title: Chromatin-dependent motif syntax defines differentiation trajectories [DAP-seq]

city: Basel

first: Sevi

last: Durdu

address: Fabrikstrasse 24

department: Genome Regulation

institute: Friedrich Miescher Institute for Biomedical Research

summary: Transcription factors (TFs) recognizing DNA motifs within regulatory regions drive cell identity. Despite recent advances, their specificity remains incompletely understood. Here, we address this by contrasting two TFs, Neurogenin-2 (NGN2) and MyoD1, which recognize ubiquitous E-box motifs yet drive distinct cell fates toward neurons and muscles, respectively. Upon induction in mouse embryonic stem cells, we monitor binding across differentiation, employing an interpretable machine learning approach that integrates preexisting DNA accessibility. This reveals a chromatin-dependent motif syntax, delineating both common and factor-specific binding, validated by cellular and in vitro assays. Shared binding sites reside in open chromatin, locally influenced by nucleosomes. In contrast, factor-specific binding in closed chromatin involves NGN2 and MyoD1 acting as pioneer factors, influenced by motif variant frequencies, motif spacing, and interaction partners, which together account for subsequent lineage divergence. Transferring our methodology to other models demonstrates how a combination of opportunistic binding and context-specific chromatin-opening underpin TF specificity, driving differentiation trajectories.

overall_design: Examining binding preferences of NGN2 and MyoD1 as homo-dimers or heterodimers with TCF family with DAPseq from DNMT-tko genomic DNA with tag-only control dataset

first: Murat

last: Iskar

first: Luke

last: Isbel

first: Leslie

last: Hoerner

first: Christiane

last: Wirbelauer

first: Lukas

last: Burger

last: Hess

first: Vytautas

last: Iesmantavicius

first: Dirk

last: Schübeler


title: Chromatin-dependent motif syntax defines differentiation trajectories [RNA-seq]

status: Public on Aug 07 2025

country: Switzerland

type: Expression profiling by high throughput sequencing

overall_design: RNA-seq of mouse embryonic stem cells upon NGN2 or MyoD1 expression induction at multiple time-points up to three days, with induction controls such as GFP expression.

first: Daniel


title: Chromatin-dependent motif syntax defines differentiation trajectories [ChIP-seq]

type: Genome binding/occupancy profiling by high throughput sequencing

overall_design: ChIP-seq of NGN2, MyoD1 with mutant and GFP IP controls upon expression induction in mESCs together with H3K27ac and Pol2 ChIP-seq to monitor downstream effect


title: VRS5 (HvTB1) binds to the promoter of tillering and floral homeotic genes to regulate their expression [RNA-seq]

city: Wageningen

first: Wilma

last: Van Esse

address: Droevendaalsesteeg 1

country: Netherlands

institute: Wageningen University & Research

summary: Variation in shoot architecture, or tillering, is an important adaptive trait targeted during the domestication of crops. A well-known regulatory factor in shoot architecture is TEOSINTE BRANCHED 1 (TB1). TB1 and its orthologs have a conserved function in integrating environmental signals to regulate axillary branching, or tillering in cereals. The barley ortholog of TB1, VULGARE ROW-TYPE SIX 5 (VRS5) does not only regulate tillering, but is also involved in regulating row-type by inhibiting lateral spikelet development. These discoveries do mainly come from genetic studies, but how VRS5 regulates these processes on a molecular level remains largely unknown. By combining transcriptome analysis between vrs5 and WT at different developmental stages and DAP-sequencing to locate the genome-wide DNA binding sites of VRS5, we identified bona fide targets of VRS5. We found that VRS5 targets in particular abscisic acid related genes to inhibit tillering in a conserved way. Later, during inflorescence development row-type gene VRS1, and some known floral development genes, like MIKCc type MADS-box genes, are targeted. The study presented here offers a solid and crucial starting point for mutant analyses of a selection of bona fide targets to gain a deeper understanding of the VRS5 network and to enlighten its mode of action in shaping barley development.

overall_design: We performed RNA-seq on barley meristems from WT (cv.Bowman) and vrs5 mutants (int-c.5) at four developmental stages, namely Vegetative Apex (VA), Transition apex (TA), triple mound (TM), and lemma and stamen primordia (LP/SP). The samples from VA and TA contain also tiller buds.

first: Ton

last: Winkelmolen

first: Pierangela

middle: E

last: Colleoni

first: Richard

middle: H

last: Immink

first: G

middle: W

last: van Esse


title: Adipocyte death drives fat redistribution from adipocytes to hepatocytes in steatotic liver disease via S100A8+ macrophages [scRNA-seq]

city: Rockville

first: Yukun

last: Guan

state: MD

address: 5625 Fishers Lane, Room 2S-30

country: USA

institute: NIAAA

summary: Both adipocytes and hepatocytes have the capacity to store fat, but the factor(s) that determine fat distribution between these cell types remain unknown. In mice fed a high-fat diet, fat initially accumulates predominantly in adipocytes, while hepatic fat accumulation mainly emerges after the onset of epididymal adipocyte death that results in elevated free fatty acids to promote lipid accumulation in hepatocytes. However, it remains unclear whether other signals following adipocyte death are required to direct and/or promote hepatocytes to store fat and subsequently trigger metabolic dysfunction-associated steatotic liver disease (MASLD, formerly known as nonalcoholic fatty liver disease). Using genetically modified mouse models combined with bulk and single-cell RNA sequencing analysis, we demonstrated that adipocyte death induces an accumulation of S100A8+ macrophages in the liver, which is partially induced by fatty acids. Macrophage-specific deletion of the S100a8 gene reduced hepatic fat accumulation and MASLD severity in mice. Mechanistically, S100A8+ macrophages suppress cellular communication network factor (CCN3), a negative regulator of CD36, thereby enhancing CD36 expression in hepatocytes. In conclusion, adipocyte death promotes hepatic infiltration of S100A8+ macrophages, which drive hepatocyte lipid storage and subsequently promote MASLD progression through CD36 upregulation, partially mediated by CCN3 suppression.

overall_design: WT or Bcl2TG male mice aged 8 weeks were placed on an HFD (60% kcal fat, D12492; Research Diets, New Brunswick, NJ) for 4 months, liver cells were isolated for scRNA.

last: GUAN

first: Seonghwan

last: HWANG

first: Dechun

last: FENG

first: Bin

last: GAO


title: Distinguishing identical twins using DNA methylation analysis

city: Daejeon

first: MUNJEONG

last: CHO

address: 125 Gwahak-ro, Yuseong-gu, Daejeon, Republic of Korea

country: South Korea

institute: Korea Research Institute of Bioscience &Biotechnology

type: Methylation profiling by genome tiling array

summary: Genome wide DNA methylation profiling of identical twins to select markers for distinguishing identical twins

overall_design: 54 samples were analyzed using Illumina Infinium MethylationEPIC version 2

first: Seung-Jin

last: Park

first: Mun-Jeong

last: Cho


title: Adhesion-Related Macrophages Regulates Metabolic Homeostasis through CAV-1 Dependency

city: Changsha

first: Hu

last: Wanyu

address: 长沙市芙蓉区人民中路139号

country: China

institute: 中南大学湘雅二医院

summary: The significance of adipose tissue macrophages (ATMs) in regulating adipose tissue function is well-established. However, our investigation revealed a previously overlooked subpopulation of macrophages adhered to adipocytes, which we term adhesion-related macrophages (ARMs). We developed an approach to isolate ARMs and compared them with traditional macrophages from the stromal vascular fraction. Our findings demonstrate that ARMs constitute the predominant expanded subpopulation of adipose tissue macrophages during obesity, exhibiting heightened adhesion, proliferation, and lipid-processing capacities. Notably, ARMs can be characterized by a key functional marker, Caveolin-1. Genetic ablation of Caveolin-1 in immune cells significantly diminishes ARM abundance, disrupting their adhesion capacity and lipid content, leading to adipocyte hypertrophy, adipose tissue expansion, and impaired glucose homeostasis. Reintroducing ARMs from lean mice into eWAT mitigate obesity-induced adipose tissue inflammation and insulin resistance. Our studies uncover a previously unexplored macrophage subpopulation, ARMs, revealing potential therapeutic targets for obesity-induced insulin resistance and opening avenues for identifying similar paradigms in other tissues and diseases.

overall_design: To investigate the function of ARM, we collected ARM and SM from ewat in ND mice and HFD mice


title: The feather moss Hylocomium splendens affects the transcriptional profile of a symbiotic cyanobacterium in relation to acquisition and turnover of key nutrients

city: Copenhagen

first: Danillo

middle: Oliveira

last: Alvarenga

address: Universitetsparken 15

country: Denmark

institute: University of Copenhagen

summary: We isolated a cyanobacterium from H. splendens and co-incubated it with H. splendens shoots. Cyanobacterium and moss were also incubated separately. After one week, we performed acetylene reduction assays to estimate N2 fixation and RNAseq to evaluate transcriptomes.

sample_organism:
Hylocomium splendens
Nostocaceae cyanobacterium

overall_design: Three treatments: 1) cyanobacterium alone; 2) cyanobacteria and feather moss incubated together; and 3) feather moss alone.

last: Oliveira Alvarenga

first: Anders

last: Priemé

first: Kathrin

last: Rousk


title: Chromatin-dependent motif syntax defines differentiation trajectories

first: Sevi

last: Durdu

overall_design: Refer to individual Series


title: Myocardin-related transcription factors regulate F-actin organization during gastrulation and neural tube closure in Xenopus embryos

city: New York

first: Mark

middle: E.

last: Corkins

state: NY

address: 1468 Madison Ave, Annenburg 25-86

department: CDRB

institute: Mount Sinai

summary: Myocardin-related transcription factors (Mrtfa and Mrtfb), also known as megakaryoblastic leukemia (Mkl1/MAL and Mkl2), associate with serum response factor (Srf) to regulate transcription in response to actin dynamics, however, the functions of Mrtfs in early development are poorly characterized.  In this study, we assessed functional roles of Mrtfs in Xenopus embryos. Modulation of Mrtf-dependent transcription in gain- and loss-of-function experiments caused gastrulation and neural tube closure defects, indicating an essential function of Mrtfs in morphogenesis. Notably, both activation and inhibition of Mrtf activity had similar morphological phenotypes. However, dominant interfering Mrtf construct reduced overall F-actin levels, whereas a constitutively active Mrtf construct specifically disrupted F-actin accumulation at the tricellular junctions. Both Mrtf constructs inhibited elongation of ectodermal explants in response to activin, but did not affect RhoA-dependent apical constriction.  RNA sequencing of ectoderm with manipulated Mrtf levels confirmed already known Mrtf target genes encoding actins and actin-binding proteins and revealed new Srf-dependent or -independent putative targets. Our findings highlight the importance of Mrtfs in early morphogenetic processes and elucidate their potential targets.

