title: Novel MST1 inhibitor accelerates murine liver regeneration following partial hepatectomy with improved survival in models of steatohepatitis

status: Public on Nov 01 2023

city: Rochester

first: Ryan

last: Watkins

state: MN

address: 200 First St SW

department: Surgery

institute: Mayo Clinic

summary: Disrupted hepatocyte regeneration following surgical resection remains a clinical problem with few therapeutic options. The current treatment paradigm relies on supportive therapy until homeostasis can be achieved. Pharmacologic acceleration of regeneration may provide an alternative therapeutic avenue. Therefore, we aimed to generate a small molecule inhibitor that could accelerate liver regeneration in both normal and diseased murine preclinical models.  mCLC846 demonstrated on target inhibition of MST1 and reduced EGFR inhibition which resulted in Yes-associated protein (YAP) activation. Oral delivery of mCLC846 perioperatively resulted in accelerated liver regeneration and improved survival in diet induced NASH models. Transcriptional analysis suggested that mCLC846 enhanced the normal regenerative pathways induced following liver resection. Overall, pharmacological acceleration of liver regeneration with mCLC846 was feasible, had an acceptable therapeutic index, and provided survival benefit in models of diet induced non-alcoholic steatohepatitis.

overall_design: Utilizing a clinically approved small molecule inhibitor as a parent compound, standard medicinal chemistry approaches were utilized to generate a novel small molecule inhibitor targeting MST1 with reduced off target effects. This compound, mCLC846, was then applied to preclinical models of murine partial hepatectomy which included models of diet induced NASH.

first: EeeLN

middle: H

last: Buckarma

first: Chantal

last: McCabe

first: Jennifer

last: Tomlinson

first: David

last: Pereya

first: Chen

last: Wang

first: Patrick

last: Starlinger

first: Gregory

middle: J

last: Gores

first: Weijun

last: Shen

first: Rory

last: Smoot


title: Optimized protocol for assay for transposase-accessible chromatin by sequencing (ATAC-seq) from Drosophila melanogaster brain tissue

status: Public on Oct 24 2023

city: Bangalore

first: Jsmine

last: Kaur

state: Karnatka

address: GKVK Campus

department: Biological Sciences

country: India

institute: NCBS

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Transposase-accessible chromatin by sequencing (ATAC-seq) has emerged as an advantageous technique to assess chromatin accessibility owing to the robustness of "tagmentation" process and a relatively faster library preparation. A comprehensive ATAC-seq protocol from Drosophila brain tissue is currently unavailable. Here, we have provided a detailed protocol of ATAC-seq assay from Drosophila brain tissue. Starting from dissection and transposition to amplification of libraries has been elaborated. Furthermore, a robust ATAC-seq analysis pipeline has been presented. The protocol can be easily adapted for other soft tissues.

overall_design:
ATAC-seq assay from Drosophila brain tissue
Nuclei isolated from brain tissue of 2-5 days old adult CS Drosophila flies

first: Jasmine

last: Kaur Dhall

first: Awadhesh

last: Pandit

first: Nandashree

last: Kasturacharya

first: Lakshminarayan

last: CP


title: Response of the transcriptome to changing light regimes.

city: Trebon

first: Karel

middle: Vaclav

last: Kopejtka

address: Novohradska

department: Centre Algatech

country: Czech Republic

institute: Institute of Microbiology Czech Acad. Sci.

type: Expression profiling by high throughput sequencing

summary: Expression dynamics for all genes significantly regulated after the transition from dark to light and vice versa.

overall_design: Cultures were first adapted to continuous illumination and to dark for 72 hours. Following the control sampling (time points 0 hours), the cultures’ light regime was reversed and cells from both treatments were harvested at 2, 4, 8, and 24 hours after the change.

first: Jürgen

last: Tomasch

first: Tomáš

last: Bílý

first: Alastair

middle: T

last: Gardiner

first: Zdenko

last: Gardian

first: Michal

last: Koblížek

last: Kaftan


title: Identification and validation of PCDHGA12 and PRRX1 methylation for detecting lung cancer in bronchial washing sample

status: Public on Oct 31 2023

city: Daejeon

first: Tae Jeong

last: Oh

address: 44-6 10-ro Yuseong-gu

country: South Korea

institute: Genomictree, Inc.

type: Methylation profiling by genome tiling array

summary: Bronchoscopy is a frequently used initial diagnostic procedure for patients with suspected lung cancer (LC). Cytological examinations of bronchial washing (BW) samples obtained during bronchoscopy often yield inconclusive results regarding LC diagnosis. The present study aimed to identify molecular biomarkers as a non-invasive method for LC diagnosis. Aberrant DNA methylation is used as a useful biomarker for LC. Therefore, microarray-based methylation profiling analyses on 13 patient-matched tumor tissues at stages I-III vs. non-tumor tissues were performed, and a group of highly differentially methylated genes was identified. A subsequent analysis using bisulfite-pyrosequencing with additional tissues and cell lines revealed six methylated genes [ADAM metallopeptidase with thrombospondin type 1 motif 20, forkhead box C2 (mesenchyme forkhead 1), NK2 transcription factor related, locus 5 (Drosophila), oligodendrocyte transcription factor 3, protocadherin γ subfamily A 12 (PCDHGA12) and paired related homeobox 1 (PRRX1)] associated with LC. Next, a highly sensitive and accurate detection method, linear target enrichment-quantitative methylation-specific PCR in a single closed tube, was applied for clinical validation using BW samples from patients with LC (n=68) and individuals with benign diseases (n=33). PCDHGA12 and PRRX1 methylation were identified as the best-performing biomarkers to detect LC. The two-marker combination showed a sensitivity of 82.4% and a specificity of 87.9%, with an area under the curve of 0.891. Notably, the sensitivity for small cell LC was 100%. The two-marker combination had a positive predictive value of 93.3% and a negative predictive value of 70.7%. The sensitivity was higher than that of cytology, which only had a sensitivity of 50%. The methylation status of the two-marker combination showed no association with sex, age or stage, but was associated with tumor location and histology. In conclusion, the present study showed that the regulatory regions of PCDHGA12 and PRRX1 are highly methylated in LC and can be used to detect LC in BW specimens as a diagnostic adjunct to cytology in clinical practice.

overall_design: indirect comparison experiment : CRD (common reference DNA) vs. adjacent normal tissues or lung tumor tissues

first: Sungwhan

last: An

first: TaeJeong


title: Pharmacological targeting of histone H3K27 acetylation/BRD4-dependent induction of ALDH1A3 for early-phase drug tolerance of gastric cancer

city: Koto-ku

first: Jin

last: Lee

state: Tokyo

country: Japan

institute: Japanese Foundation for Cancer Research

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

overall_design: Refer to individual Series


title: Function of ARGONAUTE proteins in the Arabidopsis shoot apical meristem (SAM).

city: Vienna

first: Ruben

last: Gutzat

state: Vienna

address: Dr. Bohr-Gasse 3

country: Austria

institute: Gregor Mendel Institute GmbH

summary: In sexually propagating organisms, genetic and epigenetic mutations are evolutionarily relevant only if they occur in the germline and provide inherited information to the next generation. In contrast to most animals, plants are thought to lack an early segregating germline, implying that somatic cells can contribute genetic information to the progeny if they differentiate into meiocytes. Here we demonstrate that two ARGONAUTE proteins, AGO5 and AGO9, are expressed in the reproductive lineage throughout development and mark an early-segregating germline. Furthermore, both AGOs are loaded with dynamic populations of small RNAs derived from highly methylated, pericentromeric, long transposons. Sequencing single nuclei revealed that many of these transposons are highly expressed within a central stem cell domain. This indicates a host-parasite tug of war and specific silencing pathways in plant shoot apical meristems (SAMs). Our results open the path to investigate transposon biology and epigenome dynamics at cellular resolution in the SAM stem cell niche.

overall_design: mRNA analysis of Stem Cell Domain

first: Vu

last: Nguyen

first: Gabriele

last: Bradamante


title: Transcriptomic atlas of midbrain dopamine neurons uncovers differential vulnerability in a Parkinsonism lesion model

status: Public on Oct 26 2023

city: Stockholm

first: Behzad

last: Yaghmaeian Salmani

state: Solna, Stockholm

address: Department of Cell and Molecular Biology (CMB) Karolinska Institute,, Biomedicum, Solnavägen 9, Solna, Stockholm, P.O. Box 285 SE-171 77 Stockholm, Sweden

department: CMB

country: Sweden

institute: Karolinska Institute

summary: Midbrain dopamine (mDA) neurons comprise a diverse group of cells with unique innervation targets and functions. This is illustrated by the selective sensitivity of mDA neurons of the substantia nigra compacta (SNc) in patients with Parkinson’s disease, while those in the ventral tegmental area (VTA) are relatively spared. Here we used single nuclei RNA sequencing (snRNA-seq) of approximately 70,000 mouse midbrain cells to build a high-resolution atlas of mouse mDA neuron diversity at the molecular level. The results showed that differences between mDA neuron groups could best be understood as a continuum without sharp differences between subtypes. Thus, we assigned mDA neurons to several “territories” and “neighborhoods” within a shifting gene expression landscape where boundaries are gradual rather than discrete. Based on the enriched gene expression patterns of these territories and neighborhoods, we were able to localize them in the adult mouse midbrain. Moreover, because the underlying mechanisms for the variable sensitivities of diverse mDA neurons to pathological insults are not well understood, we analyzed surviving neurons after partial 6-hydroxydopamine (6-OHDA) lesions to unravel gene expression patterns that correlate with mDA neuron vulnerability and resilience. Together, this atlas provides a basis for further studies on the neurophysiological role of mDA neurons in health and disease.

overall_design: Molecular charachterization of midbrain dopamine neurons in untreated, unlesioned (intact) and partial 6-hydroxydopamine (6-OHDA) lesioned conditions using single nuclei RNA sequencing.

last: Yaghmaeian-Salmani

first: Laura

last: Lahti

first: Linda

last: Gillberg

first: Jesper

middle: K

last: Jacobsen

first: Ioannis

last: Mantas

first: Per

last: Svenningsson

first: Thomas

last: Perlmann


title: A maternal-effect Padi6 variant causes nuclear and cytoplasmic abnormalities in oocytes as well as failure of epigenetic reprogramming and zygotic genome activation in embryos [scRNA-seq]

status: Public on Oct 18 2023

city: Caserta

first: Francesco

last: Cecere

state: Caserta

address: Via A. Vivaldi, n. 43

department: DiStABiF

country: Italy

institute: University of Campania "Luigi Vanvitelli"

summary: Maternal inactivation of genes encoding components of the sub-cortical maternal complex (SCMC) and its associated member PADI6 generally results in early embryo lethality. In humans, SCMC gene variants were found in the healthy mothers of children affected by multi-locus imprinting disturbances (MLID). However, how the SCMC controls the DNA methylation required to regulate imprinting remains poorly defined. We generated a mouse line carrying a Padi6 missense variant that was identified in a family with Beckwith-Wiedemann syndrome and MLID. If homozygous in female mice this variant resulted in interruption of embryo development at the 2-cell stage. Single-cell multi-omic analyses demonstrated defective maturation of Padi6-mutant oocytes and incomplete DNA demethylation, down-regulation of zygotic genome activation (ZGA) genes, up-regulation of maternal decay genes and developmental delay in 2-cell embryos developing from Padi6-mutant oocytes, but little effect on genomic imprinting. Western blotting and immunofluorescence analyses showed reduced level of UHRF1 in oocytes and abnormal localization of DNMT1 and UHRF1 in both oocytes and zygotes. Treatment with 5-azacytidine reverted DNA hypermethylation but did not rescue the developmental arrest of mutant embryos. Taken together, this study demonstrates that PADI6 controls both nuclear and cytoplasmic oocyte processes that are necessary for pre-implantation epigenetic reprogramming and ZGA.

