title: An integrated multi-omics analysis of identiﬁes distinct molecular characteristics in pulmonary infections of Pseudomonas aeruginosa

city: chengdu

first: yang

last: yang

address: No.17, Section 3, South Renmin Road

institute: Sichuan University

summary: Pseudomonas aeruginosa (P. aeruginosa) can cause severe acute infections, including pneumonia and sepsis, and also cause chronic infections commonly in patients with structural respiratory diseases. However, the molecular and pathophysiological mechanisms of P. aeruginosa respiratory infection are largely unknown. Here, we profiled performed to assay for transposase-accessible chromatin using sequencing (ATAC-seq), transcriptomics, and quantitative mass spectrometry-based proteomics and ubiquitin-proteomics in P. aeruginosa-infected lung tissues for multi-omics analysis, while ATAC-seq and transcriptomics were also examined in P. aeruginosa-infected mouse macrophages. To find the pivotal transcription factors that are likely involved in host immune defense, we integrally investigated systematic changes in chromatin accessibility and gene expression in P. aeruginosa-infected lung tissues combined with proteomics and ubiquitin-proteomics studies. We discovered that Stat1 and Stat3 were altered in various omics and found similar results in mouse alveolar macrophages. Taken together, these findings indicate that these crucial transcription factors and their downstream signaling molecules play a critical role in the mobilization of host immune response against P. aeruginosa infection and may serve as potential targets for bacterial infections and inflammatory diseases, as well as provide clear insights and resources for using integrative histological analyses.

overall_design: RNA-seq_TC1, RNA-seq_TC2,RNA-seq_TC3, RNA-seq_CC1, RNA-seq_CC2, and RNA-seq_CC3 served as the control group. RNA-seq_TP1 and RNA-seq_TP2,RNA-seq_TP3, RNA-seq_CP1, RNA-seq_CP2, and RNA-seq_CP3 served as the infection group. ATAC-seq_TC1, ATAC-seq _TC2, ATAC-seq_CC1, and ATAC-seq_CC2 served as the control group. ATAC-seq_TP1, ATAC-seq_TP2, ATAC-seq_CP1, and ATAC-seq_CP2 served as the infection group.

first: Yang

last: Yang

first: Teng

last: Ma

first: Jun

last: Zhang

first: Jing

first: Taiwen

first: Xikun

last: Zhou


title: Massively-parallel Microbial mRNA Sequencing (M3-Seq) reveals heterogenous behaviors in bacteria at single-cell resolution

status: Public on Jul 27 2023

city: Princeton

first: Bruce

last: Wang

state: NJ

address: Lewis Thomas Lab

country: USA

institute: Prinnceton University

type: Expression profiling by high throughput sequencing

summary: M3-Seq is a single-cell RNA-sequencing platform for bacteria that pairs combinatorial cell indexing with post hoc rRNA depletion. We use M3-Seq to profile hundreds of thousands of bacterial cells and reveal rare populations of bacteria that include bet-hedging strategies, prophage induction, and phage-infected cells in E. coli and B. subtilis.

sample_organism:
Pseudomonas aeruginosa
Escherichia coli
Bacillus subtilis

overall_design: Bacterial cells were grown and treated as described in the M3-Seq manuscript. Each named experiment corresponds to a different dataset.

first: Aaron

middle: E

last: Lin

first: Jiayi

last: Yuan

first: Matthias

middle: D

last: Koch

first: Britt

middle: S

last: Adamson

first: Ned

last: Wingreen

first: Zemer

last: Gitai


title: FUBP1 is a core splicing factor that facilitates 3' splice site recognition and splicing of long introns [In vitro iCLIP]

city: Mainz

first: Stefanie

last: Ebersberger

address: Ackermannweg 4

country: Germany

institute: Institute of Molecular Biology

summary: Splicing is a central process in metazoans and greatly expands their proteome by alternative splicing of pre-mRNA transcripts. An essential regulatory step during early spliceosome assembly is the recognition of cis-regulatory RNA motifs in pre-mRNAs. Here, we identified the RNA binding protein FUBP1 as a novel core splicing factor with a ubiquitous footprint on pre-mRNAs. FUBP1 binds to a previously unknown cis-regulatory motif upstream of the branch point of human introns. We show that FUBP1 binds and stabilises known 3' splice site components such as the essential splicing factor U2AF2. FUBP1 mutant cell lines and patient data indicate that FUBP1 is particularly relevant for efficient splicing of exons flanked by long introns. In addition to its role at the 3’ splice site, FUBP1 shows multiple interactions with U1 snRNP- associated proteins. This demonstrates an important role for FUBP1 in splice site bridging in the context of long introns.

overall_design: In vitro iCLIP measures the intrinsic binding affinity of a given RBP.  Recombinant protein and minigene in vitro transcripts (PMID: 29643205) or a large-scale oligo library are mixed and subjected to crosslinking and immunoprecipitation of the RBP of interest. For all in vitro iCLIPs, the in vitro transcript pool or oligo library was preheated for 5 min at 70°C to minimise RNA secondary structures.For minigene in vitro iCLIP, a final concentration of 2 nM of in vitro transcript pool were added to 50 nM U2AF2 RRM12 alone, or to 50 nM U2AF2 RRM12 supplemented with either 50 nM of FUBP1FL, FUBP1ΔN, or FUBP1N74 (imb_koenig_2018_01_sub12). The mixtures were incubated at 37°C for 5 min before UV-irradiation (50 mJ/cm^2). Minigene in vitro iCLIP was spiked with 10 µl crosslinked mixture containing 250 nM U2AF2RRM12 and 6 nM NUP133 in vitro transcript for normalisation.For the oligo in vitro iCLIP, the large-scale oligo library of 200mers was used. Here, 50 nM of the oligo library was incubated with 50 nM U2AF2 RRM12 alone or with either 50 nM or 300 nM FUBP1 (imb_koenig_2018_01_sub16). The mixtures were incubated at 37°C for 5 min before UV-irradiation (50 mJ/cm^2). Partial RNase digestion and L3 linker ligation was skipped as they do not apply here. Oligo in vitro iCLIP was spiked with a mix of ten 150 nt long spike-in oligonucleotides for normalisation.

first: Andreas

last: Buchbender

first: Anke

last: Busch

first: Julian

last: König


title: Intradermal but not intramuscular MVA immunizations protect against intravaginal tier2 SHIV challenges in macaques

city: Atlanta

first: Gregory

middle: K

last: Tharp

state: GA

address: 954 Gatewood Dr

department: Developmental and Cognitive Neuroscience

institute: Yerkes National Primate Research Center

summary: Route of immunization can markedly influence the quality of immune response. Here, we show that intradermal (ID) but not intramuscular (IM) modified vaccinia Ankara (MVA) vaccinations provide protection from acquisition of intravaginal tier2 SHIV challenges in female macaques. Both routes of vaccination induced comparable levels of serum IgG with neutralizing and non-neutralizing activities. The protection in MVA-ID group correlated positively with serum neutralizing and antibody-dependent phagocytic activities, and envelope-specific vaginal IgA; while the limited protection in MVA-IM group correlated only with serum neutralizing activity. MVA-ID immunizations induced greater germinal center Tfh and B cell responses, reduced the ratio of Th1 to Tfh cells in blood and showed lower activation of intermediate monocytes and inflammasome compared to MVA-IM immunizations. This lower innate activation correlated negatively with induction of Tfh responses. These data demonstrate that the MVA-ID vaccinations protect against intravaginal SHIV challenges by modulating the innate and T helper responses.

overall_design: We vaccinated two groups of rhesus macaques with 10 animals per group using the DNA/MVA/Protein vaccine regimen. Animals in both groups were primed with DNA/SHIV-BG505-CD40L via the ID route and electroporation at weeks 0 and 8. At weeks 16 and 24, all animals were boosted with MVA/SHIV-BG505 delivered through either ID or IM routes.

first: Venkata

last: Bollimpelli

first: Pradeep

middle: B

last: Reddy

first: Sailaja

last: Gangadhara

first: Tysheena

middle: P

last: Charles

first: Samantha

middle: L

last: Burton

first: Tiffany

middle: M

last: Styles

first: Celia

middle: C

last: Labranche

first: Justin

last: Smith

first: Amit

middle: A

last: Upadhyay

first: Anusmita

last: Sahoo

first: Traci

last: Legere

first: Ayalnesh

last: Shiferaw

first: Vijayakumar

last: Velu

first: Tianwei

last: Yu

first: Mark

last: Tomai

first: John

last: Vasilakos

first: Sudhir

last: Kasturi

first: George

last: Shaw

first: David

last: Montefiori

first: Steven

last: Bosinger

first: Pamela

last: Kozlowski

first: Bali

last: Pulendran

first: Cynthia

last: Derdeyn

first: Eric

last: Hunter

first: Rama

middle: R

last: Amara


title: Expression data changes induced by OICR-9429 in 3D conditions in T-ALL cells

city: Madrid

first: Javier

last: Redondo-Muñoz

address: C/ Ramiro de Maeztu 9

country: Spain

institute: Centro de Investigaciones Biológicas Margarita Salas - CSIC

summary: 3D constrained conditions promoted H3K4 methylation in ALL cells that altered the global chromatin configuration and transcriptional changes related to cell cycle and DNA replication.

overall_design: CCRF-CEM cells (T-ALL cell line) were used for RNA extraction and hybridization on Affymetrix microarrays. CCRF-CEM cells were pretreated or not with 1 µM OICR-9429 for 1 hour. Then, cells were cultured in 3D conditions (a type I collagen matrix of 1,7 mg/mL) for 1 hour. Cells were lised and mRNA was extracted to compare expression profiles. We used 4 replicates of each sample.

first: Raquel

last: González-Novo

first: Ana

last: de Lope Planelles

first: África

last: González-Murillo

first: Elena

last: Madrazo

last: Acitores

first: Mario

last: García de Lacoba

first: Manuel

last: Ramírez


title: FUBP1 is a core splicing factor that facilitates 3' splice site recognition and splicing of long introns

first: Stefanie

last: Ebersberger

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

sample_organism:
Homo sapiens
synthetic construct

platform_organism:
Homo sapiens
synthetic construct

overall_design: Refer to individual Series


title: Single cell RNA-sequencing of C134 oHSV-treated murine medulloblastoma

city: Columbus

first: Katherine

last: Miller

state: OH

address: 700 Children's Dr.

department: Institute for Genomic Medicine

institute: Nationwide Children's Hospital

summary: Here, we studied the effects of oHSV C134 in two syngeneic medulloblastoma mouse models,  one that aligns with the sonic hedgehog (SHH) subtype (MYCN) and another that aligns with tumors of the group 3 subtype (CMYC).We harvested tumor tissue two and six days post-injection of C134 and performed single cell RNA-sequencing (scRNA-seq).

overall_design: To investigate if the second-generation, chimeric HCMV/HSV-1 oncolytic virus C134 (Cassady, 2005, J Virol) is a promising therapeutic treatment for medulloblastoma we utilized two syngeneic mouse medulloblastoma models: one that aligns with the gene expression profile associated with medulloblastoma of the sonic hedgehog subtype (MYCN; Zindy et al., 2007, Cancer Res), and another that aligns with tumors of the Group 3 subtype (CMYC; Kawauchi et al., 2012, Cancer Cell). Tumors were serially transplanted through the brains of mice and treated with either C134 oHSV or vehicle. Tumors were resected 2- and 6- days post-treatment (1 mouse per group, 8 samples total in our experiment). The Chromium Next GEM Single Cell 3’ v3.1 single index kit from 10x Genomics was used to carry out cell capture and library preparation. Targeted recovery was 8,000 cells per sample. FASTQs were generated using cellranger (v6.0.0) mkfastq with default parameters. Raw counts were generated by aligning FASTQs to a combined custom reference that included mus musculus + C134 oHSV transcripts using cellranger (v6.0.0) count with default parameters.

