title: The development of a high-plex spatial proteomic methodology for the characterisation of the head and neck tumour microenvironment

city: Brisbane

first: Arutha

last: Kulasinghe

state: QLD

address: 37 kent st

institute: University of Queensland

summary: Head and neck squamous cell carcinoma (HNSCC) is the seventh most common cancer globally. Despite advancements in treatment modalities, patient prognoses remain unimproved. Insight into the tumour microenvironment (TME) using spatially resolved approaches, and its association with clinical endpoints, may provide useful prognostic tools and refine current treatment outcomes. We profiled 84 mucosal HNSCC tissue samples using next-generation ultra-high plex spatial protein and transcriptome mapping (580-proteins, 18,000 mRNA targets). We found patient survival outcomes (both disease-free and overall survival) were associated with anatomical subsite and tumour stage. We identified proteomic and transcriptomic functional T cell markers within discrete TME regions that were associated with survival. GSEA revealed elevated glycolysis and hypoxia pathways in patients with worse outcomes. Key proteomic findings (including CD34 and CD44) were independently validated using single-cell protein profiling. This study provides a systematic workflow for high-plex protein and transcriptomic profiling in mucosal HNSCC.

overall_design: GeoMX technology [GeoMx Immuno-Oncology Proteome Assay (IPA, 580-plex); Nanostring, USA] was applied to tumour cores collected from 68 chemotherapy and/or radiotherapy treated patients with tissues acquired from a range of sites across the mucosal head and neck regions (i.e. tongue, oral cavity, pharynx and lip). Each core were segmented into tumour and stroma regions based on PanCK staining, resulting in 136 fields of view (FoVs) across 68 cores. The following cell surface markers were used for morphology visualization: SYTO-13, CD45, and PanCk

first: Chin Wee

last: Tan


title: Kielin/chordin-like protein deficiency aggravates pressure overload-induced cardiac dysfunction and remodeling via P53/P21/CCNB1 signaling in mice

status: Public on Jun 01 2025

city: Wuhan

first: MENGMENG

last: Zhao

address: Wuhan University

country: China

institute: Wuhan University

type: Expression profiling by high throughput sequencing

summary: Targeting cardiac remodeling is regarded as a key therapeutic strategy for heart failure. Kielin/chordin-like protein (KCP) is a secretory protein with 18 cysteine-rich domains and associated with kidney and liver fibrosis. However, the relationship between KCP and cardiac remodeling remains unclear. Here, we aimed to investigate the role of KCP in cardiac remodeling induced by pressure overload and explore its potential mechanisms. Left ventricular (LV) KCP expression was measured with real-time quantitative PCR, western blotting, and immunofluorescence staining in pressure overload-induced cardiac remodeling in mice. Cardiac function and remodeling were evaluated in wide-type (WT) mice and KCP knockout (KO) mice by echocardiography, which were further confirmed by histological analysis with hematoxylin and eosin and Masson staining. RNA sequence was performed with LV tissue from WT and KO mice to identify differentially expressed genes and related signaling pathways. Primary cardiac fibroblasts (CFs) were used to validate the regulatory role and potential mechanisms of KCP during fibrosis. KCP was down-regulated in the progression of cardiac remodeling induced by pressure overload, and was mainly expressed in fibroblasts. KCP deficiency significantly aggravated pressure overload-induced cardiac dysfunction and remodeling. RNA sequence revealed that the role of KCP deficiency in cardiac remodeling was associated with cell division, cell cycle, and P53 signaling pathway, while cyclin B1 (CCNB1) was the most significantly up-regulated gene. Further investigation in vivo and in vitro suggested that KCP deficiency promoted the proliferation of CFs via P53/P21/CCNB1 pathway. Taken together, these results suggested that KCP deficiency aggravates cardiac dysfunction and remodeling induced by pressure overload via P53/P21/CCNB1 signaling in mice.

overall_design: To evaluate function of KCP in pressure overload induced cardiac remodeling, we knock out KCP experssion in C57 mice to evaluate the effects of KCP deficiency on cardiac remodeling.

first: Yao

last: Xu

first: Jun

last: Wan


title: Hypoxic Pregnancy Promotes Fibrosis and Increases Stress Metabolites in the Ovine Fetal Liver

city: Aurora

first: Molly

last: McGuckin

state: Colorado

address: 13243 East 23rd Avenue, Aurora, CO 80045-0508

country: USA

institute: University of Colorado

summary: Fetal hypoxemia is a common pregnancy complication associated with fetal growth restriction (FGR). Offspring born with FGR have a higher risk for postnatal liver metabolic disease and injury. Our objective was to better understand how hypoxemia impacts the developing fetal liver. We hypothesized hypoxemia promotes hepatocellular injury, shifts nutrient metabolism, and activates energetic and oxidative stress signaling pathways in the fetal liver. To test this, we used an established ovine model of hypoxia during late gestation. Pregnant ewes were housed in isobaric chambers under normoxia (CON) or hypoxic (HOX) conditions for 30 days, and fetal liver biopsies were analyzed. Compared to CON, HOX fetuses had asymmetrical growth restriction, higher hemoglobin concentration, and greater liver collagen deposition localized in periportal regions. HOX liver had unique metabolic profiles, including shifts to amino acid and central carbon metabolism and increased stress metabolites. The hepatic transcriptome of HOX fetuses included downregulation of genes involved in glutathione synthesis and redox balance and upregulation of mitochondrial dysfunction and downregulation of oxidative phosphorylation genes. Despite predicted oxidative stress, there was no difference in lipid peroxidation, oxidative phosphorylation complex abundance, or antioxidant response proteins NRF2, NQO1 or SOD2. In summary, hypoxic pregnancy increased fetal hepatic collagen deposition, an indicator of hepatic injury, with a unique profile of liver stress metabolites and metabolic adaptations in central carbon metabolism. These results provide new insight into how fetal hypoxia may initiate fibrotic and metabolic liver disease risk in exposed offspring.

overall_design:
To investigate the effect of hypoxic pregnancy on fetal liver development and metabolism , we used a sheep model of hypoxia in late gestation. Ewes were subject to normoxia (CON) or hypoxic (HOX) conditions for 30 days and fetal livers were collected at ~day 130 of gestation.
We performed gene expression analysis on bulk RNAseq data from 9 CON and 7 HOX fetal livers.

first: Dong

last: Wang

first: Jasmine

last: Ortiz

first: Evgenia

last: Dobrinskikh

first: Wen

last: Tong

first: Kimberly

last: Botting

first: Younguo

last: Niu

first: Dino

last: Giussani

first: Stephanie

last: Wesolowski


title: The histone modifier KDM5 functions as an insulator [Targeted DamID]

city: Bronx

first: Matanel

last: Yheskel

state: NY

address: 1300 Morris Park

department: Genetics

institute: Albert Einstein College of Medicine

summary: KDM5 family proteins are best known for their demethylation of the promoter proximal chromatin mark H3K4me3. KDM5-regulated transcription is critical in the brain, with variants in the X-linked paralog KDM5C causing the intellectual disability (ID) disorder, Claes-Jensen syndrome. Although the demethylase activity of KDM5C is known to be important for neuronal function, the contribution of non-enzymatic activities remains less characterized. We therefore used Drosophila to model the ID variant Kdm5L854F that disrupts a C5HC2 zinc finger adjacent to the enzymatic JmjC domain. Kdm5L854F causes similar transcriptional changes in the brain to a demethylase dead strain, Kdm5JmjC*, despite having little effect on enzymatic activity. KDM5L854F is also distinct from KDM5JmjC* in its reduced interactions with insulator proteins and enhancement of position effect variegation. Instead, the common transcriptional deficits likely result from both the JmjC and C5HC2 domains driving proper genomic organization through their activity in promoting proper loop architecture.

overall_design: Targeted DamID in KDM5 WT, JmjC, and L854F brains

first: Melissa

last: Castiglione

first: Richard

last: Kelly

first: Julie

last: Secombe


title: Filaggrin status modulates gene transcription, DNA damage and epidermal barrier repair during clinical response to methotrexate in atopic eczema

city: Newcastle upon Tyne

first: Simon

middle: Joseph

last: Cockell

address: Framlington Place

department: School of Biomedical, Nutritional and Sport Sciences

country: United Kingdom

institute: Newcastle University

summary: Background: Methotrexate (MTX) is an effective treatment for atopic eczema (AE); however, the mechanism of action remains incompletely understood. MTX is known to induce keratinocyte differentiation, which can occur following DNA damage triggered by external stimuli. Objectives: We sought to investigate the mechanism of action of MTX in AE using a combination of in vitro models and study of AE skin during the early phases of therapy with MTX. Specifically, we investigated the relationship between MTX-induced DNA damage, keratinocyte differentiation, barrier function, filaggrin status and disease activity. Methods: Lesional and non-lesional skin from two AE cohorts and in vitro human 3-D epidermal organoids (living epidermal equivalents – [LEEs]) were used to assess response to MTX. Markers of late keratinocyte differentiation and response to purine/pyrimidine nucleosides were assessed. Keratinocyte DNA damage following MTX was evaluated through measurement of DNA damage markers and transcriptomic analysis. Results: MTX restored barrier function and induced filaggrin and loricrin expression in AE skin and LEE, through a mechanism inhibited by thymidine. MTX-induced DNA damage and DNA damage response signalling pathways in LEE and AE skin which were upstream of keratinocyte differentiation. There was an interaction between gene expression, disease severity (EASI) and FLG status (heterozygotes versus wildtype, e.g. LGALS7, SPINK7). Conclusions: MTX induces key epidermal barrier proteins through thymidine-dependent DNA damage in the absence of inflammatory cells. Findings were associated with improved disease outcomes in AE skin.

overall_design: We performed a combined bulk RNAseq analysis from 44 samples across 11 AE subjects (lesional and non-lesional skin). Skin punch biopsies from lesional and non-lesional skin were collected at week 0 (baseline), week 2 (visit 1 (V1)) and week 12 (visit 2 (V2)) following MTX initiation.

first: Amaani

middle: B

last: Hussain

first: Martina

middle: S

last: Elias

middle: J

first: John

last: Casement

first: Sara

last: Brown

first: Graham

middle: R

last: Smith

first: Esme

last: Nichols

first: Yuchun

last: Ding

first: J

last: Lumley

first: Michael

last: Donaldson

first: Nick

last: Reynolds


title: The histone modifier KDM5 functions as an insulator [ATAC-seq]

type: Genome binding/occupancy profiling by high throughput sequencing

overall_design: ATAC-Seq in KDM5 WT, JmjC, and L854F brains


title: α-Arbutin Prevents UVA-Induced Skin Photodamage via Alleviating DNA Damage and Collagen Degradation in NIH-3T3 cells

city: Shenzhen, Guangdong

last: Zhao

address: 19/F, Li Ning Center, No. 9996 Shennan Avenue, Nanshan District, Shenzhen, China

institute: Shenzhen Hujia Technology

summary: Ultraviolet radiation (UV) causes certain side effects to the skin, and their accumulation to a certain extent can lead to accelerated aging of the skin. Recent studies suggests that α-arbutin may be useful in various disorders such as hyperpigmentation disorders, wound healing, and antioxidant activity. However, the role of α-arbutin in skin photodamage is unclear. In this study, under UVA-induced photodamage conditions, α-arbutin treated mouse skin fibroblasts (NIH-3T3) can repair DNA damage and resist apoptosis by reducing the production of reactive oxygen species (ROS) and increasing the phosphorylation of glycogen synthase kinase 3 beta (GSK3β) to orchestra AKT/GSK3β pathway. Meanwhile, α-arbutin can also regulate collagen metabolism and facilitate the replenishment of collagen by targeting the phosphorylation of SMAD3 to mediate the TGFβ/SMAD pathway in NIH-3T3. In conclusion, we found that α-arbutin can mitigate the detrimental effects of skin photodamage induced by UVA irradiation, and provides a theoretical basis for the use of α-arbutin in the treatment of skin photodamage.