overall_design: scRNA-seq and scBS-seq were perfomed on MII oocytes and 2cell embryo from new mutant mouse line carrying human variant of PADI6 gene. We sequenced 44 MII oocytes, 44 2cell embryo and 8 Negative control.

first: Carlo

last: Giaccari

first: Lucia

last: Argenziano

first: Angela

last: Pagano

first: Antonio

last: Galvao

first: Dario

last: Acampora

first: Gianna

last: Rossi

first: Bruno

last: Mele

first: Basilia

last: Acurzio

first: Scott

last: Coonrod

first: Maria

middle: V

last: Cubellis

first: Flavia

last: Cerrato

first: Simon

last: Andrews

first: Sandra

last: Cecconi

first: Gavin

last: Kelsey

first: Andrea

last: Riccio


title: Epigenomic tomography for probing spatially-defined chromatin state in the brain

city: Blacksburg

first: Chang

last: Lu

state: VA

address: 235 Goodwin Hall, 635 Prices Fork Road, Virginia Tech

department: Chemical Engineering

country: USA

institute: Virginia Tech

summary: Here we demonstrate brain epigenomic tomography that is constructed via epigenomic profiling of mouse neocortex slices with sub-millimeter thickness using a low-input ChIP-seq technology. We profiled the variation in histone modification signal (H3K27me3 and H3K27ac) across these slices and grouped the spatial epigenomic features into clusters of various spatial variation patterns.

overall_design: We processed mouse neocortex into 0.5 mm thick coronal slices and separated them into left and right hemisphere pieces. We extracted nuclei from each tissue piece and sorted them into neuron and glia using FACS. Nuclei were digested with MNase for MOWChIP-seq of H3K27me3 and H3K27ac. RNA-seq libraries were generated using Smart-seq2. The filename contains information on the mouse from which the tissue for the experiment was isolated from (M1-M5), the hemisphere of the brain the tissue belonged to (left/right), the number of slice of the tissue (01-22, from anterior to posterior), the type of nuclei sorted for the experiment (neuron/glia), the target being profiled (H3K27me3, H3K27ac, RNA), and replicate number. 2 technical replicates were conducted for each sample. For example, "M1-Left-01-Glia-H3K27me3-R1" refers to technical replicate 1 of the ChIP-seq assay of H3K27me3 using glia nuclei isolated from slice 1 of left hemisphere of mouse 1.

first: Zhengzhi

last: Liu


title: txci-ATAC-seq: a massive-scale single-cell technique to profile chromatin accessibility

status: Public on Oct 20 2023

city: Portland

first: Ryan Mulqueen

last: Mulqueen

state: OR

address: 3181 S.W. Sam Jackson Park Road

department: Molecular and Medical Genetics

institute: Oregon Health and Science

summary: We develop a large-scale single-cell ATAC-seq method by combining Tn5-based pre-indexing with 10X Genomics barcoding, enabling the indexing of up to 200,000 nuclei across multiple samples in a single reaction. We profile 449,953 nuclei across diverse tissues, including the human cortex, mouse brain, human lung, mouse lung, mouse liver, and lung tissue from a club cell secretory protein knockout (CC16-/-) model. Our study of CC16-/- nuclei uncovers previously underappreciated technical artifacts derived from remnant 129 mouse strain genetic material, which cause profound cell-type-specific changes in regulatory elements near many genes, thereby confounding the interpretation of this commonly referenced mouse model.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: txci-ATAC-seq using brain tissue samples using replicates of Human Cortex and Mouse whole brain tissue loaded at ~20k and ~70k cells. Human raw data will be made available through the NeMO Archive.

first: Hao

last: Zhang

first: Ryan

first: Natalie

last: Iannuzo

first: Dominique

last: Farrera

first: Francesca

last: Polverino

first: James

last: Galligan

first: Julie

last: Ledford

first: Andrew

last: Adey

first: Darren

last: Cusanovich


title: Phenotype screens of murine pancreatic cancer identify a Tgfa-Ccl2-paxillin axis driving human-like neural invasion

status: Public on Oct 11 2023

city: Munich

first: Rouzanna

last: Istvánffy

address: Ismaningerstr. 22

department: Department of Surgery

country: Germany

institute: Klinikum rechts der Isar

summary: Solid cancers like pancreatic cancer (PDAC) frequently exploit nerves for rapid dissemination. This neural invasion (NI) is an independent prognostic factor in PDAC, but insufficiently modelled in genetically-engineered mouse models (GEMM) of PDAC.Here, we systematically screened for human-like NI in Europe’s largest repository of GEMM of PDAC comprising 295 different genotypes. This phenotype screen uncovered two GEMM of PDAC with human-like NI, which are both characterized by pancreas-specific overexpression of transforming-growth-factor-alpha (TGFa) and conditional depletion of p53. Mechanistically, cancer-cell-derived TGFa upregulated CCL2 secretion from sensory neurons, which induced hyperphosphorylation of the cytoskeletal protein paxillin via CCR4 on cancer cells. This activated the cancer migration machinery and filopodia formation toward neurons. Disrupting CCR4 or paxillin activity limited NI, and dampened tumor size and tumor innervation. In human PDAC, phospho-paxillin and TGFa-expression constituted strong prognostic factors. Therefore, TGFa-CCL2-CCR4-p-paxillin axis is a clinically actionable target for constraining NI and tumor progression in PDAC.

overall_design: In the spatial transcriptome analyses with murine DRG, DRGs T8 to T12 were extracted bilaterally from TPAC (Ela-TGFα; Ptf1a-Cre; Trp53fl/fl;RelAfl/fl , n=6, 47-54 weeks old), KPC (Ptf1a-Cre; LSL-Kras+/G12D; Trp53+/fl , n=3, 20-21 weeks old) and age-similar tumor-free control mice (Ela-TGFα; Trp53fl/fl;RelAfl/fl , n=3, 48-50 weeks old for TPAC and LSL-Kras+/G12D; Trp53+/fl , n=1, 20 weeks old; Trp53+/fl , n=1, 21 weeks old and LSL-Kras+/G12D; n=1, 22 weeks old for KPC). Following the extraction, DRGs were briefly washed in ice-cold, fresh PBS (D5262, Sigma-Aldrich) and fixated in freshly prepared 4% PFA (15710, Electron Microscopy Sciences; diluted with PBS) for 24 hours at room temperature. The samples were consecutively dehydrated, paraffine embedded and sectioned. A whole transcriptome analysis was performed on NeuN rich regions within the DRG sections using the NanoString GeoMx® digital spatial profiler. 5µm thick unstained sections on slides were subjected to incubation for 35 minutes at 60°C. Afterwards, the slides were moved into Leica BOND Rx. Proteinase K (1ug/ml, for 5 minutes) and HIER2 (20 minutes) were used for RNA antigen retrieval. NanoString GeoMx Mouse Whole Transcriptome Atlas RNA probes were added for hybridization for 16 hours at 37°C. Following the stringent washes per manufacturer’s instructions, slides were blocked with W buffer for 1 hour. The slides were then stained with AF647 anti-NeuN (1:100, ab190565, Abcam), AF594 anti-Cd68 (1:200, sc-20060, Santa Cruz) AF532 Syto83 (1:10, ThermoFisher) and AF488 anti-GFAP (1:200, NBP2-3318AF488, Novus) antibodies. After washing the slides twice with SSC buffer, slides were loaded to the DSP and scanned for the regions of interest (ROI) selection. Two NeuN rich region per mouse were selected as ROIs.For the human spatial transcriptome studies, two patients with pancreatic ductal adenocarcinoma were selected for transcriptome spatial analysis using the NanoString GeoMx Digital Spatial Profiler (DSP)/Human NGS Whole Transcriptome Atlas. Cases were selected based on the presence of perineural invasion in tissue sections. Each slide was stained with fluorescence-labeled antibodies to allow identification of tissue: pan-cytokeratin for epithelial cells and PGP9.5 for nerves. Tumor and Nerve areas of interest (AOI) were selected as follows: (i) NI Tumor: nerve-invading pancreatic (N=6); (ii) no NI Tumor: non-invading tumor cells (n=7); (iii) NI Nerve: tumor-invaded nerve (n=5); (iv) no NI Nerve: non-invaded nerve (n=4).  AOIs were drawn manually around nerves and tumor cells. Following the ROI selection, UV-photocleaved oligonucleotide barcodes from each ROI were collected and prepared by the NanoString GeoMx protocol. Sequencing was performed on the NextSeq 2000. Raw basecell files were demultiplexed into fastsq files. NanoString’s GeoMx NGS Pipeline v.2.0.0 was used to convert the fastsq files to Digital Count Conversion files. None of the 45 AOIs were below the warning sequencing saturation of 50%. Data was then normalized, and the downstream gene expression analyses were performed using the R package DESeq2.

first: Ekin

middle: I

last: Demir


first: Jin

last: Lee

summary: Drug-tolerant persister (DTP) cells arise in the early phase of chemotherapy and contribute to tumor relapse. In gastric cancer, we previously identified aldehyde dehydrogenase 1 family member A3 (ALDH1A3) as a DTP signature gene that contributes to survival after chemotherapy, while it remains elusive how the ALDH1A3-overexpressing DTP cells are enriched after chemotherapy and contribute to tumor promotion. Here, we demonstrated that ALDH1A3 was frequently overexpressed in clinical gastric cancer specimen after neoadjuvant chemotherapy and ALDH1A3 overexpression in gastric cancer patient-derived cells (PDCs) promoted tumor growth in vivo. Live imaging revealed that the ALDH1A3-overexpressing DTP cells were induced rather than selected after 5-fluorouracil (5-FU) treatment. We identified global elevation of histone H3 lysine 27 acetylation (H3K27ac) in the promoter regions of DTP cells including ALDH1A3 gene locus. Chemical screening further revealed bromodomain and extra-terminal motif (BET) protein inhibitors as efficient perturbators of the DTP phenotype. The BET inhibitors suppressed 5-FU-induced ADLH1A3 expression and selectively prevented the DTP cell proliferation. Importantly, ALDH1A3 downregulation and growth inhibition by BET inhibitors was rescued by exogenous ALDH1A3 expression. Among BET proteins, BRD4 was preferentially recruited to the ALDH1A3 promoter and promoted its expression in DTP cells. The BET inhibitor, Birabresib/OTX015, significantly suppressed ALDH1A3 expression and potentiated antitumor effect of 5-FU in gastric PDC xenograft model. These data indicate that the global H3K27ac induction and the BRD4-dependent ALDH1A3 induction is essential for gastric cancer drug tolerance.

overall_design: Examination of RNA-sequencing in (1) gastric cancer patient-derived cells (JSC15-3) treated with or without 5-FU for 5 days and (2) JSC15-3 cells were transfected with siNCT(negative control) and siBRD4 and treated with or without 3 µM 5-FU for 5 days.

first: Tetsuo

last: Mashima

first: Hiroyuki

last: Seimiya


title: Effect of gene expression changes after Erlotinib and CCR6 inhibitor treatment on PC9 and PC9GR cells

status: Public on Oct 10 2023

city: Seoul

first: Eun-Ji

last: Kwon

address: 1, Gwanak-ro

institute: Seoul National University

summary: Comparative gene expression profile analysis of RNA-seq data for PC9 GR cells after compound treatment.

overall_design: To derive factors overcoming resistance, we treated erlotinib and CCR6 inhibitor on PC9 GR cells.

first: Hyuk-Jin

last: Cha


title: A maternal-effect Padi6 variant causes nuclear and cytoplasmic abnormalities in oocytes as well as failure of epigenetic reprogramming and zygotic genome activation in embryos [WGS]

first: Francesco

last: Cecere

first: Carlo

last: Giaccari

first: Lucia

last: Argenziano

first: Angela

last: Pagano

first: Antonio

last: Galvao

first: Dario

last: Acampora

first: Gianna

last: Rossi

first: Bruno

last: Mele

first: Basilia

last: Acurzio

first: Scott

last: Coonrod

first: Maria

last: Cubellis

first: Flavia

last: Cerrato

first: Simon

last: Andrews

first: Sandra

last: Sandra

first: Gavin

last: Kelsey

first: Andrea

last: Riccio


first: Jin

last: Lee

overall_design: Examination of histone modification with H3K27ac and H3K27me3 using ChIP in gastric cancer patient derived cells (JSC15-3) treated with or without 5-FU treatment.

