title: High expression of KLK5 enhances radiation resistance of cervical cancer

city: Changchun

first: Shun-Qing

last: Zhou

address: No. 218 Ziqiang Street, Changchun City, Jilin Province, China

institute: Jilin University

summary: Studies have demonstrated the correlation between kallikrein-related peptidase 5 (KLK5) and cervical cancer prognosis, but studies on the correlation between KLK5 and radiation resistance are rare. Therefore, we aimed to investigate the role of high KLK5 expression in improving radiation resistance in cervical cancer and the underlying mechanisms. Samples from 29 patients with cervical cancer who received definitive radiotherapy without surgical treatment and other antineoplastic treatments (2014–2016) were analyzed. The patients were divided into the radiotherapy-sensitive (RS) group (18 patients without local recurrence and distant metastasis within 3 years after initial treatment) and the radiotherapy-resistant (RR) group (11 patients with uncontrolled conditions with radiotherapy, local recurrence, or distant metastasis within 3 years after initial treatment). We used high-throughput gene sequencing to detect radiosensitivity-related factors. We found that the expression of KLK5 in the RR group was higher than that in the RS group, and this result was associated with poor outcomes after radiotherapy as patients developed distant metastasis and showed recurrence at 2–3 years of follow-up. Besides, a high KLK5 expression enhances radiation resistance, indicating that, with further research, KLK5 may be used to develop novel molecular target-specific modulators for anti-cervical cancer therapy, acting as a prognostic agent in personalized precision medicine for cancer.

overall_design: mRNA profile of 10 patients with primary squamous-cell cervical cancer who were admitted to the Second Hospital of Jilin University between 2014 and 2016, radiotherapy-sensitive (RS) group and adiotherapy-resistant (RR) group

first: Yan-Nan

last: Shen

first: Li-Ying

last: Han

first: Tian

last: Geng


title: Noncoding RNAs induce TDP43 pathology in the ALS-iPSC models

status: Public on Nov 24 2024

city: Taipei

first: Yi-Ying

last: Wu

address: No. 128, Sec. 2, Academia Road, Nankang

department: ICOB

country: Taiwan

institute: Academia Sinica

type:
Expression profiling by array
Non-coding RNA profiling by array

summary:
Amyotrophic lateral sclerosis (ALS) is a progressive motor neuron (MN) degenerative disease with a major pathological feature of cytoplasmic TDP-43 aggregation. However, the mechanisms underlying TDP-43 proteinopathy are still largely unknown. We performed in vitro differentiation of ALS-induced pluripotent stem cells (ALS-iPSCs; carrying the TDP-43M337V mutation) and isogenic controls and found upregulation of paraspeckle-associated lncRNA NEAT1 isoforms in the ALS-iPSC-derived MNs (ALS-iPSC-MNs). Intriguingly, the upregulated NEAT1 isoforms were mislocalized to the cytoplasm of ALS-iPSC-MNs, and the cytoplasmic NEAT1 provoked TDP-43 and TDP-43M337V liquid-liquid phase separation, generating long-lived protein condensates. These condensates had reduced mobility and were converted into aggregates, finally co-aggregating with phospho-TDP-43.
Disruption of NEAT1 expression reduced its cytoplasmic levels and also reduced the levels of TDP-43/TDP-43M337V condensates. In 3D neuromuscular organoids with the TDP-43M337V mutation, treatment with NEAT1-antisense oligonucleotides (NEAT1-ASO) promoted neuromuscular junction formation and function, as well as muscle contractility. Furthermore, treatment of TDP-43Q331K mice with Neat1-ASO attenuated TDP-43 pathology in spinal cord and preserved motor function. These findings suggest that NEAT1 plays an important role in TDP-43-associated pathology, and NEAT1-ASO may attenuate pathological TDP-43 aggregation to prevent motor neuron degeneration and muscle weakness in ALS.

overall_design: GFP+ motor neurons were sorted from in vitro motor neuron differentiation for RNA extraction and hybridization on Affymetrix microarrays. We sought to identify consistent upregulated or downregulated noncoding RNAs in the ALS-iPSC-motor neuron as compared to isogenic counterparts, two pair set of microarray were performed from iTDP#2 and iTDP#3 and the isogenic lines

first: Hung-Chih

last: Kuo


title: BCR sequences in HDM-allergic patients

status: Public on Nov 22 2024

city: Nanjing

first: Chunan

last: Sun

address: 101 Longmian Avenue, Jiangning District

country: China

institute: nanjing medical university

summary: Light and heavy chain sequences of antibodies obtained by BCR sequencing

overall_design: B cells from HDM-allergic patients were flow-sorted and subjected to BCR sequencing to discover matching antibody light and heavy chain sequences


title: Key genes for the syndrome of intrahepatic cholestasis in pregnancy

city: yichang

first: wenfei

last: zheng

address: jiefang road，NO.2

institute: Yichang Central People's Hospital

type: Expression profiling by high throughput sequencing

summary: High-throughput sequencing approach to determine genomic expression in human placental tissue during ICP development

overall_design: Pregnant women were categorized evenly into two groups: healthy group; ICP group,

first: Wenfei

last: Zheng

first: Jingqiong

last: Zhan

first: Huarong

last: Cao

first: Min

last: Wen


title: Cigarette smoke promote the tumor progression via FASN in  bladder cancer and rewiring the fatty acid metabolism

city: St Louis

first: Tiandao

last: Li

state: MO

address: 4444 Forest Park Ave

country: USA

institute: Washington University

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Cigarette smoke has been demonstrated to stimulate the growth of existing bladder tumors by enhancing the survival and proliferation of cancer cells through the action of carcinogens present in smoke. Concurrently, Fatty Acid Synthase (FASN), a key enzyme in fatty acid synthesis crucial for lipid metabolism, exhibits dysregulation in various cancer types, often correlating with aggressive phenotypes. In this study, we reveal altered fatty acid metabolism and elevated FASN and fatty acid levels, specifically in current smokers with bladder cancer (BLCA). Notably, increased FASN levels under smoke exposure are attributed to epigenetic alterations affecting fatty acid metabolism. Cells exposed to cigarette smoke demonstrate a metabolic shift in utilizing glutamine as a carbon source and producing fatty acids. The genetic and pharmacological inhibition of FASN effectively reduces tumor growth in a CAM model exposed to smoke. FASN inhibitors like TVB 2640, currently in phase II clinical trialsclinical trials in various cancer types, may be effective for smokers with BLCA exhibiting high FASN levels. In general, FASN inhibition offers therapeutic promise for mitigating the impact of cigarette smoke on bladder cancer progression.

overall_design: UMUC3 cells were treated with cigarette smoke media  (10%) for 3 days (B7), or air exposed (B6). The chromatin was immunoprecipitated with acK130-H2A antibodies, followed by sequencing (ChIP-seq).

first: Nupam

last: Mahajan


title: Whole transcriptome sequencing explored the toxic effect of cadmiun on Caenorhabditis elegans

city: Zhengzhou

first: Panpan

last: Wang

address: 郑州市高新区科学大道100号郑州大学

department: College of Public Health

institute: 郑州大学

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

overall_design: Refer to individual Series


title: Whole transcriptome sequencing explored the toxic effect of cadmiun on Caenorhabditis elegans [miRNA-seq]

address: 郑州市高新区科学大道100号郑州大学

type: Non-coding RNA profiling by high throughput sequencing

summary:
Purpose: Cadmium (Cd) is detrimental to environment and human health, but the specific mechanism of the regulatory network is still remained unclear. Caenorhabditis elegans (C. elegans) is selected as the ideal model organism to investigate the effect of Cd exposure.
Methods: We employed the phosphoproteomic technologies, high-throughput sequencing method and bioinformatic tools (RNAhybrid, Risearch2 and Targetscan) to investigate the phosphorylated change and the regulatory networks of circRNA, lncRNA, microRNA (miRNA) and mRNA after Cd exposure in C. elegans. The Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and gene set enrichment analysis (GSEA) were also employed. The relationship between transcriptional factors (TFs) and kinases, and the kinase-substrate were predicted using Jaspar database and iGPS, respectively.
Results:26 differentially expressed circRNAs (DEcircRNAs), 143 lncRNAs (DElncRNAs), 69 miRNAs (DEmiRNAs) and 6209 mRNAs (DEmRNAs) were identified. According to qRCR, four DEcircRNAs and three DElncRNAs were found and confirmed the different expression levels between the treated and control group. 23 miRNAs and six miRNAs were the potential targets of the confirmed circRNAs and lncRNAs respectively using the predictive tools of RNAhybrid, RIsearch and Targetscan. Similarly, the target mRNAs of miRNAs were identified. Next, the regulatory network of lncRNA-miRNA-mRNA and circRNA-miRNA-mRNA were established. Furthermore, five protein-coding genes might be regulated by DElnRNAs through cis-acting and 114 by trans-acting. The GO and KEGG analysis were performing on the parents’ genes of the mRNAs. GSEA was performed on all protein-coding genes, and the correlation between these genes and validated lncRNAs were used for pre-ranking. For the phosphopromotics, 42 differentially regulative phosphopeptides were identified. In this work, four novel pairs of TFs-kinases-substrate which might be influenced by Cd exposure were firstly constructed.
Conclusions: Cd might mainly influence the reproductive function and aging of C. elegans through regulating the circRNAs and lncRNAs. Also, it might inhibit the expression of TFs to reduce the phosphorylated level of corresponding protein.

overall_design: Examination of the change of RNA and protein after cadmium exposure in Caenorhabditis elegans.

first: Zhi

last: Qu

first: Peisen

last: Guo


title: Epigenetic characterization of carbon dots functions in rice

status: Public on Nov 23 2024

first: zhenyu

last: luo

state: Jiangsu

address: Xuanwu

department: College of Agricuture

institute: Nanjing Agricultural University

type: Methylation profiling by high throughput sequencing

summary: Nanoparticles (NPs) can serve as efficient carriers for delivery of genetic materials in plants. They can regulate normal growth and development, and responses to stresses in plants. However, epigenetic mechanisms underlying NPs or carbon dots (CDs) affecting plant growth and development are still largely unknown. Here we showed that CDs reprogrammed gene transcription associated with some biological processes such as stress responses and photosynthesis. Moreover, CDs resulted in subtle but significant DNA hypermethylation, especially for CHG and CHH hypermethylation, which is possibly caused by CDs induced up-regulation of OsCMTs. Interestingly, we found that CDs stabilized i-motif in the promoter regions of OsCMTs1/2, therefore resulting in CHG and CHH hypermethylation. Thus, our study for the first time reveals possible mechanisms underlying impacts of CDs on cytosine context-dependent DNA methylation on a genome-wide scale, which may regulate genes responsible for CDs-induced phenotypic changes in plants. It will be helpful for considering future application of CDs in improving crop agronomic traits.

overall_design: Bisulfite-Seq: 2 samples examined, CD (Carbon dot treatment, 2 replicates), CK (Control, 2 replicates); RNA-Seq: 2 samples examined, CD (Carbondot treatment, 2 replicates), CK (Control, 2 replicates)

first: Zhenyu

last: Luo


title: Nasopharyngeal carcinoma (NPC)-derived exosomes reconstitute tumor microenvironment

city: Taoyuan

first: Tzu-Tung

last: Liu

address: Zhongda Rd,. Zhongli District

institute: National central university

summary: Nasopharyngeal carcinoma is characterized by Epstein-Barr virus (EBV) infection and severe immune cell infiltration. Here, we explored the effect of tumor-derived exosomes on NPC microenvironment using single-cell RNA sequencing (scRNA-seq). We investigated the phenotypic and functional alterations in macrophages, as well as exosome-mediated cellular interactions. Human NPC biopsy samples were dissociated into single-cell suspension and stimuated with exosomes derived from an EBV positive cell line (HK1EBV) for 2 hours. A total of 14,446 qualified cells were subjected to analysis. Collectively, our findings revealed that NPC-derived exosomes facilitate immunosuppressive microenvironment and contribute to NPC progression.

