title: Interrogation of endothelial and mural cells in brain metastasis by multiomic analysis reveals key immune-regulatory mechanisms

city: Lausanne

first: Nadine

last: Fournier

state: Vaud

address: Agora Cancer Research Center, Bugon 25A

institute: Agora Cancer Research Center & Swiss Institute of Bioinformatics

summary: Brain metastasis (BrM) represents the most common and aggressive brain malignancy, predominantly arising from non-small cell lung cancer, breast cancer, and melanoma. Recent studies have revealed the importance of the brain tumor microenvironment (TME), notably diverse immune cells, which play important roles in regulating cancer progression in both primary and metastatic brain malignancies. The blood-brain barrier (BBB) is another critical TME component formed by endothelial cells, mural cells, astrocytic end-feet, and closely-associated microglial cells. Metastasizing cancer cells can utilize different strategies to traverse the BBB and once they have successfully seeded and colonized the brain, they can exploit the vasculature for their own benefit, forming the blood-tumor barrier. To explore the mechanisms underlying tumor vascularization in brain metastasis we performed a comprehensive multiomic analysis of the key components of the tumor vasculature. We integrated single-cell and/or bulk RNA sequencing of sorted endothelial and mural cells isolated from human and mouse BrM and non-tumor samples; immunofluorescence imaging analysis of the spatial architecture of the TME; and functional studies using BrM mouse models to target vascular regulators of tumor immunity. Our results provide a comprehensive understanding of the biology underlying vascularization in metastatic brain tumors, specifically highlighting the importance of vascular cells as immune regulators and proposing novel therapeutic strategies for these aggressive tumors.

overall_design: Expression profile of endothelial and mural cells in BrM versus non-tumor brain tissue.

first: Annamaria

last: Kauzlaric

first: Leire

last: Bejarano

first: Johanna

last: Joyce


title: HSP70 binds RNA and regulates transcription by RNA Polymerase III [RNA-seq]

status: Public on Jan 17 2024

city: Cambridge

first: Andrés

middle: Manuel

last: Herrero-Ruiz

address: Gleeson Building, Tennis Court Road

department: MRC Toxicology Unit

country: United Kingdom

institute: MRC-University of Cambridge

type: Expression profiling by high throughput sequencing

summary: We examined the RNA binding ability of the 70-kDa heat shock protein (HSP70) family member HSPA1A and identified the RNA species bound to HSP70 in vivo. We used a UV-crosslinkiung and immunoprecipitation based approach named FLASH-seq (Aktaş et al., 2017) using 3xFLAG-Histidine-Biotin tagged ectopically expressed protein in HEK293T cells.

overall_design:
HEK293 Flp-In T-Rex with stable integration of pcDNA5-FRT-TO_3xFLAG-HBH_GFP and pcDNA5-FRT-TO_3xFLAG-HBH_HSPA1A were obtained by Flp-mediated recombination. Expression of 3xFLAG-HBH_GFP and 3xFLAG-HBH_HSPA1A proteins was induced with 1  g/ml tetracycline for 16 hours.
RNA sequencing of HEK293 Flip-In T-Rex

first: Sergio

last: Leone

first: Andres

last: Herrero


title: The role of phase separation in EBF1 pioneering function

status: Public on Jan 18 2024

city: Freiburg

first: Nikolay

last: Zolotarev

address: Stübeweg, 51

department: Cellular and Molecular Immunology

country: Germany

institute: Max Planck Institute of Immunobiology and Epigenetics

type: Genome binding/occupancy profiling by high throughput sequencing

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

overall_design: Refer to individual Series


title: HSP70 binds to specific non-coding RNA and regulates human RNA polymerase III


title: HSP70 binds RNA and regulates transcription by RNA Polymerase III [ChIP-seq]

overall_design:
HEK293 Flp-In T-Rex with stable integration of pcDNA5-FRT-TO_3xFLAG-HBH_GFP and pcDNA5-FRT-TO_3xFLAG-HBH_HSPA1A were obtained by Flp-mediated recombination. Expression of 3xFLAG-HBH_GFP and 3xFLAG-HBH_HSPA1A proteins was induced with 1  g/ml tetracycline for 16 hours.
ChIP-seq of 3xFLAG-HBH tagged GFP and HSPA1A


title: HSP70 binds RNA and regulates transcription by RNA Polymerase III [FLASH-seq]

summary: We examined the RNA binding ability of the 70-kDa heat shock protein (HSP70) family member HSPA1A and HSP8 and identified the RNA species bound to HSP70 or HSP8 in vivo. We used a UV-crosslinkiung and immunoprecipitation based approach named FLASH-seq (Akta? et al., 2017) using 3xFLAG-Histidine-Biotin tagged ectopically expressed protein in HEK293T cells.

overall_design: FLASH-seq of 3xFLAG-HBH tagged GFP, HSPA1A or HSPA8; two biological replicates each.

first: Barbara

last: Hummel


title: Lactic acid modifies gene expression and chromatin accessibility of bone marrow-derived macrophages during lipopolysaccharide stimulation (RNA-seq and ATAC-seq)

status: Public on Jan 16 2024

city: Rochester

first: Aditya

last: Bhagwate

state: MN

address: 200 1st Street SW

country: USA

institute: Mayo Clinic

summary: Lactic acid (LA) has emerged as an important modulator of immune cell function. We performed ATAC-seq to profile the chromatin landscape of bone marrow-derived macrophages (BMDMs) upon lipopolysaccharide (LPS) stimulation with or without LA. Additionally, we also report the genome-wide RNA sequencing analysis in bone marrow-derived macrophages (BMDMs) where LA effect in macrophage inflammatory response was examined in BMDMs upon lipopolysaccharide (LPS) stimulation in the presence or absence of LA. To further examine LA effect due to low pH, hydrochloric acid (HCl) was used to adjust similar pH conditions (i.e. pH 6.5) as in media with LA.

overall_design:
Triplicates each of four conditions were subjected to ATAC-seq. Bone marrow derived macrophages (BMDMs) were treated with or without  LPS, LPS+LA, LA for 3 hours
Duplicates each of five conditions were subjected to RNA-seq.  Bone marrow derived macrophages (BMDMs) were treated with or without  LPS, LPS+LA, LPS+HCl, LA for 3 hours

first: Weiwei

last: Shi

middle: V

first: W

middle: K

last: Ip


title: Synthetic reversed sequences reveal default genomic states [mESC RNAseq]

city: New York

first: Brendan

middle: R.

last: Camellato

state: New York

address: 435 E 30th St

department: Institute for Systems Genetics

institute: NYU Langone Health

summary: Understanding default genome states would help interpret whether pervasive transcriptional activity has biological meaning. The genomes of extant organism have undergone billions of years of evolution, making it unclear whether observed genomic activities represent the effects of selection or “noise”. We addressed this question by introducing a novel 101-kb locus into the genomes of S. cerevisiae and M. musculus, and characterizing genomic activity. The locus was designed by reversing but not complementing human HPRT1, including substantial flank-ing regions, retaining basic sequence features but ablating evolved coding or regulatory infor-mation. We observed widespread activity of both reversed and native HPRT1 loci in yeast, de-spite the lack of evolved yeast promoters. In contrast, the reversed locus displayed no activity at all in mouse embryonic stem cells, instead showing repressive chromatin signatures. The re-pressive signature was alleviated in a locus variant lacking CpG dinucleotides; nevertheless this variant too was transcriptionally inactive. These results show that novel genomic sequences lacking coding information are active in yeast, but inactive in mouse embryonic stem cells, con-sistent with a major difference in “default genomic states” between these two divergent eukary-otic cell types, with implications for understanding pervasive transcription, horizontal transfer of genetic information, and new gene birth.

overall_design: Exploratory RNA-seq was performed to investigate whether synthetic HPRT1 and HPRT1R sequences are transcribed in mouse embryonic stem cells, and to compare to the rest of the respective genome.

middle: R

first: Ran

last: Brosh

first: Hannah

middle: J

last: Ashe

first: Matthew

middle: T

last: Maurano

first: Jef

middle: D

last: Boeke


title: Synthetic reversed sequences reveal default genomic states [mESC_ChIP-Seq]

overall_design: Exploratory ChIP-seq was performed to investigate whether synthetic HPRT1 and HPRT1R sequences are transcribed in mouse embryonic stem cells, and to compare to the rest of the respective genome.


title: Effect of pri-1 (DNA primase) RNAi on gene expression in embryos of Caenorhaditis elegans

city: Vancouver

first: Stefan

last: Taubert

state: BC

address: 950 W 28th Ave, CMMT Room 2024

department: Medical Genetics

country: Canada

institute: UBC

summary: All animals must maintain genome and proteome integrity, especially when experiencing endogenous or exogenous stress. To cope, organisms have evolved sophisticated and conserved response systems: unfolded protein responses (UPRs) ensure proteostasis while the DNA damage response (DDR) maintains genome integrity. Emerging evidence suggests that UPRs and DDRs crosstalk, but the extent of crosstalk remains poorly understood. Here, we demonstrate that inactivation of the DNA primases pri-1 and pri-2, which synthesize RNA primers at replication forks and whose inactivation causes DNA damage, activates the UPR of the endoplasmic reticulum (UPR-ER) in Caenorhabditis elegans, with especially strong activation in the germline. We observed activation of both the inositol-requiring-enzyme 1 (ire-1) and the protein kinase RNA-like ER kinase (PEK-1) branches of the UPR-ER. Interestingly, activation of the UPR-ER output gene hsp-4/BiP was partially independent of its canonical activators, ire-1 and xbp-1, and instead required the third branch of the UPR-ER, atf-6, suggesting functional redundancy. We further found that primase depletion specifically induces the UPR-ER, but not the mechanistically distinct cytosolic or mitochondrial UPRs, suggesting that primase inactivation causes compartment-specific rather than global stress. Functionally, loss of ire-1 or pek-1 sensitized animals to replication stress caused by hydroxyurea. Finally, transcriptome analysis of pri-1 embryos revealed several deregulated processes that could cause UPR-ER activation, including protein glycosylation, calcium signaling, and fatty acid desaturation. Together, our data show that the UPR-ER, but not other UPRs, responds to replication fork stress and that the UPR-ER is required to alleviate this stress.

overall_design: Comparative gene expression profiling (RNA-seq) of embryos from C. elegans hermpahrodites fed empty vector RNAi (negative control) or pri-1 RNAi

first: Brendil

last: Sabatino

first: Jiaming

last: Xu


title: Synthetic reversed sequences reveal default genomic states [Yeast_ChIPseq]

overall_design:
Exploratory RNA-seq was performed to investigate whether synthetic HPRT1 and HPRT1R sequences are transcribed in yeast, and to compare to the rest of the respective genome.
the records are duplicated by  GSE222036


title: Synthetic reversed sequences reveal default genomic states [Yeast_RNAseq]

overall_design: Exploratory RNA-seq was performed to investigate whether synthetic HPRT1 and HPRT1R sequences are transcribed in yeast, and to compare to the rest of the respective genome.


title: KDM5C-mediated BRD4 recruitment is essential for active states of enhancers and BET inhibitor sensitivity

city: Wuhan

first: Yulong

last: Qiang

address: 185 Donghu Road

country: China

institute: Wuhan University


title: Synthetic reversed sequences reveal default genomic states [mESC_ATACseq]

overall_design: Exploratory ATAC-seq was performed to investigate whether synthetic HPRT1 and HPRT1R sequences are accessible in mouse embryonic stem cells, and to compare to the rest of the respective genome.


