title: Optimizing Cost-Effective Gene Expression Phenotyping Approaches in Cattle Using 3′ mRNA Sequencing

city: Knoxville

first: Troy

last: Rowan

state: TN

address: 2506 River Dr

department: Animal Science Department

institute: University of Tennessee

summary: Background: Genetic and genomic selection programs require large numbers of phenotypes observed for animals in shared environments. Direct measurements of phenotypes like meat quality, methane emission, and disease susceptibility are difficult and expensive to measure at scale but are critically important to livestock production. Our work leans on our understanding of the “Central Dogma” of molecular genetics to leverage molecular intermediates as cheaply-measured proxies of organism-level phenotypes. The rapidly declining cost of next-generation sequencing presents opportunities for population-level molecular phenotyping. While the cost of whole transcriptome sequencing has declined recently, its required sequencing depth still makes it an expensive choice for wide-scale molecular phenotyping. We aim to optimize 3′ mRNA sequencing (3′ mRNA-Seq) approaches for collecting cost-effective proxy molecular phenotypes for cattle from easy-to-collect tissue samples (i.e., whole blood). We used matched 3′ mRNA-Seq samples for 15 Holstein male calves in a heat stress trail to identify the 1) best library preparation kit (Takara SMART-Seq v4 3′ DE and Lexogen QuantSeq) and 2) optimal sequencing depth (0.5 to 20 million reads/sample) to capture gene expression phenotypes most cost-effectively.  Results: Takara SMART-Seq v4 3′ DE outperformed Lexogen QuantSeq libraries across all metrics: number of quality reads, expressed genes, informative genes, differentially expressed genes, and 3′ biased intragenic variants. Serial downsampling analyses identified that as few as 8.0 million reads per sample could effectively capture most of the between-sample variation in gene expression. However, progressively more reads did provide marginal increases in recall across metrics. These 3′ mRNA-Seq reads can also capture animal genotypes that could be used as the basis for downstream imputation. The 10 million read downsampled groups called an average of 104,386 SNPs and 20,131 INDELs, many of which segregate at moderate minor allele frequencies in the population.  Conclusion: This work demonstrates that 3′ mRNA-Seq with Takara SMART-Seq v4 3′ DE can provide an incredibly cost-effective (<$25/sample) approach to quantifying molecular phenotypes (gene expression) while discovering sufficient variation for use in genotype imputation. Ongoing work is evaluating the accuracy of imputation and the ability of much larger datasets to predict individual animal phenotypes.

overall_design: Comparing two different 3` mRNA-Seq library prepartion kits (Takara SMART-Seq v4 3′ DE  and Lexogen QuantSeq 3′) in capturing gene expression pattern, differentially expressed genes, and sequence variants using whole blood samples collected from 15 Holstein male calves housed under climate control rooms.

first: Mohamed

last: Ruwaa

first: Ault-Seay

last: Taylor B.

first: Moisa

last: Sonia

first: Beever

last: Jonathan

first: Ríus

last: Agustín G.

first: Rowan

last: Troy


title: Aromatase in adipose tissue exerts osteoprotective function in male mice via phosphorus regulation

status: Public on Jul 22 2024

city: Toon

first: Yuuki

last: Imai

state: Ehime

address: Shituskawa

department: Proteo-Science Center

country: Japan

institute: Ehime University

type: Expression profiling by high throughput sequencing

summary: Aromatase contributes to the maintenance of bone mass because male patients with loss-of-function mutations of CYP19A1 exhibit bone loss. Furthermore, treatment with aromatase inhibitor also causes bone loss in men as well as post-menopausal women, in whom serum estrogen level is very low, suggesting that a part of the anabolic effects of testosterone in men is dependent on estradiol (E2) biosynthesized by aromatase in non-gonadal tissues. However, it remains unclear how locally biosynthesized E2 contributes to the maintenance of bone mass. We examined the function of aromatase in local tissues rather than gonad using cell type specific aromatase KO mice. Because osteoblast-specific aromatase KO mice exhibited no bone phenotype, we focused on adipose tissue, which is known as reservoir of steroid hormones, and analyzed the bone phenotypes of adipose tissue-specific aromatase KO mice (AroΔaP2). Sixteen-week-old male AroΔaP2 mice exhibit significantly lower bone mineral density in tibia and femur, especially in trabecular bone compared to the control. Bone histomorphometry showed that AroΔaP2 mice exhibited an osteomalacia-like bone phenotype with increased osteoid volume and width, and decreased osteoclast area and numbers. Moreover, serum PI and renal phosphorus reabsorption were significantly decreased but FGF23 level was decreased in AroΔaP2, suggesting that an osteomalacia-like phenotype in AroΔaP2 was not caused by abnormal FGF23 level. Finally, we analyzed NaPi2a and NaPi2c, a phosphate transporter localized in the kidney, and found that protein levels in renal brush border membrane vesicles were decreased in AroΔaP2. These results indicate that locally biosynthesized estrogens by aromatase in adipocytes can play a significant role in bone mass maintenance via regulate phosphorus reabsorption in kidney by NaPi2.

overall_design: To clarify target gene (s) of Cyp19a1 in adipose tissue that regulate phosphorus metabolism, we performed RNA-seq analysis using total RNA from eWAT in AroΔaP2 and Aroflox/flox.

first: Aoi

last: Ikedo


title: Single-cell transcriptome profiling of radial glia cells in zebrafish adult telencephalon in response to traumatic brain injury

city: Tancha

first: Manana

last: Kutsia

state: Okinawa

address: 1919-1 Tancha, Onna, Kunigami District, Okinawa 904-0495

institute: Okinawa Institute of Science and Technology Graduate University

summary: In zebrafish brain, radial glia cells (RG) show a remarkable ability to regenerate damaged neural tissue by reentering cell proliferation and produce neural precursors. To understand how RG respond to brain damage and initiate the regenerative response, we applied single-cell transcriptome analysis to RG in zebrafish adult telencephalon.

overall_design: Radial glia cells were isolated from the telencelphalon of adult zebrafish transgenic line Tg[GFAP:dTomato]nns17Tg by Fluorescence-activated cell sorting (FACS). Collected dTomato+ radial glia cells were subjected to single cell RNA-seq analysis using Chromium Single Cell 3' Reagents Kits v3 (10x chromium). The sequencing data were analyzed using 10x Genomics Cell Ranger 3.1.0 with default settings and zebrafish reference genome (GRCz11).

first: Yuki

last: Takeuchi

first: Nishtha

last: Ranawat

first: Ichiro

last: Masai


title: Gene expression profile of cells from cochlear organoids and comparison with in vivo auditory sensory epithelium cells.

status: Public on Jul 21 2024

city: Shanghai

first: Mingyu

last: Xia

state: Shanghai

address: No. 83, Fenyang Road, Xuhui District

department: ENT Institute and Otorhinolaryngology Department of the Affiliated Eye and ENT Hospital, Sta

country: China

institute: Fudan University

summary: We sequenced the total RNA of cells from inner ear organoids generated by EFI,EFI-4C and EFI-CL expansion strategies. Transcriptional profiles revealed that CHIR and LPA significantly increased proliferative potency and contributed to the stemness maintenance. We further compared the gene expression profiles among the sorted Lgr5+ CPCs, the primary organoids of passage (P)0, the P4 organoids, and the sorted E-cadherin+ sensory epithelial cells. Inner ear organoids recapitulated genetic characteristics of auditory epithelium rather than Lgr5+ progenitors alone. Differential gene analysis of cells from primary organoids and organoids at fourth passage indicated stable transcriptional profiles among passages, which confirmed the reliability and authenticity of cochlear organoids  for modeling the properties of the cochlea.

overall_design: Transcriptional profile of inner ear organoids generated by different expansion strategies, Lgr5+ CPCs and E-cadherin+ sensory epithelial cells.

first: Jiaoyao

last: Ma

first: Mingxuan

last: Wu

first: Luo

last: Guo

first: Shan

last: Sun

first: Yan

last: Chen

first: Wenyan

last: Li

first: Huawei


title: Organoids directly derived from L-MYC immortalized human neural stem cells (LMNSC01) model cerebral development in vitro

status: Public on Jul 20 2024

city: Duarte

first: Yate-Ching

last: Yuan

state: CA

address: 1500 East Duarte

country: USA

institute: City of Hope

summary: Developing platforms for ex vivo studies of human brain development and formulation of therapeutic strategies for a variety of conditions and diseases has been hampered by limited access to relevant human tissue and the necessity of using rodent models that imperfectly recapitulate human brain physiology. A promising advance is brain organoids that, to a greater extent than monolayer or spheroid cultures, recapitulate to varying extents the patterns of cell differentiation and morphological development characteristic of human brain.  Here, we present an organoid-based research platform initiated using L-MYC immortalized human fetal neural stem cells (NSCs) (LMNSC01), and) and grown in a physiological 4% oxygen environment similar that in developing brain. Expanded LMNSC01 cells maintain genomic stability and multipotency, and are non-tumorigenic.  Using authentic human NSCs as starting material eliminates the ex vivo programming and reprogramming required to achieve directed differentiation of human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs).      LMNSC008 brain organoids were characterized using NanoString technology to profile gene expression for over 120 days in vitro and comparing these patterns to these same genes during normal brain development (BrainSpan database).  We also visualized by immunofluorescence changes in expression and distribution of genes representative of these developmental processes along with morphological changes occurring over this same period.  We observe parallel developmental patterns of gene expression between organoids and developing cortex for pathways involved in neuronal cytoskeleton, neuron-glia interaction, neural connectivity, neurotransmission, metabolism, axon and dendrite structure, tissue integrity, angiogenesis, and myelination.       We suggest that LMNSC01 organoids offer an alternative to the challenges of iPSC programming and neural induction. Notable properties of this platform are its initiation with a line of authentic neural stem cells (LMNSC01), the consistency of the organoids produced, and favorable comparison of their gene expression patterns with those observed during normal fetal development.

overall_design: RNA expression was analyzed using the NanoString nCounter platform (NanoString Research) by digitally detecting and counting in a single reaction without amplification. We utilized the NanoString Neuropathology Profiling panel, consisting of genes targeting six fundamental themes: neurotransmission, neuron-glia interactions, neuroplasticity, cell structure integrity, neuroinflammation, and metabolism (Supplemental Table 1). Each assay also includes six positive and eight negative mRNA assay controls, plus ten housekeeping mRNA controls. RNA was hybridized with the gene panel Codeset at 65 oC for 16 hr. The post-hybridization probe-target mixture was quantified using the nCounter Digital Analyzer, with subsequent data analysis performed using nSolver. Raw data was first normalized to internal positive and negative controls to eliminate variability unrelated to the samples, then normalized to the selected housekeeping genes using Geometric Means methods. Gene expression variability of less than 20% within a single organoid was the criterion for acceptance. Replicates of 4-10 organoids were used for each timepoint.       mRNA extraction and preparation for NanoString was done at the COH Pathology Core. Briefly, mRNA was extracted using the miRNeasy mini kit (HB-1277-066, Qiagen). Concentration was assessed by Nanodrop spectrophotometer ND-1000 and Qubit 3.0 Fluorometer (Thermo Scientific), with mRNA fragmentation and quality assessed by 2100 Bioanalyzer (Agilent).     NanoString data were compared against a reference database for expression of these same genes during normal fetal development drawn from the BrainSpan Atlas of the Developing Human Brain (https://www.brainspan.org/). To facilitate comparisons, organoid and fetal brain datasets were both normalized to expression of the same NanoString internal reference genes (Supplementary Table 1) using Singscore(Foroutan, Bhuva et al. 2018) (PMID: 30400809), with the score indicating deviation from these genes. Singscore ranks gene expression, and calculates a non-parametric score for individual genes in each gene set (NanoString or BrainSpan). This rank-based approach is robust to differences in the underlying data distribution, enabling scoring of gene sets across different transcriptomic datasets and for assessment of gene set activity in individual samples. In the single-sample presentation employed here, scores range between 0.0 and 1.0 and represent the extent to which each gene in the gene set is expressed compared to the distribution of reference genes, and scored as higher (score > 0.5) or lower (score < 0.5).

