title: MANCR lncRNA modulates cell-cycle progression and metastasis in breastcancer cells by regulating the isoform-specific expression of nuclear Rho-GEF incis

city: Urbana

first: Prasanth

last: Kannanganattu

address: 601 S Goodwin Ave

department: Cell and Developmental Biology

institute: University of Illinois at Urbana-Champaign

summary: A significant number of the genetic alterations observed in cancer patients lie within the non-protein-coding segments of the genome, including in regions coding for long noncoding RNAs (lncRNAs). LncRNAs display aberrant expression in cancer, including Breast cancer (BrCa), but the implication of their altered expression in BrCa progression remains to be elucidated. By performing a genome-wide transcriptome screen in a Triple negative BrCa (TNBC) progression isogenic 2D and 3D spheroid model, we identify >1000 lncRNAs that display aberrant expression during BrCa progression. MANCR, a chromatin-associated lncRNA, shows elevated expression in tumorigenic and metastatic TNBC. MANCR is up regulated in response to cellular stress, including DNA damage and modulates DNA repair and cell proliferation. In addition, MANCR promotes metastasis as MANCR-depleted cells show reduced cell migration, invasion and wound healing in vitro, and defects in lung colonization in Xenograft experiments in vivo. Transcriptome analyses reveal that MANCR modulates expression and pre-mRNA splicing of genes, controlling DNA repair and checkpoint. At the mechanistic level, MANCR promotes the transcription ofNet1A, a Rho-GEF and regulator DNA damage checkpoint and metastasis incis,by differential promoter usage. Further experiments suggest that MANCR regulates the expression of cancer-associated genes by titrating the levels of various chromatin and RNA-binding proteins. Thus, our results identified the metastasis-promoting activities of a MANCR in TNBC by regulating the expression of genes incis.

overall_design: Stable knockdown of MANCR was done using shRNA (shRNA1 and shRNA2) and CRISPRi. A Dox-inducible MANCR construct was stably expressed in M1 cells and overexpressed using Doxycycline. Isogenic M1, M3, and M4 cell lines were cultured at 37 degrees Celsius with 5% carbon dioxide. RNA was extracted using Trizol and cleaned up using Qiagen RNeasy column.

first: Deepak

last: Singh

first: Zhengmin

last: Cong

first: You

last: Song

first: Kannanganattu

middle: V

last: Prasanth


title: Cyanobacterial sigma factor controls biofilm-promoting genes through intra- and intercellular pathways

status: Public on Sep 13 2024

city: Ramat-Gan

first: Hiba

last: Waldman Ben-Asher

address: Ramat-Gan

department: Life-Sciences

country: Israel

institute: Bar-Ilan University

type: Expression profiling by high throughput sequencing

summary: Cyanobacteria frequently constitute integral components of microbial communities known as phototrophic biofilms, which are widespread in various environments and hold significant industrial relevance. Previous studies of the model cyanobacterium Synechococcus elongatus PCC 7942 revealed that its planktonic growth habit results from a biofilm-suppression mechanism that depends on an extracellular inhibitor, an observation that opens the door to investigating cyanobacterial intercellular communication. Here, we demonstrate that the RNA polymerase sigma factor SigF1, is required for this biofilm-suppression mechanism and suggest that sigF1-inactivation impairs secretion of the biofilm inhibitor. The S. elongatus paralog SigF2, however, is not involved in biofilm regulation. Comprehensive transcriptome analyses identified distinct regulons under the control of each of these sigma factors. Additional data indicate that SigF1 regulates biofilm through its involvement in transcriptional induction of genes that include those for the primary pilus subunit: sigF1 inactivation both prevents pilus assembly and abrogates secretion of the biofilm inhibitor. Consequently, expression is significantly upregulated for the ebfG-operon that encodes matrix components and the genes that encode the corresponding secretion system. Thus, this study uncovers a basic regulatory component of cyanobacterial intercellular communication, a field that is in its infancy. Elevated expression of biofilm-promoting genes in a sigF1 mutant supports an additional layer of regulation by SigF1 that operates via an intracellular mechanism.

sample_organism: Synechococcus elongatus PCC 7942 = FACHB-805

overall_design: To uncover transcriptional changes associated with biofilm formation we compared the transcriptomes of WT and the biofilm-forming strains PilB::Tn5 and SigF1::Mu. In addition, SigF2 mutant was included in the analysis to define the transcriptional space of either one of these SigF factors.


title: Chondrocytes differentiation and immune cells interactions of Endplate Cells in Human Intervertebral Disc Degeneration

city: Zhengzhou

first: Chenhao

last: Shi

state: He'nan

address: No.1 Jianshe Dong Road, Erqi District, 450052, Zhengzhou, Henan, China

country: China

institute: The first affiliated hospital of Zhengzhou university

summary: We performed the single-cell RNA-sequencing analysis (scRNA-seq) of the cells focusing on degenerative human endplates. By unsupervised clustering of the 8,534 single-cell based on the gene expression, we identified nine distinct cell types. We employed Gene ontology (GO) analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways analysis, and the single-cell regulatory network inference and clustering (SCENIC) to determine the enriched pathways and transcriptional activities across seven chondrocyte subpopulations. Furthermore, two cell fates of chondrocyte differentiation were found by trajectory analysis, one was enriched in inflammation-related genes, and the other was related to extracellular matrix (ECM). Additionally, the intercellular interactions of MAs and chondrocytes, T/NK cells and chondrocytes were examined by ligand-receptor pairs analysis, showing the important regulative function of FN1 from MAs and CD74 from T/NK cells during endplate degeneration.

overall_design: Cartilage endplate tissue was obtained from two patients diagnosed with modic changes at the levels of L3-L4 or L4-L5, who were undergoing surgery for disc herniation, and analyzed using scRNA-seq. The patients were a 58-year-old female and a 66-year-old male, respectively. All participants included were confirmed with MRI images before surgery.

first: Shuangfei

last: Ni

first: Yonggang

last: Fan

first: Xiusheng

last: Huang


title: IL-8 drives the differentiation of CD74high neutrophils to activate anti-tumor immunity and attenuate NSCLC progression [II]

city: 武汉

first: ZHEN

last: TANG

state: 湖北

address: 医学研究院

institute: 武汉大学

summary: Neutrophils infiltrated in the tumor microenvironment (TME) are heterogeneous populations associated with the prognosis of cancer and cancer immunotherapy. However, the plasticity and function of heterogeneous neutrophils in the TME of non-small cell lung cancer (NSCLC) remain poorly understood. Here, we show that neutrophils produce high levels of IL-8 which induce the differentiation of CD74highSiglecFlow neutrophils and suppress the generation of CD74lowSiglecFhigh neutrophils in the TME of IL-8-humanized (IL8-hu) KrasLSL-G12DTp53fl/fl (KP) and EGFRT790M/Del-Exon19 (EGFRTD) NSCLC mouse models. The CD74highSiglecFlow neutrophils exhibit antigen cross-presentation activity to augment anti-tumor T cell responses. Consistently, deletion of CD74 in IL8-hu neutrophils attenuates the activation of T cells and exacerbates the progression of NSCLC, whereas treatment of a CD74 agonist promotes anti-tumor T cell activation and enhances the effects of anti-PD1 or Osimertinib therapy in the IL8-hu NSCLC mouse models. In addition, we have found that the CD74highCD63low neutrophils in the TME and peripheral blood of advanced NSCLC patients pheno-copy the CD74highSiglecFlow neutrophils in the TME of NSCLC mice and correlate well with the responsiveness to anti-PD-1 plus chemotherapies. Collectively, these data demonstrate an IL-8-CD74high neutrophil axis that promotes anti-tumor immunity in NSCLC.

overall_design: Comparative gene expression profiling analysis of RNA-seq data in neutrophils sorted from tumor-burdened lungs of KrasLSL-G12D/+Tp53fl/flIL8 mice.

first: Zhen

last: Tang

first: Dandan

last: Lin

first: Conghua

last: Xie

first: Bo

last: Zhong


title: IL-8 drives the differentiation of CD74high neutrophils to activate anti-tumor immunity and attenuate NSCLC progression [I]


title: IL-8 drives the differentiation of CD74high neutrophils to activate anti-tumor immunity and attenuate NSCLC progression

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

overall_design: Refer to individual Series


title: Myeloid leukemia and melanoma genomic characterization in murine cell lines

city: lille

first: frédéric

last: leprêtre

address: Boulevard du Pr Leclercq

department: CHU of Lille, CBP

country: France

institute: Lille University

type: Genome variation profiling by genome tiling array

summary: Genome profiling of mouse clones originating from myeloid leukemia and melanoma lines compared to control from the same murine strain.

overall_design: The goal of this study was to determine gene deletions and duplications on myeloid leukemia and melanoma lines affected by tumor dormancy.

first: Thierry

last: Idziorek

first: Yasmine

last: Touil


title: Coexpression and functions of the colinear genes CD47 and IFT57 in thyroid cancer

city: Bethesda

first: Thomas

middle: Joshua

last: Meyer

state: Maryland

address: 41 Center Drive, Building 41, Room B620

department: National Cancer Institute

country: USA

institute: National Institutes of Health

summary: Higher CD47 expression in some cancers correlates with poor survival. In contrast, higher CD47 mRNA expression in papillary thyroid carcinomas correlated with significantly improved overall survival. IFT57 was the top gene coexpressed with CD47 in these tumors, and higher IFT57 mRNA correlated with significantly improved overall and disease-free survival. IFT57 is a component of the primary cilium, which also regulates cancer pathogenesis. IFT57 and CD47 are colinear genes in humans, and this synteny is highly conserved across amniote genomes. Transcriptional regulators have been identified that increase CD47 expression in cancers, but its proximity suggested that IFT57 could be coregulated. CD47 and IFT57 mRNAs were also coregulated in anaplastic thyroid carcinoma cell lines and carcinomas of multiple tissues. Anaplastic thyroid carcinoma cell line mutants with decreased IFT57 or CD47 expression were used to define IFT57-dependent phenotypes and gene expression. Loss of IFT57 expression regulated transcripts involved in cell motility and extracellular matrix pathways. Altered cell motility was confirmed in IFT57 mutants. Three IFT57-dependent genes identified in the cell lines exhibited stronger coexpression with IFT57 than with CD47 in papillary thyroid tumors. CRACD was identified as an IFT57-specific target that correlates with survival in lung as well as papillary thyroid carcinomas.

overall_design: The 8505C cell line used to generate IFT57KO and CD47KO mutants via transfecting CRISPR plasmids. The Wild-type (WT), two clones of IFT57 depleted cells (SG1F4 and SG2B6), and pooled CD47 KO cells were selected for the current study.  WT and mutant 8505C cell lines were cultured in complete RPMI 1640 medium supplemented with glutamine, 10% FBS, and penicillin/streptomycin (ThermoFisher Scientific) and cells were harvested by washing with IXPBS and  cell pellets were harvested for RNA extraction.

