title: Genome-wide bisulfite sequencing (GWBS) and RNA-seq in hippocampus of Neil1- and Neil2 deficient mice

city: Oslo

first: Mingyi

last: Yang

address: Sognsvannsveien 20

department: Department of Microbiology, Rikshospitalet

institute: Oslo University Hospital

summary: We report the bisulfite sequencing and RNA-seq in hippocampus of Neil deficient adult mice. By genome-wide bisulphite sequencing (GWBS) analysis, NEIL depletion mice (including Neil1-/-, Neil2-/- and Neil1-/- Neil2-/-) consistently displayed global hypo-methylation of 5mC in base resolution in CG context, as well as in gene feature regions including transcription start sites (TSS) in CHG and CHH context by compared to wild type.  RNA-seq reveals differentially expressed genes are mainly overlapped with differential methylation. A subset of overlap genes is enriched in synaptic function. Together, NEIL1 and NEIL2  functionally promote DNA methylation and impact gene expression.

overall_design: Examination of genome-wide 5mC and 5hmC in hippocampus of Neil deficient adult mice compared to wild type;  investigation the correlation between methylaiton and gene expression.

first: Katja

last: Scheffler

first: Magnar

last: Bjoras


title: High-throughput single-cell transcriptome profiling of tomato leaf

status: Public on May 01 2024

city: Changsha

first: Hao

last: Yue

address: No. 892 Yuanda 2nd Road, Mapoling, Furong District, Changsha City, Hunan Province

country: China

institute: Hunan university

type: Expression profiling by high throughput sequencing

summary: The occurrence of Tomato chlorosis virus (ToCV) disease seriously damages tomato growth and yield, and there is no effective way to control ToCV transmission. So far, no studies have reported exploring the interaction between ToCV and tomato at the single cellular level. In this study, single cell RNA sequence was performed on a total of 26720 individual cells from healthy and ToCV-infected tomato leaves. We through identifying cell types, the first tomato leaf cell atlas was successfully constructed. In situ hybridization experiments identified specific marker genes that can be used to identify tomato cell types. Moreover, we have characterized transcription factors that may play a key role in tomato response to ToCV infection, and described the trichome differentiation trajectory during ToCV infection through pseudotime analysis. In conclusion, we proved the feasibility of single-cell sequencing to study the response of plants to biotic stress, and put forward new insights into the interaction between ToCV and tomato from the cellular level. Our data will lay the foundation for following studies between ToCV and plants, and will also provide a valuable reference for future research on non-model plant single cells.

overall_design: sample from healthy tomato plants and ToCV-infected tomato plants

first: Xiaobin

last: Shi

first: Yong

last: Liu


title: Profiling of 2'-O-methylation in Leishmania donovani rRNA using RibOxi-seq

city: Durham

first: Christopher

middle: L

last: Holley

state: NC

address: 2301 Erwin Rd

country: USA

institute: Duke University

summary: In this study we used RibOxi-seq to identify 2’-O-methylation (Nm) present on rRNAs in single nucleotide resolution.

overall_design: 2'-O-methylation profiling on Leishmania donovani total RNA

first: Yinzhou

last: Zhu

first: K

middle: S

last: Rajan

first: Shulamit

last: Michaeli


title: Gene expression analysis of myeloid cells cultured in the presence of fibronectin and/or anti-ILT3 16C5

city: South San Francisco

first: Joshua

last: Lichtman

state: CA

address: 333 Oyster Point Boulevard

department: Translational Science

institute: NGM Biopharmaceuticals

summary: RNA sequencing of monocyte-derived dendritic cells, tolerogenic dendritic cells, unpolarized macrophages, and M1, M2a, and M2c macrophages differentiated in vitro on PBS- or fibronectin-coated wells, in the presence of control IgG1 or anti-ILT3 16C5

overall_design: Peripheral blood monocytes from healthy human donors were plated on wells pre-coated with PBS or fibronectin (5 ug/mL) in the presence of an anti-KLH control antibody or anti-ILT3 16C5 (10 ug/mL). Monocytes were differentiated into dendritic cells or macrophages as follows: Monocyte-derived DCs (GM-CSF/IL-4 for 7 days), tolerogenic DCs (GM-CSF/IL-4 for 5 days + vitamin D3/dexamethasone for 2 days), unpolarized macrophages (M-CSF for 7 days), or polarized macrophages (M-CSF for 5 days plus IFN-g/LPS for 2 days for M1 macrophages, IL-4 for 2 days for M2a macrophages, or IL-10 for 2 days for M2c macrophages). Cells were harvested in TRIzol at day 7, and RNA was purified using the Qiagen RNeasy kit. Total RNA was prepared for sequencing using the TailorMix Directional RNA Sample Preparation Kit (SeqMatic) and sequenced on an Illumina NovaSeq S1.

first: Julie

last: Roda

first: Peirong

last: Krisney Chen

first: Hung-I

middle: H

last: Chen

first: Alan

last: Kutach

first: Wei

last: Guo

first: Betty

last: Li

first: Yan

last: Wang

first: Daniel

last: Kaplan


title: Next Generation Sequencing Facilitates Quantitative Analysis of PVT1 overexpressed MDA-MB-231 cells.

city: Nanjing

first: Xingxing

state: Jiangsu

address: 123 Tianfei Road

institute: Women’s Hospital of Nanjing Medical University

summary: PVT1 can regulates glycolysis related genes expression.

overall_design: We over-expressed the PVT1 in MDA-MB-231 cells using the lentiviral construct pLVX-PVT1-EF1α-IRES-EGFP-PGK-puro. We then used RNA sequencing  to compare PVT1 over-expression cells to negative controls(empty vector).

first: Mingming

last: Lv

first: Cheng

last: Lu


title: Profiling of 2'-O-methylation in Leishmania amazonensis rRNA using RibOxi-seq

summary: In this study we report the complete repertoire of 2'-O-methylation sites present in the rRNA of Leishmania amazonensis rRNAs and a subset of small RNAs using RibOxi-seq.

overall_design: Total RNA from Leishmania amazonensis are used for generating Riboxi-seq libraries for 2'-O-methylation profiling


title: Metabolic rewiring tunes dermal macrophages in staphylococcal skin infection

city: Freiburg

first: Aaron

middle: James

last: Forde

address: Breisacher Str. 115

country: Germany

institute: Medical Center University of Freiburg

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

overall_design: Refer to individual Series


title: Metabolic rewiring tunes dermal macrophages in staphylococcal skin infection [RNA-seq dataset 2]

summary: Dermal macrophages (Macs) are essential for the timely resolution of staphylococcal skin infections. However the cell intrinsic pathways and microenvironmental cues that mediate this are unclear. Here we analyzed a strictly  intradermal ear infection model with Staphylococcus aureus (S. aureus) to explore immunometabolic regulation.

overall_design: mRNA transcriptomes of CD64hi dermal macrophages sorted from uninfected mice and mice injected intradermally in both ears with S. aureus for 1, 5 or 7 days.

middle: J


title: Comparative transcriptome analysis reveals the mechanism of cross-protection against Verticillium wilt conferred on sunflower by hypovirulent strain Gibellulopsis nigrescens Vn-1

city: Hohhot

first: Xin

last: Zhao

address: No. 306, Zhaowuda Road, Saihan District, Hohhot, Inner Mongolia

institute: Inner Mongolia Agricultural University

summary:
Background: Hypovirulent fungal strain Gibellulopsis nigrescens Vn-1 cross-protects sunflower against Verticillium wilt. To discover the mechanism of cross-protection by this hypovirulent strain, we analyzed defense enzyme activities and transcriptomes of root samples infected with virulent and hypovirulent strains.
Results: Defense enzyme activities increased after inoculation, with the highest levels observed 24 h post-inoculation. At the same time, defense enzyme gene expressions were upregulated, and H2O2 accumulation decreased. A comparative transcriptome analysis revealed that there are 23 GO terms significantly enriched in the Vn-1 group compared with the control, including three specific oxidoreductase-related and four signaling related GO terms. In addition, there were 7 KEGG pathway only enriched in V33 group compared with the control, and 3 KEGG pathway (Alanine, aspartate and glutamate metabolism, Cutin, suberine and wax biosynthesis and Ribosome) only enriched in Vn-1 compared with the control.
Conclusions: According to our results, both hypovirulent strain G. nigrescens Vn-1 and virulent strain V. dahliae V33 can reduce levels of reactive oxygen species in sunflower seedling by regulating HaCAT and HaPOD expression. Twenty three GO terms and three KEGG pathway contribute to the formation of specific resistance against virulent strain V. dahliae V33.

overall_design: 15 days of wild-type (A), attenuated strain treatment (B) and virulent strain treatment (C) sunflower root mRNA profile

first: Bao-Zhu

last: Dong

first: Ying-Jie

first: Dong

first: Lan

last: Jing

first: Hong-You

last: Zhou


title: Profiling of 2'-O-methylation in Leishmania major rRNA using RibOxi-seq

first: Christopher

last: Holley

summary: In this study we profiled the complete repertoire of 2'-O-methylation sites present in the rRNA and a subset of small RNAs of Leishmania major rRNA using RibOxi-seq.

overall_design: 2'-O-methylation profiles using Leishmania major total RNA

first: Yinzhou

last: Zhu

first: K

last: Rajan

first: Shulamit

last: Michaeli


title: Extensive Splicing across the Saccharomyces cerevisiae genome

city: Los Angeles

first: Stephen

last: Douglass

address: 5128 TLSB Terasaki Life Sciences Building, 610 Charles Young Drive East

department: Molecular, Cell and Developmental Biology

institute: University of California, Los Angeles

summary: Pre-mRNA splicing is vital for the proper function and regulation of eukaryotic gene expression.  Saccharomyces cerevisiae has been used as a model organism for studies of RNA splicing because of the striking conservation of the spliceosome and its catalytic activity.  Nonetheless, there are relatively few annotated alternative splice forms, particularly when compared to higher eukaryotes.  Here, we describe a method to combine large scale RNA sequencing data to accurately discover novel splice isoforms in Saccharomyces cerevisiae.  Using our method, we find extensive evidence for novel splicing of annotated intron-containing genes as well as genes without previously annotated introns and splicing of transcripts that are antisense to annotated genes.  By incorporating several mutant strains at varied temperatures, we find conditions which lead to differences in alternative splice form usage.  Despite this, every class and category of alternative splicing we find in our datasets is found, often at lower frequency, in wildtype cells under normal growth conditions.  Together, these findings show that there is widespread splicing in Saccharomyces cerevisiae.

overall_design: Transcriptomes of 6 different genotypes of S. cerevisiae were profiled by RNA sequencing of rRNA-depleteted libraries generated from cells grown at 25 and 37 degrees Celsius

middle: M

first: Calvin

last: Leung

first: Tracy

last: Johnson


title: Metabolic rewiring tunes dermal macrophages in staphylococcal skin infection [RNA-seq dataset 1]

overall_design: mRNA Transcriptomes of bone marrow derived macrophages (BMDMs) generated from LysMcre/+::HIF1αfl/fl mice. Either  Cre(+) mice or Cre(-) were used The BMDMs were generated for 6 days in either M-CSF or GM-CSF. The macrophages were either left untreated (control) or stimulated for 4 hours with heat fixed S. aureus.


