title: Single cell RNA-sequencing analysis of human hepatic organoids derived from human induced pluripotent stem cells

summary: Liver is a central organ for drug and xenobiotics metabolism in human body. Due to the differences in interspecies metabolism, human model system for liver metabolism is necessary. However, the hepatic model systems derived from human pluripotent stem cells for metabolic screening and assays did not recapitulate the most of metabolic capabilities of human liver or primary human hepatocytes to date. In this study, we developed human hepatic endoderm organoids which are expandable and subsequently differentiated human hepatic organoids having metabolic capabilities. To investigate the cellular composition and properties of human hepatic organoids in single cell level, we performed single cell RNA-sequencing.

overall_design: human hepatic organoid derived from hiPSCs and cultured in suspension


title: Formalin-evoked pain triggers sex-specific behavior and spinal immune response

status: Public on Jun 21 2023

city: Marseille

first: Andrew

middle: J

last: Saurin

address: Campus de Luminy

country: France

institute: IBDM / CNRS

type: Expression profiling by high throughput sequencing

summary: Mounting evidence shows sex-related differences in the experience of pain with women suffering more from chronic pain than men. Yet, our understanding of the biological basis underlying those differences remains incomplete. Using an adapted model of formalin-induced chemical/inflammatory pain, we report here that in contrast to male mice, females distinctly display two types of nocifensive responses to formalin, distinguishable by the duration of the interphase. This could be explained by female gonadal hormones fluctuating across the estrus cycle, rather than by the transcriptional content of the dorsal horn of the spinal cord (DHSC). Additionally, deep RNA-sequencing of DHSC showed that formalin-evoked pain was accompanied by a male-preponderant enrichment in genes associated with the immune modulation of pain, revealing an unanticipated contribution of neutrophils. Taking advantage of the male-enriched transcript encoding the neutrophil associated protein Lipocalin 2 (Lcn2) and using flow cytometry, we confirmed that formalin triggered the recruitment of LCN2-expressing neutrophils in the pia mater of spinal meninges, preferentially in males. Our data consolidate the contribution of female estrus cycle to pain perception and provide evidence supporting a sex-specific immune regulation of formalin-evoked pain.

overall_design: Transcriptional profiles of ipsilateral drosal horn of the spinal cord harvested from males, proestrus females and metestrus females, 2H following formalin injection into the hindpaw

first: Lucie

last: Pepino

first: Pascale

last: Malapert

first: Aziz

last: Moqrich

first: Ana

last: Reynders


title: RNA-seq analysis of Molm-13 cell with GADD45g overexpressed

status: Public on Jun 22 2023

city: Tianjin

first: Dan

last: Guo

state: Tianjin

address: 288 Nanjing Road

department: State Key Laboratory of Experimental Hematology

country: China

institute: Institute of Hematology & Blood Diseases Hospital

summary: The growth arrest and DNA-damage induced 45 gamma (GADD45g) is rapidly induced by various physiological and environmental stresses associated with growth arrest. GADD45g has been observed implicated in cell survival, apoptosis, senescence, cell cycle regulation and DNA repair in a variety of human solid tumor types, acting as either tumor promoter or tumor suppressor. To date, the role of GADD45g in hematopoietic malignancies remains completely unknown. Here, we transduced Molm-13 cells with lentiviral vectors expressing doxycycline-inducible GADD45g. Molm-13 cells with dox administration or not were collected for RNA-seq.

overall_design: Molm-13 cells were treated with or without 1 mg/ml dox for 4 days. 2 biological replicate experiments were performed.

first: Yangyang

last: Zhao


title: Defining the Sox2-independent reprogramming steps to pluripotency

city: Muenster

first: Marcos

middle: J.

last: Araúzo-Bravo

address: Rogentstrasse

department: Cell and Developmental Biology

country: Germany

institute: Max Planck Institute for Molecular Biomedicine

summary:
Several Sox family members, small molecules, and inhibitors of the TGF-β pathway can replace exogenous Sox2 during the generation
of induced pluripotent stem cells (iPSCs), suggesting that SOX2 is dispensable for initiation of reprogramming. However,
the time point at which the endogenous Sox2 locus is activated in these conditions was not thoroughly evaluated. To identify the Sox2-independent
reprogramming steps, we excluded Sox2 from the reprogramming cocktail and used Sox2-deficient mouse embryonic fibroblasts.
We show that reprogramming is initiated in the absence of SOX2 as shown by formation of pre-iPSC colonies that have undergone a
mesenchymal-to-epithelial transition and that have activated early reprogramming markers.
However, these iPSC-like intermediates cannot progress through the final stabilization phase of reprogramming in the absence of SOX2.
Overall, our results provide a better understanding of the molecular events underlying the induction of pluripotency and of the role that
SOX2 plays in the process.
Both pLV-tetO-E1A-2A-Cherry and pLV-tetO-Cherry lentiviral constructs were cloned in house and defective-lentiviral particles were
produced as previously described (Han et al. 2013).
Next, 5x10 4 ESCs containing a tetracycline-inducible transactivator (irtTA-VP16-GBD, Anastassiadis et al. 2002) plus an Oct4-GFP reporter
transgene (Yoshimizu et al. 1999) were transduced twice using 20 µl of replication-defective lentiviral particles plus polybrene (4 µg/ml, Sigma).
Cells were washed 24 h after transduction and subsequently induced with doxycycline (1 µg/ml, Sigma) and dexamethasone (10-7 M, Sigma).
Samples were harvested at several time-points, 0, 3, 6, 9, 12, 24, and 48 hours after induction,
and used for microarray analysis as previously reported (Tapia et al. 2012).
Cells transduced with pLV-tetO-Cherry and induced for 48 h were used as control.
RNA wasprocessed using the RNeasy micro and mini kits with on-column DNase digestion of QIAGEN as per the manufacturer’s instructions.
Integrity of RNA samples was quality checked using a 2100 Bioanalyzer (Agilent). When possible,
300 ng of total RNA per sample was used as starting material for linear amplification (Illumina TotalPrep RNA Amplification Kit, Ambion),
which involved synthesis of T7-linked double-stranded cDNA and 14 hr of in-vitro transcription incorporating biotin-labeled nucleotides.
Purified and labeled cRNA was then quality checked on a 2100 Bioanalyser and hybridized as biological or technical duplicates for 18 hr onto
MouseRef-8 v2 gene expression BeadChips (Illumina), following the manufacturer's instructions. After being washed,
the chips were stained with streptavidin-Cy3 (GE Healthcare) and scanned using an iScan reader and accompanying software.
Bead intensities were mapped to gene information using BeadStudio 3.2 (Illumina).
Background correction was performed using the Affymetrix Robust Multi-array Analysis (RMA) background correction model.
Variance stabilization was performed using log2 scaling, and gene expression normalization was calculated with the quantile method implemented in the
lumi package of R-Bioconductor. Data post-processing and graphics were performed with in-house developed functions in Matlab.

overall_design:
16 samples were analyzed
0h, Mouse Embryonic Stem Cells (ESC) after 0 hours of E1A induction, 2 replicates
3h, Mouse Embryonic Stem Cells (ESC) after 3 hours of E1A induction, 2 replicates
6h, Mouse Embryonic Stem Cells (ESC) after 6 hours of E1A induction, 2 replicates
9h, Mouse Embryonic Stem Cells (ESC) after 9 hours of E1A induction, 2 replicates
12h, Mouse Embryonic Stem Cells (ESC) after 12 hours of E1A induction, 2 replicates
24h, Mouse Embryonic Stem Cells (ESC) after 24 hours of E1A induction, 2 replicates
48h, Mouse Embryonic Stem Cells (ESC) after 48 hours of E1A induction, 2 replicates
48hc, Mouse Embryonic Stem Cells (ESC) after 48 hours of E1A induction, cherry control, 2 replicates

first: Adele

middle: G

last: Marthaler

first: Kenjiro

last: Adachi

first: Ulf

last: Tiemann

first: Guangming

last: Wu

first: Davood

last: Sabour

first: Sergiy

last: Velychko

first: Ingo

last: Kleiter

first: Hans

middle: R

last: Schöler

first: Natalia

last: Tapia


title: Effect of lncMGC on gene expression of kidney mesangial cells under SD or TGF-ß treatment [ChIP-seq]

city: Duarte

first: Zhuo

last: Chen

address: 1500 E. Durate Rd.

country: USA

institute: City of Hope

type: Genome binding/occupancy profiling by high throughput sequencing

summary:
To investigate the function of long noncoding RNA lncMGC in WT and lncMGC KO mouse kidney mesangial cells treated with TGF-ß.
Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) for histone modication mark H3K27ac on 4 types of kidney messangial cell samples: isolated from wild type (WT) or lncMGC knockout (lncMGC_KO) mouses, each treated with either SD or TGF-ß, namely WT_SD, WT_TGF, KO_SD and KO_TGF.

overall_design: Examine the H3K27ac enrichment  levels based on the data from ChIP-seq at genomic regions of interests to reveal the regions' enhancer activity.

first: Kato

last: Mitsuo

first: Natarajan

last: Rama


title: IL-11 induces NLRP3 inflammasome activation

city: Chapel Hill

first: John

last: Wrobel

state: NC

address: 120 Mason Farm Rd

department: Department of Biochemistry and Biophysics

institute: University of North Carolina

summary: The objective of this study is to examine IL-11-induced mechanisms of inflammatory cell migration to the CNS. We report that IL-11 is produced at highest frequency by myeloid cells among the PBMC cell subsets. Patients with relapsing-remitting multiple sclerosis (RRMS) have an increased frequency of IL-11+ monocytes, IL-11+ and IL-11R+ CD4+ lymphocytes and IL-11R+ neutrophils in comparison to matched healthy controls (HCs).  IL-11+ and GM-CSF+ monocytes, CD4+ lymphocytes, and neutrophils accumulate in the cerebrospinal fluid (CSF). The effect of IL-11 in-vitro stimulation, examined using single cell RNA sequencing (scRNAseq), revealed the highest number of differentially expressed genes (DEGs) in classical monocytes, including upregulated NFKB1, NLRP3 and IL1B.  All CD4+ cell subsets had increased expression of S100A8/9 alarmin genes involved in NLRP3 inflammasome activation.  In IL-11R+-sorted cells from the CSF, classical and intermediate monocytes significantly upregulated the expression of multiple NLRP3 inflammasome-related genes, including complement, IL18, and migratory genes (VEGFA/B) in comparison to blood-derived cells. Therapeutic targeting of this pathway with aIL-11 mAb in mice with RR experimental autoimmune encephalomyelitis (EAE) decreased clinical scores, CNS inflammatory infiltrates and demyelination.  aIL-11 mAb treatment decreased the numbers of NFkBp65+, NLRP3+ and IL-1b+ monocytes in the CNS of mice with EAE.  The results suggest that IL-11/IL-11R signaling in monocytes represents a therapeutic target in RRMS.

overall_design: IL-11R+ FACS sorted cells from peripheral blood of 3 MS patients, stimulated vs. unstimulated with IL-11. IL-11R+ FACS sorted cells from CSF vs. blood of 2 MS patients.

first: Maryamsadat

last: Seyedsadr

first: Yan

last: Wang

middle: A

first: James

last: Garifallou


title: Effect of lncMGC on gene expression of kidney mesangial cells under SD or TGF-ß treatment [RNA-seq]

summary:
To investigate the function of long noncoding RNA lncMGC in WT and lncMGC KO mouse kidney mesangial cells treated with   TGF-ß.
We then performed gene expression profiling analysis using data obtained from RNA-seq of 4 types of kidney messangial cell samples: wild type (WT) or lncMGC knockout (lncMGC_KO) mouse, each treated with either SD or TGF-ß, namely WT_SD, WT_TGF, KO_SD and KO_TGF.