first: Tetsuo

last: Mashima

first: Hiroyuki

last: Seimiya


title: microRNA sequencing for biomarker detection in the diagnosis, classification and prognosis of Diffuse Large B Cell Lymphoma

city: Leioa

first: Nerea

last: Bilbao-Aldaiturriaga

address: Sarriena neighbourhood without number

country: Spain

institute: University of the Basque Country

summary: Diffuse Large B Cell Lymphoma (DLBCL) is the most common lymphoid malignancy in adults. Despite being considered a single disease, DLBCL presents with variable backgrounds in terms of morphology, genetics, and biological behavior, which results in heterogeneous outcomes among patients. Although new tools have been developed for the classification and management of patients, 40% of them still have primary refractory disease or relapse. In addition, multiple factors regarding the pathogenesis of this disease remain unclear and identification of novel biomarkers is needed. In this context, recent investigations point to microRNAs as useful biomarkers in cancer as well as important players in the development of the disease. However, regarding DLBCL, up to date, there is inconsistency in the data reported. Therefore, in this work, the main goals were to determine a microRNA set with utility as biomarkers for DLBCL diagnosis, classification, prognosis and treatment response. To achieve these goals, we analyzed microRNA expression in a cohort of 78 DLBCL samples at diagnosis and 17 controls using small RNA sequencing. This way, we were able to define new microRNA expression signatures for diagnosis, classification, treatment response and prognosis. In summary, our study remarks that microRNAs could play an important role as biomarkers in diagnosis, classification, treatment response and prognosis in DLBCL.

overall_design: 78 DLBCL samples at diagnosis, 17 controls and 7 DLBCL relapse samples

first: Ane

last: Larrabeiti-Etxebarria

first: Javier

last: Arzuaga-Mendez

first: Maialen

last: Martin-Arruti

first: Luca

last: Cozzuto

first: Ayman

last: Gaafar

first: Irune

last: Ruiz-Diaz

first: Isabel

last: Guerra

first: Idoia

last: Martin-Guerrero

first: Elixabet

last: Lopez-Lopez

last: Gutierrez-Camino


title: Transcription factor NFYa controls cardiomyocyte metabolism and proliferation during fetal heart development [ChIP-seq]

status: Public on Oct 13 2023

city: Dallas

first: Miao

last: Cui

state: Texas

address: UT Southwestern Medical Center Department of Molecular Biology 6000 Harry Hines Blvd.

institute: UTSW

summary: Cardiomyocytes are highly metabolic cells responsible for generating the contractile force that drives heart function. During fetal development and regeneration, these cells undergo active division but lose their proliferation activity in the adult heart. The mechanisms that coordinate their metabolism and proliferation are not fully understood. Here, we study the developmental functions of the transcription factor NFYa, which we previously identified from regenerating cardiomyocytes. We show that loss of NFYa profoundly alters cardiomyocyte composition, with a decrease in immature regenerative cells and an increase in trabecular and mature cardiomyocytes, as revealed by spatial and single-cell transcriptome analyses. NFYa-deleted cardiomyocytes exhibited reduced proliferation and impaired mitochondrial metabolism, contributing to the cardiac growth defect. NFYa acts as a transcriptional activator of mitochondrial metabolic genes as well as cell-cycle genes in cardiomyocytes through its interaction with the cofactor SP2, providing a direct link between metabolism and proliferation at the gene transcriptional level. Our study reveals a key role of NFYa in regulating cardiac growth before birth and a previously unrecognized transcriptional control mechanism of metabolic genes in the heart, and highlights the importance of mitochondrial metabolism during fetal heart development and regeneration.

overall_design: Examination of chromatin binding of NFYa and H3K27ac in neonatal rat ventricular cardiomyocytes by Chromatin Immunoprecipitation followed by sequencing

first: Eric

middle: N

last: Olson


status: Public on Oct 25 2023

city: Tucson

first: Hao

last: Zhang

state: AZ

address: 1230 N Cherry Ave

department: CMM

institute: The University of Arizona

summary: We develop a large-scale single-cell ATAC-seq method (txci-ATAC-seq) by combining Tn5-based pre-indexing with 10X Genomics barcoding. Leveraging this molecular hashing strategy, we demonstrate that txci-ATAC-seq enables the indexing of up to 200,000 nuclei across multiple samples in a single emulsion reaction, representing a ~22-fold increase in throughput compared to the standard workflow at the same collision rate. To improve the multiplexing capability of this new technique, we further develop a "phased" protocol variant (Phased-txci-ATAC-seq) that effectively decouples sample processing from library preparation and has the potential to profile up to 96 samples simultaneously. In this study, we profile 449,953 nuclei across diverse tissues, including the human cortex, mouse brain, human lung, mouse lung, mouse liver, and lung tissue from a club cell secretory protein knockout (CC16-/-) model. Our study of CC16-/- nuclei uncovers previously underappreciated technical artifacts derived from remnant 129 mouse strain genetic material, which cause profound cell-type-specific changes in regulatory elements near many genes, thereby confounding the interpretation of this commonly referenced mouse model.

overall_design: We extracted nuclei from multiple sources, including GM12878 and CH12.LX cell lines, human lung tissue, native lung and liver samples from two 24-week-old male C57BL/6J mice, and lung tissue from age-matched (~8 weeks) wild-type (n=3) and CC16 deficient (n=3) mice on a C57BL/6J background. Two different barnyard settings were designed to estimate the total collisions arising from pre- and/or post-pooling events. To test the total collision rate, the human and mouse cells were mixed in the same well at a 1:1 ratio to perform barcoded transposition (“true barnyard”). The collision rate driven by events downstream of pooling was tested by performing barcoded transposition on wells containing pure species (“pseudo-barnyard”) and pooling the human and mouse nuclei afterward.

first: Ryan

middle: M

last: Mulqueen

first: Natalie

last: Iannuzo

first: Dominique

middle: O

last: Farrera

first: Francesca

last: Polverino

first: James

last: Galligan

first: Julie

middle: G

last: Ledford

first: Andrew

middle: C

last: Adey

first: Darren

middle: A

last: Cusanovich


title: A maternal-effect Padi6 variant causes nuclear and cytoplasmic abnormalities in oocytes as well 2 as failure of epigenetic reprogramming and zygotic genome activation in embryos

first: Francesco

last: Cecere

type: Methylation profiling by high throughput sequencing


title: A maternal-effect Padi6 variant causes nuclear and cytoplasmic abnormalities in oocytes as well as failure of epigenetic reprogramming and zygotic genome activation in embryos [scBS-seq]

first: Carlo

last: Giaccari

first: Lucia

last: Argenziano

first: Angela

last: Pagano

first: Antonio

last: Galvao

first: Dario

last: Acampora

first: Gianna

last: Rossi

first: Bruno

last: Mele

first: Basilia

last: Acurzio

first: Scott

last: Coonrod

first: Maria

last: Cubellis

first: Flavia

last: Cerrato

first: Simon

last: Andrews

first: Sandra

last: Cecconi

first: Gavin

last: Kelsey

first: Andrea

last: Riccio


title: Cyclophilin A supports translation of intrinsically disordered proteins and affects haematopoietic stem cell ageing

city: Houston

first: Andre

last: Catic

state: TX

address: One Baylor Plaza

institute: Baylor College of Medicine

summary: NGS (paired-end RNA-seq) of mouse hematopoietic stem and progenitor cells from young (4-6 mo) and old (31-33 mo) mice.

overall_design:
Wild-type HSPCs were isolated from the hind limb long bones of 4- to 6-month-old and 31- to 33-month-old male mice. c-Kit+ cells were stained and magnetically isolated from the lineage-depleted cell suspension using the EasySep mouse CD117 (c-Kit) positive selection kit (Stem Cell Technologies), following manufacturer’s instructions. After overnight growth in serum-free medium (StemSpan SFEM, Stem Cell Technologies), supplemented with murine TPO (20 ng/ml, PeproTech), SCF (10 ng/ml, PeproTech), and the beta-Catenin agonist CHIR99021 (250 nM, Stemgent), HSPCs were harvested as cell pellets. Immediately after, RNA extraction was carried out with the RNeasy Plus Mini kit with genomic DNA Eliminator columns (Qiagen) in combination with on-column DNaseI digestion (Qiagen), according to the vendor’s protocol.
Biological duplicates of two young and two old sets of hematopoietic stem and progenitor sets were used.


title: The snRNAseq  Analysis of JunB deficient brown adipose tissue and control samples

status: Public on Oct 03 2023

city: ALBUQUERQUE

first: Xing

last: Zhang

state: NEW MEXICO

address: 915 Camino de Salud NE

institute: University of New Mexico

summary: The coexistence of brown adipocytes with low and high thermogenic activity is a fundamental feature of brown adipose tissue (BAT) heterogeneity and plasticity. However, the mechanisms that govern thermogenic adipocyte heterogeneity and its significance in obesity and metabolic disease remain poorly understood. Here we show that in male mice, a population of JunB-enriched (JunB+) adipocytes within the BAT exhibits lower thermogenic capacity compared to high-thermogenic adipocytes. To determine if JunB plays a role in the control of brown adipocyte heterogeneity in thermogenesis, we performed single-nucleus RNA sequencing (snRNA-seq) of BAT from JunB FKO and control mice (3-month-old male mice (5/group)) housed at room temperature. Our study pointing to a potent inhibition by JunB in the conversion from low- to high-thermogenic adipocyte.

overall_design: The nuclei was isolated from newly collected brown fat tissue of 10-week-old JunB deficient and control mice (n=5/group). The same number of nuclei from each individual animal were combined within a group and used for snRNAseq.

first: Meilian

last: Liu

first: Kathryn

last: Brayer


title: Transcriptomic analysis of ddi-1(icb156) mutants in C. elegans

status: Public on Sep 30 2023

city: London

first: Michalis

last: Barkoulas

address: SAF Building, South Kensington

country: United Kingdom

institute: Imperial College London

summary: We used RNA-seq to identify gene expression changes in C. elegans ddi-1(icb156) mutants

overall_design: Comparative gene expression of ddi-1(icb156) to wild-type animals at the L4 life stage

first: Manish

last: Grover


title: Global Wnt Inhibition with the Porcupine Inhibitor, LGK974, Decreases Trabecular Bone in a Murine Model with Fibrotic Bone

status: Public on Oct 16 2023

city: San Francisco

first: Edward

last: Hsiao

address: 505 Parnassus Ave

institute: UCSF

summary: G protein-coupled receptors (GPCRs) mediate a wide spectrum of physiological functions, including the development, remodeling, and repair of the skeleton. Fibrous dysplasia (FD) of the bone is characterized by fibrotic, expansile bone lesions caused by an activating mutation in GNAS. We previously showed that ColI(2.3)+/Rs1+ mice, in which Gs-GPCR signaling was hyper-activated in osteoblastic cell lineages, developed a fibrotic bone phenotype with trabecularization that could be reversed by normalizing Gs-GPCR signaling, suggesting that targeting the Gs-GPCR or components of the downstream signaling pathway could serve as a promising therapeutic strategy for FD, for which there are no effective medical treatments.The Wnt signaling pathway has been implicated in the pathogenesis of FD-like bone, but the specific Wnts and which cells produce them remains largely unknown. Single cell RNA sequencing on long-bone stromal cells of 9-week-old male ColI(2.3)+/Rs1+ mice and littermate controls showed that fibroblastic stromal cells in ColI(2.3)+/Rs1+ mice were expanded. Multiple Wnt ligands were up- or down-regulated in different cellular populations, including in non-osteoblastic cells. Treatment with the porcupine inhibitor LGK974, which blocks Wnt signaling broadly, induced partial resorption of the trabecular bone in the femurs of ColI(2.3)+/Rs1+ mice, but no significant changes in the craniofacial skeleton. Bone fibrosis remained evident after treatment. Notably, LGK974 caused significant bone loss in control mice. These results provide new insights into the role of Wnt and Gs-signaling in fibrosis and bone formation in a mouse model of Gs-GPCR pathway overactivation.

