title: Characterization of TNG348: a selective, allosteric USP1 inhibitor that synergizes with PARP inhibitors in tumors with DNA damage repair alterations

first: Antoine

last: Simoneau

address: 201 Brookline

institute: Tango Therapeutics

summary: Inhibition of the deubiquitinating enzyme USP1 can induce synthetic lethality in tumors characterized by homologous recombination deficiency (HRD) and represents a novel therapeutic strategy for the treatment of BRCA1/2 mutant cancers, potentially including patients whose tumors have primary or acquired resistance to PARP inhibitors (PARPi). Here, we present a comprehensive characterization of TNG348, an allosteric, selective, and reversible inhibitor of USP1 (USP1i). TNG348 induces dose-dependent accumulation of ubiquitinated protein substrates both in vitro and in vivo. CRISPR screens show that TNG348 exerts its anti-tumor effect by disrupting the translesion synthesis pathway of DNA damage tolerance through RAD18-dependent ub-PCNA induction. Though TNG348 and PARPi share the ability to selectively kill HRD tumor cells, CRISPR screens reveal that TNG348 and PARPi do so through discrete mechanisms. Particularly, knocking out PARP1 causes resistance to PARPi but sensitizes cells to TNG348 treatment.  Consistent with these findings, combination of TNG348 with PARPi leads to synergistic anti-tumor effects in HRD tumors, resulting in tumor growth inhibition and regression in multiple mouse xenograft tumor models. Importantly, our data in human cancer models further show that the addition of TNG348 to PARPi treatment can overcome acquired PARPi resistance in vivo. While the clinical development of TNG348 has been discontinued due to unexpected liver toxicity in patients (NCT06065059), the present data provides preclinical and mechanistic support for the continued exploration of USP1 as a drug target for the treatment of patients with BRCA1/2 mutant or HRD cancers.

overall_design: Cas9-expressing cells were infected with a library containing guide RNAs targeting 1145 DNA damage response genes (UWB1.289 and MDAMB436) or a whole genome library (COV362 and HCC1395) and subsequently grown in the presence of DMSO, TNG348 or olaparib for 10 population doublings. Guide enrichment or depletion was determined by sequencing comparing TNG348- or olaparib- to DMSO-treated samples.

first: Charlotte

last: Pratt

first: Hsin-Jung

last: Wu

first: Shreya

last: Rajeswaran

last: Comer

first: Sirimas

last: Sudsakorn

first: Wenhai

last: Zhang

first: Shangtao

last: Liu

first: Samuel

last: Meier

first: Ashley

middle: H

last: Choi

first: Tenzing

last: Khendu

first: Hannah

last: Stowe

first: Binzhang

first: Douglas

last: Whittington

first: Yingnan

last: Chen

first: Yi

last: Yu

first: William

last: Mallender

first: Tianshu

last: Feng

first: Jannik

middle: N

last: Andersen

first: John

last: Maxwell

first: Scott

last: Throner


title: The ChAHP chromatin remodelling complex regulates a network of neurodevelopmental disorder risk genes to scale the production of neocortical layers (cut&run-seq)

status: Public on Dec 29 2024

city: Ottawa

first: Pierre

last: Mattar

state: ON

address: CCW6109, Box 511, 501 Smyth Rd.

country: Canada

institute: OHRI

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Although chromatin remodellers are among the most important risk genes associated with neurodevelopmental disorders (NDDs), the roles of these complexes during brain development are in many cases unclear. Here, we focused on the recently discovered ChAHP chromatin remodelling complex. The zinc finger and homeodomain transcription factor ADNP is a core subunit of this complex, and de novo ADNP mutations lead to intellectual disability and autism spectrum disorder. However, germline Adnp knockout mice were previously shown to exhibit early embryonic lethality, obscuring subsequent roles for the ChAHP complex in neurogenesis. Here, we employed single cell transcriptomics, cut&run-seq, and histological approaches to characterize mice conditionally ablated for the ChAHP subunits Adnp and Chd4. We show that during neocortical development, Adnp and Chd4 orchestrate the production of late-born, upper-layer neurons through a two-step process. First, Adnp is required to sustain progenitor proliferation specifically during the developmental window for upper-layer cortical neurogenesis. Accordingly, we found that Adnp recruits Chd4 to genes associated with progenitor proliferation. Second, in postmitotic differentiated neurons, we define a network of risk genes linked to NDDs that are regulated by Adnp and Chd4. Taken together, these data demonstrate that ChAHP is critical for driving the expansion upper-layer cortical neurons, and for regulating neuronal gene expression programs, suggesting that these processes may potentially contribute to NDD etiology.

overall_design: Cut&run-seq was performed using the Cutana cut&run kit (14-0500; Epicypher), following the manufacturer’s instructions. Briefly, E13.5 embryos were harvested and the dorsal telencephalon was microdissected. Cells were dissociated with mechanical trituration in order to retain the glycoproteins necessary for adhering to the concavanalin beads. 500 000 cells were used in each reaction. Cut&run seq was performed using antibodies against Chd4 (Abcam ab72418) or Ctcf (Cell Signaling Technologies 3418). Non-specific IgG was used as a negative control. Libraries were prepared using the NEBNext® UltraTM II Library Prep Kit (New England Biolabs). Paired-end 150 sequencing was performed using the NextSeq 500 platform (Illumina) to a read-depth of ~20-35 million reads per sample.   Bioinformatic analysis was performed using Galaxy. FASTQ files were processed via Fastq Groomer and Trimmomatic, and then mapped to the mm9 genome using Bowtie2. To normalize samples to the E. coli spike-in, the trimmed reads were mapped to the E. coli K12 genome using Bowtie2. Coverage was calculated using Samtools Flagstat. Mapped BAM files were then scaled using Deeptools to generate scaled Bedgraph files. Peak calling was performed with Macs2.


title: Placental Growth Factor Modulates Endothelial Nitric Oxide Production and Exacerbates Experimental Hepatopulmonary Syndrome

city: 91400 Orsay

first: Florent

last: Dumont

state: Ile de France

address: 17 avenue des sciences

country: France

institute: Université Paris-Saclay

type: Expression profiling by high throughput sequencing

summary: Exploration of the role of PlGF in the development of hepatopulmonary syndrome.

overall_design: Bulk RNA sequencing of pulmonary tissues from common bile duct ligation (CBDL) rats and SHAM operated rats.

first: Fabien

last: Robert

first: Laurent

last: Savale

first: Feriel

last: Benchenouf

first: Ly

last: Tu

first: Christophe

last: Guignabert


title: MYC ecDNA promotes intratumour heterogeneity and plasticity in PDAC

city: Verona

first: Davide

last: Pasini

state: VR

address: Piazzale Ludovico Antonio Scuro 10

country: Italy

institute: University of Verona

summary: Intratumor heterogeneity and phenotypic plasticity drive tumour progression and therapy resistance. Oncogene dosage variation contributes to cell state transitions and phenotypic heterogeneity, thereby providing a substrate for somatic evolution. Nonetheless, the genetic mechanisms underlying phenotypic heterogeneity are still poorly understood. Here, we show that extrachromosomal DNA (ecDNA) is a major source of high-level focal amplification in key oncogenes and a major contributor of MYC heterogeneity in pancreatic ductal adenocarcinoma (PDAC). We demonstrate that ecDNAs drive varying levels of MYC dosage, depending on their regulatory landscape, enabling cancer cells to rapidly and reversibly adapt to microenvironmental changes. In absence of selective pressure, a high ecDNA copy number imposes a substantial fitness cost on PDAC cells. We also show that MYC dosage affects cell morphology and dependence of cancer cells on stromal niche factors. Our work provides the first detailed analysis of ecDNAs in PDAC and describes a new genetic mechanism driving MYC heterogeneity in PDAC.

overall_design: To distinguish between adaptation and selection as mechanisms of WRi acquisition, we used a barcode sequencing method. Each parental PDO was transduced with a library of 1 million random barcodes to ensure diversity. This setup allowed for tracking the expansion of specific subclones under selective pressure.


title: 7SL RNA and SRP orchestrate a global cellular response to acute thermal stress [Ribo-seq]

city: Boston

first: Hun-Goo

last: Lee

state: MA

address: 185 Cambridge St

department: Molecular Biology

country: USA

institute: Mass General Hospital

summary: Non-coding 7SL RNA is an ancestor to mammalian Alu and B1 SINE RNAs and is thought to function exclusively within the Signal Recognition Particle (SRP), aiding in the translocation of secretory proteins into the endoplasmic reticulum for export. Here, we discover a surprising new function unrelated to protein secretion. Under acute heat shock, 7SL and SRP together selectively arrest cellular transcription and translation machineries as a crucial early response to stress. Under thermal stress, 7SL is upregulated, localizes to the nucleus, and binds to target genes repressed by heat shock. Concurrently, in the cytosol, SRP binds to ribosomes and inhibits new protein synthesis. Translational suppression occurs independently of the signal peptide and is abrogated by depleting SRP. Intriguingly, translation inhibition extends to the mitochondria, as nuclear-encoded mitochondrial genes are enriched among SRP targets. Thus, apart from its role in protein export, 7SL/SRP orchestrates a global response to acute stress that encompasses the nucleus, cytosol, and mitochondria across transcription and translation.

overall_design: To investigate the function of 7SL and SRP complex in heat shock response, NIH3T3 cells were transfected either with siScr or siSrp72, and cells were exposed to heat shock (45 ̊C, 10min) or not (37 ̊C, 10min) in 1ml medium pre-warmed to 37 ̊C or 45 ̊C.

first: Bojan

last: Bujisic

first: Jeannie

middle: T


title: Transcriptome profiling tumor cells-of-origin from Nf1 and Nf1-Arf null murine model of Neurofibromatosis Type 1

status: Public on Dec 30 2024

city: Indianapolis

first: Steven

middle: P

last: Angus

state: IN

address: 1044 W Walnut St

department: Pediatrics

institute: Indiana University School of Medicine

summary: Malignant peripheral nerve sheath tumors (MPNSTs) are the leading cause of premature death for patients with Neurofibromatosis type 1 and no approved targeted therapies are available. Transformation from Nf1-null benign plexiform neurofibromas is driven by the loss of the Cdkn2a (Arf) locus. Here, genetically engineered mouse models with floxed Nf1 and combined Nf1 and Arf alleles were used. Schwann cell precursor cells (the tumor cell-of-origin) were isolated from the nerve root (dorsal root ganglia) of embryos at day 13.5. These cells, termed DRG/nerve root neurosphere cells (DNSCs) were established, and either control adenoviral infection (GFP) was performed, or adenoviral Cre-GFP infection used to conditionally ablate Nf1 or Nf1-Arf. mRNA sequencing was performed using Nf1 (control, GFP infected or “GFP”), Nf1 (Cre-GFP infected or “CRE”), or Nf1-Arf (Cre-GFP infected or “ARF”) to evaluate the effects on the transcriptome.

overall_design: mRNA sequencing was performed using Nf1 (control, GFP infected or “GFP”), Nf1 (Cre-GFP infected or “CRE”), or Nf1-Arf (Cre-GFP infected or “ARF”) Schwann cell precursor cells to evaluate effects on the transcriptome.

