title: Analysis of gene expression patterns in brown adipose tissue

city: Nuthetal

first: Tim

middle: Julius

last: Schulz

address: Arthur-Scheunert-Allee 114-116

institute: German Institute of Human Nutrition

summary: Brown adipose tissue is a specialized fat tissue involved in heat generation by using a process termed thermogenesis. This ability to convert nutrient energy into heat sets brown adipocytes apart from the more common type of white adipocytes, which are mainly involved in energy storage. Brown adipose depots occur as a large interscapular depot in mice. Brown adipocytes are multilocular, i.e. contain many small lipid-filled vacuoles and uniquely express the uncoupling protein 1. In this study, the gene expression patterns in brown adipose tissue samples of young, healthy mice was analyzed.

overall_design: Male C57Bl6J mice were housed under controlled conditions at 22 ±2 °C in an 12/12-hour light/dark cycle maintained on a standard chow diet (Ssniff, Soest, Germany). The animals were killed at 10 weeks of age by cervical dislocation. Brown adipose tissue depots were isolated and immediately frozen in liquid nitrogen. RNA was isolated from 50 mg of tissue. NGS sequencing was conducted using Illumina HiSeq 2000 with 2 x 100 bp reads (LGC genomics, Berlin, Germany). For data processing and analysis, FASTQC v0.11.8 was used for quality assessment and trimmgalore v0.6.4 was applied for filtering adapter sequences and small reads. The clean reads were mapped to a reference genome (Genome Reference Consortium Mouse Build 38 mm10) using the alignment tools HISAT2 v2.1.0 and Bowtie2 v2.3.5.1 Expression levels were quantified Cufflinks v.2.2.1.

first: Sabrina

last: Gohlke


title: Lgr5 Marks Stem/Progenitor Cells Contributing to Epithelial and Muscle Development in the Mouse Esophagus [scRNA-Seq]

status: Public on Jul 22 2024

city: Singapore

first: Lana

last: Kostic

address: 61 Biopolis Dr, Proteos

department: IMCB

country: Singapore

institute: A*STAR

type: Expression profiling by high throughput sequencing

summary: The existence and function of Lgr5+ cells within the developing esophagus remains unknown. Here, we document multiple discrete Lgr5+ populations in the developing mouse esophagus, predominantly within nascent epithelial and external muscle layers. Lgr5 expression initially emerges in the developing proximal embryonic epithelium, but progressively extends distally and persists within the distal epithelium of neonates. Fate mapping and ablation analyses reveal a long-term contribution of epithelial Lgr5+ cells to esophageal organogenesis. Additionally, Lgr5-expressing cells are present in the developing external muscle layer, particularly during the development of the striated component. Fate mapping reveals a significant contribution of these embryonic Lgr5+ cells to the adult muscle layer. Embryonic Lgr5+ epithelial cells are also found to be important for regulating epithelial development, serving as a key source of Wnt6, among other ligands, to promote epithelial cell proliferation and formation of epithelial layers. These findings significantly enhance our understanding of esophageal development and shed light on the involvement of Lgr5+ stem/progenitor cells during organogenesis. Importantly, this study lays the foundation for investigating esophageal diseases related to the Lgr5+ stem/progenitor cell pool.

overall_design: Embryonic and neonatal esophagi harvested from WT embryos at E13.5 (n=12), E16.5 (n=9) and P0 (n=7). Samples from each time point were pooled and processed toghether.

first: Carly

last: Leung

first: Katzrin

last: Ahmad Murad

first: Snezhina

last: Kancheva

first: Stefano

last: Perna

first: Bernett

last: Lee

first: Nick

last: Barker


title: Lgr5 Marks Stem/Progenitor Cells Contributing to Epithelial and Muscle Development in the Mouse Esophagus

overall_design: Embryos were harvested from WT females at E13.5 and E16.5, and esophagi were dissected from P0 neonates. Samples were fresh frozen in Tissue Freezing Medium and kept in -80 freezer before sectioning. Sections were palced on Visium Spatial Gene Expression Slide and processed according to manufacturer instructions.


title: Neuromodulatory co-expression in cardiac vagal motor neurons of the Dorsal Motor Nucleus of the Vagus [10x scRNA]

city: Albuquerque

first: Rajanikanth

last: Vadigepalli

state: PA

address: 1 University of New Mexico

country: USA

institute: University of New Mexico

summary: The activity of the cardiac vagal innervation is well known to be crucial to the maintenance of cardiac health, and to protect and recover the heart from injury. Only recently has this role been shown to depend on the activity of the underappreciated Dorsal Motor Nucleus of the Vagus (DMV). By combining neural tracing, transcriptomics, and 3D mapping in male and female Sprague Dawley rats we characterize cardiac-specific neuronal phenotypes in DMV. We find that the DMV cardiac-projecting neurons differentially express PACAP, CART, and synucleins, as well as evidence that they participate in neuromodulatory co-expression involving catecholamines. The significance of these findings is enhanced by previous knowledge of the role of PACAP at the heart and of the other neuromodulators in peripheral vagal targets.

overall_design: We generated a single cell suspension from the dorsal medulla including the DMV by a dissociation protocol for adult rat brain (Wang et al. 2021), and viability was determined by calcein AM stain. The 4 samples included pools of 4 left and right dorsal brainstem from 12 week old male Sprague Dawley rats. We obtained sc-RNAseq data using 10x Genomics Chromium assay, available as a TJU core facility service.

first: Eden

last: Hornung

first: Shaina

last: Robbins

first: Sirisha

last: Achanta

first: James

last: Schwaber


title: Neuromodulatory co-expression in cardiac vagal motor neurons of the Dorsal Motor Nucleus of the Vagus [RNAseq]

overall_design: Single neurons were microdissected from the Dorsal Motor Nucleus of the Vagus (DMV) in the brainstem after Fast Blue retrograde tracer was injected into the intrinsic cardiac nervous system of one female Sprague Dawley rat. Half of the samples are cardiac-projecting neurons labeled by Fast Blue and the other half are non-cardiac-projecting (non-Fast Blue).


title: Neuromodulatory co-expression in cardiac vagal motor neurons of the Dorsal Motor Nucleus of the Vagus [PCR]

summary: In this study we performed high throughput microfluidic qPCR using BioMark on RNA extracted from single neurons microdissected from DMV and NA tissue of one male Sprague Dawley rat.

overall_design: High throughput microfluidic qPCR using BioMark was performed on RNA extracted from 81 single neurons microdissected from DMV and NA tissue of one male Sprague Dawley rat. Two 96 x 96 BioMark Arrays were used to assay expression of 173 genes. A serial dilution gene set was included in each chip to confirm reproducibility. Technical replicates were run on each chip to evaluate technical variability.  Data Normalization: Initial quality control included melt-curve analysis and removal of samples and genes with >30% failed reactions. Genes with >75% working reactions were used to generate a median expression level across all samples. Raw Ct values were normalized against this median expression level to obtain DCt values. DCt values were generated for each gene according to the following equation: DCt_gene = Ct_gene - (median sample Ct). The median across all samples within a gene was then used to calculate -DDCt from DCt values by use of the following equation: -DDCt_gene = -(DCt_gene - DCt_median_across_samples).


title: Effect of voluntary exercise on gene expression in subcutaneous breast cancer in mice

status: Public on Jul 21 2024

first: LIANG

last: SHENG

country: China

summary: Effects of voluntary exercise on gene expression in subcutaneous breast cancer in mice.

overall_design: Mice were injected subcutaneously with breast cancer cell line 4T1 and divided into two groups, n=5,. One group was given a voluntary exercise condition and the other group was left without intervention. Subcutaneous tumors were taken at 21 days and sequenced.

first: Zhiwen

last: Luo

first: Jie

last: Mie

first: Yangyun

last: Long


title: Endometriotic lesions exert distinct metabolic activity compared to paired eutopic endometrium at a single-cell level

city: tartu

first: Maire

last: Peters

department: Dept. of Obstetrics and gynecology

country: Estonia

institute: University of Tartu

summary: Eutopic endometrium (EuE) and endometriotic peritoneal lesions (ectopic endometrium, EcE) are heterogenous tissues according to the recent scRNA studies. We aimed to explore metabolic profile of cell populations in paired samples of EuE and EcE from four women with endometriosis. We found changes in the regulation of progesterone and estradiol signaling pathways in perivascular, stromal and endothelial cells of EcE compared to EuE, which might have a direct effect on cellular metabolism and contribute to cell proliferation and angiogenesis. The metabolic pathways were differentially regulated in perivascular, stromal and endothelial clusters, and metabolic pathway activity change between EcE and EuE was the highest for AMPK, HIF-1, glutathione metabolism, OXPHOS, and glycolysis/ gluconeogenesis. Remarkably, we identified co-activation of glycolysis and OXPHOS in perivascular and stromal cells of EcE compared to EuE. Previous studies reported a Warburg-like effect in endometriosis with a high use of glycolysis pathway over oxidative metabolism. Our observations might suggest the employment of both OXPHOS and glycolysis to meet the energy demands of proliferating cells in endometriotic lesions.

overall_design: Cryopreserved samples of EuE and EcE were enzymatically dissociated to obtain single-cell suspensions with viability >90%. ScRNA libraries were constructed using 10X chromium Next GEM Single cell 3′ reagent v3.1 kit. Barcoded full-length cDNAs were produced, and pair-end sequenced targeting 35000 reads per cell using Novaseq PE150.

first: Meruert

last: Sarsenova

first: Ankita

last: Lawarde

first: Amruta

middle: D

last: Pathare

first: Merli

last: Saare

first: Vijayachitra

last: Modhukur

first: Pille

last: Soplepmann

first: Kristina

last: Gemzell-Danielsson

first: Parameswaran

middle: G

last: Lalitkumar

first: Andres

last: Salumets


title: Antibiotic Susceptibility Influenced by Carbon Source in Bacteroides thetaiotaomicron

status: Public on Jul 20 2024

city: Providence

first: Rachael

last: Nilson

state: RI

address: 171 Meeting St

institute: Brown University

summary: Antibiotic therapy causes changes in bacterial abundance and metabolism within the gut microbiome. These changes can lead to complications in the host such as microbiome dysbiosis and opportunistic infections. Additionally, increasing multidrug resistance causes significant morbidity and mortality in patients with bacterial infections. Bacteroides thetaiotaomicron (Bth) is both a normal commensal in the gut and an opportunistic pathogen in other body sites. Our lab has shown that 𝝱-lactam treatment in mice decimates nearly all gut bacterial species except Bth, which experiences a bloom. We propose that in the gut, Bth enters a protected state linked to metabolic changes such as an increase in stress response and polysaccharide fermentation, and a decrease in hexose utilization. Bth is also protected from 𝝱-lactams in vitro when grown on fiber as opposed to glucose. The goal of this project is to understand what mechanism is responsible for this polysaccharide-mediated tolerance (PM-tolerance). Studies of other bacteria show that an increase in respiratory metabolism independent of growth rate promotes susceptibility to bactericidal antibiotics. Bth has a diverse array of metabolic machinery including an electron transport chain (ETC) that works under anaerobic or microaerobic conditions. Transcriptional analysis of Bth treated with the 𝝱-lactam amoxicillin revealed a decrease in ETC enzyme expression when given fiber vs glucose. We found that knocking out different ETC enzymes alters the PM-tolerance phenotype in vitro. Thus, it appears that respiratory capacity is linked to 𝝱-lactam susceptibility in Bth. This work improves upon our understanding of bacterial tolerance strategies and could help identify future therapeutic targets to combat multidrug resistance.

overall_design: RNA-seq of Bacteroides thetaiotaomicron grown on two different carbon sources and treated with three different doses of amoxicillin for sixty minutes.