overall_design: Stage 11 X. Laevis animal caps injected with mRNA encoding deltaN-MRTF, deltaNSRF-MRTF, DeltaNSAP-MRTF

first: Keiji

last: Itoh

first: Sergei

middle: Y

last: Sokol


title: AKAP12 variant 1 knockout enhances vascular endothelial cell motility

city: Grand Forks

first: Bryon

middle: Dennis

last: Grove

state: ND

address: 1301 N Columbia Rd

department: Biomedical Sciences

institute: University of North Dakota School of Medicine and Health Sciences

summary: This study investigates the role of AKAP12 in endothelial cell motility with a specific focus on AKAP12 variants, AKAP12v1 and AKAP12v2. Previous work has shown that AKAP12, a multivalent A-kinase anchoring protein that binds to PKA and several other proteins regulating protein phosphorylation, is expressed at low levels in most endothelia in vivo but is expressed at higher levels in cells in vitro. Here, we found that AKAP12 expression in endothelial cell (HUVEC) cultures was cell density-dependent, with the expression being highest in subconfluent cultures and lowest in confluent cultures. AKAP12 expression was also elevated in cells at the wound edge of wounded endothelial cell monolayers. Knockdown of variants 1 and 2 inhibited cell migration. However, CRISPR/Cas9 knockout of AKAP12v1 enhanced migration, indicating that the absence of this variant and the presence of AKAP12v2 likely alters the signaling events controlling cell motility. Further analysis using bulk RNA sequencing revealed that the loss of AKAP12v1 affected genes associated with cell migration and intercellular junctions. We propose that AKAP12v1 and AKAP12v2 play distinct yet complementary roles in endothelial cell migration and likely work together in controlling the signaling events associated with vascular repair and development.

overall_design: Eight bulk RNA-seq libraries were prepared from subconfluent HUVEC-TERT cells: two libraries each from the three AKAP12 v1 KO clones and two libraries from WT endothelial cells.

first: Ashrifa

last: Ali

first: Baskar

last: Roy

first: Micah

last: Schott


title: VRS5 (HvTB1) binds to the promoter of tillering and floral homeotic genes to regulate their expression [DAP-seq]

first: Wilma

last: Van Esse

overall_design: DAP-seq using barley TB1 (from cv. Bowman) fused to FLAG as bait against library generated from barley genomic DNA. GFP fused to FLAG was used as control. 6 replicates of each were performed.

first: Ton

last: Winkelmolen

first: Pierangela

last: Colleoni

first: Richard

last: Immink

first: G

last: van Esse


title: High-Dimensional Analysis of Type 2 Lymphocyte Dynamics During Mepolizumab Treatment in Severe Asthma

status: Public on Aug 06 2025

city: Huddinge

first: Lorenz

last: Wirth

state: Stockholms län

address: Alfred Nobels Allé 8

country: Sweden

institute: Karolinska Institutet

summary: Background Although the type 2 biologics mepolizumab and dupilumab show clinical efficacy in severe asthma, their influence on circulating lymphocytes is largely unknown. Here, we studied their impact on type 2 lymphocytes in severe asthma.  Methods We performed high-parameter flow cytometry analysis of peripheral blood mononuclear cells from 40 patients with severe asthma before, and after 4 and 12 months of mepolizumab (n = 33) or dupilumab (n = 7) treatment, focusing on type 2 lymphocytes. Additionally, we performed single-cell RNA sequencing (scRNA-seq) (n = 3) and stimulation experiments of type 2 lymphocytes (n = 3) to explore transcriptional and functional changes associated with mepolizumab treatment.  Results Mepolizumab treatment increased circulating type 2 innate lymphoid cell (ILC2), type 2 T helper (Th2) and type 2 cytotoxic (Tc2) cell frequencies, skewing ILC2 towards a CD117low signature with high CD62L expression, and Th2/Tc2 cells towards a CD45RA−CD62L+ central memory phenotype. Dupilumab-treated patients also showed increased frequencies of total ILC2 and CD117low ILC2. Mepolizumab treatment reduced the expression of tissue homing receptors CXCR4 in ILC2, and GPR183 in ILC2, Th2, and Tc2 cells while enhancing their type 2 cytokine producing capability in response to alarmins.  Conclusion Mepolizumab increases the frequencies of circulating ILC2, Th2, and Tc2 cells, with reduced tissue homing receptor expression but increased type 2 cytokine production potential. This reveals a potentially new mechanism for how mepolizumab reduces airway inflammation by re-directing trafficking of inflammatory type 2 lymphocytes away from airway-homing, with implications for the possibility of achieving biologics-free remission in asthma.

overall_design:
Biobanked PBMCs from 3 severe asthma patients at baseline and after 1 year of mepolizumab treatment were thawed and FACS-sorted for ILC2, Th2 and Tc2 cells. Subsequently, single cell RNA-sequencing was performed. *** Due to patient confidentiality concerns, Swedish law does not allow the raw data to be hosted outside of Sweden; contact submitter for more details. ***  Flow cytometry data referred to in the abstract is available at: https://doi.org/10.5281/zenodo.15921459

***************************************************************
Due to patient-privacy concerns, raw data files cannot be made available.
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first: Wirth

last: Lorenz

first: Stamper

last: Christopher T

first: Yasinska

last: Valentyna

first: Mjösberg

last: Jenny


title: Effect of TAM treatment on gene expression profile of microglia from Cx3cr1CreERT2 mouse lines.

city: Fukuoka

first: Yasuyuki

last: Ohkawa

address: 3-1-1 Maidashi

country: Japan

institute: Medical Institute of Bioregulation

summary: Microglia, the resident macrophages in the central nervous system (CNS), have been intensively studied using rodent genetic models, including the Cx3cr1-Cre mouse line. These mice have enabled the in-depth analyses of the biological features and functions of myeloid cells, including microglia. Occasionally, these Cx3cr1CreERT2 tools have yielded conflicting outcomes, the underlying mechanism of which remains unclear. Here, we comparatively characterize the two available Cx3cr1CreERT2 lines (Cx3cr1CreERT2(Litt) and Cx3cr1CreERT2(Jung)). We find a mouse line-specific and TAM-dependent persistent induction of CDKN1A in microglia of Cx3cr1CreERT2(Litt) mice, but not in those of Cx3cr1CreERT2(Jung) mice, which affects experimental readouts with altered proliferative capacity. Furthermore, aberrant cellular alterations observed in postnatal Cx3cr1CreERT2(Litt) microglia are mitigated by a functional inhibition of CDKN1A. Together, these findings underscore the significance of mouse line-specific phenomena that alter microglial outcomes in a CDKN1A-dependent manner, highlighting the importance of rigorous validation of genetic mouse tools.

overall_design: Transcriptome profiles of microglia from whole brains of Cx3cr1-CreERT2 mouse models with TAM-treatment were generated by deep sequencing.

first: Takahiro

last: Masuda

first: Ayato

last: Yamasaki

first: Ohkawa

last: Yasuyuki

first: Kaori

last: Tanaka


title: Hepatic CB1 receptor signaling triggers Gi/oα-mediated lipolysis in lean mice but Gsα-mediated lipogenesis in obese mice

first: Jie

last: Liu

address: 5625 Fishers Lane

institute: National Institutes of Health/National Institute on Alcohol Abuse and Alcoholism

summary:
Objectives: Obesity-induced steatotic liver disease (SLD) is driven by the uptake of adipocyte-derived fatty acids (FAs) into hepatocytes via the FA translocase CD36, which also prevents their consumption by inhibiting AMP kinase (AMPK)-mediated FA oxidation (FAO). We explored the role of hepatocyte CB1 receptors (hCB1R) in controlling hepatic triglyceride (TG) content by regulating CD36 and its downstream targets.
Methods: hCB1R knockout (hCB1Rko) mice and their control littermates kept on standard or high-fat diet were used to analyze hCB1R-mediated hepatic gene expression profile and lipid metabolism in intact mice and in cultured hepatocytes.
Results: Multi-omics data indicate that hCB1R target a distinct set of genes associated with SLD, including Cd36. In mice with diet-induced obesity, hCB1R signaling via CD36-AMPK-FAO pathway contributes to both the development of SLD and its reversal by CB1R blockade. But, in lean mice hCB1R signaling inhibits CD36 expression and activates AMPK-mediated FAO. These opposite effects were replicated in AML12 mouse hepatocytes incubated with or without oleic acid (OA). OA, an endogenous ligand of GPR3, induced a switch in hCB1R signaling from a Gi/oα-mediated reduction in cAMP to a Gsα-mediated increase in cAMP in a GPR3/Gsα -dependent manner, facilitated by increasing the ratio of Gsα:Gi/oα proteins in the steatotic compared to lean liver.
Conclusions: In the lean state, endocannabinoid activation of hCB1R increases FAO, which mitigates SLD, as reported for chronic marihuana smokers, whereas in obese mice hCB1R tonically inhibit FAO, which promotes SLD and underlies the anti-steatotic effect of peripheral CB1R blockade.

overall_design: To identify downstream targets of hCB1R, we conducted genome-wide transcriptome analyses by RNA-Seq and targeted transcriptomics of lipolytic pathways using nanoString. To link gene expression patterns to FA metabolism, we applied lipidomic analysis to quantify FAs in the liver.

first: Anna

last: Oliverio

first: Grzegorz

last: Godlewski

first: Radka

last: Kočvarová

first: Muhammad

last: Arif

first: Abhishek

last: Basu

first: Yukun

last: Guan

first: Dechun

last: Feng

first: Henry

last: Puhl

first: Malliga

last: Iyer

first: Resat

last: Cinar

first: Bin

last: Gao

first: Joseph

last: Tam

first: George

last: Kunos


title: Albendazole induces ferroptosis via the USP7-GPX4 axis and enhances CAR-T immunotherapy in B-cell acute lymphoblastic leukemia

city: Guangzhou

first: Zhongjie

last: Huang

state: Guangdong

address: No. 253, Middle Industrial Avenue

institute: Zhuujiang hospital

summary: B-cell acute lymphoblastic leukemia (B-ALL) is a hematological malignancy that remains a significant clinical challenge, highlighting the urgent need for novel therapeutic strategies. Here, we performed a high-throughput screen of compounds approved by the U.S. Food and Drug Administration (FDA) and identified albendazole (ABZ), a benzimidazole anthelmintic, as a potent anti-leukemic agent against B-ALL. ABZ demonstrated robust cytotoxicity in B-ALL cell lines and patient-derived samples while sparing normal hematopoietic cells, and significantly reduced leukemia burden and prolonged survival in both cell line-derived and patient-derived xenograft models. Mechanistically, ABZ induced ferroptosis, characterized by lipid peroxidation, glutathione depletion, and ROS accumulation. ABZ promoted the ubiquitin-proteasome-mediated degradation of glutathione peroxidase 4 (GPX4) through autophagy-dependent destabilization of the deubiquitinase ubiquitin-specific protease 7 (USP7). USP7 was shown to directly interact with and stabilize GPX4 by removing K48-linked ubiquitin chains, and both proteins were markedly overexpressed in B-ALL patient samples, correlating with poor survival. Notably, ABZ also downregulated PD-L1 expression in leukemia cells, enhancing the efficacy of CD19 chimeric antigen receptor T (CAR-T) cell therapy in vitro and in vivo. Our findings delineate a previously unrecognized USP7-GPX4 axis regulating ferroptosis and provide a rationale for clinical repurposing of ABZ, alone or in combination with immunotherapies, to improve outcomes in B-ALL.

overall_design: RNA-seq of NALM-6 cells exposed to either 1 μM ABZ or DMSO for 24 hours

first: Hao

last: Wang

first: Tianwang

first: Xiaonan

last: Lin

first: Yingqi

last: Qiu

first: Zhao

last: Liang

first: Peiyun

last: Liao


title: Dysregulation of GTPase-activating protein-binding protein1 in the pathogenesis of metabolic dysfunction-associated steatotic liver disease

city: beijing

first: qinqin

last: ouyang

state: beijing

address: No. 10 Tianxiu Road, Haidian District, Beijing

department: department of nutrition and health

institute: China agricultural university

summary: Metabolic dysfunction-associated steatotic liver disease (MASLD) and metabolic dysfunction-associated steatohepatitis (MASH) are two common liver disorders characterized by abnormal lipid accumulation. Our study found reduced levels of GTPase-activating protein-binding protein1（G3BP1）in patients with MASLD and MASH, suggesting its involvement in these liver disorders. Hepatocyte-specific G3BP1 knockout (G3BP1 HKO) mice had more severe MASLD and MASH than their corresponding controls. Intriguingly, the G3BP1 HKO MASLD model mice exhibit dysregulated autophagy, and biochemical analyses demonstrated that G3BP1 promotes autophagosome-lysosome fusion through direct interactions with the SNARE proteins STX17 and VAMP8. We also show that hepatic knockout of G3BP1 promotes de novo lipogenesis, and ultimately found that G3BP1 is required for the nuclear translocation of the well-known liver-lipid-regulating transcription factor TFE3. Taken together, our results suggest that G3BP1 should be investigated as a potential target for developing medical interventions to treat MASLD and MASH.

