title: Age- and sex- divergent translatomic responses of the retinal pigmented epithelium

city: Oklahoma City

first: Willard

last: Freeman

address: 825 NE 13th St

institute: Oklahoma Medical Research Foundation

summary: Aging is the main risk factor for age-related macular degeneration (AMD), a retinal neurodegenerative syndrome that leads to irreversible blindness, particularly in people over 60 years old. Retinal pigmented epithelium (RPE) atrophy is an AMD hallmark that often precedes photoreceptor loss. Genome-wide chromatin accessibility, DNA methylation, and gene expression studies of AMD and control RPE demonstrate epigenomic/transcriptomic changes occur during AMD onset and progression. However, mechanisms by which molecular alterations of normal aging impair RPE function and contribute to AMD pathogenesis are unclear. Here, we specifically interrogate the RPE translatome with advanced age and across sexes in a novel mouse model. We find differential age- and sex- associated transcript expression with overrepresentation of pathways related to inflammation in the RPE. Concordant with impaired RPE function, the phenotypic changes in the aged translatome suggest that aged RPE becomes immunologically active, in both males and females, with some sex-specific signatures, which supports the need for sex representation for in vivo studies.

overall_design: Aldh1l1-cre/ERT2+;NuTRAP+ RNA isolated via TRAP method- Input and Positive Fractions

first: Ana

last: Chucair-Elliott

first: Sarah

middle: R

last: Ocanas

first: Kevin

last: Pham

first: Adeline

last: Machalinski

first: Scott

last: Plafker

first: Michael

last: Stout

last: Elliott


title: Gene expression profile at the single cell level of CD45+ CD11b+ cells isolated from the brain and bone marrow of mice either untreated or conditioned and transplanted with total bone marrow.

status: Public on May 13 2024

city: Stanford

first: Natalia

last: Gomez-Ospina

address: 265 Campus Drive

country: USA

institute: Stanford

type: Expression profiling by high throughput sequencing

summary: We used single-cell RNA sequencing (scRNA-seq) to analyze the diversity of bone marrow-derived CD45+CD11b+ microglia-like cells (MGLCs) engrafted in the brain of recipient mice that were conditioned using Busulfan and PLX3397 and transplanted with total bone marrow. We compared the gene expression of MGLCs to that of developmentally-derived CD45+ CD11b+ microglia/myeloid cells isolated from the brain of recipient mice (host microglia) and untreated mice (naive microglia). We also compared the gene expression of MGLCs to that of transplant-derived CD45+ CD11b+ cells engrafted in the bone marrow (abbreviated as BM-CD11b+)

overall_design: Brain CD45+ CD11b+ cells were isolated by Fluorescence-activated cell sorting (FACS) based on the co-expression of the CD45+ and CD11b+ surface markers and the expression of the Green Fluorescent Protein (GFP). Four CD45+ CD11b+ cell samples were isolated by FACS and used for the scRNA-seq. Cells isolated from C57BL/6 mice (Jax strain #000664) conditioned with Busulfan (100 mg/kg) and PLX3397 (600 mg/kg) and transplanted with total bone marrow harvested from adult sex-matched C57BL/6-Tg(CAG-EGFP)131Osb/LeySopJ mice (Jax strain #006567) were: 1)  Sample A or MGLCs: GFP+ CD45+ CD11b+ MGLCs, isolated from the brain; 2)  Sample B or host conditioned MG or, host MG: GFP- CD45+ CD11b+ cells,  isolated from the brain;  3) Sample D or BM-CD11b+: GFP+ CD45+ CD11b+ cells, isolated from the bone marrow; the time point of the analysis is 4 months after transplant. Cells isolated from untreated age-matched donor mice [C57BL/6-Tg(CAG-EGFP)131Osb/LeySopJ, (Jax strain #006567] were: 4) Sample C or naive MG: GFP+ CD45+ CD11b+ cells isolated from the brain.

first: Pasqualina

last: Colella

first: Ruhi

last: Sayana

first: Marco

last: Corbo

first: Anay

last: Limaye


title: Rare variation in noncoding regions with evolutionary signatures contributes to autism spectrum disorder risk

city: Boston

first: Janet

last: Song

state: Massachusetts

address: 3 Blackfan St

institute: Boston Children's Hospital

summary: We examined three classes of noncoding regions with different patterns of conservation and constraint: Human Accelerated Regions (HARs), which show signatures of positive selection in the human lineage; experimentally validated neural Vista Enhancers (VEs); and candidate neural enhancers (CNEs). We interrogate the in vitro enhancer activity of HARs, VEs, and CNEs in capture-based massively parallel reporter assays (caMPRAs). We also perform random mutagenesis on HARs to assess the effeects of single nucleotide variants on enhancer activity.

overall_design: capture-based Massively Parallel Reporter Assays (caMPRAs) of HARs, VEs, and CNEs in the N2A cell line

first: Taehwan

last: Shin

middle: H

first: Ryan

last: Doan

first: Christopher

middle: A

last: Walsh


title: Interferon-γ signaling in the progression from early to end stage kidney disease in type 2 diabetes

city: Ann Arbor

first: Jennifer

last: Harder

state: MI

address: 1301 Catherine St.

department: Internal Medicine, Division of Nephrology

institute: University of Michigan Medical School

summary: The effects of circulating pro-inflammatory factors on diabetic kidney disease (DKD) progression remain poorly defined. In this study, transcriptional and proteomic profiling of kidney tissue and blood samples from individuals with type 2 diabetes mellitus and early DKD was used to identify gene expression associated with kidney disease progression.  Interferon gamma (IFNG) was both identified as an upstream regulator of gene expression and detected in the serum of individuals who progressed.  Individual IFNG activation scores (representing summary expression of 72 downstream signaling mediators) early in disease correlated with DKD progression over an average of 9.7 years.  IFNG pathway inhibition reduced gene activity associated with DKD progression in both kidney organoids generated from human pluripotent stem cells and mouse DKD models.  Thus, expression of genes associated with IFNG activity early in DKD predicted disease progression; individuals demonstrating IFNG activity may benefit from targeted therapeutics early in the disease course.

overall_design: Kidney organoids were generated from human embryonic stem cells (UM77-2, NIH registry #0278) according to published protocol (Harder et al., JCI Insight 2019).  Organoids were treated on day 23 with either vehicle control, or 25ng/ml of IFNg, or 1000nM of Baricitinib or the combination of IFNg with Baricitinib (added 1 hr prior to IFNg) for 24h. Then organoid whole wells were either RNA extracted for BulkRNA Seq processing or single cell dissociated for scRNASeq processing in the Advanced Genomics Core at the University of Michigan.

first: Wenjun

last: Ju

first: Jamal

last: Le Saghir

first: Fadhl

last: Alakwaa

first: Felix

last: Eichinger

first: Rajasree

last: Menon

first: Viji

last: Nair

first: Lalita

last: Subramanian

first: Virginia

last: Vega-Warner

first: Matthew

last: Fischer


title: bulk RNA-seq of human nucleusus pulposus from disc-degenerated patients

status: Public on May 14 2024

city: Pok Fu Lam

first: Peikai

last: Chen

address: Sassoon Road 5

department: Faculty of Medicine

country: Hong Kong

institute: The University of Hong Kong

summary: Failure of the nucleus pulposus (NP) causes intervertebral disc (IVD) disease and associated low-back pain, which are highly prevalent among the aged populations. Molecular and cellular changes underpinning the structural failures in age-associated IVD diseases (IDDs) remain poorly elucidated. Here, we first identified that TAGLN, which encodes the cytoskeleton regulator transgelin, was transcribed in healthy NPs of both human and mouse, but diminished with ageing. Immunostaining showed that TAGLN were expressed on the peripheral of mouse NP (periNP). Lineage analyses in Tagln-CreERT2 mice showed that NP cells were derived from Tagln+ cells in the periNP. The PeriNP cells were proliferative and can differentiate into the inner NP (innerNP). These Tagln+ cells and their descendants diminish with ageing or puncture-induced degeneration. Single-cell transcriptomics from neonatal and adult Tagln-CreERT2 IVDs confirmed that Tagln descendants uniquely populate the NP, wherein four sub-populations were identified: chondrocyte-like, Cd228+, Tagln+ and Car3+. Immunostaining confirmed Car3 expressed in innerNP. Computational analysis indicated the lineage trajectory from Tagln+ to Car3+ sub-population, along with the involvement of Fos/Jun/TGFβ cascades and partial epithelial-to-mesenchymal transition process. Removal of TGFβ mediator Smad4 by notochord specific Foxa2mNE-Cre resulted in decreased Tagln+ cells and abnormal disc morphology, resembling disc degeneration. Our study generates a single-cell transcriptomic atlas for healthy IVD, identifies Tagln+ PeriNP cells as a progenitor pool crucial for the IVD homeostasis, and provides potential targets for regenerative cell therapy against IVD degeneration.

overall_design: We sequenced five NP from five individuals with disc degeneration.

first: Tiffany

last: Au

first: Kathryn

middle: S

last: Cheah


title: Biochemical properties of chromatin domains define their compartmentalization [I]

city: Milan

first: Chiara

last: Lanzuolo

address: Via Francesco Sforza 35

country: Italy

institute: CNR and Istituto Nazionale Genetica Molecolare

summary:
Chromatin three-dimensional (3D) organization inside the cell nucleus determines the separation of euchromatin and heterochromatin domains. Their segregation results in the definition of active and inactive chromatin compartments, whereby the local concentration of associated proteins, RNA and DNA results in the formation of distinct subnuclear structures.
Thus, chromatin domains spatially confined in a specific 3D nuclear compartment are expected to share similar epigenetic features and biochemical properties, in terms of accessibility and solubility.
Based on this rationale, we developed the 4f-SAMMY-seq to map euchromatin and heterochromatin based on their accessibility and solubility, starting from as little as 10,000 cells. Adopting a tailored bioinformatic data analysis approach we reconstruct also their 3D segregation in active and inactive chromatin compartments and sub-compartments, thus recapitulating the characteristic properties of distinct chromatin states.
A key novelty of the new method is the capability to map both the linear segmentation of open and closed chromatin domains, as well as their 3D compartmentalization in one single experiment.

overall_design: SAMMY-seq analysis on primary fibroblast cell lines, using two different protocols with three biological replicates each.  For the 4-fractions protocol, we performed also replicates on 50k cells and on 10k cells

first: Federica

last: Lucini

first: Cristiano

last: Petrini

first: Elisa

last: Salviato

first: Koustav

last: Pal

first: Valentina

last: Rosti

first: Francesca

last: Gorini

first: Philina

last: Santarelli

first: Roberto

last: Quadri

first: Emanuele

last: Di Patrizio Soldateschi

first: Giovanni

last: Lembo

first: Ilario

last: Tagliaferri

first: Eva

last: Pinatel

first: Endre

last: Sebestyén

first: Luca

last: Rotta

first: Francesco

last: Gentile

last: Vaira

first: Giulia

last: Graziano

last: Ferrari


title: Genome wide identification of replication fork stalling/pausing sites and the interplay between RNA Pol II transcription and DNA replication progression

city: Birmingham

first: Marco

last: Saponaro

address: Vincent Drive

department: Institute of Cancer and Genomic Sciences

country: United Kingdom

institute: University of Birmingham

summary: DNA replication progression can be affected by the presence of physical barriers on the DNA, like RNA Polymerases, leading to replication stress and DNA damage. To characterize what happens at sites where DNA replication forks stall and pause, we establish a genome-wide approach to identify them. This approach uses multiple timepoints during S-phase, to identify replication fork/stalling hotspots throughout the genome. These sites are typically associated with increased DNA damage, overlap with fragile sites and with breakpoints of rearrangements identified in cancers, but do not overlap with replication origins. Overlaying these sites with a genome-wide analysis of RNA Polymerase II transcription, we found that replication fork stalling/pausing sites inside genes are directly related to transcription progression and activity. This would support data that indicate that transcription and replication can coexist over the same regions. We found instances where transcription activity by reducing histone density favors replication progression through genes, but also found that slowing down transcription elongation slows down directly replication progression through genes. Importantly, rearrangements found in cancers at transcription-replication collision sites can be detected in non-transformed cells and increased following treatment with ATM and ATR inhibitors. Altogether, our findings highlight how transcription and replication overlap during S-phase, with both positive and negative consequences for replication fork progression and genome stability.

