title: RNA-seq of a mouse primary pancreatic cancer cell line and liver metastasis from mouse model INK4.1syn_Luc

status: Public on Oct 31 2023

city: Rochester

first: Cameron

last: Baker

state: NY

address: 601 Elmwood Ave

country: USA

institute: University of Rochester

type: Expression profiling by high throughput sequencing

summary: The biology of metastatic pancreatic ductal adenocarcinoma (PDAC) is distinct from that of the primary tumor. We detected an upregulation of the secreted axon guidance molecule Netrin-1 (Ntn1) in human and mouse PDAC metastases that signals through its receptor, uncoordinated- 5b (Unc5b) to facilitate metastasis in vitro and in vivo. The mechanism of Ntn1 induction in liver metastasis is mediated by hepatic stellate cell (HSC) secreted retinoic acid through RXR/RAR and Elf3 signaling. Ntn1 is present in PDAC-derived extracellular vesicles (EVs) and facilitates metastasis by inducing HSC activation through both long and short distance intercellular communication. Importantly, administration of a neutralizing monoclonal NTN1 antibody decreased metastases and increased survival in several murine PDAC models (autochthonous and metastatic). Our studies reveal a new metabolic feed-forward loop for metastatic niche formation in the liver involving NTN1 in PDAC-secreted EVs and retinoic acid released from HSCs which is targetable using anti-NTN1 therapy.

overall_design: Ink4.1syn_Luc mice were followed for liver metastasis following orthotopic injection of the Ink4a.1 cell line into the pancreas.  Cell lines of the metastases from 4 different mice were developed in vitro.  After verification that the cell lines did originate from the primary tumor (Ink4a.1 cells), cells were grown to 90% confluency, including the parental Ink4a.1 cell line.  Cell pellets were submitted to Genewiz for RNAseq.  Analysis was completed in house.

first: Carpizo

last: Darren

first: Dudgeon

last: Crissy


title: Transcriptional analyses of neuron and glia specific knockdown of PIG-A in Drosophila melanogaster

city: Salt Lake CIty

first: Katie

middle: G

last: Owings

state: UT

address: 15 North 2030 East EIHG 5200

department: Human Genetics

institute: University of Utah

summary: We performed neuron and glia specific knockdown of PIG-A in Drosophila to understand the molecular defects that occur with loss of GPI anchored proteins. PIG-A encodes an enzyme responsible for the first step in GPI anchor biosynthesis. Loss of PIG-A in neurons and glia results in different neurological defects.

overall_design: PIGA was knocked down in the glia using REPO-GAL4 x RNAi (BDSC 62696) and PIGA was knocked down in the neuron using ELAV-GAL4 x RNAi (BDSC 62696). Whole fly heads were collected from 5-10 flies. RNAseq was used to compare the PIGA KD flies to their respective genetically matched control (AttP).

first: Clement

middle: Y

last: Chow


title: Effect of Tsc2 depletion on gene expression in isolated mouse lung cells.

city: Denver

first: Susan

middle: M

last: Majka

email: majkas@njhealth.org, susanmajka@mac.com

state: co

address: 1400 Jackson St, Goodman Bldg K810a

department: Medicine

institute: NJH

summary: To evaluate the impact of mTOR activation by Tsc2KD in MVPC on the microvascular endothelial cell population we utilized single cell transcriptomic analysis of fractionated lung tissue. Mouse lung samples were pooled and cell sorting was 2 days and 10 weeks post tamoxifen induction. Following annotation of the original 28 lung subclusters, capillary MVEC were localized to 7 subclusters. Temporal comparison of the MVEC populations demonstrated that in WT the population was transcriptionally stable from 2 days to 10 weeks postinduction, as would be expected during angiostasis. In contrast lung MVEC from the mice with mTOR+ Tsc2KD MVPC demonstrated significant increases in gene expression at 10 weeks relative to day 2 post-induction. Increased gene expression was related to mesenchymal stem cell differentiation, microvascular endothelial differentiation, mTOR activation, autophagy and apoptosis. These detailed complementary analyses identify mechanistic consequences of Tsc2KD and mTOR dysregulation in MVPC.

overall_design: To investigate how tsc2 knockdown in adult mouse lung ABCG2 positive microvascular progenitor cells we induced 2 (mixed background C57bl6/B129 strains) WILDTYPE or ABCG2 CREERT2 x floxed Tsc2 (Tsc2 knockdown) adult FEMALE mice 10-12 weeks of age with a single low dose of tamoxifen (0.5mg/18g) and isolated cells 2 days or 10 weeks later. Lungs were digested to form a single cell suspension and stained with CD45 antibody. Cells were sorted from WT and Tsc2KD strains for analysis as follows: 2 day cd45negative GFP negative, CD45 negative GFP positive and CD45 positive; 10 weeks cd45negative GFP negative, CD45 negative GFP positive .

first: Swapna

last: Menon

first: Camille

last: Moore


title: RNA-seq of plko-scramble or shBicd2 plasmid hydrodynamic-injected mice livers under ConA-induced acute autoimmune hepatitis

city: Beijing

first: Wenyu

last: Tian

address: No. 38 Xueyuan Road, Haidian District

country: China

institute: Peking University

summary: To explore the effect of Bicd2 in ConA-induced acute autoimmune hepatitis, we conducted RNA transcriptome profiling of plko-scramble or shBicd2 plasmid hydrodynamic-injected  mice livers in response to ConA injection.

overall_design: We delivered plko-scramble or shBicd2 plasmid by hydrodynamic injection to 8w wild-type C57BL/6J mice. After 3 days, mice were injected with 15mg/kg ConA i.v and 6 hours later livers were collected to perform RNA sequecing.

first: Wenyan

last: Hao

first: Xin

last: Zhang

first: Dan

last: Lv


title: ZFHX3 acts as a tumor suppressor in prostate cancer by targeting FTO-mediated m6A demethylation

city: jinan

first: qingxia

last: hu

address: qingdao street 6699

institute: Shandong First Medical University & Shandong Academy of Medical Sciences

summary: Zinc-finger homeobox 3 (ZFHX3, also known as ATBF1) suppresses prostatic tumorigenesis. ZFHX3 is frequently found to have numerous deletions in human prostate cancer (PCa). However, the underlying molecular function of ZFHX3 during prostatic tumorigenesis is not well understood. N6-methyladenosine (m6A) modification in RNA plays a critical role in the development of cancers; however, the relationship between ZFHX3 and m6A modification is largely unknown in PCa. In this study, we found that ZFHX3 knockdown decreased total m6A levels through enhancing the transcriptional activity of FTO in PCa cells. Importantly, FTO inhibition suppressed cell proliferation and rescued the promoting function of ZFHX3 knockdown on cell proliferation. In vivo, we verified that FTO was upregulated and ZFHX3 was decreased in PCa patients and that a high level of ZFHX3 is indispensable for low FTO expression and is correlated with better patient survival. Through transcriptome sequencing and Me-RIP sequencing, we revealed that E2F2 and CDKN2C were the direct targets of FTO-mediated m6A modification and ZFXH3 was required for the regulation of FTO on E2F2 and CDKN2C expression. Unexpectedly, we uncovered that ZFHX3 expression was in return regulated by FTO in a m6A-dependent way. These findings establish a novel crosstalk mechanism between ZFHX3 and FTO in prostatic tumorigenesis.

overall_design: Examination of m6A modification levels in control cells and FTO-knockdown cells

first: Qingxia

last: Hu


title: Effect of long-term education with EVs from women with obesity on MCF7 gene expression

city: New York

first: Genomics-core-facility

last: WCM

state: New York

address: 1300 York Ave

institute: Weill Cornell Medicine

summary: Breast adipose tissue is an important contributor to the obesity-breast cancer link.  Dysregulated cell metabolism is now an accepted hallmark of cancer. Extracellular vesicles (EVs) are nanosized particles containing selective cargo, such as miRNAs, that act locally or circulate to distant sites to modulate target cell functions. Here, we found that long-term education of breast cancer cells (MCF7, T47D) with EVs obtained from breast adipose tissue of women who are overweight or obese (O-EVs) leads to sustained increased proliferative potential. RNA-Seq of O-EV-educated MCF7 cells demonstrates increased expression of genes, such as ATP synthase and NADH: ubiquinone oxidoreductase, involved in oxidative phosphorylation.

overall_design: MCF7 cells were seeded at 40,000 cells/well in a 6-well plate, and treated with media containing 1mg/mL L-EVs, O-EVs or vehicle control (PBS; same volume) on day 1, 4 and 7. The culture media was refreshed with each EV treatment. On day 8, cells were harvested for further experiments. Cells were lysed using Qiazol lysis reagent (Qiagen, #79306), and total RNA was extracted from samples by Trizol lysis and RNeasy Mini Kit (Qiagen, #74106) following the manufacturer’s protocols. Samples were submitted for RNA-seq at the Genomics Resources Core Facility (GRCF, Weill Cornell Medicine). Sequencing libraries were constructed at the GRCF following the Illumina TruSeq Stranded mRNA library preparation protocol (Poly-A selection and Stranded RNA-Seq). The libraries were sequenced with single read 50 bp on the Illumina NovaSeq6000 sequencing platform. All reads were independently aligned with STAR_2.4.0f1 (Dobin et al, 2013) for sequence alignment against the human genome sequence build hg19, downloaded via the UCSC genome browser and SAMTOOLS v0.1.19 (Li et al, 2009) for sorting and indexing reads. Cufflinks (2.0.2) (Trapnell et al, 2012) was used to estimate the expression values (FPKMS), and GENCODE v19 (Derrien et al, 2012) GTF file for annotation.

first: Alberto

last: Benito-Martin

first: Shuchen

last: Liu

first: Kristy

middle: A

last: Brown


title: Integrative analysis of transcriptome and metabolome provide new insights into mechanisms of Capilliposide A against cisplatin-induced nephrotoxicity

city: Hangzhou

first: Jiaxi

last: Fang

state: zhejiang

address: 浙江省杭州市钱塘区下沙街道中科院基础医学与肿瘤研究所

institute: Zhejiang Provincial People's Hospital

summary: Objective: Cisplatin (CDDP) has been widely used for chemotherapy against tumours. However，the nephrotoxicity has limited its clinical use. Here, we reported a novel compound, Capilliposide A (CPS-A), to exhibit therapeutic effects on CDDP-induced acute kidney injury （AKI）and explored its potential mechanisms via transcriptome and metabolome.                                                                                Materials and methods: HK-2 cells were treated with CPS-A, after which cell viability, apoptosis and inflammation were investigated. A mouse model of AKI was constructed by single injection of CDDP in vivo. The renal function and morphology and mitochondrial function were assessed by pathological section and transmission electron microscope (TEM). Transcriptomics and metabolomics are used to explore possible mechanisms which was later verified in vitro. Results: CPS-A administration improved the survival rates of HK-2 cells with a significant decrease in the expression of KIM-1, NGAL, IL-6, IL-8 and IL-1β. In vivo results also suggested that CPS-A attenuates CDDP-induced kidney injury by reducing serum creatinine (Cr) and blood urea nitrogen (BUN) levels. Furthermore, TEM also showed the improvement of mitochondrial ultrastructure both in vivo and vitro. Transcriptomics analysis of the mice's renal cortex indicated the expression of ATF4 and CHOP were upregulated, which was further validated by qPCR and Western blotting in vitro. Integrative analysis of transcriptome and metabolome indicated that L-Leucine enriched in Valine, leucine and isoleucine degradation might be potential targets. Conclusions: CPS-A can effectively regulate endogenous metabolites associated with amino acid metabolism and ameliorate apoptosis and oxidative stress in CDDP-induced AKI by reducing endoplasmic reticulum stress.