overall_design: Comparative gene expression profiling analysis of RNA-seq data across the 4 groups of samples WT_SD, WT_TGF, KO_SD, KO_TGF.

last: Mituso


title: Transcriptome analysis showed that tomato-rootstock enhanced salt tolerance of grafted seedlings was accompanied by multiple metabolic processes and gene differences

city: Baoding

first: ding

last: yuan

address: lekainan

institute: Hebei Agricultural University

summary:
Purpose: Grafting is a commonly used cultural practice to counteract salt stress and is especially important for vegetable production. However, it is not clear which metabolic processes and genes are involved in the response of tomato rootstocks to salt stress. Our goals is to elucidate the regulatory mechanism through which grafting enhances salt tolerance.
Methods: The salt-sensitive tomato variety Zhongza 9 and the strongly salt-tolerant tomato rootstock variety QZ-006 (selected by the research group in the early stage) were used as experimental materials and were purchased from the Vegetable and Flower Institute of the Chinese Academy of Agricultural Sciences (Beijing, CHN) and Beijing Kaixingelin Agricultural Technology Co., Ltd. (Beijing, CHN)；The leaves of the seedlings of every sample was added to 5 mL of HNO3 (65%~68%) after they were heated in a microwave digestion system for 2~3 hours；RNA-seq libraries were sequenced on an Illumina HiSeq X Ten platform；qRT‒PCR was performed according to the instructions of a Fast Super EvaGreen qPCR Master Mix Kit (US Everbright®, Inc.)；The amino acid contents in the tomato leaves were calculated by the external standard method. The chlorophyll of the seedlings was extracted with acetone-ethanol (1:1) ；An indirect enzyme-linked immunosorbent assay (ELISA) was used to determine auxin (IAA), gibberellin (GA), cytokinin (ZR), brassinolide (BR), abscisic acid (ABA), jasmonic acid (JA) and salicylic acid (SA) contents, and kit purchased from ThermoFisher Scientific Co., Ltd. (USA). Endogenous ethylene (ETH) was collected, fixed and determined according to the methods of Ling (Ling et al.,2008) using a Shimadzu 2010 gas chromatograph equipped with a hydrogen flame ion detector. ETH standard gas was purchased from Sigma Company.
Results: Compared with the NGS, the GSs were more salt tolerant, and the Na+ content in the leaves decreased significantly. Through transcriptome sequencing data analysis of 36 samples, we found that GSs exhibited more stable gene expression patterns, with a lower number of DEGs. WRKY and PosF21 transcription factors were significantly upregulated in the GSs compared to the NGSs. Moreover, the GSs presented more amino acids, a higher photosynthetic index and a higher content of growth-promoting hormones. The main differences between GSs and NGSs were in the expression levels of genes involved in the BR signalling pathway, with significant upregulation of XTHs.
Conclusions: Our study demonstrates that grafting on salt tolerant rootstocks can bring different metabolic processes and transcription levels changes to scion leaves, thereby the scion leaves show stronger salt tolerance. This information provides new insight into the mechanism underlying tolerance to salt stress regulation and provides useful molecular biological basis for improving plant salt resistance.

overall_design: We first evaluated the salt damage index, electrolyte permeability and Na+ accumulation in tomato (Solanum lycopersicum L.) leaves of grafted seedlings (GSs) and nongrafted seedlings (NGSs) subjected to 175 mmol·L− 1 NaCl for 0-96 h，covering the front, middle and rear ranges.The experiment was conducted at the scientific research base of Hebei Agricultural University. (38 ° 10 '- 40 ° 00' N, 113 ° 40 '- 116 ° 20' E). When the tomato seedlings developed four leaves and one heart, the split grafting method was used to obtain GSs. We selected GSs and NGS that were uniform and displayed good growth, rinsed the roots, used Hoagland’s nutrient solution formula for hydroponic cultivation, and used an oxygen pump to supplement oxygen to maintain the normal growth of the grafted tomato seedlings for 15 days after grafting


title: Effect of lncMGC on gene expression of kidney mesangial cells under SD or TGF-ß treatment

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

overall_design: Refer to individual Series


title: Environmental and molecular regulation of asexual reproduction in the cnidarian Nematostella vectensis

city: Kansas City

first: Andrew

middle: Joseph

last: Price

state: MO

address: 1000 E. 50th Street

department: Computational Biology

institute: Stowers Institute for Medical Research

summary: We demonstrated that the presence of substrate is effectively required for transverse fission, that animal size does not affect fission rates, and that the position of physal pinching is fixed along the oral-aboral axis of the polyp. Gene expression analyses revealed a role for putative homeobox transcription factors, and components of TGFβ, Notch, and FGF signaling pathways in the transverse fission process.

overall_design: ntotal = 6. 3 time points (x3 replicates at each). Samples ND1,ND2,ND3 were actively undergoing asexual reproduction in substrate when sacrificed and physal tissue excised for sequencing. Samples PD1,PD2,PD3 were in a steady-state with substrate present when sacrificed and physal tissue excised for sequencing.

first: Whitney

middle: B

last: Leach

first: Matt

last: Gibson

first: Layla

last: Al-Shaer

first: Michael

last: Layden


title: Programming effects of intrauterine hyperthermia on adrenal gland development

city: Madison

first: Francisco

last: Peñagaricano

state: Wisconsin

address: 1675 Observatory Drive

department: Animal and Dairy Sciences

institute: University of Wisconsin-Madison

summary: Maternal stress during late pregnancy can lead to intrauterine hyperthermia affecting fetal hypothalamic-pituitary-adrenal axis development and function. This study aimed to characterize the impact of in utero heat stress on the postnatal offspring's adrenal gland transcriptome.

overall_design: Adrenal glands were harvested from in utero heat stressed (IUHS) and in utero cooled (IUCL) female calves at birth (day 0, n=6 per treatment) and at weaning (day 63, n=6 per treatment) for RNA Sequencing.

first: Jimena

last: Laporta


title: Characterization of serotonin-5-HTR1E signaling pathways and its role in cell survival

city: Bethesda

first: Kiersten

middle: Sierra

last: Campbell

state: MD

address: 9000 Rockville Pike

institute: NICHD

summary: 5-Hydroxy tryptamine receptor 1E (5-HTR1E) is reported to activate cAMP and ERK pathways via its ligands and binding partners, but the detailed mechanism underlying the serotonin induced 5-HTR1E signaling is still not known. In the present study, we determined the cellular regulators of ERK and cAMP signaling pathways in response to serotonin induced 5-HTR1E activation in 5-HTR1E overexpressing HEK293 cells. We found that Pertussis Toxin (PTX) treatment completely reversed the effect of serotonin-5-HTR1E mediated signaling on cAMP and ERK pathways, confirming the involvement of a Gαi-linked cascade. We also observed that Gβγ and Gq were not associated with 5-HTR1E activation, while blocking PKA inhibited ERK signaling only, and had no effect on cAMP. Additionally, serotonin-stimulated ERK1/2 phosphorylation was similar in 5-HTR1E overexpressing, β-arrestin-deficient HEK293 cells and is solely dependent on G protein signaling. siRNA mediated gene knockout studies in SH-SY5Y cells revealed that the inhibition of 5-HTR1E reduced the expression of cMyc, Cyclin D1, Cyclin E and BCL2 genes which are related to cell cycle regulation and survival. MTT assays showed that 5-HTR1E knockdown in SHSY-5Y and U118 cells inhibited cell survival significantly. In addition to the signaling mechanism, we also performed RNA-seq analysis in 5-HTR1E overexpressing HEK293 cells and found that 5-HTR1E can regulate the expression of Receptor activity modifying protein 1 (RAMP1), Nuclear receptor 1 (NR4A1) and various Cyclin genes. These findings indicate that serotonin interaction with 5-HTR1E receptor simultaneously activates cAMP and ERK pathway in HEK293 cells and its expression is important for cell survival.

overall_design: Differential expression analysis of mRNA transcriptomic profiles in control and HTR1E-overexpressing HEK293 cells. Cells were also subject to serotonin treatment to characterize the interaction between HTR1E function and serotonin. This study includes additional experiments to characterize HTR1E's role in ERK signalling and the cAMP/CREB pathway.

first: Vinay

middle: K

last: Sharma

first: Xuyu

last: Yang

first: Ryan

last: Dale

last: Loh


title: Effect of lncMGC on gene expression of kidney mesangial cells under SD or TGF-ß treatment [ATAC-seq]

first: Zhuo

last: Chen

summary:
To investigate the function of long noncoding RNA lncMGC in WT and lncMGC KO mouse kidney mesangial cells treated with TGF-ß.
Assay for Transposase-Accessible Chromatin (ATAC) sequencing on 4 types of kidney messangial cell samples: isolated from wild type (WT) or lncMGC knockout (lncMGC_KO) mouses, each treated with either SD or TGF-ß, namely WT_SD, WT_TGF, KO_SD and KO_TGF.

overall_design: Study the genomic regions with open chromatin based on the data from ATAC-seq to reveal the functions of lncMGC on chromatin structures in kidney mesangial cells.

first: Kato

last: Mitsuo

first: Natarajan

last: Rama


title: RNA binding protein IGF2BP2/IMP2 promotes tumor-supporting macrophage polarization by cancer cell-derived extracellular vesicles.

city: Rochester

first: Ernesto

last: Aparicio-Puerta

state: NY

address: 265 Crittenden Boulevard, CU 420630

department: Biostatistics and Computational Biology

institute: University of Rochester

type: Non-coding RNA profiling by high throughput sequencing

summary: High expression levels of the RNA binding protein IGF2BP2/IMP2 are correlated with increased tumor cell proliferation, invasion, and poor prognosis in the clinic. Tumor cells release extracellular vesicles (EVs) that can polarize macrophages towards tumor-associated macrophages (TAMs), which play a role in tumor progression. However, there is a lack of understanding of IMP2’s effect on TAMs. EVs were isolated from colorectal cancer HCT116 parental (WT) and CRISPR/Cas9 IMP2 knockout (KO) cells via ultracentrifugation and tangential flow filtration. EVs were characterized according to MISEV guidelines and microRNA cargo was assessed by microRNA-Seq. Primary human monocyte-derived macrophages were polarized towards a TAM-like phenotype by EVs and the expression of genes and surface markers was assessed by qPCR and flow cytometry, respectively. Morphological changes of macrophages as well as the migratory potential of cancer cells were assessed by the IncuCyte® system and macrophage matrix degradation potential by zymography. Changes in the metabolic activity of macrophages were quantified in live cells in a Seahorse® analyzer. For in vivo studies, EVs were injected into the yolk sac of zebrafish embryos, and macrophages were isolated by fluorescence-activated cell sorting. EV samples from both WT and KO EVs had a similar size and concentration and were positive or CD9, CD81, CD63, and ALIX. The expression of tumor-promoting genes was higher in macrophages polarized with WT EVs, while the expression of tumor-suppressing TNF and IL6 was reduced. WT EV-polarized macrophages showed a higher abundance of TAM-like surface markers, higher matrix degrading activity, as well as a higher promotion of cancer cell migration. They exhibited a higher basal oxygen consumption rate and a lower extracellular acidification rate. microRNA-seq revealed a significant difference in the microRNA composition of WT and KO EVs. Macrophages transfected with miR-181a-5p mimic showed a lower level of the phosphatase DUSP6. Zebrafish macrophages upon polarization with WT EVs showed lower expression of IL6 and TNF. Our results show that IMP2 can determine the cargo of EVs released by cancer cells, thereby modulating the EVs’ actions on macrophages. Expression of IMP2 is responsible for the secretion of EVs that polarize macrophages toward a tumor-promoting phenotype.