first: Raju

last: Pillai

first: Holly

last: Yin

first: Michel

last: Barish

first: Margarita

last: Gutova


title: Astrocytes modulate a specific paraventricular thalamus-prefrontal cortex projection to enhance consciousness recovery from sevoflurane anesthesia in mice

city: Chengdu

first: Cheng

last: Zhou

state: Sichuan

address: No District, Chengdu, Sichuan Province. 37 Guoxue Lane, Wuhou

institute: West China Hospital of Sichuan University

summary: Previous studies have proved that astrocytes may be a key neural substrate that regulates wakefulness and consciousness recovery from general anesthesia, while the exact molecular target in astrocytes is still unclear. Using virus injection and in vivo fiber photometry in mice, we found both activating astrocytes and knocking down astrocytic Kir4.1 in paraventricular thalamus (PVT) promotes the consciousness recovery from sevoflurane anesthesia. Single-cell RNA sequencing of PVT reveals two distinct cellular subtypes of glutamatergic neurons: PVTGRM and PVTChAT neurons. Patch-clamp recording results proved that Astrocytic Kir4.1-mediated modulation of sevoflurane on PVT mainly works on PVTChAT neurons. Moreover, we found that PVTChAT neurons project mainly to the mPFC. This specific paraventricular thalamus to prefrontal cortex projection is involved in recovery of consciousness from sevoflurane anesthesia indirectly through modulation of astrocytes. In summary, our findings support the novel conception that the volatile anesthetic sevoflurane can inhibit PVT astrocytic Kir 4.1 to maintain and/or increase neuronal firing of PVTChAT neurons, which mainly projects to mPFC and promotes consciousness recovery from anesthesia.

overall_design: Using virus injection and in vivo fiber photometry in mice, we found both activating astrocytes and knocking down astrocytic Kir4.1 in paraventricular thalamus (PVT) promotes the consciousness recovery from sevoflurane anesthesia. Single-cell RNA sequencing of PVT reveals two distinct cellular subtypes of glutamatergic neurons: PVTGRM and PVTChAT neurons. Patch-clamp recording results proved that Astrocytic Kir4.1-mediated modulation of sevoflurane on PVT mainly works on PVTChAT neurons. Moreover, using virological tracer, we found that PVTChAT neurons project mainly to the mPFC.

first: Yi

last: Zhao

first: Mengchan

last: Ou

first: Jin

last: Liu

first: Jingyao

last: Jiang

first: Donghang

last: Zhang

first: Yaoxin

last: Yang

first: Bowen

last: Ke

first: Yujie

first: Ruotian

first: Daniel

middle: K

last: Mulkey

first: Tao

last: Zhu

first: Hugh

middle: C

last: Hemmings Jr


title: Asrij/OCIAD1 depletion reduces inflammatory microglial activation and ameliorates Aβ pathology in an Alzheimer's disease mouse model

city: Bangalore

first: Maneesha

middle: S

last: Inamdar

state: Karnataka

address: JNCASR, Jakkur, Bangalore.

department: Molecular Biology and Genetics Unit (MBGU)

country: India

institute: Jawaharlal Nehru Centre for Advanced Scientific Research (JNCASR)

summary: Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by the accumulation of amyloid-beta (Ab) plaques and neurofibrillary tangles, neuroinflammation, and glial activation. Asrij/OCIAD1 (Ovarian Carcinoma Immunoreactive Antigen Domain containing protein 1) is an AD-associated factor. Increased Asrij levels in the brains of AD patients and mouse models are linked to the severity of neurodegeneration. However, the contribution of Asrij to AD progression and whether reducing Asrij levels is sufficient to mitigate Ab pathology in vivo is unclear. To explore the impact of Asrij on AD pathology, we deleted asrij in the APP/PS1 mouse model of AD and analyzed the effects on AD hallmarks. We find that Asrij depletion ameliorates cognitive impairments, Ab deposition, neuronal and synaptic damage, and reactive astrogliosis in the AD mouse. Emerging evidence indicates a critical role of microglia in influencing AD pathology. Notably, Asrij-deficient microglia exhibit reduced plaque-associated proliferation and decreased phagocytic activity. Transcriptomic analyses of AD microglia reveal upregulation of energy metabolism pathways and downregulation of innate immunity and inflammatory pathways upon Asrij depletion. Mechanistically, loss of Asrij increases mitochondrial activity and Akt/mTOR signaling and impedes the pro-inflammatory Disease-Associated Microglia (DAM) state. Reduced levels of pro-inflammatory cytokines and decreased STAT3 and NF-kB activation indicate protective changes in AD microglia. Taken together, our results suggest that increased Asrij levels reported in AD, may suppress microglial metabolic activity and promote inflammatory microglial activation, thereby exacerbating AD pathology. Our study establishes a novel role for Asrij in regulating microglial responses to Ab pathology, and could be a potential target for therapeutic intervention.

overall_design: To investigate the role of Asrij in microglial activation and Alzheimer's disease (AD), we constitutively deleted asrij in APP/PS1 mouse model of AD to generate APP/PS1+/asrij-/- (AD/KO) mice. We performed bulk RNA sequencing analysis of AD and AD/KO CD11b+ microglia isolated by Magnetic-activated cell sorting (MACS).

first: Prathamesh

last: Dongre

first: Madhu

last: Ramesh

first: Thimmaiah

last: Govindaraju


title: A PLURIPOTENT STEM CELL PLATFORM FOR in VITRO SYSTEMS GENETICS STUDIES of MOUSE DEVELOPMENT

city: New York

first: Rachel

last: Glenn

address: 1300 York Avenue

institute: Weill Cornell Medicine

summary: The directed differentiation of pluripotent stem cells (PSCs) from panels of genetically diverse individuals is emerging as a powerful experimental system for understanding genotype-phenotype relationships and modeling polygenic traits and disorders. Here, we establish a genetic reference panel of naïve and primed PSCs (n = 250) from the Diversity Outbred (DO) mouse stock to enable high resolution quantitative trait locus mapping of molecular phenotypes in developing cellular systems.  These data, methods, and cellular resources provide a roadmap for population-scale studies to dissect the genetic basis of complex traits that will complement and empower similar studies in human populations.

overall_design: Ultimately, our objective is to use the panel of genetically distinct DO PSC lines to precisely measure how genetic variants in populations modulate molecular features of pluripotency and directed differentiation using single cell genomics.    Instead of individually phenotyping each cell line, pooled cell culture (“cell villages”) has been implemented in human PSCs to analyze cellular phenotypes at scale with reduced technical variability and lower material and labor costs.  To evaluate a cell pooling strategy for DO PSCs, we selected 76 distinct EpiSC lines including 49 male and 24 female lines (n=2 sex genotype XO, n=1 sex genotype undetermined) (Fig. 4A).   We thawed 15 pools of 5-6 DO PSCs each and cultured them in parallel for 48 hours.  We then combined a total of 30,000 cells from 5 distinct pools in equal proportion for single-cell RNA-sequencing across four lanes (n=28-30 total cell lines per sequencing lane).  In total, we sequenced 120,000 cells targeting a depth of 40,000 reads per cell (Fig. 4A).  We generated a pipeline to impute each donor’s full genome and output VCF genotype files for compatibility with Dropulation.  The Dropulation pipeline was adjusted for use with the GRCm38 mouse genome assembly to assign the most likely donor genotype to each cell and perform doublet detection.

first: Meik

last: Kunz

first: Thomas

last: Vierbuchen


title: Transcriptome sequencing of tumor associated macrophages cells in HCC liver of OXCT1mKO mice.

city: Guangzhou

first: Kai

last: Yan

state: Guangdong

address: No. 106, Zhongshan 2nd Road, Guangzhou

institute: Guangdong Provincial People's Hospital

summary: 6-8 weeks male LysCre/+ OXCT1fl/fl (OXCT1mKO or mKO for short) and its littermates Lys+/+ OXCT1fl/fl (control or ctrl) mice were used to build HCC implantation model (Hep1-6 in Portal Vein injection). RNA was extracted from tumor associated macrophages of OXCT1mKO and their littermate control mice for transcriptome analysis of macrophage function.

overall_design: Tumor associated macrophages cells sorted from liver of liver of OXCT1mKO HCC mice

first: Chuxu


title: Comprehensive transcriptome analysis of osteoclasts with the SIK3 deletion and pterosin B treatment.

city: Toyama

first: Jun-Dal

last: Kim

state: Toyama

address: 2630 Sugitani

department: Department of Complex Biosystem Research (CBR), Institute of National Medicine (INM)

institute: University of Toyama

summary: Maintenance of bone homeostasis and the balance between bone resorption and formation are crucial for maintaining skeletal integrity. Osteoclasts are responsible for bone resorption and play a vital role in this process. The role of salt-inducible kinase 3 (SIK3) in osteoclast differentiation and maturation and its potential as a therapeutic target for osteoporosis remain poorly understood. Here, we generated osteoclast-specific Sik3 conditional knockout (cKO) mice and investigated the effects of SIK3 deletion on bone homeostasis. Our findings revealed increased bone mineral density and an osteopetrosis phenotype in Sik3 cKO mice, indicating the involvement of SIK3 in bone resorption. Furthermore, we examined the effect of pterosin B, an SIK3 inhibitor, on osteoclast differentiation. Pterosin B treatment inhibits osteoclast differentiation in vitro, reduces the number of multinucleated osteoclasts, and suppresses bone resorption. Therefore, in this study, to better understand the molecular mechanism of SIK3 in osteoclast differentiation and maturation, we performed gene expression profiling of bone marrow monocytes from SIK3 deletion and pterosin B treated mice using RNA sequencing (RNA-seq).

overall_design: mRNA profiles of embryonic livers were generated by RNA sequencing using the Illumina NovaSeq 6000   (Illumina).

first: Yasuhito

last: Yahara


title: Single cell and bulk RNA transcriptional profile of cells from cochlear organoids

first: Mingyu

last: Xia

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

overall_design: Refer to individual Series


title: Visium Spatial transcriptomics of murine small intestine in steady state with or without LXR agonist GW3965

status: Public on Jul 19 2024

city: Stockholm

first: Jennifer

last: Fransson

address: Karolinska Institutet

country: Sweden

institute: Karolinska Institutet

summary: In this dataset, we investigate the murine small intestine spatial transcriptomic profile at the steady state and compare the genes alteration with and without LXR activation by sythetic ligand GW3965 diet.

overall_design: Wild type C57BL/6J mice were under standard or GW3965-supplemented diet for 10 days then mouse small intestine (including jejunum and ileum) were collected on day 10. Intestines were prepared as Swiss rolls and analyzed using Visium Spatial Transcriptomics (10X).

first: Xinxin

last: Luo

first: Srustidhar

last: Das

first: Eduardo

middle: J

last: Villablanca


title: Comparing plasma donor-derived cell-free DNA to gene expression in endomyocardial biopsies in the Trifecta-Heart study.