overall_design: Single cell suspension freshly isolated from NPC tumors. For exosomal treatment experiments, processed cells were treated with 50μg exosomes derived from EBV+ HK1 cells for 2 hours under standard cell culture conditions (37°C and 5% CO2).

first: Shu-Chen


title: scRNA sequencing of mice  with or without murine astrovirus infection undergoing cage change.

city: Philadelphia

first: Ken

last: Cadwell

state: PA

address: 421 Curie Boulevard

institute: University of Pennsylvania Perelman School of Medicine

summary: Viral persistence can be disrupted by preventing re-exposure and activating immunity upon stress recovery, indicating that external factors, such as cage changes, can be manipulated to shorten the duration of a viral infection.

overall_design: Laboratory mice (C57BL/6J) with and without murine astrovirus infection that did or did not undergo a cage change (2 mice per group). After 1 day, ileum cells were harvested. Cells from 2 mice per condition were pooled and then used on individual lanes for sequenceing.

first: Christin

last: Herrmann

first: Kimberly

last: Zaldana


title: DYRK1A Is a Novel Regulator of TGFβ Signalling and Determines Sensitivity to OXPHOS Inhibition

city: Shanghai

first: Ying

address: No.25/Ln 2200 Xie-Tu Road Xuhui District

institute: Shanghai Cancer Institute

summary: Intervening in mitochondrial oxidative phosphorylation (OXPHOS) has emerged as a potential therapeutic strategy for certain types of cancers. However, the exploration of targeting OXPHOS for the treatment of liver cancer is still limited, and the regulatory network involved in OXPHOS inhibition remains largely unexplored. Here, employing kinome-based CRISPR screening, we find that OXPHOS inhibitor IACS-010759 exhibits limited effect in liver cancer cells due to the function of dual specificity tyrosine phosphorylation regulated kinase 1A (DYRK1A). Genetic inactivation or pharmacological inhibition of DYRK1A combined with OXPHOS inhibitors activates transforming growth factor β (TGFβ) signalling, which is crucial for OXPHOS inhibition-triggered cell death in liver cancer cells. Mechanistically, DYRK1A directly phosphorylates SMAD3 at Thr132, thereby suppressing the negative impact of TGFβ signalling on the transcription of the glutamine transporter solute carrier family 1 member 5 (SLC1A5). This mechanism compensates for the increased glutamine requirement (?) upon OXPHOS inhibition, leading to the resistance of liver cancer cells to OXPHOS inhibition. Finally, we validated the therapeutic efficacy of targeting OXPHOS in combination with DYRK1A inhibition in multiple liver cancer models in vivo. Our study identifies DYRK1A as a novel regulator of TGF β signalling and elucidates the regulatory mechanisms governing the response to OXPHOS inhibition in tumour cells, providing novel insights into targeting OXPHOS for liver cancer therapy.

overall_design: To elucidate the mechanism of combined IACS-010759 with DYRK1A inhibitor mediates , we conducted RNA-seq on Huh7 cells treated with IACS-010759, harmine or their combination.


title: A histone hyperacetylation pulse induces cellular memory of gene expression and 3-dimensional genome folding [microC_1]

city: Montpellier

first: Marco

last: Di Stefano

address: 141 rue de la Cardonille

country: France

institute: Institut de Génétique Humaine - CNRS

summary: Epigenetic memory enables the stable propagation of gene expression patterns in response to transient developmental and environmental stimuli. Although 3-dimensional (3D) organisation is emerging as a key regulator of genome function due its role in gene expression control, it is unknown whether it contributes to cellular memory. Here, we establish that acute perturbation of the epigenome can induce cellular memory of gene expression in mouse embryonic stem cells (mESCs). Specifically, we uncover how a pulse of histone deacetylase inhibition translates to changes in the histone acetylation and methylation landscape, as well as global and local genome folding. While most epigenomic and transcriptional changes are readily reversible once the perturbation is removed, architectural features are not fully reestablished. Upon a second transient pulse of hyperacetylation, hundreds of genes maintain their dysregulated state. Using ultra-deep Micro-C, we associate memory of gene expression with enhancer-promoter contacts and repressive chromatin topology mediated by Polycomb. These results uncover the complex interplay between different epigenetic regulatory layers and establish a novel link between genome folding and cellular memory.

overall_design: Micro-C experiments in mouse ESCs in control (DMSO), TSA-treated or 24-hour recovery (REC) conditions.

first: Flora

last: Paldi

first: Michael Florian

last: Szalay

first: Giacomo

last: Cavalli


title: Single cell sequencing of IgAN patients

city: Guangzhou

first: Yifang

last: Gao

address: NO.58 zhongshan 2nd road, YueXiu district

institute: sun yat-sen University

summary: To study the functions of PBMC subpopulations in the pathogenesis and progression of IgA nephropathy

overall_design: we performed single-cell RNA sequencing of tonsillar cells and PBMCs from patient with IgA nephropathy on days 0 (pre-operation), 7, and 150.

first: Ronghai

last: Deng


title: Expression data from Methionine normal or deprived culture glioblastoma cell line U251

city: Harbin

first: Hong

last: Hu

state: 黑龙江

address: youzheng street

institute: Harbin medical university

summary: Recent research highlights the significant impact of methionine metabolism on glioma progression. An increasing amount of compelling evidence bridges long non-coding RNAs to abnormal metabolism in gliomas. However, the specific role of long non-coding RNAs in methionine metabolism regulating glioma progression remains unclear. This study reveals that methionine deprivation inhibits the proliferation, migration, and invasion capabilities of gliomas. Interestingly, the expression of TP53TG1, a long non-coding RNA, is also suppressed. TP53TG1 is highly expressed in gliomas and associated with poor patient outcomes. Subsequently, our data proves that inhibition of TP53TG1 suppresses glioma cell proliferation and the epithelial-mesenchymal transition process both in vitro and in vivo. Ultimately, we found that the underlying mechanism involves a competing endogenous RNA regulating network, in which TP53TG1 modulates the target protein STK17B by competitively binding to miR-96-5p, thus regulating glioma progression. These findings suggest that targeting methionine deprivation could be a promising approach for the clinical treatment of glioma.

overall_design: We cultured glioblastoma cell U251 in normal culture or methionine deprivation culture for 48h, then extracted total RNA and detected it using Arraystar Human LncRNA microarray.

first: Jiafeng

last: Li

first: Ruijie

first: Yishuai

last: Huang

last: Shi

first: Honglei

first: Hao

last: Chen

first: Meng

last: Cai

first: Ning

first: Tao

last: Yan

first: Kaikai

first: Huailei


title: The roles of CLOCK and CYCLE in the reproductive system

city: Beijing

first: Pengfei

last: Lv

address: No. 2, Old Summer Palace West Road

institute: China agricultural university

summary: To investigate whether the regulatory roles of the circadian genes CLOCK and CYCLE as transcription factors in the fruit fly testes and ovaries are similar to those in the head tissue, we conducted ChIP-seq analyses on these three tissues, respectively.

overall_design: Chromatin immunoprecipitation DNA sequencing (ChIP-seq) for CLOCK and CYCLE in the head, testis, and ovary.


title: The de novo synthesis of GABA and its gene regulatory function are indispensable for HCC metastasis

city: beijing

first: li

last: li

institute: pumc

summary: To investigate the mechanism of GABA in the regulation of liver cancer metastasis, we treated Huh7 cell lines with or without GABA. We then performed gene expression profiling analysis using data obtained from RNA-seq.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for Huh7 cells with or without GABA treatment for 24h.

first: Li

first: Wenjing

last: Du


title: Bulk and single cell RNA sequencing analyses of GBM Primary and Recurrent,  early and late passages, DMSO / TMZ treatment.

city: New York

first: Mungunsarnai

last: Ganbold

state: NYC

address: 417 East 68st

department: Brain Tumor Center

institute: MSKCC

summary: We used bulk RNA sequencing to analyze unsupervised clustering of early and late passages of four GBM PDXs. Single cell RNAseq data was used to study transcriptional profiles of  DMSO and TMZ, Primary and Recurrent as well as early and late passages of GBM PDXs.

overall_design: Patient PDX cells treated with DMSO or Temozolomide for 5days and subsequently harvested (see STAR Methods section, Xiex et al., 2024).

first: Xuanhua

last: Xiex

first: Luis

middle: F

last: Parada


title: Molecular states underlying neuronal cell type development and plasticity in the whisker cortex

city: Berkeley

first: Salwan

last: Butrus

state: California

address: Stanley Hall Rm

department: Chemical and Biomolecular Engineering

institute: UC Berkeley

summary: Mouse whisker somatosensory cortex (wS1) is a major model system to study the experience-dependent plasticity of cortical neuron physiology, morphology, and sensory coding. However, the role of sensory experience in regulating neuronal cell type development and gene expression in wS1 remains poorly understood. We assembled and annotated a transcriptomic atlas of wS1 during postnatal development comprising 45 molecularly distinct neuronal types that can be grouped into eight subclasses of excitatory neurons and four subclasses of inhibitory neurons. Using this atlas, we examined the influence of whisker experience between postnatal day (P) 12, the onset of active whisking, to P22, on the maturation of molecularly distinct cell types. During this developmental period, when whisker experience was normal, ~250 genes were regulated in a neuronal subclass-specific fashion. At the resolution of neuronal types, we found that only the composition of layer (L) 2/3 glutamatergic neuronal types, but not other neuronal types, changed substantially between P12 and P22. These compositional changes resemble those observed previously in the primary visual cortex (V1), and the temporal gene expression changes were also highly conserved between the two regions. In contrast to V1, however, cell type maturation in wS1 is likely independent of sensory experience, as 10-day full-face whisker deprivation had no influence on the transcriptomic identity and composition of L2/3 neuronal types. A one-day competitive whisker deprivation protocol also did not affect cell type identity but induced moderate changes in plasticity-related gene expression. Thus, developmental maturation of cell types is similar in V1 and wS1, but sensory deprivation has a minimal effect on cell type development in wS1.

overall_design: snRNA-seq from mouse whisker cortex in two different developmental time points and two different whisker deprivation conditions.

first: Hannah

last: Monday

first: Chris

last: Yoo

first: Daniel

last: Feldman

first: Karthik

last: Shekhar


title: Single-cell transcriptomic analysis of immune responses within a microfluidic skin equivalent

city: London

first: John

last: Connelly

address: 4 Newark Street

department: Centre for Cell Biology and Cutaneous Research

country: United Kingdom

institute: Queen Mary University of London

summary: Microfluidic human skin equivalents (HSEs) were constructed by 3D printing and consisted of an endothelial lined vascular microchannel within a 3D dermal matrix embedded with fibroblasts.  Keratinocytes were cultured on the surface to mimic the epidermis.  Following exposure of the epidermis to lipopolysaccharide (LPS) and nigericin, human monocytes were injected into the vascular channel and migrated into the HSE.  Single-cell RNA sequencing was performed on control or treated HSEs after 1 or 6 days to analyse the inflammatory and immunological responses to LPS and nigericin activation.  These studies identified dynamic upregulation and resolution of inflammatory gene signatures in the tissue resident cells in response to treatment, alongside differentiation of monocytes into tissue resident macrophages.

