including the TGF-beta signalling pathway, FoxO signalling pathway, and MAPK signalling pathway, while the downregulated genes were involved in pathways related to cell proliferation and apoptosis, for example, DNA replication, cell cycle, and p53 signalling pathway. Simvastatin promoted mineral deposition in a dose-dependent manner, corresponding with the upregulation of osteogenic marker genes namely OSX, DMP1, DSPP, and OCN. Pretreatment with TGF-beta receptor inhibitor, SB431542, resulted in a moderately attenuated effect on simvastatin-induced mineralisation and osteogenic marker gene expression.
Conclusions Simvastatin enhances osteogenic differentiation in SCAPs, potentially via TGF-beta signalling, implicating its potential role as an adjunctive molecule in dental pulp healing and regeneration in vital pulp treatment approaches.

overall_design: RNA-seq profilling of simvastatin-treated stem cells isolated from apical papilla compared to growth medium

first: Suphalak

last: Phothichailert

first: Uraiwan

last: Chokechanachaisakul

first: Prim

last: Janjarussakul

first: Chatvadee

last: Kornsuthisopon

first: Lakshman

last: Samaranayake

first: Thanaphum

last: Osathanon


title: Molecular landscape of tumor-associated tissue-resident memory T cells in tumor microenvironment of hepatocellular carcinoma [HCC_scTCR]

city: Gangnam-gu

first: Hong-Hee

last: Won

address: 81, Irwon-ro

institute: SungKyunKwan University

summary: Immunotherapy for liver cancer is used to rejuvenate tumor-infiltrating lymphocytes by modulating the immune microenvironment. Thus, early protective functions of T cell subtypes with tissue-specific residency have been studied in the tumor microenvironment (TME). We identified tumor-associated tissue-resident memory T (TA-TRM) cells in hepatocellular carcinoma (HCC) and characterized their molecular signatures. We obtained single-cell RNA and single-cell TCR sequencing data from five patients with HCC. The heterogeneous characteristics of TRM cell subsets within the TME were then investigated and validated. We characterized two TRM clusters (CD69+ and CD103+) that expressed unique signature genes and validated their similar molecular patterns in an independent dataset. Risk scores based on core gene expression in TA-TRM cells were associated with survival in both datasets. Trajectory analysis revealed that the two lineages followed different trajectory paths with distinct marker gene expression across pseudo-time. CD103+ TA-TRM cells showed diverse clonotypes and shared clonotypes with other cell groups. Lower clonal diversity and distinct signaling interactions were observed in the recurrent than in the non-recurrent samples. The CXCL13-CXCR3 interaction between CD103+ TA-TRM and regulatory T cells was observed only in the recurrent samples. We identified two subtypes of TA-TRM cells in HCC and demonstrated their unique molecular signatures, relevance to survival, and distinct signaling networks according to recurrence. The study findings provide a better understanding of the molecular characteristics of TA-TRM cells in HCC and potential immunotherapeutic strategies.

overall_design:
We generated scRNA-seq of CD45+ immune cells from tumors and adjacent livers for HCC by fluorescence-activated cell sorting (FACS).

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Submitter declares that the raw data cannot be provided due to patient privacy concerns.
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first: Mi-So

last: Park

first: Hyeonbin

last: Jo

first: Hyeree

last: Kim

first: Ji Young

first: Woong-Yang

first: Yong-Han

last: Paik

first: Yeup

last: Yoon

first: Wonseok

last: Kang


title: Enhanced RNA-targeting CRISPR-Cas technology in zebrafish V

city: Kansas City

first: Gabriel

last: da Silva Pescador

state: MO

institute: Stowers Institute for Medical Research

summary: Transient elimination of RNAs by CRISPR-Cas13 systems has been widely used in basic and applied sciences over the last years. However, the efficiency of the system can be enhanced in vivo, and its application has generated controversy due to the recently described collateral activity in mammalian cells and mouse models. Here, we have optimized the CRISPR-RfxCas13d (CasRx) system for an optimized RNA targeting in vivo in zebrafish embryos, by different and compatible approaches. These strategies include the use of chemically modified guide RNAs to increase and sustain mRNA knockdown, an improved nuclear targeting, and the evaluation of ex vivo computational models for predicting gRNA efficiency in vivo. Furthermore, our study demonstrated that transient CRISPR-RfxCas13d approaches can effectively deplete endogenous mRNAs in zebrafish embryos without inducing collateral effects, except for targeting extremely abundant RNAs. For that, we have implemented alternative RNA-targeting CRISPR-Cas systems with reduced or absent collateral activity in zebrafish embryos. Altogether, these findings contribute to optimize CRISPR-Cas technology for RNA targeting in zebrafish through transient approaches and assist the progress of potential implications for knockdown therapies in vivo.

