title: Targeting T cell plasticity by pooled single cell CRISPR-screening in preclinical models of kidney and gut inflammation - Colitis experiment

city: Hamburg

first: Malte

last: Hellmig

state: Hamburg

address: Martinistraße 52

department: III. Department of Medicine

institute: University Medical Center Hamburg-Eppendorf

summary:
Treatment of autoimmune diseases demands a shift from unspecific immunosuppression towards targeted therapies. This could be achieved by turning pro-inflammatory T helper (Th) cells into anti-inflammatory subsets. However, the molecular pathways involved in T cell plasticity and stability are not fully understood. Single cell CRISPR-screens are a powerful tool to simultaneously analyze the impact of multiple genes on cellular phenotypes. To investigate the molecules involved in T cell plasticity in disease settings, we established in vivo single cell CRISPR droplet sequencing (iCROP-seq). By applying this technique to in vivo models of inflammatory diseases in the intestine, we demonstrate that CRISPR-induced alterations in T cell polarization can be identified and ranked according to corresponding transcriptional perturbations. In particular, we targeted pro-inflammatory Th17 cells in models of immune-mediated diseases and quantified polarization biases into Th1 and regulatory T cells. These findings uncover Th17 to Th1 cell plasticity in the human kidney in the context of renal autoimmunity. iCROP-seq will facilitate the identification of therapeutic targets by highly efficient functional stratification of genes and pathways in a disease- and tissue-specific manner.
This series contains the alternate sequencing of the original sample from the iCROP-seq colitis experiment. Original processed data which was used in the analysis presented in the publication can be found at Figshare (https://doi.org/10.6084/m9.figshare.28547528.v2).

overall_design: Splenocytes were obtained from Il17a-Cre x Cas9-GFP mice and enriched for CD4+ T cells. With these cells a Th17 polarization was performed. After polarization the cells were transduced with lentiviral particles containing vectors for guide-RNAs and blue fluorescent protein (eBFP2). Transduced cells were then injected in Rag-1-deficient mice. For the disease model of experimental colitis, 14 days prior the injection of the transducted cells into  Rag-1-deficient mice, the mice were gavaged with colitogenic bacteria. Intestinal lymphozytes were then isolated and analysed using scRNAseq.

first: Leon

middle: U

last: Enk

first: Kristoffer

last: Riecken

first: Christoph

last: Kilian

first: Paul

last: Datlinger

first: Saskia

last: Jauch-Speer

first: Tobias

last: Neben

first: Zeba

last: Sultana

first: Varshi

last: Sivayoganathan

first: Alina

last: Borchers

first: Hans-Joachim

last: Paus

first: Yu

last: Zhao

first: Nariaki

last: Asad

first: Shuya

last: Liu

first: Theodora

last: Agalioti

first: Penelope

last: Pelczar

first: Thorsten

last: Wiech

last: Bock

last: Huber

first: Samuel

first: Stefan

last: Bonn

first: Nicola

last: Gagliani

first: Boris

last: Fehse

first: Ulf

last: Panzer

first: Christian

middle: F

last: Krebs


title: Sensitive dissection of a genomic regulatory landscape using bulk and targeted single-cell activation [NuCaptureC]

status: Public on Jun 01 2025

city: Berlin

first: Dubravka

last: Vucicevic

address: Hannoversche Straße 28

department: Berliner Institut for Medical Systems Biology

country: Germany

institute: Max Delbrück Center for Molecular Medicine in the Helmholtz Association

summary: Transcriptional enhancers are non-coding DNA elements that regulate gene transcription in a temporal and tissue-specific manner. Despite advances in computational and experimental methods, identifying enhancers and their target genes essential for specific biological processes remains challenging. Determining target genes for enhancers is also complex and often relies on indirect, low-resolution, and/or assumptive methodologies. To identify and functionally perturb enhancers at their endogenous sites without altering their sequence, we performed a pooled tiling CRISPR activation (CRISPRa) screen surrounding PHOX2B, a master regulator of neuronal cell fate and a key player in neuroblastoma development. This screen allowed the identification of CRISPRa- responsive elements (CaREs) that alter cellular growth within the 2 Mb genomic region. To determine CaRE target genes, we developed TESLA-seq (TargEted- SingLe- cell- Activation), which combines CRISPRa screening with targeted single-cell RNA-sequencing, and identified functional CaRE-target gene pairs. While most TESLA-revealed CaRE-gene relationships involved neuroblastoma-related regulatory elements already active in the system, we found many CaREs and target connections normally active only in other tissue types or with no previous evidence and induced out of context by CRISPRa. This highlights the power of TESLA-seq to reveal gene regulatory networks active outside of a given experimental system.

overall_design: nuCapture C was performed according to (Downes et al., 2022). Single-stranded DNA probes were obtained from IDT as an xGen Lockdown Pool and are listed in the manuscript.  Paired-end sequencing (2x150nt) was performed on a NextSeq 500/550 using a HighOutput v2 Kit for 300 cycles (Illumina #FC-420-1004, discontinued).

last: Vučićević

first: Che-Wei

last: Hsu

first: Lorena

middle: S

last: Lopez Zepeda

first: Martin

last: Burkert

first: Antje

last: Hirsekorn

first: Ilija

last: Bilić

first: Nicolai

last: Kastelić

first: Markus

last: Landthaler

first: Scott

middle: A

last: Lacadie

first: Uwe

last: Ohler


title: Sensitive dissection of a genomic regulatory landscape using bulk and targeted single-cell activation [TESLA-seq]

overall_design: TESLA-seq was performed as described in details in the manuscript. We selected 222 top-scoring significant CaREs from the bulk phenotypic screen. For each of them, we selected gRNAs with the highest fold change. These gRNAs, together with 52 control gRNAs (total of 1098 in  Table S5), were ordered as oligos from Twist Bioscience, cloned, and sequenced as described for the phenotypic screen. The lentiviral production was done as in the viability screen. SHSY-5Y-VPR line was infected at <0.3 MOI and the selection with 2ug/ul of puromycin started 24h post transduction. As soon as the antibiotic control non-infected cells were dead,on day 4 after transduction, the cells were collected. The viability of the cells was > 85% in both experiments as determined by the BD Rhapsody scanner after staining with Calcein AM (Thermo Fisher Scientific #C1430) and Draq7 (Thermo Fisher Scientific #564904) according to the manufacturer's protocol. Single-cell capture and cDNA synthesis were performed using the BD RhapsodyTM Single-Cell Analysis System according to the manufacturer's instructions. Capture probes for 146 transcripts corresponding to 78 genes in the 6MB genomic space (+/- 3MB from the PHOX2B TSS) on chr4 were designed by BD. The targets were enriched and the library was prepared according to the BD mRNA Targeted Library Preparation protocol. Paired-end sequencing (2x75nt) was performed on a NextSeq 500/550 using a HighOutput v2 Kit for 150 cycles with a 20% PhiX spike-in. Two replicates of this experiment are submited: TESLA-seq replicate_1 and TESLA-seq_replicate_2.


title: Sensitive dissection of a genomic regulatory landscape using bulk and targeted single-cell activation [bulkCRISPR-screen]

sample_organism:
Homo sapiens
synthetic construct

platform_organism:
Homo sapiens
synthetic construct

overall_design:
CRISPR activation or CRISPR inhibition screen in the 2 MB genomic space around PHOX2B was performed in SHSY-5Y cell line. Cells were  collected and various time points.
Oligo pools containing 46,722 oligos (Table S3) ordered from Twist Bioscience were cloned into the CROPseq-Guide-Puro plasmid (Addgene #86708) digested with Esp3I (Thermo Fisher Scientific #FD0454) according to (Joung et al., 2017b). The library was amplified using primers and according to the protocol from (Datlinger et al., 2017) and sequenced on a NextSeq 500/550 machine according to the Illumina user manual. Lentiviral production was done as described in (Datlinger et al., 2017). The lentiviral prep was concentrated using Lenti-X Concentrator (Takara Bio #631232) and the viral titer was determined using a Crystal violet viability assay (Vučićević et al., 2016). The screen was performed as described in (Datlinger et al., 2017). For each gRNA at least 1000 cells per gRNA were seeded and they were transduced at a low multiplicity of infection (<0.3 MOI) in either SH-SY5Y-dCas9VPR (CRISPRa)  and SH-SY5Y-dCas9KRAB (CRISPRi) cell line. The cells were selected 48h post-transduction for the ones that received the gRNA using 2ug/ml of puromycin (Sigma Aldrich #P9620) for three days. They were kept under constant antibiotic selection with 1ug/ml of puromycin and  100ug/ml of hygromycin and collected at several time points (day 5, day 20, day 29 and day 33). DNA was extracted using the Quick-DNA Midiprep Plus kit (Zymo Research #D4075). The library preparation was done as described in (Datlinger et al., 2017) and sequenced on a NextSeq 500/550 according to the Illumina protocol.


title: Human myometrial transcriptome and DNA methylome of testosterone treated patients appear more similar to myometrium from fibroid patients [array]

city: Grand Rapids

first: Jose

middle: M

last: Teixeira

state: MI

address: 400 Monroe Ave NW

department: Ob/Gyn

country: USA

institute: Michigan State University

summary: Uterine fibroids, or leiomyomas, are noncancerous tumors of the myometrium and the most common reproductive tract tumors in women, with an incidence of up to 80% by age 50. Currently, hysterectomy is the only definitive cure, and effective non-hormonal therapeutics are lacking. Understanding the etiology of fibroids may lead to alternative, less invasive treatments. Several obstetric disorders, including polycystic ovary syndrome (PCOS), have been linked to uterine fibroids, and women with PCOS often exhibit hormonal imbalances, particularly elevated serum testosterone levels. However, the impact of testosterone on the myometrium remains poorly understood. We hypothesize that elevated testosterone may increase the risk of developing uterine fibroids.   Using RNA sequencing and MethylationEPIC array analysis, we compared myometrial tissue from women without fibroids (MyoN, n=33), with fibroids (MyoF, n=66), and after testosterone therapy (MyoT, n=7). The transcriptomic and methylation profiles of MyoT clustered with MyoF and were distinct from MyoN. We identified 1,321 differentially expressed protein-coding genes between MyoT and MyoN, while only 494 were found between MyoT and MyoF. Disease ontology analysis of MyoT vs. MyoN revealed enrichment of the fibroid tumor gene set. Fibroid associated genes including TGFβ3, CCND1, SERPINE1, and FGFR1 were upregulated in MyoT and MyoF samples compared to MyoN samples. The DNA methylation profiles of MyoT were closer to those of MyoF, but no correlation was observed between methylation status and gene expression. Our preliminary data suggest that testosterone induces transcriptional and methylation changes in the myometrium consistent with those observed in MyoF tissues. These findings suggest that elevated testosterone may be associated with an increased risk of developing uterine fibroids.