title: Synthetic reversed sequences reveal default genomic states [Yeast_CAGEseq]

overall_design:
Exploratory RNA-seq was performed to investigate whether synthetic HPRT1 and HPRT1R sequences are transcribed in yeast, and to compare to the rest of the respective genome.
the records are duplicated by GSE222034


title: Nucleolar detention of NONO shields DNA double-strand breaks from aberrant transcripts [mNET]

city: Wuerzburg

first: Martin

last: Eilers

address: Am Hubland

department: Chair for Biochemistry and Molecular Biology

institute: University of Wuerzburg

summary: RNA-binding proteins emerge as effectors of the DNA damage response (DDR). The multifunctional non-POU domain-containing octamer-binding protein NONO/p54nrb marks nuclear paraspeckles in unperturbed cells, but also undergoes re-localisation to the nucleolus upon induction of DNA double-strand breaks (DSBs). However, NONO nucleolar re-localisation is poorly understood. Here we show that the topoisomerase-II inhibitor etoposide stimulates the production of RNA polymerase II-dependent, DNA damage-induced nucleolar antisense RNAs (diNARs) in human cancer cells. diNARs originate from distinct nucleolar intergenic spacer regions and form DNA-RNA hybrids to tether NONO to the nucleolus in ab RRM1 domain-dependent manner. NONO occupancy at protein-coding gene promoters is reduced by etoposide, which attenuates pre-mRNA synthesis, enhances NONO binding to pre-mRNA transcripts and is accompanied by nucleolar detention of a subset of such transcripts. The depletion or mutation of NONO interferes with detention and prolongs DSB signaling. Together, we describe a nucleolar DDR pathway that shields NONO and aberrant transcripts from DSBs to promote DNA repair.

overall_design: Profile of newly transcribed RNA associated with PolII of cells, with or without etoposide treatment

first: Julia

last: Hofstetter

first: Apoorva

last: Baluapuri

first: Peter

last: Gallant

first: Kaspar

last: Burger


title: Nucleolar detention of NONO shields DNA double-strand breaks from aberrant transcripts [CUT&RUN]

overall_design: Mapping of DNA-protein interaction of cells upon siCTR/shCTR or siNONO/shNONO, with or without Etoposide treatment, with or without NONO rescue

first: Barbara

last: Trifault

first: Carsten

middle: P

last: Ade

first: Giacomo

last: Cossa


title: HBO1 catalyzes lysine lactylation and mediates histone H3K9la to regulate gene transcription

city: Tianjin

first: wang

middle: si

last: yu

address: No. 22, Meteorological Tai Road, Heping District

institute: Tianjin Medical University

summary: Lysine lactylation (Kla) links metabolism and gene regulation and plays a key role in multiple biological processes. However, the regulatory mechanism and functional consequence of Kla remain to be explored. Here, we report that HBO1 functions as a lysine lactyltransferase to regulate transcription. Interestingly, CUT&Tag assays demonstrate that HBO1 is required for histone H3K9la on TSSs and the regulated Kla can facilitate in signaling pathways and progress of tumorigenesis. Our study reveals HBO1 serves as a lactyltransferase to mediate a histone Kla-dependent gene transcription.

overall_design: To study the common gene of HBO1 and H3K9la localization in HeLa cells, and to study the differential gene of H3K9la in HeLa-WT and HeLa-HBO1-KO.

first: Ziping

last: Niu

first: Chen

last: Chen

first: Siyu

last: Wang


title: ALKBH5-mediated m6A modification of 5-HT3A mRNA in primary sensory neurons promotes neuropathic pain behaviors

city: Suzhou

first: Jin

last: Tao

state: Jiangsu

address: 199 Ren-Ai Road

department: Physiology and Neurobiology

institute: Soochow University

summary: trigeminal-mediated nerve injury increases the expression of the m6A demethylase ALKBH5 in the injured TG, which elevates the level of 5-HT3A in TG and induces pain-related behaviors.

overall_design: transcripts  m6A profiles in rat trigeminal ganglion treated with NC-up (n = 3) or ALKBH5-up  (n = 3)

first: Zitong

last: Huang


title: NRAS mutant dictates AHCYL1-governed ER calcium homeostasis for tumor growth

status: Public on Jan 15 2024

city: Chicago

first: Jing

state: IL

address: 900 E 57th St

institute: University of Chicago

summary: Calcium homeostasis is critical for cell proliferation, and emerging evidence shows that cancer cells exhibit altered calcium signals to fulfill their need for proliferation. However, it remains unclear whether there are oncogene-specific calcium homeostasis regulations that can expose novel therapeutic targets. Here, from RNAi screen, we report that adenosylhomocysteinase like protein 1 (AHCYL1), a suppressor of the endoplasmic reticulum (ER) calcium channel protein inositol trisphosphate receptor (IP3R), is selectively upregulated and critical for cell proliferation and tumor growth potential of human NRAS-mutated melanoma, but not for melanoma expressing BRAF V600E. Mechanistically, AHCYL1 deficiency results in decreased ER calcium levels, activates the unfolded protein response (UPR), and triggers downstream apoptosis. In addition, we show that AHCYL1 transcription is regulated by activating transcription factor 2 (ATF2) in NRAS-mutated melanoma. Our work provides evidence for oncogene-specific calcium regulations and suggests AHCYL1 as a novel therapeutic target for RAS mutant-expressing human cancers, including melanoma.

overall_design: To investigate why and how AHCYL1 promotes cell proliferation in NRAS mutant melanoma cells, we transfected HMCB cells, a NRAS-mutated melanoma cell line, with AHCYL1 siRNA and control siRNA for 3 days, followed by RNA extraction and RNA-sequencing analysis.

first: Rong

last: Wu

first: Chufan

last: Cai

first: Lei

last: Dong


title: Nucleolar detention of NONO shields DNA double-strand breaks from aberrant transcripts [APEX2-seq]

first: Martin

last: Eilers

overall_design: RNA localization of cells upon dox inducible shCTR or shNONO, with or without etoposide treatment


title: Streamlined identification of clinically and functionally relevant genetic regulators of lower tract urogenital development

city: St. Louis

first: Meade

last: Haller

state: MO

address: 660 S Euclid Ave

institute: Washington University School of Medicine

summary: Congenital anomalies of the lower genitourinary (LGU) tract are frequently comorbid due to genetically linked developmental pathways. Genital phenotypes represent the second most common group of congenital structural anomalies in males. Genes involved in sexual differentiation are prime candidates for developmental anomalies of multiple LGU organs, but insufficient prospective screening tools have prevented the rapid identification of causative genes. Androgen signaling is among the most influential modulators of LGU development. The present study uses SpDamID technology in vivo to generate a comprehensive map of the pathways actively regulated by androgen receptor (AR) in the genitalia in the presence of the p300 co-activator, identifying WNT signaling as a highly enriched AR-regulated pathway in the genitalia. Transcription factor (TF) hits were then assayed for sexually dimorphic expression at two critical timepoints, and also cross-referenced to the DECIPHER database of clinically relevant copy number variations (CNVs) to identify 252 TFs exhibiting copy variation in patients with LGU phenotypes. A subset of 54 TFs was identified for which LGU phenotypes are statistically overrepresented as a proportion of total observed phenotypes. The 252 TF hitlist was then subjected to a functional screen to identify hits whose silencing affects genital mesenchymal growth rates. Overlap of these datasets results in a refined list of 133 transcription factors of both functional and clinical relevance to LGU development, 31 of which are top priority candidates, including the well-documented renal progenitor regulator, Sall1. Loss of Sall1 was examined in vivo and confirmed to be a powerful regulator of LGU development

overall_design: SplitDAM in vivo sequencing of embryonic murine male genitalia with DAM halves fused to AR and p300

first: Yan

last: Yin

first: Gabe

first: Tian

last: Li

first: Qiufang

first: Laura

middle: E

last: Lamb

first: Liang

last: Ma


title: The RNA-binding protein Arpp21 interacts with and promotes Rag1 target mRNA expression to enable TCR rearrangement [RNA-seq]

city: Planegg-Martinsried

first: Taku

last: Kureha

address: Grosshaderner Str. 9

department: Institute for Immunology

institute: Ludwig-Maximilians-Universität

summary: Post-transcriptional control by RNA-binding proteins (RBPs) is an essential layer of gene regulation in lymphocytes. Here, we show how members of the R3hdm RBP family regulate gene expression and promote the development of thymocytes. R3hdm paralogs with an intact R3H/SUZ domain bound mRNAs of other RBPs including Roquin-1, Roquin-2 and Nufip2, and augmented their expression. Arpp21 (R3hdm3) expression was restricted to thymocyte stages when Rag proteins recombine gene segments of the TCR variable region. Crosslinking and immunoprecipitation of Arpp21 in thymocytes revealed prominent binding to the Rag1 mRNA, and Arpp21 increased Rag1 3'-UTR reporter expression. Consequently, Arpp21–deficient mice showed decreased Rag1 expression in thymocytes, delayed TCR rearrangement, reduced frequencies of cells with TCR expression and a partial block of thymocyte development. The Arpp21 protein was regulated by Ca2+–signals that induced phosphorylation, polyubiquitination and proteasomal degradation. These findings involve general redundant functions of R3hdm RBP family proteins and show how stage-specific upregulation of Arpp21 in thymocytes promotes Rag1 expression preceding recombination while productive TCR rearrangement ceases this function.

overall_design: Examination of transcriptome in WT or Arpp12 KO thymocytes. The Arpp21-CLIP experiment were performed on polyA+ RNA from thymocytes with 4 biological replicates.

first: Meng

last: Xu

first: Tobias

last: Straub

first: Vigo

last: Heissmeyer


title: Parasympathetic neurons derived from human pluripotent stem cells model human diseases and development

city: Athens GA

first: Nadja

last: Zeltner

state: GA

address: 325 Riverbend road

institute: University of Georgia

summary: Parasympathetic neurons (parasymNs) belong to the autonomic nervous system (ANS) and are critical for unconscious body responses, including rest-and-digest and calming the body. ParasymN dysfunction has been found involved in neural diseases such as autonomic neuropathy and neurological autoimmune disease; and parasymN innervation is important for organ development. However, human parasymN function and dysfunction is vastly understudied, due to the lack of a model system. Human pluripotent stem cell (hPSC)-derived neurons can fill this void and serve for disease modeling, drug screening, and transplantation therapy. Here, we developed a differentiation paradigm detailing for the first time, the derivation of functional human parasymNs from Schwann cell progenitors (SCP). We employ these neurons (i) to assess human ANS development paradigms, (ii) to model neuropathy in the genetic disorder Familial Dysautonomia (FD), (iii) to show parasymN dysfunction during SARS-CoV-2 infection, (iv) to model the autoimmune disease Sjörgen’s syndrome and, (v) to show that parasymN innervation of white adipocytes during development and help mature the tissue. Our model system will become instrumental for future disease mechanistic and drug discovery studies as well as for human developmental studies.

overall_design:
To understand the development of parasympathetic neurons, their function, and their missregulation in diseases, we developed a protocol to differentiate parasympathetic neurons from human pluripotent stem cells. To characterize their transcriptome at different developmental stages, we collected RNA on different days throuhgout the differentiation (day 10, 14, 16, and 30) from three biological replicates (independent differentiations). We also collected RNA from parasympathetic neurons (day 30) differentiated from iPSCs dervied from one Familial dysautonomia patient (S2). We performed RNAseq of each sample.
To characterize their cellular composition, we collected single cell RNA data on day 30 from two independent differentiations (biological replicates).