overall_design: After UVA irradiation with the addition of α-arbutin, we wanted to investigate whether α-arbutin had the ability of anti-photodamage in NIH 3T3 cell, especially in DNA repair and collagen synthesis. Then we performed gene expression profiling analysis using data obtained from RNA seq of 3 different treated ways (control group, UVA treated group, UVA and α-arbutin co-treated group)in NIH 3T3 cell.

first: Jiangming

last: Zhong


title: RNA transcriptome sequencing of  lung adenocarcinoma cell line H1975 treated with siNC or siHHLA3 oligonucleotides

city: Guangzhou

first: Chunting

last: Zhang

address: Guangzhou Avenue North

institute: Southern Medical University

summary: To figure out the role of long non-coding RNA HHLA3 in lung adenocarcinoma and the underlying mechanism, RNA transcriptome sequencing was performed to globally characterize HHLA3-regulated transcriptome changes. HHLA3 knockdown by RNA interference in H1975 cells. H1975 cells treated with siNC served as control.

overall_design: Lung adenocarcinoma cell line H1975 treated with siNC or siHHLA3

first: Yi

last: Jin

first: Xiaoting

last: Liang

first: Liang

last: Zhou


title: High Mobility Group Box protein 3 (HMGB3) facilitates DNA interstrand crosslink processing and double-strand break repair in human cells

city: Austin

first: Anna

last: Battenhouse

state: TX

address: 100 East 24th St., Stop A5000

department: Center for Biomedical Research Support

institute: The University of Texas at Austin

summary: DNA damaging agents can contribute to genetic instability, and such agents are often used in cancer chemotherapeutic regimens due to their cytotoxicity. Thus, understanding the mechanisms involved in DNA damage processing can not only enhance our knowledge of basic DNA repair mechanisms, but may also be used to develop improved chemotherapeutic strategies to treat cancer. The high mobility group box protein 1 (HMGB1) is a known nucleotide excision repair (NER) co-factor, and its family member HMGB3 has been implicated in chemo-resistance in ovarian cancer. Here, we aim to understand the potential role(s) of HMGB3 in processing DNA damage. A potential role in NER was investigated using HMGB3 knockout human cell lines in response to UV damage. Interestingly, unlike HMGB1, HMGB3 does not appear to play a role in NER. Subsequently, potential roles in DNA interstrand crosslink (ICL) and DNA double-strand break (DSB) repair were investigated using mutagenesis assays, metaphase spreads, foci formation, a variety of DNA repair assays, and Tag-Seq analyses in human cells. We found evidence to suggest that HMGB3 is involved in the processing of both DSBs and ICLs in human cells. These novel results elucidate a role for HMGB3 in DNA damage repair, and surprisingly, also indicate a distinct role of HMGB3 in DNA damage repair from that of HMGB1. These findings advance our understanding of the role of HMGB3 in chemotherapeutic drug resistance and as a target for new chemotherapeutic strategies in the treatment of cancer.

overall_design: For TagSeq sequencing (a form of 3' RNA sequencing), four replicate libraries were prepared for Wild Type UNOS cells, and for each of three HMGB3 CRISPR Knock-out clones. Resulting sequences were analyzed for differentially expressed genes between the KOs and the WT.

first: Jillian

last: Dangerfield

first: Anirban

last: Mukherjee

first: Wade

last: Reh

first: Karen

last: Vasquez


title: IL-27-induced shifts in metabolism orchestrate the polarization of alternatively activated macrophages and mitigate inflammatory pain

city: Hangzhou

last: Ma

address: No.258, kaixuan RD

institute: Zhejiang University school of medicine

summary: In a previous study, data showed that IL-27 induced the alternative-activated macrophages, independent of IL-4. We investigate transcriptomic profiling to compare the difference pattern between IL-27 and IL-4.

overall_design: The bone marrow-derived macrophages (BMDM) were harvested and distributed into three groups: the non-control group, the IL-4 treated group, and the IL-27 treated group. Each group set three replicate experiments.

first: Shuyuan

last: Huang

first: Shengmei

last: Zhu


title: UCBCs cultured with TPO

city: 重庆Chongqing

first: Zeyu

last: Fu

state: 重庆Chongqing

address: 高滩岩正街

department: College of Preventive Medicine

institute: AMU

summary: In this study, we aimed to investigate the differentiation dynamics of megakaryocyte development, utilizing human umbilical cord blood (UCB) cells

overall_design: We cultured human umbilical cord blood (UCB) cells to generate megakaryocytes by first isolating mononuclear cells from UCB using density gradient centrifugation, then plating the cells in a serum-free medium supplemented with thrombopoietin (TPO) to promote megakaryocyte differentiation, maintaining the cultures at 37°C in a humidified atmosphere with5% CO2, and periodically assessing cell morphology and surface markers using flow cytometry to confirm the generation of megakaryocytes

first: Kangfu

last: Sun

first: Baichuan

last: Xu

first: Shilei

last: Chen

first: Junping

last: Wang


title: Temporally resolved single-cell RNA sequencing reveals protective and pathological responses during herpes simplex virus 1 CNS infection

city: Aarhus

first: Soren

last: Paludan

address: CF Møllers Alle 6, Building 1115

department: Biomedicine

country: Denmark

institute: Aarhus university

summary: We profile the transcriptomic changes in the infected brain stem at the single-cell level with temporal resolution, supported by GeoMx spatial transcriptomic data. Microglia proportions increased during infection, while blood-brain barrier cells declined. Transcriptional analysis revealed strong antiviral and inflammatory responses, and impairment of physiological brain activities. This study highlights the complex immune response in virus-infected brains, balancing infection elimination with immunopathology.

overall_design: We allowed the infection of HSV-1 to progress for days 4, 6, and 8. Brainstem of HSV1 infected mice and control mice were collected for scRNA sequencing


title: Antiretroviral Therapy at Conception Leads to Lower Peripheral CD49a+ NK Cells and Higher SERPINB2.

city: Chicago

first: Jinhee

last: Kim

state: IL

address: 676 North St. Clair Street Arkes Suite 2330

department: Infectious Disease

institute: Northwestern University

summary: Antiretroviral therapy (ART) during pregnancy is essential to prevent vertical HIV transmission and preserve the health of the mother and child. However, ART in pregnancy has been associated with adverse birth outcomes linked to poor placental development. Immune dysregulation of placental development is an important factor in the development of pre-eclampsia (PE), a common hypertension disorder of pregnancy. Some studies found an association between ART use at conception or during the first trimester and PE. To investigate the immune environment in pregnant persons with HIV (PPWH) on ART at conception (N=40) compared to PPWH that started ART in the 2nd trimester (N=40) we analyzed data from the International Maternal Pediatric Adolescent AIDS Clinical Trials (IMPAACT) Perinatal Core Protocol, P1025, concluded in 2013.  No difference was found in soluble factors in circulation including PlGF and sFLt-1, associated with PE. However, upon analysis of PBMC by high dimension flow cytometry, we detected a lower frequency of circulating CD49a+ NK cells, associated with uterine tissue and pregnancy, in PPWH on ART at conception compared with PPWH who started ART in the 2nd trimester. Moreover, PBMC from PPWH on ART at conception expressed higher levels of SERPINB2 in transcriptomics analyses.  Our findings shed new insights into the potential impact of ART at conception and suggest the persistence of a dysregulated inflammatory environment compared to PPWH starting ART after the conclusion of placental development.

overall_design:
Total RNA of PBMC was extracted from a subset of 20 PPWH per group (chosen based on sample availability and to maintain matched characteristics) using the RNAeasy Kit (Qiagen) following manufacturer recommendations. Due to the low RNA yield, a low-input RNA-seq library preparation was performed, and samples were sequenced at the NUSeq Core using 3 lanes of the Illumina HiSeq 4000 (50bp, single-read) yielding >22M reads per sample. Data were analyzed using the Partek Flow software.
40 Pregnant Persons with HIV (PPWH), PBMC, 2 Treatment Groups (On cART at conception vs Initiated cART at  ≥2nd trimester)

first: Yanling

last: Huo

first: Deborah

last: Kacanek

first: Sadia

last: Samer

first: Elizabeth

middle: G

last: Livingston

last: Machado

first: Elena

last: Martinelli


title: Inactivation of Atp7b copper transporter in the intestinal epithelial cells is associated with altered lipid processing and cell growth machinery that are independent from hepatic copper accumulation and liver histology severity

city: Davis

first: Gaurav

last: Sarode

state: CA

address: 6404A GBSF, 451 E. Health Sciences Dr.

department: Internal Medicine

institute: University of California, Davis

summary: The clinical manifestations of Wilson disease (WD) are related to copper accumulation in the liver and brain, but little is known about the role of other organs expressing the ATP7B copper transporter on the metabolic changes characterizing WD. To examine the metabolic and morphological consequences of intestine Atp7b inactivation in absence of hepatic copper accumulation, we generated a new mouse strain, the Atp7bΔIEC mouse and characterized its phenotype over a time-course study. Atp7bΔIEC mice were generated using B6.Cg-Tg(Vil1-cre)997Gum/J mice from the Jackson Laboratory and Atp7bLox/Lox mice. Cre-mediated removal of a 1.6-kb fragment in exon 2 results in enterocyte-specific Atp7b inactivation. Atp7bΔIEC mice were compared to wildtype mice with same genetic background (iWT). The Atp7b null global knockout (Atp7b -/-) on a C57Bl/6 background was previously generated and compared to respective WT. Mice were studied at 9, 16, 24, and 30 weeks of age. Liver and intestine histology, lipid metabolism parameters, and hepatic copper quantification were assessed. Transcriptome analysis in Atp7bΔIEC mice revealed changes in genes involved in AMPK signaling, fatty acid metabolism and cell cycle both in the intestinal epithelial cells and in the liver. No changes in hepatic and enterocyte copper levels were seen in Atp7bΔIEC livers, whereas, as expected, hepatic copper was 20-40 times higher in Atp7b -/- mice compared to WT mice. Intestine-specific Atp7b deficit affects systemic metabolic pathways independently from hepatic copper accumulation indicating evidence that WD phenotype is affected by organ-specific ATP7B variants.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for global ATP7B and  Intestine-specific ATP7B knock-out mice model with their respective control mice in intestinal epithelial cells and liver

first: Valentina

last: Medici


title: Gut microbiome derived N,N,N-trimethyl-5-aminovaleric acid (TMAVA) is a modulator of CNS-aGVHD

city: Freiburg

first: Geoffroy

last: Andrieux

address: Breisacherstr 153

country: Germany

institute: University clinics Freiburg

summary: Acute graft-vs-host disease (aGVHD) can affect the central nervous system (CNS) through activation of microglia and infiltration of T cells. However, it is unclear how microbiome modifications impact CNS-aGVHD. Here, we analyzed the role of the microbiome in facilitating microglia activation during aGVHD using antibiotics-treated, germ-free (GF) and wildling mice. Antibiotics-mediated microbiome depletion caused infiltration of IFN-γ producing T cells in the brain, activation of microglia via the TLR4/p38MAPK pathway and deficits in neurocognitive functions in aGVHD mice. We observed similar microbiome-dependent microglia activation after allo-HCT in microbiome-disrupted wildling mice, and in GF mice compared to Oligo-Mouse-Microbiota (OMM12) mice. The bacteria-derived metabolite N,N,N-trimethyl-5-aminovalerate (TMAVA) was decreased in microglia of antibiotics-treated mice. TMAVA administration diminished the activity of TLR4/p38MAPK pathway in microglia, and reversed T cell infiltration in the brain. We also found reduced TMAVA abundance in the blood of patients after GVHD onset compared to before onset.   In summary, we identified the loss of bacteria-derived TMAVA upon antibiotics treatment as a central causative factor for CNS-aGVHD.