overall_design: Genome-wide DNA replication profile of BJ-hTERT during S phase with or without indicated treatments generated by NGS

first: Patricia

last: Rojas

first: Jianming

last: Wang


title: Cardiac Outflow tract septation defects in a DiGeorge syndrome model respond to Minoxidil treatment

status: Public on May 11 2024

city: Naples

first: Antonio

last: Baldini

state: Naples

address: Via Sergio Pansini 5

department: Department of Molecular Medicine and Medical Biotechnology

institute: University of Naples Federico II

summary: We found that Tbx1 suppresses a subset of ECM-related genes in a cell differentiation model. Treatment with a Lysyl hydroxylase inhibitor drug in vivo during pregnancy reduced the  expressivity of cardiac defects in Tbx1 mutant embryos.

overall_design: E14-Tg2a cells (parental line and Tbx1-/- line 4D) were dissociated with Trypsin-EDTA and cultured at a density of 100,000 cells/ml in serum-free differentiation media. We followed the protocol described by Andersen et al Nat Commun 9, 3140 (2018) and harvested cells at day 6 of differentiation. We processed for QuantSeq 4 biological replicates of the parental line and 4 of the 4D line.

first: Rosa

last: Ferrentino

first: Ilaria

last: Aurigemma

first: Olga

last: Lanzetta

first: Varsha

last: Poondi Krishnan

first: Claudia

last: Angelini

first: Elizabeth

last: Illingworth


title: Cyclo His-Pro halts skeletal muscle degeneration and cardiomyopathy in murine myopathy models

city: Lausanne

first: Keno

last: Strotjohann

address: Route Cantonale

department: School of Life Sciences, Institute of Bioengineering

country: Switzerland

institute: École Polytechnique Fédérale de Lausanne (EPFL)

summary: Among the inherited myopathies, a group of muscular disorders characterized by structural and metabolic impairments in skeletal muscle, Duchenne muscular dystrophy (DMD) stands out for its devastating progression. Caused by the absence of functional dystrophin, DMD pathogenesis is driven by the progressive degeneration of muscle fibers, resulting in the development of inflammation and fibrosis that ultimately affect the overall muscle biomechanics. On the opposite end of the spectrum of muscle diseases, age-related sarcopenia is a common disease that affects a growing proportion of the elderly, often leading to frailty and loss of independence. Although characterized by fundamentally different pathological mechanisms, DMD and sarcopenia share the development of progressive muscle weakness and tissue inflammation. The natural compound Cyclo Histidine-Proline (CHP) has been shown to reduce inflammation and fibrosis in a number of metabolic diseases, including steatohepatitis and diabetes. We evaluate here the effects of CHP in DMD and sarcopenia. In two independent studies in the mdx mouse model of DMD, we show that CHP restored muscle contractility and force production, accompanied by a robust reduction of fibrosis and inflammation in skeletal muscle. In the mdx model, CHP furthermore prevented the development of cardiomyopathy and fibrosis in the diaphragm, the two leading causes of death for DMD patients. CHP also attenuated muscle atrophy and functional deterioration in a mouse model of age-related sarcopenia. These data in two different models of muscle dysfunction hence warrant further study of the effects of CHP in muscle pathologies in animal models and eventually in patients.

overall_design: C57BL/10ScSn (BL10) and C57BL/10ScSn-Dmdmdx (mdx) male mice (The Jackson Laboratory) were housed at 22°C with free to access to water and chow diet ad libitum. After a week of adaptation, mice were divided into three groups: mdx received either CHP treatment or water (control), BL10 mice received water (control), three times per week by oral gavage. Mice were treated from week 7 to week 22 of age with CHP at a dose of 35 mg/kg or water as control. 18-month-old male C57BL6/J mice (Korea basic science institute [KBSI]) were housed in individual cages in constant temperature and humidity air-conditioning cabinet (JD-SY-02, Jungdo) at a temperature of 23 ± 3℃ and a humidity of 50% with a 12 hours light/dark cycle. Mice were free to access distilled water (DW) and laboratory chow diet ad libitum. Mice were divided into two groups based on equal average body weight, and DW or CHP (35mg/kg/day) was orally administered for 6 months.

first: Alessia

last: De Masi

first: Nadège

last: Zanou

first: Dohyun

last: Lee

first: Tanes

middle: I

last: Lima

first: Jongsu

last: Jeon

first: Nicolas

last: Place

first: Hoe-Yune

last: Jung

first: Johan

last: Auwerx


title: Gene expression profile at the single cell level of CD45+ CD11b+ cells isolated from the brain and bone marrow of mice either untreated or conditioned and transplanted with mouse hematopoietic stem and progenitor cells (HSPCs) expanded in culture.

first: Natalia

last: Gomez-Ospina

summary: We used single-cell RNA sequencing (scRNA-seq) to analyze the diversity of HSPC-derived CD45+CD11b+ microglia-like cells (MGLCs) engrafted in the brain of recipient mice that were conditioned using Busulfan and PLX3397 and transplanted intravenous with Lineage negative KIT+ SCA1+ mouse HSPCs. The HSPCs were isolated from adult C57BL/6-Tg(CAG-EGFP)131Osb/LeySopJ homozygous mice. We compared the gene expression of MGLCs to that of developmentally-derived CD45+ CD11b+ microglia/myeloid cells isolated from the brain of recipient mice (host microglia) and untreated mice (naive microglia). We also compared the gene expression of MGLCs to that of transplant-derived CD45+ CD11b+ cells engrafted in the bone marrow (abbreviated as BM-CD11b+)

overall_design: Brain CD45+ CD11b+ cells were isolated by Fluorescence-activated cell sorting (FACS) based on the co-expression of the CD45+ and CD11b+ surface markers and the expression of the Green Fluorescent Protein (GFP). Four CD45+ CD11b+ cell samples were isolated by FACS and used for the scRNA-seq. Cells isolated from C57BL/6 mice (Jax strain #000664) conditioned with Busulfan (125 mg/kg) and PLX3397 (600 mg/kg) and transplanted with HSPCs harvested from adult sex-matched C57BL/6-Tg(CAG-EGFP)131Osb/LeySopJ mice (Jax strain #006567) were: 1)  Sample A or MGLCs: GFP+ CD45+ CD11b+ MGLCs, isolated from the brain; 2)  Sample B or host conditioned MG or, host MG: GFP- CD45+ CD11b+ cells,  isolated from the brain;  3) Sample D or BM-CD11b+: GFP+ CD45+ CD11b+ cells, isolated from the bone marrow; the time point of the analysis is 9 months after transplant. Cells isolated from untreated age-matched donor mice [C57BL/6-Tg(CAG-EGFP)131Osb/LeySopJ, (Jax strain #006567] were: 4) Sample C or naive MG: GFP+ CD45+ CD11b+ cells isolated from the brain. Cell Hashing, using oligo-tagged antibodies directed to mouse CD45 (clone 30-F11) and mouse MHCI (clone M1/42), was used to uniquely label cells from 3 distinct mice/sample (multiplexing).  Details on the cell hashing and hashtag oligo (HTO) are included in the "extract protocol" section below.

first: Pasqualina

last: Colella

first: Ruhi

last: Sayana

first: Marco

last: Corbo


title: Biochemical properties of chromatin domains define genome compartmentalization

first: Chiara

last: Lanzuolo

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

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: Refer to individual Series


title: Systems modeling of oncogenic G-protein and oncogenic GPCR signaling reveals unexpected differences in downstream signaling pathway activation [CY_GQ_experiment]

status: Public on May 10 2024

city: La Jolla

first: Kathryn

last: Lande

state: CA

address: 10010 N Torrey Pines Rd

department: IGC

institute: Salk Institute

summary: Mathematical models of biochemical reaction networks are an important and emerging tool for the study of cell signaling networks involved in disease processes. One promising potential application of such mathematical models is the study of how disease causing mutations to one or more proteins in the network promote the signaling phenotype that contributes to the disease. It is commonly assumed that one must have a thorough characterization of the network readily available for mathematical modeling to be useful, but we hypothesized that mathematical modeling could be useful when there is incomplete knowledge and that it could be a tool for discovery that opens new areas for further exploration. In the present study, we first develop a mechanistic mathematical model of a G-protein coupled receptor signaling network that is mutated in almost all cases of uveal melanoma and use model-driven explorations to uncover and explore multiple new areas for investigating this disease. Modeling the two major, mutually-exclusive, oncogenic mutations (Gαq/11 and CysLT2R) revealed the potential for previously unknown qualitative differences between seemingly interchangeable disease-promoting mutations, and our experiments confirmed oncogenic CysLT2R was impaired at activating the FAK/YAP/TAZ pathway relative to Gαq/11. This led us to hypothesize that CYSLT2R mutations in UM must co-occur with other mutations to activate FAK/YAP/TAZ signaling, and our bioinformatic analysis uncovers a role for co-occurring mutations involving the plexin/semaphorin pathway, which has been shown capable of activating this pathway. Overall, this work highlights the power of mechanism-based computational systems biology as a discovery tool that can leverage available information to open new research areas.

overall_design: To experimentally explore model-suggested potential for divergent cellular phenotypes, we expressed WT or mutant GNAQ or CYSLTR2 constructs in HEK 293T cells and quantified downstream signaling and activation of ERK and YAP signaling via RNA sequencing.

first: Michael

last: Trogdon

first: Edward

middle: C

last: Stites


title: Proteome-scale tagging and functional screening in mammalian cells by ORFtag [RNA-seq]

city: Vienna

first: Alexander

last: Stark

address: Campus-Vienna-Biocenter 1

country: Austria

institute: The Research Institute of Molecular Pathology (IMP)

summary: The systematic determination of protein function is a key goal of modern biology, but remains challenging with current approaches. Here, we present ORFtag, a versatile, cost-effective and highly efficient method for the massively parallel tagging and functional interrogation of proteins at proteome scale. ORFtag utilizes retroviral vectors bearing a promoter, peptide tag and splice donor to generate fusions between the tag and endogenous ORFs. We demonstrate the utility of ORFtag through functional screens for transcriptional activators, repressors and post-transcriptional regulators in mouse embryonic stem cells. Each screen finds known and novel regulators, including long ORFs inaccessible by other methods, revealing that Zfp574 is a highly selective transcriptional activator and that oncogenic fusions often function as transactivators.