first: Hsueh

middle: F

first: Kenyi

last: Saito-Diaz

first: Jessica

middle: L

last: McAlpine

first: Dong

last: Seo

first: Sumner

last: Magruder

first: Fabio

last: Santori

first: William

middle: H

last: Delaney

first: Smita

last: Krishnawamy

first: Natalia

middle: B

last: Ivanova


title: Venous plexus-associated lymphoid hubs support meningeal humoral immunity

first: Colin

middle: YC

last: Lee

address: Wellcome Genome Campus, Hinxton

institute: Molecular Immunity Unit

summary: We have identified previously unappreciated dural-associated lymphoid tissue hubs, present in homeostasis, and anatomically poised to acquire local and systemic antigens. These venolymphatic hubs facilitate rapid expansion of antigen specific immune responses and likely help defend the underlying CNS parenchyma against foreign invaders.

overall_design: BCR sequencing of anatomical/immune compartments 1 week or 3 weeks following nasal VSV challenge, or in naïve mice. Sequencing was performed on paired skull base, rostral-rhinal hub, superior saggital sinus dura, skull bone marrow, cervical lymph node and spleen tissues. Anti-LFA1/VLA4 antibodies or isotype control antibodies were administered starting 6 days post infection (6dpi) where indicated.

first: Zachary

last: Fitzpatrick

first: Nagela

middle: G

last: Zanluqui

first: Menna

last: Clatworthy

first: Dorian

last: McGavern


title: The RNA-binding protein Arpp21 interacts with and promotes Rag1 target mRNA expression to enable TCR rearrangement [CLIP-seq]

first: Taku

last: Kureha

overall_design: CLIP-seq

first: Meng

last: Xu

first: Tobias

last: Ito-Kureha


title: Mixed patterns of intergenerational DNA methylation inheritance in Acropora

city: Austin

first: Christopher

last: Peterson

state: TX

address: 2415 Speedway #C0930

department: Integrative Biology

institute: University of Texas

type: Methylation profiling by high throughput sequencing

summary: For sessile organisms at high risk from climate change, phenotypic plasticity can be critical to rapid acclimation. Epigenetic markers like DNA methylation are hypothesized as mediators of plasticity; methylation is associated with the regulation of gene expression, can change in response to ecological cues, and is a proposed basis for the inheritance of acquired traits. Within reef-building corals, gene body methylation can change in response to ecological stressors. If coral DNA methylation is transmissible across generations, this could potentially facilitate rapid acclimation to environmental change. We investigated methylation heritability in Acropora, a stony reef-building coral. Two A. millepora and two A. selago adults were crossed, producing eight offspring crosses (four hybrid, two of each species). We used whole-genome bisulfite sequencing to identify methylated loci and allele-specific alignments to quantify per-locus inheritance. If methylation is heritable, differential methylation (DM) between the parents should equal DM between paired offspring alleles at a given locus. We found a mixture of heritable and non-heritable loci, with heritable portions ranging from 44% to 90% among crosses. Gene body methylation was more heritable than intergenic methylation, and most loci had a consistent degree of heritability between crosses (i.e., the deviation between parental and offspring DM were of similar magnitude and direction). Our results provide evidence that coral methylation can be inherited but that heritability is heterogenous throughout the genome. Future investigations into this heterogeneity and its phenotypic implications will be important to understanding the potential capability of intergenerational environmental acclimation in reef building corals."

sample_organism:
Acropora millepora
Acropora selago
Acropora selago x Acropora millepora

overall_design:
To assess the heritability of DNA methylation, we created hybrid crosses of Acropora millepora and A. selago.
We used allele-specific methylation analysis on whole genome bisulfite sequencing data from adults and offspring to identify inherited loci.

first: Carly

last: Scott

first: Rashin

last: Ghaffari

first: Groves

last: Dixon

first: Mikhail

last: Matz


title: Pancreatic cancer xenograft tumor RNA sequencing and pathologic differentiation of matched human tumors.

city: CHARLOTTESVILLE

first: Sara

last: Adair

state: VA

address: 345 Crispell Dr.

department: Surgical Oncology

institute: University of Virginia

summary: Epithelial to mesenchymal transition (EMT) in pancreatic cancer is believed to correlate with metastatic probability and aggressiveness. In the clinical setting, tumor staging and grading reflect tumor aggressiveness, tumor invasion, and gross metastasis and are used to guide treatment options. These data were analyzed to correlate commonly used laboratory RNA sequencing analysis with clinically defined differentiation status. This dataset provides evidence that mesenchymal genes are enriched in xenograft tumors from poorly differentiated human tumors.

overall_design: Human PDACs were resected and orthotopically implanted and propagated in immunocompromised mice. Tumors were then extracted, and RNA sequencing was performed. Tumors were analyzed for gene expression (via RNA-seq) and tumor grading (differentiation status).

first: Brown

last: Brooke

first: Myers

last: Paul

first: Adair

last: Sara

first: Kane

last: William

first: Stelow

last: Edward

first: Bauer

last: Todd

first: Lazzara

last: Matthew


title: CD81 and CD82 expressing tumor_x0002_infiltrating lymphocytes in the NSCLC tumor microenvironment play a crucial role in T-cell activation and cytokine production

city: Seoul

first: Seul

last: Lee

address: 50-1, Yonsei-ro, Seodaemun-gu, Seoul, Republic of Korea

country: South Korea

institute: Yonsei University

summary:
There are differences in the expression levels of TCR-related early signaling and cytokines associated with the expression levels of CD81 and CD82.
Therefore, we hypothesized that the transcriptome related to T-cell function may also undergo changes.

overall_design: we sorted the CD81highCD82high and CD81lowCD82low groups and perfomed RNA sequencing

first: DongKwon

last: Kim

first: Kyoung-Ho

last: Pyo


title: Universal recording of immune cell interactions in vivo

city: Princeton

first: Sarah

last: Walker

state: NJ

address: Lewis-Sigler Institute of Integrative Genomics

institute: Princeton University

summary: Immune cells rely on transient physical interactions with other immune and non-immune populations to regulate their function. To study these “kiss-and-run” interactions directly in vivo, we previously developed LIPSTIC (Labeling Immune Partnerships by SorTagging Intercellular Contacts), an approach that uses enzymatic transfer of a labeled substrate between the molecular partners CD40L and CD40 to label interacting cells. Reliance on this pathway limited the use of LIPSTIC to measuring interactions between CD4+ helper T cells and antigen presenting cells, however. Here, we report the development of a universal version of LIPSTIC (uLIPSTIC), which can record physical interactions both among immune cells and between immune and non-immune populations irrespective of the receptors and ligands involved. We show that uLIPSTIC can be used, among other things, to monitor the priming of CD8+ T cells by dendritic cells, reveal the steady-state cellular partners of regulatory T (Treg) cells, and identify germinal center (GC)-resident T follicular helper (Tfh) cells based on their ability to interact cognately with GC B cells. By coupling uLIPSTIC with single-cell transcriptomics, we build a catalog of the immune populations that physically interact with intestinal epithelial cells (IECs) at steady state and profile the evolution of the interactome of lymphocytic choriomeningitis virus (LCMV)-specific CD8+ T cells in multiple organs upon systemic infection. Thus, uLIPSTIC provides a broadly useful technology for measuring and understanding cell–cell interactions across multiple biological systems.

overall_design:
uLIPSTIC interaction-dependent transcriptomics of the immune interacting partners of intestinal epithelial cells in steady-state mice and of LCMV-specific (P14) CD8+ T cells upon infection with lymphocytic choriomeningitis virus (LCMV) Armstrong cells in mediastinal lymph nodes, spleen, liver, and lung.
Please note that the HTO, Barcode sequence, and hashtagged sample information is included in the 'readme.txt'.

first: Sandra

last: Nakandakari-Higa

first: Maria

middle: C

last: Canesso

first: Verena

last: van der Heide

first: Aleksey

last: Chudnovskiy

first: Dong-Yoon

first: Johanne

last: Jacobsen

first: Roham

last: Parsa

first: Jana

last: Bilanovic

first: S

middle: M

last: Parigi

first: Karol

last: Fiedorczuk

first: Elaine

last: Fuchs

first: Angelina

last: Bilate

first: Giulia

last: Pasqual

first: Daniel

last: Mucida

first: Alice

middle: O

last: Kamphorst

first: Yuri

last: Pritykin

first: Gabriel

last: Victora


title: Nucleolar detention of NONO shields DNA double-strand breaks from aberrant transcripts [4sU-seq]

first: Martin

last: Eilers

overall_design: Nascent RNA profiling of cells upon dox inducible shCTR or shNONO, with or without etoposide treatment

first: Barbara

last: Trifault

first: Pranjali

last: Bhandare

first: Peter

last: Gallant

first: Elmar

last: Wolf

first: Kaspar

last: Burger


title: The RNA-binding protein Arpp21 interacts with and promotes Rag1 target mRNA expression to enable TCR rearrangement

first: Taku

last: Kureha

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

overall_design: Refer to individual Series


title: Nonlinear DNA methylation trajectories in aging male mice [RNA-seq]

city: Kiel

first: Lena

last: Best

address: Michaelis-Straße 5

department: Institute of Experimental Medicine

institute: Christian-Albrechts-University Kiel

summary: Although DNA methylation data yields highly accurate age predictors, little is known about the dynamics of this quintessential epigenomic biomarker during lifespan. To narrow the gap, we investigate the methylation trajectories of male mouse colon at five different time points of aging. Our study indicates the existence of sudden hypermethylation events at specific stages of life. Precisely, we identify two epigenomic switches during early-to-midlife (3-9 months) and mid-to-late-life (15-24 months) transitions, separating the rodents' life into three stages. These nonlinear methylation dynamics predominantly affect genes associated with the nervous system and enrich in bivalently marked chromatin regions. Based on groups of nonlinearly modified loci, we construct a clock-like classifier STageR (STage of aging estimatoR) that accurately predicts murine epigenetic stage. We demonstrate the universality of our clock in an independent mouse cohort and with publicly available datasets.

overall_design: RNA sequencing with Illumina’s TruSeq stranded kit, with polyA enrichment, 100 bp paired end sequencing of 83 total RNA samples from male mice’s (C57BL/6J/Ukj) colons at different ages: 3 (n = 16), 9 (n = 16), 15 (n = 16), 24 (n = 17), and 28 (n = 18) months.

first: Maja

last: Olecka

first: Alena

last: van Boemmel

first: Madlen

last: Haase

first: Silke

last: Foerste

first: Konstantin

last: Riege

first: Thomas

last: Dost

first: Stefano

last: Flor

first: Otto

middle: W

last: Witte

first: Sören

last: Franzenburg

first: Marco

last: Groth

first: Björn

last: von Eyss

first: Christoph

last: Kaleta

first: Christiane

last: Frahm

first: Steve

last: Hoffmann


title: Lack of affinity signature for germinal center cells that have initiated plasma cell differentiation (Figure4)

city: Canberra

first: Xin

last: Gao

state: ACT

address: 131 Garran Rd

department: Immunology

country: Australia

institute: The Australian National University

summary: Long-lived plasma cells (PCs) secrete antibodies that can provide sustained immunity against infection. It has been proposed that high affinity cells are preferentially selected into this compartment, potentiating the immune response. We used single cell RNA-seq to track the germinal center (GC) development of Ighg2A10 cells, specific for the Plasmodium falciparum circumsporozoite protein (PfCSP). Following immunization with Plasmodium sporozoites we identified 3 populations of cells in the GC light zone. One population expressed a gene signature associated with the initiation of PC differentiation and had an enhanced propensity to form PCs in vitro. Unexpectedly, the estimated affinity of this putative pre-PC population was indistinguishable from cells in the GC generally. This was also true when high- or low-avidity recombinant PfCSP proteins were used as immunogens. Immunization with low-avidity PfCSP did, however, induce increased affinity maturation. Collectively these findings suggest that the initiation of PC development in the GC occurs via an affinity independent process.

