title: Role of immediate early genes in the development of salivary gland organoids in polyisocyanopeptide hydrogels

city: Groningen

first: Rob

last: Coppes

address: Ant. Deusinglaan 1

department: Biomedical Sciences of Cells and Systems

institute: University Medical Center Groningen

summary: Human salivary gland organoids have opened tremendous possibilities for regenerative medicine in patients undergoing radiotherapy for the treatment of head and neck cancer. However, their clinical translation is greatly limited by the current use of Matrigel for organoid derivation and expansion. Here, we envisage that the use of a fully, synthetic hydrogel based on the oligo(-ethylene glycol) functionalized polymer polyisocyanopeptides (PICs) can provide an environment suitable for the generation and expansion of salivary gland organoids (SGOs) after optimization of PIC polymer properties. We demonstrate that PIC hydrogels decorated with the cell-binding peptide RGD allow SGO formation from salivary gland (SG)-derived stem cells. This self-renewal potential is preserved for only 4 passages. It was found that SGOs differentiated prematurely in PIC hydrogels affecting their self-renewal capacity. Similarly, SGOs show decreased expression of immediate early genes (IEGs) after culture in PIC hydrogels. Activation of multiple signalling pathways involved in IEG expression by β-adrenergic agonist isoproterenol, led to increased stem cell self-renewal capacity as measured by organoid forming efficiency (OFE). These results indicate that PIC hydrogels are promising 3D matrices for SGOs, with the option to be used clinically, after further optimization of the hydrogel and culture conditions.

overall_design: Examination of (dis)similarities between Matrigel and PIC-cultured SGOs


title: R-loop in soybean

status: Public on Dec 10 2022

city: Beijing

first: li

last: kuan

address: zhongguancun

country: China

institute: Tsinghua University

summary: The R-loop is a common chromatin feature presented from prokaryotic to eukaryotic genomes and has been revealed to be involved in multiple cellular processes and associated with many human diseases. Here, we take the advantage of our recently developed ssDRIP-seq method to profile genome-wide R-loop levels of soybean (Glycine max).

overall_design: ssDRIP-seq using soybean leaves

first: Jincong

last: Zhou


title: G-quadruplex DNA as a target for pathogenic bacteria: efficacy of an extended naphthalene diimide ligand and mode of action

first: Anne

middle: de

last: Jong

address: Nijenborgh 7

department: Molecular Genetics

country: Netherlands

institute: university of Groningen

type: Expression profiling by high throughput sequencing

summary: Guanidine DNA quadruplex (G4-DNA) structures convey a distinctive layer of epigenetic information that is critical for the regulation of key biological activities and processes as genome transcription regulation, replication and repair. Despite several works that have been published recently, the information regarding their role and possible use as therapeutic drug targets in bacteria is still scarce. Here, we tested the biological activity of a small G4-DNA ligand library based on the naphthalene diimide (NDI) pharmacophore, against both Gram-positive and Gram-negative bacteria. For the best compound identified, NDI-10, the action mechanism was further characterized. Gram-negative bacteria were more resistant altogether due to the presence of the outer membrane, although the activity of the G4-Ligand was generally bactericidal, while it was bacteriostatic for Gram-positive bacteria. This asymmetric activity could be related to the different prevalence of putative G4-DNA structures in each group, the influence that they can exert on the gene expression (which was found more severe for the Gram-negative bacteria) and the role of the G4 structures in these bacteria, that seems to be more related to promote transcription in Gram-positive bacteria and repress transcription in Gram-negative.

sample_organism:
Escherichia coli
Staphylococcus aureus

platform_organism:
Escherichia coli
Staphylococcus aureus

overall_design: 4 samples Ecoli and 4 samples Saureus no replicates

first: Rubén

last: Cebrián

first: Valentina

last: Pirota

first: Efres

last: Belmonte-Reche

last: de Jong

first: Juan

middle: C

last: Morales

first: Mauro

last: Freccero

first: Filippo

last: Doria

first: Oscar

middle: P

last: Kuipers


title: Gene expression profiling of placental villi from intrauterine and tubal ectopic pregnancies

city: Shanghai

first: Xiaoya

last: Zhao

state: -- None selected --

address: No. 910, Hengshan Rd

institute: School of Medicine, Shanghai Jiaotong University

type: Non-coding RNA profiling by high throughput sequencing

summary: The tubal ectopic pregnancy (TEP) is a severe pregnancy complication accounted for 10-15% pregnancy-related deaths owing to the implantation and growth of embryos in fallopian tubes. Placental villi from TEP exhibit significant difference compared with that from intrauterine pregnancy (IP) in total volumes, trophoblast differentiation and intravillous vascularization.  To investigate the difference in expression profiles of placental villi from IP and TEP, placental villi were collected from each phenotype, separated from maternal tissues and analyzed by high-throughput sequencing.

overall_design: microRNA-seq was performed on placental villi from pregnant women at 6-8 gestational weeks. Exclusion criteria for all samples are: IP with EP histories, miscarriage, preeclampsia, and preterm delivery. EP with some risk factors associated with fallopian tubal abnormalities, e.g., smoking, obvious tubal inflammatory adhesions, previous fallopian tubal diseases, and tubal surgery histories, were also excluded for avoiding complications. In each case, gestational days was 40-64 days confirmed from the last menstrual period. n=5 for both IP and TEP.

first: Q

last: Zhu

first: X

middle: Y

first: J

last: Zhang


title: R-loop landscapes in Rice (Oryza sativa) [DRIP-seq]

summary: The R-loop is a common chromatin feature presented from prokaryotic to eukaryotic genomes and has been revealed to be involved in multiple cellular processes and associated with many human diseases. Here, we take the advantage of our recently developed ssDRIP-seq method to profile genome-wide R-loop levels and provided a first-hand R-loop atlas of Rice (Oryza sativa) at different developmental stages.

overall_design: ssDRIP-seq in Oryza sativa. The gDNA was extracted from different tissues, and fragmented by restriction enzymes. By using S9.6 antibody, DNA-RNA hybrids were pulled down. Based on ssDNA library kit, the ssDRIP library was prepared and sequenced.

first: Wei

last: Xu

first: Sarfraz

last: Shafi

first: Kuan

last: Li

first: Qianwen

last: Sun


title: Transcriptome analysis of tumors isolated from ovarian cancer tumor bearing mice treated with Sindbis Virus vectors and untreated controls

status: Public on Dec 20 2022

city: New York

first: Ziyan

last: Lin

state: NY

department: Applied Bioinformatics Laboratories

country: USA

institute: NYU Grossman School of Medicine

summary:
Gene expression analysis of tumors isolated from mice after they received treatments with Sindbis Virus vectors for 1 week. The hypothesis tested changes in the transcriptome profiles of tumors isolated from Sindbis virus vectors treated and untreated mice.
Expression profiling by high throughput sequencing of MOSEC.Fluc.p11 tumors

overall_design: Total RNA was obtained from tumors isolated from mice seven days after start of treatments and controls (untreated tumor bearing mice). Bulk RNAseq was performed.

first: Silvana

last: Opp

first: Daniel

last: Meruelo


title: Human alveolar macrophage metabolism is compromised during Mycobacterium tuberculosis infection

status: Public on Dec 09 2022

city: Chicago

first: Luis

middle: B

last: Barreiro

address: 900 E 57th Street

institute: University of Chicago

summary: Pulmonary macrophages have two distinct ontogenies: long-lived embryonically-seeded alveolar macrophages (AM) and bone marrow-derived macrophages (BMDM). Here, we show that after infection with a virulent strain of Mycobacterium tuberculosis (H37Rv), primary murine AM exhibit a unique transcriptomic signature characterized by metabolic reprogramming distinct from conventional BMDM. In contrast to BMDM, AM failed to shift from oxidative phosphorylation (OXPHOS) to glycolysis and consequently were unable to control infection with an avirulent strain (H37Ra). Importantly, healthy human AM infected with H37Ra equally demonstrated diminished energetics, recapitulating our observation in the murine model system. However, the results from seahorse showed that the shift towards glycolysis in both AM and BMDM was inhibited by H37Rv. We further demonstrated that pharmacological (e.g. metformin or the iron chelator desferrioxamine) reprogramming of AM towards glycolysis reduced necrosis and enhanced AM capacity to control H37Rv growth. Together, our results indicate that the unique bioenergetics of AM renders these cells a perfect target for Mtb survival and that metabolic reprogramming may be a viable host adjunctive therapy against TB.

overall_design: In this work we compare genome-wide transcriptomics of macrophages from mice (Alveolar, or Bone marrow derived polarized towards M1 or M2 phenotypes) infected, and non-infected in-vitro with Mycobacterium tuberculosis H37RV

first: Laura

middle: E

last: Mendonca

first: Erwan

last: Pernet

first: Nargis

last: Khan

first: Joaquin

last: Sanz

first: Eva

last: Kaufmann

first: Jeffrey

last: Downey

first: Alexandre

last: Grant

first: Marianna

last: Orlova

first: Erwin

last: Schurr

first: Connie

last: Krawczyk

first: Russell

middle: G

last: Jones

first: Maziar

last: Divangahi


title: Expression data of islets between 3- and 7- day old pigs

city: Edmonton

first: Gina

last: Rayat

state: Alberta

department: Faculty of Medicine and Dentistry,

country: Canada

institute: Alberta Diabetes Institute, University of Alberta

summary: The yield, cell composition, and function of islets isolated from various ages of neonatal pigs were characterized using in vitro and in vivo experimental models. Islets from 7- and 10-day old pigs showed significantly better function both in vitro and in vivo compared to islets from 3- and 5-day old pigs however, the islet yield from 10-day old pigs were significantly less than those obtained from the other pigs. Since islets from 3-day old pigs were used in our previous studies and islets from 7-day old pigs reversed diabetes more efficiently than islets from other groups, we further evaluated the function of these islets post-transplantation. B6 rag-/- mouse recipients of various numbers of islets from 7-day old pigs achieved normoglycemia faster and showed significantly improved response to glucose challenge compared to the recipients of the same numbers of islets from 3-dayold pigs. These results are in line with the findings that islets from 7-day-old pigs showed reduced voltage-dependent K+ (Kv) channel activity and their ability to recover from post-hypoxia/reoxygenation stress. Despite more resident immune cells and immunogenic characteristics detected in islets from 7-day old pigs compared to islets from 3-day old pigs, the combination of anti-LFA-1 and anti- CD154 monoclonal antibodies are equally effective at preventing the rejection of islets from both age groups of pigs. Collectively, these results suggest that islets from various ages of neonatal pigs vary in yield, cellular composition, and function. Such parameters may be considered when defining the optimal pancreas donor for islet xenotransplantation studies.

overall_design: Microarray data was used to look at immunogenic differences between 3-day and 7-day old neonatal pigs. Islets were isolated from pancreas tissue of 4 3-day old pigs and 4 7-day old pigs from which RNA was extracted.  Microarrays were ran using Affymetrix® protocols, and analysis was done using GeneSpring and Ingenuity Pathway Analysis software. Lowly expressed probes across all samples were excluded prior to the examining the differentially expressed probes between 3- and 7-day old pigs.