overall_design: Fibroid-free myometrium (MyoN, n=33), myometrium from fibroid patient (MyoF, n=63) and myometrium from patient treated with testosterone (MyoT, n=7) were sent for Infinium Methylation Array Hybridization. C: Caucasian, AA: Black/African American, H: Hispanic, A: Asian, Multi: Multi-race

first: Emmanuel

middle: N

last: Paul

first: Tyler

middle: J

last: Carpenter

first: Andrew

last: Bossick

first: Ghassan

last: Allo

first: Ganesa

middle: R

last: Wegienka


title: Catalytic-dependent and independent functions of the histone acetyltransferase CBP promote pioneer factor-mediated zygotic genome activation [ChIP-seq]

city: Madison

first: Melissa

last: Harrison

state: Wisconsin

address: 6204B Biochemical Sciences Building, 440 Henry Mall

department: Biomolecular Chemistry

institute: University of Wisconsin- Madison

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Immediately after fertilization the genome is transcriptionally quiescent. Maternally encoded pioneer transcription factors reprogram the chromatin state and facilitate the transcription of the zygotic genome. In Drosophila, transcription is initiated by the pioneer factor Zelda. While Zelda-occupied sites are enriched with histone acetylation, a post-translational mark associated with active cis-regulatory regions, the functional relationship between Zelda and histone acetylation in zygotic genome activation remained unclear. We show that Zelda-mediated recruitment of the histone acetyltransferase CBP is essential for zygotic transcription and embryonic development. CBP catalytic activity is necessary for release of RNA Polymerase II (Pol II) into transcription elongation. However, CBP largely activates zygotic transcription independent of acetylation through Pol II recruitment. Neither acetylation nor CBP are required for the pioneering function of Zelda. Our data suggest that pioneer factor-mediated recruitment of CBP is a conserved mechanism required to activate zygotic transcription but that this role is separable from the function of pioneer factors in restructuring chromatin accessibility.

overall_design:
This data set contains data for ChIP-seq experiments in stage 5 embryos or S2 cells. ChIP-seq in embryos was done using antibodies against Zld and GFP to identify Zld and GFP-nej bound regions, respectively. ChIP-seq was also performed using an anti-H3K27ac antibody to identify regions acetylated by CBP. ChIP was performed in both wild-type and zld-RNAi genetic background to assay how binding of these factors are affected in the absence of Zld. In S2 cells, ChIP-seq for Zld was performed after induction of cells with CuSO4 through a copper inducible transgene. Cells were also treated with DMSO or a CBP catalytic inhibitor. H3K27ac ChIP-seq was also performed with the same treatments to verify inactivation of CBP after A485 treatment at Zld-bound sites.
This data set contains ChIP-seq experiments in stage 5 Drosophila melanogaster embryos. ChIP-seq in embryos was done using an antibody against Zld to identify Zld bound regions. Zld ChIP-seq was performed on GFP-CBP;nos-deGrad/+;his2AV-RFP embryos to examine Zld binding upon loss of CBP and GFP-CBP;his2AV-RFP embryos were used as a control Additionally, Zld ChIP-seq was done on embryos with a zld-RNAi genetic background to ensure zld binding is depleted in the absence of Zld.

first: Audrey

last: Marsh

first: Sergei

last: Pirogov

first: Yadwinder

last: Kaur

first: Abby

last: Ruffridge

first: Suresh

last: Sajwan

last: Gibson

first: George

last: Hunt

first: Mattias

last: Mannervik


title: Sensitive dissection of a genomic regulatory landscape using bulk and targeted single-cell activation [ATAC-seq]

first: Dubravka

last: Vucicevic

overall_design: ATAC-seq experiments were performed in the SH-SY5Y, CLB-Ga, IMR-5, Kelly and NGP neuroblastoma cell lines using 100,000 cells according to the protocol (Buenrostro et al., 2015) with the following modifications: transposition time was increased from 30 min to 1 h and the cell pellets were taken directly to the transposition reaction omitting the lysis step as described in (Karabacak Calviello et al., 2019). For all samples, 12 PCR cycles were performed, and the libraries were sequenced (2x75nt) on a NextSeq 500/550 using a HighOutput v2 Kit for 150 cycles (Illumina #FC-404-2002, discontinued).

last: Vučićević

first: Che-Wei

last: Hsu

first: Lorena

last: Lopez Zepeda

first: Martin

last: Burkert

first: Antje

last: Hirsekorn

first: Ilija

last: Bilić

first: Nicolai

last: Kastelić

first: Markus

last: Landthaler

last: Lacadie


title: Genome-Wide Mapping of RNA-Protein Associations via Sequencing

city: La Jolla

first: Sheng

last: Zhong

state: California

address: 9500 Gilman Drive, MC 0412

department: Department of Bioengineering

institute: University of California, San Diego

summary: To identify RNA-binding proteins and their associated RNA-protein associations at transcriptomic scale by sequencing

overall_design:
PRIM-seq starts with the SMART-display technology that labels tens of thousands of proteins with each protein’s own mRNA to generate a library of RNA-labeled proteins (Johnson et al. 2021). SMART-display extracts total human mRNAs from input cells, appends a linker to the 3’ ends of these mRNAs, and translates the mRNAs to yield proteins that are covalently linked with the mRNAs (mRNA-linker-protein).
The second module, termed REILIS (Reverse transcription, Incubation, Ligation, and Sequencing), enables RNA-protein association by incubating the library of labeled proteins with an RNA library. The associated protein-RNA pairs are then recorded by co-sequencing the protein’s labels and the associated RNAs through paired-end sequencing.

first: Zhijie

last: Qi

first: Shuanghong

last: Xue

first: Kara

last: Johnson


title: BTK Inhibitor-Mediated Transcriptional Changes in Neutrophils Infected with Aspergillus fumigatus

city: Boston

first: Jatin

last: Vyas

state: MA

address: 55 Fruit Street

department: Medicine

institute: Massachusetts General Hospital

summary: RNA expression of 773 host response genes following Aspergillus fumigatus infection in healthy neutrophils in the presence and absence of ibrutinib and/or TNFalpha

overall_design: In the study presented here, healthy neutrophils were pretreated with 0.3 uM ibrutinib (IBT) or DMSO for 30 min followed by 4 hour infection with B5233 strain of Aspergillus fumigatus in the presence or absence of TNFalpha.


title: Astrocytic RIPK3 exerts protective anti-inflammatory activity during viral encephalitis via induction of serpin protease inhibitors

city: Piscataway

first: Brian

last: Daniels

state: NJ

address: 604 Allison Road

department: Cell Biology and Neuroscience

institute: Rutgers, The State University of New Jersey

type: Expression profiling by high throughput sequencing

summary: Flaviviruses pose a significant threat to public health due to their ability to infect the central nervous system (CNS) and cause severe neurologic disease. Astrocytes play a crucial role in the pathogenesis of flavivirus encephalitis through their maintenance of blood-brain barrier (BBB) integrity and their modulation of immune cell recruitment and activation within the CNS. We have previously shown that receptor interacting protein kinase-3 (RIPK3) is a central coordinator of neuroinflammation during CNS viral infection, a function that occurs independently of its canonical function in inducing necroptotic cell death. To date, however, roles for necroptosis-independent RIPK3 signaling in astrocytes are poorly understood. Here, we use mouse genetic tools to induce astrocyte-specific deletion, overexpression, and chemogenetic activation of RIPK3 to demonstrate an unexpected anti-inflammatory function for astrocytic RIPK3. RIPK3 activation in astrocytes was required for host survival in multiple models of flavivirus encephalitis, where it restricted neuropathogenesis by limiting immune cell recruitment to the CNS. Transcriptomic analysis revealed that, despite inducing a traditional pro-inflammatory transcriptional program, astrocytic RIPK3 paradoxically promoted neuroprotection through the upregulation of serpins, endogenous protease inhibitors with broad immunomodulatory activity. Notably, intracerebroventricular administration of SerpinA3N in infected mice preserved BBB integrity, reduced leukocyte infiltration, and improved survival outcomes in mice lacking astrocytic RIPK3. These findings highlight a previously unappreciated role for astrocytic RIPK3 in suppressing pathologic neuroinflammation and suggests new therapeutic targets for the treatment of flavivirus encephalitis.

overall_design: We performed bulk RNA sequencing on primary astrocytes isolated from adult brains of Ripk3-2XFVfl/fl Aldh1l1 Cre+ mice and Cre− littermate controls  treated intraperitoneally with B/B Homodimerizer for 24 hours.

first: Marissa

last: Lindman

first: Irving

last: Estevez

first: Eduard

last: Marmut

first: Evan

last: DaPrano

first: Tsui-Wen

last: Chou

first: Kimberly

last: Newman

first: Colm

last: Atkins

first: Natasha

last: O'Brown

middle: P


title: Identification of PHGDH protein-assocaited RNAs and their overlap with PRIM-seq derived RNAs through RIP-seq

overall_design: To further validate PHGDH as an RBP, we carried out RNA immunoprecipitation followed by sequencing (RIP-seq) experiments targeting the PHGDH protein in HEK293T cells and generated two biological replicates. The isolation of RNA-protein complexes was conducted under native conditions utilizing either the anti-PHGDH antibody for PHGDH pulldown or the anti-IgG antibody as a control pulldown.

first: Junchen

last: Chen

first: Wenxin

last: Zhao

first: Xingzhao

last: Wen


title: Trajectory modeling of first trimester placenta highlight differences between cytotrophoblast progenitors and trophoblast stem cells [scRNA-Seq 2]

status: Public on May 31 2025

first: Robert

middle: E

last: Morey

state: CA

address: 2880 Torrey Pines Scenic Dr

department: Pathology

institute: UCSD

summary: Cytotrophoblast (CTB) are bipotent progenitor epithelial cells of the human placenta, which can differentiate into invasive extravillous trophoblast (EVT) and multinucleated syncytiotrophoblast (STB). Trophoblast stem cells (TSC) have also been shown to be bipotential. In this study, we set out to probe the transcriptional diversity of first trimester CTB and compare TSC to various subgroups of CTB.  We performed single-cell RNA sequencing on six normal placentae, four 6-week placentae, two of which were separated into basal and chorionic fractions prior to sequencing, and two 13-week placentae. We also sequenced three TSC lines, derived from 6-8 week placentae, to evaluate similarities and differences between primary CTB and TSC.  CTB clusters displayed notable distinctions based on gestational age, with 6-week placentae showing enrichment for specific CTB subtypes, further influenced by origin from the basal or chorionic plate. Differential expression analysis of CTB from basal versus chorionic plate highlighted pathways associated with proliferation, unfolded protein response, and oxidative phosphorylation. We identified trophoblast states representing initial progenitor CTB, precursor STB, precursor and mature EVT, and multiple CTB subtypes. CTB progenitors were enriched at 6-weeks, with basal plate cells biased toward EVT, and chorionic plate cells toward STB, precursors. Clustering and trajectory inference analysis indicated that TSC were most like EVT precursor cells, with only a small percentage of TSC on the pre-STB differentiation trajectory. This was confirmed by flow cytometric analysis of 6 different TSC lines, which showed uniform expression of proximal column markers ITGA2 and ITGA5. Additionally, we found that only ITGA5+ CTB could be plated in 2D, and these cells formed only EVT upon spontaneous differentiation.  Our findings suggest that distinct CTB states exist in different regions of the placenta as early as six weeks gestation and that current TSC lines most closely resemble ITGA5+ CTB, biased toward the EVT lineage.

overall_design: For whole placental samples, chorionic villi were minced and subjected to three sequential enzymatic digestions. After filtration, cells were counted and loaded on the 10x Genomics chip. For basal and chorion samples, under a microscope we collected the tips (basal fraction) and the base (chorionic fraction) of the villi while the middle section was discarded. The two fractions were subjected to the same sequential enzymatic digestion and after filtration cells were separated on a Percoll (Sigma-Aldrich) gradient.