first: Sukhbir

last: Kaur

first: Kun

last: Dong

middle: K

first: Maggie

last: Cam

first: David

last: Roberts

first: Richard

last: Finney


title: Antenatal Glucocorticoid Exposure Induced Neurodegenerative Changes in the Fetal Ovine Hippocampal Transcriptome Independent of Pulmonary Immaturity

city: Singapore

first: Gokce

last: OGUZ

address: 60 Biopolis St, GENOME, #02-01

department: Bioinformatics Consulting and Training Platform

country: Singapore

institute: Genome Institute of Singapore

summary:
[Background]
Antenatal steroid (ACS) therapy conveys lifesaving benefits to the preterm fetus. However, current treatments are associated with increased risk of growth, endocrine, and neurodevelopmental abnormalities.
[Objectives]
To inform ACS therapy optimisation we used a sheep model of pregnancy to test whether adverse fetal brain effects were independent of respiratory benefit and ACS pharmacokinetics.
[Study Design]
Ewes carrying a single fetus received intramuscular injections of either: i) 2 x 12mg  dexamethasone phosphate 12 hourly (DexP) (Dex High); ii) 4 x 1.5mg DexP 12 hourly (Dex Low); iii) 1 x 0.125mg/kg betamethasone acetate (Beta-Acetate); or iv) Saline (Negative Control). Lambs were delivered for ventilation after 48 hours.  Hippocampal and lung tissues were subjected to RNA sequencing.
[Results]
Dex High treatment did not mature the preterm ovine lung. Pulsatile (Dex High and Dex Low) treatments caused hippocampal RNA changes consistent with neuronal death. Constant, low-amplitude treatment (Beta Acetate) significantly improved lung function, with few adverse RNA transcriptional changes in the fetal hippocampus.
[Conclusions]
All ACS exposures caused significant alterations in fetal homeostasis. Pulsatile exposures caused dose-independent neurodegenerative changes in the fetal hippocampus. These data highlight the importance of considering magnitude, duration and stability of fetal glucocorticoid exposures in optimising ACS therapy.

overall_design: Animal Work and Antenatal Corticosteroid Treatments These studies were reviewed and approved by the University of Western Australia’s Animal Ethics Committee prior to work commencing (2021/ET000974). Date-mated Merino ewes with singleton fetuses received an intramuscular injection (IM) of 150 mg medroxyprogesterone acetate (Depo-Provera, Pfizer, New York, NY) on 117 ± 2 d of gestational age (dGA) to decrease the risk of steroid-induced premature labour. Antenatal progesterone has been previously reported not to impact fetal lung maturation in the sheep 128. Baseline maternal blood samples were collected for analysis at 0800, 24 - 48hrs prior to allocation to treatment group.  Animals were then allocated to one of four groups (Figure 6), with first treatment given at 122 ± 2 dGA (term = 150 dGA). First injections occurring at 08:00 to control for circadian effects:    i) Saline Control Group, ewes received 4 x 2 ml sterile normal saline maternal IM injections at 12 h intervals (n=11);  ii) Dex High Group, ewes received 2 x 12 mg Dex-PO4 (DBL Dexamethasone sodium phosphate 4mg/ml, Hospira NZ, New Zealand) maternal IM injections at 12 h intervals followed by 2 x 2ml IM sterile saline injections at 12 h intervals (n=12); iii) Dex Low Group, ewes received 4 x 1.5 mg Dex-PO4 (DBL Dexamethasone sodium phosphate 4mg/ml, Hospira NZ, New Zealand) maternal IM injections 12 h intervals (n=12); or  iv) Beta Acetate Group, ewes received 1 x 0.125mg/kg Betamethasone Acetate maternal IM injection followed by 3 x 2 ml IM sterile normal saline injections at 12 h intervals (n=16).    Serial maternal plasma samples were collected for analysis at 6, 12, 18, 24, and 36 h post first maternal injection.  All preterm lambs included in this study for ventilation analysis (122 - 125 d gestation) were surgically delivered 48 hours after first maternal injection at 122 – 125 dGA.  Eight animals in the Beta-Acetate Group were delivered at 24 hours as per the study design but not included in ventilation analysis. One ewe from the Saline Control group was removed from the analysis as it was found not to be pregnant at pre-delivery ultrasound. One lamb from the Beta-Acetate Group was removed from the ventilation analysis as was incorrectly intubated and not ventilated for 15 minutes. Hippocampal and Lung mRNA extraction and RNA bulk sequencing  Twenty-four animals (Six animals from each group -Saline Control, Dex High, Dex Low, Beta-Acetate) were selected for RNA bulk sequencing based on the highest maternal steroid plasma concentration prior to delivery. The same animals were used for both lung and hippocampal RNA extraction and analysis thereby allowing for matched analysis with ventilation outcomes and ACS responsiveness.  Delivery and Ventilatory Assessment Maternal plasma samples for analysis were taken immediately prior to delivery, 48 hours after first maternal injection. Fetal plasma samples for analysis were taken during operative delivery and immediately prior to ventilation commencing. Prior to delivery, ewes received an intravenous bolus of midazolam (0.5 mg/kg) and ketamine (10 mg/kg) for the deep induction of anaesthesia. A 3 ml injection of 2% (20 mg/ml) lidocaine was given at L6/L7 for spinal analgesia. The head of the fetus was delivered through abdominal and uterine incisions and a 4.5 mm endotracheal tube was secured by tracheostomy. The fetus was then delivered, and the ewe euthanized under anaesthesia with pentobarbital. The lamb was weighed, dried, and placed in a radiant warmer (Cozy Cot, Fisher & Paykel Healthcare, New Zealand) bed under a plastic insulating wrap (Neowrap, Fisher & Paykel, NZ). Mechanical ventilation (Fabian HFO, Accutronic Medical Systems AG, Switzerland) was immediately commenced with the following settings: peak inspiratory pressure (PIP) of 35 cmH2O, positive end expiratory pressure (PEEP) of 5 cmH2O, respiratory rate (RR) of 50 breaths per minute, inspiratory time (iT) of 0.5 s, using 100% heated and humidified oxygen. The use of 100% oxygen allows the comparison of oxygenation through the partial arterial pressure of oxygen among the groups.   Investigators operating the ventilators were blinded to the animal treatment groups. An umbilical artery was catheterized for blood sampling and administration of supplemental anaesthesia with ketamine (5 mg/kg) if necessary. The tidal volume (VT) was continuously measured, and the PIP was adjusted to keep the VT between 7.0 and 8.0 ml/kg but with PIP limited to 35 cmH2O. At 10, 20, and 30 min of ventilation we measured temperature, blood pressure, ventilator data (PIP, VT, and compliance), and performed blood gas measurements; pH, PCO2 (mmHg), PO2 (mmHg), O2 saturation (SO2, %), total haemoglobin (Hb, g/dL) levels (Siemens RapidPoint 500, Munich, Germany). Dynamic compliance (Cdyn, ml/cmH2O/kg) was recorded as measured by the ventilator. The ventilation efficiency index (VEI) was calculated using the formula VEI = 3,800 / (RR × (PIP − PEEP) × PCO2 (mm Hg))129.  The ACS Responder Group for RNA bulk sequencing (total 6 animals) consisted of three animals from the Dex Low and three animals from Beta-Acetate Groups; ACS non-responder subgroup (total 12 animals) consisted of six animals from the Dex High Group, three animals from the Dex Low Group and three animals from Beta-Acetate Group.  RNA was extracted from snap-frozen hippocampal and lung tissue using RNeasy Plus Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. The concentration of extracted RNA and RNA Integrity Number (RIN) was determined using Agilent Technologies RNA Nano Chip as per manufacturer’s instructions. All RNA extracts were diluted in nuclease-free water to yield a final RNA concentration of 30 ng/μl. Hippocampal and lung tissue bulk RNA sequencing was performed by Novogene Singapore.  Bulk RNA-sequencing Data Processing and Quality Control 150-bp paired-end sequenced reads were processed using the nf-core/rnaseq v3.10.1 pipeline130  with nextflow v22.10.4131. Briefly, raw reads were trimmed using Trim Galore! v0.6.7132 to remove low-quality bases and adapters. Trimmed reads were aligned to the human reference genome, hg38 with STAR v2.6.1d aligner133.  Finally, Salmon v1.9.0134 was utilized for assigning reads. Principle component analysis was conducted for quality control assessment.  Differential Gene Expression and Gene Set Enrichment Analysis All downstream analyses were performed using R statistical software version 4.2.1135. Differential gene expression analysis was performed using the DESeq2 package v1.36.0136 . Genes with at least 1.5-fold change in expression and FDR < 0.05 were considered significant. The results were visualized using the EnhancedVolcano v1.14.0137 and ComplexHeatmap v2.12.1138. Gene set enrichment analysis require full gene list therefore, all genes were ranked by their signed log p-value for gene set enrichment analysis (GSEA) using clusterProfiler v4.9.043  and KEGG database44.

first: Sean

middle: W

last: Carter

last: Oguz

first: Adaikalavan

last: Ramasamy

first: Matthew

last: Kemp


title: Isl1 identifies the extraembryonic mesodermal / allantois progenitors and required for placenta morphogenesis and vasculature formation [RNA-Seq]

city: 上海市

first: 泽悦

last: 朱

address: 四平路1239号

institute: 同济大学

summary: The transcription factor Isl1 is expressed in the progenitors of the Ex-Mes and allantois, however its role in the placentation remains unclear. Our studies showed that deletion of Isl1 causes defects in allantois growth, chorioallantoic fusion and placenta vessel morphogenesis. RNA-seq analyses have revealed that Isl1 regulates the expression of genes important for Ex-Mes multipotency, differentiation and diversification. Isl1 promotes allantoic endothelial but inhibits mesenchymal cell differentiation. Isl1 plays an important role in regulating multiple genetic and epigenetic pathways of vascular morphogenesis. Allantoic signals regulated by Isl1 in turn mediate the inductive interactions between the allantois and chorion critical for chorionic epithelium differentiation, villous formation, and labyrinth angiogenesis. In conclusion, our study provides an integrated insight into the mechanisms underlying placental lineage differentiation, diversification, and vascular formation, highlighting the necessity of Isl1 for early placenta morphogenesis. Our study suggests the potential utility of Isl1 in regenerative medicine application using the stem cells derived from the placenta, umbilical cord and amnionic membrane.

overall_design: To understand the role of Isl1 in the inductive interactions between the allantois and chorion epithelium during chorioallantoic fusion and labyrinth formation. We generated Isl1 knock-out mice (Isl1 Cre+/-; Isletf/+) with Black Swiss background through crossed Isl1 Cre+/- females with Isl1f/+ males. Then we performed RNA-seq analysis of the feto-placentas and associated allantoises at E8.25 with Rosa-tdTomato background in order to trace Cre lineage.Comparative gene expression profiling analysis of RNA-seq data for control and Isl1-null (mut) mice littermates of the feto-placentas and associated allantoises tissue.

first: Xingqun

last: Liang

first: Yunfu

last: Sun

first: Zeyue

last: Zhu

first: Qicheng

last: Zou

first: Chunxiao

last: Wang


title: Isl1 identifies the extraembryonic mesodermal / allantois progenitors and required for placenta morphogenesis and vasculature formation [CUT&Tag]

type: Genome binding/occupancy profiling by high throughput sequencing

summary: To uncover direct targets of ISL1 that could account for its role in allantois, we performed cut tag analysis on wild type allantoic cells using ISL1 antibody at E8.75. Our analyses uncovered 5509 significant binding peaks for ISL1, mainly located at intergenic or intronic regions.

overall_design: Cut-tag analysis was performed using the allantois of pregnant mice with Black Swiss background with Rosa-tdTomato background in order to trace Isl1 Cre lineage at E8.75. Tissue samples were dissected under fluorescence microscope.


title: ChIP-seq assay for BRG1 in human SUP-B15 and Nalm-6 cell lines

city: Guiyang

first: Qian

last: Kang

address: No. 28 Guimedi Street

department: Hematology Department

institute: Affiliated Hospital of Guizhou Medical University

summary: Abstract: BRG1 is highly expressed in adult B-cells acute lymphoblastic leukemia and is associated with poor prognosis. High expression of BRG1 promotes the proliferation and resistance to apoptosis of B-ALL cells. BRG1 exerts these functions mainly through activation of the PI3K/AKT signaling pathway. In order to understand the specific regulatory mechanism, we used ChIP-seq to detect the DNA fragments bound by BRG1 in SUP-B15 and Nalm-6 cell lines.