first: Kevin

last: Cassady

first: Elaine

last: Mardis

first: Jeffrey

last: Leonard


title: FUBP1 is a core splicing factor that facilitates 3' splice site recognition and splicing of long introns [RNA-Seq]

overall_design: "imb_koenig_2018_18: NGS library prep was performed with Illumina's TruSeq stranded total RNA LT Ribo Zero Gold Sample Prep Kit following Illumina?s standard protocol (Part # 15031048 Rev. E). Libraries were prepared with a starting amount of 1000 ng and amplified in 10 PCR cycles. Libraries were profiled in a DNA 1000 Chip on a 2100 Bioanalyzer (Agilent technologies) and quantified using the Qubit dsDNA HS Assay Kit, in a Qubit 2.0 Fluorometer (Life technologies). All 4 samples were pooled in equimolar ratio and sequenced on an Illumina NextSeq 500 sequencing machine as 84 nt single-end reads. imb_koenig_2020_12: NGS library prep was performed with Illumina's TruSeq stranded Total RNA LT Sample Prep Kit following Illumina?s standard protocol (Part # 15031048 Rev. E). Libraries were prepared with a starting amount of 200 ng and amplified in 12 PCR cycles. In addition, 1 µl of ERCC spike-ins (1:250 dilution) was added to every sample. Libraries were profiled in a DNA 1000 kit on a 2100 Bioanalyzer (Agilent technologies) and quantified using the Qubit dsDNA HS Assay Kit, in a Qubit 2.0 Fluorometer (Life technologies). All 12 samples were pooled in equimolar ratio and sequenced on an Illumina NextSeq 500 sequencing machine as 159 nt single-end reads.

first: Andreas

last: Buchbender

first: F

last: Sutandy

first: Miriam

last: Mulorz

first: Anke

last: Busch

first: Julian

last: König


title: MAGI1 prevents senescence and promotes the DNA damage response in ER+ breast cancer

status: Public on Jul 26 2023

city: Fribourg

first: Rüegg

last: Curzio

address: Albert-Gockel 1

department: Département de Médecine 3

country: Switzerland

institute: Université de Fribourg

summary: MAGI1 acts as tumor suppressor in estrogen receptor positive (ER+) breast cancer (BC) and its loss correlates with a more aggressive phenotype. To identify pathways and events affected by MAGI1 loss, we deleted the MAGI1 gene in the ER+ MCF7 BC cell line and performed RNA-sequencing and functional experiments in vitro. Transcriptome analyses revealed gene sets and biological processes related to estrogen signaling, cell cycle and DNA damage response affected by MAGI1 loss. Upon exposure to TNF-alpha/IFN-gamma, MCF7 MAGI1 KO cells entered a deeper level of quiescence/senescence than MCF7 control cells and activated the AKT and MAPK signaling pathways. MCF7 MAGI1 KO cells exposed to ionizing radiations or cisplatin had reduced expression of DNA repair proteins and showed increased sensitivity towards PARP1 inhibition with olaparib. Treatment with PI3K and AKT inhibitors (alpelisib and MK-2206) restored expression of DNA repair proteins and sensitized cells to fulvestrant. Analysis of human BC patients’ transcriptomic data revealed that patients with low MAGI1 levels have a higher tumor mutational burden and homologous recombination deficiency. Also, MAGI1 expression levels correlate negatively with PI3K/AKT and MAPK signaling, confirming our in vitro observations. Pharmacological and genomic evidence indicate HDACs as regulators of MAGI1 expression. Our findings provide a new view on MAGI1 function in cancer and identify potential treatment options to improve the management of ER+ BC patients with low MAGI1 levels.

overall_design: To identify pathways and events affected by MAGI1 loss, we deleted the MAGI1 gene by CRISPR/Cas9 technology in the ER+ MCF7 BC cell line and performed RNA-sequencing and functional experiments in vitro.

first: Janine

last: Wörthmüller

first: Simona

last: Disler

first: Sylvain

last: Pradervand

first: François

last: Richard

first: Lisa

last: Haerri

first: Gustavo

last: Ruiz Buendía

first: Nadine

last: Fournier

first: Christine

last: Desmedt

first: Curzio

last: Rüegg


title: FUBP1 is a core splicing factor that facilitates 3' splice site recognition and splicing of long introns [In vivo iCLIP]

first: Stefanie

last: Ebersberger

overall_design: In vivo iCLIP is used to study protein-RNA interactions on individual nucleotide resolution by means of UV crosslinking and immunoprecipitation (PMID: 20601959). For the U2AF2 in vivo iCLIP, data from two iCLIP experiments were combined. The first U2AF2 iCLIP  and PTBP1 iCLIP were performed as previously described in (PMID: 24184352 ). The second U2AF2 iCLIP as well as in vivo iCLIPs for FUBP1, SF1, and SF3B1, were performed using the iCLIP2 protocol as described in (PMID: 31610236). Briefly, HeLa cells were irradiated with 150  mJ/cm2 in a UV-C crosslinker to covalently bind the RNA-binding proteins to the bound nucleic acids. For FUBP1 in vivo iCLIP, cross-linking was achieved by 4sU-mediated cross-linking. HeLa cells were 4sU-labelled by adding a 0.1 M 4-Thiouridine (4sU, Sigma) in DMSO to a final concentration of 100 μM/10 cm cell culture dish. Cells were incubated for 16 h at 37 °C, 5% CO2, under exclusion of light. After incubation, the cells were moved onto ice, shielded from light and irradiated at 365 nm, 800 mJ. During subsequent cell lysis, RNA was partially digested to create 50-200 nt long fragments. Immunoprecipitation of the investigated proteins was performed with antibodies listed in Table S9. The anti-PTBP1 Antibody was a kind gift of Chris Smith (PMID: 17679092). Radioactive labelling at the 3’ end of the precipitated RNA enables visualisation of the RNP-complex by SDS-PAGE and transfer to a nitrocellulose membrane. After recovery of protein-RNA complexes from the membrane, a proteinase K digestion results in protein-free RNA.

first: Heike

last: Hänel

first: Lidia

last: Allcca


title: The β1-adrenergic receptor links sympathetic nerves to T cell exhaustion

city: La Jolla

first: Susan

last: Kaech

state: CA

address: 10010 N Torrey Pines Rd

department: NOMIS Center for Immunobiology and Microbial Pathogenesis

institute: Salk Institute for Biological Studies

summary: CD8+ T cells are essential components of the body’s immune reaction against viral infections and tumors, capable of eliminating infected and cancerous cells. However, when the antigen cannot be cleared, T cells enter a state known as exhaustion. Exhausted CD8+ T cells are characterized by expression of multiple inhibitory receptors, like PD-1 and TIM3 and reduced capacity to secrete cytokines, and are commonly found in chronic viral infections, like HIV and HBV, as well as in immunosuppressive tumor microenvironments (TME). Cancer and chronic infection also place considerable stress on tissue architecture and function, and while it is clear that chronic antigen contributes to CD8+ T cell exhaustion, less is known about how stress responses in tissues regulate T cell function.   Here we show a previously undescribed link between the stress-associated catecholamines adrenaline and noradrenaline and the development of T cell exhaustion through the β1-adrenergic receptor, ADRB1. We identify that exhausted CD8+ T cells express higher levels of ADRB1, and exposure of ADRB1-expressing T cells to catecholamines suppresses cytokine production and impairs T cell proliferation. Genetic or pharmacological ablation of β-adrenergic signaling via ADRB1 reduces development of T cell exhaustion in a chronic infection model and improves cytotoxic functions in tumor-infiltrating lymphocytes (TILs) when combined with immune checkpoint blockade (ICB) therapy in a melanoma model. Extending these findings to an ICB-resistant tumor model of murine pancreatic cancer, we show that beta-blockers and ICB synergize to enhance and reprogram T cell responses. Given that malignant disease is associated with increased systemic catecholamine levels in patients and therapeutic stress reduction improves survival in cancer patients, our results demonstrate a potential link between systemic stress, tumor innervation and immune responses. Our findings show that β-adrenergic signaling constitutes a novel targetable immune checkpoint in CD8+ T cells to rejuvenate antitumor functions. More broadly, our results support further clinical investigation into the application of beta-blockers, which are clinically safe and widely prescribed, as an immunomodulating agent for cancer immunotherapy.

overall_design: Single-cell RNA sequencing of orthotopic PDAC tumors from mice treated with isotype control, ICB, propranolol or combination of ICB and propranolol.

first: Anna

last: Globig


title: An oncolytic virus–delivered TGFβ inhibitor overcomes the immunosuppressive tumor microenvironment

city: Pittsburgh

first: Greg

last: Delgoffe

state: PA

address: 5115 Centre Ave.

institute: University of Pittsburgh

summary: Oncolytic viruses (OV) are promising forms of immunotherapy that have demonstrated clinical benefit in difficult-to-treat cancers such as metastatic melanoma. However, their adoption in other malignancies has been limited, in part, due to poorly understood mechansims of therapeutic resistance. Here, bulk RNA-seq was performed on oncolytic vaccinia virus-sensitive and -resistant murine head and neck squamous cell carcinomas (MEERvvS and MEERvvR, respectively) to explore potential means of OV resistance. These results corroborated a potential role for TGF-beta mediated stabilization of immunosuppressive regulatory T cells in the tumor microenvironment of OV-resistant MEERvvR-bearing mice. Subsequently, treatment with oncolytic vaccinia virus engineered to expess a dominant negative TGF-β signaling inhibitor (VVtgfbi) restored sensitivity to OV-mediated cell death among MEERvvR tumors. Single-cell RNA seq performed on CD45+ immune cells isolated from tumors suggests TGF-β inhibition may also reduce the presence and activity of myeloid-derived suppressor cell populations within the tumor microenvironment.

overall_design: Intradermal MEERvvS and MEERvvR tumor grafts were extracted, cultured ex-vivo in triplicate and subjected to bulk RNA sequencing.

first: Kristin

last: DePeaux

first: McLane

last: Watson

first: Kevin

last: Quann


title: Transcriptomic analysis implicates ABA signalling and carbon supply in the differential outgrowth of petunia axillary buds

city: Auckland

first: Zhiwei

last: Luo

state: Auckland

address: 120 Mt Albert Road

country: New Zealand

institute: PLANT AND FOOD RESEARCH

summary: Shoot branching of flowering plants exhibits phenotypic plasticity and variability. This plasticity is determined by the activity of axillary meristems, which in turn is influenced by endogenous and exogenous cues such as nutrients and light. In many species, not all buds on the main shoot develop into branches despite favorable growing conditions. In petunia, basal buds (buds 1-3) typically do not grow out to form branches, while more apical buds (buds 6 and 7) are competent to grow. The genetic regulation of buds was explored using transcriptome analyses of petunia axillary buds at different positions on the main stem. To suppress or promote bud outgrowth, we grew the plants in media with differing phosphate (P) levels. Using RNA-seq, we found many (>5000) differentially expressed genes between bud 6 or 7, and bud 2. In addition, more genes were differentially expressed when we transferred the plants from low P to high P medium, compared with shifting from high P to low P medium. Buds 6 and 7 had increased transcript abundance of cytokinin and auxin-related genes, whereas the basal non-growing buds (bud 2 and to a lesser extent bud 3) had higher expression of strigolactone, abscisic acid, and dormancy-related genes, suggesting the outgrowth of these basal buds was actively suppressed. Consistent with this, the expression of ABA associated genes decreased significantly in apical buds after stimulating growth by switching the medium from low P to high P. Furthermore, comparisons between our data and transcriptome data from other species suggest that the suppression of outgrowth of bud 2 was correlated with a limited supply of carbon to these axillary buds. Candidate genes that might repress bud outgrowth were identified by co-expression analysis.