first: Francesca

last: Soncin

first: Mana

last: Parast


title: Temporal impact of sepsis on Alzheimer's disease pathology and neuroinflammation

city: Atlanta

first: Karen

last: McFarland

state: GA

address: 100 Woodruff Circle

department: Pharmacology

institute: Emory University

summary: Epidemiological evidence has reported an associative link between sepsis survivorship and increased risk of dementia, particularly Alzheimer's disease (AD). Paradoxically, population studies show females are less susceptible to sepsis but more vulnerable to post-sepsis dementia, including Alzheimer's disease (AD). Here, we examined the temporal impacts of sepsis on AD pathology using an AD-model (TgCRND8) and wild-type mice, assessing outcomes at 7 days and 3 months post-sepsis. Following 7-days recovery, all male and female post-septic mice showed robust systemic immune activation. At this time, the female AD-model mice accumulated higher hippocampal amyloid-beta (Aβ) and upregulated AD-type transcriptomic signature. On the other hand, male AD-model mice showed no Aβ changes. Notably, wild-type post-septic males, but not females, displayed robust astrocytosis, with little change in microglial proliferation. By 3 months post-sepsis, microgliosis was specifically elevated in wild-type females, indicating a prolonged central immune response. At this time, both male and female AD-model mice showed exacerbated Aβ and anxiety. Gene network analysis revealed a stronger female immune response, while the male response was linked to estrogen receptor (ESR) signaling, with increased ERα protein observed in the brains of post-septic AD males. Together, our data highlights a sex-dimorphic temporal response in post-sepsis neuroinflammation, with ESR signaling playing a key role in males, while Aβ burden is affected similarly in both males and females.

overall_design: RNA-sequencing of male and female TgCRND8 and wild-type forebrains at 7 days and 3 months post-sepsis

first: Vo

last: Quan

first: McFarland

last: Karen N

first: Chakrabarty

last: Parmita


title: Type 2 calreticulin mutations activate ATF6 to promote BCL-xL-mediated survival in myeloproliferative neoplasms

city: Salt Lake City

first: Marco

last: Marchetti

state: Utah

department: Human Genetics

institute: University of Utah

summary: The majority of calreticulin (CALR) mutations in myeloproliferative neoplasms (MPNs) are classified as either type 1, a 52 base-pair deletion (CALRdel52), or type 2, a 5 base-pair insertion (CALRins5). Both are gain-of-function (GOF) mutations that generate an identical mutant C-terminal tail, which mediates the binding to and activation of the thrombopoietin receptor MPL. We recently reported that despite this shared GOF, CALRdel52 but not CALRins5 mutations cause loss of calcium binding function, leading to activation of and dependency on the IRE1a/XBP1 pathway of the unfolded protein response (UPR). This led us to ask whether CALRins5 mutations activate and depend on a different UPR pathway, and whether this is likewise mediated by a mutation type-specific loss-of- function (LOF). Here, we show that CALRins5 mutations lead to activation of the ATF6 pathway of the UPR due to loss of CALR chaperone function. This LOF is caused by interference of the CALRins5 mutant C-terminus with key chaperone residue H170. Further, we show that CALRins5 cells are partially dependent on ATF6 for cytokine-independent growth, and identify BCL-xL as a transcriptional target of ATF6 that promotes type 2 CALR mutant cell survival.

overall_design: RNASeq profiling of peripheral blood mononuclear cells (PBMCs) from healthy donors or patients with myeloproliferative neoplasms (MPNs) harboring type 1 CALR mutant (CALRdel52) positive, type 2 CALR mutant (CALRins5), or JAK2 V617F  mutations.

first: Nicole

last: Arellano

first: Silvia

last: Catricalà

first: Ilaria

last: Casetti

first: Oscar

last: Borsani

first: Elisa

last: Rumi

first: Daniela

last: Pietra

first: Shannon

last: Elf


title: Blood immunomap for prediction of responses to anti-PD-1 immunotherapy in metastatic non-small cell lung cancer

city: Heraklion

first: Matthieu

middle: David

last: Lavigne

address: Nikolaou Plastira 100

country: Greece

institute: IMBB-FORTH

summary: The identification of circulating predictors of response to ICB therapy is vital as very few of them meet the demands of the clinic. Herein, by using high-dimensionality mass cytometry, we designed a blood immunomap in metastatic NSCLC individuals who underwent anti-PD-1 treatment. We identified heightened frequencies of CD8+PD-L1+ T cells in non-responders compared responders. Notably, Imaging Mass Cytometry data revealed that CD8+PD-L1+ T cells were enriched in tumor biopsies, pleural infusions and BAL of early-stage NSCLC individuals, proposing this cells subset as candidate not only for the advanced but also for early disease detection. Transcriptomic analysis unveiled that CD8+PD-L1+ T cells displayed an exhausted phenotype related to their increased frequencies to non-responders to immunotherapy, and gene signatures correlated with the overall survival of an independent cohort. Overall, our study pinpoints immune-related events which may benefit the quest for detection of predictive biomarkers of immunotherapy responses.

overall_design: RNA-seq profiling of FACS-sorted human CD8+ T cells based on PD-L1 expression (positive vs. negative).

first: Maria

last: Semitekolou

first: Domenico

last: Lo Tartaro

first: Nikolaos

last: Paschalidis

first: Panayiotis

last: Verginis


title: Catalytic-dependent and independent functions of the histone acetyltransferase CBP promote pioneer factor-mediated zygotic genome activation [ATAC-seq]

first: Melissa

last: Harrison

overall_design: This data set contains ATAC-seq data from individual Drosophila melanogaster embryos precisely staged 15mins into nuclear cycle 14 during the maternal-to-zygotic transition. Single-embryo ATAC-seq was executed on GFP-CBP;his2AV-RFP and GFP-CBP;nos-deGrad/+;his2AV-RFP embryos to measure the effect CBP knockdown has on chromatin accessibility. Single embryo ATAC-seq was performed similarly on CRY2-CBP;his2avRFP and his2AV-RFP control embryos where both genotypes were subjected to either a blue light treatment precisely from NC10 to 15mins into NC14 or left in the dark. Blue light treatment inactivates CRY2-CBP histone acetyltransferase activity and therefore these experiments identify if catalytic activity of CBP is essential for chromatin accessibility. ATAC-seq in S2 cells was performed with control drug, DMSO, or a CBP catalytic inhibitor, A485, after induction of Zld from a CuSO4 inducible promoter to assay if H3K27ac is essential for chromatin accessibility at Zld pioneered regions.

first: Audrey

last: Marsh

first: Sergei

last: Pirogov

first: Abby

last: Ruffridge

first: Suresh

last: Sajwan

first: Tyler

last: Gibson

first: George

last: Hunt

first: Mattias

last: Mannervik


title: Transcriptomic changes associated to the exposure of intestinal cells to the mycotoxin Versicolorin A

city: Toulouse

first: Laura

last: Soler Vasco

state: Haute Garonne

address: 180 Chemin de Tournefeuille

country: France

institute: INRAE

summary: Versicolorin A (VerA), a precursor of the potent carcinogen aflatoxin B1 (AFB1), is an emerging mycotoxin. Recent studies have described the mutagenic and genotoxic ability of VerA, but the mechanisms of its toxicity remain to be discovered. In the present study, we investigated the early (6 h) transcriptomic changes induced by VerA in differentiated intestinal cells at non-cytotoxic conditions (1 and 3 µM) and compared its effects to those of AFB1 at 1µM. Our results showed that VerA, but not AFB1, induced significant changes in global gene expression profiles. Both toxins induced gene expression changes related to known elements of their toxicity, such as mutagenicity, genotoxicity, oxidative stress and apoptosis. However, we also observed novel features of VerA toxicity, including the ability to induce mitochondrial dysfunction, which leads to the induction of a type-1 interferon response mediated by cGAS-STING. VerA also induced changes in the expression of genes involved in the regulation of cell shape and adhesion, transcription/translation as well as genes associated with tumor biology. Our findings provide new evidence of VerA toxicity and highlight the need to further evaluate its hazards and the risk associated with its presence in food.

overall_design: The experimental design included four biological and three technical replicates for each exposure condition. Exposure conditions included 2 toxin concentrations (1 and 3 microMolar) and 2 time-points (6 and 24h). Control samples were included at each time point, corresponding to the vehicle where toxin was diluted (DMSO)

first: Carine

last: Al-Ayoubi

last: Soler-Vasco

first: Olivier

last: Puel

first: Claire

last: Naylies

first: Yannick

last: Lippi


title: Total transcriptome analysis of immune cells isolated from the high-grade glioma tumor microenvironment driven by PDGFb

city: Columbus

first: Alessandro

last: Canella

state: OH

address: 700 Children’s Drive

department: Institute for Genomic Medicine

institute: Nationwide Children's Hospital

summary: We analysed the trascriptome of tumor infiltrating immune cells in a high-grade glioma RCAS-PDGFb murine model.

overall_design: Immune cells from the TME were isolated when the animals developed high-grade glioma, and processed for Total RNA extraction and Total RNA sequencing.


title: The influence of interleukin-27 on metabolic fitness in a murine neonatal model of bacterial sepsis

city: Morgantown

first: Gangqing

last: Hu

state: West Virginia

address: 64 Medical Center Drive

department: MicroBiology, Immunology, and Cell Biology

institute: West Virginia University

summary: Human neonates are predisposed to an increased risk of mortality from infection due to fundamental differences in the framework of innate and adaptive immune responses relative to those in the adult population. As one key difference from neonates, an increase in the immunosuppressive cytokine, IL-27, is responsible for poor outcomes in a murine neonatal model of bacterial sepsis. In our model, the absence of IL-27 signaling during infection is associated with improved maintenance of body mass, increased bacterial clearance with reduced systemic inflammation, and decreased mortality rates that correlate to preservation of glucose homeostasis and insulin production. To further elucidate the mechanisms associated with IL-27 signaling and metabolic fitness, we analyzed global transcriptomes from spleen, liver, pancreas, and hindlimb muscle during Escherichia coli-induced sepsis in wild-type (WT) and IL-27Rα-deficient (KO) mice. Metabolically important tissues such as the liver, pancreas, and hindlimb muscle exhibit a shift in differential gene expression of pathways involved in oxidative phosphorylation, glycolysis, gluconeogenesis, lipid metabolism, and fatty acid beta oxidation. The hindlimb muscle of KO pups demonstrated a significant reduction in all these pathways during infection. The KO liver showed a significant down-regulation in gluconeogenesis and glycolytic pathways. Collectively, these findings suggest a positive influence of IL-27 on the metabolic profile during early life infection. This is an important consideration for antagonization of IL-27 as a potential host-directed therapeutic opportunity and our findings point to an overall improvement in infectious disease parameters and metabolic fitness.

overall_design: To further elucidate the mechanisms associated with IL-27 signaling and metabolic fitness, RNA-seq from from liver, pancreas, and hindlimb muscle during Escherichia coli-induced sepsis in wild-type (WT) and IL-27Rα-deficient (KO) mice were performed. RNA-Seq data of the same experimental design was also geranted for spleen, but deposited to GEO previously under a seperate GSE# (GSE220050; PMID 36891292).