overall_design: This study investigates the transcriptional changes in the liver of mice with a liver-specific knockout of the G3BP1 gene in response to a high-fat diet (HFD). The study includes a total of six samples derived from two distinct groups of adult male mice, with three biological replicates for each group. Wild-Type (WT) Control Group: This group consists of three mice with a wild-type genotype for the G3BP1 gene, fed a high-fat diet. The genotype is confirmed as G3BP1^flox/flox, which serves as the proper control for the knockout group. G3BP1 Knockout (KO) Group: This group consists of three mice with a liver-specific conditional knockout of the G3BP1 gene, also fed a high-fat diet. The knockout was achieved using the Cre-LoxP system by crossing mice carrying a floxed G3BP1 allele (G3BP1^flox/flox) with mice expressing Cre recombinase under the control of the Albumin promoter (Alb-cre). The resulting genotype is Alb-cre; G3BP1^flox/flox, leading to the specific deletion of G3BP1 in hepatocytes. Total RNA was extracted from the liver tissue of all six mice. Paired-end RNA sequencing libraries were prepared and sequenced on an Illumina HiSeq platform. The raw sequencing data, provided as FASTQ files, was processed to quantify gene expression, followed by differential expression analysis to identify the transcriptional impact of G3BP1 deletion in the context of a high-fat diet.

first: Qinqin

last: Ouyang

first: Jiaqi

last: Su

first: Yixuan

last: Li

first: Haiping

first: Haiying

last: Guo

first: Yanan

last: Sun

first: Xiaoyu

first: Juan

last: Chen

first: Josephine

last: Thinwa

first: Wenxing

last: Ding

first: Herbert

last: Tilg

first: Fazheng

last: Ren

last: Zhang

first: Rong

last: Liu


title: KIRA6 abrogates the generation of myeloid-derived suppressor cells and overcomes resistance to anti-PD-1 therapy

city: Zhuhai

first: Hong-Wei

address: 79 Kangning Road

department: The Affiliated Hospital of Beijing Institute of Technology (Zhuhai People's Hospital)

institute: Beijing Institute of Technology

summary: Immune checkpoint blockade (ICB) therapy is one of the cornerstones of cancer treatment regimens in a broad range of cancers nowadays, but the overall response rates remain low as a result of alternative immunosuppressive immune cells, such as myeloid-derived suppressor cells (MDSCs). Therefore, it is unmet need to target MDSCs in the tumor microenvironment to achieve better outcome of ICB therapy. Inositol-requiring enzyme 1α (IRE1α) is identified as a key regulator for generation of MDSC. Here, we evaluated the potential of KIRA6, an inhibitor for IREα kinase activity and RNase activity, to abrogate MDSC mediated immune suppression and improve outcome of ICB therapy.

overall_design: 4T1 cells were plated overnight in complete RPMI 1640 medium and then replaced with fresh culture medium with Vehicle or KIRA6 (1 μmol/L). Cells were cultured for 4 hours before RNA sample collection with TRIzol.

first: Chun


title: Chromatin-dependent motif syntax defines differentiation trajectories [ATAC-seq]

first: Sevi

last: Durdu

overall_design: ATAC-seq of mESCs upon NGN2, MyoD1 induction at 6 and 24 hours and GFP as induction control

first: Murat

last: Iskar

first: Luke

last: Isbel

first: Leslie

last: Hoerner

first: Christiane

last: Wirbelauer

first: Lukas

last: Burger

first: Daniel

last: Hess

first: Vytautas

last: Iesmantavicius

first: Dirk

last: Schübeler


title: Investigation of LARP1 regulated cellular pathways in acute myeloid leukemia

city: Chicago

first: Elspeth

last: Beauchamp

state: IL

address: 303 E Superior St

department: Medicine

institute: Northwestern University-Feinberg School of Medicine

summary: We demonstrate that LARP1, an RNA-binding protein regulated by mTOR and CDK9, promotes leukemic cell fitness and drug resistance in acute myeloid leukemia (AML). Using CRISPR/Cas9-mediated knockout models and multi-omics profiling, we show that LARP1 uniquely regulates the translation of transcripts involved in metabolism, cell cycle, and immune signalling, independent of mTOR or CDK9 inhibition. LARP1 loss reprograms mitochondrial and amino acid metabolism, downregulates cytidine deaminase, and enhances sensitivity to azacitidine and cytarabine. These findings establish LARP1 as a critical integrator of translational and metabolic control in AML and a potential therapeutic target.

overall_design: This study investigates the role of LARP1 in acute myeloid leukemia (AML) using CRISPR/Cas9-mediated knockout in human U937 cells. Total and polysome-associated RNA-seq was performed on LARP1 knockout cells (carrying single guide (sg) RNA targeting LARP1) and control cells (carrying non-targeting sgRNA), as well as cells treated with pharmacological inhibitors of mTOR (Vistusertib), CDK9 (Enitociclib), or their combination. Control U937 cells expressing non-targeting sgRNA were treated with 500 nM of each inhibitor or DMSO as vehicle.

first: Dominik

middle: A

last: Nahotko

first: Brian

last: Lee

middle: M

first: Leonidas

middle: C

last: Platanias


title: Transcriptome analysis of liver tissue from patients with Autoimmune Hepatitis, Primary Biliary Cholangitis, and Chronic Hepatitis B

city: Huzhou

first: Yunliang

last: Yao

state: Zhejiang

address: 759 East Second Ring Road

department: Medical School

institute: Huzhou University

summary: This study aims to identify immune-related genes in Autoimmune Hepatitis (AIH) by analyzing liver tissue transcriptomes. Bulk RNA sequencing was performed on liver biopsies from patients with AIH and two disease control groups: Primary Biliary Cholangitis (PBC) and Chronic Hepatitis B (CHB). Differential expression analysis was used to compare AIH against both control groups. By intersecting the resulting differentially expressed genes (DEGs) with immune gene databases, we identified 19 common immune-related DEGs (IRDEGs). Gene Ontology (GO) analysis of these genes highlighted functions related to lymphocyte-mediated immunity and T-cell receptor signaling. This dataset provides a resource for exploring liver gene expression changes in AIH.

overall_design: Overall Design: Bulk RNA sequencing was used to compare liver tissue transcriptomes from three patient cohorts: Autoimmune Hepatitis (AIH), Primary Biliary Cholangitis (PBC), and Chronic Hepatitis B (CHB). Sample Processing and Sequencing: Total RNA was isolated from residual liver biopsy tissues. Strand-specific libraries were prepared using the Illumina TruSeq Stranded Total RNA kit and sequenced on an Illumina NovaSeq 6000 platform. Data Analysis: Differentially expressed genes (DEGs) between the AIH group and the two control groups (PBC and CHB) were identified using the DESeq2 R package. The significance threshold was set at a p-value < 0.05 and an absolute log2 fold change > 1.


title: Massively Parallel Screens Reveal Factors Influencing the Activity of a Diversity-Generating Retroelement Reconstituted in Escherichia coli [Tn-Seq]

city: Beverly

first: Hoong Chuin

last: Lim

state: MA

address: 44 Dunham Ridge Road

institute: New England Biolabs

summary: Some phages and microbes employ Diversity-Generating Retroelements (DGRs) to achieve rapid targeted adaptation. DGRs mutagenize a specific gene without harming the rest of the genome. Mutagenesis relies on the DGR reverse transcriptase (dRT), which is highly error-prone when transcribing adenines of an RNA template. The resulting mutagenized cDNA then alters the target gene through a poorly understood mechanism. Transplanting DGRs into a tractable organism, like Escherichia coli, holds promise for creating a powerful system for gene-targeted editing. Here, we successfully reconstituted the archetypal BPP DGR in E. coli, demonstrating that DGRs are "plug-and-play" systems adaptable to non-native hosts. Systematic analysis of reconstituted DGR established principles for target mutagenesis. Our results revealed that cytosine misincorporation is the most common error when dRT transcribes adenines. However, error rates peak in 5'-AAC-3' and 5'-ACC-3' contexts, driven by elevated adenine and guanosine misincorporations, respectively. Using high-throughput genetics, we discovered that cells lacking the single-stranded DNA exonuclease ExoI exhibit 15-fold higher DGR activity. Our evidence indicates that ExoI inhibits DGRs by binding a limited cellular factor, rather than through direct cDNA degradation. Finally, by profiling DGR targets across thousands of chromosomal loci, we discovered that DGRs preferentially edit targets near the replication origin and oriented in the same direction as replication. We showed that the directionality of replication underlies this bias, possibly through unwinding the target to facilitate base-pairing with incoming cDNA. These findings establish a reconstituted DGR system in E. coli and provide a foundation for optimizing targeted gene-editing applications in the future.

overall_design: Transposon sequencing data of a transposon that carried a DGR reporter to identify how genomic location and orientation affect DGR retrohoming. Transposon library sequenced before and after selection by DGR specific reporter. Reporter represents a defective kanamycin resistance gene and DGR targeting sequences transposed all over the genome.

first: Irem

last: Unlu

first: Marina

middle: K

last: Smiley

first: Vladimir

last: Potapov

first: Yoan

last: Renoux-Martin

first: Zhi-Yi

last: Sun


title: Massively Parallel Screens Reveal Factors Influencing the Activity of a Diversity-Generating Retroelement Reconstituted in Escherichia coli [mutagenesis]

overall_design: cDNA sequencing of newly synthesized and mutagenized cDNA by DGRs. Arranged all 16 possible NAN tri-nucleotides in different orientations for templates and sequenced resulting cDNA to understand if sequence context matters for DGR mutagenesis.


title: Massively Parallel Screens Reveal Factors Influencing the Activity of a Diversity-Generating Retroelement Reconstituted in Escherichia coli

sample_organism: Escherichia coli str. K-12 substr. MG1655

overall_design: Transposon sequencing data to identify cellular factors that influence diversity generating retroelements (DGR). Transposon library sequenced before and after selection by DGR specific reporter. Reporter represents a defective kanamycin resistance gene and DGR targeting sequences at 4088112 on the genome.