overall_design: Ighg2A10 B cells were adoptively transferred into WT or MD4 mice which were immunized by CSP27 or CSP9. Spleens were collected on day14 post immunization and IgD- Ighg2A10 B cells were sorted and pooled for 10X Genomics single-cell RNA sequencing

first: Hannah

last: Kelly

first: Ian

middle: A

last: Cockburn


title: Cytidine deaminase resolves replicative stress and protects pancreatic cancer from DNA-targeting drugs

city: Toulouse

first: Pierre

last: Cordelier

address: 2, avenue Hubert Curien

country: France

institute: Cancer Research Center of Toulouse

summary: Cytidine deaminase (CDA) functions in the pyrimidine salvage pathway for DNA and RNA synthesis and has been shown to protect cancer cells from deoxycytidine-based chemotherapies. In this study, we observed that CDA was overexpressed in pancreatic adenocarcinoma from patients at baseline and was essential for experimental tumor growth. Mechanistic investigations revealed that CDA localized to replication forks where it increased replication speed, improved replication fork restart efficiency, reduced endogenous replication stress, minimized DNA breaks, and regulated genetic stability during DNA replication. In cellular pancreatic cancer models, high CDA expression correlated with resistance to DNA-damaging agents. Silencing CDA in patient-derived primary cultures in vitro and in orthotopic xenografts in vivo increased replication stress and sensitized pancreatic adenocarcinoma cells to oxaliplatin. This study sheds light on the role of CDA in pancreatic adenocarcinoma, offering insights into how this tumor type modulates replication stress. These findings suggest that CDA expression could potentially predict therapeutic efficacy and that targeting CDA induces intolerable levels of replication stress in cancer cells, particularly when combined with DNA-targeted therapies

overall_design: To explore the consequences of cytidine deaminase (CDA) targeting or overexpression in human panceratic cancer cells Mia PACA-2

first: Audrey

last: Lumeau


title: Simultaneous single-cell three-dimensional genome and gene expression profiling Uncovers Dynamic Enhancer Connectivity Underlying Olfactory Receptor Choice [scATAC-Seq, scRNA-seq]

city: Beijing

first: Xiaoliang

middle: Sunney

last: Xie

state: Beijing

address: No.5 Yiheyuan Road, Haidian District

institute: Peking University

summary: The simultaneous measurement of three-dimensional (3D) genome structure and gene expression of individual cells is critical for understanding our genome’s structure–function relation, yet is extremely challenging for existing methods. Here we present Linking mRNA to Chromatin Architecture (LiMCA), which jointly profiles 3D genome and transcriptome with exceptional sensitivity and from low-input materials. Combining LiMCA and our high-resolution scATAC-seq assay, METATAC, we profiled the developing mouse olfactory epithelium and reported the first chromatin accessibility profiles and the first paired 3D genome structures and gene expression information of single neurons. We greatly expanded the repertoire of known OR enhancers, and discovered unexpected rules of their dynamics: ORs and their enhancers are most accessible during early differentiation, and the active OR typically does not associate with the largest enhancer hub. These findings offer valuable insights into how 3D connectivity of ORs and enhancers dynamically orchestrate the “one neuron–one receptor” selection process.

overall_design: 10X Genomics scRNA-seq and METATAC scATAC-seq for mouse olfactory epithelium

first: Honggui

last: Wu

first: Jiankun

last: Zhang

first: Fanchong

last: Jian


title: Simultaneous single-cell three-dimensional genome and gene expression profiling Uncovers Dynamic Enhancer Connectivity Underlying Olfactory Receptor Choice [cell line, single-cell RNA-Seq & Hi-C]


title: Nucleolar detention of NONO shields DNA double-strand breaks from aberrant transcripts [BLISS]

first: Martin

last: Eilers

summary: RNA-binding proteins emerge as effectors of the DNA damage response (DDR). The multifunctional non-POU domain-containing octamer-binding protein NONO/p54nrb marks nuclear paraspeckles in unperturbed cells, but also undergoes re-localisation to the nucleolus upon induction of DNA double-strand breaks (DSBs). However, NONO nucleolar re-localisation is poorly understood. Here we show that the topoisomerase-II inhibitor etoposide stimulates the production of RNA polymerase II-dependent, DNA damage-induced nucleolar antisense RNAs (diNARs) in human cancer cells. diNARs originate from distinct nucleolar intergenic spacer regions and form DNA-RNA hybrids to tether NONO to the nucleolus in ab RRM1 domain-dependent manner. NONO occupancy at protein-coding gene promoters is reduced by etoposide, which attenuates pre-mRNA synthesis, enhances NONO binding to pre-mRNA transcripts and is accompanied by nucleolar detention of a subset of such transcripts. The depletion or mutation of NONO interferes with detention and prolongs DSB signaling. Together, we describe a nucleolar DDR pathway that shields NONO and aberrant transcripts from DSBs to promote DNA repair.

overall_design: Profiling of DSBs upon siCTR or siNONO, with or without etoposide treatment

first: Daniel

last: Solvie

first: Carsten

last: Ade

first: Kaspar

last: Burger


title: An approach to intersectionally target mature enteroendocrine cells in the small intestine of mice

city: Cologne

first: F

middle: Thomas

last: Wunderlich

address: Gleueler Str. 50

department: Obesity and Cancer

institute: MPI for Metabolism Research

summary: Enteroendocrine cells (EECs) constitute only a small proportion of Villin-1 (Vil1)-expressing intestinal epithelial cells (IECs) of the gastrointestinal tract; yet, in sum, they build the largest endocrine organ of the body, with each of them storing and releasing a distinct set of peptides for the control of feeding behavior, glucose metabolism, and gastrointestinal motility. Like all IEC types, EECs are continuously renewed from intestinal stem cells in the crypt base and terminally differentiate into mature subtypes while moving up the crypt-villus axis. Interestingly, EECs adjust their hormonal secretion according to their migration statues as EECs receive altering differentiation signals along the crypt-villus axis and thus undergo functional readaptation. Cell-specific targeting of mature EEC subtypes by specific promoters is challenging because the expression of EEC-derived peptides and their precursors are not limited to EECs but are also found in other organs, such as the brain (e.g. Cck and Sst) as well as in the pancreas (e.g. Sst and Gcg). Here, we describe an intersectional genetic approach that enables cell type-specific targeting of functionally distinct EEC subtypes by combining a newly-generated Dre-recombinase expressing mouse line (Vil1-2A-DD-Dre) with multiple existing Cre-recombinase mice, and mouse strains with rox and loxP sites flanked stop cassettes for transgene expression. We found that transgene expression in triple-transgenic mice is highly specific in I, but not D and L cells in the apical villi of the small intestine. The targeting of EECs only in villi is likely caused by the intrinsic Vil1 expression, limiting our Vil1-2A-DD-Dre mouse line and the intersectional genetic approach described here for the investigation of mature EECs.

overall_design: Single-cell RNA sequencing of the murine intestinal epithelium was performed by isolating intestinal epithelial cells of male C57BL/6N wildtype mice from the duodenum, jejunum and ileum. Isolated cells from three 12-week-old male mice that were pooled in order to minimize artefacts originating from individual mice.

first: Christian

last: Vossen

first: Claudia

middle: T


title: Simultaneous single-cell three-dimensional genome and gene expression profiling uncovers dynamic enhancer connectivity underlying olfactory receptor choice

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus


title: A multiplexed, single-cell sequencing screen identifies compounds that increase neurogenic reprogramming of mammalian Muller glia.

city: Seattle

first: Amy

middle: Moreda

last: Tresenrider

state: WA

address: 3720 15th Ave NE

department: Genome Sciences

institute: University of Washington

summary: Neurodegeneration in the retina of mammals causes permanent loss of vision, due to their lack of regeneration. Some non-mammalian vertebrates show robust regeneration, via the Muller glia (MG), and we have recently made significant progress in stimulating adult mouse MG to regenerate functional neurons by transgenic expression of the proneural transcription factor Ascl1. While these results showed that MG can serve as an endogenous source of neuronal replacement, the efficacy of this process is limited. We designed a small molecule screen using sci-Plex, a method to multiplex up to thousands of single nucleus RNA-seq conditions into a single experiment, to screen a library of 92 compounds, identified, and validated two that promote neurogenesis in vivo. Our results demonstrate that high-throughput single-cell molecular profiling can substantially improve the discovery process for molecules and pathways that can stimulate neural regeneration and further demonstrate the potential for this approach to restore vision in patients with retinal disease.

overall_design: Harvested and then in vitro reprogrammed Muller glia were  subjected to sci-Plex after various treatments with Ascl1 OE and the addition of small molecules. MG were also reprogrammed in vivo by ANT treatment and subjected to 10x scRNA-seq 21 days after injury.

first: Cole

last: Trapnell

first: Thomas

last: Reh

first: Marcus

last: Hooper

first: Levi

last: Todd

first: Faith

last: Kierney

first: Nicolai

last: Blasdel


title: Construction of Drosophila chromatin accessibility landscape at a single-cell level using CH-ATAC-seq

city: Hanzhou

first: Guoji

last: Guo

address: Yuhangtang street 866

department: Zhejiang University School of Medicine

institute: Center for Stem Cell and Regenerative Medicine

summary: The assay for transposase-accessible chromatin using sequencing (ATAC-seq) at single-cell level can provide different perspectives of regulatory patterns based on  cell type-specific accessible regions.  While human genomic elements have been well studied, understanding how nuclear acid sequence regulates the expression of target genes in a genome-wide level in other organisms remains a major challenge. Batch effects, expensive machines are still constraints nowadays to build a cross-species landscape for further analysis, a platform with high throughput and low cost will be extremely required.Here, we constructed a cross-species accessible chromatin landscape by combinatorial-hybridization-based single-cell ATAC-seq, a single-cell ATAC-seq platform with high throughput and signal-noise ratio using fresh nuclei as input.

overall_design: Whole bodies of wild-type w1118 strain of Drosophila melanogaster at 10 days were  dissociated into single-nucleus suspensions, followed by single-cell DNA library construction, high-throughput sequencing, and bioinformatics analysis.

first: Guodong

first: Yuting

last: Fu

first: Lei

last: Yang

first: Fang

last: Ye

first: Peijing

first: Shuang

first: Xiaoping

last: Han

first: Jingjing

last: Wang


title: RNA sequencing analysis of gene expression in the mPFC of stressed DBA mice given vehicle of KDM5C inhibitor

city: Kyoto

first: Shusaku

last: Uchida

state: Kyoto

address: 53 Kawaharamachi, Sakyo-ku

country: Japan

institute: Kyoto University

summary: We performed high-throughput profiling of gene expression in the mPFC of stress-vulnerable DBA2/J mice subjected to subchronic and mild social defeat stress.DBA mice were subjected to 5-day of social defeat stress and then injected with KDM5C inhibitor (KDM5Ci) or vehicle. After behavioral tests, mice were euthanized for tissue collection. We then conducted the transcriptomics analysis. Our study provided insights into the understanding of the molecular mechanisms underlying stress susceptibility and provide the role of KDM5C in stress-induced behavioral changes.

overall_design: DBA mice were subjected to 5-day of social defeat stress (smSDS). Total 3 groups were set up: non-stressed (NS_Vehicle), SA:ANH subtype of smSDS-exposed mice given vehicle (SA:ANH-Vehicle), and SA:ANH subtype of smSDS-exposed mice given KDM5C inhibitor (SA:ANH-KDM5Ci. Three replicates were used for each group.