overall_design: 2 biological replicates each of ABX-treated allo-HCT mice and vehicle-treated allo-HCT mice are analysed.

first: Sangya

last: Chatterjee

first: Robert

last: Zeiser

first: Melanie

last: Boerries


title: The histone modifier KDM5 functions as an insulator [CUT&RUN]

first: Matanel

last: Yheskel

overall_design: CUT & RUN for H3K4me3 in KDM5 WT, JmjC, and L854F brains

first: Melissa

last: Castiglione

first: Richard

last: Kelly

first: Julie

last: Secombe


title: DNA methylation affects gene expression but not chromatin structure in Escherichia coli

city: Ann Arbor

first: Lydia

last: Freddolino

state: Michigan

address: 1150 W. Medical Center Dr., Room 3315 MSRB III

institute: University of Michigan

summary: The activity of DNA adenine methyltransferase (Dam) and DNA cytosine methyltransferase (Dcm) together account for nearly all methylated nucleotides in the Escherichia coli K-12 MG1655 genome. Previous studies have shown that perturbation of DNA methylation alters E. coli global gene expression, but it is unclear whether the methylation state of Dam or Dcm target sites regulates local transcription. We observed an underrepresentation of Dam sites in transcriptionally silent extended protein occupancy domains (EPODs), and we thus hypothesized that a methylation-deficient version of MG1655 would show large-scale aberrations in chromatin structure. To test our hypothesis, we cloned methyltransferase deletion strains and performed global protein occupancy profiling using high resolution in vivo protein occupancy display (IPOD-HR), chromatin immunoprecipitation for RNA polymerase (RNAP-ChIP), and transcriptome abundance profiling using RNA-Seq. Our results indicate that loss of DNA methylation does not result in large-scale changes in genomic protein occupancy such as the formation of EPODs. However, loci with dense clustering of Dam methylation sites show methylation-dependent changes in local RNA polymerase and total protein occupancy, but local transcription is unaffected. Our transcriptome profiling data indicates that deletion of dam and/or dcm results in significant expression changes within some functional gene categories including SOS response, flagellar synthesis, and translation, but these expression changes appear to result from indirect regulatory consequences of methyltransferase deletion. In agreement with the downregulation of genes involved in flagellar synthesis, dam deletion is characterized by a swimming motility-deficient phenotype. We thus conclude that DNA methylation does not control the overall protein occupancy landscape of the E. coli genome, and that observable changes in gene regulation are generally not resulting from regulatory consequences of local methylation state.

overall_design: Three biological replicates each of wild-type, Δdam, Δdcm, and ΔdamΔdcm E. coli K12 MG1655 were grown in MOPS Rich Defined Media (RDM) with glucose to an OD600 of .2, at which point an aliquot was taken for RNA-seq processing, and the main culture was then treated with rifampicin, crosslinked with formaldehyde, and then harvested. Each biological replicate was then split to perform IPOD interphase extraction, ChIP for RNA polymerase, and input processing.

first: Willow

last: Morgan


title: Determining the transcriptional regulation of CDK4/6 inhibitor resistance in estrogen receptor-positive breast cancer

city: Boston

first: Renyan

last: Liu

state: MA

address: 450 Brookline Avenue

institute: Dana-Farber Cancer Institute

summary: CDK4/6 inhibitor (CDK4/6i) resistance is a pressing clinical problem for patients with ER+ breast cancer.  Previous work identified CDK6 overexpression as one critical determinant of acquired resistance to CDK4/6 inhibition. This dataset interrogates CDK6 interactors/recruiters to determine their role in transcriptional regulation of CDK4/6i resistance mechanisms and will provide novel therapeutic strategies to overcome resistance.

overall_design: RNA was extracted from cultured cells treated with vehicle (Palcociclib-Resitant 100nM) or the addition of JQ1 (300nM - 24hr) using the RNeasy Mini Kit (Qiagen). Prior to RNA sequencing, RNA was treated with DNase using RNase-Free DNase Set (Qiagen). rRNA depletion was performed from 100ng of purified RNA using QIAseq FastSelect rRNA HMR reagents according to manufacturer’s protocol. Libraries were prepared using Roche Kapa Biosystems RNA HyperPrep sample preparation reagents on a Beckman Coulter Biomek i7. Finished dsDNA libraries were quantified by Qubit fluorometer and Agilent TapeStation 4200. Uniquely dual indexed libraries were pooled in an equimolar ratio and shallowly sequenced on an Illumina MiSeq to further evaluate library quality and pool balance. The final pool was sequenced with paired-end 150bp reads on an Illumina NovaSeq 6000at the Dana-Farber Cancer Institute Molecular Biology Core Facilities. Sequenced reads were aligned to the UCSC hg38 reference genome assembly and gene counts were quantified using STAR (v2.7.3a).

first: Geoffrey

last: Shapiro


title: RNA-seq of vertebral metastatic tumor samples from pan-cancer primary tumors

first: Zikun

last: Liao

address: 63 Duobao Road, Liwan District

institute: The Third Affiliated Hospital of Guangzhou Medical University

summary: To define the molecular underpinning of bone metastasis in pan-cancer, we performed high-throughput transcriptome sequencing (RNA-seq) using total RNA from veterbral metatases of LUAD, BRCA, PRAD, SCRA, LIHC, SKCM, THYM, etc. tissues. Afterwards, expression profile were used for further analysis.

overall_design:
Total RNA was isolated using MJzol animal RNA Extraction Kit (MagBeads) #T02096  (MJzol, China). Paired-end libraries were synthesized by using the TruSeq® RNA Sample Preparation Kit (Illumina, USA) following TruSeq® RNA Sample Preparation Guide. Briefly, The poly-A containing mRNA molecules were purified using poly-T oligo-attached magnetic beads. Following purification, the mRNA is fragmented into small pieces using divalent cations under 94℃ for 8 min. The cleaved RNA fragments are copied into first strand cDNA using reverse transcriptase and random primers. This is followed by second strand cDNA synthesis using DNA Polymerase I and RNase H. These cDNA fragments then go through an end repair process, the addition of a single ‘A’ base, and then ligation of the adapters. The products are then purified and enriched with PCR to create the final cDNA library. Purified libraries were quantified by Qubit® 2.0 Fluorometer (Life Technologies, USA) and validated by Agilent 2100 bioanalyzer (Agilent Technologies, USA) to confirm the insert size and calculate the mole concentration. Cluster was generated by cBot with the library diluted to 10 pM and then were sequenced on the Illumina Noveseq 6000 (Illumina, USA). The library construction and sequencing was performed at Shanghai Biotechnology Corporation.
Sequencing raw reads were preprocessed by filtering out rRNA reads, sequencing adapters, short-fragment reads and other low-quality reads. We used Hisat2（version:2.0.4）to map the cleaned reads to the GRCh38 reference genome with two mismatches. After genome mapping, Stringtie（version:1.3.0）  was run with a reference annotation to generate FPKM values for known gene models. Differentially expressed genes were identified using Stringtie]. The p-value significance threshold in multiple tests was set by the false discovery rate (FDR) . The fold-changes were also estimated according to the FPKM in each sample.The differentially expressed genes were selected using the following filter criteria: FDR ≤0.05 and fold-change ≥2.

first: Wei

last: Zhou


title: Effect of depletion of FAM129B on gene expression during skeletal muscle differentiation of C2C12 cells

first: Yushen

last: Lu

state: Hubei

address: Donghu Road 115

summary: Muscle atrophy, a decline in muscle mass and quantity caused by a variety of factors such as cancer, aging, and long-term use of glucocorticoids, manifested at the molecular level as excessive protein catabolism. Muscle atrophy severely affects the quality of life and the treatment remains an unresolved challenge. FAM129B is an antioxidant protein that plays a key role in signal transduction.FAM129B has previously been reported to be involved in muscle growth and development in cattle. The current study aims to describe the role of FAM129B in muscle atrophy, which is expected to be a potential therapeutic target for skeletal muscle atrophy.

overall_design: To investigate the function of FAM129B in the regulation of skeletal muscle atrophy, we established C2C12 cells in which FAM129B has been knocked down by siRNA

first: Wang

last: Yan

first: Jinhui

last: Hou


title: RNA-seq of vertebral metastatic tumor samples from pan-cancer primary tumors II


title: The histone modifier KDM5 functions as an insulator [Hi-C]

overall_design: HiC in KDM5 WT, JmjC, and L854F brains


title: Chil3+ immature neutrophils inhibit anti-tumor immunity and impede immune checkpoint blockade therapy in bone metastases [RNA-Seq]

city: 南京

first: shiji

last: ren

address: 南京鼓楼

institute: Nanjing University

summary: Bone metastases remain incurable and respond poorly to immune checkpoint blockade (ICB) therapy. The impact of neutrophil maturation in cancer remains largely unknown, especially in refractory tumors including bone metastases. Here, we observed the predominance of immature neutrophils in the bone metastasis microenvironment of both mouse models and cancer patients. DKK1 induces the immature-like functional state in neutrophils, which exert robust immunosuppressive capabilities to inhibit anti-tumor response of CD8+ T cells. Mechanistically, driven by the DKK1-CKAP4-STAT6 signaling, Chil3 expression is identified and required for the immune suppression mediated by immature neutrophils in bone metastases. Moreover, using multiple syngeneic and human immune system (HIS) mouse models, we found that DKK1 blockade efficiently allows neutrophil maturation to improve the immune microenvironment, controls tumor progression, and enhances ICB therapy response in bone metastases. Thus, our findings uncover an essential role for immature neutrophils in cancer, especially in bone metastases, and propose a potential strategy modulating neutrophils to improve cancer immunotherapy.

overall_design: Bulk RNA-seq analysis of CD101+Ly6G+mature neutrophils (sorted from mouse bone marrow) cultured with or without DKK1 ex vivo was performed.

first: Tao

last: Shi

first: Yuting

last: Luo

first: Kaijie

last: Liang

first: Shiji

last: Ren

first: Taha

last: Merghoub

first: Yan

last: Li

first: Jia

last: Wei


title: Suppression of the mRNA export pathway dampens viral mimicry response and promotes tumor immune evasion [RIP-Seq]

city: tianjin

first: junying

last: ma

address: Heping District Meteorological Observatory Road

institute: Tianjin Medical University

summary: Transcriptional induction of retroelement transcripts through therapeutic interventions elevates cytosolic double-stranded RNA (dsRNA) levels to trigger viral mimicry response and anti-tumor immunity. Therapeutic interventions that transcriptionally induce retroelement transcripts lead to elevated levels of cytosolic double-stranded RNA (dsRNA), thereby triggering a viral mimicry response and enhancing anti-tumor immunity. Despite the promising implications, clinical studies have revealed substantial variations in the therapeutic induction of viral mimicry response in different cancer patients, with the underlying mechanisms remaining largely elusive. In this study, we illuminate the pivotal role of the mRNA export pathway in viral mimicry response and anti-tumor T cell immunity. Notably, epigenetic and posttranscriptional induction of cytosolic dsRNAs necessitates the mRNA export pathway to transport the nuclear-transcribed retroelement transcripts into the cytoplasm, which is posttranscriptionally suppressed by the RNA exosome in coactivator-associated arginine methyltransferase 1 (CARM1)-expressing tumor cells. Mechanistically, Carm1 restricts the nuclear export of retroelement transcripts through transcriptional elevation and posttranslational stabilization of the RNA exosome, which hydrolyzes the critical nuclear RNA export adaptor transcripts Ddx39a and Alyref. Collectively, our study underscores the vital importance of the mRNA export pathway in viral mimicry response and suggests that enhancing its activity through Carm1 or RNA exosome inhibition could reinforce therapy-induced viral mimicry response, thus holding the potential to overcome tumor immune escape and immunotherapy resistance.

overall_design: We conducted RIP-seq on parental and Carm1 knockout B16-F10 cells.