first: Swathi

last: Penumutchu

first: Francesco

middle: S

last: Pagano

first: Peter

last: Belenky


title: Epigenetic landscape of advanced prostate cancer

city: Boston

first: Kei

last: Mizuno

state: MASSACHUSETTS

address: 450 Brookline Avenue

institute: Dana-Farber Cancer institute

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Prostate cancer is characterized as being histologically and molecularly heterogeneous. Additionally, epigenetic changes play an important role in regulating the progression of prostate cancer. However, epigenetic intraindividual heterogeneity is largely unknown in advanced prostate cancer. Hence, the epigenetic profiles of advanced prostate cancer, including autopsy cases, were investigated.

overall_design: CUT&Tag analysis for histone modifications on samples from advanced prostate cancer patients

first: Varadha

middle: B

last: Venkadakrishnan

first: Himisha

last: Beltran


title: Tensin 2 interactomics reveals interaction with GAPDH and a phosphorylation-mediated regulatory role in glycolysis

city: Tampere

first: Paula

last: Turkki

state: Finland

address: Arvo Ylpön katu 34 (Arvo F303)

department: Faculty of Medicine and Life Sciences

country: Finland

institute: Tampere University

summary: Integrin adaptor proteins, like tensin-2, are crucial for cell adhesion and signaling. However, the function of tensin-2 beyond localizing to focal adhesions remain poorly understood. We utilized proximity-dependent biotinylation and strep-tag affinity proteomics to identify interaction partners of tensin-2 in HEK293 cells. Interactomics linked tensin-2 to known focal adhesion proteins and the dystrophin glycoprotein complex, while also uncovering novel interaction with the glycolytic enzyme GAPDH. We demonstrated that Y483-phosphorylation of tensin-2 regulates the glycolytic rate in HEK293 and MEF cells and found that pY483 tensin-2 is enriched in adhesions in MEF cells. Our study unveils novel interaction partners for tensin-2 and further solidifies its speculated role in cell energy metabolism. These findings shed fresh insight on the functions of tensin-2, highlighting its potential as a therapeutic target for diseases associated with impaired cell adhesion and metabolism.

overall_design: Gene expression analysis using data obtained from RNA-seq of 2 different cell lines


title: The Drosophila cancer-germline, head-to-head gene pair TrxT and dhd is dispensable for normal brain development but plays a major role in l(3)malignant brain tumour growth.

status: Public on Jul 17 2024

city: Barcelona

first: Cayetano

last: Gonzalez

address: baldiri Reixac 10

country: Spain

institute: IRB Barcelona

summary: Drosophila thioredoxins Deadhead (Dhd) and Thioredoxin-T (TrxT) are encoded by a head-to-head gene pair in the X chromosome. Akin to human cancer-germline (CG) genes, dhd and TrxT expression is normally sex and germline specific, but becomes upregulated in brain tumours of either sex caused by mutation in l(3)malignant brain tumour (l(3)mbt). Using CRISPR/Cas9-mediated knock-out alleles and RNA-seq, we show that while both TrxT and dhd are dispensable for normal brain development, each of them has a significant but partial effect on l(3)mbt brain tumour development as well as on tumour-linked transcriptomic signatures. Moreover, this effect is enhanced by the concomitant removal of both thioredoxins that strongly inhibits l(3)mbt larval brain tumour traits, reduces gene expression differences between tumour and wild-type tissue, and essentially erases all traces of gene expression differences between tumours from larvae of different sex. These results show that TrxT and dhd play a major synergistic role in the emergence of l(3)mbt tumour-linked transcriptomic signatures and tumour development, which is remarkable taking into account that these two genes are never expressed together under normal conditions. We have also found that TrxT is crucial but dhd has no effect on the growth of male-derived l(3)mbt allografts, hence suggesting that the initial stages of tumour development and sustained, long-term tumour growth may depend upon different molecular pathways. In humans, head-to-head inverted gene pairs are abundant among the CG genes that map to the X chromosome, some of which have been shown to work as a unit cancer. Our results identify the first instance of an X-linked, head-to-head CG gene pair in Drosophila and underpin the potential of such CG genes that are dispensable for normal development and homeostasis of somatic tissue, as targets to curtail malignant growth with minimal impact on overall health.

overall_design:
We carried out RNA-seq to identify the transcriptomes of Df(1)J5; l(3)mbtts1, dhdKO; l(3)mbtts1, TrxTKO; l(3)mbtts1, l(3)mbtts1 and w1118.
We performed pairwaise comparisons to identify differentially expressed genes between females and males and genotypes. We used 10 Drosophila larval brains per sample, and 3 replicates per condition.

first: Cristina

last: Molnar

first: Emilio

last: Palumbo

first: Giulia

last: Pollarolo


title: Effect of RNA Polymerase I (RPOA-2) degradation on transcription in intestinal cells dissected from adult C. elegans

status: Public on Jul 19 2024

city: Mainz

first: Jan

last: Padeken

address: Ackermannweg 4

country: Germany

institute: Institute of Molecular Biology

summary: Chromatin architecture is a fundamental mediator of genome function. Fasting is a major environmental cue across the animal kingdom. Yet, how it impacts on 3D genome organization is unknown. Here, we show that fasting induces an intestine-specific, reversible, and large-scale spatial reorganization of chromatin in C. elegans. This fasting-induced 3D genome reorganization requires inhibition of the nutrient-sensing mTOR pathway, through the regulation of RNA Pol I, but not Pol II nor Pol III, and is accompanied by remodeling of the nucleolus. By uncoupling the 3D genome configuration from the animal´s nutritional status we find that the spatial reorganization of chromatin correlates with the expression of metabolic and stress-related genes, potentially supporting the transcriptional response in fasted animals. Our work documents the first large-scale chromatin reorganization triggered by fasting and reveals that mTOR and RNA Pol I shape genome architecture in response to nutrients.

overall_design: RNA was extracted from intestines dissected from day 1 adults in TIR1 and in TIR1 with RPOA-2-AID worms exposed to auxin. Briefly, synchronized L1s by hypochlorite treatment were grown on OP50-seeded NGM plates for 72 hours until day 1 of adulthood. Next, they were picked from the plates and transferred onto auxin plates seeded with OP50 and maintained for the indicated time. Three time points were analyzed 0h, 12h and 24h on auxin. DCW143 (ieSi57 [eft-3p::TIR1::mRuby::unc-54 3'UTR+Cbr-unc-119(+)] II) outcrossed 1x), DCW180 (qzIs15[rpoa-2p::degron1(TIR1)::GFP::rpoa-2] I; ieSi57 [eft-3p::TIR1::mRuby::unc-54 3'UTR+Cbr-unc-119(+)] outcrossed 1x), DCW448 (ieSi57 [eft-3p::TIR1::mRuby::unc-54 3'UTR+Cbr-unc-119(+)] II) outcrossed 3x), DCW447 (qzIs15[rpoa-2p::degron1(TIR1)::GFP::rpoa-2] I; ieSi57 [eft-3p::TIR1::mRuby::unc-54 3'UTR+Cbr-unc-119(+)] outcrossed 3x).

first: Nada

last: Al-Refaie

last: Padovani

first: Johanna

last: Hornung

first: Lorenz

last: Pudelko

first: Francesca

last: Binando

first: Andrea

last: del Carmen Fabregat

first: Qiuxia

last: Zhao

first: Benjamin

last: Towbin

first: Elif

last: Cenik

first: Nicholas

last: Stroustrup

first: Kurt

middle: M

last: Schmoller

first: Daphne

last: Cabianca


title: Gene expression profile of inflammatory monocytes in cachectic blood cancer mice

city: Hachioji

first: Yoshihiro

last: Hayashi

state: Tokyo

address: 1432-1 Horinouchi

country: Japan

institute: Tokyo University of Pharmacy and Life Sciences

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

overall_design: Refer to individual Series


title: Divergent downstream biosynthetic pathways are supported by L-cysteine synthases of Mycobacterium tuberculosis

status: Public on Jul 18 2024

city: Hyderabad

first: Nitesh

middle: Kumar

last: Singh

state: Telangana

address: Habsiguda

department: Bioinformatics

country: India

institute: Center for Cellular and Molecular Biology

summary: Mycobacterium tuberculosis's autarkic lifestyle within the host involves rewiring its transcriptional networks to combat host-induced stresses. With the help of RNA-seq, we identified Mtb pathways that are upregulated in response to oxidative, nitrosative, acidic, starvation, and surfactant stresses. Genes belonging to sulfur metabolism were found to be significantly upregulated during oxidative stress. Using an integrated approach of microbial genetics, transcriptomics, metabolomics, animal experiments, chemical inhibition, and rescue studies, we investigated the biological role and therapeutic potency of non-canonical L-cysteine synthases, CysM and CysK2. CysM and CysK2 independently facilitate Mtb survival by alleviating host-induced redox stress, suggesting they are not fully redundant within the host. While transcriptome signatures of RvΔcysM and RvΔcysK2 appear similar under normal growth conditions, when subjected to oxidative stress, we identify unique transcriptional signatures. We followed pool size and labelling (34S) of key downstream metabolites viz. mycothione and ergothionine to monitor L-cysteine biosynthesis and utilization, which revealed a significant role of distinct L-cysteine biosynthetic routes on redox stress and homeostasis. With the help of genetic mutants and chemical inhibitors, we show that CysM and CysK2 serve as unique, attractive targets for adjunct therapy to combat mycobacterial infection.

overall_design: H37Rv was either left untreated, or treated with 50 micromolar of CHP for 6 h for oxidative stress, resuspended in PBS for starvation, 0.1% SDS for surfactant stress, resuspended in pH 5.5 media for acidic stress or were treated with sodium nitrate in pH 5.5 for nitrosative stress. Comparative gene expression profiling analysis of RNA-seq data for H37Rv, RvDcysM, RvDcysK2 with and without oxidative stress. H37Rv, RvDcysM, , RvDcysK2 were either left untreated or treated with 50 micromolar CHP for 6 hours.

first: Nandicoori

last: Vinay

first: Khan

last: Mehak Zahoor

first: Singha

last: Biplab

first: Singh

last: Nitesh Kumar

first: Sowpati

last: Tej Divya


title: Gene expression profile at bulk (24h, 48h & 72h) and single nucleus (24h) level of mouse right kidney post sham operation and unilateral nephrectomy.

city: Bedford Park

first: Jonathan

last: Gleadle

state: SA

address: 3D226 Renal Lab, Flinders Medical Centre, Flinders Drive

department: College of Medicine and Public Health

country: Australia

institute: Flinders University

summary: The mechanism driving the remarkable ability of the remaining kidney to enlarge and increase its function following removal of its contralateral pair remains elusive.  To explore the pathways driving compensatory renal hypertrophy, comprehensive RNA-seq transcriptional studies in the kidneys of mice undergoing hypertrophy 24, 48 and 72 hours following nephrectomy have been undertaken and compared with mice undergoing sham operations. The results reveal substantial time dependent enhancement of cholesterol biosynthesis pathways, increases in mitochondrial gene expression and cell cycle perturbations. Single nuclei RNA-Seq 24 hours post nephrectomy was used to further explore cholesterol biosynthesis signature and its driver SREBP2. In a cell specific manner, snRNA-seq demonstrated that SREBP2 activity increases in proximal tubules and medullary thick ascending limb and is responsible for cell size regulation following IGF-1 stimulation. These results suggest a previously undescribed role for SREBP2 in the mechanism underlying compensatory renal hypertrophy. This mechanism might be amenable to therapeutic manipulation to enhance kidney size and function.