title: Lack of affinity signature for germinal center cells that have initiated plasma cell differentiation

first: Xin

last: Gao


title: Construction of zebrafish chromatin accessibility landscape at a single-cell level using CH-ATAC-seq

summary: The assay for transposase-accessible chromatin using sequencing (ATAC-seq) at single-cell level can provide different perspectives of regulatory patterns based on cell type-specific accessible regions. While human genomic elements have been well studied, understanding how nuclear acid sequence regulates the expression of target genes in a genome-wide level in other organisms remains a major challenge. Batch effects, expensive machines are still constraints nowadays to build a cross-species landscape for further analysis, a platform with high throughput and low cost will be extremely required.Here, we constructed a cross-species accessible chromatin landscape by combinatorial-hybridization-based single-cell ATAC-seq, a single-cell ATAC-seq platform with high throughput and signal-noise ratio using fresh nuclei as input.

overall_design: Whole bodies of wild-type strain AB zebrafishes at 4 months were dissociated into single-nucleus suspensions, followed by single-cell DNA library construction, high-throughput sequencing, and bioinformatics analysis.

last: Zhang


title: Simultaneous single-cell three-dimensional genome and gene expression profiling Uncovers Dynamic Enhancer Connectivity Underlying Olfactory Receptor Choice [olfactory, single-cell RNA & Hi-C]

first: Xiaoliang

last: Xie

overall_design:
simultaneous 3D genome and gene expression within the same single cell by LiMCA
Each cell was named according to the developmental stage/age and mouse strain information. For example, "p007" indicates that the developmental stage/age of the mouse from which this neuron was derived is postnatal day 7. "bc" and "bdf1" indicate the mouse strain. We labeled F1 hybrids of C57BL/6J (female) and DBA/2J (male) as bdf1 and F1 hybrids of C57BL/6J (female) and CAST/EiJ (male) as bc.

first: Honggui

last: Wu

first: Jiankun

first: Fanchong

last: Jian


title: Gene expression profiles of ASC52 cells differentiated to mature adipocytes and dedifferentiated by MDA-MB-231 cells

city: Milan

first: Loris

last: De Cecco

address: via Venezian 1

country: Italy

institute: IRCSS Istituto Nazionale Tumori

summary: It is widely recognized that cancer development and progression depend not only on tumor-cell intrinsic factors but also on its microenvironment and on the host characteristics. Adipocytes are the main stromal cells in the breast and an heterotypic interaction between breast epithelial cells and adipocytes has been demonstrated.  To date, the alterations associated with adipocyte dedifferentiation has to be further studied.  The aim of our work is to compare gene expression profile of mature adipocytes (MA), adipocytes dedifferentiated (DED) by breast cancer cells and mesenchymal stem cells (MSC) using the ASC52htert in vitro model.

overall_design: ASC52htert adipocyte derived mesenchymal stem cells (ATCC) were seeded in MesenPRO RS (GIBCO) and 3 days after differentiated or not to mature adipocytes culturing them in the StemPRO Adipogenesis Differentiation medium (GIBCO) for 14 days. Dedifferentiation  was induced by culturing ASC52 mature adipocytes for 7 days in conditioned media of MDA-MB-231 cells (RPMI, 10% FBS).  Mature adipocytes after differentiation were cultured for 7 days in control medium (RPMI,10% FBS). Cells were harvested, washed in  PBS 1x and immediately stored at -80°C until RNA extraction.


title: Macrophages derived from human induced pluripotent stem cells (iPSCs) serve as a high-fidelity cellular model for investigating HIV-1, dengue, and influenza viruses

status: Public on Jan 14 2024

city: Boulder

first: Sara

last: Sawyer

state: CO

address: 3415 Colorado Ave.,

department: BioFrontiers Institute

institute: University of Colorado Boulder

summary: Macrophages are important target cells for diverse viruses, and thus represent a valuable model system for studying virus biology. Isolation of primary human macrophages is done by culture of dissociated tissues or from differentiated blood monocytes, but these methods are both time consuming and result in low numbers of recovered macrophages. Here, we explore whether macrophages derived from human induced pluripotent stem cells (iPSCs)—which proliferate indefinitely and potentially provide unlimited starting material— could serve as a faithful model system for studying virus biology. Human iPSC-derived monocytes were differentiated into macrophages and then infected with HIV-1, dengue virus, or influenza virus as model macrophage-tropic human viruses. We show that iPSC-derived macrophages supported the replication of these viruses with similar kinetics and phenotypes to human blood monocyte-derived macrophages. These iPSC-derived macrophages were virtually indistinguishable from human blood monocyte-derived macrophages based on surface marker expression analysis (flow cytometry), transcriptomics (RNA-seq), and chromatin accessibility profiling (ATAC-seq) approaches. iPSC lines were additionally generated from nonhuman primate (chimpanzee) fibroblasts. When challenged with dengue virus, human and nonhuman primate iPSC-derived macrophages show differential susceptibility to infection, thus providing a valuable resource for studying the species-tropism of viruses. Collectively, our results substantiate iPSC-derived macrophages as an alternative to blood monocyte-derived macrophages for the study of virus biology.

overall_design: We differentiated human iPSCs into hematopoietic stem progenitor cells (HSPCs), then differentiated those HSPCs into monocytes. Monocytes were loosely adherent to the HSPCs  and were readily harvested by gentle washing of the cell monolayer with growth media every 4-5 days, followed by media replacement for continued growth.  We next confirmed that iPSC derived monocytes could differentiate into macrophages and polarize into phenotypically distinct subtypes. Adherent monocytes were treated with macrophage colony stimulating factor for four days to generate naïve macrophages. M0 macrophages were then polarized into M1 or M2 subsets using interferon-gamma and lipopolysaccharide or interleukin-4, respectively. To examine whether blood- and iPSC-derived macrophages exhibit similar transcriptional profiles, we carried out transcriptomic and chromatin accessibility analyses of all cell types during differentiation and subsequent polarization via RNA-sequencing (RNA-seq) and Assay for Transposase-Accessible Chromatin-sequencing (ATAC-seq).

first: Qing

first: Arturo

last: Barbachano-Guerrero

first: Laurence

last: Fairchild

first: Teisha

middle: J

last: Rowland

first: Mary

last: Allen

first: Robin

middle: D

last: Dowell

first: Cody

last: Warren


title: Evaluation of the effect of stresses due to weaning and immune activation on pigs

city: Urbana

first: Sandra

last: Rodriguez Zas

state: IL - Illinois

address: 1207 W. Gregory Dr.

institute: University of Illinois at Urbana

summary: Weaning is a stressor that can have significant impacts on health and physiology of the offspring. The effect of this stressor may be modulated by the exposure to other stressors during gestation such as the inflammatory signals of the mother in response to infection. The goal of this study is to determine the combined effects of weaning stress and immune stress during gestation on gene expression in the amygdala, a brain region critical for decision-making and aggression. A RNA-seq study of the amygdala of female and male pigs was undertaken. Within sex group, half of the pigs were weaned at 21 d of age and the rest remain with the gilts and were nursed. Also, half of the pigs in each group were raised from gilts exposed to a viral infection and half were raised by gilts that served as control.

overall_design: One treatment factor studied was weaning (levels Weaned or Nursed). Another treatment factor studied was Porcine Reproductive and Respiratory (P) viral infection or saline control (C). The remaining factor was sex (Female and Male). Altogether 48 individual amygdala samples were obtained 24 hours later and analyzed using the TruSeq Stranded mRNAseq Sample Prep kit’ (Illumina Inc, San Diego, CA, USA) to prepare RNAseq libraries. The libraries were sequenced on a NovaSeq 6000. Pair-ended reads of length 150bp were sequenced.


title: Stromal Pbrm1 mediates chromatin remodeling necessary for embryo implantation in mouse uterus [RNA-Seq]

city: Bethesda

first: qiliang

last: Xin

state: MD

address: 50 South Dr

department: NIDDK

institute: NIH

summary: Early gestational loss occurs in ~20% of all clinically recognized human pregnancies and is a significant cause of morbidity. Either embryonic or maternal defects can cause loss, but a functioning and receptive uterine endometrium is crucial for embryo implantation. The SWI/SNF remodeling complex containing PBRM1and BRG1 is essential for implantation of the embryonic blastocyst on the wall of the uterus in mice. Although pre-implantation development is unaffected, conditional ablation of Pbrm1 in uterine stromal cells disrupts progesterone pathway and uterine receptivity. Hand2 is a bHLH transcription factor required for embryo implantation. We report a new enhancer of Hand2 in stromal cells that requires PBRM1 for epigenetic histone modifications and coactivator recruitment. In Pbrm1cKO mice, perturbations at the enhancer site compromised Hand2 transcription, disrupts the fibroblast growth factor signaling pathway and prevents normal Stromal- Epithelial crosstalk and embryo implantation. The resultant mutant female mice are infertile and provide insight into potential causes of early pregnancy loss in humans.

overall_design: RNA-seq was performed using D4 primary uterine stromal cell between control and Pbrm1 cKO mice in order to identify the differentially expressed genes.

first: Qiliang

first: Iris

last: Feng

first: Guoyun

last: Yu

first: Jurrien

last: Dean


title: Stromal Pbrm1 mediates chromatin remodeling necessary for embryo implantation in mouse uterus [CHIC-Seq]

overall_design: CHIC-seq was performed using D4 WT uterine primary stromal cell  in order to identify the direct binding sites.


title: Inhibition of METTL3 Results in a Cell-Intrinsic Interferon Response That Enhances Antitumor Immunity [QuantSeq 3’ mRNA-Seq]

city: Cambridge

first: Georgia

last: Tsagkogeorga

address: Moneta Building, Babraham Research Campus

institute: STORM Therapeutics Ltd

summary: Therapies that enhance antitumor immunity have altered the natural history of many cancers. Consequently, leveraging nonoverlapping mechanisms to increase immunogenicity of cancer cells remains a priority. Using a novel enzymatic inhibitor of the RNA methyltransferase METTL3, we demonstrate a global decrease in N6-methyladenosine (m6A) results in double-stranded RNA (dsRNA) formation and a profound cell-intrinsic interferon response. Through unbiased CRISPR screens, we establish dsRNA-sensing and interferon signaling are primary mediators that potentiate T-cell killing of cancer cells following METTL3 inhibition. We show in a range of immunocompetent mouse models that although METTL3 inhibition is equally efficacious to anti–PD-1 therapy, the combination has far greater preclinical activity. Using SPLINTR barcoding, we demonstrate that anti–PD-1 therapy and METTL3 inhibition target distinct malignant clones, and the combination of these therapies overcomes clones insensitive to the single agents. These data provide the molecular and preclinical rationale for employing METTL3 inhibitors to promote antitumor immunity in the clinic.

overall_design:
CaOV3 cells were treated with 500 nM STM3006 or 0.3% DMSO in triplicate for 48 hours. For total RNA extraction, cells were homogenized in 700 μl Trizol (Thermo Fisher Scientific) followed by Chloroform addition and centrifugation. The aqueous phase was transferred to new tubes and RNA was precipitated using equal volumes of 100% Isopropanol followed by 2 washes with 80% Ethanol. RNA pellets were air-dried and resuspended in 25 μl nuclease-free water. RNA concentration was determined by Qubit RNA broad-range assay kit (Thermo Fisher Scientific, Q10211).
QuantSeq 3’ mRNA-Seq Library preparation was performed by Lexogen GmbH (Vienna, Austria) according to manufacturer’s instructions. Sequencing was performed using the Illumina NextSeq single-end 75 bp High Output V2 chemistry.