title: Genome-wide analysis of transcription in dek3 mutants and DEK3-over expressors [2 temperatures at 2 time points]

city: Cambridge

first: Anna

last: Brestovitsky

address: Bateman Street

country: United Kingdom

institute: Sainsbury Laboratory (SLCU), University of Cambridge

summary: The decision of whether to grow and proliferate or to restrict growth and develop resilience to stress is a key biological trade-off. Multiple tumour suppressors have roles in suppressing growth and proliferation when conditions are unfavourable, and aggressive tumours show re-wiring of the metabolic pathways and removal of these restraints on growth. In plants, constitutive growth results in increased sensitivity to environmental stress. However, the underlying mechanisms controlling this decision are not well understood. We used temperature as a cue to discover regulators of this process in plants, as it both enhances growth and development rates within a specific range, and is also a stress at extremes. We find the conserved chromatin protein DEK  plays a central role in balancing the response between growth and arrest in Arabidopsis, and it does this via H2A.Z-nucleosomes. DEK target genes show two distinct categories of chromatin architecture, and these predict induction or repression by DEK. We show that these chromatin signatures of DEK target genes might be conserved in human cells, suggesting that DEK may act through a fundamental evolutionarily conserved mechanism to control the balance between growth and arrest in plants and animals.

overall_design: RNA-seq were generated for seedlings in different temperature at 2 time points. ZT0 time point just before the lights are on (End of the night); ZT8-just before the light are off(end of the day)

first: Phil

last: Wigge


title: NACER, a liver-specific lncRNA, functions as Nrf2-activating competing endogenous RNA for Plk2 and p21cip1

city: Seoul

first: Sang Geon

last: Kim

state: Seoul

address: San56-1, Sillim-dong, Kwanak-gu

department: College of Pharmacy

country: South Korea

institute: Seoul National University

summary: LncRNA for anti-oxidative capacity control has not been discovered yet. Nrf2 is a central molecule for cellular defense by increasing anti-oxidative capacity. Here, we identified a novel lncRNA named ‘NACER’ (Nrf2 Activating Competing Endogenous RNA) transcribed from an upstream region of MIR122. NACER existed in the cytoplasm, suggestive of its function as competing endogenous RNA (ceRNA, miRNA sponge). NACER served as ceRNA for Plk2 and p21cip1 through binding for miR-128/224, inducing Plk2/Nrf2/p21cip1 complexation for Nrf2 activation in cells under p53-activating conditions (i.e., DNA damage and serum deprivation). NACER expression was suppressed when apoptosis was initiated, being a rheostat for cell fate determination. NACER levels correlated with the recurrence free survival rate of patients with hepatocellular carcinoma after surgery. Collectively, NACER promotes Plk2 and p21cip1 translation by competing for specific miRNAs, activating Nrf2 under the surviving condition from oxidative stress, implying that NACER plays a fine-tuning rheostat for cell fate decision.

overall_design: Examination of RNA expression in Huh7 cells after NACER knockdown

first: Min

last: Joo

first: Eun

first: Sang

middle: G


title: Chromosome conformation capture of Jurkat T cells during parabolic flight

city: Zurich

first: Cora

middle: S.

last: Thiel

address: Winterthurerstrasse 190

department: Institute of Anatomy

country: Switzerland

institute: University of Zurich

summary: Jurkat T cells have been fixated at different gravity conditions during the 4th Swiss Parabolic Flight Campaign (4SPFC). 1g inflight samples were generated by crosslinking 5 minutes prior to onset of the first parabola. Hypg samples were generated by crosslinking the samples at the end of the first hypergravity 1.8 g phase, 20 seconds after start of the parabola. µg/0g samples were generated by crosslinking the cells 20 seconds after the onset of microgravity during the first parabola. A ground control reference condition was generated by crosslinking cells that were stored inside the flight hardware but were not onboard during the flight.

overall_design: 3 flight conditions (1gIF inflight control 5 minutes before first parabola, hypg hypergravity 1.8g during the first parabola, 0g microgravity during the first parabola), 1 ground control condition. 3 Samples per condition.

first: Christian

last: Vahlensieck

first: Oliver

last: Ullrich


title: Genome-wide analysis of transcription in dek3 mutants and DEK3-over expressors


title: Regulation of c-Myc function by centromeric protein KLIP1

city: San Antonio

first: Yidong

last: Chen

state: Texas

address: 8403 Floyd Curl Drive, MSC 7784

department: Population Health Sciences

institute: UT Health Science Center at San Antonio

summary: In this study, we show that the centromeric Kaposi’s sarcoma-associated herpesvirus (KSHV) latent nuclear antigen (LANA)-interacting protein 1 (KLIP1) is a novel regulator of the c-Myc pathway. A series of microarray experiments were designed to demonstrate that the expression levels of KLIP1 protein are inversely correlated with cell proliferation, and the expression of c-Myc targets.

overall_design: 1 MCF7 parental cell, and 3 MCF7 cell clones with stable transfection of vector, and 4 MCF7 cell clones with stable transfection of KLIP1, all with 3 technical replicates were profiled with Phalanx microarrays to examine the differenetial expression due to the over-expression of KLIP1.

first: Shou-Jiang

last: Gao

first: Kurt

last: Kuhne


title: Maternal Schistosomaisis Early bone marrow B cell scRNA CITEseq

city: Salt Lake City

first: Keke

middle: Celeste

last: Fairfax

state: UT

address: 15 N Medical Drive East

department: Pathology

institute: University of Utah

summary: We performed scRNA sequencing to find transcriptional differences in early B cell populations in the bone marrow of mice born to mothers chronically infected with Schistosoma mansoni. Cite-seq was used to help identify cluster types using surface protein markers.

overall_design: Hematopoietic stem and progenitor cells and B cells were purified from bone marrow from the femur of mice born to S. mansoni infected and control mothers using a FACs Aria. Sorted cells were DAPI-CD3-CD4-CD8a-CD11b-CD11c-GR-1-Ter119-CD19-. CD19 was used in place of B220 to enrich for pre-pro B cells, but the antibody was ineffective, allowing for all B cell types to be sorted. After sorting, cells were stained with the cell surface proteins CD117(cKit) and Sca-1 with oligonucleotide-tagged antibodies to perform CITE-sequencing. Bone marrow from at least 3 mice were pooled before sorting. Cells were loaded into the 10X Genomics Chromium instrument according to the manufacturer's directions.

first: Gibbs

last: Lisa

first: Oviedo

last: Juan Marcos

first: Fairfax

last: Keke


title: Mutational analysis of TLK1 and MDM1

status: Public on Apr 30 2024

city: Bethesda

first: Uma

middle: T

last: Shankavaram

state: MD

address: 9000 Rockville Pike

department: NCI

institute: NIH

summary: The Tousled-like kinases 1 and 2 (TLK1/TLK2) regulate DNA replication, repair and chromatin maintenance. TLK2 variants are associated with ‘Intellectual Disability, Autosomal Dominant 57’ (MRD57), a neurodevelopmental disorder (NDD) characterized by intellectual disability (ID), autism spectrum disorder (ASD) and microcephaly. Several TLK1 variants have been reported in NDDs but their functional significance is unknown. A male patient presenting with ID, seizures, global developmental delay, hypothyroidism, and primary immunodeficiency was determined to have a heterozygous TLK1 variant (c.1435C>G, p.Q479E), as well as a mutation in MDM1 (c.1197dupT, p.K400*). Cells expressing TLK1 p.Q479E exhibited reduced cytokine responses and elevated DNA damage, but not increased radiation sensitivity or DNA repair defects. The TLK1 p.Q479E variant impaired kinase activity but not proximal protein interactions.

overall_design: duplicate PCR amplified regions of TLK1 or MDM1 from patient and sibling derived lymphoblastoid cell lines were compared.

first: Travis

last: Stracker


title: Genome-wide analysis of transcription in dek3 mutants and DEK3-over expressors [8 time points]

first: Anna

overall_design: RNA-seq were generated for seedlings grown at 27C harvested in 8 different time points. ZT0 time point just before the lights are on (End of the night); ZT8-just before the light are off(end of the day)

first: Phil


title: Mapping of mitogen and metabolic sensitivity in organoids defines requirements for human hepatocyte growth.

city: Utrecht

first: Delilah

last: Hendriks

address: Uppsalalaan 8

country: Netherlands

institute: Hubrecht Institute


title: Impact of different tissue dissociation protocols on endothelial cell recovery from developing lungs.

city: bad nauheim

first: Stefan

last: Günther

address: ludwigtr. 43

institute: MPI for heart and lung research

summary: Developing mouse lungs (day 14 of post-natal life) were dissociated by two different enzymatic methods, one employing commercially available Dispase, and another employing a commercial preparation of purified collagenase. The endothelial cell populations recovered from both approaches were characterized first by flow cytometry, to assess endothelial cell purity and viability. Subsequently, single-cell RNA-Seq was employed to characterize the constituent subpopulations of endothelial cells within each preparation. The single-cell RNA-Seq revealed that endothelial cell populations generated with Dispase exhibited reduced complexity, being enriched largely with one subpopulation of microvascular endothelial cells (gCap2) characterized by H2-Ab1 and Trf expression, whilst endothelial cell populations generated using collagenase yielded a complex endothelial cell population that included endothelial cell sub-types derived from the macro- and microvasculature, including the arterial and venous circulation, as well as the lymphatics.

overall_design: Endothelial cells were isolated from mouse lungs on the 14th day of post-natal life. Lungs were cleared by vascular perfused, and enzymatically dissociated using either Dispase of collagenase. Endothelial cells were isolated by from the resultant single-cell suspensions using positive CD31 and negative CD45 selection. Life/dead cell discrimination was undertaken using DAPI exclusion by live cells. Resultant cell preparations were subjected to single-cell RNA-Seq analysis to allow clustering of constituent endothelial cell subtypes by principal component analysis.

first: Francesco

last: Palumbo

first: Rory

middle: E

last: Morty


title: Single-cell RNAseq analysis of renal endothelial cells in murine diabetic nephropathy

city: Uppsala

first: L.

last: He

address: Dag Hammarskjölds Väg 20

country: Sweden

institute: Uppsala University

summary: We applied scRNAseq analysis to investigate the renal endothelial compartments in the murine diabetic nephropathy

overall_design: Mouse kidney single cells from control and DB samples were sorted into 384-well-plates and RNA-sequenced using the SmartSeq2 method

first: Alex-Xianghua

last: Zhou

first: Marie

last: Jeansson


title: DEK controls the trade-off between growth and arrest via H2A.Z-nucleosomes in Arabidopsis

last: Brestovitsky

type: Genome binding/occupancy profiling by high throughput sequencing

summary: The decision of whether to grow and proliferate or to restrict growth and develop resilience to stress is a key biological trade-off. Multiple tumour suppressors have roles in suppressing growth and proliferation when conditions are unfavourable, and aggressive tumours show re-wiring of the metabolic pathways and removal of these restraints on growth1,2. In plants, constitutive growth results in increased sensitivity to environmental stress3,4. However, the underlying mechanisms controlling this decision are not well understood. We used temperature as a cue to discover regulators of this process in plants, as it both enhances growth and development rates within a specific range, and is also a stress at extremes. We find the conserved chromatin protein DEK plays a central role in balancing the response between growth and arrest in Arabidopsis, and it does this via H2A.Z-nucleosomes. DEK target genes show two distinct categories of chromatin architecture, and these predict induction or repression by DEK. We show that these chromatin signatures of DEK target genes might be conserved in human cells, suggesting that DEK may act through a fundamental evolutionarily conserved mechanism to control the balance between growth and arrest in plants and animals.