city: Edmonton

first: Jessica

last: Chang

address: 250 Heritage Medical Research Centre

department: Medicine

country: Canada

institute: University of Alberta

summary:
Background. Plasma donor-derived cell-free DNA (dd-cfDNA) is used to screen for rejection in heart transplants. We launched the Trifecta-Heart (ClinicalTrials.gov #NCT04707872), an investigator-initiated, prospective trial, to examine the correlations between genome-wide molecular changes in endomyocardial biopsies (EMBs) and plasma dd-cfDNA. The present report analyzes the correlation of plasma dd-cfDNA with the gene expression in EMBs from 4 vanguard centers and compared these correlations with those in 604 kidney transplant biopsies in the Trifecta-Kidney study (ClinicalTrials.gov #NCT04239703).
Results. Top transcripts correlating with dd-cfDNA were related to genes increased in rejection such as IFNG-inducible genes (e.g., HLA-DMA), but also with genes induced by injury and expressed in macrophages (e.g., SERPINA1, HMOX1). In gene enrichment analysis, the top dd-cfDNA-correlated genes reflected inflammation and rejection pathways. Dd-cfDNA correlations with rejection genes in EMB were similar to those seen in kidney transplant biopsies, with somewhat stronger correlations for TCMR genes in hearts and ABMR genes in kidneys. However, the correlations with parenchymal injury-induced genes and macrophage genes were much stronger in hearts.
Conclusions: In heart transplants in the Trifecta-Heart study, dd-cfDNA correlates significantly  with molecular rejection but also with injury and macrophage infiltration, reflecting the  proinflammatory properties of injured cardiomyocytes. The relationship supports the utility of dd-cfDNA in the clinical management of heart transplant recipients.

overall_design: Methods. We analyzed 137 consecutive dd-cfDNA-EMB pairs from 70 patients. Plasma %dd-cfDNA was measured by the ProsperaTM test (Natera, Inc.), and gene expression in EMBs was assessed by Molecular Microscope® Diagnostic System (MMDx) using machine-learning algorithms to interpret rejection and injury states.


title: Single cell transcriptional profile of cells from cochlear organoids

summary: We collected single cell transcriptome profiles of inner ear organoids  from passage 2 (day 8) in expansion medium, differentiated day 5, and differentiated day 25. Cells from the three time points were divided into 5 distinct clusters by UMAP, indicating the presence of the basic cell types of the organ of Corti in cochlear organoid model. Deafness-related gene expression in differentiated day 25 organoid showed that the cell-type specificity related to genetic auditory disease was preserved in the organoids, and dynamic gene expression changes were observed along lineage trajectories form progenitors to mature cell types, similar to in vivo developmental process. These results showed that cochlea organoid can be used as a useful model for deciphering the effects of deafness gene mutations and understanding inner ear development.

overall_design: Single cell transcriptional profile of inner ear organoids in expansion medium, early differentiation, and late differentiation phase.

first: Jiaoyao

last: Ma

first: Mingxuan

last: Wu

first: Luo

last: Guo

first: Shan

last: Sun

first: Yan

last: Chen

first: Wenyan

last: Li

first: Huawei


title: RNAseq analysis of ClpP siRNA treated mouse primary hepatocytes

city: Busan

first: Jong-Ho

last: Lee

address: 37 Nakdong-daero 550 Beon-gil

country: South Korea

institute: Dong-A University

summary: We report the gene expression profile in mouse primary hepatocytes after treatment of ClpP siRNA.

overall_design: To analyze the changes of gene expression after treatment with ClpP siRNA in mouse primary hepatocytes, the mouse primary hepatocytes were treated with ClpP siRNA using Lipofectamine 2000. The TruSeq RNA Sample Preparation Kit v2 (Illumina, #RS-122-2001) was used to convert poly-A containing mRNAs in total RNA to a cDNA library using poly-T oligos linked to magnetic beads. Following mRNA purification, the RNA was physically fragmented at an elevated temperature prior to reverse transcription and cDNA generation. The fragmentation step resulted in the production of an RNA-Seq library containing inserts ranging in size from approximately 100-400 base pairs (bp). The average insert size in an Illumina TruSeq RNA-Seq library is approximately 200 bp. The cDNA fragments were then subjected to an end-repair process, during which a single ‘A’ base was added to the 3’ ends and then adaptors were ligated. Finally, the products were purified and enriched by PCR to generate the final double-stranded cDNA library. The libraries were quantified using KAPA Library Quantification Kits for Illumina Sequencing platforms according to the qPCR Quantification Protocol Guide (KAPA Biosystems, KK4855) and qualified using the TapeStation D1000 ScreenTape device (Agilent Technologies, 5067-5582).

first: Yup

last: Kang

first: Sung-E

last: Choi


title: Transcriptome analysis of valvular interstitial cells (VICs) treated with lipoprotein (LDL or oxLDL)

city: Seoul

first: Jae-Hoon

address: 222 Wangsimniro, Seongdong Gu

department: Department of Life Science

institute: Hanyang University

summary: We performed RNA sequencing of cultured VICs treated with LDL or oxLDL to determine the inflammatory role of VICs in lipoprotein-treated samples compared to control.

overall_design: mRNA profiles of cultured VICs treated with and without LDL or oxLDL


title: RNA-sequencing of gastric cancer PDX mice model after treated with CUS

city: Wuhan

first: sheng

last: tian

address: 169 Donghu Road, Wuchang District, Wuhan City, Hubei Province.

institute: zhongnan hospital

summary: The interaction between cancer and depressive disorder remains unclear.  In this study, we obtained patient data from TCGA and MDD-related (Major Depressive Disorder) genes from the GEO database and the sequencing data of the PDX model with chronic unpredictable stress (CUS). Molecular subtypes and a prognostic signature were generated based on the MDD-related genes obtained by differential expression analysis. According to our analysis, the gene signature was a promising biomarker in distinguishing the prognosis, the molecular or immune characteristics, and the depressive risk, as well as the therapy candidates for gastric adenocarcinoma patients.

overall_design: To investigate the impact of depression on the expression of gastric cancer, we implanted surgical tissue fragments (obtained from a 77-year-old female gastric adenocarcinoma patient at Zhongnan Hospital of Wuhan University) into female NOD-SCID mice to establish a patient-derived xenograft (PDX) model. After three successive transplantations, the xenografts were isolated and implanted into the right axilla of BALB-c/nu female mice. Chronic unpredictable stress (CUS) was then applied to the PDX model for three weeks. Subsequently, gene expression profiling analysis was performed using data obtained from RNA-seq of three tumor tissues from PDX exposed to CUS and three tumor tissues from PDX not exposed to CUS.

first: Yixin

last: Liu

first: Yongchang

last: Wei


title: Initial characterization of WDR5B reveals a role in the proliferation of retinal pigment epithelial cells

city: Santa Barbara

first: Jeffrey

middle: K.

last: Bailey

department: Department of Molecular, Cellular and Developmental Biology, Neuroscience Research Institute

institute: University of California, Santa Barbara

summary: The chromatin-associated protein WDR5 has been widely studied due to its role in histone modification and its potential as a pharmacological target for the treatment of cancer.  In humans, the protein with highest sequence homology to WDR5 is encoded by the retrogene WDR5B, which remains unexplored.  Here, we used CRISPR-Cas9 genome editing to generate WDR5B knockout and WDR5B-FLAG knock-in cell lines for further characterization.  In contrast to WDR5, WDR5B exhibits low expression in pluripotent cells and is upregulated upon neural differentiation.  Loss or shRNA depletion of WDR5B impairs cell growth and increases the fraction of non-viable cells in proliferating retinal pigment epithelial (RPE) cultures.  CUT&RUN chromatin profiling in RPE and neural progenitors indicates minimal WDR5B enrichment at established WDR5 binding sites.  These results suggest that WDR5 and WDR5B exhibit several divergent biological properties despite sharing a high degree of sequence homology.

overall_design: CUT&RUN sequencing was performed using FLAG or rabbit IgG (control) antibodies in human ARPE-19 cell lines stably expressing WDR5-FLAG or WDR5B-FLAG (2 biological replicates per condition).  ARPE-19 cells were grown in Lonza XVIVO10 medium and harvested on day 8 post-seeding. CUT&RUN sequencing was also performed using WDR5, FLAG, or rabbit IgG (control) antibodies in human H9 ESC-derived neural ectoderm progenitor cells edited using CRISPR-Cas9 to endogenously express WDR5B-FLAG (2 biological replicates per condition).  Neural ectoderm cells were differentiated for 6 days prior to CUT&RUN in DMEM/F12 medium containing 1X B-27, 1X N-2, 10mM nicotinamide, 10µM SB431542, and 5µM DMH1. All cells were cultured at 37C with 5% CO2.

first: Dzwokai

first: Dennis

middle: O

last: Clegg


title: The cellular hierarchy of acute myeloid leukemia informs personalized treatment

city: Zuerich

first: Yannik

last: Severin

state: ZH

address: Otto-Stern-Weg 3

department: Biology

country: Switzerland

institute: ETH Zurich

summary: Acute myeloid leukemia (AML) is characterized by malignant myeloid precursors that span a cellular  hierarchy from dedifferentiated leukemic stem cells to mature blasts. While the diagnostic and prognostic importance of AML blast maturation is increasingly recognized, personalized therapies are currently not tailored to a patient’s individual makeup of this cellular hierarchy. In this study, we use multiplexed image-based ex vivo drug screening (pharmacoscopy) to systematically quantify the drug sensitivity across the cellular hierarchy of AML patients. We analyzed 174 prospective and longitudinal patient samples from 44 newly diagnosed AML patients, which indicated that differences in the AML hierarchy significantly identified poor responses to first-line therapy, outperforming European LeukemiaNet (ELN) criteria. Critically, drug response profiling across the AML hierarchy of each patient improved the accuracy of predicting patient response to first-line therapy (AUC 0.91), and revealed alternative individualized treatment options targeting the complete AML hierarchy of non-responding patients. We confirmed these findings in an independent cohort of 26 relapsed/refractory AML patients, for whom pan-hierarchy response profiling improved response predictions post hoc. Overall, our results quantify the clinical importance of therapeutically targeting the complete cellular hierarchy of newly diagnosed AML, and identify multiplexed image-based ex vivo drug screening to enable quantification and targeting of the AML maturation hierarchy for improved personalized treatment.

overall_design: Baseline transcriptomes of bulk AML samples from two different tissues at 3 different time points were sequenced in a single run on a NovaSeq6000 (single read, 100bp, depth 20 Mio reads per sample)

first: Yasmin

last: Festl

first: Tobias

middle: M

last: Benoit

first: Berend

last: Snijder


title: Human intestinal stromal cells promote homeostasis in normal mucosa but inflammation in Crohn's disease in a retinoic acid-deficient manner

city: Birmingham

first: Phillip

middle: D

last: Smith

state: AL

address: 1825 University Blvd

institute: University of Alabama at Birmingham

summary: Stromal cell (SC) contributions to intestinal inflammation are inadequately understood.  Here we show that CD90+vimentin+SMA- SCs isolated from Crohn’s disease intestine (Crohn’s SCs) displayed higher levels of spontaneously released and microbiota-induced inflammatory mediators, including IL-6 and TGF-b, and surface adhesion molecules, than SCs isolated from healthy intestine (Normal SCs).  Comparative transcriptome analysis confirmed the predominance of upregulated inflammatory response and cytokine signaling genes and pathway molecules in Crohn’s SCs.  Importantly, intestinal SCs provided retinoic acid (RA), which regulated DC maturation, and in an in vitro cross-talk system E. coli LF82-stimulated Crohn’s SCs promoted differentiation of more inflammatory TNFhi/IL-12lo/RAlo DCs and IFN-ghi/IL-17hi effector T cells than Normal SCs.  Explaining this finding, Crohn’s SCs synthesized less RA and expressed more retinaldehyde reductase DHRS3, which inhibits retinol conversion to retinal, than Normal SCs.  Our findings uncover microbe-SC crosstalk in which mucosal SCs initiate and perpetuate DC-driven T-cell-mediated inflammation in Crohn’s disease.