overall_design: HSEs were treated with 100 ng/ml LPS and 10 mM nigericin or left untreated (control), and primary human monocytes were injected into the microchannel.  Control and treated samples were collected at days 1 and 6 for RNA-seq analysis, and three HSEs were pooled for each condition/timepoint.   Monocytes were enriched 1:1 with resident cells by magnetic activated cell sorting for CD45.  Approximately 15,000 viable cells were sequenced per condition/timepoint.

middle: T


title: A histone hyperacetylation pulse induces cellular memory of gene expression and 3-dimensional genome folding [microC_2]

first: Marco

last: Di Stefano

first: Flora

last: Paldi

first: Michael Florian

last: Szalay

first: Giacomo

last: Cavalli


title: Development of MAPT S305 mutation models exhibiting elevated 4R tau expression, resulting in altered neuronal and astrocytic function [Targeted MAPT ISO-seq]

city: Edinburgh

first: Kathryn

middle: R

last: Bowles

address: 49 Little France Crescent

institute: University of Edinburgh

summary: We have developed a panel of isogenic iPSC lines carrying MAPT splice-site mutations S305S, S305I or S305N, derived from four different donors. All three mutations significantly increased the proportion of 4R tau expression in iPSC-neurons and astrocytes, with up to 80% 4R transcripts in S305N neurons from as early as 4 weeks of differentiation.

overall_design: iPSCs were differentiated into neurons (4, 6 and 8 weeks of differentiation) and astrocytes, and submitted for targeted MAPT ISO-seq

first: Alison

middle: M

last: Goate


title: Development of MAPT S305 mutation models exhibiting elevated 4R tau expression, resulting in altered neuronal and astrocytic function


title: Cocaine-craving drives repressive epigenetic differential gene expression in ventral tegmental area dopamine neurons

city: Miami

first: Alexander

middle: Ve

last: Margetts

state: FL

address: 1600 NW 10th Ave, Rosenstiel Med Sci 7014A

department: Psychiatry and Behavioral Sciences

institute: University of Miami

summary: Dopamine (DA) signaling plays an essential role in reward valence attribution and in encoding the reinforcing properties of natural and artificial rewards. The adaptive responses from midbrain dopamine neurons to artificial rewards such as drugs of abuse are therefore important for understanding the development of substance use disorders. Drug-induced changes in gene expression are one such adaptation that can determine the activity of dopamine signaling in projection regions of the brain reward system. One of the major challenges to obtaining this understanding is the inherent cellular heterogeneity in the brain, where each neuron population can be defined by a distinct transcriptional profile. To bridge this gap, we have adapted a virus-based method for labeling and capture of dopamine nuclei, coupled with nuclear RNA-sequencing and bioinformatics analyses, to study the transcriptional adaptations, specifically, of dopamine neurons in the ventral tegmental area (VTA) during cocaine taking and cocaine craving, using a mouse model of cocaine intravenous self-administration (IVSA). Our results show significant changes in gene expression across non-drug operant training, cocaine taking, and cocaine craving, highlighted by an enrichment in expression of repressive epigenetic modifying enzymes during cocaine craving. Immunohistochemical validation further revealed an increase of H3K9me3 deposition in DA neurons during cocaine craving. These results demonstrate that cocaine-induced transcriptional adaptations in dopamine neurons vary by phase of self-administration and suggest that such an approach may be useful in identifying relevant phase-specific molecular targets to alter the behavioral course of substance use disorders.

overall_design: DAT-Cre mice were injected with a AAV5-DIO-KASH-HA virus. After 14 days, these mice were operantly trained to lever press for food rewards. Following food training, the remaining mice were permitted to intravenously self-administer cocaine at an acquisition dose (0.3 mg/kg/inf dissolved in 0.9% saline) for 5 daily 1-hour sessions, followed by a maintenance dose (1.0 mg/kg/inf dissolved in 0.9% saline) for 10 daily 1-hour sessions. After 15 days of IVSA (Acquisition and Maintenance), remaining mice underwent 21 days of forced home cage abstinence to incubate cocaine craving. Ventral tegmental area (VTA) dopamine nuclei were isolated immediately after the final food training session (FT), after the final cocaine maintenance session (MN), and following 21 days of forced home cage abstinence (CRV). RNA-Sequencing was conducted on purified dopaminergic and non-dopaminergic nuclei obtained from FANS.

first: Tate

middle: A

last: Pollock

middle: V

first: Samara

middle: J

last: Vilca

last: Tuesta


title: Direct mitochondrial import of lactate supports resilient carbohydrate oxidation

city: Salt Lake City

first: Joseph

last: Visker

state: UT

address: 1245 W SIENNA WAY

department: Nora Eccles Harrison Cardiovascular Research and Training Institute

institute: The University of Utah

summary: Lactate is the most abundant circulating metabolic intermediate in mammals and an important energy source for many organs. To use lactate as a fuel, it must be oxidized to pyruvate for entry into the TCA cycle. It is usually described that this reaction occurs in the cytosol and requires the mitochondrial pyruvate carrier (MPC) for pyruvate transport into the mitochondria. Here, using 13C stable isotope tracing, we report that lactate can be oxidized in the heart tissue of mice even when the MPC is genetically deleted. MPC-independent lactate import and oxidation within mitochondria is dependent upon the monocarboxylate transporter 1 (MCT1/ Slc16a1). Mitochondria isolated from Mct1iCKO hearts have impaired respiration on lactate but not pyruvate. Lactate import coupled to mitochondrial lactate dehydrogenase activity functions as an electron shuttle which can produce NADH sufficient for respiration even when the TCA cycle is blocked. Cardiac-specific loss of MCT1 leads to rapid decompensation into heart failure with reduced ejection fraction in response to diverse cardiac injuries. Thus, we identify a new mitochondrial electron shuttle that enables the oxidation of lactate and is required to support cardiac energetics under stress conditions.

overall_design: To understand the mechanisms of lactate metabolism during cardiac hypertrophy we subjected control and MCT1-KO mice to Ang/PE neurohormonal agonists causing myocardial hypertrophy.

first: Ahmad

last: Cluntun

first: Greg

last: Ducker

first: Joe

first: Jared

last: Rutter


title: Targeting Cancer Associated Fibroblasts/Tumor Cells Cross-Talk Inhibits Intrahepatic Cholangiocarcinoma Progression

city: Castellana Grotte (BA)

first: Grazia

last: Serino

address: Via Turi, 27

country: Italy

institute: National Institute of Gastroenterology “S. de Bellis”, Research Hospital

summary: Cancer associated fibroblasts (CAFs), mainly responsible for the desmoplastic reaction hallmark of intrahepatic Cholangiocarcinoma (iCCA), likely take a role in tumour aggressiveness and resistance to therapy, although the molecular mechanisms involved are unknown. Aim of the study is to investigate how targeting hCAF/iCCA cross-talk with a Notch1 inhibitor namely Crenigacestat would affect cancer progressionWe used different in vitro models in 2D and we established new 3D hetero-spheroids with iCCA cells and human (h)CAFs. Results were confirmed in a xenograft model, explanted tumoral tissues underwent transcriptomic and bioinformatic analysis. hCAFs/iCCA cross-talk sustains increased migration of both KKU-M213 and KKU-M156 cells, while Crenigacestat significantly inhibits only the cross-talk stimulated migration. Hetero-spheroids grow larger than homo-spheroids,formed by only iCCA cells. Crenigacestat significantly reduced invasion and growth of hetero- but not of homo-spheroids. In xenograft models, hCAFs/KKU-M213 tumours grew significantly larger than KKU-M213 tumours, but were significantly reduced in volume by Crenigacestat treatment, which also significantly reduced the fibrotic reaction. Interpretative phenomenological analysis of the transcriptome unveiled that genes of hCAFs/KKU-M213 but not of KKU-M213 tumours increased tumour lesions, and that Crenigacestat treatment inhibited the modulated canonical pathway. Cell cycle checkpoints were the most modulated pathway. In hetero-spheroids, the number of cells increased into G2/M cell cycle phase, while Crenigacestat significantly decreased the number into G2/M phase in hetero but not in homo-spheroids. The cross-talk hCAFs/iCCA is a new target for reducing cancer progression with drugs such as Crenigacestat.

overall_design: We used RNA sequencing technology to understand the molecular mechanisms affected by Crenigacestat in a cholangiocarcinoma xenograft tissue generated from KKU-M213 cell lines or KKU-M213:hCAFs.

first: Serena

last: Mancarella

first: Gianluigi

last: Giannelli


title: scRNAseq data from 4 ASM-AHN, 3 CMML patients and 3 healthy donors

first: Xiaomeng

last: Huang

state: Utah

department: Department of Human Genetics

institute: University of Utah

summary: Aggressive systemic mastocytosis with associated hematologic neoplasm (ASM-AHN) represents a complex myeloid neoplasm, in which KIT D816V is the phenotype-defining but not the only driver mutation. Avapritinib is a potent and specific KIT D816V inhibitor recently approved for ASM treatment. To understand the effects of inhibiting KIT D816V on MC and Non-MC compartments, we combined single cell RNA sequencing (scRNAseq) with deep whole genome sequencing (WGS) to map clonal dynamics in successive blood samples from 4 ASM-AHN patients treated with avapritinib (T1 as pretreatment sample, T2 and T3 as posttreatment samples). Since CMML is the most common AHN associated with ASM, we included samples from three treatment-naïve KIT wild type CMML patients for comparison. We also included 3 age-matched healthy donor as controls.

overall_design:
WBC were collected from 4 ASM-AHN patients (3 timepoints each), 3 CMML patients and 3 healthy donors. Single cell samples were subject to 10X 3' capture scRNAseq.