overall_design: ChIP-seq assay for BRG1 in two human B-ALL cell lines (SUP-B15 and Nalm-6 cells)

first: Dan

last: Ma

first: Peng

last: Zhao

first: Jishi


title: Comparison of transcriptomic profile of the murine B16F1-GFP-M melanoma cells with the derived dormant B16F1-GFP-D, B16F1-GFP-DB#1, B16F1-GFP-DB#2, B16F1-GFP-DB#3 melanoma cells

first: frédéric

last: leprêtre

summary: Cancer patients after successful therapy contain nested in their organs and/or circulating in the systemic fluids tumor cells that remain asymptomatic for an extended period of time. They stay dormant with no apparent immediate potential to develop into a clinically manifested tumor until activated by yet not well defined mechanisms. We previously developed tumor dormancy model of murine melanoma, a cancer with a high potential of phenotype plasticity to adapt to micro-environmental changes, in which to investigate cellular quiescence and related factors as a potential mechanism of tumour dormancy. In this study,  to explore molecular mechanism responsible for cellular dormancy, we performed a comparative transcriptome analysis of dormant B16F1-GFP-D and derived dormant brain metastasis (DB) with maternal B16F1-GFP-M cells.

overall_design: Transcriptomic profiling of myeloid and melanoma cell lines in the context of dormancy.

first: Thierry

last: Idziorek

first: Yasmine

last: Touil


title: Assessment of Rocaglates and Targeted Derivatives as Selective Inhibitors of Glioblastoma Stem Cells

city: Worcester

first: Rui

last: Li

state: Massachusetts

address: 364 Plantation Street, LRB

department: MCCB

institute: University of Massachusetts Medical School

summary: A substantial body of evidence suggests that the initiation, progression, and recurrence of glioblastomas (GBM) are driven by a small subpopulation (1-5%) of cells within the tumor known as tumor-initiating cells or cancer stem cells (CSCs). GBM CSCs play a pivotal role in orchestrating drug resistance mechanisms and tumor relapse. As a prospective avenue for GBM intervention, targeted suppression of GBM CSCs holds promise. In this study, we identified novel rocaglate derivatives (rocaglate acyl sulfamides (Roc ASFs)) that can inhibit GBM CSCs with nanomolar (nM) EC50s. Our findings, supported by comparative dose-response assays and functional characterizations, demonstrate that these derivatives exert a robust, dose-dependent cytotoxic impact on GBM CSCs. Furthermore, our investigations into target identification have unveiled the DEAD box helicase DDX3X as a specific target of Roc ASFs. Inhibiting DDX3X using Roc ASF derivatives confers selective cytotoxicity to GBM CSCs. These findings suggest a promising therapeutic strategy for targeting GBM CSCs.

overall_design: Here we performed gene expression analysis in the patient-derived GBM stem cell line, GBM0308  following treatment with rocaglate acylsulfonamide (Roc ASF) derivative.

first: Zihao

first: Ritesh

middle: P

last: Thakare

first: Shalaka

last: Chitale

first: Alok

last: Mishra

first: Stanley

middle: I

last: Goldstein

first: Alice

middle: C

last: Fan

first: Lihua

middle: J

first: Lauren

middle: E

last: Brown

first: Michael

middle: R

last: Green

first: Sunil

last: Malonia

first: John

middle: A

last: Porco


title: CircHIPK3 and FMRP compete for direct binding to BRCA1 mRNA controlling its protein levels and DNA damage response [miRNA_pulldown]

city: Rome

first: Irene

last: Bozzoni

address: Piazzale Aldo Moro 5

department: Biology and Biotechnologies "Charles Darwin"

country: Italy

institute: Sapienza University of Rome

type: Non-coding RNA profiling by high throughput sequencing

summary: Circular RNAs (circRNAs) are covalently closed RNA molecules widely expressed in eukaryotes and regulated in several pathological processes, including cancer. Many studies point to their mechanism of action as miRNA and protein sponges; however, we propose a new functionality based on circRNA-mRNA interaction to regulate mRNA fate. We show that the widely expressed circHIPK3 directly interacts in vivo with the BRCA1 mRNA through the back-splicing region. This interaction favoured the translation of BRCA1 by competing for the binding of the FMRP1 RNA-binding protein, which acts as a repressor of BRCA1 translation. CircHIPK3 depletion or disruption of the CircRNA-mRNA interaction decreased BRCA1 levels and increased DNA damage, sensitizing several cancer cell lines to DNA-damage-inducing agents and rendering them susceptible to synthetic lethality. Additionally, mimicking circHIPK3 interaction with lock-nucleic acid (LNA) oligonucleotides restores BRCA1 levels in hereditary breast cancer model cells, underscoring circRNA-mRNA interaction’s importance in regulating cell homeostasis and drug response.

overall_design: High-throughput small RNA sequencing from circHIPK3 native pulldown performed in RD cells with two sets of biotynilated antisense probes (ODD and EVEN) targeting HIPK3 exon 2 and control biotynilated antisense probes targeting LacZ bacterial mRNA. One biological replicate.

first: Chiara

last: Grelloni

first: Raffaele

last: Garraffo

first: Adriano

last: Setti

first: Francesca

last: Rossi

first: Manuel

last: Beltran Nebot


title: CircHIPK3 and FMRP compete for direct binding to BRCA1 mRNA controlling its protein levels and DNA damage response


title: CircHIPK3 and FMRP compete for direct binding to BRCA1 mRNA controlling its protein levels and DNA damage response [RNA_pulldown]

overall_design: High-throughput sequencing of total RNA from circHIPK3 AMT-crosslinked pulldown performed in RH4 cells with two sets of biotynilated antisense probes (ODD and EVEN) targeting HIPK3 exon 2 and control biotynilated antisense probes targeting LacZ bacterial mRNA. Two biological replicates.


title: Isl1 identifies the extraembryonic mesodermal / allantois progenitors and required for placenta morphogenesis and vasculature formation

first: 泽悦

last: 朱


title: RNA-sequencing of Mtb H37Rv untreated and H37Rv treated with 2XMIC CJ067

city: Bengaluru

first: Amit

last: Singh

state: KARNATAKA

address: Sir CV Raman Road

department: Microbiology and Cell Biology

country: India

institute: Indian Institute of Science

summary: The ability ofMycobacterium tuberculosis(Mtb) to tolerate nitric oxide (NO) and superoxide (O2•−) produced by phagocytes contributes to its success as a human pathogen. Recombination of NO and O2•− generates peroxynitrite (ONOO-), a potent oxidant produced inside immunologically-activated macrophages and causes lethality in diverse organisms. However, while the response ofMtbtowards NO and O2•− is well established, howMtbresponds to ONOO- remains uncertain. Filling this knowledge gap is important to understand the persistence mechanisms of Mtb during infection Hence, we used an endogenous peroxynitrite donor CJ067 and treated Mtb to identify new mechanisms of  ONOO- resistance in mycobacteria.

overall_design: This study involves determining the mRNA transcriptome profiles for the strains Mtb H37Rv untreated and treated with 2X MIC CJ067; grown under standard in vitro conditions in 7H9+ADS+Tween80 at 37°C and 180 rpm, till cultures reached ~0.4 OD600.  Three independent experiments were done for each treatment.

first: Arshiya

last: Dewan

first: Nitish

last: Malhotra

first: Aswin

middle: N

last: Seshasayee


title: A potent and selective small-molecule degrader of ENL suppresses leukemia progression in vivo (ChIP-Seq)

city: Grand Rapids

first: Xiaobing

last: Shi

address: 333 Bostwick Ave. NE

institute: Van Andel Institute

summary: ENL, a stoichiometric component of the Super Elongation Complex (SEC) as well as the histone H3K79 methyltransferase DOT1L complex, is involved in the regulation of transcription elongation. Loss-of-function studies of ENL highlighted an essential role for its chromatin reader YEATS domain in the progression of acute leukemia, suggesting ENL as an attractive therapeutic target for leukemia therapy. Proteolysis targeting chimeras (PROTACs), a class of new therapeutic modalities, have the potential to degrade target proteinsthrough the ubiquitin-proteasome system. PROTACs have the potential to deliver robust therapeutic effects at low doses and infrequent dosing regimens with less side-effects. Here, we designed and synthesized a serial of ENL PROTACs. Through screening by ENL degradation in human leukemia cells, we identified MS47 as a potent ENL PROTAC. MS47 efficiently and specifically degrades ENL in a concentration-dependent and time dependent manner. In line with the mechanism of action of PROTACs, proteasome inhibitors can block ENL degradation mediated by MS47. MS47 suppresses survival and growth of a panel of acute leukemia cells, and efficiently suppresses the expression of ENL target genes, including Hoxa9, Meis1, Myb and Myc. In disseminated xenograft model, MS47 significantly benefits mouse survival, inhibits leukemia cell growth in vivo. No obvious toxic effect shown in toxicity test in vivo. Modest changes occurred on normal hematopoiesis. Together, our study developed a potent ENL PROTAC for leukemia treatment.

overall_design: Design and synthesis ENL degraders, screen these ENL degraders via western blot to get the candidates. Then test cell growth inhibition activity of ENL degrader candidates to get the best ENL degrader. Further profile transcriptional and chromatin occupancy changes upon ENL degrader treatment to investigate the mechanism of ENL. At last, test ENL degrader efficacy in xenograft mouse model.

first: Zhaoyu

last: Xue

first: Hongwen

last: Xuan

first: Hong

last: Wen


title: A potent and selective small-molecule degrader of ENL suppresses leukemia progression in vivo (RNA-Seq)


title: Effect of depletion of Isl1 on gene expression at 8.75 days of embryo development

overall_design: To understand the role of Isl1 in the inductive interactions between the allantois and chorion epithelium during chorioallantoic fusion and labyrinth formation. We generated Isl1 knock-out mice (Isl1 Cre+/-; Isletf/+) with Black Swiss background through crossed Isl1 Cre+/- females with Isl1f/+ males. Then we performed RNA-seq analysis of the feto-placentas and associated allantoises at E8.75 with Rosa-tdTomato background in order to trace Cre lineage.Comparative gene expression profiling analysis of RNA-seq data for control and Isl1-null (mut) mice littermates of the feto-placentas and associated allantoises tissue.

first: Xingqun

last: Liang

first: Yunfu

last: Sun

first: Zeyue

last: Zhu

first: Qicheng

last: Zou

first: Chunxiao

last: Wang


title: Human RP105 monoclonal antibody enables the production of antigen-specific antibody

city: Suita

first: Daisuke

last: Motooka

state: Osaka

address: 3-1, Yamadaoka

country: Japan

institute: NGS core facility, Research Institute for Microbial Diseases, Osaka University

summary: Antibody detection is a critical approach in clinical contexts. Detecting antibodies, particularly those directed against donor HLA  in organ transplantation and self-antigens in autoimmune diseases, is crucial in diagnosis and therapy. Radioprotective 105 (RP105, CD180 ), a type I transmembrane protein with structural similarities to Toll-like receptor 4, is expressed in immune-competent cells, such as B cells. Studies involving mice have reported that the antimouse RP105 antibody strongly activates B cells and triggers an adjuvant effect against viral infections. However, the antihuman RP105 antibody (ɑhRP105) weakly activates human B cells. In this study, we established new culture conditions under which human B cells are strongly activated by ɑhRP105. When combined with CpGDNA, this activation induced a heightened production of interleukin 6. Under these culture conditions, specific antibody production against blood group carbohydrates, ɑGal, and SARS-CoV-2 within supernatants from human B-cell cultures was successfully detected. Furthermore, comprehensive analysis employing LC/MS/MS analysis and single-cell RNA sequencing revealed that ɑhRP105 triggered a different activation stimulus from CpGDNA. These findings could be applied to developing a straightforward approach for identifying antibody-producing B cells in cases involving transplant rejection and autoimmune diseases.