first: Hossein

last: Arefanian

first: Donald

last: Grynoch

first: Thomas

last: Mueller

first: Anne

last: Hutton

first: Konrad

middle: S

last: Famulski

middle: R


title: RNA-seq of murine proliferating neural stem and progenitor cells from Kat7-deleted mouse fetuses

city: Parkville

first: Gordon

middle: K

last: Smyth

state: Vic

address: 1G Royal Pde

department: Bioinformatics

country: Australia

institute: Walter and Eliza Hall Institute of Medical Research

summary: Tissue-specific deletion of the gene encoding the histone acetyltransferase Kat7 (Hbo1) in the developing mouse central nervous system using cre-recombinase expression driven by regulatory sequences of the nestin locus (NesCre transgene) resulted in defective cerebral cortex development, a complete failure of neural stem cells to give rise to neurons and oligodendrocytes during in vitro differentiation, and a failure to express the neuronal differentiation program. Proliferating neural stem and progenitor cells (NPSCs) were isolated from Kat7-deleted and Kat7-intact 14.5 day embryonic mouse fetuses.

overall_design: RNA-seq profiling was undertaken of NSPC isolates from four Kat7-deleted (Kat7lox/loxNesCreT/+) and four Kat7-intact (Kat7+/+NesCreT/+) E14.5 fetuses. For completeness, the litter number that each embryo was obtained from was also recorded.

first: Aaron

last: Lun

last: Voss


title: Massively parallel engineering of persistent CAR-Ts

city: West Haven

first: Ryan

middle: D

last: Chow

state: CT

address: 850 West Campus Drive

institute: Yale University

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Chimeric antigen receptor T cells (CAR-Ts) are transformative cellular therapies; however, current forms of CAR-T face multiple major hurdles. These include antigen loss, tumor microenvironment suppression, trafficking, proliferation, toxicity, and persistence.  An efficient way to overcome these challenges is to engineer thousands of different CAR-T variants, and systematically select the best ones. To achieve this, we develop CLASH, a versatile platform that enables high-efficiency massively parallel CAR-T engineering. In CLASH, pooled AAVs mediate simultaneous gene editing and precise CAR knock-in via massively parallel homology-directed repair (HDR). Such library-scale HDR events produce a pool of stably integrated CAR-T variants each with targeted immune gene editing. In vitro or in vivo CLASH experiments using human CD8 and CD4 T cells subject the CAR-T pools to long-term co-culture or animal models of cancer, enabling unbiased   selection of favorable CAR-T variants with enhanced cancer killing and persistence. Next-generation sequencing of genomic integrations deconvolves the time-course dynamics of knock-in CAR-T variants and identify winning CAR-Ts that survived specific selections. Validations of convergent top genes JADE1, PELI1, EHMT1, NLRP10, KDM4E, TET2 and PRDM1 show that their perturbations in CAR-Ts modulate T cell characteristics such as proliferation, exhaustion and memory phenotypes. A PRDM1 exon3-skip mutant CAR-T exhibits increased proliferation capacity, central memory cell phenotype and longevity, resulting in high-performance in vivo therapeutic efficacy in adoptive cell therapy models across multiple cancer models including solid tumor. Time-course transcriptomics and immune profiling and molecular interrogations demonstrate that PRDM1 exon3-skip CAR-Ts have multiple rewired gene expression patterns and immunological programs. Interrogations of the epigenetic mechanism via histone array, co-immunoprecipitation and genome-wide chromatin binding by Cut-and-Run reveal that deletion of exon-3 leads to disruption of histone H4 binding to the PR domain, thereby altering the regulation of critical downstream immune factors including SELL, CCR7, STAT1, STAT6, CDCA7 and IL7R. The versatility of CLASH is broadly applicable to the engineering of various desired CAR features in a wide range of cell therapies.

overall_design: Cut & Run samples from IgG controls (3 replicates), anti-H3K4me3 (1 replicate, positive control), anti-PRDM1 (2 replicates each of wildtype and mutant PRDM1 exon3del)

first: Xiaoyun

last: Dai

first: Jonathan

last: Park

first: Yaying

last: Du

first: Zhenkun

first: Stanley

last: Lam

first: Sidi

last: Chen


title: Stage- and cell type-specific requirements of ikzf1 during haematopoietic differentiation in zebrafish

city: Freiburg

first: Sagar

address: Hugstetter Straße 55

department: Department of Internal Medicine II

country: Germany

institute: University Medical Center Freiburg

summary: The zinc finger transcription factor Ikaros1 (Ikzf1) is required for lymphoid development in mammals. It is characterized by the presence of four zinc fingers in its DNA binding domain and two zinc fingers in the C-terminal protein interaction module. Here, we describe the phenotypes of zebrafish homozygous for two distinct mutant ikzf1 alleles. The IT325 variant lacks the C-terminal two zinc fingers, whereas the fr105 variant retains only the first zinc finger of the DNA binding domain. Our results indicate that an intact ikzf1 gene is required for larval T cell development, whereas low levels of adult lymphoid development recover in the mutants. By contrast, the mutants exhibit a signature of increased myelopoiesis at larval and adult stages. Of note, both mutants stimulate erythroid differentiation in larvae, indicating that the C-terminal zinc fingers negatively regulate the extent of red blood cell production. An unexpected differential effect of the two mutants on adult erythropoiesis suggests a direct requirement of an intact DNA binding domain for entry of progenitors into the red blood cell lineage. Collectively, our results reinforce the biological differences between larval and adult haematopoiesis, indicate a stage-specific function of ikzf1 in regulating the hierarchical bifurcations of differentiation, and assign distinct functions to the DNA binding domain and the C-terminal zinc fingers.

overall_design: Single cell RNA sequencing was performed to examine the cellular heterogeneity of whole kidney marrow (WKM) cells in wild-type and two distinct mutant ikzf1 alleles. The IT325 variant lacks the C-terminal two zinc fingers, whereas the fr105 variant retains only the first zinc finger of the DNA binding domain.

last: Sagar

first: Isabell

last: Hess

first: Dominic

last: Gruen

last: Boehm


title: Stem cell plasticity, acetylation of H3K14 and de novo gene activation rely on KAT7

first: Belinda

last: Phipson

state: VIC

address: 1G Royal Parade

institute: WEHI

summary: In the conventional model of transcriptional activation, transcription factors bind to response elements and recruit co-factors, including histone acetyltransferases. Contrary to this model, we show that the histone acetyltransferase KAT7 (HBO1/MYST2) is required genome-wide for histone H3 lysine 14 acetylation (H3K14ac). Examining neural stem cells, we found that KAT7 and H3K14ac were present not only at transcribed genes, but also at inactive genes, intergenic regions and in heterochromatin. KAT7 and H3K14ac were not required for the continued transcription of genes that were actively transcribed at the time of loss of KAT7, but indispensable for the activation of repressed genes. The absence of KAT7 abrogated neural stem cell plasticity, diverse differentiation pathways and cerebral cortex development. Reexpression of KAT7 restored stem cell developmental potential. Overexpression of KAT7 enhanced neuron and oligodendrocyte differentiation. Our data suggest that KAT7 prepares chromatin for transcriptional activation and is a prerequisite for gene activation.

overall_design: Gene expression profiling of E16.5 cortical neurons from three wild type and four KAT7 mutant mice.


title: Key transcription factors influence the epigenetic landscape to regulate retinal cell differentiation

city: Buffalo

first: Xiuqian

last: Mu

address: 701 Ellicott Street

department: Ophthalmology

institute: University at Buffalo

summary: How the diverse neural cell types emerge from multipotent neural progenitor cells during central nervous system development remains poorly understood. Recent scRNA-seq studies have delineated the developmental trajectories of individual neural cell types in many neural systems including the neural retina. Further understanding of the formation of neural cell diversity requires knowledge about how the epigenetic landscape shifts along individual cell lineages and how key transcription factors regulate these changes. In this study, we dissect the changes in the epigenetic landscape during early retinal cell differentiation by scATAC-seq and identify globally the enhancers, enriched motifs, and potential interacting transcription factors underlying the cell state/type specific gene expression in individual lineages. Using CUT&Tag-seq, we further identify the enhancers bound directly by four key transcription factors, Otx2, Atoh7, Pou4f2, and Isl1, and uncover their roles in shaping the epigenetic landscape and controlling gene expression in a sequential and combinatorial fashion along individual retinal cell lineages such as retinal ganglion cells (RGCs). Our results reveal a general paradigm in which transcription factors collaborate and compete to regulate the emergence of distinct retinal cell types such as RGCs from multipotent retinal progenitor cells (RPCs).

overall_design: There are 6 sample for scATAC-seq with cells isolated by FACS from E14.5 and E17.7 mouse retinas: Atoh7-Null-E14.5 (zsGreen+ cells from Atoh7-zsGreen/lacZ retinas), Atoh7-Null-E17.5 (zsGreen+ cells from Atoh7-zsGreen/lacZ retina), Atoh7-WT-E14.5 (zsGreen+ cells from Atoh7-zsGreen/+ retinas), Atoh7-WT-E17.5 (zsGreen+ cells from Atoh7-zsGreen/+ retinas), Pou4f2-WT-E14.5 (tdTomato cells from Pou4f2-tdTomato/+ retinas), Pou4f2-WT-E17.5 (tdTomato cells from Pou4f2-tdTomato/+ retinas). scRNA-seq were also performed with the same cell populations. There were also one sample of E14.5 Atoh7/Pou4f2-double negative sample (zsGreen/tdTomaton double negative cells from Atoh7-zsGreen/+;Pou4f2-tdTomaton/+ retinas) for scATAC-seq. There are 4 targets for CUT&Tag on wild type E14.5 mouse retina cells with 2 replicates: Atoh7, Pou4f2, ISL1, OTX2. For Atoh7, anti-HA was used with Atoh7-HA/HA retinas. For Pou4f2, anti-HA was used with Pou4f2-HA/HA retinas. For Otx2, multiple preparations were pooled into two independent replicates. Normal rabbit IgG was used as target blank control. Note that some libraries (Pou4f2_HA Rep1, Isl1 Rep2, and  are sequenced twice, and Rabbit IgG Rep1) were sequenced twice. These read were combined as one replicate in the analysis.

first: Yichen

last: Ge

first: Xushen

last: Bard

first: Fuguo

last: Wu

first: Donald

last: Yergeau

first: Tao

last: Liu

first: Jie

last: Wang


title: Targeted zinc-finger repressors to the oncogenic HBZ gene inhibit adult T-cell leukemia (ATL) proliferation

status: Public on Dec 08 2022

city: Farmington

first: Yuqi

last: Zhao

address: 273 Main Street A4

institute: City Of Hope

summary: Epigenetic changes at the HTLV-I LTR when targeted with ZFP repressors

overall_design: Adult T-cell leukemia (ATL) cell lines TL-Om1 were infected with HTLV-1, treated under different conditions, and then harvested for  ATAC-seq.

first: Tristan

last: Scott

first: Xiwei


title: Principles of 3D chromosome folding and evolutionary genome reshuffling in mammals

city: Cerdanyola del Valles

first: Lucía

last: Alvarez-Gonzalez

state: Barcelona

address: Campus de la UAB, Bellaterra

department: Dept. Biologia Cel.lular, Fisiologia i Immunologia

country: Spain

institute: Universitat Autonoma de Barcelona

summary: Here we describe the principles of 3D genome folding dynamics in vertebrates and show how lineage-specific patterns of genome reshuffling can result in different chromatin configurations. We (i) identified different patterns of chromosome folding across vertebrate species, (ii) reconstructed ancestral marsupial and afrotherian genomes analyzing whole-genome sequences of 10 species representative of the major therian phylogroups, (iii) detected lineage-specific chromosome rearrangements and (iv) identified the dynamics of the structural properties of genome reshuffling through therian evolution.