overall_design: HCT116 Wildtype (WT) and IMP2 Knockout (KO) cells and secreted Extracellular Vesicles were sequenced using miRNA-seq.

first: Vida

last: Mashayekhi

first: Alexandra

last: Kiemer


title: A multi-omics approach unveils an association between subclinical atherosclerosis and epigenetic age acceleration mediated by systemic inflammation

city: Madrid

first: Juan

middle: Carlos

last: Silla-Castro

address: Melchor Fernandez Almagro 3

department: Bioinformatics

country: Spain

institute: CNIC


title: Next Generation Sequencing Facilitates Quantitative Analysis of control and HPK1-M46 Transgenic Mice Transcriptomes

city: Houston TX

first: Hua

last: Wang

state: TX

address: 6565 MD Anderson

institute: MD Anderson Cancer Center

summary:
Purpose: The goals of this study are to compare cerulean treated acinar cells of HPK1-M46 transgenic mice transcriptome profiling (RNA-seq) to study HPK1 pathway
Methods: Total RNA was prepared from mouse pancreata harvested 24 hours after Cerulean treatment using RNeasy Mini/Micro Kit according to the manufacturer’s instructions. RNA quality was measured by the Agilent TapeStation 2200 (Agilent, Santa Clara, CA). RNA samples with RNA integrity numbers of ≥7 were processed for RNA-seq transcriptome analysis.
Results: The RNA-seq libraries were pooled and sequenced, 83 bp single read, on Illumina NextSeq 550 or Illumina NovaSeq 6000 (Illumina, San Diego, CA). All sequencing data were demultiplexed and converted into fastq files by Illumina bcl2fastq2 v2.17.14. Filtered sequence reads were mapped to reference genome mm10 using STAR aligner with default settings.
Conclusions: Our study represents the first detailed analysis of HPK1 transcriptomes, with biologic replicates, generated by RNA-seq technology. The optimized data analysis workflows reported here should provide a framework for comparative investigations of expression profiles.

overall_design: Total RNA profiles of Cerulean treated wild type (B) and HPK1/B (HC), M46/B (MC) mice pancreas  were generated; Total RNA profiles of KC and KMC mice pancreas  were generated

first: Huamin


title: A multi-omics approach unveils an association between subclinical atherosclerosis and epigenetic age acceleration mediated by systemic inflammation (RNA-Seq)

summary: Whole blood methylomics, transcriptomics and plasma proteomics were obtained for 391 participants of the Progression of Early Subclinical Atherosclerosis (PESA) study. Epigenetic age was calculated from methylomics data for each participant. Its divergence from chronological age is termed epigenetic age acceleration. Subclinical atherosclerosis burden was estimated by multi-territory 2D/3D vascular ultrasound and by coronary artery calcification.

overall_design: 240 PESA participants with extended SA and 240 without SA were matched by sex, age (± 3 years) and traditional CV risk factors (diabetes mellitus, current smoker, dyslipidemia, and hypertension). RNA-Seq and methylomics data were generated from whole blood samples from these individuals. Moreover, hypothesis-free proteomics were generated from plasma samples of these participants. 391 samples fulfilled all quality control requirements in all three omics techniques.

first: Fátima

last: Sánchez-Cabo

middle: C

first: Enrique

last: Lara-Pezzi


title: Functional genomics of the human epididymis: further characterization of efferent ducts and model systems by single cell RNA-seq analysis.

city: Cleveland

first: Ann

last: Harris

state: OH

address: 2109 Adelbert Road

department: Genetics

institute: Case Western Reserve University

summary: Objectives Here we used single cell RNA-seq technologies to elucidate the identity of cells within the human efferent ducts and compared them to caput epididymis cells. We also compared the cellularity of primary tissues with those of 2D and 3D (organoid) culture models used for functional studies.  Materials and Methods Human epididymis tissue was dissected to separate different anatomical regions and digested to release single cells for processing on the 10X Genomics Chromium platform. Primary human epididymis epithelial (HEE) cells and HEE organoids were grown as described previously and subjected to single cell (sc) RNA-seq. ScRNA-seq data were processed by standard bioinformatics pipelines and used for comparative analysis.  Results We define the cell types in the efferent ducts which include specialized epithelial cells, connective tissue stromal cells, vascular endothelial cells, smooth muscle cells and immune cells, but lack basal cells that are seen in the caput epididymis. Furthermore, we identify a sub-population of epithelial cells which have marker genes found in the bladder and urothelium. Comparative genomics analysis of the 2D and 3D culture models shows cellular identities adapted to the culture environment while still maintaining similarity to the primary tissue.  Discussion Our data suggest that efferent ducts are lined with a transitional epithelium, which like the urothelium is able to stretch and contract depending on luminal volume. This is consistent with its primary role in seminal fluid resorption and sperm concentration. Moreover, we describe the cellularity of models to study the human epididymis epithelium in vitro. Conclusion Single cell RNA-seq data from the human epididymis make a valuable contribution to our understanding of this highly specialized organ.

overall_design: Single cells from human primary epididmis epithelial cells (HEE), human efferent duct tissue, and human caput tissue were isolated and analyzed using scRNA-seq.

first: Alekh

last: Paranjapye

first: Shih-Hsing

last: Leir


title: An unclassified T cell lymphoma subtype with co-expression of TCR αβ and γ chains revealed by single cell sequencing

city: Beijing

first: Ruoyu

last: Zhang

address: Life Science Park building 1 B220

institute: Innovec Biotherapeutics

summary: T cell lymphomas (TCL) contains a group of heterogeneous non-Hodgkin lymphomas (NHL) with T cell origins. Therapy responses and outcomes remain poor for patients with TCL due to the absence of TCL-specific regimens 1,2. In addition, TCL is highly heterogenous and contains over 40 subtypes according to 2022 WHO classification 3, making the diagnosis and treatment more challenging. In this study, by using single cell technology, we reported a patient with a previously unreported TCL subtype, which was featured by a unique TCR presentations (co-expressing of two a chains, one b chain and one g chain). We deeply dissected the molecular signatures of this unique TCL subtype to better understand the disease biology and molecular pathogenesis, which can also accelerate the development of novel diagnosis and treatment strategies.

overall_design: Bone marrow and PBMC biopsies were collected from a TCL patient. Samples were proceed for single cell RNA-seq and T cell receptor sequencing

first: Wei

last: Song

first: Keqian

last: Shi

first: Haixi


title: Global changes in granulosa cell transcription in response to the LH surge

city: Adelaide

first: James

last: Breen

state: SA

address: 108 North Terrace (Ground Floor)

department: John Curtin School of Medical Research

country: Australia

institute: Australian National University

summary: During ovulation, the LH surge leads to the activation of various signalling cascades in granulosa cells, resulting in a critical shift in chromatin landscape and correspondingly the ovarian gene expression profile. Such large-scale genomic reprogramming involves a specific suite of ovulatory transcription factors. This study aims to characterise global changes in the chromatin landscape that are induced by the LH surge in granulosa cells during ovulation.

overall_design: RNA-seq was performed on 4 replicates of granulosa cells obtained from eCG-stimulated mice after 0 h or 8 h post-hCG treatment

first: Thao

middle: D

last: Dinh

first: Rebecca

middle: L

last: Robker

first: Darryl

last: Russell


title: Galectin-1-dependent ceRNA network in HRMECs revealed its association with retinal neovascularization

city: Wuhan

first: Ning

last: Yang

address: 238# Jiefang Road

institute: Renmin Hospital of Wuhan University

summary: Background: Retinal neovascularization (RNV) is a leading cause of blindness worldwide. Long non-coding RNA (lncRNA) and competing endogenous RNA (ceRNA) regulatory networks play vital roles in angiogenesis. The RNA-binding protein galectin-1 (Gal-1) participates in pathological RNV in oxygen-induced retinopathy mouse models. However, the molecular associations between Gal-1 and lncRNAs remain unclear. Herein, we aimed to explore the potential mechanism of action of Gal-1 as an RNA-binding protein.   Results: A comprehensive network of Gal-1, ceRNAs, and neovascularization-related genes was constructed based on transcriptome chip data and bioinformatics analysis of human retinal microvascular endothelial cells. We also conducted functional enrichment and pathway enrichment analyses. Fourteen lncRNAs, twenty-nine miRNAs, and eleven differentially expressed angiogenic genes were included in the Gal-1/ceRNA network. WT1-AS, LINC01140, and LUCAT1 were situated at the center of the network. Additionally, several key angiogenic genes, such as apelin, angiomotin, and C-X-C motif chemokine ligand 10, were found to potentionally interact with Gal-1 via the ceRNA axis. Furthermore, Gal-1 may be involved in regulating biological processes related to chemotaxis, chemokine-mediated signaling, the immune response, and the inflammatory response.  Conclusions: The Gal-1/ceRNA axis identified in this study may play a vital role in RNV. This study provides a foundation for the continued exploration of therapeutic targets and biomarkers associated with RNV.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for LGALS1 knockdown and control groups in human retinal microvascular endothelial cells.

first: Ningzhi

first: Zhiyi

first: Wenye

last: Cao

first: Xuejun

last: He

first: Wenxi

first: Yiqiao

last: Xing


title: KLF10 modulates stem cell phenotypes of pancreatic adenocarcinoma by transcriptionally regulating notch receptors

city: Miaoli County

first: Shih Sheng

last: Jiang

address: No. 35 Keyan Road, Zhunan County

department: National Institute of Cancer Research

country: Taiwan

institute: National Health Research Institutes

summary: KrÜppel-like factor 10 (KLF10), deficient in two thirds of 105 resected pancreatic adenocarcinoma (PDAC) patients, was associated with rapid loco-regional recurrence and large tumor size. Additional KLF10 depletion in KC (LSL: KrasG12D; Pdx1-CRE) mice accelerated progression from pancreatic intraepithelial neoplasia to PDAC. In Panc-1 cells with KLF10 deficiency (Panc-1-pLKO-shKLF10), increased sphere formation, stem cell markers expression, and tumor growth were noted compared with those of control. Over-expressing KLF10 genetically or pharmacologically using metformin, reversed the stem cell phenotypes induced by KLF10 depletion. Ingenuity pathway analysis and gene set enrichment analysis showed Notch signal molecules including Notch receptors 3 and 4 were over-expressed in Panc-1-pLKO-shKLF10. KLF10 transcriptionally suppressed Notch 3 and 4 receptors by competing with ELF3, a positive regulator, for promoter binding. Downregulating Notch signal genetically or pharmacologically ameliorated stem cell phenotypes of Panc-1-pLKO-shKLF10. Elevating KLF10 expression by metformin with concomitant evodiamine, a non-toxic Notch 3 methylation stimulator, delayed murine tumor growth of PDAC with KLF10 deficiency without prominent toxicity. These results demonstrated a novel signal pathway of KLF10 in modulating stem cell phenotypes of PDAC via transcriptional regulating Notch signal pathway. Elevating KLF10 and suppressing Notch signal may cooperatively reduce PDAC tumorigenesis and malignant progression.

overall_design: Two samples, including a Panc-1-pLKO cell line (vector control) and Panck-1-pLKO-shKLF10 (KLF10 knockdown) cell line, were analysed.

first: Hui-Ju

last: Ch’ang


title: Sterilization of female rats by single-injection of estrogen: proof-of-concept for a nonsurgical alternative

city: Urbana

first: CheMyong

middle: Jay

last: Ko

state: Illinois

address: 2001 S. Lincoln Ave.