overall_design: Mice were anaesthetised using 1.5-2.5% isoflurane/oxygen (1.5L/min), the left kidney was accessed with a mid-lateral skin incision and exposed. Adherent fat and the adrenal gland were bluntly dissected from the cranial pole. Renal blood supply was clamped and doubly ligated (Prolene 4-0). The clamp was removed, and the abdominal wall was closed by simple interrupted stitches (Prolene 4-0). Skin was closed with wound clips. Mice were given subcutaneous 0.5ml 0.9% sterile saline and Buprenorphine (0.05mg/kg) at time of surgery. Pain relief was given every 8-12h post-surgery as required for the first 24h. Sham operations were performed similarly without kidney removal. For bulk RNA-sequencing, RNA was extracted and quantified. Library preparation was performed using the Illumina TruSeq Stranded mRNA library preparation kit and sequenced using the NovaSeq system (Illumina) platform with 150bp paired end read length. For single nuclei RNA-sequencing, a small mid cross-section of the kidney was excised and processed for nuclei isolation using the EZ Nuclei Isolation Kit (Sigma-Aldrich) following the manufacturer's protocol. DAPI stained nuclei were isolated using FACS Aria Fusion (Becton Dickinson). The single nuclei suspension was then immediately loaded onto a Chromium Controller (10x Genomics) for single nuclei encapsulation, barcoding, and library preparation using the Chromium Single Cell 3' Reagent Kit v3 (10x Genomics) following the manufacturer's instructions. The prepared libraries were sequenced on an MGI FCL sequencer with 100bp paired end read length.

first: Darling

last: Rojas-Canales

first: Soon

middle: W

last: Wong

first: Elise

middle: J

last: Tucker


title: RNA interactome profiling in hypervirulent Klebsiella pneumoniae reveals small RNA ArcZ as an inhibitor of mucoviscosity and virulence

city: Shanghai

first: Kejing

last: Wu

address: 320 Yueyang Road

institute: Shanghai Institute of Immunity and Infection, Chinese Academy of Sciences

summary: Hypervirulent Klebsiella pneumoniae (HvKP) is an emerging human pathogen causing invasive infection in immune-competent hosts. The hypervirulence is strongly linked to the overproduction of hypermucovisous capsule, but the underlining regulatory mechanism of hypermucoviscosity (HMV) has been elusive, especially at the post-transcriptional level mediated by small RNAs (sRNAs). Using a recently developed RNA interactome profiling approach, we have investigated the Hfq-associated sRNA regulatory network and established the first in vivo RNA-RNA interactome in HvKP. Our data reveal numerous interactions between sRNAs and HMV-related mRNAs, and identify a plethora of sRNA that inhibit or promote HMV. One of the strongest repressors of HMV was ArcZ, a conserved sRNA in the Enterobacteriaceae family. We found that ArcZ is activated by the master regulator of catabolite repression Crp, and down-regulates the expression of mlaA encoding an outer-membrane lipoprotein, leading to decreased HMV and virulence attenuation in mice. ArcZ significantly reduced HMV in several carbapenem-resistant and hypervirulent clinical isolates with diverse genetic background, suggesting it is an antisense RNA inhibitor of HMV with therapeutic potential. In summary, our work provides a comprehensive map of the RNA-RNA interaction network of HvKP and identifies ArcZ as a conserved repressor of HMV, providing novel insights into the mechanisms of posttranscriptional regulations of virulence.

sample_organism: Klebsiella pneumoniae subsp. pneumoniae ATCC 43816

overall_design: To assess the the effect of expression of ArcZ and MlaA on transcriptome, we have first applied RNA-seq to biological triplicates of K. pneumoniae WT, ΔmlaA, ΔarcZ, ΔarcZ+pZE12-ArcZ and ΔarcZ+pZE12-ArcZ-M5. WT and ΔarcZ carry the empty vector. Strains were grown in LB media containing IPTG as sRNA expression inducer. Cells at OD600 3 were harvested to construct the strand-specific cDNA library and to be sequenced.

first: Yanjie

last: Chao


title: Synergistic activation by Glass and Pointed promotes neuronal identity in the Drosophila eye disc

city: New York City

first: Alireza

last: Khodadadi-Jamayran

state: NY

address: 550 1st Ave, MSB 304

department: Division of Advanced Research Technologies (DART)

institute: New York University, NYU Langone Medical Center

summary: The integration of extrinsic signaling with cell-intrinsic transcription factors can direct progenitor cells to differentiate into distinct cell fates.In the developingDrosophilaeye, differentiation of photoreceptors R1-R7 requires EGFR signaling mediated by the transcription factor Pointed, and our single-cell RNA-Seq analysis shows that the same photoreceptors require the eye-specific transcription factor Glass. We discovered that ectopic expression of Glass and activation of EGFR signaling synergistically induce neuronal gene expression in the wing disc in a Pointed-dependent manner. Targeted DamID reveals that Glass and Pointed share many binding sites in the genome of developing photoreceptors. Comparison with transcriptomic data shows that Pointed and Glass induce photoreceptor differentiation through intermediate transcription factors, including the redundant homologues Scratch and Scrape, as well as directly activating neuronal genes. Our data reveal synergistic activation of a multi-layered transcriptional network as the mechanism by which EGFR signaling induces neuronal identity in Glass-expressing cells.

overall_design:
The gl60j mutant scRNA-Seq deposited here was performed in Drosophila melanogaster on white prepupal stage eye discs. The corresponding control scRNA-Seq experiment with wild type white prepupal eye discs was published at https://doi.org/10.1038/s41467-023-43037-0 and the raw data deposited at GSE235110.
The DamID experiment was performed on wandering 3rd instar larval eye discs for two transcription factors, Gl and Pnt (PntP1 isoform), as well as Dam-only controls. The GAL4 drivers used were atonal-GAL4, which is expressed in the morphogenetic furrow, and elav-GAL4, which is expressed in the committed photoreceptors. All Dam-ID experiments were performed in triplicate biological replicates.
RNA-Seq was carried out on wandering third instar larval wing discs that contained clones expressing GFP; GFP and Gl; GFP and RasV12; GFP, Gl  and RasV12; GFP, Gl and RasV12 in pntdelta88 mutant clones; and GFP and Gl in cicQ219X mutant clones. The goal was to discover genes that are synergistically activated by Gl and EGFR signaling. This bulk RNA-Seq experiment was performed in triplicate biological replicates.
We perfomed RNA-Seq analysis on temperature sensitive mutant egfrtsla/egfrf2 wandering 3rd instar  larval eye discs. Control samples were kept at the permissive temperature of 18 degrees C and mutant samples were shifted to the restrictive temperature of 29 degrees C for 24 h. This bulk RNA-Seq experiment was performed in triplicate biological replicates.

first: Hongsu

last: Wang

first: Komal

last: Kumara Bollepogu Raja

first: Kelvin

last: Yeung

first: Phoenix

last: Chen

first: Ashley

last: Jordan

first: Cornelia

last: Fritsch

first: Simon

last: Sprecher

first: Jessica

last: Treisman

first: Graeme

last: Mardon


title: Competition shapes the landscape of X-linked genetic diversity [scRNA-seq]

city: London

first: LMS

last: Bioinformatics Core

address: Hammersmith Hospital Campus, Du Cane Road

department: Bioinformatics Core

country: United Kingdom

institute: MRC London Institute of Medical Sciences

summary: Human populations harbour sequence variants even within essential genes. As a result of random X chromosome inactivation (XCI) and epigenetically stable XCI propagation, X-linked variation gives rise to genetically diverse clones that co-exist within XX individuals. Whether interactions between such clones shape the deployment of X-linked diversity remains to be explored. To address this question, we focus on benign coding variation in the X-linked STAG2 gene. Mouse models reveal that clones expressing Stag2 variants contribute to tissues such as skin and brain at the expected frequencies, but show reduced contributions to the haematopoietic stem and progenitor cell pool, and severely defective lymphoid specification. Unexpectedly, the absence of Xvariant clones from the lymphoid compartment is due not to cell-intrinsic defects, but requires competitive interactions with Xwt clones: in the absence of Xwt, Xvariant cells generate normal numbers of functional lymphocytes. X-linked competition has hallmarks of non-cell-autonomous 'cell competition', known to operate in a range of biological processes including embryonic development, aging, and cancer. These findings show that interactions between genetically diverse clones that may operate in any XX individual can shape the contribution of X-linked diversity to specific cell types and tissues.

overall_design: Bone marrow cells isolated from heterozygous Stag2-WT x Stag2-R370Q Atrx-Luc/bGal female mice were depleted of lineage markers. FITC+ (Stag2-R370Q) and FITC- (Stag2-WT) cells were sorted and analyzed by scRNAseq.

first: Teresa

last: Perera

first: Matthias

last: Merkenschlager


title: Hyperglycemia-independent neonatal streptozotocin-induced retinopathy (NSIR) in rats

status: Public on Jul 16 2024

city: Chengdu

first: Danian

address: 37 Guoxue Xiang Street

institute: West China Hospital, Sichuan University

summary: Chemicals, such as MNU (N-methyl-N-nitrosourea) and NaIO3 (sodium iodate), are widely used to induce retinal degeneration in rodents. Streptozotocin (STZ) is an analog of N-acetyl glucosamine in which an MNU moiety is linked to a hexose and has a special toxic effect on insulin-producing pancreatic β-cells. It is commonly used to induce hyperglycemia to model diabetes. While intracerebroventricular injection of STZ can produce Alzheimer's disease independent of hyperglycemia, most retinal studies using STZ focus on the effects of hyperglycemia on the retina, but whether STZ has any impact on retinal cells independent of hyperglycemia is unknown. We aimed to investigate the role of cytotoxicity of STZ in rat retina. Intravitreal (5ug or 10ug) or subcutaneous (30mg/kg) injection of STZ at the early stage of newborn rats couldn’t induce hyperglycemia but caused NSIR (neonatal STZ-induced retinopathy), including reduced ERG amplitudes, retinal rosettes and apoptosis, cell cycle arrest, microglial activation, and delayed retinal angiogenesis. STZ did not affect the early-born retinal cell types but significantly reduced the late-born ones. Short-term and long-term hyperglycemia had no significant effects on the NSIR phenotypes. RNA sequencing revealed that STZ induces oxidative stress and activates the p53 pathway of retinal cells. Locally or systemically, STZ injection after P8 couldn’t induce NSIR when all retinal progenitors exit the cell cycle. Thus, NSIR in rats is independent of hyperglycemia but due to STZ’s direct cytotoxic effects on retinal progenitor cells. NSIR is a typical reaction to STZ-induced retinal oxidative stress and DNA damage. This significant finding suggests that NSIR may be a valuable model for studying retinal progenitor DNA damage-related diseases, potentially leading to new insights and treatments.

overall_design: To understand the underlying mechanism, we analyzed the transcriptome of the P4 retinas of rats receiving IVIT STZ (10ug) by RNA sequencing. We had two control groups: citrate buffer and saline. We found no deregulated genes (DEGs) enrichment between citrate buffer and saline. Thus, we focused on the differences between the STZ and citrate buffer groups.

first: Yu

last: Lin

first: Wenyu

last: Du

first: Xiangyu

last: Fu

first: Ling

last: Huang

first: Yiwen

last: Hong

first: Haishan

last: Tan

first: Lirong

last: Xiao

first: Xiang

last: Ren

first: Yujiao


title: Generation of 3D tubular bile duct within human iPSC-derived liver organoid by incorporating human iPSC-derived blood vessel [scRNA-Seq]

city: Minato-ku

first: Yoshiki

last: Kuse

address: Shirokanedai 4-6-1

institute: The University of Tokyo

summary: Bile duct (BD) structure is crucial for bile secretion to maintain liver homeostasis. Although several human induced pluripotent stem cell (hiPSC)-derived liver organoids have been generated, no study recapitulates the development of BD tubules. Here, we focus on an environmental cue to form tubular BDs, specifically the interaction between liver progenitors with the portal vein (PV). We co-culture hiPSC-liver progenitors with PV-like hiPSC-blood vessel (hiPSC-BV) which consists of immature hiPSC-smooth muscle cells (SMC) with a similar character to fetal PV-SMC. After two weeks, liver progenitors within hiPSC-BV-integrated liver organoid (BVLO) differentiate into the cholangiocyte-lineage and establish tubular structures with epithelial characteristics including intercellular junctions, microvilli on the apical membrane, and secretory functions. Liver surface transplanted BVLO showed further BD maturation and connection to the host BD. Taken together, our study developed a novel 3D co-culture methodo establish functional human tubular BDs by recapitulating PV-BD interaction.