first: Shan

last: Huang

first: Jinhua

first: Xinyue

last: Cheng


title: CD8(+) T cells cell-in-cell mediates colorectal cancer progression via P38 activation

city: Zhanjiang

first: wei

last: yisheng

address: No. 57 South Renmin Avenue, Xiashan District

institute: The Affiliated Hospital of Guangdong Medical University

summary: Cell-in-cell(CIC) refers to the active internalization of one or more living cells into another. However, the biological significance and clinical implications of CIC between immune cells and cancer remain unclear. Here, we showed CIC by immunohistochemistry, real-time imaging and multiplex tissue immunofluorescence staining. CD8(+) T cell CIC counts were correlated with lymph node metastasis,T stage, and differentiation in stage I-III CRC. We found CIC counts >4.25 per high-power (magnification 400×) field positively affected metastasis-free survival in stage I-III CRC. Functional experiments demonstrated CD8(+) T CICs increased the migration and invasion in vitro and in vivo. Notably, CD8(+) T CICs were accompanied by greater activation of p38. In addition, blocking p38 using SB203580 significantly suppressed the migration and invasion of CD8(+) T CICs in vitro and in vivo. Moreover, phosphorylated-p38 overexpressing CD8(+) T CICs showed increased expression of cytokeratin 13 (KRT13); however, CD8(+) T CICs pretreated with SB203580 exhibited a significant suppression of KRT13. In summary, Our findings demonstrate that CD8(+) T CIC could be one of the mechanisms of CRC progression. P38 contributes to CD8(+) T “in-cell progression,” and KRT13 may participate in P38- induced “in-cell progression.”

overall_design: HCT8 cell lines stained with 2μM Green (Invitrogen) were incubated with a 2:1 ratio of live PBMCs stained with 1μM Cell-Tracker Red (Invitrogen) at 37˚C and 5% (v/v) CO2 for 8 hours. Subsequently, double-positive cells, representing HCT8 cells that had engulfed PBMCs, were sorted using flow cytometry and designated as CIC cells. The CIC cells were then treated with 10 μM SB203580 for 24 hours, resulting in the SB_CIC cells.

first: Yisheng

first: Xikai

first: Fang

last: Su

first: Yufei

last: Zhao


title: SWR1 is recruited to activated ABA response genes to maintain gene body H2A.Z in Arabidopsis thaliana [RNA-Seq]

city: Atlanta

first: Roger

last: Deal

state: Georgia

address: 1510 Clifton Road NE, Room 2006

department: Biology

institute: Emory University

summary: The histone variant H2A.Z is important for transcriptional regulation across eukaryotes, where it can alternately promote or repress transcription. Actively transcribed genes show H2A.Z enrichment in nucleosomes immediately downstream of the transcription start site (TSS), while silent genes show H2A.Z enrichment across the gene body. In plants, previous work shows that silent genes responsive to temperature and light lose gene body H2A.Z upon activation, but whether H2A.Z loss is generally required for transcription is not clear. We profiled H2A.Z and components of its deposition complex, SWR1, before and after treating Arabidopsis thaliana with the hormone abscisic acid (ABA). Our results show that transcribed genes with TSS-enriched H2A.Z have high SWR1 binding, indicating continuous replacement of H2A.Z, while silent genes with gene body H2A.Z show lower SWR1 binding. Surprisingly, upon ABA treatment, thousands of previously silent genes activate, correlating with recruitment of SWR1 and retention of gene body H2A.Z enrichment. We also found that the SWR1-interacting protein MBD9 is not required for SWR1 recruitment to activated genes. These results provide new insights into the relationship between H2A.Z and transcription and the mechanics of H2A.Z targeting to chromatin.

overall_design: To understand how WT and mbd9-1 plants respond transcriptionally to an Abscisic Acid (ABA) stress response, we performed RNA-Seq on total RNA from whole 7-day-old WT and mbd9-1 seedlings  after 4-hour of 10 uM ABA treatment or Mock treatment (DMSO in water).

first: Ellen

last: Krall


title: SWR1 is recruited to activated ABA response genes to maintain gene body H2A.Z in Arabidopsis thaliana [ChIP-Seq]

overall_design: To understand the distrubution of H2A.Z, PIE1 and MBD9 during an Abscisic Acid (ABA) stress response, we performed ChIP-Seq against all of these proteins in 7-day-old WT plants in untreated conditions and after 4-hour 10 uM ABA treatment or Mock treatment (DMSO in water). Similarly, ChIP against H2A.Z, MBD9 and PIE1 performed in untreated and 10 uM ABA treated 7-day-old plants exclusively expresssing a mutant MBD9 (D1209A) and ChIP against H2A.Z in untreated plants with no MBD9 (mbd9-1) and against H2A.Z and PIE1 in 4-hour 10 uM ABA or Mock treatment (DMSO in water) mbd9-1 plants also performed.


title: The effect of deletion of muscarinic acetylcholine receptor 4 in Math1 expressing cells in the small intestine

city: St. Louis

first: Rodney

middle: D

last: Newberry

address: 660 S. Euclid Ave

department: Medicine

institute: Washington University School of Medicine

summary: The muscarinic acetylcholine receptor 4 was inducibly deleted in Math1 expressing lineages and alterations in gene expression evaluated by bulk RNA sequencing

overall_design: Math1Cre*PR mAChR4fl/fl mice and mAChR4fl/fl littermates were gavaged with RU486 10mg/kg) daily for 4 days and gene expression on whole small intestine was evaluated.

first: Devesha

last: Kulkarni


title: The Maternal Gut-Immune Axis Programs the Neuroimmune Landscape of the Developing Brain

first: Brian

last: Kalish

address: 220 Longwood Ave

department: Neurobiology

institute: Boston Children's Hospital

summary: Immune molecules, including cytokines, chemokines, and their cognate receptors, are developmentally regulated in the brain and dynamically expressed in the context of neurodevelopmental and neuropsychiatric disease. Here, we employed highly multiplexed in situ spatial transcriptomics to map the expression of major immune ligands and receptors in the developing mouse brain during mid and late gestation. Given the importance of the maternal environment in shaping fetal neurodevelopment, we also determined how the embryonic neuroimmune landscape was altered after maternal immune and microbiome perturbations, with notable sex-specific patterns. We discovered significant changes in the expression of the CXCL12/CXCR7 chemokine network after maternal immune activation and maternal microbiome depletion, suggesting an underlying mechanism for corticogenesis abnormalities. This resource underscores how the maternal environment programs the precise regulation of immune molecules in the developing brain, highlighting sex-specific disease vulnerability.

overall_design: We performed multiplexed error-robust fluorescence in situ hybridization (MERFISH) on n=3 male embryonic day 14.5 (E14.5) and n=3 male E18.5 mouse brains. The MERFISH was also performed on two conditions 1) E14.5 embryonic brain of mice from maternal immune activated mothers, and 2) E14.5 embryonic brain of mice from maternal microbiome depleted mothers. For both conditions embryonic brains were collected from males and females with n=3, and respectively PBS controls were also studied. Coronal sections of the entire brain were used for analysis.

first: Bharti

last: Kukreja

first: Shin

last: Jeon

first: Wuxinhao

last: Cao

first: Bianca

last: Rusu

first: Camille

middle: F

last: Harrison

first: Nareh

last: Tahmassian

first: Shiyao

last: Ning

first: Jing

last: Wang

first: Min

middle: Y

last: Feng

first: Yeong

last: Yim

middle: T


title: Single-Cell Sequencing Analysis of KLF5-Induced Multilineage Reprogramming in EpiSCs

city: Shen Zhen

first: Xinyu

last: Wu

address: BGI Space-Time Research Center

institute: BGI

summary: During early embryogenesis, Kruuppel-like factor KLF5 is expressed in inner cell  mass (ICM), trophectoderm (TE), and primordial germ cell (PGC). It is wellknow to be essential for specifying the TE fate, as well as redundantly with KLF2/4 for specifying the ICM fate during the first fate segregation. Here, we aimed to clarify whether KLF5 regulates these three fates in vitro. We firstly validated the necessity of KLF5 in mouse trophoblast stem cells (TSCs) and PGC-like cells. Mechanistically, we showed that KLF5 is deeply integrated into the transcriptional networks of ICM, TE and PGC in a context-dependent manner. Furthermore, KLF5 is almost the only factor capable of driving TSC fate from primed epiblast stem cells. It could also induce a transition from the primed to naive state just in the absence of WNT signaling. During this transition, KLF5 generates PGC-like cells, concomitantly. Taken together, our work supports KLF5 as a powerful and versatile specifier in both early embryonic lineage determination and in vitro cell fate reprogramming.

overall_design: To elucidate how KLF5 drives diverse cell fates in epiSCs, we conducted single-cell RNA-seq (scRNA-seq) analysis on EpiSCs and iKlf5-induced PNT from days 1 to 5, and iKlf2,4 at day5.

first: Yinhua

last: Huang


title: Heterochromatin Protein ERH represses alternative cell fates during early mammalian differentiation [CUT&RUN]

city: Seattle

first: Ryan

middle: Lehman

last: McCarthy

state: WA

address: 1900 9th Ave

department: Pediatrics

institute: University of Washington

summary: We investigate the role of ERH in the regulation of H3K9me3 domains which impede transcription factor binding and gene activation in mouse preimplantation, human ESC differentiation, and human fibroblast reprogramming.

overall_design: Cut and Run (CUT&RUN) for histone modification H3K9me3 in human BJ fibroblasts treated with either non-targeting siRNAs or ERH targeting siRNAs. Cut and Run (CUT&RUN) for transcription factor SOX2 in BJ fibroblast cells treated with either non-targeting siRNAs or ERH targeting siRNAs then reprogrammed with Sendai virus to express OCT4, SOX2, KLF4 and c-MYC; collected 48 hours post infection.

first: Blake

last: Hernandez

first: Andrew

last: Katznelson

first: Holly

last: Fahning

first: Jingchao

last: Zhang

first: Adam

last: Burton

first: Maria-Elena

last: Torres-Padilla

first: Nicholas

last: Plachta

first: Kenneth

last: Zaret

middle: L


title: Implant renal injury-responsive cells to supplement erythropoietin and protect kidney [Spatial Transcriptomics]

city: Shanghai

first: Wei

last: Zuo

state: Shanghai

address: No. 500 Zhennan Road, Putuo District, Shanghai

institute: University

summary: Anemia poses a life-threatening risk to individuals with chronic kidney diseases (CKDs). Insufficient production of erythropoietin (EPO) from injured kidney is the major reason for anemia, while lack of EPO would further aggravate the kidney injury and make a “vicious cycle”. In this study, we dissected the change of endogenous EPO signaling in injured kidney by spatial transcriptomic analysis and validated the dual beneficial effects of local recombinant EPO administration on both anemia correction and kidney protection. Next, we constructed an injury-responsive EPO producing (iREP) element to sense the kidney damage signal and drive the synthesis and secretion of native EPO. After intra-renal implantation of iREP engineered HEK-293T embryonic kidney cells, the mouse kidney would automatically produce more EPO when sensing injury signal, which in turn enhanced red blood cell mass and protected kidney from further injury. Moreover, aided with automated equipment, we cloned urine-derived SOX9+ epithelial cells (USCs) from both healthy donors or CKD patients. The USCs could be transplanted into mouse kidney and form tubule-like structures, which makes them an alternative candidate cell for iREP engineering. Altogether, the current work proposed a potential approach based on engineered “smart” cell to treat CKDs.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: 6.5*6.5mm fresh tissues were concurrently frozen and embedded in optical cutting tissue (OCT) compound in liquid nitrogen and delivered in dry ice for spatial transcriptome sequencing.