first: Jessica

last: Povroznik

first: Lei

last: Wang

first: Madhavi

last: Annamanedi

first: Rachael

middle: L

last: Bare

first: Halima

last: Akhter

first: Cory

last: Robinson


title: Polyphosphate-Induced Changes in Transcriptome and Root-Functional Traits Elucidate Enhanced Phosphorus Acquisition Mechanisms and Growth of Durum Wheat

city: Gembloux

first: Said

last: Khourchi

address: rue de déportes

country: Belgium

institute: University of Liège

summary: Phosphate (P) fertilization impacts many rhizosphere processes, driving plant P use efficiency. However, less is known about the induced molecular and physiological root-rhizosphere traits in responses to polyphosphates (PolyP), particularly root transcriptome and belowground functional traits responsible for P acquisition. The present study aims to investigate physiological and transcriptomic belowground mechanisms explaining the enhanced durum wheat P acquisition under PolyP (PolyB and PolyC) supply. Root molecular traits were differentially expressed in response to PolyP, where PolyB induced upregulation of OGDH, MDH, and ENO, PAP21 and downregulation of PFK, and LDH genes. The modulation of gene expression can presumably explain the PolyP-induced changes in rhizosphere (root, rhizosphere soil, soil solution) acidification (pH decreased from 8 to 6.3) and acid phosphatase activities, which were concomitant with enhanced rhizosphere soil P availability and shoot Pi content (145% and 36% compared to OrthoP, respectively) along with changes in morphological and transcriptomic root (particularly, the upregulation of AUX1 and ABA transporter genes) traits. These findings provide novel insights that P acquisition from polyphosphates involves the coordinated regulation of genes governing root-rhizosphere processes and root development, ultimately enhancing wheat P acquisition.

overall_design: To investigate the root transcriptomic changes of durum wheat in response to polyphosphates application. The collected root tissues were ground in liquid nitrogen and the resulting powder was then used for RNA extraction using NucleoSpin RNA Plant kit (Macherey-Nagel, Germany). RNA sequencing and library construction were carried out at Beijing Genomics Institute (BGI), China, at a sequencing depth of 20 million paired end reads.

first: Yordan

last: Muhovski

first: Adrien

last: Blum

first: Iván

last: Jáuregui

first: Caroline

last: De Clerck

first: Adnane

last: Bargaz

first: Pierre

last: Delaplace


title: Regional centromere configuration in the fungal pathogens of the Pneumocystis genus

city: Bethesda

first: Ousmane

middle: Hamadoun

last: Cisse

state: MD

address: 10 Center Dr,

institute: National Institutes of Health

summary: Pneumocystis is a relevant genetic system to study centromere formation in relation with host adaptation. How centromeres are formed and maintained in strongly host adapted fungal pathogens is poorly investigated. Centromeres are genomic regions that coordinate accurate chromosomal segregation during mitosis and meiosis. Yet, despite their essential function, centromeres evolve rapidly across eukaryotes. CENP-A, a variant of histone H3 is the epigenetic marker that define centromeres in most eukaryotes. Centromeres are often the sites of chromosomal breaks which contribute to genome shuffling and promote speciation by inhibiting gene flow. Genome shuffling allows genome reconfiguration suitable for survival in new environment such as pathogen adaptation to new hosts. Here, we study the evolution of centromeres in closely related species of mammalian specific pathogens of the fungal phylum of Ascomycota. Long term culture of Pneumocystis species is currently untenable. Using heterologous complementation, we show that Pneumocystis CENP-A ortholog is functionally equivalent to fission yeast Cnp1. Using a short-term in vitro culture, infected animal models and ChIP-seq, we identified centromeres in three Pneumocystis species that diverged ~100 Mya ago. Each species has 17 unique short regional centromeres (< 10kb) in 17 monocentric chromosomes. The centromeres are flanked by heterochromatin. They span active genes, lack conserved DNA sequence motifs, and repeats.These features suggest an epigenetic specification of centromere function.

overall_design: Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) for the knietochore protein Mis12 in Pneumocystis macacae. P. macacae organisms were collected from a Simian immunodeficiency virus infected macaque with Pneumocystis pneumonia

middle: H

first: Jason

last: Brenchley

first: Shelly

last: Curran

first: Joseph

last: Kovacs

first: Liang

last: Ma


title: TransTag enables simple and efficient transgene mapping in zebrafish via tagmentation

status: Public on May 30 2025

city: Denton

first: Fanju

last: Meng

state: Texas

address: 1155 Union Circle #305220

department: Department of Biological Sciences

institute: University of North Texas

summary: Zebrafish has become a preeminent model for developmental biology research, largely due to the ease of transgenesis. Despite widespread usage of transgenic lines, mapping of transgene insertion sites is rare, which raises complications involving potential local chromatin influences on transgene expression, off-target effects, and issues with allelic variation. To address these short-comings, we introduce TransTag, a simple and efficient method utilizing Tn5 transposase-mediated tagmentation, for streamlined identification of Tol2-based transgene insertion sites in zebrafish. TransTag is straightforward to perform and can identify insertion sites without the need for alignment of raw sequencing data. We also provide a detailed protocol for TransTag, a step-by-step guide for data analysis, and a user-friendly Shiny app, making transgene mapping achievable at low cost for researchers without programming expertise. Altogether, TransTag emerges as a valuable tool to enhance the precision and utility of transgenesis studies by providing essential chromosome-specific information on transgene locations.

overall_design: TransTag libraries for several transgenic zebrafish strains were generated and analyzed to identify transgene insertion sites.

first: Paige

last: Schneider

first: Xiaolu

last: Wei

first: Krishan

last: Ariyasiri

first: Marnie

last: Halpern

first: Patrick

last: Murphy


title: Single-cell RNA sequencing of breast cancer patient-derived organoids

status: Public on May 29 2025

city: Zurich

first: Haithem

last: Dakhli

address: Ramistrasse 71

country: Switzerland

institute: University of Zurich

summary: Patient-derived organoids have shown great promise as models of human tumors. However, a detailed single-cell description of the heterogeneity of these models is still lacking and limits their future clinical use. Here we performed a 10X single-cell RNA sequencing experiment to study the single-cell phenotype of breast cancer patient-derived organoids from 6 donors.

overall_design: Breast cancer organoids were grown for 1-2 weeks and were then harvested, dissociated into a single-cell suspension and sequenced.

first: Nils

last: Eling

last: Bost

first: Lasse

last: Meyer

first: Bernd

last: Bodenmiller


title: VISTA immune-checkpoint blunts radiotherapy induced anti-tumor immune response

city: Palo Alto

first: Dhanya

last: Nambiar

address: 269 campus drive, CCSR building, 1230

department: Radiation Oncology

institute: Stanford University

summary: Radiotherapy (RT) is a cornerstone treatment for cancers like head and neck cancer (HNC), but it can also unintentionally activate immune-suppressive myeloid cells, which may limit its effectiveness. This study explores the role of the immune checkpoint protein VISTA, which is found at high levels on myeloid cells in both mouse and human HNC tumors. RT further increases the number of VISTA-positive myeloid cells in the tumor and bloodstream. Mice lacking VISTA responded better to RT, with their immune cells–especially macrophages and neutrophils–shifting toward anti-tumor behavior, as revealed by single-cell RNA sequencing. Importantly, combining RT with anti-VISTA antibodies significantly shrank tumors in several cancer models (HNC, breast, colorectal), more so than either treatment alone. This combination also boosted the overall immune response, improving T cell activity through reprogramming of myeloid cells. These findings suggest that blocking VISTA could make RT more effective by counteracting immune suppression in the tumor environment.

overall_design: To investigate how VISTA modulates the immune microenvironment after radiotherapy, we performed single-cell RNA sequencing on MOC2 tumors implanted in VSIR wild-type (WT) and knockout (KO) mice. Tumors were harvested two days after the last dose of radiation (3 Gy  5 fractions). Eight libraries were generated from four experimental groups (WT, KO, WT + RT, KO + RT) with two biological replicates per group. Single-cell suspensions were prepared and sequenced using the 10x Genomics Chromium Single Cell 3 v3.1 platform on an Illumina NovaSeq 6000.


title: Anti-inflammatory, but not lipid-lowering, activity of hepatocyte PPARα improves atherosclerosis in Ldlr- deficient mice

status: Public on May 28 2025

city: Lille

first: Doriane

last: Henry

state: Haut de France

address: 1 Rue du Pr. Calmette

institute: INSERM UMR 1011

summary: Atherogenic dyslipidemia and chronic inflammation appear strongly associated with residual cardiovascular disease (CVD) risk. Peroxisome proliferator-activated receptor α (PPARα) agonists, which exert both triglyceride-lowering and anti-inflammatory properties, are potential therapeutic candidates to target residual CVD risk. However, CVD outcome studies with these drugs have yielded mixed results for currently unclear reasons. Thus, we performed single-nucleus RNA sequencing (snRNA-seq) to better understand the hepatic-specific molecular actions of PPARα and their distal effects on atherosclerotic plaque development. Hepatic PPARα activation limits a pro-inflammatory phenotype in hepatocytes and reduces leukocyte recruitment. This study highlights a non-lipid, anti-inflammatory mechanism of hepatic PPARα with implications for cardiovascular disease modulation.

overall_design: Nuclei were isolated from frozen liver tissues of three biologically independent mice per group after 8 weeks of western diet supplemented or not with pemafibrate, and purified by Fluorescence-activated cell sorting (FACS). Nuclei integrity was visually verified with Trypan blue staining and  counted before snRNA-seq analysis.

first: Eric

last: Baugé

first: Kevin

last: Ory

first: Joel

middle: T

last: Haas

first: Fanny

last: Lalloyer

first: Bart

last: Staels


title: Sister Mary Joseph's Nodule in Fallopian Tube Cancer: Exploring the Metastatic Pathway

status: Public on May 26 2025

city: Minami-ku, Hiroshima-shi

first: Takuto

last: Uyama

state: Hiroshima

address: 1-2-3 Kasumi

country: Japan

institute: Hiroshima University

summary:
Three possible metastatic pathways forSister Mary Joseph's nodule (SMJN) have been postulated: hematogenous spread, lymphatic dissemination, and direct invasion. However, detailed analyses of these metastatic pathways, particularly using gene expression profiling, remain notably absent from the literature.
we investigated the metastatic patterns of SMJN by performing RNA microarray analysis on the primary tumor and each metastatic site in a case of fallopian tube cancer presenting with SMJN and inguinal lymph node metastasis.

overall_design: We collected tumor tissue samples from the primary lesion, umbilical metastasis, and inguinal lymph node metastasis for RNA microarray analysis.

first: Yusuke

last: Matoba


title: Single cell profiling of bone metastasis ecosystems from multiple cancer types reveals convergent and divergent mechanisms of bone colonization

status: Public on May 27 2025

city: Houston

first: Xiang

last: Zhang

address: One Baylor Plaza

institute: Baylor College of Medicine

summary: Bone represents congenial soil for metastatic seeds and is frequently affected by metastasis of multiple cancer types. The histological and molecular characteristics of bone metastases (BMs) are diverse but poorly understood. Herein, we performed single-cell RNA-seq on 34 BMs from 6 cancer types and identified 3 ecosystem archetypes characterized by enrichment of macrophages/osteoclasts (Mφ-OC), regulatory/exhausted T cells (Treg-Tex), and monocytes (Mono), respectively. Breast cancer BMs are mostly the Mφ-OC archetype driven by the osteolytic vicious cycle, whereas kidney cancers BMs mainly belong to the Treg-Tex archetype that lacks osteoclasts. Lung cancers BMs evenly distributed across all archetypes. Further analyses revealed parallel mechanisms of immunosuppression and bone remodeling. Elevated estrogen signaling distinguishes macrophages in the Mφ-OC subtype, which was investigated in a companion study. Together, we elucidated that divergent mechanisms toward bone colonization and that BMs of different origins can adopt the same mechanism through convergent evolution or adaptation.

overall_design:
In this study, patient samples were collected from MD Anderson Cancer Center in collaboration with Dr. Robert L Satcher. The protocols for the collection and use of human bone metastasis samples were performed following the Declaration of Helsinki and approved by the Institutional Review Boards at Baylor College of Medicine (H-49396), The University of Texas MD Anderson Cancer Center (PA15-0225), and the University of Texas Medical Branch (H-46675). All the patients have provided written informed consent on the use of their samples for research purposes when undergoing orthopedic surgery. The selection of patient samples was driven by clinical necessity, focusing on individuals requiring surgical intervention. However, there were no restrictions on patient selection beyond this clinical consideration. Our sample collection was comprehensive, encompassing both single cell sequencing and the generation of tissue sections for pathology studies as dictated by clinical need. Specifically, we acquired bone tissue regions from 34 patients, encompassing both metastasized tumors and bone tissues. These samples were predominantly sourced from long bones within the human body. Additionally, we obtained 5 healthy individual human bone marrow from Lonza (Catalog#: 1M-105). This approach allowed us to acquire a diverse range of samples tailored to the specific requirements of the patients, ultimately contributing to the comprehensiveness of our study.