overall_design: To explore possible mechanism of CPS-A against cisplatin-induced acute kidney injury，Transcriptomics of mouse‘s kidney is conducted.

first: Luping

last: Wang

first: Di

first: Wei

last: Zhu


title: comrades-OO: An Object-Oriented R Package for Comprehensive Analysis of RNA Structure Generated using RNA crosslinking experiments

city: cambridge

first: Jonathan

middle: Lewis

last: Price

address: The Sanger Building Tennis Court Road

department: Biochemistry

country: United Kingdom

institute: University of Cambridge

summary: RNA molecules have secondary and tertiary structures in vivo which play a crucial role in various cellular processes such as the regulation of gene expression, RNA processing, and localization. Investigating these structures will enhance our understanding of their functions and contribute to the diagnosis and treatment of diseases caused by RNA dysregulation. However, there are no mature pipelines or packages for processing and analysing complex in vivo RNA structural data. Here, we present COMRADES Object-Oriented (comrades-OO), a novel software package for the comprehensive analysis of data derived from the COMRADES experiment. comrades-OO offers a comprehensive pipeline from raw sequencing reads to the identification of RNA structural features. It includes read processing and alignment, clustering of duplexes, data exploration, folding and comparisons of RNA structures. COMRADES-OO also enables comparisons between conditions, the identification of inter-RNA interactions, and the incorporation of reactivity data to improve structure prediction.

overall_design: We recently developed Crosslinking Of Matched RNAs And Deep Sequencing (COMRADES) for in-depth RNA conformation capture in living cells (Ziv et al., 2018). COMRADES can detect base-paired regions in RNA inside the cell,  using a clickable psoralen derivative to specifically crosslink double-stranded RNA, and high throughput sequencing to retrieve the base-pairing information Following in vivo crosslinking, the viral RNA is selectively captured, fragmented and subjected to a click-chemistry reaction to add a biotin tag to crosslinked regions. Crosslinked RNA duplexes are then selectively captured using streptavidin affinity purification. Half of the resulting RNA is proximity ligated, following reversal of the crosslink to create chimeric RNA templates for high throughput sequencing. The other half is used as a control, in which reversal of the crosslink precedes the proximity ligation, and accurately represents the background level of non-specific ligation.COMRADES can therefore measure (i) the structural diversity of alternative RNA conformations that coexist inside cells; (ii) short-distance, as well as long-distance (over tens of thousands of nucleotides) base-pairing within the same RNA molecule; and (iii) base-pairing between different RNA molecules, such as those of host and viral origin (Kudla et al., 2020; Ziv et al., 2018).

first: Eric

last: Miska


title: Functional and molecular profiling of hematopoietic stem cells during regeneration

city: Lund

first: Maroulio

last: Pertesi

address: Sölvegatan 19

country: Sweden

institute: Lund University

summary: Hematopoietic stem cells (HSCs) enable hematopoietic stem cell transplantation (HCT) through their ability to replenish the entire blood system. Proliferation of HSCs is linked to decreased reconstitution potential, and a precise regulation of actively dividing HSCs is thus essential to ensure long-term functionality. This regulation becomes important in the transplantation setting where HSCs undergo proliferation followed by a gradual transition to quiescence and homeostasis. While mouse HSCs have been well studied during homeostatic conditions, the mechanisms regulating HSC activation during stress remain unclear. Here, we analyzed the different phases of regeneration following transplantation.  We isolated bone marrow from mice at eight time points after transplantation and examined the reconstitution dynamics and transcriptional profiles of stem and progenitor populations. We found that regenerating HSCs initially produced rapidly expanding progenitors and displayed distinct changes in fatty acid metabolism and glycolysis. Moreover, we observed molecular changes in cell cycle, MYC and mTOR signaling in both HSCs and the progenitor subsets. We used a decay rate model to fit the temporal transcription profiles of regenerating HSCs and identified genes with progressively decreased or increased expression after transplantation. These genes overlapped to a large extent with published gene sets associated with key aspects of HSC function demonstrating the potential of this data set as a resource for identification of novel HSC regulators. Taken together, our study provides a detailed functional and molecular characterization of HSCs at different phases of regeneration and identifies a gene set associated with the transition from proliferation to quiescence.

overall_design: RNAseq of steady state HSCs and HSCs isolated 3, 4, 5, 6, 8, 10, 13, and 16 weeks after transplantation.

first: Anna

last: Rydström

first: Tan

middle: H

last: Grahn

first: Abhishek

last: Niroula

first: Els

last: Mansell

first: Mark

last: van der Garde

first: Agatheeswaran

last: Subramaniam

first: Shamit

last: Soneji

first: Roman

last: Zubarev

first: Tariq

last: Enver

first: Björn

last: Nilsson

first: Kenichi

last: Miharada

first: Jonas

last: Larsson

first: Stefan

last: Karlsson


title: Vascularized immunocompetent patient-derived tumor model to predict treatment response for lung cancer patients

city: Illkirch-Graffenstaden

first: Jules

last: Deforges

address: Boulevard Gonthier d'Andernach

country: France

institute: Transgene SA

summary: This work describes a patient-derived tumoroid model (PDTs) to support precision medicine in lung oncology. The use of human adipose tissue-derived microvasculature and patient-derived PBMCs permits to achieve a physiologically relevant tumor-microenvironment in this 3D tumor model. This study involved ten patients at various stages of tumor progression. The vascularized, immune-infiltrated PDT model could be obtained within two weeks, matching the requirements of the therapeutic decision-making process. Histological and transcriptomic analyses confirmed that the main features from the original tumor were reproduced. The 3D tumor model could be used to determine the dynamics of response to antiangiogenic therapy and platinum-based chemotherapy. Antiangiogenic therapy showed a significant decrease in vascular endothelial growth factor (VEGF)-A expression, reflecting its therapeutic effect in the model. In an immune-infiltrated PDT model, chemotherapy showed the ability to decrease the levels of lymphocyte activation gene-3 protein (LAG-3), B and T lymphocyte attenuator (BTLA) and inhibitory receptors of T cells functions.

overall_design: Patient biopsies of tumors and healthy tissues compared to patient derived organoïds / tumoroïds

first: Helene

last: Le

first: Jean-Marc

last: Balloul


title: A plasma miRNA-based classifier for small cell lung cancer diagnosis

city: Richmond

first: Michela

last: Saviana

state: Virginia

address: 1101 E Marshall St

department: Department of Internal Medicine, Division of Pulmonary Diseases and Critical Care Medicine

institute: Virginia Commonwealth University

type: Non-coding RNA profiling by high throughput sequencing

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

overall_design: Refer to individual Series


title: Next Generation Sequencing of Sorted pWER::GFP Marker Line Enables Quantitative Analysis of Arabidopsis Epidermal Root Transcriptomes in Iron Deficient Conditions

city: Raleigh

first: Terri

last: Long

state: NC

department: Plant & Microbial Biology

institute: North Carolina State University

summary:
Purpose: Identify novel genes and relationships of a temporal transcriptional landscape in Arabidopsis epidermal root cells under iron deficiency stress
Results: We identified 1,709 novel iron responsive genes which would have been missed had we identified DEGs with respect to the 0hr time point using the whole root or epidermis data.
Conclusion: Gene set comparisons allowed us to identify organ-level response versus cell-specific and to parse out developmental from iron deficiency-induced response. We identified several new iron deficiency-induced and repressed TFs and implemented a novel sampling method to generate an epidermis specific GRN that incorporates gene-level expression while maintaining computational efficiency. Using cis-elements in the promoters of DEGs, we were also able to find plausible regulators that would not have been found by exclusively looking at differentially expressed genes. The new transcriptional data provides clarity to our current understanding of root responses to -Fe triggered by the first layer of “defense,” the root epidermis.

overall_design: RNA-Seq was performed on protoplast collected from GFP sorted epidermis marker line (pWER::GFP; WEREWOLF; AT5G14750; Lee & Schiefelbein, 1999). Arabidopsis roots were collected on the 7th day of growth on +Fe media, equating to 0hr. After 7days, seedlings were transfer to +Fe and -Fe media, an additional 6hr, 12hr, 18hr, 24hr, 30hr, and 36hr of growth occurred. After which, roots were harvested from both +Fe and -Fe conditions for protoplast collection, followed by fluorescence-activated cell sorting in triplicates. RNA was extracted from FACS epidermal protoplast using RNAeasy Plant RNA Purification Kit (Qiagen). cDNA synthesis and amplification were performed using the SMARTer Low Input RNA Kit and sequenced using an Illumina HiSeq 2500 sequencing machine, with 125bp single end reads. Adapters and low quality reads were filtered out using fastQC and fastq-mcf. Clean reads were mapped against the TAIR 10 reference genome using the tophat2. RPKM were acquired using RSubread, followed by edgeR to identify differentially expressed genes (DEGs), using a maximum false discovery rate (FDR) of 0.05 and minimum log fold change threshold of 0.75.

first: DurreShahwar

last: Muhammad


title: Gene expression profile at the single cell level of the trachea of Drosophila larvae

city: beijing

first: xinyu

last: She

address: shuangqing road, haidian district

department: school of life sciences

institute: Tsinghua university

summary: We performed a single-cell analysis of the larval tracheal system of Drosophila melanogaster and identified a total of 17 distinct cell populations that are part of or closely associated with the trachea.

overall_design: L3 larvae were dissected by ventral filleting and afterward, isolated trachea. Dissection refers to the protocol Feng performed (10.21769/BioProtoc.1797)

first: Judith

last: Bossen

first: Xiao

last: Niu

first: Xinyu

first: Benedikt

last: Hofbauer

first: Lasse

last: Tiedemann

first: Sören

last: Franzenburg

first: Iris

last: Bruchhaus

first: Susanne

last: Krauss-Etschmann

first: Christian

last: Wegener

first: Thomas

last: Roeder


title: Immunomodulatory Potential of Dapphitkhai and Chanthaleela Thai Traditional Recipes in the Context of COVID-19 Pathogenesis

city: Khantharawichai

first: Nuchsupha

last: Sunthamala

state: Mahasarakham

address: Department of Biology, Faculty of Science, Mahasarakham University

department: Biology

country: Thailand

summary: This study aimed to investigate the effects of two Thai traditional recipes including Dapphitkhai and Chanthaleela on various immune parameters and signaling pathways in human white blood cells. The gene expression of whole blood stimulation was analyzed through RNA-sequencing and pathway analysis. Chanthaleela and Dapphitkhai demonstrated a significant decrease in IL-8 secretion and an increase in IFN-β secretion, suggesting their potential to modulate these signaling pathways. Gene expression analysis of Chanthaleela and Dapphitkhai at 500 µg/mL revealed distinct patterns and identified differentially expressed genes. Gene ontology analysis highlighted the impact of the recipes on immune responses and cytokine regulation, while pathway analysis identified key pathways related to cytokine-cytokine receptor interaction, chemokine signaling, and infection-related processes. This study provides valuable insights into the effects of Thai traditional recipes on immune parameters and signaling pathways in human white blood cells. The findings suggest potential therapeutic applications of Thai traditional medicine in immune-related disorders and viral infections like COVID-19.

overall_design: This study aimed to investigate the effects of two Thai traditional recipes including Dapphitkhai and Chanthaleela on various immune parameters and signaling pathways in human white blood cells. Whole blood stimulation will be employed to analyze gene expression through RNA-sequencing and pathway analysis, thereby unraveling the underlying mechanisms of antiviral and anti-inflammatory immune stimulation exerted by these Thai traditional recipes.