overall_design: Cells from the bones of two 9-week old male Col1(2.3)+/Rs1+ mice and two 9-week old control mice were isolated and processed for single-cell RNA sequencing using the 10X Genomics platform.  The transcriptomic profile of cells derived from Col1(2.3)+/Rs1+ and control mice were analyzed.

first: Hsuan

last: Lung

first: Kelly

middle: L

last: Wentworth

first: Tania

last: Moody

first: Ariane

last: Zamarioli

first: Aspara

last: Ram

first: Gauri

last: Ganesh

first: Misun

last: Kang

first: Sunita

last: Ho


title: Lupus IgA1 autoantibodies synergize with IgG to enhance pDC responses to RNA-containing immune complexes

status: Public on Oct 19 2023

city: Seattle,

first: Stephanie

last: Osmond

state: WA

address: 1201 9th Ave

institute: Benaroya Research Institute

summary: Autoantibodies to nuclear antigens are hallmarks for diagnosis of the autoimmune disease systemic lupus erythematosus (SLE) and they contribute to SLE pathogenesis. However, there remains a gap in our knowledge regarding how different isotypes of autoantibodies contribute to key disease processes, including the production of the critical type I interferon (IFN) cytokines by plasmacytoid dendritic cells (pDCs) in response to immune complexes (ICs). Here, we show that individuals with SLE have IgA autoantibodies against most nuclear antigens assessed, largely correlating with the amounts of IgG against the same antigen. We investigated whether IgA autoantibodies against a major SLE autoantigen, Smith ribonucleoproteins (Sm/RNPs), play a role in IC activation of pDCs. We found that pDCs express the IgA-specific Fc receptor, FcRI, and there was a striking ability of IgA1 isotype autoantibodies to synergize with IgG in RNA-containing ICs to generate robust pDC IFN responses. pDC responses to these ICs required both FcRI and FcRIIa, suggesting the most potent ICs for pDCs contained autoantibodies of both isotypes. Sm/RNP IC binding to and internalization by pDCs were greater when ICs contained both IgA1 and IgG. In addition, binding of Sm/RNP ICs generated with IgA1-sufficient serum correlated to pDC FcRI expression, but not FcRIIa expression. pDCs from individuals with SLE had higher binding of IgA1-containing ICs and higher expression of FcRI than pDCs from healthy control individuals, correlating with expression of IFN response genes. Taken together, our data indicate that IgA1 ANAs contribute to SLE pathology and warrant further investigation.

overall_design: Cross-sectional investigation of systematic lupus erythematosus subjects in relation to disease severity and levels of Fc receptors on pDCs and monocytes. One sample per subject, RNAseq from whole peripheral blood.

first: Hayley

middle: R

last: Waterman

first: Matthew

last: Dufort

first: Sylvia

middle: E

last: Posso

first: Minjian

last: Ni

first: Lucy

middle: Z

last: Li

first: Chengsong

last: Zhu

first: Prithvi

last: Raj

first: Jane

last: Buckner

first: Jessica

last: Hamerman


title: Transcription factor NFYa controls cardiomyocyte metabolism and proliferation during fetal heart development [Visium]

first: Miao

last: Cui

overall_design: Examination of spatial transcriptome in WT and NFYa deleted  E15.5 mouse hearts using the Visium Spatial Transcriptomics

first: Eric

last: Olson


title: Transcription factor NFYa controls cardiomyocyte metabolism and proliferation during fetal heart development [Multiome]

overall_design: Examination of transcriptome and open chromatin changes in cardiomyocytes from WT and NFYa deleted  E15.5 mouse hearts using multiome RNA and ATAC sequencing


title: Transcription factor NFYa controls cardiomyocyte metabolism and proliferation during fetal heart development

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

sample_organism:
Mus musculus
Rattus norvegicus

platform_organism:
Mus musculus
Rattus norvegicus

overall_design: Refer to individual Series


title: Function of ARGONAUTE proteins in the Arabidopsis shoot apical meristem (SAM) [snRNA-seq]

first: Ruben

last: Gutzat

summary: In sexually propagating organisms, genetic and epigenetic mutations are evolutionarily relevant only if they occur in the germline and provide inherited information to the next generation. In contrast to most animals, plants are thought to lack an early segregating germline, implying that somatic cells can contribute genetic information to the progeny if they differentiate into meiocytes. Here we demonstrate that two ARGONAUTE proteins, AGO5 and AGO9, are expressed in the reproductive lineage throughout development and mark an early-segregating germline. Furthermore, both AGOs are loaded with dynamic populations of small RNAs derived from highly methylated, pericentromeric, long transposons. Sequencing single nuclei revealed that many of these transposons are highly expressed within a central stem cell domain. This indicates a host-parasite tug of war and specific silencing pathways in plant shoot apical meristems (SAMs). Our results open the path to investigate transposon biology and epigenome dynamics at cellular resolution in the SAM stem cell niche.

overall_design: analysis of mRNA expression in single nuclei of stem cells from the shoot apical meristem of seedlings 7 days after germination.

first: Vu

middle: H

last: Nguyen

first: Gabriele

last: Bradamante


title: Transcriptome-wide analysis of the function of Ded1 in translation preinitiation complex assembly in a reconstituted in vitro system

city: Bethesda

first: Jon

middle: R.

last: Lorsch

state: MD

address: BG 49 RM 2C08, 49 Convent Dr.

department: Eunice Kennedy Shriver National Institute of Child Health and Human Development,

institute: National Institutes of Health

summary: We have developed a deep sequencing-based approach, Rec-Seq,  that allows simultaneous monitoring of ribosomal 48S pre-initiation complex (PIC) formation on every mRNA in the translatome in an in vitro reconstituted system. Rec-Seq isolates key early steps in translation initiation in the absence of all other cellular components and processes. Using this approach we show that the DEAD-box ATPase Ded1 promotes 48S PIC formation on the start codons of >1000 native mRNAs, most of which have long, structured 5’-untranslated regions (5’UTRs). Remarkably, initiation measured in Rec-Seq was enhanced by Ded1 for most mRNAs previously shown to be highly Ded1-dependent by ribosome profiling of ded1 mutants in vivo, demonstrating that the core translation functions of the factor are recapitulated in the purified system. Our data do not support a model in which Ded1acts by reducing initiation at start codons in 5’UTRs and instead indicate it functions by directly promoting mRNA recruitment to the 43S PIC and scanning to locate the start codon. We also provide evidence that eIF4A, another essential DEAD-box initiation factor, is required for efficient PIC assembly on almost all mRNAs, regardless of their structural complexity, in contrast to the preferential stimulation by Ded1 of initiation on mRNAs with long, structured 5’UTRs.

overall_design: We tested yeast Ded1 and eIF4A in promoting mRNA recruitments using Rec-Seq experiment. For Ded1 protein, we tested conditions with 0, 100 or 500 nM Ded1, for eIF4A protein, we tested 500 or 5000 nM eIF4A.

first: Fujun

last: Zhou

first: Julie

last: Bocetti

first: Meizhen

last: Hou

first: Daoming

last: Qin

first: Alan

last: Hinnebusch


title: Function of ARGONAUTE proteins in the Arabidopsis shoot apical meristem (SAM) [seedling RNA-seq]

overall_design: analysis of mRNA of shoot apical meristems in different mutants and wt.


title: Function of ARGONAUTE proteins in the Arabidopsis shoot apical meristem (SAM) [RNA-seq]

overall_design: analysis of mRNA of stem cells (of the shoot apical meristem) and sperm and vn cells (from Pollen) in  different mutants and wt.


title: Function of ARGONAUTE proteins in the Arabidopsis shoot apical meristem (SAM) [RIP-seq]

overall_design: Analysis of AGO5- and AGO9-bound sRNAs at 7 days and 35 days after germination


title: Function of ARGONAUTE proteins in the Arabidopsis shoot apical meristem (SAM) [Bisulfite-Seq]

overall_design: analysis of DNA methylation of stem and sperm cells of different mutants


title: Function of ARGONAUTE proteins in the Arabidopsis shoot apical meristem (SAM)


title: Disrupted propionate metabolism evokes transcriptional changes in the heart by increasing histone acetylation and propionylation [ChIP-Seq 1]

status: Public on Sep 28 2023

city: Oxford

first: Pawel

last: Swietach

address: Sherrington Building, Parks Road

department: Department of Physiology, Anatomy & Genetics

institute: University of Oxford

summary: Propiogenic substrate catabolism and gut bacteria produce propionate, a post-translational protein modifier. Here, using a mouse model of propionic acidaemia (PA), we characterise how disturbances to propionate metabolism modify histones and affect cardiac gene expression and function. Plasma propionate was raised in PA mice, but male hearts accumulated a smaller propionyl-CoA excess, correlating with -alanine build-up. Raising -alanine experimentally in myocytes cultured under propionate-stress reduced propionyl-CoA and partially reversed the propionate-evoked metabolic disturbance. Female PA hearts manifested a moderate diastolic dysfunction phenotype, with raised diastolic [Ca2+], expanded end-systolic ventricular volume, and reduced stroke volume. Differentially-expressed genes included Pde9a and Mme that relate to contractile dysfunction through down-scaled cGMP signalling, consistent with phosphoproteome changes. Propionate was traced to histone H3 propionylation and increased acetylation genome-wide, including at Pde9a and Mme promoters, with more pronounced effects in female PA hearts.  We link perturbed propionate metabolism to epigenetic changes that impact cardiac function.

overall_design: ChIP-seq

first: Kyung

last: Park

first: Nicholas

last: Crump

first: Thomas

last: Milne


title: Disrupted propionate metabolism evokes transcriptional changes in the heart by increasing histone acetylation and propionylation [RNA-Seq 1]

overall_design: RNA-seq


title: Disrupted propionate metabolism evokes transcriptional changes in the heart by increasing histone acetylation and propionylation [ChIP-Seq 2]


title: Disrupted propionate metabolism evokes transcriptional changes in the heart by increasing histone acetylation and propionylation


title: Disrupted propionate metabolism evokes transcriptional changes in the heart by increasing histone acetylation and propionylation [RNA-Seq 2]


title: Tuberculosis resistance protein TOLLIP controls immune pathology by resolving the integrated stress response in alveolar macrophages

status: Public on Sep 27 2023

city: Seattle

first: Kimberly

last: Dill-McFarland

address: 750 Republican St

department: Allergy and Infectious Diseases

institute: University of Washington

summary: A functional polymorphism responsible for TOLLIP deficiency is associated with increased tuberculosis risk. However, TOLLIP’s role in TB pathogenesis is unknown. Tollip-/- mice developed severe Mycobacterium tuberculosis (Mtb) disease, characterized by macrophage and T cell inflammation from multiple cell populations alongside foam cell formation and lipid accumulation. Tollip-/- alveolar macrophages (AM) selectively accumulated lipid and underwent necrosis in an autonomous manner. Transcriptional and protein analysis analysis of Mtb-infected, Tollip-/- AM demonstrated increased EIF2 signaling, a key regulator of the integrated stress response (ISR) to variable environmental conditions. The ISR, inflammation, and lipid accumulation were linked, as incubation of the Mtb lipid mycolic acid with Mtb-infected Tollip-/- AM led to ISR activation and Mtb replication. Correspondingly, ISRIB, a small-molecule ISR inhibitor, selectively reduced Mtb intracellular carriage in AM and improved Mtb control, overcoming the inflammatory phenotypeinflammation. Targeting the ISR broadly improves Mtb control and immunopathology, offering a promising target for host-directed tuberculosis therapy.