overall_design: HTA11, DEK3, H3.3 and H3 ChIP-seq as well as MNase-seq were generated in seedlings grown in different temperature

middle: A

last: Wigge


title: Cytotoxic Sigma-2 Ligands Trigger Cancer Cell Death via Cholesterol-Induced-ER-Stress

city: St Louis

first: Rony

last: Takchi

state: MO

address: 660 South Euclid Avenue

department: Surgery

institute: Washington University St Louis

summary: Sigma-2-ligands (S2L) are characterized by high-affinity interaction with their cognate sigma-2 receptor, overexpressed in rapidly proliferating tumor cells. As such, S2L were developed as imaging probes (ISO1) or as cancer therapeutics, alone (SV119 [C6], SW43 [C10]) and as delivery vehicles for cytotoxic drug cargoes following chemical conjugation (C6-Erastin, C10-SMAC). However, the exact mechanism of S2L-induced cytotoxicity remains to be fully elucidated. A series of high-affinity S2L were evaluated regarding their cytotoxicity profiles across pancreatic, ovarian, and synovial cancer cell lines. While C6 and C10 displayed distinct cytotoxicities (extended aliphatic side chains), C0 (no aliphatic side chain) and ISO1 (structurally unrelated) were essentially non-toxic, enabling us to investigate the unique mechanism of S2L cytotoxicity. Confocal microscopy and lipidomics analysis in cellular and mouse models revealed that C10 induced an increase in intralysosomal free cholesterol, a corresponding increase in cholesterol esters, suggestive of unaltered intracellular cholesterol trafficking and processing, and cytotoxicity due to cholesterol excess, a phenomenon that contrasts the effects of inhibiting NPC1 function. RNA-sequencing revealed that gene clusters involved in cholesterol homeostasis and ER stress response were activated exclusively by cytotoxic S2L. ER stress markers were confirmed on qPCR, and ER stress modulators inhibited or enhanced cytotoxicity of C10 confirming the involvement of ER stress response in the cytotoxicity of S2L. Moreover, C10 increased sterol regulatory element-binding protein 2 (SREBP2) and low-density lipoprotein receptor (LDLR) which were also confirmed with qPCR and at the protein level and were found to be a pro-survival mechanism activated by ER stress response. Moreover, inhibiting the processes downstream this adaptive response with simvastatin resulted in synergistic treatment outcomes when combined with C10 in vitro and in vivo. Of note, the conjugates retained the S2L-mediated cholesterol phenotype of their parental ligands, which suggests that disrupting cholesterol homeostasis might contribute to the overall cytotoxicity of the drug conjugates. Furthermore, these phenomena were seen exclusively with cytotoxic S2L. Based on these findings, we conclude that S2L-mediated cell death is due to free cholesterol accumulation that leads to ER stress. Consequently, the cytotoxic profiles of S2L drug conjugates are proposed to be enhanced via concurrent ER stress inducers or simvastatin, strategies that could be instrumental on the path toward tumor eradication.

overall_design: To investigate the mechanism of sigma-2-ligand mediated cytotoxicity in an unbiased fashion, RNA sequencing technology (RNAseq) was employed to establish any differences in the transcriptional landscape between cancer cells treated with either the non-cytotoxic C0 (SV95), or the cytotoxic C6 (SV119) and the more potent C10 (SW43). Following a short-term (two-hour long) drug exposure (10µM) with, SYO1 cells were harvested and processed for transcriptome and functional gene enrichment analysis.

first: Bethany

middle: C

last: Prudner

first: Qingqing

last: Gong

first: Takaomi

last: Hagi

first: Kenneth

middle: F

last: Newcomer

first: Linda

middle: X

last: Jin

first: Suwanna

last: Vangveravong

first: Brian

last: Van Tine

first: William

last: Hawkins

first: Dirk

last: Spitzer


title: Mapping of mitogen and metabolic sensitivity in organoids defines requirements for human hepatocyte growth [bulk RNA-seq]

first: Delilah

last: Hendriks

summary: Mechanisms underlying human hepatocyte growth in development and regeneration are incompletely understood. In vitro, human fetal hepatocytes (FH) can be robustly grown as organoids, while adult primary human hepatocyte (PHH) organoids remain difficult to expand, suggesting different growth requirements between fetal and adult hepatocytes. Here, we characterize hepatocyte organoid outgrowth using temporal transcriptomic and phenotypic approaches. FHs initiate reciprocal transcriptional programs involving increased proliferation and repressed lipid metabolism upon initiation of organoid growth. We exploited these insights to design maturation conditions for FH organoids, resulting in acquisition of mature hepatocyte morphological traits and increased expression of functional markers. During PHH organoid outgrowth in the same culture condition, the adult transcriptomes initially mimic the fetal transcriptomic signatures, but PHHs rapidly acquire disbalanced proliferation-lipid metabolism dynamics, resulting in steatosis and halted organoid growth. IL6 supplementation, as emerged from the fetal dataset, and simultaneous activation of the metabolic regulator FXR, prevents steatosis and promotes PHH proliferation, resulting in improved expansion of the derived organoids. Single-cell RNA sequencing analyses reveal preservation of their fetal and adult hepatocyte identities in the respective organoid cultures. Our findings uncover mitogen requirements and metabolic differences determining proliferation of hepatocytes changing from development to adulthood.

overall_design:
Bulk RNA-sequencing of human hepatocyte growth from fetal liver tissue, single fetal hepatocytes, and primary human hepatocytes
***Please note that the raw data are not provided due to donor privacy concerns.

first: Benedetta

last: Artegiani


title: Mapping of mitogen and metabolic sensitivity in organoids defines requirements for human hepatocyte growth [scRNA-seq]

overall_design:
Single-cell RNA-sequencing of expanding human fetal hepatocyte organoids and primary human hepatocyte organoids
***Please note that the raw data are not provided due to donor privacy concerns.


title: Differential Expression Data from Mice 24hrs after stroke

city: Québec

first: Hejer

last: Boutej

state: Québec

address: 2601, de la Canardière

department: NEUROBIOLOGY

country: Canada

institute: LAVAL University

summary:
Microglial activation after stroke may lead to development of inflammation-induced brain damage. Here we uncover a ribosome-based mechanism/check point involved in control of the innate immune response and microglial activation orchestrated by RNA binding protein SRSF3. Using an in vivo model-system for analysis of the dynamic translational state of microglial ribosomes with mRNAs as input and newly synthesized peptides as an output, we found a marked dissociation of microglia mRNA and protein signatures following ischemic stroke.
Highly up-regulated and ribosomes associated mRNAs were not translated 24hrs after stroke resulting in two distinct microglial molecular signatures, a highly specialized pro-inflammatory mRNA and immunomodulatory/homeostatic protein signatures. We found that this is due to specific translational suppression of highly expressed mRNAs through a 3'UTR-mediated mechanism involving the RNA binding protein SRSF3. This discovery suggests avenues for therapeutic modulation of innate immune response in resident microglia after stroke.

overall_design: Transgenic mice expressing Flag/EGFP-RPL10a under the control of CD11b promoter were used to perform transcriptomic analysis in stroked and CTL mice. RNA was extracted according to the TRAP protocol and hybridized on Affymetrix microarrays (MoGene-2_0-st). The mice were used at 2-3 months old with equal ratio of male/female. Array data was processed by Transcriptome Analysis Console 3.0 (TAC). No technical replicates were performed.

first: Reza

last: Rahimian

first: Revathy

last: Guruswamy

first: Pierre

last: Cordeau Jr

first: Yuan

last: Cheng Weng

first: Jasna

last: Kriz


title: CCR5 promotes the migration of CD8+ T cells to the leishmanial lesions (S2W)

status: Public on Apr 29 2024

city: Philadelphia

first: Camila

last: Farias Amorim

state: United States

address: 380 S University Ave

department: Pathobiology

institute: University of Pennsylvania

summary: Cytolytic CD8+ T cells mediate immunopathology in cutaneous leishmaniasis without controlling parasites. Here, we identify factors involved in CD8+ T cell migration to the lesion that could be targeted to ameliorate disease severity.CCR5 was the most highly expressed chemokine receptor in patient lesions, and the high expression of CCL3 and CCL4, CCR5 ligands, was associated with delayed healing of lesions. To test the requirement for CCR5,Leishmania-infected Rag1-/- mice were reconstituted with CCR5-/- CD8+ T cells. We found that these mice developed smaller lesions accompanied by a reduction in CD8+ T cell numbers compared to controls. We confirmed these findings by showing that the inhibition of CCR5 with maraviroc, a selective inhibitor of CCR5, reduced lesion development without affecting the parasite burden. Together, these results reveal that CD8+ T cells migrate to leishmanial lesions in a CCR5-dependent manner and that blocking CCR5 prevents CD8+ T cell-mediated pathology.

overall_design: C57BL/6 mice were topically colonized with 10^8 S. epidermidis every day for a total of five applications and then intradermally infected with 2 x 10^6 L. major parasites. Staphylococcus epidermidis strain Tu3298 expressing a fluorescent protein mCherry (S2W) was a gift of Dr. Tiffany Scharschmidt (UCSF). Mice were colonized once per week for the duration of the experiment. At 5 weeks post-infection, lesions were obtained for bulk RNA-seq.

first: Phillip

last: Scott


title: Persistent gene expression and DNA methylation alterations linked to carcinogenic effects of dichloroacetic acid [RNA-seq]

city: Research Triangle Park

last: Chorley

address: US EPA, 109 TW Alexander Dr

institute: US EPA

summary: Mechanistic understanding of transient exposures that lead to adverse health outcomes will enhance our ability to recognize biological signatures of disease. Here, we measured the transcriptomic and epigenomic alterations due to exposure to the metabolic reprogramming agent, dichloroacetic acid (DCA). Previously, we showed that exposure to DCA increased liver cancer in B6C3F1 mice after continuous or early life exposures similarly over background level. Measures from these studies did not support direct cytotoxic, mitogenic, or genotoxic modes-of-action of tumorigenesis. Using archived formalin-fixed liver samples, we utilized modern methodologies to measure gene expression and DNA methylation levels to link to previously generated phenotypic measures. Gene expression was measured by targeted RNA sequencing (TempO-seq 1500+ toxicity panel: 2754 total genes) in liver samples collected from 10-, 32-, 57-, and 78-week old mice exposed to deionized water (controls), DCA continuously at 3.5g/L in drinking water (“Direct” group), or DCA at 3.5g/L for 10-, 32-, or 57-weeks followed by control water (“Stop” groups). Genome-scaled alterations in DNA methylation were measured by Reduced Representation Bisulfite Sequencing (RRBS) at 78-weeks. Transcriptomic changes were most robust with concurrent or adjacent timepoints after exposure stoppage. DNA methylation alterations followed a similar pattern, measuring 2720 and 567 differentially methylated regions (DMRs) in 78-week Direct and 10-week “Stop” DCA exposure groups, respectively. Gene pathway analysis indicated cellular effects linked to increased oxidative metabolism, a primary mechanism of action for DCA, closer to exposure windows especially early in life. Conversely, many gene signatures and pathways reversed patterns later in life and reflected more pro-tumorigenic patterns for both current and prior DCA exposures. DNA methylation patterns linked to early gene pathway perturbations, suggesting persistence in the epigenome and possible regulatory effects. In total, results suggested that liver metabolic reprogramming effects of DCA interact with normal age mechanisms to increase tumor burden with both continuous and prior DCA exposure in the B6C3F1 rodent model.