sample_organism:
Sarcophilus harrisii
Notamacropus eugenii
Loxodonta africana
Orycteropus afer

overall_design: HiC of African elephant, aardvark, Tasmanian devil and tammar wallaby fibroblasts. H3K4me3 ChIP-seq of  African elephant, aardvark, Tasmanian devil and tammar wallaby fibroblasts. CTCF ChIP-seq of African elephant, aardvark fibroblasts. RNA-seq of Tasmanian devil fibroblasts

last: Álvarez-González

first: Laia

last: Marín-Gual

first: Paul

last: Waters

first: Aurora

last: Ruiz-Herrera


title: Heat Shock Protein Family A Member 1 Promotes Intracellular Amplification of Hepatitis B Virus Covalently Closed Circular DNA

city: Doylestown

first: Ju-Tao

last: Guo

state: PA

address: 3805 Old easton road

institute: Baruch S Blumberg Institute

summary: Hepatitis B virus (HBV) contains a partially double-stranded relaxed circular DNA (rcDNA) genome that is converted into a covalently closed circular DNA (cccDNA) in the nucleus of infected hepatocyte by cellular DNA repair machinery. cccDNA associates with nucleosomes to form a minichromosome that transcribes RNA to support the expression of viral proteins and reverse transcriptional replication of viral DNA. In addition to the de novo synthesis from incoming virion rcDNA, cccDNA can also been synthesized from rcDNA in the progeny nucleocapsids in the cytoplasm of infected hepatocytes via the intracellular amplification pathway. In our efforts to identify cellular DNA repair proteins required for cccDNA synthesis by a chemogenetic screen, we found that B02, a small molecular inhibitor of DNA homologous recombination repair protein RAD51, significantly enhanced the synthesis of cccDNA via intracellular amplification pathway in human hepatoma cells. Ironically, neither siRNA knockdown of RAD51 expression nor treatment with another structurally distinct RAD51 inhibitor or activator altered cccDNA amplification. However, further mechanistic studies revealed that B02 treatment significantly elevated the levels of multiple heat shock protein mRNA and siRNA knockdown of HSPA1A expression or treatment with HSPA1 inhibitors significantly attenuated B02 enhancement of cccDNA amplification. Moreover, B02 enhanced cccDNA amplification was efficiently inhibited by compounds that selectively inhibit DNA polymerase α or topoisomerase II, the enzymes required for cccDNA intracellular amplification. Our results thus indicate that B02 treatment induces a heat shock protein-mediated cellular response that positively regulates the conversion of rcDNA into cccDNA via the authentic intracellular amplification pathway

overall_design: mRNA from control or B02 treated HepAD38 cells are sequenced and analyzed in three biological replicates

first: Liudi

last: Tang

first: Ping

last: An

first: Qiong

first: Cheryl

middle: A

last: Winkler

first: Jinhong

last: Chang


title: The miR-497~195 cluster suppresses cell cycle regulators and in vivo leukemia growth in B-cell precursor Acute Lymphoblastic Leukemia

city: Padova

first: Stefania

last: Bortoluzzi

address: via G. Colombo 3

department: Department of Molecular Medicine

country: Italy

institute: University of Padova

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

overall_design: Refer to individual Series


title: Effect of air-liquid interface stimulation on gene expression during keratinocyte differentiation by three-dimensional culture

city: Chikusa-ku

first: Hirofumi

last: Teshima

state: Aichi

address: furo-chou

country: Japan

institute: Nagoya university

summary: To investigate how air-liquid interface stimulation induces epidermal differentiation, we carried out RNA-seq on three-dimensional culture with and without air exposure.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for culture with air-liquid interface and submerged condition.

first: Kiyotaka

last: Hitomi


title: Senescent Langerhans cell histiocytosis cells arise from both dendritic cell (DC) and monocyte/DC3 lineages

city: Stockholm

first: Egle

last: Kvedaraite

address: Tomtebodavägen 18A, floor 8

country: Sweden

institute: Karolinska Institutet

summary: Langerhans cell histiocytosis (LCH) is a potentially fatal myeloid neoplasia, stemming from the mononuclear phagocyte (MNP) system. Discrimination of neoplastic cells that share features with normal MNPs is essential for mapping disease origin and pathogenesis. Here, we performed a deep profiling of LCH cells combining single cell RNA-seq and protein analysis enabling us to assess their heterogeneity and compare to their normal counterparts within the tumor microenvironment. Downstream analysis revealed LCH signatures pointing to senescence and tumor immune surveillance escape mechanisms. Developmentally, two major LCH arms, phenotypically resembling DC2 and monocyte/DC3 lineages, were identified and validated in situ across different pathological tissue sites using high-throughput microscopy. The present results support a dual origin model of LCH with an underlying neoplastic hit prior to the fate commitment to DC2 and monocyte/DC3 lineages, and provides basis for development of future targets in LCH and other histiocytic disorders.

overall_design:
Deep profiling of LCH cells combining single cell RNA-seq and protein analysis to assess their heterogeneity and compare to their normal counterparts within the tumor microenvironment.

>>>Submitter states that raw data will be submitted to EGA due to patient privacy concerns<<<

first: Florent

last: Ginhoux


title: RNAseq in Sirt1 wild-type or conditional KO mouse T-ALLs

city: New Brunswick

first: Daniel

last: Herranz

state: New Jersey

address: 195 Little Albany Street

institute: Rutgers Cancer Institute of New Jersey

summary: C57BL6 mice harboring Sirt1 conditional knockout NOTCH1-DE-induced leukemias were treated with vehicle (control) or tamoxifen to induce isogenic deletion of Sirt1. Here we report the gene expression profile of leukemic blasts obtained from the spleen from control- or tamoxifen-treated leukemic mice.

overall_design: RNAseq analysis of leukemic spleens in two genotypes and five samples per genotype.

first: Olga

last: Lancho

first: Amartya

last: Singh

first: Hossein

last: Khiabanian


title: The miRNAs in plasma exosomes may serve as markers for non-segmental vitiligo

city: Wuhan

first: Yingying

last: Dong

address: 1095 Jie Fang Avenue

department: Department of Dermatology

institute: Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology

summary: More and more studies have showed that plasma exosomal miRNAs are biomarkers for disease. The aim of the study were to investigate the miRNA profiling in plasma exosomes of patients with non-segmental vitiligo (NSV) and to find biomarkers in plasma exosomes for patients with NSV. Plasma exosomes and exosomal RNA of 10 NSV patients and 10 health persons were purified by exoRNeasy Serum/Plasma Maxi Kit. The miRNA profiles of the 20 samples were sequenced using HiSeq 2500 (Illumina) and analyzed by Reads Per Million (RPM) values and edgeR algorithm. Some differently expressed miRNAs in plasma exosomes and skin tissues of the two sets were validated by qRT–PCR.Several miRNAs were confirmed by qRT–PCR and showed similar expression patterns between plasma exosomes and skin tissues. Our study depict the miRNAs expression profiles in plasma exosomes of NSV patients and suggest that several miRNAs in plasma exosomes may serve as biomarkers for NSV.

overall_design: To investigate the miRNA profiling in plasma exosomes of patients with non-segmental vitiligo (NSV) and health persons and to find biomarkers in plasma exosomes for NSV.

first: Yunhua

last: Deng


title: Improving disease resistance in plants by editing the epigenome

city: Los Angeles

first: Zhenhui

last: Zhong

address: 610 Charles E Young Dr East

department: Department of Molecular, Cell and Developmental Biology

institute: University of California, Los Angeles

type: Methylation profiling by high throughput sequencing

summary: Pathogens rely on expression of host susceptibility (S) genes to promote infection and disease. DNA methylation is an epigenetic modification that affects gene expression. Theoretically, blocking access to S genes through targeted methylation could increase disease resistance. Xanthomonas axonopodis pv. manihotis, the causal agent of cassava bacterial blight (CBB), uses transcription activator-like20 (TAL20) to induce expression of the S gene MeSWEET10a. We directed methylation to the TAL20 effector binding element within the MeSWEET10a promoter using a synthetic zinc-finger DNA binding domain fused to a component of the RNA-directed DNA methylation pathway. We demonstrate that this methylation prevents TAL20 binding, blocks transcriptional activation of MeSWEET10a and that these plants display increased resistance to CBB. This work establishes epigenome editing as a new strategy for crop improvement.

overall_design: This dataset includes WGBS data to understand target DNA methylation in cassava.

first: Kira

middle: M

last: Veley


title: Monitoring the 5′UTR landscape reveals isoform switches to drive translational efficiencies in cancer

city: Schlieren

first: Ramona

last: Weber

address: Wagistrasse 12

department: Institute for Regenerative Medicine

country: Switzerland

institute: University of Zurich

summary: Transcriptional and translational control are key determinants of gene expression, however, to what extent these two processes can be collectively coordinated is still poorly understood. Here, we use Nanopore long-read sequencing and cap analysis of gene expression (CAGE-seq) to document the landscape of 5′ and 3′ untranslated region (UTR) isoforms and transcription start sites of epidermal stem cells, wild-type keratinocytes and squamous cell carcinomas. Focusing on squamous cell carcinomas, we show that a small cohort of genes with alternative 5′UTR isoforms exhibit overall increased translational efficiencies and are enriched in ribosomal proteins and splicing factors. By combining polysome fractionations and CAGE-seq, we further characterize two of these UTR isoform genes with identical coding sequences and demonstrate that the underlying transcription start site heterogeneity frequently results in 5′ terminal oligopyrimidine (TOP) and pyrimidine-rich translational element (PRTE) motif switches to drive mTORC1-dependent translation of the mRNA. Genome-wide, we show that highly translated squamous cell carcinoma transcripts switch towards increased use of 5′TOP and PRTE motifs, have generally shorter 5′UTRs and expose decreased RNA secondary structures. Notably, we found that the two 5′TOP motif-containing, but not the TOP-less, RPL21 transcript isoforms strongly correlated with overall survival in human head and neck squamous cell carcinoma patients. Our findings warrant isoform-specific analyses in human cancer datasets and suggest that switching between 5′UTR isoforms is an elegant and simple way to alter protein synthesis rates, set their sensitivity to the mTORC1-dependent nutrient-sensing pathway and direct the translational potential of an mRNA by the precise 5′UTR sequence.

overall_design: RNA from mouse P56-60 epidermis, cultured WT and HRASG12V; TGFBR-2 KO keratinocytes was isolated and used for NANOPORE long-read RNA sequencing. Cultured WT and HRASG12V; TGFBR-2 KO keratinocytes were used for ribosome profiling and short-read RNA sequencing.

first: Ataman

last: Sendoel


title: Title: Elevated SGPL1 expression increases metabolic rate and reduces survival in adrenocortical carcinoma patients

city: London

first: Jack

last: Williams

state: City Of London

address: Molecular Endocrinology, William Harvey Research Institute, Charterhouse Square

country: United Kingdom

institute: Queen Mary University Of London

summary: Sphingosine-1-phosphate lyase (SGPL1) catalyses the final step in sphingolipid metabolism (Figure 1), irreversibly degrading the lipid signalling molecule sphingosine-1-phosphate (S1P).  Loss-of-function mutations in SGPL1 cause a spectrum of disorders, including primary adrenal insufficiency. Given loss of SGPL1 causes reduced steroidogenesis, while increased steroidogenesis is a hallmark of aggressive adrenocortical carcinomas (ACCs), we hypothesise increased SGPL1 expression might regulate ACCs. We identified a potential novel biomarker in ACCs, whereby increased SGPL1 expression correlates with reduced patient survival. We also saw similar correlation with other sphingolipid enzymes which diminish the ceramide pool, while those that replenish it confer a beneficial patient outcome. Increased SGPL1 expression in a cell model increased their resting proliferation and migration, although no effect on apoptosis was seen. There was a global decrease in the sphingolipid pool, possibly indicating increased flux through this pathway. RNAseq analysis identified an increase in expression of oxidative phosphorylation genes, and this correlated with an increase in basal and maximal glycolysis and oxidative phosphorylation. Finally, we showed that the increase in proliferation in the OE cells is dependent on the presence of metabolic substrates which may be used for anabolism and/or the production of ATP.