department: Comparative Biosciences

institute: University of Illinois at Urbana Champaign

summary: Reproductive sterilization via surgical gonadectomy is strongly advocated to help manage animal populations, especially domesticated pets, and to prevent reproductive diseases. The present study explored the feasibility of using a single-injection method for inducing sterility in female animals as an alternative to surgical sterilization. The idea was based upon our recent finding that repetitive daily injection of estrogen into neonatal rats disrupted hypothalamic expression of Kisspeptin, the neuropeptide that triggers and regulates pulsatile secretion of GnRH in the hypothalamus. In this study, neonatal female rats were dosed with estradiol benzoate (EB) by daily injection for 11 days or subcutaneously implanted with an EB-containing Silastic capsule designed to release EB over a 2- to 3-week period. Rats treated by either method did not exhibit estrous cyclicity, were anovulatory, and became infertile. The EB-treated rats had fewer Kisspeptin neurons in their hypothalami, but their GnRH-LH axis retained responsiveness to Kisspeptin stimulation. To simplify usage, an injectable EB carrier was developed in the forms of biodegradable microspheres or pellets with pharmacokinetics comparable to the EB-containing Silastic capsule. A single injection of EB microspheres (300 µg EB) to neonatal rats made them sterile and female beagles implanted with an EB pellet at a neonatal age had lower KISS expression in their hypothalami. No concerning health impact other than infertility was observed in animals that received EB treatment at a neonatal age. Taken together, this study shows that neonatal EB administration could be developed as a non-surgical animal sterilization method.

overall_design: Sprague Dawley rats used in this study were purchased from Charles River Laboratory and bred at the University of Illinois Division of Animal Resources and maintained under controlled lighting (12 h light/12 h dark) with continuous access to food and water. Rats were acclimatized for one week prior to beginning the experimental procedures. Breeder pairs were housed together for 2 weeks; pregnant females were housed individually, and pups were weaned at 21 days of age. Female neonates were treated with Silastic capsules containing the vehicle sesame oil implanted on postnatal day (PND) 0.5 (SC300). To investigate differential gene expression in individual cells of the hypothalamus, tissues were collected from 3 intact control and 3 SC300 rats, and pooled by group. For single-cell RNA sequencing (scRNAseq) analyses, hypothalamic tissues that contain AVPV and ARC area were cut into small pieces, dissociated into single-cells, and converted into individually barcoded cDNA libraries with the Single-Cell 3’ Chromium kit version 3 from 10X Genomics (Pleasanton, CA), following the manufacturer’s protocols. The 10X Chromium instrument separates thousands of single cells into Gel Bead Emulsions (GEMs) that add a barcode to the mRNA from each individual cell. Following ds-cDNA synthesis, individually barcoded libraries compatible with the Illumina chemistry were constructed. The final libraries were quantitated on Qubit and the average size determined on the AATI Fragment Analyzer (Advanced Analytics, Ames, IA). Libraries were pooled evenly, and the final pool was diluted to 5 nM concentration and further quantitated by qPCR on a Bio-Rad CFX Connect Real-Time System (Bio-Rad Laboratories, Inc. CA). The final library pool was sequenced on a 2x150nt S4 lane in a NovaSeq 6000. Basecalling and demultiplexing of raw data was done with the mkfastq command of the software Cell Ranger 3.0.2 (10x Genomics). Single-cell expression was initially analyzed to perform quality control, sample de-multiplexing, barcode processing, and single-cell 3′ gene counting. Sequencing reads were aligned to the Ensembl96 transcriptome using the Cell Ranger suite with default parameters. Samples were merged using Cellranger aggregate function with default parameters. A total of 4,488 and 4,524 cells from hypothalami of Control and SC300 rats, respectively, was analyzed. Mean raw reads per cell were 151,484 and 147,555 in each sample. Median numbers of expressed genes per cell were 2,479 and 2,530 in each sample. Further analysis was performed in R (v3.5) using the cellrangerRkit (v2.2.0), Seurat package (v3.0.0), and Monocle package (v2.8.0).

first: ChanJin

last: Park

first: Shiori

last: Minabe

first: Rex

last: Hess

first: Po-Ching

last: Lin

first: Sherry

last: Zhou

first: Shah

middle: T

last: Bashir

first: Radwa

last: Barakat

first: Arnon

last: Gal


title: Leveraging CD16 fusion receptors to remodel the immune response for enhancing iPSC-derived NK cell anti-tumor immunotherapy

first: fanyi

last: meng

address: haidian distinct

institute: peking university

summary: We developed a novel hnCD16FR construct that exhibit more potent cytotoxicity than reported hnCD16, which is a promising approach to treating hematologic malignancies and solid tumors with improved ADCC properties. We also offer a rationale for NK activation domains that remodel immune response to enhance CD16 signaling in NK cells.

overall_design: To expand applications of hnCD16-mediated ADCC for NK-cell–based immunotherapy in cancer, we designed the ectodomain of hnCD16 fused with NK cell-specific activating domains in the cytoplasm (hnCD16 Fusion Receptor, FR), then transduced them to CD16-negative NK cell line and human iPSCs-derived NK (iNK) cells, and tested the function of several constructs in vitro and in vivo.

first: Fanyi

last: Meng

first: Yang

last: Li


title: A multi-omics approach unveils an association between subclinical atherosclerosis and epigenetic age acceleration mediated by systemic inflammation (Methylation)

first: Juan

last: Silla-Castro

type: Methylation profiling by genome tiling array

first: Fátima

last: Sánchez-Cabo

last: Silla

first: Enrique

last: Lara-Pezzi


title: Differential small RNA-Seq analysis for active EV cargo identification

city: St. Louis

first: Hua

last: Shen

address: 660 S Euclid Ave

institute: Washington University School of Medicine

summary: Extracellular vesicles (EVs) function through their cargos. This study aims to identify active components of EVs generated from mouse adipose-derived stem cells (ASCs) by comparing the small RNA compositions between EVs and ASCs as well as EVs generated from naive ASCs and primed ASCs (iASCs).

overall_design: Comparing small RNA expression between EVs and parent ASCs as well as EVs generated from naïve ASCs and primed iASCs (i.e., EVs vs iEVs)


title: Scanning iron response regulator binding sites using RNA-seq and Dap-seq in the Brucella genome

city: Shihezi

first: Huan

last: Zhang

address: North 4th Road, Nos 221 street.

institute: Shihezi University

summary: Brucella spp. are gram-negative, facultative intracellular pathogens that cause brucellosis in humans and animals. Iron is an essential element required for all organisms. Iron response regulator (Irr) is a crucial transcriptional regulator and can affect the growth and iron uptake of Brucella. The growth rate of Brucella melitensis M5-90 irr mutant was significantly lower than that of B. melitensis M5-90 under normal or iron-sufficient conditions, however, the growth rate of the B. melitensis M5-90 irr mutant was significantly higher than that of B. melitensis M5-90 under iron-limited conditions. In addition, irr mutation significantly reduced iron uptake under iron-limited conditions. Currently, bhuA (a heme transporter), bhuQ (heme utilization oxygenase Q), and RirA (iron-responsive regulator) have been identified as iron metabolism genes and are regulated by the Irr protein. These results suggest that the Irr protein has multiple target genes in the Brucella genome that are involved in iron metabolism. Therefore, a combination of RNA-seq and Dap-seq was used to investigate the other iron metabolism genes that are also regulated by the Irr protein in Brucella. A total of seven genes were identified as target genes for Irr in this study and the expression levels of these seven genes were similar in both the RNA-seq and qRT-PCR results. Furthermore, the DNA bases C and G and amino acids asparagine, arginine, histidine, and serine were predicted as predominant docking sites for DNA and Irr using the HDOCK server. The electrophoretic mobility shift assay confirmed that six out of the seven genes, namely rirA (BME_RS13665), membrane protein (BME_RS01725), hypothetical protein (BME_RS09560), Iron transporter (BME_RS14525), cation-transporting P-type ATPase (BME_RS10660), and 2Fe-2S binding protein (BME_RS13655), interact with the Irr protein. In summary, our results identified six genes regulated by the Irr protein that may participate in iron metabolism. In addition, from the RNA-seq results, the mutation of irr resulted in the down/upregulation of many genes that are associated with the bacterial secretion system, lipopolysaccharides, and flagellar assembly, and these three components are the main virulence factors for Brucella. Collectively, these results provide valuable insights for the exploration of Brucella iron metabolism and its effects on virulence.

overall_design: In this study, transcriptome and DNA affinity purification sequencing were used to scan the potential targets of Irr protein in Brucella genome. Next, HDOCK was used to predict the interface of Irr protein and the identifid genes targets. EMSA was used to further confirm the direct interaction of Irr with the potential target genes.

first: Zhen

last: Wang


title: Engineering Pluripotent DNA Methylation by CpG Island Methylation Response (CIMR) to Synthetic CpG-free ssDNA Insertion

city: Duarte

first: wei

last: chen

state: CA

address: 1500 E Duarte Rd

department: Cancer Biology-BRI

institute: City of Hope National Medical Center

summary:
Integration of synthetic CpG Free DNA induces de novo DNAme in the flanking CpG island.
Cellular differentiation requires global changes to DNA methylation (DNAme), where it functions  to regulate transcription factor and chromatin remodeling activity, and genome interpretation.  Here, we describe a simple DNAme engineering approach in pluripotent stem cells (PSCs),  extending across large stretches of CpG dense “islands (CGIs).” Integration of synthetic CpG free  single-stranded DNA (ssDNA) induces a target CpG Island Methylation Response (CIMR) in  multiple PSC lines, Nt2d1 embryonal carcinoma cells, and mouse PSCs, but not in highly  methylated CpG Island Methylator Phenotype (CIMP) positive cancer lines. CIMR DNAme at  MLH1 spans the CGI, is robustly maintained throughout cellular differentiation, suppresses target  gene activity, and sensitizes derived cardiomyocytes and thymic epithelial cells to the  chemotherapy cisplatin. Additional CIMR DNAme is reported on at TP53 and ONECUT1 CGIs.  Collectively, this new resource enables total CpG Island DNAme engineering in pluripotency and  the genesis of novel epigenetic models of development and disease

overall_design: H1 pluripotent stem cells with and without integration of a CpG free insert which encodes for antibiotic resistance. Cells are selected, sorted for individual clones and assessed for induction of DNA methylation by Infinium DNA methylation arrays (850k). Initial comparisons have been made to larger CpG free plasmid repair template.

first: Joshua

last: Tompkins


title: Transcriptomic profiling of myogenic newt A1 cells undergoing senescence induction, differentiation or serum and senescence-induced dedifferentiation

city: Dresden

first: Maximina

last: Yun

state: Saxony

address: Fetscherstrasse 105

department: CRTD

institute: TUD

summary:
To investigate the molecular basis of senescence-induced dedifferentiation, we performed RNAseq on control proliferating cells (DMSO only) and etoposide-induced senescent cells to profile senescence induction. Additionally, we performed RNAseq on purified populations of differentiated myotubes derived from untreated A1 cells to investigate differentiation, as well as myotubes subsequently induced to dedifferentiate, either by serum exposure (10% FCS) or by exposure to 48 hour conditioned media from senescent cells (including proliferating cell conditioned media controls).
We then performed gene expression profiling analysis using data obtained from RNA-seq of all 6 populations in triplicate.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for the induction of senescence, differentiation and dedifferentiation in newt A1 cells

first: Hannah

middle: E

last: Walters

first: Konstantin

last: Troyanovskiy

middle: H


title: Autonomous IL-36R Signaling positively modulates Neutrophil Anti-tumor Effector Functions

city: Newton

first: Matt

last: Kanke

state: MA - Massachusetts

address: 112 Harding Street

institute: Amgen

summary: Despite the cancer treatment revolution brought on by the rapid advancement of immunotherapies, only a small fraction of patients derive clinical benefit. Moreover, eradication of large established tumors requires a concerted effort of complete immune system and hence identifying agents that activate and interlink both innate and adaptive immune system components is required. Here, we report that IL-36 cytokine can remodel an immune-suppressive tumor microenvironment (TME) and mediate potent anti-tumor immune response through hematopoietic cells, via both innate and adaptive immunity. Further, we demonstrate that IL-36 signaling reprograms neutrophils in a cell-intrinsic manner to greatly enhances their ability to directly kill tumor cells, their antigen presentation capability and promote T cell proliferation. Thus, the pleotropic effects of IL-36 transform neutrophils into potent effector cells to promote tumor rejection. While poor prognostic outcomes are associated with neutrophil enhancement in the TME, our results highlight the therapeutic potential of harnessing the ability of IL-36 signaling to reprogram neutrophils and link innate and adaptive immune system to enhance durable anti-tumor responses in solid tumors.