***************************************************************
Submitter states that missing raw files are due to patient privacy concerns.
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first: Anthony

middle: D

last: Pomicter

first: Jonathan

last: Ahmann

first: Gabor

last: Marth

first: Michael

middle: W

last: Deininger


title: Genomic and transcriptomic profile of HNF1A-mutated adenomas: molecular signature and potential therapeutic implications

city: Turin

first: Silvia

last: Deaglio

address: Via Santena, 19

institute: University of Turin

summary:
Background: Hepatocellular adenomas (HAs) are tumors that can develop under different conditions, including in patients harboring a germline mutation in HNF1A. However, little is known about the pathogenesis of such disease. This work aims at better defining what mechanisms lie under the development of this condition.
Results: 6 HAs were sampled from the liver of a 17-year-old male affected by diabetes and multiple hepatic adenomatosis harboring the heterozygous pathogenic germline variant c.815G>A, p.(Arg272His) in HNF1A which has a dominant negative effect. All HAs were molecularly characterized and four of them were shown to harbor a second somatic HNF1A variant, one had a mutation in the ARID1A gene while no additional somatic changes were found in the remaining HA and in normal parenchyma. A transcriptomic profile of the same HA samples was also performed. HNF1A biallelic mutations were associated with the up-regulation of several pathways including tricarboxylic acid cycle, the metabolism of fatty acids and mTOR signaling while angiogenesis, endothelial and vascular proliferation, cell migration/adhesion, and immune response were down-regulated. Contrariwise, in the tumor harboring the ARID1A variant, angiogenesis was up-modulated while fatty acid metabolism was down-modulated. Histological analyses confirmed the molecular data. Independently of the second mutation, energetic processes and cholesterol metabolism were up-modulated while immune response was down-modulated.
Conclusions: this work provides a complete molecular signature of HNF1A-associated HAs, analyzing the association between specific HNF1A variants and the development of HA, while identifying potential new therapeutic targets for non-surgical treatment.

overall_design: Gene expression profiles obtained from RNA-seq data obtained from samples of hepatic adenomas and normal parenchyma.


title: Reutilization of ancestral gill regulatory programs in outer ear evolution (human)

city: Los Angeles

first: J.

middle: Gage

last: Crump

state: CA

address: 1425 San Pablo Street, BCC 406

department: Eli and Edythe Broad CIRM Center for Stem Cell and Regenerative Medicine

institute: University of Southern California

summary: How novel structures arise during evolution has long fascinated biologists. A dramatic example is how the diminutive bones of the mammalian middle ear arose from ancestral fish jawbones. In contrast, the evolutionary origins of the outer ear, another mammalian innovation, remain a mystery, in part because it is supported by non-mineralized elastic cartilage rarely recovered in fossils. Whether the outer ear arose de novo or through reuse of ancestral developmental programs is unknown. Here we show that the outer ear shares gene regulatory programs with the gills of fishes and amphibians for both its initial outgrowth and later development of elastic cartilage. Comparative single-nuclei multiomics of the human outer ear and zebrafish gills reveals conserved gene expression and putative enhancers enriched for common transcription factor binding motifs. This is reflected by transgenic activity of human outer ear enhancers in gills, and fish gill enhancers in the outer ear. Further, single-cell multiomics of the cartilaginous book gills of horseshoe crabs reveal a shared DLX-mediated gill program with vertebrates, with a book gill distalless enhancer driving expression in zebrafish gills. We propose that elements of an invertebrate gill program were reutilized in vertebrates to generate first gills and then the outer ear.

overall_design:
From de-identified 20 week post-gestation human fetal tissue acquired from an elective abortion, we separated two outer ears and the nose for further processing. We separately dissociated the outer ear and nose tissue into single cell suspensions, extracted nuclei and then prepared 10X multiome libraries. These samples were then pooled, sequenced using the Illumina Hiseq 4000 platform, and de-multiplexed using sample-specific barcodes for further analysis.

From de-identified 22 week post-gestation human fetal tissue acquired from an elective abortion, we separated one outer ear and the epiglottis for further processing. We separately dissociated the outer ear and nose tissue into single cell suspensions, extracted nuclei and then prepared 10X multiome libraries. These samples were then pooled, sequenced using the Illumina NovaSeq X platform, and de-multiplexed using sample-specific barcodes for further analysis.

first: Mathi

last: Thiruppathy

first: Jade

last: Tassey

first: Arijita

last: Sarkar

first: Alessandra

last: Moscatello

first: Christian

last: Hochstim

first: Ya-Wen

last: Chen

first: Denis

last: Evseenko

first: Gage


title: Bulk RNA sequencing and analysis of sorted Common Lymphoid Progenitor (CLP) cells from WT, Notch1+/-, and Notch1+/ΔTAD adult mouse bone marrow

city: Chicago

first: Kostandin

last: Pajcini

state: IL

address: 909 S Wolcott Ave.

department: Pharmacology and Regenerative Medicine

institute: University of Illinois at Chicago

summary: We assessed the transcriptional profiles of sorted CLP cells from the bone marrow of WT and Notch hypomorphic genetic mouse models. The goals of the study are determine the signaling pathways downstream of Notch signaling in bone marrow CLPs to determine pathways involved in Notch dependent priming towards the T cell lineage.

overall_design: CLP mRNA expression profiles from sorted adult bone marrow CLPs of WT, Notch1+/-, and Notch1+/ΔTAD mice.

first: Kilian

last: Sottoriva


title: Global gene expression in WT and antisense-acetyl-CoA (ACLA) mutant under short day conditions

city: MISSISSIPPI STATE

first: Lei

last: Wang

state: MS

address: 414 LEE BLVD 3677

department: Department of Biological Sciences

institute: Mississippi State University

summary:
Transcriptome analysis of RNA samples from WT and antisense-acetyl-CoA (ACLA) mutant.
The antisense-ACLA mutant and WT plants were arranged according to a Randomized Complete Block Design. The plants were planted in rows with seven rows in each flat; two plants of the same genotype/pot. Plants were grown under a SD photoperiod (8 h light/16 h dark) in a growth chamber as described. Eight randomly selected rows were harvested for each time point from different flats. Plant material was harvested at 11 time points in the diurnal cycle (0, 0.5,1, 4, 6, 8,12 h in the dark condition, and 0, 0.5, 1, 4 h in the light condition; Time 0 is the beginning of the light/dark period); harvesting was conducted under a green safety light. Each sample consisted of rosette leaves (leaves 5 to 8, staged following Bowmann (1994); photosynthetically active (Stessman et al., 2002)) from sixteen six-week-old plants. Leaf samples were frozen in liquid N2 immediately after harvest and stored at -80‚°C for RNA extraction. The experiment was done twice and independent randomizations for plant growth and harvest were used for the two replicates.

overall_design: The antisense-ACLA mutant and WT plants were arranged according to a Randomized Complete Block Design. The plants were planted in rows with seven rows in each flat; two plants of the same genotype/pot. Plants were grown under a SD photoperiod (8 h light/16 h dark) in a growth chamber as described. Eight randomly selected rows were harvested for each time point from different flats. Plant material was harvested at five time points in the diurnal cycle (0, 0.5,1, 4, 6, 8,12 h; Time 0 is the beginning of the light period); harvesting was conducted under a green safety light. Each sample consisted of rosette leaves (leaves 5 to 8, staged following Bowmann (1994); photosynthetically active (Stessman et al., 2002)) from sixteen six-week-old plants. Leaf samples were frozen in liquid N2 immediately after harvest and stored at -80‚°C for RNA extraction. The experiment was done twice and independent randomizations for plant growth and harvest were used for the two replicates.

first: Wurtele

last: Eve Syrkin

first: Ling

last: Li


title: Targeting IRE1α reprograms the tumor microenvironment and enhances anti-tumor immunity in prostate cancer [scRNA-seq]

city: Oslo

first: Fahri

last: Saatcioglu

address: Blindernveien 31

department: Department of Biosciences

country: Norway

institute: University of Oslo

summary: Unfolded protein response (UPR) is a central intracellular stress response pathway that is hijacked by tumor cells for their survival. However, how activation of UPR in cancer cells may shape the tumor microenvironment (TME) remains largely unexplored. Here, we investigated the potential role of IRE1α signaling on modulation of TME dynamics in prostate cancer (PCa). We found that IRE1α is increased in PCa patient tumors and genetic inhibition of IRE1α in syngeneic mouse PCa models, as well as in an orthotopic model, dramatically reduced tumor growth. Multiomics analysis suggested that IRE1α ablation in cancer cells significantly potentiated interferon (IFN) response and activation of immune system related pathways in the TME. Single-cell RNA-sequencing (scRNA-seq) revealed that the abundance of immunosuppressive cells, such as tumor-associated macrophages (TAMs), were markedly reduced in the IRE1α deficient tumors. Geneset Enrichment Analysis demonstrated that IFN response was significantly enriched in TAMs, cancer cells, and dendritic cells. Notably, the small molecule IRE1α inhibitor MKC8866 (ORIN1001), currently in clinical trials, reprogrammed the TME and enhanced the response to anti-PD-1 blockade therapy in syngeneic PCa mouse models. Furthermore, a novel scRNA-seq-derived TAM gene signature is strongly associated with poor PCa survival, which is reduced by the MKC8866 + anti-PD-1 combination therapy. Our findings indicate that activation of IRE1α signaling not only promotes cancer cell growth and survival, but it also interferes with anti-tumor immunity in the TME. Thus, IRE1α targeting could present a novel approach for improving anti-PD-1 immunotherapy in PCa.

overall_design: scRNA-sequencing (scRNA-seq) was performed on Myc-CaP IRE1α WT and KO2 tumors. Freshly dissected tumor samples were washed with PBS and transferred into GEXSCOPE Tissue Preservation Solution (Singleron), and stored on ice. Six tumor samples from each group were pooled for scRNA-seq. The tissue samples were then washed with Hanks balanced salt solution three times and minced into small pieces which were digested with Tissue Dissociation Solution (Singleron) at 37 °C for 15 min with agitation. After digestion, cells were passed through a 40 μm strainer to remove cell debris and centrifuged at 300 × g for 5 min. The supernatant was removed and cell pellet was resuspended in 1 ml PBS, followed by red blood cell lysis using RBC lysis buffer. Next, cells were centrifuged at 500 × g for 5 min and resuspended in PBS. The single cell suspension was adjusted to a concentration of 1 × 105 / ml for library preparation. This was loaded onto microfluidics chips (GEXSCOPE Single Cell RNA-seq Kit, Singleron Biotechnologies) and GEXSCOPE 3´SD scRNAseq libraries were prepared according to manufacturer instructions (Singleron Biotechnologies). Individual libraries were diluted and pooled for next generation sequencing on Illumina NovaSeq with 150 bp paired-end reads.

first: Bilal

last: Unal

first: Omer

last: Kuzu

first: Yang

last: Jin


title: An ancient competition for the conserved branchpoint sequence influences physiological and evolutionary outcomes in splicing

city: Galveston

first: Haiping

last: Hao

state: Texas

address: 301 University Blvd

institute: UTMB NGS Core

summary: Branchpoint (bp) recognition is an early event that is required for spliceosome recruitment to a pre-mRNA substrate. The bp sequence can range from nearly invariant in yeast (UACUAAC) to quite degenerate in higher eukaryotes (YUNAY in humans), which correlates with the frequency of alternative splicing observed in the latter. The closely related KH-type RNA binding proteins (RBPs) SF1 and Quaking (QKI) can bind to a similar bp-like sequence, but the extent to which competition for the bp sequence between these two proteins might influence gene expression regulatory mechanisms is unknown. Here we find genomewide evidence for competition between these two RBPs for a subset of alternatively spliced exons that have ACUAAY-like bp sequences. Using a combination of molecular genetic and biochemical techniques with a model intron containing two bp-like sequences, we find that either can be used as a bp but high affinity binding and strong splicing repression by QKI requires both sites. This directly prevents SF1 binding and the subsequent recruitment of spliceosome-associated factors. Finally the ectopic expression of QKI in S. cerevisiae (which lacks QKI) is lethal, due at least in part to widespread splicing repression. In conclusion, QKI can function as a splicing repressor by directly competing with SF1/BBP for a subset of bp sequences that closely mirror its high affinity binding site. This suggests that QKI and degenerate bp sequences may have co-evolved which allowed for expansion of the alternative splicing repertoire in metazoans, plants, and animals.