title: Stress-Survival Pathway Profiling Reveals MCM10 as a Candidate Biomarker of Hispanic Colorectal Cancer Disparities

city: El Paso

first: Md Zahirul

last: Islam Khan

state: TX

address: 500 W University Ave

department: Biological Sciences

institute: University of Texas at El Paso

summary: Background: Colorectal cancer (CRC) remains the second leading cause of cancer mortality in the U.S., with significant racial and ethnic disparities in incidence, survival, and mortality rates. The rising incidence of early-onset CRC and the frequent presentation at advanced stages of CRC among the Hispanics makes this an important ethnic group to study. A deeper understanding of the complex interplay between molecular, genetic, and environmental factors is critical for developing targeted therapies to reduce the prevalence in specific racial and ethnic groups, such as Hispanics. This study explores the role of stress-survival pathway (SSP) genes in early-onset and late-stage CRC, primarily focusing on disparities between Hispanics and Non-Hispanic Whites (NHWs). Additionally, this study investigates the role of MCM10, in contributing to CRC disparities among the Hispanic populations with an emphasis of early-onset and late-stage CRC Hispanics. Methods: One of our previous studies had identified some SSP protein coding genes associated with CRC. The expressions of these genes were validated in CRC cell lines, cDNA arrays, tissue microarrays, and tissue samples using qRT-PCR, and immunohistochemistry. The transcript-level expressions of differentially expressed SSP genes were further evaluated in Hispanic and NHWs tumor tissues, including early onset and late-stage cohorts. Additionally, we performed cellular physiological and functional assays to explore the role of MCM10 in tumor progression, before and after siRNA mediated knockdown. High-throughput transcriptomic and proteomic analyses were performed to reveal the underlying molecular mechanism.  Results: We observed differential expressions of twelve SSP genes in CRC cell lines, at the transcript level; that of ten genes in early and late stages using cDNA arrays, and nine genes at the protein levels using tissue microarrays and immunohistochemistry. Hispanic CRC samples showed differential expression of all nine SSP genes compared to NHWs, in early-onset, and late-stage CRC, with all genes being upregulated other than CDK4 and PRDX4 in late-stage CRC and CDK4 in early-onset CRC. Our functional study demonstrated that MCM10 knockdown in CRC cell lines significantly reduced cell proliferation, growth, invasion and migration by inducing cell cycle arrest, apoptosis and reactive oxygen species pathways. The integrative RNA-sequencing and proteomics study reported that the novel gene PPFI1A could be associated with MCM10 mediated cancer progression. The role of MCM10 and PPFI1A in cancer progression has been validated using a Hispanic CRC patient derived organoid. Conclusion: This study highlights the role of SSP genes, particularly MCM10, in colorectal cancer progression and its potential as a biomarker and therapeutic target to address Hispanic CRC disparities.

overall_design: RNA-seq profiling of Caco-2 and SW480 WT cell lines and their respective MCM10 knockdowns to identify its downstream signaling genes.

first: Urbashi

last: Basnet

first: Soumya

last: Nair

first: Sourav

last: Roy


title: Th17 Cells with Regulatory Phenotype are the Main IL-17F and IL-26 Producers in Palmoplantar Pustulosis

city: Ann Arbor

first: Poulami

last: Dey

address: 1301 Catherine St

institute: Michigan Medicine

summary: Palmoplantar pustulosis (PPP) is a chronic inflammatory skin disorder marked by erythematous pustules and desquamation on the palms and soles. While IL-17 pathways are implicated in PPP, IL-17 blockers have shown modest efficacy, underscoring the need for a deeper understanding of IL-17 involvement. To dissect the cellular and spatial architecture of PPP, we performed single-cell RNA sequencing (scRNA-seq) on lesional, nonlesional, and healthy acral skin to examine cellular composition, transcriptomic profiles, and cell-cell interactions. Unbiased clustering revealed nine major cell types, including an inflammatory keratinocyte subset enriched in IL-17A/TNF signatures and marked by high IL-36G expression. Within the lymphocyte compartment, we identified a hybrid “regTh17” population co-expressing regulatory markers (FOXP3, CTLA4, TIGIT), IL17F, and IL26. This regTh17 subset was distinguished by elevated IL1R1 and CD39, suggesting a IL-1β–driven differentiation. Spatial analyses demonstrated significant neighborhood enrichment of regTh17 cells with IL-36G⁺ supraspinous keratinocytes. RegTh17 cells were the predominant source of IL-17F and IL-26 signals, whereas keratinocytes were predicted as their main receivers. We further observed regTh17 co-expressing TNFRSF4 (OX40) and TNFRSF18 (GITR) specifically at sites of IL36G+ keratinocyte interactions, implicating these pathways in amplification of the IL-17/IL-36 inflammatory loop. Together, our integrated single-cell and spatial profiling uncovers Th17 plasticity in PPP, identifies a regTh17-keratinocyte interaction and highlights IL-17F, IL-26, OX40/OX40L, and GITR/GITRL as candidate targets for precision therapies in this challenging disease.

overall_design: 4-mm lesional and non-lesional punch biopsies were collected from the acral skin of three patients with palmoplantar pustulosis (PPP) and five healthy controls. Single-cell suspensions were prepared and processed using the 10X Genomics Chromium system, followed by sequencing on an Illumina NovaSeq 6000 platform. Raw reads were aligned to the human reference genome (hg38) using Cell Ranger. Downstream analysis included quality control filtering, removal of ambient RNA contamination (SoupX), doublet detection (scDblFinder), data normalization, and batch correction (Harmony) before clustering with Seurat (v5.0.2).

first: Johann

last: Gudjonsson

first: Lam

last: Tsoi


title: Eradication of tumors with pre-existing antigenic heterogeneity by vaccine-mediated co-engagement of CAR T and endogenous T cells

city: Cambridge

first: Charles

middle: Arthur

last: Whittaker

institute: Koch Institute

summary: Chimeric Antigen Receptor (CAR) T-cell therapy can be effective in treating human cancer but, loss/downregulation of the antigen recognized by the CAR poses a major obstacle to tumor eradication. Here, we report an approach for vaccine boosting CAR T-cells, which triggers engagement of the endogenous immune system to stop antigen-negative tumor escape. Vaccine boosting enhanced CAR T-cell polyfunctionality, increased tumor antigen uptake by dendritic cells, and elicited priming of endogenous anti-tumor T cells (antigen spreading). This process required vaccine-induced enhancements in CAR T IFN-γ production and a metabolic shift in CAR T-cells toward oxidative phosphorylation. Antigen spreading induced by vaccine-boosted CAR-T therapy enabled control of solid tumors with substantial pre-existing heterogeneity, which was further enhanced by genetically amplifying CAR T IFN-γ expression. Thus, CAR T cell-derived IFN-γ plays a critical role in promoting endogenous anti-tumor immunity, and vaccine boosting provides a clinically-translatable strategy to drive such responses against solid tumors.

overall_design: Between 4 and 8 replicates of intratumor CAR-T cells and splenic CAR-T cells treated with and without vaccine boosting.

first: Leyuan

last: Ma

first: Duncan

last: Morgan

first: Ina

last: Sulkaj

first: Wuhbet

last: Abraham

last: Li

first: J

last: Love

first: Darrell

middle: J

last: Irvine


title: Oncolytic virus therapy mobilizes tumor-resident CD4+ bystander T cells to restore systemic anti-microbial immunity [ATAC-seq]

city: Chongqing

first: shuai

last: yue

state: Chongqing

address: Gaotanyan street

institute: army medical university

summary: ATAC-seq library was prepared using Hyperactive ATAC-seq Library Prep Kit for Illumina (Vazyme, TD711) to study SM CD4+ T cells in lung and spleen when undergoing OV-BYTE treatment. Here, we further demonstrate that OV-BYTE delivered dual defense against tumorigenesis and pathogen infections in pre-clinical models. This study gives a epigenetic profiles of SM CD4+ T cells in lung and spleen with administration of OV-BYTE treatment.

overall_design: Congenic CD45.1+ SM CD4+ T cells were adoptively transferred into naive C57BL/6 recipients (CD45.2+), which were then infected with LCMV Armstrong and engrafted with MC38 cells on Day 60 post infection. On days 7-12 after tumor engraftment, recipients were intratumorally administered with PBS, NDV-WT or NDV-GP. On day 15 post tumor engraftment, SM CD4+ T cells in tumor and spleen were sorted by FACS and analyzed using ATAC-seq.

first: Shuai

last: Yue

first: Junjian

last: He

first: Shusen

last: Ye

first: Yao

last: Lin


title: Oncolytic virus therapy mobilizes tumor-resident CD4+ bystander T cells to restore systemic anti-microbial immunity

summary: The library preparation for single-cell RNA sequencing (scRNA-seq) using the BD Rhapsody platform to study SM CD4+ T cells in lung and spleen when undergoing OV-BYTE treatment. Here, we further demonstrate that OV-BYTE delivered dual defense against tumorigenesis and pathogen infections in pre-clinical models. This study gives a transcriptome profiles of SM CD4+ T cells in lung and spleen with administration of OV-BYTE treatment.

overall_design: Congenic CD45.1+ SM CD4+ T cells were adoptively transferred into naive C57BL/6 recipients (CD45.2+), which were then infected with LCMV Armstrong and engrafted with MC38 cells on Day 60 post infection. On days 7-12 after tumor engraftment, recipients were intratumorally administered with PBS, NDV-WT or NDV-GP. On day 15 post tumor engraftment, SM CD4+ T cells in lung and spleen were sorted by FACS and analyzed using scRNA-seq.


title: Frail Elderly DNA Methylation and Inflammatory Connections

city: Jakarta

first: Ariel

last: Pradipta

state: Jakarta

address: Jl. Salemba Raya No.6

department: Biochemistry and Molecular Biology

country: Indonesia

institute: Faculty of Medicine Universitas Indonesia

type: Methylation profiling by high throughput sequencing

summary: Various risk factors in aging contribute to the pathogenesis of frailty. Epigenetic changes are among the factors that influence gene expression. It plays a role in regulating the immune system during aging, especially DNA methylation. DNA methylation profiles in the elderly have been widely studied. However, the differences in methylation patterns between robust and frail aging populations remain underexplored. A total of 32 subjects were recruited (age ≥ 65 years old) , consisting of 16 as the frail group and 16 as the robust group. For further methylation sequencing, only one robust subject and four frail subjects were selected. The selection of these individuals was based on their TNF-α concentrations, representing one subject with the lowest and four with the highest TNF-α levels. In this study, we identified an association between altered DNA methylation patterns, mRNA expression levels, and inflammatory conditions in frail elderly. The differential expression of certain genes is likely influenced by differences in methylation level between the frail and robust groups. In overall methylation, the differential of 5mC was slightly significant (p=0.06), while 5hmC was not significantly different (p=0.53). We aim to investigate these differences to gain a deeper understanding of the pathogenesis of frailty in some specific genes.

overall_design: There was no intervention within subjects. PBMC of frail and robust group was isolated from whole blood. The genomic DNA was extracted and underwent in DNA methylation analysis. This approach aims to show the differential methylated region between frail and robust elderly adult.

first: Hiqmah

last: Yusi Yana

first: Siti

last: Setiati

first: Faris

last: Izzatur Rahman

first: Nur

last: Ayu Ramadanti

first: Monica

last: Afrilya Putri


title: Single Cell Multiomics Across Nine Mammals Reveals Cell Type Specific Regulatory Conservation in the Brain.

city: Huntsville

first: J.