overall_design: We generated SGPL1 overexpressing H295R cells, as well as SGPL1 KO H295R cells and compared their transcriptomes by RNAseq. We used 3 biological replicates per group.

middle: L

first: Charlotte

last: Hall

first: Chris

last: Smith

first: Ruth

last: Kwong

first: Lou

last: Metherell

first: Rathi

last: Prasad


title: Single-cell RNA-Seq datasets profiling PBMCs from venous blood supply to antler velvet or to backskin 3 days post-wound

city: Calgary

first: Jeff

last: Biernaskie

address: 3330 Hospital Drive NW

department: Comparative Biology and Experimental Medicine

institute: University of Calgary

summary: Project abstract: In adult mammals, skin wound healing has evolved to favor rapid repair through the formation of fibrotic scar. These dermal scars are dysfunctional and may lead to chronic disfigurement and disability, yet the biologic mechanisms that drive fibrosis and prevent tissue regeneration remain unknown. Here, we report that reindeer (Rangifer tarandus) antler velvet exhibits regenerative wound healing, whereas identical full-thickness injury in dorsal back skin of the same animal forms fibrotic scar. This regenerative capacity is retained even following ectopic transplantation of velvet to a scar-forming site, demonstrating that this latent regenerative capacity is innate to velvet cells and independent of local factors derived from the growing antler. Single cell RNA-sequencing of uninjured skin revealed a marked divergence in resting fibroblast transcriptional states and immunomodulatory function. Uninjured velvet fibroblast shared a striking resemblance with human fetal fibroblasts whereas uninjured back skin fibroblasts exhibited an overrepresentation of pro-inflammatory genes resembling adult human fibroblasts. Identical skin injury resulted in site-specific fibroblast polarization; back fibroblasts exacerbated the inflammatory response, whereas velvet fibroblasts adopted an immunosuppressive state and reverted back to a regeneration-competent ground state. Consequently, velvet wounds exhibited an accelerated adoption of anti-inflammatory immune states and an expedited resolution of immune response. This study demonstrates reindeer as a novel comparative mammalian model to study both adult skin regeneration (velvet) and scar formation (back skin) within the same animal. Our study underscores the importance of fibroblast heterogeneity in shaping local immune cell functions that ultimately polarize wound healing outcomes. Purposeful, acute modulation of fibroblast-mediated immune signaling represents an important therapeutic avenue to mitigate scar and improve wound healing.

overall_design: PBMCs were collected from venous blood supply from saphenous vein or from distal antler velvet into EDTA tubes and briefly stored on ice.  Blood was spun at 300g for 10 min and plasma removed and stored at -80oC. Buffy coat was collected, and red blood cells were lysed with ACK buffer (1:10 ACK to blood cells) for 5 minutes and then spun at 300g for 5min and then supernatant.  Cells were washed twice with 5mL PBS, then spun at 300g 5 min.  Cells were resuspended in 2% BSA in PBS at a final concentration of 30,000 cells/100uL and run through a 40µm cell strainer, before loading into the 10X controller (~12,000 cells). All samples were processed according to 10X Genomics ChromiumTM Single Cell 3’ Reagent Guidelines NextGEM Chemistry as per the manufacturer’s protocol. In brief, cells were partitioned into Gel Bead-In-EMulsions (GEMs) using 10xTM GemCodeTM Technology. This process lysed cells and enabled barcoded reverse transcription of RNA, generating full-length cDNA from poly-adenylated mRNA. DynaBeads® MyOneTM Silane magnetic beads were used to remove leftover biochemical reagents, then cDNA was amplified by PCR. Quality control size gating was used to select cDNA amplicon size prior to library construction. Read 1 primer sequences were added to cDNA during GEM incubation. D11 primers, D12 and C12  primers, i7 sample index, and Read 2 primer sequences were added during library construction. Quality control and cDNA quantification was performed using DTape station 1000. Sequencing was performed using Illumina NovaSeq with an S2 flow cell.

first: Sarthak

last: Sinha

first: Holly

last: Sparks

first: Hayley

last: Robbins

first: Kevin

last: Gowing

first: Arzina

last: Jaffer

first: Rohit

last: Arora

first: Sam

last: Raredon

first: Leslie

last: Cao

first: Scott

last: Swanson

first: Peng

last: Jiang

first: Olivia

last: Hee

first: Hanna

last: Pope

first: Elodie

last: Labit

first: Matt

last: Workentine

first: Laura

last: Niklason

first: Nicole

last: Rosin

first: Greg

last: Muench

first: Ron

last: Stewart

first: John

last: Matyas

first: Robert

last: McCorkell


title: Single-cell chromatin landscapes supporting fibroblast polarization drives skin regeneration versus fibrosis in adult reindeer

first: Jeff

last: Biernaskie

summary: In adult mammals, skin wound healing has evolved to favor rapid repair through the formation of fibrotic scar. These dermal scars are dysfunctional and may lead to chronic disfigurement and disability, yet the biologic mechanisms that drive fibrosis and prevent tissue regeneration remain unknown. Here, we report that reindeer (Rangifer tarandus) antler velvet exhibits regenerative wound healing, whereas identical full-thickness injury in dorsal back skin of the same animal forms fibrotic scar. This regenerative capacity is retained even following ectopic transplantation of velvet to a scar-forming site, demonstrating that this latent regenerative capacity is innate to velvet cells and independent of local factors derived from the growing antler. Single cell RNA-sequencing of uninjured skin revealed a marked divergence in resting fibroblast transcriptional states and immunomodulatory function. Uninjured velvet fibroblast shared a striking resemblance with human fetal fibroblasts whereas uninjured back skin fibroblasts exhibited an overrepresentation of pro-inflammatory genes resembling adult human fibroblasts. Identical skin injury resulted in site-specific fibroblast polarization; back fibroblasts exacerbated the inflammatory response, whereas velvet fibroblasts adopted an immunosuppressive state and reverted back to a regeneration-competent ground state. Consequently, velvet wounds exhibited an accelerated adoption of anti-inflammatory immune states and an expedited resolution of immune response. This study demonstrates reindeer as a novel comparative mammalian model to study both adult skin regeneration (velvet) and scar formation (back skin) within the same animal. Our study underscores the importance of fibroblast heterogeneity in shaping local immune cell functions that ultimately polarize wound healing outcomes. Purposeful, acute modulation of fibroblast-mediated immune signaling represents an important therapeutic avenue to mitigate scar and improve wound healing.

overall_design: Back and velvet skin were wounded by making 12mm circular excisions to remove a full thickness piece of tissue. Healing back and velvet skin (along with uninjured D0 skin) were harvested at 7 days following initial injury. To investigate regenerative (velvet) and non-regenerative (back) wound healing mechanisms, we isolated single-cells from all  samples and processed according to 10X Genomics Chromium Next GEM Single Cell ATAC Library & Gel Bead Kit v1 Chemistry Guidelines as per the manufacturer’s protocol. In brief, single cells were sorted based on forward versus side scatter gating into 0.1% BSA–PBS and partitioned into Gel Bead-In-EMulsions (GEMs) using 10x GemCodeTM Technology. Next-Generation Sequencing was performed using the Illumina NovaSeq SP and S1 Flow cells using paired-end dual indexing. All raw FASTQs were aligned to the bovine (Bos taurus) reference genome generated using cellranger-atac mkref pipeline. The resulting filtered peak barcode matrix in hdf5 format of the paired back and antler samples for each time point were processed using Signac v.1.2.1.

first: Sarthak

last: Sinha

first: Holly

last: Sparks

first: Hayley

last: Robbins

first: Kevin

last: Gowing

first: Micha


title: A therapeutically targetable NOTCH1-SIRT1-KAT7 axis in T-ALL

first: Daniel

last: Herranz

summary: C57BL6 mice harboring Sirt1 conditional knockout NOTCH1-DE-induced leukemias were treated with vehicle (control) or tamoxifen to induce isogenic deletion of Sirt1. Here we report the H4K12ac ChIP-seq epigenetic profile of leukemic blasts obtained from the spleen from control- or tamoxifen-treated leukemic mice.

overall_design: Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) for histone modification H4K12ac in mice

first: Olga

last: Lancho

first: Victoria

last: da Silva-Diz

first: Hossein

last: Khiabanian

first: Amartya

last: Singh


title: Bulk RNA-Seq datasets supporting fibroblast polarization drives skin regeneration versus fibrosis in adult reindeer

summary: In adult mammals, skin wound healing has evolved to favor rapid repair through formation of fibrotic scar.  Consequently, skin wounds are dysfunctional and lead to chronic disfigurement and disability, yet the biologic mechanisms that drive fibrosis and prevent tissue regeneration remain unknown. Here, we report that reindeer (Rangifer tarandus) antler velvet exhibits regenerative wound healing, whereas identical full-thickness injury in dorsal back skin forms fibrotic scar. This regenerative capacity is retained even following ectopic transplantation of velvet to a scar-forming site, demonstrating that this latent regenerative capacity is innate to velvet cells and independent of local factors derived from the growing antler. Single cell RNA-sequencing of uninjured skin revealed a marked divergence in resting fibroblast transcriptional states and immunomodulatory function. Uninjured velvet fibroblast shared a striking resemblance with human fetal fibroblasts whereas uninjured back skin fibroblasts exhibited an overrepresentation of pro-inflammatory genes resembling adult human fibroblasts.  Identical skin injury resulted in site-specific fibroblast polarization; back fibroblasts exacerbated the inflammatory response, whereas velvet fibroblasts adopted an immunosuppressive state and reverted back to a regeneration-competent ground state. Consequently, velvet wounds exhibited reduced immune infiltrate, accelerated adoption of anti-inflammatory immune states and expedited resolution of immune response. This study demonstrates reindeer as a novel mammalian model to study adult skin regeneration (velvet) and scar formation (back skin) within the same animal. Our study underscores the importance of fibroblast heterogeneity in shaping local immune cell functions that ultimately polarize wound healing outcomes. Purposeful, acute modulation of fibroblast-mediated immune signaling represents an important therapeutic avenue to mitigate scar and improve wound healing.

overall_design: 34 samples were taken from 4 different animals, 2 wound sites, and 4 different time points. Expression values from Single-End bulk RNA-Seq.

first: Arzina

last: Jaffer

first: Rohit

last: Arora

last: Raredon

first: Leslie

last: Cao

first: Scott

last: Swanson

first: Peng

last: Jiang

first: Olivia

last: Hee

first: Hanna

last: Pope

first: Elodie

last: Labit

first: Matt

last: Workentine

first: Laura


title: Monitoring the 5′UTR landscape reveals isoform switches to drive translational efficiencies in cancer [CAGE-seq]

first: Ramona

last: Weber

overall_design: RNA from mouse P56-60 epidermis, cultured WT and HRASG12V; TGFBR-2 KO keratinocytes was isolated and used for cap analysis of gene expression (CAGE sequencing).