overall_design: Male B6C3F1 mice were treated with 3.5 g/L DCA in deionized water in a stop exposure study design over a period of 78 weeks. Mice were either provided only deionized water (control), continuous DCA (direct) or in a series of stop-dosage timepoints after which the mice were switched to deionized water for the duration of the experiment. The stop group treatments were as follows: 10 weeks of DCA followed by 68 weeks of water (S1), 32 weeks of DCA and 46 weeks of water (S2) and 57 weeks of DCA with 21 weeks of water (S3). The direct group were exposed to 3.5 g/L for the entirety of the 78-week study. At each timepoint (10, 32, 57 or 78 weeks), a subset of the control, direct and stop treatment block mice were humanely euthanized with CO2 following EPA approved Animal Care and Use Committee protocols.  Portions of the livers were fresh frozen at each sacrifice timepoint along with the preparation of FFPE paraffin blocks. Figure 1 outlines study collection points and treatments. FFPE-derived samples were used to measure gene expression and frozen samples were used for DNA methylation determination.

middle: N

first: Gleta

last: Carswell

middle: D

last: Bennett


title: Loss of tumor-derived SMAD4 enhances primary tumor growth but not metastasis following BMP4 signalling

city: Heidelberg

first: Lap Hing

last: Chi

state: Victoria

address: 145 Studley Road

department: Translational Breast Cancer Program

country: Australia

institute: Olivia Newton-John Cancer Research Institute

summary: We have reported previously that in preclinical models, BMP4 is a potent inhibitor of breast cancer metastasis and that high BMP4 protein levels predict favourable patient outcome. Here, we investigated the requirement for functional SMAD4 in mediating the anti-metastatic response of BMP4, given reports of promotion of metastasis by BMP4 in cancers where SMAD4 is frequently deleted. BMP4-induced inhibition of metastasis does not require functional SMAD4 in tumor cells. However, tumor cell intrinsic signalling using a constitutively active BMP receptor does require functional SMAD4 to suppress metastasis, thus implicating BMP4 mediated paracrine signalling as a contributor to the inhibition of metastasis.

overall_design: Comparison of the transcriptomic profiles of cancer cells retrieved from primary tumours with modified expression of BMP4 and/or SMAD4.

first: Robin

middle: L

last: Anderson

first: Asha

last: Haslem


title: CCR5 promotes the migration of CD8+ T cells to the leishmanial lesions

summary: Cytolytic CD8+ T cells mediate immunopathology in cutaneous leishmaniasis without controlling parasites. Here, we identify factors involved in CD8+ T cell migration to the lesion that could be targeted to ameliorate disease severity. CCR5 was the most highly expressed chemokine receptor in patient lesions, and the high expression of CCL3 and CCL4, CCR5 ligands, was associated with delayed healing of lesions. To test the requirement for CCR5, Leishmania-infected Rag1-/- mice were reconstituted with CCR5-/- CD8+ T cells. We found that these mice developed smaller lesions accompanied by a reduction in CD8+ T cell numbers compared to controls. We confirmed these findings by showing that the inhibition of CCR5 with maraviroc, a selective inhibitor of CCR5, reduced lesion development without affecting the parasite burden. Together, these results reveal that CD8+ T cells migrate to leishmanial lesions in a CCR5-dependent manner and that blocking CCR5 prevents CD8+ T cell-mediated pathology.

overall_design: Rag1-/- mice were infected withL. braziliensis(strain MHOM/BR/01/BA788) and subsequently reconstituted with CD8+ T cells alone or CD8+ and CD4+ T cells or did not receive any T cells. 5 weeks post-infection, mice were euthanized, and ears were collected in RNAlater


title: Persistent gene expression and DNA methylation alterations linked to carcinogenic effects of dichloroacetic acid

first: Brian

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

overall_design: Refer to individual Series


title: Effect of depletion of the serine protease inhibitors (serpina3n) on gene expression in cardiac fibroblasts after myocardial injury (MI) in rodent models

first: Feiyang

last: Ma

address: 610, Charles Young Dr East, TLSB 3000C

department: Molecular Biology Institute

institute: UCLA

summary: To investigate the function of serpina3n in cardiac fibroblast. Serpina3n is significantly upregulated after myocardial injury in rodent models. The depletion of serpina3n causes heart function weakening in rodent models.

overall_design: Single cells and single nuclei were isolated from serpina3n CKO mice injured heart tissue after MI and analyzed using scRNAseq.

first: Wei

last: Chen

first: Arjun

last: Deb


title: Single cell tracing of Pomc neurons reveals recruitment of ‘Ghost’ subtypes with atypical identity in a mouse model of obesity

city: BORDEAUX

first: Carmelo

last: QUARTA

address: 146 rue Léo Saignat

country: France

institute: Inserm U1215

summary: The hypothalamus contains a remarkable diversity of neurons that orchestrate behavioural and metabolic outputs in a highly plastic manner. Neuronal diversity is key to enabling hypothalamic functions and, according to the neuroscience dogma, it is predetermined during embryonic life. Here, by combining lineage tracing of hypothalamic pro-opiomelanocortin (Pomc) neurons with single-cell profiling approaches in adult male mice, we uncovered subpopulations of 'Ghost' neurons endowed with atypical molecular and functional identity. Compared to 'classical' Pomc neurons, Ghost neurons exhibit negligible Pomc expression and are ‘invisible’ to available neuroanatomical approaches and promoter-based reporter mice for studying Pomc biology. Ghost neuron numbers increase in diet-induced obese mice, independent of neurogenesis or cell death, but weight loss can reverse this shift. Our work challenges the notion of fixed, developmentally programmed neuronal identities in the mature hypothalamus, highlighting the ability of specialized neurons to adapt their funcitonal identity to adult-onset obesogenic stimuli

overall_design: Patch-seq, scRNA seq data, samples obtained from Pomc lineage reporter mice under chow diet conditions or diet-induced obesity

last: Quarta

first: Stephane

last: Leon

first: Alexandre

last: Brochard

first: Emeline

last: Lhuillier


title: Reversal of high-glucose-induced transcriptional and epigenetic memories through NRF2 pathway activation

city: Mexico City

first: Marti

last: Wilson-Verdugo

state: Mexico City

address: Ciudad Universitaria

country: Mexico

institute: Instituto de Fisiologia Celular


title: Reversal of high-glucose-induced transcriptional and epigenetic memories through NRF2 pathway activation (RNA-seq).

summary: Aims: Evaluate the genome-wide gene expression and epigenomic alterations caused by transient high glucose (HG) in endothelial cells (ECs), and to assess NRF2 pathway activation as a potential treatment for the diabetes-associated transcriptional memory. Methods and results: We exposed human umbilical vein endothelial cells (HUVEC) to three different glucose treatments: control (5.5 mM for 8 days), HG (30 mM for 8 days) and memory (5.5 mM for 4 days followed by 30 mM for 4 days). After these treatments we conducted a series of experiments, first we found that cells in the HG treatment had sustained alterations in both glycolytic and respiratory rates that persisted in the memory treatment, this without changes in proliferation or cell viability. Similarly, HG provoked substantial changes in the expression of genes belonging to pathways known to be impaired in diabetes, like the TGF-beta, TNF, foxO, p53 and NRF2 pathways, many of which were still present in memory. In addition, we found persistent changes in chromatin accessibility located mainly in non-promoter regions, some of which we identified as putative enhancer regions neighboring genes altered in memory. Finally, we demonstrated that activation of the NRF2 pathway through either NRF2 overexpression or sulforaphane treatment was able to substantially revert the glucose-induced transcriptional memory in ECs. Conclusions: Our results demonstrate that transient HG induces persistent changes in both the transcriptomic and chromatin accessibility profiles of ECs, and that pharmacological NRF2 pathway activation through sulforaphane supplementation is able to prevent and revert the glucose-induced transcriptional memory.

overall_design:
RNA-seq/ATAC-seq of human umbilical vein endothelial cells in three different treatments: 8 days in 5.5 mM glucose (control), 8 days in 30 mM glucose (high glucose) and 4 days in 30 mM glucose followed by 4 days in 5.5 mM glucose (memory).
Additionally, to evaluate the role of NRF2 pathway activation in the high-glucose-induced transcriptional memory, we performed RNA-seq of HUVECs exposed to our high glucose or memory treatments that were either overexpressing NRF2 or incubated with 1 μM sulforaphane.
Finally, to evaluate the role of NRF2 pathway activation in the high-glucose-induced epigenetic memory, we performed ATAC-seq of HUVECs exposed to our memory treatment plus supplementation with 1 μM sulforaphane.

first: Brandon

last: Bustos-Garcia

first: Nallely

last: Cano-Dominguez

first: Maribel

last: Soto-Nava

first: Carlos

last: Acosta

first: Santiago

last: Avila-Rios

first: Lilia

last: Noriega

first: Jaqueline

last: Hersch-Gonzalez

first: Victor

last: Valdes


title: Reversal of high-glucose-induced transcriptional and epigenetic memories through NRF2 pathway activation (ATAC-seq).


title: Transcriptome analysis by total RNA sequencing of genetically modified Rhodobacter capsulatus strains for inducible expression of cas13a

city: Gießen

first: Gabriele

last: Klug

address: Heinrich-Buff-Ring 26-32

department: Molecular- and Microbiology

institute: Justus-Liebig University Gießen

summary: In this study we analyzed the effects of an altered Cas13a expression in vivo, in presence or absence of ectopically expressed protospacer RNA (plasmid pCV2_SB6), by RNA-sequencing. The genomic modification of the cas13a controlling promoter enables altered expression of cas13a by adding either subinhibitory crystal violet (CV) concentrations (for transcriptional repression of cas13a), or additionally IPTG (for transcriptional induction of cas13a), to the mutant cultures. Transcriptome profiles were compared to corresponding wild type controls.

overall_design: Comparative gene expression profiling analysis of RNA-seq data of independent biological triplicates of Rhodobacter capsulatus strains, grown either under cas13a inducing or cas13a repressing conditions.

first: Janek

last: Börner

first: Jonas

last: Kretz

first: Tobias

last: Friedrich


title: ALK inhibitors suppress HCC and synergize with anti-PD-1 therapy and ABT-263 in preclinical models

city: Birmingham

first: Narendra

last: Wajapeyee

state: AL

address: 720 20th Street South, Kaul 540A

department: Department of Biochemistry and Molecular Genetics

institute: University of Alabama at Birmingham

summary: Hepatocellular carcinoma (HCC) currently lacks effective therapies, leaving a critical need for new treatment options. A previous study identified the Anaplastic Lymphoma Kinase (ALK) amplification in HCC patients, raising the question of whether ALK inhibitors could be a viable treatment. Here, we showed that both ALK inhibitors and ALK knockout effectively halted HCC growth in cell cultures. Lorlatinib, a potent ALK inhibitor, suppressed HCC tumor growth and metastasis across various mouse models. Additionally, in an advanced immunocompetent humanized mouse model, when combined with an anti-PD-1 antibody, lorlatinib more potently suppressed HCC tumor growth, surpassing individual drug efficacy. Lorlatinib induced apoptosis and senescence in HCC cells, and the senolytic agent ABT-263 enhanced the efficacy of lorlatinib. Additional studies identified that the apoptosis-inducing effect of lorlatinib was mediated via GGN and NRG4. These findings establish ALK inhibitors as promising HCC treatments, either alone or in combination with immunotherapies or senolytic agents.

overall_design: Total RNA was extracted from all non target expressing and ALK targeted inhibition in HepG2, SK-HEP1 and SNU449 cells, followed by poly-A selection and RNA-sequencing using Illumina Hi-Seq 2500 platform, with three biological replicates for each transformed sample.