middle: D

last: Rhodes


status: Public on Dec 28 2024

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

overall_design: Refer to individual Series


title: 7SL RNA and SRP orchestrate a global cellular response to acute thermal stress [CHART-seq]

first: Uri

last: WEISSBEIN

institute: Massachusetts General Hospital

overall_design: NIH3T3 cells were resuspended in 2ml of DMEM pre-warmed to 37 ˚C and incubated for 15min at 37 ˚C (control) or 45 ˚C (heat-shock). Cells were collected and CHART-Seq was preformed with antisense or sense probes

first: Weissbein

last: Uri


overall_design: Transcriptome analysis of 14 human pancreatic cancer patient-derived organoids

first: Vincenzo

last: Corbo

first: Antonia

last: Malinova

first: Elena

last: Fiorini


title: A mutation in RNA polymerase imparts resistance to β-lactams by preventing dysregulation of amino acid and nucleotide metabolism

status: Public on Dec 27 2024

city: Ithaca

first: John

last: Helmann

state: NY

address: 123 Wing drive

department: Microbiology

institute: Cornell University

summary: Resistance to diverse antibiotics can result from mutations in RNA polymerase (RNAP), but the underlying mechanisms remain poorly understood. In this study, we compared two Bacillus subtilis RNAP mutations: one in β’ (rpoC G1122D) that increases resistance to cefuroxime (CEF, a model β-lactam) and one in β (rpoB H482Y) that increases sensitivity. CEF resistance was mediated by a decrease in branched-chain amino acid (BCAA), methionine, and pyrimidine pathways. These same pathways are up-regulated by CEF, and their derepression increases CEF sensitivity and antibiotic-induced production of reactive oxygen species. The CEF resistant rpoC G1122D mutant evades these metabolic perturbations, and repression of the BCAA and pyrimidine pathways may function to restrict membrane biogenesis, which is beneficial when cell wall synthesis is impaired. These findings provide a vivid example of how RNAP mutations, which commonly arise in response to diverse selection conditions, can rewire cellular metabolism to enhance fitness.

overall_design: WT (HB26336), rpoB H482Y (HB26341), rpoB H482Q (HB28141), rpoC G1122D (HB26291) and rpoB H482Q-rpoC G1122D (HB26332) mutants were grown in LB medium till ~0.4 OD600nm. RNA was isolated and sent for RNA-seq analysis.

first: Yesha

last: Patel


title: AML cell line HL60 treated with WIN55

city: Sevilla

first: Alfonso

last: Rodriguez Gil

email: alfrodgil@gmail.com, arg@us.es

state: Sevilla

address: Avda. Manuel Siurot s/n

department: Oncohematology

country: Spain

institute: Instituto de Biomedicina de Sevilla (IBiS)

summary: AML cell line HL60 treated with WIN55

overall_design: The AML cell line HL60 was cultured in IMDM medium  suplemented wit100 units/mL penicillin, 100 µg /mL streptomycin and 10% fetal bovine serum (FBS). All cells were cultured in a humidified atmosphere of CO2/air (5%/95%) and 37ºC

first: Mayte

last: Medrano

first: José

middle: A

last: Pérez Simón


title: Comparison of cyclic compressive stimulation and proinflammatory cytokine stimulation of three-dimensional cultured human articular chondrocytes

city: Osaka

first: Minami

last: Hikida

state: Chuou-Ku

address: 1-17 Machikaneyama-tyo

country: Japan

institute: Medicine for Sports and Performing Arts, Dept. of Health and Sport Sciences, Osaka University

summary:
Osteoarthritis is a multifactorial disease in which not only physical tissue damage but also persistent inflammation is thought to be one of the main causes. The qualitative differences between mechanical and inflammatory cytokine stimulation in the production of OA-related molecules are not fully understood, and the biological events that occur in cells when the two stimuli are applied simultaneously remain unresolved.
　Articular cartilage tissue is formed by relatively sparse cells surrounded by a rich extracellular matrix composed mainly of collagen and proteoglycans, and three-dimensional culture using hydrogels such as collagen gel has been frequently used to reproduce chondrocytes that are three-dimensionally bound to the surrounding extracellular matrix and to evaluate physiological responses.
　In this study, we aimed to compare the responses of 3D cultured articular chondrocytes to mechanical stimuli and inflammatory cytokine stimuli, to analyze the responses to the two stimuli when they occur simultaneously, and to gain insight into the molecular mechanisms involved. Three-dimensional culture of human articular chondrocytes was performed using a stiff atelocollagen sponge and atelocollagen gel, and analysis focused on changes in gene expression due to cyclic compression stimulation.

overall_design: The 3D tissue of human chondrocytes 13 hours after stimulation with IL-1β (10 ng / ml) was used as the IL-1β group. And the 3D tissue of human chondrocytes after 12 hours of stimulation with cyclic compressive loading (CCL) (40 kPa, 0.5 hz, 1 hour) was designated as the CCL group. The 3D tissue of human chondrocytes 12 hours after stimulation with IL-1β (10 ng / ml) and CCL (40 kPa, 0.5 hz, 1 hour) was used as the CCL+ IL-1β group. Each group was compared to a control sample that was cultured for 13 hours in static culture. Data are obtained with one donor, representative of results of three technical replicates.


title: 7SL RNA and SRP orchestrate a global cellular response to acute thermal stress


title: Intestinal Mucosal Mitochondrial Oxidative Phosphorylation worsens with Cirrhosis Progression and is ameliorated with FMT

status: Public on Dec 26 2024

city: Richmond

first: Grayson

middle: Welch

last: Way

email: waygw@vcu.edu, waygw@outlook.com

state: Virginia

address: 1217 E Marshall Street Room 534

department: Microbiology and Immunology

institute: VCU

summary: Intestinal barrier dysfunction, driven by increased oxidative phosphorylation (OXPHOS)   activity that leads to tissue hypoxia, contributes to the progression of cirrhosis, particularly   impacting the upper intestine. This study explores the interplay between intestinal OXPHOS,   gut microbiota changes, and the effects of fecal microbiota transplant (FMT) in cirrhotic   patients. We investigated 32 age-matched men across three groups: healthy controls,   compensated cirrhosis, and decompensated cirrhosis. Each underwent endoscopy with   duodenal and ascending colon biopsies. Subsequently, in a follow-up study, nine patients with   hepatic encephalopathy, previously enrolled in a randomized controlled trial for FMT capsules,   underwent repeat pre and post-FMT upper endoscopy. Our bioinformatics analysis highlighted   a significant upregulation of nuclear-encoded OXPHOS genes in both intestinal regions of   cirrhosis patients compared to controls, with further dysregulation in the decompensated   group. We also observed a strong correlation between shifts in gut microbiota composition,   Model for End-Stage Liver Disease (MELD) scores, and OXPHOS activity. Following FMT,   patients displayed a significant reduction in OXPHOS gene expression in the duodenum,   suggesting that FMT may restore intestinal barrier function and offer a therapeutic avenue to   mitigate liver disease progression. The findings indicate that managing intestinal OXPHOS   and microbiota through FMT could be relevant in modulating microbially-based therapies.

first: Jing

last: Zeng

first: Andrew

last: Fagan

first: Michael

last: Fuchs

first: Puneet

last: Puri

first: Brian

middle: C

last: Davis

first: Jian-Gao

last: Fan

first: Phillip

middle: B

last: Hylemon

first: Huiping

last: Zhou

first: Jasmohan

middle: S

last: Bajaj


title: 7SL RNA and SRP orchestrate a global cellular response to acute thermal stress [RNA-seq]

first: Hun-Goo

last: Lee

first: Bojan

last: Bujisic

first: Jeannie


title: Whole blood RNA sequencing of African American kidney transplant recipients from VericiDx cohort

city: New York

first: Zeguo

last: Sun

address: 1468 Madison Avenue

institute: Ichan School of Medicine at Mount Sinai

summary: The VericiDx cohort is an ongoing clinical trial (241 patients (32.0% self-reported AA) enrolled as of 06/30/2023) with a focus on predicting early acute rejection and renal fibrosis post-transplantation (ClinicalTrials.gov: NCT04727788).

overall_design: Pretransplant RNA sequencing profiles (101 PE reads) were generated on pretransplant blood, with a subset of 77 recipients. The RNA data in these 77 AA recipients was combined with two cohorts to identify the meta-gene signatures associated with the LILRB3-4SNPs. The LILRB3-4SNPs was correlated with early eGFR values (within two years) in meta-analysis with other three cohorts. However, the relatively short follow-up duration for many patients in this cohort limited the association analysis for graft failure.

first: Weijia

last: Zhang


title: RNA sequencing of 20 whole blood with and without LILRB3-4SNPs 3 months after kidney transplantation

summary: RNA sequencing were conducted on 20 transplant patients with (n=10) and without (n=10) LILRB3-4SNPs risk allele 3 months after kidney transplantation to evaluate the potential effects of this SNP on post-transplant blood.

overall_design: RNA sequencing were performed on the post-transplant blood of 20 AA recipients (10 with and 10 without LILRB3-4SNPs) at 3 month after kidney transplantation, with matched recipient and donor’s demographic and baseline clinical features (age, gender, donor status, HLA mismatches and induction types).


title: LRG1 loss effectively restrains glomerular TGF-β signaling to attenuate DKD in diabetic mice

summary: scRNA-seq analysis confirmed that increased LRG1 is limited to GECs in early diabetic kidneys. As anticipated, LRG1 loss significantly attenuated diabetic glomerulopathy including podocyte loss, and improved renal function. scRNA-seq analysis demonstrated that LRG1 loss was sufficient to reverse all significant molecular pathway changes in diabetic mouse GECs, which were also associated with the dampening of TGF-β-induced gene expression. Moreover, LRG1 loss also led to a significant attenuation of TGF-β-mediated gene expression in podocytes and mesangial cells of diabetic mice. These results indicate that increased LRG1 directly potentiates TGF-β signaling in glomerular cells in an autocrine and paracrine manner to promote DKD, and indirectly via glomerular cross-talk.

overall_design: Type 1 diabetic OVE26 mice were crossed with Lrg1-/- mice to generate OVE26 Lrg1-/- mice, and DKD progression was assessed by renal function and histopathologic parameters. scRNA-seq was employed for kidney single-cell gene expression of control and diabetic mice.

first: Jia

last: Fu


title: Endonuclease G promotes hepatic mitochondrial respiration by selectively increasing mitochondrial tRNAThr production

city: Debrecen

first: Lorant

last: Szekvolgyi

address: Egyetem ter 1.

department: Department of Pharmacy

country: Hungary

institute: University of Debrecen

summary: Mitochondrial endonuclease G (EndoG) contributes to chromosomal degradation when it is released from mitochondria during apoptosis. It is presumed to also have a mitochondrial function because EndoG deficiency causes mitochondrial dysfunction. However, the mechanism by which EndoG regulates mitochondrial function is not known. Fat accumulation in metabolic dysfunction-associated steatotic liver disease (MASLD), which is more common in men, is caused in part by mitochondrial dysfunction. EndoG expression is reduced in MASLD liver, and EndoG deficiency causes MASLD in an obesity-independent manner but only in males. EndoG promotes mitochondrial respiration by resolving mitochondrial tRNA/DNA hybrids formed during mtDNA transcription by recruiting RNA helicase DHX30 to unwind them. EndoG also cleaves off the 3'-end of the H-strand transcript that can prevent mt-tRNAThr precursor cloverleaf-folding, and processing, which increases mt-tRNAThr production and mitochondrial translation. Using fluorescent lifetime imaging microscopy technology to visualize oxygen consumption at the individual mitochondrion level, we found that EndoG deficiency leads to the selective loss of a mitochondrial subpopulation with high-oxygen consumption. This defect was reversed with mt-tRNAThr supplementation. Thus, EndoG promotes mitochondrial respiration by selectively regulating the production of mt-tRNAThr in male mice.

overall_design: DRIP-seq experiments to determine mitochondrial R-loops in wild type and endoG KO mice

first: Jay

last: Chung


title: Deep Learning-Based Multimodal Clustering Model for Endotyping and Post-Arthroplasty Response Classification using Knee Osteoarthritis Subject-Matched Multi-Omic Data

city: Toronto

first: Mohit

last: Kapoor

state: Ontario

address: 60 Leonard Ave

institute: Schroeder Arthritis Institute

type: Non-coding RNA profiling by high throughput sequencing

summary:
Background: Primary knee osteoarthritis (KOA) is a heterogeneous disease with clinical and molecular contributors. Biofluids contain microRNAs and metabolites that can be measured by omic technologies. Deep learning captures complex non-linear associations within multimodal data but, to date, has not been used for multi-omic-based endotyping of KOA patients. We developed a novel multimodal deep learning framework for clustering of multi-omic data from three subject-matched biofluids to identify distinct KOA endotypes and classify one-year post-total knee arthroplasty (TKA) pain/function responses.