UPDATE: [Sep-29-2025] The titles of a subset of Samples were updated. The former titles are included in the Sample description field. The raw and processed data associated with each GSMxxx have not changed.

first: Fengshuo

last: Liu

first: Yunfeng

last: Ding

first: Xiaoxin

last: Hao

first: Zhan

last: Xu

first: Tianhong

last: Pan

first: Robert

last: Satcher


title: snRNA sequencing on the wat tissue of cecal ligation and puncture (CLP) and Sham mice.

city: Jena

first: Zhengyuan

last: Zhou

address: Beutenbergstraße 11a

institute: Leibniz-HKI

summary: Sepsis is a life-threatening condition that often leads to multi-organ dysfunction and energy dysruption. To explore the cellular response of the mouse wat during sepsis, we employed snRNA sequencing to profile differentially expressed genes (DEGs) across various wat cell types.

overall_design: Six wat samples were collected from six male C57BL/6 mice, aged 20-24 weeks. These mice were divided into two groups: CLP (three replicates) and Sham (three replicates), each receiving the corresponding treatment. The samples were then subjected to snRNA sequencing.


title: Multi-pronged analysis of Pediatric Low-Grade Glioma

city: Aurora

first: Andrew

last: Donson

state: CO

department: Pediatrics

institute: CU Anschutz Medical Campus

summary: Multiomic profiling of low-grade glioma

overall_design: 10x genomics single cell RNA-seq and Visium spatial analyis

first: Shadi

last: Zahedi

first: Kent

last: Riemondy

first: Jean

last: Levy


title: snRNA sequencing on the liver tissue of cecal ligation and puncture (CLP) and sham mice

summary: Sepsis is a life-threatening condition that often leads to multi-organ dysfunction and energy dysruption. To explore the cellular response of the mouse liver during sepsis, we employed snRNA sequencing to profile differentially expressed genes (DEGs) across various liver cell types.

overall_design: Six liver samples were collected from six male C57BL/6 mice, aged 20-24 weeks. These mice were divided into two groups: CLP (three replicates) and Sham (three replicates), each receiving the corresponding treatment. The samples were then subjected to snRNA sequencing.


title: Babaco Mosaic Virus (BabMV) induces genome-wide transcriptomic reprogramming in Carica papaya.

city: Guayaquil

first: Maria

middle: Gabriela

last: Maridueña

state: Guayas

address: Km 30.5 via perimetral

country: Ecuador

institute: Escuela Superior Politécnica del Litoral

summary: The Babaco Mosaic Virus (BabMV) is a Potexvirus that affects babaco (Vasconcellea heilbornii) and papaya (Carica papaya) plants. To understand the impact of the virus on the plant, we performed a genome-wide transcriptome analysis of the response of young leaves of 3-4 months old papaya plants at 2, 10, 15- and 30-days post infection (dpi) in comparison to uninfected controls. Results showed that in mock-infected plants and virus-infected plants at 2 and 10dpi more than 90% of the reads mapped to the papaya genome. In contrast, at 15 and 30 dpi only 31% mapped to the papaya genome in BabMV infected leaves, while the remaining 69% of the reads aligned to the virus genome, demonstrating a high viral load. A total of 1585 papaya genes were differentially expressed between mock and BabMV inoculated leaves, among which cell wall biogenesis, redox signaling, and ribosome Gene Ontology terms were overrepresented, interestingly, the virus induced specific responses at different time points. Sugar metabolism and cell wall modification including lignin accumulation genes were affected at 15 dpi.  At 30 days post-infection, the virus primarily induced ROS production, possibly through the activation of cytochrome P450 enzymes. This led to the upregulation of genes associated with ROS scavenging and of transcription factors (TFs) belonging to the WRKY and AP2/ERF families, which play crucial roles in activating the plant's defense mechanisms against viral pathogens. This research enhances our understanding of BabMV infections, facilitating the development of effective disease control measures.

overall_design: The experiment was done using a randomized block designed (RBD), where each biological replicate consisted of three biological units. Leave samples were collected at  2, 10, 15, and 30 dpi from each of three biological replicates of each group i.e. virus-infected or mock-inoculated plants and sequencing

middle: G

last: Mariduena

first: Hamada

last: AbdElgawad

first: Mohammad

middle: K

last: Okla

first: Juan

last: Cevallos

first: Gerrit

last: Beemster

first: Carlos

last: Noceda


title: snRNA sequencing on the brain tissue of cecal ligation and puncture (CLP) and Sham mice.

summary: Sepsis is a life-threatening condition that often leads to multi-organ dysfunction and energy dysruption. To explore the cellular response of the mouse brain during sepsis, we employed snRNA sequencing to profile differentially expressed genes (DEGs) across various brain cell types.

overall_design: Six brain samples were collected from six male C57BL/6 mice, aged 20-24 weeks. These mice were divided into two groups: CLP (three replicates) and Sham (three replicates), each receiving the corresponding treatment. The samples were then subjected to snRNA sequencing.


title: mRNA data DU145 cells sensitive or not to irradiation and transfected or not with miRNA mimics

city: Nantes

first: Delphine

last: Fradin

address: 8 quai Moncousu

institute: CRCI2NA, Inserm UMR1307, CNRS UMR6075

summary: We evaluated the transcriptome of DU145 cells sensitive to irradiation (RS) or not (RR) and the impact of two miRNAs on their transcriptome.

overall_design: We used this sequencing technology to identify mRNA differentially expressed by DU145 cells

first: Maureen

last: Labbé


title: Using plasma cell-free mRNA to profile immune response and myocardial damage in immune checkpoint inhibitor-induced myocarditis

status: Public on May 24 2025

city: Stanford

first: Alireza

last: Raissadati

address: 269 Campus Drive

department: Cardiovascular Institute

institute: Stanford

summary: Immune checkpoint inhibitor-induced myocarditis (ICI-m) is a highly fatal cancer treatment complication with challenging diagnosis and invasive diagnostic methods. We developed a plasma cell-free mRNA (cf-mRNA) platform for noninvasive characterization of the immune and myocardial landscape in ICI-m. We sequenced bulk cell-free total RNA from plasma of cancer patients treated with ICIs: without immune-related adverse events (irAE) (group A, n=5), with noncardiac irAE (group B, n=7), and with ICI-m with/without noncardiac irAE (group C, n=10). Publicly available healthy control cfRNA (n=30) served as external controls. Cell type-specific gene panels from single cell RNA-sequencing of circulating immune and cardiac cells were used for cellular deconvolution. Cancer and inflammatory pathways were enriched in ICI-treated patients versus controls. Cardiac pathways and conduction system genes were significantly enriched in group C versus A and B. Temra CD8+ T cell and cardiomyocyte signature scores, with CCL5 and MYH6 expression, were elevated in group C. Our custom ICI-m classifier, containing three cardiac- and three immune cell-specific genes, effectively differentiated group C from A (AUC 0.903, 95% CI 0.858-0.903) and B patients (AUC 0.983, 95% CI 0.970-0.983), outperforming unsupervised feature selection. outperforming unsupervised feature selection. This platform captures disease-specific signatures enabling precision diagnostics for inflammatory diseases.

overall_design: Paired-end RNAseq of plasma circulating bulk cell free RNA in ICI-treated cancer patients without irAE, with extracardiac irAE only, and with ICI-m with or without extracardiac irAE.

first: Han

last: Zhu

first: Sean

last: Wu


title: Tracing the evolution of single-cell 3D genomes in Kras-driven cancers [snRNA-seq]

city: West Haven

first: S

last: Agabiti

address: 840 West Campus Drive

department: Genetics

institute: Yale University

summary: Although three-dimensional (3D) genome structures are altered in cancer cells, little is known about how these changes evolve and diversify during cancer progression. Leveraging genome-wide chromatin tracing to visualize 3D genome folding directly in tissues, we generated 3D genome cancer atlases of oncogenic Kras-driven murine lung and pancreatic adenocarcinoma. Our data reveal stereotypical, non-monotonic, and stage-specific alterations in 3D genome folding compaction, heterogeneity, and compartmentalization as cancers progress from normal to preinvasive and ultimately to invasive tumors, discovering a potential structural bottleneck in early tumor progression. Remarkably, 3D genome architectures distinguish morphologic cancer states in single cells, despite considerable cell-to-cell heterogeneity. Gene-level analyses of evolutionary changes in 3D genome compartmentalization not only showed that compartment-associated genes are more homogeneously regulated but also elucidated prognostic and dependency genes in lung adenocarcinoma and a previously unappreciated role for polycomb-group protein Rnf2 in 3D genome regulation. Our results demonstrate the utility of mapping the single-cell cancer 3D genome in tissues and illuminate its potential to identify new diagnostic, prognostic, and therapeutic biomarkers in cancer.

overall_design: We have employed a single nucleus sequencing approach in a mouse lung cancer model to explore transcriptional differences between adenoma and adenocarcima cells in vivo.

first: Miao

last: Liu

first: Shengyan

last: Jin

first: Sherry

first: Tyler

middle: B

last: Jensen

first: Tianqi

last: Yang

first: Jonathan

last: Radda

first: Christian

middle: F

last: Ruiz

first: Gabriel

last: Baldissera

first: Moein

last: Rajaei

first: Fang-Yong

last: Li

first: Jeffrey

last: Townsend

first: Mandar

middle: D

last: Muzumdar

first: Siyuan

last: Wang


title: The effect of knockdown FAM111B on mRNA expression in human hepatocellular carcinoma cells

status: Public on May 23 2025

city: Jinan

first: Yu-Chuan

last: Yan

state: Shandong

address: 107 West Wen Hua Road

country: China

institute: Qilu Hospital of Shandong University

summary: Family with sequence similarity 111 member B (FAM111B) encodes a protein with a trypsin-like cysteine/serine peptidase domain, the aberrant expression of which has been observed to contribute to malignant behavior and poor prognosis of various cancers, such as hepatocellular carcinoma (HCC), thyroid cancer, esophageal cancer, ovarian cancer, and lung adenocarcinoma. Previous studies described the role of FAM111B as a prognostic predictor or a potential protease that may degrade certain tumor suppressor. Our data demonstrated regulatory changes at the genomic transcriptional level after interfering FAM111B expression in HCC.