overall_design: We inserted a galactose-inducible cDNA encoding either EGFP, mutant K120A;R124A Qki5, or wildtype Qki5 into BY4741

first: Karen

middle: L

last: de Castro

first: Robert

middle: E

last: Johnson

first: Jose

last: Abril

first: Liao

last: Kuo-Chieh

first: Steven

middle: G

last: Widen

first: John

middle: P

last: Donohue

first: William

middle: K

last: Russell

first: W

middle: S

last: Fagg


title: Impact of pehR transcriptional regulator on Ralstonia solanacearum F1C1 gene expression patterns

city: Tezpur

first: Aditya

last: Kumar

state: Assam

address: R107, Dept Of Molecular Biology & Biotechnology

department: Molecular Biology and Biotechnology

country: India

institute: Tezpur University

summary: Ralstonia solanacearum, recognized as a pervasive plant pathogen causing lethal wilt disease in over 450 plant species worldwide, represents a substantial threat to economically vital crops including tomato, chili, and eggplant. Within R. solanacearum, pehR serves as a transcriptional regulator, forming part of the pehSR two-component regulatory system. This system governs the production of polygalacturonase (PG), a crucial extracellular enzyme involved in plant cell wall degradation and bacterial wilt development. Our study focuses on a disruption mutant of the pehR gene from the local isolate R. solanacearum F1C1, obtained from Tezpur, Assam. Utilizing RNA-seq, we examine the gene expression profiles of R. solanacearum F1C1 under pehR regulation. Differential expression analysis between wild type and pehR mutants reveals a set of genes directly influenced by pehR. This comprehensive gene expression analysis provides insights into the system-level responses orchestrated by the pehR regulator in pathogenicity and virulence development. Moreover, it sheds light on the broader regulatory network controlled by pehR and its underlying molecular mechanisms. Our work also underscores the identification of key target genes regulated by pehR within R. solanacearum F1C1, offering a deeper understanding of its role in bacterial pathogenicity. Ultimately, unraveling the complete pehR regulome holds promise for elucidating the regulatory dynamics driving the pathogenic behavior of R. solanacearum F1C1.

overall_design:
To explore the influence of the pehR transcriptional regulator on the expression patterns of the R. solanacearum F1C1 gene, we initially developed a pehR mutant utilizing the lacZ insertion mutation technique.
We employed an RNA-Seq methodology to profile the transcriptome of both the pehR mutant and the wildtype strain. RNA samples were extracted from both strains during the logarithmic growth phase in BG medium at 28°C.
After ensuring the samples met quality standards, we proceeded to sequence them using Illumina sequencing technology.

first: Sharmilee

last: Sarkar

first: Mohit

last: Yadav


title: Pathogenic TSC2 variants alter developmental trajectories of human cortical organoids and perturb cell-cell communication networks [bulk RNA-seq]

city: Atlanta

first: Zhexing

last: Wen

address: 100 Woodruff Circle

department: Department of Psychiatry and Behavioral Sciences

institute: Emory University

summary: Tuberous Sclerosis Complex (TSC), an autosomal dominant condition, is engendered by heterozygous mutations in either TSC1 or TSC2 genes, manifesting in systemic growth of benign tumors. In addition to brain lesions, neurologic sequelae represent the greatest morbidity in TSC patients. Investigations utilizing TSC1/2-knockout animal or human stem cell models suggest that TSC deficiency-causing hyper-activation of mTOR signaling might precipitate anomalous neurodevelopmental processes. However, how the pathogenic variants of TSC1/2 genes identified in TSC patients affect the trajectory of human brain development and how they contribute to the neurological manifestations in TSC remain largely unexplored. Here, we employed 3-dimensional cortical organoids derived from induced pluripotent stem cells (iPSCs) from TSC patients harboring TSC2 mutations, alongside organoids from age- and sex-matched healthy individuals as controls. Through comprehensively longitudinal molecular and cellular analysis of TSC organoids, including transcriptomics and single cell transcriptomics, we found that TSC2 pathogenic variants led to dysregulated neurogenesis, synaptogenesis, and gliogenesis, particularly for reactive astrogliosis. The altered developmental trajectory of TSC organoids significantly resembles the molecular signatures of neuropsychiatric disorders, including autism spectrum disorders, epilepsy, and intellectual disability. Through cell-cell communication analysis at the single cell level, we identified that TSC2 pathogenic variants disrupted the cell-cell communications, in particular, neuron-astrocyte interactions within the NLGN-NRXN signaling network. Furthermore, cellular and electrophysiological assessments of TSC cortical organoids, along with proteomics and phosphoproteomics analyses of synaptosomes, revealed that pathogenic TSC2 variants precipitate perturbations in mitochondrial translational integrity, neurofilament formation, synaptic transmission, and neuronal network activity. Intriguingly, the increased neu

overall_design: bulk RNA-seq

first: Weibo

last: Niu

first: Shaojun

last: Yu


title: m6A related mRNAs in ovarian-endometriosis control vs normal endometrial tissue

city: Harbin

first: Chengcheng

last: Ren

address: Youzheng 23

institute: The first affiliated hospital of Harbin Medical University

summary: aberrant m6A- related mRNAs in EI and NE tissues were uncovered

overall_design: By arraystar m6A-mRNA epitranscriptomic microarray, biological information analysis technologies, and validation of other databases and clinical tissues, aberrant m6A- related mRNAs were uncovered

first: Yanan

last: He


title: Snorkel-tag Based Affinity Chromatography for Recombinant Extracellular Vesicle Purification

city: Aarhus N

first: Morten

last: Veno

state: DK

address: Aabogade 15

country: Denmark

institute: Omiics ApS

summary: Getting insights on tissue or cell specific EV composition in pathophysiological conditions for developing therapeutics, biomarkers and diagnostic tools in modern medicine still remains a major bottleneck. Similarly, lack of standards for EV comparison also hampers progression in the field. Here, we therefore generated a tagged CD81 fusion protein highly enriched in EVs, enabling affinity isolation of EVs from complex matrices in a non-destructive and pure form without disturbing the here tested EV characteristics.

overall_design: Conditioned media from WT and CD81-snorkel tag overexpressing WJ-MSCs were concentrated by ultrafiltration. CD81-snorkel tag EVs from concentared conditioned media were purified by StEVAC method.

first: Madhusudhan

last: Bobbili

first: Yan

last: Yan

first: Jørgen

last: Kjems

first: Johannes

last: Grillari


title: Glucan Reprograms Neutrophils to Induce Disease Tolerance Against Influenza A Virus

first: Sarah

last: Sun

state: Illinois

address: 900 E 57th Street

institute: University of Chicago

summary: Disease tolerance is an evolutionarily conserved host defence strategy that preserves tissue integrity and physiology without affecting pathogen load. Unlike host resistance, the mechanisms underlying disease tolerance remain poorly understood. In the present study, we investigated whether an adjuvant (β-glucan) can reprogram innate immunity to provide protection against Influenza A virus (IAV) infection. Here we observe that β-glucan treatment reduced the morbidity and mortality against IAV infection, independent of host resistance (viral load). Increased survival of β-glucan treated mice against IAV is associated with the accumulation of neutrophils via RORgt+ T cells in the lung tissue. Using gain-and-loss-of-function approaches, we demonstrate that β-glucan reprogrammed neutrophils are essential for promoting disease tolerance, limiting pulmonary tissue damage, and enhancing survival against IAV infection. β-glucan treatment promotes granulopoiesis in a type 1 interferon-dependent manner that leads to the generation of a unique subset of neutrophils, which are less mature with higher mitochondrial mass utilizing mitochondrial oxidative (OXPHOS) metabolism. Collectively, our data indicate that β-glucan reprograms hematopoietic stem cells (HSCs) to generate neutrophils with a novel “regulatory” function, which is required for promoting disease tolerance and maintaining lung tissue integrity against viral infection.

overall_design: RNA-sequencing of mouse neutrophils collected 7 days after beta glucan or control injection from naïve or IAV infected mice.

first: Nargis

last: Khan

first: Raphael

last: Chevre

first: Mina

last: Sadeghi

first: Erwan

last: Pernet

first: Andrea

last: Herrero

first: Alexander

last: Grant

first: Jeffrey

last: Downey

first: Luis

middle: B

last: Barreiro

first: Bryan

last: Yipp

first: Oliver

last: Soehnlein

first: Maziar

last: Divangahi


title: Reutilization of ancestral gill regulatory programs in outer ear evolution

first: J.

last: Crump

summary: How novel structures arise during evolution has long fascinated biologists. A dramatic example is how the diminutive bones of the mammalian middle ear arose from ancestral fish jawbones1. In contrast, the evolutionary origins of the outer ear, another mammalian innovation, remain a mystery, in part because it is supported by non-mineralized elastic cartilage rarely recovered in fossils. Whether the outer ear arose de novo or through reuse of ancestral developmental programs is unknown. Here we show that the outer ear shares gene regulatory programs with the gills of fishes and amphibians for both its initial outgrowth and later development of elastic cartilage. Comparative single-nuclei multiomics of the human outer ear and zebrafish gills reveals conserved gene expression and putative enhancers enriched for common transcription factor binding motifs. This is reflected by transgenic activity of human outer ear enhancers in gills, and fish gill enhancers in the outer ear. Further, single-cell multiomics of the cartilaginous book gills of horseshoe crabs reveal a shared DLX-mediated gill program with vertebrates, with a book gill distalless enhancer driving expression in zebrafish gills. We propose that elements of an invertebrate gill program were reutilized in vertebrates to generate first gills and then the outer ear.

overall_design: From the adult male horseshoe crab, book gill tissue was dissected and subjected to mechanical dissociation of nuclei. We used FANS to perform nuclei sorting and then prepared 10X multiome libraries. These samples were  pooled, sequenced using the Illumina Nextseq and Hiseq 4000 platforms, and de-multiplexed using sample-specific barcodes for further analysis.

first: Mathi

last: Thiruppathy

first: Gage


title: Pathogenic TSC2 variants alter developmental trajectories of human cortical organoids and perturb cell-cell communication networks

first: Zhexing

last: Wen

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

overall_design: Refer to individual Series


title: Single-cell analysis of L-MYC expressing neural stem cells and their extracellular vesicles revealed distinct progenitor populations with neurogenic potential [bulk RNA-seq]

status: Public on Nov 21 2024

city: Duarte

first: Yate-Ching

last: Yuan

address: 1500 East Duarte

institute: City of Hope

summary: Neural stem cell (NSC)-based therapies can exploit the inherent pathotropism of NSCs to deliver neurotrophic factors for cell replacement therapies and to stimulate endogenous neurogenesis to permanently repair damaged brain tissue after brain injury. Despite studies on the protective/restorative functions of NSCs and their extracellular vesicles (EVs), a major impediment to advancing this approach to late-stage preclinical studies and clinical practice has been the lack of molecular characterization of NSCs and NSC-EV cargo composition. To fill this knowledge gap, we characterized therapeutically relevant human fetal NSC lines expressing the L-MYC gene (LMNSC01, LMNSC02) and their EVs using single-cell RNA sequencing (scRNAseq) and proteomic analyses. LMNSC01 cells differentiate into neurons and glial cells, including oligodendrocytes, and LMNSC02 mostly differentiate into neurons and oligodendrocyte progenitor cells (OPCs) under undirected differentiation in vitro. scRNAseq analysis indicates that LMNSC01 and LMNSC02 populations are homogenous, expressing not only NSC markers such as SOX2 and nestin, but also doublecortin (DCX), OPC marker O1, and astrocyte progenitor marker vimentin. LMNSC01 cells were enriched in biomarkers for astrocytes, excitatory neurons, glial cells, and OPCs, and LMNSC02 in inhibitory neurons, glial cells and OPCs. Using Single Extracellular VEsicle Nanoscopy, we quantified the size, shape, and content of tetraspanins (CD9, CD63, and CD81) for EVs secreted by cultured LMNSC01 and LMNSC02 cells. Both cell lines produced largely circular EVs; compared to EVs from LMNSC01 cells, those from LMNSC02 cells had on average higher detected tetraspanin content and larger diameter. We also quantified protein, RNA concentration, particle number, and batch-to batch variability for each EV. Our EV cargo global proteomics results suggest that LMNSC02 cells are enriched in processes involved in neuron development, neuron protection, synaptic transmission, and axon development, while EVs derived from LMNSC01 cells were more enriched in RNA processing. Finally, we identified neuroprotective properties of LMNSC-EVs in a brain organoid model of methotrexate-induced brain tissue toxicity. Our data suggest that LMNSC01 and LMNSC02 cell lines have distinct utilities, highlighting the need for molecular characterization to identify the cell lines and EVs with the highest therapeutic potential for various neurological conditions.