Materials and Methods: In 414 KOA patients, subject-matched plasma, synovial fluid and urine were analyzed by microRNA sequencing or metabolomics. Integrating 4 high-dimensional datasets comprising metabolites from plasma (n=151 features), along with microRNAs from plasma (n=421), synovial fluid (n=930), or urine (n=1225), a multimodal deep learning variational autoencoder architecture with K-means clustering was employed. Features influencing cluster assignment were identified and pathway analyses conducted. An integrative machine learning framework combining 4 molecular domains and a clinical domain was then used to classify WOMAC pain/function responses post-TKA within each cluster.

Findings: Multimodal deep learning-based clustering of subjects across 4 domains yielded 3 distinct patient clusters. Feature signatures comprising microRNAs and metabolites across biofluids included 30, 16, and 24 features associated with Clusters 1-3, respectively. Pathway analyses revealed distinct pathways associated with each cluster. Integration of 4 multi-omic domains along with clinical data improved response classification performance, with Cluster 3 achieving AUC=0·879 for subject pain response classification and Cluster 2 reaching AUC=0·808 for subject function response, surpassing individual domain classifications by 12% and 15% respectively.

Interpretation: We have developed a deep learning-based multimodal clustering model capable of integrating complex multi-fluid, multi-omic data to assist in KOA patient endotyping and test outcome response to TKA surgery.

overall_design: MiRNA-seq data for plasma, synovial fluid, and urine from 414 matched knee OA patients were analyzed for comparative miRNA profiling.

first: Jason

last: Rockel

first: Divya

last: Sharma

first: Osvaldo

last: Espin-Garcia

first: Katrina

last: Hueniken

first: Amit

last: Sandhu

first: Chiara

last: Pastrello

first: Kala

last: Sundararajan

first: Pratibha

last: Potla

first: Noah

last: Fine

first: Starlee

last: Lively

first: Kimberly

last: Perry

first: Nizar

last: Mohamed

first: Khalid

last: Syed

first: Igor

last: Jurisica

first: Anthony

middle: V

last: Perruccio

first: Raja

last: Rampersaud

first: Rajiv

last: Gandhi


title: RNA sequencing of CTOT19 transplantation patients [bulk RNA-seq]

first: Zeguo

last: Sun

summary: The CTOT19 cohort (38.2% AA, 44% White) represents a randomized controlled trial investigating the efficacy of remicade induction therapy for deceased donor kidney transplant recipients within 2 year followed-up (NCT02495077). With a cohort of 225 patients, the study spanned 2 years to assess the effectiveness of this therapy.

overall_design: RNA sequencing was executed on pretransplant blood from 130 recipients, utilizing 75bp-long paired-end (75PE) reads. Notably, the recipients were representative of various ethnicities: 49 African American, 26 Caucasian, 41 Hispanic, and 14 individuals from other ethnic backgrounds. RNA sequencing data in this cohort was used to validate the variant expression of LILRB3-4SNPs in AA recipients. The RNA data in 49 AA recipients was combined with two cohorts to identify the meta-gene signatures associated with the LILRB3-4SNPs.


title: A foundational atlas of autism protein interactions reveals molecular convergence [CUT&Tag]

status: Public on Dec 25 2024

city: San Francisco

first: Kelsey

last: Hennick

state: CA

address: 1651 4th St room 521A

institute: UCSF

summary: Translating high-confidence (hc) autism spectrum disorder (ASD) genes into viable treatment targets remains elusive. We constructed a foundational protein-protein interaction (PPI) network in HEK293T cells involving 100 hcASD risk genes, revealing over 1,800 PPIs (87% novel). Interactors, expressed in the human brain and enriched for ASD but not schizophrenia genetic risk, converged on protein complexes involved in neurogenesis, tubulin biology, transcriptional regulation, and chromatin modification. A PPI map of 54 patient-derived missense variants identified differential physical interactions, and we leveraged AlphaFold-Multimer predictions to prioritize direct PPIs and specific variants for interrogation in Xenopus tropicalis and human forebrain organoids. A mutation in the transcription factor FOXP1 led to reconfiguration of DNA binding sites and altered development of deep cortical layer neurons in forebrain organoids. This work offers new insights into molecular mechanisms underlying ASD and describes a powerful platform to develop and test therapeutic strategies for many genetically-defined conditions.

overall_design: Dissociated forebrain organoids at D111 were used as input for CUT&Tag profiling binding of FOXP1, FOXP4, and H3K27me3 in WT and R513H organoids, with two technical replicates each.

first: Tomasz

last: Nowakowski


title: A foundational atlas of autism protein interactions reveals molecular convergence [scRNA-seq]

overall_design: Forebrain organoids were dissociated at D111 and split into two technical replicates for each sample (WT and R513H) for scRNA-seq.


title: Actin polymerization by WASp limits nuclear envelope rupture and inflammation in macrophages I

city: Trieste

first: Roberto

last: Amadio

address: Padriciano 99

institute: ICGEB Trieste

summary: Mutations in the immune-specific actin regulator WASp induce a proinflammatory state in myeloid cells, whose underlying causes remain poorly defined. Here, we applied microfabricated tools that mimic tissue mechanical forces to explore the role of WASp in connecting mechano-sensing to the activation of inflammatory responses in macrophages. We show that WASp-deficient macrophages carry nuclear structure alterations and undergo increased blebbing and nuclear rupture when exposed to mechanical confinement. High-resolution imaging indicates that WASp drives the formation of protective perinuclear actin structures in response to confinement. Functionally, WASp null macrophages respond to mechanical confinement by inducing a transcriptional profile consistent with the release of immunogenic DNA in the cytosol. The proinflammatory state of mechanically confined WASp-deficient macrophages depends, in part, on the cGAS-STING pathway of cytosolic DNA sensing. Together, these data uncover a WASp-dependent mechanism to restrict activation of inflammatory signalling in tissue macrophages and provide hints to target unabated inflammation in Wiskott-Aldrich Syndrome.

overall_design: RNA-seq profiling of six Peripheal Blood Mononuclear Cells (PBMCs) from Wiskott-Aldrich Syndrome patiets (WAS) before and five years after WASp reconstitution by gene therapy.

first: Giulia Maria

last: Piperno

first: Federica

last: Benvenuti


title: Exceptional Uptake, Limited Protein Expression: Liver Macrophages Lost in Translation of Synthetic mRNA

city: Aachen

first: Matthias

last: Bartneck

address: Pauwelsstraße 30

department: Department of Internal Medicine III

country: Germany

institute: University Hospital RWTH Aachen

summary: To investigate the translation and function of nucleic acid, we designed and prepared LNPs co-encapsulating GFP mRNA and TLR4 siRNA. These LNPs were administered via tail vein injection and primarily accumulated in the liver. Therefore, in this experiment, we isolated liver cells and used flow cytometry to sort Kupffer cells for gene sequencing, aiming to study the mRNA expression levels in different cell types.

overall_design: After treating mice with different LNPs for 16 hours, Kuffer cells and hepatocytes were isolated for RNA sequencing.

first: Cheng

last: Lin

first: Adrian

last: Kuzmanovic

last: Wang

first: Liangliang

last: Liao

first: Sabrina

last: Ernst

first: Christian

last: Penners

first: Alexander

last: Jans

first: Thomas

last: Hammoor

first: Petra

last: Stach

first: Mona

last: Peltzer

first: Ines

last: Volkert

first: Elisabeth

last: Zechendorf

first: Reham

last: Hassan

first: Maiju

last: Myllys

last: Liedtke

first: Andreas

last: Herrmann

first: Gurudas

last: Chakraborty

last: Trautwein

first: Jan

last: Hengstler

first: Gerhard

last: Müller-Newen

first: Junqing

first: Ahmed

last: Ghallab


title: A Noncanonical Intrinsic Terminator in the HicAB Toxin‒Antitoxin Operon Promotes the Transmission of Conjugative Antibiotic Resistance Plasmids

city: Guangzhou

first: lin

last: jianzhong

state: Guangzhou

address: China

country: China

institute: University of Chinese Academy of Sciences

summary: Conjugative plasmids, major vehicles for the spread of antibiotic resistance genes, often contain multiple toxin‒antitoxin (TA) systems. However, the physiological functions of TA systems remain obscure. By studying TA families commonly found on colistin-resistant IncI2 mcr-1-bearing plasmids, we discovered that the HicAB TA, acts as a crucial addiction module to increase horizontal plasmid‒plasmid competition.

overall_design: Overnight cultures of K-12/p45 were diluted to an initial OD600 of approximately 0.1 and then cultivated in LB for 6 h at 37 °C. Total RNA was extracted from the cells via TRIzol® Reagent following the manufacturer’s instructions (Invitrogen), with the genomic DNA removed via the addition of 10 μL of 5 U/μL DNase I (Takara). The RNA quality was subsequently determined using a 2100 Bioanalyzer (Agilent) and quantified using an ND-2000 (NanoDrop Technologies). High-quality RNAs (RIN>8) were utilized for the construction of sequencing libraries. The RNA-seq strand-specific libraries were prepared using a TruSeq RNA Sample Preparation Kit from Illumina (San Diego, CA) with 5 μg of total RNA. The process involved removing rRNA with the RiboZero rRNA removal kit (Epicenter), followed by fragmentation using fragmentation buffer. Further steps included cDNA synthesis, end repair, A-base addition, and ligation of Illumina-indexed adaptors, which strictly followed Illumina's protocol. Libraries were then size selected for cDNA target fragments of 200–300 bp on 2% low-range ultra agarose followed by PCR amplification via Phusion DNA polymerase (NEB) for 15 PCR cycles. After quantification with TBS380, paired-end libraries were subjected to sequencing using the Illumina NovaSeq 6000 platform (Shanghai BIOZERON Co., Ltd.).