first: Andrew

last: Guirguis

first: Yaara

last: Ofir-Rosenfeld

first: Kathy

last: Knezevic

first: Wesley

last: Blackaby

first: David

last: Hardick

first: Yih-Chih

last: Chan

first: Ali

last: Motazedian

first: Andrea

last: Gillespie

first: Dane

last: Vassiliadis

first: Enid

middle: N

last: Lam

first: Kevin

last: Tran

first: Byron

last: Andrews

first: Michael

middle: E

last: Harbour

first: Lina

last: Vasiliauskaite

first: Claire

last: Saunders

first: Aleksandra

last: Azevedo

first: Joanna

last: Obacz

first: Ewa

middle: S

last: Pilka

first: Marie

last: Carkill

first: Laura

last: MacPherson

first: Elanor

last: Wainwright

first: Brian

last: Liddicoat

first: Benjamin

last: Blyth

first: Mark

middle: R

last: Albertella

first: Oliver

last: Rausch

last: Dawson


title: Construction of a cross-species chromatin accessibility landscape at a single-cell level using CH-ATAC-seq

first: Guoji

last: Guo

sample_organism:
Drosophila melanogaster
Danio rerio
Eisenia andrei

overall_design: Refer to individual Series


title: Stromal Pbrm1 mediates chromatin remodeling necessary for embryo implantation in mouse uterus [ATAC-Seq]

first: qiliang

last: Xin

overall_design: ATAC-seq was performed using  D4 primary uterine stromal cell between control and Pbrm1 cKO mice in order to identify the differentially chromatin accessibility.

first: Qiliang

first: Iris

last: Feng

first: Guoyun

last: Yu

first: Jurrien

last: Dean


title: Stromal Pbrm1 mediates chromatin remodeling necessary for embryo implantation in mouse uterus


title: Earthworm chromatin accessibility atlas with 10x Genomics

overall_design: Whole bodies of wild-type  Eisenia andrei at 4 months were  dissociated into single-nucleus suspensions, followed by single-cell DNA library construction, high-throughput sequencing, and bioinformatics analysis.

first: Guodong

last: Zhang

first: Yuting

last: Fu

first: Lei

last: Yang

first: Fang

last: Ye

first: Jingjing

last: Wang

first: Peijing

first: Shuang


title: Methylation-based markers for the estimation of age in African Cheetah, Acinonyx jubatus

city: Bloemfontein

first: Louis

middle: Stéphane

last: Le Clercq

state: FS

address: Genetics building, 205 Nelson Mandela Dr, Park West

department: Department of Genetics

country: South Africa

institute: University of the Free State

summary: Age is a key demographic in conservation biology where individual age classes show diffuse differences in terms of important population dynamics metrics such as morbidity and mortality. Furthermore, several traits including reproductive potential show clear senescence with aging. Thus, the ability to estimate the ages for the individuals of a population as part of age class assignment is critical in understanding both the current population structure as well as in modelling and predicting the future survival of species. This study explored the utility of age-related changes in methylation for six candidate genes, EDARADD, ELOVL2, FHL2, GRIA2, ITGA2B, and PENK, to create an age estimation model in captive cheetah. Gene orthologues between humans and cheetah were retrieved from NCBI containing a hundred CpG’s. Target regions were assayed for differential methylation and fragmentation patterns in fifty samples using mass array technology for a total of seventy-seven CpG clusters. Correlation analyses between CpG methylation and chronological age identified six CpG’s with an age relationship, of which four were hypomethylated and two were hypermethylated. Regression models, fitted for different combinations of CpG’s, indicated that age models using four and six CpG’s were most accurate, with the six CpG model having superior correlation and predictive power (R2 = 0.70, Mean Absolute Error = 25 months). This model was more accurate than previous attempts using methylation sensitive Polymerase Chain Reaction and performed similarly to models created using a candidate gene approach in several other mammal species, making methylation a promising tool of age estimation in cheetah.

overall_design: 50 samples were analysed for their CpG specific levels of methylation in 6 genes for a total of at least 73 CpG's. Samples were derived from African Cheetah across all relevant age classes from young cubs to very old adults.

first: Desiré

middle: L

last: Dalton

first: Antoinette

last: Kotzé

first: Paul

last: Grobler


title: WDR5 activity regulates nuclear changes and migration of acute lymphoblastic leukemia cells across confined spaces.

city: Madrid

first: Javier

last: Redondo-Muñoz

state: Madrid

address: Ramiro de Maeztu,9

department: Molecular Biomedicine

country: Spain

institute: CIB-CSIC

summary: Purpose: Acute lymphoblastic leukemia (ALL) is the most common pediatric cancer and  infiltration of leukemic cells is critical for disease progression and relapse. In spite of the  canonical functions of epigenetics in transcription and DNA homeostasis, its contribution to  the nuclear deformability of migrating leukemic cells remains unclear. The objective was to  evaluate the role of WDR5 (a core subunit of the histone H3K4 methyltransferases) during  leukemia cell migration.  Experimental design: We used ALL cell lines and primary samples from patients to study  their response and migration in 3D environments and how they extravasate in a mouse  xenograft model. We also performed, biophysical, transcriptional and ChIP-sequencing  analyses to determine the mechanism by which WDR5 controls ALL progression.  Results: We observed that WDR5 activity is critical for ALL cell invasion into 3D collagen  matrices. Notably, blocking WDR5 activity also decreased the extravasation of ALL cells in  an in vivo model. We identified that 3D constrained conditions promoted H3K4 methylation  in ALL cells that altered the global chromatin configuration and transcriptional changes  related to cell cycle and DNA replication. WDR5 inhibition did not impair cell adhesion or  cell response to chemokines; but we demonstrated that 3D conditions affected the  deformation and biophysical behavior of the nucleus in ALL cells.  Conclusions: We conclude that confined conditions have a fundamental role in ALL cell  biology and provide novel molecular and biophysical mechanisms used by leukemia cells to  disseminate. Targeting WDR5 might be a promising therapeutic strategy against ALL  infiltration and dissemination.

overall_design: 3 replicates of CCRF-CEM control and cultured in 3D conditions for 3 hours were used for chromatin immunoprecipitation of H3K4me3 and their DNA was extracted and sequenced. DNA from a control sample of the ChIP and from cells before de ChIP (input) was also sequenced.

first: Raquel

last: González-Novo

first: África

last: González-Murillo

first: Elena

last: Madrazo

first: David

last: Acitores

first: Mario

last: García de Lacoba

first: Manuel

last: Ramírez


title: Effect of microbial β-glucuronidases on Citrobacter rodentium mice infection

status: Public on Jan 13 2024

city: Madison

first: Thibaut

last: Rosay

state: Wisconsin

department: MMI

institute: UW Madison

summary: Previous experiments have shown that hexuronates regulate EHEC virulence, here we look at glucuronic acid effect on citrobacter rodentium

overall_design: 6h culture of Citrobacter rodentium in DMEM low glucose in microaerobic condition in presence or not of Glucuronic acid 100uM. Citrobacter rodentium mice infection.

first: Rosay

last: Thibaut

first: Sperandio

last: Vanessa


title: Lack of affinity signature for germinal center cells that have initiated plasma cell differentiation (Figure2)

first: Xin

last: Gao

summary: Long-lived plasma cells (PCs) secrete antibodies that can provide sustained immunity against infection. It has been proposed that high affinity cells are preferentially selected into this compartment, potentiating the immune response. We used single cell RNA-seq to track the germinal center (GC) development of Ighg2A10 cells, specific for the Plasmodium falciparum circumsporozoite protein (PfCSP). Following immunization with Plasmodium sporozoites we identified 3 populations of cells in the GC light zone. One population expressed a gene signature associated with the initiation of PC differentiation and had an enhanced propensity to form PCs in vitro. Unexpectedly, the estimated affinity of this putative pre-PC population was indistinguishable from cells in the GC generally. This was also true when high- or low-avidity recombinant PfCSP proteins were used as immunogens. Immunization with low-avidity PfCSP did, however, induce increased affinity maturation. Collectively these findings suggest that the initiation of PC development in the GC occurs via an affinity independent process.

overall_design: Probe+ IgD- Ighg2A10 B cells stained with TotalSeq-C hashtag antibodies were isolated by Fluorescence-activated cell sorting (FACS) from recipient mice that had been immunized with irradiated sporozoites either 7 or 21 days prior. The transcriptome and VDJ sequences of sorted cells was then analyzed using scRNAseq.

first: Henry

last: Sutton

first: Ian

last: Cockburn


title: SLAM-ITseq identifies that Nrf2 induces liver regeneration through the pentose phosphate pathway [Larval SLAM-Itseq]

city: Melbourne

first: Mark

last: Dawson

address: 305 Grattan Street

department: Cancer Research

institute: Peter MacCallum Cancer Centre

summary:
We investigated the immediate transcriptional changes in hepatocytes following APAP-induced acute liver injury by exposing lf:UPRT zebrafish larvae to DMSO or APAP in the presence of 4TU.
We the performed gene expression profiling analysis on total and nascent counts using data obtained from SLAM-ITseq of WT and Nrf2KO animals.

overall_design: Comparative gene expression profiling analysis of SLAM-ITseq data for WT or Nrf2KO zebrafish larvae (on the lf:UPRT background) exposed to DMSO or APAP in the presence of 4TU for 3 and 6 hours.

first: Vicky

last: Tan

first: Talhah

last: Salmi

first: Yih-Chih

last: Chan

first: Andrea

last: Gillespie

first: Andrew

middle: G

last: Cox


title: To investigate 4-TU labelling kinetics in a novel zebrafish transgenic line (lf:UPRT). [Adult SLAM-Itseq]

summary:
To understand 4-TU labelling kinetics in a novel zebrafish transgenic line (lf:UPRT), we exposed adult lf:UPRT zebrafish (whereby animals have hepatocyte-specific uracil phosphoribosyltransferase expression) with 4-TU for 1, 3, 6 and 9 hours.
We then dissected adult livers for SLAM-ITseq to determine the optimal 4-TU labelling time in our SLAM-ITseq workflow to allow us to study hepatocyte-specific nascent transcriptional changes.

overall_design: Comparative gene expression profiling analysis of SLAM-ITseq data for lf:UPRT animals exposed to 4-TU for different durations.

middle: M


title: SLAM-ITseq identifies that Nrf2 induces liver regeneration through the pentose phosphate pathway

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


title: Defining T cell receptor repertoires using nanovial-based binding and functional screening

status: Public on Jan 12 2024

city: Los Angeles

first: Dino

last: Di Carlo

address: 420 Westwood plaza

institute: University of California, Los Angeles

summary: The ability to selectively bind to antigenic peptides and secrete effector molecules can define rare and low-affinity populations of cells with therapeutic potential in emerging T cell receptor (TCR) immunotherapies. We leverage cavity-containing hydrogel microparticles, called nanovials, each coated with peptide-major histocompatibility complex (pMHC) monomers to isolate antigen-reactive T cells. T cells are captured and activated by pMHCs inducing the secretion of effector molecules including IFN-γ and granzyme B that are accumulated on nanovials, allowing sorting based on both binding and function. The TCRs of sorted cells on nanovials are sequenced, recovering paired αβ-chains using microfluidic emulsion-based single-cell sequencing. By labeling nanovials having different pMHCs with unique oligonucleotide-barcodes and secretions with oligo-barcoded detection antibodies, we could accurately link TCR sequences to specific targets and rank each TCR based on the corresponding cell’s secretion level. Using the technique, we identified an expanded repertoire of functional TCRs targeting viral antigens with high specificity and discovered rare TCRs with activity against cancer-specific splicing enhanced epitopes.