overall_design: RNA-seq profiling of normal Huh7 and Hep3B cells, and their FAM111B knockdown derivatives (Huh7-shFAM111B and Hep3B-shFAM111B).


title: Reelin-LRP8 signaling mediates brain metastasis of breast cancer cells via abluminal migration

status: Public on May 22 2025

city: Zhan Jiang

first: Xiaoyu

state: Guangdong

address: 57 People's Avenue

institute: Affiliated Hospital of Guangdong Medical University

summary: Brain metastasis (BM) remains a significant challenge in breast cancer (BC) management. While conventional metastatic routes involve hematogenous dissemination, emerging evidence suggests that BC cells can also migrate along the abluminal surface of blood vessels, bypassing the blood-brain barrier (BBB). To investigate this phenomenon, we established a zebrafish xenograft model utilizing GFP-labeled MDA-MB-231 cells, allowing real-time observation of BC cell migration along the posterior cerebral veins. Our findings revealed that LRP8, a apolipoprotein E receptor, is upregulated in BC patients with brain metastases. Functional studies demonstrated that LRP8 knockdown significantly inhibited proliferation, migration, and invasion of triple-negative breast cancer (TNBC) cells both in vitro and in vivo. Mechanistically, LRP8 promotes the activation of CDC42, enhancing filopodia formation and cell motility, a process influenced by the neuronal extracellular matrix protein, Reelin. Furthermore, we demonstrate the therapeutic potential of MEN 10207, a neurokinin-2 receptor antagonist, in inhibiting TNBC cell migration and suppressing BM formation in both zebrafish and mouse models. These findings provide novel insights into the mechanisms underlying extravascular brain metastasis in BC, highlighting the Reelin-LRP8-CDC42 axis as a potential therapeutic target for this devastating complication.

overall_design: Following the transplantation of MDA-MB-231 (BM0) cells into zebrafish, tumor progression was assessed through daily fluorescence imaging. Brain metastatic lesions were subsequently examined, excised and minced using the M-shot fluorescence stereomicroscope (Guangzhou, China) under sterile conditions. Then the GFP-labeled tumor cells were isolated and transferred into DMEM supplemented with antibiotic-antimycotic solution (Beyotime, C0224) and 15% fetal bovine serum, denoting this cell line as first-generation of brain metastatic (BM1) cells. Following dissociation and subsequent expansion in culture, BM1 underwent additional five rounds of transplantation and isolation, denoting this cell line as BM6 cells. RNA from MDA-MB-231 cells transfected with sh-ctrl and sh-LRP8-2# plasmids, BM0 cells and BM6 cells were isolated using TRIZOL. RNA-Seq was performed in the Gene Denovo Biotechnology (Guangzhou, China). Differentially expressed genes (DEGs) were detected using DESeq2. Reactome enrichment analysis and GO enrichment analysis were performed to elucidate the functional implications of DEGs. DEGs with a false discovery rate (FDR) of less than 0.05 were selected for enrichment analysis. The protein-protein interaction (PPI) network involving CDC42 and various guanine nucleotide exchange factors (GEFs) was constructed using the STRING database (https://cn.string-db.org/).


title: Lysophosphatidic acid (LPA)-dependent propagation of neuroinflammation in an optimized model of post-hemorrhagic hydrocephalus.

city: La Jolla

first: Jerold

last: Chun

address: 1090 N. Torrey Pines Rd

institute: Sanford Burnham Prebys Medical Discovery Institute

summary: Post-hemorrhagic hydrocephalus (PHH) is a neurological disease that primarily affects premature infants and involves infiltration of blood into the brain’s ventricles followed by excessive accumulation of cerebrospinal fluid (CSF) leading to ventricular enlargement and increased intracranial pressure. However, the precise mechanisms driving PHH development and persistence remain incompletely known and lack medical and disease modifying treatments. Here we use a mouse model of PHH to identify transcriptomic, proteomic and cellular changes involving neurovascular and neuroimmunological microglial alterations as features of PHH, overlapping with those reported in human disease.  Improvements on a lysophosphatidic acid (LPA)-initiated PHH mouse model were developed and combined with unbiased proteomic and single-nucleus transcriptomics that identified microglial molecular pathways promoting PHH.  Pharmacological disruption of microglia in vivo significantly reduced PHH-associated ventriculomegaly.  These data identify microglia and neurovascular molecular elements in the development of PHH, implicating them as potentially tractable therapeutic targets towards developing new treatments for PHH.

overall_design: NeuN- nuclei were isolated from whole brains from mice treated with vehicle or LPA injection. 10x Genomics Single Cell 3’ v3 kit was then used to prepare samples targeting 10,000 profiled cells

first: Paloma

last: Sánchez-Pavón

first: Carter

last: Palmer

first: Christine

middle: S

first: Valerie

last: Tan

first: Victoria

middle: A

last: Blaho


title: A Dual-Filter Strategy Integrating CRISPR-based Target Screening and Text Mining for Hand-Foot Syndrome

status: Public on May 25 2025

city: Shanghai

first: Qiqi

address: dongchuan road

department: School of Biomedical Engineering

institute: Shanghai Jiao Tong University

summary: The experimental high-throughput screening (HTS) methods, exemplified by CRISPR-based screening, have revolutionized target identification in drug discovery. However, such screens frequently yield extensive and unrelated target lists necessitating costly and time-intensive experimental validation. Here, we propose a dual-filter strategy that integrates literature-mined targets with CRISPR/Cas9 screening outputs, systematically prioritizing the most credible candidates and thereby reducing the experimental validation burden and increasing success rate. To validate this strategy, we applied it with hand-foot syndrome (HFS), a clinically challenging side effect induced by fluoropyrimidine treatment. We identified ATF4 as a key regulator of 5-fluorouracil (5-FU) toxicity in the skin and revealed forskolin as a potential therapeutic agent of HFS through the strategy. Mechanistically, forskolin triggers MEK/ERK-dependent ATF4 induction, subsequently driving 5-FU detoxification via the ATF4-mediated eIF2α/IκB signaling pathway. Our findings demonstrate that this dual-filter strategy could notably accelerate drug discovery by reducing experimental validation burden after target screening.

overall_design: RNA-seq profiling of sh-Control HaCaT cells, ATF4 knockdown HaCaT cells, over-Control HaCaT cells, and ATF4 overexpression HaCaT cells


title: 2-hydroxyglutarate mediates whitening of brown adipocytes coupled to nuclear softening upon mitochondrial dysfunction II

city: Cologne

first: Ulrike

last: Göbel

state: NRW

address: Joseph Stelzmann Strasse, 26

department: Bioinformatics Core Facility

institute: CECAD Research Center

summary: This study reports accumulation of larger lipid droplets in an adipocyte specific manner upon loss of CLPP. The mitochondria in KO BAT have mild respiratory chain deficiency but no difference in respiratory output at tissue and cellular level. Omics analysis reveals that loss of CLPP leads to tissue remodeling, and is accompanied by pertinent cytoskeletal changes. The metabolite, 2-hydroxyglutarate accumulates in KO tissue and cells and is possibly produced by the moonlighting function of PHGDH. We further identify 2-HG H3K4me3 methylation changes in epigenome being likely contributing to larger lipid droplets. The study also reports that 2HG mediated whitning is coupled to nuclear softening and changes in nuclear mechanics.

overall_design: The experiment involved extraction of RNA from treated and untreated mBA cells. Preadipocytes were isolated from WT and CLPP KO animals and immortalized. This was followed by in vitro differentiation into mature adipocytes. The WT cells were either treated with the cell permeable form of the metabolite, octyl 2-hydroxyglutarate and KO cells were treated with the inhibitor of PHGDH, NCT503; and the results compared to their respective controls.

first: Harshita

last: Kaul

first: Aleksandra

last: Trifunovic


title: 2-hydroxyglutarate mediates whitening of brown adipocytes coupled to nuclear softening upon mitochondrial dysfunction

overall_design: The experiment involved RNA sequencing for BAT (brown adipose tissue) tissue of different genotypes for mice. In order to assess global changes in BAT of mice upon CLPP loss, we  namely, WT , full body KO, adipose specific AKO and brown adipose specific BKO mice. This experiment was aimed at determining the BAT -autonomous and non - atonomous changes upon CLPP loss mediated mitochondrial dysfunction.

first: Joo

last: Park

first: Jan-Wilhelm

last: Kornfeld


title: Epithelial fusion is mediated by a partial epithelial-mesenchymal transition

status: Public on May 21 2025

city: Bengaluru

first: Varsha

middle: N

last: Tamilkumar

state: Karnataka

address: Bellary road

department: Genetics and Development

country: India

institute: NCBS

summary: Epithelial fusion is a fundamental morphogenetic process critical for the closure and compartmentalisation of developing organs. While widely studied in systems such as neural tube and palatal closure, the cellular transitions that enable fusion remain poorly understood. Here, we investigate epithelial fusion during chick otic vesicle (OV) closure and identify a transient population of cells at the epithelial interface that mediate this process. These otic edge (OE) cells exhibit distinct morphology, reduced apicobasal polarity, and dynamic junctional remodelling, including altered distribution of ZO-1, CDH1, and RAC1. Notably, OE cells lack basal contact and display high sphericity, consistent with a partial epithelial-to-mesenchymal transition (EMT) phenotype. Transcriptomic profiling of microdissected tissues reveals that OE cells constitute a transcriptionally distinct population, enriched for EMT regulators, ECM remodelling genes, and WNT pathway components. Among these, the transcription factors Grhl2 and Sp8 were specifically expressed at the OE and exhibited opposing roles in epithelial identity. CRISPR-Cas9 mediated knockdown of either gene led to disrupted CDH1 localisation, loss of OE cell morphology, and failure in epithelial segregation. These results suggest that epithelial fusion requires a regulated, hybrid EMT state that balances junctional plasticity with tissue cohesion. Our findings demonstrate that fusion-competent epithelial cells are not merely passive participants but actively modulate their shape, polarity, adhesion, and genetic identity to enable morphogenesis.

overall_design: Transcriptome signature of cells involved in epithelial fusion in the otic vesicle: The opening of the otic vesicle (the otic edge) constitutes the border between the otic cells and the surface ectoderm. These cells undergo epithelial fusion to close the otic vesicle. RNA-seq was done on cells from the otic edge, the otic vesicle without the edge, and the surface ectoderm caudal to the otic vesicle. Differential gene expression analysis between the otic edge and otic vesicle and otic edge and the surface ectoderm will reveal genes enriched in the otic edge cells, that may have a role to play in epithelial fusion.

first: Harsha

last: Purushothama

first: Raj

last: Ladher


title: STN1 facilitates metastasis by promoting transcription of EMT-activator ZEB1 in pancreatic cancer

status: Public on May 18 2025

city: Beijing

first: Chengqi

last: Yi

address: Yiheyuan Road

department: School of Life Sciences

institute: Peking University

summary: As pancreatic ductal adenocarcinoma (PDAC) poses a formidable clinical challenge, demanding further exploration of its pathogenesis and drugs targeted for metastasis-the main cause of mortality. Here, we identified STN1, a CST complex member crucial for maintaining telomere lengths and genome stability, as a key factor in promoting PDAC metastasis. Elevated STN1 levels correlate with poor patient survival, with oncogenic protein HOXB7 as an upstream transcription factor regulating STN1. Utilizing multiple PDAC experimental models, we discovered STN1’s role in promoting metastasis by acting as a novel upstream factor in epithelial-mesenchymal transition (EMT). Mechanistically, our proposed model suggests that during transcription, STN1 binds to displaced single-stranded DNA adjacent to the R-loop, recruiting STAT3 to activate ZEB1 transcription independent of its known telomere maintenance function. Notably, STAT3 inhibitors show enhanced efficiency in restraining metastatic potential in STN1-overexpressed PDAC cells, offering promise as a therapeutic avenue for targeting metastasis in STN1-overexpressed PDAC patients facing an unfavorable prognosis.