overall_design: Spleen and bone marrow were harvested from 6–12-week-old C57BL6 mice from Charles River Laboratories. Neutrophils, NK, monocytes and DCs were isolated as described above. BMDMs were generated by culturing isolated bone marrow cells with 50ng/ml of M-CSF (R&D technologies) for 7 days. Each of the cell type was either untreated or treated with 500 ng/ml of rmIL-36γ (R&D Technologies) for 6h or 24h and RNA from extracted using RNeasy isolation kit (Qiagen) according to manufacturer’s guidelines. Depending on the RNA amount available, 0.7- 5.0 ng of total RNA was used as the starting input to generate the full-length cDNA by using the SMART-Seq v4 Ultra-low Input RNA Kit for Sequencing (Takara Catalog No.634890). RNA was primed by the 3′ SMART-Seq CDS Primer II A with the SMART-Seq v4 Oligonucleotide for template switching at the 5′ end of the transcript during the first-strand cDNA synthesis. Preparation of SMARTer cDNA was sequentially amplified by following the LD-PCR procedure, which was carried out via the thermal cyclic program according to the input amount of total RNA: 11 PCR cycles for 1.0-5.0ng RNA samples and 15 PCR cycles for 0.7-1.0 ng RNA samples. The LD-PCR amplified cDNA was purified and further validated in the Agilent High Sensitivity D5000 ScreenTape (Agilent, Catalog No.5067-5592) on the Agilent 4200 TapeStation. Then, 300pg cDNA products were used for library preparation in each reaction by using the Illumina Nextera XT kit and following the manual instruction (Illumina, Catalog No.FC-131-1096). The products of low-input cDNA libraries were then barcoded after by 12 cycles of thermal program with specific index adapters for Illumina sequencing (Nextera XT Index Set D, Illumina, Catalog No. FC-131-2004). The resulting libraries were cleaned up using 0.8x AMPure XP beads (Beckman Coulter, Catalog No. A63881) and eluted in 25 µl of resuspension buffer. After library QC and quantitation, libraries were sequenced to a minimum depth of 45 million paired-end/Dual-indexed 2x150bp reads on an Illumina HiSeq4000). RNA sequencing reads (Illumina HiSeq platform, 75 bp paired end sequencing) were aligned to Human genome build 38 and Fragments per Kilobase per Million sequenced, Quantile normalized (FPKQ) values were determined using Array Suite software (Omicsoft, Cary, NC) and in-house software.

first: Sumedha

last: Roy

first: Rajkumar

last: Noubade


title: Differentiation of human cardiomyocyte and thymic epithelial cells from H1 control hESCs and H1 hESCs uniquely modified to be DNA methylated at the promoter CpG island of MLH1.

summary:
Engineering Pluripotent DNA Methylation by CpG Island Methylation Response (CIMR) to Synthetic CpG-free ssDNA Insertion
Cellular differentiation requires global changes to DNA methylation (DNAme), where it functions  to regulate transcription factor and chromatin remodeling activity, and genome interpretation.  Here, we describe a simple DNAme engineering approach in pluripotent stem cells (PSCs),  extending across large stretches of CpG dense “islands (CGIs).” Integration of synthetic CpG free  single-stranded DNA (ssDNA) induces a target CpG Island Methylation Response (CIMR) in  multiple PSC lines, Nt2d1 embryonal carcinoma cells, and mouse PSCs, but not in highly  methylated CpG Island Methylator Phenotype (CIMP) positive cancer lines. CIMR DNAme at  MLH1 spans the CGI, is robustly maintained throughout cellular differentiation, suppresses target  gene activity, and sensitizes derived cardiomyocytes and thymic epithelial cells to the  chemotherapy cisplatin. Additional CIMR DNAme is reported on at TP53 and ONECUT1 CGIs.  Collectively, this new resource enables total CpG Island DNAme engineering in pluripotency and  the genesis of novel epigenetic models of development and disease

overall_design: Modification of the MLH1 promoter to fully DNA methylated on one allele by CIMR induction. Differentiation of this MLH1-PM HESC line into human cardiomyocytes and thymic epithelial cells.


title: ncRNA profile in peripheral blood mononuclear cells and identification novel biomarkers in coronary artery disease

city: Nanjing

first: enzhi

last: jia

state: Jiangsu Province

address: Guangzhou Road 300

institute: Department of Cardiovascular Medicine, The First Affiliated Hospital of Nanjing Medical University

summary: Total transcriptome and gene expression profile of peripheral blood mononuclear cells (PMBCs) from 5 patients with CAD and 5 healthy controls were analyzed by RNA sequencing.

overall_design: 5 CAD patients and 5 controls were selected by matching sex and age for transcriptome high throughput sequencing

first: En-Zhi

last: Jia

first: Wenfeng

last: Ji

first: Jiaxin

last: Chen

first: Shu

last: He


title: Generation of synthetic Acinetobacter baumannii specific nanobodies

city: Austin

first: Bryan

last: Davies

address: 2500 Speedway

institute: University of Texas at Austin

summary: The bacterial pathogen, Acinetobacter baumannii, is a leading cause of drug-resistant infections. Here, we investigated the potential of developing nanobodies that specifically recognize A. baumannii over other Gram-negative bacteria. Through generation and panning of a synthetic nanobody library, we identified several potential lead candidates. We demonstrate how incorporation of next generation sequencing analysis can aid in selection of lead candidates for further characterization. Using monoclonal phage display, we validated the binding of several lead nanobodies to A. baumannii. Subsequent purification and biochemical characterization revealed one particularly robust nanobody that broadly and specifically bound A. baumannii compared to other common drug resistant pathogens. These findings support the potentially for nanobodies to selectively target A. baumannii and the identification of lead candidates for possible future diagnostic and therapeutic development.

overall_design:
Sequencing of phagemid VHH inserts derived from phage display selection after the second round of phage display. Phage displayed VHHs were selected for binding to either control targets (GFP and V. cholerae) or A. baumannii.
A. baumannii binding dataset was filtered by each of the three CDRs (peptide1,2,3) to identify VHHs completely unique to the A. baumannii dataset compared to the control GFP and V. cholerae datasets.  See supplementary file "Filtered A.baumannii Binding VHH.csv" linked to the Series record.

first: Gregory

last: Knauf

middle: W


title: Genetic and epigenetic instability suggest a regulated mechanism for osteosarcoma tumorigenesis

city: Campinas

first: Mariana

last: Maschietto

state: São Paulo

address: Rua Márcia Mendes, 619

department: Research Center

country: Brazil

institute: Boldrini Children's Hospital

summary: Osteosarcomas (OS) arise more commonly during the rapid bone growth and remodeling, thus it is plausible to infer the relationship between their tumorigenesis and cell cycle disruption by genetic and epigenetic factors. Methylation data of 28 primary OS was obtained using the HM450K microarray, and compared with nine normal bone tissues. The tumor mutational burden was analyzed using data from the TruSight One sequencing panel. We identified 3,146 differentially methylated positions, and OS presented high methylation heterogeneity, with global hypomethylation and focal hypermethylation at CpG islands. There were 319 hypomethylated and 266 hypermethylated regions in tumors, located in the promoter regions of 172 and 178 genes, respectively. These genes were enriched for biological processes related to skeletal system morphogenesis, proliferation, inflammatory response and signal transduction. Six tumor suppressors presented deletions or hypermethylation (DLEC1, GJB2, HIC1, MIR149, PAX6, WNT5A), and four oncogenes presented gains or hypomethylation (ASPSCR1, NOTCH4, PRDM16, RUNX3). Most cases did not report mutations in DNMTs or TETs, but DNMT3B was recurrently gained, a possible explanation for the hypermethylation at CpG islands. Our analysis also revealed hypomethylation at 6p22, a region that contains several genes, including histones. Overall, aberrant DNA methylation was spread throughout the genome of osteosarcomas, presented with high variance, similar to other molecular alterations. While the open-sea hypomethylation likely contributes to genomic instability, enriched CpG island hypermethylation suggests the involvement of mechanisms related to silencing tumor suppressor genes in osteosarcoma tumorigenesis. These data also suggest DNMT3B probably impacting biological processes associated with early bone development.

overall_design: 37 Samples

first: Ana

last: Krepischi

first: Sara

middle: F

last: Pires

first: Daniel

middle: O

last: Vidal


title: lncRNA atlas of inflammatory diseases along the GI tract highlights regulatory mitochondrial metabolic epithelial functions of GATA6-AS1

city: Ramat Gan

first: Tzipi

last: Braun

address: Derech Sheba 2

country: Israel

institute: Sheba Medical Center at Tel Hashomer

summary: Long non-coding RNA (lncRNA) mechanisms in gut inflammation are poorly understood. Tissue-specificity of lncRNAs linked to patient outcomes may direct interventions with fewer off-target effects. Using 693 mucosal samples, we prioritize lncRNAs linked with ulcerative colitis (UC) course and define an atlas of lncRNAs expressed along the gastrointestinal tract dysregulated in celiac duodenum, Crohn Disease ileum, and UC rectum using independent test and validation cohorts.

overall_design: SOURCE cohort included terminal ileum biopsies of 18 treatment naïve Crohn disease and 25 non-IBD controls from the Sheba Medical Center.

first: Katya

last: Sosnovski

first: Amnon

last: Amir

first: Marina

last: BenShoshan

first: Kelli

last: VanDussen

first: Nina

last: Levhar

first: Haya

last: Abbas-Egbariya

first: Katia

last: Beider

first: Rakefet

last: Ben-Yishay

first: Michael

middle: J

last: Rosen

first: Anne

middle: M

last: Grifiths

first: Thomas

first: David

middle: R

last: Mack

first: Brendan

last: Boyle

first: Syed

last: Asad Ali

first: Sean

last: Moore

first: Melanie

last: Schirmer

first: Ramnik

last: Xavier

first: Subra

last: Kugathasan

first: Anil

middle: G

last: Jegga

first: Ifat

last: Abramovich

first: Efrat

last: Glick Saar

first: Batya

last: Weiss

first: Iris

last: Barshack

first: Eyal

last: Gottlieb

first: Tamar

last: Geiger

first: Shomron

last: Ben-Horin

first: Igor

last: Ulitsky

first: Jeffrey

middle: S

last: Hyams

first: Lee

last: Denson

first: Haberman

last: Yael


title: VDR-dependent signaling modulates neuroinflammation in the acute phase of ischemic stroke

first: Pan

last: Cui

address: No. 45 Changchun St, Xicheng District, Beijing, China 100053

department: Department of Neurology

institute: Xuanwu Hospital, Capital Medical University

summary: VDR deficiency in microglia markedly aggravated infarct volumes, neurological deficits, and neuroinflammation after cerebral ischemia. To investigate the role of VDR in microglia-regulated neuroinflammation, we performed RNA-seq analysis on microglia isolated from poststroke microglia-conditional VDR knockout (Vdr-cKO) mice and littermate controls. VDR elimination dramatically alters the gene expression profiles of postischemic microglia, implying that vitamin D signaling play a crucial role in microglia-modulated stroke pathogenesis.