overall_design: scRNA expression profiles of iPSC-derived cells

first: Kenji

last: Aoshima

first: Naoiki

last: Tanimizu


title: Lineage specific canonical and non-canonical EZH2 activity in advanced prostate cancer subtypes [RNA-Seq]

first: Varadha Balaji

last: Venkadakrishnan

state: MA

address: 450 Brookline Ave

department: Department of Medical Oncology

institute: Dana-Farber Cancer Institute

summary: Inhibiting EZH2 using tazemetostat led to induction of lineage-specific genes

overall_design: Tazemetostat treated PRAD and NEPC samples.

first: Himisha

last: Beltran


title: Competition shapes the landscape of X-linked genetic diversity [ChIP-seq]

first: LMS

overall_design: Immunoprecipitation and sequencing of regions targetted by anti-RAD21 in wild-type and STAG1/2 mutants

first: Roel

last: Oldenkamp

first: Benjamin

last: Rowland


title: Lineage-specific canonical and non-canonical activity of EZH2 in advanced prostate cancer subtypes


title: RNA interactome profiling in hypervirulent Klebsiella pneumoniae

first: Kejing

last: Wu

summary: To map the Hfq-mediated RNA regulatory network in HvKP, we harnessed the LiRIP-seq approach recently established in our lab, which enabled global profiling of RNA-RNA interactomes in live bacteria. Transformation of the pBAD-t4rnl1 plasmid into Klebsiella allowed us to express T4 RNA ligase 1 for proximity-ligation of RNAs in vivo, followed by co-immunoprecipitation (coIP) to enrich the Hfq-bound RNA transcripts (singleton reads) and ligated RNA fragments (chimeras: sRNA-mRNA, sRNA-sRNA, etc).

platform_organism: Klebsiella pneumoniae subsp. pneumoniae ATCC 43816

overall_design: LiRIP-seq was carried out in triplicate in cells at early stationary phase of growth in LB (OD600 of 3.0), with a monoclonal anti-FLAG antibody for IP and mouse IgG as a negative control. The resulting RNA samples were purified and subjected to Illumina paired-end sequencing.

first: Yanjie

last: Chao

first: Xingyu


title: Competition shapes the landscape of X-linked genetic diversity

last: Bioinformatics Core

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus


title: EZH2 knockout and rescue in WCM154 NEPC organoid model. [RNA-Seq]

summary: EZH2 silencing led to further maturation of neuroendocrine terminal differentiation in NEPC.

overall_design: EZH2 was silenced in WCM154 NEPC organoids using 2 independent single-guide RNAs. EZH2 expression was rescued using a recombinant dTAG-EZH2.


title: Comparative single-cell analysis reveals IFN-g as a driver of respiratory sequelae post COVID-19

city: Charlottesville

first: Chaofan

last: Li

state: VA

address: 345 Crispell Drive

department: Division of Infectious Diseases and International Medicine; Carter Immunology Center

institute: University of Virginia

summary: Post-acute sequelae of SARS-CoV-2 infection (PASC) represents an urgent public health challenge, with its impact resonating in over 60 million individuals globally. While a growing body of evidence suggests that dysregulated immune reactions may be linked with PASC symptoms, most investigations have primarily centered around blood studies, with few focusing on samples derived from post-COVID affected tissues. Further, clinical studies alone often provide correlative insights rather than causation. Thus, it is essential to compare clinical samples with relevant animal models and conduct functional experiments to truly understand the etiology of PASC. In this study, we have made comprehensive comparisons between bronchoalveolar lavage fluid (BAL) single-cell RNA sequencing (scRNAseq) data derived from clinical PASC samples and the relevant PASC mouse model. This revealed a strong pro-fibrotic monocyte-derived macrophage response in respiratory PASC (R-PASC) in both humans and mice, and abnormal interactions between pulmonary macrophages and respiratory resident T cells. IFN-g emerged as a key node mediating the immune anomalies in R-PASC. Strikingly, neutralizing IFN-g post the resolution of acute infection reduced lung inflammation and tissue fibrotic changes in a mouse model of R-PASC. Our study underscores the importance of performing comparative analysis to understand the root cause of PASC for developing effective therapies.

overall_design: To facilitate the single-cell gene expression (GEX), PBMC and BAL cells from donors and MA10 infected mice at indicated time points were harvested and barcoded with 10X 5' Library & Gel Bead Kit v1.1 or 3' Library & Gel Bead Kit v3.1 in a BSL2 lab (human samples) or a ABSL3 lab (animal samples).

first: Xiaoqin

last: Wei

first: Wei

last: Qian

first: Ying

first: Jie

last: Sun


title: Whole worm transcriptomics after optogenetically induced mechanical muscle stress (OptIMMuS)

city: Köln

first: Debasish

last: Mukherjee

address: Albertus-Magnus-Platz

institute: Universität zu Köln

summary: Mechanical stress during muscle contraction is a constant threat to proteome integrity. Experimental systems are needed to identify proteostasis regulators that are critical under mechanical stress conditions. Although Caenorhabditis elegans combines the advantages of existing organismal and cell culture models, an inducible method to stimulate muscle contraction in large populations has been lacking. Here, we present a versatile technique called OptIMMuS (Optogenetic Induction of Mechanical Muscle Stress) to study changes in the proteostasis network associated with mechanical forces. Repeated blue light exposure of a muscle-expressed Chlamydomonas rheinhardii channelrhodopsin-2 variant leads to sustained muscle contraction and mechanical stress. OptIMMuS combined with proximity labeling and mass spectrometry identified regulators that cooperate with the myosin-directed chaperone UNC-45 in muscle proteostasis. We show in genetic epistasis and co-immunoprecipitation experiments that the E3 ligase NHL-1 interacts with UNC-45 and muscle myosin, and we provide evidence that this TRIM32 orthologue affects myosin protein levels and function.

overall_design: This project includes 16 samples of transgenic C. elegans whole worm lysate after mechanical stress in 4 replicates of 4 conditions. Transgenic C. elegans strains expressing the optogenetic channelrhodopsin mutant ChR2(C128S;H134R)-FLAG in body wall muscle cells (PP3189 hhIs251[myo-3p::ChR2(C128S,H134R)-FLAG::unc-54 3'UTR, Cbrunc-119(+)]) were used to contract the worms’ muscles by blue light illumination to induce mechanical muscle stress. The optogenetic channel is activated by adding the cofactor all-trans retinal (ATR) to it’s food source E. coli OP50. In this experiment, we compared the treatment condition (1) worms after 8 h of illumination on plates with ATR (light plus ATR) to three control conditions: (2) illumination on plates without ATR (light minus ATR), (3) worms kept in the dark on plates with ATR (dark plus ATR), and (4) worms kept in the dark on plates without ATR (dark minus ATR).

first: Carl

middle: E

last: Kutzner

first: Richard

last: Acton

first: Thorsten

last: Hoppe


title: Epigenetic profiling of prostate adenocarcinoma and neuroendocrine prostate cancer models to understand EZH2 activity. [Cut & Run]

first: Varadha Balaji

summary: EZH2 has differential activity in prostate adenocarcinoma and neuroendocrine prostate cancer.

overall_design: Epigenetic profiling was performed in prostate cancer models upon EZH2 inhibiton or silencing.


title: Released Bacterial ATP Shapes Local and Systemic Inflammation during Abdominal Sepsis

city: Bern

first: Daniel

last: Sanchez-Taltavull

address: Murtenstrasse 35

department: Department of BioMedical Research

country: Switzerland

institute: University of Bern

summary: Sepsis causes millions of deaths per year worldwide and is a current global health priority declared by the WHO. Sepsis-related deaths are a result of dysregulated inflammatory immune responses indicating the need to develop strategies to target inflammation. An important mediator of inflammation is extracellular adenosine triphosphate (ATP) that is secreted by inflamed host cells and tissues, and also by bacteria in a strain-specific and growth phase-dependent manner. Here, we investigated the mechanisms by which bacteria release ATP. Using genetic mutant strains of Escherichia coli (E. coli), we demonstrate that ATP release is dependent on ATP synthase within the inner bacterial membrane. In addition, impaired integrity of the outer bacterial membrane and bacterial death notably contribute to ATP release. In a mouse model of abdominal sepsis, local effects of bacterial ATP were analysed using a transformed E. coli bearing an arabinose-inducible periplasmic apyrase hydrolyzing ATP to be released. Abrogating bacterial ATP release shows that bacterial ATP suppresses local immune responses, resulting in reduced neutrophil counts and impaired survival. In addition, bacterial ATP has systemic effects via its transport in outer membrane vesicles (OMV). ATP-loaded OMV are quickly distributed throughout the body and upregulated expression of genes activating degranulation in neutrophils, potentially contributing to the exacerbation of sepsis severity. This study reveals mechanisms of bacterial ATP release and its local and systemic roles in sepsis pathogenesis.

overall_design: RNA sequencing of bead-sorted neutrophils isolated from lungs of C57Bl6 mice one hour after intraabdominal (i.a.) injection of either mock-electroporated or ATP-loaded (via electroporation) outer membrane vesicles (OMV), which were isolated from a 24h growth culture of the E. coli tolB mutant (Keio collection)

last: Spari

first: Guido

last: Beldi


title: Generation of 3D tubular bile duct within human iPSC-derived liver organoid by incorporating human iPSC-derived blood vessel [scRNA-seq]

first: Yoshiki

last: Kuse

summary: The tubular structure of the intrahepatic bile duct (BD) is crucial for its physiological functions including bile excretion. Although several human induced pluripotent stem cell (hiPSC)-derived liver organoids were generated, no prior study could emulate the development of BD tubules. Here we focus on the environmental cue to initiate BD development, specifically the interaction between liver progenitors and the portal vein (PV). We co-cultured hiPSC-liver progenitors with hiPSC-blood vessels (hiPSC-BV) consisting of immature hiPSC-smooth muscle cells (SMC) expressing JAG1, a key factor for cholangiocyte induction. After three weeks, liver progenitors within hiPSC-BV-integrated liver organoids (BVLO) differentiate into cholangiocyte lineage and establish tubular structures with epithelial characteristics, including intercellular junctions, microvilli on the apical membrane, and secretory functions. Furthermore, liver surface transplanted-BVLO demonstrated BD graft-host connection and suggested therapeutical potential. Overall, we developed a novel 3D co-culture method to establish functional human tubular BDs by emulating PV-BD interaction.