overall_design: RNA-Seq of ORFtag activator screen

first: Filip

last: Nemčko

first: Moritz

last: Himmelsbach

first: Vincent

last: Loubiere

first: Ramesh

last: Yelagandula

first: Michaela

last: Pagani

first: Nina

last: Fasching

first: Julius

last: Brennecke

first: Ulrich

last: Elling

first: Stefan

middle: L

last: Ameres


title: Comparison of novel proteomic expression profiles for radiation exposure in male and female C57BL6 mice

city: Rockville

first: Yu

last: Fan

state: MD

address: 9609 Medical Center Dr

institute: NCI/CBIIT

summary: Purpose: There is a need for point-of-care diagnostics for future mass casualty events involving radiation exposure.  The development of radiation exposure and dose prediction algorithms for biodosimetry is needed for screening of large populations during these scenarios, and exploration of the potential effects which sex, age, genetic heterogeneity, and physiological comorbidities may have on the utility of biodosimetry diagnostics is needed.  In the current study, proteomic profiling was used to examine sex specific differences in age matched C57BL6 mice on the blood proteome following radiation exposure and the usefulness of development and application of biodosimetry algorithms using both male and female samples.  Methods: C57BL6 male and female mice between 9-11 weeks of age received a single total body radiation exposure of either 2, 4 or 8 Gy with plasma collection at days 1, 3 and 7 post-irradiation.  Plasma was then screened using the SomaScan v4.1 assay for ~7000 protein analytes. A subset panel of protein biomarkers demonstrated significant (FDR<0.05 and |logFC|>0.2) changes  in expression following radiation exposure. All proteins were used for feature selection to build predictive models of radiation exposure using different sample and sex specific cohorts. Both binary (prediction of any radiation exposure) and multidose (prediction of specific radiation dose) model series were developed using either female and male samples combined or only female or only male samples. The binary series (Models 1, 2 and 3) and multidose series (Models 4, 5 and 6) series included female/male combined, female only and male only respectively.  Results: Detectable values were obtained for all ~7000 proteins included in the SomaScan assay for all samples.  Each model algorithm built using a unique sample cohort was validated with a training set of samples and tested with a separate new sample series.  Overall predictive accuracies in the binary model series was ~100% at the model training level and when tested with fresh samples 97.9% for Model 1(Female and Male) and 100% for Model 2 (Female only) and Model 3 (Male only).  When sex specific Models’ 2 and 3 were tested with the opposite sex, the overall predictive accuracy rate dropped to 62.5% for Model 2 and remained 100% for Model 3.  The overall predictive accuracy rate in the multidose model series was 100% for all models at the model training level and when tested with fresh samples 83.3%, 75% and 83.3% for Multidose Models 4-6 respectively.  When sex specific Models’ 5 (Female only) and Model 6 (Male only) were tested with the opposite sex the overall predictive accuracy rate dropped to 52.1% and 68.8% respectively.  Conclusion:  These models represent novel predictive panels of radiation responsive proteomic biomarkers and illustrate the utility and necessity of considering sex specific differences in development of radiation biodosimetry prediction algorithms. As sex specific differences were observed in this study, and as use of point-of-care radiation diagnostics in future mass casualty settings will necessarily include persons of both sexes, consideration of sex specific variation is essential to ensure these diagnostic tools have practical utility in the field.

overall_design: C57BL6 male and female mice between 9-11 weeks of age received a single total body radiation exposure of either 2, 4 or 8 Gy with plasma collection at days 1, 3 and 7 post-irradiation. Matched male and female cohorts were used with 20 untreated controls and 6 animals per dose/timepoint for each sex, totaling 148 animals for this study.

first: Mary

last: Sproull

first: Qingrong

last: Chen

first: Daoud

last: Meerzaman

first: Kevin

last: Camphausen


title: Dermal TRPV1 innervations engage a macrophage and fibroblast containing pathway to activate hair growth [dataset2]

city: SAN FRANCISCO

first: Konrad

last: Knopper

address: 513 Parnassus Ave

institute: UCSF

summary: Pain, detected by nociceptors, is an integral part of injury, yet whether and how it can impact tissue physiology and recovery remain understudied. Here we applied chemogenetics in mice to locally activate dermal TRPV1 innervations in naïve skin and found it triggered new regenerative cycling by dormant hair follicles (HFs).This was preceded by rapid apoptosis of dermal macrophages, mediated by the neuropeptide calcitonin gene-related peptide (CGRP). TRPV1 activation also triggered a macrophage-dependent induction of Spp1-expressing dermal fibroblasts. The neuropeptide CGRP andSpp1were essential for the nociceptor-triggered hair growth. Finally, we show that epidermal abrasion injury induced Spp1-expressing dermal fibroblasts and hair growth via a TRPV1 neuron and CGRP dependent mechanism.Collectively, these data demonstrate a role for TRPV1 nociceptors in orchestrating a macrophage and fibroblast-supported mechanism to promote hair growth, and enabling the efficient restoration of this mechano- and thermo-protective barrier after wounding.

overall_design:
Back skin from TRPV1hM3Dq and control mice (three mice per group) treated daily with a CNO intradermal injection for three consecutive days, were dissected, separated into the subcutis and dermal-epidermal fractions and then digested. The dermal-epidermal and subcutis fractions were stained separately with TotalSeq™ anti-mouse Hashtag antibodies (TotalSeq™ A0301 and A0302 from BioLegend) to mark the cells’ anatomical location. Thy1+ SCA1+ stromal cells (CD45−CD31− EpCAM−) were sorted and analysed. Treatment groups (two) were run on separate lanes of a 10X Chromium chip with 3′ v.2 chemistry (10X Genomics) as per the manufacturer’s instructions by the UCSF Institute for Human Genetics Sequencing Core. Transcripts captured in all the cells encapsulated with a bead were uniquely barcoded using a combination of a 16 bp 10x Barcode and a 10 bp unique molecular identifier (UMI). cDNA libraries were generated using the Chromium™ Single Cell 3’ Library & Gel Bead Kit v3 (10x Genomics) following the detailed protocol provided by the manufacturer. Libraries were sequenced with the NovaSeq 6000 platform (S1 Cartridge, Illumina) in 150 bp paired-end mode. The hashtag library was demultiplexed using CellRanger software (version 7.0.0), discriminating between spliced and unspliced transcripts. Aligned spliced reads were used to quantify the expression level of mouse genes and generation of gene-barcode matrix. Subsequent data analysis was performed using Seurat R package (4.0.4) and Scanpy (1.8.2). Quality control was performed, and viable cells were selected by excluding cells with features lower than 200 and above 4000, as well as cells having more than 5% of mitochondrial transcripts. Dermal and epidermal derived cells were demultiplexed with the HTODemux function integrated in Seurat with standard settings and dermal derived cells were included in the analysis all doublets were removed for the analysis. 2000 most variable genes used for the anchoring process were used for downstream analysis to calculate principal components, after log-normalization and scaling.
HTO; Barcode sequence; Hashtagged sample
Hashtag_1_(A0301);  ACCCACCAGTAAGAC; epidermis+dermis
Hashtag_2_(A0302);  GGTCGAGAGCATTCA; subcutis

first: Tamar

last: Shaanan

first: Lihui

last: Duan

first: Jason

last: Cyster


title: The reverse transcriptase inhibitor 3TC modulates hippocampal transcriptome signatures of inflammation in tauopathy model mice

city: Fort Collins

first: Devin

last: Wahl

state: Colorado

address: 1582 Campus Delivery, Moby Reception

institute: Colorado State University

summary: Reducing neuroinflammation, a main hallmark of brain aging and neurodegenerative diseases, is a promising strategy for improving cognitive function in these settings. The nucleoside reverse transcriptase inhibitor 3TC (Lamivudine) has been reported to improve cognitive function in old wild-type mice and in multiple mouse models of neurodegenerative disease, but the underlying mechanisms have not been comprehensively investigated. In the current study, we used transcriptomics to broadly characterize the effects of long-term supplementation with a human-equivalent therapeutic dose of 3TC on the hippocampal transcriptome in male and female rTg4510 mice (a commonly studied model of tauopathy-associated neurodegeneration). We found that tauopathy increased hippocampal transcriptomic signatures of neuroinflammation/immune activation, but 3TC treatment reversed many of these transcriptome changes. We also found that 3TC mitigated tauopathy-associated activation of several key transcription factors that contribute to neuroinflammation and immune activation, and several of these changes were correlated with improved recognition memory performance.Our findings suggest that 3TC exerts beneficial effects on the hippocampus in the context of tauopathy by modulating pathways associated with inflammation and immune response.

overall_design: PolyA RNA-seq on hippocampus isolated from rTg4510 mice, rTg4510-3TC treated mice, and LM controls.

first: Thomas

last: LaRocca


title: Hepatic BMAL1 and HIF1a regulate a time-dependent hypoxic response and their absence leads to Hepatopulmonary Syndrome II

city: Rehovot

first: Vaishnavi

last: Dandavate

address: 26B Arthur Ruppin street

department: Biomolecular Sciences

country: Israel

institute: Weizmann Institute of Science

summary: The transcriptional response to hypoxia is temporally regulated yet the molecular underpinnings and physiological implications are unknown. We examined herein the roles of hepatic Bmal1 and Hif1a in the circadian response to hypoxia in mice. We found that the majority of the transcriptional response to hypoxia is dependent on Bmal1 or Hif1a, through shared and distinct roles that are daytime determined. We further show that HIF1a accumulation upon hypoxia is temporally regulated and Bmal1-dependent. Unexpectedly, mice lacking both hepatic Bmal1 and Hif1a are hypoxemic and exhibit increased mortality upon hypoxic exposure in a daytime-dependent manner. These mice display mild liver dysfunction with pulmonary vasodilation likely due to ERK activation, endothelial nitric oxide synthase and nitric oxide accumulation in lungs, suggestive of hepatopulmonary syndrome. Our findings indicate that hepatic BMAL1 and HIF1a are key time-dependent regulators of the response to hypoxia and provide molecular insight on the pathophysiology of hepatopulmonary syndrome.

overall_design: RNA-sequencing on livers of AlbCRE, HLKO or BLKO mice exposed to 4 hour normoxia (21% oxygen) or hypoxia (6% hypoxia) at CT4-8 or CT16-20. Lights were turned off at the transition of dark to light regiment on the day of the experiment, following regular LD regimen.

first: Marina

last: Golik

first: Gad

last: Asher


title: FADS1/2 control lipid metabolism and ferroptosis susceptibility in triple-negative breast cancer

city: Barcelona

first: Alexandra

last: Avgustinova

address: C/ Baldiri Reixac 10

country: Spain

institute: IRB Barcelona

summary:
Analysis of factors upregulated in highly aggressive 4T1 tumour cells compared to the less aggressive 4T07 tumour cells and the non-metastatic 67NR tumour cells at gene expression level. The question addressed in the present study was which metabolic pathways are differentially activated  by these cells and therefore could account for the observed difference metastatic potential of these tumours. Results provide important characterisation of the ex vivo expression profiles of highly aggressive vs. non-aggressive tumour cells.
4T07 and 4T1 tumour cell data was previously published in GSE50471.

overall_design: 67NR, 4T07 or 4T1 cells were inoculated into the mammary fat pad of Ub-GFP mice and total RNA from FACSorted GFP-negative; CD45-negative tumour cells was isolated.

first: A

first: C

middle: M

last: Isacke


title: Single-cell genomics & regulatory networks for 388 human brains

city: Irvine

first: Jing

last: Zhang

address: University of California, Irvine

institute: University of California, Irvine

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Single-cell genomics is a powerful tool for studying heterogeneous tissues such as the brain. Yet, little is understood about how genetic variants influence cell-level gene expression. Addressing this, we uniformly processed single-nuclei, multi-omics datasets into a resource comprising >2.8M nuclei from the prefrontal cortex across 388 individuals. For 28 cell types, we assessed population-level variation in expression and chromatin across gene families and drug targets. We identified >550K cell-type-specific regulatory elements and >1.4M single-cell expression-quantitative-trait loci, which we used to build cell-type regulatory and cell-to-cell communication networks. These networks manifest cellular changes in aging and neuropsychiatric disorders. We further constructed an integrative model accurately imputing single-cell expression and simulating perturbations; the model prioritized ~250 disease-risk genes and drug targets with associated cell types.