overall_design: Brain single-cell suspensions were prepared from Vdr-cKO and Vdrf/f mice 3 days after middle cerebral artery occlusion (MCAO) or sham procedures. Cells were stained with APC-conjugated anti-CD45 and BV421-conjugated anti-CD11b antibodies. CD11b+CD45int microglia were sorted using FACS Aria III flow cytometer for RNA-seq analysis.

first: P

first: F

last: Xu

first: W

last: Lu

first: J

last: Hao


title: Maternal protein rbpms2 play a crucial role in mouse blastocyst formation

city: Nanjing City

first: Cheng

last: Zhou

address: Hanzhong Road no.140

institute: Nanjing Medical University

summary: The functional role of the maternal protein during early embryo development remains to be elucidated. Here we show that the maternal protein, Rbpms2, is highly expressed in mouse oocytes and that knockdown of Rbpms2 inhibits development from the morula to blastocyst stage. To study the underlying mechanism, we firstly analysed the differential expressed genes after Rbpms2 knockdown through single-cell sequencing. Combined the bioinformatics analysis of sequencing data, we then identify the impaired location of E-cadherin after knockdown of Rbpms2 may leading to the morula arrested development . Furthermore, Rbpms2 is important for development of mouse early embryos, most likely because it activates the BMP pathway. Taken together, our study uncovers that Rbpms2 activate the BMP pathway, and thus adjust the location of E-cadherin which is important for mouse early embryos development.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for rbpms2-knckdown embryos and control.

first: cheng

last: zhou

first: lu

last: zheng


title: Single cell transcriptomic profiling of microvascular endothelial cell heterogeneity in congenital diaphragmatic hernia

first: Peter

last: Bazeley

address: 9500 Euclid Avenue

department: Lerner Research Institute

institute: Cleveland Clinic

summary: Congenital diaphragmatic hernia (CDH) is a neonatal anomaly that includes pulmonary hypoplasia and hypertension. We hypothesized that differences in microvascular endothelial cell (EC) heterogeneity may be present during CDH lung development and contribute to nderdevelopment and remodeling of the lungs. Time mated rats were gavaged nitrofen to induce CDH. Left lungs were harvested from healthy control (2HC), nitrofen exposed (NC) and nitrofen-exposed with CDH (CDH) fetuses at E21.5. Transcriptomic analysis was performed using single-cell RNA sequencing, and unbiased clustering revealed 3 distinct microvascular EC clusters. The general microvascular EC cluster (mvEC) had a transcriptomic signature suggestive of increased inflammation in CDH. mvEC differential gene expression (DGE) between NC and CDH revealed downregulation of Ca4, Apln and Ednrb, which define a subpopulation of microvascular ECs important to lung development, gas exchange and repair of alveolar injury (mvCa4+). mvCa4+ ECs were significantly reduced between 2HC (22.6%), NC (13.1%) and CDH (5.3%), demonstrating an impact from lung compression. Gene ontology and Ingenuity Pathway Analysis demonstrated that mvCa4+ ECs are primed for vasculogenesis but have DGE suggestive of impaired cell division. These data suggest that inflammation driven by mvECs and absence of mvCa4+ ECs may contribute to CDH pathogenesis.

overall_design: 4 samples, 2 healthy controls, 1 treated non-diseased, 1 treated diseased

first: Jason

last: Robertson

first: Serpil

last: Erzurum

first: Kewal

last: Asosingh


title: Immunoprofiles of human Sertoli cells infected with Zika virus

status: Public on Jun 20 2023

city: Minneapolis

first: Michael

last: Gale

state: Minnesota

address: 689 23rd Ave SE

department: Microbiology and Immunology

institute: University of Minnesota

summary: Using genome-wide RNA-seq to compare immunoprofiles of human sertoli cells, we show that the most prominent response to ZIKV at early stage of infection was suppression of cell growth and proliferation functional pathways. Peak virus replication was associated with induction of multiple antiviral defense pathways. Unique ZIKV-associated signatures included dysregulation of germ cell-Sertoli cell junction signaling.

overall_design: Human Sertoli cells (hSeC) were infected with ZIKV strain PRVABC59 at the multiplicity of infection (MOI) of 1 and the virus was detected by plaque assay at 24 hrs post infection (hpi), with peak virus titers of 6 log10 PFU/mL observed at 72 hpi

first: Richard

last: Green

first: Daniel

middle: P

last: Strange

first: Leanne

last: Whitmore

first: Saguna

last: Verma

last: Gale Jr.


title: Targeting canonical and non-canonical STAT3 signaling by WB737, a novel potent STAT3 inhibitor, in natural killer/T-cell lymphoma

city: Shanghai

first: Jianfeng

last: Chen

state: Shanghai

address: 270 Dong'an Road

institute: Fudan University Shanghai Cancer Center

summary: Activating mutations of STAT3 were frequently found in patients with natural killer/T-cell lymphoma (NKTL), suggesting that targeted inhibition of STAT3 is a potential therapeutic option for NKTL patients. Here, we have developed a novel and potent STAT3 inhibitor WB737 that directly binds to the STAT3-SH2 domain with high affinity. WB737 selectively inhibits cell growth of NKTL harbored STAT3 mutations through inducing apoptosis and blocking colony formation. Mechanistically, WB737 inhibits both canonical and non-canonical STAT3 signaling via suppression of STAT3 phosphorylation at Tyr705 and Ser727 sites, respectively. Moreover, the activity of WB737 has a more potent inhibition on STAT3 compared to Sttatic, resulting in a significant anti-tumor effect followed by almost complete tumor regression in a NKTL xenograft model harboring STAT3 activating mutation. Collectively, these findings provide a preclinical proof-of-concept for WB737 as a novel therapeutic strategy for the treatment of NKTL patients with STAT3-activating mutations.

overall_design: RNA-seq for NKYS treated with DMSO or WB737 was performed (duplicate).

first: Yali

last: Wang

first: Wenbo

first: Yihua

first: Jing

last: Tan


title: Dynamic changes in the transcriptome of tRNA-derived small RNAs related with fat metabolism I

city: Chengdu

first: mailin

last: gan

state: Sichuan

address: No. 211, Huimin Road, Wenjiang District, Chengdu, Sichuan, China

institute: Sichuan Agricultural University

summary: The prevalence of obesity and overweight is steadily rising, posing a significant global challenge for humanity. The fundamental cause of obesity and overweight lies in the abnormal accumulation of adipose tissue. While numerous regulatory factors related to fat deposition have been identified in previous studies, a considerable number of regulatory mechanisms remain unknown. tRNA-derived small RNAs (tsRNAs), a novel class of non-coding RNAs, have emerged as significant regulators in various biological processes. In this study, we obtained small RNA sequencing data from subcutaneous white adipose tissue and omental white adipose tissue of lean and obese pigs. In addition, we similarly obtained tsRNAs profiles from scapular brown adipose tissue (BAT), inguinal white adipose tissue (iWAT) and epididymal white adipose tissue (eWAT) of normal mice. Finally, we successfully identified a large number of expressed tsRNAs in each tissue type and identified tsRNAs conserved in different adipose tissues of pigs and mice. These datasets will be a valuable resource for elucidating the epigenetic mechanisms of fat deposition.

overall_design: So to investigate the role of tsRNAs in fat deposition, we collected subcutaneous white adipose tissue and omental white adipose tissue from both Qingyu pig (obese pig) and Yorkshine (lean pig) and obtained their tsRNAs sequencing data. In addition, we also obtained tsRNAs sequencing data of scapular brown adipose tissue (BAT), inguinal white adipose tissue (iWAT) and epididymal white adipose tissue (eWAT) from normal C57BL/6j mice.

first: Tianci

last: Liao

first: Mailin

last: Gan

first: Linyuan

last: Shen

first: Li

last: Zhu


title: RNA-seq analysis of FOXM1 depletion and RNF112 overexpression stable MGC803 cells together with corresponding controls

city: Chongqing

first: wei

middle: Sheng

last: Zhang

address: xinqiao street

department: gastroenterology

institute: Xinqiao hospital

summary: RNA sequencing (RNA-Seq) is a widely recognized technique for genome-wide expression analysis. In this study, we aim to illustrate the role of RNF112 in gastric cancer cells and compare the downstream networks of RNF112 and FOXM1 to further elaborate the associations between these two proteins.

overall_design: mRNA profiles of FOXM1 depletion and RNF112 overexpression stable MGC803 cells together with corresponding controls

first: Changjiang

last: Hu

first: Shiming

last: Yang

first: Shengwei


title: The diapause-like colorectal cancer cells induced by SMC4 attenuation are characterized by low proliferation and chemotherapy insensitivity[scRNA-seq]

city: hefei

first: sun

last: xuedan

address: daoxiangcun

institute: ustc

summary: We used single-cell sequencing to detect tumor cell types in mouse subcutaneous MC38 colorectal cancer

overall_design: Solid tumors from mice were excised and mechanically dissociated into single cells using Tumor Dissociation Kit (SHBio). Single cells were captured in the 10 × Genomics Chromium Single Cell  Solution, and RNA-seq libraries were prepared following the manufacturer’s protocol (10 × Genomics). The libraries were subjected to high-throughput sequencing on an Illumina Hiseq X Ten PE150 platform, and 150-bp paired-end reads were generated.

first: Xue

last: Sun

last: He

first: Mian

last: Wu

first: Lian

middle: X

last: Liu


title: Analyzing the impact of extrachromosomal rDNA circles (ERCs) on the ageing yeast transcriptome

city: Cambridge

first: Laura

last: Biggins

address: Babraham Research Campus

country: United Kingdom

institute: The Babraham Institute

summary: Here we analyzed the widespread disruption of gene expression that accompanies yeast ageing, and surprisingly observed that this is completely independent of ERCs. Furthermore, we could not find subsets of genes that are differentially regulated in the presence of ERCs. High throughput imaging showed that the accumulation of Tom70-GFP which accompanies the onset of cell division defects at the Senescence Entry Point (SEP) also correlated poorly to ERC abundance, but allowed determination of a gene expression signature for the SEP. This signature included overexpression of mRNA from the chromosome XII region between the rDNA and the telomere (ChrXIIr), which has been previously noted to amplify during ageing.

overall_design: Mother Enrichment Project (MEP) yeast were harvested at log phase or aged for 24 or 48 hours in YPD. Mother cells were purified, total RNA extracted and libraries processed using a NEBNext Ultra II directional RNAseq kit with poly(A)+ selection module.

first: Hanane

last: Hadj-Moussa

first: Prasanna

last: Channathodiyil

first: Luca

last: Love

first: Andre

last: Zylstra

first: Jon

last: Houseley


title: Transcriptional deregulation during osteoclastic differentiation due to diuron exposure affects preferentially super-enhancers regulated genes

city: Nantes

first: Benjamin

last: Ory

address: 1 rue Gaston Veil

summary: Osteoclasts are major actors in the maintenance of bone homeostasis. The full functional maturation of osteoclasts from monocyte lineage cells is essential for the degradation of old/damaged bone matrix. To better characterize the differentiation and maturation of CD14+ monocytes into functionally mature osteoclasts, we performed Chromatin Immunoprecipitation against H3K27ac followed by Sequencing (ChIP-Seq) and RNA-Sequencing at different stages of an in vitro differentiation model. The combinatorial study of the epigenetic and transcriptional dynamics taking place during the course of differentiation has allowed us to reveal a very dynamic epigenetic profile that supports the expression of genes vital for osteoclasts differentiation and function. In total, we identified 122 genes induced by dynamic super-enhancers at late days. Since diuron, a commonly detected herbicide in our environment, is suspected to affect bone development, we then sought to evaluate the potential impact of diuron exposure on bone homeostasis. We performed RNA-Seq and functional test during in vitro osteoblastic and osteoclastic differentiation. First of all, our data suggest that high-dose of diuron (50 µM) affects viability of mesenchymal stem cells and thus inhibits bone mineralization. We observed at lower dose (1 µM) an inhibitory effect of this herbicide on number of osteoclasts derived from CD14+ monocytes without affecting cell viability. RNA-Seq data analysis revealed that diuron is specifically affecting the transcriptional network dedicated to osteoclasts differentiation and activity. Among the diuron affected genes, our analysis suggests a significant enrichment of genes targeted by gained super-enhancers along the differentiation process, with an odds ratio of 5.12 (ρ = 2.59 x 10-5). Taken together our results suggest that diuron exposure disrupts osteoclasts maturation by impairing the expression of cell-identity determining genes.