overall_design: scRNA expression profiles of iPSC-derived organoid

first: Erica

last: Carolina

first: Naoiki

last: Tanimizu


title: Generation of 3D tubular bile duct within human iPSC-derived liver organoid by incorporating human iPSC-derived blood vessel

overall_design: Refer to individual Series


title: Lin28A Overexpression Triggers Inflammatory Response in Mouse Kidneys

city: Ramat-Gan

first: Hiba

last: Waldman Ben-Asher

address: Ramat-Gan

department: Life-Sciences

country: Israel

institute: Bar-Ilan University

summary: The RNA-binding proteins Lin28A and Lin28B play critical roles in regulating gene expression through distinct mechanisms, including the inhibition of Let-7 microRNA maturation and direct mRNA binding. Predominantly expressed in pluripotent and multipotent stem cells, Lin28A and Lin28B contribute to a variety of biological processes such as development, growth, and tissue regeneration. Dysregulation of Lin28A/B expression is associated with numerous types of tumors. In this study, we demonstrate that overexpression of Lin28A in the mouse nephrons leads to severe inflammation and kidney damage, rather than to tumorigenesis. To investigate the causal relationship between kidney damage and inflammation, we utilized steroid treatment to suppress inflammation. This treatment also ameliorated the kidney damage phenotype, indicating that Lin28A overexpression directly induces inflammation, which in turn leads to kidney damage. Notably, Lin28A overexpression causes inflammation only when expressed in nephrons, but not in the stromal cells of the kidneys, highlighting its cell context-dependent nature. The nephron-specific, Lin28A-induced inflammatory response is distinct from the previously described Lin28B-mediated inflammatory feedback loops, as it is not IL-6 dependent. Instead, it is associated with the rapid upregulation of cytokines such as CXCL1 and CCL2. Overall, this study reveals that the impact of abnormal Lin28A expression is broader than previously understood, demonstrating that Lin28A overexpression also plays a role in inflammatory responses. Furthermore, this transgenic mouse model offers a valuable tool for advancing our understanding of the pathophysiology of acute kidney injury (AKI), where inflammation is a key factor.

overall_design: RNA was extracted form the kidneys  of the following samples in triplicate - Lin28A OE inducibale transgenic transgenic mice (Lox-stop-Lox-TetOn-Lin28A;Six2Cre)  and control (Lox-stop-Lox-TetOn-Lin28A) upon 1,2,5 weeks of Doxycyline treatment. RNA seq analysis was preformed for each sample

first: Anna

last: Futorian

first: Leah

last: Armon

first: Achia

last: Urbach


title: ATAXIN-2 Intermediate-length Polyglutamine Expansions Elicit ALS-associated Metabolic and Immune Phenotypes

city: Utrecht

first: R. Jeroen

last: Pasterkamp

address: Universiteitsweg 100

department: Translational Neuroscience

country: Netherlands

institute: University Medical Center Utrecht

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


title: Single-cell RNA sequencing analysis of iPSC-derived motor neurons from an ALS patient carrying ATXN2 intermediate repeat expansions and a healthy control

summary: Intermediate-length repeat expansions in ATAXIN-2 (ATXN2) are a strong genetic risk factor for amyotrophic lateral sclerosis (ALS). At the molecular level, ATXN2 intermediate expansions enhance TDP-43 toxicity and pathology. However, whether this triggers ALS pathogenesis at the cellular and functional level remains unknown. Here, we developed a human iPSC-derived model to investigate whether motor neurons derived from an ALS patient carrying ATXN2 intermediate repeat expansions (ALS-G) are transcriptomically distinct from a healthy control(OH2.6). For that, we generated a single cell RNA sequencing dataset consisting of a total of 384 cells.

overall_design: iPSC differentiation into MN was performed as described previously with minor modifications (Amoroso et al., 2013). Day 12 human iPSC-derived MN cultures were dissociated and sorted into single wells, where fluorescent-activated cell sorting (FACS) into 384-well plates was combined with automated single cell sequencing (SORT-seq pipeline). 384 cells were sorted into 4 libraries consisting of 96 cells each.

first: R

last: Vieira de Sá

first: E

last: Sudria-Lopez

first: M

last: Cañizares Luna

first: O

last: Harschnitz

last: Basak


title: Transcriptomic analysis of the spinal cords of knock-in adult mice carrying the ATXN2/TDP-43 human transgenes

summary: Intermediate-length repeat expansions in ATAXIN-2 (ATXN2) are a strong genetic risk factor for amyotrophic lateral sclerosis (ALS). At the molecular level, ATXN2 intermediate expansions enhance TDP-43 toxicity and pathology. However, whether this triggers ALS pathogenesis at the cellular and functional level remains unknown. To investigate gene expression changes and deregulated pathways caused by ataxin-2 intermediate repeat expansions in presence/absence of mutant TDP-43, we performed RNA sequencing of whole spinal cords from knock-in mice harboring ATXN2 and TDP-43 human transgenes as well as non-transgenic mice (NTg)

overall_design: 10-months-old spinal cord samples were obtained from each mouse line (n = 3 mice per genotype). Total RNA was isolated and processed for downstream RNA sequencing analysis (details provided below).

last: Veno

first: S

last: Seeler

first: J

last: Kjems


title: Long noncoding RNA expression profile of chemotherapy-sensitive and resistant breast cancer tissues

city: Guangzhou

first: Rong

last: Luo

address: Yanjiangxi Road No.107

institute: Sun Yat-Sen University

summary: To explore novel lncRNAs relative to chemo-resistance in breast cancer, we performed lncRNA microarray analysis using three anthracycline-resistant tissues and three anthracycline-sensitive tissues collected before neoadjuvant chemotherapy by Arraystar Human LncRNA Microarray V3.0.

overall_design: Three anthracycline-resistant and three anthracycline-sensitive breast cancer tissues were used for microarray analysis to acquire the lncRNA differential expression profile.

first: Jiannan

last: Wu

first: Erwei

last: Song

first: Man-Li


title: Long noncoding RNA expression profile of breast cancer tissues pre- and post-neoadjuvant chemotherapy

summary: To identify the differential expression of lncRNAs, we performed the lncRNA microarray analysis with three pairs of breast cancer tissues before and after neoadjuvant chemotherapy (NAC) from partially responded patients by Arraystar Human LncRNA Microarray V3.0.

overall_design: Three paired breast cancer tissues from partially responded patients before and after neoadjuvant chemotherapy were used to acquire the lncRNA differential expression profile.


title: Transcriptomic analysis of iPSC-derived  motor neurons from ALS patients carrying ATXN2 intermediate repeat expansions and healthy controls

summary: Intermediate-length repeat expansions in ATAXIN-2 (ATXN2) are a strong genetic risk factor for amyotrophic lateral sclerosis (ALS). At the molecular level, ATXN2 intermediate expansions enhance TDP-43 toxicity and pathology. However, whether this triggers ALS pathogenesis at the cellular and functional level remains unknown. Here, we developed a human iPSC-derived model to investigate whether motor neurons derived from ALS patients carrying ATXN2 intermediate repeat expansions are transcriptomically distinct from healthy controls. For that, we performed RNA sequencing of motor neurons derived from 5 ATXN2-ALS iPSC lines and 5 healthy controls (HC).

overall_design: iPSC differentiation into MN was performed as described previously with minor modifications (Amoroso et al., 2013). Human iPSC-derived MN samples from 5 controls and 5 ATXN2-ALS  lines were isolated after 12 days of co-culture with mouse primary glia. Total RNA was isolated and extracted using the RNeasy Micro Kit and only samples with RIN ≥ 8.0 were used for RNA sequencing.

first: F

last: Giuliani


title: Motor nerves direct the development of the sympathetic nervous system

status: Public on Jul 14 2024

city: lyon

first: valérie

last: CASTELLANI

address: 8 Avenue Rockefeller

country: France

institute: MeLiS | CNRS UMR 5284 | INSERM U1314

summary: The sympathetic nervous system controls a wide spectrum of bodily functions including operation of vessels, cardiac rhythm, and the “flight or fight response”. Sympathetic neurons, which are neural crest-derived, develop in coordination with presynaptic motor nerves extending from the central nervous system (CNS). By using nerve-selective genetic ablations, we revealed that sympathetic ganglia development depends on CNS-derived motor innervation. In the absence of preganglionic motor nerves, trunk sympathetic chain ganglia were fragmented and smaller in size, while cervical ganglia were severely misshapen. Sympathetic neurons were misplaced along sensory fibers and projected towards abnormal paths, in some cases invading the sensory dorsal root ganglia. The misplaced progenitors of sympathoblasts corresponded to the nerve-associated, neural crest-derived Schwann cell precursors (SCPs). Notably, we found that SCPs activate the autonomic marker PHOX2B while migrating along motor nerves towards the region of the dorsal aorta in wildtype embryos, suggesting that SCP differentiate into sympathetic neurons while still nerve-associated in motor-ablated embryos. Ligand-receptor prediction from single cell transcriptomic data coupled with functional studies identified Semaphorin 3A/3F as candidate motor nerve-derived signals influencing neural crest migration along axons. Thus, motor nerves control the placement of sympathoblasts and their subsequent axonal navigation during critical periods of sympathetic chain development.

overall_design: dissection of the meninges, inlcuding DRG, root of dorsal and ventral nerves; and further isolation and sequencing of Egr2+ and Prss56+ cell lineages

first: Fanny

last: Coulpier

first: Piotr

last: Topilko

first: Jérémy

last: Ganofsky

first: Valerie

last: Castellani

first: Alek

last: Erickson

first: Irina

last: Poverennaya

first: Igor

last: Adameyko


title: Genetic and pharmacologic modulation of mitofusins in cellular senescence

status: Public on Jul 15 2024

city: Barcelona

first: Camille

last: Stephan Otto Attolini

state: Select a State or Province

address: Baldiri Reixac 10

department: Biostatistics and bioinformatics Unit

institute: IRB Barcelona

summary: RNA-sequencing of senescent (doxorubicin) human melanoma SK-MEL-103 cells and human fetal lung fibroblast IMR-90 with different interventions

overall_design: On one hand, senescence was induced by doxorubicin in SK-MEL-103 and IMR-90 cells lacking Mitochondrial Antiviral Signaling Protein (MAVS),  mitofusin 1 (MFN1) and/or mitofusin 2 (MFN2) via siRNA (day -1) for 7 days. RNA was harvested on day 7 of treatment. On the other hand, senescence was induced for 7 days by doxorubicin and then cells were treated with MFI8 or BI for 48h, to inhibit or activate MFN1 and MFN2, respectively. RNA was harvested on day 9 of treatment.

last: Stephan-Otto Attolini

first: Manuel

last: Serrano

first: Vanessa

last: López-Polo


title: ATR/Chk1 interacting lncRNA modulates DNA damage response to induce breast cancer chemoresistance


title: Identifying specific functional roles for senescence across cell types

status: Public on Jul 12 2024

first: zixin

last: liu

address: 320 Yueyang road, Life Science Research Building A2112

institute: Center for Excellence in Molecular Cell Science

summary: Cellular senescence plays critical roles in aging, regeneration, and disease, yet the ability to discern its contributions across various cell types to pathophysiological processes in vivo remains limited. In this study, we generated an in vivotoolbox, consisting of three p16INK4a-related intersectional genetic systems, enabling pulse- chase tracing (Sn-pTracer), Cre-based tracing and ablation (Sn-cTracer), and gene manipulation combined with tracing (Sn-gTracer) of defined senescent cell types. Using liver injury and repair as an example, we found that macrophages and endothelial cells (ECs) represent distinct senescent cell populations with different fates and functions during liver fibrosis and repair.Targeted reprogramming of p16INK4a+ ECs through Kdr overexpression markedly reduces liver fibrosis. This study illuminates the pathophysiological diversity of senescent cells and offers valuable insights for the development of future cell type-specific senolytic therapies. Furthermore, our model provides a versatile platform for investigating cellular senescence in any desired cell type.

overall_design: Hepatic endothelial cell and macrophage were isolated from p16-tdT mice and performed RNAseq analysis. Hepatic non-parenchymal cells, isolated from indicated p16-tdT, SnEC-gTracer, or SnMø-cTracer mice, were analyzed using sc-RNAseq.