overall_design: Tollip-/- mixed bone marrow chimera mice were infected with Mycobacterium tuberculosis for 28 days. Lung alveolar macrophages were isolated and sorted into Mtb-infected and bystander (uninfected) populations. Total RNA-seq was performed.

first: Sambasivan

last: Venkatasubramanian

first: Courtney

last: Plumlee

first: Sara

middle: B

last: Cohen

first: Benjamin

last: Gern

first: Divya

last: Rane

first: Mackenzie

last: Meyer

first: Aparajita

last: Saha

first: Gemma

last: Pearson

first: Anne

last: Lietzke

first: Amanda

last: Pacheco

first: Yu-Hua

last: Chow

first: Chi

middle: F

last: Hung

first: Scott

last: Soleimanpour

first: Matthew

last: Altman

first: Kevin

last: Urdahl

first: Javeed

last: Shah


title: Impact of media supplementation on the transcriptional activity of porcine embryos  produced in vitro

city: Libechov

first: Alexandra

middle: Rosenbaum

last: Bartkova

address: Rumburska 89

institute: IAPG CAS

summary: In this article, we focused on the impact of precisely chemically modified FLI maturation medium and its potential to improve the efficiency of in vitro production of porcine embryos. Based on our findings, we demonstrated the positive influence of FLI medium in the evaluation of in vitro maturation, in vitro embryo production, EGA detection, transcriptomic and proteomic profile, which was confirmed by the positive activation of the embryonal genome in the 4-cell stage of parthenogenetically activated embryos.

overall_design: chemically modified FLI maturation medium and its potential to improve the efficiency of in vitro produced embryos

first: Lucie

last: Nemcova

first: Frantisek

last: Strejcek

first: Ahmed

last: Gad

first: Veronika

last: Kinterova

first: Martin

last: Morovic

first: Michal

last: Benc

first: Radek

last: Prochazka

first: Jozef

last: Laurincik


title: Cohesin-mediated chromatin folding dynamics and transcriptional interactions

city: FARMINGTON

first: Yijun

last: Ruan

state: CT

address: 10 DISCOVERY DR

department: Ruan

institute: The Jackson Laboratory

summary: We apply ChIA-Drop, a single-molecule ligation-free mapping technique, to visualize loop formation over time. Our results demonstrated a cohesin-centric framework that coordinates with CTCF and RNAPII, respectively. At the loading (NIPBL binding) site, cohesin is highly correlated with transcriptional activities and translocates bi-directionally toward CTCF motif sites, where CTCF likely provides the anchoring point for cohesin to actively reel the chromatin template according to the motif forward orientation. Furthermore, cohesin specifically co-localizes with RNAPII and together mediates multiplex chromatin interactions that involve promoters, enhancers and super-enhancers. Intriguingly, single-molecule mapping data revealed that individual constituents of super-enhancers interact with target genes singularly and in cascade through intermediate regulatory elements, suggesting a probability-based mechanism for transcription regulation.

overall_design:
The microfluidics device (10X Genomics’ Chromium instrument) produces Gel beads in Emulsion (GEM) with chromatin complexes for complex-specific DNA barcoding and amplification. Barcoded DNA amplicons from all GEMs (droplets) are pooled for DNA sequencing and mapping for identifying chromatin interactions.
In the ChIA-Drop experiment, the microfluidics device (10X Genomics? Chromium instrument) produces Gel beads in Emulsion (GEM) with chromatin complexes for complex-specific DNA barcoding and amplification. Barcoded DNA amplicons from all GEMs (droplets) are pooled for DNA sequencing and mapping for identifying chromatin interactions.
In the Hi-C experiment, the fragmented chromatin DNA was ligated after DNA ends was repaired with biotin labeled nucleotide. The ligated chromatin was sheared and then ligated with adaptors.  The fragments were amplified after the ligated chromatin DNA was enriched by streptavidin beads. The chromatin interactions will be defined according to the mapping data of two ligated DNA fragments (tags).
In the ChIA-PET experiment, the protocol is similar with Hi-C except two points: 1). The fragmented chromatin DNAs were ligated by biotin labeled 19bp linker rather than end repairment with biotin labeled nucleotide. 2). The ligated chromatin DNAs pulled down by antibody of interested protein through chromatin immunoprecipitation was tagmented by Tn5 transponase, which will be used for PET cluster amplification.
In the ChIP-Seq experiment, the fixed chromatin was sheared by sonication and followed by chromatin immunoprecipitation with antibody of specific protein. The qualified ChIP-DNA will be used for library preparation to define the specific binding sites of target protein at the whole genome level.
In the RNA-Seq experiment, total mRNA will be enriched for the library preparation. The whole transcriptome data will be used to reveal the presence and quantity of RNA expressed in the sample.

first: Minji

last: Kim

first: Ping

last: Wang

first: Byoungkoo

last: Lee


title: KDM5C safeguards the appropriate timing of early neurodevelopment [CUT&RUN]

status: Public on Sep 25 2023

city: Boston

first: Violetta

last: Karwacki-Neisius

state: MA

address: 300 Longwood Avenue

department: Newborn Medicine

institute: Boston Children's Hospital

summary: We generated human induced pluripotent cells from intellectual disability patients carrying the c.2T>C mutation in KDM5C (Called “Mutant”). We generated a paired, isogenic human iPS cell line (called “Corrected”) using CRISPR/Cas9 and PiggyBac gene-editing technologies and conducted neuronal differentiation based on “Yichen Shi et al. Nat. Protoc. 7, 1836–1846 (2012)” to define differences in binding of the KDM5C gene in Mutant and Corrected cells.

overall_design: CUT&RUN samples for KDM5C binding  KDM5C-Mutant (Mut) and Corrected (Corr) cells at day 16 of neuronal differentiation.

first: Yang

last: Shi

first: Scott

last: Pomeroy

first: Alan

last: Jiao


title: Identification of downstream target genes of NR2F1 in gastric cancer

city: Shiyan

first: Qin

last: Shanshan

address: Renmin Road 30, Maojian District

country: China

institute: Institute of Basic Medical Sciences, School of Basic Medical Sciences, Hubei University of Medicine

summary: This study aims to identify the downstream target genes of NR2F1 in gastric cancer.

overall_design: To reveal the molecular mechanism by which NR2F1 drives gastric cancer progression, we established GC cell lines silencing/overexpressing NR2F1. The transcriptome sequencing studies were performed by deep sequencing, using Illumina HiSeq 4000.

first: Shanshan

last: Qin

first: Dandan

last: Li


title: Single-cell RNA-seq of heart from mouse-rat chimeric embryos

status: Public on Sep 20 2023

city: Pamplona

first: Asier

last: Ullate-Agote

state: Navarra

address: Av. de Pío XII, 55

department: Hemato-Oncology

institute: CIMA

summary: By combining  Nkx2.5-Cre x diphtheria toxin ablation (DTA) and interspecies blastocyst complementation we have generated rat hearts into mouse embryos at E10.5.  These hearts have been compared by single cell-RNA sequencing with the hearts of stage matched E11.5 rat embryo. All heart specific cell types were found in complemented chimeras, and they showed high correlation with the control. This suggests that the mechanisms of formation of the heart are compatible between mouse and rat, and that all cardiac cell types are specified correctly in the rat-to-mouse chimeras. However, global gene expression comparison identified some genes differentially expressed between complemented chimeras and control. Notably, the most relevant transcriptomic differences between rPSCs-derived hearts and rat embryonic hearts were associated with metabolism, specifically, with a decrease in oxidative phosphorylation and an increase in glycolysis processes in the rPSCs-derived cells of chimeras. Additionally, vascular endothelial cells of the complemented chimeras augmented the expression of genes related to vasculogenesis and vascular development. These changes are suggestive of a low level of oxygen in the embryo.

overall_design: Single-cell RNAseq of the heart region of rat-mouse heart complemented embryos and comparison with a stage matched rat heart region

first: Xabier

last: Aranguren


title: KDM5C safeguards the appropriate timing of early neurodevelopment [ATAC-seq]

first: Shannan

last: Ho Sui

state: Massachusetts

address: 401 Park Drive, Level 3 East

department: Biostatistics

institute: Harvard T.H. Chan School of Public Health

summary: We generated human induced pluripotent cells from intellectual disability patients carrying the c.2T>C mutation in KDM5C (Called “Mutant”). We generated a paired, isogenic human iPS cell line (called “Corrected”) using CRISPR/Cas9 and PiggyBac gene-editing technologies and conducted neuronal differentiation based on “Yichen Shi et al. Nat. Protoc. 7, 1836–1846 (2012)” to define differences in gene expression between the Mutant and Corrected and Mutant and Corrected treated with either a Wnt inhibitor (indicated as "+inh") or activator (indicated as "+W") during neuronal differentiation.

overall_design: ATAC-seq samples from KDM5C-Mutant, Corrected, Mutant+Inhibitor and Corrected+Activator (+W) cells were collected over the time course of neuronal differentiation. The time points used were: day 8, 12, 16, and day 32.


title: Nanostring of gene expression in intrahepatic leukocytes from wild type (WT) or myeloid-specific RAGE knockout (RAGE-MKO) mice fed a chow or high-fat, -fructose, and -cholesterol (FFC) diet

status: Public on Sep 15 2023

city: Rochester

first: Harmeet

last: Malhi

state: MN

address: 200 1st ST SW

institute: Mayo Clinic

summary: Intrahepatic macrophages in nonalcoholic steatohepatitis (NASH) are heterogenous and include proinflammatory recruited monocyte derived macrophages. RAGE is expressed on macrophages and can be activated by damage associated molecular patterns (DAMPs) upregulated in NASH, yet the role of macrophage-specific RAGE signaling in NASH is unclear. Therefore, we hypothesized that a subset of RAGE expressing macrophages are proinflammatory and mediate liver inflammation in NASH. We profiled the transcriptome of intrahepatic leukocytes in a murine model of diet induced NASH in WT and RAGE-MKO mice. By Nanostring nCounter Immunology Codeset Analysis of IHLs, we observed that myeloid-specific RAGE deletion resulted in amelioration of activation of macrophage and T cell pathways which was associated with attenuation of liver inflammation.

overall_design: C57BL/6J WT and RAGE-MKO mice were fed a high-fat, -fructose, and -cholesterol (FFC) diet to induce NASH for 6 months. After enzymatic digestion of equal amounts of liver tissue, IHLs were isolated by density Percoll-gradient ultracentrifugation. Gene expression analysis was conducted using nCounter Mouse Immunology Codeset (Nanostring, Seattle, WA) on IHLs from 4 biological replicates within each group.

first: Gopanandan

last: Parthasarathy


title: Nanostring of gene expression in intrahepatic leukocytes from mice fed a high-fat, -fructose, and -cholesterol (FFC) diet

overall_design: Refer to individual Series


title: Nanostring of gene expression in intrahepatic leukocytes from mouse fed a chow or high-fat, -fructose, and -cholesterol (FFC) diet and treated with vehicle or a pharmacological inhibitor of the receptor for advanced glycation end products (RAGE), TTP488

summary: Intrahepatic macrophages in nonalcoholic steatohepatitis (NASH) are heterogenous and include proinflammatory recruited monocyte derived macrophages. RAGE is expressed on macrophages and can be activated by damage associated molecular patterns (DAMPs) upregulated in NASH, yet the role of macrophage-specific RAGE signaling in NASH is unclear. Therefore, we hypothesized that a subset of RAGE expressing macrophages are proinflammatory and mediate liver inflammation in NASH. We profiled the transcriptome of intrahepatic leukocytes in a murine model of diet induced NASH where mice received vehicle or a pharmacological RAGE inhibitor (TTP488). By Nanostring nCounter Immunology Codeset Analysis of IHLs, we observed that RAGE inhibition resulted in amelioration of FFC diet induced activation of macrophage and T cell pathways which was associated with attenuation of liver inflammation.