overall_design:
Single nuclei were isolated from human postmortem dlPFC from normal control group and sequenced for transcription profile (snRNA-seq) and chromatin accessibility (snATAC-seq)
Please note that RT00389N is a duplicate of RT00374N, and therefore shares the same metadata for both sample IDs.

first: Declan

last: Clarke

first: Cagatay

last: Dursun

first: Prashant

last: Emani

first: Chirag

last: Gupta

first: Matthew

last: Jensen

first: Che

middle: Y

last: Lee

last: Liu

first: Shaoke

last: Lou

first: Ran

last: Meng

first: Jonathan

last: Warrell

first: Siwei

last: Xu

first: Yuhang

first: Zhiyuan

last: Chu

first: Timur

last: Galeev

first: Ahyeon

last: Hwang

first: Pengyu

last: Ni

first: Xiao

last: Zhou

first: Trygve

last: Bakken

first: Lucy

last: Bicks

first: Tanima

last: Chatterjee

first: Lijun

last: Cheng

first: Yuyan

first: Yi

last: Dai

first: Ziheng

last: Flaherty

first: Sophia

last: Gaynor

first: Jennifer

last: Grundman

first: Natalie

last: Hawken

first: Ella

last: Henry

first: Ao

last: Huang

first: Ting

last: Jin

first: Nikolas

last: Jorstad

first: Riki

last: Kawaguchi

first: Junhao

first: Jianyin

first: Shuang

first: Shaojie

last: Ma

first: Michael

last: Margolis

first: Samantha

last: Mazariegos

first: Jill

last: Moore

last: Moran

first: Eric

last: Nguyen

first: Nishigandha

last: Phalke

first: Henry

last: Pratt

first: Diana

last: Quintero

first: Ananya


title: FOXK2 targeting by the SCF-E3 ligase subunit FBXO24 for ubiquitin mediated degradation modulates mitochondrial respiration

city: Columbus

first: Rama

middle: K

last: Mallampalli

state: Ohio

address: 473 W12th Avenue.

department: Department of Internal Medicine

institute: The Ohio State University

summary: FOXK2 is a crucial transcription factor implicated in a wide array of biological activities and yet understanding of its molecular regulation at the level of protein turnover is limited. Here we identify that FOXK2 is sensitive to degradation by the virulent pathogens P. aeruginosa and K. pneumoniae in lung epithelia through ubiquitin-proteasomal processing. FOXK2 through its carboxyl-terminus (aa 428-478) binds the Skp-Cullin-F-box ubiquitin E3 ligase subunit FBXO24 that mediates multisite polyubiquitylation of the transcription factor resulting in its nuclear degradation. FOXK2 was detected within mitochondria and targeted depletion of the transcription factor or cellular expression of FOXK2 mutants devoid of key carboxyl-terminal domains significantly impaired mitochondrial function. In experimental bacterial pneumonia, Fbxo24 heterozygous mice exhibited preserved mitochondrial function and Foxk2 protein levels compared to wild-type littermates. The results suggest a new mode of regulatory control of mitochondrial energetics through modulation of FOXK2 cellular abundance.

overall_design: To investigate the biological function of FOXK2 in lung epithelial cells (Beas-2B), we knowdown FoxK2 using siRNA. We then performed gene expression profile analysis using data obtained from RNA-seq of 2 different siRNA specific for FoxK2  and a control cells with scrambled RNA

first: Jose

last: Ovando-Ricardez


title: Dynamic evolution of the heterochromatin sensing histone demethylase IBM1

city: Athens

first: Robert

middle: J

last: Schmitz

state: GA

address: B416 Davison Life Sciences

department: Genetics

institute: University of Georgia

type: Methylation profiling by high throughput sequencing

summary: Heterochromatin constitutes a fundamental aspect of genomes that is crucial for maintaining genome stability. In flowering plants, maintenance of heterochromatin relies on a positive feedback loop involving the histone 3 lysine nine methyltransferase (H3K9), KRYPTONITE (KYP), and the DNA methyltransferase, CHROMOMETHYLASE3 (CMT3). An H3K9 demethylase, INCREASED IN BONSAI METHYLATION 1 (IBM1), has evolved to modulate the activity of KYP-CMT3 within transcribed genes. The absence of IBM1 activity results in aberrant methylation of gene bodies, which is deleterious. This study demonstrates extensive genetic and gene expression variations in KYP, CMT3, and IBM1 within and between flowering plant species. IBM1 activity in Arabidopsis thaliana is uniquely regulated by the abundance of H3K9me2 in a repetitive sequence within an intron preceding the histone demethylase domain. This mechanism enables IBM1 to monitor global levels of H3K9me2. We discovered that the methylated intron is prevalent across flowering plants, however, its underlying sequence exhibits dynamic evolution. Its absence in species lacking gene body DNA methylation suggests its primary role in sensing H3K9me2 and preventing its integration into these constitutively expressed genes. Furthermore, our investigation uncovered Arabidopsis thaliana accessions resembling weak ibm1 mutants, several Brassicaceae species with reduced IBM1 expression, and a potential IBM1 deletion. Evolution towards reduced IBM1 activity in some flowering plants could explain the frequent natural occurrence of diminished or lost CMT3 activity, as cmt3 mutants in A. thaliana mitigate the deleterious effects of IBM1.

sample_organism:
Arabidopsis thaliana
Boechera stricta
Raphanus raphanistrum
Isatis lusitanica

overall_design:
4 MethylC-seq data of Arabidopsis Cnt-1 and its IBM1-ox transgenic plants and 2 MethylC-seq data of Isatis lustanica and Raphanus raphanistrum, and RNA-seq data of Isatis lustanica and Raphanus raphanistrum with three replicates
1 MethylC-seq data of B.stricta

first: Hosung

last: Jang


title: Differential expression in Muller glia of injured zebrafish in a mycb-/- context

first: Bioinformatics

last: Core

address: BRCF Bioinformatics Core, 2800 Plymouth Rd

institute: University of Michigan

summary: This experiment studies retinal regeneration using a zebrafish model with a focus on Muller glia reprogramming.

overall_design: Three groups: controls (Muller glia from uninjured retinas), injured WT (Muller glia from injured WT retina at 2 days post injury (dpi)), and injured mycb-/- (Muller glia from injured mycb-/- fish at 2dpi).

first: Daniel

last: Goldman


title: Mdivi-1: Effective but complex mitochondrial fission inhibitor

city: seoul

first: Jueng Soo

last: You

address: 120 Neungdong-ro

country: South Korea

institute: Konkuk University

summary: Mdivi-1, Mitochondrial DIVIsion inhibitor 1, has been widely employed in research under the assumption that it exclusively influences mitochondrial fusion, but effects other than mitochondrial dynamics have been underinvestigated. This paper provides transcriptome and DNA methylome-wide analysis for Mdivi-1 treated SH-SY5Y human neuroblastoma cells using RNA sequencing (RNA-seq) and methyl capture sequencing (MC-seq) methods. Gene ontology analysis of RNA sequences revealed that p53 transcriptional gene network and DNA replication initiation-related genes were significantly up and down-regulated, respectively, showing the correlation with the arrest cell cycle in the G1 phase. MC-seq, a powerful sequencing method for capturing DNA methylation status in CpG sites, revealed that although Mdivi-1 does not induce dramatic DNA methylation change, the subtle alterations were concentrated within the CpG island. Integrative analysis of both sequencing data disclosed that p53 transcriptional network was activated while Parkinson's disease pathway was halted. Next, we investigated several changes in mitochondria in response to Mdivi-1. Copy number and transcription of mitochondrial DNA were suppressed, while these results were not due to mitophagy. ROS and Ca2+ levels increased, and elevated ROS triggered mitochondrial retrograde signaling rather than inducing direct DNA damage. In this study, we could better understand the molecular network of Mdivi-1 by analyzing DNA methylation and mRNA transcription in the nucleus and further investigating various changes in mitochondria, providing inspiration for studying nuclear-mitochondrial communications.

overall_design: RNA-seq, MC-seq was conducted in SH-SY5Y human neuroblastoma cell line and the same cell line exposed to Mdivi-1.

first: Seor I

last: Ahn

first: Jueng Su


title: Skeletal muscle cystathionine γ-lyase deficiency promotes obesity and insulin resistance and results in hyperglycemia and skeletal muscle injury upon HFD in mice

city: Shanghai

first: Jiani

last: Lu

address: No.85 Wujin Rd.

country: China

institute: Shanghai General Hospital

summary: In the present study, we explored whether skeletal muscle cystathionine γ-lyase (CTH) contributes to high-fat diet (HFD)-induced metabolic disorders using skeletal muscle Cth knockout (CthΔskm) mice. Metabolomics coupled with transcriptome showed that CthΔskm mice displayed impaired energy metabolism and some signaling pathways linked to insulin resistance (IR) in skeletal muscle although the mice had normal insulin sensitivity. HFD led to reduced CTH expression and impaired energy metabolism in skeletal muscle in Cth-floxed mice (Cthf/f) mice. CTH deficiency and HFD had some common pathways enriched in the aspects of amino acid metabolism, carbon metabolism, and fatty acid metabolism. CthΔskm+HFD mice exhibited increased body weight gain, fasting blood glucose, plasma insulin, and IR, and reduced glucose transporter 4 and CD36 expression in skeletal muscle compared to Cthf/f+HFD mice. Impaired mitochondria and irregular arrangement in myofilament occurred in CthΔskm+HFD mice. Omics analysis showed differential pathways enriched between CthΔskm mice and Cthf/f mice upon HFD. More severity in impaired energy metabolism, reduced AMPK signaling, and increased oxidative stress and ferroptosis occurred in CthΔskm+HFD mice compared to Cthf/f+HFD mice. Our data indicate that skeletal muscle CTH expression dysregulation contributes to metabolism disorders upon HFD.

overall_design: The WT and CSE∆skm mice were fed with HFD or chow from 4-weeks old for 13 weeks. The mice were sacrificed under deep anesthesia at 17-weeks old. Gastronemius was obtained for RNA-seq analysis.

first: Zhengshan

last: Tang

first: Miaomiao

last: Xu

first: Jianqiang

first: Changnan

last: Wang

first: Bo

last: Yu

first: Xin

last: Ni


title: Granuloma annulare exhibits mixed immune and macrophage polarization profiles with spatial transcriptomics

status: Public on May 12 2024

city: Madison

first: Bridget

middle: E.

last: Shields

state: Wisconsin

address: 1 S Park Street

department: Department of Dermatology

institute: University of Wisconsin School of Medicine and Public Health

summary: Granuloma annulare (GA) is an idiopathic condition characterized by granulomatous inflammation in the skin. Prior studies have suggested that GA develops from various triggers, leading to a complex interplay involving innate and adaptive immunity, tissue remodeling, and fibrosis. Macrophages are the major immune cells comprising GA granulomas, however, the molecular drivers and inflammatory signaling cascade behind macrophage activation is poorly understood. Histologically, GA exhibits both palisaded and interstitial patterns on histology, however the molecular composition of GA at the spatial level remains unexplored. GA is a condition without FDA-approved therapies despite the significant impact of GA on quality of life. Spatial transcriptomics is a valuable tool for profiling localized, genome-wide gene expression changes across tissue with emerging applications in clinical medicine. To improve our understanding of the spatially localized gene expression patterns underlying GA, we profiled the spatial gene expression landscape from six patients with GA. Our findings revealed mixed Th1 and Th2 signals comprising the GA microenvironment and spatially distinct M1 and M2 macrophage polarization characteristics. IFN- and TNF signals emerged as important regulators of GA granulomatous inflammation and interleukin-32 emerged as a key driver of granulomatous inflammation. Overall, our spatial transcriptomics data indicate that GA exhibits mixed immune and macrophage polarization.