overall_design: PBMCs from 3 healthy volunteers were stimulated with ɑhRP105 for 36 hours. Activated and non-activated CD19+ B-cells were stained with six different hashtag antibodies (contorol: #1, 3, and 5 vs stimulate:#2, 4, and 6), anti-CD19, and anti-CD20. Hashtagged cells from three patients were sorted, pooled, and analyzed by single-cell RNA-seq.

first: Kenta

last: Iwasaki

first: Tatsuya

last: Yamazaki

first: Masaki

last: Imai

last: Okuzaki

first: Sachiko

last: Akashi-Takamura


title: RNAseq PC3 DMSO vs GW3965

city: clermont ferrand

first: yoan

last: renaud

address: place henri dunand

institute: igred

summary: Identify LXR target gens that are involved in migration and invasion phenotype

overall_design: PC3 prostate cancer cell were treated for 24h with DMSO or GW3965 1µM in RPMI-1640, L-Glu 2mM after overnight starvation.

first: Silvere

last: BARON

first: Yoan

last: RENAUD


title: Prominent role of gut dysbiosis in the pathogenesis of cystic fibrosis-related liver disease in mice

city: New Haven

first: Romina

last: Fiorotto

state: CT

department: Internal Medicine

institute: Yale School of Medicine

summary: Cystic fibrosis-related liver disease (CFLD) is a chronic cholangiopathy that increases morbidity and mortality in patients with CF. Current treatments are unsatisfactory, and incomplete understanding of CFLD pathogenesis hampers therapeutic development. We have previously shown that mouse CF cholangiocytes respond with excessive inflammation to LPS. Thus, we investigated the role of the gut-liver axis in the pathogenesis of CFLD.

overall_design: Wild-type (WT), whole-body CFTR knockout (CFTR-KO) and gut-corrected (CFTR-KO-GC) mice were studied. Liver changes were assessed by immunohistochemistry and single-cell transcriptomics (scRNAseq), inflammatory mediators were analyzed by proteome array, faecal microbiota by 16S rRNAseq and gut permeability by FITC-dextran assay.


title: A potent and selective small-molecule degrader of ENL suppresses leukemia progression in vivo

first: Xiaobing

last: Shi

overall_design: Refer to individual Series


title: Neuroblastoma plasticity during metastatic progression stems from the dynamics of an early sympathetic transcriptomic trajectory

city: Lyon

first: Celine

last: Delloye-Bourgeois

address: 28 Rue Laennec

institute: Centre de Recherche en Cancerologie de Lyon

summary: Despite their indisputable importance in neuroblastoma (NB) pathology, knowledge of the bases of NB plasticity and heterogeneity remains incomplete. They may be rooted in developmental trajectories of their lineage of origin, the sympatho-adrenal neural crest. We found that implanting human NB cells in the neural crest of the avian embryo allows recapitulating the metastatic sequence until bone marrow involvement. Using deep single cell RNA sequencing, we characterized transcriptome states of NB cells and their dynamics over time and space, and compared them to those of fetal sympatho-adrenal tissues and patient tumors and bone marrow samples. Here we report remarkable transcriptomic proximities restricted to an early sympathetic neuroblast branch that co-exist with phenotypical adaptations over disease progression and recapitulate intratumor and interpatient heterogeneity. Combining avian and patient datasets, we identified a list of genes upregulated upon bone marrow involvement and associated with growth dependency, validating the relevance of our multimodal approach.

overall_design: IGR-N91::GFP cells were harvested before cells engraftment and from chick embryos using a fluorescence stereomicroscope. Tumor cells were dissected out from E5 chick embryos in sympatho-adrenal tissues and from E14 chick embryos at different localizations: primary tumor sites (adrenal medulla, ADR, and sympathetic ganglia, SG), secondary metastatic site (bone marrow, BM), and NB cells on spreading routes (dorsal aorta, AOR, and embryonic peripheral nerves, PN).

first: Benjamin

last: Villalard

first: Arjan

last: Boltjes

first: Florie

last: Reynaud

first: Olivier

last: Imbaud

first: Karine

last: Thoinet

first: Severine

last: Croze

first: Gianluigi

last: Atzeni

first: Joël

last: Lachuer

first: Jan

last: Molenaar

first: Godelieve

middle: M

last: Tytgat

first: Valérie

last: Castellani

first: Céline


title: Effects of inhaled ozone on gene expression profiles in mouse lung macrophages

city: Gainesville

first: Ley

middle: Cody

last: Smith

state: FL

address: 2187 Mowry Rd

institute: University of Florida

summary: Acute exposure to inhaled ozone causes oxidative stress, lung injury, and inflammation. Activated macrophages play a key role in both the initiation and resolution of the inflammatory response to inhaled ozone; this activity is mediated by distinct subsets, broadly classified as proinflammatory (M1) and anti-inflammatory/pro-resolution (M2) macrophages. Successful resolution of inflammation and tissue repair require balanced activity of M1 and M2 macrophages. In this context, overactivation of M1 macrophages or inadequate activation of M2 macrophages results in a failure to resolve inflammation and prolonged injury. Thus, identifying signaling mechanisms contributing to aberrant activation of lung macrophages is critical to mitigating lung injury and decrements in pulmonary function caused by this ubiquitous air pollutant. The purpose of the present study was to identify mechanisms regulating macrophage activation in response to acute exposure to ozone by characterizing global transcriptional profiles using RNA-seq. We hypothesized that gene expression patterns would be distinctly regulated at 24 and 72 hr post exposure to ozone as these time points reflect different phases of the inflammatory response, namely initiation and resolution, when macrophage subpopulations derived from different origins would predominate. We identified significant enrichment of pathways involved in innate immune signaling and cytokine production among differentially expressed genes at 24 and 72 hr post exposure. In addition, we observed a preponderance of pathways involved in cell cycle regulation at 24 hr and intracellular metabolism at 72 hr post exposure. These studies are significant as they permit the identification of signaling pathways representing prospective therapeutic targets to fine tune macrophage responses and limit ozone-induced lung injury.

overall_design: Female, wild type (Jackson Labs, 12 wk) were exposed to air or ozone (0.8 ppm, 3 h) in a whole-body Plexiglas chamber. Animals were euthanized 24 and 72 h after exposure by intraperitoneal injection of ketamine (135 mg/kg) and xylazine (30 mg/kg). Lung macrophages were collected by bronchoalveolar lavage plus gentile massage from air controls (n = 3), and mice euthanized 24 (n = 3), and 72 hr (n = 3) after exposure to ozone.

first: Debra

last: Laskin


title: Shikonin inhibit progression of colon cancer

city: Xi'an

first: wen

last: liu

address: New temple Road num 1

institute: Tangdu Hospital of Air Force Medical University

summary:
To investigate the role of shikonin in the development of colon cancer, we treated HCT116 cells with shikonin for 24 hours
We then used RNA-seq data from HCT116 cells for gene expression profiling

overall_design: Compare the gene expression profiles of RNA-seq data from control group and administration group of HCT116 cells.

first: Wenna

last: Liu

first: Yin

last: Liang

first: Yujia

last: Zhao


title: Single cell RNA-sequencing reveals an important role of SRSF1 and SRSF2 in the development of diabetes.

city: Jining

first: Xue

last: You

institute: Jining Medical University

summary: Single-cell sequencing of islet cells from WT , ASF knock-out, SC35 knock-out mice reveals different mechanisms of diabetes formation and development.

overall_design: The pancreases of 9-month-old WT , ASF-KO and SC35-KO mice were perfused with collagenase, and islet cells were isolated and digested into single cell for single-cell RNA-sequencing.

first: Ying

last: Feng


title: Spatial profiling of human immune cells in sarcoidosis

city: Philadelphia

first: Satish

last: Sati

state: Pennsylvania

address: 421 Curie Blvd, BRB 1039

department: Dermatology

institute: University of Pennsylvania

summary: Sarcoidosis is a multiorgan inflammatory disease that is characterized by tissue granulomas and remains so challenging for clinicians to diagnose that it has been called “the great imitator”. Here, by performing single-cell sequencing and spatial transcriptomics on skin granulomas from sarcoid and non-sarcoid patients, we discovered increased recruitment of innate lymphoid cells in granulomas.  Sarcoid-associated cells exhibited increased cell migration compared to healthy individuals. Importantly, granuloma formation was attenuated in mice genetically lacking these cells or treated with a pharmacologic inhibitor.

overall_design: scRNA-seq of Human skin cells

first: Thomas

middle: H

last: Leung


title: Immune profiles of human cells

overall_design: scRNA-seq of Human PBMCs, Human skin cells


title: Identification of Mineralocorticoid receptor targets in peripheral blood mononuclear cells (PBMC)

city: Leipzig

first: Franziska

last: Kuestermann

address: Johannisallee 30

country: Germany

institute: University Hospital of Leipzig

summary: Genome wide analysis of expression data in PBMC of three healthy male probands upon stimulation with vehicle only, aldosterone, cortisol, aldosterone and cortisol in physiological amounts in order to identify putative MR-target genes.

overall_design: PBMC of three healthy males were isolated. Cell culture was performed incubating each probands PBMCs in adosterone (1ng/ml), cortisol (1000ng/ml), aldosterone  (1ng/ml) and cortisol  (1000ng/ml), and vehicle respectively for three hours at room temperature. RNA was then isolated, transcribed into cDNA and blotted onto two Illumina HT12 Beadchips (named A and B, with B being a thenical replicate).

first: Kathy

last: Busse

first: Knut

last: Krohn


title: LINE-1 retrotransposons are reduced in bone of osteoporotic patients, correlate positively to BMD, and stimulate bone anabolism in transfected mesenchymal stem cells

city: Barcelona

first: Alfonso

last: Saera-Vila

address: Dr. Trueta 179

country: Spain

institute: Sequientia Biotech

summary: To investigate the role of LINE-1 in bone regeneration, we delivered synthetic L1 RNA to mesenchymal stem cells (MSC), from osteoporotic patients, induced to osteogenic differentiation.

overall_design: First, mesenchymal stem cells (MSC) from femur of 4 osteoporotic patients (HUK9, HUK12, HUK16, HUK19) were isolated. MSC were then induced to osteogenic differentiation and transfected after 5 days with RFP mRNA (negative control) or L1 RNA. Samples were collected at day 6 (24h post-transfection) and day 10 (5 days post-transfection). Comparative gene expression profiling analysis of RNA-seq data for RFP mRNA and L1 RNA transfected cells was performed.

first: Arianna

last: Mangiavacchi

first: Sjur

last: Reppe

first: Benjamin

last: Eggerschwiler

first: Gabriele

last: Morelli

first: Peng

first: Nazerke

last: Atinbayeva

first: Elisa

last: Casanova

first: Huoming

last: Zhang

first: Dalila

last: Bensaddek

first: Mirko

last: Celii

first: Francesco

last: Della Valle

first: Juan

last: Izpisua Belmonte

first: Paolo

last: Cinelli

first: Kaare

last: Gautvik

first: Valerio

last: Orlando


title: Whole transcriptome analysis of white adipose tissue in high fat diet induced insulin resistance mice

city: Shijiazhuang

first: Linlin

last: Yang

state: Hebei

address: 348, Heping west road

institute: Hebei General Hospital

summary: To determine the effect of resveratrol or metformin on high fat diet induced insulin resistance mice and elucidate their underlying mechanisms, we conducted a high-throughput sequencing analysis on epididymal fat tissue. LncRNAs and mRNAs were quantified and differentially expressed genes were identified, providing a potential gene signature for pharmacological mechanism of resveratrol and metformin.