overall_design: Bulk RNA Sequencing of LMNSC01-EVs and LMNSC02-EVs, derived from two LMNSC01 and LMNSC02 cell lines. LMNSC01 and LMNSC02 human cells were grown to 80-90% confluency and cultured media was collected for EV isolation and RNA isolation to sequenced using NovaSeq 6000.

first: Patrick

last: Pirrotte

first: Isabella

last: Vasquez

last: Jiang

first: Alejandra

last: Ojeda

first: Eric

last: Alsop

first: Nathaniel

last: Hansen

first: Ritin

last: Sharma

first: Meechoovet

last: Hunsar

first: Benjamin

last: Peton

first: Dorothy

last: Palomares

first: Blake

last: Brewster

first: Michael

last: Barish

first: Corina

middle: O

last: Bondi

first: Russell

middle: C

last: Rockne

first: Tijana

last: Jovanovic-Talisman

first: Kendall

last: Van Keuren-Jensen

first: Anthony

last: Kline

first: Margarita

last: Gutova


title: RNA-seq Reveals Macrophage Polarization and Fibrosis Markers in AKI to CKD Transition After Ischemia-Reperfusion Injury

city: Kurashiki

first: HAJIME

last: NAGASU

state: Okayama

address: 577 Matsushima

department: Nephrology and Hypertension

country: Japan

institute: Kawasaki Medical School

summary: RNA-seq analysis of kidney samples from mice after ischemia-reperfusion injury (IRI) identified 1,109 genes with altered expression between mild (20 minutes) and severe (35 minutes) IRI groups. Among these, 974 genes were upregulated and 135 were downregulated in the severe group.

overall_design: The experimental design involved using C57Bl/6 mice to study the transition from acute kidney injury (AKI) to chronic kidney disease (CKD) following ischemia-reperfusion injury (IRI). Two groups were established based on the duration of ischemia: a mild IRI group with 20 minutes of ischemia and a severe IRI group with 35 minutes of ischemia. The mice were sacrificed 28 days post-IRI to assess the long-term effects of the injury.

first: Hajime

last: Nagasu

first: Megumi

last: Kondo

first: Seiji

last: Kishi

first: Masanobu

last: Takasu


title: Development of MAPT S305 mutation models exhibiting elevated 4R tau expression, resulting in altered neuronal and astrocytic function [bulkRNA-seq]

first: Kathryn

middle: R

last: Bowles

address: 49 Little France Crescent

institute: University of Edinburgh

summary: We have developed a panel of isogenic iPSC lines carrying MAPT splice-site mutations S305S, S305I or S305N, derived from four different donors. All three mutations significantly increased the proportion of 4R tau expression in iPSC-neurons and astrocytes, with up to 80% 4R transcripts in S305N neurons from as early as 4 weeks of differentiation.

overall_design: We performed bulk RNA-sequencing on isogenic sets of MAPT S305 mutation iPSC-neurons at 4, 6 and 8 weeks of differentiation, as well as iPSC-astrocytes

first: Alison

middle: M

last: Goate


title: Single-cell analysis of L-MYC expressing neural stem cells and their extracellular vesicles revealed distinct progeneitor populations with nerogenic potential [scRNA-seq]

first: Yate-Ching

last: Yuan

summary: Neural stem cell (NSC)-based therapies can exploit the inherent pathotropism of NSCs to deliver neurotrophic factors for cell replacement therapies and to stimulate endogenous neurogenesis to permanently repair damaged brain tissue after brain injury. Despite studies on the protective/restorative functions of NSCs and their extracellular vesicles (EVs), a major impediment to advancing this approach to late-stage preclinical studies and clinical practice has been the lack of molecular characterization of NSCs and NSC-EV cargo composition. To fill this knowledge gap, we characterized therapeutically relevant human fetal NSC lines expressing the L-MYC gene (LMNSC01, LMNSC02) and their EVs using single-cell RNA sequencing (scRNAseq) and proteomic analyses. LMNSC01 cells differentiate into neurons and glial cells, including oligodendrocytes, and LMNSC02 mostly differentiate into neurons and oligodendrocyte progenitor cells (OPCs) under undirected differentiation in vitro. scRNAseq analysis indicates that LMNSC01 and LMNSC02 populations are homogenous, expressing not only NSC markers such as SOX2 and nestin, but also doublecortin (DCX), OPC marker O1, and astrocyte progenitor marker vimentin. LMNSC01 cells were enriched in biomarkers for astrocytes, excitatory neurons, glial cells, and OPCs, and LMNSC02 in inhibitory neurons, glial cells and OPCs. Using Single Extracellular VEsicle Nanoscopy, we quantified the size, shape, and content of tetraspanins (CD9, CD63, and CD81) for EVs secreted by cultured LMNSC01 and LMNSC02 cells. Both cell lines produced largely circular EVs; compared to EVs from LMNSC01 cells, those from LMNSC02 cells had on average higher detected tetraspanin content and larger diameter. We also quantified protein, RNA concentration, particle number, and batch-to batch variability for each EV. Our EV cargo global proteomics results suggest that LMNSC02 cells are enriched in processes involved in neuron development, neuron protection, synaptic transmission, and axon development, while EVs derived from LMNSC01 cells were more enriched in RNA processing. Finally, we identified neuroprotective properties of LMNSC-EVs in a brain organoid model of methotrexate-induced brain tissue toxicity. Our data suggest that LMNSC01 and LMNSC02 cell lines have distinct utilities, highlighting the need for molecular characterization to identify the cell lines and EVs with the highest therapeutic potential for various neurological conditions.

overall_design: Single-cell RNA Sequencing of LMNSC01 and LMNSC02 two cell lines. LMNSC01 and LMNSC02 human cells were dissociated from T75 cell, and cultured to process using Illumina’s NovaSeq 6000 for scRNAseq analysis.

first: Patrick

last: Pirrotte

first: Isabella

last: Vasquez

last: Jiang

first: Alejandra

last: Ojeda

first: Eric

last: Alsop

first: Nathaniel

last: Hansen

first: Ritin

last: Sharma

first: Meechoovet

last: Hunsar

first: Benjamin

last: Peton


title: Extrauterine support of preterm lambs achieves similar transcriptomic profiling to late pre-term lamb brains

city: Durham

first: Nicholas

last: O'Grady

state: NC

address: 2301 Erwin Rd

department: Department of Medicine, CRU

institute: Duke University

summary: Our group has developed an extra-uterine environment for newborn development (EXTEND) using an ovine model, that aims to mimic the womb to improve short and long-term health outcomes associated with prematurity. This study’s objective was to determine the histologic and transcriptomic consequences of EXTEND on the brain. Histology and RNA-sequencing was conducted on brain tissue from three cohorts of lambs: control pre-term (106-107 days), control late pre-term (127 days), and experimental lambs who were born pre-term and supported on EXTEND until late pre-term age (125-128 days). Bioinformatic analysis determined differential gene expression among the three cohorts and across four different brain tissue sections: basal ganglia, cerebellum, hippocampus, and motor cortex. There were no clinically relevant histological differences between the control late pre-term and EXTEND ovine brain tissues. RNA-sequencing demonstrated that there was greater differential gene expression between the control pre-term lambs and EXTEND lambs than between the control late pre-term lambs and EXTEND lambs. Our study demonstrates that the use of EXTEND to support pre-term lambs until they reach late pre-term gestational age results in brain tissue gene expression that more closely resembles that of the lambs who reached late pre-term gestation within their maternal sheep’s womb than that of the lambs who were born prematurely.

overall_design:
Our study investigated four brain sections per lamb from nine individual lambs. Two lambs were delivered pre-term and underwent immediate necropsy (Pre-term control); two lambs were delivered at 127 days and underwent immediate necropsy (Late Pre-term control); five lambs were delivered pre-term, underwent immediate cannulation and were supported by the EXTEND model (Experimental) and underwent necropsy after reaching a gestational age close to 127 days (gestational age similar to the Late Pre-term control cohort). The four brain sections investigated through both histology and RNA-sequencing were: hippocampus, motor cortex, basal ganglia, and cerebellum.

Flash frozen tissue was procured from ovine brain at the time of necropsy and sent to GeneWiz for RNA extraction and RNA-sequencing. Total RNA was extracted from frozen cell pellet and fresh frozen tissue samples using Qiagen RNAeasy Plus Universal mini kit following Manufacturer’s instructions (Qiagen, Hilden, Germany). GeneWiz conducted RNA-seq library preparation as follows: A) mRNA enrichment, mRNA fragmentation, and random priming; B) first and second strand cDNA synthesis; C) end repair, 5’ phosphorylation, and dA-Tailing; D) adaptor ligation, PCR enrichment, and sequencing.

Paired end sequences were assessed for quality using SeqKit.  Adapter and poly G/X tails were removed using fastp.  Post processing quality assessment is then repeated on the paired-end reads using SeqKit.  Sequences were aligned to the ovis aries genome, version 107, using STAR.  Reference files are available from Ensembl (https://ftp.ensembl.org/pub/release-107/fasta/ovis_aries/).  Gene expression was quantified against the gene transfer format (GTF), v107 annotation file using STAR’s ‘--quantMode GeneCounts’ parameter.  The reference GTF annotation file is also available from Ensembl (https://ftp.ensembl.org/pub/release-107/gtf/ovis_aries/). Sample-level quantifications from the resulting 'ReadsPerGene.out.tab’ file, column 2 for inward unstranded (IU) reads, were combined across all samples to generate the gene count matrix.

first: Jennifer

last: Cohen

first: Alan

last: Flake


title: Macrophage depletion in rat inflamed knees prevents the activation of synovial mesenchymal stem cells via weakened Nampt and Spp1 signaling

city: Bunkyo-ku

first: Kentaro

last: Endo

state: Tokyo

address: 1-5-45 Yushima

institute: Institute of Science Tokyo

summary: To analyze the crosstalk between synovial MSCs and macrophages, we conducted scRNA-seq for synovium.

overall_design: Synovial tisuues collected from inflamed rat knees treated with control liposomes or clodronate liposomes were subjected to scRNA-seq.

first: Hayato

last: Kodama


title: Pathogenic TSC2 variants alter developmental trajectories of human cortical organoids and perturb cell-cell communication networks [scRNA-seq]

first: Zhexing

last: Wen

overall_design: scRNA-seq

first: Weibo

last: Niu

first: Shaojun

last: Yu


title: Microinjection of human ESC-derived trophoblast cells(BAP cells) to mouse blastocysts

city: Taipa, Macau

first: Siyi

last: FU

state: Taipa, Macau

address: Room 2003,  N22 Building

department: FHS

country: Macau

institute: University of Macau

summary:
Purpose: 1. Characterize hESCs and hESC-derived trophoblast cells
Results: 1. hESCs have high level of most pluripotency markers; 2. hESC-derived trophoblast cells specifically express trophoblast markers.

