title: Spatiotemporal single-cell analysis decodes cellular dynamics underlying different responses to immunotherapy in Colorectal Cancer

first: Zemin

last: Zhang

state: Beijing

address: Yiheyuan Road

department: BIOPIC

summary: Expanding the efficacy of immune checkpoint blockade (ICB) in colorectal cancer (CRC) patients presses for a comprehensive understanding of treatment responsiveness. Here, we analyzed 169 single-cell samples from CRC patients at multiple sequential time points during the course of anti-PD-1 neoadjuvant therapy to map the evolution of local and systemic immunity. In tumors, exhausted T (Tex) cells or tumor-reactive-like CD8 T (Ttr-like) cells were closely related to treatment efficacy, and we observed correlated dynamics between Tex cells and multiple other tumor-enriched cell types following the treatment. Accordingly, several coordinated cellular programs exhibiting distinct response associations were identified. From a systemic perspective, we found divergent replenishment patterns of Ttr-like cells underlying different response statuses and decoded the phenotypic transitions of Ttr-like cells as they infiltrated tissues from the periphery. Finally, a predictive signature was established using circulating CD8 T cells. Our study provides novel insights into the spatiotemporal cellular dynamics following PD-1 blockade in CRC.

overall_design: Leveraging single-cell RNA-seq and TCR-seq technologies, we generated single-cell transcriptome profiles of primary tumor tissues,  adjacent normal tissues, and peripheral blood of 22  CRC patients underwent neoadjuvant anti-PD-1 treatment. Raw sequence data were provided at: China Genomic Sequence Archive (GSA), and we will provide the accession number once the paper is accepted. **Raw data will be uploaded to China Genomic Sequence Archive (GSA), according to the Regulations on the Management of Human Genetic Resources in China.**

first: Yuqing

last: Chen

first: Dongfang

last: Wang

first: Yingjie

last: Li

first: Aiwen

last: Wu


title: Tumor cells impair immunological synapse formation via central nervous system-enriched metabolite

status: Public on Jul 08 2024

city: Guang Zhou

first: Yiwen

last: Lu

state: China

address: 107 Yanjiang West Road Guangzhou 510120, P.R.China

country: China

institute: Sun Yat-Sen University 2nd Affiliated Hospital: Sun Yat-Sen Memorial Hospital

type: Expression profiling by high throughput sequencing

summary: Tumors employ various strategies to evade immune surveillance. Central nervous system (CNS) has multiple features to restrain immune response，because  cranial inflammation can rapidly develop into lethal brain edema. However, whether tumors and CNS share similar programs of immune tolerance is elusive. Here, we analyzed transcriptomic and metabolomic profiles of tumors from patients with HER2+ breast cancer, who received trastuzumab, anti-PD-L1 antibodies and docetaxel (KN035-TH-HER2 study). We found that a neuronal metabolite N-acetylaspartate (NAA) and its synthetase N-acetyltransferase 8-like (NAT8L) are enriched in resistant tumors. In CNS, NAA is released during brain inflammation. We showed that NAT8L attenuates brain inflammation in experimental autoimmune encephalomyelitis (EAE) and  impairs anti-tumor immunity by inhibiting cytotoxicity of NK cells and CD8+ T cells via NAA. Mechanistically, NAA disrupts the formation of immunological synapse by inducing K542 acetylation of lamin A, which inhibits the integration between lamin A and SUN2 and consequently impairs polarization of lytic granules in NK cells and CD8+ T cells. Our study uncovered that tumor cells mimic the anti-inflammatory protective barrier of CNS to evade from anti-tumor immunity and NAT8L is a potential target to enhance efficacy of anti-cancer agents

overall_design: Comparative gene expression profiling analysis of RNA-seq data for combination therapeutic resistant and sensitive group.

first: Shicheng

last: Su

first: Qiyi

last: Zhao


title: Interferon subverts an AHR-JUN axis to promote CXCL13+ T cells in lupus [JUN AHR modulation]

city: Chicago

first: Calvin

last: Law

state: IL

address: 303 E Superior St Lurie 5-220

department: Dermatology

country: USA

institute: Northwestern University

type: Genome binding/occupancy profiling by high throughput sequencing

summary: SLE is prototypical autoimmune disease driven by pathologic T cell-B cell interactions. Expansion of B cell-helper T cells including T follicular helper (Tfh) and T peripheral helper (Tph) cells is a prominent feature of systemic lupus erythematosus (SLE). Human Tfh and Tph cells characteristically produce high levels of the B cell chemoattractant CXCL13, yet regulation of T cell CXCL13 production and the relationship between CXCL13+ T cells and other T cell states remains unclear. Here, we identify an imbalance in CD4 T cell phenotypes in SLE patients, with expansion of PD-1+/ICOS+ CXCL13+ T cells and reduction of CD96hi IL-22+ T cells. Using CRISPR screens, we identify the aryl hydrocarbon receptor (AHR) as a potent negative regulator of CXCL13 production by human CD4 T cells. Transcriptomic, epigenetic, and functional studies demonstrate that AHR coordinates with AP-1 family member JUN to prevent CXCL13+ Tph/Tfh cell differentiation and promote an IL-22+ phenotype. Type I interferon (IFN), a pathogenic driver of SLE, opposes AHR and JUN to promote T cell production of CXCL13. These results place CXCL13+ Tph/Tfh cells on a polarization axis opposite from Th22 cells and reveal AHR, JUN, and IFN as key regulators of these divergent T cell states.

overall_design: Primary human CD4+ memory T cells were stimulated with CD3/CD28 dynabeads with TGF-beta (2ng/mL) and either AHR inhibitor (CH-223191, 10µM) or Agonist (TCDD, 3nM) for 72 hours.

first: Jaehyuk

last: Choi

first: Deepak

middle: A

last: Rao


title: Interferon subverts an AHR-JUN axis to promote CXCL13+ T cells in lupus [JUN OE]

overall_design: Primary human CD4+ memory T cells lentivirally transduced with either JUN overexpressing or control vector were stimulated with CD3/CD28 dynabeads with TGF-beta (2ng/mL) and AHR inhibitor (CH-223191, 10µM) for 72 hours.


title: Engineering Scalable Vascularized Kidney Organoids for in vivo Glomerular Filtration with Human Endothelial Integration

city: Charlestown

first: Ryuji

last: Morizane

state: MA

address: 149 13th Street

institute: Massachusetts General Hospital

summary: We tested the effects of dynamic cell culture environments in stirred bioreactors (STR) using human kidney organoids. Enhanced glomerular vascularization was observed.

overall_design: Human kidney organoids were differentiated from pluripotent stem cells by our previously published protocol in static culture (Morizane, Nature Biotechnology, 2015). Day 14 organoids were transferred to stirred bioreactors and cultured for three weeks.

first: Murat

last: Tekguc


title: Interferon subverts an AHR-JUN axis to promote CXCL13+ T cells in lupus [AHR]

summary: SLE is prototypical autoimmune disease driven by pathologic T cell-B cell interactions. Expansion of B cell-helper T cells including T follicular helper (Tfh) and T peripheral helper (Tph) cells is a prominent feature of systemic lupus erythematosus (SLE). Human Tfh and Tph cells characteristically produce high levels of the B cell chemoattractant CXCL135,6, yet regulation of T cell CXCL13 production and the relationship between CXCL13+ T cells and other T cell states remains unclear. Here, we identify an imbalance in CD4 T cell phenotypes in SLE patients, with expansion of PD-1+/ICOS+ CXCL13+ T cells and reduction of CD96hi IL-22+ T cells. Using CRISPR screens, we identify the aryl hydrocarbon receptor (AHR) as a potent negative regulator of CXCL13 production by human CD4 T cells. Transcriptomic, epigenetic, and functional studies demonstrate that AHR coordinates with AP-1 family member JUN to prevent CXCL13+ Tph/Tfh cell differentiation and promote an IL-22+ phenotype. Type I interferon (IFN), a pathogenic driver of SLE7, opposes AHR and JUN to promote T cell production of CXCL13. These results place CXCL13+ Tph/Tfh cells on a polarization axis opposite from Th22 cells and reveal AHR, JUN, and IFN as key regulators of these divergent T cell states.

overall_design: Primary human CD4+ memory T cells nucleofected with either sgAHR or Non-targeting guide RNA were stimulated with CD3/CD28 dynabeads and AHR agonist (TCDD, 3nM) for 72 hours.


title: Transcriptome analysis of toosendanin (TSN)-treated DLBCL cells

status: Public on Jul 07 2024

city: Chengdu

first: Qian

last: Hu

address: No.37 Guoxue Alley, Wuhou District

department: Hematology

institute: West China Hospital

summary: The effects of TSN on total RNA expression were tested in DOHH2 cells using RNA-seq

overall_design: DOHH2 cells were treated with 25nM and 50nM TSN or solvent control for 24 hours, and RNA was extracted using Magzol reagent (Magen, China). RNA-sequencing (RNA-seq) libraries were created and sequenced by Novogene Corporation (Beijing, China) on the Illumina NovoSeq 6000 platform. The differential gene expression between the TSN-treated groups at varying doses and the solvent control group was evaluated with the 'DESeq' package. Genes showing significant differential expression were identified with an adjusted p-value < 0.05 and a |Log2-fold change| > 1.

first: Mengyao

first: Meng

first: Jinjin

first: Ting

last: Niu


title: Biochemical characterization, biosynthesis mechanism, and functional evaluation of selenium-enriched Aspergillus oryzae A02

city: Tianjin

first: Jinyang

address: No. 32, Xiqi Road, Tianjin Airport Economic Park

institute: Chinese Academy of Sciences

summary: The synthesis mechanisms and function evaluation of selenium(Se)-enriched microorganism remain relatively unexplored. We here report the Se biotransformation by A. oryzae A02. Comparative RNA-Seq analysis revealed the upregulation of functional genes implicated in selenium transformation, activating multiple potential pathways for selenium reduction. The assimilatory and dissimilatory reductions of Se oxyanions engaged numerous parallel and interconnected pathways, manifesting a harmonious equilibrium in overall Se biotransformation in A. oryzae A02.

overall_design: To investigate the effect of Se on the transcriptional profiles of Aspergillus oryzae. A. oryzae was treated or untreated with Se for 12h, 24h and 48h and then comparative gene expression profiling analysis was performed.

first: Le

last: Gao


title: Exploration of genes involved in mature B cells-directed inflammation caused by PTEN mutation

city: Miaoli

first: Pei-Ju

last: Tsai

state: Taiwan

address: 35, Keyan Road, Zhunan Town

department: Immunology research center

country: Taiwan

institute: NHRI

summary: To expose additional genes and signaling pathways involved in the B cell-mediated inflammation in CD23-cKO mice, we performed single-cell RNA-sequencing (scRNA-seq) transcriptomic analysis. These data suggest that the induction of TLR9-mediated IL-6 production triggered by PTEN deficiency relies on BCR-directed endocytosis.

overall_design: Splenic B cells from CD23-cre-control, CD23-cKO, mb-1-cre control and mb-1-cKO mice were left unstimulated (resting), stimulated with TLR9 ligand CpG-ODN1668, or with CpG-ODN1668 plus SB203580 for 2h. The cells were labeled with BD™ Abseq antibody-oligo-conjugates B220, CD19, IgD, CD21, CD23, CD5, CD40, CD80, CD86, CD95, CD138, MHCII. The RNA libraries were prepared by using BD Rhapsody™ WTA Reagent Kit 4 pack (Cat#633802).