middle: Nicholas

last: Cochran

state: AL

address: 601 Genome Way

institute: HudsonAlpha Institute for Biotechnology

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

sample_organism:
Danio rerio
Homo sapiens
Felis catus
Equus caballus
Bos taurus
Oryctolagus cuniculus
Rattus norvegicus

overall_design: Refer to individual Series


title: Effect of Fatty Acid Binding Protein 1 (FABP1) knockdown on gene expression profile in Caco-2 colorectal cancer cells

city: La Plata

first: Natalia

last: Scaglia

country: Argentina

institute: INIBIOLP (National University of La Plata/National Research Council of Argentina)

summary: Lipid metabolism reprogramming is a well-established hallmark of many cancer types, including colorectal cancer (CRC). Nevertheless, a clear understanding on how fatty acid (FA) metabolism is fine-tuned during CRC development and progression is still missing. Given that CRC is the second leading cause of cancer-related death, addressing these critical aspects may provide the rationale for new therapeutic approaches and early biomarker identification. Fatty acid binding protein 1 (FABP1) is a small protein that binds FA and other lipophilic compounds, acting as a lipid transporter in the intestine. Little is currently known about the function of FABP1 in CRC. Here we show that the knockdown of FABP1 in CRC cells impairs de novo FA and cholesterol synthesis, specifically, via altering the transcriptional regulation of lipid metabolism genes. FABP1 depletion suppresses the expression of FA and cholesterol synthesis-associated genes while promoting that of FA oxidation genes and mitochondrial oxidative pathways. The latter is associated with increased oxygen consumption rate and activation of the energy sensor 5’ AMP-activated kinase (AMPK). Taken together, our results show that FABP1 orchestrates the balance between FA synthesis and oxidation, most likely to prevent the cytotoxic effects of circulating unbound free fatty acids. Thus, targeting FABP1 function may represent a potential therapeutic strategy in advanced CRC

overall_design: RNA-Seq profiling of mock-transfected control (pcDNA3) and their FABP1 knockdown derivatives (hFABP1as) Caco-2 cells

first: Delfina

middle: L

last: Borús


title: Shengyu decoction ameliorates knee osteoarthritis by inhibiting endoplasmic reticulum stress via Piezo1 channels

city: jinan

first: xuyu

last: song

address: 47 Shanda South Road

institute: shandong university

summary: Background: Shengyu decoction (SYD) is a classic and excellent prescription of traditional Chinese Medicine (TCM). The innovative use of SYD by Chinese medical master Prof. Qi Shi in the treatment of knee osteoarthritis (KOA) has achieved considerable clinical outcomes. However, the current weakness is the lack of study on the active ingredients and mechanisms of SYD. Purpose: To evaluate the role of SYD in reducing KOA cartilage damage as well as to explore the active ingredients and mechanisms of SYD. Methods: The KOA rat model and chondrocyte model were established. This study employed various molecular biology techniques to clarify the role of SYD in vivo and in vitro. Furthermore, the active ingredients and mechanisms of SYD were analyzed through ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS), RNA sequencing, molecular docking and surface plasmon resonance (SPR) experiment. Finally, rescue experiments were conducted to verify the mechanisms. Results: The results revealed that SYD could significantly reduce cartilage tissue lesions, inhibit inflammation and regulate proliferation-apoptosis balance. Transcriptome analysis showed an increase in the expression of Piezo1, Xbp1, Atf6 in KOA and SYD downregulated them. UPLC-Q-TOF-MS analysis revealed four bioactive compounds of SYD, which were further confirmed to directly interact with Piezo1 through molecular docking and SPR assays. Furthermore, SYD downregulated the calcium ion concentration, Piezo1 and ERS intensity. Meanwhile, in the rescue experiment, Yoda1, the agonist of Piezo1, antagonized the pharmacological effects of SYD. Conclusions: The present results provides strong evidence that SYD protected articular cartilage via inhibiting the Piezo1-mediated ERS signaling pathway. Overall, our work emphasizes the pivotal role of TCM in addressing medical challenges and provides new ideas for the treatment of KOA.

first: Xuyu

last: Song

first: Ying

last: Liu

first: Xianhui

last: Shen

first: Lei

last: Zhang

first: Hongwei

last: Kong

first: Siyi

last: Chen

first: Lisong

last: Sheng

first: Rong

last: Sun


title: Identifying antibody escape mutations using the evolutionary capacity of trVLP-ASV

city: Beijing

first: Yihan

last: Lin

address: No.5 Yiheyuan Road, Haidian

institute: Peking University

summary: The continuous evolution of SARS-CoV-2 poses global health challenges. A safe, rapid, and versatile method for assessing functions of Spike protein mutations in ACE2 receptor binding and immune evasion would be highly valuable. To address this, we engineered a transcription- and replication-competent virus-like particle (trVLP) derived from the Sindbis virus, pseudotyped with the SARS-CoV-2 receptor binding domain (RBD). This trVLP exclusively propagates in BHK-21 cell engineered to express both RNA replicase and human ACE2, providing a controllable, safe model of SARS-CoV-2 RBD-ACE2 interaction mediated virus entry. The system enables characterization of RBD interactions with ACE2 from various mammalian hosts, demonstrating its utility for studying host-virus interactions. By leveraging the evolutionary capability of trVLP mediated by error-prone RNA replication, we screened for RBD variants that evade the antibody-mediated inhibition of cell entry. Together, these findings underscore the utility of the trVLP as a safe, rapid, and flexible platform for dissecting SARS-CoV-2 RBD evolution and identifying key adaptive mutations with implications for surveillance and countermeasure development.

overall_design: In this experiment, we applied selection pressure with a range of antibodies, including REGN10987, LY-CoV1404, SA55, and six other antibodies, on the mutant library derived from the trVLP-ASV, enabling the identification of SARS-CoV-2 RBD mutants that can escape antibody neutralization.

first: Liang

last: Ma


title: Screening for SA55 escape mutations using a DMS library based on trVLP-ASV

overall_design: Using trVLP-ASV as a vector, we constructed a saturated mutant library and applied SA55 antibody selection pressure to comprehensively screen for RBD mutants capable of evading SA55 neutralization.


title: Measuring the mutation rate of the developed trVLP-ASV system

overall_design: Charactering the mutation frequency of the developed trVLP-ASV during continuous passage and used the temporal variation in mutation frequency to estimate its mutation rate.


title: EML4-ALK variant-specific genetic interactions shape lung tumorigenesis

city: Stanford

first: Stewart

last: Kim

state: CA

address: 1291 Welch Rd

department: Genetics

institute: Stanford University

summary: Oncogenic fusions of EML4 and ALK occur in ~5% of lung adenocarcinomas. EML4- ALK variants have distinct breakpoints within EML4, but the functional differences among these variants remain poorly understood. Here we use CRISPR/Cas9 somatic genome editing to generate autochthonous mouse models of the two most common EML4-ALK variants and show that V3 is more oncogenic than V1. By employing multiplexed genome editing and quantifying the effects of 29 putative tumor suppressor genes on V1- and V3-driven lung cancer growth in vivo we show that many tumor suppressor genes have dramatically variant-specific effects on tumorigenesis. Pharmacogenomic analyses suggest that tumor genotype can also modify responses to therapy. Analysis of a large human EML4-ALK lung cancer cohort identified differences in the genomic landscape depending on the EML4-ALK variant. These findings demonstrate functional heterogeneity between EML4-ALK variants, suggesting that EML4-ALK variants behave more like distinct oncogenes than a uniform entity. More broadly, these findings highlight the dramatic impact of oncogenic fusions partner proteins and coincident tumor suppressor gene alterations on the biology of oncogenic fusion driven cancer.

overall_design: To investigate the chromatin regulatory consequences of EML4-ALK translocations with or without Setd2 inactivation in lung tumors, we used a somatic CRISPR/Cas9-based system in Rosa26-LSL-Cas9-EGFP mice. Lentiviral vectors encoding Cre recombinase and sgRNAs targeting introns of Eml4 (intron 14 for V1 or intron 6 for V3) and Alk (intron 19) were used to generate specific Eml4-Alk fusion variants via chromosomal inversion. A third sgRNA targeting Neo or Setd2 was included in each vector. Eight- to fourteen-week-old Cas9-EGFP mice were anesthetized and infected intratracheally with up to 100 μL of lentivirus. This delivery method consistently induced lung tumors harboring either V1 or V3 fusions, with or without Setd2 inactivation. EGFP-positive tumors were microdissected for downstream ATAC-Seq analyses to determine differential chromatin accessibility between samples either possessing Eml-Alk V1 and sgNeo, Eml-Alk V1 and sgSetd2, Eml-Alk V3 and sgNeo, and Eml-Alk V3 and sgSetd2.


title: Xenium Spatial Transcriptomics of Head and Neck Single Cell Carcinoma

city: Houston

first: Kelli

last: McCord

address: 1 Baylor Plz

institute: Baylor College of Medicine

summary: Recent work has shown that the spatial organization of immune responses is a critical determinant of anti-tumor immunity. Here, we profiled ten head and neck squamous cell carcinoma (HNSCC) patient tumors and one ameloblastoma tumor using Xenium V1 spatial transcriptomics. The 10X genomics human multi-tissue and cancer gene expression panel targeting 377 genes was used in combination with 100 custom Xenium probes targeting patient-specific CDR3 regions of T cell receptors (TCRs) in T cells, additoinal T cell specific genes, HPV oncoprotein genes, and tumor genes of interest. This enabled the detection of 477 transcripts within each tumor sample at a single-cell resolution. For seven of the ten HNSCC samples, different tissue sections run on different days were analyzed as technical replicates. Together, these findings introduce a scalable platform for spatial clonal T cell analysis and provide new insight into the spatial relationship of cells within the HNSCC tumor microenvironment.

overall_design: Ten fresh-frozen HNSCC tumors, five of which were HPV+, were used for analysis. Seven of the ten tumors were run in replicate on different slides on different days for a total of 17 HNSCC tumor sections analyzed via Xenium spatial transcriptomics. One ameloblastoma tumor was also analysised via Xenium spatial transcriptomics.

first: William

last: Hudson


title: Massively Parallel Screens Reveal Factors Influencing the Activity of a Diversity-Generating Retroelement Reconstituted in Escherichia coli [TranscriptionalStrength]

first: Hoong Chuin

last: Lim

type: Genome variation profiling by high throughput sequencing

overall_design: DGR reporter integrated in the chromosome at 3758105 under the control of either a strong (J23118) or weak (J23112) promoter. Activated DGR repair to and sequence for mutagenesis rates to test whether the strength of the target's promoter affects repair frequency.

first: Irem

last: Unlu

first: Marina

last: Smiley

first: Vladimir

last: Potapov

first: Yoan

last: Renoux-Martin

first: Zhi-Yi


title: Gene Expression Analysis of Papillary Thyroid Carcinoma with Lymph Node Metastasis and Radioactive Iodine Refractive

city: Cheras

first: Nurul Syakima

last: Ab Mutalib

state: Kuala Lumpur

address: Jalan Yaacob Latiff

department: UKM Medical Molecular Biology Institute

country: Malaysia

institute: Universiti Kebangsaan Malaysia

summary: Radioiodine therapy (RAI) is a safe and effective treatment for DTC. It significantly increases the life expectancy of DTC patients. However, 60% of patients with recurrent or distant metastatic PTC will be radioiodine refractory (RAI-R), where 131I uptake does not occur. Patients with RAI-R thyroid cancer have a poor outcome, with a 5-year disease-specific survival rate of 60% to 70%. Therefore, resensitization of RAI-R tumours to RAI has the potential to improve survival for DTC patients.    Therefore, we used the Agilent One-Colour SurePrint G3 Human Gene Expression V3 Microarray on primary thyroid tumours of 5 PTC patients with negative LN and RAI-avid, 5 PTC patients with positive LN and non-RAI-avid and 4 adjacent normal thyroid tissues to identify potential genes involved in iodine uptake.     The extracted microarray data was analysed using AltAnalyze and NetworkAnalyst software to determine differentially expressed probesets. We used p-value < 0.05 and log2 fold change ≥1 or ≤-1 as the criteria for chosen significantly differentially probesets. Then, we used ShinyGo to annotate the probesets, determine gene type, and distribution analysis. We also used FunRich to make VennDiagram.

overall_design: 5 tumor fresh frozen thyroid tissues were collected from PTC patients with no lymph node metastasis and RAI-avid, 5 tumor fresh frozen thyroid tissues were collected from PTC patients with lymph node metastasis and RAI refractive and 4 adjacent normal fresh frozen thyroid tissues were acquired from the  Hospital Canselor Tuanku Muhriz UKM (HCTM).