overall_design: To investigate the toxicity effect of IVTed gRNAs compared with chemically synthesized


title: Window of opportunity trial reveals mechanisms of response and resistance to navtemadlin in patients with recurrent glioblastoma [patients]

city: Uppsala

first: Veronica

last: Rendo

address: Dag Hammarskjölds väg 20, Rudbeck Laboratory, C11:3

country: Sweden

institute: Uppsala University

summary: To investigate the effectiveness of p53 reactivation in treating glioblastoma, we conducted a surgical window-of-opportunity trial (NCT03107780) of the MDM2 inhibitor navtemadlin (KRT-232) on 21 patients and determined achievable drug concentrations within tumor tissue and biological mechanisms of response and resistance. Both 120 mg and 240 mg daily dosing achieved a pharmacodynamic impact, but patient relapse was observed. Sequencing of three recurrent tumors revealed an absence of TP53-inactivating mutations, indicating alternative mechanisms of resistance. To understand the mechanisms of response and resistance associated with navtemadlin, we conducted functional and spatial analyses of human tissue and patient-derived glioblastoma (GBM) models. Navtemadlin induced partial tumor cell death as monotherapy, but combination with temozolomide enhanced apoptosis while sparing normal bone marrow cells in vitro. We additionally report upregulation of oligodendrocyte differentiation genes as an unexplored mechanism of navtemadlin tolerance in GBM. Overall, these results indicate that clinically achievable doses of navtemadlin exert pharmacodynamic effects and suggest that combined treatment with standard-of-care DNA damaging chemotherapy may be a route to more durable survival benefits.

overall_design: To detect whether navtemadlin activates the p53 pathway in patient tumor tissue, we conducted bulk RNA sequencing (RNAseq) of formalin-fixed paraffin-embedded (FFPE) biopsies obtained at diagnosis (pre-treatment) and after 30 hours of drug exposure (on-treatment).


title: Window of opportunity trial reveals mechanisms of response and resistance to navtemadlin in patients with recurrent glioblastoma

overall_design: We performed RNAseq of two patient-derived in vitro neurosphere models (BT145 and BT286), both pre- and post-treatment with navtemadlin at an equivalent dose and time point to the clinical trial. We detected altered expression of 390 and 132 genes in BT145 and BT286, respectively, with 125 genes common to both (P < 0.0001). We used these 125 genes to construct sets of genes expected to be upregulated (n=31) and downregulated (n = 94) by treatment, and applied these gene sets to the RNAseq data from our clinical trial samples.


title: DOT1L and EZH2 cooperatively repress PRC2 targets to maintain the GC B cell identity in DLBCL [ChIP-seq]

city: Amsterdam

first: Rachele

last: Niccolai

address: Plesmanlaan 121

department: Tumor Biology and Immunology

country: Netherlands

institute: The Netherlands Cancer Institute

summary: To determine whether DOT1L promotes repression of PRC2 target genes independently of EZH2, we performed H3K27me3 ChIP-seq on untreated responder Oci-Ly7 and non-responder Oci-Ly8 cell lines, and selected the top 10% of protein-coding genes with the highest H3K27me3 read counts from transcription start site +3 kilobases for subsequent analysis.

overall_design: ChIP-Seq for the histone modification H3K27me3 in Oci-Ly7 and Oci-Ly8 cell lines.

first: Camiel

last: Gobel

first: Joleen

last: Traets


title: Patient-derived Organoids and Xenografts Uncover Therapeutic Vulnerabilities in Colorectal Signet Ring Cell Carcinomas