first: Hao

last: Nie

first: Chen

last: Liang

first: Zixian

last: Zhao

first: Yingxin

last: He


title: Single-cell RNA sequencing reveals liver immune cell-endothelial cell interactions in mice with carbon tetrachloride-induced liver fibrosis

city: Nanjing

first: xue

last: fan

address: 24 Tong Jia Xiang

institute: China Pharmaceutical University

summary: Liver fibrosis is a common pathological process in chronic liver disease, reflecting the advanced stage of the disease. Liver endothelial cells (ECs), especially liver sinusoidal endothelial cells (LSECs), are recognized as critical modulators of liver homeostasis and play essential roles in the recruitment and function of liver immune cells. However, the mechanism of liver EC injury and the occurrence of intercellular crosstalk in liver fibrosis are still unclear. In this study, C57BL/6 male mice were treated with CCl4 for 6 weeks to establish a liver fibrosis model. Masson staining and immunohistochemistry were performed to assess the extent of liver fibrosis. Liver endothelial injury was detected by using scanning electron microscopy (SEM) and PCR technology. Single-cell RNA sequencing (scRNA-seq) was performed to analyze phenotypic changes in nonparenchymal cells and dissect intercellular crosstalk in liver fibrosis. A total of 24,534 cells were analyzed and clustered into 10 main cell subsets. Liver ECs decreased significantly, and macrophage recruitment increased in the CCl4 group. The LSEC fenestrae and surface receptors expression were reduced, and the expression of Cd34 was upregulated. Liver ECs exhibited dense cellular crosstalk with immune cells such as macrophages, T cells, and B cells. The analysis of intercellular signaling pathways revealed that immune cells targeted liver ECs through the Ptprc-Mrc1 and Sell-Podxl signaling pathways to maintain intercellular interactions during liver fibrosis. We described the single-cell landscape of a liver fibrosis mouse model and demonstrated the presence of tight intracellular crosstalk between ECs with macrophages, T, and B cells, revealing cell-cell communications among liver immune cells-ECs during the process of liver fibrosis. The Ptprc-Mrc1 and Sell-Podxl signaling pathways exerted prominent roles in the interaction between immune cells and liver ECs.

overall_design: There are two groups in our research. Single cells were dissociated froma normal mouse was served as control (Control, n=1) and  a fibrotic liver of  carbon tetrachloride-induced mouse( CCl4, n=1) .

first: Xue

last: Fan

first: Qinwei

last: Yu

first: Zhenzhou

last: Jiang


title: Gene expression profile at single cell level of AS-like Macaca fascicularis bone marrow mononuclear cell

city: GuangZhou

first: bian

last: zhenhua

state: GuangDong

address: Panyu University Town

institute: South China University of Technology

summary: Ankylosing Spondylitis (AS) like and control macaca fascicularis bone marrow mononuclear cell, mainly immune cells.

overall_design: These cells were isolated from wild-type and AS-like macaca fascicularis bone marrow by using ficoll, and performed scRNA sequencing with 10x genomics to analyse the immune microenvironment of control and disease bone marrow.

first: TangMing

last: Guan

first: ZhenHua

last: Bian

first: PanYue

last: Luo


title: FLT3 is genetically essential for ITD-mutated leukemic stem cells but dispensable for human HSCs

city: Toronto

first: Veronique

last: Voisin

state: ON

address: 101 College St, room 8-353

country: Canada

institute: University Health Network

summary: Leukemic stem cells (LSCs) fuel relapse in acute myeloid leukemia (AML), but therapies tailored at eradicating LSCs without harming healthy hematopoietic stem cells (HSCs) are lacking. FLT3 is frequently mutated in AML and associated with relapse; but FLT3 targeting has met with limited clinical success. This raises questions of whether more potent inhibitors would increase  effectiveness but whether toxicity to HSC would become limiting. Here, we tested the consequence of complete FLT3 ablation using CRISPR/Cas9 FLT3 knock-out (FLT3-KO) in human HSCs and LSCs followed by functional xenograft assays to test their ability to regenerate human hematopoiesis and leukemia, respectively.  FLT3-KO in HSCs from human fetal liver, cord blood and adult bone marrow showed no impairment in multilineage hematopoiesis in primary and secondary xenografts. By contrast, FLT3-KO LSCs from 6 of 7  FLT3-ITD mutated AMLs were able to generate short-term engraftment but were completely exhausted by 12 weeks. Thus, FLT3 is essential for LSC long-term propagation. This dependency was unique to FLT3-ITD AML samples as non-FLT3-ITD AML samples generated leukemic grafts upon FLT3-KO. Transcriptomic analysis revealed that FLT3-KO induced downregulation of DNA repair and cell cycle checkpoints, uniquely in FLT3-ITD AML, but not in healthy HSCs or other AMLs. Our research highlights a critical distinction between healthy HSCs and LSCs: whereas healthy hematopoiesis proceeds unperturbed upon FLT3-KO, FLT3-ITD leukemogenesis is impaired through elimination of LSCs. This evidence underscores the necessity for more potent FLT3-targeting and places FLT3 as an ideal therapeutic target to selectively eradicate LSCs, while sparing HSC.

overall_design:
We used three primary patient samples of acute myeloid leukemia (AML) with the internal tandem duplication (ITD) in FLT3 – samples ITD 1, ITD 2 and ITD 3; three primary patient samples of AML without the ITD mutation – samples WT 1, WT 2 and WT 3; and three samples of human umbilical cord blood (CB). AML samples were thawed, and the fraction previously determined (PMID: 27926740) to be enriched in leukemic stem cell activity was sorted: CD34+ population was sorted from all samples, except from WT 3 where the CD45+ CD3- population was sorted. CB samples were thawed and the CD34+CD38- population was sorted. AML samples were cultured for 24-h, while CB cells were cultured for 48-h. Subsequently, FLT3-KO and OR2W5-KO were performed using CRISPR/Cas9 technology through electroporation. After electroporation, cells were cultured for 3 additional days to achieve the maximal reduction on FLT3 expression after FLT3-KO in culture. Subsequently, cells were collected and viably sorted using SYTOX Blue Dead Cell Stain (Thermo Fisher Scientific). A minimum number of 13 x103 live cells was sorted per sample (range 13 to 800 x103). One fifth of the sample was collected for total DNA isolation and subsequent determination of KO percentage mediated by CRISPR/Cas9. The rest of the sample was used for total RNA purification and DNase treatment using the RNeasy Micro kit (Qiagen). RNA integrity was measured using the RNA 6000 Pico kit (Agilent) and the average RNA integrity scores were the following: 8 for AML-ITD samples (6.9-10), 10 for AML-WT samples (9.7-10) and 9.95 for CB samples (9.8-10). To generate RNAseq libraries, total RNA was used as input with SMART-Seq V4 Ultra Low Input RNA kit (Clontech). The cDNA profiles were confirmed on the bioanalyzer using the High Sensitivity 14 DNA kit (Agilent) and next-generation sequencing libraries were made using the Nextera XT DNA Library Preparation kit (Illumina). Sequencing was performed on the NovaSeq 6000 system using 2 x 150 bp paired-ended sequencing to obtain ~50 M reads per sample.
>>>Submitter states that raw data will be submitted to EGA<<<

first: Joana

last: Araújo

first: Elvin

last: Wagenblast

first: Jessica

last: McLeod

first: Olga

middle: I

last: Gan

first: Liqing

last: Jin

first: Amanda

last: Mitchell

first: Blaise

last: Graton

first: Sarah

last: Cutting

first: Andrea

last: Arruda

first: Monica

last: Doedens

first: Jose-Mario

last: Capo-Chichi

first: Mark

last: Minden

first: Manuel

middle: A

last: Sobrinho-Simões

first: Perpétua

last: Pinto-do-Ó

first: Eric

last: Lechman

first: John

middle: E

last: Dick


title: Heterochromatin Protein ERH represses alternative cell fates during early mammalian differentiation [RNA-Seq]

first: Ryan

last: McCarthy

overall_design: To investigate the role of ERH in gene repression we performed RNA sequencing (RNA-seq). Mouse blastocysts injected with either non-targeting siRNAs or ERH targeting siRNAs at the 1 cell stage. H1 human embryonic stem cells treated with either non-targeting siRNAs or ERH targeting siRNAs then cultured in pluripotency conditions, 1 day of endoderm differentiation, and 2 days of ectoderm differentiation. Human BJ fibroblasts treated with either non-targeting siRNAs or ERH targeting siRNAs then reprogrammed with Sendai virus to express OCT4, SOX2, KLF4 and c-MYC; collected 48 hours post infection.

first: Blake

last: Hernandez

first: Andrew

last: Katznelson

first: Holly

last: Fahning

first: Jingchao

last: Zhang

first: Adam

last: Burton

first: Maria-Elena

last: Torres-Padilla

first: Nicholas

last: Plachta

first: Kenneth

last: Zaret


title: Anti-Fibrotic Effects Of A Novel Long-Acting Fgf21 Analogue Zalfermin

city: Oxford

first: Maria

last: Dermit

state: Oxfordshire

institute: Novo nordisk research centre oxford

summary: The potential clinical benefits of FGF21 analogues for the treatment of MASH has evolved significantly within the last years. However, despite clinical meaningful effect on MASH resolution and fibrosis the body weight loss in humans have been less impressive. Thus, a combination of an FGF21 analogue with a GLP-1 receptor agonist may add additional benefit to the treatment of MASH due to complimentary mode of action compared to stand alone therapies

overall_design: RNA-sequencing data. UUO vehicle (n = 7), UUO zalfermin (n=8) and healthy vehicle (n = 4).

first: Günaj

last: Rakipovski

first: Bidda

middle: C

last: Rolin

first: Jenny

last: Norlin

first: Emma

last: Henriksson

first: Mette

middle: V

last: Østergaard

first: Kristian

last: Ørum-Sass

first: Birgitte

last: Andersen


title: Effect of high muscle and low muscle reserves on longissimus dorsi transcriptome in late gestation dairy cows and the impact of branched chain volatile fatty acid supplmentation.

city: West Lafayette

first: Jacquelyn

last: Boerman

state: Indiana

address: 270 S. Russell Street

department: Animal Sciences

institute: Purdue University

summary: The amount of muscle reserves of dairy cattle in late gestation is related to production performance including calf size and milk yield in the postpartum. Branched-chain volatile fatty acids (BCVFA) supplementation may close energy gaps experienced in late gestation dairy cows, as relative to unsupplmented control it increases blood glucose levels. Our objective was to compare longissimus dorsi muscle transcriptomes and morphology between cows with high muscle (HM) versus low muscle (LM) reserves and in response to BCVFA supplementation. At 42 d before expected calving (BEC) Holstein dairy cows were enrolled in a 2 x 2 factorial study of diet and muscle reserves, by assigning to control (CON) or BCVFA supplemented diets and HM or LM groups based on depth of longissimus dorsi at 42 d BEC. We biopsied the longisumus dorsi at 21 d BEC, isolated total RNA, ribo-depleted samples and sequenced RNA using paired end reads. After trimming, DSeq2 was used to identify differentially expressed genes (DEG). Using a raw p-value cut-off of <0.05, 713 DEG were identified between HM and LM groups and 481 DEG between BCVFA and CON. Transcriptional signatures and morphology indicated differential distribution of Type II fibers between muscle groups, with MYH1 (encods type IIx) greater in LM and MYH2 (encodes type IIA) greater in HM cattle. LM cattle relative to HM showed transcriptome evidence for activation of autophagy, ubiquitin-proteasome, and Ca2+-calpain pathways. HM cattle expressed higher levels of extracellular matrix (ECM) proteins and factors that regulate ECM proteolysis and turnover. Genes that regulate fatty acid degradation and flux of carbons into the TCA cycle as acetyl CoA were increased by BCVFA supplementation. HM and LM cattle have distinct molecular signatures that indicate differences in type II fiber distribution and potentially different metabolic strategies to close energy and amino acid gaps due to metabolic demands in late gestation, whereas BCVFA supplementation increases substrates for energy generation.