first: Jianzhong

last: Lin

first: Yunxue

last: Guo

first: Xiaoxue

last: Wang


title: A microfluidic platform for anterior-posterior human endoderm patterning via countervailing morphogen gradients in vitro

city: Stanford

first: Lay Teng

last: Ang

address: Institute for Stem Cell Biology & Regenerative Medicine, Lokey Stem Cell Bldg, 265 Campus Drive

department: Urology

institute: Stanford University

summary: Understanding how morphogen gradients spatially pattern tissues is a fundamental question in developmental biology but can be difficult to directly address using conventional approaches. Here we expose hPSC-derived endoderm cells to countervailing gradients of anteriorizing and posteriorizing signals using a widely available microfluidic device. This approach yielded spatially patterned cultures comprising anterior foregut (precursor to the thyroid, esophagus, and lungs) and mid/hindgut (precursor to the intestines) cells, whose identities were confirmed using single-cell RNA-sequencing. By exposing stem cells to externally applied signaling gradients, this widely accessible microfluidic platform should accelerate the production of spatially patterned tissues, complementing internally self-organizing organoids. Applying artificial morphogen gradients in vitro may also illuminate signaling gradient interpretation mechanisms that are otherwise challenging to study in mammalian embryos in vivo.

overall_design: Following differentiation in a microfluidic chip, human pluripotent stem cell-derived anterior and posterior endoderm populations were dissociated and analyzed using single-cell RNA-sequencing.


title: Loss of Cpt1a results in elevated glucose-fueled mitochondrial oxidative phosphorylation and defective hematopoietic stem cells [RNA-Seq]

city: San Antonio

first: Yu

last: Luan

state: Texas

address: 7703 Floyd Curl Drive

department: Cell Systems and Anatomy

institute: University of Texas Health Science Center at San Antonio

summary: Hematopoietic stem cells (HSCs) rely on self-renewal to sustain stem cell potential and undergo differentiation to generate mature blood cells. Mitochondrial fatty acid β-oxidation (FAO) is essential for HSC maintenance. However, the role of carnitine palmitoyl transferase 1a (CPT1A), a key enzyme in FAO, remains unclear in HSCs. Using a Cpt1a hematopoietic specific conditional knock-out (Cpt1aΔ/Δ) mouse model, we found that loss of Cpt1a leads to HSC defects, including loss of HSC quiescence and self-renewal, and increased differentiation. Mechanistically, we find that loss of Cpt1a results in elevated levels of mitochondrial respiratory chain complex components and their activities, as well as increased ATP production, and accumulation of mitochondrial reactive oxygen species (mitoROS) in HSCs. Taken together, this suggests hyperactivation of mitochondria and metabolic rewiring via upregulated glucose-fueled oxidative phosphorylation (OXPHOS). In summary, our findings demonstrate a novel role for Cpt1a in HSC maintenance and provide insight into the regulation of mitochondrial metabolism via control of the balance between FAO and glucose-fueled OXPHOS.

overall_design: HSCs from WT and Cpt1a-KO mice were sorted out from total bone marrow and analyzed

first: Chenhui

last: He

first: Jue

last: Li

first: Gang

last: Huang


title: Loss of Cpt1a results in elevated glucose-fueled mitochondrial oxidative phosphorylation and defective hematopoietic stem cells [ATAC-Seq]


title: PI3/Elafin expression predicts recurrence after chemotherapy with DNA-damaging drugs in gastric cancer

city: Kashiwa

first: Kenji

last: Harada

state: Chiba

address: 6-5-1 Kashiwanoha

department: Exploratory Oncology Research & Clinical Trial Center

institute: National Cancer Center

summary: Despite recent advances in gastric cancer therapy, chemotherapy resistance and lack of methods for selecting combination regimens remain major problems. Organoids, which recapitulate the in vivo micro-environment, can be established from surgical specimens with a high success rate, and are widely used for drug sensitivity assays. In this study, we aimed to discover a novel biomarker for predicting multidrug resistance using gastric cancer organoids (GCOs), which provide in vivo-like culture system that more closely resembles tumor cell organization than traditional cell lines. We evaluated 5-fluorouracil or oxaliplatin-resistant GCOs to find novel biomarkers that reflect multidrug resistance in gastric cancer. To examine the resistance mechanisms, RNA-seq analysis and ex vivo drug sensitivity testing were performed. The association of biomarkers with patient prognosis and chemotherapy efficacy was evaluated using three original cohorts with a total of 230 cases. The results were also validated with two independent public cohorts and a single-cell RNA sequence data. Increased expression of PI3/Elafin was detected in all 5-fluorouracil or oxaliplatin-resistant GCOs. Our findings suggest a potential association of PI3/Elafin expression with ribosome biosynthesis and RNA metabolism under organoid conditions. We also found that PI3/Elafin overexpression promotes 5-fluorouracil/oxaliplatin/cisplatin resistance but not paclitaxel resistance. Immunohistochemical evaluation of Elafin expression revealed that the Elafin-positive gastric cancer group had a poorer outcome, especially in terms of time-to-recurrence. Elafin positivity was also an independent predictor of relapse after chemotherapy with DNA-damaging agents. PI3/Elafin promotes DNA-damaging drug resistance through multiple downstream regulations related to RNA and ribosomal metabolism. PI3/Elafin may be useful as a biomarker for therapeutic selection of non-DNA-damaging agents.

overall_design: We performed PI3/Elafin overexpression (PI3OE) and control vector transfection (Ctrl) in three gastric cancer cell lines and three GCOs to investigate the effect of Elafin expression. Gene expression was comprehensively analyzed using RNA sequencing.

first: Naoya

last: Sakamoto


title: L-OHP resistant gastric cancer organoid

summary: The attainment of drug resistance in gastric cancer (GC) is a problematic issue. Although many studies have shown that cancer stem cells (CSCs) play an important role in the acquisition of drug resistance, there is no clinically available biomarker for predicting oxaliplatin (L-OHP) resistance in relation to CSCs. Organoid technology, a novel 3D cell culture system, allows harboring of patient-derived cancer cells containing abundant CSCs using niche factors in a dish. In this study, we established L-OHP-resistant gastric cancer organoids (GCOs) and evaluated their gene expression profile using microarray analysis. We validated the upregulated genes in the L-OHP-resistant GCOs compared to their parental GCOs to find a gene responsible for L-OHP resistance by qRT-PCR, immunohistochemistry, in vitro, and in vivo experiments. We found myoferlin (MYOF) to be a candidate gene through microarray analysis. The results from cell viability assays and qRT-PCR showed that high expression of MYOF correlated significantly with the IC50 of L-OHP in GCOs. Immunohistochemistry of MYOF in GC tissue samples revealed that high expression of MYOF was significantly associated with poor prognosis, T grade, N grade, and lymphatic invasion, and showed MYOF to be an independent prognostic indicator, especially in the GC patients treated with platinum-based chemotherapy. The knockdown of MYOF repressed L-OHP resistance, cell growth, stem cell features, migration, invasion, and in vivo tumor growth. Our results suggest that MYOF is highly involved in L-OHP resistance and tumor progression in GC. MYOF could be a promising biomarker and therapeutic target for L-OHP-resistant GC cases.

overall_design: 3 L-OHP resistant gastric cancer organoids, 1 parental gastric cancer organoid, and 1 normal gastric organoid were used for microarray. The gene expression data from this study and our previous study (GSE154127), which elucidated 5-FU-resistant GCOs, were combined and analyzed together.


title: Identification of p85beta- or BCLAF1-enriched genomic regions

city: Pokfulam

first: Lydia

last: Cheung

address: 21 Sassoon Road

department: School of Biomedical Sciences

country: Hong Kong

institute: University of Hong Kong

summary: p85beta of the PI3K can localize to the nucleus. Our immunoprecipitation data showed that it physically interacts with a transcription factor BCLAF1. To reveal the enrichment of p85beta and BCLAF1 at genomic regions and to determine whether p85beta and BCLAF1 co-occupy at genomic regions, chromatin immunoprecipitation sequencing (ChIP-seq) was performed.

overall_design: Chromatin harvested from DOV13 cells stably overexpressing HA-tagged p85beta was immunoprecipitated with either ChIP grade anti-HA antibody or anti-BCLAF1 antibody. Input DNA samples were included for background signal. Three biological replicates per condition.

middle: W

last: CHEUNG


title: Genes co-regulated by p85beta and BCLAF1 in ovarian cancer cells

summary: p85beta of the PI3K can localize to the nucleus. Our data showed that it can increase the expression of a transcription factor BCLAF1. To determine whether p85beta has any affect on gene transcription and to identify gene targets that are regulated through BCLAF1, RNAseq was performed on p85beta-expressing cells transfected with BCLAF1 siRNA .

overall_design: Transient transfection of BCLAF1 siRNA or mock transfection for 72 hours in DOV13 cells stably expressing p85beta. Stable expression of empty vector was control. Three biological replicates per condition.


title: RNA sequencing of TA skeletal muscle of mice with conditional ablation of dystrophin within the myofiber

city: Birmingham

first: Matthew

last: Alexander

state: AL

address: 1900 University Blvd. THT 929 Box# 96

institute: University of Alabama at Birmingham

summary: We generated and analyzed a conditional Dystrophin flox52/Y: human alpha-skeletal actin muscle knockout (Dmd flox52/Y: HSA mKO) model by targeting exon 52 deletion in the Dystrophin gene to study the consequences of dystrophin loss in skeletal muscle lineages. The generated DMD mouse model ablates dystrophin protein expression after mating with a Cre recombinase transgenic mouse under the control of HSA promoter element that is restricted to the skeletal muscle. The resulting Dmd mKO mice have been assessed using histopathological, phenotypical, functional and biochemical assays based on TRET-NMD standard operating protocols (SOPs) for mdx mouse models. Phenotypic analysis of these conditional Dmd mKO mice revealed a significant decline in locomotor activity and reduced muscle force, motor and muscular function. The histochemical analysis revealed an increase in centralized myonuclei and fibrotic area similar to mdx mice. Immunoassays including western blot and immunohistochemistry confirmed low expression levels of dystrophin in skeletal muscles of Dmd mKO mice. Bulk RNA sequencing analysis revealed that dystrophin loss in myofiber significantly disrupted the expression of cytokines and extracellular matrix genes.

overall_design:
To investigate the molecular changes of myofiber dystophin loss in Dmd flox52/Y: HSA mKO mice
Five mice per group were used for RNA-sequencing and gene expression analysis. Total RNA was extracted from tibialis anterior (TA) muscle using Trizol reagent. Quality of RNA was assessed by Nanodrop and Bioanalyzer. Purified RNA was used to generate cDNA. Duplicates of cDNA samples were amplified on the QuantStudio 3 Real-Time PCR system using PowerUp SYBR Green Master Mix. RNA sequencing library construction and next-generation sequencing were performed by Novogene. Purified RNA from TA muscles of Dmd flox52/Y: HSA mKO and Dmd flox52/Y mice was measured by Agilent Bioanalyzer. High quality total RNA with RNA integrity value higher than 7 was used for library construction. After mRNA isolation, RNA was fragmented in First Strand synthesis buffer. Then, first-strand CDNA synthesis using reverse transcriptase and random hexamer primer was performed. Synthesis of cDNA using second-strand master mix, end-repair and dA-tailing were performed. The library was amplified and purified using AMPure beads. The library size and mass were evaluated and quantititative PCR was performed to validate the libraries. Barcoded samples were pooled equimolarly for simultanoeus sequencing. The quality of RNA-sequencing data was assessed for downstream analysis. Low-quality sequences and reads were removed. The expected read counts and FPKM were used for further analysis.