city: Columbus

first: Alessandro

last: La Ferlita

state: OH

department: The James Comprehensive Cancer Center

institute: The Ohio State University

summary: Purpose: Identifying therapeutic targets for Signet Ring Cell Carcinoma (SRCC) of the colon and rectum is a clinical challenge due to the lack of Patient-Derived Organoids (PDO) or Xenografts (PDX). We present a robust method to establish PDO and PDX models to address this unmet need. We demonstrate that these models identify novel therapeutic strategies targeting therapy resistance and peritoneal metastasis. Experimental Design: We derived nine PDO and PDX models from colorectal SRCC patients. Detailed histopathological characterization confirmed the fidelity of these models to the original tumors. Drug sensitivity assays were conducted in vitro and in vivo to assess therapeutic efficacy and impact on peritoneal metastasis. An RNA-seq analysis was performed to identify critical pathways contributing to therapy resistance and metastatic progression. Results: We successfully developed and characterized PDO and PDX models from nine SRCC patients. The SRCC PDO and PDX models exhibited histopathological features consistent with the original tumors, including high mucin content and eccentric nuclei. They demonstrated increased sensitivity to FOLFIRI combined with paclitaxel or vincristine, reducing peritoneal metastasis. RNA-seq analysis revealed the upregulation of autophagy genes in SRCC. Treatment with chloroquine alone resulted in decreased tumor growth and peritoneal metastasis. Conclusions: Our study establishes PDO and PDX models as robust platforms for studying SRCC and identifying potential therapeutic strategies. Combining FOLFIRI with paclitaxel/ vincristine or chloroquine alone inhibits tumor growth and prevents peritoneal metastasis, showing promise for clinical translation. These findings suggest that combining FOLFIRI with IP paclitaxel warrants further investigation in Phase I clinical trials for SRCC patients. These model systems offer a valuable tool to uncover new treatments for these aggressive and therapy-resistant tumors.

overall_design:
We derived nine PDO and PDX models from colorectal SRCC patients. Detailed histopathological characterization confirmed the fidelity of these models to the original tumors.
Drug sensitivity assays were conducted in vitro and in vivo to assess therapeutic efficacy and impact on peritoneal metastasis.
An RNA-seq analysis of the tumor , PDX and the adjacent normal was performed to identify critical pathways contributing to therapy resistance and metastatic progression.

first: Nazia

last: Chaudhary

first: Lalit

last: Sehgal

first: Sorab

last: Dalal


title: Enhanced RNA-targeting CRISPR-Cas technology in zebrafish IV

overall_design: To investigate the clearance of maternal mRNAs by different molecular programs upon primary miR-430 knockdown


title: Enhanced RNA-targeting CRISPR-Cas technology in zebrafish II


title: Molecular landscape of tumor-associated tissue-resident memory T cells in tumor microenvironment of hepatocellular carcinoma [HCC_scRNA]

first: Hong-Hee

overall_design:
We generated scRNA-seq of CD45+ immune cells from tumors and adjacent livers for HCC by fluorescence-activated cell sorting (FACS).

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Submitter declares that the raw data cannot be provided due to patient  privacy concerns.
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first: Mi-So


title: Enhanced RNA-targeting CRISPR-Cas technology in zebrafish

overall_design: Refer to individual Series


title: DOT1L and EZH2 cooperatively repress PRC2 targets to maintain the GC B cell identity in DLBCL [RNA-seq]

summary: Previous published data suggested that DOT1L indirectly represses PRC2 target genes in murine B cell model. To address whether this relationship persists in human GCB-DLBCL, by integration of transcriptomic and epigenetic analysis, we defined the candidate direct targets of PRC2. GSEA of this genes list showed that DOT1L inhibition was able to significantly derepress candidate PRC2-target genes in Oci-Ly7 and Oci-Ly8. This effect was also observed upon combination treatment of DOT1Li and EZH2i, suggesting that DOT1L indirectly regulates the repression of PRC2 target genes. Moreover, it has been established that in the absence of Dot1l or Ezh2, GC B cells cannot maintain their identity and instead differentiate into a dysfunctional plasma cell-like state. We therefore, investigated whether the combined inhibition of DOT1L and EZH2 enhances plasma cell differentiation. We first established differentiation-state specific signatures unique for NBC, CB, MBC, PB, and BMPC using the GenomicScape database. Unsupervised clustering of the GSVA scores revealed that all the cell lines treated with the combination treatment were clustered together and gained a BMPC signature at the expense of a CB-signature. This crosstalk between DOT1L and EZH2 appeared to be conserved among different GCB-DLBCL cell lines and was independent of their responsiveness to the treatments.

overall_design:
To investigate the effect of DOT1L and EZH2 inhibition on cell identity, we treated for 6 days four GCB-DLBCL cell lines with DOT1L inhibitor (EPZ-5676) and EZH2 inhibitor (GSK343) as a single agent or in combination.
We then performed an unsupervised clustering anlysis using data obtained from RNA-seq analysis of four GCB-DLBCL cell lines treated for 6 days with the above mentioned inhibitors.


title: Enhanced RNA-targeting CRISPR-Cas technology in zebrafish I

overall_design: To investigate the efficacy of ex vivo computational models predicting in vivo mRNA depletion using data from 200 gRNAs in 8 cocktails of 25 gRNAs (10 pg/embryo)


title: Enhanced RNA-targeting CRISPR-Cas technology in zebrafish III

overall_design: To investigate the number of gRNAs that can be injected together to detect highly efficient gRNAs