first: Nur Fadhlina

last: Mohamad Pakarulrazy

first: Nadiah

last: Abu

first: Shahrun Niza

last: Abdullah Suhaimi

first: Nadzlee Harith

last: Paisol

first: Reena Rahayu

last: Md Zin

first: Nani

last: MD Latar

first: Nurul-Syakima


title: Aerobic exercise effects on tumor and skeletal muscle transcriptome in a DMBA-induced carcinogenesis

city: Portland

first: Rafael

last: Correia

address: 2730 S Moody Ave

institute: Oregon Health & Science University

summary: Here we sought to investigate changes caused by aerobic exercise in the skeletal muscle and tumor during the progression of a DMBA-induced carcinogenesis and cachexia. Also, we investigate the cross-talk between the tissues.

overall_design:
We used Sprague-Dawley rats starting at 50 days of age. Cancer was induced using a single dose of DMBA carcinogen (65 mg/kg), and the induction time took 3 months. After induction, we performed aerobic exercise training (treadmill running)  5 times a week for 8 weeks. Tibialis anterior muscle (n=4 per group) and tumor tissue (n=4 per group) were collected.
Snap-frozen tibialis anterior and tumor tissue were homogenized in liquid nitrogen via mortar and pestle. RNA was isolated from ∼50 mg of tissue using the miRNeasy Mini Kit (Qiagen, cat: 217004). The concentration and quality of total RNA samples were assessed using an Agilent 2100 Bioanalyzer, and an RNA Integrity Number (RIN) of 8 or higher was required to pass the quality control.
Library preparation and sequencing was completed at the Massively Parallel Sequencing Shared Resource at Oregon Health and Science University using the Illumina NovoSeq6000. 150ng of total RNA was used for each sample. cDNA library preparation includes mRNA purification/enrichment, RNA fragmentation, cDNA synthesis, and ligation of index adaptors using the KAPA mRNA Hyperprep Kit prep (cat: KK8581). Each resulting indexed library was quantified, and its quality was assessed by NovaSeq 6000. 5μL of 2nM pooled libraries per lane was denatured, neutralized, and applied to the cBot for flow cell deposition and cluster amplification before loading to 100bp paired-end sequencing (Illumina, Inc.). Approximately 30-50 million reads per library were generated.
The processed data were obtained using the software Partek Flow. The quality trimming level (Phred) was 35 (3' end). We aligned the reads to the UCSC Rattus norvegicus (rn7) reference genome using STAR (Spliced Transcripts Alignment to a Reference). To quantify gene expression, we used the annotation model Rn07. The gene counts were filtered using a noise filter reduction filter <=10. The normalization was made using Median ratio (DESeq2) and differential analysis was made using DESeq2.

middle: R

first: Teresa

last: Zimmers

first: Giovana

last: Teixeira


title: The Diabetes Gene Tcf7l2 Organizes Gene Expression in the Liver and Regulates Amino Acid Metabolism

city: Boston

first: Sudha

last: Biddinger

state: Massachusetts

address: 3 Blackfan Circle, 16th Floor (Endocrinology)

institute: Boston Children's Hospital

summary: TCF7L2 harbors the strongest genetic association with diabetes identified thus far. However, its function in liver has remained unclear. Here, we find using mice with liver-specific deletion, that Tcf7l2 plays a central role in maintaining hepatic zonation. That is, in the normal liver, many genes show gradients of expression across the liver lobule; in the absence of Tcf7l2, these gradients collapse. One major consequence is the disorganization of glutamine metabolism, with a loss of the glutamine production program, ectopic expression of the glutamine consumption program, and a decrease in glutamine levels. In parallel, metabolomic profiling shows glutamine to be the most significantly decreased metabolite in individuals harboring the rs7903146 variant in TCF7L2. Taken together, these data indicate that hepatic TCF7L2 has a secondary role in glycemic control, but a primary role in maintaining transcriptional architecture and glutamine homeostasis.

overall_design: Tcf7l2Flox/Flox mice20 were purchased from Jackson Laboratories (strain code: 031436) and group-housed at 21 °C on a 12 hour light/dark cycle (7 am/7 pm) with free access to food, unless otherwise indicated. At the end of the experiment, mice were killed in the ad libitum state at 2 pm EST to collect plasma and tissues. All animal experiments were performed under approval of the Institutional Animal Care and Research Advisory Committee (IACUC) at Boston Children’s Hospital. Tcf7l2Flox/Flox littermates were injected retro-orbitally at five to seven weeks of age with 1 x 1011 genome copies of AAV8-TBG-GFP (CON, Penn Vector Core, 105535-AAV8) or AAV8-TBG-CRE (TCF7L2 L-KO, Penn Vector Core 107787-AAV8). They were then placed on a Western Diet (Envigo, TD.88137, irradiated, 42% kcal from fat, 0.2% cholesterol, and 34% sucrose by weight) for twelve weeks.

first: Joanna

last: Krawczyk

first: William

last: O'Connor

first: Pedro

last: Vendramini

first: Mareike

last: Schell

first: Kiran

first: Matt

last: Kanke

first: George

last: Pengo

first: Tiffany

last: Fougeray

first: Praveen

last: Sethupathy

first: Marcia

last: Haigis

first: Krishna

middle: G

last: Aragam

first: Wouter

last: Lamers

first: Linus

last: Tsai


title: Single-cell RNA sequencing reveals B cell-related immunosuppressive landscape and a potential suppressor in hepatocellular carcinoma

city: Jinan

first: Wenhao

last: Hou

address: xingfu

institute: Shandong First Medical University

summary: Background Sophisticated tumor microenvironment is responsible for malignant progression and poor prognosis of hepatocellular carcinoma (HCC) patients. Discovering new therapeutic targets was desired for preferable treatments of HCC patients. Methods To uncover the HCC microenvironment, single-cell transcriptomes of HCC tissues and corresponding non-cancerous tissues were analyzed. Differentially expressed genes (DEGs) and enriched pathways were analyzed in B cells. Moreover, heterogeneity between malignant and normal hepatocytes was further investigated, which revealed potential biomarkers in HCC progression. The candidate biomarkers were further explored in datasets from The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) databases. Of which, serum amyloid A2 (SAA2) was found out and further validated in HCC tissues by immunohistochemistry (IHC) and western blot. The biological roles of SAA2 were further investigations in HCC cells. Results The results suggested that B cells in HCC tissues was significantly decreased compared to non-cancerous tissues, which might result in the immunosuppressive status of HCC microenvironment. Following differentially expressed genes (DEGs) and functional enriched analysis indicated B cells might participate in immunosuppression of HCC by regulating lipid metabolism. Further analysis on hepatocytes found out highly expressed genes in normal hepatocytes derived from non-cancerous liver tissues, which were validated in datasets from The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) databases. Interestingly, we found serum amyloid A2 (SAA2) was highly expressed in normal liver tissues compared to HCC tissues. The results were validated in clinical HCC specimens by immunohistochemistry (IHC) and western blot assays. Moreover, investigations in HCC cells revealed SAA2 acted as a tumor suppressor in HCC progression. Conclusions Taken together, our study might be of great significance for revealing B cell-related immunosuppressive landscape in HCC, as well as discovering SAA2 as a novel suppressor in HCC, which made great sense for better understanding of HCC landscape and offered a promising therapeutic target for HCC patients. Keywords: B cell-related immunosuppressive landscape, hepatocellular carcinoma, serum amyloid A2, single-cell RNA sequencing, suppressor, tumor microenvironment

overall_design: Although the HCC microenvironment has been explored extensively, the tumor ecosystem at single-cell level remains to be further resolved. To investigate the HCC microenvironment at cellular level, we designed the following study. The complete study process was shown in Figure 1A. In order to build a comprehensive single-cell reference for HCC, we recruited 5 newly treated HCC patients for scRNA-seq. Detailed clinical and pathological information including gender, age, tumor size, alpha-fetoprotein (AFP) levels, Child-Pugh stage, and Barcelona Clinic Liver Cancer (BCLC) stage were shown in Supplementary Table S1. These patients were diagnosed as HCC pathologically and their surgical HCC specimens as well as paired corresponding non-cancerous tissues were collected. Sequencing libraries of pooled single cells were obtained and subjected to a sequencing run. The Chromium platform (10×Genomics) was used to capture single cells in prepared single-cell suspensions. The single cells were collected in form of droplets and marked with specific barcodes and then subjected to a subsequent series of manipulation procedures. The captured mRNAs were reverse-transcribed into cDNA and amplified for single-cell library preparation . We applied QC with key metrics including number of genes, total counts, and percentage of mitochondrial counts . After applying the QC criteria, a total of 73707 single cells were retained and included in the downstream analysis, of which 36323 single cells were from HCC tissues and the rest were from corresponding non-cancerous tissues (Supplementary Table S2). These identified cells produced an average of 2783 detected genes. Then, the cell compositions in HCC ecosystem were resolved and cell clusters closely related to the immunosuppressive landscape of HCC especially B cells, were analyzed for DEGs and corresponding signal pathways.

first: Lihui

last: Zhu

first: Jiaqi

last: Jiang

first: Liyuan

first: Hongwei

last: Xu


title: Epigenetic silencing of interleukin-10 by host-derived oxidized phospholipids supports a lethal inflammatory response to infections supports a lethal inflammatory response to infections

city: Los Angeles

first: Roberto

last: Spreafico

address: UCLA

department: Institute for Quantitative and Computational Biosciences

institute: University of Califonia Los Angeles

summary: Phagocytes initiate immunity to invading microorganisms by detecting pathogen-associated molecular patterns via pattern recognition receptors. Pathogen encounter and consequent activation of the immune system cause tissue damage and release of host-derived damage-associated molecular patterns, contributing to shape immunity. How self-derived factors are sensed by phagocytes and impact the immune response remains poorly understood. Here we demonstrate that, in mice and humans, host-derived oxidized phospholipids (oxPLs) are formed after microbial encounter. We show that oxPLs exacerbate inflammation without affecting pathogen burden. Mechanistically, oxPLs bind and inhibit AKT, potentiating the methionine cycle and the activity of the epigenetic writer enhancer of zeste homolog 2 (EZH2). EZH2 epigenetically dampens the pluripotent anti-inflammatory cytokine interleukin-10, contributing to death of the host. Overall, we found that host-derived oxPLs set the balance between protective and detrimental anti-microbial responses and that they can be prophylactically or therapeutically targeted to protect the host against deranged inflammation and immunopathology.