first: Yu-Wen


title: Long-term culture of skin biopsies: Maintenance of fibroblast production and competency of reprogramming

city: Aurora

first: Kevin

last: Ess

state: Co

address: 12700 East 19th Avenue

department: Pediatrics

institute: University Colorado Denver

summary: Primary fibroblasts are a commonly used cell type used in a variety of experimental and therapeutic studies. Patient-derived skin biopsies are an accessible way to generate fibroblasts for use in various assays as well as reprogramming to iPSCs. To understand the potential of long-term skin biopsy culture, we cultured biopsy samples for 6 to 16 months and analyzed subsequent generation of fibroblasts. We found maintenance of morphology and physiology over time. Proliferation assays showed that older generations remained proliferative but at a decreased rate. mRNA analyses revealed transcriptional changes with long-term skin culture. Deep DNA sequencing did not reveal any large deletions or amplifications. Spontaneous DNA mutations seemed to be random and not enriched for any specific signaling pathways. Older fibroblasts generated after 16 months in culture retained competency for reprogramming into iPSCs. Our results support long term culture of skin to generate very large numbers of primary fibroblasts. These cells maintain their identity and integrity enabling the study of human disorders, particularly rare ones.

overall_design:
To determine the effect of long-term skin biopsy culture and the stability of produced fibroblasts, we cultured 2 skin biopsies for 6 to 16 generations
We extracted RNA from fibroblasts from Gen1, Gen6, and Gen16 and compared expression changes over time to determine culture stability

first: Sudiksha

last: Rathan-Kumar

first: Michael

last: Ripperger

first: Grant

last: Westlake


title: Lipid Metabolism Alterations Induce PD-L1 Internalization and Signaling to Regulate Breast Cancer Metastasis and Response to Immunotherapy [EXOSC10 RNA-IP-seq]

city: Charleston

first: Wyatt

last: Wofford

state: SC

address: 86 Jonathan Lucas St

institute: MUSC

summary: Programmed death-ligand 1, PD-L1 (CD274), is expressed by cancer cells to facilitate immune evasion. PD-L1 also exerts pro-survival functions in cancer cells, and mechanisms that regulate intracellular PD-L1 signaling remain largely unknown. Here, we report a new mechanism whereby internalization of PD-L1 in response to alterations of bioactive lipid/ceramide metabolism by ceramide synthase 4 (CerS4) induces sonic-hedgehog (Shh) and TGF-b-receptor signaling to enhance tumor metastasis in triple-negative breast cancers (TNBC), which are highly resistant to immunotherapy. Mechanistically, we show here that internalized PD-L1, mediated by its cytoplasmic domain, including the S278/S279 residues, in response to CerS4/C18/C20-ceramide downregulation or genetic loss, interacts with an RNA-binding protein Caprin-1 in various metastatic breast cancer models, including 4T1 orthotopic breast allografts and mammary tumors developed in MMTV-PyMT/CerS4-/- compared to MMTV-PyMT/CerS4+/+ mice, consistent with Shh/TGFBR1/Wnt/b-catenin activation in-vivo. Genetic loss or molecular knockdown of CerS4 in breast tumors also resulted in increased infiltration of FoxP3+ T regs that are known to suppress anti-tumor immunity. Targeting the CerS4/Shh/PD-L1 axis using Sonidegib and anti-PD-L1 antibody in mice containing metastatic breast tumors in which CerS4 is downregulated vastly decreased tumor growth and metastasis, leading to inhibition of PD-L1 internalization and Shh/Wnt signaling, restoring anti-tumor immune response. These data, validated in clinical samples and multiple patient datasets, demonstrate a mechanism to define intracellular metastatic signaling by PD-L1 internalization in response to alterations of ceramide metabolism, providing a new therapeutic strategy to improve immunotherapy responses in metastatic TNBCs.

overall_design: To investigate the role of the PD-L1/Caprin-1 complex on  mRNA degradation in the context of altered lipid metabolsim, RNA-immunprecipatation using anti-EXOSC10 antibodies was performed on LM2-4175 cells stably expressing a dox-inducible CerS4 construct. IgG was used as a control. The primary comparison was between mRNA profiles bound to EXOSC10 in untreated (no dox) versus dox-treated cells.

first: Wofford

last: Wyatt

first: Ogretmen

last: Besim


title: A classical epithelial state drives acute resistance to KRAS inhibition in pancreas cancer

city: New York

first: Anupriya

last: Singhal

state: New York

address: 1275 York Avenue

institute: Memorial Sloan Kettering

summary: Intra-tumoral heterogeneity in pancreatic ductal adenocarcinoma (PDAC) is characterized by a balance between basal and classical epithelial cancer cell states, with basal dominance associating with chemoresistance and a dismal prognosis. Targeting oncogenic KRAS, the primary driver of pancreatic cancer, shows early promise in clinical trials but efficacy is limited by acquired resistance. Using genetically engineered mouse models and patient-derived xenografts, we find that basal PDAC cells are highly sensitive to KRAS inhibitors. Employing fluorescent and bioluminescent reporter systems, we longitudinally track cell-state dynamics in vivo and reveal a rapid, KRAS inhibitor-induced enrichment of the classical state. Lineage-tracing identifies these enriched classical PDAC cells to be a reservoir for disease relapse. Genetic ablation of the classical cell-state is synergistic with KRAS inhibition, providing a pre-clinical proof-of-concept for this therapeutic strategy. Our findings motivate combining classical-state directed therapies with KRAS inhibitors to deepen responses and counteract resistance in pancreatic cancer.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: NSG mice bearing orthotopic KP mouse cell line allografts (pancreatic cancer) or human subcutaneous patient-derived-xenografts (PDXs) of pancreatic cancer were administered MRTX1133 at 30 mg/kg b.i.d. vs. vehicle. Mouse PDAC tumors were treated for 7 days. PDX experiments were treated for 10-14 days. Orthotopically transplanted and PDX tumors were dissociated into single-cell suspensions and FACS sorted for tumor cells for single-cell RNA sequencing. Each dataset represents a tumor type (mouse KP tumor, or human PDX#1-3 treated with either vehicle or MRTX1133). Biological replicates with the same treatment were combined into one lane with HTO antibodies. Vehicle-treated and MRTX1133-treated tumors for each experiment were compared.

first: Hannah

last: Styers

first: Jonathan

last: Rub

first: Tuomas

last: Tammela


title: Lipid Metabolism Alterations Induce PD-L1 Internalization and Signaling to Regulate Breast Cancer Metastasis and Response to Immunotherapy [Caprin-1 RNA-IP-seq]

overall_design: To investigate the role of the PD-L1/Caprin-1 complex on  mRNA binding in the context of altered lipid metabolsim, RNA-immunprecipatation using anti-Caprin-1 antibodies was performed on 4T1 cells stably expressing SCR or CerS4 shRNA. IgG was used as a control. The primary comparison was between mRNA profiles bound to Caprin-1 in SCR versus CerS4 KD cells.


city: Guangzhou

first: Yihong

last: Li

address: Zhong shan Er lu

institute: Sun Yat-sen University

summary: As a hallmark of cancer, altered metabolisms induce the suppression of anti-tumor immunity, contributing to immunotherapy resistance. Tumor metabolic reprogramming decreases immune effector cell infiltration, inhibits antigen priming and arrests expansion of effector cells. Therefore, we analyzed metabolism-related gene expression of tumors on a single-cell level using 3 tumor samples from breast cancer patients. We demonstrated that tumor cells with high metabolism-related expression evade immune surveillance.

overall_design: scRNA-seq for breast tumors without neoadjuvant treatment

first: Shicheng

last: Su

first: Yiwen

last: Lu

first: Peng

last: Deng


title: Redox Heterogeneity in Mouse Embryonic Stem Cells Individualizes Cell Fate Decisions II

city: Reykjavik

first: Agnes

middle: Katharina

last: Ulfig

address: Sturlugata 8

department: Translational Medicine

country: Iceland

institute: University of Iceland

summary: How cellular diversity is created is a key question in biology. Embryonic stem cells (ESCs) are pluripotent cells that can develop into any cell type in the body. Yet, the regulatory mechanisms that govern cell fate decisions remain largely unknown. Moreover, although genetically identical, individual ESCs are strikingly heterogeneous regarding their differentiation capacity but the molecular basis for this phenotypic heterogeneity is unclear. We now demonstrate that pluripotent mouse ESCs (mESCs) display large natural variations in mitochondrial reactive oxygen species (ROS) levels that individualize their nuclear redox state and redox-sensitive H3K4me3 landscape. By using intracellular redox probes, we sorted pluripotent and differentiating mESCs based on their nuclear redox environments into subpopulations and analysed their differentiation capacity. We show that more reduced mESCs are biased towards the mesendodermal fate and form the primitive streak upon differentiation. This mesendodermal specification is due to increased Wnt signaling pathway activity, mediated via a ROS-specific regulatory mechanism of p53-dependent expression of the Wnt ligand Wnt3, and increased H3K4me3 levels. We demonstrate that formation of a primitive streak-like structure by differentiating mESCs is initiated by increased mitochondrial ROS and p53 stabilization, followed by antioxidant Nrf2 response pathway activation that leads to a reduced nuclear redox environment, allowing for the accumulation of ROS-sensitive H3K4me3 marks and activation of Wnt3 and other mesendoderm-related gene expression. In summary, our study shows that natural variations in endogenous ROS levels individualize the H3K4me3 landscape of mESCs, and through gene expression changes govern their cell fate decision at the onset of gastrulation.

overall_design:
To test if clonal E14Tg2a mESCs that naturally differ in their nuclear redox state choose distinct cell fates upon differentiation, we first differentiated mESCs expressing the Grx1-roGFP2 redox sensor in their nucleus to embryoid bodies. At day 5 of differentiation, we dissociated the EBs and sorted individual cells based on their nuclear redox state into two subpopulations (D5_ox and D5_red).
We sorted the 10% most oxidized (ox) and 10% most reduced (red) cells of three distinct populations (A, B, C) and isolated their total RNA.
Comparative gene expression profiling analysis of RNAseq data for the 10% most oxidized (D5_ox) and 10% most reduced cells (D5_red) in day 5 embryoid bodies.

first: Ursula

last: Jakob


title: Analysis of the accumulation of mutations in self-replicating RNAs using barcoded EGFP library

city: Beijing

first: Yihan

last: Lin

address: No.5 Yiheyuan Road, Haidian

institute: Peking University

summary: Directed evolution in mammalian cells can facilitate the engineering of mammalian-compatible biomolecules and can enable synthetic evolvability for mammalian cells. We engineered an orthogonal alphaviral RNA replication system to evolve synthetic RNA-based devices, enabling RNA replicase-assisted continuous evolution (REPLACE) in live mammalian cells. To investigate the process of mutation accumulation in REPLACE system, we constructed a repRNA-v4 plasmid library containing 64 barcodes. Using this library, we analyzed the differences in mutation accumulation for different RNAs upon entry into cells, before and after molnupiravir treatment, and before and after FACS sorting. The results demonstrated that these barcoded RNAs undergo similar processes of mutation accumulation, providing evidence that mutations are commonly accumulated across different RNAs.