first: Muthukumar

last: Karuppasamy


title: The GTE4–EML chromatin reader complex concurrently recognizes histone acetylation and H3K4 trimethylation in Arabidopsis [RNA-Seq]

status: Public on Dec 24 2024

city: Beijing

first: xinjian

address: No. 7, zhongguancun life science park road, changping district

institute: National Institute of Biological Sciences

summary: Histone acetylation and H3K4 trimethylation (H3K4me3) are associated with active transcription. However, how they cooperate to regulate transcription in plants remains largely unclear. Our study revealed that GLOBAL TRANSCRIPTION FACTOR GROUP E 4 (GTE4) binds to acetylated histones and forms a complex with the functionally redundant H3K4me3-binding EMSY-like proteins EML1 or EML2 (EML1/2) in Arabidopsis thaliana. The eml1 eml2 (eml1/2) double mutant exhibits a similar morphological phenotype to gte4, and most of the differentially expressed genes in gte4 were coregulated in eml1/2. Through chromatin immunoprecipitation followed by deep sequencing, we found that GTE4 and EML2 co-occupy protein-coding genes enriched with both histone acetylation and H3K4me3, exerting a synergistic effect on the association of the GTE4–EML complex with chromatin. The association of GTE4 with chromatin requires both its bromodomain and EML-interacting domain. This study identified a complex and uncovered how it cooperatively recognizes histone acetylation and H3K4me3 to facilitate gene transcription at the whole-genome level in Arabidopsis.

overall_design: Examination of the mRNA expression of 20-day-old wild type and three mutants in Arabidopsis

first: Xin-Jian


title: An-Gong-Niu-Huang-Wan (AGNHW) regulates cerebral blood flow by improving hypoperfusion, cerebrovascular reactivity and microcirculation disturbances after stroke

city: Shijiazhuang

first: xiao

last: zhang

address: No. 215, West Heping Road, Xinhua District, Shijiazhuang

department: Department of Neurology

institute: Second Hospital of Hebei Medical University

summary:
Background The restoration of cerebrovascular regulation and improvement of cerebral blood flow in ischaemic
regions are crucial for improving the clinical prognosis after stroke. An-Gong-Niu-Huang-Wan (AGNHW) is a famous
traditional compound Chinese medicine that has been used for over 220 years to treat acute ischaemic stroke; however, its role in the regulation of cerebral blood flow is still unclear. The aim of the present study was to investigate
the regulatory effect of AGNHW on cerebral blood flow and microcirculation after ischaemic stroke and to elucidate
the underlying mechanisms involved.
Methods Male C57BL/6 mice were subjected to distal middle cerebral artery occlusion (dMCAO) and randomly
assigned to the sham, MCAO, or AGNHW groups. AGNHW was administered intragastrically 1 h after dMCAO. The
rotarod test was utilized to evaluate behavioural function; TTC was used to determine the infarct volume; and ischaemic injury was assessed by detecting brain levels of SOD, MDA and NO. Then, cortical perfusion and acetazolamideinduced cerebrovascular reactivity were assessed using laser speckle contrast imaging, and the velocity and flux
of red blood cells in cortical capillaries were detected using two-photon laser scanning microscopy. In addition, we
employed RNA-Seq to identify variations in gene expression profiles and assessed endothelium-dependent changes
in microcirculatory dysfunction by measuring vasoactive mediator levels.
Results AGNHW significantly increased cerebral blood flow, reduced the infarct volume, and promoted functional recovery after cerebral ischaemia. AGNHW increased the velocity and flux of red blood cells in capillaries
and improved cerebrovascular reactivity in the ischaemic cortex. Furthermore, AGNHW regulated endotheliumdependent microcirculation, as evidenced by decreases in the expression of endothelins (Edn1, Edn3 and Ednrb)
and the ratios of brain and serum TXB2/6-keto-PGF1α and ET-1/CGRP.
Conclusions AGNHW improved cerebral hypoperfusion, regulated cerebrovascular reactivity and attenuated microcirculatory dysfunction within the ischaemic cortex after stroke. This outstanding effect was achieved by modulatingthe expression of genes related to vascular endothelial cell function and regulating endothelium-dependent vasoactive mediators.

overall_design: Using the distal middle cerebral artery (dMCAO) model, C57 BL/6 mice were randomly assigned to MCAO and AGNHW groups. AGNHW was administered by gavage (450 mg/kg) 1 h after dMCAO continued once a day until sampling. The peri-infarct cortex were collected for RNA sequencing 5 days after the onset of the ischemic stroke. Each group contained 4 replicates.

first: Xiao

last: Zhang

first: Jiamin

last: Pei

first: Luping

last: Xue

first: Zhe

last: Zhao

first: Renhao

last: Xu

first: Cong

first: Xiangjian

first: Lili

last: Cui


title: Inhibition of thioredoxin reductase 1 sensitizes glucose-starved glioblastoma cells to disulfidptosis 3

status: Public on Dec 23 2024

city: Hershey

first: Wei

state: PA

address: 500 University Drive, PO Box 850, MC H085

institute: Penn State Hershey College of Medicine

summary: Disulfidptosis is a recently identified form of cell death characterized by the aberrant accumulation of cellular disulfides. This process primarily occurs in glucose-starved cells expressing higher levels of SLC7A11 and has been proposed as a therapeutic target for cancers with hyperactive SCL7A11. However, the potential for inducing disulfidptosis through other mechanisms in cancers remains unclear. Here, we found that inhibiting thioredoxin reductase 1 (TrxR1), a key enzyme in the thioredoxin system, induces disulfidptosis in glioblastoma (GBM) cells. TrxR1 expression is elevated in GBM with activated transcriptional coactivator with PDZ-binding motif (TAZ) and correlates with poor prognosis. TrxR1 inhibitors induced GBM cell death that can be rescued by disulfide reducers but not by ROS scavengers or inhibitors of apoptosis, ferroptosis, or necroptosis. Glucose-starved cells, but not those deprived of oxygen or glutamine, increased TrxR1 expression in an NRF2-dependent manner and were more sensitive to TrxR1 inhibition-induced cell death. The dying cells initially exhibited highly dynamic lamellipodia, followed by actin cytoskeleton collapse. This process involved the accumulation of cytosolic peroxisomes and micropinocytic caveolae, as well as small gaps in the plasma membrane. Depletion of the WAVE complex component NCKAP1 partially rescued the cells, whereas Rac inhibition enhanced cell death. In an orthotopic xenograft GBM mouse model, TrxR1 depletion inhibited tumor growth and improved survival. Furthermore, cells undergoing TrxR1 inhibition exhibited features of immunogenic cell death. Therefore, this study suggests the potential of targeting TrxR1 as a therapeutic strategy in GBM.

overall_design: To examine how TrxR1 inhibition and glucose deprivation can synergistically induce tumor cell death, we profiled gene expression in response to these treatments through RNA-seq

first: Tang

last: Miaolu

first: Dirks

last: Kaitlyn

first: Kim

last: Soo-Yeon

first: Qiu

last: Zhiqiang

first: Gao

last: Yan

first: Sun

last: Dongxiao

first: Peruggia

last: Gabrielle

first: Sallavanti

last: Jessica

first: Li

last: Wei


title: TFEB controls expression of human syncytins during cell-cell fusion [ChIP-Seq]

city: Berkeley

first: Meagan

middle: Nicole

last: Esbin

state: California

address: 475 Li Ka Shing Center

department: Molecular and Cell Biology

institute: University of California Berkeley

summary: During human development, a subset of differentiating fetal cells form a temporary organ, the placenta, which invades the uterine wall to support nutrient, oxygen, and waste exchange between the mother and fetus until birth. Most of the human placenta is formed by a syncytial villous structure which arises via cell-cell fusion of underlying fetal trophoblast stem cells. Genetic and functional studies have characterized the membrane protein fusogens, Syncytin-1 and Syncytin-2, that are both necessary and sufficient for human trophoblast cell-cell fusion. However, identification and characterization of upstream transcriptional regulators regulating their expression has been limited. Here, using CRISPR knockout in an in vitro cellular model of syncytiotrophoblast development (BeWo cells), we find that the transcription factor TFEB, mainly known as a regulator of autophagy and lysosomal biogenesis, is required for cell-cell fusion of syncytiotrophoblasts. TFEB translocates to the nucleus, exhibits increased chromatin interactions, and directly binds the Syncytin-1 and Syncytin-2 promoters to control their expression during differentiation. While TFEB appears to play an important role in syncytiotrophoblast differentiation, ablation of TFEB largely does not affect lysosomal gene expression or lysosomal biogenesis in differentiating BeWo cells, suggesting that TFEB plays an alternative role in placental cells.

overall_design: We examined the binding of TFEB to genomic loci using ChIP-sequencing during syncytiotrophoblast differentiation in BeWo cells. We compared binding of TFEB in BeWo cells stably expressing 3xFLAG-Halo-TFEB under a low expression promoter (L30) and an antibiotic cassette (IRES Puro). We compared genomic enrichment after immunoprecipatation using an anti-FLAG antibody versus IgG pulldown or Input samples before precipitation. We compared TFEB localization to specific genes after treatment with DMSO, Forskolin-induced differentiation, or after treatment with the mTOR inhibitor Torin1.

first: Liza

last: Dahal

first: Joseph

last: McKenna

first: Xavier

last: Darzacq

first: Robert

last: Tjian


title: Inhibition of thioredoxin reductase 1 sensitizes glucose-starved glioblastoma cells to disulfidptosis 2

last: Li

overall_design: To further characterize the cellular response to AF treatment under the glucose-deprived condition, we compared the gene expression profile of LN229-TAZ(4SA) cells cultured under AF and glucose deprivation  to those cultured under normal conditions using RNA-seq as indicated in the manuscript.


title: Inhibition of thioredoxin reductase 1 sensitizes glucose-starved glioblastoma cells to disulfidptosis 1

overall_design: To understand how TAZ activation drives GBM progression, we compared gene expression in LN229TAZ(4SA) and LN229vector tumors using RNA-sequencing (RNA-seq).


title: RNA sequencing of control, DNMT1 knockout and DNMT1/L1TD1 double knockout haploid cells (HAP1)

city: Vienna

first: Christian

last: Seiser

address: Schwarzspanierstrasse 17

department: Center for Anatomy and Cell Biology

country: Austria

institute: Medical University of Vienna

summary: To investigate the effects of L1TD1 ablation in DNMT1 knockout HAP-1 cells in comparison to DNMT1 knockout and control HAP1 cells

overall_design:
For RNA sequencing three control, three DNMT1 Knockout  and three DNMT1/L1TD1 double knockout samples were isolated and RNA was and then used for RNA sequencing. The aim was to do the analysis of differentially expressed genes across the three conditions to determine  the effects of L1TD1 ablation in DNMT1 knockout HAP1 cells  and in comparison to control cells.
Repeat samples were performed using the original libraries run in a separate lane. They were later merged in the analysis.

first: Mirjam

last: Beck

first: Kavaklioglu

last: Gülnihal

first: Ido

middle: M

last: Tamir


title: Aquaporin 5 deficiency suppresses fatty acid oxidation and delays liver regeneration through the transcription factor PPAR.