overall_design: C57BL/6J mice were fed a high-fat, -fructose, and -cholesterol (FFC) diet to induce NASH or chow diet as control for 5 months. FFC fed mice received daily intraperitoneal injections of an antagonist of the extracellular domain of RAGE (TTP488) or vehicle for 30 days. After enzymatic digestion of equal amounts of liver tissue within each dietary group, IHLs were isolated by density Percoll-gradient ultracentrifugation. Gene expression analysis was conducted using nCounter Mouse Immunology Codeset (Nanostring, Seattle, WA) on IHLs from 3 biological replicates within each group.


title: Investigating how the SPT2 histone chaperone affects chromatin structure in human osteosarcoma cells.

city: Cambridge

first: Francesco Nicola

last: Carelli

address: Henry Wellcome Building of Cancer and Developmental Biology,  Tennis Court Road

institute: University of Cambridge

summary: Histone chaperones play essential roles in the regulation of chromatin structure. Here, we investigated how the SPT2 histone chaperone controls chromatin structure and accessibility in human cells (using U-2 OS osteosarcoma cells as model; these cells also express a H3.3-SNAP transgene). To achieve this, we performed ATAC-seq comparing WT U2OS vs three independent SPT2 KO clones (KO-3, KO-4 and KO-6

overall_design: We performed ATAC-seq on asynchronously growing U-2 OS cells; three independent replicates of the experiment were performed.

first: Giulia

last: Saredi

first: Francesco

first: Julie

last: Ahringer

first: John

last: Rouse


title: IFN-λ signaling protects mice from severe HSV-1 skin disease II

status: Public on Sep 12 2023

city: Chapel Hill

first: Drake

last: Philip

state: North Carolina

address: 104 Airport Drive

institute: University of North Carolina-Chapel Hill

summary: Type III interferons (IFN-λ) are antiviral and immunomodulatory cytokines that have been best characterized in respiratory and gastrointestinal infections, but the effects of IFN-λ against skin infections have not been extensively investigated. We sought to define the skin-specific effects of IFN-λ against the highly prevalent human pathogen herpes simplex virus (HSV). We infected mice lacking the IFN-λ receptor (Ifnlr1-/-), both the IFN-λ and the IFN-αβ receptor (Ifnar1-/- Ifnlr1-/-), or IFN-λ cytokines (Ifnl2/3-/-) and found that IFN-λ restricts the severity of HSV-1 and HSV-2 skin lesions, independent of a direct effect on viral load. Using conditional knockout mice, we found that IFN-λ signaling in both keratinocytes and neutrophils was necessary to control HSV-1 skin lesion severity, and that IFN-λ signaling in keratinocytes suppressed CXCL9-mediated neutrophil recruitment to the skin. Furthermore, depleting neutrophils prevented the development of severe HSV-1 skin lesions in Ifnlr1-/- mice. Altogether, our results suggest that IFN-λ plays an immunomodulatory role in the skin that restricts neutrophil-mediated pathology during HSV infection, and suggest potential applications for IFN-λ in treating viral skin infections.

overall_design: To uncover the role for IFN-λ signaling in epithelial cells in protecting mice from severe HSV-1 skin disease. To differentiate effects that were specific to IFN-λ, and not necessarily dependent on shared canonical signaling with IFN-β, we treated A549s (RenLuc, IFNLR1, and IFNAR1) with both IFNs or media alone for 8 hours and defined differentially expressed genes.

middle: T

first: Nigel

last: Goins

first: Nicholas

middle: J

last: Catanzaro

first: Ichiro

last: Misumi

first: Jason

middle: K

last: Whitmire

first: Hannah

last: Atkins

first: Helen

last: Lazear


title: The Effect of Immunotherapy PD-1 Blockade on Acute Bone Cancer Pain: Insights from Transcriptomic and Microbiomic Profiling

status: Public on Sep 14 2023

city: Shanghai

first: Ruifeng

last: Ding

address: No. 415 Fengyang Road, Huangpu District

department: Department of Anesthesiology

institute: Changzheng Hospital, Second Affiliated Hospital of Naval Medical University

summary:
Background: The skeletal system ranks as the third most common site for cancer metastasis, often leading to pain with nociceptive and neuropathic features. PD-1-targeting therapeutic antibodies offer effective cancer treatment but can cause treatment-related acute pain. Understanding the mechanisms of this pain and identifying potential interventions is still a challenge.
Methods: A murine model of bone cancer pain was established using Lewis lung carcinoma (LLC) cells, followed by intravenous administration of nivolumab, a human anti-PD-1 monoclonal antibody. Pain thresholds were measured, and micro-CT images of the skeletal system were obtained. High-throughput sequencing of the spinal cord/colon transcriptome during the acute phase of bone cancer pain and gut microbiota analysis were performed. Immunofluorescence staining and western blot experiments assessed spinal cord microglia activation and acute pain-associated molecules.
Results: PD-1 inhibition with nivolumab protected against bone degradation initiated by LLC cell administration but consistently induced acute pain during nivolumab treatment. Spinal cord and colon transcriptomics revealed an immunopathological pattern during tumor progression and the acute pain phase, with notable changes in interleukin and S100 gene families. Gut microbiota analysis post-immunotherapy showed a decline in beneficial bacteria associated with SCFA production. Activation of spinal cord microglia and enhanced glycolytic metabolism were confirmed as key factors in inducing acute pain following immunotherapy.
Conclusions: This study reveals that nivolumab induces acute pain by activating microglia and enhancing glycolytic metabolism in the treatment of bone cancer and uncovers connections between transcriptomic changes, gut microbiota, and acute pain following ICB treatment. It offers novel insights into the relationship between immune checkpoint blockade therapies and pain management.

overall_design: mRNA profiles of colon and spinal dorsal horn from three groups (sham+IgG group, cancer+IgG group, and cancer+nivolumab group) were generated by RNAseq, with n=3, using Illumina Novaseq 6000 platform.

first: Huawei

last: Wei

first: Mengqiu

last: Deng

first: Xingshuai

last: Huang

first: Jinfang

last: Lu

first: Guowei

last: Jiang

first: Hongwei

last: Zhu

first: Hongbin

last: Yuan


title: KDM5C safeguards the appropriate timing of early neurodevelopment [RNA-seq]

first: Violetta

last: Karwacki-Neisius

summary: We generated human induced pluripotent cells from intellectual disability patient carrying the c.2T>C mutation in KDM5C (Called “Mutant”). We generated a paired, isogenic human iPS cell line (called “Corrected”) using CRISPR/Cas9 and PiggyBac gene-editing technologies and conducted neuronal differentiation based on “Yichen Shi et al. Nat. Protoc. 7, 1836–1846 (2012)” to define differences in gene expression between the Mutant and Corrected and Mutant and Corrected treated with either a Wnt inhibitor (indicated as "+inh") or activator (indicated as "+W") during neuronal differentiation.

overall_design: RNA-seq samples from KDM5C-Mutant, Corrected, Mutant+Inhibitor and Corrected+Activator (+W) cells were collected over the time course of neuronal differentiation. The time points used were: day 16 and day 32.

first: Yang

last: Shi

first: Scott

last: Pomeroy

first: Albert

last: Tai


title: IFN-λ signaling protects mice from severe HSV-1 skin disease I

first: Drake

last: Philip

overall_design: To uncover the role for IFN-λ signaling in keratinocytes in protecting mice from severe HSV-1 skin disease. To differentiate effects that were specific to IFN-λ, and not necessarily dependent on shared canonical signaling with IFN-β, we treated primary keratinocytes (WT, IFNLR1, IFNAR1, and DKO) with both IFNs or media alone for 24 hours and defined differentially expressed genes.

first: Nigel

middle: M

last: Goins

first: Nicholas

last: Catanzaro


title: Modeling Radiation-induced Cardiovascular Dysfunction with Human iPSC-Derived Engineered Heart Tissues

status: Public on Sep 05 2023

city: Stanford

first: Joseph

middle: C.

last: Wu

state: California

address: 240 Pasteur Dr

department: Stanford Cardiovascular Institute

institute: Stanford university

summary: Low-LET radiation can cause cardiovascular dysfunctions at high-dose rates. For example, photons used in thoracic radiotherapy are known to cause acute cardiac tissue damage with elevated serum cardiac Troponin I level and long-term cardiac complications when delivered as fractionated exposures at high-dose rates. However, the effects of continuous low-dose rate radiation exposure on the heart, which simulate the space radiation environment, have not been well-studied. In this study, we aim to model low-LET space radiation-induced cardiovascular dysfunction using human induced pluripotent stem cell (iPSC)-derived engineered heart tissues (EHTs) exposed to protracted γ-ray irradiation. The investigation of pathophysiological changes in this model may provide insights and guide the development of countermeasures. As a proof-of-principle for the application of this model in drug development, we also tested the protective effect of a mitochondrial-specific antioxidant, MitoTempo, on irradiated EHTs.

overall_design: In this study, EHTs were generated from two healthy iPSC lines (#1 and #2) using an established protocol, comprising of 70% cardiomyocytes (CMs), 15% endothelial cells (ECs) and 15% fibroblasts (FBs). After 10 days, the EHTs were exposed to 137Cs γ-rays continuously at a dose-rate of 9.3 mGy/hour for 5 days. Cells were collected for bulk RNA sequecning from two independent experiments.

first: Xu

last: Cao

first: Dilip

last: Thomas

first: Luke

last: Whitcombb

first: Mingqiang

last: Wang

first: Anushree

last: Chatterjee

first: Adam

last: Chicco

first: Michael

last: Weil


title: Identification of 1600 replication origins in S. cerevisiae

first: Eric

middle: Jan

last: Foss

address: 1100 Fairview Ave N

department: Clinical Research Division

institute: Fred Hutchinson Cancer Research Center

summary: There are approximately 500 known origins of replication in the yeast genome, and the process by which DNA replication initiates at these locations is well understood. In particular, these sites are made competent to initiate replication by loading of the Mcm replicative helicase prior to the start of S phase; thus, "a site to which MCM is bound in G1" might be considered to provide an operational definition of a replication origin. By fusing a subunit of Mcm to micrococcal nuclease, a technique referred to as "Chromatin Endogenous Cleavage", we previously showed that known origins are typically bound by a single Mcm double hexamer, loaded adjacent to the ARS consensus sequence (ACS). Here we extend this analysis from known origins to the entire genome, identifying candidate Mcm binding sites whose signal intensity varies over at least 3 orders of magnitude. Published data quantifying the production of ssDNA during S phase showed clear evidence of replication initiation among the most abundant 1600 of these sites, with replication activity decreasing in concert with Mcm abundance and disappearing at the limit of detection of ssDNA. Three other hallmarks of replication origins were apparent among the most abundant 5,500 sites. Specifically, these sites (1) appeared in intergenic nucleosome-free regions that were flanked on one or both sides by well-positioned nucleosomes; (2) were flanked by ACSs; and (3) exhibited a pattern of GC skew characteristic of replication initiation. Furthermore, the high resolution of this technique allowed us to demonstrate a strong bias for detecting Mcm double-hexamers downstream rather than upstream of the ACS, which is consistent with the directionality of Mcm loading by Orc that has been observed in vitro. We conclude that DNA replication origins are at least 3-fold more abundant than previously assumed, and we suggest that replication may occasionally initiate in essentially every intergenic region. These results shed light on recent reports that as many as 15% of replication events initiate outside of known origins, and this broader distribution of replication origins suggest that S phase in yeast may be less distinct from that in humans than is widely assumed.

overall_design: 1.  Chromatin Endogenous Cleavage (ChEC), in which either MCM2, MCM4 or MCM6 is fused to micrococcal nuclease (MNase), which is activated by addition of calcium after permeablizing cells with detergent.  2.  MNase-seq, in which exogenously added MNase digests DNA that is not bound to proteins, such as nucleosomes.