overall_design: To investigate the process of mutation accumulation in the REPLACE system, a repRNA-v4 plasmid library containing 64 barcodes was constructed. The library was generated by amplifying barcoded sequences from the repRNA-v4 plasmid using primers containing degenerate bases (F primer: gaagcaatccgcgaaaaatgcccggtcgacc, R primer: cattggggcgtactagtatcgatgccgctcctaggcttcScWaScWcSgWgatttaggcaccgggcttgcgggt). The original fragment was then replaced with the barcoded PCR products through enzymatic digestion using SalI and AvrII enzymes (NEB). The resulting ligation products were transformed into competent Escherichia coli cells (Trelief 5α) for plasmid amplification. Next, the plasmids were subjected to in vitro transcription, and 10 μg of RNA was electroporated into 4 million BHK-21 cells (with nsP4 expressing in trans). Twenty-four hours post-electroporation (referred to as T1), the medium was replaced with fresh medium containing 5 μg/ml puromycin. After four days of selection (referred to as T5), 10 μM of molnupiravir was added to the medium for a 2-day treatment. Subsequently, the cells were allowed to grow for four days (referred to as T3) and then subjected to flow cytometry sorting. The sorted cells were cultured for approximately one week (referred to as T4) and subsequently treated with molnupiravir for another 2 days (referred to as T5). Cells at each time point (T1 to T5) were collected, and total RNA was extracted using Trizol reagent. The extracted RNA was subjected to reverse transcription, followed by amplification using primers specific to genomic RNA targeting the EGFP-PuroR-barcode region. The purified PCR products underwent adapter ligation, fragment selection, primer and polymerase binding, and were then sequenced using a PacBio Sequel IIe instrument to obtain Circular Consensus Sequencing (CCS) reads.

first: Liang

last: Ma


title: Measurement of the degradation rate of alphaviral RNA in BHK-21 cells

summary: Directed evolution in mammalian cells can facilitate the engineering of mammalian-compatible biomolecules and can enable synthetic evolvability for mammalian cells. We engineered an orthogonal alphaviral RNA replication system to evolve synthetic RNA-based devices, enabling RNA replicase-assisted continuous evolution (REPLACE) in live mammalian cells. To determine the degradation rate of alphaviral RNA in BHK-21 cells, 10 µg of repRNA-v4 RNA was transfected into 4 million wildtype BHK-21 cells via electroporation. Samples were collected at 1-hour, 3-hour, and 12-hour time points for subsequent RNA-seq analysis. The findings revealed that the half-life of alphaviral RNA in the cells was approximately 3.5 hours.

overall_design: 10 µg of repRNA-v4 RNA was transfected into 4 million wildtype BHK-21 cells via electroporation. Samples were collected at 1-hour, 3-hour, and 12-hour time points for subsequent RNA-seq analysis. RNA-seq was performed on a DNBSEQ-T7 platform. Cleaned paired-end reads were aligned to the Mesocricetus auratus genome (GCF_017639785.1_BCM_Maur_2.0_genomic.fna) and the genome of the alphaviral RNA replication system using STAR 2.7.8a. Genome annotation file (GCF_017639785.1_BCM_Maur_2.0_genomic.gtf) was downloaded from NCBI and edited to add the annotation information of the alphaviral RNA replication system. Transcript abundances were estimated using featureCounts, then normalized to RPKM using in-house R scripts.


title: Redox Heterogeneity in Mouse Embryonic Stem Cells Individualizes Cell Fate Decisions I

overall_design:
To investigate the potential downstream effects of a naturally more oxidized nuclear redox state, we generated a more oxidizing environment in an entire population of pluripotent E14Tg2a mESCs by exposing them to a non-lethal dose of 500 mM H2O2 for 30 min.
To explore whether and how transient exposure of mESC to peroxide affects gene expression patterns, we conducted RNA seq-analysis from three independent H2O2-time-course experiments, analyzing samples before (0 h) as well as defined time points after the treatment (0.5 h, 2 h, 4 h, 6 h, 8 h, 10 h, 24 h). 0.5 h time point was collected immediately after the 30 min treatment with hydrogen peroxide. File names: numbers 1, 2 and 3 following the respective time point indicate biological replicates. Cells 24 h after the treatment with hydrogen peroxide were compared with untreated cells at the 24 h time point.
Comparative gene expression profiling analysis of RNAseq data for pluripotent E14Tg2a cells at various time points after peroxide treatment vs. untreated cells before the treatment (0 h) or 24 h after starting the experiment.


title: Analyzing the process of mutation accumulation in MEK1 evolution experiment using amplicon sequencing

summary: Directed evolution in mammalian cells can facilitate the engineering of mammalian-compatible biomolecules and can enable synthetic evolvability for mammalian cells. We engineered an orthogonal alphaviral RNA replication system to evolve synthetic RNA-based devices, enabling RNA replicase-assisted continuous evolution (REPLACE) in live mammalian cells. we employed REPLACE to drive the continuous intracellular evolution of the cancer-related protein MEK1 with the aim of conferring resistance to Cobimetinib. To investigate the accumulation of mutations during this evolutionary process, we conducted amplicon sequencing on experimental materials collected at different stages. The results revealed intricate relationships among different mutations, highlighting the complex nature of the evolutionary landscape.

overall_design: MEK1 cDNA from H. sapiens was cloned into the repRNA-v4 plasmid, and the resulting replicative RNAs were electroporated into BHK-21 cells expressing nsP4 (stable cell line containing ML700 plasmid). Medium was replaced with fresh medium containing 5 μg/ml puromycin 24 h post-electroporation (i.e., Day 1). After 4 d of selection (i.e., Day 5), 2 μM of molnupiravir was added to the medium. On Day 7, cobimetinib was added to the medium. The cells were continued to be cultured and selected in medium with molnupiravir and cobimetinib (2-5 μM) for 7 days. To analyze the enrichment process of the screened mutations, amplicon sequencing was performed on MEK1 RNA extracted from cells at Day 1, and Day 14.


title: Gene expression profile at single cell level from Glycine max nodules and roots.

city: Tucson

first: Sergio Alan

last: Cervantes-Perez

state: Arizona

address: 1140 E South Campus Dr

department: School of Plant Sciences

institute: University of Arizona

summary: The nuclei of Glycine max root seedlings (6 days-old) and nodules (28 days-old) were isolated and the library consstruction was performed applying 10x Genomics tecnology.

overall_design: The nuclei of the 6 days-old roots of Glycine max seedlings grown on B&D-N media and growth chambers in dark. For nodule sample, the 3-day-old seedlings were germinated between moist Whatman filter paper and were inoculated with 1 mL of B. japonicum suspension (OD600=0.1) on transferred 3‐day‐old seedlings. The nodules were collected after 28 days. For the nuclei isolation was used mechanical and chemical steps and the libraries were made following 10X genomics recommendations.  The pre-processing and processing of the datasets were performed with Cell Ranger, and Seurat.

first: Sergio

first: Sandra

last: Thibivilliers

first: Sutton

last: Tennant

first: Marc

last: Libault


title: Redox Heterogeneity in Mouse Embryonic Stem Cells Individualizes Cell Fate Decisions

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

overall_design: Refer to individual Series


title: Temporal recording of mammalian development and precancer [WES]

city: Nashville

first: Mirazul

last: Islam

state: TN

address: 2213 Garland Ave

department: Cell and Developmental Biology

institute: Vanderbilt University

summary: Insights into many biological phenomena requires knowing the temporal order of cellular events, which is traditionally achieved through continuous direct observations [1, 2]. An alternative solution leverages irreversible genetic changes, such as naturally occurring mutations, to create indelible markers that enables retrospective temporal ordering [3-8]. Using Native sgRNA Capture and sequencing (NSC-seq), a newly devised and validated multi-purpose single-cell CRISPR platform, we developed a molecular clock approach to record the timing of cellular events and clonality in vivo while incorporating cell state and lineage information. Using this approach, we uncovered precise timing of tissue-specific cell expansion during murine embryonic development, unconventional developmental relationships between cell types, and new epithelial progenitor states by their unique genetic histories. NSC-seq analysis of murine adenomas coupled to multi-omic and single-cell profiling of human precancers, with clonal analysis of 418 human polyps, demonstrated the occurrence of polyancestral initiation in 15-30% of colonic precancers, revealing their origins from multiple normal founders. Our study presents a multimodal framework that lays the foundation for in vivo recording, integrating synthetic or natural indelible genetic changes with single-cell analyses to explore the origins and timing of development and tumorigenesis in mammalian systems.

overall_design: DNA was extracted from resected tumors and normal epithelial tissues using DNA Extraction Kits (Qiagen). Standard whole exome sequencing (WES) library was prepared using mouse exome panel (Twist Bioscience) and sequenced on S4 flow cells on NovaSeq6000 (PE150) to the targeted coverage (50X). WES reads were aligned to the mouse reference genome (mm10) using BWA and indexed by Sambamba. Somatic variants were called using GATK Mutect2. Variants were annotated using ANNOVAR and germline variants were filtered using paired normal samples. Variants were further analyzed and visualized using Maftools.

first: Islam

last: Mirazul

first: Lau

last: Ken


title: Temporal recording of mammalian development and precancer

summary:
Insights into many biological phenomena requires knowing the temporal order of cellular events, which is traditionally achieved through continuous direct observations [1, 2]. An alternative solution leverages irreversible genetic changes, such as naturally occurring mutations, to create indelible markers that enables retrospective temporal ordering [3-8]. Using Native sgRNA Capture and sequencing (NSC-seq), a newly devised and validated multi-purpose single-cell CRISPR platform, we developed a molecular clock approach to record the timing of cellular events and clonality in vivo while incorporating cell state and lineage information. Using this approach, we uncovered precise timing of tissue-specific cell expansion during murine embryonic development, unconventional developmental relationships between cell types, and new epithelial progenitor states by their unique genetic histories. NSC-seq analysis of murine adenomas coupled to multi-omic and single-cell profiling of human precancers, with clonal analysis of 418 human polyps, demonstrated the occurrence of polyancestral initiation in 15-30% of colonic precancers, revealing their origins from multiple normal founders. Our study presents a multimodal framework that lays the foundation for in vivo recording, integrating synthetic or natural indelible genetic changes with single-cell analyses to explore the origins and timing of development and tumorigenesis in mammalian systems.
This SuperSeries is composed of the SubSeries listed below.


title: RNA sequencing of BHK-21 cells carrying alphaviral RNA replication system

summary: Directed evolution in mammalian cells can facilitate the engineering of mammalian-compatible biomolecules and can enable synthetic evolvability for mammalian cells. We engineered an orthogonal alphaviral RNA replication system to evolve synthetic RNA-based devices, enabling RNA replicase-assisted continuous evolution (REPLACE) in live mammalian cells. RNA-seq of 3 different cells was performed to analyze the effect of introducing different versions of RNA replication systems (i.e., repRNA-v3 and repRNA-v4) into BHK-21 cells on endogenous gene expression. Analysis of the transcriptome profiling data indicated that repRNA-v4 stimulated lower interferon signaling and may have lower cytopathicity.

overall_design: In this study, repRNA-v3 was electroporated into BHK-21 cells and repRNA-v4 was electroporated into BHK-21 cells expressing nsP4 in trans. The cells were cultured for ~3 weeks with the presence of 10 μg/ml of puromycin. 300,000 of BHK-21 cells expressing nsP4 in trans, 300,000 of repRNA-v3 cells and 300,000 of repRNA-v4 cells were collected for RNA isolation and library preparation. RNA-seq was performed on a DNBSEQ-T7 platform. Cleaned paired-end 150 bp reads were aligned to the Mesocricetus auratus genome (GCF_017639785.1_BCM_Maur_2.0_genomic.fna) and the alphaviral RNA replication system genome using STAR 2.7.8a. Genome annotation file (GCF_017639785.1_BCM_Maur_2.0_genomic.gtf) was downloaded from NCBI and edited to add the information of the alphaviral RNA replication system. Transcript abundances were estimated using featureCounts, then normalized and statistically compared using DESeq2 (i.e., the transcriptome difference between the repRNA-v4 cells and the BHK-21 cells expressing nsP4 in trans, the transcriptome difference between the repRNA-v3 cells and the repRNA-v4 cells).