overall_design: We performed spatial transcriptomics on lesional andanatomically-matchedskin of6GApatientswith classic clinical and histologic presentations.

first: Chitrasen

last: Mohanty

first: Chandra

last: Singh

first: Joseph

last: Daccache

first: William

last: Damsky

first: Christina

last: Kendziorski

first: Di

last: Yan

first: Aman

last: Prasad

first: Donglin

last: Zhang

first: Tom

last: Keenan

first: Beth

last: Drolet

first: Nihal

last: Ahmad

middle: E


title: Systems modeling of oncogenic G-protein and oncogenic GPCR signaling reveals unexpected differences in downstream signaling pathway activation [FAKi_experiment]

first: Kathryn

last: Lande

overall_design: To experimentally explore the effect of FAK inhibition over time, cells were treated with either FAKi or DMSO, and collected for RNA sequencing at 0, 3, 7, 14, and 21 days.

first: Michael

last: Trogdon

first: Edward

last: Stites


title: Hi-C in neural stem cells before and after Phf2 KO

city: Singapore

first: Yingying

last: Zeng

state: --- Select a state ---

address: 61 Biopolis Drive

country: Singapore

institute: Institute of Molecular and Cell Biology

summary: To model and characterize the chromatin regulatory landscape in Neural stem cell before and after Phf2 knockout, we performed HiC to map large-scale 3D architectural rewiring in WT and Phf2-KO NSC.

overall_design: Hi-C in WT and Phf2-KO NSC.

first: N

middle: O

last: Cipta

first: Y

last: Loh


title: Pericytes and perivascular fibroblasts contribute to central nervous system fibrosis in a region dependent manner(DH SmartSeq)

city: Solna

first: Christian

last: Göritz

address: Solnavägen 9

department: CMB

country: Sweden

institute: Karolinska Institutet

summary: Fibrotic scar tissue formation is conserved throughout the central nervous system in humans and mice, and impairs tissue regeneration and functional recovery. However, the origin of scar-forming stromal fibroblasts is controversial. Here, we show that stromal fibroblasts found after spinal cord injury derive from two populations of perivascular cells that are anatomically and transcriptionally defined as pericytes and perivascular fibroblasts. We identify two distinct perivascular cell populations, which activate and transcriptionally converge on the generation of stromal myofibroblasts after injury. Our results suggest potential targets to improve tissue regeneration and functional recovery after central nervous system injuries.

overall_design: We crossed GLAST-CreERT2;R26R-tdTomato mice to the Pdgfrb-eGFP reporter line, which drives EGFP expression in all PDGFRb+ perivascular cells (pericytes, vSMCs and fibroblasts). We isolated GLAST-expressing perivascular cells from the uninjured spinal cord and 5 days after spinal cord injury by fluorescence activated cell sorting (FACS) based on tdTomato and EGFP expression. To define the molecular identity and compare GLAST-expressing perivascular cells (tdTomato+EGFP+) to other perivascular cells, we also sorted dTomato-EGFP+ cells. Sorted cells were subjected single cell sequencing using the SmartSeq 3 protocol.

first: Holl

last: Daniel

first: Julien

last: Anaïs

first: Göritz

last: Christian


title: Pericytes and perivascular fibroblasts contribute to central nervous system fibrosis in a region dependent manner (DH express)


title: Dermal TRPV1 innervations engage a macrophage and fibroblast containing pathway to activate hair growth [dataset1]

first: Konrad

last: Knopper

overall_design:
Back skin from TRPV1hM3Dq expressing and control mice (two mice per group) were collected 3.5 hours after a single intradermal CNO injection. The subcutis was removed mechanically and the dermal-epidermal fractions were then digested. Then, control and TRPV1 activated samples were stained using viability dyw and CD45 antibody and sorted. Treatment groups (two) were run on separate lanes of a 10X Chromium chip with 3′ v.2 chemistry (10X Genomics) as per the manufacturer’s instructions by the UCSF Institute for Human Genetics Sequencing Core. Transcripts captured in all the cells encapsulated with a bead were uniquely barcoded using a combination of a 16 bp 10x Barcode and a 10 bp unique molecular identifier (UMI). cDNA libraries were generated using the Chromium™ Single Cell 3’ Library & Gel Bead Kit v3 (10x Genomics) following the detailed protocol provided by the manufacturer. Libraries were sequenced with the NovaSeq 6000 platform (S1 Cartridge, Illumina) in 150 bp paired-end mode. The hashtag library was demultiplexed using CellRanger software (version 7.0.0), discriminating between spliced and unspliced transcripts. Aligned spliced reads were used to quantify the expression level of mouse genes and generation of gene-barcode matrix. Subsequent data analysis was performed using Seurat R package (4.0.4) and Scanpy (1.8.2). Quality control was performed, and viable cells were selected by excluding cells with features lower than 200 and above 4000, as well as cells having more than 5% of mitochondrial transcripts. Dermal and epidermal derived cells were demultiplexed with the HTODemux function integrated in Seurat with standard settings and dermal derived cells were included in the analysis all doublets were removed for the analysis. 2000 most variable genes used for the anchoring process were used for downstream analysis to calculate principal components, after log-normalization and scaling.
HTO; Barcode sequence; Hashtagged sample
Hashtag_1_(B0301);  ACCCACCAGTAAGAC; mouse1
Hashtag_2_(B0302);  GGTCGAGAGCATTCA; mouse2
Hashtag_3_(B0303);  CTTGCCGCATGTCAT; mouse3
Hashtag_3_(B0303);  CTTGCCGCATGTCAT; mouse4

first: Tamar

last: Shaanan

first: Lihui

last: Duan

first: Jason

last: Cyster


title: Proteome-scale tagging and functional screening in mammalian cells by ORFtag [PRO-Seq]

first: Alexander

last: Stark

summary: Determining protein function in a systematic and comprehensive manner is a key goal in modern molecular biology research but is challenging with current approaches. Here, we introduce ORFtag, a versatile method for the massively parallel protein tagging and interrogation of protein function at the proteome scale with minimal effort and expense. We demonstrate the versatility of ORFtag in mouse and human cells with a series of functional screens for transcriptional activators, repressors and post-transcriptional gene regulatory proteins.

overall_design: PRO-Seq for Zfp574-depleted (AID) mESC

first: Filip

last: Nemčko

first: Moritz

last: Himmelsbach

first: Vincent

last: Loubiere

first: Ramesh

last: Yelagandula

first: Michaela

last: Pagani

first: Nina

last: Fasching

first: Julius

last: Brennecke

first: Ulrich

last: Elling

first: Stefan

last: Ameres


title: Cyclic stretching stimuli promotes HGF and MMP secretion from hESC-derived hepatoblasts and endothelial cells towards liver lobule formation in vitro.

city: Kyoto

first: Koki

last: Yoshimoto

state: Kyoto

department: iCeMS

country: Japan

institute: Kyoto university

summary: We induced hepatic and endothelial cells simultaneously for day9. We applied cyclic mechancial stimuli (cMS) from day9 to day12.  We then performed gene expression profiling analysis using data obtained from RNA-seq of 3 different cells at two time points.

overall_design: 15 samples

first: Taiji

last: Adachi

first: Ken-ichiro

last: Kamei


title: Proteome-scale tagging and functional screening in mammalian cells by ORFtag [frame_specific_screen]

summary: The systematic determination of protein function is a key goal of modern biology, but remains challenging with current approaches. Here, we present ORFtag, a versatile, cost-effective and highly efficient method for the massively parallel tagging and functional interrogation of proteins at proteome scale. ORFtag utilizes retroviral vectors bearing a promoter, peptide tag and splice donor to generate fusions between the tag and endogenous ORFs. We demonstrate the utility of ORFtag through functional screens for transcriptional activators, repressors and post-transcriptional regulators in mouse embryonic stem cells. Each screen finds known and novel regulators, including long ORFs inaccessible by other methods, revealing that Zfp574 is a highly selective transcriptional activator and that oncogenic fusions often function as transactivators.

overall_design: Activator screen was performed with frame +1, frame+2 and frame+3 ORFtag cassettes separately


title: Pericytes and perivascular fibroblasts contribute to central nervous system fibrosis in a region dependent manner

first: Christian

last: Göritz

overall_design: We crossed GLAST-CreERT2;R26R-tdTomato mice to the Pdgfrb-eGFP reporter line, which drives EGFP expression in all PDGFRb+ perivascular cells (pericytes, vSMCs and fibroblasts). We isolated GLAST-expressing perivascular cells from the uninjured spinal cord and 5 days after spinal cord injury by fluorescence activated cell sorting (FACS) based on tdTomato and EGFP expression. To define the molecular identity and compare GLAST-expressing perivascular cells (tdTomato+EGFP+) to other perivascular cells, we also sorted dTomato-EGFP+ cells. Sorted cells were subjected single cell sequencing using the SmartSeq 2 protocol.

first: Daniel

last: Holl


title: Differential expression in Muller glia of injured zebrafish in a mych-/- context

city: Ann Arbor

first: Bioinformatics

last: Core

summary: This experiment studies  retinal regeneration using a zebrafish model with a focus on Muller glia reprogramming.

overall_design: Three groups: controls (Muller glia from uninjured retinas), injured WT (Muller glia from injured WT retina at 2 days post injury (dpi)), and injured mych-/- (Muller glia from injured mych-/- fish at 2dpi). Of interest are the WT injury responsive genes, and which injury-responsive genes in the WT are differentially expressed in the mych-/- fish.

last: Goldman


title: Differential expression in Zebrafish retinal muller glia under injured and knockout

status: Public on May 09 2024

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

overall_design: Refer to individual Series


title: Identification of differentially expressed tumour-related genes regulated by UHRF1 regulated  DNA methylation [WGBS]

city: Beijing

first: Gong

middle: chun

last: xue

address: 20 East Street, Fengtai District

institute: Beijing Institute of Biotechnology

summary: UHRF1 is an epigenetic regulator that plays critical roles in tumours. However, the DNA methylation alteration patterns driven by UHRF1 and the related differentially expressed tumour-related genes remain unclear. In this study, a UHRF1-shRNA MCF-7 cell line was constructed, and whole-genome bisulfite sequencing (WGBS) and RNA sequencing were performed. The DNA methylation alteration landscape was elucidated, and DNA methylation-altered regions (DMRs) were found to be distributed in both gene bodies and adjacent regions. The DMRs were annotated and categorized into 488 hypermethylated/1696 hypomethylated promoters and 1149 hypermethylated/5501 hypomethylated gene bodies. Through an integrated analysis with the RNA sequencing data, 217 upregulated methylated genes and 288 downregulated methylated genes were identified, and these genes were primarily enriched in nervous system development and cancer signalling pathways.

overall_design: UHRF1-lentivirusVS scramble-lentivirus


title: Identification of differentially expressed tumour-related genes regulated by UHRF1 regulated  DNA methylation


title: Identification of differentially expressed tumour-related genes regulated by UHRF1 regulated  DNA methylation [RNA-Seq]

overall_design: UHRF1-shRNA MCF7 VS Scramble-shRNA MCF7


title: Relaxin Attenuates the Genomic Actions and Biological Effects of Estrogens in the Myometrium by Reducing Estrogen Receptor Alpha Phosphorylation

city: Dallas

first: W. Lee

last: Kraus

state: TX

address: 5323 Harry Hines Blvd.