first: Huan

last: Zhao

first: Zixin

last: Liu

first: Hui

last: Chen

first: Maoying

last: Han

first: Mingjun

last: Zhang

first: Kuo

first: Hengwei

last: Jin

first: Mengyang

last: Shi

first: Wenjuan

last: Pu

first: Markus

last: Werner

first: Michael

last: Meister

first: Stefan

last: Kauschke

first: Qing-Dong

last: Wang

first: Xin

last: Ma

first: Jan

last: Tchorz

first: Xiuxiu

first: Ruilin

last: Sun

first: Ruling

last: Shen

first: Bin

last: Zhou


title: Generation of 3D tubular bile duct within human iPSC-derived liver organoid by incorporating human iPSC-derived blood vessel [RNA-seq]

first: Yoshiki

last: Kuse

summary: Bile duct (BD) structure is crucial for bile secretion to maintain liver homeostasis. Although several human induced pluripotent stem cell (hiPSC)-derived liver organoids have been generated, no study recapitulates the development of BD tubules. Here, we focus on an environmental cue to form tubular BDs, specifically the interaction between liver progenitors with the portal vein (PV). We co-culture hiPSC-liver progenitors with PV-like hiPSC-blood vessel (hiPSC-BV) which consists of immature hiPSC-smooth muscle cells (SMC) with a similar character to fetal PV-SMC. After two weeks, liver progenitors within hiPSC-BV-integrated liver organoid (BVLO) differentiate into the cholangiocyte-lineage and establish tubular structures with epithelial characteristics including intercellular junctions, microvilli on the apical membrane, and secretory functions. Liver surface transplanted BVLO showed further BD maturation and connection to the host BD. Taken together, our study developed a novel 3D co-culture method o establish functional human tubular BDs by recapitulating PV-BD interaction.

overall_design: mRNA expression profiles of iPSC-derived hepatic cell lineage and organoid.

first: Erica

last: Carolina

first: Naoiki

last: Tanimizu

first: Kiyoshi

last: Yamaguchi


title: Molecular and Cellular Mechanisms of Heart Failure With Preserved Ejection Fraction

city: LOS ANGELES

first: Jijun

last: Huang

state: CA

address: 650 Charles E Young Dr S, CHS 36-100

department: Medicine

institute: UCLA

summary: To understand the cellular mechanisms of heart failure with preserved ejection fraction (HFpEF), We investigated the functional outcome and the underlying mechanisms from concurrent mechanic and metabolic stresses in the heart by applying transverse aortic constriction (TAC) in lean C57Bl/6J (HFrEF phenotype) or obese/diabetic B6.Cg-Lepob/J (ob/ob) mice (HFpEF phenotype) followed by single-nuclei RNA-seq (snRNA-seq) and targeted manipulation of a top ranked signaling pathway (glucagon receptor signaling) differentially affected in the two experimental cohorts.

overall_design: For snRNA-seq, We collected hearts from adult male C57BL/6J or B6.Cg-Lepob/J mouse at 10 weeks post TAC injury or sham with two mice of each group. Following isolation of single nuclei from the fresh whole heart tissue, we conducted library preparation with 10x genomics 3' GEM library kit v3.1. The libraries were then sequenced on one lane of NovaSeq6000 S4 at UCLA TCGB Core. Sequencing data were then processed by Cell Ranger software and R package Seurat for dimension reduction UMAP plots, cell type annotation and differential gene expression analysis.

first: Chen

last: Gao

first: Zhaojun

last: Xiong

first: Shuxun

last: Ren

first: Christoph

last: Rau

first: Tomohiro

last: Yokota

first: Yibin


title: Klf2/4-independent effects of Pcdhg on gene expression in endothelial cells exposed to Oscillatory Shear Stress (OSS)

city: New Haven

first: Divyesh

last: Joshi

state: CT

address: 300 George Street, Floor 7, YCVRC, Yale University

department: Internal Medicine, Cardiology

institute: Yale University

summary: We found that Pcdhg is a potent suppressor of Klf2/4 in endothelial cells. We performed RNAseq analysis to characterize deregulated pathways and processes by which Pcdhg may regulate Klf2/4.

overall_design: Control siRNA, Pcdhg siRNA or Pcdhg+Klf2/4 siRNA transfected HUVECs were stimulated with OSS for 16h, the RNA extracted and subjected to bulk sequencing.

first: Martin

middle: A

last: Schwartz


title: TFIIIC and MYCN link the three-dimensional chromatin structure of promoters to transcription termination of stalled RNA polymerase (RNA-Seq)

city: Wuerzburg

last: Eilers

address: Am Hubland

department: Chair for Biochemistry and Molecular Biology

institute: University of Wuerzburg

summary:
MYC proteins bind to virtually all active promoters transcribed by RNA polymerase II (RNAPII), but whether they interact with the three-dimensional chromatin architecture is unknown. Here we used HiChIP sequencing of the MYCN oncoprotein and found that MYCN localizes to three-dimensional hubs formed by active promoters and enhancers. In these hubs, MYCN interacts with TFIIIC, an architectural protein complex. MYCN recruits TFIIIC to promoters when transcription elongation is inhibited, and the complex of both proteins induces premature transcription termination.
Termination correlates closely with the TFIIIC-dependent removal of MYCN from promoter hubs and with corresponding alterations in the three-dimensional interactions of cohesin complexes. This limits DNA damage by removing RNAPII that stalls proximal to double-strand breaks. Binding of TFIIIC to MYCN is limited by competition with Aurora-A and this protects genes involved in mRNA processing from termination, arguing that MYCN contributes to the unusual proliferative capacity of neuroblastoma cells via removing stalled RNAPII from promoter hubs and via increasing the capacity for RNA processing.

overall_design: RNA-sequencing was done with SH-EP NMYC-ER cells carrying a doxycycline inducible shRNA against TF3C5. Furthermore, NMYC expression can be induced in these cells with 4-OHT. Global gene-expression analysis was carried out in these cells with or without expression of shTF3C5 and with or without expression of NMYC.

first: Raphael

last: Vidal

first: Eoin

last: Leen

first: Mareike

last: Müller

first: Christina

last: Schülein-Völk

first: Ursula

first: Dimitrios

last: Papadopoulos

first: Isabelle

last: Röschert

first: Steffi

last: Herold

first: Carsten

middle: P

last: Ade

first: Peter

last: Gallant

first: Richard

last: Bayliss

first: Gabriele

last: Büchel


title: CRISPR screen of radiation resistant genes in NSCLC

city: Atlanta

first: Xingming

last: Deng

state: GA

address: 1701 Uppergate Dr NE, Radiation Oncology, Building C, 3rd floor, bench 48-54

department: Radiation Oncology

institute: Winship Cancer Institute, Emory University

summary: Radiotherapy is an important treatment for non-small cell lung cancer (NSCLC). It not only kills tumor cells directly, but also promotes the efficacy of immunotherapy. However, resistance to radiotherapy is still an unavoidable clinical challenge. In this study, we used radiation-sensitive H460 cell line to stably express Cas9 and CRISPR GeCKO v2 library (A and B). Then a single 4GY irradiation was given, and after the cells resumed proliferation, the total genome was extracted and second-generation sequencing was performed. Genes directly associated with resistance to radiotherapy were identified in comparison to controls without any treatment.

overall_design: The Cas9 and GeCKO v2 library expressing H460 celsl were separated into Control and IR groups. Each group has three repeats. The IR group was treated with 4 Gy ionizing radiation. Then, the total genome of the 6 samples was extracted and sequenced.

first: Zhen

last: Shu


title: Transcriptomic analysis of spinal cord microglial cells from NTg and ATXN2-Q33;TDP-43 adult mice

first: R. Jeroen

last: Pasterkamp

summary: Intermediate-length repeat expansions in ATAXIN-2 (ATXN2) are a strong genetic risk factor for amyotrophic lateral sclerosis (ALS). At the molecular level, ATXN2 intermediate expansions enhance TDP-43 toxicity and pathology. However, whether this triggers ALS pathogenesis at the cellular and functional level remains unknown. Our study shows that  microglial cells might contribute to ALS-related pathology observed in mice carrying ATXN2 intermediate repeat expansions (Q33) in an ALS background (TDP-43). To investigate whether ATXN2-Q33;TDP-43 spinal cord microglia are transcriptomically different from non transgenic counterparts, we performed RNA sequencing of sorted microglial cells from knock-in (ATXN2-Q33;TDP-43) as well as non-transgenic (NTg) mice.

overall_design: Microglial cells were isolated from 7-months-old spinal cords using CD11b-coated beads. A total of 9 animals per mouse line were used (ATXN2-Q33;TDP43 and NTg). Each sample consisted of 3 pooled spinal cord isolated microglia.

first: R

last: Vieira de Sá

first: E

last: Sudria-Lopez

first: M

last: Cañizares Luna

last: Veno


title: The proteasome subunit psmb1 is essential for craniofacial cartilage maturation and morphogenesis

city: Boston

first: Bess

last: Miller

address: 77 Avenue Louis Pasteur

institute: BWH

summary: Craniofacial dysmorphisms are among the most common birth defects. Proteasome mutations frequently result in craniofacial dysmorphisms including lower jaw malformations; however, the underlying mechanisms are unknown. Here we use a zebrafish proteasome subunit beta 1 (psmb1) mutant to define the cellular mechanisms underlying proteasome mutation-induced craniofacial dysmorphisms. psmb1 mutants exhibit a flattened ceratohyal and smaller Meckel’s and palatoquadrate cartilages. Ceratohyal flattening is a result of failed chondrocyte convergent extension, accompanied by reduced numbers of chondrocytes in the lower jaw due to defects in chondrocyte differentiation. Morphogenesis of craniofacial muscles and tendons is similarly perturbed. psmb1 mutants lack the hyohyal muscles and craniofacial tendons are shortened and disorganized. We additionally identify a critical period for proteasome function in craniofacial development, specifically during chondrocyte and muscle differentiation. psmb1 overexpression in sox10+ cells of mutant embryos rescued both cartilage and tendon phenotypes but induced only a partial rescue of the muscle phenotype, indicating that psmb1 is required in both tissue-autonomous and non-autonomous fashions during craniofacial development. Overall, our work demonstrates that psmb1 is required for craniofacial cartilage, tendon, and muscle differentiation and morphogenesis.

overall_design: To investigate the role of psmb1 in craniofacial chondrocyte and muscle development, we sorted sox10+ cells or mylz2+ cells from the heads of psmb1 mutant zebrafish vs. pooled wild-type and heterozygous larvae at 72hpf. We then performed bulk RNA-sequencing and differential gene expression analysis.

first: Wolfram

last: Goessling


title: TFIIIC and MYCN link the three-dimensional chromatin structure of promoters to transcription termination of stalled RNA polymerase (ChIPseq CnR)

first: Martin

last: Eilers

overall_design: CUT&RUN, ChIP and ChIP-Rx DNA-sequencing was done with SH-EP NMYC-ER cells where the NMYC expression can be induced with 4-OHT. For all CUT&Run sequencing and some ChIP-seq and ChIP-Rx sequencing experiments, SH-EP NMYC-ER cells were used that carry a doxycycline inducible shRNA against TF3C5. For CUT&Run sequencing samples, the immunoprecipitation of digested DNA was done for EXOSC5 with or without NMYC expression and with or without expression of shTF3C5. As control, DNA was sequenced following immunoprecipitation with an unspecific IgG antibody. For ChIP-seq and ChIP-Rx sequencing experiments, the immunopresipitated antibodies are listed below. All experiments were done with or without NMYC expression and where indicated, a shRNA targeting TF3C5 was induced via doxycycline or one of the below listed inhibitors was applied.