first: Somsak

last: Nualkaew


title: Human Skin CD141+ Dendritic Cells Regulate Cutaneous Immunity via the Neuropeptide Urocortin-2

city: London

first: Chrysanthi

last: Ainali

address: Strand, London WC2R 2LS, UK

institute: King's College London

summary: Skin immune homeostasis is a multi-faceted process where dermal dendritic cells (DDCs) are key in orchestrating responses to environmental stressors. We have previously identified CD141+CD14+DDCs as a skin-resident immunoregulatory population that is vitamin-D3(VitD3) inducible from monocyte-derived DCs(moDCs), termed CD141hiVitD3 moDCs. We demonstrate CD141+DDCs and CD141hiVitD3 moDCs share key immunological features including cell surface markers, reduced T-cell stimulation, IL-10 production, and a common transcriptomic signature. Bioinformatic analysis identified the neuroactive ligand receptor pathway and the neuropeptide, urocortin-2(UCN2), as a potential immunoregulatory candidate molecule. Incubation with VitD3 up-regulated UCN2 in CD141+DCs and ultraviolet-B(UVB) irradiation induced UCN2 in CD141+DCs in healthy skin in vivo. Notably, CD141+DDC generation of suppressive Tregs was dependent upon the UCN2 pathway as in vivo administration of UCN2 reversed skin inflammation in humanized mice. We propose the neuropeptide UCN2 as a novel skin DC- derived immunoregulatory mediator with a potential role in UVB and VitD3 dependent skin immune homeostasis.

overall_design:
Total RNA was obtained from 15 human dendritic cell samples in total; 5 different cell populations with 3 replicates of each.

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first: Niwa

last: Ali


title: Identification of novel target genes for Nrf2 in Mytilus coruscus.

city: Zhoushan

first: Longmei

last: Qiu

state: Zhejiang Province

address: 1 Haida South Road, Dinghai District, Zhoushan City, Zhejiang Province

institute: Zhejiang Ocean University

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Nrf2 plays a pivotal role in regulating oxidative stress, and in mollusks, understanding how Nrf2 governs the expression of new target genes is crucial in the context of oxidative stress. In this study, we have identified solute carrier family 35 member E2 (SLC35E2) as a novel Nrf2 target gene that counteracts the oxidative stress response induced by Bap.Initially, we confirmed the Nrf2-SLC35E2 interaction through dual luciferase and qRT-PCR assays, demonstrating that Nrf2 both targeted and inhibited SLC35E2 expression. Subsequently, we further substantiated Nrf2's involvement in BAP-induced oxidative stress by assessing enzyme activity.Therefore, our study provides valuable insights that can contribute to future research on the environmental adaptation and stress responses of mollusks, signifying its significant importance.

overall_design:
To investigate novel target genes regulated by Nrf2 and their involvement in oxidative stress responses, we conducted chromatin immunoprecipitation sequencing in Mytilus coruscus.
Please note that Nrf2 antibody was custom-prepared by Hangzhou Hua'an Biotechnology Co., Ltd and the purified antibody bears the project number TP30282.

first: Pengzhi

last: Qi


title: Proline synthetase PYCR1 acts as a metabolic reprogrammer dictating breast cancer stemness under chronic stress

city: Dalian

first: jinxin

last: lu

state: Liaoning

address: lvshunkou district lvshun south goad west sector No.9

institute: Dalian Medical University

summary: Aberrant proline metabolism contributes to cancer progression, yet the underlying mechanism of proline metabolic disorders in regulating cancer stem-like cells (CSCs) remains unclear. Here, we find that proline synthetase PYCR1 is critical for breast cancer stemness and tumor growth through synthesizing proline. To explore the downstream targets responsible for the increase in proline-mediated stem-like traits in breast cancer, we performed RNA-seq based transcriptome analysis of PYCR1-knockdown (shP1-1 and shP1-2) MDA-MB-231 cells versus control (shNC) MDA-MB-231 cells. Among the differentially expressed genes (DEGs) identified in shP1-1 and shP1-2 cells, 361 genes and 214 genes were downregulated, respectively. We then performed KEGG pathways enrichment analysis in downregulated genes and found that the cGMP-PKG signaling pathway was among the top 1 enriched pathways in both shP1-1 and shP1-2 groups.

overall_design: RNA-seq data of the MDB-MA-231 cells in Ctrl, shP1, shP2 group.

first: Bai

last: Cui

first: Bin

last: He

first: Yanping

last: Huang

first: Quentin

last: Liu


title: Gene expression profile at single cell level of human T cells stimulated via antibodies against the T Cell Receptor (TCR)

city: Cambridge

first: Jonathan

last: Hsu

state: MA

address: 840 Memorial Dr 4th Floor

institute: Marengo Therapeutics

summary: We used single cell RNA sequencing (scRNAseq) to compare gene expression in human T cells following stimulation with antibodies directed against a CD3 subunit (CD3ε) versus the TCRβ chain variable (V) domain

overall_design: Purified human T cells from six healthy donors were stimulated with antibodies in vitro for 7 days and analyzed using scRNAseq

first: Pierre

last: Vantourout

first: Josephine

last: Eum

first: Maria

last: Conde Poole

middle: S

last: Hayday

first: Adam

last: Laing

first: Khiyam

last: Hussain

first: Rosamund

last: Nuamah

first: Shichna

last: Kannambath

first: Jacques

last: Moisan

first: Allart

last: Stoop

first: Sebastiano

last: Battaglia

first: Roya

last: Servattalab

first: Andrew

last: Bayliffe

first: Madan

last: Katragadda

first: Adrian

middle: C


title: RNA-seq analysis of RIP of hnRNPQ and PABPN1 from HeLa cells in nutrient-deprived media

city: Tel Aviv

first: Metsada

last: Pasmanik-Chor

address: Ramat Aviv

country: Israel

institute: Tel Aviv University

summary: RNA-seq analysis was applied in RNA immunoprecipitation (RIP) of three independent repeats (hnRNPQ), and two independent repeats (PABPN1) from HeLa cells starved in nutrient-deprived media.

overall_design: Transcriptome analysis of RNA bound to hnRNPQ (3 biological replicates) and PABPN1 (2 biological replicates) pulldown with specific antibodies from cell lysates of HeLa cells treated with starvation media (HBSS, 4h). As a control (3 biological replicates), an unrelated IgG antibody was used in the pulldown experiments.

first: Avraham

last: Ashkenazi


title: Effect of Prmt5 deletion on Dermo1 gene expression in lung mesenchymal cells of C57 mice at E18.5.

city: Hefei

first: LU

last: Qingshuang

address: 111 Kowloon Road

department: Institute of Physical Science and Information Technology

institute: Anhui University

summary: To investigate the role of Prmt5 gene in lung development, we established a Prmt5 specific knockout model in Dermo1 of C57 mouse lung mesenchymal cells using cre-loxp system. Then, the data obtained from RNA-seq in E18.5-day lung tissues were used for gene expression profiling analysis.

overall_design: E18.5 mesenchymal Dermo1 specific knockout of Prmt5 in lung tissues and the data obtained in RNA-seq of wild-type E18.5 lung group were analyzed for gene expression profile.

first: Qiuling

last: Li

first: Qingshuang

last: Lu


title: Transcriptome analysis reveals the mechanism of action of tobacco NtAN3 in regulating leaf size formation

city: Guiyang

first: Renxiang

last: Liu

state: Guizhou

address: Huaxi

institute: Guizhou University

summary: Tobacco (Nicotiana tabacum L.) is an important cash crop, and the size of its leaves significantly influences both yield and quality. However, the upper part of tobacco leaves, due to its dense tissue structure, often faces issues such as narrow and thick leaves during the production of roasted cigarettes. These problems have a severe impact on the yield and quality of the upper leaf. Although the mechanism of leaf size regulation in Arabidopsis thaliana has been extensively studied, it remains unclear for tobacco. Therefore, this research aimed to investigate the role of the NtAN3 gene in regulating tobacco leaf size by utilizing the NC82 variety. The researchers created both an overexpression mutant (G27) and a silencing mutant (M21) of the NtAN3 gene and examined their impact on leaf size using cell morphology observation and transcriptome analysis. These research findings offer valuable insights for molecular breeding aimed at improving tobacco yield and enhancing the availability of upper leaves.

overall_design: 9 samples were analyzed, including 3 plant materials, each with three replicates.

first: Shuxian

last: Yang


title: Seminal extracellular vesicle sncRNA profiles reveal miRNAs/isomiRs as predictive biomarkers of testicular sperm retrieval outcome in azoospermic patients

city: Badalona

first: Lauro

last: Sumoy

state: Barcelona

address: Ctra. Can Ruti, Camí de les escoles s/n

department: High Content Genomics and Bioinformatics

country: Spain

institute: IGTP

summary: Non-invasive molecular biomarkers for differential diagnosis of the origin of azoospermia into obstructive azoospermia (OA) or secretory azoospermia (SA), as well as for prediction of the presence of residual spermatogenesis in testicular tissue of patients presenting with SA, are of great interest for TESE outcome prediction in assisted reproduction. Our previous study of several hundreds of miRNAs suggested the use of some sEV-miRNAs from seminal plasma as biomarkers for the origin of azoospermia. In this regard, studying more in depth sncRNAs in EVs from semen of azoospermic individuals will be useful to select additional non-invasive biomarkers with diagnostic/prognostic purposes which could help in the identification of those individuals with real chances of positive sperm recovery on the testicular biopsy prior to assisted reproduction technology (ART) treatment. A high-throughput sncRNA profiling analysis using paired-end small RNA-seq was performed in seminal sEVs from azoospermia and normozoospermic individuals.

overall_design: A case and control prospective study was performed. In this pilot study we aimed at extensively characterizing seminal sEV sncRNA profiles of azoospermic individuals with different testicular sperm recovery outcome. For that, we have profiled and quantified the expression of annotated sncRNAs by paired-end small RNA-seq and determined the expression pattern of not only miRNAs (analyzed at the isomiR level), but also piRNAs, and tsRNAs within the human seminal sEVs in a small number of normozoospermic (n=4) and azoospermic [OA-N n=4; SA(Sp+) n=5; SA(Sp-) n=4] individuals. RT-qPCR validation analysis of selected miRNAs was additionally performed in a larger number of individuals.

first: Sara

last: Larriba

first: Jose-Francisco

last: Sánchez-Herrero

first: Raquel

last: Pluvinet

first: Olga

last: López-Rodrigo

first: Lluís

last: Bassas


title: A plasma miRNA-based classifier for small cell lung cancer diagnosis [NanoString]

first: Michela

last: Saviana

summary: Objectives: Small cell lung cancer (SCLC) is characterized by poor prognosis and challenging diagnosis. Screening in high-risk smokers results in a reduction in lung cancer mortality, however, screening efforts are primarily focused on non-small cell lung cancer (NSCLC). SCLC diagnosis and surveillance remain significant challenges. The aberrant expression of circulating microRNAs (miRNAs/miRs) is reported in many tumors and can provide insights into the pathogenesis of tumor development and progression. Here, we conducted a comprehensive assessment of circulating miRNAs in SCLC with a goal of developing a miRNA-based biomarker classifier to assist in SCLC diagnoses.  Materials and Methods: We profiled deregulated circulating cell-free miRNA in the plasma of SCLC patients. We tested selected miRs on a training cohort and created a classifier by integrating miRNA expression and patient clinical data. Finally, we applied the classifier on a validation dataset.   Results: We determined that miR-375-3p can discriminate between SCLC and NSCLC patients, and between SCLC and Squamous Cell Carcinoma patients.  Moreover, we found that a model comprising miR-375-3p, miR-320b, and miR-144-3p can be integrated with race and age to distinguish metastatic SCLC from a control group.   Conclusion: This study proposes a miRNA-based biomarker classifier for SCLC that considers clinical demographics with specific cut offs to inform SCLC diagnosis.