first: Suresh

last: Bugide

first: Dhana Sekhar

last: Reddy

first: Parmanand

last: Malvi

first: Romi

last: Gupta


title: single-cell chromatin accessibility profiling of mouse gonads during sex determination [scATAC-seq]

status: Public on Apr 28 2024

city: Hong Kong

first: Jinyue

last: Liao

address: Room 38016, 1/F Lui Che Woo Clinical Sciences Building

department: Department of Chemical Pathology

country: Hong Kong

institute: The Chinese University of Hong Kong

summary: Gonad development includes sex determination and divergent maturation of the testes and ovaries. Recent advances in measuring gene expression in single cells are providing new insights into this complex process. However, the underlying epigenetic regulatory mechanisms remain unclear. Here, we profiled chromatin accessibility in mouse gonadal cells of both sexes from embryonic day 11.5 to 14.5 using single-cell assay for transposase accessible chromatin by sequencing (scATAC-seq). Our results showed that individual cell types can be inferred by the chromatin landscape, and that cells can be temporally ordered along developmental trajectories. Integrative analysis of transcriptomic and chromatin-accessibility maps identified multiple putative regulatory elements proximal to key gonadal genes Nr5a1, Sox9 and Wt1. We also uncover cell type-specific regulatory factors underlying cell type specification. Overall, our results provide a better understanding of the epigenetic landscape associated with the progressive restriction of cell fates in the gonad.

overall_design: Chromatin accessibility profiles of mouse gonads at E11.5, E12.5, E13.5 and E14.5.

first: Hoi Ching

last: Suen


title: RNA sequencing of gastric organoids treated with bacteria

city: Tokyo

first: Yoku

last: Hayakawa

state: TOKYO

address: 7-3-1, Hongo, Bunkyo-ku, Clinical Research Center 4F, Room 425

country: Japan

institute: University of Tokyo

summary: We treated mouse gastric organoids with Helicobacter pylori, Fusobacterium nucleatum, and Neisseria subflava, and extracted RNA after 4 hours. RNAs were applied to RNA sequencing analysis to investigate bacteria-specific effects on gastric epithelial cells.

overall_design: Examination of transcriptomic analysis on bacteria-treated gastric organoids.

first: Ryota

last: Niikura

first: Naoyoshi

last: Nagata

first: Mitsuhiro

last: Fujishiro


title: MECP2 directly interacts with RNA polymerase II to modulate transcription in human neurons [ATAC-seq]

status: Public on Apr 26 2024

city: Cambridge

first: Yi

last: Liu

state: MA

address: 455 Main St

institute: Whitehead Institute for Biomedical Research

summary: Mutations in the methyl-DNA-binding protein MECP2 cause the neurodevelopmental disorder Rett syndrome (RTT). How MECP2 contributes to transcriptional regulation in normal and disease states is unresolved; it has been reported to be an activator and a repressor. We describe here the first integrated CUT&Tag, transcriptome, and proteome analyses using human neurons with wild-type and mutant MECP2 molecules. MECP2 occupies CpG-rich promoter-proximal regions in over four thousand genes in human neurons, including a plethora of autism risk genes, together with RNA polymerase II (Pol II). MECP2 directly interacts with Pol II, and genes occupied by both proteins showed reduced expression in neurons with MECP2 patient mutations. We conclude that MECP2 acts as a positive cofactor for Pol II gene expression at many neuronal genes that harbor CpG-islands in promoter-proximal regions, and that RTT is due, in part, to the loss of gene activity at these genes in neurons.

overall_design: We used ATAC-Seq (Assay for Transposase-Accessible Chromatin using Sequencing), based on direct in vitro transposition of sequencing adaptors into native chromatin, as a rapid epigenomic analysis to identify open chromatin regions in human neurons.

first: Anthony

last: Flamier

first: George

middle: W

last: Bell

first: Troy

last: Whitfield

first: Richard

last: Young

first: Rudolf

last: Jaenisch


title: DNA-binding protein-A promotes renal ischemia/reperfusion injury and participates in mitochondrial function

city: Braunschweig

first: Robert

last: Geffers

address: Inhoffenstr. 7

department: Dep. Molecular Bacteriology

institute: HCI - Helmholtz Centre for Infection Research

summary: DNA-binding protein-A (DbpA; gene: Ybx3) belongs to the cold shock protein family with known functions in cell cycling, transcription, translation and tight junction communication. In chronic nephritis DbpA is upregulated, however its activities in acute injury models, such as renal ischemia/reperfusion injury (IRI), are unclear. Mice harboring Ybx3+/+, Ybx3+/- or Ybx3-/- genotype were characterized over a period of 24 months and following experimental renal IRI. Mitochondrial function, number and integrity were analyzed by mito stress tests, MitoTracker staining and electron microscopy. Immunohistochemistry, Western blotting and flow cytometry were performed to quantify tubular cell damage and immune cell infiltration. DbpA is dispensable for kidney development and tissue homeostasis under healthy conditions. Furthermore, endogenous DbpA protein localizes within mitochondria in primary tubular epithelial cells (TECs). Genetic deletion of Ybx3 elevates the mitochondrial membrane potential, the lipid uptake and metabolism, the oxygen consumption rates (OCR) and glycolytic activities of TECs. Ybx3-/- mice demonstrate protection from IRI with less immune cell infiltration, ER stress and tubular cell damage. A putative protective mechanism is identified via upregulated antioxidant activities and reduced ferroptosis, when Ybx3 is deleted. Here, experimental evidences reveal DbpA acts as a mitochondrial protein with profound adverse effects on cell metabolism and highlights a protective effect against IRI when Ybx3 is genetically deleted.

overall_design: Adult wildtype mice and mice with a genetic deletion of Ybx3 were investigated under healthy conditions and following experimental renal IRI.

first: Peter

middle: R

last: Mertens

first: Charlotte

last: Reichardt

first: Sabine

last: Brandt


title: MECP2 directly interacts with RNA polymerase II to modulate transcription in human neurons. [CUT&Tag]

overall_design: To investigate MECP2 DNA-binding sites, we performed CUT&Tag assays on MECP2 wild-type (WT) and mutant neurons.


title: Effects of exogenous melatonin on NaCl stress in Eriobotrya japonica seedlings

status: Public on Apr 27 2024

city: Kunming

first: Ling

last: Zhu

address: Southwest Forestry University, No. 300, Bailong Road, Qingyun Street, Kunming, Yunnan Province, China

institute: Southwest Forestry University

overall_design: Selected loquat group cultivated seedlings with consistent growth were divided into three groups, which were expressed as CK, ST and MTST (optimal treatment concentration), respectively. plants in CK group were used as blank control and watered; plants in ST group were watered first and then NaCl stress; plants in MTST group were root-irrigated first with screened optimal concentration of melatonin solution and then NaCl stress; 10 pots were planted in each treatment group with three seedlings in each pot, and the procedure was repeated for three times.


title: MECP2 directly interacts with RNA polymerase II to modulate transcription in human neurons [RNA-Seq]

overall_design: We performed RNA-Seq of hESC-derived MECP2 WT and mutant neurons using three biologically independent samples for each assay. We added ERCC RNA spike-in controls to normalize to cell number input.


title: MECP2 directly interacts with RNA polymerase II to modulate transcription in human neurons

type: Methylation profiling by high throughput sequencing


title: Mecp2 fine-tunes quiescence exit by targeting nuclear receptors

city: Guangzhou

first: Zeyou

last: Qiu

address: Baiyun Jingxi road

institute: Southern Medical University


title: Mecp2 fine-tunes quiescence exit by targeting nuclear receptors [ChIP-Seq]

summary: To identify the Mecp2-dependent transcriptome genome-wide during the very early stage of liver regeneration, we mapped the binding landscape of Mecp2 in control and MeCP2-KO livers before and after PHx using ChIP-seq. we mapped the binding landscape of Mecp2 in control livers before and after PHx by filtering out peaks identified in Mecp2-cKO livers. we identified a total of 14640 and 15350 Mecp2-binding genes before and after PHx in the Mecp2 control liver, respectively.

overall_design: ChIP-seq profiling of MeCP2 during liver regeneration before and 6 hours after PHx in Control and MeCP2-KO mice

first: Jun

last: Yang


title: MECP2 directly interacts with RNA polymerase II to modulate transcription in human neurons [WGBS]

overall_design: To examine the relationship between MECP2 genome occupancy and DNA methylation, we conducted whole-genome bisulfite sequencing (WGBS) on MECP2 wild-type (WT) neurons using three biologically independent samples for each assay.


title: Mecp2 fine-tunes quiescence exit by targeting nuclear receptors [RNA-Seq]

summary: To identify the MeCP2 targets gene during the very early stage of liver regeneration，ChIP-seq analyses in control and Mecp2-cKO mice livers before and 6 h after PHx were performed. We found 3048 Mecp2-dependent genes that were differentially expressed in a Mecp2-dependent manner.

overall_design: Ten to 12-week-old male control and MeCP2-KO mouse liver samples were obtained at 0 and 6 hours after 2/3 PH. mRNAs differentially expressed were identified by comparing expression levels during liver regeneration in mice after 2/3 partial hepatectomy (PH) at various time points.


title: TDP1 mutation causing SCAN1 neurodegenerative syndrome hampers the repair of transcriptional DNA double-strand breaks

status: Public on Apr 25 2024

city: Toulouse

first: Estelle

last: Nicolas

address: 118 route de Narbonne

institute: INSERM

summary: TDP1 removes transcription-blocking TOP1 cleavage complexes (TOP1ccs) and its inactivating H493R mutation causes the neurodegenerative syndrome SCAN1. However, the molecular mechanism underlying SCAN1 phenotype is unclear. Here, we generate human SCAN1 cell models using CRISPR/Cas9 and show that they accumulate TOP1ccs along with changes in gene expression and genomic distribution of R-loops. SCAN1 cells also accumulate transcriptional DNA double-strand breaks (DSBs) specifically in the G1 cell population due to increased DSB formation and lack of repair, both resulting from abortive removal of transcription-blocking TOP1ccs. Deficient TDP1 activity causes increased DSB production and the presence of mutated TDP1 protein hampers DSB repair by a TDP2-dependent back-up pathway. This study provides powerful models to study TDP1 functions under physiological and pathological conditions and unravels that a gain of function of the mutated TDP1 protein, which prevents DSB repair, rather than a loss of TDP1 activity itself, could contribute to SCAN1 pathogenesis.

overall_design: DRIP-seq experiments (2 replicates) in TDP1 H493R (SCAN1) and WT U2OS cells

first: Agnese

last: Cristini

first: Virginie

last: Jouffret

first: Olivier

last: Sordet


title: Transcriptome sequencing from rhesus macaque cell lines

city: Göttingen

first: Ting

last: Sun

state: Germany (DEU)

address: Hermann-Rein-Straße 3

department: Neurogenetics

institute: Max Planck Institute for Multidisciplinary Sciencesi

summary: Non-human primate (NHP)-based model systems are highly relevant for biomedical research. However, only few NHP cell lines are available and the generation of additional cell lines is an urgent need to help in the refinement and replacement of these models. Using lentiviral transduction of c-Fos, we established cell lines from the brain of rhesus macaques (Macaca mulatta). Transcriptome analysis revealed that these cell lines are closely related to astrocytes, which was confirmed by Western blot detecting expression of the astrocyte marker glial fibrillary acidic protein (GFAP). Quantitative real-time PCR (qRT-PCR) demonstrated that major pathways of the interferon (IFN) system are intact. Using retroviral pseudotypes we found that the cell lines are susceptible to entry driven by the glycoproteins of vesicular stomatitis virus (VSV), lymphocytic choriomeningitis virus (LCMV) and to a lesser extent influenza A virus (IAV). Finally, these cells supported growth of Zika virus (ZIKV) and Papiine alphaherpesvirus 2 (PaHV2).  In summary, we developed IFN-responsive cell lines from the rhesus macaque brain that allowed entry driven by several viral glycoproteins and were permissive to infection with ZIKV and a primate simplexvirus. These cell lines will be useful for efforts to analyze neurotropic viral infections in rhesus macaque models.