institute: UT Southwestern Medical Center


title: The oncolytic adenovirus Delta-24-RGD in combination with ONC201 displays a potent antitumor effect in pediatric high-grade glioma models [RNA-seqI]

city: Pamplona

first: Marta

middle: Maria

last: Alonso Roldán

state: Navarra

address: CIMA building , Pio XII 55

department: Dpt of Pediatrics

institute: University Hospital of Navarra

summary: Background: Pediatric high-grade gliomas (pHGGs), including diffuse midline gliomas (DMGs) are aggressive pediatric tumors harboring one of the poorest prognoses. Previous preclinical and clinical studies have demonstrated that oncolytic adenovirus Delta-24-RGD and the imipridone ONC201 are two promising therapies for these type of tumors as single agents. However, the combination of both agents has not been previously evaluated. Methods: Potential negative effects of ONC201 on the production of functional viruses were assessed by immunoblotting and replication assays. Antitumor effect was evaluated in a panel of human and murine pHGG and DMG cell lines. RNAseq, mTORC1 pathway analysis, Seahorse Stress Test, mitochondrial DNA content, and pH2A.X immunofluorescence were used to perform mechanistical studies. Models of both diseases were used to assess the efficacy of the combination in vivo. Tumor immune microenvironment was evaluated using flow cytometry. Results: ONC201 cotreatment with Delta-24-RGD did not affect the virus replication capability in human and murine pHGG and DMG models in vitro. Cytotoxicity analysis showed that cotreatment was either synergistic or additive. Mechanistically, combination treatment produces an increase of nuclear DNA damage and maintains the metabolic perturbation and mitochondrial damage caused by each agent alone. Delta-24-RGD/ONC201 cotreatment extends the overall survival of mice implanted with human and murine pHGG and DMG cells, independently of H3 mutational status. Finally, combination treatment in murine DMG models revealed a proinflammatory phenotype reshaping of the tumor microenvironment. Conclusions: Delta-24-RGD/ONC201 combination improves the efficacy of each agent alone in vitro and in vivo models, maintaining transcriptional and phenotypical changes, producing exacerbated nuclear DNA damage, and leading to an augmented antitumor immune response.

overall_design: Human pHGG cells (CHLA-03-AA, SJ-GBM2) and DMG cells (TP54, SU-DIPG IV) are cell lines that represent the tumor biology of pHGG and DMG diseases. Each cell line was treated in vitro with vehicle (letter “C” is included in the name in this case), ONC201 and Delta-24-RGD. At least three biological replicates are included, and it is indicated as “_number of replicate” at the end of the name of the sample.

last: Alonso


title: The oncolytic adenovirus Delta-24-RGD in combination with ONC201 induces a potent antitumor response in pediatric high-grade and diffuse midline glioma models [RNA-seqII]

summary: Background: Pediatric high-grade gliomas (pHGGs), including diffuse midline gliomas (DMGs), are aggressive pediatric tumors with one of the poorest prognoses. Delta-24-RGD and ONC201 have shown promising efficacy as single agents for these tumors. However, the combination of both agents has not been evaluated. Methods: The production of functional viruses was assessed by immunoblotting and replication assays. The antitumor effect was evaluated in a panel of human and murine pHGG and DMG cell lines. RNAseq, the seahorse stress test, mitochondrial DNA content, and γH2A.X immunofluorescence were used to perform mechanistic studies. Mouse models of both diseases were used to assess the efficacy of the combination in vivo. The tumor immune microenvironment was evaluated using flow cytometry, RNAseq and multiplexed immunofluorescence staining. Results: The Delta-24-RGD/ONC201 combination did not affect the virus replication capability in human pHGG and DMG models in vitro. Cytotoxicity analysis showed that the combination treatment was either synergistic or additive. Mechanistically, the combination treatment increased nuclear DNA damage and maintained the metabolic perturbation and mitochondrial damage caused by each agent alone. Delta-24-RGD/ONC201 cotreatment extended the overall survival of mice implanted with human and murine pHGG and DMG cells, independent of H3 mutation status and location. Finally, combination treatment in murine DMG models revealed a reshaping of the tumor microenvironment to a proinflammatory phenotype. Conclusions: The Delta-24-RGD/ONC201 combination improved the efficacy compared to each agent alone in in vitro and in vivo models by potentiating nuclear DNA damage and in turn improving the antitumor (immune) response to each agent alone.

overall_design: XFM-bearing mice were treated with vehicle, Delta-24-RGD, ONC201 and Delta-24-RGD+ONC201 at 10^7 pfu for the viruses group and 125 mg/kg for ONC201 groups. Then, mice were sacrificed and tumor tissue was recovered. RNA extraction for RNAseq was performed.

last: de la Nava


title: The Histone Methyltransferase SUV420H2 Regulates Brown and Beige Adipocyte Thermogenesis

city: Atlanta

first: Bingzhong

last: Xue

address: 50 Decatur St. SE

department: Biology

institute: Georgia State University

summary: Activation of brown adipose tissue (BAT) thermogenesis increases energy expenditure and alleviates obesity. Epigenetic regulation has emerged as a key mechanism underlying BAT development and function. To study the epigenetic regulation of BAT thermogenesis, we surveyed the expression of epigenetic enzymes that catalyze histone modifications in developmental beige adipocytes and found a unique expression pattern of suppressor of variegation 4-20 homolog 2 (Drosophila) (Suv420h2), a histone methyltransferase that preferentially catalyzes the tri-methylation at histone H4 lysine 20 (H4K20me3), a hallmark of gene silencing. Here we discovered that Suv420h2 expression parallels that of UCP1 expression in brown and beige adipocytes and that SUV420H2 knockdown significantly reduces, whereas SUV420H2 overexpression significantly increases UCP1 levels in brown adipocytes. Suv420h2 knockout (H2KO mice exhibit impaired cold-induced thermogenesis and are prone to diet-induced obesity. In contrast, mice with specific overexpression of Suv420h2 in adipocytes display enhanced cold-induced thermogenesis and are resistant to diet-induced obesity. Further study showed that Suv420h2 catalyzes H4K20 trimethylation at eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) promoter, leading to down-regulated expression of 4E-BP1, a negative regulator of the translation initiation complex. This in turn up-regulates PGC1α protein levels, which is associated with increased expression of thermogenic program. We conclude that Suv420h2 is a key regulator of brown/beige adipocyte development and thermogenesis.

overall_design: Subcutaneous white fat mRNA profiles of 12-week-old WT and H2KO as well as WT and AH2Tg mice after 7-day cold exposure

first: Hang

last: Shi


title: Structural and functional investigation of the DHH/DHHA1 family proteins in Deinococcus radiodurans

city: Hangzhou

first: Kaiying

last: Cheng

state: Zhejiang

address: No.2318, Yuhangtang Rd

department: Department of Immunology and Pathogen Biology

institute: Hangzhou Normal University

summary: To investigate the in vivo functions of RecJ and RecJ-like, we conducted transcriptome analyses on the wild-type (WT), drrecJ deletion mutant (△J), drrecJ-like deletion mutant (△J-like), and drrecJ-like/recJ double deletion mutant (△J/△J-like) strains. The results of the transcriptome analysis reveal a nuanced interplay between these two proteins in vivo, encompassing both overlapping and antagonistic functions. Interestingly, DrRecJ is not only involved in DNA repair but also associated with cell growth and division. On the other hand, DrRecJ-like not only influences stress response and cell growth and division, but is also linked to the folding/stability of intracellular proteins, as well as the formation and stability of cell membranes/walls.

overall_design: RNAseq of D. radiodurans R1 (WT), drrecJ deletion mutant (△J), drrecJ-like deletion mutant (△J-like), and drrecJ-like/recJ double deletion mutant (△J/△J-like) strains at normal condition


title: RNA sequencing comparison of B7-H3.TMIGD2 and B7-H3.CD28.4-1BB CAR-T cells

city: Bronx

first: Christopher

last: Nishimura

state: NY

address: 1300 Morris Park Avenue

institute: Albert Einstein College of Medicine


title: Relaxin Attenuates the Genomic Actions and Biological Effects of Estrogens in the Myometrium by Reducing Estrogen Receptor Alpha Phosphorylation [RNA-seq]

summary: In this study we examine the extent and nature of functional interactions between the E2 and Rln signaling pathways in the myometrium.  In addition, we delineate the mechanisms underlying functional interplay between the E2/ERα and Rln/RXFP1 signaling pathways at the molecular and physiological levels in myometrial tissue and cells during quiescence and contractility.

overall_design: RNA-seq was performed on ovariectomized mouse myometrium treated with either saline, E2, Relaxin (Rln) or E2+Rln.

first: Sudeshna

last: Tripathy

first: Anusha

last: Nagari

first: Mala

last: Mahendroo

first: W


title: Differential regulation of immune-related genes in the developing heart.

status: Public on May 08 2024

city: St. Louis

first: Mathieu

last: Garand

state: MO

address: One Childrens Place Suite 6120

department: Surgery

institute: Washington University in St. Louis

summary: In many congenital heart defects, it can be difficult to ascertain primary pathology from secondary consequences from altered flow through the developing heart. The molecular differences between the growing right and left ventricles (RV and LV, respectively) following the completion of septation and the impact of sex on these mechanisms have not been investigated. We analyzed RNA-seq data derived from twelve RV and LVs, one with Hypoplastic Left Heart Syndrome (HLHS), to compare the transcriptomic landscape between the ventricles during development. Differential gene expression analysis revealed a large proportion of genes unique to either the RV or LV as well as sex bias. Our GO enrichment and network analysis strategy highlighted the differential role of immune functions between the RV and LV in the developing heart. Comparatively, RNA-seq analysis of data from C57Bl6/J mice hearts collected at E14 resulted in the enrichment of similar processes related to T cells and leukocyte migration and activation. Differential gene expression analysis of an HLHS case highlighted significant downregulation of chromatin organization pathways and upregulation of genes involved in muscle organ development. This analysis also identified previously unreported upregulation of genes involved in IL-17 production pathways. In conclusion, differences exist between the gene expression profiles of RV versus LV with the expression of immune-related genes significantly different between these two chambers. The pathogenesis of HLHS may involve alterations in the expression of chromatin and muscle gene organization as well as upregulation of the IL-17 response pathway.

overall_design: We performed mRNA-seq analysis to gain insight into ventricle-specific gene expression changes during gestation. Our study included 11 unaffected hearts and one heart with HLHS.

first: Pirooz

last: Eghtesady


title: Comparative physiological and transcriptomic profiling reveals the characteristics of tissue　tolerance mechanisms in Japanese rice landrace under salt stress

city: Higashi-hiroshima

first: Anisa

middle: Nazera

last: Fauzia

state: Hiroshima

address: 1-4-4 Kagamiyama

department: Graduate School of Integrated Sciences for Life

institute: Hiroshima University

summary: The aim of this study was to characterize the tissue tolerance mechanisms of rice under salt stress. Our preliminary experiment identified a japonica rice landrace Shuzenji-kokumai (SZK), which is considered to be tissue-tolerant because it can maintain better growth than salt-sensitive rice while having a high Na+ concentration in the shoots under salt stress. These mechanisms differ from those of most salt-tolerant rice varieties, which have low Na+ concentrations in the shoots. We compared the physiological and molecular characteristics of SZK with those of FL478, a salt-tolerant variety, and Kunishi, a salt-sensitive variety. Under salt stress conditions, SZK accumulated high levels of Na+ in roots, leaf sheaths, and leaf blades, which were almost as high as those in the salt-sensitive Kunishi. Simultaneously, SZK maintained better growth and physiological status, as determined by its higher dry weight, lower electrolyte leakage ratio, and lower malondialdehyde concentration. OsNHX1 and OsNHX2 were up-regulated in the leaf sheaths of SZK, suggesting that Na+ is compartmentalized in the vacuole to avoid Na+ toxicity. In contrast, FL478 showed up-regulation of OsHKT1;5 and OsSOS1 in the roots, which exclude Na+ from the shoots. RNA-seq analysis showed that 4623 and 1998 differentially expressed genes (DEGs) were detected in the leaf sheaths and leaf blades of SZK, respectively. Among them, the HSP (heat shock protein) gene expression was highly up-regulated only in SZK, indicating that SZK protects against the protein damage caused by Na+ toxicity. Our findings suggest that SZK has atypical survival mechanisms under salt-stress conditions. These mechanisms offer potential traits for improving salt tolerance in rice.