first: Ataman

last: Sendoel


title: Establishment of EGFR-inhibitor resistant triple negative breast cancer models from patient derived xenografts

status: Public on Dec 07 2022

city: Richmond

first: Wright Center for Clinical and Translational Research

last: Bioinformatics Core

state: VA

address: 203 E Cary St

institute: Virginia Commonwealth University


title: Establishment of EGFR-inhibitor resistant triple negative breast cancer models from patient derived xenografts [scRNA-seq]

summary:
Aim: The goals of this study were to identify transcriptomic changes that arise in basal-like breast cancer cells during the development of resistance to epidermal growth factor receptor inhibitors (EGFRi) and to identify drugs that are cytotoxic once EGFRi resistance occurs.
Methods: Human patient-derived xenografts (PDXs) were grown in immunodeficient mice and were treated with a set of EGFRi; the EGFRi erlotinib was selected for more expansive in vivo studies. Single-cell RNA sequencing was performed on mammary tumors from the basal-like PDX WHIM2 that was treated with vehicle or erlotinib for nine weeks. The PDX was then subjected to long-term erlotinib treatment in vivo and through serialserially passaging, an erlotinib-resistant subline of WHIM2 was generated. Bulk RNA-sequencing was performed on parental and erlotinib-resistant tumors. In vitro high-throughput drug screening with >500 clinically used compounds was performed on parental and erlotinib-resistant cells. Previously published bulk gene expression microarray data from MMTV-Wnt1 tumors were contrasted with the WHIM2 PDX data.
Results: Erlotinib effectively inhibited WHIM2 tumor growth for ~four weeks. Compared to untreated cells, single-cell RNA sequencing revealed that a greater proportion of erlotinib-treated cells were in the G1 phase of the cell cycle. Comparison of WHIM2 and MMTV-Wnt1 RNA level data revealed a set of 37 overlapping genes that were differentially expressed in the erlotinib-resistant subsets of WHIM2 and MMTV-Wnt1 tumors. Comparison of all three data types revealed five genes that were upregulated across all erlotinib-resistant samples: IL19, KLK7, LCN2, SAA1, and SAA2. Despite transcriptomic differences, parental and erlotinib-resistant WHIM2 displayed similar responses to the majority of drugs assessed for cytotoxicity in vitro.
Conclusion: This study identified transcriptomic changes arising in erlotinib-resistant basal-like breast cancer. Interestingly, four of the five genes that were found to be upregulated in the basal-like erlotinib-resistant samples were also upregulated in erlotinib-resistant non-small cell lung cancer samples. These gene signatures could be used to identify basal-like breast cancer patients that would best respond to EGFRi. Future studies should explore the predictive capacity of these gene signatures as well as how these differentially expressed genes contribute to the development of EGFRi resistance.

overall_design: Tumor scRNA profiles of untreated and erlotinib treated WHIM2 TNBC PDX grown in NSG mice

first: Narmeen

last: Rashid

first: David

last: Boyd

first: Amy

last: Olex

first: Jacqueline

last: Grible

first: Alex

last: Duoung

first: Mohammad

last: Alzubi

first: Julia

last: Altman

first: Tess

middle: J

last: Leftwich

first: Mikhail

last: Dozmorov

first: J

last: Harrell


title: Single-cell RNA-Seq datasets supporting fibroblast polarization drives skin regeneration versus fibrosis in adult reindeer

first: Jeff

last: Biernaskie

summary: In adult mammals, skin wound healing has evolved to favor rapid repair through the formation of fibrotic scar.  These dermal scars are dysfunctional and may lead to chronic disfigurement and disability, yet the biologic mechanisms that drive fibrosis and prevent tissue regeneration remain unknown. Here, we report that reindeer (Rangifer tarandus) antler velvet exhibits regenerative wound healing, whereas identical full-thickness injury in dorsal back skin of the same animal forms fibrotic scar. This regenerative capacity is retained even following ectopic transplantation of velvet to a scar-forming site, demonstrating that this latent regenerative capacity is innate to velvet cells and independent of local factors derived from the growing antler. Single cell RNA-sequencing of uninjured skin revealed a marked divergence in resting fibroblast transcriptional states and immunomodulatory function. Uninjured velvet fibroblast shared a striking resemblance with human fetal fibroblasts whereas uninjured back skin fibroblasts exhibited an overrepresentation of pro-inflammatory genes resembling adult human fibroblasts.  Identical skin injury resulted in site-specific fibroblast polarization; back fibroblasts exacerbated the inflammatory response, whereas velvet fibroblasts adopted an immunosuppressive state and reverted back to a regeneration-competent ground state. Consequently, velvet wounds exhibited an accelerated adoption of anti-inflammatory immune states and an expedited resolution of immune response. This study demonstrates reindeer as a novel comparative mammalian model to study both adult skin regeneration (velvet) and scar formation (back skin) within the same animal. Our study underscores the importance of fibroblast heterogeneity in shaping local immune cell functions that ultimately polarize wound healing outcomes. Purposeful, acute modulation of fibroblast-mediated immune signaling represents an important therapeutic avenue to mitigate scar and improve wound healing.

overall_design: Back and velvet skin were wounded by making 12mm circular excisions to remove a full thickness piece of tissue. Healing back and velvet skin (along with uninjured D0 skin) were harvested at 3, 7 and 14 days following initial injury. To investigate regenerative (velvet) and non-regenerative (back) wound healing mechanisms, we isolated single-cells from all eight samples and processed according to 10X Genomics Chromium Single Cell 3’ Reagent Guidelines v3 Chemistry as per the manufacturer’s protocol. In brief, single cells were sorted based on forward versus side scatter gating into 0.1% BSA–PBS and partitioned into Gel Bead-In-EMulsions (GEMs) using 10x GemCodeTM Technology. Next-Generation Sequencing was performed using the Illumina NovaSeq SP and S2 Flow cells. All raw FASTQs were aligned to the bovine (Bos taurus) reference genome generated using cellranger mkref pipeline. The resulting gene barcode matrix of the paired back and antler samples for each time point were processed using Seurat (v3.1.1) R tool.

first: Sarthak

last: Sinha

first: Holly

last: Sparks

first: Hayley

last: Robbins

first: Kevin

last: Gowing

first: Arzina

last: Jaffer

first: Rohit

last: Arora

first: Micha

last: Raredon

first: Leslie

last: Cao

first: Scott

last: Swanson

first: Peng

last: Jiang

first: Olivia

last: Hee

first: Hanna

last: Niklason

first: Nicole

last: Rosin

first: Greg

last: Muench

first: Ron

last: Stewart

first: John

last: Matyas

first: Robert

last: McCorkell


title: TONSOKU reads the histone H3.1 variant to mediate DNA repair during replication

city: New Haven

first: Yannick

last: Jacob

state: Connecticut

address: YSB 416 260 Whitney Avenue

department: Department of Molecular, Cellular  & Developmental Biology

institute: Yale university

summary: The histone H3.1 variant is specifically inserted on chromatin during DNA replication in eukaryotes. A single amino acid variation (position 31) in the N-terminal tail of H3.1 compared to replication-independent H3.3 is conserved in plants and animals, but no function has been assigned to amino acid 31 to justify a role for H3.1 during replication. In this study, we demonstrate that the protein TONSOKU (TSK/TONSL), which participates in rescuing broken replication forks, specifically interacts with H3.1 via recognition of alanine 31 by its tetratricopeptide repeat (TPR) domain. We show that genomic amplification in the absence of H3K27me1 in plants depends on H3.1, TSK and DNA polymerase theta (Pol θ). Overall, our study reveals similar strategies in plants and animals for regulating TSK activity and post-replicative chromatin maturation, with both clades using the H3.1 variant and different histone mono-methyltransferases (ATXR5/ATXR6 or SET8) to achieve this.

overall_design: Examination of RNA seq  in 4 different backgrounds col0, atxr5/6, tsk. atxr5/6 tsk and DNA sequencing in 6 different backgrounds (col0, atxr5/6, tsk. atxr5/6 tsk, polq and atxr5/6 polq )

first: Hossein

last: Davarinejad

first: Yi-Chun

last: Huang

first: Benoit

last: Mermaz

first: Chantal

last: LeBlanc

first: Axel

last: Poulet

first: Geoffrey

last: Thomson

first: Valentin

last: Joly

first: Marcelo

last: Munoz

first: Alexis

last: Arvanitis-Vigneault

first: Gonzalo

last: Villarino

first: Benjamin

last: Rotstein

first: Emilio

middle: I

last: Alarcon

first: Joseph

last: Brunzelle

first: Jie

last: Dong

first: Jean-François

last: Couture


title: mRNA transport, translation, and decay in adult mammalian central nervous system axons

status: Public on Dec 06 2022

city: Seoul

first: Jane

last: Jung

state: Yonsei-ro, Seodaemun-gu

department: Department of Anatomy

country: South Korea

institute: Yonsei University College of Medicine

summary: Localized mRNA translation regulates synapse function and axon maintenance, but how compartment-specific mRNA repertoires are regulated is largely unknown. We developed an axonal transcriptome capture method that allows deep sequencing of metabolically-labeled mRNAs from retinal ganglion cell axon terminals in mouse. Comparing axonal-to-somal transcriptomes and axonal translatome-to-transcriptome enables genome-wide visualization of mRNA transport and translation and unveils potential regulators tuned to each process. FMRP and TDP-43 stand out as key regulators of transport, and experiments in Fmr1 knock-out mice validate FMRP’s role in the axonal transportation of synapse-related mRNAs. Pulse-and-chase experiments enable genome-wide assessment of mRNA stability in axons and reveal a strong coupling between mRNA translation and decay. Measuring the absolute mRNA abundance per axon terminal shows that the axonal transcriptome is stably maintained in adulthood by persistent transport. Our datasets provide a rich resource for unique insights into RNA-based mechanisms in maintaining presynaptic structure and function in vivo.

overall_design: Poly-adenylated RNAs in mouse retinal ganglion cell axons terminating at the superior colliculus or lateral geniculate nucleus. For the total RNA, EU (5’-Ethynyl Uridine) was injected into the vitreous to label newly transcribed RNAs and EU-labeled RNAs were affinity purified from the superior colliculus. For the ribosome-bound RNA, axon-TRAP was performed.

first: Hosung


title: Establishment of EGFR-inhibitor resistant triple negative breast cancer models from patient derived xenografts [Bulk RNA-seq]

first: Wright Center for Clinical and Translational Research

last: Bioinformatics Core

first: Narmeen

last: Rashid

first: David

last: Boyd

first: Amy

last: Olex

first: Jacqueline

last: Grible

first: Alex

last: Duoung

first: Mohammad

last: Alzubi

first: Julia

last: Altman

first: Tess

last: Leftwich

first: Mikhail

last: Dozmorov

first: J

last: Harrell


title: p38 MAPK-dependent phosphorylation of TFEB promotes monocyte-to-macrophage differentiation

city: Bethesda

first: Jose

last: Martina

state: Maryland

address: 50 South Dr., Building 50, Room 3533

department: NHLBI, CDBC

institute: NIH

summary: In addition to the role in lysosomal biogenesis and autophagy, TFEB has been described to participate in the control of inflammation and host defense against pathogen infection  The activation of TFEB in toll-like receptor-induced or bacteria exposed macrophages is regulated by a mechanism independent of mTORC1 inactivation, suggesting that other protein kinases may participate in the regulation of TFEB function during macrophage activation  In this study, we identified a novel role of p38 MAPK in TFEB regulation. We showed p38-dependent TFEB phosphorylation at serine 401 (S401) in response to a variety of stress conditions, including oxidative stress, UVC irradiation, growth factors, and lipopolysaccharide (LPS) treatment. Furthermore, inhibition of S401 phosphorylation during PMA-induced monocyte differentiation prevented TFEB nuclear accumulation and resulted in reduced expression of multiple immune genes. TFEB-S401A expressing M0 macrophages also failed to efficiently polarize into M1 inflammatory macrophages, showing defective upregulation of cytokines and chemokines, as well as reduced inflammasome activation. We conclude that TFEB is a target of the p38 signaling pathway that is required for monocyte/macrophage differentiation and function.

overall_design: Analysis of mRNA expression in THP1-TFEB-WT or TFEB-S401A cells in the presence or absence of PMA treatment.