overall_design: RNA-seq were obtained from two hydroponic experiments: in the 1st expt, plants were grown in high phosphate (250 uM) hydroponic, then were split into two groups. One group was transferred to fresh high P hydroponic whilst the other went into low P; in the 3rd expt: plants were placed in low P at the beginning, then split into two groups, one stayed in low P but the other went into high P. 24 h after medium switching, petunia axillary buds from nodes 2, 3, 6, and 7 (node 1 is the first node after cotyledons) were harvested. 3 replicates of a pool of axillary bud tissues from 6-7 plants. In total, 24 samples for the 1st expt and 24 samples from the 3rd expt.

first: Dan

last: Jones

first: Kimberley

last: Snowden

first: Joanna

last: Putterill


title: A branching model of lineage differentiation underpinning the neurogenic potential of enteric glia  (Single-cell expression analysis of  neurogenic enteric glial cell cultures)

city: London

first: Vassilis

last: Pachnis

address: 1 Midland Rd

department: Development and Homeostasis of the Nervous System Laboratory

country: United Kingdom

institute: The Francis Crick Institute

summary: Glial cells have been proposed as an endogenous source of progenitors for the treatment of neural deficits. However, the cellular and molecular mechanisms underpinning the neurogenic potential of certain populations of adult glial cells, are not known. Using single cell transcriptomic profiling, we show here that enteric glial cells represent a cell state attained by autonomic neural crest cells as they transition during development along a linear default differentiation trajectory that allows them to retain neurogenic potential while acquiring a gene expression profile associated with their role in neuronal support and immunomodulation. Key neurogenic loci in early enteric nervous system progenitors remain in open chromatin configuration in mature enteric glia, thus facilitating neuronal differentiation under appropriate conditions. Molecular profiling and gene targeting of enteric glial cells in a novel cell culture system of enteric neurogenesis and a gut injury model, demonstrated that neuronal differentiation of glia is driven by transcriptional programs employed in vivo by early progenitors. Our work provides mechanistic insight into the dynamic regulatory landscape underpinning the development of intestinal neural circuits and generates a platform for advancing glial cells as therapeutic agents for the treatment of neural deficits.

overall_design: We performed RNA-seq of freshly isolated enteric glial cells (DIV0) and their tdT+ descendants from DIV4, DIV11 and DIV20 ganglioids

last: Laddach

first: Song

last: Hui Chng

first: Reena

last: Lasrado

first: Fränze

last: Progatzky

first: Michael

last: Shapiro

first: Alek

last: Erickson

first: Marisol

last: Sampedro-Castaneda

first: Artem

last: Artemov

first: Ana

last: Bon-Frauches

first: Eleni-Maria

last: Amaniti

first: Jens

last: Kleinjung

first: Stefan

last: Boeing

first: Sila

last: Ultanir

first: Igor

last: Adameyko


title: FOXC2 knockdown in MDA-MB-231 cells

city: Cambridge

first: Kirsty

last: Sawicka

address: Robinson Way

department: University of Cambridge

institute: CRUK Cambridge Insititute

summary: To determine gene expression changes in vasculogenic mimicry competent human breast cancer cells with loss of FOXC2 we performed RNA-seq of MDA-MB-231 cells with FOXC2 knockdown.

overall_design: MDA-MB-231 cells were transduced with a control shRNA (shREN) or two different FOXC2-targetting shRNAs (shFOXC2#1 and shFOXC2#3). Two biological replicates per shRNA were subjected to RNA-seq analysis.

first: Ian

last: Cannell


title: RNA-seq of acLDL high vs low 4T1-T cells

summary: To determine whether acLDL uptake was associated with specfifc patterns of gene expression we treated vasculogenic mimicry-competent 4T1-T mammary carcinoma cells with Alexa-488 labelled acetylated low density lipoproteins (acLDL) and sorted the cells based on high or low uptake and subjected them to RNA-Seq

overall_design: 4T1-T VM cells were incubated with Alexa-488 acLDL followed by FACS. Briefly, cells were trypsinized and resuspended in MACS buffer (1x PBS, 0.5% BSA, 2 mM EDTA) and put through a 40 mm cell strainer. Cells were then sorted using a Sony SH800 cell sorter using the EGFP gate taking the top 25% as high and bottom 25% as low uptake. RNA was extracted from both populations and subjected to RNA-Seq. The experiment was repeated on three seperate occasions.


title: Evaluation of common profibrotic pathway across interstitial lung disease

city: South San Francisco

first: Jason

middle: Anthony

last: Vander Heiden

address: 1 DNA Way

department: Biological Research AI Development

institute: Genentech

summary: Given that the etiology and clinical manifestations differ substantially between IPF and SSc-ILD, we sought to better understand the similarities and differences in various profibrotic pathways of interest.

overall_design: RNA-seq was performed on lung samples collected from IPF and SSc-ILD patients undergoing transplantation.

first: Anna

last: Guttman

first: Xia

last: Gao

first: Thirumalai

last: Ramalingam


title: Investigating vasculogenic mimicry in breast cancer

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus


title: RNA-seq of whole blood from progressing SSc-ILD patients

summary: To identifying new blood derived prognostic biomarkers of lung disease progression in systemic sclerosis patients, we performed RNA-seq on whole blood from progressing SSc-ILD patients and controls enrolled in the FaSScinate clinical trial or an independent cohort.

overall_design: Comparative gene expression profiling to identify genes associated with disease progression between stable and progressing patients based on change in percent predicted FVC or HRCT.

first: Thierry

last: Sornasse

last: Hackney


title: Single cell RNA-seq of 4T1 mammary tumors treated with Axitinib

summary: To understand the effect of hypoxia on vasculogenic mimicry we treated mammary tumour dervied from parental 4T1 cells with vehicle or Axitinib. Tumors were analyzed by scRNA-seq and the expression of endothelial genes assessed in these data with and wihtout Axitinib treatment.

overall_design: 50,000 Parental 4T1 cells labelled with CellTag vector were implanted into the mammary fat-pad of 6-8 week old female Balb/C mice. After 6 days of tumor growth, animals were treated with vehicle or 50 mg/Kg Axitinib via intraperotoneal injection on a 5 days on 3 days off schedule for 14 days. Tumors were dissociated and submitted for single cell RNA-seq to the genomics core facility at the CRUK Cambridge Institute.


title: Single-cell analysis reveals region-heterogeneous responses in rhesus monkey spinal cord with complete injury

city: Beijing

first: Xianming

last: Wu

state: Chaoyang District

address: Unit 2, No.1 West Beichen Road

country: China

institute: Institute of Genetics and Developmental Biology, Chinese Academy of Sciences

summary: Spinal cord injury (SCI) leads to severe sensory and motor dysfunction below the lesion. However, the cellular heterogeneity and dynamic responses in different regions below the lesion remain to be elusive. Here, we used single-cell transcriptomics to characterize the region-related cellular responses in complete thoracic SCI of rhesus monkeys from the acute to the chronic phase. We found that cells in the distal lumbar tissue were severely affected and generated a degenerative microenvironment dominated by sustained activation of phagocytic microglia, increased inhibitory interneurons, and demyelinated oligodendrocytes after SCI. Furthermore, we revealed scaffold implantation in the injury sites could improve the microenvironment of injury sites by regulating glial cells and fibroblasts, and remodel the spared lumbar tissues by decreasing the inhibitory neurons and enhancing phagocytosis and remyelination. Our findings provide comprehensive pathological insights into the spared distal tissues and proximal tissues after SCI, highlighting the importance of scaffold-based treatment strategies for targeting heterogeneous microenvironments.

overall_design: Different regions of the completely injured rhesus monkey spinal cord from the acute phases (7 days and 14 days) to chronic phases (30 days and 6 months) and the cellular responses with functional scaffold (LOCS + Cetuximab) implantation after 6 months were analyzed using scRNA-seq.

first: Yongheng

last: Fan

first: Zhifeng

last: Xiao

first: Yannan

last: Zhao

first: Jianwu

last: Dai


title: RNA-seq analysis of doxycycline-regulated gene expression in Aspergillus fumigatus cells containing a dox off-ffmA fusion gene.

city: Iowa City

first: Scott

last: Moye-Rowley

state: Iowa

address: 6-530 Bowen Science Bldg./Dept. of Mol Physio

department: Molecular Physiology & Biophysics

institute: University of Iowa

summary: To determine the genes responsive to FfmA in A. fumigatus, a doxycycline-repressible (dox off) gene fusion to the ffmA coding sequence was constructed. This strain was designated SPF182. These cells were grown for 16 hours in the absence of doxycycline and then shifted into medium containing doxycycline (+dox) or untreated (-dox). After 8 additional hours, the cells were harvested and total RNA prepared. Wild-type cells were grown in parallel as a control in -dox medium.

overall_design: Compare the transcriptomes from cells in which FfmA was acutely depleted by doxycycline addition to a strain containing a dox off-ffmA fusion gene to the same strain maintained in the absence of dox.

first: Sanjoy

last: Paul

first: Mark

last: Stamnes

first: W


title: Single cell RNA-seq of 4T1-T vasculogenic mimicry-competent mammary tumors

summary: We have previously described a sub-clones of the 4T1 mammary carcinoma cell line that are proficient for vasculogenic mimciry (VM), namely 4T1-E and 4T1-T. In vitro assays suggest that not all cells within these lines a VM-competent. To explore subsets of cells within tumors derived from these cells that may display VM properties we utilized single cell RNA-Seq of 4T1-T mammary fat-pad tumors.

overall_design: 50,000 Parental 4T1-T cells labelled with CellTag vector were implanted into the mammary fat-pad of 6-8 week old female BALB/c mice. At day 20 post-implantation tumors were dissociated and submitted for single cell RNA-seq to the genomics core facility at the CRUK Cambridge Institute.


title: The mRNA stability factor Khd4 defines a specific RNA regulon for membrane trafficking in the pathogen Ustilago maydis

city: Düsseldorf

first: Prof. Dr. Michael

last: Feldbrügge

state: North Rheine-Westphalia

address: Universitätsstraße 1

department: Department of Biology

institute: Heinrich Heine University of Düsseldorf

overall_design: Refer to individual Series


title: The mRNA stability factor Khd4 defines a specific RNA regulon for membrane trafficking in the pathogen Ustilago maydis [RNA-Seq]

summary:
Fungal pathogens depend on sophisticated gene expression programs for successful infection. A crucial component is RNA regulation mediated by RNA-binding proteins. In the biotrophic fungal plant pathogen Ustilago maydis, loss of the multi KH domain containing RBP, Khd4, causes aberrant cell morphology, disturbed hyphal growth and impaired virulence.  To investigate the role of Khd4 in the polar growth of infectious hyphae, we established the RNA-editing based hyperTRIBE method to detect in vivo mRNA targets.This technique is especially suited to investigate proteins of high molecular weight or low expression that are difficult to purify (McMahon et al., 2016, Xu et al., 2018, Rahman et al., 2018). HyperTRIBE exploits the RNA editing activity of the catalytic domain of ADAR (Adenosine Deaminase Acting on RNA) from D. melanogaster that additionally carries the mutation E488Q to increase editing (Kuttan and Bass, 2012; designated Ada). We fused the codon-optimized catalytic Ada domain with the green fluorescent protein (see Materials and methods; enhanced version, designated Gfp, Clontech) to the C terminus of Khd4 (Khd4-Ada-Gfp). As a control for background editing, we used the strain expressing Ada-Gfp protein with N-terminal Kat fusion in the wildtype background (control-Ada).
We then performed differential gene expression analysis to determine the pathological consequence of dysregulated Khd4-mediated mRNA regulation.