overall_design: A 2 by 2 factorial of treatments of multiparous non-lactating Holstein cows assigned to high muscle (HM; >4.6 cm) or low muscle (LM; ≤4.6 cm) at 42 days before expected calving and branched chain volatile fatty acid (BCVFA) or soyhull pellets control (CON) supplementation of diets, resulting in HM-CON (n=13), HM-BCVFA (n=8), LM-CON (n=9), and LM-BCVFA (n=9). Muscle depth assignment was based on depth of longissimus dorsi muscle captured by ultrasound at 42 days before expected calving. BCVFA supplmented cows received isobutyrate, isovalerate and 2-methylbutyrate at 39.1 g/d, 19.6 g/d, and 19.4 g/d dry matter as a calcium salt of isoacid top dressed on the diet. Feed for CON cows was top dressed with 73 g/d DM of soyhull pellets. Cows were housed in tie stalls and released for exercise twice daily. Cows were fed for ad libitum intake by dispersing gestation diets one-time each day at 110% of the previous day’s intake. Biopsy of longissimus dorsi muscle was performed at approximately 0700 h 3 weeks BEC, which was 3 weeks after beginning BCVFA supplmentation and initial assignment to HM and LM groups. Muscle biopsies were flash frozen in liquid nitrogen and sent to Azenta Life sciences for isolation of total RNA and sequencig.

first: Theresa

last: Casey


title: Transcriptomic response of Hermetia illucens L. (Diptera: Stratiomyidae) to wounding and Gram-negative bacterial infection

city: Wageningen

first: Parth

last: Shah

state: Gelderland

address: Droevendaalsesteeg 1

department: Laboratory of Entomology

country: Netherlands

institute: Wageningen University

summary: The larvae of black soldier fly (BSF), Hermetia illucens L., are of economic importance due to their potential as livestock feed. However, the knowledge on their immune response to infection and wounding remains limited. In this study, we uncover the transcriptomic response of BSF larvae to wounding as well as infection with Gram-negative pathogen, Pseudomonas protegens Pf-5 at multiple timepoints. We observed that initially the BSF larvae generated a common immune response to both wounding and infection. However, the immune response is only upregulated in infected larvae over time, while the gene expression in response to wounding reduced over time. Genes encoding for signalling molecules such as PGRP-SA, Relish as well as genes encoding antimicrobial peptides like cecropin, defensin and attacin contributed primarily to BSF larvae's immune responses to both wounding as well as infection.

overall_design: Five-day old BSF larvae were injected with a) PBS (positive-controls), or b) 5000 cells of Pseudomonas protegens Pf-5, and c) untreated BSF larvae were set-up as negative controls. Samples were collected from each treatment at 2 h, 6 h and 13 h post-treatment respectively. Total RNA was extracted and sequenced. Wounding response was quantified by comparing PBS-injected larvae to untreated larvae, and response to infection was quantified by comparing bacteria-injected larvae to PBS-injected larvae at respective timepoints.

middle: N

first: Sevasti

last: Maistrou

first: Ids

last: Willemsen

first: Joop

last: van Loon

first: Marcel

last: Dicke


title: Thermogenetics-based CXCL12 Generator Activates Intraperitoneal Immunity via PerC B cells Against Metastatic Disseminated Tumor Cells

first: Xiaoshen

last: Zhang

address: 1239 siping road

institute: Tongji University

summary: Peritoneal metastasis (PM) is commonly present in advanced gastric cancer patients. While intraperitoneal immunotherapy (IPIT) has been used in clinical investigations to relieve suffering. Here we find that the peritoneal cavity B (PerC B) cell subset plays a key role in exogenous antigen-presenting in intraperitoneal immunity, and presents a unique transcriptional signature including the B-relative regulators. These immune networks are triggered by a novel IPIT approach using a bioengineered alginate-based thermogenetics CXCL12 generator (ATCG).

first: Ling

last: Li

first: Zhiwei

last: Yin

first: Qiang

first: Rui

last: Shi

first: Xin

last: Xia

first: Zhaoxin

last: Wang

first: Shengkai

first: Mao

last: Ye

first: Yanlan

last: Liu

first: Weihong

last: Tan

first: Zhuo

last: Chen


title: Next Generation Sequencing facilitates to identify the differential expression genes of expanded MAIT cells upon the stimulation of 5-OP-RU/MR1 tetramer with/without GCDCA/TCDCA/UDCA

city: Wuhan

first: xiufang

last: weng

address: 13# Hangkong Rd, Wuhan, Hubei

institute: Huazhong University of Science and Technology

summary: We enriched expanded MAIT cells in PBMCs from healthy donor upon the stimulation of 5-OP-RU/MR1 tetramer with/without GCDCA/TCDCA/UDCA through flow sorting  to perform next-generation sequencing (NGS).  Illumina mRNA-seq sample preparation kit was used to perform paired-end library sequencing with illumina  nova seq and sequence analysis was determined using the Illumina data analysis pipeline.

overall_design: Differential gene expression between expanded MAIT cells,expanded MAIT cells with GCDCA,expanded MAIT cells with TCDCA,expanded MAIT cells with UDCA.

first: Xiufang

last: Weng


title: Viruses encode tRNA and anti-retron to evade bacterial immunity

city: Shinjuku-ku

first: Aa

middle: Haeruman

last: Azam

state: Tokyo

address: Toyama 1-23-1

department: Research Center for Therapeutic Drugs and Vaccines Development

country: Japan

institute: The National Institute of Infectious Diseases

summary: Retrons are bacterial genetic retroelements that encode reverse transcriptase capable of producing multicopy single-stranded DNA (msDNA) and function as antiphage defense systems. Phages employ several strategies to counter the host defense systems, but no mechanisms for evading retrons are known. Here, we show that tRNATyr and Rad (retron anti defense) of T5 phage family inhibit the defense activity of retron 78 and a broad range of retrons, respectively. The effector protein of retron 78, ptuAB, specifically degraded tRNATyr leading abortive infection, but phage countervailed this defense by supplying tRNATyr. Rad inhibited retron function by degrading noncoding RNA, the precursor of msDNA. In summary, we demonstrated that viruses encode at least two independent strategies for overcoming bacterial defense systems: anti-defense, such as Rad, and defense canceler, like tRNA.

overall_design: PtuAB from retron Ec83 (Eco4) was cloned under an arabinose inducible promoter pBAD. 0.2% Arabinose and 0.2% Glucose were used to induce and repress the PtuAB expression, respectively.

first: Azam

last: Aa Haeruman

first: Kotaro

last: Chihara

first: Kohei

last: Kondo

first: Tomohiro

last: Nakamura

first: Shinjiro

last: Ojima

first: Azumi

last: Tamura

first: Wakana

last: Yamashita

first: Longzhu

last: Cui

first: Yoshimasa

last: Takahashi

first: Koichi

last: Watashi

first: Kiga

last: Kotaro


title: Effect of rocking culture on keratinocyte differentiation in three-dimensional reconstitution human epidermis

city: Chikusa-ku

first: Hirofumi

last: Teshima

state: Aichi

address: furo-chou

institute: Nagoya university

summary: A three-dimensional culture system of keratinocytes is able to mimic the formation of stratified epidermis. Air-exposure treatment is essential to promote keratinocyte differentiation. During the culture, we compared the transcripts in the cells with (Air) and without (Liq; submerged) air-exposure. Then, since rocking (shaking) the culture dish enabled to recover the keratinocyte differentiation despite the submerged culture (Liq), a comparative study between with and without rocking was also carried out to investigate the characteristics of the restored differentiation by RNA-seq. Transcriptome analyses revealed the expected similar pattern of the transcripts between air-exposure and rocking culture, which differed from those in Liq culture.

overall_design: To investigate the biological significance, RNAs from three different culture systems (air-exposure, the submerged culture without air-exposure, rocking in the submerged cultures) were prepared at the same time. In each culture, RNA were prepared from three independent cultured cells and subjected to RNA-seq analysis. Comparative gene expression profiling analysis of RNA-seq data was carried out for each culture system.

first: Mayuko

last: Endo

first: Kiyotaka

last: Hitomi


title: Effect of knockout of Samsn1 on gene expression during E.coli infection in Raw264.7 cells

city: Chengdu

first: Yao

last: Li

state: Sichuan province

address: No. 37 Guoxue Lane, Wuhou District

department: critical care medicine

institute: West China Hospital of Sichuan University

summary: In sepsis, infection and immunosuppression occur simultaneously. We currently found that Samsn1 expression is increased on myeloid cells in sepsis. And, Samsn1 is currently recognized as a negative regulator of immune response. So we constructed Samsn1 knockout Raw264.7 cells to study the effect of Samsn1 on macrophage function and the gene expression of macrophages with infection.

overall_design:
To investigate the functional effects of Samsn1 on macrophages, we constructed the Samsn1 knockout Raw264.7 cell line by Crispr-cas9 technology
After we had treated the cells with E. coli, RNA-seq of 4 different group of cells were used for gene  expression profiling analysis
Comparative gene expression profiling analysis of RNA-seq between different groups


title: AV8 gene therapy and environmental exposure precipitate cholestatic liver disease in a mouse model of X-linked myotubular myopathy

first: Gaia

last: Andreoletti

address: 686 Bay St

institute: Sick Kids

summary: AAV-associated hepatotoxicity has been observed in patients, with poor concordance between preclinical studies and clinical outcomes. Gene therapy for X-linked myotubular myopathy (XLMTM) has epitomized this therapeutic dilemma, as an AAV8-MTM1 clinical trial has shown efficacy but also fatal liver failure in a subset of individuals. In the present study, we address this conundrum, and demonstrate for the first time in mice that early-life nutrition and AAV exposure can promote liver injury. Specifically, XLMTM mice fed a purified-diet manifest liver dysfunction that mirrors what is seen in patients. AAV8-therapy increases this susceptibility, and additionally promotes liver injury in wild-type mice. Notably, non-viral lipid nanoparticle delivery of MTM1 rescues the XLMTM liver phenotype, offering a potential treatment strategy to mitigate liver consequences in patients. Together, these data provide critical insight into the adverse events seen in viral-gene therapy studies and set the stage for defining their pathomechanisms and identifying potential therapies.

overall_design: In this study, we employed the widely used Mtm1-/- knockout (KO) mouse to investigate the susceptibility of XLMTM mice to cholestasis. Mtm1 KO mice fed a semi-purified ingredient diet (as opposed to Chow) develop liver dysfunction that closely resembles the biochemical and histological features of cholestatic liver disease thus far described in XLMTM patients.

first: Emanuela

last: Pannia

first: James

last: Dowling


title: Effects of WAY267464 or LIT001 on the hair growth ability of dermal papilla cells

city: Kawasaki

first: Tatsuto

last: Kageyama

state: Kanagawa

address: Life Innovation Center, 3-25-22 Tonomachi

institute: Kanagawa Institute of Industrial Science and Technology

summary: We examined effects of WAY267464 or LIT001 on the hair growth ability of dermal papilla cells.

overall_design: We annalysed gene expression of human dermal papilla cells with WAY267464 or LIT001 treatment for 3 days.

first: Junji

last: Fukuda


title: Interleukin-6 Trans Signaling and STAT-3 Activation Promote Neutrophilic Inflammation in Alcohol-Associated Hepatitis

city: Davis

first: Gaurav

last: Sarode

address: 6404A GBSF, 451 E. Health Sciences Dr.