overall_design: 14 samples were analyzed, half the samples were controls

first: Lesley

middle: E

last: Smythies

first: Marie

last: Nearing

first: Diane

last: Bimczok

first: Olga

middle: V

last: Belyaeva

first: Heidi

last: Nick

first: Carolina

middle: A

last: Serrano

first: Kayci

middle: R

last: Huff

first: Lois

last: Musgrove

first: Jaleesa

last: Garth

first: David

last: Crossman

first: Jamie

last: Cannon

first: Natalia

middle: Y

last: Kedishvili

first: Katie

middle: L

last: Alexander

first: Kirk

last: Russ

first: Kondal

last: Baig

first: Shajan

last: Peter

first: Charles

last: Elson


title: The MLL3/GRHL2 complex regulates early malignant transformation and anti-tumor immunity in squamous cancer

city: Shenzhen

first: Yueyuan

last: Zheng

state: China

address: Zhenyuan Road, 628

institute: The Seventh Affiliated Hospital of Sun Yat-sen University

type: Genome binding/occupancy profiling by high throughput sequencing


title: The MLL3/GRHL2 complex regulates early malignant transformation and anti-tumor immunity in squamous cancer [RNA-seq]

summary: We previously identified that MLL3, also known as lysine methyltransferase 2C, is frequently mutated in Upper Aerodigestive Squamous Cell Carcinoma (UASCC). TCGA dataset also demonstrated that around 10% of UASCC patients have a mutation of MLL3. However, the significance of MLL3 in the context of premalignant biology and neoplastic evolution has not been well understood. Therefore, we aim to explore the functional contribution of MLL3 to the biology of UASCC in this study. We identified that MLL3 forms a protein complex with GRHL2 in the regulation of UASCC epigenome. To investigate the regulation of the downstream signaling pathway by MLL3/GRHL2 protein complex, ChIP sequencing using either MLL3 or GRHL2 antibodies was performed in UASCC cell line. In addition, either MLL3 or GRHL2 was silenced by transfection of siRNA and performed RNA-Sequencing in UASCC cell line (KYSE510) to profile the transcriptome.

overall_design: KYSE510 cells silencing either MLL3 or GRHL2 by transfection of siRNA were generated for deep sequencing to Illumina GAIIx.

first: Chehyun

last: Nam

first: Guowei

last: Huang

first: Hua

last: Zhao

first: Ethan

last: Pan

first: Megha

last: Sheth

first: Casey

last: Collet

first: Hieu

last: Van

first: Liyan

last: Xu

first: En-min

last: Li

first: Harold

middle: P

last: Koeffler

first: Young

last: Park

first: Kai

last: Ge

first: Yali

last: Dou

first: Uttam

last: Sinha

first: De-Chen

last: Lin


title: ADAM17 mediated post-translational regulation of CD122 signaling enhances CD8+ T cell anti-tumor immunity

city: xian

first: Cangang

last: Zhang

state: State...

address: Yanta district, xi 'an city, shaanxi province

institute: xian jiaotong university

summary:
CD8+ T cell immune responses are regulated by multi-layer networks, while the post-translational regulations remain largely unknown. Transmembrane ectodomain shedding is an important post-translational process regulating receptor expression and signal transduction by proteolytic cleavage of membrane proteins. Here, by targeting sheddase A Disintegrin and Metalloprotease (ADAM)17, we defined a post-translational regulatory mechanism mediated by ectodomain shedding in CD8+ T cells. Transcriptomic and proteomic analysis revealed the involvement of post-transcriptional/translational regulations in CD8+ T cells. T cell-specific deletion of ADAM17 led to a dramatic increase of effector CD8+ T cell differentiation and enhanced cytolytic effects to eliminate pathogens and tumors. Mechanistically, ADAM17 regulated CD8+ T cells by cleavage of membrane CD122. ADAM17 deficient CD8+ T cells had elevated CD122 expression and response to cytokines IL-2 and IL-15. Intriguingly, inhibition of ADAM17 in CD8+ T cells improved the efficacy of chimeric antigen receptor (CAR) T cells in solid tumor. Our findings reveal a critical post-translational regulation in CD8+ T cells, providing a potential therapeutic strategy of targeting ADAM17 for effective anti-tumor immunity.
We then performed gene expression profiling analysis using data obtained from RNA-seq of 10 samples from two groups.

overall_design: WT and KO CD8+ T cells were sorted from the spleens of recipient mice 7 days after LM-OVA infection in the co-transfer model.

first: Baojun

first: Lina

last: sun


title: The transcriptional dynamics of Ophryocystis elektroscirrha across its host's metamorphosis

city: Atlanta

first: James

middle: Gabriel Amor

last: DuBose

state: Georgia

address: 1510 East Clifton Road Northeast 2006

institute: Emory Univeristy

summary: The objective of this study was to quantify patterns of transcriptional differentiation that Ophryocystis elektroscirrha undergoes throughtout its host's metamorphosis.

overall_design: We sequenced mRNA of homogenized monarch butterflies that we infected with Ophryocystis elektroscirrha. We sequenced two larval stages (third instar and 5th instar), two pupal time points (1 day after pupaitona and 6-8 days after pupation), and adults approximately 3-6 hours after they eclosed.

middle: G

first: Jacobus

last: de Roode


title: Bulk RNA-seq of ILC2, Tconv, Th2, Treg Gata3hi and Treg Gatalo from the lungs of IL-33 treated mice

city: Brussels

first: Julie

last: Stockis

address: Avenue Hippocrate 74

department: de Duve Institute

country: Belgium

institute: UCLouvain

summary: Regulatory T cells (Tregs) are essential for limiting adaptive immunity, and restrain type-2 inflammation in allergic disease. As Tregs function locally, the mechanisms that coordinate their suppressive role in the inflamed niche are of great interest. Here we show that group 2 innate lymphoid cells (ILC2) are critical mediators of IL-33-driven Treg expansion in allergic inflammation. ILC2-derived OX40L promotes the local expansion of Gata3high Tregs, which possess distinct transcriptional and functional programmes that enforce co-localisation with ILC2 in the inflamed airways. Using OX40 Treg-conditional mutant mice, we show that Gata3high Tregs are important for restraining adaptive type-2 immunity. Mechanistically, Gata3high Tregs modulate OX40L bioavailability on ILC2, which controls effector memory Th2 cell formation. As such, ILC2 can simultaneously engage both the effector and regulatory arms of adaptive type-2 immunity via the OX40L-OX40 signalling axis. More specifically, ILC2-Treg interactions serve as a critical feedback mechanism to control adaptive type-2 immunity.

overall_design: Around 50 000 Gata3high and Gata3low Treg cells (CD45+Lin-CD3+CD8-TCRβ+CD4+RFP+YFPhi/lo), Tconv and Th2 cells (CD45+Lin-CD3+CD8-TCRβ+CD4+RFP-YFPlo/hi) and ILC2s (CD45+Lin-CD3-CD4-YFPhi) were sorted from lungs of IL-33-sensitised Gata3YFP/YFP Foxp3RFP mice on a BD Symphony S6 sorter.

first: Thomas

last: Yip

first: Ashley

last: Sawle

first: Timotheus

last: Halim


title: Transcriptional profiling of intestinal tissue from mice with Apc and additional mutations.

city: Glasgow

first: Kathryn

last: Gilroy

address: Garscube

country: United Kingdom

institute: CRUK Scotland Institute

summary: A profile of gene expression differences in mouse small intestine with short term induction of Apc inactivation, in the presence and absence of additional mutations in combinations of Kras, Pik3ca, Pten, and Slc7a5.

overall_design: Expression data from intestine with combinations of Apc, Kras, Pik3ca, Pten and Slc7a5 mutation.

first: Lucas

last: Zeiger

first: Owen

last: Sansom


title: The effect of ITPRIPL2 knockdown on the transcriptome in nHDF and HeLa cells

city: Maastricht

first: Mike

last: Gerards

address: Paul-Henri Spaaklaan 1

department: Maastricht Centre for Systems Biology (MaCSBio)

country: Netherlands

institute: Maastricht University

summary: To investigate the general effect of ITPRIPL2 knockdown on the transcription profile, we generated transient ITPRIPL2 knockdowns in nHDF and HeLa cells. Gene expression analysis was performed on RNA-seq data from four biological replicates of cells harvested 72 hours post transfection with esiRNAs.

overall_design: Comparative gene expression analysis of RNA-seq data for nHDF and HeLa cells with and without ITPRIPL2 knockdown.

first: Irene

last: Hemel

first: Steen

last: Carlijn

first: Michiel

last: Adriaens


title: ROS1 regulates blueberry fruit ripening in a negatively ABA manner

city: Beijing

first: Zejia

last: Wang

address: Yanyuan Street

institute: Peking University

type: Methylation profiling by high throughput sequencing


title: Bulk RNA-seq of OX40 wt and KO spleen Treg and Tconv

overall_design: Around 100 000 Treg (CD45+CD3+CD4+CD8−TCRβ+YFP+) and Tconv (CD45+CD3+CD4+CD8−TCRβ+YFP-) cells were sorted from spleens of Foxp3YFP-CreTnfrsf4fl/fl mice (or Foxp3YFP-Cre Tnfrsf4wt/wt) using a BD Aria II cell sorter.


title: The MLL3/GRHL2 complex regulates early malignant transformation and anti-tumor immunity in squamous cancer [ChIP-seq]

first: Yueyuan

last: Zheng

overall_design: KYSE510 cell line was harvested and performed ChIP-Seq using either MLL3 or GRHL2 antibodies. KYSE510 cells silencing either MLL3 or GRHL2 by transfection of siRNA were generated for deep sequencing to Illumina GAIIx.

first: Chehyun

last: Nam

first: Guowei

last: Huang

first: Hua

last: Zhao

first: Ethan

last: Pan

first: Megha

last: Sheth

first: Casey

last: Collet

first: Hieu

last: Van

first: Liyan

last: Xu

first: En-min

last: Li

first: Harold

last: Koeffler

first: Young

last: Park

first: Kai

last: Ge

first: Yali

last: Dou

first: Uttam

middle: K

last: Sinha

first: De-Chen

last: Lin


title: Effect of MiaPaCa-2 cancer cells on gene expression in HEI-286 Schwann cells and c-Jun KO HEI-286 Schwan cells

city: New York

first: Bic

last: MSKCC

state: NY

address: 1275 York Ave.

institute: Memorial SLoan-Kettering Cancer Center

summary: We report the transcriptome analysis of control and c-Jun knock-out HEI-286 human Schwann cells in presence or absence of cancer cells

overall_design: HEI-286 Schwann cells and c-Jun KO HEI-286 Schwann cells transcriptome profiles in absence or presence of MiaPaCa-2 cancer cells.Biological triplicate for each condition.

first: Deborde

last: Sylvie

first: Wong

last: Richard


title: Changes in gene expression following MGH-CP1 or dinaciclib treatment.