overall_design: Examination mRNA profiles of human ESCs and ESC-derived trophoblasts

first: Borong

last: HUANG


title: Reprogramming of epidermal keratinocytes by PITX1 transforms the cutaneous cellular landscape and promotes wound healing.

city: Bethesda

first: Stephen

last: Brooks

state: MD

address: 50 SOUTH DR, RM 1140

department: Biodata Mining and Discovery Section

institute: NIAMS/NIH


title: NUSAP1 regulates Rheumatoid Arthritis Fibroblast-Like Synoviocyte Phenotypes Implicated in Joint Damage, and a cancer-associated transcriptomic signature.

city: New York

first: Teresina

last: Laragione

state: NY

address: 1468 Madison Ave

department: Medicine

institute: Icahn School of Medicine at Mount Sinai

summary: The fibroblast-like synoviocyte (FLS) has a central role in rheumatoid arthritis (RA) pathogenesis and its invasive behavior strongly correlates with disease severity and joint damage. Yet, the regulation of FLS invasiveness has been incompletely characterized. The nucleolar and spindle associated protein 1 (NUSAP1) gene has been implicated in cancer cell invasion and outcomes, and we considered that it might also be involved in the regulation of FLS invasiveness. siRNA knockdown of NUSAP1 significantly reduced RA FLS invasiveness (P=0.002) and migration in the scratch/wound healing assay (P=0.024). RNA sequencing analyses revealed that NUSAP1 knockdown significantly affected processes implicated in different types of cancer and in cancer biology, including cell cycle, DNA replication, transcription, RHO GTPase signaling, regulation of cytokinesis and cell metabolism pathways. Among the genes with the most significantly decreased expression in - NUSAP1-KO cells were ARHGAP11A, ANLN, PRC1 and RACGAP1, four genes previously implicated in cancer invasion and migration, and eighteen kinesin family genes, including KIF1c, known to regulate FLS invasion. We describe a new role for NUSAP1 in the regulation of the RA FLS invasiveness and migration, both relevant to disease pathogenesis and joint damage, and identify a new transcriptomic signature regulated by this gene. These findings raise the possibility that NUSAP1, or one of its target genes and pathways may become a new prognostic marker or target for new therapies for RA.

overall_design: siRNA was used to knockdown NUSAP1 in RA FLS, Cells were then studied in invasion, migration, proliferation and adhesion assays, and RNA was used for RNA sequencing and pathway analyses.

first: Carolyn

last: Harris

first: Percio

last: Gulko


title: Specific tRNAs promote mRNA decay by recruiting the CCR4-NOT complex to translating ribosomes

city: Dallas

first: Joshua

last: Mendell

state: TX

address: 6000 Harry Hines Blvd., NA6.200A

department: Molecular Biology

institute: UT Southwestern Medical Center


title: Two pathogenic germline truncating variants of BRCA2 confer different haploinsufficiency phenotypes

city: Paris

first: Elodie

last: Girard

address: 11-13 rue Pierre et Marie Curie

institute: Institut Curie

summary: Inactivating germline monoallelic mutations in the tumor suppressor BRCA2 predispose to breast and ovarian cancer. BRCA2 is essential in DNA repair via homologous recombination (HR). Tumorigenesis in this setting is thought to require the inactivation of the second allele; however, this event is not always observed suggesting haploinsufficiency. We investigated this question recreating two BRCA2 pathogenic truncating variants in heterozygosis in breast epithelial cells. One prompted sensitivity to PARP inhibitors (PARPi) and reduced HR capacity unlike the other. The other resulted in transcriptomic and epigenetic changes including histone H4 acetylation levels and NF-kB activation that we link to PCAF acetyl transferase. These changes impacted on cell migration and proliferation in 2D and 3D, processes that were also deregulated in tumors. Our work demonstrates that distinct truncating variants of BRCA2 are not phenotypically equivalent. It also identifies PCAF as shaping BRCA2-related gene expression. These findings may have implications in cancer prevention.

overall_design: RNA-seq experiments of 9 samples extracted from the MCF10A cell lines: 3 WT controls, 6 with a pathogenic BRCA2 truncating alteration- 3 with a c.999del5 (del5) and 3 with a c.5946delT  (delT).

first: Anna

last: Minello

first: Aura

last: Carreira


title: Remodeling of the Epigenetic Landscape in Rainbow Trout, Oncorhynchus mykiss, Offspring in Response to Maternal Choline Intake

city: Birmingham

first: Peggy

last: Biga

state: Alabama

address: 902 14th Street South

department: Biology

institute: University of Alabama at Birmingham

summary: The objective of this paper was to determine whether shifts in the methylome in rainbow trout (Oncorhynchus mykiss) are correlated with transcriptomic changes during early development in response to maternal dietary choline intake. Three experimental diets were formulated to have different levels of choline: (a) 2065 ppm choline (Low Choline, 0% supplementation), (b) 5657 ppm choline (Medium Choline, 0.6% supplementation), and (c) 9248 ppm choline (High Choline, 1.2% choline supplementation). Six rainbow trout families were fed experimental diets beginning 18 months post-hatch until spawning; their offspring were fed a commercial diet. Reduced representational bisulfite sequencing (RRBS) was utilized to measure genome-wide methylation differences in offspring immediately after hatching. When comparing to the Medium Choline offspring, differential DNA methylation occurred more in the Low Choline offspring than High Choline, especially in genic features like promoters. The differentially methylated CpGs (q ≤ 0.01) were identified evenly between CpG islands and shores in the genome, mostly found in the introns of genes. Genes such as fabp2 and leap2B associated with protein binding, fatty acid binding, DNA binding, and response to bacteria were differentially methylated and detected as differentially regulated genes by previous RNA-seq analysis. Although these findings indicate that levels of dietary choline available in broodstock diets alters offspring DNA methylation; most differentially expressed genes were not associated with differential DNA methylation, suggesting additional mechanisms playing a role in regulating gene expression in response to maternal choline intake.

overall_design:
To identify metabolic regulatory mechanisms affected by choline availability in rainbow trout broodstock diets associated with improved offspring growth performance, we fed one of three commercial diets formulated with different levels of choline: Low (2065 ppm choline), Medium (5657 ppm choline), and High (9248 ppm choline).
We then performed gene expression profiling analysis using data obtained from RNA-seq of 18 rainbow trout sac-fry at 0-days post-hatch (dph) and 14-dph through a main effect of diet and diet-by-broodstock family interaction.
Differential methylation analysis from reduced representational bisulfite sequencing (RRBS) was performed to investigate methylation levels concurrently with gene expression levels at 0-dph.

first: Khalid

last: Freij

first: Beth

last: Cleveland


title: Somatic Rescue and Cancer Mutations in Telomere Biology Disorders

city: Los Angeles

first: Feiyang

last: Ma

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

department: Molecular Biology Institute

institute: UCLA

summary: Clonal hematopoiesis (CH) in inherited bone marrow failure (BMF) is disease-specific but has been poorly characterized in telomere biology disorders (TBD).We studied the architecture, trajectories, and impact of CH in a cohort of 207 TBD patients and assessed the clinical relevance of molecular signatures linked to telomere dysfunction. Most patients (92%) had known germline mutations in TBD genes. CH was rare in asymptomatic but present in 46% of symptomatic patients, recurrently in PPM1D, POT1, TERT promoter (TERTp), and U2AF1. CH frequency increased with age and was significantly higher than in age- matched controls. CH in PPM1D/TERTp was enriched in TERT patients while CH in POT1 was enriched in TINF2 patients. CH in myelodysplastic syndromes (MDS)-related genes, most commonly splicing factors, was enriched in TERT/TERC patients. CH in TERTp, TP53 ̧ and MDS- related genes associated with poorer survival. Chromosome 1q (Chr1q) gain, and splicing factor gene (dominated by U2AF1S34/Q157R) or TP53 mutations increased the risk of MDS/acute myeloid leukemia (AML) development, regardless of allele burden. Trajectories with successive acquisition of MDS-related CH driven by U2AF1S34/Q157R were maladaptive, while adaptive CH involved branched POT1/PPM1D/TERTp trajectories. U2AF1S34/Q157R compensated aberrant TP53 and interferon-γ pathway activation that contribute to hematopoietic stem cell exhaustion in TBD.

overall_design: Single cell and bulk RNA-seq to study the impact of U2AF1-S34 mutation on HSPCs.

first: Natthakan

last: Thongon

first: Simona

last: Colla


title: Specific tRNAs promote mRNA decay by recruiting the CCR4-NOT complex to translating ribosomes (SLAM-Seq)

summary: The CCR4-NOT complex is a major regulator of eukaryotic messenger RNA (mRNA) stability. Slow decoding during translation promotes association of CCR4-NOT with ribosomes, accelerating mRNA degradation. We applied selective ribosome profiling to further investigate the determinants of CCR4-NOT recruitment to ribosomes in mammalian cells. This revealed that specific arginine codons in the P-site are strong signals for ribosomal recruitment of human CNOT3, a CCR4-NOT subunit. Cryo–electron microscopy and transfer RNA (tRNA) mutagenesis demonstrated that the D-arms of select arginine tRNAs interact with CNOT3 and promote its recruitment whereas other tRNA D-arms sterically clash with CNOT3. These effects link codon content to mRNA stability. Thus, in addition to their canonical decoding function, tRNAs directly engage regulatory complexes during translation, a mechanism we term P-site tRNA-mediated mRNA decay.

overall_design: To determine transcriptome-wide effect of CNOT3 depletion on mRNA stability, we depleted CNOT3 expression in HEK293T cells using CRISPR/Cas9, and then measured mRNA half-life in CNOT3-depleted cells and control cells using SLAM-Seq.

first: Xiaoqiang

last: Zhu

first: Victor

last: Cruz

first: He

last: Zhang

first: Jan

last: Erzberger

middle: T


title: Effect of Itape stripping in skin gene expression [TS_skin]

city: Boston

first: Manuel

last: Leyva-Castillo

state: MA

address: 300 Longwood Ave

institute: Boston Children's Hospital

summary: Mechanical skin injury promote an acute inflammation and promote the development of a dominated type 2 immune response. The mechanisms initiated by skin damage are not fully understood

overall_design: After shaving the back of Balb/c mice was left intact or tape stripped 6 times with the help of a tegaderm. After  6h skin biopsy were collected and samples were stored in RNAlater. Total skin RNA was isolated with RNeasy Mini Kit (Qiagen), followed by cDNA synthesis using the SuperScript VILO cDNA Synthesis Kit (ThermoFisher Scientific). The Ion AmpliSeq Transcriptome Mouse Gene Expression Kit was used to prepare bar-coded libraries and sequenced by using an Ion S5 next-generation sequencer. The AmpliSeqRNA plug-in (ThermoFisher Scientific) was used to calculate differential gene expression analysis

first: Juan

last: Leyva Castillo


city: ST LOUIS

first: SAPANA

middle: PRAKASHRAO

last: BANSOD

state: MO

address: 4482 LINDELL BLVD, 608

department: INTERNAL MEDICINE

institute: WASHINGTON UNIVERSITY, ST LOUIS

summary: We make Pa01c cells stable expressing a scramble or shRNA against TRIM21 and performed bulk scRNASeq to assess transcriptomic changes Pa01c cells

overall_design: Bulk RNAseq analysis of Pa01c cells stable expressing a scramble or shRNA against TRIM21.