city: Qingdao

first: Peng

last: Chen

address: 308 Ningxia road, Qingdao

institute: Qingdao University

summary: After 70% partial hepatectomy (PHx), the metabolic pathways leading to hepatocyte lipid droplet accumulation during liver regeneration remain unclear. Aquaporin 5 (Aqp5) is an aquaporin and peroporin that facilitates the transport of both water and hydrogen peroxide (H2O2). In this study, we observed a delayed liver regeneration following PHx in Aqp5 knockout (Aqp5−/−) mice. Considering the role of Aqp5 in H2O2 transport, we hypothesized that deficiency in Aqp5 may induce oxidative stress and hepatocyte injury. Through the measurement of reactive oxygen species (ROS) and redox-related indices, we observed significant alterations in ROS levels as well as malondialdehyde (MDA), superoxide dismutase (SOD), and reduced glutathione (GSH) concentrations in regenerating livers lacking Aqp5 compared to wild-type controls. Oil Red O and 4-hydroxynonenal (4-HNE) staining results indicated that Aqp5 deficiency caused lipid accumulation during liver regeneration. The transcriptome sequencing results showed that the PPAR pathway is inhibited during the liver regeneration process in Aqp5 gene-knockout mice.The administration of the WY-14643 agonist, which targets the PPAR pathway, significantly mitigated delayed liver regeneration by enhancing hepatocyte proliferation and reducing lipid accumulation caused by Aqp5 deficiency. Our findings highlight the crucial role of Aqp5 in regulating H2O2 levels and lipid metabolism through the PPAR pathway during liver regeneration.

overall_design: RNA seg profling of wldtype C57 mice and Aqp5-/- mice after 70%PHx

first: Bin

last: LI

first: Shixu

last: Liu

first: Wenshuo

last: Han

first: Peirong

last: Song

first: Hetong

last: Sun

first: Xin

last: Cao

first: Guohu

last: Di


title: The GTE4–EML chromatin reader complex concurrently recognizes histone acetylation and H3K4 trimethylation in Arabidopsis [ChIP-Seq]

first: xinjian

last: He

overall_design: Examination of protein binding levels in transgenic plants

first: Xin-Jian


title: Integrated stress response associated with dark microglia contributes to neurodegeneration [snRNA-seq]

city: New York

first: Anna

last: Flury

address: 85 St Nicholas Terrace

institute: CUNY Graduate Center

summary: Microglia, the brain’s primary resident immune cells, can assume various phenotypes with diverse functional outcomes on brain homeostasis. In Alzheimer’s disease (AD), where microglia are a leading causal cell type, the identity of microglia subsets that drive neurodegeneration remains unresolved. Here, we identify a microglia phenotype characterized by a conserved stress signaling pathway, the integrated stress response (ISR). Using mouse models to activate or inhibit ISR in microglia, we show that ISR underlies the ultrastructurally distinct “dark” microglia subset linked to pathological synapse loss. Inducing microglial ISR in murine AD models exacerbates neurodegenerative pathologies, such as Tau pathology and synaptic terminal loss. Conversely, inhibiting microglial ISR in AD models ameliorates these pathologies. Mechanistically, we present evidence that ISR promotes the secretion of toxic long- chain lipids that impair neuron and oligodendrocyte homeostasis in vitro. Accordingly, inhibition of lipid synthesis in AD models ameliorates synaptic terminal loss. Our results demonstrate that activation of ISR within microglia represents a pathway contributing to neurodegeneration and suggest that this may be sustained, at least in part, by the secretion of long-chain lipids from ISR-activated microglia.

overall_design: To determine the effect of ISR on microglial subsets, we performed single-nuclei sequencing on 5xFADTRAP, 5xFADiPKR, and 5xFADEif2ATRAP microglial nuclei.

first: Leen

last: Aljayousi

first: Siaresh

last: Aziz

first: Hye-Jin

last: Park

first: Mohammadparsa

last: Khakpour

first: Colby

last: Sandberg

first: Fernando

middle: G

last: Ibanez

first: Olivia

last: Braniff

first: Pragney

last: Deme

first: Jackson

last: McGrath

first: Thi

last: Ngo

first: Jack

last: Mechler

first: Denice

last: Ramirez

first: Dvir

last: Avnon-Klein

first: John

last: Murray

first: Jia

first: Norman

middle: J

last: Haughey

first: Sebastian

last: Werneburg

first: Marie-Eve

last: Tremblay

first: Pinar

last: Ayata


title: Multidimensional Effects of Stress on Neuronal Exosome Levels and Simultaneous Transcriptomic Profiles

first: Carla

last: Nasca

address: 1 Park Avenue

institute: NYU Grossman School of Medicine

summary: Background: An excess of exosomes, nanovesicles released from all cells and key regulators of brain plasticity, is an emerging therapeutic target for stress-related mental illnesses. The effects of chronic stress on exosome levels are unknown; even less is known about molecular drivers of exosome levels in the stress response. Methods:  We used our state-of-the-art protocol with two complementary strategies to isolate neuronal exosomes from plasma, ventral dentate gyrus, basolateral amygdala, and olfactory bulbs of male mice to determine the effects of chronic restraint stress (CRS) on exosome levels. Next, we used RNA sequencing and bioinformatic analyses to identify molecular drivers of exosome levels. Results:  We found that CRS leads to an increase in the levels of neuronal exosomes but not total (i.e., not neuronally enriched) exosome levels assayed in plasma and the ventral dentate gyrus, whereas CRS leads to a decrease in neuronal exosome levels but not total exosome levels in the basolateral amygdala. There was a further specificity of effects as shown by a lack of changes in the levels of neuronal exosomes assayed in the olfactory bulbs. In pursuit of advancing translational applications, we showed that acetyl-L-carnitine administration restores the CRS-induced increase in neuronal exosome levels assayed in plasma (the most accessible specimen). Furthermore, the CRS-induced changes in neuronal exosome levels in the ventral dentate gyrus and basolateral amygdala mirrored the opposite pattern of CRS-induced transcriptional changes in these key brain areas, with β-estradiol signaling as a potential upstream driver of neuronal exosome levels. Conclusions:  This study provides a foundation for future studies of new forms of local and distant communication in stress neurobiology by demonstrating specific relationships between neuronal exosome levels assayed in plasma and the brain and providing new candidate targets for the normalization of exosome levels.

overall_design: Basolateral amygdala RNA from 3 biological replicates per group (Not-stressed Controls or Ctrl and Chronic Restrained Stress or CRS, all male mice) was used for library prep (TruSeq Stranded Total RNA with Ribo-Zero Human/Mouse/Rat) and sequenced at the Genomic Core facility at The Rockefeller University. Each sample was provided with a unique adapter and all samples were put in the same pool and run in multiple lanes to control for lane effects with a sequencing depth of about 30M (100bp, single end).

first: Hope

last: Kronman

first: Amarjyot

last: Singh

first: Shofiul

last: Azam

first: Andrea

last: Guzman

first: Danielle

last: Zelli

first: Timothy

last: Lau

first: Josh

last: Dobbin

first: Benedetta

last: Bigio


title: Integrated stress response associated with dark microglia contributes to neurodegeneration [TRAP-seq]

first: Anna

last: Flury

overall_design: To investigate the impact of microglial ISR on microglia molecular phenotypes, we performed ribosome profiling by translating ribosome affinity purification (TRAP) sequencing on microglia from TRAP (n=5, Cx3cr1CreErt2/+;Eef1a1LSL-EGFP-L10a/+), ASV-injected 5xFAD-TRAP (n=4, Cx3cr1CreErt2/+;5xFAD;Eef1a1LSL-EGFP-L10a/+), vehicle-injected iPKR (PEG:DMSO, n=4, Cx3cr1CreErt2/+;Eef1a1LSL.NS3.eGFP-L10a.iPKR/+), ASV-injected iPKR (n=5, Cx3cr1CreErt2/+;Eef1a1LSL.NS3.eGFP-L10a.iPKR/+), Eif2A (n=4, Cx3cr1CreErt2/+;  Eif2s1A/A;Tg(flEifs1)), ASV-injected 5xFADiPKR (n=4, Cx3cr1CreErt2/+;5xFAD;Eef1a1LSL.NS3.eGFP-L10a.iPKR/+), and 5xFADEif2A (n=3, Cx3cr1CreErt2/+;5xFAD;Eif2s1A/A;Tg(flEifs1)) mice. Mice were euthanized with CO2 and brain regions of interest were dissected and flash-frozen for TRAP. The cortices were micro-dissected and GFP-tagged ribosomes from the cell type of interest were immunoprecipitated with a GFP antibody. RNA sequencing was performed on the GFP antibody-bound fractions.

first: Leen

last: Aljayousi

first: Siaresh

last: Aziz

first: Hye-Jin

last: Park

first: Mohammadparsa

last: Khakpour

first: Colby

last: Sandberg

first: Fernando

last: Ibanez

first: Olivia

last: Braniff

first: Pragney

last: Deme

first: Jackson

last: McGrath

first: Thi

last: Ngo

first: Jack

last: Mechler

first: Denice

last: Liu


title: Modeling GATA2 deficiency in mice: the R396Q mutation disrupts normal hematopoiesis

city: Memphis

first: Rashid

last: Mehmood

state: Tennessee

address: 324 NIGHT SAIL DR N APT 112, UNIT # 04-112

department: Hematology

institute: St Jude Children's Research Hospital

summary: GATA2 deficiency is an autosomal dominant germline disorder of immune dysfunction and bone marrow failure with a high propensity for leukemic transformation in adolescents, present in up to 7% of pediatric myelodysplastic syndrome (MDS) and 15% of advanced MDS cases. While sequencing studies have identified several secondary mutations thought to contribute to malignancy, the mechanisms of disease progression have been difficult to identify due to a lack of disease-specific experimental models. Here, we generated a murine model of one of the most common GATA2 mutations associated with leukemic progression in GATA2 deficiency, Gata2R396Q/+. While mutant mice exhibit mild defects in peripheral blood output throughout life, they display significant hematopoietic abnormalities in the bone marrow (BM), including a reduction in hematopoietic stem cell (HSC) function and intrinsic biases toward specific stem cell subsets that differ from previous models of GATA2 loss. Supporting this observation, single-cell RNA sequencing of BM hematopoietic progenitors revealed a loss of HSC stemness, myeloid-bias, and accelerated ageing phenotype. Importantly, we show that Gata2R396Q/+ exerts effects early in hematopoietic development, as mutant mice generate fewer HSCs in the aorta gonad mesonephros, and fetal liver HSCs have reduced function. This reduced pool of HSCs and aged phenotype could be potential contributors to leukemic transformation in patients, and our model provides a useful tool to study the mechanisms of malignant transformation in GATA2 deficiency.

overall_design: Bone marrow LSK cells were isolated from WT and mutant mice for single cell RNA-seq via the 10X Genomics 3' v3.1 kit.

first: Trent

last: Hall

first: Diana

last: Sa da Bandeira

first: Anitria

last: Cotton

first: Shai

last: Izraeli

first: John

last: Crispino


title: Senescent cell extracellular vesicles are critical elements restricting cancer recurrence [scRNA-seq]

city: Portland

first: Laura

last: Heiser

state: Oregon

address: 2730 S. Moody Ave

department: BME

institute: OHSU

summary: Senescent cell extracellular vesicles (senEVs)are novel and underappreciated components of the senescence secretome. To understand the role that senEVs play in inflammatory responses to senescence, we developed and validated a novel engraftment-based senescence model that allowed us to genetically block the release of senEVs in vivo. We found that senEVs orchestrate prompt removal of senescent cells and inhibit tumor recurrence. Recruitment of MHC-II+ antigen presenting cells into the senescence microenvironment was significantly decreased in the absence of senEVs. Inhibition of senEV release changed the primary target of senescent cell signaling from antigen presenting cells to neutrophils. Antigen presenting cells recruited and activated CCR2+ TH17 cells, which in turn inhibited B cell activation. Through multimodal transcriptional and proteomic analysis, we identified 6 ligands that were specific to senEVs, suggesting a role in promoting cell adhesion. Overall, these results indicate that senEVs complement the activity of secreted inflammatory mediators in recruiting and activating distinct immune cell subsets for efficient removal of senescent cells.