city: Cleveland

first: Ajaybabu

last: Pobbati

state: Ohio

address: 9500 Euclid Ave

department: Cancer Biology

institute: Cleveland Clinic Foundation

summary: Purpose: There are no effective treatment options for patients with aggressive epithelioid hemangioendothelioma (EHE) driven by the TAZ-CAMTA1 (TC) fusion gene. Here, we aimed to understand the regulation of TC using pharmacological tools and identify vulnerabilities that can potentially be exploited for the treatment of EHE. Design: TC is a transcriptional co-regulator; we hypothesized that compounds that reduce TC nuclear levels, either through translocation of TC to the cytoplasm, or through degradation, would render TC less oncogenic. TC localization was monitored using immunofluorescence (IF) in an EHE tumor cell line. Two target-selective libraries were used to identify small molecules that reduce TC localization in the nucleus. The ability of the shortlisted hits to affect cell viability, apoptosis, and tumorigenesis was also evaluated. Results: Basal TC remained ‘immobile’ in the nucleus; administration of cyclin-dependent kinase inhibitors (CDKi) such as CGP60474 and dinaciclib mobilized TC. ‘Mobile’ TC shuttled between the nucleus and cytoplasm; however, it was eventually degraded through proteasomes. This dramatically suppressed the levels of TC-regulated transcripts and cell viability, promoted apoptosis, and reduced the area of metastatic lesions in the allograft model of EHE. We specifically identified that the inhibition of CDK9, a transcriptional CDK, destabilizes TC. Conclusions: The CDK inhibitor dinaciclib exhibited anti-tumorigenic properties both in vitro and in vivo in EHE models.  Dinaciclib has been rigorously tested in clinical trials and displayed an acceptable toxicity profile. Therefore, there is a potential therapeutic window for repurposing dinaciclib for the treatment of EHE.

overall_design: EHE17 cells were treated with MGH-CP1  or dinaciclib; biological triplicates were used.

first: Brian

last: Rubin


title: Transcription profiling by array of mouse hepatocytes following treatment with growth factor beta 1 in a time series

city: Mannheim

first: Carolina

last: Delatorre

address: Theodor-Kutzer-Ufer 1-3

country: Germany

institute: University Heidelberg

summary: To obtain a comprehensive view on the TGF-beta effects in primary mouse hepatocytes, Affymetrix microarrays were performed. We aimed at detecting early (1h), intermediate (6h) and late (24h and 48h) target genes.

overall_design: Primary hepatocytes were used as an in vitro model to detect TGF-beta effects on the expression profile. In primary cell cultures, the majority of hepatocytes survive despite activated TGF-beta signaling. These cells display phenotypic characteristics for (partial) EMT and fibrogenesis.

first: Seddik

last: Hammad


title: Therapeutic SHPRH-146aa encoded by circ-SHPRH dynamically upregulates P21 to inhibit CDKs in neuroblastoma

first: yaqiong

last: Jin

address: No.56 Nanlishi road

institute: Beijing Children’s Hospital, Capital Medical University

summary: Recent research has underscored the significance of circular RNA (circRNA) in various cancers, including neuroblastoma (NB). Specifically, circ-SHPRH, a distinct circRNA, has emerged as an inhibitor of tumor growth through its ability to sequester miRNAs or produce the SHPRH-146aa protein. To explore circ-SHPRH's involvement in NB and its potential application in gene therapy, this study examined its expression in 94 NB tissues and cell lines (SK-N-BE(2), SH-SY5Y, CHLA-255) using real-time PCR and fluorescence in situ hybridization (FISH). Functional assays encompassing both overexpression and knockdown experiments in NB cell lines, as well as in vivo investigations, were conducted. RNA-seq analysis revealed a correlation between circ-SHPRH and the P21 (CDKN1A) pathway, a pivotal cell cycle regulator. Validation through PCR and other techniques confirmed that circ-SHPRH upregulated P21 expression. Furthermore, the regulatory role of circ-SHPRH in the P21-CDK pathway was corroborated through SHPRH-146aa expression. Notably, adenovirus-mediated overexpression of circ-SHPRH effectively curbed NB tumor growth in NSG mice, while combining circ-SHPRH with everolimus exhibited potential for NB treatment. This study elucidates the remarkable significance of circ-SHPRH in NB and its prospective utility in gene therapy, thereby paving the way for innovative therapeutic approaches.

overall_design: SH-SY5Y cells were transfected with OE-NC and OE-circ-SHPRH for 72 h. Then, total RNA from these cells was isolated using TRIzol reagent.

first: Yaqiong

first: Saishuo

last: Chang

first: Dong

last: Ren


title: PD-1 is induced on tumor-associated macrophages in obesity to directly restrain anti-tumor immunity

city: Nashville

first: Jeff

last: Rathmell

address: 1211 Medical Center Drive

department: PMI

institute: Vanderbilt University Medical Center

summary: Obesity is a leading risk factor for progression and metastasis of many cancers1,2, yet can also promote improved survival for some cancers3-5 and enhance responses to some immune checkpoint blockade therapies6-8. The role of the immune system in the obesity-cancer connection and how obesity influences immunotherapy, however, remain unclear. While PD-1 expression by macrophages has been described9-12, we found that obesity selectively induced PD-1 on macrophages and that PD-1 directly impaired macrophage function. Single cell RNA sequencing of murine colorectal carcinoma tumors showed obesity remodeled myeloid and T cell populations, with fewer clonally expanded effector T cells and increased abundance of PD-1+ tumor-associated macrophages (TAM). Cytokines and molecules associated with obesity, including IL-6, leptin, and insulin, and the unsaturated fatty acid palmitate, induced PD-1 expression on macrophages in a glycolysis-dependent manner and that MTORC1 pathway signaling is essential for PD1 expression. PD-1+ TAMs had increased mitochondrial respiration, fatty acid metabolism and cell cylewhile PD-1- TAMs had increased inflammatory signature , phagocytosis and antigen presentation to T cells. These patterns were directly regulated by PD-1, as recombinant PD-L1 reduced macrophage glycolysis, phagocytic capacity and antigen presentation, and this was reversed with blocking PD-1 antibody. Further, inhibition of glycolysis with 2-deoxyglucose prevented the increase in CD86, MHC-I and MHC-II that would otherwise occur with anti-PD-1 treatment. Conversely, PD-1-deficient Pdcd1-/- TAMs had high rates of glycolysis, phagocytosis, and expression of MHC-II. Myeloid-specific PD-1 deficiency correlated with slower tumor growth, enhanced TAM antigen presentation capability, and increased CD8 T cell activation together with reduced markers of exhaustion. These findings show metabolic signaling and type 1 inflammatory cues in obesity induces PD-1-mediated suppression of TAM function and reveal a unique macrophage-specific and glycolysis-dependent mechanism to modulate immune tumor surveillance and checkpoint blockade. This may contribute to increased cancer risk yet improved response to PD-1 blockade in TAM-enriched tumors and obesity.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design:
To investigate gene expression profile of bead isolated CD11b+ MC38 tumor associated macrophages treated with a-PD1 blocking antibody or IgG control
To investigate gene expression profile between FACS isolated PD-1 high and PD-1 low expressing MC38 TAMs
To investigate PDCD1 expression of healthy donor monocytes enriched from PBMC compared to a PTEN haplodeficient patient monocytes

first: Bader

last: Jackie

first: Ye

last: Xiang


title: The effects of adipose tissue-derived mesenchymal stem cells intranasal administration on the house dust mite-induced mixed airway inflammation in an experimental asthma model

city: Bialystok

first: Andrzej

last: Eljaszewicz

state: Podlaskie

address: Jana Kilinskiego 1

department: Centre for Regenerative Medicine

country: Poland

institute: Medical University of Bialystok

summary: The beneficial effects of mesenchymal stem cell administration have been reported in numerous preclinical studies. Briefly, decreased levels of Th2-related cytokines, namely IL-4, IL-5, and IL-13, impaired eosinophils infiltration, and mucus secretion within the lung have been recognized as hallmarks of MSC-mediated immunosuppression in OVA-Alumn- induced experimental model. However, to date, the effects of MSCs transfer on mixed airway inflammation remain elusive. Therefore, here we aimed to investigate the influence of internasal adipose tissue-derived MSCs on mixed inflammation in a house dust mite (HDM) extract-induced experimental asthma model.  To induce mixed airway inflammation, mice were challenged with 10 g of HDM extract for 5 days each week. Additionally, to investigate the short- and long-term effects, adipose tissue-derived MSCs were administrated at two-time points. To analyze MSC-mediated changes, we assessed lung morphology, frequencies of T cells effectors, epithelial barrier integrity, lung transcriptomic profiles, and the levels of cytokines and chemokine in bronchoalveolar lavage (BAL). We observed the limitation of cellular infiltration and mucus production after MSCs administration. Moreover, we noted a trend to decrease in the frequencies of T cells producing IL-4 and IFNy as a long-term effect of MSCs administration. Additionally, the expression of ZO-1 was increased in both analyzed time-points of MSCs transfer. Notably, we found the changes in the transcriptomic profiles reflecting, the inflammation of the respiratory system, the proliferation of lymphocytes, migration of leukocytes, and recruitment of neutrophils and eosinophils. Finally, we also noted the differences in the levels of CXCL and CCL chemokines in BAL between short- and long-term models, while the changes in Th2-related cytokines levels were limited. Overall, here we confirmed the therapeutic potential of adipose tissue-derived MSCs administration in a novel HDM extract-induced experimental model of mixed airway inflammation.

overall_design: Female C57BL6 mice (n=5) were challenged intranasally with house dust mite (HDM) extract in the dose of 10ug for 5 following days each of two weeks. Adipose tissue-derived MSCs (5*105 cells) were administrated to mice intranasally on the 13th and 6th day of the experiment to investigate their short- and long-term effects, respectively. Saline (0,9% NaCl) treated mice were set as control. Mice were sacrificed on the 15th day. Lung lobes were collected for histology, flow cytometry, confocal microscopy, and RNA-seq analysis, while bronchoalveolar lavage (BAL) was preserved for multiplex assay.

first: Marlena

last: Tynecka

first: Adrian

last: Janucik

first: Arkadiusz

last: Zbikowski

first: Magdalena

last: Niemira

first: Agnieszka

last: Tarasik

last: Kulczynska-Przybik

first: Anna

last: Szalkowska

first: Urszula

last: Korotko

first: Joanna

last: Reszec

first: Barbara

last: Mroczko

first: Hady

last: Razak Hady

first: Miroslaw

last: Kwasniewski

first: Adam

last: Kretowski

first: Marcin

last: Moniuszko


title: Effect of Repeated Exposure of Whole-Cigarette Smoke Condensate to Lung Damage in a Rat Model

city: Jeongeup

first: Jinhee

last: Kim

address: 30, Baekhak 1-gil

institute: Korea institute of toxicology

summary: To investigate the adverse effects in the lungs caused by WCSC exposure, a 1-week, and 2-week exposure model was established through the ITI administration method. We then performed gene expression profiling analysis using data obtained from RNA-seq of 4 different concentrations of lung tissue at two time points.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for WCSC-treated and control lung tissues.

first: Yoon

last: Cho

first: Min-Seok


title: Influence of low-input RNA from FF and FFPE samples on gene expression quantification by NanoString, microarray and RNA-seq: a comparative study

city: Paris

first: Jennifer

last: Wong

address: 12 rue lhomond

country: France

institute: Institue Curie

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

overall_design: Refer to individual Series


title: Transcriptomic analysis of the mechanism of detoxification metabolism induced by β-asarone in Laodelphax striatellus (Fallén)

status: Public on Jul 18 2024

city: Jinan

first: jianhua

last: zhang

address: gongyebei street No. 23788

institute: Shandong Academy of Agricultural Sciences

summary: In order to clarify the molecular mechanism of metabolic detoxification in Laodelphax striatellus (Fallén), the transcriptome sequencing analysis was used to screen and identify the relevant genes related to metabolic detoxification after induced by β-asarone at LC50 dose.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for β-asarone treatment.