first: Raphael

last: Vidal

first: Eoin

last: Leen

first: Mareike

last: Müller

first: Christina

last: Schülein-Völk


title: Purinergic Preconditioning Induces Epigenomic and Transcriptomic Changes Resembling Epilepsy-associated Microglial States

status: Public on Jul 11 2024

city: Badalona, Barcelona

first: Esteban

last: Ballestar

state: N/A = Not Applicable

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

institute: Josep Carreras Research Institute (IJC)

summary: Microglia are the main immune effector cells in the central nervous system (CNS) and contribute to a wide spectrum of neuropathological conditions through exacerbated activation. Microglial inflammatory responses may be conditioned by prior exposures to noxious stimuli, such as increased levels of extracellular adenosine and ATP. These levels are characteristic of brain insults like epileptic seizures and could potentially determine subsequent responses through epigenetic regulation. In this study, we investigated DNA methylation and expression changes in microglia-like cells differentiated from monocytes following ATP-mediated preconditioning. First, during microglia differentiation, we mainly observed DNA demethylation in genomic sequences enriched in binding motifs of microglia lineage transcription factors like PU.1, consistent with the relevance of this factor in in vivo microglia. TLR-mediated activation, after a first exposure to ATP, promoted exacerbated pro-inflammatory activation in comparison to cells not pre-exposed to ATP. These changes were accompanied by DNA methylation and transcriptional reprogramming of immune-related pathways, neurotransmitter activity and nucleotide phosphorylation. Finally, the epigenetic and transcriptional reprogramming associated with ATP-mediated preconditioning results in profiles found in microglia subsets linked to epilepsy. Purine-driven microglia immune pre-conditioning associates with epigenetic and transcriptional changes that might contribute to altered functions of microglia during seizure development and progression, particularly associated with neuroinflammation.

overall_design:
To investigate purinergic-driven immune preconditioning, we differentiated microglia-like cells from peripheral blood monocytes of healthy individuals. Monocytes were cultured in serum-free RPMI 1640 GlutaMAX medium supplemented with GM-CSF, M-CSF, β-BGF, CCL2, IL-34, TGF-β2 and cholesterol. We then subjected microglia-like cells to sequential stimuli with 100 uM ATP and 100 ng/uL LPS.
We performed gene expression profiling analysis using data obtained from RNA-seq of freshly isolated monocytes and in vitro microglia-like cells. We obtained four microglia-like conditions: untreated with ATP; treated with ATP; untreated with ATP and treated with LPS;  treated with ATP and treated with LPS.

first: Ricardo

last: Martins-Ferreira

first: Josep

last: Calafell-Segura

first: Chaves

last: João

first: Ciudad

last: Laura

first: António

last: Martins da Silva

first: Paulo

last: Costa

first: Bárbara

last: Leal


title: The NR4A2/VGF pathway fuels inflammation-induced neurodegeneration via promoting neuronal glycolysis

city: Hamburg

first: Marcel

middle: Seungsu

last: Woo

state: Hamburg

address: Falkenried 94

department: Neurology

institute: UKE

summary: To find differently expressed genes in VGF overexpressing neuronal cultures.

overall_design: Primary neuronal cultures that were transfected with VGF or EGFP over-expressing AAVs.

first: Lukas

middle: C

last: Bal

first: Manuel

last: Friese


title: IGF2BP3 promotes mRNA degradation through internal m7G modification

status: Public on Jul 13 2024

city: Shanghai

first: Xiaoyang

last: Dou

address: 320 Yue Yang Road

institute: CAS Center for Excellence in Molecular Cell Science, CAS

type: Methylation profiling by high throughput sequencing

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

overall_design: Refer to individual Series


title: Integration of shoot-derived polypeptide signals by root TGA transcription factors is essential for survival under fluctuating nitrogen environments [ChIP-seq]

city: Nagoya

first: Yuri

last: Ohkubo

address: Chikusa

department: Department of Biological Science

institute: Nagoya University

summary: Unlike plants in the field, which experience significant temporal fluctuations in environmental conditions, plants in the laboratory are typically grown in controlled, stable environments. Therefore, signaling pathways evolved for survival in continuously fluctuating environments often remain functionally latent in laboratory settings. Here, we show that TGA1 and TGA4 act as hub transcription factors through which the expression of genes involved in high-affinity nitrate uptake are regulated in response to shoot-derived phloem mobile polypeptides, CEP DOWNSTREAM 1 (CEPD1), CEPD2 and CEPD-like 2 (CEPDL2) as nitrogen (N) deficiency signals, and Glutaredoxin S1 (GrxS1) to GrxS8 as N sufficiency signals. CEPD1/2/CEPDL2 and GrxS1-S8 competitively bind to TGA1/4 in roots, with the former acting as transcription coactivators that enhance the uptake of nitrate, while the latter function as corepressor complexes together with TOPLESS (TPL), TPL-related 1 (TPR1) and TPR4 to limit nitrate uptake. Arabidopsis plants deficient in TGA1/4 maintain basal nitrate uptake and exhibit growth similar to wild-type plants in a stable N environment, but were impaired in regulation of nitrate acquisition in response to shoot N demand, leading to defective growth under continuously fluctuating N environments where rhizosphere nitrate ions switch periodically between deficient and sufficient states. TGA1/4 are crucial transcription factors that enable plants to survive under fluctuating and challenging N environmental conditions.

overall_design: To determine the binding sites of TGA1 across the genome in vivo, we performed ChIP-Seq using TGA1-GFP plants.

first: Ryutaro

last: Kobayashi

first: Mai

last: Izumi

first: Ryosuke

last: Ota

first: Keiko

last: Yamada

first: Yoko

last: Hayashi

first: Yasuko

last: Yamashita

first: Saki

last: Noda

first: Mari

last: Ogawa-Ohnishi

first: Yoshikatsu

last: Matsubayashi


title: Integration of shoot-derived polypeptide signals by root TGA transcription factors is essential for survival under fluctuating nitrogen environments [RNA-seq]

overall_design: To determine genes repressed by GrxS5, we performed gene expression profiling using Col-0 and GFP-GrxS5 plants.


title: Dysfunctional tumor-infiltrating Vδ1+ T lymphocytes in MSS colorectal cancer

city: Vienna

first: Georg

last: Stary

address: Spitalgasse 23

department: Dermatology

country: Austria

institute: Medical University of Vienna

summary: Impairment and exhaustion of effector functions limiting a proper immune response have been reported for conventional T cells in chronic infection and cancer. However, the functional relevance of γδ T cells influenced by the tumor microenvironment in human colorectal cancer (CRC) is still unclear. Here, we identify and characterize a distinct population of Vδ1+ T cells in human microsatellite stable CRC. Using integrated gene expression analysis and ⍺β/γδ-T cell receptor sequencing, we demonstrate activated adaptive immune responses in Vδ1+ T cells in CRC, which are accompanied by perturbances of innate immunity. However, enhanced upregulation of exhaustion genes, altered effector and cell stress molecules in Vδ1+ T cells pinpoint to impaired γδ T cells in CRC compared to distant healthy colon. Cellular interaction analysis highlights a role of cancer-associated fibroblasts in the dysregulation of Vδ1+ T cells in CRC. Immunophenotyping identified a distinct population of Vδ1+ T cells which dominates the γδ T cell subsets and displays a functionally impaired phenotype with a potential to be restored in vitro. These results define pathways operative in γδ T cells in CRC and provide a roadmap for harnessing γδ T cells for specific immunotherapeutic strategies.

overall_design:
10x Genomics RNA sequencing of healthy distant colon and colorectal cancer samples, including TCR-⍺β,-γδ sequencing and CITE-seq (as stated in description)
>>>Raw data for human samples not available due to patient privacy concerns<<<

first: Victoria

first: Ram

middle: V

last: Pandey

first: Anne

last: Vogt

first: Samuel

last: Lukowski

first: Matthias

last: Farlik


title: Integration of shoot-derived polypeptide signals by root TGA transcription factors is essential for survival under fluctuating nitrogen environments


title: BMP-dependent cellular dynamics during cranial suture establishment in zebrafish

city: Los Angeles

first: Claire

last: Arata

email: carata@usc.edu, clairearata@gmail.com

address: 1425 San Pablo St

department: Stem Cell

institute: University of Southern California

summary: Cranial sutures separate neighboring skull bones and contain skeletal stem cells that drive bone growth. A key question is how osteogenic activity is controlled to promote bone growth while preventing aberrant bone fusions during skull expansion. Here we integrate single-cell transcriptomics, in vivo expression validation, photoconversion-based lineage tracing, and a zebrafish craniosynostosis model to uncover key developmental transitions regulating bone formation during skull expansion. In addition to conservation of meninges and osteoblast lineage cells between zebrafish and mouse, single-cell transcriptomic analysis of the zebrafish skull reveals distinct subpopulations of suture mesenchyme that undergo transcriptomic changes during suture establishment. While lineage tracing with an osteoblast-specific nlsEOS reporter shows that bone formation largely occurs at suture edges, a subset of mesenchyme cells in the mid-suture region upregulate a suite of genes including BMP antagonists (e.g. grem1a) and pro-angiogenic factors. Further, lineage tracing with grem1a:nlsEOS reveals that this mid-suture subpopulation is largely non-osteogenic. In twist1b; tcf12 mutant zebrafish, a model for the coronal synostosis of Saethre-Chotzen Syndrome, reduction of grem1a+ mid-suture cells correlates with misregulated bone formation and reduced blood vessels at the coronal suture. In addition, combinatorial mutation of BMP antagonists enriched in the mid-suture subpopulation results in increased BMP signaling in the suture, misregulated bone formation, and abnormal suture morphology. These data support roles of a subset of mid-suture mesenchyme in locally promoting BMP antagonism that ensures proper suture morphology.

overall_design: Dissected calvaria of zebrafish were dissociated from juvenvile and adult zebrafish and live cells were identified by sorting for the absence of fluoresence (sp7:GFP or ecad:YFP) and the absence of DAPI using Fluorescence-activated cell sorting (FACS). Sorted cells were processed using 10X Genomics

first: D'Juan

middle: T

last: Farmer

first: Gage

last: Crump


title: Restriction of Arginine Induces Antibiotic Tolerance in Staphylococcus aureus

status: Public on Jul 10 2024

city: Nashville

first: Eric

last: Skaar

state: Tennessee

address: 1161 21st Avenue South, A5303B MCN

institute: Vanderbilt University Medical Center

summary: Staphylococcus aureus is responsible for a substantial number of invasive infections globally each year. These infections are problematic because they are frequently recalcitrant to antibiotic treatment. Antibiotic tolerance, the ability of bacteria to persist despite normally lethal doses of antibiotics, contributes to antibiotic treatment failure in S. aureus infections. To understand how antibiotic tolerance is induced, S. aureus biofilms exposed to multiple anti-staphylococcal antibiotics were examined using both quantitative proteomics and transposon sequencing. These screens indicated that arginine metabolism is involved in antibiotic tolerance within a biofilm and led to the hypothesis that depletion of arginine within S. aureus communities can induce antibiotic tolerance. Consistent with this hypothesis, inactivation of argH, the final gene in the arginine synthesis pathway, induces antibiotic tolerance. Arginine restriction was found to induce antibiotic tolerance via inhibition of protein synthesis. In a mouse skin infection model, an argH mutant has enhanced ability to survive antibiotic treatment with vancomycin, highlighting the relationship between arginine metabolism and antibiotic tolerance during S. aureus infection. Uncovering this link between arginine metabolism and antibiotic tolerance has the potential to open new therapeutic avenues targeting previously recalcitrant S. aureus infections.