last: Jan Foss

first: Carmina

last: Lichauco

first: Tonibelle

last: Gatbonton-Schwager

first: Brandon

last: Lofts

first: Uyen

last: Lao

first: Toni

last: Bedalov


title: Targeting pathogenic CD8+ tissue-resident T cells with chimeric antigen receptor therapy in murine autoimmune cholangitis

city: guangzhou

first: Hao-Xian

last: ZHU

state: Guangdong

address: No. 106, Zhongshan 2nd Road

institute: Guangdong Provincial People’s Hospital

summary: Primary biliary cholangitis (PBC) is a cholestatic autoimmune liver disease secondary to an autoreactive T cell response against intrahepatic small bile ducts. Multiple murine models have demonstrated the critical role of effector CD8+T cells in this response and our work has used IL-12p40-/-IL-2Rα-/- mice (DKO mice) to study this issue. Herein we first demonstrated that use of either a CD8a knock-out or an anti-CD8a antibody prevents/reduces biliary immunopathology. Bulk and single-cell RNA sequencing analysis identifies CD8+ tissue resident memory T cells (Trm) in the liver of DKO mice, which highly express activation and cytotoxicity associated markers and have the ability to induce apoptosis of bile duct epithelial cells. Liver CD8+Trm cells also upregulate expression of several immune checkpoint molecules and in particular we identified PD-1 as a specific liver CD8+Trm cell marker in this model. Based on these data we constructed a novel chimeric antigen receptor containing a PD-L1 extracellular domain to target PD-1-expressing CD8+Trm cells. Importantly, treatment of DKO mice with PD-1 targeting CAR-T cells selectively depleted liver CD8+Trm cells in vivo and alleviated autoimmune cholangitis.

overall_design: To access the efficacy of anti-PD-1 CAR-T treatment, we extracted the total RNA of liver tissues of DKO mice after anti-PD-1 CAR-T or Ctrl-T infusion and their control littermates, , then we build the cDNA library and performed the bulk RNA sequencing.

first: Zhu

last: Hao-Xian

first: Yang

last: Shu-Han


title: Targeted gene expression panel for meningiomas

status: Public on Sep 06 2023

first: William

last: Chen

state: CA

address: 1450 3rd Street, HD 402

institute: UCSF

summary: A custom Nanostring nCounter panel of 100 or 75 meningioma related genes and reference genes was developed, and performed on human frozen and FFPE meningioma samples. This was done as part of a study developing and validating a targeted gene expression biomarker for meningioma outcomes after surgey and benefit from raditherapy.

overall_design: 1,202 human meningioma samples were processed for this study.

middle: C

first: David

last: Raleigh


title: Wnt signaling control by KDM5C during development impacts lifelong cognition

first: Violetta

last: Karwacki-Neisius

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


title: Single cell RNA sequencing analysis of fresh resected lung tissues from tuberculosis patients

first: Zilu

last: Wen

address: No. 2901, Caolang Road, Jinshan District, Shanghai

institute: SHANGHAI PUBLIC HEALTH CLINICAL CENTER

summary: Fresh resected lung tissues were obtained from six tuberculosis patients with elevated pulmonary 18F-FDG avidity. Lung tissues with 18F-FDG avidity and nearby uninvolved tissues were profiled with single-cell RNA sequencing.

overall_design: Fresh resected human lung tissues with high 18F-FDG avidity and nearby uninvolved tissues with background or low 18F-FDG avidity were profiled with single-cell RNA sequencing.

first: Lin

last: Wang

first: Hui

last: Ma

first: Meiyi

last: Li

first: Ka-Wing

last: Wong

first: Yanzheng

last: Song


title: MYC dependency in GLS1 and NAMPT is a therapeutic vulnerability in multiple myeloma

status: Public on Sep 04 2023

city: Lille

first: Lama

last: Hasan Bou Issa

address: 1 Rue du Professeur Calmette

country: France

institute: Canther

summary: Multiple myeloma is an incurable hematological malignancy evolving from precursor states to advanced phases of the disease. MYC abnormalities play a critical role in the disease progression. Nevertheless, MYC lacks therapeutic drugability, thereby necessitating the exploration of alternative strategies aimed at circumventing the challenges associated with targeting MYC. In this study, we hypothesized that MYC upregulation induces genomic dependencies in tumor cells, creating vulnerabilities that can be exploited therapeutically. We discovered a differential dependency on glutamine metabolism in MYC overexpressing cells. We functionally explored these dependencies as a selective targetable vulnerability in vitro and in vivo. Furthermore, we uncovered a potential synergistic combinations that can exacerbated this metabolic vulnerability, Collectively, our in vitro and in vivo results revealed an effective therapeutic combinatory strategy in the context of MYC overexpressing MM.

overall_design: We generated a multiple myeloma isogeneic model overexpressing MYC in U266 cell line transduced with EF1A-C-MYC lentiviral vector and we analysed the transcriptome of the generated cell lines,

first: Salomon

last: Manier

first: Léa

last: Fléchon

first: Romanos

last: Sklavenitis Pistofidis


title: RNA-seq analysis identifies differentially expressed genes regulated by LPS exposure on HNEpC cells

status: Public on Sep 01 2023

first: Hai

last: Lin

state: Shanghai

address: No. 600, Yishan Road, Xuhui District

institute: Shanghai Sixth People's Hospital Affiliated to Shanghai Jiao Tong University School of Medicine

summary: In this study, we performed high-throughput sequencing to evaluate the gene expression profiles of  Human Nasal Epithelial Cells (HNEpC) (C-12620, PromoCell, Heidelberg, Germany) after exposure to LPS for 6 hours.

overall_design: Comparison of mRNA expression profiles between LPS exposed HNEpC cells and control cells.

first: Weitian

last: Zhang


title: Mir-9-1 controls osteoblastic regulation of lymphopoiesis

status: Public on Aug 31 2023

city: Milwaukee

first: Demin

state: WI

address: 8727 West Watertown Plank Road

institute: Versiti Blood Research Institute

summary: The highly conserved MicroRNA-9 (miR-9) family consists of three members. We discovered that miR-9-1 deletion reduced mature miR-9 expression, causing 43% of the mice to display smaller size and postweaning lethality. MiR-9-1-deficient mice with growth defects experienced severe lymphopenia, but other blood cells were unaffected. The lymphopenia wasn’t due to defects in hematopoietic progenitors, as mutant bone marrow (BM) cells underwent normal lymphopoiesis after transplantation into wild-type recipients. Additionally, miR-9-1-deficient mice exhibited impaired osteoblastic bone formation, as mutant mesenchymal stem cells (MSCs) failed to differentiate into osteoblastic cells (OBs). RNA sequencing revealed reduced expression of master transcription factors for osteoblastic differentiation, Runt-related transcription factor 2 (Runx2) and Osterix (Osx), and genes related to collagen formation, extracellular matrix organization, and cell adhesion, in miR-9-1-deficient MSCs. Follistatin (Fst), an antagonist of bone morphogenetic proteins (BMPs), was found to be a direct target of miR-9-1. Its deficiency led to the up-regulation of Fst, inhibiting BMP signaling in MSCs, and reducing IL-7 and IGF-1. Thus, miR-9-1 controls osteoblastic regulation of lymphopoiesis by targeting the Fst/BMP/Smad signaling axis.

overall_design:
To explore the potential regulation of miR-9-1 in the lymphopoietic BM niche, we used CRISPR/Cas9-meidated genome editing to generate miR-9-1-deficient mice.
We then performed a high-throughput RNA sequencing analysis of global gene expression patterns in MSCs and OBs from 4 miR-9-1-deficient and wild-type mice.
Comparative global gene expression patterns analysis of RNA-seq data for miR-9-1-deficietn and wild-type MSCs and OBs.

first: Yongguang


title: Differences in PBMC mRNA profiles between control and type 2 diabetic mice

city: Nanjing

first: Tai

last: Guangjie

state: Jiangsu

address: Longmiandadao 639

department: Clinical pharmacy

institute: China pharmaceutical university

summary: Studies on immunometabolism have indicated that metabolism and the immunological state are inherently interconnected . Recent research has highlighted the impacts of metabolic syndrome on innate and adaptive immune function, which are linked to overall negative effects on the progression of related diseases . Notably, aging of immune system can be potentiated by dysmetabolism, culminating in chronic inflammation and immune fitness loss . Several age-related immune changes such as progressive loss of naive T cells, an increase in memory cells, and increased percentages of cells with immunosenescence markers, were identified in patients with T2DM . Dysfunction of both innate and adaptive immune responses in T2DM manifests similarly to aging, including poor control of infection and reduced vaccination response, along with elevated inflammatory activity.Hence, we sought to investigate the mechanism of accelerated immunosenescence during T2DM.

overall_design: To investigate the mechanisms involved in T2DM-induced immunosenescence, we isolated PBMCs from T2DM and age-matched control mice for RNA sequencing

first: Guangjie

last: Tai

first: Yanjie

first: Junlin

last: Feng


title: Tumoral and stromal hMENA isoforms influence tertiary lymphoid structure localization and predict response to immunocheckpoint blockade in lung cancer patients

status: Public on Aug 30 2023

city: Rome

first: Lorenzo

last: D'Ambrosio

address: Via Elio Chianesi 53

department: Dept. Of Research, Advanced Diagnostic, and Technological Innovation

institute: Regina Elena National Cancer Institute, IRCCS

summary:
The advent of immunotherapy in the clinical practice of non-small cell lung cancer (NSCLC) have highlighted the impact of the tumor immune microenvironment in the control of the tumor progression, as well as in the response to immune checkpoint blockade (ICB) therapy. Tertiary Lymphoid Structures (TLS), determinants of antitumor immunity, are associated with a favorable clinical outcome in NSCLC patients and with the effectiveness of ICB in several tumors. The actin regulatory protein hMENA undergoes tissue-specific splicing, with tumors expressing hMENA11ahigh/ stromal fibronectin (FN1)low related to early N0 NSCLC patients with a favorable prognosis. hMENA/hMENA∆v6 is overexpressed in pro-tumoral CAFs related to the subtype of immunosuppressive ECM-my-CAF
Herein, by analyzing the pattern of hMENA isoform expression in tumor cells and CAFs, we highlighted the role of hMENA as a crucial factor in the communication among tumor cells CAFs, T and B cells. Mechanistically, we demonstrated that in NSCLC cell lines hMENA11a increases the expression of the TLS regulator LTβR, reduces the FN1 expression and favors CXCL13 production by tissue resident T cells. Conversely in CAFs, hMENA/hMENAΔv6 inhibits the NF-kB pathway downstream to LTβR and the secretion of CXCL13, crucial in TLS formation. Of clinical relevance, our experimental data support our finding in primary tumors that TLS localized within the tumor area (TLS-IT) are predictive of a longer disease-free survival in N0 NSCLC patients and are accompanied by the maturation of B cells in the tumor tissue. TLS-IT associate with paucity of hMENA positive CAFs, low stromal FN1 and high hMENA11a tumor cell expression. To translate these results in a clinical setting of ICB treated patients, we identified a signature based on the pattern of hMENA isoforms, FN1 and LTβR expression that may discriminate NSCLC patients responding or not-responding to ICB.

overall_design: Gene expression profiling through RNA-seq for each biological replicate of H1650 cell line transfected with siControl, sihMENA(t), siMENA11a.

first: Francesca

last: Di Modugno

first: Anna

last: Di Carlo

first: Daniel

last: D'Andrea

first: Isabella

last: Sperduti

last: De Nicola

first: Paola

last: Nisticó


title: Differences in PVAT mRNA profiles of recipient mice transplanted with splenocytes

summary: Recent investigations raise the possibility that senescent immune cells may fuel vascular damage by perivascular adipose tissue (PVAT) dysfunction, as immune cell infiltration in PVAT increases substantially in the development of vascular disease compared to the healthy state and leads to changes in PVAT-derived effector cytokine levels that comprise vascular biology.Though evidence revealed possible cross-talk among immune cells, PVAT and vasculature, it is less clear if senescent immune cells or a particular cell subset may induce PVAT dysfunction and further impair vascular biology during type 2 diabtes (T2DM). NLRP3 inflammasome plays a critical role in inflammatory response as a major component of innate immunity, it provides a molecular platform that responds to a variety of stimuli, initiating sterile inﬂammation cascade.Furthermore, there is evidence that NLRP3 inflammasome can be activated by sensing danger-associated molecular patterns (DAMPs), which in turn promotes age-related thymic demise and T cell senescence, implicating its potential role in immunosenescence. Here, we employed WT and Nlrp3 KO mice to develop T2DM, and implanted the splenocytes isolated from WT, Nlrp3 KO, WT-T2DM, Nlrp3 KO-T2DM mice that were modeled for 4 months to 3-month-old WT mice. Then, we explored the differentially expressed genes of PVAT in recipient mice implanted with splenocytes and evaluate immune infiltration of PVAT.