first: Jianhua

last: Zhang

first: Yuanxue

last: Yang

first: Aiyu

last: Wang

first: Ming

last: Zhao


title: Influence of low-input RNA from FF and FFPE samples on gene expression quantification by NanoString, microarray and RNA-seq: a comparative study [RNA-seq]

summary:
Purpose: The goals of this study is to determine the best method of gene expression quantification (RNA-seq, Microarray, NanoString) and amplification kits adapted to low-input and/or low-quality RNA samples (FFPE samples)
Methods: Mouse bladder cancer cell line (mouse bladder cancer cell line, BC57) and mouse normal mouse normal urothelium were fixed in formalin and embedded in paraffin (FFPE), andfesh frozen (FF) in liquid nitrogen. The total RNA of these 4 samples were tested in triplicate by 3 technologies (NanoString, RNA-seq and Microarray) and the results were compared to its reference (high-quality and high-input RNA of mouse bladder cancer cell line and mouse normal mouse normal urothelium). For RNA-sequencing, each sample was tested by two library contruction kits: SMARTer Stranded Total RNA-Seq Kit - Pico Input mammelian and Ovation SoLo NuGEN RNA-seq System with three input quantities: 50pg, 250pg and 2ng of total RNA, except for the SMARTer Stranded Total RNA-Seq Kit - Pico Input mammelian kit for which the minimum recommended quantity was 250pg of total RNA. RNA-seq based on poly(A) tail enrichment was considered as the reference and was done for the two FF samples at high amount (1µg of total RNA) . Results obtained with the two tested kits were compared to those based on poly(A) tail enrichment. To determine which is the best kit suitable for RNA-seq, low-input and low-quality RNA samples, we performed RNA-seq control quality metrics, principal component analysis, and a differential analysis between the mouse bladder cancer cell lines and the mouse normal mouse normal urothelium for each input quantity, library kit and method of sample preservation (FF or FFPE).
Results: The Ovation SoLo NuGEN RNA-seq System library kit are recommended for quantification of gene expression of FFPE and FF samples at low-input (down to 50pg) whereas the SMARTer Stranded Total RNA-Seq Kit - Pico Input mammelian is only suitable for FF samples from 250pg of total RNA.

overall_design: For RNA-sequencing, the 4 samples were tested in triplicate by two library contruction kits: SMARTer Stranded Total RNA-Seq Kit - Pico Input mammelian and Ovation SoLo NuGEN RNA-seq System with three input quantities: 50pg, 250pg and 2ng of total RNA, except for the SMARTer Stranded Total RNA-Seq Kit - Pico Input mammelian kit for which the minimum recommended quantity was 250pg of total RNA. RNA-seq based on poly(A) tail enrichment was considered as the reference and was done for the two FF samples at high amount (1µg of total RNA) .

first: David

last: Gentien

first: François

last: Radvanyi

first: Audrey

last: Rapinat

first: Benoit

last: Albaud


title: Influence of low-input RNA from FF and FFPE samples on gene expression quantification by NanoString, microarray and RNA-seq: a comparative study [microarray]

summary:
Purpose: The goals of this study is to determine the best method of gene expression quantification (RNA-seq, Microarray, NanoString) and amplification kits adapted to low-input and/or low-quality RNA samples (FFPE samples)
Methods: Mouse bladder cancer cell line (mouse bladder cancer cell line, BC57) and mouse normal mouse normal urothelium were fixed in formalin and embedded in paraffin (FFPE), andfesh frozen (FF) in liquid nitrogen. The total RNA of these 4 samples were tested in triplicate by 3 technologies (NanoString, RNA-seq and Microarray) and the results were compared to its reference (high-quality and high-input RNA of mouse bladder cancer cell line and mouse normal mouse normal urothelium). For microarray technology,  each sample was tested by two amplification kits: GeneChip Pico WT and SensationPlus with 2 input quantities: 500pg and 2ng of total RNA, except for the SensationPlus which the recommended quantity is 50ng of total RNA. Microarray data, obtained with the kit GeneChip WT Plus from 100ng of total RNA from fresh frozen samples, was considered as the reference. All samples were hybridized on MTA 1.0 microarrays. Results obtained with the two tested kits were compared to those from GeneChip WT Plus. To determine which is the best kit suitable for microarray from low-input and  low-quality RNA samples, we performed microarray control quality metrics, principal component analysis,  and a differential analysis between the mouse bladder cancer cell lines and the mouse normal mouse normal urothelium for each input quantity, amplification kit and method of sample preservation (FF or FFPE).
Results: According to our results, the GeneChip Pico kit are recommended for quantification of gene expression of FFPE and FF samples from 2ng of total RNA. This kit is not recommended for samples below 2ng of total RNA. The kit SensationPlus is recommended for FFPE samples at 50ng of total RNA.

overall_design: For microarray technology,  4 samples were tested by two amplification kits: GeneChip Pico WT and SensationPlus with 2 input quantities: 500pg and 2ng of total RNA, except for the SensationPlus which the recommended quantity was 50ng of total RNA. Microarray data, obtained with the kit GeneChip WT Plus from 100ng of total RNA from fresh frozen samples, was considered as the reference. Each Sample tested with the GeneChip PicoWT kit was done in triplicate and each sample tested with SensationPlus and GeneChip WT Plus was done in duplicate.


title: Iso-CUT&MAP (Isolated cell-based Cut Under Target and Mapping Apoptotic Protein-binding sequences): a novel strategy for high-resolution mapping of DNA binding sites

city: Boston

first: Christina

last: Sharkey

state: MA

address: 99 Brookline Ave Rm 389

department: Urology

institute: Beth Israel Deaconess Medical Center

summary: DNA and protein interactions are essential for cellular processes and have major implications in cancer biology. DNA binding proteins, particularly transcription factors (TF), are crucial in regulating gene expression and downstream protein functions. Studying the intricate DNA-protein interactions helps unravel specific functions of TFs that are involved in cancer pathogenesis, identify regulatory pathways, and ultimately uncover therapeutic targets. Currently, the lack of high-throughput screening techniques makes research in cancer associated-TFs challenging. Therefore, we have developed an innovative Iso-CUT&MAP (Isolated cell-based Cut Under Target and Mapping Apoptotic Protein-binding sequences). Here, K562 tumor cells were coencapsulated with NK92 and antibody-conjugated (NFkB/RelA) magnetic beads in a microfluidic droplet-based system. After NK92 immune cells killed the K562 target cells, we collected RelA-bound DNA from the dead cells and analyzed DNA-binding sites using ChIP-seq. The results reveal the RelA genome-wide association study (GWAS) of the target K562 tumor cells in response to immune cell killing. The genome-wide mapping matched the expected profile of NFkB, validating the ability of our Iso-CUT&MAP method to accurately identify NFkB binding in response to NK92 killing of K562. This novel technique holds promise for precision medicine in understanding apoptosis biology and the influence of immune cell or chemotherapy-induced tumor cell killings.

overall_design: In this study, we have developed a novel single-cell method, named Iso-CUT&MAP (Isolated cell-based Cut Under Target and Mapping Apoptotic Protein-binding sequences), to investigate DNA-protein interactions and evaluate the cytotoxicity of immune cells towards target tumor cells. This innovative method combines droplet microfluidics and imaging-based technology to facilitate the isolation and analysis of protein-bound DNA from individual apoptotic tumor cells. In this approach, an effector cell is paired with a target cell at the single-cell level and magnetic beads, conjugated to antibodies to isolate the protein-bound DNA from the apoptotic target cell in droplets (Figure 1). Subsequently, the beads are released from droplets, and the genetic material captured and subjected to sequencing using ChIP-seq.

first: Agustin

last: De Ganzó

first: Judene

last: Thomas

first: Matthew

last: Sullivan

first: Tania

last: Konry


title: An HSF1-JMJD6-HSP feedback circuit promotes cell adaptation to proteotoxic stress [RNA-Seq]

city: Chicago

first: Marc

last: Mendillo

state: IL

address: 303 East Superior Street Simpson Querrey, 7th Floo

department: Biochemistry and Molecular Genetics

institute: Northwestern University

summary: Heat Shock Factor 1 (HSF1) is best known as the master transcriptional regulator of the heat-shock response (HSR), a conserved adaptive mechanism critical for protein homeostasis (proteostasis). Combining a genome-wide RNAi library with an HSR reporter, we identified JMJD6 as an essential mediator of HSF1 activity. In follow-up studies, we found that JMJD6 is itself a non-canonical transcriptional target of HSF1 which acts as a critical regulator of proteostasis. In a positive feedback circuit, HSF1 binds and promotes JMJD6 expression, which in turn reduces HSP70 R469 monomethylation to disrupt HSP70-HSF1 repressive complexes resulting in enhanced HSF1 activation. Thus, JMJD6 is intricately wired into the proteostasis network where it plays a critical role for cellular adaptation to proteotoxic stress.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for PC3M cells transfected with siNT or siJMJD6 and PC3M HSF1 KO cell lines treated with either DMSO or MG132 (2.5uM) for 4 hours. Comparative gene expression profiling analysis of RNA-seq data for PC3M cells transfected with siNT or siJMJD6 and exposed to heat shock at 42C for 1h and recovered for 2 hours.

first: Milad

last: Alasady

first: Martina

last: Koeva

first: Seesha

last: Takagishi

first: Dmitri

last: Segal

last: Amici

first: Roger

last: Smith

first: Daniel

last: Anse

first: Susan

last: Lindquist

first: Luke

last: Whitesell

first: Elizabeth

middle: T

last: Bartom

first: Mikko

last: Taipale


title: Abnormal circadian rhythms exacerbate dilated cardiomyopathy by reducing the ventricular mechanical strength

first: Jiangping

last: Song

address: 167A Beilishi Road, Xi Cheng District

institute: Fuwai Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College

summary: Dilated cardiomyopathy (DCM) has etiological and pathophysiological heterogeneity. Abnormal circadian rhythm (ACR) is related to the development of DCM in animal models, but exploration based on clinical samples is lacking. Sleep apnea (SA) is the most common disease related to ACR, and we chose SA as the study object to explore ACR-DCM. We included a DCM cohort and divided it into SA (n=76) and without SA group (n=29). RT-qPCR was used to determine the change of rhythm gene expression pattern. We used single-nucleus RNA sequencing (snRNA-seq) to explore the abnormal transcriptional patterns in the ACR group, and we verified the findings by pathological staining, atomic force microscopy (AFM), and Rev-erbα/β knockout (KO) mice analysis. DCM patients with SA showed decreased amplitude of rhythm gene expression. SA group showed more severe dilation of left heart chambers. From snRNA-seq, ACR-DCM lost the morning transcriptional patterns, detailly, actin cytoskeleton organization of cardiomyocytes (CMs) disrupted and hypertrophy aggravated, and the proportion of activated fibroblasts (Fibs) decreased with the reduction of fibrotic area ratio. The results of pathological staining, mechanical experiments, and transcriptional feature of Rev-erbα/β KO mice supported the above findings. The severe dilation of the left ventricular (LV) wall in DCM patients with SA was associated with a decrease in structural strength, and phenotypic changes of CMs and Fibs were involved in this process. ACR-DCM was histopathologically characterized by a fluffy ventricular wall.