title: Alterations of Ceramide Synthesis Induce PD-L1 Internalization and Signaling to Regulate Tumor Metastasis and Immunotherapy Response

first: Wyatt

last: Wofford

summary: Programmed death-ligand 1, PD-L1 (CD274) facilitates immune evasion and exerts pro-survival functions in cancer cells. Here, we report a new mechanism whereby internalization of PD-L1 in response to alterations of bioactive lipid/ceramide metabolism by ceramide synthase 4 (CerS4) induces sonic-hedgehog (Shh) and TGF- receptor signaling to enhance tumor metastasis in triple-negative breast cancers (TNBC), exhibiting immunotherapy resistance. Mechanistically, data showed that internalized PD-L1 interacts with an RNA-binding protein Caprin-1 to stabilize Shh/TGFBR1/Wnt mRNAs to induce -catenin signaling and TNBC growth/metastasis, consistent with increased infiltration of FoxP3+ T regs and resistance to immunotherapy. While mammary tumors developed in MMTV-PyMT/CerS4-/- were highly metastatic, targeting the Shh/PD-L1 axis using Sonidegib and anti-PD-L1 antibody vastly decreased tumor growth and metastasis, consistent with the inhibition of PD-L1 internalization and Shh/Wnt signaling, restoring anti-tumor immune response. These data, validated in clinical samples and databases, provide a mechanism-based therapeutic strategy to improve immunotherapy responses in metastatic TNBCs.

overall_design: To investigate the effect of sonidegib plus anti-PD-L1 immunotherapy on TNBC primary tumor growth and immune infiltration, FVB mice harboring PyMT tumors were treated with vehicle, sonidegib, anti-PD-L1 immunotherapy, or sonidegib + anti-PD-L1 immunotherapy. The primary comparison was differentially expressed genes across all treatment groups.

first: Besim

last: Ogretmen


title: Analyzing the process of mutation accumulation in KRAS evolution experiment using amplicon sequencing

summary: Directed evolution in mammalian cells can facilitate the engineering of mammalian-compatible biomolecules and can enable synthetic evolvability for mammalian cells. We engineered an orthogonal alphaviral RNA replication system to evolve synthetic RNA-based devices, enabling RNA replicase-assisted continuous evolution (REPLACE) in live mammalian cells. Using REPLACE, we attempted continuous intracellular evolution of the negative dominant mutant KRAS (S17N). To analyze the process of mutation accumulation, we performed amplicon sequencing on experimental materials at different stages and under different treatment conditions. The results indicated that the mutations generated by this system were primarily induced by Monanunavir, and the addition of Monanunavir significantly accelerated the rate of evolution.

overall_design: KRAS (S17N) cDNA from H. sapiens was cloned into the repRNA-v4 plasmid and the resulting replicative RNAs were electroporated into BHK-21 cells expressing nsP4 (stable cell line containing ML700 plasmid). Medium was replaced with fresh medium containing 5 μg/ml puromycin 24 h post-electroporation (i.e., Day 1). After 4 d of selection (i.e., Day 5), 2 μM of molnupiravir was added to the medium. The cells were continued to be cultured and selected in medium with molnupiravir and puromycin for 10 days. To analyze the enrichment process of the screened mutations, amplicon sequencing was performed on KRAS (S17N) DNA, in vitro transcribed RNA, and RNA extracted from cells at Day 1, Day 5, Day 9, Day 12, and Day 14. Additionally, to compare the effect of molnupiravir treatment, amplicon sequencing was performed on RNA from cells at Day 9 that did not undergo molnupiravir (Day 5-Day 9) treatment.


title: 2-Bromo-1,4-Naphthalenedione Promoting CD8+ T Cell Expansion and Inhibits Th1/Th17 Differentiation to Mitigate Experimental Autoimmune Encephalomyelitis

status: Public on Jul 06 2024

first: Yuanchen

state: State

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

institute: SYSU

summary: In this study, we aim to explore the therapeutic potential of 2-bromo-1,4-naphthalenedione (BrQ), a derivative of naphthalenedione, in experimental autoimmune encephalomyelitis (EAE), an animal model of MS, and to elucidate its underlying mechanisms.

overall_design: 4  EAE mouse were seperated into 2 groups, two were treated with BrQ, two were treated as control

first: Cuixia

last: Yang

first: Yuantao

last: Li


title: RNA-seq in A549 cell lines.

city: Zhengzhou

first: Gu

last: Zhuoyu

address: Jianshe East No.1

institute: Zhengzhou Univeristy

summary: Purpose: The goals of this study are to invsetigate the primary biological process and signalling pathway of HNRNPD. Results: Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) analysis of the differential genes showed that the PI3K signaling pathway and HIF-1 signaling pathway were enriched after HNRNPD knockdown.

overall_design: MRNA profiles of NC cells and HNRNPD knockdown cells were used. Briefly, samples (A549 cells transfected with negtive control or HNRNPD siRNA) were used to extract total RNAs for quality control and RNA-seq loading samples. RNA-seq was performed as DGE on an Illumia HiSeq platform (Seqhealth Co., Wuhan, China).

first: Zhuoyu

last: Gu


title: Hox gene activity directs physical forces to differentially shape chick small and large intestinal epithelia

city: Boston

first: Hasreet

last: Gill

address: 77 Avenue Louis Pasteur, NRB 360

department: Genetics

institute: Harvard Medical School

summary: Hox genes are highly conserved, master regulators of spatial patterning in the embryo, but how these factors trigger regional morphogenesis has largely remained a mystery. In the developing gut, Hox genes help demarcate identities of the small and large intestines early in embryogenesis, which ultimately leads to their specialization in both form and function. While the midgut forms villi, the hindgut develops flat, brain-like sulci that resolve into heterogeneous outgrowths. Combining mechanical measurements of the embryonic chick intestine and mathematical modeling, we demonstrate that the posterior Hox gene HOXD13 regulates biophysical phenomena that shape the hindgut lumen. We further show that HOXD13 acts through the TGFβ pathway to thicken, stiffen, and promote isotropic growth of the subepithelial mesenchyme; together, these features lead to hindgut surface buckling. TGFβ, in turn, promotes collagen deposition to affect mesenchymal geometry and growth. We thus identify a cascade of events downstream of positional genetic identity that direct posterior intestinal morphogenesis.

overall_design: To more specifically identify which genes are commonly differentially expressed within the subepithelial mesenchyme of the midgut with Hoxd13 misexpression and the hindgut, relative to the midgut, a spatial sequencing approach called Light-seq  was employed (Kishi et al., 2022). In situ reverse transcription was performed in midgut, hindgut, and RCAS-Hoxd13 tissue sections on slides. Light-directed crosslinking was used to iteratively barcode manually defined regions--the subepithelial mesenchyme, inner longitudinal muscle layer, and inner superficial side of the circumferential muscle layer. Barcoded cDNA was then extracted from slides and used for downstream qPCR amplification and library preparation. Each sequenced library contained reads from all three compartments. Downstream barcode parsing then enabled assignment of each read to the tissue layer of interest. Though three layers were barcoded and sequenced, only the inner mesenchyme layer was used for differential gene expression analysis comparing conditions (midgut, hindgut, midgut + Hoxd13).

middle: K

first: Sifan

last: Yin

first: Nandan

middle: L

last: Nerurkar

first: John

middle: C

last: Lawlor

first: ChangHee

last: Lee

first: Tyler

middle: R

last: Huycke

first: L

last: Mahadevan

first: Clifford

middle: J

last: Tabin


title: Multi-inlet spheroid generator for modeling immune microenvironment of glioblastoma (GBM)

city: Suwon-si

first: Seokgyu

last: Han

state: Gyeonggi-do

address: 2066, Seobu-ro

department: Mechanical Engineering

country: South Korea

institute: Sungkyunkwan univerisity

summary: Using the multi-inlet spheroid generator (MSG) platform, we generated stable GBM spheroids and observed their interactions with various immune cells, including microglia and natural killer (NK) cells, at different time points. HMO6 and NK-92 cells were used for microglia and NK cells, respectively. We examined the interactions between tumor-immune cells as well as between different immune cells. The generation of tri-cultured spheroids successfully recapitulated the immunosuppressive microenvironment of the GBM tumor.

overall_design: We performed gene expression profiling analysis of bulk RNA-seq data obtained from patient-derived GBM spheroids at various time points. Total RNA was isolated from these spheroids using the QIAzol lysis reagent (Qiagen,79306 )following the manufacturer's protocol. Libraries for bulk RNA-seq were prepared using the TruSeq Stranded mRNA Library Prep Kit. GBM spheroids were cultured and maintained in Dulbecco’s modified Eagle’s high glucose medium (SH30243.01, Hyclone) supplemented with 10% heat-inactivated fetal bovine serum (2357664RP, Gibco) and 1% antibiotic-antimycotic (15240062, Gibco). Spheroids were maintained at 37°C in a humidified atmosphere containing 5% CO2 and tested regularly for mycoplasma contamination by mycoplasma detection kit (rep-mys-20, InvivoGen).

first: Yona

last: Kim

first: Hyo

middle: E

last: Moon

first: Sun

middle: H

last: Paek

first: Sungsu

last: Park


title: High-throughput sequencing of mouse liver mRNA after treatment with the mRNA splicing inhibitor Pladienolide B

city: Wuhan

first: RUI

last: Zhou

address: Donghu Road 185

institute: Wuhan University

summary: RNA splicing is an essential step for expression of genes commonly altered in multiple liver diseases. However, how the spliceosomal components participate in the pathogenesis of acute liver failure (ALF) remains poorly defined. Here, we report that KH-type splicing regulatory protein (KSRP) which is downregulated in ALF, regulates RNA splicing in the liver through interacting with the Splicing factor 3b subunit 1 (SF3B1). Overexpression of KSRP resulted in marked amelioration of hepatic injury in vivo. Transcriptomic analysis reveals that KSRP depletion led to severe splicing defects in genes with significant intron retention. Such defects cause substantial loss of proliferation and apoptosis related genes such as EGFR, which further exaggerated liver injury. KSRP promotes RNA splicing of splicing related genes, including SF3B1, PHF5A, and SF3B4, indicating the existence of a positive feedback loop that regulates RNA splicing. Mechanistically, KSRP directly interacts with SF3B1 and enhances binding of SF3B1 to the branch sites located upstream of the exon. Overall, our findings demonstrate a novel mechanism by which KSPR protects against acute liver injury by promoting RNA splicing through interacting with the SF3B complex.

overall_design: In order to study the effect of mRNA splicing inhibitor Pladienolide B on liver splicing in mice, we used a drug concentration of 0.75mg/kg that did not cause liver damage and injected mice intraperitoneally. One day later, mouse liver tissues were collected for RNA sequencing analysis.

first: R

first: M

first: Q

last: Fang


title: Combined analysis of temporal metabolomics and transcriptomics reveals the metabolic patterns in goat oocytes during maturation

city: Nanjing

first: Jiashuo

last: Li

address: 101Longmian Avenue, Jiangning District, Nanjing 211166, P.R. China

institute: Nanjing Medical University

summary: Well-balanced and orderly metabolism is a crucial prerequisite for promoting oogenesis. Involvement of single metabolites in oocyte development has been widely reported; however, the comprehensive metabolic framework controlling oocyte maturation is still lacking. In the present study, we employed an integrated temporal metabolomic and transcriptomic method to analyze metabolism in goat oocytes at key stages, revealing the global picture of the metabolic patterns during maturation. In particular, several significantly altered metabolic pathways during goat oocyte meiosis have been identified, including active serine metabolism, increased utilization of tryptophan, and marked accumulation of purine nucleotide. In summary, the current study not only provides multiple omics data resources for goat oocyte development, but also presents a novel perspective to understand the mechanisms regulating mammalian oogenesis.

overall_design: RNA-Seq analysis of goat oocytes at GV, GVBD, and MII stages.