overall_design: BMDMs were stimulated with LPS for 3h and then treated, or not, with oxPAPC for 3h

first: Marco

last: Di Gioia

first: Valentina

last: Poli

first: Piao J

last: Tan

first: Anne

last: Chu

first: Alex G

last: Cuenca

first: Zhongyang

last: Wu

first: Mehdi

last: Benamar

first: Laura

last: Pandolfi

first: Federica

last: Meloni

first: Fatemeh

last: Askarian

first: Jason

last: Hsiao

first: Elizaveata

last: Borroum

first: Victor

last: Nizet

first: Philip LSM

last: Gordts

first: Joseph L

last: Witztum

first: Talal A

last: Chatila

first: Janet

last: Chou

first: Xu

last: Zhou

first: James R

last: Springstead

first: Ivan

last: Zanoni


title: Elevated expression of the molecular clock suppressors Period 1 and Period 2 promote myotube atrophy in vitro with implications to the loss of limb muscle mass following androgen deprivation

city: Tallahassee

first: Cynthia

last: Vied

state: Florida

address: 1115 W. Call St

department: Translational Science Laboratory

institute: Florida State University

summary: Low testosterone levels in men (hypogonadism) are associated with loss of limb muscle mass. We previously showed molecular clock function is disrupted in limb muscle by androgen deprivation. That clock disruption coincided with higher protein levels of the molecular clock suppressor, Period 2 (Per2). The primary purpose of this study was to assess whether Per2 regulates muscle size. Transducing fully differentiated C2C12 myotubes with adenovirus overexpressing Per2 was sufficient to lower myotube diameter. Moreover, deletion of Per2 in male mice partially maintained mass of the tibialis anterior (TA) muscle following castration-induced androgen deprivation. The partial maintenance of mass was specific to the TA even though molecular clock disruption is evident in other limb muscles. Thus, the secondary purpose of this study was to assess whether Per1 also regulates muscle size. Overexpression of the other primary molecular clock suppressor, Per1, in C2C12 myotubes was also sufficient to decrease myotube diameter. The lower myotube diameter in response to Per1 or Per2 overexpression was due in part to an autocrine-mediated inflammatory signals as conditioned media from Per1 or Per2 overexpressing myotubes contained elevated cytokines/chemokines, and the Per conditioned media was sufficient to lower C2C12 myotube diameter. The mRNA contents of inflammatory genes altered following Per1 or Per2 overexpression in vitro were altered in limb muscle in vivo following castration. These data define a novel muscle mass/size regulatory function of the primary molecular clock suppressors with direct implications for their role in regulating limb muscle mass loss in response to low androgen levels

overall_design: C2C12 myotubes were transduced with adenovirus overexpressing Green Fluorescent Protein (Gfp), Period 1 (Per1), or Period 2 (Per2). Myotubes were harvested in TriReagent 48 hours post transduction. Total RNA was isolated using Zymo spin columns, and the RNA was processed and subsequently subjected to RNA sequencing analysis.

first: Grant

last: Laskin

first: Jennifer

last: Steiner

first: Wayne

last: Ayers-Creech

first: Michael

last: Rossetti

first: Kirsten

last: Dunlap

first: Choogon

last: Lee

first: Nicholas

middle: P

last: Greene

first: Dennis

last: Fix

first: Orlando

last: Laitano

first: Bradley

middle: S

last: Gordon


title: Arsenic trioxide regulates DYNAP through hsa-mir-573 and inhibits the proliferation of laryngeal cancer

city: Shandong Province

first: ren

middle: yan

last: ru

address: Rencheng District, Jining City, Shandong Province

institute: School of Clinical Medicine, Jining Medical University

type: Non-coding RNA profiling by high throughput sequencing

summary:
BACKGROUND: Laryngeal squamous cell carcinoma (LSCC) is a malignant tumor with limited treatment options and poor prognosis in advanced stages. Arsenic trioxide (ATO), a drug well-known for treating acute promyelocytic leukemia, has shown potential antitumor effects in several solid tumors. This study aimed to investigate the role of ATO on LSCC proliferation and its underlying molecular mechanisms.
METHODS: LSCC cell lines (TU212, TU686, and AMC-HN-8) were treated with varying concentrations of ATO, and cell proliferation was evaluated using CCK-8, colony formation, and EdU assays. miRNA-sequencing identified differentially expressed miRNAs after ATO treatment, and bioinformatics tools predicted hsa-miR-573 target genes. The interaction between hsa-miR-573 and dynactin-associated protein (DYNAP) was validated by dual-luciferase reporter assays. Additionally, a xenograft tumor model was established to examine the in vivo effects of ATO on tumor growth.
RESULTS: ATO significantly inhibited LSCC cell proliferation in a dose- and time-dependent manner. miRNA-sequencing identified hsa-miR-573 as significantly upregulated following ATO treatment, and functional studies demonstrated that hsa-miR-573 suppresses LSCC cell proliferation by directly targeting DYNAP. Overexpression of DYNAP promoted LSCC cell proliferation, while DYNAP knockdown reversed this effect. In vivo, ATO treatment suppressed tumor growth in nude mice without significant nephrotoxicity or cardiotoxicity. Mechanistically, ATO reduced the expression of DYNAP and inhibited the PI3K/AKT signaling pathway.
CONCLUSION: ATO inhibited LSCC progression by upregulating hsa-miR-573, which directly targets DYNAP to suppress cell proliferation and disrupt the PI3K/AKT signaling pathway. These findings supported the potential of ATO as a therapeutic agent for LSCC.

overall_design: The experimental design involves setting up an arsenic trioxide intervention group and an untreated control group for TU212 cells, with three replicate samples in each group. Then, sequencing is performed on both groups to investigate the related biological changes induced by arsenic trioxide intervention.

first: Yanru

last: Ren

first: Xiao

last: Yang

first: Yang

last: Hui

first: Weiyao

last: Chen

first: Yi

last: Cheng

first: Ning

last: Zhang

first: Tao

last: Liu

first: Xinxin

first: Xiaoyu

last: Li


first: Roberto

last: Spreafico

overall_design: BMDMs were stimulated with LPS for 3h and then treated, or not, with oxPAPC for 3h or 24h.

first: Marco

last: Di Gioia

first: Valentina

last: Poli

first: Piao J

last: Tan

first: Anne

last: Chu

first: Alex G

last: Cuenca

first: Zhongyang

last: Wu

first: Mehdi

last: Benamar

first: Laura

last: Pandolfi

first: Federica

last: Meloni

first: Fatemeh

last: Askarian

first: Jason

last: Hsiao

first: Elizaveata

last: Borroum

first: Victor

last: Nizet

first: Philip LSM

last: Gordts

first: Joseph L

last: Witztum

first: Talal A

last: Chatila

first: Janet

last: Chou

first: Xu

last: Zhou

first: James R

last: Springstead

first: Ivan

last: Zanoni


title: Genetic map of the adult carotid body stem cell niche

city: Seville

first: Ricardo

last: Pardal

address: Avda. Manuel Siurot, s/n

department: Medical Physiology and Biophysics

country: Spain

institute: University of Seville

summary: We employed a single-cell RNA sequencing (scRNA-seq) approach using the 10x Genomics platform to investigate the transcriptomic landscape of all cellular constituents within the adult carotid body germinal niche. This organ serves as the principal peripheral chemoreceptor in mammals, mediating the detection of hypoxemia. To our knowledge, this study represents the first comprehensive transcriptomic profiling of all cell types residing in this niche, including neural crest-derived progenitor cells and oxygen-sensitive glomus (type I) cells. Furthermore, we analyzed samples obtained from animals maintained under normoxic and hypoxic conditions, enabling comparative analyses that revealed molecular and cellular alterations elicited by hypoxic stimulation.

overall_design: Following 8 days of either normoxia or hypoxia (10% oxygen), young adult Wistar rats were used to dissect their carotid bodies, enzymatically disperse the cells, and analyze their transcriptomes using scRNA-seq.

first: Verónica

last: Sobrino


title: Genetic Ptbp1 depletion does not induce neuronal RNA splicing patterns in mature astrocytes (Single-cell RNA-seq)

city: Riverside

first: Sika

last: Zheng

address: University Ave 900

department: School of Medicine

institute: University of California, Riverside

summary: Neuronal reprogramming of astrocytes poses a promising therapeutic approach to replace degenerating cells with newly converted neurons. Recent studies reported depletion of polyprimidine tract binding protein PTBP1, a repressor of neuronal alternative splicing, could induce astrocyte to neuron conversion and rescue functional deficits in models of neurodegenerative disease, while others disputed the astrocytic origin of converted neurons. Mechanistic understanding of the conversion, or the lack thereof, requires investigating the transcriptomic effects of Ptbp1 loss of function in mature astrocytes on RNA splicing, in conjunction with lineage tracing, which have not yet been examined. Here, we used genetic depletion of Ptbp1 in adult Aldh1l1-Cre/ERT2 reporter mice to determine whether lineage traced Ptbp1 knockout astrocytes exhibited RNA splicing alterations underlying potential neuronal transdifferentiation. Our analysis employed an alternative genetic Ptbp1 knockout mouse model and an extended 12-week window of Ptbp1 knockout to match previous reports of positive conversion. We found no widespread induction of neuronal identity, despite detecting a minuscule fraction of Cre-positive cells with neuronal transcriptomic features following Ptbp1 deletion. Importantly, PTBP1 depletion in mature astrocytes induces splicing alternations unlike neuronal features of alternative splicing. These findings highlight the challenge of targeting astrocytes for neuronal conversion via Ptbp1 knockout and underscore the importance of cellular gene expression profiling and genetic lineage tracing for in vivo studies of cell type conversion.

overall_design: Gene expression analysis for tdTomato+ cells from brains of control (+/+;tdT+/-;Aldh1l1-Cre+/-) and (Ptbp1loxp/loxp;tdT+/-;Aldh1l1-Cre+/-) mice.

first: Min

last: Zhang

first: David

last: Nikom

first: Naoto

last: Kubota


title: Genomic imprinting at a porcine ZNF locus via a canonical imprinting mechanism [RNA-seq]

city: Columbus

first: Jinsoo

last: Ahn

state: OH

address: 2029 Fyffe Rd

institute: The Ohio State University

summary: Genomic imprinting is an epigenetic mechanism that results in parent-of-origin dependent gene expression, primarily driven by differential DNA methylation between the maternal and paternal alleles. To identify genomic imprinting in pigs, we generated parthenogenetic porcine embryos alongside biparental normal embryos and performed whole-genome bisulfite sequencing and RNA-seq on these samples. By comparing parthenogenetic and control porcine embryos, we identified a maternally methylated region at a previously uncharacterized pig locus. Our findings provide mechanistic insights into genomic imprinting at a porcine locus.

overall_design: RNA-seq was performed on parthenogenetically activated embryos and control pig embryos.

first: Seongsoo

last: Hwang

first: Kichoon

last: Lee


title: Dysregulation of protein homeostasis by mutant UBA1 in VEXAS syndrome [WES]

city: Cincinnati

first: Kwangmin

last: Choi

state: Ohio

address: 333 Burnet Avenue

department: Experimental Hematology & Cancer Biology

institute: Cincinnati Children's Hospital Medical Center

summary: VEXAS is a hematopoietic disorder characterized by hyperinflammation,high mortality, and mutations at methionine 41 (M41) in the E1 ubiquitin enzyme, UBA1. Here, we developed ahuman model of VEXAS by engineering the male THP1 cell line to express the UBA1-M41V mutation. We found that UBA1-M41V cells exhibit aberrant UBA1 isoform expression, increased vacuolization, and upregulation of the unfolded protein response, recapitulating features of VEXAS.Proteomic analyses revealed dysregulated ubiquitination and proteotoxic stress in UBA1-M41V cells, with alterations in inflammatory and stress-response pathways. UBA1-M41V cells were sensitive to genetic or pharmacological inhibition of E1 enzymes. Treatment with theE1 inhibitor TAK-243 preferentially suppressed colony formation of UBA1-M41V cells. Moreover,UBA1-M41V cells exhibited greater sensitivity to TAK-243 in competition assays and showed increased apoptosis. Interestingly, TAK-243 preferentially inhibited UBA6 activity over UBA1 ,suggesting that UBA6 may compensate for UBA1 dysfunction. Targeting UBA6 using shRNA orthe UBA6-specific inhibitor phytic acid further revealed an acquired dependency on UBA6 inUBA1-M41V cells. Phytic acid impaired UBA1-M41V cells while sparing WT cells. Together, these findings establish a novel human model of VEXAS, identify key roles for UBA1 and UBA6, and demonstrate that UBA6 inhibition represents a therapeutic strategy for selectively targeting UBA1 mutant clones.