overall_design: Heart tissue samples from 16 patients were included in the snRNA-seq. The 16 patients were divided into 4 groups: Group A (n=4, ACR, local time in the morning (6 am-10 am)), group B (n=4, ACR, local time in the afternoon (2 pm-6 pm)), group C (n=4, normal circadian rhythm (NCR), morning (6 am-10 am)), and group D (n=4, NCR, afternoon (2 pm-6 pm)). The criteria for the ACR group (groups A and B) were: (1) Diagnosed with SA; (2) Abnormal expression of circadian rhythm genes (based on Log2 (Rev-erbα/Bmal1)) 7; (3) Met the inclusion and exclusion criteria for the DCM cohort. The criteria for the NCR group (groups C and D) were: (1) Without SA history and not diagnosed with SA; (2) Normal expression of circadian rhythm genes (based on Log2 (Rev-erbα/Bmal1)) 7; (3) Met the inclusion and exclusion criteria for the DCM cohort. The local time (China standard time) of the heart sample was defined as the time of the aortic cross-clamp.

first: Hao

last: Jia

last: Cui

first: Xiao

last: Chen

first: Zijie

last: Zhao

first: Han

last: Mo

first: Ningning

last: Zhang

first: Siyuan

last: Huang

first: Yue

first: Mengda

last: Xu

first: Lei

last: Han

first: Yulin

first: Yuan

last: Chang

first: Xiumeng

last: Hua

first: Zhibo

last: Shentu

first: Yu

first: Tie

last: Xia


title: An HSF1-JMJD6-HSP feedback circuit promotes cell adaptation to proteotoxic stress [ChIP-Seq]

first: Marc

last: Mendillo

overall_design: Chromatin occupancy of HSF1 was measured in PC3M cell line by ChIPseq.

first: Milad

last: Alasady

first: Martina

last: Koeva

first: Seesha

last: Takagishi

first: Dmitri

last: Segal

first: David

last: Amici

first: Roger

middle: S

last: Smith

first: Daniel

last: Anse

first: Susan

last: Lindquist

first: Luke

last: Whitesell

first: Elizabeth

first: Mikko


title: Sub-optimal paternal diet at the time of mating perturbs maternal adaptations in the late gestation mouse

city: Manchester

first: Hannah

last: Morgan

address: Oxford Road

institute: University of Manchester

summary: Pregnancy represents a stage during which maternal physiology and homeostatic regulation undergo dramatic change and adaptation. The fundamental purpose of these adaptations is to ensure the survival of her offspring through adequate nutrient provision and an environment that is tolerant to the semi-allogenic fetus. While poor maternal diet during pregnancy is associated with perturbed maternal adaptations during pregnancy, the influence of paternal diet on maternal well-being is less clearly-defined. We fed male mice either a control (CD), low protein diet (LPD), a high fat/sugar Western diet (WD) or the LPD or WD supplemented with methyl donors (MD-LPD and MD-WD respectively) for a minimum of 8 weeks prior to mating. Females were culled at day 17 of gestation for the analysis of maternal metabolic, gut, cardiac and bone health. Paternal diet had minimal influences on maternal metabolic status or gut microbiota diversity. However, analysis of the maternal hepatic transcriptome revealed distinct profiles of differential gene expression in response to the diet of the father. Paternal LPD and MD-LPD resulted in differential expression of genes associated with lipid metabolism, transcription, ubiquitin conjugation and immunity in dams, while paternal WD and MD-WD modified the expression of genes associated with ubiquitin conjugation and cardiac morphology. Finally, we observed changes in maternal femur length, volume of trabecular bone, trabecular connectivity, volume of the cortical medullar cavity and thickness of the cortical bone in response to the father’s diets. Our current study demonstrates that poor paternal diet at the time of mating can influence the patterns of maternal metabolism and gestation-associated adaptations to her physiology. Such changes could have significant consequences for the long-term health of his offspring and the mother.

overall_design: To investigate pregnant murine liver RNA profile changes in response to matings with sub-optimal diet fed fathers. We fed male mice either control diet (NPD; 18% casein, 21% sugar, 0% milk fat, 0% cholesterol), isocaloric low protein diet (LPD; 9% casein, 24% sugar, 0% milk fat, 0% cholesterol), ‘Western’ diet (WD; 19% casein, 34% sugar, 20% milk fat, 0.15% cholesterol) or LPD or WD supplemented with methyl-donors [an addition of 5 g/kg diet choline chloride, 15 g/kg diet betaine, 7.5 g/kg diet methionine, 15 mg/kg diet folic acid, 1.5 mg/kg diet vitamin B12 to either LPD (MDL) or WD (MDWD)] for a minimum of 8 weeks before males were mated with standard chow fed females. At gestational day 17.5 females were euthanised and liver samples collected for RNA sequencing.

first: Afsaneh

last: Koshkerdar

first: Nader

last: Eid

first: Vipul

last: Batra

first: Nichola

last: Baker

first: Nadine

last: Holmes

first: Sonal

last: Henson

first: Fei

last: Sang

first: Victoria

last: Wright

first: Jane

last: McLaren

first: Kevin

last: Shakesheff

first: Katie

middle: J

last: Woad

first: Adam

last: Watkins


title: Effect of ApoE in communicating immunometabolic signaling in recipient macrophages via exosomes

city: San Francisco

first: Robert

last: Raffai

state: California

address: 4150 Clement St

department: Surgery

institute: University of California San Francisco

summary: While ApoE expression by myeloid cells is recognized to control inflammation, whether such benefits can be communicated via extracellular vesicles including exosomes is not known. Through the study of exosomes produced by macrophages derived from the bone marrow of Wildtype (WT-BMDM-exo) and ApoE deficient (EKO-BMDM-exo) mice, we uncover a critical role of ApoE in regulating cell signaling properties. We treated BMDM with EKO-BMDM-exo, WT-BMDM-exo, or PBS and performed gene expression profiling analysis. We found that BMDM treated with EKO-BMDM-exo show reductions in the cholesterol efflux gene Abca1 and the long chain fatty acid transporter Cpt1a. Furthermore, BMDM treated with EKO-BMDM-exo show reduced expressions of genes involved in oxidative stress response, particularly the glutathione peroxidases (Gpx1 & Gpx3) and the selenoproteins (Selenow, Selenom, Selenop, and Selenon). In contrast, treatments of these EKO-BMDM-exo upregulated the expressions of genes reported to drive glycolysis or involved in the glycolytic pathway (Aldh2, Pkm, Cd9, Fth1, Dio2, and Pgd). Taken together, our data unveil a novel property of macrophage ApoE in controlling the immunometabolism-regulatory properties by their secreted exosomes.

overall_design: To investigate the differential expression of mRNAs in BMDM treated with exosomes derived from the bone marrow of Wildtype (WT-BMDM-exo) and ApoE deficient (EKO-BMDM-exo) mice, or PBS as control. Bone marrow cells are taken from male mice that are 6- to 12-week-old and cultured into BMDM. Cells are then treated with EKO-BMDM-exo, or WT-BMDM-exo, or PBS for 18 hours. We then collected the total RNA and performed gene expression profiling analysis using data obtained from RNA-seq of the three different cell groups. Three biological replicates (n = 3) were used for each group.

first: Tuan

last: Phu


title: RNA-seq analysis of genes involved in flag leaf photosynthesis regulation at filling stage

city: Shanghai

first: Ma

last: Bin

state: Shanghai

address: Fenglin 300 Road, Shanghai, China

institute: National Key Laboratory of Plant Molecular Genetics, CAS Center for Excellence in Molecular Plant Sciences

summary: We observed that many pathways related tophotosynthesis and sugar metabolism were enriched

overall_design: mRNA profiles of wildtype, mutant and overexpression line flag leaves were sequenced

first: Bin

last: Ma

first: You

last: Zhang


title: Cadherins modulate the self-organizing potential of gastruloids

city: LAUSANNE

first: ALEXANDRE

last: MAYRAN

state: Waadt

address: Station 19

institute: EPFL


title: ABI1 regulates transcriptional activity of Androgen Receptor by novel DNA and AR binding mechanism [RNA-seq]

city: Syracuse

first: Leszek

last: Kotula

state: New York

address: 750 East Adams Str

institute: Upstate Medical University

summary: Transcription regulates key functions of living organisms in normal and disease states, including cell growth and development, embryonic and adult tissue organization, and tumor progression. Here we identify a novel mechanism of transcriptional regulation by an actin regulatory and signaling protein, Abelson Interactor 1(ABI1). Using prostate cancer models, we uncover a reciprocal regulation between ABI1 and the Androgen Receptor (AR). ABI1 is a direct, androgen-regulated target; in turn, ABI1 interacts with AR and its splice variant ARv7, and co-regulates a subset of specific transcriptional targets. ABI1 directs transcription through transient yet well-defined interaction of its intrinsically disordered region with DNA. Clinical evaluation shows that both the ABI1-DNA binding (through Exon 4 splicing) and ABI1-AR interaction are regulated during androgen deprivation therapy and prostate cancer progression, thus controlling tumor plasticity through connecting actin cytoskeleton and cellular signaling to transcriptional regulation. We propose that ABI1 is an epigenetic regulator of transcriptional homeostasis in AR-driven cancers.

overall_design: Total RNA was extracted from LNCaP cell lines following treatmenet with DMSO or 1 nM synthetic androgen R1881 using the Qiagen Qiashredder and RNAeasy Extraction Kit (Qiagen). Statistical and quality control analysis was performed using Partek bioinformatics software. Unaligned reads were trimmed based on quality score(min quality phred score=20); STAR alignment was performed with quantification to annotation model using refseq annotation (Partek E/M); features were filtered to remove if maximum <=10 and normalized using CPM + 0.0001; differential expression analysis was performed using treatment and cell type as attributes using DESeq2

first: Baylee

last: Porter

first: Xiang

last: Li

first: Neeru

last: Arya

first: Fan

first: Sonia

last: Kung

first: Ladan

last: Fazli

first: Htoo

last: Oo

first: Yinan

first: Kenneth

last: Marincin

first: Konsta

last: Kukkonen

first: Henna

last: Urhonen

first: Maria

last: Ortiz

first: Allysa

last: Kemraj

first: Eva

last: Corey

first: Xuensen

last: Dong

first: Vladimir

last: Kuznetsov

first: Matti

last: Nykter

first: Martin

last: Gleave

first: Gennady

last: Bratslavsky

first: Alfonso

last: Urbanucci

first: Dominique

last: Frueh

first: Alaji

last: Bah


title: Cadherins modulate the self-organizing potential of gastruloids (scRNA-seq)

first: ALEXANDRE

last: MAYRAN

summary: Gastruloids have recently emerged as an efficient four-dimensional model for studying some aspects of post-implantation embryonic patterning. Their ability to undergo gastrulation-like processes leading to the self-organization into highly reproducible biological objects, has opened new avenues to investigate early embryonic patterning. Here, we sought to uncover the molecular and cellular mechanism underlying this remarkable property. We report that self-organization competence is associated with a cell-specific coordination of a Cadherin switch. We find that N-Cadherin hinders gastruloids morphogenetic competence, for its inactivation leads to the formation of trunk-like structures without relying on the otherwise requested extra-cellular matrix analogues such as Matrigel. In contrast, the repression of E-Cadherin by Snai1 is critical for self-organization: Snai1 establishes a cell-specific repressive pace by triggering the repression of a pluripotency-associated transcription program and its chromatin landscape, thus allowing a proper transition from E- to N-Cadherin to occur. Altogether, this work establishes a molecular mechanism that integrates the exit from pluripotency and the pace of cell differentiation, leading to the observed self-organizing potential of gastruloids.