institute: University of California, Davis

summary: Alcohol associated hepatitis (AH) is characterized by neutrophilic inflammation leading to liver injury. Interleukin-6 (IL-6) is a pleiotropic cytokine that regulates hepatic regeneration and hepatic acute phase responses. In the liver IL-6 can signal through either membrane-bound or soluble IL-6 receptors (IL-6R).Aims:to determine the role of IL-6 trans signaling in the pathogenesis of alcohol-associated hepatitis.Methods:Using human liver RNA-Seq, we compared expression of IL-6 signaling pathway components in 51 patients with no liver disease (N=10), early alcohol-related steatohepatitis (early ASH, N=12), non-severe AH (N=11) and severe AH (N=18). Ingenuity Pathway Analysis was used to determine upstream regulators of IL-6 receptor expression. For in vitro studies, HepG2 cells were cultured overnight with either TGF-β1 (5 ng/ml) or 1% FBS, and STAT3 was activated with either 1) IL-6 to simulate classical signaling or 2) hyper-IL-6, a recombinant IL-6/IL-6Rα peptide that selectively activates trans-IL-6 signaling. RNA-Seq was performed on stimulated cells to define an IL-6 trans-signaling gene signature. This signature was used to define a patient subset with high IL-6TS activity (IL-6TS high). For in vivo studies, female mice 14-16 years old were treated with a 10 day chronic-plus binge ethanol liver injury model.Results:Human liver RNA-Seq data demonstrated declining IL-6R expression with progressively increasing severity, and pathway analysis of 3,513 differentially expressed genes identified TGF-β1 as the strongest negative upstream regulator of IL-6R expression in AH. Despite reduced IL-6R expression, STAT3-dependent gene expression was enhanced in severe AH. In vitro, TGF-β1 suppressed STAT3 phosphorylation by both classical and trans IL-6 signaling. However, only high-dose hyper-IL-6 (20 ng/ml) restored STAT3 activation, while TGF-β1-mediated suppression of STAT3 persisted despite high-dose IL-6 (20 ng/ml) treatment. RNA-Seq of hyper-IL-6-stimulated hepatocytes was enriched in genes associated with neutrophil recruitment, and we identified a set of 11 co-clustered genes that characterized a subset of AH patients with enhanced expression of these IL-6TS-inducible transcripts (IL-6TS high). These patients exhibited increased intrahepatic neutrophilic infiltration on liver biopsies and enrichment of pathways associated with leukocyte migration on IPA analysis. In mice, ethanol treatment increased hepatic STAT3 activation despite reduced IL-6R and gp130 expression, and ethanol enhanced expression of neutrophil chemoattractants, includingIcam1,Lbp, andLcn2.Conclusion:Together, these findings suggest that trans IL-6 signaling preserves hepatocyte STAT3-dependent expression of neutrophil chemoattractants in AH. Future studies will examine if inhibiting IL-6 trans signaling abrogates neutrophilic inflammation in AH.

overall_design:
Using human liver RNA-Seq, we compared expression of IL-6 signaling pathway components in 51 patients no liver disease early alcohol-related steatohepatitis (early ASH, N=12), non-severe AH (N=11) and severe AH (N=18). Ingenuity Pathway Analysis was used to determine upstream regulators of IL-6 receptor expression.
For in vitro studies, HepG2 cells were cultured overnight with either TGF-β1 (5 ng/ml) or 1% FBS, and STAT3 was activated with either 1) IL-6 to simulate classical signaling or 2) hyper-IL-6, a recombinant IL-6/IL-6Ra peptide that selectively activates trans-IL-6 signaling. RNA-Seq was performed on stimulated cells to define an IL-6 trans-signaling gene signature.  This signature was then used to define a subset of patients with high IL-6TS activity (IL-6TS high).
For in vivo studies, female mice 14-16 years old were treated with a 10 day chronic-plus binge ethanol model of alcohol-related liver injury, and liver tissues was assessed with Western Blot and qPCR.

first: Vikrant

last: Rachakonda


title: Anxiety-like behavior and altered hippocampal activity in a transgenic mouse model of Fabry disease

city: Innsbruck

first: Kai

last: Kummer

address: Schoepfstrasse 41/Eg

department: Institute of Physiology

country: Austria

institute: Medical University of Innsbruck

summary: Fabry disease (FD) patients are known to be at high risk of developing neuropsychiatric symptoms such as anxiety, depression and cognitive deficits. Despite this, they are underdiagnosed and inadequately treated. It is unknown whether these symptoms arise from pathological glycosphingolipid deposits or from cerebrovascular abnormalities affecting neuronal functions in the central nervous system. We therefore aimed to fill this knowledge gap by exploring a transgenic FD mouse model with a combination of behavior, transcriptomic, functional and morphological assessments, with a particular focus on the hippocampus. Male FD mice exhibited increased anxiety-like behavior in the open field test, accompanied by a reduced exploratory drive in the Barnes maze, which could be related to the increased deposition of globotriaosylceramide (Gb3) identified in the dentate gyrus (DG). Hippocampus single-cell sequencing further revealed that Gb3 accumulation was associated with differential gene expression in neuronal and non-neuronal cell populations with granule, excitatory and interneurons, as well as microglia and endothelial cells as the main clusters with the most dysregulated genes. Particularly FD hippocampal neurons showed decreased electrical baseline activity in the DG and increased activity in the CA3 region of acutely dissected hippocampal slices. Our study highlights transcriptional and functional alterations in non-neuronal and neuronal cell clusters in the hippocampus of FD mice, which are suggested to be causally related to anxiety-like behavior developing as a consequence of FD pathology in mouse models of the disease and in patients.

overall_design: Single-cell suspensions were isolated from hippocampal tissue collected from middle-aged (57 weeks) GlaKO mice and wt littermate controls for single cell RNA sequencing.

first: Theodora

last: Kalpachidou

first: Michaela

last: Kress


title: Suppression of the mRNA export pathway dampens viral mimicry response and promotes tumor immune evasion [ChIP-seq]

city: tianjin

first: junying

last: ma

address: Heping District Meteorological Observatory Road

institute: Tianjin Medical University

summary: Transcriptional induction of retroelement transcripts through therapeutic interventions elevates cytosolic double-stranded RNA (dsRNA) levels to trigger viral mimicry response and anti-tumor immunity. Therapeutic interventions that transcriptionally induce retroelement transcripts lead to elevated levels of cytosolic double-stranded RNA (dsRNA), thereby triggering a viral mimicry response and enhancing anti-tumor immunity. Despite the promising implications, clinical studies have revealed substantial variations in the therapeutic induction of viral mimicry response in different cancer patients, with the underlying mechanisms remaining largely elusive. In this study, we illuminate the pivotal role of the mRNA export pathway in viral mimicry response and anti-tumor T cell immunity. Notably, epigenetic and posttranscriptional induction of cytosolic dsRNAs necessitates the mRNA export pathway to transport the nuclear-transcribed retroelement transcripts into the cytoplasm, which is posttranscriptionally suppressed by the RNA exosome in coactivator-associated arginine methyltransferase 1 (CARM1)-expressing tumor cells. Mechanistically, Carm1 restricts the nuclear export of retroelement transcripts through transcriptional elevation and posttranslational stabilization of the RNA exosome, which hydrolyzes the critical nuclear RNA export adaptor transcripts Ddx39a and Alyref. Collectively, our study underscores the vital importance of the mRNA export pathway in viral mimicry response and suggests that enhancing its activity through Carm1 or RNA exosome inhibition could reinforce therapy-induced viral mimicry response, thus holding the potential to overcome tumor immune escape and immunotherapy resistance.

overall_design: ChIP with reference exogenous genome (ChIP-Rx) was utilized to study H3K27ac and H3K4me3 modifications in parental and Carm1-/- B16-F10 cells.

first: Shan

last: Huang

first: Jinhua

last: Liu

first: Xinyue

last: Cheng


title: Suppression of the mRNA export pathway dampens viral mimicry response and promotes tumor immune evasion

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

overall_design: Refer to individual Series


title: Suppression of the mRNA export pathway dampens viral mimicry response and promotes tumor immune evasion [RNA-seq]

overall_design:
We conducted total RNA sequencing (RNA-seq) on parental and Carm1 knockout (Carm1-/-) B16-F10 cells derived from mouse tumor tissues.
Total RNA-seq was also performed on control B16-F10 cells and those with DDX39A overexpression.
mRNA sequencing (mRNA-seq) was performed on whole cells, as well as cytoplasmic and nuclear fractions, from control, Carm1-/-, Ddx39a-/- Carm1-/-, and Alyref-/- Carm1-/- B16-F10 cell lines.
We carried out mRNA-seq on whole cells, cytoplasm, and nucleus of control, Ddx39a-/- and Alyref-/- B16-F10 cells treated with Decitabine.
mRNA-seq was executed on whole cells, along with their cytoplasmic and nuclear components, from parental, Ddx39a-/- and Alyref-/- MC38 cells.
We conducted total RNA-seq on control, Dis3-/- and Exosc1-/- B16-F10 cells.


title: Gene expression data in total mRNA and light and heavy polysome fractions from human SKMEL28 melanoma cell line shCont, shp16 [II]

city: Pittsburgh

first: Katherine

last: Aird

state: PA

address: 5051 Centre Ave.

institute: University of Pittsburgh School of Medicine

summary: Cisplatin-induced senescence affects multiple pathways in ovarian cancer cells

overall_design: To investigate the transcriptonal differences upon therapy-induced senescence, we treated human Ovcar8 ovarian cancer cells with 1uM cisplatin.  We isolated total mRNA . All samples were done in triplicate. Comparative gene expression profiling analysis for DMF (control) and cisplatin.


title: Restoration of branched chain amino acid catabolism improves kidney function in preclinical cardiovascular-kidney-metabolic syndrome models

city: Cambridge

first: Mary

last: Piper

state: Massachusetts

address: 1 Portland St

institute: Pfizer Inc

summary: Patients with metabolic syndrome and heart failure (HF) often have accompanying kidney dysfunction, which was recently defined as cardiovascular-kidney-metabolic (CKM) syndrome. Prior metabolomics profiling of metabolic syndrome patients identified a plasma branched chain amino acid (BCAA) signature, and BCAAs are elevated in HF patient myocardium. The rate limiting step of BCAA catabolism is decarboxylation by branched chain ketoacid dehydrogenase enzyme (BCKDH), which is negatively regulated by BCKDH kinase (BCKDK or BDK), and BDK inhibitors improve metabolism and heart failure preclinically. Here we show that BCAA catabolic impairment is associated with and may be causal to CKM as treatment with the BDK inhibitor BT2 improved urine protein content, glomerular filtration rate, kidney hypertrophy, and kidney pathology in CKM models. Coadministration of BT2 and empagliflozin restored renal function and gene expression signatures and improved mitochondrial density and function, suggesting that BDK inhibition could represent a therapeutic avenue for CKM.

overall_design: Bulk RNA-seq  was performed on the whole heart or on the whole kidney and kidney cortex, respectively, of 30-week-old ZSF1 obese and ZSF1 lean rats.  Rats were treated with vehicle, BDK inhibitor (BT2), SGLT2 inhibitor (emplagliflozin) or combination BT2 + SGLT2 treatment.  Sequencing was performed on 10 rats per condition.

first: Rachel

last: Roth Flach

first: Eliza

last: Bollinger

first: George

last: Williams


title: Effect of SASP on human Ovcar8 ovarian cancer cell line cultured in ultra-low attachment conditions

summary: Cellular senescence has been shown to contribute to deliterious cancer phenotyores through modulation of the tumor microenvironment through the SASP. Senescence is induced in ovarian cancer by chemotherapy. Understanding how the SASP modulates gene expression on cancer may lead to novel targets to exploit to increase chemotherapeutic efficacy.

overall_design: To investigate the role of SASP on gene expresssion, fresh media and conditioned media was collected from vehicle treated proliferative cells or cisplatin therapy-induced senescent cells and used to to culture Ovcar8 human ovarian cancer cells in ultra-low attachment conditions. As the non-protein fraction of the SASP is understudied, samples were included with boiled conditioned media or conditioned media fractionated into greater the 3 kDa or less than 3 kDa components. All samples were done in triplicate.