overall_design: Cells were dissociated from Macaca mulatta cortex for cell line immortalization and culturing. The immortalized MamuCo2888-gr and Co2880-IT cell line were subjected to total RNA isolation and bulk RNA-sequencing. The later deconvolution analysis of the bulk RNA-seq results revealed a major astrocyte composition of the cell line.

first: Stefanie

last: Reiter

first: Michael

last: Winkler


title: Single cell RNA-seq profiling of mouse brain cells at postnatal day 10

status: Public on Apr 23 2024

city: Chongqing

first: Qijing

last: Lei

address: 30 Gaotanyan Main St, Shapingba District,400038

institute: Third Military Medical University

summary: 10x single-cell RNA-sequencing to profile mouse brain cells at postnatal day 10

overall_design: Single-cell profiling of mouse brain cells were freshly isolated from P10 mice (n= 6 mice)

first: Feng

last: Mei


title: RNA-seq analysis of gene expression regulated by LSD1 in CD8+ T cells

status: Public on Apr 24 2024

city: Hangzhou

first: Wanqiang

last: Sheng

state: Zhejiang

address: 866 Yuhangtang Road

department: Department of Immunology

institute: Zhejiang University School of Medicine

summary: In the RNA-seq analyses of in vitro activated and expanded CD8+ T cells, we found that GSK treatment significantly altered the transcriptome, with 241 genes upregulated and 426 genes downregulated respectively . Gene Ontology (GO) enrichment analysis revealed that the upregulated genes were highly enriched in biological processes related to cell killing and cytokine production.

overall_design: Mouse CD8+ T cells were isolated from the spleens and lymph nodes of the mice using a biotin selection kitwith an EasySep™ Mouse CD8+ T Cell Isolation Kit according to the manufacturer’s manual. The CD8+ T cells were stimulated with 2 μg/ml plate-bound anti-CD3 (2μg/ml) and 2 μg/ml soluble anti-CD28 (2μg/ml) antibodies with the addition of 20 ng/mL hIL-2 at a density of 1 million/ml for 48 h. Activated CD8+ T cells were expanded with hIL2 in fresh medium for the indicated time before analysis. 0.5 μM GSK2879552 or vehicle control was added. Total RNA was extracted and used to poly(A)+ RNA enrichment by magnetic mRNA isolation kit for constructing library. The prepared libraries were sequenced at 150 bp from both ends.

first: Fengqi


title: Circadian Control of Tumor Immunosuppression Impacts Efficacy of Immune Checkpoint Blockade

status: Public on Apr 22 2024

city: Irvine

first: Shannon

last: Pfeiffer

address: 847 Health Sciences Rd, Room 3131

department: Biological Chemistry

institute: University of California, Irvine

summary: The circadian clock is a critical regulator of immunity, and this circadian control of immune modulation plays an essential role in host defense as well as tumor immunosurveillance. Using a single cell RNA-sequencing approach in a genetic model of colorectal cancer (CRC), we identified clock-dependent changes to the immune landscape that dictate the abundance of immunosuppressive cells and consequent suppression of cytotoxic CD8+ T cells. Of these immunosuppressive cell types, PD-L1 expressing myeloid-derived suppressor cells (MDSCs) peak in abundance in a rhythmic manner. Mechanistically, we identified that disruption of the epithelial cell clock regulates the secretion of cytokines and chemokines that promote heightened inflammation, recruitment of neutrophils, and the subsequent development of MDSCs. We leveraged these findings to demonstrate that time-of-day delivery of anti-PD-L1 immunotherapy is most effective when synchronized with the abundance of immunosuppressive MDSCs. Collectively, our results indicate that circadian gating of tumor immunosuppression informs the timing and optimal efficacy of immune checkpoint inhibitors (ICIs).

overall_design: Four C57BL/6 genotypes (wild type, Bmal1-/-, Apc+/-, and Dko (Apc+/-;Bmal1-/-)) were used to study how circadian disruption in the intestine affects the tumor microenvironment of colorectal cancer. Adult mouse intestines were collected, digested for single cell suspension, and then FACS sorted for CD45+ immune cells. scRNA-seq was performed on these immune cells and analyzed using Seurat.

first: Bridget

last: Fortin

first: Jacob

last: Insua-Rodriguez

first: Hamad

last: Alshetaiwi

first: Alexander

last: Moshensky

first: Wei

last: Song

first: Alisa

last: Mahieu

first: Sung

middle: K

last: Chun

first: Amber

last: Lewis

first: Alex

last: Hsu

first: Isam

last: Adam

first: Oliver

middle: S

last: Eng

first: Nicholas

last: Pannunzio

first: Marcus

last: Seldin

first: Ivan

last: Marazzi

first: Francesco

last: Marangoni

first: Devon

last: Lawson

first: Kai

last: Kessenbrock

first: Selma

last: Masri


title: Single-cell transcriptome analysis reveals subtype-specific clonal evolution and microenvironmental changes in liver metastasis of pancreatic adenocarcinoma and their clinical implications

city: Ulsan

first: Semin

last: Lee

address: 50, UNIST-gil

department: Biomedical Engineering

institute: Ulsan National Institute of Science and Technology

summary: Intratumoral heterogeneity (ITH) and tumor microenvironment (TME) of pancreatic ductal adenocarcinoma (PDAC) play important roles in tumor evolution and patient outcomes. However, the precise characterization of diverse cell populations and their crosstalk associated with PDAC progression and metastasis is still challenging. We performed single-cell RNA sequencing (scRNA-seq) of treatment-naive primary PDAC samples with and without paired liver metastasis samples to understand the interplay between ITH and TME in the PDAC evolution and its clinical associations. scRNA-seq analysis revealed that even a small proportion (~30%) of basal-like malignant ductal cells could lead to poor chemotherapy response and patient survival and that epithelial-mesenchymal transition programs were largely subtype-specific. The clonal homogeneity significantly increased with more prevalent and pronounced copy number gains of oncogenes, such as KRAS and ETV1, and losses of tumor suppressor genes, such as SMAD2 and MAP2K4, along PDAC progression and metastasis. Moreover, diverse immune cell populations, including naive SELLhi regulatory T cells (Tregs) and activated TIGIThi Tregs, contributed to shaping immunosuppressive TMEs of PDAC through cellular interactions with malignant ductal cells in PDAC evolution. Importantly, the proportion of basal-like ductal cells negatively correlated with that of immunoreactive cell populations, such as cytotoxic T cells, but positively correlated with that of immunosuppressive cell populations, such as Tregs. We uncover that the proportion of basal-like subtype is a determinant for chemotherapy response and patient outcome, and that PDAC clonally evolves with subtype-specific dosage changes of cancer-associated genes by forming immunosuppressive microenvironments in its progression and metastasis.

overall_design: All remaining tissues are collected in MACS Tissue Storage Solution (Miltenyi Biotec., Auburn, CA, USA) on ice, and processed to the single cell dissociation immediately. Specimens can be maintained in MACS Tissue Storage Solution (Miltenyi Biotec.) for 48 hours at 4C without showing background effects like cell activation or apoptosis induction. Biopsy tissues are placed on a petri dish with a small volume of enzyme mixture solution (Tumor Dissociation Kit, Miltenyi Biotec.), and minced into small pieces under 1-2 mm with surgical blades. Minced pieces are transferred to gentleMACSTM C-tube containing enzyme mixture solution. By choosing an appropriate program of gentleMACSTM Dissociator, Tissue pieces are further dissociated three times with two interval incubations for 30 minutes at 37C under continuous rotation using the MACSmix Tube Rotator. Resuspended samples are applied to a cell strainer (70 m) placed on a 15 ml cornical tube, and washed with 10 ml of RPMI1640 media. After a centrifugation at 400xg for 5 minutes, cells were resuspended in an appropriate volume of RPMI1640 media, and counted after trypan blue staining or acridine orange/ propidium iodide staining. Optionally, erythrocytes are removed by red blood cell lysis solution for 10 min before counting.


title: Gene-expression profiling of individuals resilient to Alzheimer's disease reveals higher expression of genes related to metallothionein and mitochondrial processes and no changes in the unfolded protein response

city: Amsterdam

first: Luuk

middle: Elard

last: de Vries

address: Meibergdreef 47

department: Neuroregeneration

institute: Netherlands Institute for Neuroscience

summary: Some individuals show a discrepancy between cognition and the amount of neuropathological changes characteristic for Alzheimer’s disease (AD). This phenomenon has been referred to as ‘resilience’. The molecular and cellular underpinnings of resilience remain poorly understood. To obtain an unbiased understanding of the molecular changes underlying resilience, we investigated global changes in gene expression in the superior frontal gyrus of a cohort of cognitively and pathologically well-defined AD patients, resilient individuals and age-matched controls (n=11-12 per group). 897 genes were significantly altered between AD and control, 1121 between resilient and control and 6 between resilient and AD. Gene set enrichment analysis (GSEA) revealed that the expression of metallothionein (MT) and of genes related to mitochondrial processes was higher in the resilient donors. Weighted gene co-expression network analysis (WGCNA) identified gene modules related to the unfolded protein response, mitochondrial processes and synaptic signaling to be differentially associated with resilience or dementia. As changes in MT, mitochondria, heat shock proteins and the unfolded protein response (UPR) were the most pronounced changes in the GSEA and/or WGCNA, immunohistochemistry was used to further validate these processes. MT was significantly increased in astrocytes in resilient individuals. A higher proportion of the mitochondrial gene MT-CO1 was detected outside the cell body versus inside the cell body in the resilient compared to the control group and there were higher levels of heat shock protein 70 (HSP70) and X-box-binding protein 1 spliced (XBP1s), two proteins related to heat shock proteins and the UPR, in the AD donors. Finally, we show evidence for putative sex-specific alterations in resilience, including gene expression differences related to autophagy in females compared to males. Taken together, these results show possible mechanisms involving MTs, mitochondrial processes and the UPR by which individuals might maintain cognition despite the presence of AD pathology.

overall_design: RNA was isolated from the grey matter in the superior frontal gyrus.

first: Aldo

last: Jongejan

first: Jennifer

last: Monteiro Fortes

first: Rawien

last: Balesar

first: Annemieke

middle: J

last: Rozemuller

first: Perry

last: Moerland

first: Inge

last: Huitinga

first: Dick

last: Swaab

first: Joost

last: Verhaagen


title: Polystyrene nanoparticles trigger aberrant condensation of  TDP-43 and amyotrophic lateral sclerosis-like symptoms

status: Public on Apr 19 2024

first: Hang

last: Sun

address: No. 2 Tiansheng Road

institute: Southwest University

summary: Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the dysfunction and progressive death of cerebral and spinal motor neurons. Preliminary epidemiological research has hinted at a relationship between environmental risks and the escalation of ALS, but the underlying reasons remain mostly mysterious. Here, we show that nano-size polystyrene plastics (PS) induce ALS-like symptoms and illustrate the related molecular mechanism. When exposed to PS, cells endure internal oxidative stress, which leads to the aggregation of TAR DNA-binding protein 43 kDa (TDP-43), triggering ALS-like characteristics. Additionally, the oxidized heat shock protein 70 fails to escort TDP-43 back to the nucleus. The cytoplasmic accumulation of TDP-43 facilitates the formation of a complex between PS and TDP-43, enhancing TDP-43’s condensation and solidification. These findings are corroborated through in silico and in vivo assays. Altogether, our work illustrates a unique toxicological mechanism induced by nanoparticles and provides insights into the connection between environmental pollution and neurodegenerative disorders.