first: Kian-Huat

last: Lim

first: Sapana

last: Bansod

first: Paarth

last: Dodhiawala


title: Skin gene expression


title: Effect of Ixodes ricinus bites in skin gene expression [skin]

summary: Tick bites often promote local allergic reactions in the skin. The mechanisms involved in this process is not fully understood. Here we investigated the local changes in the skin induced by Ixodes ricinus bites.

overall_design: Ixodes ricinus were collected on vegetation in forested areas. After shaving the back of C3H/HeN mouse, a plastic cap was fixed with wax to the mouse's back. Five female ticks were introduced into the cap which was sealed. For the control animals, plastic cap was just fixed to the back of the animal. After 4 days of blood meal, the cap was removed, and all ticks were collected. At day 4, 10 and 14 days, the skin at the site of the tick bite were removed. For the control animals, the cap was removed, and skin biopsy were collected as for the animals bitten by ticks. Samples were stored in RNAlater. Total skin RNA was isolated with RNeasy Mini Kit (Qiagen), followed by cDNA synthesis using the SuperScript VILO cDNA Synthesis Kit (ThermoFisher Scientific). The Ion AmpliSeq Transcriptome Mouse Gene Expression Kit was used to prepare bar-coded libraries and sequenced by using an Ion S5 next-generation sequencer. The AmpliSeqRNA plug-in (ThermoFisher Scientific) was used to calculate differential gene expression analysis


title: Decoding RNA metabolism by RNA-linked CRISPR screening in human cells

status: Public on Nov 20 2024

city: Seattle

first: Arvind

last: Subramaniam

state: WA

address: 1100 Fairview Ave N

department: Basic Sciences Division

institute: Fred Hutchinson Cancer Center

summary: RNAs undergo a complex choreography of metabolic processes that are regulated by thousands of RNA-associated proteins. Here we present a massively parallel RNA-linked CRISPR (ReLiC) platform to measure the responses of diverse RNA metabolic events to knockout of 2,092 human genes encoding all known RNA-associated proteins. ReLiC relies on a pooled, virus-free, serine recombinase-based strategy to integrate DNA libraries encoding Cas9, multiple sgRNAs, and barcoded reporters into a defined genomic locus. Combining ReLiC with polysome fractionation reveals distinct effects of perturbing translation and proteostasis machineries on ribosome occupancy. Isoform-specific ReLiC captures differential regulation of intron retention and exon skipping by SF3b complex subunits. Chemogenomic screens using ReLiC decipher translational regulators upstream of mRNA decay and uncover a role for the ribosome collision sensor GCN1 during treatment with the anti-leukemic drug homoharringtonine. Our work demonstrates ReLiC as a versatile platform for discovering and dissecting regulatory principles of human RNA metabolism.

overall_design: Two experimental groups. First group has CRISPR knockout of SF3B5, SF3B6, AQR, or FLUC(no-targeting). Second group has CRISPR knockout of GCN1 or FLUC, followed by harringtonine treatment for 6hrs (0hrs is no treatment). The first group does not have replicates. The second group has two replicates per sample and two different sgRNA pairs for GCN1 (1_2 and 3_4). All CRISPR knockouts use paired sgRNAs integrated at the AAVS1 locus and are induced through doxycline-mediated induction of Cas9. See manuscript for further details.

first: Patrick

last: Nugent

first: Heungwon

last: Park

first: Arvind Rasi


title: Zinc finger protein 184 prevents α-synuclein preformed fibril-mediated neurodegeneration through the interleukin enhancer binding factor 3-microRNA-7 pathway

city: Suwon

first: Jooho

last: shin

state: Gyeonggi-do, Republic of Korea

address: 2066 Seoburo

country: South Korea

institute: Sungkyunkwan University, School of Medicine

summary: Parkinson’s disease (PD) is a neurodegenerative disorder characterized by a loss of dopaminergic neurons. Recent studies suggested the association of zinc finger protein 184 (ZNF184) with PD. However, the functional role of ZNF184 in PD pathogenesis remains unclear. Therefore, we aimed to confirm this association and the effects of ZNF184 in a mouse model of PD and human patients with PD.We found that ZNF184 levels were decreased in the substantia nigra (SN) of α-synuclein preformed fibril (α-syn PFF)-injected mice and cells treated with PD toxins. Furthermore, ZNF184 was reduced in the cortex and SN of patients with PD, suggesting an association between ZNF184 and PD pathogenesis. In ZNF184-overexpressing cells, RNA-sequencing analysis revealed significant alterations in several protein-coding genes including interleukin enhancer binding factor 3 (ILF3). Bioinformatic analysis identified potential ZNF184 binding motifs within the ILF3 promoter, and ZNF184 occupancy was confirmed. Since ILF3 inhibits the biogenesis of microRNA-7 (miR-7), which regulates α-synuclein aggregation, we administered the miR-7 inducer, scutellarin to α-syn PFF-injected mice, preventing dopaminergic neuron and reinstating motor abilities. Our findings suggest that ZNF184 promotes miR-7 upregulation by suppressing ILF3 transcription, revealing a novel pathway that could serve as a promising therapeutic target for the treatment of PD.

overall_design: To identify putative target genes of ZNF184, Flag-tagged ZNF184 was transiently transfected into HEK293 cells. We then performed gene expression profiling by conducting RNA sequencing (RNA-seq) on two different cell types. The differentially expressed genes were analyzed to identify potential targets of ZNF184. To validate these results, we performed real-time quantitative PCR (qRT-PCR) on SH-SY5Y cells overexpressing GFP-tagged ZNF184.

first: Jihye

last: Kim

first: Soojeong

first: Jee-Ho

last: Lee

first: Ji-Yeong

first: Joo-Ho

last: Shin


title: Effect of Ixodes ricinus bites in skin gene expression [jejunum]

summary: Tick bites often predispose to meat allergy. The mechanisms involved in this process is not fully understood. Here we investigated the  changes in the gut induced by Ixodes ricinus bites.

overall_design: Ixodes ricinus were collected on vegetation in forested areas. After shaving the back of C3H/HeN mouse, a plastic cap was fixed with wax to the mouse's back. Five female ticks were introduced into the cap which was sealed. For the control animals, plastic cap was just fixed to the back of the animal. After 4 days of blood meal, the cap was removed, and all ticks were collected. At day 4, 10 and 14 days, the jejuna of bitten mice was collected. For the control animals, the cap was removed, and jejuna were collected as for the animals bitten by ticks. Samples were stored in RNAlater. Total skin RNA was isolated with RNeasy Mini Kit (Qiagen), followed by cDNA synthesis using the SuperScript VILO cDNA Synthesis Kit (ThermoFisher Scientific). The Ion AmpliSeq Transcriptome Mouse Gene Expression Kit was used to prepare bar-coded libraries and sequenced by using an Ion S5 next-generation sequencer. The AmpliSeqRNA plug-in (ThermoFisher Scientific) was used to calculate differential gene expression analysis


title: PROSER1 Modulates DNA Demethylation through Dual Mechanisms to Prevent Syndromic Developmental Malformations [RNA-seq]

city: Dundee

first: Kasper

middle: Dindler

last: Rasmussen

address: Dow Street

department: Division of Molecular, Cellular, and Developmental Biology (MCDB)

institute: School of Life Sciences, University of Dundee

summary: The link between DNA methylation and neurodevelopmental disorders is well established. However, how DNA methylation is fine-tuned – ensuring precise gene expression and developmental fidelity – remains poorly understood. PROSER1, a known TET2 interactor, was recently linked to a severe neurodevelopmental disorder. Here, we demonstrate that PROSER1 interacts with all TET enzymes and stabilizes chromatin-bound TET-OGT-PROSER1-DBHS (TOPD) complexes, which regulate DNA demethylation and developmental gene expression. Surprisingly, we find that PROSER1 also sequesters TET enzymes, preventing widespread demethylation and transposable element de-repression. Our findings identify PROSER1 as a key factor which both positively and negatively regulates DNA demethylation essential for mammalian neurodevelopment.

overall_design: Chromatin immunoprecipitation DNA sequencing (ChIP-seq) for endogenous PROSER1 in mESCs using PROSER1 KO as background control, and endogenous TET2 in wildtype and PROSER1 KO mESCs. ChIP-seq in wildtype and PROSER1 KO mESCs for the following histone modifications: H3K27ac, H3K4me1. Enzymatic methyl-seq (EM-seq) in wildtype, PROSER1 KO and PROSER1 KO+Rescue mESCs. RNA sequencing (RNA-seq) in wildtype, PROSER1 KO and PROSER1 KO+Rescue mESCs and wildtype, PROSER1 KO and PROSER1 KO+Rescue mESCs after 2 days differentiation in N2B27 medium ("day2" samples).

first: Anna

last: Fleming

first: Elena

middle: V

last: Knatko

first: Xiang

last: Li

first: Ansgar

last: Zoch

first: Zoe

last: Heckhausen

first: Stephanie

last: Stransky

first: Alejandro

middle: J

last: Brenes

first: Simone

last: Sidoli

first: Petra

last: Hajkova

first: Dónal

last: O'Carroll


title: Gene expression profiles in human bronchial epithelial cells exposure to cellulose nanofibrils and microcrystalline cellulose

city: Tsukuba

first: Katsuhide

last: Fujita

address: 16-1 Onogawa

department: Research Institute of Science for Safety and Sustainability

institute: National Institute of Advanced Industrial Science and Technology

summary: To evaluate the biological impact of cellulose nanofibrils (CNFs) at the transcriptional level, we conducted whole-genome microarray analyses on human bronchial epithelial cells (BEAS-2B) exposed to CNFs with different physicochemical properties. The results were compared with those from exposures to microcrystalline cellulose (MCC).

overall_design: Human bronchial epithelial cells (BEAS-2B) were exposed to eight types of cellulose nanofibrils (CNF1, CNF2, CNF3, CNF4, CNF5, CNF6, CNF7, and CNF8) at a concentration of 100 µg/mL, as well as MCC, for 48 hours. A vehicle control group (n=4) was also included. Cyanine-3-labeled cRNA generated from BEAS-2B cells exposed to CNFs and MCC was hybridized to a Whole Human Genome Microarray. Scanned images of the hybridized microarray slides were then analyzed numerically to assess the transcriptional-level biological effects of CNFs on BEAS-2B, in comparison with MCC exposures.

first: Sawae

last: Obara


title: Studies on the mechanisms underlying Angiopoietin-like 4-induced epidermal stem cell proliferation

city: Suzhou

first: Rui

middle: xing

last: Hou

state: Jiangsu

address: No.5, Tayun Road, Wuzhong District

department: Key Laboratory of Hand Surgery

institute: Suzhou Ruihua Orthopedic Hospital

summary: To elucidate the mechanism by which Angiopoietin-like 4 (Angptl4) exerts a pro-proliferative effect on epidermal stem cells (EpSCs), we conducted RNA-seq analysis of EpSCs derived from 8-week-old Angptl4 gene knockout (KO) mice and wild-type (WT) control mice to gain a global view of the transcriptome profile regulated by ANGPTL4.

overall_design: Analysis of differentially expressed genes of EpSCs from Angptl4 KO and WT mice by RNA-seq.

first: Ruixing

first: Siyuan

last: Yu