overall_design: in vivo hyperTRIBE data to study the mRNA targets of RNA-binding proteins in hyphae of the phytopathogenic fungus Ustilago maydis

first: Srimeenakshi

last: Sankaranarayanan

first: Kathi

last: Zarnack

first: Michael


title: Gene expression analysis following neurogenesis of adult enteric glia/progenitor cells

first: Vassilis

last: Pachnis

state: London

address: 1 Midland Road

summary: Enteric glia cells (EGCs) are highly plastic and has been shown to give rise to neurons in adult gut upon injury to the enteric nervous system (ENS). To better characterise the dynamic molecular changes associated with EGC activation and its subsequent neurogenic differentation trajectory, we performed bulk RNAseq experiments from FACS sorted lineage-labelled cells (Sox10::CreERT2;R26-tdTomato) from adult small intestines (DIV0) and compared their transcriptomic profiles to cultured FACS sorted tdTomato+ cells from days in vitro (DIV) 4, 11 and 20, which were subjected to a protocol to support neurogenesis in culture.

overall_design: Longitudinal muscle-myenteric plexus (LMMP) peels were isolated, washed and dissociated to release adult EGCs for FACS sorting for direct RNA isolation or subjected to a neuronal differentiation protocol before purification by FACS and subsequent RNA isolation. The dynamics of gene expression changes were analysed as a time course, where cultured cells were collected from DIV 4, 11 and 20, compared to samples collected directly from the adult tissues (DIV0).

first: Song

middle: H

last: Chng

first: Franze

last: Progatzky

first: Stefan

last: Boeing


title: ChIP-seq analysis of FfmA binding in Aspergillus fumigatus

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Two strains were analyzed by ChIP-seq to determine the genomic binding sites for the Aspergillus fumigatus transcription factor FfmA. A wild-type strain (AfS35) was subjected to ChIP-seq analysis using an antibody directed against the wild-type version of the FfmA protein. An strain containing an epitope-tagged version (FLAG-ffmA) of the ffmA gene under control of a doxycycline off (dox off) promoter was also analyzed by ChIP-seq. In this latter case, an anti-FLAG antibody was used. The negative controls in both cases consisted of a ChIP-seq reaction with the respective primary antibody omitted.

overall_design: ChIP-Seq


title: Single-nucleus analysis of intact spinal cord and spared tissue after complete injury in rhesus monkey

first: Xianming

summary: The lumbar spinal cord is fundamental for hindlimb function and serves as spared tissue after spinal cord injury (SCI). However, our knowledge about cellular heterogeneity of the intact spinal cord and spared tissues is still scarce especially in primates. Here, we used single-nucleus RNA sequencing analysis in rhesus monkey to characterize the cellular heterogeneity of the spinal cord and profile the dynamic changes of cells in spared tissues after complete thoracic SCI. We found that rhesus monkey and mice shared a highly conserved pattern of cellular constitution but with divergent gene expression. After SCI, the cellular characteristics of spared tissue changed permanently and showed a regionally diverse response. We identified an emerging oligodendrocyte subtype that responded to interferon and a new permanently activated microglia subtype that was responsible for eliminating cell debris. Overall, our findings revealed dynamic changes of cells in spared tissue, providing comprehensive insights for SCI repair.

overall_design: Single-nucleus RNA-seq analysis of rhesus monkey spinal cord and spared lumbar spinal cord after complete injury.


title: FfmA

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


title: The pseudokinase Trib1 regulates the transition of exhausted T cells to a KLR+ CD8+ effector state and its deletion improves checkpoint blockade

status: Public on Jul 25 2023

city: Philadelphia

first: Warren

last: Pear

address: 421 Curie Blvd

institute: University of Pennsylvania

summary: CD8+ T cell exhaustion (TEX) impairs the ability of T cells to clear chronic infection or cancer. While TEX are hypofunctional, some TEX retain effector gene signatures, a feature that is associated with expression of KLRs (killer lectin-like receptors). Although KLR+ TEX may improve control of chronic antigen, the signaling molecules regulating this population are poorly understood. Using scRNA-seq, flow cytometry, RNA velocity, and scTCR-seq, we demonstrated that deleting the pseudokinase Trib1 shifted TEX towards CX3CR1+ intermediates (TINT) with robust enrichment of KLR+ TEX (TKLR) via clonal T cell expansion. Adoptive transfer studies demonstrate this shift towards a CD8+ TKLR in Trib1 deficient cells is CD8-intrinsic, while CD4-depletion studies demonstrate that CD4+ T cells are required for optimal viral control in Trib1 conditional knockout mice. Further, Trib1 loss augmented anti-PD-L1 blockade to improve viral clearance. These data identify Trib1 as an important regulator of CD8+ TEX whose targeting enhances the TKLR effector state and improves checkpoint inhibitor therapy.

overall_design: Activated (CD44+) T cells (TCRb+) were sorted from the spleens of either CD4-Cre Trib1 +/+ or F/F mice at day 15 post-infection with LCMV cl13. Cells were then subjected to single cell TCR-sequencing on the droplet based 10X genomics platform


title: Tumor microenvironment modifications induced by afatinib in squamous cell carcinoma of the head and neck - a window-of-opportunity study (EORTC90111-24111)

city: Brussels

first: Jean-Pascal

last: Machiels

address: Avenue Hippocrate 10

country: Belgium

institute: Cliniques Universitaires Saint-Luc

summary: The EORTC-90111-24111 window study included treatment-naïve patients with primary HNSCC receiving afatinib versus no preoperative treatment. We found that treated patients had an increase in tumor CD3+ density and CD3+ and CD8+ T-cell infiltration compared to control patients. Afatinib- exposed tumor had upregulation of gene expression signatures predictive of response to PD-1 blockade. Also, we observed two clusters among treated patients. Cluster 1 showed a higher expression of markers and gene sets implicated in epithelial-to-mesenchymal transition and cancer-associated fibroblasts activation after afatinib compared to cluster 2 and controls. These results open perspectives to overcome resistance mechanisms to anti-HER therapy and to potentiate the activity of immune checkpoint inhibitors.

overall_design: Thirty treatment-naïve patients with primary HNSCC were randomized. Twenty-five patients received afatinib for 14 days before surgery (40mg 1x/day) and five patients were attributed to the control arm. Biopsies were taken at work-up and during surgery. Good quality RNA samples were used for omics analyses (n=13 in treated patients)

first: Simon

last: Beyaert

first: Sandra

last: Schmitz


title: Single cell RNA-seq of 4T1 mammary tumors

first: Kirsty

last: Sawicka


title: SETD2 loss and ATR inhibition synergistically promote cGAS signaling and immunotherapy response in renal cell carcinoma

city: Houston

first: Christine

middle: B.

last: Peterson

state: TX

address: 1400 Pressler St.

department: Biostatistics

institute: University of Texas MD Anderson Cancer Center

summary: Immune checkpoint blockade (ICB) demonstrates durable clinical benefit only in a minority of renal cell carcinoma (RCC) patients. Identifying molecular features that determine response and developing approaches to enhance the response remain an urgent clinical need. Here we found that, in multiple RCC cell lines, targeting the ATR-CHK1 axis with pharmacological inhibitors increased cytosolic DNA accumulation, activated the cGAS-IRF3-dependent cytosolic DNA sensing pathway, and resulted in the inflammatory cytokine expression. SETD2 mutated RCC cell lines or tumor samples were associated with preferential ATR-CHK1 activation over ATM-CHK2 activation. SETD2 knockdown promoted the cytosolic DNA sensing pathway and conferred greater sensitivity in response to ATR-CHK1 inhibition. In murine Renca tumors, Setd2 knockdown and ATR inhibitor VE822 synergistically promoted cytosolic DNA sensing pathway, immune cell infiltration, and immune checkpoint protein expression. Setd2 deficient Renca tumors demonstrated greater vulnerability to ICB monotherapy or in combination with VE822 than Setd2 proficient tumors. SETD2 mutations were associated with a higher response rate and prolonged overall survival in ICB-treated RCC patients, but not in non-ICB-treated RCC patients. This study provides a mechanism-based guidance to develop more personalized combination therapy regimens for RCC patients with SETD2 mutations.

overall_design:
Cell line data: 786-O parental cell lines were treated with LY2606368 (a CHK1 inhibitor) treatment or no treatment (control), in two separate experiments
Murine tumor data:

first: Xian-De

last: Liu


title: The mRNA stability factor Khd4 defines a specific RNA regulon for membrane trafficking in the pathogen Ustilago maydis [hyperTRIBE]

summary: Fungal pathogens depend on sophisticated gene expression programs for successful infection. A crucial component is RNA regulation mediated by RNA-binding proteins. In the biotrophic fungal plant pathogen Ustilago maydis, loss of the multi KH domain containing RBP, Khd4, causes aberrant cell morphology, disturbed hyphal growth and impaired virulence.  To investigate the role of Khd4 in the polar growth of infectious hyphae, we established the RNA-editing based hyperTRIBE method to detect in vivo mRNA targets.This technique is especially suited to investigate proteins of high molecular weight or low expression that are difficult to purify (McMahon et al., 2016, Xu et al., 2018, Rahman et al., 2018). HyperTRIBE exploits the RNA editing activity of the catalytic domain of ADAR (Adenosine Deaminase Acting on RNA) from D. melanogaster that additionally carries the mutation E488Q to increase editing (Kuttan and Bass, 2012; designated Ada). We fused the codon-optimized catalytic Ada domain with the green fluorescent protein (see Materials and methods; enhanced version, designated Gfp, Clontech) to the C terminus of Khd4 (Khd4-Ada-Gfp). As a control for background editing, we used the strain expressing Ada-Gfp protein with N-terminal Kat fusion in the wildtype background (control-Ada).


title: Neuroendocrine lineage commitment of small cell lung cancers can be leveraged into p53-independent non-cytotoxic therapy [CUT&Tag]

city: Cleveland

first: Sudipta

last: Biswas

state: Ohio

address: 9500 Euclid Ave

department: Translational Hematology & Oncology Research

institute: Cleveland Clinic

summary: Small cell lung cancers (SCLC) rapidly resist cytotoxic chemotherapy and immune-checkpoint inhibitor (ICI) treatments. New, non-cross-resistant therapies are thus needed. SCLC cells are committed into neuroendocrine-lineage then maturation-arrested. The corepressor DNA methyltransferase 1 (DNMT1) mediates the maturation-arrests: (i) the repression mark methylated-CpG, written by DNMT1, is retained at suppressed neuroendocrine-lineage genes, even as other repression marks are erased; (ii) DNMT1 is recurrently amplified, while Ten-Eleven-Translocation 2 (TET2) that functionally opposes DNMT1 is deleted; (iii) DNMT1 is recruited into neuroendocrine-lineage master transcription factor (ASCL1, NEUROD1) hubs in SCLC cells; and (iv) DNMT1 knock-down activated ASCL1-target genes and released SCLC cell cycling exits by terminal lineage-maturation, cycling exits that do not require the p53/apoptosis-pathway used by cytotoxic chemotherapy. Inhibiting DNMT1/corepressors with clinical compounds accordingly extended survival of mice with chemo- and ICI-refractory disseminated SCLC. Neuroendocrine-lineage commitment of SCLC cells can thus be leveraged into p53/apoptosis-independent therapy, active against chemo- and ICI-refractory SCLC.