overall_design: To investigate the effects of the environmental particle pollutant polystyrene nanoplastics on brain damage, we established a mouse inhalation model by continuously intranasally exposing male C57BL/6J mice. We then performed gene expression profiling using RNA-seq data from whole brain tissues of mice in the unexposed and exposed groups. Comparative gene expression profiling analysis of RNA-seq data of whole brain tissues of mice in unexposed and exposed groups.

first: Bingwei

last: Yang

first: Qiong

last: Li

first: Yang

last: Song


title: Dynamic Changes in Extracellular Vesicle-Associated miRNAs Elicited by Abdominal Ultrasound in Peripheral Blood of Inflammatory Bowel Disease Patients

city: Kiel

first: Neha

last: Mishra

address: Rosalind-Franklin-Str. 12

institute: Institute of Clinical Molecular Biology

type: Non-coding RNA profiling by high throughput sequencing

summary: Blood-based biomarkers that reliably indicate disease activity in the intestinal tract are an important unmet need in the management of patients with IBD. Extracellular vesicles (EVs) are cell-derived membranous microparticles, which reflect the cellular and functional state of their site of site of origin. As ultrasound waves may lead to an increased release of EVs, we hypothesized that application of ultrasound waves on inflamed intestinal tissue in IBD may amplify the release of tissue-specific EVs, which in turn can be detected in the peripheral blood.

overall_design: 26 patients with IBD were included in the prospective clinical study. Serum samples were collected before and 30 minutes after diagnostic transabdominal ultrasound. Differential miRNA expression was analyzed by sequencing.

first: Nathan

last: Baran

first: Florian

last: Tran

first: Philip

last: Rosenstiel


title: Gene expression analysis of Unstimulated follicular B cells and follicular B cells stimulated with IFNa, CpG and CpG plus IFNa.

city: Fukuoka

first: Ryota

last: Higuchi

address: Maidashi, 3-1-1

institute: Kyushu university

summary: We report the grobal gene expression in follicular (FO) B cells stimulated with CpG, IFNa, and CpG plus IFNa for 12 hours prior to the onset of PC differentiation. We found that stimulation with CpG plus IFNa upregulates metabolic processes compared to stimulation with CpG alone. This study highlight the pivotal role of IFNa in determining the fate of PC differentiation of TLR9-activated FO B cells.

overall_design: To examine gene expression patterns of FO B cells stimulated with CpG+IFNa, which induces plasma differentiation, FO B cells from 10-week-old mice were isolated by FACS staining. These B cell subsets were stimulated with IFNa, CpG, and CpG plus IFNa for 12 hours and then submitted to a cDNA library for RNA-seq analysis along with unstimulated FO B cells. We then performed comparative gene expression analysis to identify the characteristics of gene expression shaped in FO B cells stimulated with CpG plus IFNa.

first: R

first: K

last: Tanaka

first: Y

last: Ohkawa

last: Baba


title: CEBPA restricts alveolar type 2 cell plasticity during development and injury-repair [ChIP-seq]

city: Houston

first: Dalia

last: Hassan

state: TX

department: Pulmonary Medicine

institute: MD Anderson Cancer Center

summary: Cell plasticity theoretically extends to all possible cell types, but naturally decreases as cells differentiate, whereas injury-repair re-engages the developmental plasticity. Here we show that the lung alveolar type 2 (AT2)-specific transcription factor (TF), CEBPA, restricts AT2 cell plasticity in the mouse lung. AT2 cells undergo transcriptional and epigenetic maturation postnatally. Without CEBPA, both neonatal and mature AT2 cells reduce the AT2 program, but only the former reactivate the SOX9 progenitor program. Sendai virus infection bestows mature AT2 cells with neonatal plasticity where Cebpa mutant, but not wild type, AT2 cells express SOX9, as well as more readily proliferate and form KRT8/CLDN4+ transitional cells. CEBPA promotes the AT2 program by recruiting the lung lineage TF NKX2-1. The temporal change in CEBPA-dependent plasticity reflects AT2 cell developmental history. The ontogeny of AT2 cell plasticity and its transcriptional and epigenetic mechanisms have implications in lung regeneration and cancer.

overall_design: FACS purified epithelial cells from Cebpa mutant and littermate control were profiled for ChIP-Seq at the indicated time

first: Jichao

last: Chen


title: Fecal microbiota transplantation from female donors restores gut permeability and reduces liver injury and inflammation in middle-aged male mice exposed to alcohol

status: Public on Apr 18 2024

city: Pamplona

first: SERGIO

last: ROA

address: Irunlarrea, 1

department: Biochemistry and Genetics

country: Spain

institute: University of Navarra

summary:
Background: Alcohol misuse, binge drinking pattern, and gender-specific effects in the middle-aged population has been clearly underestimated. In the present study, we focused on understanding gender-specific effects of alcohol exposure on the gut-liver axis and the role of gut microbiota in modulating gender-specific responses to alcohol consumption.

Methods: Fifty-two-week-old female and male C57BL/6 mice were fasted for 12 h, and then administered a single oral dose of ethanol (EtOH) (6 g/kg). Controls were given a single dose of PBS. Animals were sacrificed 8 h later. Alternatively, fecal microbiota transplantation (FMT) was performed in 52-week-old male mice from female donors of the same age. Permeability of the large intestine (colon), gut microbiota, liver injury, and inflammation was thoroughly evaluated in all groups.

Results: Middle-aged male mice exposed to EtOH showed a significant increase in gut permeability in the large intestine, evaluated by FITC-dextran assay and ZO-1, OCCLUDIN and MUCIN-2 immuno-staining, compared to PBS-treated animals, whilst female mice of the same age also increased their gut permeability, but displayed a partially maintained intestinal barrier integrity. Moreover, there was a significant up-regulation of TLRs and markers of hepatocellular injury, cell death (AST, TUNEL-positive cells) and lipid accumulation (ORO) in male mice after EtOH exposure. Interestingly, FMT from female donors to male mice reduced gut leakiness, modified gut microbiota composition, ameliorated liver injury and inflammation, TLR activation and the senescence phenotype of middle-aged mice.

Conclusion: Our findings highlighted the relevance of gender in middle-aged individuals who are exposed to alcohol in the gut-liver axis. Moreover, our study revealed that gender-specific microbiota transplantation might be a plausible therapy in the management of alcohol-related disorders during aging.

overall_design: To characterize changes in gut microbiota during fecal microbiota transplantation (FMT) from female mice donors and after acute ethanol (EtOH) intoxication, we collected feces from 52-week-old female donors (n = 4, OFE), FMT male receptors (n = 3, OME+FMT), and non-FMT male mice (n = 4, OME) at the end of EtOH treatments. To evaluate changes in bacterial diversity among these three groups, we performed nanopore-based sequencing of full-length 16S rRNA amplicon libraries from DNA samples of these feces.

first: Arantza

last: Lamas-Paz

first: Mariana

last: Mesquita

first: Marcos

last: Garcia-Lacarte

first: Olga

last: Estévez-Vazquez

first: Raquel

last: Benedé-Ubieto

first: Alejandro

last: Hionides-Gutierrez

first: Hanghang

last: Wu

first: Hector

last: Leal Lasalle

first: Javier

last: Vaquero

first: Rafael

last: Bañares

first: Eduardo

last: Martinez-Naves

first: Sergio

last: Roa

first: Yulia

last: Nevzorova

first: Gonzalo

last: Jorquera

first: Franciso

last: Cubero


title: Tet Controls Axon Guidance in Early Brain Development through Glutamatergic Signaling

status: Public on Apr 20 2024

city: Piscataway

first: Ruth

last: Steward

state: New Jersey

address: 190 Frelinghuysen Rd

department: Waksman Institute of Microbiology

institute: Rutgers University

summary: Ten-eleven translocation (Tet) is an important gene in neurodevelopment, but how Tet regulates brain development is still under study. Mutations in human TET proteins have been found in individuals with neurodevelopmental disorders. Here we report a new function of Tet in regulating Drosophila early brain development. We found that mutation on the Tet DNA-binding domain (TetAXXC) resulted in axon guidance defects in the mushroom body (MB). Tet is required in early brain development during the outgrowth of MB β axons. Transcriptomic study of TetAXXC mutant and wild-type pupal brains shows that glutamine synthetase 2 (Gs2), a key enzyme in glutamatergic signaling, is the most significantly down-regulated gene in the Tet mutant brains. RNAi knockdown or CRISPR/Cas9 mutagenesis of Gs2 recapitulates the TetAXXC phenotype. Surprisingly, Tet and Gs2 act in the insulin-producing cells (IPCs) to control MB axon guidance, and overexpression of Gs2 in the IPCs rescue the axonal defects of TetAXXC. Treating TetAXXC with the metabotropic glutamate receptor antagonist MPEP can also rescue the phenotype confirming Tet function in regulating glutamatergic signaling. TetAXXC and the Drosophila homolog of Fragile X Messenger Ribonucleoprotein protein mutant (Fmr13) have similar mushroom body axonal defects and reduction in Gs2 transcription, and, importantly, overexpression of Gs2 in the IPCs of Fmr1 mutants also rescues the axonal defects. Our studies reveal a new function of Tet in regulating axon guidance in the brain via glutamatergic signaling and suggest overlapping functions between Tet and Fmr1.

overall_design: Comparative gene expression analysis by RNA-seq between Drosophila wild-type (WT) and Tet CXXC Zinc Finger domain mutant (TetAXXC) brains.

first: Hiep

last: Tran

first: Le

last: Le

first: Badri

last: Nath Singh

first: Joseph

last: Kramer


title: Cancer-stromal cell interactions in breast cancer brain metastases induce glycocalyx-mediated resistance to HER2-targeting therapies [RNA-seq]

city: Boston

first: Kornelia

last: Polyak

address: 450 Brookline Ave

department: Medical Oncology

institute: Dana-Farber Cancer Institute

summary: In a 3D coculture with stroma cells derived from breast cancer patients’ brain metastasis, HER2+ breast cancer cells were protected from HER2-targeted therapies, particularly the EGFR/HER2 small molecule inhibitor neratinib. To get insight into how this protection arises, a Synthetic Notch (SynNotch) reporter model allowed to study the effect of direct contact between stroma and cancer cells.