first: Yixin

last: Ma

first: Wei

last: Zhang

first: Ming

last: Gao

first: Qiang

last: Wang

first: Minjian

last: Chen

first: Ling


title: Melatonin mitigates Chloroquine-induced defects in porcine immature Sertoli cells

city: jingzhou

first: wu

middle: zi

last: wei

state: Hubei

department: Animal Science

institute: yangtze university

summary: Chloroquine (CQ) could function as a lysosomotropic inhibitor to impair the autophagy-lysosome pathway, and is widely used to treat malarial, tumor and recently COVID-19. However, the effect of CQ treatment on porcine immature Sertoli cells (iSCs) remains unclear.;Here we showed that CQ treatment reduced iSCs viability in a dose-dependent manner. CQ treatment (20µM) of iSCs for 36h could elevate oxidative stress, damage mitochondrial function and promote apoptosis. Melatonin (MT) (10nM) could partially rescue CQ-induced phenotypic defects. Transcriptome profiling (RNA-seq) identified 1611 differentially expressed genes (DEGs) (776 up- and 835 down-regulated) (20µM CQ vs. DMSO control group), mainly involved in MAPK cascade, cell proliferation/apoptosis, HIF-1, PI3K-Akt and lysosome signaling pathways. In contrast, MT addition identified only 467 (224 up- and 243 down-regulated) DEGs (CQ+MT vs. DMSO), enriched in cell cycle, regulation of apoptotic process, lysosome and reproduction, respectively.Collectively, CQ treatment could impair porcine iSC viability by deranging apoptosis- and autophagy related signaling pathways, which could be partially rescued by MT supplementation.

overall_design: The cells were seeded at a density of approximately 1×105 cells/mL, and cultured for 6h, before treated with   Chloroquine (CQ, HY-17589, MedChem Express, New Jersey, USA)) at 20μM and 20μM CQ + 10nM MT(Melatonin, M5250, Sigma-Aldrich).

first: Cai-Xia

last: Yang

first: Zhi-Qiang

last: Du


title: High-throughput mRNA-sequence of CCl4 injected mouse liver

summary: RNA splicing is an essential step for expression of genes commonly altered in multiple liver diseases. However, how the spliceosomal components participate in the pathogenesis of acute liver failure (ALF) remains poorly defined. Here, we report that KH-type splicing regulatory protein (KSRP) which is downregulated in ALF, regulates RNA splicing in the liver through interacting with the Splicing factor 3b subunit 1 (SF3B1). Overexpression of KSRP resulted in marked amelioration of hepatic injury in vivo. Transcriptomic analysis reveals that KSRP depletion led to severe splicing defects in genes with significant intron retention. Such defects cause substantial loss of proliferation and apoptosis related genes such as EGFR, which further exaggerated liver injury. KSRP promotes RNA splicing of splicing related genes, including SF3B1, PHF5A, and SF3B4, indicating the existence of a positive feedback loop that regulates RNA splicing. Mechanistically, KSRP directly interacts with SF3B1 and enhances binding of SF3B1 to the branch sites located upstream of the exon. Overall, our findings demonstrate a novel mechanism by which KSPR protects against acute liver injury by promoting RNA splicing through interacting with the SF3B complex.

overall_design: Examination of differential expression genes in  CCl4 injected mouse liver

first: Rui

first: Mingxuan

first: Pingping

last: Xiao

first: Qian


title: MiRNA hijacking for self-modulating oligonucleotide therapy

city: Daejeon

first: Yoosik

address: Daehak-ro

institute: KAIST

summary: Due to a pivotal role in the post-transcriptional regulation of gene expressions implicated in numerous human diseases, miRNAs have been positioned to serve as promising disease biomarkers and novel therapeutic targets. However, gene silencing by miRNAs is limited to transcripts that contain complementary sequences. Herein we describe a miRNA-hijacker capable of downregulating newly assigned target mRNAs by hijacking intended miRNA. We developed a linear miRNA-hijacker with two miRNA binding sites to achieve higher repression and a hairpin-type (HT) miRNA-hijacker gifted with the hidden target mRNA binding site to minimize off-target effects. We also verified that the repression process by the miRNA-hijacker essentially involves the active mediating miRNA and functional AGO-RISC complex. By engineering the miRNA-hijackers to downregulate the anti-apoptotic BCL-xL gene, we successfully induced apoptosis only in the breast cancer cells overexpressing specific miRNAs and further validated its therapeutic efficacy in vivo, significantly reducing the tumor volume of the xenograft mouse upon its tail-vein injection. Our miRNA-hijacker technology can establish a new platform for self-modulating oligonucleotide therapy by hijacking disease-associated miRNAs and changing their destinations

overall_design: We transfected hairpin-structured miRNA-hijacker targeting PKR gene into HeLa cells overexpressing miR-141 (HeLa-141) and investigated differentially expressed genes.

first: Hansol

first: Doyeong

last: Ku

first: Namseok

last: Lee

first: Hyun

middle: G


city: BOSTON

first: CHANGHEE

last: LEE

address: 77 AVENUE LOUIS PASTEUR

overall_design: Single-cell RNA sequencing of the chick embryonic hindgut at E15, and embryonic midgut at E15 with and without ectopic, sparse, inducible HOXD13 misexpression in the mesoderm; Single-cell RNA sequencing of the chick embryonic E14 midgut and hindguts. All samples with biological multiplicates.

first: Hasreet

last: Gill

first: Sifan

last: Yin

first: Nandan

last: Nerurkar

first: John

last: Lawlor

first: ChangHee

first: Tyler

last: Huycke

first: L

last: Mahadevan

first: Clifford

last: Tabin


title: High-throughput mRNA-sequence of KSRP knock down cells in HL7702 CELLS

first: RUI

last: Zhou

overall_design: Examination of diferential expression genes in  KSRP stablely knock down HL7702 cells.

first: Rui

first: Mingxuan

last: Li


title: Cell-Cell communication between skinkeratinocytes and fibroblats is non-redundantly coded through inflammatory cytokines

status: Public on Jul 05 2024

city: Lübeck

first: Hauke

last: Busch

state: Schleswig-Holstein

address: Ratzeburger Allee 160

department: Lübeck Institute of Experimental Dermatology

country: Germany

institute: University of Lübeck

summary: Cell-cell communication between keratinocytes and fibroblasts is essential for skin homeostasis, regulation, and regeneration. How extracellular cytokine signaling, intracellular protein signaling and transcriptional regulation initiate and maintain this communication is still largely unknown. Here, we study in vitro the secretome and transcriptome dynamics that establish double paracrine cell-cell communication between normal human keratinocytes (NHK) and human dermal fibroblasts (HDF). To mimic double paracrine stimulation, we stimulate HDF with NHK-conditioned medium for 18 hours and use the then HDF-conditioned medium to stimulate the NHK. The NHK response is quantified at different timepoints  for a duration of up to 24 hours. We find that double paracrine stimulation changes extracellular matrix composition and activates translation and enhances keratinocyte migration.

overall_design: The response of NHK to double paracrine communication with human dermal fibroblasts  were  recorded at t=[ 1,2,4,6,12,24] hours after stimulation.  Unstimulated control time points were taken at  t=[ 0,4,12] hours.

first: Melanie.

last: Boerries

first: Juliana

last: Minardi-Nascimento

first: Silke

last: Kowar


title: Temporal recording of mammalian development and precancer [rna-seq]

first: Mirazul

last: Islam

overall_design: Mouse embryonic tissues and mouse intestinal epithelial (duodenum) tissues were dissociated using standard tissue dissociation procedues (methods section of the study). Cultured cell lines were also dissociated into single cells using TrypLE (Invitrogen). Dissociated single cells were encapsulated in a custom scRNA-seq (NSC-seq) platform.

first: Ken

last: Lau


title: High-throughput mRNA-sequence of KSRP knock down mouse primary hepatocytes

overall_design: Examination of diferential expression genes in  KSRP stablely knock down mouse primary hepatocytes.

first: Pingping

last: Xiao


title: Differences in the tRF & tiRNA Profiles of Sperm after Neonatal Sevoflurane Exposure

first: Lei

last: Lei

state: Henan

address: No.1 Jianshe east road

department: Department of Anesthesiology, Pain and Perioperative Medicine

institute: The First Affiliated Hospital of Zhengzhou University

type: Non-coding RNA profiling by high throughput sequencing

summary: The ncRNAs derived from transfer RNAs (tRNAs), such as tRFs (tRNA-derived fragments) and tiRNAs (tRNA halves), play crucial roles in sperm development, maturation, and function, ultimately affecting the health of offspring. To further elucidate the changes in sperm tRF & tiRNA profiles induced by neonatal sevoflurane exposure, we collected sperm from the caudal epididymis of rats and isolated total RNA for tRF & tiRNA sequencing.

overall_design: A comparative analysis of the tRF & tiRNA profiles was performed on small RNA-seq data from sperm samples of sevoflurane-exposed rats and control rats.

first: Qingsheng

last: Meng

first: Xiaoyu

last: Hu

first: Jian-jun

last: Yang


title: Human Accelerated Regions regulate gene networks implicated in apical-to-basal neural progenitor fate transitions

city: New Haven

first: Mark

middle: Alan

last: Noble

state: CT

address: 22 Gold Street, APT 102

institute: Yale University

summary: The evolution of the human cerebral cortex involved modifications in the composition and proliferative potential of the neural stem cell (NSC) niche during brain development. Genetic changes that altered the activity of transcriptional enhancers have been linked to phenotypic differences between humans and other primates. Human Accelerated Regions (HARs) consist of >1600 highly constrained noncoding regions of the genome that show significant evolutionary acceleration on the human lineage, suggesting they encode human-specific functions. Multiple studies support that HARs include neurodevelopmental enhancers with novel activities in humans, but the regulatory roles of HARs in NSC biology has not been empirically assessed at scale. Here we conducted a direct-capture Perturb-seq screen repressing 180 neurodevelopmentally active HARs in human iPSC-derived NSCs with single-cell transcriptional readout. After profiling over 188,000 NSCs, we identified a set of HAR perturbations with convergent transcriptional effects on gene networks related to the specification of basal radial glia (bRG), a progenitor population that is expanded in humans. Processes associated with these networks included the regulation of apicobasal polarity and migration. We found convergent dysregulation of specific apicobasal polarity and adherens junction regulators, including PARD3, ABI2, SETD2, and PCM1, which are critical to apical-basal RG fate decisions. Autism- and intellectual-disability-associated genes were also enriched among genes showing changes in expression due to HAR perturbations. Our findings reveal interconnected roles for HARs in NSC biology and cortical development and link specific HARs to processes implicated in human cortical expansion.

overall_design: Polyclonal human iPSC cells (line H23555, a gift from Y. Gilad) expressing KRAB:dCas9 were inducted into neural stem cells via dual SMAD inhibition for 3 days, then transduced with a direct-capture sgRNA library targeting 180 Human Accelerated Regions, 5 positive control loci, and 6 non-targeting control sequences. Cells were recovered then sorted for lentiviral BFP, and neural induction continued to 9 days. Experiments were performed in 3 batches across 8 GEM lanes each.

first: Noble

last: Mark Alan

first: Noonan

last: James


title: ac4C RIP-seq analysis of AGS cells upon NAT10 knockout.

city: GUANGZHOU

first: Min

last: Deng

state: GUANGDONG

address: Affiliated Cancer Hospital & Institute of Guangzhou Medical University, NO.78, Hengzhigang Road.

summary: N4-acetylcytidine (ac4C), a conserved chemical modification in eukaryotic prokaryotes that is catalyzed by the N-acetyltransferase 10 (NAT10) enzyme, plays a crucial role in promoting mRNA stability and translation. However, the biological function and mechanisms of NAT10-mediated ac4C in human cancer were poorly defined. In order to investigate the regulatory mechanism of NAT10 in gastric cancer, we performed ac4C RIP-seq(acRIP-seq) analysis in AGS cells with NAT10 knockout compared with control in two repeats.