overall_design: CUT&Tag for STN1 and STAT3 in MIA PaCa-2 cell lines. STN1 and STAT3 were over-expressed respectively and anti-Flag antiboty was used.

first: Zhe

last: Zhou

first: Di

last: Dong


title: MIRO2-mediated mitochondrial transfer from cancer cells induces cancer-associated fibroblast differentiation

status: Public on May 19 2025

city: Zurich

first: Sabine

last: Werner

address: Otto-Stern-Weg 7

department: Department of Biology

institute: ETHZ

summary: Cancer-associated fibroblasts (CAFs) are key components of the tumor microenvironment that commonly support cancer development and progression. Here we show that different cancer cells transfer mitochondria to fibroblasts in co-cultures and xenograft tumors, thereby inducing pro-tumorigenic CAF features. Transplantation of functional mitochondria from cancer cells induces metabolic alterations in fibroblasts, expression of CAF markers, and release of a pro-tumorigenic secretome and matrisome. These features promote tumor formation in pre-clinical mouse models. Mechanistically, the mitochondrial transfer requires the mitochondrial trafficking protein MIRO2. Its depletion in cancer cells suppresses mitochondrial transfer and inhibits CAF differentiation and tumor growth. The clinical relevance of these findings is reflected by the overexpression of MIRO2 in tumor cells at the leading edge of epithelial skin cancers. These results identify mitochondrial transfer from cancer cells to fibroblasts as a driver of tumorigenesis and provide a rationale for targeting MIRO2 and mitochondrial transfer in different malignancies.

overall_design: To assess the functional relevance of the mitochondrial transfer, we sorted viable primary human dermal fibroblasts with high and low MitoTracker Green fluorescence intensity from co-cultures with A431 cancer cells and analyzed the two populations by RNA sequencing (RNA-seq). Control fibroblasts subjected to the sorting procedure, but not maintained in co-cultures, were included for comparison. Three replicates are included in each condition.

first: Michael

last: Cangkrama

first: Huan

last: Liu


title: snRNA sequencing on the kidney tissue of cecal ligation and puncture (CLP) and Sham mice.

first: Zhengyuan

summary: Sepsis is a life-threatening condition that often leads to multi-organ dysfunction and energy dysruption. To explore the cellular response of the mouse kidney during sepsis, we employed snRNA sequencing to profile differentially expressed genes (DEGs) across various kidney cell types.

overall_design: Six kidney samples were collected from six male C57BL/6 mice, aged 20-24 weeks. These mice were divided into two groups: CLP (three replicates) and Sham (three replicates), each receiving the corresponding treatment. The samples were then subjected to snRNA sequencing.


title: Defining Cellular Diversity at the Swine Maternal-Fetal Interface Using Spatial Transcriptomics and Organoids [MFI017_Visium]

status: Public on May 13 2025

city: Durham

first: Cole

last: McCutcheon

state: North Carolina

address: 3 Genome Court

department: Department of Integrative Immunobiology

institute: Duke University

summary:
In this study we had two primary aims: 1.) spatially define the transcriptional signatures of porcine maternal-fetal interface and 2.) develop and validate an organoid model which better recapitulated the porcine placenta. Using mid-gestation maternal-fetal interfaces of commercial landrace/large white composite gilts we performed spatial transcriptomics (n=4 interfaces) using Visium v1 spatial transcriptomics. We then went on to isolate trophoblast organoids from fresh-term placentas from crossbred sows consisting

655 of Yorkshire, Large White, and Landrace breeds. We then characterized the transcriptional profile of these organoids using bulk RNA-seq from 3 seperate lines using a standard Illumina library preparation. To characterize these organoids we performed single cell RNA-sequencing on 3 separate lines of swine trophoblast organoids using a standard 10x Genomics Single Cell 3' Gene Expression platform. All reads/samples were mapped to Sus scrofa v11.1.

overall_design:
Visium Spatial Transcriptomics was performed on porcine maternal-fetal interfaces. Spaceranger v2.1.1 was utilized for analysis
sTO were grown to high density and total RNA was collected and subjected to bulk-RNAseq.
sTO were grown to high density, digested into a single cell suspension, and subjected to single cell rna-sequencing via 10X genomics.

first: Carolyn

last: Coyne


title: Beneficial Effects of High Intensity Interval Training in a Mouse Model of Hypertrophic Cardiomyopathy

status: Public on May 20 2025

city: Philadelphia

first: Sharlene

middle: M

last: Day

state: PA

address: 3400 Civic Center Blvd

department: Medicine

institute: University of Pennsylvania

summary: High intensity exercise (HIE) has become a prominent training modality used to ameliorate cardiac disease conditions. The effects of HIE training on Hypertrophic Cardiomyopathy (HCM) have not been previously studied in animal models or in patients, largely due to concerns about triggering sudden arrhythmic events. Employing a transgenic cardiac troponin (cTnT-160E or TG) preclinical murine model that closely recapitulates human HCM, we evaluated the effects of a High Intensity Interval Training (HIIT) protocol on cardiorespiratory capacity (i.e., peak oxygen consumption or pVO2), which is a significant prognostic indicator of disease trajectory. 14 month old TG male mice had lower peak VO2 than non-transgenic (NTG) male mice of the same age at baseline, but there was no difference in pVO2 in female TG compared to nTG mice. HIIT significantly improved pVO2 in both nTG female (18%) , nTG male (18%), TG female (16%) and TG male (12%) mice compared to sedentary controls. There were no changes in structure, function or LV mass as measured by echocardiography or post mortem analyses in any of the groups, and no effect of HIIT on cardiac fibrosis. HIIT led to significant increases in lean muscle mass in both nTG and TG male mice, and nTG female mice but not TG female mice. The results of our study provide foundational evidence that HIE can improve pVO2 in HCM without adverse effects on disease phenotype.

overall_design: Total bulk RNA-Seq profiles of C57BL/6J mice after 6 weeks of HIIT vs sedentary behavior in male and female nTG and TG (cTnT-delta160E ) mice. There were 8 groups total: sedentary nTG female mice, HIIT nTG female mice, sedentary TG female mice, HIIT TG female mice, sedentary nTG male mice, HIIT nTG male mice, sedentary TG male mice, HIIT TG male mice. RNA was extracted from 7-9 samples per group for 64 samples total.

first: Jonathan

middle: J

last: Herrera

first: Vasia

last: Barka

last: Morley

first: Yifan

last: Yang

first: Danielle

last: Szczesniak

first: Kate

first: Rose-Carmel

last: Goddard

first: Christoper

last: McAllister

first: Jaime

last: Yob

first: Graham

last: Branscom


title: Protein-primed DNA homopolymer synthesis by an antiviral reverse transcriptase [RNA-seq]

status: Public on May 09 2025

city: New York

first: Samuel

last: Sternberg

address: 701 W. 168th Street, HHSC 726

institute: Columbia University

summary: Bacteria defend themselves from viral predation using diverse immune systems, many of which sense and target foreign DNA for degradation. Defense-associated reverse transcriptase (DRT) systems provide an intriguing counterpoint to this strategy by leveraging DNA synthesis instead. We and others recently showed that DRT2 systems use an RNA template to assemble a de novo gene, leading to expression of an antiviral effector protein, Neo. It remains unknown whether similar mechanisms of defense are employed by other related DRT families. Focusing on DRT9, here we uncover an unprecedented mechanism of DNA homopolymer synthesis, in which viral infection triggers polydeoxyadenylate (poly-dA) accumulation in the cell to drive abortive infection and population-level immunity. Cryo-EM structures reveal how a conserved noncoding RNA serves as both a structural scaffold and reverse transcription template to direct hexameric complex assembly and RNA-templated poly-dA synthesis. Remarkably, biochemical and functional experiments identify conserved tyrosine residues within the reverse transcriptase itself that prime DNA synthesis, leading to the formation of high-molecular weight protein-DNA covalent adducts. Synthesis of poly-dA in vivo is regulated by the competing activities of phage-encoded triggers and host-encoded silencers of DRT9. Collectively, our work unveils a novel nucleic acid-driven defense system that expands the paradigm of bacterial immunity and broadens the known functions of reverse transcriptases.

sample_organism: Escherichia coli str. K-12 substr. MG1655

overall_design: We performed total RNA-seq in E. coli cells expressing SenDRT9 infected with either WT or escape mutant T5 phage to assess phage transcript levels.

first: Stephen

last: Tang

first: Rimantė

last: Žedaveinytė

first: Nathaniel

last: Burman

first: Shishir

last: Pandey

first: Josephine

last: Ramirez

first: Louie

last: Kulber

first: Tanner

last: Wiegand

first: Royce

last: Wilkinson

first: Yanzhe

last: Ma

first: Dennis

last: Zhang

first: George

last: Lampe

first: Mirela

last: Berisa

first: Marko

last: Jovanovic

first: Blake

last: Wiedenheft


title: Erosion of X-Chromosome Inactivation in female hiPSCs is heterogeneous and persists during differentiation (RNA-Seq)

city: Lisbon

first: Paulo

last: Caldas

state: Porto Salvo

address: Campus Taguspark

department: SCERG

country: Portugal

institute: Instituto Superior Tecnico

summary: During culture, female human pluripotent stem cells (hPSCs), including human induced pluripotent stem cells (hiPSCs), exhibit a propensity for erosion of X-chromosome inactivation (XCI). This phenomenon is characterized by the loss of XIST RNA expression and reactivation of a subset of X-linked genes from the inactive X chromosome (Xi). Despite its common occurrence, XCI erosion is often overlooked by the stem cell community, hindering a complete understanding of its impact on both fundamental and translational applications of hiPSCs. Our study investigates erosion dynamics in female hiPSCs and reveals that XCI erosion is a frequent yet heterogeneous phenomenon, resulting in the reactivation of several X-linked genes. The likelihood of a gene to erode increases for those located on the short arm of the X chromosome and within H3K27me3-enriched domains. Paradoxically, genes that typically escape XCI are hypersensitive to the loss of XIST RNA and XCI erosion. This implies that XIST RNA normally restrains expression levels of these genes on the Xi. Importantly, increased X-linked gene expression upon erosion does not globally impact (hydroxy)methylation levels in hiPSCs or at imprinted regions. By exploring diverse differentiation paradigms, such as trilineage commitment and cardiac differentiation, our study reveals the persistence of abnormal XCI patterns throughout differentiation. This finding has significant implications for fundamental research, translational applications, and clinical use of stem cells. We underscore the importance of raising awareness within the stem cell community regarding XCI erosion and advocate for its inclusion in comprehensive hiPSC quality control.