overall_design: A total of 24 plasma samples were used for Extracellular Vesicles (EV) isolation. The cohort comprises 8 high-risk smokers that were used as control samples, 4 adenocarcinomas, 4 squamous cell carcinomas, and 8 SCLC patients. The RNA included in EV was purified and Nanostring was performed.

first: Mario

last: Acunzo

first: Patrick

last: Nana-Sinkam


title: Gene expression in Drosophila melanogaster in response to bacterial infection

first: Marc

last: Dionne

address: Level 0, Flowers Building

department: Life Sciences

institute: Imperial College London

summary: The goal of this study was to generate a high-quality dataset of gene expression in Drosophila melanogaster 3 hours after infection with Gram-positive (M luteus) or Gram-negative (E coli) bacteria.

overall_design: RNAseq on polyA-selected RNA extracted from whole male adult flies either uninjected or 3 hours after injection with 50nl of either: PBS (vehicle control), E coli suspended in PBS (OD600=1), M luteus suspended in PBS (OD600=1). Flies were kept at 29° after injection on our standard lab diet (10% yeast, 8% fructose, 2% polenta, 0.8% agar, supplemented with nipagin and propionic acid).


title: Comprehensive multi-omics analysis of microbiome, metabolome, and host genome in right- and left-sided colon cancer

city: shanghai

first: Kong

last: Cheng

address: 270 Dong 'an Road

institute: fudan university shanghai cancer center

summary: There are distinct interactions among microbial compositions, metabolites, and host genomes in RCC and LCC as revealed by multi-omics analysis, which brings a new dimension to understanding the role of gut microbiota in tumorigenesis of RCC and LCC.

overall_design: RNA-seq was performed using CRC tissues

first: Yanlei

last: Ma

first: Lei

last: Liang

first: Cheng

last: Kong


title: Effect of treprostinil on rabbit lungs with congenital diaphragmatic hernia

city: Leuven

first: Felix

last: De Bie

address: Herestraat 49

department: Development and Regeneration

country: Belgium

institute: KU Leuven

summary: To investigate the effect of treprostinil pulmonary development in a rabbit model of congenital diaphragmatic hernia.

overall_design: We administered treprostinil on day 24 of gestation (term 31 days) to rabbits with surgically created congenital diaphragmatic hernia and compared it to unexposed rabbits with congenital diaphragmatic hernia

middle: R

first: Marius

last: Regin

first: Francesca

last: Russo

first: Jan

last: Deprest


title: Androgen receptor requires an optimal level of condensation to assemble androgen-induced active enhancers [GRO-Seq]

city: San Antonio

first: Zhijie

middle: Jason

last: Liu

state: TEXAS

address: 7703 Floyd Curl Drive

department: Department of Molecular Medicine

institute: Universality of Texas Health Science Center at San Antonio

summary: Transcription factors (TFs) activate enhancers to drive cell-specific gene expression in response to signals, but our understanding of enhancer assembly in signaling events is incomplete. Here we show that Androgen Receptor (AR), a steroid hormone signaling-regulated transcription factor, forms phase-separated condensates in response to androgen to activate enhancers. We demonstrate that the intrinsically disordered NTD of AR drives condensate formation and that NTD deletion or aromatic residue mutation reduces AR self-association and abolishes AR transcriptional activity. AR NTD can be substituted by some IDRs from other proteins for AR condensation capacity and transactivation function. Extending the polyQ tract within AR NTD strengthens AR phase separation and also leads to impaired transcriptional activity. PolyQ expansion does not affect AR binding on enhancers, but instead impairs enhancer assembly. These results suggest that AR phase separation mediates enhancer assembly, and an optimal level of AR condensation is required for its proper function in mediating AR-AR homotypic and AR-cofactor heterotypic interactions to regulate transcription in response to signals. Our study supports that alteration of the fine-tuned protein condensation might underlie AR-related human pathologies, therefore providing novel molecular insights for potential therapeutic strategies to treat prostate cancer and other AR-involved diseases by targeting AR condensation.

overall_design: Global Run On (GRO) assay followed by high throughput sequencing (GRO-seq) for checking gene expression patterns in LNCaP cells with different treatments.

first: Zhang

last: Zhao

first: Liu

last: Zhijie


title: G-quadruplex-binding molecule based on napththalene diimides showing potent activity on pancreatic cancer cells

first: Stephen

last: Neidle

address: Brunswick Square

department: School of Pharmacy

institute: UCL

summary: Targeting quadruplexes in cancer genes and pathways by a set of napththalene diimides-based compounds in models for pancreatic cancer.

overall_design: Differential gene expression between treated and untreated MIA PaCa-2 cells after 6 h and 24 h treatment with a novel class of napththalene diimides (SOP1247), which were evaluated with RNA-seq.

first: Ahmed

last: Ahmed


title: Primary breast epithelial cells from healthy donor versus its TONSL overexpressing counterpart-enriched gene expression

city: Indianapolis

first: Harikrishna

last: Nakshatri

state: IN

address: 980 W. Walnut St. R3C218C

department: General Surgery

institute: Indiana University School of Medicine

summary: To evaluate the probable role of TONSL during tumorigenesis and to study the TONSL specific transcriptomic changes, we overexpressed TONSL  in primary bresat epithelial cells and studied differential gene expression

overall_design: Total RNA from KTB103 (Specific donor identifier) primary and TONSL overexpressing KTB103 cells in triplicates were used for RNA-sequencing.

first: H


title: Tunable Hydrogel Viscoelasticity Modulates Human Neural Maturation

city: Stanford

first: Sarah

middle: Christine

last: Heilshorn

state: CA

address: 476 Lomita Mall

department: Materials Science and Engineering

institute: Stanford University

summary: Human induced pluripotent stem cells (hiPSCs) have emerged as a promising in vitro model system for studying neurodevelopment. However, current models remain limited in their ability to incorporate tunable biochemical and biomechanical signaling cues imparted by the neural extracellular matrix (ECM). The native brain ECM is viscoelastic and stress-relaxing, exhibiting a time-dependent response to an applied force. To recapitulate the remodelability of the neural ECM, we developed a family of protein-engineered hydrogels crosslinked with either static or dynamic covalent bonds that exhibit tunable stress relaxation rates. hiPSC-derived neural progenitor cells (NPCs) encapsulated within these gels underwent relaxation rate-dependent maturation. Specifically, NPCs within hydrogels with faster stress relaxation rates extended longer, more complex neuritic projections, exhibited decreased metabolic activity, and expressed higher levels of genes associated with neural maturation. By inhibiting actin polymerization, we observed decreased neuritic projections and a concomitant decrease in the expression of neural maturation genes. Taken together, these results suggest that microenvironmental viscoelasticity is sufficient to bias human NPC maturation.

overall_design: To explore the effects of matrix viscoelasticity on hiPSC differentiation and maturation, we encapsulated hiPSC-derived NPCs in protein engineered matrices with three distinct stress relaxation profiles and collected cells at cells after seven days.

first: Julien

last: Roth

first: Michelle

last: Huang


title: DNA methylation analysis in the gastric tissue collected from health check-up cohort.

city: Chiba

first: Masaki

last: Fukuyo

address: 1-8-1 Inohana, Chuo-ku

department: Department of Molecular Oncology

country: Japan

institute: Chiba University

summary: Comprehensive DNA methylation analysis using the clinical FFPE samples of the gastric cancer, gastric mucosa surrounding cancer, and biopsy gastric mucosa.

overall_design: Bisulphite converted DNA from the 164 samples were hybridised to the Infinium MethylationEPIC BeadChip kit.

first: Genki

last: Usui

first: Atsushi

last: Kaneda


title: Gene expression differences between wild-type and Vhl mutant mice in kidney stones process

city: BEIJING

first: HAO

last: wenyan

department: School of Basic Medical Sciences

institute: Peking University Health Science Center

summary: To investigate the transcriptome differences between wild-type and Vhl mutant mice in kidney stones process, we established CaOx-induced nephrolithiasis mouse model and performed RNA-sequencing.

overall_design: We then performed gene expression profiling analysis using data obtained from RNA-seq of wild-type and Vhl mutant mice injected glyoxylate.

first: Wenyan

last: Hao

first: Dan

last: Lu


title: Gene expression profiles of Nfil3-deficient basophil progenitors from mouse bone marrow

city: Daejeon

first: Jiyeon

last: Park

address: Daehak-ro 291

country: South Korea

institute: KAIST

summary: To find genes regulated by Nfil3 during development or functional maturation, we analyzed the gene expression profiles of basophil progenitors by RNA-sequencing.

overall_design: pre-Basos and basophils of littermate control or Nfil3-deficient mice were sorted from bone marrow and subject to whole transcriptome analysis. For each cell types, biological duplicates were analyzed.

first: Yuri

last: Cho

first: Hanseul

last: Yang

first: Suk-Jo

last: Kang


title: Evaluating the utility of proteomics for the identification of circulating pharmacodynamic biomarkers of IFNβ-1 biologics

city: Silver Spring

first: Paula

middle: L

last: Hyland

state: MD

address: 10903 New Hampshire Avenue

department: CDER

institute: FDA

summary: In this study, we evaluated the utility of proteomics to identify plasma proteins in healthy participants from a phase I clinical trial with IFNβ-1a and pegIFNβ-1a biologics to identify potential pharmacodynamic (PD) biomarkers. Using a linear mixed-effects model with repeated measurement for product-time interaction, we found that 248 and 528 analytes detected by the SOMAscan® assay were differentially expressed (p-value < 6.86E-06) between therapeutic doses of IFNβ-1a or pegIFNβ-1a, and placebo, respectively. We further prioritized signals based on peak change, area under the effect curve over the study duration, and overlap in signals from the two products. Analysis of prioritized datasets indicated activation of IFNB1 signaling and an IFNB signaling node with IL-6 as upstream regulators of the plasma protein patterns from both products. Increased TNF, IL-1B, IFNG, and IFNA signaling also occurred early in response to each product suggesting a direct link between each product and these upstream regulators.  In summary, we identified longitudinal global PD changes in a large array of new and previously reported circulating proteins in healthy participants treated with IFNβ-1a and pegIFNβ-1a that may help identify novel single proteomic PD biomarkers and/or composite PD biomarker signatures as well as provide insight into the mechanism of action of these products. Independent replication is needed to confirm present proteomic results and to support further investigation of the identified candidate PD biomarkers for biosimilar product development.

overall_design:
The study includes data on >7000 proteins from 12 subjects treated with IFNβ-1a, 12 with pegIFNβ-1a and 12 with placebo. The 12 subjects on IFNβ-1a were tested at 10 timepoints over 6 days, and the rest were tested at 12 time points over 13 days. Replicates for 4 random samples were included on the array. Two covid covalacent plasma samples were also included on the array.
Submitter states: Raw data is normalized using specific plasma standards by the manufacturer. The raw data can not be used as is.


title: RNA-seq of STZ-treated mouse retinal microglia cells

status: Public on Oct 30 2023

city: Saint Louis

first: Charles

middle: W.