overall_design: Identification of the downstream targets of NAT10 by acRIP-seq analysis in AGS cells upon NAT10 knockout


title: The Upstream Sequence Transcription Complex Dictates Nucleosome Positioning and Promoter Accessibility at piRNA Genes in the C. elegans germ line [small RNA-seq]

first: Valerie

last: Reinke

address: 333 Cedar St

institute: Yale University School of Medicine

summary: The piRNA pathway is a conserved germline-specific small RNA pathway that ensures genomic integrity and continued fertility.  In C. elegans and other nematodes, Type-I piRNA precursor transcripts are expressed from >10,000 small, independently regulated genes clustered within two discrete domains of 1.5 and 3.5 MB on Chromosome IV. These large domains likely play a significant role in promoting germline-specific expression of piRNAs, but the underlying mechanisms are unclear.  By examining the chromatin environment specifically in isolated germ nuclei, we demonstrate that piRNA clusters are located in closed chromatin, and confirm the enrichment for the inactive histone modification H3K27me3.  The piRNA biogenesis factor USTC (Upstream Sequence Transcription Complex) plays two roles – it promotes a strong association of nucleosomes throughout the domain, and it organizes the local nucleosome environment to direct the exposure of individual piRNA genes. Overall, this work reveals new insight into how chromatin state coordinates transcriptional regulation over large genomic domains, which has implications for understanding global genome organization in the germ line.

overall_design: small RNA sequencing (smRNA-seq) of isolated germline nuclei of control (ETOH) and ISW-1 knockdown for 3 h in C. elegans.

first: Sanchez

last: Nancy


title: The differential expression of lncRNAs in wnt/β-catenin pathway activation or inactivation of triple-negative breast cancer.

first: Hongyan

last: Huang

state: --- Select One ---

address: 253 Industrial Avenue middle, Haizhu District, Guangzhou

institute: Southern Medical University, Zhujiang hospital

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

summary: To screen the associated lncRNAs with wnt/β-catenin pathway activation of TNBC,through lncRNA microarray profiling between activated and inactivated wnt/β-catenin pathway of TNBC tissues, lnc-WAL (wnt/β-catenin associated lncRNA; WAL) was selected as the top upregulated lncRNA in wnt/β-catenin pathway activation compared with the inactivation group.

overall_design: In TNBC, to further identify wnt/β-catenin pathway activation-associated lncRNAs, we carried out one lncRNA tissue microarray to compare the differences between the activation group (n=5) and inactivation group (n=5) of TNBC tissues.

first: Hai


title: A coordinated attack by a bacterial secretion system and a small molecule drives prey specificity

first: William

middle: P

last: Robins

address: 77 Avenue Louis Pasteur NRB 1039

department: Microbiology

summary: Vibrio species are recognized for their role in food- and water-borne diseases in humans, fish, and aquatic invertebrates. We screened bacterial strains isolated from raw food shrimp for those that are bactericidal to Vibrio strains. Here we identify and characterize Aeromonas dhakensis strain A603 which shows robust bactericidal activity specifically towards Vibrio and related taxa but less potency toward other Gram-negative species. Using the A603 genome and genetic analysis, we show that two antibacterial mechanisms account for its vibriocidal activity -- a highly potent Type Six Secretion System (T6SS) and biosynthesis of a vibriocidal phenazine-like small molecule, named here as Ad-Phen. Further analysis indicates coregulation between Ad-Phen and a pore-forming T6SS effector TseC, which potentiates V. cholerae to killing by Ad-Phen.

sample_organism:
Vibrio parahaemolyticus
Aeromonas dhakensis

overall_design: Transcription of TM1 alone and when mixed with A603 and A603 T6SS and phenazine mutants

first: Wiliiam

first: Sarah

last: Beir

first: John

last: Mekalanos


title: Synthetic lethal CRISPR screen identifies a cancer cell-intrinsic role of PD-L1 in regulation of vulnerability to ferroptosis.

city: Hubei

first: Yang-Ying-Fan

last: Feng

address: Luonan stress

institute: wuhan university

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

overall_design: Refer to individual Series


title: RNA-seq analysis evaluating effects of Adssl1 overexpression in neonatal rat ventricular myocytes (NRVM)

city: Shanghai

first: Xiaxi

last: Dong

address: No.197, Ruijin 2nd Road (Yongjia Intersection)

department: Department of Cardiovascular Medicine, Ruijin Hospital

institute: Shanghai Jiao Tong University School of Medicine

summary: Reactivating cell cycle re-entry in adult cardiomyocytes  improves cardiac function after myocardial infarction (MI). De novo purine synthesis  is a critical source of nucleotides and is involved in modulating cell proliferation. Here, using gain-of-function genetic tools, we explored the role of the de novo purine synthesis enzyme Adssl1 in cardiac regeneration. RNA sequencing analysis suggested that Adssl1 overexpression induced strong dedifferentiation and cell cycle entry.

overall_design: To explore the mechanism through which Adssl1 regulates cardiomyocyte proliferation, we overexpressed Adssl1 in primary neonatal rat ventricular myocytes (NRVMs) using adenovirus. Then we performed RNA-seq analysis on Adssl1 NRVMs infected with ad5-Adssl1 or ad5-CTR virus for 48h. Comparative gene expression profiling analysis of RNA-seq data for NRVMs infected with ad5-Adssl1 or ad5-CTR virus.

first: Zhigang

last: Li


title: MTSS1 Suppresses NF-kB Activity in Lung Adenocarcinoma through Repression of RelA/p65 Phosphorylation

city: Hershey

first: Yuka

last: Imamura Kawasawa

state: PA

address: 500 University Dr.

department: College of Medicine, Pharmacology

institute: Penn State University

summary: Metastasis suppressor 1 (MTSS1) is a 755 amino acid protein found in the cell cytoplasm which binds to actin and promotes cytoskeleton organization and is a known suppressor of lung adenocarcinoma metastasis This study demonstrated that preservation of MTSS1 protein expression in lung adenocarcinoma was associated with a 20% 5-year survival advantage in patients.   Furthermore, overexpression of MTSS1 was found to reduce metastasis and disease progression  in an in-vivo orthotopic lung adenocarcinoma mouse model. Nuclear factor kappa B (NF-kB), an important nuclear transcription factor, has been shown to be constitutively active in lung adenocarcinoma and strongly associated with the development of metastasis.  The NF-kB RelA/p65 subunit is involved in NF-kB heterodimer formation and subsequent nuclear translocation leading to activation of NF-kB responsive gene transcription. Phosphorylation and acetylation of p65 are critical post-translational modifications required for NF-kB activation. In this study, we demonstrate that MTSS1 expression leads to decreased NF-κB mediated gene transcription through inhibition of p65 phosphorylation. These findings uncover a novel mechanism through which MTSS1 may regulate lung adenocarcinoma metastasis by impairment of NF-κB regulated gene transcription.

overall_design: Total 6 samples were analyzed; 3 replicates each for wild type cells and MTSS1 overexpressing groups, respectively.

first: Oana

last: Bollt

first: Shengchen

last: Lin

first: Yunzhan

first: David

last: Marino

middle: I

last: Kawasawa

first: Shengyu

first: Vladimir

middle: S

last: Spiegelman


title: Plexin D1 emerges as a novel target in the development of neural lineage plasticity in treatment-resistant prostate cancer

city: Sacramento

first: Chengfei

last: Liu

state: CA

address: 4645 2nd Ave Research III building, suite2300

institute: University of California, Davis

summary:
To explore the gene regulatory mechanisms underlying PLXND1 knockdown in neuroendocrine prostate cancer cells.
We performed RNA sequencing analyses using PLXND1-knockdown C4-2B-MDVR and H660 cells to identify the gene programs affected by the PLXND1.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for C4-2B MDVR and H660 cells (control siRNA, si PLXND1 #1, siPLXND1 #2).


title: Single Cell RNA sequencing of the non-tumor components of the tumor mass after either empty vector control or Zbtb46 overexpressing lentiviral intratumoral treatments

city: St. Louis

first: Ashraf Ul

last: Kabir

state: Missouri

address: 660 S Euclid Ave, BJCIH 8th floor, room 8302

department: Pathology and Immunology

institute: Washington University in St. Louis

summary: Tumor-angiogenesis and -immunity play critical roles in cancer progression and outcome. An inverse correlation of these two1 hints at common regulatory mechanism(s). Here, we report that Zbtb46, a repressive transcription factor and a widely accepted marker for classical dendritic cells (DCs)2,3, constitutes one such regulatory mechanism. Zbtb46 was downregulated in both DCs and endothelial cells (ECs) by tumor-derived factors to facilitate robust tumor growth. Zbtb46 downregulation led to a hallmark pro-tumor microenvironment (TME), including dysfunctional vasculature and immunosuppressive cell accumulation. Analysis of cancer patient data revealed a similar association of low ZBTB46 expression with an immunosuppressive TME and a worse prognosis. In contrast, enforced Zbtb46 expression brought dynamic changes in the TME landscape to mitigate the pro-tumor features and to enhance anti-tumor immune components, restricting tumor growth. Mechanistically, Zbtb46-deficient ECs were highly angiogenic, and Zbtb46-deficient bone-marrow progenitors upregulated Cebpb and diverted the DC program to myeloid lineage output, potentially explaining the myeloid lineage skewing phenomenon in cancer4. Conversely, enforced Zbtb46 expression normalized tumor vessels and, by suppressing Cebpb, skewed bone-marrow precursors towards more DC generation over macrophages, leading to an immune-hot TME. Remarkably, Zbtb46 mRNA treatment synergized with anti-PD1 immunotherapy to improve tumor management in pre-clinical models. These findings identify Zbtb46 as a common regulatory mechanism for angiogenesis and for myeloid lineage skewing in cancer and suggest that maintaining its expression could have therapeutic benefits.

overall_design: scRNA sequencing of the non-tumor components of the PyMT-BO1 bearing mice on day 16 after either empty vector control or Zbtb46 overexpressing lentiviral intratumoral treatments on Days 9, 12, and 15 post-tumor-transplantation were performed. Briefly, on day 16, tumor masses from a total of 5 mice were pulled in each group. Following enzymatic digestion, the cells were FACS sorted as live non-tumor (GFP-) population. For multiplexing, cells were split into separate tubes as either mCherry+ or mCherry- cells (mCherry is a fluorescence reporter included in both the empty vector and Zbtb46 overexpressing vector), marked with distinctive hashtags (Hashtag-B0301 for mCherry+ and Hashtag-B0302 for mCherry- cells), and pulled back to make two samples: control and treatment. The cells were then processed for single-cell library generation, using the 10X Genomics platform, and sequenced with Illumina NovaSeq 6000.

first: Ashraf

middle: U

first: Carisa

last: Zeng

first: Maxim

last: Artyomov

first: Kyunghee

last: Choi


title: Mouse cortex PV interneuron transcriptome changes assessed with FACS-assisted bulk RNA-seq

city: Baltimore

first: Ingie

last: Hong

state: MD

address: Neuroscience, 725 North Wolfe Street, Hunterian 1001

department: The Solomon H. Snyder Department of Neuroscience

institute: Johns Hopkins Medical Institute

summary: To investigate the novel link between CP-AMPARs and excitability, we assessed global PV interneuron transcriptome changes with FICSR-seq (Fixation-Capture Single Cell RNA Recovery-seq) on forebrain PV interneurons. FACS-assisted PV interneuron bulk RNA-seq of PV-Cre;lsl-eGFP-GluA2 mice and PV-Cre;lsl-eGFP controls showed no expression changes in 278 out of 279 genes comprising the major classes of ion channels and excitatory/inhibitory synapse proteins. This lack of expression changes suggests a post-transcriptional regulation of intrinsic and extrinsic (synaptic) excitability. The exception was GluA2 mRNA, which was expressed ~2 fold compared to control PV interneurons, in agreement with protein measurements. GluA1, although downregulated at protein level, was unchanged at the mRNA level. These transcriptomic results suggest that the substantial changes in PV interneuron excitability after CP-AMPAR removal are not supported by changes in gene expression but likely reflect post-transcriptional regulation.