overall_design: RNA-seq analysis was conducted on biological triplicates of a diverse range of hiPSC lines representing various erosion states of the X-chromosome inactivation. Cell lines exhibiting XIST expression (XIST+) consist of ASD and F7cl15, and cell lines without XIST expression (XIST-) comprise F002, F7cl4, CD, and CE. RNA-seq was also performed before (day0- D0) and after ectodermal differentiation (day7- D7) for one replicate of the isogenic pair: F7cl4 (XIST-) and F7cl15 (XIST+).

first: Ana

middle: C

last: Raposo

first: Maria

last: Arez

first: Joana

last: Jeremias

first: Simão

last: da Rocha


title: Distinct cellular and spatial niches within the inflamed synovium of childhood arthritis  [scRNA-Seq]

status: Public on May 11 2025

city: Birmingham

first: Adam

last: Croft

state: Please Select

address: Birmingham

country: United Kingdom

institute: University of Birmingham

summary: recision use of targeted therapies is urgently needed to improve long-term clinical outcomes for children affected by inflammatory arthritis, known as Juvenile Idiopathic Arthritis. Progress has been obstructed by a lack of understanding of the cellular basis of joint inflammation in children, given the difficulties in obtaining and studying synovial tissue itself. To this end, we combine single-cell RNA-sequencing, multiplexed immunofluorescence imaging and spatial transcriptomics to define the cellular and transcriptomic landscape of the synovium in children with Juvenile Idiopathic Arthritis. We identify spatial niches of resident and infiltrating cell populations that correlate with the degree of inflammation, and gene programs associated with arthritis severity. Combined with analyses of synovial fluid and peripheral blood from the same children, we distinguish differences in cellular composition, signalling pathways and transcriptional programs across anatomical compartments. Whilst we identify several pathogenic populations shared with adult-onset arthritis, our analyses highlight increased vascularity of the inflamed developing joint and TGFb-driven stromal subsets that upregulate expression of disease risk-associated genes. Overall, these findings illustrate the need for treatment algorithms informed by a tissue-based classification of arthritis.

overall_design:
To determine the molecular and cellular architecture of the inflamed joint in JIA, nineteen children underwent minimally invasive ultrasound-guided biopsies of the synovium, early in the course of their disease.  Matched peripheral blood and synovial fluid samples were also collected.  All participants were naïve to disease-modifying anti-rheumatic drugs (DMARDs) for the treatment of their arthritis. Participants with four ILAR subtypes of JIA were included, of whom 90% had oligoarticular or Rheumatoid Factor-negative JIA. Biopsy tissue samples were disaggregated and underwent 5’ single-cell RNA-sequencing (scRNA-seq), combined with parallel histopathological assessment of formalin-fixed paraffin-embedded sections, or analysed with spatial profiling technologies (transcriptomics, multiplexed imaging). scRNA-seq was also performed on matched samples of peripheral blood mononuclear cells (PBMC) and synovial fluid mononuclear cells (SFMC), with paired detection of 137 expressed surface proteins.

UPDATE: [Sep-28-2025] The GSE278962_meta_new.tsv file was updated.

first: Chris

last: Mahony

middle: P


title: Gene reactivation upon erosion of X-chromosome inactivation in female hiPSCs is predictable yet variable and persists through differentiation

summary: Female human induced pluripotent stem cells frequently undergo X-chromosome inactivation (XCI) erosion, marked by XIST RNA loss and partial reactivation of the inactive X (Xi). This overlooked phenomenon limits our understanding of its impact on stem cell applications. Here, we show that XCI erosion is frequent and heterogeneous, leading to the reactivation of several X-linked genes. These are primarily located on the short arm of the X chromosome, particularly near escape genes and within H3K27me3-enriched domains, with reactivation linked to reduced promoter DNA methylation. Interestingly, escape genes further increase their expression from Xi upon XCI erosion, highlighting the critical role of XIST in their dosage regulation. Importantly, global (hydroxy)methylation levels and imprinted regions remain unaffected, and analysis of trilineage commitment and cardiomyocyte formation reveals that XCI erosion persists across differentiation. These findings underscore the need for greater awareness of the implications of XCI erosion for stem cell research and clinical applications.

overall_design:
1) RNA-seq analysis was conducted on biological triplicates of a diverse range of hiPSC lines representing various erosion states of the X-chromosome inactivation. Cell lines exhibiting XIST expression (XIST+) consist of ASD and F7cl15, and cell lines without XIST expression (XIST-) comprise F002, F7cl4, CD, and CE. RNA-seq was also performed before (day0- D0) and after ectodermal differentiation (day7- D7) for one replicate of the isogenic pair: F7cl4 (XIST-) and F7cl15 (XIST+).

2) IMPLICON was conducted for a diverse range of human pluripotent stem cell lines (hiPSCs) representing different erosion states of the X-chromosome inactivation. Specifically, two cell lines exhibiting XIST expression (XIST+): F7cl15, 2042cl9; and five cell lines without XIST expression (XIST-) and showing varying levels of erosion: F7cl4, CD, CE, 2040cl6, 2042cl1. As controls, two lines of human fibroblasts from which some hiPSCs lines were originated, were included: 2040 and 2042 fibroblasts. Of note, the following samples are isogenic: 2040 fibroblasts and 2040cl6 hiPSCs; F7cl15 and F7cl14; 2042 fibroblasts, 2042cl9 hiPSCs and 2042cl1 hiPSCs.

3) RNA Amplicon Sequencing was conducted for four (4) human pluripotent stem cell lines (hiPSCs) representing different erosion states of the X-chromosome inactivaton and for its respective derived cardiomyocytes (hiPSC-CMs). The cell line ASD presents XIST expression (XIST+) and the cell lines F002, CD and CE are XIST- and display varied levels of erosion.

last: Rocha


title: Erosion of X-Chromosome Inactivation in female hiPSCs is heterogeneous and persists during differentiation (RNA-AMP-Seq)

summary: During culture, female human pluripotent stem cells (hPSCs), including human induced PSCs (hiPSCs) exhibit a propensity for erosion of X-chromosome inactivation (XCI). This phenomenon is characterized by loss of XIST RNA expression and reactivation of a subset of X-linked genes from the inactive X chromosome (Xi). XCI erosion, despite its common occurrence, is often overlooked by the stem cell community, hindering a complete understanding of its impact on both fundamental and translational applications of hiPSCs. Investigating erosion dynamics in female hiPSCs, our study reveals that XCI erosion is a frequent yet heterogeneous phenomenon, resulting in reactivation of several X-linked genes. The likelihood of a gene to erode increases for those located on the short arm of the X chromosome and within H3K27me3-enriched domains. Paradoxically, genes that typically escape XCI are hypersensitive to loss of XIST RNA and XCI erosion. This implies that XIST RNA normally restrain expression levels of these genes on the Xi. Importantly, increased X-linked gene expression upon erosion does not globally impact (hydroxy)methylation levels in hiPSCs or at imprinted regions. By exploring diverse differentiation paradigms, such as trilineage commitment and cardiac differentiation, our study reveals the persistence of abnormal XCI patterns throughout differentiation. This finding has significant implications for fundamental research, translational applications, and clinical use of stem cells. We underscore the importance of raising awareness within the stem cell community regarding XCI erosion and advocate for its inclusion in comprehensive hiPSC quality control.

overall_design: RNA Amplicon Sequencing was conducted for four (4) human pluripotent stem cell lines (hiPSCs) representing different erosion states of the X-chromosome inactivaton and for its respective derived cardiomyocytes (hiPSC-CMs). The cell line ASD presents XIST expression (XIST+) and the cell lines F002, CD and CE  are  XIST- and display varied levels of erosion.


title: Protein-primed DNA homopolymer synthesis by an antiviral reverse transcriptase [RIP-seq]

first: Samuel

last: Sternberg

platform_organism: Escherichia coli str. K-12 substr. MG1655

overall_design: We performed RNA immunoprecipitation and sequencing (RIP-seq) on DRT9 reverse transcriptases in E. coli cells to profile their RNA binding activities in vivo. We also performed RIP-seq using mutant DRT9 systems.

first: Stephen

last: Tang

first: Rimantė

last: Žedaveinytė

first: Nathaniel

last: Burman

first: Shishir

last: Pandey

first: Josephine

middle: L

last: Ramirez

last: Kulber


title: Bacillus velezensis 83 transcriptional response during cocultivation with Colletotrichum gloeosporioides

status: Public on May 10 2025

city: Cuernavaca

first: Leobardo

last: Serrano Carreón

state: Morelos

address: Av Universidad 2001

department: Ingenieria Celular y Biocatálisis

country: Mexico

institute: Instituto de Biotecnología - UNAM

summary: To study microbial interaction effect on transcriptomic profile of B. velezensis 83, a comparative gene expression profiling analysis was performed.

overall_design: We performed the gene expression profiling analysis using data obtained from RNA-seq of four different conditions: B.velezensis 83 cells in axenic and coculture (interaction with C. gloeosporioides) condition at two time points.

first: Agustín

last: Luna-Bulbarela

first: Teresa

last: Romero-Gutiérrez

last: Serrano-Carreón

first: Lorena

last: Balón-Rosas


title: Erosion of X-Chromosome Inactivation in female hiPSCs is heterogeneous and persists during differentiation (Bisulfite-seq)

first: Paulo

last: Caldas

type: Methylation profiling by high throughput sequencing

overall_design: IMPLICON was conducted for a diverse range of human pluripotent stem cell lines (hiPSCs) representing different erosion states of the X-chromosome inactivation. Specifically, two cell lines exhibiting XIST expression (XIST+): F7cl15, 2042cl9; and five cell lines without XIST expression (XIST-) and showing varying levels of erosion: F7cl4, CD, CE, 2040cl6, 2042cl1. As controls, two lines of human fibroblasts from which some hiPSCs lines were originated, were included: 2040 and 2042 fibroblasts. Of note, the following samples are isogenic: 2040 fibroblasts and 2040cl6 hiPSCs; F7cl15 and F7cl14; 2042 fibroblasts, 2042cl9 hiPSCs and 2042cl1 hiPSCs.

last: Raposo

last: Arez

last: Jeremias


title: The ImmgenT Open-Source project

status: Public on May 14 2025

city: Boston

first: Liang

last: Yang

state: MA

address: 77 Avenue Louis Pasteur

department: Pathology

institute: Harvard Medical School

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

overall_design: The immgen T OpenSource Project is a collaborative effort involving 100 participants across 41 laboratories, working together to create the most comprehensive atlas of T cells in the mouse immune system. Leveraging a common data generation pipeline, the goal is to profile T cells in every body location, capturing their diverse states in response to a broad range of immunological challenges and physiological states (infection, autoimmune, tumor, aging), and how specific gene programs drive this diversity. Profiling was performed at the single-cell level, with multimodal mRNA, surface protein (CITE-seq/TotalSeq, 128 antibody panel), and TCR-VDJ (paired TCRα/TCRβ). Please refer to individual series for a description of each experiment.


title: Protein-primed DNA homopolymer synthesis by an antiviral reverse transcriptase [cDIP-seq]

last: Sternberg

overall_design: We applied a recently developed technique, termed cDNA immunoprecipitation and sequencing (cDIP-seq), to DRT9 reverse transcriptases in E. coli cells to identify their cDNA products in vivo. We also performed cDIP-seq using mutant DRT9 systems.

first: Louie

first: Tanner

last: Wiegand

first: Royce

last: Wilkinson

first: Yanzhe

last: Ma

first: Dennis

last: Zhang

first: George

last: Lampe

first: Mirela

last: Berisa

first: Marko

last: Jovanovic

first: Blake

last: Wiedenheft