last: Pfeifer

state: Missouri

address: 660 S. Euclid Ave., Campus Box 8096

department: Ophthalmology and Visual Sciences

institute: Washington University School of Medicine in St. Louis

summary: We observe morphological changes of retinal microglia during early stages of diabetic retinopathy in STZ-treated mice. Therefore, in this experiment we assess the impact of STZ-induced diabetic retinopathy on retinal microglia transcriptome during early stages of disease.

overall_design: We performed comparative gene expression profiling analysis of RNA-Seq data from flow-sorted mouse retinal microglia from two diabetic time points (4wk STZ, 8wk STZ) and littermate controls.

middle: W

first: Rajendra

last: Apte


title: RNA-seq of BV2 cultured microglia following high glucose and CD200Fc treatments

summary: We observe dyseregulated CD200-CD200R signaling in early diabetes that correlates with microglia-mediated neuroretinopathy and found that CD200 fusion protein (CD200Fc), an agonist of CD200R, attenuated high glucose-induced inflammation in cultured microglia. Therefore, we performed RNA-Sequencing on cultured BV2 microglia following exposure to normal or high glucose media (NG, HG) with or without CD200Fc supplementation (50ng/mL) to investigate broader biologic implications.

overall_design: We performed comparative gene expression profiling analysis of RNA-Seq data from BV2 microglia RNA samples following treatment with normal or high glucose media (NG, HG) that is also supplemented with or without CD200Fc (50ng/mL)


title: Gene expression differences in mouse kidneys after CaOx-induced nephrolithiasis

summary: To investigate the transcriptome differences in kidney stones process, we established CaOx-induced nephrolithiasis mouse model and performed RNA-sequencing.

overall_design: We then performed gene expression profiling analysis using data obtained from RNA-seq of mice injected with saline and glyoxylate.

last: HAO


title: Androgen receptor requires an optimal level of condensation to assemble androgen-induced active enhancers [ChIP-Seq]

first: Zhijie

last: Liu

overall_design: Chromatin Immunoprecipitation (ChIP) assay followed by high throughput sequencing (ChIP-seq) for checking the genome-wide binding patterns for AR WT, dIDR mutant, and pQ69 mutant.

first: Zhang

last: Zhao

first: Liu

last: Zhijie


title: Disruption of tuftelin 1, a desmosome associated protein,causesskin fragility, woolly hair and palmoplantar keratoderma

city: Groningen

first: Marieke

middle: C.

last: Bolling

address: Hanzeplein 1

department: Department of Dermatology

country: Netherlands

institute: University of Groningen, University Medical Centre Groningen

summary: Desmosomes are dynamic complex protein structures involved in cellular adhesion. Disruption of these structures by loss of function variants in desmosomal genes lead to a variety of skin and heart related phenotypes. Here, we report tuftelin 1 as a desmosome-associated protein, implicated in epidermal integrity. In two siblings with mild skin fragility, woolly hair and mild palmoplantar keratoderma, but without a cardiac phenotype, we identified a homozygous splice site variant in the TUFT1 gene, leading to aberrant mRNA splicing and loss of tuftelin 1 protein. Patients’ skin and keratinocytes showed acantholysis, perinuclear retraction of intermediate filaments, and reduced mechanical stress resistance. Immunolabeling and transfection studies showed that tuftelin 1 is positioned within the desmosome and its location dependent on the presence of the desmoplakin carboxy-terminal tail. A Tuft1 knock-out mouse model mimicked the patients’ phenotypes. Altogether, this study reveals tuftelin 1 as a desmosome-associated protein, whose absence causes skin fragility, woolly hair and palmoplantar keratoderma.

overall_design: Total RNA was isolated from 4 wildtype and 3 healthy-looking TUFT1 knock out mouse skin sections and RNA seq was performed on these samples. Differential expression analysis was determined on knockouts over controls, and FDR<0.05 differentially expressed genes were extracted.

first: Peter

last: van der Meer

first: Annemieke

last: Verkerk


title: Single-Cell RNA Sequencing of Mouse Fibroblasts Grown as Spheroids Reveals Presence of Mesenchymal Stem-Like Cells

city: Ottawa

first: Theodore

last: Perkins

state: ON

address: 501 Smyth Rd.

department: Cellular and Molecular Medicine

country: Canada

institute: Ottawa Hospital Research Institute

summary: Compared to conventional monolayer cell culture, three-dimensional (or spheroid) cultures are more reflective of the in vivo environment and represent a better means of representing normal tissue. Therefore, understanding the biology of spheroid-derived cells is important for a more complete appreciation of in vivo tissue function. Although it has been shown that culture conditions such as nutrients, oxygen, cell passage etc. alter gene expression patterns of the cells, the gene expression profile of spheroid as compared to conventional monolayer culture in primary cells has not been studied. To investigate the effects of spheroid culture system on gene expression of primary cells, single cell RNA sequencing (scRNAseq) was performed on mouse dermal fibroblasts (tail/ear fibroblasts, TEFs) cultured as monolayers on cell culture dishes or grown as spheroids. After quality control, a total of 11,515 cells (8,022 spheroid cells and 3,493 cells of monolayer fibroblasts) were analyzed using Uniform Manifold Approximation and Projection (UMAP) which identified 2 largely separated libraries. These libraries contained cells with 2,491 and 8722 detected genes (nFeature_RNA) for monolayer and spheroid cells respectively. UMAP clustering of the integrated datasets identified 8 significant cell clusters (resolution=0.3) in which clusters 0, 1, 2, 4 and 7 had a higher number of cells in the spheroid library. Differential gene expression analyses revealed that each cluster was characterized by a specific transcriptional profile. Among cell clusters, cluster 4 exhibited significantly increased expression of collagen family genes, including Col1a2, Col3a1, Col1a1, Col5a2, Col4a, Col4a2, Col5a3, Col5a1, Col6a1, Col12a1 and Col15a1. Furthermore, expression of mesenchymal stem cell genes [Sca-1 (Ly6a), CD29 (Itgb1), CD44, CD90.1 (Thy1)] as well as a group of genes important for stem cell self-renewal including Notch2, Sox4, Sox9 Klf2, and Foxp1 were significantly increased in cluster 4. Immunofluorescence (IF) staining was performed to validate the protein expression of selected genes. Our work provides a significant advance in characterizing the global gene expression profile of spheroid culture of mouse TEFs compared to monolayer culture. We reached a high resolution at single-cell level, which enabled us to identify specific clusters associated with mesenchymal stem-like cells.

overall_design:
To investigate the effects of spheroid culture system on gene expression of primary cells, single cell RNA sequencing (scRNAseq) was performed on mouse dermal fibroblasts (tail/ear fibroblasts, TEFs) cultured as monolayers on cell culture dishes or grown as spheroids.
To establish tail/ear fibroblasts (TEFs), the tails and ears from adult mice (8-12 week-old C57BL/6 or FVB) were peeled, minced into 1 cm pieces, placed on culture dishes, and incubated in DMEM/F-12 containing 1% Penicillin and Streptomycin and 20% heat inactivated fetal bovine serum for 7 days. Cells that migrated out of the graft pieces were transferred to new plates and maintained in DMEM/F-12 supplemented with 10% FBS (passage 2). TEFs from passage 3 were used for all experiments.
For monalayer, cells were plated on pretreated cell culture plate using DMEM/F-12 supplemented with mouse or human EGF, mbFGF and mIGF-1 (all 20 ng/ml). For the suspension culture, cells were plated at 100-300 cells per well in ULA 96 well round-bottom plates (Corning® Costar® Ultra-Low Attachment Multiple Well Plate, Cat#7007) in 200 µl DMEM/F-12 supplemented with mouse or human EGF, mbFGF and mIGF-1 (all 20 ng/ml) plus 10 µM ROCKi. Cells were maintained in 5% CO2, 21% O2 and 74% N2 at 37oC for 7 days. Half of the culture medium was removed and replaced every other day.
We then performed single cell RNA sequencing (scRNAseq) on mouse dermal fibroblasts cultured as monolayers on cell culture dishes or grown as spheroids. Between 70,000-100,000 mouse adherence-cultured fibroblasts (monolayer) and dissociated spheroid cells (single cells) were freshly prepared and their gene expression profile analyzed using single-cell 3' RNA-sequencing.


title: Profiling of Polyadenylated RNAs after Brg1/Smarca4 knock-down in murine bone marrow-derived macrophages after LPS or LPS plus Dex stimulation [RNA-seq]

city: Freising

first: Franziska

last: Greulich

address: Gregor-Mendel-Str. 2

department: Metabolic Programming

country: Germany

institute: TU München

summary: We performed Brg1 (Smarac4) knock-down by siRNA in murine primary bone marrow-derived macrophages and profiled the effect on gene expression by RNAseq after lipopolysaccharide (LPS) or LPS plus dexamethasone (Dex) treatment. Brg1 knock-down cells are compared to cells treated with a control siRNA. We analysed the requirement of Brg1/Smarca4 for the expression of inflammatory and glucocorticoid receptor target genes.

overall_design: We performed gene silencing with small interfering RNA (siRNA) against Brg1 in murine bone marrrow-derived macrophages and compare dthose cells to cell streated with a control siRNA (ctrl). Cells were treated with 50nM sicontrol or siBrg1 for 48 hours and in the last 6 hours with vehicle (0.1% EtoH, 0.1%D-PBS), LPS (100ng/ml LPS and 0.1% EtoH) or Dex+LPS (100ng/ml LPS and 1µM Dex). Three replicates were sequenced per condition and treatment.

first: Aikaterini

last: Mechtidou

first: Nina

last: Uhlenhaut


title: Expression of mammary epithelial anoikis microRNAs and target genes

city: Worcester

first: Junhui

last: Li

address: 364 Plantation St

institute: University of Massachusetts Medical School

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

overall_design: Refer to individual Series


title: HO-1 modulates ferroptosis via ROS-mediated mitochondrial dysfunction in cyclophosphamide -induced ovarian granulosa cells

city: Guangzhou

first: HUI

last: CHEN

state: Guangdong

address: Guangzhou Huangpu Road

department: medicine

institute: Jinan University School of Medicine

summary: Abnormal granulosa cells (GCs) death contributes to cyclophosphamide (CTX) -induced primary ovarian insufficiency (POI). To investigate the contribution of GCs in POI, gene profiles of GCs exposed to CTX were assessed using RNA-Seq and bioinformatics analysis. The results showed the differentially expressed genes (DEGs) are enriched in ferroptosis-related pathway, which is correlated with the upregulated Heme oxygenase 1 (HO-1) and downregulated glutathione peroxidase-4 (GPX4). Using CTX-induced cell culture (COV434 and KGN cells), the levels of iron, reactive oxygen species (ROS), lipid peroxide, mitochondrial superoxide, mitochondrial morphology and mitochondrial membrane potential (MMP) were detected by DCFDA, MitoSOX, C11-BODIPY, MitoTracker, Nonylacridine Orange (NAO), JC-1 and transmission electron microscopy respectively. The results showed that iron overload and disrupting ROS, including cytoROS, mtROS and lipROS homeostasis by HO-1 upregultion could induce ferroptosis via mitochondrial dysfunction in CTX-induced GCs. Moreover, HO-1 inhibition could suppress ferroptosis induced GPX4 depletion. This implies the role of ROS in CTX-induced ferroptosis and highlighted the effect of HO-1 modulators of improving CTX-induced ovarian damage, which may provide a theoretical basis for preventing or restoring GC and ovarian function in patients with POI.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for normal granulosa cells (GCs) and granulosa cells by using cyclophosphamide (CTX)-induced

first: Ping

first: Yongqi

last: Wu

first: Hui

last: Chen

last: Nie


title: Exhausted CD8+ T cells overexpress CCL5 during combination therapy to mediate resistance of hepatoma cells to lenvatinib: one situation may contribute to the survival of residual tumor cells

city: Tianjin

first: Yafei

last: Wang

address: No.94 Weijin Road, Nankai District

department: School of Medicine

institute: Nankai University

summary: In conclusion, we discovered that during combination therapy, tumor tissue demonstrated a more active metabolic reprogramming than normal liver tissue, as indicated by a significant upregulation of CYP1A1 expression. In addition, we validated that CD8T cells secreted more CCL5 after treatment, which upregulated CYP1A1 expression by binding to CCR5, leading to resistance of hepatoma cells to lenvatinib. Finally, we demonstrated that interference with the CCL5/CCR5/CYP1A1 pathway can enhance the efficacy of lenvatinib and combination therapy.