overall_design: Objectives were to evaluate the potential of non-cytotoxic DNMT1-targeting using decitabine and 5-azacytidine to treat chemorefractory SCLC. The central hypothesis was that DNMT1-depletion from SCLC cells would release cell cycle exits by terminal-maturation, a pathway not cross-resistant with the p53/apoptosis pathway utilized by chemo-radiation.

first: Yogen

last: Saunthararajah


title: Neuroendocrine lineage commitment of small cell lung cancers can be leveraged into p53-independent non-cytotoxic therapy


title: Neuroendocrine lineage commitment of small cell lung cancers can be leveraged into p53-independent non-cytotoxic therapy [RNA-seq]


title: Network analysis reveals strain-dependent response to misfolded tau aggregates (NanoString)

city: Indianapolis

first: Dominic

middle: James

last: Acri

state: Indiana

address: 320 W 15 St, NB Room 107

department: Stark Neurosciences Research Institute

institute: Indiana University School of Medicine

summary:
To investigate the effect of wild-derived genetic background on modeling tau aggregation, we injected either AAV-eGFP or AAV-hTauP301L into postnatal day 0 mice to drive widespread expression.
We then performed gene expression profiling analysis using data obtained from Nanostring nCounter Mouse Neuroinflammation Panel from mouse brain cortex of 6 month old hTauP301L- or eGFP-mice.

overall_design: Comparative gene expression profiling analysis of Nanostring data for eGFP or hTauP301L mice on the following genetic backgrounds: C57BL6/J, CAST/EiJ, PWK/PhJ, and WSB/EiJ (n=3/background/injection).

middle: J

first: Jungsu

last: Kim


title: Effects of zinc supplementation in the temporal transcriptomic profiling of human primary fetal RPE cell using single-cell RNA sequencing

city: Belfast

first: Imre

last: Lengyel

state: Northern Ireland

address: 97 Lisburn Road

department: Wellcome-Wolfson Institute For Experimental Medicine

institute: Queen's University Belfast

summary: This is the first publication that assessed the complex molecular changes that underpin acute zinc supplementation-induced changes at the single cell RNA sequencing level in a well-characterised age-related macular degeneration (AMD) cell model. The retinal pigment epithelial (RPE) cells were allowed to mature for up to 19 weeks, during which time they developed sub-RPE deposits, a hallmark of AMD. Comparing the scRNA-Seq data from cells after two-, nine- and 19 weeks in culture, we identified distinct clusters of cells. Pseudotemporal ordering of the combined gene lists from all time points identified genes implicated in the dynamics of RPE cell differentiation, with enrichment in biologically plausible pathways. Acute (1 week) zinc supplementation (125 µM added zinc) resulted in the differential expression of several genes, including specific transcriptional regulators.

overall_design: In this in vitro study, we compared scRNA-Seq data from fetal RPE cells after two-, nine- and 19 weeks in culture and how these were modified by acute (1 week) zinc supplementation (125 µM added zinc).

first: Eszter

last: Emri

first: Oisin

last: Cappa

first: Caoimhe

last: Kelly

first: Elod

last: Kortvely

first: John

last: SanGiovanni

first: Brian

last: McKay

first: Arthur

last: Bergen

first: David

last: Simpson


title: The sex-specific factor SOA establishes X chromosome dosage compensation in Anopheles mosquitos [SOAR-RNA-seq]

city: Mainz

first: Frank

last: Rühle

address: Ackermannweg 4

department: Bioinformatics Core Facility

institute: Institute of Molecular Biology (IMB)

summary: The Anopheles mosquito is one of thousands of species in which sex differences play a central role in their biology, as only females need a blood meal in order to produce eggs. Sex differentiation is regulated by sex chromosomes, but their presence creates a dosage imbalance between males (XY) and females (XX). Dosage compensation (DC) can re-equilibrate the expression of sex-chromosomal genes, but because DC mechanisms have only been fully characterized in a few model organisms, key questions about its evolutionary diversity and functional necessity remain unresolved. Here we report the discovery of a previously uncharacterized gene (SOA) as a master regulator of DC in the malaria mosquito Anopheles gambiae. Sex-specific alternative splicing prevents functional SOA protein expression in females. The male isoform encodes a DNA-binding protein that binds the promoters of active X chromosomal genes. Expressing male SOA is sufficient to induce DC in female cells. Male mosquitoes lacking SOA or female mosquitos ectopically expressing the male isoform exhibit X chromosome misregulation, which is compatible with viability but causes developmental delay. Thus, our molecular analysis of the first DC master regulator in a non-model organism elucidates the evolutionary steps leading to the establishment of a chromosome-specific fine-tuning mechanism.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for female pupae of wild-type and SOA-R knock-in genotype

first: Agata

middle: I

last: Kalita

first: Eric

last: Marois

first: Maria

middle: F

last: Basilicata

first: Magdalena

last: Kozielska

first: Franz

last: Weissing

first: Etienne

last: Jaouen

first: Martin

last: Möckel

first: Falk

last: Butter

first: Claudia

last: Keller Valsecchi


title: The sex-specific factor SOA establishes X chromosome dosage compensation in Anopheles mosquitos [SOAR-CUTnTag]

overall_design: CUT&Tag libraries generated from WT and SOA-R pupae are analyzed. Each group has two biological replicates (except for SOA-R female IgG with one replicate). The IgG antibody is a negative control for unspecific binding of the antibodies.


title: Gene expression profile upon EWSR1::FLI1 knockdown in Ewing sarcoma cells A673 in vitro

city: MAJADAHONDA

first: Javier

last: Alonso

state: MADRID

address: CTRA. MAJADAHONDA-POZUELO KM 2

department: AREA DE GENÉTICA HUMANA

institute: INSTITUTO DE SALUD CARLOS III - IIER

summary: Ewing sarcoma, a rare and aggressive pediatric cancer, is characterized by chromosomal translocations that give rise to chimeric transcription factors. The most frequent of these chromosomal translocations is the t(11;22) that produces the fusion of the EWSR1 and FLI1 genes to generate the chimeric transcription factor EWSR1::FLI1. EWSR1::FLI1 is the main oncogenic event in Ewing's sarcoma. Recently, it has been proposed that EWSR1::FLI1 levels may fluctuate in Ewing sarcoma cells, giving rise to two cell populations: cells expressing low levels of EWSR1::FLI1 are characterized by a more migratory and invasive phenotype, while cells expressing high levels of EWSR1::FLI1 are more proliferative. The identification and functional characterization of EWSR1::FLI1 gene targets is therefore relevant to understanding the pathobiology of Ewing sarcoma, which in turn could contribute to the identification of new therapeutic targets. Using this approach, we have observed that CD44, a transmembrane protein involved in cell adhesion and migration and associated with metastasis in various cancer types, is overexpressed in the EWSR1::FLI1-low phenotype. Our results suggest that CD44 may play a role in regulating cell migration in Ewing sarcoma cells and thus contribute to the spread of tumor cells.

overall_design: A673 cells derived from Ewing sarcoma were genetically enginereed to express specific shRNAs against EWS::FLI1 upon doxycycline stimulation (A673/TR/shEF). Three independent clones from each enginereed cell were analyzed. Cells were stimulated with doxycycline for 72 hours to induce the expression of EWSR1:FLI1 shRNA. Afterwards, total RNA was isolated and gene expression profile analyzed by RNAseq. Gene expression profile in A673 cells in which EWSR1::FLI1 was downregulated (A673 EWSR1::FLI1 low expression) were compared to control cells (A673 EWSR1::FLI1 high expression).


title: Effect of Sox7 endothelial conditional knock out on blood vessel differentiation in vivo in a mouse model

city: Darlinghurst

middle: Thomas

last: Humphreys

address: 405 Liverpool Street

department: Genome core

country: Australia

institute: Victor Chang Cardiac Research Institute


title: Effect of Sox7 endothelial conditional knock out on blood vessel differentiation in vivo in a mouse model [Bulk RNA-seq]

summary: To study the effects of the loss of Sox7 function on gene expression of the endothelium in vivo during development. We generated a endothelial specific ko using a Sox7fl/fl Cdh5 creERT2 inducible system.

overall_design:
Gene expression profiling analysis using RNA-seq (polyA) of CD31-positive endothelial cells sorted from Sox7 inducible endothelial conditional knock out or sibling control embryos at 10.5dpc
Comparative gene expression profiling analysis of RNA-seq data for wild type control and Sox7 deleted endothelial cells (homozygous deletion). There are 3 wt ctrl embryos and 3 wt homozygous embryos that have been processed in the same batch to allow for pairwise comparison.

first: Mat

last: Francois

first: Ivy

last: Chiang


title: Effect of Sox7 endothelial conditional knock out on blood vessel differentiation in vivo in a mouse model [scRNA-seq]

overall_design: Gene expression profiling analysis using single cell (10x) RNA-seq of whole embryonic heart at 12.5 dpc from Sox7 inducible endothelial conditional knock out or sibling control embryos.


title: Gene expression profiling of NUP98-HOXA9 expressing mouse embryonic stem cells

city: Bunkyo-ku

first: Yuya

last: Nagaoka

state: Tokyo

address: 1-1-1 Yayoi

department: Institute for Quantitative Biosciences

country: Japan

institute: The University of Tokyo

summary: To investigate the effect of NUP98-HOXA9 on global gene expression, we established FLAG-NUP98-HOXA9 expressing mouse embryonic stem cell lines.

overall_design: We then performed gene expression profiling analysis using data obtained from RNA-seq of different cell lines.

first: Ryuichiro

last: Nakato

first: Daisuke

last: Okuzaki

first: Mayumi

last: Otani

first: Masahrio

last: Oka


title: Modeling epigenetic lesions that cause gliomas [ChIP-Seq]

city: Boston

first: Gilbert

middle: Joseph

last: Rahme

state: MA

address: 365 Longwood Ave.

department: Cancer Biology

institute: Dana-Farber Cancer Institute

summary: Epigenetic lesions that disrupt gene regulatory elements and expression are increasingly recognized as pervasive drivers of human cancers. However, we currently lack the in vitro and in vivo models required to functionally validate such lesions and their tumorigenic impact. Here we model aberrations that arise in Isocitrate Dehydrogenase mutant (IDHmut) lower-grade gliomas, which exhibit profound DNA hypermethylation. We initially focus on a CTCF insulator downstream of the PDGFRA oncogene that is recurrently disrupted in IDHmut gliomas. We demonstrate that disruption of the syntenic insulator in mouse oligodendrocyte-progenitor cells (OPCs) allows an OPC-specific enhancer to contact and induce PDGFRA, thereby increasing proliferation. In contrast, insulator disruption did not affect PDGFRA expression in neural progenitor cells (NPCs), which lack the enhancer. We also model a second recurrent epigenetic lesion in IDHmut gliomas, the methylation-dependent silencing of the CDKN2A tumor suppressor. We show that inactivation of CDKN2A/p19ARF by de novo promoter methylation or mutation drives OPC proliferation and synergizes with PDGFRA insulator loss. Finally, we use lentiviruses to coordinately inactivate the PDGFRA insulator and CDKN2A in mouse corpus callosum, resulting in low-grade gliomagenesis in vivo. Our study recapitulates recurrent epigenetic lesions in mouse models and demonstrates that combination of PDGFRA activation and CDKN2A silencing can transform OPCs in vitro and drive gliomagenesis in vivo.