overall_design:
Diuron dataset: CD14+ pre-monocytes were differentiated into osteoclasts using RANK+M-CSF treatment in the presence of either non-cytotoxic concentration of diuron or DMSO and RNA-Sequencing was performed at early stage (day3) or late stage of differentiation (day 10).
Differentiation RNA-Seq data: CD14+ pre-monocytes were cultered with or withour differentiation medium and RNA-Sequencing was performed at early stage (day3), intermediate stage (day7) or late stage of differentiation (day 10).
Differentiation ChIP-Seq data: In order to track epigenetic modifications within enhancers and super-enhancers H3K27ac ChIP-Seq was performed on cells cultered for 3 days without differentiation medium, cultered for 7 days or 10 days with differentiation medium.

first: Robel

last: Tesfaye

first: Mélanie

last: Lavaud

first: Maxime

last: Golias

first: Céline

last: Charrier

first: Pierre-François

last: Cartron

first: Franck

last: Verrecchia

first: Marc

last: Baud’huin

first: François

last: Lamoureux

first: Steven

last: Georges


title: Exploring the signalling alteration between GBM8401 and GBM8401 supplemented with siINTS9

city: Taipei

first: YAO FENG

last: LI

address: No.161, Sec. 6, Minquan E. Rd., Neihu Dist., Taipei City 11490, Taiwan

department: Pathology

institute: National Defense Medical Center

summary: To investigate the changes of the transcriptome and relevent signalling between control and siINTS9 treated glioblastoma cell line,GBM8401.

overall_design: We then performed gene expression profiling analysis using data obtained from RNA-seq of 3 sets of GBM8401 control verus 3 sets of GBM8401 treated siINTS9

first: Yao

last: Li

first: Pei-Chi

last: Chang


city: Iowa City

first: Andrew

last: Voigt

state: IA

address: 375 Newton Road

institute: University of Iowa

summary:
Purpose: To identify transcriptomic features of hiPSC-derived cells with a good capability to generate retinal organoids
Methods: Human induced pluripotent stem cell (hiPSC) samples were collected at two time points for RNA sequencing (day zero and seven, each time point in triplicate). Cell pellets were collected and flash frozen. Total RNA was extracted by a phenol/chloroform method using TRIzol LS reagent (ThermoFisher Scientific, MA) according to the manufacturer's guidelines. tRNA was treated with DNAse I prior to library preparations being performed for RNAsequencing.
Results:  Human iPSC lines show variation in their ability to differentiate into a neuroectodermal/early eye field phenotype. This study identified a panel of signature transcripts that were differentially expressed at seven days of differentiation, between lines of low- and high-retinal propensity.
Conclusions: This study provides a gene profile that identifies which hiPSC-derived cells have a good capability to generate retinal organoids. This allows for the analysis of retinal propensity by quantitative PCR within the first seven days of differentiation.

overall_design: mRNA profiles from 15 hiPSC-derived cell lines were generated in triplicate at day 0 and day 7. Transcriptomic features of hiPSC-derived cells with a good capability to generate retinal organoids were identified.

first: Jessica

middle: A

last: Cooke

first: Todd

last: Scheetz

first: Budd

last: Tucker


title: Assessment of the molecular mechanism associated with osteogenic and angiogenic differentiation induced by FeMnSi scaffold

first: Bo

last: Yuan

address: Jiuyanqiao Wangjiang Road

institute: Sichuan University

summary: To investigate the molecular mechanism underlying osteogenic and angiogenic differentiation induced by the De-HA scaffold, we conducted an in-depth gene microarray analysis on day 3 of MC3T3-E1 culture. We found that De-HA could enhance the transcription of osteogenic and angiogenic genes by activating FAK and downstream MAPK/ERK signaling to induce MC3T3-E1 differentiation. More importantly, our findings indicated that EGFR, a kind of transmembrane glycoprotein, played a key role in the osteogenic differentiation of MC3T3-E1, suggesting that the activation of the FAK/MAPK/ERK signaling cascade might be driven by EGFR.

overall_design: The scaffold samples were segregated into two groups: the first comprised of the original FeMnSi scaffold, while the second consisted of a De-HA scaffold that had been dealloyed and coated with bioactive materials. MC3T3-E1 were seeded on each scaffold at a density of 1 × 105 per/well in 24-well plates and then cultured for 3 days. An extensive gene microarray analysis was conducted on day 3 of MC3T3-E1 culture.

first: Zhikun

last: Chen


title: Single-cell RNA sequencing reveals peripheral blood mononuclear immune cell landscape associated with operational tolerance in a kidney transplant recipient [2]

city: Baltimore

first: Amol

middle: Carl

last: Shetty

state: Maryland

address: 670 W. Baltimore Street, Floor 3

department: Institute for Genome Sciences

institute: University of Maryland Baltimore

summary: Operational tolerance (OT) after kidney transplantation is defined as stable graft acceptance without the need for immunosuppression therapy. However, it is not clear which cellular and molecular pathways are driving tolerance in these patients.  In this first-of-its-kind pilot study, we assessed the immune landscape associated with OT (Tol) using the single-cell analyses. Peripheral mononuclear cells (PBMCs) from a kidney transplant recipient (KTR) with OT, a healthy individual (HC), and a KTR with normal kidney function standard of care immunosuppression (SOC) were evaluated. B cell proportion was greatest in Tol with the predominance of TCL1A+ naïve B cells; regulatory T cell (Tregs) was also high in Tol with a relatively higher expression of LSGAL1.  Putative ligand-receptor-based cell-cell interactions between B cells and Tregs were identified. TGFB-TGFBR interaction, present in both Tol and SOC, leads to the proliferation of Tregs. However, SOC had the lowest proportion of B cells and Tregs with a G1 phase accumulation of B and T cells. Our findings instigate application of this technology on a larger cohort to identify mediators of tolerance.

overall_design: PBMCs were isolated from a healthy control, a KTR with stable graft function and on standard of care, and a KTR who attained operational tolerance case (n=1).  Single cell RNA sequencing was done on this PBMCs. The proportion of cells and the differences in their gene expression was analysed and compared across the three groups to identify molecular and cellular landscape associated with operational tolerance.

first: Shafquat

last: Azim

first: Haseeb

last: Zubair

first: Thomas

last: Rousselle

first: Jennifer

last: McDaniels

first: Canan

last: Kuscu

first: Cem

first: Manish

last: Talwar

first: Daniel

last: Maluf

first: Valeria

last: Mas


title: Mitochondrial PGAM5−Drp1 signaling regulates the metabolic reprogramming of macrophages and regulates the induction of inflammatory responses

city: Sunnyvale

first: Young

middle: Jun

last: Kang

address: 428 Oakmead Pkwy

institute: Molecular Medicine Research Institute

summary: Macrophages play a critical role in the regulation of inflammation and tissue homeostasis. In addition to their vital functions for cell survival and physiology, mitochondria play a crucial role in innate immunity as a platform for the induction of inflammatory responses by regulating cell signaling and dynamics. Dynamin-related protein 1 (Drp1) plays a role in the induction of inflammatory responses and the subsequent development of various diseases. PGAM5 (phosphoglycerate mutase member 5) is a mitochondrial outer membrane phosphatase that dephosphorylates its substrate, Drp1. Previous studies showed that PGAM5 regulates the phosphorylation of Drp1 for the activation of NKT cells and T cells. However, it is not clear how PGAM5 regulates Drp1 activity for the induction of inflammation in macrophages. Here, we demonstrate that PGAM5 activity regulates the dephosphorylation of Drp1 in macrophages, leading to the induction of proinflammatory responses in macrophages. In TLR signaling, PGAM5 regulates the expression and production of inflammatory cytokines by regulating the activation of downstream signaling pathways, including the NF-kB and MAPK pathways. Upon LPS stimulation, PGAM5 interacts with Drp1 to form a complex, leading to the production of mtROS. Furthermore, PGAM5-Drp1 signaling promotes the polarization of macrophages toward a proinflammatory phenotype. Our study further demonstrates that PGAM5-Drp1 signaling promotes metabolic reprogramming by upregulating glycolysis and mitochondrial metabolism in macrophages. Altogether, PGAM5 signaling might be a link between alterations in Drp1-mediated mitochondrial dynamics and inflammatory responses in macrophages and may be a target for the treatment of inflammatory diseases.

overall_design: To investigate the role of PGAM5 in the regulatioon of inflammatory response in macropahges, immortalized mouse bone marrow-derived macrophages (BMDMs) were infected with control or PGAM5-targeting shRNA to knock down PGAM5. Control or PGAM5 KD iBMDMs were stimulated with LPS for 4 hours and total RNAs were prepared.


title: Differential Gene Expression between YTN16 Gas6 Cells and YTN16 Cells

city: Suwon

first: Tae Hoon

last: Kim

address: 206, World cup-ro, Yeongtong-gu

department: surgery

country: South Korea

institute: Ajou university

summary: We have generated Gas6 knock-down YTN16 cells using the shRNA system to specifically target Growth arrest-specific 6 (Gas6), a gene of interest in mouse gastric cancer cells. Additionally, we aimed to determine the differences in gene expression levels between YTN16 cells and YTN16 cells with Gas6 knock-down (shGas6) through RNA-sequencing analysis.

overall_design: To investigate the immunotherapy response in the GAS6/AXL signaling, we established Gas6 knock down YTN16 cell line by shRNA

first: Hoon

last: Hur

first: Dong

last: Lee

first: Tae


title: Gene expression profile of grafts in 6-week-old and 12-week-old recipients 24 h after receiving similar donor kidneys

first: Chengjun

last: Yu

address: No.136, Zhongshan 2nd Road, Yuzhong District, Chongqing City, China

institute: Children’s Hospital of Chongqing Medical University

summary: To investigate the effect of youthful systemic milieu on acute kidney injury in a rat kidney transplantation model based on young (6-week-old) and adult (12-week-old) recipients receiving similar donor kidneys (12-week-old)

overall_design: 12-week SD rat donor kidneys were heterotopically transplanted into 6-week Wistar rats (young recipients, Y group, shown in initial data as "IM") and 12-week Wistar rats (adult recipients, A group, shown in initial data as "M").

first: Shengde

last: Wu

first: Yi

last: Hua

first: Guanghui

last: Wei


title: Liver and bone marrow response to phlebotomy in Erfe-/- mice

city: TOULOUSE cedex 3

first: Léon

last: Kautz

address: CHU Purpan - Place du Dr Baylac - CS 60 039

department: IRSD U1220

institute: INSERM

summary: We analyzed the liver and marrow mRNA profiles of Erfe-/- mice 0, 24 and 48 hours after phlebotomy.

overall_design: Mice were phlebotomized by retro-orbital puncture and were analyzed after 0, 24 and 48 hours. 4 mice per time-point.