overall_design: The Huh7 cells were divided into experimental and control groups. Three replicates were set for each group of samples.The experimental group was stimulated with 100ng/ml of CCL5, while the control group was stimulated with an equal volume of ddH2O. After 24 hours of stimulation, the Huh7 cells were washed with PBS and lysed using TRIzol for RNA extraction to perform RNAseq.

first: Guankun

last: Mao

first: Chonghui

first: Shichun

last: Lu


title: Identification of miR-203a target genes [RNA-seq]

summary: To identify targets of miR-203a we coupled RNA-seq and AGO2-RNA immunoprecipitation (RIP-seq) in MCF10A cells expressing a control pre-miR-203a or control, empty vector

overall_design: Total RNA was isolated from stably transduced MCF10A cells expressing either pre-miR-203a or empty vector control and analyzed using RNAseq.  Downstream analysis was compared to RIP-seq dataset.

first: Tessa

last: Simone

first: Michael

last: Green


title: Model-driven design of synthetic N-terminal coding sequences for fine-tuning gene expression in yeast and bacteria

city: Wuxi

first: Yanfeng

last: Liu

state: Jiangsu

address: No.1800, Lihu Avenue

institute: Jiangnan University

summary: N-terminal coding sequences (NCS) are key regulatory elements for fine-tuning gene expression during translation initiation, the rate-limiting step of translation. However, due to complex combinatory effects of NCS biophysical factors and endogenous regulation, designing NCS remains challenging. Herein, we implemented multi-view learning strategy for model-driven generation of synthetic NCS for Saccharomyces cerevisiae and Bacillus subtilis, which are model microorganisms widely used in the laboratory and industry.

sample_organism:
Bacillus subtilis
Saccharomyces cerevisiae

overall_design: NCS expression level was measured using a “FACS-seq” reporter-based assay. Libraries of yeast and bacteria cells harboring a plasmid in which GFP expression was driven by Pbs, and randomly-generated NCS were inserted after the start codon (ATG) of GFP. Cells of each strain were FACS-sorted into high and low bins on the basis of expression. Specifically, NCS libraries for S. cerevisiae and B. subtilis has nearly 150,000 cells that were sorted, respectively.

first: Chenyun

first: Wei

last: Zhang

first: Rongzhen

last: Tian

first: Jianing

first: Linpei

first: Zhaohong

last: Deng

first: Long

first: Guocheng

last: Du


title: Metabolic reprogramming of cancer cells by JMJD6-mediated alternative splicing

city: Memphis

first: JUN

last: YANG

state: TN

address: 262 Danny Thomas Place

department: Surgery

institute: St Jude Children's Research Hospital

summary: Chromosome 17q gain occurs frequently in MYC-driven tumors including high-risk neuroblastoma. However, the biological consequences of this genetic event remain elusive. Here we show that JMJD6, frequently amplified at the chromosome 17q25 locus, is one of the essential genes to neuroblastoma cells that are engaged in pathways of mitochondrial metabolism, RNA processing and protein homeostasis. JMJD6 cooperates with MYC in cellular transformation and promotes cancer cell proliferation and tumor growth. Mechanistically, JMJD6 physically interacts with RNA-processing machinery to regulate the alternative splicing and protein synthesis. Notably, JMJD6 controls the alternative splicing of glutaminase (GLS), kidney-type glutaminase (KGA) and glutaminase C (GAC), a rate-limiting enzyme of glutaminolysis, and, consequently, the central carbon metabolism in neuroblastoma. Our findings indicate that JMJD6 coordinates with MYC in tumorigenesis by regulating cancer-promoting metabolic programs through an alternative pre-mRNA splicing mechanism.

overall_design: Examination of gene expression profile changes with JMJD6 siRNA knockdown.

first: Jun

last: Yang

first: David

last: Finkelstein


title: Basal forebrain cholinergic neurons are vulnerable in a mouse model of Down syndrome and their molecular fingerprint is rescued by maternal choline supplementation

city: Orangeburg

first: Stephen

middle: D.

last: Ginsberg

state: New York

address: 140 Old Orangeburg Road

department: Centre for Dementia Research

institute: Research Foundation for Mental Hygiene

summary: Basal forebrain cholinergic neuron (BFCN) degeneration is a hallmark of Down syndrome (DS) and Alzheimer's disease (AD). Current therapeutics in these disorders have been unsuccessful in slowing disease progression, likely due to poorly understood complex pathological interactions and dysregulated pathways. The Ts65Dn trisomic mouse model recapitulates both cognitive and morphological deficits of DS and AD, including BFCN degeneration and has shown lifelong behavioral changes due to maternal choline supplementation (MCS). To test the impact of MCS on trisomic BFCN, we performed laser capture microdissection to individually isolate choline acetyltransferase-immunopositive neurons in Ts65Dn and disomic littermates, in conjunction with MCS at the onset of BFCN degeneration. We utilized single population RNA sequencing (RNA-seq) to interrogate transcriptomic changes within medial septal nucleus (MSN) BFCN. Leveraging multiple bioinformatic analysis programs on differentially expressed genes (DEGs) by genotype and diet, we identified key canonical pathways and altered physiological functions within Ts65Dn MSN BFCN, which were attenuated by MCS in trisomic offspring, including the cholinergic, glutamatergic and GABAergic pathways. We linked differential gene expression bioinformatically to multiple neurological functions, including motor dysfunction/movement disorder, early onset neurological disease, ataxia and cognitive impairment via Ingenuity Pathway Analysis. DEGs within these identified pathways may underlie aberrant behavior in the DS mice, with MCS attenuating the underlying gene expression changes. We propose MCS ameliorates aberrant BFCN gene expression within the septohippocampal circuit of trisomic mice through normalization of principally the cholinergic, glutamatergic, and GABAergic signaling pathways, resulting in attenuation of underlying neurological disease functions.

overall_design:
Pups born of Ts65Dn dams were given choline normal (1.1 g/kg; control diet) or choline supplemented (5.0 g/kg; treatment diet) chow during gestation through weaning to investigate the lifelong gene expression changes in basal forbrain cholinergic neurons due to maternal choline supplementation (MCS) in a mouse model of Down syndrome.
After weaning, pups were aged on control diet to ~6MO for analysis.  Fresh frozen brains were isolated, sectioned and immonhistochemical anlaysis was done to identify basal forebrain cholinergic neurons (BFCNs), with a rapid detection method to preserve RNA.  BFCNs were isolated by laser capture and RNA-seq analysis was performed on single population BFCNs  (~500 neurons per sample) to determine lifelong gene expression changes in the DS and control animals due to MCS treatment. Cohort were n=6 male mice per genotype and treatment (24 total). Comparisons for RNA-seq analysis was done based on genotype and treatment.

first: Melissa

middle: J

last: Alldred

first: Harshitha

last: Pidikiti

first: Adriana

last: Heguy

first: Panos

last: Roussos

middle: D


title: Identification of gene expression signatures and related microRNAs for chronic glomerulonephritis based on microarray and bioinformatic analysis

city: Hefei

first: Xianjin

last: Xu

address: Changning Street

institute: Hefei Ion Medical Center, the First Affiliated Hospital of USTC

summary: To further reveal the key genes and related microRNAs involved in the CGN and to determine the potential molecular mechanisms of CGN pathogenesis. We have employed microarray expression profiling as a discovery platform to identify genes and related microRNAs. To identify the key ceRNA in CGN, we generated a ceRNA network using data from the National Center for Biotechnology Information Gene Expression Omnibus (NCBI GEO). 2334 differentially regulated genes and a total of 673 related miRNAs were identified. We found that the ceRNA network was composed of 31 lncRNAs, 344 mRNAs, and 57 miRNAs. Expression of five genes (FOS, SYK, CYP1A1, HSD3B6, UGT2B15), five microRNAs (miR-34a-5p, miR-214-3p,  miR-155-5p,  miR-182,  miR-592), three lncRNAs (LOC102547703, NONRATT006779, FQ220365) were quantified in the same RNA samples by real-time PCR, which could be potential diagnostic biomarkers and therapeutic targets for CGN.

overall_design: Glomerular tissues from normal, experimental and TCM-treated rats were analyzed based on Agilent Rat 4 × 44 K whole genome microarray, the accumulation of high-throughput gene expression profile data has greatly promoted the application of genome microarray in functional gene studies. RNA quantity and quality were measured by NanoDrop ND-1000. RNA integrity was assessed by standard denaturing agarose gel electrophoresis. Sample labeling and array hybridization were performed according to the Agilent One-Color Microarray-Based Gene Expression Analysis protocol (Agilent Technology). Briefly, total RNA from each sample was linearly amplified and labeled with Cy3-UTP. The labeled cRNAs were purified by RNeasy Mini Kit (Qiagen). The concentration and specific activity of the labeled cRNAs (pmol Cy3/μg cRNA) were measured by NanoDrop ND-1000. 1 μg of each labeled cRNA was fragmented by adding 11 μl 10 × Blocking Agent and 2.2 μl of 25 × Fragmentation Buffer, then heated the mixture at 60°C for 30 min, finally 55 μl 2 × GE Hybridization buffer was added to dilute the labeled cRNA. 100 μl of hybridization solution was dispensed into the gasket slide and assembled to the gene expression microarray slide. The slides were incubated for 17 hours at 65°C in an Agilent Hybridization Oven. The hybridized arrays were washed, fixed and scanned with using the Agilent DNA Microarray Scanner (part number G2505C).

first: Xiujuan

last: Qin


title: Mesenchymal Stromal Cells Alleviate Depressive and Anxiety-like Behaviors via a Lung Vagal-to-Brain Axis in Male Mice

first: Yuanchen

last: Ma

state: State

address: No74,Zhongshan Road2, Yuexiu District,Guangzhou,Guangdong China

institute: SYSU

summary: Major depressive disorder (MDD) is one of the most common and disabling mental disorders, and current strategies remain inadequate. Although mesenchymal stromal cells (MSCs) have shown beneficial effects in experimental models of depression, underlying mechanisms remain elusive. Here, using murine depression models, we demonstrated that MSCs could alleviate depressive and anxiety-like behaviors not due to a reduction in proinflammatory cytokines, but rather activation of dorsal raphe nucleus (DRN) 5-hydroxytryptamine (5-HT) neurons. Mechanistically, peripheral delivery of MSCs activated pulmonary innervating vagal sensory neurons, which projected to the nucleus tractus solitarius, inducing the release of 5-HT in DRN. Furthermore, MSC-secreted brain-derived neurotrophic factor activated lung sensory neurons through tropomyosin receptor kinase B (TrkB), and inhalation of a TrkB agonist also achieved significant therapeutic effects in male mice. This study reveals a role of peripheral MSCs in regulating central nervous system function and demonstrates a potential "lung vagal-to-brain axis" strategy for MDD.