first: José

first: Eutteum

last: Jeong

first: Rosa

last: Puertollano


title: Chem-map: Profiling drug binding to chromatin in cells

city: Cambridge

first: Larry

last: Melidis

state: Not US or Canada

address: Robinson Way

department: CRUK-CI

institute: University of Cambridge

summary: We present a method of mapping binding sites of small molecules on chromatine targets in situ, in U2OS and K562 cell lines

overall_design: Chem-map to map chromatin interacting small molecule binding at the whole genome level

first: Zutao

last: Yu

first: Jochen

last: Spiegel

first: Shankar

last: Balasubramanian


summary: The transcription factor EB (TFEB) regulates energy homeostasis and cellular response to a wide variety of stress conditions, including nutrient deprivation, oxidative stress, organelle damage, and pathogens. Here we identify S401 as a novel phosphorylation site within the TFEB proline-rich domain. Phosphorylation of S401 increases significantly in response to oxidative stress, UVC light, growth factors and LPS, whereas this increase is prevented by p38 MAPK inhibition or depletion, revealing a new role for p38 MAPK in TFEB regulation. Mutation of S401 in THP1 cells demonstrates that the p38 MAPK/TFEB pathway plays a particularly relevant role during monocyte differentiation into macrophages. TFEB-S401A monocytes fail to upregulate expression of multiple immune genes in response to PMA-induced differentiation, including critical cytokines, chemokines, and growth factors. Polarization of M0 macrophages into M1 inflammatory macrophages is also aberrant in TFEB-S401A cells. These results indicate that TFEB-S401 phosphorylation links differentiation signals to the transcriptional control of monocyte differentiation.

overall_design: Analysis of TFEB binding to promoter of immune genes in THP1-TFEB-WT or TFEB-S401A cells treated with PMA.


title: RNA sequencing data of liver macrophages from ND/WD mice

city: Dallas

first: Zhenyu

last: Zhong

state: Texas

address: 5323 Harry Hines Blvd, NA7.108B

department: Immunology

institute: UT Southwestern Medical Center

summary: Genes expression profile in liver macrophages isolated from wild type C57BL/6J mice fed either a western diet or normal diet

overall_design: Two groups of n = 4 of western diet or normal diet fed mice

first: Xiaochen

last: Wang

first: Shuang

last: Liang


title: RNA sequencing data of liver macrophages

summary: Genes expression profile in liver macrophages isolated from wild type C57BL/6J mice fed either a high fat or normal diet for 18 weeks

overall_design: Two groups of n = 3 of high fat diet or normal diet fed mice for 18 weeks of treatment starting at about 6 weeks of age


title: Lumbar spinal cords: Sham rats vs. chronic constriction injury (CCI) induced chronic neuropathic pain rats

city: Zhuhai

first: Ying

state: Guangdong

address: 52 Meihua East Road

institute: The Fifth Affiliated Hospital, Sun Yat-sen University

summary: To investigate the role of DNA methylation in modulating chronic neuropathic pain (NPP), and identify possible target genes of DNA methylation involved in this process. The chronic constriction injury (CCI) induced NPP model was used. The Arraystar Rat RefSeq Promoter Arrays were used to identify the methylation profiles at the genome-wide level in the DNA promoter regions of the lumbar spinal cord in rats 14 Days following CCI surgery. The underlying genes with differential methylation were then identified and submitted to Gene Ontology and pathway analysis. Methyl-DNA immunoprecipitation quantitative PCR (MeDIP-qPCR) and quantitative reverse transcription-PCR (RT‒qPCR) were used to confirm gene methylation and expression.

overall_design: Two-condition experiment, lumbar spinal cord in sham rats vs. CCI rats. Biological replicates: 3 sham replicates, 3 CCI replicates.


title: Syntaphilin regulates neutrophil migration in cancer

city: Philadelphia

first: Priyankara

last: Wickramasinghe

address: 3601 Spruce Street

institute: The Wistar Institute

summary: Pathologically activated neutrophils (PMN) with immune suppressive activity - myeloid-derived suppressor cells (PMN-MDSC) - play a critical role in regulation of tumor progression. They were implicated in promotion of tumor metastases by contributing to pre-metastatic niche formation. This effect was facilitated by enhanced spontaneous migration of PMN in early stage cancers. The molecular mechanisms underpinning this phenomenon remained unclear. We found that syntaphilin (SNPH), a cytoskeletal protein previously known for anchoring mitochondria to the microtubule in neurons and tumor cells, could regulate migration of PMN. Expression of SNPH was decreased in PMN from tumor bearing mice and cancer patients as compared to PMN from tumor-free mice and healthy donors, respectively. Deletion of SNPH increased spontaneous migration of PMN and promoted tumor metastasis. Mechanistically, deletion of SNPH increased the speed and distance travelled by mitochondria, elevated rates of oxidative phosphorylation and glycolysis, and increased generation of adenosine. Thus, our study reveals a molecular mechanism regulating increased migratory activity of PMN during cancer progression and suggests novel targeting opportunity.

overall_design: RNA-seq experiment in SNPH WT and KO cells

first: Shuyu

last: Fu

first: Dmitry

last: Gabrilovich


title: Maternal Western Diet is Associated with Distinct Preclinical Pediatric NAFLD Phenotypes in Juvenile Nonhuman Primate Offspring [Liver RNA-seq]

status: Public on Dec 05 2022

city: Oklahoma city

first: Philip

last: ONeill

state: OK

address: 945 9th st

institute: OUHSC

summary: Pediatric nonalcoholic fatty liver disease (NAFLD) has distinct and variable pathology that affects up to 10% of children, yet causation remains unclear. We have shown that maternal Western- style diet (mWSD) compared with chow diet (mCD) consumption in nonhuman primates (NHP) produces hepatic injury and steatosis in fetal offspring. Here, we define the role of mWSD and postweaning WSD (pwWSD) exposures on potential molecular mechanisms linked to NAFLD development in a cohort of 3-year-old (3YO) juvenile NHP offspring exposed to mCD or mWSD followed by CD or WSD after weaning. We used histologic, transcriptomic, and metabolomic analyses to identify hepatic pathways regulating NAFLD. Offspring exposed to mWSD showed increased hepatic periportal collagen deposition but unchanged hepatic triglyceride levels and body weight. mWSD was associated with a down-regulation of gene expression pathways underlying HNF4 activity, antioxidant signaling, mitochondrial biogenesis, and PPAR signaling. In offspring exposed to both mWSD and pwWSD, liver RNA profiles showed up-regulation of pathways promoting fibrosis and ER stress. pwWSD increased acylcarnitines and decreased anti- inflammatory fatty acids, which was more pronounced when coupled with mWSD exposure. Further, mWSD shifted liver metabolites towards decreased purine catabolism in favor of synthesis, suggesting a mitochondrial DNA repair response.

overall_design: 3-year-old (3YO) juvenile Japanese macaque offspring exposed to maternal control diet (CD) or maternal western-style diet (WSD) during gestation and lactation.  At weaning, offspring were fed either a CD or WSD. Liver tissue samples were collected at 3 years of age and used for RNA-seq. Treatment/conditions represent: WSD/WSD = maternal WSD and postweaning WSD, CD/WSD = maternal CD and postweaning WSD, CD/CD = maternal CD and postweaning CD,  and WSD/CD = maternal WSD and postweaning CD.

first: Jacob

last: Friedman

first: Michael

last: Nash

first: Stephanie

last: Wesolowski

first: Kenneth

last: Jones

middle: H

last: O'Neill


title: Comparison of the Human Transcriptional Response to Three Hypoxic Environments

city: Oklahoma City

first: Susan

middle: Kasko

last: Munster

address: 6500 S. MacArthur Blvd.

department: Office of Aerospace Medicine

institute: Federal Aviation Admininistration

summary:
Subjects were exposed to three hypoxic environments, a hypobaric altitude chamber (AC), a restricted oxygen breathing device (ROBD), and a restricted oxygen breathing environment (ROBE), and blood was collected during each exposure for total RNA analysis.
Microarrays were used to examine the transcriptional response from subjects for each of the three conditions and results were compared.  Few differences were noted in gene expresssion when comparing AC, ROBD, and ROBE.

overall_design: Subjects were exposed to three hypoxic environments and had blood collected prior to exposure, immediately after exposure was completed, 15 minutes after exposure was completed, and 2 hours after exposure was completed.  RNA was extracted from blood samples, amplified, and hybridized onto HTA 2.0 microarrays.  Sampel data was analyzed to determine if genes were differentially expressed and also to determine if there were significantly changed genes over the timepoints tested.

first: Scott

last: Nicholson

first: Vicky

last: White

first: Dennis

last: Burian

first: Megan

last: Gallo

first: Molly

last: Wade

first: Darrin

last: Bryant

first: Bruce

last: Wright


title: Real-time quantitative PCR analysis of microRNAs expressed in adult mouse ventricular-subventricular zone cell populations

city: Basel

first: Fiona

last: Doetsch

address: Spitalstrasse 41

department: Biozentrum

institute: University of Basel

summary: Total RNA was isolated from GFAP::GFP+CD133+EGFR-CD24- (quiescent neural stem cells, qNSCs), GFAP::GFP+CD133+EGFR+CD24- (activated neural stem cells, aNSCs) and GFAP::GFP+CD133- EGFR+CD24- (transit amplifying cells, TACs) cells from the adult mouse ventricular-subventricular zone (V-SVZ) (GFAP::GFP mice, Jackson Mice Stock number 003257).

overall_design:
qPCR gene expression profiling. 8 total samples consisting of three biological replicates of qNSCs and aNSCs, and two biological replicates of TACs
miRNA expression profiling by RT-PCR

first: Fabrizio

last: Favaloro

first: Annina

last: DeLeo


title: Shared Graft Versus Leukemia Minor Histocompatibility Antigens in DISCOVeRY-BMT

city: Chapel Hill

first: Kelly

middle: Shea

last: Olsen

state: NC

address: 5230E Marsico Hall, 125 Mason Farm Road

department: Lineberger Comprehensive Cancer Center

institute: University of North Carolina at Chapel Hill

summary:
To predict shared graft versus leukemia minor histocompatibility antigens (mHAs) from the DISCOVeRY-BMT dataset.
Via mass spectrometry, we then validated predicted mHAs in cell lines with the corresponding HLA allele and nonzero RNAseq reads corresponding to the source gene of the mHA.

overall_design: Analysis of read counts from RNAseq data for five AML cell lines: Nomo1, Monomac1, NB4, OCI-AML3, and U937A2.

first: Benjamin

last: Vincent


title: Genes expression profile in BMDM challenging with S1P or DAMPs

first: Zhenyu

last: Zhong

summary: To explore the differently expressed genes in BMDM after the treatment with S1P or DAMPs (Damage associated molecular patterns). BMDM cells were incubated with apoptotic cell culture medium (ACM) or S1P (500nM) for 24 hours before harvest for RNA extraction. ACM generated from palmitic acid treated AML12 cells was used as DAMPs. We also want to see whether these genes regulated by S1P or DAMPs are Trem2 dependent or independent.

overall_design: Three different treatments, DAMPs (ACM, apoptotic cell culture medium), S1P, and untreated control, were applied to primary BMDMs from mice of two different genotypes: Trem2(-/-) and WT. Each treatment/genotype combination was executed as a replicate of 4.