overall_design: To investigate the immune infiltration profiles of PVAT among recipient mice and explore the impacts of diabetic immune cells on PVAT function.(WTS-R: recipient for splenocyte from WT mice; NS-R: recipient for splenocyte from Nlrp3 KO mice; TS-R: recipient for splenocyte from T2DM mice; NTS-R:recipient for splenocyte from Nlrp3 KO T2DM mice )

last: Ma


title: circRNAs expression profiling analysis of peripheral blood from patients with β-thalassemia intermedia and major

status: Public on Aug 20 2023

city: Fuzhou

first: Hailong

last: Huang

state: Fujian

address: No.18 Daoshan Road, Gulou District

institute: Fujian Maternity and Child Health Hospital

summary: To explore the role of circRNAs in the regulation of β-thalassemia and provide new evidence and targets for the treatment of β-thalassemia, circRNAs expression profiling was analyzed from patients with β-thalassemia intermedia and major.

overall_design: Peripheral blood samples of patients with β-thalassemia intermedia and major (n=6) and healthy controls (n=6) were collected, total RNA was extracted and digested with Rnase R to remove linear RNAs and enrich circRNAs. The differentially expressed circRNAs were screened using an Arraystar Human circRNA Array (8x15K, Arraystar).


title: Effect of increased population density on gene expression in nematode Caenorhabditis elegans

status: Public on Aug 29 2023

city: Warsaw

first: Michal

last: Turek

address: Pwinskiego 5a

country: Poland

institute: Institute of Biochemistry and Biophysics Polish Academy of Sciences

summary:
To investigate the effect of increased population density on gene expression in the nematode Caenorhabditis elegans we grew worms as solitary animals or in a ten animals population.
We then performed gene expression profiling analysis using data obtained from RNA-seq of worms grown in both types of conditions (3 biological replications per condition)

overall_design: Comparative gene expression profiling analysis of RNA-seq data for worms grown as solitary animals and in ten animals population

first: Agata

last: Szczepanska


title: FLT3-ITD mutation expands dendritic cells and alters CD4+ T cells in acute myeloid leukemia

status: Public on Aug 23 2023

city: Portland

first: Evan

middle: F.

last: Lind

state: OR

address: Mail Code L220, 3181 SW Sam Jackson Park Road

department: Molecular Microbiology and Immunology

institute: Oregon Health & Science University

summary: Fms-like tyrosine kinase 3 (FLT3) is a critical receptor for functional dendritic cell (DC) development. Mutations associated with FLT3 are commonly observed in acute myeloid leukemia (AML) patients. Internal tandem duplication (FLT3-ITD) results in ligand-independent constitutive signaling and promotes tumor survival. Basic characterization of dendritic cells in the context of AML is lacking, therefore we investigated how FLT3-ITD impacts DC homeostasis and what downstream affects on the immune system may be. During AML we found that patient bone marrow DC homesostasis is disrupted and in a mouse model of AML we found that T-bet- cDC2s are increased in vivo and both Treg and Th17 cells are increased. In vitro co-cultures of AML DCs and naive OT-II cells results in more Th17 phenotype by IL-17A secretion. We propose that in FLT3-ITD+ AML DC homeostasis is dysregulated and Th17 cells are increased as a result.

overall_design: Spleens from aged matched (36 weeks) AML mice and healthy WT mice were processed for ADT and sc-RNAseq (3' GEX using 10X Genomics Chromium Platform).

first: Flynn

last: Patrick A

first: Long

last: Mark D

first: Kosaka

last: Yoko

first: Lind

last: Evan


title: A novel human fetal lung-derived alveolar organoid model reveals mechanisms of surfactant protein C maturation relevant to interstitial lung disease

first: Kyungtae

last: Lim

email: kl492@cam.ac.uk, ktlim492@korea.ac.kr

address: Tennis Court Road

department: Gurdon Institute

summary: During human lung development the distal tip epithelial cells act as multipotent progenitors. From ~15 pcw (post conception weeks) the human tip epithelium retains progenitor marker expression, but also upregulates markers of the AT2 lineage. Immature AT2 cells appear in the tissue from 17 pcw. We have recently shown that 16-22 pcw epithelial tip cells can be expanded as organoids and differentiated to AT2 cells. However, the differentiated AT2 cells were not proliferative, limiting their use for functional studies and genetic manipulation. We have now developed a highly robust, efficient, and scalable culture condition (AT2 medium) that induces the differentiation of 16-22 pcw fetal lung tip cells into mature AT2 cells which grow as expandable 3D organoids. In this study, we characterise the fetal-derived AT2 cells, showing that they are stable over long-term passaging, efficiently process and secrete surfactant, and can differentiate into AT1 cells in vitro and in mouse lung transplantation assays. Forward genetic screen identifies candidate effectors of SFTPC trafficking which we validate using CRISPR interference (CRISPRi) in the AT2 organoids. We show that the trafficking of SFTPC requires ubiquitination by HECT domain E3 ligases, particularly Itch, and that their depletion phenocopies the pathological redistribution seen for the SFTPCI73T variant.

overall_design:
Four biological AT2 organoid lines cultured in AT2 medium were used for RNA seq analysis; each at early and late passages.
Four biological AT2 organoid lines cultured in AT2 medium were used for single cell RNA seq.

>> AT2_merged.gene_counts_length_scaled.tsv <<
A comparison between the RNA sequencing data of AT2 organoids and publicly available data of fetal organoid types and other alveolar cell types was performed:

fetal early tip progenitor organoids GSM5393370 and GSM5393371; fetal late tip progenitor organoids6 GSM5393372 and GSM5393373; PSC-iAT2s7 GSM5578511, GSM5578512 and GSM5578513; cultured adult AT2 cells7 GSM5578508, GSM5578509 and GSM5578510; freshly isolated adult AT2 cells8 GSM2537127, GSM2537128 and GSM2537129

Re-analysed Samples in GSE184143/PRJNA763363, GSE96642/PRJNA379280, GSE178528/PRJNA739556:

data column header; re-analyzed sample accns and title
adult_AT2_1; GSM2537127 SRR5345478 SAMN06603895 Adult AEC2_A
adult_AT2_2; GSM2537128 SRR5345479 SAMN06603915 Adult AEC2_B
adult_AT2_3; GSM2537129 SRR5345480 SAMN06603914 Adult AEC2_C
adult_AT2_CKDCI_1; GSM5578508 SRR15902408 SAMN21438592 adult_AT2_CKDCI_MRC5_1
adult_AT2_CKDCI_2; GSM5578509 SRR15902409 SAMN21438593 adult_AT2_CKDCI_MRC5_2
adult_AT2_CKDCI_3; GSM5578510 SRR15902410 SAMN21438594 adult_AT2_CKDCI_MRC5_3
Early_tip_progenitor_1; GSM5393372 SRR14869977 SAMN19792069 14404_folded_P2_RNA-seq
Early_tip_progenitor_2; GSM5393373 SRR14869978 SAMN19792068 14387_folded_P3_RNA-seq
Late_tip_progenitor_1; GSM5393370 SRR14869975 SAMN19792071 BRC1915_CS20_P5_RNA-seq
Late_tip_progenitor_2; GSM5393371 SRR14869976 SAMN19792070 13393_CS23_P5_RNA-seq
PSC_iAT2_3D_CKDCI_1; GSM5578511 SRR15902411 SAMN21438595 iAT2_3D_CKDCI_1
PSC_iAT2_3D_CKDCI_2; GSM5578512 SRR15902412 SAMN21438596 iAT2_3D_CKDCI_2
PSC_iAT2_3D_CKDCI_3; GSM5578513 SRR15902413 SAMN21438597 iAT2_3D_CKDCI_3

first: Peng

last: He

first: Livia

last: Delpiano

first: Emma

middle: L

last: Rawlins


title: Long-term effects of Omicron BA.2 breakthrough infection on immunity-metabolism balance: a 6-month prospective study

status: Public on Aug 16 2023

city: Beijing

first: Yanhua

last: Li

address: No.1 Beichen West Road, Chaoyang District

institute: Institute of Microbiology, Chinese Academy of Sciences

summary: There have been reports of long coronavirus disease (long COVID) and breakthrough infections (BTIs); however, the mechanisms and pathological features of long COVID after Omicron BTIs remain unclear. Assessing long COVID and immune recovery after Omicron BTIs is crucial for understanding the disease and developing and managing new-generation vaccines. Here, we followed up mild BA.2 BTI convalescents for six-month with routine blood tests and neutralization assay. Then, we applied proteomic analysis and single-cell RNA sequencing (scRNA-seq) to study the convalescents’ recovery status. Lastly, we retrospectively analyzed the clinical parameters related to immunity and metabolism of the convalescents. We found that major organs exhibited ephemeral dysfunction in double-Convidecia vaccinated persons who experienced BA.2 BTI and recovered to normal in approximately six-month. We observed durable and potent levels of neutralizing antibodies against major circulating severe acute respiratory syndrome coronavirus 2 sub-variants, including BQ.1, BQ.1.1 and BF.7, indicating that hybrid humoral immunity stays active. However, PLTs may take longer to recover. This was supported by proteomic and scRNA-seq analyses, which also showed coagulation disorder and an imbalance between anti-pathogen immunity and metabolism six-month after BA.2 BTI. The immunity-metabolism imbalance was then proved with retrospective analysis of abnormal levels of hormones, low blood glucose level and coagulation profile. The long-term malfunctional coagulation and imbalance in the material metabolism and immunity may contribute to the development of long COVID and act as useful indicators for assessing recovery and the long-term impacts after Omicron sub-variant BTIs.

overall_design:
We did single-cell RNA sequencing on 9 human samples, including 5 BA.2 breakthrough infected convalescents and 4 healthy people.
>>>Raw data for human samples have been submitted to China's Genome Sequence Archive (GSA, https://ngdc.cncb.ac.cn/gsa/) with accession number HRA004484<<<

first: George

last: Gao

first: Shijie

last: Qin

first: Lei

last: Dong


title: De novo TLK1 and MDM1 mutations in a patient with a neurodevelopmental disorder and immunodeficiency

status: Public on Aug 18 2023

city: Bethesda

first: Uma

last: Shankavaram

address: 9000 Rockville Pike

department: NCI

institute: NIH

summary: The Tousled-like kinases 1 and 2 (TLK1/TLK2) regulate DNA replication, repair and chromatin maintenance. TLK2 variants are associated with ‘Intellectual Disability, Autosomal Dominant 57’ (MRD57), a neurodevelopmental disorder (NDD) characterized by intellectual disability (ID), autism spectrum disorder (ASD) and microcephaly. Several TLK1 variants have been reported in NDDs but their functional significance is unknown. A male patient presenting with ID, seizures, global developmental delay, hypothyroidism, and primary immunodeficiency was determined to have a novel, heterozygous variant in TLK1 (c.1435C>G, p.Q479E) by genome sequencing (GS). Transcriptome sequencing in patient-derived cells confirmed expression of TLK1 transcripts carrying the p.Q479E variant and revealed alterations in genes involved in class switch recombination and cytokine signaling.

overall_design: Duplicate RNAseq samples from patient and sibling derived lymphoblastoid cell lines were compared.

first: Travis

last: Stracker