overall_design: We developed a technique to preserve neurons after dissociation, fixation-capture single-cell RNA recovery-seq (FICSR-seq), which recovers PV interneurons vulnerable to dissociation at native cell frequencies. We cut brain slices from adult PV-Cre;lsl-eGFP and PV-Cre;lsl-eGFP-GluA2 mice (113.1 ± 11.6 days old) in NMDG cutting solution + Trehalose and diced them into small pieces <1 mm3. Extracellular proteins were digested with pronase (2 mg/ml; 8 U/µl) at 34-37°C, after which the slice pieces were fixed in 4% PFA in PBS (with 0.1 U/ml RNase inhibitor, Promega) for 15 mins and dissociated into single cells through careful trituration. We filtered the single cells through a 40 μm filter, labeled them with the cell-permeable nuclear dye DRAQ5 (1:1,000 dilution) to identify nuclei-containing cells, and then subjected them to FACS. DRAQ5+/GFP+ or DRAQ5+/GFP-GluA2+ cells were sorted, and over 20K cells were collected per mouse cortex to provide extensive coverage of low-expressing PV interneuron transcripts. We treated the fixed cells with Proteinase K before RNA extraction (RecoverAll Total Nucleic Acid Isolation Kit for FFPE, Thermo Fisher Scientific) to liberate RNA from protein-protein and protein-nucleic acid crosslinks generated by fixation. We prepared cDNA libraries from PV-Cre;lsl-eGFP and PV-Cre;lsl-eGFP-GluA2 mouse brains (NEBNext Ultra RNA Library Prep Kit for Illumina, NEB) from RNA enriched with mRNA through bead-based polyA selection. cDNA libraries were barcoded and sequenced together on an Illumina Hiseq 2500 sequencer, generating 150-bp paired-end reads. We processed RNA-seq reads with bcbio-nextgen v1.2.3, aligning to GRCm38 with the STAR aligner and quantifying counts per gene with Sailfish using the Ensembl annotation. We used DESeq2 to analyze differential expression.

first: Dong Won

last: Kim


title: RAG1/2 induces double-stranded DNA breaks at non-Ig loci in the proximity of single sequence repeats in developing B cells

city: Amsterdam

first: Katarina

last: Ochodnicka

address: Meibergdreef 9

country: Netherlands

institute: AmsterdamUMC

summary: In developing B cells, V(D)J gene recombination is initiated by the RAG1/2 endonuclease complex, introducing double-stranded DNA breaks (DSBs) in V, D and J genes and resulting in the formation of the hypervariable parts of immunoglobulins (Ig). Persistent or aberrant RAG1/2 targeting is a potential threat to genome integrity. While RAG1 and RAG2 have been shown to bind various regions genome-wide, the in vivo off-target DNA damage instigated by RAG1/2 endonuclease remains less well understood. In the current study, we identified regions containing RAG1/2-induced DNA breaks in mouse pre-B cells on a genome-wide scale using a global DNA DSB detection strategy. Around 1500 putative RAG1/2-dependent DSBs were identified, most of which were located outside the Ig loci. DNA sequence motif analysis showed a specific enrichment of RAG1/2-induced DNA DSBs at GA- and CA-repeats and at GC-rich motifs. These findings provide further insights into RAG1/2 off-target activity. The ability of RAG1/2 to introduce DSBs on the non-Ig loci during the endogenous V(D)J recombination emphasizes its genotoxic potential in developing lymphocytes.

overall_design: Chromatin immunoprecipitation of NBS1 in mouse v-Abl pre-B cell lines AH2 and R2K.Artm

last: Ochodnicka-Mackovicova

first: Michal

last: Mokry

first: Martin

last: Haagmans

first: Ted

last: Bradley

first: Carel

last: van Noesel

first: Jeroen

last: Guikema


title: Bulk RNA seq of sorted CD11c cells from lungs of uninfected and Mtb infected wild type mice.

city: Chicago

first: Shabaana

last: Khader

state: Illinois

address: 920 East 58th Street

institute: The University of Chicago

summary: To understand the difference between CD11c population from the uninfected and MTB HN878 infected mice at transcriptome level.

overall_design: CD11c cells sorted from lungs of uninfected and Mtb HN878 infected (50 dpi) wild type mice.


title: Computational design of sequence-specific DNA-binding proteins

city: Durham

first: Raluca

last: Gordan

state: NC

address: 101 Science Dr, CIEMAS 2179

department: Center for Genomic and Computational Biology

institute: Duke University

summary: Sequence-specific DNA-binding proteins (DBPs) play critical roles in biology and biotechnology, and there has been considerable interest in the engineering of DBPs with new or altered specificities for genome editing and other applications. While there has been some success in reprogramming naturally occurring DBPs using selection methods, the computational design of new DBPs that recognize arbitrary target sites remains an outstanding challenge. We describe a computational method for the design of small DBPs that recognize specific target sequences through interactions with bases in the major groove.

overall_design: Universal protein-binding microarray (PBM) experiments were performed for small, recombinant, DNA-binding protein designs 1, 3, 5, 6, 9, 35, & 48. Briefly, universal PBMs involved binding of his-tagged proteins to double-stranded 15K Agilent microarrays containing a DNA library designed to cover all possible 9-bp sequences, with every 7-mer occurring in at least 16 different spots on the array. This design allows comprehensive and unbiased characterization of the binding specificity of transcription factors for all possible 7-bp sequences.

first: Olivier

last: Boivin


title: Single-cell RNA and ATAC sequencing revealed distinct molecular characteristics of B cells in systemic lupus erythematosus and primary Sjogren's syndrome (scRNA-Seq)

city: HeFei

first: zhang

middle: xiao

last: jing

state: AnHui

address: 81 Meishan Road

department: dermatology

institute: Anhui Medical University

summary: Objective: Autoimmune diseases (ADs), such as systemic lupus erythematosus (SLE) and primary Sjögren's syndrome (pSS), are complex diseases with poorly understood pathogenetic mechanisms. Epigenetic changes are crucial regulatory elements in B-cell differentiation, and disruptions to these mechanisms may contribute to the development of ADs. We hypothesized that shared mechanisms may contribute to the pathogenesis of SLE and pSS by disrupting transcriptional circuits.

overall_design: Methods: B-cells were sorted by Flow cytometry, then entered into single-cell RNA sequencing (scRNA-seq) and single-cell assay for transposase-accessible chromatin sequencing (scATAC-seq). ArchR algorithm was used to integrate scRNA-seq and scATAC-seq data to investigate the transcriptome and chromatin accessibility of five B-cell subpopulations and their differentiation trajectories.

first: Zhaobing

last: Pan

first: Jiali

last: Zhang

first: Xiaojing

first: Qiaoshan

last: Yang

first: Ying

last: Yuan

first: Zhengwei

last: Zhu

first: Leilei

last: Wen

first: Fusheng

last: Zhou


title: A female-biased gene expression signature of dominance in cooperatively breeding meerkats

first: C. Ryan

last: Campbell

address: 130 Science Dr

department: Evolutionary Anthropology

summary: Dominance is a primary determinant of social dynamics and resource access in social animals. Recent studies show that differences in dominance are also reflected in the gene regulatory profiles of peripheral immune cells. However, the strength and direction of this relationship differs across the species and sex combinations investigated so far, potentially due to variation in the predictors and energetic consequences of dominant status. To test this possibility, we investigated the association between social status and gene expression in the blood of wild meerkats in the Kalahari Desert of South Africa, including in response to lipopolysaccharide, Gardiquimod, and glucocorticoid stimulation (n=740 samples; 113 meerkats). Meerkats are cooperatively breeding social carnivores in which breeding females physically outcompete other females and reproductive skew is high. They therefore present an opportunity to disentangle the effects of social dominance from those of sex per se. We identify a sex-specific signature of dominance, including 1,045 differentially expressed genes in females but none in males. Dominant females exhibit elevated activity in innate immune pathways, as well as an exacerbated immune response to LPS challenge. Female meerkats therefore resemble male baboons, where physical competition is also central to determining rank hierarchies and mating effort is high, but differ from female primates in which social status is determined by nepotism. Our results support the hypothesis that the gene regulatory signature of social status depends on the determinants and energetic costs of social dominance. They also support potential life history trade-offs between investment in reproduction versus somatic maintenance.

overall_design:
We purified PBMCs from ~1 mL of blood per blood draw via density gradient centrifugation, typically within 3 – 4 hours of the original blood draw. For each blood sample, we plated 200,000 PBMCs in 200 µL cell suspension into each of four tissue culture wells containing 20 µL cell culture media (RPMI; 10% FBS; 1% penicillin-streptomycin), for a total volume of 220 µL.
Purified PBMCs from each sample were cultured for 4 hours in (i) media only (control condition), (ii) media with 10 ng/mL lipopolysaccharide (LPS from the E. coli O111:B4 strain), to mimic bacterial exposure; (iii) media plus 1.0 μg/mL Gardiquimod (Gard), which activates Toll-like receptor 7 signaling; or (iv) media plus 1.0 μM Dexamethasone (Dex), a synthetic glucocorticoid. The cells were then incubated in parallel for 4 hours (37 C and 5% CO2), washed with 1× PBS, lysed, and stored immediately at -80° C until library preparation.

first: Campbell

last: C Ryan

first: Tung

last: Jenny


title: Long-term downregulation of SCN8A in mouse models of developmental and epileptic encephalopathy

city: Ann Arbor

first: Sophie

last: Hill

state: MI

address: 1241 E. Catherine St

department: Human Genetics

institute: University of Michigan

summary: De novo mutations of the voltage-gated sodium channel SCN8A cause severe developmental and epileptic encephalopathy (DEE).  Since pathogenic variants have gain-of-function effects on SCN8A activity, reduction of SCN8A expression is an effective therapeutic strategy. We previously described an antisense oligonucleotide (ASO) that delays seizure onset in a mouse model of SCN8A-DEE when administered at postnatal day 2. To investigate the potential effectiveness of post-onset ASO treatment, we first examined the extent of differential gene expression in hippocampus during the pre-onset period. Hippocampal single-nucleus RNA-sequencing detected only minor expression changes after the two month pre-seizure period.  ASO treatments that were initiated after seizure onset were protective in the Scn8a mutant mice during the 12 month observation period. As an alternative treatment for down-regulation of Scn8a, we administered a single dose of an AAV10 virus expressing Scn8a shRNA.  The viral shRNA was protective against seizures and lethality during the 12 month observation period. These data indicate that reduction of SCN8A expression, either by repeated administration of ASO or a single dose of shRNA virus, may be effective for longterm control of SCN8A-DEE.

overall_design: Nuclei from adult mouse hippocampus were isolated, purified with FACS sorting, and analyzed with single-nucleus RNA-sequencing (10X Genomics 3' gene expression).

middle: F

first: Miriam

last: Meisler


title: mRNA expression in gastric cancer

city: Xuzhou

first: Yang

last: Su

address: No. 209, Tongshan Road

department: Cancer Institute

institute: Xuzhou Medical University

summary: Gastric cancer (GC) is a one of the most common gastroenterological carcinoma, but its prognostic and immunological characteristics is not fully illustrated. Here we build an immune-associated gene prognostic model to predict the prognosis of GC. We performed RNA sequencing of 33 GC patients to verified the prognostic model.