overall_design:
We compared the physiological and molecular characteristics of SZK with those of FL478, a salt-tolerant variety, and Kunishi, a salt-sensitive variety. Under salt stress conditions, SZK accumulated high levels of Na+ yet maintained better growth and physiological status, indicating a tissue tolerance ability.
Genome-wide transcriptome analysis was performed by using RNA-seq to ascertain the molecular changes associated with tissue tolerance in SZK

first: Fauzia

last: Anisa Nazera

first: Nampei

last: Mami

first: Jiadkong

last: Kamonthip

first: Shinta

last: Shinta

first: Ueda

last: Akihiro


title: Overexpressed Malat1 drives metastasis through inflammatory reprogramming of lung adenocarcinoma microenvironment

city: LOS ANGELES

first: Jesse

middle: R

last: Zamudio

address: 610 CHARLES E. YOUNG DRIVE EAST BOX 957239

department: MCDB

institute: University of California, Los Angeles

summary: Although the expression of some long noncoding RNAs (lncRNAs), including MALAT1 (metastasis-associated lung adenocarcinoma transcript 1), is predictive of metastasis, their impact and mechanism of action remain elusive. Here we use CRISPR activation (CRISPRa) to model MALAT1/Malat1 overexpression in patient-derived lung adenocarcinoma (LUAD) cell lines and in the autochthonous K-ras/p53 LUAD mouse model. The results indicate that Malat1 overexpression alone is sufficient to enable the progression of LUAD to metastatic disease. We show that overexpressed MALAT1/Malat1 enhances cell mobility and promotes the recruitment of pro-tumor macrophages to the tumor microenvironment through paracrine secretion of the CCL2/Ccl2 cytokine. We determine that Ccl2 upregulation results from an increase in global chromatin accessibility upon Malat1 overexpression. Importantly, macrophage depletion and Ccl2 blockade counteracted the effects of Malat1 overexpression. These data demonstrate that a single lncRNA can drive LUAD metastasis through reprogramming of the tumor microenvironment.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for isolated tumor cells with or without targeted Malat1 overexpression in KP mouse model of lung adenocarcinoma.

first: Nadya

last: Dimitrova


title: T cell mediated microglia activation triggers myelin pathology in a model of amyloidosis

city: Munich

first: Ruoqing

last: Feng

address: Feodor-Lynen-Strasse 17

country: Germany

institute: German Center for Neurodegenerative Diseases (DZNE)

summary: Age-related myelin damage induces inflammatory responses, yet its involvement in Alzheimer’s disease (AD) remains uncertain, despite age being a major risk factor. Using a mouse model of AD, we found that amyloidosis itself triggers age-related oligodendrocyte and myelin damage. Mechanistically, CD8+ T cells promote the progressive accumulation of abnormally interferon-activated microglia that display myelin-damaging activity. Thus, immune responses against myelinating oligodendrocytes may contribute to neurodegenerative diseases with amyloidosis.

overall_design: To determine whether CD8+ T cells contribute to oligodendrocyte and myelin pathology in 5xFAD mice, we treated 6-months old 5xFAD mice with antibodies against CD8. For the depletion of CD8+ T cells mice, 5xFAD mice aged 6 months were intraperitoneally injected with anti-CD8 antibody (BioXCell, BP0061) and their respective isotype controls (BioXCell, BE0119) twice a week for a total of 6 weeks.For 10X genomic experiments, mice were deeply anesthetized and perfused with cold PBS. Each brain was removed and individually microdissected under a dissection microscope, wherein the cortex and hippocampus were isolated from the brain and the attached choroid plexus was removed.


title: Proteome-scale tagging and functional screening in mammalian cells by ORFtag [Cut & Run]

city: Vienna

first: Alexander

last: Stark

address: Campus-Vienna-Biocenter 1

institute: The Research Institute of Molecular Pathology (IMP)

summary: The comprehensive assessment of protein function is a key aim in modern biology yet has proven difficult with existing approaches. Here, we introduce ORFtag, a versatile tool for the massively parallel protein tagging and functional screening at proteome scale. ORFtag integrates a cassette bearing a promoter, the coding sequence of a protein tag, and a splice-donor site, which allows the tag to be expressed and spliced to genomic protein-coding exons and thus enables endogenous protein tagging at minimal expenditure and effort. We demonstrate the versatility of ORFtag by three functional screens for transcriptional activators and repressors and mediators of mRNA translation and stability.

overall_design: anti-V5 CUT&RUN experiments in Zfp574-AID-V5 mESC cell line

first: Filip

last: Nemčko

first: Moritz

last: Himmelsbach

first: Vincent

last: Loubiere

first: Ramesh

last: Yelagandula

first: Michaela

last: Pagani

first: Nina

last: Fasching

first: Julius

last: Brennecke

first: Ulrich

last: Elling

first: Stefan

last: Ameres


title: Meningeal lymphatic CGRP signaling governs pain via cerebrospinal fluid efflux and neuroinflammation in migraine models

city: Chapel Hill

first: Nathan

last: Nelson-Maney

state: North Carolina

address: 111 mason farm road

department: Cell Biology and Physiology

institute: UNC Chapel Hill

summary:
Migraine is primarily mediated via CGRP signaling on various tissues to induce pain and other migraine symptoms. We characterize the role of migraine related CGRP signaling on meningeal lymphatic vessels from mouse meningeal lymphatic tissue.
In this dataset, we include the expression data obtained from lyve-1 positive meningeal lymphatic endothelial cells from Lyve-1 cre mediated HA-tagged ribosomes from mice with the NTG mediated model of migraine. These data are used to obtain 700 genes that are differentially expressed in meningeal lymphatic tissue in response to NTG induced migraine.

overall_design: 6 Total samples were analyzed. We generated the following pairwise comparisons using PowerExpress: NTG vs Vehicle

first: N

first: L

last: Balint

first: A

last: Beeson

first: D

last: Serafin

first: B

last: Kistner

first: E

last: Douglas

last: Siddiqui

last: Tauro

first: K

last: Caron


title: Effect of mitochondrial electron transport chain blockade on transcriptomic profile in human H460 cells

first: Ralph

last: DeBerardinis

address: 6000 Harryhines Blvd

department: CRI

summary: Cancer cells reprogram their metabolism to support cell growth and proliferation in harsh environments. To assess how human cells respond to defective mitochondrial respiration, we analyzed metabolomics profiles in cells with deficient electron transport chain (ETC). We found that ETC deficiency induces an accumulation of purine nucleotides. Additionally, we revealed that ETC blockade suppressed de novo purine nucleotide biosynthesis while enhanced purine salvage. This metabolic rewiring of purine nucleotide biosynthesis is not regulated at the transcriptional level. Instead, stable isotope tracing experiments showed that ETC blockade promoted the oxidative branch of pentose phosphate pathway to elevate phosphoribosyl diphosphate to drive purine salvage. In summary, our findings delineate how cells remodel purine metabolism in response to ETC blockade, and uncover a new metabolic vulnerability in tumors with low respiration.

overall_design: To assess how human non-small cell lung cancer cells respond to impaired mitochondrial respiration, we challenged human H460 cells with IACS-010759, an ETC complex I inhibitor to block oxidative phosphorylation for 24 hours. To exclude the potential side effects from the inhibitor, we expressed NADH-quinone oxidoreductase (NDI1) to restore oxidative phosphorylation in the presence of IACS-010759. For each sample group, we prepared three replicates for RNA-seq analysis.

first: Zheng

last: Wu

first: Divya

last: Bezwada

first: Yuannyu

last: Zhang

first: Min

last: Ni


title: Male autism spectrum disorder is linked to brain aromatase disruption by prenatal BPA in multimodal investigations and 10HDA ameliorates the related mouse phenotype (BPA)

city: Melbourne

first: Wah Chin

last: Boon

state: Victoria

address: 30 Royal Parade Parkville

country: Australia

institute: The Florey Institute of Neuroscience and Mental Health

summary: Pregannt Cyp19-EGFP mice (maintained on a soybean free mouse chow diet) were treated with either 50μg/kg/day Bsiphenol A or vehicle (ethinol in peanut oil) between gestational days (GD) 10.5-14.5. On GD15.5, fetuses were dissected and sex determined by SRY genotyping. Fetuses were obtained from different dams.

overall_design: To investigate the effect of prenatal BPA expsoure on offspring RNA expression, Cyp19-EGFP mice dams were treated with either 50μg/kg BPA or vehicle (peanut oil) at GD10.5-GD14.5. The cortex of the brain was rapidly dissected from the fetuses at GD15.5.


title: Increased Endothelial Sclerostin Caused by Elevated DSCAM Mediates Multiple Trisomy 21 (Down Syndrome) Phenotypes

city: Boston

first: Vi

middle: T

last: Tang

state: MA

address: 77 Avenue Louis Pasteur

institute: Harvard Medical School

summary: Trisomy 21 (T21), a recurrent aneuploidy occurring in 1:800 births, predisposes to congenital heart disease (CHD) and multiple extra-cardiac phenotypes. Despite a definitive genetic etiology, the mechanisms by which T21 perturbs development and maintenance remains poorly understood. We compared the transcriptome of CHD tissues from 49 T21 and 226 euploid (eCHD) patients. We resolved cell lineages that mis-expressed T21 transcripts by cardiac single-nucleus RNA-sequencing and RNA in situ hybridization. Compared to eCHD, T21 had increased chr21 gene expression, 11-fold greater levels (p=1.2E-8) of SOST (chr17), encoding the Wnt-inhibitor sclerostin, and 1.4-fold higher levels (p=8.7E-8) of the SOST transcriptional activator ZNF467 (chr7). Cardiac endothelial cells co-expressed SOST and ZNF467. T21 endothelial cells had 6.9-fold higher SOST (p=2.7E-27) with downregulation of Wnt pathway genes. Within the chr21 CHD critical region, the expression of DSCAM was correlated with SOST (p=1.9E-5) and ZNF467 (p=2.9E-4). Deletion of DSCAM in T21 endothelial cells derived from human induced pluripotent stem cells resulted in decreased sclerostin secretion. As Wnt signaling is critical for atrioventricular canal formation, bone health, and pulmonary vascular homeostasis, we conclude that T21-mediated increased sclerostin levels inappropriately inhibits Wnt activities and promotes Down syndrome phenotypes. These findings imply therapeutic potential for anti-sclerostin antibodies in T21.