overall_design: Mice were assigned into 4 groups, epididymal fat was collected and snapped frozen on dry ice. Total RNA was harvested using Trizol reagent and then sequenced through Illumina NovaSeq 6000.

first: Guangyao

last: Song

first: Chao

last: Wang

first: Huijuan

last: Ma


title: Establishment of a human tongue cancer organoid biobank enables early diagnosis, prognosis, and identification of therapeutic targets

city: Tokyo

first: Taku

last: Sato

address: 1-5-45 Yushima Bunkyo-ku

department: Department of Biodefense Research

institute: Medical Research Institute, Tokyo Medical and Dental University (TMDU)

summary: Tongue cancer (TC) is the most frequent and a serious type of oral cancer with major clinical challenges such as lymph node metastasis and the appearance of chemotherapy resistant cells. Thus, development of diagnostic and therapeutic strategies to target these malignant cancer cells is urgently required. Here, we describe a novel and highly efficient method to generate human TC organoids, thereby creating a TC organoid biobank with primary and metastatic TC from 28 patients with diverse ages and cancer stages. These organoid lines can be expanded, passed, stored frozen for long periods, and they can reproduce original TC tissues in vitro and in xenograft models. The organoids showed different properties among patients with respect to the proliferation capacity and susceptibility to cisplatin, a commonly used chemotherapeutic agent in TC therapy. This suggests that TC organoids are useful for accurate risk grading and predicting the prognosis at early stages of TC, which is difficult to diagnose clinically. Thus, human TC organoids are useful for profiling TCs, which will lead to drug development against metastatic and chemotherapy-resistant TCs.

overall_design: Gene expression profiles of normal tissues, cancer tissues, normal organoids and cancer organoids from TC patients were generated by deep sequencing.

first: Miwako

last: Sase

first: Toshiaki

last: Ohteki


title: Using single-cell technologies to functionally define fetal haematopoietic stem and progenitor cells

city: Parkville

first: Alexandra

last: Garnham

state: Victoria

address: 1G Royal Parade

country: Australia

institute: Walter and Eliza Hall Institute of Medical Research

summary: Using single cell RNA sequencing, cellular barcoding, and transplantation experiments, my study functionally benchmarks the cell types present in the fetal liver and has demonstrated the existence of functional MPP2 to 4 within the embryo. Although strikingly, FL MPP3 has significantly greater contribution to the lymphoid lineage than its adult counterpart, acting as lineage balance MPP.   Our study has further revealed a large pool of functional LTHSCs were immunophenotypically hidden in a STHSC immunophenotype. While additionally, CD45 surface marker can be used to set apart all long-term reconstituting activity in the foetus.   Interestingly our data indicates that the majority of a recently discovered developmentally restricted HSC population is present within our FL CD45+ STHSC compartment. Suggesting a previous unrecognised critical role for this population during development.

overall_design: Single cell FL LSK subtypes (MPP2-4/ LT/ ST) were sorted and underwent library prep and RNA-sequencing to determine the molecular differences between the populations.  Long-term and short-term transplantations of bulk sorted LSK subtype were performed to assess functional abilities of these populations.  While lentiviral barcoding and short-term transplantation was also utilised on each population to determine their functional potential at the clonal level.  Lastly, via flow cytometry and further by transplantation, a Flt3-Cre YFP reporter mouse line was utilised to investigate the presence of developmentally restricted HSCs (drHSCs) within the FL immunophenotypical STHSC compartment.

middle: L

first: Olivia

last: Stonehouse

first: Samir

last: Taoudi


title: Lung-targeted lipid nanoparticle-delivered siUSP33 attenuates SARS-CoV-2 replication and virulence by promoting envelope degradation

status: Public on Sep 12 2024

city: changsha

first: yujie

last: Liao

address: tongzipo

institute: central south university

summary: As a structural protein of SARS-CoV-2, the E protein not only plays a key role in the formation of viral particles but also forms ion channels and has pathogenic functions, including triggering cell death and inflammatory responses. The stability of E proteins is controlled by the host ubiquitin-proteasome system. By screening human deubiquitinases, we found that ubiquitin-specific protease 33 (USP33) could enhance the stability of E proteins depending on its deubiquitinase activity, thereby promoting viral replication. In the absence of USP33, E proteins are rapidly degraded, leading to a reduced viral load and inflammation. Using lipid nanoparticle (LNP) encapsulation of siUSP33 by adjusting the lipid components (ionizable cationic lipids), siUSP33 was successfully delivered to mouse lung tissues, rapidly reducing USP33 expression in the lungs and maintaining knockdown for at least 14 d, effectively suppressing viral replication and virulence. This method of delivery allows efficient targeting of the lungs and a response to acute infections without long-term USP33 deficiency. Our research, based on the deubiquitination mechanism of USP33 on the E protein, demonstrates that LNP-mediated siRNA delivery targeting USP33 plays a role in antiviral and anti-inflammatory responses, offering a novel strategy for the prevention and treatment of SARS-CoV-2.

overall_design: As the article outlines, USP33 serves as a versatile E3 ligase within the host organism. To more effectively illustrate that no significant side effects were observed in mice treated with LNP-siUSP33, we procured blood samples from each cohort on both the first and fourth day following administration. The strategy here is based on the fact that in vivo delivery of LNP reaches its peak on the day immediately after injection, while the optimal efficiency for USP33 knockdown occurs four days post-injection. These samples were subsequently examined for changes in the transcriptome. Furthermore, conventional cationic lipids such as DOTAP (trimethyl-2,3-dioloyl-propylammonium bromide) have a propensity to aggregate with negatively charged serum proteins, resulting in swift elimination of lipid nanoparticles by the immune system and a markedly reduced half-life in vivo. This aggregation also heightens the risk of adverse effects, including hemolysis. To surmount these obstacles, ionizable cationic lipids with pKa values ranging from 6.0 to 7.0 have been engineered. These ionizable lipid LNPs (iLNPs) adeptly encapsulate nucleic acids while mitigating toxicity (PMID: 28412170). Consequently, the enhanced LNP employed in this study is crafted to address the side effects of traditional cationic lipids. Our approach, which involves utilizing ionizable cationic lipids, notably Dlin-MC3-DMA, to encapsulate siRNA and incorporating an extra cationic lipid (DOTAP), proved effective in diminishing side effects. This LNP system preserves a relatively neutral surface charge at physiological pH levels. We also ensured its diameter remained under 140 nm to minimize the binding of LNP to fibrinogen and the subsequent clot formation (PMID: 38394670). Moreover, to more rigorously validate its efficacy, we collected peripheral blood from mice across various treatment groups on both day 1 and day 4 for transcriptomic analysis.

first: Zheng

last: zhang

first: Zanxian

last: Xia

first: Yuzheng

last: Zhou

first: Yujie


title: Sex-specific molecular signature of mouse podocytes in homeostasis and in response to pharmacological challenge with rapamycin

city: Berlin

first: Angelika

last: Kusch

address: Chariteplatz 1

department: Medizinische Klinik m. S. Nephrologie und internistische Intensivmedizin

institute: Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt-Universität zu Berlin

summary: The global burden of chronic kidney diseases is rapidly increasing and is projected to become the fifth most common cause of years of life lost worldwide by 2040. Sexual dimorphism in kidney diseases and transplantation is well known, yet sex-specific therapeutic strategies are still missing. One reason is the lack of knowledge due to the lack of inclusion of sex as a biological variable in study designs. This work aimed at identification of molecular signatures of male and female podocytes, gate-keepers of the glomerular filtration barrier. Like cardiomyocytes, podocytes are terminally differentiated cells which are highly susceptible towards pathological challenges. Podocytes are the decisive cell-type of the kidney to maintain the physiological blood-urine barrier, and disturbances of their homeostasis critically accelerate kidney function impairment. By help of a genomic mouse model, highly purified podocytes were obtained from male and female mice with and without pharmacological challenge of the mechanistic target of rapamycin (mTOR) signaling pathway which is known to be deregulated in major kidney diseases. Deep RNA sequencing, proteomics and metabolomics revealed strong intrinsic sex differences in the expression levels of mitochondrial, translation and structural transcripts, protein abundances and regulation of metabolic pathways which might fundamentally contribute to sex differences in kidney disease susceptibilities and progression. Remarkably, high number of previously reported kidney disease genes showed so far unknown intrinsic sexual dimorphism and/or different response patterns towards mTOR inhibition. Our work provides an in-depth database for novel targets to be tested in kidney disease models to advance with sex-specific treatment strategies.

overall_design: In order to investigate cell type specific molecular response upon mTOR inhibition, podocytes were isolated from ROSAmT/mG-NPHS2 Cre mouse female and male mice treated with and without rapamycin followed by mRNAseq.

first: Christin

last: Suenkel

first: Eric

last: Blanc


title: The role of Cebpb in Alcohol-associated Liver Disease development and resolution

city: Kansas City

first: Irina

last: Tikhanovich

state: Kansas

address: 3901 Rainbow blvd

institute: University of Kansas Medical Center

summary: Abstinence is an important therapeutic intervention for patients with alcohol-associated liver disease (ALD). However, fibrosis improvement after cessation is not uniform and some patients do not improve. We aimed to use scATAC-seq analysis in a mouse model of ALD to define the mechanism of poor ALD resolution. We analyzed differentially accessible regions in livers from control, ALD, or 4 weeks post alcohol cessation mice and identified transcription factors activated in ALD that remained activated after alcohol withdrawal. The top hit was C/EBPβ. We found that hepatocyte specific Cebpb knockout prevented ALD development, while knockout at the time of alcohol cessation promoted fibrosis resolution.

overall_design: Whole liver mRNA analysis in alcohol fed (WDA - western diet with alcohol in the drinking water) Cebpb floxed mice treated with  AAV-Cre vectors

first: Michal

last: Schonfeld


title: Hepatocyte identity and zonal position determine tumourigenic potential of mutant β-catenin

city: Glasgow

first: Joseph

middle: Alexander

last: Waldron

address: Switchback road

country: United Kingdom

institute: Beatson Institute for Cancer Research

summary: Oncogenic mutations in otherwise normal tissue are common in many adult tissues. This suggests multiple events need to converge to drive tumourigenesis and that many processes such as tissue differentiation may be protective against carcinogenesis. Within the liver Wnt/β-catenin signalling maintains zonal differentiation during liver homeostasis. However, the CTNNB1 oncogene—encoding β-catenin—is also frequently mutated in hepatocellular carcinoma, resulting in aberrant Wnt signalling that promotes cell growth. Here we investigated the antagonistic interplay between Wnt-driven growth and differentiation in zonal hepatocyte populations during liver tumorigenesis. In a model that acutely recombined mutant Wnt (Ctnnb1-ex3) and MYC alleles, we found that β-catenin mutants transiently stimulated growth in zone-1 and -2 hepatocytes within the Wnt-low region of the liver lobule – before inducing a differentiated zone-3 fate. To investigate the role of translation in this model of wnt and myc driven growth we performed Ribosome sequencing of whole liver at the early proliferative stage and the later differentiated stage. We found that Mutamt B-Catenin and MYC livers had a unique tanslational efficiency , which promted translation of RNA transcripts associated with growth.

overall_design: RPFs represent ribosome protected fragments and Totals represents the paired cytoplasmic RNA control, which was extracted with TRIzol from the lysate, prior to RNase digestion. For example, WT_day4_1_RPFs and WT_day4_1_Totals represents the RPFs and total cytoplasmic RNA sample from the first replicate of the day4 WT samples. Each sample represents an individual mouse and therefore a unique biological replicate. Day4 and Day10 mice were induced at different times so that all mice could be sacrificed and harvested at the same time.