overall_design: We apply microcavity-containing hydrogel microparticles, known as nanovials, to selectively bind to and activate target T cells and capture secreted cytokines. The TCRs of sorted cells on nanovials are sequenced, recovering paired αβ-chains using microfluidic emulsion-based single-cell sequencing. By labeling nanovials having different pMHCs with unique oligonucleotide-barcodes and secretions with oligo-barcoded detection antibodies, we could accurately link TCR sequences to specific targets and rank each TCR based on the corresponding cell’s secretion level.

first: Doyeon

last: Koo

first: Zhiyuan

last: Mao

first: Robert

last: Dimatteo

first: Miyako

last: Noguchi

first: Natalie

last: Tsubamoto

first: Jami

last: McLaughlin

first: Wendy

last: Tran

first: Sohyung

last: Lee

first: Donghui

last: Cheng

first: Joseph

last: de Rutte

first: Giselle

last: Burton Sojo

first: Owen

last: Witte


title: Effect of APAP-induced acute liver injury on WT and Nrf2KO zebrafish liver gene expression following 12 and 24 hours [RNA-Seq]

first: Mark

last: Dawson

summary:
To investigate the transcriptional changes following acute liver injury, we exposed WT and Nrf2KO zebrafish larvae to APAP for 12 and 24 hours.
We then dissected out zebrafish larval livers and pooled 20 livers per sample for RNA-seq.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for WT and Nrf2KO zebrafish larval livers following acute liver injury.

first: Vicky

last: Tan

first: Talhah

last: Salmi


title: RNA sequencing of iAM-1 cells at different stages during one cycle of differentiation and subsequent dedifferentiation

city: Leiden

first: Pim

last: van Gorp

state: Zuid-Holland

address: Albinusdreef 2

department: Experimental Cardiology

country: Netherlands

institute: Leiden University Medical Center

overall_design: Examination of transcriptome during a full cylce of cardiac differentiation and subsequent dedifferentiation of conditionally immortalized neonatal rat atrial myocytes

first: P

first: A

last: de Vries


title: The cellular landscape of the endochondral bone highlights its multipotent and immunosuppressive features during the transition to extrauterine life.

city: Sevilla

first: Juan

middle: J.

last: Tena

address: Univ. Pablo Olavide, Ctra. Utrera km1

institute: CABD/CSIC

summary: The cellular complexity of the endochondral bone underlies its essential and pleiotropic roles during organismal life. While the adult bone has received significant attention, we still lack a deep understanding of the perinatal bone cellulome. Here, we have profiled the full composition of the murine endochondral bone at the single-cell level during the transition from fetal to newborn life and in comparison to the adult organ, with particular emphasis on the mesenchymal compartment. In contrast to that of adults, the perinatal bone contains several uncommitted fibroblastic clusters with blastema-like characteristics in organizing and supporting skeletogenesis, angiogenesis, peripheral nervous system development and hematopoiesis. Our data also reveals dynamic inter- and intra-compartment interactions and a highly anti-inflammatory bone marrow milieu, promoted by specific fibroblastic progenitor populations and abundant putative Myeloid-Derived Suppressive Cells (MDSC), which may ensure the proper program of lymphopoiesis and the establishment of central and peripheral tolerance in early life.

overall_design: Mouse endochondral bone cells at E18.5 and postnatal day 1 were dissociated, labelled with antibodies and isolated by Fluorescence-activated cell sorting (FACS). Gates were defined by the use of a mix of pan-antibodies to mesenchymal (CD140a/PDGFR-a) and endothelial cells (CD31/PECAM), together with CD9, a marker highly expressed in early hematopoietic progenitors and stromal cells. Sorted cells were mixed in different proportions to represent both abundant and minor populations and processed for scRNAseq.

first: Gretel

last: Nusspaumer

first: Irepan

last: Salvador-Martínez

first: Javier

last: Lopez-Rios

first: Tena

last: Juan


title: Bulk liver transcriptomics from control and TPN-fed neonatal pigs

first: Osvaldo

last: Graña

state: Comunidad de Madrid

address: C/ Melchor Fernández Almagro, 3

department: Structural Biology

institute: CNIO

summary: RNA-seq from bulk liver was performed from orally and TPN-fed piglets

overall_design: Newborn pigs were orally fed with milk as controls, or the jugular vein was catheterized and all nutrients were supplied parenterally and under a constant rate for two weeks. Liver was collected and RNA was extracted for RNA-seq analyses

first: Ana

middle: B

last: Plata-Gómez

last: Graña-Castro

first: Alejo

last: Efeyan


title: scRNAseq from Li-TSC1KORagAGTP and wild-type livers

summary: scRNAseq from liver was performed for two Li-TSC1KORagAGTP and two wild-type mice to examine different populations of hepatic cells

overall_design: Two Li-TSC1KORagAGTP and two wild-type mice were used in this experiment. Liver cells were isolated following a two-step protocol of digestion. Cell suspension was analyzed using 10X Genomics Chromium Single-Cell 3'.


title: Compact zinc finger architecture utilizing toxin-derived cytidine deaminases for highly efficient base editing in human cells -T cells

city: Richmond

first: Jessica

last: Davis

state: CA

address: 501 Canal Blvd

institute: Sangamo Therapeutics

summary: Nucleobase editors represent an emerging technology that enables precise single-base edits to the genomes of eukaryotic cells. Most nucleobase editors use deaminase domains that act upon single-stranded DNA and require RNA-guided proteins such as Cas9 to unwind the DNA prior to editing. However, the most recent class of base editors utilizes a deaminase domain, DddAtox, that can act upon double-stranded DNA. Here, we target DddAtox fragments and a FokI-based nickase to the human CIITA gene by fusing these domains to arrays of engineered zinc fingers (ZFs). We also identify a broad variety of Toxin-Derived Deaminases (TDDs) orthologous to DddAtox that allow us to fine-tune properties such as targeting density and specificity. TDD-derived ZF base editors enable up to 73% base editing in T cells with good cell viability and favorable specificity.

overall_design: Primer pools targeting potential base edited sites identified by the ZF-CBE genome-wide specificity assay were designed and synthesized. Potential base edited sites were amplified from genomic material isolated from transfected T cells and analyzed for base editing levels.

first: Friedrich

last: Fauser

first: Bhakti

last: Kadam

first: Sebastian

last: Arangundy-Franklin

first: Vishvesha

last: Vaidya

first: Nicola

last: Schmidt

first: Garrett

last: Lew

first: Danny

middle: F

last: Xia

first: Rakshaa

last: Mureli

first: Colman

last: Ng

first: Yuanyue

last: Zhou

first: Nicholas

middle: A

last: Scarlott

first: Jason

last: Eshleman

first: Yuri

last: Bendaña

first: David

last: Shivak

first: Andreas

last: Reik

first: Patrick

last: Li

first: Gregory

first: Jeffrey

middle: C

last: Miller


title: Modulation of fecal miRNA highlights the preventive effects of a Mediterranean Low-Inflammatory Dietary Intervention in patients with Familial Adenomatous Polyposis (RNA-Seq)

city: Orbassano

first: Giulio

last: Ferrero

state: TO

address: Regione Gonzole, 10

department: Dept. Clinical and Biological Sciences

institute: University of Turin

summary:
Diet can regulate gene and microRNA (miRNA) expression and various biological processes in the gut. Dietary interventions have been proposed as therapeutic approaches for several diseases, including cancer. In a pilot study, we showed that a low-inflammatory Mediterranean diet reduced markers of local and systemic inflammation in patients with Familial Adenomatous Polyposis (FAP).
We evaluated the changes induced by a low-inflammatory Mediterranean dietary intervention on fecal miRNome and intestinal tissue transcriptome in FAP subjects and assessed whether these changes could be associated with the beneficial effects observed in the pilot study.
The diet modulated 41 fecal miRNAs, and this modulation remained for three months after the intervention. miR-5092-5p, miR-4527, and miR-3612-3p were positively correlated with adherence to the Mediterranean diet, while miR-6867-5p and miR-760-5p were negatively correlated with serum calprotectin levels. The altered miRNAs target genes mainly related to inflammatory pathways, DNA repair, metabolism, and cytoskeleton organization. Seventy genes were differentially expressed between adenoma and normal tissue. Most were different before the dietary intervention, but reached similar levels after the diet. Functional enrichment analysis identified the proinflammatory ERK1/2, cell cycle regulation and nutrient response pathways as commonly regulated by differentially expressed miRNAs and genes.
These findings suggest that fecal miRNAs modulated by the diet reflect an epigenetic regulation occurring in tissues that seems to influence inflammatory pathways. miRNAs and genes with oncogenic and tumor suppressor functions are also regulated, highlighting the potential cancer-preventive effect of the low-inflammatory Mediterranean diet.

overall_design: The study was a prospective nonrandomized pilot single-arm trial aimed at evaluating the effects of a low-inflammatory Mediterranean diet on FAP patients. Briefly, FAP individuals aged >18 years, who underwent prophylactic colectomy and were involved in the surveillance program at the Fondazione IRCCS Istituto Nazionale Tumori of Milan (INT), were eligible. Participants participated in dietary intervention activities that included 12 days of training, cookery, supportive education, and meals. The intervention lasted three months and was followed by another three months in which subjects continued the diet at home.  The participants signed informed consent and were asked to donate blood and stool samples at baseline, at the end of the three-month dietary intervention, and at six months (respectively, T0, T1 and T2). They were also asked to complete a questionnaire on Mediterranean diet adherence (MEDAS). Finally, they underwent endoscopy at T0 and T2, in which tissue samples of healthy rectal mucosa and adenomas (when present) were collected as formalin-fixed and paraffin-embedded (FFPE) specimens. Normal rectal tissue was available from 8 patients at T0 and 8 at T2, while adenomas from 10 patients at T0 and 10 at T2

first: Oscar

last: Illescas Pomposo

first: Ferrero

last: Giulio

first: Barbara

last: Pardini

first: Sonia

last: Tarallo

first: Alessio

last: Naccarati

first: Manuela

last: Gariboldi

first: Antonino

last: Belfiore

first: Chiara

last: Ciniselli

first: Sara

last: Noci

first: Elena

last: Daveri

first: Stefano

last: Signoroni

first: Laura

last: Cattaneo

first: Andrea

last: Mancini

first: Daniele

last: Morelli

first: Massimo

last: Milione

first: Francesca

last: Cordero

first: Licia

last: Rivoltini

first: Paolo

last: Verderio

first: Patrizia

last: Pasanisi

first: Marco

last: Vitellaro


title: Protection from starvation-induced liver atrophy.

status: Public on Jan 11 2024

city: Greater Noida

first: Sumeet

middle: Pal

last: Singh

state: Uttar Pradesh

address: NH91, Tehsil Dadri

department: Life Sciences

country: India

institute: Shiv Nadar Institution of Eminence

summary: Starvation causes the accumulation of lipid droplets in the liver, a somewhat counterintuitive phenomenon that is nevertheless conserved from flies to humans. Much like fatty liver resulting from overfeeding, hepatic lipid accumulation (steatosis) during undernourishment can lead to lipotoxicity and atrophy of the liver. Here, we found that while surface populations of Astyanax mexicanus undergo this evolutionarily conserved response to starvation, the starvation-resistant cavefish larvae of the same species do not display an accumulation of lipid droplets upon starvation. Moreover, cavefish are resistant to liver atrophy during starvation, providing a unique system to explore strategies for liver protection. Using comparative transcriptomics between zebrafish, surface fish, and cavefish, we identified the fatty acid transporter slc27a2a/fatp2 to be correlated with the development of fatty liver. Pharmacological inhibition of slc27a2a in zebrafish rescues steatosis and atrophy of the liver upon starvation. Further, down-regulation of FATP2 in drosophila larvae inhibits the development of starvation-induced steatosis, suggesting the evolutionary conserved importance of the gene in regulating fatty liver upon nutrition deprivation. Overall, our study identifies a conserved, druggable target to protect the liver from atrophy during starvation.