overall_design: To investigate the role of senEVs in inflammatory responses to senescence, we established a novel engraftment-based senescence model. Through this model, we genetically blocked the release of senEVs. Employing scRNA sequencing, we explored the signals that senescent cells transmit to their microenvironment in both the presence and absence of senEVs, elucidatign their impact on the recruitment of immune cells.

first: Tahereh

last: Ziglari

first: Nicholas

middle: L

last: Calistri

first: Jennifer

last: Finan

first: Daniel

last: Derrick

first: Ernesto

last: Nakayasu

first: Meagan

last: Burnet

middle: E

last: Kyle

first: Matthew

last: Hoare

first: Ferdinando

last: Pucci


title: Senescent cell extracellular vesicles are critical elements restricting cancer recurrence [bulk RNA-seq]

overall_design: To investigate the role of senEVs in inflammatory responses to senescence, we established a novel engraftment-based senescence model. Through this model, we genetically blocked the release of senEVs. Employing scRNA sequencign, we explored the signals that senescent cells transmit to their microenvironment in both the presence and absence of senEVs, elucidatign their impact on the recruitment of immune cells.


title: Decoding the immune checkpoint signatures in human atherosclerosis: implications of type 2 diabetes and lipid-lowering intervention [Human_coronary_plaque_scRNA]

first: Michael

last: Gildea

state: New York

address: 550 1st avenue

institute: NYU Langone Health

summary: Immune checkpoint inhibitor (ICI) therapies in cancer accelerate atherosclerosis progression. Here, we have charted the landscape of immune checkpoint gene expression and defined the network of disease-relevant interactions with single-cell resolution. We found that signaling through PD-1 and CTLA4 is driven by a population of dendritic cells enriched for FSCN1 that can be derived from peripheral blood cells following anti-PD-1 or -CTLA4 treatment ex vivo. Type 2 diabetes dampened plaque PD-1 and CTLA4 signaling, showing that cardiometabolic comorbidities elicit unique responses to ICIs. Lipid-lowering therapy equalized the intensity and direction of immune checkpoint interactions in human blood, while atherosclerotic mice subjected to a lipid-lowering diet displayed both increased co-inhibitory signaling and a downregulation of inflammatory transcriptional programs in plaques. Our findings underscore the potential of lipid-lowering therapies in stabilizing immune checkpoint interactions and reducing plaque inflammation, offering new insights on atherosclerosis and cardiovascular risks in cancer patients undergoing ICI treatments.

overall_design: Left and right anterior descending arteries were washed, minced, and digested then filtered using cell strainers. Following a dead cell removal step, single-cell suspensions were counted and loaded onto a Chromium Controller (10x Genomics).

first: Jose

last: Barcia Duran

first: Dayasagar

last: Das

first: Kathryn

last: Moore

first: Chiara

last: Giannarelli


title: Transcriptomic analysis of primary mouse adipocytes after Rbm43 knockdown

status: Public on Dec 22 2024

city: San Francisco

first: Phillip

middle: Anthony

last: Dumesic

address: 513 Parnassus Ave, HSW1119

department: Diabetes Center

institute: University of California, San Francisco

summary: Obesity is associated with systemic inflammation that impairs mitochondrial function. This disruption curtails oxidative metabolism, limiting adipocyte lipid metabolism and thermogenesis, a metabolically beneficial program that dissipates chemical energy as heat. Here, we show that PGC1α, a key governor of mitochondrial biogenesis, is negatively regulated at the level of its mRNA translation by the RNA-binding protein RBM43. RBM43 is induced by inflammatory cytokines and suppresses mitochondrial biogenesis in a PGC1α-dependent manner. In mice, adipocyte-selective Rbm43 disruption elevates PGC1α translation and oxidative metabolism. In obesity, Rbm43 loss improves glucose tolerance, reduces adipose inflammation, and suppresses activation of the innate immune sensor cGAS-STING in adipocytes. We further identify a role for PGC1α in safeguarding against cytoplasmic accumulation of mitochondrial DNA, a cGAS ligand. The action of RBM43 defines a translational regulatory axis by which inflammatory signals dictate cellular energy metabolism and contribute to metabolic disease pathogenesis.

overall_design: Primary Ppargc1a(fl/fl) adipocytes were transduced with adenovirus encoding Gfp (control) or Cre (to excise the Ppargc1a gene). Cells were subsequently treated with control siRNA or an siRNA targeting Rbm43. Cells were differentiated completely, then RNA was harvested in biological duplicates using trizol.

first: Bruce

last: Spiegelman


title: Overview of distinct 8-Oxoguanine profiles of messenger RNA in normal and senescent cancer cells

status: Public on Dec 21 2024

first: Yu

last: Lin

state: China

address: No. 1838, Guangzhou Dadao North, Baiyun District, Guangzhou

institute: Guangdong Provincial Key Laboratory of Gastroenterology, Department of Gastroenterology, Nanfang Hospital

summary: Background: Cellular senescence plays a key role in the development of cancer, but the underlying mechanisms are unknown.Recently, several recent studies have shown that RNA methylation is closely related to cancer cell aging. 8-Oxoguanine (o8G) is an important and widely distributed methylation modification whose role in cancer cell senescence is far from elucidated. Methods: In this study, senescent cancer cell models (CaCO2 cells) were constructed by knocking down the ADAR1 gene. RNA immunoprecipitation sequencing was used to identify the o 8 G peaks on messenger RNA (mRNA) of normal CaCO2 cells and senescent CaCO2 cells, and the distribution characteristics of mRNA o 8 G modification were identified. Further bioinformatics analysis of the sequencing data was performed to preliminarily elucidate the potential function of the o8G-modified mRNA. Results: There were significant differences in mRNA o 8 G modification distribution between normal and senescent CaCO2 cells.It is suggested that o8G modification may play a key role in inducing cancer cells or promoting cancer cell senescence. Gene ontology (GO) enrichment analysis showed that the genes modified by o 8 G were enriched in Cellular component organization or biogenesis, Focal adhesion,and RNA binding. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that the genes modified by o8G areconcentrated in Focal adhesion signaling pathway, Small cell lung cancer signaling pathway and Proteoglycans in cancer signaling pathway. Conclusion: This study preliminarily revealed the different distribution patterns of o8G modification between normal CaCO2 cells and senescent CaCO2 cells. Our study established the link between o 8 G modification and cancer cell senescence, which provides a new insight into the mechanism of cancer cell senescence and a potential therapeutic target for subsequent cancer treatment.

overall_design: Methylated RNA immunoprecipitation sequencing (MeRIP-seq) was used to investigate the O8G modification level and distribution characteristics of mRNA in normal and senescent cancer cells

first: Jingwen

last: Huang

first: Yingying

last: Zhao

first: Lingbo

last: Wei


title: Spatiotemporal single-cell architecture of gene expression in the Caenorhabditis elegans germ cells [scRNA-seq]

city: hangzhou

first: li

middle: li

last: li

state: zhejiang

address: 600 Dunyu Road

institute: westlake university

summary: Spermatogenesis is an intricate and tightly controlled process, encompassing various layers of gene expression regulation. Despite our current understanding, the developmental trajectory and its regulatory mechanisms dictating spermatogenesis remain elusive. In this study, we have generated single-cell gene expression profiles for C. elegans sperm cells and constructed gene-regulated networks alongside the developmental trajectories of these cells. Our findings indicate that each pre- and post-developmental stage is closely linked by co-expressed genes, while simultaneously being uniquely identified by the combined expression of specific gene families. To illustrate the applicability of this exhaustive gene expression catalog, we used gene-regulated networks to uncover potential transcription factors for (1) the expression of genes in the phosphorylation pathway, identifying NHR-23-to-phosphatase regulation for the meiotic cell division process, and (2) the expression of constituent components of small RNA pathways, identifying ELT-1-to-Argonaute protein regulation for siRNA maintenance and sperm activation. We expect that this sperm cell-specific gene expression directory will prompt investigations into the underlying mechanisms determining anatomy, differentiation, and function across the reproductive system. Finally, our expression data can be explored using the web application CelegansGermAtlas (https://scgerm-atlas.sjtu.edu.cn/website/#/home).

overall_design: Germ cells of L4 C. elegans (gfp::tev::flag::prg-1) were isolated using Fluorescence-activated cell sorting (FACS) based on the presence or absence of GFP signal. Subsequently, the isolated cells were analyzed using single-cell RNA sequencing (scRNA-seq).

first: Lili

last: Li

first: Xuanxuan

last: Guo

first: Rao

last: Di

first: Junchao

last: Xue


title: Neutrophils physically interact with tumor cells to form a signaling niche promoting breast cancer aggressiveness

city: Tel Aviv

first: Merav

last: Cohen

address: Chaim Levanon St 55

department: Clinical microbiology and immunology

country: Israel

institute: Tel Aviv University

summary: Tissue remodeling and cell plasticity in the mammary gland are activated by multi-lineage communications. However, the dynamic signaling promoting breast cancer remains unclear. Here, by RNA-sequencing of single cells and physically interacting cells (PIC-seq) along mammary gland development and carcinogenesis, we uncovered that neutrophils appear transiently during early development and re-emerge in physical interaction with tumor cells in advanced carcinoma. Neutrophil heterogeneity analysis characterized transcriptional states linked to age and cancer stage. Integrating ligand-receptor and PIC-seq analyses with various functional experiments unveiled a physical and secreted pro-tumorigenic signaling niche. This approach revealed that neutrophils are recruited by ductal macrophages and physically interact with tumor cells, increasing tumor cell proliferative and invasive properties, as well as endothelial proliferation and angiogenesis. The molecular program upregulated in neutrophil-PICs correlates with lower survival in advanced breast cancer patients. Our interaction-driven perspective highlights potential molecular targets and biomarkers for breast cancer treatment.