overall_design:
To investigate the role of DSCAM in regulating SOST expression in endothelial cells (EC), we derived induced pluripotent stem cells (iPSCs) from a T21 patient sample and generated a series of derivative lines with genetic deficiency of one, two, or three DSCAM alleles. We then differentiated these cells into EC and compared gene expression by RNA-seq between DSCAM+/+/+, DSCAM+/+/-, DSCAM+/-/-, and DSCAM-/-/- samples.
contributor: Pediatric Cardiac Genomics Consortium Investigators

first: David

last: McKean

first: Priyanka

last: Narayan

first: Sarah

middle: U

last: Morton

first: Qi

first: Viktoria

last: Strohmenger

first: Sophie

last: McAllister

first: Ananya

last: Sharma

first: Daniel

last: Quiat

last: Reichart

last: DeLaughter

first: Hiroko

last: Wakimoto

first: Joshua

last: Gorham

first: Kemar

last: Brown

first: Barbara

last: McDonough

first: Jon

last: Willcox

last: Jang

first: Steven

last: DePalma

first: Tarsha

last: Ward

first: Richard

last: Kim

first: John

last: Cleveland

first: J

middle: G

last: Seidman

first: Christine


title: Monitoring of melanoma patients on treatment reveals a distinct macrophage population driving targeted therapy resistance

city: Zurich

first: Lukas

last: Sommer

state: Zurich

address: Winterthurerstrasse 190

department: Institute of Anatomy

institute: University of Zurich

summary: Resistance to targeted therapy remains a major clinical challenge in melanoma patients. To uncover resistance mechanisms, we performed single cell RNA sequencing of fine needle aspirates from resistant and responding tumors from melanoma patients before and during BRAFi/MEKi treatment. Among the genes most differentially expressed between malignant cells from resistant vs. responding tumors was POSTN encoding the secreted factor periostin. POSTN was predicted to predominantly signal to a macrophage population associated with targeted therapy resistance (TTR). Accordingly, tumors from patients with fast disease progression after targeted therapy exhibited high POSTN expression levels as well as high numbers of TTR macrophages, and POSTN was able to polarize macrophages towards a TTR phenotype. In a mouse model in vivo, POSTN expression protected melanoma from targeted therapy, which was associated with a phenotype change of intratumroal macrophages. Finally, polarized TTR macrophages protected melanoma cells from MEKi-induced killing through CD44 receptor expression on melanoma cells. Thus, interfering with the protective activity of TTR macrophages might represent a strategy to overcome resistance to targeted therapy in melanoma.

overall_design: We investigated if SPP1-CD44 signaling was involved in resistance mechanisms for targeted therapy. M150543 melanoma cells were subject to MEKi alone or the following combinatorial treatments: SPP1+MEKi; CD44 blocking antibody +MEKi; or SPP1+CD44 blocking antibody+MEKi.

first: Adrian

last: Salas-Bastos

first: Jelena

last: Vasilevska


title: Bacteria conjugate ubiquitin-like proteins to interfere with phage assembly

city: Rehovot

first: Jens

last: Hör

address: 234 Herzl Street

institute: Weizmann Institute for Science

summary: Multiple immune pathways in humans conjugate ubiquitin-like proteins to virus and host molecules as a means of antiviral defense. Here we studied an anti-phage defense system in bacteria, comprising a ubiquitin-like protein, ubiquitin-conjugating enzymes E1 and E2, and a deubiquitinase. We show that during phage infection, this system specifically conjugates the ubiquitin-like protein to the phage central tail fiber, a protein at the tip of the tail that is essential for tail assembly as well as for recognition of the target host receptor. Following infection, cells encoding this defense system release a mixture of partially assembled, tailless phage particles, and fully assembled phages in which the central tail fiber is obstructed by the covalently attached ubiquitin-like protein. These phages exhibit severely impaired infectivity, explaining how the defense system protects the bacterial population from the spread of phage infection. Our findings demonstrate that conjugation of ubiquitin-like proteins is an antiviral strategy conserved across the tree of life.

sample_organism:
Escherichia coli
Caulobacter sp. Root343

overall_design: Total RNA-seq of Caulobacter sp. Root343 at different growth phases, as well as E. coli expressing the Bil system from a plasmid

first: Sharon

last: Wolf

first: Rotem

last: Sorek


title: Transcriptomics profiling of lymphocytes from patients with resolved or chronic HBV infection

city: Basel

first: Jitao David

last: Zhang

address: Grenzacherstrasse 124

department: Roche Pharmaceutical Research and Early Development, Roche Innovation Center Basel

institute: F. Hoffmann-La Roche

summary: We generated transcriptional profiles of CD4 T cells, CD8 T cells, as well as a subset of CD 8 T cells that are specific for human hepatistis B virus core protein (HBcore or HBV_core) with bulk RNA sequencing. CD4 T cells are further stratified by PD1 expression profile (low or high).

overall_design: CD4, CD8, and HBcore-specific T cells were isolated from blood of non-HBV patients (HV), patients with chronic HBV infection (cHBV), and patients with resolved HBV infection (rHBV).

first: Nadine

last: Kumpesa

first: Juliane

last: Siebourg-Polster

first: Jitao

middle: D

first: Kathrin

last: Heim

first: Robert

last: Thimme


title: Serological autoantibody profiling of emmetropic controls and highly myopic patiens

city: Shanghai

first: Xiangjia

last: Zhu

address: No.83 Fenyang Road

institute: Eye & ENT Hospital, Fudan University

summary: High myopia is a leading cause of blindness worldwide, among which pathologic myopia, characterized by typical myopic macular degeneration, is the most detrimental. However, its pathogenesis remains largely unknown. By using a protein microarray assaying more than 20,000 human proteins, we determined in this study the autoantibody profiles of highly myopic patiens and emmetropic controls.

overall_design: 36 serum samples were included, 10 from emmetropic controls and 26 from highly myopic patiens. These are dual channel arrays analyzed with each channel analyzed separately.

first: Yi

last: Lu


title: Uncoupling Histone Modification Crosstalk by Engineering Lysine Demethylase LSD1

first: Kwangwoon

last: Lee

address: 77 Avenue Louis Pasteur, NRB 164

department: Medicine

institute: Brigham and Women's Hospital

summary: Biochemical crosstalk between two or more histone modifications is often observed in epigenetic enzyme regulation but its functional significance in cells has been difficult to discern.  Prior enzymatic studies have revealed that Lys14 acetylation of histone H3 can inhibit Lys4 demethylation by lysine specific demethylase 1 (LSD1). Here we have engineered a mutant form of LSD1, Y391K, which renders the nucleosome demethylase activity of LSD1 insensitive to Lys14 acetylation. Y391K LSD1 knockin cells show increased repression of a set of genes associated with cellular adhesion. Chromatin profiling revealed that the cis-regulatory regions of these silenced genes display a higher level of H3 Lys14 acetylation than the baseline in unedited, parental cells. Y391K LSD1 knockin cells show diminished H3 mono-methyl Lys4 in the vicinity of these silenced genes, consistent with a role for enhanced LSD1 demethylase activity in these regions. These findings illuminate the functional consequences of disconnecting histone modification crosstalk for a key epigenetic enzyme in gene and chromatin regulation.

overall_design: Cleavage Under Targets & Release Using Nuclease (CUT&RUN) experiments for GFP (LSD1-GFP), H3K4me1, H3K4me2, H3K14ac, and IgG control in K562 cells with WT LSD1-GFP and Y391K LSD1-GFP knockin

first: Philip

middle: A

last: Cole


title: Blinding of virus-specific CXCR6+ CD8 T cells in the liver during chronic viral hepatitis causes loss of effector function

city: Freising

first: Gustavo

middle: P.

last: de Almeida

state: Bavaria

address: Liesel-Beckmann-Str. 1

department: Division of Animal Physiology and Immunology

institute: Technical University of Munich

summary: Chronic viral hepatitis after infection with hepatotropic viruses like hepatitis B virus (HBV) affects 300 million persons worldwide, which as the result of chronic immune-mediated hepatic inflammation cause liver cirrhosis and cancer being responsible for 800.000 deaths per year3. Chronic viral hepatitis is maintained by failure of the host´s immune response to control viral infection, but the mechanisms for this inability of virus-specific CD8 T cells to eliminate HBV-infected hepatocytes remain unclear. Here, we demonstrate that during persistent experimental infection with hepatotropic viruses and HBV replication in hepatocytes all virus-specific CD8 T cells present in the liver expressed the tissue-residency markers CXCR6 and CD69. However, RNAseq analysis revealed that CXCR6+CD8 T cells during persistent hepatotropic infection were retained in the liver because of antigen-recognition rather than a transcriptional tissue-residency program, in contrast to canonical liver-resident memory CXCR6+CD8 T cells emerging after resolved infection. Whereas during persistent infection with a model virus like lymphocytic choriomeningitis virus with broad tissue and cell tropism exhausted CD8 T cells show graded loss of effector functions, hepatic virus-specific CXCR6+CD8 T cells during persistent infection with hepatotropic viruses were blinded and completely non-responsive to stimulation in absence of a canonical tox exhaustion signature. Rather, in blinded liver CXCR6+CD8 T cells, transcription factor network analysis revealed Crem, the cAMP-responsive-element-modulator, as the only transcription factor discreetly active in CD8 T cells with complete loss of effector function during persistent hepatotropic infection. Similarly, single cell RNA-sequencing of peripheral blood HBcore-specific CD8 T cells from chronic hepatitis B patients also revealed enhanced CREM activity. Notably, knock-out of the inhibitory CREM/ICER gene in T cells failed to rescue protective T cell immunity during persistent infection with hepatotropic viruses pointing towards post-translational mechanisms relevant for enhanced Crem activity and loss of effector function. Indeed, T cell receptor-associated signalling was blocked in blinded antigen-specific CXCR6+CD8 T cells, that in situ during persistent hepatotropic infection were in close proximity to liver sinusoidal endothelial cells producing high amounts of cAMP-inducing prostanoids. Inhibitory cAMP/PKA/CSK activity increased CREM activity and disconnected CXCR6+CD8 T cells from activation signalling through the T cell receptor. Thus, enhanced CREM expression identifies blinded liver CXCR6+CD8 T cells, but loss of effector functions is caused by post-translational prevention of signalling, which identifies novel molecular targets for immune monitoring and immune therapy of chronic hepatitis B.

overall_design: mRNA profiles of P14 cells after either LCMV Clone 13 or LCMV Armstrong infection 27 dpi.

first: G

middle: P

first: D

last: Zehn


title: HBcore-specific CXCR6+CD8 T cells during persistent HBV replication in mice lose their effector function and have high CREM activity

summary: We investigated molecular profiles of CD8 T cells in persistent experimental Hepatitis B virus (HBV) infection. To this end, we established a preclinical in vivo model where HBV-replicating hepatocytes were cleared by virus-specific immunity after infection with 107 IU Ad-HBV, whereas persistent HBV replication developed after infection with 108 IU Ad-HBV, revealed by high serum HBeAg levels, high numbers of HBV genome copies and of HBcorepos hepatocytes in liver tissue. To overcome variable surface expression of the T cell receptor during chronic infection and unequivocally identify HBV-specific CD8 T cells, we adoptively transferred naïve CD45.1+HBcore-specific CD8 T cells from Cor93 transgenic mice (HBcoreCD8 T cells) the day before Ad-HBV infection. After clearance of HBV-replicating hepatocytes (d44 p.i.), liver CD45.1+HBcoreCD8 T cells expressed either CXCR6 or CX3CR1, and in the spleen only CX3CR1+ CD8 T cells were detected. Next, we evaluated the transcriptional profile of FACSorted HBcoreCD8 T cells after resolved compared to persistent infection employing SmartSeq2. The resulting dataset is reported here.

overall_design: We compared expression profiles between subtypes of HBVcore-specific CD8 T cells in acute and chronic infection in either liver or spleen, and inferred gene regulatory networks underlying the observatios.

first: Vera

last: Griesser

first: Miriam

last: Bosch