overall_design: Thirty three gastric cancer tissues were obtained through biopsy or surgical procedures. RNA sequencing were performed to verified the difference in prognosis between the low risk group and high risk group.

first: Wei

last: Yin

first: Haixiao

last: Wang

first: Bi

last: Meng

first: Xia

last: Li

first: Yusi

first: Xianzheng

last: Wei

first: Yu-hang

last: Jin

first: Xiao-xue

first: Dan

last: Liu

first: Junian

last: Zheng

first: Ming

last: Shi


title: Blood-based Genomic and Cellular Determinants of Response to Neoadjuvant PD-1 Blockade in Patients with Non-Small-Cell Lung Cancer

city: Xi'an

first: Xi

last: Zhang

state: Shanxi

address: Northwest University, 229 North Taibai Road, Xi'an

department: 1 School of Life Science, Northwest University, Xi'an, Shaanxi 710069, China 2 Key Laboratory of Resource Biology and Biotechnology in Western China, Ministry of Education, School of Medicine, Northwest University, Xi'an, Shaanxi 710069, China

institute: Northwest University

summary: The integrated analyses employed in the current study identified blood-based molecular and cellular signatures that are associated with the responsiveness and overall survival of PD-1/PD-L1 blockade therapy plus chemotherapy.

overall_design: Comparative gene expression profiling analysis of RNA Seq data for 57 whole blood samples from 31 NSCLC patients and 5 healthy donors, including both responders and non-responders received anti-PD-1 Tislelizumab plus chemotherapy.

first: Rui

last: Chen

first: Wenqing

first: Shengchao

first: Mengju

last: Jiang

first: Guodong

first: Yuru

first: Yu

last: Cai

first: Wuhao

last: Huang

first: Yuyan

last: Xiong

first: Shengguang


title: tRFs&tiRNAs sequence in LUAD tissues with micropapillary /solid (High-Risk) component

status: Public on Jul 04 2024

city: nanjing

first: Qinglin

state: jiangsu

address: Jiangsu Cancer Hospital

institute: Jiangsu Cancer Hospital

summary: Despite being the leading cause of lung cancer death, the underlying molecular mechanisms driving metastasis progression are still not fully understood. tRNAs can generate a group of 18-40nt small RNA fragments named tRNA-derived small RNAs (tsRNAs) or tRNA-derived fragments (tRFs). Transfer RNA-derived fragments (tRFs) have been implicated in various biological processes in cancer. However, the role of tRFs in the development and progression of lung adenocarcinoma (LUAD) remains unclear. In the present study, we hypothesized that certain tRFs might become induced during LUAD metastatic progression. In this study, micropapillary and solid component was defined as high-risk, other component was defined as low-risk and adjcent tissue was defined as normal control. Our data revealed a series of dysregulated tRFs in 3 paired LUAD high-risk tissues, low-risk tissues and normal controls.

overall_design: Microdissection was performed after microscopic pinpointing of the High-Risk component and Low-Risk component. To identify and characterize specific differential expressed tRFs&tiRNAs in LUAD tissues with micropapillary /solid (High-Risk) component, tRFs&tiRNAs sequence was performed using 3 pairs of LUAD tissues with micropapillary /solid.With a cutoff criteria of fold change ≧8.0 and P<0.01, 26 significantly dysregulated tRFs&tiRNAs were identified by comparing High-Risk tissues cohort and adjacent normal tissues cohort while comparison between High-Risk tissues cohort and Low-Risk tissues revealed that 57 tRFs&tiRNAs were significantly dysregulated . By overlapping the two dysregulated tRFs&tiRNAs datasets, we obtained 15 significantly dysregulated tRFs&tiRNAs, which were specific differential expressed in High-Risk tissues group, including 9 up-regulated tRFs&tiRNAs and 6 down-regulated tRFs&tiRNAs (High-Risk) component and adjacent normal tissues.

first: Qixing

last: Mao


title: Gene expression profile at single cell level of low and high pigmented B16 cells [B16_D7_LP_HP_scRNA]

city: Delhi

first: Vivek

middle: T

last: Natarajan

state: Delhi

address: Mathura Road

country: India

institute: CSIR-IGIB

summary: B16 F10 is a depigmented mouse melanoma cell line which is considered as a homogeneous cell population. These cells when cultured at a very low density of 100 cells/cm2 starts to pigment progressively for a period of 7 days. Phenotypically two broad population of low and high pigmented cells are observed at day 7 in this low density culture. We used single cell RNA sequencing (scRNA-seq) to analyze the diversity of B16 cells cultured at low density for 7 days.

overall_design: B16 F10 cells were cultured at a very low density of 100 cells/cm2 for 7 days. The low and high pigment cells were sorted using the side scatter intensity of a flow cytometer

first: Aggarwal

last: Ayush

first: Natarajan

last: Vivek T


title: Single-cell RNA sequencing of bone marrow aspirate samples from multiple myeloma and its precursor conditions

city: Little Rock

first: Yan

last: Cheng

state: Arkansas

address: 4301 W. Markham St.

department: Internal Medicine

institute: University of Arkansas for Medical Sciences

summary: To determine the cell types and their transcriptional alterations during multiple myeloma progression from its precursor conditions, ie. monoclonal gammopathy of undetermined significance (MGUS), and smoldering multiple myeloma (SMM), we used single-cell RNA sequencing (scRNA-seq) to analyze the bone marrow aspirate samples from 4 newly diagnosed multiple myeloma, 6 MGUS and 4 SMM patients as well as 5 healthy donors.

overall_design: Sequential bone marrow aspirate samples were collected from 7 patients at different disease states and processed for scRNA-seq analysis.  Bone marrow aspirate samples from 5 healthy donors were used as controls.

first: Cheng

last: Yan

first: Sun

last: Fumou

first: Visanu

last: Wanchai

first: Zhan

last: Fenghuang


title: Transcriptomic profiling of Schlemm’s canal cells reveals a lymphatic-biased identity and three major cell states

first: Taibo

last: Li

address: 1830 E Monument Street, Suite 2-300

institute: Johns Hopkins School of Medicine

summary: Schlemm’s canal (SC) is central in intraocular pressure regulation but requires much characterization. It has distinct inner and outer walls, each composed of Schlemm’s canal endothelial cells (SECs) with different morphologies and functions. Recent transcriptomic studies of the anterior segment added important knowledge, but were limited in power by SEC numbers or did not focus on SC. To gain a more comprehensive understanding of SC biology, we performed bulk RNA sequencing on C57BL/6J SC, blood vessel and lymphatic endothelial cells from limbal tissue (∼4500 SECs). We also analyzed mouse limbal tissues by single-cell and single-nucleus RNA sequencing (C57BL/6J and 129/Sj strains), successfully sequencing 903 individual SECs. Together, these datasets confirm that SC has molecular characteristics of both blood and lymphatic endothelia with a lymphatic phenotype predominating. SECs are enriched in pathways that regulate cell-cell junction formation pointing to the importance of junctions in determining SC fluid permeability. Importantly, and for the first time, our analyses characterize 3 molecular classes of SECs, molecularly distinguishing inner wall from outer wall SECs and discovering two inner wall cell states that likely result from local environmental differences. Further, and based on ligand and receptor expression patterns, we document key interactions between SECs and cells of the adjacent trabecular meshwork (TM) drainage tissue. Also, we present cell type expression for a collection of human glaucoma genes. These data provide a new molecular foundation that will enable the functional dissection of key homeostatic processes mediated by SECs as well as the development of new glaucoma therapeutics.

overall_design: The anterior segment of the eye including the limbus was dissected from 3-month-old Prox1-GFP mice.  A strip of limbal tissue 1-2 mm wide consisting of the aqueous humor outflow tissues SC and TM, ciliary body, iris, and cornea was dissected from each anterior segment in ice-cold Dulbecco’s modified Eagle’s medium DMEM. To enrich for aqueous humor outflow tissues, limbal strips from 6 C57Bl/6J eyes (4 females, 2 males) were pooled for a single run of C57BL/6J scRNA-seq. C57BL/6J scRNA-seq was performed on two separate pools of eyes for a total of 12 eyes (8 females, 4 males). For 129/Sj scRNA-seq, 6 eyes (3 females, 3 males) were pooled for a single run.

first: Revathi

last: Balasubramanian

first: Krishnakumar

last: Kizhatil

first: Jiang

last: Qian

first: Simon

last: John


title: ACLY and ACSS2 link nutrient-dependent chromatin accessibility to regulation of CD8 T cell effector responses [RNA-seq]

city: Grand Rapids

first: Brandon

last: Oswald

address: 333 Bostwick Ave NE

department: Metabolism

institute: Van Andel Institute

summary: Coordination of cellular metabolism is essential for optimal T cell responses. Here, we identify cytosolic acetyl-CoA production as an essential metabolic node for CD8 T cell function in vivo. We show that CD8 T cell responses to infection depend on acetyl-CoA derived from citrate via the enzyme ATP citrate lyase (ACLY). However, ablation of ACLY triggers an alternative, acetate-dependent pathway for acetyl-CoA production mediated by acyl-CoA synthetase short chain family member 2 (ACSS2). Mechanistically, acetate fuels both the TCA cycle and cytosolic acetyl-CoA production, impacting T cell effector responses, acetate-dependent histone acetylation, and chromatin accessibility at effector gene loci. When ACLY is functional, ACSS2 is not required, suggesting acetate is not an obligate metabolic substrate for CD8 T cell function. However, loss of ACLY renders CD8 T cells dependent on acetate (via ACSS2) to maintain acetyl-CoA production and effector function.  Together, ACLY and ACSS2 coordinate cytosolic acetyl-CoA production in CD8 T cells to maintain chromatin accessibility and T cell effector function.

overall_design: Congenically marked OT-I Wild Type (WT) or OT-I ACLY Knockout (KO) CD8 T cells were adotpively transferred into C57Bl/6 mice one day prior to infection- with Listeria Monocytogenes ovalbumin (LmOVA). At six days post infection adoptively transferred cells were isolated and total RNA was extracted and sequenced. Differential gene expression was determined between WT and ACLY KO CD8 T cells.

first: Irem

last: Kaymak

first: McLane

last: Watson

middle: M

first: Brejnev

last: Muhire

first: Russell

last: Jones


title: Identification of candidate genes associated with the double flower of Brassica napus using RNA-seq

city: Wuhan

first: Junlin

last: Chen

address: No.1, Shizishan Street

institute: Huazhong Agricultural University

summary: It revealed that DEGs associated with floral development, including BnaC06.ARGOS, BnaC09.BPE, BnaA03.AUX1 and BnaAP2, and BnaC05.ERS2 involved in ethylene signalling potentially contribute to the double flower trait in B. napus.

overall_design: F2 individuals with opposite extreme phenotypes were selected for RNA-seq. At the bud stage, the apical meristem together with little buds (< 1mm) of each plant were collected for RNA extraction.


title: Small RNA sequencing based identification of miRNAs in Cnaphalocrocis medinalis

city: wenzhou

first: zhu

last: yu

address: ouhai district, liuhongqiao road

institute: Wenzhou Vocational College of Science and Technology

summary: This study analyzed the expression profiles of miRNAs in fifth instar larvae and pupae of the rice leaf folder (Cnaphalocrocis medinalis) using high-throughput sequencing technology. Furthermore, it predicted the target genes of significantly differentially expressed miRNAs and performed functional enrichment analysis.

overall_design: To investigate miRNA expression profile and differential expression information during the development of fifth-instar larvae and pupae of Cnaphalocrocis medinalis


title: Long-term downregulation of SCN8A in mouse models of developmental and epileptic encephalopathy II

first: Sophie

last: Hill

first: Miriam

last: Meisler