overall_design: To investigate the in vivo biological characteristics of MSCs, we performed RNA-seq experiments to compare the transcriptional profiles between cultured GFP+ MSCs and GFP+ MSCs isolated from the mouse lungs 3 days after transplantation.

first: Weijun

last: Huang

first: Jing

first: Xiaoran


title: Transition of allele-specific DNA hydroxymethylation at regulatory loci is associated with phenotypic variation in monozygotic twins discordant for psychiatric disorders

city: GuangZhou

first: zhongwei

last: Li

address: No. 1023 - 1063, Shatai South Road, Baiyun District, Guangzhou, China

institute: Southern Medical University

summary: Major psychiatric disorders such as schizophrenia (SCZ) and bipolar disorder (BPD) are complex genetic mental illnesses. Their non-Mendelian features such as monozygotic twins discordant for SCZ or BPD are likely complicated by environmental modifiers of genetic effects. 5-hydroxymethylcytosine (5hmC) is an important epigenetic marker in gene regulation and whether its links with genetic variants contribute to the non-Mendelian features remain largely unexplored. Here, we performed hydroxymethylome and genome analyses of blood DNA from psychiatric disorder-discordant monozygotic twins to study how allele-specific hydroxymethylation (AShM) mediates phenotypic variations. We identified thousands of genetic variants with AShM imbalances who exhibit phenotypic variation-associated AShM transition at regulatory loci. These AShMs have plausible causal associations with psychiatric disorders through effects on interactions between transcription factors, DNA methylations, or other epigenomic markers and then contribute to dysregulated gene expression, which eventually increases disease susceptibility. We then validated that competitive binding of POU3F2 on the alternative allele of psyAShM site rs4558409 (G/T) in PLLP can enhance the PLLP expression, while hydroxymethylated alternative allele alleviating the transcription factor binding activity at rs4558409 site might be associated with downregulated PLLP expression observed in BPD or SCZ. Moreover, disruption of rs4558409 induces gain of PLLP function and promotes neural development and vesicle trafficking. Our study provides a powerful strategy for prioritizing regulatory risk variants and contributes to our understanding of the interplay between genetic and epigenetic factors in mediating complex disease susceptibility.

overall_design: To validate the inhibitory role of rs4558409-containg region in PLLP expression, we employed the Cas9/sgRNA editing to disrupt the sequences around rs4558409 in SK-N-SH cells.

first: Zhanwang

first: Junping

last: Ye


title: Differential microRNA expression in adherent versus suspended MCF10A mammary epithelial cells

first: Junhui

summary: The ability to survive following loss of adhesion underpins cancer metastasis and significantly contributes triple negative breast cancer (TNBC) morbidity.  Epithelial cells that form solid tumors must evade anoikis, a special type of cell death that occurs upon detachment from the extracellular matrix in order to colonize in distal sites.   While there have been several advances in the understanding of the mechanisms by which cells undergo anoikis, the role for noncoding RNAs (ncRNAs) in this process is less well understood.  An important role for microRNAs (miRNAs) in regulating anoikis is beginning to emerge, however in general the mechanisms by which miRNAs regulate anoikis are largely unknown.  Thus we utilized the anoikis-sensitive immortalized mammary epithelial cell line, MCF10A to unbiasedly determine the microRNAs differentially regulated upon detachment.

overall_design: Total RNA (50 µg) was extracted from MCF10A cultured for 24 hours on uncoated tissue culture plates (attached) or on tissue culture plates coated with 2% Poly(2-hydroxyethyl methacrylate) in 95% ethanol(detached). Small RNAs 18-24 nucleotides in length were size selected and gel purified.

first: Simone

last: Tessa M.

first: Green

last: Michael R.


title: The differential expression of tRF&tiRNA in eutopic and ectopic endometrial tissue of patients with ovarian endometriosis.

city: Nanjing

first: Hengmei

last: SHI

address: No. 123 Tianfei Road, Qinhuai District, Nanjing City, Jiangsu Province

institute: Women’s Hospital of Nanjing Medical University (Nanjing Maternity and Child Health Care Hospital)

summary: Endometriosis is a chronic gynecological disease that afflicts millions of women of reproductive age and has traditionally been diagnosed by laparoscopic surgery. This diagnostic barrier delays identification and treatment by years, resulting in prolonged pain and disease progression. Development of a noninvasive diagnostic test could significantly improve timely disease detection. We characterized eutopic and ectopic endometrium from five patients with ovarian endometriosis using tRF&tiRNA sequencing. We identified 294 tsRNAs by RNA microarray technique in eutopic and ectopic endometrium. Under the criteria of |log2FoldChange >2.0| and p-value < 0.05, 11 tRFs/tiRNAs were reported to be dysregulated in ectopic endometrial tissue compared with eutopic endometrial tissue, of which eight were up-regulated, and three were down-regulated.

overall_design: Using high-throughput sequencing, we detected the expression profiles of tRFs&tiRNAs in eutopic and ectopic endometrium tissues of 5 patients with ovarian endometriosis.

last: Shi

first: Danni

last: He

first: Huyang

last: Hou

first: Xiaoyan


title: Dynamic scRNA-seq of live human pancreatic slices reveals functional endocrine cell neogenesis through an intermediate ducto-acinar stage

city: Miami

first: Juan

last: Domínguez-Bendala

state: FL

address: 1450 NW 10th Ave

department: Diabetes Research Institute

institute: University of Miami Miller School of Medicine

summary: Our team has previously characterized a subpopulation of ductal progenitor cells with progenitor-like characteristics. These cells proliferate in response to stimulation of the BMP pathway by BMP-7 and, following withdrawal of this growth factor, differentiate into all three adult epithelial pancreatic lineages (ductal, acinar and endocrine), both in vitro and after transplantation. scRNAseq analysis of sorted human pancreatic ductal cells has confirmed the existence of progenitor-like clusters whose transcriptomic signature suggests a role for stress-mediated de-differentiation in the acquisition of their stemness. Importantly, the application of cell trajectory analyses upon integration of this ductal dataset with other published datasets that also include endocrine and acinar cells revealed the potential existence of ducto-acinar and ducto-endocrine differentiation axes. However, these conclusions were qualified by the fact that they came from the amalgamation of multiple datasets from many different donors, processed and analyzed in different ways, and also in the absence of a defined regenerative stimulus whose effect could be investigated in a truly continuous manner.   We hypothesized that the sequential scRNAseq of same-donor HPSs would address the above constraints and allow us, for the first time, to dissect cell fate changes at the single cell level in response to BMP-7 in a “whole human pancreas” setting. To test this hypothesis, we conducted the longitudinal scRNAseq analysis of human pancreatic slices (HPSs) subjected to a 10-day regimen consisting of 5 days of exposure to BMP-7 followed by 5 days without. The most immediately apparent observation is the transcriptomic heterogeneity of the ductal and acinar compartments, which most earlier analyses (based on isolated islet samples that at best contained a small proportion of ductal and acinar cell types) have failed to detect. With the exception of a recent single nucleus RNAseq report, no other similar study thus far has presented the field with a true whole-pancreas analysis where the different cell types of the organ retain their natural proportion.  Integration of single-cell datasets obtained from same-donor HPSs at different time points, or in the presence or absence of treatment, allowed us to conduct cell trajectory analyses using true temporal information, rather than by pseudotemporal inferences. This integration revealed population shifts consistent with BMP-7-mediated progenitor cell activation, the blurring of ductal/acinar boundaries, the formation of clear ducto-acinar-endocrine differentiation axes and, notably, the appearance of transitional insulin-producing cell populations. These findings contrast with the pattern observed in untreated controls, whose cluster composition and distribution remain largely unchanged with time –another observation that further confirms the robustness of the HPS model to faithfully mirror the cellular makeup of the human pancreas over a 10-day period.  In summary, our study provides the first longitudinal scRNAseq analysis of whole human pancreatic tissue, mapping true cell fate trajectories in a manner that was simply not possible prior to the development of long-term HPS culture techniques. These experiments unequivocally confirm pancreatic plasticity in a dynamic fashion and validate the reliability of this novel human-based model for countless other research applications.

overall_design: HPSs from non-diabetic donors (n=3) were received from the Network for Pancreatic Organ Donors with Diabetes (nPOD). Slices were placed on AirHive perfluorocarbon-based culture dishes and optimized HPS medium for 24hr. After a 24h acclimation period, slices (6/group) were assigned to one of the following two groups: Control, where HPSs were cultured for 10 days; and BMP-7, in which HPSs received 100 ng/ml of BMP-7 in the medium for 5 days and remained in culture without BMP-7 for an additional 5 days.  3 slices were collected at day 5 (time point 1) for both groups; and the remaining ones at day 10 (time point 2). Harvested slices were dissociated into single cells and scRNAseq performed.

first: Mayur

last: Doke

first: Silvia

last: Álvarez-Cubela

first: Dagmar

last: Klein

first: Mirza

middle: F

last: Qadir

first: Ricardo

last: Pastori


title: Serum IGF1 is a new prognostic marker for resistance to targeted therapies and a predictive marker for anti-IGF1R therapy in melanoma

city: Grenoble

first: Florent

last: Chuffart

state: La Tronche

address: Site Santé - Allée des Alpes

institute: IAB - INSERM (U1209) - CNRS (UMR5309)

summary: In a prospective clinical study, we show that increased serum IGF1 levels in a patient may be a predictive marker of melanoma escape to therapies targeting MAPK pathway (BRAFi/MEKi). In addition, our in vitro and in vivo studies suggest that the response of targeted therapies-resistant melanoma cells to IGF1R inhibitor appears to correlate with the level of IGF1 secretion. Overall, our results suggest that monitoring serum IGF1 levels would individually identify melanoma patients who escape BRAFi/MEKi treatment and may benefit from a combination of targeted therapies and IGF1 signaling inhibition to overcome resistance.

overall_design: To investigate the role of the IGF1 pathway in resistance to therapies targeting proteins BRAF and MEK in melanoma, we established human menlanoma cell lines resistant to BRAF and MEK inhibitors.

first: Camila

last: Castillo Ferrer

first: Théo

last: Marguet

first: Laetitia

last: Vanwonterghem

first: Sule

last: Erbek

first: Stéphane

last: Mouret

first: Sabiha

last: Trabelsi Messai

first: Anne-Sophie

last: Gauchez

first: Jean-Luc

last: Coll

first: Julie

last: Charles

first: Amandine

last: Hurbin

first: Véronique

last: Martel-Frachet


title: Hepsin promotes breast tumor growth signaling via the TGFb-EGFR axis

city: Helsinki

first: Topi

last: Tervonen

address: Haartmaninkatu 8

country: Finland

institute: University of Helsinki

summary: Hepsin, a type II transmembrane serine protease, is commonly overexpressed in prostate and breast cancer. The hepsin protein is stabilized by the Ras-MAPK pathway, and downstream, this protease regulates the degradation of extracellular matrix components and activates growth factor pathways, such as hepatocyte growth factor and transforming growth factor beta (TGFβ) pathway. However, how exactly active hepsin promotes cell proliferation machinery to sustain tumor growth is not fully understood. Here, we show that genetic deletion of Hpn in a WAP-Myc model of aggressive MYC-driven breast cancer inhibits tumor growth in the primary syngrafted sites and the growth of disseminated tumors in the lungs. The suppression of tumor growth upon loss of hepsin was accompanied by downregulation of TGFβ and EGFR signaling together with a reduction in EGFR protein levels. We further demonstrate in 3D cultures of patient-derived breast cancer explants that neutralizing antibodies and small-molecule inhibitors of hepsin can be used to mitigate the hepsin-induced TGFβ signaling and reduce EGFR protein levels.The study demonstrates a role for hepsin as a regulator of cell proliferation and tumor growth through TGFβ and EGFR pathways, warranting consideration of hepsin as a potential indirect upstream target for therapeutic inhibition of TGFβ and EGFR pathways in cancer.