overall_design: To identify stroma-contact specific changes, a SynNotch reporter was used with breast cancer cell lines constitutively expressing mCherry and transiently expressing BFP when in direct contact with BM3 cells engineered with cell surface GFP. Following coculture of organoids in 3D with or without neratinib, cells were dissociated and sorted by Fluorescence-activated cell sorting (FACS) for mCherry+BFP- (breast cancer, non-contact) and mCherry+BFP+ (DP breast cancer, stroma-contact) fractions

first: Marie-Anne

last: Goyette

first: Laura

last: Stevens


title: Sex-biased Gene Expression During Neural Differentiation of Human Embryonic Stem Cells

city: Uppsala

first: Philipp

last: Pottmeier

state: Uppland

address: Kabovagen 4

department: Evolution and Development

institute: Uppsala University

summary: Sex differences in the developing human brain are primarily attributed to hormonal influence. Recently however, genetic differences and their impact on the developing nervous system have attracted increased attention. To understand genetically driven sexual dimorphisms in neurodevelopment, we investigated genome-wide gene expression in an in vitro differentiation model of male and female human embryonic stem cell lines (hESC), independent of the effects of human sex hormones. Four male and four female-derived hESC lines were differentiated into a population of mixed neurons over 37 days. Differential gene expression and gene set enrichment analyses were conducted on bulk RNA sequencing data. While similar differentiation tendencies in all cell lines demonstrated the robustness and reproducibility of our differentiation protocol, we found sex-biased gene expression already in undifferentiated ESCs at day 0, but most profoundly after 37 days of differentiation. Male and female cell lines exhibited sex-biased expression of genes involved in neurodevelopment, suggesting that sex influences the differentiation trajectory. Interestingly, the highest contribution to sex differences was found to arise from the male transcriptome, involving both Y chromosome and autosomal genes. We propose 13 sex-biased candidate genes (10 upregulated in male cell lines and 3 in female lines) that are likely to affect neuronal development. Additionally, we confirmed gene dosage compensation of X/Y homologs escaping X chromosome inactivation through their Y homologs and identified a significant overexpression of the Y-linked demethylase UTY and KDM5D in male hESC during neuron development, confirming previous results in neural stem cells. Our results suggest that genetic sex differences affect neuronal differentiation trajectories, which could ultimately contribute to sex biases during human brain development.

overall_design: Neural differentiation of 4 male and 4 female human embryonic stem cell lines over 37 days. Six cell lines (WA14, 983a, 401, LT2e, 980, 975)  and four timepoints were submitted for bulk RNA sequencing (T0, D4, D9, D37). Cell culture experiments were performed in triplicates (A, B, C). Thus, a total of 72 samples were submitted for sequencing on the NovaSeq instrument with an S4 lane (Illumina) resulting in approximately 27.7 million 150 bp paired-end read-pairs per sample. Then, gene expression was compared between male and female cell lines using DEseq2 and GSEA analysis (MSigDB).

first: Danai

last: Nikolantonaki

first: Fredrik

last: Lanner

first: Christiane

last: Peuckert

first: Elena

last: Jazin


title: XPO1 blockade with KPT-330 promotes apoptosis in Cutaneous T- cell Lymphoma by activating the p53-p21 and p27 pathways

city: Philadelphia

first: Nitin

last: Chakravarti

state: PA

address: 233 S Tenth St

institute: Thomas Jefferson University

summary: To assess the effects of XPO1 inhibition with small molecule inhibitor KPT-330 on Cutaneous T-cell Lymphoma after 48 hours of treatment

overall_design: RNA sequencing of CTCL cell lines with different treatments - DMSO  & KPT-330

first: Amy

last: Boles

first: Rachel

last: Burzinski

first: Paola

last: Sindaco

first: Colleen

last: Isabelle

first: Kathleen

last: McConnell

first: Anjali

last: Mishra

first: Pierluigi

last: Porcu


title: Heterogeneity of Immune Cells and Their Communications Unveiled by Transcriptome Profiling in Acute Inflammatory Lung Injury

city: hangzhou

first: kang

last: zhiying

address: 浙江省杭州市西湖区余杭塘路866号

institute: zhejiang universty

summary: Background: Acute Respiratory Distress Syndrome (ARDS) or its earlier stage Acute lung injury (ALI), is a worldwide health concern that jeopardizes human well-being. Currently, the treatment strategies to mitigate the incidence and mortality of ARDS are severely restricted. This limitation can be attributed, at least in part, to the substantial variations in immunity observed in individuals with this syndrome. Methods: Bulk and single cell RNA sequencing from ALI mice and single cell RNA sequencing from ARDS patients were analyzed. We utilized the Seurat program package in R and cellmarker 2.0 to cluster and annotate the data. The differential, enrichment, protein interaction, and cell-cell communication analysis were conducted. Results: The mice with ALI caused by pulmonary and extrapulmonary factors demonstrated differential expression including Clec4e, Retnlg, S100a9, Coro1a, and Lars2. We have determined that inflammatory factors have a greater significance in extrapulmonary ALI, while multiple pathways collaborate in the development of pulmonary ALI. Clustering analysis revealed significant heterogeneity in the relative abundance of immune cells in different ALI models. The autocrine action of neutrophils plays a crucial role in pulmonary ALI. Additionally, there was a significant increase in signaling intensity between B cells and M1 macrophages, NKT cells and M1 macrophages in extrapulmonary ALI. The CXCL, CSF3 and MIF, TGFβ signaling pathways play a vital role in pulmonary and extrapulmonary ALI, respectively. Moreover, the analysis of human single-cell revealed DCs signaling to monocytes and neutrophils in COVID-19-related ARDS is stronger compared to sepsis-associated ARDS. In sepsis-associated ARDS, CD8+ T and Th cells exhibit more prominent signaling to B-cell nucleated DCs. Meanwhile, both MIF and CXCL signaling pathways are specific to sepsis-associated ARDS. Conclusions: This study has identified specific gene signatures and signaling pathways in animal models and human samples that facilitate the interaction between immune cells, which could be targeted therapeutically in ARDS patients of various etiologies.

overall_design: Lung tissues from intrapulmonary ALI models constructed by intratracheal injection of LPS, extrapulmonary ALI models by intraperitoneal injection of LPS, and control mice were collected and analyzed using scRNAseq.

first: Bao-ping

last: Tian

first: Zhi-ying

last: Kang


title: Cancer-stromal cell interactions in breast cancer brain metastases induce glycocalyx-mediated resistance to HER2-targeting therapies [ATAC-seq]

first: Kornelia

last: Polyak

summary: In a 3D coculture with stroma cells derived from breast cancer patients’ brain metastasis, HER2+ breast cancer cells were protected from HER2-targeted therapies, particularly the EGFR/HER2 small molecule inhibitor neratinib. To get insight into how this protection arises, ATAC-seq on coculture cells with or without neratinib as performed.

overall_design: ATAC-seq of sorted cancer cells from a coculture with brain stromal cells or in monoculture, with or without neratinib

first: Marie-Anne

last: Goyette

first: Laura

last: Stevens


title: miRNA array in the A549 Ago2-KO/HA-Ago2Wt, Ago2-KO/HA-Ago2KA, and Ago2-KO/HA-Ago2Δ (Dm)  cells

city: Miaoli County

first: YI-JU

last: CHEN

address: Room R1-3121, 35 keyan Road, Zhunan Town

country: Taiwan

institute: National Health Research Institutes

summary: We used miRNA microarrays to detail the miRNA content in A549 Ago2-KO/HA-Ago2Wt, Ago2-KO/HA-Ago2KA, and Ago2-KO/HA-Ago2Δ (Dm)  cells

overall_design: A549 Ago2-KO/HA-Ago2Wt, Ago2-KO/HA-Ago2KA, and Ago2-KO/HA-Ago2Δ (Dm)  cells were collected for miRNA extraction and hybridization on Affymetrix GeneChip® miRNA 4.0 array.

first: Hua-Jung

last: Li

first: Yi-Ju

last: Chen


title: Xuanfei Formula Attenuates RSV Infection

city: Wuhan

first: Huan

last: Qin

state: Hubei

address: 100 Hong Kong Road

department: Clinical Laboratory

institute: Wuhan  Children’s Hospital ,Huazhong University of Science and Technology

summary: RSV, a leading cause of severe respiratory illnesses in vulnerable populations, lacks effective, affordable treatments for pediatric use. The potential of traditional Chinese medicine for relieving viral symptoms prompted this investigation into Xuanfei Formula (XFF) as an anti-RSV agent. This study employed H&E staining, cytokine profiling, and RSV titer quantification in BALB/c mice to evaluate the impact of XFF on RSV infection. Strikingly, immediate post-treatment observation showed a precipitous drop in both serum pro-inflammatory cytokine levels and pulmonary RSV-N gene copies in comparison to infected controls, suggesting XFF’s direct anti-RSV action. Transcriptome analyses were used to pinpointed the underlying mechanism behind formula’s immune-independent anti-RSV, a leading cause of severe respiratory illnesses in vulnerable populations, lacks effective, affordable treatments for pediatric use. The potential of traditional Chinese medicine for relieving viral symptoms prompted this investigation into Xuanfei Formula (XFF) as an anti-RSV agent. This study employed H&E staining, cytokine profiling, and RSV titer quantification in BALB/c mice to evaluate the impact of XFF on RSV infection. Strikingly, immediate post-treatment observation showed a precipitous drop in both serum pro-inflammatory cytokine levels and pulmonary RSV-N gene copies in comparison to infected controls, suggesting XFF’s direct anti-RSV action. Transcriptome analyses were used to pinpointed the underlying mechanism behind formula’s immune-independent anti-RSV effects during infection.

overall_design: RSV Animal Model and GroupingsFemale SPF BALB/c mice, 6-8 weeks old, were categorized into seven cohorts. The Mock group remained untreated, while other groups experienced RSV challenge and XFF or saline treatment according to designated protocols. RSV infection commenced intranasally with 1 × 106 PFU in 50 µL. Concurrently, XFF or saline (5 g/kg/day) was administered intragastrically starting at 4 hours post-infection. RNA Sequencing ProtocolLung tissues were harvested for RNA isolation and assessed for quality via Agilent tech. Libraries, post-construction, were quantified using Qubit, followed by sequencing on the DNBSEQ-T7 system for transcriptomic analysis.

first: Jin

last: Luo

last: Zhao

first: Wange

last: Lou


title: Enhancing Precise Genome Editing in Human Pluripotent Stem Cells through Dual Inhibition of DNA Damage Response and Repair Pathways [RNA-seq]

city: Seoul

first: Hyuk-Jin

last: Cha

address: 215-20 Gwan-ak ro Gwan-ak gu

institute: Seoul National University

summary: Precise genome editing is crucial for establishing isogenic human disease models and ex vivo stem cell therapy from the patient-derived human pluripotent stem cells (hPSCs). Unlike Cas9-mediated knock-in, cytosine base editor (CBE) and prime editor (PE) achieve the desirable gene correction without inducing DNA double strand breaks. However, hPSCs possess highly active DNA repair pathways and are particularly susceptible to p53-dependent cell death. These unique characteristics impede the efficiency of gene editing in hPSCs. Here, we demonstrate that dual inhibition of p53-mediated cell death and distinct activation of the DNA damage repair system upon DNA damage by CBE or PE additively enhanced editing efficiency in hPSCs. The BE4stem system comprised of dominant negative p53 (p53DD) and three UNG inhibitor (UGI), engineered to specifically diminish base excision repair (BER), improved CBE efficiency in hPSCs. Addition of dominant negative MLH1 to inhibit mismatch repair activity and p53DD in the conventional PE system also significantly enhanced PE efficiency in hPSCs. Thus, combined inhibition of the unique cellular cascades engaged in hPSCs upon gene editing could significantly enhance precise genome editing in these cells.

overall_design: To investigate off-target effects on transcriptome level after gene editing, we established RNA of EP(Electroporation only, control group) and BE4max, BEStem, PE4, PEStem treated sample.

first: Ju-Chan

last: Park

first: Yun-Jeong

last: Kim

first: Gue-Ho

last: Hwang

first: Chan

first: Sangsu

last: Bae


title: CEBPA restricts alveolar type 2 cell plasticity during development and injury-repair [1]

first: Dalia

last: Hassan

overall_design: FACS purified epithelial cells from Cebpa mutant and littermate control were profiled at the indicated time

first: Jichao