first: Xiaochen

last: Wang

first: Shuang

last: Liang


title: RNA-seq of human induced pluripotent cells treated with short-term Met deprival

city: Yokohama

first: Hiroki

last: Ozawa

state: Kanagawa

address: 4259-B-25 Nagatsuta-cho, Midori-ku

department: School of Life Science and Technology

institute: Tokyo Institute of Technology

summary: Human iPSCs require high amounts of methionine (Met). Met deprivation results in a rapid decrease in intracellular S-adenosyl-methionine (SAM), poising human iPSCs for differentiation and leading to apoptosis of undifferentiated cells. Met deprivation triggers a rapid metabolite change, including SAM, followed by reversible epigenetic modification. We show here that short-term Met deprivation impairs pluripotency network through epigenetic modification. The trimethylation of lysine 4 on histone H3 (H3K4me3) was dramatically affected compared to other histone modifications. Transcription start site (TSS) region of key pluripotent genes such as NANOG and OCT3/4 are specifically impacted upon short-term Met deprivation. Gene expression levels of these genes decreased, correlating to the loss of H3K4me3 marks. Upon differentiation, Met deprivation triggers a loss of the H3K27me3 in many mesendodermal genes, thereby switching from a bivalent to monovalent (H3K4me3) state. We concluded that Met metabolism maintains the pluripotent network with histone marks, and their loss potentiates differentiation.

overall_design: RNA-seq (n=3) in undifferentiated Ff-I01s01, an HLA homozygote donor-derived human iPSCs and the cells initiated a differentiation state with 100ng/mL Activin A and 3 µM CHIR-99021 for 24 hr. The cells formed spheres on a rotating orbital shaker for 24 hr, followed by a pre-cultured in complete or Met deprived medium for 5hr.

first: Azusa

last: kambe

first: Satoshi

last: Murakami

first: Katsunori

last: Fujiki

first: Ryuichiro

last: Nakato

first: Katsuhiko

last: Shirahige

first: Nobuaki

last: Shiraki

first: Shoen

last: Kume


title: Efficient generation of lower induced Motor Neurons by coupling Ngn2 expression with developmental cues

first: Francesco

last: Limone

state: Massachusetts

address: 7 divinity avenue, Bauer Laboratory 008

institute: harvard university

summary: Human pluripotent stem cells (hPSCs) are a powerful tool for disease modelling of hard-to-access tissues (such as the brain). Current protocols either direct neuronal differentiation with small molecules or use transcription-factor-mediated programming. In this study, we couple overexpression of transcription factor Neurogenin2 (Ngn2) with small molecule patterning to differentiate hPSCs into lower induced Motor Neurons (liMoNes/liMNs). This approach induces canonical MN markers including motor neuron (MN) specific marker Hb9/MNX1 activation in >95% of cells. liMNs resemble bona fide hPSC-derived MN, exhibit spontaneous electrical activity, express synaptic markers and can contact muscle cells in vitro. Pooled, multiplexed single-cell RNA sequencing on 50 hPSC-lines reveals reproducible populations of distinct subtypes of cervical and brachial MNs that resemble their in vivo, embryonic counterparts. Combining small molecule patterning with Ngn2 overexpression facilitates high-yield, reproducible production of disease-relevant MN subtypes, which is fundamental in propelling our knowledge of MN biology and its disruption in disease.

overall_design:
Pooled "villages" of lower induced Motor Neurons differentited from human stem-cells (n=50, two timepoints= day 35 and day 49 post-induction) were analyzed using scRNAseq

Submitter states: Raw data are available at: https://duos.broadinstitute.org/

first: Jana

last: Mitchell

first: Irune

last: Guerra San Juan

first: Janell

last: Smith

first: Kavya

last: Raghunathan

first: Daniel

last: Meyer

first: Alexander

last: Couto

first: Joseph

last: Klim

first: Sulagna

last: Ghosh

first: Brian

last: Joseph

first: John

last: Gold

first: Curtis

last: Mello

first: James

last: Nemesh

first: Britanny

first: Matthijs

last: Verhage

first: Steven

last: McCarroll

first: Olli

last: Pietiläinen

first: Ralda

last: Nehme

first: Kevin

last: Eggan


title: Human induced pluripotent cells treated with short-term Met deprival

first: Hiroki

last: Ozawa

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

overall_design: Refer to individual Series


title: CLL loss-of-function lesions distinctly impact B cell developmental and survival trajectories in B-cell restricted mouse models

city: Boston

first: Catherine

last: Wu

state: MA

address: 450 Brookline Avenue

institute: Dana-Farber Cancer Institute

summary: Mouse models represent invaluable tools for the systematic evaluation of cancer drivers, yet those that address the impact of putative genetic drivers of chronic lymphocytic leukemia (CLL) on B cell development and function are largely lacking. To study recurrent loss-of-function (LOF) mutations observed in human CLL, we established a robust transplant model that can rapidly evaluate genetic lesions, based on CRISPR-Cas9–mediated gene knockout restricted to B cells. Using this system, we successfully generated six LOF-expressing models with B cell-specific depletion of Atm, Birc3, Trp53, Chd2, Samhd1 or Mga genes. Transcriptomic analyses identified presence of shared transcriptional networks associated with presence of the 6 modeled LOF mutations, related to increased B cell fitness in vitro and in vivo. We further identified a unique role for Mga in modulating B cell development through dysregulation of MYC and NOTCH signaling targets. None of the individual lesions led to CLL development, after 24 months of observation, supporting the relevance of combinatorial mutations for CLL development in vivo.

overall_design: Highly recurrent CLL-associated loss-of-function (LOF) driver mutations (i.e.   Atm, Trp53, Samhd1, Mga, Birc3, Chd2) were introduced into a bone marrow donor line using CRISPR-Cas9. Edited and unedited B cells from the same animals were subjected to transcriptome analysis.

first: Elisa

last: ten Hacken

first: Shanye

last: Yin


title: Genome-wide mapping of fluoroquinolone-induced gyrase cleavage sites displays drug specific effects that correlate with bacterial persistence

city: Princeton

first: Juechun

last: Tang

state: NJ

address: 25 william streeet

department: Chemical and Biological Engineering

institute: Princeton University

summary: Persisters are rare phenotypic variants that can survive bactericidal doses of antibiotics and resume growth after the conclusion of treatment. They are suspected to be culprits of recurrent infections, and are distinct from resistant mutants because they are genetically identical to bacteria that die from treatment. Persisters to fluoroquinolones (FQs) experience DNA damage from treatment in stationary-phase culture, which suggests that FQs can corrupt their primary target, DNA gyrase, even in these super-tolerant cells. Since DNA damage from FQs originates from its stabilization of DNA gyrase in complexes with cleaved DNA, we hypothesized that the characteristics of FQ-stabilized DNA gyrase cleavage sites (GCS), such as the abundance, and/or location, might be influential to persistence. To test this hypothesis, we measured genome-wide distributions of GCS after treatment with a panel of FQs.We found drug-specific effects on the location and abundances of GCSs and discovered a negative correlation between the number of GCSs and FQ persister levels. Further, additional experiments and analyses suggested that persister levels are not governed by cleavage to a single specific site, but rather survival is a more complex function with respect to GCSs. Together, these findings demonstrate that there are drug specific differences in GCSs that correlate with persister levels, and suggest that the ability of an FQ to stabilize DNA-gyrase complexes at more sites will improve its bactericidal ability.

overall_design: We applied GCS-seq to identify gyrase cleavage sites (GCSs) in stationary-phase E. coli strains treated with five different FQs:  levofloxacin (LEVO), moxifloxacin (MOXI), norfloxacin (NOR), ciprofloxacin (CIP), and gemifloxacin (GEMI). We chromosomally tagged gyrA with FLAG-tag and grew cultures to stationary-phase in minimal media containing glucose as the sole carbon source, and the FLAGless strain was used as a control. We perfomred 3 independent biological replcates for each sample.

first: Mark

last: Brynildsen


title: Dissection of Richter’s syndrome pathogenesis and treatment through multiplexed CRISPR-based mouse models

summary: Richter’s syndrome (RS) is a major obstacle to the management of patients with chronic lymphocytic leukemia (CLL). Its pathogenesis remains largely unknown, and presently, faithful cellular and mouse models are lacking. We report a novel congenic mouse model of RS, based on CRISPR-mediated introduction of multiplexed CLL loss-of-function lesions (i.e. Atm, Trp53, Samhd1, Mga, Birc3, Chd2) into del(13q) B cells, leading to recapitulation of disease features from CLL to transformation to RS, and exhibiting remarkable molecular and phenotypic similarity to human disease. By integrative genomic and functional analyses, we determine that mutation in Trp53, combined with Mga and Chd2, are necessary for RS transformation and for the dysregulation of E2F/MYC and interferon signaling pathways. We demonstrate that RS requires tonic PI3K signaling for survival, and shows vulnerability to MYC, CDK, mTOR and PI3K inhibitors. This model system presents a unique tool for the study of RS biology and therapy.

sample_organism:
Mus
Mus musculus

platform_organism:
Mus
Mus musculus

overall_design:
Highly recurrent CLL-associated loss-of-function (LOF) driver mutations (i.e.   Atm, Trp53, Samhd1, Mga, Birc3, Chd2) were simultaneously introduced into a bone marrow donor line carrying homozygous MDR, a lesion recapitulating del(13q) using CRISPR-Cas9. Different normal and malignant B cells were subjected to transcriptome analysis.
Splenocytes were obtained from three murine CLL models and sequenced using mtscATAC-seq on the 10x platform

first: Livius

last: Penter


title: ChIP-seq of human induced pluripotent cells treated with short-term Met deprival

overall_design: H3K4me3 and H3K27me3 ChIP-seq (n=2) in undifferentiated Ff-I01s01, an HLA homozygote donor-derived human iPSCs and the cells initiated a differentiation state with 100ng/mL Activin A and 3 µM CHIR-99021 for 24 hr. The cells formed spheres on a rotating orbital shaker for 24 hr, followed by a pre-cultured in complete or Met deprived medium for 5hr.

first: Azusa

last: kambe

first: Satoshi

last: Murakami

first: Katsunori

last: Fujiki

first: Ryuichiro

last: Nakato

first: Katsuhiko

last: Shirahige

first: Nobuaki

last: Shiraki

first: Shoen

last: Kume


title: Effect of fluoroquinolone-induced gyrase cleavage sites on gene expression during drug treatment period and recovery period on E. coli

overall_design: Comparative gene expression profiling analysis of RNA-seq data for E. coli MG1655 derivatives treated with different drugs (moxifloxacin, levofloxacin, or mytomycin C)


title: Discriminating activities of DEAD-Box Helicase 41 from myeloid malignancy-associated germline variants by genetic rescue

status: Public on Dec 04 2022

city: MADISON

first: Siqi

last: Shen

state: WI

address: 425 Henry Mall

institute: University of Wisconsin - Madison

summary: Although DEAD-box helicase 41 (DDX41) is implicated in oncogenic and innate immune mechanisms, there are many unanswered questions about how the ever-increasing spectrum of genetic variants impacts DDX41 activity. Here, we describe a facile genetic rescue assay that discriminates activities of DDX41 from those of human myeloid malignancy-linked germline and somatic DDX41 mutants. Our analyses revealed that the variants were impaired in their intrinsic RNA-regulatory activities and to induce monocytic differentiation markers. It will be instructive to extend these analyses to more broadly conduct structure/function assessments for clinical genetic curation and leverage the quantitative assay to elucidate mechanisms and interventions that promote and/or oppose DDX41 function, thereby influencing DDX41-linked pathogenicity.

overall_design: Hoxb8-immortalized wild type hematopoietic progenitors (hi−Ddx41+/+) infected with control (empty) retrovirus, Ddx41 heterozygotic KO progenitors (hi−Ddx41+/-) infected with empty retrovirus or empty , wild type (WT) or human disease mutants of DDX41 -expressing retrovirus were FACS isolated for GFP+.