overall_design: 3' RNA-Seq (Drop-seq-like)

first: Juha

last: Klefström

first: Denis

last: Belitskin

first: Pauliina

last: Munne


title: Hsp90 shapes adaptation by controlling the fitness consequences of regulatory variation

city: Stanford

first: Christopher

middle: M

last: Jakobson

address: 318 Campus Dr, Clark Center W350

department: Chemical and Systems Biology

institute: Stanford University School of Medicine

summary: The essential stress-responsive chaperone Hsp90 impacts development and adaptation from microbes to humans. Yet despite evidence of its role in evolution, pathogenesis, and oncogenic transformation, the molecular mechanisms by which Hsp90 alters the consequences of mutations remain vigorously debated. Here we exploit the power of nucleotide-resolution genetic mapping in Saccharomyces cerevisiae to uncover more than 1,000 natural variant-to-phenotype associations governed by this molecular chaperone. Strikingly, Hsp90 more frequently modified the phenotypic effects of cis-regulatory variation than variants that altered protein sequence. Moreover, these interactions made the largest contribution to Hsp90-dependent heredity. Nearly all interacting variants—both regulatory and protein-coding—fell within clients of Hsp90 or targets of its direct binding partners. Hsp90 activity affected mutations in evolutionarily young genes, segmental deletions, and heterozygotes, highlighting its influence on variation central to evolutionary novelty. Reconciling the diverse epistatic effects of this chaperone, synthetic transcriptional regulation and reconstructions of natural alleles by genome editing revealed a central role for Hsp90 in regulating the fundamental relationship between activity and phenotype. Our findings establish that non-coding variation is a core driver of Hsp90’s influence on heredity, offering a mechanistic explanation for the chaperone’s strong effects on evolution and development across species.

overall_design: To understand the effect of Hsp90 activity on mRNA regulation, we performed mRNA seqencing of yeast strains with and without Hsp90 inhibition by radicicol.

first: José

last: Aguilar-Rodríguez

first: Daniel

last: Jarosz


title: Kinase inhibitor GW8510 rejuvenates yeast and mice through multiple pathways including SRC, PAKs, JAKs

city: Beijing

first: yongpan

last: an

address: xueyuanlu

institute: Peking University

summary: Aging, a risk factor for many diseases, is largely attributable to cell senescence and impaired mitochondrial function. Here, we used connectivity map (CMAP) to predict the anti-aging effects of drugs based on age-related transcriptomic signatures. We found the kinase inhibitor GW8510 can extend lifespan of yeast, and C57BL/6J or ICR mice by 92.18%, 31.9%, and 142.9%, respectively. Mechanistically, GW8510 can ameliorate cellular senescence by enhancing mitochondrial function, decreasing SA-β-gal staining, p21 and SASP marker expression, and rescues age-related histomorphological changes in mouse hippocampus, heart, kidney and liver. In yeast, the action of GW8510 is dependent on STE20, and in human cells, GW8510 acts through various pathways such as SRC, PAKs, and JAKs. Furthermore, inhibiting these genes can delay cellular senescence. This work provides a valuable resource for mechanistic aging studies and suggests strong clinical potential for GW8510.

overall_design: Knockdown of the SRC gene or administration of GW8510 intervention in senescent WI-38 cells.

first: Yongpan

last: An

first: Zhengwei

last: Xie

first: Baoxue

last: Yang


title: CRISPR/Cas9 genome-wide screen and transcriptome profiling in a MYC-conditional HCC model [CRISPR]

first: Dean

last: Felsher

address: 269 Campus Drive

department: Medicine

institute: Stanford University

summary: The MYC oncogene is often dysregulated in human cancer, including hepatocellular carcinoma (HCC). However, MYC is considered undruggable to date. Here, we comprehensively identify genes essential for the survival of MYC-high but not MYC-low cells by performing a CRISPR/Cas9 genome-wide screen in a MYC-conditional HCC model. Our screen identifies novel MYC-synthetic lethal interactions, as well as most previously identified MYC-synthetic lethal genes. In particular, we found genes involved in nuclear to cytoplasmic transport to be MYC-synthetic lethal in HCC, and we show that many of these genes are transcriptionally upregulated in MYC-high murine HCC.

overall_design: Pooled CRISPR library screen was performed on conditional MYC-HCC-derived mouse cells (EC4 cells) with MYC high and MYC low.

first: Anja

last: Deutzmann

first: Yulin

last: Li

first: Renumathy

last: Dhanasekaran

first: Delaney

middle: K

last: Sullivan


title: CRISPR/Cas9 genome-wide screen and transcriptome profiling in a MYC-conditional HCC model [RNA-seq]

overall_design: RNA sequencing of FVB/N mice and conditional MYC-HCC mice with MYC on and MYC off was performed.


title: Ribosome profiling of wild-type and NSUN2 knockout flies

first: Rui

last: Zhang

address: No. 135, Xingang Xi Road

institute: Sun Yat-sen University

summary: We performed ribosome profiling for wild-type and NSUN2 knockout flies.

overall_design: Ovaries of wild-type and NSUN2 knockout flies were collected.

first: Jianheng

last: Liu

first: Tao

last: Huang

first: Yusen

first: Tianxuan

last: Zhao

first: Xueni

first: Wanying

last: Chen


title: Combined blockade of B7-H3 and CD47 immune checkpoints is a new therapeutic strategy for β-catenin driven melanomas [YUMMER1.7]

city: New York

first: Tiphaine

middle: Christiane

last: Martin

address: 1470 Madison Ave

department: Oncological Sciences

institute: Icahn School of Medicine at Mount Sinai, Tisch Cancer Institute

summary: In melanoma, immune cell infiltration into the tumor is associated with better patient outcomes and response to immunotherapy. T cell non-inflamed tumors (‘cold tumors’) are associated with tumor cell intrinsic Wnt/β-catenin activation, and are resistant to anti-PD-1 alone or in combination with anti-CTLA-4 therapy. Reversal of the ‘cold tumor’ phenotype and identifying new effective immunotherapies are challenges in melanoma. In a well-established preclinical melanoma model driven by β-catenin, we found that immune checkpoint molecule B7-H3 confers a suppressive tumor microenvironment by modulating antiviral signals and matricellular proteins. Its inhibition primes the microenvironment, and together with blockade of the macrophage checkpoint CD47, but not with anti-PD-1, results in synergistic anti-tumor responses. This study brings B7-H3 to the forefront as inducing a suppressive microenvironment when overexpressed, and it’s co-targeting with CD47 as a novel combination of immune checkpoint inhibitors in melanoma that calls for testing in clinical trials.

overall_design: To identify the composition of the immune cell infiltrate within the tumor milieu, we analyzed the major immune cell subsets and functional categories (i.e. CD4+ T cells, CD8+ T cells, NK cells, dendritic cells, B cells, and macrophages) from the dissected tumors in mice treated with isotype control, anti-B7-H3 antibody, anti-PD1, and the combination of both antibodies using bulk RNA-sequencing.

first: Min

last: Hsu

first: Julide

middle: T

last: Celebi


title: Single-cell RNA-sequencing of adipose-derived stem cells

first: Zhenxing

last: Liang

address: 26 Yuancun Erheng Rd

institute: The Sixth Affiliated Hospital, Sun Yat-sen University

summary: We performed single-cell RNA-sequencing to uncover the well-documented heterogeneity in an unbiased manner from the adipose-derived stem cells.

overall_design: Single-cell RNA-sequencing to uncover the heterogeneity in adipose-derived stem cells.

first: Ping

last: Lan


title: Zebrafish as an animal model for the antiviral RNA interference pathway

city: Shanghai

first: Yang

address: 2005 Songhu Road

institute: Fudan University

summary: We have evaluated the possible use of zebrafish to study antiviral RNAi with sindbis virus (SINV), vesicular stomatitis virus (VSV), and nodamura virus (NoV). We find that SINV and NoV viruses induce the production of virus-derived small interfering RNAs (vsiRNAs), the hallmark of antiviral RNAi, with a preference of 22 nucleotides in length after infection of larval zebrafish. Meanwhile, the suppressor of RNAi (VSR) protein, NoV B2, may affect the accumulation of the NoV virus in zebrafish.

overall_design: 5 virus-derived small RNA from zebrafish infected with different viruses was detected by small RNA-seq.

first: Yanxin

last: Ren

first: Xueyu

first: Zhonghui

last: Tian

first: Yan

last: Xu

first: Ruilin


title: Combined blockade of B7-H3 and CD47 immune checkpoints is a new therapeutic strategy for β-catenin driven melanomas [I]

summary: In melanoma, immune cell infiltration into the tumor is associated with better patient outcomes and response to immunotherapy. T cell non-inflamed tumors (‘cold tumors’) are associated with tumor cell intrinsic Wnt/β-catenin activation, and are resistant to anti-PD-1 alone or in combination with anti-CTLA-4 therapy. Reversal of the ‘cold tumor’ phenotype and identifying new effective immunotherapies are challenges in melanoma. In a well-established preclinical melanoma model driven by β-catenin, we found that immune checkpoint molecule B7-H3 confers a suppressive tumor microenvironment by modulating antiviral signals and matricellular proteins. Its inhibition primes the microenvironment, and together with blockade of the macrophage checkpoint CD47, but not with anti-PD-1, results in synergistic anti-tumor responses. This study brings B7-H3 to the forefront as inducing a suppressive microenvironment when overexpressed, and co-targeting with CD47 as a novel combination of immune checkpoint inhibitors in melanoma that calls for testing in clinical trials.


title: A benzenesulfonamide GW8510 rejuvenates mice and yeast through interaction with p21-Activated kinases

first: yongpan

last: an

summary: Aging, a risk factor for many diseases, is largely attributable to cell senescence and impaired mitochondrial function. Here, we used connectivity map (CMAP) to predict the anti-aging effects of drugs based on age-related transcriptomic signatures. We found the kinase inhibitor GW8510 can extend lifespan of yeast, and C57BL/6J or ICR mice by 92.18%, 31.9%, and 142.9%, respectively. Mechanistically, GW8510 can ameliorate cellular senescence by enhancing mitochondrial function, decreasing SA-β-gal staining, p21 and SASP marker expression, and rescues age-related histomorphological changes in mouse hippocampus, heart, kidney and liver. The GW8510 mode of action depends on STE20 (yeast PAK1 homolog), and binds to PAK1 with Kd of 0.82 μM. The crystal structure shows that GW8510 binds to the PAK1 ATP binding pocket. Furthermore, inhibition of PAKs can delay the effects of aging in mice. This work provides a valuable resource for mechanistic aging studies and suggests strong clinical potential for GW8510.

overall_design: Using male ICR mice, a young control group (2 months), an elderly control group and an elderly treatment group (18 months old) were set up, and GW8510 (2mg/kg) was continuously intervened for 2 weeks from 18 months

first: Yongpan

last: An

first: Jie

last: Zhu

first: Zhengwei

last: Xie