overall_design: CTCF occupancy characterization, histone H3K27 acetylation profiling in mouse NPC and OPC cells. DNA methylation profiling (capture bisulfite sequencing) on human glioma samples.

first: Bradley

middle: E

last: Bernstein


title: Single-cell lineage capture across genomic modalities with CellTag-multi reveals fate-specific gene regulatory changes [scRNA-seq - iEP].

city: Saint Louis

first: Samantha

last: Morris

state: Missouri

address: 660 S Euclid Ave

institute: Washington University School of Medicine

summary: Complex gene regulatory mechanisms underlie differentiation and reprogramming. Contemporary single-cell lineage tracing (scLT) methods use expressed, heritable DNA barcodes to combine cell lineage readout with single-cell transcriptomics enabling high-resolution analysis of cell states while preserving lineage relationships. However, reliance on transcriptional profiling limits their adaptation to an ever-expanding tool kit of multiomic single-cell assays. With CellTag-multi, we present a novel approach for profiling lineage barcodes with single-cell chromatin accessibility without relying on co-assay of transcriptional state, paving the way for truly multiomic lineage tracing. We validate CellTag-multi in mouse hematopoiesis, characterizing transcriptional and epigenomic lineage priming across progenitor cell populations. In direct reprogramming of fibroblasts to endoderm progenitors, we use CellTag-multi to comprehensively link early cell state with reprogramming outcomes, identifying core regulatory programs underlying on-target and off-target reprogramming. Further, we reveal the Transcription Factor (TF) Zfp281 as a novel regulator of reprogramming outcome, biasing cells towards an off-target mesenchymal fate via its regulation of TGF-β signaling. Together, these results establish CellTag-multi as a novel lineage tracing method compatible with multiple single-cell modalities and demonstrate its utility in revealing fate-specifying gene regulatory changes across diverse paradigms of differentiation and reprogramming.

overall_design: scRNA-seq was performed for CellTagged in vitro reprogramming MEFs at multiple time points to allow longitudinal lineage tracing, across 2 biological replicates. At each time point cells were split across multiple lanes of 10x GEM Chip per replicate, as indicated by sample number

first: Kunal

last: Jindal

first: Mohd

middle: T

last: Adil

first: Naoto

last: Yamaguchi

first: Helen

last: Wang

first: Xue

last: Yang

first: Kenji

last: Kamimoto

first: Guillermo

last: Rivera-Gonzalez


title: Single-cell lineage capture across genomic modalities with CellTag-multi reveals fate-specific gene regulatory changes [scATAC-seq - LSK].

overall_design: scATAC was performed for CellTagged in vitro differentiating mouse LSK cells at multiple time points to allow longitudinal lineage tracing


title: Single-cell lineage capture across genomic modalities with CellTag-multi reveals fate-specific gene regulatory changes [scRNA-seq - LSK].

overall_design: scRNA was performed for CellTagged in vitro differentiating mouse LSK cells at multiple time points to allow longitudinal lineage tracing


title: Modeling epigenetic lesions that cause gliomas [Capture-Bis-seq]

type: Methylation profiling by high throughput sequencing

overall_design:
CTCF occupancy characterization, histone H3K27 acetylation profiling in mouse NPC and OPC cells. DNA methylation profiling (capture bisulfite sequencing) on human glioma samples.
NOTE FROM SUBMITTER: The capture bis seq raw data comes from patient specimens. That is why the raw data was not deposited.


title: Single-cell lineage capture across genomic modalities with CellTag-multi reveals fate-specific gene regulatory changes [scATAC-seq - iEP].

overall_design: scATAC-seq was performed for CellTagged in vitro reprogramming MEFs at multiple time points to allow longitudinal lineage tracing, across 2 biological replicates. At each time point cells were split across multiple lanes of 10x GEM Chip per replicate, as indicated by sample number


title: Differential gene expression between Kp209_CSTR and Kp209 by RNA-seq

city: Grenoble

first: Manon

last: Janet-Maitre

address: 71 Avenue des Martyrs

country: France

institute: IBS

summary: Determination of the effect of the mutation in PhoQ on global gene expression of Kp209

overall_design: RNA-seq analysis of Kp209_CSTR vs WT in Klebsiella pneumoniae Kp209, in liquid cultures grown to OD(600nm)=0.6 at 37°C, in triplicates

first: Ina

last: Attrée

first: Suzan

last: Rooijakkers


title: Modeling epigenetic lesions that cause gliomas

overall_design: Refer to individual Series


title: Network analysis reveals strain-dependent response to misfolded tau aggregates

first: Dominic

last: Acri

summary:
To investigate the effect of wild-derived genetic background on modeling tau aggregation, we injected either AAV-eGFP or AAV-hTauP301L into postnatal day 0 mice to drive widespread expression.
We then performed gene expression profiling analysis using data obtained from mRNA-seq from mouse brain cortex of 6 month old hTauP301L- or eGFP-mice.

overall_design: Comparative gene expression profiling analysis of mRNA-seq data for eGFP or hTauP301L mice on the following genetic backgrounds: C57BL6/J, CAST/EiJ, PWK/PhJ, and WSB/EiJ (n=4/background/injection/sex=64).

first: Jungsu

last: Kim


title: SOA confers X chromosome dosage compensation in Anopheles gambiae mosquitoes (RNA-Seq II)

first: Frank

last: Rühle

summary: Differences between males and females are central to the biology of thousands of species across the tree of life. Sex chromosomes play a key role, but despite their their evolutionary diversity, the  regulatory mechanisms have been mostly elucidated in the three model species Mammals, D. melanogaster and C. elegans. Here we present the characterization of the first X chromosome dosage compensation (DC) pathway in a non-model organism, the malaria mosquito Anopheles gambiae. In mosquitos, DC corrects the imbalance in gene expression between sexes by approximately two-fold upregulation of male X-linked genes. We have identified SOA, a previously uncharacterized gene, which is specific to Anophelinae and activated concomitantly with the onset of DC. SOA displays sex-specific alternative splicing, where only males express full-length protein. SOA is a DNA-binding protein, specifically localizes to an X chromosome territory and binds promoters of active X-linked genes. Expression of the male, but not female isoform, is sufficient to induce DC. SOA knock-out mosquitos display a male-specific developmental delay, which is associated with a global downregulation of the X chromosome. Thus, SOA is the first DC factor discovered in Anopheles and provides only the fourth DC pathway ever identified to date. Since only females are blood-feeding and able to transmit malaria, identification of this sex-specific pathway is highly relevant and an important progress for malaria control programs.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for pupae of wild-type and SOA knock-in genotype

first: Agata

last: Kalita

first: Eric

last: Marois

first: Maria

last: Basilicata

first: Magdalena

last: Kozielska

first: Franz

last: Weissing

first: Etienne

last: Jaouen

first: Martin

last: Möckel

first: Falk

last: Butter

first: Claudia

last: Keller Valsecchi


title: Phase-separated nuclear bodies of nucleoporin fusions promote condensation of MLL1/CRM1 and rearrangement of 3D genome structure

first: Yuya

last: Nagaoka

summary: We performed Hi-C to analyze the effect of NUP98-HOXA9 fusion on 3D chromatin structure of mouse embryonic stem cells (mESCs). Our analysis revealed that NUP98-HOXA9 induced a drastic alteration of high-order genome structure in a phase separation-dependent manner.

overall_design: Analysis of chromosome conformation by Hi-C in mESCs expressing FLAG-NUP98-HOXA9 or its mutants.

first: Masahiro

last: Oka

first: Mayumi

last: Otani

first: Yoichi

last: Miyamoto

first: Jun

last: Adachi

first: Takeshi

last: Tomonaga

first: Munehiro

last: Asally

first: Atsushi

last: Toyoda

first: Kazuki

last: Ichikawa

first: Shinichi

last: Morishita

first: Ryuichiro

last: Nakato

first: Yasuyuki

last: Ohkawa

first: Yoshihiro

last: Yoneda


title: SOA confers X chromosome dosage compensation in Anopheles gambiae mosquitoes (CUT&Tag II)

first: Frank

last: Rühle

overall_design: CUT&Tag libraries generated from WT and SOA-KI pupae are analyzed. Each group has two biological replicates (except for Male WT groups, which have four replicates). The IgG antibody is a negative control for unspecific binding of the antibodies.

first: Agata

last: Kalita

first: Eric

last: Marois

first: Maria

last: Basilicata

first: Magdalena

last: Kozielska

first: Franz

last: Weissing

first: Etienne

last: Jaouen


title: SOA confers X chromosome dosage compensation in Anopheles gambiae mosquitoes (CUT&Tag I)

overall_design: CUT&Tag samples are analyzed. Each group has two biological replicates. The samples infected with the empty baculovirus are a negative control for SOA-HA expression, whereas the IgG antibody is a negative control for unspecific binding of the antibodies

first: Martin

last: Möckel

first: Falk

last: Butter

first: Claudia

last: Keller Valsecchi


title: Sex-specific transcriptome of Anopheles gambiae during embryonic development

summary: In many species, males and females display differences in their physiology that are not limited to the gonads. Sex differences in adult insects can comprise body size and feeding behaviour, as only female, but not male mosquitoes are blood-feeding in order to produce eggs. This is relevant since the malaria-causing parasite Plasmodium is transmitted by a female mosquito through a previous blood meal from an infected individual. The initiation of sex-specific developmental programs can be often linked to heteromorphic sex chromosomes (XX/XY in Anopheles). In males, the X-linked genes are only present in a single copy. To counteract this hemizygosity, Anopheles, like other species, exhibits dosage compensation (DC). Anopheles DC manifests by upregulation of the single male X ensuring the equal expression of sex-chromosomal genes between males and females. To date, the sex-specific differences at the transcriptome level related to the X/Y chromosomes and DC have been characterized at post-embryonic stages. Their onset during embryogenesis, especially during the early stages where the embryo switches from the maternal to the zygotic transcriptome, is largely uncharacterized to date. Here, we generate a sex-specific transcriptome atlas across Anopheles embryogenesis ending at the first larval stage. Along with the transition from the maternal to the zygotic transcriptome, many transcripts switch isoform, where the 3’ untranslated regions (UTR) of zygotic transcripts are significantly extended compared to their maternal counterpart. We also identify stage- and sex-specific splicing events. Comparing male and female embryos, we identify 120 sexually dimorphic protein coding genes and non-coding RNAs, indicating that sexually dimorphic gene expression is not widespread during embryogenesis. Furthermore, the imbalance of X-linked genes between males and females after zygotic genome activation is corrected shortly after, where the majority of genes exhibit DC around 9 hours of embryogenesis with further fine-tuning later in development. Our atlas provides a framework to explore sex differences in development and evolution and thereby, enables future functional analyses of sex-specific expression patterns and isoform usage.

overall_design: Poly(A) stranded mRNA sequencing of single embryos of predetermined sex (males and females) collected at different time points of embryonic development and upon the completion of embryonic development (L1 larvae). Biological replicates are included.


title: SOA confers X chromosome dosage compensation in Anopheles gambiae mosquitoes (RNA-Seq I)

overall_design: Comparative gene expression profiling analysis of RNA-seq data for Ag55 cells. Each group has three biological replicates. The samples infected with the empty baculovirus are a negative control for SOA-HA expression.


title: SOA confers X chromosome dosage compensation in Anopheles gambiae mosquitoes (ATAC-Seq)

overall_design: ATAC-seq libraries generated from WT and SOA-KI pupae are analyzed. Biological replicates are included. WT samples are the reference group for studying the effect of SOA-KI.