first: Jeong-Ah

last: Kim

first: Sunduz

last: Keles

first: Emery

last: Bresnick


title: Fluoroquinolone-induced gyrase cleavage sites


title: RNA sequencing of HUVECs stimulated with VEGF, OSM+VEGF, or CNTF+CNTFR+VEGF

city: Freiburg im Breisgau

first: Julian

last: Rapp

address: Killianstraße 5

institute: University Medical Center Freiburg

summary: IL-6 family cytokines as OSM and CNTF modify angiogenesis to different degree. This study investigates to which extend treatment of endothelial cells with those cytokines can modify VEGF induced angiogenic reactions. Human umbilical vein endothelial cells (HUVECs) were stimulated for 24h by cytokines followed by RNA harvesting and processed for RNA sequencing.

overall_design: mRNA profiles of HUVECs stimulated with Endothelial Basal Medium (Control, n=3), VEGF (n=3), OSM+VEGF (n=3), or CNTF+CNTFR+VEGF (n=3) for 24h.

first: Felicitas

last: Bucher


title: SUMM4 complex couples insulator function and DNA replication timing control

first: Markus

last: Nevil

address: 3350 Genome Sciences Bldg, CB 7100

department: Biology

summary: The asynchronous timing of replication of different chromosome domains is essential for eukaryotic genome stability, but the mechanisms establishing replication timing programs remain incompletely understood. Drosophila SNF2-related factor SUUR imparts under-replication (UR) of late-replicating intercalary heterochromatin (IH) domains in polytene chromosomes. SUUR negatively regulates DNA replication fork progression across IH; however, its mechanism of action remains obscure. Here we developed a novel method termed MS-Enabled Rapid protein Complex Identification (MERCI) to isolate a stable stoichiometric native complex SUMM4 that comprises SUUR and a chromatin boundary protein Mod(Mdg4)-67.2. In vitro, Mod(Mdg4) stimulates the ATPase activity of SUUR, although neither SUUR nor SUMM4 can remodel nucleosomes. Mod(Mdg4)-67.2 and SUUR distribution patterns in vivo partially overlap, and Mod(Mdg4) is required for a normal spatiotemporal distribution of SUUR in chromosomes. SUUR and Mod(Mdg4)-67.2 mediate insulator activities of the gypsy mobile element that disrupt enhancer-promoter interactions and establish euchromatin-heterochromatin barriers in the genome. Furthermore, mutations of SuUR or mod(mdg4) reverse the locus-specific UR. These findings reveal that DNA replication can be delayed by a chromatin barrier and thus, uncover a critical role for architectural proteins in replication timing control. They also provide a biochemical link between ATP-dependent motor factors and the activity of insulators in regulation of gene expression and chromatin partitioning.

overall_design: DNA-sequencing of Drosophila salivary glands to measure under replication.

first: Evgeniya

middle: N

last: Andreyeva

first: Alexander

middle: V

last: Emelyanov

first: Lu

last: Sun

first: Elena

last: Vershilova

first: Christina

last: Hill

first: Michael

middle: C

last: Keogh

first: Robert

last: Duronio

first: Arthur

last: Skoultchi

first: Dmitry

last: Fyodorov


title: RRBS (Reduced Representation Bisulfite Sequencing) analysis of liver tissue from mice

status: Public on Dec 03 2022

city: Tokyo

first: Tetsuya

last: Hirabayashi

address: 2-1-6 Kamikitazawa, Setagaya-ku

institute: Tokyo Metropolitan Institute of Medical Science

summary: Choline supplies methyl groups for regeneration of methionine and the methyl donor S-adenosylmethionine in the liver. Here we demonstrate that the catabolism of membrane phosphatidylcholine (PC) into water-soluble glycerophosphocholine (GPC) by the phospholipase/lysophospholipase PNPLA8-PNPLA7 axis enables endogenous choline stored in hepatic PC to be utilized in methyl metabolism. PNPLA7-deficient mice show marked decreases in hepatic GPC, choline, and several metabolites related to the methionine cycle, accompanied by various signs of methionine insufficiency including growth retardation, hypoglycemia, hypolipidemia, increased energy consumption, reduced adiposity, increased FGF21, and an altered histone/DNA methylation landscape. Moreover, PNPLA8-deficient mice recapitulate most of these phenotypes. In contrast to wild-type mice fed a methionine/choline-deficient diet, both knockout strains display a decreased hepatic triglyceride likely via reductions of lipogenesis and GPC-derived glycerol flux. Collectively, our findings highlight the biological importance of phospholipid catabolism driven by PNPLA8/PNPLA7 in methyl group flux and triglyceride synthesis in the liver.

overall_design: RRBS for DNA methylation analysis of liver tissues (Pnpla7 knockout mice vs control wild-type mice)

first: Sayaka

last: Harada

first: Yoshitaka

last: Taketomi

first: Makoto

last: Murakami


title: Maternal mRNA deadenylation and allocation via Rbm14 condensates facilitate vertebrate blastula development

first: Jiehui

last: Chen

address: 320 Longwood Ave

institute: Boston Children’s Hospital

summary: Early embryonic development depends on proper utilization and clearance of maternal transcriptomes. How these processes are spatiotemporally regulated remains unclear. Here we show that nuclear RNA-binding protein Rbm14 and maternal mRNAs co-phase separate into cytoplasmic condensates to facilitate vertebrate blastula-to-gastrula development. In zebrafish, Rbm14 condensates were highly abundant in blastomeres and markedly reduced after prominent activation of zygotic transcription. They concentrated at spindle poles by associating with centrosomal -tubulin puncta and displayed mainly asymmetric divisions with a global symmetry across embryonic midline in 8-cell and 16-cell embryos. Their formation was dose-dependently stimulated by m6A, but repressed by m5C modification of the maternal mRNA. Furthermore, deadenylase Parn co-phase separated with these condensates, and this was required for deadenylation of the mRNAs in early blastomeres. Depletion of Rbm14 impaired embryonic cell differentiations and full activations of the zygotic genome in both zebrafish and mouse and resulted in developmental arrest at the blastula stage. Our results suggest that cytoplasmic Rbm14 condensate formation regulates early embryogenesis by facilitating deadenylation, protection, and mitotic allocation of m6A-modified maternal mRNAs, and by releasing the poly(A)-less transcripts upon regulated disassembly to allow their re-polyadenylation and translation or clearance.

sample_organism:
Danio rerio
Mus

platform_organism:
Danio rerio
Mus

overall_design:
Zebrafish embryo mRNA profiles of ctrl-MO and 14-tMOs by deep sequencing at 2.5hpf, 4hpf and 6hpf. For each sample, 30 embryos were pooled.
Mouse embryo mRNA profiles of S2-S10, by deep sequencing at E4.5.

first: Yue

last: Xiao


title: Physiological hypoxia improves growth and functional differentiation of human intestinal epithelial organoids [scRNA-seq]

status: Public on Dec 02 2022

city: Trondheim

first: Gunnar

middle: Andreas

last: Walaas

address: Prinsesse Kristinas gate 1

department: Department of Clinical and Molecular Medicine

country: Norway

institute: Norwegian University of Science and Technology

summary: The intestinal epithelium is an immunologically active barrier adapted for a low oxygen environment. Its role is implicated in the pathophysiology of various diseases, including inflammatory bowel disease (IBD) and colorectal cancer. Patient-derived intestinal epithelial organoids (IEOs) mimic the architecture and cell type composition of the intestine and can be used for disease modeling and personalized drug screening. IEOs are usually cultured in atmospheric oxygen concentrations (20% oxygen) without accounting for the physiological hypoxia in the colonic epithelium, where the oxygen concentration range from 3% to <1%.  Previously, we showed that human colon derived IEOs (colonoids) are viable and respond to the pro-inflammatory cytokines TNF and IL17 after short-term (40 hours) cultivation in low (2%) oxygen. We suggested that recapitulating the in vivo physiological oxygen environment (i.e., physioxia) will enhance the translational value of intestinal organoids as pre-clinical models. Here we evaluate whether human colonoids can be established and cultured in physioxia for the entire in vitro culture period and compare growth, differentiation, and immunological responses in colonoids at 2% and 20% oxygen.  Growth from single cells to differentiated organoids was monitored by brightfield images captured throughout cultivation and evaluated with a linear mixed model. Cell composition in undifferentiated vs. differentiated colonoids was identified by immunofluorescence staining of cell markers and single-cell RNA-sequencing. Enrichment analysis was used to identify transcriptomic differences within cell populations. Pro-inflammatory stimuli induced chemokines and Neutrophil gelatinase-associated lipocalin (NGAL) release were analyzed by Multiplex profiling and ELISA. Direct response to a lower oxygen level was analyzed by enrichment analysis of bulk RNA sequencing data.  Colonoids established in a 2% oxygen environment acquired a significantly larger cell mass compared to a 20% oxygen environment. There were significantly more cells with proliferation potential (KI67 positive) in undifferentiated than in differentiated colonoids; and significantly higher expression of cell markers for goblet cells (MUC2) and absorptive cells (MUC2 negative, CK20 positive) in differentiated colonoids. Enteroendocrine cells (CGA positive) were only present in differentiated colonoids. No differences in expression of any of the cell marker proteins were found between colonoids cultured in 2% and 20% oxygen, but the single-cell RNA-sequencing analysis identified differences in the transcriptome within stem-, progenitor- and differentiated cell clusters. Both colonoids grown at 2% and 20% oxygen secreted CX3CL1, CXCL2, CXCL5, CXCL6, CXCL10, CXCL12, CCL20, CCL25, and NGAL upon TNF + poly(I:C) treatment. However, there appeared to be a lower pro-inflammatory response in 2% oxygen.  Reducing the oxygen environment from 20% to 2% in differentiated colonoids altered the expression of genes related to differentiation, metabolism, mucus lining, and immune networks.  Our results suggest that colonoids studies can and should be performed in physioxia when the resemblance to in vivo conditions is important.

overall_design:
Single cell RNA-sequencing data of undifferentiated and differentiated intestinal epithelial organoids (untreated) cultured in 2% oxygen and 20% oxygen throughout cultivation.
***Please note that raw data is not provided as Norwegian law does not allow public access to human sequences raw data***

first: Shreya

last: Gopalakrishnan

first: Ingunn

last: Bakke

first: Helene

last: Skovdahl

first: Arnar

last: Flatberg

first: Ann

middle: E

last: Østvik

first: Arne

last: Sandvik

first: Torunn

last: Bruland


title: Transcriptomic analysis of mRNA expression changes in the murine thymus induced by RANKL expression

city: Houston

first: Maria

last: Szwarc

address: One Baylor Plaza

institute: Baylor College of MEdicine

summary: Sequencing of mRNA isolated from the thymus of control mice and mice overexpressing RANKL in keratin 5 (K5)-positive cells for 1 weeks.

overall_design: Control (monogenic control) and K5-rtTA:TetO-RANKL mice were administered doxycycline (in food and water) at 8-weeks of age for 1 week to induce transgenic RANKL expression.

first: John

middle: P

last: Lydon


title: Expression data from control, aortic aneurysm, and aortic dissection cells

city: Linz

first: Christian

last: Doppler

address: Krankenhausstraße 5

country: Austria

institute: Johannes Kepler University Linz

summary:
Aneurysmatic and dissection cells show a specific alteration of gene expression, which allow a disease specific distinction.
We used microarrays to analyse the cellular gene expression of controls, thoracic aortic aneurysm, and aortic dissection.

overall_design: Human smooth muscle cells were isolated from aortic tissue from healthy controls, aortic aneurysm and Stanford A dissection. Cells were cultivated under identical conditions following gene expression analysis.