first: June

last: Munro

first: Alex

last: Raven

first: Martin

last: Bushell

first: Owen

last: Sansom


title: Inflammation mediated by gut microbiome alterations promotes lung cancer development and an immunosuppressed tumor microenvironment

city: Houston

first: Humam

last: Kadara

state: Texas

address: 1515 Holcombe Blvd Unit 2951

department: Translational Molecular Pathology

institute: The University of Texas MD Anderson Cancer Center

summary: This study aims to elucidate the impact of gut microbiota alterations on tumorigenesis and immune response in lung adenocarcinoma (LUAD). Using Gprc5a-/- mice as a model, we performed fecal microbiota transfer (FMT) from Gprc5a-/- and Gprc5a-/-; Lcn2-/- donors to investigate the role of gut microbiome changes in modulating tumor growth and the immune microenvironment. Single-cell RNA sequencing (scRNA-seq) was conducted on colonic lamina propria and subcutaneous tumor tissues. Our findings demonstrate that gut microbiota from Lcn2-deficient mice promotes systemic inflammation and immunosuppression, enhancing tumor progression. This study provides insights into the microbiome's influence on LUAD and potential therapeutic strategies targeting microbiome-related pathways.

overall_design: Single-cell RNA sequencing (scRNA-seq) profiling was performed on colonic lamina propria and subcutaneous tumor tissues from Gprc5a-/- syngeneic mice treated with fecal microbiota transfer (FMT) from either Gprc5a-/- or Gprc5a-/-; Lcn2-/- donors. Mice were grouped based on the source of FMT and included both male and female subjects (n = 2 mice per group). Freshly collected tissues were dissociated into single-cell suspensions, and live cells were sorted to exclude dead cells.

first: Zahraa

last: Rahal


title: Image-based spatial transcriptomics identifies molecular niche dysregulation associated with distal lung remodeling in pulmonary fibrosis [Visium]

city: Phoenix

first: Nicholas

last: Banovich

state: AZ

address: 445 North Fifth Street

department: Integrated Cancer Genomics

institute: TGen - Translational Genomics Research Institute

summary: Pulmonary fibrosis (PF) is a chronic, progressive condition that represents the end-stage of many interstitial lung diseases (ILDs). Single-cell transcriptomic studies have revealed disease-emergent epithelial, fibroblast, and macrophage cell types/states in PF lungs, but the spatial contexts wherein these cells contribute to disease pathogenesis has remained uncertain.  Using image-based spatial transcriptomics to profile gene expression changes in-situ across 28 lung samples from control and PF lungs, we characterized the expression of 343 genes in over 1 million nuclei at subcellular resolution. Using both cell-based and cell-agnostic approaches, we observed a diversity of distinct molecularly-defined spatial niches in control and PF lungs. Overlaying these computationally-defined niches with disease-associated histopathologic features, we identified novel patterns of dysregulation in alveoli informed by spatial context. We computationally segmented individual air spaces and using cell composition, we ordered airspaces from homeostatic to most dysregulated. Using this ordering we identified a series of stepwise molecular changes associated with progressive distal lung remodeling. Together, these results advance our understanding of the molecular programs underlying progressive PF.

overall_design: To characterize  the cellular and molecular architecture of the lung across health and disease, we generated a spatial transcriptomics dataset measuring the expression of 343 genes across 28 3-5 mm lung tissue cores from 6 unaffected (31.6%) and 13 PF (68.42%) donors using the 10X Genomics Xenium platform. To capture the regional heterogeneity of PF, we sampled paired sets of cores from more (MF) and less (LF) fibrotic regions of 9 individuals and larger biopsies encompassing a diversity of architectures of 3 individuals. Of the 13 PF patients, most were diagnosed with IPF (n = 7, 53.8%). The majority of donors self-reported European ancestry (78.9%), and 8 (42.1%) reported current or prior tobacco use.

first: Annika

last: Vannan

first: Ruqian

last: Lyu

first: Arianna

last: Williams

last: Negretti

first: Evan

middle: D

last: Mee

last: Hirsh

first: Samuel

first: David

middle: S

last: Nichols

first: Chase

last: Taylor

first: Vasiliy

middle: V

last: Polosukhin

first: A

last: McCall

first: Jason

last: Gokey

first: Heejung

last: Shim

first: Lorraine

middle: B

last: Ware

first: Matthew

last: Bacchetta

first: Ciara

last: Shaver

first: Timothy

last: Blackwell

first: Jennifer

last: Sucre

first: Jonathan

last: Kropski

first: Davis

last: McCarthy


title: An SLC12A9-dependent ion transport mechanism regulates lysosomal osmolarity

city: Stanford

first: Roni

last: Levin-Konigsberg

state: CA

address: 240 Pasteur Dr

department: Genetics

institute: Stanford Univeristy

summary: Ammonia is a ubiquitous, toxic by-product of cell metabolism. Its high membrane permeability and proton affinity causes ammonia to accumulate inside acidic lysosomes in its poorly membrane-permeant form: ammonium (NH4+). Ammonium buildup compromises lysosomal function, suggesting the existence of mechanisms that protect cells from ammonium toxicity. Here, we identified SLC12A9 as a lysosomal regulator of ammonium export that preserves lysosomal homeostasis. SLC12A9 knockout cells showed grossly enlarged lysosomes and elevated ammonium content. These phenotypes were reversed upon removal of the metabolic source of ammonium or dissipation of the lysosomal pH gradient. Lysosomal chloride increased in SLC12A9 knockout cells and chloride binding by SLC12A9 was required for ammonium transport. Our data indicate that SLC12A9 function is central for the handling of lysosomal ammonium and chloride, an unappreciated, fundamental mechanism of lysosomal physiology that may have special relevance in tissues with elevated ammonia, such as tumors.

overall_design: To investigate the changes in gene expression in SLC12A9 KO cells compared to control (safe-sgRNA-treated) cells we cultured cells in glutamine-free medium - cellular metabolism of glutamine produces ammonia

first: Katherine

last: Liu

first: Michael

last: Bassik


title: Pregnancy Restricts an Age-Driven Accumulation of Hybrid Cells in the Mammary Gland [bulk RNA-Seq]

city: Santa Cruz

first: Paloma

last: Medina

address: 1156 High St.

institute: University of California Santa Cruz

summary: Aging increases breast cancer risk while an early first pregnancy reduces a woman’s life-long risk. Several studies have explored the effect of either aging or pregnancy on mammary epithelial cells (MECs), but the combined effect of both remains unclear. Here, we interrogate the functional and transcriptomic changes at single cell resolution in the mammary gland of aged nulliparous and parous mice to discover that pregnancy normalizes age-related imbalances in lineage composition, while also inducing a permanently differentiated cell state. Importantly, we uncover a minority population of Il33-expressing hybrid cells with high cellular potency that accumulate in aged nulliparous mice but is significantly reduced in aged parous mice. Functionally, IL33 treatment of basal, but not luminal, epithelial cells from young mice phenocopies aged nulliparous MECs and promotes formation of organoids with Tp53 knockdown. Collectively, our study demonstrates that pregnancy blocks the age-associated loss of lineage integrity in the basal layer through a decrease in Il33+ hybrid cells, potentially contributing to pregnancy-induced breast cancer protection.

overall_design: Bulk RNA sequencing was performed and analyzed by Novogene on sorted basal (CD49fhi/EPCAMlow-med) and luminal (CD49flow/EPCAMhi) populations from 3m NP, 18m NP and 18m P mice (n=3 mice/group). Messenger RNA was purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers, followed by the second strand cDNA synthesis using either dUTP for directional library or dTTP for non-directional library80. For the non-directional library, it was ready after end repair, A-tailing, adapter ligation, size selection, amplification, and purification. For the directional library, it was ready after end repair, A-tailing, adapter ligation, size selection, USER enzyme digestion, amplification, and purification. The library was checked with Qubit and real-time PCR for quantification and bioanalyzer for size distribution detection. Quantified libraries will be pooled and sequenced on Illumina platforms, according to effective library concentration and data amount.

first: Andrew

last: Olander

first: Cynthia

last: Ramirez

first: Veronica

last: Haro Acosta

first: Sara

last: Kaushik

first: Vanessa

last: Jonsson

first: Shaheen

last: Sikandar


title: Acetalax (Oxyphenisatin acetate, NSC 59687) and Bisacodyl Cause Oncosis in Triple Negative Breast Cancer by Poisoning the Ion Exchange Membrane Protein TRPM4

city: Bethesda

first: Anjali

last: Dhall

state: MD

address: 37 Convent Drive

department: DTB

institute: NCI

summary: Triple negative breast cancer (TNBC) is generally unresponsive to current therapies, and the development of new anticancer agents is needed. Oxyphenisatin acetate, called Acetalax, which was used as a laxative in the 1950’s, has recently been reported to have anticancer activity in murine models. In this study we demonstrate that Acetalax and its diphenolic laxative structural analog bisacodyl (Dulcolax) exhibit potent antiproliferative activity in TNBC cell lines. We show that both cause oncosis, a non-apoptotic cell death characterized by cellular and nuclear swelling and cell membrane blebbing that leads to mitochondrial dysfunction and ATP depletion. Acetalax was further shown to enhance immune and inflammatory responses and inhibit protein synthesis. Mechanistically, we provide evidence that TRPM4 (Transient Receptor Potential Melastatin member 4) is poisoned by Acetalax and bisacodyl. By blocking TRPM4, a monovalent cation channel, Acetalax and Bisacodyl cause oncosis, and TNBC cells without endogenous TRPM4 expression as well as TRPM 4 knockout TNBC cells were found to be Acetalax- and Bisacodyl resistant. TRPM4 was also lost in cells with acquired Acetalax resistance. Moreover TRPM4 is rapidly degraded by the ubiquitin-proteasome system upon acute exposure to Acetalax and bisacodyl. Together, these results demonstrate that TRPM4 is a previously unknown target of Acetalax and Bisacodyl and that TRPM4 expression in cancer cells is a predictor of Acetalax and Bisacodyl efficacy, and could be used for the clinical development of these drugs as anticancer agents.

overall_design: We created Acetalax resistant cells by chronically exposing MDAMB468 and BT549, which were originally sensitive. We named them MDAMB468-CE(chronic exposure) and BT549-CE. Cells were seeded in a 10 cm dish, and when the dish was almost full confluent in 1–2 weeks, the cells were passaged with double the concentration Acetalax. Initially, the Acetalax concentration was started at 0.2 μM. When the Acetalax concentration was increased to 51.2 μM, the cells stopped growing. Therefore, cells chronically exposed to 40 μM Acetalax were designated as Acetalax chronic exposure cells and are Acetalax resistant clones. CE cells were used in the experiment after a 2-week Acetalax-free period. The RNA sequencing was done for MDAMB468 and BT549 parental cells and CE cells. The expressed RNAs of these 8 samples were compared to determine what RNAs were altered in the parental and CE cells and what RNAs were altered in the no-treatment and treatment groups.  RNA was extracted from a control group of these cells and from cells treated with Acetalax 1 uM for 6 hours. RNA was extracted as per protocol using PureLink® RNA Mini Kit (Cat:12183018A. Invitrogen). The extracted RNA was measured for RNA Integrity Number to confirm that there were no problems with RNA quality. We used 800 ng of total RNA samples to generate the library (PolyA selected). The kits used for the library prep were NEBNext PolyA mRNA Magnetic Isolation Module (NEB #E7490), NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB #E7760) and NEBNext® Multiplex Oligos for Illumina® (96 Unique Dual Index Primer Pairs, NEB, E6440) as described in the manufacturer’s manual. Sequencing was carried out in the Illumina NextSeq 2000 instrument with the NextSeq 2000 P2 kit (200 Cycles) with 101x101 pair end configuration.