overall_design: Transcriptional profiling of single cells and doublets (PIC) of of immune, non-immune and epithelial populations along different timepoints across mammary gland development and carcinogenesis

first: Sandra

last: Camargo

first: Ori

last: Moskowitz

first: Amir

last: Giladi

first: Maiia

last: Levinson

first: Roi

last: Balaban

first: Shani

last: Gola

first: Alice

last: Raizman

first: Kelly

last: Lipczyc

first: Alon

last: Richter

first: Noa

last: Keren-Khadmy

first: Oren

last: Barboy

first: Yael

last: Dugach

first: Yaron

last: Carmi

last: Sonnenblick


title: Extensive mutual influences of SMC complexes shape 3D genome folding

city: Shenzhen

first: Haoyue

last: Zhang

address: Gaoke Innovation Center, Guangqiao Road, Guangming District, Shenzhen

institute: Shenzhen Bay Lab

summary: Mammalian genomes are folded by the distinct actions of SMC complexes which include the chromatin loop-extruding cohesin, the sister-chromatid cohesive cohesin, and the mitotic chromosome-associated condensins 1-3. While these complexes function at different stages of the cell cycle, they co-exist on chromatin during the G2/M-phase transition, when genome structure undergoes a dramatic reorganization 1,2. Yet, how distinct SMC complexes affect each other and how their mutual interplay orchestrates the dynamic folding of 3D genome remains elusive. Here, we engineered all possible cohesin/condensin configurations on mitotic chromosomes to delineate the concerted, mutually influential action of SMC complexes. We find that: (i) Condensin disrupts extrusive-cohesin binding at CTCF sites, thereby promoting the disassembly of interphase TADs and loops during mitotic progression. Conversely, extrusive-cohesin impedes condensin mediated mitotic chromosome spiralization. (ii) Condensin diminishes cohesive-cohesin peaks and, conversely, cohesive-cohesin antagonizes condensin-mediated mitotic chromosome longitudinal shortening. Co-presence of extrusive- and cohesive-cohesin synergizes these effects and dramatically inhibits mitotic chromosome condensation. (iii) Extrusive-cohesin positions cohesive-cohesin at CTCF binding sites. However, cohesive-cohesin by itself cannot be arrested by CTCF molecules, is insufficient to establish TADs or loops and lacks loop extrusion capacity, implying non-overlapping function with extrusive-cohesin. (iv) Cohesive-cohesin restricts extrusive-cohesin mediated chromatin loop expansion. Collectively, our data describe a comprehensive three-way interplay among major SMC complexes that dynamically sculpts chromatin architecture during cell cycle progression.

overall_design: The G1E-ER4 derivitive cell lines: Wapl-dTag/SMC2-AID; Wapl-dTag/Sororin-AID (with or without Sororin-9A) and Wapl-dTag/SMC2-AID/Sororin-AID (with or without Sororin-9A mutant) were used to obtain mitotic populations with distinct configurations of SMC protein complexes. These samples were then subject to Hi-C for chromatin structure interrogation as well as chromatin immunoprecipitation DNA sequencing (ChIP-seq) against Rad21 and flag-tagged Sororin.

first: Fuhai

last: Liu


title: ETVs dictate hPSC differentiation by tuning biophysical properties

city: Poznan

first: Katarzyna

last: Blaszczyk

state: Wielkopolska

address: ul. Uniwersytetu Poznańskiego 6/1.87

department: Faculty of Biology, Institute of Molecular Biology and Biotechnology, Department of Gene Expression

country: Poland

institute: Adam Mickiewicz University

summary: Stem cells maintain a dynamic dialogue with their niche, integrating biochemical and biophysical cues to modulate cellular behavior. Yet, the transcriptional networks that regulate cellular biophysical properties remain poorly defined. Here, we leverage human pluripotent stem cells (hPSCs) and two morphogenesis models – gastruloids and pancreatic differentiation – to establish ETV transcription factors as critical regulators of biophysical parameters and lineage commitment. Genetic ablation of ETV1 or ETV1/ETV4/ETV5 in hPSCs enhanced cell-cell and cell-ECM adhesion, leading to aberrant multilineage differentiation including disrupted germ-layer organization, ectoderm loss, and extraembryonic cell overgrowth in gastruloids. Furthermore, ETV1 loss abolished pancreatic progenitor formation. Single-cell RNA sequencing and follow-up assays revealed dysregulated mechanotransduction via the PI3K/AKT signaling. Our findings highlight the importance of transcriptional control over cell biophysical properties and suggest that manipulating these properties may improve in vitro cell and tissue engineering strategies.

overall_design: Comparative gene expression profiling analysis of RNA-seq, scRNA-seq and ATAC-seq data for iCas9 hPSCs and its KO derivatives (KO=ETV1 KO and tKO=ETV1-, ETV4-, ETV5 KO) in pluripotency  (pluri) and pancreaatic progenitors stage.

first: Natalia

last: Ziojla

first: Magdalena

last: Socha

first: Cecilia

last: Guerra

first: Edyta

last: Urbaniak

first: Dorota

last: Kizewska

first: Sara

last: Henry

first: Malgorzata

last: Grabowska

first: Aryeh

last: Warmflash

last: Borowiak

first: Kathy

middle: K

last: Niakan


title: TERT Expression and Clinical Outcome in Pulmonary Carcinoids

city: Cologne

first: Matthias

last: Fischer

address: Kerpener Str. 62

department: Department of Experimental Pediatric Oncology

institute: University Children’s Hospital of Cologne

type: Methylation profiling by genome tiling array

summary: The clinical course of pulmonary carcinoids ranges from indolent to fatal disease, suggesting that specific molecular alterations drive progression toward the fully malignant state. A similar spectrum of clinical phenotypes occurs in pediatric neuroblastoma, in which activation of telomerase reverse transcriptase (TERT) is decisive in determining the course of disease. We therefore investigated whether TERT expression defines the clinical fate of patients with pulmonary carcinoid.

overall_design: Bisulphite converted DNA from 24 pulmonary carcinoid tumors was hybridized to the Illumina Infinium HumanMethylation850 BeadChip

first: Lisa

last: Werr


title: RNA-sequencing of melanoma celllines compared to normal human melanocytes (NHEMs)

status: Public on Dec 20 2024

city: Deggendorf

first: Melanie

last: Kappelmann-Fenzl

address: Dieter-Görlitz Platz 1

department: Applied Computer Science

institute: Deggendorf Institute of Technology

summary: Given that melanomas gain different properties during their progression, 6 different melanoma cell lines (Sbcl-2, WM3211, WM793, WM1366, WM1158, WM9) were selected and categorized by tumor stage (primary tumor (PT), metastasis (MET)) as well as the previously described BRAF and PTEN mutation status. Then, differentially expressed genes in malignant melanoma were identified by performing RNA-Sequencing (RNA-seq) with all aforementioned melanoma cell lines and normal human epidermal melanocytes (NHEM).

overall_design: Total RNA-Seq of the melanoma celllines Sbcl-2, WM3211, WM13166, WM793, WM1158, WM9 and normal human epidermal melanocytes (NHEM).

first: Anja Katrin

last: Bosserhoff

first: Michael

last: Rehli

first: Claudia

last: Gebhard


title: Single cell gene and protein expression of 2W:I-Ab specific cells from effector and memory T cells after aLm infection

city: Minneapolis

first: Marc

last: Jenkins

state: MN

address: 2101 6th St SE

department: Microbiology

institute: University of Minnesota

summary: Single-cell gene expression of CD4+ tetramer+ cells from WT B6 animals was determined by sRNAseq after aLm infection.

overall_design: B6 mice were infected with aLm for 6, 21, or 60 days, treated wth an anti-ARTC2 nanobody before harvest, and sorted for CD4+ Tetramer+ cells. Post sort cells were hashtagged for each timepoint and feature barcoded for cell surface proteins and immune receptor mapping. Gene expression was determined by scRNAseq.

first: Kevin

last: Osum

first: Samuel

middle: H

last: Becker


title: The acute stress response is mediated by prefrontal 5α-reductase 2 in males via allopregnanolone synthesis

city: Salt Lake City

first: Philip

last: Moos

state: UT

department: Pharmacology & Toxicology

institute: University of Utah

summary: Acute stress triggers rapid brain responses, including increased allopregnanolone (AP) synthesis via 5α-reductase (5αR). However, the involvement of this enzyme in stress response remains elusive. Here, we investigated the roles of the two major 5αR isoenzymes in acute stress. Acute stress increased mRNA and protein levels of 5αR2, but not 5αR1, in the medial prefrontal cortex (mPFC) of male, but not female, rats. Targeted mPFC downregulation of 5αR2, but not 5αR1, markedly reduced the responsiveness of males, but not females, to both stressful and arousing stimuli. Similar sex differences were observed in 5αR2 knockout rats. While 5αR1 regulated AP synthesis under baseline conditions, 5αR2 enabled this process in response to acute stress. Single-nucleus transcriptomic analyses revealed that 5αR2 enabled stress-induced protein synthesis in pyramidal neurons and glia of the mPFC. These findings underscore the pivotal role of 5αR2 in shaping sex-related differences in acute stress reactivity.

overall_design: single-nuclei RNA sequencing from flash-frozen dissected prefrontal cortex from wild-type rats and 5-alpha-reductase-2 knockdown rats

first: Marco

last: Bortolato


title: Ribosome Profiling Reveals Differences in Global Translational vs. Transcriptional Gene Expression Changes During Early Candida albicans Biofilm Formation

city: San Antonio

first: David

last: Kadosh

address: 7703 Floyd Curl Dr., MC: 7758

department: Department of Microbiology, Immunology & Molecular Genetics

institute: University of Texas Health Science Center at San Antonio

summary: Candida albicans, a major human fungal pathogen, can form biofilms on a variety of inert and biological surfaces. C. albicans biofilms allow for immune evasion, are highly resistant to antifungal therapies, and represent a significant complication for a wide variety of immunocompromised patients in clinical settings. While transcriptional regulators and global transcriptional profiles of C. albicans biofilm formation have been well-characterized, much less is known about translational regulation of this important C. albicans virulence property. Here, using ribosome profiling, we define the first global translational profile of genes that are expressed during early biofilm development in a human fungal pathogen, C. albicans.  We show that C. albicans biofilm formation involves altered translational regulation of genes and gene classes associated with protein synthesis, pathogenesis, transport, plasma membrane, polarized growth, the cell cycle, secretion and signal transduction. Interestingly, while similar, but not identical, classes of genes showed transcriptional alterations during early C. albicans biofilm development, we observed very little overlap between specific genes that are up-regulated or down-regulated at the translational vs. transcriptional levels. Our results suggest that distinct translational mechanisms play an important role in regulating early biofilm development of a major human fungal pathogen.  These mechanisms, in turn, could serve as potential targets for novel antifungal strategies.

overall_design: C. albicans cells were grown under either biofilm or planktonic conditions using 3 biological replicates. Separate samples from cells grown under each condition were prepared for both Ribo-seq and RNA-seq analysis. Overall there are 12 total samples: 3 replicates of biofilm Ribo-seq, 3 replicates of planktonic Ribo-seq, 3 replicates of biofilm RNA-seq and 3 replicates of planktonic RNA-seq.

first: Saket

last: Choudhary


title: Expression data from WT and Neu1 deficient 1 mo and 5 mo hippocampi

first: Alessandra

last: d'Azzo

address: 262 Danny Thomas Place

institute: St. Jude Children's Research Hospital

summary:
Microarray data was used to assess progressive upregulation of genes during course of disease in Neu1 deficient mice and use for GSEA studies to assess upregulated pathways.
Top upregulated genes often belonged to the myeloid lineage. Data was used to guide assessment of the neuroinflammatory response that occurs in Neu1 deficient hippocampi.

overall_design: Total RNA (100 ng) was extracted from WT and Neu1 deficient hippocampi at 1 and 5 months, converted into biotin-labeled cRNA, and hybridized to a mouse Affymetrix GeneChip. Probe signals from microarrays were normalized and transformed into log2 transcript expression values by using the Robust Multiarray Average algorithm. Differentially expressed transcripts were identified by ANOVA, and the false discovery rate (FDR) was estimated. GSEA were completed using xtools software.

first: Leigh

last: Fremuth

first: Ida

last: Annunziata

first: Geoff

last: Neale