first: Aidan

last: Cole


title: Gene expression during PMA-induced neutrophil NETs formation with or without knockout of neutrophils of Src kinase

city: Yangzhou

first: Guotao

last: Lu

address: 368 Hanjiang Middle Road, Yangzhou

institute: The Affiliated Hospital of Yangzhou University, Yangzhou University

summary: Src kinase has functions in regulating reactive oxygen species production, degranulation, NETs formation, integrin activation, and migration to human infection sites in neutrophils. RNA sequencing was performed by extracting mRNA from PMA-induced mouse neutrophils with or without Src kinase knockout(Src fl/fl S100a8 Cre or Src fl/fl  mice). Gene enrichment analysis by Genomes (Reactome) identified RAF/MAP kinase cascade in the top ten significantly changed pathways.

overall_design: Fresh primary  neutrophils were quickly isolated from neutrophil Src kinase knockout or non knockout mice(Src fl/fl S100a8 Cre or Src fl/fl  ) using  the mouse bone marrow neutrophil isolation kit. NETs formation was induced by adding 100 nM Phorbol 12-myristate 13-acetate (PMA) for 4 h.

first: Fei

last: Han

first: Yaodong

last: Wang

first: Chenchen

last: Yuan

first: Lianghao

last: Hu

first: Weijuan

last: Gong


title: Physical exercise improves the neuronal function in ischemic stroke via microglial CB2R/P2Y12 signaling

city: Guangzhou

first: Xiaofei

last: He

state: Guangdong

address: 600 Tianhe Road

department: Department of Rehabilitation Medicine

institute: The Third Affiliated Hospital of Sun Yat-sen University

summary: Physical exercise (PE) may be the single most important and accessible lifestyle habit throughout life, it inhibits the neuroinflammatory response and protects the brain against damage. As the innate cells in brain, microglia undergo morphological and functional changes to communicate with neurons protecting other neurons from injury. Herein, aiming at exploring the effect of PE on the communication microglia - neuron during acute ischemic cerebral infarction, we carried out running wheel training before the conduction of transient middle cerebral artery occlusion (tMCAO) in Cx3cr1-GFP mice. We found that microglial P2Y12 expression in the peri-infarct area was decreased, microglial dynamics and microglia - neuron communications were impaired, using in vivo two-photon imaging. PE upregulated microglial P2Y12 expression, increased microglial dynamics, and promoted the contact of microglia with neurons. As a result, PE inhibited neuronal Ca2+ overloads and protected against damage of the neuronal mitochondria in acute tMCAO. Mechanistically, PE increased the cannabinoid receptor 2 (CB2R) in microglia, promoted the phosphorylation of Nrf2 (NF-E2-related factor 2) at ser-344, increased the transcription factor level of Mafk, and up-regulated the level of P2Y12, whereby PE increased the levels of CB2R to promote microglia-neuron contacts to monitor and protect neuronal functioning.

overall_design: To investigate the regulative mechnism and explore the potential target to protect against the ischemic stroke, C57BL/6J mice were suffered from transient middle cerebral artery occlusion.

first: Xiao-fei

first: Xiao-feng

last: Yan

first: Ge

last: Li

first: Yun

last: Zhao

first: Jing

last: Luo

first: Jing-hui

last: Xu

first: Hai-qing

last: Zheng

first: Li-ying

last: Zhang


title: SOX9 suppresses colon cancer via inhibiting epithelial-mesenchymal transition and SOX2 induction

city: Ann Arbor

first: Karan

last: Bedi

state: MI

address: 1600 Huron Pkwy

department: Biostatistics

institute: University of Michigan

summary: The Wnt/β-catenin pathway regulates expression of the SOX9 gene, which encodes SRY-box transcription factor 9, a differentiation factor and potential β-catenin regulator. Because APC tumor suppressor defects in ~80% of colorectal cancers (CRCs) activate the Wnt/β-catenin pathway, we studied SOX9 inactivation in CRC biology. Compared to effects of Apc inactivation in mouse colon tumors, combined Apc and Sox9 inactivation instigated more invasive tumors with epithelial-mesenchymal transition (EMT) and SOX2 stem cell factor upregulation. In an independent mouse CRC model with combined Apc, Kras, and Trp53 defects, Sox9 inactivation promoted SOX2 induction and distant metastases. About 20% of 171 human CRCs showed loss of SOX9 protein expression, which correlated with higher tumor grade. In an independent group of 376 CRC patients, low SOX9 gene expression was linked to poor survival, earlier age at diagnosis, and increased lymph node involvement. SOX9 expression reductions in human CRC were linked to promoter methylation. EMT pathway gene expression changes were prominent in human CRCs with low SOX9 expression and in a mouse cancer model with high SOX2 expression. Our results indicate SOX9 has tumor suppressor function in CRC; its loss may promote progression, invasion, and poor prognosis by enhancing EMT and stem cell phenotypes.

overall_design: To identify genes regulated by SOX9, we investigated gene expression profiles in mouse colon tissues and organoids using RNA sequencing (RNA-seq). The mouse colon tissues were obtained from five different groups of mice, each with specific genotypes in the Sox9 and Apc genes: 1) control mice: these are littermates without Cre or Apc/Sox9 floxed alleles; 2) CDX2P-CreERT2 Sox9flox/flox (abbreviated as S) mice; 3) CDX2P-CreERT2 Apcflox/flox (abbreviated as A) mice; 4) CDX2P-CreERT2 Apcflox/flox Sox9flox/+ (abbreviated as AShet) mice; 5) CDX2P-CreERT2 Apcflox/flox Sox9flox/flox (abbreviated as AS) mice. Each group consisted of six replicate mice except AShet group (which has 7 replicates), resulting in a total of 31 mice being analyzed in the study. The mice in each group were treated with tamoxifen (TAM) to induce deletions in the Apc and Sox9 genes in the colon. Total RNAs were collected from proximal colon tissues of these mice after 30-40 days post TAM treatment for RNA-seq analysis. Paired comparisons of gene expression profiles (e.g., AS versus A, AS versus AShet, and S versus control) were performed using the RNA-seq data obtained from these RNA samples. We also performed gene expression profiling analysis using RNA-seq data for organoids derived from the colon of A mice or AS mice that have been treated with TAM to induce gene inactivation in Apc and/or Sox9. The study aimed to compare gene expression between the four organoids derived from four TAM-treated AS mice and the four control organoids from four TAM-treated A mice.

first: Ying

last: Feng

first: Ningxin

last: Zhu

first: Jinju

first: Chamila

last: Perera

first: Maranne

last: Green

first: Naziheh

last: Assarzadegan

first: Yali

last: Zhai

first: Qingzhi

last: Liu

first: Veera

last: Baladandayuthapani

first: Jason

last: Spence

first: Kathleen

last: Cho

first: Eric

last: Fearon


title: Chromatin Reprogramming of In Vitro Fertilized and Somatic Cell Nuclear Transfer Bovine Embryos During Embryonic Genome Activation [WGS-bovine-EJG]

city: dallas

first: Edward

last: Grow

state: TEXAS

address: 7053 beltau lane

institute: UTSW

summary: Reprogramming of the gamete into a developmentally competent embryo identity is a fundamental aspect of preimplantation development. One of the most important processes of this reprogramming is the transcriptional awakening during embryonic genome activation (EGA), which robustly occurs in fertilized embryos but is defective in most somatic cell nuclear transfer (SCNT) embryos. However, little is known about the genome-wide underlying chromatin landscape during EGA in SCNT embryos and how it differs from a fertilized embryo. By profiling open chromatin genome-wide in both types of bovine embryos, we find that SCNT embryos fail to reprogram a subset of the EGA gene targets that are normally activated in fertilized embryos. Importantly, a small number of transcription factor (TF) motifs explain most chromatin regions that fail to open in SCNT embryos suggesting that over-expression of a limited number of TFs may provide more robust reprogramming. One such TF, the zygotically-expressed bovine gene DUXC which is a homologue of EGA factors DUX/DUX4 in mouse/human, is alone capable of activating ~84% of all EGA transcripts that fail to activate normally in SCNT embryos. Additionally, single-cell chromatin profiling revealed low intra-embryo heterogeneity but high inter-embryo heterogeneity in SCNT embryos and an uncoupling of cell division and open chromatin reprogramming during EGA. Surprisingly, our data also indicate that transcriptional defects may arise downstream of promoter chromatin opening in SCNT embryos, suggesting additional mechanistic insights into how and why transcription at EGA is dysregulated. We anticipate that our work will lead to altered SCNT protocols to increase the developmental competency of bovine SCNT embryos.

sample_organism:
Bos taurus
Bos indicus

platform_organism:
Bos taurus
Bos indicus

overall_design: Whole genome sequencing of Dream Boy (Bos Indicus sperm donor), Crushertime fibroblast (Bos Indicus), and cumulus cell pool (Bos Taurus)

first: Grow

last: Edward


title: Chromatin Reprogramming of In Vitro Fertilized and Somatic Cell Nuclear Transfer Bovine Embryos During Embryonic Genome Activation [single cell ATAC-seq]

sample_organism:
Bos taurus
Bos indicus x Bos taurus

platform_organism:
Bos taurus
Bos indicus x Bos taurus

overall_design: single cell ATAC-seq on pooled bovine IVR or SCNT preimplantation embryos


title: Chromatin Reprogramming of In Vitro Fertilized and Somatic Cell Nuclear Transfer Bovine Embryos During Embryonic Genome Activation [20230618-SMART-seq]

overall_design: ATAC-seq on pooled bovine IVR or SCNT preimplantation embryos


title: Chromatin Reprogramming of In Vitro Fertilized and Somatic Cell Nuclear Transfer Bovine Embryos During Embryonic Genome Activation

sample_organism:
Bos taurus
Bos indicus
Bos indicus x Bos taurus


title: Chromatin Reprogramming of In Vitro Fertilized and Somatic Cell Nuclear Transfer Bovine Embryos During Embryonic Genome Activation [ATAC-Seq]


title: Over-expression of Trib1 overcomes self-degradation to induce myeloid leukemia

city: Tokyo

first: Takuro

last: Nakamura

address: 6-1-1 Shinjuku, Shinjuku-ku

institute: Tokyo Medical University

summary: Cop1 encodes a ubiquitin E3 ligase that is well preserved during evolution both in plants and metazoan. In metazoan, C/EBP family transcription factors are Cop1 targets for degradation, and the process is regulated by Tribbles family pseudokinases. Over-expression of Tribbles homolog 1 (Trib1) induces acute myeloid leukemia (AML) via Cop1-dependent degradation of the C/EBP p42 isoform. Here we induce rapid growth arrest and granulocytic differentiation of Trib1-expressing AML by Cop1 conditional knockout (KO) that is associated with transient increase of the C/EBP p42 isoform. The growth suppressive effect by Cop1 KO was canceled by silencing of Cebpa and was reinforced by exogenous expression of p42 isoform. We further identify remarkable increase of the Trib1 protein by Cop1 KO, indicating that the Trib1 protein is self-degraded by the Cop1 degradosome. Cop1 KO modulates gene expression profiles of Trib1-expressing AML depicting hematopoietic cell proliferation and maturation pathways. COP1 downregulation also inhibited proliferation of human AML cells in a TRIB1-dependent manner. Taken together, our results provide new insights into the role of Trib1/Cop1 machinery in the C/EBPa p42-dependent leukemogenic activity, and a novel idea to develop new therapeutics.

overall_design: Mouse bone marrow cells were prepared from an 8-week-old Cop1 cKO/Rosa26-Cre-ERT2 mice five days after injecting 150 mg/kg body weight of 5-fluorouracil (Kyowa Kirin). Bone marrow cells were transduced with the pMYs retrovirus containing Trib1. Transduced cells were maintained in Iscove modified Dulbecco medium containing 10% fetal bovine serum and 5 ng/mL interleukin-3.

first: Yoshitaka

last: Sunami