first: Makito

last: Mizunuma

first: Christophe

last: Redon

first: Liton

middle: K

last: Saha

first: Andy

last: Tran

first: Robin

last: Sebastian

last: Kruhalk

first: William

last: Reinhold

first: Naoko

last: Takebe

first: Yves

last: Pommier


title: Neural circuit basis of placebo pain relief [10X]

city: Chapel Hill

first: Jesse

last: Niehaus

state: NC

address: 116 Manning Drive, 7008B Mary Ellen Jones Building

department: Neuroscience Center

institute: University of North Carolina at Chapel Hill

summary: For over a millennium, mind-body interactions have fascinated scientists and doctors for their abilities to shape human perceptions of the external world 1,2. Placebo effects are striking demonstrations of mind-body interactions in which, in the absence of any treatment, a positive expectation of pain relief can reduce or even abolish the experience of pain 3–6. However, despite widespread recognition of the strength of placebo effects and their impact on everyday human experience and clinical trials for new analgesics, the neural circuit basis of the placebo effect has remained a mystery. Here, we show that analgesia from the expectation of pain relief is mediated by a distinct population of rostral anterior cingulate cortex (rACC) neurons that project to the pontine nuclei (rACC→Pn), a pair of brainstem pre-cerebellar nuclei with no established function in pain processing. To do this, we created a behavioral assay that models placebo analgesia by conditioning mice to expect pain relief when moving from a chamber with a heated floor to a second chamber. In this assay, an expectation of pain relief induces an analgesic effect that, like placebo analgesia in humans, is mediated by endogenous opioids. Calcium imaging of neural activity in freely moving mice and electrophysiological studies in cingulate cortical brain slices showed that expectations of pain relief boost the activity of rACC→Pn neurons and potentiate neurotransmission in this pathway. Transcriptomic studies of Pn neurons revealed an unusual abundance of opioid receptors in these cells, further suggesting a role in pain modulation. Selective inhibition of either the rACC→Pn pathway or of opioid-receptor-expressing Pn neurons disrupted placebo analgesia and decreased pain thresholds. Finally, a subset of cerebellar Purkinje cells exhibits activity patterns resembling those of rACC→Pn neurons during pain relief expectation, providing cellular-level evidence of a role for the cerebellum in cognitive pain modulation. Altogether, these findings elucidate longstanding mysteries surrounding the placebo effect by identifying a specific neural pathway that mediates expectation-based pain relief. This discovery opens the possibility of targeting this novel pathway with drugs or neurostimulation methods to treat pain. More broadly, our studies provide a framework for investigating the neural circuit basis of other mind-body interactions beyond those involving pain, and point to prefrontocortical-cerebellar communication as a potential basis for such effects.

overall_design: For 10x Genomics data, two scRNA-seq runs profiling the pontine nucleus were conducted on nuclear suspensions pooled from two female mice aged 8 weeks (C57BL/6 background).

first: Chong

last: Chen

first: Gregory

last: Scherrer


title: Calorie restriction increases insulin sensitivity to promote beta cell homeostasis and longevity.

city: Nashville

first: Rafael

last: Arrojo e Drigo

state: TN

address: 2201 West End Ave, Molecular Physiology and Biophysics, LH718

department: Molecular Physiology and Biophysics

institute: Vanderbilt University

summary: Caloric restriction (CR) extends organismal life- and health-span by improving glucose homeostasis. How CR affect the structure-function of pancreatic beta cells remains unknown. We used single nucleus transcriptomics to show that CR increases the expression of genes for beta cell identity, protein processing, and organelle homeostasis. Gene regulatory network analysis reveal that CR activates transcription factors important for beta cell identity and homeostasis, while imaging metabolomics demonstrates that CR beta cells are more energetically competent. In fact, high-resolution microscopy show that CR reduces beta cell mitophagy to increase mitochondria mass and the potential for ATP generation. However, CR beta cells have impaired adaptive pro liferation in response to high fat diet feeding. Finally, we show that long-term CR delays the onset of beta cell aging and promotes cell longevity by reducing beta cell turnover. Therefore, CR could be a feasible approach to preserve compromised beta cell structure-function during aging and diabetes. 27 diabetes.

overall_design: Pancreatic islets were isolated from mice on ad-libitum diet (AL), 20% caloric restriction (CR) or 60% high-fat diet (HF) for 2 or 12 months. Islets were snap-frozen immediately. Single nuclei were isolated from snap-frozen isolated islet pellets and were processed for transposition, barcoding, library construction and sequencing according to 10x Genomics Guidelines for Single Cell Multiome ATAC + Gene Expression.

first: Cristiane

last: Santos

first: Amanda

last: Cambraia

first: Shristi

last: Shrestha

first: Melanie

last: Cutler

first: Matthew

last: Cottam

first: Guy

last: Perkins

first: Varda

last: Lev-Ram

first: Birbickram

last: Roy

first: Christopher

last: Acree

first: Keun-Young

last: Kim

first: Thomas

last: Deerinck

first: Danielle

last: Dean

first: Jean-Philippe

last: Cartailler

first: Patrick

last: MacDonald

first: Martin

last: Hetzer

first: Mark

last: Ellisman

first: Rafael Arrojo e

last: Drigo


title: Vimentin regulates mitochondrial ROS production and inflammatory responses of neutrophils

city: Aurora

first: Tonya

last: Brunetti

state: CO

address: 12800 East 19th Avenue

department: Immunology and Microbiology

institute: University of Colorado Anschutz Medical Campus

summary: The intermediate filament vimentin is present in immune cells. It provides structural support to cells and functions in macrophages to coordinate inflammatory responses. Neutrophils are an abundance innate immune cell that function by distinct mechanisms from macrophages to mediate antimicrobial activities. The role of vimentin in neutrophils is not established. Here, we developed an immortalized neutrophil model to examine the requirement of vimentin. We demonstrate that vimentin restricts proinflammatory cytokine production and reactive oxygen species production, but enhances phagocytosis and swarming. We observe that vimentin is dispensable for neutrophil extracellular trap (NET) formation, degranulation, and for inflammasome activation.  Moreover, gene expression analysis defined vimentin presence was associated with expression changes in multiple genes required for mitochondrial function and ROS overproduction in the absence of vimentin. By treatment of cells producing vimentin with rotenone, an inhibitor for complex I of the electron transport chain, or by treatment of cells lacking vimentin with mitoTEMPO, a SOD mimetic, we complement the changes in ROS levels through manipulation of mitochondrial function. Together, we suggest vimentin regulates neutrophil antimicrobial functions and alters ROS levels through regulation of mitochondrial activity.

overall_design: RNAs from WT and vimentin KO cells with and without 25ng LPS treatment were extracted using RNeasy micro kit and DNAse treated. Library was prepared for RNA-sequencing

first: Thao

last: Huynh

first: Jody

last: Toperzer

first: Allison

last: Scherer

first: Anne

last: Gumina

first: Michael

last: Mansour

first: David

last: Markovitz

first: Brian

last: Russo


title: Spatial Mapping of Mouse Brain Aging through Indexed Sequencing

city: New York

first: Abdulraouf

last: Abdulraouf

state: NY

address: 1230 York Ave

institute: Rockefeller University

summary: Here, we have developed a novel methodology called IRIS (Imaging Reconstruction using Indexed Sequencing) that enables cost-effective spatial transcriptomics profiling without relying on optical imaging. Through neighborhood interaction-based reconstruction, IRIS allows extensive analysis of large tissue sections and many replicates with adjustable mapping resolution at only a fraction of the cost of other commercial platforms. With the IRIS platform, we reconstructed a large area spatial area with two whole mouse brain coronal sections. Moreover, we also created a spatially resolved transcriptome atlas of the mouse brain and identified aging-associated changes in gene expression and spatial organization across various brain cell types. Further analysis of cell-cell interaction changes identified aging-associated foci in white matter regions enriched with inflammatory subtypes of microglia and oligodendrocytes. Overall, the IRIS methodology cost-effective and ease-of-use approach makes it broadly applicable to the studies of spatial gene expression changes in various systems.

overall_design: Utilizing the IRIS platform, we conducted a comprehensive spatial analysis of molecular profiles and spatial arrangements of cell types in mouse brains. To study the aging-associated transcriptome changes, here we collected brain tissues from adult (4-month) and the aged (23-month) wild-type C57BL/6 mice, with three replicates per age group.

first: Weirong

last: Jiang

first: Zihan

last: Xu

first: Zehao

last: Zhang

first: Tanvir

last: Raihan

first: Wei

last: Zhou

first: Junyue

last: Cao


title: Cancer-associated SF3B1 Mutations Inhibit mRNA Nuclear Export by Disrupting SF3B1–THOC5 Interactions

city: Chang Chun

first: Xin

last: Hu

address: 2699 Qianjin road,china

institute: Jilin university

summary: Pre-mRNA splicing and nuclear export are coordinated processes that ensure efficient and accurate gene expression. In recent years, disruptions in this process have been found to result in substantial gene expression aberrations, with potential contributions from SF3B1 mutations, whose mechanism remains elusive. The present study reveals that the K700E mutation in SF3B1 attenuates its interaction with THOC5, resulting in reduced mRNA binding by THOC5, and ultimately, the inhibition of mRNA nuclear export. Interestingly, overexpressing THOC5 can restore the attenuated interaction, facilitating mRNA nuclear export. Importantly, other cancer-associated SF3B1 mutations may exert a similar impact. Our research highlights the critical role of the THOC5–SF3B1 interaction in regulating mRNA nuclear export and provides valuable insights into the impact of SF3B1 mutations on this process.

overall_design: Cytoplasmic and nuclear RNA obtained from HEK293T cells transiently transfected with pIRES2-SF3B1 WT (WT) or pIRES2-SF3B1 K700E (K700E).

first: Gang

last: Liu

first: Bo

last: Zhao

first: Yueru

last: Shi

first: Youzhong

last: Wan


title: PHF2 maintains neural progenitor genome stability by preserving pericentric heterochromatin integrity (ATAC-Seq)

city: Barcelona

first: Samuel

last: Aguirre Infantes

state: Barcelona

address: Baldiri Reixac 15-21

department: Department of Structural and Molecular Biology

institute: IBMB-CSIC

summary: Heterochromatin stability is crucial for progenitor proliferation during development. It relays on the maintenance of local hubs of H3K9me. However, the mechanisms underlying the establishment of competent local levels of H3K9me remain poorly understood. To address this intriguing question, we used a neural stem cell (NSC) model to analyze the significance of the H3K9me2 demethylase PHF2, which is crucial for progenitor proliferation. Through mass spectroscopy and genome-wide assays, we uncovered that PHF2 interacts with heterochromatin components and it is enriched at pericentromeric heterochromatin (PcH) boundaries. This binding is essential for maintaining silenced the satellite repeats and to prevent DNA damage and genome instability. To do that, PHF2 balances H3K9me3 levels at these boundaries to ensure high H3K9me3 levels at satellite repeats. Mechanistically, we discover that while its catalytic and PHD domains are indispensable, the intrinsically disordered region within PHF2 is dispensable for stabilizing PcH. Altogether, our study sheds light on the intricate relationship between heterochromatin stability and progenitor proliferation during mammalian neurogenesis.

overall_design: Assay for Transposase Accesible Chromatin (ATAC-seq) was performed on Neural Stem Cells (NSCs) under shControl and shPHF2 conditions by duplicate.