first: Ugo

last: Sardo

first: Anthony

last: Emile

first: Yannick

last: Lippi


title: Single-cell RNA sequencing reveals peripheral blood mononuclear immune cell landscape associated with operational tolerance in a kidney transplant recipient [1]

first: Amol

last: Shetty

overall_design: PBMCs were isolated from a healthy control, a KTR with stable graft function and on standard of care, and a KTR who attained operational tolerance case (n=1).  Single cell RNA sequencing was done on these PBMCs. The proportion of cells and the differences in their gene expression was analyzed and compared across the three groups to identify molecular and cellular landscape associated with operational tolerance.

first: Shafquat

last: Azim

first: Haseeb

last: Zubair

first: Thomas

last: Rousselle

first: Jennifer

last: McDaniels

first: Canan

first: Cem


title: Effect of cigarette smoking on gene expression of peripheral blood B cells and on transcriptome changes by naïve-to-memory B cell shift

city: Research Triangle Park

first: Douglas

middle: A.

last: Bell

state: NC

address: PO Box 12233

department: Immunity Inflammation and Disease Lab

institute: National Institute of Environmental Health Sciences

summary: To investigate blood biomarkers of smokers and to provide molecular basis of B cell subtype shift from naïve to memory in smokers, total RNA from peripheral CD19+ B cells isolated from healthy adult smokers and nonsmokers was subjected to RNA-Seq and data were analyzed with covariates including age, race, sex, BMI, estimated CD4+T cell%, CD8+T cell%, natural killer (NK) cell%, monocyte%, granulocyte%, or naive B cell%.

overall_design: Gene expression profiling analysis was done using data obtained from RNA-seq of 2 condition groups, smoking and nonsmoking.

first: Michelle

last: Campbell

first: Hye-Youn

last: Cho


title: Modelling idiopathic intracranial hypertension in rats: contributions of high fat diet and testosterone to intracranial pressure and cerebrospinal fluid production

city: Copenhagen

first: Søren

middle: Norge

last: Andreassen

state: Region Hovedstaden

address: Blegdamsvej 3

department: Department of Neuroscience

country: Denmark

institute: University of Copenhagen

summary: Female Wistar rats were either fed nutrient source-matched control diet (control) or a high fat diet (HFD). HFD-fed rats presented with increased ICP, which was not accompanied by altered CSF dynamics or modified choroid plexus function. HFD-induced ICP elevation in experimental rats did not originate from an increased rate of CSF secretion.

overall_design: Differential expression analysis of female wistar rats fed either nutrient source-matched control diet (control) or high fat diet (HFD).

middle: N

first: Jonathan

last: Wardman

first: Nanna

last: MacAulay


title: The hyperconnected 3D clique at the Ets1 locus is required for T cell function (RNA-Seq)

city: Philadelphia

first: Golnaz

last: Vahedi

state: PA

address: 421 Curie Blvd

department: Department of Genetics

institute: University of Pennsylvania

summary: The spatiotemporal control of gene-expression programs relies on selection and nuclear organization of enhancers. Despite mounting evidence on the formation of multi-enhancer hubs with extensive spatial connectivity, the functional importance of these architectural units remains elusive. Here, we generated a new mouse model and genetically perturbed a genomic locus harboring the Ets1 gene which is hyperconnected in T cells and has strong association with immune-mediated diseases. Although T cell development was intact, competence towards CD4+ T helper 1 specialization was lost in vitro and in vivo after genetic perturbation of the Ets1 locus. Extensive high-resolution molecular mapping of 3D genome organization revealed that the hyperconnectivity of the Ets1 locus controlled the expression level of Ets1 which was required for the Th1-specific genome topology. Our work illustrates the functional relevance of multi-enhancer hub formation in T cells.

overall_design: RNA-Seq

first: Aditi

last: Chandra

first: Sora

last: Yoon

first: Michael

last: Michieletto

first: Naomi

last: Goldman

first: Emily

middle: K

last: Ferrari

first: Leonel

last: Joannas

first: Jorge

last: Henao-Mejia


title: The hyperconnected 3D clique at the Ets1 locus is required for T cell function (CUT&RUN)

overall_design: CUT&RUN

middle: F


title: Predictive signature of response to neoadjuvant chemotherapy in muscle-invasive bladder cancer integrating mRNA expression, taxonomic subtypes, and clinicopathological features

city: Madrid

first: Jaime

last: Martinez de Villarreal

state: Madrid

address: Melchor Fernández Almagro, 3

department: Molecular Oncology Programme

institute: CNIO

summary: Background and Objective: Neoadjuvant chemotherapy (NAC) followed by cystectomy is the standard of care in muscle-invasive bladder cancer (MIBC). Pathological response has been associated with longer survival, but no currently available clinicopathological variables can identify patients likely to respond, highlighting the need for predictive biomarkers. We sought to identify a predictive signature of response to NAC integrating clinical score, taxonomic subtype, and gene expression.     Material and Methods: From 1994 to 2014, pre-treatment tumor samples were collected from MIBC patients (stage T2-4N0/+M0) at two Spanish hospitals. A clinical score was determined based on stage, hydronephrosis and histology. Taxonomic subtypes (BASQ, luminal, and mixed) were identified by immunohistochemistry. A custom set of 41 genes involved in DNA damage repair and immune response was analyzed in 84 patients with the NanoString nCounter platform. Genes related to pathological response were identified by LASSO penalized logistic regression. NAC consisted of cisplatin/methotrexate/vinblastine until 2000, after which most patients received cisplatin/gemcitabine. The capacity of the integrated signature to predict pathological response was assessed with AUC. Overall survival (OS) and disease-specific survival (DSS) were analyzed with the Kaplan-Meier method.  Results: LASSO selected eight genes to be included in the signature (RAD51, IFNγ, CHEK1, CXCL9, c-MET, KRT14, HERC2, FOXA1). The highest predictive accuracy was observed with the inclusion in the model of only three genes (RAD51, IFNɣ, CHEK1). The integrated clinical-taxonomic-gene expression signature including these three genes had a higher predictive ability (AUC=0.71) than only clinical score plus taxonomic subtype (AUC=0.58) or clinical score alone (AUC=0.56). This integrated signature was also significantly associated with OS (p=0.02) and DSS (p=0.02).   Conclusions: We have identified a predictive signature for response to NAC in MIBC patients that integrates the expression of three genes with clinicopathological characteristics and taxonomic subtypes. Prospective studies to validate these results are ongoing.

first: Albert

last: Font

first: F

last: Real


title: Smoking effect on DNA methylation in human blood cell types

first: Xuting

last: Wang

address: 111 TW Alexander Dr

institute: NIEHS/NIH

summary: Tobacco smoking alters DNA methylation profiles of immune cells which may underpin some of the pathogenesis of smoking-associated diseases. However, approaches linking smoking-driven epigenetic effects in specific immune cell types with disease risk are limited. We isolated six leukocyte subtypes, CD14+ monocytes, CD15+ granulocytes, CD19+ B cells, CD4+ T cells, CD8+ T cells, and CD56+ natural killer cells, from whole blood of healthy adult smokers and nonsmokers for epigenome-wide association study (EWAS).

overall_design: As part of the Epigenetic Biomarkers of Tobacco Smoke Exposure project, a group of healthy volunteers (68 smokers and 74 nonsmokers) was recruited at the NIEHS Clinical Research Unit (protocol 10-E-0063) between March 2013 and January 2018 from the Raleigh, Durham and Chapel Hill, NC area. All nonsmokers were self-reported as not having smoked >100 cigarettes in their lifetime. Smokers reported their average daily cigarette consumption for the past 3 months [Su D et al. 2016].   For each individual, granulocytes were isolated directly from whole blood using CD15 Dynabeads (Invitrogen, Waltham, MA), magnetic beads covalently coupled with an anti-human CD15 antibody, according to the manufacturer’s protocol. Additionally, whole blood was layered on Histopaque-1077 Ficoll medium in Accuspin™ Tubes (Sigma-Aldrich, St. Louis, MO) and density gradient centrifugation was performed to isolate the mononuclear layer.  Peripheral blood mononuclear cells (PBMCs) were then counted for viability and incubated with CD34 MicroBeads magnetic beads (Miltenyi Biotec, San Jose, CA).  CD34 negative cells were collected in the flowthrough, recounted on a Cellometer Auto T4 Bright Field Cell Counter (Nexcelom, St. Lawrence, MA), and incubated with CD14 Dynabeads to isolate CD14+ monocytes according to the manufacturer’s protocol.  Flowthrough (CD14-) cells were again counted and split by volume.  One half of the cells were incubated with CD19 PanB Dynabeads for isolation of CD19+ B cells. The other flowthrough half was split in two, with half incubating with CD4 Dynabeads (for CD4+ T cells) and half incubating in CD8 Dynabeads (for CD8+ T cells).  All flowthrough (CD19-, CD4-, or CD8- cells) was then combined and incubated with CD56 MicroBeads (Miltenyi Biotec) according to the manufacturer’s protocol to isolate CD56+ NK cells.

first: Douglas

last: Bell

first: Gary

last: Pittman


title: Repair of Acute Respiratory Distress Syndrome in COVID-19 by Stromal Cells (REALIST-COVID trial).

city: Edinburgh

first: Jennifer

last: Rynne

email: jrynne@ed.ac.uk, jenrynne@gmail.com

address: QMRI, Edinburgh BioQuarter, Little France Crescent

institute: University of Edinburgh

summary: Mesenchymal stromal cells are a potential therapeutic for Acute Respiratory Distress Syndrome due to COVID-19, with pleiotropic immunomodulatory and reparative properties.This study investigated the safety and efficacy of ORBCEL-C (CD362 enriched umbilical cord-derived Mesenchymal Stromal Cells) in this patient population.

overall_design: In this multicentre, randomised, double-blind, allocation concealed, placebo-controlled trial (registration number NCT03042143), mechanically ventilated patients with moderate to severe Acute Respiratory Distress Syndrome due to COVID-19 were randomised to receive a single intravenous infusion of ORBCEL-C (400 x 106 cells) or placebo (Plasma-Lyte 148). Whole blood RNA sequencing was performed on samples at day 0, 4, and 7.

first: Ellen

last: Gorman

first: Hannah

last: Gardiner

first: Anthony

last: Rostron

last: Bannard-Smith

first: Andrew

last: Bentley

first: David

last: Brealey

first: Christina

last: Campbell

first: Gerard

last: Curley

first: Mike

last: Clarke

first: Ahilanadan

last: Dushianthan

first: Phillip

last: Hopkins

first: Colette

last: Jackson

first: Kallirroi

last: Kefela

first: Anna

last: Krasnodembskaya

first: John

last: Laffey

first: Cliona

last: McDowell

first: Margaret

last: McFarland

first: Jamie

last: McFerran

first: Peter

last: McGuigan

first: Gavin

last: Perkins

last: Silversides

first: Jon

last: Smythe

first: Jacqui

last: Thompson

first: William

last: Tunnicliffe

first: Ingeborg

last: Welters

first: Laura

last: Amado-Rodriguez

first: Guillermo

last: Albaiceta

first: Barry

last: Williams

first: Manu

last: Shankar-Hari

first: Daniel

last: McAuley

first: Cecilia

last: O'Kane


title: Multi-omic signatures of Chronic Beryllium Disease bronchoalveolar lavage cells relate to T cell function and innate immunity [Expression]

city: Aurora

first: Ivana

last: Yang

state: CO

institute: University of Colorado - Anschutz Medical Campus

summary: We performed gene expression profiling between Chronic Beryllium Disease, beryllium-sensitized, and healthy control bronchoalveolar cells, and integration with methylation and proteomic data

overall_design: Bronchoalveolar lavage cells were collected from 30 Chronic Beryllium Disease, 30 Beryllium Sensitized, and 12 healthy control lungs

first: Iain

last: Konigsberg

first: Li

last: Li

middle: V

first: Lisa

last: Maier