overall_design: scRNA-seq on gastruloids from WT and mutant conditions

first: Alexandre

last: Mayran

first: Lucille

last: Lopez-Delisle

last: Kolly

first: Denis

last: Duboule


title: ABI1 regulates transcriptional activity of Androgen Receptor by novel DNA and AR binding mechanism

first: Leszek

last: Kotula


title: ABI1 regulates transcriptional activity of Androgen Receptor by novel DNA and AR binding mechanism [ChIP-seq]

overall_design: A ChIP reaction was carried out using 40 ug of each DNA sample with antibodies to either Abi1 (MBL International, D147-3) or AR (N20, Santa Cruz Bio Technology, sc-816). The immunoprecipitated DNA was processed into a standard Illumina ChIP-Seq library and sequenced.

first: Baylee

last: Porter

first: Xiang

last: Li

first: Neeru

last: Arya

first: Fan

last: Zhang

first: Sonia

last: Kung

first: Ladan

last: Fazli

first: Htoo

last: Oo

first: Yinan

first: Kenneth

last: Marincin

first: Konsta

last: Kukkonen

first: Henna

last: Urhonen

last: Ortiz


title: Cadherins modulate the self-organizing potential of gastruloids (RNA-seq)

last: MAYRAN

overall_design: Bulk RNA-seq on gastruloids from WT and mutant conditions

first: Anne-Catherine

last: Cossy


title: Cadherins modulate the self-organizing potential of gastruloids (re-analysis)

first: ALEXANDRE

overall_design:
reanalysis of single gastruloid RNA-seq
this is a reanalysis of some datasets from GSE106227 sample reanalysed are: GSM2832454, GSM2832455, GSM2832456, GSM2832457, GSM2832458, GSM2832459, GSM2832460, GSM2832461, GSM2832462, GSM2832463

Single-reads fastqs were retrieved from the SRA server using fasterq-dump version 3.0.5. They were trimmed for bad quality and Truseq adapters with Cutadapt v4.0 (-a CTGTCTCTTATACACATCTCCGAGCCCACGAGAC -q 30 -m 15). Mapping and counting were performed on filtered reads on the mouse genome mm10 with STAR version 2.7.8a44 with a custom gtf based on Ensembl version 10245 and ENCODE parameters. FPKM values were obtained by Cufflinks version 2.2.146,47 with options --no-effective-length-correction -b 'mm10.fa' --multiread-correct --library-type fr-firststrand --mask-file 'chrM_mm10.gtf' --max-bundle-length 10000000 --max-bundle-frags 1000000 (where chrM_mm10.gtf contains a transcript on each strand of the whole chrM). Uniquely mapped reads were filtered with bamFilter48 version 2.5.1. Coverages were computed with BEDTools49 version 2.30.0 for each strand using the '-scale' parameter to normalize to million uniquely mapped reads.
All the code required to reproduce the analysis is available here (https://github.com/MayranA/allScriptsFromMayranEtAl2023)

Processed data:
.bw: normalized coverage (to million of uniquely mapped reads) with both strands combined
AllCufflinks_Simplified.txt.gz: single table with all FPKM values from cufflinks
AllHTSeqCounts.txt.gz: single table with all counts from STAR

last: Mayran

last: Lopez-Delisle


title: RNA-seq of IMR90 senescence with CCF nuclear egress inhibition

status: Public on Jul 17 2024

first: Yanxin

last: Xu

address: 185 Cambridge Street

institute: Massachusetts General Hospital/ Harvard Medical School

summary: We report that CCF nucleus-to-cytoplasm shuttling in senescence is mediated by nuclear egress, mediated by ESCRT-III and Torsin complex. We inhibit ESCRT-III by KO ALIX, and Torsin by KO TOR1A.

overall_design: IMR90 cells were stably expressing CRISPR construct encoding sgPRM1 (control), sgALIX, or sgTOR1A, and were induced to senescence by IR. Three replicates for each sample.

first: Zhixun

last: Dou

first: Tara

middle: C

last: O’Brien

first: Takuya

last: Kumazawa

first: Murat

last: Cetinbas

first: Ruslan

middle: I

last: Sadreyev


title: Effect of chronic epinephrine exposure on gene expression in MIN6 cells

city: Chongqing

first: Bingpeng

last: Yao

state: -- Please Select --

address: Tiansheng Road 2

institute: southwest university

summary: Chronic exposure to epinephrine enhances the compensatory insulin secretion in MIN6 cells while persistently inhibiting cell proliferation. The molecular mechanisms underlying these phenomena were investigated through transcriptome sequencing, which identified differential mRNA transcriptional expression. This analysis has uncovered the pivotal signaling pathways that govern cellular function and destiny.

overall_design:
To explore the effects of chronic epinephrine on the functionality and fate of islet beta cells, we conducted an investigation using the MIN6 cell line. The cells were incubated with epinephrine, after which we performed an analysis of their gene expression profiles.

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Submitter states that missing raw files are due to file loss.
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title: iPSC-NSCs-Derived Exosomal let-7b-5p Improves Motor Function After Spinal Cord Injury by Modulating Microglial/Macrophage Pyroptosis

city: nanjing

first: Jie

last: Liu

state: Jiang Su

address: 101 Longmian Avenue, Jiangning District, Nanjing

institute: Nanjing Medical University

type: Non-coding RNA profiling by high throughput sequencing

summary:
Background: Following spinal cord injury (SCI), the inflammatory storm initiated by microglia/macrophages poses a significant impediment to the recovery process. Exosomes play a crucial role in the transport of miRNAs, facilitating essential cellular communication through the transfer of genetic material. However, the miRNAs from iPSC-NSCs-Exos and their potential mechanisms leading to repair after SCI remain unclear. This study aims to explore the role of iPSC-NSCs-Exos in microglia/macrophage pyroptosis and reveal their potential mechanisms.
Methods: iPSC-NSCs-Exos were characterized and identified using transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and Western blot. A mouse SCI model and a series of in vivo and in vitro experiments were conducted to investigate the therapeutic effects of iPSC-NSCs-Exos. Subsequently, miRNA microarray analysis and rescue experiments were performed to confirm the role of miRNAs in iPSC-NSCs-Exos in SCI. Mechanistic studies were carried out using Western blot, luciferase activity assays, and RNA-ChIP.
Results: Our findings revealed that iPSC-NSCs-derived exosomes inhibited microglia/macrophage pyroptosis at 7 days post-SCI, maintaining myelin integrity and promoting axonal growth, ultimately improving mice motor function. The miRNA microarray showed let-7b-5p to be highly enriched in iPSC-NSCs-Exos, and LRIG3 was identified as the target gene of let-7b-5p. Through a series of rescue experiments, we uncovered the connection between iPSC-NSCs and microglia/macrophages, revealing a novel target for treating SCI.
Conclusion: In conclusion, we discovered that iPSC-NSCs-derived exosomes can package and deliver let-7b-5p, regulating the expression of LRIG3 to ameliorate microglia/macrophage pyroptosis and enhance motor function in mice after SCI. This highlights the potential of combined therapy with iPSC-NSCs-Exos and let-7b-5p in promoting functional recovery and limiting inflammation following SCI.

overall_design: In our study, we used advanced miRNA sequencing technology to explore the underlying biological mechanisms influencing the effect of iPSC-NSCs-Exos on microglia/macrophage pyroptosis, resulting in the identification  differentially expressed miRNAs between iPSC-NSCs-Exos and iPSC-NSCs.

first: Jin

last: Fan

first: Guang

last: Kong

first: Wu

last: Xiong


title: Single cell transcriptomics reveals distinct transcriptional responses to oxycodone and buprenorphine by iPSC-derived brain organoids from patients with opioid use disorder

city: Rochester

first: Hu

last: Li

address: 200 1st St SW

institute: Mayo Clinic

summary: The opioid epidemic represents a national crisis. Oxycodone is one of the most prescribed opioid medications in the United States, whereas buprenorphine is currently the most prescribed medication for opioid use disorder (OUD) pharmacotherapy. Given the extensive use of prescription opioids and the global opioid epidemic, it is essential to understand how opioids modulate brain cell type function at the single-cell level. We performed single nucleus RNA-seq (snRNA-seq) using iPSC-derived forebrain organoids from three male OUD subjects in response to oxycodone, buprenorphine, or vehicle for seven days. We utilized the snRNA-seq data to identify differentially expressed genes following drug treatment using the Seurat integrative analysis pipeline. We utilized iPSC-derived forebrain organoids and single-cell sequencing technology as an unbiased tool to study cell-type-specific and drug-specific transcriptional responses. After quality control filtering, we analyzed 25787 cells and identified sixteen clusters using unsupervised clustering analysis. Our results reveal distinct transcriptional responses to oxycodone and buprenorphine by iPSC-derived brain organoids from patients with OUD. Specifically, buprenorphine displayed a significant influence on transcription regulation in glial cells. However, oxycodone induced type I interferon signaling in many cell types, including neural cells in brain organoids. Finally, we demonstrate that oxycodone, but not buprenorphine activated STAT1 and induced the type I interferon signaling in patients with OUD. These data suggest that elevation of STAT1 expression associated with OUD might play a role in transcriptional regulation in response to oxycodone. In summary, our results provide novel mechanistic insight into drug action at single-cell resolution.

overall_design: This study was designed to identify gene expression profiles associated with drug treatment with oxycodone or buprenorphine drug treatment in iPSC-derived brain organoids from OUD patients.  Drug treatment was conducted at 83-90 days of forebrain organoid differentiation and at 28-34 days of forebrain neuron differentiation. The concentrations of oxycodone (50 µg/L) and buprenorphine (2 ng/mL) used to perform these experiments were selected to fall within the range of blood drug concentrations in patients taking standard clinical doses of these two drugs. 3-D iPSC-derived brain organoids were cultured in the bioreactors with the drugs for seven days, and the medium was changed daily.

first: Ming-Fen

last: Ho

first: Richard

last: Weinshilboum


title: Next generation sequencing analysis of tea plant transcriptomes in response to Colletotrichum camelliae infection.

city: Changchun

first: Shouan

address: Xian Da Road

institute: Jilin University

summary: Next generation sequencing (NGS) was performed to identify genes changed in tea plant upon Colletotrichum camelliae infection. The goal of the work is to find interesting genes involved in tea plant in response to fungi infection. The object is to reveal the molecular mechanism of tea plant defense.

overall_design: mRNA profiling of tea plants in response to Colletotrichum camelliae infection.


title: The ROS Regulates NCF2, Key Metabolic Enzymes and MDA Levels to Affect the Growth of Fusarium solani

city: Lanzhou

first: jie

last: li

state: Gansu

address: No. 1, yingmen village

institute: Gansu Agricultural University

summary:
To investigate the expression level of NCF2, SOD1, CTA1 and PXMP4.
We then performed gene expression profiling analysis using data obtained from RNA-seq of 3 different treatments.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for pure culture , DPI treatment and H2O2 treatment.

first: Li-dan

last: Feng


title: Transcriptomic study in human glioblastoma multiforme by DNA microarray

city: Elche

first: Víctor M.

last: Barberá

state: Not US or Canada

address: Camí de l'Almazara, 11

department: Laboratorio de Genética Molecular

country: Spain

institute: Hospital General Universitario de Elche

summary: Transcriptional profiling of primary human glioblastoma tumor samples compared to a non-tumor brain RNA control from the frontal lobe anatomical region (pool from 5 donors).

overall_design: Two-condition experiment, glioblastoma tumor samples vs. non-tumor brain RNA (pool from five donors). Biological replicates: 9 tumor replicates 1 control replicate.

first: Victor

middle: M

last: Barbera

first: Eduardo

last: Larriba