overall_design: THP1 UBA1 WT and M41V mutant UBA1 cells were collected, and DNA was extracted using  Qiagen Dneasy kit. Following initial sample dsDNA quality control for quantity with a Qubit fluorometer and size distribution with an Agilent Fragment Analyzer, 50 ng of gDNA is enzymatically fragmented using Twist Bioscience’s Library Preparation EF Kit 2.0 (Twist Bioscience, 104207). Index adapters (Twist Bioscience, 100577) are ligated onto the DNA fragments, and then amplified with 10 cycles of PCR. Libraries are purified with Binding and Purification beads (Twist Bioscience, 100983), quantified with a Qubit fluorometer, and checked again on a Fragment Analyzer. Up to 8 libraries of like organisms are pooled together with a total mass of 1500 ng/pool. Human DNA is then hybridized with Twist’s Comprehensive Exome probes (Twist Bioscience, 102032). Samples are hybridized for 30 minutes with Twist’s Fast Hybridization Reagents (Twist Bioscience, 104180) before being bound to streptavidin beads and washed with Twist’s Fast Hybridization and Wash Kit (Twist Bioscience, 104180). Following hybridization, hybridized pools are amplified with 8 cycles of PCR using primers and master mix from the Library Preparation Kit EF. Final libraries are purified with Twist’s Binding and Purification beads, quantified with a Qubit fluorometer, and their size checked with a Fragment Analyzer. Libraries are sequenced on an Illumina NovaSeq X Plus, generating > 25 million 100 bp read pairs and analyzed using Illumina’s Dragen enrichment pipeline. Data Alignment and Analysis Methods Reads were aligned using a functionally equivalent pipeline, as previously defined70. Specifically, each read pair was separately aligned to the appropriate reference (GRCh38) genome using bwa-mem version 0.7.17 with the -M option. The resulting SAM files are first sorted and converted to BAM with Picard SortSam (V2.18.22), then all resulting BAMs are merged and duplicate marked with Picard MarkDuplicates (https://broadinstitute.github.io/picard/) using the “OPTICAL_DUPLICATE_PIXEL_DISTANCE=2500” command line flag. Variants were filtered and subsetted using BCFtools (v1.9, https://samtools.github.io/bcftools/) and annotated with Variant Effect Predictor (VEP, https://useast.ensembl.org/info/docs/tools/vep/).

first: Courtnee

last: Clough

first: Claire

last: Cunningham

first: Sophia

last: Philbrook

first: Kathleen

last: Hueneman

first: Avery

last: Sampson

first: Choi

last: Kwangmin

first: Kenneth

last: Greis

first: Daniel

last: Starczynowski


title: Mechanistic basis for protection against fatty liver disease by CIDEB loss-of-function mutations

city: Dallas

first: Qiyu

last: Zeng

address: 5323 Harry Hines Blvd

institute: UTSW

summary: Background & Aims: Somatic and germline CIDEB mutations are associated with protection from chronic liver diseases. The mechanistic basis and whether CIDEB suppression would be an effective therapy against fatty liver disease remain unclear. Methods: 21 CIDEB somatic mutations were introduced into cells to assess functionality. In vivo screening was used to trace Cideb mutant clones in mice fed normal chow, western (WD), and choline-deficient, L-amino acid-defined, high-fat (CDA-HFD) diets. Constitutive and conditional Cideb knockout mice were generated to study Cideb in liver disease. Isotope tracing was used to evaluate fatty acid oxidation and de novo lipogenesis. Transcriptomics, lipidomics, and metabolic analyses were utilized to explore molecular mechanisms. Double knockout models (Cideb/Atgl and Cideb/Ppara) tested mechanisms underlying Cideb loss. Results: Most CIDEB mutations showed that they impair function, and lineage-tracing showed that loss-of-function clones were positively selected with some, but not all fatty liver inducing diets. Cideb KO mice were protected from WD, CDA-HFD, and alcohol diets, but had the greatest impact on CDA-HFD induced liver disease. Hepatocyte-specific Cideb deletion could ameliorate disease after MASLD establishment, modeling the impact of therapeutic siRNAs. Cideb loss protected livers via increased β-oxidation, specifically through ATGL and PPARa activation. Conclusions: Cideb deletion is more protective in some types of fatty liver disease. β-oxidation is an important component of the Cideb protective mechanism. CIDEB inhibition represents a promising approach, and somatic mutations in CIDEB might predict the patient populations that might benefit the most.

overall_design: RNAseq profiling of Cidebfl/fl mice injected with AAV-TBG-GFP or Cre on Western diet and CDA-HFD for 3 months.


title: Dysregulation of protein homeostasis by mutant UBA1 in VEXAS syndrome [RNA-seq]

overall_design: THP1 WT, M41V, Scramble non-targetting, UBA1 knockdown, and UBA6 knockdown cells were collected in triplicate, and RNA was extracted using ZYMO RESEARCH Quick-RNA Miniprep kit (cat #R1055). After the confirmation of the RNA quality using Agilent 2100 Bioanalyzer, the libraries were prepared with polyA selection using the Truseq RNA Library Prep Kit, and the libraries were sequenced at an average depth of 30M paired-end 100bp nucleotide reads. After the quality of reads was examined using FastQC (v0.11.7, https://www.bioinformatics.babraham.ac.uk/projects/fastqc), paired-end reads were aligned against human GRCh38 genome (iGenome,https://support.illumina.com/sequencing/sequencing_software/igenome.html) using HISAT2 (v2.0.5,http://daehwankimlab.github.io/hisat2). The raw gene counts were calculated using featureCounts (v1.5.2, http://subread.sourceforge.net/) and normalized using edgeR (v3.16.5, https://bioconductor.org/packages/release/bioc/html/edgeR.html) in iGEAK (https://pubmed.ncbi.nlm.nih.gov/30841853/). Differentially expressed genes were predicted using limma/voom (v3.30.6, https://bioconductor.org/packages/release/bioc/html/limma.html).


title: Genomic imprinting at a porcine ZNF locus via a canonical imprinting mechanism [WGBS]

overall_design: Whole genome bisulfite sequencing was performed on parthenogenetically activated embryos and control pig embryos.


title: Function and Mechanism of METTL5-mediated translation in multiple myeloma

city: Nanchang

first: Junyao

last: Jiang

address: 1 Minde Road, Nanchang City, Jiangxi Province

department: Department of Clinical Laboratory

institute: The Second Affiliated Hospital, Jiangxi Medical College, Nanchang University

summary: The abnormal expression of m6A methyltransferase is a significant factor in the occurrence and progression of tumors. The 18S ribosomal RNA (rRNA) m6A methyltransferase Methyltransferase-like 5 (METTL5) is upregulated in various cancers, leading to adverse prognosis by abnormally regulating protein translation in tumor cells. However, the functionality and molecular mechanisms of METTL5 in the progression of multiple myeloma (MM) remains unknown. In this study, we demonstrate that the expression of METTL5 in the bone marrow (BM) of newly diagnosed MM patients is significantly higher than in healthy individuals and patients in remission following treatment. Importantly, we found that MM patients with upregulated METTL5 expression had a poorer prognosis. Additionally, we show that METTL5 plays a key role in promoting MM progression both in vitro and in a orthotopical xenograft model. Mechanistically, the depletion of METTL5 expression mediates a decrease in overall translation efficiency and selenium metabolism-related signaling pathway levels. We further revealed that the reduction in selenophosphate synthetase 2 (SEPHS2) translation efficiency mediated by METTL5 depletion can lead to diminished synthesis of selenoprotein and lead to increased reactive oxygen species (ROS), thereby inducing apoptosis in MM. Salvianolic Acid C (SAC) was identified as a potential METTL5 inhibitor, demonstrating significant pro-apoptotic effects during the treatment of MM both in vitro and in vivo. In summary, our research highlights the critical role of METTL5 in the progression of MM cells. Our data indicate METTL5’s function is to influence the overall translation efficiency and reprogram selenium metabolism to inhibit apoptosis.

overall_design: RNA-seq and Ribo-seq were used to study the differential translated genes in the METTL5 knockout and control cells.

first: Xiaozhong

last: Wang

first: Bo

last: Huang

first: Fangmin

last: Zhong


title: Upregulated Natriuretic Peptide B expression as a hallmark of chronic itch

city: Baltimore

first: Pang-Yen

last: Tseng

state: MD

address: 655 W. Lombard St.

department: Pain and Translational Symptom Science

institute: University of Maryland Baltimore

summary: To examine transcriptional plasticity in sensory neurons, we performed bulk RNA-sequencing studies on dorsal root ganglia from an atopic dermatitis-like mouse  and a contact dermatitis monkey model.

sample_organism:
Macaca nemestrina
Mus musculus

platform_organism:
Macaca nemestrina
Mus musculus

overall_design: Dorsal root ganglia of ADAM17 knockout mice and Squaric acid dibutyl ester (SADBE) treated pig-tailed monkeys (Macaca nemestrina) were harvested to perform bulk RNA-sequencing analysis.

first: Mark

last: Hoon


title: Insulator BEAF32 regulates expression of tissue-specific genes and piRNA source loci in the Drosophila ovaries

city: Moscow

first: Anastasiya

last: Kobelyatskaya

address: Vavilov str, 32

country: Russia

institute: Engelhardt Institute of Molecular Biology, RAS

summary:
Background: Insulators are the multifunctional DNA binding proteins that perform architectural functions and regulate gene transcription. Although insulators have a well-established role in genome organization, it is still unclear how insulator proteins affect the control of tissue-specific processes. Drosophila insulator BEAF32 (Boundary Element-Associated Factor of 32 kD) is a component of chromatin complexes found in the open chromatin regions containing promoters of housekeeping genes. BEAF32 knockout impairs oogenesis and female fertility suggesting its specific functions during oogenesis
Results: To get a better understanding of BEAF32 roles in oogenesis, we first examined its ovarian binding targets and discovered an enrichment of its localization sites in the promoters of both housekeeping and tissue-specific genes. Differential expression gene analysis revealed that BEAF32 knockout resulted in abnormal activation of non-ovarian tissue-specific genes in the ovaries, implying that BEAF32 regulates tissue-specific patterns of gene expression. Among upregulated genes there were not only BEAF32-associated genes suggesting direct and indirect mechanisms of BEAF32-mediated expression control. We discovered that BEAF32 occupied many ovary-specific gene promoters and acted as a positive regulator of the cell-cycle regulatory kinase Polo expression. To reveal the possible role of BEAF32 in the pathway mediated by Piwi-interacting RNAs (piRNAs) we performed an analysis of ovarian small RNAs in the BEAF32 null mutants and revealed a strong decrease in the production of piRNAs from the 3R subtelomeric region. Our data suggest that BEAF32-containing chromatin complex located upstream of the subtelomeric repeats preserves transcriptional and chromatin integrity of this domain in the germline. BEAF32 was also found upstream of flamenco, a largest piRNA source locus in the follicular cells, and is required for the cell-specific transcription of the flam locus.
Conclusions: These findings suggest that BEAF32 represses the ectopic expression of developmental and tissue-specific genes in the ovaries. BEAF32 regulates polo kinase and other oogenesis-related genes. BEAF32 insulators have been revealed to play a specific ovarian role in the maintenance of piRNA-producing loci. Our results provide strong support for the importance of BEAF32 insulator protein in determining the proper landscape of tissue-specific gene expression and locus-specific function realization.

overall_design: Deep-sequencing of chromatin-associated RNAs of Drosophila melanogaster ovaries

first: Alla

last: Kalmykova


title: Genetic Ptbp1 depletion does not induce neuronal RNA splicing patterns in mature astrocytes (Bulk RNA-seq)

first: Sika

last: Zheng

overall_design: Comparative gene expression and splicing analysis for tdTomato+ cells from brains of control (+/+;tdT+/-;Aldh1l1-Cre+/-) and (Ptbp1loxp/loxp;tdT+/-;Aldh1l1-Cre+/-) mice.

first: Min

last: Zhang

first: David

last: Nikom

first: Naoto

last: Kubota