last: Aguirre

first: Stella

last: Pappa

first: Núria

last: Serna-Pujol

first: Natalia

last: Padilla

first: Silvina

last: Nacht

first: Guillermo

middle: P

last: Vicent

first: Albert

last: Jordan

first: Xavier

last: de la Cruz

first: Marian

last: Martínez-Balbás


title: Trans-differentiation takes place without reprogramming of developmental-specific DNA methylation, a key determinant of stable cell identity [RNA-seq]

city: Jerusalem

first: Batia

last: Azria

state: Israel

address: Jerusalem

institute: Hebrew university of Jerusalem

summary: A number of studies have demonstrated that it is possible to directly convert one cell type to another by factor-mediated trans-differentiation, but in the vast majority of cases, the resulting reprogrammed cells are unstable in the sense that they are unable to maintain their new cell identity.  To better understand this phenomenon, we have developed a new analytical approach for better characterizing trans-differentiation-associated changes in DNA methylation, a major determinant of long-term cell identity.  By examining various models of trans-differentiation both in vitro and in vivo, our studies indicate that despite convincing expression changes, trans-differentiated cells seem unable to alter their original developmentally-mandated methylation patterns.  We propose that this blockage is due to basic developmental limitations built into the regulatory-sequence that govern epigenetic programming of cell identity. These results shed new light on the molecular rules of stable somatic cell reprogramming.

overall_design: Expression profile of direct reprogramming cells.

first: Ahmed

last: Radwan

first: Jason

last: Eccleston

first: Ofra

last: Sabag

first: Howard

last: Marcus

first: Jonathan

last: Sussman

first: Alberto

last: Ouro

first: Moran

last: Rahamim

first: Meir

last: Azagury

first: Ben

middle: Z

last: Stanger

last: Cedar

first: Yosef

last: Buganim


title: In vivo functional genomics identifies essentiality of potassium homeostasis in medulloblastoma

city: Changsha

first: Siyi

last: Wanggou

state: Hunan

address: Xiangya Road, No.87

department: Departmeng of Neurosurgery

institute: Xiangya Hospital, Central South University

summary: This dataset includes the raw single-cell RNAseq data from Math1-Cre;SmoM2 mouse medulloblastoma model at P7.

overall_design: Single-Cell RNAseq from Math1-Cre;SmoM2 mouse medulloblastoma model at P7.

first: Xi

last: Huang

first: Jerry

last: Fan


title: Mitochondrial Dysfunction and Increased Reactive Oxygen Species Production in MECP2 Mutant Astrocytes and their Impact on Neurons

city: Cambridge

first: Danielle

last: Tomasello

state: Massachusetts

address: 455 Main St

institute: Whitehead Institute

summary: Studies on MECP2 function and its implications in Rett Syndrome (RTT) have traditionally centered on neurons. Here, using human embryonic stem cell (hESC) lines, we modeled MECP2 loss-of-function (LOF) to explore its effects on astrocyte (AST) development and dysfunction in the brain. Ultrastructural analysis of RTT hESC-derived cerebral organoids revealed significantly smaller mitochondria compared to controls (CTR), particularly pronounced in glia versus neurons. Employing a multiomics approach, we observed increased gene expression and accessibility of a subset of nuclear-encoded mitochondrial genes upon mutation of MECP2 in ASTs compared to neurons. Analysis of hESC-derived ASTs showed reduced mitochondrial respiration and altered key protein in the tricarboxylic acid cycle and electron transport chain in RTT versus controls. Additionally, RTT ASTs exhibited increased cytosolic amino acids under basal conditions, depleted upon increased energy demand. Notably, mitochondria isolated from RTT ASTs exhibited increased reactive oxygen species and influenced neuronal activity when transferred to cortical neurons. These findings underscore MECP2 mutation's differential impact on mitochondrial and metabolic pathways in ASTs versus neurons, suggesting that dysfunctional AST mitochondria may contribute to RTT pathophysiology by affecting neuronal health.

overall_design: Human stem cell-derived astrocytes, cortical neurons and cerebral organoids

first: M

middle: I

last: Barrasa

first: David

last: Mankus

first: Katia

last: Alarcon

first: Abigail

last: Lytton-Jean

first: X

last: Liu

first: Rudolf

last: Jaenisch


title: Transcriptomic analysis of cell lines derived from a metaplastic breast cancer with squamous metaplasia (MBC-SM) tissue

city: Miaoli Conunty

first: Su-Fang

last: Lin

address: No 35, Keyan Road, Zhunan Town

department: NICR

country: Taiwan

institute: National Health Research Institutes, Taiwan

summary: Although epithelial–mesenchymal transition (EMT) is the favored hypothesis for why cancer metastasis occurs, the cause–effect relationship remains unclear due to inconsistent clinical evidence. In this study, we establised epithelial– and mesenhcymal–predominant cell clones from a patient derived-metaplastic breast carcinoma with squamous metaplasia tissue (MBC-SM), followed by in-depth characterization of the derived cell clones.

overall_design: Before cells reaching confluence, total RNA of each cell clone was extracted with TRIzol reagent (Thermo Fischer Scientific, Waltham, MA, USA), including epithelial-predominant (n=3), E/M hybrid (n=2) and mesenchymal-predominant  (n=3). The quality of RNA was validated by a Bioanalyzer (Agilent, Santa Clara, CA, USA)  and subjected to Tri-I Biotech Inc. (Taipei, Taiwan) for RNA sequencing. Briefly, transcriptome libraries from the mRNA fractions were generated by MGIEasy RNA Library kit (SE50) and sequenced on a BGI-seq 500 machine. After reads trimming and adaptor removal, clean reads were used for downstream bioinformatic analysis including CLC Genomic Workbench (v10, Qiagen, Hilden, Germany), R limma (v 3.40.6), and GSEA (hallmark v7.4, Broad Institute, MA, USA).

first: Huang-Chun

last: Lien

first: Yen-Shen

last: Lu


title: Neural circuit basis of placebo pain relief [SMART-seq]

first: Jesse

last: Niehaus

overall_design: For SMART-seq v2 data, pontine nucleus cell from 8-week-old Snap25-IRES2-Cre;Ai14 mice (1 male and 1 female) were sorted for tdTomato into PCR tubes for library generation.

first: Chong

last: Chen

first: Gregory

last: Scherrer


title: Trans-differentiation takes place without reprogramming of developmental-specific DNA methylation, a key determinant of stable cell identity [RRBS]

first: Batia

last: Azria

type: Methylation profiling by high throughput sequencing

overall_design: DNA methylation profile of Direct reprogramming cells.

first: Ahmed

last: Radwan

first: Jason

last: Eccleston

first: Ofra

last: Sabag

first: Howard

last: Marcus

first: Jonathan

last: Sussman

first: Alberto

last: Ouro

first: Moran

last: Rahamim

first: Meir

last: Azagury

first: Ben

last: Stanger

last: Cedar

first: Yosef

last: Buganim


title: FGF8-mediated gene regulation affects regional identity in human cerebral organoids

status: Public on Sep 11 2024

city: Nice

first: Michele

last: Bertacchi

state: Provence Alpes Côte d Azur

address: 28, avenue Valrose

institute: iBV - Institut de Biologie Valrose

summary: Single cell RNA sequencing

overall_design: Human cortical organoids (day 69 of differentiation); 2 control batches (3 organoids each) and 2 FGF8-treated batches (3 organoids each). 17,400 cells per channel (corresponding to an estimated recovery of 10,000 cells per channel) were loaded onto a Chromium Single Cell 3′ Chip (10x Genomics, PN 2000177). scRNA-seq libraries were prepared with the Chromium Single Cell 3′ Library & Gel Bead Kit v.2 (10x Genomics, PN-120237). Libraries were sequenced using Illumina HiSeq 4000 system in Paired-End mode with 100 or 28 bases for read 1 and 100 bases for read 2, following Illumina's instructions. Image analysis and base calling were performed using RTA version 2.7.2 and Cell Ranger version 3.0.2.

first: Matthieu

last: Jung

first: Michèle

last: Studer


title: Pacbio long read Iso-sequencing is performed to acquire a high-precision transcription landscape after STAMBPL1 knockingdown in Huh7 cells（Long-read）

city: beijing

first: hongli

last: zhang

state: beijing

address: Beijing University of Chemical Technology

summary: STAMBPL1 plays a previously unappreciated role in post-transcriptional regulation, especially in RNA splicing pathways. Thus, PacBio long-read iso-sequencing was performed to acquire a high-precision transcription landscape  after STAMBPL1 knockingdown in Huh7 cells

overall_design: Negative control group versus STAMBPL1 konck down group

first: Hongli

last: Zhang


title: GENOMES UNCOUPLED PROTEIN1 binds to plastid RNAs and promotes their maturation

city: Planegg

first: Tatjana

last: Kleine

address: Großhaderner Straße 2

department: Biologie I, Botanik

institute: Ludwig-Maximilians-Universität München

summary:
Investigation of the function of GUN1 in the chloroplast (post)-transcriptome
We performed gene expression profiling analysis using data obtained from lncRNA-seq of gun1 seedlings with white, marble or green cotyledons

overall_design: Comparative gene expression profiling analysis of RNA-seq data for gun1W/Col-0, gun1M/Col-0, and gun1G/Col-0

first: Kleine

last: Tatjana


title: Cholangiocyte Ciliary Defects Induce Sustained Epidermal Growth Factor Receptor Signaling

city: Austin

first: Sergio

last: Gradilone

address: 801 16th Avenue NE

department: The Hormel Institute

institute: University of Minnesota

summary: Background & Aims: The primary cilium, an organelle that protrudes from cell surfaces, is essential for sensing extracellular signals. With disturbed cellular communication and chronic liver pathologies, this organelle's dysfunctions have been linked to disorders, including polycystic liver disease (PLD) and Cholangiocarcinoma (CCA). This study aimed to clarify the interaction between primary cilia and the critical regulator of cellular proliferation known as the epidermal growth factor receptor (EGFR) signaling pathway, which has been linked to several clinical conditions. Approach & Results: The study identified abnormal EGFR signaling pathways inside cholangiocytes lacking functioning primary cilia using liver-specific IFT88 knockout mice, a Pkhd1 mutant rat model, and human cell lines with ciliary deficiencies. Cilia-deficient cholangiocytes showed persistent EGFR activation because of impaired receptor degradation, in contrast to their normal counterparts where EGFR localization to cilia promotes appropriate signaling. By using HDAC6 inhibitors to restore primary cilia, EGFR degradation was accelerated, which in turn reduced the maladaptive signaling. Importantly, EGFR was moved to cilia because of experimental intervention with the HDAC6 inhibitor tubastatin A in an orthotopic rat model, along with a reduction in the phosphorylation of ERK. In cholangiocarcinoma and polycystic liver disease cells, concurrent administration of EGFR and HDAC6 inhibitors displayed synergistic anti-proliferative effects that were related to the restoration of functioning primary cilia. Conclusion: The findings of this study shed light on defective ciliary function and robust EGFR signaling with slower receptor turnover. A potential method for treating EGFR-driven pathologies in PLD and CCA is the pharmacological restoration of primary cilia function.

overall_design: Normal Human Cholangiocytes cell line (NHC) were stably transfected with non-target (NT) and IFT88 specific shRNAs in induce deciliation. Next-generation sequencing (RNAseq) was used to further study the consequences of ciliary lost.

first: Kishor

last: Pant

first: Seth

last: Richard

first: Estanislao

last: Peixoto

first: Subheksha

last: Baral

first: Rendong

last: Yang

first: Yanan

last: Ren

first: Tatyana

last: Masyuk

first: Nicholas F

last: LaRusso