title: Cell-type-specific regulation of APOE and CLU levels in human neurons by the Alzheimer’s disease risk gene SORL1

status: Public on Jul 24 2023

first: Richard

middle: VanDyke

last: Pearse

address: 60 Fenwood Rd.

department: Neurology

institute: Brigham and Womens Hospital

summary: SORL1 is strongly implicated in the pathogenesis of Alzheimer’s disease (AD) through human genetic studies that point to an association of reduced SORL1 levels with higher risk for AD. To interrogate the role(s) of SORL1 in human brain cells, SORL1 null iPSCs were generated, followed by differentiation to neuron, astrocyte, microglial, and endothelial cell fates. Loss of SORL1 led to alterations in both overlapping and distinct pathways across cell types, with the greatest effects in neurons and astrocytes. Intriguingly, SORL1 loss led to a dramatic neuron-specific reduction in APOE and CLU levels, an increase in the accumulation of lipid droplets and altered lipid profiles. Pathway analysis implicated intracellular transport pathways and TGF-β/SMAD signaling in the function of SORL1 in neurons. Enhancement of retromer-mediated trafficking and autophagy rescued tau phenotypes observed in SORL1 null neurons but did not rescue APOE levels. However, stimulation and inhibition of SMAD signaling in neurons modulated APOE RNA levels in a SORL1-dependent manner. Further, analyses of iPSCs derived from a human aging cohort revealed a neuron-specific linear correlation between SORL1 and APOE RNA and protein levels, a finding validated in human post-mortem brain. These studies provide a mechanistic link between two of the strongest genetic risk factors for AD.

overall_design: The CVB (Ventner) iPSC line was CRISPR modified to eliminate expression of the SORL1 gene (SORL1-KO), control lines went through the CRISPR process but resulted in no modification of SORL1 (SORL1-WT). Both SORL1 WT and KO lines were harvested in 3 replicates either as indifferentiated iPSC cells or as differentiated neurons (iN), astrocytes (iAst), microglia (iMGL), or endothelial cells (iEC). Briefly iNs were generated by direct differentiation by expression of neurogenin-2 using a modified version of the process originally described in Zhang et al. (Neuron 78(5), 2013). Astrocytes were generate by expression of SOX9 and NIFB using a modified version of the protocol described in Canals et al. (Nature Methods 15, 2018). Differentiated neurons and astrocytes were both harvested on d21 of differentiation. Microglia were differentiated by first exposing iPSC colonies to hematopoietic differentiation conditions using the STEMdiff Kit from STEMCELL technologies and then exposing the resultant CD34+, CD45+, CD43+ cells to exogenous differentition factors as thoroughly described in Abud et al. (Neuron 94(2), 2017). Differentiated microglia were harvested at d41 of the protocol. Endothelial Like Cells (iECs), were differentiated with minor modification according to the protocols of Lippmann et al., 2012 , Lippmann et al., 2015 , and Park et al., 2019.

first: Hyo

last: Lee

middle: V

first: Tracy


title: A Phase I, First-in-Human Study to Evaluate the Safety and Tolerability, Pharmacokinetics and Pharmacodynamics of MRG-001 in Healthy Subjects

city: Baltimore

first: Ali

last: Ahmadi

address: 855 N Wolfe Street

institute: MedRegen LLC

summary: BACKGROUND: Preclinical studies have demonstrated that pharmacological mobilization and recruitment of endogenous bone marrow stem cells and immunoregulatory cells by a combination of plerixafor and low-dose tacrolimus (MRG-001) improves wound healing, promotes tissue regeneration and prevents allograft rejection. This first‐in‐human phase I dose‐escalation study evaluates the safety, tolerability, pharmacokinetics and pharmacodynamics of MRG-001, a novel fixed-dose combination drug.  METHODS: In this Phase 1, double‐blind, randomized, placebo-controlled study, multiple ascending dose (MAD) cohorts are randomized to receive MRG-001 containing up to 0.02 mL/kg (plerixafor 24 mg/mL and tacrolimus 0.5 mg/mL) or saline placebo, subcutaneously every other day (SC, QAD) for 5 days (ClinicalTrials.gov: NCT04646603)The primary outcome is safety and tolerability. Safety and functional assessments are performed throughout the study. Blood samples are collected to evaluate systemic exposure. Fluorescence-activated cell sorting analysis and RNA expression of peripheral blood mononuclear cells (PBMCs) are used to evaluate the pharmacodynamics.  RESULTS: Fourteen subjects received MRG-001 and 7 received a placebo. MRG-001 is safe and well-tolerated over the selected dose range. No deaths or severe adverse events are reported. There are no clinically significant laboratory changes after MRG-001 administration, apart from the predicted generalized leukocytosis. The intermediate dose group (0.01 mL/kg) showed the most significant white blood cell mobilization over time and increased by 2-4 fold from baseline and returned to baseline levels prior to the next injection. Circulating immune cells including FOXP3+ regulatory T cells and hematopoietic stem cells (CD45IntCD34+) increased significantly after MRG-001 injection. PBMC RNA sequencing and gene set enrichment analysis revealeds 31 down-regulated pathways in the intermediate dose MRG-001 group compared to no changes in the placebo group.  CONCLUSION: MRG-001 is safe and well-tolerated across the full dose ranges tested. MRG-001 may be a clinically useful therapy for immunoregulation and tissue regeneration. A Phase II trial to treat severely and critically ill COVID-19 patients with MRG-001 has been initiated (NCT04646603) and a second phase II trial will explore the potential of MRG-001 to accelerate wound healing (NCT05844527),

overall_design: To investigate the dynamic differences of gene expression of the peripheral mononuclear blood cells after injection of placebo compared to different dosages of the investigational drug MRG-001.

last: Mecking

first: Falko

last: Hoffman


title: The sex-specific factor SOA establishes X chromosome dosage compensation in Anopheles mosquitos [Mybless-CUTnTag]


title: Transcriptome analysis identifies genes dysregulated in Sox7-null embryos at E8.5

first: Chad

middle: A.

last: Shaw

address: 1 Baylor Plaza

department: Molecular and Human Genetics

institute: BCM

summary: To identify genes that are differentially expressed in the developing mouse embryo as a result of SOX7 deficiency, we performed bulk RNA-seq on Sox7-null and wild-type embryos harvested at E8.5.

overall_design: RNA-seq differential gene expression analysis was accomplished using E8.5 Sox7-null mouse embryos (n = 3) and E8.5 wild-type mouse embryos (n = 3).

first: Andres

last: Hernandez-Garcia

first: Daryl

last: Scott

first: Alexander

last: Renwick


title: Identification and characterization of striking multiple-insecticide resistance in a Tetranychus urticae field population from Greece.

city: Athens

first: John

last: Vontas

state: Attica

address: Iera Odos 75

department: Department of Crop Science

country: Greece

institute: Agricultural University of Athens

summary: We identified and characterized a striking case of multiple acaricide resistance in a field population of T. urticae. Exceptionally strong resistance phenotypes, with accumulation of multiple resistance mutations and over-expression of P450s and other detoxification genes in the same field population is reported.

overall_design: We compared whole-body transcriptome of the multi-resistant T. urticae Trizina strain with the transcriptomes of two susceptible strains, London and BC2018-14.

first: Kyriaki-Maria

last: Papapostolou

last: Riga

first: Jason

last: Charamis

first: Evangelia

last: Skoufa

first: Vassilis

last: Souchlas

first: Aris

last: Ilias

first: Wannes

last: Dermauw

first: Panagiotis

last: Ioannidis

first: Thomas

last: Va Leeuwen


title: Liver transcriptome analysis of obese mice expressing a SUMO-deficient Prox1 (K556R) mutant in hepatocytes

city: Munich

first: Mauricio

last: Berriel Diaz

state: Bavaria

address: Ingolstädter Landstraße 1

department: Institute for Diabetes and Cancer

institute: Helmholtz Center Munich

summary: Accumulation of excess nutrients hampers proper liver function and is linked to non-alcoholic fatty liver disease (NAFLD) in obesity. However, the signals responsible for an impaired adaptation of hepatocytes to obesogenic dietary cues remain still largely unknown. Post-translational modification by the Small Ubiquitin-like Modifier (SUMO), allows for a dynamic regulation of numerous processes including transcriptional reprograming. We demonstrate that specific SUMOylation of transcription factor Prox1 represents a nutrient-sensitive determinant of hepatic fasting metabolism. Prox1 is highly SUMOylated on lysine 556 in the liver of ad libitum and re-fed mice, while this modification is abolished upon fasting. In the context of diet-induced obesity, Prox1 SUMOylation becomes less sensitive to fasting cues. The hepatocyte-selective knock-in of a SUMOylation-deficient Prox1 mutant into mice fed a high-fat/high-fructose diet leads to a reduction of systemic cholesterol levels, associated with the induction of liver bile acid detoxifying pathways during fasting. The generation of tools to maintain the nutrient-sensitive SUMO-switch on Prox1 may thus contribute to the development of “fasting-based” approaches for the preservation of metabolic health.

overall_design: To explore the function of Prox1 SUMOylation in the liver we generated a conditional hepatocyte specific SUMO-deficient Prox1 (K556R) knock-in mouse model. The expression of Prox1 (K556R) was induced via Cre recombination in 8 weeks old male mice. Mice were then fed a high-fat (45%)/high-fructose (20%) diet for 18 weeks. Liver samples were collected in the fasted and re-fed states (ZT 15-17) and subjected to RNA-seq analysis.

first: Ana

last: Alfaro

first: Amit

last: Mhamane

first: Stephan

last: Herzig


title: Regulatory T cells targeting a pathogenic MHC class II: Insulin peptide epitope postpone spontaneous autoimmune diabetes

first: Joshua

last: Kulas

state: IN

address: 1210 Waterway Blvd Ste. 2000

institute: IBRI

summary: In spontaneous type 1 diabetes (T1D) non-obese diabetic (NOD) mice, the insulin B chain peptide 9-23 (B:9-23) can bind to the MHC class II molecule (IAg7) in register 3 (R3), creating a bimolecular IAg7/InsulinB:9-23 register 3 conformational epitope (InsB:R3). Previously, we showed that the InsB:R3-specific chimeric antigen receptor (CAR), constructed using an InsB:R3-monoclonal antibody, could guide CAR-expressing CD8 T cells to migrate to the islets and pancreatic lymph nodes. Regulatory T cells (Tregs) specific for an islet antigen can broadly suppress various pathogenic immune cells in the islets and effectively halt the progression of islet destruction. Therefore, we hypothesized that InsB:R3 specific Tregs would suppress autoimmune reactivity in islets and efficiently protect against T1D. To test our hypothesis, we produced InsB:R3-Tregs and tested their disease-protective effects in spontaneous T1D NOD CD28-/- mice. InsB:R3-CAR expressing Tregs secrete IL-10 dominated cytokines upon engagement with InsB:R3 antigens. A single infusion of InsB:R3 Tregs delayed the onset of T1D in 95% of treated mice, with 35% maintaining euglycemia for two healthy lifespans, while whereas control Tregs did not. Our data demonstrate that Tregs specific for MHC class II: Insulin peptide epitope (MHCII/Insulin) protect mice against T1D more efficiently than polyclonal Tregs lacking islet antigen specificity, suggesting that the MHC II/insulin-specific Treg approach is a promising immune therapy for safely preventing T1D.