overall_design: Zebrafish larvae at 4 dpf were treated with Lipofermata (5 uM) or vehicle (DMSO) for 48 hours. During treatment, no exogenous food was added. At 6 dpf, the livers were isolated for RNA-Sequencing. Experiment was performed in duplicate.

first: Singh

middle: P

last: Sumeet


title: Loss of GTF2I promotes neuronal apoptosis and synaptic reduction in human cellular models of neurodevelopment

city: La Jolla

first: Alysson

middle: R.

last: Muotri

address: 2880 Torrey Pines Scenic Road MC 0695

institute: UNIVERSITY OF CALIFORNIA, SAN DIEGO

summary: Individuals with Williams syndrome (WS), a multisystemic neurodevelopmental disorder, characteristically portray a hypersocial phenotype. WS is caused by the hemizygous loss of 26-28 genes at chromosomal locus 7q11.23, one of which is GTF2I. Copy number variations and mutations in GTF2I are associated with altered sociality and have been proposed to underlie the hypersocial expression of WS. However, the contribution of GTF2I to human neurodevelopment remains poorly understood. Here, human cellular models of neurodevelopment, including neural progenitors, neurons, and three-dimensional cortical organoids, were differentiated from CRISPR-Ca9-edited GTF2I-knockout (GTF2I-KO) pluripotent stem cells (hiPSCs) to investigate the role of GTF2I in human neurodevelopment. Compared to controls, GTF2I-KO progenitors exhibited an increased proliferation rate and an altered cell cycle profile. Cortical organoids and neurons demonstrated increased cell death and synaptic dysregulation, including synaptic structural dysfunction and decreased electrophysiological activity on a multi-electrode array. Overall, our findings suggest that changes in synaptic circuit integrity may be a prominent mediator of the link between alterations in GTF2I and variation in the phenotypic expression of human sociality.

overall_design: Samples are two biological replicates each of two-month control and GTF2I-KO cortical organoids generated using the CVB hiPS cell line. Gene expression was compared between control and GTF2I-KO conditions.

first: Jason

middle: W

last: Adams

first: Annabelle

last: Vinokur

first: Janaina

last: de Souza

first: Charles

last: Austria

first: Bruno

last: Guerra

first: Roberto

middle: H

last: Herai

first: Karl

middle: J

last: Wahlin


title: Modulation of fecal miRNA highlights the preventive effects of a Mediterranean Low-Inflammatory Dietary Intervention in patients with Familial Adenomatous Polyposis (sRNA-Seq)

first: Giulio

last: Ferrero

type: Non-coding RNA profiling by high throughput sequencing

summary:
Diet can regulate gene and microRNA (miRNA) expression and various biological processes in the gut. Dietary interventions have been proposed as therapeutic approaches for several diseases, including cancer. In a pilot study, we showed that a low-inflammatory Mediterranean diet reduced markers of local and systemic inflammation in 27 patients with Familial Adenomatous Polyposis (FAP).
We evaluated the changes induced by a low-inflammatory Mediterranean dietary intervention on fecal miRNome and intestinal tissue transcriptome in FAP subjects and assessed whether these changes could be associated with the beneficial effects observed in the pilot study.
The diet modulated 41 fecal miRNAs, and this modulation remained for three months after the intervention. miR-5092-5p, miR-4527, and miR-3612-3p were positively correlated with adherence to the Mediterranean diet, while miR-6867-5p and miR-760-5p were negatively correlated with serum calprotectin levels. The altered miRNAs target genes mainly related to inflammatory pathways, DNA repair, metabolism, and cytoskeleton organization. Seventy genes were differentially expressed between adenoma and normal tissue. Most were different before the dietary intervention, but reached similar levels after the diet. Functional enrichment analysis identified the proinflammatory ERK1/2, cell cycle regulation and nutrient response pathways as commonly regulated by differentially expressed miRNAs and genes.
These findings suggest that fecal miRNAs modulated by the diet reflect an epigenetic regulation occurring in tissues that seems to influence inflammatory pathways. miRNAs and genes with oncogenic and tumor suppressor functions are also regulated, highlighting the potential cancer-preventive effect of the low-inflammatory Mediterranean diet.

overall_design: The study was a prospective nonrandomized pilot single-arm trial aimed at evaluating the effects of a low-inflammatory Mediterranean diet on FAP patients. Briefly, FAP individuals aged >18 years, who underwent prophylactic colectomy and were involved in the surveillance program at the Fondazione IRCCS Istituto Nazionale Tumori of Milan (INT), were eligible. Participants participated in dietary intervention activities that included 12 days of training, cookery, supportive education, and meals. The intervention lasted three months and was followed by another three months in which subjects continued the diet at home. The participants signed informed consent and were asked to donate blood and stool samples at baseline, at the end of the three-month dietary intervention, and at six months (respectively, T0, T1 and T2). They were also asked to complete a questionnaire on Mediterranean diet adherence (MEDAS). Finally, they underwent endoscopy at T0 and T2, in which tissue samples of healthy rectal mucosa and adenomas (when present) were collected as formalin-fixed and paraffin-embedded (FFPE) specimens.

first: Oscar

last: Ilescas Pomposo

first: Sonia

last: Tarallo

first: Barbara

last: Pardini

first: Alessio

last: Naccarati

first: Manuela

last: Gariboldi

first: Antonino

last: Belfiore

first: Chiara

last: Ciniselli

first: Sara

last: Noci

first: Elena

last: Daveri

first: Stefano

last: Signoroni

first: Laura

last: Cattaneo

first: Andrea

last: Mancini

first: Daniele

last: Morelli


title: ATAC-Seq of Batf-deficient pDC Transcriptomes (CpG 0h, 2h)

city: Duesseldorf

first: Stefanie

last: Scheu

state: Nordrhein-Westfalen

address: Universitaetsstr. 1

department: Institute of Medical Microbiology and Hospital Hygiene

institute: University of Duesseldorf

summary:
Purpose: The goals of this study are to compare the pDC chromatin structure (ATAC-seq) at steady state and at 2h after CpG activation.
Methods: Cells were harvested and frozen in culture media containing FBS and 5% DMSO. Cryopreserved cells were sent to Active Motif to perform the ATAC-seq assay. The cells were then thawed in a 37°C water bath, pelleted, washed with cold PBS, and tagmented as previously described (Buenrostro et al., 2013), with some modifications based on (Corces et al., 2017). Briefly, cell pellets were resuspended in lysis buffer, pelleted, and tagmented using the enzyme and buffer provided in the Nextera Library Prep Kit (Illumina). Tagmented DNA was then purified using the MinElute PCR purification kit (Qiagen), amplified with 10 cycles of PCR, and purified using Agencourt AMPure SPRI beads (Beckman Coulter). Resulting material was quantified using the KAPA Library Quantification Kit for Illumina platforms (KAPA Biosystems), and sequenced with PE42 sequencing on the NextSeq 500 sequencer (Illumina).
Results: Reads were aligned using the BWA algorithm (mem mode; default settings). Duplicate reads were removed, only reads mapping as matched pairs and only uniquely mapped reads (mapping quality >= 1) were used for further analysis. Alignments were extended in silico at their 3’-ends to a length of 200 bp and assigned to 32-nt bins along the genome. The resulting histograms (genomic “signal maps”) were stored in bigWig files. Peaks were identified using the MACS 2.1.0 algorithm at a cut off of p-value 1e-7, without control file, and with the –nomodel option. Peaks that were on the ENCODE blacklist of known false ChIP-Seq peaks were removed. Signal maps and peak locations were used as input data to Active Motifs proprietary analysis program, which creates Excel tables containing detailed information on sample comparison, peak metrics, peak locations and gene annotations. For differential analysis, reads were counted in all merged peak regions (using Subread), and the replicates for each condition were compared using DESeq2.
Conclusions: Our study represents a detailed analysis of naive and activated pDC chromatin that analyzes the role of the transcription factor BATF for chromatin structure in pDCs.

overall_design: Chromatin profiles of bone marrow-derived Flt3-L FACS purified pDCs from Batf-deficient mice that were left naïve or stimulated with TLR9 agonist CpG for 2h.

first: Shafaqat

last: Ali

first: Ritu

last: Mann-Nüttel


title: Next Generation Sequencing  of  Batf-knock out pDC Transcriptomes in a longitudinal activation study (CpG 0h, 2h, 6h, 12)

summary:
Purpose: The goals of this study are to compare NGS-derived pDC transcriptome profiling (RNA-seq) normalized counts and differential expression of genes between different pDC states (steady state, or TLR9 activated for 2h, 6h, or 12h with CpG) in Batf presence and absence.
Methods: mRNA profiles of Bone marrow-derived Flt3-L cultured FACS purified pDCs from wild-type and Batf-knock out mice that were left naive or stimulated with TLR9 agonist CpG for 2h, 6h r 12h were generated by deep sequencing, in triplicate, using the Illumina HiSeq3000 platform. DNase digested total RNA samples used for transcriptome analyses were quantified (Qubit RNA HS Assay, Thermo Fisher Scientific) and quality measured by capillary electrophoresis using the Fragment Analyzer and the ‘Total RNA Standard Sensitivity Assay’ (Agilent Technologies, Inc. Santa Clara, USA). All samples in this study showed high quality RNA Quality Numbers (RQN; mean = 9.9). The library preparation was performed according to the manufacturer’s protocol using the Illumina® ‘TruSeq Stranded mRNA Library Prep Kit’. Briefly, 200 ng total RNA were used for mRNA capturing, fragmentation, the synthesis of cDNA, adapter ligation and library amplification. Bead purified libraries were normalized and finally sequenced on the HiSeq 3000/4000 system (Illumina Inc. San Diego, USA) with a read setup of SR 1x150 bp. The bcl2fastq tool was used to convert the bcl files to fastq files as well for adapter trimming and demultiplexing.
Results: Data analyses on fastq files were conducted with CLC Genomics Workbench (version 11.0.1, QIAGEN, Venlo. NL). The reads of all probes were adapter trimmed (Illumina TruSeq) and quality trimmed (using the default parameters: bases below Q13 were trimmed from the end of the reads, ambiguous nucleotides maximal 2). Mapping was done against the Mus musculus (mm10; GRCm38.86) (March 24, 2017) genome sequence. After grouping of samples (three biological replicates each) according to their respective experimental condition, multi-group comparisons were made and statistically determined using edgeR on usegalaxy.org The Resulting pvalues were corrected for multiple testing by FDR. A pvalue of ≤0.05 was considered significant.
Conclusions: Our study represents the first detailed analysis of Batf-dependent gene expression in naive and activated pDC transcriptomes in a longitduinal study. Our results show that Batf absence significantly altered the global gene expression patterns in pDCs, modulating many biological pathways important for cell development and effector function.

overall_design: mRNA profiles of Bone marrow-derived Flt3-L FACS purified pDCs from wild type and Batf-deficient mice that were left naïve or stimulated with TLR9 agonist CpG for 2h, 6h or 12h.