overall_design: This study performed a genome wide TnSeq screen comparing S. aureus grown as a filter colony biofilm for 48 hours and exposed to 5 different conditions (no antibiotics or 4 different antibiotics) for another 48 hours. S. aureus grown as a biofilm for 48 or 96 hours was also compared to the input

first: Jeffrey

last: Freiberg

first: Valeria

last: Ruiz

first: Brittney

last: Gimza

first: Caitlin

last: Murdoch

first: Erin

middle: R

last: Green

first: Jacob

last: Curry

first: James

last: Cassat


title: Single cell RNA-seq of thymic cells from the Treg lineage from Foxp3eGFP and Foxp3eGFPxLta-/- mice

city: Marseille

first: Magali

last: IRLA

address: 163, avenue de Luminy - Case 906

institute: Center of Immunology Marseille Luminy (CIML)

summary: Single cell transcriptomic analyses reveal that Lta regulates the metabolic profile of thymic Treg cells

overall_design: Expression profiling by high throughput sequencing

last: Irla

first: Alexia

last: Borelli

first: Jeremy

last: Santamaria

first: Lionel

last: Spinelli


title: Nucleophosmin 1 Promotes Mucosal Immunity by Supporting Mitochondrial Oxidative Phosphorylation and ILC3 Activity

status: Public on Jul 09 2024

city: Suzhou

first: Minxuan

last: Sun

address: Keling Road

institute: Chinese Academy of Sciences

summary: NPM1 is commonly mutated in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Concurrent inflammatory bowel diseases (IBD) and MDS have been reported frequently, indicating a close relationship between IBD and MDS. Here, we examined the role of NPM1 in IBD and colitis-associated colorectal cancer (CAC). NPM1 was reduced in IBD patients. Npm1+/- mice were more susceptible to acute colitis and experimentally induced CAC than littermate controls. Npm1 deficiency impaired the function of IL-22-producing group 3 innate lymphoid cells (ILC3s). Mice lacking Npm1 in ILC3s exhibited decreased IL-22 production and accelerated development of colitis. NPM1 was important for mitochondrial biogenesis and metabolism by oxidative phosphorylation in ILC3s. Further experiments revealed that NPM1 cooperates with p65 to promote TFAM transcription in ILC3s. Overexpression of Npm1 in mice enhanced ILC3 function and reduced severity of DSS-induced colitis. Thus, our findings suggest the protective function of NPM1 in ILC3s against IBD by regulating mitochondrial metabolism through p65-TFAM axis.

overall_design: To uncover mechanisms by which NPM1 regulates ILC3 expansion and function, we performed RNA sequencing (smart-seq2) of Live+Lin-CD45lowCD90high LPLs from colon of WT and Npm1+/- mice with colitis induced by DSS treatment.

first: Rongchuan

last: Zhao

first: Jiao

last: Yang

first: Yunjiao

last: Zhai

first: Hong

last: Zhang

first: Yuanshuai

last: Zhou

first: Lei

last: Hong

first: Detian

last: Yuan

first: Ruilong

last: Xia

first: Yanxiang

last: Liu

first: Jinlin

last: Pan

first: Shaheryar

last: Shafi

first: Guohua

last: Shi

first: Ruobing

first: Dingsan

last: Luo

first: Jinyun

first: Dejing

first: Changgeng

last: Peng

first: Shiyang

last: Li


first: Esteban

last: Ballestar

summary: DNA methylation profiling of monocytes and monocyte-derived microglia like cells. Innate immune memory was explored in microglia-like cells through consecutive exposure to ATP and LPS.The Illumina Infinium MethylationEPIC Beadchip was used to obtain DNA methylation profiles across approximately 850,000 CpGs.

overall_design: Bisulphite converted DNA from the 20 samples were hybridised to the Illumina Infinium MethylationEPIC Beadchip.

first: Ricardo

last: Martins-Ferreira

first: Josep

last: Calafell-Segura

first: João

last: Chaves

first: Laura

last: Ciudad

first: António

last: Martins da Silva

first: Paulo

last: Costa

first: Bárbara

last: Leal


title: 53BP1 loss elicits cGAS-STING-dependent antitumor immunity in ovarian and pancreatic cancer

first: Jeffrey

last: Patterson-Fortin

address: 450 Brookline Avenue

department: Medical Oncology

institute: Dana-Farber Cancer Institute

summary: 53BP1 nucleates the anti-end resection machinery at DNA double-strand breaks (DSBs), thereby countering BRCA1 activity. Loss of 53BP1 leads to DNA end processing and homologous recombination (HR) in BRCA1-deficient cells. Consequently, BRCA1-mutant tumors, typically sensitive to PARP inhibitors (PARPi), become resistant in the absence of 53BP1. Here, we demonstrate that the 'leaky' DNA end resection in the absence of 53BP1 results in increased micronuclei and cytoplasmic dsDNA, leading to activation of the cGAS-STING pathway and pro-inflammatory signaling. This enhances CD8+ T cell infiltration, activates macrophages and natural killer (NK) cells, and impedes tumor growth. Loss of 53BP1 correlates with a response to immune checkpoint blockade (ICB) and improved overall survival. Immunohistochemical assessment of 53BP1 in two malignancies, high grade serous ovarian cancer (HGSOC)s and pancreatic ductal adenocarcinoma (PDAC)s, which are refractory to ICBs, revealed that lower 53BP1 levels correlated with an increased adaptive and innate immune response. Finally, BRCA1-deficient tumors that develop resistance to PARPi due to the loss of 53BP1 are susceptible to ICB. Therefore, we conclude that 53BP1 is critical for tumor immunogenicity and underpins the response to ICB. Our results support including 53BP1 expression as an exploratory biomarker in ICB trials for malignancies typically refractory to immunotherapy.

overall_design: To investigate the clinical role of 53BP1 and its effect on the immune response, we established mouse Trp53bp1 knockout cell lines (ID8, KPC). We implanted these cell lines into syngeneic mice resulting in tumor formation. We then performed gene expression profiling analysis using data obtained from RNA-seq of 16 different tumors. Comparative gene expression profiling analysis of RNA-seq data for KPC tumors and its 53BP1 KO counterparts, or ID8 tumors and its 53BP1 KO counterparts.

first: Yajie

last: Sun

first: Sen

last: Han

first: Zhe

first: Zuzanna

last: Nowicka

first: Yuna

last: Hirohashi

first: Susan

last: Kilgas

first: Michelle

middle: L

last: Swift

first: Jae

last: Kyo Yi

first: Alexander

last: Spektor

first: Wojciech

last: Fendler

first: Panagiotis

last: Konstantinopoulos

first: Dipanjan

last: Chowdhury


title: In vivo CRISPR screens identify a dual function of MEN1 in regulating tumor-microenvironment interactions [CRISPR screening]

first: Yin

last: Liu

state: Shanghai

address: Building #2, 507 Zhengmin Road

department: Department of Laboratory Medicine

institute: Shanghai Pulmonary Hospital, Tongji University School of Medicine

summary: In this study, we used a targeted CRISPR/Cas9 screen to identify genes that determine growth of A549 cells in vivo and in vitro respectively.

overall_design: The CRISPR screen employed in our pooled Epidrug library consisted of ~12,500 sgRNAs targeting 317 epigenetic regulators, 657 FDA-approved drug targets based on Drugbank v4.3, and control genes, with 10 sgRNAs per gene on average. The sgRNAs were designed using the CRISPR-DO tool. Stable Cas9-expressing A549 cell line was generated. Cas9-expressing A549 cell line was infected with the library at an MOI of ~ 0.3 and coverage of 300x. 24-48h post-infection, cells were selected with puromycin for 72h and part of cells were sujected to DNA extraction as day 0. Other cells were inoculated in NOD/SCID mice or cultured in vitro, maintaining 300x coverage prior to our screening assay. Genomic DNA was extracted from replicate samples and sgRNA inserts were amplified by PCR. The input amount of genomic DNA was calculated to achieve 250x coverage of the library and resulting libraries were sequenced on an Illumina HiSeq2500.

first: Peiran

last: Su

first: Tianyi

last: Chen

first: Ming-Sound

last: Tsao

first: Housheng

last: He


title: In vivo CRISPR screens identify a dual function of MEN1 in regulating tumor-microenvironment interactions [ChIP-seq]

summary: In this study, we used a targeted CRISPR/Cas9 screen to identify genes that determine growth of A549 cells in vivo and in vitro.

overall_design: Examination of MLL and menin chromatin binding and H3K4me3 histone modifications in MEN1 knockout A549 cells.


title: In vivo CRISPR screens identify a dual function of MEN1 in regulating tumor-microenvironment interactions [CUT & RUN]


title: A Lactobacilli-Based Inhaled Live Biotherapeutic Product Attenuates Pulmonary Neutrophilic Inflammation

status: Public on Jul 08 2024

city: Birmingham

first: Tamas

last: Jilling

state: AL

address: UAB Station

department: Department of Pediatrics

institute: The University of Alabama at Birmingham

summary: Bronchopulmonary dysplasia (BPD) is a chronic lung disease of prematurity. Exposure to noxious stimuli such as hyperoxia, volutrauma, and infection in infancy can have long-reaching impacts on lung health and predispose towards the development of conditions such as chronic obstructive pulmonary disease (COPD) in adulthood. BPD and COPD are both marked by lung tissue degradation, neutrophil influx, and decreased lung function. Both diseases also express a change in microbial signature characterized by firmicute depletion. However, the relationship between pulmonary bacteria and the mechanisms of downstream disease development has yet to be elucidated. We hypothesized that murine models of BPD would show heightened acetylated proline-glycine-proline (Ac-PGP) pathway and neutrophil activity, and through gain- and loss-of-function studies we show that Ac-PGP plays a critical role in driving BPD development. We further test a inhaled live biotherapeutic (LBP) using active Lactobacillus strains in in vitro and in vivo models of BPD and COPD. The Lactobacillus-based LBP is effective in improving lung structure and function, mitigating neutrophil influx, and reducing a broad swath of pro-inflammatory markers in these models of chronic pulmonary disease via the MMP-9/PGP (matrix metalloproteinase/proline-glycine-proline) pathway. Inhaled LBPs show promise in addressing common pathways of disease progression that in the future can be targeted in a variety of chronic lung diseases.

overall_design: To determine the effect of Lactobacillus live biotherapeutic product on POA and smoke-exposed HBE cells.

first: Nicola

last: Teodora

first: Wenger

last: Nancy

first: Jilling

last: Tamas

first: Lal

last: Charitharth Vivek


title: Epstein-Barr virus induces host shutoff extensively via BGLF5-independent mechanisms [RNA-seq]

city: Madison

first: Alejandro

last: Casco

state: WI

address: 1111 Highland Ave

institute: University of Wisconsin-Madison

summary: This study characterizes the effects of Epstein-Barr virus (EBV) reactivation on the host and virus transcriptomes. For this work, a dual-fluorescent lytic reporter (DFLR) EBV was constructed based on the M81 EBV strain and used to transform primary B cells into lymphoblastoid cell lines (LCL). Deposited here is RNA-seq data from these DFLR LCLs that were induced by BCR crosslinking and sorted into latent, early lytic, and late lytic fractions. All samples consist of 40,000 sorted cells and include equal quantities of ERCC spike-in RNAs for normalization. We also performed the same experimental approach on a second DFLR LCL in which the EBV BGLF5 host shutoff nuclease was deleted. This allowed us to define which changes in the transcriptome during EBV replication were due to BGLF5. This approach demonstrated extensive shutoff of host genes during EBV replication and, unexpectedly, revealed that most of those changes were independent of BGLF5.

overall_design: DFLR and DFLR BGLF5-knockout (dBGLF5) LCLs induced into lytic EBV replication were FAC-sorted and RNA-sequenced (5 and 3 biological replicates, respectively) to characterize transcriptomic changes during early and late lytic phases and investigate BGLF5's role in host shutoff. Differential gene expression and alternative splicing/readthrough transcription were analyzed between latent and lytic fractions in WT and BGLF5-deficient LCLs.

first: Ohashi

last: Makoto

first: Eric

last: Johannsen