overall_design: The pancreas of mice treated with InsB:R3-Tregs, Ctl-Tregs, or saline buffer (Non-T), three mice per group, was digested, and islets were handpicked on day 8 post-transfer. Using independent parametric significance testing between two groups and assuming a large effect size (Cohen’s d) equal to 4, equal variances, and power of 0.80, significant results (can be achieved with three samples per group. Three diabetic NOD.CD28-/- mice (DM) diagnosed within one week were included for comparison. Fresh islets from each group were pooled, and RNAs were extracted using an RNA Miniprep kit (Zymo Research, R1054). High-quality RNA samples that passed the quality control test were subjected to RNA-Seq analysis (Novogene Corporation, Inc.).

first: Nyerhovwo

last: Obarorakpor

first: Deep

last: Patel

first: Reni

last: Boyarov

first: Nansalmaa

last: Amarsaikhan

first: Joseph

last: Ray Cepeda

first: Doreen

last: Eastes

first: Sylvia

last: Robertson

first: Travis

last: Johnson

first: Kai

last: Yang

first: Qizhi

last: Tang

first: Li

last: Zhang


title: Liver transcriptome analysis of lean mice expressing a SUMO-deficient Prox1 (K556R) mutant in hepatocytes

overall_design: To explore the function of Prox1 SUMOylation in the liver we generated a conditional hepatocyte specific SUMO-deficient Prox1 (K556R) knock-in mouse model. The expression of Prox1 (K556R) was induced via Cre recombination in 8 weeks old male mice. 11 weeks later, liver samples were collected in the fasted and re-fed states (ZT 15-17) and subjected to RNA-seq analysis.


title: Bi-directional interactions of omental adipocytes and SKOV3ip1 cells

city: Chicago

first: Abir

last: Mukherjee

state: Illinois

address: 929 E 57th street, Gordon center for integrative sciences, room w107K

institute: university of Chicago

summary:
Primary human omental adipocytes were isolated from patients with non-cancer conditions. Subsequently, gene expression profiled in adipocytes and ovarian cancer cell line (SKOV3ip1)  alone (control) or  under co-culture conditions.
Following co-culture cells were separated and total RNA isolated for microarray analysis.

overall_design: Total RNA was isolated from adipocytes and SKOV3ip1 cells (alone or in co-culture) and expression profiling carried out using Illumina microarray.


title: SOA confers X chromosome dosage compensation in Anopheles gambiae mosquitoes

first: Frank

last: Rühle

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

overall_design: Refer to individual Series


title: Oral spirochete Treponema denticola intraoral infection and periodontitis in C57BL/6J mice [16_weeks_Td_data]

city: GAINESVILLE

first: LAKSHMYYA

last: KESAVALU

state: FLORIDA

address: 1600 SW ARCHER ROAD Rm D10-23

department: PERIODONTOLOGY/DENTISTRY

institute: UNIVERSITY OF FLORIDA

summary: Individual miRNA analyzed were successfully constructed through nanostring technology of a total of 577 mouse miRNAs in 20 number of SHAM mice and 20 number of T. denticola infected mice, which have been euthanized on the end of 16 weeks infection study.

overall_design: The study depicted here, is a 16 weeks of total, 10 mice/group, Sham infected and T. denticola infected groups both in 8 weeks and 16 weeks, followed 16 weeks, euthanized and extracted left mandibles for miRNA expression profiles, of a total of 577 unique Nanostring miRNA panels. This leading to successful construction of supervised.

first: Aravindraja

last: Chairmandurai

first: Lakshmyya

last: Kesavalu


title: Oral spirochete Treponema denticola intraoral infection and periodontitis in C57BL/6J mice


title: Oral spirochete Treponema denticola intraoral infection and periodontitis in C57BL/6J mice [16_weeks_sham_data]


title: Oral spirochete Treponema denticola intraoral infection and periodontitis in C57BL/6J mice [8_weeks_Td_data]


title: Oral spirochete Treponema denticola intraoral infection and periodontitis in C57BL/6J mice [8_weeks_sham_data]


title: Acquisition of a Glycosylated B-Cell Receptor Drives Follicular Lymphoma Towards a Dark Zone Phenotype

city: Leiden

first: Cornelis

last: van Bergen

address: Albinusdreef 2

department: Hematology

country: Netherlands

institute: Leiden University Medical Center

summary: A balanced number of NliGpos and NliGneg FL B cells from a single lymph node were processed and analyzed. We conclude that a distinction can be made between NliGpos and NliGneg FL cells and centroblast/centrocyte phenotype.

overall_design: 10X Genomics of flowcytometry purified Follicular Lymphoma cells, 5' chemistry, full transcriptome and BCR enrichment

first: C

first: J

last: Veelken


title: A novel covalent inhibitor of CDK7  synergizes with Venetoclax in acute myeloid leukemia

city: Mumbai

first: Syed

last: Hasan

state: Maharashtra

address: Sector 22 Kharghar Navi Mumbai

country: India

institute: Advanced Centre for Treatment Research and Education in Cancer (ACTREC)

summary: To assess the synergistic effects of CDK7 inhibitor with Venetoclax, we performed RNA sequencing and gene set enrichment analysis using Venetoclax sensitive and resistant model systems. RNA-sequencing from paired Venetoclax-sensitive and Venetoclax-resistant cells treated with CDK7 inhibitor showed downregulation of genes involved in proliferation and apoptosis. CDK7 inhibitor induces apoptosis in AML cells and combined treatment with Venetoclax led to synergistic effects in overcoming resistance.

overall_design: We have labeled parental Venetoclax-sensitive MOLM13 cells with CDK7 inhibitor treatment PT while untreated cells were marked as PU. Similarly, Venetoclax-resistant MOLM13 cells with and without CDK7 inhibitor treatment were labeled as RT and RU respectively. cDNA libraries in duplicates from PT, PU RT and RU were pooled and diluted to final optimal loading concentration before cluster amplification on the Illumina flow cell. Once the cluster generation is completed, the clustered flow cell is loaded on Illumina HiSeq X instrument to generate 167 million, 150bp paired end reads per sample. Image analysis, base calling, and quality check was performed with Illumina data analysis pipeline RTA v1.18.64. RNAseq reads were provided in compressed Sanger FASTQ format.

first: Gaur

last: Tarang

first: Hasan

last: Syed


title: CEBPA phase separation links transcriptional activity and 3D chromatin hubs

city: Barcelona

first: Gregoire

last: Stik

state: select a state

address: 88 Dr Aiguader

institute: CRG

summary: Cell identity is orchestrated through an interplay between transcription factor (TF) action and genome architecture. The mechanisms used by TFs to shape three-dimensional (3D) genome organization remain incompletely understood. Here we present evidence that the lineage-instructive TF CEBPA drives extensive chromatin compartment switching and promotes the formation of long-range chromatin hubs during induced B cell to macrophage transdifferentiation. A large intrinsically disordered region (IDR) enables CEBPA to undergo in vitro phase separation and to co-condense with transcriptional partners, which is at least partially mediated by aromatic residues. Furthermore, CEBPA forms visible nuclear condensates in transdifferentiating B cells that co-localize with co-activator condensates and recover rapidly upon photobleaching. Finally, native CEBPA-expressing cell types such as primary granulocyte-macrophage progenitors (GMPs), liver cells and trophectoderm cells also reveal nuclear CEBPA condensates and long-range 3D chromatin hubs at CEBPA-bound regions. These findings support a model in which CEBPA acts as a 3D genome structural organizer and suggest that this effect is mediated at least in part by its phase-separation capacity.

overall_design: HiC datasets were generated from BLaER cells knocked out for a CEBPA binding site in the FGGY locus  before and after 24h of CEBPA induction (n=2)

first: Julia

last: Erber

first: Thomas

last: Graf


title: Chilling stress drives organ specific transcriptional cascades and dampens diurnal oscillation in Tomato

city: West Lafayette

first: Kranthi

last: Varala

address: 625 Agriculture Mall Dr.

department: Horticulture and Landscape Architecture

institute: Purdue University

summary: This study assayed the gene expression changes in response to chilling stress in the shoots and roots of tomato seedlings. Both a pretreatment and a concurrent ambient control were sampled to contrast gene expression.

overall_design: Tomato seedlings were grown for 18 days in a hydroponic setup and split into two groups. One group remained at 24C while the other group was moved to 4C. Shoots and roots were sampled (3 replicates) from seedlings prior to start of treatment, from shoots at 30 minutes after treatment and from shoots and roots after 3 hours of treatment.


title: Effect of deletion of the ipdc gene, involved in indole-3-acetic acid synthesis, in the biocontrol agent Serratia plymuthica A153

city: Granada

first: Míriam

last: Rico Jiménez

state: Granada

address: Profesor albareda, 1

department: Biotechnology and environmental protection

institute: CSIC

summary: To explore the role of indole-3-acetic acid (IAA) as signalling molecule in plant-associated bacteria we analyzed the whole transcriptome of S. plymuthica A153 wild type and its Δipdc mutant in vitro

overall_design: We compared the transcriptome profiles of the parental strain S. plymuthica A153 relative to that of the ipdc mutant by transcriptomic sequencing (RNA-seq). RNA samples from cultures grown in Lysogeny broth medium supplemented with 1mg/mL L-tryptophan until early stationary phase of growth were prepared.

first: Miguel

last: Matilla

first: Miriam

last: Rico-Jiménez


title: Continuous pioneer activity is required for maintaining pluripotency specific gene expression

city: Amsterdam

first: Elzo

last: de Wit

state: Noord-Holland

address: Plesmanlaan 121

department: Gene Regulation

institute: Netherlands Cancer Institute


title: RNA-seq of human prostate cancer cell line PC-3M trpv6 knockout versus wild type and PC3M-Luc-C6 wild type versus trpv6 overexpression.

city: Villeneuve d'Ascq

first: clement

last: cordier

address: Batiment SN3, cité Scientifique

institute: PhyCell

summary: TRPV6 is a membrane protein, a member of the transient receptor potential (TRP) ion channel family. The TRPV6 cation channel is highly selective for Ca2+, with PCa/PNa values greater than 100, playing a crucial role in calcium homeostasis in various cell types. Previous transcriptome analysis indicated that TRPV6 mRNAs were expressed in PCa and that channel expression correlates strongly with the pathology stage and metastasis status. The aim of the present study was to examine calcium-regulated signaling pathways in castration-resistant prostate cancers.

overall_design: Three biological replicates were analyzed for each of 2 conditions of PC3M cell line or two biological replicates were analyzed for each of 2 conditions of PC3M-Luc-C6 cell line with different trpv6 expression.

first: Clement

last: Cordier

first: V'yacheslav

last: Lehen'kyi


title: Multidimensional epigenomic priming of inflammatory genes enables rapid recall in human memory T cells

summary: During T cell responses, a fraction of activated cells adopts a memory phenotype and returns to quiescence. These long-lived memory T cells retain an irreversible molecular imprint that enables them to mount a secondary recall response to the same antigen that is faster and greater in magnitude than the primary response of a naive cell. Memory T cells provide long-lasting immunological protection, forming the foundation for vaccination strategies and representing prime targets for immunotherapies to treat diseases characterized by dysfunctional memory T cells - including cancer and chronic inflammation. The molecular circuitry that endows memory T cells with their rapid recall ability remains incompletely understood. Here, we applied a multi-layered 1D-3D epigenomics approach to systematically dissect the molecular program driving rapid recall in primary human Th2 cells.

overall_design: Various omics datasets were generated from purified human naive and memory CD4+ T cells (n=3 donors) prior to and after T cell receptor stimulation. Findings were validated using flowcytometry, inhibitor studies and CRISPR-Cas9 genome editing in primary human CD4+T cells.

first: Anne

last: Onrust-van Schoonhoven

first: Ralph

last: Stadhouders